Prevalence and characterization of aminoglycoside resistance gene aph(2")-If-carrying Campylobacter jejuni.

Campylobacter jejuni is recognized as a significant foodborne pathogen, and recent studies have indicated a rising trend of aminoglycosides resistance gene aph(2″)-If among C. jejuni isolates from food-producing animals in China. However, systematic information about aph(2″)-If-positive C. jejuni from food-producing animals and other sources worldwide based on whole-genome analysis remains a knowledge gap. In this study, we aimed to analyze the worldwide distribution, genetic environment and phylogenetic tree of aph(2″)-If by utilizing Whole Genome Sequencing (WGS) data obtained, coupled with information in the GenBank database. A total of 160C. jejuni isolates in the GenBank database and 14C. jejuni isolates in our laboratory carrying aph(2″)-If gene were performed for further analysis. WGS analysis revealed the global distribution of aph(2″)-If among C. jejuni from 6 countries. Multilocus Sequence Typing(MLST) results indicated that 70 STs were involved in the dissemination of aph(2″)-If, with ST10086 being the predominant ST. Whole-genome Multilocus Sequence Typing(wg-MLST) analysis according to times, countries, and origins of C. jejuni isolation further demonstrated a close relationship between aph(2″)-If carrying C. jejuni isolates from farm and food. The findings also revealed the existence of 32 distinct types of genetic environments surrounding aph(2″)-If among these isolates. Notably, Type 30, characterized by the arrangement ISsag10-deoD-ant(9)-hp-hp-aph(2″)-If, emerged as the predominant genetic environment. In conclusion, our analysis provides the inaugural perspective on the worldwide distribution of aph(2″)-If. This resistance gene demonstrates horizontal transferability and regional diffusion in a clonal pattern. The close association observed among aph(2″)-If-positive C. jejuni strains isolated from poultry, food, and clinical environments underscores the potential for zoonotic transmission from these isolates.

Tandem amplification of a plasmid-borne tet(A) variant gene confers tigecycline resistance in Escherichia coli.

OBJECTIVES: To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene. METHODS: E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5 mg/L). When exposed to increasing concentrations of tigecycline (0.25-8 mg/L), mutants growing at 2, 4 and 8 mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS). RESULTS: Tigecycline resistance with maximum MICs of 16 mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8 mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline. CONCLUSIONS: Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.

Laccase Lac-W detoxifies aflatoxin B1 and degrades five other major mycotoxins in the absence of redox mediators.

A multicopper oxidase Lac-W from Weizmannia coagulans 36D1 was identified and characterized as a laccase (Lac-W) with a robust enzymatic activity, which was used in various mycotoxins degradation. We demonstrated that Lac-W could directly degrade six major mycotoxins in the absence of redox mediators in pH 9.0, 24h static incubation at room temperature, including aflatoxin B1 (AFB1, 88%), zearalenone (60%), deoxynivalenol (34%), T-2 toxin (19%), fumonisin B1 (18%), and ochratoxin A (12%). The optimal condition for Lac-W to degrade AFB1 was 30 °C, pH 9.0, enzyme-substrate ratio 3U/µg in 24h static condition. Furthermore, we characterized aflatoxin Q1 as a Lac-W-mediated degradation product of AFB1 using UHPLC-MS/MS. Interestingly, degradation products of AFB1 failed to generate cell death and apoptosis of intestinal porcine epithelial cells. Finally, our molecular docking simulation results revealed that the substrate-binding pocket of Lac-W was large enough to allow the entry of six mycotoxins with different structures, and their degradation rates were positively correlated to their interacting affinity with Lac-W. In summary, the unique properties of the Lac-W make it a great candidate for detoxifying multiple mycotoxins contaminated food and feed cost-effectively and eco-friendly. Our study provides new insights into development of versatile enzymes which could simultaneously degrade multiple mycotoxins.

Emergence of high-level tigecycline resistance due to the amplification of a tet(A) gene variant in clinical carbapenem-resistant Klebsiella pneumoniae.

OBJECTIVE: To investigate the prevalence of a tet(A) gene variant and its role in developing high-level tigecycline resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates. METHODS: The mechanism of high-level tigecycline resistance in CRKP mediated by a tet(A) variant was explored by induction experiments, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis. The amplification and overexpression of the tet(A) variant were measured by the determination of sequencing depth, gene copy numbers, and qRT-PCR. RESULTS: A high rate (62.1%, 998/1607) of tet(A) variant carriage was observed among 1607 CRKP clinical isolates from Henan Province, China. High-level tigecycline resistance could rapidly develop by the amplification of the tet(A) variant in these isolates. The analysis of the raw sequencing data and the plasmid mapping depth revealed that the ΔtnpA homologous sequence of Tn1721 supports the amplification of the region that harbours the tet(A) variant by forming a large number of repeat arrays through translocatable units (TUs). Moreover, the epidemiological analysis of tet(A) variant-carrying structures among 1607 clinical CRKPs showed that the TU structure is widely present. CONCLUSION: The presence of a tigecycline resistance-mediating tet(A) variant in CRKP clinical isolates represents a greater health concern than initially thought and should be monitored consistently.

IS257-mediated amplification of tet(L) variant as a novel mechanism of enhanced tigecycline resistance in Staphylococcus cohnii.

The mechanism of enhanced tigecycline MIC in Staphylococcus cohnii after in vitro tigecycline exposure was investigated. S. cohnii 11-B-312 was exposed to incremental concentrations of tigecycline (2-32 mg/L) and the mutants growing at 8, 16 and 32 mg/L were determined by AST and WGS. Copy number and relative transcription level of the tet(L) gene were determined by quantitative PCR. The fitness cost was evaluated by growth kinetics and competition assays. The results revealed that enhanced tigecycline MIC was identified in S. cohnii mutants. Copy number and relative transcription level of tet(L) in the mutants increased 8-, 20-, and 23-fold and 20-, 34-, and 39-fold in the presence of 8, 16, and 32 mg/L tigecycline, respectively. The read-mapping depth ratio analysis indicated that a multidrug resistance region carrying the tet(L) variant has a gradually increased copy number, correlating with the tigecycline selection pressure. S. cohnii strain 11-B-312_32 had a fitness cost, and enhanced tigecycline MIC can revert to the initial level in the absence of tigecycline. In summary, enhanced tigecycline MIC develops with extensive amplification of an IS257-flanked tet(L)-carrying segment in S. cohnii. IS257 seems to play a vital role in the gain and loss of the amplification product.

Genomic insights into linezolid-resistant Enterococci revealed its evolutionary diversity and poxtA copy number heterogeneity.

OBJECTIVES: This study aimed to determine the molecular mechanisms of linezolid-resistant enterococci (LRE) in swine slaughterhouses in China and apply the "One Health" perspective to analyse the evolutionary dynamics of poxtA-positive E. faecium in clinical and non-clinical settings worldwide. METHODS: The phenotypic and genomic characteristics of multiple LRE isolates were systematically investigated using antimicrobial susceptibility testing, transfer assays, evolutionary experiments, quantitative RT-PCR assays, whole-genome sequencing, and bioinformatics analyses. RESULTS: Swine faeces served as a significant reservoir for LRE isolates, and optrA and poxtA were the primary contributors to linezolid resistance. Co-occurrence network analysis revealed a significant interconnection between optrA and several other ARGs. The poxtA copy number heterogeneity and polymorphism were initially observed in E. faecium parental and evolved isolates. The poxtA-carrying tandem repeat region exhibits high mobility and has undergone extensive duplication owing to linezolid pressure. The poxtA copy number varies from four copies on the plasmid of E. faecium IC25 to 11 copies on the plasmid and six copies on the chromosome in the evolved isolate IC25-50_poxtA. Furthermore, phylogenetic analysis of 185 poxtA-positive E. faecium strains worldwide found that one isolate from a French patient in 2018 shared only two SNPs with CC17 E. faecium isolates IC25 and IC7-2 from this study, highlighting the potential global transmission of CC17 poxtA-positive E. faecium between humans and animals. CONCLUSION: This study identified amplification of poxtA as a response of E. faecium to linezolid pressure. Phylogenetic analysis shed light on the potential global transmission of hospital-associated CC17 poxtA-positive E. faecium in clinical and non-clinical settings.

Pathogen-Targeting Bimetallic Nanozymes as Ultrasonic-Augmented ROS Generator against Multidrug Resistant Bacterial Infection.

Clinical treatment of multidrug resistant (MDR) pathogens-induced infection is emerging as a growing challenge in global public health due to the limited selection of clinically available antibiotics. Nanozymes as artificial enzymes that mimicked natural enzyme-like activities, are received great attention for combating MDR pathogens. However, the relatively deficient catalytic activity in the infectious microenvironment and inability to precisely targeting pathogen restrains their clinical anti-MDR applications. Here, pathogen-targeting bimetallic BiPt nanozymes for nanocatalytic therapy against MDR pathogen are reported. Benefiting from electronic coordination effect, BiPt nanozymes exhibit dual-enzymatic activities, including peroxidase-mimic and oxidase-mimic activities. Moreover, the catalytic efficiency can be efficiently increased 300-fold by ultrasound under inflammatory microenvironment. Notably, BiPt nanozyme is further cloaked with a platelet-bacteria hybrid membrane (BiPt@HMVs), thus presenting excellent homing effect to infectious sites and precise homologous targeting to pathogen. By integrating accurate targeting with highly efficient catalytic, BiPt@HMVs can eliminate carbapenem-resistant Enterobacterales and methicillin-resistant Staphylococcus aureus in osteomyelitis rats model, muscle-infected mice model, and pneumonia mice model. The work provides an alternative strategy based on nanozymes for clinically addressing MDR bacteria-induced infections.

Linezolid-Resistant Enterococcus faecalis of Chicken Origin Harbored Chromosome-Borne optrA and Plasmid-Borne cfr, cfr(D), and poxtA2 Genes.

The aim of this study was to investigate the transferability of acquired linezolid resistance genes and associated mobile genetic elements in an Enterococcus faecalis isolate QZ076, cocarrying optrA, cfr, cfr(D), and poxtA2 genes. MICs were determined by broth microdilution. Whole-genome sequencing (WGS) was performed using the Illumina and Nanopore platforms. The transfer of linezolid resistance genes was investigated by conjugation, using E. faecalis JH2-2 and clinical methicillin-resistant Staphylococcus aureus (MRSA) 109 as recipients. E. faecalis QZ076 harbors four plasmids, designated pQZ076-1 to pQZ076-4, with optrA located in the chromosomal DNA. The gene cfr was located on a novel pseudocompound transposon, designated Tn7515, integrated into the 65,961-bp pCF10-like pheromone-responsive conjugative plasmid pQZ076-1. Tn7515 generated 8-bp direct target duplications (5'-GATACGTA-3'). The genes cfr(D) and poxtA2 were colocated on the 16,397-bp mobilizable broad-host-range Inc18 plasmid pQZ076-4. The cfr-carrying plasmid pQZ076-1 could transfer from E. faecalis QZ076 to E. faecalis JH2-2, along with the cfr(D)- and poxtA2-cocarrying plasmid pQZ076-4, conferring the corresponding resistant phenotype to the recipient. Moreover, pQZ076-4 could also transfer to MRSA 109. To the best of our knowledge, this study presented the first report of four acquired linezolid resistance genes [optrA, cfr, cfr(D), and poxtA2] being simultaneously present in the same E. faecalis isolate. The location of the cfr gene on a pseudocompound transposon in a pheromone-responsive conjugative plasmid will accelerate its rapid dissemination. In addition, the cfr-carrying pheromone-responsive conjugative plasmid in E. faecalis was also able to mobilize the interspecies transfer of the cfr(D)- and poxtA2-cocarrying plasmid between enterococci and staphylococci. IMPORTANCE In this study, the simultaneous occurrence of four acquired oxazolidinone resistance genes [optrA, cfr, cfr(D), and poxtA2] was identified in an E. faecalis isolate of chicken origin. The association of the cfr gene with a novel pseudocompound transposon Tn7515 integrated into a pCF10-like pheromone-responsive conjugative plasmid will accelerate its dissemination. Moreover, the location of the resistance genes cfr(D) and poxtA2 on a mobilizable broad-host-range Inc18 family plasmid represents the basis for their intra- and interspecies dissemination with the aid of a conjugative plasmid and further accelerates the spreading of acquired oxazolidinone resistance genes, such as cfr, cfr(D), and poxtA2, among Gram-positive pathogens.

Emergence of lnu(C) variant conferring lincomycin resistance in Campylobacter coli of chicken origin.

Lincomycin is widely used in respiratory and gastrointestinal infection in veterinary medicine and food animal production. Campylobacter members are vital foodborne pathogens causing campylobacteriosis, and the resistance to lincosamides is seldom reported. To date, only the rRNA methyltransferase Erm(B) has been confirmed to be associated with lincosamides resistance in Campylobacter. In this study, we identified a lnu(C) variant conferring lincomycin resistance in this pathogen of chicken origin. The Lnu(C) encoded by this gene variant showed substitution at position 8 (Asn8Lys), 11 (Phe11Leu) and 112 (Leu112Phe), when compared with the firstly reported Lnu(C) from Streptococcus agalactiae. Cloning of the lnu(C) variant into lincosamide-susceptible Campylobacter jejuni NCTC 11168 confirmed its function in conferring resistance to lincomycin with the 32-fold increased MICs. Sequencing analysis showed that the lnu(C) variant was located within a MTnSag1-like transposon together with insLNU, which is inserted between panB and cj0299 genes on the chromosome. lnu(C) gene was distributed among C. coli globally, and various STs were involved in the dissemination of lnu(C). Although transposition mediated by MTnSag1-like transposon failed to occur, the horizontal transfer mediated by natural transformation and reservoir for resistance genes may facilitate their adaptation to the antimicrobial selection pressure in chickens, which should not be ignored.

Characterization of a genomic Island carrying the tet(X4) gene in porcine Acinetobacter towneri co-harboring plasmid-borne bla NDM-1 and bla OXA-58 genes.

Tigecycline and carbapenems are last-resort antimicrobial agents to treat serious infections caused by multi-drug resistant bacterial pathogens. However, the co-occurrence of tigecycline and carbapenem resistance determinants challenges the clinical efficacy of these antimicrobial agents. In this study, we report the co-existence of tet(X4), bla NDM-1 and bla OXA-58 genes in the porcine Acinetobacter towneri isolate 19110F47. Sequence analysis revealed that tet(X4) gene, along with the florfenicol resistance gene floR, was flanked by three copies of IS91-like elements, which can form three different translocatable units (TUs), and were located in a 41,098-bp multidrug resistance region (MDRR) within a novel 100,354-bp genomic island (GI) region. TUs comprising floR-virD2-ISVsa3, hp-abh-tet(X4)-ISVsa3 and virD2-floR-ISVsa3-hp-abh-tet(X4)-ISVsa3 can be looped out from the chromosomal DNA and facilitate the transfer of the TU-based resistance genes into other plasmidic or chromosomal sites. In addition, the carbapenemase genes bla NDM-1 and bla OXA-58 were found on different non-conjugative multiresistance plasmids in this isolate, with the genetic contexts ISAba125-bla NDM-1-ble MBL-tnpR and ΔISAba3-bla OXA-58 -ISAba3, respectively. The simultaneous occurrence of tet(X4), bla NDM-1 and bla OXA-58 in the same porcine A. towneri isolate emphasizes the importance of antimicrobial resistance surveillance in food-producing animals.

Hybrid IncFIA/FIB/FIC(FII) plasmid co-carrying blaNDM-5 and fosA3 from an Escherichia coli ST117 strain of retail chicken.

Carbapenems and fosfomycin are important antibiotics used to treat Enterobacteriaceae-associated infections. This study aimed to characterize the co-resistance and co-dissemination mechanism of carbapenem and fosfomycin resistance in an Escherichia coli ST117 strain isolated from retail chicken meat. Antimicrobial susceptibility testing showed that an E. coli CS18F strain had a multidrug resistance profile, including carbapenem and fosfomycin resistance. The presence of blaNDM-5 and fosA3 genes was confirmed by PCR and Sanger sequencing. The blaNDM-5 and fosA3 genes were successfully transferred to the recipient strain E. coli J53 via conjugation, and the transconjugants had elevated minimum inhibitory concentrations (MICs) for meropenem and fosfomycin. Whole genome sequencing (WGS) of E. coli CS18F revealed that blaNDM-5 and fosA3 were colocalized on an IncFIA/FIB/FIC(FII) type plasmid of 189,141 bp, which was designated as pCS18F-NDM-Fos. A novel structure with five IS26 sequences flanking the multiple drug resistance region (MDRR) was identified, and three copies of IS26 were found to be flanked blaNDM-5, fosA3, dfrA12, aadA2, and sul1. Three types of translocation units (TUs) were identified by PCR, containing either the resistance gene blaNDM-5 and an IS26 sequence, fosA3, and an IS26 sequence, or both, indicating their potential co-transfer via TUs. Thus, this is an unprecedented report of the presence of a plasmid co-carrying blaNDM-5 and fosA3 and TUs potentially mediating their simultaneous transfer.

Excision and integration of unconventional circularizable structures involving the erm(B) gene in enterococci.

Traditionally, insertion sequences (ISs) play a major role in disseminating antimicrobial resistance genes (ARGs) in bacteria through transposition and translocation, forming regions that contain multiple ARGs flanked by single or multiple copies of IS. In addition, unconventional circularizable structures (UCSs), lacking recombinase genes but being surrounded by directly repeated sequences (DRs) of various sizes which do not contain transposase genes, were reported to be involved in the dissemination of ARGs. In this study, a novel UCS was identified on plasmid pE508-2 in E. faecalis E508, which carried a 24,411 bp multiresistance gene cluster, consisting of the resistance genes aphA3, lnu(B), lsa(E), spw, aac(A)-aph(D), lnu(B), dfrG, and two copies of aadE flanked by copies of erm(B). PCR assays revealed that three types of UCSs with lengths of 7235, 16,437, and 23,673 bp were formed, each of which contained the respective resistance genes and one copy of erm(B). Using erm(B)-negative and -positive strains, we demonstrated that erm(B)-carrying UCSs failed to transfer into an erm(B)-negative strain, but could integrate into an erm(B)-positive strain in a new site adjacent to a pre-existing erm(B) gene by natural transformation. Database searches revealed that erm(B)-flanked multiresistance gene regions, which might be able to form the respective UCSs, are present among various bacteria from different sources in various countries. In summary, this study experimentally demonstrated the excision and integration of UCS involving structures that include erm(B). The widespread presence of these UCSs in various Gram-positive bacteria highlights its role in the dissemination of ARGs among bacterial pathogens.

Plasmid-Assisted Horizontal Transfer of a Large lsa(E)-Carrying Genomic Island in Enterococcus faecalis.

The horizontal transfer of genomic islands is essential for the adaptation and evolution of Enterococcus faecalis. In this study, three porcine E. faecalis strains, each harboring a large lsa(E)-carrying genomic island, were identified. When using the E. faecalis OG1RF as the recipient, the horizontal transfer of the lsa(E)-carrying genomic island occurred only from E. faecalis E512, which also harbored a pheromone-responsive conjugative plasmid, but not from the other two E. faecalis strains, E533 and E509, which lacked such a plasmid. Subsequently, through plasmid curing of E. faecalis E512 and plasmid introduction into E. faecalis E533, the pheromone-responsive conjugative plasmid was identified to be indispensable for the horizontal transfer of the lsa(E)-carrying genomic island and a subsequent homologous recombination between the chromosomal DNA of the donor and the recipient. In addition, the presence of a chromosomally-located conjugative transposon, Tn916, in E. faecalis E509 could not mediate the horizontal transfer of the lsa(E)-carrying genomic island, although Tn916 itself could transfer by conjugation. Thus, these data highlight the role of the pheromone-responsive conjugative plasmid in the transfer of the lsa(E)-carrying genomic island in E. faecalis, thereby establishing the dual role of pheromone-responsive conjugative plasmids in contributing to the dissemination of both plasmid-borne resistance genes and chromosomally-located genomic islands. IMPORTANCE In this study, it was shown that a pheromone-responsive conjugative plasmid played an indispensable role in the horizontal transfer of a lsa(E)-carrying genomic island. This finding indicates a dual role of the pheromone-responsive conjugative plasmid in disseminating both plasmid-borne resistance genes and chromosomally-located genomic islands. The role of the pheromone-responsive conjugative plasmid in disseminating chromosomal genomic islands is suggested to be essential in the genomic evolution of E. faecalis, which has become one of the leading nosocomial pathogens worldwide.

A Small Multihost Plasmid Carrying erm(T) Identified in Enterococcus faecalis.

The aim of this study was to determine the mobile genetic elements involved in the horizontal transfer of erm(T) in Enterococcus faecalis, and its transmission ability in heterologous hosts. A total of 159 erythromycin-resistant enterococci isolates were screened for the presence of macrolide resistance genes by PCR. Whole genome sequencing for erm(T)-carrying E. faecalis E165 was performed. The transmission ability in heterologous hosts was explored by conjugation, transformation, and fitness cost. The erm(T) gene was detected only in an E. faecalis isolate E165 (1/159), which was located on a 4,244-bp small plasmid, designed pE165. Using E. faecalis OG1RF as the recipient strain, pE165 is transferable. Natural transformation experiments using Streptococcus suis P1/7 and Streptococcus mutans UA159 as the recipients indicated it is transmissible, which was also observed by electrotransformation using Staphylococcus aureus RN4220 as a recipient. The erm(T)-carrying pE165 can replicate in the heterologous host including E. faecalis OG1RF, S. suis P1/7, S. mutans UA159, and S. aureus RN4220 and conferred resistance to erythromycin and clindamycin to all hosts. Although there is no disadvantage of pE165 in the recipient strains in growth curve experiments, all the pE165-carrying recipients had a fitness cost compared to the corresponding original recipients in growth competition experiments. In brief, an erm(T)-carrying plasmid was for the first time described in E. faecalis and as transmissible to heterologous hosts.

Studies on the Transmission of a Tigecycline Resistance-Mediating tet(A) Gene Variant from Enterobacter hormaechei via a Two-Step Recombination Process.

To investigate the contribution of a tet(A) variant to tigecycline resistance in Enterobacter hormaechei and the recombination events that occurred during transmission of this variant. MICs were determined by broth microdilution. E. hormaechei G17 was characterized by PCR, transfer assay, S1-PFGE, Southern blot hybridization, and WGS analysis. A tet(A) variant conferring resistance to tigecycline was present in E. hormaechei G17. This strain harbored two resistance plasmids (pG17-1, 264,084 bp and pG17-2, 68,610 bp) and its E. coli transformant Tm-G17TGC one resistance plasmid (pTm-G17, 93,013 bp). The comparative analysis of pG17-1, pG17-2, and pTm-G17 showed that a tet(A) variant-carrying multiresistance gene cluster (~23 kb) originating from pG17-1 had integrated into pG17-2, forming the novel plasmid pTm-G17. In a first step, this multiresistance gene cluster was excised from pG17-1 by recombination of homologous sequences, including △TnAs1 at both termini, thereby generating an unconventional circularizable structure (UCS). In a second step, this UCS integrated into pG17-2 via recombination between homologous sequences, including IS26 present on both, the UCS and pG17-2, thereby giving rise to the new plasmid pTm-G17. In summary, a tet(A) variant conferring resistance to tigecycline was reported in E. hormaechei. Transfer of a tet(A) variant-carrying multiresistance gene cluster between plasmids occurred in a two-step recombination process, in which homologous sequences, including either △TnAs1 or IS26, were involved. IMPORTANCE Tigecycline is an important last-resort broad spectrum antimicrobial agent. This study describes the two-step recombination processes resulting in the transfer of the tet(A) variant gene between different plasmids in E. hormaechei, which depicts the role of recombination processes in the generation of UCSs and new plasmids, both carrying a tet(A) variant conferring resistance to tigecycline. Such processes enhance the dissemination of resistance genes, which is of particular relevance for resistance genes, such as the tet(A) variant. The presence and transmission of a tet(A) variant in E. hormaechei will compromise the efficacy of tigecycline treatment for E. hormaechei associated infection.

A novel lysin Ply1228 provides efficient protection against Streptococcus suis type 2 infection in a murine bacteremia model.

Streptococcus suis is an important zoonotic pathogen that is difficult to control with antibiotics due to the widespread development of multidrug-resistant strains. Phage lysin is considered a potential therapeutic agent to combat S. suis. In this study, the novel lysin Ply1228 derived from the prophage of S. suis type 12 was identified. Bioinformatics analysis showed that Ply1228 contains a CHAP catalytic domain, which is a binding domain composed of a CW-7 binding motif and an amidase-2 catalytic domain. The CHAP catalytic domain is essential for the bactericidal function of lysin Ply1228 and does not depend on the presence of Ca2+. C34 and H99 of the CHAP domain were identified as the key active sites. The CW-7 binding motif plays a key binding role in Ply1228. Ply1228 can specifically lyse S. suis, including types 2, 3, 7, 9, 10, 12, 14, and 27. Within 10 min, Ply1228 killed 4 log of the S. suis population, which had a starting concentration of approximately 107 CFU/mL. In addition, Ply1228 showed favourable thermal and pH stability. The therapeutic effect of Ply1228 was further investigated in a mouse model of S. suis bacteremia. The administration of the lysin Ply1228 (200 µg/mouse) 1 h after the intraperitoneal injection of 2 × MLD of SS2 strain SC225 was sufficient to protect the mice (P < 0.0001) and significantly reduced the bacterial loads in the blood and organs (livers, spleens, lungs and kidneys). The levels of inflammation and histopathological damage in infected mice were effectively relieved after the Ply1228 treatment. These results indicate that Ply1228 might represent a new enzybiotic candidate for S. suis infection.

Recombination events that occur in a poxtA-carrying Enterococcus faecium during the conjugation process.

OBJECTIVES: To investigate transferability of the poxtA-carrying plasmids in Enterococcus faecium and the mechanism of recombination that occurs during the conjugation process. METHODS: MICs were determined by broth microdilution. Transferability of the poxtA-carrying plasmids in E. faecium was investigated by conjugation. The mechanism of recombination that occurred during the conjugation process was explored by S1-PFGE and WGS. RESULTS: E. faecium strain Fac90 carries two plasmids, designated pFac90-154 and pFac90-54, respectively. Six transconjugants with different characteristics were obtained. In transconjugant T90-1, a plasmid-chromosome fusion event led to the integration of plasmid pFac90-154 from the donor E. faecium strain Fac90 into the chromosomal DNA of the recipient strain Enterococcus faecalis JH2-2. In transconjugants T90-2, -3 and -4, losses or additions of different-sized plasmid segments most likely occurred due to IS1216-mediated recombination. In transconjugants T90-5 and -6, two large plasmids with sizes of 101 656 and 149 526 bp were formed by plasmid fusion. CONCLUSIONS: To the best of our knowledge, this is the first report showing the integration of pFac90-154 from E. faecium Fac90 into the chromosomal DNA of recipient E. faecalis JH2-2 via homologous recombination. Besides, we showed that five new plasmid types were formed by genetic rearrangements. These recombination events resulted simultaneously in the formation of various types of mosaic plasmids with multiple resistance genes and/or conjugation characteristics, which might promote the transmission of diverse plasmids encoding resistance genes among enterococci. Thus, these data significantly expand our knowledge regarding conjugative events, establishing a dual role of conjugation in both dissemination of resistance genes and plasmid evolution.

Plasmid Fusion and Recombination Events That Occurred during Conjugation of poxtA-Carrying Plasmids in Enterococci.

Linezolid plays a crucial role in the treatment of infections caused by multiresistant Gram-positive bacteria. The poxtA gene not only confers oxazolidinone and phenicol resistance but also decreases susceptibility to tetracycline. In this study, we investigated structural changes in mobilizable poxtA-carrying plasmids in enterococci which occurred during conjugation experiments using S1-PFGE (pulsed-field gel electrophoresis), Southern blot hybridization, and whole-genome sequencing (WGS) analysis. Two poxtA-carrying strains were identified in Enterococcus faecalis E006 and Enterococcus lactis E843, respectively. E. faecalis E006 contains the 121,520-bp conjugative plasmid pE006-121 and the 19,832-bp mobilizable poxtA-carrying plasmid pE006-19, while E. lactis E843 contains the 171,930-bp conjugative plasmid pE843-171 and the 27,847-bp mobilizable poxtA-carrying plasmid pE843-27. Moreover, both poxtA-carrying plasmids were mobilized by their respective conjugative plasmid in enterococci by plasmid fusion; one was generated by homologous recombination in E. faecalis through an identical 864-bp homologous region in the plasmids of the parental strain, while another was generated by an IS1216E-mediated plasmid integration in E. lactis, involving a replicative transposition. IMPORTANCE Until now, all the poxtA genes described in enterococci, including E. faecalis, E. faecium, and E. hirae, are plasmid-borne, suggesting that plasmids play an important role in the dissemination of the poxtA gene among enterococci. This study showed that the mobilizable poxtA-carrying plasmid could transfer with the help of conjugative plasmid in enterococci via plasmid fusion, with one generated by homologous recombination in E. faecalis, and another by replicative transposition in E. lactis. During both the fusion events, the poxtA-carrying plasmids changed from nonconjugative to conjugative, leading to the generation and enhanced dissemination of the larger phenicol-oxazolidinone-tetracycline resistance-encoding plasmids in enterococci.

erm(T)-Mediated Macrolide-Lincosamide Resistance in Streptococcus suis.

To investigate the presence and location of erm(T) in clinical Streptococcus suis isolates and explore the transmission ability and fitness cost of erm(T)-carrying mobile genetic elements among S. suis isolates, MICs were determined by broth microdilution. The presence of erm(T) in S. suis was detected by PCR. The genetic environment of erm(T) in S. suis was explored by whole-genome sequencing (WGS) analysis. Intraspecies and interspecies transmission were examined by electrotransformation. The fitness cost associated with the carriage of an erm(T)-harboring plasmid or an integrative and conjugative element (ICE) was examined by competition experiments. Of 237 nonduplicate strains, erm(T) was detected in 2 S. suis strains (SC262-ST954 and SC117-ST1314), with its location on a 5,125-bp plasmid in S. suis SC262 and on a 64,013-bp ICESsuSC117 in S. suis SC117, respectively. Both the erm(T)-carrying plasmid pSC262 and the ICESsuSC117 were transmissible by transformation. Plasmid pSC262 can replicate and express macrolide-lincosamide resistance in heterologous hosts, including S. aureus and S. pneumoniae. Both the erm(T)-carrying plasmid and the ICE posed a fitness cost to the host S. suis isolate. To the best of our knowledge, this is the first report of the macrolide-lincosamide-streptogramin B resistance gene erm(T) in S. suis. Its location on a plasmid or an ICE will aid in its transmission. The low detection rate of erm(T) gene among the S. suis population might be due to the fitness cost of the erm(T)-carrying plasmid and ICE. IMPORTANCE Macrolide and lincosamide resistance due to the presence of erm(T) have posed a challenge for the treatment of Gram-positive pathogens. Although the low detection rate of erm(T) gene among the S. suis population due to the fitness cost of the erm(T)-carrying plasmid and ICE, the presence of erm(T) in S. suis and its potential transmission to other Gram-positive pathogens will be of important significance.