Phenotypic and Genetic Characteristics of Carbapenemase-Producing Enterobacterales Isolates at Okayama University Hospital.

Spread of carbapenemase-producing Enterobacterales (CPE) is an ongoing public health issue worldwide, including in Japan. In this study, we investigated the phenotypic and genetic characteristics of CPE isolates at Okayama University Hospital over the 5 years (2013-2018) prior to the outbreak of the 2019 coronavirus pandemic. Of 24 carbapenem-resistant Enterobacterales isolated during the study period, we identified 8 CPE isolates harboring blaIMP-1 (5 isolates) and blaIMP-6 genes (3 isolates). Bacterial species and carbapenem susceptibility patterns exhibited diversity. Minimum inhibitory concentrations (MICs) of meropenem were generally higher than those of imipenem and biapenem. Results of pulsed-field gel electrophoresis demonstrated that neither clonal nor plasmid-mediated outbreaks of blaIMP-harboring CPE isolates have developed at our hospital. One Klebsiella oxytoca isolate showed a high MIC (128 µg/mL) of meropenem, which could be explained by the high plasmid copy number. Subsequent analysis of this isolate may elucidate the intricacies of carbapenem resistance profiles among CPE isolates. Collectively, our findings underscore the necessity for ongoing genetic surveillance of CPE, complemented by tailored approaches for infection prevention and control.

Molecular Epidemiology of Carbapenem-Resistant Klebsiella aerogenes in Japan.

Information regarding Klebsiella aerogenes haboring carbapenemase in Japan is limited. A comprehensive nationwide survey was conducted from September 2014 to December 2022, and 67 non-duplicate strains of carbapenem-resistant K. aerogenes were isolated from 57 healthcare facilities in Japan. Through genetic testing and whole-genome sequencing, six strains were found to possess carbapenemases, including imipenemase (IMP)-1, IMP-6, New Delhi metallo-ß-lactamase (NDM)-1, and NDM-5. The strain harboring blaNDM-5 was the novel strain ST709, which belongs to the clonal complex of the predominant ST4 in China. The novel integron containing blaIMP-1 featured the oxacillinase-101 gene, which is a previously unreported structure, with an IncN4 plasmid type. However, integrons found in the strains possessing blaIMP-6, which were the most commonly identified, matched those reported domestically in Klebsiella pneumoniae, suggesting the prevalence of identical integrons. Transposons containing blaNDM are similar or identical to the transposon structure of K. aerogenes harboring blaNDM-5 previously reported in Japan, suggesting that the same type of transposon could have been transmitted to K. aerogenes in Japan. This investigation analyzed mobile genetic elements, such as integrons and transposons, to understand the spread of carbapenemases, highlighting the growing challenge of carbapenem-resistant Enterobacterales in Japan and underscoring the critical need for ongoing surveillance to control these pathogens.

Detection and characterization of carbapenem resistant Gram-negative bacilli isolates recovered from hospitalized patients at Soba University Hospital, Sudan.

BACKGROUND: Antimicrobial resistance (AMR) poses a complex threat to global health security and universal health coverage. Recently, nosocomial infections with carbapenemase-producing Gram-negative bacilli (GNB) is increasing worldwide. We report the molecular characterization and detection of genes associated with carbapenemase producing Gram negative bacteria isolated from hospitalized patients at Soba University Hospital (SUH) in Khartoum State, Sudan. RESULTS: Between October 2016 and February 2017, a total of 206 GNB clinical specimens were collected from hospitalized patients in SUH. Of 206 carbapenem resistance isolates, 171 (83 %) were confirmed as phenotypically resistant and 121 (58.7 %) isolates harboured one or more carbapenemase genes. New Delhi metallo-ß-lactamase (NDM) types were the most predominant genes, blaNDM 107(52 %), followed by blaIMP 7 (3.4 %), blaOXA-48 5(2.4 %) and blaVIM 2 (0.9 %). Co-resistance genes with NDM producing GNB were detected in 87 (81.3 %) of all blaNDM producing isolates. NDM-1 was the most frequent subtype observed in 75 (70 %) blaNDM producing isolates. The highest percentage of resistance was recorded in ampicillin (98 %), cephalexin (93.5 %) amoxicillin clavulanic acid (90 %), cefotaxime (89.7 %), ceftriaxone (88.4 %), ceftazidime (84.2 %), sulfamethoxazole-trimethoprim (78.4 %) and nitrofurantoin (75.2 %), aztreonam (66 %) and temocillin (64 %). A close correlation between phenotypic and carbapenemase genes detection in all GNB was observed. CONCLUSIONS: The frequency of carbapenemase producing bacilli was found to be high in SUH. NDM was found to be the most prevalent carbapenemase gene among clinical isolates. Close surveillance across all hospitals in Sudan is required. The relative distribution of carbapenemase genes among GNB in nosocomial infections in Africa needs to be defined.

Performance of the BD Phoenix CPO detect assay for detection and classification of carbapenemase-producing organisms.

Increasing worldwide, prevalence of carbapenem-resistant gram-negative bacteria demands urgent a need for rapid detection and accurate identification of carbapenemases. The BD Phoenix CPO detect (PCD) assay possesses an in-built capacity for parallel susceptibility testing and detection of carbapenemases. Here, the ability of the assay to detect and classify carbapenemase production was tested in a collection of carbapenem-resistant Enterobacterales and non-fermentative gram-negative rods. The ability of the PCD assay to detect and classify carbapenemases was investigated in a collection of 194 clinical, carbapenem-resistant isolates (Enterobacterales [n = 65]; non-fermentative gram-negative rods [n = 129]). AST results were compared to MICS determined by gradient diffusion to determine accuracy of the PCD assay. The accuracy of the PCD assay to detect carbapenemases was compared to the results of molecular isolate characterization using a LDT multiplex carbapenemase PCR assay. All 194 isolates classified as carbapenem-resistant by reference susceptibility testing were also classified correctly as CRO by the PCD assay. Performance analysis of the PCD assay to detect carbapenemase production revealed an overall sensitivity of 98.29% and specificity of 17.95% for the detection of carbapenemase production. For the classification of carbapenemases classes A, B, and D, the PCD correctly classified 79.17% Enterobacterales and 67.16% non-fermentative gram-negative rods. The PCD assay is a reliable tool for the detection of carbapenem resistance and allows for parallel analysis of carbapenemase production. However, while sensitivity is high, low specificity in carbapenemase detection and erroneous classification demands mandatory confirmation by alternative methods, especially in non-fermentative gram-negative bacteria.

Multicenter Evaluation of the BD Phoenix CPO Detect Test for Detection and Classification of Carbapenemase-Producing Organisms in Clinical Isolates.

Limited treatment options contribute to high morbidity/mortality rates with carbapenem-resistant, Gram-negative bacterial infections. New approaches for carbapenemase-producing organism (CPO) detection may help inform clinician decision-making on patient treatment and infection control. BD Phoenix CPO detect (CPO detect) detects and classifies carbapenemases in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa during susceptibility testing. The clinical performance of CPO detect is reported here. Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates were evaluated across three sites using CPO detect and a composite reference method (RM); the latter was comprised of the modified carbapenem inactivation method and a MIC screen for ertapenem, imipenem, and meropenem. Multiplex PCR testing was also utilized for Ambler class determination. Positive and negative percentages of agreement (PPA and NPA, respectively) between CPO detect and the RM were determined. The PPA and NPA for Enterobacterales were 98.5% (confidence intervals, 96.6%, 99.4%) and 97.2% (95.8%, 98.2%), respectively. The A. baumannii PPA and NPA, respectively, were 97.1% (90.2%, 99.2%) and 97.1% (89.9%, 99.2%). The P. aeruginosa PPA and NPA, respectively, were 95.9% (88.6%, 98.6%) and 92.3% (86.7%, 95.6%). The PPA values for carbapenemase class designations for all organisms combined and Enterobacterales alone, respectively, were 95.3% (90.2%, 97.8%) and 94.6% (88.8%, 97.5%) for class A, 94.0% (88.7%, 96.6%) and 96.4% (90.0%, 98.8%) for class B, and 95.0% (90.1%, 97.6%) and 99.0% (94.4%, 99.8%) for class D carbapenemases. NPA values for all organisms and Enterobacterales alone ranged from 98.5% to 100%. CPO detect provided accurate detection and classification of CPOs for the majority of isolates of Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa tested.

First Report of the Carbapenemase Gene blaOXA-499 in Acinetobacter pittii.

We identified the carbapenemase gene blaOXA-499, a variant of blaOXA-143, from a clinical isolate of Acinetobacter pittii for the first time. OXA-499 shared 93.1% amino acid identity with OXA-143, and the gene was located on the chromosome. By cloning the OXA-499-encoding gene into the pWH1266 vector and transforming it into susceptible Acinetobacter spp., we were able to show that OXA-499 confers resistance to carbapenems.

Using Quantitative Polymerase Chain Reaction (qPCR) to Identify a Myriad of Carbapenemase Genes in Fresh Cow Dung in Bangladesh.

Introduction The emergence of antimicrobial resistance (AMR) is driven by the selection pressure of frequent uses of antimicrobial agents in healthcare, the food chain, agriculture, fishery, and the food animal industry, which poses a serious health risk for transmission-linked humans and the surrounding environment. Livestock, particularly cattle, play an essential role in the food sector in Bangladesh. The food-animal chains can be the potential routes of exposure to AMR-microorganisms for every domain of one health. Antimicrobial resistance genes (ARGs) can impart a reservoir of AMR within the food supply chain, even without pathogenic microorganisms. This study investigated the history of infection for the last six-month period of antimicrobials utilized in cattle farms and the distribution of selected carbapenemase resistance genes, namely, bla-KPC, bla-IMP, bla-VIM, bla-NDM-1, bla-SIM, bla-GIM, bla-SPM, and bla-SME, in cattle feces in Bangladesh. Methods A cross-sectional study was designed to analyze ARGs in fresh cow dung samples collected from commercial farms and individual houses in four Bangladesh districts, namely, Dhaka, Gazipur, Manikganj, and Tangail. Types of cattle breeds, their existing diseases, recent antimicrobial uses, and vaccine uses were recorded. DNA was extracted from each cow dung sample using commercial kits (Qiagen GmbH, Germany). Real-time quantitative polymerase chain reaction (RT-qPCR) was employed to assess the eight carbapenem resistance genes in the extracted DNA. The eight carbapenem resistance genes in the extracted DNA were assessed by RT-qPCR using the qTOWER3 thermal cycler (Analytik Jena GmbH, Konrad-Zuse-Straße 1, 07745 Jena, Germany). Results Group A carbapenemase, bla-KPC, was detected in 66.7% of the samples. However, no bla-SME was identified in all of the test samples. Group B metallo carbapenemase, bla-IMP, bla-NDM-1, bla-VIM, bla-SIM, bla-GIM, and bla-SPM, were in 66.7% (80/120), 49.2% (59/120), 48.3% (58/120), 68.3% (82/120), 58.3% (70/120), and 12.5% (15/120), respectively. Only 8.3% of the tested samples contained no MBL gene; 10% carried a single-type carbapenemase gene; and the remaining 81.7% carried two or more carbapenemase genes concurrently. Co-carriage of four or more genes was found in over 59% of samples. As many as seven genes were found together in 6.7% of samples. ARG detection in commercial cattle samples and household feces is not statistically significant. Conclusions Substantial carbapenem-resistance ARGs were detected in commercially farmed cow dung and household cattle samples. Frequent use of antibiotics for cattle for treatment and prophylactic purposes may influence the high acquisition of ARGs. Bangladeshi cattle farms are reservoirs and routes of AMR, posing a significant threat to the country's public health.

Surveillance in a Neonatal Intensive Care Unit Allowed the Isolation of a Strain of VIM-Producing Pantoea brenneri.

Here, we describe the isolation of a strain of the genus Pantoea encoding a VIM carbapenemase, the first to our knowledge. The strain, isolated from a rectal swab of a 10-day-old newborn admitted to a neonatal intensive care unit (NICU), was identified through whole-genome sequencing analyses as Pantoea brenneri. The strain harbored the carbapenemases gene blaVIM-1. The prompt application of contact measures and the isolation of the newborn prevented the dissemination of VIM-producing P. brenneri and of the plasmid carrying the VIM-1 gene to other newborns.

Genetic Diversity of Polymyxin-Resistance Mechanisms in Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae: a Multicenter Study in China.

Polymyxin has been the last resort to treat multidrug-resistant Klebsiella pneumonia. However, recent studies have revealed that polymyxin-resistant carbapenem-resistant Klebsiella pneumonia (PR-CRKP) emerged due to the mutations in chromosomal genes or the plasmid-harboring mcr gene, leading to lipopolysaccharide modification or efflux of polymyxin through pumps. Further surveillance was required. In the present study we collected PR-CRKP strains from 8 hospitals in 6 provinces/cities across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features by whole-genome sequencing (WGS). The broth microdilution method (BMD) was performed to determine the MIC of polymyxin. Of 662 nonduplicate CRKP strains, 15.26% (101/662) were defined as PR-CRKP; 10 (9.90%) were confirmed as Klebsiella quasipneumoniae by WGS. The strains were further classified into 21 individual sequence types (STs) by using multilocus sequence typing (MLST), with ST11 being prevalent (68/101, 67.33%). Five carbapenemase types were identified among 92 CR-PRKP, blaKPC-2 (66.67%), blaNDM-1 (16.83%), blaNDM-5 (0.99%), blaIMP-4 (4.95%), and blaIMP-38 (0.99%). Notably, 2 PR-CRKP strains harbored both blaKPC-2 and blaNDM-1. The inactivation of mgrB, associated significantly with high-level polymyxin resistance, was mainly caused by the insertion sequence (IS) insertion (62.96%, 17/27). Furthermore, acrR was inserted coincidently by ISkpn26 (67/101, 66.33%). The deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47 (capsule locus types), and diverse mutations of the ramR gene were identified. Only one strain carried the mcr gene. In summary, the high IS-inserted mgrB inactivation, the close relationship between ST11 and the deletion or splicing mutations of the crrCAB, and the specific features of PR-K. quasipneumoniae constituted notable features of our PR-CRKP strains in China. IMPORTANCE Polymyxin-resistant CRKP is a serious public health threat whose resistance mechanisms should be under continuous surveillance. Here, we collected 662 nonduplicate CRKP strains across China to identify the carbapenemase and polymyxin resistance genes and epidemiological features. Polymyxin resistance mechanism in 101 PR-CRKP strains in China were also investigated, 9.8% of which (10/101) were K. quasipneumoniae, as determined via WGS, and inactivation of mgrB remained the most crucial polymyxin resistance mechanism, significantly related to high-level resistance. Deletion or splicing mutations of crrCAB were significantly associated with ST11 and KL47. Diverse mutations of the ramR gene were identified. The plasmid complementation experiment and mRNA expression analysis further confirmed that the mgrB promoter and ramR played a critical role in polymyxin resistance. This multicenter study contributed to the understanding of antibiotic resistance forms in China.

Performance of a loop-mediated isothermal amplification assay (Isoplex CRE-ART) to detect common carbapenemase-encoding genes in Gram-negative bacteria.

Carbapenem-resistant Gram-negative bacteria (CR-GNB) are a major source of nosocomial infections worldwide. In this study, the ability of a loop-mediated isothermal amplification (LAMP)-based method (Isoplex CRE-ART) to rapidly detect carbapenemase-encoding genes bla OXA-48-like, bla OXA-23-like, bla OXA-24-like, bla KPC, bla VIM, bla NDM and bla IMP in 231 carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii isolates was investigated. The accuracy of the LAMP test was compared to results of molecular isolate characterization using a Laboratory Developed Test multiplex carbapenemase PCR assay. The LAMP test correctly identified the presence of on-panel carbapenemases with a sensitivity of 99.16 % [95 % confidence interval (CI): 95.39-99.96 %] and a specificity of 98.21 % (95 % CI: 93.72-99.68 %) in 60 min. Our findings suggest that the Isoplex CRE-ART assay is able to rapidly identify carbapenemase genes in CR-GNB and improves options for pathogen characterization in the context of clinical microbiological and infection control diagnostics.

Carbapenemase detection by NG-Test CARBA 5-a rapid immunochromatographic assay in carbapenem-resistant Enterobacterales diagnosis.

BACKGROUND: The global spread of carbapenem-resistant Enterobacterales (CRE) represents a serious public health concern as these organisms are associated with limited treatment options, high mortality rate and rapid transmissibility. The identification of carbapenemase remains a challenge in microbiological laboratories as no single method is perfect when considering cost, carbapenemase coverage, accuracy, handling complexity and TATs together. METHODS: NG-Test CARBA 5 assay and modified carbapenem inactivation method in conjunction with EDTA carbapenem inactivation method (mCIM/eCIM) were challenged with a collection of 299 molecularly characterized CRE isolates in China in order to evaluate the performance in detecting five major carbapenemases (bla KPC, bla NDM, bla VIM, bla IMP, and bla OXA-48) among Enterobacterales. RESULTS: NG-Test CARBA 5 detected all KPC-, NDM-, VIM- and OXA-48-producing isolates perfectly with a weak false-positive signal for NDM in an IMP-4 producer, which makes the specificity for NDM decreases to 99.6%. The overall specificity/sensitivity were 99.9%/100% for NG-Test CARBA 5. mCIM/eCIM achieved high specificity of 100%/100% and sensitivity of 99.6%/97.4%, with one S. marcescens isolate harboring VIM-2 undetected. CONCLUSIONS: Both NG-Test CARBA 5 and mCIM/eCIM showed excellent results in the tested carbapenemase (bla KPC, bla NDM, bla VIM, bla IMP, and bla OXA-48) detection compared with molecular genotypic test. As every assay has its own limitations, suitable methods should be combined for the establishment of the CRE diagnostic pathways.

Adenosine triphosphate bioluminescence to validate decontamination of endoscopes.

The reports of outbreaks involving carbapenemase-resistant Enterobacteriaceae (CRE) associated with gastrointestinal endoscopy prompted a review and study of a novel method of assessing cleaning. This study assessed adenosine triphosphate (ATP) bioluminescence to demonstrate cleanliness prior to endoscopy. ATP testing was compared with microbiological monitoring for 127 endoscopes. Samples were taken after cleaning, reprocessing and storage, but immediately before the endoscopy procedure. We recommend implementing ATP testing prior to endoscopy procedures as an alternative to microbiological testing at periodic intervals. ATP testing provides a convenient assessment of endoscopy hygiene to demonstrate safety and quality assurance.