J Med Microbiol 56 (2007), 988-992; DOI: 10.1099/jmm.0.47186-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Repeated bacteraemia caused by Streptococcus mutans in a patient with Sjögren's syndrome
Ryota Nomura1,
Masakazu Hamada2,
Kazuhiko Nakano1,
Hirotoshi Nemoto1,
Koji Fujimoto2 and
Takashi Ooshima1
1 Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan
2 Department of Dentistry and Oral Surgery, Itami City Hospital, Itami, Hyogo 664-8540, Japan
Correspondence
Kazuhiko Nakano
nakano{at}dent.osaka-u.ac.jp
Received 25 January 2007
Accepted 7 March 2007
Streptococcus mutans, considered to be a pathogen for dental caries, is known to cause bacteraemia and infective endocarditis. Herein, an unusual case of repeated bacteraemia caused by S. mutans identified in a 71-year-old male is described. The patient visited Itami City Hospital with the major complaint of a fever, and a subsequent clinical examination led to a diagnosis of possible infective endocarditis without specific vegetation formation around the heart valve. A bacteriological examination of blood taken at the first visit showed the presence of S. mutans. Antimicrobial treatment was provided, which successfully eliminated the pathogenic bacteria from the blood. However, the patient returned and was hospitalized twice more with a recurrent fever, and S. mutans was again detected. Analyses of the biological properties of the S. mutans isolates showed that they possessed cariogenic properties and had a low susceptibility to phagocytosis by human polymorphonuclear leukocytes. Since the patient had Sjögren's syndrome, in which a reduction of saliva secretion is a characteristic feature, a great number of dental caries lesions were identified. The findings indicated that S. mutans present in those dental caries lesions caused repeated bacteraemia in this case.
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Case report
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A 71-year-old male with Sjögren's syndrome came to the Department of Internal Medicine, Itami City Hospital, Hyogo, Japan, with a persistent fever on 14 April 2006. Fig. 1
summarizes the period of hospitalization, antibiotic therapy and sampling times. A transoesophageal echocardiograph examination on day 1 led to a diagnosis of atrial fibrillation, mitral valve prolapse, mitral regurgitation and possible infective endocarditis without specific vegetation formation around the heart valve, while a bacteriological examination of blood taken at the first visit showed the presence of Streptococcus mutans. A routine antibiotic susceptibility test for six antibiotics (ampicillin, cefazolin, meropenem, erythromycin, minocycline and vancomycin) was carried out, which showed that the S. mutans strain was susceptible to all. The patient was immediately hospitalized, with antibiotic therapy initiated on the second day using intravenous cefazolin at 2 g per day for 10 days (days 2–11). The patient was discharged from the hospital on day 19; however, he suffered from recurrent fever and returned on day 21. Antibiotic therapy with intravenous cefazolin at 2 g per day was initiated again for 9 days (days 21–29), after which he was discharged on day 34. Nevertheless, the patient returned again due to a persistent fever on day 39, after which he was prescribed intravenous meropenem at 20.5 g per day for 10 days (days 41–50), followed by cefepime at 1 g per day for 11 days (days 68–78).
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Fig. 1. Timetable of the present case of repeated bacteraemia. The patient was hospitalized on three separate occasions from April to July 2006. During that period, he was referred to the Department of Dentistry and Oral Surgery on June 6, where S. mutans was identified in three blood culture examinations. In addition, oral specimens were collected six times; however, S. mutans could not be isolated from any of those samples. CEZ, cefazolin; MEPM, meropenem; SBTPC, sultamicillin; CFPM, cefepime; CFPN-PI, cefcapene. Numbers written in italics refer to the number of days from the first visit to the hospital.
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During the third hospitalization (days 39–86), the patient was referred to the Department of Dentistry and Oral Surgery of that hospital for analysis of possible pathogens related to the persistent fever. The attending physicians considered that the reduced function of saliva secretion due to Sjögren's syndrome in the patient may have caused an occurrence of dental caries, and they requested us to look for the presence of possible pathogens related to repeated bacteraemia in the oral cavity. An intraoral examination showed that the patient had 24 teeth, of which 15 had been treated due to dental caries, though some of these 15 were found to have secondary caries, including previously treated teeth and new active caries lesions. Further, since the tooth crowns of three of these teeth were completely decayed, the teeth were extracted on days 53 and 80. An orthopantomographic examination showed that the amount of supportive bone was not extremely reduced, indicating that the patient was not affected by severe periodontitis.
We decided to analyse the properties of S. mutans in blood samples as well as the oral S. mutans strains. Blood was collected on day 68 and directly streaked onto Mitis-Salivarius (MS) agar plates (Difco Laboratories) containing bacitracin (0.2 U ml–1; Sigma) and 15 % (w/v) sucrose (MSB plates), after which 50 isolates were sampled and designated MH1–MH50. The colony morphology of all strains on the MSB agar was rough, which was similar to that of the reference strain MT8148 (serotype c), which was isolated from the oral cavity of a Japanese child. In addition, supra- and subgingival plaque samples were taken from mesial and buccal subgingival sites of all teeth of the patient with sterile Gracey curettes, placed in sterile PBS, then streaked onto MSB agar plates. However, no S. mutans oral strains could be isolated, even after repeating the procedure six times between days 62 and 80.
Genomic DNA was extracted from the blood isolate strains (MH1–50) and the 16S rRNA gene sequence of the strains was found to be completely identical to that of S. mutans NCTC 10449 (GenBank accession no. X14409) (Fujiwara et al., 2001). Further, an immunodiffusion method that utilized rabbit antisera specific for S. mutans serotypes c, e, f and k (Nakano et al., 2004) showed that all of the MH strains were of serotype e. Arbitrarily primed (AP)-PCR fingerprinting and random amplified polymorphic DNA (RAPD) analysis (Nomura et al., 2006) also demonstrated that all 50 strains had the same fingerprinting pattern, indicating that they were of the same genotype. Western blot analyses of whole cells of MH1–10 using antisera against major cell surface proteins (GTFB, GTFC, GTFD, PAc, GbpA, GbpB and GbpC) (Fujiwara et al., 2001; Matsumoto-Nakano et al., 2007) identified no significant differences between the MH strains and MT8148 (data not shown).
Next, a sucrose-dependent adhesion assay, regarded as the most essential biological test for S. mutans when considering cariogenic properties, was carried out using a method described previously (Kawabata & Hamada, 1999). The sucrose-dependent adhesion rate of MH1–10 (84.6±2.1 %; range 81.4–87.2 %) was significantly lower than that of MT8148 (89.3±0.7 %; range 88.3–90.0 %) (Fisher's protected least-significant-difference test, P <0.001). However, cellular hydrophobicity findings, determined using the method of Rosenberg et al. (1980), showed that MH1–10 were not significantly different from MT8148 (data not shown). These results indicated that the MH strains isolated from our patient possessed cariogenic properties and we considered them to be oral derivatives.
Phagocytosis susceptibility was then determined by evaluating the interactions between the tested strains and human polymorphonuclear leukocytes, as described previously (Nakano et al., 2004). Fig. 2(a) shows the phagocytosis rates after 10 min, with the MH strains found to be significantly less susceptible than MT8148 to phagocytosis. The phagocytosis rates were also monitored at 15, 30, 60, 90 and 120 min; significant differences between MT8148 and MH1 were shown at 15, 30, 60 and 90 min, while the rates of both strains reached 100 % at 120 min (Fig. 2b).
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Fig. 2. Phagocytosis rates of S. mutans isolates. The results shown represent the mean±SD from five experiments. (a) The phagocytosis rates of MT8148 and 10 MH strains were examined following 10 min of incubation. (b) Changes in phagocytosis rates of strains MT8148 and MH1 after 15, 30, 60, 90 and 120 min of incubation. There were statistically significant differences between MT8148 and MH1, as shown by Fisher's protected least-significant-difference analysis (*, P <0.001).
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S. mutans detection was carried out by PCR with an S. mutans-specific set of primers, as described previously (Hoshino et al., 2004). The blood specimens showed a positive reaction for S. mutans; however, no positive reaction was identified in the oral specimens. We also conducted broad-range PCR assays targeting eubacterial 16S rRNA with direct sequencing using whole DNA extracted from dental plaque and blood to define the bacterial species, as described previously (Rovery et al., 2005). The 16S rRNA sequences obtained were compared with those available in the GenBank, EMBL and DDBJ databases using the gapped BLASTN 2.0.5 program obtained from the National Center for Biotechnology Information server (http:/www.ncbi.nlm.nih.gov/BLAST/). The dental plaque sampling times were fairly compressed, because we attempted to isolate S. mutans from the oral cavity for comparison with the blood isolates. However, S. mutans could not be isolated in six different trials conducted between days 62 and 80. All 50 clones amplified from the blood specimens taken on day 68 showed the 16S rRNA sequence of S. mutans exclusively, whereas only 1 of 300 clones from the dental plaque specimens (50 clones per specimen) showed that of S. mutans (Table 1). The most frequently detected species throughout the examinations was Pseudomonas, which had the highest numbers in the third (day 69) and sixth (day 80) dental plaque sample. In addition, Enterobacter species were the most prevalent in the first dental plaque sampling (day 62) and second most prevalent in the third (day 69), while Streptococcus species, including Streptococcus sanguinis, Streptococcus parasanguinis and Streptococcus anginosus, were the most prevalent in the second sampling (day 67).
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Table 1. Characteristics of bacterial species in oral specimens by sequence analysis of fragments amplified using broad-range PCR
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Discussion
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S. mutans, a pathogen related to dental caries, is known to invade the bloodstream following professional dental treatment and daily oral care, and has been reported to cause transient bacteraemia (Seymour et al., 2000). However, there are few reports describing the properties of S. mutans in samples from bacteraemia patients. The major characteristic of the present case is that the patient repeatedly suffered from fever after the systemic condition had been stabilized with antibiotic therapy. The local physician ordered blood culture examinations on four occasions, in which S. mutans was identified three times. Although the patient displayed valvulitis without vegetation on day 1, it is possible that vegetative growth might have appeared later during the treatment period. Another possibility is that the repeated bacteraemia in this case may have been due to an undiscovered focus of infection. We considered that the S. mutans blood isolates in this study were disease-associated and examined their various biological properties.
An antibiotic susceptibility test was performed on day 1, which showed that S. mutans isolated on that day was susceptible to cefazolin. On day 11, the blood culture was negative, which was possibly derived from the 10-day intravenous cefazolin administration. However, the patient returned to the hospital with a fever. Although no blood culture examination was carried out during the second hospitalization, intravenous cefazolin was given and the fever was resolved. Surprisingly, the patient returned to the hospital on day 39, 5 days after leaving the hospital, during which time a blood culture was performed and S. mutans was detected. Another antibiotic (meropenem) was selected, as the physician considered that an unknown pathogen might be present in the oral cavity, since the patient had severe dental decay. We first examined the patient on day 53 and extracted two severely decayed teeth. However, S. mutans was again identified in a blood culture examination on day 68 and antibiotic therapy with cefepime was initiated.
In order to determine whether bacterial species other than S. mutans were present in the blood samples, broad-range PCR and sequencing analysis were carried out; however, only S. mutans could be detected among all of the clones generated from the blood samples. Therefore, S. mutans was concluded to be the only pathogenic organism that caused bacteraemia in this case. The S. mutans isolates (MH1–50) were easily obtained from the blood specimen taken on day 68, whereas no S. mutans strains were isolated from the oral cavity of the patient in six different trials conducted between days 62 and 80 (Fig. 1
and Table 1).
AP-PCR and RAPD analyses showed that all 50 S. mutans isolates from blood were of the same genotype, which indicated that the bacteraemia in this case was caused by a single genotype of S. mutans. Further, our findings in Western blot analyses, as well as with regard to sucrose-dependent adhesion rate and cellular hydrophobicity, showed that the blood isolates possessed cariogenic properties, thus we considered them to be oral derivatives.
No S. mutans strains could be isolated from the oral cavity in the present case, even though we have found it generally quite easy to isolate S. mutans strains from oral samples of most subjects. One possible reason is that most of the bacteria may have been eliminated by the antibiotic therapy, with only very few remaining and located deep in the caries lesions.
We previously analysed the phagocytosis susceptibility of four blood isolates from patients with bacteraemia after tooth extraction or infective endocarditis with the same method used in this study, and the blood isolates showed significantly lower susceptibility to phagocytosis than those from the oral cavity (Nakano et al., 2004). Therefore, it is reasonable to speculate that strains with a low susceptibility to phagocytosis possess an ability to survive in blood. In addition, the repeated bacteraemia in the present patient was thought to be caused by the pathogens persistently present in the oral cavity, as S. mutans was presumably present in the severely decayed teeth as well as in the active dental caries lesion.
In another report, a patient with Sjögren's syndrome was characterized by a high number of dental caries lesions as compared with healthy individuals, due to the dry mouth condition associated with that disease (Almstahl et al., 2003). Various studies have reported that higher numbers of oral streptococci, including S. mutans, are detected in subjects with Sjögren's syndrome (Almstahl et al., 2003). However, S. mutans could not be isolated from dental plaque samples taken from the patient in this study between days 62 and 80, and our molecular analysis showed that S. mutans DNA was rarely present (Table 1). On the other hand, nearly half of the species (43.7 %) detected in those samples were Pseudomonas or Enterobacter species, which are not typically detected in healthy individuals or those with Sjögren's syndrome. Multidrug-resistant Pseudomonas and Enterobacter species have been reported in a number of studies, and microbial substitution has been observed when antibiotics are used (Poirel et al., 2004; Horii et al., 2005). We speculated that microbial substitution to Pseudomonas and Enterobacter species following the use of antibiotics might have occurred in the oral cavity of our patient. Thus it is important to carefully determine which bacterial species have infected patients with bacteraemia or heart disease, and then choose the appropriate antibiotics for their elimination.
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ACKNOWLEDGEMENTS
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This study was supported by the 21st Century COE program entitled ‘Origination of Frontier BioDentistry’ at Osaka University Graduate School of Dentistry supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research (B) 16390605 from the Japan Society for Promotion of Science, a Grant-in-Aid for Exploratory Research 17659647 and a Grant-in-Aid for Young Scientists (A) 18689050 from the Ministry of Education, Culture, Sports, Science and Technology.
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