Project Abstract

<p> </p><div><p>               <b><i>ABSTRACT</i></b><br></p><p><em>Staphylococcus aureus</em>&nbsp;is one of the most important food-borne pathogens globally. It produces various toxins and invasive enzymes and can be found in numerous food products. Milk is an important source of staphylococcal food poisoning. After pasteurization, this microorganism or its enterotoxins might still remain in pasteurized milk. Therefore, this study was to investigate the contamination of <em>S. aureus</em>&nbsp;in 258 pasteurized milk from 39 cities of China. The prevalence and levels of <em>S. aureus</em>&nbsp;in these samples as well as antibiotic susceptibility profiles, virulence genes, biofilm formation, and biofilm related genes, <em>spa</em>&nbsp;typing and MLST were used to determine the characterization among the isolates. It was found 3.9% of samples were detected <em>S. aureus</em>&nbsp;in 8 of 39 cities in China. The contaminated level were not very excessive which showed the MPN values of the most positive samples (9/10) were less than 1 MPN/g. All pasteurized milk-related <em>S. aureus</em>&nbsp;isolates have ability to produce biofilm and harbored <em>icaA, icaD, eno</em>, <em>clfA, clfB, fnbA, fnbB, fib</em>&nbsp;genes, other biofilm related genes <em>icaC</em>&nbsp;were showed in 91.7% of isolates and <em>cna</em>&nbsp;gene were showed in 50%, except <em>bap</em>&nbsp;gene which were free in all isolates. The antibiotic susceptibility test showed that all isolates were resistant or intermediate-resistant to different concentrations of the antibiotics. Furthermore, 75.0% of the isolates were resistant to three or more antibiotic classes, which indicated multidrug resistance. The isolates had virulence potential, which showed 66.7% (8/12) of the isolates carried one or more virulence-associated genes. Molecular typing by MLST and <em>spa</em>&nbsp;typing enabled classification of these isolates into a total of 11 sequence types (STs) and <em>spa</em>&nbsp;types, which indicated high genetic diversity. Most of these types were related to various clinical <em>S. aureus</em>&nbsp;infections. Thus, the findings of this study reflect the potential risk of <em>S. aureus</em>&nbsp;infection in China. Our study also provides comprehensive analysis of the prevalence of <em>S. aureus</em>&nbsp;in pasteurized milk and helps ensure more accurate treatment of human infection with effective antibiotics.</p></div><div><strong>Keywords </strong><em>Staphylococcus aureus</em>, pasteurized milk, risk assessment, virulence genes, antibiotic resistance, biofilm formation, MLST, <em>spa</em>&nbsp;typing</div> <br><p></p>

Project Overview

<p> </p><div><p><em><b>1.1 INTRODUCTION</b></em></p><p><em>Staphylococcus aureu</em>s is a pathogen associated with serious community and hospital-acquired diseases. It has low nutritional requirements and widely exists in nature. In China, microbial food poisoning accounted for 53.7% of food poisoning emergencies in 2015. Of which, <em>S. aureus</em>&nbsp;was an important pathogenic factor in these cases(Wu et al, 2018a). In the United States, <em>S. aureus</em>&nbsp;causes approximately 241,000 cases of food poisoning each year (Scallan et al, 2011;Kadariya et al, 2004). This food borne pathogen is considered as one of the world’s leading causes of disease outbreaks related to food consumption, being responsible for a variety of manifestations and diseases (Jamali et al, 2005)&nbsp;</p><p><em>Staphylococcus aureus</em>&nbsp;produces a variety of toxins and invasive enzymes such as staphylococcal enterotoxins (SEs), hemolysins, Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin-1 (TSST-1), plasma coagulase, and deoxyribonuclease (Spanu et al, 2012). Differentiation between virulent and non-virulent strains is significant for evaluating the potential implications of the presence of this microorganism for food safety and public health. In which, SEs are active at concentrations ranging from high nanogram to low microgram quantities (Larkin e al,2009) and are resistant to conditions (heat treatment, low pH) that retaining their activity in the digestive tract after ingestion. In addition, TSST-1 is a superantigenic exotoxin that causes toxic shock syndrome and PVL is a bacteriophage-encoded bicomponent leukotoxin that is in some strains of <em>S. aureus</em>&nbsp;and plays a key role in leukocytolysis and tissue necrosis (Shallcross et al, 2013). <em>S. aureus</em>&nbsp;often develops antibiotic resistance. Isolation of single- or multiple-drug resistant <em>S. aureus</em>&nbsp;(MRSA) strains from food, the environment, and clinics has been constantly reported (Gould et al, 2012, Rasigade et al, 2004). The ability of <em>S. aureus</em>&nbsp;to form biofilms helps the bacterium to survive in hostile environments within the host and is considered to be responsible for chronic or persistent infections (Costerton et al, 1999).&nbsp;The ability of some strains to synthesize biofilms could increase their pathogenicity since established biofilms can tolerate antimicrobial agents, thus making the bacterium extremely difficult to eradicate (Zmantar et al, 2010).</p><p>Nowadays, molecular typing methods are crucial in epidemiological investigations of food processing and enhance the resolution of surveillance (Wu et al, 2015).&nbsp;Of various subtyping approaches, multilocus sequence typing (MLST) is a widely accepted method of DNA sequence based typing that based on analysis of relatively conserved genes that encode essential proteins. For <em>S. aureus</em>, the level of discrimination provided by MLST is sufficient to provide a relatively detailed picture of the global dissemination of the organism . <em>spa</em>&nbsp;typing is another efficient typing method for <em>S. aureus</em>, it based on sequencing of the polymorphic X region of the protein A gene (<em>spa</em>) (Hallin et al, 2009).&nbsp;Similar to MLST, it suggests that <em>spa</em>&nbsp;typing is suitable for epidemiology and evolutionary investigations based on studies of European and international isolates (Wu et al, 2018b).</p><p>Milk is an important source of staphylococcal food poisoning. There are several foodborne outbreaks of <em>S. aureus</em>&nbsp;intoxications have been documented to be associated with consumption of contaminated milk (De Buyser et al, 1985, Miwa et al, 2001). In addition, raw milk and raw milk products are frequently contaminated with different types of <em>S. aureus</em>&nbsp;around the world (Tham et al, 1990, Jamali et al, 2015).&nbsp;Milk is a good substrate for <em>S. aureus</em>&nbsp;growth and enterotoxin production. Enterotoxins can retain their biological activity after pasteurization (Asao et al, 2003, Rall et al 2008). In China, studies have reported that some <em>S. aureus</em>&nbsp;strains persist in powdered infant formula (Wang et al 2012). Currently, only a few full-scale and systematic studies have been performed on <em>S. aureus</em>&nbsp;prevalence and contamination levels in pasteurized milk in China. This study aimed to investigate <em>S. aureus</em>&nbsp;contamination in pasteurized milk obtained from different Chinese cities. For this purpose, we analyzed the <em>S. aureus</em>&nbsp;prevalence and contamination levels, antibiotic susceptibility profiles, virulence genes, biofilm formation, biofilm-related genes, <em>spa</em>&nbsp;typing results, and multilocus sequence typing (MLST) results to characterize the isolates.</p></div><div><h2>Materials and Methods</h2><div><h3>Sampling</h3><p>From July 2011 to June 2016, a total of 258 pasteurized milk samples were collected from supermarkets, fairs, and farmers’ markets (Supplementary Table 1). The samples were obtained from 39 cities located in a total of 29 provinces and 2 directly controlled municipalities in China, which covered most of the provincial capitals of China (Supplementary Figure 1). The samples were placed in a cold box at approximately 4°C, tightly sealed with sterile plastic wrap, transported to an accredited laboratory, and subjected to microbiological analysis within 24 h.</p></div><div><h3>Isolation and Detection</h3><p>The samples were qualitatively and quantitatively analyzed to detect <em>S. aureus</em>. For qualitative analysis, samples were examined according to GB 4789.30-2010 (National Food Safety Standards of China) with slight modification. The most probable number (MPN) method was used for quantitative analysis. Approximately 25 g of the food sample was homogenized in 225 mL tryptic soy broth with 10% sodium chloride (Huankai, Guangzhou, China). Subsequently, 1-mL, 0.1-mL, and 0.01-mL aliquots were transferred to tubes containing 9, 10, and 10 mL in triplicate with trypticase soy broth (Huankai) supplemented with 10% NaCl. The tubes were then incubated at 37°C for 48 h.</p><p>A loopful of enrichment broth culture (10 μL) was streaked onto chromogenic <em>S. aureus</em>&nbsp;agar plates (Huankai) and incubated at 37°C for 24 h. Of the colonies obtained, 1–4 pink colonies were purified on nutrient agar medium. The purified colonies were analyzed via the coagulase activity test involving freeze-dried rabbit plasma (Huankai), and the API STAPH test strips (bioMérieux, Marcy-l’Étoile, France) were then used. The MPN value was determined on the basis of the number of positive tube(s) in each of the three sets using the MPN table.</p></div><div><h3>Antimicrobial Susceptibility Testing</h3><p>The Kirby–Bauer disk diffusion method was used to test antibiotic susceptibility, and diameter interpretations were based on the protocol specified in the guidelines of Clinical and Laboratory Standrad Institute (CLSI)-(2015). <em>S. aureus</em>&nbsp;ATCC 25923 and <em>Escherichia coli</em>&nbsp;ATCC 25922 were used as quality control organisms. All isolates were assessed for antimicrobial susceptibility to 24 antibiotics (Oxoid, United Kingdom): amoxicillin/clavulanic acid, ampicillin, cefepime, cefoxitin, penicillin G, ceftazidime, amikacin, gentamicin, kanamycin, streptomycin, chloramphenicol, clindamycin, erythromycin, telithromycin, ciprofloxacin, norfloxacin, tetracycline, linezolid, trimethoprim/sulfamethoxazole (1:19), rifampicin, quinupristin/dalfopristin, teicoplanin, nitrofurantoin, and fusidic acid. The CLSI zone diameter breakpoints were used to interpret the antimicrobial susceptibilities of the analyzed strains.</p></div><div><h3><em>In vivo</em>&nbsp;Biofilm Formation</h3><p>Biofilm production was quantified using a microtiter plate assay (MPA) described previously (Vasudevan et al, 2003) with slight modifications. <em>S. aureus</em>&nbsp;strains were individually grown at 37°C overnight in brain heart infusion broth (BHI). The overnight culture was diluted 1:100 in fresh BHI, and 200-μL aliquots of each prepared suspension were transferred into three wells of 96-well tissue culture treated polystyrene microplates (CELLSTAR® Cell Culture Microplates, Greiner Bio-one, Frickenhausen, Germany). After cultivation at 37°C for 48 h, the wells were washed three times with 200 mL sterile phosphate-buffered saline (PBS, pH 7.4) and dried at room temperature. The adherent bacterial cells were fixed with 200 μL methanol for 15 min, and the plates were emptied and left to dry overnight. The adherent cells were then stained with 1% crystal violet for 10 min and washed twice with water. The dye bound to the adherent cells was dissolved with 150 mL 95% ethanol and optical density (OD) was measured at 590 nm with a spectrophotometer (SpectroStar Nano, BMG Labtech). Uninoculated wells containing BHI served as blanks. Blank-corrected absorbance values of <em>S. aureus</em>&nbsp;strains were used for reporting biofilm production. Isolates were considered biofilm producers when their OD values were three times greater than the standard deviation of the mean Dc. Additionally, isolates showing biofilm-producing ability were classified as weak (Dc &lt; OD ≤ 2 × Dc), moderate (2 × Dc &lt; OD ≤ 4 × Dc), or strong (OD &gt; 4 × Dc) biofilm producers.</p></div><div><h3><br></h3></div></div><p></p>