Journal of Agriculture and Food Research 1 (2019) 100006

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Journal of Agriculture and Food Research

journal homepage: www.journals.elsevier.com/journal-of-agriculture-and-food-research/

Antibiotic resistance of lactic acid bacteria isolated from dairy products in

Tianjin, China
Kaidi Wang a, Hongwei Zhang a, b, Jinsong Feng a, c, d, Luyao Ma a, C
esar de la Fuente-Nún
~ez e,
f a, *
Shuo Wang , Xiaonan Lu
a
Food, Nutrition and Health Program, Faculty of Land and Food Systems, The University of British Columbia, Vancouver, BC, V6T1Z4, Canada
b
Animal & Plant & Foodstuffs Inspection Center of Tianjin Customs District, Tianjin, 300387, China
c
Michael Smith Laboratories, The University of British Columbia, Vancouver, BC, V6T1Z4, Canada
d
Department of Microbiology and Immunology, The University of British Columbia, Vancouver, BC, V6T1Z3, Canada
e
Synthetic Biology Group, MIT Synthetic Biology Center, The Center for Microbiome Informatics and Therapeutics, Research Laboratory of Electronics, Department of
Biological Engineering, Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, 02139, United States
f
Tianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai University, Tianjin, 300071, China

A R T I C L E I N F O A B S T R A C T

Keywords: Antibiotic resistance poses safety risk to public health. Limited studies have considered the spread of resistance
Antibiotic resistance due to bacteria used in food production. We conducted a study investigating the antibiotic resistance of lactic acid
Lactic acid bacteria bacteria from fermented dairy products in Tianjin. A total of 9 strains (3 Lactobacillus bulgaricus and 6 Streptococcus
Dairy products
thermophilus) were isolated from commercial yogurt and cheese. Antibiotic resistance to 4 antibiotics of all isolates
Resistance genes
was analyzed by disc diffusion method and the corresponding resistance genes were determined using PCR and
sequencing. Eight of 9 isolates were identified to be resistant to at least one antibiotic and 6 isolates displayed
multi-drug resistance. Occurrence rate of antibiotic resistant strains to vancomycin, neomycin, gentamycin and
streptomycin were 11.1%, 77.8%, 66.7% and 44.4%, respectively. The presence of antibiotic resistance genes van,
aph or aadA2 were identified in 6 resistant strains. Sequencing results of aph and aadA2 amplicon demonstrated
100% and 99% identity to the resistant genes in vector pEXKm4 and Lactococcus lactis subsp. cremoris, respec-
tively. L. bulgaricus and S. thermophilus used in dairy products can harbour antibiotic resistance genes and
disseminate the resistance through food. Screening for antibiotic resistance in fermented foods should be a routine
inspection for food safety.

1. Introduction to chromosomal mutation have a low potential for horizontal dissemi-

nation, whereas resistance due to the acquisition of mobile genetic ele-
Antibiotic resistance has been identified as one of the three greatest ments (e.g. plasmids or transposons) can be more easily disseminated
threats to human health [1]. Global dissemination of bacterial antibiotic [4].
resistance is associated with a high medical cost, increased hospitaliza- Previous epidemiological studies on antibiotic resistance have mainly
tion cases and mortality rates. It is estimated that in the United States at focused on clinically relevant pathogenic bacteria [5]. However, recent
least 2 million people are infected with antibiotic-resistant bacteria studies indicate that non-pathogenic bacteria, such as Lactobacillus,
annually, with over 23,000 deaths as a result [2]. Therefore, there is a which is a well-known probiotic, may also exhibit antibiotic resistance
demanding need to control the spread and reduce the risk of antibiotic and contribute to the dissemination of antibiotic resistance genes to other
resistant bacteria in various sectors, such as agri-foods. microorganisms, including human pathogens [6,7]. With this in mind,
Bacteria can be intrinsically resistant to antibiotics as a result of food products can serve as a critical dissemination channel leading to the
inherent structural or functional characteristics but can also develop transmission of antibiotic resistance from non-pathogenic bacteria to
antibiotic resistance through the mutation of chromosomal genes or consumers. Particularly, fermentation food may serve as a vehicle to
acquisition of exogenous DNA [3]. Intrinsic resistance and resistance due introduce large numbers of non-pathogenic antibiotic resistance bacteria

* Corresponding author.
E-mail address: xiaonan.lu@ubc.ca (X. Lu).

https://doi.org/10.1016/j.jafr.2019.100006
Received 17 October 2019; Received in revised form 7 November 2019; Accepted 8 November 2019
2666-1543/© 2019 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
K. Wang et al. Journal of Agriculture and Food Research 1 (2019) 100006

into the human gastrointestinal tract, interact with the gut microflora and M17 agar plates (Oxoid, Italy) in aerobic environment at 37  C for 48 h.
disseminate antibiotic resistant genes [8]. Presumptive colonies with typical characteristics, namely pure white,
Fermentation is often enabled by lactic acid bacteria (LAB), a group of small, and containing entire margins, were selected from each plate and
Gram-positive, facultative anaerobic and fermentative bacteria that are sub-cultured onto the fresh plates to ensure the purity.
widespread in the natural environment and frequently used in the food
industry [9]. Recent studies indicate that LAB can potentially disseminate 2.2. Identification of LAB
antibiotic resistance through the entire food chain [5]. For example, LAB
can acquire antibiotic resistant genes from the resistant bacteria in the Biochemical assays were adopted for the identification of Lactobacilli
raw milk and subsequently transferred mobile resistant gene to other and Streptococci [12]. All the isolates were tested for Gram staining,
bacteria during food processing [6]. Over the last decade, catalase and oxidase activity, cell morphology, and spore formation. The
antibiotic-resistant LAB have been frequently isolated from fermented putative strains were then further tested for the production of acids from
foods, such as dairy products, wine and meat [10]. Further, carbohydrates and related compounds by using API 50 CH and API 20
antibiotic-resistant genes on conjugative plasmids or transposons in LAB STREp kits (BioMerieux, France). API tests were performed according to
have also been reported, which potentially lead to horizontal gene the manufacturer's instruction. The results were collected after incuba-
transfer [11]. Therefore, there is an urgent need to investigate the tion for 24 h and 48 h at 37  C and analyzed for species identification.
resistance profile of LAB in food industry. Purified isolates were stored in MRS broth (for Lactobacilli) or M  17
Little is known about the antibiotic resistance of LAB isolated from broth (for Streptococci) with 15% (w/v) glycerol at -80  C.
fermented dairy products in China. Tianjin is one of the major port cities
as well as the economic center of north China with a population of over 2.3. Antibiotic susceptibility testing
15.6 million. In addition, it has one of the largest dairy industries in Asia.
In the current study, we aimed to investigate the antibiotic-resistance Antibiotic susceptibility of 9 isolated LAB strains was evaluated by
profiles of most commonly used LAB starters, namely Lactobacillus bul- using the standard disk diffusion method. Briefly, single colony of each
garicus and Streptococcus thermophilus, isolated from fermented dairy isolate was picked up to prepare the overnight culture in the corre-
products in Tianjin, China and characterize the corresponding resistant sponding media. A total of 200 μL of the inoculum (diluted to approxi-
genes. This preliminary study serves as a sample of the current situation mately 105 CFU/mL) were evenly spread on MRS or M  17 agar plates
regarding antibiotic resistance profiles of dairy products associated with and dried at room temperature for 15 min. Antibiotic-containing disks
LAB and provides important information for further monitoring and (Oxoid, Italy) were placed onto the plates. The diameters of inhibition
controlling the dissemination of drug-resistant genes via the food chain at zones were measured after incubation of 16 h at 37  C. The resistance
a large scale. tests were conducted with 4 different antibiotics, including vancomycin
(30 μg per disk), neomycin (30 μg per disk), gentamycin (10 μg per disk),
2. Materials and methods and streptomycin (10 μg per disk). The results were interpreted following
the breakpoints proposed in a previous study [13,14].
2.1. Isolation of LAB and growth conditions
2.4. PCR detection of antibiotic resistance genes
LAB strains were isolated from commercial dairy products including
yogurt (n ¼ 7) and cheese (n ¼ 2). The detailed information is shown in The genomic DNA of LAB strains was extracted using Presto™ Mini
Table 1. Dairy samples were obtained from several local markets in gDNA Bacteria Kit (FroggaBio, Ontario, Canada) according to the man-
Tianjin, China. The isolation was performed following the methods ufacturer's instruction. Genes responsible for the resistance to vanco-
described in a previous study with some modifications [3]. Briefly, the mycin (van), gentamycin (aadB), neomycin (aph), and streptomycin
dairy samples were homogenized and then serially diluted in the sterile (aadA2) were investigated by using PCR with the primers described in
saline water. For the isolation of L. bulgaricus, diluted samples were Table 2. PCR reaction was performed in a total volume of 50 μL that
plated on De Man Rogosa Sharpe (MRS) agar plates (Oxoid, Italy) and contained 0.5 μM of each primer, 0.3 mM of dNTP, 1 buffer (MgCl2
incubated anaerobically at 37  C for 48 h. S. thermophilus were isolated on included), 3 μL of purified gDNA, and 2.5 U of Taq DNA Polymerase.
PCR amplification was performed using a thermal cycler (Eppendorf,
Hamburg, Germany), according to the following program: initial dena-
Table 1 turation at 94  C for 3 min, followed by 35 cycles of 94  C for 40 s, 56  C
The origin of lactic acid bacteria isolated from commercial fermented dairy
for 40 s, 72  C for 50 s, and ended with 72  C for 8 min. The PCR
products in Tianjin, China.
amplicons were analyzed by using 1% (w/v) agarose gel electrophoresis.
No. Species Strains Origin Producer

1 S. S1 Yili original probiotic Inner Mongolia Yili 2.5. Sequencing and analysis of antibiotic resistance genes
thermophilus yogurt Industrial Group Co.,
Ltd
Positive amplicons of aph and aadA2 were purified using QIAGEN
2 S. S2 Mengniu original China Mengniu Dairy
thermophilus yogurt Co., Ltd PCR purification kit (Hilden, Germany) and sequenced by Beijing
3 S. S3 Bright JianNeng Bright Dairy & Food Genomic Institute (Beijing, China). The sequencing results were analyzed
thermophilus AB100 probiotic Co., Ltd by using the BLAST program available at the National Center for
yogurt Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.
4 S. S4 Mengniu LABS China Mengniu Dairy
thermophilus probiotic yogurt Co., Ltd
gov/BLAST).
5 S. S5 Inner Mongolian Farmers' market
thermophilus cheese 3. Results
6 S. S6 Inner Mongolian Farmers' market
thermophilus cheese
3.1. Isolation and identification of LAB
7 L. bulgaricus L1 Hehai BIO-2 probiotic Tianjin Haihe Dairy
yogurt Co., Ltd
8 L. bulgaricus L2 Mengniu original China Mengniu Dairy A total of 9 LAB strains including S. thermophilus (n ¼ 6) and
yogurt Co., Ltd L. bulgaricus (n ¼ 3) were isolated from commercial yogurt and cheese
9 L. bulgaricus L3 Sanyuan Yijunduo Beijing Sanyuan Foods products purchased in Tianjin, China (Table 1). All the tested commercial
original yogurt Co., Ltd
samples claimed the presence of L. bulgaricus and S. thermophilus on their

2
K. Wang et al. Journal of Agriculture and Food Research 1 (2019) 100006

Table 2
PCR primers used for the detection of antibiotic resistance genes in lactic acid bacterial strains.
Antibiotic resistance Target gene Primer sequence (5'to 30 ) Ta/ C Size/bp Accession number

vancomycin van Uv1: TTGGCGTCGTGTTAGGGA 57.2 503 FM200053

Lv2: CTGATGCTGCGTGGGAATAGG
gentamycin aadB Ug1: CACAACGCAGGTCACATTGATA 55.4 414 EU042136
Lg2: GGTACTTCATCGGCATA
neomycin aph Un1: ACAAGATGGATTGCACGCAGGT 59.7 624 AB255435
Ln2: CGGCCACAGTCGATGAAT
streptomycin aadA2 Ust1: GCTTACCTCGCCCGTTAGACAT 58.6 762 AB297447
Lst2: CCAAGTGATCTGCGCGTGA

van: coding for vancomycin resistance protein.

aadB: coding for aminoglycoside 20 -O- adenylyl transferase.
aph: coding for aminoglycoside-30 -O- phospho transferase.
aadA2: coding for streptomycin/spectinomycin3'adenyl transferase.

product labels. Both L. bulgaricus and S. thermophilus were isolated from investigated using PCR to amplify the known resistance genes listed in
Mengniu original yogurt, while other dairy products were identified to Table 2. PCR amplicons of van (503bp), aadB (414bp), aph (624bp) and
contain either L. bulgaricus or S. thermophilus. aadA2 (762bp) are shown in Fig. S1. The results of PCR amplification are
summarized and compared with the phenotypic antibiotic resistance in
3.2. Phenotypic profiles of antibiotic resistance of LAB Table 3. The gene encoding vancomycin resistance protein was detected
in 3 strains of LAB. However, only L. bulgaricus L1 showed phenotypic
The antibiotic susceptibility of 9 LAB isolates to 4 antibiotics (i.e. resistance to vancomycin while the other 2 stains were susceptible.
vancomycin, gentamycin, neomycin and streptomycin) was evaluated Among the 7 neomycin-resistant LAB strains, corresponding resistance
using disc diffusion method. The average diameters of the inhibition zone gene aph was identified in 4 strains whereas negative results were
are provided in Table S1. The antibiotic susceptibility results are sum- observed for the remaining 3 strains. The gene aadB is known to be
marized in Table 3, where isolates were categorized as resistant (R) or responsible for gentamycin resistance. However, none of the LAB strains
sensitive (S), according to the breakpoints proposed in other previous possessed this gene although a high level of gentamycin-resistant strains
studies [13,14]. (66.7%) was identified using the disc diffusion method. Similarly, for the
Eight LAB isolates (88.9%) were identified to be resistant to at least 4 strains that exhibited streptomycin resistance, no positive PCR result of
one antibiotic tested, while only S. thermophilus S4 was susceptible to all aadA2 was detected. Nevertheless, 2 strains resistant to neomycin and
the tested antibiotics. One S. thermophilus strain (S1) and one L. bulgaricus gentamycin were positive for aadA2. The sequencing results for aph and
strains (L3) were identified to be multi-drug resistant against both gen- aadA2 were analyzed by BLAST and identification to the genes in GEN-
tamycin and neomycin. Two S. thermophilus strains (S3 and S5) and two BANK is described in Table 4. Sequences for amplicons of aph showed
L. bulgaricus (L1 and L2) displayed multi-drug resistant to 3 different 100% similarity to the gene encoding neomycin phosphotransferase II in
antibiotics. Resistant strains with the highest occurrence were against gene replacement vector pEXKm4. Sequences of aadA2 amplicons were
neomycin (77.8%), including 5 out of 6 S. thermophilus and 2 out of 3 validated to be 99% identical to the gene responsible for tRNA adenosine
L. bulgaricus. Six LAB strains (66.7%) displayed resistance to gentamycin. deaminases in Lactococcus lactis subsp. cremoris.
For streptomycin, 2 S. thermophilus strains S3, S5 and 2 L. bulgaricus
strains L1, L2 (44.4%) were characterized as resistant while all the other 4. Discussion
6 strains were susceptible to this antibiotic. Besides, antibiotic resistance
to vancomycin was observed only from one L. bulgaricus strain L1 In the current study, a total of 9 strains of L. bulgaricus and
(11.1%). S. thermophilus were isolated from commercial yogurts and cheese. The
resistance profiles to different types of antibiotics including 1) inhibitors
of cell wall synthesis such as glycopeptides (vancomycin); 2) inhibitors of
3.3. Detection of antibiotic resistance genes
bacterial synthesis on the 30S ribosomal subunit such as aminoglycosides
(neomycin, gentamycin and streptomycin) and their corresponding
The genetic basis for the observed phenotypic resistance was

Table 3
Comparison between phenotypic antibiotic resistance analyzed by the disc diffusion method and the presence of genotypic determinants determined by using PCR.
Species Strains Glycopeptides Aminoglycosides

VAN van NEO aph GEN aadB STR aadA2

S. thermophilus S1 S þ R þ R – S –
S2 S – R – S – S –
S3 S – R – R – R –
S4 S – S – S – S þ
S5 S – R þ R – R –
S6 S – R – S – S þ
L. bulgaricus L1 R þ S þ R – R –
L2 S – R þ R – R –
L3 S þ R – R – S þ
Occurrence rate 11.1% 77.8% 66.7% 44.4%

VANr, NEOr, GENr, STRr: phenotypic antibiotic resistance to vancomycin, neomycin, gentamycin and streptomycin, respectively.
R: resistant; S: susceptive.
þ: positive results; -: negative results.
van: coding for vancomycin resistance protein; aadB: coding for aminoglycoside 20 -O- adenylyl transferase; aph: coding for aminoglycoside-30 -O- phospho transferase;
aadA2: coding for streptomycin/spectinomycin3'adenyl transferase.

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K. Wang et al. Journal of Agriculture and Food Research 1 (2019) 100006

Table 4
Identification of sequencing results for antibiotic-resistance genes in lactic acid bacteria.
Genes Length (bp) Accession number Organism Similarity (%) Encoding products

aph 589 FJ797516 Gene replacement vector pEXKm4 100% Neomycin Phosphotransferase II
aadA2 663 AM406671 Lactococcus lactis subsp. cremoris 99% tRNA adenosine deaminases

resistance genes (van, aph, aadA2 and aadB) were analyzed in this study. with the phenotypic results (Table 3). Some resistant strains in the cur-
The presence of antibiotic resistance was identified in 8 out of 9 LAB rent study did not carry associated resistance genes. For example, aadB
strains isolated from the fermented dairy products, and the resistance was not detected in any of the gentamycin resistant strains. There are
genes were identified in 6 resistant strains, namely van in L. bulgaricus L1, many possible factors to explain this result. For instance, the emergence
aph in S. thermophilus S1, S5 and L. bulgaricus L1, L2 and aadA2 in of resistance in these strains may be caused by mutations and the un-
S. thermophilus S6 and L. bulgaricus L3. derlying genetic determinants may not be well understood. Another
Antibiotic resistance to aminoglycosides was identified in 8 out of 9 possible explanation is that the acquired resistance genes carried by
LAB isolates except for S. thermophilus S4. The occurrence rate of the transposon or plasmid were not detected by the method used in this
resistant strains to neomycin, gentamycin and streptomycin was 77.8%, study. On the other hand, some strains with positive results of resistance
66.7% and 44.4%, respectively. Although a limited number of isolates genes did not demonstrate phenotypic antibiotic resistance. For instance,
was included in this study, these data were in accordance with others two S. thermophilus strains (S4 and S6) were susceptible to streptomycin
who have also reported a high percentage of LAB strains that were although they harbour the aadA2 gene that encodes for streptomycin
resistant to aminoglycosides [15–17]. Membrane impermeability has resistance. These results may be explained by the low levels, or down
been regarded as the main mechanism behind the resistance of LAB to regulation, of gene expression or by an inactive gene product [26].
aminoglycosides because most species of this genus lack the Antibiotic resistant LAB might be beneficial for the patients who are
cytochrome-mediated electron transport that can mediate drug uptake suffering the antibiotic-induced diarrhea as these strains can survive
[18]. Some non-specific mechanisms, such as multidrug transporters better under antibiotic pressure and contribute to the maintenance of the
[19] and defective cell wall autolytic systems [20], may contribute to the gastrointestinal stasis [14,27]. However, from the food safety perspec-
antibiotic resistance. Besides, resistance to aminoglycosides might also tive, LAB used in food fermentation should not be antibiotic resistant.
be mediated by other genes, such as aph, ant(6), aph(3) and When the resistant LAB are used as probiotics or starter cultures, a large
aac(6)-aph(2) [10,21,22]. In addition, the influence of low pH of the test number of cells enter the human intestine and interact with the indige-
media (MRS agar, 6.2  0.2) might result in the reduced antimicrobial nous intestinal microbiota. Previous research indicated that antibiotic
effect of aminoglycosides (optimum pH, 7.8) [17]. In contrast to the resistant genes showed the potential to be transferred to commensal
current study, Zhou and coauthors reported that a high portion of bacteria or enteric pathogenic bacteria through horizontal gene transfer
L. bulgaricus and S. thermophilus tested was susceptible to gentamycin [5,28], and may pose a serious threat to food safety and public health. To
[17]. Moreover, the susceptibility to neomycin was demonstrated in prevent the undesirable resistant gene transfer, LAB used in food industry
another study [23]. The occurrence of antibiotic resistance among LAB should not carry resistance other than that specifically required. There-
from food sources usually varies among different studies. Firstly, various fore, routine inspection on the antibiotic resistance profile of commonly
methods were applied in different studies to evaluate antibiotic resis- used LAB strains is critical.
tance, including E-test, agar dilution, microbroth culture and disk We found a high prevalence of antibiotic-resistant LAB isolated from
diffusion, the results of which could not be directly compared [15]. In dairy products in Tianjin, China, thus highlighting the need for strict
addition, culture conditions such as culture media or inoculum volume monitoring and regulation in the food industry. Only one strain was
may also influence the susceptibility test and outcome. In addition, the susceptible to all antibiotics and multi-resistance to most antibiotics was
location of a particular resistance gene (in the chromosome or in a identified in others. Antibiotic-resistant genes were detectable in some
plasmid) [8] or the involvement of other non-specific mechanisms may strains with resistant phenotypes. Further work should focus on testing
also account for different results. the transferability of genetic determinants. Evaluation of the safety of
Resistance to vancomycin of Lactobacilli was generally considered as LAB consumption must be guided by establishing criteria and regulation,
intrinsic due to the presence of D-Ala-D-lactate instead of natural D-Ala-D- and standardized methods for pre-market biosafety testing and post-
Ala dipeptide in their peptidoglycan [24]. For example, all of the Lacto- market surveillance.
bacillus strains were resistant to vancomycin as reported by Morandi and
co-workers [7]. However, only one strain of Lactobacilli detected in the Author contributions statement
current study was resistant to vancomycin, which did not support the
intrinsic resistance of LAB to this antibiotic. Similar study has been re- XL and KW designed the study. KW and HZ conducted the experi-
ported by Nawaz and coworkers, where all 13 L. bulgaricus and 11 ments and interpreted the results. KW drafted the manuscript. JF, LM, CF
S. thermophilus strains were susceptible to vancomycin [3]. As afore- and SW provided critical feedback and helped revise the paper.
mentioned, the differences may be due to different methods, media used
in the antibiotic susceptibility testing or different origin of the isolates Declaration of competing interest
investigated.
Multidrug resistance of LAB strains was identified in the current The authors declare no conflict of interest.
study. This was in agreement with various reports indicating that LAB are
normally resistant to the several commonly used types of antibiotics, Acknowledgement
such as aminoglycosides, quinolones and fluoroquinolones [25]. In a
recent study conducted by Zhou and others [17], both L. bulgaricus and Financial support to X.L. in the form of a Discovery Grant from the
S. thermophilus exhibited combined resistance to streptomycin, neomycin Natural Sciences and Engineering Research Council of Canada (NSERC
and gentamycin. RGPIN-2014-05487) is greatly acknowledged.
Regarding the genotypic determinants, one or more common resis-
tance genes (van for glycopeptides; aph and aadA2 for aminoglycosides) Appendix A. Supplementary data
were detected in 6 out of 8 resistant LAB strains. However, the presence
of antibiotic resistance genes detected by PCR was not fully consistent Supplementary data to this article can be found online at https://do

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