Antibiotic Resistance of Lactobacillus spp. and Streptococcus thermophilus Isolated from Chinese Fermented Milk Products

Abstract

The aim of the present study was to investigate the phenotypic and genotypic antimicrobial resistance and the transferability of resistance markers in 87 lactic acid bacterial strains recovered from fermented milk products obtained from different areas of China. The isolates were identified as 21 Lactobacillus bulgaricus, 8 Lactobacillus casei, 6 Lactobacillus rhamnosus, 3 Lactobacillus paracasei, 2 Lactobacillus acidophilus, and 47 Streptococcus thermophilus strains. High levels of intrinsic resistance were revealed among the tested species. The following resistance genes were detected in strains isolated from fermented milk products: tet(M) in two L. bulgaricus and two S. thermophilus isolates, strA and strB in nine and seven S. thermophilus isolates, respectively; sul1 in six L. bulgaricus and seven S. thermophilus isolates, sul2 in one S. thermophilus isolate, aac(6′)-aph(2″) in two L. bulgaricus isolates, and aph(3″)-II and aph(3″)-III in one S. thermophilus and two L. bulgaricus isolates, respectively. Transfer of the monitored antibiotic resistance genes was not observed in the filter mating assays of this study. To our knowledge, the strA, strB, sul1, sul2, and aph(3″)-II genes in S. thermophilus, and the sul1 and aac(6′)-aph(2″) genes in L. bulgaricus were identified for the first time. These results indicate the potential risks posed by lactic acid bacteria (LAB) in fermented milk products in expanding the antibiotic resistance gene reservoir and transferring antibiotic resistance genes among bacteria. Further investigations are required to identify the potential sources of contamination and the dissemination routes of antibiotic resistance genes among LAB in fermented milk products.

Introduction

L actobacillus spp. and Streptococcus thermophilus have been used in producing several probiotic products, including yogurt, fermented milk, cheese, and ice cream (Abriouel et al., 2015; Lollo et al., 2015; Batista et al., 2017; Balthazar et al., 2018; Silva et al., 2018). These food products rich in lactic acid bacteria (LAB) are very popular among consumers and are often ingested in large quantities due to a myriad of potential health benefits, such as alleviation of lactose intolerance, peptic ulcers, improving the intestinal microecological balance, prevention of colon cancer, antiallergic effects, antifungal actions, stimulation of the immune system, and delay of the aging process (Masood et al., 2011; Sperry et al., 2018).

LAB are often considered to be probiotics, live microorganisms that when administered in adequate amounts confer a health benefit on the host (Hill et al., 2014). However, as bacterial antibiotic resistance is increasing worldwide, intrinsic antibiotic resistance and the nature of the acquisition and distribution of antimicrobial resistance by LAB in food production have raised serious concerns (Abriouel et al., 2015).

Fermented milk products found in retail markets are often consumed without any heat treatment, and therefore, the LAB in these products can potentially become part of the indigenous human microbiome and interact directly with the human gastrointestinal tract and the microbiota (Chiara et al., 2013). Starter cultures and LAB in fermented food products, such as S. thermophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus reuteri, and Lactobacillus acidophilus, that are resistant to one or multiple antibiotics have previously been reported (Guo et al., 2017; Ledina et al., 2018). Furthermore, these antibiotic resistance genes present in LAB have been known to spread between different LAB species via horizontal gene transfer (Marshall et al., 2009; Nawaz et al., 2011). As the number of LAB in fermented food products is usually very high, the frequency of antibiotic resistance gene transfer between LAB and pathogenic bacteria during the passage through the intestinal tract could be high and potentially pose a threat to human health (Flórez et al., 2005). Therefore, studies on the presence and extent of antimicrobial-resistant LAB in fermented milk products are critically important.

Guidelines for the safety evaluation of probiotics have been produced by the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO/WHO, 2002). According to these guidelines, assessment of antibiotic resistance in the starter cultures for fermented food products is required before any use in commercial food production. However, assessment of the antibiotic resistance status of LAB in fermented food products on the retail market is usually ignored. As far as the authors are aware, despite a high consumption level of fermented milk products in China, there are limited reports available on the prevalence and characteristics of antibiotic-resistant LAB in these products. Thus, the present study aimed to evaluate antibiotic susceptibility and the presence of selected antibiotic resistance genes within LAB isolated from fermented milk products manufactured in different parts of China, and to analyze the transferability of detected markers of resistance genes. This study may provide important data for further investigations into the potential risks associated with the handling and consumption of fermented milk products containing antibiotic-resistant LAB.

Materials and Methods

Sample collection

Between September 2013 and April 2014, a total of 72 fermented milk products were collected. One to 8 different fermented milk products containing two or more species of LAB were obtained from 11 major producers in 3 main supermarkets of Guangzhou City, China (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/fpd). All fermented milk products were lactic acid bacterium drinks or yogurts with different flavors. All samples were transported to the laboratory under refrigerated conditions and were processed for bacterial isolation immediately after arrival.

Bacterial isolation, identification, and cultivation

Isolation of LAB was performed according to the standard procedures described in the National Standards of the People's Republic of China (GB4789.35-2010). Briefly, 25 mL of each sample was collected aseptically and mixed thoroughly with 225 mL of 0.85% NaCl solution. The mixture was then serially diluted, spread onto de Man, Rogosa, and Sharpe (MRS) and M17 (NA; Huankai Ltd., Guangzhou, Guangdong, China) agar plates, and incubated anaerobically at 37°C for 24–48 h. After incubation, five morphologically different colonies were choosen and subcultured for further identification by 16S rRNA gene sequencing and comparison with sequences in GenBank using BLAST, and further confirmed by conventional biochemical methods using the Microbial Biochem Identification Tube System (HKM, Inc., China). The polymerase chain reaction (PCR) primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used to amplify 1465 bp 16S rRNA gene fragment. PCR conditions were as follows: 95°C for 5 min; 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min; and then 72°C for 10 min. For the same species from the same sample, only one isolate was chosen for further analysis. All isolates were stored in MRS or M17 broth containing 15% glycerol at −80°C.

The strains used as recipients in the gene transfer experiments were cultured as follows: Streptococcus mutans UA159 was cultured anaerobically utilizing Brain Heart Infusion (BHI, NA; Huankai Ltd.), Enterococcus faecium GE1 and Enterococcus faecalis OG1RF using BHI broth or agar plates containing 100 μg/mL rifampin, and Escherichia coli J53 was cultured utilizing Luria-Bertani (LB, NA; Huankai Ltd.) broth or agar plates containing 150 μg/mL sodium azide.

Antibiotic susceptibility testing

The phenotypic susceptibility of all strains to 11 antibiotic compounds, belonging to 9 different classes, was determined as the minimum inhibitory concentration (MIC) defined as the lowest antibiotic concentration able to inhibit the visible growth of a microorganism after incubation (Andrews, 2001). The MIC of the antibiotics (ampicillin, streptomycin, gentamicin, kanamycin, tetracycline, chloramphenicol, erythromycin, ciprofloxacin, clindamycin, trimethoprim, and vancomycin) was determined by the microdilution method on LAB susceptibility test medium (NA; Huankai Ltd.) according to a modification of the Clinical and Laboratory Standards Institute (CLSI) standards (CLSI, 2012). The assays were performed in duplicate and repeated three times. Susceptibility to antibiotics was determined according to the European Food Safety Authority (EFSA, 2012) guidelines and European Commission (EC, 2001) (Supplementary Table S2). E. faecalis ATCC 29212 was included as a quality control organism.

Detection of antibiotic resistance genes, class 1 integron, and the Tn916 transposon

Extraction of bacterial DNA was performed utilizing the QIAGEN DNeasy 96 Blood & Tissue Kit (QIAGEN). A concentration of 50 to 100 ng/μL was used in each PCR as templates. The presence of genes associated with aminoglycoside resistance [aac(6′)-aph(2″), aphA3, aph(3″)-I, aph(3″)-II, aph(3″)-III, aadA, aadE, ant6, strA, and strB], chloramphenicol resistance (catA), glycopeptide resistance (vanA, vanH, vanR, vanS, vanX, vanY, vanZ, vanB, and vanC), tetracycline resistance [tet(M), tet(L), tet(S), tet(K), and tet(W)], sulfonamide resistance (sul1 and sul2), the tet(M) gene-associated transposon Tn916, and the gene encoding the class 1 integron (intI1) was analyzed by conventional PCR using a commercial amplification kit (TAKARA). PCR amplicons were confirmed by Sanger sequencing. Primers and annealing temperatures are described in Supplementary Table S3.

Conjugation assays

Conjugation assays were performed by the membrane filter mating method as described previously (Li et al., 2016). The four tetracycline-resistant strains [containing the tet(M) gene] isolated in the present study were used as donors, while E. coli J53 (sodium azide^R), S. mutans UA159, E. faecium GE1 (rifampin^R), and E. faecalis OG1RF (rifampin^R) were used as recipients. All matings were repeated three times in duplicate.

Selection of E. faecium GE1 and E. faecalis OG1RF transconjugants was performed by cultivation on BHI plates supplemented with rifampin (25 μg/mL) and tetracycline (30 μg/mL). LB plates supplemented with sodium azide (150 μg/mL) and tetracycline (30 μg/mL) were used for the selection of E. coli J53 transconjugants. BHI agar plates supplemented with tetracycline (30 μg/mL) were used for the selection of S. mutans UA159 transconjugants. All plates were placed at 37°C for 48 h. Transconjugants were subjected to examination of their antibiotic susceptibility, plasmid contents, and the presence of the transposon-associated tet(M) gene by PCR amplification followed by a DNA sequence analysis of the products.

Results

Bacterial species

A total of 87 LAB isolates, belonging to 6 different species, were isolated in this study. Strains isolated from fermented milk products included 21 L. bulgaricus, 8 Lactobacillus casei, 6 Lactobacillus rhamnosus, 3 L. paracasei, 2 L. acidophilus, and 47 S. thermophilus.

Antibiotic susceptibility

Antibiotic susceptibility of the Lactobacillus spp. and S. thermophilus strains was analyzed and classified either as resistant (R) or sensitive (S) based on the break points provided by the EFSA and EC guidelines.

All Lactobacillus spp. strains from the fermented milk products were determined to be susceptible to ampicillin, erythromycin, and clindamycin, but resistant to streptomycin, kanamycin, and ciprofloxacin with MIC values up to 1024, 512, and 64 μg/mL, respectively. The majority of Lactobacillus strains were susceptible to chloramphenicol and tetracycline. Only one isolate of L. bulgaricus strain was resistant to chloramphenicol and one L. casei and 3 L. acidophilus strains were resistant to tetracycline. Most of the lactobacillus spp. were resistant to gentamicin with MIC values ranging from 16 to 512 μg/mL, only with the exception of one L. casei and L. acidophilus strain, which were found susceptible. For vancomycin, all L. rhamnosus and L. paracasei strains as well as 75% (6/8) of the L. casei and 57.1% (12/21) of L. bulgaricus strains were resistant, whereas the L. acidophilus strains were susceptible. All of the L. rhamnosus and L. acidophilus strains and most of the L. casei (75%, 6/8), L. bulgaricus (66.7%, 14/21), and L. paracasei (66.7%, 2/3) strains were resistant to trimethoprim (Table 1).

Table 1.

Minimum Inhibitory Concentration Distributions of Lactobacillus spp. and Streptococcus thermophilus Isolated from Fermented Milk Products

			Number of strains with MIC (μg/mL) as follows:
Antibiotic agent	Species	Number of strains	≤0.125	0.25	0.5	1	2	4	8	16	32	64	128	256	512	1024	>1024	Resistance (%)
Ampicillin	Lactobacillus casei	8	2	3	3													0
	Lactobacillus rhamnosus	6		1	1	2	2											0
	Lactobacillus bulgaricus	21	5	10	6													0
	Lactobacillus acidophilus	2	1	1														0
	Lactobacillus paracasei	3		2	1													0
	Streptococcus thermophilus	47		3	5	18	15	2	2	2								12.8
Chloramphenicol	L. casei	8																0
	L. rhamnosus	6		6														0
	L. bulgaricus	21		1	4	2	5	8	1									4.8
	L. acidophilus	2		2														0
	L. paracasei	3		3														0
	S. thermophilus	47		23		15	9											0
Streptomycin	L. casei	8											3	2	2	1		100
	L. rhamnosus	6										4	1	1				100
	L. bulgaricus	21									5	6	2	5	2	1		100
	L. acidophilus	2											2					100
	L. paracasei	3											1	2				100
	S. thermophilus	47								2	1	1	12	10	15	6		91.5
Gentamicin	L. casei	8									1	2	2	1	2			87.5
	L. rhamnosus	6									2	2	1	1				100
	L. bulgaricus	21								1	8	6	4	1	1			95.2
	L. acidophilus	2										2						100
	L. paracasei	3											3					100
	S. thermophilus	47						9	18	15	1	1	2	1				8.5
Kanamycin	L. casei	8											3	2	3			100
	L. rhamnosus	6											4	1	1			100
	L. bulgaricus	21									8	4	4	2	3			100
	L. acidophilus	2											2					100
	L. paracasei	3											1	2				100
	S. thermophilus	47											22	10	8	5	2	100
Tetracycline	L. casei	8				1	4	2		1								12.5
	L. rhamnosus	6			2	1	2	1										0
	L. bulgaricus	21		2	5	2	4	5	1	2								14.3
	L. acidophilus	2		2														0
	L. paracasei	3			3													0
	S. thermophilus	47			2	8	20	15		1	1							4.3
Ciprofloxacin	L. casei	8							2	4	1	1						100
	L. rhamnosus	6							1	2	2	1						100
	L. bulgaricus	21							5	4	6	6						100
	L. acidophilus	2							2									100
	L. paracasei	3							3									100
	S. thermophilus	47			2	10	10	25										0
Trimethoprim	L. casei	8								1	1	4	1	1				75
	L. rhamnosus	6										6						100
	L. bulgaricus	21								2	5	12	2					66.7
	L. acidophilus	2										1	1					100
	L. paracasei	3									1	2						66.7
	S. thermophilus	47							1	22	16	1	5	2				17
Vancomycin	L. casei	8						2								1	5	75
	L. rhamnosus	6												1			5	100
	L. bulgaricus	21		3	5	1						6	6					57.1
	L. acidophilus	2				2												0
	L. paracasei	3													3			100
	S. thermophilus	47				19	25								2		1	6.4

MIC, minimum inhibitory concentration.

All of the S. thermophilus isolates from fermented milk products were found to be susceptible to chloramphenicol, ciprofloxacin, erythromycin, and clindamycin, whereas these isolates were resistant to kanamycin with MIC values up to 1024 μg/mL. A high resistance rate to streptomycin (43/47, 91.5%) was also observed among the S. thermophilus isolates, whereas the resistance frequency to vancomycin (3/47, 6.4%), ampicillin (6/47, 12.8%), gentamicin (4/47, 8.5%), tetracycline (2/47, 4.3%), and trimethoprim (8/47, 17%) was low (Table 1).

Detection of antibiotic resistance genes, class 1 integron, and the Tn916 transposon

The prevalence and distribution of one gentamicin, four kanamycin or neomycin, five streptomycin, one chloramphenicol, nine vancomycin, five tetracycline, and two sulfonamide resistance genes, along with Tn916 and intI1, were determined in all isolates. The sul1 gene was identified in six L. bulgaricus and seven S. thermophilus strains isolated from fermented milk products. The tet(M) gene was detected in two tetracycline-resistant L. bulgaricus and two tetracycline-resistant S. thermophilus strains. The strA and strB genes were found to reside in nine and seven streptomycin-resistant S. thermophilus isolates, respectively. Six S. thermophilus isolates coharbored the strA and strB genes. The aac(6′)-aph(2″) and aph(3″)-III genes were found in two L. bulgaricus strains. The aph(3″)-II gene was detected in one S. thermophilus isolate (Table 2). Neither intI1 nor Tn916 transposon gene was detected.

Table 2.

Distribution of Resistance Genes Within Lactobacillus spp. and Streptococcus thermophilus Isolates from Fermented Milk Products

		Antibiotic resistance
Bacterial species	Strain	Phenotypic resistance ^a	Genes
Lactobacillus bulgaricus	LB2013N12	STR-GEN-TMP-KAN	sul1
	LB2013N18	STR-GEN-CIP-TMP-KAN	sul1
	LB2013N19	STR-TMP-KAN	sul1
	LB2013N5	STR-VAN-GEN-CIP-SXT-KAN	sul1
	LB2013N26	STR-GEN-CIP-TMP-KAN	sul1
	LB2013N2	STR-GEN-TET-CIP-TMP-KAN	sul1, tet(M)
	LB2014N28	STR-GEN-CIP-TMP-KAN	aac(6′)-aph(2″), aph(3″)-III
	LB2014N21	STR-GEN-TET-CHL-CIP-TMP-KAN	aac(6′)-aph(2″), aph(3″)-III, tet(M)
Streptococcus thermophilus	ST2013N7	STR-TMP-KAN	sul1
	ST2013N12	STR-GEN-TMP-KAN	sul1
	ST2014N31	AMP-STR-TET-CIP-TMP-KAN	sul1
	ST2014N6	STR-CIP-KAN	sul1, sul2
	ST2013N29	STR-GEN-CIP-TMP-KAN	sul1, tet(M)
	ST2014N5	STR-CIP-TMP-KAN	strA
	ST2014N29	STR-GEN-CIP-TMP-KAN	strA
	ST2014N30	STR-KAN	strA
	ST2014N16	STR-GEN-KAN	strB
	ST2014N2	AMP-STR-TET-CIP-KAN	strA, strB, tet(M)
	ST2013N2	AMP-STR-GEN-TET-CIP-TMP-KAN	strA, strB, sul1
	ST2014N8	STR-GEN-KAN	strA, strB, sul1
	ST2014N1	STR-GEN-TMP-KAN	strA, strB
	ST2014N11	AMP-STR-GEN-TMP-KAN	strA, strB
	ST2014N26	STR-CIP-KAN	aph(3″)-II, strA, strB

AMP, ampicillin; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; TET, tetracycline; TMP, trimethoprim; VAN, vancomycin.

Conjugation and transfer of tet(M)

The capacity of lateral transfer of the tet(M) gene was tested. The two tet(M) containing L. bulgaricus and two S. thermophilus isolates were conjugally mated with E. coli J53, S. mutans UA159, E. faecium GE1, and E. faecalis OG1RF by filter mating. No colonies of presumptive tet(M) transconjugants were found on the selective plates after mating. Thus, our results indicated that the tet(M) gene was not transferred between donor strains and recipient strains in this study.

Discussion

Owing to their potential health benefits, LAB are increasingly being used as probiotic agents and added to food products (Champagne et al., 2018; Ranadheera et al., 2018). However, the safety of these LAB in relation to antibiotic resistance and the potential lateral transfer of antibiotic resistance genes from LAB to pathogenic bacteria in the gut are largely unknown. Therefore, it is necessary to characterize antibiotic resistance among commonly isolated LAB in food products to ensure public health.

In the present study, the substantial presence of antibiotic resistance genes was revealed within LAB isolated from retail fermented milk products. This is in accordance with other recent studies reporting that Lactobacillus spp. and S. thermophilus in retail fermented milk products could be a reservoir for antibiotic resistance determinants (Chiara et al., 2013; Abriouel et al., 2015; Zhou et al., 2012); suggesting that dairy fermentation could be a susceptible process during which antibiotic-resistant bacteria could possibly evolve and proliferate. Thus, the high number of LAB in fermented milk products and their ability to horizontally transfer antimicrobial/antibiotic resistance genes could be a serious health concern.

In this study, most of the Lactobacillus spp. from fermented milk products in the retail market were resistant to a wide range of antibiotics, including streptomycin, kanamycin, ciprofloxacin, gentamicin, vancomycin, and trimethoprim. This observation was not surprising, as Lactobacillus spp. have previously been reported to possess a natural resistance to aminoglycosides (neomycin, kanamycin, streptomycin, and gentamicin), glycopeptides (vancomycin and teicoplanin), inhibitors of the nucleic acid synthesis (ciprofloxacin, enoxacin, pefloxacin, norfloxacin, nalidixic acid, and metronidazole), and inhibitors of folic acid synthesis (sulfamethoxazole, trimethoprim, and cotrimoxazole) (Zhou et al., 2005; Patel et al., 2012).

Data from various studies have shown that Lactobacillus spp. are generally quite sensitive to clinically relevant antibiotics, such as penicillin G, ampicillin, tetracycline, erythromycin, chloramphenicol, and clindamycin (Danielsen and Wind 2003; Delgado et al., 2005; Ammor et al., 2008; Patel et al., 2012). In accordance with these results, all Lactobacillus spp. isolates in the present study were sensitive to ampicillin, erythromycin, and clindamycin, and exhibited only low resistance rates to chloramphenicol and tetracycline. In contrast, it has previously been reported that a considerable share of the L. bulgaricus strains isolated from Chinese yogurts were resistant to ampicillin, chloramphenicol, and tetracycline (Zhou et al., 2012). Generally, tetracycline and chloramphenicol resistance is associated with mobile genetic elements, which may potentially contribute to the dissemination of resistance genes through horizontal gene transfer (Abriouel et al., 2015). Consequently, the reduced susceptibility of Lactobacillus spp. to tetracycline and chloramphenicol deserves further investigation.

Different resistance rates to ciprofloxacin (60–70%), gentamicin (0–100%), and streptomycin (70–80%) have been reported among Lactobacillus spp., including Lactobacillus helveticus, L. casei, and L. plantarum, and a similar tendency has been reported in Streptococcus, Lactococcus, Pediococcus, and Leuconostoc (Hummel et al., 2007; Li et al., 2015; Guo et al., 2017). This indicates the existence of considerable differences in antibiotic resistance both on an intergenus and interspecies level as well as a species dependency in Lactobacillus spp. As the number of species encountered in the Lactobacillus spp. group of the present study was limited to six, more data concerning antibiotic resistance within other species of Lactobacillus are needed to establish the antibiotic resistance profiles of the general Lactobacillus spp. populations.

The current study observed that S. thermophilus strains isolated from fermented milk products were resistant to tetracycline, vancomycin, gentamicin, ampicillin, trimethoprim, streptomycin, and kanamycin. Previously, even higher resistance rates to ampicillin, chloramphenicol, tetracycline, kanamycin, streptomycin, and gentamicin have been reported in S. thermophilus isolated from fermented dairy products within both China and Europe (Temmerman et al., 2002; Zhou et al., 2012). The broad resistance spectrum of S. thermophilus strains isolated from fermented milk products may be partly due to evolutionary events occurring along the processing chain, however, other potential causes leading to the emergence of resistance genes need to be clarified.

Detection of antibiotic resistance genes showed that the sul1, sul2, tet(M), strA, strB, aac(6′)-aph(2″), aph(3″)-II, and aph(3″)-III genes were present in both L. bulgaricus and S. thermophilus strains isolated from fermented milk products. The tet(M) gene has previously been widely detected in many LAB species isolated from dairy products, including Lactobacillus spp. (L. bulgaricus, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus curvatus, L. paracasei, L. plantarum, Lactobacillus sakei, and Lactobacillus salivarius) and S. thermophilus (Gfeller et al., 2003; van Hoek et al., 2008; Nawaz et al., 2011; Zhou et al., 2012). This wide distribution of the tet(M) gene has been hypothesized to be a result of its association with mobile genetic elements (Wozniak and Waldor, 2010). In addition, the aph(3″)-III gene has previously been demonstrated in both Lactobacillus spp. (L. bulgaricus, L. casei, and Lactobacillus pentosus) and S. thermophilus isolated from dairy products (Ouoba et al., 2008; Zhou et al., 2012; Han et al., 2013). The sul1 and sul2 genes, which are also associated with mobile genetic elements, have been reported to be widespread within many bacterial species found in food products, clinical isolates, and isolates from environmental sources (Machado et al., 2013; Li et al., 2016). However, sul1 and sul2 have not previously been reported in L. bulgaricus and S. thermophilus isolates. Furthermore, the aac(6′)-aph(2″), strA, strB, and aph(3″)-II genes, conferring resistance to gentamicin, streptomycin, and neomycin, have not previously been demonstrated in L. bulgaricus and S. thermophilus isolated from fermented dairy products. Therefore, to the best of our knowledge, the strA, strB, sul1, sul2, and aph(3″)-II genes in S. thermophilus (MH361002, MH361003, MH361004, MH361005, MH361008) as well as the sul1 and aac(6′)-aph(2″) genes in L. bulgaricus (MH361006, MH361007) were detected for the first time in the present study.

Although some strains exhibited high MIC values toward gentamicin, kanamycin, and vancomycin, none of the strains was found to possess the genes [aac(6′)-aph(2″), aphA3, aph(3″), vanA, vanH, vanR, vanS, vanX, vanY, vanZ, vanB, and vanC] known to confer resistance to these compounds. Also, many of the target antibiotic genes have not been detected in phenotypically resistant Lactobacillus strains (Toomey et al., 2010; Guo et al., 2017), which strongly indicates that the genetic basis and mechanisms of resistance to certain antibiotics are still largely unknown in LAB. Moreover, due to nucleotide sequence divergence within many resistance genes in different Lactobacillus spp., such as cat and vanX, it would be impossible to detect these genes among different species of this genus by the utilization of PCR (Abriouel et al., 2015). Thus, to detect similar genes implicated in antibiotic resistance of Lactobacillus spp. and S. thermophilus strains, especially intrinsic resistance, whole-genome sequence analysis is needed to clarify the resistance mechanisms further.

Several studies have reported the transfer of antibiotic resistance genes, such as tet(M) and ermB, from Lactobacillus spp. to both the same species and other species (Ouoba et al., 2008; Nawaz et al., 2011). However, the present study did not succeed in demonstrating the transfer of the tet(M) gene. The assessment of the risk of antibiotic resistance gene transfer from Lactobacillus spp. to other bacteria might be limited in the present study due to the small numbers of bacteria tested. Future studies are needed to examine more LAB species and to further analyze the transferability of the antibiotic resistance genes possessed by these widespread bacteria.

Conclusion

This study demonstrated that Lactobacillus spp. and S. thermophilus strains, naturally present in fermented milk products, may represent an important reservoir for antibiotic resistance genes, which could potentially spread directly to the human intestinal microbiota by horizontal gene transfer. Therefore, increased attention should be focused on the safety of antibiotic-resistant LAB in fermented milk products, and the risk of horizontal transfer of resistance genes to human pathogens. In addition, future studies are required to identify the factors contributing to the emergence of antibiotic-resistant LAB during the manufacturing of fermented milk products, hereby enabling a targeted prevention strategy against this concerning issue.

Footnotes

Acknowledgments

This work was supported by the National key research and development plan (2016YFD0500600), Fundamental Research Funds for the Central Universities (2017MS104, 21618309), Guangdong provincial science and technology plan project (2017B020207004), and the Open Project Program of Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety.

Disclosure Statement

No competing financial interests exist.

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