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Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella and, as a result of the continuous emergence of multi-drug resistant (MDR) strains, the cause of severe nosocomial infections. The decline in the effectiveness of antibiotic treatments for infections caused by MDR bacteria has generated particular interest in the study of bacteriophages. In this study, we characterized a total of 40 temperate bacteriophages (prophages) with a genome range of 11.454-84.199 kb, predicted from 16 carbapenemase-producing clinical strains of K. pneumoniae belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order Caudovirales (27 prophages belonging to the family Myoviridae , 10 prophages belonging to the family Siphoviridae and 3 prophages belonging to the family Podoviridae ). Genomic comparison of the 40 prophage genomes led to the identification of four prophages isolated from different strains and of genome sizes of around 33.3, 36.1, 39.6 and 42.6 kb. These prophages showed sequence similarities (query cover >90 %, identity >99.9 %) with international Microbe Versus Phage (MVP) (http://mvp.medgenius.info/home) clusters 4762, 4901, 3499 and 4280, respectively. Phylogenetic analysis revealed the evolutionary proximity among the members of the four groups of the most frequently identified prophages in the bacterial genomes studied (33.3, 36.1, 39.6 and 42.6 kb), with bootstrap values of 100 %. This allowed the prophages to be classified into three clusters: A, B and C. Interestingly, these temperate bacteriophages did not infect the highest number of strains as indicated by a host-range assay, these results could be explained by the development of superinfection exclusion mechanisms. In addition, bioinformatic analysis of the 40 identified prophages revealed the presence of 2363 proteins. In total, 59.7 % of the proteins identified had a predicted function, mainly involving viral structure, transcription, replication and regulation (lysogenic/lysis). Interestingly, some proteins had putative functions associated with bacterial virulence (toxin expression and efflux pump regulators), phage defence profiles such as toxin-antitoxin modules, an anti-CRISPR/Cas9 protein, TerB protein (from ter ZABCDE operon) and methyltransferase proteins.

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with bla CMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid., (Copyright © 2020 American Society for Microbiology.)

Background: Quinolone resistance in Enterobacteriaceae species has increased over the past few years, and is significantly associated to beta-lactam resistance. The aim of this study was to evaluate the prevalence of chromosomal- and plasmid-mediated quinolone resistance in acquired AmpC β-lactamase and/or carbapenemase-producing Enterobacteriaceae isolates., Methods: The presence of chromosomal- and plasmid-mediated quinolone resistance mechanisms [mutations in the quinolone resistance determining region (QRDR) of gyrA and parC and qnr, aac(6')-Ib-cr and qepA genes] was evaluated in 289 isolates of acquired AmpC β-lactamase- and/or carbapenemase-producing Enterobacteriaceae collected between February and July 2009 in 35 Spanish hospitals., Results: Plasmid mediated quinolone resistance (PMQR) genes were detected in 92 isolates (31.8%), qnr genes were detected in 83 isolates (28.7%), and the aac(6')-Ib-cr gene was detected in 20 isolates (7%). qnrB4 gene was the most prevalent qnr gene detected (20%), associated, in most cases, with DHA-1. Only 14.6% of isolates showed no mutations in gyrA or parC with a ciprofloxacin MIC of 0.5mg/L or higher, whereas PMQR genes were detected in 90% of such isolates., Conclusion: qnrB4 gene was the most prevalent PMQR gene detected, and was significantly associated with acquired AmpC β-lactamase DHA-1. PMQR determinants in association with other chromosomal-mediated quinolone resistance mechanisms, different to mutations in gyrA and parC (increased energy-dependent efflux, altered lipopolysaccharide or porin loss), could lead to ciprofloxacin MIC values that exceed breakpoints established by the main international committees to define clinical antimicrobial susceptibility breakpoints., (Copyright © 2016 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

We studied in parallel the population structure of 90 carbapenemase-producing and 88 carbapenemase-susceptible Klebsiella pneumoniae isolates collected in 20 Spanish hospitals, in the context of the EuSCAPE project. Fourteen and 50 multilocus sequence types (MLSTs) were detected among the carbapenemase-producing and carbapenem-susceptible isolates, respectively. ST11 and ST15 clones were more frequent in the carbapenemase-producing group than in the carbapenemase-susceptible group ( P < 0.0001). Among the members of the carbapenem-suceptible group, the cefotaxime-resistant population showed population parameters that differed between the populations of the wild-type strains and the carbapenemase producers., (Copyright © 2017 American Society for Microbiology.)

Escherichia coli recovered from three hospitals in Barcelona (Spain) were studied to determine the prevalence of isolates with acquired AmpC (ac-AmpC) and/or overproduced chromosomal AmpC (c-AmpC). Mechanisms involved in blac-AmpC overexpression, blaac-AmpC and the plasmids associated with their distribution as well as the prevalence of plasmid-mediated quinolone resistance (PMQR) in AmpC-producing isolates were also determined. Isolates were selected according to their resistance phenotype. blaac-AmpC, alterations in the blac-AmpC promoter/attenuator, and PMQR genes [qnrA, qnrB, qnrS, aac(6')-Ib-cr and qepA] were characterised by PCR and sequencing. blac-AmpC expression was determined by qRT-PCR. Population structure analysis was performed using PFGE, MLST and phylogenetic group PCR. Plasmids carrying blaac-AmpC were characterised by PCR-based replicon typing and S1-PFGE. IncI1 and IncF plasmids were also analysed by plasmid MLST and replicon sequence typing, respectively. Among 21563 E. coli isolates, 240 (1.1%) overproduced AmpC β-lactamases, including 180 (75.0%) harbouring ac-AmpC (132 CMY-2 variants and 48 DHA-1) and 60 (25.0%) c-AmpC enzymes. Three mutation profiles in the blac-AmpC promoter/attenuator were associated with a 72.5-, 19.9- and 5.8-fold increased expression, respectively. Moreover, 63.3% of ac-AmpC and 43.3% of c-AmpC isolates belonged to B2, D, E or F phylogenetic groups. PMQR was found in 31% of ac-AmpC isolates [38 qnrB4, 8 aac(6')-Ib-cr, 6 qnrS1 and 3 qnrB19] and in 10% of c-AmpC isolates [5 aac(6')-Ib-cr and 1 qnrS1]. IncI1-ST12 and IncF were associated with blaCMY-2 and blaDHA-1, respectively. These results suggest that ac-AmpC β-lactamases were the main mechanism of AmpC production. Isolates and plasmids both showed high genetic diversity., (Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Objectives: To describe the prevalence and risk factors for infection due to AmpC β-lactamase-producing Escherichia coli (AmpC-EC)., Methods: For the prevalence study, all clinical isolates of E. coli with reduced susceptibility to third-generation cephalosporins were prospectively included from June 2010 to November 2011. For risk factor analysis, a case-control study was conducted. Cases were patients with an infection due to AmpC-EC. Controls were patients infected with cephalosporin-susceptible E. coli, matched 1 : 2. Detection of blaAmpC genes was done with a multiplex AmpC-PCR, and hyperproduction of E. coli chromosomal blaAmpC by quantitative RT-PCR. Alteration of the blaAmpC promoter was studied by PCR and sequencing., Results: We identified 243 (1.1%) AmpC-EC strains out of 21 563 clinical isolates. Three cases with strains carrying ESBLs, 18 strains that were considered due to colonization and 8 cases lost to clinical follow-up were excluded. Finally, 214 cases were included in the analysis. Ninety-one cases (42.5%) and 269 (62.8%) controls were strictly community acquired (P < 0.001). Thirty-five (16.3%) cases and 186 controls (43.5%) did not have any identifiable risk factor (P < 0.001). Among cases, 158 (73.8%) were found to harbour an acquired AmpC (73.4% CMY-2). Previous use of fluoroquinolones [OR 2.6 (95% CI 1.12-3.36); P = 0.008] was independently associated with AmpC-EC in the multivariate analysis., Conclusions: Prevalence of AmpC in E. coli remains low in our area. Plasmid acquisition (CMY type) represents the main mechanism of AmpC production. A high proportion of community-acquired isolates and patients with no identifiable risk factors were found. Previous use of fluoroquinolones was identified as a risk factor., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The aim of this study was to determine the impact of carbapenemase-producing Enterobacteriaceae (CPE) in Spain in 2013 by describing the prevalence, dissemination, and geographic distribution of CPE clones, and their population structure and antibiotic susceptibility. From February 2013 to May 2013, 83 hospitals (about 40,000 hospital beds) prospectively collected nonduplicate Enterobacteriaceae using the screening cutoff recommended by EUCAST. Carbapenemase characterization was performed by phenotypic methods and confirmed by PCR and sequencing. Multilocus sequencing types (MLST) were determined for Klebsiella pneumoniae and Escherichia coli. A total of 702 Enterobacteriaceae isolates met the inclusion criteria; 379 (54%) were CPE. OXA-48 (71.5%) and VIM-1 (25.3%) were the most frequent carbapenemases, and K. pneumoniae (74.4%), Enterobacter cloacae (10.3%), and E. coli (8.4%) were the species most affected. Susceptibility to colistin, amikacin, and meropenem was 95.5%, 81.3%, and 74.7%, respectively. The most prevalent sequence types (STs) were ST11 and ST405 for K. pneumoniae and ST131 for E. coli. Forty-five (54.1%) of the hospitals had at least one CPE case. For K. pneumoniae, ST11/OXA-48, ST15/OXA-48, ST405/OXA-48, and ST11/VIM-1 were detected in two or more Spanish provinces. ST11 isolates carried four carbapenemases (VIM-1, OXA-48, KPC-2, and OXA-245), but ST405 isolates carried OXA-48 only. A wide interregional spread of CPE in Spain was observed, mainly due to a few successful clones of OXA-48-producing K. pneumoniae (e.g., ST11 and ST405). The dissemination of OXA-48-producing E. coli is a new finding of public health concern. According to the susceptibilities determined in vitro, most of the CPE (94.5%) had three or more options for antibiotic treatment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

In recent years, Enterobacteriaceae isolates have increased their potential to become highly drug resistant by acquiring resistance to carbapenems, primarily due to the production of acquired carbapenemases. The carbapenemases detected in Enterobacteriaceae are largely of the KPC, VIM, NDM, IMP and OXA-48 types. Although the epidemiological origin and geographic distribution of carbapenemases are clearly different, they all first appeared in the late 20th Century. Only a decade later, these enzymes have already become established and have expanded globally. An important epidemiological change has occurred in Spain in recent years, characterized by a rapid increase in the number of cases of carbapenemase-producing Enterobacteriaceae (CPE), causing both nosocomial outbreaks and single infections. The impact of CPE in Spain is primarily due to OXA-48-producing and VIM-1-producing Klebsiella pneumoniae isolates, although other species such as Escherichia coli and Enterobacter cloacae are also increasing. The emergence of CPE as a cause of community-onset infections is a matter of great concern. Taking into account recent experience, and considering the fact that increasing numbers of patients are becoming infected by CPE and reservoirs of carbapenemases are growing globally, the trend of the CPE epidemic points toward a rise in its incidence. To prevent a massive CPE pandemic, a well-coordinated response from all health professionals and national and supranational authorities is clearly needed., (Copyright © 2014 Elsevier España, S.L.U. All rights reserved.)

During a Spanish surveillance study, two natural variants of DHA β-lactamases, DHA-6 and DHA-7, were found, with the replacements Ala226Thr and Phe322Ser, respectively, with respect to DHA-1. The DHA-6 and DHA-7 enzymes were isolated from Escherichia coli and Enterobacter cloacae clinical isolates, respectively. The aim of this study was to genetically, microbiologically, and biochemically characterize the DHA-6 and DHA-7 β-lactamases. The blaDHA-6 and blaDHA-7 genes were located in the I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The genetic contexts of blaDHA-6 and blaDHA-7 were similar to that already described for the blaDHA-1 gene and included the qnrB4 and aadA genes. The MICs for cephalothin, aztreonam, cefotaxime, and ceftazidime were 8- to 32-fold lower for DHA-6 than for DHA-1 or DHA-7 expressed in the same isogenic E. coli TG1 strain. Interestingly, the MIC for cefoxitin was higher in the DHA-6-expressing transformant than in DHA-1 or DHA-7. Biochemical studies with pure β-lactamases revealed slightly lower catalytic efficiencies of DHA-6 against cephalothin, ceftazidime, and cefotaxime than those of DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed that the DHA-6 protein displayed significantly higher stability than the DHA-1 and DHA-7 enzymes. The proximity of Thr226 to the N terminus in the tertiary protein structure in DHA-6 may promote this stabilization and, consequently, may induce a slight reduction in the dynamic of this enzyme that primarily affects the hydrolysis of some of the bulkiest antibiotics., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A novel class C β-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8-fold reduction in resistance to ceftazidime relative to FOX-3. In a kinetic analysis, FOX-8 displayed a 33-fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E. coli strains carrying this variant.

A total of 788 clinical Enterobacteriaceae were collected to describe the aminoglycoside-modifying genes (AME genes) and to characterize the plasmids that carry these genes. Among the 788 strains collected, 330 (41.8%) were aminoglycoside-resistant: 264 Escherichia coli (80%), 33 Proteus mirabilis (10%), 10 Klebsiella pneumoniae (3%), six K. oxytoca (1.8%), five Enterobacter cloacae (1.5%), three Morganella morganii (0.9%), three Providencia stuartii (0.9%), two Salmonella enterica (0.6%), and one each Citrobacter freundii, C. koseri, Proteus vulgaris, and Shigella sonnei. The most affected aminoglycoside was streptomycin (92.7%), followed by kanamycin (26.3%), gentamicin (18%), tobramycin (16.9%), netilmicin (3.6%), and amikacin (1.5%). The AME genes found were aph(3″)-Ib (65.4%), ant(3″)-Ia (37.5%), aph(3')-Ia (13.9%), aac(3)-IIa (12.4%), aac(6')-Ib (4.2%), ant(2″)-Ia (3.6%), and aph(3')-IIa (1.2%). Thirty-four percent of the strains showed more than one enzyme. The most frequent association was ant(3″)-Ia plus aph(3″)-Ib (35 strains). From 66 selected AME genes, 24 were plasmid located: 12 aac(3)-IIa, six aph(3')-Ia, three ant(3″)-Ia, two ant(2″)-Ia, and one aac(6')-Ib. These genes were located in plasmids belonging to incompatibility groups F, FIA, FIB, or HI2. In conclusion, the AME genes involved in aminoglycoside-clinical resistance were aac(3)-IIa, aac(6')-Ib, and ant(2″)-Ia, genes that confer resistance to tobramycin, gentamicin, and amikacin.

An isolate of Klebsiella oxytoca carrying a novel IMP metallo-β-lactamase was discovered in Madrid, Spain. The bla(IMP-28) gene is part of a chromosomally located class I integron. The IMP-28 k(cat)/K(m) values for ampicillin, ceftazidime, and cefepime and, to a lesser extent, imipenem and meropenem, are clearly lower than those of IMP-1. The His306Gln mutation may induce important modifications of the L3 loop and thus of substrate accessibility and hydrolysis and be the main reason for this behavior.

Objectives: To investigate the epidemiology and clinical features of infections caused by Enterobacteria producing plasmid-mediated AmpC β-lactamases (pAmpC), which are emerging as a cause of resistance to extended-spectrum cephalosporins., Methods: A prospective multicentre cohort of patients with infection/colonisation due to pAmpC-producing Enterobacteriaceae was performed in 7 Spanish hospitals from February throughout July 2009. pAmpCs were characterised by PCR and sequencing., Results: 140 patients were included; organisms isolated were Escherichia coli (n = 100), Proteus mirabilis (n = 20), Klebsiella pneumoniae (n = 17), and others (n = 3). Overall, 90% had a chronic underlying condition. The acquisition was nosocomial in 43%, healthcare-associated in 41% (14% of those were nursing home residents), and community in 16%. Only 5% of patients had no predisposing feature for infection with multidrug-resistant bacteria. Nineteen percent of patients were bacteraemic. Inappropriate empirical therapy was administered to 81% of bacteraemic patients, who had a crude mortality rate of 48%. The most frequent enzyme was CMY-2 (70%, predominantly in E. coli and P. mirabilis) followed by DHA-1 (19%, predominantly in K. pneumoniae)., Conclusion: pAmpC-producing Enterobacteriaceae caused nosocomial, healthcare-associated and community infections mainly in predisposed patients. Invasive infections were associated with high mortality which might be partly related to inappropriate empirical therapy., (Copyright © 2011 The British Infection Association. Published by Elsevier Ltd. 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 bla(CMY-2), while L/M replicons were associated with bla(DHA-1). bla(ACC-1) was linked to I1 and MOB(F11) plasmids; bla(CMY-27) was associated with IncF and MOB(P12) plasmids; the plasmid carrying bla(CMY-25) could not be typed, and bla(CMY-40) was chromosomally located. All 87 isolates carrying bla(CMY-2), bla(CMY-4), bla(CMY-25), bla(CMY-27), bla(CMY-40) or bla(ACC-1) displayed the transposon-like structures ISEcp1/ΔISEcp1-bla(CMY)-blc-sugE or ΔISEcp1-bla(ACC-1)-gdha. The most prevalent structure in bla(DHA-1) (93.3% of cases) was identical to that described in the Klebsiella pneumoniae pTN60013 plasmid. Remarkably, in three isolates containing chromosomal bla(CMY-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 localisation and genetic organisation of bla(CTX-M-15) were studied in 37 CTX-M-15-producing Klebsiella pneumoniae isolates collected from 2005 to 2008 within the Barcelona metropolitan area. Polymerase chain reaction (PCR)-based replicon typing and Southern hybridisations were used to identify the bla(CTX-M-15) location. The genetic environment was analysed by PCR mapping and sequencing, and transferability of bla(CTX-M-15) was evaluated by conjugation and transformation assays. The majority of the 37 isolates carried bla(CTX-M-15) in a plasmid location, frequently associated with the aac(6')-Ib-cr gene. Plasmids encoding bla(CTX-M-15) carried three distinct replicons, i.e. IncFII, IncR and IncFIIk, the latter two not having been described previously in association with bla(CTX-M-15). Several of these plasmids were not self-transferable. Furthermore, in all isolates belonging to sequence type ST-1, bla(CTX-M-15) was found integrated into the K. pneumoniae chromosome. In all the studied isolates, the mobile element ISEcp1 was found upstream of bla(CTX-M-15), whereas IS26 was found inserted within ISEcp1 in several isolates, in previously unreported positions. In conclusion, these findings indicate that among K. pneumoniae strains isolated in the Barcelona metropolitan area, bla(CTX-M-15) is associated with diverse genetic elements, including the IncR and IncFIIk replicons, as reported for the first time here, and the chromosome., ((c) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Objectives: We describe 12 VIM-1-producing strains (7 Enterobacter cloacae, 2 Klebsiella pneumoniae and 3 clonal Klebsiella oxytoca strains) detected among clinically relevant Enterobacteriaceae isolates from routine cultures at the Hospital del Mar (Barcelona, Spain) from December 2006 to May 2007., Methods: Susceptibility to carbapenems was evaluated with the MicroScan system. beta-Lactamases were identified by PCR and sequencing. Clonal relationships between the isolates were analysed by PFGE. Transferability of the enzymes was tested by conjugation. Plasmid characterization was performed by PCR-based replicon typing and PFGE with S1 nuclease digestion of whole genomic DNA. The PFGE gels were then transferred and hybridized., Results: The disc diffusion method correctly identified five of the seven E. cloacae isolates as intermediate or resistant strains. All isolates produced the VIM-1 enzyme. Three E. cloacae and three K. oxytoca strains were also CTX-M-9-producing strains, and one E. cloacae was also a CTX-M-3-producing strain. The plasmids carrying the bla(VIM) gene, of unknown incompatibility group, had a size of approximately 75 kb (eight strains) or 40 kb (three strains) and also contained the qnrS and the aac(6')-Ib-cr genes. In the remaining strain the bla(VIM-1) gene was found in an HI2 plasmid of 290 kb together with bla(CTX-M-9), qnrA, qnrS and the aac(6')-Ib-cr genes., Conclusions: The results showed a linkage between the bla(VIM-1) and the qnrS and the aac(6')-Ib-cr genes, and between the bla(CTX-M-9) and the qnrA genes.

Background: Screening methods for the detection of plasmid-mediated AmpC beta-lactamases are technically demanding. The purpose of this study was to assess screening methods for the detection of these enzymes in clinical isolates of Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis., Methods: Isolates were selected according to a resistance phenotype consistent with production of an AmpC-type beta-lactamase. Detection of acquired ampC genes was done with a multiplex ampC-PCR and sequencing. The phenotypic detection methods evaluated included visual examination of antibiogram plates to identify the presence of scattered colonies located near the edge of the inhibition halo of cefoxitin, cefotaxime, ceftazidime and aztreonam, and a double-disc synergy test using cloxacillin (500 mg) to inhibit AmpC enzymes., Results: Seventy-seven isolates were selected from among 6,209 isolates recovered. Acquired ampC genes (blaCMY-2, blaDHA-1, blaCMY-4 and blaACC-1) were found in 19 (24.7%) of these isolates, including 14 E. coli, two K. pneumoniae and three P. mirabilis isolates. The differential trait for the presence of colonies in the inhibition halo was 100% sensitive and specific. Similar results were obtained for the cloxacillin test, except for the E. coli isolates in which specificity was 10.3%., Conclusion: The phenotypic trait described here can be considered useful for suspecting the presence of these enzymes. The cloxacillin test was only useful in isolates lacking a natural AmpC beta-lactamase.