Antimicrobial susceptibility testing of Bilophila wadsworthia by using triphenyltetrazolium chloride to facilitate endpoint determination

Summanen, Paula; Wexler, Hannah M.; Finegold, Sydney M.

doi:10.1128/aac.36.8.1658

Cited by 39 publications

(42 citation statements)

References 19 publications

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“…TTC has been widely used in microbiology to rapidly determine drug susceptibilities of microorganisms (5,29) and for rapid detection of bacteria in urine (7) or bacterial contamination in water (21). It has also been used to identify a pathological subspecies of Campylobacter (14) as well as to identify some Candida species (18).…”

Section: Discussionmentioning

confidence: 99%

Efficiency of a Pneumococcal Opsonophagocytic Killing Assay Improved by Multiplexing and by Coloring Colonies

Kim

Nahm

2003

Clin Vaccine Immunol

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For evaluating pneumococcal vaccines, the opsonophagocytic killing assay (OPKA) is useful as a supplement to the pneumococcal antibody enzyme-linked immunosorbent assay (ELISA). However, evaluations of pneumococcal vaccines require the determination of antibody responses to 7 to 11 serotypes, and the OPKA is tedious to perform and requires more serum than the ELISA. Consequently, the OPKA is infrequently used for evaluating pneumococcal vaccines. To overcome these limitations, we have developed a simple multiplexed (double-serotype) OPKA by using antibiotic-resistant pneumococci for nine serotypes. Serotype 6B, 9V, 19A, and 23F strains were made streptomycin resistant, and serotype 4, 6A, 14, 18C, and 19F strains were made optochin resistant. The multiplexed OPKA was the same as the single-serotype OPKA except for two changes. First, the target bacteria were a mixture of one streptomycin-resistant strain and one optochin-resistant strain. Second, the surviving bacteria of each serotype were enumerated by plating on Todd-Hewitt agar plates with yeast extract and an agar overlay containing the appropriate antibiotics and 2,3,5-triphenyl tetrazolium chloride. The performance of the multiplexed OPKA was evaluated by analyzing 28 serum samples from adults immunized with a 23-valent polysaccharide vaccine by using the single-serotype OPKA and the multiplexed OPKA. The multiplexed OPKA was specific for the desired serotypes. The multiplexed and conventional OPKAs had comparable assay sensitivities and produced results that were highly correlated (r 2 values ranging from 0.92 to 0.98) for all nine serotypes. A simple modification of the conventional OPKA produces a multiplexed assay that greatly reduces effort, reagents, and the necessary amount of serum.

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Section: Discussionmentioning

confidence: 99%

Efficiency of a Pneumococcal Opsonophagocytic Killing Assay Improved by Multiplexing and by Coloring Colonies

Kim

Nahm

2003

Clin Vaccine Immunol

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“…MICs were defined as the lowest concentration of antimicrobial agent resulting in no growth or a marked change in the appearance of growth compared to the control plate, as described in the CLSI protocol. Triphenyltetrazolium chloride was used as an aid in interpreting the growth endpoints of Bilophila wadsworthia (10). Reference strains of Bacteroides fragilis (ATCC 25285), Bacteroides thetaiotaomicron (ATCC 29741), and Eggerthella lenta (ATCC 43055) were used as controls in each test.…”

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confidence: 99%

In Vitro Activities of DX-619 and Four Comparator Agents against 376 Anaerobic Bacterial Isolates

Molitoris

Väisänen

Bolaños

et al. 2006

Antimicrob Agents Chemother

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The activity of DX-619 was evaluated against 376 anaerobic isolates using the reference CLSI agar dilution method. Overall, 90% of the strains were susceptible to DX-619 at <1 g/ml. It was more active than the other four compounds tested except for meropenem, which showed virtually identical overall activity.Resistance to antimicrobial agents has been observed in many if not most clinically significant pathogenic bacteria and is increasing in prevalence. New classes of antimicrobial agents and modifications of existing agents, as well as of methods of blocking bacterial resistance mechanisms, are essential for improving activity against these resistant organisms. DX-619 is a newly developed des-F(6)-quinolone ( Fig. 1) that has been shown to be effective against multiresistant gram-positive bacteria including methicillin-, ciprofloxacin-, and vancomycinresistant Staphylococcus aureus, ciprofloxacin-resistant Streptococcus pneumoniae, and vancomycin-resistant enterococci (1, 4). It is currently under development for use in gram-positive infections. Garenoxacin, another desfluoroquinolone, has shown good broad-spectrum activity against gram-positive and gramnegative aerobes and anaerobes (3,5,8). This study compared the activity of DX-619 and that of four comparator agents (amoxicillin-clavulanate, linezolid, meropenem, and moxifloxacin, chosen from different classes of antimicrobials that are effective against anaerobes) against 376 strains of anaerobic bacteria.The bacteria included in this study were recent isolates from the Greater Los Angeles Veterans Administration Healthcare Center. Bacteria were identified according to established procedures (6), supplemented in a number of cases by 16S rRNA sequence analysis. Most of the organisms studied are involved in a great variety of infections. MICs with regard to anaerobes were determined by the Clinical and Laboratory Standards Institute (CLSI) (formerly the National Committee for Clinical Laboratory Standards)-approved Wadsworth agar dilution technique (2). A suspension of colonies taken from 48-h blood agar plates was used to achieve a final inoculum of 10 5 CFU/ spot. The basal medium was brucella base laked-blood agar (Anaerobe Systems, Morgan Hill, CA) with hemin, vitamin K 1 , and 5% laked sheep blood, supplemented with pyruvic acid (1% final concentration) for the growth of Bilophila wadsworthia and formic and fumaric acids (0.3%/0.3%) for Sutterella wadsworthensis. Plates were incubated in an anaerobic chamber (Anaerobe Systems) for 48 h at 37°C. MICs were defined as the lowest concentration of antimicrobial agent resulting in no growth or a marked change in the appearance of growth compared to the control plate, as described in the CLSI protocol. Triphenyltetrazolium chloride was used as an aid in interpreting the growth endpoints of Bilophila wadsworthia (10). Reference strains of Bacteroides fragilis (ATCC 25285), Bacteroides thetaiotaomicron (ATCC 29741), and Eggerthella lenta (ATCC 43055) were used as controls in each test. The antimicrobial agents t...

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“…While B. wadsworthia is found in the stool in mean counts of only 105 to 106/g (where total bacterial counts are -10'/g), it was the third most common anaerobe isolated during a study of infection related to gangrenous and perforated appendices (1,5). Most strains are ,-lactamase positive (24). Our early studies (27) indicated significant resistance to ,B-lactamase-stable ,B-lactams, such as imipenem and cefoxitin, among strains of B. wadsworthia.…”

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confidence: 99%

“…However, susceptibility results were difficult to interpret because many strains of B. wadsworthia produced a heavy haze on agar dilution plates, even in the presence of high concentrations of antimicrobial agents. Studies that used viability staining (triphenyltetrazolium chloride [TTC]), viability counts, and microscopy of the hazy areas showed that no net increase in viable cells occurred and that the haze consisted of spheroplasts (24).…”

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confidence: 99%

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Morphological response of Bilophila wadsworthia to imipenem: correlation with properties of penicillin-binding proteins

Summanen

Wexler

Lee

et al. 1993

Antimicrob Agents Chemother

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The penicillin-binding protein (PBP) patterns of six strains of Biophila wadsworthia were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and subsequent fluorography of membrane preparations labelled with 13H]benzylpenicillin. The PBP profiles among the strains were similar, generally, seven to nine PBP-reactive bands could be visualized; their molecular weights ranged from 31 to 137 kDa. The relative affinities of the PBPs of four strains ofB. wadsworthia for imipenem were examined and correlated with the morphological responses of the cells to imipenem. Morphological changes were examined by light and electron microscopies. Light microscopy revealed that at low concentrations (less than the MIC), imipenem induced the formation of rounded and bulging cells; rarely, elongation without filamentation was observed. In the presence of imipenem at the MIC, spheroplast formation was observed. Scanning and transmission electron microscopies revealed round forms together with larger, multilobate cells in the presence of subinhibitory concentrations of imipenem, suggesting that new growth sites were initiated while cell division was inhibited. Peeling of the outer membrane was also seen. Spheroplasts were very large (up to 30 ,um in diameter) and stable in aqueous solution. Inhibition of the PBPs could be seen in the presence of low imipenem concentrations.

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Antimicrobial susceptibility testing of Bilophila wadsworthia by using triphenyltetrazolium chloride to facilitate endpoint determination

Cited by 39 publications

References 19 publications

Efficiency of a Pneumococcal Opsonophagocytic Killing Assay Improved by Multiplexing and by Coloring Colonies

Efficiency of a Pneumococcal Opsonophagocytic Killing Assay Improved by Multiplexing and by Coloring Colonies

In Vitro Activities of DX-619 and Four Comparator Agents against 376 Anaerobic Bacterial Isolates

Morphological response of Bilophila wadsworthia to imipenem: correlation with properties of penicillin-binding proteins

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