Description of Campylobacter curvus and C. curvus-Like Strains Associated with Sporadic Episodes of Bloody Gastroenteritis and Brainerd's Diarrhea

Campylobacter curvus is a rarely encountered Campylobacter speciesin human, animal, and environmental samples. During the courseof two investigations, one involving a search for possible bacterialagents causing bloody gastroenteritis and a second concerninga small outbreak of Brainerd's diarrhea in northern California,20 strains of C. curvus or C. curvus-like organisms were isolatedby a microfiltration technique and prolonged incubation. Theresults suggest that C. curvus may be an underappreciated Campylobacterthat may be involved in sporadic and outbreak cases of bloodyor chronic diarrhea in humans.

INTRODUCTION

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Introduction

The genus Campylobacter consists of curved or S-shaped gram-negative,oxidase-positive microaerophilic bacilli with a respiratorytype of metabolism (15). The genus has undergone several taxonomicrevisions over the past decade, including the transfer of anumber of species to either the genus Arcobacter or the genusHelicobacter (12). Sixteen species presently reside in the genusCampylobacter. Of these 16 species, Campylobacter jejuni, C.coli, and C. fetus are the major species of medical, publichealth, or veterinary interest involved in disease processes(12).

Of the 13 remaining Campylobacter species, very little informationregarding their pathogenicity is available, although some species,such as C. upsaliensis, C. hyointestinalis, and C. lari, havebeen associated with various infectious syndromes (1, 3, 13).Even less information about campylobacters such as C. curvusis available. Originally described in 1984 ("Wolinella curva")(14), only four strains of C. curvus were described in thatreport; two strains were associated with the oral cavity, oneisolate was from blood, and the fourth was a clinical isolateof unknown origin. Since that initial report, few studies havereported on the isolation of C. curvus. C. curvus has been isolatedfrom the stools of patients who subsequently presented witheither Guillain-Barré or Fisher's syndrome, althoughno role in the development of these neurologic conditions couldbe associated with this species (8). In a 2000 study of morethan 1,300 human stool specimens conducted to study the prevalenceof campylobacters (other than C. jejuni and C. coli), only 3of 48 campylobacters recovered were tentatively identified asC. curvus-like (4). A study from 2003 (11) reported that 320consecutive liquid or semisolid fecal specimens submitted toa laboratory for enteric pathogen detection were culture negativefor C. curvus, although one stool specimen was positive forC. curvus by 16S rRNA gene PCR. These cumulative results suggestthat the frequency of C. curvus in the gastrointestinal tractsof symptomatic individuals is exceedingly low.

Over a 4-year period, we have isolated 20 strains of C. curvusfrom two separate and distinct clinical settings, namely, froma hospital survey of infectious causes of bloody diarrhea andfrom an outbreak of Brainerd's diarrhea in northern California.These isolations serve as the basis of this report.

MATERIALS AND METHODS

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Materials and Methods

Settings. The first setting from which C. curvus was isolated involvedan analysis of 142 consecutive fecal specimens with a diagnosisof bloody diarrhea submitted to the Microbial Diseases Laboratoryby a large regional microbiology laboratory in a major medicalcenter in Oakland, Calif. These samples originated from individualsin all age groups, although the majority came from adults. Noneof these symptomatic patients had unusual risk factors. All142 stool specimens were either grossly bloody or were positivefor occult blood by the Hemoccult SENSA Fecal Occult Blood Test(Smith Kline Diagnostics, San Jose, Calif.). The period of sampleanalysis ran from October 1995 through February 1996. In thesecond setting, C. curvus was isolated in conjunction with asmall outbreak of Brainerd's diarrhea in Humboldt County innorthern California in March and April 1999. These cases ofwatery diarrhea involved individuals who had had symptoms formore than 4 weeks.

Isolation of C. curvus. Liquid stools or emulsified suspensions of semisolid or solidfeces in phosphate-buffered saline were inoculated onto 0.45-µm-pore-sizefilters placed on sheep blood agar plates (BAPs). The plateswere incubated at room temperature (25°C) for 30 min, afterwhich the filters and any excess fluid were removed and theBAPs were incubated under microaerophilic conditions for 7 days.The cultures were observed daily for evidence of growth.

Biochemical tests and 16S rRNA gene sequencing. A number of biochemical tests were performed with all isolates,based on the tests recommended for use in the differentiationof aerobic, microaerophilic, motile, or helical vibrios or gram-negativebacteria (6). These included tests for catalase, oxidase, andnitrate reductase activities; H₂S production from triple sugariron agar (TSI) and lead acetate paper strips; growth in 1%glycine and 3.5% NaCl; growth at 25, 35, and 42°C; hydrolysisof indoxyl acetate; and sensitivity to cephalothin (30-µgdisk) and nalidixic acid (30-µg disk). The cellular fattyacid (CFA) profiles for all isolates were determined by fattyacid methyl ester analysis (MIS system software, version 3.2C;Microbial Identification Systems, Newark, Del.) (10).

Phylogenetic relatedness studies were performed with all campylobacter-likeisolates by 16S rRNA gene sequencing (500-bp alignment), asdescribed previously (7). Sequence assembly and analysis wereaccomplished with MicroSeq Identification software (AppliedBiosystems, Foster City, Calif.). An isolate was defined asbelonging to a particular species if it exhibited $≤$ 1.2% sequencedivergence from the type strain of a given species.

RESULTS

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Results

Sporadic cases of bloody diarrhea. Of 142 consecutive stool specimens containing macroscopic amountsof blood or occult blood, 15 (10.6%) yielded a campylobacteror a campylobacter-like isolate upon subculture on BAPs. Thesecampylobacter-like isolates typically took between 3 and 7 daysof incubation under microaerophilic conditions before they firstappeared as pinpoint colonies on BAPs. Of the 15 patients fromwhich campylobacter isolates were detected, 3 presented withfrankly bloody stools, while the remainder were occult bloodpositive. No other bacterial pathogens were identified in 12patients, including Salmonella, Shigella, Escherichia coli (sevenpathogenic groups), Yersinia enterocolitica, Vibrio spp., otherCampylobacter species, Aeromonas, Plesiomonas, and Edwardsiellatarda. The stool of one person who was occult blood positivecontained Cryptosporidium, as determined by enzyme immunoassay,while the stools of two other individuals (one of whom presentedwith frankly bloody diarrhea) also harbored diffusely adheringE. coli. The average age of a person from whom a campylobacterisolate was recovered was 42.8 years (age range, 18 months to78 years). The ratio of males to females was 1:2.

Biochemically, these 15 strains met the definition of membersof the genus Campylobacter, as they were nonfermentative, microaerophilicgram-negative rods that were oxidase positive and that grewat both 35 and 42°C (9). Consistent with the biochemicaldata was the result of CFA analysis: a lack of branched-chainfatty acids but the presence of hydroxy acids, with the majorityof CFAs present as straight-chained saturated and monounsaturatedfatty acids. However, neither the biochemical nor the CFA profilesexactly matched the phenotypic and CFA profiles described forexisting taxa in the genus Campylobacter or in the genera Helicobacteror Arcobacter (Table 1). C. curvus strains have been describedas catalase negative and indoxyl acetate positive. However,in our study 50% of the C. curvus strains were catalase positive,while only 15% were indoxyl acetate positive. On TSI, only 40%of our strains produced H₂S, although all were positive by thelead acetate paper method.

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TABLE 1. Phenotypic features of C. curvus and C. curvus-like strains

Brainerd's diarrhea outbreak. During the early part of 1999 an outbreak of Brainerd's diarrhea,characterized by three or more loose bowel movements in a 24-hperiod for $≥$ 4 consecutive weeks, occurred in northern California.At least 23 cases were identified in this outbreak, and stoolsamples from 6 of the symptomatic individuals were submittedfor analysis. Of these six stool samples, five (83.3%) yieldeda campylobacter-like organism with phenotypic properties similarto those associated with bloody diarrhea described above. Noother enteric bacterial or enteric viruses (some samples) wereisolated from these 23 samples, although only some samples weretested for enteric viruses. Five fecal samples obtained fromasymptomatic household or close contacts of these individualswere negative for all enteric pathogens, including Campylobacterspecies.

16S rRNA gene sequencing. Because phenotypic tests, including CFA analysis, failed toyield a definitive answer regarding species identification,the 20 campylobacter-like strains were subjected to 16S rRNAgene sequencing. Overall, 18 of these 20 strains were identifiedas C. curvus by 16S rRNA gene sequencing analysis. These 18strains had a sequence divergence from the type strain of C.curvus (ATCC 35224) ranging from 0 to 1.2% (average divergence,0.64%). One additional strain (MDL 99A2133) had a nonexpressed $~$ 50-bp intervening sequence in the rRNA gene, which complicatedthe analysis, but the strain was also subsequently identifiedas C. curvus by 16S rRNA gene sequencing analysis. A final isolate(MDL 95M387) was unrelated to the genus Campylobacter and hadthe closest match to Aquaspirillum autotrophicum (10.88% divergence)upon alignment of a 500-bp sequence. Strain MDL 95M387 had propertiesidentical or very similar to those of the other strains, withthe exception that it was indoxyl acetate positive. The resultsof an unweighted pair group method analysis with the 15 strainsrecovered from patients with bloody diarrhea is depicted inFig. 1.

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FIG. 1. Dendrogram depicting the linkage of 16 C. curvus or C. curvus-like strains by 16S rRNA gene sequencing by the neighbor-joining method.

DISCUSSION

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Discussion

Since the original description of C. curvus, which includedonly four strains, there have been only a couple of citationsin the literature regarding the isolation of a single strainof this bacterium (4, 8) or the detection of its genetic material(11) from clinical samples. The present report describes forthe first time a large collection of C. curvus strains associatedwith two infectious processes, namely, bloody diarrhea (n =14) and Brainerd's diarrhea (n = 5). Although it cannot be conclusivelyproven that C. curvus was the etiologic agent of either disease,there is compelling evidence that suggests that this was, infact, the case. With the exception of diffusely adhering E.coli as a potential but still controversial copathogen in twopatients and a Cryptosporidium isolate that infected one individual,no other enteric pathogens were identified in the feces of symptomaticindividuals in either group containing C. curvus. The fact that>10% of persons who presented with frankly bloody stoolsor who were positive for occult blood were positive for C. curvusand the fact that this species is rarely isolated from gastrointestinalsamples in large-scale studies (4, 8) strongly suggest a causalrole in this setting. Furthermore, in the Brainerd's diarrheaoutbreak, C. curvus was isolated from five of six sick patientsbut not from any of five symptomless household or close contactsof the infected individuals.

The phenotypes of the strains identified as C. curvus in thisstudy deviated from the ideal phenotype in several characteristics(Table 1). These deviations are because of the small subset(n = 4) of C. curvus strains used to establish the biochemicalprofile of this species in the original publication (14) orbecause of methodological differences. The latter possibilitycan be seen in the test results for H₂S production by two differentmethods. Sequencing of the 16S rRNA gene indicates that severalstrains generated 100% sequence matches (no divergence) to thetype strain of C. curvus, further supporting their identification.While there are no criteria or established values for 16S rRNAgene sequence similarity for species identification, one recentstudy established a similarity score of $≥$ 99% (2), which is verysimilar to that used in the present study. Whether strains withdivergence values of $~$ 1% are in fact C. curvus or a phylogeneticallyrelated and biochemically similar but distinct taxon await DNA-DNAhybridization studies. One strain of C. curvus (MDL 99A2133)was difficult to identify because of an intervening sequence.However, this phenomenon has previously been reported in Campylobacterspecies, including C. rectus, C. sputorum, and C. curvus (5).The exact reason why these intron-like DNA sequences are insertedwithin ribosomal genes is unknown.

The large number of C. curvus strains identified in the presentstudy appears to result from two laboratory procedures, namely,inoculation of stool suspensions onto a 0.45-µm-pore-sizefilter and prolonged incubation (>3 days) of BAPs in a microaerophilicenvironment. The significance of the isolation of C. curvusfrom gastrointestinal specimens will require additional prospectivestudies evaluating the frequency and occurrence of this speciesin distinct diarrheal disease syndromes.

FOOTNOTES

* Corresponding author. Mailing address: Microbial Diseases Laboratory, California Department of Health Services, Room E164, 850 Marina Bay Parkway, Richmond, CA 94804. Phone: (510) 412-3700. Fax: (510) 412-3722. E-mail: jjanda{at}dhs.ca.gov.

Present address: Accugenix, Newark, DE 19702.

REFERENCES

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