Phenotypic, Technological, Safety, and Genomic Profiles of Gamma-Aminobutyric Acid-Producing Lactococcus lactis and Streptococcus thermophilus Strains Isolated from Cow's Milk.

Gamma-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) can be used as starters in the development of GABA-enriched functional fermented foods. In this work, four GABA-producing strains each of Lactococcus lactis and Streptococcus thermophilus species were isolated from cow's milk, and their phenotypic, technological, and safety profiles determined. Genome analysis provided genetic support for the majority of the analyzed traits, namely, GABA production, growth in milk, and the absence of genes of concern. The operon harboring the glutamate decarboxylase gene (gadB) was chromosomally encoded in all strains and showed the same gene content and gene order as those reported, respectively, for L. lactis and S. thermophilus. In the latter species, the operon was flanked (as in most strains of this species) by complete or truncated copies of insertion sequences (IS), suggesting recent acquisition through horizontal gene transfer. The genomes of three L. lactis and two S. thermophilus strains showed a gene encoding a caseinolytic proteinase (PrtP in L. lactis and PrtS in S. thermophilus). Of these, all but one grew in milk, forming a coagulum of good appearance and an appealing acidic flavor and taste. They also produced GABA in milk supplemented with monosodium glutamate. Two L. lactis strains were identified as belonging to the biovar. diacetylactis, utilized citrate from milk, and produced significant amounts of acetoin. None of the strains showed any noticeable antibiotic resistance, nor did their genomes harbor transferable antibiotic resistance genes or genes involved in toxicity, virulence, or pathogenicity. Altogether these results suggest that all eight strains may be considered candidates for use as starters or components of mixed LAB cultures for the manufacture of GABA-enriched fermented dairy products.

Antibiotic Resistance/Susceptibility Profiles of Staphylococcus equorum Strains from Cheese, and Genome Analysis for Antibiotic Resistance Genes.

In food, bacteria carrying antibiotic resistance genes could play a prominent role in the spread of resistance. Staphylococcus equorum populations can become large in a number of fermented foods, yet the antibiotic resistance properties of this species have been little studied. In this work, the resistance/susceptibility (R/S) profile of S. equorum strains (n = 30) from cheese to 16 antibiotics was determined by broth microdilution. The minimum inhibitory concentration (MIC) for all antibiotics was low in most strains, although higher MICs compatible with acquired genes were also noted. Genome analysis of 13 strains showed the S. equorum resistome to be composed of intrinsic mechanisms, acquired mutations, and acquired genes. As such, a plasmidic cat gene providing resistance to chloramphenicol was found in one strain; this was able to provide resistance to Staphylococcus aureus after electroporation. An msr(A) polymorphic gene was identified in five strains. The Mrs(A) variants were associated with variable resistance to erythromycin. However, the genetic data did not always correlate with the phenotype. As such, all strains harbored a polymorphic fosB/fosD gene, although only one acquired copy was associated with strong resistance to fosfomycin. Similarly, a plasmid-associated blaR1-blaZI operon encoding a penicillinase system was identified in five ampicillin- and penicillin G-susceptible strains. Identified genes not associated with phenotypic resistance further included mph(C) in two strains and norA in all strains. The antibiotic R/S status and gene content of S. equorum strains intended to be employed in food systems should be carefully determined.

Phenotypic and Safety Assessment of the Cheese Strain Lactiplantibacillus plantarum LL441, and Sequence Analysis of its Complete Genome and Plasmidome.

This work describes the phenotypic typing and complete genome analysis of LL441, a dairy Lactiplantibacillus plantarum strain. LL441 utilized a large range of carbohydrates and showed strong activity of some carbohydrate-degrading enzymes. The strain grew slowly in milk and produced acids and ketones along with other volatile compounds. The genome of LL441 included eight circular molecules, the bacterial chromosome, and seven plasmids (pLL441-1 through pLL441-7), ranging in size from 8.7 to 53.3 kbp. Genome analysis revealed vast arrays of genes involved in carbohydrate utilization and flavor formation in milk, as well as genes providing acid and bile resistance. No genes coding for virulence traits or pathogenicity factors were detected. Chromosome and plasmids were packed with insertion sequence (IS) elements. Plasmids were also abundant in genes encoding heavy metal resistance traits and plasmid maintenance functions. Technologically relevant phenotypes linked to plasmids, such as the production of plantaricin C (pLL441-1), lactose utilization (pLL441-2), and bacteriophage resistance (pLL441-4), were also identified. The absence of acquired antibiotic resistance and of phenotypes and genes of concern suggests L. plantarum LL441 be safe. The strain might therefore have a use as a starter or starter component in dairy and other food fermentations or as a probiotic.

Directed Recovery and Molecular Characterization of Antibiotic Resistance Plasmids from Cheese Bacteria.

Resistance to antimicrobials is a growing problem of worldwide concern. Plasmids are thought to be major drivers of antibiotic resistance spread. The present work reports a simple way to recover replicative plasmids conferring antibiotic resistance from the bacteria in cheese. Purified plasmid DNA from colonies grown in the presence of tetracycline and erythromycin was introduced into plasmid-free strains of Lactococcus lactis, Lactiplantibacillus plantarum and Lacticaseibacillus casei. Following antibiotic selection, the plasmids from resistant transformants were isolated, analyzed by restriction enzyme digestion, and sequenced. Seven patterns were obtained for the tetracycline-resistant colonies, five from L. lactis, and one each from the lactobacilli strains, as well as a single digestion profile for the erythromycin-resistant transformants obtained in L. lactis. Sequence analysis respectively identified tet(S) and ermB in the tetracycline- and erythromycin-resistance plasmids from L. lactis. No dedicated resistance genes were detected in plasmids conferring tetracycline resistance to L. casei and L. plantarum. The present results highlight the usefulness of the proposed methodology for isolating functional plasmids that confer antibiotic resistance to LAB species, widen our knowledge of antibiotic resistance in the bacteria that inhabit cheese, and emphasize the leading role of plasmids in the spread of resistance genes via the food chain.

Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances.

Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances.

Effect of different starter cultures on chemical and microbial parameters of buckwheat honey fermentation.

The aim of this study was to analyze the microbiology of buckwheat honey fermentation inoculated with different starter cultures by culturing and PCR-DGGE, taking as a model for comparison a spontaneously fermented batch. The inoculants tested were (i) cider lees (from a cider factory), (ii) sourdough (from a bakery), and (iii) a commercial Saccharomyces cerevisiae strain. The results of the culturing and culture-independent techniques agreed well and detected the same dominant species along the fermentations. Our results suggest that S. cerevisiae strains, which constituted a majority population in all batches including the uninoculated one, carried out the fermentations. The highest microbial diversity was found at the beginning of the fermentation in the uninoculated batch; this contained in addition to S. cerevisiae bacteria (Paracoccus sp., Staphylococcus sp., and Bacillus sp.) and yeast (Candida sp.) species. Candida sp. was also common in batches inoculated with sourdough and cider lees cultures. Lactobacillus species were found throughout the fermentation of the sourdough-inoculated batch. Basic chemical analysis and testing trials demonstrated that the overall sensory acceptance of the four meads were highly similar. Yeast and bacteria isolated in this study could serve as a source of technologically relevant microorganisms for mead production.

Corrigendum: Phenotype testing, genome analysis, and metabolic interactions of three lactic acid bacteria strains existing as a consortium in a naturally fermented milk.

[This corrects the article DOI: 10.3389/fmicb.2022.1000683.].

Epicoccum sp. as the causative agent of a reddish-brown spot defect on the surface of a hard cheese made of raw ewe milk.

Colour defects can affect the appearance of cheese, its flavour, the safety of its consumption, and the price it can demand. This work reports the identification of five fungal isolates from a dairy plant where the surface of most cheeses was affected by patent, reddish-to-brown stains. One of these isolates was obtained from cheese, two from brine, and two from a bulk tank containing ewe milk. Molecular identification by partial amplification, sequencing, and database comparison of the concatenated sequence of the genes coding for the largest subunit of RNA polymerase II (RPB2), ß-tubulin (ß-TUB), and the large subunit of the rRNA molecule (LSU), plus the internal transcribed sequence (ITS) regions, assigned the isolates to Epicoccum layuense, Epicoccum italicum, and Epicoccum mezzettii. Features of the growth of these different species on different agar-based media, and of the morphology of their conidia following sporulation, are also reported. The strain isolated from cheese, E. layuense IPLA 35011, was able to recreate the reddish-brown stains on slices of Gouda-like cheese, which linked the fungus with the colour defect. In addition, two other strains, E. italicum IPLA 35013 from brine and E. italicum IPLA 35014 from milk, also produced stains on cheese slices. Epicoccum species are widely recognized as plant pathogens but have seldom been reported in the dairy setting, and never as human or animal pathogens.

Prodigiosin-Producing Serratia marcescens as the Causal Agent of a Red Colour Defect in a Blue Cheese.

Technological defects in the organoleptic characteristics of cheese (odour, colour, texture, and flavour) reduce quality and consumer acceptance. A red colour defect in Cabrales cheese (a traditional, blue-veined, Spanish cheese made from raw milk) occurs infrequently but can have a notable economic impact on family-owned, artisanal cheesemaking businesses. This work reports the culture-based determination of Serratia marcescens as the microbe involved in the appearance of red spots on the surface and nearby inner areas of such cheese. Sequencing and analysis of the genome of one S. marcescens isolate, RO1, revealed a cluster of 16 genes involved in the production of prodigiosin, a tripyrrole red pigment. HPLC analysis confirmed the presence of prodigiosin in methanol extracts of S. marcescens RO1 cultures. The same was also observed in extracts from red areas of affected cheeses. The strain showed low survival rates under acidic conditions but was not affected by concentrations of up to 5% NaCl (the usual value for blue cheese). The optimal conditions for prodigiosin production by S. marscescens RO1 on agar plates were 32 °C and aerobic conditions. Prodigiosin has been reported to possess antimicrobial activity, which agrees with the here-observed inhibitory effect of RO1 supernatants on different bacteria, the inhibition of Enterobacteriaceae, and the delayed development of Penicillium roqueforti during cheesemaking. The association between S. marcescens and the red colour defect was strengthened by recreating the fault in experimental cheeses inoculated with RO1. The data gathered in this study point towards the starting milk as the origin of this bacterium in cheese. These findings should help in the development of strategies that minimize the incidence of pigmenting S. marcescens in milk, the red defect the bacterium causes in cheese, and its associated economic losses.

Genome sequence of Lactococcus garvieae IPLA 31405, a bacteriocin-producing, tetracycline-resistant strain isolated from a raw-milk cheese.

This work describes the draft genome sequence of Lactococcus garvieae IPLA 31405, isolated from a traditional Spanish cheese. The genome contains a lactose-galactose operon, a bacteriocin locus, two integrated phages, a transposon harboring an active tet(M) gene, and two theta-type plasmid replicons. Genes encoding virulence factors were not recorded.

Phenotype testing, genome analysis, and metabolic interactions of three lactic acid bacteria strains existing as a consortium in a naturally fermented milk.

This work reports the characterization of three lactic acid bacteria (LAB) strains -Lactococcus lactis LA1, Lactococcus cremoris LA10, and Lactiplantibacillus plantarum LA30- existing as a stable consortium in a backslopping-inoculated, naturally fermented milk (NFM). This study aimed at uncovering the biochemical and genetic basis of the stability of the consortium and the cooperativity among the strains during milk fermentation. All three strains were subjected to phenotyping, covering the utilization of carbohydrates, enzyme activity, and antibiotic resistance. The strains were grown in milk individually, as well as in all possible combinations, and the resulting fermented product was analyzed for sugars, organic acids, and volatile compounds. Finally, the genomes of the three strains were sequenced and analyzed for genes associated with technological and safety properties. As expected, wide phenotypic diversity was seen between the strains. Lactococcus cremoris LA10 was the only strain to reach high cell densities and coagulate milk alone after incubation at 22°C for 24 h; congruently, it possessed a gene coding for a PrtP type II caseinolytic protease. Compared to any other fermentation, acetaldehyde concentrations were greater by a factor of six when all three strains grew together in milk, suggesting that its production might be the result of an interaction between them. Lactococcus lactis LA1, which carried a plasmid-encoded citQRP operon, was able to utilize milk citrate producing diacetyl and acetoin. No genes encoding virulence traits or pathogenicity factors were identified in any of the strains, and none produced biogenic amines from amino acid precursors, suggesting them to be safe. Lactiplantibacillus plantarum LA30 was susceptible to tetracycline, although it harbors a disrupted antibiotic resistance gene belonging to the tetM/tetW/tetO/tetS family. All three strains contained large numbers of pseudogenes, suggesting that they are well adapted ("domesticated") to the milk environment. The consortium as a whole or its individual strains might have a use as a starter or as starter components for dairy fermentations. The study of simple consortia, such as that existing in this NFM, can help reveal how microorganisms interact with one another, and what influence they may have on the sensorial properties of fermented products.

Isolation and phenotypic and genomic characterization of Tetragenococcus spp. from two Spanish traditional blue-veined cheeses made of raw milk.

High throughput sequencing has recently revealed the presence of Tetragenococcus-related DNA sequences in dairy environments such as brine and cheeses. In the present work, a selective medium was developed to isolate Tetragenococcus spp. from two ripened, traditional, Spanish, blue-veined cheese varieties made from raw milk. The strains recovered belonged to either Tetragenococcus koreensis or Tetragenococcus halophilus species. Twenty of these isolates (15 of T. koreensis and 5 of T. halophilus) were then subjected to a battery of phenotypic and genetic tests, and six strains (4 T. koreensis and 2 T. halophilus) to genome sequencing. Wide genetic and phenotypic diversity was noted. All strains grew poorly in milk, producing small quantities of lactic and acetic acids. Most strains used lactose as a carbon source and ferment milk citrate. In agreement, genome analysis detected in the genome of the six strains analyzed gene clusters harboring several lactose/galactose-related genes and genes encoding citrate metabolic enzymes (permease, citrate lyase, and oxaloacetate decarboxylase). Most of the tested strains were resistant to erythromycin and clindamycin, and a few to other antimicrobial agents, but neither known mutations nor acquired genes conferring resistance to antibiotics were identified in their genomes. Neither were genes coding for pathogenicity or virulence factors detected. Decarboxylase-encoding genes involved in biogenic amine production were not identified, in keeping with the strains' negative biogenic amine-producer phenotype. Genome comparison revealed vast arrays of genes (similar in number to those described in other lactic acid bacteria) coding for components of proteolytic and lipolytic systems. Tetragenococcus strains showing desirable traits plus the absence of detrimental features might be exploitable in the form of secondary, adjunct or ripening cultures to ensure the typical bouquet of traditional blue-veined cheeses is obtained, or to diversify the final flavor in other varieties.

Microbial Interactions within the Cheese Ecosystem and Their Application to Improve Quality and Safety.

The cheese microbiota comprises a consortium of prokaryotic, eukaryotic and viral populations, among which lactic acid bacteria (LAB) are majority components with a prominent role during manufacturing and ripening. The assortment, numbers and proportions of LAB and other microbial biotypes making up the microbiota of cheese are affected by a range of biotic and abiotic factors. Cooperative and competitive interactions between distinct members of the microbiota may occur, with rheological, organoleptic and safety implications for ripened cheese. However, the mechanistic details of these interactions, and their functional consequences, are largely unknown. Acquiring such knowledge is important if we are to predict when fermentations will be successful and understand the causes of technological failures. The experimental use of "synthetic" microbial communities might help throw light on the dynamics of different cheese microbiota components and the interplay between them. Although synthetic communities cannot reproduce entirely the natural microbial diversity in cheese, they could help reveal basic principles governing the interactions between microbial types and perhaps allow multi-species microbial communities to be developed as functional starters. By occupying the whole ecosystem taxonomically and functionally, microbiota-based cultures might be expected to be more resilient and efficient than conventional starters in the development of unique sensorial properties.

Heterologous expression of equol biosynthesis genes from Adlercreutzia equolifaciens.

Equol is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. It is produced from daidzein by fastidious and oxygen-susceptible intestinal bacteria, which hinders their use at an industrial scale. Therefore, expressing the equol production machinery into easily-cultivable hosts would expedite the heterologous production of this compound. In this work, four genes (racemase, tdr, ddr and dzr) coding for key enzymes involved in equol production in Adlercreutzia equolifaciens DSM19450T were synthesized and cloned in a pUC-derived vector (pUC57-equol) that was introduced in Escherichia coli. Recombinant clones of E. coli produced equol in cultures supplemented with daidzein (equol precursor) and dihydrodaidzein (intermediate compound). To check whether equol genes were expressed in Gram-positive bacteria, the pUC57-equol construct was cloned into the low-copy-number vector pIL252, and the new construct (pIL252-pUC57-equol) introduced into model strains of Lacticaseibacillus casei and Lactococcus lactis. L. casei clones carrying pIL252-pUC57-equol produced a small amount of equol from dihydrodaidzein but not from daidzein, while L. lactis recombinant clones produced no equol from either of the substrates. This is the first time that A. equolifaciens equol genes have been cloned and expressed in heterologous hosts. E. coli clones harboring pUC57-equol could be used for biotechnological production of equol.

Intra- and interlaboratory performances of two commercial antimicrobial susceptibility testing methods for bifidobacteria and nonenterococcal lactic acid bacteria.

In a small-scale harmonization study involving nine laboratories in eight European countries, the intra- and interlaboratory performances of two commercially available systems, i.e., the VetMIC microplate system and Etest, for antimicrobial susceptibility testing of nonenterococcal lactic acid bacteria (NELAB) and bifidobacteria were analyzed. In addition, one laboratory also performed standard broth microdilution as a reference method. MICs of tetracycline, erythromycin, ampicillin, gentamicin, clindamycin, and streptomycin for the type strains of 25 species of NELAB and bifidobacteria and MICs of vancomycin for a selection of relevant taxa were determined. The previously described lactic acid bacterium susceptibility test medium (LSM) and related mixed-medium formulations, all including Iso-Sensitest broth as a basic component, were used as test media. The overall agreement of median MIC ranges +/- 1 log(2) dilution determined by the VetMIC and Etest methods with the median MICs determined by the reference method was very good for tetracycline, ampicillin, and streptomycin (92.3 to 100%) but low for erythromycin (19.5 to 30.7%) and clindamycin (50.0 to 80.8%). There was a consensus among the participating laboratories that VetMIC was preferred over Etest because of its lower cost, better growth support, and more uniform criteria for MIC end point reading. With the range for acceptable intralaboratory reproducibility being defined as the median MIC +/- 1 log(2) dilution, VetMIC results (with 69.2% of all data sets in the acceptable range) were shown to display greater reproducibility than Etest results (with 58.8% of all data sets in the acceptable range). Also at the interlaboratory level, the proportion of MIC values obtained with VetMIC that belonged to the complete agreement category (60.0%) was higher than the proportion of such values obtained with Etest (47.0%), which indicates a higher degree of interlaboratory reproducibility for the former method. Apart from some agent-specific effects, the majority of VetMIC and Etest replicate data sets were situated within a 1- to 2-log(2) dilution range, suggesting that the two methods can be considered to be equivalent for recognizing resistance phenotypes. This multicenter study has further validated the standard use of LSM and related mixed-medium formulations with commercially available systems and formed the basis for the ongoing development of the ISO 10932/IDF 223 standard for susceptibility testing of NELAB and bifidobacteria.

Genetic basis of tetracycline resistance in Bifidobacterium animalis subsp. lactis.

All strains of Bifidobacterium animalis subsp. lactis described to date show medium level resistance to tetracycline. Screening of 26 strains from a variety of sources revealed the presence of tet(W) in all isolates. A transposase gene upstream of tet(W) was found in all strains, and both genes were cotranscribed in strain IPLAIC4. Mutants with increased tetracycline resistance as well as tetracycline-sensitive mutants of IPLAIC4 were isolated and genetically characterized. The native tet(W) gene was able to restore the resistance phenotype to a mutant with an alteration in tet(W) by functional complementation, indicating that tet(W) is necessary and sufficient for the tetracycline resistance seen in B. animalis subsp. lactis.

Candida cabralensis sp. nov., a yeast species isolated from traditional Spanish blue-veined Cabrales cheese.

Three yeast strains, 1AD8(T), 3AD15 and 3AD23, belonging to a previously unknown yeast species were isolated from two independent batches of the Spanish blue-veined Cabrales cheese, a traditional cheese manufactured without the addition of starter and mould cultures. Physiological characterization revealed that the unknown yeast is not fermentative and does not assimilate lactose; rather it assimilates dl-lactic acid and ethanol, major end products of lactic acid bacteria metabolism in cheese. The novel yeast is anamorphic. Phylogenetic tree reconstruction based on nucleotide sequence comparison of the D1/D2 region of the 26S rRNA gene showed that Pichia terricola and Pichia fermentans are the closest relatives of the unknown species. The name Candida cabralensis sp. nov. is proposed, and the isolate 1AD8(T) (=CECT 13027(T) =CBS 11679(T)) is the type strain of this novel taxon.

Draft Genome Sequence of Adlercreutzia equolifaciens IPLA 37004, a Human Intestinal Strain That Does Not Produce Equol from Daidzein.

Equol is an intestinal bacterial metabolite derived from the isoflavone daidzein and has beneficial health effects. Most equol producers belong to the family Coriobacteriaceae, which includes species such as Adlercreutzia equolifaciens Here, we report the draft genome sequence of A. equolifaciens IPLA 37004, a human isolate that does not produce equol.

Metabolism of Soy Isoflavones by Intestinal Bacteria: Genome Analysis of an Adlercreutzia Equolifaciens Strain That Does Not Produce Equol.

Isoflavones are transformed in the gut into more estrogen-like compounds or into inactive molecules. However, neither the intestinal microbes nor the pathways leading to the synthesis of isoflavone-derived metabolites are fully known. In the present work, 73 fecal isolates from three women with an equol-producing phenotype were considered to harbor equol-related genes by qPCR. After typing, 57 different strains of different taxa were tested for their ability to act on the isoflavones daidzein and genistein. Strains producing small to moderate amounts of dihydrodaidzein and/or O-desmethylangolensin (O-DMA) from daidzein and dihydrogenistein from genistein were recorded. However, either alone or in several strain combinations, equol producers were not found, even though one of the strains, W18.34a (also known as IPLA37004), was identified as Adlercreutzia equolifaciens, a well-described equol-producing species. Analysis and comparison of A. equolifaciens W18.34a and A. equolifaciens DSM19450T (an equol producer bacterium) genome sequences suggested a deletion in the former involving a large part of the equol operon. Furthermore, genome comparison of A. equolifaciens and Asaccharobacter celatus (other equol-producing species) strains from databases indicated many of these also showed deletions within the equol operon. The present results contribute to our knowledge to the activity of gut bacteria on soy isoflavones.

Antibiotic Resistance-Susceptibility Profiles of Enterococcus faecalis and Streptococcus spp. From the Human Vagina, and Genome Analysis of the Genetic Basis of Intrinsic and Acquired Resistances.

The spread of antibiotic resistance is a major public health concern worldwide. Commensal bacteria from the human genitourinary tract can act as reservoirs of resistance genes playing a role in their transfer to pathogens. In this study, the minimum inhibitory concentration of 16 antibiotics to 15 isolates from the human vagina, identified as Enterococcus faecalis, Streptococcus anginosus, and Streptococcus salivarius, was determined. Eight isolates were considered resistant to tetracycline, five to clindamycin and quinupristin-dalfopristin, and four to rifampicin. To investigate the presence of antimicrobial resistance genes, PCR analysis was performed in all isolates, and five were subjected to whole-genome sequencing analysis. PCR reactions identified tet(M) in all tetracycline-resistant E. faecalis isolates, while both tet(M) and tet(L) were found in tetracycline-resistant S. anginosus isolates. The tet(M) gene in E. faecalis VA02-2 was carried within an entire copy of the transposon Tn916. In S. anginosus VA01-10AN and VA01-14AN, the tet(M) and tet(L) genes were found contiguous with one another and flanked by genes encoding DNA mobilization and plasmid replication proteins. Amplification and sequencing suggested the lsaA gene to be complete in all E. faecalis isolates resistant to clindamycin and quinupristin-dalfopristin, while the gene contain mutations rendering to a non-functional LsaA in susceptible isolates. These results were subsequently confirmed by genome analysis of clindamycin and quinupristin-dalfopristin resistant and susceptible E. faecalis strains. Although a clinical breakpoint to kanamycin for S. salivarius has yet to be established, S. salivarius VA08-2AN showed an MIC to this antibiotic of 128 µg mL-1. However, genes involved in kanamycin resistance were not identified. Under the assayed conditions, neither tet(L) nor tet(M) from either E. faecalis or S. anginosus was transferred by conjugation to recipient strains of E. faecalis, Lactococcus lactis, or Lactobacillus plantarum. Nonetheless, the tet(L) gene from S. anginosus VA01-10AN was amplified by PCR, and cloned and expressed in Escherichia coli, to which it provided a resistance of 48-64 µg mL-1 to tetracycline. Our results expand the knowledge of the antibiotic resistance-susceptibility profiles of vaginal bacteria and provide the genetic basis of their intrinsic and acquired resistance.