Characterization and classification of Actinobacillus (Haemophilus) pleuropneumoniae plasmids.

Actinobacillus (Haemophilus) pleuropneumoniae plasmids were characterized and classified. They were isolated from A pleuropneumoniae strains different in serotype, year isolated, or location from which isolated. Six of 8 plasmids encoded streptomycin (Sm) and sulfonamide (Su) resistance (SmSu). One of the other plasmids, pVM105, encoded ampicillin (Ap) resistance and another, pHM0, encoded no drug resistance. All SmSu plasmids were transferred to Escherichia coli strains by transformation. Among them, pABO and pMS260 were 8.1 kb and incompatible with each other; they were stable in E coli. The other SmSu plasmids, pHM1, pVM104, pVM106, and pKD25, were 4.3 kb and did not replicate stably in E coli. The former SmSu plasmids were mobilized in E coli strains by a plasmid RP4, which belonged to incompatibility (Inc) group P, but the latter plasmids were not. Further, each 8.1-kb SmSu plasmid and each 4.3-kb plasmid had the same respective restriction pattern. These results indicated that there were at least 2 types of SmSu plasmids in A pleuropneumoniae. The 2 types were classified in 2 groups: H1(pMS260 and pABO) and H2(pHM1, pVM104, pVM106, and pKD25). The H1 and H2 plasmids belonged to different Inc groups, and H2 plasmids belonged to a different Inc group from that of pHMO and pVM105.     

Relation of plasmids to virulence and other properties of salmonellae from avian sources

A collection of 185 isolates of 34 serovars of Salmonella from avian sources was examined for plasmids, drug resistance, biochemical properties, serum resistance, and virulence. No serovars other than S. enteritidis, S. typhimurium, and S. heidelberg showed evidence of serovar-associated plasmids. All S. enteritidis isolates carried a single plasmid of 36 Mdal and were resistant to guinea pig serum; one strain that was tested was virulent. Of 27 isolates of S. typhimurium, 11 possessed a 60-Mdal plasmid and 17 harbored a 2.3-Mdal plasmid. Among isolates of S. heidelberg, 21 of 24 carried a 2.2-Mdal plasmid. The only biochemical property that varied was fermentation of inositol, which tended to be related to serovar. Of 172 isolates, 54 were resistant to at least one drug. Multiple drug resistance was usually associated with R plasmids, and transmissible plasmids that encoded resistance to chloramphenicol and gentamicin were demonstrated. Of 117 isolates tested, 43 were resistant to guinea pig serum. Resistance appeared to be a characteristic of isolates rather than serovar and could not be related to plasmids. Twenty-five isolates highly resistant to guinea pig serum were all susceptible to the bactericidal action of chicken serum. In tests for virulence using intraperitoneally (i.p.) and orally inoculated Balb/c mice and day-old chicks, only i.p.-inoculated chicks proved useful in demonstrating large differences among isolates: LD50's ranged from 10(0) to 10(8).     

Antimicrobial susceptibility and plasmid analysis of Actinobacillus pleuropneumoniae isolated in Taiwan.

Sixty Actinobacillus pleuropneumoniae (App) strains from pigs in Taiwan were examined. Serotyping revealed that these belonged to serovars 1 (n=53), 2 (n=3), and 5 (n=4). Agar disk diffusion susceptibility testing of the isolates showed 55 (92%) were resistant to three or more antimicrobial agents. Six resistance patterns were observed. Ampicillin-chloramphenicol-flumequine-nalidixic acid-streptomycin-sulfonamide/trimethoprim-tetracycline was the most common multi-resistance pattern. Minimal inhibitory concentration of 14 antimicrobial agents was determined. The isolates were highly susceptible to ceftiofur and trimethoprim in vitro. Isolates were resistant to streptomycin, ampicillin, and nalidixic acid. All isolates were examined for the presence of plasmids using the alkaline lysis method. Forty three (72%) isolates had four plasmid bands with an approximate sizes of 3.5, 4.3, 5.8 and 6.0 kb; 12 (20%) had three bands at 3.5, 4.3 and 5.2 kb, and 5 (8%) had no plasmid bands. Antimicrobial resistance plasmids were detected in resistant strains of App. Three antimicrobial resistance plasmids were transformed into E. coli DH5 alpha. pTMY1 (4.3 kb) encoded a streptomycin kinase and a dihydropteroate synthase; pTMY2 (6.0 kb) encoded ROB-1 beta-lactamase and aminoglycoside 3'-phosphotransferase; pTMY3 (5.2 kb) encoded only ROB-1 beta-lactamase. The 4.3 kb plasmid was sequenced and consisted of 4242 bp with 42.9% GC content. The 4.3 kb plasmid DNA sequence was 98% homologous to a plasmid previously isolated from Pasteurella haemolytica.     

Plasmid profiles of six species of Campylobacter from human beings, swine, and sheep

Twenty-four isolates representing 6 species of Campylobacter were screened for plasmids. A large plasmid with an approximate molecular weight of 38 Mdal was detected in 5 C jejuni isolates originally recovered from diarrheic human beings, in one isolate of C coli recovered from diarrheic pigs, and in 1 isolate of C sputorum ssp mucosalis and 2 isolates of C hyointestinalis recovered from pigs with proliferative enteritis. One isolate of C coli and 1 isolate of C hyointestinalis contained an additional smaller plasmid with an approximate molecular weight of 1.6 Mdal; this plasmid was partially mapped by restriction endonuclease digestion. Fifteen Campylobacter isolates contained no detectable plasmids: 2 C coli, 2 C sputorum ssp mucosalis, 2 C fecalis, 1 C fetus ssp fetus, and 8 C hyointestinalis isolates. In summary, 37.5% of the Campylobacter isolates contained a 38-Mdal plasmid, with 8% having both 38 Mdal and 1.6-Mdal plasmids; 62.5% contained no detectable plasmids.     

Isolation, characterization, and molecular cloning of cryptic plasmids isolated from Edwardsiella ictaluri

Fifty-five isolates of Edwardsiella ictaluri were examined for the presence of plasmid DNA by a rapid alkaline extraction procedure. All 49 isolates from channel catfish and a single isolate from Bengal danio carried 2 plasmids with molecular masses of approximately 3.2 and 3.7 megadaltons (Mdal). Five E ictaluri isolates from other fish contained 1 to 3 plasmids, which had molecular masses ranging from 2.5 to 45 Mdal. The 2 plasmids (3.2 and 3.7 Mdal) from the type strain of E ictaluri (ATCC 33202) were ligated into pUC19 cloning vectors, and restriction endonuclease maps of each insert were prepared.     

Linkage of genes for heat-stable enterotoxin, drug resistance, K99 antigen, and colicin in bovine and porcine strains of enterotoxigenic Escherichia coli

Linkages among genes for production of Escherichia coli heat-stable enterotoxin (ST), drug resistance, K99 antigen, and colicin were investigated in 2 bovine and 3 porcine strains of enterotoxigenic E coli. In conjugation experiments, all 5 isolates transferred enterotoxigenicity and the ability to produce K99; 4 of the 5 isolates also transferred antibiotic resistance markers, and 3 colicinogenic strains transmitted the ability to produce colicin. In 2 of the 3 colicin-producing strains, the genes for colicin were located along with those for K99 and ST on a single plasmid. One of these 2 strains transferred the genes for tetracycline resistance and production of both mouse-active ST (STa) and mouse-inactive ST, whereas the other transmitted the gene(s) for STa only. Transformation studies with the 3rd strain revealed that the K99 determinant resided on a 22-megadalton (Mdal)R plasmid, and that STa and colicin production were on a 65-Mdal plasmid. Analysis of the plasmids from the transformation experiments revealed that the larger plasmid was conjugative and the smaller plasmid was nonconjugative; stable cointegrate formation occurred between these 2 plasmids. Genetic information coding for the production of STa and K99 were also present on a single plasmid of approximately 80 Mdal in both noncolicinogenic strains.     

Antimicrobial susceptibility of Salmonella isolates from the broiler chicken industry in Ontario.

Antibacterial drug resistance among 219 salmonella isolates recovered during 1974 from poultry and poultry environments at the various production stages of broiler chickens in three integrated Ontario companies are recorded. All isolates were susceptible to ampicillin, trimethroprim-sulfamethoxazole complex, furazolidone, cephaloridine and amoxicillin. A relative increase in resistance to tetracycline and streptomycin with an accompanying decrease in resistance to triple sulfa compound was recorded when compared to a previous investigation of avian salmonella isolates in Ontario. The percentage and patterns of antimicrobial resistance were comparable at the various stages of production. Resistance to tetracycline and streptomycin was the most common pattern found among both Salmonella typhimurium and other serotypes. A notably high prevalence of resistance was found among Salmonella enteritidis isolates including some isolates with R factors for chloramphenicol resistance. This latter finding is of particular concern because of the high prevalence of this serotype in poultry and in human salmonellosis.     

Analysis of southern Ontario Actinobacillus (Haemophilus) pleuropneumoniae isolates by restriction endonuclease fingerprinting.

Isolates of Actinobacillus (Haemophilus) pleuropheumoniae were studied by restriction endonuclease fingerprinting (REF) analysis using the enzymes BamHI and HindIII. Restriction fragments were resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Except for serotypes 1 and 9, reference strains of A. pleuropneumoniae serotypes 1 to 10 had clearly distinguishable REF profiles. Analysis of REF profiles of southern Ontario field isolates revealed limited heterogeneity amongst isolates of serotype 1 or serotype 5. The REF profiles of the serotype 7 isolates studied showed greater variation. Heterogeneity could not be correlated with the presence of plasmids nor with antibiotic resistance. Limited heterogeneity could also be detected amongst REF profiles of A. pleuropneumoniae isolates recovered from a closed herd suggesting that there is a small amount of genetic variation within clonal populations.     

Transformation of Actinobacillus pleuropneumoniae and analysis of R factors by electroporation

An efficient method for DNA transfer is essential for the genetic manipulation of any organism. Such a capacity will be required for the genetic analysis of Actinobacillus pleuropneumoniae as a swine pathogen, as well as for its manipulation for vaccination purposes. For this reason, the use of electroporation as a means of plasmid DNA introduction into this species was examined. The multiple antibiotic-resistant strain 80-8141 of Actinobacillus pleuropneumoniae harbors 3 plasmids: pYG10, pYG15, and pYG12 of 5.0, 2.7, and 2.5 kb, respectively. Electroporation of A pleuropneumoniae strain 4074 with a plasmid extract of strain 80-8141 showed that pYG10 encodes chloramphenicol resistance and that pYG12 encodes ampicillin resistance. Electrical pulse conditions for efficient electroporation of strain 4074 were examined by use of pYG10 DNA isolated from a 4074 transformant. Efficiency, expressed as transformants per microgram of plasmid DNA, increased directly with pulse amplitude. However, high efficiencies were only observed in a narrow window of pulse duration (tau = 12 to 22 ms at 6.25 kV/cm). Longer pulse durations resulted in cell death. Electroporation efficiencies increased with cell density. Yield of transformants increased directly with DNA concentration. Results indicate that electroporation can be used to efficiently transform A pleuropneumoniae and that pYG10 and pYG12 are suitable plasmid vectors for use in the genetic manipulation of this organism.     

Antimicrobial resistance of Actinobacillus pleuropneumoniae isolated from swine

The aim of this retrospective study was to evaluate the antimicrobial resistance rates and the trend in resistance of Actinobacillus pleuropneumoniae isolated from pigs in Italy from 1994 to 2009. A total of 992 A. pleuropneumoniae isolates were tested for their susceptibility to a panel of antimicrobial agents in a disk diffusion method. Resistance to 7 drugs (amoxicillin, amoxicillin/clavulanic acid, ampicillin, cefquinome, cotrimoxazole, penicillin G and tilmicosin) showed a significant increasing trend over the time, while for 2 drugs (gentamycin and marbofloxacin) a significant decrease was observed. Resistance to the remaining 14 antimicrobial agents tested did not change significantly over the study period. Most of the isolates retained high susceptibility to antimicrobials usually effective against A. pleuropneumoniae such as amphenicols, fluoroquinolones and ceftiofur. However, high rates of resistance were observed for potentiated sulfa drugs, tetracyclines and penicillins which are currently recommended antimicrobials for pig pleuropneumonia therapy. Our results suggest the importance of continued monitoring of A. pleuropneumoniae clinical isolates in order to choose the most appropriate treatment of infections and to control the increase of resistance to currently used antimicrobials.     

Hybridization studies with a DNA probe derived from the virulence region of the 60 Mdal plasmid of Salmonella typhimurium.

Plasmid DNA of 68 strains of Salmonella that belonged to 18 serovars and exhibited 48 different plasmid profiles was examined for hybridization with a 32P-labelled DNA probe which consisted of a 3750 base pairs (bp) HindIII-HindIII fragment derived from the virulence region of the 60 megadalton (Mdal) plasmid of Salmonella typhimurium. The 32 Mdal plasmid of S. cholerae-suis, the 50 Mdal plasmid of S. dublin, the 36 Mdal plasmid of S. enteritidis, the 60 Mdal plasmid of S. gallinarum, the 60 Mdal plasmid of S. pullorum, and the 60 Mdal plasmid of S. typhimurium, plasmids that have been associated with virulence, all hybridized with the probe. Digestion of plasmid DNA of these strains with PvuII and hybridization with the probe revealed that the plasmids of strains of all six serovars contained fragments of approximately 2520 and 1520 bp that hybridized with the probe. Similarly, hybridization with BglI digests of DNA of the virulence-associated plasmids of strains of these six serovars showed that all six plasmids contained a fragment of approximately 3690 bp that hybridized with the probe. No other plasmids of these strains nor any plasmids of 12 other Salmonella serovars hybridized with the probe. Chromosomal DNA did not hybridize with the probe. The 60 Mdal plasmids of S. gallinarum and S. pullorum showed similar digestion patterns with restriction endonucleases BglI, BglII and PvuII.     

Possession of identical nonconjugative plasmids by different isolates of Pasteurella multocida does not imply clonality

Experiments were performed to test the hypothesis that possession of the same nonconjugative R plasmid by different isolates of Pasteurella multocida implied that they were of the same clone. Seven isolates of P. multocida were studied, two possessed an identical nonconjugative R plasmid (pVM109), four possessed another (pVM110), and one isolate possessed a nonconjugative R plasmid related to pVM110. Phenotypic and genotypic characteristics of the isolates were determined and compared. Isolates possessing the same nonconjugative R plasmid were shown to be different, and isolates possessing a different nonconjugative R plasmid were shown to be the same. We conclude that possession of an identical nonconjugative R plasmid by two isolates of P. multocida does not imply clonality.     

Detection of florfenicol resistance genes in Riemerella anatipestifer isolated from ducks and geese

The cat gene, coding for chloramphenicol acetyltransferase has been reported for conferring the chloramphenicol resistance for Riemerella anatipestifer. Chloramphenicol acetyltransferases, however, are unable to inactivate florfenicol. In this study, 66 R. anatipestifer isolates were investigated for their susceptibility to chloramphenicol and florfenicol and the presence of floR gene. Results showed nine florfenicol intermediate or resistant R. anatipestifer isolates were all floR positive. The expression of floR gene in E. coli and inhibition studies with PAβN indicated that the floR gene was as an efflux pump conferring resistance to both chloramphenicol and florfenicol. Southern hybridization revealed the floR was located in the plasmid DNA of five isolates and in the chromosomal DNA of four isolates. Furthermore, two novel floR-carrying plasmids designated pRA0726 and pRA0846 were sequenced completely. pRA0726 was 11,704 bp in size with 10 putative open reading frames which included the floR, catB and bla(OXA-209) resistance genes. The most differences between sequences of pRA0846 and pRA0726 were the absence of a bla(OXA-209) gene and the deletion of 321 nucleotides of orf1 in pRA0846. Plasmid curing tests demonstrated that pRA0726 carried functional coding proteins for resistance to phenicol and β-lactam antimicrobials. To the best of our knowledge, this is the first report of presence of the floR and bla(OXA-209) resistance genes in R. anatipestifer.     

替米考星、红霉素体外诱导猪胸膜肺炎放线杆菌耐药性的初步研究

采用微量稀释法测定了替米考星和红霉素对5株临床分离猪胸膜肺炎放线杆菌(App)的最小抑菌浓度,并用药物浓度递增法体外诱导App对两种药物的耐药性。结果表明替米考星和红霉素对App都具有很高的体外抑菌活性;经15代诱导,App对替米考星的最高耐受浓度没发生明显变化,而对红霉素的最高耐受浓度有了较大程度的提高,表明App对替米考星不易产生耐药,而对红霉素可缓慢产生耐药。试验结果提示替米考星是治疗App感染的理想药物。     

Molecular epidemiology and antimicrobial resistance of Salmonella typhimurium DTI04 on Ontario swine farms.

This study was conducted to examine antimicrobial resistances, plasmid profiles, and pulsed-field gel electrophoresis patterns of 80 Salmonella Typhimurium (including var. Copenhagen) DT104 strains (including DT104a and DT104b) recovered from pig and environmental fecal samples on 17 swine farms in Ontario. No resistance was observed to amoxicillin/clavulanic acid, apramycin, carbadox, cephalothin, ceftriaxone, ceftiofur, cefoxitin, ciprofloxacin, nalidixic acid, trimethoprim, and tobramycin. However, the isolates exhibited resistance against 4 to 10 antimicrobials with the most frequent resistance being to sulfonamides (Su), ampicillin (A), streptomycin (S), spectinomycin (Sp), chloramphenicol (C), tetracycline (T), and florfenicol (F). Thirteen distinct resistance patterns were determined but 88% of isolates shared the typical resistance pattern "ACSpSSuT." Twelve different plasmid profiles were observed; the 62 MDa virulence-associated plasmid was detected in 95% of the isolates. The 2.1 MDa plasmid was the second most frequent one, which was harbored by 65% isolates. The isolates were classified into 23 distinct genotypes by PFGE-SpeI + BlnI when difference in at least one fragment was defined as a distinct genotype. In total, 39 distinct "types" were observed when defining a "type" based on the combination of antimicrobial resistance, plasmid pattern, and PFGE-SpeI + BlnI for each isolate. The highest diversity was 0.96 (95% CI: 0.92, 0.96) for the "type" described above followed by 0.92 (95% CI: 0.88, 0.93) for PFGE-SpeI + BlnI. The diversity of DT104 isolates indicates there might be multiple sources for this microorganism on swine farms. This knowledge might be used to track these sources, as well as to study the extent of human salmonellosis attributed to pork compared to food products derived from other food-producing animals.     

Distribution of drug resistance among enterococci and <Emphasis Type="Italic">Salmonella</Emphasis> from poultry and cattle in Ethiopia

Enterococci and Salmonella were isolated from feces of chicken in intensive poultry farms and cattle which are maintained following traditional practices. Their resistance to different antibiotics was also determined. A total of 298 enterococcal isolates consisting of Enterococcus faecium (49.6%), Enterococcus durans (26.9%), Enterococcus hirea (11.9%), and Enterococcus faecalis (11.5%) were obtained. Among the enterococci, resistance to erythromycin (Ery), clindamicin (Cli), amoxicillin (Amo), ampicillin (Amp), and cephalothin (Cep) was high. Resistance to vancomycin (Van) was detected in all enterococcal species. Over 80% of the isolates showed multiple drug resistance. The most dominant patterns in poultry were Amo/Amp/Cep/Pen and Amo/Amp/Cep/Cli/Pen/Van. Among isolates from cattle, Amo/Amp/Cep/Cli/Ery/Pen/Van and Amo/Amp/Cli/Ery/Pen/Van constituted the most dominant multiple resistance patterns. A total of 51 Salmonella isolates were obtained from poultry (43/280) and cattle (8/450). About 70% of the isolates had varying resistance to the tested antibiotics. Multiple drug resistance was observed in over 30% of the Salmonella isolates. The most frequent resistance pattern was Amo/Amp/Cip/Gen/Str in cattle and Amo/Amp/Cep/Cip/Gen/Kan/Str in poultry. Enteroccoccal and Salmonella isolates showed multiple resistance to those antibiotics used in human and veterinary medicine. The high frequency of isolation of resistant enterococci is indicative of the wide dissemination of antibiotic resistant bacteria in the farm environment.     

Penetration of drug resistance in Escherichia coli isolated from laboratory animals

A total of 713 strains of fecal Escherichia coli (E. coli) isolated from laboratory animals in the colonies of 4 research laboratories and 4 commercial breeders in Japan in 1994 were examined in regard to resistance to 8 antibacterial agents. The incidence of resistance to sulfadimethoxine (Su), streptomycin (Sm), ampicillin, cephaloridine, tetracycline, chloramphenicol, kanamycin, and gentamicin was 99.9%, 32.5%, 6.7%, 0.7%, 7.0%, 2.6%, 6.6% and 0.7%, respectively. These results indicated that Su and Sm resistance are penetrating into normal E. coli strains isolated from laboratory animals.     

Antimicrobial drug susceptibility of Staphylococcus intermedius clinical isolates from canine pyoderma

A total of 50 Staphylococcus intermedius strains isolated in France from canine pyodermas in 2002 were investigated for their susceptibility to various antimicrobial drugs. Antimicrobial susceptibility was assessed using a 2-fold serial dilution method in Mueller-Hinton agar, and the minimal inhibitory concentrations (MICs) were determined. About 62% of the 50 strains tested were producers of beta-lactamase and categorized as penicillin-resistant. About 26% demonstrated resistance to sulphonamides, 46% to oxytetracycline, 30% to chloramphenicol, 28% to streptomycin, kanamycin, neomycin or erythromycin, 22% to clindamycin, 6% to doxycycline, 2% to gentamicin, enrofloxacin, marbofloxacin or pradofloxacin. Acquired resistance was not observed to a clavulanic acid-amoxicillin combination, oxacillin, cephalosporins (cephalexin, ceftiofur and cefquinome), trimethoprim, a sulphamethoxazole-trimethoprim combination and florfenicol. About 42% were simultaneously resistant to three or more antimicrobial classes (multiresistance). All isolates with acquired resistance to erythromycin were also resistant to streptomycin and neomycin/kanamycin. About 22% of isolates exhibited cross-resistance between erythromycin and clindamycin and all clindamycin-resistant isolates also exhibited resistance to erythromycin. Resistance to penicillin, oxytetracycline and chloramphenicol was also positively associated with resistance to erythromycin and streptomycin.     

Antimicrobial resistance patterns and plasmid profiles of Streptococcus suis isolates.

Streptococcus suis isolates recovered from diseased animals in Quebec and western Canada and from human cases in Europe were tested for their susceptibility to different antimicrobial agents and screened for their plasmid content. Most isolates from Quebec were clindamycin, erythromycin, and tetracycline resistant; animal isolates from western Canada were notably less resistant to clindamycin and erythromycin, whereas human isolates were considerably more susceptible to most antimicrobials tested. More than 60% of isolates had plasmids that ranged from 1.5 to 35 kilobases (kb). Of the 7 plasmid profiles found, 2 were particularly frequent in isolates from Quebec and western Canada, suggesting the presence of epidemic strains in the swine population. A particular plasmid band of about 5 kb was present in most Canadian isolates. When this band was used as a probe in colony and Southern blot hybridization, most isolates harboring the 5-kb plasmid hybridized, even though their plasmid profiles were different. Human isolates from Europe differed in their plasmid content from Canadian isolates of animal origin. Although a high degree of antimicrobial resistance was associated with the presence of plasmids in most isolates, it was not possible to establish a causative relationship.     

Isolation of Streptococcus suis from diseased pigs in Canada

A total of 260 isolates of streptococci collected over a 9-year period from diseased pigs submitted for necropsy were studied. Seventy-seven percent of isolates were identified as S. suis and 32% of S. suis isolates were retrieved in pure culture. S. suis was found more frequently in lungs and was often isolated in conjunction with Actinobacillus pleuropneumoniae, Pasteurella multocida, Escherichia coli and other microorganisms. A total of 151 (76%) of S. suis isolates could be serotyped within the 9 recognized serotypes. Serotype 2 was the most prevalent with 33%, followed by serotypes 3, 5 and 7. All isolates were sensitive to ampicillin, penicillin, cephradine, chloramphenicol and trimethoprim-sulfamethoxazole. Resistance to streptomycin, neomycin and tetracycline appeared to be very high.