Emergence of pandrug-resistant carbapenemase-producing Enterobacterales in dogs and cats: a cross-sectional study in Egypt.

One of the most important emerging health problems is the increasing role of animals in the rapid global rise in resistance to last-resort antibiotics, such as carbapenems. However, there is limited information on the role of pet animals in harboring and spreading pandrug-resistant (PDR) carbapenemase-producing Enterobacterales (CPE), especially in Egypt. This cross-sectional study was conducted to screen for CPE in healthy and diseased pets using phenotypic and molecular methods and the NG-Test CARBA 5 immunochromatographic assay. Rectal swabs were collected from 62 dogs and 48 cats, incubated overnight in tryptic soy broth containing 10 µg of meropenem disc and subsequently cultured on MacConkey agar supplemented with meropenem (1 mg/L). Sixty-six isolates (60.6%), including 56 Klebsiella pneumoniae, seven Escherichia coli, and three K. oxytoca isolates, were confirmed to be carbapenem-resistant Enterobacterales (CRE) by the disc diffusion method, broth microdilution test, CNPt-direct, and PCR assay targeting carbapenemase genes. Forty-three (65.2%) dogs and 23 (34.8%) cats carried CPE. Of these, 35 (70.0%) were healthy (including 27 dogs and 8 cats) and 31 (52.5%) were diseased (including 16 dogs and 15 cats). bla OXA-181 was the most common gene detected (42/66, 63.6%), followed by bla IMP (40/66, 60.6%), bla OXA-48-like (29/66, 43.9%), bla KPC and bla VIM (20/66, 30.3% each), and bla NDM (17/66, 25.8%). The identified genotypes were bla KPC-2, bla IMP-1, bla VIM-1, bla NDM-1, and bla NDM-5. The CARBA 5 assay showed higher sensitivity and specificity for the detection of NDM, OXA and KPC than that for VIM and IMP genes. Antimicrobial resistance profiles of CRE isolates revealed 20 PDR, 30 extensively drug-resistant (XDR), and 16 multidrug-resistant (MDR) phenotypes. This study provides evidence of colonization with PDR CPE in dogs and cats. To manage the infection or colonization of pets in veterinary clinical settings, extended surveillance systems should be considered, and the use of critical antibiotics should be strictly controlled.

Evaluation of Phenotypic Tests for Carbapenemase Detection in Enterobacteriaceae in Tunisia.

Introduction: Resistance to carbapenems in Enterobacteriaceae is a challenge for public health. Carbapenemase production is the leading mechanism. This work aims to evaluate four phenotypic methods for carbapenemase detection in comparison with a molecular method. Materials and Methods: Thirty-seven nonrepeating Enterobacteriaceae strains with decreased susceptibility to ertapenem were included. Imipenem MIC, Modified Hodge Test (MHT), Neo-Rapid Carb Kit® and KPC, MBL, and OXA-48 Confirm Kit® were performed. Isolates were tested for blaOXA-48, blaNDM, and blaVIM genes by end-point polymerase chain reaction. The results of the molecular study were used as a reference test to determine the performances of the phenotypic tests. Results: Imipenem resistance does not seem to be a good marker for carbapenemase production with a sensitivity of 54% (95% CI: 38-71). MHT showed 82% sensitivity (95% CI: 65-91). Overall, the enzymatic test showed the best performances for carbapenemase detection with 100% sensitivity (95% CI: 89-100) and the best turnaround time. The characterization of carbapenemases classes by the combined discs test demonstrated 88% overall sensitivity (95% CI: 72-95). Conclusion: The results of this study support the combination of the enzymatic and the combined disc tests for carbapenemase detection in Enterobacteria.

Dynamics of blaOXA-23 gene transmission in Acinetobacter spp. from contaminated veterinary environmental surfaces: an emerging One Health threat?

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii is a common pathogen associated with healthcare-acquired infections, and robust infection prevention and control protocols exist in human healthcare settings. In contrast, infection prevention and control (IPC) standards are limited in veterinary medicine, necessitating further investigation. AIM: Examine the possible transmission of carbapenem-resistant Acinetobacter spp. in a veterinary practice where a cat was diagnosed with an OXA-23-producing A. baumannii ST2 strain. METHODS: Environmental samples together with nasal and hand swabs from the veterinary personnel were collected. All swabs were screened for the presence of extended-spectrum-ß-lactamase- and carbapenemase-producing Enterobacterales, meticillin-resistant staphylococcus and multi-drug-resistant Acinetobacter spp. Whole-genome sequencing was performed for carbapenemase-producing strains. RESULTS: Of the veterinary staff, 60% carried meticillin-resistant Staphylococcus epidermidis. Environmental evaluation showed that 40% (N=6/15) of the surfaces analysed by contact plates and 40% (N=8/20) by swabs failed the hygiene criteria. Assessment of the surfaces revealed contamination with five OXA-23-producing Acinetobacter spp. strains: an OXA-23-producing Acinetobacter schindleri on the weight scale in the waiting room; and four OXA-23-producing Acinetobacter lwoffii strains, on different surfaces of the treatment room. The blaOXA-23 gene was located on the same plasmid-carrying Tn2008 across the different Acinetobacter spp. strains. These plasmids closely resemble a previously described OXA-23-encoding plasmid from a human Portuguese nosocomial Acinetobacter pittii isolate. Distinctly, the OXA-23-producing A. baumannii ST2 clinical strain had the resistant gene located on Tn2006, possibly inserted on the chromosome. CONCLUSION: The detection of an OXA-23-producing A. baumannii ST2 veterinary clinical strain is of concern for companion animal health and infection, prevention and control. This study established the dynamic of transmission of the plasmid-mediated blaOXA-23 gene on critical surfaces of a small animal veterinary practice. The genetic resemblance to a plasmid found in human nosocomial settings suggests a potential One Health link.

Multicenter study to assess the use of BL-DetecTool for the detection of CTX-M-type ESBLs and carbapenemases directly from clinical specimens.

Antimicrobial resistance (AMR) is one of the major public health problems worldwide. Multiple strategies have been put in place to address this problem. One of them is the rapid detection of the mechanisms of resistance, such as extended-spectrum beta-lactamases (ESBLs) and/or carbapenemases. We conducted a multicenter study that included nine European centers for the assessment of prototypes of a novel lateral flow immunoassay-based device (BL-DetecTool) for a rapid detection of ESBL (NG-Test CTX-M-MULTI DetecTool) and/or carbapenemases (NG-Test CARBA 5 DetecTool) from Enterobacterales and Pseudomonas aeruginosa in positive urine, positive blood cultures, and rectal swabs. We performed a prospective analysis between January 2021 and June 2022, including overall 22,010 samples. Based on each hospital information, the sensitivity to detect CTX-M was 84%-100%, 90.9%-100%, and 75%-100% for urine, positive blood cultures, and enriched rectal swabs, respectively. On the other hand, the sensitivity to detect carbapenemases was 42.8%-100%, 75%-100%, and 66.6%-100% for urine, positive blood cultures, and enriched rectal swab, respectively. BL-DetecTool allows a rapid and reliable detection of ESBL and carbapenemases directly from urine, positive blood cultures, or enriched rectal swabs, being an easy technique to implement in the workflow of clinical microbiology laboratories. IMPORTANCE: The assessed rapid assay to detect CTX-M beta-lactamases and carbapenemases directly from clinical samples can favor in the rapid detection of these mechanisms of resistance and hence the administration of a more adequate antimicrobial treatment.

Detection of rare carbapenemases in Enterobacterales-comparison of two colorimetric and three CIM-based carbapenemase assays.

Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) is crucial for prompt treatment and infection control. Most assays target the primary four enzymes (KPC, OXA-48-like, VIM, and NDM), often missing less common variants (e.g., GES, IMI, OXA-23, and OXA-58). Therefore, assays based on the hydrolysis of carbapenems are recommended in addition to differentiation tests such as PCR or immunochromatographic assays. The aim of this study was to compare the currently Clinical and Laboratory Standards Institute (CLSI)-recommended tests mCIM (modified carbapenem inactivation method) and Carba NP with new colorimetric tests (NitroSpeed-Carba NP) and novel variations of the carbapenem inactivation method (CIM) such as simplified CIM (sCIM) or modified zinc-supplemented CIM (mzCIM). The challenge collection included 205 clinical isolates, 139 CPE vs 66 non-CPE. Among all 205 isolates, the sensitivity/specificity of mCIM was 81.3%/98.5%, Carba NP 76.3%/100%, NitroSpeed-Carba NP 86.3%/78.8%, sCIM 100%/94%, and mzCIM 97.8%/98.5%. For rare carbapenemases (n = 48), the sensitivity of mzCIM (98.3%) and sCIM (100%) was higher than that of mCIM (60.4%), Carba NP (50%), or NitroSpeed-Carba NP (70.2%). Most indeterminate results occurred for mCIM (14.4%), Carba NP (8.2%), and sCIM (6.3%). The detection of rare carbapenemases remains challenging with the currently recommended assays. The CIM-based tests demonstrated superior sensitivity, with sCIM and mzCIM outperforming the currently recommended mCIM and Carba NP, especially among isolates with weakly hydrolyzing carbapenemases (e.g., OXA-23 and OXA-58). Although colorimetric assays provide more rapid results, laboratories have to be aware of the low sensitivity for rare carbapenemases. Both sCIM and the new mzCIM performed well, are cost-effective, and can easily be implemented in any laboratory.IMPORTANCEDetection of so-called rare carbapenemases (e.g., GES, IMI, OXA-23, and OXA-58) in Enterobacterales is challenging, and data on the performance of currently available assays are scarce. This study systematically assessed the performance of currently recommended and novel hydrolysis-based assays on a set of molecularly characterized isolates. It demonstrates that the currently recommended assays mCIM and Carba NP perform well on isolates producing common carbapenemases such as KPC, VIM, NDM, and OXA-48, but have only a moderate sensitivity in the detection of rare carbapenemases. In contrast, the newer CIM-based variants, sCIM and mzCIM, are equally capable of detecting frequent and uncommon carbapenemases. These assays could potentially help to improve our knowledge on the epidemiology of these "rare" enzymes.

The role of the plasmid-mediated fluoroquinolone resistance genes as resistance mechanisms in pediatric infections due to Enterobacterales.

Introduction: Fluoroquinolones (FQs) are not commonly prescribed in children, yet the increasing incidence of multidrug-resistant (MDR) Enterobacterales (Ent) infections in this population often reveals FQ resistance. We sought to define the role of FQ resistance in the epidemiology of MDR Ent in children, with an overall goal to devise treatment and prevention strategies. Methods: A case-control study of children (0-18 years) at three Chicago hospitals was performed. Cases had infections by FQ-susceptible, ß-lactamase-producing (bla) Ent harboring a non- or low-level expression of PMFQR genes (PMFQS Ent). Controls had FQR infections due to bla Ent with expressed PMFQR genes (PMFQR Ent). We sought bla genes by PCR or DNA (BD Max Check-Points assay®) and PMFQR genes by PCR. We performed rep-PCR, MLST, and E. coli phylogenetic grouping. Whole genome sequencing was additionally performed on PMFQS Ent positive isolates. Demographics, comorbidities, and device, antibiotic, and healthcare exposures were evaluated. Predictors of infection were assessed. Results: Of 170 ß-lactamase-producing Ent isolates, 85 (50%) were FQS; 23 (27%) had PMFQR genes (PMFQS cases). Eighty-five (50%) were FQR; 53 (62%) had PMFQR genes (PMFQR controls). The median age for children with PMFQS Ent and PMFQR Ent was 4.3 and 6.2 years, respectively (p = NS). Of 23 PMFQS Ent, 56% were Klebsiella spp., and of 53 PMFQR Ent, 76% were E. coli. The most common bla and PMFQR genes detected in PMFQS Ent were bla SHV ESBL (44%) and oqxAB (57%), and the corresponding genes detected in PMFQR Ent were bla CTX-M-1-group ESBL (79%) and aac(6')-Ib-cr (83%). Whole genome sequencing of PMFQS Ent revealed the additional presence of mcr-9, a transferable polymyxin resistance gene, in 47% of isolates, along with multiple plasmids and mobile genetic elements propagating drug resistance. Multivariable regression analysis showed that children with PMFQS Ent infections were more likely to have hospital onset infection (OR 5.7, 95% CI 1.6-22) and isolates containing multiple bla genes (OR 3.8, 95% CI 1.1-14.5). The presence of invasive devices mediated the effects of healthcare setting in the final model. Differences in demographics, comorbidities, or antibiotic use were not found. Conclusions: Paradoxically, PMFQS Ent infections were often hospital onset and PMFQR Ent infections were community onset. PMFQS Ent commonly co-harbored multiple bla and PMFQR genes, and additional silent, yet transferrable antibiotic resistance genes such as mcr-9, affecting therapeutic options and suggesting the need to address infection prevention strategies to control spread. Control of PMFQS Ent infections will require validating community and healthcare-based sources and risk factors associated with acquisition.

Molecular epidemiology of clinical isolation of carbapenem-resistant Enterobacterales and application of carbapenemase inhibitor enhancement test. / ä¸´åºŠåˆ†ç¦»ç¢³é’éœ‰çƒ¯ç±»è€è¯è‚ æ†èŒç›®ç»†èŒèŒæ ªçš„åˆ†å­æµè¡Œç— å­¦åŠç¢³é’éœ‰çƒ¯é ¶æŠ‘åˆ¶å‰‚å¢žå¼ºè¯•éªŒçš„åº”ç”¨.

OBJECTIVES: The prevalence of carbapenem-resistant Enterobacterales (CRE) presents a significant challenge in clinical anti-infective treatment. This study aims to investigate drug resistance and the molecular epidemiological characteristics of CRE in our area. Additionally, we seek to evaluate practicality of utilizing carbapenemase inhibitor enhancement test in clinical laboratory. METHODS: Non-repeated CREs isolated from clinical specimens at Xiangya Hospital, Central South University, were collected. Minimum inhibitory concentration (MIC) combined with Kirby-Bauer (KB) assay was used to detect the drug susceptibility of the strains, and 13 carbapenemase-producing genes were detected by PCR. The phenotype of 126 strains of carbapenemase-producing Enterobacterales identified by PCR was detected by the carbapenemase inhibitor enhancement test to understand the agreement between the method and the gold standard PCR results. RESULTS: Among 704 CRE strains examined, we observed significant drug resistance in 501 strains dentified as carbapenemase-producing Enterobacterales (CPE). Klebsiella pneumoniae was the predominant CPE strain, followed by Enterobacter cloacae and Escherichia coli. A total of 9 carbapenemase types were detected, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-ß-lactamase (NDM), Verona integron- encoded metallo-ß-lactamases (VIM), imipenemase (IMP), oxacillinase-48 (OXA-48), and rare imipenem-hydrolyzing ß-lactamase (IMI), adelaide imipenemase (AIM), Bicêtre carbapenemase (BIC), and guiana extended-spectrum ß-lactamase (GES). The detection rate of KPC serine carbapenemase was 61.7% (309/501). The carbapenemase inhibitor enhancement test exhibited a 100% consistency rate for the strains producing Class A serine carbapenemase and/or Class B metallo-ß-lactamases. CONCLUSIONS: CRE strains in Changsha, Hunan, China, are wide distribution and exhibit carbapenemase production. The main mechanism of carbapenem resistance in these bacterias is predominatly attributed to the production of KPC serine carbapenemase. The presence of GES and IMI genes carried by Enterobacterales has been detected for the first time in this region. The carbapenemase inhibitor enhancement test has been proven to be an accurate method for detecting CRE producing Class A serine carbapenemase and/or Class B metallo-ß-lactamases. This method offers simpicity of operation and ease of results interpretation, making it weel-suited meeting the clinical microbiology laboratory's reguirements for the detection of serine carbapenemase and metallo-ß-lactamases.

Antibiotic resistance and genetic diversity among Pseudomonas aeruginosa isolated from urinary tract infections in Iran.

Aims: To determine the antibiotic resistance and genetic diversity of Pseudomonas aeruginosa isolates. Methods: The antibiotic resistance, genetic diversity and the conjugate transformation among Pseudomonas aeruginosa collected from patients with urinary tract infection in Tehran, Iran, was investigated. Results: Antibiotic resistance against cefepime was seen in 51.74% of the isolates, followed by amikacin (47.76%). blaOXA-10 and blaVIM were the most prevalent extended-spectrum ß-lactamase and metallo-ß-lactamases genes, respectively. Five clusters (C1-C5) were obtained by pulse field gel electrophoresis and multilocus sequence typing revealed two strain types, ST235 and ST664. Conjugation detected blaOXA-48 and blaNDM genes were transferred to Escherichia coli K12. Conclusion: The resistance of P. aeruginosa to antibiotics is increasing, which highlights the need to determine the resistance patterns to design better treatment strategies.

[Evaluation of two phenotypic techniques and a commercial multiplex molecular panel for the detection of different carbapenemases]. / Evaluación de dos técnicas fenotípicas y un panel molecular multiplex comercial para la detección de diferentes carbapenemasas.

OBJECTIVE: The prevalence of carbapenemase-producing Enterobacterales has increased in recent years and is considered an important public health problem. METHODS: A total of 106 clinical samples were analyzed by different carbapenemase detection techniques: inhibition discs (ID), immunochromatographic test (ICT) and a genotypic method, comparing them with a multiplex RT-PCR as a reference method. RESULTS: Overall, all 3 techniques exceeded 90% sensitivity, although with differences in the performance of some of them by carbapenemase type. DI had low specificity (62%) for OXA-48, while with TIC the sensitivity for NDM-type metallo-beta-lactamase (93%) was slightly lower than for OXA-48 (95%). The best results were obtained with the genotypic technique (100% overall performance). CONCLUSIONS: Despite the lower sensitivity of TICs (especially in NDM carbapenemases) compared to molecular techniques, with the modification of the protocol we managed to increase this sensitivity and, together with the lower price, simplicity and speed, it makes this technique a good screening option.

Characterization of carbapenemase-producing Gram-negative bacilli: first report of blaNDM-1 in Enterobacter cloacae.

INTRODUCTION: The spread of multidrug-resistant bacteria, particularly carbapenem-resistant Gram-negative bacilli (CR-GNB), has become a serious challenge for clinicians due to limited therapeutic options. The aim of the study was to investigate the prevalence of carbapenemase production among clinical isolates recovered from 352 samples collected in Tebessa hospital, Algeria. METHODOLOGY: Bacterial isolates were identified by 16S RNA gene sequencing and susceptibility to antibiotics was determined by disk diffusion method. Carbapenem-resistant isolates were screened for carbapenemase production using modified carba Nordmann-Poirel test, modified Hodge test and imipenem-EDTA combined disc test. Extended-spectrum ß-lactamases (ESBL) were detected using double-disk synergy test. Molecular characterization of carbapenemases and ESBL genes was performed by polymerase chain reaction (PCR) and sequencing. RESULTS: A total of 85 Gram-negative bacilli isolates were recovered mainly from urine samples and were identified as: Klebsiella pneumoniae (17.65%), Serratia odorifera (15.29%), Escherichia coli (12.94%), Raoultella ornithinolytica, Enterobacter cloacae (11.76%), Serratia marcescens (10.59%), Morganella morganii (7.06%), Proteus mirabilis (5.88%), Acinetobacter baumannii (4.70%) and Pseudomonas aeruginosa (2.35%). All strains were resistant or intermediate to imipenem and/or ertapenem. ESBL, carbapenemase and metallo-beta-lactamases (MBL) phenotypes were detected in 19 (22.35%), 9 (10.59%) and 2 (2.35%) GNB isolates, respectively. PCR results in nine carbapenemase-producing GNB strains chosen showed the presence of one to four carbapenemase genes (blaGES, blaSME, blaNDM-1, blaVIM, blaGIM, blaSPM, blaOXA-48) in four strains; however, seven strains had at least one ESBL gene (blaTEM-1, blaCTXM-15, blaSHV). CONCLUSIONS: In this study, we report the first incidence of blaNDM-1 gene in Enterobacter cloacae isolated from urine sample in Algeria.

Resist Acineto rapid immunological test for the detection of acquired carbapenemase producers among Acinetobacter spp.

The Resist Acineto from Coris Bioconcept is a novel immunochromatographic test for detection of the major acquired carbapenemases (OXA-23, OXA-40, OXA-58, and NDM) identified in Acinetobacter spp. This rapid and easy-to-perform test showed an excellent specificity and sensitivity, with positive and negatives predictive values of 100% in both cases.

Evaluation of real-time PCR and flow cytometry efficiency in rapid detection of carbapenemase-producing Enterobacteriales.

INTRODUCTION: Infections due to carbapenem-resistant Enterobacteriales, which have increased worldwide in recent years, cause concern. This study aimed to rapidly detect carbapenemase gene region by using flow cytometry in Enterobacteriales isolates and to evaluate its efficiency and susceptibility by comparing it with polymerase chain reaction (PCR). METHODOLOGY: In the study, 21 isolates obtained from the blood cultures of patients hospitalized in intensive care units and found to intermediate or resistant to at least one carbapenem in the automated system, and 14 isolates belonging to the carbapenem-susceptible Enterobacteriales family were included. Carbapenemase gene regions were investigated by PCR after their susceptibility was determined by disk diffusion method. Bacterial suspensions were treated with meropenem + specific carbapenemase inhibitors (EDTA or APBA) and Temocillin and stained with thiazole orange (TO) and propidium iodide (PI) to show dead/live cell differentiation. Dead/live cell percentages were calculated after reading on the flow cytometer device. RESULTS: In the ROC analysis of the flow cytometry method, the cut-off value, specificity, and susceptibility of PI staining rates for meropenem were found as 14.37%, 100%, and 65%, respectively. It was found that the flow cytometry method was well-compatible with PCR in the detection of the carbapenemase gene region. CONCLUSIONS: Flow cytometry will continue to be a promising method for the detection of antimicrobial susceptibility and resistance due to its rapid analysis of many cells and its high compatibility with PCR results.

Utility of the MBT STAR-Cepha kit in detecting extended-spectrum ß-lactamase producers in clinical urine samples and positive blood cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Urinary tract infection (UTI) and bloodstream infection (BSI) caused by extended-spectrum ß-lactamase (ESBL)-producing bacteria are important healthcare problems. Appropriate infection management requires the direct detection of organisms in clinical specimens. We assessed the capability of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based MBT STAR-Cepha kit to detect ESBL producers in clinical urine and blood samples. Over a 1-year period, 90 urine samples and 55 positive monomicrobial (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, or Proteus mirabilis) blood cultures were collected from patients with UTI or BSI at Hamamatsu University Hospital. ß-lactamase activity in these samples was directly detected using the MBT STAR-Cepha kit, and the results were compared with those of antimicrobial susceptibility testing and polymerase chain reaction detection assay for the isolates. In receiver operating characteristic curve analysis, the kit assay showed low accuracy in detecting ESBL producers in urine samples (area under the curve [AUC], 0.69). Meanwhile, the AUC for detecting all ESBL-producing bacteria in positive blood cultures was 0.81. The kit assay detected cefotaxime (CTX) resistance (mainly in CTX-M-type ESBL producers) with high accuracy in positive blood cultures; however, it did not accurately detect ESBL producers in urine samples and CTX-susceptible isolates with other ESBL-associated genes (e.g., TEM and SHV types) in positive blood cultures. MBT STAR-Cepha testing can accurately discriminate CTX-resistant ESBL producers in BSI cases and thus can contribute to effective infection management. The results suggest that different sample types, antibiotic resistance profiles, and resistance genes can affect the kit performance.

Comparing the broth enrichment-multiplex lateral flow immunochromatographic assay with real time quantitative PCR for the rapid detection of carbapenemase-producing organisms in rectal swabs.

BACKGROUND: Rapid and accurate identification of carbapenemase-producing organism (CPO) intestinal carriers is essential for infection prevention and control. Molecular diagnostic methods can produce results in as little as 1 h, but require special instrumentation and are expensive. Therefore, it is urgent to find an alternative method. The broth enrichment-multiplex lateral flow immunochromatographic assay was recently reported, but using it to directly detect CPO intestinal carriers in rectal swabs still requires the evaluation of many samples. The aim of this study was to compare the performance of these two methods, and to explore the control measures of CPO infection. METHODS: Through CPO selective culture, PCR and DNA sequencing, 100 rectal swabs confirmed to be CPO-positive and 100 rectal swabs with negative results were collected continuously. After eluting the rectal swabs with saline, three aliquots were used: one for counting, one for detection by Xpert Carba-R, and one for culture in broth for 0 h, 1 h, 2 h, 3 h and 4 h, followed by NG-Test CARBA 5 assessment. The sensitivity and specificity of the NG-Test CARBA 5 method after different incubation times were calculated. The limit of detection (LoD) of this assay after 4 h broth incubation was estimated by examining the bacterial suspensions and simulated faecal suspensions prepared with CPOs producing different types of carbapenemases. RESULTS: Xpert Carba-R demonstrated a combined sensitivity of 99.0% and specificity of 98.0%. The sensitivity and specificity were higher than 90.0% for the different enzyme types. The specificities of five common carbapenemases detected by the broth enrichment NG-Test CARBA 5 combined method after different incubation times were 100%. The sensitivities increased with increasing incubation time. At 4 h, the Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), imipenemase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM), and oxacillinase (OXA) -48 detection sensitivities were 93.0%, 96.3%, 100%, 100% and 85.7%, respectively. The LoDs were between 102 and 104 CFU/mL for all five enzymes after 4 h of incubation. CONCLUSIONS: This investigation highlighted that the broth enrichment-multiplex lateral flow immunochromatographic assay can be used as a new method for screening CPOs in rectal swabs.

One Health compartment analysis of ESBL-producing Escherichia coli reveals multiple transmission events in a rural area of Madagascar.

BACKGROUND: ESBL-producing Escherichia coli (ESBL-Ec) is considered a key indicator for antimicrobial resistance (AMR) epidemiological surveillance in animal, human and environment compartments. There is likelihood of ESBL-Ec animal-human transmission but proof of cross-compartment transmission is still unclear. OBJECTIVES: To characterize ESBL-Ec genetic similarity in various compartments (humans, animals and environment) from a rural area of Madagascar. METHODS: We collected ESBL-Ec isolates prospectively from humans, animals and the environment (water) between April and October 2018. These isolates were subject to WGS and analysed with cutting-edge phylogenomic methods to characterize population genetic structure and infer putative transmission events among compartments. RESULTS: Of the 1454 samples collected, 512 tested positive for ESBL-Ec. We successfully sequenced 510 samples, and a phylogenomic tree based on 179 365 SNPs was produced. Phylogenetic distances between and amongst compartments were indistinguishable, and 104 clusters of recent transmission events between compartments were highlighted. Amongst a large diversity of ESBL-Ec genotypes, no lineage host specificity was observed, indicating the regular occurrence of ESBL-Ec transfer among compartments in rural Madagascar. CONCLUSIONS: Our findings stress the importance of using a phylogenomic approach on ESBL-Ec samples in various putative compartments to obtain a clear baseline of AMR transmissions in rural settings, where one wants to identify risk factors associated with transmission or to measure the effect of 'One Health' interventions in low- and middle-income countries.

Proteus mirabilis - analysis of a concealed source of carbapenemases and development of a diagnostic algorithm for detection.

OBJECTIVES: To analyse carbapenemases in Proteus mirabilis and assess the performance of carbapenemase detection assays. METHODS: Eighty-one clinical P. mirabilis isolates with high-level resistance at least to ampicillin (>32 mg/L) or previous detection of carbapenemases were selected and investigated by three susceptibility testing methods (microdilution, automated susceptibility testing, and disk diffusion), six phenotypic carbapenemase assays (CARBA NP, modified carbapenemase inactivation method [CIM], modified zinc-supplemented CIM, simplified CIM, faropenem, and carbapenem-containing agar), two immunochromatographic assays, and whole-genome sequencing. RESULTS: Carbapenemases were detected in 43 of 81 isolates (OXA-48-like [n = 13]; OXA-23 [n = 12]; OXA-58 [n = 12]; New Delhi metallo-ß-lactamase (NDM) [n = 2]; Verona integron-encoded metallo-ß-lactamase (VIM) [n = 2]; Imipenemase (IMP) [n = 1]; Klebsiella pneumoniae carbapenemase (KPC) [n = 1]). Carbapenemase-producing Proteus were frequently susceptible to ertapenem (26/43; 60%), meropenem (28/43; 65%), ceftazidime (33/43; 77%), and some even to piperacillin-tazobactam (9/43; 21%). Sensitivity/specificity of phenotypic tests were 30% (CI: 17-46%)/89% (CI: 75-97%) for CARBA NP, 74% (CI: 60-85%)/82% (CI: 67-91%) for faropenem, 91% (CI: 78-97%)/82% (CI: 66-92%) for simplified CIM, and 93% (CI: 81-99%)/100% (CI: 91-100%) for modified zinc-supplemented CIM. An algorithm for improved detection was developed, which demonstrated sensitivity/specificity of 100% (CI: 92-100%)/100% (CI: 91-100%) on the 81 isolates, and 100% (CI: 29-100%)/100% (CI: 96-100%) in a prospective analysis of additional 91 isolates. Interestingly, several OXA-23-producing isolates belonged to the same clonal lineage reported previously from France. DISCUSSION: Current susceptibility testing methods and phenotypic tests frequently fail to detect carbapenemases in P. mirabilis, which could result in inadequate antibiotic treatment. In addition, the non-inclusion of blaOXA-23/OXA-58 in many molecular carbapenemase assays further impedes their detection. Therefore, the prevalence of carbapenemases in P. mirabilis is likely underestimated. With the herein proposed algorithm, carbapenemase-producing Proteus can be easily identified.