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Objectives: To characterize the vectors involved in the dissemination of bla CMY-2 genes in clinical isolates of Proteus mirabilis collected between 1999 and 2007. Methods: Plasmid analysis of 19 P. mirabilis carrying ampC genes was performed by PCR-based replicon typing, S1-PFGE and Southern hybridization with ampC and replicon probes. Isolates that could not be characterized were examined for the presence of SXT/R391-like elements. To demonstrate the involvement of these elements in the dissemination of bla CMY-2, we performed a PCR amplification of the integrase (int) and toxin/antitoxin (TA) genes from SXT/R391-like integrative conjugative elements (ICEs). Later on, I-Ceu-I PFGE gels and hybridization with bla CMY-2, int and prfC probes were performed. The genetic organization of bla CMY-2 was also studied. Results: ampC genes were located on large conjugative plasmids in 11 of the 19 (58%) P. mirabilis studied. However, in eight of these isolates a plasmid was not involved in the mobilization of ampC genes. I-Ceu-I PFGE and hybridization analyses revealed thatbla CMY-2 were chromosomally located in these eight P. mirabilis isolates. The genetic organization of bla CMY-2 and hybridization analyses revealed that bla CMY-2 was carried by an ICE almost identical to ICEPmiJpan1 in seven out of these eight isolates. Conclusions: The prevalence of ICEs carrying bla CMY-2 was surprisingly high [37% (7 out of 19)]. This is the first study giving prevalence data on ICEs carrying bla CMY-2 genes. These results suggest the need to study these mobile genetic elements in the context of dissemination of acquired AmpC ß-lactamases and also of other ß-lactamases, such as extended-spectrum ß-lactamases and carbapenemases. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.

[Objectives]: To gain insights into ampC transmission between bacterial strains. Methods: We examined the genetic context of 117 acquired ampC genes from 27119 Enterobacteriaceae collected between 1999 and 2007. Plasmid analysis was carried out by PCR-based replicon or relaxase typing, S1-PFGE and Southern hybridization. I-CeuI/PFGE was used for isolates not characterized by plasmid analysis. PCR reactions were used to map the genetic organization of the ampC genes. [Results]: Among the isolates studied, 81.2% of ampC genes were located on plasmids of known Inc/MOB groups, 7.7% were chromosomally located and 11.1% were not determined. A/C, I1 and K were the most commonly found replicons in plasmids carrying blaCMY-2, while L/M replicons were associated with blaDHA-1. blaACC-1 was linked to I1 and MOBF11 plasmids; blaCMY-27 was associated with IncF and MOBP12 plasmids; the plasmid carrying blaCMY-25 could not be typed, and blaCMY-40 was chromosomally located. All 87 isolates carrying blaCMY-2, blaCMY-4, blaCMY-25, blaCMY-27, blaCMY-40 or blaACC-1 displayed the transposon-like structures ISEcp1/δISEcp1-blaCMY--blc-sugE or δISEcp1-blaACC-1--gdha. The most prevalent structure in blaDHA-1 (93.3% of cases) was identical to that described in the Klebsiella pneumoniae pTN60013 plasmid. Remarkably, in three isolates containing chromosomal blaCMY-2, this gene was mobilized by conjugation. [Conclusions]: Although plasmids are the main cause of the rapid dissemination of ampC genes among bacteria,we need to be aware that other mobile genetic elements such as integrative and conjugative elements (ICEs) can be involved in the mobilization of these genes. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved., This study was partially supported by the Ministry of Health and Consumer Affairs, Instituto de Salud Carlos III-Feder, the Spanish Network for Research in Infectious Diseases (REIPI/RD06/0008/0013 and RD06/0008/1012), BFU2008-00995/BMC (Spanish Ministry of Education) and the European Union Seventh Framework Programme under grant agreement number 241476 (PAR project). A. A. was partially funded by the I Plan Regional of I+D+I from Cantabria. M. P. G.-B. is the recipient of a JAE contract from Consejo Superior de Investigaciones Cientificas (CSIC).

A collection of 30 DHA-1-Enterobacteriaceae producers was examined for the presence of qnr genes. PCR-based replicon typing, plasmid profile and Southern hybridisation analyses revealed that all isolates co-harboured blaDHA-1 and qnrB genes on the same plasmid. All but one of these plasmids belonged to the L/M group. Genetic organization analyses of a randomly selected isolate revealed the co-localization of both genes on an IS26-composite transposon. As plasmids carrying both genes seem to have a high prevalence and a worldwide distribution, care should be taken when quinolones are used to treat infections caused by DHA-1 producers. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

We report a Serratia marcescens and an Escherichia coli isolate simultaneously detected in the same patient. Both isolates showed susceptibility patterns suggestive of harbouring a plasmid-mediated AmpC beta-lactamase (pACBL) and a plasmid-encoded quinolone resistance (PMQR). PCR-based replicon, MOB typing, plasmid profile and Southern hybridization analyses revealed that both isolates coharboured bla(DHA-1) and qnrB genes on the same IncL/M-MOB(P13) plasmid approximately 70 kb in size. Together with the fact that both plasmids were conjugative in the laboratory, these results strongly suggest that a horizontal transfer event could take place in vivo. This is the first report of an isolate of S. marcescens harbouring a pACBL. The only phenotypic method that suggests the presence of a pACBL in an isolate harbouring an inducible chromosomal AmpC enzyme is the observation of scattered colonies near the edge of the inhibition zones of some beta-lactams. The presence of both resistance genes on the same plasmid and the reported increase in PMQR could perhaps explain the widespread distribution of bla(DHA-1) genes.

In 2007, a significant increase in acquired ampC genes in Enterobacteriaceae from 0.06% in 1999 to 1.3% was observed. Proteus mirabilis showed the highest prevalence (0.95%) and CMY-2 was the most prevalent AmpC enzyme (66.7%). Other enzymes such as CMY-4, DHA-1, ACC-1, and three new enzymes called CMY-25, CMY-27 and CMY-40 were detected. Seven out of the 117 isolates (6%) also produced an extended-spectrum β-lactamase. As acquired AmpC enzymes are likely to become a serious public health issue worldwide, close surveillance is necessary to curb their spread.

Sepsis is a serious health condition worldwide, affecting more than 30 million people globally each year. Blood culture (BC) is generally used to diagnose sepsis because of the low quantity of microbes occurring in the blood during such infections. However, ~50% of bloodstream infections (BSI) give negative BC, this figure being higher for sepsis, which delays the start of appropriate antimicrobial therapy. This prospective study evaluated a multiplex real-time polymerase chain reaction, the MagicplexTM Sepsis test (MP), for the detection of pathogens from whole blood, comparing it to routine BC. We analyzed 809 blood samples from 636 adult patients, with 132/809 (16.3%) of the samples positive for one or more relevant microorganism according to BC and/or MP. The sensitivity and specificity of MP were 29 and 95%, respectively, while the level of agreement between BC and MP was 87%. The rate of contaminated samples was higher for BC (10%) than MP (4.8%) (P < 0.001). Patients with only MP-positive samples were more likely to be on antimicrobial treatment (47%) than those with only BC-positive samples (18%) (P = 0.002). In summary, the MP test could be useful in some clinical setting, such as among patients on antibiotic therapy. Nevertheless, a low sensitivity demonstrated impairs its use as a part of a routine diagnostic algorithm.