Phenotypic and genotypic characterization of PVL-positive Staphylococcus aureus isolated from retail foods in China

https://doi.org/10.1016/j.ijfoodmicro.2019.05.021Get rights and content

Highlights

  • Food-related S. aureus in China had detected PVL gene.

  • The PVL-positive S. aureus isolates covering different food types and cities.

  • The presence of different isolates harbouring important resistance and virulence determinants.

  • The dominant type of PVL-positive MRSA isolates belonged to CC59-t437, which is the predominant type of CA-MRSA in China.

Abstract

Staphylococcus aureus encodes numerous toxins that are known or strongly suspected to cause specific diseases or symptoms. Panton-Valentine leukocidin (PVL) is one of these important toxins that is associated with high mortality rates. In our previous study, 1581 S. aureus strains were isolated from 4300 samples of retail foods obtained from most of the provincial capitals in China from 2011 to 2016. This study aimed to investigate the prevalence of PVL-positive S. aureus isolates from retail foods in China and characterize these isolates by antibiotic resistance testing, spa typing, multilocus sequence typing (MLST) and enterotoxin gene analyses. In total, seventy-two isolates (72/1581, 4.6%) possessed pvl genes, including 24.1% MRSA isolates (26/108) and 3.1% MSSA isolates (46/1473), covering different types of food. The strains were divided into seventeen sequence types (STs) and twenty-seven spa types, and 43.1% (31/72) of the PVL-positive S. aureus isolates belonged to CC59-t437. These isolates contained at least one of the following enterotoxin genes: sei (97.2%), sem (86.1%), seq (80.6%), seg (68.1%), sek (68.1%), seb (62.5%), sel (52.8%), sej (50.0%), seh (48.6%), sep (45.8%), sea (38.9%), ser (37.5%), sen (27.8%), sec (16.7%), see (16.7%), sed (6.9%), seo (6.9%) and seu (6.7%). A total of 87.5% of the S. aureus isolates (63/72) harboured the classic SE genes (sea, seb, sec, sed, and see), whereas all the S. aureus isolates harboured the genes of the egc cluster (seg, sei, sem, sen, seo, and seu). In antimicrobial susceptibility tests, 98.6% of the isolates (71/72) exhibited resistance to at least one antibiotic, including 47 multi-drug-resistant isolates. Resistance to penicillin (94.4%), erythromycin (83.4%), clindamycin (63.9%), kanamycin (61.1%), telithromycin (58.3%), streptomycin (51.4%), tetracycline (47.2%), chloramphenicol (27.8%), fusidic acid (27.8%) and other antibiotics (<20%) was observed. All the PVL-positive MRSA isolates belonged to CC59-t437, which is the predominant type of community-associated (CA)-MRSA in China. The presence of these isolates in food represents a potential health risk for consumers and warrants further attention.

Introduction

Staphylococcus aureus is a bacterial pathogen that can infect humans, animals, plants and contaminate foods that is responsible for a wide variety of diseases. Despite the ubiquitous distribution of S. aureus in nature, food remains the most important source of infection, and staphylococcal food poisoning is reported to be the third most prevalent cause of foodborne illness worldwide (Aydin et al., 2011). Every year, approximately 241,000 foodborne illnesses are caused in the United States by S. aureus (Scallan et al., 2011). In China, it is estimated that approximately 20–25% of foodborne bacterial outbreaks are caused by S. aureus, which ranks just next to Salmonella and Vibrio parahaemolyticus in the list of important foodborne pathogens (Wang et al., 2013).

As an important pathogen, S. aureus encompasses a spectrum of strains with varying virulence and pathogenicity. Differentiation between virulent and non-virulent strains is important for evaluation of the potential implications of the presence of this microorganism for food safety and public health. There are many putative virulence markers in S. aureus, such as staphylococcal enterotoxins (SEs), exfoliatins, toxic shock syndrome toxin (TSST), haemolysins, and Panton-Valentine leukocidin (PVL) (Grumann et al., 2014). Among these virulence markers, PVL is a bacteriophage-encoded bicomponent leukotoxin that is present in some strains of S. aureus and plays a key role in leukocytolysis and tissue necrosis (Shallcross et al., 2013). This toxin belongs to a family of synergohymenotropic toxins that consist of two non-associated components that act synergistically on cell membranes. PVL is encoded by the lukS-PV and lukF-PV bacteriophage-transmitted genes, the detection of which has been used in epidemiological studies to detect and determine the prevalence of PVL-positive S. aureus (Melles et al., 2006). Nowadays, many studies have reported an association between PVL genes and invasive disease (Ellis et al., 2004; Gillet et al., 2002; Department of Health and Human Services, Centers for Disease Control and Prevention, 1999; Kaplan, 2006; Lee et al., 2005). In fact, the risk of death associated with PVL-positive S. aureus has been reported to be higher than that associated with non-PVL-producing S. aureus (Gillet et al., 2002).

In recent years, antibiotic-resistant S. aureus clones have rapidly emerged. Isolation of single- or multiple-drug resistant S. aureus strains from food, the environment and clinics has been continuously reported (Gould et al., 2012; Rasigade et al., 2014). Methicillin-resistant S. aureus (MRSA) is one of most important types of multi-drug-resistant S. aureus and has spread globally in recent years. As we know, MRSA can be resistant to several antibiotics and leading causes of death due to infection by any single infectious agent (Deleo et al., 2010). Distinct epidemiological groups of MRSA have been identified, such as hospital-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). By contrast, CA-MRSA has been reported to carry the loci for PVL at high frequency (Dufour, 2002). In addition to CA-MRSA, PVL is also carried by some methicillin-susceptible S. aureus (MSSA) strains, which exhibit similar disease potentials and epidemiological characteristics to those of MRSA (Rasigade et al., 2010). In some countries, such as the UK, PVL-positive MSSAs are more common than MRSA in community-acquired infections (Holmes et al., 2005).

Globally, the reported incidence of PVL-positive S. aureus is variable, and the presence of this pathogen is strongly dependent on strain types/lineages as well as geographical differences. There have been a limited number of studies reported on PVL-positive S. aureus isolated from retail food. Previously, to study S. aureus present in retail foods in China from July 2011 to June 2016, we collected 4300 retail food samples from supermarkets, fairs and farmers' markets, covering most of the provincial capitals of China, and isolated 1581 S. aureus strains from 1063 positive samples from all the sampling sites (Wu et al., 2018a; Wu et al., 2018b). In order to further understand these food related S. aureus isolates in China, this study was to identify PVL-positive S. aureus isolates from our previous study and to characterize these isolates by phenotyping (antimicrobial susceptibility testing) and genotyping (enterotoxin gene typing, multi-locus sequence typing (MLST) and spa typing) methods to understand the genetic background of food-related PVL-positive S. aureus in China.

Section snippets

Bacterial isolates

A total of 1581 S. aureus isolates, including 108 MRSA isolates and 1473 MSSA isolates, collected from retail food samples in 39 Chinese cities were analysed, comprising 469 isolates from meat and meat products (bacon/sausage, poultry, pork, mutton and beef), 511 isolates from aquatic products (freshwater fish, shrimp and seafood), 368 isolates from quick-frozen products (frozen dumplings/steamed stuffed buns and frozen meat), 148 isolates from ready-to-eat (RTE) food, 13 isolates from

Results

Overall, of the 1581 S. aureus isolates from retail foods in China, 72 (4.6%) isolates tested positive for the pvl gene, including 20 isolates (20/469, 4.3%) from raw meat, 22 isolates (22/511, 4.3%) from aquatic products, 17 isolates (17/368, 4.6%) from quick-frozen food, 7 isolates (7/148, 4.7%) from RTE food, 5 isolates (5/42, 11.9%) from edible mushrooms and one isolate (1/30, 3.3%) from vegetables, whereas all the isolates from pasteurized milk lacked the pvl gene (Table 1). Twenty-six

Discussion

Recent years, PVL-positive S. aureus has received a significant amount of attention which can cause highly necrotizing pneumonia, necrotizing dermatitis, and other primary diseases in humans (Shallcross et al., 2013). Approximately, the mortality of PVL-positive S. aureus infection were ranges from 40% to 60% (Lina et al., 1999). PVL-positive S. aureus frequently detected in clinical (Asiimwe et al., 2017; Brown et al., 2011; Hewagama et al., 2016; Krziwanek et al., 2007; Monecke et al., 2014;

Conclusion

In conclusion, this study shows the existence of PVL-positive S. aureus in retail food and the presence of different S. aureus strains harbouring important resistance and virulence determinants. The molecular epidemiological study conducted herein showed the relationships between these food-related PVL-positive S. aureus strains and hospital-acquired isolates. Notably, 24.1% of the MRSA isolates were PVL positive and belonged to the predominant type of CA-MRSA (CC59-t437) in China. Our study is

Funding

This work was supported by the National Natural Science Foundation of China (31801657), China Postdoctoral Science Foundation (2017M612623), and GDAS' GDAS' Special Project of Science and Technology Development (2019GDASYL-0201001).

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