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Objectives: The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious worldwide medical problem. This study was designed to clarify the genetic and epidemiological properties of MDR A. baumannii clinical isolates. Methods: A total of 66 MDR A. baumannii isolates were obtained from 66 inpatients between May 2019 and February 2020 in a university hospital in Nepal. Whole genomes of these isolates were sequenced using next-generation sequencing. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence typing (MLST) and clonal complex (CC) analysis were conducted, and drug-resistance genes were identified. Results: Of the 66 isolates, 26 harboured a gene encoding NDM-type metallo-β-lactamase, and 55 harboured a gene encoding the 16S rRNA methyltransferase, ArmA. All isolates had point mutations in the quinolone-resistance-determining regions of gyrA and parC. Phylogenetic analysis showed that 55 isolates harboured armA, 26 harboured blaNDM-1, and14 harboured blaPER-7. Multilocus sequence typing and CC analysis revealed that 34 isolates belonged to CC2 (ST2), 10 to CC1 (nine ST1 and one ST623), and eight to CC149 (ST149). Compared to our previous study on MDR A. baumannii in Nepal in 2012, the isolation rate of CC2 increased, whereas that of CC149 decreased between 2012 and 2020. Conclusions: This study indicates that MDR A. baumannii producing carbapenemase and 16S rRNA methyltransferase, with high resistance to carbapenems and/or aminoglycosides, are spreading in medical settings in Nepal. The genetic backgrounds of MDR A. baumannii isolates have shifted to international clone 2 over several years.

Objective: Multidrug-resistant (MDR) Acinetobacter baumannii is a major issue within healthcare facilities in Bangladesh due to its frequent association with hospital-acquired infections. In this study we report on a carbapenem-resistant draft genome sequence of an A. baumannii BD20 sample isolated from an infected wound in Bangladesh. Methods: A. baumannii BD20 was isolated from an infected burn wound. Whole-genome sequencing was carried out and annotated using PGAP and Prokka. Sequence type, antimicrobial resistance genes, virulence factor genes, and metal resistance genes were investigated. Core genome multilocus sequence typing–based phylogenomic analysis between A. baumannii BD20 and 213 A. baumannii strains retrieved from the NCBI GenBank database was performed using the BacWGSTdb 2.0 server. Results: A. baumannii BD20 (MLST 491) was resistant to all the antibiotics tested, except for colistin and polymyxin B. Along with many other antibiotic resistance genes, the isolate harbored three classes of beta lactamase–producing genes: blaGES-11 (class A), blaOXA-69 (class D), blaADC-10 (class C), and blaADC-11 (class C). Additionally, the strain carried several virulence genes and metal resistance determinants, which may contribute to its increased virulence. Core genome MLST–based phylogenomic analysis revealed that A. baumannii BD20 was closely related to another ST491 strain isolated from Singapore. Conclusions: The findings of this study underscore the growing challenge of MDR A. baumannii, emphasizing the need for vigilant surveillance and infection-control measures in healthcare settings in order to address these emerging threats effectively.

Objectives: The aim of this study was to characterize an NDM-1-producing Acinetobacter seifertii isolates from a patient in South Korea. Methods: Antibiotic susceptibility testing and genotyping using multigene sequencing were performed and whole plasmid sequences were determined. Results: The genotype of A. seifertii was ST1899 and was resistant to ceftazidime, trimethoprim–sulfamethoxazole, and piperacillin–tazobactam, in addition to carbapenem. blaNDM-1 was surrounded by the ISAba125 insertion sequence within the structure of Tn125 in the 47 kb-sized plasmid. The plasmid exhibited a structure similar to that of other plasmids of diverse Acinetobacter sp. found worldwide. Transconjugation and the growth curve indicated that the plasmid was adapted to A. seifertii rather than other closely related Acinetobacter sp. Conclusions: Acquisition of carbapenem resistance by horizontal transfer of the blaNDM-1-carrying plasmid from another Acinetobacter species was found with no growth defect.

This work explores the potential of a triple combination of meropenem (MEM), a novel metallo-β-lactamase (MBL) inhibitor (indole-2-carboxylate 58 (InC58)), and a serine-β-lactamase (SBL) inhibitor (avibactam (AVI)) for broad-spectrum activity against carbapenemase-producing bacteria. A diverse panel comprising MBL- and SBL-producing strains was used for susceptibility testing of the triple combination using the agar dilution method. The frequency of resistance (FoR) to MEM combined with InC58 was investigated. Mutants were sequenced and tested for cross resistance, fitness, and the stability of the resistance phenotype. Compared with the double combinations of MEM plus an SBL or MBL inhibitor, the triple combination extended the spectrum of activity to most of the isolates bearing SBLs (oxacillinase-48 (OXA-48) and Klebsiella pneumoniae carbapenemase-2 (KPC-2)) and MBLs (New Delhi metallo-β-lactamases (NDMs)), although it was not effective against Verona integron-encoded metallo-β-lactamase (VIM)-carrying Pseudomonas aeruginosa (P. aeruginosa) and OXA-23-carrying Acinetobacter baumannii (A. baumannii). The FoR to MEM plus InC58 ranged from 2.22 × 10−7 to 1.13 × 10−6. The resistance correlated with mutations to ompC and comR, affecting porin C and copper permeability, respectively. The mutants manifested a fitness cost, a decreased level of resistance during passage without antibiotic pressure, and cross resistance to another carbapenem (imipenem) and a β-lactamase inhibitor (taniborbactam). In conclusion, compared with the dual combinations, the triple combination of MEM with InC58 and AVI showed a much wider spectrum of activity against different carbapenemase-producing bacteria, revealing a new strategy to combat β-lactamase-mediated antimicrobial resistance.

Objectives: Despite clinical relevance, commercially available molecular tools for accurate β-lactamase detection are limited. In this study, we evaluated the performance of the ARM-DⓇ Kit, β-Lactamase, a commercially available multiplex PCR assay designed to detect nine β-lactamase genes, including the five major plasmid-mediated carbapenemases, ESBL and AmpC genes circulating in the United States. Methods: A diverse collection of 113 Gram-negative isolates, including 42 with multiple β-lactamases genes, was selected from the U.S. Centers for Disease Control and Prevention (CDC) & Food and Drug Administration (FDA) Antimicrobial Resistance Isolate Bank, to represent the most frequently detected bacterial species carrying plasmid-mediated β-lactam resistance genes. Results: Results were compared with whole genome sequence data. Of 164 β-lactamase gene targets with 49 unique variants, all were detected correctly without any cross-reactivity. The sensitivity and specificity were 100% (164/164) and 99.9% (852/853), respectively. Conclusion: The ARM-DⓇ Kit, β-Lactamase detected a wide range of β-lactamase genotypes at a low upfront cost. The Streck assay represents a suitable, comprehensive tool for the detection of key β-lactamase resistance genes of public health concern in the United States.

Background: This study aimed to characterize carbapenem-nonsusceptible Acinetobacter (CNSA) isolated from patients with bacteremia from 1997 to 2015. Methods: A total of 173 CNSA (12.3%) was recovered from 1403 Acinetobacter isolates. The presence of selected β-lactamase genes in CNSA was determined by PCR amplification. The conjugation test was used to determine the transferability of metallo-β-lactamase (MBL)-carrying plasmids. Whole genome sequencing in combination with phenotypic assays was carried out to characterize MBL-plasmids. Results: In general, a trend of increasing numbers of CNSA was observed. Among the 173 CNSA, A. baumannii (54.9%) was the most common species, followed by A. nosocomialis (23.1%) and A. soli (12.1%). A total of 49 (28.3%) CNSA were extensively drug-resistant, and all were A. baumannii. The most common class D carbapenemase gene in 173 CNSA was blaOXA-24-like (32.4%), followed by ISAba1-blaOXA-51-like (20.8%), ISAba1-blaOXA-23 (20.2%), and IS1006/IS1008-blaOXA-58 (11.6%). MBL genes, blaVIM-11, blaIMP-1, and blaIMP-19 were detected in 9 (5.2%), 20 (11.6%), and 1 (0.6%) CNSA isolates, respectively. Transfer of MBL genes to AB218 and AN254 recipient cells was successful for 7 and 6 of the 30 MBL-plasmids, respectively. The seven AB218-derived transconjugants carrying MBL-plasmids produced less biofilm but showed higher virulence to larvae than recipient AB218. Conclusions: Our 19-year longitudinal study revealed a stable increase in CNSA during 2005–2015. blaOXA-24-like, ISAba1-blaOXA-51-like, and ISAba1-blaOXA-23 were the major determinants of Acinetobacter carbapenem resistance. MBL-carrying plasmids contribute not only to the carbapenem resistance but also to A. baumannii virulence.

Background: Detection of K. pneumoniae superbugs carrying Extended-spectrum β-lactamase (ESBL) and Carbapenemase resistance genes among hospitalized patients is crucial for infection control and prevention. The aim of this molecular study was to investigate the spread of ESBL and Carbapenemase-producing K. pneumoniae in two hospitals located in Southwest Iran. Methods: One hundred clinical isolates of K. pneumoniae were randomly collected from two hospitals over a period of five months, from November 2023. The isolates were confirmed using biochemical and genotypic tests. According to the CLSI 2022 guidelines, K. pneumoniae isolates that exhibited resistance to at least one of the three indicator cephalosporins or carbapenems were selected for evaluation of ESBL and carbapenemase production. This was done using a combination disk confirmatory test and the modified carbapenem inactivation method (mCIM). Finally, the presence of ESBLs and carbapenemase resistance encoding genes was assessed using PCR and specific primers. Results: Out of the 100 isolates, the percentage of antibiotic resistance was cefoxitin (29 %), cefixime (28 %), ceftazidime (26 %), cefotaxime (24 %), cefepime (22 %), ceftriaxone (21 %), imipenem (20 %), and meropenem (17 %). Additionally, thirty isolated strains were found to be multidrug-resistant. Out of these, twenty-seven strains demonstrated a potential for ESBLs, twenty strains for Carbapenemase, and seventeen strains for both ESBLs and Carbapenemase production. Moreover, the occurrence of ESBLs and carbapenemase genes was as follows: blaSHV (25 %), blaTEM (23 %), blaCTX-M (20 %), blaOXA-48 (17 %), and blaVIM (13 %). It is important to mention that we did not detect the blaIMP and blaKPC. resistant genes among clinical isolates. Conclusion: Based on the results, the existence of this type of resistance in hospital centers needs to be reevaluated in terms of empirical antibiotic prescribing. Additionally, it is recommended that infection control measures should be taken for public health. Also, it's suggested that hospital-acquired infections caused by superbug K. pneumoniae resistant strains should be addressed.

Objectives: To evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing. Methods: DNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST). Results: We processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (

The known intrinsic and polymorphic blaOXA-51-like genes of Acinetobacter baumannii were recently reported in other non-A. baumannii Gram-negative pathogens. Accurate detection of this potentially transferrable carbapenemase gene in the clinical setting is critical. This study developed a loop-mediated isothermal amplification (LAMP) assay targetting multiple alleles of blaOXA-51-like genes. Specifically, an alignment-based primer design, in silico primer screening, and in vitro assay confirmation were conducted. Both in silico and in vitro results revealed the tolerance of the LAMP assay to up to five primer-template mismatches outside the 3′-end primer regions. Within 90 min, the LAMP assay also detected the gene targets in other Gram-negative bacteria with known and novel blaOXA-51-like genes. Finally, it showed a superior limit of detection (as low as 101 CFU/mL) compared with polymerase chain reaction, and high specificity against non-targets. This study developed a highly adaptable LAMP assay to monitor blaOXA-51-like genes in the clinical setting and provided important insights into LAMP primer design and screening.

ABSTRACT: Objectives: Klebsiella pneumoniae is an important opportunistic Gram-negative pathogen. This study describes an outbreak due to colistin-resistant and carbapenem-resistant Klebsiella pneumoniae (ColR-CRKP) in a tertiary hospital related to six patients successively admitted to the department of medical intensive care unit (MICU) between March 11 and April 29, 2021. Methods: Phenotypic characterization was conducted on 16 ColR-CRKP strains obtained from six infected patients and five ColR-CRKP strains isolated from 48 environmental samples, followed by whole-genome sequencing (WGS) and polymerase chain reaction (PCR) analysis. Results: All ColR-CRKP strains showed resistance to commonly used antibiotics. Whole-genome sequencing revealed a variety of resistance genes such as blaKPC-2, blaCTX-M-65, and blaTEM-4 present in all strains, which is consistent with their antimicrobial resistance profile. All isolates were identified as the high-risk sequence type 11 (ST11) clonal lineage by multilocus sequencing typing (MLST) and subsequently clustered into a single clonal type by core genome MLST (cgMLST). IS5-like element ISKpn26 family transposase insertion mutations at positions 74 nucleotides in the mgrB gene were the main cause of colistin resistance in these ColR-CRKP. The variations of genes were verified by PCR. SCOTTI analysis demonstrated the transmission pathway of the ColR-CRKP between the patients. Conclusion: Our study highlights the importance of coordinated efforts between clinical microbiologists and infection control teams to implement aggressive surveillance cultures and proper bacterial genotyping to diagnose nosocomial infections and take control measures. Routine surveillance and the use of advanced sequencing technologies should be implemented to enhance nosocomial infection control and prevention measures.

ABSTRACT: Background: Carbapenemase-producing Citrobacter freundii has been reported as a leading cause of healthcare-associated infections. Particularly, C. freundii belonging to the sequence type (ST) 18 is considered to be an emerging nosocomial clone. Objectives: To report the genomic background and phylogenomic analysis of a multidrug-resistant NDM-1-producing C. freundii ST18 (strain CF135931) isolated from an endangered green sea turtle affected by plastic pollution in Brazil. Methods: Genomic DNA was extracted and sequenced using the Illumina NextSeq platform. De novo assembly was performed by CLC Workbench, and in silico analysis accomplished by bioinformatics tools. For phylogenomic analysis, publicly available C. freundii (txid:546) genome assemblies were retrieved from the NCBI database. Results: The genome size was calculated at 5 290 351 bp, comprising 5263 total genes, 4 rRNAs, 77 tRNAs, 11ncRNAs, and 176 pseudogenes. The strain belonged to C. freundii ST18, whereas resistome analysis predicted genes encoding resistance to β-lactams (blaNDM-1, blaOXA-1, blaCMY-117, and blaTEM-1C), aminoglycosides (aph(3′')-Ib, aadA16, aph(3′)-VI, aac(6′)-Ib-cr, and aph(6)-Id), quinolones (aac(6′)-Ib-cr), macrolides (mph(A) and erm(B)), sulphonamides (sul1 and sul2), tetracyclines (tetA and tetD), and trimethoprim (dfrA27). The phylogenomic analysis revealed that CF135931 strain is closely related to international human-associated ST18 clones producing NDM-1. Conclusion: Genomic surveillance efforts are necessary for robust monitoring of the emergence of drug-resistant strains and WHO critical priority pathogens within a One Health framework. In this regard, this draft genome and associated data can improve understanding of dissemination dynamics of nosocomial clones of carbapenemase-producing C. freundii beyond hospital walls. In fact, the emergence of NDM-1-producing C. freundii of global ST18 in wildlife deserves considerable attention.

Objectives: Since its discovery, blaNDM-5 has spread widely amongst Escherichia coli strains in clinical patients, causing carbapenem resistance. Here we report the complete genome sequence of an NDM-5-producing E. coli strain isolated from the faecal sample of a healthy individual in Hangzhou, China. Methods: The whole-genome sequence of E. coli CREC8 was obtained utilising both the Nanopore sequencer and the Illumina NovaSeq 6000 platform. Antimicrobial resistance genes, multilocus sequence typing, and plasmid replicons were identified using the BacWGSTdb server. The phylogenetic relationship between CREC8 and other E. coli strains was investigated using the core genome multilocus sequence typing (cgMLST) strategy. Results: The complete genome sequence of E. coli CREC8 consists of one chromosome and 7 plasmids. CREC8 belongs to ST167 according to the MLST scheme. Seven ARGs were identified, including carbapenem resistance gene blaNDM-5 which was located in an IncFIA/IncFII type plasmid. A total of 164 E. coli ST167 strains related to 25 countries across four continents can be retrieved from the NCBI database, 95 of them carrying the blaNDM gene with blaNDM-5 the most (N = 79). Phylogenetic analysis revealed a worldwide distribution of E. coli ST167 strains, with China having the highest prevalence (37%, 61/165). Conclusion: In summary, we reported a blaNDM-5-carrying E. coli ST167 strain isolated from a healthy individual in China. Such strains are more commonly isolated from hospitalised patients but are rarely isolated from healthy individuals. This indicates a further epidemic of carbapenem-resistant E. coli strains in the healthy population which needs our attention.

Summary: Background: The presence of carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) around the world is increasing, particularly in healthcare settings. Surveillance testing for plasmid-mediated carbapenemase genes is necessary to tracking CP-CRE infections. Aim: In the state of Ohio, surveillance of carbapenem-resistant Enterobacterales (CRE) began in 2018, and to the authors' knowledge data on these cases has not been published to date. This study analyzed data on CRE from a large teaching hospital in Ohio, and by the Ohio Department of Health Laboratory (ODHL). Methods: Carbapenemase production was detected using mCIM, and plasmid-mediated carbapenemase genes were detected using rtPCR. Data was collected on 344 standard-of-care isolates from a large teaching hospital in Ohio, including data collected from chart review. Deidentified surveillance data on 4,391 CRE isolates was provided by the ODHL. Statistical analysis was performed using binary logistic regression. Findings: While KPC was the most common carbapenemase gene (n=1590), NDM (n=98), VIM (n=10), IMP (n=39) and OXA-48 (n=35) were also detected in the isolates studied. Klebsiella pneumoniae and Enterobacter cloacae were the most common CRE, and carbapenemase genes were most commonly detected in K. pneumoniae. Inpatient hospital stays and long-term care were associated with CP-CRE and were more common in women. Conclusion: Surveillance data shows that CP-CRE are present in Ohio, most commonly in Klebsiella pneumoniae. A better understanding of the prevalence of CRE, plasmid-mediated carbapenemase genes present, and the populations affected are important when tracking the spread of disease. Further study and surveillance of carbapenem-resistant organisms can provide a better understanding of their prevalence in the state.

ABSTRACT: Objectives: Our aim was to characterize and compare contemporary carbapenem-resistant Enterobacterales (CRE) isolates from gulls, the River Danube, and humans in Hungary, Budapest. Methods: Multiresistant Enterobacterales were sought for in 227 gull faecal and 24 Danube water samples from 2019 to 2020. Eosin-methylene blue agar containing 2 mg/L cefotaxime and Colilert-test containing 10 mg/L cefotaxime were used for gull and water samples, respectively. Isolates were characterized by polymerase chain reactions (PCRs); acquired carbapenemase producers were further analysed by whole-genome sequencing, together with 21 Hungarian human CR Escherichia coli (CREc) isolates. Results: Gull and water samples exhibited a CRE prevalence of 7.4% (9/122) and 6.7% (7/105), none and 5/12 water samples yielded CRE from 2019 and 2020, respectively; CRE were found only in samples taken downstream of Budapest. The dominant species was Escherichia coli and the most prevalent carbapenemase was blaNDM-1. High-risk CREc clones were found both in gulls (ST224, ST372, ST744) and the Danube (ST10, ST354, ST410); the closest associations were between ST410 from humans and the Danube, among ST1437 among gulls, and between ST1437 in gulls and the Danube (46, 0, and 22–24 allelic distances, respectively). Direct links between human and gull isolates were not demonstrated. Conclusion: The study demonstrates potential epidemiological links among humans, a river crossing a city, and urbanised birds, suggesting a local transmission network. Water bodies receiving influent wastewater, together with animals using such habitats, may serve as a local reservoir system for CRE, highlighting the importance of One Health in CRE transmission, even in a country with a low CRE prevalence in humans.

ABSTRACT: Objectives: Acinetobacter bereziniae has been found to cause health care–associated infections, especially in immunocompromised patients. The emergence of two carbapenemase-producing A. bereziniae strains complicates clinical management. Here, we present the genome sequence of a clinical A. bereziniae strain from China co-carrying blaOXA-301 and blaNDM-1. Methods: The genomic DNA of BZAB1 was subjected to whole-genome sequencing using the Illumina NovaSeq 6000 system and assembled using SPAdes 3.13.0. Using the resfinder database of ABRicate V1.01, antimicrobial resistance genes were identified. The Snippy application was used to carry out the phylogenetic analysis. Results: The genome sequence of A. bereziniae BZAB1 consists of 122 contigs consisting of 4 596 983 bp. A total of nine antimicrobial resistance genes were predicted in BZAB1, including two carbapenemase genes: blaOXA-301 and blaNDM-1. Sixty-nine A. bereziniae strains can be retrieved from the National Centre for Biotechnology Information database, 29 of which possess the blaOXA-301 gene and five of which contain the blaNDM-1 gene. Only three strains carry both blaNDM-1 and blaOXA-301. It is worth noting that all three strains carrying both blaNDM-1 and blaOXA-301 are from China, two of which are clonally related to BZAB1. Conclusion: We report the genome sequence of a multidrug-resistant A. bereziniae strain co-carrying blaOXA-301 and blaNDM-1. A. bereziniae strains carrying various beta-lactam resistance genes have been identified sporadically over the world. Our findings could help us aid in understanding the genomic insights of this pathogen. Their future prevalence must be given more consideration.

Objective: This work aimed to describe the in vitro performance of the combined activity of ceftazidime-avibactam (CZA) plus aztreonam (ATM) against carbapenemase-producing Enterobacterales (CPE). Methods: We studied 44 CPE clinical isolates: NDM-1 (31), KPC-2 (5), KPC-3 (3), VIM-2 (2), NDM-1+KPC-2 (2), and OXA-48 (1). The efficacy of CZA in combination with were determined by two methods: (i) Kirby-Bauer's double disk synergy test and; (ii) Determination of the minimum inhibitory concentration to CZA by E-test, in either Mueller-Hinton agar alone or, supplemented with ATM 4 mg/L. Additionally, the Fractional inhibitory concentration index (FICI) was determined; values of ≤ 0.5 were interpreted as synergistic, while FICI > 0.5 were considered indifferent. Results: All isolates were carbapenem-resistant, 14 were resistant to CZA and ATM, 15 were only CZA resistant, 12 were only ATM resistant, and three were susceptible to both. 34/44 isolates presented positive double disk synergy tests between CZA and ATM regardless of their susceptibility profile, the isolates with negative synergy tests were susceptible to at least one of the agents. On the other hand, the 21 isolates selected to compare the MIC to CZA alone and CZA plus 4 mg/L ATM of exhibited FICI values between 0.016 and 0.125, indicating a synergistic effect. Conclusions: This method is available to clinical laboratories and would provide valuable information to guide the treatment of infections with CZA and ATM. In this sense, the use of CZA together with ATM is a potentially suitable combination for the treatment of carbapenemase-producing microorganisms.

Background: Carbapenemase-producing Enterobacterales (CPE) are global concerns in infection control, and the number of CPE outbreaks in hospitals is increasing despite the strengthening of contact precautions. This study aimed to confirm the prevalence and transition rate of CPE infection from stool surveillance culture and to identify the acquisition pathway of CPE. Methods: This is a longitudinal review of patients with stool surveillance cultures at a tertiary center in Seoul, South Korea, from July 2018 to June 2020. Pulsed-field gel electrophoresis, multi-locus sequence typing, and whole genome sequencing were performed for carbapenemase-producing Klebsiella pneumoniae and Escherichia coli strains. Results: Among 1620 patients who had undergone stool CPE surveillance cultures, only 7.1% of active surveillance at the Emergency Room (ER) and 4.4% of universal surveillance in the Intensive Care Unit (ICU) were stool CPE positive. The transition rates from stool carriers to clinical CPE infections were 29.4% in the ER and 31.3% in the ICU. However, it was significantly high (55.0%) in the initial stool CPE-negative ICU patients. Among the initial stool CPE-positive patients, hypertension (61% vs. 92.3%, P = 0.004), malignancy (28.8% vs. 53.8%, P = 0.027), and mechanical ventilation (25.4% vs. 53.8%, P = 0.011) were significant risk factors for clinical CPE infection. Molecular typing revealed that sequence type (ST) 307 and ST 395 were dominant in K. pneumoniae, and ST 410 was dominant in E. coli isolates. Conclusions: Active surveillance showed a higher detection rate than universal stool CPE screening, and one-third of positive stool CPE specimens ultimately developed subsquent clinical CPE infection. According to the molecular typing of the identified CPE strains, in-hospital spread prevailed over external inflow, and the transition rate to clinical CPE was particularly high in the ICU. Therefore, in order to control CPE propagation, not only active surveillance to block inflow from outside, but also continuous ICU monitoring within the hospital is necessary.

Background: Enterobacterales carrying blaNDM represent an emerging challenge in treating infectious diseases. In this study, we aimed to investigate the characteristics of blaNDM-producing Enterobacterales from three hospitals in southern Taiwan. Methods: Enterobacterales strains that were nonsusceptible to more than one carbapenem (ertapenem, meropenem, imipenem, or doripenem) were collected from hospitalized patients. Molecular typing for New Delhi metallo-β-lactamase (NDM) and antibiotic susceptibility tests were performed, followed by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and plasmid analysis by PCR-based replicon typing. Results: A total of 1311 carbapenem-nonsusceptible Enterobacterales were isolated from 2017 to 2021. blaNDM-encoding genes were detected in 108 isolates, with 53 (49.1%) harboring blaNDM-1 and 55 (50.9%) harboring blaNDM-5. The rate of blaNDM-1 detection among isolates decreased to 2% in 2021. However, the rate of E. coli harboring blaNDM-5 increased from 1% to 12% of total isolates during the study period. Of 47 NDM-5-positive E. coli isolates, 44 (93.6%) were ST8346 with high genetic relatedness. E. coli ST8346 isolates showed high-level resistance to both carbapenems and aminoglycosides. Most (38 out of 47, 80.9%) blaNDM-5-harboring E. coli isolates co-harbored blaOXA-181. We analyzed the regions harboring blaNDM-5 in E. coli ST8346 via PCR amplification. blaNDM-5 and blaOXA-181 were located on two separate plasmids, IncF and IncX3, respectively. Conclusion: The dissemination of E. coli ST8346 caused an increase in blaNDM-5 and blaOXA-181 co-harboring Enterobacterales in southern Taiwan, which show high-level resistance to both carbapenems and aminoglycosides. We identified a distinct IncF plasmid encoding blaNDM-5 that has the potential for rapid spread and needs further surveillance.

ABSTRACT: Objectives: A higher diversity of species, clones and genes have been increasingly implicated in carbapenemases spread, though the mobile genetic elements responsible for their acquisition and dispersion at local and global levels are less explored, particularly in species other than Klebsiella pneumoniae or Escherichia coli. We aim to explain the emergence of NDM-1 and KPC-3 carbapenemases in a Kluyvera cryocrescens isolate, and to shed light on the heterogeneity of genetic platforms and acquisition routes of blaNDM-1 in diverse Enterobacterales species in Portugal. Methods: A KPC-3 and NDM-1-producing K. cryocrescens colonizing a hospitalized patient in 2019 was characterized by whole-genome sequencing and antibiotic resistance profiling following standard methods. Conjugative transfer of carbapenemases genes was assessed by filter mating. Plasmids were reconstructed with in silico and in vitro approaches. blaNDM-1 genetic context was compared with that of diverse NDM-1-producing Enterobacterales species, previously described in Portugal. Results: K. cryocrescens K629 showed a multidrug resistance profile. Resistance gene blaKPC-3 was harboured by a Tn4401d transposon within a worldwide-spread IncN-ST15 plasmid (pKLU-KPC3), whereas blaNDM-1 was located in a Tn3000 within a non-typeable mosaic plasmid (pKLU-NDM1). The heterogeneous blaNDM-1 genetic platforms and variable plasmid backbones identified in various Enterobacterales species suggested multiple introductions of blaNDM-1 in Portugal, mediated by variable insertion sequences. Conclusions: We report the convergence of KPC-3 and NDM-1 in K. cryocrescens and the variable dissemination modes of these carbapenemases in different Enterobacterales species, underlining the need to track down genetic platforms responsible for carbapenemases diffusion.

Background: Worldwide observations revealed increased frequencies of multi-resistant Enterobacterales and resistance genes in hospital wastewater compared to any other type of wastewater. Despite the description of clonal lineages possibly adapted to hospital wastewater, little is known about long term persistence as well as evolution of these lineages. Methods: In this study, wastewater isolates of different Enterobacterales species from a tertiary care hospital were investigated with 2.5 years distance. Whole Genome Sequencing (WGS) and resistance gene identification were performed for E. coli, C. freundii, S. marcescens, K. pneumoniae, K. oxytoca, and E. cloacae isolates (n = 59), isolated in 2022 and compared with strains isolated from the same wastewater pipeline in 2019 (n = 240). Results: Individual clonal lineages with highly related isolates could be identified in all species identified more than once in 2022 that appear to persist in the wastewater drainage. A common motif of all persistent clonal lineages was the carriage of mobile genetic elements encoding carbapenemase genes with hints for horizontal gene transfer in persistent clones in this environment observed over the 2.5-year period. Multiple plasmid replicons could be detected in both years. In 2022 isolates blaVIM-1 replaced blaOXA-48 as the most common carbapenemase gene compared to 2019. Interestingly, despite a similar abundance of carbapenemase genes (>80% of all isolates) at both time points genes encoding extended spectrum β-lactamases decreased over time. Conclusions: This data indicates that hospital wastewater continuously releases genes encoding carbapenemases to the urban wastewater system. The evolution of the resident clones as well as the reasons for the selection advantage in this specific ecological niche needs to be further investigated in the future.

We analyzed consecutive clinical cases of infections due to carbapenemase-producing gram-negative bacteria detected in war-wounded patients from Ukraine who were treated at one university medical center in southwest Germany between June and December 2022. The isolates of multiresistant gram-negative bacteria were subjected to a thorough microbiological characterization and whole genome sequencing (WGS). We identified five war-wounded Ukrainian patients who developed infections with New Delhi metallo-β-lactamase 1-positive Klebsiella pneumoniae. Two isolates also carried OXA-48 carbapenemases. The bacteria were resistant to novel antibiotics, such as ceftazidime/avibactam and cefiderocol. The used treatment strategies included combinations of ceftazidime/avibactam + aztreonam, colistin, or tigecycline. WGS suggested transmission during primary care in Ukraine. We conclude that there is an urgent need for thorough surveillance of multiresistant pathogens in patients from war zones.

Background: Presently, community-associated carbapenemase-producing Enterobacterales (CPE) remains largely unknown and require public attention. This study aimed to investigate the presence of CPE from outpatients in Thailand. Methods: Non-duplicate stool (n = 886) and urine (n = 289) samples were collected from outpatients with diarrhea and urinary tract infection, respectively. Demographic data and characteristics of patients were collected. Isolation of CPE was performed by plating enrichment culture on agar supplemented with meropenem. Carbapenemase genes were screened by PCR and sequencing. CPE isolates were phenotypically and genotypically characterized. Results: Fifteen samples (1.3%, 14 stool and 1 urine) yielded blaNDM-1-positive carbapenemase-producing Klebsiella pneumoniae (CPKP). Additional resistance to colistin and tigecycline was observed in 53.3% and 46.7% of isolates, respectively. Age >60 years was identified as a risk factor for patients with CPKP (P

ABSTRACT: Objectives: This study aimed to characterise compounds with activity against carbapenemase-expressing Gram-negative bacteria and nematodes and evaluate their cytotoxicity to non-cancerous human cells. Methods: The antimicrobial activity and toxicity of a series of phenyl-substituted urea derivatives were evaluated using broth microdilution, chitinase, and resazurin reduction assays. Results: The effects of different substitutions present on the nitrogen atoms of the urea backbone were investigated. Several compounds were active against Staphylococcus aureus and Escherichia coli control strains. Specifically, derivatives 7b, 11b, and 67d exhibited antimicrobial activity against Klebsiella pneumoniae 16, a carbapenemase-producing Enterobacteriaceae species, with minimum inhibitory concentration (MIC) values of 100, 50, and 72 µM (32, 64, and 32 mg/L), respectively. In addition, the MICs obtained against a multidrug-resistant E. coli strain were 100, 50, and 36 µM (32, 16, and 16 mg/L) for the same compounds, respectively. Furthermore, the urea derivatives 18b, 29b, 50c, 51c, 52c, 55c–59c, and 62c were very active towards the nematode Caenorhabditis elegans. Conclusions: Testing on non-cancerous human cell lines suggested that some of the compounds have the potential to affect bacteria, especially helminths, with limited cytotoxicity to humans. Given the simplicity of synthesis for this class of compounds and their potency against Gram-negative, carbapenemase-expressing K. pneumoniae, aryl ureas possessing the 3,5-dichloro-phenyl group certainly warrant further investigation to exploit their selectivity.

Summary: Background: Acquisition of carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) are associated with negative health outcomes. Our adult intensive care unit (ICU) population has experienced low levels of CP-CRE acquisition; however, specific risk factors for this population at our medical facility have not been studied. Aims: To identify risk factors of CP-CRE acquisition and describe CP-CRE epidemiology among adult ICU patients at our medical facility. Methods: A retrospective cohort study was performed at a Kentucky Academic Medical Center. Surveillance specimens were collected at admission and weekly thereafter to identify CP-CRE colonization. Clinical data were extracted from patient medical records. Cases were defined as those who tested positive for CP-CRE on ICU admission day 3 or greater. Risk of CP-CRE acquisition was calculated using Modified Poisson regression. Findings: Independent risk factors of CP-CRE acquisition included administration of enteral tube feeds (risk ratio [RR], 4.46; 95% confidence interval [CI], 1.74–11.43); diagnosis of Clostridioides difficile enterocolitis (RR, 3.51; 95% CI, 1.27–9.68), pressure ulcer (RR, 3.48; 95% CI, 1.91–6.36), and morbid obesity (RR, 2.10; 95% CI, 1.12–3.95); having a drainage tube (RR, 2.63; 95% CI, 1.38–4.98); admission to a medical ICU (RR, 2.39; 95% CI, 1.32–4.35); 90-day use of a carbapenem (RR, 2.27; 95% CI, 1.21–4.26); and dialysis procedure (RR, 2.22; 95% CI, 1.15–4.27). Conclusion: Most CP-CRE risk factors were associated with alteration of colon microbiota and/or invasive procedures/devices. These results will assist in creating a more targeted CP-CRE active surveillance system and highlight areas for infection prevention intervention.

Olive ridley (Lepidochelys olivacea) turtles migrate across tropical regions of the Atlantic, Pacific, and Indian Oceans. Worryingly, olive ridley populations have been declining substantially and is now considered a threatened species. In this regard, habitat degradation, anthropogenic pollution, and infectious diseases have been the most notorious threats for this species. We isolated a metallo-β-lactamase (NDM-1)-producing Citrobacter portucalensis from the blood sample of an infected migratory olive ridley turtle found stranded sick in the coast of Brazil. Genomic analysis of C. portucalensis confirmed a novel sequence type (ST), named ST264, and a wide resistome to broad-spectrum antibiotics. The production of NDM-1 by the strain contributed to treatment failure and death of the animal. Phylogenomic relationship with environmental and human strains from African, European and Asian countries confirmed that critical priority clones of C. portucalensis are spreading beyond hospital settings, representing an emerging ecological threat to marine ecosystems.

Introdução/objetivo: Os hospitais são ambientes propícios para seleção de Microrganismos (MO) resistentes devido a superexposição aos antimicrobianos. Entre esses MO, alguns Bacilos Gram-Negativos (BGN) multirresistentes estão na lista de agentes prioritários para a OMS. O objetivo dessa pesquisa foi descrever o perfil epidemiológico das culturas positivas por BGN produtoras de Carbapenemases (CARB) isoladas de pacientes hospitalizados em um hospital pediátrico. Métodos: Estudo quantitativo, documental retrospectivo conduzido em um hospital pediátrico em Curitiba. Foram coletados os resultados de culturas e testes fenotípicos (MCIM e ECIM) em amostras de sangue, aspirado traqueal, lavado broncoalveolar, líquidos nobres (líquor, ascítico) e urina, provenientes de pacientes (0 a 18 anos) atendidos entre Jan/20 a Dez/21. O total de amostras analisadas foi 1441 (em 2020) e 1796 (em 2021). O projeto foi submetido e aprovado pelo Comitê de Ética com o número 5.690.088. Resultados: Para a família Enterobacteriaceae, as maiores frequências de isolados de CARB em 2020 e 2021 foram, respectivamente, líquidos nobres (17%) e amostras respiratórias (75%). A Klebsiella pneumoniae foi a principal espécie identificada. Para os isolados não-fermentadores de glicose (NFG), em 2020 e 2021, a distribuição de resistência por CARB detectadas foi em culturas de líquidos nobres (20%) e urina (25%), respectivamente. Acinetobacter baumannii e a Pseudomonas aeruginosa foram as espécies mais comumente recuperadas. Quanto as classes de CARB mais frequentes, entre os anos de 2020 e 2021, as metalo-betalactamases representaram 35% e 44% e as serino-carbapenemases, 26% e 56%. Com relação a identificação de CARB por biologia molecular, os resultados foram em 2020 os genes bla-NDM (28%) e bla-KPC (9%); em 2021, o perfil muda para genes bla-SPM (25%) e bla-NDM (7%). Outro resultado é a detecção de isolados resistentes aos CARB por mecanismos não enzimáticos, tais como perda de porinas e ativação de bombas de efluxo. Em 2020 tivemos 14% e 2021 foi 12%. Conclusão: Acompanhar anualmente o perfil epidemiológico de multirresistência atende a uma das principais ações dos programas de gerenciamento de antimicrobianos, porque permite observar se os resultados seguem o padrão local e nacional de resistência. Nessa casuística, as cepas produtoras de CARB e genes de resistência são semelhantes ao encontrado no estado do Paraná e Brasil, segundo os boletins epidemiológicos do período.

Introdução: Pseudomonas sp é um patógeno oportunista que está associado a diversos casos de Infecções Relacionadas à Assistência à Saúde (IRAS). A produção de enzimas carbapenamases incluíndo a coprodução destas enzimas por estes patógenos se tornou uma preocupação em saúde pública, principalmente após a pandemia da COVID-19, uma vez que os carbapenêmicos estão entre os antimicrobianos de último recurso para o tratamento de infecções multirresistentes e novos antimicrobianos, como ceftazidima-avibactam, dependem do tipo de carbapenemase envolvido. Objetivo: Avaliar a prevalência dos genes de enzimas carbapenemases mais prevalentes em 208 isolados de Pseudomonas sp não sensíveis ao meropenem, no período de julho de 2022 a junho 2023 no Hospital de Clínicas de Porto Alegre. Métodos: A identificação bacteriana foi realizada pela técnica de MALDI TOF pelo Vitek MS, Biomerieux. O teste de suscetibilidade aos antimicrobianos foi realizado pelo método de disco-difusão conforme o BrCAST, 2022. A Reação em Cadeia da Polimerase (PCR) convencional para a identificação do gene blaSPM e o teste de RT-qPCR por High-Resolution Melt (HRM) para a detectação de seis diferentes tipos genéticos de carbapenemases blaKPC, blaNDM, blaGES, blaOXA-48, blaIMP e blaVIM foram realizados. Resultados: Um total de 52,9% dos isolados de Pseudomonas spp. meropenem resistentes não apresentou nenhum dos genes pesquisados; 37,0% apresentaram o gene blaNDM; 3,4% apresentaram o gene blaVIM, 1,4% apresentaram o gene blaIMP e 1,0% apresentaram o gene blaSPM; 1,9% apresentaram concomitantemente os genes blaIMP e blaVIM; 0,5% apresentaram os genes blaVIM e blaSPM; e 0,5% apresentaram os genes blaNDM e blaGES. Conclusão: Mais da metade dos isolados de Pseudomonas spp. não apresentaram produção das carbapenemases pesquisadas, podendo haver outros mecanismos de resistência aos carbapenêmicos que não foram pesquisados. A enzima mais prevalente foi a blaNDM sendo detectada em mais de um terço dos isolados, excluindo a possibilidade de tratamento com ceftazidima-avibactam. Portanto, evidencia-se a importância em pesquisar a produção de carbapenemases em Pseudomonas spp. tanto para o sucesso do tratamento como para o conhecimento da epidemiologia local.

Objectives: Bacterial persister cells are a sub-population of cells that are tolerant to high concentrations of antibiotics. In this study, we investigated the effect of plasmids bearing carbapenemase genes on persister cell formation. Methods: Three plasmids, IncX3-type plasmid with blaNDM-1, IncN-type plasmid with blaKPC-2, and ColE-type plasmid with blaOXA-232, were transformed into Escherichia coli MG1655. For the ColE-type plasmid (pM5_OXA232), gene-deletion plasmids were constructed and transformed into the MG1655. Persister assays were performed against ciprofloxacin and amikacin, and expression levels of relA and spoT were measured for the wild-type E. coli and all transformants. Results: Unlike the other two plasmids, transformation of ColE-type plasmid (pM5_OXA232) caused a significant increase in the formation of persister cells. Compared with transformants that harboured intact pM5_OXA232, transformants that harboured plasmids with deletions of gene(s), vbhA, hypothetical gene, or a mobile gene cassette showed decreased persister cell formation. Expression levels of relA and spoT exhibited patterns similar to those of persister cell formation rates, particularly against ciprofloxacin. Conclusion: In this study, we showed that a small ColE-type plasmid bearing blaOXA-232 has an effect on persister cell formation, possibly contributing to the dissemination of low-level carbapanemase.

ABSTRACT: Objectives: Ceftolozane-tazobactam (C/T) proved its efficacy for the treatment of infections caused by non-carbapenemase producing Pseudomonas aeruginosa and Enterobacterales. Here, we aimed to provide susceptibility data on a large series of Enterobacterales since the revision of EUCAST categorization breakpoints in 2020. Methods: First, C/T susceptibility was determined on characterized Enterobacterales resistant to third generation cephalosporins (3GCs) (extended spectrum β-lactamase [ESBL] production or different levels of AmpC overexpression) (n = 213) and carbapenem-resistant Enterobacterales (CRE) (n = 259), including 170 carbapenemase producers (CPE). Then, 1632 consecutive clinical Enterobacterales responsible for infection were prospectively collected in 23 French hospitals. C/T susceptibility was determined by E-test® (biomérieux) and broth microdilution (BMD) (Sensititre™, Thermo Scientific) to perform method comparison. Results: Within the collection isolates, 88% of 3GC resistant strains were susceptible to C/T, with important variation depending on the resistance mechanism: 93% vs. 13% susceptibility for CTX-M and SHV-ESBL producers, respectively. Only 20% of the CRE were susceptible to C/T. Among CPE, 80% of OXA-48-like producers were susceptible to C/T, whereas all metallo-β-lactamase producers were resistant. The prospective study revealed that 95.6% of clinical isolates were susceptible to C/T. Method comparison performed on these 1632 clinical isolates demonstrated 99% of categorization agreement between MIC to C/T determined by E-test® in comparison with the BMD (reference) and only 74% of essential agreement. Conclusion: Overall, C/T showed good activity against wild-type Enterobacterales, AmpC producers, and ESBL-producing Escherichia coli but is less active against ESBL-producing Klebsiella pneumoniae, and CRE. E-test® led to an underestimation of the MICs in comparison to the BMD reference.

ABSTRACT: Objectives: A novel ß-lactam-β-lactamase inhibitor (BLBI), meropenem (MEM), combined with the boronate-based inhibitor vaborbactam (VAB), has recently been introduced for the treatment of infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. The purpose of this study was to select for MEM-VAB resistance using a collection of eight KPC-producing K. pneumoniae clinical isolates, including three that produce KPC variants conferring ceftazidime-avibactam (CAZ-AVI) resistance, and subsequently decipher the corresponding resistance mechanisms. Methods: Mutants were selected in a stepwise process on agar plates containing different MEM-VAB concentrations. Susceptibility testing was performed by broth microdilution, and complementation assays were performed with wildtype ompK36. Whole genome sequencing was performed on mutants, and KPC copy number was assessed by quantitative polymerase chain reaction . Results: Mutants were obtained from 6/8 tested isolates and reduced susceptibility to all tested β-lactams, and BLBIs, including CAZ-AVI, imipenem-relebactam, and aztreonam-AVI, were observed. No mutations were identified in the blaKPC. However, mutations in ompK36 were observed in four mutant lineages, and complementation with a wild-type ompK36 resulted in a reduction of minimal inhibitory concentrations to both MEM-VAB and other ß-lactams/BLBIs. blaKPC gene copy numbers were significantly increased in four mutant lineages. Whole genome sequencing identified genomic rearrangements in two lineages comprising mutations in the plasmid replicon encoding gene and duplication of the Tn4401 transposon bearing the blaKPC gene into a ColE-like, high copy number plasmid. Conclusions: In contrast to what is observed with KPC-producing mutants exhibiting resistance to CAZ-AVI, mainly corresponding to mutated KPC enzymes, here the MEM-VAB-resistant mutants showed permeability defects combined with increased KPC production, resulting from genomic rearrangement.

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

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) co-carrying multiple carbapenemases is complicating clinical treatment. This study aimed to investigate the global dissemination trends of CRKP strains that co-carry multiple carbapenemases. The CRKP isolate KP424 co-carrying blaNDM-1 and blaKPC-2, recovered from a stool specimen, was identified by the NG-Test Carba 5 test, and the genome sequence was further determined by using Nanopore MinION and Illumina NovaSeq 6000 technologies. The genome sequences of the CRKP strains carrying multiple carbapenemase genes were further retrieved from the NCBI GenBank database. Thirteen antimicrobial resistance genes, including blaNDM-1 and blaKPC-2, have been identified in KP424, with blaNDM-1 and blaKPC-2 located on different plasmids. In total, 832 genome sequences of CRKP strains co-carrying two carbapenemase genes were retrieved from the NCBI database. Strains carrying both blaNDM and blaOXA-48-like accounted for 665 (79.9 %) of the total strains, ranking first, and those carrying both blaKPC and blaNDM accounted for 103 (12.4 %), ranking second. The prevalence of CRKP strains co-carrying two carbapenemase genes increased significantly over time, from 0.40 % in 2010 to 9.67 % in 2021. The proportion of strains carrying both blaKPC and blaNDM has also increased, from 0.00 % in 2010 to 4.40 % in 2021. The strains carrying both blaKPC and blaNDM had the highest prevalence (66.7 %, 52/78) in China, while those carrying both blaNDM and blaOXA-48-like had the highest prevalence worldwide. Multiple-carbapenemase producers pose a great threat to public health; further research on the mechanisms underlying multiple carbapenemase gene occurrence is required to prevent their global dissemination.

Purpose: In this study, we aimed to assess the geographic distribution and molecular characteristics of β-lactamases among Enterobacterales isolates causing intra-abdominal infections (IAIs) from 2015 to 2018 in the Asia–Pacific region. Method: Isolates were investigated for extended-spectrum β-lactamases (ESBLs), AmpC β-lactamases, and carbapenemases using multiplex PCR assays and full-gene DNA sequencing. Result: A total of 832 Enterobacterales isolates from 8 different countries with β-lactamase genes were analysed. Plasmid-mediated ESBLs and AmpC β-lactamases were encoded in 598 (71.9 %) and 314 (37.7 %) isolates, respectively. In 710 (85.3 %) carbapenemase-negative isolates, positivity for both AmpC β-lactamases and ESBLs was identified in 51 (8.5 %) Escherichia coli and 24 (3.4 %) Klebsiella pneumoniae isolates. The most prevalent countries were Taiwan and Vietnam, and the co-occurrence of CMY/CTX-M in E. coli and DHA-1/ESBLs in K. pneumoniae was predominant. All isolates showed high susceptibility to colistin, but susceptibility to carbapenems varied among different resistance mechanism combinations. Among 122 (14.7 %) isolates encoding carbapenemase, NDM (n = 67, including 64.2 % NDM-1) was the most common, followed by the OXA-48-type (n = 49), KPC (n = 24) and IMP (n = 4). The most prevalent country was Thailand (n = 44), followed by Vietnam (n = 35) and the Philippines (n = 21). Twenty-two isolates were found to encode multiple carbapenemases, 16 of which were collected from Thailand and harbored NDM-1, OXA-232 and CTX-M-15. Despite high susceptibility to amikacin, susceptibility to colistin was only 56 %. Conclusion: The emergence of carbapenem-non-susceptible AmpC/ESBL co-occurring Enterobacterales and colistin non-susceptible carbapenemases co-occurring K. pneumoniae highlights potential therapeutic challenges in the Asia–Pacific region.

ABSTRACT: Objectives: Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections globally. High-risk carbapenemase-encoding P. aeruginosa clones are disseminating in many regions. The aim of this study was to learn more about the lineages and mechanisms of resistance of P. aeruginosa circulating in Peru. Methods: A total of 141 carbapenemase-producing isolates recovered from hospitalized and ambulatory patients in Lima were sequenced and analyzed to infer their lineages through whole-genome sequence typing (wgST) and to identify their antimicrobial resistance genes. Results: wgST identified nine sequence types (STs); ST111 and ST357 were the most frequently encountered (44.0% and 38.3%, respectively), followed by ST179 (8.5%), with the remaining six detected only sporadically. Among ST357 isolates, 96.3% carried the novel blaIMP-93 allele, whereas the remainder harbored blaIMP-74. 74.2% of ST111 isolates co-harbored blaIMP-18 and blaVIM-2, while the rest carried either of these genes individually. All other ST lineages carried a single carbapenemase, which was either blaIMP-16, blaIMP-74, or blaVIM-2. Conclusion: Our study shows that the high-risk P. aeruginosa clones ST357, which harbors the novel blaIMP-93, and ST111, which carries blaIMP-18 and blaVIM-2, have apparently become endemic in the region.

ABSTRACT: Objectives: We characterised the complex surrounding regions of blaGES-16 in a Pseudomonas aeruginosa exoU+ strain (P-10.226) in Brazil. Methods: Species identification was performed by MALDI-TOF MS, and the antimicrobial susceptibility profile was determined by broth microdilution based on European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The whole genome sequencing (WGS) of P-10.226 strain was performed using both short-read paired-end sequencing on the Illumina MiSeq platform as well as the long-read Oxford Nanopore MinION. Results: WGS analysis showed that P-10.226 carried blaGES-16, which was found as a gene cassette inserted into a novel class I integron, In1992 (aadB-blaOXA-56-blaGES-16-aadB-aadA6c), whose 3′-CS was truncated by a nested transposable element, IS5564::ISPa157. The structure was even more complex since IS6100-ΔIS6100 structure and a TnAs2-like harbouring the operon merRTPADE was found downstream In1992. Fragments of TnAs3 harbouring 25-bp imperfect inverted repeats were identified bordering the intl1 of In1992 and also flanking IS6100-ΔIS6100, which might be genetic marks of its previous presence in the genome. Interestingly, In1992 also shows a distinct cassette array from In581 (blaGES-16-dfrA22-aacA27-aadA1), which was previously reported in Serratia marcescens strains recovered in Brazil. Finally, exoU gene, which encodes a potent cytotoxin of type III secretion systems (T3SS) effector proteins from P. aeruginosa and is associated to severe infections, was also detected. Conclusion: We described the novel In1992 carrying blaGES-16 surrounded by complex transposition events in a XDR P. aeruginosa strain. The identification of many sets of direct repeats adjacent to TnAs3 fragments indicates a major past of transposition events that shaped the current genetic environment of In1992.

ABSTRACT: Objectives: Multidrug-resistant Acinetobacter baumannii (MDR-Ab), particularly strains producing oxacillinase (OXA)-type carbapenemases, have rapidly emerged in health care settings as a frequent cause of serious infections with limited treatment options. This study evaluated the in vitro activity of sulbactam (SUL) combined with durlobactam (DUR) against a collection of carbapenemase-producing A. baumannii, and investigated the mechanisms of resistance. Methods: Susceptibility testing was performed on 100 isolates by either broth microdilution or by the Epsilometer test. Isolates were screened for the insertion sequence ISAba1 upstream of the intrinsic chromosomal blaADC by polymerase chain reaction (PCR). Whole genome sequencing was performed on 25 SUL-DUR resistant isolates, and analyses were performed using the Center for Genomic Epidemiology platform. Target gene sequences were compared to A. baumannii American Type Culture Collection (ATCC) 17978. Results: SUL-DUR exhibited excellent activity against A. baumannii isolates with susceptibility levels as follows: amikacin, 18%; colistin, 91%; cefepime, 5%; imipenem, 0%; minocycline, 46%; SUL, 3%; sulbactam-cefoperazone, 8%; SUL-DUR, 71% (based on a breakpoint at 4 mg/L). Twenty-five non-New Delhi metallo-ß–lactamase (NDM)-producing isolates had SUL-DUR MIC values >4 mg/L, amongst which 14 isolates showed substitutions in penicillin-binding protein (PBP)3, previously shown to be associated with SUL-DUR resistance. Substitutions that have not previously been described were detected in SUL-DUR targets, namely PBP1a, PBP1b, PBP2, and PBP3. By contrast, there was no evidence of the involvement of permeability or efflux. Conclusions: SUL-DUR exhibited excellent in vitro antibacterial activity against carbapenemase-producing A. baumannii isolates. Amongst the 25 resistant isolates, we identified a number of mechanisms which may be contributing factors, in particular PBP substitutions and the production of specific beta-lactamases.

ABSTRACT: Objectives: Although Sri Lanka belongs to a region with a high prevalence of extended-spectrum β-lactamase (ESBL), AmpC β-lactamase and carbapenemase-producing Enterobacteriaceae, data regarding antimicrobial resistance (AMR) is limited. We studied the prevalence and diversity of β-lactamases produced by Enterobacteriaceae urinary pathogens from two hospitals in the Western Province of Sri Lanka. Methods: ESBL, AmpC β-lactamase and carbapenemase production was detected by phenotypic testing followed by genotyping. Results: The species responsible for urinary tract infections (UTI) were Escherichia coli (69%), Klebsiella pneumoniae (16%) and Enterobacter sp (6%). The prevalence of ESBL (50%), AmpC β-lactamase (19%) and carbapenemase (11%) phenotypes was high, and greater in hospital-acquired (HA-UTI) (75%) than in community-acquired UTI (CA-UTI) (42%). Identification of CA-UTI caused by carbapenemase-producing Enterobacteriaceae (5%) is alarming. Only one ESBL gene, blaCTX- M-15, was detected. AmpC β-lactamase genes found in E. coli and K. pneumoniae were blaCMY-2, blaCMY-42 and blaDHA-1, while Enterobacter sp. carried blaACT-1. Carbapenemase genes were blaNDM-1, blaNDM-4, blaOXA-181 and blaOXA-232, while blaKPC, blaIMP and blaVIM were absent. Co-occurrence of multiple bla genes, with some isolates harbouring six different bla genes, was common. Carbapenem-resistant isolates without carbapenemase genes displayed mutations in the outer membrane porin genes, ompF of E. coli and ompK36 of K. pneumoniae. Factors associated with UTI with β-lactamase-producing Enterobacteriaceae were age ≥50 years, previous hospitalization, presence of an indwelling urinary catheter, history of diabetes mellitus or other chronic illness and recurrent urinary tract infections. Conclusion: This study adds to the currently scarce data on AMR in Sri Lanka.

Objectives: Carbapenem-resistant Enterobacterales (CRE) remain an urgent public health priority in the United States. CRE poses a major threat to patients in healthcare and a potential risk to the community. This study examined the epidemiological trends, clinical, and microbiological data of CRE in the Greater Houston region of Texas. Methods: A multi-institutional retrospective observational study was conducted using surveillance data collected from 2015 to 2020. Predictors of incidence rates of CRE were determined by a negative binomial regression fit using a generalized estimation equation. Results: Over a 6-year period, 4236 CRE cases were reported, of which Klebsiella pneumoniae accounted for 84.8%. The results show a steady increase in CRE cases, with a sharp rise since 2018. The majority of carbapenemase-producing Enterobacterales were Klebsiella pneumoniae carbapenemase (KPC)-producing (77.2%), followed by other rare carbapenemases, which includes OXA-48, NDM, IMP, VIM, coproduction of KPC with OXA-48, KPC with NDM, and NDM with OXA-48. Acute care hospitals (ACH) accounted for 68.5% of the source of CRE cases. The incidence rate of CRE cases reported from ACH and long-term acute care (LTAC) facilities was 1.16 times that of long-term care facilities (adjusted rate ratio [ARR] = 1.16, 95% confidence interval [CI]:1.04–1.30). The incidence rate of CRE among patients with indwelling devices was 15% (ARR = 0.85, 95% CI: 0.79–0.92) lower than that of patients without indwelling devices. Conclusion: The rise in the rate of CRE cases despite aggressive infection prevention and control strategies in the region is alarming. Evaluating and improving the current infection control strategies may be warranted.

Summary: Purpose: This study identified the epidemiological characteristics, including the size and major strains, of carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) and CP-CRE-related factors by comparing the characteristics of patients in the CP-CRE and non-CP-CRE groups and the CP-CRE and non-CRE groups. Methods: This secondary data analysis study included 24 patients in the CP-CRE group, 113 patients in the non-CP-CRE group, and 113 in the non-CRE group. The size and type of CP-CRE were analyzed in terms of frequency and percentage, and CP-CRE risk factors were identified using multiple logistic regression analysis. Results: The rate of CP-CRE positivity among patients with CRE was 17.5%, and the most common causative organism in the CP-CRE group was Klebsiella pneumoniae (81.8%). There were no significant differences between patients in the CP-CRE and non-CP-CRE groups. When compared with the non-CRE group, the isolation of multidrug-resistant organisms except for CRE, particularly vancomycin-resistant Enterococcus, was confirmed as a major risk factor. Conclusion: To prevent CP-CRE acquisition, patients with multidrug-resistant organisms require treatment with more thorough adherence to CRE prevention and management guidelines.

Carbapenemase enzyme production is responsible for resistance to carbapenem among Gram-negative bacteria. This study aimed to detect common carbapenemase and oxacilinase genes among uropathogenic E. coli (UPEC) isolated from hospitalized patients in Rasht, north of Iran. In the present study, from 2000 urine samples, 263 UPEC strains were isolated from inpatients with urinary tract infections (UTI) in 2020. The Kirby-Bauer disk diffusion susceptibility test was used to determine the sensitivity or resistance of isolates to antimicrobial compounds. The double-disk test confirmed extended-spectrum β lactamase (ESBL) production phenotypically, and the presence and distribution of genes encoding carbapenemase and oxacilinase were investigated using polymerase chain reaction (PCR). Based on the findings, 13/263 isolates (8 ESBL and five non-ESBL) showed a non-susceptible phenotype to at least one of the studied carbapenem group antibiotics, and 121 (46%) isolates were ESBL-producers. PCR for oxacilinase and carbapenemase genes was done on all 126 isolates, including ESBL-positive and carbapenem-resistant strains, in which 10 (7.9%) and 25 (19.8%) isolates harbored OXA-1 and IMP genes, respectively. Also, OXA-2, OXA-10, OXA-48, VIM, and NDM genes were not found in any studied isolates. IMP and OXA-1 genes among carbapenemase-producing isolates indicate the possible spread of antibiotic-resistant strains. Hence, identification and control of ESBL and carbapenemase-producing strains, although with almost low frequency due to plasmid genes encoding carbapenemase, is essential for infection control.

ABSTRACT: Objectives: : To identify the carbapenemase and colistin resistance genes and epidemiological status of carbapenem-resistant Enterobacterales (CRE) among 7 secondary and 11 tertiary hospitals in Henan province, China. Methods: : CRE isolates and clinical data of infected patients were collected from 7 secondary and 11 tertiary hospitals in Henan from July to September 2019 and analysed retrospectively. Polymerase chain reaction (PCR) and gene sequencing were performed to detect carbapenemase and colistin resistance genes; multilocus sequence typing was also performed. Results: : In total, 238 nonduplicate CRE isolates were collected mainly from the respiratory tract (54.20%) and blood (18.91%) of CRE-infected patients, half of them aged >65 years (45.80%). Carbapenem-resistant Klebsiella pneumoniae (CRKP) was the most common CRE (184 isolates, 77.31%) with constituent ratios of 84.38% and 72.54% in secondary and tertiary hospitals, respectively. In 184 CRKP isolates, blaKPC-2 (89.13%) was the dominant carbapenemase gene, and ST11 (71.74%) was the most prevalent sequence type (ST), with constituent ratios of 83.95% and 62.14% in secondary and tertiary hospitals, respectively. In 29 carbapenem-resistant Escherichia coli (CREC) isolates, blaNDM-5 (58.62%) and ST2 (31.03%) were prevalent. Four CRKP isolates and one CREC isolate were colistin-resistant and carried the plasmid-mediated mcr-1 gene. Conclusion: : Our results showed a wide spread of CRKP-ST11 with KPC-2 carbapenemase in the analysed 18 hospitals. The CRKP constituent ratio, CRKP-STs, and CREC carbapenemase genes between secondary and tertiary hospitals showed significant differences. The emergence of a colistin-resistant CRKP with plasmid-mediated resistance gene mcr-1 is of serious concern due to the limited treatment options.

ABSTRACT: Objectives: Here we describe an IncQ1-like plasmid carrying blaKPC-2 in a new non-Tn4401 element found in Citrobacter werkmanii recovered from coastal water. Methods: In vitro and in silico approaches were used to assess antimicrobial resistance determinants, as well as blaKPC-2 vicinities. Results: The LB-887 isolate showed a multidrug-resistant phenotype and was identified as C. werkmanii. Resistome analysis identified further acquired resistance determinants to β-lactams, aminoglycosides, sulphonamides/trimethoprim, tetracyclines, chloramphenicol, macrolides, rifampicin and fluoroquinolones. Plasmidome included incompatibility groups IncA, IncC2, IncR, Col and IncQ families. The blaKPC-2 was inserted on a new variant of NTEKPC-II, called here NTEKPC-IIe, carried by an InQ1-like plasmid of 7930 kb (pKPC-LB887). NTEKPC-IIe differed from NTEKPC-IId by the complete absence of ISKpn6-tnpA. The InQ1-like backbone harbouring this element had been described in Enterobacterales recovered from clinical and environmental settings. Conclusion: Unravelling genetic structures related to blaKPC dissemination in different settings may provide clues on the main forces driving evolution of this important resistance determinant. Indeed, the occurrence of blaKPC in a new NTEKPC variant from an environmental source highlights the ongoing evolution of this mobile genetic element. In addition, blaKPC carriage on a small and highly mobilizable IncQ plasmid in C. freundii complex from recreational water, similar to others found in clinical isolates, may suggest its relevance for blaKPC-2 dissemination among different compartments.

ABSTRACT: Objectives: The resistome, virulome, mobilome and phylogenetic relationship of the Acinetobacter baumannii isolate FG121 depicting the multilocus sequence type (ST) 231 isolated from hospital effluent water in South Africa was determined using whole-genome sequence analysis. Method: A. baumannii FG121 was isolated on Leed Acinetobacter Medium (LAM) agar and the bacterial isolate was identified using the VITEK®2 platform. Antibiotic susceptibility testing was performed using Kirby–Bauer Disk diffusion method. A whole genome sequencing library was constructed from DNA extracted from the isolate using the Illumina Nextera XT library preparation kit and was sequenced using the Illumina NextSeq500 platform. Generated reads were de novo assembled using SpAdes v.3.9. The assembled contigs were annotated, and multilocus sequence type, antimicrobial resistance, and virulence genes were identified. Results: The resistome was consistent with the resistance phenotype of the isolate with resistance determinants for beta-lactams, aminoglycosides, and tetracycline (blaADC-25, blaOXA-23, blaOXA-51, blaNDM-1, aph[3’]-VIa and tet[B]). Global phylogenomic analysis using BacWGSTdb revealed that the isolate belonged to the multilocus sequence type ST-231, similar to previously reported isolates from South Africa, the United States, and related to the invasive KR3831 isolate identified from Oman in 2012, suggesting the isolate might be imported from abroad. Virulome analysis predicted both virulence and biofilm-determinants of A. baumannii, which may help to establish infections in adverse conditions. Conclusion: This is the first report on a carbapenemase-encoding A. baumannii ST-231 isolated from hospital effluent water. Our data will offer insight into the global phylogenetic, pathogenicity and distribution of A. baumannii in South Africa.

Carbapenemase-producing Enterobacteriaceae (CPE) isolates are recognized as one of the most severe threats to public health. However, the population structure and genetic characteristics of CPE isolates among bloodstream infections (BSIs) are largely unknown. To address this knowledge gap, in this study, we included patients with clinically significant BSIs due to Enterobacterales isolates, recruited from 26 sentinel hospitals in China (2014–2015). CPE isolates were microbiologically and genomically characterized, including their susceptibility profiles, molecular typing, phylogenetic features, and genetic context analysis of carbapenemase-encoding genes. Of the 2569 BSI Enterobacterales isolates enrolled, 42 (1.6%) were carbapenemase-positive. Moreover, among the 2242 investigated isolates, 1111 (49.6%) extended-spectrum β-lactamase (ESBL)-producing isolates were identified in Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Proteus mirabilis (P. mirabilis), and Klebsiella oxytoca. Whole genome sequencing analysis showed the clonal spread of K. pneumoniae carbapenemase (KPC)-2-producing K. pneumoniae sequence type (ST) 11 and New Delhi metallo-β-lactamase (NDM)-5-producing E. coli ST167 in our collection. Plasmid analysis revealed that carbapenemase-encoding genes were located on multiple plasmids. A high prevalence of biofilm-encoding type 3 fimbriae clusters and yesiniabactin-associated genes was observed in K. pneumoniae isolates. This work demonstrates the high prevalence of ESBLs and the wide dissemination of CPE among BSI isolates in China, which represent real clinical threats. Moreover, our findings first illustrate a more comprehensive genome scenario of CPE isolates among BSIs. The clonal spread of KPC-2-producing K. pneumoniae ST11 and NDM-5-producing E. coli ST167 needs to be closely monitored.

Background: Aggressive antimicrobial infections together with an increasing in resistance to antimicrobial medications cause a threat to immunocompromised individual. In this context, spiro compounds play an important role in drug discovery research due to their diverse biological activities including antimicrobial activity which is well documented in literature, because of their unique structural properties allow for easy interaction with the target enzyme of the biological system. This biological precedent encouraged us to design and synthesis of novel dispirooxindolopyrrolidine hybrid heterocycles via multicomponent cycloaddition protocol. Materials and methods: Novel class of dispirooxindolopyrrolidine hybrid heterocycles has been synthesized by multicomponent 1,3-dipolar cycloaddition methodology and the structure of the products was determined through spectroscopic analysis. The synthesized organic compounds were evaluated for their antimicrobial activities using sensitivity test with standard agar well diffusion method. Results: The synthesized new class of dispiropyrrolidine embedded indandione hybrids showed significant antimicrobial activity against carbapenemase producing Klebsiella pneumoniae (CKPs) isolated from hospital patients. Compound that possessing chlorine substituted on the aryl ring exhibited more potent antimicrobial activities and the zone of inhibitions was observed between 7.00 ± 0.09 and 18.40 ± 0.70 mm. The MIC value of compound 4b was 0.030 mg/mL against tested CKP. Conclusion: A new class of dispiropyrrolidine heterocyclic hybrids were synthesized in good to excellent yield employing multicomponent 1,3-dipolar cycloaddition strategy. Among them, compounds bearing 4-chloro substituted on the aryl ring exhibited notable activity just equal to the standard drug, ciprofloxacin. Future studies are needed to elucidate the pharmacological properties of new compounds that were synthesized in our laboratory for the novel therapeutic development.

ABSTRACT: Objectives: This study aimed to develop and evaluate a novel air-dried high-resolution melt (HRM) assay to detect eight major extended-spectrum β-lactamase (ESBL) (blaSHV and blaCTX-M groups 1 and 9) and carbapenemase (blaNDM, blaIMP, blaKPC, blaVIM and blaOXA-48-like) genes that confer resistance to cephalosporins and carbapenems. Methods: The assay was evaluated using 439 DNA samples extracted from bacterial isolates from Nepal, Malawi and the UK and 390 clinical isolates from Nepal with known antimicrobial susceptibility. Assay reproducibility was evaluated across five different real-time quantitative PCR (qPCR) instruments [Rotor-Gene® Q, QuantStudioTM 5, CFX96, LightCycler® 480 and Magnetic Induction Cycler (Mic)]. Assay stability was also assessed under different storage temperatures (6.2 ± 0.9°C, 20.4 ± 0.7°C and 29.7 ± 1.4°C) at six time points over 8 months. Results: The sensitivity and specificity (with 95% confidence intervals) for detecting ESBL and carbapenemase genes was 94.7% (92.5–96.5%) and 99.2% (98.8–99.5%) compared with the reference gel-based PCR and sequencing and 98.3% (97.0–99.3%) and 98.5% (98.0–98.9%) compared with the original HRM wet PCR mix format. Overall agreement was 91.1% (90.0–92.9%) when predicting phenotypic resistance to cefotaxime and meropenem among Enterobacteriaceae isolates. We observed almost perfect inter-machine reproducibility of the air-dried HRM assay, and no loss of sensitivity occurred under all storage conditions and time points. Conclusion: We present a ready-to-use air-dried HRM PCR assay that offers an easy, thermostable, fast and accurate tool for the detection of ESBL and carbapenemase genes in DNA samples to improve antimicrobial resistance detection.

ABSTRACT: Objectives: This study reports the antimicrobial activity of ceftazidime/avibactam (CZA) and comparators against carbapenemase-producing Enterobacterales (N = 1992) and carbapenemase-producing Pseudomonas aeruginosa (N = 784) collected in Africa/Middle East, Asia/South Pacific, Europe and Latin America (2016–2018).Methods: Minimum inhibitory concentrations (MICs) and susceptibility were determined using broth microdilution methodology and EUCAST breakpoints. Carbapenemase-encoding genes were detected using multiplex PCR.Results: No isolates of carbapenemase-producing, metallo-β-lactamase (MBL)-negative Enterobacterales from Africa/Middle East or Latin America were resistant to CZA; resistance rates in Europe and Asia/South Pacific were ≤4.5%. Colistin had the lowest resistance rate among MBL-positive isolates (6.0–11.4%). Enterobacterales isolates collected in Latin America predominantly carried a KPC carbapenemase (77.6%), whereas in Africa/Middle East OXA-48-like carbapenemases were most frequently detected (55.9%), and in Asia/South Pacific most isolates carried NDM carbapenemases (56.2%). Among all Enterobacterales carrying KPC carbapenemases, the lowest rate of resistance was to CZA (1.5%), and among isolates carrying NDM carbapenemases it was to colistin (10.8%). Among carbapenemase-producing, MBL-negative P. aeruginosa, resistance rates to CZA were 8.6% for isolates collected in Europe and 53.2% in Latin America. Isolates in each region most frequently carried VIM carbapenemases, ranging from 41.7% of isolates in Asia/South Pacific to 86.2% in Africa/Middle East. No P. aeruginosa carrying KPC or NDM carbapenemases and 1.0% of isolates carrying GES carbapenemases were resistant to colistin.Conclusion: Given the limited therapeutic options to treat infections caused by carbapenemase-positive Enterobacterales and P. aeruginosa, continued surveillance of CZA activity as well as agents such as colistin is crucial.