SummaryBackgroundTraditional methods for molecular epidemiology of Neisseria gonorrhoeae are suboptimal. Whole-genome sequencing (WGS) offers ideal resolution to describe population dynamics and to predict and infer transmission of antimicrobial resistance, and can enhance infection control through linkage with epidemiological data. We used WGS, in conjunction with linked epidemiological and phenotypic data, to describe the gonococcal population in 20 European countries. We aimed to detail changes in phenotypic antimicrobial resistance levels (and the reasons for these changes) and strain distribution (with a focus on antimicrobial resistance strains in risk groups), and to predict antimicrobial resistance from WGS data.MethodsWe carried out an observational study, in which we sequenced isolates taken from patients with gonorrhoea from the European Gonococcal Antimicrobial Surveillance Programme in 20 countries from September to November, 2013. We also developed a web platform that we used for automated antimicrobial resistance prediction, molecular typing (N gonorrhoeae multi-antigen sequence typing [NG-MAST] and multilocus sequence typing), and phylogenetic clustering in conjunction with epidemiological and phenotypic data.FindingsThe multidrug-resistant NG-MAST genogroup G1407 was predominant and accounted for the most cephalosporin resistance, but the prevalence of this genogroup decreased from 248 (23%) of 1066 isolates in a previous study from 2009–10 to 174 (17%) of 1054 isolates in this survey in 2013. This genogroup previously showed an association with men who have sex with men, but changed to an association with heterosexual people (odds ratio=4·29). WGS provided substantially improved resolution and accuracy over NG-MAST and multilocus sequence typing, predicted antimicrobial resistance relatively well, and identified discrepant isolates, mixed infections or contaminants, and multidrug-resistant clades linked to risk groups.InterpretationTo our knowledge, we provide the first use of joint analysis of WGS and epidemiological data in an international programme for regional surveillance of sexually transmitted infections. WGS provided enhanced understanding of the distribution of antimicrobial resistance clones, including replacement with clones that were more susceptible to antimicrobials, in several risk groups nationally and regionally. We provide a framework for genomic surveillance of gonococci through standardised sampling, use of WGS, and a shared information architecture for interpretation and dissemination by use of open access software.FundingThe European Centre for Disease Prevention and Control, The Centre for Genomic Pathogen Surveillance, Örebro University Hospital, and Wellcome.

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A Salmonella enterica serotype Cubana isolate exhibiting resistance to most ␤-lactam antibiotics, including oxyimino-cephalosporins and imipenem, was isolated from a 4-year-old boy with gastroenteritis in Maryland. ␤-Lactam resistance was mediated by a conjugative plasmid that encoded KPC-2, a class A carbapenemase previously found in a Klebsiella pneumoniae isolate from the Maryland area as well. Sequence analysis of the flanking regions indicated a potential association of bla KPC-2 with mobile structures.Resistance to expanded-spectrum oxyimino-cephalosporins among Salmonella strains is mostly due to acquisition of plasmids encoding various class A extended-spectrum ␤-lactamases (1,8,13,17,(19)(20)(21). Production of plasmid-mediated class C ␤-lactamases by Salmonella isolates has also been described previously (4,22,23). The emergence of such strains may have serious implications because of the limitation of therapeutic choices for patients with invasive Salmonella infections and by facilitation of the spread of bla genes in the community. We describe here an imipenem-resistant Salmonella enterica serotype Cubana isolate that produces the recently described KPC-2 ␤-lactamase (E. S. MATERIALS AND METHODSSalmonella serotype Cubana was isolated in December 1998 from a stool specimen of a 4-year-old boy with diarrhea in a hospital in Maryland. The patient was chronically ill with Wiskott-Aldrich syndrome. In the 6 months before isolation of serotype Cubana, he had been hospitalized three times and had received intravenous antibiotics. During each of those hospitalizations he had received intravenous ␤-lactams, including ceftriaxone and ceftazidime, but not carbapenems. There was no history of recent travel. The isolate (AM04707) was submitted to the Centers for Disease Control and Prevention as part of the National Antimicrobial Resistance Monitoring System for enteric bacteria (http://www .cdc.gov/NARMS/) and was subsequently forwarded to the Hellenic Pasteur Institute.Escherichia coli K-12 strain 14R525 (Nal r ) was used as the recipient in conjugation experiments. E. coli DH5␣ (GIBCO-BRL, Carlsbad, Calif.) was used for transformation. Chloramphenicol-resistant plasmid pBCSK(ϩ) (Stratagene, La Jolla, Calif.) was used for cloning and expression of bla genes.The MICs of ␤-lactams were determined by an agar dilution technique (10). Susceptibilities to other antimicrobial agents were assessed by a disk diffusion method (11) and by a partial-range broth microdilution method (Sensititre; Trek Diagnostics; Westlake, Ohio), according to the instructions of the manufacturer.Conjugation experiments were carried out in mixed broth cultures as described previously (5). Transconjugant clones were selected on Mueller-Hinton agar containing ampicillin (50 g/ml) plus nalidixic acid (200 g/ml). Plasmid DNA preparations were obtained by an alkaline lysis technique and resolved in 0.8% (wt/vol) agarose gels. Individual plasmids were excised as discrete bands from low-melting-point agarose (0.8%) and were subjected to partial di...

A transferable ␤-lactamase produced by a multidrug-resistant clinical isolate of Enterobacter cloacae was studied. The bla gene was carried by a large (>80-kb) transmissible plasmid. Nucleotide sequence analysis of cloned fragments revealed that it was part of a gene cassette carried by a class 1 integron along with other resistance genes, including aac(6)-Ib. The encoded ␤-lactamase, designated IBC-1, was a novel class A enzyme that hydrolyzed ceftazidime and cefotaxime and was inhibited by tazobactam and, to a lesser extent, by clavulanate. Also, imipenem exhibited potent inhibitory activity against IBC-1. The enzyme consisted of 287 amino acid residues, including Ser-237, cysteines at positions 69 and 237a, and Arg-244, which may be implicated in its interaction with ␤-lactams. In amino acid sequence comparisons, IBC-1 displayed the highest similarity with the chromosomal penicillinase of Yersinia enterocolitica, a carbenicillinase from Proteus mirabilis GN79, the species-specific ␤-lactamases of Klebsiella oxytoca, and the carbapenemase Sme-1. However, a phylogenetic association with established ␤-lactamase clusters could not be conclusively shown.Production of extended-spectrum (ES) ␤-lactamases (ESBLs) is one of the major causes of resistance to the newer oxyimino-␤-lactams in enterobacteria. ␤-Lactamases hydrolyzing broadspectrum ␤-lactams and inhibited by clavulanic acid are considered to have ES properties (3). Except for the class D enzymes OXA-11 (9) and OXA-18 (22), which display ES properties, the ESBLs described to date belong to molecular class A. The most clinically important enzymes in this group are the ES descendants of the common plasmid-mediated TEM-1 and SHV-1 ␤-lactamases. Various non-TEM, non-SHV class A ␤-lactamases exhibiting ES activities have also been described. CTX-M-type (1, 8) and SFO-1 (13) ␤-lactamases can be placed in a cluster of clavulanate-inhibited enzymes that preferentially hydrolyze cefotaxime and are related to the species-specific ␤-lactamases of Klebsiella oxytoca (7) and other enterobacterial species. PER (2, 19) and VEB-1 (23) ␤-lactamases and their distant relatives CME-1 from Chryseobacterium meningosepticum (28) and the cephalosporinases of Bacteroides spp. (21) also exhibit ES properties. VEB-1 is the first class A ESBL that was found to be encoded by an integron-associated gene (23). Resistance genes found in the variable region of class 1 integrons are parts of cassettes (27) that also include an integrase-specific recombination segment called the 59-base element (59-be) (35). The bla genes that are most frequently associated with these structures belong to the pse, carb, and oxa types (27).In this work we describe IBC-1, a novel class A ESBL that was encoded by a class 1 integron-associated gene. The integron was located in a multidrug-resistant transferable plasmid found in an Enterobacter cloacae clinical strain. MATERIALS AND METHODSBacterial strains and plasmids. E. cloacae HT9 was isolated in January 1999 in Hippokration General Hospital, Thessaloniki, G...