Ye LI, Xiongwei DU, Wenting DI, Jing WU, Jie GANG, Wenzhong HU

1. Liaoning Entry-Exit Inspection and Quarantine Bureau, Dalian 116001, China;

2. Dalian Nationalities University, Dalian 116600, China;

3. Tianjin University of Science and Technology, Tianjin 300457, China

Salmonella is a genus of Gramnegative, non-spore bearing,acapsular, peritrichous, movable aerobic or facultative anaerobic bacillus[1]. At present, it has been confirmed that in addition to salmonellosis caused by living Salmonella, enterotoxins produced by Salmonella can also lead to these symptoms. Salmonellosis is mainly caused by animal foodstuff; fish, milk and eggs also easily lead to serious foodborne diseases[2].However, with continuous research and identification of Salmonella, antibacterial drugs have been abused to prevent diseases caused by Salmonella in livestock breeding industry,which leads to the formation of drug resistant strains, multi-drug resistance of Salmonella, and increasingly wide resistance spectrum. Therefore, drug resistance spectrum of Salmonella should be analyzed by drug resistance test, thus guiding clinical medication,which has important significance to food safety. Salmonella and its drug resistance can be transmitted through the food chain from the food to the people. Thus, it is quite necessary to detect Salmonella in fresh meat and determine the inhibitory concentration of a certain antibacterial drug to Salmonella via drug susceptibility test.In recent years, identification and detection of foodborne Salmonella has attracted much attention in related fields at home and abroad. Ma et al.[3],Zhang et al.[4], Zhang et al.[5], Yang et al.[6]investigated foodborne Salmonella in Shanxi Province and HenanProvince, but other studies have not been reported[7]. In this study, several swine Salmonella strains in Dalian City were isolated to analyze their susceptibility to 15 antibiotics,aiming at providing reference for ensuring swine-derived food safety in Dalian City.

Materials and Methods

Materials

Strains During October 2012 to September 2013, 68 fresh pork samples were collected from several major wholesale markets in Dalian Economic& Technological Development Zone.Totally 12 suspicious strains were isolated, with E. coil ATCC25922 as a control strain.

Antibiotics Chloramphenicols: florfenicol(Flo,98.4%). Aminoglycosides:neomycin sulfate (Neo, 696 U/mg),gentamicin sulphate (Gen, 610 U/mg).Penicillins: amoxicillin (Amo, 96.4%).Quinolones: niacin norfloxacin (Nia,99%),sarafloxacin hydrochloride (Sar,98% ), ciprofloxacin lactate (CL,98.6%), enrofloxacin (Enr, 99.1%),ciprofloxacin hydrochloride (CH,98.8%).Macrolides:erythromycin thiocyanate (Thi, 780 U/mg). Sulfanilamides: sulfamonomethoxine sodium(Sul, 99.2%). Polymyxins: colistin sulphate (Col, 19 578 U/mg). Tetracyclines: oxytetracycline (Oxy, 96.1%),doxycycline hydrochloride (Dox, 92%doxycycline). Synthetic antimicrobial veterinary drugs: mequindox (Meq,99.1%).

Methods

Sampling Several sealing films were prepared and wiped with ethanolsoaked tampons for sterilization; 9 ml of nutrient broth was added into several test tubes, stoppered and autoclaved at 121 ℃for 30 min.

Fresh pork samples without miscellaneous bacteria were collected.Each sample was sealed with a sealing film and recorded accordingly. After each sampling period, ultraviolet lamp on the clean bench was turned on for 30 min. Fresh pork samples in the sealing films were cut into 2 -3 soybean-sized pieces,transferred into sterilized tubes containing nutrient broth and incubated at 36 ℃ with shaking at 150 r/min for 18-24 h.

Selective incubation and amplification culture ①Preparation:Ultraviolet lamp on the clean bench was turned on for 30 min. SS medium and dishes (wrapped) were autoclaved at 121 ℃for 30 min,and then SS medium were added into each dish and labelled under sterile conditions.The incubated samples were inoculated to SS medium with streak plate method and incubated upside down at (36±1)℃for 18-24 h[8].Subsequently,ultraviolet lamp on the clean bench was turned on for 30 min; several EP tubes, nutrient broth, pipette tips and toothpicks were wrapped and autoclaved at 121 ℃for 30 min.

②Primary culture:1 ml of nutrient broth was transferred into an EP tube with a pipette under sterile conditions.A single colony on the plate was inoculated to the EP tube with an inoculation needle. Subsequently, the EP tube was wrapped with a sealing film and incubated at 36 ℃with shaking at 150 r/min for 18-24 h.

③Secondary culture: at 18-24 h after primary culture, 1 ml of nutrient broth was transferred into a sterilized EP tube with a pipette under sterile conditions. Bacterial liquid cultured in the shaker was inoculated to the new EP tube with an inoculation ring. Subsequently, the remaining bacterial liquid in the former EP tube was mixed with 30%-50%glycerol(3∶1),wrapped with a sealing film and preserved at-20 ℃.The new EP tube was wrapped with a sealing film and incubated at 36 ℃with shaking at 150 r/min for 18-24 h.

PCR assay A bowl of ice was prepared. The water bath was heated to 100 ℃. Distilled water and pipette tips were sterilized at 121 ℃for 30 min,and illuminated under ultraviolet lamp for 30 min.After secondary culture,the EP tube was centrifuged at 1 000 r/min for 3 min; the supernatant was discarded, and the precipitate in the EP tube was washed twice with 200 μl of sterile water, added with 200 μl of sterile water,heated in a water bath for 10 min,cooled in an ice bath for 5 min,and centrifuged at 10 000 r/min for 5 min. According to the introduction of PCR kit, PCR assay was performed.The PCR amplification was started with initial denaturation at 94 ℃for 5 min, followed by 30-35 cycles of denaturation at 94 ℃,annealing at different temperatures (annealing temperature varied among different primers),and extension at 72 ℃; the amplification was completed by holding the reaction mixture at 72 ℃for 10 min to allow complete extension of PCR products. PCR products were stored at 16 ℃[9]before agarose gel electrophoresis.

Preparation of agarose gel: 12 ml of TAE, 1 g of agarose and 98 ml of distilled water were mixed and heated until agarose was dissolved completely;the mixture was poured into the electrophoresis tank, and cooled at room temperature.

Subsequently, 10 μl of sample was mixed with 2 μl of Buffer for electrophoresis. After sample loading, agarose gel electrophoresis was performed under conditions of U=400 V,I=95 mA, W=38, T=1 h. After electrophoresis, the product was soaked in staining solution for 30 min, and observed, photographed and recorded under ultraviolet lamp[10]. The result showed that the Salmonella strip was 286 bp in length.

Drug susceptibility test Preparation: nutrient broth, test tubes, petri dishes, and pipette tips were sterilized at 121 ℃for 30 min;96-well plate was sterilized with ethanol, and illuminated under ultraviolet lamp for 30 min[11].All the drugs were prepared into a final concentration of 5.12 g/L and preserved in 10 ml EP tubes at-20 ℃[12].

Subsequently, 1 ml of drug was added into a test tube containing 9 ml of nutrient broth, and drug concentration was 512 mg/L; 20 μl of bacterial liquid and 20 ml of nutrient broth were mixed in a petri dish. Each well of the 96-well plate was added with 100 μl of nutrient broth;100 μl of 512 mg/L drug solution was added into the first row of the 96-well plate and mixed evenly;100 μl of mixture in the first row was added into the second row of the 96-well plate and mixed evenly; consequently, each well contained 100 μl of mixture; finally, each well was added with 100 μl of bacterial liquid (drug concentration in the first row was 128 mg/L; drug concentration in the second row was 64 mg/L;drug concentrations in the 3rd-12throws were 32, 16,8, 4, 2, 1, 0.5, 0.25, 0.125, 0.062 5 mg/L, respectively). The 96-well platewas incubated in a constant temperature incubator at 36 ℃for 12 h, and the result was observed.

Results and Analysis

Isolation and identification of Salmonella from fresh pork samples

As shown in Table 1, totally 12 Salmonella strains were isolated from the collected fresh pork samples, indicating that the contamination rate of Salmonella in fresh pork from several wholesale markets in Dalian Economic& Technological Development Zone was relatively high, which reached 17.64%.

Drug resistance of swine Salmonella

As shown in Table 2, the resistance of Salmonella to various antibiotics was 0-75%. To be specific, the resistance of Salmonella to florfenicol,neomycin sulfate, sarafloxacin hydrochloride and doxycycline hydrochloride was the highest, which reached 75.0%, 66.7%, 66.7% and 66.7% , respectively; the resistance rate of Salmonella to other antibiotics demonstrated a descending order of oxytetracycline, gentamicin sulphate,mequindox (58.3% ) ＞ciprofloxacin lactate, enrofloxacin, ciprofloxacin hydrochloride (50.0%) ＞erythromycin thiocyanate(41.7%)＞amoxicillin,colistin sulphate (16.7%); the resistance rate of Salmonella to niacin norfloxacin and sulfamonomethoxine sodium was 0, and the susceptibility reached 100%,indicating that these drugs have extremely strong antibacterial effects on Salmonella. Overall, the resistance rate of Salmonella to different types of antibiotics demonstrated a descending order of sulfanilamides ＞penicillins,polymyxins ＞fluoroquinolones ＞tetracyclines ＞chloramphenicols.

Table 1 Identification of Salmonella from fresh pork samples

Table 2 Drug resistance of swine Salmonella

Table 3 Multi-drug resistance spectrum of swine Salmonella

Multi-drug resistance spectrum of swine Salmonella

In this study,83.33%of the isolated strains could at least resist one antibiotic, and some strains were resistant to nine or even 10 antibiotics,showing extremely high drug resistance.As shown in Table 3,among 12 Salmonella strains isolated in this study, 11 strains exhibited drug resistance;nine strains could at least resist four antibiotics; seven strains were resistant to five antibiotics; several strains were resistant to more than seven antibiotics. In addition, four strains were isolated from two identical samples, but their drug resistance spectra varied remarkably, which might be due to multiple factors. For instance,experimental errors led to differences in the drug resistance spectra of two strains from the same sample,including errors in amplification culture and errors of bacteria amount in inoculation. Moreover, these two Salmonella strains from the same sample belonged to different serotypes, resulting in different drug resistance. However, whether thesetwo strains were the same requires further verification. The emergence of multi-resistant bacteria has become a common phenomenon, which should attract sufficient attention to reduce the risk of Salmonella infection in human.

Conclusion and Discussion

In this study,totally 12 Salmonella strains were isolated from 68 fresh pork samples, and the isolation rate was 17.64%, which indicated an extremely high contamination rate of Salmonella in wholesale markets in Dalian Economic&Technological Development Zone. However, samples collected from other wholesale markets were not contaminated by Salmonella. Salmonella is one of the most common pathogenic bacteria causing foodborne illness. The result of this study indicates that Salmonella contamination in commercially available fresh meat products should attract sufficient attention. Salmonella contamination in samples collected in this study might be due to two reasons. On one hand, wholesale market environment creates good conditions for the growth of Salmonella,and fresh pork may be contaminated by unsterilized instruments; on the other hand,pigs have been infected with Salmonella before slaughtering.Therefore,commercially available pork is seriously contaminated, suggesting that the management of slaughtering,transportation, processing, storage and marketing of pork should be strengthened to prevent contamination of pathogenic bacteria.

Swine Salmonella showed extremely high drug resistance.Salmonella strains isolated in this study could at least resist one antibiotic,and some strains were resistant to 6-7 antibiotics. Despite the low drug resistance rates, multi-resistance of Salmonella to drugs has become a common phenomenon. Strong drugresistance of Salmonella can also be transmitted through the food chain from the food to the people and endows human with antibiotic resistance,which increases difficulties in the treatment of foodborne diseases.Thus,it is necessary to strengthen the monitoring of food and drugs.

[1]YANG BW, QU D, ZHANG XL, et al.Prevalence and characterization of Salmonella serovars in retail meats of marketplace in Shaanxi,China[J].International Journal of Food Microbiology,2010,141(1/2):63-72.

[2]ZHANG H(張華).Harm of Salmonella in animal producing food and precautionary measures (動(dòng)物性產(chǎn)品中沙門氏菌的危害及控制措施)[J]. China Animal Health(中國(guó)動(dòng)物保健),2004(6):8-10.

[3]MA GZ(馬國(guó)柱),WANG AL(王安禮),LIU CH (劉長(zhǎng)宏), et al. Surveillance for foodborne pathogen in foods in Shaanxi Province 2002(2002 年陜西省食品中食源性致病菌監(jiān)測(cè))[J].Chinese Journal of Food Hygiene (中國(guó)食品衛(wèi)生雜志),2003,15(6):489-491.

[4]ZHANG F(張芳),MA GZ(馬國(guó)柱),PAN L(潘立),et al.Surveillance and analysis for food-borne pathogens in retailed foods in Shaanxi Province, 2002-2006(陜西省2002-2006 年食源性致病菌污染狀況)[J]. Chinese Journal of Public Health (中國(guó)公共衛(wèi)生), 2008, 24(2):222-224.

[5]ZHANG XL(張秀麗),LIAO XG(廖興廣),HAO ZY (郝宗宇), et al. Pro-active monitoring and establishment of DNA fingerprint database of Salmonella in Henan Province during 2006 -2007(2006-2007 年河南省生肉食品中沙門菌的主動(dòng)監(jiān)測(cè)及其DNA 指紋圖譜庫(kù)的建立) [J]. Chinses Journal of Health Laboratory Technology (中國(guó)衛(wèi)生檢驗(yàn)雜志),2009,19(7):1545-1548.

[6]YANG BW(楊保偉),QU D(曲東),SHEN JL(申進(jìn)玲),et al.Antimicrobial susceptibility and related genes of Salmonella serovars from retail food in Shaanxi Province(陜西食源性沙門氏菌耐藥及相關(guān)基因)[J]. Acta Microbiologica Sinica(微生物學(xué)報(bào)),2010,50(6):788-796.

[7]YANG BW (楊保偉),ZHANG XL (張秀麗), QU D (曲東), et al. Serotypic and genotypic characterization of Salmonella serovars from retails meat in Shaanxi Province (2007-2008) (2007-2008 陜西部分零售畜禽肉沙門氏菌血清型和基因型)[J]. Acta Microbiologica Sinica (微生物學(xué)報(bào)),2010,50(5):654-660.

[8]LIU HW(劉華偉),MA LN(馬立農(nóng)),GUO AG(郭藹光),et al.Control and rapid detection of Salmonella by PCR in domestic animal, poultry and the environment (畜禽及環(huán)境中沙門氏菌的PCR快速檢測(cè)與控制)[J]. Acta Ecologiae Animalis Domastici (家畜生態(tài)學(xué)報(bào)),2005,26(2):59-62.

[9]LIU Q(劉渠),LIU HC(劉衡川),LI ZP(李灶平),et al.A study on the antibiotic resistance of Salmonella spp.(食品中沙門氏菌的耐藥性研究)[J]. Modern Preventive Medicine(現(xiàn)代預(yù)防醫(yī)學(xué)),2004,31(3):330-332.

[10]CHEN DS(陳弟詩(shī)),GUO WZ(郭萬(wàn)柱),XU ZW (徐志文), et al. Isolation and identification of Salmonella choleraesuis and the establishment of its PCR detective method(豬霍亂沙門氏菌的分離與鑒定以及PCR 檢測(cè)方法的建立)[J]. Journal of Anhui Agricultural Sciences (安徽農(nóng)業(yè)科學(xué)), 2007, 35(20):6020-6023.

[11]GUAN WY (關(guān)文英),SHEN ZX (申志新),ZHANG SH(張淑紅),et al.A study on the antibiotic resistance of Salmonella spp. in Hebei Province (河北省食品中沙門氏菌的耐藥性研究)[J].Modern Preventive Medicine(現(xiàn)代預(yù)防醫(yī)學(xué)),2006,33(10):1761-1763.

[12]WANG J (王娟), QU ZN (曲志娜),ZHAO SJ(趙思俊),et al.Study on susceptibility for avian E. coli and Salmonella(禽源大腸桿菌和沙門氏菌耐藥性研究)[J]. Chinese Journal of Animal Health Inspection(中國(guó)動(dòng)物檢疫),2009,26(5):64-65.