Importance of Medium in Demonstrating Penicillin Tolerance by Group B Streptococci

Abstract

A total of 30 clinical isolates of group B streptococci were studied for penicillin tolerance in vitro. Minimal inhibitory and bactericidal concentrations of penicillin were determined simultaneously in three test media which have been used for group B streptococci, tryptose phosphate, Muelier-Hinton, and Todd-Hewitt broths, using a logarithmic-phase inoculum of 105 colony-forming units per ml.Minimal inhibitory concentrations in the three media did not differ significantly. However, minimal bactericidal concentrations were significantly higher in tryptose phosphate broth (mean, 1.04 ,ug/ml) than in Mueller-Hinton broth (0.22 ,ug/ ml) or Todd-Hewitt broth (0.15 ,ug/ml). Similarly, ratios of minimal bactericidal to minimal inhibitory concentrations were significantly greater in tryptose phosphate broth than in Mueller-Hinton or Todd-Hewitt broth. After incubation in tryptose phosphate broth for an additional 24 h, the minimal bactericidal concentration consistently fell to levels which were only twice or equal to the minimal inhibitory concentration. This study illustrates the importance of the medium in the demonstration of penicillin tolerance and of controlling laboratory variables in the susceptibility testing of group B streptococci with penicillin.The group B streptococci are recognized as a major cause of serious neonatal infections. Poor results with penicillin therapy, despite consistent susceptibility to this group of antimicrobial agents, have prompted studies of the in vitro susceptibility ofthese streptococci to antibiotics. There are several reports of penicillin tolerance (minimal bactericidal concentration [MBC] significantly higher than minimal inhibitory concentration [MIC]), but the methodology has varied from study to study. In this report, we describe the importance of the test medium in the demonstration of this phenomenon. MATERIALS AND METHODSA total of 30 strains of group B streptococci were obtained from the clinical microbiology laboratory at Harbor/UCLA Medical Center, 12 were fsh isolates, and 18 were previous isolates which had been stored at -70°C in 10% sterile skimmed milk (Difco Laboratories, Detroit, Mich.) for up to 12 months. Each strain was from a different patient. They were identified as group B streptococci by the method of Lancefield (8) with sera provided by the Center for Disease Control, Atlanta, Ga. Twelve isolates were from throat cultures, five were from cervical swabs, two were from cerebrospinal fluid, two were from umbilical cultures, and one each was from blood, gastric aspirate, urine, stool, conjunctiva, nose, external ear, endometrium, and scalp lesion.Five colonies were subcultured from sheep blood agar to 10 ml of tryptose phosphate (TP) broth (Difco), which was incubated at 37°C for 6 h. This was diluted in fresh TP broth to a concentration of approximately 105 colony-forming units per ml, as determined by measuring the light scattering of the suspension with a Coleman model 9 nephocolorimeter (Coleman Instruments, Oak Brook, Ill.). Colony counts performe...

Bactericidal synergy between penicillin or ampicillin and aminoglycosides against antibiotic-tolerant lactobacilli

“…Clinically, lactobacillemia associated with deep-seated foci, particularly endocarditis, has been relatively refractory to high-dose parenteral therapy with the penicillins, despite MICs which are readily attainable in serum (1,13,19,20). Our previous studies have demonstrated that this discrepancy between in vitro activity and suboptimal therapeutic responses in vivo may be related to antibiotic tolerance of lactobacilli to f8-lactam and other antibiotics.…”

Evaluation of oxacillin tolerance in Staphylococcus aureus by a novel method

“…ADPC MIC plate counts fell dramatically at the MIC and were essentially absent on plates with concentrations above the MIC as long as oxacillin action was permitted to continue. ADPC MBC plate-count patterns showed significantly less variation and inoculum phase (5,18,20,21,25,29), incubation time (1,19,22,26,34,36), and carry-over of antimicrobial agents from MIC tubes to MBC plates (12,19,31,33) have been shown to influence the measured incidence of tolerance and related phenomena for a variety of bacteria and antimicrobial agents. Less intuitively obvious has been the finding that bacteria subjected to broth dilution procedures may be inadvertently protected from the action of antimicrobial agents in broth cultures, thus causing tolerance interpretations to be spuriously high and often irregular in manifestation and reproducibility (15,26,39).…”

“…Evidence has gradually accumulated indicating a significant dependency of BDPC results, especially MBCs, on technical factor variations. It is not surprising that factors such as pH (18,38), temperature (30), type of medium (22,28,32), VOL. 28, 1985 on clinical isolates and allegedly oxacillin-tolerant reference strains, Taylor and associates (36) compared results obtained by the standard broth macrodilution BDPC method as outlined in the Manual of Clinical Microbiology with those of a carefully controlled procedure patterned along the lines recommended by Ishida and associates.…”

Variables in demonstrating methicillin tolerance in Staphylococcus aureus strains

“…It is clear from our studies and from those of others (1,3,5,6,8,12) that because there is a multiplicity of methodological determinants which affect the status of a given strain, a standardized reference procedure for the determination of tolerance needs to be established.…”

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