Fangyou Yu

Molecular characterization of Staphylococcus aureus isolates causing skin and soft tissue infections (SSTIs).

BackgroundStaphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), is an important cause of pyogenic skin and soft tissue infections (SSTIs). The aim of present study is to investigate the molecular characteristic of Staphylococcus aureus isolates isolated from the pus samples from the patients with purulent skin and soft tissue infections in Wenzhou, China.MethodsBetween December 2002 and June 2008, a total of 111 nonduplicate S. aureus isolates were collected from the pus samples of the patients with SSTIs in a teaching hospital in Wenzhou, China. All the tested isolates were confirmed as S. aureus using a Staph SPA agglutination kit, Grams stain and a Vitek-60 microbiology analyzer. The homology among the tested isolates was determined by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was used to determine the sequence types (STs) of the selected isolates. The genotypes of SCCmec were determined by a multiplex PCR in the MRSA isolates. Panton-Valentine leukocidin (PVL) genes and mecA were also determined by another multiplex PCR.ResultsAmong the 111 S. aureus isolates, 48 and 63 isolates were community-acquired and hospital-acquired respectively. Sixty isolates were confirmed as MRSA harboring mecA detected by PCR. A total of 32 PFGE clonal types were obtained by PFGE, with 10 predominant patterns (types A to J). Twenty-five different STs including ST398 and three novel STs were found among 51 selected isolates. The main STs were ST239, ST1018, ST59, ST7 and ST88. Of 60 MRSA isolates, SCCmec II, III, IV and SCCmec V were found in three, 50, three and two isolates, respectively. The positive rates of PVL genes in overall isolates, HA-isolates, CA-isolates, MRSA isolates and MSSA isolates were 23.4% (26/111), 20.6% (13/63), 27.1% (13/48), 21.7% (13/60) and 25.5% (13/51), respectively. Eight (33.3%, 8/24) of 24 CA-MRSA isolates and 5 (13.9%, 5/36) of 36 HA-MRSA isolates were positive for PVL genes. ST239-MRSA-SCCmecIII and ST1018-MRSA-SCCmecIII clones were found to be main clones and spread between community and hospital.ConclusionS. aureus isolates causing SSTIs showed considerable molecular heterogeneity and harbored high prevalence of PVL genes. Clonal spread was responsible for the dissemination of the isolates of S. aureus associated with SSTIs.

Molecular Characterization and Antimicrobial Susceptibility of Nasal Staphylococcus aureus Isolates from a Chinese Medical College Campus

Staphylococcus aureus colonization and infection occur more commonly among persons living or working in crowded conditions, but characterization of S. aureus colonization within medical communities in China is lacking. A total of 144 (15.4%, 144/935) S. aureus isolates, including 28 (3.0%, 28/935) MRSA isolates, were recovered from the nares of 935 healthy human volunteers residing on a Chinese medical college campus. All S. aureus isolates were susceptible to vancomycin, quinupristin/dalfopristin and linezolid but the majority were resistant to penicillin (96.5%), ampicillin/sulbactam (83.3%) and trimethoprim/sulfamethoxazole (93.1%). 82%, (23/28) of the MRSA isolates and 66% (77/116) of the MSSA isolates were resistant to multiple antibiotics, and 3 MRSA isolates were resistant to mupirocin—an agent commonly used for nasal decolonization. 16 different sequence types (STs), as well as SCCmec genes II, III, IVd, and V, were represented among MRSA isolates. We also identified, for the first time, two novel STs (ST1778 and ST1779) and 5 novel spa types for MRSA. MRSA isolates were distributed in different sporadic clones, and ST59-MRSA-VId- t437 was found within 3 MRSA isolates. Moreover, one isolate with multidrug resistance belonging to ST398-MRSA-V- t571 associated with animal infections was identified, and 3 isolates distributed in three different clones harbored PVL genes. Collectively, these data indicate a high prevalence of nasal MRSA carriage and molecular heterogeneity of S. aureus isolates among persons residing on a Chinese medical college campus. Identification of epidemic MRSA clones associated with community infection supports the need for more effective infection control measures to reduce nasal carriage and prevent dissemination of MRSA to hospitalized patients and health care workers in this community.

Emergence of blaNDM-1 among Klebsiella pneumoniae ST15 and novel ST1031 clinical isolates in China☆

The emergence of NDM-1 has become established as a major public health threat and represents a new and major challenge in the treatment of infectious diseases. A total of 39 carbapenem-resistant Enterobacteriaceae isolates collected from patients receiving care at 5 teaching hospitals in Jiangxi province, central China, were analyzed for carriage of resistance genes, including bla(NDM-1). Two carbapenem-resistant Klebsiella pneumoniae isolates (NC12 and NC18) were found to harbor bla(NDM-1). In addition to bla(NDM-1), NC12 also carried bla(SHV-1), while NC18 harbored additional resistance genes, including bla(SHV-12), bla(CTX-M-14), armA and bla(TEM-1). NC12 and NC18 belonged to ST15 and novel ST1031 and were clonally unrelated. Carbapenem resistance for NC12 could be transferred to Escherichia coli recipients through conjugation and chemical transformation, while carbapenem resistance for NC18 was only transferred to E. coli recipients by chemical transformation. The EcoR1-digested DNA pattern of plasmids from the transformants of NC12 was identical to that for NC18. Taken together, this is the first report of bla(NDM-1) carriage by K. pneumoniae clinical isolates in mainland China, indicating that bla(NDM-1) is disseminated among Enterobacteriaceae in China. Systemic surveillance should focus on the dissemination of bla(NDM-1) among Gram-negative clinical isolates, especially some major clones, such as K. pneumoniae ST15 which is a major clone among CTX-M-15-producing isolates.

Staphylococcal Panton-Valentine Leukocidin Induces Pro-Inflammatory Cytokine Production and Nuclear Factor-Kappa B Activation in Neutrophils

Panton-Valentine leukocidin (PVL) is a cytotoxin secreted by Staphylococcus aureus and associated with severe necrotizing infections. PVL targets polymorphonuclear leukocytes, especially neutrophils, which are the first line of defense against infections. Although PVL can induce neutrophil death by necrosis or apoptosis, the specific inflammatory responses of neutrophils to this toxin are unclear. In this study, both in vivo and in vitro studies demonstrated that recombinant PVL has an important cytotoxic role in human neutrophils, leading to apoptosis at low concentrations and necrosis at high concentrations. Recombinant PVL also increased the levels of pro-inflammatory cytokine secretion from neutrophils. The up-regulation of pro-inflammatory cytokines was due to nuclear factor-kappa B (NF-κB) activation induced by PVL. Moreover, blocking NF-κB inhibited the production of inflammatory cytokines. To test the role of neutrophil immune responses during the pathogenesis of PVL-induced acute lung injury, we used immunocompetent or neutropenic rabbits to develop a model of necrotizing pneumonia. Immunocompetent rabbits challenged with PVL demonstrated increased inflammation containing neutrophilic infiltrates. In addition, there were elevated levels of inflammatory cytokines (IL-6, IL-8, TNF-α and IL-10) and NF-κB in the lung homogenate. In contrast, the lung tissues from neutropenic rabbits contained mild or moderate inflammation, and the levels of inflammatory cytokines and NF-κB increased only slightly. Data from the current study support growing evidence that neutrophils play an important role in the pathogenesis of PVL-induced tissue injury and inflammation. PVL can stimulate neutrophils to release pro-inflammatory mediators, thereby causing an acute inflammatory response. The ability of PVL to induce inflammatory cytokine release may be associated with the activation of NF-κB or its pore-forming properties.

Virulence gene profiling and molecular characterization of hospital-acquired Staphylococcus aureus isolates associated with bloodstream infection

A better understanding of virulence gene profiling and molecular characterization of Staphylococcus aureus isolates associated with bloodstream infection (BSI) may provide further insights related to clinical outcomes with these infections. We analyzed 89 S. aureus isolates including 37 MRSA isolates (41.6%) recovered from 89 adult patients with BSI from 4 hospitals in Zhejiang province, eastern China. Thirty-five (94.6%) of MRSA isolates and 4 (7.7%) of methicillin-sensitive S. aureus (MSSA) isolates were resistant to multiple antimicrobials. All isolates harbored at least 2 of 22 possible virulence genes, including sdrC (92.1%), icaA (89.9%), hla (80.9%), clf (69.7%), sea (68.5%), sdrD (67.4%), hlb (67.4%), sdrE (65.2%), sei (51.7%), seg (50.6%), and cna (50.6%). Forty-four (49.4%) of all S. aureus BSI isolates, including 23 (62.2%) of MRSA isolates, harbored ≥10 of the virulence genes evaluated in this study. Sixteen (43.2%) MRSA isolates and 5 (9.6%) MSSA isolates harbored the gene encoding Panton-Valentine leukocidin (PVL). Collective genes for pvl, sdrE, sed, seg, and sei among MRSA isolates were significantly more frequent relative to MSSA isolates (P < 0.05). A total of 22 sequence types (STs), including novel ST2184, ST2199, and ST2200, and 33 spa types, including novel spa types t9530 and t9532, were identified among S. aureus BSI isolates, among which ST188 (15.7%) and ST7 (15.7%), and t091 (12.4%) and t189 (12.4%), seldom noted for Chinese isolates previously, were major STs and spa types, respectively. In contrast to previous reports, no predominant clones were found in the present study. Among the MRSA isolates, although ST239-MRSA-SCCmecIII, predominant clone in China, still represented the most common clone, it only accounted for 18.9%. However, ST188-MRSA- SCCmecIV seldom reported before accounted for 10.8%. Among the MSSA isolates, ST7-MSSA represented the most common clone (23.1%), followed by ST188-MSSA and ST630-MSSA (9.6% each). In conclusion, simultaneous carriage of multiple virulence genes and genetically considerable diversity were common among S. aureus BSI isolates. Furthermore, MRSA isolates exhibited more frequent carriage of superantigen genes and pvl relative to MSSA isolates. Taken together, there are distinctive virulence gene profiling and molecular characteristic among S. aureus isolates associated with bloodstream infection in China.

The prevalence of carbapenemase genes and plasmid-mediated quinolone resistance determinants in carbapenem-resistant Enterobacteriaceae from five teaching hospitals in central China.

We investigated the prevalence of β-lactamase genes and plasmid-mediated quinolone resistance (PMQR) determinants in 51 carbapenem-resistant Enterobacteriaceae (CRE) from five teaching hospitals in central China. The prevalence of carbapenem resistance in Enterobacteriaceae was 1·0% (51/5012). Of 51 CRE, 31 (60·8%) isolates were positive for one tested carbapenemase gene, while 10 (19·6%) were simultaneously positive for two tested carbapenemase genes. The positive rates of bla KPC-2, bla NDM-1, bla IMP-4, bla IMP-26 and bla IMP-8 were 54·9%, 17·6%, 11·8%, 11·8% and 3·9%, respectively. Of 10 CRE with two carbapenemase genes, three, five, one and one were positive for bla KPC-2 and bla IMP-4, bla KPC-2 and bla IMP-26, bla KPC-2 and bla IMP-8, and bla KPC-2 and bla NDM-1, respectively. Eight of nine bla NDM-1-positive isolates lacked carbapenemases by the modified Hodge test, while 27/28 isolates harbouring bla KPC-2 were positive for carbapenemases determined by this test; 41·2% of the CRE-positive isolates also harboured ESBL genes in various combinations (three and two positive for bla KPC-2 also carried bla DHA-1 and bla CMY-2). The positive rates of qnrS1, qnrA1, qnrB and aac-(6/)-Ib-cr in CRE were 25·5%, 9·8%, 23·5% and 15·7%, respectively. In particular, 7/9 isolates harbouring bla NDM-1 were positive for these quinolone resistance genes, of which five carried qnrS1 and two carried qnrS1 and qnrB4. All but two of 29 Klebsiella pneumoniae isolates were grouped into 20 clonal clusters by PFGE, with the predominant cluster accounting for four bla KPC-2-positive isolates distributed in the same hospital. We conclude that there is a high prevalence of bla NDM-1 and PMQR determinants in CRE isolates in central China. Multiple resistance determinants in various combinations co-exist in these strains and we report for the first time the co-existence of bla KPC-2 and bla IMP-26 in a strain of Klebsiella oxytoca.

High Prevalence of Extended-Spectrum Beta Lactamases among Salmonella enterica Typhimurium Isolates from Pediatric Patients with Diarrhea in China

We investigated the extended-spectrum beta lactamases among 62 Salmonella enterica Typhimurium isolates recovered from children with diarrhea in a Chinese pediatric hospital. A large proportion of S. enterica Typhimurium isolates were resistant to multiple antimicrobial agents, including ampicillin (90.3%), tetracycline (80.6%), trimethoprim/sulfamethoxazole (74.2%), chloramphenicol (66.1%), cefotaxime (27.4%). Forty-nine (79.0%) of S. enterica Typhimurium isolates were positive for bla TEM-1b and resistant to ampicillin. Thirteen S. enterica Typhimurium isolates (21.0%) were positive for bla CTX-M-1-group and bla CTX-M-9-group, and all isolates harboring bla CTX-M genes were positive for ISEcp1. Two main clones (PFGE type A and D) accounted for nearly 70% of S. enterica Typhimurium isolates, and 7 CTX-M-producing isolates belonged to PFGE type D. Collectively, our data reveal multi-drug resistance and a high prevalence of extended spectrum beta lactamases among S. enterica Typhimurium isolates from children in China. In addition, we report the first identification of bla CTX-M-55 within Salmonella spp. Our data also suggest that clonal spread is responsible for the dissemination of S. enterica Typhimurium isolates.

Outbreak of pulmonary infection caused by Klebsiella pneumoniae isolates harbouring blaIMP-4 and blaDHA-1 in a neonatal intensive care unit in China.

Outbreaks caused by Klebsiella pneumoniae producing carbapenemases and other β-lactamases have been reported. Four neonates admitted to a neonatal intensive care unit (NICU) in a Chinese hospital developed respiratory infection while receiving intensive care. In all four cases, multidrug-resistant K. pneumoniae was isolated from multiple respiratory specimens, leading to additional characterization of these organisms and investigation of the local environment in the NICU. Multiple β-lactamase genes, including bla(TEM-1), bla(IMP-4), bla(DHA-1) and bla(CTX-M-14), as well as the quinolone resistance gene qnrB4, were harboured by transferable plasmids from all four clinical isolates. Furthermore, PFGE confirmed that three of the four clinical isolates from the patients and three K. pneumoniae isolates collected from the hands of health-care workers and an incubator in the NICU belonged to the same PFGE cluster, indicating that an outbreak due to multidrug-resistant K. pneumoniae carrying bla(IMP-4) and bla(DHA-1) occurred in this NICU. As far as is known, this is the first report of the co-existence of bla(IMP-4) and bla(DHA-1) in the same K. pneumoniae isolate. These data suggest that additional precautions are needed to prevent outbreaks of infection caused by multidrug-resistant K. pneumoniae resulting from environmental exposure in NICUs.

High prevalence of plasmid-mediated quinolone resistance determinant aac(6 )-Ib-cr amongst Salmonella enterica serotype Typhimurium isolates from hospitalised paediatric patients with diarrhoea in China

In this study, the antimicrobial susceptibilities and prevalence of plasmid-mediated quinolone resistance determinants amongst Salmonella enterica serotype Typhimurium isolates from hospitalised paediatric patients with diarrhoea in China were investigated. In total, 40 (64.5%) of 62 S. Typhimurium isolates were resistant to ciprofloxacin (minimum inhibitory concentration ≥0.5 μg/mL), comprising 28 isolates with low-level resistance and 12 isolates with high-level resistance. All ciprofloxacin-resistant isolates were multiresistant to other antimicrobial agents. Four pulsed-field gel electrophoresis (PFGE) clusters were found amongst the 40 ciprofloxacin-resistant isolates, amongst which PFGE clusters A, B, E and D accounted for 7, 4, 1 and 28 isolates, respectively. Two isolates with high-level ciprofloxacin resistance had two mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The remaining ciprofloxacin-resistant isolates had only one mutation in the QRDR of gyrA. All 62 S. Typhimurium isolates were negative for qnr genes and qepA and 23 (37.1%) of the isolates were positive for aac(6)-Ib-cr. Nineteen isolates harbouring aac(6)-Ib-cr belonged to PFGE cluster D. A high prevalence of ciprofloxacin resistance and aac(6)-Ib-cr was found amongst S. Typhimurium isolates in China from hospitalised paediatric patients with diarrhoea not receiving quinolones. A single mutation in the QRDR of gyrA as well as production of AAC(6)-Ib-cr contributed to ciprofloxacin resistance. Clonal spread was responsible for the dissemination of aac(6)-Ib-cr amongst S. Typhimurium isolates.

Carriage of virulence factors and molecular characteristics of Staphylococcus aureus isolates associated with bloodstream, and skin and soft tissue infections in children.

We investigated the virulence gene carriage and molecular type characteristics of Staphylococcus aureus isolates from bloodstream infections (BSIs) and skin and soft tissue infections (SSTIs) in children. A total of 71 isolates, 16 of which were methicillin-resistant S. aureus (MRSA), were investigated by PCR for virulence-associated gene profiles, sequence type and spa type. This revealed that 76.7% and 53.7% of the SSTI and BSI isolates, respectively, exhibited simultaneous carriage of o10 virulence genes. Compared to BSI isolates, carriage rates for hla, hlb, cna, clfA, seb, sec and pvl genes were significantly higher in SSTI isolates. By contrast, carriage of eta, etb and sea was significantly higher for BSI isolates. Thirty-four sequence types (STs) and 36 spa types were identified in the 71 isolates and included 14 novel STs and four novel spa types. ST59-MRSA-IV/V-t437 was the most common clone in the MRSA isolates. We concluded that virulence determinants are widely distributed in isolates of S. aureus strains from children with BSIs and SSTIs, with an unexpectedly high rate in SSTI isolates. Future profiling of S. aureus virulence determinants may allow the prediction of severity and outcome for children with these infections.