Antibiotic Susceptibility,Biofilm-Forming Ability,and Incidence of Class 1 Integron of Salmonella spp.,Escherichia coli,and Staphylococcus aureus Isolated from Various Foods in a School Canteen in China

Abstract

In recent years, the food poisoning incidents from school canteens have aroused widespread concern in China. Microbial contamination to the foods is the main factor responsible for these food poisoning events. In this study, identification of microbial pathogens including Salmonella spp., Escherichia coli, and Staphylococcus aureus in samples (frozen pork, fresh pork, fresh chicken, and different fresh vegetables) of a school canteen in China during 2017 to 2018 was performed. The antibiotic susceptibility pattern, class 1 integron, and biofilm formation ability of the isolated pathogens were also investigated. In total, 96 strains were isolated (32 Salmonella spp., 32 E. coli, and 32 S. aureus). The antibiogram study results demonstrated that 61.5% strains were found resistant to at least one type of antibiotics, and 17.7% were resistant to three or more antibiotics. In addition, 31.3% strains possessed class 1 integron. Among the integron-positive isolates, 38.9% Salmonella spp. and 87.5% E. coli contained ∼800 or/and 1500 bp size gene cassette within the integrons. However, four S. aureus strains possessing class 1 integron without gene cassette were found. Although none of the isolated strains were found strong biofilm producer, 44.8% were found to have weak or moderate biofilm formation ability. Despite biofilm formation ability or not, the Salmonella spp. containing positive class 1 integron showed significant resistance to cefazolin and gentamicin. In addition, class 1 integron-positive E. coli isolates having the biofilm formation ability hardly showed sensitive to four antibiotics, such as amikacin, amoxicillin–clavulanate, cefazolin, and gentamicin. Therefore, it is necessary to reduce the prevalence of antibiotic resistance gene cassettes containing antibiotic resistance genes by the prudent use of antibiotics in livestock farms, and the improvement of food processing and storage environment.

Introduction

A total of 1810 campus food poisoning incidents were reported by the National Foodborne Disease Surveillance Network in China during 2002 to 2016 (Geng et al., 2018). Among these incidents, 557 happened in different schools across the country, which were mainly (70.56%) caused by foodborne pathogens or biological pollutants (Pan et al., 2017). Therefore, the regulation and control of foodborne pathogenic microorganisms are necessary to reduce the occurrence of food poisoning events. Salmonella was mainly found in feeding livestock breeding industries products, such as pork, chicken, and eggs. Staphylococcus aureus strains were usually isolated from food processing, cereal products, and even egg products, whereas Escherichia coli was widely distributed in feces of human and animals. According to the report published by the national food safety monitoring and surveillance system in China (Wu and Chen, 2018), Salmonella spp. and S. aureus were still major species causing most bacterial foodborne illnesses, and E. coli was one of hygiene indicator bacteria. In Japan, there were also staphylococcal food poisoning cases occurring from 1990 to 2015 (Sato'o et al., 2014; Yuki et al., 2018).

As reported, food poisoning incidents caused by bacterial contamination were most associated with Gram-negative bacteria such as Salmonella, E. coli, and Gram-positive bacteria such as S. aureus (Ashraf et al., 2018). Therefore, effective monitoring focused on the source and the storage environment of these three harmful bacteria would be an effective way to reduce cross-infection and ensure food safety.

In the clinic, the increasing emergence of multidrug-resistant strains has made the treatment of foodborne illnesses more and more compromised (Bai et al., 2016; Yan et al., 2016; Hu et al., 2017). Commonly, the emergence of microbial resistance is always accompanied by the process of conjugation, transduction, or transformation of mobile DNA segments. For example, class 1 integron is capable of capturing one or more cassettes, which are able to mediate the expression of antibiotic resistance (Soucy et al., 2015; Carraro et al., 2017). Besides, biofilm has been causing the dilemma to treatment. Pathogens in the biofilm state could partly exhibit increased antimicrobial resistance to some antibiotics, such as tobramycin, ciprofloxacin, meropenem, imipenem–cilastatin, and ceftazidime (Høiby et al., 2010; Müsken et al., 2010; Fricks-Lima et al., 2011; Corzo-Ariyama et al., 2019). The biofilms are difficult to remove through the normal disinfection procedure during food processing. As a consequence, more food contamination would occur by contacting with equipment's surfaces (Patel and Sharma, 2010; Soni et al., 2013; Ledwoch et al., 2018).

Drug-resistant microorganisms are easily transferred from polluted environments, food raw materials, and so on to humans. To the best of our knowledge, in connection with the intraspecific or interspecific dissemination of antibiotic resistance genes, it is still unclear whether there is an underlying relationship between the integrons and biofilm formation ability. Herein, a total of 96 strains (32 Salmonella spp., 32 E. coli, and 32 S. aureus) were isolated from various foods, especially some raw meat products and vegetables, in a school canteen in China from 2017 to 2018, aiming to find some phenotypic correlation among their antibiotic susceptibility, class 1 integron, and biofilm-forming ability.

Materials and Methods

Bacterial isolates

The bacterial strains used in this study were isolated from 480 samples, which were the most common food raw materials collected during April to June and September to November from a school canteen in China during 2017 to 2018. The samples included fresh vegetables (common vegetables such as beans, lettuce, and cauliflower, n = 192), fresh pork (n = 96), frozen pork (preservation in canteen freezers, n = 96), and fresh chicken (n = 96). All samples were transported in cooler boxes and maintained at 4°C until further processing within 24 h.

Each sample was aseptically cut into slices or pieces to prepare 50 g composite specimens. Specimens from each sample were placed in sterile homogenized bags containing 225 mL phosphate-buffered saline (PBS) in a 1:10 weight-to-volume ratio. After homogenization, 0.1 mL of each of the PBS-sample mixtures was placed on three types of plates, on a violet red bile agar (Huankai Microbial, China), on a Salmonella–Shigella agar (Huankai Microbial), and a mannitol salt agar (Huankai Microbial). All plates were incubated at 37°C for 24 h. All the isolated strains were identified using colony morphology, Gram staining, and other biochemical tests. The confirmation of identified strains was carried out using API commercial kit and semi-automated Vitek 2 system (BioMerieux). As a result, a total of 96 strains were isolated (32 Salmonella spp., 32 E. coli, and 32 S. aureus).

Antimicrobial susceptibility testing

Antimicrobial susceptibility was determined by disk diffusion technique using the semi-automated Vitek 2 system. Twenty-five antibiotics including amikacin, amoxicillin–clavulanate (AMC), cefazolin, cefmetazole, trimethoprim–sulfamethoxazole (SXT), cefepime, cefoperazone–sulbactam, piperacillin–tazobactam, cefoxitin, ceftriaxone, cefuroxime, ciprofloxacin, clindamycin, doxycycline, erythromycin, gentamicin, imipenem, levofloxacin, linezolid, meropenem, moxifloxacin, oxacillin, teicoplanin, vancomycin, and penicillin were assessed (Sigma–Aldrich, St. Louis, MO). Dosages of antibiotics for the tested isolates were strictly based on the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2012).

Detection of class 1 integron and relevant gene cassettes

Integrons were detected using polymerase chain reaction (PCR) of the conserved region of integron-encoded integrase gene intI1. Genomic DNA from each strain was used as the template for PCR amplification, and DNA was extracted using a DNA extraction kit (Dongsheng Biotech, Guangzhou) according to the manufacturer's instructions. The concentration and quality of the extracted DNA were determined using a NanoDrop 2000 (Thermo Fisher Scientific, Inc, Waltham, MA). The primers used for the detection of class 1 integron were as follows (Igbinosa et al., 2013; Asadpour, 2018): intl1-U, 5′-GTTCGGT CAAGGTTCTG-3′; intl1-P, 5′-GCCAACTTTCAGCACATG-3′.

To amplify the cassette insertion region, a pair of generalized primers given previously was used, which were: PF, 5′-GGCATCCAAGCAGCAAGC-3′; PB, 5′-AAGCAGACTTGACCTAAT-3′. The purified products were subsequently sequenced by BGI Company (Shenzhen, China). The sequences were compared with reference sequences in the GenBank using a BLAST (Basic Local Alignment Search Tool) program.

Biofilm assay

Biofilm formation was quantitatively assessed by crystal violet staining assay in a 96-well polystyrene microplate described by Odeyemi et al. (2012) with modification. The absorbance in the microplate was determined at 590 nm using the microplate reader (Bio-Rad). Positive control wells contained S. aureus ATCC 25922, Salmonella enterica ATCC 14028, and E. coli ATCC 43894, respectively, and the negative control wells contained uninoculated Tryptone Soy Broth. Three independent replicates were performed for each isolate, from which the mean optical density (OD) values were calculated (OD = Mean OD − Mean ODc). The isolates were classified according to their biofilm-forming ability by comparing the OD values. Strongly adherent: OD >1, moderately adherent: 0.5 < OD <1, weakly adherent: 0.3 < OD <0.5, and nonadherent: OD <0.3.

Statistical analysis

All experiments were performed at least in triplicate. Statistically significant differences in the distribution of the three type strains among the antimicrobial susceptibility, biofilm formation, and the carriage of class 1 integron and relevant gene cassettes were assessed by either chi-squared or Fisher's exact tests (in cases with an n < 40). p < 0.05 was considered statistically significant.

Results and Discussion

Antimicrobial susceptibility of the foodborne isolates

Antibiotic resistance is one of the major problems concerning the treatment of infectious diseases worldwide. The isolates were evaluated for their antibiotic susceptibilities. As shown in Table 1, 59 isolates (61.5%) were resistant to at least one type of antibiotics, and 17 were resistant to three or more antibiotics. Among these 59 resistant strains, 56 resistant strains were derived from meat food sources. Fresh chicken samples were the dominating source, followed by fresh pork, which may be caused by the indiscriminate use of antibiotics in poultry farming. Only three strains of S. aureus were found from vegetables, and eight from frozen pork. E. coli (100.0%) had the highest resistance to trimethoprim–sulfamethoxazole, which demonstrated significant differences (p < 0.001, Fig. 1a) to Salmonella spp. (25.0%). The resistance of E. coli to amoxicillin–clavulanate, cefazolin, gentamicin, ciprofloxacin, piperacillin–tazobactam, and levofloxacin (Fig. 1a) decreased in order. All the E. coli (100.0%, p < 0.001) isolates showed moderate or strong resistance to amikacin, cefoperazone–sulbactam, and ceftriaxone, and only 6.3% of the Salmonella spp. isolates were resistant to these three antibiotics. Besides, S. aureus isolates exhibited 59.4%, 57.5%, 41.1%, 23.0%, and 18.5% nonsensitivity to penicillin, ciprofloxacin, erythromycin, gentamicin, and clindamycin, respectively (Fig. 1b). One strain exhibited resistance to vancomycin, which was used successfully for over 50 years to treat S. aureus infections (Holmes et al., 2012).

FIG. 1.

The percentage of the isolates exhibiting nonsensibility to the tested antibiotics. Salmonella spp. and Escherichia coli (a); Staphylococcus aureus (b). % (R+M), percentage of the resistant and intermediate. AMC, amoxicillin–clavulanate; AMK, amikacin; CFP, cefoperazone-sulbactam; CFZ, cefazolin; CIP, ciprofloxacin; CLI, clindamycin; CMZ, cefmetazole; CRO, ceftriaxone; CXM, cefuroxime; DOX, doxycycline; ERY, erythromycin; FEP, cefepime; FOX, cefoxitin; GEN, gentamicin; IPM, imipenem; LEV, levofloxacin; LNZ, linezolid; MEM, meropenem; MXF, moxifloxacin; OXA, oxacillin; PEN, penicillin; SXT, trimethoprim–sulfamethoxazole; TEI, teicoplanin; TZP, piperacillin–tazobac; VAN, vancomycin.

Table 1.

Antimicrobial-Resistant Isolates Isolated from Different Food Samples

Bacteria	No. of resistant isolates			No. of resistant isolates in different food types				No. of isolates about the ability of biofilm formation
Bacteria	One antibiotic	Two antibiotics	Three or more antibiotics	Frozen pork	Fresh chicken	Fresh pork	Vegetables	NA	WA	MA	SA
Salmonella spp. (n = 32)	10	2	3	2	8	5	0	18	10	4	0
Escherichia coli (n = 32)	1	5	8	2	6	6	0	16	11	5	0
Staphylococcus aureus (n = 32)	17	7	6	4	13	10	3	19	10	3	0
Total	28	14	17	8	27	21	3	53	31	12	0

MA, moderately adherent; NA, nonadherent; SA, strongly adherent; WA, weakly adherent.

Detection of class 1 integron and characterization of gene cassettes

To investigate the potential dissemination of antibiotic resistance determinants in its processing and storage environments, class 1 integron and antibiotic resistance genes harbored in strains of Salmonella, E. coli, and S. aureus were determined. Table 2 displays the features of the 30 foodborne isolates carrying class 1 integron (31.3%). Among the 18 integron-positive Salmonella spp. isolates, 3 isolated strains from fresh chicken exhibited multiple antibiotics resistance, of which 2 had both aadA2 and aadB antibiotic resistance genes (1500, 800 bp) and 1 had only aadA2 antibiotic resistance gene. The results were similar to the previous research (Zhao et al., 2017) that Salmonella isolated from farm animals in Shandong Province of China contained gene cassettes aadA2. There were only two Salmonella spp. strains carrying class 1 integron in frozen pork, which were resistant to AMC or SXT. The possible reason may be that cold storage prevented the spread of foodborne bacteria with reducing biological activity under low temperature (Matthew and Boris, 2019). Report from another research showed similar results that 155 Salmonella strains isolated from pork obtained from open markets in Xuzhou city of Jiangsu Province of China, 97.4% of the isolates were resistant to at least one type of antibiotics, and 66.1% exhibited resistance to at least four antibiotics (Zhu et al., 2019).

Table 2.

Features of the Foodborne Isolates Carrying Class 1 Integron

Number	Strains	Origin	Ability of biofilm formation	Resistance phenotype pattern		Class 1 integron
Number	Strains	Origin	Ability of biofilm formation	Resistance	Intermediate	Gene cassettes size, bp	Antibiotic resistant genes
S01	Salmonella spp.	Frozen pork	−	AMC	ND	Empty	ND
S03	Salmonella spp.	Frozen pork	−	SXT	ND	Empty	ND
S04	Salmonella spp.	Fresh pork	−	CXM	ND	Empty	ND
S06	Salmonella spp.	Fresh pork	+	CIP	ND	Empty	ND
S07	Salmonella spp.	Fresh pork	+	GEN	CIP	1500	aadA2
S08	Salmonella spp.	Fresh pork	+	LEV	CFZ	Empty	ND
S09	Salmonella spp.	Fresh pork	+	SXT	CFZ	Empty	ND
S10	Salmonella spp.	Fresh chicken	++	GEN	AMC, CFZ	1500	aadA2
S11	Salmonella spp.	Fresh chicken	−	SXT	TZP	Empty	ND
S13	Salmonella spp.	Fresh chicken	−	CFZ	CXM	Empty	ND
S15	Salmonella spp.	Fresh chicken	−	AMC, SXT	ND	Empty	ND
S17	Salmonella spp.	Fresh chicken	++	GEN, SXT	CFZ	1500	aadA2
S19	Salmonella spp.	Fresh chicken	++	AMC, GEN, SXT	CFZ	1500, 800	aadA2 and aadB
S20	Salmonella spp.	Fresh chicken	+	AMC, CIP, SXT	ND	1500	aadA2
S21	Salmonella spp.	Fresh chicken	++	AMC, GEN, CFZ	TZP	1500, 800	aadA2 and aadB
S27	Salmonella spp.	Fresh chicken	−	ND	AMC, CFZ, LEV	1500	aadA2
S28	Salmonella spp.	Fresh chicken	−	ND	CIP, SXT	Empty	ND
S29	Salmonella spp.	Fresh chicken	−	ND	CIP, TZP	Empty	ND
E2	Escherichia coli	Fresh pork	++	AMK, AMC, CFZ, CIP, GEN, LEV	ND	1500, 800	aadA2 and aadB
E3	E. coli	Fresh pork	++	AMK, AMC, CFZ, CIP, GEN, LEV	ND	1500, 800	aadA2 and aadB
E4	E. coli	Fresh pork	+	AMK, CFZ, CRO	AMC, CRO	1500, 800	aadA2 and aadB
E7	E. coli	Fresh chicken	++	AMC, CFZ, CXM	CRO	1500, 800	aadA2 and aadB
E9	E. coli	Fresh chicken	++	AMC, CRO, CXM, GEN	ND	1500	aadA2
E10	E. coli	Fresh chicken	+	AMC, CFZ, CRO	GEN	1500, 800	aadA2 and aadB
E12	E. coli	Fresh chicken	+	AMK, AMC, CFZ	GEN	800	aadB
E14	E. coli	Fresh chicken	++	CFP, GEN, TZP	AMC	Empty	ND
G1	Staphylococcus aureus	Fresh chicken	−	PEN, ERY, CLI	CIP	Empty	ND
G4	S. aureus	Fresh chicken	−	PEN, ERY, CIP	ND	Empty	ND
G7	S. aureus	Fresh chicken	−	PEN, ERY, CLI, CIP	ND	Empty	ND
G12	S. aureus	Fresh chicken	−	PEN, GEN, CLI, CIP	ND	Empty	ND

−, no adhere ability; +, weak ability of biofilm formation; ++, intermediate ability of biofilm formation.

AMC, amoxicillin–clavulanate; AMK, amikacin; CFP, cefoperazone–sulbactam; CFZ, cefazolin; CIP, ciprofloxacin; CLI, clindamycin; CRO, ceftriaxone; CXM, cefuroxime; ERY, erythromycin; FOX, cefoxitin; GEN, gentamicin; LEV, levofloxacin; ND, not determined; PEN, penicillin; SXT, trimethoprim–sulfamethoxazole; TZP, piperacillin–tazobac.

For E. coli, eight class 1 integron-positive isolates from fresh chicken and fresh pork all were resistant to at least three or more antibiotics. Similarly, Cheng et al. reported that among the 182 E. coli strains from Penaeus vannamei at a Freshwater Shrimp Farm in Zhejiang Province of China, 28.6% isolates showed multiple antibiotics resistance (Cheng et al., 2019). As shown in Table 2, five of these E. coli strains had both aadA2 and aadB antibiotic resistance genes, and two contained at least one of these antimicrobial resistance genes. Four S. aureus strains carrying class 1 integron were found without gene cassette within integrons. The aadA2 and aadB gene cassettes detected in this study were commonly found in different Enterobacteriaceae species and nonfermentative bacteria located on the Genomic Island 1 and plasmids (Lee et al., 2009; Hsiao et al., 2014). They are potentially transferrable and are usually connected with resistance to aminoglycosides (Glenn et al., 2011; Jacoby et al., 2014).

Some developed European countries, such as Latvia, Poland, and Greece, also have food contamination issues. Despite low overall usage of antimicrobials in these countries, Salmonella spp. and E. coli with one or multi-antibiotic resistance, and enterotoxigenic S. aureus or methicillin-resistant Staphylococcus were frequently found in food industries (Margarita et al., 2018; Korpysa-Dzirba and Osek, 2018; Panagiotis et al., 2019). As a consequence, simultaneous surveillance should be conducted, focusing on the antibiotic resistance profiles and DNA fingerprinting of drug-resistant pathogens for the food raw materials (raw meat products, particularly).

Abilities of biofilm formation and its correlation with class 1 integron

Biofilm formation is considered an environmental adaptation strategy by microorganism. High humidity or moisture content in the environment favors the formation of bacterial biofilm. Commonly, it is difficult to remove through the normal disinfection procedure, such as detergents or sanitizing agents (Chuah et al., 2018). The biofilm formation ability of these pathogens was determined in this study. The OD values of the 96 foodborne pathogens ranged from 0.016 to 0.665. Based on the criteria applied in this study, 31 isolates had weak biofilm formation abilities, 12 harbored moderate abilities, and none of these isolates was strong biofilm producer (Table 1). Fisher's exact test was applied to analyze the qualitative variables of the 18 class 1 integron-positive Salmonella spp. strains, in terms of their ability and inability of biofilm formation and the antimicrobial susceptibility.

As shown in Table 3, statistically significant difference (p < 0.05) was only observed in two antibiotics, which were cefazolin and gentamicin. Previous report suggested that biofilm formation could be induced by the aminoglycoside antibiotics including gentamicin. All the eight class 1 integron-positive E. coli isolates that harbored the abilities of biofilm formation were hardly sensitive to four antibiotics, such as amikacin, amoxicillin–clavulanate, cefazolin, and gentamicin. It was reported that the horizontal transfer and acquisition of antibiotic resistance plasmids or determinants had enhanced their survivability and pathogenicity of Salmonella and E. coli biofilms under certain conditions (Król et al., 2013; Lukasz Mąka, 2016; De Campos et al., 2018). The plasmids with class 1 integron in the biofilm state would become a more effectively transmissible agent on dissemination of antibiotic resistance genes.

Table 3.

Correlation Analysis Between Biofilm Formation and Antimicrobial Susceptibility of the Salmonella spp. Isolates Carrying Class 1 Integron

Antibiotics	Salmonella spp. isolates carrying class 1 integron				p
	Ability of biofilm formation (n = 9)		Inability of biofilm formation (n = 9)
	% (S)	% (R+M)	% (S)	% (R+M)
AMK	0.0	0.0	0.0	0.0	NS
AMC	55.6	44.4	66.7	33.3	NS
CFZ	33.3	66.7	77.8	22.2	<0.05
FEP	0.0	0.0	0.0	0.0	NS
CFP	0.0	0.0	0.0	0.0	NS
CRO	0.0	0.0	0.0	0.0	NS
CXM	0.0	0.0	0.0	0.0	NS
CIP	66.7	33.3	77.8	22.2	NS
GEN	44.4	55.6	100.0	0.0	<0.05
IPM	0.0	0.0	0.0	0.0	NS
LEV	88.9	11.1	88.9	11.1	NS
MEM	0.0	0.0	0.0	0.0	NS
TZP	88.9	11.1	77.8	22.2	NS
SXT	55.6	44.4	66.7	33.3	NS

% (S), percentage of the susceptible; % (R+M), percentage of the resistant and intermediate; NS, not statistically significant.

AMC, amoxicillin–clavulanate; AMK, amikacin; CFP, cefoperazone–sulbactam; CFZ, cefazolin; CIP, ciprofloxacin; CRO, ceftriaxone; CXM, cefuroxime; FEP, cefepime; GEN, gentamicin; IPM, imipenem; LEV, levofloxacin; MEM, meropenem; SXT, trimethoprim–sulfamethoxazole; TZP, piperacillin–tazobac.

Conclusion

Our study reported the antibiotic resistance profile, biofilm formation ability, and class 1 integron of Salmonella spp., E. coli, and S. aureus, isolated from raw food samples of a school canteen in China. The antibiogram study results demonstrated that 61.5% strains were found resistant to at least one type of antibiotics, and 17.7% were resistant to three or more antibiotics. In addition, 31.3% strains possessed class 1 integron. Among the integron-positive isolates, 38.9% Salmonella spp. and 87.5% E. coli contained ∼800 or/and 1500 bp size gene cassette within the integrons. Furthermore, among the isolated strains, 44.8% strains had the ability to bind to surfaces and then formed biofilms, which would not be removed by the conventional antimicrobial agents and thus infection would persist. The antibiotic resistance patterns of the isolates and the presence of class 1 integron revealed the possibility of horizontal gene transfer of antibiotic resistance determinants. Therefore, to avoid food contamination issues happened in school canteen, it is necessary to ensure food raw materials traceable, normative food processing, and storage environment dry and sanitation. And the prudent use of antimicrobials in livestock farms should also be practiced.

Footnotes

Acknowledgment

The authors wish to thank Navindra P. Seeram for his technical support.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the National Key Research and Development Program of China (2017YFD0601303), the Science and Technology Program of Guangzhou (201707010129), and the Science and Technology Planning Project of Jieyang (2017xm020).

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