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Journal of Clinical Microbiology, August 2003, p. 3926-3928, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3926-3928.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Typing of Campylobacter jejuni Isolates Involved in a Neonatal Outbreak Indicates Nosocomial Transmission

José Llovo,1 Estíbaliz Mateo,2 Angeles Muñoz,3 María Urquijo,2 Stephen L. W. On,4 and Aurora Fernández-Astorga2*

Servicio de Microbiología, Complexo Hospitalario Universitario de Santiago de Compostela, 15706 Santiago de Compostela,1 Departamento de Microbiología y Parasitología, Facultad de Biología, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, A Coruña,3 Departamento de Inmunología, Microbiología y Parasitología, Facultad de Farmacia, Universidad del País Vasco, E-01080 Vitoria-Gasteiz, Spain,2 Danish Veterinary Institute, DK-1790 Copenhagen V, Denmark4

Received 19 December 2002/ Returned for modification 28 March 2003/ Accepted 1 May 2003


   ABSTRACT
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Genotypic typing by restriction fragment length polymorphism and pulsed-field gel electrophoresis showed that two neonates in a neonatal ward were infected with the same Campylobacter jejuni strain. Isolates from the mother and brother of the index patient were identical to each other but distinct from the neonatal type. Genotyping results therefore suggested that the neonatal C. jejuni infection was nosocomial in origin.


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Although Campylobacter jejuni is one of the most common pathogens causing acute bacterial enteritis in children and adults, neonatal enteritis is relatively rare (1, 14, 15). However, neonate bacteremia and meningitis have been documented, mainly in hospital outbreaks (5, 10). Although mothers often have no history of diarrhea during pregnancy, contamination during delivery is the most likely route of transmission in neonates (1, 14, 15). However, accurate strain differentiation and careful analysis of patient data are necessary for the identification of other sources and routes of contamination.

Among the many systems described for typing Campylobacter, genotypic methods are the most discriminatory (13). The aim of the present study was to apply pulsed-field gel electrophoresis (PFGE) and fla typing by restriction fragment length polymorphism (RFLP) to C. jejuni isolates during an outbreak of gastroenteritis in a neonatal ward in order to characterize and contain the incident.

Epidemiological and clinical data. Between 7 and 13 December 2000, two cases of C. jejuni infection occurred in the neonatal ward in the Hospital Clínico Universitario in Santiago (A Coruña), a city in northwestern Spain. Two neonates were affected, a 2-day-old female (index patient) and a 30-day-old female. On the second day after birth, the index patient passed blood and mucus in the stool. No other clinical symptoms such as fever or vomiting were noted. Direct microscopic observation of a fresh fecal smear revealed the presence of large numbers of leukocytes, erythrocytes, and motile spiral bacteria, suggesting the presence of Campylobacter spp. Five days after microscopic diagnosis, treatment with erythromycin was initiated.

The second infant had respiratory distress at birth, and she was admitted to the intensive care nursery. In the neonatal ward, she received orogastric feedings, and on day 30 of life, 6 days after the index case, the baby passed blood and mucus. Direct microscopic observation of a fresh fecal smear revealed findings consistent with those of the index case, after which erythromycin therapy was initiated. Early administration of erythromycin led to improvement and rapid clearance of bacteria from the infant stools, as well as preventing spread within the nursery.

Microbiology. Definitive diagnosis of campylobacteriosis was established by isolation of the microorganism from stool samples by the use of two selective media, charcoal-cefoperazone deoxycholate agar (Becton Dickinson) and 10% sheep blood agar containing vancomycin, trimethoprim, polymyxin, amphotericin B, and cephalothin (Becton Dickinson). Plates were incubated at 37°C under microaerobic conditions (7% CO2, 8% O2, and 85% N2) for 48 h. Species-level identification of strains was based upon biochemical tests and PCR. Biochemical tests included cytochrome oxidase production, catalase activity, hydrolysis of hippurate and indoxyl acetate, H2S production on triple sugar iron, and susceptibility to nalidixic acid and cephalothin. PCR identification was based upon assays for mapA for C. jejuni (12) and ceuE for Campylobacter coli (4). Amplification was carried out in a RoboCycler Gradient 96 (Stratagene) thermocycler as described by Denis et al. (2) but with an increased concentration (200 µM) of each deoxynucleoside triphosphate. Amplification generated 589- and 462-bp fragments corresponding to C. jejuni and C. coli, respectively.

Antimicrobial susceptibility (Table 1) was assayed by the Kirby-Bauer agar diffusion method with disks (Oxoid) and E-test strips (AB Biodisk) on Mueller-Hinton agar supplemented with 5% sheep blood (Becton Dickinson). Susceptibilities to nalidixic acid (30 µg), ciprofloxacin (5 µg), erythromycin (15 µg), clindamycin (2 µg), tetracycline (30 µg), ampicillin (10 µg), imipenem (10 µg), cephalothin (30 µg), chloramphenicol (30 µg), gentamicin (10 µg), and trimethoprim-sulfamethoxazole (25 µg) were assayed.


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  		TABLE 1. Antimicrobial susceptibilities of Campylobacter species isolated from stools

 
Once the index case was confirmed as C. jejuni infection, stools from the patient's mother and brother, as well as from all 16 babies admitted at the same time to the neonatal ward, were examined. The mother had diarrhea prior to delivery. The brother, 2 years old, had also had a recent gastrointestinal disturbance. C. jejuni was recovered from stools of both the mother and the brother of the index patient. However, stools from the other 16 neonates present in the ward proved Campylobacter culture negative.

Epidemiological study. A prevalence study was conducted in order to evaluate the potential origin of infection in the index case and the other neonate, such as fecal-oral transmission during delivery and postnatal person-to-person spread. In the epidemiological study, four additional isolates of Campylobacter, drawn from persons originally not connected to the patients, were used as epidemiologically unrelated controls to test the value of the genotypic methods and the validity of the hypotheses.

All isolates but one were confirmed as C. jejuni by PCR. Strain E-871 was C. coli. These two species are differentiated principally by the ability of C. jejuni to hydrolyze hippurate, although certain atypical strains fail to do so (11). Well-designed PCR tests are insensitive to such problems and can be considered useful adjunctive tools for use in epidemiological studies (6).

Genotyping. All eight isolates were submitted to PCR-RFLP and PFGE profiling. For PCR-RFLP, a 1.7-kb fragment of the flaA gene was amplified and analyzed after digestion with two restriction enzymes, DdeI and HinfI, by the procedure described by Nachamkin et al. (7). For PFGE profiling, DNA-agarose samples were prepared from formaldehyde-treated bacterial cells by the protocol of Gibson et al. (3), modified as described previously (8, 9). DNA was digested with SmaI, and fragments were separated by use of the CHEF-DR III PFGE system (Bio-Rad Laboratories). In both methods, standardized parameters were as proposed by CAMPYNET (http://www.campynet.vetinst.dk). Any differences between PFGE profiles of strains were considered significant.

The genotyping was carried out as two independent assays. PCR-RFLP analysis was done at the Faculty of Pharmacy (UPV/EHU), and PFGE was done at the Danish Veterinary Institute. There was a strict correlation between the PFGE and the fla genotypes (Fig. 1). Results from each laboratory did not confirm our original hypothesis about the maternal origin of infection but instead identified a nosocomial outbreak in the neonatal ward, the duration and spread of which were probably severely restricted by early antibiotic therapy. Of equal note was the finding that the mother and brother of the 2-day-old neonate harbored genotypically indistinguishable strains that were clearly different from those found in the neonates. The validity of our findings was confirmed by clear discrimination of these strains from all other epidemiologically unrelated isolates examined (Fig. 1).



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  		FIG. 1. (A) RFLP patterns of the Campylobacter flagellin gene flaA, obtained with DdeI digestion. Lanes 2, 3, 7, and 8, related strains E-868, E-869, E-875, and E-876, respectively. Lanes 4, 5, 6, and 9, unrelated strains E-872, E-870, E-871, and E-873, respectively. Lanes M, 100-bp DNA ladder (Promega). (B) PFGE profiles obtained with SmaI chromosomal digestion of Campylobacter strains. Lanes 1, 6, and 11, the normalization standard pattern. Lanes 2 to 5, related strains E-868, E-869, E-875, and E-876, respectively. Lanes 7 to 10, unrelated strains E-870, E-871, E-872, and E-873, respectively. Lane M, molecular size markers.

 
This study comes from a retrospective review of gastroenteritis cases recorded at the Hospital Clínico Universitario in Santiago (Spain), and thus only clinical isolates were available. This was a serious limitation for determining the source of the two groups of strains. Nonetheless, the use of genotypic methods was vital in determining that C. jejuni isolates in both groups came from different sources, converting an apparent, somewhat ordinary case of vertical transmission into an exceptional case of horizontal transmission. Our results highlight the value of genetic methods for identification and typing of bacteria in the epidemiological study of C. jejuni infection in neonates.


   ACKNOWLEDGMENTS
 
We thank Santiago Méndez and Pedro Vázquez for their technical assistance at the Microbiology Laboratory in the Hospital Clínico Universitario and Penelope Jordan at the Danish Veterinary Institute for performing the PFGE typing.

This work was supported in part by grants of the Comisión Interministerial de Ciencia y Tecnología (ALI99-0477-CO2-01) and the Departamento de Sanidad of the Basque country government (Exp. 200012004).


   FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Inmunología, Microbiología y Parasitología, Facultad de Farmacia (UPV/EHU), Apdo. 450, E-01080 Vitoria-Gasteiz, Spain. Phone: 34 945 013909. Fax: 34 945 013014. E-mail: oipfeasa{at}vf.ehu.es. Back


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Journal of Clinical Microbiology, August 2003, p. 3926-3928, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3926-3928.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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