The Virulence Markers of Salmonella enterica Serovar TyphimuriumDifferentPhage-Type Strains Isolated

Folia Microbiol. 50 (6), 519–523 (2005) http://www.biomed.cas.cz/mbu/folia/

The Virulence Markers of Salmonella enterica Serovar

Typhimurium. Different Phage-Type Strains

Isolated in Slovakia

V. MAJTÁN, ď. MAJTÁNOVÁ, M. SZABÓOVÁ

Department of Microbiology, Slovak Medical University, 833 03 Bratislava, Slovakia

fax +421 259 369 245 e-mail viktor.majtan@szu.sk

Received 9 May 2005 Revised version 28 July 2005

ABSTRACT. Phage-typing determination of cell-surface hydrophobicity, motility, and serovar-specific viru-

lence plasmid was performed in a collection of 154 clinical isolates of S. enterica serovar Typhimurium (SeT) isolated in Slovakia. All isolates were also examined in PCR for the presence of both stn (enterotoxin) and iroB (siderophore) genes. The DT104 was the definitive phage type most frequently identified (37.7 %), the second most frequently isolated phage type was DT41 (5.8 %); the occurrence of other phage types was not epidemiologically significant. On the basis of virulence-marker investigation, 46.1 % of isolates were hydrophobic in the assay of bacterial adherence to xylene, and 97.4 % were hydrophobic in salt-aggregation test. Motility of more than 50 mm was expressed by 20.8 % isolates. The serovar-specific 90-kb virulence plasmid was contained in 138 (89.6 %) of isolates. All SeT isolates were found (according to PCR) to carry the Salmonella-enterotoxin (stn) gene and the siderophore (iroB) gene. The increasing incidence of SeT DT104 human strains in Slovakia requires continuous attention; this can be markedly improved by sur-veillance efficiency and made possible by determining relationships between sporadic isolates.

Salmonella, as a pathogen of humans and animals, is of ubiquitous nature with multiple modes of

transmission and exists as an ever persisting problem for public health (Wray et al. 2000; Asheg et al. 2003).

In many European countries S. enterica serovar Enteritidis (SeE) and S. enterica serovar Typhimurium (SeT)

are among the serovars most commonly associated with human salmonellosis (Anonymous 2000; Scuderi et

al. 2000; Usera et al. 1995). However, the epidemic clone of SeE PT8 still remains the most frequently iso-

lated Salmonella clone in Slovakia (Majtánová 2004). The prevalence of SeT strains is low and they are asso-

ciated mainly with sporadic human infections.

From 2003 we observed moderate increase of this serovar and therefore our aim was to investigate

(1) the phage type, (2) the cell-surface hydrophobicity, motility and the serovar-specific virulence plasmid(s)

as the markers of virulence, (3) the presence of virulence genes for enterotoxin and siderophores of the SeT

strains isolated from humans.

MATERIALS AND METHODS

Bacterial strains. The collection included 154 SeT strains isolated in 2003–2004 from humans at

the departments of microbiology of hospitals or other clinical microbiology laboratories in Slovakia; the iso-

lates were epidemiologically unrelated.

Phage typing was done using 31 typing phages according to Anderson et al. (1977) in the National

Reference Center for Phage Typing of Salmonellae in Slovak Medical University (Bratislava, Slovakia).

Culture media. The synthetic minimal medium (Staples et al. 1980) was used for strain preparation

to hydrophobicity and motility tests; for detection of the plasmid the strains were cultivated in Luria–Ber-

tani (‘Miller’) broth.

Surface hydrophobicity was assessed using bacterial adhesion to xylene (BATH) and the salt aggre-

gation test (SAT). BATH was done according to Rosenberg et al. (1980); the exposed strain was considered

hydrophobic when it exhibited a fraction of adsorption to xylene of 󰀃35 %. SAT was done according to

Blanco et al. (1990) with diammonium sulfate solutions; the strain was considered hydrophobic if it aggre-

gated at a concentration <1.4 mol/L. 520 V. MAJTÁN et al. Vol. 50

Assay of motility was carried out according to Braga et al. (1995) using semisolid agar medium

(0.35 %). The plates were incubated for 6 h at 37 °C and a growth-zone diameter was measured

Plasmid DNA profile. Plasmid DNA was isolated by alkaline lysis according to Birnboim and Doly

(1979) with a modification: all strains were grown on blood agar plates overnight at 37 ºC. Inoculum of corn

size was used for plasmid isolation. Ammonium acetate (7.7 mol/L) was used for neutralization and the phe-

nol–chloroform step was omitted. Samples were analyzed electrophoretically in 1×TBE buffer in 0.7 % agar-

ose gel. The plasmid size was determined according to the reference strains E. coli V517 and S. enterica sv.

Typhimurium LT2, and supercoiled DNA ladder (Gibco BLL, UK).