blaNDM-1 Gene Research Articles

Molecular epidemiology of carbapenemase-producing Klebsiella pneumoniae in Gauteng South Africa

Klebsiella pneumoniae multidrug-resistant (MDR) high-risk clones drive the spread of antimicrobial resistance (AMR) associated infections, resulting in limited therapeutic options. This study described the genomic characteristics of K. pneumoniae MDR high-risk clones in Gauteng, South Africa. Representative carbapenem-resistant [K. pneumoniae carbapenemase (KPC)-2, New-Delhi metallo-beta (β)-lactamase (NDM)-1, oxacillinase (OXA)-181, OXA-232, OXA-48, Verona integron-encoded metallo-β-lactamase (VIM)-1] K. pneumoniae isolates (n = 22) obtained from inpatient and outpatient’s urine (n = 9) and inpatients rectal carriage (n = 13) were selected for short-read whole genome sequencing. Klebsiella pneumoniae population include sequence type (ST)-307 (n = 3), ST2497 (n = 5) and ST17 (n = 4). The ST17 strains were exclusively obtained from rectal screening. Ten isolates co-harboured carbapenemase genes including β-lactamase gene encoding KPC-2 + OXA-181, NDM-1 + OXA-48 and NDM-1 + OXA-181. One ST307 isolate (UP-KT-73CKP) co-harboured three carbapenemase genes (blaNDM-1 + blaOXA-48 + blaOXA-181), while all the ST2497 strains co-harboured (blaNDM-1 + blaOXA-232). Phenotypically, hypermucoviscosity was observed in a single ST307 isolate. The ST307 isolate UP-KT-151UKP harboured colibactin genotoxins. The following mobile genetic elements were detected: plasmids [incompatibility group (Inc)-FIB(K), IncX3], and bacteriophages [e.g. Klebsi_ST16_OXA48phi5.4_NC_049450, Klebsi_3LV2017_NC_047817(36)]. The study highlights the importance of local genomic surveillance systems to characterise K. pneumoniae MDR high-risk clones. This data will aid in designing infection and prevention measures for limiting the spread of carbapenemase-producing K. pneumoniae in Gauteng, South Africa.

Molecular Characterization of Multidrug-Resistant and Hypervirulent New Delhi Metallo-Beta-Lactamase Klebsiella pneumoniae in Lazio, Italy: A Five-Year Retrospective Study

Background/Objectives: Antimicrobial resistance represents a challenge to public health systems because of the array of resistance and virulence mechanisms that lead to treatment failure and increased mortality rates. Although for years the main driver of carbapenem resistance in Italy has been the Klebsiella pneumoniae KPC carbapenemase, recent years have seen an increase in VIM and NDM metallo-beta-lactamases (MBLs). We conducted a five-year survey of New Delhi Metallo-beta-Lactamase (NDM)-producing Klebsiella pneumoniae (NDM-Kpn) clinical isolates from the Lazio region, Italy; the study aimed to elucidate the molecular mechanisms underpinning their resistant and virulent phenotype. Methods: Antimicrobial susceptibility was evaluated by automated systems and broth microdilution. In silico analysis of acquired resistance and virulence genes was performed using whole-genome sequencing (WGS), molecular typing through MLST, and core genome multi-locus sequence typing (cgMLST). Conclusions: A total of 126 clinical NDM-Kpn isolates were collected from 19 distinct hospitals in the Lazio region. Molecular analysis highlighted the existence of NDM-1 (108/126) and NDM-5 (18/126) variants, 18 Sequence Types (STs), and 15 Cluster Types (CTs). Notably, 31/126 isolates displayed a virulence score of 4, carrying ybt, ICEKp, iuc, and rmp genes. This study identified a variety of NDM-Kpn STs, mainly carrying the blaNDM-1 gene, with a significant number linked to high-risk clones. Of these isolates, 24.6% showed high-level resistance and virulence, emphasizing the risk of the spread of strains that combine multi-drug-resistance (MDR) and virulence. Proactive surveillance and international collaborations are needed to prevent the spread of high-risk clones, as well as further research into new antimicrobial agents to fight antibiotic resistance.

Analysis of Acinetobacter P-type type IV secretion system-encoding plasmid diversity uncovers extensive secretion system conservation and diverse antibiotic resistance determinants.

Acinetobacter baumannii is globally recognized as a multi-drug-resistant pathogen of critical concern due to its capacity for horizontal gene transfer and resistance to antibiotics. Phylogenetically diverse Acinetobacter species mediate human infection, including many considered as important emerging pathogens. While globally recognized as a pathogen of concern, pathogenesis mechanisms are poorly understood. P-type type IV secretion systems (T4SSs) represent important drivers of pathogen evolution, responsible for horizontal gene transfer and secretion of proteins that mediate host-pathogen interactions, contributing to pathogen survival, antibiotic resistance, virulence, and biofilm formation. Genes encoding a P-type T4SS were previously identified on plasmids harboring the carbapenemase gene blaNDM-1 in several clinically problematic Acinetobacter; however, their prevalence among the genus, geographical distribution, the conservation of T4SS proteins, and full capacity for resistance genes remain unclear. Using systematic analyses, we show that these plasmids belong to a group of 53 P-type T4SS-encoding plasmids in 20 established Acinetobacter species, the majority of clinical relevance, including diverse A. baumannii sequence types and one strain of Providencia rettgeri. The strains were globally distributed in 14 countries spanning five continents, and the conjugative operon's T4SS proteins were highly conserved in most plasmids. A high proportion of plasmids harbored resistance genes, with 17 different genes spanning seven drug classes. Collectively, this demonstrates that P-type T4SS-encoding plasmids are more widespread among the Acinetobacter genus than previously anticipated, including strains of both clinical and environmental importance. This research provides insight into the spread of resistance genes among Acinetobacter and highlights a group of plasmids of importance for future surveillance.

Longitudinal genomics reveals carbapenem-resistant Acinetobacter baumannii population changes with emergence of highly resistant ST164 clone

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a persistent nosocomial pathogen that poses a significant threat to global public health, particularly in intensive care units (ICUs). Here we report a three-month longitudinal genomic surveillance study conducted in a Hangzhou ICU in 2021. This followed a three-month study conducted in the same ICU in 2019, and infection prevention and control (IPC) interventions targeting patients, staff and the ICU environment. Most A. baumannii isolated in this ICU in 2021 were CRAB (80.9%; 419/518) with higher-level resistance to carbapenems. This was accompanied by the proportion of global clone 2 (GC2) isolates falling from 99.5% in 2019 to 50.8% (213/419) in 2021. The phylogenetic diversity of GC2 increased, apparently driven by regular introductions of distinct clusters in association with patients. The remaining CRAB (40.2%; 206/419) were a highly clonal population of ST164. Isolates of ST164 carried blaNDM-1 and blaOXA-23 carbapenemase genes, and exhibited higher carbapenem MIC50/MIC90 values than GC2. Comparative analysis of publicly available genomes from 26 countries (five continents) revealed that ST164 has evolved towards carbapenem resistance on multiple independent occasions. Its success in this ICU and global capacity for acquiring resistance determinants indicate that ST164 CRAB is an emerging high-risk lineage of global concern.

Emerging blaNDM-positive Salmonella enterica in Chinese pediatric infections.

Non-typhoidal Salmonella (NTS) is a common zoonotic foodborne pathogen, whose rising antimicrobial resistance has been an urgent threat to global public health. Here, we reported two carbapenem-resistant Salmonella enterica (CRSE) strains (NBFE-049 and NBFE-164) carrying the blaNDM genes, which were recovered from two Chinese children, belonged to Salmonella enterica serovar Typhimurium (S. Typhimurium) monophasic variant (S. 1,4,[5],12:i:-) ST34 (a sequence type) and S. Typhimurium ST19, respectively. Genes blaNDM-5 and blaNDM-13 were detected in NBFE-049 and NBFE-164, respectively. The blaNDM-5 in NBFE-049 was located in an IncHI2-type plasmid, named pNBFE-049. In NBFE-164, the blaNDM-13 was located in an IncI1-type plasmid, named pNBFE-164. The plasmid pNBFE-164 successfully transferred its resistance phenotype into the recipient strain Escherichia coli J53 with a high efficiency of 1.1 × 10-2, while no transconjugants were obtained in pNBFE-049 conjugation assays. We further elucidated the genetic relationships of globally occurring New Delhi Metallo-β-lactamase (NDM)-positive strains and locally distributed clinical strains within the same serovar. The closest relative of NBFE-049 was clinical Salmonella strain 1722, which was recovered in 2020 and differed by only three Single Nucleotide Polymorphisms (SNPs). No NDM-positive ST19 could be found in the National Center for Biotechnology Information (NCBI) database, and NBFE-164 showed a close genetic relationship with the other ST19 in this area. To sum up, we suggested the potential contributions of clonal spread and plasmid-mediated blaNDM transfer in CRSE dissemination. This study reported the complete genome of two blaNDM-carrying S. Typhimurium isolates, shedding new insights into the antimicrobial resistance mechanisms and dissemination patterns of the emerging CRSE.IMPORTANCENTS is one of the most common zoonotic pathogens that causes foodborne illnesses, while S. Typhimurium is one of the most common serovars. With the rising prevalence of multi-resistant Salmonella worldwide, carbapenems have emerged as the last-line antibiotics for treating severe bacterial infections. In this study, we reported the genomic characteristics of two carbapenem-resistant S. Typhimurium strains, which were recovered from two pediatric patients, carrying blaNDM-5 and blaNDM-13, providing new insights into the antimicrobial resistance deteriminants and transmission risk of blaNDM-positive NTS in China. We suggested the potential contributions of clonal spread and plasmid-mediated blaNDM transfer in CRSE dissemination. Future enhanced surveillance policy should mitigate CRSE spreading, and more importantly, clinical antimicrobial therapeutic regimens should be adjusted accordingly.

Transmission of carbapenem-resistant enterobacterales producing NDM-5 during the broiler breeding process in China

This study conducted a four-month monitoring of carbapenem resistance in a broiler breeding farm in China. A total of 185 carbapenem-resistant bacterial isolates were obtained from 2298 cloacal swabs from broiler breeders and their offspring within a production cycle. The detection rate of carbapenem-resistant isolates was higher during the brooding period. Whole-genome sequencing (WGS) was performed on 133 isolates based on sampling stages, including 113 carbapenem-resistant Enterobacterales (CRE) isolates and 20 Stenotrophomonas pavanii isolates, which have intrinsic resistance to carbapenems. A total of 69 antibiotic resistance genes (ARGs), including blaNDM-1, mcr-1, and blaNDM-5, were identified among the sequenced CRE isolates. Notably, blaNDM-5 (92.0 %, 104/113) was the primary contributor to carbapenem resistance. CRE isolates from the same breeding stage exhibited close genomic relationships, and the blaNDM-5 genes were observed in similar genetic backgrounds, indicating the transmission of CRE strains and blaNDM-5 during the broiler breeding process. No CRE was isolated from 0 d broiler offspring, suggesting that broiler breeders were not the direct source of CRE in their offspring. Tracing the feeding process revealed that brooder and rearing houses were likely key factors in the cross-transmission of CRE between broiler breeders and their offspring. CRE pose a significant threat to public health and food safety. China is one of the world's leading poultry producing and consuming countries. This study provided insights into the epidemiological trends and key transmission nodes of carbapenem resistance and CRE within the broiler breeding process, which could help the control of antibiotic resistance and bacterial infections in the broiler industry.

Genomic Characterization of 16S rRNA Methyltransferase-Producing Enterobacterales Reveals the Emergence of Klebsiella pneumoniae ST6260 Harboring rmtF, rmtB, blaNDM-5, blaOXA-232 and blaSFO-1 Genes in a Cancer Hospital in Bulgaria.

Background: Acquired 16S rRNA methyltransferases (16S-RMTases) confer high-level resistance to aminoglycosides and are often associated with β-lactam and quinolone resistance determinants. Methods: Using PCR, whole-genome sequencing and conjugation experiments, we conducted a retrospective genomic surveillance study of 16S-RMTase-producing Enterobacterales, collected between 2006 and 2023, to explore transmission dynamics of methyltransferase and associated antibiotic resistance genes. Results: Among the 10,731 consecutive isolates, 150 (1.4%) from 13 species carried armA (92.7%), rmtB (4.7%), and rmtF + rmtB (2.7%) methyltransferase genes. The coexistence of extended-spectrum β-lactamase (blaCTX-M-3/15, blaSHV-12, blaSFO-1), carbapenemase (blaNDM-1/5, blaVIM-1/4/86, blaOXA-48), acquired AmpC (blaCMY-2/4/99, blaDHA-1, blaAAC-1), and plasmid-mediated quinolone resistance (qnrB, qnrS, aac(6')-Ib-cr) genes within these isolates was also detected. Methyltransferase genes were carried by different plasmids (IncL/M, IncA/C, IncR, IncFIB, and IncFII), suggesting diverse origins and sources of acquisition. armA was co-transferred with blaCTX-M-3/15, blaNDM-1, blaVIM-4/86, blaOXA-48, blaCMY-4, aac(6')-Ib-cr, qnrB, and qnrS, while rmtF1 was co-transferred with blaSFO-1, highlighting the multidrug-resistant nature of these plasmids. Long-read sequencing of ST6260 K. pneumoniae isolates revealed a novel resistance association, with rmtB1 and blaNDM-5 on the chromosome, blaOXA-232 on a conjugative ColKP3 plasmid, and rmtF1 with blaSFO-1 on self-transmissible IncFIB and IncFII plasmids. Conclusions: The genetic plasticity of plasmids carrying methyltransferase genes suggests their potential to acquire additional resistance genes, turning 16S-RMTase-producing Enterobacterales into a persistent public health threat.

High frequency of acquired virulence factors in carbapenemase-producing Klebsiella pneumoniae isolates from a large German university hospital, 2013-2021.

Carbapenemase-producing Klebsiella pneumoniae (CP-Kp) isolates are a public health concern as they can cause severe hospital-acquired infections that are difficult to treat. It has recently been shown that CP-Kp can take up virulence factors from hypervirulent K. pneumoniae lineages. In this study, 109 clinical CP-Kp isolates from the University Hospital Cologne were examined for the presence of acquired virulence factors using whole-genome sequencing and phenotypic tests, and results were linked to clinical data. The virulence factor iuc was present in 18/109 of the CP-Kp isolates. Other acquired virulence factors, such as ybt, cbt, iro, rmpA/rmpA2, peg-344, and hypervirulence-associated capsule types were detected in various combinations among these isolates. The iuc-positive isolates produced OXA-232 (n = 7), OXA-48 (n = 6), OXA-48+NDM (n = 3), NDM, and KPC (each n = 1), and 7/18 isolates were resistant to ceftazidime-avibactam, colistin, and/or cefiderocol. Four isolates carried hybrid plasmids that harbored acquired virulence factors alongside the carbapenemase genes blaNDM-1/5 or blaOXA-48. In 15/18 patients, iuc-positive CP-Kp were isolated from a clinically manifest infection site. Among these, four patients had osteomyelitis, and four patients died from pneumonia with OXA-232-producing ST231 isolates, three of them as part of an outbreak. In conclusion, acquired virulence factors are frequently detected in various combinations in carbapenemase-producing K. pneumoniae isolates in Germany, warranting continuous monitoring of infections caused by these strains.

Detection of antibiogram pattern and prevalence of blaNDM and blaVIM genes among carbapenem resistant Eschericia coli isolates in a tertiary care hospital

Background: Clinical significance of Eschericia coli is due to its capacity to develop resistance against many antibiotics specially carbapenem by producing carbapenemase which limits treatment options. blaNDM and blaVIM genes are responsible for this multi-drug-resistance. This is an alarming condition for society as well as clinician because they are very difficult to treat due to limited options of antibiotics and its high virulence. Aims and Objectives: The study was conducted to detect antibiogram pattern and prevalence of blaNDM and blaVIM genes among carbapenem resistant E. coli isolates in a tertiary care hospital. Our study will also help to identify molecular basis of Carbapenem resistance including treatment guideline. Materials and Methods: Cross-sectional study was performed during January’2021 to June’2022 time period. Identification of E. coli was followed by standard protocol and antibiotic sensitivity was done by Kirby-Bauer disk-diffusion method following CLSI guideline, using VITEK-2D-SYSTEM. Detection of genes was done by polymerase chain reaction (PCR). Results: Out of 1867 samples, 161 isolates were identified as E. coli, among which 90 were resistant to carbapenem. Among those, 50 (31%) were resistant to all three carbapenems. Among those, 100% were resistant to ampicillin, amoxycillin-clavulanic acid, cefuroxime, cefuroxime-axetil, cefoperazone-sulbactam, ceftriaxone, cefepime, piperacillin-tazobactam, nalidixic acid, ciprofloxacin; 52% were resistant to amikacin; 54% were resistant to gentamicin; 86% were resistant to cotrimoxazole; 22% were resistant to nitrofurantoin and 96% were sensitive to tigecycline; 100% were intermediate sensitive to colistin. blaNDM gene is detected in 17 (34%) isolates and blaVIM gene is detected in 2 (4%) isolates by PCR. Conclusion: E. coli leads to common clinical presentations such as urinary tract infection, blood stream infection, pneumonia, and wound infection. High burden of carbapenem resistant E. coli was associated with urinary indoor samples. Rising incidence of multi-drug-resistance occurs due to lack of infection control measures, irrational use of antibiotics. Continuous surveillance will prevent development of multi-drug-resistance and help to improve treatment guideline.

Coexistence of seven different carbapenemase producers in a single hospital admission screening confirmed by whole-genome sequencing

ObjectiveTo molecularly characterize several extensively drug-resistant isolates from a single hospital admission screening of a war injured patient from the Ukraine. MethodsAdmission screening included swabs from skin, wounds, catheters, nasopharyngeum and rectum. Bacterial identification, antimicrobial susceptibility testing (AST) and rapid multiplex PCR assays targeting resistance genes were performed during routine diagnostics. Isolates positive by PCR had their genomes sequenced using short- and long read-platforms (MiSeq and MinION) to confirm species, identify resistance genes and plasmids and investigate clonality with core genome MLST (cgMLST). ResultsSeven Gram-negative pathogens (Acinetobacter baumannii (n=2; ST78, ST2), Klebsiella pneumoniae (n=2; ST395), Pseudomonas aeruginosa (n=1; ST1047), Escherichia coli (n=1; ST46), Enterobacter cloacae complex (n=1; ST231)) were molecularly confirmed non-identical. AST showed resistance to carbapenems (7/7 isolates) and last resort treatment options such as ceftazidime-avibactam (6/7 isolates) and cefiderocol (4/7 isolates). All isolates were colistin susceptible. Sequencing identified the E. cloacae complex as E. hormaechei subsp. xiangfangensis. Six acquired carbapenemase genes (blaIMP-1, blaNDM-1, blaOXA-48, blaNDM-5, blaOXA-23 and blaOXA-72) were detected. Both A. baumannii isolates differed in sequence type, carbapenemases and cefiderocol susceptibility. Both K. pneumoniae isolates shared sequence type and some resistance genes on an IncR plasmid but were different in cgMLST and carbapenemases (OXA-48 or NDM-1). One vancomycin-resistant Enterococcus faecium was also detected (VanA). ConclusionWar injured patients from Ukraine may carry different clones of multidrug-resistant pathogens with limited treatment options and diverse resistance genes at risk for dissemination. Infection control measures should include early molecular characterization of isolates for detection of routes of transmission.

Epidemiological and genomic analysis revealed the significant role of flies in dissemination of carbapenem-resistant Enterobacteriaceae (CRE) in China

While the role of flies as potential vectors for bacterial transmission is well recognized, the epidemiological features and genomic characteristics of associated antimicrobial-resistant strains remain underexplored. This study conducted a nationwide surveillance including 3689 flies and 838 isolates from sixteen human communities (HCs) and eight animal farms (AFs) across 21 provinces in China. Our findings revealed high carriage rates of carbapenem-resistant Enterobacteriaceae (CRE) in flies, with 27.0 % in AFs and 15.3 % in HCs. Among the 705 CRE strains identified, Providencia spp. (n = 356) and Escherichia coli (n = 231) were predominant, with 90.6 % attributed to the dissemination of the blaNDM gene. Phylogenetic tree analysis highlighted frequent clonal transmission events, with over 18 and 13 clonal evolutionary clades in Providencia spp. and E. coli, respectively. Notably, spearman analysis linked the increase of CRE prevalence in AFs flies to environmental factors like precipitation (p < 0.028) and temperature (p < 0.037), while in HCs, it positively corelated with the total meat production (p < 0.027) in each province except for Hainan. Despite significant differences in ciprofloxacin resistance (p ≤ 0.001) among E. coli strains from HCs and AFs, core genome cluster analysis and Uniform Manifold Approximation and Projection (UMAP) approach indicated these strains exhibiting close relatedness to clinical strains. This comprehensive study underscores the critical role of flies in the escalation of CRE spread within the ecosystem in China. Flies found in both HCs and AFs contribute to the dissemination of CRE, highlighting the need for urgent interventions to address this pressing issue.

Investigation of in vitro susceptibility and resistance mechanisms to amikacin among diverse carbapenemase-producing Enterobacteriaceae

ObjectiveThis study aims to assess the in vitro drug susceptibility of various Carbapenemase-Producing Enterobacteriaceae (CPE) genotypes and elucidate the underlying mechanisms of amikacin resistance.MethodsA total of 72 unique CPE strains were collected from the Second Hospital of Jiaxing between 2019 and 2022, including 51 strains of Klebsiella pneumoniae, 11 strains of Escherichia coli, 6 strains of Enterobacter cloacae, 2 strains of Klebsiella aerogenes, 1 strain of Citrobacter freundii, and 1strain of Citrobacter werkmanii. Among these strains, 24 carried blaKPC gene, 20 carried blaNDM gene, 23 carried blaOXA−48−like gene, and 5 carried both blaKPC and blaNDM. We measured the in vitro activity of amikacin and other common antibiotics. Strains carrying blaOXA−48-like gene were selected for whole genome sequencing (WGS) via next-generation sequencing to identify genes related to antimicrobial resistance (AMR) and virulence factor (VF).ResultsOut of the 72 CPE strains tested, 41.7% exhibited resistance to amikacin. The drug resistance rates for K. pneumoniae, E. coli, and Enterobacter spp. were 51.0%, 27.3%, and 10.0%, respectively. The majority of the CPE strains (> 90%) displayed resistance to cephalosporins and carbapenems, while most of them were sensitive to polymyxin B and tigecycline (97.2% and 94.4%). The amikacin resistance rate was 100% for strains carrying blaOXA−48, 20.8% for those with blaKPC, 5.0% for those with blaNDM, and 20.0% for those with both blaKPC and blaNDM. These differences were statistically significant (P < 0.05). Through sequencing, we detected aminoglycoside resistance genes rmtF and aac(6’)-Ib, VF genes iucABCD and rmpA2 in OXA-48-producing multidrug resistance and highly virulent strains. These genes were located on a IncFIB- and IncHI1B-type plasmid, respectively. Both plasmids were highly homologous to the plasmid from OXA-232 strains in Zhejiang province and Shanghai province. Integration of these resistance genes into the IncFIB plasmid, facilitated by the IS6 and/or Tn3 transposons, resulted in OXA232-producing K. pneumoniae with amikacin resistance.ConclusionThis study identified significant amikacin resistance in CPE strains, particularly in those carrying the blaOXA−48 gene. Resistance genes rmtF and aac(6’)-Ib were identified on plasmids. These results highlight the need for careful monitoring of amikacin resistance.

Evaluation of gradient strip diffusion for susceptibility testing of aztreonam-avibactam in metallo-β-lactamase-producing Enterobacterales.

The emergence of metallo-β-lactamase (MBL)-producing Enterobacterales presents unique clinical treatment challenges. Recently developed β-lactam/ β-lactamase inhibitor combination agents, while effective against other carbapenemase-producing organisms, are notably ineffective against MBL producers. While MBLs do not hydrolyze monobactams (aztreonam), many MBL-producing organisms are resistant to aztreonam through alternate mechanisms, leaving cefiderocol as the sole monotherapy treatment option recommended for MBL producers. Recent guidelines for the treatment of MBL-harboring organisms have added combination therapy with aztreonam and ceftazidime-avibactam, using ceftazidime-avibactam as a source of the β-lactamase inhibitor avibactam. Current laboratory testing options for the combination of aztreonam-avibactam are limited to broth microdilution (BMD) and broth disk elution (BDE) methods, which are not practical in most clinical laboratories. In this study, we evaluated the performance of aztreonam/avibactam gradient strips on 103 MBL-producing Enterobacterales patient isolates as well as an additional 31 isolates from the CDC AR Bank. All MBL Enterobacterales patient isolates included in this study harbored a New Delhi metallo-β-lactamase (blaNDM) gene. Essential agreement of gradient strip minimal inhibitory concentrations (MICs) for patient isolates compared to BMD was 93.2%. While there are no established breakpoints for aztreonam-avibactam, category agreement (CA) for patient isolates was 97.1% when using the CLSI aztreonam breakpoints. There were no major or very major errors observed. There were three minor errors. Precision for aztreonam-avibactam gradient strip diffusion was 100%. These data demonstrate that the use of gradient strip diffusion for aztreonam-avibactam MIC determination in MBL-producing Enterobacterales is a viable option for clinical laboratories.

Characterization of colistin-resistant carbapenemase producing Klebsiella pneumoniae in a river receiving wastewater treatment plant effluent.

Genes conferring antibiotic resistance phenotype, particularly to last resort antibiotics, pose a significant concern globally. Wastewater treatment plant (WWTP) effluent substantially contributes to antibiotic resistance in receiving rivers, threatening human health. Globally, colistin- and carbapenem-resistant Klebsiella pneumoniae infections cause high morbidity and mortality. We investigated colistin-resistant carbapenemase-producing K. pneumoniae (Co-CRKP) isolates in Kathajodi river receiving WWTP effluent, their resistance genes, and pathogenic potential. Four isolates (Co-CRKP-7, Co-CRKP-8, Co-CRKP-10, and Co-CRKP-15) exhibited extensively drug-resistant (XDR) phenotype, harbouring blaTEM-1, blaCTX-M-15, blaNDM-5, and blaOXA-48 genes. Colistin resistance was attributed to mutations in the pmrA and pmrB genes. Virulence genes (fimH, mrkD, entB, iucA, iutA, and irp1), capsular serotypes (K1, K2) and biofilm formation in the isolates explicated their pathogenicity. Furthermore, Inc plasmid replicons (Y, FrepB, P, K/B, L/M, N, FIA, A/C, and FIB) indicated the dissemination potential of the resistance genes in Co-CRKP isolates. The multi-locus sequence typing showed that Co-CRKP-7 and Co-CRKP-8 belonged to ST42, while Co-CRKP-10 and Co-CRKP-15 were ST16 and ST231, respectively. These high-risk clones carrying multidrug resistance and virulence genes, implicated in numerous outbreaks, have spread worldwide. Our findings emphasize the necessity for effective treatment of hospital wastes to restrict the spread of clinical isolates into aquatic environments.

Predominance of blaNDM- and blaIMP-Harboring Escherichia coli Belonging to Clonal Complexes 131 and 23 in a Major University Hospital.

Background and Objectives: Carbapenem resistance is a growing global challenge for healthcare, and, therefore, monitoring its prevalence and patterns is crucial for implementing targeted interventions to mitigate its impact on patient outcomes and public health. This study aimed to determine the prevalence of carbapenem resistance among Escherichia coli (E. coli) strains in the largest tertiary care hospital of the capital territory of Pakistan and to characterize the isolates for the presence of antimicrobial resistance genes. Additionally, the most prevalent sequence types were analyzed. Materials and Methods: A total of 15,467 clinical samples were collected from November 2020 to May 2022, underwent antimicrobial susceptibility testing, and were analyzed for antimicrobial resistance genes through conventional PCR and sequence typing using MLST. Results: In carbapenem-resistant E. coli (CR-EC), 74.19% of isolates harbored the blaNDM gene, with blaNDM-1 (66.96%), blaNDM-5 (12.17%), and blaNDM-7 (20.87%) variants detected. Additionally, blaIMP was found in 25.81% and blaOXA-48 in 35.48% of isolates. The presence of blaCTX-M15 and blaTEM was identified in 83.87% and 73.55% of CR-EC isolates, respectively, while armA and rmtB were detected in 40% and 65.16% of isolates, respectively. Colistin and tigecycline were the most effective drugs against CR-EC isolates, with both showing an MIC50 of 0.5 µg/mL. The MIC90 for colistin was 1 µg/mL, while for tigecycline, it was 2 µg/mL. MLST analysis revealed that the CR-EC isolates belonged to ST131 (24.52%), ST2279 (23.87%), ST3499 (16.13%), ST8051 (15.48%), ST8900 (9.68%), ST3329 (7.10%), ST88 (1.94%), and ST6293 (1.29%). The ST131 complex (70.97%) was the most prevalent, harboring 95.65% of the blaNDM gene, while the ST23 complex (18.06%) harbored 62.50% of the blaIMP gene. Conclusions: Implementing large-scale surveillance studies to monitor the spread of specific pathogens, along with active infection control policies, is crucial for the effective containment and prevention of future epidemics.

Antimicrobial resistance profiling of bacteria isolated from wastewater and samples of pharmaceutical industries in South India

The study was aimed to determine the phenotypic and genotypic antimicrobial resistance in the isolated bacteria from the influent (25), effluent (15), surface and ground water samples (15) surrounding the pharmaceutical industries located in south India. From 55 samples, 48 isolates of 10 different bacteria were obtained. The identified bacterial isolates were viz. Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, Corynebacterium sp., Acinetobacter sp., Aeromonas punctata, Ralstonia picketti, Staphylococcus aureus, Stenotrophomonas maltophillia, and Citrobacter freundii. The phenotypic profile of resistance through antibiotic susceptibility test was carried out against sixteen different antibiotics. Standard PCR technique was used for the detection of 12 resistance genes encoding carbapenems, quinoline, aminoglycoside, β-lactam belonging blaOXA-58,blaOXA-22,qnrA, qnrB, aac(6)-Ib-cr, aac (3)-XI, mec A, qepA, aadB, blaVIM, blaOXA-48 and blaNDM. Pseudomonas aeruginosa (1: TN/I/2020) showed presence of 3 resistance genes. qnrB (489 bp) gene was present in maximum of 7 isolates while blaVIM (196 bp) gene was present in 6 isolates. The resistance genes blaNDM (621 bp) was present in three different isolates; aac (X):6)-lb-cr (482 bp), qepA (495 bp), aadB (500 bp), blaOXA-58 (843 bp) resistant genes were present in two different isolates each among the bacterial isolates obtained in this study. In phenotypic resistance profiling by AST method, out of 16 antibiotics tested, 14 showed resistance. Similarly, in genotypic resistance profiling, among 12 resistance genes tested, a maximum of three resistance genes were noticed in Pseudomonas aeruginosa. There were positive and negative correlations observed between phenotypic and genotypic resistance among different antibiotics and their resistance genes indicating the variations in the resistance gene expression.

Co-Occurrence of Two Plasmids Encoding Transferable blaNDM-1 and tet(Y) Genes in Carbapenem-Resistant Acinetobacter bereziniae.

Acinetobacter bereziniae has emerged as a significant human pathogen, acquiring multiple antibiotic resistance genes, including carbapenemases. This study focuses on characterizing the plasmids harboring the blaNDM-1 and tet(Y) genes in two carbapenem-resistant A. bereziniae isolates (UCO-553 and UCO-554) obtained in Chile during the COVID-19 pandemic. Methods: Antibiotic susceptibility testing was conducted on UCO-553 and UCO-554. Both isolates underwent whole-genome sequencing to ascertain their sequence type (ST), core genome multilocus sequence-typing (cgMLST) profile, antibiotic resistance genes, plasmids, and mobile genetic elements. Conjugation experiments were performed for both isolates. Results: Both isolates exhibited broad resistance, including resistance to carbapenems, third-generation cephalosporins, fluoroquinolones, tetracycline, cotrimoxazole, and aminoglycosides. Both isolates belong to sequence type STPAS1761, with a difference of 17 out of 2984 alleles. Each isolate carried a 47,274 bp plasmid with blaNDM-1 and aph(3')-VI genes and two highly similar plasmids: a 35,184 bp plasmid with tet(Y), sul2, aph(6)-Id, and aph(3″)-Ib genes, and a 6078 bp plasmid containing the ant(2″)-Ia gene. Quinolone-resistance mutations were identified in the gyrA and parC genes of both isolates. Importantly, blaNDM-1 was located within a Tn125 transposon, and tet(Y) was embedded in a Tn5393 transposon. Conjugation experiments successfully transferred blaNDM-1 and tet(Y) into the A. baumannii ATCC 19606 strain, indicating the potential for horizontal gene transfer. Conclusions: This study highlights the critical role of plasmids in disseminating resistance genes in A. bereziniae and underscores the need for the continued genomic surveillance of this emerging pathogen. The findings emphasize the importance of monitoring A. bereziniae for its potential to cause difficult-to-treat infections and its capacity to spread resistance determinants against clinically significant antibiotics.

Microbiological Quality of Coconut Water Sold in the Grande Vitória Region, Brazil, and Phenogenotypic Antimicrobial Resistance of Associated Enterobacteria.

This study aimed to evaluate the microbiological quality of coconut water sold from street carts equipped with cooling coils or refrigerated at bakeries in the Grande Vitória Region, Brazil. Additionally, it assessed the phenotypic and genotypic antimicrobial resistance profiles of isolated enterobacteria. The results indicated that coconut water sold at street carts had lower microbiological quality compared to refrigerated samples, as evidenced by significantly higher counts of mesophilic microorganisms. Using MALDI-TOF, the following opportunistic pathogens were identified: Citrobacter freundii, Enterobacter bugandensis, E. kobei, E. roggenkampii, Klebsiella pneumoniae, and Kluyvera ascorbata. Three isolates-E. bugandensis, K. pneumoniae, and K. ascorbata-were classified as multidrug-resistant (MDR). Widespread resistance to β-lactams and cephalosporins was detected, and some isolates were resistant to quinolones, nitrofurans, and phosphonic acids. The gene blaCTX-M-2 was detected in C. freundii, E. bugandensis, E. kobei, and K. ascorbata. However, genes blaNDM, blaKPC, blaCMY-1, and blaCMY-2 were not detected in any isolate. The findings underscore the need to enhance good manufacturing practices in this sector to control the spread of antimicrobial resistance (AMR). To our knowledge, this is the first study documenting the presence of potentially pathogenic enterobacteria in coconut water samples and their associated phenotypic and genotypic AMR profiles.

In-host intra- and inter-species transfer of blaKPC-2 and blaNDM-1 in Serratia marcescens and its local and global epidemiology

ObjectivesThe aim of this study was to investigate interspecies transfer of resistance gene blaNDM-1 and intraspecies transfer of resistance gene blaKPC-2 in Serratia marcescens, and explore the epidemical and evolutionary characteristics of carbapenemase-producing S. marcescens (CPSM) regionally and globally. MethodsInterspecies and intraspecies transfer of blaKPC-2- or blaNDM-1 were identified by antimicrobial susceptibility testing, plasmid conjugation and curing, discovery of transposable units (TUs), outer membrane vesicles (OMVs), qPCR, whole-genome sequencing (WGS) and bioinformatic analysis. The genomic evolution of CPSM strains was explored by cgSNP and maximum-likelihood phylogenetic tree. ResultsCPSM S50079 strain, co-carrying blaKPC-2 and blaNDM-1 on one plasmid, was isolated from the blood of a patient with acute pancreatitis and could generate TUs carrying either blaKPC-2 or blaNDM-1. The interspecies transfer of blaNDM-1-carrying plasmid from Providencia rettgeri P50213, producing the identical blaNDM-1-carrying TUs, to S. marcescens S50079K, an S50079 variant via plasmid curing, was identified through blaNDM-1-harbouring plasmid conjugation and OMVs transfer. Moreover, the intraspecies transfer of blaKPC-2, mediated by IS26 from plasmid to chromosome in S50079, was also identified. In another patient, who underwent lung transplantation, interspecies transfer of blaNDM-1 carried by IncX3 plasmid was identified among S. marcescens and Citrobacter freundii as well as Enterobacter hormaechei via plasmid transfer. Furthermore, 11 CPSM from 349 non-repetitive S. marcescens strains were identified in the same hospital, and clonal dissemination, with carbapenemase evolution from blaKPC-2 to both blaKPC-2 and blaNDM-1, was found in the 8 CPSM across 4 years. Finally, the analysis of 236 global CPSM from 835 non-repetitive S. marcescens genomes, retrieved from the NCBI database, revealed long-term spread and evolution worldwide, and would cause the convergence of more carbapenemase genes. ConclusionsInterspecies transfer of resistance gene blaNDM-1 and intraspecies transfer of resistance gene blaKPC-2 in CPSM were identified. Nosocomial and global dissemination of CPSM were revealed and more urgent surveillance was acquired.

Presence of extended-spectrum beta-lactamase-producing Escherichia coli and carbapenem resistance in ready-to-eat stuffed mussels in Istanbul

AbstractFoodborne pathogens' transmission is essential in the spread of antibiotic resistance, and extended-spectrum beta-lactamase-producing Escherichia coli especially threatens public health. E. coli plays an essential role in the resistance to commonly used beta-lactam group antibiotics. Ready-to-eat (RTE) stuffed mussels are among many restaurants and street vendors, presenting potential health risks of food hygiene origin. 200 RTE stuffed mussels were collected from the Asian and European sides of Istanbul and analysed for the presence of E. coli. As a result of PCR analysis, E. coli was detected in 7 (3.5%) samples. An antibiotic susceptibility test was performed using the disc diffusion method to determine ESBL and carbapenem resistance. All isolates were resistant to ampicillin. The double-disk synergy test was performed as an ESBL phenotypic confirmation test, and no phenotypically ESBL-producing E. coli were detected. The blaTEM gene was detected in one isolate (14.2%) by mPCR, but blaCTX-M, blaSHV, and blaOXA genes were not observed. Meropenem and imipenem were used with the disk diffusion method for carbapenem resistance study, and no resistant isolate was found. Carbapenem resistance genes were investigated by monoplex PCR, and blaNDM-1, blaOXA-48, blaVIM, and blaIMP resistance genes were not detected. This is the first report on ESBL-producing E. coli in RTE stuffed mussels in Türkiye, which draws attention to a public health risk.