Abstract
Recent years have witnessed an explosion in genome sequencing of probiotic strains for accurate identification and characterization. Regulatory bodies are emphasizing on the need for performing phase I safety studies for probiotics. The main hypothesis of this study was to explore the feasibility of using genome databases for safety screening of strains. In this study, we attempted to develop a framework for the safety assessment of a potential probiotic strain, Lactobacillus helveticus MTCC 5463 based on genome mining for genes associated with antibiotic resistance, production of harmful metabolites, and virulence. The sequencing of MTCC 5463 was performed using GS-FLX Titanium reagents. Genes coding for antibiotic resistance and virulence were identified using Antibiotic Resistance Genes Database and Virulence Factors Database. Results indicated that MTCC 5463 carried antibiotic resistance genes associated with beta-lactam and fluoroquinolone. There is no threat of transfer of these genes to host gut commensals because the genes are not plasmid encoded. The presence of genes for adhesion, biofilm, surface proteins, and stress-related proteins provides robustness to the strain. The presence of hemolysin gene in the genome revealed a theoretical risk of virulence. The results of in silico analysis complemented the in vitro studies and human clinical trials, confirming the safety of the probiotic strain. We propose that the safety assessment of probiotic strains administered live at high doses using a genome-wide screening could be an effective and time-saving tool for identifying prognostic biomarkers of biosafety.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
The awareness of the beneficial effects of probiotics in promoting gut and general health has grown over the decade, leading to a surge in the consumption of probiotic foods worldwide. Probiotic bacteria, especially strains of Lactobacillus and Bifidobacterium, isolated from fermented foods are “Generally Regarded As Safe” (GRAS), according to the American Food and Drug Administration, due to their long history of safe use in fermented foods. Lactobacillus helveticus strains are given the Qualified Presumption of Safety (QPS) status as they are readily identified to the species level and have rarely been indicated in opportunistic infections [1]. With new strains being identified from diverse niches like gastrointestinal tract, vagina, and honey bee stomach, the efficacy of novel strains calls for a careful case-by-case evaluation to determine whether they share the safety status like the traditional food-grade organisms [2]. Illnesses caused by lactobacilli are reported in isolated cases in elderly and immune-compromised patients, rather than as collective foodborne diseases [3–9]. Further, the risk of infection with probiotic lactobacilli is similar to that of commensal strains, presenting negligible risk to consumers [7]. With the recent developments in tools and techniques to track specific strains, the reports of infections and other adverse incidents traced to probiotics have surfaced. Infections caused by lactobacilli and bifidobacteria make up 0.05–0.4 % of cases of endocarditis and bacteremia, respectively. About 1.7 % of infections reported were linked directly with intensive dairy probiotic consumption by patients and not healthy individuals [10]. A number of well-controlled human studies have tracked adverse incidents and have provided data about the safety assessment of probiotic cultures [11]. Classical risk assessment approaches, like microbiological risk assessment, are not warranted for lactobacilli because they are ubiquitous in nature [12]. The safety of probiotics is dependent on the strain, intended use, host health status, dose and duration of consumption, and both the manner and frequency of administration. The degree of risk that is regarded as acceptable can vary between countries, depending on the safety standards and carriers, like food, feed, and supplements. Emerging safety risks of probiotic strains include acquisition of antibiotic resistance and virulence determinants, genetic stability, deleterious metabolic activities, potential for pathogenicity, and immunological effects [11]. To address these risks, it would be better to do a premarket risk assessment of the strain for the above-mentioned theoretical safety concerns.
Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for testing safety-related genes [13]. L. helveticus strains, used largely in the cheese industry as a starter culture with high proteolytic activity, have been recently characterized at the genomic level [14–18]. Safety assessment can be greatly improvised using mechanistic omics-based data [19, 20]. L. helveticus MTCC 5463 (from now on referred to as strain MTCC 5463) is a potential probiotic strain and the first in India to be fully sequenced. It displayed antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enterica serovar Typhi, and Escherichia coli [21], immunomodulation in a chicken model [22] and hypocholesterolemic effects in human volunteers [23]. The strain MTCC 5463 is a vaginal isolate, initially identified as L. acidophilus LBKV3 by preliminary biochemical tests. The partial 16S rRNA sequencing (accession number GQ253959) (1053 bp) of the vaginal isolate showed similarities of above 97 % with that of other L. helveticus strains, and it was confirmed by API 50 CHL results that the strain was L. helveticus. The genomic library contained 119,569 reads, and assembly generated a 1,911,350-bp single chromosome. In total, 2046 coding sequence (CDS) regions and 71 RNA genes were reported. MTCC 5463 showed resistance to phenol (0.4 %), bile salt (4 %), and low pH (pH 3) with a significant antimicrobial activity against B. cereus, S. aureus, P. aeruginosa, S. enterica serovar Typhi, and E. coli [21]. Studies on the strain’s safety, dose response, and effect on host intestinal well-being [24] have also been conducted. The strain also produced extracellular polysaccharide and was able to adhere to cells of the human carcinoma cell line HT 29 [25]. The above-mentioned adaptive and probiotic characteristics of the strain were confirmed through trait and gene matching confirming that the strain possesses the genetic arsenal required to adapt to the gut milieu [26]. The predictions of functional genes further validate the experimental evidence of adaptation and probiosis. On comparing the stress-responsive genes of MTCC 5463 and a cheese starter strain, L. helveticus DPC 4571, gene sets specific to the gut and dairy niche could be identified [27]. Upon completion of genome sequencing of the strain in question [17], we attempted a bioinformatic analysis to provide additional insight into the genetic basis of its safety. The criteria for safety were delineated similarly to the QPS approach (defining the taxonomic unit, collecting the body of knowledge, safety concerns like the presence of antibiotic resistance, likelihood of gene transfer, tolerance to heavy metals, virulence determinants, and production of harmful metabolites). Our approach enables rapid screening of the gene set that determines the safety of a probiotic strain and its potential to become an opportunistic pathogen. We illustrate this strategy for MTCC 5463, but it can be readily adapted for biosafety assessment of other potential probiotic strains.
Materials and Methods
Bacterial Strains, Growth Conditions, and Pyrosequencing
The culture maintenance and pyrosequencing methodology is explained in our previous publication [17, 26]. Each DNA sample was subjected to pyrosequencing (454 Life Sciences technology) based on a high-throughput sequencer (GS-FLX, Roche) according to the manufacturer’s instructions. The genome was sequenced to a depth of 10X.
Identification of Antibiotic Resistance and Virulence-Related Genes
Putative virulence factors and putative antibiotic resistance genes were identified by Basic Local Alignment Search Tool (BLAST) with the Virulence Factors Database (VFDB) (http://www.mgc.ac.cn/VFs/main.htm) and Antibiotic Resistance Genes Database (ARDB) (http://ardb.cbcb.umd.edu/), respectively. Safety genes were exhaustively searched based on published literature [20, 28–30].
Results
Mobilomes
Mobile genetic elements (mobilome), such as bacteriophages, plasmids, transposons, and ISs, are important for the colonization of ecological niches, symbiosis, host–cell interaction, and pathogenicity [31].
Plasmids
The strain MTCC 5463 showed a lack of plasmids. Scanning the genome for toxin–antitoxin (TA) proteins for plasmid maintenance showed the presence of an antitoxin of TA system that was present as a nonfunctional hypothetical protein.
Prophages and Integrases
The strain lacked the presence of a complete prophage but revealed the presence of structural and regulatory genes associated with prophages such as putative prophage repressor, DNA-packaging protein NU1, and a phage-associated protein. The strain MTCC5 463 further showed the presence of group II intron-encoded maturase, integrase recombinase, and putative integrases with integrase catalytic region encoding genes (Table 1).
Transposases and Insertion Sequence (ISs) Elements
Annotations showed the presence of a large number of putative transposase genes (154 copies). Six copies of IS 1201, one of IS lhe15, and two coding sequences for the IS 4 family were observed in the strain MTCC 5463. Further analysis revealed that no core gene was clustered with the ISs restricting its transferability.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Restriction/Modification (R/M) Systems
CRISPR provides acquired immunity against foreign genetic elements. The probiotic bacteria did not exhibit the presence of CRISPRs and associated proteins. A range of R/M systems like the type I, type II, and type III restriction/modification system has been observed in the genome.
Resistome: Antibiotic and Heavy-Metal Resistance
A growing biosafety concern is of the transfer of antibiotic resistance genes from commensals to pathogens in the gut (reservoir hypothesis). The genome in question was mined for the presence of resistance genes against common antibiotics.
Beta-lactam
The most common and important mechanism through which bacteria becomes resistant to β-lactams is by the expression of β-lactamases. The strain MTCC 5463 harbored three homologs of beta-lactamases of various classes. The presence of penicillin-binding proteins (PBPs) could also have been contributing to beta-lactam resistance. Although five homologs of PBPs were present in the genome, BLAST analysis showed no mutation in the sequence implying that the altered PBPs were not the resistance-conferring agents.
Fluoroquinolone
The molecular targets of fluoroquinolone in the genome, like DNA gyrase, and topoisomerase IV, showed no mutation, and aminoglycoside N3′-acetyltransferase gene was present as a hypothetical protein. Hence, it is unclear whether they encode actual proteins. The ABC transporter, ATPase, permease components, and efflux pumps could be the probable determinants of resistance against quinolones (Table 2).
Other Antibiotic Resistance Genes
Tetracycline resistance determinants, like the NADP requiring oxidoreductase and xanthine–guanine phosphoribosyltransferase, were absent in the genome. Ribosomal protection proteins having homology to elongation factors were identified.
The additional presence of ABC transporter, ATPase, permease components, and efflux pumps mentioned above may contribute toward tetracycline resistance. Other antibiotic resistance genes mined in the strain MTCC 5463 genome included macrolide-lincosamide-streptogramin B (MLSB) resistance-conferring rRNA methylases, aminoglycoside acetyltransferases, and members of the GCN5 superfamily of proteins that include the histone acetyltransferases.
Bacteriocins and Immunity Genes
Genes expressing bacteriocin helveticin, bacteriocin production-related histidine kinase, bacteriocin response regulator, and a bacteriocin ABC transporter along with homologs of immunity proteins, like PlnI, were present in the genome.
Tolerance to Heavy Metals
The strain MTCC5 463 genome carried genes involved in copper homeostasis, like the copper-transporting ATPase and the copper chaperone. It has also developed systems for the removal of excess cobalt from cells by efflux system genes to avoid toxicity. Cadmium efflux mechanisms are also exhibited by the probiotic strain, as evidenced by the presence of cadmium efflux ATPase and cadmium-translocating P-type ATPase. In addition, genes to acquire iron and nickel transport system permease proteins are present in the genome (Table 2).
Adverse Metabolic Genes
The potential of the strain MTCC5 463 to produce the biogenic amines agmatine and putrescine from arginine and its amino acid derivative, ornithine, was investigated. It was found that sequences coding for the enzymes arginine deiminase and ornithine transcarbamylase existed as pseudogenes, rendering the pathway nonfunctional (Table 2). The strain encoded ornithine decarboxylase, which catalyzed the conversion of ornithine to putrescine, while both the spermidine/putrescine ABC transporter permease component and the spermidine/putrescine import related ATP-binding protein, PotA-accommodated ornithine uptake into the cell. Putrescine itself did possess a direct harmful biologic activity. Instead, it enhanced the toxic effects of histamine and tyramine, which were not produced by the strain. The strain MTCC 5463 exhibited the orthologs for beta-glucuronidase, Lac L, and Lac M. The presence of conjugated bile salt hydrolase gene in the genome in question is a fitness factor facilitating resistance against intestinal conditions rather than a risk factor.
Virulence- and Stress-Related Genes
Factors required to survive the physiological stresses and host interaction may be termed as virulence mechanisms in the case of undesirable bacteria. The same factors are considered crucial for a symbiotic interaction in the case of probiotics and termed as fitness factors or “host interaction factors” [32]. Offensive virulence factors are an invasion, mucin degradation, cytotoxicity, hemolysis, and biofilm production. We used a set of virulence factors from the VFDB to search for the presence of such classical and defensive virulence factors in MTCC 5463 genome. Data mining the probiotic strain’s genome revealed adhesion factors, including a putative sortase gene, GroEL, aggregation promoting protein, two copies of fibronectin-binding protein, S-layer protein, and a mucus-binding protein (Table 3).
Exopolysaccharide (EPS) is the probiotic effector molecules and biofilm-forming ability help in colonizing the gut. The strain MTCC 5463 also carried genes like glycosyltransferases that are important for the biosynthesis of exopolysaccharide (EPS) including bactoprenol glucosyl transferase and putative hexosyl transferase YtcC. Probiotic MTCC 5463 strain displayed the biosynthesis of glycosyltransferase protein for capsular polysaccharide synthesis and four exopolysaccharide biosynthesis proteins. The epsE gene-encoding phosphoribosyltransferase was present as a single copy. The genome also encoded ten potential uncharacterized glycosyltransferases and one galactosyltransferase. General stress adaptation genes exhibited by the strain included the universal stress protein, UspA, chaperones GroES and GroEL, Clp protease, heat-shock proteins, HtpX, GrpE, DnaK, FtsY and DnaJ, HtrA-like serine protease, and F0F1 ATP synthase subunits (Table 3).
Discussion
The use of lactobacilli in food and food production is generally accepted as safe by the scientific community. Even so, the authors attempted to conduct an exhaustive safety risk analysis for two main reasons: (1) to assess the suitability of the strain MTCC 5463 for the use with immunocompromised human hosts of all ages and (2) to provide a platform whereby all future potential probiotic isolates could undergo an exhaustive safety genes assessment. The safety of a strain cannot be assessed until it is taxonomically characterized. This characterization facilitates the comparison with known variants and helps track the strain in case of outbreaks. Using a polyphasic approach, the strain under study was identified as L. helveticus MTCC 5463. The genomic sequence of the strain MTCC 5463 (1.91 Mb) was smaller than those of the reference dairy strain L. helveticus DPC 4571 (2.08 Mb) [15], but larger than the smallest reported genome L. iners AB-1 (1.3 MB) [33]. This is suggestive of probable alterations in the MTCC 5463 genome to adapt to the nutritionally rich milk medium and for probiotic functionality [34]. An example of this adaptability is the presence of conjugated bile salt hydrolase in the gut strain, MTCC 5463, and a nonfunctional homolog in the dairy strain L. helveticus DPC 4571 [27]. The RAST server predicted the closest six neighbors of the MTCC5 463 strain to be of lactobacilli, and the first three significant hits (E value of 1e-20 or less) to the NCBI database were of the genus Lactobacillus. Thus, it can be concluded that the strain has not acquired a large number of genes from other organisms. This indicates the excellent genomic stability and minimum influence of horizontal gene transfer [8]. Being devoid of plasmids, strain MTCC 5463 loses its capacity to transfer antibiotic resistance genes to gut commensals. The strain maintains an unpaired antitoxin that is rendered nonfunctional. The nonfunctionality could be due to lack of plasmids and lower competition in a milk matrix against pathogens compared to the vaginal ecosystem. The strain MTCC 5463 harbors 154 copies of IS elements with no evidence of chromosomal rearrangements, similar to the 213 IS element-loaded DPC 4571 genome. Another reason for the maintenance of the high number of IS elements could be because the protective milk/food matrix with a defined strain inoculum used for propagation promotes low levels of intra-species competition which favors clones with increased numbers of IS elements to survive [35]. Genomic stability is a required trait to ensure that probiotic attributes are not affected by long-term preservation and production. The strain MTCC 5463 has exhibited a stable technological performance for 25 years, and the whole-genome order is conserved. This provides conclusive evidence that the large number of IS elements did not cause genetic instability. Further analysis revealed that no core genes were clustered with ISs, thus restricting transfer of antibiotic resistance. In silico analysis of the genome of the strain MTCC 5463 did not reveal any complete prophages but showed the presence of isolated prophage remnants. This suggested an inactivation or elimination of integrated prophages and evolution toward a stable genome. R/M systems present in the strain further act as barriers against horizontal gene transfer. Strain MTCC 5463 lacks CRISPRs that exclude foreign DNA. The absence of CRISPRs although does not result in an increase in foreign genes. Antibiotic resistance markers for penicillin are evidently the beta-lactamases, as no alterations in penicillin-binding (PBP) sites were observed. The molecular basis for the resistance to quinolones could not be established due to the absence of mutations in the gyrA or parC genes. The chromosomal nature of the resistance determinants does not pose any risk. Lactobacilli are known to possess intrinsic resistances to tetracycline, quinolones, vancomycin, and erythromycin that support the safety of the strain MTCC 5463 [36, 37]. The stable antibiotic resistance profile of the strain MTCC 5463 is beneficial during administration along with antibiotic therapy. The antibiotic resistance observed in strain MTCC 5463 was not acquired but intrinsic resistance. According to the QPS criteria, these results provide safety assurance for the ongoing use of the strain MTCC 5463 as a probiotic. The theoretical resistance markers need to be further subjected to expression studies and transferability tests, like bacterial mating experiments. As the resistance genes are not located on the plasmid, standard protocols for showing genetic transfer are not available in the literature [38]. Further research in this area is warranted. Lactic acid bacteria can add further functionality as heavy-metal-detoxifying agents by binding and effluxing heavy metals from food and water [39]. A growing concern lies in the co-occurrence of genes conferring metal resistance and antibiotic resistance genes, which could lead to the selection of antibiotic-resistant organisms in the human gut. This concern is mitigated in strain MTCC 5463 due to the absence of mobilome-associated resistant determinants. The suggested roles of heavy-metal-transporting ATPases and copper homeostasis elements in the acid tolerance mechanisms [40] further support the role of these related genes as a survival factor for the strain in gut rather than making the strain unsafe for human consumption.
The possibility of biogenic amine-induced discomfort during probiotic intervention depends on the number of strains in the product and the sensitivity levels of the host. No cases of biogenic toxicity have been reported during the three-controlled human clinical studies undertaken with strain MTCC 5463. The presence of ornithine decarboxylase and its expression could be a defense mechanism used by bacteria to withstand acidic environments [41] rather than for the production of biogenic amines. Similarly, the presence of D-lactate dehydrogenase in MTCC 5463 has not caused any complaint of D-lactic acidosis in human subjects. Beta-glucuronidase (BG) activity is considered a cancer risk biomarker [42], as well as an anti-tumor factor [43]. Therefore, the presence of BG in the strain MTCC 5463 genome can be concluded to be beneficial, as no detrimental activities have been reported in its long history of use. Functional metagenomic approaches have identified BG as a part of the functional core of the human microbiome of the gut ecosystem [44].
Among the vaginal microflora, bacteriocin producers achieve a competitive edge over uropathogens. This explains the presence and maintenance of bacteriocin genes, along with immunity proteins in the strain MTCC 5463. The strain MTCC 5463 did not produce bacteriocins in in vitro studies, but the presence of helveticin homologs suggests that expression of such genes may occur only under stress. The presence of pore-forming hemolysin genes may have been conserved due to the bacteria’s need for sustenance, defense, and survival through menstruation in the vaginal ecosystem. Virulence factors are universally differentiated into defensive factors (that protect the bacteria from the host immune system) and offensive factors (those that harm the host). In the case of probiotics, this classification is blurred. The vast number of genes identified through VRDB correlates with virulence factors of some pathogens but relates to fitness factors in the case of probiotics. Adaptation factors like adhesins and stress proteins are important to the characteristics of probiotics and should be considered as fitness factors. Fibronectin-binding protein, mucus-binding and sortase-dependent proteins involved in bacterial–host interactions are common to lactobacilli that colonize the gastrointestinal tract [41]. Thus, we can conclude that they are not acquired virulence genes, but genes that confer niche adaptation. The molecular chaperones present in the genome include DnaK, GroEL, and GroES, and they are pivotal for long-term acid stress resistance. Clp ATPase is particularly important for the fast response of lactobacilli in adverse conditions [45]. The use of databases like ARDB and VRDB is meaningful only in case of high-coverage assemblies and high sequencing depths. The safety of the strain MTCC 5463 is further supported by human clinical evidence to claim that the strain is not associated with any intestinal disorders or discomfort.
Conclusion
Traditional methods of analyzing the safety of a strain are replaced by cost-effective next-generation sequencing based on annotations. These methods reveal the maximum potential risk. The authors second the need put up by Zhang et al. [20] for a safety-associated gene database specifically for probiotics. This database could act as a preliminary defense against unsafe and uncharacterized strains entering the market and hence the food chain. Because probiotic strains are free living in natural niches and rarely the primary cause of an infection, it is often difficult to identify specific traits that contribute to pathogenesis. Certain virulence and antimicrobial characteristics are bound to be present in the genome, as lactobacilli are known to prevent microbial spoilage. The authors further emphasize the need for using multiple databases before drawing any conclusion. The detection of the genes, or homologs thereof, does not show whether they are intact and functional. The sequences may have premature stop codons, insertions, or deletions, making them nonfunctional. Expression studies of genes could further strengthen the safety assessments. The transferability of traits needs to be further validated using conjugation and filter mating experiments. The strain under study shows no known transferable determinants for antibiotic resistance, and the resistance traits are intrinsic in nature. The strain exhibits a specific pattern of fitness and adaptive factors. The study findings provide safety assurance for the ongoing use of the strain MTCC 5463 as a probiotic.
References
European Food Safety Authority (2007) Scientific Opinion of the scientific committee on the introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. EFSA J 578:1–16
Salminen S, Nybom S, Meriluoto J, Carmen M, Vesterlund S, El-Nezami H (2010) Interaction of probiotics and pathogens: benefits to human health? Curr Opin Biotechnol 21:157–167. doi:10.1016/j.copbio.2010.03.016
Brughton RA, Gruber WC, Haffar AA, Baker CJ (1983) Neonatal meningitis due to Lactobacillus. Pediatr Infect Dis J 2:382–384
Harty DW, Oakey HJ, Patrikakis M, Hume HJ, Knox KW (1994) Pathogenic potential of lactobacilli. Int J Food Microbiol 24:179–189
Rautio MH, Jousimies-Somer H, Kauma H, Pietarinen M, Saxelin S, Tynkkynen S, Koskela M (1999) Liver abscess due to a Lactobacillus rhamnosus strain indistinguishable from L. rhamnosus strain GG. Clin Infect Dis 28:1159–1160
Adams M, Mitchell R (2002) Fermentation and pathogen control: a risk assessment approach. Int J Food Microbiol 79:75–78
Borriello SP, Hammes WP, Holzapfel W (2003) Safety of probiotics that contain lactobacilli or bifidobacteria. Clin Infect Dis 36:775–780
Lee SY, Chang MT, Lee MH, Wu MS (2004) Lactobacillus peritonitis: a rare cause of peritonitis in peritoneal dialysis patients. Ren Fail 26:419–423
Tleyjeh IM, Routh M, Qutub MO, Lischer G (2004) Lactobacillus gasseri causing Fournier’s gangrene. Scand J Infect Dis 36:501–503
Kubiszewska I, Januszewska M, Rybka J, Gackowska L (2014) Lactic acid bacteria and health: are probiotics safe for human? Postepy Hig Med Dosw 17:1325–1334
Sanders ME, Akkermans LMA, Haller D, Hammerman C, Heimbach J, Huys G, Levy D, Mack D, Phothirath P, Constable A, Solano-Aguilar G, Vaughan E (2010) Assessment of probiotic safety for human use. Gut Microbes 1:1–22
International Life Sciences Institute Risk Science Institute Pathogen Risk Assessment Working Group (1996) A conceptual framework to assess the risks of human disease following exposure to pathogens. Risk Anal 16:841–848
Didelot X, Bowden R, Wilson DJ, Peto TA, Crook DW (2012) Transforming clinical microbiology with bacterial genome sequencing. Nat Rev Genet 13:601–612. doi:10.1038/nrg3226
Klaenhammer TR, Azcarate-Peril MA, Altermann E, Barrangou R (2007) Influence of the dairy environment on gene expression and substrate utilization in lactic acid bacteria. J Nutr 137:748–750
Callanan M, Kaleta P, O’Callaghan J, O’Sullivan O, Jordan K (2008) Genome sequence of Lactobacillus helveticus, an organism distinguished by selective gene loss and insertion sequence element expansion. J Bacteriol 190:727–735. doi:10.1128/JB.01295-07
Zhao W, Chen Y, Sun Z, Wang J, Zhou Z, Sun T, Wang L, Chen W, Zhang H (2011) Complete genome sequence of Lactobacillus helveticus H10. J Bacteriol 193:2666–2667. doi:10.1128/JB.00166-11
Prajapati JB, Khedkar CD, Chitra J (2011) Whole genome shotgun sequencing of Indian origin strain of Lactobacillus helveticus strain MTCC 5463 with probiotic potential. J Bacteriol 193:4282–4283. doi:10.1128/JB.05449-11
Tompkins TA, Barreau G, Broadbent JR (2012) Complete genome sequence of Lactobacillus helveticus R0052, a commercial probiotic strain. J Bacteriol 194:6349. doi:10.1128/JB.01638-12
Brul S, Bassett JP, Cook P (2012) Omics’ technologies in quantitative microbial risk assessment. Trends Food Sci Technol 27:12–24
Zhang ZY, Liu C, Zhu YZ, Wei YX, Tian F, Zhao GP, Guo XK (2012) Safety assessment of Lactobacillus plantarum JDM1 based on the complete genome. Int J Food Microbiol 153:166–170. doi:10.1016/j.ijfoodmicro.2011.11.003
Khedekar CD, Dave JM, Sannabhadti SS (1990) Inhibition of growth of pathogenic microorganisms during production and storage of cultured milk. J Food Sci Technol 27:214–217
Patidar SK, Prajapati JB (1999) Effect of feeding Lactobacilli on serum antibody titre and faecal flora in chicks. Microbiogie Aliments Nutr 17:145–154
Ashar N, Prajapati JB (1999) Evaluation of hypolipemic effect of feeding Lactobacillus acidophilus V3 in human subjects. J Dairy Foods Home Sci 18:78–84
Prajapati JB, Senan S, Momin JK, Damor R (2012) A randomised double blind placebo controlled trial of potential probiotic strain Lactobacillus helveticus MTCC 5463: assessment of its safety, tolerance and influence on intestinal well-being and humoral immune response in healthy human volunteers. Int J Health Pharm Sci 3:1–12
Kodaikkal V, Prajapati JB, Ljungh A (2012) Evaluation of adhesion of Lactobacillus strains to HT 29 cells by a flow cytometric assay. Int J Appl Anim Sci 1:1–7
Senan S, Prajapati JB, Joshi CG (2015) Whole-genome based validation of the adaptive properties of Indian origin probiotic Lactobacillus helveticus strain MTCC 5463. J Sci Food Agric 95:321–328
Senan S, Prajapati JB, Joshi CG (2014) Comparative genome-scale analysis of niche-based stress-responsive genes in Lactobacillus helveticus strains. Genome 57:185–192. doi:10.1139/gen-2014-0020
Wei YX, Zhang ZY, Liu C (2012) Safety assessment of Bifidobacterium longum JDM301 based on complete genome sequences. World J Gastroenterol 18:479–488. doi:10.3748/wjg.v18.i5.479
Bourdichon F, Casaregola S, Farrokh C (2012) Food fermentations: microorganisms with technological beneficial use. Int J Food Microbiol 154:87–97. doi:10.1016/j.ijfoodmicro.2011.12.030
Bennedsen M, Stuer-Lauridsen B, Danielsen M, Johansen E (2011) Screening for antimicrobial resistance genes and virulence factors via genome sequencing. Appl Environ Microbiol 77:2785–2787. doi:10.1128/AEM.02493-10
Hacker J, Kaper JB (1999) The concept of pathogenicity islands. In: Kaper JB, Hacker J (eds) Pathogenicity islands and other mobile virulence elements. ASM Press, Washington, DC, pp 1–11
Sui SJ, Fedynak A, Hsiao WML (2009) The association of virulence factors with genomic islands. PLoS ONE 4:8094. doi:10.1371/journal.pone.0008094
Macklaim JM, Gloor GB, Anukam KC, Cribby S, Reid G (2011) At the crossroads of vaginal health and disease, the genome sequence of Lactobacillus iners AB-1. Proc Natl Acad Sci USA 108:4688–4695. doi:10.1073/pnas.1000086107
Siezen RJ, van Hylckama Vlieg LET (2011) Genomic diversity and versatility of Lactobacillus plantarum, a natural metabolic engineer. Microb Cell Fact 10:S1–S3. doi:10.1186/1475-2859-10-S1-S3
Parkhill J, Sebaihia M, Preston A, Murphy LD (2003) Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Nat Genet 35:32–40
Zarazaga M, Sáenz Y, Portillo A, Tenorio C, Ruiz-Larrea F, Del Campo R, Baquero F, Torres C (1999) In vitro activities of ketolide HMR3647, macrolides, and other antibiotics against Lactobacillus, Leuconostoc, and Pediococcus isolates. Antimicrob Agents Chemother 43:3039–3041
Temmerman R, Pot B, Huys G, Swings J (2003) Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int J Food Microbiol 81(1):1–10
Hummel A, Holzapfel WH, Franz CM (2007) Characterization and transfer of antibiotic resistance genes from enterococci isolated from food. Syst Appl Microbiol 30:1–7
Halttunen TMC, Collado H, El-Nezami H (2008) Combining strains of lactic acid bacteria may reduce their toxin and heavy metal removal efficiency from aqueous solution. Lett Appl Microbiol 46:160–165
Penaud S, Fernandez A, Boudebbouze S (2006) Induction of heavy-metal-transporting CPXtype ATPases during acid adaptation in Lactobacillus bulgaricus. Appl Environ Microbiol 72:7445–7454
Azcarate-Peril MAE, Altermann LG, Goh YJ, Tallon R (2008) Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism. Appl Environ Microbiol 74:4610–4625. doi:10.1128/AEM.00054-08
Kim DH, Jung EA, Sohng IS, Han JA, Kim TH, Han MJ (1998) Intestinal bacterial metabolism of flavonoids and its relation to some biological activities. Arch Pharm Res 21:17–23
Chen X, Wu B, Wang PG (2003) Glucuronides in anti-cancer therapy. Curr Med Chem Anti-Cancer Agents 3:139–150
Gloux K, Berteau O, Oumami HE, Béguet F, Leclerc M, Doré J (2011) A metagenomic β-glucuronidase uncovers a core adaptive function of the human intestinal microbiome. PNAS 108:4539–4546. doi:10.1073/pnas.1000066107
Lebeer S, Vanderleyden J, De Keersmaecker SC (2008) Genes and molecules of lactobacilli supporting probiotic action. Microbiol Mol Biol Rev 72:728–764. doi:10.1128/MMBR.00017-08
Acknowledgments
The project was funded by the Indian Council of Agricultural Research under the Niche Area of Excellence program.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
Senan, Prajapati, and Joshi have no affiliations with or involvement in any organization or entity with any financial interest or nonfinancial interest in the subject matter or materials discussed in this manuscript.
Ethical Statement
The research has been conducted with integrity, and intellectual honesty, and human or animal subjects have not been a part of this study.
Rights and permissions
About this article
Cite this article
Senan, S., Prajapati, J.B. & Joshi, C.G. Feasibility of Genome-Wide Screening for Biosafety Assessment of Probiotics: A Case Study of Lactobacillus helveticus MTCC 5463. Probiotics & Antimicro. Prot. 7, 249–258 (2015). https://doi.org/10.1007/s12602-015-9199-1
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12602-015-9199-1