Yan Yan Hu

Molecular Typing of CTX-M-Producing Escherichia coli Isolates from Environmental Water, Swine Feces, Specimens from Healthy Humans, and Human Patients

ABSTRACT CTX-M-producing Escherichia coli is the predominant type of extended-spectrum β-lactamase (ESBL)-producing E. coli worldwide. In this study, molecular typing was conducted for 139 CTX-M-producing E. coli isolates, phenotypically positive for ESBLs, isolated from environmental water, swine, healthy humans, and hospitalized patients in Hangzhou, China. The antibiotic resistance profiles of the isolates for the cephalosporins and fluoroquinolones were determined. The isolates showed 100% resistance to cefotaxime and ceftriaxone while maintaining relatively high susceptibility to cefoxitin, cefepime, and ceftazidime. A total of 61.9% (86/139) of the isolates, regardless of origin, showed high resistance to fluoroquinolones. PCRs and DNA sequencing indicated that bla CTX-M-14 was the most prevalent CTX-M-9 group gene and that bla CTX-M-15 and bla CTX-M-55 were the dominant CTX-M-1 group genes. Isolates from all sources with CTX-M types belonging to the CTX-M-1 or CTX-M-9 group were most frequently associated with epidemics. Molecular homology analysis of the isolates, conducted by phylogenetic grouping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST), demonstrated that the dominant clones belonged to B2-ST131, D-ST648, D-ST38, or A-CC10. These four sequence types (STs) were discovered in E. coli isolates both from humans and from environmental water, suggesting frequent and continuous intercompartment transmission between humans and the aquatic environment. Seven novel sequence types were identified in the current study. In conclusion, this study is the first to report the molecular homology analysis of CTX-M-producing E. coli isolates collected from water, swine, and healthy and hospitalized humans, suggesting that pathogens in the environment might originate both from humans and from animals.

Emergence of Escherichia coli Sequence Type 131 Isolates Producing KPC-2 Carbapenemase in China

ABSTRACT Twenty-two KPC-2-producing Escherichia coli isolates were obtained from three hospitals in Hangzhou, China, from 2007 to 2011. One isolate, with OmpC porin deficiency, exhibited high-level carbapenem resistance. Pulsed-field gel electrophoresis showed that few isolates were indistinguishable or closely related. Multilocus sequence typing indicated that sequence type 131 (ST131) was the predominant type (9 isolates, 40.9%), followed by ST648 (5 isolates), ST405 (2 isolates), ST38 (2 isolates), and 4 single STs, ST69, ST2003, ST2179, and ST744. Phylogenetic analysis indicated that 9 group B2 isolates belonged to ST131, and 5 of 11 group D isolates belonged to ST648. Only one group B1 isolate and one group A isolate were identified. A representative plasmid (pE1) was partially sequenced, and a 7,788-bp DNA fragment encoding Tn3 transposase, Tn3 resolvase, ISKpn8 transposase, KPC-2, and ISKpn6-like transposase was obtained. The blaKPC-2-surrounding sequence was amplified by a series of primers. The PCR results showed that 13 isolates were consistent with the genetic environment in pE1. It is the first report of rapid emergence of KPC-2-producing E. coli ST131 in China. The blaKPC-2 gene of most isolates was located on a similar genetic structure.

Emergence of Proteus mirabilis Harboring blaKPC-2 and qnrD in a Chinese Hospital

ABSTRACT Nineteen carbapenem-nonsusceptible Proteus mirabilis isolates were recovered from intensive care units in the Second Affiliated Hospital of Zhejiang University during a 3-month period. The isolates showed a high level of resistance against ciprofloxacin, in addition to their resistance against the carbapenems. Pulsed-field gel electrophoresis (PFGE) analysis showed that these isolates belonged to three clonal strains. PCRs and DNA sequence analysis of the carbapenemase and other β-lactamase genes indicated that all the isolates harbored the blaKPC-2 gene. Twelve of 19 isolates harbored the plasmid-mediated quinolone resistance (PMQR) genes, both the qnrD and aac(6′)-Ib-cr genes. Eight representative isolates with high levels of quinolone resistance carried the similar mutation profiles of S83I in gyrA, E466D in gyrB, and S80I in parC. Reduced carbapenem susceptibility was transferred to Escherichia coli (EC600) in a conjugation experiment, while the quinolone resistance was not. DNA hybridization showed that qnrD was located on a plasmid of approximately 4.5 kb. In summary, large clonally related isolates of KPC-2-producing P. mirabilis emerged in a Chinese hospital, and qnrD was detected in KPC-producing P. mirabilis for the first time.

Linezolid-resistant clinical isolates of meticillin- resistant coagulase-negative staphylococci and Enterococcus faecium from China

Seventeen meticillin-resistant coagulase-negative staphylococci (MRCoNS), including ten Staphylococcus capitis, four Staphylococcus cohnii, two Staphylococcus haemolyticus and one Staphylococcus sciuri, and an Enterococcus faecium isolate with various levels of linezolid resistance were isolated from intensive care units in a Chinese hospital. PFGE indicated that the four S. cohnii isolates belonged to a clonal strain, and that nine of the S. capitis isolates were indistinguishable (clone A1) and the other one was closely related (clone A2). A G2576T mutation was identified in domain V of the 23S rRNA gene in the E. faecium isolate. Besides the G2576T mutation, a novel C2104T mutation was detected in the nine clone A1 S. capitis isolates. The cfr gene was detected in all the staphylococci except an S. sciuri isolate, whose 23S rRNA gene contained the G2576T mutation. There was a clonal dissemination of linezolid-resistant MRCoNS in intensive care units of our hospital, and this is the first report, to our knowledge, of linezolid-resistant staphylococci and enterococci in China.

Detection of OmpK36 porin loss in Klebsiella spp. by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry

The extensive use of carbapenems has led to the rapid dissemination of carbapenem-resistant Enterobacteriaceae ([9][1]), especially KPC-2-producing Klebsiella pneumoniae ([3][2], [8][3], [12][4], [13][5]). In addition to carbapenemase production, porin loss plays an important role in carbapenem

Detection of Colistin Resistance Gene mcr-1 in Hypervirulent Klebsiella pneumoniae and Escherichia coli Isolates from an Infant with Diarrhea in China

In recent years, antimicrobial resistance has increasingly been recognized as a significant problem that transcends international boundaries. With the global increase in the prevalence of carbapenem-resistant Enterobacteriaceae, polymyxins have been resurrected as a last-resort treatment option ([1

Detection of the Smqnr quinolone protection gene and its prevalence in clinical isolates of Stenotrophomonas maltophilia in China.

The aim of this study was to detect novel variants of the Stenotrophomonas maltophilia Smqnr gene family and analyse the prevalence of Smqnr genes in clinical isolates of S. maltophilia in China. In total, 442 clinical isolates of S. maltophilia were collected from nine hospitals in four provinces in China. Antimicrobial susceptibility testing against six commonly used antibiotics was performed on these isolates. The sequences of the Smqnr genes amplified by PCR were aligned with those of known Smqnr genes in GenBank and an Smqnr database. The resistance rate against co-trimoxazole was highest at 48.6 %, followed by resistance rates against ceftazidime, chloramphenicol, ticarcillin/clavulanate and tigecycline at 28.7, 21.3, 19.0 and 16.1 %, respectively. The highest susceptibility was shown to levofloxacin, with a resistance rate of just 6.1 %. Smqnr genes were detected in 114 isolates, and comprised 11 previously identified genes and 20 new variants, bringing the total number of known Smqnr genes to 47. The 20 novel Smqnr genes were designated Smqnr28-47 and the encoded proteins showed only 1-12 amino acid differences among each other. The most common Smqnr genes in China were Smqnr8 and its variant Smqnr35 with prevalences of 17.5 % (20/114) and 13.2 % (15/114), respectively. Both the known and the novel Smqnr genes were discovered in both quinolone non-sensitive and sensitive isolates with similar frequency, suggesting that the Smqnr gene makes little contribution to quinolone resistance in this organism.

Dissemination of the Same cfr-Carrying Plasmid among Methicillin-Resistant Staphylococcus aureus and Coagulase-Negative Staphylococcal Isolates in China

ABSTRACT Six cfr-harboring methicillin-resistant Staphylococcus aureus (MRSA) isolates, which belonged to the same clone of sequence type 5 (ST5)-staphylococcal cassette chromosome mec element II (SCCmec II)-spa t311, were investigated in this study. Complete sequencing of a cfr-carrying plasmid, pLRSA417, revealed an 8,487-bp fragment containing a Tn4001-like transposon, cfr, orf1, and ISEnfa4. This segment, first identified in an animal plasmid, pSS-01, was observed in several plasmids from clinical coagulase-negative staphylococci in China, suggesting that the cfr gene, which might originate from livestock, was located in the same mobile element and disseminated among different clinical staphylococcal species.

Colistin resistance gene mcr-1 in gut flora of children

The aim of this study was to determine the prevalence and transmission mechanism(s) of mcr-1 in the gut flora of children. Faecal samples (n = 173) were obtained from non-diarrhoea patients at the Childrens Hospital of Zhejiang University (Hangzhou, China). PCR-based analysis indicated that 17 isolates from 9.8% of the samples were positive for mcr-1, comprising 16 Escherichia coli and 1 Citrobacter freundii. Nine mcr-1-bearing isolates co-expressed extended-spectrum β-lactamase (ESBL) genes, but plasmid-mediated quinolone resistance (PMQR) genes were not detected. Transconjugation followed by Southern hybridisation analysis revealed that 14 of the E. coli isolates were able to transfer their colistin-resistant phenotype to E. coli EC600. All 14 of these E. coli strains contained a major mcr-1-containing conjugative plasmid with a size of ca. 33 kb or 55 kb. All but two of the E. coli isolates presented distinct pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing (MLST) analysis revealed 11 sequence types (STs) among the E. coli 16 isolates, with ST117 being the most common. The finding of a high prevalence of mcr-1 in the intestinal flora of children, with the majority of mcr-1-positive isolates being E. coli, highlights the need for more rational use of polymyxins to prevent polymyxin resistance from becoming disseminated among different microbial pathogens. Given the high detection rate of mcr-1 in children, we recommend that polymyxin is no longer used as a last-resort antimicrobial agent and that alternative strategies are developed to treat infections caused by such pathogens.

Detection of KPC-2 and qnrS1 in clinical isolates of Morganella morganii from China

Seven carbapenem-nonsusceptible Morganella morganii isolates, which have similar antibiotic susceptibility profiles, were isolated over a 5-month period. MICs of imipenem, meropenem, and ertapenem were 8, 1, and 0.25 to 0.5 μg/mL, respectively. Pulsed-field gel electrophoresis indicated that 6 isolates were indistinguishable or closely related. Carbapenem resistance can be transferred from M. morganii to Escherichia coli by conjugation. All M. morganii isolates and E. coli transconjugants produced KPC-2 and carried the qnrS1 gene. Production of KPC-2 mainly contributed to the carbapenem resistance in M. morganii. KPC-2-producing M. morganii clonally spread in a hospital in China.