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.     

Study of Klebsiella pneumoniae producing extendedspectrum β-lactamases against aminoglycosides

Klebsiella pneumoniae （ K. pneumoniae） is one of the main gmn-negative bacilli in clinical practice. Nosocomial infections caused by K. pneumoniae producing extended-spectrum β-lactamases （ESBLs） are very difficult to treat. This paper investigated the resistant characteristics of K. pneumoniae producing ESBLs and their aminoglycoside-modifying enzyme gene expressions including Nacetyltransferases and O-adenyltransferases. Bacteria identification and ESBLs confirmatory tests were performed by Phoenix^TM-100 system. And minimum inhibitory concentrations （MICs） of gentamicin, amikacin, kanamycin, tobranycin, netilmicin and neomycin in 53 K. pneumoniae isolates were detected by agar dilution. In addition, six aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction （PCR） and verified by DNA sequencer. It was found that imipenem and meropenem against 120 K. pneumoniae isolates produced powerful antimicrobial activities. The resistant rates of gentamicin and amikacin were 55.0% and 46.7%, respectively. Except neomycin, MIC50 and MIC90 of gentamicin, amikacin, kanamycin, tobramycin and netilmicin in 53 K. pneumoniae were all 〉 128 μg/ml, and the resistant rates were 83.0%, 52.3%, 75.5%, 81.1% and 69.8%, respectively. However, neomycin was only 39.6%. In addition, five modifying enzyme genes, including aac（3）-Ⅰ, aac（3）-Ⅱ, aac（6＇）-Ⅰb, ant（3＂）-Ⅰ, ant（2＂）-Ⅰ genes, were found in 53 isoaltes except aac （6＇）-Ⅱ, and their positive rates were 11.3%, 67.9%, 47.2%, 1.9% and 39.6%, respectively. It was also confirmed by nucleotide sequence analysis that the above resistant genes shared nearly 100% identities with GenBank published genes. The results obtained in the present study indicated that K. pneumoniae producing ESBLs strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycoside-modifying enzyme gene expressions.     

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.     

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.     

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.     

Prevalence of OXA-type β-lactamases among Acinetobacter baumannii isolates from Northwest of Iran

Carbapenems have been considered as last line antibiotics for treatment of multidrug-resistant (MDR) Acinetobacter baumannii but carbapenem resistant A. baumannii has been increased during the last decade in many parts of the world. OXA-type β-lactamase enzymes are the most common cause of carbapenem resistance in A. baumannii and presence of ISAba1 in upstream of these genes may increase the expression of these OXA genes. The aim of this study was to determine, for the first time, the antibiotic resistance pattern and prevalence of OXA type β-lactamases among nosocomial A. baumannii isolates from northwest of Iran. A total of 100 A. baumannii isolates were recovered from hospitalized patients in a university hospital in northwest of Iran. Sixty-two percent of isolates were resistant to imipenem. All isolates carried bla(OXA-51)-like gene. Among imipenem resistant isolates, 88.7% carried bla(OXA-23)-like, 1.6% carried bla(OXA-40)-like, and 3.2% had bla(OXA-58)-like resistance genes. Ninety percent of isolates contained ISAba1 element and in 74.2% of imipenem resistant isolates, ISAba1 was located in upstream of bla(OXA-23)-like. The results of this study demonstrated high prevalence of OXA-type carbapenemase among MDR A. bumanii in the Northwest of Iran.     

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.     

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.     

Evaluation of four phenotypic methods to detect plasmid-mediated AmpC β-lactamases in clinical isolates

Four phenotypic methods (three dimensional test, AmpC test, cloxacillin synergy test and cefotetan/cefotetan-cloxacillin E-test) to detect plasmid-mediated AmpC β-lactamases (pAmpC) were compared in 125 clinical Enterobacteriaceae isolates with AmpC profile: 74 E. coli (bla (CMY-2): 70; bla (DHA-1): 4), five K. pneumoniae (bla (CMY-2): 2; bla (DHA-1): 3), six P. mirabilis (bla (CMY-2): 6) and 40 negative isolates for pAmpC β-lactamases. All evaluated methods showed a good sensitivity (>95%) but low values of specificity (<60%) in E. coli, explained by an increase of AmpC expression caused by chromosomal ampC promoter/attenuator mutations (-42, -18, -1, +58, predominantly). The cefotetan/cefotetan-cloxacillin or cloxacillin synergy test may be advocated as phenotypic screening test, and the AmpC test as confirmatory test for detection of pAmpC in isolates that lack or minimally express chromosomally encoded AmpC β-lactamases. In the case of E. coli, the phenotypic evaluated tests were not able to differentiate between chromosomal ampC overexpression or acquisition of plasmid-encoded ampC genes.     

Observation on integron carriage among clinical isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamases

Purpose: Klebsiella pneumoniae is considered an important pathogen causing nosocomial and community-acquired infections and is often associated with the production of extended-spectrum β-lactamases (ESBL) belonging to SHV and CTX-M families, which are frequently described as a part of complex integrons, facilitate their horizontal transfer to other related as well as unrelated microbes. The present study was undertaken to investigate the occurrence and characterization of integrons among K pneumoniae isolates producing ESBL in a tertiary referral hospital. Materials and Methods: A total of 136 clinical isolates of K pneumoniae were investigated for the presence of ESBL. Their ESBL genes were characterized by multiplex polymerase chain reaction (PCR). Integrase gene PCR was performed to detect the presence of integron. The isolates were further typed by random amplification of polymorphic DNA (RAPD). Result: Out of 136 K pneumoniae isolates, 63 (46%) were confirmed to be ESBL producers. SHV (68%) and CTX–M (67%) ESBL genes were the most common in our study. Of the 63 ESBL-positive isolates, 58 (92%) strains carried integrons; 52 strains (82%) carried only class 1 integron, whereas 6 (9%) isolates harboured both class 2 integrons and the class 1 gene. However, in ESBL negatives, only 29 (40%) strains were positive for class 1 integron and none for class 2 integron. Conclusion: The presence of class 2 integron amongst ESBL-producing K pneumoniae is being described for the first time in this part of the world. The findings of this study strongly suggest that integrons have a role in the dissemination of ESBL-mediated resistance among the nosocomial isolates of K pneumonia.     

Comparison of isoelectric focusing and polymerase chain reaction for the detection of β-lactamases

Extended spectrum β-lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. Threat posed by antibiotic resistance because of ESBLs is more serious as a number of technical problems are associated with the detection of these enzymes. Although a number of detection methods have been designed for ESBLs, every method has its own benefits and shortcomings as well. In earlier days, isoelectric focusing (IEF) was used as the gold standard for ESBL detection. This study was undertaken to compare IEF with polymerase chain reaction, a method which has been extensively used for ESBL detection these days.     

Real-time polymerase chain reaction for rapid detection of genes encoding SHV extended-spectrum β-lactamases

Purpose: This study aimed to develop an improved method for the detection of bacterial SHV-type extended-spectrum β-lactamases (ESBLs). Materials and Methods: Our method was based on real-time polymerase chain reaction (PCR) in which the amplification of the product was monitored with a fluorescent probe. This method enabled the detection of bla_SHV genes with high degrees of sensitivity and specificity. Results: Based on ESBL phenotyping methods and bla gene DNA sequencing, we identified 240 bla genes from 662 Enterobacteriaceae isolated from clinical culture specimens. Of these 240 isolates, 26 had the blaSHV-28 genotype and three had the blaSHV-1 genotype. With our new real-time PCR assay, we detected 29 out of 29 bla_SHV genes in ESBL-producing isolates. Conclusion: This method represents a powerful tool for epidemiological studies of SHV ESBLs. Furthermore, it has potential for use in diagnostic microbiology.     

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.     

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.     

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.