Restoring colistin sensitivity in colistin-resistant Salmonella and Escherichia coli: combinatorial use of berberine and EDTA with colistin.

The appearance and prevalence of multidrug-resistance (MDR) Gram-negative bacteria (GNB) have limited our antibiotic capacity to control bacterial infections. The clinical efficacy of colistin (COL), considered as the "last resort" for treating GNB infections, has been severely hindered by its increased use as well as the emergence and prevalence of mobile colistin resistance (MCR)-mediated acquired drug resistance. Identifying promising compounds to restore antibiotic activity is becoming an effective strategy to alleviate the crisis of increasing MDR. We first demonstrated that the combination of berberine (BBR) and EDTA substantially restored COL sensitivity against COL-resistant Salmonella and Escherichia coli. Molecular docking indicated that BBR can interact with MCR-1 and the efflux pump system AcrAB-TolC, and BBR combined with EDTA downregulated the expression level of mcr-1 and tolC. Mechanically, BBR combined with EDTA could increase bacterial membrane damage, inhibit the function of multidrug efflux pump, and promote oxidative damage, thereby boosting the action of COL. In addition, transcriptome analysis found that the combination of BBR and EDTA can accelerate the tricarboxylic acid cycle, inhibit cationic antimicrobial peptide (CAMP) resistance, and attenuate Salmonella virulence. Notably, the combination of BBR and EDTA with COL significantly reduced the bacterial load in the liver and spleen of a mice model infected with Salmonella. Our findings revealed that BBR and EDTA can be used as adjuvants collectively with COL to synergistically reverse the COL resistance of bacteria. IMPORTANCE: Colistin is last-resort antibiotic used to treat serious clinical infections caused by MDR bacterial pathogens. The recent emergence of transferable plasmid-mediated COL resistance gene mcr-1 has raised the specter of a rapid worldwide spread of COL resistance. Coupled with the fact of barren antibiotic development pipeline nowadays, a critical approach is to revitalize existing antibiotics using antibiotic adjuvants. Our research showed that berberine combined with EDTA effectively reversed COL resistance both in vivo and in vitro through multiple modes of action. The discovery of berberine in combination with EDTA as a new and safe COL adjuvant provides a therapeutic regimen for combating Gram-negative bacteria infections. Our findings provide a potential therapeutic option using existing antibiotics in combination with antibiotic adjuvants and address the prevalent infections caused by MDR Gram-negative pathogens worldwide.

Upregulation of outer membrane porin gene ompC contributed to enhancement of azithromycin susceptibility in multidrug-resistant Escherichia coli.

The outer membrane (OM) in gram-negative bacteria contains proteins that regulate the passive or active uptake of small molecules for growth and cell function, as well as mediate the emergence of antibiotic resistance. This study aims to explore the potential mechanisms for restoring bacteria to azithromycin susceptibility based on transcriptome analysis of bacterial membrane-related genes. Transcriptome sequencing was performed by treating multidrug-resistant Escherichia coli T28R with azithromycin or in combination with colistin and confirmed by reverse transcription-quantitative PCR (RT-qPCR). Azithromycin enzyme-linked immunosorbent assay (ELISA) test, ompC gene overexpression, and molecular docking were utilized to conduct the confirmatory research of the potential mechanisms. We found that colistin combined with azithromycin led to 48 differentially expressed genes, compared to azithromycin alone, such as downregulation of tolA, eptB, lpxP, and opgE and upregulation of ompC gene. Interestingly, the addition of colistin to azithromycin differentially downregulated the mph(A) gene mediating azithromycin resistance, facilitating the intracellular accumulation of azithromycin. Also, overexpression of the ompC elevated azithromycin susceptibility, and colistin contributed to further suppression of the Mph(A) activity in the presence of azithromycin. These findings suggested that colistin firstly enhanced the permeability of bacterial OM, causing intracellular drug accumulation, and then had a repressive effect on the Mph(A) activity along with azithromycin. Our study provides a novel perspective that the improvement of azithromycin susceptibility is related not only to the downregulation of the mph(A) gene and conformational remodeling of the Mph(A) protein but also the upregulation of the membrane porin gene ompC.IMPORTANCEUsually, active efflux via efflux pumps is an important mechanism of antimicrobial resistance, such as the AcrAB-TolC complex and MdtEF. Also, bacterial porins exhibited a substantial fraction of the total number of outer membrane proteins in Enterobacteriaceae, which are involved in mediating the development of the resistance. We found that the upregulation or overexpression of the ompC gene contributed to the enhancement of resistant bacteria to azithromycin susceptibility, probably due to the augment of drug uptakes caused and the opportunity of Mph(A) function suppressed by azithromycin with colistin. Under the combination of colistin and azithromycin treatment, OmpC exhibited an increased selectivity for cationic molecules and played a key role in the restoral of the antibiotic susceptibility. Investigations on the regulation of porin expression that mediated drug resistance would be important in clinical isolates treated with antibiotics.

Analysis of Regulatory Mechanism of AcrB and CpxR on Colistin Susceptibility Based on Transcriptome and Metabolome of Salmonella Typhimurium.

With the increasing and inappropriate use of colistin, the emerging colistin-resistant isolates have been frequently reported during the last few decades. Therefore, new potential targets and adjuvants to reverse colistin resistance are urgently needed. Our previous study has confirmed a marked increase of colistin susceptibility (16-fold compared to the wild-type Salmonella strain) of cpxR overexpression strain JSΔacrBΔcpxR::kan/pcpxR (simplified as JSΔΔ/pR). To searching for potential new drug targets, the transcriptome and metabolome analysis were carried out in this study. We found that the more susceptible strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels. The virulence-related genes and colistin resistance-related genes (CRRGs) were significantly downregulated in JSΔΔ/pR. There were significant accumulation of citrate, α-ketoglutaric acid, and agmatine sulfate in JSΔΔ/pR, and exogenous supplement of them could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. Additionally, we also demonstrated that AcrB and CpxR could target the ATP and reactive oxygen species (ROS) generation, but not proton motive force (PMF) production pathway to potentiate antibacterial activity of colistin. Collectively, these findings have revealed several previously unknown mechanisms contributing to increased colistin susceptibility and identified potential targets and adjuvants for potentiating colistin treatment of Salmonella infections. IMPORTANCE Emergence of multidrug-resistant (MDR) Gram-negative (G-) bacteria have led to the reconsideration of colistin as the last-resort therapeutic option for health care-associated infections. Finding new drug targets and strategies against the spread of MDR G- bacteria are global challenges for the life sciences community and public health. In this paper, we demonstrated the more susceptibility strain JSΔΔ/pR displayed striking perturbations at both the transcriptomics and metabolomics levels and revealed several previously unknown regulatory mechanisms of AcrB and CpxR on the colistin susceptibility. Importantly, we found that exogenous supplement of citrate, α-ketoglutaric acid, and agmatine sulfate could synergistically enhance the bactericidal effect of colistin, indicating that these metabolites may serve as potential adjuvants for colistin therapy. These results provide a theoretical basis for finding potential new drug targets and adjuvants.

Synergistic antibacterial activity of baicalin and EDTA in combination with colistin against colistin-resistant Salmonella.

The emergence and rapid spread of multidrug resistant (MDR) Gram-negative bacteria have posed a serious threat to global health and security. Because of the time-consuming, high cost and high risk of developing new antibiotics, a significant method is to use antibiotic adjuvants to revitalize the existing antibiotics. The purpose of the study is to research the traditional Chinese medicine baicalin with the function of inhibiting the efflux pump and EDTA whether their single or combination can increase the activity of colistin against colistin-resistant Salmonella in vitro and in vivo, and to explore its molecular mechanisms. In vitro antibacterial experiments, we have observed that baicalin and EDTA alone could enhance the antibacterial activity of colistin. At the same time, the combination of baicalin and EDTA also showed a stronger synergistic effect on colistin, reversing the colistin resistance of all Salmonella strains. Molecular docking and RT-PCR results showed that the combination of baicalin and EDTA not only affected the expression of mcr-1, but also was an effective inhibitor of MCR-1. In-depth synergistic mechanism analysis revealed that baicalin and EDTA enhanced colistin activity through multiple pathways, including accelerating the tricarboxylic acid cycle (TCA cycle), inhibiting the bacterial antioxidant system and lipopolysaccharide (LPS) modification, depriving multidrug efflux pump functions and attenuating bacterial virulence. In addition, the combinational therapy of colistin, baicalin and EDTA displayed an obvious reduction in bacterial loads cfus of liver and spleen compared with monotherapy and 2-drug combination therapy. In conclusion, our study indicates that the combination of baicalin and EDTA as a novel colistin adjuvant can provide a reliable basis for formulating the therapeutic regimen for colistin resistant bacterial infection.

Decreased cyclic-AMP caused by ATP contributes to fosfomycin heteroresistance in avian Escherichia coli.

BACKGROUND: Fosfomycin is an important broad-spectrum bactericidal antibiotic to treat multidrug-resistant bacteria infections. It is generally accepted that heteroresistant bacteria are an intermediate stage in the formation of drug resistance, but there are few studies on the formation mechanism underlying fosfomycin heteroresistance (FHR). OBJECTIVES: To reveal the characteristics and formation mechanisms of FHR in Escherichia coli isolates obtained from chickens. METHODS: We identified the FHR according to the population analysis profile (PAP) test and in vitro time-kill assay. Growth curves for FHR E. coli and their subpopulations were measured. Also, the subpopulations were repeatedly cultured in fosfomycin-free medium for 5-20 overnight incubation periods. The formation mechanisms of FHR in E. coli isolates were identified through accumulation assay, carbohydrate utilization testing, real-time relative quantitative PCR analysis, DNA sequencing, transcriptomic analysis, intracellular ATP and cAMP-level assessment. RESULTS: Four of six E. coli strains were confirmed to show FHR, with a total of six subpopulations. The subpopulations restored phenotypic susceptibilities to fosfomycin within 5-20 overnight incubation sessions, but four of six subpopulations still maintained FHR characteristics. Differing from their parental isolates, the uptake of fosfomycin in the subpopulations through GlpT was reduced remarkably. Further studies identified that the low expression of glpT was due to the decrease of intracellular cAMP levels in the subpopulations, which was caused by the decreased ATP levels in cells. CONCLUSIONS: Our findings revealed the formation mechanism of E. coli isolates showing FHR obtained from chicken in China and characterized the dynamic change traits in vitro of the subpopulations.

Characterization of IncI1/ST71 and IncF18:A-:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate.

To characterize IncI1 and IncF18:A-:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate, antibiotic susceptibility testing, conjugation assays, transformation assays, S1-PFGE, and WGS analysis were performed. The 119,457-bp plasmid pEC014-1 with a multidrug-resistance region (MRR) containing four different segments interspersed with six IS26 elements, belonged to incompatibility group I1 and sequence type 71. The 154,516-bp plasmid pEC014-2 with two replicons, typed as FII-18 and FIB-1, carried 14 resistance determinants including blaTEM-1b, blaOXA-1, oqxAB, dfrA17, aac(6')-Ib-cr, sul1, sul2, tet(A), floR, catB3, hph(aph(4)-Ia), aacC4(aac(3)-IV), aadA5, arr-3, and a merEDACPTR loci in MRR, and additionally encoded three virulence loci: iroNEDCB, sitABCD, and iucABCD-iutA. Plasmid stability assays showed that pEC014-1 and pEC014-2 were stable in recipient E. coli C600 for at least 15 days of passage. Competition assays were carried out to evaluate the fitness impact of pEC014-2 carriage in vitro, revealing a decrease in host fitness. Growth kinetics showed that the growth rate for pEC014-1 or/and pEC014-2 bearing cells was significantly slower than that of the E. coli C600 host strain in the exponential stage (p < 0.01), with only cells carrying pEC014-1 sustaining rapid growth after 6 h of exponential growth. Our findings highlight the mosaic structures of epidemic plasmid IncI1/ST71 and F18:A-:B1 lineages and contribute to a better understanding of the evolution and dissemination of these multidrug resistance and virulence plasmids.

Multidrug Resistance Genes Carried by a Novel Transposon Tn7376 and a Genomic Island Named MMGI-4 in a Pathogenic Morganella morganii Isolate.

Antimicrobial resistance in Morganella morganii is increasing in recent years, which is mainly introduced via extra genetic and mobile elements. The aim of our study is to analyze the multidrug resistance (MDR) and characterize the mobile genetic elements (MGEs) in M. morganii isolates. Here, we report the characteristic of a pathogenic M. morganii isolate containing multidrug resistance genes that are mainly carried by a novel transposon Tn7376 and a genomic island. Sequence analysis suggested that the Tn7376 could be generated through homologous recombination between two different IS26-bounded translocatable units (TUs), namely, module A (IS26-Hp-IS26-mph(A)-mrx(A)-mphR-IS6100-chrA-sul1-qacEΔ1) and module B (ISCR1-sul1-qacEΔ1-cmlA1-aadA1-aadB-intI1-IS26), and the genomic island named MMGI-4 might derive from a partial structure of different original genomic islands that also carried IS26-mediated TUs. Notably, a 2,518-bp sequence linked to the module A and B contains a 570-bp dfrA24 gene. To the best of our knowledge, this is the first report of the novel Tn7376 possessing a complex class 1 integron that carried an infrequent gene dfrA24 in M. morganii. IMPORTANCE Mobile genetic elements (MGEs), especially for IS26-bounded translocatable units, may act as a reservoir for a variety of antimicrobial resistance genes in clinically important pathogenic bacteria. We expounded this significant genetic characteristic by investigating a representative M. morganii isolate containing multidrug resistance genes, including the infrequent dfrA24. Our study suggested that these acquired resistance genes were mainly driven by IS26-flanked important MGEs, such as the novel Tn7376 and the MMGI-4. We demonstrated that IS26-related MGEs contributed to the emergence of the extra gene dfrA24 in M. morganii through some potential genetic events like recombination, transposition, and integration. Therefore, it is of importance to investigate persistently the prevalence these MEGs in the clinical pathogens to provide risk assessment of emergence and development of novel resistance genes.

Characterization of a Novel Linezolid Resistance Gene optrA and Bacitracin Resistance Locus-Carrying Multiple Antibiotic Resistant Integrative and Conjugative Element ICESsu1112S in Streptococccus Suis.

Streptococcus suis strain 1112S was isolated from a diseased pig in a feedlot from Henan, China, in 2019. The isolate harbored a linezolid resistance gene optrA. WGS data revealed that the optrA gene was associated with a single copy ETAf ISS1S, in tandem with erm(B) and tet(O), located in a novel 72,587 bp integrative and conjugative element (ICE). Notably, this novel element, designated ICESsu1112S, also carried a novel bacitracin resistance locus. ICESsu1112S could be excised from chromosome and transferred to the recipient strain S. suis P1/7 with a frequency of 5.9 × 10-6 transconjugants per donor cell. This study provided the first description of the coexistence of optrA and a novel bacitracin locus on a multiple antibiotic resistant ICE and highlighted that ICE were major vehicle and contribute to the potential transfer of clinically relevant antibiotic resistance genes. IMPORTANCE Antimicrobial resistance (AMR) caused by the imprudent use of antimicrobials has become a global problem, which poses a serious threat to treatment of S. suis infection in pigs and humans. Importantly, AMR genes can horizontally spread among commensal organisms and pathogenic microbiota, thereby accelerating the dissemination of AMR determinants. These transfers are mainly mediated by mobile genetic elements, including ICEs. In S. suis, ICEs are the major vehicles that contribute to the natural transfers of AMR genes among different bacterial pathogens. However, ICEs that carry optrA and bacitracin resistance locus are rarely investigated in S. suis isolates. Here, we investigated a S. suis isolate carrying an optrA and a novel bacitracin resistance locus, which were co-located on a novel multiple antibiotic resistant ICESsu1112S. Our study suggests that more research is needed to access the real significance of ICEs that horizontally spread clinical important resistance genes.

Double deletion of cpxR and tolC significantly increases the susceptibility of Salmonella enterica serovar Typhimurium to colistin.

BACKGROUND: The increasing use of colistin causes a serious breach in our last line of defence against MDR Gram-negative pathogens. Our previous study showed that CpxR overexpression increases the susceptibility of acrB and cpxR double-deleted Salmonella enterica serovar Typhimurium to colistin. OBJECTIVES: To identify the mechanism of CpxAR and efflux pumps that synergistically enhance the susceptibility of S. Typhimurium to colistin. METHODS: A series of cpxR- and tolC-deleted mutants and a cpxR-complemented strain from a multidrug-susceptible standard strain of S. Typhimurium (JS) were generated in our previous study. Herein, we investigated the susceptibility of these strains to colistin through the broth microdilution method, time-kill curves and survival assays. Growth curves were measured by OD600 in LB broth, tryptone-soy broth (TSB) and M9-glucose (0.2%) minimal media. Finally, molecular mechanisms underlying the mode of action were elucidated by transcriptomic analysis. RESULTS: We found that in contrast to JS (0.8 mg/L), the MIC of colistin for JSΔtolC::kan showed a 16-fold decrease (0.05 mg/L). Notably, JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were associated with a 256-fold decrease (0.0031 mg/L) compared with JS. Growth curves identified that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR displayed a markedly lower growth rate and poorer adaptability. In addition, time-kill curves and survival assays showed that JSΔcpxRΔtolC and JSΔcpxRΔtolC/pcpxR were more susceptible to colistin. Lastly, double deletion of cpxR and tolC enhanced oxidative damage through promoting oxidative phosphorylation, the tricarboxylic acid (TCA) cycle and trimethylamine N-oxide (TMAO) respiration. CONCLUSIONS: Our findings revealed that double deletion of cpxR and tolC significantly increases the susceptibility of S. Typhimurium to colistin.

The Formation of Two Hybrid Plasmids Mediated by IS26 and Tn6952 in Salmonella enterica Serotype Enteritidis.

To characterize the formation mechanism and characteristics of two cointegrate plasmids in Salmonella enterica serotype Enteritidis strain S13, plasmids from strain S13 and three corresponding transconjugants were subjected to whole genome sequencing and analyzed using bioinformatics tools. The traits of two fusion plasmids in transconjugants were characterized by stability and conjugation experiments. Sequence analysis indicated that strain S13 contained four plasmids, including mcr-1-bearing pS13-1, bla CTX-M-55-carrying pS13-2, tet(M)-bearing pS13-3, and floR-carrying pS13-4. IncN1-F33:A-:B- plasmid pS13-2, respectively, fused with IncFI:A-:B- plasmid pS13-3 and IncX1 plasmid pS13-4, which generated two cointegrate plasmids, designated pS13D and pS13F, which involved in two intermolecular replicative mechanisms mediated by IS26 and the novel transposon Tn6952 (ΔTnAS3-IS26-ΔISEcp1-ramA-ΔIS26-ΔTnAS1), respectively. This is the first report of the fusion of the IncN1-F33:A-:B- plasmid and IncFI:A-:B- plasmid mediated by IS26, and with IncX1 plasmid mediated by Tn6952. The formation and evolution of cointegrate plasmids could expand the resistance and host spectrum of fusion plasmids.

Characterization of blaCMY-2-carrying IncC and rmtB-carrying IncI1/ST136 plasmids in an avian Escherichia coli ST224 strain.

To analyze characteristics and underlying evolutionary processes of IncC and IncI1 plasmids in a multidrug-resistant avian E. coli strain, antibiotic susceptibility testing, PCR, conjugation assays, and next-generation sequencing were performed. The type 1 IncC plasmid pEC009.1 harbored three antimicrobial resistance regions including ISEcp1-blaCMY-2-blc-sugE, ARI-B resistance island, and ARI-A island that was a mosaic multidrug resistance region (MRR) comprised of a class 1 integron with cassette array |aac(6')-II(aacA7)|qacE∆1|sul1|, IS26-mphR(A)-mrx-mph(A)-IS26, IS26-fosA3-IS26, and mercury resistance cluster merRTPABDE. It is the first report of three different size circular forms derived from IS26-mphR(A)-mrx-mph(A)-IS26-fosA3-IS26 in ARI-A of type 1 IncC plasmid. In IncI1/ST136 pEC009.2, the truncated transposon Tn1722 carrying blaTEM-1b, rmtB, aac(3)-IId(aacC2d), and a class 1 integron with cassette array |dfrA12|orfF|aadA2|, inserted into the plasmid backbone generating 5-bp direct repeats (DRs, TATAA) at the boundaries of the region, which was highly similar to that of other IncI1 plasmids, and differed by the arrangements of resistance determinants. Comparison among two epidemic plasmid lineages showed complex MRRs respectively located in the specific position in type 1 IncC and IncI1/ST136 plasmids with conserved backbones, and these have evolved via multiple events involved in mobile elements-mediated loss and gain of resistance genes and accessory genes. Strains harboring these plasmids may serve as a reservoir for antibiotic resistance genes, thereby contributing to the rapid spread of resistance genes and posing a public health threat.

Molecular Characterization of a Novel Integrative Conjugative Element ICEHpa1 in Haemophilus parasuis.

ICEHpa1 was identified in the genome of a serovar 8 Haemophilus parasuis ST288 isolate YHP170504 from a case of swine lower respiratory tract infection. The aim of the present study was to characterize the integrative conjugative element ICEHpa1 and its multiresistance region. Susceptibility testing was determined by broth microdilution and the complete ICEHpa1 was identified by WGS analysis. The full sequence of ICEHpa1 was analyzed with bioinformatic tools. The presence of ICEHpa1, its circular intermediate and integration site were confirmed by PCR and sequence analysis. Transfer of ICEHpa1 was confirmed by conjugation. ICEHpa1 has a size of 68,922 bp with 37.42% GC content and harbors 81 genes responsible for replication and stabilization, transfer, integration, and accessory functions, as well as seven different resistance genes [bla Rob- 3, tet(B), aphA1, strA, strB, aac(6)'-Ie-aph(2')-Ia, and sul2]. Conjugation experiments showed that ICEHpa1 could be transferred to H. parasuis V43 with frequencies of 6.1 × 10-6. This is the first time a multidrug-resistance ICE has been reported in H. parasuis. Seven different resistance genes were located on a novel integrative conjugative element ICEHpa1, which suggests that the ICEHpa1 is capable of acquiring foreign genes and serving as a carrier for various resistance genes.

Characterization of pTS14, an IncF2:A1:B1 Plasmid Carrying tet(M) in a Salmonella enterica Isolate.

The objective of this study was to explore the genetic and biological features of the tet(M)-harboring plasmid pTS14 in Salmonella enterica strain S14 isolated from a chicken fecal sample. Plasmid pTS14 was identified by conjugation, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and plasmid sequencing. The biological characteristics of pTS14 were assessed via stability, growth kinetics, and starvation survival experiments. Strain S14, belonging to ST3007, harbored a 119-kb tet(M)-bearing IncF2:A1:B1 conjugative plasmid pTS14. The plasmid pTS14 contained a novel transposon Tn6709 with the genetic structure IS26-tnpA1-tnpA2-Δorf13-LP-tet(M)-tnpX-ΔtnpR-IS26, and the resistance genes tet(B), tet(D), strAB, sul2, and bla TEM-1b. In addition, pTS14 was found to be highly stable in the recipient strain E. coli J53. The transconjugant TS14 exhibited a higher survival ratio than E. coli J53 under permanent starvation-induced stress. The tet(M)-bearing IncF2 epidemic plasmid lineage may accelerate the dissemination of tet(M) and other genes by coselection, which could constitute a potentially serious threat to clinical treatment regimens.

CpxR regulates the colistin susceptibility of Salmonella Typhimurium by a multitarget mechanism.

BACKGROUND: The two-component signalling systems PmrAB and PhoPQ of Salmonella have been extensively studied with regard to colistin resistance. We previously showed that overexpressed CpxR could significantly increase the colistin susceptibility (16-fold compared with the WT strain) of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) through PmrAB and PhoPQ. OBJECTIVES: To identify the potential target genes of CpxR in PmrAB- and PhoPQ-related signalling pathways. METHODS: His6-CpxR was prokaryotically expressed and purified by Ni-NTA resin affinity chromatography. ß-Galactosidase activity assays were conducted to investigate whether CpxR could regulate the promoters of colistin resistance-related genes (CRRGs). Electrophoretic mobility shift assays (EMSAs) were used to further detect His6-CpxR complexes with promoters of CRRGs. RESULTS: We demonstrated for the first time (to the best of our knowledge) that CpxR and the AcrAB-TolC efflux pump have reciprocal effects on CRRG transcription. Additionally, CpxR could regulate the colistin susceptibility of Salmonella Typhimurium by binding directly to the promoters of phoPQ, pmrC, pmrH and pmrD at the CpxR box-like sequences or indirectly through other regulators including pmrAB and mgrB. CONCLUSIONS: CpxR could regulate the colistin susceptibility of Salmonella Typhimurium by a multitarget mechanism.

Characterization of the IncHI2 plasmid pTW4 harboring tet(M) from an isolate of Escherichia coli ST162.

To investigate the genetic features and biological costs of the plasmid pTW4 harboring tet(M) in an isolate of Escherichia coli ST162 from a duck. The complete nucleotide sequence of plasmid pTW4 was determined. The characteristics of plasmid pTW4 in E. coli were investigated by stability and direct competition assays. pTW4 is an IncHI2-type plasmid that contained the resistant genes tet(M), floR, strAB, sul2, rmtB, and blaCMY-2. Tet(M) is located in the composite transposon Tn6539 within the multidrug resistant (MDR) region on this plasmid. Furthermore, the resistance gene rmtB and blaCMY-2 were found outside the MDR region. The plasmid pTW4 remained stable in the host strain E. coli J53 after passage under an antibiotic-free environment for 7 days. However, the strain E. coli J53/pTW4 showed a fitness disadvantage of 6% per ten generations in the process of growth competition with E. coli J53. In conclusion, the plasmid pTW4, a mobile MDR vehicle, may promote the dissemination of tet(M), floR, rmtB, strAB, sul2, and blaCMY-2 among bacteria and then, but it appears to confer growth disadvantage to the host.

Antimicrobial resistance-encoding plasmid clusters with heterogeneous MDR regions driven by IS26 in a single Escherichia coli isolate.

BACKGROUND: IS26-flanked transposons played an increasingly important part in the mobilization and development of resistance determinants. Heterogeneous resistance-encoding plasmid clusters with polymorphic MDR regions (MRRs) conferred by IS26 in an individual Escherichia coli isolate have not yet been detected. OBJECTIVES: To characterize the complete sequence of a novel blaCTX-M-65- and fosA3-carrying IncZ-7 plasmid with dynamic MRRs from an E. coli isolate, and to depict the mechanism underlying the spread of resistance determinants and genetic polymorphisms. METHODS: The molecular characterization of a strain carrying blaCTX-M-65 and fosA3 was analysed by antimicrobial susceptibility testing and MLST. The transferability of a plasmid bearing blaCTX-M-65 and fosA3 was determined by conjugation assays, and the complete structure of the plasmid was obtained by Illumina, PacBio and conventional PCR mapping, respectively. The circular forms derived from IS26-flanked transposons were detected by reverse PCR and sequencing. RESULTS: A novel IncZ-7 plasmid pEC013 (∼118kb) harbouring the blaCTX-M-65 and fosA3 genes was recovered from E. coli isolate EC013 belonging to D-ST117. The plasmid was found to have heterogeneous and dynamic MRRs in an individual strain and the IS26-flanked composite transposon-derived circular intermediates were identified and characterized in pEC013. CONCLUSIONS: The heterogeneous MRRs suggested that a single plasmid may actually be a cluster of plasmids with the same backbone but varied MRRs, reflecting the plasmid's heterogeneity and the survival benefits of having a response to antimicrobial-related threatening conditions in an individual strain.

Identification and prevalence of RND family multidrug efflux pump oqxAB genes in Enterococci isolates from swine manure in China.

PURPOSE: The resistance/nodulation/cell division (RND) family multidrug efflux pump, OqxAB, has been identified as one of the leading mechanisms of plasmid-mediated quinolone resistance and has become increasingly prevalent among Enterobacteriaceae in recent years. However, oqxAB genes have not yet been reported in Enterococcus isolates. The aim of the present study was to identify the oqxAB genes and investigate their prevalence among Enterococcus from swine manure in China. METHODOLOGY: The oqxAB genes were screened in 87 Enterococcus isolates by PCR. The transferability of the oqxAB genes in Enterococcus was determined by conjugation experiments. The genetic environment of oqxAB genes was investigated by cloning experiments, PCR mapping and sequencing. RESULTS: A high prevalence (86.2 %) of olaquindox resistance was observed in Enterococcus and 98.9 % isolates exhibited multidrug-resistance phenotypes. The occurrence of oqxA and oqxB in Enterococcus was also high (79.3 and 65.5 %, respectively). Sequence analysis of the cloned fragment indicated that the oqxAB cassette was linked to an incomplete Tn5 transposon containing aph(3')-IIa and flanked by IS26 [IS26-oqxAB-IS26-aph(3')-IIa]. The oqxAB-aph(3')-IIa-positive transconjugant or transformant showed resistance or reduced susceptibility to enrofloxacin, ciprofloxacin, olaquindox, mequindox, florfenicol, neomycin and kanamycin. CONCLUSION: This is the first time that the oqxAB genes have been identified in Enterococcus faecalis from swine manure. The genetic linkage of oqxAB-aph(3')-IIa in Enterococcus has not been described before. The high prevalence of oqxAB genes in Enterococcus suggests that it may constitute a reservoir for oqxAB genes and pose a potential threat to public health.

The prevalence of mcr-1 and resistance characteristics of Escherichia coli isolates from diseased and healthy pigs.

Colistin has been used as the last-line antibiotic for Escherichia coli infections. Herein, we collected 102 E. coli isolates from diseased pigs and 204 from healthy ones in Henan province of China. Then, we screened antimicrobial resistance and mcr-1 of bacteria. There was 25.5% (78/306) mcr-1-positive porcine E. coli, in which 46 isolates (45.1%, 46/102) were obtained from diseased pigs; the others (15.7%, 32/204) were collected from healthy pigs (45.1% versus 15.7%, P=0.000). Meanwhile, the former presented more serious resistance to colistin, ceftiofur, cefquinome, gentamicin, amikacin, doxycycline, florfenicol, enrofloxacin, and olaquindox than those from healthy pigs, which were similar to the relations between isolates with or without mcr-1, except for amikacin and doxycycline. Also, the resistance profiles of mcr-1-positive E. coli were more extensive than those of mcr-1-negative isolates.

Comparative genomics of rmtB-carrying IncI1 ST136 plasmids in avian escherichia coli isolates from chickens in China.

Eight rmtB-carrying avian Escherichia coli strains from a farm in China were characterised in our previous study, but little is known about the backbones and entire multiresistance regions (MRRs) of these plasmids. Here, three rmtB-carrying IncI1 ST136 plasmids were analysed by whole-plasmid sequencing and were compared. These plasmids were composed of an 83 470-bp IncI1 backbone carrying genes responsible for plasmid replication, transfer, maintenance and stability functions, as well as a 17 330-bp MRR for pEC006 and pEC007, and a 34 626-bp MRR for pEC008. Plasmid pEC006 was not transferable, thus truncation of the traI gene may explain the inability to conjugate. pEC008 harboured the blaTEM-1, rmtB, aacC2, tetA, floR and strAB genes as well as a class 1 integron cassette array (|dfrA12|orfF|aadA2|), which were interspersed with different mobile elements, including Tn2, Tn1721, Tn1722, Tn5393, ISCfr1, IS5057, ISCR1 and ISCR2, and three copies of IS26. The MRR of pEC008 may have resulted from transposition of Tn1722 into the plasmid backbone. Acquisition and rearrangement of MRRs demonstrated the accumulation of different resistance determinants.

IS26-Flanked Composite Transposon Tn6539 Carrying the tet(M) Gene in IncHI2-Type Conjugative Plasmids From Escherichia coli Isolated From Ducks in China.

Tet(M)-type proteins confer resistance to tetracycline and related antibiotics by interacting with the ribosome. Genes encoding Tet(M) have been found in a range of bacteria, including Escherichia coli. In the current study, conjugation experiments were performed between seven different tetracycline-resistant, azide-susceptible E. coli strains isolated from ducks and tetracycline-sensitive, azide-resistant E.coli J53. Transconjugants were obtained from two of the strains at a frequency of 1.2 × 10-8. PCR, southern blotting and sequencing demonstrated that tet(M) in the transconjugants was located on a ~50 kb IncHI2-type plasmid and was part of a composite transposon, designated Tn6539. This transposon is flanked by two IS26 elements in opposite orientation and contains the Tn3ΔtnpA+Δorf13-lp-tet(M)+gamma delta+tnpX+ΔtnpR sequences. The Δorf13-lp-tet(M) sequence was a highly conserved genetic fragment in E. coli harboring tet(M) and mainly located in the composite transposons flanked by IS6-family elements. In summary, Tn6539 is a new composite transposon capable of horizontal transfer of tet(M) among E. coli isolates.