We have identified a second resistance-nodulation-cell division (RND)-type efflux pump, AdeIJK, in clinical isolate Acinetobacter baumannii BM4454. The adeI, adeJ, and adeK genes encode, respectively, the membrane fusion, RND, and outer membrane components of the pump. AdeJ belongs to the AcrB protein family (57% identity with AcrB from Escherichia coli). mRNA analysis by Northern blotting and reverse transcription-PCR indicated that the genes were cotranscribed. Overexpression of the cloned adeIJK operon was toxic in both E. coli and Acinetobacter. The adeIJK genes were detected in all of the 60 strains of A. baumannii tested. The two latter observations suggest that the AdeIJK complex might contribute to intrinsic but not to acquired antibiotic resistance in Acinetobacter. To characterize the substrate specificity of the pump, we have constructed derivatives of BM4454 in which adeIJK (strain BM4579), adeABC (strain BM4561), or both groups of genes (strain BM4652) were inactivated by deletion-insertion. Determination of the antibiotic susceptibility of these strains and of BM4652 and BM4579, in which the adeIJK operon was provided in trans, indicated that the AdeIJK pump contributes to resistance to ␤-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, trimethoprim, acridine, safranin, pyronine, and sodium dodecyl sulfate. The chemical structure of these molecules suggests that amphiphilic compounds are the preferred substrates. The AdeABC and AdeIJK efflux systems contributed in a more than additive fashion to tigecycline resistance.

Susceptibility to antibiotics of 4,816 clinical L. monocytogenes strains isolated since 1926 was studied, and the temporal evolution of susceptibility to antibiotics was analyzed through several decades. The mechanisms of resistance in each resistant strain were studied. The prevalence of resistant strains was estimated at 1.27% among isolates from humans. Resistance to tetracyclines؉ and fluoroquinolones was more common and has recently emerged. Although acquired resistance in clinical L. monocytogenes did not implicate clinically relevant antibiotics, the possibility of resistance gene transfers, the description of the first clinical isolate with high-level resistance to trimethoprim, and the recent increase in penicillin MICs up to 2 g/ml reinforce the need for microbiological surveillance.

Leptospirosis is considered an underdiagnosed disease. Although several PCR-based methods are currently in use, there is little information on their comparability. In this study, four quantitative real-time PCR (qPCR) assays (SYBR green and TaqMan chemistries) targeting the secY, lfb1, and lipL32 genes were evaluated as diagnostic assays. In our hands, these assays can detect between 10 2 and 10 3 bacteria/ml of pure culture, whole-blood, plasma, and serum samples. In three independent experiments, we found a slightly higher sensitivity of the PCR assays in plasma than in whole blood and serum. We also evaluated the specificity of the PCR assays on reference Leptospira strains, including newly described Leptospira species, and clinical isolates. No amplification was detected for DNA obtained from saprophytic or intermediate Leptospira species. However, among the pathogens, we identified sequence polymorphisms in target genes that result in primer and probe mismatches and affect qPCR assay performance. In conclusion, most of these assays are sensitive and specific tools for routine diagnosis of leptospirosis. However, it is important to continually evaluate and, if necessary, modify the primers and/or probes used to ensure effective detection of the circulating Leptospira isolates.

New extended-spectrum ␤-lactamase GES-11 was detected in Acinetobacter baumannii BM4674. The enzyme conferred resistance to ␤-lactams, including aztreonam, and reduced susceptibility to carbapenems. The structural gene was part of a class 1 integron borne by self-transferable plasmid pIP847. GES-type ␤-lactamases have not been reported previously in A. baumannii.Acinetobacter baumannii is a predominant species associated with outbreaks of nosocomial infections, such as pneumonia, urinary tract infections, septicemia, and meningitis. Its clinical significance is due to its ability either to upregulate indigenous efflux pumps (5) or to acquire numerous resistance mechanisms (7) that lead to therapeutic failure. A rapid, global emergence of A. baumannii strains resistant to all ␤-lactams, including carbapenems, aminoglycosides, quinolones, tetracyclines-glycylcyclines, polymyxins, and trimethoprim-sulfamethoxazole, has been observed (1). A. baumannii clinical specimens resistant to all known antibiotics, including polymyxins, have been reported, illustrating the genetic flexibility of this pathogen (13).Resistance to ␤-lactams in A. baumannii is due mainly to the production of ␤-lactamases but can also result from several other mechanisms, including changes in outer membrane proteins, overexpression of multidrug efflux pumps, and alterations in the affinity or production of penicillin-binding proteins (9). ␤-Lactamases with carbapenemase activity, i.e., class D carbapenem-hydrolyzing oxacillinases or, less frequently, class B metallo-␤-lactamases, represent the major clinical concern. To the best of our knowledge, Ambler class A carbapenemases KPC, GES, SME, NMC, and IMI have not yet been reported in A. baumannii (18).A. baumannii BM4674 was isolated from the tibia fracture of a patient hospitalized at the Centre Hospitalier Universitaire in Nancy, France, in September 2008. MICs of antimicrobial agents for this strain were determined by Etest (AB Biodisk, Combourg, France) on Mueller-Hinton agar (bioMerieux, Marcy l'Etoile, France), and the breakpoints delivered by the Comité de l'Antibiogramme de la Société Française de Microbiologie were used for interpretations of results (3). A. baumannii BM4674 was resistant to all ␤-lactams, with decreased susceptibility to carbapenems (MIC imipenem ϭ 4 g/ ml; MIC meropenem ϭ 8 g/ml). It was also resistant to aminoglycosides, co-trimoxazole, quinolones, and chloramphenicol but remained susceptible to tetracyclines-glycylcyclines, colistin, and rifampin (rifampicin).The transfer of ␤-lactam resistance from A. baumannii BM4674 to A. baumannii BM4652 was performed by conjugation on solid medium, as described previously (11). Transconjugants selected on agar containing apramycin (80 g/ml) and ceftazidime (16 g/ml) were obtained at a high frequency of ca. 1 ϫ 10 Ϫ3 per recipient cell. They exhibited a broad spectrum of resistance to ␤-lactams, including aztreonam, diminished susceptibility to imipenem (MIC ϭ 0.75 g/ml) and meropenem (MIC ϭ 1.5 g/ml) compared to that of the recipi...

BackgroundLeptospirosis is one of the most important neglected tropical bacterial diseases in Latin America and the Caribbean. However, very little is known about the circulating etiological agents of leptospirosis in this region. In this study, we describe the serological and molecular features of leptospires isolated from 104 leptospirosis patients in Guadeloupe (n = 85) and Martinique (n = 19) and six rats captured in Guadeloupe, between 2004 and 2012.Methods and FindingsStrains were studied by serogrouping, PFGE, MLVA, and sequencing 16SrRNA and secY. DNA extracts from blood samples collected from 36 patients in Martinique were also used for molecular typing of leptospires via PCR. Phylogenetic analyses revealed thirteen different genotypes clustered into five main clades that corresponded to the species: L. interrogans, L. kirschneri, L. borgpetersenii, L. noguchi, and L. santarosai. We also identified L. kmetyi in at least two patients with acute leptospirosis. This is the first time, to our knowledge, that this species has been identified in humans. The most prevalent genotypes were associated with L. interrogans serovars Icterohaemorrhagiae and Copenhageni, L. kirschneri serovar Bogvere, and L. borgpetersenii serovar Arborea. We were unable to identify nine strains at the serovar level and comparison of genotyping results to the MLST database revealed new secY alleles.ConclusionsThe overall serovar distribution in the French West Indies was unique compared to the neighboring islands. Typing of leptospiral isolates also suggested the existence of previously undescribed serovars.

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Prevention and control of leptospirosis are based on the knowledge of locally circulating strains. Thus, efforts to obtain local isolates are paramount to the epidemiological understanding of leptospirosis. We report and discuss here the first isolation of members of serogroups Autumnalis and Panama from cattle, both belonging to Leptospira noguchii species. Urine samples (n = 167) were collected directly by puncture of the bladder from randomly selected cows from a slaughterhouse in Rio de Janeiro, Brazil, for bacteriological culture. Isolates were characterized by serogrouping and sequencing (rrs and secY genes). Overall, 10/167 positive urine samples (6%) were obtained. Sequencing of amplicons targeting for both rrs and secY genes identified two of them (2013_U73 and 2013_U232) as L. noguchii. Serogrouping of those strains indicated that 2013_U73 belonged to the Panama serogroup (titre 1600), and 2013_U232 to the Autumnalis serogroup (titre 12800). Both Panama and Autumnalis are known agents of incidental leptospirosis in cattle. This group of leptospires could be particularly important in tropical countries. This is the first report of members of serogroups Autumnalis and Panama belonging to L. noguchii species from cattle. Although related to previously reported strains, these isolates have been shown to be genetically diverse from them.