Zhongju Chen

Characterization of a novel Klebsiella pneumoniae sequence type 476 carrying both bla KPC-2 and bla IMP-4.

Carbapenemase-producing Klebsiella pneumoniae has recently spread rapidly throughout China. In this study, we characterized a carbapenem-resistant K. pneumoniae isolate that produced both KPC-2 and IMP-4 type carbapenemases. A clinical isolate of K. pneumoniae, resistant to both meropenem and imipenem, was recovered from a urine sample. Antibiotic susceptibility was determined using the broth microdilution method and Etest (bioMérieux, France). Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) were used for gene type analysis. blaKPC and the encoding genes of ESBLs and plasmid-mediated AmpC enzymes were polymerase chain reaction (PCR) amplified and sequenced. Plasmids were analyzed by transformation, enzyme restriction and Southern blot. PCR analysis revealed that the isolate was simultaneously carrying blaKPC-2, blaIMP-4, blaTEM-1, and blaOKP-B genes. MLST assigned the isolate to a novel sequence type, ST476. blaKPC-2-harbouring plasmids of the isolate and comparative strains had similar EcoRI and HindIII restriction maps, while IMP-4-harbouring plasmids had variable HindIII restriction maps. Coexistence of blaKPC-2 and blaIMP-4 was probably due to blaIMP-4-harbouring plasmid transmission into KPC-2-producing K. pneumoniae (ST476). The concomitant presence of these genes is alarming and poses both therapeutic and infection control problems.

Comparison of 16S rRNA gene PCR and blood culture for diagnosis of neonatal sepsis

UNLABELLED Septicemia is a common cause of morbidity and mortality among newborns in the developing world. However, accurate clinical diagnosis of neonatal sepsis is often difficult because symptoms and signs are often nonspecific. Blood culture has been the gold standard for confirmation of the diagnosis. However, the sensitivity is low and results are usually not promptly obtained. Therefore, the diagnosis of sepsis is often based on clinical signs in association with laboratory tests such as platelets count, immature/total neutrophils ratio (I/T), and a rise in C-reactive protein (CRP). Polymerase chain reaction (PCR) methods for the detection of neonatal sepsis represent new diagnostic tools for the early identification of pathogens. METHODS During a 4-month prospective study, 16S rRNA PCR was compared with conventional blood culture for the diagnosis of neonatal bacterial sepsis. In addition, the relationship between known risk factors, clinical signs, laboratory parameters, and the diagnosis of sepsis was considered. RESULTS Sepsis was suspected in 706 infants from the intensive neonatal care unit. They all were included in the study. The number of positive cultures and positive PCR results were 95 (13.5%) and 123 (17.4%), respectively. Compared with blood culture, the diagnosis of bacterial sepsis by PCR revealed a 100.0% sensitivity, 95.4% specificity, 77.2% positive predictive value, and 100.0% negative predictive value. In this study, Apgar scores at 5 min, weight, icterus, irritability, feeding difficulties, gestational age (GA), premature rupture of membrane (PRM), platelets count, I/T, and a marked rise in CRP were important in establishing the diagnosis of sepsis in the newborn. In addition, weight, GA, PRM, irritability, duration of antibiotic usage, mortality rate, and number of purulent meningitis cases were significantly different between early-onset sepsis and late-onset sepsis. CONCLUSION 16S rRNA PCR increased the sensitivity in detecting bacterial DNA in newborns with signs of sepsis, allowed a rapid detection of the pathogens, and led to shorter antibiotic courses. However, uncertainty about the bacterial cause of sepsis was not reduced by this method. 16S rRNA PCR needs to be further developed and improved. Blood culture is currently irreplaceable, since pure isolates are essential for antimicrobial drug susceptibility testing.

Molecular characteristics and virulence factors in methicillin-susceptible, resistant, and heterogeneous vancomycin- intermediate Staphylococcus aureus from central-southern China

BACKGROUND Staphylococcus aureus is a leading cause of nosocomial infections. The purpose of this study was to evaluate the prevalence of methicillin-resistant S. aureus (MRSA) and heterogeneous vancomycin-intermediate S. aureus (hVISA), and compare the antimicrobial susceptibility, molecular characteristic, and virulence factors in methicillin-susceptible S. aureus (MSSA), MRSA, and hVISA from central-southern China. METHODS A total of 184 S. aureus were isolated from sterile body fluids. All isolates were subjected to population analysis profiling for the identification of hVISA phenotype and polymerase chain reaction analysis for genotyping and 31 virulence genes. RESULTS The prevalence of MRSA isolates was 41.8% in central-southern China. Of 77 MRSA isolates, 17 (22.1%) were identified as hVISA. The most common MRSA and MSSA clones were ST239-MRSA-SCCmecIII-t030-agr-I (55.8%) and ST188-MSSA-t189-agr-I (20.6%), respectively. The frequency of carriage of pvl, hemolysins, tst, and staphylococcal enterotoxin genes among MSSA isolates was significantly higher than that for MRSA isolates (p < 0.05); 98 MSSA isolates (53.3%) carried ≥ 10 tested virulence genes simultaneously, which was significantly higher than that of MRSA isolates (33.8%; p = 0.004). The 17 hVISA isolates carried a significantly small number of virulence genes; only two hVISA isolates carried ≥ 10 tested virulence genes simultaneously, and two hVISA isolates harbored only four virulence genes. Compared with other clonal complexes (CCs), CC1 and CC398 isolates harbored a higher frequency of exfoliatin genes, CC1 and CC59 harbored a higher frequency of pvl gene, and only CC1 isolates harbored lukED. CONCLUSION The prevalence of hVISA was considerably high in central-southern China. Simultaneous carriage of multiple virulence genes was common in S. aureus isolates; the virulence genes were more diverse and frequent among MSSA isolates than among MRSA isolates. Furthermore, the distribution of some virulence genes was correlated with the different S. aureus CCs.

First report in China of Enterobacteriaceae clinical isolates coharboring blaNDM-1 and blaIMP-4 drug resistance genes.

AIMS To describe the identification of two carbapenem-resistant, NDM-1 and IMP-4, carbapenemases coproducing Enterobacteriaceae isolates recovered from hospitalized patients in China. Both Klebsiella pneumoniae clinical isolates (Kpn922 and Kpn9599) were resistant to meropenem and imipenem and were subjected to additional antibiotic susceptibility testing. Polymerase chain reaction (PCR) and sequence analyses were used to characterize bacterial carbapenemase resistance genes, extended-spectrum β-lactamases, plasmid-mediated AmpC enzymes, quinolone resistance, and 16s RNA methylase. Genetic relatedness was determined using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmids were analyzed by S1-PFGE and Southern blot. RESULTS PCR analyses revealed that the Kpn922 isolate carried blaNDM-1, blaIMP-4, blaTEM-1, and blaSHV-1 genes, while Kpn9599 carried blaNDM-1, blaIMP-4, blaTEM-1, and blaSHV-12 genes. MLST determined that the two isolates were ST1043 and ST571 sequence types. Southern blot analyses revealed that metallo-β-lactamase genes were plasmid borne in both isolates. Plasmids ∼300 kb simultaneously carried blaNDM-1 and blaIMP-4. CONCLUSIONS Coexistence of blaNDM-1 and blaIMP-4 in these clinical isolates may herald the emergence of a new pattern of drug resistance. Surveillance of carbapenemases, particularly metallo-β-lactamases, in Enterobacteriaceae is urgently needed to control and prevent the spread of these resistance determinants in China.

Vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolated from a patient who never received vancomycin treatment.

BACKGROUND With the abuse of antibiotics, the methicillin-resistant Staphylococcus aureus (MRSA) strain became prevalent. Furthermore, Staphylococcus aureus with a character of vancomycin intermediate-resistance (VISA) has been found globally since the first report in Japan. The main objectives of this study were to report a case of VISA isolated from a Chinese patient who had never undergone Vancomycin treatment, and to determine its molecular character. METHODS A total of 9 strains were recovered from a patient during the therapeutic process. Antimicrobial susceptibility testing was performed to determine their antibiotic susceptibility patterns. To detect the VISA strains molecular epidemiological features, growth and morphological characters, we used multilocus sequence typing, autolysis assay and transmission electric microscope tests. Pulsed-field gel electrophoresis (PFGE) was performed to characterize the heterogeneities of all isolates. RESULTS One isolate was found to exhibit vancomycin intermediated-resistant with MIC of 8 μg/ml. It was ST239-T030-agr-1, had thickened cell wall, and displayed a slower growth rate and reduced susceptibility to Triton X-100-induced autolysis than other strains. All 9 strains exhibited the same PFGE pattern. CONCLUSION This is the first report of VISA found in central China from a patient who had never received vancomycin treatment.

The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1 gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1 and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1 gene. However, this study is the first to report the presence of the vanC1 gene in E. faecium of human origin. Additionally, our research showed the vanC1 gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1 gene from different species.The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

Outbreak of nosocomial NDM-1-producing Klebsiella pneumoniae ST1419 in a neonatal unit

OBJECTIVES The aim of this study was to characterise carbapenem-resistant Klebsiella pneumoniae isolates recovered from neonatal clinical specimens over a 4-month period. METHODS Seven carbapenem-resistant K. pneumoniae isolates were analysed. Antibiotic susceptibilities of the isolates were determined by the agar dilution method, and the drug resistance genes were evaluated by PCR. Clonal relatedness of the isolates was assessed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Conjugation experiments and Southern blot hybridisation were performed to determine the transferability of the plasmids. RESULTS All of the K. pneumoniae isolates carried the blaNDM-1 gene but were negative for all other carbapenemases tested. All of the isolates harboured blaSHV-12, and five isolates also carried blaCTX-M-15 and/or blaTEM-1. All of the isolates exhibited multidrug resistance. The isolates belonged to sequence types ST1419 and ST101 and formed three different PFGE patterns. Plasmids carrying blaNDM-1 were successfully transferred from six of the seven isolates to the Escherichia coli recipient. These six NDM-1-producing K. pneumoniae were clonal and carried blaNDM-1 on the same plasmid, but one isolate possibly carried chromosomal blaNDM-1. CONCLUSIONS This is the first report of NDM-1-positive K. pneumoniae ST1419 from neonates in China. Closer attention should be paid to monitoring blaNDM-1 gene dissemination because it is potentially transferred horizontally.