Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6)-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25 g/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindIII, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%, 42%, 59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.Plasmid-mediated quinolone resistance was first described for a ciprofloxacin-resistant strain of Klebsiella pneumoniae in 1998 (15). The responsible gene, qnr (later named qnrA), was located on plasmid pMG252, which encodes multidrug resistance proteins. qnrB and qnrS were discovered in 2005 and 2006, respectively, and mediated similar levels of ciprofloxacin resistance (9, 11). Qnr proteins belong to the pentapeptide repeat protein (PRP) family and protect DNA gyrase and topoisomerase IV from quinolone inhibition (26,27,28). qnr genes show a high level of diversity; there are at least 6 qnrA, 20 qnrB, and 3 qnrS alleles reported, with one or more amino acid alterations within each family (12; http://www.lahey.org /qnrStudies). More recently, qnrD was found in Salmonella isolates (3). qnr genes are widely distributed in clinical Enterobacteriaceae isolates around the world and are usually associated with mobile elements (21). There were also qnr-like genes found on the chromosomes of Vibrio vulnificus, Vibrio parahaemolyticus, Photobacterium profundum, Stenotrophomonas maltophilia, and gram-positive genera such as Enterococcus, Listeria, Clostridium, and Bacillus (1,17,22,24). The wide distribution of qnr genes in different species of Enterobacteriaceae and their high degree of diversity raise the concern that there might be more qnr genes that have not yet been discovered. In this study, a new plasmid-mediated quinolone resistance gene, qnrC, was found on and cloned from a transferable plasmid, pHS10, in a clinical ...

Three kinds of plasmid-mediated quinolone resistance (PMQR) determinants have been discovered and have been shown to be widely distributed among clinical isolates: qnr genes, aac(6)-Ib-cr, and qepA. Few data on the prevalence of these determinants in strains from animals are available. The presence of PMQR genes in isolates from animals was determined by PCR amplification and DNA sequencing. The production of extended-spectrum ␤-lactamases (ESBLs) and AmpC ␤-lactamases in the strains was detected, and their genotypes were determined. The genetic environment of PMQR determinants in selected plasmids was analyzed. All samples of ceftiofur-resistant (MICs > 8 g/ml) isolates of the family Enterobacteriaceae were selected from 36 companion animals and 65 food-producing animals in Guangdong Province, China, between November 2003 and April 2007, including 89 Escherichia coli isolates, 9 Klebsiella pneumoniae isolates, and isolates of three other genera. A total of 68.3% (69/101) of the isolates produced ESBLs and/or AmpC ␤-lactamases, mainly those of the CTX-M and CMY types. Of the 101 strains, PMQR determinants were present in 35 (34.7%) isolates, with qnr, aac(6)-Ib-cr, and qepA detected alone or in combination in 8 (7.9%), 19 (18.8%), and 16 (15.8%) strains, respectively. The qnr genes detected included one qnrB4 gene, four qnrB6 genes, and three qnrS1 genes. Five strains were positive for both aac(6)-Ib-cr and qepA, while one strain was positive for qnrS1, aac(6)-Ib-cr, and qepA. qnrB6 was flanked by two copies of ISCR1 with an intervening dfr gene downstream and sul1 and qacE⌬1 genes upstream. In another plasmid, aac(6)-Ib-cr followed intI1 and arr-3 was downstream. PMQR determinants are highly prevalent in ceftiofur-resistant Enterobacteriaceae strains isolated from animals in China. This is the first report of the occurrence of PMQR determinants among isolates from companion animals.

Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcus faecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 g/ml) and teicoplanin (MIC, 96 g/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated

Fifty-three Mycoplasma pneumoniae strains were isolated from pediatric patients in Shanghai, China, from October 2005 to February 2008. Of 53 clinical isolates, 44 (83%) were resistant to erythromycin (MICs of >128 g/ml for all 44 strains), azithromycin, and clarithromycin. All macrolide-resistant M. pneumoniae strains harbored an A-to-G transition mutation at position 2063 in 23S rRNA genes. Forty-five (85%) clinical isolates were classified into the P1 gene restriction fragment length polymorphism type I, and six (11%) were type II.Mycoplasma pneumoniae is increasingly recognized as common and important pathogen in community-acquired respiratory tract infections (RTIs) and pneumonia, particularly in school-age children and young adults (9, 13). M. pneumoniae isolates are usually susceptible to macrolides; thus, macrolides are the most important drugs for treatment of RTI infections, especially in children (13). Fluoroquinolones and tetracyclines, which may be used as alternatives to macrolides, are not recommended for children. Macrolide-resistant M. pneumoniae strains have been reported in Japan, China, France, and the United States (6-8, 12, 16, 17).In this study, 53 strains of M. pneumoniae were collected from pediatric patients with RTIs in Shanghai, China. The in vitro activities of macrolides, tetracyclines, and fluoroquinolones against M. pneumoniae isolates were determined, and the mechanisms of resistance for macrolide-resistant stains were investigated. In addition, the type of M. pneumoniae clinical isolates was determined by PCR-restriction fragment length polymorphism (RFLP) typing of the P1 gene. pneumoniae was performed as described previously by Waites (14). All of the strains were identified by colony morphology and PCR assay. PCR amplification of 16S rRNA genes was done to identify M. pneumoniae using primers with sequences 5Ј-GCCACCCTCGGGGGCAGTCAG-3Ј and 5Ј-GAGTCGG GATTCCCCGCGGAGG-3Ј as previously described (4).Antimicrobial susceptibility of M. pneumoniae. To determine the MICs, the broth microdilution method with SP4 broth (Remel, Lenexa, KS) was performed as described previously by Waites (14). Every susceptibility testing was repeated three times. M. pneumoniae reference strain MPFH (ATCC 15531) was also included. Comparative in vitro activities of 10 antimicrobials are listed in Table 1. All of the M. pneumoniae isolates were susceptible to the tetracyclines and fluoroquinolones tested in this study. Moxifloxacin was more active than ciprofloxacin and levofloxacin. The MIC 50 and MIC 90 of moxifloxacin were 0.03 g/ml and 0.06 g/ml, respectively-much lower than those of ciprofloxacin and levofloxacin.Among 53 strains, 44 (83%) were resistant to erythromycin with a MIC 90 of Ͼ128 g/ml to either erythromycin or clarithromycin. These strains also showed high MICs to azithromycin and josamycin, with MIC 90 s of 64 g/ml and 4 g/ml, respectively (Table 1). All 44 macrolide-resistant strains had erythromycin MICs of Ͼ128 g/ml. The 16-member macrolide josamycin possessed lower MICs than the 14-a...

Hypervirulent variants of Klebsiella pneumoniae (hvKp) that cause invasive community-acquired pyogenic liver abscess (PLA) have emerged globally. Little is known about the virulence determinants associated with hvKp, except for the virulence genes rmpA/A2 and siderophores (iroBCD/iucABCD) carried by the pK2044-like large virulence plasmid. Here, we collected most recent clinical isolates of hvKp from PLA samples in China, and performed clinical, molecular, and genomic sequencing analyses. We found that 90.9% (40/44) of the pathogens causing PLA were K. pneumoniae. Among the 40 LA-Kp, K1 (62.5%), and K2 (17.5%) were the dominant serotypes, and ST23 (47.5%) was the major sequence type. S1-PFGE analyses demonstrated that although 77.5% (31/40) of the LA-Kp isolates harbored a single large virulence plasmid varied in size, 5 (12.5%) isolates had no plasmid and 4 (10%) had two or three plasmids. Whole genome sequencing and comparative analysis of 3 LA-Kp and 3 non-LA-Kp identified 133 genes present only in LA-Kp. Further, large scale screening of the 133 genes in 45 LA-Kp and 103 non-LA-Kp genome sequences from public databases identified 30 genes that were highly associated with LA-Kp, including iroBCD, iucABCD and rmpA/A2 and 21 new genes. Then, these 21 new genes were analyzed in 40 LA-Kp and 86 non-LA-Kp clinical isolates collected in this study by PCR, showing that new genes were present 80–100% among LA-Kp isolates while 2–11% in K. pneumoniae isolates from sputum and urine. Several of the 21 genes have been proposed as virulence factors in other bacteria, such as the gene encoding SAM-dependent methyltransferase and pagO which protects bacteria from phagocytosis. Taken together, these genes are likely new virulence factors contributing to the hypervirulence phenotype of hvKp, and may deepen our understanding of virulence mechanism of hvKp.

Decreased ceftazidime/avibactam susceptibility in KPC-KP clinical isolates is caused by high ceftazidime hydrolysis activity and OmpK35 porin deficiency and the majority of isolates belong to ST11.

Variants of the oqxAB gene, oqxA2, oqxB2 and oqxB3, were identified in two E. coli strains. The oqxAB gene was present in all K. pneumoniae strains studied and is likely located on the chromosome, thus identifying the genome of K. pneumoniae as a possible reservoir of oqxAB.

The purpose of this study was to investigate the epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) in Shanghai Children's Hospital in China. Twenty-two non-duplicate CRKP strains were collected from pediatric patients between March and June in 2014. Antimicrobial susceptibility testing was conducted by the agar dilution method. Beta-lactamases were characterized by polymerase chain reaction (PCR) and DNA sequencing. The transferability of bla NDM-1 was investigated by conjugation experiment. The plasmids bearing antibiotic resistance genes were characterized by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization. Clonal relatedness was evaluated by PFGE and multilocus sequence typing (MLST). The clinical data of patients were retrospectively reviewed. The 22 CRKP strains were resistant to most of the antimicrobial agents tested, except tigecycline and colistin. Overall, 59, 77, and 100 % of these strains were resistant to imipenem, meropenem, and ertapenem, respectively. The bla NDM-1 was positive in 77.3 % (17/22) of the CRKP strains, of which the 16 isolates from inpatients were designated as ST37 (n = 9) and ST76 (n =7) and one isolate from an outpatient belonged to ST846. The 17 bla NDM-1-positive isolates belonged to PFGE type A (n = 9), type C (n = 7), or type B (n = 1). The plasmids bearing bla NDM-1 could be transferred into recipient Escherichia coli J53 through conjugation in 88.2 % (15/17) of the strains. The hybridization results showed that the plasmids carrying the bla NDM-1 gene were approximately 50-240 kb in size. This is the first report of an outbreak caused by NDM-1-producing K. pneumoniae ST76 and ST37 among neonates.