Carbapenemases In Enterobacteriaceae Research Articles

BlaNDM gene identified from clinical samples of companion animals: First description in Brazil

Background: Emergence of acquired carbapenemases and their worldwide dissemination are a major global threat to antibiotic era and represent a major clinical challenge in both human and veterinary contexts. Carbapenems are broad-spectrum beta-lactam antibiotics employed for treatment of serious infections, usually considered as the last-line available therapy against multidrug-resistant (MDR) Gram-negative bacterial infections like those caused by extended spectrum beta-lactamase (ESBL) pathogens. The New Delhi metallo-beta-lactamase (NDM) was named after being discovered in India, in 2009, and has been isolated in several parts of the world. Until now, the occurrence of NDM appeared to be restricted mainly to humans, however the prevalence and characteristics of MDR genes in Enterobacteriaceae originating from companion animals are still not well known. Thus, this study aimed to investigate the occurrence of carbapenemase encoding genes in bacteria resistant to carbapenems, isolated from clinical samples of companion animals. Methods and materials: There were considered bacterial isolates from dogs (97 strains) and cats (24 isolates) showing phenotypic resistance to carbapenems, identified during routine microbiological investigation between July 2018 and April 2019 in Joinville, Southern Brazil. Phenotypic resistance to carbapenems and the susceptibility profile to other common antimicrobials were determined by the disc-diffusion agar method. In addition, Modified Carbapenem Inactivation Method (mCIM) was used for phenotypic confirmation of carbapenemases in Enterobacteriaceae. The presence of blaNDM gene was confirmed by Real-time Polymerase Chain Reaction employing TaqMan probes. Results: In this study, there were identified two MDR isolates (Klebsiella pneumoniae and Enterobacter cloacae) carrying the blaNDM gene derived from dogs urine. Conclusion: To our knowledge, this is the first report of NDM identified in clinical isolates derived from companion animals in Brazil. Of note, the blaNDM gene may be located in readily transferable plasmids, increasing the variety of transmission vectors of resistance to carbapenems, also considering that the species carrying the gene identified in this study are widely distributed and shared among the environment, animals and humans.

Combination of modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) for phenotypic detection of carbapenemase-producing Enterobacteriaceae

BackgroundCarbapenemase-resistant Enterobacteriaceae (CRE) cause many serious infections resulting in increasing treatment cost, prolonged hospitalization, and mortality rate. Reduced expression and/or mutations of porins and the presence of carbapenemase promote Enterobacteriaceae survival under carbapenem treatments. Development of accurate methods for the detection of antimicrobial resistance is required not only for therapy but also to monitor the spread of resistant bacteria or resistance genes throughout the hospital and community. In this study, we aimed to evaluate the phenotypic methods, Modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), and EDTA-CIM (eCIM) for the detection of carbapenemase-producing Enterobacteriaceae (CPE).ResultsThe results showed that mCIM had a sensitivity of 100% and a specificity of 100%, whereas the MHT had a sensitivity of 84.8% and a specificity of 97.8% for the 195 CRE isolates tested (105 CPE and 90 non-CPE isolates). The sensitivity of the mCIM/eCIM to detect metallo-carbapenemases in this study was 89.3% and the specificity was 98.7% as compared to the genotypic PCR detection.ConclusionsThese findings indicate that the mCIM combined with eCIM is useful for detecting and distinguishing different types of carbapenemase in Enterobacteriaceae.

Molecular Epidemiology of Carbapenemases in Enterobacteriales from Humans, Animals, Food and the Environment.

The Enterobacteriales order consists of seven families including Enterobacteriaceae, Erwiniaceae, Pectobacteriaceae, Yersiniaceae, Hafniaceae, Morganellaceae, and Budviciaceae and 60 genera encompassing over 250 species. The Enterobacteriaceae is currently considered as the most taxonomically diverse among all seven recognized families. The emergence of carbapenem resistance (CR) in Enterobacteriaceae caused by hydrolytic enzymes called carbapenemases has become a major concern worldwide. Carbapenem-resistant Enterobacteriaceae (CRE) isolates have been reported not only in nosocomial and community-acquired pathogens but also in food-producing animals, companion animals, and the environment. The reported carbapenemases in Enterobacteriaceae from different sources belong to the Ambler class A (blaKPC), class B (blaIMP, blaVIM, blaNDM), and class D (blaOXA-48) β-lactamases. The carbapenem encoding genes are often located on plasmids or associated with various mobile genetic elements (MGEs) like transposons and integrons, which contribute significantly to their spread. These genes are most of the time associated with other antimicrobial resistance genes such as other β-lactamases, as well as aminoglycosides and fluoroquinolones resistance genes leading to multidrug resistance phenotypes. Control strategies to prevent infections due to CRE and their dissemination in human, animal and food have become necessary. Several factors involved in the emergence of CRE have been described. This review mainly focuses on the molecular epidemiology of carbapenemases in members of Enterobacteriaceae family from humans, animals, food and the environment.

Evaluation of Modified Rapid Carbapenem Inactivation Method (mrCIM) Combined with Rapid EDTA-Modified Carbapenem Inactivation Method (reCIM) to Detect Carbapenemase and Distinguish Metallo-Carbapenemase in Enterobacteriaceae Within Four Hours.

PurposeTo develop a rapid EDTA-modified carbapenem inactivation method (reCIM) combined with modified rapid carbapenem inactivation method (mrCIM) to detect carbapenemase and distinguish metallo-β-lactamases from carbapenemases in Enterobacteriaceae in 4 hrs.Materials and MethodsThe sensitivities and specificities of mrCIM and reCIM were retrospectively evaluated in 247 carbapenem-resistant Enterobacteriaceae of which 107 were carbapenemase producers confirmed by PCR and sequencing. In addition, mrCIM and reCIM were prospectively evaluated with 47 carbapenem-resistant enterobacterial isolates.ResultsThe sensitivity and specificity of mrCIM were 96.3% and 97.1% at 2.5 hrs post incubation, and the specificity increased to 98.6% at 3 hrs. The combined mrCIM and reCIM showed a sensitivity of 95.4% and a specificity of 100% at 2.5 hrs post incubation in identifying metallo-β-lactamases, and the sensitivity increased to 97.0% at 3 hrs. These performance characteristics are comparable to mCIM and eCIM; however, compared with mCIM and reCIM tests which need at least 24 hrs to detect results, the mrCIM and reCIM required less than 4 hrs of total work time.ConclusionThe combined mrCIM and reCIM can be used to accurately and quickly detect carbapenemase and metallo-β-lactamases in Enterobacteriaceae in 4 hrs and are suitable for routine use in most clinical microbiology laboratories.

Differences between meropenem and imipenem disk to detect carbapenemase in gram-negative bacilli using simplified carbapenem inactivation method

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a major threat to public health. In the present study, we compared the difference between meropenem and imipenem disk for detecting carbapenemase-producing gram-negative bacilli using simplified carbapenem inactivation method (sCIM). 106 Enterobacteriaceae, including 74 CPE, 17 Pseudomonas aeruginosa including 10 carbapenemase-producing isolates and 36 Acinetobacter baumannii including 20 carbapenem-resistant isolates preserved in our laboratory were tested. Based on sCIM method, the test bacteria were tested with both meropenem and imipenem disk, respectively. In Enterobacteriaceae, the usage of both meropenem and imipenem disk showed high concordance (99.1%). Meropenem disk cannot identify positive isolates among the 10 P. aeruginosa and 20 A. baumannii isolates due to low carbapenem hydrolytic ability of the carbapenemase produced by these strains. Thus, meropenem disk was found to be similar to imipenem disk, presenting high specificity and sensitivity in the detection of carbapenemase in Enterobacteriaceae, but it cannot be used for the detection of carbapenemase in P. aeruginosa and A. baumannii.

Detection of Acinetobacter pittii ST220 co-producing NDM-1 and OXA-820 carbapenemases from a hospital sink in a non-endemic country of NDM

ObjectivesNDM-1 is by far one of the most commonly prevalent carbapenemases in Enterobacteriaceae and Acinetobacter baumannii. This study presented an Acinetobacter pittii (A. pittii) isolate co-harboring blaNDM-1 and blaOXA-820 from a university hospital sink, where New Delhi metallo-β-lactamase (NDM) producers have not been found in either patients or their environments. MethodsWhole-genome sequencing was performed on the HiSeq 4000 platform, and the reads were de novo assembled using the A5-miSeq Assembly pipeline. Annotation of the resulting scaffolds were performed by using the DDBJ Fast Annotation and Submission Tool (DFAST). The blaNDM-1-carrying plasmid was determined. ResultsThe A. pittii ST220 strain SU1805 detected from a sink strainer in the treatment room was resistant to imipenem and meropenem. Antimicrobial resistance genes blaNDM-1, blaOXA-820, blaADC-43, and aphA6 were found in this strain. The blaNDM-1 was found to be located downstream of an ISAba125 element on a plasmid pSU1805NDM with a size of 41,022 bp, and GC content of 38.3% harbouring 48 protein-coding genes. The aphA6 gene was also located upstream of the ISAba125 on the same plasmid. The A. pittii intrinsic blaOXA-213-like gene blaOXA-820 was located between fxsA and yncA genes in the chromosome. The strain also harboured biofilm-associated genes such as ompA, the csu operon and their regulating genes bfmRS. ConclusionThis study described the first isolation of NDM-1-producing A. pittii in Japan, and highlighted the importance of proper implementation of measures against AMR for sink drainage systems, since NDM producers may have already been hidden in such environments in a non-endemic country of NDM.

Evaluation of the automated BD Phoenix CPO Detect test for detection and classification of carbapenemases in Gram negatives

We evaluated the performance of the automated BD Phoenix CPO Detect test (BD-CPO test) for detection and Ambler classification of carbapenemases in Enterobacteriaceae, P.aeruginosa and A.baumannii complex. A collection of 287 Gram-negative clinical isolates, with a reduced susceptibility to at least one carbapenem including 184 carbapenemase-producing organisms (CPO) and 103 non-CPO, was tested.The BD-CPO test showed an overall sensitivity of 89.7% and specificity of 83.5% for carbapenemase detection. 1/7 of class A, 82.9% of class B, and 89.8% of class D carbapenemases were correctly classified. Poor detection sensitivity of 68.9% and specificity of 62.1% in P.aeruginosa was observed. However, combination with ceftazidime/avibactam susceptibility, provided by this panel, increased the performances for P.aeruginosa. The integration of an automated carbapenemase detection and classification in routine susceptibility panels would save time and help for therapeutic management. Further developments are needed to improve the accuracy of the BD-CPO test.

Complex Class 1 Integron in a Clinical Escherichia coli Strain From Vietnam Carrying Both mcr-1 and bla NDM-1.

The co-production of MCR and carbapenemase in Enterobacteriaceae has been previously reported. Here, we describe a clinical strain of Escherichia coli from Vietnam carrying both mcr-1 and blaNDM–1. Whole-genome sequencing showed that the genome of this strain consists of a 4,975,832-bp chromosome and four plasmids. The mcr-1 and blaNDM–1 genes are located on IncI2 and IncA/C2-type plasmids, respectively. Genetic analysis revealed the presence of a multidrug-resistant region with the structure of a novel complex class 1 integron including a class 1 integron region bearing two 5′ conserved segments and one 3′ conserved segment and two complete structures of ISCR1. The complex integron contains aminoglycoside resistance genes aadA2, aadB, strA, strB, and aphA6, quinolone resistance gene qnrA1, extended-spectrum β-lactamase gene blaOXA–4, and a Tn125-like transposon bearing blaNDM–1. In addition, the dfrA12-gcuF-aadA2-cmlA1-aadA1-qacH gene cassette array belonging to the sul3-type integron was also identified, but the region found downstream of the gene cassette array is the IS440-tet(M)-IS26 element instead of the sul3 gene. The results further support that Enterobacteriaceae isolates co-harboring mcr and blaNDM are widely being distributed. The structural characteristics of the complex integron reveal that ISCR1 elements play an important role in the mobilization of blaNDM–1 and the development of multidrug-resistant regions.

Plasmid-Mediated Transmission of KPC-2 Carbapenemase in Enterobacteriaceae in Critically Ill Patients.

Carbapenem-resistant Enterobacteriaceae (CRE) cause health care-associated infections worldwide, and they are of severe concern due to limited treatment options. We report an outbreak of KPC-2-producing CRE that was caused by horizontal transmission of a promiscuous plasmid across different genera of bacteria and hospitals in Germany. Eleven isolates (8 Citrobacter freundii, 2 Klebsiella oxytoca, and 1 Escherichia coli) were obtained from seven critically ill patients during the six months of the outbreak in 2016. One patient developed a CRE infection while the other six patients were CRE-colonized. Three patients died in the course of the hospital stay. Six of the seven patients carried the same C. freundii clone; one K. oxytoca clone was found in two patients, and one patient carried E. coli and C. freundii. Molecular analysis confirmed the presence of a conjugative, blaKPC-2-carrying 70 kb-IncN plasmid in C. freundii and E. coli and an 80 kb-IncN plasmid in K. oxytoca. All transconjugants harbored either the 70 or 80 kb plasmid with blaKPC-2, embedded within transposon variant Tn4401g. Whole genome sequencing and downstream bioinformatics analyses of all plasmid sequences showed an almost perfect match when compared to a blaKPC-2-carrying plasmid of a large outbreak in another German hospital two years earlier. Differences in plasmid sizes and open reading frames point to the presence of inserted mobile genetic elements. There are few outbreak reports worldwide on the transmission of blaKPC-2-carrying plasmids across different bacterial genera. Our data suggest a regional and supraregional spread of blaKPC-2-carrying IncN-plasmids harboring the Tn4401g isoform in Germany.

Evaluation of the RESIST-4 K-SeT assay, a multiplex immunochromatographic assay for the rapid detection of OXA-48-like, KPC, VIM and NDM carbapenemases.

Accurate and fast identification of carbapenemase producers is essential for optimal patient management. Here, a new lateral flow immunochromatographic RESIST-4 K-SeT assay was assessed for the detection of carbapenemases in Enterobacteriaceae and non-fermenters. The RESIST-4 K-SeT assay targets OXA-48-like, KPC, VIM and NDM, but not IMP carbapenemases. The assay was first evaluated using a collection of isolates with well-characterized resistance mechanisms to β-lactams (n = 134) and against an international external quality assessment carbapenemase panel (n = 8). The assay was then challenged prospectively using 345 consecutive, non-duplicate isolates including 279 Enterobacteriaceae and 66 non-fermenters (mostly Pseudomonas spp.) that were sent to the Belgian National Reference Centre for identification of the mechanisms related to carbapenem resistance. Globally, for the collection of retrospective and prospective clinical isolates (n = 479), the assay showed a sensitivity ranging from 99% for the detection of VIM to 100% for the detection of OXA-48-like, KPC and NDM carbapenemase-producing strains. The specificity was 100% for each carbapenemase and a perfect match in results was observed for the external quality assessment for the carbapenemases targeted by the assay. The RESIST-4 K-SeT assay is a valuable alternative for detection and identification of carbapenemases from culture isolates compared with the more costly molecular assays, which may also further require skilled staff and dedicated facilities.

Epidemiology of carbapenemase-producing Enterobacteriaceae in a pediatric hospital in a country with high endemicity

BackgroundLittle is known about epidemiology of carbapenemase-producing Enterobacteriaceae (CPE) in children. Aim of this study was to describe CPE epidemiology in a tertiary care pediatric hospital in Italy that admits patients coming from geographic areas with high diffusion of CPE. MethodsProspective evaluation of the proportion and rates per 100,000 hospital discharges (D) or hospitalization-days (HD) of invasive infections due to CPE from 2013 to 2017 and of CPE infections and colonizations from 2014 to 2017. Disease-preventing strategies comprised patients’ screening at admission, pre-emptive contact isolation precautions pending cultures results, and bundles for prevention of healthcare associated infections. ResultsFrom 2013 to 2017 CPE represented 3.5% of all invasive infections due to Enterobacteriaceae, with rates ranging 7.30–14.33 for D and 1.03–2.06 for HD, without major changes over time. On the contrary, overall rates of isolates increased from 83.03 to 191.34 for D and from 12.21 to 28.35 for HD. The intra-hospital diffusion consisted of 2 small outbreaks without invasive diseases in 2014–2015, and sporadic, not epidemiologically-related cases in 2016–2017. Globally, Escherichia coli and Klebsiella pneumoniae represented 64% of identified CPE, while 70% of carbapenemases identified were metallo-beta-lactamases (VIM or NDM), with changes over time. ConclusionsIn our center metallo-beta lactamases were the most frequently identified carbapenemases in Enterobacteriaceae and E. coli and K. pneumoniae the most frequently isolated pathogens carrying these enzymes. A proactive management strategy was effective in containing in-hospital spreading.

Draft genome sequence of KPC-2-producing Pseudomonas aeruginosa recovered from a bloodstream infection sample in Brazil.

Klebsiella pneumoniae carbapenemase (KPC) is the most widespread carbapenemase in Enterobacteriaceae in Brazil. Although its presence is not common in Pseudomonas aeruginosa, it has been increasingly reported. Here we report a draft genome sequence of a KPC-producing P. aeruginosa strain recovered from a bloodstream infection sample in Brazil. The antimicrobial susceptibility of KPC-producing P. aeruginosa CCBH17348 was evaluated by the disk diffusion method, Etest and broth microdilution. Carbapenemase production was confirmed by colorimetric assay (Carba NP) and PCR. Genomic DNA was sequenced by Illumina MiSeq sequencing and was assembled using the A5-Miseq pipeline, and gene annotation was performed using RAST 2.0. The database ResFinder 2.1, CRISPRFinder and MLST website were used to identify resistance genes, clustered regularly interspaced short palindromic repeats (CRISPRs) and sequence types (STs), respectively. Isolate CCBH17348 was considered multidrug-resistant, was susceptible to fluoroquinolones, gentamicin and polymyxin, and belonged to a newly described ST (ST2584), carrying an IncQ1 plasmid with blaKPC-2 and aph(3')-VI genes. Other genes associated with resistance and virulence found in the genome were blaOXA-50, blaPAO, fosA, catB, mutation in oprD and mexT (MexEF-OprN efflux regulator), and exotoxin-encoding genes (exoS, exoY and exoT). This study highlights the potential risk of new STs of P. aeruginosa carrying blaKPC-2 and the potential spread of blaKPC-2 in an IncQ1 plasmid.

High Rate of Association of 16S rRNA Methylases and Carbapenemases in Enterobacteriaceae Recovered from Hospitalized Children in Angola.

Acquired 16S rRNA methylases (RMTases) conferring pan-drug resistance to aminoglycosides were searched among enterobacterial isolates recovered in Angola. A total of 36 hospitalized children were screened for rectal colonization using the Superaminoglycoside selective medium. Twenty-two pan-aminoglycoside-resistant enterobacterial isolates were recovered, all of which produced RMTases, i.e., RmtB, ArmA, and RmtC. Highly diverse genetic backgrounds were identified for Escherichia coli and Klebsiella pneumoniae isolates, most of which coproduced carbapenemases NDM-1 or NDM-5, respectively.

Prevalence of carbapenemase in Enterobacteriaceae with decreased susceptibility to carbapenems isolated in a tertiary referral hospital

ABSTRACT Infections caused by carbapenem-resistant Enterobacteriaceae are a serious public health issue. This work aims to analyze the resistance mechanisms related to Enterobacteriaceae with decreased susceptibility to carbapenems isolated at the Hospital Julia Kubitschek (HJK) of the Minas Gerais State Founding Hospital [Fundacao Hospitalar do Estado de Minas Gerais (FHEMIG)], Brazil. Seventy-one strains with reduced susceptibility to carbapenems were found, and 45/71 (63.4%) were Klebsiella pneumoniae carbapenems (KPC) producers.

Results of the national surveillance of antimicrobial resistance of Enterobacteriaceae and Gram negative bacilli in health care-associated infections in Colombia, 2012-2014.

The Colombian National Antimicrobial Resistance Monitoring System for the surveillance of healthcare-associated infections was set up to meet this problem in the third quarter of 2012. To describe resistance profiles and laboratory-based surveillance based on the information collected by the System. We conducted a retrospective and descriptive study of the information notified to the Colombian Public Health Surveillance System (Sivigila), and in the Whonet databases covering the period from July 2012 to December 2014 provided by the primary data-generating units in the country, as well as laboratory surveillance results from 1,642 phenotypic and genotypic tests on carbapenemase isolates (927 from Enterobacteriaceae, 614 from Pseudomonas spp. and 101 from Acinetobacter spp.). There was a significant increase in Escherichia coli resistance to third-generation cephalosporins (reaching 26.3% in ICUs and 22.5% in other hospital wards), and Klebsiella pneumoniae resistance to ertapenem also increased (reaching 14.6% in ICUs). Acinetobacter baumannii carbapenem resistance exceeded 50% in ICUs whereas Pseudomonas aeruginosa had lower carbapenem resistance (38.8%). KPC (n = 574) and NDM (n=57) were the most frequently occurring carbapenemases in Enterobacteriaceae, VIM (n=229) and KPC (n=114) in P. aeruginosa, and OXA-23 in A. baumannii (n=87); several carbapenemase combinations were identified, KPC + VIM being the most common in Pseudomonas spp. and Enterobacteriaceae. The data from the surveillance of healthcare-associated infections revealed significant carbapenem resistance profiles and antimicrobial resistance mechanisms circulating in Colombian healthcare institutions.

A novel transposon, Tn6306, mediates the spread of blaIMI in Enterobacteriaceae in hospitals

The increasing incidence of carbapenem-resistant Enterobacteriaceae has become a challenge for clinical therapy. In our study, we analysed the molecular characteristics of imipenem-hydrolyzing β-lactamase (IMI) in Enterobacteriaceae isolates. Two reported clinical isolates, the IMI-3-producing Raoultella ornithinolytica RJ46C and the IMI-2-producing Escherichia coli RJ18 were identified in our retrospective review of isolates collected from June 2010 to June 2013, both isolates were resistant to carbapenem but sensitive to expanded-spectrum cephalosporins. The blaIMI gene was located on different ∼170-kb plasmids in both isolates. The blaIMI-3 gene was carried by the plasmid pRJ46C, which was extracted from the transconjugant and identified to be a 166,620-bp conjugative IncFIIY plasmid that contained 193 open reading frames, including replication-, plasmid conjugal transfer-, partitioning-, and mobilization-associated structures. The blaIMI-3 gene was located on a 15-kb region with a completely inverted sequence relative to that of plasmid pGA45, two ISEcl1-like elements containing two 33-bp complete inverted repeats were in an inverted orientation on both sides of the 15-kb region. We identified this typical structure as a novel composite transposon named Tn6306, indicating the occurrence of transposition. In addition, the blaIMI-2-carrying pRJ18 was an IncFIB plasmid, and a similar ISEcl1-like element was identified in an inverted direction upstream of IMI-2 in pRJ18. The identification of blaIMI in R. ornithinolytica and E. coli highlights the diversity of spreading carbapenemases in Enterobacteriaceae between hospitals and the environment in China. The novel transposon Tn6306, and other insert sequences, may play important roles in blaIMI mobilization.

Evaluation of the Carba NP test for carbapenemase detection in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., and its practical use in the routine work of a national reference laboratory for susceptibility testing

The aim of this study was to evaluate the Carba NP test (and CarbAcineto) for the detection of carbapenemases in Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp., and to assess its usefulness in the routine work of the National Reference Centre for Susceptibility Testing (NRCST) in Poland. The evaluation of the Carba NP/CarbAcineto tests was carried out on a group of 81 Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. isolates producing KPC-, NDM-, VIM-, IMP- or OXA-48, -23, -24/40, -58-type carbapenemases, and on 26 carbapenemase-negative strains cultivated on a broad panel of microbiological media. Subsequently, the performance of the Carba NP/CarbAcineto tests was assessed on 1282 isolates of Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. from Polish hospitals, submitted to the NRCST during a 9-month period in 2014. The Carba NP/CarbAcineto results were compared with other phenotypic tests and/or polymerase chain reaction (PCR). The impact of the media on the results of the Carba NP/CarbAcineto tests was observed, with the Columbia blood agar yielding the highest sensitivity and clarity of the results. Furthermore, the Carba NP/CarbAcineto tests were included in the NRCST routine procedure for carbapenemase identification. The sensitivity and specificity of the Carba NP test were 95.8% and 93.3%, respectively, for Enterobacteriaceae, and 97.5% and 99.0%, respectively, for Pseudomonas spp. The sensitivity of the CarbAcineto test for Acinetobacter spp. was 88.9%. This study confirmed the usefulness of the Carba NP/CarbAcineto tests for the rapid detection of various types of carbapenemases.

Evaluation of the RAPIDEC® CARBA NP and β-CARBA® tests for rapid detection of Carbapenemase-producing Enterobacteriaceae

The analytical performances of the RAPIDEC CARBA NP® (bioMérieux) and the β-CARBA® (Bio-Rad) tests were evaluated for the rapid detection of any known type of carbapenemases in Enterobacteriaceae. An international collection of 149 enterobacterial isolates comprising 111 Carbapenemase-Producing Enterobacteriaceae (CPE) and 38 non-carbapenemase producers was used. CPE included 32 carbapenemase producers of class A (18 KPC-2, 1 FRI-1, 5 SME and 8 IMI), 33 of class B (13 NDM, 11 VIM and 9 IMP) and 46 of class D (15 OXA-48, 14 OXA-181, 10 OXA-204, 3 OXA-232 and 4 OXA-162). The RAPIDEC CARBA NP® and the β-CARBA® tests were performed in strict accordance to the manufacturer's instructions and results were read within 2 h and 30 min, respectively. RAPIDEC CARBA NP® detected 104/111 CPE isolates compared to 72/111 for the β-CARBA® test. The overall sensitivity and specificity were 93.7% and 100%, respectively, for the RAPIDEC CARBA NP® test, and 64.9% and 90%, respectively, for the β-CARBA® test. The β-CARBA® test failed to detect all the non-KPC class A carbapenemases (14/14) and most (24/31) of the OXA-48-like producers (OXA-162, OXA-181, OXA-204 and OXA-232), and detected 1/1 OXA-163 and 1/1 OXA-405 as carbapenemase producers whereas these enzymes are rather defined as non carbapenemases. RAPIDEC CARBA NP® test exhibited better performances than those of the β-CARBA® test and confirmed to be a reliable tool for the detection of CPE, especially for OXA-48-like producers.

Detection of OXA-181/OXA-48 carbapenemase producing Enterobacteriaceae in Bangladesh

Carbapenem resistant Enterobacteriaceae (CRE) is becoming a major public health concern globally. Detection of carbapenem hydrolyzing enzyme carbapenemase in Enterobacteriaceae is important to institute appropriate therapy and to initiate preventive measures. This study was designed to determine the presence of carbapenemase producers among the CRE isolated from patients at Dhaka Medical College Hospital, Bangladesh. Twenty-nine CRE strains detected by disk diffusion technique were included in the study. Minimum inhibitory concentration of imipenem and tigecycline was determined by agar dilution method. Carbapenemase production was phenotypically detected by Modified Hodge test while MBL producers were detected by combined disk and double disk synergy tests. Genes encoding blaNDM-1, blaOXA-181, blaOXA-48, blaKPC, blaCTX-M-15, blaOXA-1-group were identified by polymerase chain reaction (PCR). Out of 29 CRE, nineteen (65.6%) were positive for carbapenemase by any of the three phenotypic tests namely MHT, CD or DD tests. Those 19 isolates were also positive either for blaNDM-1 or blaOXA-181/blaOXA-48 by PCR. Of the 19 PCR positive isolates, the rate of positivity for blaNDM- 1, blaOXA-181/blaOXA-48 and blaNDM-1+ blaOXA-181/blaOXA-48 was 73.7% (14/19), 57.9% (11/19) and 31.6% (6/19) respectively. Both blaOXA-181 and blaOXA-48 co-existed. All the carbapenemase producing organisms harboured blaCTX-M-15 except one C. freundii strain. The rate of resistance to different classes of antibiotics ranged from 63.2% to 100% except colistin and tigecycline. Organisms positive for OXA-181/OXA-48 had a low level of resistance to carbapenem (MIC 1 - 4 ì g/ml) while with NDM-1 had high level resistance to imipenem (MICs 16 - ? 32 ì g/ ml). Out of 19 carbapenemase positive isolates, 12 (63.16%) were extensively drug-resistant (XDR) and were only sensitive to tigecycline and colistin. The result of this study showed the presence of blaOXA-181/ blaOXA-48, blaNDM-1 positive strains in Bangladesh and colistin and tigecycline were the most effective drugs against carbapenemase producing Enterobacteriaceae (CPE). Epidemiological monitoring of carbapenemase producing organisms in Bangladesh is important to prevent their dissemination.Ibrahim Med. Coll. J. 2015; 9(2): 45-51

Molecular characterization of class b carbapenemases in advanced stage of dissemination and emergence of class d carbapenemases in Enterobacteriaceae from Croatia

Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin β-lactamases, class B metallo-β-lactamases (MBL) or class D OXA-48-like β-lactamases. The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia.In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST.Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 β-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae, Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively.Three different types of carbapenemases were identified showing the complexity of CRE in Croatia.