Anand Manoharan

Population Genetics and Linkage Analysis of Loci within the FCT Region of Streptococcus pyogenes

The FCT regions of Streptococcus pyogenes strains encode a variety of cell wall-anchored surface proteins that bind the extracellular matrix of the human host and/or give rise to pilus-like appendages. Strong linkage is evident between transcription-regulatory loci positioned within the FCT and emm regions and the emm pattern genotype marker for preferred infection of the throat or skin. These findings provide a basis for the hypothesis that FCT region gene products contribute to tissue-specific infection. In an initial series of steps to address this possibility, the FCT regions of 13 strains underwent comparative sequence analysis, the gene content of the FCT region was characterized for 113 strains via PCR, and genetic linkage was assessed. A history of extensive recombination within FCT regions was evident. The emm pattern D-defined skin specialist strains were highly homogenous in their FCT region gene contents, whereas the emm pattern A-C-defined throat specialist strains displayed a greater variety of forms. Most pattern A-C strains harbored prtF1 (75%) but lacked cpa (75%). In contrast, the majority of emm pattern D strains had cpa (92%) but lacked prtF1 (79%). Models based on FCT and emm region genotypes revealed the most parsimonious pathways of evolution. Using niche-determining candidate genes to infer phylogeny, emm pattern E strains--the so-called generalists, which lack a strong tissue site preference--occupied a transition zone separating most throat specialists from skin specialists. Overall, population genetic analysis supports the possibility that the FCT region gives rise to surface proteins that are largely necessary, but not always sufficient, to confer tissue site preference for infection.

Population structure of a hybrid clonal group of methicillin-resistant Staphylococcus aureus, ST239-MRSA-III.

The methicillin-resistant Staphylococcus aureus (MRSA) clonal group known as ST239-MRSA-III is notable for its hybrid origin and for causing sustained hospital epidemics worldwide since the late 1970s. We studied the population structure of this MRSA clonal group using a sample of 111 isolates that were collected over 34 years from 29 countries. Genetic variation was assessed using typing methods and novel ascertainment methods, resulting in approximately 15 kb of sequence from 32 loci for all isolates. A single most parsimonious tree, free of homoplasy, partitioned 28 haplotypes into geographically-associated clades, including prominent European, Asian, and South American clades. The rate of evolution was estimated to be approximately 100× faster than standard estimates for bacteria, and dated the most recent common ancestor of these isolates to the mid-20th century. Associations were discovered between the ST239 phylogeny and the ccrB and dru loci of the methicillin resistance genetic element, SCCmec type III, but not with the accessory components of the element that are targeted by multiplex PCR subtyping tools. In summary, the evolutionary history of ST239 can be characterized by rapid clonal diversification that has left strong evidence of geographic and temporal population structure. SCCmec type III has remained linked to the ST239 chromosome during clonal diversification, but it has undergone homoplasious losses of accessory components. These results provide a population genetics framework for the precise identification of emerging ST239 variants, and invite a re-evaluation of the markers used for subtyping SCCmec.

Evolution and Global Dissemination of Macrolide-Resistant Group A Streptococci

ABSTRACT Macrolide-resistant group A streptococci (MRGAS) have been recovered from many countries worldwide. However, the strain typing information that is available has been insufficient for estimating the total number of macrolide-resistant clones, their geographic distributions, and their evolutionary relationships. In this study, sequence-based strain typing was used to characterize 212 MRGAS isolates from 34 countries. Evaluation of clonal complexes, emm type, and resistance gene content [erm(A), erm(B), mef(A), and undefined] indicate that macrolide resistance was acquired by GAS organisms via ≥49 independent genetic events. In contrast to other collections of mostly susceptible GAS, genetic diversification of MRGAS clones has occurred primarily by mutation rather than by recombination. Twenty-two MRGAS clonal complexes were recovered from more than one continent; intercontinental strains represent nearly 80% of the MRGAS isolates under study. The findings suggest that horizontal transfer of macrolide resistance genes to numerous genetic backgrounds and global dissemination of resistant clones and their descendants are both major components of the present-day macrolide resistance problem found within this species.

Evolution of Transcription Regulatory Genes Is Linked to Niche Specialization in the Bacterial Pathogen Streptococcus pyogenes

Streptococcus pyogenes is a highly prevalent bacterial pathogen, most often giving rise to superficial infections at the throat or skin of its human host. Three genotype-defined subpopulations of strains exhibiting strong tropisms for either the throat or skin (specialists) or having no obvious tissue site preference (generalists) are recognized. Since the microenvironments at the throat and skin are distinct, the signal transduction pathways leading to the control of gene expression may also differ for throat versus skin strains of S. pyogenes. Two loci (mga and rofA/nra) encoding global regulators of virulence gene expression are positioned 300 kb apart on the genome; each contains alleles forming two major sequence clusters of approximately 25 to 30% divergence that are under balancing selection. Strong linkage disequilibrium is observed between sequence clusters of the transcription regulatory loci and the subpopulations of throat and skin specialists, against a background of high recombination rates among housekeeping genes. A taxonomically distinct commensal species (Streptococcus dysgalactiae subspecies equisimilus) shares highly homologous rof alleles. The findings provide strong support for a mechanism underlying niche specialization that involves orthologous replacement of regulatory genes following interspecies horizontal transfer, although the directionality of gene exchange remains unknown.

Correlation of TEM, SHV and CTX-M extended-spectrum beta lactamases among Enterobacteriaceae with their in vitro antimicrobial susceptibility

PURPOSE The present study was carried out to characterize the ESBL types and evaluated their in vitro activity against a collection of Gram negative bacteria (GNB) from a multicentric Indian surveillance study. MATERIAL AND METHODS During January 2005 to June 2006, six tertiary care centres in India forwarded 778 non-duplicate GNB to our reference laboratory. Three hundred GNB from this collection were selected based on clinical significance and were used in the present study. Tested isolates included Escherichia coli (167), Klebsiella spp. (122) and Enterobacter spp. (11). ESBL screening and confirmation was performed for all the isolates. Minimum inhibitory concentration of imipenem, meropenem, ertapenem, levofloxacin, amikacin, piperacillin/tazobactam and ceftriaxone was determined by the E-test method. Molecular typing of the ESBLs was performed by polymerase chain reaction among the 121 selected isolates. RESULTS The study showed excellent susceptibility among the strains to imipenem (100%), meropenem (100%) and ertapenem (98.7%); good susceptibility to amikacin (89.7%) and piperacillin/tazobactam (85.3%) was observed. TEM and CTX-M were predominantly found in E. coli (39.2%) while, among the Klebsiella spp., TEM, SHV and CTX-M occurred together in 42.6% of the isolates. CONCLUSION More than one ESBL was produced by many strains, and this was correlated with increased resistance levels. Carbapenems continue to show good in vitro activity and ertapenem is a potential alternative to imipenem and meropenem. Continued antimicrobial resistance surveillance is warranted in light of these findings.

Detection and characterization of metallo beta lactamases producing Pseudomonas aeruginosa

This study was undertaken to evaluate phenotypic and genotypic methods for detection of Metallo-Beta-Lactamases (MBLs) among nosocomial Pseudomonas aeruginosa. Sixty one among 176 P. aeruginosa isolates, collected as part of a multicentric study (2005-2007), were evaluated for carbapenem resistance (CARB-R; resistant to either imipenem/meropenem) and screened for MBL by Combination Disk Diffusion Test (CDDT) using imipenem (IMP), meropenem (MER) and ceftazidime (CAZ) with EDTA. MBL positives were further confirmed by IMP + EDTA Etest. Twenty strains (42.6%) were found to be MBL producers among the 61 P. aeruginosa. PCR for IMP and VIM MBL was performed on 48 of the 61, 15 were positive for VIM MBL type. CDDT using IMP + EDTA had the highest sensitivity and specificity of 87.8% and 84.4% when compared to Etest, which was higher than the values obtained for CAZ + EDTA and MER + EDTA. CDDT using IMP + EDTA also compared very well with the PCR (specificity = 90.9%, sensitivity = 93.3%). CARB-R among P. aeruginosa is mediated predominantly via MBL production. Clinical P. aeruginosa isolates can be screened routinely using the less expensive IMP + EDTA CDDT in clinical microbiology laboratories.

Detection of Oxacillin-Susceptible mecA-Positive Staphylococcus aureus Isolates by Use of Chromogenic Medium MRSA ID

ABSTRACT Reports of oxacillin-susceptible mecA-positive Staphylococcus aureus strains are on the rise. Because of their susceptibility to oxacillin and cefoxitin, it is very difficult to detect them by using routine phenotypic methods. We describe two such isolates that were detected by chromogenic medium and confirmed by characterization of the mecA gene element.

Tetracycline Resistance in Group A Streptococci: Emergence on a Global Scale and Influence on Multiple-Drug Resistance

ABSTRACT A global sample of group A streptococci (GAS) revealed ≥80 separate acquisitions of tetracycline resistance. Of 244 clones, 38 and 25% displayed resistance to tetracycline and erythromycin, respectively; a relatively high proportion (15%) were resistant to both classes of drugs. tet(M) displayed a highly significant association with erm(B).

Evaluation of tigecycline activity in clinical isolates among Indian medical centers

BACKGROUND Resistance to multiple antibiotics among Gram-positive cocci (GPC) and Gram negative bacilli (GNB) is high in India. Tigecycline, a glycylcycline antibiotic is a newer treatment option for emerging single or multidrug-resistant (MDR) GPC and GNB. MATERIAL AND METHOD We evaluated the in vitro activity of tigecycline and compared it against other antimicrobials. Between 2005-2007, seven Indian medical centers from diverse geographic regions forwarded 727 isolates [Escherichia coli (166), Staphylococcus aureus (125), Klebsiella spp (120), Streptococcus pneumoniae (102), Enterococcus spp. (100), Pseudomonas aeruginosa (50), Acinetobacter spp. (50) and Enterobacter spp. (14)] from patients with blood stream (BSI), skin and soft tissue (SSTI) including surgical site, urinary tract and respiratory infections to our reference laboratory. Susceptibility to 11 antimicrobials besides tigecycline included: vancomycin, linezolid, teicoplanin, quinopristin-dalfopristin, daptomycin, amikacin, imipenem, levofloxacin, meropenem, and piperacillin/tazobactam was determined by agar dilution and Etest method. RESULT Tigecycline was active against all GPC (MIC 90 < 0.25 μg/ml), E. coli and Klebsiella spp. (MIC 90 ≤1 μg/ml). MDR Acinetobacter spp. showed lower susceptibility (70.6%) to tigecycline. Tigecycline MIC 90 values were not influenced by oxacillin resistance among S. aureus, S. pneumoniae, vancomycin resistance in Enterococci (VRE) and ESBL producing E. coli, Klebsiella spp. and Enterobacter spp. Increased resistance was seen to other antimicrobials among ESBL producing E. coli, Klebsiella spp., Metallo Beta Lactamase (MBL) producing P. aeruginosa and VRE. CONCLUSION Tigecycline is an alternative option for emerging multidrug-resistant (MDR) pathogens exhibiting promising spectrum/potency exceeding currently available agents seen in India.

Fecal carriage rates of extended-spectrum β-lactamase-producing Escherichia coli among antibiotic naive healthy human volunteers.

INTRODUCTION Higher prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli fecal carriage has been reported in the nosocomial setting than in the community. We tried to determine the fecal carriage of ESBL-producing E. coli among healthy volunteers in a relatively isolated community. MATERIALS AND METHODS This study was conducted on 115 healthy adult volunteers from whom one fecal sample was collected and was plated on selective media. Each morphotypes were identified, characterized, and ESBL phenotype was confirmed by double-disk potentiation method. Molecular characterization of ESBL gene was done using multiplex polymerase chain reaction and pulse-field gel electrophoresis (PFGE) was done to identify their clonal relation. RESULTS ESBL-producing E. coli had a prevalence of 19% (22/115) among the healthy volunteers in the community. CTX-M was the predominant type, showed a presence 95.5% (21/22), TEM 63%, SHV 9%, and both TEM and CTX-M were present in 63.6% (14/22), all three present in 4.5% (1/22). The lineage using PFGE showed a single clone in 17 isolates. Seven isolates were type A (all TEM & CTX-M), six were type A1 (all TEM & CTX-M except 2), four were type A2 (all CTX-M), and three belonged to types B, C, and D respectively Conclusion: High prevalence rate of 19% in the community indicated by this study implies the possibility of sustained ESBL carriage even among isolated population, which could serve as a reservoir for enriching the ESBL pool in the hospital. Clonal relations also indicate a possible epidemiological source that needs to be evaluated.