Characterization of Salmonella Dublin and Salmonella Typhimurium (Copenhagen) Isolates from Cattle

Brackelsberg, C.A., Nolan, L.K. and Brown, J., 1997. Characterization of Salmonella dublin and Salmonella typhimurium (Copenhagen) isolates from cattle. Veterinary Research Communications, 21 (6), 409-420Eight Salmonella typhimurium (Copenhagen) and eight Salmonella dublin isolates from cattle were compared by their antibiotic resistance patterns, by their production of colicin, aerobactin, haemolysin and capsule, by their possession of transmissible R plasmids and the spvC gene, and by their ability to invade and replicate within cultured epithelial cells. The two groups differed in their antibiotic resistance profiles, with more of the host-adapted S. dublin isolates resistant to tetracycline than were the non-host-adapted S. typhimurium (Copenhagen) group, but more of the S. typhimurium (Copenhagen) isolates resistant to the other antibiotics tested. None of the isolates produced colicin, but all produced aerobactin. One isolate in each group was encapsulated. All of the S. typhimurium (Copenhagen) and S. dublin isolates contained plasmids, and all of them contained the spvC-homologous sequences. Four of the S. typhimurium (Copenhagen) isolates were able to transfer an R plasmid to a recipient organism by conjugation. One of the five S. dublin isolates, which showed resistance to some of the antibiotics tested, was able to transfer an R plasmid by conjugation. Both groups of isolates invaded cultured epithelial cells to a similar degree after 1 h, but the S. dublin isolates reached significantly higher levels within the cells than did S. typhimurium (Copenhagen) after 9 h. This ability may, in part, explain the association of S. dublin with more severe forms of salmonellosis and prolonged carrier states. Further study of the intracellular growth of these isolates seems warranted.     

A molecular epidemiologic investigation of Salmonella from a meat source to the feces of captive cheetah (Acinonyx jubatus).

Low cheetah (Acinonyx jubatus) birth rates were observed for a long time in a captive breeding facility in which Salmonella, which was possibly present in contaminated beef, was isolated from still-born lion (Panthera leo) cubs. Salmonella, including 14 isolates of Salmonella serovar typhimurium and 19 isolates of Salmonella serovar muenchen, was subsequently isolated 47 times from 378 meat samples at the facility during a 13-mo period. Salmonella, including 26 isolates of S. serovar typhimurium, 10 of S. serovar muenchen, and 11 other serovars, also was isolated 54 times from 119 fecal samples. Only three plasmid profiles were identified in 59 S. typhimurium isolates from both meat and fecal samples. Although random-amplified polymorphic DNA fingerprinting using different primers in the polymerase chain reaction was able to distinguish between S. typhimurium and S. muenchen and to demonstrate similar chromosomal DNA fingerprints in some of the isolates from meat and feces, the results were not consistent enough to prove that the Salmonella in the feces originated from contaminated meat. However, the predominance of only two serovars in the meat fed to carnivores and in the feces of these animals suggests that the meat was the source of the Salmonella organisms in the feces.     

Transmissible antibiotic resistance in Salmonella isolated from random-source cats purchased for use in research.

Salmonella isolates from random-source cats designated for use in research were examined for antibiotic susceptibilities and the presence of plasmids containing R factors. The serotypes studied were Salmonella derby, S typhimurium, S anatum, S enteritidis, and S bredeney. Eighty percent of the isolates were resistant to one or more antibiotics. The greatest frequency of resistance was to streptomycin. The majority of the salmonella isolates transferred all or a part of their antibiotic resistance to an Escherichia coli K-12 recipient. Thermosensitive R factors were found in two S typhimurium isolates.     

Aggregation of animal lactobacilli with O157 enterohemorrhagic Escherichia coli

The effect of selected autoaggregative lactobacilli on enterohemorragic O157 Escherichia coli was studied. A total of 27 Lactobacillus sp. strains were examined for expression of autoaggregation and cell surface hydrophobicity. Autoaggregative isolates were obtained from the crops of chickens and vaginas of calves. Aggregative activity was seen between eight autoaggregative lactobacilli and six strains of enterohemorrhagic E. coli O157. The presence of aggregation-promoting factor (apf gene) was confirmed by polymerase chain reaction (PCR). We propose that autoaggregative lactobacilli that aggregate with enterohemorrhagic E. coli can express a class of APF proteins that exhibit the function of an aggregative mediator. The results of this study suggest that APF-producing lactobacilli could represent a further mechanism in the interaction of comensal microflora with enterohemorrhagic E. coli.     

Proposal of screening method for intestinal mucus adhesive lactobacilli using the enzymatic activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)

Adhesion tests are complex, time‐consuming and expensive, while the most important criterion for a probiotic lactobacilli is the ability to adhere to the human intestine. Thirty lactobacilli isolates from human intestinal tissues were measured for cell surface glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) activity using a microtiter plate screening method. GAPDH activities were detected in 21 out of 30 samples from 12 h cultures and in all samples from 18 h cultures. This suggests GAPDH is universally expressed on the bacterial cell surfaces from many lactobacilli. A statistically significant positive correlation was shown between GAPDH activity and adhesion using the BIACORE adhesion assay (P < 0.01). The new screening method using GAPDH enzymatic activity without an adhesion test may be possible due to the significant positive correlation of GAPDH activity with adhesion of lactobacilli derived from the human intestine.     

Phage types, ribotypes and tetracycline resistance genes of Salmonella enterica subsp. enterica serovar Typhimurium strains isolated from different origins in Italy

The tetracycline resistance (tet) gene patterns of 52 tetracycline resistant Salmonella enterica subsp. enterica (S.) serovar Typhimurium isolates collected from animals, food of animal origin, and humans in Italy, were investigated to evaluate whether the tet gene patterns could be used for strain differentiation in addition to phage typing and ribotyping. The detection of tet genes was performed by specific PCR assays. Ribotyping was performed automatically using PvuII as restriction enzyme. Ten different ribotyping patterns were detected. All isolates were positive for at least one of the tet genes studied and six different tet gene patterns were observed. Ribotyping and tet gene patterns showed discriminatory indices of 0.741 and 0.812, respectively. Multiple tet genes were commonly found among tetracycline resistant S. typhimurium isolates from various sources. The resulting tet gene patterns allowed further discrimination of strains which were otherwise indistinguishable by their phage type, ribotype and origin. Thus, the analysis of tet gene patterns might represent an additional tool for the differentiation of S. typhimurium isolates.     

Comparison of Salmonella enterica subspecies enterica serovar Typhimurium isolates from dairy cattle and humans using in vitro assays of virulence

Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella typhimurium) can infect and cause disease in a wide range of host species however there have been suggestions that this serovar may have genes involved with host range or specificity [Tsolis, R.M., Townsend, S.M., Miao, E.A., Miller, S.I., Ficht, T.A., Adams, L.G., Baumler, A.J., 1999. Identification of a putative S. enterica serotype Typhimurium host range factor with homology to IpaH and YopM by signature-tagged mutagenesis. Infect. Immun. 67 (12), 6385-6393]. Our goal in this study was to determine if in vitro virulence assays would support this suggestion. Twelve human and 10 bovine isolates of S. typhimurium from a single county in California were evaluated using in vitro virulence assays of adhesion and invasion. The resulting data was combined with results from previously reported genotypic and phenotypic testing of the isolates and statistical analysis performed using multivariate general linear models. Human isolates had higher adhesion values in each of the statistical models tested (p<0.05) but no statistical differences were found in the invasion values of human and bovine source isolates. Both adhesion and invasion values differed between the two largest groups of isolates segregated on the basis of pulsed-field gel patterns. The findings suggest there may be genetically defined in vitro virulence attributes in S. typhimurium that are associated with host species.     

Multiple drug-resistant Salmonella typhimurium DT104 and DT104b isolated in bobwhite quail (Colinus virginianus)

Multiple drug-resistant Salmonella typhimurium, definitive type (DT) 104 and DT104b, were isolated in three separate hunting preserve bobwhite quail (Colinus virginianus) outbreaks. The cases involved 4-day-old and 3-wk-old quail with increased mortality of 5%-8.6%, respectively. Postmortem lesions included emaciation, distended abdomens, and dark colon contents, which were gaseous and fluid in consistency. Salmonella typhimurium isolated from the intestines and/or livers was resistant to ampicillin, chloramphenicol, sulfonamides, and tetracycline. The isolate involving the 3-wk-old quail was phage typed as S. typhimurium DT104. The isolates involving the two cases of 4-day-old quail were phage typed as S. typhimurium DT104b.     

Effect of sodium nitrite and sodium chloride on growth of lactic acid bacteria.

The effect of NaNO2 and NaCl on the growth of 24 lactic acid bacteria strains isolated from vacuum-packed cooked ring sausages were examined by analyzing different growth parameters with Bioscreen. NaNO2 had a very limited effect on the growth of lactic acid bacteria at 50 and 100 mg/l but at 400 mg/l a more pronounced inhibitory effect was found. Bacterial growth was enhanced by 1-2% (w/v) of added NaCl, while NaCl concentrations above 3% (w/v) had a clear inhibitory effect. Leuconostoc isolates seemed to be more sensitive to sodium nitrite and sodium chloride than homofermentative lactobacilli strains. Among homofermentative lactobacilli, the strains resembling Lactobacillus curvatus were more sensitive to NaCl than those resembling Lactobacillus sake.     

Detection and characterization of Salmonella strains from laughing gulls (Larus ridibundus)]

25 and 17 Salmonella strains could be isolated from 429 and 423 blackheaded gulls (Larus ridibundus), respectively, during two years of examination. S. typhimurium was the most frequent serovar. All strains of S. typhimurium belonged to the biochemovar c (inosite and rhamnose negative), nearly a third of isolates caused a mannose-sensitive hemagglutination of guinea pig erythrocytes. This result is in contradiction to the literature. Furthermore the phagovars, the plasmid profiles and the resistance against chemotherapeutics were tested. The Salmonella carriage by gulls presumably reflects the contamination of the environment.     

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).     

Dynamics of Salmonella contamination in a commercial quail operation.

Control of carcass contamination requires knowledge of the source and dynamics of spread of Salmonella in commercial poultry production. We examined Salmonella contamination at a U.S. commercial quail operation. Pulsed-field gel electrophoresis (PFGE) was used to type isolates in order to trace Salmonella throughout this production environment. During a 6-mo survey, Salmonella serotypes hadar, typhimurium, typhimurium variant Copenhagen, and paratyphi were encountered within this poultry operation. Ninety-four percent of the Salmonella isolated from breeder and production houses and from carcass rinses belonged to Salmonella serotypes typhimurium variant Copenhagen and hadar. There were six distinct S. typhimurium variant Copenhagen genetic types, as identified by PFGE, present within this particular poultry operation. Seventy-nine percent of S. typhimurium variant Copenhagen identified from the environment of the breeder and production houses produced the same PFGE pattern. Thirty-eight percent of S. typhimurium Copenhagen isolated from carcass rinses and the breeder house shared the same PFGE DNA pattern. This study demonstrates the transmission of salmonellae throughout this production environment, from the breeders to their progeny and to the birds ultimately processed for human consumption.     

Intestinal bacterial flora of the household lizard, Gecko gecko

A total of 114 isolates was recovered from the intestines of 43 househould lizards, Gecko gecko. Among the important ones were Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, Proteus mirabilis and Edwardsiella tarda.     

Screening for probiotic characters in lactobacilli isolated from chickens revealed the intra-species diversity of Lactobacillus brevis

Considering the importance of the poultry industry and the increasing interest in alternative growth promoters, probiotics are considered as a potential candidate for use in the poultry industry. In this study, Lactobacillus species were isolated from 21 rectal swabs of 11 healthy 6-day-old and 10 healthy 21-day-old chickens and their fecal and feed samples. The isolates were characterized and their probiotic characteristics, including resistance to gastric acid and bile salts, biofilm formation and adherence to epithelium or mucus, amylase and protease activity and production of inhibitory compounds, were assessed. From 31 acid and bile resistant lactobacilli, only 2 Lactobacillus brevis and 1 Lactobacillus reuteri strains showed significant probiotic properties. These isolates indicated detectable attachment to Caco-2 cells and significant antibacterial activities against Gram-positive and Gram-negative pathogens. Additionally, phenotypic and genotypic diversity of lactobacilli isolates were studied by Phene Plate (PhP) system (PhP-LB) and random amplified polymorphic DNA (RAPD)-PCR, respectively. PhP-LB results of 24 L. brevis isolates showed a high phenotypic variation among the isolates. In comparison, results of RAPD-PCR highlighted a low diversity. Therefore, it seems that combination of the 2 techniques (PhP and RAPD-PCR) could result in a significant discriminatory power than each of them used alone.     

Pathogenicity of different serogroups of avian salmonellae in specific-pathogen-free chickens.

The pathogenicity of one isolate of Salmonella typhimurium, four isolates of Salmonella heidelberg, three isolates of Salmonella kentucky, two isolates of Salmonella montevideo, one isolate of Salmonella hadar, and two isolates of Salmonella enteritidis (SE), one belonging to phage type (PT)13a and the other to PT34, was investigated in specific-pathogen-free chicks. Three hundred eighty-four chicks were separated into 16 equal groups of 24 chicks. Thirteen groups were inoculated individually with 0.5 ml of broth culture containing 1 x 10(7) colony-forming units (CFU) of either S. typhimurium (one source), S. heidelberg (four sources), S. montevideo (two sources), S. hadar (one source), S. kentucky (three sources), SE PT 13a (one source) or SE PT 34 (one source) by crop gavage. Two groups of 24 chicks were inoculated in the same way with 1 x 10(7) CFU of SE PT4 (chicken-CA) and Salmonella pullorum. Another group of 24 chicks was kept as an uninoculated control group. The chicks were observed daily for clinical signs and mortality. Isolation of salmonella was done from different organs at 7 and 28 days postinoculation (DPI). All the chicks were weighed individually at 7, 14, 21, and 28 DPI. Two chicks chosen at random from each group were euthanatized and necropsied at 7 and 14 DPI and all the remaining live chickens, at 28 DPI. Selected tissues were taken for histopathology at 7 and 14 DPI. Dead chicks were examined for gross lesions and tissues were collected for histopathology. Chicks inoculated with S. pullorum had the highest mortality (66.66%), followed by S. typhimurium (33.33%). Chicks inoculated with S. heidelberg (00-1105-2) and SE PT4 (chicken-CA) had 12.5% mortality and 8.3% mortality, respectively, with SE PT 13a. Ceca were 100% positive for salmonellae at acute or chronic infection compared with other organs. Mean body weight reduction ranged from 0.67% (inoculated with S. kentucky 00-926-2) to 33.23% (inoculated with S. typhimurium 00-372) in the inoculated groups at different weeks compared with uninoculated controls. Gross and microscopic lesions included peritonitis, perihepatitis, yolk sac infection, typhilitis, pneumonia, and enteritis in some groups, especially those inoculated with S. typhimurium, S. heidelberg (00-1 105-2), SE PT4 (chicken-CA), and S. pullorum.     

Differentiation of Salmonella typhimurium DT204c by plasmid profile and biotyping

Seventy per cent of the United Kingdom isolates of Salmonella typhimurium from calves are phage type 204c and the study of the epidemiology of this organism requires additional methods of strain characterisation. This paper describes the applications of biotyping and plasmid-profile analysis for this purpose. One hundred and eleven isolates from 73 outbreaks of disease were examined. All belonged to the same primary biotype, although strains from 39 of the outbreaks differed in secondary tests in failing to ferment m-inositol at 25 degrees C. Four different antibiotic resistance patterns were detected among the isolates, which possessed seven distinct plasmid profiles. The spread of a distinct type through the calf marketing chain was investigated by using these techniques.     

Salmonella-associated conjunctivitis in a cat.

Salmonella typhimurium-associated conjunctivitis in an adult female cat was characterized by epiphora and blepharospasm. The cat also harbored intestinal S typhimurium, as did one other cat in the same shipment of 7 cats obtained from a commercial cat dealer. The 3 isolates were resistant to ampicillin, cephaloridine, cephalothin, streptomycin, triple sulfonamides, and tetracycline. All 3 isolates were capable of transferring a part of the antibiotic resistance pattern to an Escherichia coli K-12 recipient. Salmonella were not recovered from the conjunctiva or fecal flora of either cat after 10 days of therapy with chloramphenicol.     

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.     

Investigation and control of an outbreak of salmonellosis caused by multidrug-resistant Salmonella typhimurium in a population of hospitalized horses

An outbreak of salmonellosis in a population of hospitalized horses resulted in the closure of a teaching hospital for a period of 10 weeks. Fecal samples were collected from suspected cases and cultured for Salmonella. Salmonella isolates were characterized using antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and phage typing. Thirty-three cases of infection by a multidrug-resistant strain of S. typhimurium were detected. The index case was admitted on 26 August 1999. Fifteen (45%) cases occurred between April and June 2000. PFGE results suggested that this strain of S. typhimurium might have been introduced into the hospital environment by a foal presenting with diarrhea. The hospital was closed on June 13, and intensive environmental cleaning and disinfection were completed. Enforcement of infectious disease control protocols in hospitals and environmental and patient surveillance is needed to prevent outbreaks of salmonellosis.     

Collagen (Cn-I) binding by gut lactobacilli.

Four gut Lactobacillus strains displaying the features which make them particularly promising for the preparation of probiotic products were investigated together with 5 fresh isolates and one collection strain of Lactobacillus plantarum for their ability to bind type I collagen (Cn-I). Immobilised Cn-I in microtitre plates was bound only by 3 strains of gut lactobacilli from piglets and the collection strain Lactobacillus plantarum LHI 10 from Prague in range of A570nm readings 0.114-0.221. Six strains (isolates from turkeys and a calf) did not bind Cn-I (A570nm < 0.1) in this assay. An influence of cultivation medium on Cn-I binding was significant (P < 0.001) in all four adherent strains. Significantly higher (P < 0.001) binding of Cn-I was observed for Lactobacillus casei L 81 and Lactobacillus plantarum LHI 10 grown on solid medium (MRS agar) than for MRS broth-grown cells, however, Lactobacillus plantarum L 5 and Lactobacillus fermentum L 435 expressed significantly (P < 0.001) higher Cn-I binding during cultivation in MRS broth. The specificity of the binding was confirmed because the Cn-I binding by lactobacilli after their preincubation with this protein was completely abolished. Three selected inhibitors (fucoidan, heparan sulphate and hyaluronic acid) significantly (P < 0.001) reduced Cn-I binding by the Lactobacillus plantarum L 5 strain. Following up on some earlier strain characteristics, these results suggest that the selected piglets lactobacilli are also able to bind Cn-I and therefore should antagonize collagen niche colonization by various enteropathogens when used for probiotic purposes.