poxtA amplification and mutations in 23S rRNA confer enhanced linezolid resistance in Enterococcus faecalis.

OBJECTIVES: This study aimed to explore the evolutionary patterns and resistance mechanisms of an Enterococcus faecalis strain harbouring poxtA under linezolid exposure. METHODS: A poxtA-carrying E. faecalis electrotransformant DJH702 with a linezolid minimum inhibitory concentration of 4 mg/L was exposed to increasing concentrations of linezolid (8-64 mg/L). The derived strains growing at 8, 16, 32 and 64 mg/L, designed DJH702_8, DJH702_16, DJH702_32 and DJH702_64, were obtained. The amplification and overexpression of poxtA were measured using sequencing and RT-PCR, the fitness cost by competition assays and the stability of the repeat units by serial passage. RESULTS: In all derived strains, high-level linezolid resistance develops through poxtA amplification. The relative copy numbers and transcription levels of poxtA were significantly increased. However, in the presence of higher linezolid concentrations, DJH702_32 and DJH702_64 showed reduced poxtA copy numbers and transcription levels compared with DJH702_8 and DJH702_16, but additional mutations in the 23S rRNA (G2505A). IS1216E-mediated formation of translocatable units with subsequent tandem amplification of these translocatable units supported the gain of poxtA segments. However, these amplicons were not stable and were lost frequently in the absence of a linezolid selection pressure. The amplification of the poxtA region did not result in a fitness cost, but mutations in 23S rRNA did. CONCLUSIONS: poxtA-carrying E. faecalis electrotransformants used two distinct mechanisms to resist linezolid selection pressure: at lower concentrations, strains prioritized increasing poxtA expression levels, while at higher concentrations, a combination of increased poxtA expression and mutations in 23S rRNA was observed.

Research note: characteristics of blaNDM and mcr-1 co-producing Escherichia coli from retail chicken meat.

Carbapenems and colistin are vital antimicrobials used to treat Enterobacteriaceae-caused infections. The present study aimed to characterize the coexistence mechanism of carbapenem and colistin resistance in an Escherichia coli isolated from retail chicken meat. A total of 4 E. coli isolates co-harboring carbapenem resistance gene blaNDM (2 E. coli isolates with blaNDM-5 and 2 with blaNDM-9) and colistin resistance gene mcr-1. Antimicrobial susceptibility testing exhibited that all the 4 E. coli strains had multidrug resistance profile and consistent with the resistance genes they carried. MLST showed that 3 E. coli isolates belonged to a pathogenic E. coli lineage ST354, which is closely associated with human infections and pose a serious threat to public health. Whole genome sequencing (WGS) showed that 4 mcr-1-positive plasmids with sizes of 60.4 kb to 67.4 kb all belonged to the IncI2 type. A total of 5 blaNDM-harboring plasmids ranged from 99.0 kb to 138.3 kb, among which 4 plasmids belonged to unknow type and only pCS5L-NDM belonged to IncFIA/IncFIB group of hybrid plasmids, a novel carrier for blaNDM. Comparative analysis exhibited that the mcr-1 or blaNDM-carrying plasmids of E. coli strains from chicken meat showed high identity with that from Enterobacteriaceae of human origin, which indicated the risk of mcr-1 or blaNDM dissemination from retail meat to human. The simultaneous occurrence of mcr-1 and blaNDM in E. coli emphasizes the significant of antimicrobial resistance surveillance in retail meat.

Dual-band frequency reconfigurable metasurface antenna for millimeter wave joint communication and radar sensing systems.

This paper introduces an innovative, compact, and high-gain metasurface antenna, covering both the 24 GHz millimeter wave (mmWave) radar band and the 5 G n257 and n258 bands. The proposed metasurface antenna consists of a wideband stacked patch antenna and a dual-layer metasurface to focus its radiation beams for multiple mmWave bands. The operating frequency can be slightly shifted by altering the distance between the feeder and the metasurface. The distribution of the metasurface unit cells is designed based on a simplified phase compensation formula. The dimension of the fabricated feeder is 6 mm × 6 mm, and the metasurface occupies a 65 mm × 65 mm radome area. Experimental results demonstrate a wide bandwidth from 23.5 GHz to 29.1 GHz for the feeder, and impressive maximum gains of 19.7 dBi and 19.5 dBi for the lower band and higher band of the metasurface antenna are achieved simultaneously. The frequency reconfiguration ability was characterized by a 750 MHz frequency shift with every 1 mm distance adjustment. The compact size and high gain performance of the proposed design underscore its potential for practical applications in millimeter wave joint communication and radar sensing systems.

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.

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.

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.

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.

Case report: POLE (P286R) mutation in a case of recurrent intestinal leakage and its treatment.

In recent years, although new drugs and molecular markers have been used to treat metastatic colorectal cancer, there has been little progress in the immunotherapy of advanced colon cancer. The development of sequencing and multiomics technology helps us classify patients more accurately, and then find patients who may benefit from immunotherapy. The development of this advanced technology and immunotherapy based on new targets may herald a new era in the treatment of metastatic colorectal cancer. It is well known that colorectal cancer with dmmr/msi-h phenotype is sensitive to immunotherapy, yet the POLE mutation is the MSS phenotype in colorectal tumors but is also an effective target for immunotherapy. This paper describes a case of recurrent intestinal leakage that required multiple surgical procedures. A high-grade colon adenocarcinoma was identified on surgical histopathology after 18 months, and bevacizumab combined with oxaliplatin and capecitabine proved ineffective against this cancer. An analysis of gene expression indicated that POLE (P286R) mutation, TMB 119.333 mutation per 100 MB, and immune checkpoint inhibitor treatment had a significant impact. This case reminds us that the existence of malignant tumors should be considered for patients with repeated intestinal leakage, and emphasizes the importance of gene detection in the treatment of malignant tumors and the significance of POLE mutations in colorectal cancer.

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.

Relapsed primary extraskeletal osteosarcoma of liver: A case report and review of literature.

BACKGROUND: Extraskeletal osteosarcoma (ESOS) is a highly malignant osteosarcoma that occurs in extraskeletal tissues. It often affects the soft tissues of the limbs. ESOS is classified as primary or secondary. Here, we report a case of primary hepatic osteosarcoma in a 76-year-old male patient, which is very rare. CASE SUMMARY: Here, we report a case of primary hepatic osteosarcoma in a 76-year-old male patient. The patient had a giant cystic-solid mass in the right hepatic lobe that was evident on ultrasound and computed tomography. Postoperative pathology and immunohistochemistry of the mass, which was surgically removed, suggested fibroblastic osteosarcoma. Hepatic osteosarcoma reoccurred 48 d after surgery, resulting in significant compression and narrowing of the hepatic segment of the inferior vena cava. Consequently, the patient underwent stent implantation in the inferior vena cava and transcatheter arterial chemoembolization. Unfortunately, the patient died of multiple organ failure postoperatively. CONCLUSION: ESOS is a rare mesenchymal tumor with a short course and a high likelihood of metastasis and recurrence. The combination of surgical resection and chemotherapy may be the best treatment.

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.