Bla NDM-1 Research Articles

Characterization of a Novel Sequence Type (ST) 6758 Klebsiella Pneumoniae and the Role of IncX3 Plasmid in the Transmission of bla NDM.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a significant public health threat, particularly as a superbug responsible for nosocomial infections. In this study, we report a novel sequence type 6758 of K. pneumoniae harboring the bla NDM-1 gene. Antimicrobial susceptibility testing was conducted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The complete genome sequence of the strain was determined using the Illumina NovaSeq 6000 platform and long-read MinION sequencer. Genomic features and resistance mechanisms of the strain were further comprehensively analysed using various bioinformatics approaches. Antimicrobial susceptibility testing revealed that this strain exhibited resistance to multiple antimicrobials, including ceftazidime, ceftriaxone, cefazolin, cefepime, imipenem, meropenem, ampicillin/sulbactam, and sulfamethoxazole/trimethoprim. The genome analysis identified sixteen resistance genes. The bla NDM-1 carbapenemase gene is located on a 47,823 bp IncX3-type plasmid (pNDM-CRKP331). A total of 41 K. pneumoniae strains carrying similar IncX3-type plasmids were retrieved from the NCBI database, representing 20 sequence types (STs) across 11 countries. The most common resistance gene carried by these IncX3-type plasmids is bla NDM, and all these plasmids contain only the bla NDM gene. The bla NDM-carrying IncX3-type plasmids are widely prevalent in K. pneumoniae in China, spanning 15 STs. In summary, our study reports the first genome sequence of an ST 6758 K. pneumoniae strain containing the class B β-lactamase bla NDM-1 isolated from a clinical sample. Given the global emergence of bla NDM, measures should be taken to prevent the spread of these bla NDM-carrying IncX3-type plasmids. Our findings contribute to the understanding of the transmission mechanisms of bla NDM in K. pneumoniae.

Carbapenem resistance in gram-negative pathogens in an Iranian hospital: high prevalence of OXA-type carbapenemase genes.

The widespread dissemination of carbapenem-resistant Gram-negative bacteria poses a significant threat to global public health. This study aimed to investigate the prevalence of carbapenem resistance in Gram-negative bacteria isolated from patients at the Children's Medical Center Hospital, Tehran, Iran, to understand the molecular mechanisms underlying this resistance. During the period spanning from June 2019 to June 2020, 777 Gram-negative bacterial strains were isolated. Antibiotic susceptibility testing was performed according to Clinical and Laboratory Standards Institute. Polymerase chain reaction was used to detect carbapenem resistance genes including bla OXA23, bla OXA24, bla OXA48, bla OXA51, bla OXA58, bla OXA143, bla KPC, bla IMP, bla VIM, and bla NDM. Among the total bacterial isolates, 141 (18.1%) exhibited carbapenem resistance. Escherichia coli was the most prevalent (57.4%), followed by Klebsiella pneumoniae (11.3%) and Acinetobacter baumannii (10.6%). Other notable contributors included Enterobacter spp. (5.7%), Salmonella spp. (3.5%), and Stenotrophomonas maltophilia (2.8%). Citrobacter spp., Proteus mirabilis, and Pseudomonas aeruginosa contributed to the distributions of two, one, and three isolates, respectively. Notably, bla OXA48 showed the highest prevalence (33%), followed by bla OXA143 and bla OXA58 (27% and 24%, respectively). In addition, bla OXA24 was present in 11% of the total isolates, bla OXA23 in 10%, and bla NDM in 10%, whereas bla KPC, bla VIM, and bla IMP were not detected. Our study highlights the prevalence of carbapenemase-producing Gram-negative isolates among pediatric patients. Notable resistance patterns, especially in K. pneumoniae and E. coli, underline the urgent need for proactive interventions, including appropriate antibiotic prescription practices and strengthening of antibiotic stewardship programs.

Characterization of bla NDM in two Escherichia coli ST1193 clinical isolates in the Gulf region.

Escherichia coli ST1193 is an emerging high-risk clone associated with the production of plasmid-mediated CTX-type extended-spectrum β-lactamases. However, this clone has seldom been found to contain plasmids carrying carbapenemase genes. We report two epidemiologically unlinked multidrug-resistant (MDR) clinical isolates of E. coli ST1193 with plasmids harbouring NDM-type carbapenemase genes from the Gulf region. The isolates were identified by MALDI-TOF MS and antibiotic susceptibility testing was performed using the VITEK 2/Phoenix system. A conjugation experiment was performed to assess the transferability of the resistance plasmids. Genomic DNA of both isolates was subject to Illumina sequencing; one isolate was also sequenced using Oxford Nanopore technology. Bioinformatics analyses were performed to detect antimicrobial resistance genes, and to annotate the genetic context of the NDM genes. Both isolates were resistant to carbapenems using phenotypic tests. Conjugation experiment confirmed that NDM-5-encoding plasmids of both strains could be transferred to the recipient cells. The completed NDM-5-encoding plasmid of E. coli isolate FQ71 was highly similar to several plasmids from ST410 isolates in the NCBI database. Genomic analysis revealed the presence of transposase genes and transposons in the flanking regions of the NDM genes in the plasmids. Since carbapenems constitute first-line agents for the treatment of serious infections caused by ESBL producers, E. coli ST1193 isolates co-producing ESBL and NDM-type carbapenemases represent a serious challenge for antimicrobial stewardship and infection control programmes.

Natural products from food sources can alter the spread of antimicrobial resistance plasmids in Enterobacterales.

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum β-lactam antibiotics in Gram-negative bacteria is a major impediment to treating infections. Genes responsible for antibiotic resistance are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the conjugation of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based flow cytometry assay, we found that the natural products were more active in reducing transmission of the IncK extended-spectrum β-lactamase-encoding plasmid pCT in Escherichia coli EC958c, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII carbapenemase-encoding plasmid pKpQIL. The exception was the natural product rottlerin, also active in K. pneumoniae. In classical conjugation assays, rottlerin also reduced the conjugation frequency of the IncFII bla NDM-1 carrying plasmid pCPE16_3 from a clinical K. pneumoniae isolate. Our data indicate that the natural products tested here, in their current molecular structure, reduced conjugation by a small amount, which is unlikely to achieve a large-scale reduction in AMR in bacterial populations. However, certain natural products like rottlerin could provide a foundation for further research into compounds with effective anti-plasmid activity.

Characterisation of carbapenemase-producing Gram-negative bacilli among clinical isolates in a tertiary care centre in Kerala, South India

Background: Infections caused by carbapenem-resistant Gram-negative bacteria are a cause for concern due to the limited choice of antibiotics available for their treatment. Aims, Settings and Design: This study screened multidrug-resistant (MDR) Gram-negative bacilli isolated from clinical samples over a period of one year, for carbapenem resistance and characterised them using phenotypic methods such as combined disc diffusion test (CDDT), modified Hodge test (MHT), E-test for metallo-beta-lactamase (MBL) and molecular method, PCR. Materials and Methods: Two hundred and ten MDR Gram-negative bacilli were screened for carbapenem resistance using Imipenem and Meropenem disc diffusion. These were further checked for carbapenemase production by CDDT, MHT and E-test for MBL. Those positive by E-test were subjected to PCR. Uniplex PCR for New Delhi metallo-beta-lactamase-1 was used for Escherichia coli and Klebsiella pneumoniae , and multiplex PCR for Imipenemase and Verona imipenemase was used for Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Results: Twenty-three (11%) isolates were found to be carbapenem-resistant and included E. coli (six) K. pneumoniae (three), P. aeruginosa (five) and A. baumannii (nine). Seventeen (74%) isolates were positive by phenotypic methods and were subjected to PCR. Out of eight Enterobacteriaceae isolates subjected to PCR, all were positive for bla NDM gene. All were negative for bla KPC gene. All five A. baumannii isolates subjected to PCR were found to contain bla VIM gene. Two out of four P. aeruginosa isolates were positive for bla IMP , one was positive for bla VIM gene. One P. aeruginosa isolate was positive for both bla IMP and bla VIM gene. Conclusions: In view of the increasing resistance of Gram-negative bacilli to carbapenems, rational use of antibiotics needs to be emphasised.

Emergence of bla NDM-1-carrying Enterobacter chengduensis in China.

Enterobacter chengduensis was defined as a novel species in the genus. Enterobacter in 2019, however, antimicrobial resistance, such as carbapenem resistance, has rarely been described in E. chengduensis. This study described the molecular features of four carbapenem-resistant E. chengduensis strains collected from a tertiary health care hospital in Southwest China. Whole genome sequencing (WGS) was used to determine the genome sequence of four E. chengduensis strains. The precise species of strains were identified by average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH). The clonal relatedness of four E. chengduensis strains and additional 15 ones from NCBI were examined through phylogenetic analysis. The molecular features of E. chengduensis and genetic structure of carbapenemase- encoding plasmids were characterized through genomic annotation and analysis. The results revealed the emergence of bla NDM-1-carrying E. chengduensis strains in China. Multilocus sequence typing (MLST) analysis showed that all 19 E. chengduensis belonged to the same sequence type of ST414. Core SNP analysis suggested the potential intrahospital clonal transmission of ST414 E. chengduensis. The carbapenemase-encoding gene bla NDM-1 was harbored by an IncC-type plasmid, which was experimentally confirmed to be able to conjugate. This study reports the first emergence and potential clonal transmission of bla NDM-1-carrying E. chengduensis. Further surveillance should be advocated to monitor the dissemination of carbapenem-resistant E. chengduensis and bla NDM-1-harboring IncC-type plasmids in China.

Limited transmission of carbapenem-resistant Klebsiella pneumoniae between animals and humans: a study in Qingdao

ABSTRACT Despite no carbapenem use in food animals, carbapenem-resistant Klebsiella pneumoniae (CRKP) perseveres within food animals, rising significant concerns regarding public health risks originating from these non-clinical reservoirs. To investigate the potential link between CRKP in food animals and its infections in humans, we conducted a cross-sectional study encompassing human clinical, meat products, and farm animals, in Qingdao city, Shandong province, China. We observed a relatively higher presence of CRKP among hospital inpatients (7.3%) compared to that in the meat products (2.7%) and farm animals (pig, 4.6%; chicken, 0.63%). Multilocus sequence typing and core-genome phylogenetic analyses confirm there is no evidence of farm animals and meat products in the clinical acquisition of K. pneumoniae isolates and carbapenem-resistant genes. However, potential transmission of K. pneumoniae of ST659 and IncX3 plasmid harbouring bla NDM-5 gene from pigs to pork and farm workers was observed. Our findings suggest a limited role of farm animals and meat products in the human clinical acquisition of K. pneumoniae, and the transmission of K. pneumoniae is more common within settings, than between them.

Genomic study of Acinetobacter baumannii strains co-harboring bla OXA-58 and bla NDM-1 reveals a large multidrug-resistant plasmid encoding these carbapenemases in Brazil.

Acinetobacter baumannii contributes significantly to the global issue of multidrug-resistant (MDR) nosocomial infections. Often, these strains demonstrate resistance to carbapenems (MDR-CRAB), the first-line treatment for infections instigated by MDR A. baumannii. Our study focused on the antimicrobial susceptibility and genomic sequences related to plasmids from 12 clinical isolates of A. baumannii that carry both the blaOXA-58 and bla NDM-1 carbapenemase genes. Whole-genome sequencing with long-read technology was employed for the characterization of an A. baumannii plasmid that harbors the bla OXA-58 and blaNDM-1 genes. The location of the bla OXA-58 and bla NDM-1 genes was confirmed through Southern blot hybridization assays. Antimicrobial susceptibility tests were conducted, and molecular characterization was performed using PCR and PFGE. Multilocus Sequence Typing analysis revealed considerable genetic diversity among bla OXA-58 and bla NDM-1 positive strains in Brazil. It was confirmed that these genes were located on a plasmid larger than 300 kb in isolates from the same hospital, which also carry other antimicrobial resistance genes. Different genetic contexts were observed for the co-occurrence of these carbapenemase-encoding genes in Brazilian strains. The propagation of bla OXA-58 and bla NDM-1 genes on the same plasmid, which also carries other resistance determinants, could potentially lead to the emergence of bacterial strains resistant to multiple classes of antimicrobials. Therefore, the characterization of these strains is of paramount importance for monitoring resistance evolution, curbing their rapid global dissemination, averting outbreaks, and optimizing therapy.

Coexistence of a novel NDM-1-encoding MDR plasmid and an IMP-4-encoding IncN-IncU hybrid plasmid in a clinical isolate of Citrobacter freundii BC73.

To investigate the genetic characteristics and transmission mechanism of the NDM-1-, IMP-4-, and SHV-12-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii BC73. C. freundii BC73 was isolated from a urine specimen of a urological patient diagnosed with bladder cancer at a Chinese teaching hospital. Antimicrobial susceptibility testing was carried out using DL-120E susceptibility cards and DL-96A system. Whole genome sequencing (WGS) of the isolate was performed using the Illumina and Oxford Nanopore platforms to analyze the genetic context of drug resistance genes and plasmid characteristics. The phylogenetic tree was constructed and visualized by KSNP3.0 software and iTOL5.0 online database. C. freundii isolate BC73 co-carrying bla NDM-1, bla IMP-4 and bla SHV-12 were multidrug-resistant. bla NDM-1 and bla IMP-4 were located on a novel IncFIB-like plasmid, pCFBC1, and an IncN-IncU hybrid plasmid, pCFBC2, respectively. The transferability of bla NDM-1 and bla IMP-4 from C. freundii BC73 to E. coli J53 was successfully demonstrated. The genetic context of the bla NDM-1 and bla IMP-4 genes were ISCR27-groEL-∆groES-cutA-dsbD-trpF-ble MBL-bla NDM-1-∆ISAba125-IS3000 and intI1-bla IMP-4-Kl.pn.13-mobC-IS6100, respectively. Additionally, two extensive transposition units (MGE1 in pCFBC1, MGE2 in pCFBC2) were identified and numerous antimicrobial resistance genes were discovered on it. To our knowledge, our study represents the first characterization of a ST22 C. freundii isolate co-harboring bla NDM-1, bla IMP-4, and bla SHV-12, obtained from a urine sample. The dissemination of this MDR isolate should be of close concern in future clinical surveillance.

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.

The bla NDM-1 and mcr-1 genes coexist in Escherichia coli strain isolated from public trash cans.

The bla NDM-1 and mcr-1 genes coexist in Escherichia coli strain isolated from public trash cans.

Nosocomial transmission of tet(x3), bla NDM-1 and bla OXA-97-carrying Acinetobacter baumannii conferring resistance to eravacycline and omadacycline, the Netherlands, March to August 2021.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important pathogen causing serious nosocomial infections. We describe an outbreak of CRAb in an intensive care unit in the Netherlands in 2021. During an outbreak of non-resistant A. baumannii, while infection control measures were in place, CRAb isolates carrying highly similar bla NDM-1 - and tet(x3)-encoding plasmids were isolated from three patients over a period of several months. The chromosomal and plasmid sequences of the CRAb and non-carbapenemase-carrying A. baumannii isolates cultured from patient materials were analysed using hybrid assemblies of short-read and long-read sequences. The CRAb isolates revealed that the CRAb outbreak consisted of two different strains, carrying similar plasmids. The plasmids contained multiple antibiotic resistance genes including the tetracycline resistance gene tet(x3), and the bla NDM-1 and bla OXA-97 carbapenemase genes. We determined minimal inhibitory concentrations (MICs) for 13 antibiotics, including the newly registered tetracycline antibiotics eravacycline and omadacycline. The CRAb isolates showed high MICs for tetracycline antibiotics including eravacycline and omadacycline, except for minocycline which had a low MIC. In this study we show the value of sequencing multidrug-resistant A. baumannii for outbreak tracking and guiding outbreak mitigation measures.

Molecular characterization of carbapenem and ceftazidime-avibactam-resistant Enterobacterales and horizontal spread of bla NDM-5 gene at a Lebanese medical center.

In the battle against multidrug-resistant bacterial infections, ceftazidime- avibactam (CZA) stands as a pivotal defense, particularly against carbapenemresistant (CR) Gram-negative pathogens. However, the rise in resistance against this drug poses a significant threat to its effectiveness, highlighting the critical need for in-depth studies about its resistance mechanisms. This research focuses on the genomic characterization of CR- and CZA-resistant Escherichia coli (n=26) and Klebsiella pneumoniae (n=34) strains, harboring the blaNDM and/or blaOXA-48-like genes, at a major Lebanese tertiary care medical center, using whole genome sequencing (WGS). Our findings revealed a notable prevalence of blaNDM in all K. pneumoniae strains isolates, with 27 of these also harboring blaOXA-48. On the other hand, E. coli strains predominantly carried the blaNDM-5 gene. Whole genome sequencing (WGS) identified a predominance of ST383 among K. pneumoniae strains, which possessed a multi-replicon IncFIB-IncHI1B plasmid harboring the blaNDM-5. Additionally, various Inc group plasmids in K. pneumoniae across multiple sequence types were found to carry the blaNDM. Similarly, diverse STs of E. coli were observed to carry blaNDM-5 on different plasmids. The study underscores NDM carbapenemases as a paramount resistance mechanism in Lebanon,jeopardizing critical last-resort treatments. It also illuminates the role of varied sequence types and mobile genetic elements in the spread of NDM resistance,stressing the urgent need for strategies to mitigate this threat, especially in nosocomial infections.

Detection of carbapenemase producing Acinetobacter baumannii ST19 from Georgia and Ukraine carrying bla OXA-23, bla OXA-72, and/or bla NDM-5, December 2019 to June 2023.

In 2003-2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the Multidrug-Resistant Organism Repository and Surveillance Network, 97 were ST19PAS, 34 of which carbapenem-resistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23, bla OXA-72, and/or bla NDM-5. Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub-lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.

Dissemination of extensively drug-resistant NDM-producing Providencia stuartii in Europe linked to patients transferred from Ukraine, March 2022 to March 2023

Background The war in Ukraine led to migration of Ukrainian people. Early 2022, several European national surveillance systems detected multidrug-resistant (MDR) bacteria related to Ukrainian patients. Aim To investigate the genomic epidemiology of New Delhi metallo-β-lactamase (NDM)-producing Providencia stuartii from Ukrainian patients among European countries. Methods Whole-genome sequencing of 66 isolates sampled in 2022–2023 in 10 European countries enabled whole-genome multilocus sequence typing (wgMLST), identification of resistance genes, replicons, and plasmid reconstructions. Five bla NDM-1-carrying-P. stuartii isolates underwent antimicrobial susceptibility testing (AST). Transferability to Escherichia coli of a bla NDM-1-carrying plasmid from a patient strain was assessed. Epidemiological characteristics of patients with NDM-producing P. stuartii were gathered by questionnaire. Results wgMLST of the 66 isolates revealed two genetic clusters unrelated to Ukraine and three linked to Ukrainian patients. Of these three, two comprised bla NDM-1-carrying-P. stuartii and the third bla NDM-5-carrying-P. stuartii. The bla NDM-1 clusters (PstCluster-001, n = 22 isolates; PstCluster-002, n = 8 isolates) comprised strains from seven and four countries, respectively. The bla NDM-5 cluster (PstCluster-003) included 13 isolates from six countries. PstCluster-001 and PstCluster-002 isolates carried an MDR plasmid harbouring bla NDM-1, bla OXA-10, bla CMY-16, rmtC and armA, which was transferrable in vitro and, for some Ukrainian patients, shared by other Enterobacterales. AST revealed PstCluster-001 isolates to be extensively drug-resistant (XDR), but susceptible to cefiderocol and aztreonam–avibactam. Patients with data on age (n = 41) were 19–74 years old; of 49 with information on sex, 38 were male. Conclusion XDR P. stuartii were introduced into European countries, requiring increased awareness and precautions when treating patients from conflict-affected areas.

First Record of the Isolation of blaNDM-1-Producing Enterotoxigenic Enterobacter cloacae ssp. cloacae in Baghdad/Iraq.

Antibiotic resistance is a major problem threatening human beings. The genetic determinants that carry resistance genes can be transmitted in several ways in clinical and food environments. Hence, this research study aimed to investigate the presence of New Delhi metallo-beta-lactamase-1 (blaNDM-1) produced by enterotoxigenic Enterobacter cloacae in both clinical and food samples. Twenty-four isolates of Enterobacter spp. were isolated, seven isolates from food samples and 17 isolates from blood taken from neonates and children (1 day - 10 years old) resident in a children's hospital. Antibiotic susceptibility test to 14 antibiotics was performed for all isolates. Enterotoxigenicity of the clinical and foodborne isolates was detected phenotypically using Suckling mouse bioassay. Genomic deoxyribonucleic acid (DNA) was extracted from the isolated Enterobacter spp. that were detected resistant to imipenem. Polymerase chain reaction (PCR) was used to amplify blaNDM-1 gene followed by sequencing. The results of the bioassay revealed that 64.28% of E. cloacae ssp. cloacae isolates were enterotoxigenic. Two E. cloacae ssp. cloacae were imipenem resistant. This study showed that one isolate from a male child 1 < year was bla NDM-1 positive that was con-firmed by sequencing. This is the first report that revealed blaNDM-1 producing Enterobacter cloacae in Iraq.

Study of the Epidemiological and Mechanistic Differences Between Carbapenem-Resistant Klebsiella pneumoniae Infections in Children and Adults.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has garnered international concern due to its significant antibiotic resistance. Notably, children exhibit distinct resistance mechanisms compared to adults, necessitating a differential approach to antibiotic selection. A thorough analysis of CRKP's epidemiology and drug resistance mechanisms is essential for establishing a robust foundation for clinical anti-infection strategies and precise prevention and control measures. This study involved the collection of 31 non-repetitive strains from pediatric and adult patients at a tertiary hospital in China, spanning from July 2016 to July 2022, testing for resistance genes, antimicrobial susceptibility, and homology analysis. Infants (0-1 year) were the largest pediatric CRKP group, with 61.3% of cases. The neonatal intensive care unit (NICU) and pediatrics were the main departments affected. Adults with CRKP had a mean age of 67 years, with the highest prevalence in neurology and emergency ICU. Antimicrobial susceptibility testing revealed that adult CRKP strains exhibited higher resistance to amikacin, ciprofloxacin, cotrimoxazole, and aztreonam compared to pediatric strains. Conversely, pediatric strains showed a higher rate of resistance to ceftazidime/avibactam. The predominant resistance genes identified were bla NDM-5 in children (58.1%) and bla KPC-2 in adults (87.1%), with over 93% of both groups testing positive for extended-spectrum beta-lactamase (ESBL) genes. Multilocus Sequence Typing (MLST) indicated ST2735 and ST11 as the predominant types in children and adults, respectively. Pulsed-field gel electrophoresis (PFGE) identified clonal transmission patterns of ST11 bla KPC-2 and ST15 bla OXA-232 across both age groups. Notably, this study reports the first instance of ST1114-type CRKP co-producing bla NDM-5 and bla OXA-181 in the NICU. This study reveals distinct resistance mechanisms and epidemiology in CRKP from children and adults. The identified clonal transmission patterns emphasize the need for improved infection control to prevent the spread of resistant strains.

Analysis of the Class 1 Integrons, Carbapenemase Genes and Biofilm Formation Genes Occurrence in Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is a non-fermentative Gram-negative bacterium that can cause nosocomial infections in critically ill patients. Carbapenem-resistant A. baumannii (CRAB) has spread rapidly in clinical settings and has become a key concern. The main objective of this study was to identify the distribution of integrons and biofilm-formation-related virulence genes in CRAB isolates. A total of 269 A. baumannii isolates (219 isolates of CRAB and 50 isolates of carbapenem-sensitive A. baumannii (CSAB)) were collected. Carbapenemase genes (bla KPC, bla VIM, bla IMP, bla NDM, and bla OXA-23-like) and biofilm-formation-related virulence genes (abal, bfms, bap, and cusE) were screened with PCR. Class 1 integron was screened with PCR, and common promoters and gene cassette arrays were determined with restriction pattern analysis combined with primer walking sequencing. Whole-genome sequencing was conducted, and data were analyzed for a bla OXA-23-like-negative isolate. All 219 CRAB isolates were negative for bla KPC, bla VIM, bla IMP, and bla NDM, while bla OXA-23-like was detected in 218 isolates. The detection rates for abal, bfms, bap, and cusE in 219 CRAB were 93.15%, 63.93%, 88.13%, and 77.63%, respectively. Class 1 integron was detected in 75 CRAB (34.25%) and in 3 CSAB. The single gene cassette array aacA4-catB8-aadA1 with relatively strong PcH2 promoter was detected in class 1 integrons. The bla OXA-23-like-negative CRAB isolate was revealed to be a new sequence type (Oxford 3272, Pasteur 2520) carrying bla OXA-72, bla OXA-259, and bla ADC-26. In conclusion, bla OXA-23-like was the main reason for CRAB's resistance to carbapenems. A new (Oxford 3272, Pasteur 2520) CRAB sequence type carrying the bla OXA-72, bla OXA-259, and bla ADC-26 was reported.

Characterizing carbapenemase-producing Escherichia coli isolates from Spain: high genetic heterogeneity and wide geographical spread.

Carbapenemase-Producing Escherichia coli (CP-Eco) isolates, though less prevalent than other CP-Enterobacterales, have the capacity to rapidly disseminate antibiotic resistance genes (ARGs) and cause serious difficult-to-treat infections. The aim of this study is phenotypically and genotypically characterizing CP-Eco isolates collected from Spain to better understand their resistance mechanisms and population structure. Ninety representative isolates received from 2015 to 2020 from 25 provinces and 59 hospitals Spanish hospitals were included. Antibiotic susceptibility was determined according to EUCAST guidelines and whole-genome sequencing was performed. Antibiotic resistance and virulence-associated genes, phylogeny and population structure, and carbapenemase genes-carrying plasmids were analyzed. The 90 CP-Eco isolates were highly polyclonal, where the most prevalent was ST131, detected in 14 (15.6%) of the isolates. The carbapenemase genes detected were bla OXA-48 (45.6%), bla VIM-1 (23.3%), bla NDM-1 (7.8%), bla KPC-3 (6.7%), and bla NDM-5 (6.7%). Forty (44.4%) were resistant to 6 or more antibiotic groups and the most active antibiotics were colistin (98.9%), plazomicin (92.2%) and cefiderocol (92.2%). Four of the seven cefiderocol-resistant isolates belonged to ST167 and six harbored bla NDM. Five of the plazomicin-resistant isolates harbored rmt. IncL plasmids were the most frequent (45.7%) and eight of these harbored bla VIM-1. bla OXA-48 was found in IncF plasmids in eight isolates. Metallo-β-lactamases were more frequent in isolates with resistance to six or more antibiotic groups, with their genes often present on the same plasmid/integron. ST131 isolates were associated with sat and pap virulence genes. This study highlights the genetic versatility of CP-Eco and its potential to disseminate ARGs and cause community and nosocomial infections.

Co-occurrence of triple carbapenemase genes, bla VIM-2, bla NDM-1, and bla OXA-48 in Enterobacter hormaechei clinical isolates –first report from Croatia

Two Enterobacter hormaechei isolates harbouring three carbapenemase genes each, were isolated from two patients from different ICUs at University Hospital Centre Zagreb, Croatia, which is to our knowledge, the first report of triple carbapenemase (bla VIM-2, bla NDM-1, and bla OXA-48 ) co-existence in E. hormachei strains and also among Enterobacterales members in Croatia. Antimicrobial susceptibility testing showed susceptibility only to colistin and amikacin. The production of carbapenemases was phenotypically tested by immunochromatographic assay and confirmed by PCR. Detailed analysis by Whole Genome Sequencing (WGS) of short reads by Illumina and long reads by Oxford Nanopore Technologies (ONT) was additionally performed and showed that both isolates belonged to ST200. They were separated by 98 Single Nucleotide Polymorphisms (SNPs) having variations in the number of bla VIM-2 genes on the chromosome, the number of bla NDM-1 genes on the plasmid, non-identical bla NDM-1 plasmids, different plasmid content in general, and only one isolate carried a 94 kb prophage.