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PubMed2005

Characteristics of Haemophilus influenzae type b responsible for meningitis in Poland from 1997 to 2004.

Two hundred forty-five H. influenzae isolates responsible for meningitis in Poland from 1997 to 2004 were studied. Among these, 233 (95.1%) belonged to serotype b (Hib), 2 belonged to serotype f, and 10 were noncapsulated. The relatedness of all isolates was evaluated by pulsed-field gel electrophoresis (PFGE), and selected representatives were evaluated by multilocus sequence typing. Resistance to ampicillin was identified in 34 (14.6%) of the Hib isolates and was associated with the production of beta-lactamase only. Except for four isolates nonsusceptible to chloramphenicol, all isolates were susceptible to cefotaxime, ciprofloxacin, and rifampin. The PFGE analysis divided the Hib isolates into five PFGE types; however, all of them were possibly related. The most common PFGE type, with 25 subtypes, was characteristic for 97.4% of the isolates. The most prevalent PFGE subtype found in our study was also the most common among the Hib isolates responsible for invasive disease in Italy and the Czech Republic and was found among isolates causing lower respiratory tract infections in Poland. The most prevalent sequence types (STs) in the studied group were ST6 and ST92. Four new STs were found: ST188, ST189, ST190, and ST268. Results of this study support the evidence that the genetic structure of encapsulated H. influenzae is clonal. The continuing high number of meningitis cases due to Hib in Poland underlines the need for mass vaccination against Hib in Poland.

Ampicillin↗

PubMed2002

Molecular epidemiological analysis of the changing nature of a meningococcal outbreak following a vaccination campaign.

A serogroup C meningococcal outbreak that occurred in an Israeli Arab village led to a massive vaccination campaign. During the subsequent 18 months, new cases of type B Neisseria meningitidis infection were revealed. To investigate the influence of vaccination on bacteriological epidemiology, bacteria were isolated from individuals at the outbreak location, patients with several additional other sporadic cases, and patients involved in another outbreak. Haploid bacterial genomic DNA was mixed with a consensus PCR product to form a heteroduplex state that enabled multilocus sequence typing (MLST) to be combined with denaturing high-performance liquid chromatography (DHPLC) for a novel high-throughput molecular typing method called MLST-DHPLC. A 100% correlation was found to exist between the sequencing by MLST alone and the MLST-DHPLC method. Independent molecular typing by repetitive extragenic palindromic PCR discriminated the neisserial clones as well as the MLST-DHPLC method did. The occurrence of type B N. meningitidis in the postvaccination period might be attributed to the selection pressure applied to the bacteria by vaccination, suggesting a possible unwarranted outcome of vaccination with the quadrivalent vaccine for control of a serogroup C meningococcal outbreak. This is the first time that DHPLC has been applied to the genotyping of bacteria, and it proved to be more efficient than MLST alone.

Adolescent↗

PubMed2005

Pyrosequencing: nucleotide sequencing technology with bacterial genotyping applications.

Pyrosequencing is a relatively new method for real-time nucleotide sequencing. It has rapidly found applications in DNA sequencing, genotyping, single nucleotide polymorphism analysis, allele quantification and whole-genome sequencing within the areas of microbiology, clinical genetics and pharmacogenetics. It is fast becoming a real alternative to the traditional Sanger sequencing method although, at present, read lengths are normally limited to approximately 70 nucleotides. The pyrosequencing method involves four main stages: first, target DNA is amplified using PCR; second, double-stranded DNA is converted to single-stranded DNA templates; third, oligonucleotide primers are hybridized to a complementary sequence of interest; and, finally, the pyrosequencing reaction itself, in which a reaction mixture of enzymes and substrates catalyses the synthesis of complementary nucleotides. Data are shown as a collection of signal peaks in a pyrogram. Pyrosequencing is increasingly used for bacterial detection, identification and typing, and, recently, a commercial system became available for the identification of bacterial isolates. Pyrosequencing can also be partially or fully automated, thus enabling the high-throughput analysis of samples. Wider use of pyrosequencing may occur in the future if longer nucleotide reads are made possible, which will enable its expansion into larger nucleotide sequencing such as multilocus sequence typing and whole-genome sequencing.

Animals↗

PubMed2004

Multiple-locus variable-number tandem repeat analysis, a novel typing scheme to study the genetic relatedness and epidemiology of Enterococcus faecium isolates.

Multiresistant Enterococcus faecium is a major cause of hospital acquired infections and outbreaks. Here, we describe the development of multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) as a novel typing method to assess the genetic relatedness of E. faecium isolates. Six VNTR loci were used to genotype 392 isolates recovered from different animals and human community, hospital survey, and clinical isolates. From 3 to 13 alleles were found per locus, resulting in 127 different MLVA profiles. Clustering of MLVA profiles confirmed the host-specific genogroups found by multilocus sequence typing (MLST) and showed the grouping of clinical and epidemic isolates that belonged to the MLST-C1 cluster in a distinct MLVA-C1 cluster (sensitivity of 97% and specificity of 90%). Furthermore, the discriminatory power of MLVA is comparable to MLST. MLVA profiles appeared to be relatively stable, since isolates from a single outbreak shared the same MLVA profile, which is a prerequisite when MLVA is used to study hospital outbreaks. Our data show that MLVA is a highly reproducible and portable typing method; in contrast to MLST, it is fast, relatively cheap, and easy to perform. Furthermore, it has the abilities of MLST to recognize genetically related and potential epidemic isolates. Submission of MLVA profiles is possible via a Web-based database for international comparison.

Bacterial Typing Techniques↗

PubMed2026

Genomic characterization of novel human-associated CTX-M-15-producing Serratia nevei ST625 lineage infecting a vulnerable loggerhead sea turtle.

BACKGROUND: Serratia nevei is a newly classified and opportunistic bacterial species belonging to the Serratia marcescens complex (SMC). Genomic data from this species is highly relevant for public health and epidemiological tracking. OBJECTIVE: To report the first identification and genomic characterization of extended-spectrum β-lactamase (CTX-M-15)-producing S. nevei sequence type (ST) ST625 lineage infecting a vulnerable loggerhead sea turtle. METHODS: Strain BP02 was recovered from the coelomic cavity of a loggerhead sea turtle (Caretta caretta) admitted to a rehabilitation center in southeastern Brazil. MALDI-TOF MS was initially used for species identification and was further confirmed by whole-genome sequencing on the Illumina HiSeq platform, followed by ANI, dDDH, multilocus sequence typing, resistome, plasmidome, virulome, and SNP-based phylogenomic analyses. RESULTS: Strain BP02 exhibited a multidrug-resistant profile, including resistance to third- and fourth-generation cephalosporins. Genomic analyses identified BP02 as S. nevei ST625 carrying blaCTX-M-15 within the ISEcp1-blaCTX-M-15-wbuC-ΔTn2 genetic environment, in addition to multiple AMR determinants and the IncC plasmid replicon. Phylogenomic analysis demonstrated close relatedness between BP02 and human clinical ST625 strains, previously reported in São Paulo, Brazil, including a urine-derived strain isolated in 2019, differing by only 27 SNPs. Notably, all publicly available ST625 genomes were associated with human clinical sources and displayed multidrug resistance genotypes. CONCLUSION: This study expands the current knowledge regarding the ecology and genomic features of S. nevei, demonstrating the emergence of a human multidrug-resistant clone in marine wildlife. Our findings reinforce the importance of monitoring clinically relevant SMC members across distinct ecological niches within a One Health perspective.

ESBL↗

PubMed2002

Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus.

Full characterization of methicillin-resistant Staphylococcus aureus (MRSA) requires definition of not only the bacterial genetic background but also the structure of the complex and heterologous mec element these bacteria carry, which is associated with drug resistance determinant mecA. We report the development, validation, and application of a multiplex PCR strategy that allows quick presumptive characterization of the mec element types based on the structural features that were shown to be typical of mec elements carried by several MRSA clones. The strategy was validated by using a representative collection of pandemic MRSA clones in which the full structure of the associated mec elements was previously determined by hybridization and PCR screenings and also by DNA sequencing. The method was tested together with multilocus sequence typing and other typing methods for the characterization of 18 isolates representative of the MRSA clones recovered during a hospital outbreak in Barcelona, Spain. The multiplex PCR was shown to be rapid, robust, and capable in a single assay of identifying five structural types of the mec element among these strains, three major and two minor variants, each one of which has been already been seen among MRSA characterized earlier. This technique should be a useful addition to the armamentarium of molecular typing tools for the characterization of MRSA clonal types and for the rapid tentative identification of structural variants of the mec element.

Bacterial Proteins↗

PubMed2026

Drug-resistant genes, virulence characteristics, and molecular typing of clindamycin-resistant Streptococcus agalactiae in late pregnancy.

BACKGROUND: Streptococcus agalactiae increases the risk of adverse pregnancy outcomes and neonatal infections. Clindamycin is a key alternative for intrapartum prophylaxis in penicillin-allergic women, but the prevalence of clindamycin-resistant S. agalactiae is increasing, posing a significant clinical challenge. METHODS: A total of 178 strains isolated from tertiary hospitals in Jinan and Qingdao, Shandong Province, China, were characterized using antimicrobial susceptibility testing, whole-genome sequencing, multilocus sequence typing, serotyping, and analysis of resistance and virulence genes. RESULTS: All strains were susceptible to penicillin, ampicillin, linezolid, vancomycin, and tigecycline. In contrast, resistance rates to erythromycin, levofloxacin, and tetracycline were 95.5%, 60.1%, and 56.7%, respectively. Six serotypes and 15 sequence types belonging to eight clonal complexes were identified. Notable regional differences were observed. The Ib-ST10-CC12 lineage dominated in Jinan, whereas V-ST529-CC327 was predominant in Qingdao. The resistance gene mreA was ubiquitous (100%), followed by ermB (80.3%). The key virulence genes cylE, hylB, and pavA, were detected in all strains. fbsA (99.4%), the alpha protein family (98.9%), cfb (98.3%), the Pilus Island gene cluster (94.9%), and lmb (92.7%) were also highly prevalent. The two major clindamycin resistance genes, erm and lnuB, exhibited distinctly different enrichment patterns among S. agalactiae clonal complexes, despite a certain overlap in CC19 and CC327. Specifically, erm was significantly enriched in CC12 (serotype Ib), CC19 (III/V), and CC327 (III/V). In contrast, lnuB was predominantly restricted to CC19 and CC327, where it defined a unique phylogenetic subcluster. Significant differences in resistance and virulence gene profiles were observed across different clonal complexes. CONCLUSION: Clindamycin-resistant S. agalactiae in late-pregnancy women in Shandong Province, China exhibits a broad resistance spectrum, diverse molecular types, and significant regional heterogeneity. These findings underscore the need for continued surveillance and region-specific strategies for preventing neonatal S. agalactiae infections.

Humans↗

PubMed2006

The important role of nontypable Streptococcus pneumoniae international clones in acute conjunctivitis.

BACKGROUND: In a recent epidemiological study in southern Israel, nontypable Streptococcus pneumoniae isolates were found to be highly associated with sporadic cases of acute conjunctivitis (AC). The purpose of the present study was to evaluate the relative importance in causing AC of the absence of capsule versus genotype properties. METHODS: DNA typing by pulsed-field gel electrophoresis (PFGE) was performed on 148 nontypable organisms isolated from 3 sites: nasopharynx of healthy children, middle-ear fluid, and conjunctiva. RESULTS: Analysis of the PFGE patterns revealed the presence of 6 clusters; 2 clusters that included 44% of the isolates (65/148) were associated with AC, and the remaining 4 were frequently isolated from the nasopharynx. Multilocus sequence typing, performed on representative isolates of the 2 major clusters, confirmed that the organisms were pneumococci; one is a single-locus variant of sequence type (ST) 448, and the other is related to ST344. Both types appear to be members of pneumococcal lineages that have lost capsular loci. The nontypable isolates showed high rates of resistance to antimicrobial agents. CONCLUSIONS: Our data suggest that the absence of the capsule--along with other, yet-unidentified genetic characteristics--provide S. pneumoniae with a selective virulence advantage in conjunctivitis.

Bacterial Typing Techniques↗

PubMed2001

Interpreting the rising incidence of meningococcal disease in Belgium: the contribution of molecular typing.

During a period of increasing meningococcal disease incidence in Belgium, all 538 serogroup B and all 87 serogroup C strains isolated between 1996 and 1998 were investigated by PCR with the arbitrary primer D8635, which is able to identify lineage III strains. In all, 399 strains (64%) were attributed to lineage III on the basis of PCR-based typing. Since their introduction in the Belgian population in the early 1990s, lineage III strains have become increasingly variable in phenotype. Currently, they are represented by strains belonging to 38 different phenotypes, of which 25 were not found in the period 1990-1995. The 87 serogroup C strains were further investigated by pulsed-field gel electrophoresis (PFGE), and a subset of 30 strains was also investigated by multilocus sequence typing (MLST). Strains of phenotype C:2b:P1.5,2, which currently constitute the majority of the serogroup C strains, were demonstrated to belong to cluster A4. Comparison of the discriminatory ability of D8635-primed PCR, PFGE and MLST revealed that D8635-primed PCR was the least discriminatory method and PFGE the most discriminatory method. However, the MLST data were more readily interpreted than the PFGE fingerprint patterns and can be compared easily with data obtained in other studies. In conclusion, the ongoing increase of meningococcal disease in Belgium could be attributed not only to the further expansion of lineage III, but also to the introduction of C:2b:P1.5,2 strains of cluster A4 in to the Belgian population.

Bacterial Typing Techniques↗

PubMed2004

Molecular characteristics of nosocomial and Native American community-associated methicillin-resistant Staphylococcus aureus clones from rural Wisconsin.

In central and northern Wisconsin methicillin-resistant Staphylococcus aureus (MRSA) was first detected in 1989. Over the next 10-year period, 581 MRSA isolates were collected, 17.2% of which came from patients who were treated at five Native American clinics. These isolates were typed by SmaI-macrorestricted pulsed-field gel electrophoresis (PFGE). The PFGE patterns clustered the isolates into six major clonal groups (MCGs), i.e., MCGs 1 to 6, and 19 minor clonal groups (mCGs). The 25 clonal groups were represented by 109 unique PFGE types. Sixty-five percent of the MCG-2 isolates were recovered from patients who were treated at Native American clinics. Ninety-four percent of the MCG-2 isolates harbored the staphylococcal cassette chromosome mec (SCCmec) IVa. These isolates also had PFGE profiles that were clonally related to the midwestern community-associated MRSA (CA-MRSA) strain, MW2. The representative isolates from MCG-2 had the multilocus sequence type allelic profile 1-1-1-1-1-1-1 and contained pvl genes. They were also susceptible to various antibiotics, a finding consistent with the CA-MRSA phenotype. SCCmec IV was also present in other mCGs. Unlike MCG-2, isolates from the remaining five MCGs harbored SCCmec II and were resistant to multiple antibiotics, suggesting their nosocomial origin. The 19 mCGs were represented by diverse SCCmec types and three putative new variants referred to as SCCmec Ib, IIa, and IIb.

Community-Acquired Infections↗

PubMed2026

Global Diffusion of IncC Plasmid Harboring blaNDM-1in the High-Risk Escherichia coli ST131 Clone.

AIMS: The global expansion of quinolone-resistant Escherichia coli (QR-EC) is increasingly associated with β-lactam resistance and mobile genetic elements that facilitate resistance dissemination. This study investigated the molecular mechanisms underlying fluoroquinolone and β-lactam resistance in clinical QR-EC isolates and explored the plasmid type associated. METHODS AND RESULTS: A total of 123 non-duplicate QR-EC clinical isolates responsible mainly for gastrointestinal colonization were collected between 2019 and 2021. Plasmid-mediated quinolone resistance (PMQR), extended-spectrum β-lactamase (ESBL), and carbapenemase genes were screened by PCR. Mutations in the quinolone resistance-determining regions (QRDR) of gyrA and parC were analyzed using sequencing and mismatch amplification mutation assay PCR (MAMA-PCR). Selected isolates underwent multilocus sequence typing (MLST). Whole-genome sequencing (WGS) of a representative extensively drug-resistant strain carrying multiple quinolone resistance determinants, ESBL genes, and carbapenemase genes, was performed. PMQR genes were prevalent among QR-EC, dominated by aac(6')-Ib-cr (60.9% of isolates). ESBL genes were identified in 93.5% of isolates, predominantly blaCTX-M (95.7%). Among ertapenem-resistant isolates (QCR-EC) (n=18), blaNDM-1 and blaOXA-48 were detected in 13 and 11 isolates, respectively. QRDR mutations were highly frequent, particularly gyrA83 (98.4%) and parC80 (30.9%). Major QCR-EC genotypes belonged to sequence types ST167 (n=2), ST1196, ST469, and ST410. High-risk E. coli ST131 clone harboring IncC plasmid encoding blaNDM-1 was described for the first time in Africa, following its emergence, in two continents, Asia and America. Despite the very rare description of these strains worldwide, their description in three continents sign their global diffusion. CONCLUSIONS: This finding highlights the ongoing spread of carbapenem resistance and underscores the urgent need for strengthened genomic surveillance.

Escherichia coli↗

PubMed2003

Clonal analysis of Streptococcus pneumoniae nonsusceptible to penicillin at day-care centers with index cases, in a region with low incidence of resistance: emergence of an invasive type 35B clone among carriers.

The nasopharyngeal carriage rate of potential respiratory pathogens was studied in 36 index children with a pneumococci nonsusceptible to penicillin (PNSP), in 595 healthy children, and in 123 personnel at 16 day-care centers (DCCs) with index cases in the Stockholm area, an urban area with a low incidence of antibiotic resistant pneumococci, during the winter of 1997-1998. The spread and clonality of PNSP, Haemophilus influenzae and Moraxella catarrhalis, were studied by analyzing antibiotic susceptibility and serotype, and for PSNP also by using pulsed-field electrophoresis (PFGE) and multilocus sequence typing (MLST). In contrast to the low carriage rate found among the adult contacts (2%), 40% of the children harbored pneumococci, of which 20% were PNSP. Nasopharyngeal colonization decreased with age. The 49 PNSP isolates consisted of 20 clones, of which 10 could be identified in more than one child attending the same or different DCCs. In five DCCs, we observed a spread of PNSP from the index case. A novel PNSP clone of type 35B, found to cause invasive disease in several states in the United States, was found to emerge among several carriers at two DCCs . A high proportion of PNSP isolates were multiresistant to antibiotics (34%), which has implications for treatment regimens, even in a country like Sweden where the proportion of PNSP currently is low (3-4%). One PNSP clone of type 9V found among the carriers, has been shown to cause invasive disease in Sweden as well as in other countries, suggesting that one reason for the occurrence of invasive PNSP clones may be their ability to colonize and spread among healthy carriers. Other internationally spread antibiotic resistant pneumococcal clones found were of types 9V, 19F, and 23F.

Anti-Bacterial Agents↗

PubMed2007

Amplified fragment length polymorphism of Streptococcus suis strains correlates with their profile of virulence-associated genes and clinical background.

Amplified fragment length polymorphism (AFLP) typing was applied to 116 Streptococcus suis isolates with different clinical backgrounds (invasive/pneumonia/carrier/human) and with known profiles of virulence-associated genes (cps1, -2, -7 and -9, as well as mrp, epf and sly). A dendrogram was generated that allowed identification of two clusters (A and C) with different subclusters (A1, A2, C1 and C2) and two heterogeneous groups of strains (B and D). For comparison, three strains from each AFLP subcluster and group were subjected to multilocus sequence typing (MLST) analysis. The closest relationship and lowest diversity were found for patterns clustering within AFLP subcluster A1, which corresponded with sequence type (ST) complex 1. Strains within subcluster A1 were mainly invasive cps1 and mrp+ epf+ (or epf*) sly+ cps2+ strains of porcine or human origin. A new finding of this study was the clustering of invasive mrp* cps9 isolates within subcluster A2. MLST analysis suggested that A2 correlates with a single ST complex (ST87). In contrast to A1 and A2, subclusters C1 and C2 contained mainly pneumonia isolates of genotype cps7 or cps2 and epf- sly-. In conclusion, this study demonstrates that AFLP allows identification of clusters of S. suis strains with clinical relevance.

Animals↗

PubMed2006

Molecular typing of meningococci: recommendations for target choice and nomenclature.

The diversity and dynamics of Neisseria meningitidis populations generate a requirement for high resolution, comprehensive, and portable typing schemes for meningococcal disease surveillance. Molecular approaches, specifically DNA amplification and sequencing, are the methods of choice for various reasons, including: their generic nature and portability, comprehensive coverage, and ready implementation to culture negative clinical specimens. The following target genes are recommended: (1) the variable regions of the antigen-encoding genes porA and fetA and, if additional resolution is required, the porB gene for rapid investigation of disease outbreaks and investigating the distribution of antigenic variants; (2) the seven multilocus sequence typing loci-these data are essential for the most effective national, and international management of meningococcal disease, as well as being invaluable in studies of meningococcal population biology and evolution. These targets have been employed extensively in reference laboratories throughout the world and validated protocols have been published. It is further recommended that a modified nomenclature be adopted of the form: serogroup: PorA type: FetA type: sequence type (clonal complex), thus: B: P1.19,15: F5-1: ST-33 (cc32).

Bacterial Outer Membrane Proteins↗

PubMed2006

Nucleotide sequence-based analysis for determining the molecular epidemiology of Penicillium marneffei.

The dimorphic fungus, Penicillium marneffei, is an emerging opportunistic pathogen endemic in Southeast Asia, especially for those with impaired cellular immunity such as human immunodeficiency virus-infected persons. A discriminatory and reproducible method based on the analysis of nucleotide sequences would facilitate epidemiologic investigations of this fungus. Twenty-four clinical or environmental isolates of P. marneffei obtained from China, Thailand, and Vietnam were analyzed by nucleotide sequence analysis. A total of 3,803 bp, consisting of eight nuclear gene fragments (transcription factor [AbaA], catalase [CpeA]], homodomain transcription factor [StlA], isocitrate lyase [Icl1], polyaromatic amino acid biosynthesis [PAA], NADH-dependent glutamate synthase [NGS], lovastatin nonaketide synthase [LNS], a cell wall mannoprotein [MP1], and a gene fragment of the cytochrome oxidase subunit 1 gene [COX1] of the P. marneffei mitochondrial genome) were amplified by PCR and then sequenced. No polymorphic sites within the Cox1 gene fragment were observed. Likewise, no nucleotide sequence polymorphisms were observed for three gene fragments: StlA, AbaA, and NGS. Seven single-nucleotide polymorphisms were observed for three gene fragments, Icl1, CpeA, and PAA, providing only a low degree of discriminatory power (D = 0.747). In contrast, the gene fragment for an antigenic cell wall glycoprotein, MP1, a useful immunologic marker for infection, was observed to be highly polymorphic with 12 different MP1 types (D = 0.887). Single-nucleotide polymorphisms were observed at 21 different locations in the MP1 gene fragment. Indels of 3, 21, 24, and 42 bp were observed and were in frame for protein translation. The relatively high degree of MP1 polymorphisms suggests the sequence is rapidly evolving in order to evade host immune responses. After all polymorphic gene sequences were combined, a high degree of genetic variation was observed (D = 0.949) for a total of 16 different haploid sequence types with 11 genotypes represented by single isolates. Phylogenetic analysis detected clusters composed of isolates obtained only from China or Thailand, as well as clusters with a combination of isolates from these two countries, indicating some mixing or common descent. Identical sequences were observed for isolates passed in vitro for 8 weeks, suggesting good reproducibility. The low degree of nucleotide diversity in housekeeping and regulatory genes suggests the recent emergence and spread as a species or an evolutionary bottleneck. In summary, multilocus sequence typing demonstrated a high degree of discriminatory power and reproducibility and may provide a robust and reliable adjunct method for genotyping isolates of P. marneffei and facilitating interlaboratory comparisons.

Amino Acid Sequence↗

PubMed2004

Prevalence and molecular analysis of macrolide and fluoroquinolone resistance among isolates of Streptococcus pneumoniae collected during the 2000-2001 PROTEKT US Study.

The PROTEKT US (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin in the United States) surveillance program was established to determine the prevalence and mechanisms of antibacterial resistance among bacterial pathogens from patients with community-acquired respiratory tract infections. In year 1 of the PROTEKT US study, 10,103 isolates of Streptococcus pneumoniae, including 3,133 erythromycin-resistant strains and 81 levofloxacin-resistant strains, were collected from 206 centers. We report on the molecular analyses of these resistant strains. The resistance genotypes among the 3,044 typed macrolide-resistant isolates overall were mef(A) (n = 2,157; 70.9%), erm(B) (n = 530; 17.4%), mef(A) erm(B) (n = 304; 10.0%), and erm(A) subclass erm(TR) (n = 5; 0.2%). Fifty (1.6%) macrolide-resistant isolates were negative for the mef and the erm resistance genes. Seventy-eight (96.3%) of the 81 levofloxacin-resistant isolates analyzed possessed multiple mutations in the gyrA, gyrB, parC, and/or parE quinolone resistance-determining regions. A total of 43 known multilocus sequence typing (MLST) profiles (or single- or double-locus variants) accounted for 75 of 81 isolates. There was no evidence of dissemination of fluoroquinolone-resistant clones within the United States; however, 12 isolates with the same MLST profile were located in one center in Massachusetts. Almost 90% of the erythromycin-resistant isolates and approximately one-third of the levofloxacin-resistant isolates were multidrug resistant.

Anti-Bacterial Agents↗

PubMed2006

Comparison of subtyping methods for differentiating Salmonella enterica serovar Typhimurium isolates obtained from food animal sources.

Molecular characterization (e.g., DNA-based typing methods) of Salmonella isolates is frequently employed to compare and distinguish clinical isolates recovered from animals and from patients with food-borne disease and nosocomial infections. In this study, we compared the abilities of different phenotyping and genotyping methods to distinguish isolates of Salmonella enterica serovar Typhimurium from different food animal sources. One hundred twenty-eight S. enterica serovar Typhimurium strains isolated from cattle, pigs, chickens, and turkeys or derived food products were characterized using pulsed-field gel electrophoresis (PFGE), repetitive element PCR (Rep-PCR), multilocus sequence typing (MLST), plasmid profiling, and antimicrobial susceptibility testing. Among the 128 Salmonella isolates tested, we observed 84 Rep-PCR profiles, 86 PFGE patterns, 89 MLST patterns, 36 plasmid profiles, and 38 susceptibility profiles. The molecular typing methods, i.e., PFGE, MLST, and Rep-PCR, demonstrated the best discriminatory power among Salmonella isolates. However, no apparent correlation was evident between the results of one molecular typing method and those of the others, suggesting that a combination of multiple methods is needed to differentiate S. enterica serovar Typhimurium isolates that genetically cluster according to one particular typing method.

Animals↗

PubMed2005

Single-nucleotide polymorphism phylotyping of Escherichia coli.

We describe a rapid and easily automated phylogenetic grouping technique based on analysis of bacterial genome single-nucleotide polymorphisms (SNPs). We selected 13 SNPs derived from a complete sequence analysis of 11 essential genes previously used for multilocus sequence typing (MLST) of 30 Escherichia coli strains representing the genetic diversity of the species. The 13 SNPs were localized in five genes, trpA, trpB, putP, icdA, and polB, and were selected to allow recovery of the main phylogenetic groups (groups A, B1, E, D, and B2) and subgroups of the species. In the first step, we validated the SNP approach in silico by extracting SNP data from the complete sequences of the five genes for a panel of 65 pathogenic strains belonging to different E. coli pathovars, which were previously analyzed by MLST. In the second step, we determined these SNPs by dideoxy single-base extension of unlabeled oligonucleotide primers for a collection of 183 commensal and extraintestinal clinical E. coli isolates and compared the SNP phylotyping method to previous well-established typing methods. This SNP phylotyping method proved to be consistent with the other methods for assigning phylogenetic groups to the different E. coli strains. In contrast to the other typing methods, such as multilocus enzyme electrophoresis, ribotyping, or PCR phylotyping using the presence/absence of three genomic DNA fragments, the SNP typing method described here is derived from a solid phylogenetic analysis, and the results obtained by this method are more meaningful. Our results indicate that similar approaches may be used for a wide variety of bacterial species.

Bacterial Typing Techniques↗