Resistance Plasmid Research Articles

Multidrug-resistant ESBL-producing Klebsiella pneumoniae complex in Czech hospitals, wastewaters and surface waters

BackgroundMultidrug-resistant (MDR) bacteria pose a significant challenge to the treatment of infectious diseases. Of particular concern are members of the Klebsiella pneumoniae species complex (KpSC), which are frequently associated with hospital-acquired infections and have the potential to spread outside hospitals via wastewaters. In this study, we aimed to investigate the occurrence and phylogenetic relatedness of MDR KpSC from patients with urinary tract infections (UTIs), hospital sewage, municipal wastewater treatment plants (mWWTPs) and surface waters and to evaluate the clinical relevance of the KpSC subspecies.MethodsA total of 372 KpSC isolates resistant to third-generation cephalosporins and/or meropenem were collected from patients (n = 130), hospital sewage (n = 95), inflow (n = 54) and outflow from the mWWTPs (n = 63), river upstream (n = 13) and downstream mWWTPs (n = 17) from three cities in the Czech Republic. The isolates were characterized by antimicrobial susceptibility testing and whole-genome sequencing (Illumina). The presence of antibiotic resistance genes, plasmid replicons and virulence-associated factors was determined. A phylogenetic tree and single nucleotide polymorphism matrix were created to reveal the relatedness between isolates.ResultsThe presence of MDR KpSC isolates (95%) was identified in all water sources and locations. Most isolates (99.7%) produced extended-spectrum beta-lactamases encoded by blaCTX−M−15. Resistance to carbapenems (5%) was observed mostly in wastewaters, but carbapenemase genes, such as blaGES−51 (n = 10), blaOXA−48 (n = 4), blaNDM−1 (n = 4) and blaKPC−3 (n = 1), were found in isolates from all tested locations and different sources except rivers. Among the 73 different sequence types (STs), phylogenetically related isolates were observed only among the ST307 lineage. Phylogenetic analysis revealed the transmission of this lineage from patients to the mWWTP and from the mWWTP to the adjacent river and the presence of the ST307 clone in the mWWTP over eight months. We confirmed the frequent abundance of K. pneumoniae (K. pneumoniae sensu stricto and K. pneumoniae subsp. ozaenae) in patients suffering from UTIs. K. variicola isolates formed only a minor proportion of UTIs, and K. quasipneumoniae was not found among UTIs isolates; however, these subspecies were frequently observed in hospital sewage communities during the first sampling period.ConclusionThis study provides evidence of the transmission and persistence of the ST307 lineage from UTIs isolates via mWWTPs to surface waters. Isolates from UTIs consisted mostly of K. pneumoniae. Other isolates of KpSC were observed in hospital wastewaters, which implies the impact of sources other than UTIs. This study highlights the influence of urban wastewaters on the spread of MDR KpSC to receiving environments.

Phenotypic and genomic characterization of ST11-K1 CR-hvKP with highly homologous bla KPC-2 -bearing plasmids in China

ABSTRACT Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains present a significant global public health threat due to their high mortality rates. This study investigated the genomic characteristics of seven ST11-K1 CR-hvKP isolates harboring highly homologous KPC-2-encoding multidrug-resistance plasmids. The strains were isolated from a Chinese tertiary hospital between 2017 and 2020. Whole-genome sequencing and bioinformatic analysis revealed various antibiotic resistance genes (ARGs) and virulence determinants. The bla KPC-2 -bearing plasmids that contain multiple antibiotic-resistance genes were also identified in these strains. ISfinder and Orifinder were applied to identify insertion sequences (IS) and conjugation-related factors among these bla KPC-2 -bearing plasmids. The bla KPC-2 was highly consistent in seven bla KPC-2 -bearing plasmids (IS Kpn6-bla KPC-2 -IS Kpn27 -IS Yps3 -IS 26 ). In addition, we found a region composed of IS IR , Tn 5393 , and IS 26 . It was located upstream of the bla CTX-M-15 gene and presented in six bla KPC-2 -bearing plasmids, with pCR-hvKP221-KPC-P3 as an exception. Conjugation experiments demonstrated the horizontal transfer of resistance plasmids pCR-hvKP128-KPC-P1 and pCR-hvKP132-KPC-P1 across species. Notably, pLVPK-like virulence plasmids carrying virulence gene clusters pCR-hvKP173-Vir-P1, and pCR-hvKP221-Vir-P1 were also detected. A fusional plasmid pCR-hvKP221-Vir-P2, which carries virulence gene clusters and ARGs, was also identified. Five CR-hvKP strains displayed enhanced biofilm formation and high virulence in vivo infection models. Phylogenetic and single nucleotide polymorphism (SNP) analyses indicated a close genetic relationship among the isolates, suggesting a subclade. These findings highlight the complex genetic profiles and potential transmission mechanisms of CR-hvKP strains. IMPORTANCE We reported seven CR-hvKP strains all carried a highly homologous bla KPC-2 integrated IncFⅡ-resistant plasmid, and two strains harbored virulence plasmids. Conjugation experiments confirmed the transferability of these plasmids, indicating a potential for resistance spread. Phylogenetic analysis clarified the relationship among the CR-hvKP isolates. This study provides insights into the phenotypic and genomic characteristics of seven ST11-K1 CR-hvKP strains. The high prevalence and potential for local outbreaks emphasize the need for effective control measures.

Emergence of multidrug-resistant E. coli ST8346 isolates carrying three distinct plasmids with NDM-5, KPC-2, and OXA-181

BackgroundE. coli ST8346 is a unique strain associated with the potential carriage of multiple carbapenemases. Three unique E. coli ST8346 isolates were identified, each concurrently harboring multiple carbapenemases, including blaNDM-5, blaKPC-2, and/or blaOXA-181. This study aimed to characterize the genetic and plasmid structures of these isolates to understand their transmission and resistance mechanisms. MethodsAntibiotic resistance profiles, genetic relatedness, and plasmid structures were determined using antibiotic susceptibility testing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), S1 nuclease PFGE, and long-read sequencing. ResultsAll the strains were carbapenem resistant. PFGE revealed close genetic relationships among the isolates, despite the lack of geographical or epidemiological connections. blaNDM-5, blaKPC-2, and blaOXA-181 were located on separate plasmids. Plasmids harboring blaNDM-5 showed genetic similarities to blaNDM-5-bearing IncF plasmids in the United Kingdom. The IncA/C plasmids harboring blaKPC-2 had identical sequences resembling a plasmid from a K. pneumoniae strain in Taiwan, except for the blaKPC-2 region, which matched a strain from China, indicating a hybrid plasmid. ConclusionThis study is the first to identify and characterize the coexistence of blaNDM-5, blaKPC-2, and blaOXA-181 in E. coli ST8346 isolates. The spread appears to be due to independent acquisition events. We identified the putative origins of these plasmids and detected a possible recombination event in a novel IncA/C plasmid. These findings emphasize the importance of ongoing surveillance and further investigations.

Interrelation Between Pathoadaptability Factors and Crispr-Element Patterns in the Genomes of Escherichia coli Isolates Collected from Healthy Puerperant Women in Ural Region, Russia.

Escherichia coli is a commensal and opportunistic bacterium widely distributed around the world in different niches including intestinal of humans and animals, and its extraordinary genome plasticity led to the emergence of pathogenic strains causing a wide range of diseases. E. coli is one of the monitored species in maternity hospitals, being the main etiological agent of urogenital infections, endometriosis, puerperal sepsis, and neonatal diseases. This study presents a comprehensive analysis of E. coli isolates obtained from the maternal birth canal of healthy puerperant women 3-4 days after labor. According to whole genome sequencing data, 31 sequence types and six phylogenetic groups characterized the collection containing 53 isolates. The majority of the isolates belonged to the B2 phylogroup. The data also includes phenotypic and genotypic antibiotic resistance profiles, virulence factors, and plasmid replicons. Phenotypic and genotypic antibiotic resistance testing did not demonstrate extensive drug resistance traits except for two multidrug-resistant E. coli isolates. The pathogenic factors revealed in silico were assessed with respect to CRISPR-element patterns. Multiparametric and correlation analyses were conducted to study the interrelation of different pathoadaptability factors, including antimicrobial resistance and virulence genomic determinants carried by the isolates under investigation. The data presented will serve as a valuable addition to further scientific investigations in the field of bacterial pathoadaptability, especially in studying the role of CRISPR/Cas systems in the E. coli genome plasticity and evolution.

Investigation of Citrobacter freundii clinical isolates in a Chinese hospital during 2020-2022 revealed genomic characterization of an extremely drug-resistant C. freundii ST257 clinical strain GMU8049 co-carrying blaNDM-1 and a novel blaCMY variant.

The emergence of multidrug-resistant Citrobacter freundii poses a significant threat to public health. C. freundii isolates were collected from clinical patients in a Chinese hospital during 2020-2022. An unusual strain, GMU8049, was not susceptible to any of the antibiotics tested, including the novel β-lactam/β-lactamase inhibitor combination ceftazidime-avibactam. Whole-genome sequencing (WGS) revealed that GMU8049 harbors a circular chromosome belonging to the rare ST257 and an IncX3 resistance plasmid. Genomic analysis revealed the coexistence of two β-lactamase genes, including plasmid-mediated blaNDM-1 and chromosomal blaCMY encoding a novel CMY variant, combined with an outer membrane porin deficiency, which may account for the extreme resistance to β-lactams. Conjugation experiment confirmed that the blaNDM-1 resistance gene located on pGMU8049 could be successfully transferred to Escherichia coli EC600. The novel CMY variant had an amino acid substitution at position 106 (N106S) compared to the closely related CMY-51. Additionally, a GMU8049-specific truncation in an OmpK37 variant that produces a premature stop codon. Moreover, a variety of chromosome-located efflux pump coding genes and virulence-related genes were also identified. Analysis of strain GMU8049 in the context of other C. freundii strains reveals an open pan-genome and the presence of mobile genetic elements that can mediate horizontal gene transfer of antimicrobial resistance and virulence genes. Our work provides comprehensive insights into the genetic mechanisms of highly resistant C. freundii, highlighting the importance of genomic surveillance of this opportunistic pathogen as a high-risk population for emerging resistance and pathogenicity.IMPORTANCEEmerging pathogens exhibiting multi-, extremely, and pan-drug resistance are a major concern for hospitalized patients and the healthcare community due to limited antimicrobial treatment options and the potential for spread. Genomic technologies have enabled clinical surveillance of emerging pathogens and modeling of the evolution and transmission of antimicrobial resistance and virulence. Here, we report the genomic characterization of an extremely drug-resistant ST257 Citrobacter freundii clinical isolate. Genomic analysis of GMU8049 with a rare ST type and unusual phenotypes can provide information on how this extremely resistant clinical isolate has evolved, including the acquisition of blaNDM-1 via the IncX3 plasmid and accumulation through chromosomal mutations leading to a novel CMY variant and deficiency of the outer membrane porin OmpK37. Our work highlights that the emergence of extremely resistant C. freundii poses a significant challenge to the treatment of clinical infections. Therefore, great efforts must be made to specifically monitor this opportunistic pathogen.

Versatile silicate-modified hydrochar based on Scutellaria baicalensis-residue for efficiently removing heavy metal-antibiotic co-contamination and relevant bio-contaminants from wastewater

In this study, we utilized typical Chinese medicine herbal residues (CMHRs)－Scutellaria baicalensis (Scutellaria baicalensis Georgi) residue (SR), as the raw material and employed Na2SiO3 and Fe2(SO4)3 as modifying agents to fabricate a novel and multifunctional hydrochar (FeSi-SRHC), which was designed for comprehensive removal of typical contaminants such as Cu2+, Zn2+, tetracycline (TC), and ciprofloxacin (CIP), along with relevant bio-contaminants (resistant bacteria (RBs) and resistance genes (RGs)) present in wastewater. Based on the analysis of adsorption kinetics and Freundlich isotherm, it was found that FeSi-SRHC exhibited physical monolayer adsorption for Cu2+/Zn2+ while mainly chemical multilayer adsorption for TC/CIP in single-contamination system. Furthermore, Langmuir isotherm demonstrated excellent adsorption capacity of FeSi-SRHC towards Cu2+/Zn2+/TC/CIP with maximum capacities of 255.75, 265.26, 425.53, and 404.86 mg/g, respectively. In the co-contamination system, the presence of Cu2+, Zn2+, TC, and CIP exhibited varying degrees of inhibitory or promotive effects on the mutual adsorption by FeSi-SRHC. This divergence stemmed from differences in complexation intensities and concentration ratios among diverse co-existing contaminants. Based on XPS and Density Functional Theory (DFT) analyses, the adsorption process for Cu2+, Zn2+, TC, and CIP by FeSi-SRHC primarily involves pore fill, complexation reactions, ion exchange, hydrogen bonding, π-π stacking interactions, and electrostatic interactions. Bio-contaminant removal experiments revealed that the release of baicalin and wogonoside from FeSi-SRHC disrupts the structure of RBs cells and compromises the integrity of resistance plasmids. The practical application experiment showed that FeSi-SRHC displayed favorable performance for removing heavy metals, antibiotics, and bio-contaminants in actual wastewater. This study presented a “Treating waste with waste” strategy, which provided a method with low carbon, eco-friendly, and inexpensive for CMHRs resources and turning waste into treasure while proposing a idea to address the challenges associated with treating heavy metal, antibiotic, and bio-contaminant contamination in wastewater.

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

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

Dietary zinc supplementation inhibits bacterial plasmid conjugation in vitro by regulating plasmid replication (rep) and transfer (tra) genes.

Humans use dietary supplements for several intended effects, such as supplementing malnutrition. While these compounds have been developed for host end benefits, their ancillary impact on the gut microbiota remains unclear. The human gut has been proposed as a reservoir for the prevalent lateral transfer of antimicrobial resistance and virulence genes in bacteria through plasmid conjugation. Here, we studied the effect of dietary zinc supplements on the incidence of plasmid conjugation in vitro. Supplement effects were analyzed through standardized broth conjugation assays. The avian pathogenic Escherichia coli (APEC) strain APEC-O2-211 was a donor of the multidrug resistance plasmid pAPEC-O2-211A-ColV, and the human commensal isolate E. coli HS-4 was the plasmid-free recipient. Bacterial strains were standardized and mixed 1:1 and supplemented 1:10 with water, or zinc derived from either commercial zinc supplements or zinc gluconate reagent at varying concentrations. We observed a significant reduction in donors, recipients, and transconjugant populations in conjugations supplemented with zinc, with a dose-dependent relationship. Additionally, we observed a significant reduction (P < 0.05) in log conjugation efficiency in zinc-treated reactions. Upregulation of the mRNA for the plasmid replication initiation gene repA and the subset of transfer genes M, J, E, K, B, P, C, W, U, N, F, Q, D, I, and X was observed. Furthermore, we observed a downregulation of the conjugal propilin gene traA and the entry exclusion gene traS. This study demonstrates the effect of dietary zinc supplements on the conjugal transfer of a multidrug resistance plasmid between pathogenic and commensal bacteria during in vitro conditions.IMPORTANCEThis study identifies dietary zinc supplementation as a potential novel intervention for mitigating the emergence of multidrug resistance in bacteria, thus preventing antibiotic treatment failure and death in patients and animals. Further studies are required to determine the applicability of this approach in an in vivo model.

A novel chicken-origin colistin-resistant Klebsiella pneumoniae ST5982 Co-harboring mcr-3.11 and CTX-M-27

Klebsiella pneumoniae (K. pneumoniae) is an important zoonotic opportunistic pathogen of Enterobacteriaceae that has become one of the most common infectious diseases causing Enterobacteriaceae after Escherichia coli. In this study, we identified a colistin-resistant, multidrug-resistant ST5982 K. pneumoniae strain of broiler origin. The isolate carried 35 resistance genes of 10 antibiotics classes, detected by whole genome sequencing (WGS); 11.4 % (4/35) of the resistance genes were distributed in the chromosome, and 88.6 % (31/35) of the resistance genes were located in four different resistance plasmids. Among the four plasmids, we found for the first time that CTX-M-27 and mcr-3.11 simultaneously coexisted in K. pneumoniae, and a resistance plasmid of IncI1 carrying a combination of mcr-3.11 and qnrS1 was identified. We successfully transferred mcr-3.11, qnrS1 and CTX-M-27 genes into E. coli J53 through conjugation experiments. In the present study, the co-occurrence of CTX-M-27 and mcr-3.11 in multidrug-resistant K. pneumoniae strain ST5982 was detected for the first time; its drug resistance was evaluated, and the risk of its transmission was assessed to provide a reference for further prevention and treatment of K. pneumoniae.

Genomic epidemiology of ceftriaxone-resistant non-typhoidal Salmonella enterica strain in China

Non-typhoidal Salmonella (NTS) is one of the top causes of diarrhea worldwide. Ceftriaxone is commonly recommended as the initial treatment option for Salmonella infections due to its antibacterial effectiveness. The objective of this study was to investigate the molecular epidemiological characteristics of NTS and to compare the phenotypic and genotypic profiles of antimicrobial resistance in multidrug-resistant Salmonella strains by sequencing 329 NTS strains collected from a county-level hospital between 2018 and 2021. Multi-locus sequence typing (MLST), antimicrobial resistance genes and plasmid types were identified by BacWGSTdb 2.0 webserver. Phylogenetic analysis of all NTS strains was carried out using Snippy and Gubbins software. The transferability of ceftriaxone resistant plasmids was confirmed through plasmid conjugation assays, and verified by S1-PFGE-Southern blot assays. The predominant serotypes among all NTS strains were Typhimurium (161/329), Enteritidis (49/329) and London (45/329). The most common sequence type observed was ST34 (86/329), followed by ST19 (72/329) and ST11 (47/329). The antimicrobial resistance of Salmonella to a wide range of antimicrobials showed an overall increase. Out of these 37 (11.24%) ceftriaxone-resistant strains, with the majority of them (33/37) being blaCTX−M. The predominant plasmid types identified were IncHI2 (14/21) and IncI1 (6/21), ranging in size from 70 kb to 360 kb. The conjugation efficiency was calculated with the high conjugation efficiency of 1.1 × 10− 5 to 9.3 × 10− 2. The strains varied widely, ranging from 3 to 45,024 single nucleotide polymorphisms (SNPs). There are close linkages observed among the predominant lineage, with an average of 78 SNPs between each pair of ST34 strains. The findings contribute to our understanding of the transmission and resistance mechanisms of multidrug-resistant Salmonella, thereby facilitating the development of effective control strategies.

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 Ukraine. MethodsAdmission screening included swabs from skin, wounds, catheters, nasopharyngeum and rectum. Bacterial identification, antimicrobial susceptibility testing 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. 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. Antimicrobial susceptibility testing 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 Enterobacter 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 core-genome MLST and carbapenemases (OXA-48 or NDM-1). One vancomycin-resistant Enterococcus faecium was also detected (VanA). ConclusionsWar-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.

Whole genome analysis of Pantoea species identified from sepsis patients in selected Ethiopian referral hospitals: emerging pathogens

BackgroundThe burden of sepsis worsens due to the continuation of emerging pathogens such as multidrug-resistant Pantoea species.MethodsA multicenter study was conducted between October 2019 and September 2020 at four hospitals located in central, southern, and northern parts of Ethiopia. A total of 1416 sepsis patients were recruited and blood cultures were performed. At each study site, positive cultures were characterized by their colony characteristics, gram stain, and conventional biochemical tests. All Pantoea species were identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF) and subjected to whole genome sequencing (WGS) using Illumina HiSeq 2500. The phylogeny structure of Pantoea isolates was calculated using IQ-TREE v1.6.12 from single-nucleotide polymorphisms detected by Snippy v.4.6.0 and filtered by Gubbins v.2.3.4. Average nucleotide identity was estimated by using OrthoANI v.0.93.1 on Shovill v.1.1.0 assemblies. Antimicrobial resistance genes and plasmid replicons were detected using ARIBA v.2.14.6. Phylogenetic trees were visualized using iTOLv.6.5.2.ResultsMultiple Pantoea species include: P. dispersa (n = 19), P. septica (n = 1), and a novel Pantoea spp. (n = 1), were identified among sepsis patients. All P. dispersa isolates and the novel Pantoea species were isolated at Dessie Referral Hospital and displayed phylogenetic clonality, including the ubiquity of an IncM1 plasmid and identical antimicrobial resistance (AMR) gene profiles, encoding blaCTX−M−15, blaTEM−1D, blaSCO−1, and aac(3)-lla. The novel Pantoea spp. isolate harboured blaCTX−M−9 and blaTEM−1D and carried an IncN3 plasmid replicon. The P. septica was isolated at Tikur Anbessa Specialized Hospital in Addis Ababa and carried no detectable acquired AMR genes.ConclusionThe emerging Pantoea spp. carrying multiple AMR genes were identified from sepsis patients. Implementation of strong infection prevention strategies and building surveillance capacity with advanced bacteriology laboratories capable of identifying multidrug-resistant emerging pathogens is strongly recommended.

Drug-resistant Pantoea agglomerans Causing Bacteremia at a Tertiary Care Hospital in Kolkata, India: First Report of Carbapenem Resistance Mediated by OXA-181.

The spread of antibiotic resistance (ABR) in uncommon human pathogens endangers global public health, escalating morbidity, death, and healthcare expenditures. Pantoea agglomerans, a member of the Erwiniaceae family that rarely infects humans, is emerging as a drug-resistant nosocomial pathogen. Seven P. agglomerans isolates were recovered from bacteremia patients at a tertiary care hospital in Kolkata, West Bengal, between March 2022 and October 2022. The isolates were evaluated for phenotypic resistance, β-lactamase and plasmid-mediated quinolone resistance (PMQR) genes, plasmid profiling, and clonality assessment. All isolates were resistant to fluoroquinolones and third-generation cephalosporins, with four resistant to carbapenems. The following β-lactamases and PMQR genes were identified: blaOXA-1 (n = 1), blaTEM (n = 1), blaCTX-M-1 (n = 2), blaNDM (n = 5), blaOXA-181 (n = 1), qnrB (n = 2), and qnrS (n = 4). Six isolates carried up to seven plasmids ranging in size from 2kb to > 212kb. IncFI, FII, HI, and X3 plasmid types were detected in three isolates, while the rest remained untypable. Four different genetic patterns were noted. Four isolates were clonally related, with three being clonal. The swap of environmental isolates to human pathogens exacerbates the ABR dilemma, periling patient care and outcomes. This is the first report in India of a carbapenem-resistant P. agglomerans blood isolate carrying blaOXA-181. In-depth genomic research of drug-resistant microbes adapted to the environment-human interfaces might underpin the source-route-containment of ABR.

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.

Antibiotic resistance plasmids in Enterobacteriaceae isolated from fresh produce in northern Germany.

This study showed that Enterobacteriaceae from raw vegetables carried plasmids ranging in size from 2,715 to 326,286 bp, of which about less than one-third carried antibiotic resistance genes encoding resistance toward antibiotics such as tetracyclines, aminoglycosides, fosfomycins, sulfonamides, quinolones, and β-lactam antibiotics. Some strains encoded multiple resistances, and some encoded extended-spectrum β-lactamases. The study highlights the potential of produce, which may be eaten raw, as a potential vehicle for the transfer of antibiotic-resistant bacteria.

Geneticand phenotypic characterization of a novel ST45-K43 carbapenem-resistant Klebsiella pneumoniae strain causing bloodstream infection: a potential clinical threat.

Klebsiella pneumoniae is a common pathogen of nosocomial infection, which can cause pneumonia, urinary tract infection, cystitis, and bloodstream infections (BSIs). Here, we genetically characterize a novel carbapenem-resistant K. pneumoniae (CRKP) strain recovered from the blood of a 44-year-old male patient with severe acute necrotizing pancreatitis and septic shock in China. The strain is a ST45 K. pneumoniae with a novel serotype of K43, named 18SHX166. The susceptibility testing results showed that 18SHX166 was resistant to cephalosporin and carbapenems but still susceptible to ceftazidime-avibactam, quinolones, colistin, and amikacin. Genomic sequencing revealed that 18SHX166 contains three plasmids, namely pSHX166-Hv, pSHX166-KPC, and pSHX166-3. pSHX166-Hv harbored the iucABCD operon, encoding the siderophore of aerobactin. pSHX166-KPC harbored blaKPC-2 gene and possessed complete conjugative regions. The conjugation experiment verified pSHX166-KPC as a self-transmissible plasmid mediating the dissemination of antibiotic resistance, with a conjugation rate of 2.21 × 10-5. Additionally, the growth curve showed that 18SHX166 demonstrates a higher growth rate than the control strains. The characteristics of 18SHX166 indicate a potential high risk of clinical transmission.IMPORTANCEST45-K43 carbapenem-resistant Klebsiella pneumoniae isolate, 18SHX166, carries a carbapenem resistance plasmid and virulence plasmid. It has the characteristics of multidrug resistance, high transmissibility, and a fast growth rate, which could pose a threat to the control of antimicrobial resistance and clinical transmission, causing a severe challenge to public health.

Comparison of Plasmid Curing Efficiency across Five Lactic Acid Bacterial Species.

With the recent stringent criteria for antibiotic susceptibility in probiotics, the presence of antibiotic resistance genes and plasmids associated with their transfer has become a limiting factor in the approval of probiotics. The need to remove genes related to antibiotic resistance and virulence through plasmid curing for the authorization of probiotics is increasing. In this study, we investigated the curing efficiency of ethidium bromide, acridine orange, and novobiocin at different concentrations and durations in five strains of plasmid-bearing lactic acid bacteria and examined the curing characteristics in each strain. Limosibacillus reuteri and Lacticaseibacillus paracasei exhibited curing efficiencies ranging from 5% to 44% following treatment with ethidium bromide (10-50 μg/ml) for 24-72 h, while Lactobacillus gasseri showed the highest efficiency at 14% following treatment with 10 μg/ml novobiocin for 24 h. Lactiplantibacillus plantarum, which harbors two or more plasmids, demonstrated curing efficiencies ranging from 1% to 8% after an additional 72-h treatment of partially cured strains with 10 μg/ml novobiocin. Plasmid curing in strains with larger plasmids exhibited lower efficiencies and required longer durations. In strains harboring two or more plasmids, a relatively low curing efficiency with a single treatment and a high frequency of false positives, wherein recovery occurred after curing, were observed. Although certain strains exhibited altered susceptibilities to specific antibiotics after curing, these outcomes could not be attributed to the loss of antibiotic resistance genes. Furthermore, the genomic data from the cured strains revealed minimal changes throughout the genome that did not lead to gene mutations.

Analysis of the Association Between Antimicrobial Resistance Genes and Virulence Factors in ST11 and Non-ST11 CR-KP Bloodstream Infections in the Intensive Care Unit.

This study aims to investigate the association between antimicrobial resistance genes and virulence factors in ST11 and non-ST11 types of CR-KP in bloodstream infections in the intensive care unit, providing a theoretical basis for infection control and clinical diagnosis and treatment. From January 2021 to June 2023, samples of Klebsiella pneumoniae from bloodstream infections were collected at our hospital, focusing on those resistant to carbapenems. The resistance genes, housekeeping genes, and virulence genes were identified through PCR and analyzed using the GrapeTree software to perform MLST-based minimum spanning tree typing. Among the 85 CR-KP cases, 61.18% were of the ST11 type, predominantly of the KL64 capsular type; non-ST11 types were mainly ST15, accounting for 25.88%, predominantly of the KL5 capsular type. The carriage rates of virulence genes such as rmpA2, entB, silS, kpn, iucA, peg-344, and terB were significantly higher in the ST11 group than in the non-ST11 group. The primary carbapenemase identified was class A enzyme bla KPC-2, with a higher carriage rate in the ST11 group. Drug susceptibility tests showed that the resistance rates for cefepime, ertapenem, nitrofurantoin, amikacin, and gentamicin were also higher in the ST11 group, consistent with the resistance genotype findings. The study reveals that ST11 type CR-KP in intensive care unit bloodstream infections exhibits stronger resistance and higher virulence compared to non-ST11 types, posing significant challenges to clinical treatment. Thus, strict control over the use of carbapenem antibiotics is essential to prevent the spread of resistant plasmids.

Loss of Antibiotic Resistance Transposons if grown in the absence of Antibiotics

In a recent article I have proven F is not a single replicon but co-integrate of two complete replicons – F and R. These two replicons are not allowed to function simultaneously but their individual functional states are stringently controlled by the transposon Tn 1000(5.7 Kb). Besides these two replicons we have previously mentioned the Ent plasmid P307. In 1977 we characterized the antibiotic resistance plasmid pCG86 in ETEC isolated from the stool of a piglet with diarrhoeal disease. The genes for heat-labile enterotoxin and heat-stable enterotoxin were also carried by the same plasmid pCG86. In the year 1975 we received this same bacterial isolate of Carlton Gyles (Department of Veterinary Microbiology and Immunology, University of Guelph, Ontario Canada) from Professor S. Falkow (University of California) who saved the stab at room temperature but without adding antibiotics. The same strain can’t grow in the presence of antibiotics. After the discovery of transposons, I felt the re-interpretation of this heteroduplex molecule formed between pCG86 and Ent P307 is absolutely necessary.

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

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