Carba NP Test Research Articles

Characterization of NDM-1- and OXA-23-producing Acinetobacter baumannii isolates from inanimate surfaces in a hospital environment in Algeria

Investigation of several outbreaks of multidrug-resistant Acinetobacter baumannii infection has demonstrated that contamination of the inanimate hospital environment could be implicated in the spread of these multidrug-resistant strains. To investigate the occurrence of carbapenem-resistant A. baumannii on inanimate surfaces and possible dissemination in the hospital environment in Algeria as a potential source of infection in humans. A. baumannii strains were isolated from the hospital environment and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antimicrobial susceptibility was determined using disc diffusion and E-test methods. Carbapenemase activity was detected using microbiological tests, including modified Hodge test, modified Carba NP test, and EDTA test. Carbapenem resistance determinants were studied by polymerase chain reaction (PCR) and sequencing. Clonal relatedness was determined using multi-locus sequence typing (MLST). A total of 67 A. baumannii isolates were obtained from 868 environmental samples and identified by MALDI-TOF MS. Among them, 61 isolates were resistant to imipenem with minimum inhibitory concentration >32 μg/mL and positive by the modified Hodge test and modified Carba NP test. In addition, the activity of carbapenemase was inhibited by EDTA in 32 strains. PCR and sequencing showed the presence of blaOXA-23 gene in 29 strains, and the blaNDM-1 gene in 32 isolates. MLST demonstrated the presence of five types of ST (ST19, ST2, ST85, ST98, and ST115). Our study demonstrated the dissemination of carbapenemase-producing A. baumannii strains recovered from inanimate surfaces in a hospital environment, surrounding patients, healthcare workers and visitors, in Algeria as a potential source for nosocomial infection.

Evaluation of the Rapidec Carba NP Test Kit for Detection of Carbapenemase-Producing Gram-Negative Bacteria.

Recently, bioMérieux, France, introduced the Rapidec Carba NP test kit for rapid detection of carbapenemase-producing Gram-negative bacteria. This kit was evaluated in this study, and we report sensitivity, specificity, and positive and negative predictive values of 92.6%, 96.2%, 95.83%, and 92.6%, respectively. The test was easy to perform and interpret and relatively inexpensive ($5/Rs 300 per test) and provides a practical solution for early detection of carbapenemase-producing, multidrug-resistant Gram-negative bacteria.

Review of established and innovative detection methods for carbapenemase-producing Gram-negative bacteria.

The minimal antibiotic options for carbapenemase-producing Gram-negative bacteria necessitate their rapid detection. A literature review of a variety of phenotypic and genotypic methods is presented. Advances in culture methods and screening media are still subject to long incubation hours. Biochemical methods have shorter turnaround times and higher sensitivities and specificities, but cannot differentiate between various types and variants. Spectrophotometric methods are cheap and efficient, but are uncommon in many clinical settings, while the MALDI-TOF MS is promising for species identification, typing and resistance gene determination. Although next generation sequencing (NGS) technologies provide a better platform to detect, type and characterize carbapenem-resistant bacteria, the different NGS platforms, the large computer memories and space needed to process and store genomic data and the nonuniformity in data analysis platforms are still a challenge. The sensitivities, specificities and turnaround times recorded in the various studies reviewed favours the use of the biochemical tests (Carba NP or Rapid Carb screen tests) for the detection of putative carbapenemase-producing isolates. MALDI-TOF MS and/or molecular methods like microarray, loop-mediated isothermal amplification and real-time multiplex PCR assays could be used for further characterization in a reference laboratory. NGS may be used for advanced epidemiological and molecular studies.

Genetic and Biochemical Characterization of FRI-1, a Carbapenem-Hydrolyzing Class A β-Lactamase from Enterobacter cloacae

An Enterobacter cloacae isolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI-1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified β-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (IC50s) of clavulanic acid and tazobactam were 10-fold higher than those found for Klebsiella pneumoniae carbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to β-lactamase inhibitors. The blaFRI-1 gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5' end of the β-lactamase gene was identified, leading to inducible expression of the blaFRI-1 gene.

First report of NDM-5-producing Escherichia coli ST1284 isolated from dog in Bejaia, Algeria.

Carbapenemase-producing isolates have been isolated from humans, environmental samples and, more recently, companion animals [1]. Among the newly emerged carbapenemases, New Delhi metallo-β-lactamase (NDM) represents the latest threat for public health [2]. Sixteen variants of NDM-type carbapenemase (NDM-1 to NDM-16) have been reported (http://www.lahey.org/Studies). Here we report what is to our knowledge the first case of Escherichia coli carrying the blaNDM-5 gene isolated from a dog in Algeria. In March 2015 a rectal swab was collected from a 3-year-old German Shepherd dog with a tumor at its third finger. The sample was cultured in 10 mL nutrient broth containing half a disc of ertapenem. After incubation at 37°C for 24 hours, 200 μL was streaked on MacConkey agar plates containing imipenem at a concentration of 0.5 μg/mL. An Escherichia coli isolate was identified by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Microflex, Bruker Daltonics, Bremen, Germany). Antibiotic susceptibility testing was performed by the disk diffusion method and interpreted according to the European Committee on Antimicrobial Susceptibility Testing guidelines (http://www.eucast.org/). The minimum inhibitory concentration (MIC) values of β-lactams were determined using Etest strips according to the manufacturer's recommendations (AB bioMerieux, Solna, Sweden). The E. coli isolate was found resistant to β-lactams, including carbapenems (MICs >32 μg/mL) and extended-spectrum cephalosporins (MICs >256 μg/mL). The isolate was also resistant to tetracycline and fluoroquinolones but was susceptible to amikacin, cotrimoxazole, colistin and tigecycline. The isolate was screened for carbapenemase production using the modified Carba NP test [3], and the production of metallo-β-lactamases was detected by the inhibition of the metallo-β-lactamase activity by ethylenediaminetetraacetic acid (EDTA) as previously described [4]. The E. coli isolate was positive for production of carbapenemase, which is inhibited by EDTA. PCR was used to screen for the presence of genes encoding for class A carbapenemases (blaKPC and blaGES), class D carbapenemases (blaOXA-48) and metallo-β-lactamases (blaNDM-1 and blaVIM) as previously described [5], followed by sequencing, which led to the identification of blaNDM-5. A review the literature at PubMed found 12 reports on NDM-5, which were all recovered from clinical specimens; no reports were from veterinary isolates, however. Among these 12 reports, one was from Algeria [6]. The authors of this study reported three isolates of E. coli sequence type (ST) 2659 recovered from urine and blood specimen isolated between January 2012 and February 2013 in the university hospital of Annaba (east Algeria). Thus, this is the first report of NDM-5-producing E. coli isolated from companion animals. Furthermore, other types of carbapenemases have been reported from domesticated animals, including NDM-1 in multidrug-resistant E. coli strains recovered from companion animals in the United States [7], and OXA-48 carbapenemase-producing Klebsiella pneumoniae and E. coli isolates obtained from a dog in Germany [8]. The E. coli isolate also carried the blaTEM-33 gene. The transferability of the resistance phenotype was studied by a conjugation experiment using sodium azide–resistant E. coli strain J53 as a recipient. The transconjugants were selected on Luria agar plates containing ceftazidime (8 μg/mL) and sodium azide (200 μg/mL) [2]. Only transconjugants carrying the blaTEM-33 gene were obtained. To determine the localization of the blaNDM-5 gene the isolate was subjected to plasmid extraction using the High Purity Plasmid Miniprep Kit (Neobiotech, Los Angeles, CA, USA) and the QIAquick Gel extraction kit (Qiagen, Germantown, MD, USA), followed by PCR amplification targeting blaNDM-5 performed on the extracted plasmid. The result showed that the isolate harboured only one plasmid which did not contain the blaNDM-5 gene, indicating that this gene is probably integrated into the chromosome. Genotyping of the isolate was performed by multilocus sequence typing (MLST) using seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA), as described at the E. coli MLST Database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). According to MLST analysis, the E. coli isolate was attributed to ST1284. This ST has been identified previously in E. coli isolates producing extended-spectrum β-lactamase recovered from animals [9]. In summary, this report documents the emergence of NDM-5 among E. coli ST1284 isolated for the first time from a companion animal in the North African countries.

Rapid identification of carbapenemase-producing Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii using a modified Carba NP test.

Biochemical tests have been previously developed to identify carbapenemase-producing Enterobacteriaceae, Pseudomonas spp. (Carba NP test) and Acinetobacter spp. (CarbAcineto NP test). We evaluated a modified Carba NP test to detect carbapenemase production in Enterobacteriaceae, Pseudomonas and Acinetobacter species using a single protocol with rapid results and found good reliability and speed.

MUS-2, a novel variant of the chromosome-encoded β-lactamase MUS-1, from Myroides odoratimimus.

The aim of the present study was to investigate the molecular mechanism of carbapenem resistance of three imipenem-resistant isolates of Myroides odoratimimus recovered from two livestock farms of cows and pigeons by rectal swab in Lebanon in January 2014. Investigation of imipenem resistance of these isolates using the modified Hodge test, the EDTA test, the modified CarbaNP test and the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Ultraflex assay showed a carbapenemase activity due to the presence of a chromosome-encoded β-lactamase MUS, verified by PCR. However amplification and sequencing of this chromosomal gene showed a novel variant of it designated MUS-2 by the curators of the Lahey database of β-lactamases (http://www.lahey.org/Studies/webt.asp). Cloning of the blaMUS-2 was performed, followed by protein expression in Escherichia coli TOP 10. Pulsed-field gel electrophoresis clearly showed that the three isolates belonged to the same clone. This study reports a novel variant of the chromosome-encoded blaMUS-1 associated with carbapenem resistance in Myroides odoratimimus and shows that animals may represent a reservoir of bacteria harbouring several variants of resistance genes.

Evaluation of several phenotypic methods for the detection of carbapenemase-producing Pseudomonas aeruginosa.

The purpose of this investigation was to compare several phenotypic methods, including combined disk tests (CDT) containing metallo-β-lactamase (MBL) inhibitors or cloxacillin, and the Carba NP test for the detection of carbapenemase-producing Pseudomonas aeruginosa (CPPA). A new CDT using imipenem (10 μg) ± cloxacillin 4,000 μg and the Carba NP test were evaluated to detect CPPA. In addition, four commercially available combined disks containing a carbapenem and ethylene-diamine-tetra-acetic acid (EDTA) or dipicolinic acid (DPA) as the inhibitor were tested in order to detect MBL-positive P. aeruginosa. All these phenotypic methods were evaluated on 188 imipenem non-susceptible P. aeruginosa (CPPA, n = 75) isolates divided into 26 well-characterized collection strains and 162 non-duplicate clinical isolates referred to the national reference laboratory in 2013. For the total of 188 isolates tested, CDT containing EDTA or DPA displayed high sensitivities (99%) and specificities (95%) for detecting MBL-producing isolates. CDT with cloxacillin showed a sensitivity and specificity of 97%/96% compared to 88%/99% for the Carba NP test in order to detect CPPA. For the 162 clinical isolates, CDT containing EDTA or DPA displayed a high negative predictive value (NPV) (99%) for detecting MBL-producing isolates. CDT with cloxacillin showed an NPV of 98%, compared to 95% for the Carba NP test in order to detect CPPA. In our setting, CDT associating imipenem ± EDTA or ± DPA performed best for the detection of MBL-producing P. aeruginosa. Imipenem/imipenem-cloxacillin test yielded good NPV to exclude the presence of MBL in imipenem non-susceptible isolates.

Genetic and biochemical characterization of OXA-405, an OXA-48-type extended-spectrum β-lactamase without significant carbapenemase activity.

The epidemiology of carbapenemases worldwide is showing that OXA-48 variants are becoming the predominant carbapenemase type in Enterobacteriaceae in many countries. However, not all OXA-48 variants possess significant activity toward carbapenems (e.g., OXA-163). Two Serratia marcescens isolates with resistance either to carbapenems or to extended-spectrum cephalosporins were successively recovered from the same patient. A genomic comparison using pulsed-field gel electrophoresis and automated Rep-PCR typing identified a 97.8% similarity between the two isolates. Both strains were resistant to penicillins and first-generation cephalosporins. The first isolate was susceptible to expanded-spectrum cephalosporins, was resistant to carbapenems, and had a significant carbapenemase activity (positive Carba NP test) related to the expression of OXA-48. The second isolate was resistant to expanded-spectrum cephalosporins, was susceptible to carbapenems, and did not express a significant imipenemase activity, (negative for the Carba NP test) despite possessing a blaOXA-48-type gene. Sequencing identified a novel OXA-48-type β-lactamase, OXA-405, with a four-amino-acid deletion compared to OXA-48. The blaOXA-405 gene was located on a ca. 46-kb plasmid identical to the prototype IncL/M blaOXA-48-carrying plasmid except for a ca. 16.4-kb deletion in the tra operon, leading to the suppression of self-conjugation properties. Biochemical analysis showed that OXA-405 has clavulanic acid-inhibited activity toward expanded-spectrum activity without significant imipenemase activity. This is the first identification of a successive switch of catalytic activity in OXA-48-like β-lactamases, suggesting their plasticity. Therefore, this report suggests that the first-line screening of carbapenemase producers in Enterobacteriaceae may be based on the biochemical detection of carbapenemase activity in clinical settings.

War wound treatment complications due to transfer of an IncN plasmid harboring bla(OXA-181) from Morganella morganii to CTX-M-27-producing sequence type 131 Escherichia coli.

A 22-year-old male developed a recurrent sacral abscess associated with embedded shrapnel following a blast injury. Cultures grew extended-spectrum β-lactamase (ESBL)-producing, carbapenem-susceptible Escherichia coli. Ertapenem was administered, but the infection recurred after each course of antibiotics. Initial surgical interventions were unsuccessful, and subsequent cultures yielded E. coli and Morganella morganii, both nonsusceptible to carbapenems. The isolates were Carba NP test negative, gave ambiguous results with the modified Hodge test, and amplified the bla(OXA48)-like gene by real-time PCR. All E. coli isolates were sequence type 131 (ST131), carried nine resistance genes (including bla(CTX-M-27)) on an IncF plasmid, and were identical by genome sequencing, except for 150 kb of plasmid DNA in carbapenem-nonsusceptible isolates only. Sixty kilobases of this was shared by M. morganii and represented an IncN plasmid harboring bla(OXA-181). In M. morganii, the gene was flanked by IS3000 and ISKpn19, but in all but one of the E. coli isolates containing bla(OXA-181), a second copy of ISKpn19 had inserted adjacent to IS3000. To the best of our knowledge, this is the first report of bla(OXA-181) in the virulent ST131 clonal group and carried by the promiscuous IncN family of plasmids. The tendency of M. morganii to have high MICs of imipenem, a bla(OXA-181) substrate profile that includes penicillins but not extended-spectrum cephalosporins, and weak carbapenemase activity almost resulted in the presence of bla(OXA-181) being overlooked. We highlight the importance of surveillance for carbapenem resistance in all species, even those with intrinsic resistances, and the value of advanced molecular techniques in detecting subtle genetic changes.

The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods.

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.

First case of NDM-1 producing Klebsiella pneumoniae in Caribbean islands

Characterize a NDM-1 producing K. pneumoniae isolate recovered from a patient hospitalized in Guadeloupe, French West Indies, after its transfer from Cuba Antibiotic susceptibilities were determined by the disk diffusion method, and E-test. Carbapenemase production was assessed using the Carba NP test. Antibiotic resistance determinants and their surrounding structures were characterized by PCR mapping and DNA sequencing. Transfer of the β-lactam resistance marker was attempted by liquid mating-out assays Here we reported the first NDM-1 producing enterobacterial isolate recovered from Caribbean islands. This K. pneumoniae isolate belongs to a new sequence type (ST1649). The blaNDM-1 gene together with the aacA4 gene were carried on a self conjugative IncR plasmid of c.a. 80kb. This study describes the first identification of a NDM-1 producer in Caribbean islands. The uncommon incompatibility group of the blaNDM-1 carrying plasmid and the uncommon ST type of the K. pneumoniae strain suggest a possible local emergence of NDM producers.

Evaluation of phenotypic screening tests for carbapenemase production in Pseudomonas aeruginosa from patients with cystic fibrosis

Carbapenemase phenotypic tests (inhibitor-based, modified Hodge and Carba NP) for Pseudomonas aeruginosa isolates from patients with cystic fibrosis (CF) were evaluated with PCR as a reference assay. Except for Carba NP, all methods demonstrated high false positives. Carba NP test presents a promising screening method for CF P. aeruginosa isolates.

Epidemiological Characteristics, Resistance Patterns and Spread of Gram-Negative Bacteria Related to Colonization of Patients in Intensive Care Units

Our aim was to determine the epidemiological characteristics, the resistance patterns and the spread of Gram negative bacteria related to colonization of patients in adult Intensive Care Units. Methods: A prospective cohort of patients colonized and/or infected with Gram negative bacteria was conducted at two adult ICUs from hospitals in Brazil (April 2012 to February 2013). Nasal, groin and perineum swabs were performed. Samples were incubated on MacConkey and cetrimide agar (48 h at 37℃) and identification tests (Vitek-BioMerieux), antibiogram (Bauer-Kirby method), Carba NP test, Polymerase Chain Reaction (PCR) and sequencing were performed. The patterns of resistant microorganisms were compared by rep-PCR (Diversilab). Results: There were 53 cases of colonization. In these cases, we identified imipenem-resistant Acinetobacter baumannii (51%), Pseudomonas aeruginosa (32%), Klebsiella pneumoniae ESBL (38%) or imipenem resistant (5.6%). The use of antimicrobials and medical devices were related to colonization (p < 0.05). The resistance patterns expressed by Klebsiella pneumoniae were ESBL (CTX-M, SHV e TEM) and KPC2. A verified profile of Acinetobacter baumannii was related to OXA-23 and OXA-253 (OXA-143 variant). The profiles ESBL and KPC2 expressed by Klebsiella pneumoniae were distributed between the both ICUs. The distribution of OXA-23 and OXA-253 was verified only in one ICU. The similarity of strains ranged from 80% to 95%, highlighting the horizontal transference of these microorganisms.

Genotypic and phenotypic identification of aeromonas species and carbapenem resistance in Queensland, Australia

Aims This study defines the population of Aeromonas spp. in Queensland and carbapenem resistance due to carbapenemase production, CphA. Phenotypic tests were correlated with the presence of the cphA gene. Methods Isolates from blood (22), wound (46), sterile sites (11), stool (18), eye (2) and sputum (1) were characterised by rpoB and gyrB sequencing. Disc diffusion and meropenem E-test MIC were determined. Carbapenemase production was assessed by Carba NP test and cphA by PCR. Results Isolates identified as A. dhakensis (39), A. veronii (21), A. hydrophila (20), A. caviae (14), A. jandaei (4), A. bestiarum (1), and A. sanarellii (1). Disc diffusion and E-test failed to demonstrate carbapenem resistance. Carba NP test performed with 97.4% sensitivity and 95.7% specificity. The cphA gene was detected in A. veronii (21; 100%), A. hydrophila (18; 90%), A. dhakensis (34; 87.2%), A. jandaei (2; 50%), A. bestiarum (1; 100%) and not A. caviae . Discussion Phylogenetic analysis revealed A. dhakensis caused majority of infection, an unrecognised pathogen in this region. Carbapenem resistance due to cphA was prevalent. The Carba NP was a reliable phenotypic test. Adequate speciation and detection of carbapenem resistance in Aeromonas spp. is essential to guide appropriate antimicrobial therapy.

Clinical Importance of Carbapenemase Production in Gram-Negative Bacteria

Carbapenem resistance is one of the major threats faced in antimicrobial treatment of infections caused by Gram negative organisms. In India and throughout the world the use of carbapenems has increased resulting in an increased resistance of pathogens to this class of antibiotics. Bacteria are capable enough to become resistant to antibiotics by a number of mechanisms both intrinsic and acquired, most common of which include enzymatic degradation of antibiotics. Carbapenem resistant Gram-negative bacteria usually spreads in the hospital settings to other patients and caregivers or relatives by unwashed hands or from contact with soiled equipment and surfaces such as bedrails, tables, chairs, countertops and door handles. These carriers are the ultimate sources of dissemination in the community. Detection of carbapenemase is a crucial infection control issue because they are often associated with extensive antibiotic resistance, treatment failures and infection-associated mortality. For the identification of carbapenemase producers different nonmolecular methods are used such as E- test (Epsilometer Test), Modified hodge test, MIC by Agar Dilution Method, Carba NP Test, EDTA disk synergy test, Boronic Acid Test, 2-mercaptopropionic acid inhibition (2-MPA) test. Molecular techniques remain the gold standard for the precise identification of carbapenemase genes. Most of these techniques are based on PCR and may be followed by a sequencing step if a precise identification of the carbapenemase gene is needed. This review article describes clinical importance and methods used for the identification of carbapenemase producers.

Virulence of carbapenem-resistant Escherichia coli isolated in the Arabian Peninsula

Introduction: It is generally assumed that multidrug-resistant bacteria are less virulent. However, little is known about the virulence traits of carbapenem-resistant Escherichia coli . Objectives: To reveal the distribution of virulence factors of carbapenem-resistant extraintestinal pathogenic E. coli isolated in the Arabian Peninsula. Materials and methods: Thirty independent carbapenem-resistant E. coli samples were collected between 2009 and 2013 from Saudi Arabia (n =3), Kuwait (n =5), Oman (n =7) and the United Arab Emirates (UAE) (n =15). Carbapenemase production was confirmed by the carba-NP test. Genes of KPC, NDM, OXA-48-like, VIM, IMP carbapenemases, the lipopolysaccharide core types and the phylogenetic types were identified by polymerase chain reaction (PCR). Clonal relations were established by pulsed-field gel electrophoresis (PFGE) using an 80% similarity threshold. Virulence factor-encoding genes were detected by multiplex PCRs. Biofilm production was assessed in a stationary model. Results: Of the 30 isolates, 11 produced OXA-48-like, seven NDM-1 and one VIM-4 carbapenemases, and 11 of them did not produce carbapenemases. The strains exhibited 27 different PFGE patterns, with only three pairs of strains exhibiting clustering. Although strains belonged to all four phylogenetic groups and all five core types were detected, remarkably all OXA-48-like positive isolates belonged to phylogenetic group D and carried core type R4. The virulence factor score for the carbapenemase producers did not differ from that of the carbapenemase non-producer strains. However, members of the former group were significantly more likely to carry the yersiniabactin siderophore gene (P =0.01), to possess the virulence associated K5 capsular type (P =0.02) and to produce biofilm in anaerobic conditions (P =0.02). Conclusions: Carbapenem-resistant E. coli strains exhibit a variety of virulence factors. Those promoting intestinal colonization, i.e. K5 capsular type, and biofilm production under anaerobic conditions show some association with carbapenemase production.

First Report of Klebsiella pneumoniae-Carbapenemase-3-Producing Escherichia coli ST479 in Poland.

An increase in the antibiotic resistance among members of the Enterobacteriaceae family has been observed worldwide. Multidrug-resistant Gram-negative rods are increasingly reported. The treatment of infections caused by Escherichia coli and other Enterobacteriaceae has become an important clinical problem associated with reduced therapeutic possibilities. Antimicrobial carbapenems are considered the last line of defense against multidrug-resistant Gram-negative bacteria. Unfortunately, an increase of carbapenem resistance due to the production of Klebsiella pneumoniae carbapenemase (KPC) enzymes has been observed. In this study we describe the ability of E. coli to produce carbapenemase enzymes based on the results of the combination disc assay with boronic acid performed according to guidelines established by the European Community on Antimicrobial Susceptibility Testing (EUCAST) and the biochemical Carba NP test. Moreover, we evaluated the presence of genes responsible for the production of carbapenemases (bla KPC, bla VIM, bla IMP, bla OXA-48) and genes encoding other β-lactamases (bla SHV, bla TEM, bla CTX-M) among E. coli isolate. The tested isolate of E. coli that possessed the bla KPC-3 and bla TEM-34 genes was identified. The tested strain exhibited susceptibility to colistin (0.38 μg/mL) and tigecycline (1 μg/mL). This is the first detection of bla KPC-3 in an E. coli ST479 in Poland.

Evaluation of the Carba NP test for carbapenemase detection.

The Carba NP test was evaluated against a panel of 61 carbapenemase-producing bacterial species (15 producing class A carbapenemases, 15 producing class D carbapenemases, and 31 producing metallo-β-lactamases) and against 111 isolates with non-wild-type carbapenem susceptibility but not producing carbapenemase. Carbapenemase production was verified by PCR and UV-spectrophotometric measurement of imipenem hydrolysis. No false positives were seen, but there were consistent problems with the detection of OXA-48-like enzymes and also some rarer class A enzymes.

Persistence of Klebsiella pneumoniae ST258 as the predominant clone of carbapenemase-producing Enterobacteriaceae in post-acute-care hospitals in Israel, 2008-13.

To study the molecular characteristics of carbapenemase-producing Enterobacteriaceae (CPE) in post-acute-care hospitals (PACHs) in Israel and to analyse the temporal changes between 2008 and 2013. CPE isolates were obtained during two cross-sectional, point prevalence national surveys of PACHs in Israel performed in 2008 and 2013. Surveillance cultures were collected by streaking rectal swabs onto selective media. Isolates were identified to species level and tested for blaKPC, blaNDM and blaOXA-48 by PCR and by the Carba NP test. Molecular typing was done by PCR for the pilv-l gene, designed for the ST258 KPC-producing Klebsiella pneumoniae (KPC-KP) clone, BOX-PCR and MLST. The prevalence of CPE carriage in the first survey was 184/1147 (16%); all of the isolates were KPC-KP. The prevalence of CPE carriage in the second survey was 127/1287 (9.9%); of these isolates, 113 (89%) were KPC-KP, 9 (7%) were other KPC-producing species and 5 (4%) were NDM- and OXA-48-producing CPE (n = 1 and 4, respectively). The proportion of the KPC-KP population represented by the ST258 clone increased from 120/184 (65%) in 2008 to 91/113 (80%) in 2013. In 58% (71/122) of the KPC-CPE carriers identified in the 2013 survey, the source of acquisition was determined to be the PACH itself. All four OXA-48 CPE were acquired either directly or indirectly from patients arriving from the Palestinian Authority or Syria. Despite the decreased prevalence of CPE in Israeli PACHs, and the emergence of new types of CPE, the KPC-KP ST258 clone remains the predominant clone represented.