Coli C600 Research Articles

The effect of triclosan on intergeneric horizontal transmission of plasmid-mediated tigecycline resistance gene tet(X4) from Citrobacter freundii isolated from grass carp gut

The transmission of antibiotic resistance genes (ARGs) in pathogenic bacteria affects culture animal health, endangers food safety, and thus gravely threatens public health. However, information about the effect of disinfectants - triclosan (TCS) on ARGs dissemination of bacterial pathogens in aquatic animals is still limited. One Citrobacter freundii (C. freundii) strain harboring tet(X4)-resistant plasmid was isolated from farmed grass carp guts, and subsequently conjugative transfer frequency from C. freundii to Escherichia coli C600 (E. coli C600) was analyzed under different mating time, temperature, and ratio. The effect of different concentrations of TCS (0.02, 0.2, 2, 20, 200 and 2000 μg/L) on the conjugative transfer was detected. The optimum conditions for conjugative transfer were at 37 °C for 8h with mating ratio of 2:1 or 1:1 (C. freundii: E. coli C600). The conjugative transfer frequency was significantly promoted under TCS treatment and reached the maximum value under 2.00 μg/L TCS with 18.39 times that of the control group. Reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) activities, cell membrane permeability of C. freundii and E. coli C600 were obviously increased under TCS stress. Scanning electron microscope showed that the cell membrane surface of the conjugative strains was wrinkled and pitted, even broken at 2.00 μg/L TCS, while lysed or even ruptured at 200.00 μg/L TCS. In addition, TCS up-regulated expression levels of oxidative stress genes (katE, hemF, bcp, hemA, katG, ahpF, and ahpC) and cell membrane-related genes (fimC, bamE and ompA) of donor and recipient bacteria. Gene Ontology (GO) enrichment demonstrated significant changes in categories relevant to pilus, porin activity, transmembrane transporter activity, transferase activity, hydrolase activity, material transport and metabolism. Taken together, a tet(X4)-resistant plasmid could horizontal transmission among different pathogens, while TCS can promote the propagation of the resistant plasmid.

High prevalence of multidrug-resistant Escherichia coli in retail aquatic products in China and the first report of mcr-1-positive extended-spectrum β-lactamase-producing E. coli ST2705 and ST10 in fish

Contamination of food by multidrug-resistant (MDR) bacteria is a potential threat to consumers. Aquatic products are increasingly consumed due to their high value and rich nutrient. Nevertheless, the prevalence of multi-drug resistant (MDR) E. coli in retail aquatic products has not been systematically investigated in China. In this study, we conducted a national investigation on the prevalence of E. coli and MDR E. coli in retail aquatic products and the characteristics of the MDR E. coli isolates. A total of 849 samples consisting of 680 fish, 143 shrimp, and 26 shellfish were purchased from markets in 39 cities in China and investigated for the presence of E. coli. Overall, 340 (40.0 %) and 169 (19.9 %) samples were contaminated with E. coli and MDR E. coli, indicating poor hygiene conditions of retail aquatic products. A total of 190 MDR E. coli were isolated, which were recovered from 38.5 % shellfish, 20.1 % shrimp, and 19.0 % fish. Thirty-two ESBL-producing E. coli were identified from 3.5 % fish, 3.5 % shrimp, and 3.8 % shellfish. The MDR E. coli isolates showed a high prevalence of resistance to tetracycline (93.7 %), trimethoprim-sulfamethoxazole (78.9 %), ampicillin (78.4 %), chloramphenicol (72.1 %), nalidixic acid (73.2 %), cephalothin (65.3 %), and streptomycin (65.8 %). Resistances to kanamycin (42.1 %), gentamicin (37.9 %), ciprofloxacin (42.6 %), and norfloxacin (45.8 %) were also common. Further, 15.3 % and 8.4 % of the isolates were resistant to cefotaxime and ceftazidime, respectively. Four isolates were resistant to colistin. More than 85.0 % of the isolates were resistant to 5–15 antibiotics. Of the antibiotic resistance genes, TEM-1, tetA, strA/B, sul2, aadA, floR, and qnrS were the most prevalent, followed by sul1, aac(6′)-Ib, oqxA/B, cmlA and aphA1. Six CTX-M-types were found, among which CTX-M-55 and CTX-M-14 were predominant. All 4 colistin-resistant isolates carried the mcr-1 gene, of which three were ESBL strains. WGS indicated that the mcr-1 gene was located on two types of plasmids (IncHI2 and IncX4). Conjugation experiments showed the mcr-1 gene could be transferred to E. coli C600. To our knowledge, this is the first report of mcr-1-positive ESBL E. coli (ST10 and ST2705) in retail aquatic products. The emergence of such strains poses a serious threat to consumers, with the potential to spread resistance genes to humans. Our results indicate that retail aquatic products are important reservoirs of MDR E. coli and facilitate the dissemination of the resistance genes. Continuous surveillance and interventions of restricting antibiotic use in aquatic environments should also be applied to reduce the prevalence of MDR bacteria.

Effect of Dimethyl Sulfoxide on the Bioluminescence Expression of the Inducible SOS-Lux gene Biosensor E. coli C600 pPSL-1 and E. coli DPD1718 elicited by Mitomycin-C

Effect of Dimethyl Sulfoxide on the Bioluminescence Expression of the Inducible SOS-Lux gene Biosensor E. coli C600 pPSL-1 and E. coli DPD1718 elicited by Mitomycin-C

Prevalence of colistin-resistant mcr-1-positive Escherichia coli isolated from children patients with diarrhoea in Shanghai, 2016–2021

The emergence of the plasmid-mediated colistin resistance 1 (mcr-1) of Escherichia coli has become a global health concern. This study reports the prevalence of mcr-1 among E. coli isolates from diarrheal children under 5 years old in Shanghai and the genetic characterization of mcr-1-harbouring plasmid. A total of 1,723 E. coli strains were collected from the feces of diarrheal patients in all sentinel hospitals in Shanghai during 2016-2021. Antimicrobial susceptibility testing was performed by broth microdilution and plasmid conjunction transfer assay was carried out using E. coli C600 as the recipient. The mcr-1-positive E. coli strains (MCRPEC) were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. Only 5 (0.28%) strains were found to harbour the mcr-1 gene using PCR screening. Plasmid conjugation assay and whole genome sequencing indicated EC16500, one MCRPEC strain, co-exhibited mcr-1, blaTEM-1, blaOXA-1, qnrS1, qnrS2, arr-3, and catB3, could be conjugated to EC C600 by horizontal transfer with an average efficiency of 3.2 × 10-5. The plasmid pEC16500 harboured the similar backbone as p70_2_15, pECGD-8-33, pNCYU-29-19-1_MCR1 and pIBMC_mcr1, and was shown to be encoded within a type IV secretion system (T4SS)-containing 32.6 kbp IncX4, next to the pap2-like membrane-associated gene to form a 2.4-kb cassette. Furthermore, sequencing and phylogenetic analyses revealed a similarity between other MCR-1-homolog proteins, indicating that the 5 E. coli isolates were colistin resistant. Our data represents a significant snapshot of colistin resistance mcr-1 genes and highlights the need to increase active surveillance especially among the children under 5 years old in Shanghai. Great effort needs to be taken to avoid further dissemination of plasmid-mediated colistin resistance among clinically relevant Gram-negative bacterial pathogens.

Tracing ΦX174 bacteriophage spreading during aerosol-generating procedures in a dental clinic

ObjectiveThe aim of this study was to test the plausibility of using the ΦX174 bacteriophage as a tracer of viral aerosols spreading in a dental aerosol-generating procedure (AGP) model.MethodsΦX174 bacteriophage (~ 108 plaque-forming units (PFU)/mL) was added into instrument irrigation reservoirs and aerosolized during class-IV cavity preparations followed by composite fillings on natural upper-anterior teeth (n = 3) in a phantom head. Droplets/aerosols were sampled through a passive approach that consisted of Escherichia coli strain C600 cultures immersed in a LB top agar layer in Petri dishes (PDs) in a double-layer technique. In addition, an active approach consisted of E coli C600 on PDs sets mounted in a six-stage cascade Andersen impactor (AI) (simulating human inhalation). The AI was located at 30 cm from the mannequin during AGP and afterwards at 1.5 m. After collection PDs were incubated overnight (18 h at 37 °C) and bacterial lysis was quantified.ResultsThe passive approach disclosed PFUs mainly concentrated over the dental practitioner, on the mannequin’s chest and shoulder and up to 90 cm apart, facing the opposite side of the AGP’s source (around the spittoon). The maximum aerosol spreading distance was 1.5 m in front of the mannequin’s mouth. The active approach disclosed collection of PFUs corresponding to stages (and aerodynamic diameters) 5 (1.1–2.1 µm) and 6 (0.65–1.1 µm), mimicking access to the lower respiratory airways.ConclusionThe ΦX174 bacteriophage can be used as a traceable viral surrogate in simulated studies contributing to understand dental bioaerosol’s behavior, its spreading, and its potential threat for upper and lower respiratory tract.Clinical relevanceThe probability to find infectious virus during AGPs is high. This suggests the need to continue characterizing the spreading viral agents in different clinical settings through combination of passive and active approaches. In addition, subsequent identification and implementation of virus-related mitigation strategies is relevant to avoid occupational virus infections.

Antimicrobial Resistance and Genomic Epidemiology of tet(X4)-Bearing Bacteria of Pork Origin in Jiangsu, China.

The emergence of tigecycline-resistant bacteria in agri-food chains poses a public health concern. Recently, plasmid-mediated tet(X4) was found to be resistant to tigecycline. However, genome differences between tet(X4)-positive Escherichia coli of human and pork origins are still under-investigated. In this study, 53 pork samples were collected from markets in Jiangsu, China, and 23 tet(X4)-positive isolates were identified and shown to confer resistance to multiple antibiotics, including tigecycline. tet(X4)-positive isolates were mainly distributed in E. coli (n = 22), followed by Klebsiella pneumoniae (n = 1). More than half of the tet(X4) genes were able to be successfully transferred into E. coli C600. We downloaded all tet(X4)-positive E. coli isolates from humans and pork found in China from the NCBI database. A total of 42 known STs were identified, of which ST10 was the dominant ST. The number of ARGs and plasmid replicons carried by E. coli of human origin were not significantly different from those carried by E. coli of pork origin. However, the numbers of insertion sequences and virulence genes carried by E. coli of human origin were significantly higher than those carried by E. coli of pork origin. In addition to E. coli, we analyzed all 23 tet(X4)-positive K. pneumoniae strains currently reported. We found that these tet(X4)-positive K. pneumoniae were mainly distributed in China and had no dominant STs. This study systematically investigated the tet(X4)-positive isolates, emphasizing the importance of the continuous surveillance of tet(X4) in pork.

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.

Conjugative transfer of mcr-1-bearing plasmid from Salmonella to Escherichia coli in vitro on chicken meat and in mouse gut

Since mcr-1 was first discovered in 2015, this gene has shown excellent transmission ability and evolutionary characteristics worldwide, leading to major public health and food safety concerns. In this study, chicken meat was used as a food vehicle for the conjugation of mcr-1-bearing Salmonella at different storage temperatures (4 °C, 25 °C, and 37 °C) to simulate mcr-1 transmission during food transportation and storage and determine its efficiency and mechanism. In addition, conjugation experiments were performed in mouse gut to further confirm that mcr-1 is horizontally transferred in vivo during food consumption. 16S rDNA sequencing of mouse stool samples was performed to understand the effect of horizontal transfer of mcr-1 on mouse gut bacteria. mcr-1-bearing plasmids were characterized using pulsed-field gel electrophoresis (PFGE) and S1 nuclease-PFGE and sequenced by Illumina sequencing. Our results showed that mcr-1-bearing plasmids in donors are successfully transferred to recipients on chicken meat at not only 25 °C and 37 °C but also 4 °C with conjugation frequencies between 1.32 × 10-6 and 3.85 × 10-4 per recipient cell. In mouse gut, mcr-1 was transferred not only to the recipient bacteria introduced by intragastric administration but also to the intestinal bacteria (E. coli strain named as E6353). Horizontal transfer of mcr-1-bearing plasmid in mouse gut negatively affected the mouse intestinal microbiota. In a constant conjugative environment, plasmid replicon type is the most decisive factor affecting the conjugation frequency. The peak number of transconjugants in group D6-E. coli C600 with an IncHI2-type mcr-1-bearing plasmid (1.43 × 102 colony-forming units [CFU]/g feces) was significantly higher than that of transconjugants in group D7-E. coli C600 with an IncX4-type mcr-1-bearing plasmid (0.3 × 102 CFU/g feces). The upstream and downstream genetic environment of mcr-1 in different plasmid replicon types in Salmonella varied during conjugation in different horizontal transfer environments. An IncI2 plasmid (p25-D4R7S1_mcr-1) lost the insertion sequence ISApl1, which originally existed upstream of mcr-1, when this plasmid transferred from donor to recipient cells on chicken meat at 25 °C. An IncHI2 plasmid was more active than IncI2 and IncX4 plasmids during bacterial reproduction and evolution; an IncFIB–IncHI2 hybrid plasmid (p6176253_mcr-1) was formed in mouse gut during conjugation from pD6_tet(M) and pD6_mcr-1. mcr-1 is captured by mobile genetic elements IS26 in IncX4 plasmids and ISApl1 in IncI2, IncHI2, and IncFIB–IncHI2 hybrid plasmids and is disseminated among bacteria.

Coexistence of tet(X4), mcr-1, and blaNDM-5 in ST6775 Escherichia coli Isolates of Animal Origin in China.

ABSTRACTEmergence of pathogens harboring multiple resistance genes incurs great concerns. Cooccurrence of mobile resistance genes conferring resistance to tigecycline, colistin, and carbapenems in Escherichia coli has not been investigated. This study aimed to characterize three E. coli isolates coharboring tet(X4), mcr-1, and blaNDM-5. Isolates coharboring tet(X4), mcr-1, and blaNDM-5 were identified and characterized by PCR, Sanger sequencing, antimicrobial susceptibility testing, conjugation assays, Illumina sequencing, nanopore sequencing, and bioinformatic analysis. Three E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 were identified from pigeons in China. They were resistant to almost all antimicrobials except enrofloxacin. tet(X4) and blaNDM-5 could be conjugated into E. coli C600, but mcr-1 was nontransferable in three isolates. Three isolates belonged to sequence type 6775 (ST6775), and clonal dissemination of isolates carrying tet(X4), mcr-1, and blaNDM-5 existed in the pigeon farm. Genetic analysis revealed that mcr-1 mediated by the Tn6330 was located on the chromosome, tet(X4) was located on the IncFII plasmid, and blaNDM-5 was located on the IncX3 plasmid. We first characterized the E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 simultaneously. Relevant measures should be taken to decrease the prevalence of pathogens carrying tet(X4), mcr-1, and blaNDM-5.IMPORTANCE Tigecycline and colistin are regarded as vital antimicrobials to treat multidrug-resistant (MDR) bacterial infections, such as that caused by carbapenemase-producing Enterobacteriaceae (CPE). Cooccurrence of mobile resistance genes conferring resistance to last-resort antimicrobials in E. coli remains unknown. Here, we characterized E. coli strains coharboring tet(X4), mcr-1, and blaNDM-5 phenotypically and genetically. Resistance genes tet(X4), mcr-1, and blaNDM-5 were located on transposons or plasmids that were mobile genetic elements related to the capture, accumulation, and dissemination of such important resistance genes. The emergence of E. coli isolates carrying tet(X4), mcr-1, and blaNDM-5 highlights the importance of monitoring the coexistence of novel mobile resistance genes in different settings with a One Health approach. Risk of transmission of such MDR pathogens from animals to humans should be evaluated comprehensively.

Genetic context diversity of plasmid-borne blaCTX-M-55 in Escherichia coli isolated from waterfowl

The aim of this study was to explore the prevalence of blaCTX-M and elucidate the characteristics of transferable blaCTX-M-55 plasmids. Escherichia coli were isolated from 10 large-scale duck farms from nine counties and cities in Guizhou Province. They were identified, subjected to antimicrobial susceptibility testing and screened for antibiotic resistance genes. blaCTX-M-positive strains were subtyped, and blaCTX-M-55-containing strains were further analysed by multilocus sequence typing, conjugation experiments and polymerase chain reaction-based replicon typing. Three transferable plasmids were selected for whole genome sequencing sequencing and further analysis of genetic context. In total, 128 E. coli strains were identified, and 23 were blaCTX-M-positive. Subtype analysis of blaCTX-M-positive strains revealed that 23 were blaCTX-M-55 and one of the strains contained both blaCTX-M-65 and blaCTX-M-55. They were divided into 12 multilocus sequence typing types, and 6 were undetermined. blaCTX-M-55-bearing plasmids in seven were transferred to E. coli C600 at frequencies between 10-6 and 10-9, and blaCTX-M-55 was found to be located on IncN, IncFⅡ and IncFII-N fusion type plasmids. Three plasmids (IncFⅡ plasmids pEC99_Plas3 and pS1_Plas2 and IncFII-N plasmid pD2_plas2) shared a homologous region of IS26-ARGs-IS26-blaTEM-orf477-blaCTX-M-55-ISEcp1△-IS26. Our study demonstrates the environmental presence of blaCTX-M-55 harbouring E. coli in Guizhou. Analysis of the genetic context of blaCTX-M-55 aids in understanding possible dissemination routes of ESBL genes and controlling widespread antibiotic resistance.

Characterization of Erysipelothrix rhusiopathiae Isolates from Diseased Pigs in 15 Chinese Provinces from 2012 to 2018.

Erysipelothrix rhusiopathiae can cause erysipelas in animals and erysipeloid in humans. Since its recurrence in 2012, swine erysipelas has caused serious losses within the pig industry in China. The aim of this study was to perform multilocus sequence typing and understand the virulence and antimicrobial susceptibility of E. rhusiopathiae isolates in China. Multilocus sequence typing (MLST) of a total of 120 strains was performed, and as a result, three different sequence types were identified, of which ST48 was the main one. Five isolates of each MLST type were randomly selected to be used to challenge mice. ST48 was associated with a higher virulence. Antimicrobial susceptibility was tested using a microdilution technique and, to analyze the resistance mechanism, six strains were selected for genome sequencing. A comparison of the six genomes indicated the presence of a suspected macrolide resistance gene, namely, Erm(A)-like, in erythromycin-resistant strains, which increased the minimum inhibitory concentration (MIC) of erythromycin against E. coli C600 at least four-fold. In addition, three mutations (gyrA86T-I, gyrA90D-N, and parC81S-I) were observed in the quinolone resistance-determining regions (QRDRs) of gyrA and parC in quinolone-resistant strains. After the gyrA gene with the 86T-I mutation or the parC gene with the 81S-I mutation was transfected into E. coli C600, the MIC of enrofloxacin against this strain increased at least two-fold. Our findings provide a theoretical basis for developing antibacterial drugs and may contribute to the clinical prevention and control of E. rhusiopathiae.

High prevalence of multidrug-resistant Escherichia coli and first detection of IncHI2/IncX4-plasmid carrying mcr-1 E. coli in retail ready-to-eat foods in China

Antibiotic-resistant bacteria in food pose an important threat to public health. Multidrug-resistant strains in ready-to-eat (RTE) foods can be transferred to humans through diet, which increases their health risk. This study systematically investigated antibiotic resistance and antibiotic resistance genes in E. coli isolated from retail RTE foods and characterized plasmid-mediated colistin-resistant E. coli strains. A total of 1118 RTE food samples were collected from markets in 39 cities in China, and 126 E. coli strains, >95% of which were multidrug-resistant, were isolated. The isolates showed a high prevalence of resistance to tetracycline (95.24%), ampicillin (82.54%), trimethoprim-sulfamethoxazole (77.78%), nalidixic acid (74.60%), cephalothin (72.22%), chloramphenicol (66.67%), and streptomycin (53.97%). Twenty-two extended-spectrum β-lactamase (ESBL)-producing E. coli and four colistin-resistant E. coli were identified. The resistance genes TEM, CTX-M, tetA, sul2, strA/strB, aadA, and qnrS were the most frequently detected. CTX-M-55 and CTX-M-14 were the predominant CTX-M types. All the four colistin-resistant E. coli isolates were positive for mcr-1. The mcr-1 gene can be transferred to E. coli C600 through conjugation and transformation. Whole-genome sequencing revealed that the mcr-1 genes were found in IncX4 and IncHI2 plasmids. To the best of our knowledge, this is the first report of IncHI2/IncX4 plasmid-bearing mcr-1-positive E. coli strains in RTE foods sold in markets, and the first report of the isolation of the international epidemic E. coli clone ST101 and mcr-1-carrying ESBL-producing E. coli from RTE foods. These results provide valuable information for assessing antibiotic-resistant E. coli infections and controlling antibiotic-resistant E. coli.

Rapid Increase in the IS26-Mediated cfr Gene in E. coli Isolates with IncP and IncX4 Plasmids and Co-Existing cfr and mcr-1 Genes in a Swine Farm.

This paper aimed to investigate the molecular epidemiological features of the cfr gene in E. coli isolates in a typical swine farm during 2014–2017. A total of 617 E. coli isolates were screened for the cfr gene using PCR amplification. A susceptibility test, pulsed-field gel electrophoresis (PFGE), S1-PFGE, southern blotting hybridization, and the genetic context of the cfr gene were all used for analyzing all cfr-positive E. coli isolates. A conjugation experiment was conducted with the broth mating method using E. coli C600 as the recipient strain and 45 mcr-1-cfr-bearing E. coli isolates as the donor strain. Plasmids pHNEP124 and pHNEP129 were revealed by Illumina Miseq 2500. Eighty-five (13.7%) E. coli isolates were positive for the cfr gene and the prevalence of the cfr gene had significantly increased from 1.6% in 2014 to 29.1% in 2017. The Pulsed-Field Gel Electrophoresis (PFGE) analysis indicated that the spread of the cfr gene among E. coli isolates was mainly due to horizontal transfer. In addition, the cfr gene was primarily located on the plasmids between 28.8-kb to 60-kb in size, and the cfr gene was flanked by two copies of IS26 with the same orientation. Sequence analysis suggested that the plasmids pHNEP124 and pHNEP129 co-harboring the cfr and mcr-1 genes belonged to the plasmids IncP plasmid and IncX4 plasmid, respectively. In conclusion, this is the first study to report the high prevalence of the cfr gene among E. coli isolates and the first report of the complete genome sequence of IncP and IncX4 plasmids carrying the mcr-1 and cfr genes. The occurrence and dissemination of the cfr/mcr-1-carrying plasmids among E. coli isolates need further surveillance.

Identification of fosA10, a Novel Plasmid-Mediated Fosfomycin Resistance Gene of Klebsiella pneumoniae Origin, in Escherichia coli.

PurposeSeveral subtypes of plasmid-mediated fosfomycin resistance gene fosA in Enterobacteriaceae have been reported worldwide and have caused concern. The present study characterized a novel member of fosA gene located on a plasmid from Escherichia coli.Materials and MethodsA fosfomycin-resistant E. coli isolate PK9 was recovered from a chicken meat sample in 2018. The presence of fosA genes was detected by PCR and sequencing. Whole-genome sequencing (WGS), conjugation, and cloning were performed to identify the mechanism responsible for fosfomycin resistance. Oxford Nanopore MinION sequencing was carried out to characterize the plasmid carrying fosfomycin resistance gene and the genetic context of the novel fosA variant.ResultsA novel fosA gene with significant homology (>98%) with fosA6 and fosA5 genes was identified by WGS and was named fosA10. FosA10 shared 56.1% to 98.6% amino acid sequence identity with other reported plasmid-mediated FosA enzymes. Fosfomycin resistance and fosA10 gene were successfully transferred to E. coli C600 by conjugation. Cloning confirmed that FosA10 could confer fosfomycin resistance (MIC > 128 μg/mL). The fosA10 gene was localized on a 53kb IncFII (F35:A-:B-) plasmid. The ∆lysR-fosA10-∆hp fragment (4328 bp), located between two copies of IS10R, showed 100% identity with the chromosomal sequences of 17 Klebsiella pneumoniae strains of ST664 and one of ST3821 in GenBank.ConclusionOur findings indicated that the fosA10 gene of E. coli might be captured from the chromosome of K. pneumoniae by IS10, which further demonstrated that K. pneumoniae might act as a reservoir of fosA-like genes acquired by E. coli.

Antibiotic-resistance gene transfer in antibiotic-resistance bacteria under different light irradiation: Implications from oxidative stress and gene expression

Due to the significant public health risks, there is substantial scientific interest in the increasing abundance of antibiotic-resistance bacteria (ARB) and the spread of antibiotic-resistance genes (ARGs) in aquatic environments. To clearly understand the mechanism of ARG transfer, this study examined the conjugative transfer of genes encoding resistance to cephalosporin (blaCTX) and polymyxin (mcr-1) from two antibiotic-resistant donor strains, namely E. coli DH5α (CTX) and E. coli DH5α (MCR), and to a streptomycin-resistant receptor strain (E. coli C600 (Sm)). Conjugative transfer was specifically studied under different light irradiation conditions including visible light (VL), simulated sunlight (SS) and ultraviolet light (UV254nm). Results show that the conjugative transfer frequency was not affected by VL irradiation, while it was slightly improved (2–10 fold) by SS irradiation and extremely accelerated (up to 100 fold) by UV irradiation. Furthermore, this study also explored the link between ARG transfer and stress conditions. This was done by studying physiological and biochemical changes; oxidative stress response; and functional gene expression of co-cultured AR-E. coli strains under stress conditions. When correlated with the transfer frequency results, we found that VL irradiation did not affect the physiological and biochemical characteristics of the bacteria, or induce oxidative stress and gene expression. For SS irradiation, oxidative stress occurred slowly, with a slight increase in the expression of target genes in the bacterial cells. In contrast, UV irradiation, rapidly inactivated the bacteria, the degree of oxidative stress was very severe and the expression of the target genes was markedly up-regulated. Our study could provide new insight into the underlying mechanisms and links between accelerated conjugative transfer and oxidative stress, as well as the altered expression of genes relevant to conjugation and other stress responses in bacterial cells.

The effect of antimicrobials on verocytotoxin bacteriophage transduction under bovine rumen fluid and broth conditions

AbstractThe verocytotoxin genes in verocytotoxigenicEscherichia coli(VTEC) are carried by bacteriophages, incorporated into the bacterial genome (prophage). Antibiotics may promote phage replication and release to infect other cells (transduction), thus leading to the emergence of new VTEC strains. This study investigated transduction of a verocytotoxin2-encoding bacteriophage (3538(vtx2::cat)) under laboratory conditions, including the effect of antibiotic treatments. Luria-Bertani Miller broth and rumen fluid (raw and sterilised by irradiation) were inoculated with the donor (C600φ3538(Δvtx2::cat)) and recipient (E. coli C600::kanamycinR) strains (4 log10cfu/mL) and incubated at 38°C. Antibiotic treatments (minimal inhibitory and sub-inhibitory concentrations of ampicillin, cefquinome, oxytetracycline and sodium sulfamethazine) were applied after 3 h. Samples were tested for donor, recipient, cell-free phage and transductants at times t = 0, 3, 4, 6, 27 (24 h post-antibiotic treatment) and 51 h. Free phage was detected in the untreated broth and rumen samples, as were the transductants confirmed by polymerase chain reaction. The antibiotic treatments did not significantly (P &gt; 0.01) increase the concentrations of free phage or transductants detected. It was therefore concluded that, under laboratory conditions, the antibiotics tested did not induce bacteriophage lysis, release and infection of new bacterial cells beyond that constitutively found in the phage population.

Contribution of cropland to the spread of Shiga toxin phages and the emergence of new Shiga toxin-producing strains

A growing interest in healthy eating has lead to an increase in the consumption of vegetables, associated with a rising number of bacterial outbreaks related to fresh produce. This is the case of the outbreak in Germany, caused by a O104:H4 enteroaggregative E. coli strain lysogenic for a Stx phage. Temperate Stx phages released from their hosts occur as free particles in various environments. This study reports the occurrence of Stx phages in vegetables (lettuce, cucumber, and spinach) and cropland soil samples. Infectious Stx2 phages were found in all samples and many carried also Stx1 phages. Their persistence in vegetables, including germinated sprouts, of Stx phage 933 W and an E. coli C600 (933 W∆stx::gfp-cat) lysogen used as surrogate, showed reductions below 2 log10 units of both microorganisms at 23 °C and 4 °C over 10 days. Higher reductions (up to 3.9 log10) units were observed in cropland soils at both temperatures. Transduction of a recombinant 933 W∆stx::kan phage was observed in all matrices. Protecting against microbial contamination of vegetables is imperative to ensure a safe food chain. Since the emergence of new Stx strains by Stx phage transduction is possible in vegetable matrices, methods aimed at reducing microbial risks in vegetables should not neglect phages.

Association of antibiotic resistance with SHV-12 extended-spectrum β-lactamase in Enterobacter cloacae.

The association between antibiotic resistance and SHV-12 extended-spectrum β-lactamase (ESBL) in Enterobacter cloacae remains unknown. The aim of the present study was to investigate the prevalence of both chromosome- and plasmid-borne SHV-12 ESBL genes in Enterobacter cloacae. Transmission of the SHV-12 ESBL gene was explored, and the risk factors for antibiotic resistance in E. cloacae were analyzed. Polymerase chain reaction (PCR) results showed that 58 out of the 100 isolates carried the SHV-12 ESBL gene: 34.48% of them occurred in the chromosome, 48.28% were plasmid-borne and 17.24% appeared in both. Enterobacterial repetitive intergenic consensus-PCR tests detected 82 chromosomal genotypes. Conjugation assays showed that 70.00% of plasmid-borne SHV-12 ESBL genes were successfully transconjugated into E. coli C600 and that the antibiotic resistance phenotype of E. cloacae was partially (84%) or completely (10%) transferred. A significantly higher SHV-12 ESBL detection rate was found in patients with underlying conditions and/or complications compared with those without (P<0.05). The detection of SHV-12 ESBL-producing E. cloacae from vertical transmission varied significantly across clinical departments and age groups (P<0.05), with the highest rates in the intensive care unit and the group of patients aged ≥60 years. The present results indicate that the location and transmission efficiency of SHV-12 ESBL are closely correlated with the antibiotic resistance of E. cloacae.

Coupling dTTP Hydrolysis with DNA Unwinding by the DNA Helicase of Bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.

Molecular modeling and docking analysis of beta-lactamases with inhibitors: a comparative study.

Beta-lactamases are bacterial enzymes which impart resistance against β-lactam-antibiotics. CTX-Ms are the β-lactamases that target cephalosporin antibiotics (e.g. cefotaxime and ceftazidime) while SME-1, KPC-2, IMI-1 and SFC-1 target carbapenems. Clavulanic acid, sulbactam and tazobactam are traditional β-lactamase inhibitors while LN1-255 and NXL-104 whereas novel inhibitors, inhibiting the activity of these enzymes. Studying the binding pattern of these drugs is helpful in predicting the versatile inhibitors for betalactamases. The aims of the study were: describing the mode of interaction of CTX-M (modeled from the blaCTX-M gene of this study) and the said carbapenemases with their respective target drugs and inhibitors and to perform an in silico comparison of the efficacies of traditional and novel β-lactamase-inhibitors based on fitness score. The blaCTX-M marker was PCR-amplified from plasmid DNA of E. coli strain isolated from community-acquired urinary tract infection. E. coli C600 cells (harboring cloned blaCTX-M) were found positive for extended-spectrum-β-lactamase (ESBL) production by the double-disk-synergy test. The three dimensional structures of CTX-M-15, SME-1 and IMI-1 were predicted by Swiss Model Server. The interaction between selected structures and inhibitors was performed by GOLD 5.0. On the basis of the docking score and binding pattern, we conclude that compound LN1-255 followed by tazobactam is best inhibitor against all the selected target enzymes as compared to clavulanate, sulbactam and NXL-104. Five conserved amino acids, Ser70, Ser130, Lys235, Thr236 and Gly237 were found crucial in stabilizing the complexes through hydrogen bonding and hydrophobic interactions.