NDM Genes Research Articles

Carbapenemase-producing Klebsiella pneumoniae 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.

Real-time PCR assays for the detection and quantification of carbapenemase genes (bla KPC, bla NDM, and bla OXA-48) in environmental samples.

In this study, we have developed real-time PCR assays using SYBR Green chemistry to detect all known alleles of bla KPC, bla NDM, and bla OXA-48-like carbapenemase genes in water, sediment, and biofilm samples collected from hospital and wastewater treatment plant (WWTP) effluents and rivers receiving chronic WWTP discharges. The amplification of bla KPC, bla NDM, and bla OXA-48 DNA was linear over 7 log dilutions (R 2 between 0.995 and 0.997) and showing efficiencies ranging from 92.6% to 100.3%. The analytical sensitivity indicated that the reaction for bla KPC, bla NDM, and bla OXA-48-like genes was able to detect 35, 16, and 19 copy numbers per assay, respectively. The three carbapenemase genes were detected in hospital effluents, whereas only the bla KPC and bla NDM genes were detected in biofilm and sediment samples collected from wastewater-impacted rivers. The detection of bla KPC, bla NDM, and bla OXA-48-like genes in different matrices suggests that carbapenem-resistant bacteria occur in both planktonic and benthic habitats thus expanding the range of resistance reservoirs for last-resort antibiotics. We believe that these real-time PCR assays would be a powerful tool for the rapid detection and quantification of bla KPC, bla NDM, and bla OXA-48-like genes in complex environmental samples.

The Prevalence of Extended-Spectrum β-lactamase (ESBL) and Carbapenem-Resistant Enterobacteriaceae (CRE) Isolates in Positive Blood Cultures of Patients in A Teaching Hospital in Kuwait Over A 2-year Period

Extended spectrum β-lactamase (ESBL)-producing organisms and carbapenem resistant Enterobacteriaceae (CRE) pose unique challenges to clinical microbiologists, infectious diseases physicians, infection control professionals and antibacterial-discovery scientists. This study was undertaken to determine the prevalence of extended-spectrum β-lactamase (ESBL)-producing and carbapenemase-positive members of the family Enterobacteriaceae isolated from positive blood cultures of patients in our hospital. Enterobacteriaceae isolates from proven cases of blood stream infections (BSI) from January 2014 to December 2015 were included in the study. They were identified and tested for their susceptibilities to 18 antibiotics by conventional methods and semi-automated VITEK-2 system. ESBL and carbapenem resistance (CR) were detected by phenotypic and molecular PCR methods, and characterized by sequencing. A total of 4133 blood cultures were positive. Of these, 543 (13.1%) were positive for Enterobacteriaceae, 135 (24.9%) of which were ESBL producers and 28 (5.2%) identified as CRE. The most common of the ESBL-producing isolates were Escherichia coli (81:60%) and Klebsiella pneumoniae (44:32.6%). Resistance rates of E. coli to the cephalosporins ranged from 47–100%, ciprofloxacin 70.4%, imipenem 2.5% and meropenem 1.2%. Higher proportions of K. pneumoniae were resistant to the cephalosporins (68.2–100%), imipenem (27.3%) and meropenem (22.7%). Sequence analysis of the ESBL revealed CTX-M-15 as the commonest enzyme (75%). The CRE harbored mainly the VIM (28.6%) and NDM-1 (35.7%) genes. Our study demonstrates a high prevalence of ESBL-producing Enterobacteriaceae isolates of the CTX-M-15 variety and equally high prevalence of CRE, predominantly VIM and NDM-1 producers, among blood culture isolates at our hospital. All authors: No reported disclosures.

Emergence of Gram-Negative Bacilli with Concomitant <i>bla</i>NDM-1- and <i>bla</i>OXA-48-Like Genes in Egypt

Multidrug-resistant Gram-negative organisms have emerged as a major threat to hospitalized patients, and are associated with serious morbidity and mortality. This study aimed to characterize carbapenem resistance genes among Gram-negative bacilli isolated from clinical samples from patients in the intensive care unit of Cairo University Hospital. A total of 211 samples were collected from patients showing clinical evidence of infection. Bacteria were isolated and identified by conventional microbiological methods. <i>Acinetobacter baumannii</i> isolates were furtherly characterized by polymerase chain reaction (PCR), using primers specific for <i>bla</i>_OXA-51-like genes. The Kirby Bauer disc diffusion method was used to determine susceptibility patterns of isolates, and carbapenem resistance was further examined by a modified Hodge test. Positive isolates were tested for the presence of <i>bla</i>_KPC, <i>bla</i>_OXA-48, and <i>bla</i>_NDM-like genes by PCR. NDM gene types were determined by direct sequencing. From the 211 samples, 229 Gram-negative bacilli were isolated. Fifty isolates (21.2%) were resistant to carbapenem. PCR analysis showed that none of the 50 isolates carried <i>bla</i>_KPC-like genes, while 24 (48%) isolates carried <i>bla</i>_OXA-48-like genes, 8 (16%) carried <i>bla</i>_NDM-1, and five isolates (10%) carried both <i>bla</i>_NDM-1 and <i>bla</i>_OXA-48-like genes. These results indicate that continuous surveillance of these multidrug-resistant pathogens is urgently required. And that is very important is to activate the antimicrobial stewardship programs of which the most important is restriction of the big gun antibiotics like carbapenems, colistin, tigecyclin and vancomycin and restricting their prescription to privileged specialties.

Multidrug-resistant Enterobacteriaceae nosocomial uropathogens at Menoufia University Hospitals: phenotypic characterization and detection of resistance genes using real-time PCR

Objectives The aims of this study were as follows: to determine the incidence of multidrug-resistant Enterobacteriaceae isolated from the urine of patients admitted in different departments at Menoufia University Hospitals; to detect the presence of extended spectrum β-lactamases (ESβLs), AmpC β-lactamases, and carbapenemases among the isolated pathogens using phenotypic methods; and to investigate the presence of bla KPC and bla NDM genes using real-time PCR. Background Urinary tract infections are the most common type of nosocomial infections. ESβLs and AmpC β-lactamases are clinically significant because they may confer resistance to multiple classes of antibiotics. Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams. Materials and methods This study included 260 urine samples collected from 260 patients. ESβL production was detected on the basis of resistance to third-generation cephalosporins and aztreonam and then confirmed using the combined disk test. AmpC production was detected using the cefoxitin disk test and confirmed using the AmpC disk test. Carbapenemases were detected on the basis of resistance to imipenem, meropenem, and ertapenem, and then confirmed using the modified Hodge test, the phenylboronic acid combined disk test, and the imipenem/EDTA combined disk test. Carbapenemase gene (bla KPC and bla NDM) detection was performed using real-time PCR. Results Carbapenem resistance was 42.5% to ertapenem, 40.8% to meropenem, and 45% to imipenem. Risk factors associated with Hospital aquired infection were hospital stay of 14 days or more, exposure to invasive procedures, and comorbid conditions. ESβL production occurred in 55.8% and AmpC occurred in 30.8% of Enterobacteriaceae spp. Carbapenemase production was detected in 87%, Klebsiella pneumoniae carbapenemase (KPC) was detected in 37%, metallo-β-lactamase was detected in 54%, and bla KPCgene was detected in 24% of imipenem-resistant Enterobacteriaceae spp. However, bla NDM gene was not detected in all tested isolates. Conclusion Surveys of the prevalence, antibacterial susceptibility patterns, and identification of resistance patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients.

Molecular characterization of extended-spectrum β-lactamase, plasmid-mediated AmpC cephalosporinase and carbapenemase genes among Enterobacteriaceae isolates in five medical centres of East and West Azerbaijan, Iran

Very little is known about the occurrence and various types of extended-spectrum β-lactamase (ESBL), AmpC and carbapenemase in Iran. The aims of this study were to determine the prevalence of ESBLs, AmpCs and carbapenemase genes among Enterobacteriaceae in Azerbaijan and to characterize the genetic composition of the detected genes. A total of 307 Enterobacteriaceae isolates, recovered from five medical centres, were screened for ESBL, AmpC and carbapenemase activities by the disc diffusion method and phenotypic confirmatory tests. The 162 selected strains (third-generation cephalosporins, cefoxitin- or carbapenem-resistant strains with positive or negative phenotypic confirmatory tests) were selected for multiplex PCR screening for β-lactamase genes, and detected genes were confirmed by sequencing. Of 162 isolates, 156 harboured 1 to 6 β-lactamase genes of 41 types. The most prevalent genes were blaTEM-1 (29.9 %), followed by blaCTX-M-15 (25.7 %). Plasmid-mediated AmpC was detected in 66 strains (21.5 %) alone or in combination with other genes. Carbapenemase-encoding genes were detected in 18 strains (5.8 %) of 27 carbapenem-non-susceptible isolates including 11, 7, 3 and 1 cases of blaOXA-48, blaNDM-1, blaKPC-2 and blaKPC-3 genes, respectively. Interestingly, 148 (94.8 %) of 156 strains with any β-lactamase gene were found to have a multidrug-resistant pattern. The rate of resistance to β-lactams and multidrug-resistant Enterobacteriaceae is high in Azerbaijan. All positive strains for carbapenemase genes were resistant to all β-lactams. The present study reveals the high occurrence of CTX-M-type ESBLs followed by TEM and SHV variants among Enterobacteriaceae isolates. East Azerbaijan seems to be an alarming focus for OXA-48, NDM-1 and KPC dissemination.

Molecular Epidemiology and Genome Dynamics of New Delhi Metallo-β-Lactamase-Producing Extraintestinal Pathogenic Escherichia coli Strains from India.

The global dissemination and increasing incidence of carbapenem-resistant, Gram-negative organisms have resulted in acute public health concerns. Here, we present a retrospective multicenter study on molecular characterization of metallo-β-lactamase (MBL)-producing clinical Escherichia coli isolates recovered from extraintestinal infections in two hospitals in Pune, India. We screened a large sample size of 510 E. coli isolates for MBL production wherein we profiled their molecular determinants, antimicrobial resistance phenotypes, functional virulence properties, genomic features, and transmission dynamics. Approximately 8% of these isolates were MBL producers, the majority of which were of the NDM-1 (69%) type, followed by NDM-5 (19%), NDM-4 (5.5%), and NDM-7 (5.5%). MBL producers were resistant to all antibiotics tested except for colistin, fosfomycin, and chloramphenicol, which were effective to various extents. Plasmids were found to be an effective means of dissemination of NDM genes and other resistance traits. All MBL producers adhered to and invaded bladder epithelial (T24) cells and demonstrated significant serum resistance. Genomic analysis of MBL-producing E. coli isolates revealed higher resistance but a moderate virulence gene repertoire. A subset of NDM-1-positive E. coli isolates was identified as dominant sequence type 101 (ST101) while two strains belonging to ST167 and ST405 harbored NDM-5. A majority of MBL-producing E. coli strains revealed unique genotypes, suggesting that they were clonally unrelated. Overall, the coexistence of virulence and carbapenem resistance in clinical E. coli isolates is of serious concern. Moreover, the emergence of NDM-1 among the globally dominant E. coli ST101 isolates warrants stringent surveillance and control measures.

Molecular epidemiology of carbapenem resistant gram-negative bacilli from infected pediatric population in tertiary - care hospitals in Medellín, Colombia: an increasing problem.

BackgroundGram-negative bacilli are a cause of serious infections in the pediatric population. Carbapenem are the treatment of choice for infections caused by multidrug-resistant Gram-negative bacilli, but the emergence of carbapenem resistance has substantially reduced access to effective antimicrobial regimens. Children are a population vulnerable to bacterial infections and the emergence of resistance can worsen prognosis. The aim of this study is to describe the clinical and molecular characteristics of infections caused by carbapenem-resistant Gram-negative bacilli in pediatric patients from five tertiary-care hospitals in Medellín, Colombia.MethodsA cross-sectional study was conducted in five tertiary-care hospitals from June 2012 to June 2014. All pediatric patients infected by carbapenem-resistant Gram-negative bacilli were included. Clinical information for each patient was obtained from medical records. Molecular analyses included PCR for detection of blaVIM, blaIMPblaNDM,blaOXA-48 and blaKPC genes and PFGE and MLST for molecular typing.ResultsA total of 59 patients were enrolled, most of them less than 1 year old (40.7 % n = 24), with a previous history of antibiotic use (94.9 %; n = 56) and healthcare-associated infections - predominately urinary tract infections (31.0 %; n = 18). Klebsiella pneumoniae was the most frequent bacteria (47.4 %), followed by Enterobacter cloacae (40.7 %) and Pseudomonas aeruginosa (11.9 %). For K. pneumoniae, KPC was the predominant resistance mechanism (85.7 %; n = 24) and ST14 was the most common clone (39.3 % n = 11), which included strains closely related by PFGE. In contrast, E. cloacae and P. aeruginosa were prevailing non-carbapenemase-producing isolates (only KPC and VIM were detected in 1 and 3 isolates, respectively) and high genetic diversity according to PFGE and MLST was found in the majority of the cases.ConclusionsIn recent years, increasing carbapenem-resistant bacilli in children has become in a matter of great concern. It is important to conduct systemic surveillance and take measures to prevent dissemination of multidrug-resistant bacteria.

Karbapeneme Dirençli Acinetobacter baumannii Suşlarında Kolistin/Sulbaktam Kombinasyonu Etkinliğinin Değerlendirilmesi

Acinetobacter baumannii strains, are opportunistic pathogens that cause severe nosocomial infections that are difficult to treat due to development of resistance to multiple antibiotics. As the antibiotic choices to be used in treatment are limited, combinations of a variety of antibiotics are used. The aims of this study were to identify the minimal inhibitory concentration (MIC) values of colistin and sulbactam against A.baumannii isolates and to determine the in vitro activity of colistin-sulbactam combination. A total of 50 A.baumannii strains isolated from different clinical specimens (32 tracheal aspirates, 10 blood, 6 urine and 2 wound samples) were included in the study. The identification of bacteria was performed by traditional methods and Vitek-2 (BioMerieux, France) automated system. Antibiotic susceptibilities were detected by Mueller-Hinton agar disk diffusion method and Vitek-2 automated system and the results were interpreted according to the CLSI standards. MIC values of colistin and sulbactam against A.baumannii strains and in vitro interactions of colistin-sulbactam combinations were determined with the E-test (BioMerieux, France). Fractional inhibitory concentration (FIC) index was used for the detection of efficacy of drug combinations. The presence of oxacillinase and metallo-beta-lactamase (MBL) genes that lead carbapenem resistance was investigated by polymerase chain reaction (PCR), and pulsed-field gel electrophoresis (PFGE) was performed for the determination of clonal relationship. In our study, all strains (100%) were detected as susceptible to colistin, 48 (96%) to trimethoprim/sulphamethoxazole and 18 to (36%) tigecyclin; however all of them were resistant to the other studied antibiotics, including sulbactam and carbapenem. When the colistin-sulbactam combination was assessed according to FIC index, all strains were found to have antagonistic effect. All of the carbapenem-resistant strains were positive for OXA-51 and OXA-23, and 3 (6%) were positive for OXA-24. Among MBLs, OXA-58, OXA-48, IPM, SPM, SIM, GIM, VIM and NDM-1 genes were not detected. In the evaluation of PFGE results it was found that the clonal distribution of the strains, except one, were all pulsotype A. In the assessment of in vitro efficacy of the colistin-sulbactam combination against A.baumannii strains with multidrug resistance, antagonistic effect was observed in all strains. In the resistance and clonal analysis it was determined that the strains belonged to the same clone, and they had the same resistance genes and therefore the result of the in vitro activity was considered to have similar effect among all strains. It was decided that especially in units where critical patients are monitored and where resistant strains that are difficult to treat are isolated, performing synergy studies may be beneficial for the selection of combination treatment and the determination of the treatment combination to be chosen specifically for the hospital or even the unit.

Phenotypic and Genotypic Detection of Klebsiella Pneumoniae Carbapenemase and New Delhi Metallo-β-Lactamase of Enterobacteriaceae in Benha University Hospitals

Background: The prevalence of carbapenem-resistant Enterobacteriacea is on the rise worldwide, posing a major public health threat as well as a serious concern for infection control management. The accurate identification and reporting of carbapenemase producing Enterobacteriacea (CPE) will aid infection control practitioners in preventing the spread of these multidrug- resistant isolates. Objectives: This study aimed to detect the prevalence of CPE in Benha university hospitals and to confirm the presence of k. Pneumoniae carbapenemase (KPC) and New Delhi Metallo-β- Lactamase (NDM) in Enterobacteriacea. Methodology: This study was conducted on 100 Enterobacteriacea strains collected from patients admitted to Benha University Hospitals. The isolated Enterobacteriacea strains were screened for CPE by chromID CARBA agar with species identification by using API 20 E test strips as a biochemical identification system. KPC and NDM carbapenemases detection was confirmed \phenotypically by Mast Disc ID carbapenemase detection set (MDI) and genotypically by multiplex PCR. Results: Out of 100 Enterobacteriacea isolates, 40 strains (40 %) are carbapenemase producers and 27strains of them (67.5 %) are carrying genes responsible for carbapenem resistant. Twenty six strains (65%) are carrying KPC gene and only one k. Pneumoniae strain (2.5%) is carrying NDM-1 gene for MβL production. MDI detected 60% of KPC and 25% of MβL producers with high sensitivity (92.6 %) in comparison to PCR. Intensive Care Unit patients harbored most of carbapenem-resistant isolates with the highest percentage of carbapenemases genes in k. Pneumoniae. Conclusion: Emergence of CPE pathogens in our setting create an important challenge for clinicians and hospital epidemiologists with the possibility of outbreak eruption by these difficult-to-treat pathogens, in the future.

Local transmission and global dissemination of New Delhi Metallo-Beta-Lactamase (NDM): a whole genome analysis.

BackgroundNew Delhi metallo-β-lactamase (blaNDM), a plasmid-borne carbapenemase gene associated with significant mortality and severely limited treatment options, is of global public health concern as it is found in extremely diverse Gram-negative bacterial strains. This study thus aims to genetically characterize local and global spread of blaNDM.MethodsTo investigate local transmission patterns in the context of a single hospital, whole genome sequencing data of the first 11 blaNDM-positive bacteria isolated in a local hospital were analyzed to: (1) identify and compare blaNDM-positive plasmids; and (2) study the phylogenetic relationship of the bacteria chromosomes. The global analysis was conducted by analyzing 2749 complete plasmid sequences (including 39 blaNDM-positive plasmids) in the NCBI database, where: (1) the plasmids were clustered based on their gene composition similarity; (2) phylogenetic study was conducted for each blaNDM-positive plasmid cluster to infer the phylogenetic relationship within each cluster; (3) gene transposition events introducing blaNDM into different plasmid backbones were identified; and (4) clustering pattern was correlated with the plasmids’ incompatibility group and geographical distribution.ResultsAnalysis of the first 11 blaNDM-positive isolates from a single hospital revealed very low blaNDM-positive plasmid diversity. Local transmission was characterized by clonal spread of a predominant plasmid with 2 sporadic instances of plasmid introduction. In contrast to the low diversity locally, global blaNDM spread involved marked plasmid diversity with no predominant bacterial clone. Thirty-nine (1.4 %) out of the 2749 complete plasmid sequences were blaNDM-positive, and could be resolved into 7 clusters, which were associated with plasmid incompatibility group and geographical distribution. The blaNDM gene module was witnessed to mobilize between different plasmid backbones on at least 6 independent occasions.ConclusionsOur analysis revealed the complex genetic pathways of blaNDM spread, with global dissemination characterized mainly by transposition of the blaNDM gene cassette into varied plasmids. Early local transmission following plasmid introduction is characterized by plasmid conjugation and bacterial spread. Our findings emphasize the importance of plasmid molecular epidemiology in understanding blaNDM spread.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2740-0) contains supplementary material, which is available to authorized users.

Detection of Antibiotic Resistance Genes in Source and Drinking Water Samples from a First Nations Community in Canada.

Access to safe drinking water is now recognized as a human right by the United Nations. In developed countries like Canada, access to clean water is generally not a matter of concern. However, one in every five First Nations reserves is under a drinking water advisory, often due to unacceptable microbiological quality. In this study, we analyzed source and potable water from a First Nations community for the presence of coliform bacteria as well as various antibiotic resistance genes. Samples, including those from drinking water sources, were found to be positive for various antibiotic resistance genes, namely, ampC, tet(A), mecA, β-lactamase genes (SHV-type, TEM-type, CTX-M-type, OXA-1, and CMY-2-type), and carbapenemase genes (KPC, IMP, VIM, NDM, GES, and OXA-48 genes). Not surprisingly, substantial numbers of total coliforms, including Escherichia coli, were recovered from these samples, and this result was also confirmed using Illumina sequencing of the 16S rRNA gene. These findings deserve further attention, as the presence of coliforms and antibiotic resistance genes potentially puts the health of the community members at risk. In this study, we highlight the poor microbiological quality of drinking water in a First Nations community in Canada. We examined the coliform load as well as the presence of antibiotic resistance genes in these samples. This study examined the presence of antibiotic-resistant genes in drinking water samples from a First Nations Community in Canada. We believe that our findings are of considerable significance, since the issue of poor water quality in First Nations communities in Canada is often ignored, and our findings will help shed some light on this important issue.

Phenotypic and genotypic characteristics of carbapenem-resistant Enterobacteriaceae in a tertiary-level reference hospital in Turkey.

BackgroundEnterobacteriaceae are among the most common pathogens that are responsible for serious community-acquired, hospital-acquired, and health-care associated infections. The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) have become an increasing concern for healthcare services worldwide. Infections caused by these bacteria have been associated with significant morbidity and mortality and treatment options have been limited. The rapid and accurate detection of carbapenem resistance in these bacteria is important for infection control. The aim of this study was to investigate the phenotypic and genotypic features of CRE strains isolated in a tertiary-level reference hospital in Turkey.MethodsA total of 181 CRE strains were included in the study. Antimicrobial susceptibility rates were tested using Vitek 2 system. Modified Hodge test (MHT) was performed using meropenem and ertapenem discs. Metallo-β-lactamase antimicrobial gradient test (E-test MBL strips) were used for evaluation of metallo-β-lactamase production. A multiplex PCR was used for detection of carbapenems resistance genes (IMP, VIM, KPC, NDM-1 and OXA-48).ResultsThe OXA-48 gene was detected in 86 strains, NDM-1 gene in six strains, VIM gene in one strain. IMP and KPC genes were not identified. Three strains produced both OXA-48 and NDM-1 and one strain produced both OXA-48 and VIM. In two patients more than one genus of OXA-48 positive CREs was isolated. Ninety-two of the isolates were multidrug-resistant. One hundred and ten isolates were MHT with meropenem (MEM-MHT) positive and 109 isolates were MHT with ertapenem (ERT-MHT) positive. Nine of the isolates were positive with E-test MBL strips. The sensitivity of MEM-MHT and ERT-MHT for detection of OXA-48 was 70.9 and 70.6 %, respectively. MEM-MHT was found highly discriminative for OXA-48 Escherichia coli (p < 0.001). The sensitivity of E-test MBL for NDM-1 was 66.7 %. A statistically significant correlation was observed between OXA-48 gene and MHT positivity and between NDM-1, VIM gene and E-test MBL positivity (p < 0.001).ConclusionsOXA-48 gene is spreading rapidly to many different species of Enterobacteriaceae in the hospital environment. While OXA-48 is still the most common source of carbapenem resistance in Enterobacteriaceae in our country, NDM-1 is increasingly being isolated from patients without a history of foreign contact.

Type 1 Diabetes Genetic Risk Score: A Novel Tool to Discriminate Monogenic and Type 1 Diabetes.

Distinguishing patients with monogenic diabetes from those with type 1 diabetes (T1D) is important for correct diagnosis, treatment, and selection of patients for gene discovery studies. We assessed whether a T1D genetic risk score (T1D-GRS) generated from T1D-associated common genetic variants provides a novel way to discriminate monogenic diabetes from T1D. The T1D-GRS was highly discriminative of proven maturity-onset diabetes of young (MODY) (n = 805) and T1D (n = 1,963) (receiver operating characteristic area under the curve 0.87). A T1D-GRS of >0.280 (>50th T1D centile) was indicative of T1D (94% specificity, 50% sensitivity). We then analyzed the T1D-GRS of 242 white European patients with neonatal diabetes (NDM) who had been tested for all known NDM genes. Monogenic NDM was confirmed in 90, 59, and 8% of patients with GRS <5th T1D centile, 50-75th T1D centile, and >75th T1D centile, respectively. Applying a GRS 50th T1D centile cutoff in 48 NDM patients with no known genetic cause identified those most likely to have a novel monogenic etiology by highlighting patients with probable early-onset T1D (GRS >50th T1D centile) who were diagnosed later and had less syndromic presentation but additional autoimmune features compared with those with proven monogenic NDM. The T1D-GRS is a novel tool to improve the use of biomarkers in the discrimination of monogenic diabetes from T1D.

The identification of carbapenemase types in Enterobacteriaceae by using molecular assay and phenotyping confirmation tests

In this study, we aimed to identify the molecular carbapenemase types of the Enterobacteriaceae isolates and to evaluate the performance of manually prepared and commercially available combination disc methods and the modified Hodge test. One hundred and forty carbapenemase producing isolates and 45 isolates as control group were included in our study. The Xpert CARBA-R test was used as the molecular method. Antibiotic susceptibility tests were performed using combined discs, manually prepared with APBA (3-aminophenyl boronic acid), DPA (dipicolinic acid), EDTA (Ethylene diamine tetra acetic acid), cloxacillin supplements and Mastdiscs Combi-D70C that includes four antibiotic discs with specific inhibitors and temocillin discs. The modified Hodge test was performed on all isolates. OXA-48 gene was identified in 129 isolates , the NDM gene was identified in 10 isolates and VIM in one isolate. Thirty inaccurate results (30/185, 16%) were detected by using the manually prepared confirmation test. The sensitivity and specificity of this test were identified respectively 85% and 73%. Also, the sensitivity and specificity of the Mastdiscs Combi-D70C were identified as 100%. Negative results were detected in 3 NDM isolates with the use of a modified Hodge test. Sensitivity and specificity were calculated for the modified Hodge test respectively 97% and 100%. Finally, molecular methods provide results rapidly but they are not always easily accessible. The modified Hodge test can be used only for screening as a first step test and is not one of the tests that can identify the type of the carbapenemase. When carbapenem-resistant Enterobacteriaceae are detected, a commercial kit like Mastdiscs Combi-D70 may be preferred instead of the manually prepared phenotypic verification tests.

English

The β-lactams are frequently used antibiotics and are essential in treatment of serious bacterial infections. The emergence of β-lactamases has been an ongoing serious therapeutic problem. To overcome this problem, scientists utilized several β-lactamases inhibitors such as clavulanic acid and tazobactam that are used in combination with antibiotics. However, pathogenic bacteria have acquired additional mechanisms of resistance such as the acquisition of extended spectrum beta-lactamases (ESBL) and carbapenemases that cannot be inhibited by current inhibitors. Carbapenem-resistant Enterobacteriaceae (CRE) has become an international health threat. In the present work, 100 clinical Klebsiella strains were isolated, identified, and their antibiotic profiles were determined by the disc diffusion method. β-lactam resistance was evaluated using phenotypic and genotypic methods. More than 50% of the Klebsiella isolates exhibited resistance to tetracyclin, ceftriaxone, (pipracillin/tazobactam), azetreonam, ofloxacin, cefepime and cefoperazone/sulbactam. Klebsiella isolates were less resistant to gentamicin (32.4%) and amikacin (14.7%). Few isolates were resistant to meropenem and imipenem (6.86%). Among the 100 clinical isolates, 50% were ESBL producers and 32% were AmpC producers. The 7 imipenem resistant isolates were carbapenemase producers. PCR showed that carbapenem resistance may be due to NDM gene that was present in 43% of the isolates and OXA gene that was found in 28% of the isolates. KPC genes were not detected in any of the isolates. Antibiotic resistance is a worldwide problem that poses a major threat to therapeutic efficacy of available antibiotics, including carbapenem. Determination of causes of resistance is essential for better treatment options. Key words: Klebsiella, beta-lactams, ESBLs, carbapenem-resistant, NDM, OXA, antibiotic resistance

Repeated local emergence of carbapenem-resistant Acinetobacter baumannii in a single hospital ward

We recently reported a dramatic increase in the prevalence of carbapenem-resistant Acinetobacter baumannii infections in the intensive care unit (ICU) of a Vietnamese hospital. This upsurge was associated with a specific oxa23-positive clone that was identified by multilocus VNTR analysis. Here, we used whole-genome sequence analysis to dissect the emergence of carbapenem-resistant A. baumannii causing ventilator-associated pneumonia (VAP) in the ICU during 2009–2012. To provide historical context and distinguish microevolution from strain introduction, we compared these genomes with those of A. baumannii asymptomatic carriage and VAP isolates from this same ICU collected during 2003–2007. We identified diverse lineages co-circulating over many years. Carbapenem resistance was associated with the presence of oxa23, oxa40, oxa58 and ndm1 genes in multiple lineages. The majority of resistant isolates were oxa23-positive global clone GC2; fine-scale phylogenomic analysis revealed five distinct GC2 sublineages within the ICU that had evolved locally via independent chromosomal insertions of oxa23 transposons. The increase in infections caused by carbapenem-resistant A. baumannii was associated with transposon-mediated transmission of a carbapenemase gene, rather than clonal expansion or spread of a carbapenemase-harbouring plasmid. Additionally, we found evidence of homologous recombination creating diversity within the local GC2 population, including several events resulting in replacement of the capsule locus. We identified likely donors of the imported capsule locus sequences amongst the A. baumannii isolated on the same ward, suggesting that diversification was largely facilitated via reassortment and sharing of genetic material within the localized A. baumannii population.

SPECTRUM OF ANTIBIOTIC RESISTANCE AND PREVALENCE OF OXA-CARBAPENEMASES AMONG ACINETOBACTER BAUMANNII STRAINS, ISOLATED FROM PATIENTS OF SURGICAL AND REANIMATION DEPARTMENTS IN MOSCOW

Characterize spectrum of antibiotics resistance of Acinetobacter baumannii strains, isolated from patients of 8 surgical and reanimation departments of 3 medical institution of Moscow, and determine molecular-genetic mechanisms of stability of their carbapenem-resistant forms. 95 strains of A. baumannii, isolated from patients of reanimation and surgical departments of Moscow in 2012-2014, were studied. Sensitivity of strains to antibiotics was tested phenotypically according to recommendations of EUCAST. The presence of VIM, IMP, OXA-23, OXA-40, OXA-48, OXA-58 and NDM genes in the studied strains was determined by polymerase chain reaction in real time. 86.3% of strains turnedout to be non-sensitive to carbapenems, sensitive--13.7%. 80.0% of strains were non-sensitive to gentamicin, 80.0% of strains--to netilmicin, 94.7% of strains--to ciprofloxacin 2.1%--to colistin. 91.6% of isolates have shown non-sensitivity to members of 2 and more classes of antibiotics, 78.9% of strains--to members of 3 classes. 2 strains were panresistant, 4.2% (4/95) of the isolates were sensitive to all the classes of antibiotics. Metallo-β-lactamases were not detected. Genes of carbapenemases (OXA-23 and/or OXA-40) were detected in 85.3% (81/95) of strains, characterized phenotypically as non-sensitive to carbapenems. The results obtained shown an increase of resistance to carbapenems and multiple resistance in clinically significant strains of A. baumannii. Resistance to carbapenems is associated with OXA-23 and OXA-40 genes. The conclusions allow to justify perspectives of introduction of technologies of molecular-genetic testing of antibiotics resistance.

Outbreak of NDM-1-producing Klebsiella pneumoniae 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 50-240kb in size. This is the first report of an outbreak caused by NDM-1-producing K. pneumoniae ST76 and ST37 among neonates.

Türkiye’de 2014 Yılı İçinde İzole Edilen Karbapeneme Dirençli Escherichia coli ve Klebsiella pneumoniae İzolatlarında Karbapenemaz Varlığının Araştırılması

Carbapenems are the choice of treatment in infections caused by multidrug resistant Enterobacteriaceae. In recent years carbapenem-resistant Enterobacteriaceae isolates due to carbapenemases have been increasingly reported worldwide. Multicenter studies on carbapenemases are scarce in Turkey. The aim of this study was to determine the distribution of carbapenemases from different parts of Turkey as a part of the European Survey of Carbapenemase Producing Enterobacteriaceae (EuSCAPE) project. Beginning in November 2013, carbapenem-resistant isolates resistant to at least one of the agents, namely imipenem, meropenem, and ertapenem were sent to the coordinating center. Minimum inhibitory concentrations for these carbapenems were determined by microdilution tests following EUCAST guidelines. Production of carbapenemase was confirmed by combination disk synergy tests. Types of carbapenemases were investigated using specific primers for VIM, IMP; NDM, KPC and OXA-48 genes by multiplex polymerase chain reaction. In a six month period, 155 suspected carbapenemase-positive isolates were sent to the coordinating center of which 21 (13.5%) were E.coli and 134 (86.5%) were K.pneumoniae. Nineteen (90.5%) strains among E.coli and 124 (92.5%) strains among K.pneumoniae were shown to harbour at least one carbapenemase gene by molecular tests, with a total of 92.3% (143/155). Carbapenemases were determined as a single enzyme in 136 strains (OXA-48: 84.6%; NDM: 6.3%; VIM: 2.8%; IMP: 1.4%) and as a combination in seven isolates (OXA-48 + NDM: 2.1%; OXA-48 + VIM: 2.1%; VIM + NDM: 0.7%). KPC was not detected in any of the isolates. According to the microdilution test results, resistance to imipenem, meropenem and ertapenem in OXA-48 isolates were 59.5%, 52.9% and 100%, respectively. The combination disk synergy test was 100% compatible with the molecular test results. As most of the OXA-48 producing isolates were susceptible to meropenem but all were resistant to ertapenem, ertapenem seems to be the most sensitive agent in screening carbapenemases in areas where OXA-48 is prevalent and phenotypic combination tests can be useful in centers where molecular tests are not available.