Drug Resistance and R Plasmids in Vibrio anguillarum Isolated in Cultured Ayu (Plecoglossus altivelis)

Two hundred twenty-six strains of Vibrio anguillarum collected from cultured ayu (Plecoglossus altivelis) between 1978 and 1980 were studied for their sensitivities to 10 chemotherapeutics. In order to determine whether the drug-resistant strains possessed transferable R plasmids, they were conjugated with Escherichia coli. Almost all the strains isolated during the 3 years showed resistance to nalidixic acid (NA) and/or furazolidone (NF). NA and NF resistance were not transferred to Escherichia coli from any of the strains. Chloramphenicol-resistant strains were isolated in every year and almost all of them carried transferable R plasmids. Only one strain with tetracycline resistance was found among the strains tested. Strains resistant to sulfonamides, streptomycin, ampicillin (ABP), and trimethoprim (TMP) increased rapidly in 1980, and a large number of them carried transferable R plasmids. Transferable R plasmids encoded with resistance to ABP and TMP were detected for the first time in V. anguillarum strains. The R plasmids detected in the strains isolated in 1980 were classified into incompatibility groups E, A, and an untypable group. The R plasmid DNAs were cleaved by EcoRI to yield 11 to 13 fragments. The estimated molecular weights of the R plasmids from the five strains ranged from 97 to 104 M daltons.     

通辽地区犊牛腹泻大肠杆菌耐药性检测及一株多重耐药菌全基因组测序分析

【背景】大肠杆菌(Escherichia coli)是引起犊牛腹泻的最主要病原菌,其耐药性菌株的不断出现引起广泛关注。【目的】了解内蒙古自治区通辽市犊牛腹泻大肠杆菌耐药性及耐药基因流行情况。【方法】从通辽市多个旗县采集犊牛腹泻样品40份,经细菌分离纯化及16S rRNA基因测序,最终鉴定出20株大肠杆菌。采用药敏试验和PCR方法对分离菌进行耐药性及耐药基因检测分析,并对其中1株多重耐药菌株进行全基因组测序。【结果】20株分离菌均具有多重耐药性,对链霉素、环丙沙星、恩诺沙星和复方新诺明的耐药率达80%以上。所检耐药基因中,aphA1、strB、TEM-1和qnrS检出率达100%。通过对代表性菌株TL-13全基因组测序发现,其基因组大小为4897185bp,GC含量为50.68%,同时携带2个质粒,大小分别为108288bp(pTL13-1)和64018bp(pTL13-2)。质粒中共携带18个可移动耐药基因。【结论】通辽地区犊牛腹泻大肠杆菌多重耐药性普遍存在,4种常见耐药基因普遍流行。     

Prevalence and antibiotic resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis> in tropical seafood

Summary The occurrence and antibiotic resistance of Escherichia coli in tropical seafood was studied. A 3-tube MPN method was used for determining the level of faecal contamination of fresh and processed seafood. Of the 188 samples tested which included finfish, shellfish, water and ice, 155 were positive for the presence of faecal coliforms following incubation at 44.5 °C. However, E. coli was isolated from only 47% of the samples positive for faecal coliforms. The antibiotic resistance of 116 strains isolated from seafood was tested using 14 different antibiotics including ampicillin, cephalothin, chloramphenicol, ciprofloxacin, gentamycin, nalidixic acid, streptomycin and vancomycin. Seven strains were resistant to more than five antibiotics of which one was resistant to eight antibiotics. The multiple drug resistant strains harboured plasmids of varying sizes. Antibiotic susceptibility studies revealed that seafood from India contains multiple antibiotic resistant strains of E. coli which may serve as a reservoir for antibiotic resistance genes in the aquatic environment. All the strains used in this study did not harbour any virulence genes commonly associated with pathogenic E. coli, when tested by polymerase chain reaction (PCR).     

Low occurrence of multi-antimicrobial and heavy metal resistance in Salmonella enterica from wild birds in the United States

Wild birds are common reservoirs of Salmonella enterica. Wild birds carrying resistant S. enterica may pose a risk to public health as they can spread the resistant bacteria across large spatial scales within a short time. Here, we whole-genome sequenced 375 S. enterica strains from wild birds collected in 41 U.S. states during 1978–2019 to examine bacterial resistance to antibiotics and heavy metals. We found that Typhimurium was the dominant S. enterica serovar, accounting for 68.3% (256/375) of the bird isolates. Furthermore, the proportions of the isolates identified as multi-antimicrobial resistant (multi-AMR: resistant to at least three antimicrobial classes) or multi-heavy metal resistant (multi-HMR: resistant to at least three heavy metals) were both 1.87% (7/375). Interestingly, all the multi-resistant S. enterica (n = 12) were isolated from water birds or raptors; none of them was isolated from songbirds. Plasmid profiling demonstrated that 75% (9/12) of the multi-resistant strains carried resistance plasmids. Our study indicates that wild birds do not serve as important reservoirs of multi-resistant S. enterica strains. Nonetheless, continuous surveillance for bacterial resistance in wild birds is necessary because the multi-resistant isolates identified in this study also showed close genetic relatedness with those from humans and domestic animals.     

Drug Resistance and Broad Geographical Distribution of Identical R Plasmids of Pasteurella piscicida Isolated from Cultured Yellowtail in Japan

An MIC test of 12 chemotherapeutic agents performed on 175 strains of Pasteurella piscicida collected from cultured yellowtail (Seriola quinqueradiata) in different areas of Japan from 1989 to 1991 revealed 152 strains (87%) with resistance to combinations of ampicillin (AP), chloramphenicol (CP), kanamycin (KM), nalidixic acid (NA), sulfamonomethoxine (SA), tetracycline (TC), and/or trimethoprim (TMP). The remaining 23 strains were sensitive to all the drugs tested: AP, cefazolin, CP, florfenicol (FF), furazolidone, KM, NA, novobiocin, SA, streptomycin, TC, and TMP. FF showed the most effective antibacterial activity against P. piscicida with MICs ranging from 0.004 to 0.6 μg/ml. One hundred and forty-nine of the 152 resistant strains carried transferable R plasmids encoding one of the Cp Km Sa Tc, Km Sa Tc, Km Sa, and Sa resistance. The most common resistance marker of transferable R plasmids identified in P. piscicida was Km Sa Tc. R plasmids encoding three different resistant markers were very similar on the basis of their digestion patterns with restriction endonucleases. There was homology among the DNAs of nine transferable R plasmids selected. Our findings suggest that multiple drug resistant strains of P. piscicida carrying transferable R plasmids with the same DNA structure are common in yellowtail farms and that the R plasmid has been retained within the P. piscicida population without change in their DNA structure according to geography and year.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a “common” plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with >80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

Effects of different alleles of the E. coli K12 polA gene on the replication of non-transferring plasmids

Summary The effects of eight different polA ^-alleles on the replication of six different non-transferring enterobacterial plasmids have been tested. Using phage P1CM transduction, different allelic polA ^- mutations were introduced into E. coli K12 strains carrying one of several antibiotic resistance plasmids. Plasmid stability in the transductants was examined by testing clones for drug resistance after growth under various conditions. From the results, the R factors may be divided into three different classes. One plasmid is only affected by PolA^- conditions which inhibit host cell growth, three plasmids (from the same compatibility group) are unstable under conditions in which the cells are severely deficient in DNA polymerase I and two other plasmids (compatible with each other and with the other four) are immediately lost from such transductants and are unstable in a number of others. Furthermore, the plasmids which are most dependent on DNA polymerase I have been shown to replicate in the presence of chloramphenicol and therefore typify a class of plasmids which includes bacteriocinogenic factors such as ColE1 and CloDF13, resistance determinant RSF1030 and the E. coli 15 minicircular plasmid.     

HIGH LEVELS OF COLICIN RESISTANCE IN ESCHERICHIA COLI

Colicins are plasmid-encoded antibiotics that are produced by and kill Escherichia coli and other related species. The frequency of colicinogeny is high, on average 30% of E. coli isolates produce colicins. Initial observations from one collection of 72 strains of E. coli (the ECOR collection) suggest that resistance to colicin killing is also ubiquitous, with over 70% of strains resistant to one or more colicins. To determine whether resistance is a common trait in E. coli, three additional strain collections were surveyed. In each of these collections levels of colicin production were high (from 15 to 50% of the strains produce colicins). Levels of colicin resistance were even higher, with most strains resistant to over 10 colicins. A survey of 137 non-E. coli isolates revealed even higher levels of resistance. We discuss a mechanism (pleiotropy) that could result in the co-occurrence of such high levels of colicin production and colicin resistance.     

Transfer and stability of drug resistance plasmids inEscherichia coli K12

Mating experiments between pairs of strains ofEscherichia coli containing either the compatible plasmids TP120 (Inc N) and R1 (Inc FII) or the incompatible plasmids TP125 (Inc B) and TP113 (Inc B) were undertaken in mixed continuous-flow cultures and in dialysis sacs suspended in pond water. Plasmid transfer was readily demonstrated between strains carrying compatible plasmids TP120 and R1 in both continuous-flow culture and pond water. In mixed cultures of strains carrying plasmids TP125 and TP113, transfer was only observed in continuous-flow culture systems. Strains ofE. coli containing aggregates of plasmids TP120 and R1 were shown to be stable over 5 months continuous cultivation under carbon limited conditions at a growth rate of 0.1 hours^–1 in the presence of drugs which select for the maintenance of both plasmids. In the strains containing plasmid aggregates, a gene dosage effect was observed with respect to the levels of resistance to drugs whose resistance was encoded by both plasmids. Chemostat experiments showed that no cointegrate plasmids were found from the strains ofE. coli initially containing both plasmid TP120 and plasmid R1.     

A Possible Role of R Plasmids in Bacterial Permeability for β-Lactam Antibiotics

Four strains of clinical isolates of Serratia marcescens (13039, 13090, 13093, 14093) harboring R plasmids were highly resistant to ampicillin (ABPC) and cephaloridine (CER). With elimination of R plasmids from these strains by acriflavine treatment, ABPC-resistance levels of these strains were markedly reduced. Reduction of CER-resistance levels was also demonstrated in strains 13039 and 13093, but not in strains 13090 and 14093. The permeability of the former strains for CER was also decreased, but not in the latter strains. At the same time, β-lactamase activity of these strains also almost completely disappeared when the R plasmids were eliminated. By broth matings with these strains, the recipient strains of S. marcescens 13031 (rif), Escherichia coli K-12 (rif), and E. coli 15046 (rif) all acquired a high permeability barrier against CER with inheritance of the R plasmids from strains 13039 and 13093, but not from strains 13090 and 14093. The transconjugant of strain 13031 that inherited R plasmid 13093 was resistant not only to CER but also to cefazolin, cephalothin, and cephalexin. Its permeability to these antibiotics was significantly lower than that of the original strain. This fact suggests the possibility that the R plasmid from strain 13093 may be involved not only in production of β-lactamases, but also in regulation of bacterial permeability for cephalosporins.     

Diarrheagenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Strains Recovered from Urban Pigeons (<Emphasis Type="Italic">Columba livia</Emphasis>) in Brazil and Their Antimicrobial Susceptibility Patterns

Urban pigeons (Columba livia) come into close contact with humans and animals, and may contribute to the spread of infectious agents. These may include human pathogens such as diarrheagenic Escherichia coli strains, which are able to survive in pigeon feces, thus creating potential for human exposure and infection. Our objectives were to determine the occurrence of diarrheagenic E. coli strains in fresh feces from urban pigeons and their drug susceptibility patterns. E. coli strains were isolated from 100 fresh feces samples and presumptive phenotypic species identification was carried out, confirmed by amplification of specific 16S ribosomal RNA encoding DNA. Multiplex PCR was performed to characterize pathogenic strains. Drug susceptibility patterns were determined by the agar dilution method. Enteroinvasive E. coli, Shiga toxin-producing E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli were detected at an overall rate of 12.1%. Among the isolated E. coli strains, 62.1% were susceptible to all tested drugs, whereas 37.9% were resistant to at least one of the antimicrobials tested. Amikacin was the less effective drug (36.8% resistance), followed by ampicillin (7.8%). No resistance was detected to gentamicin, ceftriaxone, and ceftazidime and almost all the isolates were susceptible to ampicillin-sulbactam (98.4%), levofloxacin (97.8%), and trimethoprim-sulfametoxazole (96.1%). Since these pigeons may harbor multidrug-resistant pathogens, their presence in an urban environment could be an important component of infection spread, with impact on public health.     

Studies on the Establishment of Multi-Drug-Resistant Strain BIO-4R of Streptococcus faecalis in the Intestinal Tract of Germ-Free Mice

Germ-free ICR mice were mono- or dicontaminated with a multi-drug-resistant strain BIO-4R of Streptococcus faecalis (BIO-4R) and Escherichia coli 026 : K60 (E. coli) and administered aminobenzyl penicillin (ABPC). BIO-4R was established in the intestinal tract at a level of 10⁸ viable cells per gram of stool on the fourth day following oral inoculation and the BIO-4R population was stably maintained thereafter. The drug resistance of BIO-4R remained unchanged in the intestinal tract of gnotobiotes throughout the experiment. Highly resistant cells of E. coli were isolated from the feces of some dicontaminated mice after ABPC administration. However, it seems that the high resistance of these E. coli is not due to the transfer of resistance of BIO-4R to E. coli. All animals given a large amount of BIO-4R (10⁸ cells) per os survived throughout the study period of two weeks without symptoms.     

VIM‐1 carbapenemase‐producing Escherichia coli in gulls from southern France

Acquired carbapenemases currently pose one of the most worrying public health threats related to antimicrobial resistance. A NDM‐1‐producing Salmonella Corvallis was reported in 2013 in a wild raptor. Further research was needed to understand the role of wild birds in the transmission of bacteria resistant to carbapenems. Our aim was to investigate the presence of carbapenem‐resistant Escherichia coli in gulls from southern France. In 2012, we collected 158 cloacal swabs samples from two gull species: yellow‐legged gulls (Larus michahellis) that live in close contact with humans and slender‐billed gulls (Chroicocephalus genei) that feed at sea. We molecularly compared the carbapenem‐resistant bacteria we isolated through culture on selective media with the carbapenem‐susceptible strains sampled from both gull species and from stool samples of humans hospitalized in the study area. The genes coding for carbapenemases were tested by multiplex PCR. We isolated 22 carbapenem‐resistant E. coli strains from yellow‐legged gulls while none were isolated from slender‐billed gulls. All carbapenem‐resistant isolates were positive for bla_VIM‐1 gene. VIM‐1‐producing E. coli were closely related to carbapenem‐susceptible strains isolated from the two gull species but also to human strains. Our results are alarming enough to make it urgently necessary to determine the contamination source of the bacteria we identified. More generally, our work highlights the need to develop more bridges between studies focusing on wildlife and humans in order to improve our knowledge of resistant bacteria transmission routes.     

Use of Peracetic Acid as a Disinfectant in a Water-Treatment Plant: Effect on the Plasmid Contents of Escherichia coli Strains

Total thermotolerant coliforms (TTC) and Escherichia coli strains were isolated from sewage from a treatment plant before and after peracetic acid (PAA) disinfection. The plasmid profiles of 120 E. coli strains were analyzed. Although PAA disinfection effectively reduced the number of TTC and E. coli strains, the percentage of E. coli strains containing plasmids was not statistically different among water samples. The sizes of the plasmids found ranged from <3 kb to >56 kb, but plasmids of between 3 and 5 kb were encountered most frequently.     

Drug resistance in Indian isolates of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Five different strains of enteropathogenic Escherichia coli (EPEC) and one non-pathogenic strain of E. coli were studied for the determination of resistance to various antibiotics by disc inhibition test. Our results demonstrated multiple drug resistance among the selected E. coli isolates with all of them including the non-pathogenic control strain showing high resistance towards ampicillin. Our results from conjugation test clearly showed the presence of most of the antibiotic markers on the transferred plasmids. Simultaneously, the plasmid profile of conjugants also indicated the presence of more than one plasmid along with the expected megaplasmid of ~90 Kb present in them. Thus our results amply demonstrate that these drug markers are associated with these mobile plasmids.     

Transformation of Pseudomonas aeruginosa and Escherichia coli with resistance plasmid DNA: formation of smaller conjugative plasmids from RPI.

Summary Sheared DNA from RPI, an R plasmid from a strain of Pseudomonas aeruginosa, was used to transform other strains of P. aeruginosa and Escherichia coli. From transformed cells other plasmids like RPI were isolated. These deletion plasmids were conjugally transferrable and confer resistance mainly against carbenicillin and tetracycline.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

血培养病原菌构成及耐药性分析

了解2008年至2012年哈尔滨医科大学附属第一医院血培养常见病原菌构成及耐药性。对血培养分离出的病原菌进行鉴定,用MIC法、K-B法测定药物敏感性,用WHONET 5.6统计软件进行细菌菌谱及耐药性分析。共分离出病原菌4 245株;其中凝固酶阴性葡萄球菌最多,947株占22.3%;其次为大肠埃希菌822株,肺炎克雷伯菌520株,鲍曼不动杆菌195株,金黄色葡萄球菌142株;耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)和耐甲氧西林金黄色葡萄球菌(MRSA)检出率分别为81.1%、38.8%,未发现耐万古霉素、利奈唑胺及替考拉宁的凝固酶阴性葡萄球菌和金黄色葡萄球菌;大肠埃希菌及肺炎克雷伯菌产ESBLs检出率分别为55.2%、53.8%,对头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、头孢替坦、头孢西丁、阿米卡星的耐药率较低,对碳青霉烯类抗菌药物存在耐药现象;鲍曼不动杆菌对抗菌药物的耐药率普遍较高。及时、准确地对血培养分离出的病原菌进行监测,以便指导临床合理用药,控制耐药株的产生。     

Escherichia coli isolated from seafood: toxicity and plasmid profiles

Thirty-two Escherichia coli strains were isolated from red snapper (Lutjanus purpureus) and from seabob shrimp (Xiphopenaeus kroyeri). The strains were numbered S1–S16, and F1–F16, which corresponds to the isolation origin from shrimp (S) and fish (F). The isolates were biologically and antigenically characterized by agglutination tests with enteropathogenic E. coli (EPEC)-, enteroinvasive E. coli (EIEC)- and enterohemorrhagic E. coli (EHEC) O157-specific antisera. The ETEC enterotoxins were characterized by GMI-ELISA for enterotoxin LT-1 (thermolabile) and by inoculation of supernatants prepared from newly born mice for enterotoxin Sta. A total of 14 strains produced exotoxins, of which seven were thermolabile (LT) and seven were thermostable (ST). Antimicrobial susceptibility profiles were determined by disc diffusion in agar using ampicillin, cephalothin, cefoxitin, ceftriaxone, imipenem, nalidixic acid, ciprofloxacin, chloramphenicol, gentamicin, nitrofurantoin, sulfamethoxazole–trimethoprim, and tetracycline. Four isolates showed lower susceptibility to some antibiotics, two strains were resistant to ampicillin, tetracycline, and sulfamethoxazole-trimethoprim, and two were resistant to tetracycline and nitrofurantoin. Plasmids were extracted in the four resistant isolates; two of them contained plasmids whose molecular weight varied from low to high. The characterization of LT- and ST-toxin-producing E. coli strains displaying multiresistance and containing plasmids suggests the need for tightening current control measures for the use of antimicrobials. Electronic Publication     

一株污水源多重耐药大肠杆菌的耐药性检测及全基因组测序分析

【背景】水体环境分布广、流动性强,是耐药菌和耐药基因传播的主要媒介。【目的】了解北方污水厂大肠杆菌携带的耐药基因及可移动遗传元件情况。【方法】从北方污水厂筛选出一株多重耐药大肠杆菌,通过药敏试验进行耐药性检验,采用96孔板法测定菌株的最小抑菌浓度,利用酶标仪探究亚抑菌浓度抗生素对菌株生长的影响,并对菌株进行全基因组测序,对其携带的耐药基因及可移动遗传元件进行预测。【结果】大肠杆菌WEC对四环素、环丙沙星、诺氟沙星和红霉素具有耐药性,亚抑菌浓度的四环素、环丙沙星和诺氟沙星能够延缓或抑制菌株的生长。WEC菌株的基因组中包含一条大小为4 782 114 bp的环状染色体和2个大小分别为60 306 bp (pWEC-1)和92 065 bp (pWEC-2)的环状质粒。菌株共携带129个耐药基因,其中128个位于染色体上,在染色体上预测到原噬菌体、基因岛及插入序列的存在,部分可移动遗传元件携带有耐药基因。质粒pWEC-1中无耐药基因,pWEC-2含有1个耐药基因,在质粒基因组中预测到原噬菌体和插入序列。【结论】污水源大肠杆菌WEC是一株多重耐药菌株,其基因组中携带耐药基因和多种可移动遗传元件...