Salmonella Strains Research Articles

Variations in biochemical phenotypes and phage types ofSalmonella enteritidisin Germany 1980–92

The Phene Plate system for typing Salmonella serotypes (PhP-S) is a simple automated typing method based on biochemical fingerprinting. It gives a quantitative value of the metabolism of various substrates by measuring the speed and intensity of each reaction. The 'biochemical fingerprint' of each isolate is used to calculate similarities among the tested strains with a personal computer program. We used this system to examine a collection of 86 strains of Salmonella enteritidis isolated from human sporadic cases in Germany between 1980 and 1992. Twenty-three biochemical phenotypes (BPTs) consisting of 9 common (C) and 14 single (S) BPTs were identified. BPTs C2 and C4 containing 20 and 36 strains respectively accounted for 65% of the isolates. Strains of BPT C2 were found over a wide period of time whereas strains of BPT C4 were isolated during the period between 1988 and 1992. With phage typing, 11 discrete phage types (PTs) and 18 strains designated as non-specific type (NST) were identified. PTs 4 and 8 with 39 and 17 strains respectively were the dominant PTs. Strains of PT 8 were isolated over a wide period of time whereas all (except one) strains of PT 4 were isolated between 1988 and 1992. Combination of biochemical fingerprinting and phage typing divided the strains into 25 phenotypes (BPT:PTs). Whilst phenotype C2:8 was found over a number of different years, phenotype C4:4 was isolated only between 1988 and 1992. These findings indicate the presence of one persistent and one recently emerged phenotype among S. enteritidis strains in Germany.(ABSTRACT TRUNCATED AT 250 WORDS)

Replicon typing characterization of plasmids encoding resistance to gentamicin and apramycin inEscherichia coliandSalmonella typhimuriumisolated from human and animal sources in Belgium

Escherichia coli and salmonella strains with plasmids conferring resistance to gentamicin and apramycin have been isolated with increasing frequency both from cattle and hospital patients in Belgium. The apramycin-gentamicin resistance plasmids were characterized in recipient strains by their profiles and molecular weights using agarose gel electrophoresis, by their antimicrobial resistance patterns and by replicon typing using a series of DNA probes specific for the genes controlling their systems of replication. Overall, most of the plasmids differed in their DNA electrophoretic patterns. Seventeen different antimicrobial resistance profiles were observed, and there were six different types of replicons. However, two replication genes predominated and had a preferential distribution in different bacterial species. The rep FIC.a plus rep Q multireplicon was found mainly in plasmids recovered from gentamicin- and apramycin-resistant E. coli while replicon of the type rep FIC.b largely prevailed in S. typhimurium. Identical replication genes were found in most animal and human strains, hence suggesting a high homology between apramycin-gentamicin plasmids in these communities. Finally, our results indicate that the rapid spread of apramycin-gentamicin-resistance in several species of Enterobacteriaceae isolated from animals and from humans in Belgium is not due to a single plasmid, but rather that the gene encoding AAC(3)-IV is carried by various replicons.

Salmonella identification by the polymerase chain reaction

Polymerase chain reaction (PCR) primers for genus specific detection of Salmonella have been selected from a Salmonella-specific fragment of 2·3 kilobases (kb). Due to interserovar sequence diversity within this fragment, primer selection was based on DNA sequence alignment of sequences from 20 different Salmonella serovars. The specific PCR product of 429 base pairs (bp) was formed from 144 of 146 salmonella strains tested (116 of 118 serovars). The two false-negative strains belonged to two different serovars of the rarely isolated subspecies IIIa (monophasic S. arizonae). No product was produced in any of 86 non-Salmonella Enterobacteriacea strains tested, covering 41 species from 21 genera.

Isolation of a multiresistant strain ofSalmonella typhi in Italy

Isolation of a multiresistant strain ofSalmonella typhi in Italy

The prevalence of Salmonella infections in camels ( Camelus dromedarius) in the United Arab Emirates

One hundred and eighty-seven salmonella strains were isolated from 4006 samples collected from camels in the United Arab Emirates between 1987 and 1991. One hundred and sixty-five (4.3%) strains were isolated from 3801 faecal and 22 from 205 organ samples of 62 autopsied camels. In total, 28 different serotypes were identified with S. saintpaul being the most frequent (69), followed by S. frintrop (31) and S. hindmarsh (15). Salmonella typhimurium was isolated from only two faecal specimens. All salmonellas isolated from faecal samples originated from carrier camels, and those isolated from organs were secondary findings. The camels from which salmonella organisms were found died from diseases other than salmonellosis. Most of the S. saintpaul isolates originated from one herd suffering from Clostridium perfringens type A enterotoxaemia which suggests that salmonella infections may predispose camels to clostridial enterotoxaemias.

Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.

Bacteriophage lytic patterns for identification of salmonellae, shigellae, Escherichia coli, Citrobacter freundii, and Enterobacter cloacae.

A series of bacteriophages specific for Escherichia coli (E-1, E-2, E-3, and E-4), Citrobacter freundii (phi I, phi II, and phi III), Enterobacter cloacae (Ent), and Shigella spp. (Sh) have been isolated from hospital sewage. These bacteriophages, in combination with Felix Salmonella phage O-I, were used as a diagnostic phage typing set which included seven phage preparations: O-I, C (phi I and phi III), Sh, E (E-1 and E-2), CE (phi II and E-3), E-4, and Ent. After 20,280 cultures of 27 species and 9 biogroups of 15 genera of the family Enterobacteriaceae and 276 cultures of 8 species of 6 genera outside the Enterobacteriaceae were tested, it was shown that most strains of salmonellae, E. coli, C. freundii, and E. cloacae can be identified accurately. The sensitivities of identification were 83.6% for E. cloacae, 88.8% for C. freundii, 90.3% for E. coli, and 95.76% for salmonellae. The specificities were 99.78% for salmonellae, 99.84% for E. cloacae, 99.89% for E. coli, and 99.97% for C. freundii. The results of bacteriophage lytic patterns were highly correlated with Shigella serotypes. Therefore, such a phage typing set may be used routinely in public hygiene and clinical laboratories.

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas.

A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10(6)/ml of a smooth wild strain of Salm. typhimurium, and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.

Use of Salmonella typhimurium WG49 to enumerate male-specific coliphages in an estuary and watershed subject to nonpoint pollution

The occurrence of male-specific RNA (FRNA) coliphages, proposed as indicators of enteric viruses, was determined in an estuary subject to nonpoint pollution that included fecal inputs from livestock. A host originally developed for detecting FRNA phages in sewage was applied to water and sediment samples. Phages were enumerated using the host Salmonella typhimurium WG49 containing an Escherichia coli plasmid coding for sex pili, and the female parent strain WG45. FRNA phages and fecal coliforms were enumerated in samples collected seasonally from an estuary and associated feeder streams and densities related to selected environmental parameters. Mean phage densities enumerated on WG49 ranged from < 1 to 50 100 ml −1 water and < 13 to 7200 100 g −1 dry sediment. Examination of 300 phages from estuarine and freshwater samples showed that ⩾ 99% were RNase-resistant, ⩾ 94% were lytic to the female parent salmonella strain (WG45), ⩽ 9% were lytic to male E. coli C3000, and none were lytic to female E. coli C. RNase resistant phages lytic to both salmonella strains were noncontractile flexible tailed phages and those lytic to male salmonella or E. coli hosts were filamentous phages. Electron micrographs of the only RNase-sensitive phage recovered that plaqued only male hosts showed cubic phage particles adsorbed to sex pili. Parallel enumerations of environmental samples on WG45 and WG49 yielded equal or greater phage densities on the former host. Purified phages from these samples were lytic to certain salmonella serovars recovered from the environment but did not cross react with fecal coliform or heterotrophic bacteria isolated from the environment. Although the WG49 host was inappropriate to estuarine and freshwater samples examined because of interference by somatic phages, WG45 and WG49 should be examined as hosts for enumerating salmonella phages. Similarly, the public health significance of somatic phages detected by these hosts should be determined. FRNA phages, with a single exception ( 1 187 samples), were not detected in a condemned shellfish growing area subject to nonpoint pollution. This observation questions the application of FRNA phages as indicators of fecal contamination in waters impacted by diffuse fecal inputs.

Cockroaches (BlattaandPeriplanetaspecies) as reservoirs of drug-resistant salmonellas

A total of 221 cockroaches (Blatta and Periplaneta spp.), collected in hospitals, houses, animal sheds, grocery stores and restaurants, in various parts of South Kanara District, a south-west coastal region of India, were studied bacteriologically for the presence of various salmonellas. Salmonellas were isolated from 4.1% of these cockroaches. Nine strains of salmonellas were recovered, belonging to five serotypes--Salmonella bovismorbificans, S. oslo, S. typhimurium, S. mbandaka and S. braenderup, the former two being the commonest serotypes. All salmonellas were resistant to one or other of 11 antibacterial drugs used in the susceptibility test. Isolation of salmonellas from cockroaches collected from the livestock premises and human dwellings suggested that they may act as significant reservoirs of salmonella in nature. Recovery of serotypes, phage types and R-types that were commonly isolated from humans and animals of this locality, suggested a transmission role for cockroaches. By harbouring potentially pathogenic, drug-resistant salmonellas, these wandering arthropods may pose dangerous infective hazards to humans and animals.

Characterization of murine monoclonal antibodies against serogroup B salmonellae and application as serotyping reagents

Six murine hybridoma monoclonal antibodies reactive with lipopolysaccharide antigens of Salmonella typhimurium were obtained from a fusion of immune spleen cells from mice immunized with S. typhimurium and NS1 myeloma cells. Four antibodies appeared to be specific for serogroup B salmonellae, while the remaining two antibodies were found to be cross-reactive with Salmonella paratyphi A. The exquisite specificities of the Salmonella serogroup B monoclonal antibodies were demonstrated by their unique reactivities with different serotypes of group B salmonellae but with neither other O serogroups of salmonellae nor a wide spectrum of standard strains of other bacterial species. Serotyping of salmonella strains by the slide agglutination method with two of the serogroup B-specific monoclonal antibodies demonstrated their usefulness as serotyping reagents for the identification of serogroup B salmonellae in a routine diagnostic bacteriology laboratory.

A survey of antimicrobial resistance in salmonellae isolated from animals in England and Wales during 1984–1987

Resistance to 14 antimicrobial substances was tested in 18 647 salmonella strains isolated from animals, their environment and from animal feeds during the period 1984–1987. Of the 2284 Salmonella dublin strains the percentage sensitive to all the antimicrobial substances ranged from 18.1 to 26.8. Resistance to the higher concentration of streptomycin (S25) ranged from 1.9 to 6.4%, whereas the corresponding figures when the lower concentration (S10) was used were 32.3 and 63.8%. Resistance to the higher sulphonamide concentration (Su 500) never exceeded 3.3%, although in 1987 70.3% of strains showed resistance to the lower concentration. In general, less than 1% of strains showed resistance to the other antibacterial substances. No strains resistant to amikacin (AK), apramycin (Apr), gentamicin (CN) and colistin (CT) were detected. Of the 8677 S. typhimurium strains the percentage sensitive to all the antimicrobial agents ranged from 6.6 in 1985 to 13.6 in 1987. Resistance to tetracycline (T), ampicillin (PN), trimethoprim (TM) and chloramphenicol (C) ranged from 36.5 to 58.8%, the highest percentages being detected with tetracyclines. Less than 1% of strains showed resistance to furazolidone and none was resistant to amikacin and colistin. Resistance to apramycin ranged from 3.1% in 1984 to 11.6% in 1985; the figures for gentamicin were approximately half that of apramycin. In 1984, 41.6% of strains showed resistance to neomycin but only 8.5% in 1987. The fall in neomycin resistance was associated with the epidemic spread of the commonest phage-type DT204C becoming sensitive to neomycin. Of the 7687 strains of serotypes other than S. typhimurium and S. dublin the percentage sensitive to all antimicrobial agents ranged from 23.7 in 1985 to 14.7 in 1987. Resistance to tetracyclines and sulphonamides (Su 500) ranged from 5.2 to 12.1% and 5.8 to 13.3% respectively. Resistance to the other antimicrobial agents was usually less than 5%.

Escherichia coli O157: H7 and its significance in foods

Escherichia coli O157:H7 was conclusively identified as a pathogen in 1982 following its association with two food-related outbreaks of an unusual gastrointestinal illness. The organism is now recognized as an important cause of foodborne disease, with outbreaks reported in the U.S.A., Canada, and the United Kingdom. Illness is generally quite severe, and can include three different syndromes, i.e., hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Most outbreaks have been associated with eating undercooked ground beef or, less frequently, drinking raw milk. Surveys of retail raw meats and poultry revealed E. coli O157:H7 in 1.5 to 3.5% of ground beef, pork, poultry, and lamb. Dairy cattle, especially young animals, have been identified as a reservoir. The organism is typical of most E. coli, but does possess distinguishing characteristics. For example, E. coli O157:H7 does not ferment sorbitol within 24 h, does not possess β-glucuronidase activity, and does not grow well or at all at 44–45.5°C. The organism has no unusual heat resistance; heating ground beef sufficiently to kill typical strains of salmonellae will also kill E. coli O157:H7. The mechanism of pathogenicity has not been fully elucidated, but clinical isolates produce one or more verotoxins which are believed to be important virulence factors. Little is known about the significance of pre-formed verotoxins in foods. The use of proper hygienic practices in handling food of animal origin and proper heating of such foods before consumption are important control measures for the prevention of E. coli O157:H7 infections.

Instability of multiple drug resistance plasmids inSalmonella typhimuriumisolated from poultry

Plasmids in five strains of Salmonella typhimurium resistant to ampicillin, chloramphenicol, gentamicin, neomycin/kanamycin, streptomycin, sulphonamides, tetracyclines and trimethoprim (ACGKSSuTTm), CGKSSuTTm, ACSSuT or CSSuT which had been isolated from poultry in the first 3 months of 1989 have been characterized and compared with plasmids in two strains of R-types ACGKSSuTTm and ASSuTTm isolated from two patients later in the year. With the exception of the human isolate of R-type ASSuTTm, all strains carried two non-conjugative plasmids, one coding for SSu and belonging to incompatibility group Q, and a second coding for multiple resistance and belonging to the FIme incompatibility group. The human isolate of R-type ASSuTTm did not carry the IncQ SSu plasmid but like the poultry isolates, carried a non-conjugative FIme plasmid. Restriction endonuclease digestion with the enzymes EcoR I, Pst I and Hind III demonstrated that the FIme plasmids from strains of different R-types showed a high degree of homology but exhibited numerous fragment size polymorphisms. The restriction digest fingerprint of plasmids in the human isolate of R-type ACGKSSuTTm was indistinguishable from a poultry isolate of the same R-type. Analysis of segregants of one of the poultry isolates of R-type ACGKSSuTTm demonstrated that resistance determinants could be rapidly lost from the FIme plasmid to give rise to a number of R-types and fingerprint patterns. Loss of tetracycline resistance from this plasmid appeared to be correlated with the integration of other plasmid-mediated resistances into the bacterial chromosome. Evidence is presented for the rapid loss of antimicrobial resistance determinants from a multiple resistance plasmid of the FIme incompatibility group in response to withdrawal of antibiotic selective pressure.

Mouse hepatitis virus strain UAB infection enhances resistance to Salmonella typhimurium in mice by inducing suppression of bacterial growth

We have previously shown that intranasal infection of mice with mouse hepatitis virus (MHV) strain UAB (MHV-UAB) increases their resistance to Salmonella typhimurium injected intravenously 6 days later. To study how salmonella resistance was induced, BALB/cAnNCr mice were infected with salmonella strains carrying specific genetic alterations. One set of studies compared the effect of MHV infection on subsequent salmonella infections with AroA- (avirulent) and Aro+ (virulent) salmonellae. Unlike its effect on Aro+ salmonellae, MHV failed to reduce the number of AroA- salmonellae recovered from mice. Because AroA- S. typhimurium shows almost no growth in vivo, this failure indicated that the effect of MHV on salmonella resistance required growth of the infecting salmonellae. In other studies, the effect of MHV infection on both growth and killing were monitored simultaneously in mice with growing salmonellae carrying a single copy of the temperature-sensitive pHSG422 plasmid, which is unable to replicate in vivo. MHV infection reduced salmonella growth but caused no increase in salmonella killing. MHV infection of mice given wild-type salmonellae also resulted in no increase in salmonella killing 4 h after salmonella challenge. These studies demonstrate that MHV-UAB infection increases host resistance to salmonellae by enhancing suppression of bacterial growth instead of by increasing the amount of salmonella killing.

Salmonella dublin Experimental Infection in Calves: Protection after Oral Immunization with an Auxotrophic aroA Live vaccine

Salmonella dublin strain SL5631, which is auxotrophic for p-amino-benzoic acid and 2,3-dihydroxybenzoate because of a deleted aroA gene, was given orally in a dose of 10(10) live bacteria to 6 calves 5-7 weeks old. The calves tolerated the strain well, had a transient mucoid diarrhea and sacrificed animals showed a moderate acute inflammation in the ileum on day 2. The salmonella strain was seen lining the mucosal epithelium using immunohistopathology. Already in calves sacrificed on day 6 the damage was less pronounced and signs of regeneration were obvious. The healing process was more accentuated in calves sacrificed on day 14. The results demonstrated the attenuating effect of the deleted aroA gene. Groups of 5-7 weeks old calves (n = 25) orally immunized with 10(8), 10(9) and 10(10) S. dublin SL5631 at weekly intervals were challenged 2, 6 or 15 weeks after the immunization. All calves were protected against oral challenge with 10(10) bacteria of the virulent S. dublin strain, which equals 1,000 LD50 doses. At autopsy, calves were sacrificed 3 weeks after challenge, all calves had normal intestinal findings with only slightly enlarged mesenteric lymph nodes. The protective effect is surmised to involve cell-mediated as well as humoral defense mechanisms.

Hepatitis B virus nucleocapsid/pre-S2 fusion proteins expressed in attenuated Salmonella for oral vaccination.

Hybrid HBV nucleocapsid-pre-S(2) fusion proteins were stably expressed in several aromatic-dependent attenuated Salmonella typhimurium and Salmonella dublin strains. When these live recombinant bacteria were administered i.p. to BALB/c mice they induced high titer anti-hepatitis B virus core Ag (HBc) and detectable anti-pre-S2 serum antibodies. Upon oral feeding of the recombinant salmonellae to mice, the rate of seroconversion to anti-HBc was dependent on the salmonella strain used. With the best carrier strain high titer anti-HBc antibodies and lower titer anti-pre-S2 serum IgG antibodies were observed two weeks after a single oral immunization. The Ig class and IgG subclass distribution of anti-HBc antibodies after i.p. and oral immunization is consistent with the induction of functional T cell help.

Radiation resistance ofSalmonella

The ionizing radiation resistances of sixSalmonella species were examined. The experimental variables were the suspending medium, the presence or absence of air, and the temperature during the irradiation process.S. typhimurium ATCC 14028,S. enteritidis ATCC 9186,S. newport ATCC 6962,S. dublin ATCC 15480,S. anatum ATCC 9270, andS. arizonae ATCC 29933 were suspended in phosphate buffer (0.1 M, pH 7.0), brain heart infusion broth (BHI) or mechanically deboned chicken and exposed to gamma radiation from cesium-137 at 0.12 kGy per min. The radiation resistance of theSalmonella increased approximately two-fold when assayed in sterile mechanically deboned chicken rather than in buffer or BHI. The average radiation (0.30 to 1.20 kGy) D-value for all sixSalmonella strains was 0.56 kGy in mechanically deboned chicken.S. enteritidis was significantly more resistant to ionizing radiation than the other five strains ofSalmonella tested on mechanically deboned chicken. The temperature of irradiation but not the presence or absence of air significantly influenced the survival ofS. typhimurium andS. enteritidis in mechanically deboned chicken. Treatment of chicken meat with ionizing radiation would be an effective means for control ofSalmonella contamination.

Plasmid profile analysis and restriction enzyme fingerprinting of Salmonella DO‐group strains

In Sweden Salmonella dublin is the most common serotype within the DO-group isolated from animals. In recent years also salmonella strains belonging to the DO-group but lacking H-antigen have been isolated from cattle in different areas. It was not possible to further differentiate the latter strains by serological methods. However, all Salmonella dublin strains and those strains lacking H-antigen carried a 50 Mdal plasmid exhibiting the same EcoRI and Hind III restriction enzyme digestion pattern. Two of the 26 strains contained an additional 5 Mdal plasmid. Other serotypes within the DO-group investigated, namely Salmonella enteritidis and Salmonella panama, carried plasmids smaller than 50 Mdal. The plasmid profiles and restriction enzyme digestion patterns indicated that the salmonella strains lacking H-antigen were variants of Salmonella dublin. Thus, analysis of plasmid profile and restriction enzyme fingerprinting are useful complements to serological methods in the differentiation of salmonella DO-group strains.

山梨県における散発性下痢症患者由来サルモネラの血清型薬剤感受性およびRプラスミド保有状況 (1985～1989)

A total of 391 strains of salmonella isolated from patients with sporadic cases of diarrhea during the period from April 1985 to June 1989 in Yamanashi Prefecture, were tested for their serovars, drug susceptibility and conjugative transmissible R plasmids. The results can be summarized as follows: 1) The isolates were serologically classified into 35 different serovars. The predominant serovars were S. Typhimurium (24.8%), S. Litchfield (9.2%), S. Oranienburg (9.0%), S. Enteritidis (7.9%) and S. Hadar (6.1%). 2) Serovars of S. Hadar, S. Brandenburg, S. Chester, S. Chailey, S. Oslo and S. Inchpark were isolated from human sources for the first time in Yamanashi Prefecture. 3) Monthly frequency of isolates were August (17.3 strains), September (11.3 strains) and May (10.6 strains). 4) The predominant ages of isolates were 2 years of age (17.1%), 1 years of age (15.1%), 3 years of age (11.2%), under 1 years of age (10.7%) and 4 years of age (9.8%). 5) The rate of isolates from male were higher than female. (Male: 57.5%, Female: 42.5%). 6) The frequency of resistant isolates was 45.8% in the period. The most predominant resistance pattern was SA.SM.TC.CP.KM.ABPC resistance (11.2%). Conjugative transmissible R plasmids appeared in 41.3% of the resistant strains. 7) The number of resistant strains of S. Typhimurium was 57 out of 97 strains (58.8%).