chromID CARBA Research Articles

Prevalence and molecular epidemiology of carbapenemase-producing Enterobacterales isolated from dog and cat faeces submitted to veterinary laboratories in the USA.

To estimate the prevalence of carbapenemase-producing Enterobacterales (CPE) carriage among pets using faecal specimens submitted to veterinary diagnostic laboratories throughout the US. A secondary aim was to employ whole-genome sequencing (WGS) to characterize isolates of CPE from companion animals and compare them to publicly available CPE genomes. To estimate the prevalence of CPE in companion animals in the USA, a multicenter surveillance study including 8 different veterinary diagnostic laboratories from across the USA was conducted. Briefly, remnant faecal specimens from dogs and cats were screened using two selective agar plates (CHROMID Carba and MacConkey with 1 mg/L cefotaxime and 0.125 mg/L meropenem) and presumptive CPE isolates screened by the modified carbapenemase inactivation method for carbapenemase production. A total of 2393 specimens were screened and yielded 196 isolates for carbapenemase screening. A total of 5 isolates from 4 dogs and 1 cat at 3 different veterinary diagnostic laboratories were confirmed to produce a carbapenemase (0.21%). Whole-genome sequencing (WGS) revealed two E. coli (ST167) isolates that both produced an NDM-5 carbapenemase, two Enterobacter hormaechei (ST171) isolates that produced an NDM-5 carbapenemase and a KPC-4 carbapenemase respectively and one Klebsiella oxytoca (ST199) that produced an Oxa-48-type carbapenemase. Both E. coli isolates were found to be within at least 22 SNPs of previously characterized canine and human CPE isolates. This study demonstrates that the prevalence of CPE among companion animals is relatively low (0.21%) but that given the genetic relatedness of animal isolates to human isolates, additional surveillance is needed.

Carbapenemase Detection and Identification: Which method should be Chosen?

Carbapenemase production constitutes the key mechanism of resistance to carbapenems. Rapid detection or confirmation of the involvement of carbapenemase-producing bacteria (CPB) in infections is crucial for adequate antibiotic therapy and infection control, particularly during epidemics or for surveillance purposes. The Ambler classification proposes four classes of carbapenemases: classes A, B, C, and D. Carbapenemases A, C, and D are called serine carbapenemases because they use serine in their active site to catalyze the hydrolysis of carbapenems. Class B carbapenemases are metallo-beta-lactamases (MBLs) that use a cation (Zn2+) to hydrolyze the beta-lactam ring. Biochemical, chromogenic, immunochromatographic, mass spectrometric, and molecular methods have both advantages and limitations for carbapenemase detection. Although there is no single method that meets all specifications of an ideal test, it is important to explore methods to identify the most suitable ones. Thus, according to the research articles reported in this review and the criteria (cost, turnaround time, sensitivity, specificity, expertise needs, target carbapenemases), chromogenic tests such as ChromID CARBA SMART and CHROMagar™ mSuperCARBA™ would be the best candidates for the rapid and effective detection of carbapenemase-producing bacteria in developing countries. Moreover, Carba NP, SUPERCARBA and Carbapenem Inactivation Method (CIM) could also be considered good carbapenemase-detecting methods. WGS may be reserved for large-scale funded studies of carbapenemase-producing bacteria.

Evaluation and validation of laboratory procedures for the surveillance of ESBL-, AmpC-, and carbapenemase-producing Escherichia coli from fresh meat and caecal samples.

Extended-spectrum β-lactamase- (ESBL) and AmpC- β-lactamase-producing Enterobacterales are widely distributed and emerging in both human and animal reservoirs worldwide. A growing concern has emerged in Europe following the appearance of carbapenemase-producing Escherichia coli (E. coli) in the primary production of food animals. In 2013, the European Commission (EC) issued the Implementing Decision on the monitoring and reporting of antimicrobial resistance in zoonotic and commensal bacteria. The European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) was tasked with providing two laboratory protocols for samples derived from meat and caecal content, respectively, for the isolation of ESBL- and AmpC-producing E. coli (part 1) and carbapenemase-producing (CP) E. coli (part 2). In this study, we describe the current protocols, including the preparatory work for the development. Up to nine laboratory procedures were tested using minced meat as the matrix from beef, pork, and chicken as well as six procedures for the caecal content of cattle, pigs, and chicken. Variables included sample volume, pre-enrichment volume, pre-enrichment broth with and without antimicrobial supplementation, and incubation time/temperature. The procedures were evaluated against up to nine E. coli strains harboring different AMR genes and belonging to the three β-lactamase groups. The laboratory procedures tested revealed that the most sensitive and specific methodologies were based on a Buffered Peptone Water pre-enrichment of 225 ml to 25 g or 9 ml to 1 g for minced meat and caecal content, respectively, incubated at 37°C overnight, followed by inoculation onto MacConkey agar supplemented with 1 mg/L cefotaxime for detecting ESBL- and AmpC-producing E. coli and Chrom ID SMART (Chrom ID CARBA and OXA) for CP E. coli, incubated overnight at 37 and 44°C, respectively. We provided two isolation protocols for the EU-specific monitoring of ESBL- and AmpC- producing E. coli (part 1) and CP E. coli (part 2) from fresh meat (protocol 1) and caecal (protocol 2) samples, which have been successfully implemented by all EU Member States for the monitoring period 2014-2027 (EU 2020/1729).

Screening for carriers of carbapenemase producing Enterobacteriaceae in critical care units

Background: The increasing cases of carbapenemase resistant Enterobacteriaceae (CRE) across the world is a cause of concern. Asymptomatic carriage of CRE in critical care units is a menace to infection control. Aims: This study determines the carriage rate of CRE in patients admitted to the intensive care units (ICU's) and evaluates the potential risk factors, leading to colonization in patients with CRE. Materials and Methods: Sixty rectal swabs from patients in the ICU's were screened for carriage of CRE. The samples were inoculated onto ChromID CARBA SMART bi-plate. The organisms showing color appearances as per the manufacturer's instructions were considered as CRE. Routine disk diffusion technique was also employed and CRE was defined as an organism belonging to the Enterobacteriaceae family which was resistant to either imipenem or meropenem. Results: The organisms isolated were identified and the percentage of carriage of carbapenem-resistant organisms was 12 (20%), of which Klebsiella pneumoniae was 4 (33.3%), Escherichia coli 6 (50%), Citrobacter freundii 1 (8.3%), and Enterobacter spp. 1 (8.3%). Out of these, 2 (3.3%) showed OXA 48 type resistance seen with K. pneumoniae and E. coli. Prior hospitalization, the use of high-end antibiotics and patients who have undergone surgeries were the most common potential risk factors for colonization with CRE. Conclusion: The prompt detection of CRE by routine screening using cost-effective methods and reduction of potential risk factors for gut colonization reduce the transmission of drug resistance in any hospital setting and pave the way for better antibiotic stewardship and appropriate contact isolation precautions.

Comparison of Classical methods and Chromogen Media for Detection of Stool Colonisation by Carbapenem-resistant Enterobacteriaceae

Abstract Objectives: We aimed to compare classical methods and chromogenic media to detect carbapenem-resistant Enterobacteriaceae (CRE) colonization among hospitalized patients and determine the risk factors causing infection in colonized patients. Methods: Between January and August 2017, 100 patients over the age of 18 who were hospitalized in the Reanimation Intensive Care Unit and the Trauma Emergency Intensive Care Unit of a university hospital were examined. From the first day of intensive care unit (ICU) admission, rectal swabs were collected once every week and were tested for the presence of CRE by using the classical method defined by the Centers for Disease Control and Prevention (CDC), ChromID CARBA chromogenic medium, and direct inoculation into MacConkey agar plates. In addition, MIC values for imipenem, ertapenem, meropenem and colistin were determined by using the Etest. Results: Rectal BDE carriage was detected by at least one method in 46 (46%) of 100 patients included in the study. Sensitivity and specificity values of the CDC classical method, direct MacConkey inoculation, and ChromID CARBA medium in the first 24 hours were found as 78%-42%, 87%-80%, and 91%-98%, respectively. Sensitivity and specificity values of these methods after 72 hours were determined as 78%-100%, 87%-100%, and 91%-100%, respectively. Conclusion: We observed that, although the ChromID CARBA method performed better than classical CDC and direct MacConkey inoculation methods, direct MacConkey inoculation can still be employed, especially in areas with limited resources. Keywords: carbapenem, carbapenemase, rectal colonization

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 blaNDM-1 and 33 blaOXA48. 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).

532. Can Saccharomyces boulardii Therapy Be Effective in Decolonizing Rectal Carbapenem-Resistant Enterobacteriaceae (CRE) Colonization?

BackgroundCRE are globally important pathogens associated with significant morbidity and mortality. The problem of carrying CRE may continue to create a problem in discharged cases in the community. Saccharomyces boulardii sachet therapy (SBST) is reported to cause decolonization in several MDR bacteria carriers. Herein, it is aimed to present the decolonizing rates of rectal CRE colonized cases after SBST treatment.MethodsThe study period was August 2018–March 2019. Inclusion criteria were: (i) age >18, (ii) receiving Saccharomyces boulardii 250 mg sachets q12h for 7 days, (iii) being proven CRE carrier on rectal swab culture (RSC) up to 5 days period before SBST. The first repeated RSC was performed 3–5 days after the end of SBST. Data were retrieved from the hospital electronic database. Cases with three consecutive weekly performed negative RSC were considered to be decolonized. RSC were processed according to CDC protocol; briefly, the swab was inoculated into 10 mL of trypticase soy broth (bioMérieux Inc., Marcy-l’Étoile, France) with the addition of one 10-μg ertapenem disk (Oxoid, Altrincham, UK) and incubated at 35°C for 18–20 h. The next day, after vortexing, 100 μL of the inoculum was subcultured (8) onto chromID CARBA agar plates (bioMérieux) and incubated at 35°C for 18–20 h. Suspected CRE colonies on chromID CARBA (blue/green to blue/gray in color) were identified by the VITEK MS system (bioMérieux). Susceptibility testing of the isolates was performed with the VITEK 2 system (bioMérieux). Isolates were tested for their resistance phenotypes to imipenem, ertapenem, and meropenem by E-test (bioMérieux). The results were interpreted according to the EUCAST criteria.ResultsFifteen cases [2 women, mean age 60.6 ± 18.3 (min. 18–max. 83)] fulfilled the inclusion criteria. All had a history of carbapenem usage. Five cases (33%) had three consequent negative RSC after SBST and were considered to be decolonized. Twelve cases were receiving concomitant antibiotic during SBST (10 carbapenem based regimens). Three cases who received no concomitant antibiotic were decolonized.ConclusionSBST may be a promising tool for decolonizing CRE carriers. These data need to be validated in larger cohorts preferably via randomized-controlled trials.Disclosures All authors: No reported disclosures.

Diagnostic performance of the Xpert Carba-R assay for active surveillance of rectal carbapenemase-producing organisms in intensive care unit patients

BackgroundThere are growing concerns regarding the spread of carbapenemase-producing organisms (CPOs) among patients in long-term care facilities (LTCFs) and hospitals in South Korea. We have established a screening protocol for the detection of CPOs in high-risk patients upon admission to intensive care units (ICUs). The diagnostic performance of the Xpert Carba-R assay was compared to that of rectal culture for CPO detection in high-risk patients upon ICU admission.MethodsA total of 408 consecutive rectal swabs were obtained from December 2016 to December 2017. CPO screening was performed using the Xpert Carba-R assay (Cepheid, Sunnyvale, CA, USA). When a carbapenemase gene was detected, additional rectal swabs were incubated overnight and inoculated on chromID CARBA medium (bioMérieux, Marcy l’Etoile, France). Bacterial carbapenemase genes, including blaKPC, blaNDM, blaVIM, blaIMP-1, and blaOXA-48, were confirmed by conventional PCR. The diagnostic performance of the Carba-R assay was ascertained based on the culture results.ResultsThe prevalence of CPO carriage was 7.4% according to the Carba-R assay and 3.7% according to rectal culture. The median Ct values of IMP-1 and KPC were significantly different (35.2 vs. 26.6, P = 0.0143). The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Carba-R assay were 100.0% (95% confidence interval [CI], 78.2–100.0), 96.7% (94.4–98.2), 53.6% (40.4–66.4) and 100.0% (99.0–100.0), respectively.ConclusionsWe demonstrated the prevalence of CPO carriage in high-risk patients upon ICU admission and evaluated the diagnostic performance of the Carba-R assay. The combined use of the Xpert Carba-R assay and culture produces rapid and reliable results for the active surveillance of rectal CPO in ICU patients.

A comparative evaluation of two chromogenic media for surveillance of carbapenem-resistant Enterobacterales with non-carbapenemase-producing or carbapenemase-producing strains other than blaKPC: blaGES-5, blaNDM-1 or blaVIM-2

Abstract Background Infections caused by carbapenem-resistant Enterobacterales (CREs) are an emerging problem associated with high rates of morbidity and mortality. CREs are divided into two categories (carbapenemase-producing [CP] CRE and non-CP CRE). The most prevalent carbapenemase produced by Enterobacterales is Klebsiella pneumoniae carbapenemase (KPC) in Korea. Rapid identification of CREs is clinically important in infection control precaution. We compared the performance of two chromogenic media (chromID CARBA agar and CHROMagar KPC agar) for non-CP CREs or CP CREs with blaGES-5, blaNDM-1 or blaVIM-2 in a Korean hospital. Methods The study was carried out during a 3-month period from April to June 2017 during the surveillance program for CRE colonization. Antimicrobial susceptibility testing (AST) and polymerase chain reaction (PCR) were performed at the Korean Centers for Disease Control and Prevention. Results A total of 45 rectal swabs from 42 hospitalized patients were examined. Sensitivity of both chromID CARBA and CHROMagar KPC were 100% for CP CREs; and 50% and 100% for non-CP CREs, respectively. Specificity of chromID CARBA and CHROMagar KPC were 89.2% and 70.3% for CP CRE, respectively; and 76.9% and 66.7% for non-CP CRE, respectively. Conclusions The CHROMagar KPC is useful to monitor non-CP and CP CREs. The chromID CARBA is efficient for rapid detection of CP CREs requiring high contact precaution.

2560. Multispecies Outbreak of KPC-2 Producing Enterobacteriaceae in a Chilean Pediatric Hospital

BackgroundCarbapenem-resistant Enterobacteriaceae (CRE) are a critical global health problem. We detected a surge of CRE cases in a pediatric hospital in Chile, a country with a low endemicity of KPC-producing organisms. Herein, we describe the molecular epidemiology of this outbreak.MethodsCRE isolates from clinical specimens and surveillance rectal swabs (obtained using chromID CARBA SMART agar, BioMerieux) of pediatric patients were collected from July 2015 to January 2017. Species identity was confirmed by MALDI-TOF. Carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like) were detected by multiplex PCR, followed by amplification and sequencing of the blaKPC allele. Conjugation experiments were conducted with representative species as donors and sodium azide-resistant E. coli J53 as recipient. PCR-based plasmid typing (PBRT Diatheva kit) was then performed on donors and recipients. For K. pneumoniae, genetic relatedness was investigated by PFGE, multilocus sequence typing and wzi typing.ResultsSixty-one CRE clinical and surveillance isolates were obtained from 49 patients aged 17 days to 16 years. blaKPC-2 was present in 57/62 isolates; no other carbapenemases were found. For 11 patients, multiple cultures were obtained; 4/11 had more than one KPC-harboring species. KPC-harboring isolates displayed ertapenem MICs ranging from 1 to >8 mg/L. Preliminary analyses suggest that blaKPC-2 is contained within a nonclassical Tn4401 structure (lacking the upstream promoter). Mating experiments indicate that blaKPC-2 is carried by a conjugative IncN backbone plasmid. Interestingly, K. pneumoniae isolates were nonclonal by PFGE and belonged to multiple STs unrelated to CG258 (ST34, ST36, among others) and different wzi types (37, 154, among others). ConclusionWe report a multispecies outbreak of KPC-2 producing CRE in children mainly driven by horizontal dissemination of a promiscuous IncN plasmid. The nonclonal, multispecies nature of this outbreak provides insights into the complex dynamics of KPC dissemination in countries like Chile, where the clonal spread of highly successful clones like CG258 is not the predominant dissemination vehicle, and instead HGT-related spread could be playing a more important role.Disclosures All authors: No reported disclosures.

Pseudomonas aeruginosa - Modified Hodge Test (PAE-MHT) and ChromID Carba Agar for Detection of Carbapenemase Producing Pseudomonas Aeruginosa Recovered from Clinical Specimens

AIMS:This study aims to evaluate the ability of ChromID Carba agar, and Pseudomonas aeruginosa modified Hodge test (PAE-MHT) for detection of carbapenemase-producing P. aeruginosa and to determine the associated carbapenemase gene classes by PCR.METHODS:One hundred Carbapenem-resistant P. aeruginosa (CRPA) isolates were tested for: i) carbapenemases production by ChromID carba agar, Modified Hodge test (MHT) and (PAE-MHT) and ii) detection of some carbapenemase genes by PCR.RESULTS:All (100%) of the isolates showed growth on ChromID Carba agar with 100% sensitivity. Using MHT, 54% of isolates were positive, 3% were indeterminate, and 43% were negative, demonstrating 58.9% sensitivity and 80% specificity. On performing PAE-MHT, 91% of the strains were positive, 3% were intermediate, and 6% were negative, demonstrating 97.9% sensitivity and 80% specificity. The most prevalent gene was blaKPC (81%), followed by blaVIM (74%); blaIMP was detected in only one isolate, and blaOXA-48 in 34% of the isolates.CONCLUSIONS:We conclude that PAE-MHT and ChromID Carba are sensitive, specific, simple and cost-effective screening tests for detection of CRPA isolates compared to the traditional MHT.

Chromogenic culture media or rapid immunochromatographic test: Which is better for detecting Klebsiella pneumoniae that produce OXA-48 and can they be used in blood and urine specimens

Our goal was to compare a rapid test (OXA-48K-SeT) and four different chromogenic media (CHROMagar KPC, CHROMagar mSuperCARBA, ChromID Carba and ChromID OXA-48) for the detection of OXA-48 producing Klebsiella pneumoniae isolates and spiked urine/blood samples with these bacteria. In total 100 K.pneumoniae isolates, including 60 OXA-48 positive, 15 other carbapenemase producing, 15 Extended spectrum betalactamases (ESBL) positive and 10 carbapenem sensitive K.pneumoniae were included in the study. After all samples were inoculated into all chromogenic media, temocillin discs were placed onto the media. OXA-48K-SeT was studied according to the manufacturer's instructions and the lower detection limit was determined. Sensitivities and specificities of all chromogenic media and rapid test were detected as 100%. All of the OXA-48 producers were found resistant to temocillin on all chromogenic media. The lower detection limit of the rapid assay was determined as 106 in both direct bacterial samples and in spiked urine/blood samples. As a result, four chromogenic culture media and OXA-48 K-SeT can be used safely for detection of OXA-48 positive K.pneumoniae isolates. Although direct clinical specimens were not used, our study suggests that this media and OXA-48 K-SeT may be used in patient samples like blood and urine. Further studies are needed to assess this suggestion.

Evaluation of three screening methods for detection of carbapenemase-producing Enterobacteriaceae in rectal swabs

Carbapenemase-producing Enterobacteriaceae (CPE) have taken great importance on public health at global scale, which makes it necessary to implement rapid test for its prompt detection. To evaluate three screening methods to detect CPE in rectal swabs. Transverse study, prospective. Seventy three rectal swabs were evaluated by three methodologies. Microorganism identification and susceptibility testing were made using automated systems. Carbapenemase production was confirmed by modified Hodge test and synergy tests using boronic acid and EDTA. The method 1 (ChromID CARBA®) detected 20 positive samples (27.4%), 5 false positives (6.9 %), with concordance index of 93.2%, sensitivity 100% and specificity of 90%. Method 2 (HB&L Carbapenemase®) detected 17 positive samples (23.3%) and 3 false negatives (4.1%). The sensitivity and specificity of the assay were 85% and 100%, with concordance index of 95.9%. Method 3 (Xpert Carba-R®) detected 19 positive samples (57.5%) and 1 false negatives (3.1%), sensitivity 95%, specificity 100% and concordance index of 97%. There is a wide variety of methodologies for rapid detection of carbapenemase-producing microorganisms. Choosing the best method must have as requirement a good sensitivity, specificity, and cost-effectiveness.

OXA-244-Producing Escherichia coli Isolates, a Challenge for Clinical Microbiology Laboratories.

OXA-244 is a single-point-mutant derivative of OXA-48 displaying reduced carbapenemase activity. Here, we report the microbiological features of seven OXA-244-producing Escherichia coli isolates. Only one isolate grew on ChromID Carba Smart medium (bioMérieux), but six of the seven isolates grew on ChromID extended-spectrum-β-lactamase (ESBL) medium (bioMérieux), as they coproduced an ESBL and/or a plasmid-encoded cephalosporinase. The production of a carbapenemase was detected in 57.1%, 71.4%, 71.4%, and 100% of the E. coli isolates using the Carba NP test, the Rapidec Carba NP test (bioMérieux), a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) hydrolysis assay (Bruker), and the OXA-48 K-SeT assay (Coris BioConcept), respectively. Our results indicate that OXA-244-producing E. coli isolates are difficult to detect, which may lead to their silent spread.

Development of selective medium for IMP-type carbapenemase-producing Enterobacteriaceae in stool specimens

BackgroundIdentification of carbapenemase-producing Enterobacteriaceae (CPE) in faecal specimens is challenging. This fact is particularly critical because low-level carbapenem-resistant organisms such as IMP-producing CPE are most prevalent in Japan. We developed a modified selective medium more suitable for IMP-type CPE.MethodsFifteen reference CPE strains producing different types of β-lactamases were used to evaluate the commercially available CHROMagar KPC and chromID CARBA as well as the newly prepared MC-ECC medium (CHROMagar ECC supplemented with meropenem, cloxacillin, and ZnSO4) and M-ECC medium (CHROMagar ECC supplemented with meropenem and ZnSO4). A total of 1035 clinical samples were then examined to detect CPE using chromID CARBA and M-ECC medium.ResultsAll tested strains producing NDM-, KPC-, and OXA-48-carbapenemases were successfully cultured in the media employed. Although most of the IMP-positive strains did not grow in CHROMagar KPC, chromID CARBA, or MC-ECC, all tested strains grew on M-ECC. When faecal samples were applied to the media, M-ECC medium allowed the best growth of IMP-type CPE with a significantly higher sensitivity (99.3%) than that of chromID CARBA (13.9%).ConclusionsM-ECC medium was determined as the most favourable selective medium for the detection of IMP-type CPE as well as other types of CPE.

Evaluation of Genotypic and Phenotypic Methods to Detect Carbapenemase Production in Gram-Negative Bacilli.

Carbapenemase-producing gram-negative bacteria (CP-GNB) are an urgent and expanding public health threat. Rapid and accurate identification of these organisms facilitates infection prevention efforts in healthcare facilities. The objective of our study was to evaluate methods to detect and identify CP-GNB. We examined 189 carbapenem-resistant GNB(CR-GNB), including Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii complex, using 3 different methods: 2 methods to screen isolates of GNB for carbapenemase production [the carbapenem inactivation method (CIM) and 2 chromogenic agars] and a molecular method (Cepheid GeneXpert Carba-R) to identify the mechanism of carbapenem resistance and the associated resistance genes (blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM). The CIM was a simple and inexpensive phenotypic screen to differentiate between CR-GNB and CP-GNB, with improved analytical performance characteristics and inter-reader correlation compared to the modified Hodge test. Both chromogenic agars evaluated (HardyCHROM CRE and chromID CARBA) were able to support growth of most of the organisms tested, including isolates possessing the blaOXA-48-like gene. However, these media had a low analytical specificity for carbapenemase production, with breakthrough of CR-GNB that did not produce a carbapenemase. The Xpert Carba-R assay was rapid and easy to perform, and demonstrated 100% positive and negative agreement for characterization of genetic determinants of carbapenem resistance. Screening by CIM followed by the Xpert Carba-R PCR is an accurate method for detecting and characterizing CP-GNB, including Enterobacteriaceae, P. aeruginosa, and A. baumannii complex.

High Prevalence of Faecal Carriage of ESBL-Producing Enterobacteriaceae among Children in Dar es Salaam, Tanzania.

BackgroundFaecal carriage of ESBL-producing bacteria is a potential risk for transmission and infection. Little is known about faecal carriage of antibiotic resistance in Tanzania. This study aimed to investigate the prevalence of faecal carriage of ESBL-producing Enterobacteriaceae and to identify risk factors for carriage among young children in Tanzania.Methodology/Principal FindingsFrom August 2010 to July 2011, children below 2 years of age were recruited in Dar es Salaam, including healthy community children (n = 250) and children hospitalized due to diarrhoea (n = 250) or other diseases (n = 103). ChromID ESBL agar and ChromID CARBA SMART agar were used for screening. Antimicrobial susceptibility testing was performed by the disk diffusion method. ESBL genotypes were identified by Real-Time PCR and sequencing.The overall prevalence of ESBL carriage was 34.3% (207/ 603). The prevalence of ESBL carriage was significantly higher among hospitalized children (50.4%), compared to community children (11.6%; P < 0.001; OR = 7.75; 95% CI: 4.99–12.03). We found high prevalence of Multidrug-resistance (94%) among Escherichia coli and Klebsiella pneumoniae isolates. No resistance to carbapenems was detected. For the majority of isolates (94.7%) we detected a blaCTX-M-15-like gene. In addition, the plasmid mediated AmpC beta-lactamase CMY-2 was detected for the first time in Tanzania. ESBL prevalence was significantly higher among HIV positive (89.7%) than HIV negative (16.9%) children (P = 0.001; OR = 9.99; 95% CI: 2.52–39.57). Use of antibiotics during the past 14 days and age below 1 year was also associated with ESBL carriage.Conclusions/SignificanceWe report a high rate of faecal carriage of ESBL-producing Enterobacteriaceae among children below 2 years of age in Tanzania, particularly those with HIV-infection. Resistance to a majority of the available antimicrobials commonly used for children in Tanzania leaves few treatment options for infections when caused by these bacteria.

Laboratory detection of intestinal carriage of carbapenemase-producing Enterobacteriaceae – A comparison of algorithms using the Carba NP test

Stool specimens spiked with a panel of 46 carbapenemase-producing Enterobacteriaceae (CPE) and 59 non-carbapenemase producers were used to compare the diagnostic accuracy of 4 testing algorithms for the detection of intestinal carriage of CPE: (1) culture on Brilliance ESBL agar followed by the Carba NP test; (2) Brilliance ESBL followed by the Carba NP test, plus chromID OXA-48 agar with no Carba NP test; (3) chromID CARBA agar followed by the Carba NP test; (4) chromID CARBA followed by the Carba NP test, plus chromID OXA-48 with no Carba NP test. All algorithms were 100% specific. When comparing algorithms (1) and (3), Brilliance ESBL agar followed by the Carba NP test was significantly more sensitive than the equivalent chromID CARBA algorithm at the lower of 2 inoculum strengths tested (84.8% versus 63.0%, respectively [P<0.02]). With the addition of chromID OXA-48 agar, the sensitivity of these algorithms was marginally increased.

Phenotypic and Genotypic Detection of Klebsiella Pneumoniae Carbapenemase and New Delhi Metallo-β-Lactamase of Enterobacteriaceae in Benha University Hospitals

Background: The prevalence of carbapenem-resistant Enterobacteriacea is on the rise worldwide, posing a major public health threat as well as a serious concern for infection control management. The accurate identification and reporting of carbapenemase producing Enterobacteriacea (CPE) will aid infection control practitioners in preventing the spread of these multidrug- resistant isolates. Objectives: This study aimed to detect the prevalence of CPE in Benha university hospitals and to confirm the presence of k. Pneumoniae carbapenemase (KPC) and New Delhi Metallo-β- Lactamase (NDM) in Enterobacteriacea. Methodology: This study was conducted on 100 Enterobacteriacea strains collected from patients admitted to Benha University Hospitals. The isolated Enterobacteriacea strains were screened for CPE by chromID CARBA agar with species identification by using API 20 E test strips as a biochemical identification system. KPC and NDM carbapenemases detection was confirmed \phenotypically by Mast Disc ID carbapenemase detection set (MDI) and genotypically by multiplex PCR. Results: Out of 100 Enterobacteriacea isolates, 40 strains (40 %) are carbapenemase producers and 27strains of them (67.5 %) are carrying genes responsible for carbapenem resistant. Twenty six strains (65%) are carrying KPC gene and only one k. Pneumoniae strain (2.5%) is carrying NDM-1 gene for MβL production. MDI detected 60% of KPC and 25% of MβL producers with high sensitivity (92.6 %) in comparison to PCR. Intensive Care Unit patients harbored most of carbapenem-resistant isolates with the highest percentage of carbapenemases genes in k. Pneumoniae. Conclusion: Emergence of CPE pathogens in our setting create an important challenge for clinicians and hospital epidemiologists with the possibility of outbreak eruption by these difficult-to-treat pathogens, in the future.

Carbapenemase-producing Enterobacteriaceae in hospital wastewater: a reservoir that may be unrelated to clinical isolates

Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging infection control problem in hospitals worldwide. Identifying carriers may help reduce potential spread and infections. To assess whether testing hospital wastewater for CPE can supplement patient-based screening for infection prevention purposes in a hospital without a recognized endemic CPE problem. Wastewater collected from hospital pipework on 16 occasions during February to March 2014 was screened for CPE using chromID(®) CARBA agar and chromID(®) CPS agar with a 10μg ertapenem disc and combination disc testing. Minimum inhibitory concentrations were determined using British Society for Antimicrobial Chemotherapy methodology and carbapenemase genes detected by polymerase chain reaction or whole-genome sequencing. Selected isolates were typed by pulsed-field gel electrophoresis. Suspected CPE were recovered from all 16 wastewater samples. Of 17 isolates sent to the Antimicrobial Resistance and Healthcare Associated Infections Reference Unit, six (four Citrobacter freundii and two Enterobacter cloacae complex) were New Delhi metallo-β-lactamase (NDM) producers and the remaining 11 (six Klebsiella oxytoca and five Enterobacter cloacae complex) were Guiana-Extended-Spectrum-5 (GES-5) producers, the first to be described among Enterobacteriaceae in the UK. The four NDM-producing C.freundii, two NDM-producing E.cloacae complex, and four out of five GES-5-producing E.cloacae complex were each indistinguishable isolates of the same three strains, whereas the six GES-5-producing K.oxytoca overall shared 79% similarity. CPE are readily isolated from hospital wastewater using simple culture methods. There are either undetected carriers of CPE excreting into the wastewater, or these CPE represent colonization of the pipework from other sources. Surveillance of hospital wastewater for CPE does not appear helpful for infection control purposes within acute hospitals.