Prevalence,Antimicrobial Resistance,and Molecular Characteristics of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus from Retail Ice Cream in Shaanxi Province,China

Abstract

Staphylococcus aureus (S. aureus) is one of the major opportunistic foodborne pathogens as well as a source of human and animal infections. As surveillance of S. aureus and methicillin-resistant Staphylococcus aureus (MRSA) is limited in ice cream, a total of 240 ice cream samples were collected from three cities in Shaanxi province, China, and screened for S. aureus. All isolates were characterized by antimicrobial susceptibility testing, staphylococcal protein A typing, multilocus sequence typing, enterobacterial repetitive intergenic consensus typing, virulence, and resistance genes. S. aureus was recovered from 10 (4.2%) ice cream samples (13 isolates) with average count from 10 to 100 colony-forming units per gram in all cases. Resistance to amoxicillin/clavulanic acid, penicillin, and trimethoprim/sulfamethoxazole (each 100.0%) was most frequently observed, followed by ampicillin (76.9%), erythromycin (46.2%), ceftriaxone (30.8%), and cefoxitin (15.4%). A total of five types of antimicrobial resistance genes were detected, including β-lactam (blaZ and mecA), macrolide (ermB and ermC), tetracycline (tetK), aminoglycoside [aac(6′)/aph(2′) and aph(3′)-III], and trimethoprim (dfrG). All of the strains harbored at least one staphylococcal enterotoxins gene. The commonly detected virulence genes were selw and hld (100.0%), followed by selx (92.3%); hla (84.6%); pvl (76.9%); seg, sem, and sen (each 38.5%); sei, seo, and hlb (each 30.8%); sea, seb, selu, and sely (each 23.1%); sed, sej, sek, sep, and seq (each 15.4%); and ser (7.7%). ST5-t002, ST7-t091, and ST5225-t4911 (each 15.4%) were the predominant clones, followed by ST5-t045/t105, ST6-t701/t15417, ST25-t078, ST188-t189, and ST398-t034 (each 7.7%). Among the 13 strains of S. aureus, 2 isolates were detected as MRSA (15.4%), and the molecular type belonged to ST5225-IVa-t4911. Using a 98.8% similarity cutoff, the 13 isolates were divided into 5 clusters (I-1 to I-5). These results demonstrated that the prevalence of S. aureus and MRSA was low in ice cream. However, these isolates exhibited a high level of potential pathogenicity, which represents a potential health hazard for consumers.

Introduction

S taphylococcus aureus is an important foodborne pathogen that can express a large number of virulence factors in various foods, including staphylococcal enterotoxins (SEs) and staphylococcal like enterotoxins (SEls), exfoliative toxins A and B, toxic shock syndrome toxin-1, and Panton–Valentine leukocidin (PVL). Among these virulence factors, SEs and SEls are known to play a key role in staphylococcal food poisoning (SFP) (Argudin et al., 2010). To date, 29 types of SEs and SEls have been identified, including classical enterotoxin (sea-see) and new enterotoxin (seg-sel27) (Ono et al., 2015; Aung et al., 2020). SFP outbreaks are mainly caused by classical SEs (SEA to SEE) (accounting for 95%), and SEA is considered as the main cause of SFP outbreaks (accounting for 80%) (Argudín et al., 2010).

In recent years, reports of the emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) isolates in food have greatly increased. (Li et al., 2021; Omwenga et al., 2021). S. aureus carrying mecA gene or exhibiting an oxacillin minimum inhibitory concentration of ≥4 μg/mL is defined as MRSA (VanEperen and Segreti, 2016). With the changes in the use of antibiotics, the emergence and wide spread of oxacillin-susceptible mecA-positive S. aureus (OS-MRSA) in both humans and animals have been increasingly reported and become a serious public health concern (Oniciuc et al., 2017).

Owing to the possession of mecA, it could be easily misdiagnosed by antibiotic phenotypic laboratory testing and potentially triggers the development of highly new resistant MRSA variants under antibiotic selection (Quijada et al., 2019). In addition, molecular typing methods of bacterial pathogens, such as staphylococcal protein A (SPA), multilocus sequence typing (MLST), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and so on, are not only critical for outbreak investigation but also useful to determine the source of foodborne illness and to monitor the spread of resistant strains (Wang et al., 2011).

As a kind of frozen food, ice cream is deeply loved by the public, especially children in summer. However, ice cream is greatly affected by temperature and easily melts during storage and transportation. The process of repeated freezing and thawing of ice cream may cause the proliferation of pathogenic microorganisms, including S. aureus (Fetsch et al., 2014). In addition, due to its rich nutrients, appropriate pH value, and long shelf life, it can support the presence of S. aureus (Mirzaei et al., 2012; Gucukoglu et al., 2013).

At present, there are reports of food poisoning incidents caused by eating ice cream contaminated with S. aureus in China (Ma et al., 2019) and other countries (Cerqueiracampos et al., 1993; Fetsch et al., 2014). China is the largest producer and consumer of ice cream in the world. The weather of June to September every year in china is relatively hot, and these three months is the peak period of ice cream consumption. However, there is a paucity of data regarding the characteristics of S. aureus from ice cream. Therefore, the purpose of this study was to evaluate the contamination of S. aureus in ice cream in three cities in Shaanxi province, and to further evaluate molecular characterization and antimicrobial resistance of S. aureus in ice cream.

Materials and Methods

Sample collection

From June to September 2015, 240 ice cream samples were collected from retail stores and supermarkets in Shaanxi province, China (Xi'an, Baoji, and Yangling): 79 samples in Yangling city, 82 samples in Baoji city, and 79 samples in Xi'an city. In each city, five to six retail stores and three to four supermarkets were selected with the high pedestrian flow in railway stations and main urban roads. Within each retail store and supermarket, one to five individually packaged ice cream samples, which were sold in frozen condition (−20°C), were collected each month. All samples were immediately transported in an icebox to the laboratory at Northwest A&F University (Yangling, Shaanxi, China) and processed within <4 h.

Count, isolation, and identification of S. aureus

The number of S. aureus isolates was counted according to GB 4789.10-2016 China National Food Safety Standard Food Microbiological Examination. S. aureus was further confirmed by PCR detection using the thermonuclease gene (nuc) primers nucF (5-GCGATTGATG GTGATACGGTT-3) and nucR (5-AGCCAAGCCTTGAC GAACTAAAGC-3). Staphylococcus aureus ATCC 25923 and double-distilled water (ddH₂O) were the positive and negative control, respectively. All identified strains were stored at −80°C in trypticase soy broth (TSB, Beijing Land Bridge Technology Ltd.) containing 50% (v/v) glycerol for further use.

PCR detection of virulence and resistance genes

The presence of 34 virulence and 27 resistance genes in the strains was analyzed. Thirty-four virulence genes were detected, including 5 classic enterotoxin genes (sea, seb, sec, sed, and see), 22 novel enterotoxin genes (seg, seh, sei, selj, sek, sel, sem, sen, seo, sep, seq, ser, ses, set, selu, selv, selw, selx, sely, selz, sel26, and sel27), 4 hemolysin genes (hla, hlb, hld, and hlg), pvl, and 2 epidermal detoxifying toxins A and B (eta and etb).

Twenty-seven antibiotic resistance genes were screened: 6 erythromycin resistance genes (ermA, ermB, ermC, ermT, msrA, and msrB), 4 tetracycline resistance genes (tetK, tetM, tetO, and tetL), 3 aminoglycoside resistance genes [aac(6′)/aph(2″), ant(4′)-Ia, and aph(3′)-III], 4 trimethoprim resistance genes (dfrD, dfrK, dfrG, and dfrS1), 5 chloramphenicol resistance genes (cat:pC211, cat:pC194, cat:pC223, catpIp-501, and fexA), 2 β-lactam resistance genes (blaZ and mecA), and 3 glycopeptide-resistant genes (VanB, VanC1, and VanC2/3). The primers and annealing temperature for the PCR assays are shown in Supplementary Table S1.

Antimicrobial susceptibility testing

Susceptibility of the isolates to penicillin G/P (resistance breakpoint, 0.25 μg/mL), ampicillin/AMP (0.5 μg/mL), trimethoprim/sulfamethoxazole/SXT (4/76 μg/mL), oxacillin/OX (4 μg/mL), rifampin/RIP (4 μg/mL), ciprofloxacin/CIP (4 μg/mL), gentamicin/GEN (8 μg/mL), erythromycin/ERY (8 μg/mL), cefoxitin/FOX (8 μg/mL), amoxicillin/clavulanic acid/AMC (8/4 μg/mL), tetracyclines/TE (16 μg/mL), chloramphenicol/C (32 μg/mL), ceftriaxone/CRO (64 μg/mL), and amikacin/AK (64 μg/mL) was performed by the agar dilution method according to the Clinical and Laboratory Standards Institute (CLSI, 2016). Staphylococcus Aureus ATCC 29213 and Escherichia Coli ATCC 25922 were used as the quality control strains.

SPA typing

The amplification and sequencing primers of SPA typing were acquired from the website (www.seqnet.org). The primers are spa1113f (5′-TAAAGACGATCCT TCGGTGAGC-3′) and spa1514r (5′-CAGCAGTAGTGCCGTTTGCTT-3′). PCR products of the spa locus were sequenced and submitted to the Ridom Spa Server (http://spaserver.ridom.de) to determine the SPA types based on the number and arrangement of tandem repeat sequences.

Multilocus sequence typing

Seven MLST housekeeping genes (ÿrc, aroE, glpF, gmk, pta, tpi, and yqiL) were obtained from the MLST database (www.mlst.net). The PCR products of seven housekeeping gene fragments were sequenced and submitted to the MLST database to determine the sequence typing (ST) types.

Staphylococcal Cassette Chromosome mec typing

The Staphylococcal Cassette Chromosome mec (SCCmec) type was determined by using a multiplex PCR as described previously by Zhang et al. (2005), which generates a specific amplification pattern for each SCCmec structure type. Then the PCR products were analyzed by gel electrophoresis.

ERIC typing

The ERIC-PCR typing was performed according to Ye et al. (2012), using primers ERIC-F (5′-ATGTAAGCTCC TGGGGATTCAC-3′) and ERIC-R (5′-AAGTAAGTGACTGGGGTGAGCG-3′). The PCR conditions were as follows: incubation at 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, 35°C for 1 min, and 72°C for 4 min; and a final extension step at 72°C for 10 min.

The images of the gels stained with ethidium bromide were taken under ultraviolet transillumination. The images were used for cluster analysis by BioNumerics Software 3.0 (Applied Maths, Kortrijk, Belgium). Percentage similarities were identified on a dendrogram derived from the unweighted pair group method with arithmetic mean and based on Dice coefficients. Band position tolerance and optimization were set at 1.5% and 0.0%, respectively. A similarity coefficient of 94.51% was selected to define the ERIC-PCR type clusters. Mark 2000 was used as a molecular size marker.

Statistical analysis

The chi-square tests were analyzed using Minitab^® 15 statistical software (Minitab, State College, PA, USA), and a probability value of <0.05 was considered to be significant.

Results and Discussion

S. aureus prevalence and count

Among the 240 samples, 10 (4.2%, 10/240) samples were contaminated by S. aureus, including 5.1% (4/79) in Yangling, 4.9% (4/82) in Baoji, and 2.5% (2/79) in Xi'an. The detection rate of contaminated samples was low compared with the 5–25% of ice cream samples contaminated with S. aureus in Egypt, Turkey, and Iran (Gucukoglu et al., 2013; Rahimi, 2013; Abdeen et al., 2020). Furthermore, 13 S. aureus isolates were collected from 10 S. aureus-positive samples (1–2 isolates per sample). The number of 10 S. aureus-positive samples ranged from ˂10 to 100 colony-forming units per gram (CFU/g), and the lower frequency distribution was <10 CFU/g in 4 samples in Yangling and 4 samples in Baoji.

However, the frequency distribution of the counting area was lower from 10 to 100 CFU/g in only 2 samples in Xi'an (Table 1). According to GB 29921-2013 China National Food Safety Standard: Maximum limits of pathogenic bacteria in food, the acceptable limit value of S. aureus in ice cream is 100 CFU/g (mL), and the maximum safe limit for S. aureus in ice cream is 1000 CFU/g (mL) (National Standard of the People's Republic of China, 2013). Therefore, all ice cream samples did not exceed the permissible limits of S. aureus count reported in the Chinese Standards in this study.

Table 1.

Prevalence of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus Strains Isolated from Ice Cream

City	No. of samples	No. of S. aureus positive samples			No. of S. aureus isolates recovered^a	No. of MRSA strains ^b (%)
		Range (CFU/g)		Total (%)
		<10¹	10¹–10²	Total (%)
Yang Ling	79	4	—	4 (5.1)	4	1 (25.0)
Baoji	82	4	0	4 (4.9)	6	1 (16.7)
Xi'an	79	0	2	2 (2.5)	3	0 (0.0)
Total	240	8	2	10 (4.2)	13	2 (15.4)

One to two S. aureus strains were collected from each positive sample.

The percentage of MRSA-positive strains among S. aureus strains.

CFU/g, colony-forming units per gram; MRSA, methicillin-resistant Staphylococcus aureus.

Besides, the ice cream samples contaminated with S. aureus were evenly distributed in August (8.1%, 5/62), September (5.1%, 3/59), and July (3.3%, 2/60), June (0.0%, 0/59) (Table 2). The detection rate of S. aureus in August was significantly higher than that in June (p = 0.026). In Shaanxi, the weather in August is warmer than that in June. It may be due to high external temperatures and improper storage that promote the growth of microorganisms, but other factors cannot be ruled out. The test results of ice cream contamination at two types of retail markets showed that the contamination rates of ice cream in retail stores and supermarkets were 5.2% (6/116) and 3.2% (4/124), respectively (Table 2).

Table 2.

Prevalence of Staphylococcus aureus in Ice Cream at Retail Stores and Supermarkets with Different Packaging Methods and Months in Three Cities

City	Retail store		Supermarket		Different packaging		Different months
	Retail store		Supermarket		No. of S. aureus positive samples		No. of S. aureus-positive samples
	No. of samples	No. of S. aureus-positive samples	No. of samples	No. of S. aureus-positive samples	Bagged samples	Boxed samples	June	July	August	September
Yang Ling	27	1	52	3	3/53	1/26	0/19	1/22	2/20	1/18
Baoji	37	3	45	1	3/58	1/24	0/20	1/18	2/22	1/22
Xi'an	52	2	27	0	1/45	1/34	0/20	0/20	1/20	1/19
Total	116	6	124	4	7/156	3/84	0/59	2/60	5/62	3/59

There was no significant difference in the contamination rate of S. aureus between samples purchased from retail stores and supermarkets (p = 0.451). In this study, 10 positive samples are 7 bagged samples (4.5%, 7/156) and 3 boxed samples (3.6%, 3/84) (Table 2). Our results confirmed that the effects of different packing methods on S. aureus contamination were not significant among samples from retail stores and supermarkets (p = 0.735). Warke et al. (2000) also report that S. aureus could be detected in different forms of ice cream.

Among the 10 positive samples, the samples from the same plants or different plants of the same manufacturers are found to be contaminated by S. aureus (Table 3). Human handling of food products and infection/colonization of livestock or farm workers have been described as main reasons for the contamination of dairy products with S. aureus (Greig et al., 2007; Crago et al., 2012). This is most likely due to the use of the same batch raw materials or contamination in the processing.

Table 3.

Antimicrobial Resistance, Virulence Genes Profilies and Resistance Gene Profiles of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus Strains from Ice Cream

Strain ID	Sample ID	The site of the samples	Manufacturer	Processing plants	Production date	Antimicrobial resistance profiles	Virulence gene profiles	Resistance gene profiles	mecA
1	24	Yangling	A	Zhengzhou, Henan	January 6, 2015	AMP-CRO-SXT-P-AMC	seg-sei-sem-sen-seo-seu-selw-selx-pvl-hla-hlb-hld	blaZ	−
2	23	Yangling	B	Xi'an, Shaanxi	March 6, 2015	CRO-SXT-P-AMC	sed-seg-sei-sej-sem-sen-seo-seu-selw-selx-pvl-hld	blaZ	−
3	44	Yangling	C	Shanghai	May 5, 2015	CRO-SXT-P-AMC	sea-seg-sei-sem-sen-seo-seu-selw-selx-pvl-hla-hld	blaZ	−
4	233	Yangling	D	Yangling Shaanxi	July 18, 2015	AMP-FOX-ERY-SXT-P-AMC	seb-sek-seq-selw-selx-sely-mecA-pvl-hla-hlb-hld	blaZ-erm(B)-tet(K)-aph(3’)-III	+
5	105	Baoji	B	Xi'an, Shaanxi	March 4, 2015	ERY-CRO-SXT-P-AMC	sed-seg-sej-sem-sen-ser-selw-selx-pvl-hla-hld	blaZ-erm(C)	−
6	128	Baoji	E	Jiaozuo, Henan	May 9, 2015	AMP-SXT-P-AMC	sea-selw-selx-pvl-hla-hld	blaZ	−
7	128	Baoji	E	Jiaozuo, Henan	May 9, 2015	AMP-SXT-P-AMC	sea-selw-selx-pvl-hla-hld	blaZ	−
8	203	Baoji	E	Inner Mongolia	January 22, 2015	AMP-FOX-ERY-SXT-P-AMC	seb-sek-seq-selw-selx-sely-mecA-pvl-hla-hlb-hld	blaZ-erm(B)-tet(K)-aph(3’)-III	+
9	203	Baoji	E	Inner Mongolia	January 22, 2015	AMP-ERY-SXT-P-AMC	sep-selw-selx-sely-hla-hld	blaZ-erm(B)-tet(K)
10	209	Baoji	F	Xi'an, Shaanxi	March 24, 2015	AMP-ERY-SXT-P-AMC	selw-hlb-hld	blaZ-erm(B)-aac(6’)/aph(2’’)-dfrG	−
11	76	Xi'an	B	Xi'an, Shaanxi	April 16, 2015	AMP-SXT-P-AMC	selw-selx-pvl-hla-hld	blaZ	−
12	179	Xi'an	G	Fushun, Liaoning	February 4, 2015	AMP-ERY-SXT-P-AMC	sep-pvl-selw-selx-hla-hld	blaZ-erm(C)-tet(K)	−
13	179	Xi'an	G	Fushun, Liaoning	February 4, 2015	AMP-ERY-SXT-P-AMC	seb-seg-sei-sem-sen-seo-seu-selw-selx-hla-hld	blaZ-erm(C)-dfrG	−

−, mecA gene negative; +, mecA gene positive; A–G, represent the manufacturer of ice cream; AMC, amoxicillin/clavulanic acid; AMP, ampicillin; CRO, ceftriaxone; ERY, erythromycin; FOX, cefoxitin; P, penicillin G; SXT, trimethoprim/sulfamethoxazole; TET, tetracyclines.

Antimicrobial susceptibility and resistance genes testing

Among the 13 S. aureus isolates tested, resistance was most frequently observed with AMC, P, and SXT (each 100.0%), followed by AMP (76.9%), ERY (46.2%), CRO (30.8%), and FOX (15.4%) (Table 3). All isolates were sensitive to AK, GEN, RIP, C, CIP, and TE. In particular, six antimicrobial resistance profiles were identified. The most common antimicrobial susceptibility testing profile was AMP-ERY-SXT-P- AMC (30.8%, 4/13), followed by AMP-SXT-P-AMC (23.1%, 3/13), AMP-FOX-ERY- SXT-P-AMC, and CRO-SXT-P- AMC (15.4%, 2/13 each), AMP-CRO-SXT-P-AMC, and ERY-CRO-SXT-P-AMC (7.7%, 1/13 each).

In addition, 53.8% (7/13) of isolates were multidrug resistance (MDR) bacteria (resistant to three or more types of antibiotics simultaneously). The most common detected resistance gene was blaZ (100.0%, 13/13), followed by ermB and tetK (30.8%, 4/13 each); ermC (23.1%, 3/13); mecA, dfrG, and aph(3′)-III (15.4%, 2/13 each); and aac(6′)/aph(2′) (7.7%, 1/13) (Table 3).

In summary, resistance to β-lactam, sulfonamide, and macrolide antibiotics was most frequently observed in this study. This result was similar to that reported by Rahimi (2013). This result may be due to the widespread use of these three classes of antibiotics, and the emergence of MDR strains also increases the risk of infection in humans (Wang et al., 2017; Jia et al., 2020). Therefore, a growing number of food source isolates more resistant to multiple antibiotics have caused concerns (Li et al., 2018).

In this study, the phenotype and genotype of the drug-resistant isolates are inconsistent. For example, the isolates carrying aph(3′)-III and aac(6′)/aph(2′) did not show resistance to aminoglycoside drugs. Seyedi-Marghaki et al. (2018) have also found that some isolates carry aminoglycoside antibiotic-related genes, but are susceptible to aminoglycoside. These studies have indicated that aminoglycoside sensitive strains carried aminoglycoside antibiotic-related genes may be due to lower enzymatic activity of aminoglycoside-modifying enzymes of these strains.

In addition, two MRSA isolates from different samples were identified. Although the detection rate of ice cream samples was low (0.8%, 2/240), its spread in the food chain cannot be ignored. Because ice cream is not heated before consumption, MRSA can enter into the digestive tract by ingesting MRSA-contaminated ice cream and may colonize in the human intestinal tract, which may cause MRSA infection and spread of drug-resistant genes (Lee, 2003).

Oxacillin, as an indicator antibiotic for the detection of MRSA isolates, has been unable to accurately identify MRSA isolates. In recent years, mecA-positive S. aureus susceptible to oxacillin has appeared and there is a growing trend (Oniciuc et al., 2017; Quijada et al., 2019). In this study, two mecA-positive isolates were sensitive to oxacillin. Such strains may trigger the development of highly resistant MRSA variants under antibiotic selection, due to the possession of mecA (Oniciuc et al., 2017; Quijada et al., 2019).

Detection of virulence genes

In this study, all isolates (100.0%) carried at least one of the enterotoxin genes. Among them, the most frequently detected gene was selw (100.0%, 13/13), followed by selx (92.3%, 12/13); seg, sem, and sen (38.5%, 5/13 each); sei and seo (30.8%, 4/13 each); sea, seb, selu, and sely (23.1%, 3/13 each); sed, selj, sek, sep, and seq (15.4%, 2/13 each); and ser (7.7%, 1/13). None of the isolates carried sec, see, seh, sel, ses, set, selv, selz, sel26, sel27, and tst genes (Table 3). This result was similar to previous reports examining enterotoxin carrying rate of S. aureus isolates in dairy products by Rahimi (2013) and Suzuki (2019), but higher than the SEs carrying rate of S. aureus isolates from quick-frozen dumplings (56.4%) by Hao et al. (2015).

This difference may be due to differences in food materials, processing environment, and processing personnel. It has been reported that the processing process is the main cause of food contamination (Crago et al., 2012). Notably, the isolates carried selw (100.0%), selx (92.3%), and sely (23.1%), as well as a relatively high carrying rate in this study. It is consistent with the result of a previous study for food handlers and colonizing isolates in Myanmar, in which the detection rates of selw, selx, and sely were 98%, 97%, and 28%, respectively (Aung et al., 2017). As far as we know, this is the first reported that isolates from food samples carried newly described SEs genes.

Although the pathogenicity of newly described toxins has not been determined, the health risks cannot be ignored. In this study, the detection rate of classical enterotoxin genes (63.2%) was higher than that of other isolates from food sources described previously (Wang et al., 2014; Li et al., 2018). However, ice cream is a nonheated food, which means that the classic enterotoxins are a potential threat to human health. It has been reported that humans eating ice cream contaminated with S. aureus cause food poisoning (Cerqueiracampos et al., 1993; Fetsch et al., 2014).

In addition, hemolysin genes (hla, hlb, hld, and hlg) and pvl genes were highly detected in this study. Among them, all the isolates carried hld genes (100.0%, 13/13), followed by hla (84.6%, 11/13), pvl (76.9%, 10/13), and hlb (30.8%, 4/13). None of the isolates carried hlg, eta, and etb genes (Table 3). Hemolysin is a strong pathogenic toxin factor secreted by S. aureus, and almost all S. aureus isolates express hemolysin (Ren et al., 2020). It has been reported that the vast majority of S. aureus isolates from humans are hlb negative due to insertional inactivation of the hlb structural gene by lysogenic prophages (Hau et al., 2015).

In contrast, S. aureus isolates of animal origin are usually hlb positive (van Wamel et al., 2006; Hau et al., 2015). The sea or sep gene is usually detected in hlb-negative isolates, as this gene is often encoded by hlb-inactivating prophages (van Wamel et al., 2006; Hau et al., 2015). This result has also confirmed that 100.0% (4/4) hlb-positive isolates did not carry sea or sep genes, and 55.6% (5/9) of the hlb-negative isolates carried sea or sep genes in this study. And it is found that the molecular type of hlb-positive strains is more common in animal isolates, such as t034, whereas the molecular type of hlb-negative strains is more common in human isolates, such as t002 and t091 (Bystroń et al., 2010; Yan et al., 2012; Gu et al., 2020).

To sum up, raw milk and processing link may be the main reasons for the contamination of ice cream samples. In addition, the two MRSA strains carry both pvl and hemolysin (hla, hlb, and hld) genes in this study. The finding of pvl-positive isolates is important, which have significant potential to cause skin and soft tissue infections (Shrestha, 2013). Therefore, whether food-borne MRSA can be spread to humans through the food chain requires more attention.

Molecular typing

High prevalence of genotypes frequently occurring in humans and animals was demonstrated in food isolates characterized so far (Zhang et al., 2020). In this study, 10 clone types were detected: ST5-t002/t045/t105, ST6-t701/t15417, ST7-t091, ST25-t078, ST188-t189, ST398-t034, and ST5225-t4911 (Fig. 1). The most predominant ST type was ST5 (30.8%). ST5 isolates are widely prevalent in human infections and different types of food (Oliveira et al., 2002; Zhang et al., 2020).

FIG. 1.

Dendrogram of ERIC, SPA, and ST of Staphylococcus aureus isolates from ice cream. ERIC, enterobacterial repetitive intergenic consensus; SPA, staphylococcal protein A; ST, sequence typing.

In recent years, Gu et al. (2020) reported that ST5 strain has replaced ST239 as the main molecular strain in Shanghai hospital. ST6-t701 and ST7-t091 were the dominant molecular types of food poisoning isolates in China (Yan et al., 2012; Li et al., 2015). This is due to the high carrying rate of sea gene in ST6 and ST7 strains. In addition, the clone types of MRSA were ST5225-IVa-t4911 in this study. It is worth noting that the ST5225-IVa-t4911 clonal strain carries the seb-sek-seq gene profile. The seb-sek-seq gene profile is located in SaPI3, and is often detected in ST59-t437 strains (Bae et al., 2020).

Recently, Bae et al. (2020) have found that SEB contributes to the systemic infection in the widespread CA-MRSA lineage ST59, which may be the cause for the increased case fatality rate of ST59 infections in China. Whether the clone strain of ST5225-IVa-t4911 has the same infection ability as ST59-t437 still needs attention and further verification. Recent data suggested that selw contributes to the pathogenesis of ST398 strains (Vrieling et al., 2020). ST398 strains also carried selw in this study. Therefore, it also suggests that we should not neglect the monitoring of newly described enterotoxins (selw, selx, sely, selz, sel26, and sel27).

The profiles produced seven different banding patterns with amplicons ranging from five to eight by the ERIC-PCR typing technique (Supplementary Fig. S1). When using a 98.8% similarity cutoff, the 13 isolates were divided into 5 clusters (designated by I-1 to I-5) (Fig. 1). The major ERIC clusters were observed in four (30.8%, 4/13) strains grouped in I-4, and contained three clones (ST5-t002, ST5-t105, and ST5-t405). Another two major ERIC clusters were observed in three (23.1%, 3/13 each) strains grouped in I-3 and I-5, and contained three clones (ST6-t701, ST6-t15417, and ST25-t078) and two clones (ST5225-t4911 and ST398-t034), respectively.

The clustering of isolates was correlated with ST types in this study. Yet, there was no clear relationship between fingerprinting pattern and SPA typing. Thus, the combination of those techniques could be very useful to differentiate S. aureus isolates for the purpose of epidemiological surveillance from food samples. In addition, ST5225-IVa-t4911 and ST398 strains revealed a high similarity of banding patterns (Fig. 1), which has not been reported in a new molecular type at present. Moreover, these MRSA isolates exhibited MDR and harbored multiple toxin genes, which is a potential threat to human health.

Conclusions

Our results indicate the presence of S. aureus and MRSA contamination in retail ice cream in Shaanxi province, China. Most S. aureus isolates carried high levels of the newly described toxin genes (selw, selx, and sely), which should be followed by concern about the emergence of novel toxin genes. In addition, attention should also be paid to the presence of these potentially pathogenic S. aureus and MRSA isolates in retail ice cream, and further research to explore their role in causing food poisoning and infections in humans is warranted.

Footnotes

Disclosure Statement

The authors of this article declare that they have no conflicts of interest.

Funding Information

This research was supported by the National Natural Science Foundation of China (Grant Nos. 31871894, U1703119, and 31271858).

Supplementary Material

Supplementary Figure S1

Supplementary Table S1

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