Indicator Medium for Isolation of Campylobacter pylori

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1987, p. 2378-2379 0095-1137/87/122378-02$02.00/0 Copyright C 1987, American Society for Microbiology

Vol. 25, No. 12

Indicator Medium for Isolation of Campylobacter pylori DULCIENE M. M. QUEIROZ,* EDILBERTO N. MENDES, AND GIFONE A. ROCHA Laboratory of Research in Bacteriology, Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil Received 1 July 1987/Accepted 11 September 1987

The use of a new indicator culture medium, Belo Horizonte medium, is proposed for better colony recognition and a presumptive identification of Campylobacter pylori. This medium, containing brain heart infusion sheep blood agar, was supplemented with 40 mg of 2,3,5-triphenyltetrazolium chloride per liter in addition to vancomycin, nalidixic acid, and amphotericin B. On Belo Horizonte medium, Campylobacter pylori present unique golden colonies.

Spiral bacteria have long been found in human stomachs (3, 4, 10) but were overlooked until Marshall cultured Campylobacter-like organisms from antral biopsy specimens by using microaerophilic isolation techniques (8). Since then, there has been considerable interest in the relationship of this microorganism, named Campylobacter pylori, with gastritis and peptic ulceration (1, 5, 6, 9, 11). However, the culture methods used for the isolation of this bacterium are cumbersome and may not permit the detection of colonies when few are present. Also, biochemical tests are required for the identification of C. pylori. We have undertaken this investigation with the objective of developing a culture medium which would facilitate the detection and presumptive identification of C. pylori. We used tetrazolium salts, which are reduced to insoluble red formazan complexes by growing bacteria (7), resulting in pigmented colonies. We decided on the use of these salts since in a previous study we observed that the colonies of this microorganism displayed a distinct appearance that differentiated C. pylori from other bacteria.

nas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Candida albicans, Alcaligenes faecalis, Serratia marcescens, Streptococcusfaecalis, Streptococcus pyogenes, and Streptococcus spp. (Table 2) for colony appearance on BHM without antimicrobial agents. The inocula were prepared as described above for C. pylori, but the plates were incubated in a candle jar for 24 h at 37°C. Thereafter, 58 antral and 16 duodenal biopsy specimens were obtained from patients with symptoms referable to the upper gastrointestinal tract and duodenal ulcer, respectively. These fragments were kept in 0.5 ml of thioglycolate broth without indicator (Difco) at 4°C for no longer than 5 h before processing. Each specimen was plated on BHM and on Campylobacter-selective medium (CSM) containing brain heart infusion agar with yeast extract (Difco) (5 g/liter), 15% sheep blood, vancomycin (6 mg/liter), nalidixic acid (20 mg/liter), and amphotericin B (2 mg/liter) (Table 1) as growth control. The sequence in which plates were inoculated was varied so that each medium was randomly inoculated first. The plates were then incubated at 37°C in a microaerophilic environment (GasPak disposable H2 and C02 generator envelope; BBL) without palladium catalyst and examined daily, for 7 days, to detect growth and colony appearance of C. pylori; if they were negative, they were discarded. C. pylori was identified by gram-negative appearance, positive oxidase and catalase tests, and a rapidly positive urease test (9, 12).

MATERIALS AND METHODS Initially, we tested 54 strains of C. pylori, isolated from 40 previously studied patients (2) and stored in liquid nitrogen in brucella broth (Difco Laboratories, Detroit, Mich.). All strains were tested for 2,3,5-triphenyltetrazolium chloride (TTC) tolerance and for colonial appearance on a medium named Belo Horizonte medium (BHM). This medium was prepared with a brain heart infusion agar base (Difco), supplemented with 10% sheep blood and containing a final concentration of 6 mg of vancomycin (Sigma Chemical Co., St. Louis, Mo.) per liter, 20 mg of nalidixic acid (Sigma) per liter, 2 mg of amphotericin B (Sigma) per liter, and 40 mg of TTC (Riedel, De Haën, AG Seelze, Hannover, Federal Republic of Germany) per liter (Table 1). For each strain, a saline suspension (equivalent to a McFarland no. 1 standard, ca. 3 x 108 organisms per ml) (7) was made from a 72-h-old culture on brain heart infusion agar (Difco), supplemented with 10% sheep blood, incubated at 37°C in a microaerophilic environment (GasPak disposable H2 and C02 generator envelope; BBL Microbiology Systems, Cockeysville, Md.) without palladium catalyst. This suspension was then inoculated onto BHM. Also, we tested strains of Campylobacterjejuni, Staphylococcus aureus, Staphylococcus epidermidis, Proteus spp., Salmonella spp., Pseudomo-

RESULTS All 54 strains of C. pylori tested for tolerance to TTC (40 mg/liter) grew and exhibited on BHM nonhemolytic tiny circular convex colonies with entire edges and a brilliant golden pigment. Other microorganisms, such as C. jejuni, S. aureus, S. epidermidis, Proteus spp., Salmonella spp., Shigella spp., P. aeruginosa, E. coli, K. pneumoniae, Enterobacter spp., C. albicans, A. faecalis, S. marcescens, S. TABLE 1. Composition of media Components

BHM

CSM

Yeast extract (g/liter) Sheep blood (%)

0 10 6 2 20 40

5 15 6 2

Vancomycin Amphotericin B Nalidixic acid TTC a

*

Corresponding author.

Both media had a brain heart infusion agar base. Amounts are in

milligrams per liter unless otherwise noted.

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INDICATOR MEDIUM FOR ISOLATION OF C. PYLORI

VOL. 25, 1987

TABLE 2. Strains of microorganisms examined for TTC tolerance and for colony appearance on BHM (without antimicrobial agents)a No. of Microorganism strains Strainb Sourceb tested

Campylobacterjejuni Staphylococcus aureus Staphylococcus epidermidis Streptococcus faecalis Streptococcus pyogenes Streptococcus spp. Alcaligenes faecalis Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa Salmonella typhimurium Serratia marcescens Shigella sonnei Candida albicans

32 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Human isolate ATCC 25923 ATCC 14990 ATCC 33186 ATCC 10389 Human isolate Human isolate ATCC 13047 ATCC 25922 ATCC 27736 ATCC 6380 ATCC 27853 ATCC 13311 Human isolate ATCC 11060 Human isolate

HC Difco Roche Difco Roche HC HC Roche Difco Roche Roche Difco Roche HC Roche HC

a All strains were lyophilized except for the C. jejuni strains, which were stored in liquid nitrogen. b HC, Hospital das Clfnicas/Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (isolated by standard methods [13] from 1984 to 1986); ATCC, American Type Culture Collection; Roche, Roche Diagnostics (Nutley, N.J.).

faecalis, S. pyogenes, and Streptococcus spp., were able to grow on the BHM without antimicrobial agents, but their colonies showed neither the typical form nor the color of C. pylori colonies. C. pylori was isolated from 46 of 58 antral biopsy specimens regardless of the culture medium employed. On the other hand, all 16 patients with duodenal ulcer had C. pylori isolated from duodenal biopsies cultured on BHM. With CSM, 13 biopsies were positive and 3 were negative. All the strains first isolated on BHM grew on CSM when subcultured. DISCUSSION When antral biopsies were cultured on BHM, C. pylori was isolated from 46 of 58 biopsies (79.3%). The same result was obtained when CSM was employed. In these cases, the plates showed moderate or heavy growth of the microorganisms. However, the 16 (100%) duodenal biopsies were positive for C. pylori on BHM, while 13 (81.0%) were positive on CSM. When cultures wère only positive on BHM, the density of growth was low (1 to 4 colonies per plate). Furthermore, on CSM the colonies of C. pylori are very small and translucent. In the presence of relatively large colonies of other bacteria, it may be difficult to detect these campylobacters. With BHM, most of the colonies of C. pylori are also small, but they are easily identified by their unique appearance, which differs from that of contaminating microorganisms. The initially colorless tetrazolium salts become pigmented when reduced to insoluble formazan complexes by growing bacteria (7). However, none of the other microorganisms studied exhibited golden colonies despite the fact that they have reduced TTC to a pink-red precipitate of formazan. None of the C. pylori strains was inhibited by TTC.

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Because all the tiny golden colonies on BHM were identified by biochemical tests and Gram morphology as being C. pylori, this medium can facilitate not only C. pylori isolation but also its presumptive identification. It should be pointed out that some C. pylori colonies were seen earlier on BHM than on CSM. For instance, in some cases the colonies could be seen as soon as 48 h after inoculation, and all the positive cases grew in the first 4 days. After 48 h of incubation, bacteria from the colonies of C. pylori showed darting or corkscrew-like motion in the direct microscopic examination and, when stained with a solution of carbolfuchsin diluted 1:2 in distilled water, the smears of these colonies revealed comma-, gull wing-, and S-shaped rods. ACKNOWLEDGMENTS We are grateful to Celso Affonso de Oliveira and Geraldo Ferreira Lima, Jr., who performed the endoscopic biopsies. This study was supported by grants from the Brazilian National Research Council (Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico) Brasil.

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