| Fluoroquinolones are widely used for treating human and animal infections, as one of the most important and efficient antimicrobial classes. However, the widely use of fluoroquinolones in human clinics and animals for therapy or growth promotion has led to significantly increasing resistance to quinolones, especially with the emergence of the plasmid-mediated quinolone resistance(PMQR) genes. Although oqx AB and qep A, both of them belong to PMQR genes and encode efflux pump, were previously investigated among animals or humans, the transmission mechanisms of oqx AB and qep A genes in E. coli isolates from different sources(animal, food, and human origin) are still unclear and needed to be analyzed. This study was carried out to investigate the prevalence of PMQR genes,transmission mechanisms of oqx AB and qep A, the characteristics of plasmids carrying oqx AB or qep A genes among E. coli isolates from different sources, including animals, food,pets, and clinical humans, in order to provide basic scientific research data for the prevention and control of quinolones resistance, to guide clinical rational drug use, and to support the development of new drugs.1740 E. coli isolates from various sources were tested for their susceptibility to eight antimicrobials classes using agar dilution method. The results showed that 80% of isolates were resistant to trimethoprim/sulfamethoxazole, which was the highest resistance rate,followed by ampicillin(73%), tetracycline(73%), streptomycin(67%), and olaquindox(64%), respectively. However, only 25% to 53% of isolates exhibited resistance to cefotaxime, ceftazidime, neomycin, gentamicin, apramycin, chloramphenicol, florfenicol,and ciprofloxacion. The resistance rates to cefoxitin, amikacin, and colistin were only 7%,4%, and 11%, respectively. More than 55% isolates of animal, food, and human origin showed high resistance rates to ampicillin and tetracycline. In addition, isolates of animal origin exhibited highest resistance rates to streptomycin, neomycin, apramycin,chloramphenicol, florfenicol, colistin, and olaquindox compared with isolates from food,pets and human, while isolates of human origin showed highest resistance rates to cefoxitin,cefotaxime, and ceftazidime. Of note, more than 95% of isolates from pigs and chickenwere resistant to olaquindox, significantly higher than isolates from food(39%) and human(38%). Meanwhile, more than 50% of strains of company animal and chicken origin exhibited resistance to ciprofloxacin, followed by isolates of food, human, and pig origin.Interestingly, isolates from company animals showed highest resistance to amikacin, the resistance rate was 27%, which was notably higher than isolates from food animals, food,and human.A total of 1034 E. coli strains from various sources were randomly selected to screen for PMQR genes by polymerase chain reaction(PCR) amplification. The prevalences of oqx AB in the isolates obtained from both food animals and food were 22.9%, significantly higher than that in isolates from humans and company animals. Isolates of food animal origin showed the highest prevalence of qnr S(30.3%), followed by isolates from animal-derived food(21.9%), while isolates from human and company animals showed distinctly lower prevalence, namely 7.86% and 8.01%, respecitively. However, isolates from human(4.08%) and company animals(6.0%) showed much higher prevalence of qep A than that in isolates of food animal(1.41%) and food(0.07%) origin. The prevalences of qnr B in strains isolated from animal, pets, and food were1.76%, 2.0%, and 0.35%,respectively. The qnr A, qnr C, and qnr D genes were not detected.50 oqx AB-positive E. coli from food animals, food, and patients were randomly selected to analyze their relateness by pulsed-field gel electrophoresis(PFGE). PFGE analysis of 45 oqx AB-positive E. coli strains revealed 32 Xba I patterns, most of which were clonally unrelated. But the clonal transmission of oqx AB-carrying E. coli occurred among animals, animal-derived food, and patients. 21 oqx AB transformants were obtained by electrotransformation, all of them showed multidrug-resistant, except transformant SNJ43-1.S1-PFGE and Southern blot hybridization indicated that the 21 oqx AB transformants carried1 to 4 plasmids ranged in size from 28 to 500 kb, oqx AB was located on one single plasmid in 16 transformants, four transformants with two plasmids showed two hybridization signals. Additionally, oqx AB genes were located on plasmids belonged to Inc N(n=1),Inc HI2(n=3), and Inc F(n=9), including five multiple-replicon plasmids. Meanwhile, seven of ten plasmids carrying oqx AB obtained from pigs, chicken, or food showed identical RFLP patterns, another two oqx AB-carrying plamids isolated from chicken and patient also demonstrated indistinguishable RFLP patterns, indicating that horizontal transfer of similar plasmids contributed to oqx AB dissemination among E. coli isolates obtained from different sources. Then the 21 oqx AB-positive transformants were investigated the sequences surrounding oqx AB by inverse PCR combined with PCR mapping strategy. In 19 transformants, oqx AB was flanked by two copies of IS26 located in the same orientation.The genetic environments of oqx AB in E. coli strains obtained from animals, human, and food in the present study were similar to previously reported oqx AB-carrying plasmids from E. coli or Salmonella strains of human or animal origin. Thus, IS26 may play an important role in oqx AB transmission among various plasmids and strains from different sources. The qep A genes from two isolates were successfully transferred by conjugation and were co-located on F2:A-:B- type multidrug resistance plasmids with rmt B. They showed indistinguishable RFLP patterns compared with transconjugants 3A11-16 J obtained from pet in 2008, suggesting that the dissemination of qep A and rmt B genes in E. coli isolates from various sources is mainly mediated by the F2:A-:B- plasmids.The complete sequences of the multiresistant oqx AB-carrying plasmid p HNZY32 were obtained through Pac Bio sequencing technology. Plasmid p HNZY32 harbored multiple replicons, including Inc FII, Inc N and Inc X replicons. The transfer area and adjacent leading area of p HNZY32 were highly identical to F33:A:B- type plasmids, and it also carried four plasmid addiction systems, namely pem KI, vag CD, hok-sok and srn BC. Plasmid p HNZY32 contained two multiresistance regions, including resistance genes aph(3’)-IIa, Δble, oqx AB,bla TEM-1, rmt B, fos A3 and bla CTX-M-55, as well as flo R, tet A, str A, str B and sul2. p HNZY32 appears to have evolved from N1- F33:A-:B- plasmid p HNFP460-1 or its similar plasmids through the stepwise acquisition, recombination associated with strunctural rearrangement of Inc X plasmid segment including oqx AB and replication region, as well as 20-Kb segment containing multiple antimicrobial resistance determinants. The replication region belongs to Inc X plasmid of p HNZY32 was also identified in 15 oqx AB-carrying transformants,suggesting that Inc X plasmid may play a role in the spread of oqx AB gene between E. coli isolates from different sources.The qep A-harbouring F2:A-:B- plasmid p HN3A11 was fully sequenced by using a whole-genome shotgun approach and compared with other Inc FII plasmids. The backbone of p HN3A11 was similar to that of the Inc FII plasmids obtained from China(p HK23 a,p FOS-HK151325, p XZ) and Canada(p C15-1a). It contained plasmid addiction systems(pem I/pem K, hok/mok/sok) and partitioning systems(par M, par B, and stb B) that promoted plasmid maintenance during vertical transmission. rmt B qep A, bla TEM-1 and dfr genes were found in multiresistance region of plasmid p HN3A11, interspersed with different complete or truncated insertion sequences and transposons(ΔIS1, ΔTn2, Δint I1, ISCR33, IS26 and Tn21). Further analysis confirmed that p HN3A11-like plasmids have disseminated in E.coli isolates from pets, food animals and farm environments.In summary, oqx AB was widely distributed in E. coli strains from food animals, pets,animal-derived food, and patients. Clonal spread of oqx AB-positive isolates as well as horizontal transmission mediated by plasmids and insertion sequence IS26, contributed to the dissemination of oqx AB among E. coli isolates from different sources. As for qep A, it was co-located on F2:A-:B- plasmid with rmt B and their dissemination in E. coli isolates from various sources is mainly mediated by the F2:A-:B- plasmids. The spread of oqx AB and qep A among E. coli from various sources would not only influence the prevention and therapy in veterinary clinical, but may also pose a potential challenge to the public health. |
PDF Full Text Request