Prosthetic hip joint infection with a Streptococcus agalactiae isolate not susceptible to penicillin G and ceftriaxone

Sir, Streptococcus agalactiae [group B Streptococcus (GBS)] colonize and cause various infections in neonates and adults. GBS are reported to be universally susceptible to penicillin G.1 Four recent studies documented amino acid substitutions in penicillin-binding protein (PBP) in GBS clinical isolates with increased penicillin MICs.2–5 Here we report a case of invasive penicillin G-non-susceptible GBS infection. In 2002, a 55-year-old woman with a history of treated ovarian carcinoma had a right hip prosthesis for a fractured femoral neck. In 2004, the culture of the articular fluid from the right hip was positive for GBS. The patient was treated intravenously with 12 × 106 units of penicillin G Na daily for 6 weeks followed by prolonged oral therapy with 300 mg of penicillin V every 24 h. In 2007, the culture of the pus, from a para-articular collection near the right hip, grew GBS, and penicillin V was increased to 600 mg every 8 h. In 2008, the abscess was drained without surgical debridement, the culture of the pus was negative and the patient was treated with 500 mg of cefadroxil every 12 h for 14 days. Two months later, the technetium and gallium scans were negative for infection, the sedimentation rate and protein electrophoresis were normal and the patient had a normal right hip exam. Identification of the two GBS isolates was confirmed at the provincial reference laboratory LSPQ/INSPQ. The susceptibility of GBS isolated in 2004 (GBS 2004) and 2007 (GBS 2007) was tested at Hôpital Saint-Luc (Montréal) by Etest (oxacillin, ampicillin and meropenem), by the CLSI disc diffusion method with linezolid 30 µg discs and at LSPQ/INSPQ by the CLSI microdilution method with penicillin G, ceftriaxone, erythromycin, clindamycin, levofloxacin, chloramphenicol and vancomycin.1,6 MICs of 11 antimicrobial agents for the two GBS isolates are reported in Table 1. The GBS 2007 was not susceptible to ceftriaxone with increased MICs of three dilutions and to penicillin G, ampicillin and oxacillin with increased MICs of two dilutions. MICs of meropenem, even if still susceptible, increased from 0.03 mg/L for GBS 2004 to 0.25 mg/L for GBS 2007. The two GBS isolates were susceptible to erythromycin, clindamycin, vancomycin, levofloxacin, chloramphenicol and linezolid, but were resistant to tetracycline. β-Lactamase was negative for both GBS isolates with nitrocefin discs (BD BBL Cefinase discs). PFGE showed that the two GBS isolates were identical. MICs (mg/L) of antimicrobial agents for GBS isolated in 2004 and 2007 GBS, group B Streptococcus; CLSI S and CLSI R, MIC breakpoints for susceptibility (S) and resistance (R);1 NA, not available. aNo breakpoints for GBS, but breakpoints for Staphylococcus spp. are: ≥4 mg/L, resistant; and ≤2 mg/L, susceptible. MICs (mg/L) of antimicrobial agents for GBS isolated in 2004 and 2007 GBS, group B Streptococcus; CLSI S and CLSI R, MIC breakpoints for susceptibility (S) and resistance (R);1 NA, not available. aNo breakpoints for GBS, but breakpoints for Staphylococcus spp. are: ≥4 mg/L, resistant; and ≤2 mg/L, susceptible. The coding regions of the transpeptidase domain of the pbp1a, pbp2b and pbp2x genes of the two GBS isolates were amplified and sequenced according to previously outlined methods.4 Amino acid sequences were deduced and analysed using the ClustalW alignment tool included in the Lasergene software (DNAstar, Madison, WI, USA). Nucleotide and deduced amino acid sequences were compared with those of the reference penicillin-susceptible strains 2603V/R (GenBank accession number: NC_004116) and NEM316 (GenBank accession number: NC_004368). DNA analysis revealed that the pbp genes of the clinical GBS isolates possessed many amino acid substitutions compared with the corresponding genes of the reference strains 2603V/R and NEM316. Five previously described amino acid substitutions were observed in both the penicillin-susceptible GBS and penicillin G-non-susceptible GBS isolates (S453N and N682D in PBP1a, V625I in PBP2b, and I377V and G627V in PBP2x).4 However, there were three novel substitutions (T526A in PBP1a, P278L in PBP2b and N575D in PBP2x) found exclusively in the penicillin G-non-susceptible GBS 2007 isolate. In previous studies, the V405A and Q557E substitutions adjacent to the conserved SSN and KSG motifs in PBP2x, considered to form the active site of the enzyme, were found in 4 invasive GBS with elevated, but still susceptible, MICs of one or multiple β-lactam antibiotics, in 21 penicillin G-non-susceptible GBS isolated from the respiratory tract and in a penicillin G-non-susceptible GBS recurrently isolated from a sacral ulcer.2–5 Several amino acid substitutions in PBP1a, PBP2a and PBP2b were also found in penicillin G-non-susceptible GBS isolated from the respiratory tract.4 However, the PBP2a amino acid substitutions were documented in only two GBS with penicillin MICs of 1 mg/L.4 In the present study, the penicillin G-non-susceptible GBS 2007 isolate did not harbour the PBP2x V405A and Q557E substitutions previously associated with reduced susceptibility to penicillin.2–5 Instead, the penicillin G-non-susceptible GBS 2007 isolate possessed three novel substitutions (T526A in PBP1a, P278L in PBP2b and N575D in PBP2x). At this time it is not known whether these substitutions can actually increase resistance to penicillin since they were not found within or in the proximity of the putative conserved motifs, and their significance needs to be assessed in future studies. Moreover, it is possible that the penicillin G-non-susceptible GBS 2007 isolate harboured mutations in other pbp or non-pbp genes that are responsible for the observed phenotype. To our knowledge, that is the first report of development of invasive GBS not susceptible to penicillin G and ceftriaxone after prolonged low-dose oral penicillin V. The data were generated as part of the routine work of the microbiology laboratory of CHUM and LSPQ/INSPQ. M. R. is the recipient of a Research Scholar award from the Fonds de recherche en santé du Québec (FRSQ). None to declare. We thank the personnel of the bacteriology section of the Medical Microbiology Laboratory of CHUM-Hôpital Saint-Luc for their technical assistance. The editorial work on this manuscript by Ovid Da Silva, Research Support Office, Research Centre, CHUM, is appreciated.

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