Staphylococcus aureus ST6-t701 Isolates from Food-Poisoning Outbreaks (2006–2013) in Xi'an,China

Introduction

Staphylococcal food poisoning (SFP) is one of the most common foodborne diseases. It is often caused by the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. In China, it is estimated that approximately 20–25% of foodborne bacterial outbreaks are caused by S. aureus, which ranked just near Salmonella and Vibrio parahaemolyticus among the list of important foodborne pathogens (Liu et al., 2006).

In recent years, an increasing number of foodborne poisonings with S. aureus multilocus sequence type (ST) 6 and/or spa type (t701) have been reported in Shenzhen and Ma'anshan, China (Wang et al., 2011; Yan et al., 2012), and Turin, Italy (Gallina et al., 2013), Korea (Cha et al., 2006; Hyeon et al., 2013), and Japan (Sato'o et al., 2014; Suzuki et al., 2014). We described five foodborne poisoning outbreaks with S. aureus ST6-t701 in the Xi'an city of China during an 8-year period (2006–2013). Two foodborne poisoning outbreaks associated with ST5-t002 and ST59-t172 were also identified. However, very little is known about the characteristic of S. aureus ST6-t701 isolates collected from food-poisoning outbreaks. The aim of this study was to characterize these strains by determining antimicrobial susceptibility, resistance, and toxin genes.

Materials and Methods

Results

As shown in Figure 1, three sequence types (STs), namely, ST6 (71.4%, 5/7), ST59 (14.3%, 1/7), and ST5 (14.3%, 1/7) were found by MLST, and spa typing of all of the isolates yielded three spa types: t701 (71.4%, 5/7), t002 (14.3%, 1/7), and t172 (14.3%, 1/7). Moreover, the result of PFGE confirmed that seven different patterns were found in this study. In addition, we found that all isolates were mecA negative as confirmed by PCR.

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FIG. 1.

Molecular characterization (PFGE dendrogram, agr type, toxin and resistance gene profiles, MLST, spa type) and antimicrobial resistance patterns of Staphylococcus aureus isolates from seven outbreaks. PFGE, pulsed-field gel electrophoresis; MLST, multilocus sequence typing; TMP, trimethoprim; ERY, erythromycin; TET, tetracycline; CD, clindamycin; PEN, penicillin; AMP, ampicillin.

Of S. aureus isolates, resistance was most frequently observed to TMP and PEN (71.4%, 5/7 each), followed by ERY (28.6%, 2/7), AMP, CD, and TET (14.3%, 1/7 each). All strains were susceptible to CHL, FOX, OXA, AMK, and VAN. Four resistance profiles were identified: TMP-PEN (28.6%, 2/7), TMP-AMP-PEN, TMP-ERY-PEN, and TMP-ERY-CD-TET-PEN (14.3%, 1/7 each). Resistance to lincosamides was conferred by the linA/linA′ (100%, 7/7), ermC, ermT, and ermB (14.3%, 1/7 each). Resistance to AMP-PEN was mainly mediated by blaZ (85.7%, 6/7). Tetracycline resistance was encoded by tet(K) genes in 1 (14.3%, 1/7) isolate. All strains were negative for aac(6′)/aph(2″), aph(3′)-IIIa, ant(4′)-Ia, tet(L), tet(M), tet(O), ermA, msrA, msrB, fexA, cat::pC194, cat::pC221, cat::pC223, CatpIp-501, dfrD, dfrK, dfrG, dfrS, and cfr (Table 1).

Of S. aureus isolates, the most frequently identified exotoxin gene was seu, lukED, hla, hlb, hld (100%, 7/7 each), followed by hlg and hlgv (85.7%, 6/7 each), lukPV (71.4%, 5/7), sea (57.1%, 4/7), see (42.9%, 3/7 each), and etd (28.6%, 2/7). Each gene of seb, sec, sed, sej, and sek was detected in a single strain. No isolates harbored seg, seh, sei, sel, sem, sen, seo, sep, seq, ser, lukM, eta, etb, and tst. For the agr locus, only agrI (42.9%, 3/7) was found (Table 1).

Discussion

SFP is one of the most prevalent causes of foodborne intoxication worldwide. Genetic aspects of those isolates from SFP outbreaks may differ in different countries. For example, ST1 in South Korea (Cha et al., 2006) and ST81 in Japan (Sato'o et al., 2014; Suzuki et al., 2014) are the most epidemic clone of SFP outbreaks. This may be due to the differences in food consumption and eating habits (Cha et al., 2006). In this study, S. aureus ST6-t701 is the predominant S. aureus lineage isolated from outbreaks, which agreed with previous outbreaks in Shenzhen and Ma'anshan, China (Wang et al., 2011; Yan et al., 2012). However, S. aureus ST6 and/or t701 are not the predominant S. aureus lineage that caused illness in hospitals and animals in China (Yan et al., 2012). Why S. aureus ST6 and/or t701 became the dominant molecular typing that causes SFP and whether ST6 and/or t701 was the predominant molecular typing in food products or among SFP isolates in China should be further investigated (Yan et al., 2012).

In this study, resistance to TMP and PEN was most frequently observed, which differed from the report by Yan et al. (2012), showing that resistance to PEN and TET was common (96.2% and 28.8%, respectively) among S. aureus isolates from SFP outbreaks. Folate pathway inhibitors are commonly used as first-line treatment options to cure infection caused by community-associated methicillin-resistant S. aureus, which has emerged as the predominant cause of skin infections (Frei et al., 2010). This result suggested that TMP-resistant S. aureus from S. aureus foodborne disease cases could constitute a potential risk to consumers. In addition, the linA/linA′, ermC, ermT, and ermB, blaZ, tet(K) genes were commonly detected in these isolates. It is demonstrated that S. aureus associated with SFP outbreaks exhibited multidrug resistance and contained some resistance genes. This confirmed the results by Yan et al. (2012) and Hyeon et al. (2013).

Staphylococcal enterotoxin A, associated with other staphylococcal enterotoxins, is most frequently involved in food-poisoning outbreaks (Cha et al., 2006; Kérouanton et al., 2007; Yan et al., 2012; Johler et al., 2013), which was in agreement with our study. Our results show that the sea gene is often associated with seu in S. aureus strains linked to SFP outbreaks in Xi'an, China, which disagreed with Kérouanton et al. (2007) and Cha et al. (2006). They both showed that the sea gene is often associated with seh in S. aureus stains linked to SFP outbreaks. In addition, seu, lukED, hla, hlb, and hld genes were the most frequently detected in these isolates. Those results demonstrated that S. aureus associated with SFP outbreaks harbored several important virulence factors.

In conclusion, S. aureus ST6-t701 isolates were detected at a high prevalence (71.4%) in the outbreaks in Xi'an, China. The majority of the isolates exhibited multidrug resistance and harbored multiple toxin and resistance genes. Therefore, good manufacturing practice, as well as hazard analysis and critical control points systems should be taken in order to avoid the contamination of food by S. aureus and toxin production in foods. Further studies are required to elucidate the possible role of S. aureus ST6-t701 strains as a source of SPF.