Presence and characterization of blaNDM-1-positive carbapenemase-producing Klebsiella pneumoniae from outpatients in Thailand

BackgroundPresently, community-associated carbapenemase-producing Enterobacterales (CPE) remains largely unknown and require public attention. This study aimed to investigate the presence of CPE from outpatients in Thailand.MethodsNon-duplicate stool (n = 886) and urine (n = 289) samples were collected from outpatients with diarrhea and urinary tract infection, respectively. Demographic data and characteristics of patients were collected. Isolation of CPE was performed by plating enrichment culture on agar supplemented with meropenem. Carbapenemase genes were screened by PCR and sequencing. CPE isolates were phenotypically and genotypically characterized.ResultsFifteen samples (1.3%, 14 stool and 1 urine) yielded bla_NDM-1-positive carbapenemase-producing Klebsiella pneumoniae (CPKP). Additional resistance to colistin and tigecycline was observed in 53.3% and 46.7% of isolates, respectively. Age >60 years was identified as a risk factor for patients with CPKP (P < 0.001, adjusted odds ratio = 11.500, 95% confidence interval = 3.223–41.034). Pulsed field gel electrophoresis revealed genetic diversity of CPKP isolates; however, clonal spread has been observed. ST70 (n = 4) was common, followed by ST147 (n = 3). bla_NDM-1 from all isolates were transferable and mainly resided on IncA/C plasmid (80%). All bla_NDM-1 plasmids remained stable in bacterial host for at least 10 days in antibiotic-free environments, regardless of replicon types.ConclusionThis study demonstrates that the prevalence of CPE among outpatients in Thailand remains low and the spread of bla_NDM-1-positive CPKP may be driven by IncA/C plasmid. Our results emphasize the need for a large-scale surveillance study to limit further spread of CPE in community.     

Alcaligenes faecalis metallo-β-lactamase in extensively drug-resistant Pseudomonas aeruginosa isolates

ObjectivesTo characterize Alcaligenes faecalis metallo-β-lactamase (MBL) AFM-2 and AFM-3 from clinical Pseudomonas aeruginosa isolates NDTH10366, NDTH9845 and WTJH17.MethodsClinical isolates were whole-genome sequenced using the Illumina and Oxford Nanopore platforms. MICs of clinical isolates and transformants containing MBL genes were determined using broth microdilution methods. Kinetic parameters of purified AFM and NDM-1 were measured using a spectrophotometer. The AFM structure was modelled with SWISS-MODEL.ResultsNDTH10366 and NDTH9845 were extensively drug-resistant (XDR) isolates carrying bla_AFM-2 and multiple copies of bla_KPC-2, whereas WTJH17 was an XDR isolate carrying bla_AFM-3. The plasmid-borne bla_AFM-2 and bla_AFM-3 genes are associated with a novel ISCR element, ISCR29. AFM-2 and AFM-3, differing from AFM-1 by one amino acid substitution each, shared 86.2% and 86.6% amino acid sequence identity with NDM-1, respectively. Phylogenetic analysis confirmed the close relationship between AFM and NDM. Expression of AFM and NDM-1 under their native promoters in DH5α and PAO1 led to elevated MICs for all tested β-lactams except aztreonam. Comparable catalytic abilities were observed for AFM and NDM-1 when hydrolysing nitrocefin, cefepime, imipenem and biapenem, whereas for other tested β-lactams AFM displayed weaker enzymatic activities. Modelling AFM structure revealed a characteristic αβ/βα fold with two zinc-binding active sites.ConclusionsAFM from clinical P. aeruginosa isolates demonstrated β-lactamase activity comparable to NDM-1. Co-carriage of bla_AFM and blaKPC renders clinical P. aeruginosa isolates non-susceptible to all antipseudomonal β-lactams. The association of bla_AFM genes with translocatable genetic elements and plasmids highlights their concerning potential for dissemination.     

Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014–2018 national laboratory surveillance

ObjectivesCarbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system.MethodsMeropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS).ResultsIn total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with bla_OXA-48 being predominant (38%, 336/892), followed by bla_NDM-1 (16%, 145/892). For the first time in the Netherlands, bla_OXA-181, bla_OXA-232 and bla_VIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse.ConclusionsThe CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are bla_OXA-48 and bla_NDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem.     

The evaluation of vancomycin-resistant enterococci and carbapenamase producing Klebsiella colonization among ICU-Hospitalized Patients

BackgroundMulti-drug resistant organisms, especially Vancomycin-Resistant Enterococcus (VRE) and Carbapenam Resistant Klebsiella pneumoniae (KPC), are serious health threat. Early detection of resistant bacteria colonization among patients in intensive care units (ICUs) not only enables effective treatment but more importantly prevents disease and limits transmission. Therefore, we aimed to to assess the frequency of VRE and KPC colonization via rectal swab sampling.MethodsThe study was carried out in ICUs of a tertiary hospital. Two rectal swab samples were collected within the first 24 hours of admission and another one was taken every subsequent 15 days to test for for VRE and KPC carriage.ResultsA total 316 rectal swab samples taken from 230 patients. Forty-seven patients were screened at least 2 times. 183 patients were not further screened due to discharge, exitus or transfer to other wards. Thirty-six patients (16%) were determined to be VRE (+). The most frequently isolated strain was E. faecium (80.5%) and its most common genotype was VanA (87.5%). Seven patients (3%) were identified as KPC (+). OXA-48 type crbapenamase was confirmed in all KPC isolates.ConclusionThis study shows that VRE and KPC colonization continues to be a serious threat in ICUs.     

Prevalence of faecal carriage of Carbapenemase Producing Enterobacteriaceae in healthy Indian subjects from the community

PurposeFaecal carriage of carbapenemase-producing Enterobacterales (CPE) has been extensively investigated in hospitalized patients, but limited data is available on the carriage rate in healthy individuals in India.MethodsA total of 1000 stool samples were screened for CPE from healthy individuals in Chennai (n ?= ?50), Hyderabad (n ?= ?184) and Mumbai (n ?= ?766). Diluted stool samples were cultured on chromID CARBA SMART plates. Growing colonies were screened for CPE by RAPIDEC® CARBA NP Test and minimum inhibitory concentration (MIC) of imipenem by E-Test. PCR was performed for confirmation of CPE genes.ResultsOut of the 1000 stool samples tested, 6.1% were positive for CPE. A total of 64 carbapenem resistant isolates (56 ?E.coli, 4 Klebsiella pneumoniae, 3 Enterobacter cloacae and 1 Citrobacter freundii) were recovered from ChromID CARBA SMART biplate. Carbapenemase production was identified in 57/64 isolates by RAPIDEC® CARBA NP test. PCR analysis showed 28 bla_NDM-1 and 33 bla_OXA48. Three remaining isolates (2 ?E.coli, 1 ?K.pneumoniae) were negative for the tested carbapenemase genes. Interestingly, out of these 61 PCR positive isolates, 49.1% displayed imipenem MIC within the susceptibility range on the basis of CLSI interpretative criteria.ConclusionsFaecal carriage of CPE among healthy individuals was 6.1%. Comprehensive measures to improve the sanitation scenario and implementation of National AMR action plan are needed to prevent further generation and dissemination of carbapenem resistant Enterobacterales (CRE).     

Bloodstream infection caused by KPC-producing Klebsiella pneumoniae resistant to ceftazidime/avibactam: epidemiology and genomic characterization

ObjectivesThe aim of this study was to evaluate the incidence of ceftazidime/avibactam resistance among Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) strains isolated from patients with bloodstream infection.MethodsWe collected 120 carbapenemase producing Enterobacteriaceae (CPE) strains from unique patients hospitalized in two Italian hospitals between January 2018 to February 2019. Strains were phenotypically characterized for the type of carbapenemase production and susceptibility to ceftazidime/avibactam. Ceftazidime/avibactam-resistant strains were characterized by whole-genome sequencing.ResultsDuring the study period, we characterized 105 (87.5%) KPC producers among a total of 120 CPE strains. Ceftazidime/avibactam resistance was found in three KPC-Kp strains isolated from patients with no history of previous ceftazidime/avibactam-based treatment. Of note, two out of three ceftazidime–avibactam-resistant KPC-Kp were also resistant to meropenem/vaborbactam. Genomic characterization showed that a ceftazidime/avibactam-resistant KPC-Kp harboured a mixed population with D179Y mutated KPC-2, while the other two ceftazidime–avibactam-resistant KPC-Kp possessed non-functional ompK35-ompK37 and mutated ompK36 porins associated with higher copy number of bla_KPC gene.ConclusionsOur results showed that incidence of ceftazidime/avibactam resistance emerged in KCP-Kp strains independently from previous antimicrobial exposure. Resistance to ceftazidime/avibactam was associated with mutations within the bla_KPC gene or porin deficiency associated with higher bla_KPC copy number and is also related to the meropenem/vaborbactam resistance.     

The molecular and epidemiological characteristics of carbapenemase-producing Enterobacteriaceae isolated from children in Shanghai,China, 2016–2021

BackgroundWe isolated the carbapenemase-producing Enterobacteriaceae (CPE) strains from children during 2016–2021 in Shanghai, China and investigated the antimicrobial resistance, molecular and epidemiological features of these isolates.MethodsAntimicrobial susceptibility tests were performed to confirm the carbapenem resistance. Carbapenemase production was assessed by the rapid phenotypic identification of five major carbapenemases (KPC, NDM, VIM, IMP, and OXA-48), which were further confirmed by PCR amplification and sequencing. Multilocus sequence typing (MLST) was conducted for phylogenetic analyses.ResultsA total of 320 CPE strains were collected from 2016 to 2021, consisting of carbapenemase-producing Klebsiella pneumoniae (CP-Kpn, 55.0%), Escherichia coli (CP-Eco, 24.5%) and Enterobacter cloacae (CP-Ecl, 20.4%) and others (2, 0.1%). NDM was the primary carbapenemase (67.6%) in children, followed by KPC(26.4%), IMP(5.3%) and OXA-48 (0.6%). The minimum inhibitory concentration (MIC) for imipenem has been increasing from 2016 to 2021. NDM and KPC isolates are high resistant while IMP strains show the lower resistant to imipenem. Invasive infection accounted for 10.7% of CPE-related infections and was mainly caused by CP-Kpn (70.6%). NDM-Kpn was detected in 51.8% of infants (70.8% of neonates), while KPC-Kpn was mainly isolated from non-infants (56.3%～64.3%). ST11 was the primary clone (64.6%) of KPC-Kpn and presented an increasing trend from 2016 to 2021.ConclusionNDM is widely prevalent and transfers among CPE strains in children. NDM-Kpn shows the most serious threat to infants, especially to neonates. High-risk clone of ST11 KPC-Kpn should be paid more attention and monitored continuously in children.     

Evaluation of the Oxoid Brilliance™ CRE Agar for the detection of carbapenemase-producing Enterobacteriaceae

The adequate detection of carbapenemase-producing Enterobacteriaceae (CPE) is essential for adequate antibiotic therapy and for infection control purposes, especially in an outbreak setting. Selective agars play an important role in the detection of CPE. The Oxoid Brilliance? CRE Agar (Thermo Fisher Scientific) was evaluated for the detection of CPE using 255 non-repetitive Enterobacteriaceae isolates, including 95 CPE (36 KPC, 4 KPC plus VIM, 4 NDM, 6 GIM, 20 VIM, and 25 OXA-48-producing isolates). The sensitivity of the CRE agar for the detection of CPE was 94 % (89/95), but differed per carbapenemase gene (100 % for KPC, NDM, and GIM, 90 % for VIM, and 84 % for OXA-48-producing isolates). The specificity of the CRE agar was 71 %, due to the growth of AmpC- and/or ESBL-producing isolates. The CRE agar is a sensitive tool for the detection of KPC and metallo-carbapenemase-producing Enterobacteriaceae, although the detection of OXA-48 producers is less optimal. The relatively low specificity requires confirmation of carbapenemase production for isolates recovered from the CRE agar.     

Does the presence of Klebsiella pneumoniae carbapenemase and New Delhi metallo-β-lactamase-1 genes in pathogens lead to fatal outcome?

Introduction: Infections due to multidrug-resistant (MDR) pathogens are a medical challenge. There is considerable apprehension among clinicians regarding pathogens reported as carrying New Delhi metallo-β-lactamase-1 (NDM) and Klebsiella pneumoniae carbapenemase (KPC) genes from their patients. In the face of extremely high rates of antimicrobial resistance, it is essential to gauge the clinical significance of isolation of pathogens carrying these genes from clinical samples. This study compares the outcome of patients infected with pathogens carrying NDM/KPC genes versus those without these genes. Methods: The study was conducted over a 1-year period at a Level-1 trauma centre. Hospital-acquired infections were diagnosed on the basis of CDC’s criteria. The correlation of isolation of a multi-resistant pathogen carrying KPC or NDM genes with the clinical outcome was ascertained. Results: A total of 276 consecutive patients admitted to the Intensive Care Units/wards of the JPNA Trauma Centre were included in this study. Of the 371 isolates recovered from these patients, 116 were from patients who had a fatal outcome. The difference in prevalence of bla_NDM and bla_KPC was not significant in any genera of Gram-negative pathogens isolated from patients who survived versus those who had a fatal outcome. Conclusion: Isolation of MDR pathogens carrying NDM/KPC genes from clinical samples is not always a harbinger of a fatal outcome. Efforts should be made to prevent cross-transmission of these pathogens.     

Disparities between Uptake of Germline BRCA1/2 Gene Tests and Implementation of Post-test Management Strategies in Epithelial Ovarian,Fallopian Tube,or Primary Peritoneal Cancer Patients

BackgroundTo assess the rate of germline BRCA gene tests in epithelial ovarian cancer (EOC) patients and uptake of post-test risk management strategies in BRCA1/2-mutated patients.MethodsInstitutional databases were searched to identify patients who were diagnosed with epithelial ovarian, fallopian tube, or primary peritoneal cancer (EOC) between 2009 and 2019 in two academic hospitals. Retrospective review on medical records was performed to collect clinico-pathologic variables, including performance of germline BRCA gene test and its results, as well as conduct of breast cancer screening tests and cascade testing. If annual mammography +/− breast ultrasonography was performed, it was considered that regular breast cancer surveillance was done.ResultsA total of 840 women with EOC were identified during the study period. Of these, 454 patients (54.0%) received BRCA gene testing and 106 patients (106/454, 23.3%) were positive for BRCA1/2 mutations. The rate of BRCA tests has markedly increased from 25.8% in 2009-2012 to 62.7% in 2017-2019. Among the 93 patients with BRCA1/2 mutation without previous personal breast cancer history, 20 patients (21.5%) received annual mammography with or without breast ultrasonography for regular surveillance. Among the 106 BRCA1/2-mutated EOC patients, cascade testing on family members was performed only in 13 patients (12.3%).ConclusionAlthough BRCA1/2 gene tests have been substantially expanded, the uptake of post-test risk management strategies, including breast cancer screening for BRCA1/2-mutated patients and cascade testing for family members, has remained low. Strategies to increase its uptake and education about the importance of post-test risk managements are needed.     

A 5-day course of oral antibiotics followed by faecal transplantation to eradicate carriage of multidrug-resistant Enterobacteriaceae: a randomized clinical trial

ObjectivesIntestinal carriage with extended spectrum β-lactamase Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) can persist for months. We aimed to evaluate whether oral antibiotics followed by faecal microbiota transplantation (FMT) can eradicate intestinal carriage with ESBL-E/CPE.MethodsRandomized, open-label, superiority trial in four tertiary-care centres (Geneva (G), Paris (P), Utrecht (U), Tel Aviv (T)). Non-immunocompromised adult patients were randomized 1: 1 to either no intervention (control) or a 5-day course of oral antibiotics (colistin sulphate 2 × 10⁶ IU 4×/day; neomycin sulphate 500 mg 4×/day) followed by frozen FMT obtained from unrelated healthy donors. The primary outcome was detectable intestinal carriage of ESBL-E/CPE by stool culture 35–48 days after randomization (V4). ClinicalTrials.gov NCT02472600. The trial was funded by the European Commission (FP7).ResultsThirty-nine patients (G = 14; P = 16; U = 7; T = 2) colonized by ESBL-E (n = 36) and/or CPE (n = 11) were enrolled between February 2016 and June 2017. In the intention-to-treat analysis 9/22 (41%) patients assigned to the intervention arm were negative for ESBL-E/CPE at V4 (1/22 not receiving the intervention imputed as positive) whereas in the control arm 5/17 (29%) patients were negative (one lost to follow up imputed as negative) resulting in an OR for decolonization success of 1.7 (95% CI 0.4–6.4). Study drugs were well tolerated overall but three patients in the intervention group prematurely stopped the study antibiotics because of diarrhoea (all received FMT).ConclusionsNon-absorbable antibiotics followed by FMT slightly decreased ESBL-E/CPE carriage compared with controls; this difference was not statistically significant, potentially due to early trial termination. Further clinical investigations seem warranted.     

Gastrointestinal colonization by KPC-producing Klebsiella pneumoniae following hospital discharge: duration of carriage and risk factors for persistent carriage

The natural history of KPC-producing Klebsiella pneumoniae (KPC KP) carriage is unknown. We aimed to examine the duration of KPC KP carriage following hospital discharge and to study the risk factors for persistent carriage. A cohort of 125 KPC KP carriers was followed monthly for between 3 and 6 months after discharge from an acute-care hospital. Rectal swabs and data were collected at baseline and at each visit. KPC KP was detected by culture and direct bla_KPC PCR. Acquisition time was regarded as the earliest date of KPC KP isolation. Resolution of carriage was defined as a negative KPC KP test in at least two consecutive samples. Analyses were separated for recent (<4 months) (REC, 75 patients) and remote (≥4 months) (REM, 50 patients) acquisition groups. Risk factors for persistent carriage were examined by survival analyses for the REC group and by prevalence methods for the REM group. The mean age of patients was 67.5 years and 49.6% were male. Forty-six (61%) patients in the REC group and 14 (28%) in the REM group were persistent carriers (p < 0.001). A significant risk factor for persistent carriage identified in both the REC and REM groups was the presence of any catheter (p < 0.05). Unique risk factor groups included long-term care facility (LTCF) residence (p < 0.01) and a low functional status as measured by the Barthel’s index (p < 0.05) in the REC group and high Charlson’s score in the REM group (p < 0.05). Out of the entire 100 patients who had at least one negative sample, only 65 remained negative on subsequent cultures. In conclusion, persistent carriage of KPC KP is associated with catheter use and a low functional status; it is more common in patients with recent acquisition and is related to LTCF stay. A single negative KPC KP test is insufficient to exclude persistent carriage.     

Carbapenemases in Klebsiella pneumoniae and Other Enterobacteriaceae: an Evolving Crisis of Global Dimensions

Summary: The spread of Enterobacteriaceae, primarily Klebsiella pneumoniae, producing KPC, VIM, IMP, and NDM carbapenemases, is causing an unprecedented public health crisis. Carbapenemase-producing enterobacteria (CPE) infect mainly hospitalized patients but also have been spreading in long-term care facilities. Given their multidrug resistance, therapeutic options are limited and, as discussed here, should be reevaluated and optimized. Based on susceptibility data, colistin and tigecycline are commonly used to treat CPE infections. Nevertheless, a review of the literature revealed high failure rates in cases of monotherapy with these drugs, whilst monotherapy with either a carbapenem or an aminoglycoside appeared to be more effective. Combination therapies not including carbapenems were comparable to aminoglycoside and carbapenem monotherapies. Higher success rates have been achieved with carbapenem-containing combinations. Pharmacodynamic simulations and experimental infections indicate that modification of the current patterns of carbapenem use against CPE warrants further attention. Epidemiological data, though fragmentary in many countries, indicate CPE foci and transmission routes, to some extent, whilst also underlining the lack of international collaborative systems that could react promptly and effectively. Fortunately, there are sound studies showing successful containment of CPE by bundles of measures, among which the most important are active surveillance cultures, separation of carriers, and assignment of dedicated nursing staff.     

Emergence of New Delhi metallo-beta-lactamase 1 and other carbapenemase-producing <Emphasis Type="Italic">Acinetobacter calcoaceticus-baumannii</Emphasis> complex among patients in hospitals in Ha Noi,Viet Nam

Acinetobacter baumannii is an important cause of multidrug-resistant hospital acquired infections in the world. Here, we investigate the presence of NDM-1 and other carbapenemases among carbapenem-resistant A. baumannii isolated between August 2010 and December 2014 from three large hospitals in Hanoi, Vietnam. We identified 23/582 isolates (4 %) (11 from hospital A, five from hospital B, and seven from hospital C) that were NDM-1 positive, and among them 18 carried additional carbapenemase genes, including seven isolates carrying NDM-1, IMP-1, and OXA-58 with high MICs for carbapenems. Genotyping indicated that NDM-1 carrying A. baumannii have expanded clonally in these hospitals. Five new STs (ST1135, ST1136, ST1137, ST1138, and ST1139) were identified. One isolate carried NDM-1 on a plasmid belonging to the N-repA replicon type; no NDM-1-positive plasmids were identified in the other isolates. We have shown the extent of the carbapenem resistance and the local clonal spread of A. baumannii carrying NDM-1 in these hospitals; coexistence of NDM-1 and IMP-1 is reported for the first time from Vietnam here, and this will further seriously limit future therapeutic options.     

The Israeli national policy for discontinuation of isolation of carbapenem-resistant Enterobacterales carriers by carbapenemase type: a retrospective cohort study

ObjectivesThe Israeli national policy for containing carbapenemase-producing Enterobacterales (CPE) includes a protocol allowing for discontinuation of carrier status following spontaneous decolonization. We examined the strategy's effectiveness based on carbapenemase type.MethodsWe performed a retrospective cohort study comparing individuals colonized with KPC- or NDM-producing Enterobacterales who underwent the process of isolation discontinuation. The primary outcome was reversion of carrier status, i.e. re-identification of the same CPE species following isolation discontinuation. We used survival analysis to estimate overall hazard ratio and performed competing-risks analysis using a Fine–Gray subdistribution hazard model and cause-specific hazard ratios.ResultsBetween 1 January 2006 and 1 January 2019 we identified 1694 individuals who met inclusion criteria, including 1337 (78.9%) carriers of KPC-producing Enterobacterales, 305 (18.0%) carriers of NDM-producing Enterobacterales and 52 (3.1%) carriers of dual KPC-/NDM-producing Enterobacterales. A total of 134 individuals (7.9%) had reversion of carrier status: 9.1% (121/1337) and 4.3% (13/305) of individuals with KPC- and NDM-producing Enterobacterales, respectively. The subdistribution hazard ratio of status reversion was not increased among carriers of NDM producers compared with KPC producers (0.567, 95% CI 0.320–1.000], p 0.052). Cause-specific hazard ratios yielded similar results (0.522, 95% CI 0.291–0.937, p 0.029.ConclusionsCarriage of NDM-producing Enterobacterales was not associated with higher rates of reversion to carrier status following spontaneous decolonization than was carriage of KPC-producing Enterobacterales.     

Evaluation of Two Phenotypic Screening Tests for Carbapenemase-Producing Enterobacteriaceae

We evaluated the performance of two rapid tests for detection of carbapenemase-producing Enterobacteriaceae (CPE) strains. The sensitivities and the specificities were 97.6% and 94.4% for the Rapid CARB Screen and 98.8% and 93.1% for the KPC/MBL & OXA-48 Confirm tests, providing the usefulness of these tools for screening CPE in microbiology wards.     

Reliability of carbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) for detection of OXA-48-like and NDM-1

PurposeCarbapenem inactivation method (CIM) and modified carbapenem inactivation method (mCIM) were recently developed for rapid detection of carbapenemase producing Gram negative bacilli (CP-GNB). In this study we compared the ability of modified Hodge test (MHT), CIM and mCIM to identify CP-GNB in Oman and India.MethodsFifty fully characterized and genotyped CP-GNB (26 OXA-48-like, 2 NDM-1 from Oman and 22 NDM-1 from India) and 8 AmpC as controls in India were subjected to MHT, CIM, mCIM and mCIM with in-house modifications. Wilcoxon paired test and receiver operating characteristics (ROC) were utilised for statistical analysis.ResultsIsolates were predominantly OXA-48-like genes producing Klebsiella pneumoniae from Oman and NDM-1 producing Escherichia coli from India. MHT was positive in all except one OXA-48-like producers and in 70.8 ​% of the NDM-1 isolates. The sensitivity of CIM in detecting 0XA-48 like and NDM-1 carbapenemases were 39.2% and 87.5% respectively. mCIM at 4 ​h detected 92.3 ​% and 79.1% of 0XA-48 and NDM-1 respectively. Using receiver operative characteristics (ROC), highest sensitivity and specificity for detection of OXA-48-like was obtained by mCIM at 4 ​h at cut off 17 ​mm while for NDM-1 CIM was the test of choice at 16 ​mm.ConclusionCIM and mCIM are simple, cheap and easy tests to perform. CIM gave excellent results with NDM1 strains while it was quite poor in predicting OXA-48-like. We recommend CIM and eCIM for rapid identification of NDM-1 producers and mCIM at 4 ​h and MHT for detection of OXA-48-like. No one method can correctly detect both genotypes. As determined by ROC curves a zone of inhibition of 17 ​mm was considered adequate for detection of OXA-48-like and 16 ​mm of NDM-1 by mCIM at 4 ​h and CIM respectively.     

Multidrug-resistant Enterobacteriaceae including metallo-β-lactamase producers are predominant pathogens of healthcare-associated infections in an Indian teaching hospital

Purpose: A study was carried out in an Indian teaching hospital in 2009 to detect the rate of surgical site infections (SSI) and peripheral vascular access site infections. Materials and Methods: The study was a point-prevalence study involving over 300 patients. The presence of infection was determined according to the CDC criteria. Swabs were taken from the infected sites and identification and sensitivity were carried out using VITEK® 2 automated system. Characterisation of β-lactamase was carried out at ARRML, Colindale, London. Results: The rate of SSI was 15% for the clean and clean-contaminated categories while that for the dirty contaminated category was 85% (NNIS risk index 0). Cultures yielded definite or probable pathogens from 64% (9/14) of the patients with SSI. In 1/3^rd of the cultures, Staphylococcus aureus was grown and the rest had Enterobacteriaceae, either extended-spectrum β-lactamase (ESBL) producers or Amp-C hyperproducers and, alarmingly, three isolates were positive for newly recognised New Delhi metallo-β-lactamase-1 (NDM-1). In medicine, 87% (n = 99) of the patients had a peripheral IV access device, 55% developed associated phlebitis/infection and, in seven, probable pathogens were isolated (Candida species and Escherichia coli producing ESBL and NDM-1, respectively, Staphylococcus aureus and Enterococcus faecium). All ESBL and metallo-β-lactamase producers were resistant to multiple classes of antimicrobials, the latter being sensitive only to colistin and tigecycline. The study also found that all post-operative patients were on antibiotics, 92% on IV [213 defined daily doses (DDD)/100 post-op patients] limited mainly to the third-generation cephalosporins (26%) and aminoglycosides (24%) and imidazole derivatives (30%). In medicine, 83% (n = 82) were on IV antibiotics (123 DDD/100 bed-days), limited mainly to the third-generation cephalosporins (74%). Conclusion: Indiscriminate use of antibiotics is a major problem predisposing patients to harm by multi-resistant pathogens. Carbapenems were in little use in this hospital, but the selection pressure exerted by cephalosporins and other unrelated classes was sufficient to select NDM-1-producing strains due to co-selection, suggesting a role of single plasmid carrying resistance genes to multiple classes.     

Evaluation of a DNA microarray (Check-MDR CT102) for rapid detection of TEM, SHV, and CTX-M extended-spectrum β-lactamases and of KPC, OXA-48, VIM, IMP, and NDM-1 carbapenemases

The Check-MDR CT102 microarray, aimed at identifying bacteria producing extended-spectrum β-lactamase (ESBL) (SHV, TEM, and CTX-M) and carbapenemase (KPC, OXA-48, VIM, IMP, and NDM-1), was evaluated on a total of 144 Gram-negative strains expressing various β-lactamases. The sensitivity and specificity were 100% for most tested genes, suggesting that this assay allows accurate identification of common ESBL and carbapenemase producers from bacterial cultures.     

Phenotypic and Molecular Characteristics of Carbapenem-Resistant Enterobacteriaceae in a Health Care System in Los Angeles,California, from 2011 to 2013

Carbapenem-resistant Enterobacteriaceae (CRE) are a concern for health care in the United States but remain relatively uncommon in California. We describe the phenotype, clonality, and carbapenemase-encoding genes present in CRE isolated from patients at a Californian tertiary health care system. CRE for this study were identified by evaluating the antibiograms of Enterobacteriaceae isolated in the UCLA Health System from 2011 to 2013 for isolates that were not susceptible to meropenem and/or imipenem. The identification of these isolates was subsequently confirmed by matrix-associated laser desorption ionization–time of flight, and broth microdilution tests were repeated to confirm the CRE phenotype. Real-time PCR for bla_KPC, bla_SME, bla_IMP, bla_NDM-1, bla_VIM, and bla_OXA-48 was performed. Clonality was assessed by repetitive sequence-based PCR (repPCR) and multilocus sequence typing (MLST). Of 15,839 nonduplicate clinical Enterobacteriaceae isolates, 115 (0.73%) met the study definition for CRE. This number increased from 0.5% (44/8165) in the first half of the study to 0.9% (71/7674) in the second (P = 0.004). The most common CRE species were Klebsiella pneumoniae, Enterobacter aerogenes, and Escherichia coli. A carbapenemase-encoding gene was found in 81.7% (94/115) of CRE and included bla_KPC (78.3%), bla_NDM-1 (0.9%), and bla_SME (2.6%). The majority of bla_KPC genes were in K. pneumoniae isolates, which fell into 14 clonal groups on typing. bla_KPC was identified in more than one species of CRE cultured from the same patient in four cases. Three bla_SME-carrying Serratia marcescens isolates and one bla_NDM-1 carrying Providencia rettgeri isolate were detected. CRE are increasing in California, and carbapenemases, particularly KPC, are a common mechanism for carbapenem resistance in this region.