Prevalence and molecular epidemiology of acquired AmpC β-lactamases and carbapenemases in Enterobacteriaceae isolates from 35 hospitals in Spain

The purpose of this investigation was to determine the prevalence of plasmid-mediated AmpC (pAmpC) and carbapenemases in Enterobacteriaceae collected from 35 hospitals in Spain and to establish their epidemiological relationships. We conducted a prospective multi-centre study on pAmpC- or carbapenemase-producing Enterobacteriaceae isolates from clinical samples collected from February to July 2009. The strains suspected to carry pAmpC were resistant or showed intermediate susceptibility to co-amoxiclav and second- or third-generation cephalosporins. Strains suspected to carry a carbapenemase were selected because they showed a minimum inhibitory concentration (MIC) to imipenem >1 mg/L. Polymerase chain reaction (PCR) and a sequencing strategy were used to characterise the enzymes. The clonal relationships between isolates was analysed by pulsed field gel electrophoresis (PFGE). Among 100,132 Enterobacteriaceae isolates collected, 1,654 were compatible with the production of pAmpC or carbapenemases. We found a prevalence of 0.64 % of pAmpC (n?=?635) and 0.04 % of carbapenemases (n?=?43). The most prevalent pAmpC enzymes were CMY-type (78.3 %), DHA-type (19.5 %), ACC-type (1.6 %) and FOX-type (0.6 %). The CMY-type was the most frequent in Escherichia coli and Proteus mirabilis species, whereas the DHA-type was mainly found in Klebsiella spp. The enzymes involved in carbapenem resistance were VIM-1, IMP-22 and the new IMP-28. Nine new bla genes were described: bla _CMY-54, bla _CMY-55, bla _CMY-56, bla _CMY-57, bla _CMY-96, bla _DHA-6, bla _DHA-7, bla _FOX-8 and bla _IMP-28. The prevalence of pAmpC or carbapenemases found is not negligible. The CMY-types were the predominant pAmpC, whereas the VIM or IMP enzymes were the predominant carbapenemases. Furthermore, we observed a great genetic diversity among pAmpC-producing strains and a close clonal relationship between carbapenemase-producing strains.     

Clinical characteristics of bacteraemia caused by extended-spectrum β-lactamase-producing Enterobacteriaceae in the era of CTX-M-type and KPC-type β-lactamases

Clin Microbiol Infect 2012; 18: 887-893 ABSTRACT: A multicentre, case-control study was conducted to assess risk factors and patient outcomes of bacteraemia caused by Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs) and Klebsiella pneumoniae carbapenemases (KPCs). One hundred and five and 20 patients with bacteraemia caused by ESBL-producing and KPC-producing organisms were matched to controls who had bacteraemia caused by non-ESBL/KPC-producing organisms, respectively. Independent risk factors for ESBL production included admission from a nursing home (OR 4.64; 95% CI 2.64-8.16), chronic renal failure (OR 2.09; 95% CI 1.11-3.92), the presence of a gastrostomy tube (OR 3.36; 95% CI 1.38-8.18), length of hospital stay before infection (OR 1.02; 95% CI 1.01-1.03), transplant receipt (OR 2.48; 95% CI 1.24-4.95), and receipt of antibiotics with Gram-negative activity in the preceding 30 days (OR 1.76; 95% CI 1.00-3.08). Twenty-eight-day crude mortality rates for patients infected with ESBL-producing or KPC-producing organisms and controls were 29.1% (34/117) and 19.5% (53/272), respectively (OR 1.70; 95% CI 1.04-2.80). On multivariate analysis, inadequate empirical therapy (OR 2.26; 95% CI 1.18-4.34), onset of bacteraemia while in the intensive-care unit (OR 2.74; 95% CI 1.47-5.11), Apache II score (OR 1.17; 95% CI 1.12-1.23) and malignancy (OR 2.66; 95% CI 1.31-5.41) were independent risk factors for mortality. CTX-M was the most common ESBL type in Escherichia coli, whereas SHV predominated in Klebsiella spp. and Enterobacter spp.     

Current trends in culture-based and molecular detection of extended-spectrum-β-lactamase-harboring and carbapenem-resistant Enterobacteriaceae

The ever-expanding role of extended-spectrum-β-lactamase (ESBL)-harboring and carbapenem-resistant Enterobacteriaceae in causing serious infections poses a grave public health threat because these organisms also tend to be multidrug resistant, for which very few (if any) antibiotic options remain available. Rapid detection of such panresistant organisms offers one of the best solutions to improve patient screening and hospital infection control practices as well as curb inappropriate antibiotic use. This review discusses and compares primarily the current state-of-the-art culture-based and molecular methods that are commercially available for detection or screening of ESBL-harboring and carbapenem-resistant Enterobacteriaceae.     

Persistence of TEM-52/TEM-92 and SHV-12 extended-spectrum β-lactamases in clinical isolates of Enterobacteriaceae in Italy

Extended-spectrum β-lactamases (ESBLs) belonging to the TEM and SHV families were investigated in 583 ESBL-producing Enterobacteriaceae collected at the clinical microbiology laboratories of 11 teaching Italian hospitals. By molecular analysis TEM-type and SHV-type ESBLs were confirmed on 154 and 74 isolates, respectively. High variability was found among TEM-types β-lactamases with the following variants: TEM-5, TEM-6, TEM-12, TEM-15, TEM-24, TEM-26, TEM-29, TEM-52, TEM-92, TEM-134, and TEM-149. Among SHV variants, SHV-2a, SHV-5, SHV-12, and SHV-28 have been detected. The most widespread variants are TEM-52/92 and SHV-12.     

Evaluation of a diagnostic flow chart for detection and confirmation of extended spectrum β-lactamases (ESBL) in Enterobacteriaceae

This study aimed to develop a modular, diagnostic algorithm for extended spectrum β-lactamase (ESBL) detection in Enterobacteriaceae. Clinical Enterobacteriaceae strains (n = 2518) were screened for ESBL production using Clinical and Laboratory Standards Institute (CLSI) breakpoints for third-generation cephalosporins and by synergy image detection (clavulanic acid/extended-spectrum cephalosporins). Isolates screening positive for ESBL (n = 242, 108 by critical CLSI diameters alone, five by double disk synergy test (DDST) alone, and 129 by both critical diameters and DDST) and 138 ESBL screening negative isolates (control group) were investigated by molecular methods considered to be the reference standard (multiplex CTX-M type PCR, TEM and SHV type sequence characterization). One hundred and twenty-four out of 242 Enterobacteriaceae isolates screening positive for ESBL were confirmed to be ESBL positive by the reference standard, the majority of them in E. coli, K. pneumoniae and E. cloacae (94, 17 and nine isolates, respectively). Prevalence of ESBL production ranged from < 1% for P. mirabilis to 4.7%, 5.1% and 6.6%, for K. pneumoniae, E. cloacae and E. coli, respectively. Combining CLSI ceftriaxone and cefpodoxime critical ESBL diameters was found to be the most sensitive phenotypic screening method (sensitivity 99.2%). Combining critical diameters of cefpodoxime and ceftriaxone with DDST for cefpodoxime resulted in a sensitivity of 100%. For phenotypic confirmation, combining the CLSI recommended combined disk test (CDT) for ceftazidime and cefotaxime amended with a cefepime CDT was highly sensitive (100%) and specific (97.5%). With respect to the studied population, the diagnostic ESBL algorithm developed would have resulted in sensitivity and specificity of 100%. The corresponding flow chart is simple, easy to use, inexpensive and applicable in the routine diagnostic laboratory.     

Detection and characterization of Enterobacteriaceae producing metallo-β-lactamases in a tertiary-care hospital in Spain

Carbapenem-resistant or intermediate (MIC ≥1 mg/L) clinical isolates (n = 12) of three species of Enterobacteriaceae (Klebsiella pneumoniae, Klebsiella oxytoca and Escherichia coli) were characterized. The isolates harboured integrons containing the VIM-1 metallo-β-lactamase gene together with other resistance gene cassettes. In particular, the CTX-M-2 gene was detected in four of the K. pneumoniae isolates. The patient population was mostly paediatric and characterized by severe underlying illnesses that involved long-term hospitalization, major surgery and/or immunosuppressive and broad-spectrum antibiotic therapy.     

Occurrence and regional distribution of SHV-type extended-spectrum β-lactamases in Hungary

In order to determine the presence and geographical distribution of SHV-type extended-spectrum -lactamase genes among Enterobacteriaceae strains in Hungary, isolates from 25 microbiology laboratories throughout the country were collected between January 2002 and August 2003 and examined. Sequencing of the genes showed that SHV-5 and SHV-2a are the dominant SHV-types in extended-spectrum -lactamase-producing Enterobacteriaceae strains in this country. The SHV-2 gene, which is prevalent in many European countries, was not detected, but one isolate carried the SHV-12 gene. The results show that these genes are circulating among Enterobacteriaceae strains in Hungary and indicate that strict infection control measures are warranted in order to prevent their spread.     

Diversity of carbapenemases in clinical isolates of Enterobacteriaceae in Croatia—the results of a multicentre study

Since the first carbapenem-resistant Klebsiella pneumoniae strain was isolated in 2008, Enterobacteriaceae with reduced susceptibility to one or more carbapenems have emerged sporadically in different geographical regions in Croatia. These observations gave rise to a multicenter study on carbapenem resistance in Enterobacteriaceae from Croatia. Fifty-seven carbapenem-non-susceptible strains of Enterobacteriaceae were collected during 2011–2012 from four large hospital centres in Croatia. Overall, 36 strains produced VIM-1 β-lactamase, three produced NDM-1, and one produced KPC-2. A high degree of clonal relatedness was observed in Enterobacter cloacae and Citrobacter freundii strains, in contrast to K. pneumoniae strains. Bla_VIM genes were located within class1 integron which contained genes encoding resistance to aminoglycosides (aac_A4). The study found strong association between bla_VIM and qnr_B6 and between bla_NDM and qnr_A6 genes.     

Prevalence and spread of extended-spectrum β-lactamase-producing Enterobacteriaceae in Ngaoundere,Cameroon

During April 2010 and June 2010, 334 Enterobacteriaceae isolates from 590 participants (outpatients, inpatients, inpatient carers, hospital workers and members of their households) were collected from faecal samples. Based on β-lactamase pattern, origin of strains and the relationship between participants, 44 isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae were selected from 44 participants (in Ngaoundere Protestant Hospital and Ngaoundere Regional Hospital, Cameroon). To determine the relatedness of bacterial strains, these isolates were fingerprinted using the automated, repetitive-sequenced-based PCR-based DiversiLab system. Subsequently, E. coli isolates that had undergone DiversiLab analysis were examined with respect to their phylogenetic group and detection of the ST131 clone to shed light on the epidemiology of these isolates in the Ngaoundere hospitals. The prevalence of faecal carriage of ESBL-producing Enterobacteriaceae among the study participants was 54.06%. According to participant groups, the prevalence of faecal carriage was also high (outpatients 45%; inpatients 67%; inpatient carers 57%; hospital workers 44%; and members of their households 46%). Analysis of the molecular epidemiology of ESBL-producing E. coli and K. pneumoniae showed a close relationship of the isolates between related and non-related individuals. In addition, DiversiLab results of E. coli identified four related isolates (4/22) from cluster III belonging to the epidemiologically important clone ST131. Our results highlight the importance of outpatients, inpatients, their carers, hospital workers and their families as reservoirs of ESBL-producing Enterobacteriaceae     

Prevalence and characterization of plasmid-mediated quinolone resistance genes in extended-spectrum β-lactamase-producing Enterobacteriaceae isolates in Mexico

The objectives of this study were to investigate the prevalence of plasmid-mediated quinolone resistance genes in a collection of 226 extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates and characterize the qnr-positive isolates. The rate of qnr-positive isolates was 21.6% (49/226), 49.5% for aac(6')-Ib-cr (112/226), and 1.7% for qepA1 (4/226). Those isolates carried qnr genes corresponding to types qnrB (71.4%), qnrS1 (24.4%), and qnrA1 (18.3%). The distribution among bacterial species was as follows: 55.8% (19/34) to Enterobacter cloacae, 50% (28/56) to Klebsiella pneumoniae, and 1.4% (2/136) to Escherichia coli. The characterization of qnr-positive isolates indicated the ESBL SHV-types as the most prevalent (81.6%), including the ESBLs SHV-12, SHV-5, and SHV-2a, followed by CTX-M-15 (44.9%) and TLA-1 (8.1%). In addition, for qnr-positive isolates, the prevalence of aac(6')-Ib-cr was 55.1%, but qepA was not identified. Alterations at codons Ser-83 and Asp-87 in GyrA and at codons Ser-80 in ParC were observed in 69% and 80% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a cotransmission of bla(CTX-M-15) with qepA1 or aac(6')-Ib-cr and/or qnrA1 and bla(SHV-type) with qnrB5 and qnrB6 genes. To conclude, these findings indicate a high prevalence of qnr and aac(6')-Ib-cr among ESBL-producing isolates from Mexican hospitals and point to the wide spread of qnr-like determinants associated to ESBLs SHV- and CTX-M-type in Mexican clinical isolates.     

Extended-spectrum β-lactamase-producing Enterobacteriaceae in Cameroonian hospitals

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have been described worldwide, but there are few reports on the carriage of these bacteria in Cameroon. In order to investigate the types of ESBLs and to analyse some risk factors associated with ESBL carriage, faecal samples were collected between 3 January and 3 April 2009 from hospitalised patients at Yaounde Central Hospital and at two hospitals in Ngaoundere, Cameroon. Enterobacterial isolates resistant to third-generation cephalosporins were screened for ESBL production using the double-disk synergy test. Polymerase chain reaction (PCR) and DNA sequencing were performed in order to find out the different types of ESBL genes in presumptive ESBL-positive isolates. During the study period, a total of 121 different patients were screened for ESBL carriage. The prevalence among these patients whose faecal samples were found to contain ESBL-producers was 55.3 % (67/121). According to a univariate analysis, hospitalisation during the previous year was found to be associated with ESBL carriage. Of the 71 bacteria isolated, Escherichia coli was predominant and represented 48 % of all isolates. ESBL characterisation revealed two types of ESBLs, CTX-M-15 (96 %) and SHV-12 (4 %). The present study emphasises the importance of screening for ESBLs in laboratories in African countries. The monitoring and detection of ESBL-producing bacteria are important in the setting up of appropriate treatment of patients and to ensure effective infection control efforts.     

Prevalence of acquired AmpC β-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes at a Spanish hospital from 1999 to 2007

In 2007, a significant increase in acquired ampC genes in Enterobacteriaceae from 0.06% in 1999 to 1.3% was observed. Proteus mirabilis showed the highest prevalence (0.95%) and CMY-2 was the most prevalent AmpC enzyme (66.7%). Other enzymes such as CMY-4, DHA-1, ACC-1, and three new enzymes called CMY-25, CMY-27 and CMY-40 were detected. Seven out of the 117 isolates (6%) also produced an extended-spectrum β-lactamase. As acquired AmpC enzymes are likely to become a serious public health issue worldwide, close surveillance is necessary to curb their spread.     

Cessation of screening for intestinal carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae in a low-endemicity intensive care unit with universal contact precautions

ObjectivesThe usefulness of screening for carriage of extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) with active surveillance cultures (ASC) remains equivocal in low-endemicity intensive care units (ICUs). Our primary objective was to appraise the impact of ceasing ASC on the incidence of ICU-acquired ESBL-E infections in an ICU with universal contact precautions (CP). Patient outcomes and carbapenem consumption were also investigated.MethodsA single-ICU, retrospective, uncontrolled before-and-after study including all patients admitted for ≥3 days during two consecutive 1-year periods with and without ASC.ResultsA total of 524 and 545 patients were included during the ASC and the no-ASC periods, respectively. Twenty-eight patients (5.3%) from the ASC period were ESBL-E carriers. An ICU-acquired ESBL-E infection (median duration of risk exposure, 4 (range 2–9) days for both periods) occurred in 1.1% and 1.5% of patients admitted during the ASC and the no-ASC periods (p = 0.64), with no inter-period variation in incidence after adjustment on competing risks of death and ICU discharge (standardized hazard ratio (SHR) 2.32, 95% CI 0.80–6.73, p = 0.12). An admission during the no-ASC period exerted no independent impact on the hazards of ESBL-E infections (adjusted OR 1.16, 95% CI 0.38–3.50, p = 0.79), in-ICU death (SHR 1.22, 95% CI 0.93–1.59, p = 0.15) and extended length of stay (SHR for discharge 0.89, 95% CI 0.79–1.01, p = 0.08). Carbapenem exposure in patients without ESBL-E infection decreased between the ASC and no-ASC periods (75 versus 61 carbapenem-days per 1000 patient-days, p = 0.01).ConclusionsIn a low-endemicity ICU with universal CP, the withdrawal of routine screening for ESBL-E carriage had no significant effect on the incidence of ICU-acquired ESBL-E infections and patient outcomes. Carbapenem consumption decreased in patients without ESBL-E infection.     

Modified CLSI Extended-Spectrum β-Lactamase (ESBL) Confirmatory Test for Phenotypic Detection of ESBLs among Enterobacteriaceae Producing Various β-Lactamases

The worldwide dissemination of Enterobacteriaceae producing AmpC β-lactamases and carbapenemases makes difficult the phenotypic detection of extended-spectrum β-lactamases (ESBLs), as they may be masked by these additional enzymes. A modification of the CLSI ESBL confirmatory test was developed and evaluated in a comparative study for its ability to successfully detect ESBLs among Enterobacteriaceae producing various carbapenemases (Klebsiella pneumoniae carbapenemase [KPC], VIM, NDM, and OXA-48) and plasmidic or derepressed AmpCs. The modified CLSI ESBL confirmatory test was performed with cefotaxime and ceftazidime disks with and without clavulanate, on which both boronic acid (BA) and EDTA were dispensed. A total of 162 genotypically confirmed ESBL-positive Enterobacteriaceae isolates (83 carbapenemase/ESBL producers, 25 AmpC/ESBL producers, and 54 ESBL-only producers) were examined. For comparison, 139 genotypically confirmed ESBL-negative Enterobacteriaceae isolates (94 of them possessed carbapenemases and 20 possessed AmpCs) were also tested. The standard CLSI ESBL confirmatory test was positive for 106 of the 162 ESBL producers (sensitivity, 65.4%) and showed false-positive results for 4 of the 139 non-ESBL producers (specificity, 97.1%). The modified CLSI ESBL confirmatory test detected 158 of 162 ESBL producers (sensitivity, 97.5%) and showed no false-positive results for non-ESBL producers (specificity, 100%). The findings of the study demonstrate that the modified CLSI ESBL confirmatory test using antibiotic disks containing both BA and EDTA accurately detects ESBLs in Enterobacteriaceae regardless of the coexistence of additional β-lactam resistance mechanisms.     

VIM and IMP metallo-β-lactamases and other extended-spectrum β-lactamases in Escherichia coli and Klebsiella pneumoniae from environmental samples in a Tunisian hospital

An extremely drug-resistant Enterobacteriaceae species emerged in Kasserine Hospital, Tunisia between 2009 and 2010 causing a local outbreak. We aimed to characterize extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL)-producing Enterobacteriaceae from the hospital environment. Swabs were collected from ten different wards from Kasserine Hospital, Tunisia. A total of 46 isolates were cultured onto MacConkey agar supplemented with ceftazidime to select for ESBL-producing Enterobacteriaceae. Identification and susceptibility patterns were performed using Phoenix-automated phenotypic identification criteria. Extended spectrum β-lactamases (ESBLs) were detected using cefepime ESBL E-test. Colony blotting was first used to detect the occurrence of bla(SHV) , bla(CTX-M) , bla(CMY) , bla(IMP) , and bla(VIM) genes. PCR was used to amplify these genes, and the amplicons were sequenced and analyzed. Total DNA was digested with XbaI, and PFGE was used to type the major isolates that produced IMP-1. Among the 46 isolates, 63% were Klebsiella pneumoniae, 13% were Escherichia coli, 8.7% were Proteus mirabilis, 6% were Enterobacter cloaceae, 4.3% were Providencia rettgeri, 2.5% were Serratia marcescens, and 2.5% were Pantoea agglomerans. PCR amplification and DNA sequencing showed that hospital environment isolates produced SHV-125, CTX-M-15, CMY-2 ESBLs, and IMP-1 and VIM-2 MBLs. PFGE typing showed the emergence of IMP-1 MBL-producing K. pneumoniae isolates that were not clonal. In this study, we report the first characterization of IMP-1 and VIM-2 MBL-producing K. pneumoniae and E. coli isolates collected from Kasserine Hospital, Tunisia.     

Prevalence and types of extended spectrum β-lactamases among urinary Escherichia coli isolates in Moroccan community

This study was designed to characterize extended-spectrum-β-lactamases (ESBL) produced by Escherichia coli isolates causing community urinary tract infections over a 2-year period (2010 and 2011) in a Moroccan large geographical region. Molecular characterization was done by using PCR and sequencing of the β-lactamases genes and plasmid-mediated quinolone resistance determinants. Among 1174 isolates, 49 (4.1%) were ESBL producers. The bla_CTx-M-15 (n = 31) was the most frequent ESBL gene detected, followed by bla_CTx-M-1 (n = 5), bla_SHV-12 (n = 6), bla_PER-2 (n = 3), then bla_TEM-3, bla_TEM-20, bla_TEM-158, bla_SHV-27, bla_SHV-28, bla_SHV-36, bla_SHV-125, bla_CTx-M-14 and bla_CTx-M-27 with one isolate for each. The non-ESBL genes detected were bla_TEM-70 (n = 1), bla_TEM-176 (n = 1), bla_TEM-104 (n = 6), bla_TEM-1 (n = 15) and bla_OxA-1 (n = 12). Plasmid mediated AmpC β-lactamases genes; bla_ACT-5 (n = 1), bla_DHA-1(n = 2) and bla_CMY-2 (n = 4) were detected in seven isolates (14.2%). The bla_OxA-48 (n = 1) and bla_IMP-1 (n = 1) carbapenemases genes were detected among five carbapenem-resistant E. coli. Five isolates (10.2%) harboured qnr genes, qnrB1 (n = 3), qnrB2 (n = 1) and qnrS1 (n = 1) type were detected. Thirty isolates (61.2%) were positive for aac(6′)-Ib-cr gene. The class 1 integron was detected in twenty two (44.8%) isolates. Phylogenetic grouping revealed that 22 (44.8%) isolates belonged to group A, while 15 (30.6%), 11 (22.4%) and 1 (2%) belonged to B2, D and B1. Results of conjugation experiments indicated that bla_CTx-M-15, bla_TEM-1, bla_OxA-1, aac(6′)-Ib-cr and qnrB1 genes were co-transferred and that these genes were carried by a conjugative plasmid of high molecular weight. The results of this work reports the genetic diversity of ESBL genes, with the CTX-M-15 enzyme being the most common among ESBL-producing E. coli in Moroccan community.     

Connexin-26–associated deafness: Phenotypic variability and progression of hearing loss

PurposeTo evaluate genotype-phenotype correlation over time for a cohort of children with connexin-26 (GJB2)–associated autosomal recessive hearing loss.MethodsFifty-two children were identified from a database of individuals with homozygous or compound heterozygous mutations in GJB2 and subjected to chart review of their otolaryngologic and serial audiometric evaluations. Genotype-phenotype correlations were identified among the members of this group by appropriate statistical analyses.ResultsHearing loss was most severe in individuals with two truncating mutations in GJB2 and mildest in those with two nontruncating mutations. Progressive hearing loss was seen directly by serial audiometry in 24% of all subjects, and suggested in a total of 28% when those with normal newborn hearing screens and subsequent hearing loss were included. Progression was particularly common among carriers of the p.V37I allele either in homozygosity or in compound heterozygosity with a truncating allele; these children are primarily of Asian descent and demonstrate mild, slowly progressive hearing loss.ConclusionsPhenotype in GJB2-associated hearing loss is correlated with genotype, with truncating mutations giving rise to more severe hearing loss. Progression of hearing loss is not uncommon, especially in association with the p.V37I allele. These results suggest that close audiometric follow-up is warranted for patients with GJB2-associated recessive hearing loss.