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Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen that has diverse molecular heterogeneity. Most MRSA strains in the United States are pulsed-field gel electrophoresis USA100 sequence type (ST) 5 and USA300 ST8. Infections with MRSA ST239-III are common and found during health care-associated outbreaks. However, this strain has been rarely reported in the United States. As part of a study supported by the Prevention Epicenter Program of the Centers for Disease Control and Prevention (Atlanta, GA, USA), which evaluated transmission of MRSA among hospitals in Ohio, molecular typing identified 78 (6%) of 1,286 patients with MRSA ST239-III infections. Ninety-five percent (74/78) of these infections were health care associated, and 65% (51/78) of patients had histories of invasive device use. The crude case-fatality rate was 22% (17/78). Identification of these strains, which belong to a virulent clonal group, emphasizes the need for molecular surveillance. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus infection and colonization in patients may be transmitted to healthcare providers and the environment and subsequently cause healthcare-associated infections in other patients. Pathogenic S. aureus strains produce virulence factors, such as Panton-Valentine Leukocidin (PVL), that contribute to the severity of infections and aid in their spread. The emergence of antimicrobial resistance (AMR) is additional concern with respect to S. aureus infection. In this study, the virulence genes and antibiotic resistance profiles of S. aureus were characterized from patients' clinical isolates, healthcare workers' (HCWs') nasal colonization screenings, and the environment at a tertiary healthcare hospital in Addis Ababa, Ethiopia. A total of 365 samples were collected from September 2021 to September 2022: 73 patients' clinical specimens, 202 colonization screenings from HCWs, and 90 hospital environment's swabs. Fifty-one (25.2%) HCW and 10/90 (11.1%) environment S. aureus isolates were identified. Among the 134 isolates, 10 (7.5%) were methicillin-resistant S. aureus (MRSA). Three (4.1%), five (9.8%), and two (20.0%) of the MRSA isolates were identified from the patients, HCWs, and the environment, respectively. Overall, 118 (88.1%) were ampicillin and penicillin resistant; 70 (52.2%) were trimethoprim sulfamethoxazole resistant; and 28 (20.9%) were erythromycin resistant. S. aureus isolates from patients were more resistant to antibiotics than isolates from HCWs or the hospital environment (p<0.05). A total of 92/134 (68.6%) isolates possessed the lukfF-PV gene, which was identified in 62 (85.0%), 26 (51.0%), and 4 (40.0%) of the patient, HCWs, and the environment, respectively. The proportion of lukfF-PV gene containing S. aureus isolated from patient samples was statistically significant. Four (40.0%) of the MRSA isolates also had the lukfF-PV gene. The identification of highly AMR and virulence factors from patients, HCWs and the environment is concerning. Further studies are needed to identify potential transmission links and improve infection prevention and control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Purpose: To identify the predictors of morbidity and mortality in matched COVID-19 positive and negative patients who were septic with Gram positive or Gram negative infections., Methods: We conducted a retrospective review, from March to October 2020, of matched septic patients at five Hackensack Meridian Health hospitals who had bacteremia with Staphylococcus aureus, Klebsiella pneumoniae or Escherichia coli with and without COVID-19. We extracted patient demographics, comorbidities and clinical outcomes data using ICD-10 codes. Bacterial isolates were compared by whole genome sequencing analysis. Multivariate logistic regression was used to analyze independent predictors of morbidity and mortality., Results: A total of 208 patients were grouped by positive bloodstream infection (BSI) with COVID-19 (n = 104) and without COVID-19 (n = 104). Most patients were over age 50 (90% vs. 89%) and Caucasian (78% vs. 86%). Inpatient mortality was higher in patients with COVID-19 for both GP (35% vs. 8%, p < 0.05) and GN (28% vs. 10%, p < 0.05) BSIs. Patients with Gram positive (GP) BSIs had a significant increase in mortality risk (OR 4.5, CI 1.4-14.5, p < 0.05) in contrast to those with Gram negative (GN) infections (OR 0.4, CI 0.4-4.0, p = 0.4)., Conclusion: Concurrent COVID-19 infection is associated with a significant increase in morbidity and mortality in patients with GP and GN BSIs. Patients with S. aureus BSIs with COVID-19 are more likely to develop shock and respiratory failure and have higher rates and odds of mortality than those without COVID-19. These findings provide an essential insight into the care of these patients, especially those co-infected with Staphylococcus aureus., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

New Jersey was among the first states impacted by the COVID-19 pandemic, with one of the highest overall death rates in the nation. Nevertheless, relatively few reports have been published focusing specifically on New Jersey. Here we report on molecular, clinical, and epidemiologic observations, from the largest healthcare network in the state, in a cohort of vaccinated and unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection. We conducted molecular surveillance of SARS-CoV-2-positive nasopharyngeal swabs collected in nine hospitals from December 2020 through June 2022, using both whole genome sequencing (WGS) and a real-time RT-PCR screening assay targeting spike protein mutations found in variants of concern (VOCs) within our region. De-identified clinical data were obtained retrospectively, including demographics, COVID-19 vaccination status, ICU admission, ventilator support, mortality, and medical history. Statistical analyses were performed to identify associations between SARS-CoV-2 variants, vaccination status, clinical outcomes, and medical risk factors. A total of 5007 SARS-CoV-2-positive nasopharyngeal swabs were successfully screened and/or sequenced. Variant screening identified three predominant VOCs, including Alpha ( n = 714), Delta ( n = 1877), and Omicron ( n = 1802). Omicron isolates were further sub-typed as BA.1 ( n = 899), BA.2 ( n = 853), or BA.4/BA.5 ( n = 50); the remaining 614 isolates were classified as "Other". Approximately 31.5% (1577/5007) of the samples were associated with vaccine breakthrough infections, which increased in frequency following the emergence of Delta and Omicron. Severe clinical outcomes included ICU admission (336/5007 = 6.7%), ventilator support (236/5007 = 4.7%), and mortality (430/5007 = 8.6%), with increasing age being the most significant contributor to each ( p < 0.001). Unvaccinated individuals accounted for 79.7% (268/336) of ICU admissions, 78.3% (185/236) of ventilator cases, and 74.4% (320/430) of deaths. Highly significant ( p < 0.001) increases in mortality were observed in individuals with cardiovascular disease, hypertension, cancer, diabetes, and hyperlipidemia, but not with obesity, thyroid disease, or respiratory disease. Significant differences ( p < 0.001) in clinical outcomes were also noted between SARS-CoV-2 variants, including Delta, Omicron BA.1, and Omicron BA.2. Vaccination was associated with significantly improved clinical outcomes in our study, despite an increase in breakthrough infections associated with waning immunity, greater antigenic variability, or both. Underlying comorbidities contributed significantly to mortality in both vaccinated and unvaccinated individuals, with increasing risk based on the total number of comorbidities. Real-time RT-PCR-based screening facilitated timely identification of predominant variants using a minimal number of spike protein mutations, with faster turnaround time and reduced cost compared to WGS. Continued evolution of SARS-CoV-2 variants will likely require ongoing surveillance for new VOCs, with real-time assessment of clinical impact.

Staphylococcus aureus is among the top three causative agents of nosocomial infection in Ethiopia. The majority of studies in Ethiopia have focused on the epidemiology of S. aureus in hospital settings, with limited molecular genotyping results. Molecular characterization of S. aureus is essential for identification of strains, and contributes to the control and prevention of S. aureus infection. The aim of the current study was to determine the molecular epidemiology of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from clinical specimens in Ethiopia. A total of 161 MSSA and 9 MRSA isolates were characterized using pulsed-field gel electrophoresis (PFGE) and staphylococcal protein A (spa) typing. Based on the PFGE analysis, MSSA isolates were grouped into eight pulso-types groups (from A to I), while MRSA isolates clustered into three (A, B and C) pulso-types with more than 80% similarity. The spa typing analysis showed diversity of S. aureus with 56 distinct spa types. Spa type t355 was most prevalent (56/170, 32.9%), while eleven new spa types were detected including t20038, t20039, and t20042. The identified spa types were clustered into 15 spa-clonal complexes (spa-CCs) using BURP analysis; novel/unknown spa types were further subjected to MLST analysis. The majority of isolates belonged to spa-CC 152 (62/170, 36.4%), followed by spa-CC 121 (19/170, 11.2%), and spa-CC 005 (18 /170, 10.6%). Of the nine MRSA isolates, 2 (22.2%) were spa-CC 239 with staphylococcal cassette chromosome (SCC)mec III. These findings highlight the diversity of S. aureus strains in Ethiopia, as well as the presence of potentially epidemic strains circulating in the country necessitating further characterization of S. aureus for antimicrobial resistance detection and infection prevention purposes., (© 2023. The Author(s).)

Examining the neutralizing capacity of monoclonal antibodies (MAbs) used to treat COVID-19, as well as antibodies recovered from unvaccinated, previously vaccinated, and infected individuals, against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) remains critical to study. Here, we report on a SARS-CoV-2 nosocomial outbreak caused by the SARS-CoV-2 R.1 variant harboring the E484K mutation in a 281-bed psychiatric facility in New Jersey among unvaccinated inpatients and health care professionals (HCPs). A total of 81 inpatients and HCPs tested positive for SARS-Cov-2 by reverse transcription (RT)-PCR from 29 October 9 to 30 November 2020. The R.1 variant exhibits partial or complete resistance to two MAbs in clinical use, as well as 2 receptor binding domain MAbs and 4 N-terminal domain (NTD) MAbs. NTD MAbs against pseudovirus harboring single characteristic R.1 mutations highlight the role of S255F in loss of activity. Additionally, we note dampened neutralization capacity by plasma from individuals with previous SARS-CoV-2 infection or sera from vaccinated individuals. The relative resistance of the R.1 variant is likely lower than that of B.1.351 and closer to that of P.1 and B.1.526. The R.1 lineage has been reported in 47 states in the United States and 40 countries. Although high proportions exhibited symptoms (26% and 61% among patients and HCPs, respectively) and relative antibody resistance, we detected only 10 R.1 variants from over 2,900 samples (~0.34%) collected from January to October 2021. Among 3 vaccinated individuals previously infected with R.1, we observed robust neutralizing antibody responses against SARS-CoV-2 wild type and VOCs. IMPORTANCE The neutralizing capacities of monoclonal antibodies used to treat COVID-19 and of those recovered from previously infected and vaccinated individuals against SARS-CoV-2 variants of concern (VOCs) remain important questions. We report on a nosocomial outbreak caused by a SARS-CoV-2 R.1 variant harboring an E484K mutation among 81 unvaccinated inpatients and health care professionals. We note high attack rates with symptoms in nearly 50% of infected individuals, in sharp contrast to an unrelated institutional outbreak caused by the R.1 variant among a vaccinated population. We found little evidence of significant community spillover. This variant exhibits partial or complete resistance to two monoclonal antibodies in clinical use and dampened the neutralization capacity of convalescent-phase plasma from individuals with previous infection or sera from vaccinated individuals. Among three vaccinated individuals previously infected with R.1, we observed robust neutralizing antibody responses against SARS-CoV-2 wild type and VOCs. These findings underscore the importance of vaccination for prevention of symptomatic COVID-19 disease.

Emergence of SARS-CoV-2 with high transmission and immune evasion potential, the so-called variants of concern (VOC), is a major concern. We describe the early genomic epidemiology of SARS-CoV-2 recovered from vaccinated health care professionals (HCP). Our postvaccination COVID-19 symptoms-based surveillance program among HCPs in a 17-hospital network identified all vaccinated HCPs who tested positive for COVID-19 after routine screening or after self-reporting. From 1 January 2021 to 30 April 2021, 23,687 HCPs received either mRNA-1273 or BNT162b2 mRNA vaccine. All available postvaccination SARS-CoV-2 samples and a random collection from nonvaccinated patients during the similar time frame were subjected to VOC screening and whole-genome sequencing (WGS). Sixty-two percent (23,697/37,500) of HCPs received at least one vaccine dose, with 60% (22,458) fully vaccinated. We detected 138 (0.58%, 138/23,697) COVID-19 cases, 105 among partially vaccinated and 33 (0.15%, 33/22,458) among fully vaccinated. Five partially vaccinated required hospitalization, four with supplemental oxygen. VOC screening from 16 fully vaccinated HCPs identified 6 (38%) harboring N501Y and 1 (6%) with E484K polymorphisms; percentage of concurrent nonvaccinated samples was 37% (523/1,404) and 20% (284/1,394), respectively. There was an upward trend from January to April for E484K/Q (3% to 26%) and N501Y (1% to 49%). WGS analysis from vaccinated and nonvaccinated individuals indicated highly congruent phylogenies. We did not detect an increased frequency of any receptor-binding domain (RBD)/N-terminal domain (NTD) polymorphism between groups ( P  > 0.05). Our results support robust protection by vaccination, particularly among recipients of both doses. Despite VOCs accounting for over 40% of SARS-CoV-2 from fully vaccinated individuals, the genomic diversity appears to proportionally represent VOCs among nonvaccinated populations. IMPORTANCE A number of highly effective vaccines have been developed and deployed to combat the COVID-19 pandemic. The emergence and epidemiological dominance of SARS-CoV-2 mutants with high transmission potential and immune evasion properties, the so-called variants of concern (VOC), continue to be a major concern. Whether these VOCs alter the efficacy of the administered vaccines is of great concern and a critical question to study. We describe the initial genomic epidemiology of SARS-CoV-2 recovered from partial/fully vaccinated health care professionals and probe specifically for VOC enrichment. Our findings support the high level of protection provided by full vaccination despite a steep increase in the prevalence of polymorphisms associated with increased transmission potential (N501Y) and immune evasion (E484K) in the nonvaccinated population. Thus, we do not find evidence of VOC enrichment among vaccinated groups. Overall, the genomic diversity of SARS-CoV-2 recovered postvaccination appears to proportionally represent the observed viral diversity within the community.

This study reported the involvement of a gene cluster from a conjugative plasmid in the biofilm formation of Escherichia coli . We used a novel EZ-Tn 5 transposon technique to generate a transposon library and used arbitrarily primed PCR to detect the insertion sites in biofilm formation-deficient mutants. To validate the function of candidate biofilm formation genes, the genes were cloned into plasmid pBluescript II SK (+) and transformed into E. coil DH5α. Biofilm production from the transformants was then assessed by phenotypic biofilm formation using Crystal Violet staining and microscopy. A total of 3,000 transposon mutants of E. coli DH5α-p253 were screened, of which 28 were found to be deficient in biofilm formation. Further characterization revealed that 24/28 mutations were detected with their insertions in chromosome, while the remaining 4 mutations were evidenced that the functional genes for biofilm formation were harbored in the plasmid. Interestingly, the plasmid sequencing showed that these four transposon mutations were all inserted into a fimbriae-associated gene cluster ( fim -cluster). This fim -cluster is a hybrid segment spanning a 7,949 bp sequence, with a terminal inverted repeat sequence and two coding regions. In summary, we performed a high-efficiency screening to a library constructed with the EZ-Tn5-based transposon approach and identified the gene clusters responsible for the biofilm production of E. coli , especially the genes harbored in the plasmid. Further studies are needed to understand the spread of this novel plasmid-mediated biofilm formation gene in clinical E. coli isolates and the clinical impacts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs), harboring spike protein N-terminal domain (NTD) or receptor-binding domain (RBD) mutations, exhibit reduced in vitro susceptibility to convalescent-phase serum, commercial antibody cocktails, and vaccine neutralization and have been associated with reinfections. The accumulation of these mutations could be the consequence of intrahost viral evolution due to prolonged infection in immunocompromised hosts. In this study, we document the microevolution of SARS-CoV-2 recovered from sequential tracheal aspirates from an immunosuppressed patient on steroids and convalescent plasma therapy and identify the emergence of multiple NTD and RBD mutations. SARS-CoV-2 genomes from the first swab (day 0) and from three tracheal aspirates (days 7, 21, and 27) were compared at the sequence level. We identified a mixed viral population with five different S protein mutations (141 to 144 deletion, 243 to 244 deletion, E484K, Q493K, and Q493R) at the NTD or RBD region from the second tracheal aspirate sample (day 21) and a predominance of the S protein 141 to 144 LGVY deletion and E484K mutant on day 27. The neutralizing antibodies against various S protein lentiviral pseudovirus mutants, as well as the anti-SARS-CoV-2 total Ig and IgG, showed "U" shape dynamics, in support of the endogenous development of neutralizing antibodies. The patient's compromised immune status, the antirejection regiment, convalescent plasma treatment, and the development of neutralizing antibodies may have resulted in unique selective pressures on the intrahost genomic evolution, and this observation supports the hypotheses that VOCs can independently arise and that immunocompromised patients on convalescent plasma therapy are potential breeding grounds for immune escape mutants. IMPORTANCE Over a year of the COVID-19 pandemic, distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages have arisen in multiple geographic areas around the world. SARS-CoV-2 variants of concern (VOCs), i.e., B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), and B.1.617.2 (delta), harboring mutations and/or deletions in spike protein N-terminal domain (NTD) or receptor-binding domain (RBD) regions showed evidence of increased transmissibility and disease severity and possible reduced vaccine efficacy. In this study, we report the emergence of five different NTD and RBD mutations in an uncommon SARS-CoV-2 B.1.369 lineage from an immunosuppressed patient undergoing steroid and convalescent plasma therapy. The observation highlighted that VOCs can independently arise in immunocompromised populations undergoing anti-SARS-CoV-2 therapy, and enhanced measures will be required to reduce the transmission.

Fungal infections are common complications of respiratory viral infections and are associated with the increased need for intensive care and elevated mortality. Data regarding microbiological and molecular characteristics of such infections in COVID-19 patients are scarce. Here, we performed a comprehensive analysis, including species identification, antifungal susceptibility testing, molecular resistance determinants analysis, typing, and retrospective clinical data review, of fungal isolates recovered from 19 COVID-19 patients, who were hospitalized at the Hackensack University Medical Center (HUMC) in Hackensack, New Jersey, USA, in the initial phase of the pandemic from April-May 2020. In total, 17 Candida albicans , two C. parapsilosis , and two Aspergillus fumigatus were analyzed. All Candida spp. isolates were susceptible to micafungin and azole drugs (fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole). A. fumigatus isolates were susceptible to micafungin and all triazole drugs except fluconazole (intrinsic resistance). Multilocus sequence typing (MLST) of C. albicans isolates revealed 15 different sequence types (STs), which clustered below the clade-defining limit of p-distance < 0.04. Pulsed-field gel electrophoresis (PFGE) karyotyping revealed no chromosomal rearrangements in these isolates. A. fumigatus isolates were of different, non-related genotypes. We speculate that virus- and drug-induced immunosuppression (94.7% of the patients received corticosteroids), together with prolonged hospital stay (median duration of 29 days) and mechanical ventilation (median duration of 24 days) likely increased the susceptibility to secondary respiratory and bloodstream infections in the studied patient population. The presence of fungi in blood or respiratory tract fluid was a prognosticator for poor clinical outcome, which presented as an 89.5% 30-day mortality in our patient cohort.

New Jersey was an early epicenter for the COVID-19 pandemic in the United States, yet information on hospitalized COVID-19 patients from this area is scarce. This study aimed to provide data on demographics and clinical features of a hospitalized patient population who were confirmed with infection by our in-house (CDI) real-time reverse-transcription polymerase chain reaction (RT-PCR) test. We included consecutive patients who were admitted to Hackensack Meridian Health system hospitals with laboratory-confirmed diagnoses of COVID-19 at Hackensack University Medical Center by the CDI virus test between March 12, 2020, and April 8, 2020. Clinical data and viral testing results were collected and analyzed for characteristics associated with outcomes, as well as the correlation with viral load. A total of 722 patients were included in the study, with a median age of 63 (interquartile range (IQR), 51-75) and 272 (37.7%) females. Mortality of this case series was 25.8%, with a statistically significant linear increase observed from age 40 to ≥ 80 by 10-year intervals. Viral load, as indicated by the cycle of threshold (Ct) values from the RT-PCR test, was significantly higher in the oldest patient group (≥ 80), and inversely correlated with survival. This is the first report to describe the clinical characteristics and outcomes in a large hospitalized COVID-19 patient series from New Jersey. Findings from this study are valuable to the ongoing response of both nationwide healthcare networks and the medical research community.

The treatment of bacterial infections is hindered by the presence of biofilms and metabolically inactive persisters. Here, we report the synthesis of an enantiomeric block co-beta-peptide, poly(amido-D-glucose)-block-poly(beta-L-lysine), with high yield and purity by one-shot one-pot anionic-ring opening (co)polymerization. The co-beta-peptide is bactericidal against methicillin-resistant Staphylococcus aureus (MRSA), including replicating, biofilm and persister bacterial cells, and also disperses biofilm biomass. It is active towards community-acquired and hospital-associated MRSA strains which are resistant to multiple drugs including vancomycin and daptomycin. Its antibacterial activity is superior to that of vancomycin in MRSA mouse and human ex vivo skin infection models, with no acute in vivo toxicity in repeated dosing in mice at above therapeutic levels. The copolymer displays bacteria-activated surfactant-like properties, resulting from contact with the bacterial envelope. Our results indicate that this class of non-toxic molecule, effective against different bacterial sub-populations, has promising potential for the treatment of S. aureus infections.

Background: Overcoming β-lactam resistance in pathogens such as Pseudomonas aeruginosa is a major clinical challenge. Rapid molecular diagnostics (RMDs) have the potential to inform selection of empiric therapy in patients infected by P. aeruginosa., Methods: In this study, we used a heterogeneous collection of 197 P. aeruginosa that included multidrug-resistant isolates to determine whether 2 representative RMDs (Acuitas Resistome test and VERIGENE gram-negative blood culture test) could identify susceptibility to 2 newer β-lactam/β-lactamase inhibitor (BL-BLI) combinations, ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (TOL/TAZO)., Results: We found that the studied RMD platforms were able to correctly identify BL-BLI susceptibility (susceptibility sensitivity, 100%; 95% confidence interval [CI], 97%, 100%) for both BLs-BLIs. However, their ability to detect resistance to these BLs-BLIs was lower (resistance sensitivity, 66%; 95% CI, 52%, 78% for TOL/TAZO and 33%; 95% CI, 20%, 49% for CZA)., Conclusions: The diagnostic platforms studied showed the most potential in scenarios where a resistance gene was detected or in scenarios where a resistance gene was not detected and the prevalence of resistance to TOL/TAZO or CZA is known to be low. Clinicians need to be mindful of the benefits and risks that result from empiric treatment decisions that are based on resistance gene detection in P. aeruginosa, acknowledging that such decisions are impacted by the prevalence of resistance, which varies temporally and geographically., (Published by Oxford University Press for the Infectious Diseases Society of America 2018.)

The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is increasing globally, with different molecular mechanisms described. Here we studied the molecular mechanisms of carbapenem resistance, including clonal and plasmid dissemination, of 67 CRE isolates collected between 2012 and 2016 from a tertiary hospital in Eastern China, an CRE endemic region. Species identification and susceptibility testing were performed using the BD Phoenix Automated Microbiology System. Isolates were characterized by PCR (for carbapenemases, ESBLs, AmpC and porin genes), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and conjugation transfer experiments. Selected bla KPC-2 -harboring plasmids were subjected to next-generation sequencing using the Illumina Miseq platform. Among the 67 CRE isolates, 42 Klebsiella pneumoniae , 10 Serratia marcescens , 6 Enterobacter cloacae , 2 Raoultella ornithinolytica , 2 K. oxytoca , 1 K. aerogenes , and 4 Escherichia coli isolates were identified. Six different carbapenemases were detected, including bla KPC-2 ( n = 45), bla KPC-3 ( n = 1), bla NDM-1 ( n = 6), bla NDM-5 ( n = 1), bla IMP-4 ( n = 2), and bla VIM-1 ( n = 2); bla OXA-48 -like genes were not detected. One E. cloacae strain possessed both bla NDM-1 and bla KPC-3 , while two E. cloacae isolates harbored bla NDM-1 and bla VIM-1 . ESBLs (CTX-M, SHV, and TEM) and/or AmpC (CMY, DHA, and ACT/MIR) genes were also identified in 59 isolates, including 13 strains that lacked carbapenemases. Several insertions or stop codon mutations were found within porin genes of K. pneumoniae, E. coli and S. marcescens isolates, both with and without carbapenemases. The 42 K. pneumoniae isolates belonged to 12 different sequence types (ST), with ST11 being the most common, while the 6 E. cloacae isolates comprised 4 different STs. The 10 S. marcescens all shared the same PFGE pulsotype, suggestive of clonal spread. Complete plasmid sequencing and PCR screening revealed both intra-strain and inter-species spread of a common bla KPC-2 -harboring plasmid in our hospital. Taken together, our study revealed extensive genetic diversity among CRE isolates form a single Chinese hospital. CRE isolates circulating in the hospital differ significantly in their species, STs, porin genes, carbapenemase genes, and their plasmid content, highlighting the complex dissemination of CRE in this endemic region.

The USA300 North American epidemic (USA300-NAE) clone of methicillin-resistant Staphylococcus aureus has caused a wave of severe skin and soft tissue infections in the United States since it emerged in the early 2000s, but its geographic origin is obscure. Here we use the population genomic signatures expected from the serial founder effects of a geographic range expansion to infer the origin of USA300-NAE and identify polymorphisms associated with its spread. Genome sequences from 357 isolates from 22 U.S. states and territories and seven other countries are compared. We observe two significant signatures of range expansion, including decreases in genetic diversity and increases in derived allele frequency with geographic distance from the Pennsylvania region. These signatures account for approximately half of the core nucleotide variation of this clone, occur genome wide, and are robust to heterogeneity in temporal sampling of isolates, human population density, and recombination detection methods. The potential for positive selection of a gyrA fluoroquinolone resistance allele and several intergenic regions, along with a 2.4 times higher recombination rate in a resistant subclade, is noted. These results are the first to show a pattern of genetic variation that is consistent with a range expansion of an epidemic bacterial clone, and they highlight a rarely considered but potentially common mechanism by which genetic drift may profoundly influence bacterial genetic variation. IMPORTANCE The process of geographic spread of an origin population by a series of smaller populations can result in distinctive patterns of genetic variation. We detect these patterns for the first time with an epidemic bacterial clone and use them to uncover the clone's geographic origin and variants associated with its spread. We study the USA300 clone of methicillin-resistant Staphylococcus aureus , which was first noticed in the early 2000s and subsequently became the leading cause of skin and soft tissue infections in the United States. The eastern United States is the most likely origin of epidemic USA300. Relatively few variants, which include an antibiotic resistance mutation, have persisted during this clone's spread. Our study suggests that an early chapter in the genetic history of this epidemic bacterial clone was greatly influenced by random subsampling of isolates during the clone's geographic spread., (Copyright © 2018 Challagundla et al.)

We characterized spa types, SCCmec types, and antimicrobial resistance patterns of 516 methicillin-resistant Staphylococcus aureus (MRSA) isolates, collected between 2011 and 2014 from nares and blood cultures of United States patients. Among nares isolates, 45 spa types were observed; 29.9% were t002/SCCmec II and 30.9% were t008/SCCmec IV. Among blood isolates, 40 spa types were identified; 24.4% were t002/SCCmec II and 39.9% were type t008/SCCmec IV. Compared to data from our 2009-2010 survey, the percentage of t008/SCCmec IV isolates from nares increased significantly (20.4%-30.9%; P=0.004) while the percentage from positive blood cultures remained similar (39.2% versus 39.9%; P=0.921). There were also significant changes in the overall antimicrobial resistance patterns observed, including the decrease of the clindamycin, erythromycin, levofloxacin and moxifloxacin multidrug resistance pattern, likely the result of t002/SCCmec II strains being displaced by t008/SCCmec IV strains. Rates of high-level mupirocin resistance did not change significantly from our past study (4.1% compared to 4.7%; P=0.758) but an increase in low-level resistance, particularly among t002/SCCmec II isolates, was observed., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA), is one of the most prevalent clinical pathogens isolated from hospital settings, and has increasingly identified in community settings. In China, the SCC mec III-ST239 strains are disseminated in different geographic regions, accounting for >75% of all MRSA isolates in some national studies. Here we characterized 150 non-duplicate MRSA isolates collected from February 2012 to May 2013 in a tertiary hospital in Suzhou, Eastern China, to explore the molecular epidemiology. All isolates were characterized by spa typing, SCC mec typing, and detection of genes encoding Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin (TSST-1). Representative genotypes were also subjected to multilocus sequence typing (MLST). Antibiotic susceptibility testing was performed using BD Phoenix™ Automated Microbiology System. Molecular typing identified 11 clonal complex (CC) and 28 spa types, with the CC5- spa t002 (29.3%) and CC239- spa t037 (14.7%) being the most prevalent. SCC mec types II, III, IV, and V were identified in 33.3, 21.3, 23.3, and 21.3% of all isolates, respectively. PVL genes ( lukF/S-PV ) were detected in 11.3% of all isolates and from 6 CCs (5, 8, 59, 88, 239, and 398). The TSST-1 gene ( tst ) was detected in 18.0% of the all isolates, predominantly in CC5 (96.3%). All the tst-1 -positve CC5 isolates were spa t002. Eighteen patients died within 30 days of hospitalization, and the in-hospital 30-day mortality was 12.0%. Multivariable analysis showed that 60 years old (odds ratio [OR] = 7.2, P = 0.026), cancer diagnosis (OR = 9.6, P = 0.022), and MRSA isolate carriage of tst-1 (OR = 62.5, P < 0.001) were independent factors associated with 30-day mortality. Our study revealed unique MRSA dissemination patterns in our hospital in comparison to those of other regions in China. The finding that tst-1 -positive CC5 strains were associated with higher mortality highlights the need for strict infection control measures in order to prevent further spread of these strains in our hospital, as well as others.

The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., bla OXA-23 , bla OXA-24/40 , and bla OXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen., (Copyright © 2016 American Society for Microbiology.)

Oritavancin is a lipoglycopeptide antibiotic with bactericidal activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). The phase 3 studies SOLO I and SOLO II demonstrated comparable efficacy and safety of a single dose of oritavancin compared with 7-10 days of twice-daily vancomycin in adults with acute bacterial skin and skin-structure infections (ABSSSIs). The present analysis assessed clinical responses by pathogen at 48-72 h and at study days 14-24 in SOLO patients within the pooled data set. Of the 1959 patients in the pooled SOLO studies, 1067 had at least one baseline Gram-positive pathogen and 405 had MRSA. Clinical response rates were similar for oritavancin- and vancomycin-treated patients by pathogen, including Staphylococcus aureus with or without the Panton-Valentine leukocidin (pvl) gene and from different clonal complexes, and were similar for pathogens within each treatment group. Oritavancin exhibited potent in vitro activity against all baseline pathogens, with MIC 90 values (minimum inhibitory concentration required to inhibit 90% of the isolates) of 0.12 µg/mL for Staphylococcus  aureus, 0.25 µg/mL for Streptococcus pyogenes and 0.06 µg/mL for Enterococcus faecalis. Whereas both oritavancin and vancomycin achieved similarly high rates of clinical response by pathogen, including methicillin-susceptible and -resistant Staphylococcus aureus, oritavancin provides a single-dose alternative to 7-10 days of twice-daily vancomycin to treat ABSSSIs., (Copyright © 2016 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Unlabelled: Colistin is increasingly used as an antibiotic of last resort for the treatment of carbapenem-resistant Gram-negative infections. The plasmid-borne colistin resistance gene mcr-1 was initially identified in animal and clinical samples from China and subsequently reported worldwide, including in the United States. Of particular concern is the spread of mcr-1 into carbapenem-resistant bacteria, thereby creating strains that approach pan-resistance. While several reports of mcr-1 have involved carbapenem-resistant strains, no such isolates have been described in the United States. Here, we report the isolation and identification of an Escherichia coli strain harboring both mcr-1 and carbapenemase gene blaNDM-5 from a urine sample in a patient without recent travel outside the United States. The isolate exhibited resistance to both colistin and carbapenems, but was susceptible to amikacin, aztreonam, gentamicin, nitrofurantoin, tigecycline, and trimethoprim-sulfamethoxazole. The mcr-1- and blaNDM-5-harboring plasmids were completely sequenced and shown to be highly similar to plasmids previously reported from China. The strain in this report was first isolated in August 2014, highlighting an earlier presence of mcr-1 within the United States than previously recognized., Importance: Colistin has become the last line of defense for the treatment of infections caused by Gram-negative bacteria resistant to multiple classes of antibiotics, in particular carbapenem-resistant Enterobacteriaceae (CRE). Resistance to colistin, encoded by the plasmid-borne gene mcr-1, was first identified in animal and clinical samples from China in November 2015 and has subsequently been reported from numerous other countries. In April 2016, mcr-1 was identified in a carbapenem-susceptible Escherichia coli strain from a clinical sample in the United States, followed by a second report from a carbapenem-susceptible E. coli strain originally isolated in May 2015. We report the isolation and identification of an E. coli strain harboring both colistin (mcr-1) and carbapenem (blaNDM-5) resistance genes, originally isolated in August 2014 from urine of a patient with recurrent urinary tract infections. To our knowledge, this is the first report in the United States of a clinical bacterial isolate with both colistin and carbapenem resistance, highlighting the importance of active surveillance efforts for colistin- and carbapenem-resistant organisms., (Copyright © 2016 Mediavilla et al.)

Here we completely sequenced four mcr-1-haboring plasmids, isolated from two extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli and two carbapenemase-producing Klebsiella pneumoniae clinical isolates. The mcr-1-harboring plasmids from an E. coli sequence type 2448 (ST2448) isolate and two K. pneumoniae ST25 isolates were identical (all pMCR1-IncX4), belonging to the IncX4 incompatibility group, while the plasmid from an E. coli ST2085 isolate (pMCR1-IncI2) belongs to the IncI2 group. A nearly identical 2.6-kb mcr-1-pap2 element was found to be shared by all mcr-1-carrying plasmids., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background: Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians., Methods: We compared the performance characteristics of 4 RMD platforms for detecting resistance against β-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against β-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making., Results: In PRIMERS I, the 4 RMD platforms detected β-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem., Conclusions: RMD platforms can help inform empiric β-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions., (© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

We report the first multidrug-resistant Proteus mirabilis strain producing the carbapenemase OXA-48 (Pm-OXA-48) isolated at Al-Shifa hospital in Gaza, Palestine. Draft genome sequencing of Pm-OXA-48 identified 16 antimicrobial resistance genes, encoding resistance to β-lactams, aminoglycosides, fluoroquinolones, phenicols, streptothricin, tetracycline, and trimethoprim-sulfamethoxazole. Complete sequencing of the bla(OXA-48)-harboring plasmid revealed that it is a 72 kb long IncL/M plasmid, harboring carbapenemase gene bla(OXA-48), extended spectrum β-lactamase gene bla(CTX-M-14), and aminoglycoside resistance genes strA, strB, and aph(3')-VIb., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections (BSI) are classified epidemiologically as health care-associated hospital onset (HAHO)-, health care-associated community onset (HACO)-, or community-associated (CA)-MRSA. Clinical and molecular differences between HAHO- and HACO-MRSA BSI are not well known. Thus, we evaluated clinical and molecular characteristics of MRSA BSI to determine if distinct features are associated with HAHO- or HACO-MRSA strains. Molecular genotyping and medical record reviews were conducted on 282 MRSA BSI isolates from January 2007 to December 2009. MRSA classifications were 38% HAHO-, 54% HACO-, and 8% CA-MRSA. Comparing patients with HAHO-MRSA to those with HACO-MRSA, HAHO-MRSA patients had significantly higher rates of malignancy, surgery, recent invasive devices, and mortality and longer hospital stays. Patients with HACO-MRSA were more likely to have a history of renal failure, hemodialysis, residence in a long-term-care facility, long-term invasive devices, and higher rate of MRSA relapse. Distinct MRSA molecular strain differences also were seen between HAHO-MRSA (60% staphylococcal cassette chromosome mec type II [SCCmec II], 30% SCCmec III, and 9% SCCmec IV) and HACO-MRSA (47% SCCmec II, 35% SCCmec III, and 16% SCCmec IV) (P < 0.001). In summary, our study reveals significant clinical and molecular differences between patients with HAHO- and HACO-MRSA BSI. In order to decrease rates of MRSA infection, preventive efforts need to be directed toward patients in the community with health care-associated risk factors in addition to inpatient infection control., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in both community and healthcare-related settings worldwide. Current knowledge regarding the epidemiology of S. aureus and MRSA in Gaza is based on a single community-based carriage study. Here we describe a cross-sectional analysis of 215 clinical isolates collected from Al-Shifa Hospital in Gaza during 2008 and 2012., Methods: All isolates were characterized by spa typing, SCCmec typing, and detection of genes encoding Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin (TSST-1). Representative genotypes were also subjected to multilocus sequence typing (MLST). Antibiotic susceptibility testing was performed using VITEK2 and MicroScan., Results: MRSA represented 56.3% of all S. aureus strains, and increased in frequency from 2008 (54.8%) to 2012 (58.4%). Aside from beta-lactams, resistance was observed to tetracycline, erythromycin, clindamycin, gentamicin, and fluoroquinolones. Molecular typing identified 35 spa types representing 17 MLST clonal complexes (CC), with spa 998 (Ridom t223, CC22) and spa 70 (Ridom t044, CC80) being the most prevalent. SCCmec types I, III, IV, V and VI were identified among MRSA isolates, while type II was not detected. PVL genes (lukF/S-PV) were detected in 40.0% of all isolates, while the TSST-1 gene (tst) was detected in 27.4% of all isolates, with surprisingly high frequency within CC22 (70.4%). Both PVL and TSST-1 genes were found in several isolates from 2012., Conclusions: Molecular typing of clinical isolates from Gaza hospitals revealed unusually high prevalence of TSST-1 genes among CC22 MRSA, which is noteworthy given a recent community study describing widespread carriage of a CC22 MRSA clone known as the 'Gaza strain'. While the latter did not address TSST-1, tst-positive spa 998 (Ridom t223) has been detected in several neighboring countries, and described as endemic in an Italian NICU, suggesting international spread of a 'Middle Eastern variant' of pandemic CC22 strain EMRSA-15.

Repetitive element polymerase chain reaction (rep-PCR) typing has been used for methicillin-resistant Staphylococcus aureus (MRSA) strain characterization. The goal of this study was to determine if a rapid commercial rep-PCR system, DiversiLab™ (DL; bioMérieux, Durham, NC, USA), could be used for MRSA surveillance at a large medical center and community hospitals. A total of 1286 MRSA isolates genotyped by the DL system were distributed into 84 distinct rep-PCR patterns: 737/1286 (57%) were clustered into 6 major rep-PCR patterns. A subset of 220 isolates was further typed by pulsed-field gel electrophoresis (PFGE), spa typing, and SCCmec typing. The 220 isolates were distributed into 80 rep-PCR patterns, 94 PFGE pulsotypes, 27 spa, and 3 SCCmec types. The DL rep-PCR system is sufficient for surveillance, but the DL system alone cannot be used to compare data to other institutions until a standardized nomenclature is established and the DL MRSA reference library is expanded., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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.

Most studies on Staphylococcus aureus have focused on the molecular epidemiology of methicillin-resistant S. aureus (MRSA) infections. In contrast, little information is available regarding the molecular epidemiology of currently circulating methicillin-susceptible S. aureus (MSSA) isolates in hospital settings, an epoch when the epidemiology of S. aureus has undergone significant changes. We conducted a cross-sectional study to compare the clinical, epidemiological, and genetic characteristics of MSSA and MRSA isolates at 3 tertiary-care hospitals in Medellín, Colombia, from February 2008 to June 2010. The infections were classified according to the Centers for Disease Control and Prevention (CDC) definitions. Genotypic analysis included spa typing, multilocus sequence typing (MLST) and staphylococcal cassette chromosome (mec) (SCCmec) typing. A total of 810 patients was enrolled. One hundred infections (12.3%) were classified as community-associated (31 CA-MSSA, 69 CA-MRSA), 379 (46.8%) as healthcare-associated community-onset (136 HACO-MSSA, 243 HACO-MRSA), and 331 (40.9%) as healthcare-associated hospital-onset (104 HAHO-MSSA, 227 HAHO-MRSA). Genotype analyses showed a higher diversity and a more varied spa type repertoire in MSSA than in MRSA strains. Most of the clinical-epidemiological characteristics and risk factors evaluated did not allow for discriminating MRSA- from MSSA-infected patients. The lack of equivalence among the genetic backgrounds of the major MSSA and MRSA clones would suggest that the MRSA clones are imported instead of arising from successful MSSA clones. This study emphasizes the importance of local surveillance to create public awareness on the changing S. aureus epidemiology., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have emerged as major nosocomial pathogens. bla(KPC), commonly located on Tn4401, is found in Gram-negative bacterial strains, with the two most common variants, bla(KPC-2) and bla(KPC-3), identified in plasmids with diverse genetic backgrounds. In this study, we examined bla(KPC-4)- and bla(KPC-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 bla(KPC-4), bla(TEM-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 bla(KPC-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 bla(KPC-5) gene is carried on a Tn4401 element and differs from the genetic environment of bla(KPC-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 bla(KPC) 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.

During the 1990s, various reports of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections appeared in the literature, caused by novel strains genetically distinct from traditional healthcare-associated MRSA (HA-MRSA). Numerous lineages of CA-MRSA have since emerged on every continent, several of which have spread internationally, most notably USA300. CA-MRSA strains are increasingly implicated in nosocomial infections, and may eventually displace HA-MRSA strains in hospitals. Consequently, distinctions based on clinical epidemiology and susceptibility are becoming less relevant, arguing in favor of genotypic definitions. We review the current molecular epidemiology of CA-MRSA with respect to genetic diversity, global distribution, and factors related to its emergence and spread., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We describe a multiplex real-time PCR assay capable of identifying both the epidemic Klebsiella pneumoniae ST258 clone and bla(KPC) carbapenemase genes in a single reaction. The assay displayed excellent sensitivity (100%) and specificity (100%) for identification of ST258 clone and bla(KPC) 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.

We describe a novel Tn4401 variant (Tn4401d) in epidemic Klebsiella pneumoniae clone ST258, from which a partial bla(KPC) 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 bla(KPC) is undergoing recombination and that Tn4401 itself is heterogeneous and highly plastic.

A total of 299 nares and 194 blood isolates of methicillin-resistant Staphylococcus aureus (MRSA), each recovered from a unique patient, were collected from 23 U.S. hospitals from May 2009 to March 2010. All isolates underwent spa and staphylococcal cassette chromosome mec element (SCCmec) typing and antimicrobial susceptibility testing; a subset of 84 isolates was typed by pulsed-field gel electrophoresis (PFGE) using SmaI. Seventy-six spa types were observed among the isolates. Overall, for nasal isolates, spa type t002-SCCmec type II (USA100) was the most common strain type (37% of isolates), while among blood isolates, spa type t008-SCCmec type IV (USA300) was the most common (39%). However, the proportion of all USA100 and USA300 isolates varied by United States census region. Nasal isolates were more resistant to tobramycin and clindamycin than blood isolates (55.9% and 48.8% of isolates versus 36.6% and 39.7%, respectively; for both, P < 0.05). The USA300 isolates were largely resistant to fluoroquinolones. High-level mupirocin resistance was low among all spa types (<5%). SCCmec types III and VIII, which are rare in the United States, were observed along with several unusual PFGE types, including CMRSA9, EMRSA15, and the PFGE profile associated with sequence type 239 (ST239) isolates. Typing data from this convenience sample suggest that in U.S. hospitalized patients, USA100 isolates of multiple spa types, while still common in the nares, have been replaced by USA300 isolates as the predominant MRSA strain type in positive blood cultures.

Background: We performed this study 1) to determine the prevalence of community-associated extended spectrum beta-lactamase producing Enterobacteriaceae (ESBLPE) colonization and infection in New York City (NYC); 2) to determine the prevalence of newly-acquired ESBLPE during travel; 3) to look for similarities in contemporaneous hospital-associated bloodstream ESBLPE and travel-associated ESBLPE., Methods: Subjects were recruited from a travel medicine practice and consented to submit pre- and post-travel stools, which were assessed for the presence of ESBLPE. Pre-travel stools and stools submitted for culture were used to estimate the prevalence of community-associated ESBLPE. The prevalence of ESBLPE-associated urinary tract infections was calculated from available retrospective data. Hospital-associated ESBLPE were acquired from saved bloodstream isolates. All ESBLPE underwent multilocus sequence typing (MLST) and ESBL characterization., Results: One of 60 (1.7%) pre- or non-travel associated stool was colonized with ESBLPE. Among community-associated urine specimens, 1.3% of Escherichia coli and 1.4% of Klebsiella pneumoniae were identified as ESBLPE. Seven of 28 travelers (25.0%) acquired a new ESBLPE during travel. No similarities were found between travel-associated ESBLPE and hospital-associated ESBLPE. A range of imported ESBL genes were found, including CTX-M-14 and CTX-15., Conclusion: ESBL colonization and infection were relatively low during the study period in NYC. A significant minority of travelers acquired new ESBLPE during travel.

Background: Recent reports highlight the incursion of community-associated MRSA within healthcare settings. However, knowledge of this phenomenon remains limited in Latin America. The aim of this study was to evaluate the molecular epidemiology of MRSA in three tertiary-care hospitals in Medellín, Colombia., Methods: An observational cross-sectional study was conducted from 2008-2010. MRSA infections were classified as either community-associated (CA-MRSA) or healthcare-associated (HA-MRSA), with HA-MRSA further classified as hospital-onset (HAHO-MRSA) or community-onset (HACO-MRSA) according to standard epidemiological definitions established by the U.S. Centers for Disease Control and Prevention (CDC). Genotypic analysis included SCCmec typing, spa typing, PFGE and MLST., Results: Out of 538 total MRSA isolates, 68 (12.6%) were defined as CA-MRSA, 243 (45.2%) as HACO-MRSA and 227 (42.2%) as HAHO-MRSA. The majority harbored SCCmec type IVc (306, 58.7%), followed by SCCmec type I (174, 33.4%). The prevalence of type IVc among CA-, HACO- and HAHO-MRSA isolates was 92.4%, 65.1% and 43.6%, respectively. From 2008 to 2010, the prevalence of type IVc-bearing strains increased significantly, from 50.0% to 68.2% (p = 0.004). Strains harboring SCCmec IVc were mainly associated with spa types t1610, t008 and t024 (MLST clonal complex 8), while PFGE confirmed that the t008 and t1610 strains were closely related to the USA300-0114 CA-MRSA clone. Notably, strains belonging to these three spa types exhibited high levels of tetracycline resistance (45.9%)., Conclusion: CC8 MRSA strains harboring SCCmec type IVc are becoming predominant in Medellín hospitals, displacing previously reported CC5 HA-MRSA clones. Based on shared characteristics including SCCmec IVc, absence of the ACME element and tetracycline resistance, the USA300-related isolates in this study are most likely related to USA300-LV, the recently-described 'Latin American variant' of USA300.

Objective: To describe the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) carriage and transmission in an ambulatory hemodialysis population., Design: Prospective cohort study., Setting: Outpatient hemodialysis facility affiliated with a large academic medical center., Participants: Of the 170 facility patients, 103 (61%) participated in the study., Methods: Swab specimens of the nares, axillae, and vascular access site were collected from participants weekly for 3 weeks and then monthly for 5 months. Demographic and clinical data were collected monthly for 12 months. Molecular analysis of MRSA isolates was performed., Results: The baseline MRSA carriage prevalence was 12%. Factors associated with MRSA carriage included a history of MRSA; failed renal transplantation; hospital admission within 6 months; and receipt of a first-generation cephalosporin, cefepime, or vancomycin. Six subjects acquired MRSA after enrollment (incidence, 1.2 per 100 patient-months at-risk; overall prevalence, 18%). Molecular analysis suggested that transmission occurred within the facility. The incidence of MRSA infection among carriers was 1.76 per 100 patient-months. Community-associated strains (ie, USA300) were isolated from 28% of carriers and at least 25% of infections., Conclusions: The prevalence of MRSA carriage and the incidence of infection among carriers were high among ambulatory hemodialysis patients, and community-associated MRSA was responsible for a large portion of the MRSA burden. A relatively high rate of MRSA acquisition was observed, with indirect evidence of intrafacility transmission. Additional studies are needed to confirm these findings and to identify effective and feasible methods to prevent MRSA transmission and infection among hemodialysis patients.

lukF-PV was present in 36% of skin and soft tissue infection (SSTI)-derived methicillin-susceptible Staphylococcus aureus (MSSA) strains and comprised six distinct clones, which contained fewer enterotoxin genes than strains without lukF-PV. Clinical presentations and outcomes of lukF-PV(+) methicillin-resistant S. aureus (MRSA) and MSSA SSTIs were comparable. In multivariable analysis, the presence of lukF-PV remained a significant predictor for incision and drainage among MSSA strains.

The study aimed to determine the natural history of Staphylococcus aureus nasal colonization in hemodialysis outpatients. Surveillance cultures were taken from patients presenting for hemodialysis or routine care to identify S. aureus nasal carriers. A prospective cohort study was performed to identify risks for persistent colonization. Detailed microbiologic and molecular studies of colonizing isolates were performed. Only 23/145 (15.9%) dialysis patients were persistently colonized, and only HIV-positive status was associated with persistence (P = 0.05). Prior hospitalization was the only risk factor for methicillin-resistant S. aureus carriage (OR 2.5, P = 0.03). In isolates from patients with ≤ 42 days of vancomycin exposure, vancomycin minimum bactericidal concentrations (MBCs) increased with duration of exposure. Among dialysis patients, S. aureus colonization was limited and transient; only HIV status was associated with persistence. Nevertheless, duration of vancomycin exposure was associated with increasing vancomycin MBCs. Vancomycin exposure in S. aureus carriers may be involved in increasing resistance., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. Multiple KPC gene variants (bla(KPC)) have been reported, with KPC-2 (bla(KPC-2)) and KPC-3 (bla(KPC-3)) associated with epidemic outbreaks in New York City and various international settings. Here, we describe the development of a multiplex real-time PCR assay using molecular beacons (MB-PCR) for rapid and accurate identification of bla(KPC) variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described bla(KPC) variants (bla(KPC-2) to bla(KPC-11)). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each bla(KPC) variant, and cross-reactivity was not observed using DNA isolated from several bacterial species. A total of 457 clinical Gram-negative isolates were successfully characterized by our MB-PCR assay, with bla(KPC-3) and bla(KPC-2) identified as the most common types in the New York/New Jersey metropolitan region. The MB-PCR assay described herein is rapid, sensitive, and specific and should be useful for understanding the ongoing evolution of carbapenem resistance in Gram-negative bacteria. As novel bla(KPC) variants continue to emerge, the MB-PCR assay can be modified in response to epidemiologic developments.

From 2004 to 2005, 60%-72% of invasive Staphylococcus aureus isolates from Romanian hospitals were resistant to methicillin (methicillin-resistant S. aureus [MRSA]), the highest frequency for any European nation. Few reports, however, have addressed the molecular characteristics of S. aureus in Romania. In this study, we utilized spa typing, multilocus sequence typing, staphylococcal cassette chromosome mec (SCCmec) typing, dru typing, pulsed-field gel electrophoresis, and detection of virulence factors to characterize 146 S. aureus strains isolated from 2004 to 2005 at the Clinic County Hospital in Braşov. Antibiotic susceptibility patterns for all MRSA isolates and patient demographic data were also obtained. Fifty-six strains (38.4%) were determined to be MRSA by susceptibility testing and SCCmec typing. All MRSA strains were resistant to beta-lactams and tetracycline, but susceptible to nitrofurans, vancomycin, and clindamycin, with inducible clindamycin resistance in 23/28 clindamycin-sensitive/erythromycin-resistant isolates. Molecular typing identified 15 clonal backgrounds (CC 1, 5, 8, 8/239, 9, 15, 20, 22, 25, 30, 45, 80, 97, 101, and 121), only 4 of which were associated with MRSA (CC 1, 8/239, 30, and 80). Spa types 35 (t127, CC 1) and 351 (t030, CC 8/239) accounted for 27.4% and 21.9% of all S. aureus strains, respectively, and 19.6% and 57.1% of all MRSA strains. Both hospital-associated (SCCmec type III) and community-associated (SCCmec type IV) elements were identified within MRSA strains, whereas Panton-Valentine leukocidin was detected in 10 MRSA and 12 methicillin-sensitive S. aureus strains. These results demonstrate the presence of various endemic S. aureus clones within the Clinic County Hospital in Braşov, suggestive of ongoing nosocomial and community transmission.

A novel composite transposon (Tn6072) resembling staphylococcal cassette chromosome mercury (SCCHg) was identified in a collection of sequence type (ST) 239 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates from Romanian hospitals. Tn6072 is homologous to the 5' region of SCCHg found in staphylococcal cassette chromosome mec (SCCmec) type III prototype strain 85/2082 but lacks the characteristic mer operon. SCCHg has previously been reported to integrate downstream of orfX, at the same chromosomal location as SCCmec. Tn6072, by contrast, is demarcated by two IS431 elements, flanked by 8-bp direct repeats, and inserted upstream of the origin of replication, within an open reading frame homologous to SAR2700 of S. aureus strain MRSA252. Analysis of a geographically and temporally diverse collection of 111 strains from the ST239 clonal group uncovered 11 additional strains harboring Tn6072, demonstrating a lineage-specific insertion pattern. Complete sequence analysis of the SCCmec regions of two representative Romanian strains (BK16704, BK16691) revealed two additional novel structures derived from a type III SCCmec background. BK16704 possesses an SCCmec 3A.1.4 structure, with an IS256 insertion downstream of the right chromosomal junction. In contrast, the SCCmec element of BK16691 is truncated downstream of the mec gene complex, with a 24-kb deletion encompassing the right chromosomal junction and an inverted downstream IS256 element. This structure, tentatively named "psiSCCmec16691," confers methicillin resistance but lacks most of the J1/J2 region, including the ccr gene complex. Taken together, these findings provide evidence for the continuing evolution of SCC elements, as well as the ST239 clonal group.

Objective: To determine the prevalence of asymptomatic carriage of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) among healthcare professionals (HCPs) who experience varying degrees of exposure to ambulatory patients and to genetically characterize isolates., Methods: This single-center, cross-sectional study enrolled 256 staff from the intensive care units, emergency department, and prehospital services of an urban tertiary care university hospital in 2008. Occupational histories and nasal samples for S. aureus cultures were obtained. S. aureus isolates were genetically characterized with the use of spa typing and screened for mecA. MRSA isolates underwent further characterization., Results: S. aureus was isolated from 112 of 256 (43.8%) HCPs, including 30 of 52 (57.7%) paramedics, 51 of 124 (41.1%) nurses, 11 of 28 (39.3%) clerical workers, and 20 of 52 (38.5%) physicians. MRSA was isolated from 17 (6.6%) HCPs, including 1 (1.9%) paramedic, 13 (10.5%) nurses, 1 (3.6%) clerical worker, and 2 (3.8%) physicians. Among S. aureus isolates, 15.2% were MRSA. MRSA prevalence was 9.6% (12/125) in emergency department workers, 5.1% (4/79) in intensive care unit workers, and 1.9% (1/52) in emergency medical services workers. Compared with paramedics, who had the lowest prevalence of methicillin resistance among S. aureus isolates (1 of 30 [3.3%] isolates), nurses, who had the highest prevalence (13 of 51 [25.4%] isolates), had an odds ratio of 9.92 (95% confidence interval, 1.32-435.86; P = .02) for methicillin resistance. Analysis of 15 MRSA isolates revealed 7 USA100 strains, 6 USA300 strains, 1 USA800 strain, and 1 EMRSA-15 strain. All USA300 strains were isolated from emergency department personnel., Conclusions: The observed prevalence of S. aureus and MRSA colonization among HCPs exceeds previously reported prevalences in the general population. The proportion of community-associated MRSA among all MRSA in this colonized HCP cohort reflects the distribution of the USA300 community-associated strain observed increasingly among US hospitalized patients.

Staphylococcal enterotoxins (SE) can cause toxin-mediated disease, and those that function as superantigens are implicated in the pathogenesis of allergic diseases. The prevalence of 19 enterotoxin genes was determined by PCR in clinical S. aureus strains derived from wounds (108) and blood (99). We performed spa typing and multilocus sequence typing (MLST) to determine clonal origin, and for selected strains staphylococcal enterotoxin B (SEB) production was measured by enzyme-linked immunosorbent assay. Strains carried a median of five SE genes. For most SE genes, the prevalence rates among methicillin-resistant and methicillin-sensitive S. aureus isolates, as well as wound- and blood-derived isolates, did not differ. At least one SE gene was detected in all except two S. aureus isolates (>99%). Complete egc clusters were found in only 11% of S. aureus isolates, whereas the combination of sed, sej, and ser was detected in 24% of clinical strains. S. aureus strains exhibited distinct combinations of SE genes, even if their pulsed-field gel electrophoresis and MLST patterns demonstrated clonality. USA300 strains also showed considerable variability in SE content, although they contained a lower number of SE genes (mean, 3). By contrast, SE content was unchanged in five pairs of serial isolates. SEB production by individual strains varied up to 200-fold, and even up to 15-fold in a pair of serial isolates. In conclusion, our results illustrate the genetic diversity of S. aureus strains with respect to enterotoxin genes and suggest that horizontal transfer of mobile genetic elements encoding virulence genes occurs frequently.

Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the DeltamecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the DeltamecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates.

We report a case of recurrent Staphylococcus aureus bacteremia in a patient who failed vancomycin due to a vancomycin-heteroresistant strain lacking methicillin resistance. Although initial isolates were susceptible, isolates obtained after vancomycin chemotherapy were vancomycin heteroresistant. This case thus illustrates the clinical emergence of vancomycin heteroresistance.

Methicillin-resistant Staphylococcus aureus (MRSA) carriage and subsequent infection were prospectively compared among a well-defined group of 107 individuals infected with human immunodeficiency virus type 1 (HIV-1) who had no evidence of immune suppression and 52 epidemiologically matched, uninfected individuals. The carriage strains and infecting strains were genetically characterized. The cumulative prevalence of MRSA carriage was significantly higher among HIV-infected individuals (16.8%) than among individuals without HIV infection (5.8%) (P = .04; odds ratio, 3.3 [95% confidence interval, 1.3-14.7]). Fifteen of 21 MRSA isolates recovered from colonized individuals were identified as strain USA300. Of the 10 MRSA skin and soft tissue infections observed in this study, all occurred in HIV-infected individuals who were colonized with the same strain that caused the infection. Previous antibiotic use was the only statistically significant risk factor for MRSA carriage. These data highlight the fact that MRSA colonization and infection are important clinical issues among asymptomatic HIV-1-infected individuals.