Genus-Wide Assessment of Antibiotic Resistance in Lactobacillus spp.

Lactobacillus species are generally considered to be nonpathogenic and are used in a wide variety of foods and products for humans and animals. However, many of the species examined in this study have antibiotic resistance levels which exceed those recommended by the EFSA, suggesting that these cutoff values should be reexamined in light of the genetic basis for resistance discussed here. Our data provide evidence for rationally revising the regulatory guidelines for safety assessment of lactobacilli entering the food chain as starter cultures, food preservatives, or probiotics and will facilitate comprehensive genotype-based assessment of strains for safety screening.

ABSTRACT

Lactobacillus species are widely used as probiotics and starter cultures for a variety of foods, supported by a long history of safe usage. Although more than 35 species meet the European Food Safety Authority (EFSA) criteria for qualified presumption of safety status, the safety of Lactobacillus species and their carriage of antibiotic resistance (AR) genes is under continuing ad hoc review. To comprehensively update the identification of AR in the genus Lactobacillus, we determined the antibiotic susceptibility patterns of 182 Lactobacillus type strains and compared these phenotypes to their genotypes based on genome-wide annotations of AR genes. Resistances to trimethoprim, vancomycin, and kanamycin were the most common phenotypes. A combination of homology-based screening and manual annotation identified genes encoding resistance to aminoglycosides (20 sequences), tetracycline (18), erythromycin (6), clindamycin (60), and chloramphenicol (42). In particular, the genes aac(3) and lsa, involved in resistance to aminoglycosides and clindamycin, respectively, were found in Lactobacillus spp. Acquired determinants predicted to code for tetracycline and erythromycin resistance were detected in Lactobacillus ingluviei, Lactobacillus amylophilus, and Lactobacillus amylotrophicus, flanked in the genome by mobile genetic elements with potential for horizontal transfer.

IMPORTANCE Lactobacillus species are generally considered to be nonpathogenic and are used in a wide variety of foods and products for humans and animals. However, many of the species examined in this study have antibiotic resistance levels which exceed those recommended by the EFSA, suggesting that these cutoff values should be reexamined in light of the genetic basis for resistance discussed here. Our data provide evidence for rationally revising the regulatory guidelines for safety assessment of lactobacilli entering the food chain as starter cultures, food preservatives, or probiotics and will facilitate comprehensive genotype-based assessment of strains for safety screening.

INTRODUCTION

Antibiotics represent one of the most powerful therapeutic options in human and veterinary medicine for the treatment of infectious diseases caused by bacterial agents (1, 2). However, overprescribing and misuse of antibiotics in medicine, animal feed, aquaculture, and agriculture have led to the emergence and spread of antibiotic-resistant bacteria, which constitutes a serious problem for the health of both humans and animals (3). Today, the ever-increasing prevalence of antibiotic resistance (AR) in infectious microbes is a global public health emergency (4, 5).

Lactic acid bacteria (LAB) have been extensively used as probiotics and starter cultures due to their long history of safe use, and many species have qualified presumption of safety (QPS) status (6, 7) including members of the genus Lactobacillus, Lactococcus, Leuconostoc, and Pediococcus (8). In Europe, the absence of acquired or transferable resistance factors must be established for a candidate probiotic or starter culture in order for them to be declared safe for human and animal consumption and to achieve QPS status from the European Food Safety Authority (EFSA) (9).

The significant economic and scientific impact of the members of the genus Lactobacillus includes many strains commonly used as probiotics and others marketed as probiotic cosmetics, drug supplements, or even medical devices (10). Furthermore, Lactobacillus species are probably those most widely used as starter cultures for industrial and agricultural applications (e.g., fermented foods and silage cultures), due to their long history of safe and technological use (11–13). Despite their safety status, many lactobacilli have been reported as being antibiotic resistant (12, 14, 15), where a vancomycin-resistant phenotype is perhaps the best-characterized intrinsic resistance mechanism (16). Most Lactobacillus species are intrinsically resistant to aminoglycosides (gentamicin, kanamycin, streptomycin, and neomycin), ciprofloxacin, and trimethoprim, and they are susceptible to penicillin and β-lactams, chloramphenicol, tetracycline, erythromycin, linezolid, and quinupristin-dalfopristin (14). However, acquired resistance to tetracycline, erythromycin, clindamycin, and chloramphenicol has been detected in lactobacilli isolated from fermented foods (17–20). Given the widespread use of some species of this genus in fermented food production and functional foods/probiotics, lactobacilli could act as donors or reservoirs for AR genes, with the potential risk of transferring these genes to pathogenic bacteria in food matrices as well as in the gastrointestinal tract (GIT) (21). Thus, even though more than 35 species meet the criteria of QPS status proposed by the EFSA (8), the safety of Lactobacillus species and their possible involvement in the spread of AR determinants along the food chain warrant investigation. QPS is an attribute of a species rather than a strain, and it is noteworthy that genome content often varies widely within species, including in lactobacilli (22–24).

The recent determination of the genome sequences of almost all Lactobacillus type strains (25, 26) allows the safety assessment of the genus Lactobacillus by surveillance of the presence of AR genes, as well as their potential for transfer to other microorganisms. Within the limits of database quality and annotation, whole-genome sequencing (WGS) potentially allows the identification of all possible genetic determinants of antimicrobial resistance in a microbial genome (27). WGS could revolutionize food safety assessments, resulting in a paradigm shift from phenotype-based to genotype-based assays of AR (28).

The aim of the current study was to determine the antibiotic susceptibility patterns of 197 type strains representing the whole Lactobacillus genus and to compare these phenotypes to their genotypes based on genome-wide annotation of AR genes. Parallel analysis of phenotype and genotype would provide the definitive knowledge base for the distribution, origins, and mechanisms of AR in the genus and would facilitate rational discussions about regulating strains or species harboring intrinsic, acquired, or transmissible AR mechanisms.

RESULTS

Determination of MICs.

The MIC values of 16 antibiotics belonging to the most important antimicrobial classes used in human and veterinary medicine were tested using broth microdilution VetMIC plates for 197 Lactobacillus strains representing the whole Lactobacillus genus. The MIC profiles were obtained for 182 strains (because 15 strains were not capable of growth in the VetMIC medium) and were analyzed in the context of the Lactobacillus phylogroups described in references 25 and 29 and shown in Table 1.

TABLE 1.

Features of the 197 type strains of the genus Lactobacillus analyzed, including genome accession number and growth condition applied for the determination of MIC values

Species	Strain	Metabolism phenotypea	Phylogroupb	GenBank accession no.c	Source	Niche category	Growth conditions	Temp (°C)	Medium
Lactobacillus acetotolerans	DSM 20749T	FHE	L. delbrueckii	AYZC00000000	Fermented vinegar broth	Food	Anaerobic	30	MRS + 0.05% cysteine
Lactobacillus acidifarinae	DSM 19394T	OHE	L. brevis	AZDV00000000	Artisanal wheat sourdough	Food	Microaerophilic	30	MRS + 0.05% cysteine (pH 5.2)
Lactobacillus acidipiscis	DSM 15836T	FHE	L. salivarius	AZFI00000000	Fermented fish	Food	Microaerophilic	37	MRS
Lactobacillus acidophilus	ATCC 4356T	OHO	L. delbrueckii	AZCS00000000	Human	Animal	Microaerophilic	37	MRS
Lactobacillus agilis	DSM 20509T	FHE	L. salivarius	AYYP00000000	Municipal sewage	Environment	Microaerophilic	37	MRS + 0.05% cysteine
Lactobacillus algidus	DSM 15638T	FHE	L. salivarius	AZDI00000000	Vacuum-packed beef	Food	Anaerobic	20	MRS (pH 5.7)
Lactobacillus alimentarius	DSM 20249T	FHE	L. alimentarius	AZDQ00000000	Marinated fish product	Food	Aerobic	30	MRS
Lactobacillus amylolyticus	DSM 11664T	OHO	L. delbrueckii	AZEP00000000	Acidified beer wort	Wine product	Preferably anaerobic	37	MRS
Lactobacillus amylophilus	DSM 20533T	OHO	L. delbrueckii	AYYS00000000	Swine waste-corn fermentation	Animal	Aerobic	30	MRS + 1% glucose
Lactobacillus amylotrophicus	DSM 20534T	OHO	L. delbrueckii	AZCV00000000	Swine waste-corn fermentation	Animal	Aerobic	30	MRS
Lactobacillus amylovorus	DSM 20531T	OHO	L. delbrueckii	AZCM00000000	Cattle waste-corn fermentation	Animal	Microaerophilic-anaerobic	37	MRS
Lactobacillus animalis	DSM 20602T	OHO	L. salivarius	AYYW00000000	Dental plaque of baboon	Animal	Aerobic	37	MRS
Lactobacillus antri	LMG 22111T	OHE	L. reuteri-L. vaccinostercus	AZDK00000000	Gastric biopsy specimens, human stomach mucosa	Animal	Anaerobic	37	MRS
Lactobacillus apinorum	DSM 26257T	OHE	L. fructivorans	JXCT00000000	Honey stomach of honeybee	Animal	Anaerobic	37	MRS + 2% fructose
Lactobacillus apis	LMG 26964T	OHO	L. delbrueckii	JXLG00000000	Stomachs of honeybees	Animal	Anaerobic	37	MRS
Lactobacillus apodemi	DSM 16634T	OHE	L. salivarius	AZFT00000000	Feces of wild Japanese wood mouse	Animal	Microaerophilic	37	MRS
Lactobacillus aquaticus	DSM 21051T	OHO	L. salivarius	AYZD00000000	Surface of a eutrophic freshwater pond	Environment	Microaerophilic	37	MRS
Lactobacillus aviarius subsp. araffinosus	DSM 20653T	OHO	L. salivarius	AYYZ00000000	Intestine of chicken	Animal	Microaerophilic	37	MRS + 0.05% cysteine-hydrochloride
Lactobacillus aviarius subsp. aviarius	DSM 20655T	OHO	L. salivarius	AYZA00000000	Chicken feces	Animal	Microaerophilic	37	MRS + 0.05% cysteine
Lactobacillus backii	DSM 18080T	OHO	L. coryniformis	NA	Orchardgrass silage	Plant	Preferably anaerobic	28	MRS
Lactobacillus bifermentans	DSM 20003T	FHE	L. coryniformis	AZDA00000000	Blown cheese	Food	Anaerobic	30	MRS
Lactobacillus bombi	DSM 26517T	FHE	L. alimentarius	NA	Digestive tracts of bumblebee queens	Animal	Anaerobic	37	MRS
Lactobacillus bombicola	DSM 28793T	FHE	L. delbrueckii	NA	Bumble bee gut	Animal	Strictly anaerobic	37	MRS + 0.05% cysteine-hydrochloride
Lactobacillus brantae	DSM 23927T	FHE	L. casei-L. manihotivorans	AYZQ00000000	Feces of Canada goose	Animal	Microaerophilic	37	MRS
Lactobacillus brevis	DSM 20054T	OHE	L. brevis	AZCP00000000	Feces	Animal	Microaerophilic	30	MRS
Lactobacillus buchneri	DSM 20057T	OHE	L. buchneri	AZDM00000000	Tomato pulp	Plant	Microaerophilic	37	MRS
Lactobacillus cacaonum	DSM 21116T	FHE	L. salivarius	AYZE00000000	Cocoa bean heap fermentation	Plant	Microaerophilic	30	MRS
Lactobacillus camelliae	DSM 22697T	OHO	L. casei-L. manihotivorans	AYZJ00000000	Fermented tea leaves (miang)	Plant	Microaerophilic	37	MRS
Lactobacilllus capillatus	DSM 19910T	FHE	L. salivarius	AZEF00000000	Fermented brine used for stinky tofu production	Food	Microaerophilic	30	MRS
Lactobacillus casei	DSM 20011T	FHE	L. casei-L. manihotivorans	AZCO00000000	Cheese	Food	Microaerophilic	30	MRS
Lactobacillus ceti	DSM 22408T	FHE	L. salivarius	JQBZ00000000	Lungs of a beaked whale	Animal	Strictly anaerobic	37	MRS
Lactobacillus colehominis	DSM 14060T	FHE	L. reuteri-L. vaccinostercus	AZEW00000000	Human vagina	Animal	Microaerophilic	37	MRS
Lactobacillus collinoides	DSM 20515T	OHE	L. collinoides	AYYR00000000	Fermenting apple juice	Food	Anaerobic	26	MRS
Lactobacillus composti	DSM 18527T	FHE	Other	AZGA00000000	Composting material of distilled shochu residue	Wine product	Microaerophilic	30	MRS
Lactobacillus concavus	DSM 17758T	OHO	Other	AZFX00000000	Walls of a distilled spirit fermenting cellar	Environment	Microaerophilic	30	MRS
Lactobacillus coryniformis subsp. coryniformis	LMG 9196T	FHE	L. coryniformis	AZCN00000000	Silage	Plant	Microaerophilic	30	MRS
Lactobacillus coryniformis subsp. torquens	DSM 20004T	FHE	L. coryniformis	AZDC00000000	Air of cow shed	Environment	Microaerophilic	30	MRS
Lactobacillus crispatus	DSM 20584T	OHO	L. delbrueckii	AZCW00000000	Eye	Animal	Preferably anaerobic	37	MRS
Lactobacillus crustorum	LMG 23699T	OHO	L. alimentarius	JQCK00000000	Wheat sourdough	Food	Aerobic	30	MRS
Lactobacillus curieae	JCM 18524T	OHE	L. buchneri	CP018906	Tofu brine	Food	Facultatively anaerobic	30	MRS
Lactobacillus curvatus	DSM 20019T	FHE	L. sakei	AZDL00000000	Milk	Food	Microaerophilic	30	MRS
Lactobacillus delbrueckii subsp. bulgaricus	DSM 20081T	OHO	L. delbrueckii	JQAV00000000	Bulgarian yogurt	Food	Microaerophilic	37	MRS
Lactobacillus delbrueckii subsp. delbrueckii	DSM 20074T	OHO	L. delbrueckii	AZCR00000000	Sour grain mash	Food	Microaerophilic	37	MRS
Lactobacillus delbrueckii subsp. indicus	DSM 15996T	OHO	L. delbrueckii	AZFL00000000	Traditional dairy fermented product (Dahi type)	Food	Microaerophilic	37	MRS
Lactobacillus delbrueckii subsp. jakobsenii	DSM 26046T	OHO	L. delbrueckii	JQCG00000000	Dolo wort (alcoholic fermented beverage)	Wine product	Anaerobic	37	MRS
Lactobacillus delbrueckii subsp. lactis	DSM 20072T	OHO	L. delbrueckii	AZDE00000000	Emmental cheese	Food	Microaerophilic	37	MRS
Lactobacillus dextrinicus	DSM 20335T	OHE	Other	AYYK00000000	Silage	Plant	Aerobic	30	MRS
Lactobacillus diolivorans	DSM 14421T	OHE	L. buchneri	AZEY00000000	Maize silage	Plant	Aerobic	30	MRS
Lactobacillus equi	DSM 15833T	OHO	L. salivarius	AZFH00000000	Horse feces	Animal	Microaerophilic	37	MRS
Lactobacillus equicursoris	DSM 19284T	OHO	L. delbrueckii	AZDU00000000	Healthy thoroughbred racehorse	Animal	Microaerophilic	37	MRS
Lactobacillus equigenerosi	DSM 18793T	OHE	L. reuteri-L. vaccinostercus	AZGC00000000	Feces of thoroughbred horse	Animal	Microaerophilic	37	MRS
Lactobacillus fabifermentans	DSM 21115T	FHE	L. plantarum	AYGX00000000	Cocoa bean heap fermentation	Plant	Microaerophilic	30	MRS
Lactobacillus faecis	DSM 23956T	OHO	L. salivarius	NA	Animal feces	Animal	Microaerophilic	37	MRS
Lactobacillus farciminis	LMG 9189	OHO	L. alimentarius	AZDR00000000	Sausage	Food	Microaerophilic	30	MRS
Lactobacillus farraginis	DSM 18382T	FHE	L. buchneri	AZFY00000000	Composting material of distilled shochu residue	Wine product	Microaerophilic	30	MRS
Lactobacillus fermentum	DSM 20055T	OHE	L. reuteri-L. vaccinostercus	JQAU00000000	Human saliva	Animal	Aerobic	30	MRS
Lactobacillus floricola	DSM 23037T	OHO	L. delbrueckii	AYZL00000000	Flower of Caltha palustris	Plant	Aerobic	30	MRS
Lactobacillus florum	DSM 22689T	OHE	L. fructivorans	AYZI00000000	Peony (Paeonia suffruticosa)	Plant	Anaerobic	28	MRS + 1% fructose
Lactobacillus formosensis	NBRC 109509T	OHO	L. alimentarius	NA	Fermented soybean	Food	Anaerobic	37	MRS (pH 6.2)
Lactobacillus fornicalis	JCM 12512T	?	Other	NA	Human vagina	Animal	Aerobic	37	MRS
Lactobacillus fructivorans	DSM 20203T	OHE	L. fructivorans	AZDS00000000	NA	Unknown	Microaerophilic	30	MRS
Lactobacillus frumenti	DSM 13145T	OHE	L. reuteri-L. vaccinostercus	AZER00000000	Rye bran sourdough	Food	Anaerobic	40	MRS (pH 6.2)
Lactobacillus fuchuensis	DSM 14340T	FHE	L. sakei	AZEX00000000	Vacuum-packaged beef	Food	Aerobic	20	MRS
Lactobacillus furfuricola	DSM 27174T	OHO	L. alimentarius	NA	Rice bran paste	Plant	Microaerophilic	30	MRS
Lactobacillus futsaii	JCM 17355T	OHO	L. alimentarius	AZDO00000000	Fu-tsai, a traditional fermented mustard product	Food	Aerobic	30	MRS
Lactobacillus gallinarum	DSM 10532T	OHO	L. delbrueckii	AZEL00000000	Chicken crop	Animal	Microaerophilic	37	MRS
Lactobacillus gasseri	LMG 9203T	OHO	L. delbrueckii	NC_008530	Human	Animal	Preferably anaerobic	30	MRS
Lactobacillus gastricus	DSM 16045T	OHE	L. reuteri-L. vaccinostercus	AZFN00000000	Gastric biopsy specimens, human stomach mucosa	Animal	Anaerobic	37	MRS
Lactobacillus ghanensis	DSM 18630T	OHO	L. salivarius	AZGB00000000	Cocoa fermentation	Plant	Anaerobic	30	MRS
Lactobacillus gigeriorum	DSM 23908T	OHO	L. delbrueckii	AYZO00000000	Chicken crop	Animal	Microaerophilic	37	MRS
Lactobacillus ginsenosidimutans	DSM 24154T	OHO	L. alimentarius	CP012034	Kimchi	Food	Anaerobic	30	MRS
Lactobacillus gorillae	DSM 28356T	FHE	L. reuteri-L. vaccinostercus	NA	Gorilla feces	Animal	Microaerophilic	37	MRS
Lactobacillus graminis	DSM 20719T	FHE	L. sakei	AYZB00000000	Grass silage	Plant	Microaerophilic	30	MRS
Lactobacillus hammesii	DSM 16381T	FHE	L. brevis	AZFS00000000	Wheat sourdough	Food	Microaerophilic	30	MRS + 1% maltose + 0.5% yeast extract
Lactobacillus hamsteri	DSM 5661T	FHE	L. delbrueckii	AZGI00000000	Hamster feces	Animal	Anaerobic	37	MRS
Lactobacillus harbinensis	DSM 16991T	FHE	L. perolens	AZFW00000000	Chinese traditional fermented vegetable	Food	Microaerophilic	37	MRS
Lactobacillus hayakitensis	DSM 18933T	OHO	L. salivarius	AZGD00000000	Feces of thoroughbred horse	Animal	Microaerophilic	37	MRS
Lactobacillus heilongjiangensis	LMG 26166T	OHO	L. alimentarius	CP012559	Chinese pickle	Plant	Microaerophilic	28	MRS
Lactobacillus helsingborgensis	DSM 26265T	OHO	L. delbrueckii	JXJR00000000	Honey stomach of honeybee	Animal	Anaerobic	35	MRS + fructose (20 g/liter)
Lactobacillus helveticus	LMG 22464	OHO	L. delbrueckii	JQCJ00000000	Malt whiskey fermentation	Wine product	Anaerobic	37	MRS
Lactobacillus herbarum	DSM 100358T	OHE	L. plantarum	LFEE00000000	Fermented white radish	Food	Microaerophilic	25	MRS
Lactobacillus hilgardii	LMG 6895T	OHE	L. buchneri	AZDF00000000	Wine	Wine product	Preferably anaerobic	37	MRS
Lactobacillus hokkaidonensis	DSM 26202T	OHE	L. reuteri-L. vaccinostercus	JQCH00000000	Timothy grass silage	Plant	Microaerophilic	25	MRS
Lactobacillus hominis	DSM 23910T	OHO	L. delbrueckii	AYZP00000000	Human intestine	Animal	Microaerophilic	37	MRS
Lactobacillus homohiochii	DSM 20571T	FHE	L. fructivorans	JQBN00000000	Spoiled sake	Wine product	Microaerophilic	26	MRS
Lactobacillus hordei	DSM 19519T	OHO	L. salivarius	AZDX00000000	Malted barley	Plant	Anaerobic	37	MRS
Lactobacillus iners	DSM 13335T	OHO	L. delbrueckii	AZET00000000	Human urine	Animal	Anaerobic	37	MRS
Lactobacillus ingluviei	DSM 15946T	OHE	L. reuteri-L. vaccinostercus	AZFK00000000	Pigeon, crop	Animal	Microaerophilic	37	MRS
Lactobacillus insicii	DSM 29801T	OHO	L. alimentarius	NA	Fermented raw meat	Animal	Microaerophilic	30	MRS
Lactobacillus intestinalis	DSM 6629T	FHE	L. delbrueckii	AZGN00000000	Intestine of rat	Animal	Aerobic	37	MRS
Lactobacillus iwatensis	DSM 26942T	OHO	L. coryniformis	NA	Orchardgrass silage	Plant	Microaerophilic	30	MRS
Lactobacillus jensenii	DSM 20557T	FHE	L. delbrueckii	AYYU00000000	Human vaginal discharge	Animal	Microaerophilic	37	MRS
Lactobacillus johnsonii	LMG 9436T	OHO	L. delbrueckii	AZCY00000000	Human blood	Animal	Preferably anaerobic	30	MRS
Lactobacillus kalixensis	DSM 16043T	OHO	L. delbrueckii	AZFM00000000	Gastric biopsy specimens, human stomach mucosa	Animal	Microaerophilic	37	MRS
Lactobacillus kefiranofaciens subsp. kefiranofaciens	LMG 19149T	OHO	L. delbrueckii	AZGG00000000	Kefir grains	Plant	Anaerobic	30	MRS
Lactobacillus kefiranofaciens subsp. kefirgranum	DSM 10550T	OHO	L. delbrueckii	AZEM00000000	Kefir grains	Plant	Anaerobic	30	MRS
Lactobacillus kefiri	DSM 20587T	OHE	L. buchneri	AYYV00000000	Kefir grains	Plant	Aerobic	30	MRS
Lactobacillus kimbladii	DSM 26263T	FHE	L. delbrueckii	JXLH00000000	Honey stomach of honeybee	Animal	Anaerobic	30	MRS
Lactobacillus kimchicus	JCM 15530T	FHE	L. collinoides	AZCX00000000	Kimchi	Food	Aerobic	37	MRS
Lactobacillus kimchiensis	DSM 24716T	OHO	L. alimentarius	JQCF00000000	Kimchi	Food	Microaerophilic	25	MRS
Lactobacillus kisonensis	DSM 19906T	OHE	L. buchneri	AZEB00000000	Sunki, a Japanese traditional pickle	Food	Microaerophilic	30	MRS + maltose (10 g/liter) + l-arabinose (10 g/liter)
Lactobacilus kitasatonis	DSM 16761T	OHO	L. delbrueckii	AZFU00000000	Chicken intestine	Animal	Microaerophilic	37	MRS
Lactobacillus koreensis	JCM 16448T	OHE	L. brevis	AZDP00000000	Cabbage kimchi	Food	Aerobic	30	MRS (pH 5.5)
Lactobacillus kullabergensis	DSM 26262T	FHE	L. delbrueckii	JXBY00000000	Honey stomach of honeybee	Animal	Strictly anaerobic	30	MRS
Lactobacillus kunkeei	DSM 12361T	OHE	L. fructivorans	AZCK00000000	Commercial grape wine	Wine product	Aerobic	30	MRS + 0.05% cysteine (pH 5.2)
Lactobacillus lindneri	DSM 20690T	OHE	L. fructivorans	JQBT00000000	Spoiled beer	Wine product	Aerobic	30	MRS + 0.05% cysteine (pH 5.2)
Lactobacillus malefermentans	LMG 11455T	OHE	L. collinoides	AZGJ00000000	Sour beer	Wine product	Microaerophilic	30	MRS
Lactobacillus mali	ATCC 27304T	OHO	L. salivarius	JQAR00000000	Wine must	Wine product	Aerobic	30	MRS
Lactobacillus manihotivorans	DSM 13343T	OHO	L. casei-L. manihotivorans	AZEU00000000	Cassava sour starch fermentation	Plant	Aerobic	30	MRS
Lactobacillus mellifer	DSM 26254T	FHE	L. alimentarius	JXJQ00000000	Honey stomach of honeybee	Animal	Anaerobic	35	MRS + fructose (20 g/liter)
Lactobacillus mellis	DSM 26255T	FHE	L. alimentarius	JXBZ00000000	Honey stomach of honeybee	Animal	Strictly anaerobic	30	MRS + fructose (20 g/liter)
Lactobacillus melliventris	DSM 26256T	FHE	L. delbrueckii	JXLI00000000	Honey stomach of honeybee	Animal	Strictly anaerobic	30	MRS + fructose (20 g/liter)
Lactobacillus mindensis	DSM 14500T	OHO	L. alimentarius	AZEZ00000000	Sourdough	Food	Microaerophilic	30	MRS + 0.05% cysteine-hydrochloride (pH 5.2)
Lactobacillus mixtipabuli	DSM 28580T	OHE	L. collinoides	NA	Silage	Plant	Microaerophilic	30	MRS
Lactobacillus modestisalitolerans	NBRC 107235T	OHO	L. plantarum	NA	Fermented fish	Food	Facultatively anaerobic	30	MRS
Lactobacillus mucosae	DSM 13345T	OHE	L. reuteri-L. vaccinostercus	AZEQ00000000	Pig small intestine	Animal	Preferably anaerobic	37	MRS
Lactobacillus mudanjiangensis	LMG 27194T	OHO	L. plantarum	NA	Pickle	Plant	Microaerophilic	28	MRS
Lactobacillus murinus	DSM 20452T	FHE	L. salivarius	AYYN00000000	Intestine of rat	Animal	Microaerophilic	37	MRS
Lactobacillus nagelii	DSM 13675T	OHO	L. salivarius	AZEV00000000	Partially fermented wine	Wine product	Anaerobic	30	MRS
Lactobacillus namurensis	DSM 19117T	OHE	L. brevis	AZDT00000000	Sourdough	Food	Microaerophilic	30	MRS + 0.05% cysteine + 0.7 % maltose (pH 5.2)
Lactobacillus nantensis	DSM 16982T	FHE	L. alimentarius	AZFV00000000	Wheat sourdough	Food	Microaerophilic	30	MRS + 0.05% cysteine-hydrochloride + 1% maltose + 0.5% fresh yeast extract
Lactobacillus nasuensis	JCM 17158T	OHO	L. casei-L. manihotivorans	AZDJ00000000	Sudangrass silage sample	Plant	Anaerobic	30	MRS
Lactobacillus nenjiangensis	LMG 27192T	OHE	L. reuteri-L. vaccinostercus	NA	Pickle	Plant	Microaerophilic	28	MRS
Lactobacillus nodensis	DSM 19682T	FHE	L. alimentarius	AZDZ00000000	Japanese pickles	Food	Microaerophilic	30	MRS
Lactobacillus odoratitofui	DSM 19909T	OHE	L. collinoides	AZEE00000000	Fermented brine used for stinky tofu production	Food	Microaerophilic	30	MRS
Lactobacillus oeni	DSM 19972T	OHO	L. salivarius	AZEH00000000	Bobal wine	Wine product	Microaerophilic	30	MRS
Lactobacillus oligofermentans	DSM 15707T	OHE	L. reuteri-L. vaccinostercus	AZFE00000000	Broiler leg	Animal	Microaerophilic	25	MRS
Lactobacillus oris	DSM 4864T	OHE	L. reuteri-L. vaccinostercus	AZGE00000000	Human saliva	Animal	Anaerobic	37	MRS
Lactobacillus oryzae	DSM 26518T	OHE	L. collinoides	BBJM00000000	Fermented rice grain	Food	Anaerobic	30	MRS
Lactobacillus otakiensis	DSM 19908T	OHE	L. buchneri	AZED00000000	Sunki, a Japanese traditional pickle	Food	Microaerophilic	30	MRS
Lactobacillus ozensis	DSM 23829T	OHE	L. fructivorans	AYYQ00000000	Chrysanthemum, Oze National Park	Plant	Microaerophilic	30	MRS
Lactobacillus panis	DSM 6035T	OHE	L. reuteri-L. vaccinostercus	AZGM00000000	Sourdough	Food	Aerobic	37	MRS
Lactobacillus pantheris	DSM 15945T	OHO	L. casei-L. manihotivorans	AZFJ00000000	Jaguar feces	Animal	Microaerophilic	37	MRS
Lactobacillus parabrevis	LMG 11984T	OHE	L. brevis	JQCI00000000	Wheat	Food	Anaerobic	30	MRS
Lactobacillus parabuchneri	DSM 5707T	OHE	L. buchneri	AZGK00000000	Human saliva	Animal	Aerobic	30	MRS
Lactobacillus paracasei subsp. paracasei	DSM 5622T	FHE	L. casei-L. manihotivorans	AZGH00000000	NA	Unknown	Microaerophilic	30	MRS
Lactobacillus paracasei subsp. tolerans	DSM 20258T	FHE	L. casei-L. manihotivorans	AYYJ00000000	Pasteurized milk	Food	Aerobic	30	MRS
Lactobacillus paracollinoides	DSM 15502T	OHE	L. collinoides	AZFD00000000	Brewery environment	Environment	Anaerobic	25	MRS (pH 5.8)
Lactobacillus parafarraginis	LMG 24141T	FHE	L. buchneri	AZFZ00000000	Composting material of distilled shochun residue	Wine product	Microaerophilic	28	MRS
Lactobacillus parakefiri	DSM 10551T	OHE	L. buchneri	AZEN00000000	Kefir grain	Plant	Anaerobic	30	MRS
Lactobacillus paralimentarius	DSM 13238T	FHE	L. alimentarius	AZES00000000	Sourdough	Food	Microaerophilic	30	MRS
Lactobacillus paraplantarum	DSM 10667T	FHE	L. plantarum	AZEO00000000	Beer contaminant	Wine product	Aerobic	30	MRS
Lactobacillus pasteurii	DSM 23907T	FHE	L. delbrueckii	AYZN00000000	NA	Unknown	Microaerophilic	37	MRS
Lactobacillus paucivorans	DSM 22467T	FHE	L. brevis	JQCA00000000	Yeast storage tank containing lager beer	Wine product	Anaerobic	28	MRS + 0.05% cysteine-hydrochloride + 1% fructose (pH 5.8)
Lactobacillus pentosus	DSM 20314T	FHE	L. plantarum	AZCU00000000	NA	Unknown	Microaerophilic	30	MRS
Lactobacillus perolens	DSM 12744T	FHE	L. perolens	AZEC00000000	Orange lemonade	Food	Anaerobic	30	MRS
Lactobacillus plajomi	NBRC 107333T	FHE	L. plantarum	NA	Fermented fish	Food	Facultatively anaerobic	30	MRS
Lactobacillus plantarum (formerly Lactobacillus arizonensis)	DSM 20174T	FHE	L. plantarum	AZEJ00000000	Pickled cabbage	Food	Aerobic	37	MRS
Lactobacillus plantarum subsp. argentoratensis	DSM 16365T	FHE	L. plantarum	AZFR00000000	Fermented cassava roots (fufu)	Plant	Microaerophilic	30	MRS
Lactobacillus pobuzihii	NBRC 103219T	FHE	L. salivarius	JQCN00000000	Pobuzih (fermented cummingcordia), Cordia dichotoma	Plant	Microaerophilic	37	MRS + 5% NaCl
Lactobacillus pontis	DSM 8475T	OHE	L. reuteri-L. vaccinostercus	AZGO00000000	Rye sourdough	Food	Aerobic	30	MRS
Lactobacillus porcinae	LMG 26767T	FHE	L. casei-L. manihotivorans	NA	Nem chua (fermented meat)	Food	Microaerophilic	28	MRS
Lactobacillus psittaci	DSM 15354T	OHE	L. delbrueckii	AZFB00000000	Lung of parrot	Animal	Anaerobic	37	MRS
Lactobacillus rapi	DSM 19907T	OHE	L. buchneri	AZEI00000000	Sunki, a Japanese traditional pickle	Food	Microaerophilic	30	MRS
Lactobacillus rennini	DSM 20253T	FHE	L. coryniformis	AYYI00000000	Rennin	Animal	Microaerophilic	30	MRS
Lactobacillus reuteri	DSM 20016T	OHE	L. reuteri-L. vaccinostercus	AZDD00000000	Intestine of adult	Animal	Aerobic	37	MRS
Lactobacillus rhamnosus	DSM 20021T	FHE	L. casei-L. manihotivorans	AZCQ00000000	NA	Unknown	Anaerobic	37	MRS
Lactobacillus rodentium	DSM 24759T	OHO	L. delbrueckii	NA	Digestive tract of rodents	Animal	Microaerophilic	37	MRS
Lactobacillus rossiae	DSM 15814T	OHE	Other	AZFF00000000	Wheat sourdough	Food	Microaerophilic	30	MRS + 1% maltose + 1% yeast extract (pH 5.6)
Lactobacillus ruminis	DSM 20403T	OHO	L. salivarius	AYYL00000000	Bovine rumen	Animal	Anaerobic	37	MRS + 2% glucose
Lactobacillus saerimneri	DSM 16049T	OHO	L. salivarius	AZFP00000000	Pig feces	Animal	Aerobic	37	MRS
Lactobacillus sakei subsp. carnosus	DSM 15831T	FHE	L. sakei	AZFG00000000	Fermented meat product	Food	Microaerophilic	37	MRS
Lactobacillus sakei subsp. sakei	CECT 4591T	FHE	L. sakei	AZDN00000000	“Moto” starter of sake	Wine product	Aerobic	30	MRS
Lactobacillus salivarius	DSM 20555T	FHE	L. salivarius	AYYT00000000	Saliva	Animal	Microaerophilic	37	MRS
Lactobacillus sanfranciscensis	LMG 16002T	OHE	L. fructivorans	AYYM00000000	Sourdough	Food	Anaerobic	28	MRS + 1% fructose (pH 5.5)
Lactobacillus saniviri	DSM 24301T	FHE	L. casei-L. manihotivorans	JQCE00000000	Feces of a Japanese healthy adult male	Animal	Microaerophilic	37	MRS
Lactobacillus satsumensis	DSM 16230T	OHO	L. salivarius	AZFQ00000000	Shochu mash	Wine product	Microaerophilic	30	MRS
Lactobacillus secaliphilus	DSM 17896T	FHE	L. reuteri-L. vaccinostercus	JQBW00000000	Sourdough	Food	Microaerophilic	37	MRS
Lactobacillus selangorensis	ATCC BAA66T	OHO	Other	JQAT00000000	Chili bo	Food	Aerobic	30	MRS
Lactobacillus senioris	DSM 24302T	FHE	L. buchneri	AYZR00000000	Feces of a healthy 100-yr-old Japanese female	Animal	Microaerophilic	37	MRS
Lactobacillus senmaizukei	DSM 21775T	FHE	L. brevis	AYZH00000000	Senmaizuke, a Japanese pickle	Food	Microaerophilic	30	MRS
Lactobacillus sharpeae	DSM 20505T	OHO	L. casei-L. manihotivorans	AYYO00000000	Municipal sewage	Environment	Microaerophilic	30	MRS + 0.05% cysteine
Lactobacillus shenzhenensis	DSM 28193T	OHE	L. perolens	AVAA00000000	Fermented dairy beverage	Food	Microaerophilic	37	MRS
Lactobacillus sicerae	KCTC 21012T	OHO	L. salivarius	NA	Spanish natural cider	Food	Anaerobic	37	MRS
Lactobacillus silagei	DSM 27022T	OHE	L. collinoides	NA	Orchardgrass silage	Plant	Microaerophilic	30	MRS
Lactobacillus siliginis	DSM 22696T	OHE	Other	JQCB00000000	Wheat sourdough	Food	Anaerobic	37	MRS
Lactobacillus similis	DSM 23365T	OHE	L. collinoides	AYZM00000000	Fermented cane molasses at alcohol plants	Wine product	Microaerophilic	37	MRS
Lactobacillus songhuajiangensis	DSM 28401T	OHO	L. casei-L. manihotivorans	NA	Sourdough	Food	Microaerophilic	28	MRS
Lactobacillus spicheri	DSM 15429T	FHE	L. brevis	AZFC00000000	Rice sourdough	Food	Microaerophilic	30	MRS (pH 5.8)
Lactobacillus sucicola	DSM 21376T	OHO	L. salivarius	AYZF00000000	Sap of an oak tree	Plant	Microaerophilic	30	MRS
Lactobacillus suebicus	DSM 5007T	OHE	L. reuteri-L. vaccinostercus	AZGF00000000	Apple mash	Food	Microaerophilic	30	MRS
Lactobacillus sunkii	DSM 19904T	OHE	L. buchneri	AZEA00000000	Sunki, a Japanese traditional pickle	Food	Microaerophilic	30	MRS
Lactobacillus taiwanensis	DSM 21401T	OHO	L. delbrueckii	AYZG00000000	Silage cattle feed	Plant	Microaerophilic	37	MRS
Lactobacillus thailandensis	DSM 22698T	OHO	L. casei-L. manihotivorans	AYZK00000000	Fermented tea leaves (miang)	Plant	Microaerophilic	30	MRS
Lactobacillus tucceti	DSM 20183T	OHO	L. alimentarius	AZDG00000000	Sausage	Food	Aerobic	30	MRS
Lactobacillus ultunensis	DSM 16047T	OHO	L. delbrueckii	AZFO00000000	Gastric biopsy specimens, human stomach mucosa	Animal	Anaerobic	37	MRS
Lactobacillus uvarum	DSM 19971T	OHO	L. salivarius	AZEG00000000	Must of Bobal grape variety	Plant	Microaerophilic	30	MRS
Lactobacillus vaccinostercus	DSM 20634T	OHE	L. reuteri-L. vaccinostercus	AYYY00000000	Cow dung	Animal	Microaerophilic	30	MRS
Lactobacillus vaginalis	LMG 12891T	OHE	L. reuteri-L. vaccinostercus	AZGL00000000	Vaginal swab	Animal	Anaerobic	28	MRS
Lactobacillus versmoldensis	DSM 14857T	OHO	L. alimentarius	AZFA00000000	Poultry salami	Food	Microaerophilic	30	MRS
Lactobacillus vespulae	DSM 103408T	OHE	L. fructivorans	NA	Gut of queen wasp	Animal	Microaerophilic	30	MRS
Lactobacillus vini	DSM 20605T	FHE	L. salivarius	AYYX00000000	Must of grape	Plant	Aerobic	37	MRS
Lactobacillus wasatchensis	LMG 28678T	OHE	L. reuteri-L. vaccinostercus	AWTT00000000	Cheddar cheese	Food	Anaerobic	25	MRS
Lactobacillus xiangfangensis	LMG 26013T	FHE	L. plantarum	JQCL00000000	Pickles	Food	Aerobic	30	MRS
Lactobacillus yonginensis	DSM 29216T	FHE	L. brevis	NA	Kimchi	Food	Microaerophilic	30	MRS
Lactobacillus zeae	DSM 20178T	FHE	L. brevis	AZCT00000000	Corn steep liquor	Wine product	Anaerobic	37	MRS
Lactobacillus zymae	DSM 19395T	OHE	L. casei-L. manihotivorans	AZDW00000000	Artisanal wheat sourdough	Food	Microaerophilic	30	MRS + 0.05% cysteine (pH 5.2)

^aOHO, obligately homofermentative; FHE, facultatively heterofermentative; OHE, obligately heterofermentative.

^bThe phylogroups correspond to those described in references 25 and 29.

^cNA, not available.

A wide range of MIC values was exhibited by all phylogroups for most antibiotics analyzed, except for linezolid, quinupristin-dalfopristin, and chloramphenicol (see Table S1 in the supplemental material). In particular, a unimodal MIC distribution was generally observed for these antibiotics, which covered four 2-fold dilutions for most strains analyzed (89% of strains for quinupristin-dalfopristin and 96% for chloramphenicol and linezolid), ranging in actual MIC values from 2 to 16 µg/ml for chloramphenicol, 1 to 8 µg/ml for linezolid, and 0.5 to 4 µg/ml for quinupristin-dalfopristin.

High MIC values were observed for trimethroprim and vancomycin, at ≥64 and >128 µg/ml, respectively, in the most strains analyzed (81 and 73%, respectively). Interestingly, strains belonging to the Lactobacillus delbrueckii group showed susceptibility to low concentrations of vancomycin (concentrations lower than 1 μg/ml inhibited the growth of 92% of strains belonging to the L. delbrueckii group), despite the general consensus in the literature that Lactobacillus spp. are not inhibited by vancomycin.

The MICs of the remaining antibiotics showed variability across the genus. In particular, the MIC values for aminoglycosides covered more than nine 2-fold dilution steps, ranging from 2 to >1,024 μg/ml for kanamycin, and from 0.2, 0.5, and 2 to >256 μg/ml for gentamicin, neomycin, and streptomycin, respectively. Bimodal MIC distributions were observed for the Lactobacillus alimentarius, Lactobacillus collinoides, Lactobacillus fructivorans, Lactobacillus plantarum, and Lactobacillus reuteri-L. vaccinostercus groups for all aminoglycosides tested (Table S1). Moreover, strains of the L. reuteri-L. vaccinostercus phylogroup showed MIC values distributed across the whole concentration range tested for gentamicin (0.5 to 256 μg/ml), kanamycin (2 to 1,024 μg/ml), and neomycin (0.5 to 256 μg/ml).

The L. alimentarius, L. collinoides, L. delbrueckii, L. fructivorans, L. plantarum, and L. reuteri-L. vaccinostercus groups displayed bimodal MIC value distributions for tetracycline, erythromycin, and clindamycin, antibiotics which notably affect the function of the ribosome. In contrast, a unimodal MIC distribution was observed for β-lactams, including ampicillin and penicillin, except for some strains belonging to the Lactobacillus brevis group (Lactobacillus spicheri DSM 15429^T and Lactobacillus zymae DSM 19395^T), L. collinoides group (Lactobacillus similis DSM 23365^T), L. plantarum group (Lactobacillus pentosus DSM 20314^T), Lactobacillus salivarius group (Lactobacillus ghanensis DSM 18630^T), and other (Lactobacillus selangorensis ATCC BAA66^T) groups, which displayed MIC values of >16 μg/ml.

The distribution of rifampin and ciprofloxacin MIC values was broad; they trended toward the low-end concentration range tested for this antibiotic (0.12 to 16 μg/ml), except for Lactobacillus jensenii DSM 20557^T and Lactobacillus oris DSM 4864^T, belonging to the L. delbrueckii and L. reuteri-L. vaccinostercus groups, respectively, which showed MIC values higher than 64 μg/ml.

Identification of resistance phenotypes.

Phenotypic resistance was interpreted based on the epidemiological cutoff (ECOFF) values reported in references 9, 30, and 31, classifying a strain as resistant when the MIC value for a specific antibiotic was higher than the corresponding ECOFF. EFSA specified the ECOFF value for the species Lactobacillus casei/L. paracasei, Lactobacillus plantarum/L. pentosus, and Lactobacillus rhamnosus. For other species of the genus Lactobacillus, EFSA refers to the fermentation metabolic categories, defining ECOFF values for Lactobacillus obligate heterofermentative (OHE), Lactobacillus obligate homofermentative (OHO), and Lactobacillus facultative heterofermentative (FHE) metabolic patterns (9). For comparative clarity, the fermentation metabolism for the strains analyzed are thus presented in Table 1.

Trimethoprim resistance was the most common phenotype observed (84% [152/182 strains]), and most of the Lactobacillus strains were not susceptible to vancomycin (77% [141/182 strains]) and kanamycin (61% [111/181 strains]) (Fig. 1). Multidrug resistance, defined as resistance to three or more different antimicrobials, was observed in 152 strains (84%). Interestingly, Lactobacillus thailandensis DSM 22698^T showed resistance to all 16 antibiotics tested (Fig. 1). In contrast, Lactobacillus sanfranciscensis LMG 16002^T and Lactobacillus pobuzihii NBRC 103219^T were identified as susceptible to all 16 antibiotics tested, including vancomycin. Lactobacillus ozensis DSM 23829^T, Lactobacillus equigenerosi DSM 18793^T, Lactobacillus capillatus DSM 19910^T, and Lactobacillus vini DSM 20605^T showed resistance only toward vancomycin.

FIG 1.

Resistance profiles of 182 type strains of the genus Lactobacillus compared with epidemiological cutoff values provided in references 9, 30, and 31. Resistant strains with MIC values higher than the ECOFF are indicated in green, whereas sensitive strains are depicted in gray. Strains are clustered by the phylogroups reported by references 25 and 29 and are demarcated by the colored bar on the left of the heat plot. GM, gentamicin; KM, kanamycin; SM, streptomycin; NM, neomycin; TC, tetracycline; EM, erythromycin; CL, clindamycin; CM, chloramphenicol; AM, ampicillin; PC, penicillin; VA, vancomycin; QD, quinupristin-dalfopristin; LZ, linezolid; TM, trimethoprim; CI, ciprofloxacin; RI, rifampin.

Overall, the 182 type strains showed high susceptibility to gentamicin, erythromycin, penicillin, quinupristin-dalfopristin, linezolid, and rifampin (Fig. 1). In fact, only 5% of the strains investigated (10 out of 182) were resistant to quinupristin-dalfopristin. In contrast, the resistance levels toward ampicillin were higher than those identified for penicillin, and they were mainly detected in members of the phylogroups L. brevis (73%), L. alimentarius (58%), and L. collinoides (57%) (Fig. 2).

FIG 2.

Prevalence of antibiotic-resistant (blue) and antibiotic-susceptible (green) strains within the Lactobacillus phylogroups tested for antimicrobial agents specified in reference 9, including inhibitors of cell wall synthesis (ampicillin and vancomycin), inhibitors of protein synthesis (erythromycin, clindamycin, chloramphenicol, and tetracycline), and aminoglycosides (gentamicin, kanamycin, and streptomycin).

Notably, 50% and 49% of the type strains examined in this study were resistant to tetracycline and chloramphenicol, respectively, and 31% were resistant to both antibiotics. Tetracycline resistance phenotypes were mainly observed in species of the phylogroups Lactobacillus buchneri, L. collinoides, L. plantarum, L. reuteri-L. vaccinostercus, L. fructivorans, and L. brevis, while members of the phylogroups L. brevis, Lactobacillus casei-L. manihotivorans, and Lactobacillus perolens showed the highest prevalences of resistance to chloramphenicol (Fig. 2). For clindamycin and streptomycin, the resistance levels were low, at 20% and 18%, respectively, out of the 182 Lactobacillus strains analyzed. Finally, with regard to aminoglycosides, kanamycin resistance occurred in at least 50% of the members of all Lactobacillus phylogroups except for L. perolens, which was very susceptible to this antibiotic. Streptomycin resistance was observed in 35% of the strains examined.

Identification of AR genes.

According to the EFSA guidelines, lactobacilli intended for human consumption should be tested for their resistance to gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, ampicillin, and vancomycin, and they should be genetically investigated for the absence of acquired or transferable AR determinants (9). Thus, the genome sequences for 161 out of the 182 type strains (21 genome sequences were not available at the time of the study) tested for phenotypic resistance were aligned against the protein sequences of AR genes in the Comprehensive Antibiotic Resistance Database (CARD). Based on the selection criteria and manual annotation (see Materials and Methods), a total of 146 gene sequences were identified among the type strains analyzed, which are predicted to encode resistance to aminoglycosides (20 sequences), tetracycline (18), erythromycin (6), clindamycin (60), and chloramphenicol (42) (Fig. S1).

Genes encoding penicillin binding proteins (PBPs) and d-alanine d-alanine ligase (Ddl) involved in ampicillin and vancomycin resistance, respectively, were found in all genomes investigated. Furthermore, amino acid sequence analysis of the PBPs for the 161 type strains confirmed the presence of conserved amino acid residues in the known binding site motif for β-lactams. The Ddl enzyme of all vancomycin-resistant type strains exhibited a conserved phenylalanine (F) residue in the active site of the enzyme, while all members of the phylogroup L. delbrueckii, which were susceptible to vancomycin, were characterized by the presence of a tyrosine (Y) residue at this position, with the exception of strains L. jensenii DSM 20557^T, Lactobacillus amylophilus DSM 20533^T, and Lactobacillus amylotrophicus DSM 20534^T. Their respective proteins carried the Y-type motif, even though they were resistant to vancomycin. However, Lactobacillus amylophilus DSM 20533^T and Lactobacillus amylotrophicus DSM 20534^T harbored specific d-alanine-d-lactate ligase sequences in their genomes, which could explain their vancomycin resistance phenotypes. In addition, L. sanfranciscensis LMG 16002^T, Lactobacillus hilgardii LMG 6895^T, Lactobacillus composti DSM 18527^T, L. pobuzihii NBRC 103219^T, Lactobacillus farciminis LMG 9189, Lactobacillus ceti DSM 22408^T, and Lactobacillus algidus DSM 15638^T were characterized by the presence of Ddl of the F type, despite their susceptibility to vancomycin (Fig. S2).

Aminoglycoside resistance genes.

Twenty different sequences predicted to encode aminoglycoside-modifying enzymes were identified among the 161 Lactobacillus genomes, which were mainly acetyltransferases (AACs) (7 sequences), nucleotidyltransferases (ANTs) (8 sequences), and phosphotransferases (APHs) (5 sequences) (Fig. S1). In particular, the AAC(3) family N-acetyltransferase was found in five L. brevis phylogroup genomes and two L. delbrueckii phylogroup members. All these strains showed resistance to kanamycin, and some of them were also resistant to streptomycin, such as Lactobacillus acidifarinae DSM 19394^T, Lactobacillus koreensis JCM 16448^T, L. spicheri DSM 15429^T, and Lactobacillus hominis DSM 23910^T. Moreover, L. zymae DSM 19395^T showed resistance to gentamicin, whereas Lactobacillus namurensis DSM 19117^T was susceptible to aminoglycosides despite harboring the aac(3) gene.

Gene sequences coding for nucleotidyltranferase enzymes, such as ant(6) and ant(9), were identified in 7 type strains, as follows: L. amylophilus DSM 20533^T and L. amylotrophicus DSM 20534^T (L. delbrueckii phylogroup), Lactobacillus fabifermentas DSM 21115^T (L. plantarum phylogroup), Lactobacillus animalis DSM 20602^T, and L. pobuzihii NBRC 103219^T (L. salivarius phylogroup), Lactobacillus sharpeae DSM 20505^T (L. casei-L. manihotivorans phylogroup), and Lactobacillus rossiae DSM 15814^T (“other” phylogroup). In particular, the ant(9) gene was only found in the genome of L. pobuzihii NBRC 103219^T, despite its phenotypic susceptibility toward aminoglycosides. Similarly, the ant(6) gene was found in L. sharpeae DSM 20505^T and L. rossiae DSM 15814^T, which were susceptible to aminoglycosides. Conversely, the presence of this AR determinant in L. amylophilus DSM 20533^T, L. amylotrophicus DSM 20534^T, L. fabifermentas DSM 21115^T, and L. animalis DSM 20602^T might explain their resistance to kanamycin and streptomycin. Interestingly, in these strains, the predicted amino acid sequence encoded by the ant(6) gene found in the genomes of L. animalis DSM 20602^T and L. amylophilus DSM 20533^T shared 99% similarity with the ANT6 aminoglycoside nucleotidyltransferases of Streptococcus suis (NCBI RefSeq accession no. WP_044770667.1) and Clostridium difficile (NCBI RefSeq accession no. WP_077726164.1). Regarding phosphotransferases, the aph(3) gene was found in five members of the L. delbrueckii phylogroup (Lactobacillus acidophilus ATCC 4356^T, Lactobacillus gasseri LMG 9203^T, Lactobacillus johnsonii LMG 9436^T, Lactobacillus kalixensis DSM 16043^T, and Lactobacillus pasteurii DSM 23907^T), which showed resistance to kanamycin, except for ATCC 4356^T and LMG 9203^T.

Clindamycin resistance genes.

The lsa gene encoding a lincosamide efflux protein was found in 60 strains, 13 of which displayed resistance to clindamycin. Alignment of the amino acid sequence of the Lsa efflux protein revealed a truncated carboxy terminus for the predicted Lsa proteins in L. hominis DSM 23910^T, Lactobacillus gallinarum DSM 10532^T, and Lactobacillus iners DSM 13335^T. Four conserved amino acid domains were found in the remaining 57 sequences, corresponding to two copies of Walker A and B motifs, which play an important role in ATP binding and hydrolysis that energizes efflux (Fig. S3).

Chloramphenicol resistance genes.

Among the 161 type strains of the genus Lactobacillus, 36 chloramphenicol resistance-related sequences were found coding for chloramphenicol acetyltransferase, like the cat gene, and for specific membrane-associated transporters, like CmlA. The cat gene was detected in 34 lactobacilli, two of which (Lactobacillus kimchicus JCM15530^T and L. similis DSM 23365^T) carried two copies of this gene. Moreover, Lactobacillus hammesii DSM 16381^T, L. koreensis JCM 16448^T, L. namurensis DSM 19117^T, and L. zymae DSM 19395^T (belonging to the phylogroup L. brevis) were characterized by the presence of either the cmlA or cat gene. Conversely, L. acidifarinae DSM 19394^T and L. selangorensis ATCC BAA66^T displayed only the presence of cmlA.

Tetracycline resistance genes.

The 18 gene sequences found among Lactobacillus strains code for ribosomal protection proteins [tet(M), tet(S), tet(Q), and tet(W)] and efflux pumps [tet(L) and tet(P)]. The tet(L) gene was found in the tetracycline-resistant strains Lactobacillus suebicus DSM 5007^T and Lactobacillus ingluviei DSM 15946^T. L. ingluviei DSM 15946^T was also characterized by the presence of tet(W) and tet(M). Interestingly, tet(M) in DSM 15946^T exhibited 99% residue identity with the corresponding sequences of Enterococcus faecalis (NCBI RefSeq accession no. WP_049098680.1), Enterococcus faecium (NCBI RefSeq accession no. WP_010777232.1), Streptococcus pneumoniae PT814 (GenBank accession no. HG799502.1), and Staphylococcus epidermidis (NCBI RefSeq accession no. WP_002403674.1). Similarly, the sequence of tet(L) displayed 99% residue identity with those carried by E. faecalis (NCBI RefSeq accession no. WP_002387933.1), E. faecium (NCBI RefSeq accession no. WP_096541192.1), and Streptococcus agalactiae (NCBI RefSeq accession no. WP_041974946.1). tet(W) showed 99% residue identity with the sequences of Trueperella pyogenes OX9, Bifidobacterium longum subsp. longum F21, and C. difficile CI7. tet(W) was also identified in L. pasteurii DSM 23907^T, while tet(M) was found in L. sharpeae DSM 20505^T (L. casei-L. manihotivorans group), L. acidophilus ATCC 4356^T, Lactobacillus crispatus DSM 20584^T, L. gallinarum DSM 10532^T, L. amylophilus DSM 20533^T, L. amylotrophicus DSM 20534^T (L. delbrueckii group), and L. equigenerosi DSM 18793^T (L. reuteri-L. vaccinostercus group). Strains DSM 20584^T, ATCC 4356^T, and DSM 18793^T showed susceptibility to tetracycline. Regarding other ribosomal protection proteins, the tet(Q) and tet(S) genes were found in L. brevis DSM 20054^T and Lactobacillus heilongjiangensis LMG 26166^T, respectively, both being resistant to tetracycline, while tet(P) was found in L. gasseri LMG 9203^T, Lactobacillus taiwanensis DSM 21401^T, Lactobacillus ruminis DSM 20403^T, and L. johnsonii LMG 9436^T. Only LMG 9436^T showed phenotypic resistance to tetracycline.

Erythromycin resistance genes.

Across the 161 Lactobacillus genomes, six gene sequences predicted as contributing to erythromycin resistance were detected, which include the erm(B) gene coding for a predicted rRNA methylase, and two variants of the mef, mef(E), and mef(B) genes, encoding macrolide efflux pumps. These genes were identified in three erythromycin-susceptible strains, namely L. delbrueckii subsp. lactis DSM 20072^T [mef(E)], L. casei DSM 20041^T, and Lactobacillus paracasei subsp. paracasei DSM 5622^T [mef(B)]. erm(B) was found in the erythromycin-resistant strains L. amylophilus DSM 20533^T, L. amylotrophicus DSM 20534^T, and L. ingluviei DSM 15946^T, which also showed the presence of tetracycline resistance determinants.

Analysis of ORFs in regions flanking tetracycline and erythromycin resistance genes.

Tetracycline and erythromycin are two of the most widely used antimicrobials in human and animal therapy, and genetic determinants involved in the resistance to those antibiotics are usually associated with mobile genetic elements. Therefore, upstream and downstream regions of the tetracycline and erythromycin resistance genes were further investigated in L. ingluviei DSM 15946^T, L. amylophilus DSM 20533^T, and L. amylotrophicus DSM 20534^T, the only three strains that simultaneously harbored erythromycin and tetracycline resistance genes within the data set. Sequence analysis of the L. ingluviei DSM 15946^T genome revealed that tet(M) and tet(L) were closely located in the same genomic region, separated by 146 nucleotides. Upstream of tet(M), 12 open reading frames (ORFs) encoding conjugation transfer elements were identified that shared 99% residue identity with the corresponding sequences of the Tn5251 transposon carried by S. pneumoniae DP1322. Moreover, a sequence predicted to encode a transposase was found downstream of the tet(L) determinant (Fig. 3). This is a common configuration for a Tn916-like transposon. The analysis of the distribution of these genetic elements in the available genome sequences of Lactobacillus isolates revealed that the predicted transposon carried by L. ingluviei DSM 15946^T (GenBank accession no. CP016400.1) is essentially identical to that harbored by L. johnsonii BS15, except for the presence of a recombinase downstream of the tet(L) gene. In contrast, L. salivarius JCM 1046 (GenBank accession no. CP007650.1) and L. salivarius JCM 1047 (GenBank accession no. NBEF01000044.1) lack the tet(L) determinant, and the regulation region has been retained, similar to that of the Tn5251 transposon in S. pneumoniae DP1322. These two transposons are essentially identical except for the presence of ORF 23 in L. salivarius JCM 1047, which was not identified in the transposon harbored by L. salivarius JCM 1046. Interestingly, L. iners UMB1051 (GenBank accession no. PNGO01000001.1) was characterized by the presence of a Tn916-like transposon in which a Tn917 carrying an erm(B) gene was inserted, resulting in a Tn3872-like transposon (Fig. S4). The tet(W) and erm(B) genes were located in the same genomic region and shared 99% similarity with the corresponding sequences of the strain S. suis SsCA. Moreover, these AR determinants were flanked by regions with high similarity (99%) to replication proteins and to the integrase of the plasmid pLR581 of Lactobacillus reuteri SD2112 (GenBank accession no. CP002845.1) (Fig. 4).

FIG 3.

Diagram showing the genetic organization of the Tn916-like transposon (transp) identified in L. ingluviei DSM 15946^T. Red, AR genes; yellow, genes involved in genetic transfer; gray, ORFs involved in the conjugation process; green, regulatory sequences. Numbers above the diagram refer to loci in Tn916; numbers below are a base pair scale.

FIG 4.

Genetic organization of sequences surrounding the tet(W) and erm(B) genes identified in L. ingluviei DSM 15946^T. Red, AR genes; yellow, genes involved in genetic transfer; pink, genes encoding plasmid-associated replication proteins; gray, gene coding for hypothetical proteins. Numbers above the diagram refer to loci; numbers below are a base pair scale. Rep, plasmid replication; int; plasmid integration.

L. amylophilus and L. amylotrophicus harbored the tet(M) and erm(B) genes in two different genomic regions, and they shared 99% residue identity with the corresponding sequences of S. agalactiae SG-M4 and Staphylococcus hyicus HW17, respectively. The flanking region structures of these AR determinants were identical at the amino acid level in the two type strains. In particular, the up- and downstream sequences surrounding tet(M) were characterized by the presence of several genes predicted to encode conjugation proteins and transposases (Fig. 5A). For erm(B), the upstream region showed an 83-bp sequence corresponding to a 27-amino-acid leader peptide. A gene encoding a mobilization protein was found upstream of the leader peptide sequence, sharing 99% residue identity with the corresponding sequence in the S. hyicus plasmid pSTE1 (GenBank accession no. HE662694.1) (Fig. 5B). Unfortunately, the location of erm(B) at the 3′ end of the contig did not allow for the characterization of the downstream region.

FIG 5.

Genetic organization of sequences surrounding the tet(M) (A) and erm(B) (B) genes identified in L. amylophilus DSM 20533^T and L. amylotrophicus DSM 20534^T. Red, AR genes; yellow, genes involved in genetic transfer; green, genes encoding regulatory proteins; gray, gene coding for hypothetical proteins. int, integrase. Numbers below the diagram are a base pair scale.

Phenotype-genotype correlation.

Overall, phenotypic resistance correlated with genotypes for 67% of the cases examined with genomic data. In detail, the genotype was in accordance with the phenotype for 892 out of 1,449 phenotypic tests investigated, which included 782 cases representing a susceptible phenotype toward a specific antibiotic linked to the absence of AR determinants and 110 cases for which the resistance phenotype correlated with the presence of one or more AR genes. Most notably, the F-type Ddl enzyme was found in 99% of the vancomycin-resistant Lactobacillus strains, revealing the high relationship between genotype and phenotype for this antibiotic. For aminoglycosides, the 20 AR determinants identified in this study might explain the resistance phenotype for 13.7%, 14.2%, and 20.8% of the strains for kanamycin, streptomycin, and gentamicin, respectively. For chloramphenicol, the cat and cmlA genes were found in 20 strains out of 79 chloramphenicol-resistant lactobacilli and in 59 strains out of 82 lactobacilli susceptible to this antibiotic. However, the genetic basis of chloramphenicol resistance was not revealed for 74.7% of the resistant strains. The presence of the lsa gene positively correlated with the resistance phenotype for 40.6% of the strains. However, 47 strains harbored the lsa gene in their genomes even though they showed susceptibility to clindamycin. For tetracycline and erythromycin, the presence of AR genes explained the resistance phenotype for 12.7% and 10.3% of the strains, respectively. However, in a significant number of examples (364 cases), a genetic basis was not detected for a demonstrated resistance phenotype (Fig. 6).

FIG 6.

Phenotype-genotype correlation analysis for the 161 type strains of the genus Lactobacillus. Positive correlations between genomic data and phenotypes observed are presented in green and gray, whereas negative correlations are indicated in yellow and blue. GM, gentamicin; KM, kanamycin; SM, streptomycin; TC, tetracycline; EM, erythromycin, CL, clindamycin; CM, chloramphenicol; AM, ampicillin; VA, vancomycin.

DISCUSSION

The data reported in the present study provide evidence at phenotype and genotype levels for rationally revising the regulatory guidelines for safety assessment of lactobacilli, showing that certain resistances are widespread within the genus. For example, low-level resistance to linezolid, rifampin, and quinupristin-dalfopristin was generally observed for type strains, in line with previous reports (30–35). We also confirmed low-level resistance to rifampin, as previously reported for Lactobacillus species isolated from the human GIT (36), traditional dairy products (37), and in probiotic strains in marketed foods and drugs (38). Generally, we found Lactobacillus species to be susceptible to low concentrations of β-lactams, including penicillin and ampicillin (16), even though atypical insensitivity to higher concentrations of these cell wall inhibitors has been reported for some L. crispatus and L. johnsonii strains isolated from healthy chickens (39). Higher levels of resistance to ampicillin compared to penicillin were detected for the type strains analyzed, in line with some previous studies (40–42). Interestingly, ampicillin resistance in L. reuteri strains has been attributed to point mutations in the genes encoding the penicillin binding proteins (PBPs) (43), but the PBP sequences for the 161 type strains in this study all retained conserved amino acid residues (i.e., linked to sensitivity) in the binding sites for these antibiotics.

Conversely, the Lactobacillus strains showed high intrinsic (as opposed to acquired) resistance to trimethoprim and vancomycin. Folate auxotrophic lactobacilli have been reported as being intrinsically resistant to trimethoprim (44), including L. johnsonii, L. acidophilus, L. salivarius, L. brevis, L. casei, L. gasseri, L. rhamnosus, L. delbrueckii, Lactobacillus fermentum, Lactobacillus helveticus, L. plantarum, L. reuteri, Lactobacillus sakei, and L. crispatus (45), where the vancomycin-resistant phenotypes are perhaps the best-characterized resistance mechanisms described in lactobacilli (16), with many species being intrinsically resistant (30). Exceptions to this are L. delbrueckii, L. acidophilus, L. johnsonii, and L. crispatus, where the vancomycin-susceptible phenotype has been associated with the presence of a Y-type Ddl enzyme (46). In this genus-wide analysis, the vancomycin-susceptible strains were mainly represented by almost all members of the L. delbrueckii phylogroup, which were characterized by the presence of a Y-type Ddl. The substitution of tyrosine 261 in the Ddl enzyme by a phenylalanine residue (F-type enzyme) is associated with the synthesis of peptidoglycan precursors containing a d-Ala-d-Lac residue conferring vancomycin resistance (46). Notably, almost all vancomycin-resistant strains in this study carried a Ddl of the F type. However, we noted inconsistencies between phenotypes and genotypes for vancomycin in 8 of the species (L. jensenii, L. sanfraciscensis, L. hilgardii, L. composti, L. pobuzihii, L. farciminis, L. ceti, and L. algidus), which could be due to the presence of alternative resistance mechanisms or to alteration of gene expression.

Overall, Lactobacillus type strains displayed higher resistance toward kanamycin and streptomycin than to gentamicin and neomycin. Aminoglycoside resistance has been described as an intrinsic feature for some Lactobacillus species (i.e., L. rhamnosus, L. acidophilus, L. delbrueckii subsp. bulgaricus, and L. helveticus) (47, 48) due to the lack of cytochrome-mediated drug transport (15). In contrast, high-level susceptibility to gentamicin is most likely linked to the superior ability of this antibiotic to cross the membrane compared to other aminoglycosides (49).

We found genes for nucleotidyltransferases, such as ant(6) and ant(9), in 7 type strains, 4 of which showed resistance to kanamycin and streptomycin (L. amylophilus, L. amylotrophicus, L. fabifermentas, and L. animalis), in accordance with the high affinity of ANT6 for streptomycin (50). Moreover, the nucleotide sequence of the ant(6) gene harbored by L. animalis DSM 20602^T and L. amylophilus DSM 20533^T was 99% identical to the sequences in S. suis and C. difficile, of which S. suis is usually associated with the animal GIT (51–53), from which L. animalis strains can be isolated (54). The GIT is characterized by a high cell density, which can lead to microbial interactions and facilitates horizontal gene transfer (HGT) events (55, 56). These findings suggest that the ant(6) gene is undergoing HGT between commensal/food bacteria and pathogenic species, which in and of itself is a particular concern. As such, these lactobacilli could be acting as vectors for AR genes in the food chain and the gut. Furthermore, the ant(6) and aph(3) genes are usually found on plasmids or transposons, increasing the risk of resistance dissemination between different bacteria (57). We found the aph(3) and aac(3) genes in 9 Lactobacillus strains conferring resistance to kanamycin, in accordance with the substrate specificity of these AR determinants for aminoglycosides (46). Moreover, the aph(3) gene has been identified in Lactobacillus strains resistant to kanamycin, streptomycin, and gentamicin in a previous study (50).

Although the majority of Lactobacillus species are susceptible to antibiotics that inhibit protein synthesis, including erythromycin, tetracycline, clindamycin, and chloramphenicol (14), resistance to tetracycline and chloramphenicol was the most common phenotype detected. Even though this Lactobacillus type strain panel was characterized by low-level resistance to clindamycin, some strains carried the lsa gene, which in E. faecium plays a key role in resistance to clindamycin and quinupristin-dalfopristin (58). The ABC transporter Walker A and B motifs (59) are conserved in the Lsa protein predicted for the strains in this study, suggesting that it is functional.

Chloramphenicol (cat genes) and tetracycline resistance determinants [tet(M), tet(S), tet(W), tet(O), and tet(Q)] are the most commonly acquired resistance genes found in lactobacilli (15, 19, 39, 60, 61). In particular, the most widespread resistance genes are tet(M) and tet(S) in foodborne and probiotic bacteria (12, 14, 62) due to the frequent association of tet(M) with conjugative transposons, such as Tn916 (63), which was corroborated by this study. Furthermore, our data revealed the association of tet(M) with a Tn916-like transposon in the genome of L. ingluviei DSM 15946^T. This transposon sequence is 99% identical to the Tn5251 of S. pneumoniae DP1322 (64), except for the presence of tet(L) in Tn916. Notably, this study reports a Tn916-like transposon in L. ingluviei, and the transferability of this element warrants further investigation. A Tn916-like transposon has been previously identified in L. paracasei and L. sakei isolated from Italian traditional cheese (65, 66) and in L. salivarius JCM 1046 (22).

Notably, we detected the simultaneous presence of tet(M) and erm(B) genes in L. amylophilus DSM 20533^T and L. amylotrophicus DSM 20534^T, which were flanked by transposases and conjugative proteins in both organisms. This is probably reflective of/arising from the close phylogenetic relatedness between L. amylophilus and L. amylotrophicus (67). The erm(B) gene (associated with erythromycin resistance) has been reported in Lactobacillus species in several studies (19, 30, 60, 68–70), where it is generally found in conjugative transposons located in chromosomes, in plasmids, and in nonconjugative transposons, such as Tn917 and Tn551 (15). These observations suggest that L. ingluviei, L. amylophilus, and L. amylotrophicus could act as dissemination vectors for tetracycline and erythromycin resistance. Strains of L. ingluviei and L. amylophilus are commonly isolated from animal GIT (71), whereas L. amylotrophicus has been found in swine waste (67); these are considered potential hotspots for promoting the dissemination of AR genes in the environment. Indeed, the transfer of erm(B) and tet(M) from lactobacilli to other microorganisms has been previously demonstrated in vitro (62, 68, 72). The present study reveals that in many cases, the genetic basis for AR remains unknown, as no known genes were identified which could explain the associated phenotype. This could be due to the inherent insensitivity of some of the strains to particular antibiotics due to intrinsic factors, such as cell envelope (wall/membrane) structure or the ability to produce polysaccharides. Indeed, it would be very difficult to predict these phenotypes based on just database comparisons alone, and as such, functional studies would be required to probe the basis of these resistances more deeply. Overall, this pangenomic study reveals that while AR is widespread in lactobacilli, the level of resistance to widely used antibiotics in human clinical medicine is low or not so common (with the possible exception of erythromycin and tetracycline). However, the vast majority (88%) of these strains were found to fail the EFSA guidelines for AR and as such might encounter problems in obtaining regulatory approval for food use unless the resistance is proven to be nontransferable. As such, opportunities to develop new applications for novel Lactobacillus strains could be delayed or lost, which highlights the urgent need to fundamentally understand AR and its spread within members of the genus. Only then can a proper framework to regulate their use for human and animal purposes be implemented.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The 197 type strains of the Lactobacillus genus used in this study are listed in Table 1 and were obtained from the American Type Culture Collection (ATCC, Manassas, VA), BCCM/LMG Bacteria Collection (Ghent, Belgium), the Spanish Type Culture Collection (CECT, Valencia, Spain), the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), the Korean Collection for Type Cultures (KCTC, Jeollabuk-do, South Korea), the Japan Collection of Microorganisms (JCM, Koyadai Tsukuba, Japan), and the NITE Biological Resource Centre (NBRC, Nishihara, Japan). Lactobacillus strains were grown in de Man-Rogosa-Sharpe (MRS) medium (Thermo Fisher Scientific, Waltham, MA, USA) under specific conditions reported in Table 1 and kept in liquid cultures with 20% (w/vol) glycerol at −80°C for long-term storage.

Antimicrobial susceptibility testing.

The MICs of several antibiotics were determined using broth microdilution methods according to the Clinical and Laboratory Standards Institute (CLSI; www.clsi.org), the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.eucast.org), and ISO standards. In particular, VetMIC plates (National Veterinary Institute, Uppsala, Sweden) for LAB were used containing serial 2-fold dilutions of 16 antibiotics (ampicillin, ciprofloxacin, clindamycin, chloramphenicol, erythromycin, gentamicin, kanamycin, linezolid, neomycin, penicillin, quinupristin-dalfopristin, rifampin, streptomycin, tetracycline, trimethoprim, and vancomycin). These antibiotics represent the main classes of antimicrobials employed in human and veterinary treatments and overlap the main antibiotics of interest to the EFSA.

MICs were evaluated in LAB susceptibility test medium (LSM) (73), a mixed formulation containing 90% Iso-Sensitest broth and 10% MRS Difco broth (Beckton, Dickinson and Company, Le Pont-de-Claix, France) supplemented with 0.05% (wt/vol) l-cysteine, as described in the ISO 10932 (IDF 223) document (74) and recommended by the EFSA (9). L. paracasei LMG 12586 was used as a control strain. Briefly, individual Lactobacillus strains were grown on MRS agar (with supplements as required for the specific strains and incubation for 24 to 48 h depending on the strain). One-microliter sterile loops with biomass from approximately 3 to 5 colonies were suspended in 4 ml maximum recovery diluent (MRD; Oxoid Ltd., Basingstoke, Hants, UK) sterile saline solution to obtain a concentration of approximately 3 × 10⁸ CFU/ml in each case. This suspension was diluted 1:1,000 in LSM broth (final concentration, approximately 3 × 10⁵ CFU/ml), and then 100 μl was added to each well of the VetMIC plate. This test was performed using a minimum of three biological replicates for each strain. Plates were incubated under anaerobic conditions at the recommended temperatures for 48 h. MICs were read as the lowest concentration of an antimicrobial agent at which visible growth was inhibited. Epidemiological cutoff (ECOFF) values were retrieved from reference 9. Breakpoints for antibiotics not covered by EFSA were adopted from references 30 and 31.

Identification of resistance genes.

The annotated sequences of the available genomes for the type strains of the genus Lactobacillus (25) were downloaded from NCBI using the accession numbers reported in Table 1. These sequences were employed to query the Comprehensive Antibiotic Resistance Database (CARD, version 1.0.6; http://arpcard.mcmaster.ca) (75) through the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov) in order to identify all AR genes involved in the resistance phenotypes observed. A gene was annotated as a putative AR determinant according to its best BLASTP hit in CARD, with a threshold of amino acid sequence identity of >30% and query coverage of >70%. In addition, the amino acid sequences of all AR genes retrieved from CARD, resulting in a reference data set of 2,163 amino acid sequences, were aligned against the annotated genome sequences of the collection, and the best BLASTP hits were filtered as described above. In order to minimize putative false-negative or false-positive outputs, only the putative AR determinants obtained from both approaches were considered for subsequent analyses. Specifically, each putative AR determinant was manually annotated querying the NCBI nonredundant (NR) protein database to verify its putative function in the resistome and to determine its involvement in acquired phenotypes.

Phenotype-genotype correlation.

By focusing on the nine antibiotics for which the EFSA defined reference ECOFFs (9) and the genome sequences available for 161 Lactobacillus strains, a total of 1,449 phenotypic tests were considered for the phenotype-genotype correlation. Each interpretation of a resistant or susceptible phenotype to a given antimicrobial agent was compared with the presence or absence of a known corresponding resistance gene(s) manually annotated and/or structural gene mutations identified through genome sequence analysis (76–78). The overall correlation between phenotype and genotype was classified as positive when genomic data agreed with phenotypic testing; thus, resistance and susceptible phenotypes correlated with the presence or absence of one or more AR genes, respectively. Otherwise, the correlation was considered negative.

Flanking regions of the AR genes.

The genetic composition of upstream and downstream sequences flanking tetracycline and erythromycin resistance genes was characterized by performing a BLASTN and BLASTX alignment of the contigs carrying the AR genes against the NCBI NR database. This analysis was carried out for L. ingluviei DSM 15946^T, L. amylophilus DSM 20533^T, and L. amylotrophicus DSM 20534^T and allowed us to identify mobile genetic elements which might be involved in the spread of AR determinants.