IDENTIFICATION OF LACTIC ACID BACTERIA AND

ENTEROBACTERIA. A TECHNIQUE IN MICROPLATES

Carlos Gusils, Rolando Figueroa, Adriana Pérez Chaia, Silvia González and Guillermo Oliver

Carlos Gusils. Researcher, National Research Council of Argentina and Centro de Referencia para Lactobacilos (CONICET-CERELA). Instructor of Virology and Microbiology, National University of Tucumán (UNT).

Rolando Figueroa. Instructor of Mathematics, UNT.

Adriana Pérez Chaia. Researcher, CONICET-CERELA. Instructor, General Microbiology, UNT.

Silvia González. Researcher, CONICET-CERELA. Associate Professor, Public Health, UNT.

Guillermo Oliver. Researcher, CONICET-CERELA. Emeritus Professor, UNT. Member of National Academy of Medicine, National Academy of Agronomy and Veterinary and the New York Academy of Sciences. Address: Chacabuco 145, S.M. de Tucumán, 4000 - Tucumán, Argentina. e-mail: oliver@cerela.org.ar

Summary

In this work a microtechnique for the identification of lactic acid and enteropathogenic bacteria is proposed. Results obtained were compared with those from the conventional method. The final volume and the incubation time for studying sugar fermentation and other biochemical properties were reduced when compared with traditional tests. The correlation between the microplate test and the conventional method is more than 90%. The preparation of microplates with reagents can be stored under refrigeration 30 days before use. The microtechnique could be considered an easy, swift and thrifty method.

Resumen

En este trabajo se desarrolla una microtécnica para la identificación de bacterias lácticas y bacterias enteropatogénicas. Los resultados fueron comparados con los obtenidos por el método convencional. Se logró una reducción del volumen final y del tiempo de incubación para el estudio de fermentación de azúcares y otras propiedades bioquímicas en comparación con el ensayo tradicional. La correlación obtenida entre el ensayo en microplaca y el método convencional fue superior al 90%. La preparación de las microplacas con los diferentes reactivos pueden ser conservadas bajo condiciones de refrigeración durante 30 días antes de su empleo. La microtécnica puede ser considerada como un método sencillo, rápido y económico.

Resumo

Neste trabalho se desenvolve uma microtécnica para a identificação de bactérias lácticas e bactérias enteropatogênicas. Os resultados foram comparados com os obtidos pelo método convencional. Foi conseguida a redução do volume final e do tempo de incubação para o estudo de fermentação de açúcares e outras propriedades bioquímicas em comparação com a experiência tradicional. A correlação obtida entre a experiência em microplaca e o método convencional foi superior ao 90%. A preparação das microplacas com os diferentes reativos podem ser conservadas sob condições de refrigeração durante 30 dias antes de seu emprego. A microtécnica pode ser considerada como um método simples, rápido e econômico.

Keywords / Microtechnique / Lactic Acid Bacteria / Enteropathogenic Bacteria /

Recibido: 09/08/2001. Modificado: 02/10/2001. Aceptado: 17/10/2001

Introduction

Certain gut species are pathogens but a number of other resident bacteria may be of some benefit to host health. Examples include enterococci, lactobacilli, propionibacteria and bifidobacteria which are present in the colon in significant numbers. Probiotics, prebiotics and synbiotics are all mechanisms to improve host health through fortification of selected bacteria in the gut (Macfarlane and Gibson, 1994). The nutritional benefits of probiotics have been investigated in regard to the fermentation, on the quantity, availability and digestion of nutrients.

Identification and classification objectives are not identical. An identification description can be carried out only after that group has first been classified. It is based on a pattern of properties shown by all the members of the group and which other groups do not posses. The properties used in identification are often different from those used in the classification of the group. Biochemical, nutritional and physiological characterization tests, usually carried out in bottles and tubes of solid and liquid media and on plates have been developed and modified since the earliest days of bacteriology. Generally, the characteristics chosen for an identification plan should be easily determinable, whereas those used for classification may be quite difficult to determine such as DNA homology. Genera and species identification might not be based on only a few tests, but rather on the pattern given by a whole battery of tests. The members of the family Lactobacillaceae represent one example of this (Fagnant et al., 1982). Some probiotic strains can be selected for their beneficial properties as active antimicrobial agents against pathogenic microorganisms, hydrophobic ability, presence of substances with capacity for adherence to epithelium, etc. (Gusils et al., 1999). After isolating, the identification is an important step before selecting probiotic strains. These identifications can be facilitated through microtechniques. To alleviate the need for inoculating large numbers of tube media (Conventional test), some rapid multitest systems have been devised and are commercially available (such as API or Biolog systems). Each manufacturer provides charts, tables, codes and characterization profiles for use of the particular system. Although expensive to use for large studies, and not always sufficiently versatile, these kits do offer the advantages of convenience, miniaturization, rapidity and, above all, strict standarization. In this work we propose a simple, rapid and economical technique in microplates to identify lactic acid bacteria and enterobacteria based on general metabolic characters adapted for studying routinely a large number of strains. Other methods (Du Plessis and Dicks, 1995) such as ribotyping or randomly amplified polymorphic DNA (RAPD) do not take into account the phenotypic characteristics (biotyping).

Materials and Methods

Microorganisms

The lactic acid bacteria used in this study were obtained from the Cerela Collection (CRL) and were isolated in our laboratory and identified according to the criteria of the Bergey’s Manual of Systematic Bacteriology (1984). Enterobacteria were provided by the Microbiology Institute Culture Collection of UNT, Tucumán, Argentina. Microorganisms used in this study were from different origins such as human, cheese, chicken, sausage, soil of mountain, pig, yogurt and ATCC (American Type Culture Collection).

Culture media. LAPTG (Raibaud et al., 1961) and Brain Heart Infusion (Merck) broth were used as culture media for lactic acid bacteria and enterobacteria respectively. After adequate incubation the cells were harvested, washed twice in saline solution and then suspended in distilled water (2% w/v). For biochemical studies, these suspensions were used ten fold diluted in adequate sugar fermentation media.

Sugar Fermentations. Sugar solutions (20% w/v) were prepared and sterilized by filtration. The media for sugar fermentation were MRS (De Man et al., 1960) for lactobacilli and medium for cocci fermentation (Larpent and Larpent-Gourgaud, 1975) but in both cases ten fold concentration (0.02mgml-1) of pH indicator (Phenol red for cocci and Bromcresol purple for lactobacilli) were used.

Preparation of microplates: The microplates added with 10µl of each sugar solution (2% final concentration) were stored under sterile and refrigeration conditions (10°C). They were used after storage during 7, 14, 30 and 60 days. Sugar fermentation was studied after addition of 200µl of the specific inoculated medium. The microaerophilic conditions can be obtained by adding 50µl of sterile vaseline or by incubation in a controlled atmosphere and were incubated at 37ºC during 48 h. The fermentation test was considered positive when the pH indicator in the medium became yellow after incubation. An orange color was considered as a weak result, any shade of red being considered as negative.

Biochemicals assays

Studies of the biochemical properties of lactobacilli included hydrolysis of arginine, and esculin (Raibaud et al., 1973), growth in litmus milk (Harrigan and McCance, 1976), growth in the presence of 0.1 and 0.3% methylene blue added to milk (Larpent and Larpent-Gourgaud, 1975), indol, arginine, esculin, reduction of nitrate, gluconate, Gibson medium to study formation of carbon dioxide from glucose (Gibson and Abd-El-Malek, 1945) and growth at different temperatures (15, 37, 45 and 50°C). Studies of the biochemical properties of cocci included arginine, esculin and hippurate (Seeley and Dain, 1960) hydrolisis, 2,3,5-triphenyltetrazolium chloride (Barnes, 1956), potassium tellurite (Larpent and Larpent-Gourgaud, 1975) and nitrate reductions, growth in litmus milk, growth in LAPTG at initial pH of 9.2, 9.4 and 9.6, growth at 4 and 6.5% sodium chloride (Facklam and Wilkinson, 1981), growth in the presence of 0.1 and 0.3% methylene blue added to milk, growth in 0.02% of sodium azide, growth in the presence of 0.1 and 0.4% Oxgall (Difco), growth at different temperatures (15, 37, 45 and 50°C) and acetyl-methylcarbinol production (Harrigan and McCance, 1976) were included for identifying cocci.

For enterobacteria the tests were: urea, citrate, triple sugar iron (TSI medium) and individual fermentation of glucose, sucrose and lactose in broth medium. Preparation of microplates: the microplates to which 200µl of each sterile medium was added, were stored at 4ºC for 7, 14 and 30 days.

Bacteria were harvested at the early logarithmic growth phase, washed twice, and resuspended in physiological solution (PS) to DO560 (optical density at 560nm) of 0.5-0.7. Bacterial suspensions were added into test tubes and microplates containing media, and incubated at 37°C. Microbial growths after incubation were considered as reaction positive.

Statistical analysis

All the experiments were performed in triplicate. The correlation of the results between test tubes and microplates were analyzed by use of a two-way analysis of variance.

Results and Discussion

A novel approach commercially available as the Biolog system, uses redox indicators to detect colorimetrically the increased metabolic rate of a bacterial suspension. However, this system is not appropriate to identify lactic acid bacteria because of errors that make difficult the bacterial identification. Given its importance, the genus Lactobacillus has been somewhat neglected by numerical taxonomists, with most studies concentrating on strains from special environments such as wine, cheese, meat, human epithelia rather than taking a broader view. The proposed microtechnique has shown a correlation of more than 95% with the conventional method (P<0.001) for lactic acid bacteria and their biochemical properties profiles can be obtained and analyzed easily within 24 hours (Figures 1 and 2). A correlation of 100% between conventional and microplate methods was observed for lactobacilli such as Lactobacillus. animalis, L. casei subsp. casei, L. casei subsp. rhamnosus, L. acidophilus, L. plantarum and L. buchnerii. L. fermentum presented a correlation of 84% when esculin hydrolisis and/or decarboxylation from glucose were studied.

Figure 1. Sugar fermentations by cocci. A: control sugars; B: Lactococcus lactis subsp. Lactis; C: Enterococcus faecium; D: E. faecalis; E: Streptococcus salivarus subsp. termophilus. Sugars: 1) bacteria control, 2) arabinose, 3) cellobiose, 4) fructose, 5) galactose, 6 and 7) glucose, 8) lactose, 9) maltose, 10) mannitol, 11) mannose, 12) melezitose, 13) melibiose, 14) raffinose, 15) rhamnose, 16) ribose, 17) sucrose, 18) salicine, 19) sorbitol, 20) trehalose and 21) xilose.

Figure 2. Sugar fermentation by lactobacilli. A: control sugars; B: Lactobacillus casei subsp. Rahmnosus; C: L. buchnerii; D: L. plantarum; E: L. casei subsp. Casei; F: L. fermentum; G: L. animalis; H: L. acidophilus. Sugars: 1) bacteria control, 2) arabinose, 3) cellobiose, 4) fructose, 5) galactose, 6) glucose, 7) lactose, 8) maltose, 9) mannitol, 10) mannose, 11) melezitose, 12) melibiose, 13) raffinose, 14) rhamnose, 15) ribose, 16) salicine, 17) sorbitol, 18) trehalose y 19) xilose.

The enterococci are differentiated by routine physiological and biochemical tests, but because many characters are inconsistent within individual species, the identification is based upon overall patterns of results over a relatively large number of characters rather than being limited to a few unreliable key characters. The micromethod for Enterococcus faecalis showed a total correlation with the conventional method while a correlation of 84% for Lactococcus lactis subsp. lactis was obtained about esculin hydrolysis. E. faecium in 0.02% sodium azide and/or Streptococcus salivarus subsp. termophilus in 40% oxgall presented a correlation of 87% and 75%, respectively.

The incubation time used in these tests represented a reduction of about 7 or 3 times respectively, when compared with traditional tests where incubations for one week or more were necessary.

The results from sugar fermentation studies with all strains showed that a storage of microplates for 60 days is possible, but recently cell suspensions must be used.

In the case of enterobacteria, a good differentiation between genera using the proposed microthechnique is possible (Figure 3). Moreover no significant differences between the two methods (P<0.001) were determined. A total correlation was observed for Serratia marcescens, Proteus vulgaris, Shigella sonnei, Klebsiella and Pseudomona, but when we studied citrate utilization in E. coli and/or H2S production in Salmonella a correlation of 87% was obtained.

Figure 3: Biochemical assays of enterobacteria. A: control of different media; B: Serratia marcescens; C: E. coli; D: Proteus vulgaris; E: Shigella sonnei; F: Salmonella paratyphimurium; G: Klebsiella; H: Pseudomona. Probes: 1) TSI; 2, 3 and 4) TSI with different sugars (glucose, lactose and sucrose, respectively), 5) urea, 6) citrate.

When the other biochemical characters were studied, microplates stored for 7, 14 and 30 days showed identical results (P<0.05). However, reliable data were not obtained from microplates stored for more than one month.

The correlation between hexose monophosphate shunt and fermentation-type in lactic acid bacteria (Buyze et al., 1957) cannot be studied by this micromethod; as also the formation of carbon dioxide by lactic acid bacteria in a culture medium with 5% glucose (Harrigan and McCance, 1976). The API-system is able to determine the utilization of gluconate as single carbon source but not decarboxylations from glucose. Some classical tests as nitrate reduction are not well standardized and are not available in kit form.

The reproducibility of the proposed method was more than 90% (P<0.001) and offers a rapid and inexpensive identification of microorganisms.

In a previous work (Font de Valdez, 1993) a microsystem was developed but this technique only optimized the characterization of lactobacilli after sugar fermentation. Moreover the method included vials for each test and the incubation was carried out in a Gaspack anaerobic jar.

To establish highly reliable identification schemes, phenotypes generated with biochemical tests must be considered in combination with genotypes determined in studies of DNA content and relatedness (Brenner, 1980). However, the purpose of this study was not to establish a precise taxonomic description of the lactobacilli; rather it was to develop a practical approach to their identification. To this aim, our method can be considered convenient for assaying daily identifications. It is easy, swift, thrifty and the microplates can be stored under refrigeration at 10°C for 30 days before use.

ACKNOWLEDGEMENTS

The authors acknowledge the support through Grant CIUNT D-126 CONICET.

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