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New Delhi metallo-β-lactamase, a metallo-β-lactamase carbapenemase type, mediates resistance to most β-lactam antibiotics including penicillins, cephalosporins, and carbapenems. Therefore, it is important to detect blaNDM genes in children’s clinical samples as quickly as possible and analyze their characteristics. Here, a recombinase-aided amplification (RAA) assay, which operates in a single one-step reaction tube at 39°C in 5−15 min, was established to target blaNDM genes in children’s clinical samples. The analytical sensitivity of the RAA assay was 20 copies, and the various bacterial types without blaNDM genes did not amplify. This method was used to detect blaNDM genes in 112 children’s stool samples, 10 of which were tested positive by both RAA and standard PCR. To further investigate the characteristics of carbapenem-resistant bacteria carrying blaNDM in children, 15 carbapenem-resistant bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Citrobacter freundii, Klebsiella oxytoca, Acinetobacter junii, and Proteus mirabilis) were isolated from the 10 samples. Notably, more than one bacterial type was isolated from three samples. Most of these isolates were resistant to cephalosporins, cefoperazone-sulbactam, piperacillin-tazobactam, ticarcillin-clavulanic acid, aztreonam, co-trimoxazole, and carbapenems. blaNDM–1 and blaNDM–5 were the two main types in these samples. These data show that the RAA assay has potential to be a sensitive and rapid blaNDM gene screening test for clinical samples. The common existence of blaNDM and multi-drug resistance genes presents major challenges for pediatric treatment.

BackgroundIncreasing use of colistin has led to the world-wide emergence of mobile colistin resistant gene (mcr). The present study aimed to identify and characterise mcr and other drug-resistant genes in colistin resistant Klebsiella pneumoniae clinical isolates.MethodsTwenty-two colistin resistant K. pneumoniae were analysed for mcr and other drug-resistant genes, efflux pumps, and virulence genes, and for their biofilm forming ability. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed for all mcr-1 positive isolates. S1-PFGE and Southern hybridisation were performed for localisation of mcr-1 and blaNDM.ResultsNineteen colistin resistant K. pneumoniae harboured mcr-1 and 3 had mgrB disruption. All isolates harboured blaOXA-48-type and ESBL genes; eight strains (five with mcr-1 and three with mgrB disruption) co-harboured blaNDM. Efflux pumps genes AcrAB and mdtK were detected in all 22 and tol-C in 21 isolates. Virulence-related genes entB and irp-1 were detected in all 22, mrkD in 20, and fimH-1 in 18 isolates; 11 isolates were strong biofilm producers. PFGE clustered mcr-1 positive isolates into eight groups based on ≥90% similarity; MLST revealed diverse sequence types, predominant being ST-15 (n = 4) and ST-16 (n = 4). Both mcr-1 and blaNDM were localised on plasmid and chromosome; mcr-1 was present on IncFII type and blaNDM on IncFIB and IncA/C type plasmids.ConclusionsColistin resistance in K. pneumoniae was predominantly mediated by mcr-1. Co-existence of colistin, carbapenem, and other drug-resistant genes along with efflux pumps indicates towards enormous genomic plasticity in K. pneumoniae with ability to emerge as super-spreader of drug-resistance.

In this study, a detailed genetic dissection of the huge and complex blaNDM-carrying genetic elements and their related mobile genetic elements was performed in Enterobacteriaceae. An extensive comparison was applied to 12 chromosomal genetic elements, including six sequenced in this study and the other six from GenBank. These 12 genetic elements were divided into five groups: a novel IME Tn6588; two related IMEs Tn6523 (SGI1) and Tn6589; four related ICEs Tn6512 (R391), Tn6575 (ICEPvuChnBC22), Tn6576, and Tn6577; Tn7 and its derivatives Tn6726 and 40.7-kb Tn7-related element; and two related IMEs Tn6591 (GIsul2) and Tn6590. At least 51 resistance genes, involved in resistance to 18 different categories of antibiotics and heavy metals, were found in these 12 genetic elements. Notably, Tn6576 carried another ICE Tn6582. In particular, the six blaNDM-carrying genetic elements Tn6588, Tn6589, Tn6575, Tn6576, Tn6726, and 40.7-kb Tn7-related element contained large accessory multidrug resistance (MDR) regions, each of which had a very complex mosaic structure that comprised intact or residual mobile genetic elements including insertion sequences, unit or composite transposons, integrons, and putative resistance units. Core blaNDM genetic environments manifested as four different Tn125 derivatives and, notably, two or more copies of relevant Tn125 derivatives were found in each of Tn6576, Tn6588, Tn6589, and 40.7-kb Tn7-related element. The huge and complex blaNDM-carrying genetic elements were assembled from complex transposition and homolog recombination. Firstly identified were eight novel mobile elements, including three ICEs Tn6576, Tn6577, and Tn6582, two IMEs, Tn6588 and Tn6589, two composite transposons Tn6580a and Tn6580b, and one integron In1718.

Background: Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract infection (UTI); however, treatment of UTI has been challenging due to increased antimicrobial resistance (AMR). One of the most important types of AMR is carbapenem resistance (CR). CR bacteria are known as an important threat to global public health today. Class B metallo-beta-lactamases (MBLs) are one of the major factors for resistance against carbapenems. We aimed to investigate the characteristics of UPEC isolates producing MBL. Methods: A cross-sectional study was conducted from October 2018 to December 2019 in Ahvaz; Iran. UPEC isolates were identified by biochemical and molecular methods. Metallo-beta-lactamase-producing isolates were detected using modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) tests. MBL genes, phylogenetic group, and virulence genes profile of carbapenem resistant isolates were determined. Conjugation assay and plasmid profiling were conducted to evaluate the ability of transferring of CR to other E. coli isolates. Clonal similarity of isolates were assessed using Enterobacterial intergenic repetitive element sequence (ERIC)-PCR. Results: Among 406 UPEC isolates, 12 (2.95%) carbapenem-resistant were detected of which 11 were phenotypically MBL-producing strains. Four isolates were resistant to all investigated antimicrobial agents and were considered possible pandrug-resistant (PDR). blaNDM, blaOXA-48, blaIMP-1, and blaIMP-2 genes were found in 9, 5, 1, and 1 isolates, respectively. Among 30 virulence genes investigated, the traT, fyuA followed by fimH, and iutA with the frequency of 8 (66.7%), 8 (66.7%), 7 (58.3%), and 7 (58.3%) were the most identified genes, respectively. Siderophore production was the main virulence trait among carbapenem-resistant UPEC isolates. Except for two, all other isolates showed weak to moderate virulence index. In all recovered isolates, CR was readily transmitted via plasmids to other isolates during conjugation experiments. Conclusion: MBL and carbapenemase genes, especially blaNDM and blaOXA-48 are spreading rapidly among bacteria, which can be a threat to global public health. Therefore monitoring the emergence and dissemination of new AMR is necessary to continuously refine guidelines for empiric antimicrobial therapy. Understanding the mechanisms of resistance and virulence in this group of bacteria can play an effective role in providing new therapeutic methods. [ABSTRACT FROM AUTHOR]

Background: Increasing use of colistin has led to the world-wide emergence of mobile colistin resistant gene (mcr). The present study aimed to identify and characterise mcr and other drug-resistant genes in colistin resistant Klebsiella pneumoniae clinical isolates. Methods: Twenty-two colistin resistant K. pneumoniae were analysed for mcr and other drug-resistant genes, efflux pumps, and virulence genes, and for their biofilm forming ability. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed for all mcr-1 positive isolates. S1-PFGE and Southern hybridisation were performed for localisation of mcr-1 and bla NDM. Results: Nineteen colistin resistant K. pneumoniae harboured mcr-1 and 3 had mgrB disruption. All isolates harboured bla OXA-48-type and ESBL genes; eight strains (five with mcr-1 and three with mgrB disruption) co-harboured bla NDM. Efflux pumps genes AcrAB and mdtK were detected in all 22 and tol-C in 21 isolates. Virulence-related genes entB and irp-1 were detected in all 22, mrkD in 20, and fimH-1 in 18 isolates; 11 isolates were strong biofilm producers. PFGE clustered mcr-1 positive isolates into eight groups based on ≥90% similarity; MLST revealed diverse sequence types, predominant being ST-15 (n = 4) and ST-16 (n = 4). Both mcr-1 and bla NDM were localised on plasmid and chromosome; mcr-1 was present on IncFII type and bla NDM on IncFIB and IncA/C type plasmids. Conclusions: Colistin resistance in K. pneumoniae was predominantly mediated by mcr-1. Co-existence of colistin, carbapenem, and other drug-resistant genes along with efflux pumps indicates towards enormous genomic plasticity in K. pneumoniae with ability to emerge as super-spreader of drug-resistance. [ABSTRACT FROM AUTHOR]

The occurrence and characterization of carbapenemase-producing Enterobacteriaceae from companion animals in Guangzhou, China, are investigated. Six isolates (2.3%, 6/257) were positive for blaNDM-5, that is, one Enterobacter cloacae, one Citrobacter freundii, and four Escherichia coli. Three E. coli isolates obtained from the same animal hospital were ST410 and showed identical pulse field gel electrophoresis pattern, resistance profiles, and resistance genes. blaNDM-5 was located on IncX3 (n = 5) and IncK2 (n = 1) plasmid, respectively. The presence of carbapenemase-producing Enterobacteriaceae among companion animals needs continued surveillance. [ABSTRACT FROM AUTHOR]

The emergence of carbapenem-resistant Acinetobacter calcoaceticus-baumannii complex (CRACB) in clinical environments is a significant global concern. These critical pathogens have shown resistance to a broad spectrum of antibacterial drugs, including carbapenems, mostly due to the acquisition of various β-lactamase genes. Clinical samples (n = 1985) were collected aseptically from multiple sources and grown on blood and MacConkey agar. Isolates and antimicrobial susceptibility were confirmed with the VITEK-2 system. The modified Hodge test confirmed the CRACB phenotype, and specific PCR primers were used for the molecular identification of blaOXA and blaNDM genes. Of the 1985 samples, 1250 (62.9%) were culture-positive and 200 (43.9%) were CRACB isolates. Of these isolates, 35.4% were recovered from pus samples and 23.5% from tracheal secretions obtained from patients in intensive care units (49.3%) and medical wards (20.2%). An antibiogram indicated that 100% of the CRACB isolates were resistant to β-lactam antibiotics and β-lactam inhibitors, 86.5% to ciprofloxacin, and 83.5% to amikacin, while the most effective antibiotics were tigecycline and colistin. The CRACB isolates displayed resistance to eight different AWaRe classes of antibiotics. All isolates exhibited the blaOXA-51 gene, while blaOXA-23 was present in 94.5%, blaVIM in 37%, and blaNDM in 14% of the isolates. The blaOXA-51, blaOXA-23, and blaOXA-24 genes co-existed in 13 (6.5%) isolates. CRACB isolates with co-existing blaOXA-23, blaOXA-24, blaNDM, blaOXA-51 and blaVIM genes were highly prevalent in clinical samples from Pakistan. CRACB strains were highly critical pathogens and presented resistance to virtually all antibacterial drugs, except tigecycline and colistin.

The advent of carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a significant challenge to public health, as carbapenems are typically employed as a last resort to treat nosocomial infections caused by such organisms, especially in intensive care units (ICUs). This study aims to characterize the CRKP isolated from patients admitted to the Zagazig University Hospitals (ZUHs) ICU in Egypt. About 56.2%, 41.0%, and 32.4% of the isolates indicated the presence of blaNDM, blaOXA-48, and blaKPC, respectively. Carbapenemase-encoding genes were found in many isolates, and blaNDM was the most predominant gene. Nevertheless, this situation has become a heavy burden in developing countries, including Egypt, and is associated with substantial morbidity, mortality, and increased healthcare expenses.

Wild and feral birds are known to be involved in the maintenance and dissemination of clinically-important antimicrobial-resistant pathogens, such as extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae. The aim of our study was to evaluate the presence of ESBL- and carbapenemase-producing Escherichia coli among wild and feral birds from Greece and to describe their antimicrobial resistance characteristics. In this context, fecal samples of 362 birds were collected and cultured. Subsequently, the antimicrobial resistance pheno- and geno-type of all the obtained E. coli isolates were determined. A total of 12 multidrug-resistant (MDR), ESBL-producing E. coli were recovered from eight different wild bird species. Eleven of these isolates carried a blaCTX-M-1 group gene alone or in combination with blaTEM and one carried only blaTEM. AmpC, fluoroquinolone, trimethoprim/sulfamethoxazole, aminoglycoside and macrolide resistance genes were also detected. Additionally, one carbapenemase-producing E. coli was identified, harboring blaNDM along with a combination of additional resistance genes. This report describes the occurrence of ESBL- and carbapenemase-producing E. coli among wild avian species in Greece, emphasizing the importance of incorporating wild birds in the assessment of AMR circulation in non-clinical settings.

Purpose: Carbapenemase-producing Enterobacteriaceae (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt and effective therapy. In order to quickly and comprehensively detect the five major families of carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like), colorimetric loop-mediated isothermal amplification (LAMP) was employed. Materials and Methods: Five sets of LAMP primers were designed, each of which can, respectively, amplify all the carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-"standard strains", including 1 blaNDM-1, 1 blaNDM-5, 1 blaNDM-6, 1 blaNDM-7, 2 blaIMP-4, 1 blaIMP-8, 2 blaKPC-2, 1 blaKPC-3, 1 blaKPC-4, 1 blaKPC-5, 1 blaKPC-6, 1 blaKPC-7, 1 blaOXA-48 and 1 blaOXA-181 carrier, and 1 blaVIM and blaOXA-244, 1 blaKPC-2 and blaIMP-4, 1 blaKPC-2 and blaVIM-1 and 1 blaKPC-2 and blaNDM-1-co-carriers, were used to establish a 25-microliter visual LAMP reaction system (kept at 65°C for 30 minutes in water bath). Color change from bright pink to yellow indicated positive amplification. In addition, 126 pre-verified clinical carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 65 CPE (23 blaNDM, 2 blaOXA-48-like, 1 blaKPC and blaVIM, 2 blaIMP, and 37 blaKPC carriers) and 61 non-CPE, were also detected. Results: With the lowest detection limit of 10 colony forming units (CFU) per reaction for LAMP and 103 CFU per reaction for PCR, the LAMP system demonstrated dramatically higher sensitivity while retaining the same specificity. Furthermore, we demonstrated concordant results between the two methods for the 126 clinical isolates. Conclusion: Therefore, LAMP could be used for rapid identification of the five major carbapenemase gene families in routine clinical laboratories. [ABSTRACT FROM AUTHOR]

Background: The therapeutic management of carbapenem-resistant Acinetobacter baumannii (CR-AB) represents a serious challenge to the public health sector because these pathogens are resistant to a wide range of antibiotics, resulting in limited treatment options. The present study was planned to investigate the clonal spread of CR-AB in a clinical setting. Methodology: A total of 174 A. baumannii clinical isolates were collected from a tertiary care hospitals in Lahore, Pakistan. The isolates were confirmed by VITEK 2 compact system and molecular identification of recA and blaOXA-51. Antimicrobial profile and the screening of carbapenem-resistant genes were carried out using VITEK 2 system and PCR, respectively. The molecular typing of the isolates was performed according to the Pasteur scheme. Results: Of the 174 A. baumannii isolates collected, the majority were isolated from sputum samples (46.5%) and in the intensive care unit (ICU, 75%). Among these, 113/174 (64.9%) were identified as CR-AB, and 49.5% and 24.7% harbored blaOXA-23 and blaNDM-1, respectively. A total of 11 (9.7%) isolates co-harbored blaOXA-51, blaNDM-1, and blaOXA-23. Interestingly, 46.9% of the CR-AB belonged to sequence type 2 (ST2; CC1), whereas 15.9% belonged to ST1 (CC1). All of the CR-AB isolates showed extensive resistance to clinically relevant antibiotics, except colistin. Conclusion: The study concluded CR-AB ST2 clone harboring blaOXA-23 and blaNDM-1 are widely distributed in Pakistan's clinical settings, which could result in increased mortality. Strict compliance with the National Action Plan on Antimicrobial Resistance is necessary to reduce the impacts of these strains. [ABSTRACT FROM AUTHOR]

Background: To explore the plasmid characteristics and transfer mechanisms of an extensive drug resistant (XDR) clinical isolate, Citrobacter portucalensis L2724hy, co-producing bla SFO-1 , bla NDM-1 , and bla KPC-2 ., Methods: Species confirmation of L2724hy was achieved through 16S rRNA sequencing and Average Nucleotide Identity (ANI) analysis. Antimicrobial susceptibility testing (AST) employed the agar dilution and micro broth dilution methods. Identification of resistance genes was carried out by PCR and whole-genome sequencing (WGS). Essential resistance gene locations were verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern hybridization experiments. Subsequent WGS data analysis delved into drug resistance genes and plasmids., Results: The confirmation of the strain L2724hy as an extensive drug-resistant Citrobacter portucalensis, resistant to almost all antibiotics tested except polymyxin B and tigecycline, was achieved through 16S rRNA sequencing, ANI analysis and AST results. WGS and subsequent analysis revealed L2724hy carrying bla SFO-1 , bla NDM-1 , and bla KPC-2 on plasmids of various sizes. The uncommon ESBL gene bla SFO-1 coexists with the fosA3 gene on an IncFII plasmid, featuring the genetic environment IS 26-fosA3 -IS 26-ampR-bla SFO-1 -IS 26 . The bla NDM-1 was found on an IncX3 plasmid, coexisting with bla SHV-12 , displaying the sequence IS 5 -IS 3000 -IS 3000 -Tn 2-bla NDM-1 - ble-trpF-dsbD-cutA-gros-groL , lacking IS Aa125 . The bla KPC-2 is located on an unclassified plasmid, exhibiting the sequence Tn 2-tnpR -IS Kpn27-bla KPC-2 -IS Kpn6-korC . Conjugation assays confirmed the transferability of both bla NDM-1 and bla KPC-2 ., Conclusion: We discovered the coexistence of bla SFO-1 , bla NDM-1 , and bla KPC-2 in C. portucalensis for the first time, delving into plasmid characteristics and transfer mechanisms. Our finding highlights the importance of vigilant monitoring of drug-resistance genes and insertion elements in uncommon strains., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Guo et al.)

This study aimed to investigate the dissemination and characteristics of blaKPC, blaNDM, blaOXA-48-like, blaIMP, and blaVIM among the carbapenem-resistant Enterobacteriaceae (CRE) strains isolated from adult and children patients. A total of 935 non-duplicate CRE strains were collected from 36 hospitals in 24 provinces or cities across China from 2016 to 2018. Antimicrobial susceptibility testing was performed by broth microdilution method and carbapenemase genes blaKPC, blaNDM, blaOXA-48-like, blaIMP, and blaVIM were screened by PCR and confirmed by DNA sequencing. Overall, carbapenemases were produced in 97.4% (911/935) of CRE strains, including KPC-2 (51.6%, 482/935), NDM (35.7%, 334/935), and OXA-48-like carbapenemases (7.3%, 68/935). Overall, the most prevalent carbapenemase gene was blaKPC-2 among Klebsiella pneumoniae (64.6%, 457/709) and the CRE strains isolated from adult patients (70.3%, 307/437), and blaNDM among Escherichia coli (96.0%, 143/149) and the CRE strains from children (49.0%, 247/498). The blaOXA-232-positive carbapenem-resistant K. pneumoniae (9.3%, 66/709) were all isolated from children. Sixteen strains were positive for blaIMP and 9 strains produced multiple carbapenemases. No strain was positive for blaVIM. Most of the CRE strains (>90%) were resistant to cephalosporins and carbapenems, more than half (>50%) were resistant to aminoglycosides and fluoroquinolones, but the majority (95.8 and 98.4%) were susceptible to polymyxin B and tigecycline. Ceftazidime-avibactam showed excellent in vitro activity against blaKPC-2 and blaOXA-48-like positive strains (100% susceptible). In China, KPC-2, NDM, and OXA-48-like carbapenemases were predominant among CRE clinical isolates. The most prevalent carbapenemase gene was blaKPC-2 among K. pneumoniae isolates from adult patients, and blaNDM among E. coli isolates from children.

Somayeh Kiaei,1,2 Mohammad Moradi,2 Hossein Hosseini-Nave,2 Mahsa Ziasistani,3 Davood Kalantar-Neyestanaki2 1Department of Microbiology and Virology, Student Research Committee, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 2Department of Microbiology and Virology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran; 3Pathology and Stem Cell Research Center, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran Introduction: The emergence and spread of Klebsiella pneumoniae strains resistant to multiple antimicrobial agents are considered as a serious challenge for nosocomial infections. Materials and methods: In this study, 175 nonrepetitive clinical isolates of K. pneumoniae were collected from hospitalized patients in Kerman, Iran. Extended-spectrum β-lactamases (ESBLs), AmpC, and carbapenemase-producing isolates were recognized by phenotypic methods. The resistance genes including efflux pumps oqxA/oqxB, 16S rRNA methylase, ESBL, AmpC, and carbapenemase were detected by PCR-sequencing method. Molecular typing was performed by enterobacterial repetitive intergenic consensus-PCR and multilocus sequence typing methods among blaNDM-positive isolates. Results: Thirty-seven (21.14%) isolates along with sequence types (STs): ST43, ST268, ST340, ST392, ST147, and ST16 were harbored blaNDM. ST43 in 2015 and ST268 during 2016–2017 were the most frequent STs among New Delhi metallo-beta-lactamase (NDM)-positive isolates. We found the distribution of some isolates with blaNDM, blaCTX-M, blaSHV, blaOXA, blaTEM, blaCMY, rmtC, and oqxA/oqxB. Enterobacterial repetitive intergenic consensus-PCR represented seven clusters (A–G) plus four singletons among NDM-positive isolates. This study provides the first report of blaNDM-1-positve K. pneumoniae along with ST268 as well as the spread of nosocomial infections with six different STs harboring blaNDM-1 and other resistance genes in hospital settings especially neonatal intensive care unit. Conclusion: The dissemination of various clones of NDM-producing K. pneumoniae can contribute to increase the rate of their spread in health care settings. Therefore, molecular typing and detection of resistance genes have an important role in preventing and controlling infection by limiting the dissemination of multidrug-resistant isolates. Keywords: blaNDM, 16S rRNA methylase, MLST, ERIC-PCR

New Delhi metallo-β-lactamase (blaNDM) acquisition by Gram-negative bacteria is a primary concern due to its broad-host-range distribution. This study investigated two potential in-vivo horizontal gene transfers (HGTs) of blaNDM between Enterobacterales and Pseudomonas aeruginosa, initially indicated by polymerase chain reaction. Whole-genome sequencing showed independent parallel acquisition of two different blaNDM variants (NDM-1 and NDM-5) in P. aeruginosa and Enterobacterales, respectively. The data show that short-read sequencing provides the necessary resolution to confirm or dispute HGT by the comparison of genetic elements surrounding the gene of interest, and thus provide a timely response to potential outbreaks. [ABSTRACT FROM AUTHOR]

Carbapenem resistance among Enterobacteriaceae is a major concern that is increasingly reported worldwide. The objective of this study is to determine the incidence of carbapenem resistance as well as to investigate for carbapenemase-encoding genes among Enterobacteriaceae clinical isolates from cancer patients at different cancer institutes in Egypt. This determination was a cross-sectional study with a total of 135 clinical isolates collected over a period of 1 year. All isolates were sub-cultured on ChromID agar and subjected to phenotypic and molecular detection of carbapenemases. Most of the Enterobacteriaceae isolates were MDR with high resistance rates against tested antimicrobials. Overall, the results of PCR assays revealed that 89.62% (121/135) of isolates harbored one or more of the carbapenemase-encoding genes, while phenotypic assays revealed the production of carbapenemases in 68.88% (93/135) of isolates. BlastN analysis against the non-redundant genome sequences available in the GenBank database revealed that the blaNDM-1 gene was the most prevalent genotype of carbapenemases in 93/135 (68.88%), followed by blaOXA-48 44/135 (32.59%), blaOXA-23 42/135 (31.11%), and blaKPC-2 2/135 (1.48%). Klebsiella pneumoniae isolates harbored the highest number of carbapenemase-encoding genes 34/121 (28.09%). The high prevalence of carbapenemases and/or their encoding genes among MDR Enterobacteriaceae bacteria in Egypt is alarming, thus, the management of serious infections caused by Enterobacteriaceae, particularly in cancer patients will be challenging to clinicians. Carbapenemase blaNDM genotype is emerging in cancer healthcare settings in Egypt, which could be the cause of the current increase in carbapenemase-producing Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

To characterize carbapenemase-producing Enterobacteriaceae (CPE) from vegetables, 185 vegetable samples were collected from farmers' markets and screened for carbapenem-resistant Enterobacterales (CRE). Two Citrobacter freundii and seven Escherichia coli isolates (ST10, ST93, ST206, ST746, ST6736, ST7058, and ST7458) recovered from eight (4.32%) vegetable samples were positive for bla NDM (one bla NDM-1 and eight bla NDM-5). The bla NDM-5 gene was identified on three plasmid types, including IncX3 (n = 2), IncHI2 (n = 4, three ST3-type and one ST2-type), and phage-like p0111 type plasmid (n = 1), while the bla NDM-1 gene was located on IncX3 plasmids. Conjugation experiments confirmed the transferability of all bla NDM -positive plasmids except for the p0111-type plasmid pHNGDSC2239PM-1. Subsequent genomic analysis revealed p0111 and IncHI2/ST2 as two novel bla NDM-5 carrying plasmids vehicles. Notably, the bla NDM-5 -carrying IncHI2/ST3 plasmids exhibited high structural similarity to plasmids isolated from diverse sources, and were extensively disseminated in China. The acquisition of the bla NDM-5 gene in the p0111-type plasmid pHNGDSC2239PM-1 was mediated by IS 26 -flanked pseudocomposite transposons. The capture of bla NDM-5 by the IncHI2/ST2 plasmid pHNBY4G-1 was likely attributed to the homologous recombination events mediated by IS Kpn19 and ΔTn 2. Overall, the emergence of bla NDM-5 in IncHI2/ST2 and p0111 plasmids, coupled with the widespread distribution of bla NDM-5 -bearing IncHI2/ST3 multidrug-resistance plasmids in animals and foods, indicates a potential for wider dissemination of bla NDM-5 , which necessitates attention. • Two novel types of bla NDM-5 -bearing plasmid, p0111 and IncHI2/ST2, were emerged. • Wide spread of similar ST3-type IncHI2 plasmids carrying bla NDM-5 across different niche was detected in China. • A tet (X4)- and bla NDM -coharboring E. coli was firstly identified in vegetables. [ABSTRACT FROM AUTHOR]

Escherichia coli is one of the important reservoirs of antimicrobial resistance genes (ARG), which often causes food-borne diseases and clinical infections. Contamination with E. coli carrying clinically important antimicrobial resistance genes in retail meat products can be transmitted to humans through the food chain, posing a serious threat to public health. In this study, a total of 330 E. coli strains were isolated from 464 fresh meat samples from 17 food markets in China, two of which were identified as enterotoxigenic and enteropathogenic E. coli. Whole genome sequencing revealed the presence of 146 different sequence types (STs) including 20 new STs, and 315 different clones based on the phylogenetic analysis, indicating the high genetic diversity of E. coli from retail meat products. Antimicrobial resistance profiles showed that 82.42 % E. coli were multidrug-resistant strains. A total of 89 antimicrobial resistance genes were detected and 12 E. coli strains carried clinically important antimicrobial resistance genes bla NDM-1 , bla NDM-5 , mcr-1 , mcr-10 and tet (X4), respectively. Nanopore sequencing revealed that these resistance genes are located on different plasmids with the ability of horizontal transfer, and their genetic structure and environment are closely related to plasmids isolated from humans. Importantly, we reported for the first time the presence of plasmid-mediated mcr-10 in E. coli from retail meat. This study revealed the high genetic diversity of food-borne E. coli in retail meat and emphasized their risk of spreading clinically important antimicrobial resistance genes. • A total of 330 E. coli strains were isolated from retail meat in 17 food markets in China. • WGS revealed that there were 146 different STs including 20 novel STs, with high genetic diversity. • A total of 89 antimicrobial resistance genes were detected, including bla NDM , mcr and tet (X4). • Ten plasmids carrying clinically important antimicrobial resistance genes were characterized. • It is the first time to report plasmid-mediated mcr-10 in E. coli from retail meat. [ABSTRACT FROM AUTHOR]

The increasing incidence of carbapenem-resistant Enterobacteriaceae (CRE) - mediated hospital infections in China prompted a need to investigate the genetic basis of emergence of such strains. A nationwide survey was conducted in China covering a total of 1105 CRE strains collected from 25 geographical locales with results showing that acquisition of two carbapenemase genes, blaKPC-2 and blaNDM, was responsible for phenotypic resistance in 90% of the CRE strains tested (58% and 32% respectively), among which several major strain types, such as ST11 of K. pneumoniae and ST131/ST167 of E. coli, were identified, suggesting that dissemination of specific resistant clones is mainly responsible for emergence of new CRE strains. Prevalence of the fosA3 gene which mediates fosfomycin resistance, was high, while the colistin resistance determinant mcr-1 was rarely present in these isolates. Consistently, the majority of the blaNDM-bearing plasmids recoverable from the test strains belonged to IncX3, which contained a common core structure, blaNDM-blaMBL-trpF. Likewise, the core structure of ISKpn27-blaKPC-2-ISKpn2 was observed among plasmids harboring the blaKPC-2 gene, although they were genetically more divergent. In conclusion, the increasing prevalence of CRE strains in China is attributed to dissemination of conservative mobile elements carrying blaNDM or blaKPC-2 on conjugative and non-conjugative plasmids.

The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) is a new threat to healthcare. In this study, we analyzed nine CR-hvKp isolates of different sequence-types (ST) recovered from patients with nosocomial infections in two hospitals in Saint Petersburg. Whole-genome sequencing showed that eight of them harbored large mosaic plasmids carrying resistance to carbapenems and hypervirulence simultaneously, and four different types of hybrid plasmids were identified. BLAST analysis showed a high identity with two hybrid plasmids originating in the UK and Czech Republic. We demonstrated that hybrid plasmids emerged due to the acquisition of resistance genes by virulent plasmids. Moreover, one of the hybrid plasmids carried a novel New Delhi metallo-beta-lactamase (NDM) variant, differing from NDM-1 by one amino acid substitution (D130N), which did not provide significant evolutionary advantages compared to NDM-1. The discovery of structurally similar plasmids in geographically distant regions suggests that the actual distribution of hybrid plasmids carrying virulence and resistance genes is much wider than expected.

The study aimed to determine the prevalence of bla-genes (blaVIM, blaIMP and blaNDM) encoding MBLs among the isolates of P. aeruginosa, which were recovered from various clinical samples from hospitalized patients in Diyala hospitals. This study was carried out during the period from February 2018 to August 2018. Out of 326 specimens, 81 isolates of P. aeruginosa were recovered. All isolates were tested toward the different class of clinically important antipseudomonal agents(18) by using agar diffusion method. The results of resistance were as following: piperacillin 74.07, ticarcillin 85.18%, Amoxicillin-Clavulanic acid 93.82%, Ticarcillin/clavulanic acid 71.60%, ceftriaxone 87.65%, cefotaxime 85.18% and ceftazidime 75.30%, cefepime 80.24%, streptomycin 90.12%, gentamicin 85.18%, tobramycin 65.43%, amikacin 56.79%, ciprofloxacin 67.90%, levofloxacin 72.83%, oflaxacin 69.13%, Aztreonam 50.61%, Imipenem 11.11% and Meropenem 23.45%.In this investigation, antibiotic susceptibility testing of the P. aeruginosa isolates showed that 20(24.69%), 25(30.8%), 27(33.33%), and 9(11.11%) of the isolates were MDS, MDR, XDR and PDR, respectively. Based on the results from susceptibility testing, 19 (23.45%) of P. aeruginosa isolates were found to be resistant to at least one of carbapenems. Out of 19 of carbapenem resistant P. aeruginosa isolates, 16 (84.21%) were found to produce MBL. Among 16 phenotypic Metallo β-lactamase isolates the results achieved by using PCR revealed that 9 (56.25%) isolates have blaVIMgenes, while 4 (25%) isolates carried blaNDM genes. No blaIMP was detected among carbapenem resistant strains in this study. [ABSTRACT FROM AUTHOR]

The New Delhi Metallo-β-lactamase (NDM) producing Enterobacteriaceae is spreading worldwide. Although the blaNDM gene has been identified in animal associated Enterobacteriaceae isolates in many countries, little is known about its occurrence in animal products in Pakistan. In this study, 13 Escherichia coli isolates were collected from chicken meat samples in Pakistan. Two isolates, 15978 and C4109, exhibited reduced susceptibility (MIC ≥1 μg/mL) to imipenem, and carried blaNDM-5 and blaNDM-7 gene, respectively. Whole-genome sequencing and Oxford Nanopore MinION sequencing revealed that 15978 and C4109 belonged to ST156 and ST167, respectively. blaNDM-7 was carried by an IncX3 plasmid that has disseminated worldwide, whereas blaNDM-5 was located on an F36: A-: B32 plasmid, which shared high identity with two plasmids carried by E. coli isolates from other countries (one from a patient in Canada). To the best of our knowledge, this is the first report characterizing blaNDM-carrying plasmids from chicken meat samples in Pakistan. The dissemination of almost identical blaNDM-5-bearing F36:A-:B32 and blaNDM-7-bearing IncX3 plasmids in different countries highlights the importance of international trade and travel in the spread of antimicrobial resistance strains and plasmids worldwide. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: The carbapenemase-encoding genes, blaNDM- and blaOXA-48-like, confer resistance to all the known beta-lactams and are encountered along with other beta-lactamase-encoding genes and/or 16S ribosomal RNA (rRNA)-methylating genes. The co-occurrence of blaNDMand blaOXA-48-likeon a single plasmid is a rare occurrence. AIM AND OBJECTIVE: The purpose of the study was to characterize the plasmids in Klebsiella pneumoniae isolates producing 16S rRNA methyltransferase along with blaNDM, blaOXA-48-like, and other resistance encoding genes. MATERIALS AND METHODS: One-hundred and seventeen K. pneumoniae clinical isolates which were resistant to aminoglycosides were collected. Polymerase chain reaction-based screening for 16S rRNA methyltransferase genes armA, rmtB, and rmtC; carbapenamase genes blaNDM, blaOXA-48-like, blaIMP,blaVIM,and blaKPC; and other resistance genes such as blaTEM,blaSHV,blaCTX-M, and qnr (A, B, and S) determinants acc (6') Ib-cr was performed. Conjugation experiment was carried out for seven isolates that anchored blaNDMand blaOXA-48-likealong with any one of the 16S rRNA methyltransferases. The plasmid-based replicon typing for different plasmid-incompatible (Inc) group was performed on the conjugatively transferable plasmids. RESULTS: Among the 16S rRNA methyltransferases, armA was more predominant. blaNDMand blaOXA-48-likewere present in 56 (47.86%) and 22 (18.80%) isolates, respectively. Out of seven isolates which were conjugatively transferable, only four had blaNDMand blaOXA-48-likeon the same plasmid and they belonged to Inc N and A/C replicon. Three isolates co-harbored 16S rRNA methyltransferases armA, rmtB, and rmtC, and out of the them, one isolate harbored two 16S rRNA methyltransferases armA and rmtB, on the single-plasmid replicon A/C. CONCLUSION: This is the first report revealing the coexistence of blaNDMand blaOXA-48-likeco-harboring two 16S rRNA methylases on a single conjugative plasmid replicon belonging to incompatibility group A/C. [ABSTRACT FROM AUTHOR]

Multidrug resistance associated with pigs not only affects pig production but also threatens human health by influencing the farm surrounding and contaminating the food chain. This paper focused on the occurrence and prevalence of high-risk resistance genes (using bla NDM and mcr-1 as marker genes) in two Chinese swine farms, and investigated their fate and seasonal changes in piggery wastewater treatment systems (PWWTSs). Results revealed that bla NDM and mcr-1 were prevalent in both confined swine farms, and even prevailed through various processing stages of PWWTSs. Moreover, the abundance of bla NDM and mcr-1 in winter was higher than that in summer, with 0.01–1.01 logs variation in piggery wastewater. Of concern is that considerable amounts of bla NDM and mcr-1 were present in final effluent that is applied to farmland (up to 102–104 copies/mL), raising the risk of propagation to indigenous bacteria. Worse still, those pig-derived isolates harboring the bla NDM / mcr-1 gene were confirmed to spread multidrug resistance to other bacteria, which further increased their dissemination potential in agricultural environment. This study highlights the prevalence of bla NDM and mcr-1 in swine farms, meanwhile, also emphasizes the necessary to mitigate the release and propagation of these high-risk genes from swine farms following land fertilization and wastewater usage. Unlabelled Image • The level and fate of high-risk bla NDM and mcr-1 were addressed in swine farms. • This paper demonstrated the prevalence of bla NDM and mcr-1 in swine farms. • Bla NDM and mcr-1 in swine waste were more abundant in winter than in summer. • Swine waste is an important reservoir for high-risk bla NDM and mcr-1 genes. [ABSTRACT FROM AUTHOR]

Background: Emergence of blaKPC and blaNDM co-producing Klebsiella pneumoniae strains have led to the limited therapeutic options for clinical treatment. Understanding the diversity and frequency of resistance and virulence genes of these isolates is of great significance. Purpose: The aim of this study is to research the diversity and frequency of resistance and virulence genes in the blaKPC and blaNDM co-producing Klebsiella pneumoniae strains. Methods and Results: In this study, 117 K. pneumonia strains were isolated from China, and among of which, 24 were found to be blaKPC and blaNDM co-producing with significant resistance against almost all the commonly used antibiotics. Additionally, 4 strains were hypermucoviscous and 8 showed high serum resistance. Overall, blaSHV, blaCTX-M, tetA and sul1 resistance genes found in 100% of the isolates, followed by blaTEM (95.8%), oqxA/B (91.7%), qnrB (87.5%), aac(6')Ib-cr (83.3%), blaDHA (79.2%), rmtB (66.7%), qnrS (54.2%), cat(54.2%), floR (50.0%), sul2 (45.8%) cmlA (20.8%)andblaCMY (8.33%), respectively. What' more, seven blaCTX-M subtypes [blaCTX-M-14 (n=18), blaCTX-M-3(n=11), blaCTX-M-65 (n=4), blaCTX-M-15 (n=3), blaCTX-M-28 (n=2), blaCTX-M-55 (n=2), blaCTX-M-22 (n=1)] and six blaSHV subtypes [blaSHV-12(n=16), blaSHV-11 (n=4), blaSHV-2a(n=1), blaSHV-1(n=1), blaSHV-38(n=1) and blaSHV-28(n=1)] were detected. The frequency of virulence genes was as follows: 100% for entB, ybtS and irp, 95.8% for mrkD, 91.66% for fimH, 79.2% for iutA, 62.5% for iroBCDE, aerobactin and kfu, 66.7% for allS, 45.8% for wcaG, 37.5% for rmpA, 20.8% for pagO and 16.7% for magA. Conclusion: From this study, we concluded that the blaKPC and blaNDM co-producing Klebsiella pneumoniae strains have a high diversity and frequency of resistance and virulence genes. This study may offer hospitals important information about the control of infections caused by blaKPC and blaNDM co-producing Klebsiella pneumoniae. [ABSTRACT FROM AUTHOR]

Literature regarding multidrug resistant (MDR) carbapenemase-producing Escherichia coli (CPE) recovered from food-producing animals is lacking from Pakistan. Here, we report on the isolation and characterization of MDR-CPE recovered from retail-poultry meat in Khyber Pakhtunkhwa Pakistan. A total of 101 retail poultry-meat samples were screened for Escherichia coli and tested against 17 different antibiotics through disc diffusion test to define MDRs. MDR isolates were genotyped by PCR for carbapenemase-encoding elements. Finally, integron typing, insertion sequence common region 1 (ISCR1) and its association with carbapenemaseencoding genes was established through PCR. Of the 33 E. coli isolates, 28 (Phylogroup B2=39.2% (11/28), A=32.1 % (9/28) and group D=28.5% (8/28) were MDR displaying resistance to cefotaxime, tetracycline, ampicillin and trimethoprimsulfamethoxazole, while, 96.4, 92.8, 89.2 and 85.7% were also found resistant to colistin-sulphate, enrofloxacin, doxycycline and ciprofloxacin, respectively. The blaVIM was predominantly identified in 8 (28.5%) isolates followed by blaNDM 4/28 (14.2%) and 2 isolates were carrying a combination of blaVIM+blaNDM. Class-1 integron was detected in 21 (75%), class-2 and class-3 integrons were detected in 25% and 7.1%, respectively. ISCR1 element was identified among 11 isolates (39.28%) that was found associated with blaVIM gene among 3 isolates (27.25%) suggesting involvement in its mobilization. We report the incidence of E. coli carrying carbapenemase-encoding genes located on the plasmids carrying integron 1 recovered from retail-poultry-meat in Mardan, Pakistan. [ABSTRACT FROM AUTHOR]

Background & objectives: The global spread of carbapenem-resistant Enterobacteriaceae (CRE) is an emerging clinical problem. Hence, in this study, the plausible role of extended-spectrum beta-lactamases (ESBLs)/carbapenemases, OmpC/Ompk36, acrB and their combinations was explored among CRE. Methods: The minimum inhibitory concentration (MIC) of meropenem, enzyme-phenotypes (ESBLs/ IR and metallo-beta-lactamase (MBL)/non-MBL carbapenemase), genotypes (blaTEM, blaSHV and blaCTX-M; blaNDM and blaVIM; blaKPC and blaOXA-48-like variants), acrB and outer membrane protein (OMP) expressions were analyzed with a total of 101 non-duplicate clinical isolates, obtained from various samples of patients visiting two tertiary care units of Eastern India during May 2013 - October 2016. This included Escherichia coli (n=36) and Klebsiella pneumoniae (n=65), categorized into two groups, namely Group I (resistant to all carbapenems; n=93; E. coli=34 and Klebsiella spp.=59) and Group II (non-resistant to all the carbapenems; n=8; E. coli=2 and Klebsiella spp.=6). Results: Though 88.17 per cent of Group I isolates exhibited ESBL property, the presence of carbapenemase activity (70.96%) and that of blaNDM gene (42/66: 63.63%) indicated their contributions towards the emergence of CRE. Further, porin loss and/or efflux pump activation among ESBL/carbapenemase-producing isolates heightened the MIC of meropenem from 64 to 256 mg/l (range exhibited by only ESBL/carbapenemase-producing isolates) to >256 mg/l. Interpretation & conclusions: These findings implied the major contribution of porin loss and/or efflux pump activation over the presence of ESBLs/carbapenemases in imparting carbapenem resistance in pathogenic bacteria. [ABSTRACT FROM AUTHOR]

Abstract Carbapenem-resistant Enterobacteriaceae strains as a new serious threat for the public health have been increasingly reported worldwide. In this study, one multi-resistant Escherichia coli strain Z SH6 which co-carried bla KPC-2 , bla NDM-5 and bla CTX-M , was isolated from human blood sample. By using plasmid conjugation experiments, ZSH6 was found to harbor three plasmids carrying the bla NDM-5 gene, the bla KPC-2 and bla CTX-M gene, respectively. Whole-genome sequencing of Z SH6 yielded 122 scaffolds of chromosomal DNA and three circular plasmids including pZSH6- bla KPC-2 (46,319 bp), pZSH6- bla NDM-5 (46,161bp) and pZSH6- bla CTX-M (184,723). The isolate was classified to Sequence Type 2 and to the O89: H10 serotype. The results of genome analyses revealed that Z SH6 carried three virulence factors (capU , gad and iss) and twenty resistance genes [ bla KPC-2 bla NDM-5 , bla CTX-M-3 , bla CTX-M-65 , bla TEM-1 , floR , tet(A) , tet(B) , dfrA17 , aadA5 , sul1 , mdf(A) , mph(A) , erm(B) , aph(3′)-Ia , aph(3′)-Ib , aph(4)-Ia , aph(6)-Id , aac(3)-Iva , aac(3)-IId ]. Therefore, the co-existence of such a large number of resistance genes in multiple plasmids making Z SH6 highly resistant to almost all kinds of commonly used antibiotics, and brings a serious challenge for resistance control and clinical treatment of infections caused by this bacterium. Highlights • O89: H10 E.coli Z SH6 strain co-carried a large number resistance genes. • E.coli Z SH6 strain co-carried three plasmids. • Three plasmids were pZSH6- bla KPC-2 , pZSH6- bla NDM-5 and pZSH6- bla CTX-M. [ABSTRACT FROM AUTHOR]

Large cointegrate plasmids recruit genetic features of their parental plasmids and serve as important vectors in the spread of antibiotic resistance. They are now frequently found in clinical settings, raising the issue of how to limit their further transmission. Here, we conducted evolutionary research of a large blaNDM-positive cointegrate within Escherichia coli C600, and discovered that adaptive evolution of chromosome and plasmid jointly improved bacterial fitness, which was manifested as enhanced survival ability for in vivo and in vitro pairwise competition, biofilm formation, and gut colonization ability. From the plasmid aspect, large-scale DNA fragment loss is observed in an evolved clone. Although the evolved plasmid imposes a negligible fitness cost on host bacteria, its conjugation frequency is greatly reduced, and the deficiency of anti-SOS gene psiB is found responsible for the impaired horizontal transferability rather than the reduced fitness cost. These findings unveil an evolutionary strategy in which the plasmid horizontal transferability and fitness cost are balanced. From the chromosome perspective, all evolved clones exhibit parallel mutations in the transcriptional regulatory stringent starvation Protein A gene sspA. Through a sspA knockout mutant, transcriptome analysis, in vitro transcriptional activity assay, RT-qPCR, motility test, and scanning electron microscopy techniques, we demonstrated that the mutation in sspA reduces its transcriptional inhibitory capacity, thereby improving bacterial fitness, biofilm formation ability, and gut colonization ability by promoting bacterial flagella synthesis. These findings expand our knowledge of how cointegrate plasmids adapt to new bacterial hosts., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

Non-typhoidal Salmonella (NTS) are foodborne pathogens that are responsible for self-limiting gastroenteritis in humans. The present study aims at the molecular characterisation and comparative genomics of Salmonella enterica serovar Senftenberg strain P5558 isolated from the pus samples of a patient suffering from stump infection. The isolate was subjected to serotyping and antimicrobial susceptibility test to understand the phenotypical characteristics. Whole genome sequencing (WGS) was carried out and comparative genomics using computational tools showed the antimicrobial resistance and virulence gene profile of the isolates from the genome sequence data. Typing experiments confirmed that the isolate belong to S. Senftenberg with sequence type ST14. Resistance against β-lactams is associated with the presence of blaTEM-1, blaOXA-9, blaCMY-2 and blaNDM-1 genes. Similarly resistance to aminoglycoside was associated with five aminoglycoside modifying enzymes aac(6ʹ)-Ia, aac(6ʹ)-Ib, aph(3ʹ)-Ib, aph(6ʹ)-Ib and ant(3ʹʹ)-Ia, sulfonamide with sul-1 and sul-2 and chloramphenicol with florR gene. Substitutions in gyrA (S83Y, D87G) and parC (S80I) genes found to be the reason for fluoroquinolone resistance. The plasmid profiling showed the isolate has four resistance plasmids in which plasmid p5558-NDM (IncA/C) harbours major resistance genes including blaNDM-1 and blaCMY-2. Determination of virulence gene profile revealed that the genome carries all major Salmonella pathogenicity islands and virulence factors. From our findings it is clear that the isolate possess characteristic pathogenicity islands (SPI 1-6, 13, 14), major virulence factors and acquired resistance genes. Comparative analysis suggests the evolution and distribution of the MDR gene encoding plasmids in NTS. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Enterobacteriaceae (CRE) have evolved into a major challenge to antibiotic therapy worldwide. The problem is more confounding when wider dissemination of CRE occurs in the community and the environment. In this study, six blaNDM-harboring Enterobacteriaceae, four Klebsiella pneumoniae, and two Escherichia coli, isolated from seafood, were characterized with respect to their antibiotic resistance and the genetic factors responsible for these resistances. The isolates were resistant to all β-lactam antibiotics, quinolones, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline and were susceptible to colistin, polymixin B, fosfomycin, and tigecycline. Four isolates harbored New Delhi metallo β-lactamase (blaNDM-5 genes, while one isolate each harbored blaNDM-1 and blaNDM-2 genes, respectively. The blaNDM genes in all the isolates were flanked by an upstream, truncated ISAba125, and downstream bleMBL-trpF genes. Conjugation experiments showed that the NDM plasmids were readily transmissible. Further, the two blaNDM-positive E. coli isolates belonged to the multidrug-resistant ST131 clone. This study highlights the growing danger of seafood as carriers of multidrug-resistant bacteria leading to their wider dissemination in the community. [ABSTRACT FROM AUTHOR]

Abstract A multidrug resistant isolate, identified as Citrobacter amalonaticus using MALDI-TOF MS and confirmed by genomic analysis, was recovered from a pediatric patient in a hospital from Buenos Aires, Argentina. By whole-genome sequencing a total of 16 resistance genes were detected, including bla NDM-1 and mcr-1.5. To the best of our knowledge this is the first description of these two genes together in a clinical isolate of the Citrobacter genus. Highlights • Multidrug resistant Citrobacter amalonaticus clinical isolate • C. amalonaticus harboring bla NDM-1 and mcr-1.5 genes • Specie identification was performed using MALDI-TOF MS and confirmed by sequencing. [ABSTRACT FROM AUTHOR]

Carbapenems are administered as the final drug of choice for treating complicated nosocomial infections caused by multidrug-resistant Acinetobacter baumannii strains. It is currently a worldwide issue that metallo-β-lactamases (MBLs) as carbapenem-hydrolyzing enzymes are one of the major drug resistance mechanisms. This investigation is thus aimed to assess the prevalence and characterize the MBL-producing strains of A. baumannii both by phenotypic assays and by genotypic characterization. A total of 73 isolates of A. baumannii were phenotypically and genotypically characterized from patients (N = 1,000) with severe urinary tract infection. Tested strains were subjected to double disc synergy testing (DDST) by Kirby–Bauer disc diffusion method with imipenem (IMP) and IMP/EDTA combination discs. Plasmid DNA was molecularly screened for MBL-encoding bla IMP, bla VIM, bla GIM, and bla NDM genes by PCR for the genetic relatedness of the MBL genes with carbapenem resistance. Carbapenem resistance profile showed 100%, 45%, and 49% non-susceptibility against imipenem, doripenem, and meropenem, respectively. Altogether 42.46% (n = 31) of the isolates showed MBL production upon double disc phenotypic test with IMP and IMP/EDTA discs. The bla VIM and bla GIM were detected in 34.24% (n = 25) and 16.43% (n = 12) of the isolates, respectively, while the co-occurrence of bla VIM and bla GIM was 2.73% among the isolates. DDST-positive isolates showed 21.19% and 9.58% strains positive for bla VIM and bla GIM, respectively, whereas 1.36% of the strains for both genes. None of the strains yielded bla IMP and bla NDM genes. The findings of this study showed prevalence of carbapenem resistance among A. baumannii from urine samples and the frequency of bla VIM and bla GIM. [ABSTRACT FROM AUTHOR]

In recent years, the bla NDM gene, which mediate resistance to carbapenems, has disseminated all over the world, and has also been detected in animals. Understanding the dissemination and accumulation of antibiotic resistance genes (ARGs) in a human-impacted environment is essential to solve the food safety problems caused by antibiotics. In this study, two strains of carbapenem bacteria carrying bla NDM were screened from 244 strains isolated from two T. sinensis farms in Zhejiang province, China. After their plasmids were isolated and sequenced, their structure and gene environment were analyzed and the mechanism of bla NDM gene transfer was explored. The study measured the fitness cost of plasmids carrying different bla NDM subtypes by four biological characteristics experiments. The results showed that the fitness cost of IncC plasmid carrying bla NDM-1 was higher than that of IncX3 plasmid carrying bla NDM-5. Furthermore, the real-time PCR showed that the decrease of transcription level of fitness-related genes lead to the different fitness cost of plasmids carrying different bla NDM subtypes. Fitness of many bla NDM -harboring plasmids enhanced the further dissemination of this gene and increase the risk of bla NDM gene spreading in aquatic environment, and thus further investigation of carbapenem-resistant bacterias among food animals are in urgent need. • ISAba125 is the main mobile element to promote horizontal transmission of bla NDM. • The fitness cost of plasmids carrying different bla NDM subtypes was measured by biological characteristics test. • Different fitness costs will lead to different communication capabilities. • Exploring the reasons for the difference of fitness cost from the transcription level. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance poses a great challenge to clinicians leaving very limited treatment options available. A panel of carbapenem-resistant bacterial isolates was selected based on Rapidec Carba NP test from a total of 900 samples, which were collected from different specialities hospitals of Kanpur, India. Carba NP-positive isolates were screened for carbapenemases, extended-spectrum beta-lactamases (ESBLs), quinolone resistance, and 16s methyltransferase genes. Presence of diverse blaNDM ( blaNDM-1 and blaNDM-5) were detected and horizontal transferability was determined by transformation and conjugation assay. Elimination of blaNDM-1 and blaNDM-5 harboring plasmid was done by treating the isolates with sodium dodecyl sulfate. The transcriptional response of blaNDM-1 and blaNDM-5 under the exposure of imipenem, meropenem, and ertapenem stress was determined by quantitative real-time polymerase chain reaction. blaNDM harboring isolates were found to be horizontally transferable through IncFrepB and K type plasmid and could be successfully eliminated after the single treatment with sodium dodecyl sulfate. A distinct pattern of transcriptional response was observed for blaNDM-1 and blaNDM-5 under the pressure of carbapenem antibiotics where an upregulated expression of both blaNDM-1 and blaNDM-5 was observed. Minimum inhibitory concentration (MIC) results revealed that blaNDM harboring strains showed a high MIC range against imipenem, meropenem, ertapenem, cefepime, and aztreonam. Thus, prudent action should be taken to control the spread of these multidrug-resistant determinants or at least slowing down their lateral transfer in the hospital settings. [ABSTRACT FROM AUTHOR]

Members of the family Enterobacteriaceae include several human pathogens that can be acquired through contaminated food and water. In this study, the incidence of extended spectrum β-lactamase (ESBL)-producing enterobacteria was investigated in fresh seafood sold in retail markets. The ESBL-positive phenotype was detected in 169 (78.60%) isolates, with Escherichia coli being the predominant species (53), followed by Klebsiella oxytoca (27), and K. pneumoniae (23). More than 90% of the isolates were resistant to third generation cephalosporins, cefotaxime, ceftazidime, and cefpodoxime. Sixty-five percent of the isolates were resistant to the monobactam drug aztreonam, 40.82% to ertapenem, and 31.36% to meropenem. Resistance to at least five antibiotics was observed in 38.46% of the isolates. Polymerase Chain Reaction (PCR) analysis of ESBL-encoding genes detected blaCTX, blaSHV, and blaTEM genes in 76.92%, 63.3%, and 44.37% of the isolates, respectively. Multiple ESBL genes were detected in majority of the isolates. The recently discovered New Delhi metallo-β-lactamase gene (blaNDM-1) was detected in two ESBL+ isolates. Our study shows that secondary contamination of fresh seafood with enteric bacteria resistant to multiple antibiotics may implicate seafood as a potential carrier of antibiotic resistant bacteria and emphasizes an urgent need to prevent environmental contamination and dissemination of such bacteria. [ABSTRACT FROM AUTHOR]

Aim A cross-sectional study was conducted in 10 government-organized pig farms between 2014 and 2016 representing seven states of India to understand the epidemiology of carbapenem resistance in the Escherichia coli. Methods and Results In this study, fecal sample ( n = 673) from non-diarrheic ( n = 501) and diarrheic ( n = 172) piglets were processed for isolation of carbapenem resistant E. coli. Of 673, E. coli isolate ( n = 112) was genotyped for confirming the carbapenem resistance and associated virulence factors. Of the 112 isolates, 23 were phenotypically resistant to carbapenem and 8 were carrying the New Delhi metallo beta-lactamase ( bla NDM) gene. The carbapenem-resistant isolates also produced extended spectrum beta-lactamases and were multidrug resistant. The PCR-based pathotyping revealed the presence of stx1 , stx2, eae and hlyA genes. The enterobacterial repetitive intergenic consensus PCR dendrogram analysis of the isolates yielded three distinct clusters. The statistical analysis revealed no association between carriages of carbapenem-resistant E. coli in different breed of piglets however, location, sex, health status of piglets and age showed significant difference. The spatial analysis with Sa TScan helped in identification of carbapenem-resistant clusters. Conclusions The presence of carbapenem resistant E. coli isolates with virulence genes in the piglet poses a potential public health risk through possible access and spread via the food chain and environment. Efflux pump may also play an important role in carbapenem resistance in piglet E. coli isolates. Furthermore, identification of risk factors in relation to spatial clusters will help in designing preventive strategies for reducing the risk of spread of carbapenem resistant bacteria. Significance and Impact of the Study 1. Piglets harbor carbapenem resistant E. coli and have great public health significance. 2. Apart from carbapenemase, efflux pump is also important for carbapenem resistance. 3. This is the first report of blaNDM in the piglets from India. [ABSTRACT FROM AUTHOR]

Carbapenem resistance among Enterobacterales has become a global health concern. Clinical Escherichia coli isolates producing the metallo β-lactamase NDM have been isolated from two hospitals in Faisalabad, Pakistan. These E. coli strains were characterized by MALDI-TOF, PCR, antimicrobial susceptibility testing, XbaI and S1 nuclease pulsed-field gel electrophoresis (PFGE), conjugation assay, DNA hybridization, whole genome sequencing, bioinformatic analysis, and Galleria mellonella experiments. Thirty-four bla NDM producing E. coli strains were identified among 52 nonduplicate carbapenem-resistant strains. More than 90% of the isolates were found to be multidrug resistant by antimicrobial susceptibility testing. S1 PFGE confirmed the presence of bla NDM gene on plasmids ranging from 40 kbps to 250 kbps, and conjugation assays demonstrated transfer frequencies of bla NDM harboring plasmids ranging from 1.59 × 10 -1 to 6.46 × 10 -8 per donor. Whole genome sequencing analysis revealed bla NDM-5 as the prominent NDM subtype with the highest prevalence of bla OXA-1 , bla CTX-M-15 , aadA2 , aac(6')-Ib-cr , and tet(A ) associated resistant determinants. E. coli sequence types: ST405, ST361, and ST167 were prominent, and plasmid Inc types: FII, FIA, FIB, FIC, X3, R, and Y, were observed among all isolates. The genetic environment of bla NDM region on IncF plasmids included partial IS Aba125 , the bleomycin ble gene, and a class I integron. The virulence genes terC, traT, gad, fyuA, irp2, capU, and sitA were frequently observed, and G. mellonella experiments showed that virulence correlated with the number of virulence determinants. A strong infection control management in the hospital is necessary to check the emergence of carbapenem resistance in Gram-negative bacteria.IMPORTANCEWe describe a detailed analysis of highly resistant clinical E. coli isolates from two tertiary care centers in Pakistan including carbapenem resistance as well as common co-resistance mechanisms. South Asia has a huge problem with highly resistant E. coli . However, we find that though these isolates are very difficult to treat they are of low virulence. Thus the Western world has an increasing problem with virulent E. coli that are mostly of low antibiotic resistance, whereas, South Asia has an increasing problem with highly resistant E. coli that are of low virulence potential. These observations allow us to start to devise methodologies to limit both virulence and resistance and combat problems in developing nations as well as the Western world.

Purpose: This research aims to profile ten novel strains of carbapenem-resistant Enterobacteriaceae (CRE) co-carrying bla KPC and bla NDM ., Methods: Clinical CRE strains, along with corresponding medical records, were gathered. To ascertain the susceptibility of the strains to antibiotics, antimicrobial susceptibility tests were conducted. To validate the transferability and cost of fitness of plasmids, conjugation experiments and growth curves were employed. For determining the similarity between different strains, ERIC-PCR was utilised. Meanwhile, whole genome sequencing (WGS) was performed to characterise the features of plasmids and their evolutionary characteristics., Results: During the course of this research, ten clinical CRE strains co-carrying bla KPC and bla NDM were gathered. It was discovered that five out of these ten strains exhibited resistance to tigecycline. A closer examination of the mechanisms underlying tigecycline resistance revealed that tmexCD 1 -toprJ 1, bla KPC-2 , and bla NDM-1 existed concurrently within a single Citrobacter freundii strain (CF10). This strain, with a minimum inhibitory concentration (MIC) of 32 mg/L to tigecycline, was obtained from a sepsis patient. Furthermore, an investigation of genome evolution implied that CF10 belonged to a novel ST type 696, which lacked analogous strains. Aligning plasmids exposed that similar plasmids all had less than 70% coverage when compared to pCF10-tmexCD1, pCF10-KPC, and pCF10-NDM. It was also found that tmexCD 1 -toprJ 1, bla KPC-2 , and bla NDM-1 were transferred by Tn 5393, IS 5, and Tn 6296, respectively., Conclusion: This research presents the first report of coexistence of tmexCD 1 -toprJ 1, bla KPC-2 , and bla NDM-1 in a carbapenem and tigecycline-resistant C. freundii strain, CF10., Importance: Tigecycline is considered a "last resort" antibiotic for treating CRE infections. The ongoing evolution of resistance mechanisms to both carbapenem and tigecycline presents an alarming situation. Moreover, the repeated reporting of both these resistance mechanisms within a single strain poses a significant risk to public health. The research revealed that the genes tmexCD 1 -toprJ 1, bla KPC-2 , and bla NDM-1 , which cause carbapenem and tigecycline-resistance in the same strain, were located on mobile elements, suggesting a potential for horizontal transmission to other Gram-negative bacteria. The emergence of such a multi-resistant strain within hospitals should raise significant concern due to the scarcity of effective antimicrobial treatments for these "superbugs"., Competing Interests: The authors report no conflicts of interest in this work., (© 2023 Huang et al.)

Introduction. Carbapenemase-producing Enterobacteriaceae (CPE) have emerged as a global threat to public health and clinical practice. Hypothesis/Gap Statement. In Thailand, reports describing CPEs carrying bla NDM and bla OXA-48 -like genes have been increasing recently; however, data on detailed plasmid analysis and temporal shift of sequence type and carbapenemase type are limited. Aim. In this study, we analysed whole-genome sequencing (WGS) data of clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) to reveal the molecular epidemiology of CPKP in a tertiary-care hospital in Bangkok, Thailand. Methodology. Seventy-seven non-duplicated CPKP isolates collected during 2013-2016 were examined for their drug-resistance genes, sequence types and phylogenetic relationships. Results. All the tested isolates possessed carbapenemase gene(s), and the major type of carbapenemase gene in 2014-2015 was bla NDM-1 , whereas isolates in 2016 harboured more bla OXA-232 than bla NDM-1 . Other carbapenemase gene variants, such as bla NDM-4 , bla NDM-5 , bla OXA-48 , bla OXA-181 and bla IMP-14 were detected in some CPKP isolates. Furthermore, this study revealed that CPKP co-harbouring two genes, bla NDM-1 and bla OXA-232 or bla OXA-181 , emerged during this period. Notably, such isolates co-carrying the two carbapenemase genes emerged in three different sequence types, even in a single hospital, and then spread clonally. The WGS of CPKP revealed a temporal shift of the predominant carbapenemase genes from bla NDM-1 to bla OXA-232 along with a variation in other carbapenemase gene types within a span of 4 years. Conclusion. Our findings suggest that a substantial change in CPE types occurred in Thailand and potentially in Southeast Asian countries.

The growing threat posed by antimicrobial resistance on the healthcare and economic well-being of mankind is pushing the need to develop novel and improved diagnostic platforms for its rapid detection at point of care, facilitating better patient management strategies during antibiotic therapy. In this paper, we present the manufacturing and characterisation of a low-cost carbon screen-printed electrochemical sensor on a ceramic substrate. Using label-free electrochemical impedance spectroscopy, the sensor is demonstrated for the detection of blaNDM, which is one of the main antimicrobial resistance factors in carbapenem-resistant Enterobacteriaceae. The electrochemical performance of the newly fabricated sensor was initially investigated in relation to the function of its underlying composite materials, evaluating the choice of carbon and dielectric pastes by characterising properties like surface roughness, wetting and susceptibility of unspecific DNA binding. Subsequently, the sensor was used in an electrochemical impedance spectroscopy assay for the sensitive and specific detection of synthetic blaNDM targets achieving a detection limit of 200 nM. The sensor properties and performance demonstrated in this study proved the suitability of the new electrode materials and manufacturing for further point-of-care test development as an inexpensive and effective alternative to gold electrodes sensor. [ABSTRACT FROM AUTHOR]

Members of the family Enterobacteriaceae include several human pathogens that can be acquired through contaminated food and water. In this study, the incidence of extended spectrum β-lactamase (ESBL)-producing enterobacteria was investigated in fresh seafood sold in retail markets. The ESBL-positive phenotype was detected in 169 (78.60%) isolates, with Escherichia coli being the predominant species (53), followed by Klebsiella oxytoca (27), and K. pneumoniae (23). More than 90% of the isolates were resistant to third generation cephalosporins, cefotaxime, ceftazidime, and cefpodoxime. Sixty-five percent of the isolates were resistant to the monobactam drug aztreonam, 40.82% to ertapenem, and 31.36% to meropenem. Resistance to at least five antibiotics was observed in 38.46% of the isolates. Polymerase Chain Reaction (PCR) analysis of ESBL-encoding genes detected blaCTX, blaSHV, and blaTEM genes in 76.92%, 63.3%, and 44.37% of the isolates, respectively. Multiple ESBL genes were detected in majority of the isolates. The recently discovered New Delhi metallo-β-lactamase gene (blaNDM-1) was detected in two ESBL+ isolates. Our study shows that secondary contamination of fresh seafood with enteric bacteria resistant to multiple antibiotics may implicate seafood as a potential carrier of antibiotic resistant bacteria and emphasizes an urgent need to prevent environmental contamination and dissemination of such bacteria.