The prevalence of antimicrobial resistance and carriage of virulence genes in Staphylococcus aureusisolated from food handlers in Kuwait City restaurants

Staphylococcus aureus can cause localized and invasive infections in humans. This is attributed to its ability to produce a variety of virulence factors such as capsular polysaccharides, staphylococcal enterotoxins (SEs), toxic shock syndrome toxin 1 (TSST-1) [1, 2], panton – valentine leukocidin (PVL) [3] and accessory gene regulators (agr) [1, 4–6]. Although S. aureus isolates produce one of 11 serotypes of capsular polysaccharides, most clinical isolates belong to serotypes 5 and 8 [2]. Enterotoxin-producing S. aureus are common causes of food poisoning in many parts of the world. The ingestion of the preformed toxins in food often leads to the development of food poisoning. The symptoms typically have a rapid onset (2–6 h) and may include nausea, vomiting, diarrhea and abdominal pain [1]. Nasal and hand carriage of enterotoxigenic S. aureus by food handlers is an important source of staphylococcal food contamination in restaurants and fast food outlets [7]. Therefore it is important to detect S. aureus carriage among food handlers to prevent possible food contamination by them resulting in food poisoning.

Food poisoning outbreaks result in huge financial losses to restaurants, in addition to the loss of reputation and confidence among the public. Staphylococcal food-borne diseases are estimated to cause 6 – 81 million illnesses and up to 9000 deaths, and accounts for 14–20% of outbreaks involving contaminated food in the USA [8].

Most of the studies on S. aureus associated with food poisoning have focused on screening of the isolates for enterotoxins [1, 9–13] with only sparse data on the carriage of other virulence factors and antimicrobial resistance among S. aureus obtained from food handlers [14–17] especially in the Arabian Gulf countries. Although we previously studied the prevalence of staphylococcal enterotoxins in S. aureus isolated from food handlers in Kuwait city [10], their susceptibility to antibacterial agents were not investigated. Therefore in the present study, we characterized S. aureus isolated from food handlers in Kuwait City restaurants for their susceptibility to antibacterial agents and the carriage of virulence associated genes.

One hundred and eighty five (92.5%) of the 200 S. aureus isolates were resistant to antibacterial agents. The majority, 164 (82.0%) and 136 (68.0%) of them were resistant to penicillin G and cadmium acetate respectively. Forty-two isolates (21.0%) were resistant to tetracycline, 15 isolates (7.5%) were resistant to trimethoprim and six isolates (3%) were resistant to kanamycin. Five and four isolates were resistant to erythromycin and streptomycin respectively. Two isolates were resistant to fusidic acid and only one isolate was resistant to methicillin and gentamicin. They were all susceptible to mupirocin, rifampicin, vancomycin, teicoplanin and linezolid. Sixty-five percent of them were resistant to two or more antibacterial agents and 23% were multiresistant with resistance to three or more classes of antibacterial agents.

The distribution of genes for SE, TSST-1 and PVL is summarized in Figure 1 and Table 2. In total, 142 (71.0%) isolates were positive for SE, 8 (4.0%) were positive for TSST-1 and, 18 isolates, including the only MRSA isolate, were positive for PVL. Gene for SEI was the most common SE gene. It was detected in 77 isolates. Fifty isolates contained single SE genes, 54 isolates carried two SE genes, 28 and 10 isolates contained three and four SE genes respectively.

The characteristics of the 18 lukS-lukF positive isolates are presented in Table 3. They had different resistance patterns, belonged to different agr and cap types and harbored different SE genes but none carried gene for TSST-1. When typed by PFGE to determine if they were related, they yielded eight PFGE patterns designated types A to H (Figure 2, Table 3). Eight isolates obtained from stool, hand and nasal samples had the same PFGE pattern (type A) and 10 isolates belonged to seven PFGE patterns.

When the isolates obtained during the screening exercise (screening samples) were compared with those obtained during routine investigation of food poisoning cases (routine samples), the results were not significantly different for the distribution of genes for agr and capsular polysaccharides (data not shown). Similarly, 72.9% of the screening sample isolates (97/133) and 67.1% (45/67) of the routine samples isolates contained genes for SEs showing no significant difference in the distribution of genes for SE between the two sets of isolates (P value = 0.50). Furthermore tst was detected in 5.2% (7/133) of the screening samples isolates and in 1.5% (1/67) of the routine sample isolates (P value = 0.4)

This study has demonstrated a high prevalence of antibacterial resistance and diversity in the carriage of virulence genes in S. aureus obtained from food handlers employed in restaurants in Kuwait City. Our results that 92.5% of the isolates expressed resistance to antibacterial agents is similar to the prevalence of antibacterial resistance in methicillin-susceptible S. aureus obtained from patients in Kuwait hospitals [24] and to that found in S. aureus isolates from food handlers in Chile [11] and Botswana [17]. Although only one isolate was methicillin-resistant, the finding is significant because food handlers carrying MRSA had initiated outbreaks in hospitals in the Netherlands [14], USA [25] and Brazil [15]. As food handlers represent a section of the healthy population in the community, besides working in restaurants, the detection of high prevalence of antibiotic resistance in S. aureus isolated from them also highlights the growing problem of antibiotic resistance in the community.

The ability of S. aureus to colonize or infect its host is related to its ability to express virulence factors that facilitate their adherence to surfaces, cause damage or its ability to evade host's immune system [1, 2]. Our isolates carried genes for a range of virulence factors including three of the four accessory gene regulators that regulate staphylococcal virulence factors [6]. Our result that 50.5% of the isolates were agr type I concurs with results of other studies that the majority of S. aureus from clinical sources belong to agr type I [4, 23]. Similarly, our results that 57% of the isolates belonged to serotype 8 and 38% belonged to serotype 5 are in agreement with previous studies that showed that the majority of S. aureus obtained from clinical samples belonged to capsular polysaccharide serotypes 5 or 8 [2, 23].

With regards to the carriage of SE determinants, the results of this study differ from that of a previous study in Kuwait [10]. The 71.0% prevalence of SE genes in this study was lower than the 86.6% prevalence detected previously in S. aureus from food handlers in Kuwait restaurants [10]. However, it was similar to the prevalence of 74.1% obtained from S. aureus isolated from food poisoning cases in Taiwan [26] and higher than the prevalence of SE reported in S. aureus from food handlers in Chile [19%, [11]] and from humans, food and animal sources in Malaysia [20.8%, [27]]. In addition, the demonstration of SEI as the most common SE gene in this study contrasted with the results of a previous study of SE in S. aureus isolated from food handlers in Kuwait restaurants [10] and other studies [9, 11, 12] where SEA was the most common SE. Furthermore, whereas 64.7% of the SE positive isolates in this study carried genes for two to four SEs, only 8.6% of S. aureus in the previous study from Kuwait carried more than one SE gene [10]. This is consistent with recent reports in the literature that also document the carriage of multiple SE gene in enterotoxigenic S. aureus [9, 12, 13]. The detection of multiple SE genes in recent S. aureus isolates could be due to the improvement in detection methods following the application of PCR technology and the discovery of new SE genes which were not tested in the previous study. It could also be due to frequent horizontal transmission of phage-mediated toxin genes among the staphylococcal populations [21].

PVL is widely distributed among some clones of community-associated MRSA (CA-MRSA) isolated in Kuwait and other countries [3, 28, 29] and was thought to contribute to the increased virulence of CA-MRSA isolates [3, 28]. However, PVL has recently also been detected in healthcare-associated MRSA and in methicillin-susceptible S. aureus from patients [29]. Our report is the first in S. aureus obtained from food handlers in Kuwait and elsewhere. Its detection in 9.0% of the isolates in this study supports the suggestion that PVL-bearing phages are widespread among S. aureus of different genetic backgrounds and are not a specific characteristic of CA-MRSA [19, 29]. The observation that eight of the 18 lukS-lukF positive isolates had same PFGE patterns suggested a transmission of a common strain among these workers. The detection of genes for PVL in S. aureus obtained from food handlers is of public health interest since these food handlers can serve as sources of transmission of PVL producing S. aureus in the community especially among family members.

Given that the majority of the isolates in this study contained genes for an array of virulence factors, they are potential causes of serious S. aureus infections. Therefore food handlers carrying these strains can contaminate food that can lead to food poisoning [7]. Thus, our results support the current practice of screening restaurant workers to identify S. aureus carriers and referring them to the appropriate health authorities for decolonization. Although the S. aureus carriage rate and their antibacterial resistance in this study may be similar to those found among healthy individuals in the general population [30], it is important to decontaminate food handlers carrying S. aureus because their exposure to, and possible contamination of food prepared and served in restaurants is a public health concern.

This study has provided updated data on the carriage of SE and other virulence genes, and initial information on the prevalence of antibacterial resistance in S. aureus obtained from food handlers in restaurants in Kuwait and in the Gulf region. Our results should contribute to better management of S. aureus carriers among the food handlers and enhance the safety of restaurant customers.