Assessment of antibiotic resistance of lactic acid bacteria in Chinese fermented foods

Food Control 22 (2011) 1316e1321

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Assessment of antibiotic resistance of lactic acid bacteria in Chinese fermented foods Lu Pan, Xiaoqing Hu, Xiaoyuan Wang* State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; and Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 August 2010 Received in revised form 28 January 2011 Accepted 7 February 2011

Lactic acid bacteria isolated from 11 Chinese fermented foods were investigated for their resistance incidences of 7 clinically important antibiotics, including chloramphenicol, kanamycin, tetracycline, ciprofloxacin, ampicillin, clindamicin and erythromycin. It was found that antibiotic resistant lactic acid bacteria are widespread among traditional Chinese fermented foods and their resistance incidences depended on the raw material and manufacturing area of the foods. Resistance incidences of fermented sausages were much higher than that of fermented vegetables. Among 202 antibiotic resistant lactic acid bacterial isolates, 14 were further identified and their minimum inhibitory concentrations against 7 antibiotics were examined. Multi-resistance was observed in these 14 strains, and the presence of genes leading resistance was examined by PCR. Genes tetM and ermB were detected on both plasmid and chromosome in certain strains; gene aphA3 was only detected on plasmid while gene mefA was only on chromosome; genes gyrA, blaZ and catpIP501 were not detected. These results indicate the possible role of lactic acid bacteria as reservoirs for dissemination of antibiotic resistance in foods and the environment. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Antibiotic resistance Chinese fermented food Lactic acid bacteria Food safety

1. Introduction The overuse and misuse of antibiotics has created a tremendous selective pressure toward antibiotic resistant bacteria in the environment (Levy, 1992). Different mechanisms for the resistance to various antibiotics have been found in bacteria. Some bacteria make proteins to decrease the uptake or to increase the export of antibiotics (Sutcliffe, Grebe, Tait-Kamradt, & Wondrack, 1996); some bacteria modify the structure of the target molecules for antibiotics (Aarestrup, Agerso, Gerner-Smidt, Madsen, & Jensen, 2000; Schmitz et al., 1998; Sutcliffe, Grebe, Tait-Kamradt, & Wondrack, 1996); some bacteria make proteins to inactivate the antibiotic itself (Navarro et al., 2001; Van De Klundert & Vliegenthart, 1993; Normark & Normark, 2002). The emergence of antibiotic resistance (AR) is a global threat because it reduces the efficiency of the antibiotic therapy, which is getting worse by the horizontal transfer of antibiotic resistant genes between bacteria (Schlegelová et al., 2002). Studies on AR used to be mainly focused on clinical bacterial species however, recently such studies on food microbes are getting more interesting (Mathur & Singh, 2005).

Fermented foods may be important vehicles for enormous amounts of living bacteria to enter human body. These bacteria may carry transferable ARs which could be transferred to commensal or pathogenic bacteria (Mathur & Singh, 2005). Although lactic acid bacteria (LAB) have been widely used in the production of fermented foods for a long history (Leroy & De Vuyst, 2004) and were generally recognized as safe, some of them showed intrinsic or acquired AR (Levy, 1997). Therefore, it is necessary to evaluate the AR of LAB in different fermented foods. Chinese fermented foods have been consumed for centuries, but there is little information on AR of LAB colonizing these products. Here we have studied the resistance of LAB in two categories of Chinese fermented food (pickles and fermented pork sausages) to 7 clinically important antibiotics, chloramphenicol (Cam), kanamycin (Kan), tetracycline (Tet), ciprofloxacin (Cip), ampicillin (Amp), clindamicin (Cli) and erythromycin (Ery). This study would be very useful for safety evaluation of LAB strains in Chinese fermented food. 2. Materials and methods 2.1. Growth media

* Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China. Tel./fax: þ86 510 85329239. E-mail address: [email protected] (X. Wang). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.02.006

LSM broth (90% Isosensitest; 10% MRS, Oxoid) and antibiotic containing LSM (ALSM) were used for minimum inhibitory concentration (MIC) assays. LSM with 0.02% (w/v) bromocresol purple and 50 mg/ml nystatin (Generay, Shanghai, China), defined

L. Pan et al. / Food Control 22 (2011) 1316e1321

as LBN, and antibiotic containing LBN (ALBN), were used for determination of LAB’s AR incidences (ARI) and strain purification. Bromocresol purple as pH indicator would turn yellow when pH lowers below 5.2 and nystatin would inhibit yeast growth. Antibiotics (Sangon Biotech Co., Ltd, Shanghai, China) were added to LBN at the following concentrations equal to their highest breakpoints as suggested in EFSA (2008): 8 mg/ml Cam, 512 mg/ml Kan, 32 mg/ml Tet, 4 mg/ml Cip, 4 mg/ml Amp, 4 mg/ml Cli or 4 mg/ml Ery. MRS broth or agar was used for storing LAB strains.

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(2.5 mM each), 1 ml chromosome (5e100 ng/ml), 1 ml PCR primer fD1 (5 mM), 1 ml PCR primer rP2 (5 mM) and 17.25 ml ultra pure water. The reaction mixture was first heated to 94  C for 5 min, then the cycle of denaturation at 94  C for 30 s, annealing at 53  C for 30 s, and elongation at 72  C for 90 s were run 35 times. At the end, the reaction was incubated at 72  C for 10 min. Approximate 1500 bp fragments of the 16S rDNA were amplified by PCR, and were sequenced by Sangon Biotech (Shanghai, China). 2.4. MIC assays

2.2. Determination of ARI Sixteen Chinese fermented foods, including 8 fermented pork sausages and 8 pickles, were purchased from RT-Mark and refrigerated at 4  C before use. For sausage samples, 10 g were chopped and homogenized in a blender (BagMixer 400 CC, Interscience, France) with 90 ml of 0.85% (w/v) sterile physiological saline for 2 min, and serially diluted in ratio of 1:10. For pickle samples, the juice was directly used for the serial dilution. Next, 100 ml of appropriate dilutions were spread on LBN agar plate and another 100 ml on ALBN agar plate, and the plates were incubated at 30  C for 24e48 h. Colonies turned the media yellow on both plates were counted as LAB. The ratio of LAB numbers grown on ALBN to LBN agar plates was defined as ARI. All experiments were performed in triplicate. 2.3. Identification of antibiotic resistant LAB Antibiotic resistant colonies, which had different colonial morphology and turned the media yellow, were randomly selected from different ALBN plate and purified by re-streaking. All the purified isolates were further characterized by Gram staining, catalase testing and cell morphology analyzing. Their ability to produce lactic acid was also analyzed by RP-HPLC (Agilent 1200, USA) (Liang, Wang, & Jiang, 2006). The gram-positive, catalase negative, nonsporing, cocci or rod isolates which produced lactic acid as the major end product during glucose fermentation were identified as LAB (Axelsson, 2004), and stored at 80  C in MRS broth containing 20% glycerol. Several LAB colonies were randomly selected for molecular strain typing. Chromosome DNAs extracted from LAB using TIANamp Bacteria DNA kit (Tian Gen Biotech, Beijing, China) were used as template, and 16S rDNAs were amplified by PCR (Eppendorf, Hamburg, Germany). The 25 ml PCR reaction mixture contained 2.5 ml 10Ultra-Pfu PCR Buffer (with Mg2þ), 0.25 ml Ultra-Pfu DNA Polymerase (Bioedlfy Biotech, Nanjing, China), 2 ml dNTP mixture

The broth microdilution method (Klare et al., 2005) was used to evaluate MICs for each antibiotic. Briefly, a 96-well plate was inoculated with 198 ml ALSM broth containing serial (1:2) concentrations of antibiotics (0.25e128 mg/ml Tet; 1e512 mg/ml Ery; 1e2048 mg/ml Kan; 0.25e64 mg/ml Cam; 0.5e64 mg/ml Cip; 0.5e128 mg/ml Cli; 0.25e64 mg/ml Amp) and 2 ml fresh LAB samples. LAB cultures were first grown in 2 ml MRS for 24 h at 30  C and then subsequently diluted in 0.85% (w/v) physiological saline to the concentration of approximately 1  107 cell/ml. Bacteria inoculated in LSM were used as positive control, and a bacteria-free well was used as negative control. Plates were incubated under anaerobic conditions at 30  C for 48 h. MIC values of each antibiotic were visually evaluated as the lowest concentrations at which no growth was observed. Interpretation for susceptibility status was based on EFSA (2008). All the tests were repeated at least twice. In duplicate experiments, the differences of MIC for independent sample never exceeded 1 order of dilution. 2.5. PCR detection of AR genes Chromosomes and plasmids, if existed, were isolated from the 14 strains of LAB and used as templates for PCR to detect the known antibiotic resistance genes. Plasmid DNA was extracted from LAB using EZ-10Spin Column Plasmid Mini-Preps Kit (Bio Basic Inc, Beijing, China). PCR conditions were the same as described above except for the differences of primer sequences, annealing temperature and elongation time which are listed in Table 1. 3. Results and discussion 3.1. Diverse antibiotic resistance incidences were observed in different Chinese fermented foods Antibiotics had been spread in the environment when used as growth promoters in livestock years ago, leading to the selection of

Table 1 List of primers and PCR conditions used in this study. Primer pairs and their sequences (50 e30 )

Annealing temperature ( C)

Amplicon size (bp)

Reference

fD1: AGA GTT TGA TCC TGG CTC AG rP2: ACG GCT ACC TTG TTA CGA CTT catpIP501-F: GGA TAT GAA ATT TAT CCC TC catpIP501-R: CAA TCA TCT ACC CTA TGA AT aphA3-F: GCC GAT GTG GAT TGC GAA AA aphA3-R: GCT TGA TCC CCA GTA AGT CA tetM-F: GTT AAA TAG TGT TCT TGG AG tetM-R: CTA AGA TAT GGC TCT AAC AA gyrA-F: ACT TGA AGA TGT TTT AGG TGA T gyrA-R: TTA GGA AAT CTT GAT GGC AA blaZ-F: ACTTCAACACCTGCTGCTTTC blaZ-R: TAGGTTCAGATTGGCCCTTAG ermB-F: CAT TTA ACG ACG AAA CTG GC ermB-R: GGA ACA TCT GTG GTA TGG CG erm-F: CCG GGC CCA AAA TTT GTT TGA T erm-R: AGT CGG CAG CGA CTC ATA GAA T mefA-F: AGT ATC ATT AAT CAC TAG TGC mefA-R: TTC TTC TGG TAC TAA AAG TGG

53

1506

Weisburg, et al.,1991

50

486

Aarestrup, Agerso, Gerner-Smidt, et al., 2000

55

292

45

657

Van Asselt, Vliegenthart, Petit, Van de Klundert, & Mouton, 1992 Aarestrup, Agerso, Ahrens, et al., 2000

55

559

Schmitz et al., 1998

52

173

Martineau et al., 2000

52

405

Jensen, Frimodt-Moller, & Aarestrup, 1999

55

726

Lee & Morrison, 1999

52

348

Sutcliffe, Grebe, Tait-Kamradt, & Wondrack, 1996

1318

L. Pan et al. / Food Control 22 (2011) 1316e1321

antibiotic resistant bacteria (Devirgiliis, Caravelli, Coppola, Barile, & Perozzi, 2008). These bacteria may reside in or on fruits, vegetables and animal feeds (Pernezny, K udela, Kokosková, & Hládká, 1995; Umesha, 2006) and end up in the fermented foods (Fu, Xi, & Liu, 2008). Therefore, it is important to evaluate the ARI of bacteria in different fermented foods. In this study, 16 fermented Chinese foods, 8 sausages and 8 pickles, were initially chosen from different sources, but 3 sausage samples and 2 pickle samples were eliminated from assessment because there were few LAB isolated in them. Limited LAB population in these samples could be due to the presence of antiseptic or the pasteurization before marketing. Thus the ARI evaluation was only carried out to the remaining 11 Chinese foods, including fermented radish (RD), Chinese cabbage (CC), cucumbers (CB), mustard tuber (MT), assorted pickle (AP), sour and hot cabbage

(SHC) and five fermented sausages from Sichuan (SC), Yunnan (YN), Guangzhou (GZ), Shanghai (SH) and Jiangsu (JS) provinces, respectively. The concentrations of antibiotic added in ALBN were the same as the highest breakpoint suggested by FESA. As shown in Fig. 1, LABs in pickles and sausages have different patterns of ARI. The average ARI of the pickle samples were found to be less than that of sausage samples. LABs in the pickle samples were susceptible to Tet, Amp, and Cli with ARIs less than 5%, but were resistant to the other four antibiotics with ARIs up to 31%. LABs in SH sausage have ARI to Cli as low as 9.43% but to Kan as high as 43.22%. For sausage samples, LABs in JS, SH and GZ have higher ARI to Amp of more than 22%; while LABs in SC and YN have ARIs to Amp lower than 6%. All antibiotics used in this study are human or veterinary drugs and they are not supposed to present in food production. However, due to the overuse or misuse of antibiotics in China, antibiotic

Fig. 1. Incidences of resistance of LABs in different Chinese fermented foods to tetracycline(A), ampicillin (B), clindamicin (C), erythromycin (D), ciprofloxacin (E), chloramphenicol (F), and kanamycin (G). Total 11 food samples, including six pickles (black bars) and five fermented pork sausages (white bars) were tested.

L. Pan et al. / Food Control 22 (2011) 1316e1321

residues were detected in livestock, earth and water (Deng, Chen, & Zeng, 2009; Li, Liu, & Li, 2008; Wang & Ma, 2008), which might be the reasons for the high ARIs of LABs in Chinese fermented foods. The antibiotic residues in meat products mainly come from injection, eating and drinking (Shi, 2009; Zhang, Wang, Gao, & Li, 2006). For fermented sausages making, casing which was from small intestines of animal was requisite. Though it must be washed and air-dried before use, a few intestinal bacteria, including some antibiotic resistant ones, could still remain. One reason for high antibiotic residue in vegetables might be using antibiotic contaminated water, animal or human feces when growing them (Na et al., 2009; Whitlock, Jones, & Harwood, 2002). Recent study showed that Vetiver grass could take up 95.5% of the drugs from the water into its tissue (Casey, 2010), suggesting that vegetables could enrich antibiotics from environment. Since samples were collected from different cities and processed by different factories, materials could be harvested from various regions and processed in different conditions of antibiotic pollution (Devirgiliis et al., 2008), resulting in the diversity of ARIs. Furthermore, the reuse of some starter culture, such as some pickle juice, could make the spread of AR inescapable. 3.2. Various levels of antibiotic resistance exist in different LAB isolates Total 202 isolates were identified as LAB by physiological and biochemical methods. Among the 202 isolates, 14 were randomly selected and further analyzed by amplification and sequencing of 16S rDNA. They turned out to be 6 strains of Lactobacillus plantarum, 2 strains of L. fermentium, 2 strains of Enterococcus faecium, 1 strain of L. brevis, 1 strain of L. mali, 1 strain of L. helveticus and 1 strain of L. namurensis. MICs of these 14 strains to 7 antibiotics were assessed by broth microdilution method (Table 2). Each strain was able to grow on media added with different antibiotics. For each antibiotic, more than half of the 14 strains were resistant. There were 8 strains resistant to Kan, 9 strains to Ery, 9 strains to Cam, 9 strains to Tet, 11 strains to Amp, 12 strains to Cli and 12 strains to Cip. In addition,

1319

super-high MICs were observed for some strains, such as more than 1024 mg/ml for Kan, more than 512 mg/ml for Ery, more than 128 mg/ ml for Cli and Tet. All the 6 strains of Lactobacillus plantarum were resistant to Cli, Cip and Amp. Furthermore, plasmids were isolated from 13 out of the 14 LAB (Table 2). Multi-antibiotic resistance could be divided into cross-resistance and co-resistance (Chapman, 2003). Cross-resistance has the potential to occur when different antibiotics attack the same target or share the similar mechanisms of action and arises through the identical genetic mutations (Duval, 1985; Jeong et al., 2003; Min et al., 2003; Roberts, 2004). In this study, Ery-Cli cross-resistant LAB was found, such as L. plantarum SHC062, SHC154, SHC167. Co-resistance occurs when the resistance determinants locate together on a mobile genetic element, such as a plasmid, transposon, or an integron. L. plantarum SHC062, SHC096, SHC154, and E. faecium SZ109 might have co-resistance genes. 3.3. New antibiotic resistant genes might exist in these antibiotic resistant LABs These 14 antibiotic resistant LAB strains were tested by PCR for the presence of the most frequently detected AR genes: aphA3, catpIP501, ermB, erm, mefA, tetM, blaZ and gyrA. Genes aphA3 and catpIP501 encode enzymes modifying Kan and Cam (Aarestrup, Agerso, Ahrens, Jorgensen, Madsen, & Jensen, 2000; Van De Klundert & Vliegenthart, 1993), respectively; genes ermB and erm encode rRNA methylase which could prevent lincosamides and/or macrolides from binding rRNA (Sutcliffe, Grebe, Tait-Kamradt, & Wondrack, 1996); the gene mefA encodes a membrane efflux pump which could extrude macrolides (Sutcliffe, Grebe, Tait-Kamradt, & Wondrack, 1996); the product of tetM catalyzes the release of Tet from the ribosome (Aarestrup, Agerso, Ahrens, et al., 2000); the gene blaZ encodes betalactamase (McLaughlin, Murray, & Rabinowitz, 1981); and the gene gyrA produces the DNA gyrase subunit A which could combine fluoroquinolones (Schmitz et al., 1998). As shown in Table 3, genes erm, ermB, and tetM were detected on either plasmid or chromosomal DNA. tetM was detected in all the tetracycline resistant LAB strains, 4 of which carried tetM on both

Table 2 Antibiotic susceptibilities and MICs for 14 LAB isolates form Chinese fermented foods. MICs (mg/ml)

Strains Kan Lactobacillus plantarum SHC062 2048 SHC096 32 SHC124 64 HI134 8 SHC154 64 SHC167 128 L. brevis CC085 4 L. mali CB100 <1 L. fermentium CB101 >2048 MT133 4 L. helveticus CC135 >2048 L. namurensis CB136 128 Enterococcus faecium WX108 1024 SZ109 512

Cli

Ery

Cip

Plasmid Amp

Cam

Tet

R S R S R R

>128 4 128 4 >128 >128

R R R R R R

32 8 512 <1 32 256

R R R S R R

8 4 8 8 4 8

R R R R R R

8 4 16 8 32 8

R R R R R R

8 8 16 2 8 4

R R R S R S

>128 32 16 16 64 32

R R S S R R

þ þ þ þ þ þ

S

<0.5

S

4

R

8

R

4

S

2

S

1

S

þ

S

4

R

<1

S

<0.5

S

<0.25

S

S

32

R

þ

R S

16 <0.5

R S

512 <1

R S

>64 8

R R

32 8

R R

>64 2

R S

>128 32

R R

þ þ

R

2

R

<1

S

1

S

2

R

64

R

2

S

þ

R

2

R

<1

S

4

R

0.5

S

4

R

32

R

þ

S R

4 >128

R R

32 >512

R R

8 8

R R

R R

8 32

R R

<0.25 >128

S R

e þ

4 16

<0.25

The MIC breakpoints were chosen as suggested by EFSA (2008). Because no MIC breakpoint was given for Cip by the EEDAP, the MIC breakpoints of its class representatives (quinupristim and dalfopristin) were chosen. For the LAB strain that no specific MIC breakpoints have been published, the ones published for their group were chosen. For example, the MIC breakpoint L. helveticus was chosen for L. mali (obligate homofermentative group), L. fermentium for L. namurensis (obligate heterofermentative), and L. plantarum for L. brevis (facultative heterofermentative). R and S represent “resistant” and “sensitive”, respectively.

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L. Pan et al. / Food Control 22 (2011) 1316e1321

Table 3 PCR detection of AR genes in plasmids or chromosomes of the 14 LAB isolates. Strains

aphA3 P

Lactobacillus plantarum SHC062 e SHC096 / SHC124 e HI134 / SHC154 þ SHC167 e L. brevis CC085 / L. mali CB100 / L. fermentium CB101 e MT133 / L. helveticus CC135 e L. namurensis CB136 þ Enterococcus faecium WX108 / SZ109 þ

erm

ermB

mefA

gyrA

tetM

blaZ

catpIP501

C

P

C

P

C

P

C

P

C

P

C

P

C

P

C

e / e / e e

þ e e e e e

e e e e e e

e þ e e e þ

e e e e e e

e e e e e e

e e e þ e e

e e e e e e

e e e e e e

þ þ / e þ þ

þ þ / þ e þ

e e e / e /

e e e / e /

e e e / e /

e e e / e /

/

e

e

þ

e

e

e

e

e

/

/

/

/

/

/

/

e

e

e

e

e

e

e

e

e

þ

/

/

/

/

e /

e /

e /

e /

e /

e /

e /

e e

e e

e e

þ þ

e /

e /

e e

e e

e

e

þ

e

e

e

e

e

e

/

/

e

e

e

e

e

e

e

e

e

e

e

e

e

e

þ

/

/

/

/

/ e

/ e

e e

/ þ

e þ

/ e

e e

/ e

e e

/ þ

/ þ

/ /

e /

/ /

e /

P and C represent “Plasmid” and “Chromosome”, respectively; “þ” and “e” represent positive and negative results of PCR detections, respectively. “/” means that PCR was not performed because either no plasmid was isolated from the strain or the strain was not resistant to the relevant antibiotic.

plasmid and chromosome. Gene ermB was also detected on both plasmid and chromosome isolated from the strain Enterococcus faecium SZ109. Gene aphA3 was only plasmid detected and mefA was only detected on chromosome. Genes tetM and erm were detected in the same plasmid isolated from L. plantarum SHC062; genes tetM, ermB and aphA3 were detected in the same plasmid from E. faecium SZ109, suggesting these genes might transfer together through plasmid between different strains. Genes gyrA, blaZ and catpIP501 were not detected, suggesting that other AR genes might exist in these LAB strains. 4. Conclusion Traditional fermented foods play an important role in the food systems in China. However, no investigation has been conducted to assess the ARI of LAB in Chinese fermented foods. In this study, LAB have been isolated from 11 traditional Chinese fermented foods. High ARIs to clinically important antibiotics of LAB in some Chinese fermented food were observed, indicating the high gene transfer frequency of the microbes in Chinese food. Antibiotics, like Tet, Amp, Cli, showed strong bactericidal effect to LAB from pickles but they were not effective to LAB isolated from fermented sausages. The ARI of LAB from fermented sausages was much higher than that from pickles. In addition, genes erm, ermB, and tetM were detected on either plasmid or chromosomal DNA of certain LAB isolates, but not genes gyrA, blaZ and catpIP501, suggesting that new antibiotic resistant mechanism might exist in these LAB strains. The present research showed that AR, even multi-resistance, existed in LAB from Chinese traditional fermented food. Even though the genetic basis and associated resistance mechanisms of LAB in fermented foods need to be further studied, our study is useful for understanding of intrinsic and transferable AR of bacteria. Such studies will be conducive to safety assessment and control of fermented food in China. Acknowledgments Funding was provided by grants from the Program of State Key Laboratory of Food Science and Technology (SKLF-MB-200801), and the Basic Research Programs of Jiangsu Province (BK2009003).

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