Carbapenem-nonsusceptible Enterobacteriaceae Research Articles

Risk factors and clinical impact of bacteremia due to carbapenem-nonsusceptible Enterobacteriaceae: A multicenter study in southern Taiwan.

The emergence of carbapenem-non-susceptible Enterobacteriaceae (CnSE) infections is a public health threat. This study investigated the risk factors and clinical impact of bacteremia due to CnSE. The study was conducted at three hospitals in southern Taiwan between January 1, 2017, and October 31, 2019. Only the first episode of CnSE bacteremia from each adult was included. For one episode of CnSE bacteremia, two subsequent bacteremic episodes due to carbapenem-susceptible Enterobacteriaceae isolates in each hospital were included as the controls. Among a total of 641 episodes of monomicrobial Enterobacteriaceae bacteremia were noted, 47 (7.3%) of which were of CnSE bacteremia. Ninety-four episodes of carbapenem-susceptible Enterobacteriaceae (CSE) bacteremia were selected as the controls for further analyses. In the multivariate analysis, hypertension (odds ratio [OR], 4.21; P=0.005), Pitt bacteremia score (OR, 1.61; P=0.002), and nosocomial bacteremia (OR, 3.30; P=0.01) were associated with carbapenem nonsusceptibility among Enterobacteriaceae bacteremia. The most abundant CnSE isolate was Klebsiella pneumoniae (91.5%), followed by Klebsiella oxytoca (6.4%) and Escherichia coli (2.1%). Patients with CnSE bacteremia had a higher overall in-hospital mortality rate than those with CSE bacteremia (53.2% vs. 23.4%, P=0.001). Moreover, in the multivariate analysis, the in-hospital mortality was significantly associated with higher Pitt bacteremia score (OR, 1.38; P=0.02) and marginally associated with CnSE infections (OR, 2.44; P=0.06). Among adults with Enterobacteriaceae bacteremia, carbapenem nonsusceptibility, male sex, and the presence of hypertension or chronic kidney disease indicate a poor prognosis during hospitalization.

Activities of imipenem-relebactam combination against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

Imipenem-relebactam is a new β-lactam and β-lactamase inhibitor combination to treat carbapenem-resistant gram-negative bacteria infections. However, difference in carbapenem resistant mechanisms existed with geographic variations. To evaluate the susceptibility of imipenem-relebactam to 660 carbapenem-nonsusceptible Enterobacteriaceae isolates in Taiwan and to identify the invivo efficacy with a Caenorhabditis elegans model. 188 carbapenem-nonsusceptible Escherichia coli isolates and 472 carbapenem-nonsusceptible Klebsiella pneumoniae isolates were collected from a national surveillance study in Taiwan. The antimicrobial susceptibility profiles and carbapenemase distributions were determined. An agar dilution method was performed to evaluate the invitro activities of imipenem monotherapy and imipenem-relebactam combination. Contributions of metallo-carbapenemase to imipenem-relebactam susceptibility was investigated via EDTA treatment. A C. elegans model was used to evaluate the invivo efficacy of imipenem-relebactam combination. 87.8% and 82.2% susceptibility to imipenem-relebactam was observed for 188 carbapenem-nonsusceptible E. coli and 472 carbapenem-nonsusceptible K. pneumoniae, respectively. However, poor activities of imipenem-relebactam was observed against 23 metallo-carbapenemase producers tested in this study. In the invivo C. elegans model, imipenem-relebactam significantly rescued nematodes from the infection of a blaKPC-producing K. pneumoniae isolate. Our study supports that imipenem-relebactam is a potential therapy against carbapenem-nonsusceptible Enterobacteriaceae, and to our knowledge, this is the first report of evaluation for imipenem-relebactam efficacy against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

In Vitro and In Vivo Evaluations of β-Lactam/β-Lactamase Mono- and Combined Therapies against Carbapenem-Nonsusceptible Enterobacteriaceae in Taiwan.

Increasing carbapenem resistance rates worldwide underscored the urgent need of novel antimicrobials. Ceftazidime–avibactam and aztreonam–avibactam combinations are developed to combat carbapenem resistance, but biological and geographic variations must be considered for antibiotic susceptibility patterns varied. Thus, we sought to assess the susceptibilities of ceftazidime–avibactam and aztreonam–avibactam against 660 carbapenem-nonsusceptible Enterobacteriaceae isolates (472 Klebsiella pneumoniae and 188 Escherichia coli) collected during an earlier Taiwan surveillance study. Agar dilution method was used to determine ceftazidime–avibactam and aztreonam–avibactam susceptibility. Metallo-carbapenemase’s contribution to resistance were investigated with EDTA addition. The in vivo efficacies were evaluated using a Caenorhabditis elegans model. High susceptibility rates were observed for ceftazidime–avibactam and aztreonam–avibactam against the 472 carbapenem-nonsusceptible K. pneumoniae (CnsKP) (85.2% and 95.3%, respectively) and 188 carbapenem-nonsusceptible E. coli (CnsEC) isolates (91.5% and 94.1%, respectively). For non-metallo-carbapenemase producers, the susceptibility rates for ceftazidime–avibactam were 93.6% for CnsKP and 97.7% for CnsEC, whereas only 7.1% CnsKP and 11.1% CnsEC in metallo-carbapenemase producers were susceptible to ceftazidime–avibactam. Of all isolates, 95.3% CnsKP and 94.1% CnsEC were susceptible to aztreonam–avibactam. In C. elegans model, ceftazidime–avibactam and aztreonam–avibactam revealed effective against a blaKPC-producing K. pneumoniae isolate in vivo. Our results propose a positive therapeutic approach for both combinations against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

Evaluation of modified carbapenem inactivation method with CARBA NP, MHT and real-time PCR for detection of carbapenemase-producing Enterobacteriaceae

Background: Carbapenemase-producing Enterobacteriaceae (CPE) are a global emerging threat with high transmissibility, multidrug-resistance and limited treatment options. Reliable detection is important for prevention, containment of these pathogens as well as for antimicrobial stewardship. The modified carbapenem inactivation method (mCIM) is an inexpensive and straightforward phenotypic tool for detecting carbapenemase activity. The aim of this study was to evaluate the performance of mCIM for detecting carbapenemase activity by comparing with CARBA NP, Modified Hodge Test (MHT) and Real-time PCR. Methods and materials: A total of 94 carbapenem-nonsusceptible Enterobacteriaceae comprising of E. coli (n = 43), Klebsiella species (n = 33) and Enterobacter species (n = 18) were analyzed by mCIM, MHT and CARBA NP according to the Clinical and Standard Institute (CLSI) guidelines (M100-S27). In-house Real-time PCR was performed to confirm carbapenemase genes (KPC, NDM, VIM, IMP, OXA-48). The sensitivity and specificity of mCIM, MHT and CARBA NP was determined by comparing the results with Real-time PCR. Klebsiella pneumoniae BAA-1706 strain and Klebsiella pneumoniae ATCC 700603 both carbapenemase non-producers were used as negative controls. Results: Of the ninety-four isolates analyzed by real-time PCR, 47 isolates expressed NDM and 5 isolates expressed OXA. 38 isolates showed co-expression of NDM and OXA. Two showed co-expression of KPC and NDM. One Enterobacter aerogenes showed co-expression of NDM and VIM, while another showed co-expression of NDM and IMP. The sensitivity of mCIM, CARBA NP and MHT were 100%, 84% and 74% respectively. mCIM showed higher sensitivity than CARBA NP & MHT. The mCIM assay perfectly correlated with the genotypic findings of the 94 isolates with 100% sensitivity and specificity for CPE detection. Conclusion: The results of our study show that mCIM is suitable and reliable for detecting carbapenemase-producing Enterobacteriaceae than CARBA NP and MHT. Compared to molecular method, mCIM is less expensive and can be readily adopted in clinical microbiology laboratory.

Identification of AbaR4 Acinetobacter baumannii resistance island in clinical isolates of blaOXA-23-positive Proteus mirabilis.

bla OXA-23 is a class D carbapenemase-encoding gene typical of the Acinetobacter genus. However, its occurrence in the Enterobacteriaceae is uncommon. Here we provide the genome characterization of blaOXA-23-positive Proteus mirabilis. In Singapore, a national surveillance of carbapenem non-susceptible clinical Enterobacteriaceae has enabled the collection of OXA-23 bearing isolates. Three clinical P. mirabilis were whole-genome sequenced using Oxford Nanopore MinION and Illumina platforms. The sequence accuracy of MinION long-read contigs was enhanced by polishing with Illumina-derived short-read data. In two P. mirabilis genomes, blaOXA-23 was detected as two copies, present on the chromosome and on a 60018 bp plasmid. blaOXA-23 was associated with the classic Acinetobacter composite transposon Tn2006, bounded by two copies of ISAba1 bracketing the carbapenemase gene. The Tn2006 itself was embedded within an Acinetobacter baumannii AbaR4 resistance island. In the chromosome, the AbaR4 was found integrated into the comM gene, which is also the preferred 'hotspot' in A. baumannii. In the plasmid, AbaR4 integrated into a putative colicin gene. Our description of an A. baumannii AbaR4 encoding blaOXA-23 in P. mirabilis is to our knowledge the first description of an Acinetobacter resistance island in Proteus and suggests that P. mirabilis may be a reservoir for this class D carbapenemase gene.

Antecedent Carbapenem Exposure as a Risk Factor for Non-Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae and Carbapenemase-Producing Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) can be mechanistically classified into carbapenemase-producing Enterobacteriaceae (CPE) and non-carbapenemase-producing carbapenem nonsusceptible Enterobacteriaceae (NCPCRE). We sought to investigate the effect of antecedent carbapenem exposure as a risk factor for NCPCRE versus CPE. Among all patients with CRE colonization and infection, we conducted a case-control study comparing patients with NCPCRE (cases) and patients with CPE (controls). The presence of carbapenemases was investigated with phenotypic tests followed by PCR for predominant carbapenemase genes. We included 843 unique patients with first-episode CRE, including 387 (45.9%) NCPCRE and 456 (54.1%) CPE. The resistance genes detected in CPEs were blaNDM (42.8%), blaKPC (38.4%), and blaOXA-48-like (12.1%). After adjusting for confounders and clustering at the institutional level, the odds of prior 30-day carbapenem exposure was three times higher among NCPCRE than CPE patients (adjusted odds ratio [aOR], 3.48; 95% confidence interval [CI], 2.39 to 5.09; P < 0.001). The odds of prior carbapenem exposure and NCPCRE detection persisted in stratified analyses by Enterobacteriaceae species (Klebsiella pneumoniae and Escherichia coli) and carbapenemase gene (blaNDM and blaKPC). CPE was associated with male gender (aOR, 1.45; 95% CI, 1.07 to 1.97; P = 0.02), intensive care unit stay (aOR, 1.84; 95% CI, 1.24 to 2.74; P = 0.003), and hospitalization in the preceding 1 year (aOR, 1.42; 95% CI, 1.01 to 2.02; P = 0.05). In a large nationwide study, antecedent carbapenem exposure was a significant risk factor for NCPCRE versus CPE, suggesting a differential effect of antibiotic selection pressure.

1372. In Vitro Activity of Novel Ceftazidime–Avibactam and Aztreonam–Avibactam Combinations Against Carbapenem-Nonsusceptible Enterobacteriaceae Isolates by Phenotype Collected in Latin America From 2014 to 2017 as Part of the INFORM Surveillance Program

BackgroundCarbapenem-nonsusceptible Enterobacteriaceae (CRE) are often multidrug-resistant and infections caused by these organisms are associated with increased morbidity and mortality. The combination of avibactam (AVI), a non-β-lactam/β-lactamase inhibitor of Class A, C, and some D serine β-lactamases, with ceftazidime (CAZ) and aztreonam (ATM) is being developed to treat infections caused by CRE. CAZ-AVI reveals potent in vitro activity against CRE, except those producing metallo-β-lactamases (MBLs), whereas ATM-AVI inhibits growth of both MBL-positive and MBL-negative CRE. We evaluated the in vitro activity of CAZ-AVI and ATM-AVI against Enterobacteriaceae isolates nonsusceptible to meropenem (MEM-NS) collected in 2014–2017 in Latin America through the INFORM global surveillance program.MethodsNonduplicate clinically significant isolates were collected from 29 hospital laboratories located in Argentina, Brazil, Chile, Colombia, Mexico, and Venezuela. Susceptibility testing was performed by CLSI broth microdilution. AVI was tested at a fixed concentration of 4 µg/mL in combination with CAZ and ATM. MEM-NS Eba (MIC >1 µg/mL) were screened for the presence of β-lactamase genes by PCR and sequencing.ResultsFive hundred fifty-seven MEM-NS isolates were identified (440 Klebsiella pneumoniae and 117 isolates of 13 other species). Of these, 441 (79.2%) carried carbapenemases (Cpase) (KPC only, n = 383; MBL only, n = 48; OXA-48-like only, n = 5; KPC and OXA-48-like, n = 2; MBL and GES, n = 2; MBL and KPC, n = 1). CAZ-AVI showed potent in vitro activity against Cpase-positive MBL-negative and Cpase-negative Eba and against all MEM-NS Eba, but was not active against MBL-positive Eba. 100% of MEM-NS Eba were inhibited by ≤8 µg/mL of ATM-AVI. ConclusionCAZ-AVI and ATM-AVI displayed potent in vitro activity against MEM-NS Eba collected in LA. These agents could serve as promising options for treatment of infections caused by CRE.Disclosures K. Kazmierczak, Pfizer Inc.: Consultant, Consulting fee. IHMA, Inc.: Employee, Salary. B. De Jonge, AstraZeneca: Shareholder, Dividends. Pfizer Inc: Employee, Salary. G. G. Stone, Pfizer Inc.: Employee, Salary. AstraZeneca: Former Employee and Shareholder, Salary. D. Sahm, Pfizer Inc.: Consultant, Consulting fee.IHMA, Inc.: Employee, Salary.

Molecular characterization and epidemiology of carbapenem non-susceptible Enterobacteriaceae isolated from the Eastern region of Heilongjiang Province, China

BackgroundThe aim of this study was to elucidate the molecular epidemiology of carbapenem non-susceptible Enterobacteriaceae(CNSE) isolated in the Eastern region of Heilongjiang Province, China, and the mechanism of carbapenem resistance.MethodsA total of 53 CNSE isolates were collected in a grade-3 hospital in Heilongjiang province. Sensitivity to antibiotics was determined using the VITEK-2 Compact automatic system. The modified Hodge test (MHT) and modified carbapenem inactivation test (mCIM) were performed for phenotypic identification. Beta-lactamases gene were detected by Polymerase chain reaction(PCR) and DNA sequencing. The transfer of blaNDM and blaKPC was investigated through conjugation experiment. The clinical data of patients were retrospectively reviewed. Homology of Carbapenem-resistant Klebsiella pneumoniae(CRKP) was conducted by multilocus sequence typing (MLST).ResultsCNSE were highly resistant to the majority of antimicrobial agents. The resistance rate was 100% for first, third, fourth generation cephalosporins and enzyme inhibitor compounds. Gentamicin and tobramycin recorded a resistance rate higher than 80%. Less than 30% resistance was detected for amikacin and levofloxacin. Among CNSE 52(98.1%) and 48(90.6%) of CNSE were positive for mCIM and MHT respectively. There were 42 positive blaKPC genes, three blaNDM-1 genes, three blaNDM-5 genes, one blaNDM-7 gene, and six blaIMP-4 genes. Most isolates harbored multiple drug resistance gene, especially as related to extended-spectrum-β-lactamases, blaSHV, blaTEM and blaCTX-M-15 genes.The resistant gene was transferred into recipient Escherichia coli J53 through conjugation in 21.3% (10/47) of the strains. MLST revealed that ST76 (n = 36) was the most predominant clone, followed by ST896, ST323 and ST11. A new one ST 2946 was identity by this study.ConclusionThe carbapenem resistance phenomenon is alarming and blaKPC-2 is the main resistant gene of CNSE in our hospital. This is the first report of an outbreak caused by blaKPC-2 positive K. pneumoniae ST76 in the Eastern region of Heilongjiang Province, China. Relevant departments should implement infection control and prevention measures to avoid further dissemination of the multi drug-resistant bacteria (MDR).

Carbapenemase-producing Enterobacteriaceae in South Korea: a report from the National Laboratory Surveillance System.

To assess the epidemiology of carbapenemase-producing Enterobacteriaceae (CPE) in South Korea. From 2011 to 2015, 2487 carbapenem-nonsusceptible Enterobacteriaceae were collected through the Korean National Laboratory Surveillance System. Disk-diffusion for antimicrobial susceptibility, PCR/sequencing to detect carbapenemase genes and multilocus sequence typing for molecular epidemiology were carried out. The number of carbapenem-nonsusceptible Enterobacteriaceae was increasing approximately 1.5-fold per year and the proportion of CPEs was exponentially confirmed from 2014. KPC was the most dominant, mostly associated with Klebsiella pneumoniae ST11 and ST307, NDM was the second and OXA-48-like was the third dominant carbapenemases. The IMP, VIM and GES-5 CPEs were identified sporadically. The nation-wide spreads of KPC, NDM and OXA-48-like CPEs were in an alarming epidemiological stage.

Dissemination of bla OXA-370 gene among several Enterobacteriaceae species in Brazil.

OXA-370 is a recently described OXA-48 variant that has only been described in a few Enterobacter spp. and Klebsiella pneumoniae isolates. The purpose of this study is to assess the prevalence of OXA-370-producing isolates in carbapenem-nonsusceptible Enterobacteriaceae recovered from 28 hospitals from Brazil. Real-time PCR was used to determine the presence of bla NDM-1, bla KPC-2, bla VIM-type, bla GES-type, bla OXA-48-like, and bla IMP-type genes. A total of 4,451 Enterobacteriaceae were screened. The gene bla OXA-48-like was detected in 74 (2.5%) isolates, mostly of Enterobacter spp. (44.6% E. cloacae and 2.7% E. aerogenes) and Klebsiella spp. (31.1% K. pneumoniae and 6.7% K. oxytoca), followed by Escherichia coli, (6.7%), Morganella morganii, (2.7%), Citrobacter freundii (1.3%), Proteus mirabilis (1.3%), Providencia stuartii (1.3%), and Serratia spp. (1.3%). These isolates were from five hospitals, 67 (90.5%) from the hospital where the bla OXA-370 was first described. Sequencing of bla OXA-48-like was performed in 52 isolates, including E. cloacae, E. aerogenes, K. pneumoniae, K. oxytoca, E. coli, and C. freundii; all presenting 100% identity with bla OXA-370. PFGE revealed the presence of distinct clones among K. pneumoniae, E. cloacae, K. oxytoca, and E. coli. Susceptibility rates to meropenem, imipenem, and ertapenem among OXA-370-producing isolates were 92.3%, 78.8%, 7.7% respectively; the MIC50 /MIC90 were 0.38/2mg/L and 1/3mg/L for meropenem and imipenem respectively. Overall, antimicrobial susceptibility analysis suggests that OXA-370 lacks carbapenemase activity. Our study demonstrated that the bla OXA-370 gene is disseminated among several Enterobacteriaceae species and clones, indicating a high potential for dissemination.

Factors associated with carriage of carbapenem-non-susceptible Enterobacteriaceaein North-Eastern France and outcomes of infected patients.

Carbapenems are frequently used as a last resort to treat infections caused by multidrug-resistant Gram-negative organisms, thus carbapenem-non-susceptible Enterobacteriaceae (CNSE) is an emerging health threat. To assess risk factors and outcomes of CNSE carriage. We conducted a matched case-control study in six hospitals in North-Eastern France. The controls were patients harbouring carbapenem-susceptible Enterobacteriaceae. Fifty-five cases and 110 controls were included. Most of the CNSE isolates were Enterobacter cloacae and Klebsiella pneumoniae . Carbapenemase production was observed in 40% of isolates and they produced OXA-48 only. CNSE carriage was significantly associated with recent antibiotic use ( P = 0.014), particularly carbapenems ( P = 0.03) and fluoroquinolones ( P = 0.016). A multivariate analysis using conditional logistic regression showed that the presence of concomitant infection(s) (OR: 9.83; 95% CI 3.04-21.39, P = 0.0031), nosocomial infections (OR: 7.84; 95% CI 2.00-12.54, P = 0.0063) and a high age (OR: 1.07; 95% CI 1.01-1.06, P = 0.038) were independently associated with CNSE carriage. Moreover, patients infected with CNSE had worse outcomes: fewer resolved infections at 1 month ( P = 0.02), and they had a higher mortality rate ( P = 0.0004) and longer hospital stays ( P = 0.02). We identified three independent risk factors for CNSE carriage as well as worse outcomes in infected patients in North-Eastern France. This highlights the importance of early detection of CNSE and the need for antimicrobial therapy re-evaluation after bacteriological analysis has been performed.

Development of an algorithm for phenotypic screening of carbapenemase-producing Enterobacteriaceae in the routine laboratory

BackgroundCarbapenemase-producing Enterobacteriaceae (CPE) are difficult to identify among carbapenem non-susceptible Enterobacteriaceae (NSE). We designed phenotypic strategies giving priority to high sensitivity for screening putative CPE before further testing.MethodsPresence of carbapenemase-encoding genes in ertapenem NSE (MIC > 0.5 mg/l) consecutively isolated in 80 French laboratories between November 2011 and April 2012 was determined by the Check-MDR-CT103 array method. Using the Mueller-Hinton (MH) disk diffusion method, clinical diameter breakpoints of carbapenems other than ertapenem, piperazicillin+tazobactam, ticarcillin+clavulanate and cefepime as well as diameter cut-offs for these antibiotics and temocillin were evaluated alone or combined to determine their performances (sensitivity, specificity, positive and negative likelihood ratios) for identifying putative CPE among these ertapenem-NSE isolates. To increase the screening specificity, these antibiotics were also tested on cloxacillin-containing MH when carbapenem NSE isolates belonged to species producing chromosomal cephalosporinase (AmpC) but Escherichia coli.ResultsOut of the 349 ertapenem NSE, 52 (14.9%) were CPE, including 39 producing OXA-48 group carbapenemase, eight KPC and five MBL. A screening strategy based on the following diameter cut offs, ticarcillin+clavulanate <15 mm, temocillin <15 mm, meropenem or imipenem <22 mm, and cefepime <26 mm, showed 100% sensitivity and 68.1% specificity with the better likelihood ratios combination. The specificity increased when a diameter cut-off <32 mm for imipenem (76.1%) or meropenem (78.8%) further tested on cloxacillin-containing MH was added to the previous strategy for AmpC-producing isolates.ConclusionThe proposed strategies that allowed for increasing the likelihood of CPE among ertapenem-NSE isolates should be considered as a surrogate for carbapenemase production before further CPE confirmatory testing.

Heterogeneity of Carbapenem Resistance Mechanisms Among Gram-Negative Pathogens in Lebanon: Results of the First Cross-Sectional Countrywide Study.

Carbapenem-resistant Gram-negative pathogens have progressively disseminated to different countries worldwide, presenting a serious public health concern. The aims of this study were to determine the prevalence of carbapenem resistance in Gram-negative bacteria in Lebanon, to elucidate molecular mechanisms, and to identify genetic relatedness of incriminated strains. Carbapenem nonsusceptible Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas were collected from 11 Lebanese hospitals in 2012. Antimicrobial susceptibility was assessed with phenotypic tests, genes encoding carbapenemases were screened via PCR-sequencing, and genetic relatedness was examined by PGFE and ERIC-PCR. A total of 398 nonrepetitive carbapenem nonsusceptible isolates were studied, of which 44 were Enterobacteriaceae, 142 were A. baumannii, and 212 were Pseudomonas. Among Enterobacteriaceae, 70.4% carried blaOXA-48-like gene on IncL/M-type plasmids, while acquired AmpC cephalosporinases, extended-spectrum-β-lactamases, and efflux-pump were additional contributors to carbapenem resistance. Among A. baumannii, 90% produced OXA-23 and GES-11 and carried insertion sequence ISAba1 upstream and adjacent to blaOXA-23 and blaAcinetobacter-derived cephalosporinases. Among Pseudomonas, 16% harbored VIM-2, 4.2% IMP-2, and 1.4% IMP-1 metallo-β-lactamases. Fingerprint analysis indicated that the spread of OXA-48-like carbapenemases was mostly mediated by horizontal transfer, while OXA-23 and GES-11 diffusion in A. baumannii and VIM-2 diffusion in P. aeruginosa were primarily due to clonal dissemination. This study is the first nationwide investigation of carbapenem resistance in Lebanon, showing low level of resistance in Enterobacteriaceae, and higher levels in A. baumannii and Pseudomonas. With current changes in the region, continuous surveillance of carbapenem resistance is crucial.

The First Enterobacter cloacae Co-Producing NDM and OXA-48 Carbapenemases and Interhospital Spread of OXA-48 and NDM-Producing Klebsiella pneumoniae in Turkey.

The aim of this study was to investigate the occurrence of carbapenemase-producing Enterobacteriaceae. A total of 54 carbapenem nonsusceptible Enterobacteriaceae (CRE) isolates were recovered from clinical samples sent to the Dr. Lutfi Kirdar Kartal Training and Research Hospital from the period 2011 through 2014. Forty-four isolates were Klebsiella pneumoniae (CRKP) and the other 10 were Enterobacter cloacae (CREC).The isolate identifications and antibiotic sensitivity tests were performed using a Vitek2 automatic system. The clonality of isolates was determined using rep-PCR Diversilab. Presence of blaOXA-48, blaNDM, blaVIM, blaIMP, and blaKPC genes were screened using polymerase chain reaction (PCR) with specific primers. CRKP were isolated from blood, urine, wounds, catheter tips, and tracheal aspirate samples; a total 44 isolates were evaluated. All isolates were nonsusceptible to ertapenem/imipenem or meropenem. Eighteen percent of the isolates were resistant to colistin. CREC were isolated from blood, urine, cerebrospinal fluid and sputum; a total of 10 isolates were evaluated. They were resistant to all carbapenems and 90% were resistant to cefoperazone/sulbactam and trimethoprim/sulfamethoxazole, and 50 - 70% isolates were resistant to gentamicin, amikacin, and ciprofloxacin. Thirty-three (75%) OXA-48 producing CRKP were identified. Thirteen (29.5%) were positive and two (4.5%) NDM-producing K. pneumoniae were co-producing OXA-48. Of the ten CREC strains tested, eight were positive for blaNDM, one isolate was positive for blaVIM and another for blaIMP genes. rep-PCR typing revealed the presence of a clonal dissemination in CRKP and CREC in the hospital. To our knowledge, this is the first identification of blaNDM in E. cloacae isolates in Turkey. These findings describe an interhospital spread of CRKP-producing OXA-48 and NDM carbapenemases that started in 2011. Continuous monitoring is necessary to better understand their dissemination in the hospital, which probably occurred as a result of transmission from an environmental reservoir. These findings emphasize the need for intensive surveillance and precautions.

Global Dissemination of blaKPC into Bacterial Species beyond Klebsiella pneumoniae and In Vitro Susceptibility to Ceftazidime-Avibactam and Aztreonam-Avibactam.

The Klebsiella pneumoniae carbapenemase (KPC), first described in the United States in 1996, is now a widespread global problem in several Gram-negative species. A worldwide surveillance study collected Gram-negative pathogens from 202 global sites in 40 countries during 2012 to 2014 and determined susceptibility to β-lactams and other class agents by broth microdilution testing. Molecular mechanisms of β-lactam resistance among carbapenem-nonsusceptible Enterobacteriaceae and Pseudomonas aeruginosa were determined using PCR and sequencing. Genes encoding KPC enzymes were found in 586 isolates from 22 countries (76 medical centers), including countries in the Asia-Pacific region (32 isolates), Europe (264 isolates), Latin America (210 isolates), and the Middle East (19 isolates, Israel only) and the United States (61 isolates). The majority of isolates were K. pneumoniae (83.4%); however, KPC was detected in 13 additional species. KPC-2 (69.6%) was more common than KPC-3 (29.5%), with regional variation observed. A novel KPC variant, KPC-18 (KPC-3[V8I]), was identified during the study. Few antimicrobial agents tested remained effective in vitro against KPC-producing isolates, with ceftazidime-avibactam (MIC90, 4 μg/ml), aztreonam-avibactam (MIC90, 0.5 μg/ml), and tigecycline (MIC90, 2 μg/ml) retaining the greatest activity against Enterobacteriaceae cocarrying KPC and other β-lactamases, whereas colistin (MIC90, 2 μg/ml) demonstrated the greatest in vitro activity against KPC-positive P. aeruginosa This analysis of surveillance data demonstrated that KPC is widely disseminated. KPC was found in multiple species of Enterobacteriaceae and P. aeruginosa and has now become a global problem.

Molecular Characterization of Carbapenem-Nonsusceptible Enterobacterial Isolates Collected during a Prospective Interregional Survey in France and Susceptibility to the Novel Ceftazidime-Avibactam and Aztreonam-Avibactam Combinations.

An interregional surveillance program was conducted in the northwestern part of France to determine the prevalence of carbapenem-nonsusceptible Enterobacteriaceae (CNSE) isolates and their susceptibility to ceftazidime-avibactam and aztreonam-avibactam combinations. Nonduplicate CNSE clinical isolates were prospectively collected from six hospitals between June 2012 and November 2013. MICs of ceftazidime and aztreonam, alone or combined with a fixed concentration of avibactam (4 μg/ml), and those of carbapenems (comparator agents) were determined. MICs of ertapenem in combination with phenylalanine arginine-naphthylamide dihydrochloride (PAβN) were also determined to assess active efflux. Genes encoding carbapenemases, plasmid-mediated AmpC enzymes, extended-spectrum β-lactamases (ESBLs), and major outer membrane proteins (OMPs) were amplified and sequenced. OMPs were also extracted for SDS-PAGE analysis. Among the 139 CNSE isolates, mainly Enterobacter spp. and Klebsiella pneumoniae, 123 (88.4%) were ertapenem nonsusceptible, 12 (8.6%) exhibited reduced susceptibility to all carbapenems, and 4 Proteeae isolates (2.9%) were resistant to imipenem. Carbapenemase production was detected in only two isolates (producing OXA-48 and IMI-3). In contrast, OMP deficiency, in association with AmpCs and/or ESBLs (mainly CTX-M-9, SHV-12, and CTX-M-15), was largely identified among CNSE isolates. The ceftazidime-avibactam and aztreonam-avibactam combinations exhibited potent activity against CNSE isolates (MIC50/MIC90, 1/1 μg/ml and 0.5/0.5 μg/ml, respectively) compared to that of ceftazidime and aztreonam alone (MIC50/MIC90, 512/512 μg/ml and 128/512 μg/ml, respectively). This study reveals the in vitro activity of ceftazidime-avibactam and aztreonam-avibactam combinations against a large collection of porin-deficient enterobacterial isolates that are representative of the CNSE recovered in the northern part of France.

Characterization of Carbapenem-Resistant Enterobacteriaceae with High Rate of Autochthonous Transmission in the Arabian Peninsula.

To establish the role of local transmission versus possible pathogen import due to previous foreign exposure in infections caused by carbapenem non-susceptible Enterobacteriaceae in the Arabian Peninsula, 200 independent isolates collected in 16 hospitals of Saudi Arabia, Kuwait, Oman and the United Arab Emirates were studied. All strains were multidrug resistant; 42.5% of them also qualified as extremely drug resistant. The frequency of various carbapenemases varied according to the participating countries, but in the collection, as a whole, bla NDM-1 was the most frequently encountered carbapenemase gene (46.5%) followed by bla OXA-48-like gene (32.5%). A comparatively high rate (8.9%) of multi-clonal strains carrying both bla NDM and bla OXA-48-like genes in the United Arab Emirates, representing the most resistant subgroup, was encountered. No KPC-expressing isolates were detected. Three major clones of bla NDM-1 carrying Klebsiella pneumoniae of ST152 (n = 22, Saudi Arabia), ST14 (n = 7, United Arab Emirates) and ST147 types (n = 9, Oman) were identified, the latter two clones carrying similar, but not identical HI1b incompatibility type plasmids of >170kb. While from 78.6% of the cases with documented foreign hospitalization bla NDM positive strains were isolated, these strains formed only 25.6% of all the isolates expressing this enzyme. In fact, 56.8% of the NDM, 75.7% of OXA-48-like and 90.9% of VIM positive strains were recovered from patients without documented foreign exposure, neither in the form of travel or prior hospitalization abroad, suggesting a high rate of autochthonous infections. This, considering the extensive links of these countries to the rest of the world, predicts that trends in the local epidemiology of carbapenem resistant strains may increasingly affect the spread of these pathogens on the global scale. These results call for improved surveillance of carbapenem resistant Enterobacteriaceae in the countries of the Arabian Peninsula.

Antimicrobial resistance surveillance of doripenem in China.

To investigate the antibacterial resistance to doripenem in China and to understand the distribution trends of resistant bacteria. All the clinical isolates were collected from hospitals and the susceptibility tests were performed using the agar dilution method recommended by the Clinical Laboratory Standards Institute (CLSI) central laboratory. The susceptibility of the isolates to antimicrobial agents was determined using the CLSI (2014) or European Committee on Antimicrobial Susceptibility Testing (EUCAST) (2013) guidelines. A total of 4047 pathogenic strains were isolated from 18 tertiary hospitals in 18 cities across China between July 2011 and June 2012. MIC results indicated that the vast majority of Enterobacteriaceae maintained high susceptibility to doripenem, with a lower resistance rate (1.9%) than that observed for other drugs tested. In the case of non-fermenting Gram-negative isolates, the resistance rate of Pseudomonas aeruginosa was 16.2%, which was less than that of imipenem and meropenem, and the Acinetobacter baumannii doripenem resistance rate was 67.4%. Doripenem also showed good in vitro activity against other the bacteria tested. This study suggests that the gradual increase in carbapenem nonsusceptible Enterobacteriaceae should be monitored carefully alongside the increasing multidrug-resistant and extensively drug-resistant A. baumannii.

Colistin resistance superimposed to endemic carbapenem-resistant Klebsiella pneumoniae: a rapidly evolving problem in Italy, November 2013 to April 2014

Consecutive non-replicate clinical isolates (n=191) of carbapenem non-susceptible Enterobacteriaceae were collected from 21 hospital laboratories across Italy from November 2013 to April 2014 as part of the European Survey on Carbapenemase-producing Enterobacteriaceae (EuSCAPE) project. Klebsiella pneumonia carbapenemase-producing K. pneumoniae (KPC-KP) represented 178 (93%) isolates with 76 (43%) respectively resistant to colistin, a key drug for treating carbapenamase-producing Enterobacteriaceae. KPC-KP colistin-resistant isolates were detected in all participating laboratories. This underscores a concerning evolution of colistin resistance in a setting of high KPC-KP endemicity.

Comparison of phenotypic methods for the detection of carbapenem non-susceptible Enterobacteriaceae.

BackgroundMultidrug resistance and, in particular, carbapenem resistance is spreading worldwide at an alarming rate, comprehending a variety of bacterial species and causing both nosocomial and community acquired outbursts. Early and efficient detection of infected patients or colonized carriers are mandatory steps in infection control and prevention of multidrug resistance diffusion. The latest EUCAST guidelines for detection of carbapenemase-producing Enterobacteriaceae have set low clinical breakpoints to ensure the maximum detection sensitivity of positive samples. Current workflows involve an initial screening step for species and resistance pattern detection, followed by phenotypic and/or genotypic confirmation. The aim of the present study was to assess the efficiency of six widely used and validated phenotypic assays for the detection of carbapenemases/AmpC in Enterobacteriaceae, to estimate the best workflow in the routine characterization of Enterobacteriaceae isolates.MethodsA panel of 108 non-repetitive Enterobacteriaceae isolates with reduced susceptibility to carbapenems was analyzed by means of 1) Modified Hodge Test, 2) Metallo Beta Lactamase Etest, 3) Double disk test with EDTA, 4) Rosco Diagnostica KPC and MBL confirm kit (RDCK™), 5) AmpC Etest and 6) Cloxacillin inhibition test. Confirmation and validation of results was achieved by genotypic analysis.ResultsThe most accurate identification of resistance determinants was obtained with the combined disc test (Rosco Diagnostica KPC and MBL confirm kit) which had to be coupled with the cloxacillin inhibition test for correct detection of AmpC enzymes. However, in general, phenotypic tests failed to characterize isolates harboring multiple carbapenem resistance determinants, which were successfully assessed only by PCR-based analysis.ConclusionsTo detect and control the spread of pathogens with complicated resistance patterns, both optimized phenotypic analysis (i.e. Rosco Diagnostica KPC and MBL confirm kit coupled with the cloxacillin inhibition test) and genotypic assays are recommended in the routine diagnostic of clinical laboratories.