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Journal of Clinical Microbiology, February 2008, p. 807-809, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01963-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

CASE REPORT

Heterogeneity of Hemolysin Expression during Neonatal Streptococcus agalactiae Sepsis

Anja Sigge,¹ Manuel Schmid,² Stefanie Mauerer,¹ and Barbara Spellerberg^1*

Institute of Medical Microbiology and Hygiene, Robert Koch Str. 8, Ulm D-89081, Germany,¹ University Children's Hospital, Ulm, Eythstr. 24, Ulm D-89081, Germany²

Received 4 October 2007/ Returned for modification 3 November 2007/ Accepted 26 November 2007

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The β-hemolysin of Streptococcus agalactiae is a major virulence factor; consequently, nonhemolytic strains rarely cause infections. We report on a case of neonatal sepsis caused by a strain displaying heterogeneous hemolysin expression. It was detected by the simultaneous isolation of hemolytic and nonhemolytic colonies from cultures of the infant's blood.

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A term neonate was born at 41 weeks of gestation weighing 4,300 g. Due to a failure of adequate labor progress and pathological cardiotocographic readings, the infant was delivered by vacuum extraction. During delivery green amniotic fluid was noted. The Apgar score was 7/9/9, and the umbilical arterial pH was 7.17. Due to progressing signs of dyspnea, a gray skin color, a prolonged capillary refill time, and a further fall in the pH to 7.02, the child was transferred to the neonatal intensive care unit of the Pediatrics University Clinics 2 h after birth. The prenatal group B streptococcus colonization status of the mother was unknown. At admission, a blood sample for aerobic culture (PLUS Pediatric; BD, Heidelberg, Germany) was obtained and treatment with mezlocillin (200 mg/kg of body weight /day) and gentamicin (5 mg/kg/day) was initiated for suspected neonatal sepsis. The infant was negative for C-reactive protein (CRP) at admission, but the interleukin-8 level was elevated to 1,517 ng/liter. The aerobic blood culture bottle registered positive after 24 h of incubation in an automated blood culture system (Bactec 9240; BD). Microscopic analysis revealed the presence of gram-positive cocci growing in chains and displaying a typical streptococcal morphology. Following subculture on 5% sheep blood agar plates and overnight incubation at 35°C, the growth of two different colony types was detected. Gray colonies surrounded by a small zone of beta-hemolysis were present, as were colonies of the same morphology lacking any type of hemolysis. Both colony types were catalase negative. Further species identification of both strains was achieved biochemically with the API Strep system (BioMerioeux) and resulted in the detection of Streptococcus agalactiae isolates with identical API profiles. The isolate identities were confirmed by CAMP testing and serological testing with the Streptex system (Murex Streptex; Abbott, Wiesbaden, Germany), which showed the presence of the group B streptococcal antigen in both strains.

During the next days the infant's clinical situation quickly improved. The CRP concentration reached a maximum of 19.7 mg/liter. The cerebrospinal fluid did not reveal any signs of meningitis, and the child was discharged on day 4 in excellent clinical condition.

To assess the colonization status of the mother, a vaginal swab was obtained postpartum and was cultured in selective LIM broth, as recommended by CDC guidelines (11). Typical beta-hemolytic colonies were detected, and species identification revealed S. agalactiae. Despite a thorough inspection of the subcultures for evidence of nonhemolytic S. agalactiae colonies, only the beta-hemolytic phenotype could be isolated from the maternal vaginal tract.

To investigate the genetic basis for the loss of hemolysis in the nonhemolytic strain, genes of the cyl gene cluster were amplified by PCR, as described previously (12). Interestingly, the PCR product of the cylA gene amplified from the nonhemolytic strain was considerably larger than the same PCR product generated from the hemolytic blood culture isolate. The sizes of the PCR products of all the other cyl genes from the hemolytic and the nonhemolytic isolate were indistinguishable on visual inspection of the agarose gels and matched the expected sizes of the corresponding genes (Fig. 1). The cylA PCR product of the nonhemolytic isolate was further investigated by DNA sequencing, which revealed the presence of the insertion element IS1381 after nucleotide 467 of the cylA gene. CylA represents the ATP binding component of the S. agalactiae hemolysin transporter. Targeted mutagenesis of the transporter results in the loss of the hemolytic phenotype (6), and it has previously been noted that some naturally occurring nonhemolytic S. agalactiae mutants harbor an additional copy of IS1381 inserted at this location in cylA (12). The presence of an additional IS1381 element in the nonhemolytic strain could also be demonstrated by Southern blot hybridization. For this purpose, the genomic DNA of the hemolytic strains from the mother and the infant and the nonhemolytic blood culture isolate were digested with EcoRI and BamH1 and subsequently hybridized with a probe to the nucleotide sequence of IS1381. The nonhemoytic strain clearly showed six IS1381-specific bands, whereas the maternal and the neonatal hemolytic strains appeared to be identical and each strain harbored five IS1381-specific bands (Fig. 2). However, the banding pattern of the nonhemolytic isolate differed from the pattern for the hemolytic strains by more than just one additional band. Therefore, it remains unclear if all of the changes can be attributed to the insertion of an additional IS1381 copy or if additional mutations account for these differences. IS1381 is an insertion element that was first detected in Streptococcus pneumoniae (10) and that is commonly found in clinical S. pneumoniae strains. Its host range is, however, not limited to S. pneumoniae, and it has been described as a potential tool for the subtyping of S. agalactiae isolates (13).

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FIG. 1. PCRs of S. agalactiae cyl genes, as described previously (12). PCR was performed with the genomic DNA of nonhemolytic blood culture isolate BSU25 and hemolytic blood culture isolate BSU26. Lane M, molecular size marker. Genes that were amplified by a specific PCR are indicated. The numbers on the left are in nucleotides.

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FIG. 2. Southern blot analysis for the presence of IS1381 in both neonatal strains (strains BSU25 and BSU26) and the maternal vaginal isolate (strain BSU27). An IS1381-specific probe was amplified with primers 5'-CTT GTT GGT GTT CAG CGC AC-3' and 5'-TGA ACA ACT TCA TAT CGT GGC-3'. Southern blotting was carried out after digestion of the genomic DNA with EcoRI and BamHI. Lane M, molecular size marker; lane 1, strain BSU25 (nonhemolytic neonatal isolate); lane 2, strain BSU26 (hemolytic neonatal isolate); lane 3, strain BSU27 (hemolytic maternal isolate). The numbers on the left are in nucleotides.

To elucidate if the nonhemolytic strain did originate from the hemolytic strain found in the blood culture of the infant and the maternal vaginal tract, we performed molecular subtyping of all three strains. The capsular serotype was detected as described by Kong et al. (8) and showed that all three isolates were serotype III strains. Surface protein antigens were detected by PCR (2) and showed the presence of rib in all strains. Further subtyping was achieved by multilocus sequence typing (MLST) (7) and revealed that both beta-hemolytic strains belong to sequence type (ST) 19 (ST19), which represents a major serotype III subclone. Interestingly, the nonhemolytic isolate belonged to ST27, which differs from ST19 only by a single nucleotide exchange in the glnA gene. ST27 is therefore regarded as a member of the ST19 complex, which consists of several closely related STs (4). The fact that, aside from the loss of hemolysis, a point mutation occurred in the nonhemolytic strain underlines the idea of a high mutation rate in the respective beta-hemolytic blood culture isolate. The glnA gene itself has not been associated with a loss of hemolysis. The vast majority of S. agalactiae strains carrying allele 4 of glnA, which is found in ST27 strains, are hemolytic. In summary, all subtyping methods supported the conclusion that the nonhemolytic strain represents a spontaneous mutant of the beta-hemolytic blood culture isolate (Table 1).

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TABLE 1. Molecular characteristics of the S. agalactiae isolatesa

Mutations of S. agalactiae isolates caused by the insertion of IS1381 have repeatedly been reported (3, 9), and the presence of different copy numbers of IS1381 elements in pairs of maternal-neonatal strains is not unusual (13). It is interesting to speculate when the insertion of the additional IS1381 element in the cylA gene occurred in our case. Thorough investigation of the vaginal swab from the mother only revealed a strain displaying the typical beta-hemolytic phenotype of S. agalactiae. No evidence for the presence of a nonhemolytic strain was found in the vaginal swab material. Molecular typing showed the maternal strain to be identical to the beta-hemolytic blood culture isolate of the infant. The most likely scenario is therefore the transmission of the maternal beta-hemolytic strain from the mother to the infant and the subsequent invasion of the beta-hemolytic strain into the vascular system. Mutation of the cylA gene through the insertion of an additional IS1381 copy probably occurred afterwards. This hypothesis would be consistent with the fact that β-hemolysin is regarded as a virulence factor important for the invasion of host tissues (5). It seems unlikely that the hemolytic strain and the nonhemolytic strain independently invaded the vascular system, especially since no evidence for the presence of a nonhemolytic strain in the maternal vaginal tract was found. However, we cannot, of course, exclude this possibility.

The identification of nonhemolytic S. agalactiae isolates in blood cultures is rare, since these strains are of reduced virulence and the hemolysin is considered important for invasion processes. Among colonizing strains, less than 5% of strains are nonhemolytic (1). The identification of nonhemolytic S. agalactiae isolates is complicated by the unusual phenotype but can be achieved on the basis of biochemical tests and the CAMP reaction, which is positive for nonhemolytic strains. This is, to the best of our knowledge, the first report on the simultaneous detection of a hemolytic and a nonhemolytic S. agalactiae strain in invasive infections.

 
* Corresponding author. Mailing address: Institute of Medical Microbiology and Hygiene, University of Ulm, Robert Koch Str. 8, Ulm D-89081, Germany. Phone: (49)-731-50065333. Fax: (49)-731-50065302. E-mail: barbara.spellerberg{at}uniklinik-ulm.de

Published ahead of print on 12 December 2007.

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Journal of Clinical Microbiology, February 2008, p. 807-809, Vol. 46, No. 2
0095-1137/08/$08.00+0     doi:10.1128/JCM.01963-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sigge, A. Articles by Spellerberg, B. Search for Related Content PubMed PubMed Citation Articles by Sigge, A. Articles by Spellerberg, B.