- Email: [email protected]
Bacterial communication between Lactobacillus spp. isolated from poultry in the inhibition of Salmonella Heidelberg—proof of concept
Bacterial communication between Lactobacillus spp. isolated from poultry in the inhibition of Salmonella Heidelberg—proof of concept Adriano Sakai Okamoto,1 Raphael Lucio Andreatti Filho, Elisane Lenita Milbradt, Ana Carolina Izidoro Moraes, Igor Henrique Bastos Vellano, and Priscylla Tatiana Chalfun Guimar˜ aes-Okamoto Veterinary Clinic Department, FMVZ/UNESP Botucatu, S˜ ao Paulo, Brazil, 18618-681 ABSTRACT Bacterial communication has become an increasingly studied topic aiming at the discovery of new products to aid the treatment of diseases for which conventional options do not work. The production of safe foods, free of pathogens, has been receiving increasing attention due to market demands for food products of high quality and free of residues. This study assessed the communication between Lactobacillus spp. during the in vitro inhibition of Salmonella Heidelberg (SH) and the impact an autoinducer produced by a strain of Lactobacillus plantarum has on communication in the normal microbiota and inhibiting SH in newborn chicks. For this purpose, the isolates of Lactobacillus spp. were isolated cloacal swabs of broilers and identified through biochemical and molecular assays and were obtained from broiler farms. They later had their inhibitory potential against SH stimulated after contact with the autoinducer. For assessing bacterial
communication (quorum sensing) during the inhibition of SH by Lactobacillus spp., a spot on the lawn assay was conducted. For the in vivo, 75 one-day-old chicks were divided in 5 experimental groups: control with no treatment; treatment with a Lactobacillus spp. pool on the first day; treatment with autoinducer on the second day; treatment with Lactobacillus spp. on the first day; and autoinducer on the second day and treatment without autoinducer. The autoinducer was assessed through an SH count in the ceca of the birds. The autoinducer produced by the strain of L. plantarum proved to be efficient for communicating with the other Lactobacillus spp. isolates as previous contact with SH induced the production of an autoinducer capable of increasing inhibition of SH both in vitro (in average 132.73%) and in vivo, acting similarly to the Lactobacillus spp. pool (probiotic) by decreasing the SH count in the ceca (64%–24 h, 42%–96 h, and 46%–168 h).
Key words: autoinducer, chicken, microbiota, quorum sensing, probiotic 2018 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pey141
INTRODUCTION AND JUSTIFICATION
diseases and, despite technological breakthroughs, remains an important and global problem (Boyle et al., 2007). In 1993, Salmonella spp. emerged as a great health hazard for both poultry and humans in Brazil. The occurrence of asymptomatic carriers facilitates dissemination and increases the amount of contaminated food, proving the importance of this agent as a worldwide public health issue (Shinohara et al., 2008). An indiscriminate use of antibiotics in birds allowed the maintenance of batches positive for Salmonella spp., causing a marked increase in antimicrobial resistance and multiresistance (Palmeira, 2007; Ribeiro et al., 2008). Over 2,500 serovars are already known, among which paratyphi salmonellas are considerably important (Berchieri Junior, 2000), as they are not specific to birds and present a more serious health risk to the population by increasing the contamination of carcasses and eggs. Among these, Salmonella Heidelberg (SH) has been most widely reported in poultry worldwide, including Brazil, and is important due to how difficult it is to control and eradicate once it has colonized the bird (Cury, 2013). Aside from biosafety, alternative
Brazil has a rapidly growing poultry industry, as shown by the high production of chickens in 2016, reaching 13,146 million tons, awarding the country second place worldwide in overall production and first place in exports with 4,304 million tons (ABPA, 2016). This population increase in the country has created favorable conditions for the proliferation of several avian pathogens, particularly Salmonella spp. (Silva and Duarte, 2002), which is considered a food-safety pathogen. The occurrence of diseases transmitted by food has been widely discussed in the past few years due to the worldwide concern with strategies ensuring they are controlled and, therefore, the entry of safe products in the world market. Salmonella spp. is a gram-negative bacillus which usually moves through a flagellum. It is the most important agent involved in food-transmitted C 2018 Poultry Science Association Inc. Received December 18, 2017. Accepted March 21, 2018. 1 Corresponding author: [email protected]
1 Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey141/4967666 by Durham University user on 11 April 2018
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measures should be taken to control these bacteria, such as specific vaccination for laying and breeding hens, acquisition of batches free of the pathogen, use of organic acids and probiotics (Santos and Gil-Turnes, 2005). Probiotics are products composed of bacteria that are part of the bird’s natural microbiota and, therefore, are considered natural products, and do not present residues in animal products or stimulate resistance to drugs administered to humans (Da Silva and Andreatti Filho, 2000). An example of probiotic is Lactobacillus spp., gram-positive, and non-spore-forming bacteria whose shape may vary from a long and thin bacillus to a short coccobacillus. They are mostly motionless (Koneman et al., 2011) and induce the intestinal microbiota into equilibrium by producing several metabolites with antimicrobial properties and competing for binding sites with pathogenic bacteria. They also help immunomodulate the animal’s microbiota and aid nutrient absorption, allowing their use as enhancing agents by helping the birds in several unfavorable conditions (Da Silva and Andreatti Filho, 2000; Kuritza et al., 2014). One of the ways through which Lactobacillus spp. inhibit Salmonella spp. is through intraspecies and interspecies communication, termed quorum sensing. The bacteria note differences in the environment through signaling substances secreted by individual cells that are recognized by specific receptors in another bacterium, leading to a modification in their behavior (Hawver et al., 2016). This behavioral modification results in a collective response that modulates physiological activity in a positive way to survive, such as promoting change to a location with more nutrients and the formation of a biofilm granting protection against environmental actions; monitoring population density, gene expression, and virulence factors; producing bacteriocins with antibiotic activity; sporulation; and bioluminescence (Hawver et al., 2016). The bacteria also use this mechanism to produce, release, and recognize autoinducers that modulate these activities (Miller and Bassler, 2001; Parker and Sperandio, 2009; Sola et al., 2012). Some strains of Lactobacillus spp. produce substances with protein origin, such as bacteriocins. These substances can inhibit growth or deactivate other bacteria through the quorum sensing mechanism and, therefore, present antagonism against Salmonella spp. (Rumjanek et al., 2004). This study aims at assessing the ability of Lactobacillus spp. isolated from chicken to inhibit the multiplication of Salmonella Heidelberg through the quorum sensing mechanism both in vitro and in vivo.
MATERIALS AND METHODS In vitro Experiment The in vitro experiment consisted of proving the occurrence of quorum sensing between 17 isolates of
Lactobacillus spp. by evaluating the inhibition against Salmonella Heidelberg. For this purpose, a Spot on the Lawn test was conducted on a pure culture of Lactobacillus spp. isolates, both before and after an autoinducer produced by a strain of Lactobacillus plantarum that was previously in contact with Salmonella Heidelberg. Bacterial Samples. Salmonella Heidelberg—the sample of S. Heidelberg used in the study belongs to the Avian Pathology Service’s Bacteria Collection at Universidade Estadual Paulista—UNESP, Botucatu campus, obtained from the liver of 1-day-old chicks (Cobb). Lactobacillus spp.—the Lactobacillus spp. used as donor for the autoinducer belongs to species L. plantarum resistant to Rifamficin (Rif) and nalidixic acid (Nal) obtained from the Avian Pathology Service’s Bacteria Collection at Universidade Estadual Paulista— UNESP, Botucatu campus. The 17 isolates of Lactobacillus spp. used to assess quorum sensing in the inhibition of S. Heidelberg were isolated from a cloacal swab from 52-wk-old Cobb broilers. After collection, the swabs were cultivated in deMan Rogosa and Sharp (MRS) broth at 38◦ C for 24 h, and then seeded in MRS agar under the same growth conditions. Thirty characteristic Lactobacillus spp. colonies were stained by Gram’s method and the Gram-negative colonies were discarded. After this screening process, the isolates were identified through biochemical assays (catalase production, potassium hydroxide production, gas production through glucose fermentation and hydrogen sulfide production in Triple Sugar Iron). Genus identification was confirmed through polymerase chain reaction (PCR), and the DNA was extracted with a commercially available extraction kit (QiagenR ). The primers used were Forw R16-1 5 -CTT GTA CAC ACC GCC CGT CA-3 and Rev LbLMA15 -CTC AAA ACT AAA CAA AGT TTC -3 amplifying a 250-bp product (18). The reaction was comprised of 20 pmol of each primer, 12.5 μL of GoTaq Master Green, 2.5 μL of ultrapure water, and 5 μL of DNA, totaling 25 μL. Amplification was initiated at 95◦ C for 5 min, followed by 20 cycles at 95◦ C for 30 s, 55◦ C for 30 s, and 72◦ C for 30 s, followed by a final extension at 72◦ C for 7 min. The PCR products underwent electrophoresis in agarose gel at 1.4%, stained and viewed in an ultraviolet transilluminator. Spot on the Lawn Test. Among the 30 colonies possessing characteristics of genus Lactobacillus spp., 17 were identified and submitted to the spot on the lawn test so that the inhibition halo against S. Heidelberg could be assessed. The isolates of Lactobacillus spp. were cultivated individually at 38◦ C for 18 h in MRS agar (10 μL) as dots while the sample of S. Heidelberg was cultivated in brain heart infusion (BHI) broth and then incubated at 38◦ C for 18 h. Then, 200 μL of S. Heidelberg were transferred to new tubes containing 20 mL of BHI with 0.75% homogenized agar-agar poured on the respective dish for each isolates of Lactobacillus spp.
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QUORUM SENSING BETWEEN LACTOBACILLUS
After complete solidification, the dishes were incubated again at 38◦ C for 12 h. The inhibition halo formed starting from the edge of the Lactobacillus spp. colony was then measured. Autoinducer Production. The autoinducer was produced through the growth of L. plantarum in MRS broth and SH in BHI, both incubated at 38◦ C for 24 h. One milliliter of each sterile medium (MRS and BHI) was then mixed at a 1:1 proportion and seeded the samples of L. plantarum and S. Heidelberg, which were incubated at 38◦ C for 24 h. One milliliter was taken and seeded in 5 mL of MRS Nal/Rif to eliminate S. Heidelberg. The sample of L. plantarum was incubated again, this time without S. Heidelberg, in MRS broth to eliminate the antimicrobial residue (Nal/Rif). The sample to eliminate L. plantarum was then sterilized with a 0.22-μm cellulose membrane filter. A product without the autoinducer was obtained by incubating L. plantarum in MRS broth at 38◦ C for 24 h, with subsequent sterilization with a 0.22-μm cellulose membrane filter. This product was used as a negative control of the autoinducer, and was considered a L. plantarum supernatant, free of bacterial cells and autoinducers, produced before contact with Salmonella Heidelberg. Quorum Sensing Verification. The isolates of Lactobacillus spp. (Rumjanek et al., 2004) isolated were reincubated at 38◦ C for 24 h in MRS broth together with the autoinducer (1:1) and were submitted again to the spot on the lawn test for a comparison between the halos observed before and after contact with the autoinducer. Concomitantly, the spot on the lawn test was performed with the product without the autoinducer (L. plantarum supernatant), as a negative control.
In vivo Experiment Lactobacillus spp. Inoculum. The isolates of Lactobacillus spp. that presented inhibitory action against Salmonella Heidelberg in the spot on the lawn test before contact with the autoinducer were individually grown in 5 mL of MRS broth (5 colonies) at 38◦ C for 24 h and then mixed (equal volume), creating a Lactobacillus spp. inoculum with 1.8 × 108 colony-forming units (CFU)/mL. Experiment Design. A total of 75 one-day-old chicks obtained from a commercial hatchery were used, all male and belonging to the Cobb lineage. The birds were separated in 5 groups of 15, which were placed in wire cages and received water and feed ad libitum in an acclimatized environment. - Group (C): control (no treatment); - Group (L): treatment with a Lactobacillus spp. pool on the first day; - Group (A+): treatment with the autoinducer on the second day;
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- Group (LA): treatment with a Lactobacillus spp. pool on the first day and autoinducer on the second day. - Group (A–): treatment without autoinducer product (only L. plantarum supernatant). Negative control autoinducer. Challenge with Salmonella Heidelberg. On the third day, all groups were challenged with an inoculum of S. Heidelberg which was previously seeded in BHI broth and incubated at 38◦ C until a turbidity of 0.5 on the MacFarland scale (confirmed by plating and determining 2.5 × 108 CFU/mL). The inoculum was administered intraesophageally at a dosage of 1 mL per chick. Salmonella Heidelberg Count. The assessment of these treatments was conducted by counting the colonies of Salmonella Heidelberg in the ceca of 5 birds in each group at 3 different points in time: 24, 96 and 168 h after the challenge with S. Heidelberg. The birds were euthanized through cervical dislocation, and the ceca were aseptically removed, individually stored in sterile plastic containers, and kept in a refrigerated environment until being processed in the laboratory. After weighting, the ceca of each bird was macerated and diluted (1:10) in phosphate buffered saline, giving us the first dilution (10−1 ). Another 7 serial dilutions at the 1:10 proportion until dilution 10−8 were then obtained. All dilutions were seeded in petri dishes containing brilliant green agar in duplicate and then incubated at 38◦ C for 24 h.
Ethics Commission This experiment was approved by the Commission for Ethics on the Use of Animals—CEUA/FMVZ— UNESP, under Protocol Number 0115/2017.
RESULTS AND DISCUSSION In vitro Analysis All tested isolates of Lactobacillus spp. increased their inhibition potential in contrast to the inhibition halos observed in the spot on the lawn test before contact with the autoinducer, highlighting the expected quorum sensing between L. plantarum and the isolates of Lactobacillus spp. in the fight against Salmonella Heidelberg (Table 1). According to Khmel and Metlitskaya (2006), the autoinducers are responsible for cell-to-cell communication in bacteria and may induce the activation of genes responsible for producing bactericidal substances, such as the bacteriocins produced by some strains of Lactobacillus spp. The isolate of Lactobacillus spp. that presented the lowest increase in Salmonella Heidelberg inhibition was Lactobacillus spp. “G”, which presented an increase of 18% in the inhibition after contact with the
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In vivo Analysis
Table 1. Diameters of the inhibition halos observed in Lactobacillus spp. against Salmonella Heidelberg before and after quorum sensing (millimeters). Lactobacillus spp.
Measurement before AI1
A B C D E F G H I J K L M N O P Q
5.6 1.6 3.0 5.0 3.3 4.6 7.6 5.0 5.0 4.6 2.7 2.3 3.3 2.3 3.3 2.3 3.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.46 0.11b 0.05b 0.12b 0.11b 0.15b 0.15 0.05 0.06b 0.11b 0.11b 0.05b 0.06b 0.06b 0.12b 0.11b 0.15b
Measurement after AI 7.0 6.5 8.3 9.5 9.3 7.3 9.0 6.7 10.0 7.7 6.0 8.0 6.3 7.0 8.3 6.7 8.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.11 0.05a 0.11a 0.06a 0.05a 0.05a 0.05 0.11 1.1a 0.05a 0.11a 0.05a 0.05a 0.05a 0.11a 0.12a 0.15a
Toward the end of the 1960s, Antunes (2003) believed that bacteria, similarly to other unicellular organisms, presented individual behavior. Presently, we know about the collective behavior of these microorganisms, which allows them to act socially and synchronize the behavior of all members of a group, acting like multicellular entities and increasing their survival rate in highly complex environments (Bassler and Losick, 2006; Hooshangi and Bentley, 2008). Therefore, with an in vivo experiment, we can observe what really happens inside the birds and how their gut microbiota is modulated. All groups treated presented comparable results and were less colonized by Salmonella Heidelberg in contrast with the control group (Group C), especially 24 h after the challenge. Observing the results presented on Table 2, note that both the group treated only with autoinducer on the second day (Group A+) and the group treated with the Lactobacillus spp. pool on the first day and the autoinducer on the second day (Group LA) presented comparable results at all points in time to those observed on the group treated only with the Lactobacillus spp. pool on the first day (Group L). The S. Heidelberg count on these groups was lower than the one observed on the control group (Group C), which received no treatment, showing the efficiency of the autoinducer in controlling Salmonella Heidelberg under these experimental conditions. Despite the autoinducer not being related to Lactobacillus spp., it presented a similar action and revealed the existence of some substance responsible for communicating with other bacteria colonizing the birds that, somehow, promoted the inhibition of Salmonella Heidelberg. In this study, this can be inferred from the fact that the negative control group (Group A–) presented no such inhibition of Salmonella Heidelberg. This highlights the quorum sensing happening between the bacteria present in the normal gut microbiota, which results in the activation or suppression of the genes responsible for alterations in metabolic activity, morphological activity, motility and association/aggregation with other bacteria (Adak et al., 2011).
Negative control 5.6 1.8 3.0 5.5 3.8 4.8 7.6 5.3 5.5 5.0 2.9 2.5 3.3 2.3 3.5 2.5 3.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.55 0.12b 0.06b 0.13b 0.11b 0.55b 0.12 0.55 0.07b 0.12a,b 0.05b 0.06b 0.05b 0.05b 0.13b 0.12b 0.06b
Values are mean ± SEM. 1 Autoinducer. a,b Means followed by different lowercase letters in the same row are significantly different; Tukey’s multiple comparison test (P < 0.05).
autoinducer. On the other hand, the sample of L. spp. that presented the highest increase in S. Heidelberg inhibition after contact with the autoinducer was L. spp. “B”, which had its inhibition halo increased in 306%. This highlights that L. plantarum produces signaling substances that can stimulate the samples of Lactobacillus spp. to reach an inhibition increased against Salmonella Heidelberg after contact with the pathogen (Table 1). According to Khmel and Metlitskaya (2006), gene expression during quorum sensing depends on the bacterial population density, and the amount of autoinducers produced is directly proportional to the amount of bacteria. The production only starts after a minimal stimulation level is reached (Rutherford and Bassler, 2012). In this study, the minimum stimulation level of autoinducers or bacteria (data not shown) was verified. However, at a concentration of 108 CFU/mL of the medium L. plantarum could produce enough signaling substances to increase the inhibition of SH on the isolates of Lactobacillus spp. in vitro.
Table 2. Mean of log10 colony-forming units of Salmonella Heidelberg per gram of ceca at different collection times and different groups. Time (hours) PI1 No treatment Lactobacillus spp. pool Autoinducer Lactobacillus spp. pool + Autoinducer Negative control autoinducer
24 11.34 8.29 6.92 7.61 9.26
± ± ± ± ±
1.65a 0.56b,c 0.05c 0.05b,c 0.05a,b,c
96 (log10 CFU of SE/g ± SEM) 10.38 ± 0.97a 8.69 ± 1.12a,b 7.33 ± 1.65b 7.69 ± 0.08b 9.76 ± 0.99a
168 8.66 7.74 5.91 6.47 8.42
± ± ± ± ±
2.25a 1.33a,b 0.05b 0.05a,b 1.85a
Values are mean ± SEM. 1 PI—post-infection. a–c Means followed by different lowercase letters in the same column are significantly different; Tukey’s multiple comparison test (P < 0.05). Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey141/4967666 by Durham University user on 11 April 2018
QUORUM SENSING BETWEEN LACTOBACILLUS
According to Jayaraman and Wood (Jayaraman and Wood, 2008), the autoinducers produced by Grampositive bacteria are peptides synthesized by peptide precursors which are modified and exported by a transport protein, resulting in adenosine triphosphate expenditure. These results highlight the role of the autoinducer in the quorum sensing process, acting between bacteria in the highly complex and dynamic microenvironment of the gut microbiota. Therefore, a more detailed understanding regarding the processes involved and the composition of this substance is critically important for the development of new alternatives to antimicrobial agents. In the present study, quorum sensing happens between Lactobacillus spp. during the inhibition of Salmonella Heidelberg both in vitro and in vivo through the use of an autoinducer, which present results similar to those observed with the use of a probiotic agent. Younger birds have a developing intestinal microbiota in comparison to older animals (Lu et al., 2003). Under this light, the positive results obtained in this study in 1-day-old chicks may prove to be even better if applied to older birds since their microbiota are more dynamic, more complex, and more stable. Therefore, the usage of this autoinducer in adult broilers, while still needing further studies, may be considered a promising new strategy to deal with the problem of Salmonella Heidelberg in these birds.
ACKNOWLEDGMENTS This study was financed by FAPESP - Funda¸c˜ao de Amparo a` Pesquisa do Estado de S˜ ao Paulo (2017/17712-4).
REFERENCES Adak, S., L. Upadrasta, S. P. J. Kumar, R. Soni, and R. Banerjee. 2011. Quorum quenching – an alternative antimicrobial therapeutics. Pages 586–593 in Science Against Microbial Pathogens. A. Mendez-Vilas ed. Formatex Research Center, Badajoz, Spain. Associa¸c˜ao Brasileira de Prote´ına Animal – ABPA. 2016. Relat´ orio Anual 2016. Accessed Oct. 2016. http://abpa-br. com.br/setores/avicultura/publicacoes/relatorios-anuais/2016. Antunes, L. C. M. 2003. A linguagem. Ciˆencia hoje. 33:17–20. Bassler, B. L., and R. Losick. 2006. Bacterially Speaking. Cell. 125:237–246. ˆ 2000. Salmonellose Avi´ Berchieri Junior, A. aria. Pages 185–195 in A. Berchieri J´ unior, and M. Macari. Doen¸ca das aves. Campinas: Facta. Boyle, E. C., J. L. Bishop, G. A. Grassl, and B. B. Finlay. 2007. Salmonella: from pathogenesis to therapeutics. J. Bacteriol. 189:1489–1495.
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Cury, H. S. 2013. Avalia¸c˜ao de aditivos na a´gua de bebida para controle de Salmonella enterica subespecie enterica sorovar Heidelberg em frangos de corte. Msc. Diss. Ciˆencias Veterin´arias. Univ. Federal de Pelotas, Pelotas - Rio Grande do Sul. Da Silva, E. N., and R. L. Andreatti Filho. 2000. Pr´ obi´ oticos e prebi´ otico na avicultura. Proc. 2nd Simp´ osio de Sanidade Av´ıcola, Santa Maria, Rio Grande do Sul. Accessed Dec. 2016. http://www.cnpsa.embrapa.br/sgc/sgc publicacoes/anais9000 edir.pdf. Dubernet, S., N. Desmasures, and M. A. Gu´eguen. 2002. PCR- based method for identification of lactobacilli at the genus level. Microbiol. Let. 214:271–275. Hawver, L. A., S. A. Jung, and W. L. Ng. 2016. Specificity and complexity in bacterial quorum-sensing systems. Federaton of European Microbiological Societies. Microbiol. Rev. 40:738–752. Hooshangi, S., and W. E. Bentley. 2008. From unicellular properties to multicellular behavior: bacteria quorum sensing circuitry and applications. Curr. Opin. Biotechnol. 19:550–555. Jayaraman, A., and T. K. Wood. 2008. Bacterial quorum sensing: signals, circuits, and implications for biofilms and disease. Annu. Rev. Biomed. Eng. 10:145–167. Khmel, I. A., and A. Z. Metlitskaya. 2006. Quorum sensing regulation of gene expression: a promising target for drugs against bacterial pathogenicity. Mol. Biol. 40:169–182. Koneman, E. W., S. D. Allen, W. M. Janda, P. C. Schreckenberger, and W. C. Winn, Jr. 2011. Diagn´ ostioco Mocrobiol´ ogico. Pages 679–680 in Texto e Atlas Colorido. E. W. Koneman ed. Guanabara Koognan, Rio de Janeiro. Kuritza, L. N., P. Westphal, and E. Santin. 2014. Probi´ oticos na avicultura. Cienc. Rural. 44:1457–1465. Lu, J., U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee. 2003. Diversity and succession of the intestinal bacterial community of the maturing broiler chicken. Appl. Environ. Microbiol. 69:6816–6824 Miller, M. B., and B. L. Bassler. 2001. Quorum Sensing in bacteria. Annu. Rev. Microbiol. 55:165–199. Palmeira, A. L. B. 2007. Prevalˆencia e perfil de resistˆencia aos antimicrobianos dos sorovares de Salmonella sp isolados das carca¸cas de frango e peru na regi˜ ao sul do Brasil no per´ıodo de 2004 a 2006. Msc. Diss. Ciˆencias Veterin´aria. Univ. Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul. Parker, C. T., and V. Sperandio. 2009. Cell-to-cell signalling during pathogenesis. Cell Microbiol. 11:363–369. Ribeiro, A. R., A. Kellermann, L. R. Santos, and V. P. Nascimento. 2008. Resistˆencia antimicrobiana em Salmonella Enteritidis isoladas de amostras cl´ınicas e ambientais de frangos de corte e matrizes pesadas. Arq. Bras. Med. Vet. Zootec. 60:1259–1262. Rumjanek, N. G., M. C. C. Fonseca, and G. R. Xavier. 2004. Quorum Sensing em sistemas agr´ıcolas. Rev. Biotecnol. Ciˆenc. Desenvol. 33:35–50. Rutherford, S. T., and B. L. Bassler. 2012. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb. Perspect. Med. 2:1–26. Santos, J. R. G., and C. Gil-Turnes. 2005. Probi´ oticos em avicultura. Cienc. Rural. 35:741–747. Shinohara, N. K. S., V. B. Barros, S. M. C. Jimenez, E. C. L. Machado, R. A. F. Dutra, and J. L. Lima Filho. 2008. Salmonella spp., importante agente patogˆenico veiculado em alimentos. Ciˆenc. Sa´ ude Coletiva. 13:1675–1683. Silva, E. N., and A. Duarte. 2002. Salmonella Enteritidis em Aves: Retrospectiva no Brasil. Rev. Bras. Cienc. Avic. 4:85–100. Sola, M. C., A. P. Oliveira, J. C. Feistel, and C. S. M. Rezende. 2012. Mecanismo de Quorum Sensing e sua relevˆancia na microbiologia de alimentos. Enciclop´edia biosfera. 8:14–19.
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communication (quorum sensing) during the inhibition of SH by Lactobacillus spp., a spot on the lawn assay was conducted. For the in vivo, 75 one-day-old chicks were divided in 5 experimental groups: control with no treatment; treatment with a Lactobacillus spp. pool on the first day; treatment with autoinducer on the second day; treatment with Lactobacillus spp. on the first day; and autoinducer on the second day and treatment without autoinducer. The autoinducer was assessed through an SH count in the ceca of the birds. The autoinducer produced by the strain of L. plantarum proved to be efficient for communicating with the other Lactobacillus spp. isolates as previous contact with SH induced the production of an autoinducer capable of increasing inhibition of SH both in vitro (in average 132.73%) and in vivo, acting similarly to the Lactobacillus spp. pool (probiotic) by decreasing the SH count in the ceca (64%–24 h, 42%–96 h, and 46%–168 h).
Key words: autoinducer, chicken, microbiota, quorum sensing, probiotic 2018 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pey141
INTRODUCTION AND JUSTIFICATION
diseases and, despite technological breakthroughs, remains an important and global problem (Boyle et al., 2007). In 1993, Salmonella spp. emerged as a great health hazard for both poultry and humans in Brazil. The occurrence of asymptomatic carriers facilitates dissemination and increases the amount of contaminated food, proving the importance of this agent as a worldwide public health issue (Shinohara et al., 2008). An indiscriminate use of antibiotics in birds allowed the maintenance of batches positive for Salmonella spp., causing a marked increase in antimicrobial resistance and multiresistance (Palmeira, 2007; Ribeiro et al., 2008). Over 2,500 serovars are already known, among which paratyphi salmonellas are considerably important (Berchieri Junior, 2000), as they are not specific to birds and present a more serious health risk to the population by increasing the contamination of carcasses and eggs. Among these, Salmonella Heidelberg (SH) has been most widely reported in poultry worldwide, including Brazil, and is important due to how difficult it is to control and eradicate once it has colonized the bird (Cury, 2013). Aside from biosafety, alternative
Brazil has a rapidly growing poultry industry, as shown by the high production of chickens in 2016, reaching 13,146 million tons, awarding the country second place worldwide in overall production and first place in exports with 4,304 million tons (ABPA, 2016). This population increase in the country has created favorable conditions for the proliferation of several avian pathogens, particularly Salmonella spp. (Silva and Duarte, 2002), which is considered a food-safety pathogen. The occurrence of diseases transmitted by food has been widely discussed in the past few years due to the worldwide concern with strategies ensuring they are controlled and, therefore, the entry of safe products in the world market. Salmonella spp. is a gram-negative bacillus which usually moves through a flagellum. It is the most important agent involved in food-transmitted C 2018 Poultry Science Association Inc. Received December 18, 2017. Accepted March 21, 2018. 1 Corresponding author: [email protected]
1 Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey141/4967666 by Durham University user on 11 April 2018
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measures should be taken to control these bacteria, such as specific vaccination for laying and breeding hens, acquisition of batches free of the pathogen, use of organic acids and probiotics (Santos and Gil-Turnes, 2005). Probiotics are products composed of bacteria that are part of the bird’s natural microbiota and, therefore, are considered natural products, and do not present residues in animal products or stimulate resistance to drugs administered to humans (Da Silva and Andreatti Filho, 2000). An example of probiotic is Lactobacillus spp., gram-positive, and non-spore-forming bacteria whose shape may vary from a long and thin bacillus to a short coccobacillus. They are mostly motionless (Koneman et al., 2011) and induce the intestinal microbiota into equilibrium by producing several metabolites with antimicrobial properties and competing for binding sites with pathogenic bacteria. They also help immunomodulate the animal’s microbiota and aid nutrient absorption, allowing their use as enhancing agents by helping the birds in several unfavorable conditions (Da Silva and Andreatti Filho, 2000; Kuritza et al., 2014). One of the ways through which Lactobacillus spp. inhibit Salmonella spp. is through intraspecies and interspecies communication, termed quorum sensing. The bacteria note differences in the environment through signaling substances secreted by individual cells that are recognized by specific receptors in another bacterium, leading to a modification in their behavior (Hawver et al., 2016). This behavioral modification results in a collective response that modulates physiological activity in a positive way to survive, such as promoting change to a location with more nutrients and the formation of a biofilm granting protection against environmental actions; monitoring population density, gene expression, and virulence factors; producing bacteriocins with antibiotic activity; sporulation; and bioluminescence (Hawver et al., 2016). The bacteria also use this mechanism to produce, release, and recognize autoinducers that modulate these activities (Miller and Bassler, 2001; Parker and Sperandio, 2009; Sola et al., 2012). Some strains of Lactobacillus spp. produce substances with protein origin, such as bacteriocins. These substances can inhibit growth or deactivate other bacteria through the quorum sensing mechanism and, therefore, present antagonism against Salmonella spp. (Rumjanek et al., 2004). This study aims at assessing the ability of Lactobacillus spp. isolated from chicken to inhibit the multiplication of Salmonella Heidelberg through the quorum sensing mechanism both in vitro and in vivo.
MATERIALS AND METHODS In vitro Experiment The in vitro experiment consisted of proving the occurrence of quorum sensing between 17 isolates of
Lactobacillus spp. by evaluating the inhibition against Salmonella Heidelberg. For this purpose, a Spot on the Lawn test was conducted on a pure culture of Lactobacillus spp. isolates, both before and after an autoinducer produced by a strain of Lactobacillus plantarum that was previously in contact with Salmonella Heidelberg. Bacterial Samples. Salmonella Heidelberg—the sample of S. Heidelberg used in the study belongs to the Avian Pathology Service’s Bacteria Collection at Universidade Estadual Paulista—UNESP, Botucatu campus, obtained from the liver of 1-day-old chicks (Cobb). Lactobacillus spp.—the Lactobacillus spp. used as donor for the autoinducer belongs to species L. plantarum resistant to Rifamficin (Rif) and nalidixic acid (Nal) obtained from the Avian Pathology Service’s Bacteria Collection at Universidade Estadual Paulista— UNESP, Botucatu campus. The 17 isolates of Lactobacillus spp. used to assess quorum sensing in the inhibition of S. Heidelberg were isolated from a cloacal swab from 52-wk-old Cobb broilers. After collection, the swabs were cultivated in deMan Rogosa and Sharp (MRS) broth at 38◦ C for 24 h, and then seeded in MRS agar under the same growth conditions. Thirty characteristic Lactobacillus spp. colonies were stained by Gram’s method and the Gram-negative colonies were discarded. After this screening process, the isolates were identified through biochemical assays (catalase production, potassium hydroxide production, gas production through glucose fermentation and hydrogen sulfide production in Triple Sugar Iron). Genus identification was confirmed through polymerase chain reaction (PCR), and the DNA was extracted with a commercially available extraction kit (QiagenR ). The primers used were Forw R16-1 5 -CTT GTA CAC ACC GCC CGT CA-3 and Rev LbLMA15 -CTC AAA ACT AAA CAA AGT TTC -3 amplifying a 250-bp product (18). The reaction was comprised of 20 pmol of each primer, 12.5 μL of GoTaq Master Green, 2.5 μL of ultrapure water, and 5 μL of DNA, totaling 25 μL. Amplification was initiated at 95◦ C for 5 min, followed by 20 cycles at 95◦ C for 30 s, 55◦ C for 30 s, and 72◦ C for 30 s, followed by a final extension at 72◦ C for 7 min. The PCR products underwent electrophoresis in agarose gel at 1.4%, stained and viewed in an ultraviolet transilluminator. Spot on the Lawn Test. Among the 30 colonies possessing characteristics of genus Lactobacillus spp., 17 were identified and submitted to the spot on the lawn test so that the inhibition halo against S. Heidelberg could be assessed. The isolates of Lactobacillus spp. were cultivated individually at 38◦ C for 18 h in MRS agar (10 μL) as dots while the sample of S. Heidelberg was cultivated in brain heart infusion (BHI) broth and then incubated at 38◦ C for 18 h. Then, 200 μL of S. Heidelberg were transferred to new tubes containing 20 mL of BHI with 0.75% homogenized agar-agar poured on the respective dish for each isolates of Lactobacillus spp.
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After complete solidification, the dishes were incubated again at 38◦ C for 12 h. The inhibition halo formed starting from the edge of the Lactobacillus spp. colony was then measured. Autoinducer Production. The autoinducer was produced through the growth of L. plantarum in MRS broth and SH in BHI, both incubated at 38◦ C for 24 h. One milliliter of each sterile medium (MRS and BHI) was then mixed at a 1:1 proportion and seeded the samples of L. plantarum and S. Heidelberg, which were incubated at 38◦ C for 24 h. One milliliter was taken and seeded in 5 mL of MRS Nal/Rif to eliminate S. Heidelberg. The sample of L. plantarum was incubated again, this time without S. Heidelberg, in MRS broth to eliminate the antimicrobial residue (Nal/Rif). The sample to eliminate L. plantarum was then sterilized with a 0.22-μm cellulose membrane filter. A product without the autoinducer was obtained by incubating L. plantarum in MRS broth at 38◦ C for 24 h, with subsequent sterilization with a 0.22-μm cellulose membrane filter. This product was used as a negative control of the autoinducer, and was considered a L. plantarum supernatant, free of bacterial cells and autoinducers, produced before contact with Salmonella Heidelberg. Quorum Sensing Verification. The isolates of Lactobacillus spp. (Rumjanek et al., 2004) isolated were reincubated at 38◦ C for 24 h in MRS broth together with the autoinducer (1:1) and were submitted again to the spot on the lawn test for a comparison between the halos observed before and after contact with the autoinducer. Concomitantly, the spot on the lawn test was performed with the product without the autoinducer (L. plantarum supernatant), as a negative control.
In vivo Experiment Lactobacillus spp. Inoculum. The isolates of Lactobacillus spp. that presented inhibitory action against Salmonella Heidelberg in the spot on the lawn test before contact with the autoinducer were individually grown in 5 mL of MRS broth (5 colonies) at 38◦ C for 24 h and then mixed (equal volume), creating a Lactobacillus spp. inoculum with 1.8 × 108 colony-forming units (CFU)/mL. Experiment Design. A total of 75 one-day-old chicks obtained from a commercial hatchery were used, all male and belonging to the Cobb lineage. The birds were separated in 5 groups of 15, which were placed in wire cages and received water and feed ad libitum in an acclimatized environment. - Group (C): control (no treatment); - Group (L): treatment with a Lactobacillus spp. pool on the first day; - Group (A+): treatment with the autoinducer on the second day;
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- Group (LA): treatment with a Lactobacillus spp. pool on the first day and autoinducer on the second day. - Group (A–): treatment without autoinducer product (only L. plantarum supernatant). Negative control autoinducer. Challenge with Salmonella Heidelberg. On the third day, all groups were challenged with an inoculum of S. Heidelberg which was previously seeded in BHI broth and incubated at 38◦ C until a turbidity of 0.5 on the MacFarland scale (confirmed by plating and determining 2.5 × 108 CFU/mL). The inoculum was administered intraesophageally at a dosage of 1 mL per chick. Salmonella Heidelberg Count. The assessment of these treatments was conducted by counting the colonies of Salmonella Heidelberg in the ceca of 5 birds in each group at 3 different points in time: 24, 96 and 168 h after the challenge with S. Heidelberg. The birds were euthanized through cervical dislocation, and the ceca were aseptically removed, individually stored in sterile plastic containers, and kept in a refrigerated environment until being processed in the laboratory. After weighting, the ceca of each bird was macerated and diluted (1:10) in phosphate buffered saline, giving us the first dilution (10−1 ). Another 7 serial dilutions at the 1:10 proportion until dilution 10−8 were then obtained. All dilutions were seeded in petri dishes containing brilliant green agar in duplicate and then incubated at 38◦ C for 24 h.
Ethics Commission This experiment was approved by the Commission for Ethics on the Use of Animals—CEUA/FMVZ— UNESP, under Protocol Number 0115/2017.
RESULTS AND DISCUSSION In vitro Analysis All tested isolates of Lactobacillus spp. increased their inhibition potential in contrast to the inhibition halos observed in the spot on the lawn test before contact with the autoinducer, highlighting the expected quorum sensing between L. plantarum and the isolates of Lactobacillus spp. in the fight against Salmonella Heidelberg (Table 1). According to Khmel and Metlitskaya (2006), the autoinducers are responsible for cell-to-cell communication in bacteria and may induce the activation of genes responsible for producing bactericidal substances, such as the bacteriocins produced by some strains of Lactobacillus spp. The isolate of Lactobacillus spp. that presented the lowest increase in Salmonella Heidelberg inhibition was Lactobacillus spp. “G”, which presented an increase of 18% in the inhibition after contact with the
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In vivo Analysis
Table 1. Diameters of the inhibition halos observed in Lactobacillus spp. against Salmonella Heidelberg before and after quorum sensing (millimeters). Lactobacillus spp.
Measurement before AI1
A B C D E F G H I J K L M N O P Q
5.6 1.6 3.0 5.0 3.3 4.6 7.6 5.0 5.0 4.6 2.7 2.3 3.3 2.3 3.3 2.3 3.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.46 0.11b 0.05b 0.12b 0.11b 0.15b 0.15 0.05 0.06b 0.11b 0.11b 0.05b 0.06b 0.06b 0.12b 0.11b 0.15b
Measurement after AI 7.0 6.5 8.3 9.5 9.3 7.3 9.0 6.7 10.0 7.7 6.0 8.0 6.3 7.0 8.3 6.7 8.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.11 0.05a 0.11a 0.06a 0.05a 0.05a 0.05 0.11 1.1a 0.05a 0.11a 0.05a 0.05a 0.05a 0.11a 0.12a 0.15a
Toward the end of the 1960s, Antunes (2003) believed that bacteria, similarly to other unicellular organisms, presented individual behavior. Presently, we know about the collective behavior of these microorganisms, which allows them to act socially and synchronize the behavior of all members of a group, acting like multicellular entities and increasing their survival rate in highly complex environments (Bassler and Losick, 2006; Hooshangi and Bentley, 2008). Therefore, with an in vivo experiment, we can observe what really happens inside the birds and how their gut microbiota is modulated. All groups treated presented comparable results and were less colonized by Salmonella Heidelberg in contrast with the control group (Group C), especially 24 h after the challenge. Observing the results presented on Table 2, note that both the group treated only with autoinducer on the second day (Group A+) and the group treated with the Lactobacillus spp. pool on the first day and the autoinducer on the second day (Group LA) presented comparable results at all points in time to those observed on the group treated only with the Lactobacillus spp. pool on the first day (Group L). The S. Heidelberg count on these groups was lower than the one observed on the control group (Group C), which received no treatment, showing the efficiency of the autoinducer in controlling Salmonella Heidelberg under these experimental conditions. Despite the autoinducer not being related to Lactobacillus spp., it presented a similar action and revealed the existence of some substance responsible for communicating with other bacteria colonizing the birds that, somehow, promoted the inhibition of Salmonella Heidelberg. In this study, this can be inferred from the fact that the negative control group (Group A–) presented no such inhibition of Salmonella Heidelberg. This highlights the quorum sensing happening between the bacteria present in the normal gut microbiota, which results in the activation or suppression of the genes responsible for alterations in metabolic activity, morphological activity, motility and association/aggregation with other bacteria (Adak et al., 2011).
Negative control 5.6 1.8 3.0 5.5 3.8 4.8 7.6 5.3 5.5 5.0 2.9 2.5 3.3 2.3 3.5 2.5 3.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.55 0.12b 0.06b 0.13b 0.11b 0.55b 0.12 0.55 0.07b 0.12a,b 0.05b 0.06b 0.05b 0.05b 0.13b 0.12b 0.06b
Values are mean ± SEM. 1 Autoinducer. a,b Means followed by different lowercase letters in the same row are significantly different; Tukey’s multiple comparison test (P < 0.05).
autoinducer. On the other hand, the sample of L. spp. that presented the highest increase in S. Heidelberg inhibition after contact with the autoinducer was L. spp. “B”, which had its inhibition halo increased in 306%. This highlights that L. plantarum produces signaling substances that can stimulate the samples of Lactobacillus spp. to reach an inhibition increased against Salmonella Heidelberg after contact with the pathogen (Table 1). According to Khmel and Metlitskaya (2006), gene expression during quorum sensing depends on the bacterial population density, and the amount of autoinducers produced is directly proportional to the amount of bacteria. The production only starts after a minimal stimulation level is reached (Rutherford and Bassler, 2012). In this study, the minimum stimulation level of autoinducers or bacteria (data not shown) was verified. However, at a concentration of 108 CFU/mL of the medium L. plantarum could produce enough signaling substances to increase the inhibition of SH on the isolates of Lactobacillus spp. in vitro.
Table 2. Mean of log10 colony-forming units of Salmonella Heidelberg per gram of ceca at different collection times and different groups. Time (hours) PI1 No treatment Lactobacillus spp. pool Autoinducer Lactobacillus spp. pool + Autoinducer Negative control autoinducer
24 11.34 8.29 6.92 7.61 9.26
± ± ± ± ±
1.65a 0.56b,c 0.05c 0.05b,c 0.05a,b,c
96 (log10 CFU of SE/g ± SEM) 10.38 ± 0.97a 8.69 ± 1.12a,b 7.33 ± 1.65b 7.69 ± 0.08b 9.76 ± 0.99a
168 8.66 7.74 5.91 6.47 8.42
± ± ± ± ±
2.25a 1.33a,b 0.05b 0.05a,b 1.85a
Values are mean ± SEM. 1 PI—post-infection. a–c Means followed by different lowercase letters in the same column are significantly different; Tukey’s multiple comparison test (P < 0.05). Downloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey141/4967666 by Durham University user on 11 April 2018
QUORUM SENSING BETWEEN LACTOBACILLUS
According to Jayaraman and Wood (Jayaraman and Wood, 2008), the autoinducers produced by Grampositive bacteria are peptides synthesized by peptide precursors which are modified and exported by a transport protein, resulting in adenosine triphosphate expenditure. These results highlight the role of the autoinducer in the quorum sensing process, acting between bacteria in the highly complex and dynamic microenvironment of the gut microbiota. Therefore, a more detailed understanding regarding the processes involved and the composition of this substance is critically important for the development of new alternatives to antimicrobial agents. In the present study, quorum sensing happens between Lactobacillus spp. during the inhibition of Salmonella Heidelberg both in vitro and in vivo through the use of an autoinducer, which present results similar to those observed with the use of a probiotic agent. Younger birds have a developing intestinal microbiota in comparison to older animals (Lu et al., 2003). Under this light, the positive results obtained in this study in 1-day-old chicks may prove to be even better if applied to older birds since their microbiota are more dynamic, more complex, and more stable. Therefore, the usage of this autoinducer in adult broilers, while still needing further studies, may be considered a promising new strategy to deal with the problem of Salmonella Heidelberg in these birds.
ACKNOWLEDGMENTS This study was financed by FAPESP - Funda¸c˜ao de Amparo a` Pesquisa do Estado de S˜ ao Paulo (2017/17712-4).
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