Molecular characterization of group B streptococci with reduced penicillin susceptibility recurrently isolated from a sacral decubitus ulcer

Sir, Penicillin is the first-line antibiotic for group B streptococci (GBS) in disease therapy as well as in intrapartum chemoprophylaxis because resistance to this agent has so far not been reported among GBS clinical isolates. However, we have recently reported GBS isolates with reduced penicillin susceptibility (PRGBS), where altered penicillin-binding protein (PBP) 2X demonstrates a major contribution to the reduction of β-lactam susceptibility.1 We have also shown a diversity of mutations in PBP genes among PRGBS, while those genes of the penicillin-susceptible strains were highly conserved, irrespective of their isolation dates.2 Those PRGBS isolates have been mostly recovered from respiratory specimens. Here, we report a case of a lasting sacral decubitus ulcer from which PRGBS were isolated repeatedly, together with the molecular characteristics of the isolates. A 58-year-old man underwent descending graft replacement of a Stanford type B dissecting aortic aneurysm at our medical centre. After the operation, the patient developed paraplegia, which was not improved after axillofemoral bypass surgery. He was then transferred to a special physical rehabilitation centre. During his prolonged rehabilitation stay, a severe decubitus ulcer developed in the sacral area, wound bleeding was seen and he was referred to our centre. He had not received antimicrobial treatment for more than 3 months except for a 1 day intravenous injection of piperacillin (2000 mg/day) due to the occurrence of a fever just 3 days before hospital admission. Bacterial culture of the specimen taken at the surface of the decubitus ulcer at his initial visit yielded heavy growth of GBS as well as small numbers of group G streptococci and Proteus mirabilis. GBS was still predominant together with group G streptococci, and a small number of Staphylococcus aureus, in a similar sample taken on the day of admission, which corresponded to 22 days after his initial visit. On day 6, operative treatment of the decubitus ulcer by debridement followed by a rotation flap was performed. After 4 day intravenous administration of cefazolin (2000 mg/day) starting on day 6, an agent used for conventional post-operative treatment in our medical centre, only S. aureus was recovered from post-operative fistula exudates. The GBS serotype Ia isolates, which were detected at intervals, were PRGBS with a penicillin MIC of 0.25 mg/L determined by a broth microdilution method with a MicroScan MICroFAST panel type 3J system (Siemens Healthcare Diagnostics Inc., Tokyo, Japan) as described previously.2 MICs of ampicillin, cefazolin and cefotaxime were 0.25, 2 and 1 mg/L, respectively (Table 1). PFGE patterns of ApaI-digested DNA1 of these two isolates were identical. Multilocus sequence typing (MLST) was performed using specific primers as described previously.3 Amplification of seven housekeeping genes, adhP, pheS, atr, glnA, sdhA, glcK and tkt, by PCR was carried out using PrimeSTAR HS DNA polymerase (Takara Shuzo Co., Kyoto, Japan) with reaction conditions of one cycle of 98°C for 1 min, followed by 30 cycles of 98°C for 10 s, 55°C for 15 s and 72°C for 1 min, and finally one cycle of 72°C for 7 min. PCR products were purified and sequenced.2 Allelic profile assignment and sequence type (ST) determinations were made using the GBS MLST databases (http://pubmlst.org/sagalactiae). Both PRGBS were found to have ST-1 with allelic profile 1121122, which has been a heterogeneous lineage comprising mainly serotype V and various other serotypes, and has been identified among carriage and invasive isolates.4 Thus, those PRGBS were regarded as genetically identical by PFGE and MLST. MICs of antimicrobials for PRGBS isolates MICs of antimicrobials for PRGBS isolates The nucleotide and deduced amino acid sequences of PBP 2X from the two PRGBS isolates (GenBank accession number AB512415) were compared with those from the 2603V/R reference strain (ATCC BAA-611; GenBank accession number NC_004116). The PRGBS isolates shared amino acid substitution V405A, which has been found to be a key substitution, together with Q557E, responsible for penicillin non-susceptibility in PBP 2X.1,2 In addition, the isolates shared three substitutions, G398A,1 G329V and G429D, where the latter two were new substitutions for PRGBS. The G429D substitution, which corresponds to the G422D substitution in Streptococcus pneumoniae, may possibly be a compensatory mutation, as has been suggested for S. pneumoniae.5 In this case, PRGBS was consecutively and predominantly isolated from a sacral decubitus ulcer as a cause of mixed infection, and thus was found to be capable of surviving persistently at the site of infection, for >3 weeks. It remains unclear whether or not the results of in vitro penicillin non-susceptibility of PRGBS would predict the clinical significance of this kind of microbe in antimicrobial chemotherapy. Despite the relatively high MIC of cefazolin, 2 mg/L for this PRGBS, 4 day intravenous administration of cefazolin (2000 mg/day) seemed effective in this case, but accumulation of clinical data on an appropriate antimicrobial therapeutic strategy would be essential, especially in the cases of sepsis or meningitis in both neonates and elderly individuals. This work was supported by a grant (grant number H18-Shinkou-011) from the Ministry of Health, Labour and Welfare of Japan. None to declare. We are grateful for the valuable scientific advice of Satowa Suzuki, Kunikazu Yamane and Jun-ichi Wachino, Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases.