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This article discusses the detection of the cfr gene in Citrobacter braakii and the characterization of two cfr-carrying plasmids from a chicken farm in Jiangxi, China. The cfr gene is a multidrug resistance gene that has spread globally in Gram-positive and Gram-negative bacteria. The study identified three cfr-positive strains, including one C. braakii and two E. coli strains, and found that the cfr gene was located on different plasmids in these strains. The article highlights the widespread distribution and rapid dissemination of cfr in animal-derived bacteria and emphasizes the need for monitoring and control of cfr plasmids. [Extracted from the article]

Extended-spectrum- β -lactamases (ESBLs)-producing Escherichia coli conferred resistance to most β -lactams, except for carbapenems. To date, the transmission mechanism of bla CTX-M , as the most common ESBLs subtype, in E. coli has received sustained attention around the worldwide, but the research on the pathogenicity of bla CTX-M -bearing E. coli is still scarce. The aims of this study were to discern the spread characteristics of ColV (encoding colicin V) plasmids in bla CTX-M -positive E. coli. The multi-drug resistance traits, phylogroups, and ColV plasmid profilings were screened in 76 bla CTX-M -positive E. coli. Thereafter, the genetic profiles of E. coli G12 and GZM7 were determined by whole genome sequencing, conjugation and S1-pulsed-field gel electrophoresis. The median lethal dose was analyzed in E. coli G12 and TG12A, the ColV-plasmid transconjugant of G12. Of all 76 bla CTX-M -bearing E. coli , 67.11% exhibited resistance to at least 2 drugs in addition to ceftiofur, 14.47% carried ColV-positive plasmids, and 53.95% were phylogroup C. Further studies demonstrated that the bla CTX-M -bearing E. coli G12 was assigned to the predominant lineage O78:H4-ST117 of phylogroup G. In addition, its ColV-positive plasmid simultaneously carried multiple resistance genes, and could be independently transferred to confer partial pathogenicity on its host by plasmid mating. E. coli GZM7 was O53:H9-ST23 of phylogroup C, which belonged to another representative lineage of APEC (avian pathogenic E. coli). Its ColV-positive plasmid could complete conjugation with the help of the other coexisting-resistance conjugative plasmid, although it failed to transfer alone. Our findings highlight the flexibly horizontal transfer of ColV plasmids along with multidrug-resistant genes among bla CTX-M -bearing E. coli poses a threat to poultry health and food safety, which contributes to elucidate the concept of "One Health" and deserves particular concern. [ABSTRACT FROM AUTHOR]

Objectives: To characterize a novel tigecycline resistance gene, tet(X6), and a novel SXT-related integrative and conjugative element (ICE), ICEPgs6Chn1, found in a tigecycline-resistant Proteus genomospecies 6 strain, T60.Methods: Strain T60 was identified by the VITEK 2 system, biochemical reactions and an SNP-based approach. The genetic profile of strain T60 was determined by WGS analysis. ICEPgs6Chn1 was analysed by PCR, conjugation experiments and bioinformatics tools. tet(X6) was characterized by cloning and protein structure prediction.Results: Strain T60 was resistant to ampicillin, tetracycline, tigecycline, florfenicol, colistin and kanamycin, but susceptible to cefotaxime; it also exhibited high MICs of eravacycline (32 mg/L) and omadacycline (>64 mg/L). Only one chromosome was identified and tet(X6) was located in chromosomal ICEPgs6Chn1, a member of the SXT/R391 ICE family, of 114 368 bp and encoding the antimicrobial resistance genes floR, strB, strA, aph(3')-Ia, aac(3)-IV, aph(4)-Ia, tet(X6) and sul2. The circular intermediate of ICEPgs6Chn1 was detected by PCR and sequencing, but conjugation experiments showed that it was not self-transmissible. Cloning of the novel gene tet(X6) and protein structure prediction revealed that Tet(X6) confers tigecycline resistance.Conclusions: To our knowledge, this is the first report of a novel SXT/R391 ICE in a Proteus genomospecies 6 strain. Importantly, a novel high-level tigecycline resistance gene, tet(X6), emerged for the first time in the SXT/R391 element of Proteus genomospecies 6, revealing that ICEs may serve as an important platform for the accumulation of antibiotic resistance genes. [ABSTRACT FROM AUTHOR]

Objectives: To characterize the complete sequences of four plasmids in MCR-1-producing clinical Escherichia coli strain D72, and to depict the formation mechanism and characteristics of the cointegrate plasmid derived from the pD72-mcr1 and pD72-F33 plasmids.Methods: The genetic profiles of plasmids in strain D72 and its transconjugant were determined by conjugation, S1-PFGE, Southern hybridization, WGS analysis and PCR. Plasmid sequences were analysed with bioinformatic tools. The traits of the fusion plasmid were characterized by cointegration, stability and conjugation assays.Results: Strain D72, belonging to ST1114, contained four plasmids, including mcr-1-carrying pD72-mcr1, blaCTX-M-55-carrying pD72-F33, blaTEM-238-bearing pD72-IncP and pD72-IncX1 carrying aph(3')-Ia, qnrS2 and floR. A single plasmid, pD72C, in the transconjugant was found to be larger than any plasmid in the original strain D72. Sequence analysis showed that pD72C was the fusion product of pD72-mcr1 and pD72-F33, and the recombinant event involved an intermolecular replicative mechanism. Plasmid fusion occurred at a frequency of 1.75 × 10-4 cointegrates per transconjugant. The fusion plasmid presented a high stability and conjugation frequency of 8.00 × 10-3.Conclusions: To our knowledge, this is the first report of the IS26-mediated fusion of an IncN1-F33:A-:B- plasmid and an mcr-1-carrying phage-like plasmid, providing evidence for the important role of IS26 in the recombination of plasmids. The biological advantages of the fusion plasmid indicated that the fusion event presumably plays a potential role in the dissemination of mcr-1. [ABSTRACT FROM AUTHOR]

Background: Colistin has been used as the last therapeutic resort for treatment of MDR Gram-negative bacteria infections in humans. The two-component system CpxAR has been reported to contribute to the MDR of bacteria. There may be a more complex network mediated by CpxAR contributing to colistin susceptibility than previously understood.Methods: A series of AcrB or CpxR deletion mutants of a multidrug-susceptible standard strain of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) was constructed in our previous study. MICs of colistin were determined by the 2-fold serial broth microdilution method. Time-kill and survival assays were carried out with various concentrations of colistin. Growth curves and starvation survival were measured by OD600 or cfu count in LB and M9-glucose (0.2%) minimum media. Quantitative RT-PCR was used to determine the mRNA expression levels of target genes.Results: The results showed that the MIC of colistin for the CpxR-overexpressed strain JSΔacrBΔcpxR::kan/pcpxR was dramatically decreased (0.05 mg/L) by 16-fold compared with JS (0.8 mg/L) and JSΔacrBΔcpxR::kan (0.8 mg/L). Colistin time-kill and survival assays showed that JSΔacrBΔcpxR::kan/pcpxR was more susceptible to colistin (0.05 mg/L), but had a considerably higher survivability regarding prolonged starvation stress compared with JSΔacrBΔcpxR::kan. Furthermore, the expression levels of colistin resistance-related genes (phoP, phoQ, pmrB, pmrC, pmrH and pmrD) were found to be remarkably down-regulated and the negative regulatory protein mgrB was significantly up-regulated.Conclusions: This study demonstrated that CpxR may regulate the colistin susceptibility of Salmonella Typhimurium through the PmrAB and PhoPQ regulatory systems. [ABSTRACT FROM AUTHOR]

Aim of this study was to investigate the prevalence and genetic environment of the oxazolidinone resistance gene optrA in Streptococcus suis (S. suis) isolates from diseased pigs in China. A total of 178 S. suis isolates were screened for the optrA gene by PCR. The phenotypes and genotypes of optrA -positive isolates were investigated by antimicrobial susceptibility testing, core genome Multilocus Sequence Typing (cgMLST), capsular serotypes determination and whole-genome sequencing (WGS). Fifty-one (28.7%) S. suis isolates were positive for optrA. Phylogenetic analysis indicated that the spread of the optrA among S. suis isolates was primarily due to horizontal transfer. Analysis of S. suis serotypes from diseased pigs revealed substantial diversity. The genetic environment of optrA was complex and diverse and could be divided into 12 different types. Interestingly, we identified a novel integrative and conjugative element ICE Ssu 988S, carrying optrA and erm (T) genes. This is to the best of our knowledge the first report of the optrA and erm (T) co-located on an ICE in S. suis. Our results showed a high prevalence of optrA gene in S. suis isolates in China. Further research is needed to evaluate the importance of ICEs, as they horizontally propagate important clinical resistance genes. [ABSTRACT FROM AUTHOR]

This study was designed to depict prevalence and antimicrobial resistance characteristics of Proteus mirabilis ( P. mirabilis ) strains in 4 chicken farms and to probe the transfer mechanism of resistance genes. A total of 187 P. mirabilis isolates were isolated from 4 chicken farms. The susceptibility testing of these isolates to 14 antimicrobials showed that the multidrug resistance (MDR) rate was as high as 100%. The β-lactamase resistance genes bla OXA-1 , bla CTX-M-1G , bla CTX-M-9G and colistin resistance gene mcr-1 were highly carried in the P. mirabilis isolates. An MDR strain W47 was selected for whole genome sequencing (WGS) and conjugation experiment. The results showed that W47 carried 23 resistance genes and 64 virulence genes, and an SXT/R391 integrated conjugative elements (ICEs) named ICE Pmi Chn5 carrying 17 genes was identified in chromosome. ICE Pmi Chn5 was able to be excised from the chromosome of W47 forming a circular intermediate, but repeated conjugation experiments were unsuccessful. Among 187 P. mirabilis isolates, 144 (77.01%, 144/187) isolates carried ICE Pmi Chn5-like ICEs, suggesting that ICEs may be the major vector for the transmission of resistance genes among MDR chicken P. mirabilis strains in this study. The findings were conducive to insight into the resistance mechanism of chicken P. mirabilis strains and provide a theoretical basis for the use of antibiotics for the treatment of MDR P. mirabilis infections in veterinary clinic. [ABSTRACT FROM AUTHOR]

• This study characterized a novel integrative and conjugative element, carrying eight resistance genes and seven IS elements, from a serovar 2 Glaesserella parasuis ST279 isolate. • Tn6743, a novel transposon carrying six resistance genes, was identified. • This study characterized a novel insertion element ISGpa1, which is the first characterized example in G. parasuis. ICE Gpa1804 was identified in the genome of a serovar 2, ST279 isolate EHP1804 carrying eight different resistance genes from 200 Glaesserella parasuis strains isolated from swine with lower respiratory tract infection in seven provinces of China. Susceptibility testing for EHP1804 was determined by broth microdilution, and its genetic profile was determined by whole-genome sequencing. The complete ICE Gpa1804 was analysed by polymerase chain reaction, conjugation assay and bioinformatics tools. The conjugation assay was performed using EHP1804 as the donor and G. parasuis V43 (rifampicin-resistant) as the recipient. ICE Gpa1804 has a size of 71,880 bp and contains 83 genes, including eight resistance genes [ tet (B), bla Rob-1 , aphA1, strA, strB, aac(3)-IId, catA3 and sul2 ]. The conjugation assay showed that ICE Gpa1804 could be transferred to G. parasuis V43 with frequencies of 4.3 × 10−7. To the best of the authors' knowledge, this is the first study to identify a novel integrative and conjugative element (ICE) carrying eight resistance genes and seven insertion sequence (IS) elements from a G. parasuis isolate. Tn 6743 , a novel transposon carrying six resistance genes, was identified. Moreover, IS Gpa1 , a novel IS 256 family insertion element, is the first characterized example of a G. parasuis insertion element. Multiple mobile genetic elements involved in resistance genes were located in chromosomal ICE Gpa1804 , which showed that ICEs may serve as a vital platform for the accumulation of resistance genes. [ABSTRACT FROM AUTHOR]

It has been recognized that colistin resistance is a growing problem that seriously impairs the clinical efficacy of colistin against bacterial infections. One strategy that has been proven to have therapeutic effect is to overcome the widespread emergence of antibiotic-resistant pathogens by combining existing antibiotics with promising non-antibiotic agents. In this work, antibiotic susceptibility testing, checkerboard assays and time-kill curves were used to investigate the antibacterial activity of the individual drugs and the potential synergistic activity of the combination. The molecular mechanisms of tetrandrine in combination with colistin were analyzed using fluorometric assay and Real-time PCR. To predict possible interactions between tetrandrine and MCR-1, molecular docking assay was taken. Finally, we evaluated the in vivo efficacy of tetrandrine in combination with colistin against MCR-positive Salmonella. Overall, the combination of tetrandrine and colistin showed significant synergistic activity. In-depth mechanistic analysis showed that the combination of tetrandrine with colistin enhances the membrane-damaging ability of colistin, undermines the functions of proton motive force (PMF) and efflux pumps in MCR-positive bacteria. The results of molecular docking and RT-PCR analyses showed that tetrandrine not only affects the expression of mcr -1 but is also an effective MCR-1 inhibitor. Compared with colistin monotherapy, the combination of tetrandrine with colistin significantly reduced the bacterial load in vivo. Our findings demonstrated that tetrandrine serves as a potential colistin adjuvant against MCR-positive Salmonella. • In our study, the combination of tetrandrine and colistin drastically enhanced colistin bactericidal activity compared with monotreatment. • In-depth mechanistic analysis showed that tetrandrine potentiated colistin activity through enhancing the membrane-damaging ability of colistin. • Tetrandrine dramatically undermines the function of PMF and result in decreased intracellular ATP levels. In addition, the activity of efflux pump was significantly inhibited by the addition of tetrandrine in both single or combination treatments. • Tetrandrine not only affects the expression of mcr -1 but also as an effective MCR-1 inhibitor. • The discovery of tetrandrine as a potential adjuvant for colistin therapy in combination therapy for severe infections caused by MCR-1 positive Salmonella. [ABSTRACT FROM AUTHOR]

This study was undertaken to discern the transmission characteristics of mcr-1 and bla CTX-M-type in one multidrug-resistant Escherichia coli LWY24 from chicken in China. The genetic profiles of LWY24 isolate were determined by conjugation, S1-pulsed-field gel electrophoresis, southern blot hybridization, and whole genome sequencing analysis. Meanwhile, co-transfer of plasmids in LWY24 isolate was screened by dual conjugation assays. The LWY24 isolate was identified as ST93, and harbored 3 conjugative plasmids, pLWY24J-3 (bla CTX-M-55 -bearing IncFⅡ), pLWY24J- mcr-1 (mcr-1 -carrying IncI2), and pLWY24J-4 (non-resistance-conferring IncI1), and one nonconjugative plasmid pLWY24 (bla CTX-M-14 -containing IncHI2/IncHI2A). Numerous resistance genes, insertion sequences (especially IS 26), and transposons were found in the 4 plasmids, suggesting that horizontal transmission have occurred by plasmid mating, homologous recombination, and transpositions. Under the selection pressure of cefotaxime and colistin or cefotaxime alone, the mcr-1 -bearing plasmid and the bla CTX-M-55 -harboring plasmid could be co-transferred at a similar frequency, with 8.00 × 10−4 or 9.00 × 10−4 transconjugants per donor cell, respectively. The specific shufflon region in mcr-1 -encoding plasmid could generate up to 6 diverse PilV structures, which may further accelerate the horizontal transfer of plasmid. In conclusion, the transmission characteristics of mcr-1 and bla CTX-M-type in LWY24 isolate could due to clonal spread of ST93, selective pressure of cefotaxime, IS 26 -mediate homologous recombination and transposition, and the specific shufflon region. [ABSTRACT FROM AUTHOR]

Background: Streptococcus pluranimalium is a new member of the Streptococcus genus isolated from multiple different animal hosts. It has been identified as a pathogen associated with subclinical mastitis, valvular endocarditis and septicaemia in animals. Moreover, this bacterium has emerged as a new pathogen for human infective endocarditis and brain abscess. However, the patho-biological properties of S. pluranimalium remain virtually unknown. The aim of this study was to determine the complete genome sequence of S. pluranimalium strain TH11417 isolated from a cattle with mastitis, and to characterize its antimicrobial resistance, virulence, and carbon catabolism. Results: The genome of S. pluranimalium TH11417, determined by single-molecule real-time (SMRT) sequencing, consists of 2,065,522 base pair (bp) with a G + C content of 38.65%, 2,007 predicted coding sequence (CDS), 58 transfer RNA (tRNA) genes and five ribosome RNA (rRNA) operons. It contains a novel ISSpl1 element (a memeber of the IS3 family) and a Ф11417.1 prophage that carries the mef(A), msr(D) and lnu(C) genes. Consistently, our antimicrobial susceptibility test confirmed that S. pluranimalium TH11417 was resistant to erythromycin and lincomycin. However, this strain did not show virulence in murine pneumonia (intranasal inoculation, 107 colony forming unit – CFU) and sepsis (intraperitoneal inoculation, 107 CFU) models. Additionally, this strain is able to grow with glucose, lactose or galactose as the sole carbon source, and possesses a lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS). Conclusions: We reported the first whole genome sequence of S. pluranimalium isolated from a cattle with mastitis. It harbors a prophage carrying the mef(A), msr(D) and lnu(C) genes, and is avirulent in the murine infection model. [ABSTRACT FROM AUTHOR]

Background: Staphylococcus aureus is one of the most prevalent pathogens and a causative agent of a variety of infections in humans and animals. A total of 640 samples were collected from healthy animals and patients from 2013 to 2014 in Henan Province, China, to investigate the prevalence and perform molecular characterization of S. aureus. Antimicrobial resistance and virulence genes were determined and pulsed-field gel electrophoresis (PFGE) and staphylococcal cassette chromosome mec (SCC mec ) typing were performed. Results: Overall, 22.3% (n = 143) of the samples were positive for S. aureus. The prevalence of methicillin-resistant S. aureus (MRSA) was 5.59%. Capsular polysaccharide locus type 5 ( Cap 5; 56.64%) was the dominant serotype. S. aureus strains showed high resistance to penicillin (96.50%), ciprofloxacin (52.45%), amikacin (67.83%), erythromycin (96.50%), lincomycin (97.20%), and tetracycline (68.53%) and 109 (76.2%) isolates harbored six or more tested resistance genes. The most predominant resistance genes were aphA (52.45%), ermC (53.15%), and tetM (52.45%). Eighty-seven (60.8%) isolates harbored six or more tested virulence genes. The most predominant enterotoxin genes were sed (20.28%), sej (20.98%), sep (14.69%), and set (37.76%). The prevalence of lukED gene was (57.34%), and a small number of isolates carried pvl (5.59%) and TSST - 1 (2.80%). A total of 130 (82.52%) isolates could be typed by PFGE with SmaI digestion. PFGE demonstrated that 45 different patterns (P) that were grouped into 17 pulsotypes and 28 separate pulsotypes using a 90% cut-off value. A total of 118 (82.52%) isolates were successfully typed by spa , and 26 spa types were identified, t15075 (14.00%) and t189 (12.59%) were the most common types. SCC mec types were detected from eight MRSA isolates, with the most prevalent type being SCC mec IVa. MRSA-SCC mec Iva- t437 was observed in human isolates. Conclusion: This study revealed a high prevalence of S. aureus in healthy animals and patients from Henan Province, China. Resistant S. aureus exhibited varying degrees of multidrug resistance. The presence of antibiotic resistance and virulence genes may facilitate the spread of S. aureus strains and pose a potential threat to public health, highlighting the need for vigilant monitoring of these isolates at the human–animal interface. [ABSTRACT FROM AUTHOR]

Background: The plasmid-encoded multidrug efflux pump oqxAB confers bacterial resistance primarily to olaquindox, quinolones, and chloramphenicol. The aims of this study were to investigate the prevalence of oqxAB among Escherichia coli isolates from dogs, cats, and humans in Henan, China and the susceptibilities of E. coli isolates to common antibiotics. Methods: From 2012 to 2014, a total of 600 samples which included 400 rectal samples and 200 clinical human specimens were tested for the presence of E. coli. All isolates were screened for oqxAB genes by PCR and sequencing. The MICs of 11 antimicrobial agents were determined by the broth microdilution method. A total of 30 representative oqxAB-positive isolates were subjected to ERIC-PCR and MLST. Additionally, conjugation experiments and southern hybridizations were performed. Results: Of 270 isolates, 58.5% (62/106) of the isolates from dogs, 56.25% (36/64) of the isolates from cats, and 42.0% (42/100) of the isolates from humans were positive for the oqxAB. Olaquindox resistance was found for 85.7%-100% of oqxAB-positive isolates. Of oqxAB-positive isolates from dogs, cats, and humans, ciprofloxacin resistance was inspected for 85.8%, 59.1%, and 93.8%, respectively. Several oqxAB-positive isolates were demonstrated by ERIC-PCR and MLST, and have high similarity. Phylogenetic analysis showed that oqxAB-positive isolates could be divided into 7 major clusters. OqxAB-positive conjugants were obtained, southern hybridization verified that the oqxAB gene complex was primarily located on plasmids. Conclusion: In conclusion, oqxAB-positive isolates were widespread in animals and humans in Henan, China. Carriage of oqxAB on plasmids of E. coli isolates may facilitate the emergence of multidrug resistant and its transmission via horizontal transfer, and might pose a potential threat to public health. [ABSTRACT FROM AUTHOR]