Isolation and characterization of catalase-negative and catalase-weak ...

Vol. 27, No. 9

JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1989, p. 2042-2045

0095-1137/89/092042-04$02.00/0 Copyright © 1989, American Society for Microbiology

Isolation and Characterization of Catalase-Negative and CatalaseWeak Strains of Campylobacter Species, Including "Campylobacter upsaliensis," from Humans with Gastroenteritis DIANE E. TAYLOR,'.2* KOJI HIRATSUKA,2 AND L. MUELLER3

Departments of Microbiology' and Medical Microbiology and Infectious Diseases,2 University of Alberta, Edmonton, Alberta, Canada T6G 2H7, and Provincial Laboratory of Northern Alberta, Edmonton, Alberta, Canada T6G 2J23 Received 13 March 1989/Accepted 7 June 1989

During 1987 and 1988, nine strains of catalase-negative or -weak Campylobacter species were isolated in Alberta, Canada. DNA hybridization studies demonstrated that seven strains were "Campylobacter upsaliensis," one strain was highly homologous with Campylobacterjejuni DNA, and one strain was a campylobacter unrelated to the other two species. Ali "C. upsaliensis" strains were hippurate negative, and six of seven were susceptible to cephalothin. The unusual variant of C. jejuni was hippurate positive and cephalothin resistant, whereas the unclassified strain was hippurate negative and resistant to intermediate levels of cephalothin. All patients from whom "C. upsaliensis" was isolated had diarrhea. Five of the patients were children two years old or younger, and two were adults. In this study, all catalase-negative and -weak strains were isolated from stool specimens by using a charcoal-based selective medium containing 32 ,ug of cefaperazone per ml and which was described by Hutchinson and Bolton (D. N. Hutchinson and F. J. Bolton, J. Clin. Pathol. 37:956-957, 1984). The

genus Campylobacter comprises 14 species, which recently reviewed in detail (19). Of these species, Campylobacterjejuni is responsible for the majority of cases of campylobacter enteritis in developed countries, with Campylobacter coli responsible for about 2 to 5% of the cases (10, 28). In contrast, C. coli is isolated with higher frequency in underdeveloped nations (7). Both produce catalase, and for many years it has been convenient to divide the Campylobacter genus into pathogens and nonpathogens based on catalase production (2). As discussed by Penner, this grouping has become less relevant since the discovery of the nonpathogenic, catalase-positive species Campylobacter cryaerophila and Campylobacter nitrofigilis (19). However, many clinical laboratories continue to test Campylobacter species for catalase production. In addition to C. jejuni and C. coli, several other catalase-positive Campylobacter species, Campylobacter fetus subsp. fetus (3, 4), Campylobacter laridis (22, 25), and Campylobacter hyointestinalis (5), are occasionally responsible for cases of human gastroenteritis. Catalase-negative or -weak (CNW) strains of Campylobacter were first isolated from dogs, both with and without diarrhea, by Sandstedt et al. (21). DNA hybridization studies demonstrated that they belonged to a new species (20, 21, 24), and the name "Campylobacter upsaliensis" has been proposed for them (K. Sandstedt and J. Ursing, Abstr. XIV Int. Congr. Microbiol., P.B8-19, 1986). CNW strains have been isolated from stools of children in Bordeaux, France (13), and in Central Australia (24). Recently, Patton and co-workers isolated and characterized 11 "C. upsaliensis" strains from human patients. They demonstrated that "C. upsaliensis" is a potential human pathogen associated with gastroenteritis and bacteremia in normal individuals, as well as with opportunistic infections in immunocompromised patients (18). Strains of Campylobacter have been isolated and mainwere

*

tained in storage at the Provincial Laboratory of Northern Alberta since October 1977. Over the last 2 years, several CNW strains have been identified. Therefore, data on Campylobacter strains isolated over the past 3 years were examined to determine the frequency of various Campylobacter species, including CNW strains, isolated. DNA from CNW strains was hybridized with DNA probes specific for C. jejuni or "C. upsaliensis." Patient records, when available, were reviewed to determine details of the ages of patients and their symptoms. Most CNW strains were shown to be "C. upsaliensis" by DNA homology studies. However, one catalase-negative variant of C. jejuni was also identified. "C. upsaliensis" was isolated from both adults and children with gastroenteritis.

MATERIALS AND METHODS Bacterial strains. Within a 3-year period, 915 strains of Campylobacter were isolated from stool specimens at the Northern Alberta Provincial Laboratory. Some stool specimens were submitted in duplicate, but repeated cultures were not counted in the total figures quoted in Table 1. Isolation procedure. Fecal samples were spread directly onto the blood-free medium with charcoal devised by Hutchinson and Bolton (8) and marketed by Oxoid Ltd., Basingstoke, United Kingdom, as Campylobacter blood-free selective medium (modified CCDA-Preston), which contains 32 ,ug of cefoperazone per ml. Plates were incubated in a microaerophilic gas mixture containing 85% N2, 10% C02, and 5% °2 at 42°C. Plates were examined daily and held for 2 days before being discarded. Antibiotic susceptibilities. Resistance to antibiotics was determined by the disk diffusion method of Bauer et al. (1). The antibiotics used per disk were cephalothin (30 ,ug), nalidixic acid (30 ,ug), erythromycin (2 p.g), tetracycline (30 ,ug), and kanamycin (30 ,ug). Phenotypic characterization. Gram-negative, motile-curved, or spiral rods forming typical Campylobacter colonies on charcoal agar were tested for oxidase, catalase, and hippu-

Corresponding author. 2042

CHARACTERIZATION OF CNW CAMPYLOBACTER SPP.

VOL. 27, 1989

TABLE 1. Isolation of Campylobacter species in Alberta, Canada, from 1986 to 1988 No. (%) of strains isolated in:

Species

1986 (n = 340)

1987 (n = 280)

1988 (n = 295)

C. jejuni C. coli C. fetus subsp. fetus "C. upsaliensis" C. laridis

312 (91.8) 27 (7.9) 0 0 1(0.3)

251 (89) 21 (7.5) 3 (1.1) 4 (1.4) 1 (0.4)

279 (95) 10 (3.4) 1 (0.3) 3 (1.0) 2 (0.7)

C. jejuni probe

A

RESULTS Frequency of different Campylobacter species isolated from stool specimens from 1986 to 1988. In Northern Alberta, Canada, 89 to 95% of campylobacters isolated from stool specimens were C. jejuni (Table 1). C. coli accounted for 3 to 8% of the isolates, whereas 1% or less were C. fetus subsp. fetus. Since 1987, CNW strains of Campylobacter species have been isolated in Alberta, accounting for about 1% of the Campylobacter isolates. Nalidixic acid-resistant, thermo-

B

1 2 *

C .

'-

D E

F

*

*

G

H

i

*

3

C. 'upsaliensis probe

A t

rate hydrolysis. The oxidase test was performed by spotting colonies onto filter paper impregnated with tetramethylp-phenylenediamine. Catalase was detected by using 3% H202 in a slide test. Catalase tests were considered positive if definite bubbles were formed rapidly. If bubbles formed very slowly, strains were considered to be weak catalase producers. The absence of bubbles was considered negative. The test was run in duplicate for each isolate. Hippurate hydrolysis was determined as described previously (9). All Campylobacter species were tested for growth at 25, 37, and 42°C. C. fetus subsp. fetus was identified by growth at 25°C (but not at 42°C), resistance to nalidixic acid, and susceptibility to cephalothin. "C. upsaliensis" E11173-87 was confirmed previously as "C. upsaliensis" by H. Lior, National Reference Service for Campylobacters, Laboratory Centre for Disease Control, Tunney's Pasture, Ottawa. Definitive tests for "C. upsaliensis" are growth at 42 but not at 25°C, phenotypes that are CNW, hippurate negative, and negative for the production of H2S in triple sugar iron medium, inability to grow anaerobically in trimethylamine-N-oxide, inability to grow in the presence of 1% glycine, and inability to grow in 3.5% NaCl (21). DNA hybridization studies. Total DNA from Campylobacter species was extracted as described previously (6) by a modification of the method of Marmur and Doty (12). DNA was applied to nitrocellulose filters with a Bio-Dot apparatus (Bio-Rad Laboratories, Richmond, Calif.). The filters were processed and hybridized with DNA probes as described previously by Ng et al. (15). Hybridization was performed both at 37 and 42°C in 50% formamide for 16 h. DNA probes. The probes used were the following: (i) C. jejuni chromosomal DNA from strain NCTC 11168 (UA580), (ii) a C. jejuni-specific DNA probe consisting of a 1,475base-pair fragment of C. jejuni DNA encoding a major outer membrane protein (D. E. Taylor and K. Hiratsuka, manuscript in preparation), (iii) chromosomal DNA from C. laridis NCTC 111352 (UA577), and (iv) "C. upsaliensis" chromosomal DNA from strain E11173-87. DNA probes were labeled in vitro with [at-32P]dATP (New England Nuclear Corp., Boston, Mass.) by nick translation (11). The labeled DNA had specific activities of 107 to 108 cpm/,lg of DNA.

2043

B

*-

C

D

E

.*

F

G

H

e

2

3 FIG. 1. DNA hybridization of Campylobacter species with DNA probes for C. jejuni and "C. upsaliensis." Autoradiograms of dot blot hybridization with a C. jejuni DNA probe (a 1,475-base-pair fragment of C. jejuni DNA, described in text) and a "C. upsaliensis" total chromosomal DNA probe from strain E11173-87 as shown on the figure. CNW strains are in the following positions: E4116-88 (1A), E5520-88 (lB), E6755-87 (1C), E6787-87 (1D), E7603-88 (lE), E8230-87 (1F), E8258-87 (1G), E11117-88 (1H), and E11173-87 (11); see Table 2 for details of strain characteristics. Additional strains of C. jejuni (2A through 2H), C. coli (3A through 3D), C. laridis (3F and 3G), and C. fetus subsp. fetus (3H and 31) are included in the blots. Each dot represents 5 x 10' cells, except for 2E and 2G, in which the cell number was about 10-fold lower. Hybridization was performed at 37°C in 50% formamide for 16 h.

philic campylobacters were isolated infrequently during the 3-year period, making up 0.3 to 0.7% of the total number of isolates (Table 1). DNA hybridization studies. All catalase-negative and catalase-weak campylobacters were hybridized with DNA probes for "C. upsaliensis" and C. jejuni. The "C. upsaliensis" probe consisted of total chromosomal DNA from strain E11173-87. C. jejuni probes were total chromosomal DNA from C. jejuni UA580 (15) and a specific 1,475base-pair probe for C. jejuni which does not hybridize with C. coli (D. E. Taylor and K. Hiratsuka, unpublished data). Seven of nine CNW strains hybridized to "C. upsaliensis" DNA (Fig. 1). No DNA homology was observed between these seven strains and either of the C. jejuni DNA probes. Thus, seven of nine CNW strains belong to "C. upsaliensis." One of the CNW strains, E6755-87, which did not hybridize to "C. upsaliensis" DNA, hybridized to both C. jejuni DNA probes. The other CNW strain, E11117-88, hybridized to neither C. jejuni probes nor "C. upsaliensis" probes and, therefore, does not belong to either species. Hybridizations were performed at both high (42°C) and low (37°C) stringencies as determined by Ng et al. (16) with similar results. Moreover, both C. jejuni total chromosomal DNA (data not shown) and the 1,475-base-pair C. jejunispecific probe (Fig. 1) gave similar results with DNA from CNW strains. To ensure that the C. laridis strains shown in Table 1 belong to that species rather than being nalidixic acidresistant variants of C. coli, which may be selected by mutation (27), all four putative C. laridis strains were hybridized with C. laridis UA577 DNA. The four strains were identified as belonging to C. laridis by their homology with UA577 DNA (data not shown). Characteristics of CNW strains of Campylobacter species. All strains grew at 37 and 42°C, but none grew at 25°C, except for E6755-87, which showed weak growth after 48 h.

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TAYLOR ET AL.

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TABLE 2. Characteristics of CNW Campylobacter spp. Strain

Catalase

Strainproductiona

E6755-87 E6787-87 E8230-87 E8258-87 E11173-87 E4116-88 E5520-88 E7603-88 E11117-88

W w

W W -

Hippurate hydrolysis

Cephalothin

+ -

R R S S S S S S I

resistance

Hybridization with DNA probes" C. jejuni "C. upsaliensis" + -

+ + + + + + + -

Age 4 18 15 6 2 18 38 31 31

yr mo mo mo yr mo yr yr yr

Patient details Symptoms

Diarrhea Diarrhea Diarrhea Diarrhea Diarrhea Diarrhea Diarrhea Diarrhea Abdominal pain

a Catalase production was absent (-) or weak (W). b Isolates were determined to be resistant (R) or susceptible (S) to cephalothin (as determined by use of a 30-p.g disk), whereas the intermediate (1) level gave a zone of 14 to 15 mm. c See text for details of probes used. d Details of patients from whom strains were isolated.

Of the nine strains isolated during 1987 and 1988 (Table 2), five produced no catalase and four were weak catalase producers. All strains except one were hippurate negative. The single catalase-negative, hippurate-positive strain (E6755-87), which hybridized with C. jejuni DNA but not with "C. upsaliensis" DNA, has been included with the C. jejuni strains in Table 1. All catalase-negative, hippuratenegative strains except one hybridized with "C. upsaliensis" DNA but not with C. jejuni DNA. The exception was E11117-88, a hippurate-negative, catalase-negative strain, which appears to be unrelated to either C. jejuni or "C. upsaliensis." Antibiotic susceptibilities. Of the nine CNW strains examined, six were susceptible to cephalothin as determined by use of a 30-,ug disk, whereas strain E11117-88 was resistant to an intermediate level of cephalothin. The hybridization studies above demonstrated that all but one (E6787-87) of the "C. upsaliensis" strains were susceptible to cephalothin. The C. jejuni strain E6755-87 was resistant to 30 ,ug of cephalothin. All CNW strains were susceptible to nalidixic acid, erythromycin, kanamycin, and tetracycline. Clinical features associated with isolation of "C. upsaliensis" from stool specimens. Of the seven CNW catalaseproducing strains shown by DNA hybridization studies to belong to "C. upsaliensis," five were isolated from specimens from children 2 years of age or younger and two were isolated from specimens from adults (Table 2). All of the patients were known to have diarrhea. No other potentially pathogenic bacteria were found in any of the fecal specimens. However, two of the children from whom "C. upsaliensis" was isolated also harbored Giardia lamblia cysts in their stools. The catalase-negative C. jejuni strain was isolated from a stool specimen from a patient who was heavily infested with various intestinal parasites. This patient had recently settled in Alberta and had originally lived in South America. However, in three other stool specimens from which "C. upsaliensis" was isolated, there was no evidence of intestinal parasites. The physician did not request examination of the two adults for intestinal parasites (Table 2). DISCUSSION Several studies have implicated CNW strains of Campylobacter species as a cause of diarrhea. Megraud and Bonnet (13) isolated CNW strains of Campylobacter from several children with intestinal disturbances and diarrhea in Bordeaux, France. However, DNA hybridization was not per-

formed on these strains. Steel et al. (24) demonstrated CNW strains in fecal specimens from children in Central Australia, and these strains were shown to hybridize with CNW strains isolated in Sweden (21). More recently, Patton et al. demonstrated definitively that "C. upsaliensis" is responsible for enteritis in both adults and children (18). Our study presents additional evidence that "C. upsaliensis" can cause diarrhea and that both children and adults are affected. Antimicrobial agent susceptibility testing demonstrated that all CNW strains were susceptible to nalidixic acid, kanamycin, and tetracycline. Plasmid-mediated resistance to kanamycin and tetracycline has been reported in C. jejuni and C. coli (26), but neither appears to have spread yet to "C. iupsaliensis." Erythromycin is the drug of choice for treatment of Campylobacter infections. All the CNW strains in this study were susceptible to erythromycin, although Patton et al. (18) reported that two of their isolates were resistant to erythromycin (MIC, 8 Fxg/ml). Resistance to erythromycin in "C. upsaliensis," as well as in C. jejuni and C. coli, should continue to be monitored. Although "C. upsaliensis" is catalase negative or demonstrates only weak catalase production, a catalase test alone cannot be used to differentiate this species. Unusual variants of C. jejuni, such as E6755-87, can also be catalase negative. The former may be differentiated superficially from the majority of the "C. upsaliensis" strains by its ability to hydrolyze hippurate and its resistance to cephalothin, although some variants of "C. upsaliensis," such as strain E6787-87, are also resistant to cephalothin. In the case of E11117-88, which is catalase negative, hippurate negative, and resistant to intermediate levels of cephalothin, DNA hybridization studies demonstrated that the strain is a member of neither C. jejuni nor "C. upsaliensis" and may belong to a new species of Campylobacter. Prior to 1987, CNW strains, including those of "C. upsaliensis," were not isolated in Alberta. Does this mean that a new species has emerged with pathogenic potential, or does the increase in isolation frequency depend on some other factor(s), such as temperature of isolation or selection medium used? We have observed previously that C. jejuni and especially C. coli are inhibited by antibiotics used in selective media (14, 17). In the previous studies in which CNW strains were isolated, investigators used a filtration technique to obtain campylobacters from fecal specimens in conjunction with a nonselective culture medium (13, 21, 24). Moreover, Patton et al. also reported that none of their 12

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CHARACTERIZATION OF CNW CAMPYLOBACTER SPP.

isolates nor the "C. upsaliensis" reference strain grew on the Campylobacter-selective medium (18). All "C. upsaliensis" strains in this study, as well as the catalase-weak C. jejuni and the unclassified CNW strain, were isolated on a charcoal medium (modified CCDA-Preston) which contains 32 ,ug of cefoperazone per ml as the only antibiotic (8). This medium has been in use at the Provincial Laboratory of Northern Alberta since August 1985; prior to this, Skirrow medium (23) had been in use since 1977. Plates for selection of Campylobacter species were incubated at 42°C, as they had been since 1977. It seems that the modified CCDAPreston medium preferentially selected for "C. upsaliensis" strains, comparable to Skirrow medium. Yet why were none of these strains isolated during 1986? Although the reasons for the enhanced isolation frequency of "C. upsaliensis" strains are not yet completely understood, our study demonstrates that CNW strains can be isolated by using the blood-free, charcoal-based medium of Hutchinson and Bolton (8) as well as by the filtration technique. ACKNOWLEDGMENTS We thank D. L. J. Tyrrell for his interest in this study. This work was supported in part by a strategic grant (Biotechnology) from the Natural Sciences and Engineering Research Council of Canada to D.E.T., who received a scholarship from the Alberta Heritage Foundation for Medical Research. LITERATURE CITED 1. Bauer, A. W., W. M. Kirby, J. C. Sherris, and M. Turek. 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45:493-496. 2. Bryner, J. H., and A. H. Frank. 1955. A preliminary report on the identification of Vibrio fetus. Am. J. Vet. Res. 16:76-78. 3. Deviin, H. R., and L. McIntyre. 1983. Campylobacter fetus subsp. fetus in homosexual males. J. Clin. Microbiol. 18: 999-1000. 4. Edmonds, P., C. M. Patton, T. J. Barrett, G. M. Morris, A. G. Steigerwalt, and D. J. Brenner. 1986. Biochemical and genetic

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