Transmission of blaNDM in Enterobacteriaceae among animals, food and human.

Despite carbapenems not being used in animals, carbapenem-resistant Enterobacterales (CRE), particularly New Delhi metallo-ß-lactamase-producing CRE (NDM-CRE), are prevalent in livestock. Concurrently, the incidence of human infections caused by NDM-CRE is rising, particularly in children. Although a positive association between livestock production and human NDM-CRE infections at the national level was identified, the evidence of direct transmission of NDM originating from livestock to humans remains largely unknown. Here, we conducted a cross-sectional study in Chengdu, Sichuan Province, to examine the prevalence of NDM-CRE in chickens and pigs along the breeding-slaughtering-retail chains, in pork in cafeterias of schools, and in colonizations and infections from children's hospital and examined the correlation of NDM-CRE among animals, foods and humans. Overall, the blaNDM increases gradually along the chicken and pig breeding (4.70%/2.0%) -slaughtering (7.60%/22.40%) -retail (65.56%/34.26%) chains. The slaughterhouse has become a hotspot for cross-contamination and amplifier of blaNDM. Notably, 63.11% of pork from the school cafeteria was positive for blaNDM. The prevalence of blaNDM in intestinal and infection samples from children's hospitals was 21.68% and 19.80%, respectively. whole genome sequencing (WGS) analysis revealed the sporadic, not large-scale, clonal spread of NDM-CRE along the chicken and pig breeding-slaughtering-retail chain, with further spreading via IncX3-blaNDM plasmid within each stage of whole chains. Clonal transmission of NDM-CRE is predominant in children's hospitals. The IncX3-blaNDM plasmid was highly prevalent among animals and humans and accounted for 57.7% of Escherichia coli and 91.3% of Klebsiella pneumoniae. Attention should be directed towards the IncX3 plasmid to control the transmission of blaNDM between animals and humans.

MC-PRPA-HLFIA Cascade Detection System for Point-of-Care Testing Pan-Drug-Resistant Genes in Urinary Tract Infection Samples.

Recently, urinary tract infection (UTI) triggered by bacteria carrying pan-drug-resistant genes, including carbapenem resistance gene blaNDM and blaKPC, colistin resistance gene mcr-1, and tet(X) for tigecycline resistance, have been reported, posing a serious challenge to the treatment of clinical UTI. Therefore, point-of-care (POC) detection of these genes in UTI samples without the need for pre-culturing is urgently needed. Based on PEG 200-enhanced recombinase polymerase amplification (RPA) and a refined Chelex-100 lysis method with HRP-catalyzed lateral flow immunoassay (LFIA), we developed an MCL-PRPA-HLFIA cascade assay system for detecting these genes in UTI samples. The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 min without high-speed centrifugation or pre-incubation of urine samples. Following optimization, the cascade detection system achieved an LOD of 102 CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. It takes less than an hour to complete the process without the use of high-speed centrifuges or other specialized equipment, such as PCR amplifiers. The MCL-PRPA-HLFIA cascade assay system provides new ideas for the construction of rapid detection methods for pan-drug-resistant genes in clinical UTI samples and provides the necessary medication guidance for UTI treatment.

Aeromonas spp. from hospital sewage act as a reservoir of genes resistant to last-line antibiotics.

BACKGROUND: Aeromonas species are opportunistic pathogens distributed widely in the ecosystem. They are known to be capable of acquiring antibiotic resistance genes, including those encoding proteins against last-line antibiotics, such as the tmexCD-toprJ, mcr and carbapenemase genes. We investigated the genomic and phenotypic characteristics of tmexCD-toprJ-positive Aeromonas strains collected from human, animals, and water samples, particularly those from hospital wastewater in China. METHODS: Samples were collected from living animals, meat, water and human. Aeromonas strains in these samples were isolated in selective media. Antimicrobial resistance profiles of all Aeromonas strains were tested by the broth microdilution method. The presence of tmexCD-toprJ was verified by polymerase chain reaction (PCR). All tmexCD-toprJ-positive (n = 36) and selected tmexCD-toprJ-negative (n = 18) Aeromonas strains were subjected to whole genome sequencing. Carriage of antimicrobial resistance genes, the genetic environment of tmexCD-toprJ and genetic diversity of tmexCD-toprJ-positive Aeromonas strains were determined by bioinformatics analysis. Phylogenetic tree of the Aeromonas strains was built by using the Harvest Suite. FINDINGS: Among the 636 Aeromonas strains isolated from different sources, 36 were positive for tmexCD-toprJ, with the highest prevalence of tmexCD-toprJ being found in fishes (8.8%, 95 CI% 3.6-17.2%), followed by hospital wastewater (6.5%, 95 CI% 4.3-9.3%), river water (2.0%, 0.1-10.9) and duck (1.2%, 95 CI% 3.6-17.2%). All tmexCD-toprJ-positive Aeromonas strains carried multiple antimicrobial resistance genes and exhibited resistance to different classes of antibiotics. Co-existence of tmexCD-toprJ, mcr and blaKPC-2 were identified in 21 strains. The tmexCD-toprJ-positive Aeromonas strains were genetically diverse and found to belong to four different species that could be clustered into three major lineages. The tmexCD-toprJ gene clusters were predominantly located in the chromosome (35/36) of Aeromonas spp., with only one strain carrying the plasmid-borne tmexCD-toprJ cluster. The tmexCD-toprJ genes were associated with seven different types of genetic environments, each of which carried distinct types of mobile elements that may be responsible for mediating transmission of this gene cluster.

Spreading of cfr-Carrying Plasmids among Staphylococci from Humans and Animals.

The multidrug resistance gene cfr mediates resistance to multiple antimicrobial agents, including linezolid. Plasmids are the preferred vector for the dissemination of cfr. However, the presence and transmission of cfr-carrying plasmids among staphylococci from humans and animals have rarely been studied. Here, we investigated the presence of the cfr gene in 2,250 staphylococci of human clinical origin collected in Zhejiang, China, in 1998 to 2021 and in 3,329 porcine staphylococci preserved in our laboratories. The cfr gene was detected in 38 human isolates; its presence in Staphylococcus haemolyticus and Staphylococcus cohnii in 2003 was earlier than that identified in 2005, and Staphylococcus capitis (n = 30) was the predominant species. The cfr-carrying fragment in 38 isolates exhibited >99% nucleotide sequence similarity to plasmid pLRSA417 (39,504 bp), which was identified in 2015 and originated from a human clinical methicillin-resistant Staphylococcus aureus isolate from Zhejiang, China. The cfr-carrying plasmids in 18 MinION-sequenced staphylococci ranged in size from 32,697 bp to 43,457 bp. Fifteen plasmids were identical to pLRSA417, except for the inversion of an 8.4-kb segment comprising IS256-aacA/aphD-ISEnfa4_1-cfr-ISEnfa4_2, while the remaining 3 plasmids exhibited slightly different structures. Among the 114 cfr-positive staphylococci from pigs, pLRSA417-like plasmids were detected in 3 isolates. Intraspecies and interspecies conjugation occurred in human-derived pLRSA417-like plasmids. The presence of pLRSA417-like plasmids in staphylococci from multiple geographic regions and different hosts implied the possible transmission of the respective isolates between humans and animals. IMPORTANCE The therapeutic efficacy of the oxazolidinone antimicrobial linezolid is reduced by the emergence and dissemination of the multidrug resistance gene cfr. The cfr-carrying plasmid pLRSA417 was first identified in a clinical methicillin-resistant Staphylococcus aureus isolate, but its presence in staphylococci of human and animal origin has not been reported previously. This study showed that conjugative plasmids similar to pLRSA417 were detected mainly in Staphylococcus capitis and existed in different staphylococci in 2003 to 2021 in various clinical departments in the same hospital. pLRSA417-like plasmids were also present in staphylococci of food animal sources from different geographic regions, which suggested possible transmission among humans and animals.

Rapid On-Site Detection of Extensively Drug-Resistant Genes in Enterobacteriaceae via Enhanced Recombinase Polymerase Amplification and Lateral Flow Biosensor.

The widespread emergence of transferable extensively drug-resistant (XDR) genes, including blaNDM and blaKPC for carbapenem resistance, mcr-1 for colistin resistance, and tet(X4) and tet(X6) for tigecycline resistance, in Enterobacteriaceae poses a major threat to public health. Thus, rapid on-site detection of these XDR genes is urgently needed. We developed a cascade system with a unitary polyethylene glycol (PEG) 200-enhanced recombinase polymerase amplification (RPA) as the core, combined with a modified Chelex-100 lysis method and a horseradish peroxidase (HRP)-catalyzed lateral flow immunoassay (LFIA) biosensor, to accurately detect these genes in Enterobacteriaceae. The conventional Chelex-100 lysis method was modified to allow in situ extraction of bacterial DNA in 20 min without requiring bulky high-speed centrifuges. Using PEG 200 increased the amplification efficiency of the RPA by 13%, and the HRP-catalyzed LFIA biosensor intensified the colorimetric signal of the test line. Following optimization, the sensitivity of the cascade system was <10 copies/µL with satisfactory specificity, allowing for highly sensitive detection of these XDR genes in Enterobacteriaceae. The complete detection procedure can be completed in less than 1 h without using large-scale instruments. This assay is conducive to rapid on-site visual detection of these XDR genes in Enterobacteriaceae in practical applications, thus providing better technical support for clinical surveillance of these genes and better treatment of XDR pathogens. IMPORTANCE Carbapenem, colistin, and tigecycline are considered the last resorts for treating severe bacterial infections caused by extensively drug-resistant (XDR) pathogens. A major threat to public health is the emergence and prevalence of transferable XDR genes in Enterobacteriaceae, such as blaNDM and blaKPC for carbapenem resistance, mcr-1 for colistin resistance, and tet(X4) and tet(X6) for tigecycline resistance. Therefore, it is imperative to develop rapid on-site methods to detect these XDR genes. In this study, we constructed a cascade system for detecting these genes based on PEG 200-enhanced recombinase polymerase amplification combined with a modified Chelex-100 lysis method and HRP-catalyzed lateral flow immunoassay. The current method is capable of detecting the above-mentioned XDR genes in situ with satisfactory specificity and sensitivity, which could provide technical support for the surveillance of these genes and provide medication recommendations for the treatment of relevant clinical infections.

Fecal Carriage of Escherichia coli Harboring the tet(X4)-IncX1 Plasmid from a Tertiary Class-A Hospital in Beijing, China.

The emergence of the mobile tigecycline-resistance gene, tet(X4), poses a significant threat to public health. To investigate the prevalence and genetic characteristics of the tet(X4)-positive Escherichia coli in humans, 1101 human stool samples were collected from a tertiary class-A hospital in Beijing, China, in 2019. Eight E. coli isolates that were positive for tet(X4) were identified from clinical departments of oncology (n = 3), hepatology (n = 2), nephrology (n = 1), urology (n = 1), and general surgery (n = 1). They exhibited resistance to multiple antibiotics, including tigecycline, but remained susceptible to meropenem and polymyxin B. A phylogenetic analysis revealed that the clonal spread of four tet(X4)-positive E. coli from different periods of time or departments existed in this hospital, and three isolates were phylogenetically close to the tet(X4)-positive E. coli from animals and the environment. All tet(X4)-positive E. coli isolates contained the IncX1-plasmid replicon. Three isolates successfully transferred their tigecycline resistance to the recipient strain, C600, demonstrating that the plasmid-mediated horizontal gene transfer constitutes another critical mechanism for transmitting tet(X4). Notably, all tet(X4)-bearing plasmids identified in this study had a high similarity to several plasmids recovered from animal-derived strains. Our findings revealed the importance of both the clonal spread and horizontal gene transfer in the spread of tet(X4) within human clinics and between different sources.

Clonal relationship of tet(X4)-positive Escherichia coli ST761 isolates between animals and humans.

OBJECTIVES: To characterize the relationship of tet(X4)-positive isolates from different hosts and environments. METHODS: PCR and MALDI-TOF MS were used to identify the tet(X4)-positive isolates. The MICs of 13 antimicrobial agents were determined by broth microdilution. Illumina technology was used to sequence all of the isolates. One isolate was randomly selected from Escherichia coli ST761 clones for long-read sequencing to obtain plasmid sequences. Bioinformatics analysis was used to determine the phylogeny of 46 tet(X4)-positive E. coli ST761 strains. RESULTS: A total of 12 tet(X4)-positive isolates, 8 E. coli and 4 Aeromonas simiae, were obtained from six lairages of a slaughterhouse. These isolates exhibited resistance to at least three classes of antimicrobials, including tigecycline. The majority of them, seven E. coli and three A. simiae, represent separate clonal groups. Notably, the seven E. coli isolates belonged to ST761, a common ST carrying the tet(X4) gene that has been identified in 39 isolates from animals, meat, wastewater and humans from seven Chinese provinces. All 46 tet(X4)-positive E. coli ST761 strains from various sources have a close phylogenetic relationship (0-72 SNPs), with a high nucleotide sequence similarity of resistance genes and the tet(X4)-carrying IncX1-IncFIA(HI1)-IncFIB(K) hybrid plasmid, indicating a clonal relationship of tet(X4)-positive E. coli ST761 among animals, food, the environment and humans. CONCLUSIONS: The clonal relationship of tet(X4)-positive E. coli ST761 between humans and animals poses a previously underestimated threat to public health. To the best of our knowledge, this is the first description of tet(X4)-positive A. simiae.

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

The 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.

Structural diversity of the ISCR2-mediated rolling-cycle transferable unit carrying tet(X4).

BACKGROUND: Mobile tigecycline-resistance gene tet(X) variants have emerged as diverse pathogens from animal, human as well as their associated environments, which could potentially threaten public health. The insertion sequence, ISCR2, carries tet(X4) for horizontal transfer by rolling-cycle (RC) transposition. However, the diversity of ISCR2 and tet(X4) isolated from different sources is largely unknown. METHODS: The tet(X4)-carrying isolates were collected from human and livestock in several multiple regions of China. The whole genomic sequences of these isolates were either obtained from NCBI GenBank or determined by Illumina Hiseq 2500 and the MinION platform. The intact transposon region, ISCR2-tet(X4)-ISCR2, observed in a small number of isolates as the reference sequence to construct the transposon phylogeny. The diversity of the genetic environments of all ISCR2-tet(X4) elements were analyzed. RESULTS: A 2760-bp element encompassing the tet(X4)-hydrolase-encoding gene, catD, located between two ISCR2 elements was highly conserved in all isolates and could form an RC transposable unit (RC-TU). ISCR2 could also capture more resistance genes and formed a larger RC-TU base on RC transposition. However, the ISCR2-mediated RC-TUs were constantly truncated and inserted by other IS elements, indicating frequent recombination events. Of these elements, IS26 disrupted both the upstream and downstream ISCR2-mediated RC-TUs, indicating that IS26 captured tet(X4), thus leading to a wider spread of tet(X4). CONCLUSIONS: These results confirmed the critical role of ISCR2 for dissemination and co-transmission of tet(X4) and other resistance genes. More effort is needed to monitor the variation tendencies of tet(X4)-carrying mobile elements and determine the driving factors for disseminating transferable tigecycline resistance.

Abundance of tigecycline resistance genes and association with antibiotic residues in Chinese livestock farms.

The discovery of plasmid-mediated tet(X) variants and efflux pump gene tmexCD1-toprJ1 conferring bacteria resistance to tigecycline has compromised glycylcycline as the last line of defense against infection, which poses serious threat to public health. Herein, real-time quantitative PCR was used to detect the abundance of seven tigecycline resistance genes (TRGs), including six tet(X) variants and tmexCD1-toprJ1, and insertion sequences ISCR2 and IS26. Then, the concentrations of nine antibiotics were quantified in fecal samples collected from 157 livestock farms in four Chinese provinces. TRGs, especially tet(X4), tmexCD1-toprJ1, and insertion sequences ISCR2 and IS26, were more abundant in chicken feces than in pig and cattle feces, suggesting the greater risk for the propagation of TRGs in chicken feces. Positive correlations (ρ = 0.3741-0.8275, P < 0.0001) between ISCR2/IS26 and TRGs (except tet(X1)) further demonstrated that ISCR2 mediates the transfer of tet(X3), tet(X4), and tet(X5) and that IS26 plays a certain role for the mobilization of tet(X4) and tmexCD1-toprJ1. Tetracyclines had no positive correlation with the abundance of TRGs (except tet(X1)), meanwhile florfenicol and tiamulin were positively correlated with TRGs. However, further research is needed to confirm whether or not florfenicol and tiamulin are potential driving factors of TRG accumulation.

A Novel Transposon, Tn6518, Mediated Transfer of mcr-3 Variant in ESBL-Producing Aeromonas veronii.

PURPOSE: The aim of this study was to determine the prevalence and transmission mechanism of mcr-3 in Aeromonas spp. isolated from chicken cloaca. MATERIALS AND METHODS: A. veronii w55 was isolated from chicken in 2008. PCR assay was used to detect mcr genes and putative circular intermediate. Susceptibility testing was identified by the microdilution method. WGS was performed to obtain the whole sequence. S1-PFGE and DNA southern hybridization were used to study the location of mcr-3.6. RESULTS: PCR-based analysis indicated that 1 out of 55 Aeromonas spp. isolates was mcr-3-positive. Whole-genome sequencing revealed that the strain A. veronii w55 belonged to novel sequence type ST514 and had two adjacent chromosomally located mcr variants, mcr-3.6 and mcr-3-like. The mcr-3.6 and mcr-3-like genes showed 93.67% and 82.84% nucleotide sequence identity, respectively, to original mcr-3 from E. coli. A. veronii w55 also exhibited resistance to extended-spectrum ß-lactams and was positive for bla PER-3, and this is the first time to report bla PER-3 in A. veronii. Genetic environment analysis revealed that the segment of mcr-3.6-mcr-3-like-dgkA was flanked by five insertion sequence elements originated from Aeromonas species, and the structure of ISAs2-ISAhy2-ISAs20-mcr-3.6-mcr-3-like-dgkA-ISAs2 was designated as a novel transposon Tn6518, in which an 8405-bp circular intermediate carrying two mcr-3 variants can be looped out. CONCLUSION: This result suggested the mcr-3 variant genes could be disseminated between various Aeromonas species via transposon-mediated transmission.

Fitness Cost of blaNDM-5-Carrying p3R-IncX3 Plasmids in Wild-Type NDM-Free Enterobacteriaceae.

The wide dissemination of New Delhi metallo-ß-lactamase genes (blaNDM) has resulted in the treatment failure of most available ß-lactam antibiotics, with IncX3-type blaNDM-5-carrying plasmids recognised as having spread worldwide. In China, bacteria carrying these plasmids are increasingly being detected from diverse samples, including hospitals, communities, livestock and poultry, and the environment, suggesting that IncX3 plasmids are becoming a vital vehicle for blaNDM dissemination. To elucidate the fitness cost of these plasmids on the bacterial host, we collected blaNDM-negative strains from different sources and tested their ability to acquire the blaNDM-5-harboring p3R-IncX3 plasmid. We then measured changes in antimicrobial susceptibility, growth kinetics, and biofilm formation following plasmid acquisition. Overall, 70.7% (29/41) of our Enterobacteriaceae recipients successfully acquired the blaNDM-5-harboring p3R-IncX3 plasmid. Contrary to previous plasmid burden theory, 75.9% (22/29) of the transconjugates showed little fitness cost as a result of plasmid acquisition, with 6.9% (2/29) of strains exhibiting enhanced growth compared with their respective wild-type strains. Following plasmid acquisition, all transconjugates demonstrated resistance to most ß-lactams, while several strains showed enhanced biofilm formation, further complicating treatment and prevention measures. Moreover, the highly virulent Escherichia coli sequence type 131 strain that already harbored mcr-1 also demonstrated the ability to acquire the blaNDM-5-carrying p3R-IncX3 plasmid, resulting in further limited therapeutic options. This low fitness cost may partly explain the rapid global dissemination of blaNDM-5-harboring IncX3 plasmids. Our study highlights the growing threat of IncX3 plasmids in spreading blaNDM-5.

Identification of the novel tigecycline resistance gene tet(X6) and its variants in Myroides, Acinetobacter and Proteus of food animal origin.

OBJECTIVES: To report a novel tigecycline resistance gene, tet(X6), and its variants in four bacterial species isolated from chickens and pigs in China. METHODS: WGS was conducted to identify the suspected resistance genes in the tigecycline-resistant Myroides phaeus 18QD1AZ29W. Functional cloning, homology modelling and molecular docking were performed to compare the function with other Tet(X) variants. Retrospective screening for tet(X6) was conducted for 80 isolates in our WGS data collection, and all genomic environments of tet(X6)-positive isolates were analysed. RESULTS: The tigecycline-resistant M. phaeus 18QD1AZ29W isolated from a pig farm in Shandong in 2018 was positive for tet(X2) and a novel tet(X) gene, designated tet(X6). Tet(X6) could increase the MICs of all tested tetracyclines/glycylcyclines for Escherichia coli only 2- to 4-fold, which was possibly due to a lower tetracycline binding capacity of Tet(X6) compared with that of other Tet(X) variants. Retrospective screening showed that seven other isolates (7/80, 8.8%), comprising four Proteus spp. and three Acinetobacter spp. from chickens and pigs in Shandong and Guangdong, were positive for three different variants of tet(X6). The analysis of the genomic environment revealed that two tet(X6)-positive isolates from M. phaeus and Proteus cibarius, respectively, contained ISCR2, which may play a role in tet(X6) transmission. CONCLUSIONS: This study identified a novel type of tigecycline resistance gene, tet(X6), in Myroides, Acinetobacter and Proteus from chickens and swine. Tet(X6) conferred lower tetracycline/glycylcycline MICs than other Tet(X) variants, and ISCR2 may play a role in the transmission of tet(X6).