Common Carbapenemase Research Articles

Phenotypic and molecular characterization of Acinetobacter baumannii isolates causing lower respiratory infections among ICU patients

BackgroundMulti-drug resistant Acinetobacter baumannii has emerged as important nosocomial pathogen associated with various infections including lower respiratory tract. Limited therapeutic options contribute to increased morbidity and mortality. Acinetobacter baumannii has the ability to persist in the environment for prolonged periods. Breach in infection control practices increases the chances of cross transmission between patients and inter/intraspecies transmission of resistance elements. The present prospective work was conducted among patients with lower respiratory tract infections (LRTI) in the intensive care unit (ICU) to study the etiology with special reference to Acinetobacter baumannii and the role of immediate patient environment in the ICU as possible source of infection. Acinetobacter baumannii were characterized for antimicrobial susceptibility, mechanism of carbapenem resistance and virulence determinants. Molecular typing of the clinical and environmental isolates was undertaken to study the probable modes of transmission. Materials and methodsAppropriate respiratory samples from 107 patients with LRTI admitted to ICU during September 2016 to March 2017 were studied for likely bacterial pathogens. Environmental samples (n = 71) were also screened. All the samples were processed using conventional microbiological methods. Consecutive Acinetobacter spp. isolated from clinical and environmental (health care workers and environment from ICU) samples were included in the study. Antimicrobial susceptibility was performed as per CLSI guidelines. Carbapenem resistance, mediated by carbapenemase genes (blaOXA-23-like,blaOXA-24-like,blaOXA-58-like and blaNDM-1) were studied by PCR. Biofilm forming ability was tested phenotypically using microtitre plate method. Pulse Field Gel Electrophoresis (PFGE) was used to study clonality of the clinical and environmental isolates. ResultsThe prevalence of Acinetobacter baumannii was 26.2% (28/107) and 11.26% (8/71) among patients with LRTI and environmental samples respectively. The carbapenem resistance was high, 96.42% (27/28) and 87.5% (7/8) in clinical and environmental isolates respectively. The most common carbapenemase associated with resistance was blaOXA-23-like gene followed by blaNDM-1 among both the clinical and environmental isolates. All isolates were sensitive to colistin (MIC ≤ 1 μg/ml). Biofilm production was observed among all clinical (n = 28) and 87.5% (7/8) of the environmental isolates. Line listing of the cases suggests the occurrence of infections throughout the study period with no significant clustering. On PFGE, 12 clusters were observed and 16/36 isolates were present in one single cluster that included both clinical and environmental isolates which were either carbapenem resistant or sensitive. DiscussionCarbapenem resistant Acinetobacter baumannii (CRAB) is an important cause of LRTI in the ICU. PFGE suggests spread of carbapenem resistant isolates via cross transmission among patients and the environment. The detection of blaNDM-1 gene among Acinetobacter baumannii and existence of carbapenem resistant and sensitive isolates within the same clones suggests horizontal transmission of resistant genes among various bacterial species. The ability of Acinetobacter baumannii to form biofilms may contribute to its persistence in the environment. This along with breach in infection control practices are the likely factors contributing to this transmission. This information can be used to strengthen and monitor infection control (IC) and the hospital cleaning and disinfection practices to prevent spread of resistant organisms within the ICU. Colistin remains drug of choice for management of CRAB.

2064. Rapid Detection of Carbapenmase-Producing Klebsiella pneumoniae (CPK) Directly from Respiratory Secretion and Clinical Characteristics of Patients with CPK from a 1,200-Bed Tertiary Care Hospital in Thailand

BackgroundThere is an unmet need for rapid detection of carbapenemase-producing Klebsiella pneumoniae (CPK) for patient care and infection control. The BD MAX™ CRE (automated PCR, Geneohm, Canada) test is the fully automated sample-to-result platform for detection of resistance genes, blaKPC, blaOXA-48 and blaNDM within 2 hours. It has been validated for using with colonies and rectal swab. We aimed to evaluate its performance for detection of carbapenemases genes directly from respiratory samples of the patients whom K. pneumoniae (KP) were identified.MethodsA total of 169 KP isolates and respiratory samples were collected from patients admitted to King Chulalongkorn Memorial Hospital, Bangkok, Thailand from June 2017 to December 2017. The automated PCR test was performed directly from respiratory specimens. The results were compared with in-house PCR for detection of carbapenemase genes performed on KP colonies isolated from respiratory specimens as our reference method. Patient and clinical characteristics between patients with CPK and non-CPK were also analyzed.ResultsThe prevalence of CPK was 10.6% (18/169 isolates). The automated PCR test had 91.12% accuracy, 66.7% sensitivity (95% CI, 40.9–86.6), 94.0% specificity (95% CI, 88.9–86.6), 57.1% positive predictive value (95% CI, 39.5–73.1) and 95.9% negative predictive value (95% CI, 92.48–97.85). Of 18 isolates, blaOXA-48 was the most common carbapenemase gene (17 isolates; 94.4%), followed by blaNDM-1 (7 isolates; 38.9%). A combination of blaOXA-48 and blaNDM-1 was detected in 6 isolates (33.3%). There were 7 (38.8%) colonizations and 11 (61.1%) infections. The significant risk factors for CPK included post-surgery (P = 0.04) and prior antibiotics exposure (P = 0.04). There was a trend toward higher mortality in patients with CPK albeit not significantly (33% vs. 24.5%, P = 0.41).ConclusionThe automated PCR test has an acceptable accuracy with fair sensitivity for the detection of carbapenemase genes. It is unique that OXA-48 and OXA-48/NDM-1 are the most common carbapenemases in our institute. This diagnostic test may be use for rapid diagnosis or infection control purposes. Exposure to antibiotics associated with colonization or infection with CPK. Patients with CPK had higher mortality.Disclosures All authors: No reported disclosures.

1176. Risk Factors for ESBL Enterobacteriaceae Colonization Identified by Universal Admission Screening in a London Teaching Hospital

BackgroundIndividual risk factors such as antibiotic use and foreign travel are typically associated with ESBL-producing Enterobacteriaceae (ESBL-E) carriage. Few studies have evaluated variations in community demographics or social and material deprivation as risk factors for ESBL-E carriage.MethodsAll admissions to a London hospital group were screened for ESBL-E carriage from rectal swabs for four months in 2015. Patients completed a risk factor questionnaire, and those with a residential postcode in the catchment area were linked to a database containing community-based risk factor data. Risk factors for ESBL-E carriage were determined by binary logistic regression. Isolates that were ESBL-E phenotypically were confirmed by microarray (CT103XL Check-MDR, CheckPoint). The Check-MDR array simlutaneously detects common ESBL and carbapenemase genes, including plasmid-mediated AmpC. Genotypic ESBL-E were split into three groups: CTX-M-15 (Group 1), CTX-M-9 (Group 2), and other (Group 3) for further analysis.ResultsThree hundred sixty (9.0%) of 4,006 patients carried ESBL-E; of which, 1,633 had a residential postcode within the catchment area. In multivariable analysis, risk factors for phenotypic ESBL-E carriage included travel to Asia (OR 5.0, CI 2.5–10.0) or Africa (OR 2.9, CI 1.2–7.0) in the past 12 months, and two or more courses of antibiotics in the past 6 months (OR 2.2, CI 1.5–3.4). Residence in an area with a high proportion of Arab residents and residence in an area with a low proportion of houses with two or more bedrooms were associated with ESBL-E carriage in univariable but not multivariable analysis. Risk factors for the three genotypic ESBL groups were broadly similar to the analysis of phenotypic ESBL carriage, although the number of days traveling abroad in the past 12 months was more associated with Group 1 (CTX-M-15) and Group 3 (other), and older age was associated with Group 3 (other) ESBLs.ConclusionWe linked individual risk factor information with community-based risk factor information, concluding that individual risk factors (including antibiotic use and overseas travel) were more important than community-based risk factors for predicting colonization with ESBL-E at the time of hospital admission. This information is useful when identifying risk groups for targeted screening.Disclosures All authors: No reported disclosures.

1546. Incidence of Carbapenemase-Producing Klebsiella pneumoniae Colonization in Hematopoietic Stem Cell Transplant Recipients in King Chulalongkorn Memorial Hospital (KCMH), Thailand

BackgroundCarbapenemase-producing Klebsiella pneumoniae (CPKP) is an emerging pathogen which had the serious clinical infections, high mortality and difficult to control. Hematopoietic stem cell transplantation (HSCT) patients are particularly susceptible to multidrug-resistant bacteria especially carbapenemase-producing Enterobacteriacae. CPKP infections are an emerging cause of death after HSCT and the mortality rate was reported up to 60%. The major risk factors of CPKP infections were colonization these organism before transplantation. However, in Thailand, the incidence rate of CPKP colonization and clinical outcome in HSCT was limited. Objectives. To determine the incidence rate of CPKP colonizationand risk factors of 30-day-hospital mortality in HSCT patients at King Chulalongkorn Memorial Hospital.MethodsA prospective study was conducted in total of 96 consecutive HSCT patients at King Chulalongkorn Memorial Hospital, Bangkok, Thailand, from July 2016 to March 31, 2018.ResultsIncidence rate of CPKP colonizationin HSCT patients was 22.2% (18/96 patients) and incidence rate of CPKP infections was 5.2% (5/96 patients). Both blaOXA-48 and blaNDM were the most common carbapenemase gene (50%). Patients with CPKP infection were more likely in ICU setting than colonization group. CPKP colonization was more significantly found in urinary specimens (P = 0.029) whereas CPKP infections were common found in respiratory tract, but not significantly (P = 0.583). In CPKP infection group, the 30-day mortality rate was significantly higher than colonization group; 80% (4/5) vs. 23% (3/13), P = 0.047). Using univariable analysis, ICU setting was associated with CPKP infection (RR = 6.27 95% CI, 0.87–45.34) and had a worse outcome.The risk factor associated with 30-day mortality was CPKP infection (RR = 3.47 95% CI 1.17–10.26).ConclusionIn our study, the incidence of CPKP colonization in HSCT patients was 22.2%. The incidence of CPKP infections found only 5.2% in HSCT patients, but there was significantly associated with increased 30-day-hospital mortality.Disclosures All authors: No reported disclosures.

Preliminary study on resistance mechanism and virulence features in carbapenems-resistant Klebsiella pneumoniae from burn patients

Objective: To investigate resistance mechanism and virulence features of carbapenems-resistant Klebsiella pneumoniae (CRKP) and to provide theoretical foundation for the prevention and control of nosocomial infections in burn wards as well as clinical therapy of CRKP. Methods: Strains of Klebsiella pneumoniae isolated from clinical samples of burn patients hospitalized in our unit from February to December 2016 were collected. The diameters of inhibition zone of imipemen or meropenem susceptibility disk of the above strains were≤22 mm. Kirby-Bauer disk diffusion method and automated instrument were applied to test drug resistance of the strains to 17 antibiotics of cephalosporins, aminoglycosides, sulfonamides, quinolones, and carbapenems. Pulsed-field gel electrophoresis (PFGE) was used to analyze homology of the strains. Crystal violet staining was applied to assess ability of biofilm formation of the strains. The hypermucoviscosity (HM) phenotype of the strains was determined by string test. Polymerase chain reaction was used to detect common carbapenemase genes of bla(KPC), bla(IMP), bla(OXA-48), and bla(NDM), capsular serotype genes of K1, K2, and K57, and virulence-associated gene of rmpA. Results: (1) A total of 29 CRKP strains were isolated. The 29 CRKP strains were with high drug resistance rate to carbapenems antibiotics of imipenen, meropenem, and ertapenem, and cephalosporins, aminoglycosides, and quinolones antibiotics, while the strains were with low drug resistance rate to sulfonamides antibiotics and tegafycline. (2) The 29 CRKP strains had 4 types of A, B, C, and D according to PFGE, with 11 strains of type A, 10 strains of type B, 6 strains of type C, and 2 strains of type D. (3) Among the 29 CRKP strains, 25 strains were positive in biofilm formation, with positive rate of 86.2%. (4) None of the 29 CRKP strains was positive in the string test with HM phenotype. (5) All the 29 CRKP strains carried carbapenemase genes. Among the 29 CRKP strains, 11 strains carried both bla(NDM-1) and bla(OXA-48) genes, 1 strain carried both bla(NDM-1) and bla(KPC-2) genes, 12 strains only carried bla(KPC-2) gene, and 5 strains only carried bla(NDM-1) gene. The 79.3% (23/29) strains were completely resistant to 3 kinds of carbapenems antibiotics. (6) Among the 29 CRKP strains, 3 strains carried K2 gene, 2 of which were completely resistant to 3 kinds of carbapenems antibiotics, 1 strain carried rmpA gene, and no strain carried K1 or K57 gene. Conclusions: The detection rate of CRKP isolated from burn patients hospitalized in our unit is high. The strains are mostly extensively drug-resistant bacteria, and the mechanism of drug resistance of which is complicated. There may have clonal spread of CRKP. And appropriate measures should be taken timely and effectively to prevent nosocomial spread of CRKP.

Detection of high-risk carbapenem-resistant Klebsiella pneumoniae and Enterobacter cloacae isolates using volatile molecular profiles

Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are alarming in the clinical setting, as CRE isolates often exhibit resistance to most clinically-available antibiotics. Klebsiella pneumoniae carbapenemase (KPC) is the most common carbapenemase carried by CRE in North America and Europe, frequently detected in isolates of K. pneumoniae, Escherichia coli, and Enterobacter cloacae. Notably, KPC-expressing strains often arise from clonal lineages, with sequence type 258 (ST258) representing the dominant lineage in K. pneumoniae, ST131 in E. coli, and ST78 and ST171 in E. cloacae. Prior studies have demonstrated that carbapenem-resistant K. pneumoniae differs from carbapenem-susceptible K. pneumoniae at both the transcriptomic and soluble metabolomic levels. In the present study, we sought to determine whether carbapenem-resistant and carbapenem-susceptible isolates of K. pneumoniae, E. coli, and E. cloacae produce distinct volatile metabolic profiles. We were able to identify a volatile metabolic fingerprint that could discriminate between CRE and non-CRE with an area under the receiver operating characteristic curve (AUROC) as high as 0.912. Species-specific AUROCs were as high as 0.988 for K. pneumoniae and 1.000 for E. cloacae. Paradoxically, curing of KPC-expressing plasmids from a subset of K. pneumoniae isolates further accentuated the metabolic differences observed between ST258 and non-ST258.

Phenotypic screening and molecular characterization of carbapenemase-producing Gram-negative bacilli recovered from febrile neutropenic pediatric cancer patients in Egypt.

AimInfections with carbapenem-resistant Gram-negative bacteria (GNB) are among the most frequent complications in the immunocompromised cancer patients because of their considerable morbidity and mortality. Therefore, the aim of the current study was to characterize the prevalence of carbapenemase-producing GNB recovered from febrile neutropenic pediatric cancer patients in Egypt.MethodsStandard methods were used for identification, sensitivity testing (Kirby-Bauer and broth microdilution method according to CLSI guidelines). Standard methods were applied for both phenotypic and genotypic detection of the carbapenemase-producing GNB.ResultsA total of 185 GNB were recovered from different clinical specimens, Escherichia (E.) coli (86; 46.48%), followed by Klebsiella spp. (71; 38.37%), Acinetobacter (A.) baumannii (7; 3.78%) and others including Pseudomonas spp., Enterobacter (Ent.) cloacae and Proteus spp. (21; 11.35%). It is a matter of concern that 116 out of 171 enterobacterial isolates (94.15%) showed resistance to three or more antimicrobial classes and were considered multidrug resistant. Additionally, the rate of carbapenem-resistance displayed a worrisome trend as 113 out of 171 enterobacterial isolates (66.08%) and 12 out of 14 non fermenting bacilli (85.71%) showed resistance pattern to at least one of the tested carbapenems. After performing a series of phenotypic tests for initial screening of potential carbapenemase producers, molecular characterization to the 29 extracted plasmids were subjected to PCR (using 5 common carbapenemase primers). The results revealed that blaOXA-48 was the most prevalent 17 (58.62%), followed by blaNDM 8(27.58%), then blaVIM 3 (10.3%) and blaKPC 2 (6.89%).ConclusionThese results are an alarming threat to public health that calls for urgent application of antimicrobial stewardship programs along with routine surveillance for controlling outbreaks.

Risk Factors for Gastrointestinal Colonization and Acquisition of Carbapenem-Resistant Gram-Negative Bacteria among Patients in Intensive Care Units in Thailand.

This study was conducted to investigate the prevalence of and risk factors for colonization and acquisition of carbapenem-resistant (CR) Gram-negative bacteria (GNB) among patients admitted to intensive care units (ICUs) in two tertiary care hospitals in northern Thailand. Screening of rectal swab specimens for CR-GNB was performed on patients at ICU admission and discharge. The phenotypes and genotypes of all isolates were determined. Risk factors were analyzed by logistic regression analysis. The overall carriage rate of CR-GNB at admission was 11.6% (32/275), with the most predominant species carried being Acinetobacter baumannii (n = 15), followed by Klebsiella pneumoniae (n = 9). The risk factor for CR-GNB colonization was hospitalization within the previous 6 months (P = 0.002). During the ICU stay, the rate of CR-GNB acquisition was 25.2% (52/206), with the most predominant species carried being A. baumannii (n = 28) and K. pneumoniae (n = 13). Risk factors associated with CR-GNB acquisition were the use of an enteral feeding tube (P = 0.008) and administration of third-generation cephalosporins (P = 0.032) and carbapenems (P = 0.045). The most common carbapenemase genes in A. baumannii and K. pneumoniae were blaOXA-23/51 and blaNDM, respectively. Patient-to-patient transmission was demonstrated in three cases, resulting in the acquisition of CR A. baumannii (2 cases) and K. pneumoniae (1 case) isolates from other patients who were admitted during the same period of time. This is the first Indochinese study screening patients, examining patients for the carriage of CR-GNB, and further demonstrating the transfer of CR-GNB isolates in ICUs. Our study suggests that effective infection control measures are required to limit the spread of CR-GNB within hospitals.

Carbapenem-resistant Gram-negative bacteria associated with catheter-related bloodstream infections in three intensive care units in Egypt.

We aimed to identify the carbapenem-resistant Gram-negative bacteria (GNB) causing catheter-related bloodstream infections (CRBSI) in intensive care units (ICU) in a tertiary care Egyptian hospital, to study their resistance mechanisms by phenotypic and genetic tests, and to use ERIC-PCR for assessing their relatedness. The study was conducted over 2years in three ICUs in a tertiary care hospital in Egypt during 2015-2016. We identified 194 bloodstream infections (BSIs); 130 (67.01%) were caused by GNB, of which 57 were isolated from CRBSI patients (73.84%). Identification of isolates was performed using conventional methods and MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was done by disc diffusion following CLSI guidelines. Phenotypic detection of carbapenemases enzymes activity was by modified Hodge test and the Carba-NP method. Isolates were investigated for the most common carbapenemases encoding genes blaKPC, blaNDM, and blaOXA-48 using multiplex PCR. Molecular typing of carbapenem-resistant isolates was done by ERIC-PCR followed by sequencing of common resistance genes. The overall rate of CRBSI in our study was 3.6 per 1000 central venous catheter (CVC) days. Among 57 Gram-negative CRBSI isolates, Klebsiella pneumoniae (K. pneumoniae) was the most frequently isolated (27/57; 47.4%), of which more than 70% were resistant to Meropenem. Phenotypic tests for carbapenemases showed that 37.9% of isolates were positive by modified Hodge test and 63.8% by Carba-NP detection. Multiplex PCR assay detected the blaNDM in 28.6% of the isolates and blaKPC in 26.8%, blaNDM and blaKPC were detected together in the same isolate in 5.6%, while blaOXA-48-like were not detected. ERIC-PCR detected limited genetic relatedness between K. pneumoniae isolates. Elevated resistance rates were observed to all antibiotics including carbapenems among K. pneumoniae isolates causing CRBSI. ERIC-PCR showed that the resistant isolates were mainly polyclonal. Our results call for reinforcement of antimicrobial stewardship and measures to prevent CRBSI.

Genomic Epidemiology of Global Carbapenemase-Producing Enterobacter spp., 2008-2014.

We performed whole-genome sequencing on 170 clinical carbapenemase-producing Enterobacter spp. isolates collected globally during 2008–2014. The most common carbapenemase was VIM, followed by New Delhi metallo-β-lactamase (NDM), Klebsiella pneumoniae carbapenemase, oxacillin 48, and IMP. The isolates were of predominantly 2 species (E. xiangfangensis and E. hormaechei subsp. steigerwaltii) and 4 global clones (sequence type [ST] 114, ST93, ST90, and ST78) with different clades within ST114 and ST90. Particular genetic structures surrounding carbapenemase genes were circulating locally in various institutions within the same or between different STs in Greece, Guatemala, Italy, Spain, Serbia, and Vietnam. We found a common NDM genetic structure (NDM-GE-U.S.), previously described on pNDM-U.S. from Klebsiella pneumoniae ATCC BAA-214, in 14 different clones obtained from 6 countries spanning 4 continents. Our study highlights the importance of surveillance programs using whole-genome sequencing in providing insight into the molecular epidemiology of carbapenemase-producing Enterobacter spp.

Clonal diversity and genetic profiling of antibiotic resistance among multidrug/carbapenem-resistant Klebsiella pneumoniae isolates from a tertiary care hospital in Saudi Arabia

BackgroundThe nexus between resistance determinants, plasmid type, and clonality appears to play a crucial role in the dissemination and survival of carbapenem-resistant Klebsiella pneumoniae (CRKP). The incidence of infections involving CRKP in Saudi Arabia is increasing and there is a need for detailed molecular profiling of this pathogen for CRKP surveillance and control.MethodsThe resistance determinants of 71 non-redundant CRKP isolates were investigated by polymerase chain reaction (PCR) and sequencing. Plasmid typing was performed using PCR-based replicon typing and the clonality of isolates was determined by multilocus sequence typing. Capsular polysaccharide synthesis genes and other virulence factors were examined using multiplex PCR. Diversity was calculated using DIVEIN, clonal relationship was determined using eBURST, and phylogenetic analysis was performed using SplitsTree4.ResultsA polyclonal OXA-48 gene alone was the most common carbapenemase detected in 48/71 (67.6%) isolates followed by NDM-1 alone in 9/71 (12.7%) isolates. Coproduction of OXA-48 and NDM-1 was observed in 6/71 (8.5%) isolates. Both carbapenemase genes could be transferred into an Escherichia coli recipient. CTX-M-15 was the most abundant extended-spectrum β-lactamase gene detected in 47/71 (66.2%) isolates, whereas clone-specific CTX-M-14 (ST-199 and -709) was found in 15/71 (21%) isolates. Sixty-seven of 71 isolates were positive for one or more plasmid replicons. The replicons detected were: IncFII; IncFIIK; IncFIA; IncFIB; L/M; IncI1; and IncN. FIIK and L/M were predominant, with 69 and 67% positivity, respectively. All isolates were negative for the magA (K1), rmpA, and K2 genes and presented a non-hypermucoviscous phenotype.ConclusionA polyclonal CRKP reservoir of sequence types (STs)-37, − 199, and − 152 was observed and ST-152 appeared to be a “frequent carrier” of the NDM-1 gene. ST-199, a singleton not previously reported, showed a sequence diversity suggestive of positive selection. A significant association was evident between resistance determinants and the clonal types of K. pneumoniae: all ST-152 isolates were positive for NDM-1 but negative for OXA-48; ST-199 isolates were positive for OXA-48 but negative for NDM-1; and ST-709 and -199 isolates were positive for CTX-M-14. The incidence of certain clonal types in large numbers predicts an outbreak-like situation and warrants stringent surveillance and infection control.

A multicenter study of hospital infection with carbapenem-resistant Acinetobacter baumannii

Objective To investigate the epidemiologic features of interhospital, intrahospital, interdepartmental and intradepartmental infections of carbapenem-resistant Acinetobacter baumannii (CRAB) isolated from clinical specimens. Methods From September 2015 to September 2016, 540 non-repetitive strains of Acinetobacter baumannii were clinically isolated from 38 tertiary hospitals in Anhui Province, of which 460 strains were confirmed by antimicrobial resistance surveillance in Anhui Province. Agar plate dilution method was used to determine the MIC of 16 antibiotics and to select 312 CRAB isolates for drug sensitivity test. Pulsed field gel electrophoresis (PFGE) was used to analyze homology of 145 strains. PCR and clonal sequencing were used to determine the corresponding genotypes. Results All the 312 CRAB strains were multiple antibiotic resistant strains. The drug resistance rates to imipenem and meropenem were 67.9% and 70.6%, respectively. All the 145 strains were divided into 80 different types (PT1-PT80) by PFGE. Only one strain was found in the 60 types and two or more strains were found in the other 20 types. Genotype blaOXA-51 was detected in 145 strains (100%), genotype blaOXA-23 was detected in 134 of strains (92.41%), genotype blaOXA-24 was detected in 1 strain (0.69%), and subtype of blaIMP-4 was detected in 4 strains (2.76%). Genotype blaOXA-58, blaVIM, blaSIM, blaNDM and blaKPC were undetectable. Conclusions Homologous analysis indicates that all 145 strains of CRAB exhibit the interhospital, intrahospital, interdepartmental and intradepartmental transmission. Genotype blaOXA-51 is the inherent drug resistant associated gene in the chromosome of AB and genotype blaOXA-23 is the most common carbapenemase gene in Anhui Province and the main genotypes of resistance transmission. Key words: Acinetobacter baumannii; Drug resistance; Cross infection; Carbapenemase; PFGE

Phenotypic and molecular identification of carbapenemase-producing Enterobacteriaceae - challenges in diagnosis and treatment

Abstract Introduction: Infections due to carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CPCRE) are an emerging global public health threat. The purpose of this study was to investigate phenotypic and genotypic features of CP-CRE strains isolated from hospitalized patients. Material and methods: Between 1st of January - 1st of July 2017, in the Department of Microbiology, “Dr. Constantin Opriş” County Emergency Hospital Baia Mare, Romania, 1110 strains of Enterobacteriaceae were isolated from bronchial secretions, urine, wounds and blood cultures. Bacterial identification and antimicrobial susceptibility tests were performed by conventional methods, Vitek 2 Compact and M.I.C.E. strips. We analysed all Enterobacteriaceae strains non-susceptible to carbapenems according to CLSI 2017 criteria. The modified Hodge test (MHT), the modified carbapenem inactivation method (mCIM) and the combination disks test (KPC, MBL, OXA-48 Confirm kit, Rosco Diagnostica) were used for phenotypic confirmation, whereas a multiplex PCR assay for genes blaKPC, blaNDM and blaOXA-48 was used for genetic confirmation. Results: 19 non-duplicate strains isolated from 16 patients were phenotypically identified as CP-CRE: Klebsiella pneumoniae (n=14), Escherichia coli (n=2), Providencia stuartii (n=2) and Serratia marcescens (n=1). Most strains were isolated from bronchial secretions (n=9). The carbapenem-hydrolizing enzymes were identified by the combination disks test as: KPC (n=9), OXA-48-like (n=5) and MBL (n=5). Molecular confirmation was performed in 18 phenotypically positive isolates with 100% concordant results with mCIM and combination disks test. Discrepant results were noticed with the MHT in case of 4 NDM-producers confirmed by PCR. All CP-CRE strains were resistant to all tested cephems. Three out of 9 K. pneumoniae strains tested against colistin were found resistant. Conclusions: The most common carbapenemase detected was KPC. Therapeutic options were limited in all positive cases. Rapid and reliable detection of CP-CRE is critical for preventing the spread of these pathogens

Antibacterial and Antibiofilm Activity of Grape Seed Extract Against Carbapenem Resistant and Biofilm Producer Enterobacteriaceae

Background and objectives: Carbapenem-resistant and biofilm producing Enterobacteriaceaeare a major health problem. This study was carried to determine the antibacterial and antibiofilm activity of grape seed extract (GSE) against carbapenem-resistant and biofilm producing Enterobacteriaceae isolates. Methods: Antibiotics susceptibility patterns were detected by the disk diffusion method. carbapenem-resistant Enterobacteriaceae (CRE) isolates were screened by carbapenems disks and imipenem minimum inhibitory concentrations (MIC). The biofilm formation was detected by the microplate method. The carbapenemase genes were detected by PCR. The total polyphenolic content of GSE was determinate by Folin Ciocalteu technique. The antibacterial and antibiofilm effects of GSE were tested by the MIC and biofilm inhibitory concentration (BIC), respectively. Results: In this study, total phenolic content of extracted 1 gram of GSE was equivalent to 700 mg gallic acid. Eighteen non-duplicated CRE isolates were selected. All isolates were fosfomycin susceptible. Variable frequency of resistance to the other tested antibiotics was observed. The blaOXA-48 was the most common carbapenemase type. Among 18 isolates, 13 were biofilm producer while GSE inhibited CRE growth at 1024 µg/mL for 15 isolates and 2048 µg/mL for three isolates. Biofilm production was inhibited by GSE in 2000 µg/mL, 4000 µg/mL and 8000 µg/mL after 72 h incubation. Conclusion: The significant antibacterial and antibiofilm effects of GSE suggested GSE as a promising candidate for treatment of infections caused by these organisms.

Overproduction of efflux pumps caused reduced susceptibility to carbapenem under consecutive imipenem-selected stress in Acinetobacter baumannii.

PurposeAcinetobacter baumannii is an important pathogen in the nosocomial infections worldwide. Combining with carbapenemases, efflux pumps and outer membrane proteins (OMPs) have been thought to affect the development of carbapenem resistance in A. baumannii. This study aimed to investigate the contributions of different efflux pumps and OMPs in developing carbapenem resistance in a clinical isolate of A. baumannii and reveal the possible mechanism of overproduction of main efflux pumps.Patients and methodsIn this study, an imipenem-susceptible clinical isolate was identified as A. baumannii and named SZE. Several common carbapenemases were detected by polymerase chain reaction (PCR). Imipenem-selected mutants were selected from SZE by serial subcultivations on Mueller–Hinton agar, and the minimum inhibitory concentration (MIC) was detected. Gene expressions of four families of efflux pumps, five OMPs, and blaOXA-51 were determined by reverse transcription quantitative PCR, and comparisons were made between SZE strain and the imipenem-selected mutants. The adeRS system in SZE and its mutant was sequenced and aligned.ResultsUnder consecutive imipenem-selected stress, the MIC to imipenem increased gradually from 0.125 μg/mL to 8 μg/mL. The effect of resistance inducement was almost neutralized when treated with an efflux pump inhibitor. The expression of efflux pumps, adeB, adeG, and adeJ, was increased by 6.9-, 4.0-, and 2.1-fold in mutants, respectively, compared to SZE. A single mutation (G to A) at position 58 was detected in the regulatory adeRS system and possibly upregulated the adeB expression, and then affected the carbapenem resistance in A. baumannii strains.ConclusionIn conclusion, under consecutive imipenem-selected stress in vitro, A. baumannii strain evolved the ability to reduce susceptibility to a variety of antimicrobials by overproduction of efflux pumps. Especially, the resistance-nodulation-cell division super family and a nucleotide mutant in adeRS regulating system caused the overexpression of adeABC.

Ceftazidime/avibactam susceptibility by three different susceptibility testing methods in carbapenemase-producing Gram-negative bacteria from Australia

Avibactam (AVI) is a novel β-lactamase inhibitor active against class A, class C and some class D β-lactamases. In combination with ceftazidime, AVI may be useful for the treatment of infections due to Gram-negative bacteria producing carbapenemases from these classes; however, susceptibility data for some of the less common carbapenemases are limited. To assess the in vitro activity of ceftazidime/avibactam (CZA), a panel of 50 diverse carbapenemase-producing Gram-negative bacteria collected from clinical samples in Victoria, Australia, containing KPC, GES, SME, OXA-23 and OXA-48-like carbapenemases were tested for susceptibility to CZA using the broth microdilution (BMD), Etest and disk diffusion methods. All isolates were susceptible to CZA. Etest correlated well with BMD, although Etest minimum inhibitory concentrations (MICs) were generally lower than BMD. Disk diffusion correlated moderately well with BMD, with two interpretive errors. This study confirms phenotypic CZA susceptibility in the carbapenemase groups tested, including the less common OXA-23-producing Escherichia coli, SME-producing Serratia marcescens and GES-5-producing Pseudomonas aeruginosa.

Activity of ceftazidime-avibactam against carbapenemase-producing Enterobacteriaceae from urine specimens obtained during the infection-carbapenem resistance evaluation surveillance trial (iCREST) in Spain

The increasing rates of carbapenemase-producing Enterobacteriaceae (CPE) represent an important threat to health care systems and treatment of CPE infections is a challenge. The aim of the infection-carbapenem resistance evaluation surveillance trial (iCREST) was to determinate the prevalence of CPE in urine specimens in Spain and to evaluate the in vitro activity of ceftazidime-avibactam. Urine specimens (n = 11 826) were included and activity of ceftazidime-avibactam and comparators were investigated by broth microdilution in CPE. Carbapenemases were characterised by polymerase chain reaction (PCR) and sequencing as well as by whole genome sequencing (WGS). Overall prevalence of CPE was 1.6%. OXA-48 was the most prevalent (86.8%), followed by KPC (6.9%), VIM (4.8%), NDM (1.1%) and IMP (0.6%) carbapenemases. Klebsiella pneumoniae was the most common carbapenemase producer (87.8%). An uncommon carbapenemase type (IMP-8) in Spain was identify by WGS in an Enterobacter cloacae isolate, reinforcing the utility of surveillance programmes as effectives tools to detect unexpected genes that encode antimicrobial resistance. Ceftazidime-avibactam showed 100% susceptibility in KPC and OXA-48 producers and the rates of susceptibility in CPE non-susceptible to ceftazidime or meropenem were 92.1% and 96.9%, respectively. Ceftazidime-avibactam could be considered an adequate treatment option for urinary tract infections caused by KPC and OXA-48 producers.

The emergence of carbapenem-resistant Klebsiella pneumoniae isolates producing OXA-48 and NDM in the Southern (Asir) province, Saudi Arabia.

Objectives:To identify the prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and the most common types of cabapenemases among CRKP in the Southern (Asir) province hospitals, Saudi Arabia.Methods:The cross-sectional study was conducted between late April and September in 2015. A total of 54 Klebsiella pneumoniae (K. pneumoniae) isolates with reduced sensitivity to carbapenems were obtained from various clinical specimens of the 2 largest hospitals in the Southern province. Minimum inhibitory concentrations (MICs) of carbapenems were confirmed using E-test. Molecular detection of the most common carbapenemase genes (blaIMP, bla-carbapenem-hydrolyzing oxacillinase [OXA-48], blaVIM, bla-New Delhi metallo-ß-lactamas [NDM], and blaKPC) was performed using multiplex-polymerase chain reaction.Results:The current study found that increasing age and intensive care unit admission were associated with CRKP isolation. The major type of carbapenemases was OXA-48 with 81.5% (n=44) and it seems to reach an endemic level. New Delhi metallo-ß-lactamas (NDM) was the second most frequent carbapenemase by 7.4% (n=4) of isolates while Verona integron-encoded metallo-ß-lactamase (VIM) was reported only in one isolate.Conclusion:Saudi Arabia receives large numbers of visitors and migrant workers from OXA-48 and NDM endemic countries such as Turkey, India, and Pakistan every year.

Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria.

PURPOSE:Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB.MATERIALS AND METHODS:The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR.RESULTS:A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-β-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes.CONCLUSION:The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.

Identifying Spectra of Activity and Therapeutic Niches for Ceftazidime-Avibactam and Imipenem-Relebactam against Carbapenem-Resistant Enterobacteriaceae

We determined imipenem, imipenem-relebactam, ceftazidime, and ceftazidime-avibactam MICs against 100 CRE isolates that underwent whole-genome sequencing. Klebsiella pneumoniae carbapenemases (KPCs) were the most common carbapenemases. Forty-six isolates carried extended-spectrum β-lactamases (ESBLs). With the addition of relebactam, imipenem susceptibility increased from 8% to 88%. With the addition of avibactam, ceftazidime susceptibility increased from 0% to 85%. Neither imipenem-relebactam nor ceftazidime-avibactam was active against metallo-β-lactamase (MBL) producers. Ceftazidime-avibactam (but not imipenem-relebactam) was active against OXA-48-like producers, including a strain not harboring any ESBL. Major OmpK36 porin mutations were independently associated with higher imipenem-relebactam MICs (P < 0.0001) and showed a trend toward independent association with higher ceftazidime-avibactam MICs (P = 0.07). The presence of variant KPC-3 was associated with ceftazidime-avibactam resistance (P < 0.0001). In conclusion, imipenem-relebactam and ceftazidime-avibactam had overlapping spectra of activity and niches in which each was superior. Major OmpK36 mutations in KPC-K. pneumoniae may provide a foundation for stepwise emergence of imipenem-relebactam and ceftazidime-avibactam resistance.