Plasmid Transfer Research Articles

How Gut Microbiome Perturbation Caused by Antibiotic Pre-Treatments Affected the Conjugative Transfer of Antimicrobial Resistance Genes

The global spread of antimicrobial resistance genes (ARGs) poses a significant threat to public health. While antibiotics effectively treat bacterial infections, they can also induce gut dysbiosis, the severity of which varies depending on the specific antibiotic treatment used. However, it remains unclear how gut dysbiosis affects the mobility and dynamics of ARGs. To address this, mice were pre-treated with streptomycin, ampicillin, or sulfamethazine, and then orally inoculated with Salmonella enterica serovar Typhimurium and S. Heidelberg carrying a multi-drug resistance IncA/C plasmid. The streptomycin pre-treatment caused severe microbiome perturbation, promoting the high-density colonization of S. Heidelberg and S. Typhimurium, and enabling an IncA/C transfer from S. Heidelberg to S. Typhimurium and a commensal Escherichia coli. The ampicillin pre-treatment induced moderate microbiome perturbation, supporting only S. Heidelberg colonization and the IncA/C transfer to commensal E. coli. The sulfamethazine pre-treatment led to mild microbiome perturbation, favoring neither Salmonella spp. colonization nor a conjugative plasmid transfer. The degree of gut dysbiosis also influenced the enrichment or depletion of the ARGs associated with mobile plasmids or core commensal bacteria, respectively. These findings underscore the significance of pre-existing gut dysbiosis induced by various antibiotic treatments on ARG dissemination and may inform prudent antibiotic use practices.

Klebsiella pneumoniae, a human-dog shuttle organism for the genes of CTX-M ESBL.

Antimicrobials reserved for human medicines are permitted for companion animals and it is important to understand multidrug-resistant pathogens recovered from companion animals in terms of epidemiological correlation with human pathogens and possibility of transmission to human-beings. Seventeen of each CTX-M-type extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) and Klebsiella pneumoniae (ESBL-KP) canine isolates were assessed. Entire genomes of the 34 isolates were sequenced. Plasmid transfer and relative growth rates were assessed at differed temperature conditions indicating the body temperature of dogs, that of human-beings, and environment. ESBL-ECs were clonally diverse, while ESBL-KPs were not. The ESBL-ECs carried the blaCTX-M-15 gene in plasmids and the blaCTX-M-14-like gene either in chromosomes or in plasmids. The ESBL-KPs possessed the blaCTX-M-15 gene in plasmids (n = 15). One of the isolates carried another blaCTX-M-15 gene in a chromosome simultaneously and the other isolate had an additional blaCTX-M-9 gene-harbouring plasmid, together. Two ESBL-KP isolates carried the blaCTX-M-14 gene in plasmids. Plasmid transfer ESBL-EC to K. pneumoniae was efficient and the differed biological costs by temperature was much more in ESBL-EC than in ESBL-KP. Intersectoral dissemination of ESBL-ECs occurred mainly by horizontal gene transfer, while that of ESBL-KPs occurred by clonal dissemination.

Tn4661-mediated transfer of bla CTX-M-15 from Klebsiella michiganensis to an outbreak clone of Pseudomonas aeruginosa.

Carriage of CTX-M-type extended-spectrum β-lactamase (ESBL) is rare in Pseudomonas aeruginosa. During routine surveillance of an endemic ST-621 P. aeruginosa at a large hospital, isolate MRSN 100690 carrying bla CTX-M-15 was cultured from a patient (P2). This was the first detection of this ESBL in the endemic ST-621 lineage. All 1 488 bacterial isolates collected from the same facility in the 12 months prior to the incidence of 100 690 were screened for the presence of bla CTX-M-15. A set of 183 isolates was identified, in which corresponding patient metadata was evaluated for spatiotemporal overlaps with P2. The resulting three isolates, along with 100 690, were long-read sequenced using the Oxford Nanopore MinION platform to determine a potential donor of bla CTX-M-15. The screen revealed a single Klebsiella michiganensis isolate, MRSN 895358, which carried an IncA/C2 plasmid harbouring bla CTX-M-15. Notably, the patient harbouring 895358, P1, occupied the same hospital room as P2 9 months prior. Genomic alignment revealed that both isolates shared an identical 80.8 kb region containing the IncA/C2 plasmid replicon and bla CTX-M-15. This region was plasmid bound in 895 358, but chromosomally bound in 100 690 due to Tn4661-mediated transposition. ESBL bla CTX-M-15 was acquired and subsequently integrated into the chromosome of a ST-621 P. aeruginosa, likely initiated by plasmid transfer from a K. michiganensis strain.

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.

Various plasmid strategies limit the effect of bacterial restriction-modification systems against conjugation.

In bacteria, genes conferring antibiotic resistance are mostly carried on conjugative plasmids, mobile genetic elements that spread horizontally between bacterial hosts. Bacteria carry defence systems that defend them against genetic parasites, but how effective these are against plasmid conjugation is poorly understood. Here, we study to what extent restriction-modification (RM) systems-by far the most prevalent bacterial defence systems-act as a barrier against plasmids. Using 10 different RM systems and 13 natural plasmids conferring antibiotic resistance in Escherichia coli, we uncovered variation in defence efficiency ranging from none to 105-fold protection. Further analysis revealed genetic features of plasmids that explain the observed variation in defence levels. First, the number of RM recognition sites present on the plasmids generally correlates with defence levels, with higher numbers of sites being associated with stronger defence. Second, some plasmids encode methylases that protect against restriction activity. Finally, we show that a high number of plasmids in our collection encode anti-restriction genes that provide protection against several types of RM systems. Overall, our results show that it is common for plasmids to encode anti-RM strategies, and that, as a consequence, RM systems form only a weak barrier for plasmid transfer by conjugation.

Signaling Molecules in Pseudomonas aeruginosa Response to Antibiotics at Sub-Inhibitory Concentrations

Signaling Molecules are N-acylhomoserine lactones (AHLs) that form the Pseudomonas aeruginosa cell information system which determines gene expressions in a population dependent manner called quorum sensing (QS). Signal molecules, which are chemically varied, control genes expressions to antibiotics resistance, pathogenicity, motility, biofilm development, bioluminescence, secondary metabolite production, and plasmid transfer. This research was aimed to identify the signaling molecules in Pseudomonas aeruginosa response to antibiotics at sub-inhibitory concentrations. One hundred and fifty (150) clinical swab specimens were collected from urinary catheter wound and ear infection of patients and the swabs inoculated using standard microbiology method. The isolates were characterized based on the bacteriological methods such as morphology and biochemical tests. The isolates were further confirmed by species specific PCR by amplification of 16S rRNA and the amplicons were analyzed by gel electrophoresis; and further genomic sequencing was done and blast with NCBI database mining. Disc diffusion method was applied for the antibiotics susceptibility pattern. The isolates cell suspensions were analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). The result of the isolation showed 22(59.46%) from wound infections, 12(32.43%) from ear infections and 3(8.11%) from urinary catheter. The isolates were Gram negative, produced β-hemolysis on blood agar and the morphology is small pigmented circular in form. The isolates showed positive result to catalase, oxidase, citrate, nitrate and indole tests. The amplification of 16S rRNA gene region resulted in the band size of 1500bp PCR product and the BLAST analysis gave 99% similarity. The resistant antibiotics susceptibility showed that Pseudomonas aeruginosa is multidrug resistant bacteria. The GC-MS results obtained from urinary catheter isolates showed four (4) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone, N-Dodecanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone. Three (3) signaling molecules such as N-Octanoyl-L-homoserine Lactone, N-Decanoyl-DL-Homoserine Lactone and N-Tetradecanoyl-L-Homoserine Lactone were obtained from wound isolates. N-Tetradecanoyl-L-Homoserine Lactone was the only signaling molecule obtained from ear isolates. Further research should be done to develop rapid tests that could be possible to detect acyl homoserine specific to Pseudomonsa aeruginosa present to human samples and deliver results in real time. Signaling molecules inhibitors (SMI) are possible potential therapeutics that could produce the subsequent class of antibacterial drugs. The fundamental role of each signal molecule in the production of biofilms and virulent factors should also be investigated, as well as whether Pseudomonas aeruginosa needs one or more signaling molecules to form quorum sensing. Identification of the signals molecules (AHLs) of P. aeruginosa and developing signaling molecules inhibitors (SMI) can have an important prognostic, diagnostic and therapeutic value in human medicine for the treatment of P. aeruginosa infections.

An outbreak of blaKPC−4- and blaVIM−1-producing Klebsiella pneumoniae and Klebsiella variicola at a single hospital in South Korea

BackgroundThe dissemination of Klebsiella spp. producing multiple carbapenemases has been increasingly recognized. Between July 2019 and August 2021, ten patients were found to carry Klebsiella spp. co-harboring blaKPC−4 and blaVIM−1 across multiple wards at a Korean hospital, and one isolate was recovered from a hand-washing sink, more than a year after the outbreak. This study aimed to investigate the outbreak and conduct a genomic study of these isolates.MethodsWhole-genome sequencing, including long-read sequencing, was performed to analyze plasmid structures and mobile genetic elements (MGEs). Bioinformatics analyses were performed to trace clonal transmission chains and horizontal gene transfer.ResultsThe findings suggested that the inter-ward spread of Klebsiella spp. seemed to be facilitated by healthcare worker contact or patient movement. Of the nine isolates collected (eight clinical and one environmental), seven (including the environmental isolate) were identified as K. pneumoniae (ST3680) and two were K. variicola (single-locus variant of ST5252). These isolates showed high genetic relatedness within their species and harbored the IncHI5B plasmid carrying both blaKPC−4 and blaVIM−1 (pKPCVIM.1). On this plasmid, blaVIM−1 was located in the Class 1 integron associated with IS1326::IS1353 (In2), and Tn4401b carrying blaKPC−4 was inserted into IS1326::IS1353, creating a novel MGE construct (In2_blaVIM−1-Tn4401b_blaKPC−4).ConclusionThe hospital-wide spread of blaKPC−4 and blaVIM−1 was facilitated by clonal spread and horizontal plasmid transfer. The persistence of this strain in the hospital sink suggests a potential reservoir of the strain. Understanding the transmission mechanisms of persistent pathogens is important for improving infection control strategies in hospitals.

Molecular epidemiological analysis of blaNDM-5-producing Klebsiella pneumoniae ST2407-K25 causing infection outbreaks in pediatric patients based on whole genome sequencing

BackgroundPediatric patients are vulnerable to the threat of carbapenem-resistant Klebsiella pneumoniae (CRKP) due to their limited immunity and few available antibiotics. Especially when these pathogens exhibit hypervirulent phenotypes, they are often associated with poor clinical outcomes.MethodsIn this study, we investigated a CRKP outbreak in pediatric patients from 2019 to 2021 in a teaching hospital in China based on whole genome sequencing. We sequenced twenty-nine CRKP isolates isolated from unduplicated pediatric patients to understand their genetic relationships, virulence factors, resistance mechanisms, and transmission trajectories. Conjugation experiments were performed to evaluate the horizontal transfer ability of carbapenem resistance determinants in twenty-nine CRKP isolates. We then characterized these isolates for biofilm formation ability and serum resistance. Genetic relatedness, comparison of plasmids, and chromosomal locus variation of CRKP isolates were analyzed by bioinformatics.ResultsAll the isolates were carbapenemase-producers harbouring blaNDM−5. Among them, twenty-eight isolates belonged to the ST2407 group, with the consistent capsular serotype K25. The virulence-related factors: ureA, fim, ybtA, irp1/irp2, and mrkA were prevalent in these isolates. Additionally, most CRKP isolates showed moderately adherent biofilm formation. Although the ST2407 clonal group did not exhibit serum resistance, the heterogeneous level of serum resistance was related to the disruption of oqxR. Conjugation and WGS revealed that the blaNDM−5 carried by the twenty-eight CRKP ST2407 isolates was located on nonconjugative IncX3 plasmids associated with deleting the T4SS-encoding genes. Clonal transmission of CRKP ST2407 in pediatric patients was suggested by the phylogenetic tree.ConclusionsOur study provides evidence of the clonal spread of blaNDM−5-producing K. pneumoniae in pediatric patients and the necessity for the T4SS system for horizontal transfer of the IncX3 plasmid carrying blaNDM−5. Additionally, the disruption of oqxR may have affected the serum resistance of CRKP. The results of this study emphasize the importance of continuously monitoring for CRKP infection in pediatric patients to prevent recurrent infections.

IncC plasmid genome rearrangements influence the vertical and horizontal transmission tradeoff in Escherichia coli.

It has been shown that an evolutionary tradeoff between vertical (host growth rate) and horizontal (plasmid conjugation) transmissions contributes to global plasmid fitness. As conjugative IncC plasmids are important for the spread of multidrug resistance (MDR), in a broad range of bacterial hosts, we investigated vertical and horizontal transmissions of two multidrug-resistant IncC plasmids according to their backbones and MDR-region rearrangements, upon plasmid entry into a new host. We observed plasmid genome deletions after conjugation in three diverse natural Escherichia coli clinical strains, varying from null to high number depending on the plasmid, all occurring in the MDR region. The plasmid burden on bacterial fitness depended more on the strain background than on the structure of the MDR region, with deletions appearing to have no impact. Besides, we observed an increase in plasmid transfer rate, from ancestral host to new clinical recipient strains, when the IncC plasmid was rearranged. Finally, using a second set of conjugation experiments, we investigated the evolutionary tradeoff of the IncC plasmid during the critical period of plasmid establishment in E. coli K-12, by correlating the transfer rates of deleted or non-deleted IncC plasmids and their costs on the recipient strain. Plasmid deletions strongly improved conjugation efficiency with no negative growth effect. Our findings indicate that the flexibility of the MDR-region of the IncC plasmids can promote their dissemination, and provide diverse opportunities to capture new resistance genes. In a broader view, they suggest that the vertical-horizontal transmission tradeoff can be manipulated by the plasmid to improve its fitness.

Characterization of Chlorhexidine Resistance Among Clinical Isolates of Coagulase-Negative Staphylococcus Species in a Tertiary Care Center, Chennai.

Background Coagulase-negativeStaphylococci(CoNS) are potential pathogens and are often associated with healthcare-associated infections (HAIs). Chlorhexidine (CHX) is the most widely used antiseptic to reduce colonization and infection by allStaphylococci, including CoNS. Resistance to CHXamong CoNS has been observed over the past few years, consequent to its widespread use. Phenotypic tolerance or reduced susceptibility to CHX is conferred by plasmid-mediatedqacgroup of genes, mainlyqacA/Bandsmr, which cause activation of efflux pumps over the bacterial cell wall. This study aims to characterize the phenotypic and genotypic resistance exhibited by CoNS species against CHX. Methods After ethical approval, 148 consecutive, non-repetitive isolates of clinically significant CoNS species of hospitalized patients, isolated from blood samples and exudative specimens, were included in the study. Speciation was performed by conventional biochemical identification and automated methods. Antimicrobial susceptibility testing was performed by disc diffusion technique and for vancomycin by minimum inhibitory concentration (MIC) determination, as per Clinical Laboratory Standards Institute (CLSI) M-100 2023 guidelines. Methicillin resistance was detected using a cefoxitin disc. MIC for CHX was performed by agar dilution method; reduced susceptibility was considered when MIC to CHX ≥4 µg/mL. The simplex polymerase chain reaction (PCR) was carried out with suitable controls to detectqacA/Bandsmr. Statistical analysis was conducted to determine the association ofqacA/Bandsmrgenes with MIC of CHXin the study isolates. Results Fifteen different species of CoNS were obtained from clinical samples. A high percentage of resistance was observed against various classes of antibiotics. Methicillin resistance was observed in 69.6% (103/148) of isolates. Of 148 CoNS, 52.7% (78/148) of isolates exhibited reduced susceptibility to CHX with an MIC ≥4 µg/mL. These isolates exhibited a higher percentage of methicillin resistance (75.6%, 59/78). By PCR, 34.5% (51/148) of isolates carried either or both genes. GeneqacA/Bwas solely detected in 27.02% (40/148) of isolates, of which 14 were CHX-tolerant and the remaining 26 were CHX-susceptible. Genesmrwas solely detected in 4.1% (6/148) of isolates comprising three isolates each in CHX-tolerant and susceptible categories.There were 3.4% (5/148) of isolates that harbored bothgenes, of which only one isolate was CHX-susceptible, while the other four were CHX-tolerant. A proportion of isolates that were phenotypically tolerant to CHX did not carry either or both genes. A significant statistical association was found between reduced susceptibility to CHX and the presence of antiseptic resistance genesin the study isolates (p-value=0.033942). Conclusion To our knowledge, this is the first study from South India to investigate CHX resistance among CoNS using phenotypic and genotypic methods. The rise of antiseptic resistance among CoNS is an emerging threat to current infection control practices. The presence ofqacA/Bandsmrgenes, especially in CHX susceptible isolates, is concerning since these resistance genes are located on transferable plasmids, and the isolates can develop resistance eventually upon exposure to CHX.

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.

Colistin, doxycycline and Labetalol-meropenem combination are the most active against XDR-Carbapenem-resistant Acinetobacter baumannii: role of a novel transferrable plasmid conferring carbapenem resistance

This study aimed to evaluate the antimicrobial susceptibility and combination of a beta-blocker, labetalol (LAB) and meropenem (MEM) on Carbapenem-resistant (CR) A. baumannii clinical isolates. A total of 43 CR- A. baumannii were isolated of which 37 (86.6%) and 28 (65%) exhibited MDR and XDR phenotypes, respectively. Colistin and doxycycline still retain their activities in 93.1% and 72.1% of the isolates, respectively. Combining MEM with LAB at 0.25 mg /mL, decreased MIC values in 91.4% (32/35) however, at 0.5 mg /mL, it decreased MIC value and restored susceptibility to MEM in 100% and 91.4% of the tested isolates, respectively. A novel transferable plasmid pAcbGIM3 harboring aph-3’, blaoxa-58,blaGIM3 and blaCTX-M3 and eight mobile genetic elements were successfully isolated from a pan-drug resistant (PDR) isolate. In conclusion, LAB-MEM is a promising combination and should be clinically examined. This is the first report of a transmissible plasmid harboring blaGIM3 gene in Egypt.

Optimizing the Procedure for Manufacturing Clinical-grade Genetically Manipulated NK cells for Adoptive Immunotherapy

BackgroundEx vivo expanded natural killer (NK) cells hold significant potential as antitumor effector cells for adoptive immunotherapy. However, producing clinical-grade genetically modified NK cells in sufficient quantities presents a considerable challenge. MethodsWe tested RPMI 1640, KBM581, SCGM, NK MACS, X-VIVO 15, and AIM-V, each supplemented with fetal bovine serum (FBS), human AB serum, human platelet lysate (HPL), or Immune Cell Serum Replacement (SR) combined with feeder cells, to produce cytotoxic NK cells. Subsequent analyses were conducted to assess cell viability, expansion folds, cytotoxicity, immunophenotype, and transcriptome profile of NK cells under certain conditions. Furthermore, transfer plasmids varying in transgene size, promoter elements, backbones and packaging plasmids with different envelopes were used to transduce NK cells and differences in transduction efficiency were compared. Nucleofection was performed every two days from Day 0 to Day 12 to determine the optimal time window for gene editing. ResultsNK cells cultured in KBM581 medium supplemented with SR exhibited the best expansion, achieving over 5000-fold increase within two weeks and exceeding 25,000-fold expansion within three weeks. Additionally, NK cells cultured in KBM581 medium with human AB serum demonstrated the highest cytolytic activities and exhibited higher expression of NKp30, 2B4, PRF1, Granzyme B, and IL2RG. Baboon envelope (BaEV) pseudotyped lentivirus outperformed BaEV-Vesicular Stomatitis Virus type-G (VSVG) hybrid envelope lentivirus, achieving robust NK cell transduction. Additionally, efficient gene knockout efficiency was achieved in NK cells on day 4 to day 6 post feeder cell activation using LONZA DN-100 program, which can strike a balance between editing efficiency and cell expansion. ConclusionsThis research presents a Good Manufacturing Practice (GMP)-compliant protocol utilizing a feeder cell expansion system for the large-scale production of highly cytotoxic Natural Killer (NK) cells. The protocol facilitates genetic modification of these cells, positioning them as promising candidates for universal therapeutic applications in immunotherapy.

Risk factors and molecular characterization of carbapenem resistant Escherichia coli recovered from a tertiary hospital in Fujian, China from 2021 to 2023

BackgroundThere is a serious public health concern regarding the emergence of carbapenem-resistant Escherichia coli (CREC). The purpose of this study is to identify the molecular characterization and risk factors of CREC in Fujian province, China.MethodsA total of 48 CREC isolates were collected from various clinical samples. The strains were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Susceptibility to antibiotics was determined by the standard broth microdilution method. Polymerase chain reaction (PCR) was used to screen common drug resistance genes. Multilocus sequence typing (MLST) was used to type isolates. RT-qPCR was used to detect gene expression of acrA, acrB, and tolC. Conjugation assays were used to analyze the transferability of plasmids carrying mcr-1 or blaNDM. Risk factors for CREC infection were identified by logistic regression analysis.Results48 CREC strains were collected, with 81.25% producing carbapenemase (CP-CREC), and 18.75% were not producing carbapenemase (no-CP-CREC). They belonged to 21 sequence type (STs) and five unknown STs. Perianal swabs were the main sample type, with 25 patients found to have hematological malignancies. All isolates of CP-CREC were found to contain blaNDM (blaNDM−5 (n = 32), blaNDM−1 (n = 5), blaNDM−4 (n = 1), and blaNDM−13 (n = 1)), among which one isolate co-existence blaNDM−5 and blaOXA−48. Two blaNDM-positive strains, specifically blaNDM−5 and blaNDM−4, were found to co-habor mcr-1 with ST617. Conjugation assays confirmed that blaNDM−1, blaNDM−13, and most blaNDM−5(68.75%, 22/32) could be transferred between E. coli strains. Four of the 9 non-CP-CREC isolates had deletions in ompC and ompF with blaCTX−M production, while the other five showed high expression of acrA, acrB, and tolC. Antibiotics usage, antifungal treatment, detection of other pathogens (prior to CREC infection), and respiratory disease were identified as independent risk factors for CREC infection. The area under the receiver operating characteristic curve for the scoring system was 0.937. Youden’s index, with sensitivity and specificity of 0.96 and 0.78, was maximal when 2 points were scored.ConclusionsIn CP-CREC, carbapenem resistance is caused primarily by multiple types of blaNDM, while non-CP-CREC is caused by loss of porin protein or high expression of efflux pumps coupled with carrying blaCTX−M. CREC isolates were highly diverse in terms of ST, with a total of 21 STs identified. Here, we first describe a clinical strain of CREC from China both mcr-1 and blaNDM −4 with ST617. An easy-to-use scoring system was developed to diagnose CREC infections.

Prevalence and Characteristics of Plasmid-Mediated Fosfomycin Resistance Gene fosA3 among Salmonella Enteritidis Isolates from Retail Chickens and Children with Gastroenteritis in China.

A total of 265 Salmonella Enteritidis isolates collected from retail markets and children's hospitals in Shanghai were used to investigate the prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes. Nine of the isolates-7 from the 146 (4.79%) retail chicken-related samples and 2 from the 119 (1.68%) samples from clinical children-were fosfomycin-resistant (FosR). The fosA3 gene was detected in all of the nine FosR isolates, which were located on Inc F-type (8/9, 88.9%) and unknown-type (1/9, 11.1%) transferable plasmids. In total, five plasmid types, namely Inc HI2 (1/9, 11.1%), Inc I1 (3/9, 33.3%), Inc X (8/9, 88.9%), Inc FIIs (9/9, 100%), and Inc FIB (9/9, 100%), were detected in these FosR isolates, which possessed five S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) profiles. The extended-spectrum β-lactamase determinant blaCTX-M-14 subtype was identified in one FosRS. Enteritidis isolate, which was located in a transferable unknown-type plasmid co-carrying fosA3 and tetR genes. Sequence homology analysis showed that this plasmid possessed high sequence similarity to previously reported blaCTX-M-14- and fosA3-positive plasmids from E. coli strains, implying that plasmids carrying the fosA3 gene might be disseminated among Enterobacterales. These findings highlight further challenges in the prevention and treatment of Enterobacteriaceae infections caused by plasmids containing fosA3.

Characterization of pKPN945B, a novel transferable IncR plasmid from hypervirulent carbapenem-resistant Klebsiella pneumoniae, harboring blaIMP-4 and qnrS1.

Up to now, IncR replicons carrying blaIMP-4 have not been reported, and the IncR plasmids described in previous studies have been found to be non-transferrable to other bacteria through conjugation. Moreover, there have been no extensive phylogenetic analyses of strains carrying blaIMP in the published papers. The lack of data in these studies is noteworthy because blaIMP appears in the novel transferable fusion plasmid IncR. Although the IncR plasmid has no tra operon, it can still be transferred to Escherichia coli EC600 or Klebsiella pneumoniae ATCC13883 (RIFR) without high fitness cost, but it only affects the MIC of imipenem. blaIMP integrates with other resistance mechanisms leading to the formation of multidrug-resistant strains. Notably, the high prevalence of blaIMP-4 in Asia and the presence of blaIMP-4 on novel transferable IncR plasmids suggest the urgent need to monitor the emergence of such plasmids and control their spread.

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

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

Removing multiple refractory organic pollutants in wastewater by cell bioaugmentation and genetic bioaugmentation with Rhodococcus sp. strain p52

Bioaugmentation is an appealing remediation strategy to treat refractory organic pollutants in contaminated environments. In this study, activated sludge was bioaugmented with Rhodococcus sp. strain p52 to treat synthetic wastewater containing multiple refractory organic pollutants (dibenzofuran, dibenzo-p-dioxin, and phenanthrene). Bioaugmentation of activated sludge reactors with strain p52 alleviated the adverse effects of refractory pollutants on the chemical oxygen demand and ammonia nitrogen removal capacity of the reactors and enhanced the removal of the refractory organic pollutants, especially highly refractory compound dibenzo-p-dioxin. Toxicity of the refractory pollutants to activated sludge bacteria was attenuated due to contaminant degradation by strain p52. Genetic bioaugmentation mediated by catabolic plasmid transfer from strain p52 to activated sludge bacteria and cell bioaugmentation occurred along with successful colonization and proliferation of strain p52. In addition, a potential metabolically mutualistic relationship existed between strain p52 and activated sludge bacteria for refractory pollutants degradation. Both refractory organic pollutants input and strain p52 bioaugmentation profoundly influenced the structure of the activated sludge bacterial community and drove bacterial community shifts in different directions. This study provides insights into the activities of bioaugmented bacteria and bioaugmentation technology for environmental contaminant remediation.

Molecular Epidemiology and Horizontal Transfer Mechanism of optrA-Carrying Linezolid-Resistant Enterococcus faecalis.

The aim of this work was to provide a theoretical and scientific basis for the treatment, prevention, and control of clinical drug-resistant bacterial infections by studying the molecular epidemiology and horizontal transfer mechanism of optrA-carrying linezolid-resistant Enterococcus faecalis strains (LREfs) that were clinically isolated in a tertiary hospital in Kunming, China. Non-repetitive LREfs retained in a tertiary A hospital in Kunming, China. The strains were identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The transferability and horizontal transfer mechanism of optrA gene were analyzed using polymerase chain reaction (PCR), whole-genome sequencing (WGS), and conjugation experiments. A total of 39 LREfs strains were collected, and all of them were multi-drug resistant. There were 30 LREfs strains (76.9%) carrying the optrA gene, The cfr, poxtA genes and mutations in the 23S rRNA gene were not detected. The conjugation experiments showed that only three of 10 randomly selected optrA-carrying LREfs were successfully conjugated with JH2-2. Further analysis of one successfully conjugated strain revealed that the optrA gene, located in the donor bacterium, formed the IS1216E-erm(A)-optrA-fexA-IS1216E transferable fragment under the mediation of the mobile genetic element (MGE) IS1216E, which was then transferred to the recipient bacterium via horizontal plasmid transfer. Carrying the optrA gene is the primary resistance mechanism of LREfs strains. The optrA gene could carry the erm(A) and fexA genes to co-transfer among E. faecalis. MGEs such as insertion sequence IS1216E play an important role in the horizontal transfer of the optrA gene.

Metabolic interactions control the transfer and spread of plasmid-encoded antibiotic resistance during surface-associated microbial growth

Surface-associated microbial systems are hotspots for the spread of plasmid-encoded antibiotic resistance, but how surface association affects plasmid transfer and proliferation remains unclear. Surface association enables prolonged spatial proximities between different populations, which promotes plasmid transfer between them. However, surface association also fosters strong metabolic interactions between different populations, which can direct their spatial self-organization with consequences for plasmid transfer and proliferation. Here, we hypothesize that metabolic interactions direct the spatial self-organization of different populations and, in turn, regulate the spread of plasmid-encoded antibiotic resistance. We show that resource competition causes populations to spatially segregate, which represses plasmid transfer. In contrast, resource cross-feeding causes populations to spatially intermix, which promotes plasmid transfer. We further show that the spatial positionings that emerge from metabolic interactions determine the proliferation of plasmid recipients. Our results demonstrate that metabolic interactions are important regulators of both the transfer and proliferation of plasmid-encoded antibiotic resistance.