Epithelial cell adhesion and gastrointestinal colonization of in poultry

Megan A. Spivey , Sadie L. Dunn-Horrocks , and Tri Duong 1

Department of Poultry Science, Texas A&M University, College Station 77843-2472

ABSTRACT Administration of probiotic Lactobacillus 78. However, when colonization was evaluated in the cultures is an important alternative to the use of an- ileum and cecum of broiler chicks, L. crispatus TDCC Downloaded from https://academic.oup.com/ps/article-abstract/93/11/2910/2730493 by guest on 13 November 2018 tibiotic growth promoters and has been demonstrated 75 and L. gallinarum TDCC 77 were more persistent to improve animal health, growth performance, and than L. crispatus TDCC 76 and L. gallinarum TDCC preharvest food safety in poultry production. Where- 78. The reduction of growth in medium supplemented as gastrointestinal colonization is thought to be criti- with oxgal was greater for L. gallinarum TDCC 78 than cal to their probiotic functionality, factors important L. gallinarum TDCC 77, suggesting that whereas adhe- to Lactobacillus colonization in chickens are not well sion was similar for the 2 strains, the difference in colo- understood. In this study we investigate epithelial cell nization between L. gallinarum strains may be due in adhesion in vitro and colonization of Lactobacillus in part to their bile sensitivity. This study demonstrates vivo in broiler chickens. Adhesion of Lactobacillus cul- that whereas adhesion to epithelial cells may be impor- tures to epithelial cells was evaluated using the chicken tant in predicting gastrointestinal colonization, other LMH cell line. Lactobacillus cultures were able adhere factors including bile tolerance may also contribute to effectively to LMH cells relative to Bacillus subtilis and the colonization of Lactobacillus in poultry. Addition- Salmonella Typhimurium. Epithelial cell adhesion was ally, the chicken LMH cell line is expected to provide a similar for TDCC 75, L. cristpa- platform for investigating mechanisms of Lactobacillus tus TDCC 76, and Lactobacillus gallinarum TDCC 77, adhesion to epithelial tissue and evaluating the probi- and all 3 were more adherent than L. gallinarum TDCC otic potential Lactobacillus in poultry.

Key words: Lactobacillus , probiotic , adhesion , colonization , LMH cell 2014 Poultry Science 93 :2910–2919 http://dx.doi.org/10.3382/ps.2014-04076

INTRODUCTION istration has also been shown to reduce colonization by Campylobacter (Ghareeb et al., 2012; Neal-McKinney Lactobacillus species are common inhabitants of the et al., 2012), Clostridium (La Ragione et al., 2004), and gastrointestinal tract and are often used as probiot- Salmonella (Chen et al., 2012; Ghareeb et al., 2012), ics due to their health promoting properties (Klaen- improving the microbial food safety of poultry. hammer et al., 2008). Probiotics, sometimes called Colonization and persistence in the gastrointestinal direct-fed microbials when used in livestock animals tract are thought to be critical for the realization of (Sanders, 2008), are often seen as alternatives to the probiotic benefits (Bernet et al., 1994; Mack et al., use of antibiotic growth promoters in poultry produc- 1999) and serve as important criteria for the selection of tion (Patterson and Burkholder, 2003; o’Bryan et al., probiotic microorganisms (Klaenhammer et al., 2005). 2008; Huyghebaert et al., 2011). The administration of Understanding how Lactobacillus species colonize chick- probiotic lactobacilli has been demonstrated to stimu- ens will be important in the selection of more effec- late immune responses (Haghighi et al., 2005; Sato et tive probiotic cultures and their application in poultry al., 2009; Brisbin et al., 2011), improve digestive health production. While the ability of probiotic lactobacilli (Gusils et al., 1999; Kim et al., 2012), and improve to colonize the gastrointestinal tract of poultry is mul- growth performance (Loh et al., 2010; Shim et al., 2012; tifactorial, adherence to epithelial cells is thought to Askelson et al., 2014) in poultry. Lactobacillus admin- contribute to colonization. The ability of Lactobacillus species to adhere to epi- thelial tissues and colonize poultry has been reported © 2014 Poultry Science Association Inc. (Jin et al., 1996; Edelman et al., 2002; Bouzaine et al., Received April 1, 2014. Accepted August 8, 2014. 2005). However, microbial factors important to gastro- 1 Corresponding author: [email protected] intestinal persistence of Lactobacillus in poultry are not

2910 LACTOBACILLUS ADHESION AND COLONIZATION 2911 well characterized. Aggregation, cell wall hydrophobic- try (Larson et al., 2008). The LMH cell line has also ity, and adhesion to extracellular components including been used to identify genes important in cellular inva- intestinal mucus, fibronectin, and basement membrane sion by Salmonella Enteritidis (Shah et al., 2012) and matrix have been used as in vitro measures of the ad- characterize the association of Clostridium perfringens herence of Lactobacillus strains to poultry epithelia NetB toxin to necrotic enteritis in chickens (Keyburn et (Edelman et al., 2002; Ehrmann et al., 2002; Gusils et al., 2008). Thus, utility of the chicken LMH epithelial al., 2003; Bouzaine et al., 2005; Taheri et al., 2009; Ro- cell line for the investigation of host-microbe interac- cha et al., 2012). Additionally, dissected tissue sections tions in the gastrointestinal tract of poultry has been (Fuller and Brooker, 1974; Edelman et al., 2002), pri- well established. mary chicken intestinal epithelial cells (Jin et al., 1996; In this study, we evaluated the adhesion of poultry- Garriga et al., 1998; Lin et al., 2007), and the HEp- derived strains of Lactobacillus crispatus and Lactoba- 2 (Ehrmann et al., 2002) and Caco-2 (Messaoudi et cillus gallinarum to the LMH epithelial cell line in vitro Downloaded from https://academic.oup.com/ps/article-abstract/93/11/2910/2730493 by guest on 13 November 2018 al., 2012) human epithelial cell lines have been used to and their colonization in vivo in broiler chicks to gain evaluate the potential of Lactobacillus cultures as poul- a better understanding of bacterium-host interactions try probiotics. Whereas cultured epithelial cell lines in chickens. are the most widely applied models for evaluating the adhesion of microorganisms (Ouwehand and Salminen, MATERIALS AND METHODS 2003), adherence of Lactobacillus cultures to a poultry- specific epithelial cell line has not been reported previ- Bacterial Strains ously. The lack of a species-specific cell culture model has been an obstacle to investigations of Lactobacillus The bacterial strains used in this study are listed adhesion and its contribution to gastrointestinal coloni- in Table 1. Lactobacillus strains were cultured using zation in poultry. deMan, Rogosa, and Sharpe (MRS) medium (Difco, The chicken LMH epithelial cell line (Kawaguchi et Franklin Lakes, NJ) in 10% CO2 at 37°C with rifampi- al., 1987), derived from a hepatocellular carcinoma, cin (Rif; EMD Chemicals Inc., San Diego, CA) added represents a cell line potentially useful for the investi- when appropriate. Salmonella Typhimurium and Ba- gation of probiotic functionality in poultry. The LMH cillus subtilis were cultured using tryptic soy medium cell line has been used previously as an in vitro model (TSA/TSB; Difco) aerobically at 37°C. For adhesion to investigate mechanisms of Campylobacter jejuni colo- assays, 18 h cultures of were harvested by cen- nization in poultry. Competitive inhibition between C. trifugation, washed 3 times with PBS, and subsequent- jejuni F38011 and C. jejuni 02–833L for adherence to ly resuspended in PBS to achieve the desired concen- gastrointestinal epithelia was demonstrated using the tration for inoculation onto LMH cells. chicken LMH epithelial cell line and verified in broiler chicks in vivo by Konkel et al. (2007). Additionally, Selection of Rifampicin-Resistant LMH cells were used to demonstrate that the adhesins Lactobacillus Cultures CadF and FlpA contribute to C. jejuni colonization in broiler chicks by mediating adherence to epithelial cells To generate rifampicin resistant (Rifr) cultures for (Flanagan et al., 2009), that autoinducer-2 expression use in in vitro adhesion and in vivo colonization assays, contributes to C. jejuni colonization by regulating ad- Lactobacillus cultures were serially transferred in MRS hesion to host tissues (Quinones et al., 2009), and that broth supplemented with up to 100 µg∙mL−1 Rif and the cytokine response to C. jejuni in chicken compared subsequently plated onto MRS agar supplemented with −1 with humans contributes to its commensalism in poul- 100 µg∙mL Rif. Following incubation in 10% CO2 at

Table 1. Bacterial strains used in this study

Strain Relevant characteristic Source or reference Lactobacillus crispatus ST1 Chicken crop isolate Ojala et al., 2010 JCM 5810 Chicken fecal isolate JCM1 TDCC 75 Rifr variant of L. crispatus ST1 This study TDCC 76 Rifr variant of L. crispatus JCM 5810 This study Lactobacillus gallinarum ATCC 33199T Chicken crop isolate, type strain ATCC2 JCM 8782 Chicken fecal isolate JCM TDCC 77 Rifr variant of L. gallinarum ATCC 33199 This study TDCC 78 Rifr variant of L. gallinarum JCM 8782 This study Bacillus subtilis ATCC 23857 Strain 168, genome sequenced variant ATCC Salmonella Typhimurium USDA-ARS,3 primary poultry isolate Byrd et al., 1998 1JCM = Japan Collection of Microorganisms. 2ATCC = American Type Culture Collection. 3ARS = Agricultural Research Service. 2912 Spivey et al. 37°C, resistant colonies were selected and their Rif re- the Texas A&M University Institutional Animal Care sistance was confirmed before use in this study. and Use Committee.

Culture of LMH Cells Lactobacillus Broiler Chick Chicken LMH hepatocellular carcinoma epithelial Colonization Assay cells (ATCC CRL-2117) were cultured in flasks coated Lactobacillus crispatus TDCC 75 (ST1-Rifr), L. cris- with 0.1% gelatin (Sigma Chemical Co., St. Louis, MO) patus TDCC 76 (JCM 5810-Rifr), L. gallinarum TDCC using Waymouth’s MB 752/1 medium (Life Technolo- 77 (ATCC 33199-Rifr), and L. gallinarum TDCC 78 gies, Grand Island, NY) supplemented with 10% fetal (JCM 5810-Rifr) were cultured in MRS broth supple- bovine serum (Atlanta Biologicals, Lawrenceville, GA). mented with Rif (100 µg∙mL−1) and incubated for 18 h

The cells were maintained in a humidified 5% CO2 in- in 10% CO2 at 37°C. Cultures were centrifuged at 700 Downloaded from https://academic.oup.com/ps/article-abstract/93/11/2910/2730493 by guest on 13 November 2018 cubator at 37°C. × g for 5 min at 20°C, washed 3 times with PBS to remove Rif, and resuspended in PBS. On day of hatch Lactobacillus-LMH Cell Binding Assay and 3 d posthatch, broiler chicks were administered 0.5 mL of Lactobacillus (108 cfu) culture suspended in PBS Adhesion of Lactobacillus cultures to LMH cells was by oral gavage. A mock inoculated group was admin- evaluated using methods adapted from Konkel et al. istered sterile PBS. After inoculation, the remaining (2007). Unless otherwise stated, assays were performed bacterial suspensions were enumerated using MRS agar as follows. Gelatin coated (0.1%) 24-well tissue culture supplemented with Rif (100 µg∙mL−1) to confirm the plates were seeded with 1.5 × 105 LMH cells per well count (cfu) of Lactobacillus in each dose. At 4, 6, and 8 and incubated for 18 h at 37°C in a humidified 5% CO2 d posthatch, 5 chicks from each group were euthanized incubator. The LMH cells were rinsed 3 times with PBS by CO2 asphyxiation and necropsied to evaluate gas- and inoculated with approximately 1.5 × 107 cfu bac- trointestinal colonization of poultry. At each sampling, teria suspended in PBS (100:1 bacteria per LMH cell). a section of ileum and a cecum were dissected from Plates were centrifuged at 600 × g for 5 min at 20°C to each chick and placed in separate Whirlpak bags. Gas- promote bacterium-host cell contact and incubated for trointestinal specimens were weighed, thoroughly ho- 30 min at 37°C in a humidified 5% CO2 incubator. Non- mogenized, diluted serially, and plated onto MRS agar adherent bacteria were removed by rinsing the wells 5 and MRS agar supplemented with Rif (100 µg∙mL−1) times with PBS, and LMH cells were lysed with 0.1% for enumeration of total (LAB) and Triton X-100 (Sigma Chemical Co.) in PBS. Bacterial Rifr Lactobacillus, respectively. The number of viable −1 suspensions were diluted in PBS and enumerated using bacteria recovered was reported as mean log10 cfu∙g the appropriate solid medium. To develop standard as- ± SEM gastrointestinal contents from 5 broiler chicks. say conditions, cultures of Lactobacillus crispatus ST1 Gastrointestinal samples in which Rifr Lactobacillus were used to evaluate the effects of various assay condi- was not detected were assigned the lower limit of de- −1 tions using the above methodology while varying the tection, 2 log10 cfu∙g . Lactobacillus colonization over number of postincubation washes and the count (cfu) of time was analyzed using a 2-factor ANOVA for strain bacteria per LMH cell. Bacillus subtilis and Salmonella and day. Significantly different means (P ≤ 0.05) were Typhimurium were used as low and high adhering con- separated using Duncan’s multiple range test. trols, respectively. Lactobacillus cultures were enumer- ated using MRS agar, whereas B. subtilis and Salmonel- Bile Tolerance Assay la were enumerated using TSA. For each experimental condition or strain evaluated, bacterial counts from 4 Growth curve analysis was used to evaluate bile tol- replicate wells from 3 independent assays were log10 erance (O’Flaherty and Klaenhammer al., 2010) of L. transformed and analyzed using a 2-factor ANOVA for crispatus TDCC 75 (Rifr ST1), L. crispatus TDCC 76 experimental treatment and assay. Significantly differ- (Rifr JCM 5810), L. gallinarum TDCC 77 (Rifr ATCC ent means (P ≤ 0.05) were separated using Duncan’s 33199), and L. gallinarum TDCC 78 (Rifr JCM 8782). multiple range test. Lactobacillus cultures were inoculated into triplicate wells of a 96-well plate containing 200 µL of MRS broth Broiler Chicks supplemented with 0.0, 0.1, 0.25, 0.5, and 1.0% (wt/ vol) oxgall (Difco). These concentrations were selected Male broiler chicks (Ross × Ross) were obtained from to emulate the 0.1 to 1% bile concentration range found a commercial hatchery on day-of-hatch and randomly in the poultry gastrointestinal tract, which have been assigned to 1 of 5 treatment groups of 15 birds each. reported to be approximately 0.25% in the ileum and Each group of chickens was reared in separate battery 0.1% in the cecum (Green and Kellogg, 1987; Lin et brooder pens. Water and chick starter feed were provid- al., 2003; Iwata et al., 2013). Wells containing sterile ed ad libitum. All of the experimental procedures were medium (uninoculated) were used for blank correction. performed in compliance with protocols approved by The cultures were incubated under microaerobic con- LACTOBACILLUS ADHESION AND COLONIZATION 2913 ditions at 37°C, and the absorbance (optical density LMH cells than B. subtilis and less adherent than Sal- at 600 nm; OD600nm) was recorded at 15-min inter- monella. Lactobacillus crispatus TDCC 76 was the most vals for 24 h using a Spectrafluor Plus (Tecan Systems weakly adherent of the lactobacilli, but no difference in Inc., San Jose, CA) microtiter plate reader. Maximum the number of viable adherent bacteria was observed growth rate (µmax) was determined by fitting blank- between the remaining Lactobacillus cultures. Within corrected growth curves to a modified Gompertz model each species, L. crispatus TDCC 75 adhered more ef- (Zwietering et al., 1990). Growth rates were reported fectively to the LMH cells than L. crispatus TDCC 76, as the mean µmax ± SEM from 3 independent wells. whereas there was no difference between the 2 L. gal- Growth rate of Lactobacillus cultures was analyzed us- linarum strains. ing a 2-factor ANOVA for strain and treatment. Sig- nificant differences (P ≤ 0.05) in growth rate between Lactobacillus Colonization of Broiler Chicks strains from the same species for each treatment were Downloaded from https://academic.oup.com/ps/article-abstract/93/11/2910/2730493 by guest on 13 November 2018 determined using a t-test with Bonferroni correction. Colonization of broiler chickens by L. crispatus TDCC 75(Rifr ST1), L. crispatus TDCC 76 (Rifr JCM r RESULTS 5810), L. gallinarum TDCC 77 (Rif ATCC 33199), or L. gallinarum TDCC 78 (Rifr JCM 8782) was evalu- Evaluation of Factors Affecting the Adhesion of Lactobacillus to Chicken LMH Cells Lactobacillus crispatus ST1 was used to evaluate the effect of the number of postincubation washes (Figure 1A) and dose (cfu bacteria per LMH cell; Figure 1B) on the number of adherent bacteria. After incubating L. crispatus ST1 with LMH cells, individual wells were washed 0, 1, 3, 5, or 8 times before being enumerat- ed. The number of viable-adherent bacteria decreased with each successive wash up to 5 total washes but did not significantly decrease from 5 to 8 washes. Thus, 5 postincubation washes were sufficient to remove the nonadherent bacteria from chicken LMH cells. Lacto- bacillus cultures were co-incubated for with LMH cells at ratios of 100:1, 250:1, 500:1, and 1,000:1 cfu bacte- ria per LMH cell. The number of adherent bacteria in- creased with the count (cfu) per cell. However, the per- centage of viable adherent bacteria did not change with increasing dose. Thus, the adhesion of L. crispatus ST1 to LMH cells was determined to be dose dependent and linear within the range evaluated. Based on these data, Lactobacillus cultures were incubated with LMH cells at a ratio of 100 cfu per LMH cell and washed 5 times before enumeration to determine the number of viable adherent bacteria in the subsequent assays. These rep- resent a standard set of conditions for in vitro adhesion assays using the chicken LMH epithelial cell line.

Adhesion of Lactobacillus Cultures to Chicken LMH Cells The adhesion Lactobacillus cultures to chicken LMH cells was evaluated (Figure 2). In preliminary assays Figure 1. Evaluation of assay conditions. Adhesion of Lactobacil- evaluating the adhesion of B. subtilis and Salmonella lus crispatus ST1 to chicken LMH epithelial cells was evaluated under varying assay conditions. (A) L. crispatus cultures were incubated with Typhimurium to LMH cells, these cultures were consis- LMH cells (100 cfu per LMH cell) and washed 0, 1, 3, 5, and 8 times 5 7 tently observed to adhere at approximately 10 and 10 before enumeration. (B) L. crispatus cultures were added to LMH cells cfu per well, respectively (data not shown). Thus, B. using ratios of 100:1, 250:1, and 500:1, and 1,000:1 cfu per LMH cell. subtilis was selected as a low-adherent and Salmonella The geometric mean ± 95% CI adherent bacteria (bars) per well and percent adherent bacteria ± SEM (■) of inoculum from 4 replicate Typhimurium was selected as a high-adherent control wells from 3 independent assays are shown. Different letters (a–d) culture. Lactobacillus cultures were more adherent to indicate that means (adhesion) are significantly different (P ≤ 0.05). 2914 Spivey et al. Bile Tolerance of Lactobacillus Cultures Bile tolerance was evaluated by determining the µmax of Lactobacillus strains cultured in MRS broth supplemented with increasing concentrations of oxgall. The growth of all 4 Lactobacillus strains was reduced as oxgall concentration increased. Although the growth rate of the L. crispatus strains was greater than the L. gallinarum strains, there was no difference in growth rate between stains of the same Lactobacillus species when cultured in MRS broth alone. However, when the lactobacilli were cultured in 0.1 and 0.25% oxgall, the growth rate of L. crispatus TDCC 75 was greater than Downloaded from https://academic.oup.com/ps/article-abstract/93/11/2910/2730493 by guest on 13 November 2018

Figure 2. Adhesion of Lactobacillus cultures to chicken LMH epi- thelial cells. The adherence of Rifr Lactobacillus crispatus ST1 (TDCC 75), L. crispatus JCM 5810 (TDCC 76), Lactobacillus gallinarum ATCC 33199 (TDCC 77), and L. gallinarum JCM 8782 (TDCC 78) to chicken LMH epithelial cells was evaluated. Bacillus subtilis ATCC 23857 and Salmonella Typhimurium were used as low- and high-ad- herent controls, respectively. The geometric mean ± 95% CI adherent bacteria (bars) per well and percent adherent bacteria ± SEM (■) of inoculum from 4 replicate wells from 3 independent assays are shown. Different letters (a–d) indicate that means (adhesion) are significantly different (P ≤ 0.05). ated. The recovery of viable Rifr Lactobacillus in our time course study showed the administered Lactoba- cillus cultures transiently colonized the ileum and ce- cum of broiler chickens (Figure 3). The numbers of Rifr Lactobacillus recovered from the cecum and intestine of the inoculated groups was similar at 4 d posthatch and decreased over the time-course of our study. Lac- tobacillus gallinarum TDCC 77 was the most persistent colonizer, followed by L. crispatus TDCC 75, having been recovered both from the gastrointestinal contents of a greater number of birds and at higher numbers than L. crispatus TDCC 76 and L. gallinarum TDCC 78 at each time point. There was no difference observed between L. crispatus TDCC 76 and L. gallinarum JCM TDCC 78. Although there were some differences at in- dividual time points, administration of Lactobacillus cultures did not appear to affect the number of total LAB recovered from the gastrointestinal specimens of broiler chickens overall (Table 2). However, L. crispatus TDCC 75 and L. gallinarum TDCC 77 did comprise a larger proportion of the total LAB at 6 d posthatch in the intestine and 6 and 8 d posthatch in the cecum as compared with the other lactobacilli. Additionally, Figure 3. Gastrointestinal colonization of poultry by Lactobacillus. r counts of Rif Lactobacillus, when recovered, and total Viable Rifr Lactobacillus were enumerated from the (A) ileum and (B) LAB were generally higher in the cecum than the il- cecum of broiler chicks administered Lactobacillus crispatus TDCC r 75 (●), L. crispatus TDCC 76 (○), Lactobacillus gallinarum TDCC eum. When Rif LAB were recovered from mock inocu- 77 (■), L. gallinarum TDCC 78 (□), or a mock inoculation (▲). Tis- lated broiler chicks, they were recovered near the limit sue samples were collected at 4-, 6-, and 8 d posthatch. Lactobacillus −1 of detection (2 log10 cfu g ), representing the natural colonization of broiler chicks is reported as the mean log10 ± SEM cfu∙g−1 recovered from 5 chicks sampled at each time point (limit of background level of Rif resistance. Thus our contain- −1 detection, 2 log10 cfu∙g ). Bracketed data points with different letters ment procedures were sufficient to prevent cross-con- (a,b) at each time point indicate that means are significantly different tamination over the course of the study. (P ≤ 0.05). LACTOBACILLUS ADHESION AND COLONIZATION 2915

Table 2. Enumeration of total lactic acid bacteria (LAB) from broiler chickens

Lactobacillus Lactobacillus Mock crispatus L. crispatus gallinarum L. gallinarum Treatment inoculated TDCC 75 TDCC 76 TDCC 77 TDCC 78 Ileal specimen −1 1 Total LAB (log10 cfu∙g ± SEM) d 42 7.60 ± 0.24 8.00 ± 0.28 7.20 ± 0.37 8.11 ± 0.25 8.51 ± 0.20 d 6 8.85 ± 0.22a 9.23 ± 0.12a 7.67 ± 0.30b 9.67 ± 0.22a 8.66 ± 0.53a d 8 8.51 ± 0.89 7.35 ± 0.37 7.47 ± 0.35 8.23 ± 0.23 7.26 ± 0.21 % Rifr of total LAB d 4 26.4 ± 0.8b 73.3 ± 8.7a 68.3 ± 8.2a 82.4 ± 2.8a 56.7 ± 9.8a d 6 23.7 ± 0.9b 55.9 ± 5.0a 29.5 ± 2.8b 65.9 ± 4.3a 31.8 ± 5.2b d 8 24.6 ± 2.8 37.3 ± 3.5 28.3 ± 1.8 55.0 ± 9.3 27.7 ± 0.8 Rifr LAB-positive chicks3 (n) d 4 1 5 5 5 5 Downloaded from https://academic.oup.com/ps/article-abstract/93/11/2910/2730493 by guest on 13 November 2018 d 6 3 5 1 5 5 d 8 0 3 1 5 1 Cecal specimen −1 Total LAB (log10 cfu∙g ± SEM) d 4 9.83 ± 0.22 9.42 ± 0.14 9.02 ± 0.29 9.09 ± 0.44 9.70 ± 0.07 d 6 9.15 ± 0.24a 7.88 ± 0.24b 9.08 ± 0.13a 7.78 ± 0.24b 9.22 ± 0.27a d 8 8.42 ± 0.17c 8.77 ± 0.21b 8.88 ± 0.46b 9.41 ± 0.06a 8.96 ± 0.08b % Rifr of total LAB d 4 23.4 ± 1.6b 74.8 ± 6.8a 76.5 ± 3.6a 88.4 ± 7.8a 65.4 ± 6.1a d 6 24.8 ± 3.4b 85.7 ± 9.1a 36.8 ± 5.7b 99.0 ± 2.7a 31.0 ± 4.9b d 8 23.8 ± 0.5ab 53.0 ± 8.3a 24.0 ± 1.0b 57.1 ± 10.3a 22.3 ± 0.2b Rifr LAB-positive chicks (n) d 4 4 5 5 5 5 d 6 2 5 5 5 4 d 8 1 4 3 5 2 a–cDifferent superscripts within rows indicate that means differ significantly (P ≤ 0.05). 1 −1 Limit of detection, 2 log10 cfu∙g . 2Days posthatch. 3 r −1 Number of chicks from which Rif LAB were recovered ≥2 log10 cfu∙g .

L. crispatus TDCC 76 (Figure 4A), and the growth rate Quinones et al., 2009) and pathogenesis of C. perfrin- of L. gallinarum TDCC 77 was greater than that of L. gens (Keyburn et al., 2008) and Salmonella Enteritidis gallinarum TDCC 78 (Figure 4B). There was no differ- (Shah et al., 2012) in poultry. Finally, the LMH cell line ence in growth rate between strains of the same spe- was selected to better reflect the behavior of Lactobacil- cies at 0.5% oxgall or greater. Because the reduction in lus cultures in the digestive tract of chickens (Konkel growth rate was greater at lower oxgall concentrations et al., 2007). for L. crispatus TDCC 76 and L. gallinarum TDCC 78, Lactobacillus crispatus ST1 was originally isolated they are more sensitive to bile than the other strain from the crop of a chicken (Edelman et al., 2002) and from their respective species. has been demonstrated to adhere to frozen epithelial tis- sue sections from the gastrointestinal tract of chickens DISCUSSION and to inhibit adhesion of avian pathogenic Escherichia coli to intestinal surfaces and ileal mucus of chickens The goal of this study was to investigate epithelial (Edelman et al., 2003). The availability of a complete cell adhesion and its potential contribution to gastro- genome sequence (Ojala et al., 2010) has allowed the intestinal colonization of Lactobacillus in poultry. Al- characterization of several potentially important adhe- though previous studies have evaluated cellular adhe- sive proteins in vitro. Lactobacillus crispatus ST1 Lac- sion of Lactobacillus cultures for use in poultry using tobacillus epithelium adhesin (LEA) has been shown to human cell lines (Ehrmann et al., 2002; Lin et al., 2007; bind stratified squamous epithelium of frozen chicken Messaoudi et al., 2012), we are unaware of any studies crop sections and mediate binding of L. crispatus ST1 in which the binding potential of Lactobacillus cultures to the VK2/E6E7 human vaginal cell line (Edelman et has been assessed using an epithelial cell line of chicken al., 2012). Additionally, the extracellular enzymes eno- origin. Although, the LMH chicken epithelial cell line lase, glucose-6-phosphate isomerase, glutamine synthe- was derived from the liver (Kawaguchi et al., 1987), its tase, and glyceraldehyde-3-phosphate dehydrogenase utility and in vivo relevance as an in vitro model for have been demonstrated to bind murine and human host-microbe interactions in the gastrointestinal tract extracellular matrix proteins (Hurmalainen et al., 2007; of poultry has been established previously in studies Kainulainen et al., 2012), suggesting they may serve as of C. jejuni colonization and commensalism (Konkel et moonlighting proteins to promote adhesion to epithelial al., 2007; Larson et al., 2008; Flanagan et al., 2009; tissue. Thus, L. crispatus ST1 was used to evaluate the 2916 Spivey et al. tissue culture cells (Greene and Klaenhammer, 1994; Lehto and Salminen, 1997). Additionally, because the gastrointestinal pH has been reported to be 6.3 to 6.4 in the ileum and 7.0 or greater in the cecum of chickens (Denbow, 2000), PBS (pH 7.2) is appropriate for in vitro adhesion assays. It was determined that 5 washes were sufficient to remove nonadherent lactobacilli before disruption of LMH cells for enumeration of bacteria (Figure 1A). Previous studies evaluating adherence of Lactobacillus isolates to human epithelial cell lines report the use of 3 to 5 postincubation washes (Kapczynski et al., 2000; Downloaded from https://academic.oup.com/ps/article-abstract/93/11/2910/2730493 by guest on 13 November 2018 Medellin-Peña and Griffiths, 2009; Duary et al., 2011), whereas studies performed using epithelial tissues dis- sected from poultry reported using 3 washes (Gusils et al., 1999; Ben Salah et al., 2012). The use of 3 postincu- bation washes has also been reported previously in the evaluation of C. jejuni adherence to LMH cells (Konkel et al., 2007; Larson et al., 2008; Flanagan et al., 2009). Because this bacterium is known to be a particularly strong colonizer of the poultry gastrointestinal tract, it is likely that there are fewer nonadherent bacteria as a percentage of the inoculum than for Lactobacillus. Adhesion of Lactobacillus ST1 to chicken LMH cells was determined to be dose dependent, increasing with the count (cfu) per LMH cell ratio from 100:1 to 1,000:1 (Figure 1B). However, the percentage of the inoculum which adhered to the LMH cell was unchanged. Thus, 100:1 cfu per LMH cell was selected for subsequent assays in this study. Ratios of bacteria (cfu) to cells ranging from 100:1 to 2,000:1 have been reported in studies of Lactobacillus adhesion to human cell lines (Ehrmann et al., 2002; Lin et al., 2007; Medellin-Peña and Griffiths, 2009; Duary et al., 2011). A study evalu- Figure 4. Lactobacillus bile tolerance. The maximum growth rate ating Lactobacillus adhesion to dissected tissue sections (µmax) was determined for (A) Lactobacillus crispatus TDCC 75 (●), from poultry reported a cfu per cell ratio of 200:1 (Ben L. crispatus TDCC 76 (○), (B) Lactobacillus gallinarum TDCC 77 (■), and L. gallinarum TDCC 78 (□). Growth rate is shown as mean Salah et al., 2012). A multiplicity of infection of 100:1 ± SEM µmax from 3 independent cultures. *P ≤ 0.05, **P ≤ 0.01, is commonly used in studies of C. jejuni adhesion to ***P ≤ 0.001. chicken LMH cells (Konkel et al., 2007; Flanagan et al., 2009). Although we did not investigate whether adherence was saturable at cfu per cell ratios greater effect of various experimental factors on the adhesion of than 1,000:1, the dose-dependent nature of Lactobacil- Lactobacillus to the LMH epithelial cell line to establish lus adherence to the chicken LMH cell line suggests a a standard reproducible set of conditions that could mechanism of adherence dependent upon specific inter- be used to evaluate probiotic adhesion and potentially actions of cell surface factors (Konkel et al., 2010). The mimic their colonization in chickens. adhesive properties of several surface and extracellular The effect of buffer pH on in vitro adhesion of Lac- proteins of L. crispatus ST1 have been characterized tobacillus cultures (Ouwehand and Salminen, 2003) has previously (Hurmalainen et al., 2007; Edelman et al., been demonstrated with adhesion being enhanced in 2012; Kainulainen et al., 2012). However, their role in acidic buffers (Greene and Klaenhammer, 1994; Leh- vivo in mediating adherence to epithelial tissues or gas- to and Salminen, 1997; Roos and Jonsson, 2002) and trointestinal colonization in poultry has not yet been moonlighting surface proteins becoming detached at demonstrated. neutral to basic pH (Kainulainen et al., 2012). Howev- The Lactobacillus-LMH cell binding assay was used er, when working with tissue culture cells the adhesion to evaluate the adhesion of Lactobacillus cultures to a buffer should have neutral pH due to the negative effect poultry-derived epithelial cell line (Figure 2). In addi- of low pH on the viability of tissue culture cells and the tion to L. crispatus ST1, 3 additional poultry isolates, potential aberrant exposure of potential binding sites L. crispatus JCM 5810, and Lactobacillus gallinarum due to pH dependent changes in surface proteins of (ATCC 33199 and JCM 8782), were selected to serve LACTOBACILLUS ADHESION AND COLONIZATION 2917 as model probiotic Lactobacillus cultures. Lactobacillus Lactobacillus cultures as probiotics for use in humans crispatus JCM 5810 and L. gallinarum ATCC 33199 (Azcarate-Peril et al., 2008; Klaenhammer et al., 2008) have been demonstrated previously to inhibit the and livestock animals (Ehrmann et al., 2002; Shin et growth of C. jejuni in vitro and reduce gastrointestinal al., 2002; Brashears et al., 2003; Taheri et al., 2009). colonization of C. jejuni in broiler chickens (Neal-McK- In this study, we characterized adhesion to epithelial inney et al., 2012). Because they would be used to fa- cells and gastrointestinal colonization of 4 model probi- cilitate differentiation of the administered Lactobacillus otic Lactobacillus cultures in poultry. We have demon- cultures from the background LAB in the in vivo colo- strated the value of the chicken LMH cell line for use in nization studies, adhesion assays were performed using cell culture models of Lactobacillus adhesion to chicken Rifr derivative cultures of each Lactobacillus strain as epithelial tissues. However, its relevance to gastrointes- well. All Lactobacillus cultures were found to adhere ef- tinal colonization is limited due to the complexity of fectively to the chicken LMH cells when compared with the gastrointestinal environment and the multifactorial Downloaded from https://academic.oup.com/ps/article-abstract/93/11/2910/2730493 by guest on 13 November 2018 B. subtilis and Salmonella Typhimurium, which were nature of colonization. Evaluating the adhesion of Lac- used as low- and high-adherent controls, respectively. tobacillus cultures to other substrates including intes- Lactobacillus crispatus TDCC 75 and the 2 L. gallina- tinal mucus and extracellular matrix components will rum strains adhered to the chicken LMH cells at similar continue to be important given the limitations of the levels, whereas adhesion of L. crispatus TDCC 76 was LMH cell line. Additionally, our results demonstrate lower than the other strains. The number of adherent that factors unrelated to adhesion such as bile tolerance Lactobacillus was found to be 1.5 to 3.5% of the cul- also contribute to the ability of lactobacilli to persist tures used to inoculate the chicken LMH cells. Studies in the gastrointestinal tract of poultry. Studies using evaluating Lactobacillus adherence to human epithelial isogenic mutant strains differing in cellular adhesion, cell lines report adherence of 1 to 14% of the inoculum bile tolerance, and other properties are needed to elu- (Ehrmann et al., 2002; Messaoudi et al., 2012). cidate the role of these factors in the gastrointestinal Lactobacillus gallinarum TDCC 77, followed by L. persistence and colonization of probiotic Lactobacillus crispatus TDCC 75, were found to colonize broiler cultures in poultry (Ouwehand and Salminen, 2003). chicks more persistently and at greater levels than the The availability of a chicken-specific cell culture mod- other lactobacilli in our time course study (Figure 3). el will contribute to these future studies. Finally, the Although we are hesitant to speculate on the role of LMH cell line has also been demonstrated previously to tissue-specific tropism on the greater persistence of L. be useful for the investigation of host-microbe interac- crispatus TDCC 75 and L. gallinarum TDCC 77, it is tions of human foodborne pathogens in poultry, includ- interesting to note that their wild-type parent strains ing Campylobacter, Clostridium, and Salmonella. This were both reported to be isolated from the crop of suggests the utility of this cell culture model for investi- chickens, whereas the other strains were reported to gation of Lactobacillus-mediated competitive exclusion be isolated from chicken feces (Table 1). In addition to and virulence inhibition of pathogenic microorganisms differences in gastrointestinal persistence of Lactobacil- in poultry. These studies are expected to contribute to lus cultures, our study demonstrates the transient na- a mechanistic understanding of probiotic functionality ture of colonization by allochthonous bacteria (Walter, in chickens, the development of more effective screen- 2008). The inability of probiotic cultures to establish ing tools for the selection of probiotic cultures, and the permanent colonization in the gastrointestinal tract has more effective application of probiotics in poultry pro- been well documented (Jacobsen et al., 1999; Tannock duction. et al., 2000; Frese et al., 2012). These data suggest that continued repeated administration is required to main- ACKNOWLEDGMENTS tain the presence of probiotic cultures in the gastroin- testinal tract. We thank Dale Hyatt, Jason T. Lee, Joseph M. Stur- The similar in vitro adhesion of L. crispatus TDCC ino, and David J. Caldwell (Texas A&M University, 75, L. gallinarum TDCC 77, and L. gallinarum TDCC College Station) for insightful discussions and techni- 78 suggested that their colonization of broiler chicks cal assistance in conducting this study. We also thank might also be comparable. However, colonization of L. J. Allen Byrd (USDA-Agricultural Research Service, gallinarum TDCC 78 was similar to the less adherent College Station, TX) and Benita Westerlund-Wikstrom L. crispatus TDCC 76. L. gallinarum TDCC 78 was (University of Helsinki, Helsinki, Finland) for providing determined to be more sensitive to bile than L. gal- the Salmonella Typhimurium and Lactobacillus crispa- linarum TDCC 77, suggesting that although adhesion tus ST1 cultures used in this study, respectively. of the L. gallinarum strains was comparable, the dif- This research was supported by Texas A&M AgriL- ferences in persistence may be due to reduced surviv- ife Research and USDA-NIFA Hatch project number ability of L. gallinarum TDCC 78. Bile tolerance has TEX09405. Megan A. Spivey was supported by the Pa- been suggested previously as an additional factor likely mela Hargis-Plantation Foods Endowed Fellowship and to be important in gastrointestinal persistence and has a graduate assistantship from the Texas A&M Univer- been used previously as a criterion for the selection of sity Department of Poultry Science. 2918 Spivey et al. REFERENCES Garriga, M., M. Pascual, J. M. Monfort, and M. Hugas. 1998. 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