Serovar Strains Research Articles

Studies of the phenomenon of host adaptation in Salmonella.

To study the phenomenon of host adaptation in Salmonella, a mathematical model has been developed which permits a definition and experimental investigation of the specific interaction between the adapted serovar and the adequate host. After experimental infection using a mixture of equal parts of two Salmonella strains, A and B, the bacterial concentrations CA and CB were determined in the organs of the animals infected. If an animal of species a and an animal of species b are infected with the same mixture of strain A adapted to a and strain B adapted to b, an expression: log10Qab = log10CaA + log10CbB - log10CaB - log10CbA may be calculated which describes the influence of the specific serovar-host interaction on the dynamics of the bacterial count. The variable Qab was determined using four Salmonella dublin, four Salmonella choleraesuis and five Salmonella gallinarum/pullorum strains in a total of 63 pairs of different hosts (calf and pig, calf and chicken, or pig and chicken). On this basis, the following statements can be made. The epidemiologically defined host adaptation of Salmonellas is accompanied by a specific agent-host interaction between the adapted serovar and adequate host. It promotes adherence and spreading of the agent in the adequate host. The effect was particularly expressed on day 3 post-infection and could be detected both in the lumen of the anterior sections of the intestine and in the intestinal lymph nodes and the liver. In part, the host-independent strain characteristics had a greater influence than the specific serovar-host interaction on the dynamics of the bacterial count. Strains of non-adapted serovars may thus result in a more intense colonization and invasion of the host than simultaneously administered bacteria of a serovar which is adapted to the respective host. The effects of a specific serovar-host interaction on colonization of and spreading in the host should be considered only as a component which contributes to the phenomenon of host adaptation.

Detection and subtyping of Actinobacillus pleuropneumoniae strainsby PCR-RFLP analysis of the tbpA and tbpB genes

A PCR-based procedure for detection and serotype identification of Actinobacillus pleuropneumoniae strains was developed and evaluated. The A. pleuropneumoniae tbpA and tbpB genes were used as targets for amplification of DNA fragments, with a pair of specific primers for each gene. Amplification with tbpA primers rendered a 2.8-kb PCR product from all 12 A. pleuropneumoniae reference strains as well as from Actinobacillus suis strain CCM 5586, while amplification of a 1.9-kb PCR product was observed when testing ten Haemophilus parasuis strains of different serovars. Amplification of the tbpB gene from A. pleuropneumoniae serotypes 1, 6, 8 and 12, and A. suis CCM 5586 rendered an identical 1.8-kb fragment, while from A. pleuropneumoniae serotypes 2, 3, 4, 7, 9, 10 and 11, and H. parasuis strains it produced a 1.7-kb fragment. No PCR amplification product was observed when examining strains of 19 other swine pathogens or closely related species. The minimal detection limit for whole-cell A. pleuropneumoniae templates was between 5–50 and 3 × 10 2–3 × 10 3 CFU when tbpA and tbpB specific primers, respectively, were used. Restriction fragment length polymorphism (RFLP) analysis of the PCR-generated products rendered different patterns, easily allowing us to discriminate between A. pleuropneumoniae, H. parasuis and A. suis and, more importantly, to distinguish ten RFLP A. pleuropneumoniae groups (the highest discrimination reported so far for a PCR assay with A. pleuropneumoniae), in such a way that the only serotypes with profiles identical to each other were 4 to 11 and 7 to 9. Moreover, the PCR-RFLP analysis was assayed in 36 A. pleuropneumoniae field isolates and in porcine samples (lungs and nasal swabs from experimentally infected animals). In both cases the system proved to be very efficient in A. pleuropneumoniae identification and serotype discrimination.

Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis.

Seventy-four strains of Bacillus thuringiensis thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction. The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains. There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection. Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B. cereus isolated from incidents of food poisoning. Microbiological Societies.

Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis

Seventy-four strains of Bacillus thuringiensis representing 24 serovars were examined for the presence of three enterotoxin genes/operons; the non-haemolytic enterotoxin Nhe, the haemolytic enterotoxin hbl and the Bacillus cereus toxin bceT using polymerase chain reaction. The nheBC genes were found in all strains examined, the hblCD genes in 65 of the 74 strains and bceT in 63 strains. There was little consistency of the distribution of enterotoxin loci among strains of the same serovar in serovars that were well represented in our collection. Culture supernatants from all but one strain inhibited protein synthesis in Vero cells, generally with a toxicity equivalent to that seen in strains of B. cereus isolated from incidents of food poisoning.

Clonal dissemination of Vibrio parahaemolyticus displaying similar DNA fingerprint but belonging to two different serovars (O3[ratio ]K6 and O4[ratio ]K68) in Thailand and India

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.

Probability of detection of Salmonella using different analytical procedures, with emphasis on subspecies diarizonae serovar 61:k:1,5,(7) [ S. IIIb 61:k:1,5,(7)

A parameter for assessing qualitative methods is suggested: ‘The probability of detection’ has been used in this study to compare the efficiency of commonly used media for detecting Salmonella in mutton and faeces. Mutton and ovine faeces spiked with strains of Salmonella IIIb 61:k:1,5,(7) and Salmonella Typhimurium were analysed and the performance of each combination of media was estimated. Extensive variation between serovars and strains of identical serovars were recorded. In meat, an inoculum of up to 10 6/g was needed to detect salmonellae with 90% probability, while in faeces even 10 7/g was not enough for some strains of S. IIIb 61:k:1,5,(7). All method combinations thus had a rather low efficiency. Based on our experiments, we recommend using a combination of the XLD medium and any of the three selective broths for detection of S. IIIb 61:k:l,5,(7) from mutton, while we recommend using the SC broth with any of the three plating media for detection from ovine faeces.

Taxonomic evidence that serovar 7 of Erysipelothrix strains isolated from dogs with endocarditis are Erysipelothrix tonsillarum.

The levels of relatedness among strains of Erysipelothrix serovar 7 isolated from dogs with endocarditis were estimated by performing DNA-DNA hybridization experiments with the type strains of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum. All the canine strains exhibited more than 81% hybridization with the type strain of E. tonsillarum but less than 13% hybridization with the type strain of E. rhusiopathiae. Based on DNA-DNA hybridization results we confirmed that serovar 7 of the isolates from dogs with endocarditis were conclusively identified as E. tonsillarum. These results strongly indicate that some strains of genomic E. tonsillarum are a canine pathogen.

Amplified Fragment Length Polymorphism (AFLP) Analysis of Listeria monocytogenes

AFLP analysis using four selective primers was performed on a set of 33 Listeria monocytogenes including strains from patients and foods implicated in outbreaks, human sporadic cases or foods. Strains were tested belonging to serovars 1/2a, 1/2b, 1/2c, 3b, and 4b. Using one of the primers, the AFLP technique generated 20 different sized DNA fragments. The 33 cultures segregated into 14 different patterns, each comprising 7-12 different fragments. Although the method was not sufficiently discriminatory for epidemiological typing, AFLP analysis reconfirmed the observation that L. monocytogenes comprises two major genetic groups: group 1 includes strains of serovars 1/2a and 1/2c, while group 2 serovars 1/2b, 3b and 4b.

Molecular grouping of Listeria monocytogenes based on the sequence of the inlB gene

The major part of the gene inlB was sequenced in 24 strains of Listeria monocytogenes belonging to serovars 1/2a, 1/2b, 1/2c, 3b and 4b. A phylogenetic analysis based on the inlB nucleotide sequences showed that strains of serovars 1/2a and 1/2c were closely related, as well as those of serovars 1/2b and 3b. Strains sharing serovar 4b could be divided into two distinct groups. There were differences in amino-acid sequence between all serovars except between serovars 1/2b and 3b. Differences in amino-acid sequence were also seen within each of the serovars 1/2a and 4b. The data presented indicate that the inlB gene may be useful for typing purposes as an alternative or complement to serotyping.

Bacterial Microflora of Wild Brown Trout (Salmo trutta), Wild Pike (Esox lucius), and Aquacultured Rainbow Trout (Oncorhynchus mykiss)

Initial numbers of bacteria associated with wild (brown trout and pike) and cultured (rainbow trout) freshwater fish as well as with the water in which they were caught were determined. Subsequently, a total of 979 randomly selected isolates were characterized and identified to the genus level. For all counts performed (aerobes, psychrotrophs, anaerobes, Enterobacteriaceae, and enterococci), no significant differences were observed in water samples, the highest level corresponding to psychrotrophs in pike environments (4.23 × 103 CFU/ml). Overall, the skin and intestinal content of brown trout were the most contaminated, while rainbow trout specimens (gills and gut) yielded the lowest numbers. For all bacterial groups, pike gills had the highest numbers. Counts for all of the sampling sites compare well with findings in other temperate geographical environments. Biological characteristics (feeding and skin properties) and the use of antimicrobials in aquaculture might have influenced these results. Motile and nonmotile aerobic gram-negative bacteria together with Enterobacteriaceae accounted for 50 to 70% of the psychrotrophs isolated from water. Micrococcaceae, lactic acid bacteria, Bacillus, and coryneforms were also found. The groups represented in psychrotrophic isolates from the outer surfaces do not reflected those detected in water, so it was common that those organisms recovered in significant numbers from fish were not detected in surrounding habitat of the fish. Motile aeromonads and Carnobacterium were the dominant psychrotrophs in the guts of pike and brown trout, respectively. The intestinal content of reared fish gave a high incidence of Bacillus and coryneforms, while Enterobacteriaceae was absent. Again, rearing practices could have influenced this finding. Listeria monocytogenes was not detected in any of the examined samples. Two strains of Salmonella, which belonged to the same serovar and lysotype, were recovered from pond-water samples taken from one facility on different sampling days. From the gut of a pike specimen and from the pike's environment, two Plesiomonas shigelloides strains of different serovars were recovered. These latter four strains were resistant to a considerable number of antimicrobial compounds (multiple antibiotic resistance indices > 0.2).

Phylogenetic analysis of Leptospira strains of pathogenic serovars using 23S rDNA gene sequences

The 23S ribosomal DNAs were amplified from 11 strains of Leptospira interrogans sensu lato by polymerase chain reaction (PCR) and sequenced. The PCR products of about 290-bp DNA fragments indicated more than 97% sequence similarity to each other. The phylogenetic tree based on the 23S ribosomal DNAs obtained in this study revealed that 11 strains of L. interrogans examined composed a cluster distinct to that of L. weilii and L. borgpetersenii, confirming that these strains were similar to strain Moulton of L. interrogans serovar canicola in 23S rDNA sequence.

Biotin-streptavidin enzyme-linked immunosorbent assay for the detection of antibodies to Campylobacter jejuni and C. coli in chickens.

An enzyme-linked immunosorbent assay (ELISA) was developed in a homologous system with bacterial ultrasonic-treated proteins as the antigen and antisera from chickens infected orally and subcutaneously with the strain Campylobacter jejuni serovar 6 (CJ 6). The cut-off level was determined using antisera from non-infected specific-pathogen-free chickens up to the age of 10 weeks. The suitability of the ELISA system was verified using antisera taken from chickens orally infected at the age of 4 weeks with CJ 1, 6, 28 or 36 or with Campylobacter coli serovar 28 (CC 28). The development of antibodies was monitored up to 6 weeks post-infection (p.i.). Sera from chickens infected with CJ 1, 6, 36 or CC 28 contained specific antibodies to Campylobacter, whereas in those infected with CJ 28 no specific antibodies were found. Distinct cross-reactions were observed between CJ 6, 28 and CC 28 antigens and their antisera 6 weeks p.i., while poor cross-reactions were found with antisera to CJ 1 and 28. Antibodies to strains of all heterologous serovars were successfully detected with an antigen pool comprised of CJ 1, 6 and 36 antigens. In 11 out of the 12 field sera obtained from 5- and 9-week-old broiler chickens suffering from campylobacteriosis, high specific antibody titres to Campylobacter jejuni were found.

Isolation of Vibrio vulnificus serovar E from aquatic habitats in Taiwan.

The existence of strains of Vibrio vulnificus serovar E that are avirulent for eels is reported in this work. These isolates were recovered from water and oysters and differed from eel virulent strains in (i) fermentation and utilization of mannitol, (ii) ribotyping after HindIII digestion, and (iii) susceptibility to eel serum. Lipopolysaccharide of these strains lacked the highest molecular weight immunoreactive bands, which are probably involved in serum resistance.

Similarity in moth-fly specific larvicidal activity between two serologically unrelated Bacillus thuringiensis strains.

Parasporal inclusions of a Bacillus thuringiensis isolate designated 92-KU-105-9 (H14/19) exhibited unusual larvicidal activity, specific for the moth-fly, Telmatoscopus albipunctatus (Diptera: Psychodidae), similar to that of a previously reported B. thuringiensis serovar leesis (H33) strain. The LC50 value of the purified inclusions was 4.92 micrograms ml-1 for the moth-fly larvae, while no mortality was shown in the mosquitoes Culex pipiens molestus and Anopheles stephensi, at protein concentrations up to 10 mg ml-1. Morphologically, the inclusion was a homogeneous globular body surrounded by an electron-dense, thick envelope. Multilamellar inner structure was evident between envelope membrane and inclusion matrix. SDS-PAGE revealed that the inclusions consist of five proteins with molecular masses of 72, 70, 68, 56 and 30 kDa. These proteins cross-reacted with the antibodies against inclusion proteins of the serovar leesis strain. High homologies existed in N-terminal amino acid sequences between the three major proteins (72, 70 and 68 kDa) and the two established protein classes, Cry4A and Cry10A.

The phylogeny of Ureaplasma urealyticum based on the mba gene fragment.

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14 and 8, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types, which have been designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3 and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a and 6b), thus facilitating high resolution typing of U. urealyticum. Inclusion of reference strains for serovars 1, 3, 6 and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition, a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however, this fragment was invariant within the parvo biovar. This study has shown that the mba sequence can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Physical and genetic maps of the Leptospira interrogans serovar icterohaemorrhagiae strain Ictero no.1 chromosome and sequencing of a 19-kb region of the genome containing the 5S rRNA gene.

We report the construction of physical and genetic maps of the chromosome of Leptospira interrogans serovar icterohaemorrhagiae strain Ictero No.1 using pulsed-field gel electrophoresis of DNA fragments generated by digestion with enzymes SrfI, AscI, FseI, and NotI and using reciprocal hybridization. We also sequenced the 19-kilobase (kb) DNA segment including the one gene for 5S rRNA (rrf) of pathogenic Leptospira. The size of the chromosome of the strain Ictero No.1 was estimated to be 4673kb and was found to be similar to those of the chromosomes of the leptospira strains Verdun (serovar icterohaemorrhagiae) and RZ11 (serovar pomona). The strains Verdun and RZ11 carry a small 350-kb replicon (minichromosome), and the strain Ictero No.1 also contained the same kind of molecule together with the chromosome. The physical maps of the strains Ictero No.1 and Verdun were almost identical, as were the locations of the selected genes, except for the location of one of the 16S rRNA genes. Overall, the genetic organization appeared to be conserved within the serovar icterohaemorrhagiae strains. In the sequenced region, we identified 10 putative ORFs and one rrf sequence, and the transcription orientations were all the same. A homology search for the products deduced from the sequenced data revealed that the orf H exhibited high similarity to malic acid enzyme of Haemophilus influenzae and fumarate hydratase of Escherichia coli (orf J). The rest of the putative products encoded by ORFs in the sequenced region showed little similarity with the proteins contained in the databases and were considered to be unknown proteins.

Studies of the Presence of the Virulence Factors, Adhesion, Invasion, Intracellular Multiplication and Toxin Formation in Salmonellas of Different Origin

Salmonellas of different origin were classified into two groups (11 strains of common serovars which had been isolated from organs of calves having died from salmonellosis and 18 strains belonging to rare serovars which showed uncommon metabolic characteristics and had been isolated from spices and spiced foods). The strains were examined with regard to different virulence parameters. All salmonellas investigated possessed the genetic information on invasion (invA) and toxin formation (stn). They adhered equally well to epithelial cells, could penetrate into these and survive and multiply inside the cells. The formation of toxic substances could be detected in all strains after co-cultivation with epithelial cells in the CHO-K1 test. Significant differences between the groups of strains could be demonstrated only for the invasion of epithelial cell monolayers. Since adhesion, invasion and the ability of intracellular survival and multiplication as well as toxin formation constitute virulence parameters of salmonellas, it must be assumed that also the Salmonella serotypes studied which have been rarely observed epidemiologically constitute a risk for humans.

Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan.

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, was initiated in January 1994. The incidence of cases of V. parahaemolyticus infection suddenly increased in February 1996 and has remained high since then. One hundred thirty-four strains of V. parahaemolyticus isolated from January 1994 to August 1996 were examined for serovar, the presence of the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin genes (trh1 and trh2), production of urease, and antibiogram. Strains of the O3:K6 serovar appeared for the first time in February 1996. The O3:K6 serovar strains accounted for 50 to 80% of the strains isolated during the high-incidence period (February to August 1996). All of the serovar O3:K6 strains carried the tdh gene but not the trh genes and did not produce urease. All of the isolates except two were sensitive to all of the antibiotics tested. These and the results of analysis by an arbitrarily primed PCR method indicated that the O3:K6 serovar strains belong to a unique clone. When the O3:K6 serovar strains, isolated from travelers arriving in Japan from Southeast Asian countries, were compared by the arbitrarily primed PCR method, the strains isolated between 1982 and 1993 were distinct from Calcutta O3:K6 while the strains isolated in 1995 and 1996 were indistinguishable from the Calcutta O3:K6 strains. The results suggest that this unique O3:K6 clone may have become prevalent not only in Calcutta but also in Southeast Asian countries very recently. Not only the O3:K6 strains but also the non-O3:K6, tdh-bearing strains isolated in 1996 produced thermostable direct hemolysin at high levels, and thus the level of hemolysin produced does not appear to have influenced the high incidence of serovar O3:K6 strains.

Importance of the Serovar-Specific Plasmid for Virulence of Salmonella Strains in Calves

To evaluate the influence of serovar-specific plasmids on salmonella virulence in calves, experiments were performed involving infection, by the oral route, with mixtures of strains containing equal counts of a plasmid-carrying and a plasmid-free strain of the same serovar. The concentration ratio between the plasmid-carrying and the plasmid-free strain which had developed in the organs of the infected animals was used for a comparative evaluation of virulence and pathogenetic behaviour of the strains. While in the S. typhimurium strains studied, the presence of the plasmid was accompanied by a significantly increased colonization and multiplication of the agent in the host's body, examination of S. enteritidis and S. dublin revealed that the plasmid-free strains exhibited identical or even significantly higher bacterial counts than the plasmid-carrying strains in organs. The fact that plasmid-free salmonella strains with a high virulence for calves have been found demonstrates that the presence of a serovar-specific plasmid is not an indispensable requirement for the development of salmonellosis in calves.

Colicin U, a novel colicin produced by Shigella boydii.

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.