Virulence studies of Salmonella enteritidis phage types

Salmonella enteritidis phage types (PTs) 8, 13a and 24 could be distinguished by their virulence for BALB/c mice, their plasmid content and plasmid fingerprint. Virulent strains expressed long-chain lipopolysaccharide and carried a 38 MDa plasmid indistinguishable from that in Salm. enteritidis PT 4. Avirulent strains were smooth but did not carry the 38 MDa plasmid. Possession of antibiotic resistance factors by some strains of Salm. enteritidis PT 24 did not contribute to the virulence of their host strains.     

Virulence of Salmonella enteritidis phage type 4 is related to the possession of a 38 MDa plasmid

Nine strains of Salmonella enteritidis phage type 4 were examined for virulence in BALB/c mice. The possession of a 38 MDa plasmid was necessary for full virulence. Strains carrying this plasmid had LD50 values of less than 20 bacteria whilst plasmid-free strains had LD50 values of greater than 10(6) bacteria when challenged intraperitoneally. Pathogenesis of disease involved the widespread distribution of bacteria throughout the tissues. Possession of the 38 MDa plasmid could not be linked with the ability of strains to express novel outer membrane proteins, to produce toxins affecting Vero, Y1, HeLa, Henle or HEp-2 cells, or to invade HEp-2 cells. Furthermore, the 38 MDa plasmid did not encode an aerobactin-mediated iron uptake system or the production of a haemolysin. Strains of S. enteritidis PT4 isolated in 1967, 1978 or 1979 and possessing the 38 MDa plasmid showed the same virulence properties as the current plasmid-carrying strains. This suggests that the enhanced virulence of the current strains for poultry is unlikely to be the result of changes in the 38 MDa plasmid.     

Conversion of Salmonella enteritidis phage type 4 to phage type 7 involves loss of lipopolysaccharide with concomitant loss of virulence

Three strains of Salmonella enteritidis phage type 4 (PT4) and 33 strains of S. enteritidis phage type 7 (PT7) were examined for the ability to produce lipopolysaccharide (LPS) and for plasmid carriage. The LPS of all strains of PT4 gave a typical 'ladder' pattern by SDS-PAGE and silver staining, and on serotyping these strains were shown to express the O-antigens 9, 12. In contrast, strains of PT7 did not express long-chain LPS and were autoagglutinable. All strains of PT4 and the majority of strains of PT7 carried a single plasmid of 38 MDa, indistinguishable when characterised by restriction endonuclease fragmentation analysis. Epidemiological and experimental observations have demonstrated a relationship between strains of S. enteritidis PT4 and PT7, and our results, using mice, show that the loss of ability of strains of PT4 to snythesise LPS is responsible for the conversion of highly virulent strains of PT4 to avirulent strains of PT7. From epidemiological data of human infections in England and Wales, we suggest that strains of S. enteritidis PT7 may be less virulent for humans.     

Unusual expression of lipopolysaccharide by strains of Salmonella enteritidis phage type 30

An investigation of 83 strains of Salmonella enteritidis phage type 30 showed that these bacteria were unusual in expressing either trace amounts of, or no, lipopolysaccharide (LPS). Regardless of whether strains expressed LPS or not, or carried a 38 MDa mouse virulence' plasmid, these strains were avirulent for BALB/c mice.     

Interrelationships between strains of Salmonella enteritidis belonging to phage types 4, 7, 7a, 8, 13, 13a, 23, 24 and 30

Salmonella enteritidis strain P278849 expressed long-chain lipopolysaccharide (LPS, termed 'smooth'), carried plasmids of 38, 34 (pDEP 44, incompatibility group N, R-type AS), 2.0 and 1 MDa, and belonged to phage type (PT) 23. Introduction of pDEP 44 into a smooth strain of Salm. enteritidis PT 4 produced a smooth strain of Salm. enteritidis of PT 24. Transfer of this plasmid into a strain of PT 8 also resulted in formation of a smooth strain of Salm. enteritidis of PT 24. Moving pDEP 44 into strains of Salm. enteritidis of PTs 7 or 7a which did not express LPS (termed 'rough') resulted in rough strains of PT 23. In contrast, transfer of this plasmid into a smooth strain of Salm. enteritidis PT 7a resulted in a smooth strain of PT 23. Introduction of pDEP 44 into strains of Salm. enteritidis of PT 13 or PT 13a did not change the phage type, whereas transferring the plasmid into strains of PT 30 caused strains to become resistant to lysis by the typing phages and therefore untypable. The possibility of strains of Salm. enteritidis of PT 8 being the progenitors of strains of Salm. enteritidis of PT 24 must now be considered when investigating the epidemiology of Salm. enteritidis of PT 24 infections in areas where Salm. enteritidis PT 8 is common.     

Identification of contemporary plasmid virulence genes in ancestral isolates of Salmonella enteritidis and Salmonella typhimurium

Six epidemiologically distinct ancestral strains of Salmonella enteritidis and 5 of S. typhimurium from the pre-antibiotic era were examined for plasmid content, and for presence of plasmid genes implicated in mouse-virulence. Five sizes of plasmid were detected in S. enteritidis varying from 1 to 60 MDa. Two sizes of plasmid were found in S. typhimurium, 28 and 60 MDa. Plasmids of the same size were not common to both serovars. The HindIII restriction patterns of 3 of the ancestral S. enteritidis plasmids were identical to the modern 38 MDa plasmid, while all contained identical bands of 3.5, 2.7 and 1.9 kb. All the 60-MDa S. typhimurium plasmids, ancestral and contemporary, had an identical restriction pattern. Three different sized S. enteritidis plasmids and one size S. typhimurium plasmid contained a 3.5-kb DNA fragment carrying the virulence locus VirA. The VirB virulence locus was located on a 2.7-kb DNA fragment in S. enteritidis and on a 2.5-kb fragment in S. typhimurium. Both loci were precisely conserved between the ancestral strains and the modern representatives of both serovars.     

The invasiveness of different strains of Salmonella enteritidis phage type 4 for young chickens

Five strains of Salmonella enteritidis phage type 4 (PT4) isolated in 1978, 1984 and 1988 were examined for their ability to colonise the caecum and invade the liver of day-old chickens. All strains were capable of caecal colonisation and there were no differences in their colonisation ability in this respect. In contrast there was a gradation in the ability of strains to invade the liver, with strains isolated in 1988 proving the most invasive. Absence of a 38 megadalton (Md) plasmid, which has been shown to be involved in the virulence of S. enteritidis PT4 for BALBc mice, had little effect on the ability of strains of this phage type to colonise the caecum or invade the liver of day-old chickens. These results suggest that recent isolates of PT4 may have enhanced virulence for chickens which is not necessarily associated with the carriage of a 38 Md plasmid.     

Distribution of virulence plasmids within Salmonellae

The virulence region of the Salmonella dublin 50 MDa plasmid shared homology with 678 of 1021 salmonellae tested in colony hybridization experiments. The majority of S. dublin, S. typhimurium and S. enteritidis isolates tested hybridized with the region whereas, with the exception of S. hessarek, S. pullorum and S. gallinarum, other serotypes did not. Homologous virulence regions were plasmid encoded. In S. typhimurium a common 60 MDa plasmid was present in all phage types tested but not in DT4, DT37 and DT170. Smaller plasmids showing partial homology were found in DT12, DT18, DT193 and DT204C. In S. enteritidis a distinct plasmid profile for each of eight phage types was observed. Hybridizing plasmids were found in DT3, DT4, DT8, DT9 and DT11 whereas DT7, which was plasmid free, and DT10 and DT14, which harboured plasmids, did not hybridize. The extent of homology shared between S. dublin, S. typhimurium and S. enteritidis virulence plasmids was about 10 MDa and appeared conserved. Virulence plasmids from S. typhimurium and S. enteritidis did not show homology with a region of the S. dublin 50 MDa plasmid which was not associated with virulence functions whereas plasmids of about 24 MDa and 38 MDa in some S. typhimurium phage types did. The association of conserved virulence regions upon differing plasmids within salmonellae is discussed with reference to possible mechanisms of distribution and evolution of virulence genes.     

Expression of lipopolysaccharide by phage types of Salmonella enteritidis

Examination of 44 type strains of Salmonella enteritidis for the ability to express long-chain lipopolysaccharide showed that strains belonging to phage types (PTs) 7, 23 and 30 were rough variants, a phenotype correlating with avirulence for BALB/c mice. Strains of Salm. enteritidis belonging to PTs 23 and 30 were thought to originate from smooth strains associated with poultry.     

Plasmid characterization and pulsed-field electrophoretic analysis demonstrate that ampicillin-resistant strains of Salmonella enteritidis phage type 6a are derived from Salm. enteritidis phage type 4

Plasmid incompatibility studies have demonstrated that strains of Salmonella enteritidis phage type (PT) 6a resistant to ampicillin possess a 36 megadalton incompatibility group (Inc) X plasmid coding for resistance to ampicillin which is capable of converting strains of Salm. enteritidis belonging to PTs 1 and 4 to PT 6a, and PT 8 to PT 13. However, pulsed-field gel electrophoresis (PFGE) has demonstrated that all clinical isolates of PT 6a have a characteristic Xba I pulsed-field profile which is distinct from that of PT 1 and which can only be differentiated from that of PT 4 by the presence of plasmid-associated fragments of less than 45 kb. It is concluded that ampicillin-resistant strains of Salm. enteritidis PT 6a are derived from strains of Salm. enteritidis PT 4 by acquisition of an Inc X ampicillin resistance plasmid.     

Expression of a new porin 'OmpE' by strains of Salmonella enteritidis

Abstract Strains of Salmonella enteritidis expressed a novel porin with a subunit size of 35.5 kDa as shown by SDS-PAGE. This protein was expressed as a major outer membrane protein (MOMP) when grown on nutrient agar, McConkey agar or blood agar, or in Tris-succinate medium; but was only produced in trace amounts when strains were grown in nutrient broth. This OMP was produced by all strains of S. enteritidis examined, regardless of phage type, and expression was not related to the possession of a 38-MDa mouse-virulence plasmid or the ability of strains to make long-chain lipopolysaccharide. This new porin has been tentatively called OmpE.     

Neisseria meningitidis: heterogenicity in the outer membrane proteins released into the growth medium

Eight strains of Neisseria meningitidis belonging to different serogroups were analysed for their virulence in mice and their release of outer membrane proteins into the medium during growth. All strains released proteins. No detectable lipopolysaccharide was observed. However, SDS-PAGE showed a heterogenicity in the protein number and profile among the different strains of N. meningitidis tested.     

In vitro and in vivo pathogenicity of <Emphasis Type="Italic">Salmonella enteritidis</Emphasis> clinical strains isolated from North America

Salmonella enteritidis is a leading cause of food-borne gastroenteritis worldwide. In this study, 48 strains of S. enteritidis isolated from clinical cases of salmonellosis in North America were tested for their virulence-associated traits including cell invasiveness, biofilm, motility, presence of a virulence plasmid, and virulence in orally challenged mice. The majority of strains exhibited high invasiveness (n = 45), whereas only few strains (n = 3) exhibited low invasiveness. All low-invasive strains (100%, 3/3) were biofilm negative, whereas the distribution of biofilm positive and negative phenotypes among high-invasive strains was 53.4% (24/45) and 46.6% (21/45), respectively. The in vitro cell invasiveness was not associated with biofilm formation (Fisher's exact test, P = 0.23) or the presence of a spvB gene, a marker for the virulence-associated plasmid (Fisher's exact test, P = 1). There was no correlation between cell invasiveness and motility (Spearman's rank test, r = -0.15; P = 0.27). Virulence testing in orally challenged mice revealed that the low-invasive strains were as virulent as high-invasive strains, indicating that in vitro cell invasiveness did not correlate with in vivo virulence. In conclusion, we show that despite phenotypic diversity among clinical strains of S. enteritidis, the majority of strains are highly invasive in vitro and in vivo.     

Siderophore production by Salmonella species isolated from different sources

A total of 230 Salmonella strains were screened for enterobactin and aerobactin production, sensitivity to bacteriocins and resistance to antibiotics. All the isolates produced the phenolate siderophore enterobactin. Amongst these, 74 strains, most belonging to S. enteritidis, were sensitive to colicin B. Only 26 isolates, all belonging to S. wien, produced an additional iron chelator, i.e. the siderophore aerobactin, and 22 out of these were sensitive to cloacin DF13. Analysis of iron repressible outer membrane proteins and plasmid profiles in S. wien strains showed that the expression of a 74-kDa iron-repressible outer membrane protein and the presence of large plasmids were associated with multiple antibiotic resistance, aerobactin production and sensitivity to cloacin DF13. The incidence of aerobactin-producing strains among S. wien isolates was higher during years 1974-1985; the epidemiological implications of these results are discussed.     

Leishmania amazonensis: humoral response to amastigote excreted-antigens in murine leishmaniasis

With the purpose of studying the antigenic role that factors excreted by Leishmania amastigotes might have during murine infection, immunoblots were carried out with sera from C57BL/6 and BALB/c mice infected with two strains of Leishmania (L.) amazonensis, NR and IFLA/BR. Both strains differ widely in virulence in BALB/c mice. BALB/c but not C57BL/6 sera recognized several excretion products. The excreted antigens showed a strong response towards IgG1 and IgG2a isotypes whilst they reacted only weakly against IgG2b and IgG3. A low-molecular weight antigen (about 20 kDa) excreted by both Leishmania strains was strongly recognized by IgG1 from BALB/c mice sera infected with IFLA/BR, the most virulent strain. Sera from NR infected mice were incapable of recognizing this antigen in spite of its presence in NR excreted products. The results indicate that the humoral immune response to excreted antigens of amastigotes depends on both the host genetic background and the parasite strain.     

Phenotypic Characteristics and Virulence of Vibrio anguillarum-Related Organisms

The phenotypic, molecular, and virulence properties of 46 Vibrio anguillarum-related (VAR) strains isolated from diseased fish and shellfish and from the environment were investigated. Twelve reference strains belonging to the 10 serotypes of V. anguillarum and the Vibrio splendidus type strain were included for comparison. Numerical taxonomy studies allowed us to group the isolates into four phena. The main phenotypic traits to differentiate VAR strains from V. anguillarum were fermentation of arabinose and mannitol, indole and Voges-Proskauer reactions, gelatin and casein hydrolysis, hemolytic activity, growth at 37 and 4°C, and resistance to ampicillin. Serological analysis confirmed that phena I and II were composed mainly of strains of V. anguillarum, while phena III and IV included VAR strains. Excluding the reference strains, the typeable isolates belonged to serotypes O3 (15 strains), O4 (3 strains), and O5 (2 strains) of V. anguillarum. The infectivity trials showed that only 9 of a total of 24 strains tested displayed virulence for rainbow trout. Virulent strains (50% lethal dose ranging from 10² to 10⁶ cells) included V. anguillarum strains belonging to serotypes O1 (one strain), O2 (one strain), O3 (three isolates), and O4 (one isolate) and only three strains of the VAR group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of lipopolysaccharide and outer membrane proteins showed heterogeneity not only among the 10 V. anguillarum serotypes but also within the VAR group. Immunoblot assays demonstrated a close relationship among V. anguillarum strains from the same serotype, while strains from different serotypes were not antigenically related. The VAR strains did not share antigenic components with the serotypes of V. anguillarum tested (serotypes O1 to O5). Plasmids were detected in only 19 of the total of 59 strains. The majority of the strains carrying plasmids were grouped within phenon IV, in which plasmid bands of 27 and 36 MDa were found in all the isolates. No correlation between the plasmid content of VAR microorganisms and their phenotypic or virulence characteristics was observed. From these results it can be concluded that VAR strains associated with disease should be included together with V. anguillarum in the formulation of vaccines against vibriosis.     

Experimental Infection of Mice with Yersinia enterocolitica Serotype O9 by Oral and Parenteral Routes: Spreading and Enterotropism of Virulent Yersiniae

An isogenic pair of Yersinia enterocolitica serotype O9 strains, with and without virulence plasmid, was used to study the plasmid role in the infection of BALB/c mice by oral, intraperitoneal, and intravenous routes. The plasmid-bearing strain, but not its plasmid-less derivative, caused enteric infection after challenge by all three routes. The virulence plasmid did not influence the peritoneal clearance of yersiniae, but only the plasmid-bearing yersiniae were able to move from the peritoneal cavity to the bloodstream, and thus they spread to spleen and liver. Moreover, plasmid-bearing yersiniae were able to move from the liver to the gallbladder, and they shed in bile into the intestine. Western blot analysis of antibody responses to chromosomally encoded outer membrane proteins revealed similar patterns with sera from mice challenged with each one of the two strains by intraperitoneal route. In contrast, only the plasmid-bearing strain elicited an antibody response to these antigens in mice challenged by oral route. Although mice experimentally infected with plasmid-bearing O9 yersiniae developed an enteric infection, irrespective of the inoculation route, differences between the first steps in infection by oral and parenteral routes may be important, especially when the infection model is used as an approach to study the yersinia-host interactions. Received: 31 August 1998 / Accepted: 13 November 1998     

Virulence and immunogenicity in experimental animals of Bacillus anthracis strains harbouring or lacking 110 MDa and 60 MDa plasmids

A comparative study was made of the virulence and immunogenicity in mice or guinea pigs of Bacillus anthracis strains harbouring 110 MDa and/or 60 MDa plasmids. Strains cured of the 110 MDa or the 60 MDa plasmid were more than 100-fold less virulent to mice than were the parental strains harbouring these plasmids. Guinea-pigs immunized with plasmid-free derivatives of the non-encapsulated vaccine strain 34F2 showed no resistance to challenge with strain 17JB, which harbours both 110 MDa and 60 MDa plasmids, suggesting that the derivative strains had lost their immunizing ability against anthrax.     

Lipopolysaccharide expression and virulence in mice of Australian isolates of Salmonella enteritidis

The lipopolysaccharide (LPS) of 54 Australian isolates, nine isolates acquired or isolated overseas, and two reference strains of Salmonella enteritidis was studied to assess its relation to pathogenicity. LPS was extracted by proteinase K digestion of whole cells, and analysed by polyacrylamide gel electrophoresis. All isolates possessed an LPS structure identical to that of a reference strain of Salm. enteritidis phage type 4. Representative strains of the clinically prevalent phage types 4, 14 and 26, which express long chain LPS, were assessed for their pathogenicity in mice. Salmonella enteritidis phage type 4 produced a lethal infection in BALB/c mice, but not in C3H/HeJ or Quackenbush (outbred) strains. Phage types 14 and 26 did not produce an obvious infection in any mice, suggesting Australian strains of phage type 4 are more virulent than phage types 14 and 26.     

Plasmid pC present in Salmonella enterica serovar Enteritidis PT14b strains encodes a restriction modification system

Salmonella enterica serovar Enteritidis (S. Enteritidis) possesses plasmids of different sizes and roles. Besides the serovar-specific virulence plasmid present in most field strains, S. Enteritidis can harbour plasmids of low molecular mass whose biological role is poorly understood. We therefore sequenced plasmid pC present in S. Enteritidis strains belonging to phage type PT14b. The size of plasmid was determined to be 5,269 bp and it was predicted to encode four open reading frames (ORFs). The first two ORFs were found (initial 3,230 bp) to be highly homologous to rom and mbeA genes of ColE1 plasmid of Escherichia coli. Proteins encoded by the other two ORFs were 99% homologous to a restriction methylase and restriction endonuclease encoded by plasmid pECO29 of a field strain of E. coli. Using insertional mutagenesis we confirmed experimentally that the plasmid pC-encoded restriction modification system was functional and could explain the high resistance of S. Enteritidis PT14b strains to phage infection.