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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.)

Introduction: To characterize OXA-48 carbapenemase-producing Klebsiella pneumoniae strains isolated after an increase in carbapenem resistance in Catalonia., Methodology: K. pneumoniae identification, antimicrobial susceptibility studies, the Modified Hodge Test method, amplification of antimicrobial resistance genes (against β-lactamases, quinolones and aminoglycosides), molecular typing (by PFGE and MLST), conjugation assays, plasmid characterization (PBRT-PCR and Southern blot), a description of mobile genetic elements and statistical analysis were done., Results: OXA-48 was the only carbapenemase detected, with a prevalence of 1.9%. The bla OXA-48 gene was located in an IncL conjugative plasmid of 62kb and integrated into the transposons Tn1999.2 (91.7%) or Tn1999.1. Five PFGE profiles (A to E) were found, which exactly matched the MLST: ST101, ST17, ST1233, ST14 and ST405, respectively. ST1233 is described here for the first time. K. pneumoniae OXA-48-producing strains were also CTX-M-15 carriers, some producing OXA-1 and TEM-1 penicillinases. The acquired qnrB66 and qnrB1 and aac(3')-IIa, aac(6')-Ib genes were also identified., Conclusion: The K. pneumoniae ST405 clone has played an important role in the growing prevalence of OXA-48 in Catalonia. All clones described preserved the bla OXA-48 genetic environment and mobile genetic elements (Tn1999). Notably, the three strains with minor sequence types in this study are not multiresistant strains. These strains are expanding in elderly patients (average age of 76 years) with serious underlying diseases, mainly women (61.2%)., (Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Phage particles have emerged as elements with the potential to mobilise antibiotic resistance genes (ARGs) in different environments, including the intestinal habitat. This study aimed to determine the occurrence of ARGs in phage particles present in faecal matter and induced from strains isolated from faeces. Nine ARGs (bla TEM , bla CTX-M-1 group , bla CTX-M-9 group , bla OXA-48 , qnrA, qnrS, mecA, sul1 and armA) were quantified by qPCR in the phage DNA fractions of 150 faecal samples obtained from healthy individuals who had not received antibiotic treatment or travelled abroad in the 3 months prior to sample collection. On the suspicion that the detected particles originated from bacterial flora, 82 Escherichia coli and Klebsiella pneumoniae isolates possessing at least one identified ARG (bla TEM , bla CTX-M-1 group , bla CTX-M-9 group , armA, qnrA, qnrS and sul1) were isolated and their capacity to produce phage particles carrying these ARGs following induction was evaluated. Of 150 samples, 72.7% were positive for at least one ARG, with bla TEM and bla CTX-M-9 group being the most prevalent and abundant. Of the 82 isolates, 51 (62%) showed an increase in the number of copies of the respective ARG in the phage fraction following induction, with bla TEM , bla CTX-M-1 group , bla CTX-M-9 group and sul1 being the most abundant. Phages induced from the isolates were further purified and visualised using microscopy and their DNA showed ARG levels of up to 10 10 gene copies/mL. This study highlights the abundance of phage particles harbouring ARGs and indicates that bacterial strains in the intestinal habitat could be source of these particles., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. 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.)

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.

Objectives: The aim of the study was to characterize plasmids that harbour blaESBL genes and their genetic environment in Escherichia coli and Klebsiella pneumoniae clones circulating in Spain., Methods: The incompatibility group of plasmids within 58 strains harbouring blaCTX-M (n=45) and blaSHV (n=15) genes was determined by rep-typing-PCR and hybridization. The blaESBL genetic environment was determined by PCR and sequencing., Results: The blaCTX-M-9 genes (n=14) were linked to In60 located in IncI1 (50%) or IncHI2 plasmids (28%). All blaCTX-M-14 genes (n=13) were flanked by ISEcp1 and IS903 and 12 were associated with IncK plasmids. One of two blaCTX-M-10 genes was present in an IncK plasmid, but both genes were linked to a phage-related element. Five of seven blaCTX-M-1 (71%), all three blaCTX-M-32 and one of two blaCTX-M-3 genes were linked to IncN plasmids. The other blaCTX-M-3 gene was linked to IncA/C and the remaining two blaCTX-M-1 genes to IncFII plasmids. Three blaCTX-M-15 genes were associated with IncF (repFIA) and one with IncFII plasmids. All these genes from blaCTX-M group-1 showed the ISEcp1 upstream truncated by different insertion sequences. Forty-three percent of blaSHV-12 genes (n=14) were located in IncI1 plasmids, all flanked by the IS26 and DEOR region. The only detected blaSHV-5 gene was located in an IncFII plasmid and flanked by recF and DEOR regions., Conclusions: A diversity of the plasmid incompatibility groups that harbour blaESBL genes was observed, except for the blaCTX-M-14 gene. Moreover, a high variability was confirmed in the genetic environment of these genes as a result of insertion and deletion events.

Introduction: The epidemiological distribution of extended-spectrum beta-lactamase (ESBL) types in Escherichia coli and Klebsiella pneumoniae was evaluated in various hospitals in Spain and compared with previous studies., Methods: A total of 11 Spanish hospitals participated in this study. Each center collected the first 15 isolates of E. coli and the first 5 of K. pneumoniae suspected of being ESBL-producers and isolated during the first quarter of 2004. Clonal study was done by PFGE after total DNA digestion with XbaI and by ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus Sequences-Polymerase Chain Reaction), typing. ESBL-producers were characterized by isoelectric focusing (IEF), PCR and sequencing., Results: A total of 124 strains were collected. PFGE restriction patterns showed considerable diversity among E. coli strains; 4 clusters of 2 strains each were detected. ESBL characterization of 92 E. coli strains showed a predominance of CTX-M-14 (45.7%), CTX-M-9 (20.6%) and SHV-12 (21.7%). Clonal diversity among the 32 K. pneumoniae strains was less pronounced than in E. coli; 3 clusters included 53.1% of strains. The ESBL detected in these strains included a CTX-M type in 20 cases (62.5%) (CTX-M-1, CTX-M-9, CTX-M-14 and CTX-M-15); a SHV type in 11 (34.4%) (SHV-12 and SHV-5) and TEM-4 (3.1%) in 1 case., Conclusion: The E. coli and K. pneumoniae strains analyzed in this period displayed a greater diversity of ESBL than has been observed in previous epidemiological studies. Analysis of clonal relationships revealed a greater diversity in E. coli than in K. pneumoniae.

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.

Two strains of Enterobacteriaceae, Escherichia coli and Klebsiella pneumoniae, producing VIM-1 were isolated for the first time in Spain. In both strains, bla(VIM-1) was found to be carried on a gene cassette inserted into a class 1 integron. The bla(VIM-1)-containing integron was located on a transferable plasmid.

Objectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain. Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA−48 (263/377), blaKPC−3 (62/377), blaVIM−1 (28/377), and blaNDM−1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3. [ABSTRACT FROM AUTHOR]

Objectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain. Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis. Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were bla OXA–48 (263/377), bla KPC–3 (62/377), bla VIM–1 (28/377), and bla NDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5). Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3. [ABSTRACT FROM AUTHOR]

Whole-genome sequencing (WGS)-based typing methods have emerged as promising and highly discriminative epidemiological tools. In this study, we combined gene-by-gene allele calling and core genome single nucleotide polymorphism (cgSNP) approaches to investigate the genetic relatedness of a well-characterized collection of OXA-48 - producing Klebsiella pneumoniae isolates. We included isolates from the predominant sequence type ST405 (n = 31) OXA-48-producing K. pneumoniae clone and isolates from ST101 (n = 3), ST14 (n = 1), ST17 (n = 1), and ST1233 (n = 1), obtained from eight Catalan hospitals. Core-genome multilocus sequence typing (cgMLST) schemes from Institut Pasteur's BIGSdb-Kp (634 genes) and SeqSphere+ (2,365 genes), and a SeqSphere+ whole-genome MLST (wgMLST) scheme (4,891 genes) were used. Allele differences or allelic mismatches and the genetic distance, as the proportion of allele differences, were used to interpret the results from a gene-by-gene approach, whereas the number of SNPs was used for the cgSNP analysis. We observed between 0–10 and 0–14 allele differences among the predominant ST405 using cgMLST and wgMLST from SeqSphere+, respectively, and <2 allelic mismatches when using Institut Pasteur's BIGSdb-Kp cgMLST scheme. For ST101, we observed 14 and 54 allele differences when using cgMLST and wgMLST SeqSphere+, respectively, and 2–5 allelic mismatches for BIGSdb-Kp cgMLST. A low genetic distance (<0.0035, a previously established threshold for epidemiological link) was generally in concordance with a low number of allele differences (<8) when using the SeqSphere+ cgMLST scheme. The cgSNP analysis showed 6–29 SNPs in isolates with identical allelic SeqSphere+ cgMLST profiles and 16–61 cgSNPs among ST405 isolates. Furthermore, comparison of WGS-based typing results with previously obtained MLST and pulsed-field gel electrophoresis (PFGE) data showed some differences, demonstrating the different molecular principles underlying these techniques. In conclusion, the use of the different WGS-based typing methods that were used to elucidate the genetic relatedness of clonal OXA-48-producing K. pneumoniae all led to the same conclusions. Furthermore, threshold parameters in WGS-based typing methods should be applied with caution and should be used in combination with clinical epidemiological data and population and species characteristics. [ABSTRACT FROM AUTHOR]

Summary: 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 &y& Elsevier]

Some Aeromonas species, potentially pathogenic for humans, are known to express up to three different classes of chromosomal β-lactamases, which may become hyperproduced and cause treatment failure. The aim of this study was to assess the utility of these species-specific β-lactamase genes as phylogenetic markers using whole-genome sequencing data. Core-genome alignments were generated for 36 Aeromonas genomes from seven different species and scanned for antimicrobial resistance genes. Core-genome alignment confirmed the MALDI-TOF identification of most of the isolates and re-identified an A. hydrophila isolate as A. dhakensis. Three (B, C and D) of the four Ambler classes of β-lactamase genes were found in A. sobria, A. allosacharophila, A. hydrophila and A. dhakensis (blaCphA, blaAmpC and blaOXA). A. veronii only showed class-B- and class-D-like matches (blaCphA and blaOXA), whereas those for A. media, A. rivipollensis and A. caviae were class C and D (blaCMY, blaMOX and blaOXA427). The phylogenetic tree derived from concatenated sequences of β-lactamase genes successfully clustered each species. Some isolates also had resistance to sulfonamides, quinolones and aminoglycosides. Whole-genome sequencing proved to be a useful method to identify Aeromonas at the species level, which led to the unexpected identification of A. dhakensis and A.rivipollensis and revealed the resistome of each isolate. [ABSTRACT FROM AUTHOR]

A correction to the article "Detection of Three Stable Genetic Clones of CTX-M-15-Producing Klebsiella pneumoniae in the Barcelona Metropolitan Area, Spain" that was published in a 2009 issue of the journal is presented.