The pUM505 plasmid, isolated from a clinical isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat infections. analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the gene in pUM505 displays 40% amino acid identity to the aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned (renamed, for iprofloxacinesistance rotein,lasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-, conferred resistance to CIP on strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in, which involves phosphorylation of the antibiotic.

Acinetobacter baumannii is an emergent bacterial pathogen that provokes many types of infections in hospitals around the world. The genome of this organism consists of a chromosome and plasmids. These plasmids vary over a wide size range and many of them have been linked to the acquisition of antibiotic-resistance genes. Our bioinformatic analyses indicate that A. baumannii plasmids belong to a small number of plasmid lineages. The general structure of these lineages seems to be very stable and consists not only of genes involved in plasmid maintenance functions but of gene sets encoding poorly characterized proteins, not obviously linked to survival in the hospital setting, and opening the possibility that they improve the parasitic properties of plasmids. An analysis of genes involved in replication, suggests that members of the same plasmid lineage are part of the same plasmid incompatibility group. The same analysis showed the necessity of classifying the Rep proteins in ten new groups, under the scheme proposed by Bertini et al. (2010). Also, we show that some plasmid lineages have the potential capacity to replicate in many bacterial genera including those embracing human pathogen species, while others seem to replicate only within the limits of the Acinetobacter genus. Moreover, some plasmid lineages are widely distributed along the A. baumannii phylogenetic tree. Despite this, a number of them lack genes involved in conjugation or mobilization functions. Interestingly, only 34.6% of the plasmids analyzed here possess antibiotic resistance genes and most of them belong to fourteen plasmid lineages of the twenty one described here. Gene flux between plasmid lineages appears primarily limited to transposable elements, which sometimes carry antibiotic resistance genes. In most plasmid lineages transposable elements and antibiotic resistance genes are secondary acquisitions. Finally, broad host-range plasmids appear to have played a crucial role.

ObjectivesTo characterize the microbiological, molecular and epidemiological data of an outbreak of carbapenem-resistant Enterobacteriaceae (CRE) in a tertiary-care hospital in Mexico.MethodsFrom September 2014 to July 2015, all CRE clinical isolates recovered during an outbreak in the Hospital Civil "Fray Antonio Alcalde" in Jalisco, Mexico were screened for antimicrobial susceptibility, carbapenemase production, carbapenemase-encoding genes, and plasmid profiles. Horizontal transfer of imipenem resistance; and clonal diversity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST); as well as biofilm production and the presence of 14 virulence genes were analyzed in selected isolates.ResultsFifty-two carbapenem-resistant isolates corresponding to 5 species were detected, i.e., Klebsiella pneumoniae (n = 46), Enterobacter cloacae (n = 3), Escherichia coli (n = 1), Providencia rettgeri (n = 1) and Citrobacter freundii (n = 1) with carbapenemase encoding genes blaNDM-1 (n = 48), blaVIM (n = 3), blaIMP (n = 1) and blaKPC (n = 1) detected in these isolates.The blaNDM-1 gene was detected in plasmids from 130- to 170-kb in K. pneumoniae (n = 46); E. cloacae (n = 3), E. coli (n = 1) and P. rettgeri (n = 1). The transfer of plasmids harboring the blaNDM-1 gene was obtained in eight transconjugants. One plasmid restriction pattern was detected, with the blaNDM-1 identified in different restriction fragments.Predominant clone A of K. pneumoniae isolates archived 28/46 (60%) isolates and belongs to ST392. Besides, ST307, ST309, ST846, ST2399, and ST2400 were detected for K. pneumoniae; as well as E. cloacae ST182 and E. coli ST10.The fimA and uge genes were more likely to be identified in K. pneumoniae carbapenem-susceptible isolates (p = <0.001) and biofilm production was more liable to be observed in carbapenem-resistant isolates (p = <0.05).ConclusionsFour Enterobacteriaceae species harboring the blaNDM-1 gene were detected in a nosocomial outbreak in Mexico; horizontal transfer and strain transmission were demonstrated for the blaNDM-1 gene. Given the variation in the size of the plasmid harboring blaNDM-1, complex rearrangements must also be occurring.

BackgroundKlebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.ResultThis work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.ConclusionsThis multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0396-6) contains supplementary material, which is available to authorized users.

IntroductionPatients with hematologic malignancies have greater risk-factors for primary bloodstream infections (BSI).MethodsFrom 2004–2009, we analyzed bacteremia caused by extended-spectrum beta-lactamase Escherichia coli (ESBL-EC) (n = 100) and we compared with bacteremia caused by cephalosporin-susceptible E. coli (n = 100) in patients with hematologic malignancies.ObjectiveTo assess the clinical features, risk factors, and outcome of ESBL-EC BSI in patients with hematologic malignancies, and to study the molecular epidemiology of ESBL-EC isolates.ResultsThe main diagnosis was acute leukemia in 115 patients (57.5%). Death-related E. coli infection was significantly increased with ESBL-EC (34% vs. control group, 19%; p = 0.03). Treatment for BSI was considered appropriate in 64 patients with ESBL-EC (mean survival, 245±345 days), and in 45 control patients this was 443±613 (p = 0.03). In patients not receiving appropriate antimicrobial treatment, survival was significantly decreased in cases compared with controls (26±122 vs. 276±442; p = 0.001). Fifty six of the ESBL-EC isolates were characterized by molecular analysis: 47 (84%) expressed CTX-M-15, two (3.6%) SHV, and seven (12.5%) did not correspond to either of these two ESBL enzymes. No TLA-1 enzyme was detected.ConclusionsPatients who had been previously hospitalized and who received cephalosporins during the previous month, have an increased risk of ESBL-EC bacteremia. Mortality was significantly increased in patients with ESBL-EC BSI. A polyclonal trend was detected, which reflects non-cross transmission of multiresistant E.coli isolates.

A novel QnrB-like plasmid-mediated resistance determinant, QnrB19, was identified from an Escherichia coli clinical isolate from Colombia. Its gene was associated with an ISEcp1-like insertion element that did not act as a promoter for its expression. Using an in vitro model of transposition, we showed that the ISEcp1-like element was able to mobilize the qnrB19 gene.Resistance to quinolones in Enterobacteriaceae most commonly arises stepwise as a result of chromosomal mutations responsible for the modification of target enzymes (DNA gyrase and topoisomerase IV) or decreased intracellular drug accumulation by the upregulation of efflux pumps and/or modified outer-membrane porins (27). Since the discovery of the first plasmid-mediated quinolone resistance (PMQR) determinant, QnrA, in 1998 (15), four other PMQR determinants have been identified to date: the QnrB (12) and QnrS (10) proteins, the aminoglycoside acetyltransferase AAC(6Ј)-Ib-cr (25), and the efflux pump QepA (19,30). Resistance due to Qnr determinants is increasingly reported worldwide in enterobacterial isolates (18,26) and has been recently identified outside Enterobacteriaceae in environmental Aeromonas isolates from France (3). The three types of Qnr determinants, QnrA, QnrB, and QnrS, belong to the pentapeptide repeat family of proteins (18,26). By protecting DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones, Qnr proteins confer resistance to quinolones (e.g., nalidixic acid) and decreased susceptibility to fluoroquinolones, therefore facilitating the recovery of chromosome-encoded target mutants with a higher level of resistance to fluoroquinolones (18, 26). Whereas qnrAlike genes have only been identified as part of the complex sul1-type integrons in association with the orf513 transposase gene (18,26), which is part of a region redefined as ISCR1 (28), qnrB-like genes have been found to be associated with either the orf1005 gene encoding a putative transposase (12) or the ISCR1 element (8,24).The aim of this study was (i) to investigate the genetic environment of a qnrB-like gene from an Escherichia coli clinical isolate, (ii) to evaluate experimentally the mobility of that putative transposon in E. coli, and (iii) to determine the promoter sequences of the qnrB-like gene responsible for the expression of that gene.E. coli R4525 expressing an extended-spectrum ␤-lactamase (ESBL) phenotype had been isolated in 2002 from a wound culture of a patient hospitalized at the Hospital San Jeronimo in Monteria, Colombia. In the course of studying the genetic support of the ESBL determinant in E. coli R4525, conjugation experiments followed by selection with sodium azide (100 g/ ml) and amoxicillin (50 g/ml) as previously described (3) gave E. coli J53 transconjugants displaying an ESBL phenotype and decreased susceptibility to fluoroquinolones. This result prompted us to search for the presence of PMQR determinants. The screening of qnr genes using a multiplex strategy (5) identified a qnrB-like gene in both E. coli R4525 and it...

Fecal colonization by ESBL-EC is associated with increased risk of BSI by this strain, longer hospital stay, and higher related costs.

During 2003, 40 carbapenem-resistant Pseudomonas aeruginosa clinical isolates collected in a Mexican tertiary-care hospital were screened for metallo-␤-lactamase production. Thirteen isolates produced IMP-15, and 12 had a single pulsed-field gel electrophoresis pattern. The bla IMP-15 gene cassette was inserted in a plasmid-borne integron with a unique array of gene cassettes and was named In95.