Application of a multiplex immunochromatographic assay for rapid identification of carbapenemases in a clinical microbiology laboratory: performance and turn-around-time evaluation of NG-test Carba 5.

BACKGROUND: Prompt and accurate identification of carbapenemase production is essential for appropriate treatment and infection control. NG-Test Carba 5 (termed herein "Carba 5"; NG Biotech, Guipry, France) is a multiplex immunochromatographic assay for the rapid phenotypic identification of five major carbapenemases (KPC, NDM, VIM, IMP, and OXA-48-like) from bacterial isolates. This study aimed to evaluate the diagnostic performance of Carba 5 and its impact on the turn-around-time in a clinical microbiology laboratory. RESULTS: Carba 5 was retrospectively evaluated using 78 carbapenemase producers and 23 non-carbapenemase producers confirmed by PCR and sequencing. The performance and time required for carbapenemase identification were prospectively evaluated using 47 carbapenem resistant Enterobacteriaceae isolates, and the results were compared to those obtained using Xpert Carba-R (Cepheid, Sunnyvale, CA, USA). For the bacterial isolates included in retrospective and prospective evaluation, the Carba 5 assay correctly identified 147 isolates except one isolate with a sensitivity of 99.13% (95% CI 95.25-99.98%) and specificity of 100% (95% CI 89.42-100%). The Carba 5 assay missed one VIM-1 among 13 VIM producers. The assay showed a sensitivity of 92.31% (95% CI 63.97-99.81%) for detecting VIM and 100% for detecting KPC, NDM, OXA-48-like, and IMP. Compared to the Xpert Carba-R assay, Carba 5 exhibited 100% agreement and was more time-efficient (median time 24 min vs. 1 h 11 min). CONCLUSIONS: The Carba 5 assay has potential as an alternative to molecular methods for detecting major carbapenemases from bacterial isolates in a clinical microbiology laboratory. Compared to the Xpert Carba-R, Carba 5 turns out to be more affordable and time-efficient while showing a comparable performance, and may accelerate therapeutic and infection control decisions.

Genomic and clinical characterisation of multidrug-resistant carbapenemase-producing ST231 and ST16 Klebsiella pneumoniae isolates colonising patients at Siriraj hospital, Bangkok, Thailand from 2015 to 2017.

BACKGROUND: Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) have continually grown as a global public health threat, with significant mortality rates observed across the world. We examined the clinical data from patients with CPE infections and their outcomes, concentrating on Klebsiella pneumoniae isolates. We analysed the clinical information, performed antimicrobial susceptibility testing, and conducted molecular epidemiological and genomic analyses on the isolates to identify patterns in the data. METHODS: The clinical characteristics of 33 hospitalised patients with confirmed CPE, including patient-related factors associated with the development of CPE infections, were examined. Patients were divided according to whether they were "colonised" or "infected" with CPE and by the timing and frequency of their rectal swab collections, from which 45 swabs were randomly selected for analysis. CPE isolates were purified, and antimicrobial susceptibility tests performed. Whole genome sequences of these isolates were determined and analysed to compute bacterial multilocus sequence types and plasmid replicon types, infer phylogenetic relationships, and identify antimicrobial resistance and virulence genes. RESULTS: Altogether, 88.9% (40/45) of the CPE isolates were K. pneumoniae. The most abundant carbapenemase gene family in the K. pneumoniae isolates (33/39) was blaOXA-232, with blaNDM-1 additionally identified in 19 of them. All CPE isolates carrying either blaOXA-232 or blaNDM-1 were resistant to meropenem, but only 40 from 45 were susceptible to colistin. Among the CPE-infected patients (n = 18) and CPE-colonised patients who developed CPE infections during the study (n = 3), all but one received standard colistin-based combination therapy. Phylogenetic analysis revealed the polyclonal spread of carbapenemase-producing K. pneumoniae (CPKP) within the patient population, with the following two major subclades identified: ST16 (n = 15) and ST231 (n = 14). CPKP-ST231 had the highest virulence score of 4 and was associated with primary bacteraemia. The siderophores yersiniabactin and aerobactin, considered to be important virulence factors, were only identified in the CPKP-ST231 genomes. CONCLUSIONS: This study has revealed the genomic features of colonising CPE isolates, focusing on antimicrobial resistance and virulence determinants. This type of multi-layered analysis can be further exploited in Thailand and elsewhere to modify the regimes used for empirical antibiotic treatment and improve the management strategies for CPE infections in hospitalised patients.

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

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 ß-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.

A novel potent metal-binding NDM-1 inhibitor was identified by fragment virtual, SPR and NMR screening.

NDM-1 can hydrolyze nearly all available ß-lactam antibiotics, including carbapenems. NDM-1 producing bacterial strains are worldwide threats. It is still very challenging to find a potent NDM-1 inhibitor for clinical use. In our study, we used a metal-binding pharmacophore (MBP) enriched virtual fragment library to screen NDM-1 hits. SPR screening helped to verify the MBP virtual hits and identified a new NDM-1 binder and weak inhibitor A1. A solution NMR study of 15N-labeled NDM-1 showed that A1 disturbed all three residues coordinating the second zinc ion (Zn2) in the active pocket of NDM-1. The perturbation only happened in the presence of zinc ion, indicating that A1 bound to Zn2. Based on the scaffold of A1, we designed and synthesized a series of NDM-1 inhibitors. Several compounds showed synergistic antibacterial activity with meropenem against NDM-1 producing K. pneumoniae.

Trends and molecular characteristics of carbapenemase-producing Enterobacteriaceae in Japanese hospital from 2006 to 2015.

BACKGROUND: The increasing number of carbapenemase-producing Enterobacteriaceae (CPE) has become a global problem. Most carbapenemases detected in Japan are imipenemase, which is an imipenem-degrading enzyme with low ability; thus, CPE could have been overlooked. Therefore, this study aimed to detect and analyze CPE, without overlooking CPE showing the low minimum inhibitory concentration phenotype. METHODS: CPE screening was conducted on 531 ceftazidime-resistant Enterobacteriaceae isolated from Kitasato University Hospital during 2006-2015. We confirmed the presence of the carbapenemase genes (blaIMP, blaVIM, blaKPC, blaNDM, and blaOXA-48) by multiplex polymerase chain reaction. The detected CPE strains were analyzed by antimicrobial susceptibility testing, multilocus sequence typing, conjugal experiments, replicon typing, and plasmid profiling by restriction enzyme treatment. RESULTS: The CPE detection rate in Kitasato University Hospital within the past 10 years was 0.0003% (nine CPE strains). These nine CPE strains were identified to harbor 8 blaIMP-1 or 1 blaNDM-5. The CPE strains consisted of five species including Klebsiella pneumoniae and Citrobacter freundii. Six of eight blaIMP-1 were coded by IncHI2 plasmid, and the other two were coded by IncA/C plasmid. Plasmid profiling revealed that K. pneumoniae and C. freundii isolated from the same patient harbored the same plasmid. CONCLUSION: The CPE detection rate in this study was significantly lower than those previously reported in Japan. In one case, IncA/C plasmid transmission through different bacterial species within the body was speculated. Although the number of CPE detected was low, these results indicated that the resistance plasmid could spread to other bacterial species.

Evolution of New Delhi metallo-ß-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used ß-lactamase inhibitors to counter the activity of metallo-ß-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-ß-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, 1H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ∼10 °C increased Tm ) and improved zinc affinity (up to ∼10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km = 5 × 106 m-1 s-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of ß-lactam antibiotics and zinc deprivation.

Transplant tourism complicated by life-threatening New Delhi metallo-ß-lactamase-1 infection.

Transplant tourism, which is the practice of traveling to other countries for transplant, continues to be a major problem worldwide. We describe a patient who traveled to Pakistan and underwent commercial kidney transplant. He developed life-threatening infections from New Delhi metallo-ß-lactamase-1-producing Enterobacter cloacae and Rhizopus oryzae, resulting in a necrotizing kidney allograft infection and subsequent external iliac artery rupture. He survived after a prolonged course of nonstandardized antimicrobial therapy, including a combination of aztreonam and ceftazidime-avibactam, and aggressive surgical debridement with allograft nephrectomy. The early timing of infection with these unusual organisms localized to the allograft suggests contamination and substandard care at the time of transplant. This case highlights the challenges of caring for these infections and serves as a cautionary tale for the potential complications of commercial transplant tourism.

Rapid Identification of New Delhi Metallo-ß-Lactamase (NDM) Using Tryptic Peptides and LC-MS/MS.

There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-ß-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 blaNDM-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/µg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the blaNDM-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

EDTA-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Metallo-ß-Lactamase-Producing Enterobacteriaceae.

The increase in the prevalence and impact of infections caused by carbapenemase-producing Enterobacteriaceae is a global health concern. Therefore, rapid and accurate methods to detect these organisms in any clinical microbiology laboratory, including those in resource-limited settings, are essential to prevent and contain their spread. It is also important to differentiate between serine- and metal-dependent carbapenemases elaborated by carbapenemase-producing isolates for epidemiologic, infection control and prevention, and therapeutic purposes. Here, we describe the development and evaluation of the EDTA-modified carbapenem inactivation method (eCIM), an assay for discriminating between serine- and metal-dependent (i.e., metallo-ß-lactamases [MBLs]) carbapenemases when used in conjunction with the modified carbapenem inactivation method (mCIM). The eCIM had an overall sensitivity and specificity of 100% and was adopted by the Clinical and Laboratory Standards Institute as a method to use in combination with the mCIM to identify MBL-producing Enterobacteriaceae.

Wastewater as a Probable Environmental Reservoir of Extended-Spectrum-ß-Lactamase Genes: Detection of Chimeric ß-Lactamases CTX-M-64 and CTX-M-123.

The presence of antimicrobial-resistant bacteria and resistance genes in aquatic environments is a serious public health concern. This study focused on Escherichia coli possessing blaCTX-M genes in wastewater inflows. Twelve crude inflow water samples from wastewater treatment plant (WWTP) A and two samples each from three other WWTPs were collected in 2017 and 2018. A total of 73 E. coli isolates with 31 different sequence types (STs) harboring distinctive blaCTX-M gene repertoires were detected. In WWTP A influents, blaCTX-M-14 (14 isolates) was dominant, followed by blaCTX-M-15 (12 isolates) and blaCTX-M-27 (10 isolates). The chimeric blaCTX-M-64 and blaCTX-M-123 genes were each identified in one of the E. coli isolates from the same WWTP A inflow port. The blaCTX-M-27 gene was associated with five of seven B2-ST131 isolates, including three isolates of the B2-O25b-ST131-H30R/non-Rx lineage. One of the remaining two isolates belonged to the B2-O25b-ST131-H30R/Rx lineage harboring the blaCTX-M-15 gene. As for the B2-O25b-ST131-H30R/non-Rx lineage, two isolates with blaCTX-M-27 were recovered from each of the WWTP B and D influents, and one isolate with blaCTX-M-174 was also recovered from WWTP B influent. Whole-genome sequencing of chimeric blaCTX-M-harboring E. coli isolates revealed that the blaCTX-M-64 gene was integrated into the chromosome of ST10 E. coli B22 via ISEcp1-mediated transposition of a 9,467-bp sequence. The blaCTX-M-123-carrying IncI1 plasmid pB64 was 109,169 bp in length with pST108. The overall findings suggest that wastewater may act as a probable reservoir of clinically significant clonal lineages mediating antimicrobial resistance genes and chimeric genes that have not yet been identified from human isolates of domestic origin in Japan.IMPORTANCE Global spread of CTX-M-type extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is a critical concern in both clinical and community settings. This dominance of CTX-M-type ESBL producers may be largely due to the successful international spread of epidemic clones, as represented by the extraintestinal pathogenic Escherichia coli (ExPEC) ST131. Our findings highlight the worrisome presence of diverse E. coli clones associated with humans, including ExPEC lineages harboring the most common blaCTX-M variants in untreated wastewater samples. Moreover, the chimeric genes blaCTX-M-64 and blaCTX-M-123, which have not yet been identified from human isolates of domestic origin in Japan, were identified. Exposure to untreated wastewater through combined sewer overflow caused by heavy rains derived from abnormal weather change could pose a risk for human health due to ingesting those antimicrobial-resistant bacteria.

Diversity of carbapenemases in clinical isolates: The emergence of blaVIM-5 in Bangladesh.

Global emergence and dissemination of carbapenemases are clinically threatening, notably in countries with endemic blaNDM. To analyze the extent of carbapenemases in Bangladesh, 71 isolates were collected from 7 different clinical sources: wound swab (n = 38), pus (n = 13), urine (n = 9), blood (n = 4), tracheal aspirate (n = 3), pleural fluid (n = 1) and vaginal swab (n = 3) from Dhaka Medical College Hospital, Bangladesh. Among the isolates, 25 were resistant to at least one of the three carbapenems (imipenem, meropenem and doripenem), including 15 being resistant to all. These resistant isolates were identified as Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, P. hibiscicola, Proteus mirabilis, Providencia stuartii and Citrobacter sedlakii. Carbapenemase detection among these 25 isolates varied in individual phenotypic assays (83% in Modified Hodge Test, 50% in Combined Disk Test for Metallo-ß-lactamase prediction) as compared with the genotypes observed (96% prevalence of various carbapenemases including blaOXA-1,48, blaNDM-1,5, blaVIM-2,5). blaOXA-48 was the most prevalent gene (84%) followed by blaNDM (72%). Coexistence of multiple gene combination such as blaNDM+blaOXA-48+blaOXA-1 was prevalent (48%). Harborage of blaVIM-5 (n = 1) was characterized for the first time, while blaNDM-5 (n = 5) was reported contemporarily with a recent study in Bangladesh. Presence of plasmids (64%) and integron class 1 (100%) signifies the transferable potential of resistant traits. The emergence of such new variants along with the presence of the mobile genetic elements demands strict surveillance and combating strategies.

Diversity of metallo-ß-lactamase-encoding genes found in distinct species of Acinetobacter isolated from the Brazilian Amazon Region.

BACKGROUND: The multidrug resistance (MDR) phenotype is frequently observed in Acinetobacter baumannii, the most clinically relevant pathogenic species of its genus; recently, other species belonging to the A. calcoaceticus-A. baumannii complex have emerged as important MDR nosocomial pathogens. OBJECTIVES: The present study aimed to verify the occurrence of metallo-ß-lactamase genes among distinct Acinetobacter species in a hospital located in the Brazilian Amazon Region. METHODS: Antimicrobial susceptibility profiles were determined by broth microdilution. The genetic relationships among these isolates were assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Pyrosequencing reads of plasmids carrying the bla NDM-1 gene were generated using the Ion Torrent™ platform sequencing. FINDINGS: A total of six isolates carried bla NDM-1: A. baumannii (n = 2), A. nosocomialis (n = 3), and A. pittii (n = 1); three carried bla IMP-1: A. baumannii, A. nosocomialis, and A. bereziniae. Resistance to colistin was observed for an NDM-1-producing A. nosocomialis isolate. Diverse PFGE patterns and sequence types were found among A. nosocomialis and A. baumannii isolates. The bla NDM-1 sequence was inserted in a Tn125 transposon, while the bla IMP-1 was found as a gene cassette of the class 1 integron In86. MAIN CONCLUSIONS: To the best of our knowledge, this is the first report describing the dissemination of bla NDM-1 among distinct Acinetobacter species recovered from the same hospital in South America.

Tracing local and regional clusters of carbapenemase-producing Klebsiella pneumoniae ST512 with whole genome sequencing, Finland, 2013 to 2018.

BackgroundTwo epidemiologically-unrelated clusters of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae were detected among several healthcare facilities (HCF) in Finland by routine surveillance using whole genome sequencing (WGS).AimThe objective was to investigate transmission chains to stop further spread of the responsible strain.MethodsIn this observational retrospective study, cases were defined as patients with K. pneumoniae KPC-3 sequence type (ST)512 strain detected in Finland from August 2013 to May 2018. Environmental specimens were obtained from surfaces, sinks and toilets in affected wards. WGS was performed on K. pneumoniae cultures using Illumina MiSeq platform and data were analysed using Ridom SeqShere software K. pneumoniae core genome multilocus sequence typing (cgMLST) scheme. Epidemiological information of the cases was provided by HCFs.ResultsWe identified 20 cases in six HCFs: cluster 1 included 18 cases in five HCFs and cluster 2 two cases in one HCF. In cluster 1, a link with a foreign country was unclear, 6/18 cases without overlapping stay had occupied the same room in one of the five HCFs within > 3 years. In cluster 2, the index case was transferred from abroad, both cases occupied the same room 8 months apart. A strain identical to that of the two cases in cgMLST was isolated from the toilet of the room, suggesting a clonal origin.ConclusionsThe clusters were mostly related to case transfer between facilities and likely involved environmental transmission. We show that CPE surveillance using WGS and collaboration between hospitals are crucial to identify clusters and trace transmission chains.

Antimicrobial Resistance and Molecular Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolated from Ducks in South Korea.

Ducks are potential carriers of pathogenic bacteria, which are capable of transmitting zoonotic diseases to humans. The global spread of Enterobacteriaceae carrying extended-spectrum ß-lactamase (ESBL) genes is a public health concern. This study investigated the prevalence of antimicrobial resistance in Escherichia coli isolated from ducks in Korea and described the molecular characteristics of the ESBLs they produced. A total of 146 E. coli isolates from 404 duck fecal and carcass samples in 85 duck farms were tested for antimicrobial resistance using the broth dilution method and were further characterized using molecular methods. We observed high resistance rates to tetracycline, trimethoprim/sulfamethoxazole, nalidixic acid, ampicillin, and ciprofloxacin. In total, six ceftiofur-resistant isolates (4.1%) were observed, which produced CTX-M-55 (n = 3) or CTX-M-65 ß-lactamase (n = 3). All CTX-M-producing E. coli isolates were also resistant to ciprofloxacin, with mutations in the quinolone resistance determining region of GyrA (S83L with or without D87N) and ParC (S80I), and three CTX-M-producing E. coli isolates carried plasmid-mediated quinolone resistance (PMQR) genes, qepA (n = 1), qnrS, and acc(6')-Ib-cr (n = 2). The transfer of blaCTX-M genes was observed in one isolate mediated by IncF-family plasmids but not in the co-resistant isolates carrying both blaCTX-M and PMQR genes. Pulsed-field gel electrophoresis and multilocus sequence typing demonstrated that CTX-M-producing isolates were heterogeneous; however, identical isolates were found in different farms and slaughterhouses. This study presents baseline data on antimicrobial resistance of E. coli derived from duck samples and is the first report of CTX-M-55 and CTX-M-65 ß-lactamase-producing E. coli isolated from ducks in Korea. The dissemination of ESBL-producing E. coli poses a potential risk to public health and therefore should be monitored.

FALSE POSITIVE PHENOTYPIC DETECTION OF METALLO-BETA-LACTAMASES IN ACINETOBACTER BAUMANNII.

Phenotypic detection of metallo-ß-lactamases (MBLs) in Acinetobacter (A.) baumannii is a serious challenge to clinical microbiologists. MBLs are inhibited by metal chelators such as ethylenediaminetetraacetic acid) (EDTA). Production of MBLs cannot be recognized based on resistance phenotype. Therefore, phenotypic tests using EDTA are recommended. The aim of this study was to investigate the sensitivity and specificity of inhibitor based tests (EDTA) for detection of MBL. A total of 172 A. baumannii strains (123 carbapenemase positive and 49 carbapenemase negative) were analyzed. Phenotypic detection of MBLs was performed by the combined disk test with EDTA (CDT-EDTA) and EPI-dilution test (EPI-DT). Both tests were positive in all 11 isolates possessing VIM-1 MBL, showing 100% sensitivity. However, false positive results were observed in strains with class D carbapenemases using both tests, i.e. all OXA-23 and OXA-24/40 producing organisms and most OXA-58 positive strains (77% with CDT-EDTA vs. 65% with EPI-DT). False positive results can occur because oxacillinases are converted to a less active state in the presence of EDTA, leading to augmentation of the inhibition zone around the carbapenem disk or reduction of carbapenem minimum inhibitory concentrations. This study showed high sensitivity but low specificity of phenotypic methods in the detection of MBLs.

FRI-4 carbapenemase-producing Enterobacter cloacae complex isolated in Tokyo, Japan.

Objectives: A carbapenem-resistant Enterobacter cloacae complex isolated in Tokyo, Japan, produced a carbapenemase that was detected by a Carba NP test and a modified carbapenem inactivation method, but none of the 'Big Five' carbapenemase genes was detected by PCR. This study aimed to identify the carbapenemase. Methods: Carbapenemase genes were screened by WGS. Next, we generated a recombinant plasmid in which the carbapenemase gene was inserted. We also extracted the carbapenemase gene-carrying plasmid from the E. cloacae complex. The effects of both plasmids on the antibiotic susceptibility of Escherichia coli were then tested. The carbapenemase gene-carrying plasmid in the E. cloacae complex was completely sequenced. Results: A novel carbapenemase gene, blaFRI-4, encoded an amino acid sequence that was 93.2% identical to French imipenemase (FRI-1). E. coli transformed with blaFRI-4 showed reduced carbapenem susceptibility. A complete sequence of the blaFRI-4-carrying 98 508 bp IncFII/IncR plasmid (pTMTA61661) showed that blaFRI-4 and the surrounding region (18.7 kb) were duplicated. Conclusions: The FRI-4-producing E. cloacae complex was isolated in Japan, whereas all other FRI variants have been found in Europe, suggesting that the spread of FRI carbapenemases is global.

Evaluation of the AusDiagnostics MT CRE EU assay for the detection of carbapenemase genes and transferable colistin resistance determinants mcr-1/-2 in MDR Gram-negative bacteria.

Objectives: To evaluate the AusDiagnostics MT CRE EU assay for the detection of carbapenemase and acquired colistin resistance genes in Gram-negative bacteria. Methods: The assay allows the detection of blaKPC, blaOXA-48-like, blaNDM, blaVIM, blaIMP, blaSIM, blaGIM, blaSPM, blaFRI, blaIMI, blaGES (differentiating ESBL and carbapenemase variants), blaSME and mcr-1/-2. It was evaluated against a panel of isolates including Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. retrospectively (n = 210) and prospectively (n = 182). Results: The CRE EU assay was able to detect 268/268 carbapenemase genes, with 239 belonging to the 'big five' families (KPC, OXA-48-like, NDM, VIM and IMP) and 29 carbapenemase genes of the SIM, GIM, SPM, FRI, IMI, SME and GES families. It could distinguish between ESBL and carbapenemase variants of GES. It also allowed detection of mcr-1/-2 colistin resistance genes on their own or in isolates co-producing a carbapenemase. Conclusions: The AusDiagnostics MT CRE EU assay offered wide coverage for detection of acquired carbapenemase genes. It required minimal hands-on time and delivered results in less than 4 h from bacterial culture.

Detection of CTX-M-15 harboring Escherichia coli isolated from wild birds in Tunisia.

BACKGROUND: The spreading of antibiotic resistant bacteria is becoming nowadays an alarming threat to human and animal health. There is increasing evidence showing that wild birds could significantly contribute to the transmission and spreading of drug-resistant bacteria. However, data for antimicrobial resistance in wild birds remain scarce, especially throughout Africa. The aims of this investigation were to analyze the prevalence of ESBL-producing E. coli in faecal samples of wild birds in Tunisia and to characterize the recovered isolates. RESULTS: One hundred and eleven samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 µg/ml). ESBL-producing E. coli isolates were detected in 12 of 111 faecal samples (10.81%) and one isolate per sample was further characterized. ß-lactamase detected genes were as follows: blaCTX-M-15 (8 isolates), blaCTX-M-15 + blaTEM-1b (4 isolates). The ISEcp1 and orf477 sequences were found respectively in the regions upstream and downstream of all blaCTX-M-15 genes. Seven different plasmid profiles were observed among the isolates. IncF (FII, FIA, FIB) and IncW replicons were identified in 11 CTX-M-15 producing isolates, and mostly, other replicons were also identified: IncHI2, IncA/C, IncP, IncI1 and IncX. All ESBL-producing E. coli isolates were integron positive and possessed "empty" integron structures with no inserted region of DNA. The following detected virulence genes were: (number of isolates in parentheses): fimA (ten); papC (seven); aer (five); eae (one); and papGIII, hly, cnf, and bfp (none). Molecular typing using pulsed-field gel electrophoresis and multilocus sequence typing showed a low genetic heterogeneity among the 12 ESBL-producing strains with five unrelated PFGE types and five different sequence types (STs) respectively. CTX-M-15-producing isolates were ascribed to phylogroup A (eleven isolates) and B2 (one isolate). CONCLUSION: To our knowledge, this study provides the first insight into the contribution of wild birds to the dynamics of ESBL-producing E. coli in Tunisia.

Microbial infection pattern, pathogenic features and resistance mechanism of carbapenem-resistant Gram negative bacilli during long-term hospitalization.

BACKGROUND: Carbapenem-resistant Gram-negative bacilli (GNB) have become an important cause of nosocomial infections of hospitalized patients. METHODS: To investigate the microbial infection patterns and molecular epidemiology characteristics of the carbapenem-resistant GNB isolates from a long-term hospitalized patient, antimicrobial susceptibility testing, phenotypic screening test for carbapenemase production, PCR screening and DNA sequencing of carbapenemase genes, repetitive extragenic palindromic sequence-based PCR (REP-PCR), multilocus sequencing typing (MLST) and genetic environment analysis were performed. RESULTS: Twelve strains with carbapenemase genes were detected from 63 carbapenem-resistant isolates, including two blaIMP-25-carrying Pseudomonas aeruginosa, one blaNDM-1-carrying Citrobacter freundii, three blaNDM-1-carrying Klebsiella pneumoniae and six blaKPC-2-carrying K. pneumoniae. Only the blaNDM-1 genes were successfully transferred from three K. pneumoniae strains to Escherichia coli C600 by conjugation. Genetic environment of blaIMP-25, blaNDM-1 and blaKPC-2 genes in our study were consistent with previous reports. Molecular typing of K. pneumoniae performed by MLST revealed that most of the isolates belonged to ST11. blaNDM-1-carrying K. pneumoniae sequencing type 1416 was first reported in our study. CONCLUSIONS: Carbapenem-resistant GNB are common pathogens during long-term hospitalization, and ST11 blaKPC-2-carrying K. pneumoniae is the dominant bacterium in our study. Colonization and horizontal transmission of resistance by plasmids of carbapenem-resistant GNB have increased the risks of persistent infection and mortality of long-term hospitalized patients.

Warning: spread of NDM-1 in two border towns of Iran.

NDM-1 producing gram-negative bacteria can be resistant to every beta-lactam antibiotic, including carbapenem which is one of the last-lines of antibiotic therapy against multi-drug resistant bacteria. This study aimed to detect the metallo-beta-lactamase in the isolated gram-negative bacteria of the Iranian clinical specimens collected from two major cities in Iran. In this cross sectional study 171 Acinetobacter baumannii, 120 Enterobacter spp. and 145 Klebsiella pneumoniae isolated from clinical specimens of two training hospitals in Tabriz and Mashhad were evaluated. Carbapenem resistant screening was performed according to CLSI guide line. The antibiotic susceptibility testing was prepared for carbapenem resistant strains. Then, the metallo-beta- lactamase genes detection was also carried out by PCR assay and confirmed by sequencing. Sixty-eight, 12 and 22 carbapenem resistant Acinetobacter baumannii, Klebsiella pneumoniae and Enterobacter spp. were respectively confirmed, respectively. blaVIM in 9% and blaNDM-1  in 4% of isolated A. baumannii were observed. blaNDM-1 was also detected in 18% and 25% of K. pneumoniae and Enterobacter spp. isolates, respectively. This is the first report of NDM-1 producer A. baumannii and Enterobacter pp. in Iran. NDM-1 producing gram-negative bacteria can be resistant to all beta-lactam antibiotics and cause complicated challenges in health care systems.