Characterization of Escherichia coli strains associated with canine pyometra

The purpose of this study was to characterize E. coli isolates from canine pyometra which were isolated in pure culture. The E. coli strains were obtained in 128 cases, from 143 animals which were submitted to ovariohisterectomy. Biochemical analysis of all strains examined was possible on separation of 10 primary biotypes. The majority of the strains (87.5%) belonged to biotype 9, 1, 13 and 15. Dulcitol was fermented by 93% of all isolates. Haemolysin and colicin production was found in 53.9% and 26.6% of the strains, respectively. Approximately 37% of strains expressed resistance to two or more antibacterial agents. No plasmid was detected in 4.6% of the isolates. Plasmid profiles of all plasmid-containing isolates revealed plasmid bands corresponding to molecular weight ranging from 1 kb to 160 kb. Many of the strains examined had a single plasmid of 110 kb (46.1%), or two plasmids 110:65 kb (18.8%). Both plasmids appearing alone or in combination with other plasmids were detected in 90.1% of isolates with plasmid content. It was established that among haemolytic, colicinogenic and motile strains, the presence of both plasmids was 91.3, 94.1 and 91.4%, respectively. The appearance of both plasmids among dulcitol-positive and raffinose-negative strains was estimated at 88.2 and 88.3%, respectively. In a group of colicinogenic strains the presence of a single plasmid of molecular weight 110 kb was estimated at 5.9%. When both plasmids were present (profile 110:65), the percentage of these strains was 70.6%.     

55 kb Plasmid and Virulence-Associated Genes are Positively Correlated with Salmonella enteritidis Pathogenicity in Mice and Chickens

Twenty-four strains of Salmonella enteritidis, isolated from several outbreaks of salmonellosis from different poultry farms in India, were checked for the plasmid profile and detection of virulence gene(s) by PCR. Most of the strains contained only a single plasmid of 55 kb. Additional plasmids of 23.2 kb and 8.7 kb were seen in one of the strains, and another strain carried only two plasmids of 23.2 kb and 8.7 kb. Four strains did not carry any plasmid. PCR amplification showed the presence of virulence-associated genes in all the isolates harbouring the 55 kb plasmid. Intraperitoneal inoculation of mice, with most of the strains carrying the 55 kb plasmid, caused 100% mortality. Most strains lacking the 55 kb plasmid were avirulent. In chickens, oral inoculation of the S. enteritidis strains carrying the 55 kb plasmid produced 40–100% mortality, with characteristic signs of salmonellosis. Oral inoculation of strains lacking the 55 kb plasmid did not cause any mortality. Hence, it appears that the large plasmid of S. enteritidis probably contributes towards virulence in mice and chickens.     

Genomic lineage of Salmonella enterica serovar Dublin

Thirty five strains of the host adapted Salmonella serotype Dublin (S. Dublin) have been characterized by IS200 patterns, ribotyping, pulsed-field gel electrophoresis (PFGE), restriction fragment polymorphism after hybridization with five randomly cloned DNA-framents of S. enteridis (RFLP), and plasmid profiling in order to divide the strains into ‘genomic lines’. For comparison, 20 other strains of 9 different group-D serotypes were included. The IS200 patterns were identical in all strains of S. Dublin examined. These patterns were different from those observed in other group-D Salmonella with the exception of one strain S. Enteritidis phage type 11 and a strain of S. Rostock. The insertion element IS200 was not detected in strains of S. Dar-es-Salam, S. (II) 9,12:z -, and S. Panama. RFLP, based on probing with five random cloned chromosomal fragments gave the same pattern in all strains except for one isolate from the UK. This strain was also found to have an unique PFGE pattern and ribotype. Among the remaining strains, three different PFGE patterns and 7 different ribotypes were observed. Based on all four typing methods, 8 different ‘genomic lines’ of S. Dublin were identified. The same grouping could be obtained from the use of ribotyping alone, but PFGE and RFLP were found to provide valuable information on possible relationships between ribotypes. Seven different plasmid profiles and a group of strains without plasmids were observed. In several cases, the same plasmid profile was shown to be present in more than one ‘genomic line’.     

Occurrence and diversity of plasmids in ovine isolates of Pasteurella haemolytica.

160 ovine isolates of Pasteurella haemolytica, representing each of the 16 serotypes and also untypable strains, were examined for plasmid content. Plasmid DNA was identified in, and prepared from, strains of serotypes A2, T3, A14 and A16 and also from an untypable strain. The relationship between the plasmids present in the different strains was examined both by restriction fragment profile analysis and by DNA/DNA hybridisation. Both methods gave broadly similar results and showed that each serotype tended to contain either a single plasmid species, or a limited range of species, and that structural similarities could traverse serotype boundaries. None of the plasmid-bearing strains showed any significant level of resistance to a range of antibiotics.     

Characterization of Salmonella isolates from beef cattle, broiler chickens and human sources on Prince Edward Island

Non-typhoid Salmonella serovars remain a potential threat to human health, and beef cattle and broiler chickens are possible sources of these organisms on Prince Edward Island (PEI). In this study, the ceca of beef cattle belonging to fasted and non-fasted groups, and broiler chickens were examined for Salmonella at the time of slaughter. The characteristics of the isolates, including antimicrobial resistance patterns and virulence genes, were studied along with the isolates obtained from cases of human salmonellosis on PEI during the study period (1996–97). The prevalence of Salmonella in beef cattle was 4.6% (11/240). The rate was significantly higher in fasted cattle (7.46%), than in non-fasted cattle (0.94%). The prevalence rate in chickens was 32.5% (39/120). In beef cattle, Salmonella typhimurium phage type (PT) or definitive type (DT) 104 which was resistant to ampicillin, chloramphenicol, streptomycin, sulfisoxazole and tetracycline, was the most predominant type (64%). In chickens, S. heidelberg, with resistance to gentamicin, streptomycin and sulfisoxazole, predominated. Of 26 isolates from humans, the most common serovar was S. typhimurium, including a multidrug-resistant strain of DT104. Examination by PCR revealed presence of the virulence gene invA in all serovars, and the spvC gene in all S. typhimurium isolates, of both beef cattle and human origin. Among the other serovars the latter gene was found in 7 human isolates, but in none of the chicken or beef isolates. All but 3 of the spvC-positive isolates possessed a 90 kilobasepair (kbp) plasmid suggesting that the 3 isolates had the spvC gene on their chromosome. These findings were confirmed by plasmid DNA isolation using 3 different protocols and by sequence analysis of the spvC-PCR product.     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul, Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Plasmid analysis and epidemiology of Salmonella enteritidis infection in three commercial layer flocks.

Ninety-six S. enteritidis isolates obtained from three commercial layer flocks in 1988-90 were examined following DNA extraction, restriction enzyme digestion, and gel electrophoresis for plasmid size profiles and restriction fragment length polymorphisms (RFLPs). The S. enteritidis isolates from the three flocks had three, eight, and two different plasmid profiles, respectively. Only four isolates from one flock lacked plasmids. A 36-megadalton (mDa) (54-kilobase) plasmid was present in 73% of the isolates, either alone or in combination with other plasmids. Isolates with only the 36-mDa plasmid had identical RFLPs. The diversity of plasmid profiles was greater than that of phage-types among isolates from the three flocks: 12 unique plasmid profiles vs. four phage-types. Mixed infections with S. enteritidis strains having distinct plasmid profiles occurred in all three flocks. Reinfection of these flocks in 1990 with one or more of the strains obtained earlier was evident, because some of the original isolates and the 1990 isolates had matching plasmid profiles and were of the same phage-types. Isolates from both environmental and tissue samples, examined from one flock, were found to share the same plasmid profile and phage-type.     

Analysis of Salmonella enterica serotype Enteritidis isolated from human and chickens by repetitive sequence-PCR fingerprinting, antibiotic resistance and plasmid profiles

A total of 22 Salmonella enterica serotype Enteritidis (S. Enteritidis) strains isolated from human and chicken were subjected to DNA fingerprinting by repetitive sequence PCR using ERIC and BOX primers, antibiotic resistance and plasmid patterns. Both ERIC and BOX PCR amplification data revealed a highly genetic homogeneity between isolates from human and chicken except one isolate, which originated from chicken and showed a different DNA band pattern from others. Eleven of 22 S. Enteritidis isolates (50%) were resistant to more than one antibiotics and characterized by 5 resistance patterns. The most common pattern was penicillin resistant (63.6%). Only one isolate from chicken showed a multiple drug resistance patterns to 4 antibiotics. All 22 S. Enteritidis isolates harbored more than two plasmids with eight different plasmid profiles including two to six plasmids with approximate molecular size ranging from 1.9 to 21 kb. A band of 15 kb size was detected in all isolates tested, however, the band sizes smaller than 15 kb were found only in isolates from chicken.     

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.     

Analysis of gene cassettes of streptomycin-spectinomycin resistance of Hungarian Salmonella enterica serotype Typhimurium strains

By PCR using the ant(3")-Ia primer pair the aadA gene was detected in 34 streptomycin- and spectinomycin-resistant Salmonella enterica serotype Typhimurium strains. Out of them 12 belonged to DT104 and 22 to non-DT104 phage type. Using different primer combinations it was demonstrated that this gene was integron-associated in all cases: in the DT104 strains it was generally contained by a 1 kb integron while in the majority of the non-DT104 strains by a 2.05 kb (less often by a 1.9 or 1 kb) integron. In the case of integrons carrying multiple cassettes the cassette containing the aadA gene was located closer to the 3' end of the integron. The aadA genes of DT104 and non-DT104 strains were different: in the former group the aadA2 gene, while in the latter group (constituted by strains of five different phages types as well as unclassifiable and untypable strains) the aadA1 gene could be identified. The RH50/RH51 primer pair described by Collis and Hall (1992) proved to be suitable for rapid discrimination between the aadA1 and aadA2 genes on the basis that the RH51 primer bound exclusively to the aadA2 gene.     

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.     

Epidemiology of Salmonella derby strains isolated from swine, pork and pork products

A total of 35 Salmonella derby strains, isolated from 6 types of samples of porcine origin from 9 different places in Hungary were examined for their characteristics. Thirty-two strains (91%) were of phage type 25, 2 (6%) of phage type 15 and 1 (3%) of phage type 58. Colicin production was observed in 3 (9%) strains. Five strains (14%) were found to be resistant to tetracycline (Tc). The strains harboured plasmids of 2.2, 2.4, 3.4, 4.2 and 72 Md. The 72 Md plasmid appears to be characteristic of S. derby and possibly encodes Tc resistance. The 72 Md plasmid belonged partly to incompatibility (Inc) group I1, while the other plasmid of the same size belonged to Inc. group B. The findings suggest that healthy salmonella carrier pigs carried the infection from the farm to the abattoir. Slaughtering of infected pigs may have led to contamination of the carcasses and, thereafter, that of the pork and pork products.     

用质粒DNA指纹图谱法分析猪水肿病大肠杆菌的流行病学特征

从广西各地病死猪器官、组织中分离到17株细菌,经细菌形态、培养特性及生化反应鉴定为大肠杆菌。作本动物回归试验、小白鼠致病性试验和溶血试验结果表明,全部为致病性大肠杆菌。血清定型鉴定结果发现,菌株血清型分布不规律。17株致病性大肠杆菌的质粒指纹图谱显示:来源于不同猪群的菌株GP20-3和GP25-1以及GP18-9和GP8-3分别具有相同的质粒图谱,说明发病猪群传染源的首发菌相同。所有菌株都含有1条大小相同的质粒带,广西流行的猪水肿病主要是由在遗传学上具有高度同源相关性的大肠杆菌的广泛流行传播所引起。菌株GP18-9和GP8-3的质粒指纹图谱相同,但它们的血清型却不一致的现象,可作为自然条件下,同一质粒在不同血清型大肠杆菌中扩散转化的一个生物学依据。     

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.     

鸡源大肠杆菌的耐药性监测及生物学特性研究

从山东省诸城地区6个集约化养鸡场采集50只病死鸡病料,从中分离鉴定得到32株大肠杆菌,对其进行了12种常规药敏试验,发现分离的菌株有不同程度的耐药性。对14株优势血清型菌株进行了质粒DNA提取分析,然后用Hind Ⅲ限制性内切酶对质粒进行了酶切。结果表明,质粒的得率为100%,来源相同的菌株具有相同或相似的质粒图谱,也就是说它们有共同的起源;来源不同的菌株的质粒图谱一般不同,即有不同的起源。将血清型和质粒图谱比较,结果发现,同一血清型可以有不同的质粒图谱,不同血清型可以有相似的质粒图谱,血清型与质粒图谱没有直接的联系。     

Phenotypic and genetic characterization of Salmonella enterica subsp. enterica serovar Typhimurium isolated from pigs in Rio Grande do Sul,Brazil

This study aimed to investigate the relatedness of porcine Salmonella enterica subsp. enterica (S.) serovar Typhimurium strains isolated in Southern Brazil. Sixty-six isolates from pigs belonging to three commercial companies were submitted to phage typing, XbaI-macrorestriction (PFGE), IS200 hybridization, rep-PCR, antimicrobial susceptibility testing, and PCR assay targeting the spvR region. All strains presented a unique rep-PCR pattern and 63 strains had a common IS200 profile. One pulse-type (XA) was the most prevalent (39/66 strains) and included strains of phage types DT177, DT192, DT194 and RDNC. The spvR region was detected in three strains, which harboured plasmids of 90 kb. High rates of tetracycline, sulfonamide and streptomycin resistance were found. Isolates from farms located in different geographic regions but associated to the same commercial companies clustered together and presented a common resistance profile. Results suggested that clonal groups of S. Typhimurium are present in pig commercial companies in Southern Brazil.     

Characterization of Vibrio anguillarum Strains Isolated from Diseased Fish in Finland

Characterization of V anguillarum strains (n=109) isolated from diseased salmonids was performed. Eight O serovars were found among the strains. Serovar Ol was predominant (90 %), while serovars O2, O3, O5, O8, O9, and a new serovar Va NT2, were represented by 1 or 2 strains. Two strains remained non-typeable. One of these was cross-reactive with several antisera, but had a LPS profile identical to that of serovar O8. All serovars showed specific LPS profiles. All but 1 of the Ol strains had a plasmid comparable in size to the pJMl virulence plasmid, while plasmids of different sizes were found in O2, Va NT2 and the non-typeable strains. Apart from a single strain resistant to tetracycline, all the strains were sensitive to oxolinic acid, tetracycline, and trimethoprim-sulfonamides. By their biochemical and antigenic properties strains causing vibriosis among salmonids in Finland closely resemble Scandinavian strains. Predominance of the serovars Ol and O2 suggests that commercial vaccines containing these serovars should afford sufficient protection against vibriosis in Finland.     

Nationwide surveillance of salmonella in the faeces of pigs in Japan

The prevalence of faecal carriage of salmonella in 5393 pigs reared on 218 pig farms located in 31 of 47 prefectures in Japan over the period July 2003 to June 2005 was investigated. We isolated 172 strains belonging to 20 serovars and one untypable Salmonella enterica from 169 pig faecal samples (3.1%) collected from 48 farms (22.0%). The most prevalent type of S. enterica was untypable O4,12:d:- which lacks phase 2 flagellar antigen, representing 29.1% (50/172) of all isolates. Of 26 S. enterica serovar Typhimurium isolates, 16 strains appeared to be definitive phage type 104 (DT104) by polymerase chain reaction.     

Lytic activities, protein profiles and morphologic characteristics of new bacteriophages isolated from canine and human Staphylococcus aureus strains.

The lytic activity, protein profile and morphology of five newly isolated phages from canine Staphylococcus aureus strains and one from a human S. aureus strain were compared with those of selected phages in the international phage sets (IPS). Five canine phages lysed 57 (76.0%) of 75 canine isolates of Staphylococcus aureus from Nigeria at routine test dilution (RTD) while 34 (IPS) phages typed only 31 (41.3%) strains at RTD or/and 100-RTD. The new human phage lysed 11 (14.7%) of 75 strains isolated from human diarrhoea. The new phages were readily propagated, specific in activity and stable during storage at 4 degrees C. Prominent proteins detected by SDS-PAGE indicated similarities between some of the phages but one canine phage was distinctly different, as was its morphology which was an isometric head with a short tail compared to oval heads and long tails which characterized others. IPS phages in the same serologic group had similar protein profiles but no correlation was observed with lytic groups. The use of protein profile and electron micrographs allowed classification of the phages into serogroups. It is concluded that the newly isolated canine phages could be very useful in typing Nigerian canine strains of S. aureus.     

Phenotypic and genotypic differentiation of porcine Salmonella enterica subsp. enterica serovar Derby isolates

Sixty-two Salmonella enterica subsp. enterica serovar Derby isolates from slaughter pigs and meat products isolated in Southern Brazil were analyzed for their genomic relationships and for the presence of antimicrobial resistance genes. Twenty-four S. Derby isolates were indistinguishable by their subtracted restriction fingerprinting (SRF) pattern, XbaI- and BlnI-macrorestriction patterns, phage type, plasmid profile, and resistance pattern. In contrast to the BlnI-macrorestriction patterns, the XbaI-macrorestriction patterns were in good agreement with the results of SRF analysis and phage typing. Among the four phage types detected, PT10 and PT21 were the most common. The combination of all typing methods revealed a great diversity among the S. Derby isolates. All strains carried plasmids and the 60 resistant isolates showed at least tetracycline resistance. The resistance genes found were sul1 and/or sul2 (sulfonamide resistance), aadA2 (streptomycin/spectinomycin resistance), tet(A) (tetracycline resistance), tet(B) (tetracycline/minocycline resistance), bla(TEM) (ampicillin resistance), and dfrA14 (trimethoprim resistance). A correlation of the geno- and phenotypic characteristics with the origin of the isolates revealed a substantial temporal variation in the occurrence of specific S. Derby isolates in different independent pig production lines in Southern Brazil. The large number of resistant isolates underlined the potential risk that S. Derby isolates can pose to human health when they enter the food chain.