Molecular epidemiology of carbapenem-resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in an endemic area: comparison with global data

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is an endemic problem in certain countries including Greece. CRPA and multidrug-resistant P. aeruginosa (MDRPA) firstly emerged in our region during the 80s, right after the launch of imipenem and meropenem as therapeutic agents against P. aeruginosa infections. The role of outer membrane protein (Opr) inactivation has been known to contribute to imipenem resistance since many years, while efflux overexpression systems have been mainly associated with meropenem resistance. Among carbapenemases, metallo-β-lactamases (MBL) and mostly Verona integron-mediated (VIM) MBL’s have played the most crucial role in CRPA emergence. VIM-2 and VIM-4 producing CRPA, usually belonging to clonal complexes (CC) 111 and 235 respectively, have most frequently been isolated. Bla_VIM-2 and bla_VIM-4 are usually associated with a class 1 integron. VIM-17 also has appeared in Greece. On the other hand, other VIM subtypes detected in a global level, such as VIM-3, VIM-5, VIM-6, VIM-7, VIM-11, VIM-14, VIM-15, VIM-16 and VIM-18 have not yet emerged in Greece. However, new VIM subtypes will probably emerge in the future. In addition, MBL carbapenemases other than VIM, detected worldwide have not yet appeared. A single CRPA isolate producing KPC has emerged in our region several years ago. The study of the molecular basis of Opr deficiency and efflux overexpression remains a challenge for the future. In this article, we review the molecular epidemiology of CRPA in an endemic area, compared to global data.     

Related carbapenemase-producing <Emphasis Type="Italic">Klebsiella</Emphasis> isolates detected in both a hospital and associated aquatic environment in Sweden

Carbapenem antibiotics are one of the last-resort agents against multidrug-resistant (MDR) bacteria. The occurrence of carbapenemase-producing Enterobacteriaceae (CPE) in wastewater and aquatic environments is an indication of MDR bacteria in the community. This study evaluated CPE in aquatic environments and compared them to the local hospital isolates in Sweden. Phenotypic and genotypic analyses of antibiotic resistance of environmental and clinical CPE were performed. The relatedness of the isolates and possible clonal dissemination was evaluated using phylogenetic and phyloproteomic analysis. Klebsiella oxytoca carrying carbapenemase genes (bla_VIM-1, bla_IMP-29) were isolated from wastewater and the recipient river, while K. oxytoca (bla_VIM-1) and Klebsiella pneumoniae (bla_VIM-1, bla_OXA-48, bla_NDM-1, bla_KPC-3) were isolated from patients at the local clinics or hospital. The K. oxytoca classified as sequence type 172 (ST172) isolated from the river was genotypically related to two clinical isolates recovered from patients. The similarity between environmental and clinical isolates suggests the dispersion of bla_VIM-1 producing K. oxytoca ST172 from hospital to aquatic environment and the likelihood of its presence in the community. This is the first report of CPE in aquatic environments in Sweden; therefore, surveillance of aquatic and hospital environments for CPE in other urban areas is important to determine the major transfer routes in order to formulate strategies to prevent the spread of MDR bacteria.     

Nosocomial dissemination of VIM-2-producing ST235 <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in Lithuania

Pseudomonas aeruginosa multidrug resistance, and particularly the production of carbapenemases linked to international high-risk clones, is of growing concern. While high levels of carbapenem resistance (>60 %) have been reported in Lithuania, so far, there is no information on the underlying mechanisms. Thus, the aim of this work was to determine the molecular epidemiology and prevalence of acquired carbapenemases among 73 carbapenem-resistant P. aeruginosa isolates recovered in a hospital from Kaunas, Lithuania in 2011–2012. The presence of acquired carbapenemases was evaluated through phenotypic (modified Hodge test, cloxacillin inhibition test, double-disc synergy test) and genetic methods [polymerase chain reaction (PCR) and sequencing]. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Acquired β-lactamases were detected in 19 (26 %) of the isolates, whereas resistance was exclusively chromosomal (OprD inactivation?±?AmpC hyperproduction) in the remaining 54 (74 %) isolates. The acquired β-lactamases detected included 16 VIM-2, one PER-1 and two GES enzymes. PFGE revealed that 15 of the 16 VIM-2 isolates belonged to a single clone, identified as the international high-risk clone ST235 by MLST. bla _VIM-2 was preceded by aacA7 in a class I integron, similar to epidemic ST235 isolates described in nearby countries. Additionally, sequencing of bla _GES revealed the presence of the carbapenem-hydrolysing enzyme GES-5 in one of the isolates and a novel GES variant, designated GES-27, in the other. GES-27 differed from GES-5 by a single amino acid substitution, proline 167, that was replaced by glutamine. Increasing emergence and dissemination of concerning resistance mechanisms and international clones warrants global surveillance and control strategies.     

Characterization of carbapenem-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> from a healthcare region in Hong Kong

Carbapenem-resistant Enterobacteriaceae represents a major public health issue. This study investigated the clonality and resistance mechanisms of 92 carbapenem-resistant E. coli (n?=?21) and K. pneumoniae (n?=?71) isolates collected consecutively from clinical specimens and patients at high risk of carriage between 2010 and 2012 in a healthcare region in Hong Kong. Combined disk tests (CDTs) and the Carba NP test were used for phenotypic detection of carbapenemases. PCR assays were used to detect carbapenemase genes. All isolates were intermediate or resistant to at least one carbapenem. Nine (9.8 %) isolates were genotypic carbapenemase producers and included six K. pneumoniae (one ST1306/bla _IMP-4, one ST889/bla _IMP-4, two ST11/bla _KPC-2, one ST258/bla _KPC-2, one ST483/bla _NDM-1) and three E. coli (one ST131/bla _IMP-4, two ST744/ bla _NDM-1) isolates. All nine isolates carrying carbapenemase genes could be detected by the CDTs and the Carba NP test. PCR identified bla _CTX-M and bla _AmpC alone or in combination in 77.8 % (7/9) and 96.4 % (80/83) of the carbapenemase-producers and non-producers, respectively. Porin loss was detected in 22.2 % (2/9) and 59.0 % (49/83) of the carbapenemase-producers and non-producers, respectively. Overall, the E. coli clones were diverse (14 different STs), but 36.6 % (26/71) of the K. pneumoniae isolates belonged to ST11. In conclusion, the prevalence of carbapenemases among carbapenem-nonsusceptible E. coli and K. pneumoniae remained low in Hong Kong. Porin loss combined with AmpC and/or CTX-M type ESBL was the major mechanism of carbapenem resistance in the study population.     

Prevalence and molecular characterization of class 1 integrons among clinical isolates of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> in Northwest of Iran

Introduction

Integrons are introduced as the most common mechanism for resistance gene dissemination in Pseudomonas aeruginosa.

Aims and tasks

The purpose of the present study was to determine the diversity of the gene cassettes carried with class 1 integrons in clinical isolates of Pseudomonas aeruginosa.

Methods and results

One hundred clinical isolates of Pseudomonas aeruginosa from four hospitals in Northwest of Iran were investigated for antimicrobial susceptibility, presence of class 1 integrons and associated resistance gene cassettes. Seventy-one percent of isolates were multidrug resistant (MDR) and colistin was the most effective antibiotic in this study. Sixty-six isolates (66%) were contained class 1 integrons which all of them carried gene cassettes. There was a strong correlation between integron presence and multidrug resistance phenotype (p < 0.05). Amplification of the internal variable regions of class 1 integrons revealed 10 different arrays ranging in size from 0.6 to 3.5 kb. DNA sequencing analysis of class 1 integrons revealed the presence of several gene cassettes associated with resistance to aminoglycosides (aac and aad), β-lactams (bla _OXA), chloramphenicol (cmlA and catB), hypothetical gene cassette encoding a hypothetical protein and an open reading frame with unknown function (orfD). The most prevalent gene cassette found within class 1 integrons was aadB (68.18%), followed by aacA4 (48.48%). To the best of our knowledge, some of the gene cassettes arrays such as aac(3)-Ic-aacA5-cmlA5 and aacA5-aadA1-cmlA5 were first identified in this study. Hospital ward was demonstrated as significant risk factors for the acquisition of class 1 integrons harboring isolates in this study (P < 0.05).

Conclusions

Most of gene cassette arrays in this study are reported for the first time in Iran. These show the role of integrons in dissemination of resistance genes among clinical isolates of P. aeruginosa.

     

Multilocus Sequence Types of Carbapenem-Resistant Pseudomonas aeruginosa in Singapore Carrying Metallo-β-Lactamase Genes,Including the Novel blaIMP-26 Gene

Nine imipenem-resistant Pseudomonas aeruginosa isolates were found to contain a variety of metallo-β-lactamase genes, including bla_IMP-1, bla_IMP-7, bla_VIM-2, bla_VIM-6, and the novel bla_IMP-26. Multilocus sequence typing showed a diversity of sequence types. Comparison with isolates from an earlier study showed that the epidemic clones from 2000 have not become established.Carbapenem-resistant Pseudomonas aeruginosa is an increasing problem worldwide. While many underlying mechanisms may account for carbapenem resistance in this species, the possession of metallo-β-lactamase (MBL) genes is of particular concern because these enzymes are able to hydrolyze all β-lactam antimicrobials with the exception of aztreonam. In addition, these genes may be mobilized and transferred between different species of bacteria. We conducted a study in 2008 to investigate if there were any changes in the epidemiology of P. aeruginosa isolates containing MBL genes in our hospital compared to results from an earlier survey carried out in 2000 (3).Of 2,552 nonduplicate P. aeruginosa organisms isolated in 2008, 123 isolates were imipenem resistant. Of these, 11 were positive for MBL production by imipenem-EDTA disk diffusion (5). Nine of these yielded a product by multiplex PCR for MBL genes (2). The individual MBL genes were then amplified and sequenced. The clonal relationship between isolates with MBL genes was determined by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA restricted with SpeI (3). The PFGE band patterns were analyzed with Bionumerics (Applied Maths NV, Sint-Martens-Latem, Belgium), and all strains with more than 85% similarity were considered to belong to the same clone. All strains were further subjected to multilocus sequence typing (MLST) (1). Because it is a nucleic acid sequence-based method, MLST is able to characterize bacterial types in an unambiguous fashion and establish evolutionary relationships between strains better than band-based methods like PFGE. Representative MBL-producing P. aeruginosa isolates from the 2000 survey were also subjected to PFGE and MLST. MLST profiles were submitted to eBURST V3 (http://eburst.mlst.net/) on 10 March 2010. Isolates sharing six out of seven alleles were assigned to the same BURST group and can be considered to belong to the same clonal complex descended from a common founder genotype. The PFGE, MBL gene sequence, and MLST results are summarized in Fig. ​Fig.11.Open in a separate windowFIG. 1.Dendrogram of PFGE patterns of P. aeruginosa isolates with metallo-β-lactamase genes, showing the year of isolation, MLST sequence type, and BURST group.In our previous study, 21 of 2,094 (1.0%) of all nonduplicate P. aeruginosa isolates in our hospital had MBL genes (3). With the exception of one isolate with bla_IMP-7, all other isolates had bla_IMP-1 and belonged to one of two PFGE clones. Isolates belonging to clone A had sequences identical to that of the original bla_IMP-1 first reported in Japan. Four representatives of clone A isolated from our hospital in 2000 had sequence type 964 (ST964) by MLST. Isolates belonging to clone B isolated in 2000 had sequences for variant bla_IMP-1 (bla_IMP-1v) with four silent mutations. Three representatives of this clone from 2000 had ST233 and one had ST742 based on MLST. All four representatives of clone B belong to the same BURST group, which was different from that of clone A.In contrast, in the 2008 survey, 9 of 2,552 (0.35%) nonduplicate P. aeruginosa isolates had MBL genes. Unlike the earlier study, there were no large clonal outbreaks. Two isolates with bla_IMP-1v had similar PFGE patterns and belonged to the same BURST group as representative isolates from clone B in 2000.Two isolates from 2008 with bla_IMP-7 had similar PFGE patterns and shared the same BURST group. The rest of the isolates from 2008 had distinct PFGE patterns.There was a greater diversity of MBL genes compared to the 2000 survey results. In particular, this is the first time that bla_VIM-2 and bla_VIM-6 have been found in P. aeruginosa in Singapore. bla_IMP-26 is a novel MBL gene that differs from bla_IMP-4 at position 145 (G-to-T change). The translated amino acid sequence differs from IMP-4 at residue 49 (phenylalanine for valine). This sequence has been previously deposited in the GenBank database as IMP-4 from an Acinetobacter calcoaceticus isolate from Malaysia (accession number {"type":"entrez-protein","attrs":{"text":"ABC24668.1","term_id":"83583501"}}ABC24668.1).Three of the isolates in this study (separately containing bla_VIM-2, bla_IMP-1, and bla_IMP-7) belonged to ST235. This sequence type has been described in a VIM-producing P. aeruginosa isolate in Belgrade and is the founder of an international clonal complex of isolates bearing MBL genes found in several countries in Europe (6). Recently, an increasing prevalence of IMP-1-producing P. aeruginosa has been found in Hiroshima, Japan. This was due entirely to the clonal expansion of only two lineages, ST235 (BURST group 3) and ST357 (BURST group 108) (4). This is similar to the situation that existed in Singapore in 2000, where only two lineages (BURST groups 29 and 44) accounted for the majority of MBL-producing P. aeruginosa (3).It is noteworthy that the original fear that a clone of MBL-producing P. aeruginosa would become established in Singapore has not been realized. The BURST group 29 and 44 lineages from 2000 were represented by only one to two isolates in 2008. The two P. aeruginosa isolates with bla_IMP-7 in 2008 are unrelated to the solitary isolate with bla_IMP-7 from 2000. It has been suggested that P. aeruginosa displays an epidemic population structure, with a limited number of clones emerging from a large number of unrelated genotypes (7). Although we did not correlate our study with hospital infection control measures, the Japanese data and our own seem to suggest that controlling the prevalence of MBL-producing P. aeruginosa may be achieved by preventing the transmission of specific epidemic clones.While it is reassuring to note that the prevalence of MBL producers in carbapenem-resistant P. aeruginosa has not increased, the increased diversity of MBL genes represents a new cause for concern. We were unable to characterize the gene responsible for the MBL phenotype in two isolates in this study, and these may represent novel resistance determinants. Although clones of MBL-producing P. aeruginosa have not become established, it seems likely, given the variation of MBL genes and MLST types in this study, that MBL-producing P. aeruginosa continues to be introduced to our hospital from diverse sources.     

Spread and exchange of <Emphasis Type="Italic">bla</Emphasis><Subscript>NDM-1</Subscript> in hospitalized neonates: role of mobilizable genetic elements

To investigate the mobilizable elements associated with bla _NDM-1 in Enterobacteriaceae isolated from septicaemic neonates at a NICU in India, during December, 2008–2011. An attempt was also made to understand whether there was a pattern in the temporal acquisition of bla _NDM-1 within the unit. Transferability of carbapenem resistance was tested by conjugation and transformation. Plasmid types and addiction systems were analysed. The genetic background of bla _NDM-1 and association with class 1 integron were evaluated by PCR mapping. RFLP was carried out to discriminate plasmids of same incompatibility group. Transfer of carbapenem resistance was successful in 13/15 cases. bla _NDM-1 was associated with different plasmid scaffolds (IncFII, IncL/M, IncN, IncR, IncHIB-M/FIB-M), IncF type being the prevalent one. Addiction systems ccdAB and hok/sok were associated with transferable plasmids. Genetic structures surrounding bla _NDM-1 showed its association with at least a remnant of ISAba125 at its 5′-end. The spread of NDM-1 was not related to class 1 integron which possessed resistance determinants against trimethoprim (dfrA12, dfrA1, dfrA5), streptomycin (aadA2, aacA4), and rifampicin (arr-3). RFLP showed that three isolates possessed the same FII/FIIs plasmid; two of these three isolates were from a single neonate, implying interspecies transfer of bla _NDM-1. The predominance of FII plasmids and ISAba125 along with bla _NDM-1 was noted, but no specific pattern in the temporal acquisition of mobile genetic elements could be identified. To the best of our knowledge, this report is the first to inform the in-vivo interspecies plasmid transfer event of bla _NDM-1 in a neonate.     

Outbreak of NDM-1-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> ST76 and ST37 isolates in neonates

The purpose of this study was to investigate the epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) in Shanghai Children’s Hospital in China. Twenty-two non-duplicate CRKP strains were collected from pediatric patients between March and June in 2014. Antimicrobial susceptibility testing was conducted by the agar dilution method. Beta-lactamases were characterized by polymerase chain reaction (PCR) and DNA sequencing. The transferability of bla _NDM-1 was investigated by conjugation experiment. The plasmids bearing antibiotic resistance genes were characterized by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization. Clonal relatedness was evaluated by PFGE and multilocus sequence typing (MLST). The clinical data of patients were retrospectively reviewed. The 22 CRKP strains were resistant to most of the antimicrobial agents tested, except tigecycline and colistin. Overall, 59, 77, and 100 % of these strains were resistant to imipenem, meropenem, and ertapenem, respectively. The bla _NDM-1 was positive in 77.3 % (17/22) of the CRKP strains, of which the 16 isolates from inpatients were designated as ST37 (n?=?9) and ST76 (n?=7) and one isolate from an outpatient belonged to ST846. The 17 bla _NDM-1-positive isolates belonged to PFGE type A (n?=?9), type C (n?=?7), or type B (n?=?1). The plasmids bearing bla _NDM-1 could be transferred into recipient Escherichia coli J53 through conjugation in 88.2 % (15/17) of the strains. The hybridization results showed that the plasmids carrying the bla _NDM-1 gene were approximately 50–240 kb in size. This is the first report of an outbreak caused by NDM-1-producing K. pneumoniae ST76 and ST37 among neonates.     

Plasmids carrying DHA-1 β-lactamases

The aim of this review is to provide an update on the plasmids mediating DHA-1 cephalosporinase in Klebsiella pneumoniae. These plasmids have been mainly found in this bacterium but not only. The first was isolated from Salmonella sp. in France in the early 1990s. They are currently reported worldwide. Bla_DHA-1 beta-lactamase gene is usually co-expressed with many other antibiotic resistance genes such as extended-spectrum β-lactamases (bla_CTX-M-, bla _SHV -types), oxacillinases (bla_OXA-1, bla_OXA-30), penicillinases (bla _TEM -type), carbapenemases (bla _OXA48 , bla_KPC-2), aminoglycosides (aacA, aadA, armA), fluoroquinolones (qnrB4, aac6′-1b-cr), and sulfonamide (sul1) resistance genes. Plasmids carrying DHA-1 cephalosporinase have different sizes (22 to 313 kb), belong to diverse groups of incompatibility (R, L/M, FII(k), FIB, A/C2, HI2, HIB), and are self-transferable or not. The multidrug resistance region consists of a mosaic structure composed of resistance genes, insertion sequences, composite transposon, and integrons.     

Clonal relationship between human and avian ciprofloxacin-resistant <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates in North-Eastern Algeria

The objectives of this study were to determine rates, patterns, and mechanisms of antibiotic resistance, and to assess connections between chicken commensal, human commensal, and pathogenic ciprofloxacin-resistant Escherichia coli isolates. All E. coli isolates collected from chickens, their farmers, and patients in the Constantine region (North-east Algeria) were analyzed for bla and plasmid-mediated quinolone resistance (PMQR) gene contents, phylogroups, Rep-PCR profiles, and multilocus sequence types. A high prevalence of resistance to fluoroquinolones (51.4 % to ciprofloxacin) was recorded in avian isolates. Of these, 22.2 % carried the aac(6’)-Ib-cr gene, whereas lower resistance levels to these antibiotics were recorded in chicken farmers’ isolates. None of the commensal isolates harbored the qnr, qepA, or oqxAB genes. One human pathogenic isolate was ertapenem-resistant and harbored the bla _OXA-48 gene, 84 showed an extended-spectrum β-lactamase phenotype, with bla _CTX-M-15 gene prevalent in 87.2 % of them. Seventy isolates were resistant to fluoroquinolones, with aac(6’)-Ib-cr present in 72.8 %, qnrB in 5.7 %, and qnrS in 10 %. Three Rep-PCR profiles were common to chicken commensal and human pathogenic isolates (phylogroups D and B1; ST21, ST48, and ST471 respectively); one was found in both chicken and chicken-farmer commensal strains (D; ST108), while another profile was identified in a chicken-farmer commensal strain and a human pathogenic one (B1; ST19). These findings suggest clonal and epidemiologic links between chicken and human ciprofloxacin-resistant E. coli isolates and the important role that poultry may play in the epidemiology of human E. coli infections in the Constantine region.     

High prevalence of the PER-1 gene among carbapenem-resistant <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> in Riyadh,Saudi Arabia

The prevalence of carbapenem-resistant Acinetobacter baumannii in Saudi Arabia and their resistance genetic mechanisms are yet to be identified. We studied the prevalence and genetic diversity of extended-spectrum beta-lactamase genes, particularly the PER-1 gene, among carbapenem-resistant A. baumannii strains from patients at a tertiary care hospital in Riyadh, Saudi Arabia between 2006 and 2014. Fresh subcultured samples were tested for antimicrobial susceptibility minimum inhibitory concentration (MIC). Total genomic DNA was extracted from each isolate and further used for polymerase chain reaction (PCR) genotyping, sequence-based typing (SBT) of PER-1 and OXA-51-like gene, and multilocus sequence typing (MLST) of positive isolates. Randomly selected clinical isolates (n = 100) were subjected to MLST. A total of 503 isolates were characterized as multidrug-resistant (MDR) using the MIC. Isolates were further PCR tested for bla _-TEM and bla _-PER-1 resistance genes (n = 503). The genotyping results showed that 68/503 (14 %) isolates were positive to bla TEM. The genotyping results of PER-1-like genes showed that 384/503 (76.3 %) were positive among MDR Acinetobacter isolates. Based on SBT, the majority of these isolates were clustered into three main groups including isolates harboring PER-1: AB11 (bla _-PER-1), isolate AB16 (bla _-PER-1), and, finally, the plasmid pAB154 (bla _-PER-7). Remarkably, many isolates were concealing the PER-1 gene and harboring the TEM resistance genes as well. MLST results for selected isolates (n = 100) identified four main sequence types (STs: 2, 19, 20, and 25) and four novel isolates (ST 486–489). We report 76.3 % prevalence of the PER-1 resistance gene among Acinetobacter clinical isolates from Riyadh, Saudi Arabia. Further work is needed to explore the clinical risks and patient outcome with such resistance related to healthcare-associated infections and investigate the genetic and molecular mechanisms that confer the MDR phenotype.     

Molecular epidemiology of virulence and antimicrobial resistance determinants in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> from hospitalised patients in Kilimanjaro,Tanzania

This study aimed to use whole-genome sequencing to determine virulence and antimicrobial resistance genes in K. pneumoniae isolated from patients in a tertiary care hospital in Kilimanjaro. K. pneumoniae isolates from patients attending Kilimanjaro Christian Medical Centre between August 2013 and August 2015 were fully genome-sequenced and analysed locally. Sequence analysis was done for identification of virulence and AMR genes. Plasmid and multi-locus sequence typing and capsular or capsular (K) typing were performed and phylogeny was done to ascertain K. pneumoniae relatedness. Stata 13 (College Station, TX, 77845, USA) was used to determine Cohen’s kappa coefficient of agreement between the phenotypically tested and sequence-predicted resistance. A total of 16 (47.1%) sequence types (STs) and 10 (29.4%) K types were identified in 30 (88.2%) and 17 (50.0%) of all analysed isolates, respectively. K. pneumoniae ST17 were 6 (17.6%). The commonest determinants were bla_CTX-M-15 in 16 (47.1%) isolates_, bla_SHV in 30 (88.2%), bla_OXA-1 in 8 (23.5%) and bla_TEM-1 in 18 (52.9%) isolates. Resistance genes for aminoglycosides were detected in 21 (61.8%) isolates, fluoroquinolones in 13 (38.2%) and quinolones 34 (100%). Ceftazidime and ceftriaxone showed the strongest agreement between phenotype- and sequence-based resistance results: 93.8%, kappa?=?0.87 and p?=?0.0002. Yersiniabactin determinant was detected in 12 (35.3%) of K. pneumoniae. The proportion of AMR and virulence determinants detected in K. pneumoniae is alarming. WGS-based diagnostic approach has showed promising potentials in clinical microbiology, hospital outbreak source tracing virulence and AMR detection at KCMC.     

Spread of carbapenem-resistant international clones of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> in Turkey and Azerbaijan: a collaborative study

Epidemic clones of Acinetobacter baumannii, described as European clones I, II, and III, are associated with hospital epidemics throughout the world. We aimed to determine the molecular characteristics and genetic diversity between European clones I, II, and III from Turkey and Azerbaijan. In this study, a total of 112 bloodstream isolates of carbapenem-resistant Acinetobacter spp. were collected from 11 hospitals across Turkey and Azerbaijan. The identification of Acinetobacter spp. using conventional and sensitivity tests was performed by standard criteria. Multiplex polymerase chain reaction (PCR) was used to detect OXA carbapenemase-encoding genes (bla _OXA-23-like, bla _OXA-24-like, bla _OXA-51-like, and bla _OXA-58-like). Pulsed-field gel electrophoresis (PFGE) typing was used to investigate genetic diversity. The bla _OXA-51-like gene was present in all 112 isolates, 75 (67 %) carried bla _OXA-23-like, 7 (6.2 %) carried bla _OXA-58-like genes, and 5 (4.5 %) carried bla _OXA-24-like genes. With a 90 % similarity cut-off value, 15 clones and eight unique isolates were identified. The largest clone was cluster D, with six subtypes. Isolates from clusters D and I were widely spread in seven different geographical regions throughout Turkey. However, F cluster was found in the northern and eastern regions of Turkey. EU clone I was grouped within J cluster with three isolates found in Antalya, Istanbul, and Erzurum. EU clone II was grouped in the U cluster with 15 isolates and found in Kayseri and Diyarbak?r. The bla _OXA-24-like gene in carbapenemases was identified rarely in Turkey and has been reported for the first time from Azerbaijan. Furthermore, this is the first multicenter study in Turkey and Azerbaijan to identify several major clusters belonging to European clones I and II of A. baumannii.     

Spread of Multidrug-Resistant Pseudomonas aeruginosa Clones in a University Hospital

An outbreak of multidrug-resistant Pseudomonas aeruginosa (MDRPA) infections in a university hospital is described. Phenotypic and genotypic analysis of 240 isolates revealed that 152 patients, mainly in the intensive care unit (ICU), were colonized or infected with MDRPA, the majority with O11. All metallo-β-lactamase (MBL)-positive isolates carried the bla_VIM-2 or bla_VIM-1 gene. One or more type III secretion system toxin genes were detected in most isolates. Five dominant pulsed-field gel electrophoresis (PFGE) types were characterized, associated with ST235, ST111, ST253, ST309, and ST639.     

Dissemination of <Emphasis Type="Italic">bla</Emphasis><Subscript>OXA-370</Subscript> is mediated by IncX plasmids and the Tn6435 transposon

In Enterobacteriaceae, the bla_OXA-48-like genes have been identified on plasmids in different regions of the world. The OXA-370 is a plasmid-encoded OXA-_48-like enzyme reported in two distinct regions of Brazil. Recently, we demonstrate that the bla_OXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination. In this work, we described for the first time the complete nucleotide sequence of six plasmids harboring the bla_OXA-370 gene. Complete DNA sequencing using the Illumina platform and annotation of the plasmids showed that they belonged to incompatibility groups IncX and had in average 70 kbp. The bla_OXA-370 gene is located in a composite transposon containing four genes encoding transposases, named Tn6435. In this study, highly similar plasmids were detected in different Enterobacteriaceae genera.     

Emergence of Pseudomonas aeruginosa strains producing metallo-β-lactamases of the IMP-15 and VIM-2 types in Mexico

Eighty-six carbapenem non-susceptible Pseudomonas aeruginosa isolates collected in the National Institute of Respiratory Diseases of Mexico City were screened for the presence of metallo-β-lactamase (MBL) activity using both E-test strips and a microbiological assay with EDTA-imipenem. Genomic comparisons and sequence analyses conducted with these isolates revealed the presence of bla_VIM-2 in two clonally related isolates, and bla_IMP-15 in a clonally unrelated isolate. Both genes were found to be carried by class 1 integrons, and bla_IMP-15 was additionally present on a broad host-range plasmid. This is the first report of co-existing P. aeruginosa strains producing different MBLs in a Mexican hospital, highlighting the necessity of appropriate surveillance to prevent dissemination of carbapenem resistance.     

Carbapenemase-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> bloodstream infection in critically ill patients: risk factors and predictors of mortality

A significant increase in carbapenemase-producing Klebsiella pneumoniae (CP-Kp) bacteraemias has been observed worldwide. The objective of the present work was to study the risk factors and predictors of mortality of CP-Kp bacteraemias among critically ill patients. During a 4-year period (2012–3015), a matched 1:2 case-control study was conducted. Klebsiella pneumoniae was identified by Vitek 2 technology. Antibiotic susceptibility was performed by the agar disc diffusion method and Etest. The presence of the bla _KPC, bla _VIM and bla _NDM genes was confirmed by polymerase chain reaction (PCR). Epidemiologic data were collected from the intensive care unit (ICU) computerised database. One hundred and thirty-nine patients who developed a CP-Kp bacteraemia were matched with 278 patients. The majority of isolates (128; 92.1%) carried the bla _KPC gene, seven carried both bla _KPC and bla _VIM, three bla _VIM and one carried bla _NDM. Risk factors for the development of CP-Kp bacteraemia were administration of tigecycline and number of antibiotics administered prior to CP-Kp bacteraemia. Overall, the 30-day mortality was 36.0%. Multivariate analysis revealed septic shock, Simplified Acute Physiology Score II (SAPS II) upon infection onset, adjunctive corticosteroid administration and parenteral nutrition as independent predictors of mortality, while treatment with a combination of appropriate antibiotics was identified as a predictor of good prognosis. Among septic shock patients (n?=?74), Sequential Organ Failure Assessment (SOFA) score upon infection onset, adjunctive corticosteroid administration and strain carrying the bla _KPC gene were independently associated with mortality, while the administration of combination treatment was identified as a predictor of a good prognosis. The administration of tigecycline predisposes to the induction of bacteraemia. Appropriate antibiotic treatment is associated with better survival, while concomitant corticosteroid treatment is associated with mortality.     

Emergence and establishment of KPC-2-producing ST11 <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in a general hospital in Shanghai,China

The aim of this study was to investigate the characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) collected during an outbreak in a Chinese teaching hospital and to provide insights into the prevention and control of nosocomial infection. We collected unique CRKP clinical isolates from 2009 to 2013. Antibiotic-resistant genes were identified by polymerase chain reaction (PCR) and sequencing. The isolates were typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmids were classified using a PCR-based incompatibility/replicon typing method and a replicon sequence typing method. Conjugation experiments were performed to evaluate the transferability of carbapenem-resistant genes. Whole genome sequencing (WGS) was conducted to further investigate the genetic background of the isolates. Infection control practices were reviewed throughout the study period. Klebsiella pneumoniae sequence type (ST) 11 emerged in 2010 and acquired the bla _KPC-2 gene by 2011. From 2011 to 2013, ST11 KPC-2-producing CRKP (G type) prevailed as the most common CRKP in our hospital, causing a prolonged outbreak. The majority of these CRKP strains possess an IncFII plasmid, with Tn1721-bla _KPC-2-ΔTn3-IS26 bearing the genetic structure for bla _KPC-2. Infection prevention control measures available at the time contained the initial outbreak, but had no effect on the spread of CRKP later. This study demonstrated the seriousness concerning the spread of KPC-2-producing ST11 CRKP in a Chinese hospital, indicating that current prevention and control strategies for carbapenem-resistant Enterobacteriaceae (CRE) nosocomial infection need to be investigated and adjusted.     

Molecular epidemiology of <Emphasis Type="Italic">Escherichia coli</Emphasis> sequence type 131 and its H30/H30-Rx subclones recovered from extra-intestinal infections: first report of OXA-48 producing ST131 clone from Iran

Multidrug-resistant (MDR) O25b-ST131 clone of Escherichia coli is well established as a significant cause of extra-intestinal infections worldwide. However, there have been no studies about the prevalence of ST131 and its H30/H30Rx subclones from Iran. The prevalence of ST131 was 29.8% among phylogroups B2, D, and F of E.coli isolates recovered from extra-intestinal infections. Fifty-seven (90.4%) and six (9.6%) of isolates belonged to serogroups O25b and O16 respectively, and exhibited high rates of MDR (98.4% and 83.3%) and extended spectrum β-lactamase (ESBL) production (96.8% and 83.3%). The majority (56/57, 98.2%) of O25b isolates belonged to H30 lineage; of those, 24 isolates (42.8%) belonged to H30-Rx subclone. O16-ST131 isolates were H30-negative. The resistance rate values of O16-ST131subgroup were lower for fluoroquinolones/aminoglycosides and higher for carbapenems, cephalosporins, β-lactam/β-lactamase inhibitors and trimethoprim/sulfamethoxazole, as compared to O25b-ST131 isolates. Among H30 sub lineage and in comparison with non-Rx isolates, H30-Rx subclone showed higher resistance score and virulence genes (papA and papC), and was also associated with CTX-M group 1. bla _OXA-48 carbapenemase was detected in seven O25b and one O16 isolates; of those, one O25b-ST131 isolate was carbapenem-susceptible. The ST131 isolates comprised 15 ‘enterobacterial repetitive intergenic consensus’ (ERIC) clusters, and O16 isolates remained distributed in five groups in cluster with O25b-ST131 isolates. In conclusion, this is the first report of the presence of MDR, bla _OXA-48/CTX-M-positive O25b/O16-ST131 isolates in Iran. Contrary to lower prevalence of O16-ST131 subgroup, higher resistance rates to β-lactam antibiotics may indicate the importance of this subgroup in the spread of MDR E.coli isolates.     

Diversity in VIM-2-encoding class 1 integrons and occasional blaSHV2a carriage in isolates of a persistent,multidrug-resistant Pseudomonas aeruginosa clone from Tunis

From 2002 to 2006, 35 of 73 multidrug-resistant Pseudomonas aeruginosa isolates from different wards at Charles Nicolle hospital of Tunis were positive for class B carbapenemase (using the imipenem-EDTA test), owing to a bla_VIM-2 gene cassette in a class 1 integron. Twenty-three isolates additionally produced the extended-spectrum β-lactamase SHV2a. DNA sequences immediately surrounding bla_SHV2a shared extensive identity with a Klebsiella pneumoniae plasmid sequence. Despite belonging to the same chromosomal type, as shown by pulsed-field gel electrophoresis (PFGE), the VIM-2 producing P. aeruginosa isolates prevalent at Charles Nicolle hospital displayed a diversity of VIM-2-carrying integrons.