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1.  Methicillin-Resistant Staphylococcus aureus Sequence Type 239-III, Ohio, USA, 2007–20091 
Emerging Infectious Diseases  2012;18(10):1557-1565.
Identification of virulent strains emphasizes the need for molecular surveillance.
PMCID: PMC3471631  PMID: 23018025
Staphylococcus aureus; methicillin-resistant Staphylococcus aureus; MRSA; MRSA ST239-III; bacteria; sequence type; virulent clones; Brazilian clone; Portuguese clone; Ohio; United States
2.  Multiplex PCR for Identification of Two Capsular Types in Epidemic KPC-Producing Klebsiella pneumoniae Sequence Type 258 Strains 
We developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258] cps-1 and cps-2) in epidemic Klebsiella pneumoniae ST258 strains. The assay performed with excellent sensitivity (100%) and specificity (100%) for identifying cps types in 60 ST258 K. pneumoniae sequenced isolates. The screening of 419 ST258 clonal isolates revealed a significant association between cps type and K. pneumoniae carbapenemase (KPC) variant: cps-1 is largely associated with KPC-2, while cps-2 is primarily associated with KPC-3.
PMCID: PMC4068549  PMID: 24733470
3.  Comparative Genomic Analysis of KPC-Encoding pKpQIL-Like Plasmids and Their Distribution in New Jersey and New York Hospitals 
The global spread of Klebsiella pneumoniae carbapenemase (KPC) is predominately associated with K. pneumoniae strains genotyped as sequence type 258 (ST258). The first ST258-associated plasmid, pKpQIL, was described in Israel in 2006, but its history in the northeastern United States remains unknown. Six pKpQIL-like plasmids from four K. pneumoniae isolates (three ST258 and one ST234), one Escherichia coli isolate, and one Enterobacter aerogenes isolate, collected from 2003 to 2010 in New York (NY) and New Jersey (NJ) hospitals, were completely sequenced. The sequences and overall sizes of the six plasmids are highly similar to those of pKpQIL; the major difference is that five of six NJ/NY strains harbor blaKPC-2, while pKpQIL contains blaKPC-3. Moreover, a 26.7-kb fragment was inverted in pKpQIL-234 (from ST234 K. pneumoniae), while a 14.5-kb region was deleted in pKpQIL-Ec (from ST131 E. coli). PCR screening of 284 other clinical K. pneumoniae isolates identified 101 (35.6%) harboring pKpQIL-like plasmids from 9 of 10 surveyed hospitals, demonstrating the wide dissemination of pKpQIL in this region of endemicity. Among the positive isolates, 87.1% were typed as ST258 and 88.1% carried blaKPC-2. The finding of pKpQIL-like plasmid in this study from strains that predate the initial report of KPC in Israel provides evidence that pKpQIL may have originated in the United States. Our findings demonstrate that pKpQIL plasmids are both spreading clonally in ST258 strains and spreading horizontally to different sequence types and species, further highlighting the clinical and public health concerns associated with carbapenem resistance.
PMCID: PMC3993205  PMID: 24614371
4.  Molecular Survey of the Dissemination of Two blaKPC-Harboring IncFIA Plasmids in New Jersey and New York Hospitals 
Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains have spread worldwide and become a major threat in health care facilities. Transmission of blaKPC, the plasmid-borne KPC gene, can be mediated by clonal spread and horizontal transfer. Here, we report the complete nucleotide sequences of two novel blaKPC-3-harboring IncFIA plasmids, pBK30661 and pBK30683. pBK30661 is 74 kb in length, with a mosaic plasmid structure; it exhibits homologies to several other plasmids but lacks the plasmid transfer operon (tra) and the origin of transfer (oriT) that are required for plasmid transfer. pBK30683 is a conjugative plasmid with a cointegrated plasmid structure, comprising a 72-kb element that highly resembles pBK30661 (>99.9% nucleotide identities) and an extra 68-kb element that harbors tra and oriT. A PCR scheme was designed to detect the distribution of blaKPC-harboring IncFIA (pBK30661-like and pBK30683-like) plasmids in a collection of clinical Enterobacteriaceae isolates from 10 hospitals in New Jersey and New York. KPC-harboring IncFIA plasmids were found in 20% of 491 K. pneumoniae isolates, and all carried blaKPC-3. pBK30661-like plasmids were identified mainly in the epidemic sequence type 258 (ST258) K. pneumoniae clone, while pBK30683-like plasmids were widely distributed in ST258 and other K. pneumoniae sequence types and among non-K. pneumoniae Enterobacteriaceae species. This suggests that both clonal spread and horizontal plasmid transfer contributed to the dissemination of blaKPC-harboring IncFIA plasmids in our area. Further studies are needed to understand the distribution of this plasmid group in other health care regions and to decipher the origins of pBK30661-like and pBK30683-like plasmids.
PMCID: PMC4023724  PMID: 24492370
5.  Complete Sequence of a KPC-Producing IncN Multidrug-Resistant Plasmid from an Epidemic Escherichia coli Sequence Type 131 Strain in China 
We report here the nucleotide sequence of a novel blaKPC-2-harboring incompatibility group N (IncN) plasmid, pECN580, from a multidrug-resistant Escherichia coli sequence type 131 (ST131) isolate recovered from Beijing, China. pECN580 harbors β-lactam resistance genes blaKPC-2, blaCTX-M-3, and blaTEM-1; aminoglycoside acetyltransferase gene aac(6′)-Ib-cr; quinolone resistance gene qnrS1; rifampin resistance gene arr-3; and trimethoprim resistance gene dfrA14. The emergence of a blaKPC-2-harboring multidrug-resistant plasmid in an epidemic E. coli ST131 clone poses a significant potential threat in community and hospital settings.
PMCID: PMC4023777  PMID: 24395232
6.  Epidemic Klebsiella pneumoniae ST258 Is a Hybrid Strain 
mBio  2014;5(3):e01355-14.
Carbapenem-resistant Enterobacteriaceae (CRE), especially Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, pose an urgent threat in health facilities in the United States and worldwide. K. pneumoniae isolates classified as sequence type 258 (ST258) by multilocus sequence typing are largely responsible for the global spread of KPC. A recent comparative genome study revealed that ST258 K. pneumoniae strains are two distinct genetic clades; however, the molecular origin of ST258 largely remains unknown, and our understanding of the evolution of the two genetic clades is incomplete. Here we compared the genetic structures and single-nucleotide polymorphism (SNP) distributions in the core genomes of strains from two ST258 clades and other STs (ST11, ST442, and ST42). We identified an ~1.1-Mbp region on ST258 genomes that is homogeneous to that of ST442, while the rest of the ST258 genome resembles that of ST11. Our results suggest ST258 is a hybrid clone—80% of the genome originated from ST11-like strains and 20% from ST442-like strains. Meanwhile, we sequenced an ST42 strain that carries the same K-antigen-encoding capsule polysaccharide biosynthesis gene (cps) region as ST258 clade I strains. Comparison of the cps-harboring regions between the ST42 and ST258 strains (clades I and II) suggests the ST258 clade I strains evolved from a clade II strain as a result of cps region replacement. Our findings unravel the molecular evolution history of ST258 strains, an important first step toward the development of diagnostic, therapeutic, and vaccine strategies to combat infections caused by multidrug-resistant K. pneumoniae.
Recombination events and replacement of chromosomal regions have been documented in various bacteria, and these events have given rise to successful pathogenic clones. Here we used comparative genomic analyses to discover that the ST258 K. pneumoniae genome is a hybrid—80% of the chromosome is homologous to ST11 strains, while the remaining 20% is homologous to that of ST442. Meanwhile, a recent study indicated that ST258 strains can be segregated into two ST258 clades, with distinct capsule polysaccharide gene (cps) regions. Our analysis suggests ST258 clade I strains evolved from clade II through homologous recombination of cps region. Horizontal transfer of the cps region appears to be a key element driving the molecular diversification in K. pneumoniae strains. These findings not only extend our understanding of the molecular evolution of ST258 but are an important step toward the development of effective control and treatment strategies for multidrug-resistant K. pneumoniae.
PMCID: PMC4073492  PMID: 24961694
7.  Complete Nucleotide Sequence of a blaKPC-Harboring IncI2 Plasmid and Its Dissemination in New Jersey and New York Hospitals 
Antimicrobial Agents and Chemotherapy  2013;57(10):5019-5025.
Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains have spread worldwide and become a significant public health threat. blaKPC, the plasmid-borne KPC gene, was frequently identified on numerous transferable plasmids in different incompatibility replicon groups. Here we report the complete nucleotide sequence of a novel blaKPC-3-harboring IncI2 plasmid, pBK15692, isolated from a multidrug-resistant K. pneumoniae ST258 strain isolated from a New Jersey hospital in 2005. pBK15692 is 78 kb in length and carries a backbone that is similar to those of other IncI2 plasmids (pR721, pChi7122-3, pHN1122-1, and pSH146-65), including the genes encoding type IV pili and shufflon regions. Comparative genomics analysis of IncI2 plasmids reveals that they possess a conserved plasmid backbone but are divergent with respect to the integration sites of resistance genes. In pBK15692, the blaKPC-3-harboring Tn4401 was inserted into a Tn1331 element and formed a nested transposon. A PCR scheme was designed to detect the prevalence of IncI2 and pBK15692-like plasmids from a collection of clinical strains from six New Jersey and New York hospitals isolated between 2007 and 2011. IncI2 plasmids were found in 46.2% isolates from 318 clinical K. pneumoniae strains. Notably, 59 pBK15692-like plasmids (23%) have been identified in 256 KPC-bearing K. pneumoniae strains, and all carried KPC-3 and belong to the epidemic ST258 clone. Our study revealed that the prevalence of IncI2 plasmids has been considerably underestimated. Further studies are needed to understand the distribution of this plasmid group in other health care regions and decipher the association between IncI2 plasmids and blaKPC-3-bearing ST258 strains.
PMCID: PMC3811408  PMID: 23896467
8.  Complete Sequence of a blaKPC-2-Harboring IncFIIK1 Plasmid from a Klebsiella pneumoniae Sequence Type 258 Strain 
We report the nucleotide sequence of a novel blaKPC-2-harboring IncFIIK1 plasmid, pBK32179, isolated from a carbapenem-resistant Klebsiella pneumoniae ST258 strain from a New York City patient. pBK32179 is 165 kb long, consists of a large backbone of pKPN3-like plasmid, and carries an 18.5-kb blaKPC-2-containing element that is highly similar to plasmid pKpQIL. pBK32179-like plasmids were identified in 8.3% of strains in a collection of 96 K. pneumoniae isolates from hospitals in the New York City area.
PMCID: PMC3591897  PMID: 23295924
9.  Genetic Variation among Panton-Valentine Leukocidin-Encoding Bacteriophages in Staphylococcus aureus Clonal Complex 30 Strains 
Journal of Clinical Microbiology  2013;51(3):914-919.
Clonal complex 30 (CC30), one of the major Staphylococcus aureus lineages, has caused extensive hospital-acquired and community-acquired infections worldwide. Recent comparative genomics studies have demonstrated that three CC30 clones—phage type 80/81, Southwest Pacific (SWP), and contemporary EMRSA-16 associated (Con) strains—shared a recent common ancestor more than 100 years ago. Panton-Valentine leukocidin (PVL), a bacteriophage encoded toxin that has been epidemiologically linked with community-associated methicillin-resistant S. aureus (CA-MRSA), has frequently been identified in CC30 clones, although the pvl gene variation and distribution of PVL-encoding phages are poorly understood. We determined here the distribution of PVL phages, PVL gene sequences, and chromosomal phage insertion sites in 52 S. aureus CC30 PVL-harboring isolates, collected from four continents over a 75-year period. Our results indicate that PVL phages with icosahedral heads, including Φ108PVL and ΦPVL, were mainly associated with phage 80/81 strains, whereas phages with elongated heads were predominantly found in SWP (ΦSa2958 and ΦTCH60) and Con (ΦSa2USA) strains. Nine single-nucleotide polymorphisms were identified in the lukSF-PV gene, with six isolates harboring the R variant that has been previously associated with CA-MRSA strains. Interestingly, all six R variant strains belonged to the same Con CC30 clone and carried a ΦSa2USA-like phage. Similar chromosomal phage insertion sites were also identified in all 52 PVL-harboring CC30 strains. These analyses provide important insights into the microepidemiology of PVL-harboring CC30 strains, while the discovery of ΦSa2USA-associated R variant strains sheds further light on the evolution of PVL-positive CA-MRSA.
PMCID: PMC3592069  PMID: 23284024
10.  Complete Nucleotide Sequences of blaKPC-4- and blaKPC-5-Harboring IncN and IncX Plasmids from Klebsiella pneumoniae Strains Isolated in New Jersey 
Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have emerged as major nosocomial pathogens. blaKPC, commonly located on Tn4401, is found in Gram-negative bacterial strains, with the two most common variants, blaKPC-2 and blaKPC-3, identified in plasmids with diverse genetic backgrounds. In this study, we examined blaKPC-4- and blaKPC-5-bearing plasmids recovered from two K. pneumoniae strains, which were isolated from a single New Jersey hospital in 2005 and 2006, respectively. IncN plasmid pBK31551 is 84 kb in length and harbors blaKPC-4, blaTEM-1, qnrB2, aac(3)-Ib, aph(3′)-I, qacF, qacEΔ1, sul1, and dfrA14, which confer resistance to β-lactams, quinolones, aminoglycosides, quaternary ammonium compounds, and co-trimoxazole. The conserved regions within pBK31551 are similar to those of other IncN plasmids. Surprisingly, analysis of the Tn4401 sequence revealed a large IS110- and Tn6901-carrying element (8.3 kb) inserted into the istA gene, encoding glyoxalase/bleomycin resistance, alcohol dehydrogenase, and S-formylglutathione hydrolase. Plasmid pBK31567 is 47 kb in length and harbors blaKPC-5, dfrA5, qacEΔ1, and sul1. pBK31567 belongs to a novel IncX subgroup (IncX5) and possesses a highly syntenic plasmid backbone like other IncX plasmids; however, sequence similarity at the nucleotide level is divergent. The blaKPC-5 gene is carried on a Tn4401 element and differs from the genetic environment of blaKPC-5 described in Pseudomonas aeruginosa strain P28 from Puerto Rico. This study underscores the genetic diversity of multidrug-resistant plasmids involved in the spread of blaKPC genes and highlights the mobility and plasticity of Tn4401. Comparative genomic analysis provides new insights into the evolution and dissemination of KPC plasmids belonging to different incompatibility groups.
PMCID: PMC3535970  PMID: 23114770
11.  Epidemiologic Consequences of Microvariation in Mycobacterium tuberculosis 
The Journal of Infectious Diseases  2012;205(6):964-974.
Background. Evidence from genotype-phenotype studies suggests that genetic diversity in pathogens have clinically relevant manifestations that can impact outcome of infection and epidemiologic success. We studied 5 closely related Mycobacterium tuberculosis strains that collectively caused extensive disease (n = 862), particularly among US-born tuberculosis patients.
Methods. Representative isolates were selected using population-based genotyping data from New York City and New Jersey. Growth and cytokine/chemokine response were measured in infected human monocytes. Survival was determined in aerosol-infected guinea pigs.
Results. Multiple genotyping methods and phylogenetically informative synonymous single nucleotide polymorphisms showed that all strains were related by descent. In axenic culture, all strains grew similarly. However, infection of monocytes revealed 2 growth phenotypes, slower (doubling ∼55 hours) and faster (∼25 hours). The faster growing strains elicited more tumor necrosis factor α and interleukin 1β than the slower growing strains, even after heat killing, and caused accelerated death of infected guinea pigs (∼9 weeks vs 24 weeks) associated with increased lung inflammation/pathology. Epidemiologically, the faster growing strains were associated with human immunodeficiency virus and more limited in spread, possibly related to their inherent ability to induce a strong protective innate immune response in immune competent hosts.
Conclusions. Natural variation, with detectable phenotypic changes, among closely related clinical isolates of M. tuberculosis may alter epidemiologic patterns in human populations.
PMCID: PMC3415951  PMID: 22315279
12.  Multiplex Real-Time PCR for Detection of an Epidemic KPC-Producing Klebsiella pneumoniae ST258 Clone 
We describe a multiplex real-time PCR assay capable of identifying both the epidemic Klebsiella pneumoniae ST258 clone and blaKPC carbapenemase genes in a single reaction. The assay displayed excellent sensitivity (100%) and specificity (100%) for identification of ST258 clone and blaKPC in a collection of 75 K. pneumoniae isolates comprising 41 sequence types. Our results suggest that this assay is an effective tool for surveillance of this clone among carbapenem-resistant K. pneumoniae clinical isolates.
PMCID: PMC3370784  PMID: 22450983
13.  Partial Excision of blaKPC from Tn4401 in Carbapenem-Resistant Klebsiella pneumoniae 
We describe a novel Tn4401 variant (Tn4401d) in epidemic Klebsiella pneumoniae clone ST258, from which a partial blaKPC fragment has been excised along with ISKpn7 and a partial tnpA fragment. Nested-PCR experiments confirmed that this region can be removed from distinct Tn4401 isoforms in both K. pneumoniae and Escherichia coli. This study highlights that the region surrounding blaKPC is undergoing recombination and that Tn4401 itself is heterogeneous and highly plastic.
PMCID: PMC3294926  PMID: 22203593
14.  Molecular Characterization of an Early Invasive Staphylococcus epidermidis Prosthetic Joint Infection 
Microbial Drug Resistance  2011;17(3):345-350.
Historically regarded as a skin commensal, Staphylococcus epidermidis has been increasingly implicated in invasive foreign body infections such as catheter-related bloodstream infections, indwelling device infections, and prosthetic joint infections. We report a case of an aggressive, difficult-to-eradicate, invasive prosthetic hip infection occurring early after hardware implant and associated with a high-grade bacteremia and assess its salient molecular characteristics. The clinical and molecular characteristics of this isolate mirror the pathogenesis and persistence commonly seen with invasive methicillin-resistant S. aureus and may be attributed to the combination of resistance genes (SCCmec type IV), putative virulence factors (arcA and opp3a), cytolytic peptide production (α-type phenol-soluble modulins), and biofilm adhesion, interaction, and maturation (bhp, aap, and β-type phenol-soluble modulins).
PMCID: PMC3161624  PMID: 21510745
15.  Methicillin-Susceptible Staphylococcus aureus ST398, New York and New Jersey, USA 
Emerging Infectious Diseases  2012;18(4):700-702.
PMCID: PMC3309677  PMID: 22469250
Staphylococcus aureus; MSSA; ST398; CC398; livestock-associated; human; infection; bacteria; New York; New Jersey; staphylococci; United States
16.  Doripenem, Gentamicin, and Colistin, Alone and in Combinations, against Gentamicin-Susceptible, KPC-Producing Klebsiella pneumoniae Strains with Various ompK36 Genotypes 
Gentamicin doses of 2 and 10 μg/ml were bactericidal against 64% and 100%, respectively, of gentamicin-susceptible KPC-2-producing Klebsiella pneumoniae strains. Treatment with the combination of doripenem (8 μg/ml) plus colistin (2 μg/ml) was inferior to treatment with gentamicin (2 μg/ml), doripenem-gentamicin, gentamicin-colistin, and doripenem-gentamicin-colistin against strains with glycine and aspartic acid insertions in OpmK36 porin at amino acid (aa) positions 134 and 135 (n = 9). Doripenem-colistin was comparable to other 2- or 3-drug regimens and superior to single drugs against wild-type/minor ompK36 mutants (n = 5). An algorithm incorporating ompK36 genotypes and susceptibility to gentamicin and doripenem may predict antimicrobial activity against KPC-producing K. pneumoniae.
PMCID: PMC4068457  PMID: 24566172
17.  Seasonal H3N2 influenza A virus fails to enhance Staphylococcus aureus co-infection in a non-human primate respiratory tract infection model 
Virulence  2013;4(8):707-715.
Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.
PMCID: PMC3925702  PMID: 24104465
Staphylococcus aureus; influenza a virus; coinfection; USA300; MRSA; pneumonia
18.  Tackling antibiotic resistance 
Nature reviews. Microbiology  2011;9(12):894-896.
The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals that is generally not preventable, but can nevertheless be controlled and must be tackled in the most effective ways possible. To explore how the problem of antibiotic resistance might best be addressed, a group of thirty scientists from academia and industry gathered at the Banbury Conference Centre in Cold Spring Harbor, New York, May 16-18, 2011. From these discussions emerged a priority list of steps that need to be taken to resolve this global crisis.
PMCID: PMC4206945  PMID: 22048738
19.  Growth-inhibitory activity of natural and synthetic isothiocyanates against representative human microbial pathogens 
Journal of applied microbiology  2013;115(4):943-954.
The aim of this study was to test the growth inhibition activity of isothiocyanates (ITC), defense compounds of plants, against common human microbial pathogens.
Methods and Results
In this study we have tested the growth inhibitory activity of a diverse collection of new and previously known representative ITC of various structural classes against pathogenic bacteria, fungi and molds by a serial dilution method. Generally, the compounds were more active against Gram-positive bacteria and fungi exhibiting species-specific bacteriostatic or bactericidal effect. The most active compounds inhibited the growth of both drug-susceptible and multi drug resistant (MDR) pathogens at micromolar concentrations. In the case of Mycobacterium tuberculosis some compounds were more active against MDR, rather than against susceptible strains. The average anti-microbial activity for some of new derivatives was significantly higher than previously reported for the most active ITC compounds. The structure-activity relationship (SAR) established for various classes of ITC with Bacillus cereus (model organism for B. anthracis) followed a distinct pattern, thereby enabling prediction of new more efficient inhibitors. Remarkably, tested bacteria failed to develop resistance to ITC. While effectively inhibiting microbial growth, ITCs displayed moderate toxicity towards eukaryotic cells.
High antimicrobial activity coupled with moderate toxicity grants further thorough studies of the ITC compounds aimed at elucidation of their cellular targets and inhibitory mechanism.
Significance and impact of the study
This systematic study identified new ITC compounds highly active against common human microbial pathogens at the concentrations comparable with those for currently used antimicrobial drugs (e.g. rifampicin, fluconazole). Tested representative pathogens do not develop resistance to the inhibitors. These properties justify further evaluation of ITC compounds as potential antimicrobial agents for medicinal use and for industrial applications.
PMCID: PMC3787980  PMID: 23789822
Isothiocyanate; natural; synthetic; bacteria; fungi; growth; inhibition; bacteriostatic; bactericidal
20.  Naturally occurring IgG antibody levels to the Staphylococcus aureus protein IsdB in humans 
Human Vaccines & Immunotherapeutics  2013;9(9):1857-1864.
Staphylococcus aureus is a well-recognized, clinically important cause of nosocomial infections, and as such, a vaccine to prevent S. aureus infections would be an important achievement. A Phase IIB/III study of V710, a vaccine containing iron-regulated surface determinant B (IsdB), demonstrated significant sero-conversion rates in cardiovascular surgery patients following a single pre-surgery immunization. However, the vaccine was not efficacious in preventing bacteremia or deep sternal wound infection post-surgery, thus raising the possibility that IsdB might not be available for immune recognition during infection. The purpose of the work described herein was to evaluate and quantify the naturally occurring anti-IsdB levels at baseline and over time during infection, to understand whether IsdB is expressed during a S. aureus infection in hospitalized non-vaccinated patients. We evaluated baseline and follow-up titers in 3 populations: (1) healthy subjects, (2) hospitalized patients with non-S. aureus infections, and (3) hospitalized patients with S. aureus infections. Baseline anti-IsdB levels generally overlapped between the 3 groups, but were highly variable within each group. In healthy subjects, baseline and follow-up levels were highly correlated (Spearman's rho = 0.93), and the geometric mean fold-rise (GMFR) in anti-IsdB levels between study entry and last value was 0.9-fold (95% confidence interval (CI): 0.8 to 1.0 ; p = 0.09), showing no trend over time. The convalescent GMFR in anti-IsdB levels from baseline was 1.7-fold (95% CI: 1.3 to 2.2, p = 0.0008) during S. aureus infection, significantly different from the 1.0-fold GMFR (95% CI: 0.9–1.2, p = 0.60) in non-S. aureus infection, p = 0.005. Additionally, S. aureus isolates (51) obtained from the hospitalized patient group expressed the IsdB protein in vitro. Collectively, these data suggest that IsdB expression levels rise substantially following infection with S. aureus, but not with other pathogens, and IsdB is likely well-conserved across S. aureus strains.
PMCID: PMC3906349  PMID: 23778314
S. aureus infection; IsdB; antibody levels; healthy subjects; hospitalized patients
21.  Production of an Attenuated Phenol-Soluble Modulin Variant Unique to the MRSA Clonal Complex 30 Increases Severity of Bloodstream Infection 
PLoS Pathogens  2014;10(8):e1004298.
Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of morbidity and death. Phenol-soluble modulins (PSMs) are recently-discovered toxins with a key impact on the development of Staphylococcus aureus infections. Allelic variants of PSMs and their potential impact on pathogen success during infection have not yet been described. Here we show that the clonal complex (CC) 30 lineage, a major cause of hospital-associated sepsis and hematogenous complications, expresses an allelic variant of the PSMα3 peptide. We found that this variant, PSMα3N22Y, is characteristic of CC30 strains and has significantly reduced cytolytic and pro-inflammatory potential. Notably, CC30 strains showed reduced cytolytic and chemotactic potential toward human neutrophils, and increased hematogenous seeding in a bacteremia model, compared to strains in which the genome was altered to express non-CC30 PSMα3. Our findings describe a molecular mechanism contributing to attenuated pro-inflammatory potential in a main MRSA lineage. They suggest that reduced pathogen recognition via PSMs allows the bacteria to evade elimination by innate host defenses during bloodstream infections. Furthermore, they underscore the role of point mutations in key S. aureus toxin genes in that adaptation and the pivotal importance PSMs have in defining key S. aureus immune evasion and virulence mechanisms.
Author Summary
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality and a great concern for public health. The CC30 MRSA lineage is especially notorious for causing bloodstream infections with complications such as seeding into organs. In our study, we show that this lineage produces an attenuated form of a key S. aureus toxin with decreased pro-inflammatory features. Our results suggest that attenuation of this toxin allows the bacteria to evade recognition and subsequent elimination by host defenses, thereby increasing pathogen success during blood infection.
PMCID: PMC4140855  PMID: 25144687
22.  Death of Woman with Peripartum Influenza B Virus Infection and Necrotizing Pneumonia 
Emerging Infectious Diseases  2014;20(7):1258-1260.
PMCID: PMC4073880  PMID: 24960253
Influenza; Methicillin-Resistant Staphylococcus aureus; MRSA; Panton-Valentine leukocidin; pneumonia; pregnancy; peripartum; Respiratory Distress Syndrome; antibiotic; antimicrobial drug
23.  Mutations of the ompK36 Porin Gene and Promoter Impact Responses of Sequence Type 258, KPC-2-Producing Klebsiella pneumoniae Strains to Doripenem and Doripenem-Colistin 
Antimicrobial Agents and Chemotherapy  2013;57(11):5258-5265.
Doripenem-colistin exerts synergy against some, but not all, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains in vitro. We determined if doripenem MICs and/or ompK36 porin gene mutations impacted the responses of 23 sequence type 258 (ST258), KPC-2-producing strains to the combination of doripenem (8 μg/ml) and colistin (2 μg/ml) during time-kill assays. The median doripenem and colistin MICs were 32 and 4 μg/ml. Doripenem MICs did not correlate with KPC-2 expression levels. Five and 18 strains had wild-type and mutant ompK36, respectively. The most common mutations were IS5 promoter insertions (n = 7) and insertions encoding glycine and aspartic acid at amino acid (aa) positions 134 and 135 (ins aa134-135 GD; n = 8), which were associated with higher doripenem MICs than other mutations or wild-type ompK36 (all P values ≤ 0.04). Bactericidal activity (24 h) was achieved by doripenem-colistin against 12%, 43%, and 75% of ins aa134-135 GD, IS5, and wild-type/other mutants, respectively (P = 0.04). Doripenem-colistin was more active in time-kill studies than colistin at 12 and 24 h if the doripenem MIC was ≤8 μg/ml (P = 0.0007 and 0.09, respectively), but not if the MIC was >8 μg/ml (P = 0.10 and 0.16). Likewise, doripenem-colistin was more active at 12 and 24 h against the wild type/other mutants than ins aa134-135 GD or IS5 mutants (P = 0.007 and 0.0007). By multivariate analysis, the absence of ins aa134-135 GD or IS5 mutations was the only independent predictor of doripenem-colistin responses at 24 h (P = 0.002). In conclusion, ompK36 genotypes identified ST258 KPC-K. pneumoniae strains that were most likely to respond to doripenem-colistin.
PMCID: PMC3811244  PMID: 23939888
24.  Active Surveillance for Carbapenem-Resistant Enterobacteriaceae Using Stool Specimens Submitted for Testing for Clostridium difficile 
Active surveillance to identify asymptomatic carriers of carbapenem-resistant Enterobacteriaceae (CRE) is a recommended strategy for CRE control in healthcare facilities. Active surveillance using stool specimens tested for Clostridium difficile is a relatively low-cost strategy to detect CRE carriers. Further evaluation of this and other risk factor–based active surveillance strategies is warranted.
PMCID: PMC3984911  PMID: 24334803
25.  Genomic Analysis Identifies Targets of Convergent Positive Selection in Drug Resistant Mycobacterium tuberculosis 
Nature genetics  2013;45(10):10.1038/ng.2747.
Mycobacterium tuberculosis is successfully evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including the genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly and 7 previously sequenced M. tuberculosis genomes, we identified genomewide signatures of positive selection specific to the 47 resistant genomes. By searching for convergent evolution, the independent fixation of mutations at the same nucleotide site or gene, we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode pathways of cell wall biosynthesis, transcriptional regulation and DNA repair. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.
PMCID: PMC3887553  PMID: 23995135

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