Dissertations / Theses on the topic 'Antibiotic susceptibility of ureaplasmas'

Bibliography
Thesis (PhD (Pathology. Medical Microbiology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Overview: Mycoplasmas and ureaplasmas are not routinely diagnosed and are under researched in South Africa. Prevalence, population shifts especially concerning genital flora and implications in infection or other conditions are unknown. Information pertaining to Mycoplasma pneumoniae in respiratory disease is similarly lacking. There is little information on antimicrobial susceptibilities and resistance development against Sexually Transmitted Infections (STI) syndromic management approaches. Aims: a) Elucidate mycoplasmal and ureaplasmal prevalence and contributing factors concerning cervical colonisation or preterm delivery in conjunction with HIV and Chlamydia trachomatis b) Investigate prevalence of M. pneumoniae in respiratory infections in conjunction with HIV, Mycobacterium tuberculosis and Pneumocystis jiroveci. c) Determine antimicrobial susceptibilities of mycoplasmas and ureaplasmas and analyse resistance genes. d) Assess the inter-generic transfer potential of resistance gene (tetM) between Ureaplasma spp. and Neisseria gonorrhea. Genital setting: The prevalence of genital mycoplasmas, ureaplasmas and Chlamydia on women attending their first prenatal visit, in conjunction with preterm labour or HIV status was investigated. For preterm labour (2003), 199 women were monitored for preterm delivery (<37 weeks); for colonisation and HIV (2005), 219 women were screened. Microbial detection was performed on DNA extracted from endocervical swabs employing PCR techniques. Colonisation was seen to be highest in the 14-20 year group from 2003. In women aged ±21 years, co-colonisation was 13% although there was a shift from co-colonisation with Mycoplasma hominis and Ureaplasma spp. in 2003 to other dual/triple combinations in 2005. Overall major trends from both collection periods were that the prevalence of Ureaplasma spp. tended to be higher in women ±26 years, whilst prevalence of C. trachomatis and M. hominis were lower. No association was evident between colonisation with M. hominis, U. urealyticum, Ureaplasma parvum and labour outcome. HIV status had no effect on the prevalence/co-colonisation of M. hominis, Ureaplasma spp. or C. trachomatis. Respiratory setting: Studies were conducted to determine the prevalence of community acquired atypical pneumonias in adults (M. pneumoniae and P. jiroveci) and neonates (mycoplasmas, ureaplasmas and Chlamydia trachomatis) in order to improve treatment management programmes in the Port Elizabeth region. Sputum specimens from 102 adult patients presenting with pneumonia/symptoms of pneumonia admitted to hospitals were assessed by PCR. Details of patient’s gender, age, HIV and Mycobacterium tuberculosis status were provided by the hospitals. Women were seen to be at high risk for community-acquired P. jiroveci colonisation. Overall, prevalence of P. jiroveci was 52.9% (54/102 patients). P. jiroveci was mainly associated with HIV (25/74) (P. jiroveci and HIV positive patients in patient sample for which clinical data and HIV status was available) and co-infection with M. tuberculosis was observed in 12 HIV cases and one HIV negative patient. No DHPS (20) or DHFR (17) resistance associated mutations were found in P. jiroveci. M. pneumoniae was detected in one patient. For prevalence studies (2007-2008) on atypical pneumonia in neonates, 69 endotracheal aspirates were obtained. PCR detection of M. hominis, U. urealyticum and C. trachomatis was performed and U. parvum detected in two specimens. Antibiotic susceptibilities and resistance genes: The following investigations on clinical isolates of U. parvum and U. urealyticum were conducted (i) antibiotic susceptibility profiles, (ii) detection of drug target gene mutations, or gene acquisitions and (iii) inter-generic resistance gene transfer potential to Neisseria gonorrhoeae. Culture techniques applied to 132 endocervical specimens provided 66 Ureaplasma cultures (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIC determinations to ofloxacin, erythromycin, tetracycline, doxycycline, azithromycin and josamycin were performed. Thirty-seven ureaplasma cultures were fully susceptible to all antibiotics tested; 21 showed intermediate resistance to erythromycin, azithromycin and ofloxacin; while seven were resistant to tetracycline, three of which were also resistant to doxycycline and one also resistant to azithromycin. Concerning ofloxacin resistance directed at quinolone resistance determining regions, a substitution of Ser83Leu in ParC was demonstrated in one intermediately-resistant Ureaplasma (MIC 4 µg/ml) while a triple substitution of Asp112Glu in GyrA along with Ala125Thr and Ala136Thr in ParC was found in six further intermediately-resistant strains. No mutations were found in strains with MICs 1 µg/ml. No mutations were detected in 23S rRNA operons, L4 or L22 proteins. TetM and int-Tn genes were found in seven tetracycline-resistant strains. On screening 59 tetracycline-susceptible and -intermediate strains, eleven whilst possessing an int-Tn gene lacked a large region of tetM and 48 only contained small regions of tetM. The tetM genes of the seven tetracycline-resistant strains were sequenced and comparisons performed against GenBank sequences of Neisseria gonorrhoeae, Streptococcus pneumoniae and U. urealyticum. For five strains tetM was seen to be highly mosaic in structure containing regions that were similar to those of the GenBank strains and others that were unique. In the tetM leader region, four hot spot recombination sites were identified that could certainly influence the formation of the mosaic structures, upstream insertion sequences/open reading frames and transposon regions that regulate expression. On characterising the int-Tn genes of the seven tetracycline-resistant strains, three types were present indicating transposons from different origins had integrated into ureaplasma genomes. Reciprocal tetracycline resistance gene transfer between ureaplasmas and N. gonorrhoeae were unsuccessful. However, low-level tetracycline resistance (MICs 4-8 µg/ml) was transferred to a U. parvum recipient from one U. urealyticum and three U. parvum donors that carried tetM with MICs 16-64 µg/ml. On tetM PCR analysis, tetM was not detected in the transformants. Conclusions: The importance of genital mycoplasmas, ureaplasmas and C. trachomatis in long term aetiologies requires further investigations, certainly in relation with syndromic management regimens that fail to reduce colonisation rates. The high prevalence of P. jiroveci, the presence of M. pneumoniae in cases of pneumonia and detection of U. parvum in two cases of neonatal pneumonia investigated emphasises that in the absence of definitive diagnoses, it is crucial to monitor treatment responses carefully, especially when first line antibiotic preferences are ß-lactams, in order to ensure adequate and informed delivery of medical care. The finding of transposon and/or tetM regions in all ureaplasmas investigated with or without full expression of tetracycline resistance, in conjunction with tetM gene diversity, certainly places ureaplasmas strongly in the picture for intra- and inter-generic exchange of antibiotic resistance genes.
AFRIKAANSE OPSOMMING: Oorsig: Mikoplasma en ureaplasma word nie roetineweg gediagnoseer nie en in Suid Afrika is nog min navorsing daaroor gedoen. Prevalensie, populasie verskuiwings, veral in genital flora, en die impliksies van infeksie en ander toestande is onbekend. Inligting rakende Mycoplasma pneumoniae in respiratoriese siekte is ook gebrekkig. Daar is min inligting beskikbaar rakende die antimikrobiale vatbaarheid en die ontwikkeling van weerstandigheid gesien teen die benadering tot sindromiese hantering van seksueel oordraagbare siektes. Doelwitte: a) Om inligting te verskaf oor die prevalensie van mikoplasma en ureaplasma en bydraende faktore betreffende voortydige kraam tesame met MIV en Chlamydia trachomatis. b) Ondersoek van die prevalensie van M. pneumoniae in respiratoriese infeksies tesame met MIV, Mycobacterium tuberculosis en Pneumocystis jiroveci. c) Bepaling van die antimikrobiale vatbaarheid van mikoplasma en ureaplasma en analisevan weerstandigheids gene. d) Bereken die inter-genetiese oordrag potensiaal van weerstandigheids gene (tetM) tussen Ureaplasma spp. en Naisseria gonorrhoeae. Genitale omgewing: Die prevalensie van genitale mikoplasma, ureaplasma en Chlamydia in vroue tydens hul eerste prenatale besoek, tesame met vroegtydige kraam en MIV status is ondersoek. In voortydige kraam (2003), is 199 vroue gemonitor vir voortydige kraam (<37 weke); vir kolonisasie en MIV (2005), is 219 vroue getoets. Mikrobiale toetsing is gedoen deur DNS te win vanaf endoservikale deppers met PKR tegnieke. Kolonisasie was die hoogste in die ouderdomsgroep 14.20 jaar, in 2003. In vroue van ±21 jaar was medekolonisasie 13% alhoewel daar en verskuiwing was van mede-kolonisasie met Mycoplasma hominis en Ureaplasma spp. in 2003 tot ander dubbel/trippel kombinasies in 2005. Die oorkoepelende tendens in altwee die tydperke van waarneming was dat die prevalensie van Ureoplasma spp. geneig was om hoër te wees in vroue ±26 jaar, terwyl prevalensie van C. trachomatis en M. hominis laer was. Geen assosiasie kon getoon word tussen koloniesasie met M. hominis, U. urealyticum, Ureaplasma parvum en uitkoms van kraam nie. MIV status het geen effek gehad op die prevalensie/mede-kolonisasie van M. hominis, Ureaplasma spp. of C. Trachomatis nie. Respiratories: Studies is gedoen om die prevalensie van gemeenskaps verworwe atipiese pneumonie in volwassenes (M. pneumoniae en P. jiroveci) en neonate (mikoplasma, ureaplasma en Chlamydia trachomatis) te bepaal om behandeling en hantering programme in die Port Elizabeth area te verbeter. Sputum monsters van 102 volwasse pasiënte wat presenteer het met pneumonie of simptome van pneumonie en wat tot hospitale toegelaat was, is ontleed. Besonderhede van die pasiënte se geslag, ouderdom, MIV en Mycobacterium tuberculosis status is deur die hospitale verskaf. PKR is gedoen met inleiers gerig teen die volgende gene: P. jiroveci vir die aantoning van mitokondriale groot subeenheid RNS en vir die analise van mutasies vir ko-trimoksasool weerstandigheid dihydropteroaat sintetase (DHPS) en dihydrofolaat reduktase (DHFR); M. pneumoniae vir die aantoning van P1 adhesien en 16S rRNS. Vroue het ‘n hoë risiko vir gemeenskapsverworwe P. jiroveci kolonisasie gehad. In die algemeen was die prevalensie van P. jiroveci 52.9% (54/102 pasiënte). P. jiroveci was hoofsaaklik geassosieerd met MIV (25/74) (P. jiroveci en MIV positiewe pasiënte in die pasiënt monster waarvoor daar kliniese data en MIV status bekend was) en mede-infeksie met M. tuberculosis is gesien in 12 MIV gevalle en een MIV negatiewe pasiënt. Geen DHPS (20) of DHFR (17) weerstandigheids geassosieerde mutasies is gevind in P. Jiroveci nie. M. pneumoniae was aangetoon in een pasiënt. Vir prevalensie studies (2007-2008) op atipiese pneumonie in neonate is 69 endotrageale aspirate verkry. PKR toetsing vir M. hominis, U. urealyticum en C. trachomatis is gedoen met ‘primers’ soos voorheen gepubliseer. Ureaplasma parvum is aangetoon in twee neonate met PKR met negatiewe kultuur resultate. Antibiotika sensitiwiteite en weerstandigheids gene: Die volgende toetse is gedoen op kliniese isolate van U. parvum en U. urealyticum (i) antibiotika sensitiwiteits profiele, (ii) aantoning van teiken geen mutasies, of geen aanwinste en (iii) potensiaal vir inter-generiese weerstandigheids geen oordrag na Neisseria gonorrhoeae. Kultuur tegnieke toegepas op 132 endoservikale monsters het 66 Ureaplasma kulture gelewer (35 U. parvum, 9 U. urealyticum, 22 U. parvum + U. urealyticum). MIK bepaling vir ofloksasien, eritromisien, tetrasiklien, doksisiklien, azitromisien en josamisien is gedoen. Sewe-en-dertig kulture was ten volle sensitief vir alle antibiotika wat getoets is; een-en twintig het intermediere weerstandigheid teenoor eritromisien, azitromisien en ofloksasien getoon, terwyl sewe weerstandig was vir tetrasiklien, drie daarvan was ook weerstandig vir doksisiklien. Wat betref ofloksasien weerstandigheid gemik teen kwinoloon weerstandigheids bepalende gebiede, is vervanging van Ser83Leu in ParC gedemonstreer in een intermedier weerstandige Ureaplasma (MIK 4 µml) terwyl en trippel vervanging van Asp112Glu in GyrA saam met Ala125Thr en Ala136Thr in ParC gevind is in ses ander intermedier weerstandige stamme. Geen mutasies is gevind in stamme met MIKs van MICs 1 µg/ml nie. Geeneen van die ureaplasma was weerstandig vir eritromisien/azitromisien nie en geen mutasies is gevind in 23S rRNA operons , L4 of L22 proteine nie. TetM en int- Tn gene is gevind in sewe tetrasiklien weerstandige stamme. 58 Tetrasiklien sensitiewe en .intermediere stamme is getoets, waarvan elf en int-Tn geen gekort het sowel as en groot deel van tetM, terwyl 48 slegs klein dele van TetM bevat het. Die tetM gene van die sewe tetrasiklein-werstandige stamme se geenvolgorde is bepaal en vergelykings is getref teenoor die GenBank volgordes van Neisseria gonorrhoeae, Streptococcus pneumoniae en U. urealyticum. In vyf stamme is gevind dat die tetM geen hoogs mosaiek in struktuur was met areas wat ooreenstem met die in GenBank stamme, en ander areas wat uniek is. In die tetM leier area, is vier ehot spot f herkombinasie areas geidentifiseer wat sekerlik die vorming van die mosaiiek strukture kon beinvloed, asook transposon areas wat geenuitdrukking bepaal. Met karakterisering van die int-Tn gene van die sewe tetrasikleinweerstandlige stamme, was drie tipes teenwoordig waarin transposons vanaf verskillende oorsprong aangedui was, geintegreerd met die ureaplama genome. Resiprokale tetrasiklien weerstandigheids geen oordrag tussen ureaplasma en n. gonorrhoea was nie suksesvol nie. Lae-vlak tetrasiklien weerstandigheid (MIK fs van 4 . 8 µg/ml) is wel suksesvol oorgedra na en U. parvum ontvanger vanaf een U. urealyticum en drie U. parvum ontvangers wat tetM gedra het met MIKs van 16-64 µg/ml. Met die analise van tetM met PKR, kon tetM nie aangetoon word in die transformante nie. Gevolgtrekkings: Die belang van genitale mykoplasma, ureaplasma en C. trachomatis in langtermyn etologie benodig verdere ondersoek, veral in die lig van die sindromiese behandeling regimes wat nie kolonisasie verminder nie. Die hoe prevalensie van P. jiroveci, die teenwoordigheid van M. pneumoniae in gevalle van pneumonie en die aantoning van U. parvum in twee gevalle van neonatale pneumonie benadruk dat, in die afwesigheid van en definitiewe diagnose, dit noodsaaklik is om respons tot behandeling sorgvuldig te moniteer, veral indien die eerste lyn antibiotika keuse ß-laktam antimikrobiale middels of kefalosporiene is, sodat behoorlike en ingeligde gesondheidsorg gelewer kan word. Die bevinding van transposon en/of tetM gebiede in alle ureaplasma wat ondersoek is met of sonder volle uitdrukking van tetrasiklien weerstandigheid, in samehang met tetM diversiteit, plaas verseker ureaplasma sterk in die prentjie vir intra- en inter-generiese uitruiling van antibiotika weerstandigheids gene.
Nelson Mandela Metropolitan University
National Research Foundation (NRF Thuthuka)
Medical Research Council

Os Mollicutes causam doenças em várias espécies animais de importância econômica, inclusive em bovinos. Neste estudo, foi avaliada por concentração inibitória mínima (CIM) e citometria de fluxo, a atividade de oito agentes antibacterianos (enrofloxacina, ciprofloxacina, gentamicina, claritromicina, cloranfenicol, oxitetraclina, tiamulina e tilosina) contra Ureaplasma diversum. Foram analisadas 24 amostras de isolados de campo oriundas da mucosa genital de fêmeas bovinas. As amostras foram confirmadas por crescimento em caldo, placa e por PCR. Os inóculos foram submetidos à analise de suscetibilidade aos antibióticos pelo método da microdiluição em microplaca e posteriormente analisados pelo citômetro de fluxo a fim de avaliar a atividade antimicrobiana nas células. A claritromicina apresentou os maiores índices de inibição in vitro, sendo a gentamicina considerada o antibiótico de menor espectro de ação nesse estudo. De acordo com as análises do citômetro, a gentamicina apresentou o menor número de células viáveis enquanto a tiamulina apresentou o maior número. Embora haja resultados destoantes entre as técnicas utilizadas, o citômetro de fluxo pode ser utilizado como uma boa ferramenta para auxiliar a avaliação da suscetibilidade desses microrganismos a antibióticos.
The Mollicutes cause disease in several economically important species, including cattle. In this study, was evaluated by minimum inhibitory concentration (MIC) and flow cytometry, the activity of eight antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, clarithromycin, chloramphenicol, oxitetraclina, tiamulin and tylosin) against Ureaplasma diversum. We analyzed 24 samples of field isolates originating from the genital mucosa of cows. The samples were confirmed by growth in broth, plate, and PCR. The inoculations were subjected to analysis of susceptibility to antibiotics by the method of micro-dilution plate and then analyzed by flow cytometry to assess the antimicrobial activity in cells. Clarithromycin showed the highest levels of inhibition in vitro, the antibiotic gentamicin considered lower spectrum of action in this study. According to the analysis of the flow cytometer, gentamicin showed the lowest number of viable cells as tiamulin showed the greatest number. Although there are divergent results between the techniques used, flow cytometry can be used as a good tool even help assess the susceptibility of microorganisms to antibiotics.

Treatment of tuberculosis requires months of antimycobacterial therapy. This tolerance to antibiotics displayed by Mycobacterium tuberculosis could be attributed to biofilm formation. Biofilms are the cause of many chronic infections. The aim of this thesis was to apply laboratory methods for the culture of Mycobacterium smegmatis and M. tuberculosis H37Rv biofilms and to further characterise these bacterial phenotypes in terms of their physiology, gene expression and drug susceptibility The Modified Robbins Device (MRD) and the Constant Depth Film Fermenter (CDFF) laboratory models were applied alongside the previously established well-plate pellicle model. Antibiotic efficacy studies of M. tuberculosis pellicles identified drug-tolerant bacteria. These pellicle biofilms exhibited tolerance to rifampicin and isoniazid many times above the planktonic minimum inhibitory concentration (MIC). CDFF biofilms were tolerant to a planktonic MIC of isoniazid. CDFF and pellicle biofilms of M. tuberculosis and pellicle biofilms of M. smegmatis were investigated in terms of their gene expression using microarrays to determine the underpinning molecular mechanisms behind biofilm formation Biofilms of both mycobacterial species upregulated lipid metabolism and biosynthesis. M. tuberculosis biofilms upregulated genes involved in the type seven secretion system (T7SS) and genes which code for PE/PPE proteins. T7SS is known to interact with some PE/PPE proteins, many of which are cell-surface associated. These gene expression profiles suggested significant restructuring of the cell wall and provides a genetic basis for extra-cellular matrix formation. Also, low levels of metabolic activity were identified within these biofilms using fluorescent staining with viability dyes and flow cytometry. Overall, this thesis provides a controlled method for mycobacterial biofilm formation using the CDFF and confirms that M. tuberculosis H37Rv biofilms comprise antibiotic tolerant cells.

The proposed project presents a methodology to detect how susceptible or resistant certain bacteria are to an applied antibiotic. This detection is achieved by calculating the area of Zone of Inhibition (ZOI) regions present in the petri dish and comparing the results to the prescribed standards. The ZOI regions are empty areas formed around an antibiotic disc when placed on a petri dish containing a sample of the bacterial culture. Digital image processing techniques are employed to automate the process of ZOI detection. Experimental results show that the proposed project is successful in detecting ZOI regions of various shapes, such as perfectly circular, irregular, and overlapping. The experimental results also show that the accuracy of detection is typically over 95%, and it remains above 90%, even when the image is degraded by additive Gaussian noise.

Staphylococci are opportunistic pathogens responsible for a range of infections. Many staphylococcal species are frequently found to be resistant to antibiotics. The environment is considered a potential reservoir of genes conferring antibiotic resistance, which known as the 'resistomes'. Monitoring the dissemination of antibiotic resistant staphylococci is instrumental to mitigating this global health risk. The overall aim of this study was to generate informative data regarding dissemination of antibiotic resistance in environmental and public settings. This included looking into the distribution, epidemiology characteristic and transfer of oxacillin resistant determinant mecA; gaining an insight into genomic features that contribute to multiple antibiotic resistance and pathogenicity of one S. epidermidis isolate; and understanding the stress responses in mediating oxacillin resistance in S. aureus. The use of MALDI-TOF MS allowed identification of staphylococci to species level. MALDI-TOF MS data were used for taxonomic analysis of staphylococci, and taxonomic data were then combined with isolation sites and antimicrobial susceptibility profiles to aid the understanding of dissemination of environmental resistant staphylococci. The widespread dissemination of antibiotic resistant staphylococci in the environment was demonstrated. 12% of staphylococci harboured mecA gene. Community associated SCCmec types IV and V were more prevalent than nosocomial associated SCCmec types I, II, and III in the environment. 52% of SCCmec were non-typable. In addition, 14 new environmental S. epidermidis MLST types were reported. 9 antibiotic resistant determinants that were responsible for the resistant to 7 antimicrobial classes have been identified in environmental S. epidermidis 118 (G6_2). Proteomic analysis revealed that stress responses, including SOS response, stringent response and heat shock response, mediate oxacillin resistance in S. aureus. These results demonstrate widespread multiple drug resistance in different staphylococcal species isolated from non-healthcare environments. This uncontrolled dissemination of multidrug resistant bacteria poses a potential public health threats.

Neisseria meningitidis, also known as the meningococcus, is a globally spread obligate human bacterium causing meningitis and/or septicaemia. It is responsible for epidemics in both developed and developing countries. Untreated invasive meningococcal disease is often fatal, and despite modern intensive care units, the mortality is still remarkably high (approximately 10%). The continuously increasing antibiotic resistance in many bacterial pathogens is a serious public health threat worldwide and there have been numerous reports of emerging resistance in meningococci during the past decades. In paper I, the gene linked to reduced susceptibility to penicillins, the penA gene, was examined. The totally reported variation in all published penA genes was described. The penA gene was highly variable (in total 130 variants were identified). By examination of clinical meningococcal isolates, the association between penA gene sequences and penicillin susceptibility could be determined. Isolates with reduced susceptibility displayed mosaic structures in the penA gene. Two closely positioned nucleotide polymorphisms were identified in all isolates with reduced penicillin susceptibility and mosaic structured penA genes. These alterations were absent in all susceptible isolates and were successfully used to detect reduced penicillin susceptibility by real-time PCR and pyrosequencing in paper II. In papers III and IV, antibiotic susceptibility and characteristics of Swedish and African meningitis belt meningococcal isolates were comprehensively described. Although both populations were mainly susceptible to the antibiotics used for treatment and prophylaxis, the proportion of meningococci with reduced penicillin susceptibility was slightly higher in Sweden. A large proportion of the African isolates was resistant to tetracycline and erythromycin. In paper V, the gene linked to rifampicin resistance, the rpoB gene, was examined in meningococci from 12 mainly European countries. Alterations of three amino acids in the RpoB protein were found to always and directly lead to rifampicin resistance. A new breakpoint for rifampicin resistance in meningococci was suggested. The biological cost of the RpoB alterations was investigated in mice. The pathogenicity/virulence was significantly lower in rifampicin resistant mutants as compared with susceptible wild-type bacteria.

Corynebacterium glutamicum is a Gram positive soil bacterium with high industrial importance in ton scale production of amino acids. Apart from that, it becomes more and more important for medical studies, where it serves as model organism due to its close relation to bacteria causing several pathogens such as tuberculosis, diphtheria and leprosy. C. glutamicum, like Mycobacterium tuberculosis, has a distinct cell wall which is composed of a peptidoglycan layer (murein) with covalently bound polysaccharide layers that are capped with mycolic acids. In addition, both organisms have a polar cell wall synthesis machinery which is spatially regulated by DivIVA (Wag31 in M. tuberculosis). The present study shows that DivIVA regulates cell wall synthesis upon direct interaction with the lipid II flippase RodA. RodA determines morphology and growth in C. glutamicum and is localized to the poles and septa. The absence of rodA results in growth defects and cell shape alterations as well as altered lipid II proliferation of the poles (polar cell growth is sustained). DivIVA is furthermore involved in chromosome segregation upon direct interaction with the partitioning ParB protein, which binds to parS sites on the chromosome, thus tethering the replicated nucleoids to the cell poles. Interactions of DivIVA with ParB and RodA were identified in a synthetic in vivo protein-protein interaction assay where fluorescently labeled proteins of interest are expressed in E. coli cells and interaction is analyzed microscopically. A decisive improvement of this assay is the application of FRET, which is more sensitive and allows quantification of interaction. In order to test whether ParB and RodA compete for the same interaction site in DivIVA, we mapped interaction sites of both proteins. It turned out that ParB binds to a middle region of DivIVA, whereas RodA binds to the N-terminal domain of DivIVA where one lysine residue is essential for interaction. To fight bacterial infections, that cause thousands of casualties each year, it is mandatory to understand mechanisms in cellular processes, such as cell division and growth, to find new targets for antibiotic intervention. Unfortunately, bacteria are able to develop resistances against many antibiotics. The mycolic acid or arabinan layer and synthesis machinery are good candidates for new antibiotics. Amongst others, two of them have emerged as useful drugs against M. tuberculosis, ethambutol (EMB) and BTZ043. In this study, we investigated the modes of action and antibiotic susceptibility of C. glutamicum after EMB and BTZ043 treatment. We found that both antibiotics, which target the arabinan synthesis pathway, affect exclusively polar elongation growth, as demonstrated in different staining assays. Interestingly, only 10% of the cells were killed and cells in stationary phase were not affected by EMB or BTZ043. Moreover, we used a chromosomal DivIVA-mCherry fusion and found that DivIVA protein level is drastically increased. The cells show asymmetric recovery after treatment, in which one daughter cell acquires the excess DivIVA whereas the other daughter cell exhibits normal cell growth.
Corynebacterium glutamicum ist ein Gram-positives Bodenbakterium mit großer industrieller Bedeutung für die Herstellung von Aminosäuren im Tonnenmaßstab. Des Weiteren bekommt es zunehmende Bedeutung für die medizinische Forschung, wo es aufgrund seiner engen Verwandtschaft zu den pathogenen Erregern von Tuberkulose, Diphtherie und Lepra als idealer Modellorganismus dient. Besonders die Zellwand von C. glutamicum hat große Ähnlichkeit zu der vieler pathogener Vertreter wie Mykobakterium tuberculosis. Sie besteht aus einer Peptidoglycan-Schicht (Murein), an der über weitere Polysaccharid-Schichten die charakteristischen Mycolsäuren gebunden sind. Darüber hinaus besitzen beide Organismen eine polare Zellwandsynthese, die von DivIVA (Wag31 in M. tuberculosis) räumlich reguliert wird. Die Rolle von DivIVA am Zellwachstum wurde vor Jahren erstmals beschrieben, jedoch war seine exakte Funktion bis zuletzt unbekannt. In dieser Studie wird erstmals die Funktion von DivIVA am polaren Zellwachstum durch Interaktion mit der Lipid II-Flippase RodA gezeigt. RodA beeinflusst die Morphologie und das Wachstum von C. glutamicum und wird von DivIVA an die Zellpole lokalisiert. Deletion von rodA resultiert in reduziertem Wachstum und veränderter Morphologie, sowie einer alternativen Lipid II Versorgung der Zellpole, da das polare Zellwachstum erhalten bleibt. DivIVA ist darüber hinaus an der Chromosomensegregation beteiligt, wo es direkt mit ParB interagiert, das über parS-Seiten an die replizierten Chromosomen bindet um sie an die Zellpole zu fixieren. Die Interaktionen zwischen DivIVA und ParB bzw. RodA wurden mit Hilfe eines synthetischen in vivo Assays identifiziert, worin die zu untersuchenden Gene an Fluorophore gekoppelt und in E. coli Zellen exprimiert werden. Somit lässt sich eine Co-Lokalisation nach individueller und Co-Expression der Fusionsproteine mikroskopisch analysieren. Eine entscheidende Verbesserung dieses Assays ist die Verwendung von FRET, das sensitiver ist und eine Quantifizierung der Interaktion ermöglicht. Um herauszufinden, ob ParB und RodA um die gleiche Bindungsstelle an DivIVA konkurrieren, wurden die Interaktionsdomänen beider Proteine ermittelt. Während ParB an eine mittlere Region in DivIVA bindet, bindet RodA an die N-terminale Domäne von DivIVA, in der ein Lysin-Rest für die Bindung essenziell ist. Für den Kampf gegen bakterielle Infektionskrankheiten, die jährlich tausende Todesfälle verursachen, ist es dringend notwendig zelluläre Mechanismen, beispielsweise der Zellteilung und des Wachstums, zu entschlüsseln um Targets für neue Antibiotika zu finden. Insbesondere die kontinuierliche Entstehung neue Resistenzen macht diese Aufgabe wichtiger denn je. Die Mykolsäureschicht und ihre Synthese sind vielversprechende Targets, da bisher nur wenige Antibiotika, wie Ethambutol (EMB) oder BTZ043, dagegen existieren. Wir haben die Wirkungsweise und antibiotische Suszeptibilität von C. glutamicum nach EMB und BTZ043 Behandlung untersucht. Beide Antibiotika, die in die Arabinogalactan-Synthese eingreifen, beeinflussen ausschließlich das polare Zellwachstum, wie in mehrerer Färbeassays gezeigt. Lediglich 10% der Zellen wurden getötet. Zellen, die sich in der stationären Phase befanden, wurde nicht beeinflusst. Darüber hinaus zeigte die Verwendung eines Stammes mit chromosomaler DivIVA-mCherry Fusion, dass das DivIVA Protein Level stark erhöht ist. Erholungsexperimente nach Antibiotikazugabe zeigten, dass die Zellen asymmetrisch reagieren, wobei eine Tochterzelle das überschüssige DivIVA übernimmt, während die andere Zelle normales Wachstum erfährt.

During the last decades, newly developed medical devices often came along with unappropriate designs, increasing the likelihood of misuse through the operator. Part of the root cause was that no sufficient measures were applied to assimilate user needs. Consequently, usability engineering approaches are now stronger emphasized to ensure that new devices are not only safe to use but are also designed for users’ needs. Besides, testing processes in clinical microbiology laboratories are currently reshaped due to new generations of rapid testing methods. Hence, it is particularly important to apply usability engineering frameworks during the development phase to make sure devices address users’ needs and also fit into the new work- and communication flows. Based on that, this research project applies a usability engineering approach to the design process of a new rapid antibiotic susceptibility testing system of Astrego Diagnostic AB that is supposed to be used in clinical microbiology laboratories in the near future. The research questions focus on how this device can be designed to enable integration into clinical laboratories. -       How can a rapid AST testing system be integrated into the workflow of clinical microbiology laboratories? -       What are the remaining uncertainties for integrating a rapid AST system into the workflow of a clinical microbiology laboratory on the example of Astrego’s AST system? Several methods were used to address these questions, which include literature research, a competitive audit, subject matter interviews and semi-structured interviews, and observations of targeted users. The findings show that a rapid antibiotic susceptibility testing system may be used in several different ways, which also impacts its design. Process-wise, it could be used after Gram staining and bacterial identification has been conducted and, more realistically, simultaneously bacterial identification to pave the way for additional time savings further. However, uncertainties remain regarding the design of the new testing system. Depending on the number of devices that targeted laboratories need to implement to accommodate their testing volume, it makes sense to design a built-in user interface or an external one that can be accessed through a tablet or desktop. Thus, it is uncertain to what extent manual input of bacteria ID is relevant as the dRAST system fully enables manual input of Gram type and bacteria IDs while it might also be possible to avoid manual interaction by receiving this information through software interfaces.

Effective treatment of tuberculosis requires at least six months of combination therapy involving four antibiotics. Alterations in the physiological state of Mycobacterium tuberculosis during infection may reduce drug efficacy and prolong treatment, but these adaptations are incompletely defined. To investigate the mechanisms limiting antibiotic efficacy, I performed a comprehensive genetic study to identify M. tuberculosis genes and pathways important for bacterial survival during antibiotic treatment in vivo. First, I identified mutants in the glycerol kinase enzyme, GlpK, that promote survival under combination therapy. Similar glycerol catabolic mutants are enriched in extensively drug-resistant clinical isolates, indicating that these mutations may promote survival and the development of resistance in humans. A majority of these mutations are frameshifts within a homopolymeric region of the glpK gene, leading to the hypothesis that M. tuberculosis may reversibly produce drug-tolerant phenotypes through genetic variation introduced at homopolymer sites as a strategy for survival during antibiotic treatment. Second, I identified bacterial mutants with altered susceptibility to individual first-line anti-mycobacterial drugs. Many of these mutations did not have obvious effects in vitro, demonstrating that a wide variety of natural genetic variants can influence drug efficacy in vivo without altering standard drug-susceptibility tests. A number of these genes are enriched in drug-resistant clinical isolates, indicating that these genetic variants influence treatment outcome. Together, these data suggest new targets for improving therapy, as well as mechanisms of genetic adaptations that can reduce antibiotic efficacy and contribute to the evolution of resistance.

Thesis advisor: Jianmin Gao
With the rapid evolution of antibiotic resistance, the need for more effective antibiotics is imminent. Bacterial membranes are an appealing target due to their accessibility and relatively conserved structures. Membrane targeting antibiotics, especially cationic antimicrobial peptides (CAMPs) such as host defense peptides, have been increasingly explored as novel antibiotics and tunable innate antimicrobials. The latter could be achieved by treatment with an antibiotic adjuvant: a compound that would increase the potency of host CAMPs without killing the bacteria on its own. Boosting the host’s own immune system with an adjuvant is beneficial over using antibiotics and would theoretically avoid triggering bacterial resistance. One mechanism of bacterial resistance is increasing the cationic charge of the membrane. As CAMPs are electrostatically attracted to anionic bacterial membranes, making the membrane more cationic decreases that attraction, rendering CAMPs less effective. To target this resistance mechanism chemically, two antibiotic adjuvant strategies were explored as co-treatments with various CAMPs: membrane targeting peptides used to bind and block surface amines, and small molecules used to either acetylate surface amines or convert a cationic membrane phospholipid to an anionic phospholipid. Co-treatment of the Staphylococcus aureus (S. aureus) membrane targeting peptide KAM-CT and various CAMPs increased S. aureus susceptibility to those CAMPs. Bacterial surface acetylation using sulfo-NHS-acetate followed by CAMP treatment caused up to 10 times increased CAMP potency. Hydrazine and hydroxylamine were shown to cleave the lysine moiety from the lysyl-phosphatidylglycerol (Lys-PG) phospholipid to generate phosphatidylglycerol (PG) in liposome models. S. aureus was treated with a hydroxylamine-CAMP conjugate, but it showed decreased antibiotic activity compared to the CAMP alone. To better understand what was happening in the bacteria, a novel Lys-PG quantification protocol was created by fluorophore labeling Lys-PG and quantifying the labeled Lys-PG via normal phase high-performance liquid chromatography (NP-HPLC). Cyclic peptides, such as KAM-CT, represent complex yet synthetically attainable moieties that could be used as novel antibiotics adjuvants. Expanding the repertoire of reversible covalent chemistries, especially those applied to peptide cyclization, is desirable due to the high potency and selectivity of such interactions. Herein, we also describe a novel reversible covalent chemistry between 2-formylphenylboronic acid (FPBA) and 2,3-diaminopropionic acid (Dap): the imidazolidino boronate (IzB) conjugate. It was found to be potent (Kd = 100 μM) and quickly reversible (t1 = ~6 sec) under physiological conditions. IzB formation was successfully employed as a peptide cyclization strategy as there was little interference from biologically relevant small molecules, except cysteine. Cysteine interference was utilized to create “smart” peptides that can linearize upon increasing cysteine concentrations via thiazolidino boronate (TzB) formation with the FPBA moiety in the peptide. Such “smart” peptides could be used as pH-responsive peptides or cysteine sensors able to report on the cysteine concentration in complex media
Thesis (MS) — Boston College, 2017
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

Rapid and accurate species identification and antibiotic susceptibility testing are of great importance for patients with sepsis and to stop over- and misuse of antibiotics contributing to antibiotic resistance. QuickMIC™ is a rapid antibiotic susceptibility testig system based on a microfluidic technology solution developed by Gradientech that measure MICs on bacteria from positive blood culture bottles. By combining QuickMIC™ with a rapid system for detection and identification, the time to detection, identification and antibiotic susceptibiolity testing could be shortened with days compared to pipelines used today which could mean the difference of life and death for patients. The T2Bacteria® panel and T2Dx® instrument developed by T2 biosystems is an FDA-cleared test for rapid detection and identification of bacteria from whole blood based on magnetic molecular resonance technology. The time to result of the T2Dx® instrument is 3-4 hours and the time to result for QuickMIC™ is 2-4 hours. In this project, the possibilities and benefits of such a pipeline have been studied by comparison to a pipeline typically used today. Time, accuracy and practical aspects have been investigated during the project and the results are promising for future further studies.

Uropathogenic E. coli (UPEC) is the most frequent cause of urinary tract infection (UTI), being responsible for up to 85% of community acquired and 40% of nosocomial cases. UPEC strains harbour various virulence factors that contribute to their ability to cause disease. The high prevalence across the globe of multidrug resistant UPEC is a significant threat to therapy. Virulent and resistant UPEC strains have been recognised as belonging to major lineages and we have only recently begun to understand the factors contributing to their successful global dissemination. Work in this thesis was carried out to identify the population structure of E. coli isolates recovered from urosepsis and biliary sepsis, to reveal any differences in genetic background. A total of 100 isolates from the blood and urine of 50 patients presenting with urosepsis and 27 isolates from cases of biliary sepsis were subjected to genotypic and phenotypic analysis, including MLST, virulence gene detection and antibiogram and metabolic profiling. Urosepsis paired isolates showed identical genotypes and antimicrobial resistance profiles. However, several pairs of isolates showed discrepant metabolic activity profiles suggesting niche specific regulation of metabolism. Members of the ST131 clone were significantly associated with antibiotic resistance and ST38 isolates were associated with the highest level of metabolic activity. An in vivo infection model was used to investigate the virulence potential of isolates from the major UPEC lineages. Galleria mellonella larvae inoculated with ST69 and ST127 isolates showed significantly higher mortality rates than those infected with other strains. However, one isolate of ST127 (strain EC18) was avirulent and comparative genomic analyses with a single virulent ST127 strain revealed an IS1 mediated deletion in the O-antigen cluster in strain EC18, which is likely to explain the lack of virulence in the larvae and demonstrates the importance of this cell surface molecule in the model system. Finally, a total of 202 UPEC isolates were recovered from community and hospital urine samples from a tertiary care hospital in Riyadh, Saudi Arabia. Molecular epidemiological investigation of the strains was carried out to examine the overall UPEC population structure, for the first time in any part of Saudi Arabia. The most common lineages were ST131 (17.3%), ST73 (11.4%), ST38 (7.4%), ST69 (7.4%) and ST10 (6.4%). The findings highlight the successful spread of multidrug resistant, CTX-M positive ST38, ST131 and ST405 UPEC in Saudi Arabia. The high proportion (35%) of ESBL producing E. coli isolates is a particular concern and is driving frequent prescription of carbapenem antibiotics. A total of four isolates of ST38 were positive for aggR, which is a virulence marker of enteroaggregative E. coli (EAEC); ST38 strains that cause UTI but have an EAEC genetic background are becoming recognised as novel UPEC and this clonal group warrants further study.

Staphylococcus aureus is one of the leading causes of hospital acquired infections. The ability of S. aureus to acquire resistance to a diverse range of antimicrobial compounds, results in limited treatment options, particularly in methicillin-resistant S. aureus. A mechanism by which S. aureus develops reduced susceptibility to antimicrobials is through the formation of small colony variants (SCVs). Reduced antimicrobial susceptibility in S. aureus SCVs is not related to ‘classical’ mechanisms of resistance, but occurs as a direct result of the development of the SCV phenotype. S. aureus SCVs are frequently associated with defects in the bacterial electron transport chain and these defects are responsible for the characteristics associated with the SCV phenotype. This study aimed to investigate and characterise the selection of S. aureus SCVs in the presence of various antibiotics and also to examine their biofilm forming capabilities. Four members of the aminoglycoside family of antibiotics were shown to select for S. aureus SCVs. In addition, a broad range (X 0.25 MIC – X 4 MIC) of aminoglycoside concentrations were shown to select for S. aureus SCVs. Characterisation of these isolates revealed that differences in auxotrophy, biochemical profiles, carotenoid production, haemolysis, levels of intracellular ATP, mutation frequency and reversion rate were present. Members of the tetracycline family of antibiotics were also shown to select for S. aureus SCVs. Tetracycline selected S. aureus SCVs show attenuated catalase, coagulase and heamolysis activity and reduced production of extracellular DNase and lipase and reduced susceptibility to various antimicrobial agents. As SCVs have been linked to persistent and recurrent infections their ability to form biofilms was also investigated. A range of S. aureus SCVs isolated from various backgrounds were shown to form greater biofilms in comparison to parent strains, which was attributed to increased production of polysaccharide intracellular adhesin. In addition S. aureus SCV biofilms displayed a more pronounced reduction in antimicrobial susceptibility, which was attributed to a reduction in antimicrobial penetration through SCV biofilms. Limited discovery of novel antibiotics in recent years and the observation that S. aureus SCVs can be selected for by various antimicrobial compounds highlights the need for novel antimicrobial compounds. Accordingly, an investigation into the susceptibility of S. aureus to various plant compounds was undertaken. Both S. aureus SCVs and parent strains showed susceptibility to five plant antimicrobials tested, of which SCVs were more susceptible to cinnamon bark, green tea and oregano. Resistance to these plant antimicrobials could not be induced and synergistic relationships between certain plant antimicrobials and antibiotics were demonstrated. Finally, formation of SCVs in bacterial species other than S. aureus was examined. Gentamicin induced SCV selection in Escherichia coli, Pseudomonas aeruginosa and S. epidermidis as well as chloroamphenicol and ciprofloxacin in E. coli and tetracycline in S. epidermidis. SCVs from these bacterial species shared common characteristics associated with the SCV phenotype including altered growth and biochemical profiles, auxotrophy for compounds involved in electron transport, reduction in expression of virulence factors and reduced antimicrobial susceptibility. Additionally all SCVs showed an increased capacity to form biofilms. The ability of certain antibiotics to select for SCVs and their increased capacity to form biofilms suggest that SCV are an important adaptation to aid survival and persistence in times of stress. Reduced susceptibility to commonly used antibiotics in SCVs signifies that the development of new antimicrobial compounds is required. Harnessing naturally occurring plant antimicrobials and their synergistic relationship with antibiotics may offer a novel approach to treating antibiotic resistant infections whilst overcoming antibiotic selection for SCVs.

Mycobacterium avium and Mycobacterium intracellulare are environmental opportunistic pathogens whose source for human infection is water and soil. M. avium and M. intracellulare cause pulmonary infections (tuberculosis) in immunocompetent individuals and bacteremia in immunodeficient individuals (e.g. AIDS). One factor likely influencing the lack of success of antibiotic therapy in patients would be their ability to form biofilms. Growth in biofilms might result in antimicrobial resistance because (1) cells are protected by layers of other cells and extracellular material (2) and differences in physiologic state of cells as a consequence of growing on surfaces. The objectives of the work were to (1) establish methods for reproducible growth of mycobacterial biofilms (2) measure the formation of biofilms on surfaces by cells of M. avium and M. intracellulare (3) measure the antibiotic- and chlorine- susceptibility of M. avium and M. intracellulare strain TMC1406T in cell grown in suspension, cells grown in biofilms and suspended and of cells grown in biofilms (4) measure the hydrophobicity of M. avium and M. intracellulare grown in suspension and in biofilms. Methods were developed for growing mycobacteria in biofilms in polystyrene flasks and on glass beads. Although both strains formed biofilms, M. intracellulare strain TMC 1406T more readily formed biofilms than M. avium strain A5 in polystyrene flasks. The majority of M. intracellulare strain TMC 1406T cells grew on the walls of the flasks rather than in suspension like M. avium strain A5. The susceptibility of M7H9 medium-grown cells of M. avium strain A5 and M. intracellulare strain TMC 1406T cells grown in suspension, cells grown in biofilms and suspended and cells grown in biofilms was measured against clarithromycin, ethambutol, kanamycin, rifampicin and streptomycin. Cells grown in biofilms and exposed to antibiotics in biofilms were five-fold resistant to antibiotics than were cells grown in biofilms and exposed in suspension. Cells grown and exposed in suspension were ten-fold more sensitive to antibiotics than were cells grown in biofilms and exposed in suspension. The chlorine susceptibility of cells grown in medium and water was also measured. Cells grown in biofilms were more resistant to chlorine than cells grown in biofilms and suspended. Cells grown in suspension were more sensitive to chlorine than cells grown in biofilms and suspended. The hydrophobicity data (i.e., hexadecane adherence and contact angle measurements) showed that cells grown in biofilms are more hydrophobic than cells grown in biofilms and suspended and cells grown in suspension. It is clear that there are physiological changes between cells grown in suspension, cells grown in biofilms and suspended and cells in biofilms.
Master of Science

Antibiotic resistance is a problem of growing importance in veterinary medicine. In order to ensure that antibiotics are used appropriately, antibiograms are generated to monitor bacterial susceptibility to antibiotics. The computer program Biomic was used to generate antibiograms for bacterial isolates from canine, feline, and equine samples sent to the Arizona Veterinary Diagnostic Laboratory between January 1st, 2015 and December 31st, 2016. The most common specimen types were urine (n=125), ear cultures (n=92), wounds (n=63), and skin cultures (n=30) for canines, uterine cultures (n=44) and wounds (n=41) for equines, and urine (n=16) and wounds (n=17) from felines. Of the canine isolates, the most common urine isolate E. coli was most susceptible to amikacin and chloramphenicol (92%), the most common ear isolate P. aeruginosa was most susceptible to amikacin (74%), the most common skin isolate Staphylococcus coagulase-negative was most susceptible to marbofloxacin, amikacin, amoxicillin-clavulanate, and cephalothin (60%), and the most common wound isolate E. coli was most susceptible to trimethoprim-sulfamethoxazole (100%). Of the equine isolates, the most common uterine isolate S. equi spp zooepidemicus was most susceptible to penicillin G (92%) and the most common wound isolate S. equi spp zooepidemicus were most susceptible to penicillin G (100%).

Background Broth microdilution (BMD) is a gold-standard reference method to determine minimum inhibitory concentration (MIC) of antibiotics. For this, a standardized concentration of bacterial inoculum (2e5–8e5 colony-forming units, CFU/ml) is added to progressively higher concentrations of antibiotics. Bacteria stop growing at a particular antibiotic concentration termed MIC. Like other assays, various biological and/or technical factors can affect BMD results.   Aims To investigate the effects of inoculum concentration (5e4–5e6 CFU/ml), growth-medium concentration (cation-adjusted Mueller-Hinton Broth (CAMHB)), ranging 0.5x to 2x (1x as standard)) and age (<6-months or >1-year old) of fastidious medium on MIC results. And to compare BMD results using 5 different brands of CAMHBs and 1 cation-non-adjusted MH-broth (non-CAMHB).   Methods 12 isolates of bacteria (gram-positive (n=3), gram-negative(n=5), fastidious isolates (n=7)) and custom-made antibiotics-containing plates for gram-positive (11 antibiotics) or gram-negative bacteria (10 antibiotics) were used. Overnight-grown colonies were used to prepare BMD solutions (MH-broth + inoculum +/- fastidious) which were plated on antibiotic-plates as well as diluted prior to plating on agar-plates. Antibiotic- and agar-plates were incubated (18–20hr, 35°C) and used to determine MICs (following European Committee on Antimicrobial Susceptibility Testing instructions) and actual number of viable bacteria in BMD solutions, respectively.   Results Increasing inoculum concentration increased MICs of all antibiotics except cefoxitin. Piperacillin–tazobactam, levofloxacin, benzylpenicillin and ampicillin were especially sensitive to increase in inoculum and showed a 4-fold increase in >50% isolates. MICs for tobramycin, tigecycline and gentamicin increased by 2-fold in >50% isolates every time MH-broth concentration increased. Age of fastidious medium had no decipherable pattern of effects on MIC. All MH-broths gave similar results except when testing daptomycin which gave higher MICs with non-CAMHB compared to CAMHB.    Conclusion This research reveals some technical factors affecting MIC results. These results could help define parameters for automated BMD-performing-systems. However, this research shows only trends as more replicates are needed to determine statistically significant results.

Infectious diseases resulting from bacterial pathogens are the most common causes of patient morbidity and mortality worldwide. The rapid identification of the pathogens and their antibiotic resistances is crucial for proper clinical management. However, the standard culture-based diagnostic approach requires a minimum of two days from the initial specimen collection to result reporting. As a consequence, broad-spectrum antibiotics are often prescribed under the worst-case assumption without knowledge of the pathogens or their resistances. The current clinical practice results in improper treatment of the patient and causes the rapid emergence of multi-drug resistant pathogens. A rapid diagnostics system has therefore been developed which performs hybrid electrokinetic sample preparation and volume reduction, for single-cell antimicrobial susceptibility testing (AST). The system combines multiple electrokinetic forces for sample preparation, which reduces the sample volume for over 3 orders of magnitude and minimizes the matrix effects of physiological samples for enhanced sensitivity. The device is integrated with a single-cell AST system with microfluidic confinement and electrokinetic loading to phenotypically determine the bacterial antibiotic resistance at the single-cell level. The applicability of the system has been demonstrated for performing direct AST with urine and blood samples within one hour, enabling rapid infectious disease diagnostics in non-traditional healthcare settings.

Oral Biology
M.S.
Objectives: Systemic antibiotics are generally recognized as providing a beneficial impact in treatment of both aggressive and chronic periodontitis. Since strains of periodontal pathogens among periodontitis patients may vary in their antibiotic drug resistance, the American Academy of Periodontology recommends antimicrobial susceptibility testing of suspected periodontal pathogens prior to administration of systemic periodontal antibiotic therapy, to reduce the risk of a treatment failure due to pathogen antibiotic resistance. E-test and MIC Test Strip assays are two in vitro antimicrobial susceptibility testing systems employing plastic- and paper-based, respectively, carriers loaded with predefined antibiotic gradients covering 15 two-fold dilutions. To date, no performance evaluations have been carried out comparing the Etest and MIC Test Strip assays in their ability to assess the in vitro antimicrobial susceptibility of periodontal bacterial pathogens. As a result, the purpose of this study was to compare the in vitro performance of E-test and MIC Test Strip assays in assessing minimal inhibitory concentration (MIC) values of four antibiotics frequently utilized in systemic periodontal antibiotic therapy against 11 fresh clinical subgingival isolates of the putative periodontal pathogen, Prevotella intermedia/ nigrescens, and to compare the distribution of P. intermedia/ nigrescens strains identified with interpretative criteria as "susceptible" and "resistant" to each of the four antibiotics using MIC values determined by the two antimicrobial susceptibility testing methods. Methods: Standardized cell suspensions, equivalent to a 2.0 McFarland turbidity standard, were prepared with 11 fresh clinical isolates of P. intermedia/nigrescens, each recovered from the subgingival microbiota of United States chronic periodontitis subjects, and plated onto to the surfaces of culture plates containing enriched Brucella blood agar. After drying, pairs of antibiotic-impregnated, quantitative, gradient diffusion strips from two manufacturers (E-test, bioMérieux, Durham, NC, USA, and MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, clindamycin, metronidazole, and doxycycline were each placed apart from each other onto the inoculated enriched Brucella blood agar surfaces, so that an antibiotic test strip from each manufacturer was employed per plate against each P. intermedia/ nigrescens clinical isolate for antibiotic susceptibility testing. After 48-72 hours anaerobic jar incubation, individual MIC values for each antibiotic test strip against P. intermedia/nigrescens were read in μg/ml at the point where the edge of the bacterial inhibition ellipse intersected with the antibiotic test strip. MIC50, MIC90, and MIC range were calculated and compared for each of the test antibiotics, with essential agreement (EA) values determined per test antibiotic for the level of outcome agreement between two antimicrobial susceptibility testing methods. In addition, the identification of antibiotic "susceptible" and "resistant" strains among the P. intermedia/nigrescens clinical isolates was determined for each test antibiotic using MIC interpretative criteria from the MIC interpretative standards developed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for gram-negative anaerobic bacteria for amoxicillin, clindamycin, and metronidazole findings, and from the French Society of Microbiology breakpoint values for anaerobic disk diffusion testing for doxycycline data. Results: For amoxicillin, higher MIC50 and MIC90 values against the P. intermedia/ nigrescens strains were found with the MIC Test Strip assay than with E-test strips, resulting in a relatively low EA value of 45.5% between the two susceptibility testing methods. A higher percentage of amoxicillin "resistant" P. intermedia/nigrescens strains (72.7%) were identified by MIC Test Strips as compared to E-test strips (54.5%), although both methods found the same proportion of amoxicillin "susceptible" strains (27.3%). For clindamycin, both susceptibility testing methods provided identical MIC values (EA value = 100%), and exactly the same distributions of "susceptible" and "resistant" strains of P. intermedia/nigrescens. For metronidazole, only very poor agreement (EA value = 9.1%) was found between the two susceptibility testing methods, with MIC Test Strips exhibiting markedly higher MIC50 and MIC90 values against P. intermedia/nigrescens as compared to E-test strips. However, the distribution of "susceptible" and "resistant" P. intermedia/ nigrescens were identical between the two susceptibility testing methods. For doxycycline, relatively good agreement (EA value = 72.7%) was found in MIC concentrations between the two susceptibility testing methods, although generally lower MIC values were associated with MIC Test Strips. In addition, identical distributions of "susceptible" and "resistant" P. intermedia/nigrescens were provided by both susceptibility testing methods. Conclusions: Relative to MIC values measured against periodontal strains of P. intermedia/nigrescens, MIC Test Strips gave higher MIC values with amoxicillin and metronidazole, equal MIC values with clindamycin, and lower MIC values with doxycycline, as compared to MIC values measured with the E-test assay. Relative to the identification of antibiotic "susceptible" periodontal P. intermedia/ nigrescens strains, both susceptibility testing methods provided identical findings, suggesting that both methods appear to be interchangeable for clinical decision making in regard to identification of antibiotic-sensitive strains of periodontal P. intermedia/nigrescens. However, for epidemiologic surveillance of drug susceptibility trends, where exact MIC values are important to track over time, the relatively higher proportion of non-exact MIC differences between the two susceptibility testing methods argues against using them interchangeably. Instead, one or the other method should be used consistently for such studies. Further comparative studies of the E-test and MIC Test Strip assays are indicated using other periodontopathic bacterial species besides P. intermedia/ nigrescens, and to assess the reproducibility of MIC values provided by both in vitro susceptibility testing methods over time.
Temple University--Theses

Instead of planktonic growth in nature, many species of bacteria form biofilm to survive in harsh conditions. Although many chronic bacterial infections are caused by bacterial species in a biofilm lifestyle, previous research has focused on studying antibiotic resistance in planktonic growth. Here we used a modified MBEC assay, i.e. biofilm growth on pegs, to determine Escherichia coli biofilm inhibitory concentrations (BIC) of ciprofloxacin, streptomycin and rifampicin and to study the minimal selective concentration (MSC) for ciprofloxacin in E. coli biofilm. We could observe high inhibitory concentrations for all antibiotics in the biofilm pre-formed in media without antibiotics compared to the biofilm formed in antibiotics. We also show preliminary result indicating that sublethal concentrations of ciprofloxacin lead to the selection of ciprofloxacin resistant mutants in biofilm and that the selection level is lower than what was observed in planktonic growing E. coli. With more knowledge in how the biofilm formation precedes in different antibiotic settings, the treatment for chronic biofilm infections used today could be evaluated and changed so that the infections could be eradicated.

Klebsiella pneumoniae is one of the top eight pathogens in hospitals, causing around 10% of hospital-acquired infections (nosocomial infections). It often produces extended-spectrum β-lactamase enzymes (ESBLs). This has led to numerous outbreaks, especially in intensive care, neonatal and surgical wards, associated with increases in morbidity and mortality. In order to reduce the number of infections caused by multi-resistant K. pneumoniae and improve standards of infection control within hospitals, there is extensive use of biocides as disinfectants and antiseptics. However this raises concerns that intensive exposure of hospital pathogens to biocides may result in the emergence of resistance not just to themselves but also to antibiotics. The reduced susceptibility to biocides and their relationship with resistance to antibiotics was assessed in this thesis. The susceptibility of 64 isolates of K. pneumoniae to five biocides preparations, Chlorhexidine (CHX), Benzalkonium chloride (BZK), Trigene, MediHex-4 (MH-4), Mediscrub (MS) and 17 antibiotics, were tested. The isolates of K. pneumoniae were collected from Royal Infirmary Hospital in Edinburgh (RIE) between 2006 and 2008 from different sites of infection. Antimicrobial susceptibility was tested by the agar double dilution method (DDM) and disc diffusion methods following the British Society for Antimicrobial Chemotherapy (BSAC) guidelines. A few isolates of K. pneumoniae showed insusceptibility to cephalosporins, colistin, rifampicin, trimethoprim and penicillin but not to carbapenems. Biocide susceptibility testing showed that 57, 55 and 61 strains had reduced susceptibility to Chlorhexidine, Trigene and Benzalkonium chloride, respectively, but not to MediHex-4 and Mediscrub. The effect of efflux pumps were determined by carbonyl cyanide m-chlorophenylhydrazone (CCCP) (10mg/L), which decreased the MICs of Chlorhexidine and Medihex-4 by 2 – 128 fold but had no impact on the MICs of Benzalkonium chloride, Trigene and Mediscrub. Six isolates of K. pneumoniae were chosen for their varying sensitivity to Chlorhexidine (CHX), and were tested for their minimum bactericidal concentration (MBC) to biocides. The high MBCs of Mediscrub and Trigene, over 500-fold greater than the minimum inhibitory concentration (MICs), indicates that these compounds are mainly bacteriostatic. Conversely, the MBCs of Chlorhexidine and MediHex-4, which contains chlorhexidine, were less than 10-times the MIC value indicating they are effective in killing the organism. However, this thesis showed how the killing capability of Chlorhexidine was hindered by the presence of organic matter, which compromised its effect. The relationship between reduced susceptibility to biocides and the carriage of antiseptic resistance genes, cepA, qacΔE1 and qacE was determined by polymerase chain reaction. The antiseptic resistance genes cepA, qacΔE1 and qacE were found in 56, 34 and 1 isolates respectively, and the levels of gene expression were detected by the reverse transcription polymerase chain reaction (RT-PCR). These results have shown that there was a close link between carriage of efflux pump genes, cepA, qacΔE1 and qacE genes and reduced susceptibility to biocides. Most strains showed decreased susceptibility to Chlorhexidine, Trigene and Benzalkonium chloride and this correlated with the carriage of the cepA, qacΔE1 and qacE genes encoding efflux. There was no correlation between the reduced susceptibility to biocides and antibiotic resistance in clinical isolates of K. pneumoniae.

Orientador: Caio Cezar Randi Ferraz
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O objetivo deste estudo foi investigar a composição da microbiota de canais radiculares infectados associados a abscessos periapicais; analisar a correlação entre espécies bacterianas específicas com a localização clínica dos abscessos periapicais, sinais e sintomas e testar a suscetibilidade de bactérias anaeróbias estritas prevalentes. As amostras microbiológicas foram coletadas de 60 canais radiculares usando pontas de papel estéreis, transportadas em RTF, diluídas, plaqueadas e incubadas em câmara de anaerobiose. Colônias microbianas foram isoladas, caracterizadas e identificadas por métodos padronizados. Das 287 diferentes espécies bacterianas, 201 eram anaeróbias estritas. Uma ou mais (máximo de 14) espécies bacterianas foram recuperadas de 59 (98,3%) canais radiculares, confirmando a característica polimicrobiana das infecções endodônticas. As bactérias anaeróbias estritas mais freqüentemente isoladas foram: Peptostreptococcus prevotii (22/60), Peptostreptococcus micros (19/60), Fusobacterium necrophorum (19/60). Embora menos freqüentes, bactérias facultativas como Gemella morbillorum (19/60), Streptococcus mitis (13/60), e Streptococcus sanguis (11/60)também foram encontradas. O teste de Pearson ou teste exato de Fisher mostrou que houve relação positiva de algumas espécies bacterianas com a localização do abscesso periapical, bem como, com alguns sinais e sintomas de origem endodôntica (p<0,05). Os resultados indicaram predominância de bactérias anaeróbias Gram-positivas e a presença de microbiota mista nos canais radiculares infectados associados a abscessos periapicais. O método do E-test revelou sensibilidade bacteriana das espécies testadas aos antibióticos benzilpenicilina, amoxicilina, amoxicilinalclavulanato de potássio, metronidazol, clindamicina e cefaclor, contudo, certos microrganismos foram resistentes a azitromicina e eritromicina. Enquanto que, nenhum dos microrganismos testados produziu 'beta¿-lactamase
Abstract: The aim of this study was to investigate the composition of the microbiota of infected root canaIs associated with periapical abscesses, to investigate the correlation of specific species of bacteria with the clinical localization of the periapical abscesses, signs, and symptoms, and to test the susceptibility of prevalent strict anaerobic bacteria isolated. Microbiological samples were collected from 60 root canals using sterile paper points, transported in RTF and diluted, plated and incubated in an anaerobic chamber. Microbial colonies were then purified, characterized and identified by established methods. Of the 287 different bacterial species recovered, 201 were strict anaerobes or microphilic species. One or more (maximum of 14) bacterial species were recovered from 59 (98.3%) root canals, showing the polymicrobial characteristic of endodontic infections. The most frequently strict anaerobes isolated were: Peptostreptococcus prevotii (22/60), Peptostreptococcus micros (19/60), Fusobacterium necrophorum (19/60). Although less frequent, facultative bacteria such as Gemella morbillorum (19/60), Streptococcus mitis (13/60), and Streptococcus sanguis (11/60) were also found. The Pearson X 'POT. 2¿ test or Fisher's exat test showed positive relationship among some bacterial species and the localization of periapical abscesses as well as with some endodontic signs and symptoms (p<0.05). Results indicated predominance of Gram-positive anaerobic bacteria in the mixed microbiota of dental root canals associated with periapical abscesses. The E-test revealed bacterial susceptibility to benzylpenicillin, amoxicillin, amoxicillin/potassium clavulanate, clindamycin and cefaclor. All microorganisms tested did not produce 'beta¿-lactamase
Doutorado
Endodontia
Doutor em Clínica Odontológica

Host defense peptides (HDPs) provide innate immune defense against invasive S. aureus infection. Recent studies suggest potential cross-resistance between HDPs and the lipopeptide antibiotic, daptomycin (DAP). Isolates that exhibit DAP non-susceptible phenotypes may have virulence advantages and pose challenges to effective treatment. The current studies were performed to compare the efficacies and mechanisms of action of native and engineered HDPs vs. clinical S. aureus strain pairs which differed in susceptibility to daptomycin in vitro. Ultrasensitive radial diffusion and multi-colored flow cytometry were employed to analyze distinctive susceptibilities and mechanisms of resistance, respectively. Overall efficacies were greater vs. DAP-susceptible (DSSA) vs. DAP non-susceptible (DNSA) S. aureus isolates for some but not all HDPs. Efficacy profiles of certain HDPs were influenced by pH, regardless of whether the particular isolate was DSSA or DNSA phenotype. Mechanistically, DSSA and DNSA isolates differed in responses to specific HDPs regarding cell energetics, membrane permeability, cytoplasm membrane turnover, and cell death protease induction. DSSA and DNSA strain pairs exhibited non-identical mechanisms of resistance to HDPs. At pH 7.5, as expected, HDPs hNP-1 and RP-1 exerted significantly greater efficacy on susceptible control strain ISP479C vs. its resistant counterpart ISP479R. These data suggest different mechanisms of HDP resistance are active in differing DNSA strains. These preliminary results are under further investigation, as are the genetic determinant(s) that may emerge during infection. If substantiated, these findings would imply multiple modes of survival of S. aureus in the face of DAP or HDPs.

Negli ultimi decenni l'utilizzo degli antibiotici a scopo terapeutico o come promotori della crescita nell'allevamento animale ha portato alla comparsa e alla diffusione di microrganismi resistenti. In questo contesto, la presenza di Lattobacilli (LAB) antibiotico resistenti non rappresentano di per sé un rischio clinico. Tuttavia la possibilità che essi ma possono essere veicolo di geni codificanti l'antibiotico-resistenza verso batteri patogeni presenti negli alimenti o nel tratto gastro-intestinale umano (inclusi enterococchi, streptococchi e listeria), costituisce un possibile rischio per la salute umana che deve essere attentamente valutato. Obiettivo di questo lavoro è stato quello di valutare attraverso metodi di indagine fenotipica con le tecniche delle microdiluizioni in brodo, Etest e disc-diffusion, i livelli di antibiotico resistenza per le specie S. thermophilus e L. plantarum verso gli antibiotici tetraciclina, eritromicina, clindamicina, streptomicina, gentamicina, ampicillina. Ceppi atipici appartenenti alla specie S. thermophilus sono stati sottoposti ad analisi genetiche con lo scopo di caratterizzare e localizzare i geni responsabili della resistenza. E' stato inoltre testato il possibile trasferimento orizzontale dei geni di antibiotico resistenza nativi da S. thermophilus verso i batteri Gram-positivi E. faecalis e Listeria monocytogenes. In alcuni ceppi di S. thermophilus resistenti si sono infine osservati e studiati particolari caratteri fenotipici ( fitness ) correlati alla presenza delle determinanti genetiche di antibiotico resistenza nell'ospite batterico.
In the last decades, the use of antibiotics in human therapy or in animal husbandry as growth promoters has induced the development and the diffusion in antibiotic resistant micro-organisms. In this context antibiotic resistant Lactic Acid Bacteria (LAB) do not represent a clinical risk in themselves. However, the possibility that S. thermophilus cultures might transfer antibiotic resistance genes to pathogenic species either present in food or in the gastrointestinal tract (including enterococci, streptococci and listeria) represents a potential clinical risk that needs to be carefully evaluated. The aim of this study was to evaluate by means of phenotypic methods (microdilution, E-test, disc-diffusion) the levels of antibiotic resistance for S. thermophilus and L. plantarum species against the antibiotic tetracycline, erythromycin, clyndamicin, streptomycin, gentamycin and ampicillin. The atypical resistant S. thermophilus strains were subjected to genetic analyses in order to characterise and to localise the antibiotic resistance determinants. Furthermore the ability of the resistant S. thermophilus strains in transferring the antibiotic resistant determinant was assessed in mating experiments using as recipients the Gram-positive bacteria E. faecalis and Listeria monocytogenes. In same resistant S. thermophilus strains, special bacterial fitness related with the presence of the antibiotic resistance determinants in the bacterial hosts were observed and studied.

Staphylococcus epidermidis has emerged in recent years as an important nosocomial pathogen, especially in infections associated with implanted foreign body materials (e.g., prosthetic joints and heart valves) and in individuals with a compromised immune system (e.g., cancer patients and neonates). Although rare, implant infections are long lasting and cause severe suffering for the patient that includes pain and disability and even increased mortality. One aim of the present thesis was to develop and evaluate a genetic method for species identification and simultaneous detection of rifampicin resistance in staphylococci. A second aim was to examine S. epidermidis isolated from prosthetic joint infections (PJIs) and from wrists and nares of healthy individuals regarding their antibiotic susceptibility, biofilm production, virulence factors, and epidemiology. Comparison with phenotypic diagnostics revealed that 8 (16%) of 49 isolates differed in their species identification in favour of the genetic method. In addition, mutations associated with rifampicin resistance, including two not previously reported, were possible to detect in all isolates resistant to rifampicin. Antibiotic susceptibility testing of 61 PJI isolates showed multi-drug resistance in 91%. Furthermore, the results of the synergy testing revealed that no antibiotic combination was significantly better than the others. Hence, the effects that were possible to detect were isolate dependent. To find a method for discriminating between invasive (n=61) and commensal (n=24) isolates of S. epidermidis genotypic and phenotypic characterisations of biofilm production (including the ica and aap genes), antibiotic susceptibility, virulence-related genes (such as agr and ACME) and epidemiology were performed (using multilocus sequence typing [MLST], typing of the staphylococcal chromosome cassette mec [SCCmec] and PhenePlate). Significant differences were found in antibiotic susceptibility, i.e. there was more resistance among invasive isolates. MLST sequence types (ST) ST2 and ST215 dominated the invasive isolates.

Orientador: Caio Cezar Randi Ferraz
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Canais radiculares infectados tipicamente abrigam uma microbiota mista, composta principalmente por espécies anaeróbias. O papel das bactérias e seus subprodutos no desenvolvimento e perpetuação das infecções endodônticas já está bem estabelecido. Pesquisas têm sido realizadas para correlacionar a presença de bactérias em canais radiculares infectados com sinais e sintomas clínicos. A proposta do presente estudo foi investigar a composição da microbiota de canais radiculares infectados, associados a abscessos periapicais, e a possível correlação entre espécies bacterianas específicas com as fases dos abscessos, além de realizar testes de sensibilidade antimicrobiana das bactérias isoladas. As amostras microbiológica foram coletadas de 30 canais radiculares usando pontas de papel estéreis, transportadas em VMGA, diluídas, plaqueadas e incubadas em câmara de anaerobiose. Colônias microbianas foram isoladas, caracterizadas e identificadas por métodos padronizados. Cento e dezessete bactérias foram encontradas, sendo 75 (64,1 %) anaeróbias estritas. Uma ou mais (máximo de 10) espécies bacterianas foram encontradas em 29 (96,6%) canais radiculares infectados associados a abscessos periapicais, confirmando a característica polimicrobiana das infecções endodônticas. As bactérias anaeróbias mais freqüentemente isoladas foram: Peptostreptococcus prevotii (43,3%), Peptostreptococcus micros (30%), Fusobacterium necrophorum (23,3%). Embora menos freqüentes, bactérias facultativas como Gemella morbillorum (30%) e Streptococcus mitis (20%) também foram encontradas. Contudo, a análise estatística não encontrou relação entre presença dos abscessos periapicais com qualquer das espécies bacterianas identificadas (p>0,05). As espécies mais prevalentes Peptostreptococcus prevotii e Fusobacterium necrophorum foram testadas quanto à suscetibilidade antimicrobiana através do método do E­test, utilizando os seguintes antibióticos: benzilpenicilina, amoxicilina, amoxicilina + ácido clavulânico, metronidazol e clindamicina. Ps. prevotii e F. necrophorum apresentaram-se sensíveis a todos os antibióticos testados. Apesar da ausência de significância estatística, nossos resultados indicaram predominância de bactérias anaeróbias Gram-positivas e a presença de microbiota mista nos canais radiculares infectados associados a abscessos periapicais. Os testes de suscetibilidade antimicrobiana revelaram a presença de sensibilidade bacteriana entre as espécies Peptostreptococcus prevotii e Fusobacterium necrophorum aos antibióticos benzilpenicilina, amoxicilina, amoxicilina + ácido clavulânico, metronidazol e clindamicina
Abstract: Infected dental root canais typically harbour a mixed flora, including many anaerobic species. The role of these microorganisms and their by-products in the development and perpetuation of pulp and periapical diseases has already been well established. Efforts have been made in order to correlate bacteria present in infected dental root canais to clinical signs and symptoms. The present study was outlined to identify microorganisms present in root canais associated to periapical a bscesses , the correlation specific bacteria species with the phases of this inflammatory acute process, signs and symtoms and, to test the susceptibility of this microbiota to antibiotics. Microbiological samples were collected from 30 root canais using sterile paper points, transported in VMGA and diluted, plated and incubated in an anaerobic chamber. Microbial colonies were then purified, characterised and identified by established methods. One hundred seven different bacterial species were recovered, being 75 (64.1 %) strict anaerobes or microphilic species. One or more (maximum of 10) bacterial species were recovered from 29 (96.6%) root canais, showing the polymicrobial characteristic of dental infections. The anaerobes most frequently isolated were: Peptostreptococcus prevotii (43.3%), Peptostreptococcus micros (30%), Fusobacterium necrophorum (23.3%). Although less frequent, facultative bacteria as Gemella morbillorum (30%) e Streptococcus mitis (20%) were also found. However, statistical analysis by a Pearson X2 test or a one-sided Fisher's exact test did not find statistical relationship between any bacterial specie identified and the presence of periapical abscesses (p>0.05). Antibiotic sensitivity of Peptostreptococcus prevotii e Fusobacterium necrophorum was accomplished with the E-test System. These bacterial isolates were tested for their susceptibility/resistance to benzylpenicilin, amoxiciln, amoxicilin combined with clavulanate and clindamycin. The species Peptostreptococcus prevotii and Fusobacterium necrophorum were susceptible to ali tested antibiotics. In spite of the lack of statistical significance, our results indicated predominance of Gram-positive anaerobic bacteria at the mixed microflora present in dental root canais associated with periapical abscesses. Antibiotic susceptibility data showed Peptostreptococcus prevotii and Fusobacterium necrophorum susceptibility to ali tested antibiotics
Mestrado
Endodontia
Mestre em Clínica Odontológica

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
The marketing of fish has shown substantial increase; however, their place of origin and ways of handling and storage may pose health risk. The microorganisms in the food chain and in capture environments cause concern over the risk of disease transmission and the multiple resistance to several drugs. This study aimed at characterizing the antimicrobial resistance of 191 strains of Escherichia coli isolated from 20 fish samples, ten saltwater samples: (5) mackerel (S. cavalla) and (5) snapper (L. purpureus) and ten freshwater samples: (5) curimatà (P. cearenses) and (5) tilapia (O. niloticus), sold in street markets of the city of Fortaleza (Cearà State), Brazil. The strains were isolated, identified and then subjected to susceptibility testing (20 antibiotics) in order to provide the resistance profile. Moreover, it was also performed: Multiple Resistance Index (MRI), the Antimicrobial Resistance Index (ARI), Minimum Inhibitory Concentration (MIC), plasmid "cure" and analysis of plasmid profile through DNA extraction. Strains isolated from marine and freshwater fish species showed distinct multidrug resistance profiles, but with broad resistance to penicillins and tetracyclines. There was high frequency of saltwater isolates resistant to chloramphenicol. Overall, low resistance to betalactamase inhibitors (ampicillin/sulbactam, and piperacillin/tazobactam) and aminoglycosides was observed, excepting streptomycin. The MRI has shown that about 50% of the isolates were resistant to five of the twenty tested antimicrobials, highlighting the strains from mackerel (55.5%). As for MIC, the percentage of isolates resistant to NAL, CIP, MFX, SUT and AMO stood out in saltwater fish strains. For bacteria from freshwater, greater resistance to high concentrations of antimicrobials were most significant for AMO, SUL and NAL. Chromosomal profiles in freshwater isolates were mostly composed of amoxicillin, ampicillin, streptomycin, and sulfametazol/trimethoprim. For saltwater fish, there was predominance of quinolones. Analysis of the plasmid DNA showed the occurrence of a heterogeneous population of small plasmids distributed in various profiles. The greater diversity and lower molecular weight were observed in strains of marine fish samples. Thus, it is emphasized that the circulation of pathogenic E. coli with antimicrobial resistance characteristics poses a risk to the aquatic ecosystem and the marketing environment, there being need for continued vigilance to contaminant bacterial to fish so that the safety of these foods is guaranteed.
A comercializaÃÃo de pescado tem indicado aumento substancial, contudo, seu local de origem e formas de manipulaÃÃo e armazenamento, podem representar risco sanitÃrio. Os micro-organismos presentes na cadeia produtiva de alimentos e nos ambientes de captura ocasionam preocupaÃÃo pelo risco de transmissÃo de doenÃas e pela mÃltipla resistÃncia apresentada a diversos fÃrmacos. Este trabalho objetivou caracterizar a resistÃncia antimicrobiana de 191 cepas de Escherichia coli isoladas de 20 amostras de pescado, sendo dez amostras de pescado de Ãgua salgada: (5) cavala (S. cavalla) e (5) pargo (L. purpureus) e dez amostras de Ãgua doce: (5) curimatà (P. cearenses) e (5) tilÃpia (O.niloticus), comercializadas em feiras livres da cidade de Fortaleza, CE. As cepas foram isoladas, identificadas e posteriormente submetidas a testes de susceptibilidade (20 antimicrobianos) a fim de conferir o perfil de resistÃncia. AlÃm disso, foram realizados: Ãndice de MÃltipla ResistÃncia (IMR), Ãndice de ResistÃncia a Antimicrobianos (IRA), ConcentraÃÃo InibitÃria MÃnina (CIM), âcuraâ plasmidial e anÃlise de perfil plasmidial, atravÃs de extraÃÃo de DNA. As cepas isoladas das espÃcies de pescado marinho e de Ãgua doce apresentaram perfis de multirresistÃncia distintos, porÃm com ampla resistÃncia Ãs penicilinas e Ãs tetraciclinas. Houve elevada frequÃncia de isolados de pescado marinho resistentes ao cloranfenicol. Foi observada, de forma geral, baixa resistÃncia para inibidores de betalactamases (ampicilina/ sulbactam e piperacilina/ tazobactam) e aminoglicosÃdeos, excetuando, estreptomicina. O IMR demonstrou que cerca de 50% dos isolados foram resistentes a cinco dos vinte antimicrobianos testados, destacando as cepas provenientes de cavala (55,5%). Nos testes de CIM observou-se que, cepas isoladas de pescados de Ãgua salgada apresentaram maiores percentuais de isolados resistentes a NAL, CIP, MFX, SUT e AMO. Maiores resistÃncias a elevadas concentraÃÃes de antimicrobianos, quando testadas bactÃrias oriundas de Ãgua doce, foram observadas para AMO, SUL e NAL. Os perfis cromossomiais em isolados de Ãgua doce foram na maioria compostos por amoxicilina, ampicilina, estreptomicina e sulfametazol ∕ trimetropim. Para o pescado de Ãgua salgada, foi verificado a predominÃncia de quinolonas. A anÃlise do DNA plasmidial mostrou a ocorrÃncia de uma populaÃÃo heterogÃnea de pequenos plasmÃdios distribuÃdos em vÃrios perfis. A maior diversidade e menor peso molecular foram observados em cepas de amostras de pescado de origem marinha. Dessa forma, destaca-se que a circulaÃÃo de E. coli patogÃnicas com caracterÃsticas de resistÃncia antimicrobiana representa um risco ao ecossistema aquÃtico e ao ambiente de comercializaÃÃo, havendo necessidade de vigilÃncia contÃnua a bactÃrias contaminantes do pescado, para que a seguranÃa desses alimentos seja garantida.

Suite à la découverte des antibiotiques, les succès thérapeutiques ont laissé présager un avenir où les maladies infectieuses d’origine bactérienne seraient éradiquées. Cependant, en moins d’un siècle, l’utilisation massive des antibiotiques à large spectre a conduit à l’émergence de résistances réduisant ainsi les options thérapeutiques. Mon projet de recherche vise à comprendre les altérations métaboliques et morphologiques bactériennes induites précocement par les antibiotiques et à contribuer au développement de tests diagnostiques rapides et fiables pour favoriser la mise en place d’antibiothérapies plus ciblées. Grâce au suivi des modifications des divers paramètres métaboliques et morphologiques des bactéries après traitement aux antibiotiques, nous avons montré l’intérêt des marqueurs de viabilité tels que le DiBAC4(3), le TOPRO®-3 ou encore l’Alexa FluorTM Hydrazide pour la détection rapide (<3h) de la sensibilité des bactéries aux antibiotiques. Nous avons notamment montré, pour la première fois, que la carbonylation des protéines, qui est induite dans des conditions de stress oxydatif et de vieillissement cellulaire, est un marqueur précoce universel de la sensibilité aux antibiotiques bactéricides. Suite à cette première partie de l’étude, nous avons souhaité comprendre les mécanismes mis en jeu par les bactéries en réponse au stress létal causé pas les antibiotiques. Au cours de nos expériences, il a été observé que lorsque les conditions ne permettaient plus la survie de l’organisme, un signal de fluorescence intrinsèquement lié à la bactérie permettait de prédire l’issue fatale après seulement 2 heures d’incubation avec l’antibiotique. En effet, suite à un traitement avec un antibiotique bactéricide ciblant la synthèse du peptidoglycane bactérien (ampicilline), nous avons observé une fluorescence maximale des cellules à la dose d’antibiotique correspondant à la Concentration Minimale Inhibitrice (CMI). L’augmentation de la fluorescence des cellules bactériennes a aussi été observé lors du traitement létal avec un agent biocide (hypochlorite de sodium). Cependant, ce phénomène n’est plus observable avec des antibiotiques bactériostatiques ou bactéricides qui inhibent la synthèse protéique indiquant l’importance d’un métabolisme bactérien actif. Les corrélations de propriétés spectrales nous ont permis de suspecter les molécules de flavines comme étant responsables du phénomène d’autofluorescence observé. De plus, nous avons montré une suractivation de la voie de biosynthèse des cofacteurs de type flavines et des flavoprotéines en présence d’ampicilline. Finalement, nous avons effectué des expériences de tri et de survie cellulaire de populations bactériennes traitées à l’ampicilline. Nos résultats ont montré que les cellules très fluorescentes ont une survie moyenne 5 fois supérieure aux cellules peu fluorescentes. Ceci suggère que le signal de fluorescence observé est une réponse cellulaire médiée par les composés flavonoïdes pour tenter de survivre au traitement antibiotique. Des travaux exploratoires suggèrent que le phénomène étudié chez les bactéries est conservé chez les levures et chez les cellules humaines. Ces résultats ouvrent de nouvelles perspectives dans la compréhension de la physiologie bactérienne, l’étude de la réponse bactérienne face à un stress exogène et la surveillance rapide de la viabilité des cellules
Following the discovery of antibiotics, the therapeutic successes foreshadowed a future where infectious diseases of bacterial origin would be eradicated. However, in less than a century, the massive use of broad-spectrum antibiotics led to the emergence of resistance thus reducing therapeutic options. My research project aims to understand early bacterial metabolic and morphological changes induced by antibiotics and to contribute to the development of rapid and reliable diagnostic tests to promote the implementation of more targeted antibiotic treatments. By monitoring changes in various metabolic and morphological parameters of bacteria after antibiotic treatment, we have shown the interest of viability markers such as DiBAC4(3), TOPRO®-3 or Alexa FluorTM Hydrazide for rapid detection (<3h) of bacterial susceptibility to antibiotics. In particular, we have shown for the first time that protein carbonylation, which is induced under conditions of oxidative stress and cellular aging, is a universal early marker of bactericidal antibiotic susceptibility. Following this first part of the study, we wanted to understand the mechanisms involved in bacterial response to lethal stress caused by antibiotics. Following this first part of the study, we wanted to understand the bacterial mechanisms involved in response to lethal stress caused by antibiotics. In our experiments, it was observed that when the conditions no longer allowed the organism survival, a fluorescence signal intrinsically linked to the bacterium allowed to predict the fatal outcome after only 2 hours of incubation. Indeed, following a treatment with a bactericidal antibiotic targeting the synthesis of bacterial peptidoglycan (ampicillin), we observed a maximum fluorescence of the cells at the dose of antibiotic corresponding to the Minimum Inhibitory Concentration (MIC). The fluorescence increase of bacterial cells was also observed during the lethal treatment with a biocidal agent (sodium hypochlorite). However, this phenomenon is no longer observable with bacteriostatic or bactericidal antibiotics that inhibit protein synthesis indicating active bacterial metabolism importance. The correlations of spectral properties allowed us to suspect the flavin molecules as responsible for the observed autofluorescence phenomenon. In addition, we showed an overactivation of the biosynthesis pathway of flavin-type cofactors and flavoproteins occurring during ampicillin treatment. Finally, we performed cell sorting and cell survival experiments of ampicillin-treated bacterial populations. Our results showed that highly fluorescent cells have an average survival 5 times higher than low fluorescent cells. This suggests that the fluorescence signal observed is a cellular response mediated by flavonoid compounds in an attempt to survive to antibiotic treatment. Exploratory work suggests that the phenomenon studied in bacteria is conserved among yeasts and human cells. These results open new perspectives in bacterial physiology understanding, the study of bacterial response to exogenous stress and the rapid monitoring of cell viability

Introdução - Vibrio metschnikovii é um bacilo gram-negativo, potencialmente patogênico, amplamente distribuído e isolado de ecossistemas aquáticos e raramente de amostras clínicas de humanos. Na triagem laboratorial para espécies do gênero Vibrio, Vibrio metschnikovii é descartado por ser oxidase negativa, característica que o diferencia das demais espécies patogênicas. Em relação à pesquisa do perfil de suscetibilidade a antibióticos, esta é pouco realizada com isolados de origem ambiental. Objetivos - Isolar e identificar genotipicamente Vibrio metschnikovii de amostras ambientais e caracterizar seu perfil de suscetibilidade a antibióticos. Métodos - Um total de dez amostras foram obtidas de Março a Agosto de 2009, sendo uma de molusco (vôngole), três de peixe (pescada, sardinha e tainha) e seis de esgoto (três de esgoto bruto e três de esgoto tratado). O isolamento foi inicialmente realizado em meio seletivo para Vibrio (ágar TCBS) e a confirmação da espécie foi feita com iniciadores específicos através da PCR utilizando o DNA extraído pela técnica de choque térmico. O antibiograma foi realizado com base no documento M45-A CLSI 2006, seguindo a técnica de disco difusão, empregando quinze antibióticos. Foi realizada a pesquisa de genes de resistência a beta-lactâmicos e aminoglicosídeos. Resultados - De 123 isolados com características típicas para a espécie, 70 foram confirmados como Vibrio metschnikovii através da PCR, sendo 43 de amostras de molusco e peixes e 27 de esgoto. Um total de 66 isolados foi resistente a pelo menos um antibiótico, mas nenhum gene de resistência foi detectado. Conclusões - Os resultados indicam que Vibrio metschnikovii pode ser isolado de diferentes amostras de origem ambiental, demonstrando que esses microrganismos podem ter distribuição mais ampla que o descrito na literatura. A PCR foi uma ferramenta fundamental para superar a fragilidade da identificação da espécie segundo o método fenotípico. Além disso, a resistência observada mostra que Vibrio metschnikovii pode atuar como um reservatório de genes de resistência que podem ser facilmente transferidos entre microrganismos em um mesmo ambiente. O perfil de suscetibilidade a antibióticos pode ser utilizado como referência na caracterização clínica deste patógeno em potencial, auxiliando na conduta terapêutica em casos de ocorrência de doentes acometidos pelo microrganismo em questão
Introduction - Vibrio metschnikovii is a gram-negative bacillus, potentially pathogenic, widely distributed and isolated from aquatic ecosystems and rarely from human clinical samples. At laboratorial screening for the species of the Vibrio genus, Vibrio metschnikovii is discarded for being oxidase negative, characteristic that differentiates it from the others pathogenic species. With regard to the research of antibiotic susceptibility profile, this is seldom done with environmental isolates. Objectives - To isolate and genotypically identify Vibrio metschnikovii from environmental samples and characterize the antibiotic susceptibility profile. Method - A total of ten samples were obtained from March to August 2009, with one of them originated from mussel (vongole), tree of fish (whitefish, sardine and mullet) and six from sewage (three from raw sewage and three from treated sewage). The isolation was initially performed in a selective medium for Vibrio (TCBS agar) and the specie confirmation was done with specific primers through PCR using DNA extracted by the thermal shock technique. The antibiogram was performed according to the document M45-A from CLSI 2006, following the technique of disk diffusion, using fifteen antibiotics. The research for beta-lactams and aminoglycosides resistance genes was also performed. Results Seventy out of 123 isolates, with typical characteristics for the specie, were confirmed as Vibrio metschnikovii by PCR, with 43 originated from mussel and fish and 27 from sewage. A total of 66 isolates were resistant to at least one antibiotic, however no resistance gene was detected. Conclusions The results indicate that Vibrio metschnikovii can be isolated from different samples of ambient origin, demonstrating that these microorganisms can have wider distribution than the described in literature. The PCR was a fundamental tool to overcome the fragility of the specie identification according to the phenotypic method. Furthermore, the resistance found shows that Vibrio metschnikovii can act as a reservoir of resistance genes that can easily be transferred between microorganisms in the same environment. The antibiotic susceptibility profile can be used as reference at the clinical characterization of this potential pathogen, assisting therapeutic management in cases of patients affected by this microorganism

Au cours des dernières années, l’automatisation en bactériologie clinique est devenue très importante du fait de l’augmentation constante du nombre d’échantillons mais nécessite tout de même des améliorations. Une revue a été rédigée pour présenter les nouveaux challenges et opportunités dans la surveillance et la détection des bactéries multi-résistantes. Premièrement nous avons testé de nouveaux outils technologiques innovants permettant la lecture plus précoce des antibiogrammes grâce à des scanners de haute résolution d’images: scanner Advencis Bio-System Incubator et le Scan® 1200 et de réaliser l’ensemencement des antibiogrammes en automatique sur un automate déjà existant le PREVI® Isola (bioMérieux, Marcy l’Etoile, France). Dans un second temps, une analyse rétrospective de la résistance aux antibiotiques observés sur l’hôpital la Timone à Marseille a été réalisé entre 2014 et 2016. Nous avons développer un logiciel d’interprétation automatique des phénotypes basé sur la reconnaissance d’images pour lequel un brevet a été rédigé : le logiciel « ANTI-LOGIC ». Le but de notre troisième travail a été de participer au développement d’une PCR en temps réel permettant de détecter le gène mcr-1. Puis, nous avons travaillé sur des souches multi-résistantes afin de tester un panel constitué de 29 à 32 antibiotiques. Pour la détection des souches résistantes à la colistine, un milieu de culture sélectif a été mis au point et nous avons essayé de trouver une alternative thérapeutique par l’étude de combinaisons d’anciens antibiotiques (colistine-sulfadiazine) pour le traitement et la décolonisation avant greffe fécale de patients infectés ou colonisés par des BMR
In the last few years, automation in clinical microbiology has become very important due to the constant increase of samples but they need to be improved. A review of the literature was performed to expose the new challenges and the new opportunities in the surveillance and the detection of multi-drug resistance bacteria.For that purpose, we conduct a thorough search to test new innovative technological tools for reading earlier AST with high-resolution image scanner: Advencis Bio-System Incubator and the Scan® 1200 and to carry out the automatic seeding of AST with the PREVI® Isola system (bioMérieux, Marcy l’Etoile, France). In a second step, the retrospective analysis of antibiotic resistance and phenotypes observed at La Timone hospital in Marseille was carried out between 2014 and 2016. We have developped a new software for the automatic interpretation of phenotypes based on image recognition protected by a patent: the "ANTI-LOGIC" software. The aim of our third work was to participate on the development of a real-time PCR to detect the mcr-1 gene. We then worked on multi-resistant strains with Gram-negative bacteria, we have tested a large panel of 29-32 antibiotics to see if we are in a therapeutic stalemate. For the detection of colistin resistant strains, a selective culture medium has been developed. In order to finish this part, with the appearance of colistin-resistant strains, we tried to find a therapeutic alternative by studying combinations of old antibiotics (colistin-sulfadiazine) for the treatment and decolonization before faecal transplantation of patients infected or colonized by multi-resistant bacteria

Escherichia coli patogênica aviária (APEC) é a causa de doenças extra-intestinais em aves, que se manifestam na forma de doenças localizadas ou infecções sistêmicas, gerando grandes perdas econômicas para a indústria aviária. O objetivo desse trabalho foi estudar isolados de APEC do sul do Brasil em relação à resistência a agentes antimicrobianos; a prevalência de fatores associados à virulência; a tipagem filogenética e o perfil filogenético. Na avicultura, os agentes antimicrobianos são muito utilizados na prevenção de infecções e na forma de promotores de crescimento. Foram utilizadas 41 isolados de E. coli obtidos de infecções sistêmicas e 144 isolados de celulite, utilizando o método de difusão com discos. Isolados APEC apresentaram baixos níveis de resistência, com excessão da tetraciclina e das sulfonamidas; e para isolados de colissepticemia, também foi observado uma resistência aos antimicrobianos que atuam na parede celular (ampicilina, cefalotina, bacitracina e ceftiofur). Vários fatores de virulência têm sido investigados em cepas APEC, os que têm sido mais frequentemente associados com a patogenicidade são as fímbrias de aderência F1 e Tsh (hemaglutinina sensível à temperatura); o sistema sideróforo aerobactina, a proteína iss (increased serum survival), a cápsula K e a produção de colicina V. Foram realizadas reações da polimerase em cadeia multiplex (PCR), testando um total de 33 fatores nos isolados APEC. Os fatores relacionados a adesão, sideróforos e resistência ao soro foram presentes em todas as amostras. Os fatores que apresentaram maior prevalência nas amostras foram, fimC, ompA, crlA e traT. A tipagem filogenética foi realizada através do método descrito por Clermont et al., (2000), que permite separar os isolados em 4 grupos filogenéticos principais (A, B1, B2 e D). Observamos a maior presença de isolados APEC pertencentes ao grupo D, tanto para celulite quanto para colissepticemia. A análise do perfil filogenético foi realizada pelo método ARDRA, que é baseado na variação da região do espaçamento intergênico (ISR) na região 16S-23S do DNA ribossômico. Os resultados foram analisados formando um dendrograma que mostra a proximidade filogenética dos isolados, indicando que não foi possível separar os clones de celulite dos de colissepticemia e não existem clones endêmicos nas regiões analisadas. Os resultados obtidos no presente estudo, fornecem um panorama geral da resistência aos agentes antimicrobianos, prevalência dos fatores de virulência, tipagem filogenética e perfil filogenético encontrados nos isolados de E. coli aviária do sul do Brasil de infecções de colissepticemia e celulite.
extra-intestinal infections in poultry, called colibacilose, and cause great economic losses to the poultry industry. The aim of this work was to examine APEC strains, isolated from avian cellulitis and colisepticemic chickens from South Brazil, for antibiotic resistance, presence of virulence-associated genes (VAGs), phylogenetic typing and phylogenetic analyses. In poultry, antibiotics are routinely used to prevent infection and promote growth. We analyzed the susceptibility to 15 antibiotics in 144 E. coli isolates collected from cellulitis lesions and in 41 isolates from septicemic chickens. APEC isolates have shown low levels of resistance excepting tetracycline and sulphonamides, and colisepticemic isolates were resistant to antibiotics that act in the cell wall, such as ampicilin, cephalotin, ceftiofur and bacitracin. The virulence factors most frequently associated with APEC pathogenicity are the adhesins F1 and Tsh (Temperature sensitive hemagglutinin), the iron acquisition system aerobactin, the protein Iss (increased serum survival), K1 capsule and production of colicin V. To investigate the presence of VAGs in the isolates, we performed multiplex polymerase chain reactions to test 33 virulence factors. Virulence factors related to adhesion, iron acquisition and serum resistance were present in almost all strains. The factors fimC, ompA, crlA and traT were the most frequent in APEC isolates. The phylogenetic typing were done using the Clermont et al. (2000) method, which classifies E. coli strains into four main phylogenetic groups (A, B1, B2, and D). Our phylogenetic typing has shown that cellulitis and colisseptisemic isolates were more related to group D. Amplified ribossomal restriction analysis (ARDRA) was performed in colissepticemic and celullitis isolates from broiler chickens from Southern Brasil. The similarity among isolates were observed with a dendrogram based on the band pattern. Our results have shown that clones of celullitis and colissepticemic isolates could not be distinguished and there is no endemic clones in regions observed analised. The results of the present work give us a panorama of the susceptibility to antibiotics, virulence factors, phylogenetic typing and phylogenetic analyses currently found in APEC isolates from severe lesions of cellulites and colibacillosis in south Brazil. Besides that, these analyses could be helpful for monitoring and preventing outbreakes of colibacilosis. Controling the first stages of the disease and detecting more prevalent clones in this region may reduce the incidence of the disease.

Dissertação de Mestrado Integrado em Medicina Veterinária
Tendo em conta que a colocação de bypass ureterais subcutâneos, como forma de tratamento da ureterolitíase obstrutiva em gatos, é uma crescente realidade e considerando que a infeção do trato urinário poderá ser uma complicação deste procedimento, o conhecimento dos agentes etiológicos e padrões de suscetibilidade nestas circunstâncias é importante para o seu tratamento. Neste estudo pretendeu-se contribuir para a caracterização da infeção do trato urinário felino num hospital veterinário de Lisboa, comparando dois grupos distinguíveis pela ausência ou presença de bypass ureteral subcutâneo de modo a percecionar as diferenças existentes entre estes dois grupos. Para o efeito realizou-se um estudo retrospetivo, englobando todos os gatos submetidos a urocultura no Hospital Veterinário do Restelo, durante o período de 2012-2017. Avaliaram-se vários parâmetros, em especial os agentes etiológicos isolados, padrões de suscetibilidade e antibioterapia prescrita. Os agentes uropatogénicos isolados com maior frequência foram a Escherichia coli, o Staphylococcus spp. e o Enterococcus spp. Perante a presença de bypass, verificou-se uma maior infeção do trato urinário por Staphylococcus spp. (p<0.05) e menor infeção por E. coli (p<0.05). Não se identificaram diferenças estatisticamente significativas entre os perfis de suscetibilidade dos dois grupos. De entre os antibióticos testados, a gentamicina foi a substância ativa com maior proporção de agentes patogénicos sensíveis e a penicilina foi a molécula que apresentou maior resistência. Observou-se 25% de estirpes multirresistentes na totalidade de agentes uropatogénicos isolados nos dois grupos. Neste estudo, observou-se a prescrição de antibioterapia empírica em cerca de 30% dos animais. De modo a instituir uma antibioterapia adequada e minimizar a emergência de resistência bacteriana, é fundamental que se monitorize o uso de antibióticos e que se conheça os principais agentes etiológicos e padrões de suscetibilidade em cada região geográfica. Este estudo pode assim contribuir para o aumento do conhecimento da infeção urinária felina e auxiliar a prescrição antibiótica, principalmente em animais submetidos à colocação de um bypass ureteral subcutâneo, na região de Lisboa.
ABSTRACT - CONTRIBUTION TO THE CHARACTERIZATION OF URINARY TRACT INFECTION IN CATS: A RETROSPECTIVE STUDY IN ANIMALS WITH AND WITHOUT SUBCUTANEOUS URETERAL BYPASS - Subcutaneous urethral bypass placement as a form of obstructive ureterolithiasis treatment in cats is a growing reality. Considering that urinary tract infection may be a complication of this procedure, knowledge of the etiologic agents and patterns of susceptibility in these circumstances is important for their treatment. This study aimed to contribute to the characterization of feline urinary tract infection in a veterinary hospital in Lisbon, comparing two groups distinguishable by the absence or presence of subcutaneous ureteral bypass in order to perceive the differences between both of them. For this purpose, a retrospective study was carried out, including all cats submitted to urine culture at the Veterinary Hospital of Restelo, from 2012 to 2017. Several parameters such as uropathogens, susceptibility patterns and prescribed antibiotherapy were evaluated. The most common pathogens identified were Escherichia coli, Staphylococcus spp. and Enterococcus spp. A greater infection of the urinary tract by Staphylococcus spp. (p<0.05) and lower infection by E. coli (p<0.05) was observed in the presence of bypass. No statistically significant differences were found between susceptibility patterns of the two groups. Of the antimicrobial agents tested in the present study, gentamicin had the highest proportions of susceptible bacterial isolates while penicillin showed the highest resistance. It was observed a 25% of multiresistant strains in all the uropathogens isolated in both groups. Empirical antibiotherapy was prescribed in 30% of the animals. Monitoring of the antibiotic use and the knowledge of the main pathogens and susceptibility patterns in each geographic region are essential to institute an adequate antibiotherapy and to minimize the emergence of antimicrobial resistance. So the results of the present study may thus contribute to an increase in the knowledge of feline urinary infection and to help the antibiotic prescription, especially in animals submitted to a subcutaneous ureteral bypass in the Lisbon region.
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Chlamydia trachomatis est une bactérie à développement intracellulaire obligatoire, divisée en 19 sérovars parmi lesquels les sérovars D-K sont responsables d’infections oculo-génitales et les sérovars L de la lymphogranulomatose vénérienne (LGV). En France, C. trachomatis est le principal agent bactérien responsable d’infections sexuellement transmissibles (IST). Les méthodes moléculaires occupent une place de choix dans le dépistage et l’épidémiologie des infections à C. trachomatis. Grâce à leur utilisation à partir de prélèvements non invasifs, nous disposons de chiffres de prévalence qui s’élèvent à 1,5% dans la population générale, 3,6% chez les femmes âgées de 18 à 24 ans sexuellement actives et 10 à 15% dans les centres à vocation de dépistage des IST. N’ayant aucune donnée chez la femme enceinte, le programme hospitalier de recherche clinique (MATIST) que nous avons mis en place chez les femmes enceintes suivies au CHU de Bordeaux a montré une prévalence de l’infection à C. trachomatis de 2,5%, à M. genitalium de 0,8% et à N. gonorrhoeae de 0%. Chez les femmes de moins de 24 ans, la prévalence était respectivement de 7,9% et 2,4%. La compréhension de l’épidémiologie et de la dissémination des infections à C. trachomatis nécessite la mise au point de techniques de typage performantes d’autant qu’un seul sérovar, le sérovar E, est rencontré dans près de la moitié des cas. Nous avons développé une méthode de typage moléculaire, la MLVA (MultiLocus Variable Number of Tandem Repeat Analysis), qui analyse le polymorphisme associé aux répétitions en tandem et permet un typage intra-sérovar. Cinq VNTRs ont été identifiés. La méthode a été automatisée puis appliquée à 220 souches et échantillons cliniques de C. trachomatis de génovar E, permettant d’identifier 25 types MLVA. Les souches d’origine ano-rectale isolées de patients homosexuels et les souches suédoises appartenant au nouveau variant ont été individualisées au sein de deux types MLVA uniques et distincts, suggérant une origine clonale. L’ensemble des résultats obtenus ont montré que la MLVA est un outil de typage moléculaire performant, plus discriminant que les autres méthodes auxquelles nous l’avons comparée. De plus, dans le cadre de la surveillance épidémiologique de la LGV ano-rectale due au variant L2b qui sévit en Europe depuis 2003 presque exclusivement chez les homosexuels, nous avons identifié le premier cas de LGV ano-rectale chez une femme. Enfin, nous avons développé une technique de PCR en temps réel permettant une détermination objective de la concentration minimale inhibitrice d’un antibiotique donné vis-vis de C. trachomatis. Cette technique a également montré que les antibiotiques étudiés n’avaient qu’une activité bactériostatique sur C. trachomatis
Chlamydia trachomatis is an obligate intracellular bacterium, divided into 19 serovars, among which serovars D-K are responsible for oculo-genital infections and serovars L of lymphogranuloma venereum (LGV). In France, C. trachomatis is the main bacterial cause of sexually transmitted diseases (STI). Molecular methods are the methods of choice for the C. trachomatis detection and epidemiology. Through their use, it has been shown that the prevalence of C. trachomatis infection rise up to 1.5% in the general population, to 3.6% for sexually experienced women aged 18-24 and to 10-15% in STI medical settings. As no data were available for pregnant women, we conducted a clinical research study (MATIST) in pregnant women at the Bordeaux University hospital. The prevalence of C. trachomatis, M. genitalium and N. gonorrhoeae infections was 2.5%, 0.8% and 0%, respectively. In women under 24 years, the prevalence of C. trachomatis, and M. genitalium infections was 7.9% and 2.4%, respectively. Understanding the epidemiology and the spread of C. trachomatis infection requires the development of efficient typing techniques knowing that a single serovar, serovar E, is found in nearly half the cases. We developed a MLVA (MultiLocus Variable-Number of Tandem Repeat Analysis) method which analyzes the genome polymorphism associated to tandem repeats and allowed intra-serovar subtyping. Five VNTRs were identified. The automated method was applied on 220 C. trachomatis genovar E clinical specimens and isolates, yielding 25 MLVA types. All anorectal isolates from men who have sex with men exhibited the same MLVA type, suggesting clonal spread. In the same way, we confirmed the clonal origin of the Swedish new variant of C. trachomatis. MLVA appears to be a good tool for molecular typing, with a higher discriminatory power than those of other methods used for comparison. Since 2003, a LGV proctitis outbreak caused by the new variant L2b has been reported in Europe in men who have had sex with HIV-positive men. We reported the first case of C. trachomatis L2b proctitis diagnosed in a woman. Finally, we developed a real-time PCR method allowing an objective determination of minimum inhibitory concentration of antibiotics for C. trachomatis. Our results also showed that all antibiotics studied only had bacteriostatic activity on C. trachomatis

Escherichia coli é um bacilo Gram-negativo, anaeróbico facultativo e de distribuição cosmopolita. E. coli coloniza o intestino de humanos e outros animais endotérmicos logo após o nascimento, estabelecendo-se como um importante membro da microbiota intestinal. Algumas cepas de E. coli podem adquirir fatores de virulência, assumindo assim, uma natureza patogênica, como é o caso das E. coli patogênicas extraintestinais (ExPEC). As cepas ExPEC apresentam a capacidade de colonizar e se disseminar em diversos nichos no hospedeiro, e são divididas em UPEC (E. coli uropatogênica), NMEC (E. coli causadora de meningite neonatal) e APEC (E. coli patogênica para aves). UPEC, NMEC e APEC compartilham fatores associados à virulência. Para serem aptas a causar doença, cepas ExPEC devem apresentar pelo menos um fator associado à adesão, um fator para captação de ferro (sideróforo) e um fator de resistência ao soro, podendo, também, apresentar genes que codificam toxinas e invasinas. Embora sejam conhecidos muitos fatores de virulência associados à patogenicidade de cepas ExPEC, a regulação da expressão de tais fatores ainda não foi elucidada. Fumarato-nitrato-redutase (FNR) é uma proteína que atua como regulador global, agindo como um sensor da presença de oxigênio em bactérias gramnegativas. Já foi demonstrado que FNR está relacionada à regulação da virulência de bactérias patogênicas como Shigella flexneri e Salmonella enterica serovar Typhimurium. Este trabalho teve como objetivo a análise epidemiológica e caracterização de cepas APEC, bem como a investigação do controle da expressão de fatores associados à virulência de ExPEC pelo regulador global FNR. Os resultados da análise epidemiológica das cepas APEC mostram o perfil de resistência aos agentes antimicrobianos, a prevalência dos fatores de virulência e dos grupos filogenéticos (de acordo com a classificação EcoR) e a relação filogenética dos isolados, fornecendo um panorama da caracterização de E. coli patogênicas aviárias de lesões severas de celulite e de infecção sistêmica oriundas da região sul do Brasil. Em relação ao FNR, este estudo mostrou a influência deste regulador sobre importantes fatores associados à virulência, estando envolvido no controle de várias etapas do estabelecimento da infecção por cepas ExPEC. A deleção de fnr na cepa UPEC CFT 073 reduziu a motilidade, a expressão das fimbrias tipo I e tipo P, reduziu a expressão da hemolisina e controlou a expressão da ilha de patogenicidade do α- cetoglutarato. Além disso, a deleção de fnr fez tornou as bactérias incapazes de invadir células dos rins e da bexiga e de causar doença in vivo em camundongos de 6 semanas. FNR também foi capaz de controlar as etapas da infecção por NMEC 56. Uma vez deletado, as bactérias perderam a capacidade de causar bacteremia, de crescer no fluido cerebrospinal e de causar doença in vivo em ratos de 5 dias de idade. A deleção de fnr em APEC O1 resultou na diminuição da expressão da proteína OmpT plasmidial, da fímbria do tipo I e do auto-transportador AatA. A principal contribuição deste trabalho foi demonstrar que FNR atua na regulação da expressão de importantes fatores associados à virulência de cepas ExPEC (UPEC, NMEC e APEC), sendo importante para o estabelecimento da infecção por essas cepas. Neste trabalho, verificamos que, além da função já conhecida de regular os genes envolvidos na manutenção de um meio anaeróbico, FNR também atua no controle de genes associados à virulência de cepas ExPEC, refletindo na capacidade de causar doença que tais cepas apresentam.
Escherichia coli is a Gram-negative bacillus, facultative anaerobic and has cosmopolitan distribution. E. coli colonizes the intestine of humans and other endothermic animals immediately after birth, establishing as an important member of the intestinal microbiota. Some strains of E. coli can acquire virulence factors thereby assuming a pathogenic nature, as in the case of extraintestinal pathogenic E. coli (ExPEC). ExPEC strains have the ability to colonize and spread out in different niches of the host, and are divided into UPEC (uropathogenic E. coli), NMEC (newborn meningitis E. coli) and APEC (avian pathogenic E. coli). UPEC, NMEC and APEC share virulence factors. To be able to cause disease, ExPEC strains must produce virulence factors required for adherence, for iron uptake (siderophore) and for resistance to serum and may also contain genes encoding toxins and invasins. Although many virulence factors associated with the pathogenicity of ExPEC strains are known, the regulation of the expression of these factors has not yet been fully elucidated. Fumarate nitrate reductase (FNR) is a global regulatory protein, acting as a sensor of oxygen in Gram- negative bacteria. It has been shown that FNR relates virulence of pathogenic bacteria such as Shigella flexneri and Salmonella enterica serovar Typhimurium. The aim of this study was to do an epidemiological analysis and characterization of APEC strains as well as the investigation of regulation of ExPEC’s virulence factors by the global FNR regulator. The results of epidemiological analysis of APEC strains showed the profile of antimicrobial resistance , the prevalence of virulence factors and phylogenetic groups (according to the EcoR group) and the phylogenetic relationship of the isolates, providing an overview of the characterization of avian pathogenic E. coli causing severe cellulitis lesions and systemic infection originating from southern Brazil. In relation to FNR, this study showed the influence of this important regulator of virulence factors that is involved in controlling various stages of establishment of infection by ExPEC strains. Deletion of fnr in UPEC strain CFT 073 reduced motility and expression of type I and type P fimbriae, reduced the expression of hemolysin and control the expression of the pathogenicity island of α -ketoglutarate. Furthermore, fnr mutant strains were unable to invade cells of kidney and bladder, and to colonize the urinary tract of 6 weeks-old mice. FNR was also able to control the stages of infection of NMEC 56. The fnr mutant lost its ability to cause bacteremia, grow in cerebrospinal fluid, cause disease in 5 days old rats. Deletion of fnr in APEC O1 resulted in decreased expression of genes corresponding to the plasmid encoded OmpT protein, type I fimbriae and autotransporter AatA. The main contribution of this work was to demonstrate that FNR regulates expression of important virulence factors of ExPEC strains (UPEC, NMEC and APEC), which is important for the establishment of infection by these strains. In this work, we found that, besides the already known function in regulating genes involved in maintaining an anaerobic environment, FNR also acts in the control of virulenceassociated genes of ExPEC strains, reflecting the ability of these strains to cause disease.

Resistensbestämningar som genererar ett kvantitativt MIC-värde är värdefullt för att kunna välja adekvat antibiotikabehandling. Referensmetoden för MIC-bestämning är mikrobuljongspädning där kommersiella antibiotikapaneler underlättar handhavandet av metoden på kliniska laboratorier. Syftet med studien var att verifiera mikrobuljongspädning med Sensititre™-panelerna SEMSE7 och SEMST7 avsedda för grampositiva kocker, genom att jämföra erhållet resultat med tidigare uppmätta referensvärden av MIC samt SIR-kategorier. Mikrobuljongspädning utfördes med SEMSE7-panel och MH-buljong för Staphylococcus spp. (n=21) och Enterococcus spp. (n=13) samt med SEMST7-panel och MH-F buljong för Streptococcus pneumoniae (n=20). Avläsning av MIC utfördes samt tolkades enligt SIR-systemet. Resultaten jämfördes mot kända referensvärden tidigare uppmätta med mikrobuljongspädning. Isolat analyserade med SEMSE7-panel och SEMST7-panel erhöll en överensstämmelse av MIC på 88,2% respektive 96,7%. Motsvarande kategorisk överensstämmelse av SIR var 92,5% respektive 92,6%. Resultatet visar att Sensititre™-panelen SEMST7 är godkänd i verifieringen av mikrobuljongspädning med god överensstämmelse med referensvärden av MIC samt SIR-kategorier. Vidare bedöms att fortsatt verifiering krävs av SEMSE7-panelen eftersom ej tillfredställande överenstämmelse med referensvärden av MIC erhölls, trots god överenstämmelse av SIR-kategorier. Studien visar att SEMST7-panelen kan implementeras för Streptococcus pneumoniae i den kliniska verksamheten på mikrobiologilaboratoriet, Jönköping.
Antimicrobial susceptibility testing methods generating a quantitative MIC are valuable for choosing adequate antibiotic treatment. Broth microdilution is the reference method for MIC determination and commercial panels with antibiotics facilitate the procedure in clinical laboratories. The aim of the study was to verify broth microdilution with the Sensititre™ panels SEMSE7 and SEMST7 designed for gram-positive cocci, by comparing results with previously measured reference values of MIC and SIR. Broth microdilution was performed using the SEMSE7 panel with MH broth for Staphylococcus spp. (n=21) and Enterococcus spp. (n=13), and using the SEMST7 panel with MH-F broth for Streptococcus pneumoniae (n=20). Reading MIC endpoints was performed and interpreted according to SIR system. Isolates analysed with the SEMSE7 and SEMST7 panel obtained essential agreement of 88,2% and 96,7%, and categorical agreement of 92,5% and 92,6% respectively. In conclusion, the Sensititre™ panel SEMST7 is approved in the verification of broth microdilution with satisfactory essential agreement and categorical agreement. Furthermore, it is considered that further verification is required for the SEMSE7 panel as unsatisfactory essential agreement was obtained, despite satisfactory categorical agreement. The study shows that the SEMST7 panel can be implemented for Streptococcus pneumoniae in the clinical practice at the microbiology laboratory, Jönköping.

Dissertação de Mestrado Integrado em Medicina Veterinária
Hospital-acquired infections (HAIs) are a rising problem worldwide. The best way of coping with these HAIs is through infection tracking and surveillance systems combined with prevention strategies namely efficient disinfection protocols. These considerations, associated with increasing reports about reductions in biocide susceptibility and development of cross-resistance to antimicrobials, emphasise the need of identifying how different factors can influence a biocide’s efficiency. In this study, 29 bacterial isolates (E. coli n=3, Pseudomonas spp. n=2, Enterococcus spp. n=23 and Staphylococcus pseudintermedius n=1), obtained from environmental samples collected from the Biological Isolation and Containment Unit (BICU), of the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine, University of Lisbon, were characterized regarding their antimicrobial susceptibility profile. From these isolates, 13 were further selected to investigate their susceptibility to Virkon® S, with and without the presence of organic matter. After this determination, 7 isolates (all enterococci) were submitted to sub-lethal concentrations of this formulation and, subsequently, new antimicrobial susceptibility profiles were determined. Fourteen of the 29 isolates (48.3%), all previously identified as enterococci, were classified as Multi-Drug Resistant. Concerning Virkon® S’s susceptibility in the absence of organic matter, the Minimal Bactericidal Concentration (MBC) of this biocide regarding all isolates was lower than the concentration regularly used. However, when organic matter was added, MBC values experienced a significant rise. After submission to sub-lethal concentrations of Virkon® S, 4 enterococci presented a phenotypic change in their susceptibility towards gentamicin. Virkon® S also presented higher MBC values in the presence of low concentrations of organic matter, but no rise in these values was observed in assays without interfering substance. Virkon® S proved to be an efficient formulation in eliminating the bacterial isolates collected from the BICU. However, organic matter may impair this efficiency, which reinforces the importance of proper sanitization before disinfection procedures. The changes observed in antimicrobial susceptibility profile may be due to a stress induced response, promoted by the sub-lethal concentration of Virkon® S and not necessarily due to the occurrence of mutations or other alterations in the isolates’ genome.
RESUMO - As infeções adquiridas em ambiente hospitalar são um problema emergente a nível mundial. O combate a estas infeções tem sido feito principalmente através da aplicação de sistemas de rastreio de infeções e de sistemas de vigilância, conjuntamente com estratégias de prevenção eficazes, como são os protocolos de desinfeção. A associação destes fatores com o aumento de estudos referentes a diminuições de suscetibilidade a biocidas e também a uma possível resistência cruzada a vários antibióticos, enfatizam a necessidade de entender de que forma certos fatores podem influenciar a eficiência destes compostos. Neste estudo, foram caracterizados 29 isolados bacterianos (E. coli n=3, Pseudomonas spp. n=2, Enterococcus spp. n=23 e Staphylococcus pseudintermedius n=1), obtidos de amostras ambientais de várias superfícies da Unidade de Isolamento e Contenção Biológica (UICB) pertencente ao Hospital Escolar da Faculdade de Medicina Veterinária, Universidade de Lisboa, relativamente ao seu perfil de suscetibilidade a antimicrobianos. Deste isolados, 13 foram selecionados de maneira a investigar a sua suscetibilidade ao Virkon® S, aquando a presença/ausência de matéria orgânica. Após esta determinação, 7 isolados (todos enterococos) foram incubados conjuntamente com uma concentração sub-letal de Virkon® S e foram determinados novos perfis de suscetibilidade. Catorze dos 29 isolados (48,3%), todos identificados como enterococos, foram classificados como multirresistentes. Na ausência de matéria orgânica, os valores de Concentração Mínima Bactericida (CMB) do Virkon® S foram inferiores à concentração regularmente utilizada. No entanto, aquando a adição de matéria orgânica, estes valores sofreram um grande aumento. Após submissão das bactérias a concentrações sub-letais de Virkon® S, 4 enterococos apresentaram uma alteração fenotípica relativamente à sua suscetibilidade à gentamicina. Em ensaios realizados na presença de matéria orgânica, observou-se um aumento dos valores de CMB do Virkon® S, enquanto que, quando esta estava ausente, este mesmo aumento não foi observado. O Virkon® S parece ser uma formulação eficiente, uma vez que conseguiu eliminar todos os isolados bacterianos testados. A matéria orgânica pode, no entanto, ser um entrave a esta eficiência, o que reforça a importância da limpeza pré-desinfeção. As alterações observadas relativamente ao perfil de suscetibilidade a antimicrobianos podem ser explicadas por uma adaptação ao stress químico incitado sobre as bactérias, devido à presença da concentração sub-letal de Virkon® S, e não necessariamente por mutações ou outras alterações genómicas.
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