Journal articles: 'Environmental microbiology (Analysis; Identification)'

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Slany, Michal, Petr Jezek, Vera Fiserova, Monika Bodnarova, Jiri Stork, Marta Havelkova, Frantisek Kalat, and Ivo Pavlik. "Mycobacterium marinum infections in humans and tracing of its possible environmental sources." Canadian Journal of Microbiology 58, no. 1 (January 2012): 39–44. http://dx.doi.org/10.1139/w11-104.

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The low frequency of nontuberculous mycobacterial infections, nonspecific symptoms for individual mycobacteria, and the lack of specific identification methods could alter correct diagnosis. This study presents a combined microbiology and molecular-based approach for Mycobacterium marinum detection in four aquarists with cutaneous mycobacterial infection. Simultaneously, ecology screening for M. marinum presence in the aquarists’ fish tanks was performed. A total of 38 mycobacterial isolates originated from four human patients (n = 20), aquarium animals (n = 8), and an aquarium environment (n = 10). Isolate identification was carried out using 16S rRNA sequence analysis. A microbiology-based approach, followed by 16S rRNA sequence analysis, was successfully used for detection of M. marinum in all four patients. Animal and environmental samples were simultaneously examined, and a total of seven mycobacterial species were isolated: Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium kansasii , Mycobacterium mantenii , Mycobacterium marinum , and Mycobacterium peregrinum . The presence of M. marinum was proven in the aquarium environments of two patients. Although M. marinum is described as being present in water, it was detected only in fish.

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Drancourt, Michel, Claude Bollet, Antoine Carlioz, Rolland Martelin, Jean-Pierre Gayral, and Didier Raoult. "16S Ribosomal DNA Sequence Analysis of a Large Collection of Environmental and Clinical Unidentifiable Bacterial Isolates." Journal of Clinical Microbiology 38, no. 10 (2000): 3623–30. http://dx.doi.org/10.1128/jcm.38.10.3623-3630.2000.

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Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter andPantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

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Torkko, Pirjo, Marja-Leena Katila, and Merja Kontro. "Gas-chromatographic lipid profiles in identification of currently known slowly growing environmental mycobacteria." Journal of Medical Microbiology 52, no. 4 (April 1, 2003): 315–23. http://dx.doi.org/10.1099/jmm.0.05113-0.

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Cellular fatty acid analysis by GLC is widely used in the species identification of mycobacteria. Combining mycolic acid cleavage products with shorter cellular fatty acids increases the informative value of the analysis. A key has been created to aid in the identification of all currently known slowly growing environmental species. In this scheme, the species are classified into six categories, each characterized by a combination of fatty markers shared by those species. Within each category, individual species may be distinguished by the presence or absence of specific marker substances, such as methyl-branched fatty acids or secondary alcohols. This study also describes earlier unpublished GLC profiles of 14 rare, slowly growing, environmental mycobacteria, Mycobacterium asiaticum, Mycobacterium botniense, Mycobacterium branderi, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium doricum, Mycobacterium heckeshornense, Mycobacterium heidelbergense, Mycobacterium hiberniae, Mycobacterium kubicae, Mycobacterium lentiflavum, Mycobacterium scrofulaceum, Mycobacterium triplex and Mycobacterium tusciae. Though no single identification technique alone, even sequencing of an entire single gene such as 16S rRNA, can identify all mycobacterial species accurately, GLC has proven to be both reliable and reproducible in the identification of slowly growing mycobacteria. In cases of earlier unknown species, it generates useful information that allows their further classification and may lead to the description of novel species.

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Burton, J. E., O. J. Oshota, and N. J. Silman. "Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis." Journal of Applied Microbiology 101, no. 4 (October 2006): 754–63. http://dx.doi.org/10.1111/j.1365-2672.2006.02991.x.

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MACALUSO, ALESSIA, ANNARITA PETRINCA, LUIGI LANNI, STEFANO SACCARES, SONIA AMITI, ROSANNA GABRIELI, and MAURIZIO DIVIZIA. "Identification and Sequence Analysis of Hepatitis A Virus Detected in Market and Environmental Bivalve Molluscs." Journal of Food Protection 69, no. 2 (February 1, 2006): 449–52. http://dx.doi.org/10.4315/0362-028x-69.2.449.

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In Italy in 1998, hepatitis A virus (HAV) was responsible for an infectious disease transmitted by contaminated bivalve molluscs. To determine the presence of HAV in the bivalves collected during a 1-year follow-up study, hepatitis A RNA was extracted and amplified by a nested reverse transcriptase–PCR method overlapping the VP1/2A region. The HAV genome was detected in 24 (14.1%) of 170 samples: 19 clams (Tapes decussates and Tapes semidecussatus), 1 oyster (Crossostea gigas), and 4 mussels (Mytillus galloprovincialis). Eleven positive samples were collected from marketing areas, and 13 positive samples were collected from growing areas. Seventeen of the 24 positive samples had been taken from domestic products, and 7 had been imported. Sequence analysis showed the presence of genotypes IA and IB. Our results suggest significant presence of HAV in bivalves from both marketing (public consumption) and environmental (growing) areas.

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Reffuveille, Fany, Charlène Leneveu, Sylvie Chevalier, Yanick Auffray, and Alain Rincé. "Lipoproteins of Enterococcus faecalis: bioinformatic identification, expression analysis and relation to virulence." Microbiology 157, no. 11 (November 1, 2011): 3001–13. http://dx.doi.org/10.1099/mic.0.053314-0.

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Enterococcus faecalis is a ubiquitous bacterium that is capable of surviving in a broad range of natural environments, including the human host, as either a natural commensal or an opportunistic pathogen involved in severe hospital-acquired infections. How such opportunistic pathogens cause fatal infections is largely unknown but it is likely that they are equipped with sophisticated systems to perceive external signals and interact with eukaryotic cells. Accordingly, being partially exposed at the cell exterior, some surface-associated proteins are involved in several steps of the infection process. Among them are lipoproteins, representing about 25 % of the surface-associated proteins, which could play a major role in bacterial virulence processes. This review focuses on the identification of 90 lipoprotein-encoding genes in the genome of the E. faecalis V583 clinical strain and their putative roles, and provides a transcriptional comparison of microarray data performed in environmental conditions including blood and urine. Taken together, these data suggest a potential involvement of lipoproteins in E. faecalis virulence, making them serious candidates for vaccine production.

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Beau, Frédéric, Claude Bollet, Thierry Coton, Eric Garnotel, and Michel Drancourt. "Molecular Identification of a Nocardiopsis dassonvillei Blood Isolate." Journal of Clinical Microbiology 37, no. 10 (1999): 3366–68. http://dx.doi.org/10.1128/jcm.37.10.3366-3368.1999.

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Nocardiopsis dassonvillei is an environmental aerobic actinomycete seldom isolated in cutaneous and pulmonary infections. We herein report the first N. dassonvillei blood isolate in a patient hospitalized for cholangitis. Although morphological characteristics and biochemical tests allowed a presumptive identification of this isolate, cell wall fatty acid chromatographic analysis confirmed identification at the genus level, and 16S rRNA gene sequencing achieved definite identification. This study illustrates the usefulness of 16S rRNA gene sequencing as a routine method for the identification of actinomycetes.

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Clarridge, Jill E. "Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria on Clinical Microbiology and Infectious Diseases." Clinical Microbiology Reviews 17, no. 4 (October 2004): 840–62. http://dx.doi.org/10.1128/cmr.17.4.840-862.2004.

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SUMMARY The traditional identification of bacteria on the basis of phenotypic characteristics is generally not as accurate as identification based on genotypic methods. Comparison of the bacterial 16S rRNA gene sequence has emerged as a preferred genetic technique. 16S rRNA gene sequence analysis can better identify poorly described, rarely isolated, or phenotypically aberrant strains, can be routinely used for identification of mycobacteria, and can lead to the recognition of novel pathogens and noncultured bacteria. Problems remain in that the sequences in some databases are not accurate, there is no consensus quantitative definition of genus or species based on 16S rRNA gene sequence data, the proliferation of species names based on minimal genetic and phenotypic differences raises communication difficulties, and microheterogeneity in 16S rRNA gene sequence within a species is common. Despite its accuracy, 16S rRNA gene sequence analysis lacks widespread use beyond the large and reference laboratories because of technical and cost considerations. Thus, a future challenge is to translate information from 16S rRNA gene sequencing into convenient biochemical testing schemes, making the accuracy of the genotypic identification available to the smaller and routine clinical microbiology laboratories.

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Wintzingerode, Friedrich, Frederick A. Rainey, Reiner M. Kroppenstedt, and Erko Stackebrandt. "Identification of environmental strains of Bacillus mycoides by fatty acid analysis and species-specific 16S rDNA oligonucleotide probe." FEMS Microbiology Ecology 24, no. 3 (January 17, 2006): 201–9. http://dx.doi.org/10.1111/j.1574-6941.1997.tb00437.x.

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Horz, Hans-Peter, Jan-Henrich Rotthauwe, Thomas Lukow, and Werner Liesack. "Identification of major subgroups of ammonia-oxidizing bacteria in environmental samples by T-RFLP analysis of amoA PCR products." Journal of Microbiological Methods 39, no. 3 (February 2000): 197–204. http://dx.doi.org/10.1016/s0167-7012(99)00119-0.

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van Pelt, Cindy, Cees M. Verduin, Wil H. F. Goessens, Margreet C. Vos, Burkhard Tümmler, Christine Segonds, Frans Reubsaet, Henri Verbrugh, and Alex van Belkum. "Identification of Burkholderia spp. in the Clinical Microbiology Laboratory: Comparison of Conventional and Molecular Methods." Journal of Clinical Microbiology 37, no. 7 (1999): 2158–64. http://dx.doi.org/10.1128/jcm.37.7.2158-2164.1999.

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Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli(n = 14), Ralstonia pickettii(n = 6), B. multivorans(n = 2), Stenotrophomonas maltophilia(n = 3), and Pseudomonas aeruginosa(n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped theB. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderiaphenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.

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Fung, Daniel Y. C. "Predictions for Rapid Methods and Automation in Food Microbiology." Journal of AOAC INTERNATIONAL 85, no. 4 (July 1, 2002): 1000–1002. http://dx.doi.org/10.1093/jaoac/85.4.1000.

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Abstract A discussion is presented on the present status of rapid methods and automation in microbiology. Predictions are also presented for development in the following areas: viable cell counts; real-time monitoring of hygiene; polymerase chain reaction, ribotyping, and genetic tests in food laboratories; automated enzyme-linked immunosorbent assay and immunotests; rapid dipstick technology; biosensors for Hazard Analysis Critical Control Point programs; instant detection of target pathogens by computer-generated matrix; effective separation and concentration for rapid identification of target cells; microbiological alert systems in food packages; and rapid alert kits for detecting pathogens at home.

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Feng, Xiaobo, Zhirong Yao, Daming Ren, and Wanqing Liao. "Simultaneous identification of molecular and mating types within the Cryptococcus species complex by PCR-RFLP analysis." Journal of Medical Microbiology 57, no. 12 (December 1, 2008): 1481–90. http://dx.doi.org/10.1099/jmm.0.2008/003665-0.

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The Cryptococcus species complex consists of two species, Cryptococcus neoformans and Cryptococcus gattii, which cause systemic infections in both immunocompromised and immunocompetent patients. Both species have a bipolar mating system, with mating type (MAT) α being predominant in clinical and environmental isolates. The strains of the Cryptococcus species complex have been divided into eight major molecular types, which show differences in epidemiology, biology and pathogenicity. In this study, two PCR-RFLP analyses, based on the CAP1 and GEF1 genes, which are both located at the MAT locus, were developed for simultaneous identification of the molecular and mating types of isolates of the Cryptococcus species complex. The molecular and mating types of all 144 cryptococcal isolates, including rare subtypes, were successfully determined by both PCR-RFLP approaches. Pattern analysis of the AD hybrids revealed that the serotype A MAT a allele in strains of AaDα derived from genotype VNB, whereas the serotype A MATα allele among strains of AαDa and AαDα derived from molecular type VNI.

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SÃ¸rensen, Jan, Jan Skouv, Anita JÃ¸rgensen, and Ole Nybroe. "Rapid identification of environmental isolates ofPseudomonas aeruginosa, P. fluorescensandP. putidaby SDS-PAGE analysis of whole-cell protein patterns." FEMS Microbiology Letters 101, no. 1 (June 1992): 41–50. http://dx.doi.org/10.1111/j.1574-6968.1992.tb05760.x.

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Wang, Qinning, Nadine Holmes, Elena Martinez, Peter Howard, Grant Hill-Cawthorne, and Vitali Sintchenko. "It Is Not All about Single Nucleotide Polymorphisms: Comparison of Mobile Genetic Elements and Deletions in Listeria monocytogenes Genomes Links Cases of Hospital-Acquired Listeriosis to the Environmental Source." Journal of Clinical Microbiology 53, no. 11 (August 26, 2015): 3492–500. http://dx.doi.org/10.1128/jcm.00202-15.

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The control of food-borne outbreaks caused byListeria monocytogenesin humans relies on the timely identification of food or environmental sources and the differentiation of outbreak-related isolates from unrelated ones. This study illustrates the utility of whole-genome sequencing for examining the link between clinical and environmental isolates ofL. monocytogenesassociated with an outbreak of hospital-acquired listeriosis in Sydney, Australia. Comparative genomic analysis confirmed an epidemiological link between the three clinical and two environmental isolates. Single nucleotide polymorphism (SNP) analysis showed that only two SNPs separated the three human outbreak isolates, which differed by 19 to 20 SNPs from the environmental isolates and 71 to >10,000 SNPs from sporadicL. monocytogenesisolates. The chromosomes of all human outbreak isolates and the two suspected environmental isolates were syntenic. In contrast to the genomes of background sporadic isolates, all epidemiologically linked isolates contained two novel prophages and a previously unreported clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) locus subtype sequence. The mobile genetic element (MGE) profile of these isolates was distinct from that of the other serotype 1/2b reference strains and sporadic isolates. The identification of SNPs and clonally distinctive MGEs strengthened evidence to distinguish outbreak-related isolates ofL. monocytogenesfrom cocirculating endemic strains.

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Fergusson, Kim M., and Christopher P. Saint. "Molecular Phylogeny of Anabaena circinalis and Its Identification in Environmental Samples by PCR." Applied and Environmental Microbiology 66, no. 9 (September 1, 2000): 4145–48. http://dx.doi.org/10.1128/aem.66.9.4145-4148.2000.

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ABSTRACT Although the cyanobacterium Anabaena circinalis occurs worldwide, Australian isolates are believed to exclusively possess the saxitoxin group neurotoxins (paralytic shellfish poisons). Identification of A. circinalis in a mixed population is complicated due to limited morphological differences betweenAnabaena species. Sequence analysis of the DNA-dependent RNA polymerase (rpoC1) gene from 24 Anabaenaisolates, including 12 designated A. circinalis, permitted a phylogenetic analysis to be performed. In addition, an A. circinalis-specific PCR was developed and tested successfully on environmental samples.

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Schulze, W. X. "Protein analysis in dissolved organic matter: What proteins from organic debris, soil leachate and surface water can tell us - a perspective." Biogeosciences 2, no. 1 (March 4, 2005): 75–86. http://dx.doi.org/10.5194/bg-2-75-2005.

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Abstract. Mass spectrometry based analysis of proteins is widely used to study cellular processes in model organisms. However, it has not yet routinely been applied in environmental research. Based on observations that protein can readily be detected as a component of dissolved organic matter (DOM), this article gives an example about the possible use of protein analysis in ecology and environmental sciences focusing on different terrestrial ecosystems. At this stage, there are two areas of interest: (1) the identification of phylogenetic groups contributing to the environmental protein pool, and (2) identification of the organismic origin of specific enzymes that are important for ecosystem processes. In this paper, mass spectrometric protein analysis was applied to identify proteins from decomposing plant material and DOM of soil leachates and surface water samples derived from different environments. It is concluded, that mass spectrometric protein analysis is capable of distinguishing phylogenetic origin of proteins from litter protein extracts, leachates of different soil horizons, and from various sources of terrestrial surface water. Current limitation is imposed by the limited knowledge of complete genomes of soil organisms. The protein analysis allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific active enzymes. Further applications, such as in pollution research are conceivable. In summary, the analysis of proteins opens a new area of research between the fields of microbiology and biogeochemistry.

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Sebat, Jonathan L., Frederick S. Colwell, and Ronald L. Crawford. "Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library." Applied and Environmental Microbiology 69, no. 8 (August 2003): 4927–34. http://dx.doi.org/10.1128/aem.69.8.4927-4934.2003.

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ABSTRACT Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ∼1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.

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Miller, Melissa B., and Yi-Wei Tang. "Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology." Clinical Microbiology Reviews 22, no. 4 (October 2009): 611–33. http://dx.doi.org/10.1128/cmr.00019-09.

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SUMMARY The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles.

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Park, Sarah Y., Karen L. Kelminson, Anthea K. Lee, Peng Zhang, Rachel E. Warner, David H. Rehkopf, Stephen B. Calderwood, and Jane E. Koehler. "Identification, Characterization, and Functional Analysis of a Gene Encoding the Ferric Uptake Regulation Protein inBartonella Species." Journal of Bacteriology 183, no. 19 (October 1, 2001): 5751–55. http://dx.doi.org/10.1128/jb.183.19.5751-5755.2001.

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ABSTRACT Environmental iron concentrations coordinately regulate transcription of genes involved in iron acquisition and virulence via the ferric uptake regulation (fur) system. We identified and sequenced the fur gene and flanking regions of threeBartonella species. The most notable difference betweenBartonella Fur and other Fur proteins was a substantially higher predicted isoelectric point. No promoter activity or Fur autoregulation was detected using a gfp reporter gene fused to the 204 nucleotides immediately upstream of the Bartonella fur gene. Bartonella henselae fur gene expression complemented a Vibrio cholerae fur mutant.

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Naser, Sabri M., Fabiano L. Thompson, Bart Hoste, Dirk Gevers, Peter Dawyndt, Marc Vancanneyt, and Jean Swings. "Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes." Microbiology 151, no. 7 (July 1, 2005): 2141–50. http://dx.doi.org/10.1099/mic.0.27840-0.

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The aim of this study was to evaluate the use of RNA polymerase α subunit (rpoA) and phenylalanyl-tRNA synthase (pheS) gene sequences as species identification tools for enterococci. Ninety-six representative strains comprising all currently recognized Enterococcus species were examined. rpoA gene sequences generated a robust classification into species groups similar to the one based on 16S rRNA gene sequence analysis. On the other hand, the pheS gene is a fast-evolving clock even better suited for species delineation than the rpoA gene, but not for recognition of species groups within Enterococcus as determined by both rpoA and 16S rRNA genes. All enterococcal species were clearly differentiated on the basis of their rpoA and pheS sequences. Evaluation of intraspecies variation showed that both rpoA and pheS genes have a high degree of homogeneity among strains of the same species. Strains of the same enterococcal species have at least 99 % rpoA and 97 % pheS gene sequence similarity, whereas, different enterococcal species have at maximum 97 % rpoA and 86 % pheS gene sequence similarity. It was concluded that both genes can be used as reliable tools for identification of clinical and environmental species of Enterococcus and are efficient screening methods for the detection of novel species. The sequence data obtained in this study were compared to the available atpA and 16S rRNA gene sequences. The MLSA approach to Enterococcus taxonomy provides portable, highly reproducible data with lower costs for rapid identification of all enterococcal species.

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Saleh, Tahreer Hadi, Majeed Arsheed Sabbah, Kifah A. Jasem, and Zuhair N. Hammad. "Identification of virulence factors in Vibrio cholerae isolated from Iraq during the 2007–2009 outbreak." Canadian Journal of Microbiology 57, no. 12 (December 2011): 1024–31. http://dx.doi.org/10.1139/w11-094.

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Thousands of people were infected with Vibrio cholerae during the outbreak in Iraq in 2007–2009. Vibrio cholerae was shown to be variable in its content of virulence determinants and in its antibiotic sensitivity. This study was designed to isolate and characterize clinical and environmental V. cholerae isolates and to determine antibiotic sensitivity, enzyme and toxin production, and the presence of virulence genes. Eighty clinical and five environmental bacterial isolates were collected and diagnosed by subjecting them to microscopic, biochemical, serological, and molecular analysis. The results revealed that 55% of clinical isolates belonged to the Inaba serotype, 32.5% to the Ogawa serotypes, and 12.5% to the Non-O1 serotype. All environmental V. cholerae isolates belonged to the Non-O1 serotype. All environmental isolates were sensitive to all examined antimicrobial agents, while all clinical isolates showed a high sensitivity (100%) to ampicillin, gentamicin, cephalothin, tetracycline, erythromycin, and ciprofloxacin, and a high resistance (97.5%) to co-trimoxazole, nalidixic acid, and chloramphenicol. It was found that all V. cholerae (O1) isolates were resistant to the Vibrio static O129 and all Non-O1 V. cholerae isolates were sensitive to the Vibrio static O129. All clinical and environmental isolates produced hemolysin (100%) and lecithinase (100%), while they showed various production rates of protease (90% of clinical and 60% of environmental) and lipase (50% of clinical and 20% of environmental). The ompW gene was amplified in all the clinical and environmental V. cholerae isolates, but not in other related and nonrelated bacteria. Multiplex PCR analysis showed that the toxR gene was amplified in all clinical and environmental isolates, while ctxA, ctxB, tcpA genes were amplified only in clinical (O1) isolates. This study indicates the differences in the production of some enzymes and toxins and in the content of virulence genes between clinical and environmental isolates in Iraq during the outbreak (2007–2009).

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Lo Presti, François, Serge Riffard, François Vandenesch, and Jerome Etienne. "Identification of Legionella Species by Random Amplified Polymorphic DNA Profiles." Journal of Clinical Microbiology 36, no. 11 (1998): 3193–97. http://dx.doi.org/10.1128/jcm.36.11.3193-3197.1998.

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Random amplified polymorphic DNA (RAPD) was used for the identification of Legionella species. Primer SK2 (5′-CGGCGGCGGCGG-3′) and standardized RAPD conditions gave the technique a reproducibility of 93 to 100%, depending on the species tested. Species-specific patterns corresponding to the 42Legionella species were consequently defined by this method; the patterns were dependent on the recognition of a core of common bands for each species. This specificity was demonstrated by testing 65 type strains and 265 environmental and clinical isolates. No serogroup-specific profiles were obtained. A number of unidentifiedLegionella isolates potentially corresponding to new species were clustered in four groups. RAPD analysis appears to be a rapid and reproducible technique for identification ofLegionella isolates to the species level without further restriction or hybridization.

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Loy, Alexander, Claudia Schulz, Sebastian Lücker, Andreas Schöpfer-Wendels, Kilian Stoecker, Christian Baranyi, Angelika Lehner, and Michael Wagner. "16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales”." Applied and Environmental Microbiology 71, no. 3 (March 2005): 1373–86. http://dx.doi.org/10.1128/aem.71.3.1373-1386.2005.

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ABSTRACT For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.

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Schatz, Michael C., Adam M. Phillippy, Pawel Gajer, Todd Z. DeSantis, Gary L. Andersen, and Jacques Ravel. "Integrated Microbial Survey Analysis of Prokaryotic Communities for the PhyloChip Microarray." Applied and Environmental Microbiology 76, no. 16 (June 25, 2010): 5636–38. http://dx.doi.org/10.1128/aem.00303-10.

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ABSTRACT PhyloTrac is an integrated desktop application for analysis of PhyloChip microarray data. PhyloTrac combined with PhyloChip provides turnkey and comprehensive identification and analysis of bacterial and archaeal communities in complex environmental samples. PhyloTrac is free for noncommercial organizations and is available for all major operating systems at http://www.phylotrac.org/ .

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Pfaller, Stacy L., Timothy W. Aronson, Alan E. Holtzman, and Terry C. Covert. "Amplified fragment length polymorphism analysis of Mycobacterium avium complex isolates recovered from southern California." Journal of Medical Microbiology 56, no. 9 (September 1, 2007): 1152–60. http://dx.doi.org/10.1099/jmm.0.47075-0.

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Fine-scale genotyping methods are necessary in order to identify possible sources of human exposure to opportunistic pathogens belonging to the Mycobacterium avium complex (MAC). In this study, amplified fragment length polymorphism (AFLP) analysis was evaluated for fingerprinting 159 patient and environmental MAC isolates from southern California. AFLP analysis accurately identified strains belonging to M. avium and Mycobacterium intracellulare and differentiated between strains within each species. The method was also able to differentiate strains that were presumed to be genetically identical in two previous studies using large RFLP analysis with PFGE, or PCR-amplification of DNA segments located between insertion sequences IS1245 and IS1311. For M. avium, drinking-water isolates clustered more closely with each other than with patient or food isolates. Patient isolates were more genetically diverse. None of the environmental isolates shared identical AFLP patterns with patient isolates for either species. There were, however, environmental isolates that shared identical patterns, and patient isolates that shared identical patterns. A subset of the isolates, which are referred to as MX isolates due to their ambiguous identification with the Gen-Probe system, produced AFLP patterns similar to those obtained from M. intracellulare isolates. Sequence analysis of 16S rDNA obtained from the MX isolates suggests that they are strains of M. intracellulare that were not correctly identified by the M. intracellulare AccuProbe from Gen-Probe.

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Grover, Amit, and Rakesh Sharma. "Identification and Characterization of a Major Zn(II) Resistance Determinant of Mycobacterium smegmatis." Journal of Bacteriology 188, no. 19 (October 1, 2006): 7026–32. http://dx.doi.org/10.1128/jb.00643-06.

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ABSTRACT A zinc ion-sensitive mutant of Mycobacterium smegmatis was isolated. The transposon insertion was located in zitA (MSMEG0750), a gene coding for a cation diffusion facilitator family protein. Zinc ions specifically induced expression of zitA. In silico analysis revealed that environmental and opportunistic pathogenic species contain higher numbers of cation diffusion facilitator genes than do obligate pathogens.

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Lamy, Brigitte, Fréderic Laurent, and Angeli Kodjo. "Validation of a partialrpoBgene sequence as a tool for phylogenetic identification of aeromonads isolated from environmental sources." Canadian Journal of Microbiology 56, no. 3 (March 2010): 217–28. http://dx.doi.org/10.1139/w10-006.

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A collection of 50 aeromonads isolated from environmental sources were studied, together with all known Aeromonas nomenspecies, by phenotypic, amplified 16S rDNA restriction analysis (16S rDNA RFLP) and by partial sequence alignment of both 16S rDNA and rpoB genes. Although most of the type strain showed a unique phenotypic pattern, a database constructed on type strain phenotype allowed the identification of only 24% of the isolates. Analysis of 16S rDNA RFLP and the rpoB sequence were almost concordant in identifying environmental isolates at the species level, except for strains belonging to Aeromonas caviae spp., which were not differentiated from Aeromonas aquariorum , nor Aeromonas hydrophila susbsp. dhakensis by 16S rDNA RFLP. In addition, rpoB gene analysis clustered separately a group of isolates found in snails within the A. hydrophila species. In contrast to 16S rDNA RFLP and rpoB, the partial 16S rDNA sequence analysis was weak in resolving species identity. Part of these results, phenotypic and phylogenetic data, showed that Aeromonas molluscorum and Aeromonas sharmana are distant from all other Aeromonas species and that the type species of A. hydrophila subsp. anaerogenes is similar to A. caviae and should be considered synonymous.

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La Rosa, G., S. Fontana, A. Di Grazia, M. Iaconelli, M. Pourshaban, and M. Muscillo. "Molecular Identification and Genetic Analysis of Norovirus Genogroups I and II in Water Environments: Comparative Analysis of Different Reverse Transcription-PCR Assays." Applied and Environmental Microbiology 73, no. 13 (May 4, 2007): 4152–61. http://dx.doi.org/10.1128/aem.00222-07.

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ABSTRACT Noroviruses have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Our inability to grow them in cell culture and the lack of an animal model hinder the characterization of these viruses. More recently, molecular approaches have been used to study the genetic relationships that exist among them. In the present study, environmental samples from seawater, estuarine water, and effluents of sewage treatment plants were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for transmission of norovirus genogroups I and II. Novel broad-range reverse transcription-PCR/nested assays targeting the region coding for the RNA-dependent RNA polymerase were developed, amplifying fragments of 516 bp and 687 bp in the nested reactions for genogroups II and I, respectively. The assays were evaluated and compared against widely used published assays. The newly designed assays provide long regions for high-confidence BLAST searches in public databases and therefore are useful diagnostic tools for molecular diagnosis and typing of human noroviruses in clinical and environmental samples, as well as for the study of molecular epidemiology and the evolution of these viruses.

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Whitby, Paul W., Timothy M. VanWagoner, Ashlee A. Taylor, Thomas W. Seale, Daniel J. Morton, John J. LiPuma, and Terrence L. Stull. "Identification of an RTX determinant of Burkholderia cenocepacia J2315 by subtractive hybridization." Journal of Medical Microbiology 55, no. 1 (January 1, 2006): 11–21. http://dx.doi.org/10.1099/jmm.0.46138-0.

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This study utilized suppressive subtractive hybridization between the clinical isolate Burkholderia cenocepacia J2315 and the closely related environmental isolate Burkholderia cepacia ATCC 25416T to isolate DNA fragments specific to B. cenocepacia J2315. Analysis of the resulting pools of B. cenocepacia-specific DNAs identified several fragments that may be part of putative virulence factors. Further in silico analysis of a single fragment indicated that it was internal to a gene of which the predicted product had characteristics of repeat in toxin (RTX)-like proteins and high similarity to proteins in other human or plant pathogens. In conjunction with this finding, phenotypic traits associated with known RTX proteins were assessed. A haemagglutinating activity of B. cenocepacia J2315 was identified that was absent in B. cepacia ATCC 25416T. The expression of this activity appeared to be growth phase-dependent. Analysis of the gene presence and haemagglutinating activity across the species of the B. cepacia complex showed that both were common to the ET12 lineage of B. cenocepacia, but were absent in the other species examined. Haemagglutinating activity was limited to isolates with the RTX-like gene. Expression studies utilizing quantitative PCR demonstrated an association between onset of haemagglutinating activity and increased expression of the gene, which suggests that the putative RTX determinant encodes a haemagglutinating activity.

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Sørensen, Jan, Jan Skouv, Anita Jørgensen, and Ole Nybroe. "Rapid identification of environmental isolates of Pseudomonas aeruginosa, P. fluorescens and P. putida by SDS-PAGE analysis of whole-cell protein patterns." FEMS Microbiology Ecology 10, no. 1 (June 1992): 41–50. http://dx.doi.org/10.1111/j.1574-6941.1992.tb01647.x.

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Sørensen, J. "Rapid identification of environmental isolates of Pseudomonas aeruginosa, P. fluorescens and P. putida by SDS-PAGE analysis of whole-cell protein patterns." FEMS Microbiology Ecology 101, no. 1 (June 1992): 41–50. http://dx.doi.org/10.1016/0168-6496(92)90070-a.

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Schulze, W. "Protein analysis in dissolved organic matter: what free proteins from soil leachate and surface water can tell us – a perspective." Biogeosciences Discussions 1, no. 1 (December 20, 2004): 825–53. http://dx.doi.org/10.5194/bgd-1-825-2004.

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Abstract. Mass spectrometry based analysis of proteins is widely used to study cellular processes in model organisms. However, it has not yet routinely been applied in environmental research. Based on observations that protein can readily be detected as a component of dissolved organic matter (DOM), this article gives an example about the possible use of protein analysis in ecology and environmental sciences focusing on different terrestrial ecosystems. At this stage, there are two areas of interest: (1) the identification of phylogenetic groups contributing to the DOM protein pool, and (2) identification of the organismic origin of specific enzymes that are important for ecosystem processes. In this paper, mass spectrometric protein analysis was applied to identify proteins from DOM and organism-free surface water samples derived from different environments. It is concluded, that mass spectrometric protein analysis is capable of distinguishing phylogenetic origin of proteins from leachates of different soil horizons, and from various sources of terrestrial surface water. Current limitation is imposed by the limited knowledge of complete genomes of soil organisms. The protein analysis allows to relate protein presence to biogeochemical processes, and to identify the source organisms for specific active enzymes. Further applications, such as in pollution research are conceivable. In summary, the analysis of proteins opens a new area of research between the fields of microbiology and biogeochemistry.

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Iatta, Roberta, Federica Nuccio, Davide Immediato, Adriana Mosca, Carmela De Carlo, Giuseppe Miragliotta, Antonio Parisi, Giuseppe Crescenzo, Domenico Otranto, and Claudia Cafarchia. "Species Distribution andIn VitroAzole Susceptibility of Aspergillus SectionNigriIsolates from Clinical and Environmental Settings." Journal of Clinical Microbiology 54, no. 9 (July 13, 2016): 2365–72. http://dx.doi.org/10.1128/jcm.01075-16.

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AspergillussectionNigriincludes species of interest for animal and human health, although studies on species distribution are limited to human cases. Data on the antifungal susceptibilities and the molecular mechanism of triazole resistance in strains belonging to this section are scant. Forty-two blackAspergillusstrains from human patients (16 isolates), animals (14 isolates), and the environment (12 isolates) were molecularly characterized and theirin vitrotriazole susceptibilities investigated.Aspergillus tubingensiswas isolated from humans, animals, and environmental settings, whereasAspergillus awamoriandAspergillus nigerwere isolated exclusively from humans. Phylogenetic analyses of β-tubulin and calmodulin gene sequences were concordant in differentiatingA. tubingensisfromA. awamoriandA. niger. Voriconazole and posaconazole (PSZ) were the most active triazoles. OneA. tubingensisstrain was resistant to itraconazole and PSZ and oneA. nigerstrain to PSZ. Sequence analysis of thecyp51Agene revealed different sequence types within a species, andA. tubingensisstrains were also phylogenetically distinct fromA. awamori/A. nigerstrains according to the strain origin and susceptibility profile. Genetic analysis of thecyp51Asequences suggests that two nonsynonymous mutations resulting in amino acid substitutions in the CYP51A protein (changes of L to R at position 21 [L21R] and of Q to R at position 228 [Q228R]) might be involved in azole resistance. Though azole resistance in blackAspergillusisolates from animals and rural environments does not represent a threat to public health in Southern Italy, the use of triazoles in the clinical setting needs to better monitored. Thecyp51Asequence is useful for the molecular identification of blackAspergillus, and point mutations in protein sequences could be responsible for azole resistance phenomena.

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HUANG, YING, LU ZHANG, and HUA H. WANG. "Identification of a New Tetracycline Resistance Determinant tet47 from Fish Intestine." Journal of Food Protection 78, no. 8 (August 1, 2015): 1581–85. http://dx.doi.org/10.4315/0362-028x.jfp-15-025.

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To better understand food safety risks, functional genomic analysis was conducted to identify undescribed antibiotic resistance genes in fish samples from an aquaculture fish farm in Ohio. A fosmid genomic library from pooled DNA of antibiotic-resistant isolates was used to screen for resistance genes against tetracycline (Tet). A new Tet-resistant determinant designated as tet47 was identified, with the original hosts being Providencia spp. from fish intestine. The new gene was also found to confer Tet resistance in Escherichia coli. Fish and byproducts were shown to be possible carriers that may disseminate new, functional, and potentially transmissible antibiotic resistance determinants through food, feed, and environmental contacts.

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Franzen, Caspar, and Andreas Müller. "Molecular Techniques for Detection, Species Differentiation, and Phylogenetic Analysis of Microsporidia." Clinical Microbiology Reviews 12, no. 2 (April 1, 1999): 243–85. http://dx.doi.org/10.1128/cmr.12.2.243.

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SUMMARY Microsporidia are obligate intracellular protozoan parasites that infect a broad range of vertebrates and invertebrates. These parasites are now recognized as one of the most common pathogens in human immunodeficiency virus-infected patients. For most patients with infectious diseases, microbiological isolation and identification techniques offer the most rapid and specific determination of the etiologic agent. This is not a suitable procedure for microsporidia, which are obligate intracellular parasites requiring cell culture systems for growth. Therefore, the diagnosis of microsporidiosis currently depends on morphological demonstration of the organisms themselves. Although the diagnosis of microsporidiosis and identification of microsporidia by light microscopy have greatly improved during the last few years, species differentiation by these techniques is usually impossible and transmission electron microscopy may be necessary. Immunfluorescent-staining techniques have been developed for species differentiation of microsporidia, but the antibodies used in these procedures are available only at research laboratories at present. During the last 10 years, the detection of infectious disease agents has begun to include the use of nucleic acid-based technologies. Diagnosis of infection caused by parasitic organisms is the last field of clinical microbiology to incorporate these techniques and molecular techniques (e.g., PCR and hybridization assays) have recently been developed for the detection, species differentiation, and phylogenetic analysis of microsporidia. In this paper we review human microsporidial infections and describe and discuss these newly developed molecular techniques.

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Moreira, David, Francisco Rodríguez-Valera, and Purificación López-García. "Metagenomic analysis of mesopelagic Antarctic plankton reveals a novel deltaproteobacterial group." Microbiology 152, no. 2 (February 1, 2006): 505–17. http://dx.doi.org/10.1099/mic.0.28254-0.

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Phylogenetic screening of 3200 clones from a metagenomic library of Antarctic mesopelagic picoplankton allowed the identification of two bacterial 16S-rDNA-containing clones belonging to the Deltaproteobacteria, DeepAnt-1F12 and DeepAnt-32C6. These clones were very divergent, forming a monophyletic cluster with the environmental sequence GR-WP33-58 that branched at the base of the myxobacteria. Except for the possession of complete rrn operons without associated tRNA genes, DeepAnt-1F12 and DeepAnt-32C6 were very different in gene content and organization. Gene density was much higher in DeepAnt-32C6, whereas nearly one-third of DeepAnt-1F12 corresponded to intergenic regions. Many of the predicted genes encoded by these metagenomic clones were informational (i.e. involved in replication, transcription, translation and related processes). Despite this, a few putative cases of horizontal gene transfer were detected, including a transposase. DeepAnt-1F12 contained one putative gene encoding a long cysteine-rich protein, probably membrane-bound and Ca2+-binding, with only eukaryotic homologues. DeepAnt-32C6 carried some predicted genes involved in metabolic pathways that suggested this organism may be anaerobic and able to ferment and to degrade complex compounds extracellularly.

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ROZWADOWSKA, BEATA, MARTA ALBERTYŃSKA, GRZEGORZ HUDZIK, HUBERT OKŁA, KRZYSZTOF P. JASIK, and JAN SŁODKI. "Application of a Real-Time PCR Method for Salmonella spp., Escherichia coli, Staphylococcus aureus and Clostridium perfringens Detection in Water Samples." Polish Journal of Microbiology 62, no. 4 (2013): 439–43. http://dx.doi.org/10.33073/pjm-2013-060.

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The diagnostic assessment of water sanitary state is based mainly on the cultivation of bacteria retained on membrane filters. However classical microbiology methods have a lot of disadvantages. More and more frequently, rapid detection and identification of pathogens present in water is based on molecular biology techniques. The aim of this study was to determine the effectiveness and usefulness of a real-time PCR method, when compared to the recommended bacteria culture method, in diagnostics of pathogens in water samples. The research concerned the detection and identification of main sanitary indicators of water such as: Salmonella spp., Escherichia coli, Staphylococcus aureus and Clostridium perfringens. The analyses were conducted in water samples contaminated with the reference material (the aforementioned bacteria) and real environmental samples, which were examined for the presence of nucleic acid of: Salmonella spp., E. coli, S. aureus and C. perfringens using a real-time PCR method.

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Brisse, Sylvain, Cees M. Verduin, Dana Milatovic, Ad Fluit, Jan Verhoef, Severine Laevens, Peter Vandamme, Burkhard Tümmler, Henri A. Verbrugh, and Alex van Belkum. "Distinguishing Species of the Burkholderia cepacia Complex and Burkholderia gladioli by Automated Ribotyping." Journal of Clinical Microbiology 38, no. 5 (2000): 1876–84. http://dx.doi.org/10.1128/jcm.38.5.1876-1884.2000.

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Several species belonging to the genus Burkholderia are clinically relevant, opportunistic pathogens that inhabit major environmental reservoirs. Consequently, the availability of means for adequate identification and epidemiological characterization of individual environmental or clinical isolates is mandatory. In the present communication we describe the use of the Riboprinter microbial characterization system (Qualicon, Warwick, United Kingdom) for automated ribotyping of 104 strains of Burkholderia species from diverse sources, including several publicly accessible collections. The main outcome of this analysis was that all strains were typeable and that strains of Burkholderia gladioli and of each species of the B. cepacia complex, includingB. multivorans, B. stabilis, and B. vietnamiensis, were effectively discriminated. Furthermore, different ribotypes were discerned within each species. Ribotyping results were in general agreement with strain classification based on restriction fragment analysis of 16S ribosomal amplicons, but the resolution of ribotyping was much higher. This enabled automated molecular typing below the species level. Cluster analysis of the patterns obtained by ribotyping (riboprints) showed that withinB. gladioli, B. multivorans, and B. cepacia genomovar VI, the different riboprints identified always clustered together. Riboprints of B. cepacia genomovars I and III, B. stabilis, and B. vietnamiensis did not show distinct clustering but rather exhibited the formation of loose assemblages within which several smaller, genomovar-specific clusters were delineated. Therefore, ribotyping proved useful for genomovar identification. Analysis of serial isolates from individual patients demonstrated that infection with a single ribotype had occurred, despite minor genetic differences that were detected by pulsed-field gel electrophoresis of DNA macrorestriction fragments. The automated approach allows very rapid and reliable identification and epidemiological characterization of strains and generates an easily manageable database suited for expansion with information on additional bacterial isolates.

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Butler, W. Ray, and Linda S. Guthertz. "Mycolic Acid Analysis by High-Performance Liquid Chromatography for Identification of Mycobacterium Species." Clinical Microbiology Reviews 14, no. 4 (October 1, 2001): 704–26. http://dx.doi.org/10.1128/cmr.14.4.704-726.2001.

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SUMMARY Mycobacterium tuberculosis is the etiologic agent of tuberculosis and can be accurately detected by laboratories using commercial genetic tests. Nontuberculosis mycobacteria (NTM) causing other mycobacterioses can be difficult to identify. The identification processes are confounded by an increasing diversity of newly characterized NTM species. The ubiquitous nature of NTM, combined with their potential to be opportunistic pathogens in immunocompromised as well as nonimmunodeficient patients, further complicates the problem of their identification. Since clinical case management varies depending on the etiologic agent, laboratories must identify the species in a timely manner. However, only a few identification methods can detect the species diversity within the Mycobacterium genus. Over the last decade, high-performance liquid chromatography analysis of the mycolic acids has become an accepted method for identification of mycobacteria. In this review, we assess its development and usefulness as an identification technique for Mycobacterium species.

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Kï¿½ser, Michael, Julia Hauser, Pamela Small, and Gerd Pluschke. "Large Sequence Polymorphisms Unveil the Phylogenetic Relationship of Environmental and Pathogenic Mycobacteria Related to Mycobacterium ulcerans." Applied and Environmental Microbiology 75, no. 17 (July 10, 2009): 5667–75. http://dx.doi.org/10.1128/aem.00446-09.

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ABSTRACT Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

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Zuccaro, Alga, Conrad L. Schoch, Joseph W. Spatafora, Jan Kohlmeyer, Siegfried Draeger, and Julian I. Mitchell. "Detection and Identification of Fungi Intimately Associated with the Brown Seaweed Fucus serratus." Applied and Environmental Microbiology 74, no. 4 (December 14, 2007): 931–41. http://dx.doi.org/10.1128/aem.01158-07.

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ABSTRACT The filamentous fungi associated with healthy and decaying Fucus serratus thalli were studied over a 1-year period using isolation methods and molecular techniques such as 28S rRNA gene PCR-denaturing gradient gel electrophoresis (DGGE) and phylogenetic and real-time PCR analyses. The predominant DGGE bands obtained from healthy algal thalli belonged to the Lindra, Lulworthia, Engyodontium, Sigmoidea/Corollospora complex, and Emericellopsis/Acremonium-like ribotypes. In the culture-based analysis the incidence of recovery was highest for Sigmoidea marina isolates. In general, the environmental sequences retrieved could be matched unambiguously to isolates recovered from the seaweed except for the Emericellopsis/Acremonium-like ribotype, which showed 99% homology with the sequences of four different isolates, including that of Acremonium fuci. To estimate the extent of colonization of A. fuci, we used a TaqMan real-time quantitative PCR assay for intron 3 of the beta-tubulin gene, the probe for which proved to be species specific even when it was used in amplifications with high background concentrations of other eukaryotic DNAs. The A. fuci sequence was detected with both healthy and decaying thalli, but the signal was stronger for the latter. Additional sequence types, representing members from the Dothideomycetes, were recovered from the decaying thallus DNA, which suggested that a change in fungal community structure had occurred. Phylogenetic analysis of these environmental sequences and the sequences of isolates and type species indicated that the environmental sequences were novel in the Dothideomycetes.

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Liu, Zongmin, Lingzhi Li, Qianwen Wang, Faizan Ahmed Sadiq, Yuankun Lee, Jianxin Zhao, Hao Zhang, Wei Chen, Haitao Li, and Wenwei Lu. "Transcriptome Analysis Reveals the Genes Involved in Bifidobacterium Longum FGSZY16M3 Biofilm Formation." Microorganisms 9, no. 2 (February 14, 2021): 385. http://dx.doi.org/10.3390/microorganisms9020385.

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Biofilm formation has evolved as an adaptive strategy for bacteria to cope with harsh environmental conditions. Currently, little is known about the molecular mechanisms of biofilm formation in bifidobacteria. A time series transcriptome sequencing analysis of both biofilm and planktonic cells of Bifidobacterium longum FGSZY16M3 was performed to identify candidate genes involved in biofilm formation. Protein–protein interaction network analysis of 1296 differentially expressed genes during biofilm formation yielded 15 clusters of highly interconnected nodes, indicating that genes related to the SOS response (dnaK, groS, guaB, ruvA, recA, radA, recN, recF, pstA, and sufD) associated with the early stage of biofilm formation. Genes involved in extracellular polymeric substances were upregulated (epsH, epsK, efp, frr, pheT, rfbA, rfbJ, rfbP, rpmF, secY and yidC) in the stage of biofilm maturation. To further investigate the genes related to biofilm formation, weighted gene co-expression network analysis (WGCNA) was performed with 2032 transcript genes, leading to the identification of nine WGCNA modules and 133 genes associated with response to stress, regulation of gene expression, quorum sensing, and two-component system. These results indicate that biofilm formation in B. longum is a multifactorial process, involving stress response, structural development, and regulatory processes.

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Leão, Sylvia Cardoso, Marcelo R. S. Briones, Marcelo Palma Sircili, Simone Carvalho Balian, Nelson Mores, and José Soares Ferreira-Neto. "Identification of Two Novel Mycobacterium avium Allelic Variants in Pig and Human Isolates from Brazil by PCR-Restriction Enzyme Analysis." Journal of Clinical Microbiology 37, no. 8 (1999): 2592–97. http://dx.doi.org/10.1128/jcm.37.8.2592-2597.1999.

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Mycobacterium avium complex (MAC) is composed of environmental mycobacteria found widely in soil, water, and aerosols that can cause disease in animals and humans, especially disseminated infections in AIDS patients. MAC consists of two closely related species, M. avium and M. intracellulare, and may also include other, less-defined groups. The precise differentiation of MAC species is a fundamental step in epidemiological studies and for the evaluation of possible reservoirs for MAC infection in humans and animals. In this study, which included 111 pig and 26 clinical MAC isolates, two novel allelic M. aviumPCR-restriction enzyme analysis (PRA) variants were identified, differing from the M. avium PRA prototype in theHaeIII digestion pattern. Mutations in HaeIII sites were confirmed by DNA sequencing. Identification of these isolates as M. avium was confirmed by PCR with DT1-DT6 and IS1245 primers, nucleic acid hybridization with the AccuProbe system, 16S ribosomal DNA sequencing, and biochemical tests. The characterization of M. avium PRA variants can be useful in the elucidation of factors involved in mycobacterial virulence and routes of infection and also has diagnostic significance, since they can be misidentified as M. simiae II and M. kansasii I if the PRA method is used in the clinical laboratory for identification of mycobacteria.

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Graf, Joerg. "Diverse Restriction Fragment Length Polymorphism Patterns of the PCR-Amplified 16S rRNA Genes in Aeromonas veronii Strains and Possible Misidentification ofAeromonas Species." Journal of Clinical Microbiology 37, no. 10 (1999): 3194–97. http://dx.doi.org/10.1128/jcm.37.10.3194-3197.1999.

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Restriction fragment length polymorphism analysis after PCR amplification (RFLP-PCR) of the 16S rRNA gene has been previously proposed as a rapid method to identify Aeromonas species. In the present study, the precision of RFLP-PCR was evaluated with 62Aeromonas reference strains. The analysis revealed thatAeromonas veronii biovar sobria strains produce various patterns, possibly leading to its misidentification as an environmental species. For most other Aeromonas species little variation was noted. This study supports the usefulness of RFLP-PCR analysis to separate three clinically important species but also reveals possible misidentifications that necessitate further biochemical tests to validate the preliminary identification.

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Cerezer, Vinicius Godoy, Silvia Yumi Bando, Jacyr Pasternak, Marcia Regina Franzolin, and Carlos Alberto Moreira-Filho. "Phylogenetic Analysis ofStenotrophomonasspp. Isolates Contributes to the Identification of Nosocomial and Community-Acquired Infections." BioMed Research International 2014 (2014): 1–13. http://dx.doi.org/10.1155/2014/151405.

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Stenotrophomonasssp. has a wide environmental distribution and is also found as an opportunistic pathogen, causing nosocomial or community-acquired infections. One species,S. maltophilia, presents multidrug resistance and has been associated with serious infections in pediatric and immunocompromised patients. Therefore, it is relevant to conduct resistance profile and phylogenetic studies in clinical isolates for identifying infection origins and isolates with augmented pathogenic potential. Here, multilocus sequence typing was performed for phylogenetic analysis of nosocomial isolates ofStenotrophomonasspp. and, environmental and clinical strains ofS. maltophilia. Biochemical and multidrug resistance profiles of nosocomial and clinical strains were determined. The inferred phylogenetic profile showed high clonal variability, what correlates with the adaptability process ofStenotrophomonasto different habitats. Two clinical isolates subgroups ofS. maltophiliasharing high phylogenetic homogeneity presented intergroup recombination, thus indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. For most of the clinical strains, phylogenetic inference was made using only partialppsA gene sequence. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, determining whether the infection withS. maltophiliawas nosocomial or community-acquired.

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Papapetropoulou, M., A. Tsintzou, and A. Vantarakis. "Environmental mycobacteria in bottled table waters in Greece." Canadian Journal of Microbiology 43, no. 5 (May 1, 1997): 499–502. http://dx.doi.org/10.1139/m97-071.

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A hundred and fifty samples of bottled table water sold by Greek factories were examined for the presence of environmental mycobacteria. Environmental mycobacteria were found in 23 (15.6%) of the 150 tested samples. Bacterial numbers of 1–100, 101–300, 301–1000, and > 103CFU/L were found in 8, 2, 1, and 4% of the samples, respectively. The identification of the environmental mycobacteria was performed by both polymerase chain reaction – restriction enzyme analysis (PCR–REA) and biochemical methods. The environmental mycobacteria found were 14 Mycobacterium chelonae, 3 Mycobacterium phlei, 4 Mycobacterium gordonae, and 2 Mycobacterium flavescens. The relatively high number of environmental mycobacteria in bottled table water leads us to believe that the search of these opportunistic microorganisms in bottled water could be a useful index of their hygienic quality when this water is to be consumed by immunologically compromised patients. No statistically significant correlation was found between the presence of mycobacteria and the bacteriological faecal indicators (P < 0.005).Key words: environmental mycobacteria, bottled water, PCR–REA, Greece.

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Pontes, Daniela Santos, Cláudia Iracema Lima-Bittencourt, Marcela Santiago Pacheco Azevedo, Edmar Chartone-Souza, and Andréa Maria Amaral Nascimento. "Phenotypic and genetic analysis of Enterobacter spp. from a Brazilian oligotrophic freshwater lake." Canadian Journal of Microbiology 53, no. 8 (August 2007): 983–91. http://dx.doi.org/10.1139/w07-060.

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We characterized a population of Enterobacter spp. of the Enterobacter cloacae complex isolated from an oligotrophic lake; most isolates were identified as E. cloacae. Fingerprinting polymerase chain reaction (PCR), along with morphological, biochemical, physiological, and plasmid profiles analyses, including antimicrobial susceptibility testing, were performed on 22 environmental isolates. Misidentification occurred when using the API 20E identification system. Analysis of 16S rDNA sequences confirmed the close relatedness between species of the E. cloacae complex. The tDNA PCR allowed the differentiation and identification of the E. cloacae isolates. Evaluation of genetic diversity by 16S rDNA sequence, tDNA, internal transcribed spacers, and enterobacterial repetitive intergenic concensus profiles revealed nearly identical isolates, although they exhibited different physiological and antimicrobial resistance profiles. Among the Enterobacter isolates, 96% were resistant to at least one antimicrobial; multiple resistance was also found at a high frequency (86%). The antimicrobials against which resistance was found most frequently were β-lactams, chloramphenicol, and streptomycin. Plasmids were found in 21 of the 22 Enterobacter isolates. This confirms the conception that antibiotic resistance can occur in oligotrophic freshwater lake bacteria, which has important implications for public health.

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Chisanga, Malama, Howbeer Muhamadali, David I. Ellis, and Royston Goodacre. "Surface-Enhanced Raman Scattering (SERS) in Microbiology: Illumination and Enhancement of the Microbial World." Applied Spectroscopy 72, no. 7 (March 23, 2018): 987–1000. http://dx.doi.org/10.1177/0003702818764672.

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The microbial world forms a huge family of organisms that exhibit the greatest phylogenetic diversity on Earth and thus colonize virtually our entire planet. Due to this diversity and subsequent complex interactions, the vast majority of microorganisms are involved in innumerable natural bioprocesses and contribute an absolutely vital role toward the maintenance of life on Earth, whilst a small minority cause various infectious diseases. The ever-increasing demand for environmental monitoring, sustainable ecosystems, food security, and improved healthcare systems drives the continuous search for inexpensive but reproducible, automated and portable techniques for detection of microbial isolates and understanding their interactions for clinical, environmental, and industrial applications and benefits. Surface-enhanced Raman scattering (SERS) is attracting significant attention for the accurate identification, discrimination and characterization and functional assessment of microbial cells at the single cell level. In this review, we briefly discuss the technological advances in Raman and Fourier transform infrared (FT-IR) instrumentation and their application for the analysis of clinically and industrially relevant microorganisms, biofilms, and biological warfare agents. In addition, we summarize the current trends and future prospects of integrating Raman/SERS-isotopic labeling and cell sorting technologies in parallel, to link genotype-to-phenotype in order to define community function of unculturable microbial cells in mixed microbial communities which possess admirable traits such as detoxification of pollutants and recycling of essential metals.

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Hale, W. B., M. W. van der Woude, and D. A. Low. "Analysis of nonmethylated GATC sites in the Escherichia coli chromosome and identification of sites that are differentially methylated in response to environmental stimuli." Journal of Bacteriology 176, no. 11 (1994): 3438–41. http://dx.doi.org/10.1128/jb.176.11.3438-3441.1994.

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Journal articles on the topic 'Environmental microbiology (Analysis; Identification)'

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