Carbapenemase-producing Enterobacterales Isolates Research Articles

A comparison of two MALDI-TOF MS based assays for the detection of carbapenemases in Enterobacterales

Carbapenem resistant (CRE) and carbapenemase producing Enterobacterales (CPE) in particular, represent a major threat for healthcare systems worldwide. Rapid, reliable, and easy to perform assays are required to enable targeted and effective therapy. MALDI-TOF MS based carbapenemase diagnostics has potential advantages over molecular and phenotypic sensitivity tests, especially in terms of time to result. So far, only one mass spectrometry (MS)-based carbapenemase test system is commercially available for routine use. The aim of this study was to compare the performance of the established system to a novel MS-based test to identify CPE isolates. Forty consecutive CRE isolates (70% CPEs) were pre-screened for carbapenemase activity by routine laboratory methods. Isolates then were tested using the to date only IVD CE certified MALDI-TOF MS carbapenemase detection assay (MBT STAR-Carba IVD Kit; Bruker Daltonics) and a novel test designed for the recently launched EXS2600 MALDI-TOF MS system (Carbapenemase Activity Kit; Zybio). Valid results were obtained for 93% and 85% isolates by the Bruker and the Zybio assay, respectively. Sensitivities, specificities, positive and negative predictive values were 92%, 91%, 96%, and 83% for the Bruker assay and 96%, 64%, 85%, and 88% for the Zybio assay. There are notable differences concerning the handling of the assays. In summary, both systems featured high sensitivities for the detection of carbapenemases, but the Bruker assay yielded less false-positive results. There are advantages and disadvantages concerning the handling for each system, but both proved to be suitable for the use in a routine laboratory.

Resistance profiles of carbapenemase-producing Enterobacterales in a large centre in England: are we already losing cefiderocol?

Carbapenemase-producing Enterobacterales (CPE) pose difficult therapeutic challenges. We aimed to characterize antimicrobial resistance profiles of CPE in our centre. All non-duplicate CPE isolates between 1 August 2020 and 31 August 2023 in a large teaching trust in England were retrospectively studied. Cefiderocol antimicrobial susceptibility testing (AST) was performed using disc diffusion, ceftazidime/avibactam using disc diffusion and gradient diffusion, and ceftazidime/avibactam aztreonam synergy using the double disc diffusion method. EUCAST version 14.0 breakpoints were used. A total of 158 CPE from 136 patients were isolated. Most patients were colonized with CPE, but only 16.9% had active infections. Thirty-day all-cause mortality was 10.3%, increasing to 13% for patients with infections and to 18.2% for bacteraemias. OXA-48 was the most prevalent carbapenemase (48.1%), followed by NDM (38%). All isolates exhibited MDR profiles, with high levels of resistance to meropenem (41.1%). Resistance to cefiderocol was found in 69.7% of NDM-producing isolates, with a further 18.2% in the area of technical uncertainty. Ceftazidime/avibactam and aztreonam synergy was seen in 87.5% of isolates, whereas colistin and fosfomycin susceptibility remained high (98.1% and 97.2%, respectively). All OXA-48-producing isolates were susceptible to ceftazidime/avibactam, and 15.3% were resistant to cefiderocol. No patients had been exposed to cefiderocol beforehand, whereas three had been exposed to ceftazidime/avibactam. The most common risk factor for CPE isolation was travel and receiving healthcare abroad, especially in Asia. We found high rates of resistance to cefiderocol in CPE isolates without prior cefiderocol exposure. Our results prohibit empirical use of cefiderocol for the treatment of CPE infections in our setting.

In Vitro Synergistic Activity of Ceftazidime-Avibactam and Aztreonam against New Delhi Metallo-β-Lactamase-Producing Clinical Enterobacterales Isolates

Background: Carbapenemase-producing-Enterobacterales (CPE) infections are on the rise and associated with increased morbidity and mortality, due to a limited number of therapeutic options. The goal of this study was to assess the synergistic activity of ceftazidime-avibactam (CZA) and aztreonam (ATM) combination against phenotypically and genetically characterized blaMBL-producing Enterobacterales.Methods: In this study, forty (n=40) non-repeat, CPE clinical isolates, including: n=35 Klebsiella pneumoniae, n=2 each of Escherichia coli and Klebsiella oxytoca, and n=1 Enterobacter cloacae isolates were identified and susceptibilities were assessed using the Vitek-II compact (bioMérieux, Inc., France) and Microscan Walkaway (Beckman Coulter) systems. Genotypic analysis of clinically relevant carbapenemases was performed by using Xpert-Carba-R assay on GeneXpert (Cepheid, USA). The minimum inhibitory concentrations (MICs) and synergy testing of CZA and ATM, were tested by the Etest fixed ratio method (bioMérieux, Inc., France). The fractional inhibitory concentration index (FICI) was calculated for each antibiotic.Results: Our results showed that 97.5% of blaMBL-producing Enterobacterales isolates were susceptible to an in-vitro CZA + ATM combination regimen. The fractional inhibitory concentration index (FICI) ranged from 0.001 to 1.001. Among the tested CPE isolates, Synergy was observed in 36/40 (90%), an additive effect was observed in 5% (n=2)’ while two (5%) isolates showed indifference. There was no antagonism observed in our study.Conclusion: Our study exhibited a potent activity of CZA and ATM combination synergy against clinical CPE metallo- β-lactamase producers. More extensive studies involving a variety of Gram-negative pathogens with different resistance mechanisms are required to determine the efficacy of this combination regimen.Keywords: Synergy; Aztreonam; Ceftazidime-Avibactam; Carbapenem-Resistant Enterobacterales; Etest

Geographic differences in susceptibility profiles of potential non-class B carbapenemase-producing Enterobacterales isolates against ceftazidime-avibactam, meropenem-vaborbactam, colistin, amikacin, gentamicin, and tigecycline: Data from the Antimicrobial Testing Leadership and Surveillance, 2018–2022

To evaluate the susceptibility profiles of regional meropenem-resistant (MEM-R) potential non-class B carbapenemase-producing Enterobacterales (CPE) isolates (without confirmation by phenotypic tests) against important antibiotics, we extracted data from the 2018-2022 Antimicrobial Testing Leadership and Surveillance. This data included susceptibility information of MEM-R potential non-class B CPE isolates against indicated antibiotics - amikacin [AMK], gentamicin [GM], ceftazidime-avibactam [CZA], colistin [CST], meropenem-vaborbactam [MVB], and tigecycline [TGC] - from sepsis patients hospitalized in ICUs across six major regions. Carbapenemase-encoding genes of the tested CPE isolates, determined by multiplex PCR and Sanger sequencing, were also analyzed. Susceptibility breakpoints recommended by CLSI 2024 and US FDA criteria (for TGC only) against Enterobacterales were employed. A total of 1,500 potential non-class B CPE isolates (89% of which were Klebsiella pneumoniae) were tested globally. Resistance rates to AMK and GM against the evaluated isolates were statistically higher in Africa/the Middle East, Europe, and India compared to other regions. A similar pattern was observed in the susceptibility of these potential CPE isolates to CZA and MVB. High CST resistance rates were noted in Asia, Latin America, and Europe (29%-35%). Furthermore, the proportions of potential CPE isolates carrying genes encoding blaOXA variants were notably higher among the tested CPE isolates in India, Europe, and Africa/the Middle East regions (99.2%, 53.3%, and 96.7%, respectively) compared to other regions. Trends in resistance to important antibiotics among potential non-class B CPE isolates warrant close monitoring.

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.

Phenotypic antimicrobial resistance associated with different <em>bla</em> genotypes and their combinations in Enterobacterales

Introduction: Varying combinations of β-lactamase genes (bla) in Enterobacterales manifest different resistant phenotypes.Objectives: This study aimed to identify bla gene types and combinations resulting in variations in phenotypic antimicrobial resistance.Methods: Minimum inhibitory concentrations (MICs) of 75 extended spectrum β-lactamase (ESBL), AmpC β-lactamase and carbapenemase-producing Entero-bacterales isolates from a variety of clinical specimens were determined by Vitek micro-broth dilution. Bla genes were detected by 16-plex tandem PCR panel and Xpert Carba-R assay. BlaCTX-M group-1 and blaOXA-48 were the most common ESBL and carbapenemase genes, respectively.Results: Majority of the isolates (92%, 69/75) had bla gene combinations. Thirty-eight isolates (51%) carried ESBL, AmpC genes and/or narrow spectrum bla genes without any carbapenemase genes. The common bla gene combination in these isolates was blaCTX-M group-1 + blaOXA1 which is known to confer decreased susce-ptibility to piperacillin-tazobactam. Antibiotics with potential activity against ESBL-producing bacteria such as fourth generation cephalosporins, β-lactam/β-lactam-inhibitor combinations, quinolones and gentamicin also showed low rates of susceptibility. Highest susceptibility rates were to meropenem (92%) and amikacin (92%). Thirty-seven isolates (49%) carried carbapenemase genes as well. These isolates showed even higher resistance rates to all antibiotics, except amikacin (92%susceptible). Significantly higher MIC values for cefoxitin, ceftazidime, ceftriaxone, cefepime, meropenem, amikacin, tobramycin and trimethoprim-sulfamethoxazole were seen in isolates carrying blaNDM than blaOXA-48. However, a higher MIC for norfloxacin was seen in isolates with blaOXA-48.Conclusions: In conclusion, this study showed that Enterobacterales with different bla gene combinations manifest variations in phenotypic resistance to both β-lactam and non-β-lactam antibiotics.

Genomic characterization of carbapenemase-producing Enterobacterales from Dhaka food markets unveils the spread of high-risk antimicrobial-resistant clones and plasmids co-carrying bla NDM and mcr-1.1.

The transmission of carbapenemase-producing Enterobacterales (CPE) in the external environment, especially through food, presents a significant public health risk. To investigate the prevalence and genetic characteristics of CPE in food markets of Dhaka, Bangladesh, using WGS. CPE isolates were obtained from different food and water samples collected from food markets in the southern part of Dhaka, Bangladesh. The isolates subsequently underwent molecular typing, WGS employing both short- and long-read sequencers, and plasmid analysis. This study unveiled an extensive spread of CPE, with no significant difference in contamination rates observed in samples (N = 136), including meat (n = 8), fish (n = 5), vegetables (n = 36) or various food-washed water (n = 65) from markets near hospitals or residential areas. Thirty-eight Enterobacterales from 33 samples carried carbapenemase genes (bla NDM-1, -4, -7, bla KPC-2, bla OXA-181 or bla IMI-1). Among these, the high-risk Escherichia coli ST410 clone was the most prevalent and distributed across various locations. Furthermore, the identification of IncHI2 plasmids co-harbouring resistance genes like bla NDM-5 and mcr-1.1, without discernible epidemiological connections, is a unique finding, suggesting their widespread dissemination. The analysis unveils a dynamic landscape of CPE dissemination in food markets, underscored by the proliferation of novel IncHI2 hybrid plasmids carrying both colistin- and carbapenem-resistance genes. This illuminates the ever-evolving landscape of antimicrobial resistance in Dhaka, urging us to confront its emergent challenges.

Prevalence of carbapenem-resistant Enterobacterales with bla IMP-6 predominance in hospitals from 2018 to 2021 in Nara, Japan.

Despite the global health risk of carbapenem-resistant Enterobacterales (CRE), especially carbapenemase-producing Enterobacterales (CPE), Japan reports a significantly low frequency of CRE with a predominance of IMP-type carbapenemases. This study aimed to investigate the prevalence and characteristics of CRE isolated from hospitals in the city of Nara, Japan. We obtained 171 CRE isolates from 16 791 Enterobacterales isolated at 23 hospitals in Nara between January 2018 and December 2021. Isolates of CPE were characterized through antimicrobial susceptibility testing, the carbapenem inactivation method, PCR and DNA sequencing. Genotypic diversity of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae was determined via MLST and PFGE. The prevalence of CRE between 2018 and 2021 was 1.02%, gradually decreasing from 1.13% to 0.74%. Ninety-nine isolates were identified as CPE, representing six species. Ninety-seven CPE isolates harboured bla IMP-6, while the remaining two carried either bla IMP-1 or bla IMP-19. Genotype analysis identified ST131 as the dominant genotype for E. coli, but none for K. pneumoniae. PFGE results suggested clonal spread of CPE in Hospital A, where CRE was isolated in high numbers (n = 44). In this study, CRE prevalence was marginally higher than previously reported in Japan, but still low in frequency. A predominance of Enterobacterales harbouring bla IMP-6 was confirmed in Nara. The spread of CPE at Hospital A suggested the possibility of a nosocomial outbreak due to bla IMP-6 transmission via plasmids or clonal spread. Continued monitoring is crucial for effective management of CRE prevalence in the region.

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.

Epidemiology and clinical significance of carbapenemases in Australia: a narrative review.

Carbapenemase-producing gram-negative bacteria (CP-GNB) infections threaten public health with high mortality, morbidity and treatment costs. Although frequencies remain low in Australia (total number of CP-GNB infections reported was 907 in 2022), blaIMP-4 has established low levels of endemicity in many states. Imipenemase metallo-β-lactamase types alone accounted for more than half of all carbapenemases in carbapenemase-producing Enterobacterales isolates in Australia, particularly in Enterobacter cloacae complex. New Delhi metallo-β-lactamase constitutes almost 25% of all carbapenemases in Australia and was identified predominantly in Escherichia coli. The OXA-48-like carbapenemases include almost 10% of all carbapenemases and are mainly seen in Klebsiella pneumoniae and E. coli. Although K. pneumoniae carbapenemase-type carbapenemases are rare in Australia, some local outbreaks have occurred. Most carbapenem-resistant (CR) Pseudomonas aeruginosa strains in Australia do not produce carbapenemases. Finally, OXA-23-like carbapenemases are overwhelmingly positive in CR-Acinetobacter baumannii strains in Australia. Treatment of CR-GNB infections challenges physicians. Of 10 new antibiotics active against at least some CR-GNB infections that are approved by the USFood and Drug Administration, just three are approved for use in Australia. In this context, there is still an unmet need for novel antibacterials that can be used for the treatmentof CR-GNB infections in Australia, as well as a pressing requirement for new mechanisms to 'de-link' antibiotic sales from their availability. In this narrative review, we aim to overview the epidemiology and clinical significance of carbapenem resistance in Australia as it pertains to Enterobacterales, P. aeruginosa and A. baumannii.

Integrated Analysis of Patient Networks and Plasmid Genomes to Investigate a Regional, Multispecies Outbreak of Carbapenemase-Producing Enterobacterales Carrying Both blaIMP and mcr-9 Genes.

Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of imipenemase (IMP)-encoding CPE among diverse Enterobacterales species between 2016 and 2019 across a London regional network. We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE-positive patients. Genomes of IMP-encoding CPE isolates were overlaid with patient contacts to imply potential transmission events. Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, and Escherichia coli); 86% (72 of 84) harbored an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68 of 72). Phylogenetic analysis of IncHI2 plasmids identified 3 lineages showing significant association with patient contacts and movements between 4 hospital sites and across medical specialties, which was missed in initial investigations. Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multimodal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.SummaryThis was an investigation, using integrated pathway networks and genomics methods, of the emergence of imipenemase-encoding carbapenemase-producing Enterobacterales among diverse Enterobacterales species between 2016 and 2019 in patients across a London regional hospital network, which was missed on routine investigations.

Carbapenemase-producing Enterobacterales isolated from hospital sinks: molecular relationships with isolates from patients and the change in contamination status after daily disinfection with sodium hypochlorite.

This study aimed to investigate the contamination status of hospital sinks with carbapenemase-producing Enterobacterales (CPE), the efficacy of daily cleaning with sodium hypochlorite, and the relationships between CPEs isolated from contaminated sinks and patients. Pre/postintervention surveys of the CPE-contaminated sinks. Hospital wards including pediatric intensive care unit in a children's hospital. Consenting CPE-colonized patients admitted between November 2018 and June 2021 in our hospital. Environmental culture of 180 sinks from nine wards in our hospital was performed three times with an interval of 2 years (2019, 2021, 2023). Molecular typing of the isolated strains from the sinks and patients was performed. After the first surveillance culture, we initiated daily disinfection of the sinks using sodium hypochlorite. Before the intervention, we detected 30 CPE-positive sinks in 2019. After the intervention with sodium hypochlorite, we observed a substantial decline in the number of sinks contaminated with CPE; 13 in 2021 and 6 in 2023. However, the intervention did not significantly reduce the number of CPE-contaminated sinks used for the disposal of nutrition-rich substances. The CPE isolates from the patients and those from the sinks of the wards or floors where they were admitted tended to have similar pulse-field gel electrophoresis patterns. Contaminated sinks could be reservoirs of disseminating CPE to the patients. Daily disinfection of sinks with sodium hypochlorite may be effective in eliminating CPE, although the effect could be weaker in sinks with a greater risk of contact with nutrition-rich substances.

Deciphering mechanisms affecting cefepime-taniborbactam in vitro activity in carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas spp. isolates recovered during a surveillance study in Spain.

To characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates. CPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing. A reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers. The reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

Active surveillance of carbapenemase-producing Enterobacterales using genomic sequencing for hospital-based infection control interventions.

Whole-genome sequencing (WGS) is increasingly used to characterize hospital outbreaks of carbapenemase-producing Enterobacterales (CPE). However, access to WGS is variable and testing is often centralized, leading to delays in reporting of results. We describe the utility of a local sequencing service to promptly respond to facility needs over an 8-year period. The study was conducted at Royal Prince Alfred Hospital in Sydney, Australia. All CPE isolated from patient (screening and clinical) and environmental samples from 2015 onward underwent prospective WGS. Results were notified to the infection control unit in real time. When outbreaks were identified, WGS reports were also provided to senior clinicians and the hospital executive administration. Enhanced infection control interventions were refined based on the genomic data. In total, 141 CPE isolates were detected from 123 patients and 5 environmental samples. We identified 9 outbreaks, 4 of which occurred in high-risk wards (intensive care unit and/or solid-organ transplant ward). The largest outbreak involved Enterobacterales containing an NDM gene. WGS detected unexpected links among patients, which led to further investigation of epidemiological data that uncovered the outpatient setting and contaminated equipment as reservoirs for ongoing transmission. Targeted interventions as part of outbreak management halted further transmission. WGS has transitioned from an emerging technology to an integral part of local CPE control strategies. Our results show the value of embedding this technology in routine surveillance, with timely reports generated in clinically relevant timeframes to inform and optimize local control measures for greatest impact.

Predominance of OXA-48 Carbapenemase-Producing Enterobacterales in a Moroccan Hospital.

The emergence of carbapenemase-producing Enterobacterales (CPE) is a major concern that is increasingly reported worldwide. Our study aimed at investigating the resistance of CPE isolates in a Moroccan teaching hospital using phenotypic and genotypic methods. Enterobacterales strains from March to June 2018 were collected from different clinical samples. The Enterobacterales isolates resistant to third-generation cephalosporins (3GC) and/or carbapenems were subjected to the Carba NP test and an immunochromatographic test for phenotypic detection. Detection of extended-spectrum β-lactamases (ESBL) was also performed following standards. Molecular screening of carbapenemases genes (OXA-48, NDM, blaKPC, blaIMP, blaVIM, and blaOXA-24, blaOXA-23, OXA-51, OXA-58) using conventional multiplex PCR assays was also performed on 143 isolates. Enterobacterales represented 52.7% with a proportion of 21.8% of bacteria resistant to 3GC and/or carbapenems. Within 143 isolates MDR to 3GC, K. pneumoniae, E. coli, and E. cloacae represent 53.1%, 40.6%, and 6.3%, respectively. These strains were isolated mainly from urinary samples (74.8%) in patients admitted to emergency and surgical units. 81.1% of strains are producing ESBL and 29% are carbapenemase producers as confirmed by the Carba NP test, immunochromatographic test, and molecular testing. OXA-48 carriers represent 83.3% of these strains, followed by NDM with 16.7%. blaKPC, blaIMP, blaVIM, and blaOXA-24, blaOXA-23, OXA-51, OXA-58 were not detected in any of these bacteria. A high rate of CPE carrying OXA-48 among Enterobacterales resistant to 3GC and/or carbapenems isolates was found. Strict observance of hospital hygiene measures and more rational use of antibiotics are mandatory. Implantation of carbapenemases detection should be encouraged in our hospital settings to estimate the true burden of the CPE.

First Report of OXA-181-Producing Enterobacterales Isolates in Latin America.

We characterized five carbapenemase-producing Enterobacterales (CPE) isolates from two health care institutions in Lima, Peru. The isolates were identified as Klebsiella pneumoniae (n = 3), Citrobacter portucalensis (n = 1), and Escherichia coli (n = 1). All were identified as blaOXA-48-like gene carriers using conventional PCR. Whole-genome sequencing found the presence of the blaOXA-181 gene as the only carbapenemase gene in all isolates. Genes associated with resistance to aminoglycosides, quinolones, amphenicols, fosfomycins, macrolides, tetracyclines, sulfonamides, and trimethoprim were also found. The plasmid incompatibility group IncX3 was identified in all genomes in a truncated Tn6361 transposon flanked by ΔIS26 insertion sequences. The qnrS1 gene was also found downstream of blaOXA-181, conferring fluoroquinolone resistance to all isolates. CPE isolates harboring blaOXA-like genes are an increasing public health problem in health care settings worldwide. The IncX3 plasmid is involved in the worldwide dissemination of blaOXA-181, and its presence in these CPE isolates suggests the wide dissemination of blaOXA-181 in Peru. IMPORTANCE Reports of carbapenemase-producing Enterobacterales (CPE) isolates are increasing worldwide. Accurate detection of the β-lactamase OXA-181 (a variant of OXA-48) is important to initiate therapy and preventive measures in the clinic. OXA-181 has been described in CPE isolates in many countries, often associated with nosocomial outbreaks. However, the circulation of this carbapenemase has yet to be reported in Peru. Here, we report the detection of five multidrug-resistant CPE clinical isolates harboring blaOXA-181 in the IncX3-type plasmid, a potential driver of dissemination in Peru.

Resistance to critically important antibiotics in hospital wastewater from the largest Croatian city

The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10−1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate.

MALDI-TOF mass spectrometry has become widely used in clinical microbiology and has proved highly accurate for detection of carbapenemases in Gram-negative bacteria. However, the use of carbapenem-hydrolysis assays in routine diagnostics is hampered by the need for antibiotic substances and for making their fresh solutions each time an assay is conducted. Here, we evaluated the use of commercial antibiotic susceptibility-testing disks as source of ertapenem substrate in MALDI-TOF MS-based assay for detection of carbapenemase-producing Enterobacterales (CPE). The assay was validated on 48 CPE isolates of 8 different species expressing NDM-, VIM-, KPC- and OXA-48-type carbapenemases and exhibiting various levels of resistance to carbapenems (MIC range: 0.25- > 32 mg/l), as well as on 48 carbapenemase-non-producing isolates. The assay conditions were optimized as follows: 10-μl loopful of bacterial colonies was suspended in 150 μl 0.01 M Na-PBS buffer, pH 7.4, a 10 μg ertapenem susceptibility-testing disk was immersed in the suspension and incubated 3 h at 35°C, after which supernatant was obtained by centrifugation and applied on a target plate with alpha-cyano-4-hydroxycinnamic acid matrix. Mass spectra were analyzed between 440 and 560 m/z. Carbapenemase activity was detected in all tested CPE isolates by the appearance of m/z peaks corresponding to ertapenem hydrolysis products: [Mh + H]+:494.2, [Mh + Na]+:516.2, [Mh + 2Na]+:538.2, [Mh/d + H]+:450.2, [Mh/d + Na]+:472.2, and simultaneous decrease or loss of peaks of intact antibiotic: [M + H]+:476.2, [M + Na]+:498.1, [M + 2Na]+:520.1. No hydrolysis peaks or loss of intact ertapenem peaks were observed for carbapenemase-negative strains. We therefore report the development of a sensitive, specific and cost-effective MALDI-TOF MS-based assay for detection of CPE, which makes use of antibiotic disks readily available in most laboratories.

Effect of Colistin, Fosfomycin and Meropenem/Vaborbactam on Carbapenem-Resistant Enterobacterales in Egypt: A Cross-Sectional Study.

The increasing multi-drug carbapenem resistance among Enterobacterales are a severe health problem limiting therapeutic options and worsen the prognosis. This study characterizes carbapenemase genes and integrons among uropathogenic carbapenem resistant Enterobacterales (CRE) isolates recovered from Mansoura University Hospitals and evaluates the effect of colistin, fosfomycin and meropenem-vaborbactam on these isolates. A total of 200 Enterobacterales isolates were collected from patients with urinary tract infections. Antimicrobial susceptibility testing was performed by the disc diffusion method. Colistin susceptibility was tested using the broth microdilution method and fosfomycin and meropenem/vaborbactam susceptibility were tested by MIC Test Strips. Carbapenem resistant isolates were screened for carbapenemase activity phenotypically using the modified carbapenem inactivation method and EDTA-modified carbapenem inactivation method and genotypically by multiplex PCR. Integrons class 1 and 2 and fosA gene were assayed by PCR. Data were statistically analyzed using the Statistical Package for Social Sciences (SPSS) version 16. The Chi-square or Fisher's exact test was used to compare groups, as appropriate. Ninety-two Enterobacterales isolates were resistant to meropenem (46%); 52 E. coli and 40 K. pneumoniae strains. All CRE isolates were multi-drug resistant (MDR). Sensitivity of CRE isolates to colistin, fosfomycin and meropenem/vaborbactam were 67.4%, 82.6% and 58.7%, respectively. Carbapenemase genes were detected by multiplex PCR in 69.6% of CRE isolates (Carbapenemase producing Enterobacterales (CPE) mainly blaNDM (37%). CPE isolates were significantly more resistant to meropenem/vaborbactam than non-CPE isolates; 51.6% vs 17.8%, respectively (P = 0.003) especially blaNDM carrying isolates (70.6%). Class 1 integrons and fosA gene were detected in 91.3% and 11.9% of CRE isolates, respectively. This study revealed that about half of the uropathogenic Enterobacterales isolates were MDR CRE. Carbapenemase gene blaNDM was the main gene among CRE isolates. Meropenem/vaborbactam sensitivity was significantly higher on non-CPE than CPE isolates and limited by the predominance of blaNDM .

Implementation of Chromatic Super CAZ/AVI® medium for active surveillance of ceftazidime-avibactam resistance: preventing the loop from becoming a spiral

Acquired resistance towards ceftazidime-avibactam (CAZ-AVI) is increasingly reported. Several mechanisms can be involved, but mutations in the Ω-loop region of β-lactamases are the most described. Herein, we assessed the implementation of Chromatic Super CAZ/AVI® medium in rectal swab surveillance cultures in a geographic area with endemic distribution of KPC-producing Klebsiella pneumoniae. Routine rectal swabs collected from the intensive care unit (ICU) and non-ICU patients were screened for carbapenemase-producing Enterobacterales (CPE), carbapenem-resistant Gram-negative organisms (CR-GN) and CAZ-AVI-resistant organisms by Chromatic CRE and Super CAZ/AVI® media. Among the 1839 patients screened, 146 (7.9%) were found to be colonized by one or more CPE and/or CR-GN isolates during hospitalization. Overall, among colonized patients the most common bacteria encountered were KPC-producing Enterobacterales (n = 60; 41.1%), carbapenem-resistant Pseudomonas aeruginosa (n = 41; 28.1%) and carbapenem-resistant A. baumannii (n = 34; 23.3%). Among patients colonized by KPC-producing Enterobacterales, thirty-five (58.3%) had CAZ-AVI-resistant strains. A 30.5% rate of faecal carriage of CAZ-AVI-resistant KPC-producing K. pneumoniae, substantially higher than that of susceptible isolates (2.8%), was observed in the COVID-19 ICU. Prevalence of faecal carriage of metallo-β-lactamase-producing organisms was low (0.5% and 0.2% for Enterobacterales and P. aeruginosa, respectively). Chromatic Super CAZ/AVI® medium showed 100% sensitivity in detecting CPE or CR-GN isolates resistant to CAZ-AVI regardless of both MIC values and carbapenemase content. Specificity was 86.8%. The Chromatic Super CAZ/AVI® medium might be implemented in rectal swab surveillance cultures for identification of patients carrying CAZ-AVI-resistant organisms to contain the spread of these difficult-to-treat pathogens.