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J Clin Microbiol. Jun 2006; 44(6): 2019–2024.
PMCID: PMC1489434

Identification of Campylobacter fetus Subspecies by Phenotypic Differentiation and PCR

Abstract

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis and C. fetus subsp. fetus, which differ in their epidemiologies and clinical importance. The differences between these subspecies make accurate distinction between the two essential. First, the value of seven key tests for the traditional differentiation of C. fetus was investigated. Afterwards, the results of the phenotypic differentiation and PCR were compared to address the question of the reliability of this PCR assay. Altogether, 103 C. fetus isolates were investigated, including the type strains of C. fetus subsp. fetus and C. fetus subsp. venerealis. Depending on the result of the glycine tolerance test, the isolates could be separated into 81 C. fetus subsp. venerealis isolates (glycine intolerant) and 22 C. fetus subsp. fetus isolates (glycine tolerant). For all C. fetus subsp. venerealis strains tested, the results of the selenite reduction assay and sensitivity to metronidazole and cefoperazone completely agreed with the results of the glycine tolerance test (correspondence, 100%). Seventy-three C. fetus subsp. venerealis isolates did not grow at 42°C (correspondence, 90.1%), but eight isolates showed a faintly discernible, flat, dark gray growth. For 22 C. fetus subsp. fetus isolates, the results of additional phenotypic tests only partly agreed with the results of the glycine tolerance test. For C. fetus subsp. fetus the results of the glycine tolerance test showed a relatively good correspondence with those of the selenite reduction assay (correspondence, 81.8%), assays for cefoperazone resistance (correspondence, 86.4%), and assays for growth at 42°C (correspondence, 81.8%). The results of the glycine tolerance test and PCR completely agreed for the 103 C. fetus isolates tested. We conclude that at present the traditional phenotypic characterization of C. fetus subspecies under strongly defined conditions remains indispensable, but this PCR assay constitutes a valuable adjunctive technique for the confirmation of phenotypic results.

The species Campylobacter fetus is divided into the subspecies C. fetus subsp. venerealis and C. fetus subsp. fetus, which differ in their epidemiologies and clinical importance. C. fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis (bgC). This bacterium is characterized by a strong tropism for the genital tract of cattle and causes abortions (enzootic abortion). Infections caused by C. fetus subsp. fetus must be separated from those caused by C. fetus subsp. venerealis. The natural habitat of C. fetus subsp. fetus is the intestinal tract of cattle, but it can also cause abortions (sporadic abortion). The differences between these subspecies make accurate distinction essential (7). At present, the traditional differentiation between both subspecies is based on a single phenotypic test (tolerance to 1% glycine) in current phenotypic identification schemes, including the recommended international standard (1). Chang and Ogg (3) have shown that the glycine tolerance characteristic may not be reliable for separation of the two subspecies because glycine tolerance can be acquired by transduction or mutation. Additionally, glycine-tolerant variants of C. fetus subsp. venerealis (referred to as biovar “intermedius”) have been described (14), although some evidence suggests that the strain characteristics are artifacts due to the inappropriate standardization of biochemical tests (13, 9). Furthermore, glycine-sensitive C. fetus subsp. fetus isolates have been observed (13). Recommendations for performing most phenotypic tests have been made but have not yet been widely adopted (8). The extent to which the divergent results in some laboratories may be due to the use of inappropriate methods is unknown (7). This situation emphasizes the need to base subspecies-level designations of C. fetus on more than one phenotypic reaction.

In recent years, molecular methods for distinguishing between the C. fetus subspecies (including PCR and pulsed-field gel electrophoresis [PFGE]) have been developed to substantiate the results of phenotypic differentiation and to avoid misidentification. In 2003, we reported on a specific PCR-based assay for identifying and differentiating the two C. fetus subspecies (6). This assay was originally described by Hum et al. (5) in 1997 and was adopted in our laboratory. In brief, a species-specific 764-bp amplicon is produced with primers MG3F and MG4R with the DNA of both subspecies of C. fetus. Afterwards, primers VenSF and VenSR are used for differentiation. The identification of C. fetus subsp. venerealis is based on the presence of a 142-bp amplicon, which is not formed by C. fetus subsp. fetus. Seventy-three field strains were investigated by this PCR assay (including 24 C. fetus subsp. venerealis strains and 26 C. fetus subsp. fetus strains). No discrepancies could be observed between the results of phenotypic differentiation and those of PCR. However, the reliability of this PCR was questioned (1). The subspecies of up to 10% of the strains were not correctly identified by this PCR assay, as described in the original paper (5), and this observation was confirmed in a later study (17). Therefore, we started an investigation to address this question. First, we investigated the value of seven key tests for the traditional differentiation of C. fetus isolates (sensitivity to glycine, metronidazole, cefoperazone, KMnO4, basic fuchsin and selenite reduction, and growth at 42°C), as recommended by On and Harrington (13). Afterwards, we compared the results of the phenotypic differentiation and PCR assays to answer the question of the reliability of the PCR assay that is described.

MATERIALS AND METHODS

Bacterial isolates.

Table Table11 shows details of the origins of the isolates tested. All C. fetus subsp. venerealis strains were isolated from bovine specimens. The C. fetus subsp. fetus isolates came from different sources. The organism can cause sporadic abortions in cattle, but it is also one of the causative agents of ovine abortion. In our collection, 9 of 22 strains were isolated from ovine fetuses. Altogether, 103 C. fetus strains were investigated, including the type strains C. fetus subsp. fetus (DSMZ 5361) and C. fetus subsp. venerealis (NCTC 010354). Seventy-seven C. fetus field strains were isolated in the Federal Republic of Germany between 1999 and 2005 and were submitted to the National Reference Laboratory of bgC. Twenty-six C. fetus strains had been already isolated between 1985 and 1994 and derived from different collections. The primary phenotypic identifications of the strains were made by the laboratories submitting the strains.

TABLE 1.
Origins of C. fetus isolates and results of phenotypic and PCR typing

Phenotypic differentiation.

No standard methods for the phenotypic differentiation of C. fetus subspecies have been published. Most guidelines do not give explicit details on the minimum test requirements or test methodologies, and many workers use methods peculiar to their own laboratories. Differences in methods may lead to differences in the outcome of nominally the same test. Recommendations for performing the tests are given by On and Holmes (10, 11, 12). These recommendations were considered in our investigations.

Culture conditions.

The basal media used were as follows: (i) Mueller-Hinton (MH) agar (TN 1162; Institut für Immunpräparate und Nährmedien, Berlin, Germany) supplemented with 10% calf blood and (ii) nutrient broth no. 2 (CM 67; Oxoid, Wesel, Germany).

The glycine medium (1%; 17-1323-01; Pharmacia Biotech, Uppsala, Sweden) was prepared by adding the test substance to MH agar before it was melted at 100°C. Metronidazole (4 mg/liter; M-1547; Sigma, Taufkirchen, Germany) as a stock solution of 4 mg per 10 ml N,N-dimethylacetamide (803235; Merck, Darmstadt, Germany), cefoperazone (64 mg/liter; C-4292; Sigma), and fuchsin (160 mg/liter; 1.15937; Merck) as a stock solution of 160 mg per 10 ml 96% ethanol (filter sterilized) were added to autoclaved MH agar after the agar was cooled to approximately 56°C.

Selenite (0.1%; sodium selenite pentahydrate; 1.06607; Merck,) and KMnO4 (0.1% potassium permanganate; 1.59231; Merck) media were prepared by adding filter-sterilized solutions of these substances aseptically to nutrient broth no. 2.

The Campylobacter strains were grown microaerobically on MH agar plates for 72 h at 37°C. The bacteria were harvested from the plates with phosphate-buffered saline, and the suspensions were adjusted spectrophotometrically (CADAS 30 photometer; Lange GmbH, Berlin, Germany) to an optical density of 0.1. This density corresponded to 1.1 × 107 to 9.8 × 107 CFU per ml for C. fetus subsp. venerealis and 1.0 × 108 to 3.9 × 108 CFU per ml for C. fetus subsp. fetus. The suspensions of bacteria were diluted 1:10 for C. fetus subsp. venerealis and 1:100 for C. fetus subsp. fetus. The agar plates were inoculated with 20 μl of these dilutions in duplicate, and the spots were allowed to dry (for no more than 15 min). In this way, the number of culturable bacteria was used as a comparable basis for inoculation of the solid media.

In a similar way, liquid media (5 ml) were inoculated with 100 μl of undiluted suspension for C. fetus subsp. venerealis (optical density, 0.1 [see above]) and with 100 μl of a 1:10 dilution of suspension for C. fetus subsp. fetus.

All solid media supplemented with the test substances and the KMnO4 medium were microaerobically incubated for 72 h (selenite medium was incubated for 120 h) at 37°C. In the same way, inoculated MH agar plates without test substances were incubated at 42°C and 37°C (control).

Evaluation.

In all series of investigations, type strains of both C. fetus subspecies were used as controls. Each test was performed at least two times on separate occasions and on freshly prepared media in order to assess its reproducibility. Any growth on the test plates was compared with that on the accompanying unsupplemented basal medium (control plates). Table Table11 shows all criteria of evaluation. The growth of the bacteria on the agar plates was evaluated with a stereomicroscope, and the growth was allocated to a scale of − to ++++. After inoculation of the MH agar plates (incubation for 48 h at 37°C), the inhibition of the bacteria in the liquid medium supplemented with KMnO4 was assessed in the same way. Selenite reduction was evaluated on the basis of the reddening of the bouillon.

Identification of C. fetus subspecies by PCR.

For DNA extraction, 2 ml of broth culture was centrifuged at 16,100 × g for 10 min. The pellet was resuspended in 200 μl of phosphate-buffered saline. DNA was extracted by using a High Pure PCR template preparation kit (Roche Diagnostics, Mannheim, Germany), according to the instructions of the manufacturer.

PCRs were carried out in two separate reactions. For detection of the C. fetus species, primer pair MG3F-MG4R (5) was used with a modified program. An initial denaturation (96°C for 60 s) was followed by 35 cycles of denaturation (96°C for 15 s), primer annealing (60°C for 60 s), and primer extension (72°C for 90 s). The reaction was terminated by a final extension step (72°C for 180 s). The same PCR program was used for the differentiation of C. fetus subsp. venerealis with primers VenSF and VenSR (5). Amplifications were carried out with a T3 thermocycler (Biometra, Göttingen, Germany) and the following conditions: a 50-μl PCR mixture contained 5 μl 10× PCR buffer (Roche Diagnostics), 2 μl deoxynucleoside triphosphate mixture (each deoxynucleoside triphosphate at 2 mM; Roche Diagnostics), 0.2 μl Taq DNA polymerase (Roche Diagnostics), 1 μl of the forward and reverse primers (10 pmol/μl; JenaBioScience, Jena, Germany), and 1 μl of DNA extract.

Analysis of the PCR products was carried out by agarose gel electrophoresis with 1.5% agarose gels. After ethidium bromide staining, visualization of the bands was done under UV light. A 100-bp DNA ladder (Genaxxon BioScience, Biberach, Germany) was used as a size marker.

RESULTS

Identification of C. fetus subspecies by phenotypic testing.

Table Table11 shows the details of the identification of the C. fetus subspecies isolates by phenotypic tests. In the course of the evaluation, the results of the phenotypic tests were summarized by the following classification: −, (+), and + reactions were classified as negative and ++, +++, and ++++ reactions were classified as positive. Depending on the result of the glycine tolerance test, the isolates were designated as C. fetus subsp. venerealis (glycine intolerant) and C. fetus subsp. fetus (glycine tolerant). In this way, the 103 C. fetus isolates could be separated into 81 C. fetus subsp. venerealis isolates and 22 C. fetus subsp. fetus isolates. Glycine-tolerant variants of C. fetus subsp. venerealis and glycine-sensitive variants of C. fetus subsp. fetus could not be observed.

Table Table22 shows the expected reactions of the C. fetus subspecies in the key tests in comparison to our results. For all C. fetus subsp. venerealis field strains, the results of selenite reduction and sensitivity to metronidazole and cefoperazone completely agreed with glycine tolerance (correspondence, 100%) (Table (Table2).2). Seventy-three C. fetus subsp. venerealis isolates did not grow [−, (+), and + reactions] at 42°C (correspondence, 90.1%), but 8 isolates showed a faintly discernible, flat, dark gray growth, indicated by ++ in Table Table1.1. Unlike the C. fetus subsp. venerealis isolates, the C. fetus subsp. fetus isolates showed a luxurious, light gray growth (with three exceptions), which enabled easy differentiation of the isolates.

TABLE 2.
Correspondence of glycine tolerance with other phenotypic tests and PCR in C. fetus isolatesa

For 22 C. fetus subsp. fetus isolates, the results of four additional phenotypic tests only partly agreed with the glycine tolerance test results. The numbers of isolates with the expected results were as follows: 18 for selenite reduction (correspondence, 81.8%), 9 for metronidazole resistance (correspondence, 40.9%), 19 for cefoperazone resistance (correspondence, 86.4%), and 18 for growth at 42°C (correspondence, 81.8%).

The inhibitory effects of basic fuchsin and KMnO4 against the C. fetus isolates were not reproducible. Numerous modifications of these tests did not improve the reproducibility (results not shown).

Identification of C. fetus subspecies by PCR.

All C. fetus strains were tested by PCR for their subspecies identity by using the primer systems described by Hum et al. (5). Eighty-one of 103 isolates were identified as C. fetus subsp. venerealis; the others were identified as C. fetus subsp. fetus. All PCR results confirmed the subspecies identities obtained by the glycine tolerance tests (Table (Table11).

It was remarkable that the amplicons obtained by the species-specific C. fetus PCR were about 750 bp (Fig. (Fig.1),1), nearly 200 bp smaller in fragment length, as originally described by Hum et al. (5).

FIG. 1.
Agarose gel electrophoresis of PCR products obtained by using primer pairs MG3F-MG4R (lanes 1 to 7) and VenSF-VenSR (lanes 9 to 15). Lanes: 1 and 9, C. fetus subsp. venerealis 69/05; 2 and 10, C. fetus subsp. venerealis 70/05; 3 and 11, C. fetus subsp. ...

DISCUSSION

Resistance to 1% glycine was used by Florent (4) as a criterion to separate C. fetus into the two subspecies C. fetus subsp. fetus and C. fetus subsp. venerealis (former designation, Vibrio fetus with the varieties Vibrio fetus var. intestinalis and Vibrio fetus var. venerealis). For decades the glycine tolerance test has been the only reaction (“gold standard”) for the differentiation of C. fetus subspecies. Some months ago, the international standard for the diagnosis of bgC was revised (1), but this test remains the only reaction recommended for this purpose. However, the phenotypic differentiation of C. fetus isolates can be a cumbersome task. In some laboratories, variants of both C. fetus subspecies have been observed. At present, it cannot be excluded that these divergences are based on insufficiently standardized methods. In many aspects, the C. fetus subspecies are very similar (7). Therefore, the differences are more quantitative than qualitative in most reactions. For that reason, the standardization of phenotypic tests is a decisive requirement. It is well established in antimicrobial susceptibility testing that the inadequate or inappropriate standardization of the inocula can result in false-positive or -negative results. In studies on the phenotypic characterization of Campylobacter species, false-positive results were obtained by several key tests, generally when the inoculum size exceeded 107 to 108 CFU per ml (10). They indicated that for some commonly used tolerance tests, an inoculum size of approximately 106 CFU per ml is the most appropriate when blood agar is used as the basal medium. Therefore, we used a suspension of this density of bacteria to perform the tests. Furthermore, blood agar would appear to be the medium of choice for most tolerance tests used for the identification of Campylobacter isolates, provided that other important factors (such as the size of the inoculum and atmospheric conditions) are also considered (11). Whenever possible, we preferred blood agar to liquid medium to avoid misinterpretation of the phenotypic test results.

The tolerance to 1% glycine has been the only internationally accepted phenotypic test for the differentiation of C. fetus subspecies (1). Depending on the result of this test, the 103 field isolates investigated were classified as 81 C. fetus subsp. venerealis isolates (glycine intolerant) and 22 C. fetus subsp. fetus isolates (glycine tolerant). Glycine-tolerant variants of C. fetus subsp. venerealis and glycine-sensitive C. fetus subsp. fetus could not be observed.

The C. fetus subsp. venerealis strains were isolated over a long period of time and came from different sources. Despite this fact, these isolates showed relatively uniform reactions by the phenotypic tests. For all isolates of this subspecies, the results for selenite reduction and sensitivity to metronidazole and cefoperazone completely agreed with the results for glycine tolerance. Only in the parameter of growth at 42°C did the isolates reacted differently: 73 isolates had no growth and 8 isolates had faintly discernible, flat, dark gray growth. However, these eight isolates could be also separated from C. fetus subsp. fetus, because this subspecies (with three exceptions) showed a luxurious, light gray growth.

For C. fetus subsp. fetus, the four additional phenotypic tests only partly agreed with the glycine tolerance test results and a certain degree of nonuniformity was observed. C. fetus subsp. fetus showed a relatively good correspondence between the glycine tolerance test results with the results of selenite reduction, cefoperazone resistance, and growth at 42°C (correspondences, 81.8%, 86.4%, and 81.8%, respectively). In general, C. fetus is considered a nonthermophilic Campylobacter, but in our studies the majority of the C. fetus subsp. fetus isolates showed abundant growth at 42°C. In other investigations, the proportion of strains able to grow at 42°C varied from 0% (17) to 59% (2) and 62% (13).

In the last decade, progress has been made in the development of molecular methods for the identification of C. fetus. In 1997, Hum et al. (5) described a specific PCR-based assay for the identification and differentiation of the two C. fetus subspecies. This assay was evaluated, and 97 C. fetus field isolates were investigated. The initial identifications obtained by conventional phenotypic methods agreed with those suggested by PCR for 78 isolates, of which 56 were C. fetus subsp. venerealis isolates and 22 were C. fetus subsp. fetus isolates. The characterization of 19 isolates for which the results of conventional phenotyping and genotyping by PCR differed suggest that misidentification of C. fetus by phenotypic testing may be relatively common. Repeat testing of these 19 problem isolates revealed that for only 2 (2.1%) of 97 isolates were the identifications suggested by the PCR assay (as C. fetus subsp. venerealis) discordant with those made by macrorestriction profile analysis and/or conventional and probabilistic phenotypic methods. On and Harrington (13) examined 31 C. fetus strains by phenotypic, PCR-based, and PFGE-based methods; and the 16S rRNA gene sequences of 18 strains were compared. For 28 of the 31 strains, the results of the subspecies-level identifications by phenotypic, PCR-based, and PFGE-based methods concurred. Discrepancies between the results of the methods were observed for three strains. Wagenaar et al. (18) investigated 69 C. fetus isolates from three geographical regions by phenotypic testing, PCR, and amplified fragment length polymorphism (AFLP) analysis. Depending on the result of the glycine tolerance test, 47 of the strains were typed as C. fetus subsp. fetus and 22 were typed as C. fetus subsp. venerealis. The investigation by PCR resulted in 54 C. fetus subsp. fetus strains, and 7 of these strains were negative by the glycine tolerance test. In these cases, the PCR results were supported by data from the AFLP analysis, suggesting that the strains were mistyped by phenotypic testing. These seven strains were part of a group of nine strains from South Africa, and the results may indicate some evolutionary distinction between C. fetus subsp. fetus strains from South Africa and those from other geographical regions. Conversely, 1 of the 15 C. fetus subsp. venerealis strains (strain 98/v445) identified by PCR was positive for growth on 1% glycine. Vargas et al. (16) investigated 31 bovine C. fetus isolates. Four isolates were tolerant to glycine and, therefore, were classified as C. fetus subsp. fetus but were considered C. fetus subsp. venerealis when molecular diagnostic methods (including PCR) were used. Willoughby et al. (19) reported that phenotyping of C. fetus subsp. venerealis isolates could not be confirmed reliably by multiplex PCR. A specific amplicon of 142 bp was obtained for only 14 of 32 C. fetus subsp. venerealis isolates. They suspected that the presence of an unusual clone of this subspecies in the United Kingdom could have been the reason for the failure of PCR genotyping. Despite these discrepancies, the PCR assay is recommended by Hum et al. (5), On and Harrington (13), Wagenaar et al. (18), and Vargas et al. (16) as a rapid adjunctive technique for the identification and differentiation of C. fetus subspecies.

In our investigations, 103 C. fetus isolates were typed by phenotypic testing and the results were confirmed by PCR. For evaluation of the PCR results, the glycine tolerance test was used as reference method. It is noteworthy that the phenotypic methods used in these investigations have been standardized as far as possible, especially concerning the number of culturable C. fetus organisms per ml of inoculum. In each investigation, the number of culturable bacteria per ml of inoculum was checked. Additionally, blood agar was preferred to liquid medium in most reactions. Under these stringent conditions, the results of the glycine tolerance test and PCR were compared, and there was a complete correspondence between the results of both methods. Some groups (5, 13, 16, 19) used a multiplex PCR assay to identify and differentiate the C. fetus isolates. In our experience, problems can occur because of the insufficient optimization of the assay, such as by the use of different primer concentrations. To avoid any complications from the use of a multiplex PCR system, two separate PCRs were used. Our investigations with the described PCR assay resulted in one remarkable observation. In other investigations (5, 13, 16), which used primers MG3F and MG4R, amplicons with fragment lengths of 960 bp were observed. In our work with these primers, as well as in the studies of Wagenaar et al. (18) and Willoughby et al. (19), the fragment length of the amplicon was approximately 750 bp. The differences in the amplicon lengths in different studies cannot be explained. The C. fetus strains used in these investigations were exclusively isolated in Germany, but bgC is of significant importance in countries that breed cattle all over the world. Consequently, the possibility of the existence of C. fetus variants in other countries cannot be excluded. For a further evaluation of phenotypic tests and PCR, these investigations should be extended and isolates from different countries should be included. Recently, the possibilities for the identification of C. fetus subspecies have been improved. Van Bergen et al. (15) used AFLP analysis to identify C. fetus subspecies-specific markers and designed a novel PCR primer set (primer Cf C05) for the identification of C. fetus subsp. venerealis.

We conclude that at present the traditional phenotypic characterization of C. fetus subspecies remains indispensable, but the PCR assay described by Hum et al. (5) constitutes a valuable method for confirmation of the results of phenotypic tests.

Acknowledgments

We thank Kerstin Kluge and Byrgit Hofmann for excellent technical assistance.

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