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PubMed2026

Efficacy of the NMIC-150 system in identifying extended-spectrum beta-lactamases in clinical isolates.

Extended-spectrum beta-lactamases (ESBLs) are significant contributors to the growing global crisis of antimicrobial resistance. This study evaluated the performance of the NMIC-150 System for susceptibility testing of third-generation cephalosporins (3GCs) and assessed whether ceftazidime-avibactam and aztreonam-avibactam could identify ESBL-producing carbapenem-resistant Enterobacterales (CREs). A total of 278 non-duplicate clinical isolates (Klebsiella pneumoniae, E. coli, and Proteus mirabilis) were analyzed. Antimicrobial susceptibility was determined using reference broth microdilution (BMD) and the NMIC-150 System. ESBL production was defined as an ≥eight-fold reduction in the minimum inhibitory concentration (MIC) of 3GCs in the presence of clavulanic acid, according to CLSI criteria. Whole-genome sequencing was performed to characterize ESBL and carbapenemase genes among 3GC-resistant isolates. A Random Forest model was used to predict ESBL-producing isolates based on MIC values. The NMIC-150 System demonstrated over 90% categorical and essential agreement with BMD for ceftazidime and ceftriaxone, along with robust predictive performance via Random Forest analysis. These findings suggest that the NMIC-150 System is a reliable platform for 3GC susceptibility testing and that an ≥eight-fold MIC reduction with ceftazidime-avibactam or aztreonam-avibactam may serve as a phenotypic indicator of ESBL production in CRE isolates. In conclusion, the NMIC-150 System shows potential for routine antimicrobial resistance surveillance and may facilitate the rapid identification of ESBL-producing CREs in clinical settings.

Microbial Sensitivity Tests↗
PubMed1992

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3.

The genetic environment of plasmid-borne blaTEM mutant genes, encoding nine distinct TEM-type extended-spectrum beta-lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine blaTEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes blaTEM-1 or -2. A 6.6 kb DNA fragment of pCFF04 containing blaTEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that blaTEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with blaTEM-3.

Amino Acid Sequence↗
PubMed1992

Transposition of the gene encoding a TEM-12 extended-spectrum beta-lactamase.

An isolate of Klebsiella oxytoca from the blood culture of a child with leukemia was found to produce two beta-lactamases, at least one of which conferred resistance to ceftazidime. Genes encoding both enzymes were located on a single self-transmissible 100-kb plasmid, pOZ201. This plasmid was introduced into Escherichia coli UB5201 (pACYC184), and the gene encoding one beta-lactamase was transposed onto plasmid pACYC184 by exploiting a gene dosage effect. The transposable gene was found to encode a TEM-12 enzyme as determined by nucleotide sequencing. This gene was subsequently transposed onto plasmid pUB307. The transposable element encoding the TEM-12 enzyme has been designated Tn841. Both plasmids pACYC184::Tn841 and pUB307::Tn841 were shown to encode a beta-lactamase with the same isoelectric point and substrate profile as the TEM-12 beta-lactamase. Transposon Tn841, at approximately 7 kb, is larger than TnA (4.8 kb) and transposes at a lower frequency. Although it produced a resolvase which can complement the resolvase of Tn3, its transposase function was not able to complement the transposition of a TnA element which lacked transposase. The occurrence of a gene encoding an extended-spectrum beta-lactamase on a transposable element in a clinically significant bacterium is potentially a cause for concern for the spread of resistance to the extended-spectrum cephalosporins.

Base Sequence↗
PubMed2024

Optimized methods for the targeted surveillance of extended-spectrum beta-lactamase-producing Escherichia coli in human stool.

Understanding transmission pathways of important opportunistic, drug-resistant pathogens, such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producing E. coli (ESBL-EC) between two sources, single-nucleotide resolution of E. coli strains, as well as E. coli diversity within and between samples, is required. However, the microbiological methods to best track these pathogens are unclear. Here, we compared different steps in the microbiological workflow to determine the impact different pre-enrichment broths, pre-enrichment incubation times, selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high-quality DNA for sequencing and genomic epidemiology. We demonstrate that using a 4-h pre-enrichment in Buffered Peptone Water, plating on cefotaxime-supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole-genome sequencing. We conclude that our optimized workflow can be applied for single-nucleotide variant analysis of an ESBL-EC from stool.IMPORTANCEDrug-resistant infections are increasingly difficult to treat with antibiotics. Preventing infections is thus highly beneficial. To do this, we need to understand how drug-resistant bacteria spread to take action to stop infection and transmission. This requires us to accurately trace these bacteria between different sources. In this study, we compared different laboratory methods to see which worked best for detecting extended-spectrum beta-lactamase (ESBL)-producing E. coli, a common cause of urinary tract or bloodstream infections, from human stool samples. We found that enriching stool in a nutrient broth for 4 h, then plating the bacterial suspension on antibiotic-selective MacConkey agar, and finally extracting DNA from the bacteria using a specific DNA purification kit resulted in improved recovery of ESBL E. coli and high-quality DNA. Sequencing multiple isolates from stool allowed us to distinguish unambiguously and at high resolution between different variants of ESBL E. coli present in stool.

Humans↗
PubMed1992

Piperacillin, tazobactam, and gentamicin alone or combined in an endocarditis model of infection by a TEM-3-producing strain of Klebsiella pneumoniae or its susceptible variant.

The efficacy of tazobactam, a beta-lactamase inhibitor, in combination with piperacillin, was studied in vitro and in rabbit experimental endocarditis due to a Klebsiella pneumoniae strain (KpR) producing an extended-spectrum beta-lactamase, TEM-3, or its nonproducing variant (KpS). In vitro, piperacillin was active against KpS (MIC = 4 micrograms/ml, MBC = 8 micrograms/ml with 10(7)-CFU/ml inoculum) but not against KpR (MIC = MBC = 256 micrograms/ml). Tazobactam (1 microgram/ml) restored the activity of piperacillin against KpR (MIC = 2 micrograms/ml, MBC = 4 micrograms/ml). Gentamicin was active against both strains (MIC = 0.25 and 0.5 micrograms/ml for KpS and KpR, respectively). The piperacillin-tazobactam-gentamicin combination was synergistic in vitro. The piperacillin/tazobactam ratio in plasma and in vegetations was always lower than the 4/1 injected dose ratio. In vivo, piperacillin (300 mg/kg of body weight four times a day [QID]) was active against KpS but not against KpR. Tazobactam (75 mg/kg QID) was able to restore the in vivo effect of piperacillin (300 mg/kg QID) against KpR (-3.0 log10 CFU/g of vegetation versus that of controls). Gentamicin (4 mg/kg twice a day [BID]) was active against both strains. Compared with controls, the combination of gentamicin plus piperacillin against KpS (-5.6 log10 CFU/g of vegetation), and the gentamicin-piperacillin-tazobactam combination against KpR (-4.4 log10 CFU/g of vegetation) achieved the greatest decrease in bacterial counts in vegetations and were the only regimens that significantly increased the proportion of sterile vegetations. It is concluded that (i) tazobactam was able to restore the effect of piperacillin against a TEM-3 extended-spectrum Beta-lactamase-producing strain of K. pneumoniae, both in vitro and in a severe experimental infection with high inoculum, when used in a 4/1 piperacillin/tazobactam dose ratio; (ii) gentamicin alone was effective because of the high peak/MBC ratio in plasma; (iii) piperacillin-tazobactam-gentamicin, probably because of the effect of gentamicin in reducing bacterial inoculum in vivo, as stressed by the results obtained by piperacillin-gentamicin against KpS, may be the most effective regimen against KpR.

Animals↗
PubMed1992

Outbreak of infection in two UK hospitals caused by a strain of Klebsiella pneumoniae resistant to cefotaxime and ceftazidime.

During an 8-month period, Klebsiella pneumoniae resistant to cefotaxime and ceftazidime were isolated from 18 elderly patients in two closely-situated UK hospitals. Amongst these 18 patients, the organisms were isolated from urine samples of 17, from blood cultures of two and from a wound swab of one. The infected patients were located in nine different wards and several of the patients had been transferred between wards, within and between the two hospitals. All the bacterial isolates belonged to serotype K62, were non-typable or reacted only weakly with bacteriophage, showed similar plasmid profiles and were resistant to tetracycline and trimethoprim, thus indicating they were the same strain. Resistance to cefotaxime and ceftazidime was inhibited by clavulanic acid suggesting the involvement of extended-spectrum beta-lactamase (ESBL) enzyme activity. This was confirmed by analytical isoelectric focusing, which showed that isolates each produced two beta-lactamases with isoelectric points of 7.0(SHV-3) and 7.6 (SHV-1/2) respectively.

Cefotaxime↗
PubMed2025

Integrated metagenomic, culture-based, and whole genome sequencing analyses of antimicrobial resistance in wastewater and drinking water treatment plants in Barcelona, Spain.

The misuse and overuse of antimicrobials drive the emergence of antimicrobial resistance (AMR), a critical global health concern. While wastewater treatment plants (WWTPs) are essential for removing microorganisms and contaminants, they also serve as hotspots for antibiotic-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs), facilitating their persistence and dissemination. This study investigated AMR in two WWTPs and one drinking water treatment plant (DWTP) in the Baix Llobregat area of Barcelona, Spain. Four sampling campaigns were conducted during winter and summer 2023 across different treatment stages. Due to drought conditions, reclaimed water from the Baix Llobregat WWTP was discharged upstream of the DWTP intake to supplement water resources for indirect potable reuse. A total of 991 cultivable ARB were obtained, enabling phenotypic and genotypic characterisation. The most prevalent included Aeromonas spp. (44.3 %), Enterobacterales (27.9 %), Pseudomonas spp. (19.1 %), Acinetobacter spp. (4.8 %), Shewanella spp. (2.2 %), Stenotrophomonas spp. (1 %), and others (0.7 %). Among these, 57.3 % were multidrug-resistant and 2.7 % were extensively drug-resistant. Furthermore, 34.6 % produced extended-spectrum beta-lactamases, 14.1 % harboured carbapenemase genes, and 2.9 % exhibited colistin resistance. Shotgun metagenomic analysis revealed high taxonomic diversity, without dominant genera across treatment stages. The resistome was dominated by ARGs conferring resistance to beta-lactams, aminoglycosides, and macrolides, alongside genes linked to biocide resistance and heavy metal tolerance. Spearman correlation analysis of selected sequenced strains suggested a weak to moderate co-occurrence between ARGs and biocide or heavy metal tolerance genes. These findings underline WWTPs as AMR hotspots and reinforce the need to monitor DWTP source water within the One Health framework.

Wastewater↗
PubMed2025

Snapshot Genomic Surveillance Reveals Insights into Antimicrobial Resistance and Lineage Diversity of Uropathogens in Older Adults in Queensland.

BACKGROUND: Community-acquired urinary tract infections (UTIs) are a significant health concern in older adults. However, few studies have investigated the epidemiology of uropathogens across diverse settings where older adults reside. METHODS: In this study, we whole-genome sequenced urinary isolates of Escherichia coli, Klebsiella species, and Enterobacter cloacae complex collected from individuals aged 70 and over living in the community and residential aged care facilities (RACFs) in Queensland, Australia. We investigated the prevalence of antimicrobial resistance (AMR), pathogen population structure, and transmission dynamics across the settings. RESULTS: E. coli was most frequently identified (82.2%, 447/544), followed by K. pneumoniae (10.8%, 59/544) and E. hormaechei (2.6%, 14/544). Intraspecies lineages were diverse, and a total of 177 sequence types were identified. The three predominant lineages were E. coli ST73 (10.7%, 54/504), ST95 (8.1%, 41/504), and ST131 (5.4%, 27/504). Transmission events were minimal, being identified in 13 patients (2.5%), mainly from the community. The resistance rate to antibiotics was low, with only a small proportion (9.7%) of multidrug-resistant (MDR) isolates. The predominant MDR lineage was E. coli ST131, which carried extended-spectrum beta-lactamase bla CTX-M genes. CONCLUSIONS: Community-acquired UTIs in older adults are predominantly caused by diverse E. coli lineages, with limited evidence of transmission within aged care facilities. The low rates of AMR in the community, along with the absence of strains adapted to a particular setting, suggest that current empiric therapy guidelines remain appropriate. Our prospective genomic surveillance offers valuable insights for monitoring UTIs in this population. It demonstrates the importance of an unbiased approach to accurately capture the prevalence and diversity of uropathogen lineages.

Enterobacteriaceae↗
PubMed2026

Dynamics and virulence of Enterobacteriaceae reservoirs harboring blaCTX-M group 1 in community wastewater.

UNLABELLED: Extended-spectrum beta-lactamase (ESBL)-producing bacteria are ubiquitous and can cause serious infections. Here, we examined untreated community wastewater influent as a reservoir for blaCTX-M group 1 organisms and their virulence potential. Raw influent samples (n = 268) were collected from four wastewater treatment plants (WWTPs) representing dense urban populations. We observed that blaCTX-M group 1 levels were high at all WWTPs and only ~1-2 log10 lower and not correlated to common human-specific microbiome fecal markers, Lachno3 and HF183, indicating a lack of connection to human fecal inputs. Concentrations of blaCTX-M group 1 genes and markers for presumptive host organisms Escherichia coli and Klebsiella pneumoniae were influenced by travel time and season. Amplicon sequencing revealed high diversity of blaCTX-M group 1-9 genes, with 63% belonging to group 1. Selective culture and 16S rRNA gene sequencing showed blaCTX-M group 1 isolates were 26% E. coli, 26% K. pneumoniae, 40% other Enterobacteriaceae, and 8% Aeromonas. Overall, E. coli averaged 3.6E7 cells/L, with 3% of all E. coli found to contain blaCTX-M group 1. Whole-genome sequencing of blaCTX-M group 1 E. coli from wastewater revealed resistance and virulence gene profiles similar to clinical isolates and distinct from other wastewater ESBL-resistant and non-resistant E. coli. Interpretation of wastewater data needs to consider both the existence of environmental reservoirs that contain potentially pathogenic organisms and the strong influence the dynamics of the conveyance system can have on final concentrations measured at the WWTP. IMPORTANCE: The CTX-M enzyme family is highly abundant in nosocomial, community, and environmental settings and is leading to treatment of infections with carbapenem antibiotics, a last-line therapeutic option. The progressive increase of the clinically relevant blaCTX-M group 1 resistance genes in the human population warrants investigation, particularly to understand the establishment and dynamics of environmental reservoirs. This study utilized molecular and culture methods to gain insight into the possible origin, abundance, and dynamics of blaCTX-M group 1 genes in untreated wastewater influent samples. We found extremely high levels of these genes, with Escherichia coli as a major host organism that closely resembled clinical strains, suggesting they are seeded and propagate in sewer pipe systems. The significance of our research is in developing approaches to monitor antimicrobial resistance reservoirs in community wastewater, which could shed light on global burdens and potential transmission cycles and indicate increasing inputs of clinically relevant strains originating from human populations.

E. coli↗
PubMed2026

Emergence and persistence of ESBL- and carbapenemase-producing Klebsiella pneumoniae-related species in Barcelona wastewater treatment plants.

The World Health Organization classifies extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing Klebsiella pneumoniae as critical-priority pathogens due to their high incidence, mortality, transmissibility, rapid resistance acquisition, and limited treatment options. Beyond clinical settings, their detection in wastewater treatment plants (WWTPs) provides an opportunity to assess their prevalence, persistence, and circulation within wastewater systems. This study characterized 37 antibiotic-resistant K. pneumoniae-related species strains isolated from two WWTPs in the metropolitan area of Barcelona, analyzing their antimicrobial resistance (AMR) profiles, antimicrobial resistance genes (ARGs), biocide and heavy metal tolerance genes (HMTGs), virulence factor genes (VFGs), biofilm-forming capacity, and conjugation ability. Among them, 70.3% were multidrug-resistant (MDR), and 16.2% were extensively drug-resistant. Whole-genome sequencing revealed diverse ARGs; all strains carried β-lactam resistance genes (14 ESBL and 12 carbapenemase producers), nearly all (96.9%) carried biocide or HMTGs, 64.9% harbored integrases, and all carried VFGs. Core-genome SNP analysis identified closely related strains across sampling periods and treatment stages, suggesting long-term persistence within the wastewater treatment system, despite biological and chemical processes in secondary treatment. Most strains (67.6%) displayed biofilm-forming capacity, and conjugation assays confirmed horizontal gene transfer in five of the seven ESBL-producing strains tested. High-risk clones were predominantly detected in the IFAS secondary treatment stage of the Gavà-Viladecans WWTP. The three strains recovered from the reclaimed water of the Baix Llobregat WWTP were ESBL or carbapenemase producers. Altogether, these results provide genomic and phenotypic evidence of the persistence and circulation of antibiotic-resistant K. pneumoniae-related species within wastewater treatment systems.IMPORTANCEWWTPs are essential for urban sanitation and environmental protection. Understanding how clinically relevant pathogens, such as ESBL and carbapenemase-producing K. pneumoniae-related species strains, behave in these settings may inform public health considerations. Investigating the presence and persistence of high-risk MDR pathogens in WWTPs helps identify circulation of AMR, assess the risk of gene transfer, and evaluate the potential for co-selection with other contaminants. This knowledge supports efforts to improve wastewater treatments, strengthen environmental surveillance, and develop integrated One Health strategies to limit the spread of AMR across human, animal, and environmental sectors.

Wastewater↗
PubMed2026

Frequency and characteristics of extended-spectrum beta-lactamase-producing Escherichia coli in wastewater in Dakar, Senegal.

OBJECTIVE: This study aimed to investigate the occurrence, antimicrobial resistance profiles, and genetic characteristics of extended-spectrum beta-lactamase (ESBL)-producing E. coli in wastewater collected in Dakar, Senegal. RESULTS DESCRIPTION: All samples (n = 48) carried ESBL-producing isolates. The concentrations of ESBL-producing E. coli ranged from 1.4 × 10⁴ to 3.3 × 10⁵ CFU/100 mL, whereas the ratio of ESBL-producing E. coli among the total E. coli population varied between 0.2% and 16.3%. All the 107 isolated ESBL-producing E. coli isolates were multidrug resistant (MDR), with 100% resistance to beta-lactams (ampicillin, cefalotin, cefotaxime, ceftazidime, cefepime, aztreonam) and high resistance rates to non-beta-lactam antibiotics, including ciprofloxacin (77.6%). No resistance was observed to imipenem. CTX-M-type genes were present in all isolates, with blaCTX-M-1 group (89.7%) and blaCTX-M-8 group (88.8%) being the most prevalent. The blaCTX-M-15 variant, a subgroup of blaCTX-M- group1, was detected in 96.9% of blaCTX-M-1 positive isolates. Additionally, blaTEM (31.8%) and blaOXA-1 (34.6%) were detected, while blaSHV and blaCTX-M-25 group were absent. Phylogenetic analysis of 107 isolates revealed a diverse distribution across four phylogroups: A (37.4%), D (22.4%), B1 (14.0%), and B2 (5.7%). The predominance of phylogroup A, mainly associated with intestinal commensal carriage, suggests fecal contamination as a primary source.

Senegal↗
PubMed2026

Prevalence and risk factors for persistent faecal carriage of extended spectrum beta-lactamase producing Escherichia coli in a paediatric community population.

OBJECTIVE: To investigate clinical and microbiological factors associated with persistent faecal carriage of extended spectrum beta-lactamase (ESBL) producing Escherichia coli in infants. METHODS: Between 2010 and 2022, children aged 3 months to 2 years old were sampled in a community setting in France, at two visits, V1 and V2, 3-24 months apart, to screen for prolonged faecal carriage of ESBL-producing E. coli. Patient clinical information and whole genome sequence of each isolate were used for association studies. RESULTS: A total of 4641 children were sampled. 375 (8%) carried an ESBL-producing Enterobacterales, among which 142 of ESBL-producing E. coli carriers were once again sampled at V2 and included in this study. 21.8% (n = 31/142) and 18.4% (n = 16/99) carried the same ESBL-producing E. coli clone for at least 3 and 6 months, respectively. B2 phylogroup, and among which ST131 clones were associated with an increased risk of persistent carriage. Multivariate analysis identified virulence associated genes involved in adhesion (papC/papGII allele and a tia-like gene) and encoding toxin (senB) as major risk factors for persistence. A genome wide association study highlighted the potential role of the frz metabolic operon, known to be involved in enterocytes adhesion/internalisation. CONCLUSION: Main extraintestinal pathogenic E. coli genomic features (phylogenetic background, adhesion properties) are associated with ESBL-producing E. coli gut colonisation persistence in infants, which could potentially lead to an increased risk of febrile urinary tract infection in these patients.

Child↗
PubMed2026

Molecular characterization and antimicrobial resistance profiles of Shigella flexneri isolates from pediatric clinical cases in Ahvaz, Iran.

Shigella is a highly invasive pathogen that causes dysentery and is associated with significant morbidity and mortality in children under five years of age. This agent is a major public health problem in developing countries. Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a reliable, cost-effective typing method with high discriminatory power and reproducible results. The rise of drug resistance in Shigella strains is a growing global health threat. Despite the significance of Shigella in Iran, there is limited knowledge about genetic diversity and drug resistance profiles of local strains. Therefore, the purpose of this study was to characterize the genetic diversity and drug resistance profiles of Shigella strains isolated in Ahvaz, Iran. A total of 49 Shigella flexneri isolates were recovered from 500 stool samples of pediatric patients. Routine biochemical tests were used to identify all isolates. Antimicrobial susceptibility testing was performed, and resistance genes were detected by polymerase chain reaction (PCR). Extended-spectrum β-lactamases (ESBL), carbapenemase, and Metallo-β-lactamase (MBL) production were detected phenotypically using combination disk assays and confirmed by the CLSI-recommended modified Carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM). MLVA based on seven VNTR loci was performed to characterize the genetic diversity of the isolates. All 49 isolates were resistant to ceftazidime, trimethoprim/sulfamethoxazole, ampicillin, and ceftriaxone (100% each). High resistance rates were also observed for imipenem 36/49 (73.5%), meropenem 36/49 (73.5%), azithromycin 21/49 (42.9%), and ciprofloxacin 16/49 (32.7%). Furthermore, phenotypic testing revealed ESBL production in 46/49 (93.9%) isolates and carbapenemase activity in 36/49 (73.5%), of which 22/49 (44.9%) were MBL. PCR analysis identified blaCTX-M 38/49 (77.6%) and blaSHV 35/49 (71.4%) as the most prevalent ESBL genes, whereas blaNDM 14/49 (28.6%), and blaOXA-48 14/49 (28.6%) were the most common carbapenemase genes. MLVA typing divided the isolates into 22 different MLVA types, including 10 clusters and 12 singletons, and locus ms21 showed the highest discriminatory power. The isolates exhibited high genetic diversity with a non-clonal distribution of resistance, which indicates dissemination through horizontal gene transfer. Our results demonstrated that mCIM/eCIM and MLVA are viable methods for investigating Shigella species as they are cost-effective, provide quick results, and allow for easy sharing of numerical data between laboratories.

Humans↗
PubMed2025

Chromosomal resistance mutations facilitate acquisition of multidrug-resistant plasmids in Escherichia coli.

Bacteria can gain multiple resistance mechanisms in a single step by the acquisition of multidrug-resistant (MDR) plasmids, but it is unclear how antibiotic selection during the acquisition of MDR plasmids affects the evolution of additional resistance mechanisms. Through conjugating separate extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing MDR plasmids into plasmid-naive Escherichia coli hosts, we examine the effects of acquisition of a single plasmid or co-acquisition of multiple plasmids upon fitness costs, resistance and subsequent genomic adaptation. We show that acquisition of pOXA-48, encoding OXA-48 carbapenemase, is associated with highly variable fitness costs and levels of resistance to ertapenem in transconjugants independent of the presence of pLL35. This phenomenon was not observed during the acquisition of ESBL CTX-M-15-encoding pLL35 alone. Within a single growth cycle, transconjugants receiving pOXA-48 rapidly gained parallel mutations affecting the membrane porin OmpF, or its regulators OmpR or EnvZ. These chromosomal mutations were not compensatory for the fitness costs imposed by the plasmid, nor did they provide significant increases in resistance to carbapenems in the absence of the pOXA-48. Rather, they acted synergistically with the plasmid-encoded carbapenemase, which alone only provided marginal resistance, together providing high-level resistance to ertapenem. Such rapid evolutionary processes may play an important role in plasmid dynamics within environments with strong antibiotic selection for plasmid-encoded antimicrobial resistance genes (ARGs), particularly when these ARGs provide only marginal resistance.

Escherichia coli↗
PubMed2026

Evaluation of carbapenem inactivation method-based phenotypic assays for the detection of GES-type carbapenemases in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii.

UNLABELLED: Detection of GES-type carbapenemases remains challenging because of their low prevalence and frequently weak hydrolytic activity against carbapenems. Carbapenem inactivation method (CIM)-based assays are widely used as phenotypic screening tools for carbapenemase detection; however, their performance in large collections of GES producers has not been systematically evaluated. We assessed the performance of CIM, modified CIM (mCIM), and CIM-Tris in a diverse collection of GES-producing clinical isolates, including 110 Enterobacterales and 108 Pseudomonas aeruginosa, recovered from Spanish hospitals (2010-2024), and 10 Acinetobacter baumannii isolates, mostly obtained from a hospital in Egypt. Whole-genome sequencing was carried out for species confirmation and resistome analysis. Meropenem MICs were determined by broth microdilution. Overall, 92.1% of isolates were GES-carbapenemase producers (CP), whereas 7.9% expressed GES-type extended-spectrum β-lactamases (ESBLs). In Enterobacterales (predominantly carrying blaGES-6), mCIM improved sensitivity compared with CIM (63.6% vs 40.0%), although many isolates remained undetected due to low meropenem MICs (MIC50, 0.5 µg/mL). In CP-P. aeruginosa (mainly blaGES-5), CIM, mCIM, and CIM-Tris showed sensitivities of 89.1%, 94.6%, and 100%, respectively; however, CIM-Tris yielded false-positive results in 50% of non-CP isolates (mostly blaGES-1 producers). Meropenem MICs in P. aeruginosa were higher (MIC50, >32 µg/mL). In A. baumannii, CIM-Tris improved sensitivity compared with CIM (100% vs 25.0%). These findings indicate that CIM-based methods can detect GES-type carbapenemases, but performance varies according to bacterial species and GES variant, and reduced specificity may occur in isolates producing GES-type ESBLs. Complementary molecular testing may therefore be necessary to ensure accurate detection of GES-type carbapenemases in routine clinical laboratories. IMPORTANCE: GES-type carbapenemases represent an important but underrecognized diagnostic challenge due to their low global prevalence, heterogeneous hydrolytic activity, and the limited performance data available for routine phenotypic detection methods. Although CIM-based assays are widely implemented in clinical microbiology laboratories for carbapenemase screening, their performance against GES-producing organisms has not been comprehensively evaluated across different bacterial genera and GES variants. In this study, we evaluated the performance of CIM, modified CIM (mCIM), and CIM-Tris in a large multicenter collection of well-characterized GES-producing clinical isolates, including Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. Our findings demonstrate substantial variability in assay performance according to bacterial species and GES variant. Notably, mCIM improved sensitivity among Enterobacterales with low meropenem MICs, whereas CIM-Tris achieved excellent sensitivity in P. aeruginosa and A. baumannii but at the expense of reduced specificity in isolates producing GES-type ESBLs. To the best of our knowledge, this is the first study directly comparing multiple CIM-based approaches in such a large and taxonomically diverse collection of GES-producing isolates.

beta-Lactamases↗
PubMed2026

Genomic analysis of community-associated multidrug-resistant Klebsiella quasipneumoniae subsp. similipneumoniae and the identification of the ST2059-KL1 clone in the U.S.

UNLABELLED: Klebsiella quasipneumoniae subsp. similipneumoniae is an important member of the K. pneumoniae species complex (KpSC) and is increasingly reported as multidrug-resistant (MDR) in healthcare- and community-associated infections. Since clinical laboratories do not routinely distinguish K. quasipneumoniae subsp. similipneumoniae from K. pneumoniae, national prevalence estimates, particularly for MDR, are lacking. In this study, a total of 2,006 community-associated MDR KpSC isolates were collected from 42 U.S. states, with 30 K. quasipneumoniae subsp. similipneumoniae isolates originating from 12 states identified using whole genome sequencing. All isolates were resistant to ceftriaxone and exhibited high rates of resistance to other antimicrobial agents, including ampicillin-sulbactam (56.7%, 17/30), levofloxacin (75.9%, 22/29), and trimethoprim-sulfamethoxazole (53.3%, 16/30). Notably, five isolates were also carbapenem-resistant. Genomic analysis resolved 10 sequence types (STs), with ST2059 (n = 13) and ST414 (n = 9) predominating. Ceftriaxone resistance in most isolates (90%, 27/30) was conferred by an extended-spectrum β-lactamase gene, predominantly blaCTX-M-15 (73.3%, 22/30); the remaining isolates carried either a carbapenemase (blaKPC-3) or an AmpC β-lactamase (blaCMY-2). Nanopore sequencing identified blaCTX-M-15 harbored on two types of IncFIB(Kpn3) antimicrobial resistance (AMR) plasmids, either with or without the conjugative tra gene cluster. Interestingly, the KL1 locus, associated with canonical hypervirulent K. pneumoniae strains, was detected in all ST2059 isolates. Further analysis of public genomic data showed that the KL1 locus is widely distributed across KpSC. KL1 phylogenetic analyses indicated frequent intrasubspecies recombination but limited intersubspecies exchange of KL1. The identification of the dominant MDR K. quasipneumoniae subsp. similipneumoniae KL1-ST2059 clone in the U.S. underscores the importance of ongoing genomic surveillance. IMPORTANCE: Klebsiella quasipneumoniae subsp. similipneumoniae is an underrecognized member of the Klebsiella pneumoniae species complex that is frequently misidentified in clinical laboratories, leading to an incomplete understanding of its role in antimicrobial resistance. In this study, we used large-scale genomic surveillance of community-associated multidrug-resistant isolates across the U.S. to identify this subspecies as a reservoir of clinically relevant resistance plasmids. Notably, we detected a widely distributed ST2059 lineage carrying the K1 capsular locus, a feature traditionally associated with hypervirulent K. pneumoniae. These findings highlight the convergence of resistance and virulence-associated traits in an overlooked species and underscore the need for genomic surveillance to monitor emerging high-risk lineages in community settings.

Drug Resistance, Multiple, Bacterial↗
PubMed2026

Escalation of CTX-M-producing extensively drug-resistant Shigella spp. in Kolkata, India, following the COVID-19 pandemic.

Shigella spp. is recognized by the World Health Organization as a high-priority pathogen due to its global prevalence, unique pathogenic mechanisms, and growing antimicrobial resistance (AMR). Nearly half of all Shigella strains worldwide are now multidrug-resistant (MDR), and the emergence of extensively drug-resistant (XDR) variants-resistant to ciprofloxacin, ceftriaxone, and azithromycin-has severely limited effective treatment options. The present study is based on prospective laboratory surveillance involving 323 Shigella isolates collected during 2021-2023, with pre-COVID-19 pandemic data included from a previously published study solely for historical comparison. The presence of antibiotic resistance genes (ARGs) was investigated, and whole-genome sequencing (WGS) was performed on representative isolates to assess phylogenetic relatedness with global isolates. Approximately 10% of isolates exhibited resistance to third-generation cephalosporins. While only 3% of Shigella isolates carried the blaCTX-M-15 gene from 2013 to 2019, its prevalence increased to 26% by 2022-2023. Among 38 ceftriaxone-resistant S. sonnei isolates, 33 were also resistant to azithromycin, categorizing them as XDR. These isolates showed 48% clonal similarity and high phylogenetic resemblance to the isolates reported from England. Hybrid genome assembly revealed a plasmid harboring both the blaCTX-M-15 and mphA ARGs. Conjugation experiments and plasmid profiling confirmed the plasmid's transferability. We report a rising trend in third-generation cephalosporin resistance among Shigella spp., primarily driven by the spread of extended-spectrum β-lactamase-producing S. flexneri and the emergence of XDR S. sonnei. These findings underscore the urgent need for strengthened national AMR containment strategies and enhanced international surveillance of cephalosporin-resistant Shigella to mitigate this growing public health threat.IMPORTANCEShigella is a leading cause of diarrheal disease globally and has been prioritized by the World Health Organization due to its rapid acquisition of antimicrobial resistance. Our prospective surveillance in Kolkata, India, reveals a worrisome escalation of third-generation cephalosporin resistance over the past decade, primarily associated with the spread of blaCTX-M-15 and the emergence of extensively drug-resistant (XDR) S. sonnei. The detection of plasmids carrying both blaCTX-M-15 and mphA, coupled with evidence of their transferability, highlights the potential for accelerated dissemination of multidrug resistance. When compared with a global data set of international genomes, the Kolkata XDR isolates were found to cluster closely with isolates reported from England. By linking local surveillance with global genomic context, our findings provide critical insights for treatment guidelines, antimicrobial stewardship, and the design of international containment strategies aimed at curbing the rise of cephalosporin- and azithromycin-resistant Shigella.

India↗