Elsevier

Food Microbiology

Volume 93, February 2021, 103602
Food Microbiology

Molecular interaction between methicillin-resistant Staphylococcus aureus (MRSA) and chicken breast reveals enhancement of pathogenesis and toxicity for food-borne outbreak

https://doi.org/10.1016/j.fm.2020.103602Get rights and content

Highlights

  • Clinical MRSA can be transmitted to humans via contaminated food for infection.

  • S. aureus FORC_062 is a clinical MRSA with a mobile gene elements for pathogenesis.

  • RNA-Seq analysis with chicken breast revealed amino acid utilization from the chicken.

  • The chicken breast enhanced virulence factors of MRSA for more fatal human infections.

Abstract

To study pathogenesis and toxicity of Staphylococcus aureus in foods, FORC_062 was isolated from a human blood sample and complete genome sequence has a type II SCCmec gene cluster and a type II toxin-antitoxin system, indicating an MRSA strain. Its mobile gene elements has many pathogenic genes involved in host infection, biofilm formation, and various enterotoxin and hemolysin genes. Clinical MRSA is often found in animal foods and ingestion of MRSA-contaminated foods causes human infection. Therefore, it is very important to understand the role of contaminated foods. To elucidate the interaction between clinical MRSA FORC_062 and raw chicken breast, transcriptome analysis was conducted, showing that gene expressions of amino acid biosynthesis and metabolism were specifically down-regulated, suggesting that the strain may import and utilize amino acids from the chicken breast, but not able to synthesize them. However, toxin gene expressions were up-regulated, suggesting that human infection of S. aureus via contaminated food may be more fatal. In addition, the contaminated foods enhance multiple-antibiotic resistance activities and virulence factors in this clinical MRSA. Consequently, MRSA-contaminated food may play a role as a nutritional reservoir as well as in enhancing factor for pathogenesis and toxicity of clinical MRSA for severe food-borne outbreaks.

Introduction

Staphylococcus aureus is a well-known pathogen for human infection via cross contamination in hospitals or communities, causing cutaneous lesions, pneumonia, osteomyelitis, toxic shock syndrome, nausea, violent vomiting, and diarrhea (Lowy, 1998). Due to these virulence factors, clinical outbreaks were reported with 119,000 infections and almost 20,000 deaths in 2017 (Kavanagh, 2019). In addition, Staphylococcal food poisoning caused about 241,000 illnesses per year in the United States by the consumption of contaminated animal foods (Zeaki et al., 2019). In particular, it was reported that methicillin-resistant S. aureus (MRSA) strains are responsible for approximately 44% of cases and over 20% of excess mortality. Furthermore, the MRSA strains account for a proportion of more than 20% among Staphylococcus aureus isolates causing infections (Di Ruscio et al., 2019). Due to its pathogenicity and antibiotic resistance activity, understanding of its virulence factors at the genome level and the transcriptome level is urgently required to control the outbreaks caused by the pathogen (Baba et al., 2008).

Virulence factors of S. aureus are associated with adherence and invasion to a host cell surface, immune evasion system, type VII secretion system (T7SS), hemolysis, enterotoxin production, antibiotic resistance, and a toxin-antitoxin (TA) system. Host cell adherence to membrane glycoproteins and its invasion via host membrane by host tissue damage play a role in penetration to a host cell surface for bacterial infection, which is important as the first step in host cell infection (Foster et al., 2014; Priest et al., 2012). S. aureus has a specific immune evasion system to thwart the neutrophil in various ways and to produce capsular polysaccharides for efficient disruption of a host primary defense system (Foster, 2005; Nanra et al., 2013; van Kessel et al., 2014). In addition, T7SS is a complex protein system for secretion of several virulence factors including hemolysin, enterotoxin, and even antibiotic resistance proteins, which may be associated with promoting bacterial survival and the long-term persistence of staphylococcal abscess communities (Cao et al., 2016).

Hemolysin of S. aureus consists of three subunits: alpha (α), beta (β) and delta (δ) hemolysin proteins (Kielian et al., 2001). The α-hemolysin has an activity for pore formation in the blood cell membrane and δ-hemolysin for membrane lysis, associated with the formation of a spheroplast or a protoplast of blood cells (Bhakdi and Tranum-Jensen, 1991; Bhakoo et al., 1982; Freer and Birkbeck, 1982; Husmann et al., 2009). β-Hemolysin disrupts the formation by lysis activity (Glenny and Stevens, 1935; Huseby et al., 2007; Projan et al., 1989).

Recently, 17 types of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ and SEU) were identified (Aydin et al., 2011). It has been suggested that they may have the biological effects of superantigens, causing toxic shock syndrome by initiating the activation and proliferation of T cells, pyrogenicity, enhancement of lethal endotoxin shock, and induction of inflammatory cytokines (Bohach et al., 1990; Marrack and Kappler, 1990; Miethke et al., 1992). In particular, SEA has been known to be associated with food poisoning causing gastroenteric syndrome in humans (Balaban and Rasooly, 2000; Letertre et al., 2003).

The staphylococcal cassette chromosome mec (SCCmec) is a mobile genetic element specific for Staphylococcus, associated with methicillin resistance activity. SCCmec contains two essential components, methicillin-resistant gene (mecA) complex and cassette chromosome recombinase (ccr) gene complex (Sani et al., 2014). The mec gene complex consists of mecA gene encoding a penicillin binding protein 2 A (PBP2A) with low affinity to beta-lactam antibiotics and regulatory genes. This low binding affinity to antibiotics endows the host with its antibiotic resistance activity by protection from the inhibition of cell wall synthesis, substantiating that the mecA gene is responsible for staphylococci resistant to penicillin-like antibiotics (Fogarty et al., 2015). The ccr gene complex (ccrC or the pair of ccrA and ccrB) encoding recombinases provides the mobility of a SCCmec genetic element on the host chromosome via excision, integration, and ligation (Huda et al., 2017). According to the combination of mec gene complex and ccr gene complex, SCCmec genetic element could be classified into types I to VIII (Kennedy and DeLeo, 2009; Zhang et al., 2009).

To further understand the virulence, pathogenesis, and antibiotic resistance of S. aureus, their genomes have been sequenced and their functionalities were analyzed according to the development of next-generation sequencing (NGS) technology (Durand et al., 2018; Nair et al., 2011; Zubair et al., 2015). To date (Sep 2019), complete genome sequences of 443 S. aureus strains are available in the GenBank database. Recently, the genome of S. aureus MCRF184 was completely sequenced and analyzed with bioinformatics, revealing that this genome has an enterotoxin gene cluster, a superantigen/hemolysin gene cluster, an immune evasion gene cluster, and a putative antimicrobial resistance gene cluster, regarding its pathogenesis and antibiotic resistance, but not a SCCmec genetic element (Aswani et al., 2019). This putative antimicrobial gene cluster consists of a type III restriction-modification (RM) system, efflux pump, acetyltrasferase, regulators, and mobile elements, probably instead of a SCCmec cluster for antibiotic resistance. Therefore, this S. aureus genome study is important to extend our knowledge on S. aureus virulence and pathogenesis activities for control of this pathogen.

According to the sources of MRSA, it categorized into three groups: healthcare-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA), and livestock-associated MRSA (LA-MRSA) (Abolghait et al., 2020). HA-MRSA has been well-known human pathogen for infections in the hospitals. Because of its various antibiotic resistance, clinical treatment of HA-MRSA has been serious issues in the patients. Although it is a primary MRSA for human infections, it has decreased for the last decade, probably due to development of healthcare technology. However, CA-MRSA and LA-MRSA have increased every year. Therefore, clinical MRSA human infection via ingestion of the contaminated foods is probably getting worse and it is one of the major safety topics for human infections (Kluytmans, 2010). In particular, MRSA is generally transmitted to humans via various contaminated food, but it is not clearly understood the role of the MRSA for survival, propagation and even toxicity of clinical MRSA in the food environments, even though those properties of HA-MRSA has been widely studied for human infections (Sergelidis and Angelidis, 2017). In addition to clinical MRSA infection to human in hospitals, it has been often detected in contaminated chicken, indicating that clinical MRSA propagation and infection to human via food, which could be a threat to food safety and public health (Fox et al., 2017; Hennekinne et al., 2012). Therefore, it is necessary to understand the interaction between clinical MRSA and the food environment to control food poisoning. Due to the development of NGS technology and the accumulation of S. aureus genome information, transcriptome analysis has recently been available to extend our understanding of their interaction for food poisoning at the genomic level. The most recent paper reported that S. aureus increased utilization activities of amino acids and sugars from chicken breast, comparing with Luria-Bertani medium, suggesting that S. aureus can propagate in specific food as a nutrient reservoir and then could cause food poisoning via enterotoxin production after ingestion of the contaminated chicken breast (Dupre et al., 2019). Therefore, understanding of the behavior of S. aureus when exposed to raw chicken breast may be important to elucidate the survival and pathogenesis of S. aureus in frequently contaminated raw chicken for food safety.

In this study, a clinical isolate, S. aureus FORC_062, was isolated from an infected patient's blood sample. To understand its pathogenesis and antibiotic resistance at the genomic level, its genome was completely sequenced and compared with other S. aureus genomes, and its genome functionality was analyzed using bioinformatics tools. In addition, transcriptome analysis of FORC_062 with raw chicken breast was performed to elucidate its interaction and adaptation with a frequently contaminated food sample for survival and pathogenesis in the food as a reservoir or carrier for propagation. This result would be useful for providing a novel insight into pathogenic characteristics of S. aureus and the development of a new regulation approach for food safety.

Section snippets

Strain isolation, growth conditions, and identification

S. aureus FORC_062 was obtained from Samsung Medical Center (Seoul, South Korea) and the strain was designated as FORC_062 by its deposition to the culture collection of the Food-borne Pathogen Omics Research Center (FORC). The methicillin-resistant S. aureus (MRSA) N315 and methicillin-sensitive S. aureus (MSSA) ATCC 29213 were obtained from a culture collection of the Department of Food Science and Biotechnology, Seoul National University (Seoul, South Korea). They were cultivated at 37 °C

General characteristics and genome properties of S. aureus FORC_062

FORC_062 was isolated from human patient blood and identified as S. aureus by 16 S rRNA sequence analysis (data not shown). In addition, PCR was performed with mecA and nuc gene-targeting primer sets (Fang and Hedin, 2003; Mehrotra et al., 2000), showing that this strain has methicillin-resistant Staphylococcus aureus (MRSA) specific genes (Fig. S1). A subsequent antibiotic screening test with methicillin resistance (oxacillin resistance) confirmed its methicillin resistance activity, following

Discussion

So far, pathogenesis and toxicity of S. aureus as a clinical or food-borne pathogen have been performed in vitro in the laboratories. Although hundreds of complete or draft genome sequences of S. aureus are available in public databases, its molecular mechanisms for infection, pathogenicity, and cytotoxicity remain unknown. According to the rapid development of next-generation sequencing (NGS) technologies and their broad applications, it is now possible to extend our knowledge on its host

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work was supported by the Ministry of Food and Drug Safety, South Korea (19162MFDS037 to JL), and by the National Research Foundation of Korea funded by the Ministry of Science, ICT, and Future Planning (2017R1E1A1A01074639 to SC) in 2019.

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