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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

S. SOOKKHEE, M. CHULASIRI AND W. PRACHYABRUED. 2001. The aims of the present study were to screen and characterize the antimicrobial lactic acid bacteria which were isolated from healthy oral cavities of Thai volunteers, and to characterize their inhibiting substances. Among 3790 isolates (suspected to be lactic acid bacteria) from 130 volunteers, five showed an appreciable effect against Sarcina lutea ATCC 9341, Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Streptococcus mutans DTMU 1, Strep. salivarius DTMU 1, Strep. sanguis DTMU 1, Candida albicans ATCC 13803 and C. albicans DTMU 2, as well as the oral pathogens. These antimicrobial isolates included L17 and N14 which showed the antibacterial activity, D14 which showed the anticandidal activity, and D6 and N8 which showed both the antibacterial and anticandidal activities. The isolates were later found to be facultative anaerobic, Gram-positive, non-spore-forming, non-capsule-forming and catalase-negative bacilli. They could utilize casein but could not hydrolyse starch, and they produced hydrogen peroxide and bacteriocins. Their antimicrobial potentials were found to be affected by pH, catalase, proteolytic enzymes and temperature. The activity was partially inactivated after catalase treatment, significantly declined at pH ≥9·0 or after trypsin and pepsin treatments, and also reduced after heating at ≥100°C. However, the antimicrobial activity of these five isolates was somewhat resistant to heat. When the isolates were tested for their antimicrobial sensitivity, they were shown to be sensitive to a number of antimicrobial agents. The final identification revealed that D6, D14 and N14 were Lactobacillus paracasei subsp. paracasei, and L17 and N8 were Lact. rhamnosus.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Lactic acid bacteria (LAB) can be found as human flora in the mouth, intestine and vagina (Ahrne et al. 1998). The genera belonging to this group can produce organic acids such as lactic acid and acetic acid from carbohydrate fermentation. These acids render a low ecological pH which can interfere with the growth of surrounding micro-organisms. Additionally, some bacteria of the group produce hydrogen peroxide (oxidizing agent) and/or bacteriocins (proteinaceous compounds) which are antimicrobial substances (McGroarty et al. 1992; Kanatani et al. 1995; Green et al. 1997; van Reenen et al. 1998). Due to their antimicrobial and interference activities, LAB have shown an inhibitory effect on a variety of micro-organisms. Studies have revealed the role of LAB in the protection of intestinal and vaginal infections (Tannock 1990; McGroarty et al. 1992; Andreu et al. 1995), and the protective ability on oral infections (Arihara et al. 1996). As they possess an antimicrobial property, these bacteria may be beneficial as bioprotective agents to control infections in either the intestine, vagina or mouth. In the current study, antimicrobial-producing LAB were isolated from the oral cavities of Thai volunteers, and their inhibitory effect was characterized for the purpose of applying them as oral bioprotectants.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Microbial isolation

Volunteers (130) classified as having good oral hygiene according to the simplified oral hygiene index (Greene and Vermillion 1964) were recruited. Saliva from each volunteer was collected using the paraffin-stimulating technique. Mitis-Salivarius Agar (MSA; Difco) and Rogosa SL Agar (RSA; Difco) plates were used as the selective media. All catalase-negative isolates, which were suspected to be LAB, were selected for further study. The antimicrobial isolates selected from the study were resuspended in 20% Skim Milk Broth (Difco) before freeze-drying for storage. Prior to use, each lyophilized aliquot was propagated twice. The laboratory and clinical strains used in the antimicrobial study are shown in Table 1.

Table 1.  Micro-organisms used in the study Thumbnail image of

Screening of antimicrobial isolates

The primary antimicrobial screen of the above LAB isolates was performed by an agar spot test (Fleming et al. 1975). Sarcina lutea ATCC 9341 and Candida albicans ATCC 13803 were the tested organisms. The test was performed on 0·1% w/v glucose–MSA for streptococci screening, and 0·1% w/v glucose–Lactobacillus de Mann–Rogosa–Sharpe Agar (LMA; Difco) for lactobacilli screening. These agar media were overlaid with the molten agar media, as shown in Table 1 seeded with the above tested micro-organisms previously grown at 37°C for 24 h. After incubation at 37°C for 24 h, zone-producing colonies were selected. The selected isolates were repeatedly screened for antimicrobial activity by an agar-well diffusion method (Schillinger and Lucke 1989). Each of the suspected antimicrobial isolates was tested against additional laboratory strains. The isolates were each grown in Tryptone–Glucose–Yeast Extract Broth at 37°C under CO2 for 48 h, and their filtrates were placed in the wells of Mueller Hinton Agar (MHA; Difco) which had been previously spread with the test organisms. These bacterial and candidal strains were those grown in media as shown in Table 1 and adjusted to a turbidity equivalent to McFarland no. 0·5. The tested plates were incubated at 37°C for 24 h, except the plates with streptococci which were incubated at 37°C under CO2 for 48 h. The selected isolates from this study were further tested against the clinical strains, which were those isolated from oral infections of HIV-seronegative and -positive patients. The test was also performed by the agar well-diffusion method as described above. In the experiment with obligate anaerobes, MHA plates were replaced by Anaerobic Plaque Agar (APA) plates, and incubated at 37°C under anaerobic conditions for 72 h. The final selected antimicrobial isolates were used for further study.

Microbial characterization

Morphology.

The selected isolates were primarily characterized by Gram, spore and capsule staining.

Casein utilization.

The selected isolates were each spotted on Skim Milk Agar (Difco) plates. The clear zones were measured to assess casein utilization activity after incubation at 37°C under CO2 for 24 h.

Starch hydrolysis.

The selected isolates were each spotted on Starch Agar (Difco) plates. After incubation at 37°C under CO2 for 24 h, the clear zones of hydrolysed starch surrounded by a blue background after flooding with iodine solution were measured to assess starch hydrolysis activity.

Antimicrobial characterization

Hydrogen peroxide production.

Hydrogen peroxide from the selected isolates was determined using a modified method described by Eschenbach et al. (1989). These isolates were cultured on LMA plates containing 0·25 g l–1 tetramethylbenzidine (Sigma) and 0·01 g l–1 horseradish peroxidase (Sigma) at 37°C under CO2 for 48 h. The colony which had a blue colour around it was interpreted to be the H2O2 producer.

Bacteriocin production.

Antimicrobial substances from the selected isolates were detected by the method described by Mortvedt and Nes (1990). Briefly, the supernatant fluids from the cultures grown in Lactobacillus de Mann–Rogosa–Sharpe Broth (LMB; Difco) at 37°C under CO2 for 48 h were adjusted to pH 7·0 and treated with catalase. The treated supernatant fluids were twofold serially diluted and placed in the 96-well, flat-bottom microtitre plate. Afterwards, the test organisms Strep. mutans DTMU 1 and C. albicans DTMU 2 were added. One bacteriocin unit (BU ml–1) was defined as the reciprocal of the highest dilution which inhibited 50% of the growth of the test organisms.

pH sensitivity.

Supernatant fluids from antimicrobial cultures grown in LMB for 48 h were divided into portions. These portions were each adjusted with 1N HCl or 1N NaOH to pH 4, 5, 6, 7, 8, 9 and 10 and placed in the 96-well, flat-bottom microtitre plate. The same volume of Strep. mutans DTMU 1 and C. albicans DTMU 2 as the corresponding antimicrobial cultures was added. After incubating the supernatant fluid test cultures at 37°C under CO2 for 12 h, growth was measured by a microtitre plate reader. The inhibitory effect of the antimicrobial supernatant fluids towards the test organisms was calculated as percentage of inhibition (Miteva et al. 1998).

Enzyme sensitivity.

Supernatant fluids from antimicrobial cultures as above were separately treated with 1 g l–1 catalase, 0·25 g l–1 trypsin and 0·25 g l–1 pepsin at 37°C for 5 min, before the enzymes were inactivated with heat-inactivated fetal calf serum. They were then processed as described above.

Heat sensitivity.

Supernatant fluids from antimicrobial cultures, as above, were separately treated at 60, 80 and 100°C for 30 min, and at 121°C for 15 min. They were then processed as described above.

Antimicrobial sensitivity

The sensitivity of the test antimicrobial isolates to antimicrobial agents was performed by an agar-disc diffusion method as described by Kirby et al. (1966), with slight modification. Briefly, Tryptone–Glucose–Yeast Extract Agar (TGYA) plates were spread with grown cultures of the isolates which had been previously adjusted to a turbidity equivalent to McFarland No. 0·5. The test antimicrobial discs were each placed on the cultured TGYA. After incubation at 37°C under CO2 for 48 h, the clear zones were measured and the sensitivity was interpreted according to a Table of Antibiotic Susceptibility provided by Difco. In this study, Staph. aureus ATCC 6538 and E. coli ATCC 25922 were tested in parallel.

Identification

The antimicrobial isolates tested were each identified on the basis of morphological examination, as stated above, and biochemical profiles according to the API-50CHL test kit (bioMerieux Vitek Inc., Hazel Wood, MO, USA).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

Antimicrobial screening

Among 3790 colonies grown on RSA and MSA, which were those isolated from healthy oral cavities of 130 volunteers, 1094 and 53 were shown to have primarily antibacterial and anticandidal activities, respectively. Results are shown in Table 2. In the following screen, nine isolates which were designated as B12, D6, D14, H19, JY, J8, L17, N8 and N14, showed appreciable antimicrobial effects towards the test laboratory strains. These isolates were those grown on RSA. Results in Table 3 show that J8, L17 and N14 had an antibacterial effect while D14, H19 and JY had an anticandidal effect; B12, D6 and N8 had both effects. These isolates were then selected for additional antimicrobial study against the clinical strains which were freshly isolated from oral infections of HIV-seronegative and -seropositive patients. Results shown in Figure 1 reveal that J8, L17 and N14 inhibited 68·8, 68·8 and 75·0% of 15 bacterial pathogens isolated from the mouth of HIV-seronegative patients, and 27·3, 86·4 and 86·4% of 22 pathogens from HIV-seropositive patients, respectively; D14, H19 and J7 inhibited 50·0, 31·8 and 22·7% of 22 candidal pathogens isolated from the mouth of HIV-seronegative patients, and 66·7, 11·1 and 22·2% of 18 pathogens from HIV-seropositive patients, respectively. B12, D6 and N8 inhibited 68·8, 68·8 and 68·8% of 15 bacterial pathogens isolated from the mouth of HIV-seronegative patients, and 40·9, 77·3 and 90·9% of 22 pathogens from HIV-seropositive patients, respectively; they also inhibited 27·3, 50·5 and 36·4% of 22 candidal pathogens isolated from the mouth of HIV-seronegative patients, and 38·9, 55·6 and 50·0% of 18 pathogens from HIV-seropositive patients, respectively. In view of the significantly antimicrobial results, D6, D14, L17, N8 and N14 were selected for further study. Table 4 shows the width of the inhibition zones exhibited by filtrates from the grown cultures of selected isolates towards the clinical pathogens. It can be seen that there was no difference between the sensitivity of the oral pathogens from HIV-seronegative and -positive patients to the tested antimicrobial isolates.

Table 2.  The first antimicrobial screen of the isolates from oral cavities using Sarcina lutea ATCC 9341 and Candida albicans ATCC 13803 as the tested strains. Results are expressed as the numbers and percentage of the oral isolates which could inhibit each tested strain Thumbnail image of
Table 3.  The second antimicrobial screen of the selected antimicrobial isolates from oral cavities using additional laboratory strains as the test micro-organisms. Results are expressed as the average diameter of inhibition zone (in mm) caused by each of the selected isolates Thumbnail image of
image

Figure . 1. The third antimicrobial screen of the selected antimicrobial isolates towards oral pathogens isolated from (▪) HIV-seronegative (15 bacterial and 22 candidal strains) and (□) HIV-seropositive patients (22 bacterial and 18 candidal strains). Results are expressed as the percentage of sensitive oral pathogens to each supernatant fluid from antimicrobial isolate. (a) bacteria; (b) candida

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Table 4.  The third antimicrobial screen of the selected antimicrobial isolates from oral cavities using oral pathogens as the test micro-organisms. Results are expressed as the diameter of inhibition zone (mean ± standard deviation) in mm caused by each of the antimicrobial isolates Thumbnail image of

Microbial characterization

Results demonstrated that D6, D14, L17, N8 and N14 were Gram-positive, catalase-negative rods without capsules and spores. This microbial characterization study suggested that the oral isolates belonged to the genus Lactobacillus. They were shown to be able to utilize casein but to be unable to hydrolyse starch (data not shown).

Antimicrobial characterization

Results shown in Table 5 demonstrate that the test antimicrobial isolates produced hydrogen peroxide and bacteriocins. Table 6 shows that the antimicrobial activity of D6, D14, L17, N8 and N14 was affected by pH, catalase, proteolytic enzymes and temperature. Activity was reduced after treatment with pH values above 9, trypsin and pepsin, as well as after heat treatment at temperatures over 100°C. However, the antimicrobial activity of these isolates was seen to be somewhat resistant to heat.

Table 5.  Antimicrobial production of the selected antimicrobial isolates from oral cavities Thumbnail image of
Table 6.  Effects of pH, enzymes and heat on antimicrobial activity of the selected oral isolates using Stretpcoccus mutans DTMU 1 and Candida albicans DTMU 2 as the test strains. Results are expressed as the percentage of antimicrobial activity of each oral isolate against the test micro-organisms Thumbnail image of

Antimicrobial sensitivity

All the tested antimicrobial isolates were sensitive to a number of antimicrobial agents. Results in Table 7 show that these isolates were sensitive to amoxicillin, amoxicillin/clavulanic acid, ampicillin, bacitracin, chloramphenicol, clarithromycin, clindamycin, erythromycin, imipenem, nitrofurantoin, norfloxacin, penicillin G, rifampicin and tetracycline, but resistant to amikacin, ceftazidime, cloxacillin, colistin, gentamycin, kanamycin, nalidixic acid, polymyxin B, streptomycin, sulphanilamide/trimetroprim, trimetroprim and vancomycin.

Table 7.  Antimicrobial sensitivity of the selected oral isolates Thumbnail image of

Identification

After identification using the API-50CHL kit, D6, D14 and N14 were found to be Lact. paracasei subsp. paracasei, while L17 and N8 were Lact. rhamnosus (data not shown).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

LAB is a group of bacteria, e.g. streptococci and lactobacilli, which are found as part of the normal flora in humans. They are present predominantly in the oral cavity, intestinal tract and vagina (Ahrne et al. 1998). As they are producers of lactic acid and some other organic acids, these micro-organisms are known to have antimicrobial activity (Taniguchi et al. 1998). Some LAB produce bacteriocins which are proteinaceous substances with antimicrobial activity (Arihara et al. 1996; van Reenen et al. 1998). The present investigation revealed that both streptococci and lactobacilli could be isolated from the healthy oral cavities of Thai volunteers. Results showed that five oral lactobacillus isolates were good antimicrobial producers which could inhibit a number of oral pathogens. The present study therefore demonstrates that oral lactobacilli also possess antimicrobial activity, as had been found in intestinal and vaginal lactobacilli (Tannock 1990; McGroarty et al. 1992; van Reenen et al. 1998).

As stated above, LAB can produce lactic acid and some other organic acids which produce the antimicrobial activity of the micro-organisms (Taniguchi et al. 1998). Therefore, the existence of antimicrobial substances in the oral isolates studied would be partly due to organic acids, because the present investigation showed that the antimicrobial activity of the test isolates was more active at acidic pH than at alkali pH. After adjusting the supernatant fluids from these cultures to pH 7·0, there was a slight reduction (10–20%) of the antimicrobial activity. This indicated that organic acids were not the only antimicrobial substances in the studied oral isolates. It was shown in the present study that the antimicrobial activity of the oral isolates was reduced after treatment with catalase, and their growing colonies demonstrated a blue pigment around the colonies on the medium supplemented with TMB and horseradish peroxidase. These results showed that the isolates could produce hydrogen peroxide, which is another type of antimicrobial substance (McGroarty et al. 1992). The tested isolates also showed a significant reduction in inhibitory activity after treatment with trypsin and pepsin. This suggested that the additional type of antimicrobial substances present in these isolates were bacteriocins, which are proteinaceous antimicrobial agents (Klaenhammer 1988). The present study corroborated many other studies which have revealed that LAB could produce organic acids, hydrogen peroxide and bacteriocins (McGroarty et al. 1992; Kanatani et al. 1995; Green et al. 1997; van Reenen et al. 1998). The bacteriocins found in the test isolates were class II bacteriocins because they could tolerate heat to some extent. Examples of class II bacteriocins which have been found previously include salivacin 140 from Lact. salivarius (Arihara et al. 1996), plantaricin 423 from Lact. plantarum (van Reenen et al. 1998) and acidocin J1229 from Lact. acidophilus (Tahara et al. 1996). The activity of the bacteriocins of this class is due to pore formation in the cytoplasmic membrane (Ojcius and Young 1991; Tahara and Kanatani 1996).

Following examination, three isolates were identified as Lact. paracasei subsp. paracasei and two were found to be Lact. rhamnosus. These species have been shown to be the common human flora in the mouth and intestine (Ahrne et al. 1998), and were able to inhibit oral and intestinal pathogens. Results from this investigation support this finding because these species were from healthy oral cavities of Thai volunteers and could inhibit a number of oral pathogens.

As the current study was aimed at investigating the LAB from the healthy oral cavity, and needed to apply those with appreciable antimicrobial activity as the bioprotective agents for control of oral infections, especially in HIV-positive patients, other important characteristics, e.g. the ability to colonize the mouth, production of some useful enzymes/substances to promote oral health, and safety of use, need to be further studied.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography

This work was supported by the National Science and Technology Development Agency (NSTDA) of Thailand.

Footnotes
  1. Present address: Department of Odontology and Oral Pathology, Faculty of Dentistry, Chiang Mai University, Suthep Road, Chiang Mai 50202, Thailand.

Bibliography

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. Bibliography
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