Antilisterial efficacy of Lactobacillus brevis MF179529 from cow: an in vivo evidence

Pathogenic microbes are important due to their role in health complications and food spoilage. Listeria monocytogenes (LM) is a facultative intracellular food borne pathogen which is causative agent of human listeriosis, gastrointritous, abortion, meningoencephalits and septicemia [1]. Immuno-compromised individuals, pregnant women and fetus are more prone to listeriosis [2]. Ingestion of LM contaminated food results in translocation of pathogen from intestinal lumen to different organs [3]. Listeriosis is serious public health issue and lead to 20 to 30% mortality [4].

Probiotics are defined as live nonpathogenic microorgansims, when used in adequate amount give health benefits to the host [5]. They play an important role in maintaining the microbial balance in gastrointestinal tract of the host by promoting growth of beneficial microbes and also modulate immune responses [6]. Mostly probiotics belong to a group of Lactic acid bacteria (LAB), among them Lactobacillus and Bifidobacterium genera are used commercially [7, 8]. Probiotic strains use different mechanisms to combat infection viz., production of inhibitory substances, blocking of adhesion sites on the intestinal surface, dislocation of the initially adhered pathogen, competition for nutrients and stimulation of mucosal and systemic immunity [9]. The antagonistic and co-aggregation potential of probiotic strains against pathogen is strain specific [10]. Probiotic supplements are used singly or in combination to combat pathogen but mixture of probiotic strains is reported to be more effective [11]. Probiotics are recommended by health professionals to cure gastroenteritis including mild diarrhea, ulcerative colitis, antibiotic associated diarrhea when standard theropies did not work [12].

Can be used as an alternative to chemical growth promoter, and growth performance of the host. LAB are reported to produce antimicrobial compound such as lactic acid, hydrogen peroxide, reuterin, diacetyl, acetoin reutricyclin, antifungal peptides and bacteriocins [13]. Bacteriocins are small peptides produced as secondary metabolites which have the ability to inhibit the growth of closely related species, allowing competition among different niches. LAB are known to produce fermented products as well as bacteriocins that have the inhibitory effect against clinical and foodborne pathogens including LM [14, 15]. Thus LAB are recommended to be used as food supplement and therapeutic agent against infectious diseases [16].

To the best of our knowledge, this is the first report on antimicrobial efficacy of L. brevis (LB) MF179529 against LM using a murine infection model.

The probiotic strain used in the study was Gram positive rod, non-spore former, non-motile and catalase negative. It was identified as Lactobacillus brevis on the basis of 16S rRNA sequencing and assigned accession number MF179529 by National Center for Biotechnology Information (NCBI). In pilot study, LM could not be re-isolated from tissues including liver, spleen and intestine of animals exposed to lower bacterial density (10⁵ CFU/ml). Animal exposed to a dose of 10⁷ CFU/ml showed presence of LM in few tissues. It was reisolated from liver and intestine of 2/6 and spleen of 1/6 animals. A single intragastric dose of 10⁹CFU/ml of LM resulted in translocation of pathogen in all tissues of sacrificed animals (Table 1).

In the main study, protective effect of orally administered LB MF179529 (10⁹CFU/ml) against Listeria infection (10⁹ CFU/ml) was monitored. General health conditions of LB fed group were better than positive control group (P ≤ 0.05). The comparison of GSH scores in LB treated and infected group (IV and II) is presented in Table 2.

Initial body weight among groups was between 30 to 34 g. Among groups there was no significant difference in body weight of animals at the start of experiment. During course of experiment the body weight of negative control (I) and LB fed (III) group remained unaltered, while continuous decrease was observed in positive control (II) group where it reduced from 34 to 28 g. LM + LB treated group (IV) showed initial decrease in body weight at 3rd dpi but later on it increased to level of negative control (Additional file 1: Figure S1). Feed intake of positive control group (II) also decreased in comparison with all other groups (Additional file 2: Figure S2). Body temperature of negative control and LB fed animals (I and III) group remained normal throughout the experiment. Body temperature of LB + LM group (IV) increases up to 3rd dpi and then become equal to negative control (I) group. While body temperature remained high in LM group (II) (Fig. 1).

Data on LM translocation in organs revealed that LM load in liver, spleen and intestine was significantly reduced in LB + LM treated (IV) group as compared to positive control (II) group. LM count of group IV was 4.45 CFU/g, 4.15 CFU/g in liver and 4.45 CFU/g and 4.15 CFU/g in spleen at 3rd and 5th dpi respectively. However, it could not be re-isolated at 7th dpi LM from both tissues. On the other hand, its value was 5.3, 5.1 and 4.8 log CFU/g at 3rd, 5th and 7th dpi respectively in intestine. The LM count in group II remained high in all tissues (4.9–5.1 CFU/g in liver, 4.4–4.6 CFU/g in spleen and 6.7–7.3 log CFU/g in intestine) during course of experiment (Fig. 2).

Influence of LB treatment on the microbial composition of intestine of mice was analyzed by comparing coliform, LAB, TPC and APC count of group I-IV. LB supplementation lead to significant reduction in TPC and coliform count in group III and IV (Fig. 3a and d). However, significant elevation in LAB count was detected in group III and IV. While infection with LM reduced LAB count in group II when compared with negative control group I (Fig. 3b). The anaerobic plate count (APC) was recorded higher in group III while, in other groups it was similar (Fig. 3c). The pattern of change in TPC, APC, LAB and coliform count was similar at all time points (3rd, 5th and 7th dpi).

Effect of LB treatment on course of infection was further determined by comparing hematological picture of the four groups. A significant decrease in hemoglobin (Hb) and elevation in TLC was noticed at 3rd dpi in positive control (II) group. Platelets count of group II also decreased significantly as compared to negative control group. But LB treated groups (III and IV) showed higher platelets count as compared to negative control group. A significant increase in neutrophils and decrease in lymphocytes was also observed in positive control group (II) while no such alteration was observed in LB treated groups (III and IV). The variation in eosinophils and monocytes levels was not significant among groups (p > 0.05) (Table 3). Serological parameters including ALP, ALT/GPT, AST/GOT, TBil, Urea, creatinine, CHOL, TG, HDL and LDL were analyzed during experiment at 3rd, 5th and 7th dpi. No significant change was observed in serological parameters of infected and uninfected groups (data not mentioned).

Listeria monocytogenes is a worldwide public health threat and cause listeriosis by consumption of contaminated food. The current study was performed to comprehend the impact of Lactobacillus brevis MF179529 against orally acquired Listeriosis in mice.Listeria monocytogenes ATCC 19115 was chosen as a model of bacterial infection in mice. First obstacle in the study was the establishment of LM ATCC 19115 infection in mice. The bacterial density of pathogen for induction of infection varies depending on virulence of strain, route of exposure and dose of pathogen [17]. Since LM is not acid tolerant and most likely to be killed due to low pH in stomach [18]. Higher bacterial density of pathogen is required to induce infection through oral route as compared to intraperitoneal and subcutaneous. Previously, various authors reported successful infection in experimental animals at the dose of 10⁹ of LM [19‐21]. Therefore, a pilot study was conducted to determine the infective dose of LM ATCC 19115 using oral transmission route. The oral route was preferred due to its mimicry with human fecal oral route of Listeriosis [22]. Mice were infected with LM ATCC 19115 at three doses (200 μl of 0, 10⁵, 10⁷, 10⁹ CFU/ml) and monitored for morbidity as well as mortality up to 7 dpi. Mice receiving 10⁹CFU/ml displayed clinical signs of infection including circumflex back, ruffled fur and sluggish movement. Additionally, LM was successfully re-isolated from liver, spleen and intestine indicating successful establishment of clinical infections and dissemination of pathogens. On the other hand, in animals exposed to dose of 10⁷ CFU/ml, re-isolation frequency was very low indicating slight infection. In light of these results bacterial density of 10⁹ CFU/ml was decided for main study.

In an effort to analyze the protective effect of local strain L. brevis MF179529 against LM ATCC 19115 infection in mice, main study was conducted. Previously, most of the literature reports the inhibitory effects of Pediococcus acidilactici, Lactobacillus casei, Lactobacillus brevis and Lactobacillus paracasei on LM. L. brevis MF179529 was one of our lab isolates with significant antimicrobial activity against both Gram positive and Gram negative pathogenic isolates (data not shown). To the best of our knowledge, no data exists on antilisterial effect of LB.

On the basis of previous literature on LAB species, 200 μl of 10⁹ CFU/ml bacterial density of LB MF179529 was offered to mice three days prior to LM infection. [23, 24]. A significant reduction in general health score (GHS) of LB treated animals was noticed throughout. LB treated groups manifested a significant increase in feed intake and gain in body weight as compared to positive control. Likewise, body temperature of LB treated group remained high up to 3 dpi and later on dropped to normal level. These positive effects may be due to limiting growth or dissemination of LM, or change in intestinal microbial equilibrium by LB.

To further strengthen the role of LB in decreasing LM infection, the pathogen load in different organs was quantified. LB treated group displayed low tissue invasion in liver, spleen and intestine relative to positive control group. Interestingly, complete elimination of pathogen was noticed in liver and spleen at 7th dpi. Consistently, LM counts in intestinal tract also reduced gradually from 3rd to 7th dpi, although it was not totaly eliminated. It may be due to secretions of antimicrobial compounds, competition among LB and LM for colonization in GI tract etc. Our findings are consistent with Becattini et al. [25] who reported that commensal gut microbiota play a defensive role against LM infection by preventing its systemic dissemination. Our results are in accordance with previous observations on reduction of LM count among liver, spleen and intestine by pre feeding with Lactobacillus casei [26‐29].

Of note, another important contribution of LB MF179529 consumption on GI tract was noticed by alteration in intestinal microbial equilibrium. A significant increase in lactic acid bacteria along with reduction of other intestinal bacteria including LM proposed antilisterial activity of LB. It may be due to competitive survival of LB in GI tract [30]. Recovery of high quantity of LAB from the intestine further indicates that LB could successfully withstand the stressed conditions of gastrointestinal tract of mice supporting its probiotic nature. In fact, use of LAB to ablate the intestinal pathogens is pertinent to previous observations on the synergistic effects of antibiotics and corticosteroids in listeriosis [31]. Additionally, antilisterial activity of LB may be an outcome of antimicrobial peptide secretion as reported previously [32].

Hematological studies provide important information about multiple disorders including bacterial infections. Phagocytes especially macrophages and neutrophils plays an important role against bacterial infections. To further confirm the remodeling role of LB, the hematological picture of all groups was compared. Our results indicated that Hb level decreased in infection group making it a high risk factor to the health [33]. It is known since long that LM produces soluble hemolysin which causes lysis of erythrocytes and LM utilize the iron present in hemoglobin to thrive in the host [34]. Absence of anemia in LB treated mice (group III and IV) confirms the antilisterial efficiency of our strain. It may be due to absence of hemolysin production by our strain. Further evidence was provided by TLC, neutrophil and platelet count. Increased TLC and neutrophil count along with decreased platelets counts are indication of establishment and persistence of infection [35, 36]. In LB treated group (III and IV) TLC, neutrophil and platelets counts remained equal to negative control. Similarly, lymphocytes count remained unaltered in all groups except infection group where it decreased significantly. This indicates the clinical importance and non-pathogenic nature of LB.

In vivo safety assessment of LB was performed by comparing hematological and serum biochemical profiles in control and LB fed group. No significant variation in any serological parameter could be recorded indicating safe nature of LB MF179529. In culmination, our results suggest that LB MF179529 from cow intestine is useful for prevention of invasion and facilitates suppression of LM ATCC19115 infection in mice.