Immunomodulatory effects of live and pasteurized Lactobacillus crispatus strain RIGLD-1 on Helicobacter pylori-triggered inflammation in gastric epithelial cells in vitro.

BACKGROUND: Helicobacter pylori infection is considered as the major risk factor for gastric adenocarcinoma. Today, the increasing emergence of antibiotic-resistant strains has drastically decreased the eradication rate of H. pylori infection. This study was aimed to investigate the inhibitory and modulatory effects of live and pasteurized Lactobacillus crispatus strain RIGLD-1 on H. pylori adhesion, invasion, and inflammatory response in AGS cell line. METHODS AND RESULTS: The probiotic potential and properties of L. crispatus were evaluated using several functional and safety tests. Cell viability of AGS cells exposed to varying concentrations of live and pasteurized L. crispatus was assessed by MTT assay. The adhesion and invasion abilities of H. pylori exposed to either live or pasteurized L. crispatus were examined by gentamycin protection assay. The mRNA expression of IL-1ß, IL-6, IL-8, TNF-α, IL-10, and TGF-ß genes was determined by RT-qPCR from coinfected AGS cells. ELISA was used for the detection of IL-8 secretion from treated cells. Both live and pasteurized L. crispatus significantly decreased H. pylori adhesion/invasion to AGS cells. In addition, both live and pasteurized L. crispatus modulated H. pylori-induced inflammation by downregulating the mRNA expression of IL-1ß, IL-6, IL-8, and TNF-α and upregulating the expression of IL-10, and TGF-ß cytokines in AGS cells. Furthermore, H. pylori-induced IL-8 production was dramatically decreased after treatment with live and pasteurized L. crispatus. CONCLUSIONS: In conclusion, our findings demonstrated that live and pasteurized L. crispatus strain RIGLD-1 are safe, and could be suggested as a potential probiotic candidate against H. pylori colonization and inflammation.

The core genome evolution of Lactobacillus crispatus as a driving force for niche competition in the human vaginal tract.

The lower female reproductive tract is notoriously dominated by Lactobacillus species, among which Lactobacillus crispatus emerges for its protective and health-promoting activities. Although previous comparative genome analyses highlighted genetic and phenotypic diversity within the L. crispatus species, most studies have focused on the presence/absence of accessory genes. Here, we investigated the variation at the single nucleotide level within protein-encoding genes shared across a human-derived L. crispatus strain selection, which includes 200 currently available human-derived L. crispatus genomes as well as 41 chromosome sequences of such taxon that have been decoded in the framework of this study. Such data clearly pointed out the presence of intra-species micro-diversities that could have evolutionary significance contributing to phenotypical diversification by affecting protein domains. Specifically, two single nucleotide variations in the type II pullulanase gene sequence led to specific amino acid substitutions, possibly explaining the substantial differences in the growth performances and competition abilities observed in a multi-strain bioreactor culture simulating the vaginal environment. Accordingly, L. crispatus strains display different growth performances, suggesting that the colonisation and stable persistence in the female reproductive tract between the members of this taxon is highly variable.

Genetic Elements Orchestrating Lactobacillus crispatus Glycogen Metabolism in the Vagina.

Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus. Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatuspulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

Characterization of a novel type of glycogen-degrading amylopullulanase from Lactobacillus crispatus.

Glycogen is one of the major carbohydrates utilized by the human vaginal microbiota, which is commonly dominated by Lactobacillus, especially L. crispatus. An in silico analysis predicted that a type I pullulanase was involved in glycogen degradation in L. crispatus. The biochemical and genetic properties of the pullulanase still need to be determined. Here, we de novo identified the glycogen (Glg)-utilization enzyme (named GlgU) from L. crispatus through a biochemical assay. GlgU was optimally active at acidic pH, approximately 4.0 ~ 4.5, and was able to hydrolyze glycogen into low-molecular-weight malto-oligosaccharides. Actually, GlgU was a type II pullulanase (amylopullulanase) with just one catalytic domain that possessed substrate specificity toward both α-1,4 and α-1,6-glucosidic bonds. Phylogenetically, GlgU was obviously divergent from the known amylases and pullulanases (including amylopullulanases) in lactobacilli. In addition, we confirmed the catalytic activity of glgU in a nonglycogen-utilizing lactobacilli strain, demonstrating the essential role of glgU in glycogen metabolism. Overall, this study characterized a novel type of amylopullulanases, contributing to the knowledge of the glycogen utilization mechanism of the dominant species of human vaginal microbiota. KEY POINTS: • GlgU was a type II pullulanase, not a type I pullulanase predicted before. • GlgU was able to completely hydrolyze glycogen into malto-oligosaccharides. • GlgU played a key role in the metabolism of extracellular glycogen.

Lactobacillus johnsonii 3-1 and Lactobacillus crispatus 7-4 promote the growth performance and ileum development and participate in lipid metabolism of broilers.

Long-term use of antibiotic growth promoter (AGP) in animal production is the main cause of antimicrobial resistance of pathogenic bacteria. Therefore, seeking alternatives to AGP is crucial for animal husbandry. Among all AGP alternatives, probiotics are promising candidates. In this study, two strains of lactic acid bacteria, L. johnsonii 3-1 and L. crispatus 7-4, were isolated from the feces of wild Gallus gallus, which exhibited obvious anti-pathogenic activity and improved the growth performance of broilers. Furthermore, we found that these two strains participated in the lipid metabolism of broilers by reducing the content of TC and TG in ileal epithelial cells and up-regulating the liver AMPKα/PPARα/CPT-1 pathway, which affects abdominal fat deposition. In summary, L. johnsonii 3-1 and L. crispatus 7-4 have the potential to be used as AGP substitutes and participate in the lipid metabolism of broilers to reduce abdominal fat deposition. Importantly, our study reveals for the first time that L. crispatus participates in liver lipid metabolism to reduce abdominal fat deposition in broilers.

The Vaginal Microbiome of Nonhuman Primates Can Be Only Transiently Altered to Become Lactobacillus Dominant without Reducing Inflammation.

The vaginal microbiome composition in humans is categorized based upon the degree to which one of four species of Lactobacillus is dominant (Lactobacillus crispatus, community state type I [CST I], Lactobacillus gasseri, CST II, Lactobacillus iners, CST III, and Lactobacillus jensenii, CST V). Women with a vaginal microbiome not dominated by one of the four Lactobacillus species tend to have a more diverse microbiome, CST IV. CSTs I, II, III, and V are common in North America and Europe and are associated with lower incidences of some pathogens, such as human immunodeficiency virus (HIV), human papillomavirus (HPV), and Gardnerella vaginalis. As a result, therapeutic interventions to change the composition of the vaginal microbiomes are under development. However, Homo sapiens is the only mammalian species which has high frequencies of Lactobacillus-dominated vaginal microbiomes. Here, we treated female nonhuman primates (NHPs) with regimens of metronidazole and high levels of L. crispatus to determine how well these animals could be colonized with L. crispatus, how this influenced the immunological milieu, and how Lactobacillus treatment influenced or was influenced by the endogenous vaginal microbiome. We find that NHPs can transiently be colonized with L. crispatus, that beta diversity and not the number of doses of L. crispatus or pretreatment with metronidazole predicts subsequent L. crispatus colonization, that L. crispatus does not alter the local immunological milieu, and that the vaginal microbiome composition was resilient, normalizing by 4 weeks after our manipulations. Overall, this study suggests these animals are not amenable to long-term L. crispatus colonization. IMPORTANCE NHPs have proven to be invaluable animal models for the study of many human infectious diseases. The use of NHPs to study the effect of the microbiome on disease transmission and susceptibility is limited due to differences between the native microbiomes of humans and NHPs. In particular, Lactobacillus dominance of the vaginal microbiome is unique to humans and remains an important risk factor in reproductive health. By assessing the extent to which NHPs can be colonized with exogenously applied L. crispatus to resemble a human vaginal microbiome and examining the effects on the vaginal microenvironment, we highlight the utility of NHPs in analysis of vaginal microbiome manipulations in the context of human disease.

Interstrain Variability of Human Vaginal Lactobacillus crispatus for Metabolism of Biogenic Amines and Antimicrobial Activity against Urogenital Pathogens.

Lactobacillus crispatus is the dominant species in the vagina of many women. With the potential for strains of this species to be used as a probiotic to help prevent and treat dysbiosis, we investigated isolates from vaginal swabs with Lactobacillus-dominated and a dysbiotic microbiota. A comparative genome analysis led to the identification of metabolic pathways for synthesis and degradation of three major biogenic amines in most strains. However, targeted metabolomic analysis of the production and degradation of biogenic amines showed that certain strains have either the ability to produce or to degrade these compounds. Notably, six strains produced cadaverine, one produced putrescine, and two produced tyramine. These biogenic amines are known to raise vaginal pH, cause malodour, and make the environment more favourable to vaginal pathogens. In vitro experiments confirmed that strains isolated from women with a dysbiotic vaginal microbiota have higher antimicrobial effects against the common urogenital pathogens Escherichia coli and Enterococcus faecium. The results indicate that not all L. crispatus vaginal strains appear suitable for probiotic application and the basis for selection should not be only the overall composition of the vaginal microbiota of the host from which they came, but specific biochemical and genetic traits.

Cervicovaginal microbiota and metabolome predict preterm birth risk in an ethnically diverse cohort.

The syndrome of spontaneous preterm birth (sPTB) presents a challenge to mechanistic understanding, effective risk stratification, and clinical management. Individual associations between sPTB, self-reported ethnic ancestry, vaginal microbiota, metabolome, and innate immune response are known but not fully understood, and knowledge has yet to impact clinical practice. Here, we used multi-data type integration and composite statistical models to gain insight into sPTB risk by exploring the cervicovaginal environment of an ethnically heterogenous pregnant population (n = 346 women; n = 60 sPTB < 37 weeks' gestation, including n = 27 sPTB < 34 weeks). Analysis of cervicovaginal samples (10-15+6 weeks) identified potentially novel interactions between risk of sPTB and microbiota, metabolite, and maternal host defense molecules. Statistical modeling identified a composite of metabolites (leucine, tyrosine, aspartate, lactate, betaine, acetate, and Ca2+) associated with risk of sPTB < 37 weeks (AUC 0.752). A combination of glucose, aspartate, Ca2+, Lactobacillus crispatus, and L. acidophilus relative abundance identified risk of early sPTB < 34 weeks (AUC 0.758), improved by stratification by ethnicity (AUC 0.835). Increased relative abundance of L. acidophilus appeared protective against sPTB < 34 weeks. By using cervicovaginal fluid samples, we demonstrate the potential of multi-data type integration for developing composite models toward understanding the contribution of the vaginal environment to risk of sPTB.

Phenyl-Lactic Acid Is an Active Ingredient in Bactericidal Supernatants of Lactobacillus crispatus.

Lactobacillus crispatus is a well-established probiotic with antimicrobial activity against pathogens across several niches of the human body generally attributed to the production of bacteriostatic molecules, including hydrogen peroxide and lactic acid. Here, we show that the cell-free supernatants of clinical isolates of L. crispatus harbor robust bactericidal activity. We further identify phenyl-lactic acid as a bactericidal compound with properties and a susceptibility range nearly identical to that of the cell-free supernatant. As such, we hypothesize that phenyl-lactic acid is a key active ingredient in L. crispatus supernatant. IMPORTANCE Although Lactobacillus crispatus is an established commensal microbe frequently used in probiotics, its protective role in the bladder microbiome has not been clarified. We report here that some urinary isolates of L. crispatus exhibit bactericidal activity, primarily due to its ability to excrete phenyl-lactic acid into its environment. Both cell-free supernatants of L. crispatus isolates and phenyl-lactic acid exhibit bactericidal activity against a wide range of pathogens, including several that are resistant to multiple antibiotics.

Variability of the response of human vaginal Lactobacillus crispatus to 17ß-estradiol.

We previously showed that the physiological concentration of 17ß-estradiol in the vaginal environment is sufficient to affect the membrane dynamics and adhesion phenotype of the Lactobacillus crispatus strain CIP104459. However, L. crispatus is a heterogeneous species. Here, we investigated the effect of 17ß-estradiol on the recently isolated L. crispatus vaginal strain V4, related to a cluster distant from CIP104459 and at the limit of being a different subspecies. Grown in the same medium, the two strains expressed a highly similar pool of proteins. However, in contrast to CIP104459, L. crispatus V4 showed high aggregation potential and 17ß-estradiol promoted this phenotype. This effect was associated with large changes in cell-surface polarity and Lewis acid/base properties. In addition, we observed no effect on the membrane dynamics, contrary to CIP104459. These results can be explained by differences in the properties and organization of the S layer between the two strains. However, as for CIP104459, 17ß-estradiol increased biosurfactant production of L. crispatus V4 and their adhesion to vaginal cells. This suggests that 17ß-estradiol agonists would be valuable tools to favor a stable re-implantation of L. crispatus in the vaginal mucosa.

Surface-layer protein produced by Lactobacillus crispatus JCM 2009 ameliorates lipopolysaccharide-induced inflammation through autophagy cross-talk with the NF-κB signaling pathway.

In recent years, studies on immunomodulation by surface-layer proteins (Slps) have mainly focused on Lactobacillus acidophilus, there is little information on Slp from L. crispatus and its intestinal immunomodulatory mechanisms in macrophages. In our study, the anti-inflammatory actions of Slp derived from L. crispatus JCM 2009 and its related molecular mechanisms were investigated. We initially found that incubation with Slp (5-10 µg/mL) for 4 h significantly inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-stimulated RAW264.7 cells (P < 0.001). We then found that Slp inhibited the inflammatory response by regulating the PI3K/AKT/mTOR signaling pathway and activating autophagy in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Furthermore, ELISA and Western blotting results demonstrated that the NF-κB signaling pathway positively regulated autophagic activity to inhibit the productions of PGE2 and NO during this inflammatory response. And p65 was identified as a potentially important NF-κB signaling pathway molecule mediating the effects of Slp on the LPS-induced inflammatory response in RAW264.7 cells. Our findings provide the novel perspective that Slp exerts its anti-inflammatory effects through the activation of autophagy, making it a promising bioactive ingredient for the development of functional foods.

Characterization of the Extracellular Fructanase FruA in Lactobacillus crispatus and Its Contribution to Fructan Hydrolysis in Breadmaking.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) trigger symptoms of irritable bowel syndrome (IBS). Fructan degradation during bread making reduces FODMAPs in bread while maintaining the content of dietary fiber. This study explored the presence of the fructanases FruA in lactobacilli and characterized its use in bread making. FruA was exclusively present in vertebrate-adapted lactobacilli. In Lactobacillus crispatus DSM29598, FruA was located in cell wall fractions and includes a SLAP domain. FruA hydrolyzed levan or inulin; expression of fruA was not subject to catabolite repression. Fructans in bread were reduced by less than 50% in a straight dough process; conventional sourdough fermentation reduced fructans in bread by 65-70%. Sourdough fermentation with L. crispatus reduced fructans in bread by more than 90%. In conclusion, reduction of FODMAP by sourdough fermentation may improve tolerance in many IBS patients. Fermentation with FruA-expressing L. crispatus DSM29598 produces a low FODMAP bread.

Vaginal Biomarkers That Predict Cervical Length and Dominant Bacteria in the Vaginal Microbiomes of Pregnant Women.

In many impoverished regions of the world, it may not be possible to assess two major risk factors for preterm birth: a short cervical length and the depletion of vaginal lactobacilli. We determined whether measuring specific compounds in vaginal fluid might be a simple, noninvasive, and cost-effective way to predict the bacteria that dominate the vaginal microbiome and indicate the presence of a shortened cervix (<25 mm). Vaginal fluid samples were prospectively collected from mid-trimester pregnant women, and the concentrations of d- and l-lactic acid, tissue inhibitor of matrix metalloproteinases TIMP-1 and TIMP-2, matrix metalloproteinases MMP-2 and MMP-8, the 70-kDa heat shock protein, a2 isoform of vacuolar ATPase, and sequestrome-1 were quantified by an enzyme-linked immunosorbent assay (ELISA). The compositions of vaginal microbiomes were assessed by analysis of the V1-V3 regions of 16S rRNA genes, while cervical length was determined by transvaginal ultrasonography. The vaginal microbiomes could be clustered into five community state types (CSTs), four of which were dominated by a single Lactobacillus species. The dominance of Lactobacillus crispatus or Lactobacillus jensenii in the vaginal microbiome predicted the level of d-lactic acid present. Several of the biomarkers, especially TIMP-1, in combination with the subject's age and race, were significantly associated with cervical length. Using piecewise structural equation modeling, we established a causal network that links CST to cervical length via biomarkers. We concluded that measuring levels of TIMP-1 and d-lactic acid in vaginal secretions might be a straightforward way to assess the risk for preterm birth due to a short cervix and microbiome composition.IMPORTANCE Premature birth and its complications are the largest contributors to infant death in the United States and globally. A short cervical length and the depletion of Lactobacillus species are known risk factors for preterm birth. However, in many resource-poor areas of the world, the technology to test for their occurrence is unavailable, and pregnant women with these risk factors are neither identified nor treated. In this study, we used path analysis to gain an unprecedented understanding of interactions between vaginal microbiome composition, the concentrations of various compounds in vaginal secretions, and cervical length. We identified low-cost point-of-care measures that might be used to identify pregnant women at risk for preterm birth. The use of these measures coupled with appropriate preventative or treatment strategies could reduce the incidence of preterm births in poor areas of the world that lack access to more sophisticated diagnostic methods.

Comparative genomics of human Lactobacillus crispatus isolates reveals genes for glycosylation and glycogen degradation: implications for in vivo dominance of the vaginal microbiota.

BACKGROUND: A vaginal microbiota dominated by lactobacilli (particularly Lactobacillus crispatus) is associated with vaginal health, whereas a vaginal microbiota not dominated by lactobacilli is considered dysbiotic. Here we investigated whether L. crispatus strains isolated from the vaginal tract of women with Lactobacillus-dominated vaginal microbiota (LVM) are pheno- or genotypically distinct from L. crispatus strains isolated from vaginal samples with dysbiotic vaginal microbiota (DVM). RESULTS: We studied 33 L. crispatus strains (n = 16 from LVM; n = 17 from DVM). Comparison of these two groups of strains showed that, although strain differences existed, both groups degraded various carbohydrates, produced similar amounts of organic acids, inhibited Neisseria gonorrhoeae growth, and did not produce biofilms. Comparative genomics analyses of 28 strains (n = 12 LVM; n = 16 DVM) revealed a novel, 3-fragmented glycosyltransferase gene that was more prevalent among strains isolated from DVM. Most L. crispatus strains showed growth on glycogen-supplemented growth media. Strains that showed less-efficient (n = 6) or no (n = 1) growth on glycogen all carried N-terminal deletions (respectively, 29 and 37 amino acid deletions) in a putative pullulanase type I protein. DISCUSSION: L. crispatus strains isolated from LVM were not phenotypically distinct from L. crispatus strains isolated from DVM; however, the finding that the latter were more likely to carry a 3-fragmented glycosyltransferase gene may indicate a role for cell surface glycoconjugates, which may shape vaginal microbiota-host interactions. Furthermore, the observation that variation in the pullulanase type I gene is associated with growth on glycogen discourages previous claims that L. crispatus cannot directly utilize glycogen.

Identifying probiotic characteristics of Lactobacillus crispatus isolated from the vagina

Lactobacilli prevent overproduction of pathogenic microorganisms and contribute protecting vaginal microbiota. Many probiotic microorganisms are categorized as Lactic Acid Bacteria. In this study, it was aimed identifying probiotic characteristics of Lactobacillus crispatus isolated from the vagina of a healthy woman. For this purpose, lactic acid, hydrogen peroxide and proteolytic activity quantities and auto-aggregation, co-aggregation and hydrophobicity abilities of Lactobacillus crispatus, which has been isolated and identified by 16s rRNA sequence analysis, were determined. Additionally, bile salt and acid resistance, along with antibiotic susceptibility of Lactobacillus crispatus were analyzed by the end of 3 hours. Lactic acid, hydrogen peroxide and proteolytic activity quantities of Lactobacillus crispatus were measured 2.275%, 0.334±0.075 µg/mL and 2.131±0,000 mg/mL respectively. The findings include existence of co-aggregation and auto-aggregation ability, but not hydrophobicity. By the end of 3 hours, the viability was preserved in 0.1% and 0.3% bile salt medium and, at pH 3. L. crispatus exhibited resistance to methicillin, metronidazole, oxacillin, and sulfamethoxazole + trimethoprim, but the bacteria exhibited susceptibility to tested the other antibiotics. This study will make an important contribution to the literature about probiotic characteristics of L. crispatus and our strain isolated from the vagina might be considered as a candidate probiotic.

Culture Supernatants of Lactobacillus gasseri and L. crispatus Inhibit Candida albicans Biofilm Formation and Adhesion to HeLa Cells.

PURPOSE: Vulvovaginal candidiasis (VVC) is a common superficial infection of the vaginal mucous membranes caused by the fungus Candida albicans. The aim of this study was to assess the mechanisms underlying the inhibitory effects of the culture supernatants of Lactobacillus gasseri and L. crispatus, the predominant microbiota in Asian healthy women, on C. albicans biofilm formation. The inhibition of C. albicans adhesion to HeLa cells by Lactobacillus culture supernatant was also investigated. METHODS: Candida albicans biofilm was formed on polystyrene flat-bottomed 96-well plates, and the inhibitory effects on the initial colonization and maturation phases were determined using the XTT reduction assay. The expression levels of biofilm formation-associated genes (HWP1, ECE1, ALS3, BCR1, EFG1, TEC1, and CPH1) were determined by reverse transcription quantitative polymerase chain reaction. The inhibition of C. albicans adhesion to HeLa cells by Lactobacillus culture supernatant was evaluated by enumerating viable C. albicans cells. RESULTS: The culture supernatants of both Lactobacillus species inhibited the initial colonization and maturation of C. albicans biofilm. The expression levels of all biofilm formation-related genes were downregulated in the presence of Lactobacillus culture supernatant. The culture supernatant also inhibited C. albicans adhesion to HeLa cells. CONCLUSION: The culture supernatants of L. gasseri and L. crispatus inhibited C. albicans biofilm formation by downregulating biofilm formation-related genes and C. albicans adhesion to HeLa cells. These findings support the notion that Lactobacillus metabolites may be useful alternatives to antifungal drugs for the management of VVC.

Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane.

Helveticin-M, a novel Class III bacteriocin produced by Lactobacillus crispatus exhibited an antimicrobial activity against Staphylococcus aureus, S. saprophyticus, and Enterobacter cloacae. To understand how Helveticin-M injured target cells, Helveticin-M was cloned and heterologously expressed in Escherichia coli. Subsequently, the cell wall organization and cell membrane integrity of target cells were determined. The mechanism of cellular damage differed according to bacterial species. Based on morphology analysis, Helveticin-M disrupted the cell wall of Gram-positive bacteria and disorganized the outer membrane of Gram-negative bacteria, therefore, altering surface structure. Helveticin-M also disrupted the inner membrane, as confirmed by leakage of intracellular ATP from cells and depolarization of membrane potential of target bacteria. Based on cell population analysis, Helveticin-M treatment caused the increase of cell membrane permeability, but the cytosolic enzymes were not influenced, indicating that it was the sublethal injury. Therefore, the mode of Helveticin-M action is bacteriostatic rather than bactericidal.