Phenotypic screening of carbapenemases and associated β-lactamases in carbapenem-resistant Enterobacteriaceae

Dissemination of carbapenem resistance among Enterobacteriaceae poses a considerable threat to public health. Carbapenemase gene detection by molecular methods is the gold standard but is available in only a few laboratories. The aim of this study was to test phenotypic methods for the detection of metallo-β-lactamase (MBL)- or Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and associated mechanisms of β-lactam resistance against a panel of 30 genotypically characterized carbapenem-resistant Enterobacteriaceae : 9 MBL, 7 KPC, 6 OXA-48, and 8 extended-spectrum β-lactamase (ESBL) or AmpC β-lactamases associated with decreased permeability. We used carbapenemase inhibitor-impregnated agar to test for carbapenem-resistant strains. Differences in the inhibition zone sizes of the meropenem, imipenem, ertapenem, and doripenem disks were measured between control and inhibitor (EDTA or phenylboronic acid [PBA] with or without cloxacillin)-impregnated Mueller-Hinton agar with a cutoff of 10 mm. All 9 MBL- and 7 KPC-producing Enterobacteriaceae were identified from the differences in zone size in the presence and absence of specific inhibitors, regardless of the carbapenem MICs and including isolates with low-level resistance to carbapenems. We also detected their associated β-lactam resistance mechanisms (11 ESBL-type and 5 class A β-lactamase 2b). No differences in zone size were observed for OXA-48-producing strains or other carbapenem resistance mechanisms such as ESBL and decreased permeability. We propose a new strategy to detect carbapenemases (MBL- and KPC-type) and associated mechanisms of β-lactam resistance (ESBL or class A β-lactamase 2b) by the use of inhibitor-impregnated agar. A rapid phenotypic detection of resistance mechanisms is important for epidemiological purposes and for limiting the spread of resistant strains by implementing specific infection control measures.     

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.     

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.     

IMP-type metallo-β-lactamases in Gram-negative bacilli: distribution, phylogeny, and association with integrons

Twenty-nine IMP-type β-lactamases (IMPs) have been identified in at least 26 species of clinically important Gram-negative bacilli from more than 24 countries/regions. Most of bla(IMP) genes are harbored by class 1 integrons that are usually embedded in transposons and/or plasmids, footnoting their horizontal transfer and worldwide distribution. bla(IMP) genes usually co-exist with other resistance genes, such as aacA, catB, and bla(OXA), resulting in multi-drug resistance. Compared to other gene cassettes, 76.3% of the bla(IMP) gene cassettes are located adjacent to Pc promoter of the class 1 integrons, indicating that the bla(IMP) genes are readily expressed in most of bacterial hosts.     

Molecular and phenotypic characterization of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases with focus on CTX-M in a low-endemic area in Sweden

During the last decade increasing prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has been detected worldwide, mainly due to dissemination of Escherichia coli and Klebsiella pneumoniae producing CTX-M-type ESBLs. CTX-M-15 is the most widespread CTX-M type, and the predominant type in various countries. Dissemination of ESBL-producing organisms is caused not only by horizontal transfer of plasmids, but also by clonal spread of ESBL-producing strains. In this study, the molecular epidemiology of class A ESBL (ESBL(A))-producing E. coli and K. pneumoniae isolated in ?rebro County, Sweden, was investigated. Out of 200 ESBL(A) -producing E. coli and K. pneumoniae isolates, collected over a 10-year period, 87% were producing CTX-M, belonging to subgroup CTX-M-1 (64%), CTX-M-9 (34%), or CTX-M-2 (2%). The remaining isolates were producing variants of SHV and TEM. Sequencing of the bla(CTX-M) genes revealed 10 different CTX-M types, with a dominance of CTX-M-15 (E. coli 54%, K. pneumoniae 50%) followed by CTX-M-14 (E. coli 28%, K. pneumoniae 27%). Phenotypic characterization of the CTX-M-producing isolates was performed using the PhenePlate system. Although a few minor clusters of CTX-M-15 and CTX-M-14 producers were identified, the majority of the isolates did not appear to be clonally related.     

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.     

OXA- and GES-type β-lactamases predominate in extensively drug-resistant Acinetobacter baumannii isolates from a Turkish University Hospital

We determined the antibiotic susceptibility and genetic mechanisms of resistance in clinical strains of Acinetobacter baumannii from Istanbul, Turkey. A total of 101 clinical strains were collected between November 2011 and July 2012. Antimicrobial susceptibility was performed using the Vitek 2 Compact system and E-test. Multiplex PCR was used for detecting bla_OXA-51-like, bla_OXA.-_23-like, bla_OXA-40-like and bla_OXA-58-like genes. ISAba1, bla_IMP-like, bla_VIM-like, bla_GES, bla_VEB, bla_PER-2, aac-3-Ia and aac-6'-Ib and NDM-1 genes were detected by PCR and sequencing. By multiplex PCR, all strains were positive for bla_OXA-51, 79 strains carried bla_OXA-23 and one strain carried bla_OXA-40. bla_OXA-51 and bla_OXA-23 were found together in 79 strains. ISAba1 element was detected in 81 strains, and in all cases it was found upstream of bla_OXA-51. GES-type carbapenemases were found in 24 strains (GES-11 in 16 strains and GES-22 in 8 strains) while bla_PER-2, bla_VEB-1, bla_NDM-1, bla_IMP- and bla_VIM-type carbapenemases were not observed. Aminoglycoside modifying enzyme (aac-3-Ia and aac-6'Ib) genes were detected in 13 and 15 strains, respectively. Ninety-seven (96%) A. baumannii strains were defined as MDR and of these, 98% were extensively drug resistant (sensitive only to colistin). Colistin remains the only active compound against all clinical strains. As seen in other regions, OXA-type carbapenemases, with or without an upstream ISAba1, predominate but GES-type carbapenemases also appear to have a significant presence. REP-PCR analysis was performed for molecular typing and all strains were collected into 12 different groups. To our knowledge, this is the first report of GES-11 and OXA-40 in A. baumannii from Turkey.     

Acinetobacter: a potential reservoir and dispenser for β-lactamases

Innate resistance and remarkable ability to acquire additional resistance determinants underline the clinical importance of Acinetobacter. Over 210 β-lactamases belonging to 16 families have been identified in the genus, mostly in clinical isolates of A. baumannii. In this review, we update the current taxonomy of the genus Acinetobacter and summarize the β-lactamases detected in Acinetobacter spp. with an emphasis on Acinetobacter-derived cephalosporinases (ADCs) and carbapenem-hydrolysing class D β-lactamases (CHDLs). We also discuss the roles of integrons and insertion sequence (IS) elements in the expression and dissemination of such resistance determinants.     

CTX-M-9 group extended-spectrum β-lactamases in neonatal stool isolates: emergence in India

The study reports for the first time the identification of CTX-M-14-like and CTX-M-27-like extended-spectrum β-lactamases (ESBLs) belonging to the CTX-M-9 group in Klebsiella pneumoniae and Escherichia coli isolated from the neonatal stool in India. The plasmid carrying the blaCTX-M-9 group in both the isolates was transferable. Till date, no other CTX-M group, except the CTX-M-1 group, has been reported from India. A total of 77% of the neonates had ESBL-producing K. pneumoniae or E. coli in their stool, and blaCTX-M-15 was the predominant ESBL gene. Although the CTX-M-9 group was found in the stool and did not cause infection, the detection of the CTX-M-9 group might be a prelude to future infections.     

Prevalence of extended-spectrum β-lactamases in the local farm environment and livestock: challenges to mitigate antimicrobial resistance

Abstract

The effectiveness of antibiotics has been challenged by the increasing frequency of antimicrobial resistance (AR), which has emerged as a major threat to global health. Despite the negative impact of AR on health, there are few effective strategies for reducing AR in food-producing animals. Of the antimicrobial resistant microorganisms (ARMs), extended-spectrum β-lactamases (ESBLs)-producing Enterobacteriaceae are an emerging global threat due to their increasing prevalence in livestock, even in animals raised without antibiotics. Many reviews are available for the positive selection of AR associated with antibiotic use in livestock, but less attention has been given to how other factors including soil, water, manure, wildlife, and farm workers, are associated with the emergence of ESBL-producing bacteria. Understanding of antibiotic resistance genes and bacteria transfer at the interfaces of livestock and other potential reservoirs will provide insights for the development of mitigation strategies for AR.     

Identification of blaOXA-128 and blaOXA-129, two novel OXA-type extended-spectrum-β-lactamases in Pseudomonas aeruginosa, in Hunan Province, China

We collected 97 non-repetitive carbapenemases-sensitive clinical isolates of Pseudomonas aeruginosa in Human Province, China, during the period of October 2006 to January 2007. From these isolates, we identified two novel oxacillin-hydrolysing (OXA) type extended-spectrum-β-lactamases (ESBLs): bla OXA-128 and bla OXA-129, which contain the mutations of I89V from bla OXA-56 and K134N from bla OXA-10, respectively. Clinical isolates containing either bla OXA-128 or bla OXA-129 show resistance to cephamycin-class antibiotics but sensitive to carbapenem-class antibiotics. The occurrence of novel OXA-type lactamases suggests a regional prevalent pattern of ESBLs Pseudomonas aeruginosa in this area.     

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.     

Genetic diversity of carbapenem-hydrolysing β-lactamases in Acinetobacter baumannii from Romanian hospitals

Thirteen carbapenem-resistant Acinetobacter baumannii isolates, collected in Romania during 2009-2010, were investigated to identify the mechanism(s) responsible for carbapenem resistance. Genotyping was performed by pulsed-field gel electrophoresis, multiplex PCR sequence typing and multilocus sequence typing. Eleven non-clonally related isolates harboured the bla(OXA-23) gene on their chromosome within a Tn2008 transposon structure. The two remaining isolates harboured a bla(OXA-58) gene that was either plasmid or chromosome borne. Two isolates co-expressed OXA-23 together with the extended-spectrum β-lactamase PER-1. This study constitutes the first report of OXA-58 and OXA-23-producing A. baumannii isolates in Romania.     

Diversity of β-lactamases in Pseudomonas aeruginosa isolates producing metallo-β-lactamase in two Tunisian hospitals

This study was conducted to identify the β-lactamase content of 30 metallo-β-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) were identified in 17, 5, 21, and 1 isolates, respectively. These enzymes were often associated in the same isolate: 26 isolates had at least two β-lactamases. The predominant serotype was O12. Pulsed-field gel electrophoresis revealed genetic diversity among the metallo-β-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) in Tunisia.     

Detection of AmpC β-lactamases in Escherichia coli,Klebsiella spp., Salmonella spp. and Proteus mirabilis in a regional clinical microbiology laboratory

There are currently no standardized diagnostic tests available for the reliable detection of AmpC β-lactamases in Klebsiella spp., Escherichia coli, Proteus mirabilis and Salmonella spp. A study was designed to evaluate a confirmation disk test using cefotetan (CTT) and cefoxitin (FOX) with phenylboronic acid (PBA). It also investigated the most suitable screening concentrations of FOX, ceftriaxone (CRO) and ceftazidime (CAZ) for the detection of AmpC β-lactamases. A total of 126 control (consisting of 11 laboratory and 115 well-characterized clinical strains) and 29 840 non-repeat clinical isolates were included. FOX with PBA used in a confirmation test and CRO and CAZ as screening agents were found to be unreliable. FOX at ≥ 32 mg/L was the best screening agent and CTT with PBA was the best confirmation test. Of the clinical isolates 635 (2%) were found to be resistant to cefoxitin (MIC ≥ 32 ug/mL) and 332 (52%) were AmpC positive. E. coli was the most common organism with AmpC β-lactamases and was mostly present in urines from community patients. It is recommended that laboratories use FOX at 32 mg/L as a screening agent and perform a disk test with CTT and PBA to confirm the presence of an AmpC cephalosporinase in isolates of Klebsiella spp., E. coli, Salmonella spp. and P. mirabilis. This approach is convenient, practical and easy to incorporate into the workflow of a clinical laboratory. False-positive AmpC detection may occur with KPC-producing bacteria when inhibitor-based methods are used.     

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.     

Using nucleic acid microarrays to perform molecular epidemiology and detect novel β-lactamases: a snapshot of extended-spectrum β-lactamases throughout the world

The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008-2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla(ESBL) (bla(SHV), bla(TEM), and bla(CTX-M) genes of groups 1, 2, 9, and 8/25) and bla(KPC) genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla(ESBL) and bla(KPC) genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla(ESBL) gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla(TEM) (n = 5), bla(SHV) (n = 11), bla(CTX-M) (n = 19), and bla(KPC) (n = 3) were detected. A new bla(SHV) variant, bla(SHV-129), and a new bla(KPC) variant, bla(KPC-11), were also identified. The most common bla genes found in this study were bla(CTX-M-15), bla(CTX-M-14), and bla(SHV-12). Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla(ESBL) genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla(SHV-129) and bla(KPC-11).