Short communicationThe distribution of enterotoxin and enterotoxin-like genes in Staphylococcus aureus strains isolated from nasal carriers and food samples
Abstract
Staphylococcus aureus strains were examined for the presence of 18 enterotoxin (se) and enterotoxin-like (sel) genes by PCR and four classic enterotoxins SEA to SED by reverse passive latex agglutination. We screened three groups of isolates: 53 recovered from food samples collected in years 2004–2005, 18 — from food samples collected in the 1960s and 1970s and 30 — from nasal carrier samples collected in the years 2000–2002. Eighty percent of all tested strains were se and sel positive, but the frequency of these genes was higher among nasal carrier strains (93%) than among food samples isolates (circa 76%). The enterotoxin genes cluster (egc) was the most prevalent among carrier strains (18/30–60%) and the least — among food strains isolated in the 1960s and 1970s (6/18 strains — 33%). sea coding enterotoxin A, which was recognised as the major cause of staphylococcal food poisoning so far, was more often found among carrier strains than among the years 2004–2005 food strains (27% versus 11%), but it was the most frequent se/sel gene among food strains isolated in the 1960s and 1970s (10/18 strains). Moreover in our results certain se and sel genes coexisted, which was in accordance with current knowledge about movable genetic elements carrying those genes. The exception was for only one strain, which harboured the sole selr gene on a possibly new genetic element not yet described. As we found both types of egc, as well as seh (especially food samples strains) occurred alone in a majority of positive strains for each of those genes.
The analysis of the results obtained by SET-RPLA method for the productivities of classical enterotoxins A–D and the results obtained by PCR for the presence of sea–sed genes revealed the correlation between each other. Only three of all sea–sed positive strains had silent genes — sed.
Introduction
Staphylococcus aureus is an important pathogen of humans in both community-acquired as well as nosocomial infections (Chambers, 2001, Karlowsky et al., 2004). S. aureus is also among the four most common cause of food-borne illnesses. S. aureus food-borne disease is a typical intoxication due to enterotoxins ingestion preformed in food by the enterotoxinogenic strain (Tenover and Gaynes, 2000). Staphylococcal food poisoning is widespread and quite frequent. The number of staphyloenterotoxicosis cases is probably underestimated significantly. There may be a lot of reasons for that: not calling medical services by many ill people due to the short duration of the disease or mild symptoms, improper both sample collection and laboratory examination.
Humans, rarely cows with infected udders, or other animals, may serve as reservoir of S. aureus. S. aureus colonizes 20–30% of human population persistently and 60% of population intermittently. In fact only 20% of people almost never carry S. aureus (Kluytmans and Wertheim, 2005). Carriage of that bacteria concerns anterior nares, axillae, perineum and vagina. People who have minute non-septic cuts and abrasions of the body, e.g. hands, are also predisposed to be colonized with S. aureus (Gilbert and Humphrey, 1998). S. aureus colonization or septic infection in any part of the body of a food handler finally leads to the carriage of this organism on hands. The source of S. aureus intoxication is any kind of food which has come into contact with a food handlers' hands contaminated with S. aureus, and subsequently not properly stored for longer than 4 h at temperature range 4–60 °C. Such conditions promote massive multiplication (105 colony forming units/g) of S. aureus and subsequent enterotoxins production, which are responsible for characteristic symptoms of staphyloenterotoxicosis.
Clinical characteristics of the staphylococcal food-poisoning (SFP) comprise: acute and rapid onset of symptoms (2–6 h after the consumption of contaminated food), with the most common nausea, vomiting, prostration, sometimes diarrhea (never diarrhea alone), lowered body temperature (never fever) and lowered blood pressure (Gilbert and Humphrey, 1998). Recovery usually takes 1–2 days. The demonstration of each symptom, incubation time, severity and duration of the illness depends on individual characteristic of the human. Sometimes hospitalization for intravenous therapy is needed because of dehydration. Deaths occur rarely and mostly concern infants, the elderly and debilitated people (Do Carmo et al., 2004).
There are 18 serologically distinct staphylococcal enterotoxins (SE) and enterotoxin-like (SEl) toxins of S. aureus known so far. They are designated as A–U according to alphabetical order, excluding F, S and T (Holtfreter and Bröker, 2005). For toxins C, G, I and U several variants have been described (Blaiotta et al., 2004). The genes: seg, sei, selm, seln, selo and optionally selu compose enterotoxin genes cluster (egc) (Jarraud et al., 2001, Letertre et al., 2003). Some authors use names egc2 and egc1 for determining egc with selu and without selu, respectively (Fueyo et al., 2005b, Bania et al., 2006b); we follow this approach in this paper.
All enterotoxins and enterotoxin-like exotoxins of S. aureus share similar properties. They are small simple proteins with similar three-dimensional structures, stable at boiling temperature, resistant to proteolysis, all have superantigenic properties. Those of them which cause emesis after oral administration in laboratory monkeys are called staphyloenterotoxins (SE) and refer to A–I toxins' types. The other related toxins are called enterotoxin-like toxins (SEl), because they do not have emetic properties (SElL, SElK, SElQ) or they have not been tested so far (SElJ, SElM–SElP, SElR, SElU) (Lina et al, 2004). The enterotoxins and enterotoxin-like exotoxins of S. aureus can also be devided into two groups: classical (SEA-E) and new ones (SEG-SElU).
As it has been reported, 15–80% of S. aureus strains isolated from various sources are enterotonoxigenic (Gilbert and Humphrey, 1998, Omoe et al., 2005, Fueyo et al., 2005a, Fueyo et al., 2005b, Bania et al., 2006a, Bania et al., 2006b). Enterotoxinogenic S. aureus have different profiles of se/sel genes and may simultaneously harbour several of them.
The SEs and SEls genes are carried on movable genetic elements such as: phages (SEA, SEE, SElP), plasmids (SED, SElJ, SElR), and pathogenicity islands (SaPIs) (all other SEs, excluding SEH and egc2) (Holtfreter and Bröker, 2005). Most of SaPIs contain several SEs genes, which have a great impact on ease of horizontal transfer and abundance of them among staphylococcal strains.
The serological methods are no longer sufficient enough now for identifying types of SEs produced by S. aureus strains because of costs and antigenic similarities among SEs and SEls which may cause cross-reactions in tests (Edwin et al., 1986). Besides, there have, as yet, not been immunological tests against SEG-U commercially available. Thus PCR method of SE and SEls genes detection in S. aureus strains may be a very useful device for pathogenicity, diagnostic and epidemiologic analyses (Van Belkum, 2003).
The aim of this study was to determine the distribution of se/sel genotypes among S. aureus strains isolated from healthy humans and food samples in conjunction with the probable profile of movable genetic elements, which could carry them. We also tried to assess the frequency of coexistence of se and sel genes.
Section snippets
Bacterial strains and culture media
There were 101 S. aureus strains used for purposes of this work. Thirty of them were isolated from the nasal swabs of healthy humans in the years 2000–2002. Fifty three and 18 strains were isolated from various kinds of food all over Poland during a routine national survey of food in years 2004–2005 and in the 1960s and 1970s, respectively. All strains were identified as S. aureus according to standard microbiological procedures (Kloos and Bannerman, 1999).
Stock cultures of the strains were
Results and discussion
We screened three groups of isolates: 53 recovered from food samples collected in years 2004–2005, 18 — from food samples collected in the 1960s and 1970s and 30 — from nasal carrier samples collected in the years 2000–2002 for the presence of se/sel genes by PCR method.
As far as the isolates from nasal carriers and food samples collected in the years 2004–2005 were concerned, we obtained similar results to the ones described by Bania et al., 2006a, Bania et al., 2006b, Fueyo et al. (2005b) and
Acknowledgments
We are very grateful to Dr. J. Bania for the help and a lot of valuable information at this work, and Dr. G. Lina for providing us with the S. aureus reference strains used in this study.
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