Spontaneous peritonitis and bacteremia caused by Streptococcus equinus: Value of 16S rRNA gene sequencing analysis

Streptococcus equinus constitutes an uncommon Gram-positive cocci that can cause infections in humans rarely. We have recently encountered a case of S. equinus peritonitis, established by 16S rRNA gene sequence analysis, in a peritoneal dialysis (PD) patient which, in our knowledge, is the first reported case in a PD child complicated with bacteremia. Correspondence to: Stella Stabouli, MD, PhD, 1st Department of Pediatrics, Hippokratio Hospital, Konstantinoupoleos 49, 546 42 Thessaloniki, Greece, Tel:003


Introduction
Streptococcus equinus is Gram-positive cocci, catalase negative, which has seldom been reported to cause infections in humans [1]. Even rarer are the peritonitis cases presented in the bibliography in previous decades [2,3]. We have recently encountered a case of S. equinus peritonitis in a peritoneal dialysis (PD) patient that, in our knowledge, is the first reported case in a PD child complicated with bacteremia.

Case report
A 9-year old boy who had end stage renal disease, due to infantiletype polycystic kidney disease, was treated with continuous ambulatory PD from the age of 10-months old. He was admitted to the hospital with cloudy effluent, fever (39°C), vomiting and diarrhea. Empirically treatment with intraperitoneal (i.p.) vancomycin (30 mg/L) plus ceftazidime (125 mg/L) was initiated in addition to intravenous (i.v.) cefouroxime (50 mg/kg). The PD fluid obtained, was slightly opalescent, and was sent immediately to the microbiological laboratory for further analysis. In parallel, blood cultures were also obtained. The PD fluid analysis revealed the presence of 650 cells/µL with 85% neutrophils, while, Gram stain was negative for microorganism. PD fluid was injected into both aerobic and anaerobic blood culture bottles which were processed by automated BACTEC 9120 System (Becton Dickinson). Furthermore, 20 ml of the PD fluid was collected in rubber sealed pyrogen-free tubes for direct detection of bacterial DNA (Endo Tube ET; Chromogenix AB, Vienna, Austria). A broad-range 16S rRNA PCR with subsequent DNA sequencing, detecting bacterial DNA directly in clinical specimens of patients with serious infections is applied on a routine basis. The isolation and the detection of bacterial DNA in the PD fluid were performed as previously reported [4]. The PCR amplicons (520 bp) were sequenced in both directions by the same pair of primers used for amplification, in an ABI PRISM310 automated sequencer according to the manufacturer's instructions, and the obtained sequences (approximately 460 bp) were compared with 16S rRNA sequences available, both with the advanced BLAST search.
Specifically, primers 5'-GACGAACGCTGGCGGCGTGCCTA-3' and 5'-CGCTCGTTGCGGGACTTAACC-3' were used. Analysis of the sequences results demonstrated 100% identity of the microorganism with Streptococcus equines (Gen Bank accession No KJ803948.1) and was available one day after admission (Figure 1). Three days after admission and two days after the molecular    [5,6].

Discussion
Published cases of human infections due to S. equinus are rare with only two peritonitis cases presented till now. In the first case, a patient with cirrhosis developed spontaneous bacterial peritonitis plus bacteremia and was treated with aztreonam [2]. The second case was in a 63-year-old man with end-stage renal disease of unknown origin treated with CAPD. The isolate was identified as a S. equinus by an API 32 (bioMerieux, Lyon, France) automated identification system and the patient was treated with i.p. cefalotin for a total of 14 days [3]. However, 16S rRNA gene sequence analysis was not available for both cases.
The scarcity of reports concerning S. equinus infections may be due to low pathogenicity, but it also may be due to difficulties in culturing and identifying the bacteria. If it is available a 16S rRNA gene sequence analysis is useful to establish certain biotypes and subspecies when a S. equinus is isolated from a culture, and although S. equinus is rare as a cause of human infections, it is very easy to be treated even in severe cases of both bacteremia and peritonitis such in our case.