Plasmid-mediated Resistance Genes Research Articles

Dissemination and prevalence of plasmid-mediated high-level tigecycline resistance gene tet (X4).

With the large-scale use of antibiotics, antibiotic resistant bacteria (ARB) continue to rise, and antibiotic resistance genes (ARGs) are regarded as emerging environmental pollutants. The new tetracycline-class antibiotic, tigecycline is the last resort for treating multidrug-resistant (MDR) bacteria. Plasmid-mediated horizontal transfer enables the sharing of genetic information among different bacteria. The tigecycline resistance gene tet(X) threatens the efficacy of tigecycline, and the adjacent ISCR2 or IS26 are often detected upstream and downstream of the tet(X) gene, which may play a crucial driving role in the transmission of the tet(X) gene. Since the first discovery of the plasmid-mediated high-level tigecycline resistance gene tet(X4) in China in 2019, the tet(X) genes, especially tet(X4), have been reported within various reservoirs worldwide, such as ducks, geese, migratory birds, chickens, pigs, cattle, aquatic animals, agricultural field, meat, and humans. Further, our current researches also mentioned viruses as novel environmental reservoirs of antibiotic resistance, which will probably become a focus of studying the transmission of ARGs. Overall, this article mainly aims to discuss the current status of plasmid-mediated transmission of different tet(X) genes, in particular tet(X4), as environmental pollutants, which will risk to public health for the “One Health” concept.

Widespread Dissemination of Plasmid-Mediated Tigecycline Resistance Gene tet(X4) in Enterobacterales of Porcine Origin.

ABSTRACTThe emergence of the plasmid-mediated high levels of the tigecycline resistance gene has drawn worldwide attention and has posed a major threat to public health. In this study, we investigated the prevalence of the tet(X4)-positive Enterobacterales isolates collected from a pig slaughterhouse and farms. A total of 101 tigecycline resistance strains were isolated from 353 samples via a medium with tigecycline, of which 33 carried tet(X4) (9.35%, 33/353) and 2 carried tet(X6) (0.57%, 2/353). These strains belong to seven different species, with Escherichia coli being the main host bacteria. Importantly, this report is the first one to demonstrate that tet(X4) was observed in Morganella morganii. Whole-genome sequencing results revealed that tet(X4)-positive bacteria can coexist with other resistance genes, such as blaNDM-1 and cfr. Additionally, we were the first to report that tet(X4) and blaNDM-1 coexist in a Klebsiella quasipneumoniae strain. The phylogenetic tree of 533 tet(X4)-positive E. coli strains was constructed using 509 strains from the NCBI genome assembly database and 24 strains from this study, which arose from 8 sources and belonged to 135 sequence types (STs) worldwide. We used Nanopore sequencing to interpret the selected 21 nonclonal and representative strains and observed that 19 tet(X4)-harboring plasmids were classified into 8 replicon types, and 2 tet(X6) genes were located on integrating conjugative elements. A total of 68.42% of plasmids carrying tet(X4) were transferred successfully with a conjugation frequency of 10−2 to 10−7. These findings highlight that diverse plasmids drive the widespread dissemination of the tigecycline resistance gene tet(X4) in Enterobacterales of porcine origin.IMPORTANCE Tigecycline is considered to be the last resort of defense against diseases caused by broad-spectrum resistant Gram-negative bacteria. In this study, we systematically analyzed the prevalence and genetic environments of the resistance gene tet(X4) in a pig slaughterhouse and farms and the evolutionary relationship of 533 tet(X4)-positive Escherichia coli strains, including 509 tet(X4)-positive E. coli strains selected from the 27,802 assembled genomes of E. coli from the NCBI between 2002 and 2022. The drug resistance of tigecycline is widely prevalent in pig farms where tetracycline is used as a veterinary drug. This prevalence suggests that pigs are a large reservoir of tet(X4) and that tet(X4) can spread horizontally through the food chain via mobile genetic elements. Furthermore, tetracycline resistance may drive tigecycline resistance through some mechanisms. Therefore, it is important to monitor tigecycline resistance, develop effective control measures, and focus on tetracycline use in the pig farms.

Investigation of In Vitro Susceptibility and Resistance Mechanisms in Skin Pathogens: Perspectives for Fluoroquinolone Therapy in Canine Pyoderma.

Fluoroquinolones (FQ) are commonly used in dogs with bacterial skin infections. Their use as first choice, along with the increased incidence of FQ-resistance, represents a risk to animal and public health. Our study determined minimum inhibitory (MIC) and bactericidal (MBC) concentrations of five FQs in Staphylococcus aureus, Staphylococcus pseudintermedius, and Escherichia coli, together with FQ-resistance mechanisms. MICs, efflux pump (EP) overexpression and MBCs were measured in 249 skin infection isolates following CLSI guidelines (CLSI VET01-A4, CLSI M26-A). Chromosomal and plasmid-mediated resistance genes were investigated after DNA extraction and sequencing. FQ-resistance was detected in 10% of methicillin-susceptible (MS), 90% of methicillin-resistant (MR) staphylococci and in 36% of E. coli. Bactericidal effect was observed except in 50% of MRSA/P for ciprofloxacin and in 20% of MRSPs for enrofloxacin. Highest MICs were associated with double mutation in gyrA (Ser83Leu + Asp87Asn), efflux pumps and three PMQR genes in E. coli, and grlA (Ser80Phe + Glu84Lys) in S. aureus. EP overexpression was high among E. coli (96%), low in S. aureus (1%) and absent in S. pseudintermedius. Pradofloxacin and moxifloxacin showed low MICs with bactericidal effect. Since in vitro FQ resistance was associated with MR, FQ use should be prudently guided by susceptibility testing.

Environmentally relevant concentrations of mercury facilitate the horizontal transfer of plasmid-mediated antibiotic resistance genes

Abundant antibiotic resistance genes (ARGs) are typically found in mercury (Hg)-contaminated aquatic environments. This phenomenon is partly attributed to the co-resistance, cross-resistance, and shared regulatory responses to Hg and antibiotics. However, it remains unclear whether and how Hg influences the conjugative transfer of ARGs mediated by mobilizable plasmids. In the present study, we found that Hg2+ at the environmentally relevant concentrations (0.001–0.5 mg L−1) facilitated the conjugative transfer of ARGs through the mobilizable plasmid RP4 from the donor Escherichia coli HB101 to the recipient E. coli K12. Exposure to Hg2+ significantly increases the formation of reactive oxygen species, malondialdehyde production, antioxidant enzyme activities, and cell membrane permeability, while decreasing the concentration of glutathione. Scanning electron microscopy and transmission electron microscopy showed that the cell membrane suffered from oxidative damage, which is beneficial for conjugative transfer. The expression of global regulatory genes (korA, korB, and trbA) negatively regulating conjugative transfer was restrained by Hg2+, while promoting the expression of positive regulatory genes involved in the mating pair formation system (trbBp and traF) and the DNA transfer and replication systems (trfAp and traJ). Although a high Hg2+ concentration (1.0 mg L−1) suppressed ARGs conjugative transfer, our results suggest that Hg2+ facilitates the dissemination of ARGs in aquatic environments at environmentally relevant concentrations. This study improves our understanding of ARGs dissemination in Hg-contaminated aquatic environments.

Emergence of a Novel Plasmid-Mediated Tigecycline Resistance Gene Cluster, tmexCD4-toprJ4, in Klebsiella quasipneumoniae and Enterobacter roggenkampii.

ABSTRACTThe occurrence of transferable tigecycline resistance determinants, tmexCD1-toprJ1, tmexCD2-toprJ2, tmexCD3-toprJ1b, and multiple tet(A) and tet(X) variants, presents an unprecedented challenge to clinical therapeutic options. tmexCD-toprJ-like gene clusters can mediate multidrug resistance and have been detected in a variety of bacteria. Here, we characterized the fourth tmexCD-toprJ-like gene cluster, tmexCD4-toprJ4, identified on untypeable plasmids of Klebsiella quasipneumoniae and Enterobacter roggenkampii isolated from chicken meat and environmental samples from farm markets, respectively. TMexCD4-TOprJ4 was closely related (92 to 99% amino acid identity) to TMexCD1-TOprJ1, TMexCD2-TOprJ2, and TMexCD3-TOprJ1. Phylogenetic analysis revealed that tmexCD4-toprJ4 was not in the same branch as the other three variants. Expression of tmexCD4-toprJ4 increased tigecycline efflux in Escherichia coli and resulted in a 4- to 8-fold increase in MICs of tigecycline in E. coli and Klebsiella pneumoniae. Moreover, tmexCD4-toprJ4 can act synergistically with its upstream gene tet(A) to reduce the susceptibility of E. coli and K. pneumoniae strains to tigecycline. The tmexCD4-toprJ4-containing plasmid is a novel plasmid type and can be transferred to E. coli and K. pneumoniae only via electrotransformation. The increasing emergence of plasmid-mediated tigecycline resistance gene clusters suggests that the spread of tmexCD-toprJ-like gene clusters requires widespread attention.IMPORTANCE The plasmid-mediated tigecycline resistance gene cluster tmexCD1-toprJ1 and other variants have been detected in a variety of strains from multiple sources, including human-derived strains. In addition to tigecycline, these tmexCD-toprJ-like gene clusters reduce susceptibility of the host strain to many other antimicrobials. Here, we identified tmexCD4-toprJ4 in K. quasipneumoniae and E. roggenkampii, suggesting that this gene cluster is already present in the human-associated environment and the risk of transmission to humans is increased. Monitoring tigecycline-resistant Gram-negative bacteria is essential for understanding and addressing the spread of this gene cluster in agriculture and health care.

Emergence of plasmid-mediated tigecycline resistance gene, tet(X4), in Escherichia fergusonii from pigs

This study aimed to identify tigecycline-resistant tet(X4)-bearing Escherichia fergusonii isolated from pigs in China with a complete genome sequence. E. fergusonii was isolated by selective medium and screened from tigecycline-supplemented agar plates. The microbroth dilution method was used for antimicrobial susceptibility testing, and the minimal inhibitory concentration (MIC) results refer to the interpretation standard in the Clinical and Laboratory Standards Institute of America (CLSI). Whole-genome sequencing was performed on the Illumina HiSeq and Nanopore GridION platforms. The antimicrobial resistance (AMR) genes virulence genes and replicon types of plasmids were predicted by the CGE server. E. fergusonii EF21QZZ116 was identified from 760 faecal and caecal content samples and was resistant to tigecycline, tetracycline, ampicillin, sulfisoxazole, trimethoprim-sulfamethoxazole, spectinomycin, and florfenicol. The AMR genes tet(X4), blaTEM-1B, and floR are located on the hybrid IncFIA-IncHI1A-IncHI1B plasmid pQZZ166-tetX-190K and are 190 391 bp in length. This hybrid plasmid has high homology with the plasmids of Escherichia coli, Enterobacter cloacae, Klebsiella, Citrobacter, and Salmonella based on comparison and analysis with the NCBI database. Notably, conjugative transfer and S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) assays show that the plasmid can transfer horizontally. To the best of our knowledge, this is the first report of E. fergusonii carrying the tet(X4) gene isolated from a pig; this report provides insight into the AMR characteristics of E. fergusonii and offers insight into public health.

Effect of sulfamethazine on the horizontal transfer of plasmid-mediated antibiotic resistance genes and its mechanism of action

As a new type of environmental pollutant, antibiotic resistance genes (ARGs) pose a huge challenge to global health. Horizontal gene transfer (HGT) represents an important route for the spread of ARGs. The widespread use of sulfamethazine (SM2) as a broad-spectrum bacteriostatic agent leads to high residual levels in the environment, thereby increasing the spread of ARGs. Therefore, we chose to study the effect of SM2 on the HGT of ARGs mediated by plasmid RP4 from Escherichia coli (E. coli) HB101 to E. coli NK5449 as well as its mechanism of action. The results showed that compared with the control group, SM2 at concentrations of 10 mg/L and 200 mg/L promoted the HGT of ARGs, but transfer frequency decreased at concentrations of 100 mg/L and 500 mg/L. The transfer frequency at 200 mg/L was 3.04 × 10−5, which was 1.34-fold of the control group. The mechanism of SM2 improving conjugation transfer is via enhancement of the mRNA expression of conjugation genes (trbBP, trfAP) and oxidative stress genes, inhibition of the mRNA expression of vertical transfer genes, up regulation of the outer membrane protein genes (ompC, ompA), promotion of the formation of cell pores, and improvement of the permeability of cell membrane to promote the conjugation transfer of plasmid RP4. The results of this study provide theoretical support for studying the spread of ARGs in the environment.

Large-Scale Analysis of Fitness Cost of tet(X4)-Positive Plasmids in Escherichia coli.

Tigecycline is one of important antimicrobial agents for the treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, the emergence and prevalence of plasmid-mediated tigecycline resistance gene tet(X4) are threatening human and animal health. Fitness cost elicited by resistance plasmids is a key factor affecting the maintenance and transmission of antibiotic resistance genes (ARGs) in the host. A comparative analysis of the fitness cost of different types of tet(X4)-positive plasmids is helpful to understand and predict the prevalence of dominant plasmids. In this study, we performed a large-scale analysis of fitness cost of tet(X4)-positive plasmids origin from clinical isolates. These plasmids were successfully electroporated into a reference strain Escherichia coli TOP10, and a series of transformants carrying the tet(X) gene were obtained. The effects of tet(X4)-positive plasmids on the growth rate, plasmid stability, relative fitness, biofilm formation, and virulence in a Galleria mellonella model were evaluated. Consequently, we found that these plasmids resulted in varying degrees of fitness cost on TOP10, including delayed bacterial growth and attenuated virulence. Out of these plasmids, tet(X4)-harboring IncFII plasmids showed the lowest fitness cost on the host. Furthermore, by means of experimental evolution in the presence of commonly used drugs in clinic, the fitness cost of tet(X4)-positive plasmids was substantially alleviated, accompanied by increased plasmid stability. Collectively, our data reveal the differential fitness cost caused by different types of tet(X4)-positive plasmids and suggest that the wide use of tetracycline antibiotics may promote the evolution of plasmids.

Genetic and epidemiological analysis of ESBL-producing Klebsiella pneumoniae in three Japanese university hospitals

IntroductionWe aimed to clarify the genetic background and molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) at three geographically separated university hospitals in Japan. MethodsFrom January 2014 to December 2016, 118 ESBL-producing K. pneumoniae (EPKP) strains that were detected and stored at three university hospitals were collected. Molecular epidemiological analysis was performed using enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) and multi-locus sequence typing (MLST). The ESBL type was determined using the PCR-sequence method. The presence of plasmid-mediated fluoroquinolone resistance (PMQR) genes was analyzed by PCR. We compared the relationships between PMQR gene possession/quinolone resistance-determining region (QRDR) mutation and levofloxacin (LVFX)/ciprofloxacin (CPFX) susceptibility. ResultsThe detection rate of EPKP was 4.8% (144/2987 patients). MLST analysis revealed 62 distinct sequence types (STs). The distribution of STs was diverse, and only some EPKP strains had the same STs. ERIC-PCR showed discriminatory power similar to that of MLST. The major ESBL genotypes were CTX-M-15-, CTX-M-14-, and SHV-types, which were detected in 47, 30, and 27 strains, respectively. Ninety-one out of 118 strains had PMQR genes and 14 out of 65 strains which were not susceptible to CPFX had QRDR mutations, and the accumulation of PMQR genes and QRDR mutations tended to lead to higher minimum inhibitory concentrations (MICs) of LVFX. ConclusionsAt three geographically separated university hospitals in Japan, the epidemiology of EPKP was quite diverse, and no epidemic strains were found, whereas CTX-M-14 and CTX-M-15 were predominant.

Expression of plasmid-mediated resistance genes ESBLs and PMQR among uropathogens, isolated from non-dialysis CKD patients with pyelonephritis

Urinary tract infections (UTIs) are thought to be the most common group of bacterial infections, worldwide. It is estimated that the proportion of UTIs remains at a high level reaching 150 million episodes per year worldwide and accounting for $6 billion in health care expenditures. In USA more than 100,000 hospital admissions per year are the UTIs patients, among them the patients with pyelonephritis are the most frequent. For the US outpatients, approximately 15 % of antibiotics used are prescribed for UTIs. However, recent studies in Europe and the United States have demonstrated a steady increase in the rate of uropathogen resistance to commonly prescribed antibiotics, and this obviously will lead to a reduction in therapeutic possibilities of UTI. CDC has estimated that more thаn 2 million infections and 23,000 deaths are due to antibiotic resistance each year. By 2050, it is estimated, that antibiotic resistance will cause 10 million deaths every year. Infection is an importаnt cause of mоrbidity and mоrtality amоng patients with kidney failurе and is the sеcond lеading causе of death following CVD. At the EU/EEA level, more than half (54 %) of the E.coli isolates reported to EARS-Net for 2020 were resistant to at least one of the antimicrobial groups under regular surveillance, i.e. aminopenicillins, fluoroquinolones, third-generation cephalosporins, aminoglycosides and carbapenems. A majority (87.4 %) of the third-generation cephalosporin-resistant E.coli isolates from 2020 were extended-spectrum beta-lactamase (ESBL) positive. Use of broad-spectrum antimicrobials is a known risk factor for colonization and spread of resistant Enterobacteriaceae, including E.coli. The high levels of ESBLs and increasing resistance to key antimicrobial groups might also lead to an increased consumption of carbapenems, which in turn can increase the selection pressure and facilitated the spread of carbapenem-resistant Enterobacteriaceae. The article presents data on the antibacterial susceptibility of isolated uropathogens, and their association with the presence or absence of plasmid-mediated resistance genes ESBLs and PMQR.

Plasmid-Mediated Fluoroquinolone Resistance Genes in Quinolone-Susceptible Aeromonas spp. Phenotypes Isolated From Recreational Surface Freshwater Reservoir

Aeromonas spp. are recognized as opportunistic pathogens causing diseases. Infections in humans can result mainly in gastrointestinal and wound diseases with or without progression to septicemia. Although Aeromonas spp. are not known uropathogens and they rarely cause urinary tract infection, we hypothesize that the presence of these bacteria in the water and the contact during, e.g., recreational and bathing activity can create the conditions for the colonization of the human body and may result to diseases in various locations, including the urinary tract. Our study presents the occurrence of aeromonad fluoroquinolone-susceptible phenotypes with the presence of plasmid-mediated fluoroquinolone resistance (PMQR) genes in a natural freshwater reservoir occasionally used for recreational activities. Sixty-nine isolates collected during the bathing period were identified by mass spectrometry and screened for the presence of fluoroquinolone-resistant phenotypes and genotypes. Fluoroquinolone susceptibility was determined as minimal inhibitory concentration values. PMQR qnr genes were detected by PCR. Isolates comprising eight species, namely, mainly Aeromonas veronii (50.7% isolates) and Aeromonas media (24.6% isolates) and rarely Aeromonas eucrenophila, Aeromonas caviae, Aeromonas bestiarum, Aeromonas ichthiosmia, and Aeromonas hydrophila, were selected. All isolates were phenotypically susceptible either to ciprofloxacin or levofloxacin. Unexpectedly, at least one to three of the PMQR genes were detected in 42.0% of the fluoroquinolone-susceptible Aeromonas spp. phenotypes. Mainly the qnrS (34.8% isolates) and qnrA (14.5% isolates) determinants were detected. In conclusion, the freshwater reservoir occasionally used for bathing was tainted with aeromonads, with a high occurrence of opportunistic pathogens such as A. veronii and A. media. MALDI‐TOF MS is a powerful technique for aeromonad identification. Our data reveals the mismatch phenomenon between fluoroquinolone-susceptible aeromonad phenotypes and the presence of plasmid-mediated qnr resistance genes. It suggests that phenotypically susceptible bacteria might be a potential source for the storage and transmission of these genes. The exposure during, e.g., a recreational activity may create the potential risk for causing infections, both diagnostically and therapeutically difficult, after expressing the resistance genes and quinolone-resistant strain selection.

Extensive spread of tet(X4) in multidrug-resistant Escherichia coli of animal origin in western China

The recent emergence of plasmid-mediated tigecycline resistance gene tet(X) variants represents a threat to public health. tet(X4) is highly endemic in northwestern China. Here, we extended the region to western China to further understand its epidemiology and distribution. During 2018–2020, 1497 faecal samples from pigs, sheep and goats in western China were screened for tet(X4)-positive strains. A total of 134 tigecycline-nonsusceptible Escherichia coli were isolated; among them, the largest number were isolated from Guangxi (66.42%, 89/134), Shaanxi (17.16%, 23/134) and Ningxia (8.21%, 11/134). Antibiotic susceptibility testing showed that all of these isolates were multidrug-resistant bacteria that were also resistant to florfenicol. Eighty-nine of 134 tet(X4)-positive Escherichia coli were analysed by whole-genome sequencing. They belonged to twenty-seven sequence types, and ST10, ST48, ST189 and ST2223 were the main types of Escherichia coli. Seventy-six of the 134 tet(X4)-positive Escherichia coli could transfer tet(X4) into the recipient Escherichia coli 26 R 793. Southern blot analysis showed that there were multiple plasmids carrying the tet(X4) gene in a single strain. The plasmids of the 89 tet(X4)-bearing isolates in this study were analysed. tet(X4)-bearing plasmids ranged from 20 to 400 kb, and the main plasmid types were categorised as IncX1, IncY, and ColRNAI. Guangxi, Ningxia, and Shanxi are likely to be the seminal and most affected areas. Additional efforts are needed to evaluate the impact of the spread of these tigecycline resistance genes and tigecycline-resistant isolates.

Uncovering antimicrobial resistance in three agricultural biogas plants using plant-based substrates

Antimicrobial resistance (AMR) is becoming an increasing global concern and the anaerobic digestion (AD) process represents a potential transmission route when digestates are used as fertilizing agents. AMR contaminants, e.g. antibiotic-resistant bacteria (ARB) and plasmid-mediated antibiotic resistance genes (ARGs) have been found in different substrates and AD systems, but not yet been investigated in plant-based substrates. AMR transfer from soils to vegetable microbiomes has been observed, and thus crop material potentially represents a so far neglected AMR load in agricultural AD processes, contributing to AMR spread. In order to test this hypothesis, this study examined the AMR situation throughout the process of three biogas plants using plant-based substrates only, or a mixture of plant-based and manure substrates. The evaluation included a combination of culture-independent and –dependent methods, i.e., identification of ARGs, plasmids, and pathogenic bacteria by DNA arrays, and phylogenetic classification of bacterial isolates and their phenotypic resistance pattern. To our knowledge, this is the first study on AMR in plant-based substrates and the corresponding biogas plant. The results showed that the bacterial community isolated from the investigated substrates and the AD processing facilities were mainly Gram-positive Bacillus spp. Apart from Pantoea agglomerans, no other Gram-negative species were found, either by bacteria culturing or by DNA typing array. In contrast, the presence of ARGs and plasmids clearly indicated the existence of Gram-negative pathogenic bacteria, in both substrate and AD process. Compared with substrates, digestates had lower levels of ARGs, plasmids, and culturable ARB. Thus, digestate could pose a lower risk of spreading AMR than substrates per se. In conclusion, plant-based substrates are associated with AMR, including culturable Gram-positive ARB and Gram-negative pathogenic bacteria-associated ARGs and plasmids. Thus, the AMR load from plant-based substrates should be taken into consideration in agricultural biogas processing.

Presence of Mobile Tigecycline Resistance Gene tet(X4) in Clinical Klebsiella pneumoniae.

ABSTRACTThe recently emerged plasmid-mediated tigecycline resistance gene tet(X4) has mainly been detected in Escherichia coli but never in Klebsiella pneumoniae. Herein, we identified a clinical K. pneumoniae isolate that harbored the tet(X4) gene located on a non-self-transferable IncFII-type plasmid, which could be cotransferred with a conjugative plasmid to E. coli C600. The extending of bacterial species carrying tet(X4) suggested the increasing risk of spreading mobile tigecycline resistance genes among important pathogens in clinical settings.IMPORTANCE Tigecycline, the first member of glycylcycline class antibiotic, is often considered one of the effective antibiotics against multidrug-resistant (MDR) infections. However, the emergence and wide distribution of two novel plasmid-mediated tigecycline resistance genes, tet(X3) and tet(X4), pose a great threat to the clinical use of tigecycline. The newly tet(X) variants have been identified from multiple different bacterial species, but the tet(X) variant in the Klebsiella pneumoniae strain has been reported only once before. In this study, we identified a clinical K. pneumoniae isolate that harbored a non-self-transferable tet(X4)-carrying plasmid. This plasmid has never been found in other tet(X4)-harboring strains and could be cotransferred with a conjugative plasmid to the recipient strain. Our findings indicate that the tet(X4) gene breaks through its original bacterial species and spreads to some important nosocomial pathogens, which posed a serious threat to public health.

Epidemiology and genotypic characteristics of carbapenem resistant Enterobacterales in Henan, China: a multicentre study

To identify the carbapenemase and colistin resistance genes and epidemiological status of carbapenem-resistant Enterobacterales (CRE) among 7 secondary and 11 tertiary hospitals in Henan province, China. CRE isolates and clinical data of infected patients were collected from 7 secondary and 11 tertiary hospitals in Henan from July to September 2019 and analysed retrospectively. Polymerase chain reaction (PCR) and gene sequencing were performed to detect carbapenemase and colistin resistance genes; multilocus sequence typing was also performed. In total, 238 nonduplicate CRE isolates were collected mainly from the respiratory tract (54.20%) and blood (18.91%) of CRE-infected patients, half of them aged >65 years (45.80%). Carbapenem-resistant Klebsiella pneumoniae (CRKP) was the most common CRE (184 isolates, 77.31%) with constituent ratios of 84.38% and 72.54% in secondary and tertiary hospitals, respectively. In 184 CRKP isolates, blaKPC-2 (89.13%) was the dominant carbapenemase gene, and ST11 (71.74%) was the most prevalent sequence type (ST), with constituent ratios of 83.95% and 62.14% in secondary and tertiary hospitals, respectively. In 29 carbapenem-resistant Escherichia coli (CREC) isolates, blaNDM-5 (58.62%) and ST2 (31.03%) were prevalent. Four CRKP isolates and one CREC isolate were colistin-resistant and carried the plasmid-mediated mcr-1 gene. Our results showed a wide spread of CRKP-ST11 with KPC-2 carbapenemase in the analysed 18 hospitals. The CRKP constituent ratio, CRKP-STs, and CREC carbapenemase genes between secondary and tertiary hospitals showed significant differences. The emergence of a colistin-resistant CRKP with plasmid-mediated resistance gene mcr-1 is of serious concern due to the limited treatment options.

Virulence Genotype and Phenotype of Multiple Antimicrobial-Resistant Escherichia coli Isolates from Broilers Assessed from a “One-Health” Perspective

Extraintestinal pathogenic Escherichia coli (ExPEC) include several serotypes that have been associated with colibacillosis in poultry and with urinary tract infections (UTIs) and newborn meningitis in humans. In this study, 57 antimicrobial-resistant E. coli from apparently healthy broiler chickens were characterized for their health and safety risks. These isolates belonged to 12 serotypes, and isolates of the same serotype were clonal based on single nucleotide variant analysis. Most of the isolates harbored plasmids; IncC and IncFIA were frequently detected. The majority of the resistant isolates harbored plasmid-mediated resistance genes, including aph(3″)-Ib, aph(6)-Id, blaCMY-2, floR, sul1, sul2, tet(A), and tet(B), in agreement with their resistant phenotypes. The class 1 integron was detected in all E. coli serotypes except O124:H25 and O7:H6. Of the 57 broiler E. coli isolates, 27 were avian pathogenic, among which 18 were also uropathogenic E. coli and the remainder were other ExPEC. The two isolates of serotype O161:H4 (ST117) were genetically related to the control avian pathogenic strains and a clinical isolate associated with UTIs. A strain of serotype O159:H45 (ST101) also was closely related to a UTI isolate. The detected virulence factors included adhesins, invasins, siderophores, type III secretion systems, and toxins in combination with other virulence determinants. A broiler isolate of serotype O7:H18 (ST38) carried the ibeA gene encoding a protein involved in invasion of brain endothelium on a 102-kbp genetic island. This isolate moderately adhered and invaded Caco-2 cells and induced mortality (42.5%) in a day-old-chick infection model. The results of this study suggest that multiple antimicrobial-resistant E. coli isolates recovered from apparent healthy broilers can be pathogenic and act as reservoirs for antimicrobial resistance genes, highlighting the necessity of their assessment in a "One-Heath" context.

Environmentally Relevant Concentrations of Mercury Facilitate the Horizontal Transfer of Plasmid-Mediated Antibiotic Resistance Genes

Environmentally Relevant Concentrations of Mercury Facilitate the Horizontal Transfer of Plasmid-Mediated Antibiotic Resistance Genes

Hormesis Effect of Pyroligneous Acids on Conjugative Transfer of Plasmid-Mediated Multi-Antibiotic Resistance Genes within Bacterial Genus

Hormesis Effect of Pyroligneous Acids on Conjugative Transfer of Plasmid-Mediated Multi-Antibiotic Resistance Genes within Bacterial Genus

Comprehensive Genomic Investigation of Tigecycline Resistance Gene tet(X4)-Bearing Strains Expanding among Different Settings.

ABSTRACTThe emergence of plasmid-mediated tigecycline resistance genes has attracted a great deal of attention globally. Currently, no comprehensive in-depth genomic epidemiology study of tet(X4)-bearing pathogens present of pork origin as the One Health approach has been performed. Herein, 139 fresh pork samples were collected from multiple regions in China and 58 tet(X4)-positive strains were identified. The tet(X4) gene mainly distributed in Escherichia coli (n = 55). Besides, 4 novel tet(X4)-positive bacterial species Klebsiella pneumoniae (n = 2), Klebsiella quasipneumoniae (n = 1), Citrobacter braakii (n = 1) and Citrobacter freundii (n = 1) were first characterized here. Four different core tet(X4)-bearing genetic environments and five types of tet(X4)-bearing tandem duplications were discovered among 58 strains. The results of the phylogenetic tree showed that there was some correlation between E. coli strains from pork, human, pig farms, and slaughterhouses. A total of seven types of plasmid replicons were found in tet(X4)-positive plasmids, among which multireplicon plasmids were observed. Notably, two tet(X4)-positive fusion plasmids pCSZ11R (IncX1-IncFIA-IncFIB-IncFIC) and pCSX5G-tetX4 (IncX1-IncFII-IncFIA) were formed by IS26 in the hot spot. Besides, six samples were identified to harbor two different tet(X4)-bearing strains. More interestingly, the absolute quantitative results showed that the expression levels of tet(X4) between different strains with different tet(X4) copies were approximate. In this study, the genetic environment of tet(X4)-positive plasmids containing different plasmid replicons was analyzed to provide a basis for the further development of effective control measures. It is also highlighted that animal-borne tet(X4)-bearing pathogens incur a transmission risk to consumed food. Therefore, there is an urgent need for large-scale monitoring as well as the development of effective control measures.IMPORTANCE Tigecycline was considered the last-line drug against serious infections caused by multidrug-resistant Gram-negative bacteria. However, the plasmid-mediated tigecycline resistance gene tet(X) has been widely reported in different sources of Enterobacterales and Acinetobacter in China. China is one of the largest pig-producing nations in the world, and in-depth investigation of gene in pork is vital to figure out the fundamental dissemination of these genes and set up a reasonable control framework. In this study, we conducted an in-depth and systematic analysis of the diversity of tet(X4)-positive plasmids and the genetic environment of tet(X4) contained in pork samples from multiple regions of China, providing a basis for further development of effective control measures. It is also highlighted that animal-borne tet(X4)-bearing pathogens incur a transmission risk to consumed food. Therefore, there is an urgent need for large-scale monitoring as well as the development of effective control measures.

Paclitaxel and its derivative facilitate the transmission of plasmid-mediated antibiotic resistance genes through conjugative transfer

The rapid dissemination of antibiotic resistance by horizontal gene transfer (HGT) renders the global resistance crisis more tense and urgent as few effective antimicrobials are available to combat multidrug-resistant (MDR) pathogens at present. Conjugation is one of the most dominant and representative pathways of HGT. Antibiotic residue in environment is recognized as an important accelerator for conjugal transfer, whereas the roles of non-antibiotic pharmaceuticals in this process are not fully understood. Here we found that environmentally relevant concentrations of paclitaxel as well as its derivative docetaxel, two commonly used anticancer drugs, remarkably facilitated the conjugative transfer of resistance plasmids carrying multiple antibiotic resistance genes (ARGs). The underlying mechanisms accounting for the enhanced conjugation were investigated by detecting the activity of RpoS regulon, membrane permeability, SOS response and gene expression of conjugative transfer systems. Our results showed that paclitaxel induced a series of cellular responses, including up-regulation of rpoS expression, activated SOS response, increased cell membrane permeability, enhanced plasmid replication and mating pilus formation. Collectively, our data provide new insight on the roles of paclitaxel and its derivative in promoting the conjugal transfer of ARGs, highlighting the importance of good antimicrobial stewardship.