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Iranian Journal of Veterinary Research logoLink to Iranian Journal of Veterinary Research
. 2019 Summer;20(3):192–198.

The effect of Lactobacillus reuteri cell free supernatant on growth and biofilm formation of Paenibacillus larvae

R Betesho Babrud 1,2, R Kasra Kermanshahi 3,*, F Motamedi Sede 4, S Z Moosavinejad 5
PMCID: PMC6811706  PMID: 31656524

Abstract

Background:

Paenibacillus larvae is the etiological agent of American foulbrood (AFB) disease, the most lethal disease in honeybee (Apis mellifera) larvae.

Aims:

The aim of the present work was to study the antimicrobial effect of cell free supernatant (CFS) of probiotics on an Iranian isolate’s biofilm formation.

Methods:

A local strain was identified by 16S rRNA sequencing. The antibacterial effect of some probiotics was evaluated through drop plate method, minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC) and time-kill assay. The biofilm formation ability of P. larvae and the inhibition of biofilm formation by CFS were studied by microplate and scanning electron microscopy (SEM). The nature of the secondary metabolites in CFS was examined by microscale optical density assay (MODA).

Results:

Alignment of the results of P. larvae KB10 (GenBank accession number MH000685.1) 16S rRNA with the database revealed more than 97% identity with P. larvae. The most antibacterial effect was observed in the CFS of Lactobacillus reuteri ATCC23272 with 12.75 ± 3.2 mm for zone of inhibition (ZOI) at 1000 µL/ml for MIC and MBC. Time-kill assay revealed that CFS eliminated 1.5 × 108 CFU/ml P. larvae KB10 at 2 h of exposure. Microtitre plate and SEM results revealed that CFS (at sub-MIC concentration) was able to inhibit biofilm formation by P. larvae. The results of MODA assay showed that antimicrobial activity were related to the production of organic acids.

Conclusion:

Cell free supernatant from L. reuteri ATCC 23272 had inhibitory effects on P. larvae KB10 growth and biofilm production due to its acidic nature. The obtained results can be used for antibiotic substitution in AFB control and treatment.

Key Words: Anti-bacterial agents, Biofilm, Lactobacillus reuteri, Paenibacillus larvae, Virulence factors

Introduction

American foulbrood (AFB) disease, a devastating disease caused by Paenibacillus larvae spores, affects honeybee (Apis mellifera) larvae (Hamdi et al., 2013 ▶). Antibiotics treatment of AFB has risk of the residues in honey and also increases antibiotics resistance level in the pathogen (Alippi et al., 2007 ▶).

Bacteria in microbial populations exist in floating state and inside the biofilm. A biofilm is a growing microbial population on surfaces within a biopolymer matrix consisting of microbial organic materials. Regulation of gene expressions within the biofilm by phenotypic variation, food shortage and antimicrobial agents cause the bacteria to adapt to existing conditions    (Garrett et al., 2008  ▶; Wei and Ma, 2013 ▶). It is essential to find a new therapeutic approach for the treatment of biofilm-related infections without using antibiotics. Previous research shows the prohibition of pathogen biofilms with the use of probiotic products (Fang et al., 2018 ▶).

Probiotics adopt different strategies such as producing organic acids          (Mudroňová et al., 2011 ▶), antimicrobial compounds (Cleusix et al., 2007 ▶) and colonization (Dhanani and Bagchi, 2013 ▶) to inhibit or kill pathogens. Usually, probiotics do not have side effects on the host, cause environmental hazards or induce resistance. Many articles are published about the effects of probiotics on the in vitro prevention of P. larvae growth            (Jaouani et al., 2014 ▶). Lactobacillus spp. produce several secondary metabolites which have inhibitory effects on biofilm formations of pathogens    (Vahedi Shahandashti et al., 2016  ▶; Zamani et al., 2017 ▶). The possible effects of Lactibacillus spp. secondary metabolites on the growth and biofilm formation of P. larvae has not been studied earlier. The aim of this research was to evaluate the effect of Lactobacillus reuteri ATCC23272 cell free supernatant (CFS) on the biofilm formation, growth and viability of the P. larvae local isolate. Presumptive identification of effective secondary metabolites with inhibitory properties in the crude CFS was also undertaken.

Materials and Methods

Isolation and identification of P. larvae

Paenibacillus larvae were isolated from comb pieces of Isfahan apiaries according to Dingman and Stahly    (1983)  ▶ on Muller Hinton yeast pyruvate glucose phosphate agar (MYPGP-agar) and Columbia sheep blood (CSA) agar by 7 days incubation at 37°C in a CO2 gas pack containing jar (Cornman et al., 2013 ▶). Gram-positive, catalase negative, spore-forming bacilli were further examined for biochemical characteristics (Ayoub et al., 2013 ▶). Extracted DNA (Pouya Gene Azma kit) was amplified using the universal primers (Sinaclon) 27F (5´-AGA GTT TGA TGM TGG CTC AG-3´) and 1492R (5´-GGT TAC CTT GTT ACG ACT T-3´)    (Mao et al., 2012 ▶). Polymerase chain reactions (PCR) were performed in a final volume of 50 µL (1 µM of each oligonucleotide primer, 200 µM dNTPs, 1 mM MgCl2, 0.05 U/µL DNA Taq polymerase, 19 µL deionized water, 5 µL from 6× buffer) (all from Sinaclon) and 2 µL DNA (295.9 ng/µL). Two tubes without DNA were used as negative control. Polymerase chain reaction was performed as follows; 2 min denaturing step at 94°C followed by 30 cycles consisting of DNA denaturation at 94°C, 50 s, primer annealing at 59.1°C, 30 s; DNA elongation at 72°C, 1 min; final extension at 72°C, 10 min). Polymerase chain reaction products were then mixed with SYBR-gold and loaded in 1% w/v agarose gel (Sinaclon) prepared in 1× TBE buffer (Tris base 0.446 M, Boric acid 0.445 M, 20 ml EDTA 0.5 M, pH = 8). After electrophoresis with 100 v DC for 1 h, bands were visualized with Gel Doc (VILBER E-Box VX5). Gene sequencing was later performed using automated DNA sequencing (Bioneer, Korea). Pair wise and multiple DNA sequence alignments were then carried out comparing 16S rRNA gene sequences available by basic local alignment search tool (BLAST) search in the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/) using Bioedit 7.2.5 software. The sequences were submitted to GenBank for accession numbers. The phylogenetic tree was constructed by the neighbor-joining (NJ) method using the Molecular Evolutionary Genetic Analysis 7.0 (Mega version 7.0).

Preparation and antimicrobial activity of CFS from different probiotics

Overnight cultures of Lactobacillus acidophilus ATCC4356, L. plantarum ATCC8014, L. reuteri ATCC23272, L. fermentum ATCC9338, L. rhamnosus ATCC7469 in de Man, Rogosa and Sharpe (MRS)-broth (Merck)(pH = 5.4) in 5% CO2 at 37°C were centrifuged and filter sterilized (0.22 µm) (Millipore) to achieve CFS. Paenibacillus larvae in MYPGP-broth (OD595=0.1) was lawn cultured on MYPGP-agar after which a drop of 20 µL CFS was put on the culture. The zone of inhibition (ZOI) on the droplets’ place was evaluated after 48 h incubation at 37°C. Sterile MRS-broth was used as negative control. Broth microdilution method was carried out to determine minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of CFS. One hundred fifty µL of CFS two-fold serially diluted in MRS-broth was dispensed in wells of a 96 well polystyrene microtitre plate (Sorfa). Each well was inoculated with 150 µL of overnight culture of P. larvae in MYPGP-broth (OD595=0.1) then diluted 1/100. Minimum inhibitory concentrations and MBC were defined as Balouiri (Balouiri et al., 2016) comparing positive (MRS-broth instead of CFS) and negative (sterile MYPGP-broth) controls after 24 h at 37°C (Balouiri et al., 2016). A time-kill test was performed according to Bajpai with CFS at MIC concentration compared to the positive control (bacterial culture in MYPGP-broth).

Biofilm formation ability

Microplate technique was used to assay biofilm forming according to Cai et al.    (2013)  ▶. Optical density (OD) was read at 595 nm by a microplate reader (Epoch BioTek). The cut-off (ODC) was defined as the mean OD value of the negative control (sterile MYPGP-broth). Biofilms were classified as no (OD≤ODC), weak (ODC<OD≤2×ODC), moderate (2×ODC<OD≤4×ODC) or strong (4×ODC<OD) (Gómez et al., 2016 ▶).

Inhibition of biofilm formation by CFS

The effect of CFS on biofilm formation was assessed by co-incubation of P. larvae in MYPGP-broth (OD595=0.1) with the same volume of CFS at sub-MIC concentration (1/2 CFS prepared in MRS-broth). For positive control, MRS-broth was used instead of CFS (Wojnicz et al., 2013). The inhibition of biofilm formation was calculated according to Vahedi Shahandashti et al.    (2016)  ▶.

Effect of CFS removal on biofilm

After 24 and 48 h of biofilm formation, its removal by CFS was assessed according to Vahedi Shahandashti    et al. (2016)  ▶. In the positive control, phosphate buffered saline )PBS( was used instead of CFS.

Microscopic observation techniques

Microscopic observation of biofilms on glass slides was evaluated using MYPGP-broth culture samples with CFS, comparing biofilms formed in sterile MRS-broth (positive control) and MYPGP-broth (untreated biofilm), according to Vahedi Shahandashti et al.    (2016)  ▶ after 48 and 96 h at 37°C and studied using scanning electron microscopy (SEM) (Zeiss).

Effects of different treatments on CFS

The growth inhibitory effect of CFS was examined by microscale optical density assay (MODA) following different treatments of NaOH, acetic acid, trypsin, pepsin and catalase after 24 h at 37°C by absorbance at 595 nm according to Lash et al.    (2005)  ▶. The growth was evaluated as the difference in absorbance between the control and the samples (Vahedi Shahandashti et al., 2016 ▶).

Statistical analysis

Statistical analyses were conducted using SPSS 20. One-way analysis of variance (ANOVA) and independent samples t-tests were performed after assumptions of normality and variances of homogeneity were checked. Significance levels were set at P<0.05.

Results

Isolation and identification of P. larvae

Isolates of P. larvae were identified on the basis of cultural and biochemical characteristics. Sequence alignment of a partial 16S rRNA amplified gene revealed that the bacterial strain was P. larvae with 100% identity to P. larvae strains and 97.48% identity with standard strains of P. larvae larvae (DSM 25430) in the database (NCBI). The sequences were submitted to GenBank. Accession numbers are mentioned on phylogenetic tree in Fig. 1.

Fig. 1.

Fig. 1

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Evolutionary relationships of taxa: the evolutionary history was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 24.22610255 is shown (next to the branches). The stars represent Iranian isolated strains. The strain in the box was studied in this research

Antimicrobial activity of CFS

According to the results of five repetitions, CFS of L. reuteri ATCC23272 was able to inhibit the growth of P. larvae KB10, with a larger ZOI (12.75 ± 3.2 mm) comparing to other tested probiotics. No growth inhibition was seen at the the negative control drop. Broth microdilution method showed that CFS from L. reuteri ATCC23272 with a concentration of 1000 µL/ml (1/1 v/v CFS/MRS) inhibited the growth of P. larvae KB10 and destroyed 99.9% of the living cells. Therefore, this concentration was considered as MIC and MBC of CFS from L. reuteri ATCC23272 (Fig. 2A). A time-kill test of CFS revealed significant inhibitory effects of CFS on P. larvae KB10 by the complete inhibition of cell viabilities during the first 2 h of exposure. The inhibition effect was directly related to CFS as compared to the control tube containing MRS-broth (Fig. 2B).

Fig. 2.

Fig. 2

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Antimicrobial activity of cell free supernatant (CFS): (A) Microdilution plate of two fold dilutions of CFS in sterile de Man, Rogosa and Sharpe (MRS)-broth: 1000 µL/ml (a), 500 µL/ml (b), sterile Muller Hinton yeast pyruvate glucose phosphate agar (MYPGP) broth (negative control) (c), P. larvae KB10 in MYPGP broth (positive control) (d), and P. larvae KB10 in (MYPGP broth+MRS broth) (positive control) (e). (B) Time-kill test of CFS on P. larvae KB10. Data are the average of four replications ± SD

CFS effects on biofilm formation by P. larvae KB10

Biofilm formation ability, investigated by the microplate method, indicated that P. larvae KB10 was able to form biofilm. The biofilms in 24 and 48 h were weak (OD=0.181) and moderate (OD=0.243), respectively; compared to the negative control (sterile MRS-broth) (OD=0.117) (Fig. 3A). Inhibition of biofilm formation by CFS (at sub-MIC concentration) showed a significant reduction of 50% and 37.29% in 24 and 48 h exposure to CFS, respectively (P<0.05), compared to the positive control (biofilm in exposure to MRS-broth) (Fig. 3B).

Fig. 3.

Fig. 3

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Effects of cell free supernatant (CFS) of L. reuteri ATCC23272 at sub-MIC concentration (500 µL/ml) on: biofilm formation (A), and biofilm removal (B) of P. larvae KB10. Results are the average of three replications ± SD

The evaluation of CFS removal effects on formed biofilms showed no significant differences with the control (PBS treated biofilm) (P>0.05) (Fig. 3A). Microscopic observations confirmed biofilm formation in MYPGP-broth (Fig. 4A) and MYPGP/MRS-broth (Figs. 4B and C). Inhibition of biofilm formation in exposure to CFS was also confirmed in microscopic observations (Figs. 4D and E).

Fig. 4.

Fig. 4

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Scanning electron microscopy images from biofilms of P. larvae KB10 on glass slides. (A) 24 h in MYPGP broth, (B) 48 h in a mix of MYPGP/MRS broth (1/1), (C) 48 h in MYPGP/CFS, (D) 96 h in MYPGP/MRS broth, and (E) 96 h in MYPGP/CFS (×30000)

Effects of different treatments on CFS anti-microbial activity

The results of MODA revealed that neutralizing CFS with NaOH caused an increase in the growth of P. larvae KB10 compared to untreated CFS (P<0.05). Bacteria growth reduced significantly in acid treated MRS-broth compared to untreated CFS as the control (P<0.05). Elimination of probable H2O2 (by catalase) and proteins (by pepsin and trypsin) in CFS did not change the growth rate (P>0.05). Boiled CFS showed a slight but not significant increase in growth compared to untreated CFS as control (P<0.05) (Fig. 5).

Fig. 5.

Fig. 5

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Microscale optical density assay results of cell free supernatant (CFS) from L. reuteri ATCC23272 on the growth of 1/100 diluted of P. larvae KB10 (OD=0.1) following different treatments of NaOH, acetic acid, trypsin, pepsin catalase and boiling. Results are the average of five replications ± SD

Discussion

Sequence alignment of P. larvae KB10 showed 97.48% identity to the ERIC II genotype of the P. larvae larvae standard strain (DSM25430). The authors have shown the presence of a crystalline surface layer in this strain (unpublished data), given that this layer has been identified exclusively in the ERIC II genotype (Poppinga et al., 2012 ▶). Determining the genotype group in this strain is necessary for the strain.

The quantified biofilm strength indicated an increase from 24 to 48 h incubation which was in accordance with biofilm formation dynamics (Cai et al., 2013 ▶). Scanning electron microscopy images of 24-h biofilm revealed a production of some extracellular scaffold-like matrices, which are the basis of adhesion in biofilm production and extension (Fig. 4A). Extracellular polymeric substance consists of exopolysaccharides, proteins, extracellular DNA and other compounds (Zamani et al., 2017 ▶). Bacteria in the 48-h biofilm bonded together and created a multi-layer structure (Fig. 4B). The created biofilm after 96-h was less three-dimensional (Fig. 4C) and had lower numbers of cells in the cementing mass compared to the 48-h biofilm (Fig. 4B); however, it still had an extracellular matrix. This is the detachment stage, in which the production of some secondary metabolites cause bacteria to disperse and leave a mass of extracellular material (Joo and Otto, 2012 ▶). This is the first report on the kinetics of biofilm formation in a local isolate of P. larvae.

Biofilm formation reduced when exposed to CFS compared to the control. The reduction of biofilm after 48 h was not significant compared to the reduction at 24 h of exposure (Fig. 3A) (P>0.05). This might be due to the strength (higher amount of extracellular polymeric substances) and lower bacterial metabolism in the 48 h biofilm comparing to the biofilm of 24 h.

The 48-h biofilm created in contact with CFS (Fig. 4D), encountered a decrease in attached numbers of bacteria and biofilm production. In the meanwhile, the 48-h P. larvae KB10 culture, exposed to MRS-broth (control) (Fig. 4B), caused the bacteria to gather, attach to the surface and form micro-colonies. Biofilm formation in contact with CFS (OD595=0.227) reduced about 37.29% compared to the control (OD595=0.362) (Fig. 3B). Similar findings regarding the effect of CFS on reducing biofilm production from other bacteria were reported by    Zamani et al. (2017)  ▶. Exposure to CFS (pH = 4.18) was more stressful for P. larvae KB10 than the control medium containing MRS-broth (pH = 5.4) (Fig. 4B). Therefore, the number of bacteria decreased and consequently, a very weak biofilm was produced. This significant effect may contribute to the acidity or other unknown compounds in CFS. In the present study, CFS at sub-MIC concentrations had no effect on P. larvae KB10 cell wall lysis. The biofilm in exposure to CFS after 96 h (Fig. 4E) differed from that formed when exposed to the control sterile MRS-broth. This difference consisted of fewer bacteria without extracellular materials on the surface and a soft and loose connection to the matrix in the biofilm. The matrix appeared weak and porous and seems to be too unsuitable in amount or quality to bind to the substrate. P. larvae KB10 in CFS seems to have weak adhesion to glass surfaces. Lactobacillus sp. suppressive effects have been reported earlier by other researchers only with regards to the growth of P. larvae (Pehrson et al., 2015 ▶) Our research is, therefore, the first to represent the inhibitory effect of CFS on the biofilm formation of P. larvae. Although CFS ability in removing biofilm has been reported earlier (Vahedi Shahandashti et al., 2016 ▶) the results of our research showed that CFS had no significant removal effects on the formed biofilm (Fig. 3A). The reasons may be attributed to biofilm structure, CFS concentration and its mode of action on bacterial cells or penetration into the matrix barrier.

The time-kill assay showed that the 4.5 log CFU/ml viable cell count of P. larvae KB10 was completely eliminated after the first 2 h of contact with the CFS at MIC concentrations (Fig. 2B), which confirm the results of MBC. The results are acceptable comparing other similar investigations (Dasari et al., 2014 ▶).

According to the results obtained from the MODA test, it can be concluded that the inhibitory effect of CFS was related to acidity rather than H2O2 or protein compounds. Boiling CFS caused a slight pH increase (0.77 units) which caused a non-significant increase in the growth of P. larvae KB10 (P>0.05) (Fig. 5). It is reported that L. reuteri produces a number of heat-sensitive metabolites such as carbon dioxide (Morita et al., 2008 ▶) and ethanol (Chen et al., 2016 ▶). The evaporation of such materials in boiling temperatures might be the reason of increased P. larvae KB10 growth.

Acknowledgements

Authors would like to acknowledge Dr. K. Ahmadi’s efforts for preparing combs, H. Akbari, B. Mosayebiyan, and F. Nabardi for microbial culture technical support.

Conflict of interest

The Faculty of Biological Sciences of Alzahra University supported this work. All authors disclose any financial and personal relationships with other people or organizations that might inappropriately influence or bias their work.

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