Supplemented Mueller-Hinton Agar Research Articles

Detection of N526K-substituted penicillin-binding protein 3 conferring low-level mutational resistance to -lactam antibiotics in Haemophilus influenzae by disc diffusion testing on Mueller-Hinton agar according to EUCAST guidelines

EUCAST has recently authorized a new disc diffusion test for routine antimicrobial susceptibility testing of Haemophilus influenzae, calibrated to EUCAST MIC breakpoints. We investigated whether disc diffusion testing as recommended by EUCAST could discriminate strains of H. influenzae carrying the N526K substitution in penicillin-binding protein 3 from the wild-type population. A total of 170 recent clinical isolates, genetically characterized for the presence of acquired and mutational resistance mechanisms, were tested by disc diffusion of β-lactam antibiotics on supplemented Mueller-Hinton agar. Tentative epidemiological breakpoint values for the presence of the N526K substitution were suggested for various β-lactams, and the performances were calculated. Epidemiological cut-off values of 19/20 mm for ampicillin (2 μg) and 11/12 mm for benzylpenicillin (1 U) accurately categorized 96% of the study strains, and outperformed cephalosporin-containing discs in the discrimination of mutational resistance in β-lactamase-non-producing isolates. Current EUCAST interpretative criteria for the categorization of clinical resistance showed concordance between resistance rates based on MIC and zone diameter breakpoints for both ampicillin and cefuroxime, but categorization of individual isolates was not consistent. Disc diffusion testing of H. influenzae accurately identified β-lactamase-non-producing isolates with the N526K substitution by use of discs containing low amounts of penicillins. Cephalosporin-containing discs could detect mutational resistance in β-lactamase-producing isolates, but performed with reduced specificity.

Comparison between Disk Diffusion and Microdilution Methods for Determining Susceptibility of Clinical Fungal Isolates to Caspofungin

We compared the caspofungin (CAS) susceptibility testing results generated by the disk diffusion (DD) assay with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BD) reference method for 106 yeast isolates. The isolates represented 11 different fungal species, including Candida albicans (n = 50), C. parapsilosis (n = 10), C. glabrata (n = 10), C. tropicalis (n = 10), C. guillermondii (n = 6), C. rugosa (n = 5), C. krusei (n = 5), C. kefyr (n = 2), C. pelliculosa (n = 2), Saccharomyces cerevisiae (n = 3), and Geotrichum candidum (n = 3). The DD assay was performed in supplemented Mueller-Hinton agar with CAS, which was tested at concentrations of 2, 10, and 25 mug per disk. MICs and inhibition zone diameters were evaluated at 24 and 48 h. In general, the results obtained by the DD assay correlated well with those obtained by the BD method. In particular, a significant correlation between methods was observed when CAS was used at concentration of 2 mug/disk at a reading time of either 24 or 48 h.

Multicenter Evaluation of a New Disk Agar Diffusion Method for Susceptibility Testing of Filamentous Fungi with Voriconazole, Posaconazole, Itraconazole, Amphotericin B, and Caspofungin

The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera, Aspergillus sp. (five species), Alternaria sp., Bipolaris spicifera, Fusarium sp. (three species), Mucor sp. (two species), Paecilomyces lilacinus, Rhizopus sp. (two species), and Scedosporium sp. (two species): (i) two media (supplemented Mueller-Hinton agar [2% dextrose and 0.5 microg/ml methylene blue] and plain Mueller-Hinton [MH] agar), (ii) three incubation times (16 to 24, 48, and 72 h), and (iii) seven disks (amphotericin B and itraconazole 10-microg disks, voriconazole 1- and 10-microg disks, two sources of caspofungin 5-microg disks [BBL and Oxoid], and posaconazole 5-microg disks). MH agar supported better growth of all of the species tested (24 to 48 h). The reproducibility of zone diameters and their correlation with either MICs or MECs (caspofungin) were superior on MH agar (91 to 100% versus 82 to 100%; R, 0.71 to 0.93 versus 0.53 to 0.96 for four of the five agents). Based on these results, the optimal testing conditions for mold disk diffusion testing were (i) plain MH agar; (ii) incubation times of 16 to 24 h (zygomycetes), 24 h (Aspergillus fumigatus, A. flavus, and A. niger), and 48 h (other species); and (iii) the posaconazole 5-microg disk, voriconazole 1-microg disk, itraconazole 10-microg disk (for all except zygomycetes), BBL caspofungin 5-microg disk, and amphotericin B 10-microg (zygomycetes only).

Synergy of daptomycin with oxacillin and other beta-lactams against methicillin-resistant Staphylococcus aureus.

We previously observed marked synergy between daptomycin and both rifampin and ampicillin against vancomycin-resistant enterococci (VRE). Because the synergy between daptomycin and ampicillin was observed for 100% of VRE strains with high-level ampicillin resistance (ampicillin MIC of > or =128 microg/ml), we looked for synergy between daptomycin and other beta-lactams against 18 strains of methicillin-resistant Staphylococcus aureus (MRSA) by employing a time-kill method using Mueller-Hinton broth supplemented to 50 mg of Ca2+/liter. All strains were resistant to oxacillin (16 of 18 strains were resistant at drug concentrations of > or =256 microg/ml), and all strains were susceptible to daptomycin (the MIC at which 90% of the tested isolates were inhibited was 1 microg/ml). Daptomycin was tested at concentrations of 2, 1, 0.5, 0.25, 0.125, and 0.0625 microg/ml alone or in combination with oxacillin at a fixed concentration of 32 microg/ml. Synergy was found for all 18 strains with daptomycin at one-half the MIC in combination with 32 microg of oxacillin/ml, and synergy was found for 11 of 18 strains (61%) with daptomycin at one-fourth the MIC or less in combination with oxacillin. At 24 h, the daptomycin-oxacillin combination with daptomycin at one-half the MIC showed bactericidal activity against all 18 strains, and the combination with one-fourth the daptomycin MIC showed bactericidal activity against 9 of 18 strains. We also used a novel screening method to look for synergy between daptomycin and other beta-lactams. In this approach, daptomycin was incorporated into Ca(2+)-supplemented Mueller-Hinton agar at subinhibitory concentrations, and synergy was screened by comparing test antibiotic Kirby-Bauer disks on agar with and without daptomycin. By this method, daptomycin with ampicillin-sulbactam, ticarcillin-clavulanate, or piperacillin-tazobactam showed synergy comparable to or greater than daptomycin with oxacillin. For seven of the eight strains tested, time-kill studies confirmed synergy between daptomycin and ampicillin-sulbactam with ampicillin in the range of 2 to 8 microg/ml. The combination of daptomycin and beta-lactams may be useful for the treatment of MRSA infection, but further studies are needed to elucidate the mechanisms and to determine the in vivo efficacy of the combination.

Daptomycin synergy with rifampicin and ampicillin against vancomycin-resistant enterococci.

We used a novel screening method to look for synergy between daptomycin and 18 other antibiotics against 19 strains of high-level vancomycin-resistant enterococci (VRE) (vancomycin MIC > or = 256 mg/L). In this approach, daptomycin was incorporated into Ca(2+)-supplemented Mueller-Hinton agar at subinhibitory concentrations, and synergy was screened by comparing test antibiotic Etest MICs on agar with and without daptomycin. A striking reduction in the rifampicin MIC was seen in 11/15 (73.3%) VRE that were resistant to rifampicin, from > or =12 mg/L to a mean +/- s.d. of 0.22 +/- 0.21 mg/L at daptomycin 0.25 x MIC and 0.85 +/- 0.90 mg/L at daptomycin 0.125 x MIC. Synergy was also observed for 13/19 (68%) isolates with ampicillin (MIC > or = 128 mg/L). There was no significant synergy between daptomycin and any other antibiotic by this screening method. If confirmed by further studies, daptomycin with either rifampicin or ampicillin may be useful in the management of infections caused by VRE.

Cytotoxicity of extracellular Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaire's disease and Pontiac fever, is known to produce a cytopathic effect on macrophages. The capacity of extracellular L. pneumophila to mediate toxicity for guinea pig peritoneal macrophages and J774 mouse macrophages was assessed. Extracellular organisms were found to be capable of mediating toxicity; however, toxic activity appeared to require close proximity with the mononuclear cell surface. Serogroup 1 strains grown on supplemented Mueller-Hinton agar exhibited variable expression of toxic activity. One strain positive on supplemented Mueller-Hinton agar was cytotoxic and unable to replicate in J774 macrophages but remained virulent for guinea pigs at high doses.

Correlation ofLegionella pneumophila virulence with the presence of a plasmid

Legionella pneumophila is the causative agent of Legionellosis in man and considered an opportunistic intracellular Gram-negative bacterium that preferentially infects macrophages. The presence of a plasmid in these organisms was determined in cultures of the bacteria grown in vitro. A correlation was observed between the growth of virulent strains of theLegionella in murine macrophages and growth on standard buffered charcoal yeast extract agar supplemented with 0.1% α-ketoglutarate (BCYEα) agar medium rich in cysteine and widely used for growth of the bacteria in vitro. In contrast, the avirulent isolates of these strains grew well on supplemented Mueller-Hinton (SMH) agar utilized for differentiating virulent from avirulentLegionella. However, one virulent strain ofLegionella (the Iowa strain) was found to grow moderately well on the SMH agar. In addition, test strains ofLegionella that infect in vitro human monocytes were found to grow moderately well on the BCYE-α agar, but did not grow on the SMH agar. Examination of these strains for plasmid DNA expression showed that extra chromosomal DNA-containing molecules were present in theL. pneumophila strains characterized as virulent for in vitro growth in macrophages. However, the avirulent strains that replicated in the human monocytes readily but only poorly in the permissive murine macrophages did not show evidence of similar plasmid DNA expression.

Characterization of the selective inhibition of growth of virulent Legionella pneumophila by supplemented Mueller-Hinton medium.

The phenotypic difference between virulent and avirulent Legionella pneumophila with regard to growth on supplemented Mueller-Hinton (SMH) agar was investigated. Defined populations of virulent and avirulent L. pneumophila were inoculated onto hybrid growth media containing the combination of SMH agar components with potassium phosphate-buffered charcoal-yeast extract agar. The casein acid hydrolysate component of SMH agar was found to be inhibitory to the growth of virulent but not avirulent cells. The selectively inhibitory component of the casein acid hydrolysate was identified as NaCl.

Virulence conversion of Legionella pneumophila: a one-way phenomenon

Previous investigations have shown that Legionella pneumophila converts from virulence to avirulence after passage on supplemented Mueller-Hinton (SMH) agar and may convert back to virulence after passage in guinea pigs. However, there is no additional information concerning the apparent interconversion of virulent and avirulent derivatives of L. pneumophila cultures. We investigated the stability of a parental virulent culture and its avirulent derivatives and the growth and viability of these cultures on charcoal-yeast extract (CYE) and SMH agars. Avirulent derivatives of a highly virulent L. pneumophila culture were obtained by passage of the virulent parent culture on SMH agar. The only time a virulent L. pneumophila culture was recoverable from an avirulent culture was when the avirulent culture was derived from a saline suspension of a virulent culture which had been passaged only five times on SMH agar. When an avirulent culture was derived from a virulent culture passaged 25 times on SMH agar or from an isolated colony which grew on a SMH agar plate, we were unable to recover a virulent culture after successive passage through guinea pigs. These results suggest that the conversion process which occurs between virulent and avirulent forms of L. pneumophila is a one-way phenomenon from virulence to avirulence and that stable avirulent derivatives can be isolated. Furthermore, our findings suggest that SMH agar acts as a selective medium for the growth of avirulent L. pneumophila, and growth on SMH agar may be a phenotypic marker for avirulence. Virulent cells, although unable to grow on SMH agar, may remain viable for several passages on SMH agar and propagate when inoculated into guinea pigs.

Agar disk elution method for susceptibility testing of Mycobacterium marinum and Mycobacterium fortuitum complex to sulfonamides and antibiotics.

An agar disk elution method using round well plates, supplemented Mueller-Hinton agar, and commercial drug disks is described for susceptibility testing of Mycobacterium marinum and the rapidly growing mycobacteria to antibiotics and sulfonamides. By this method, 14 of 14 strains of M. marinum were susceptible to rifampin, doxycycline, minocycline, and trimethoprim-sulfamethoxazole. Identical results were obtained with Middlebrook 7H10 agar and drugs prepared from standard powders. With 58 isolates of Mycobacterium fortuitum and Mycobacterium chelonei, this method had a 92% correlation with broth minimal inhibitory concentration determinations for cefoxitin and greater than 98% for doxycycline, kanamycin, amikacin, and the sulfonamides. Sixty-nine percent of isolates of M. chelonei susceptible to amikacin on supplemented Mueller-Hinton agar were resistant on 7H10 agar, and 15 of 16 M. chelonei isolates susceptible to erythromycin in broth were resistant by disk elution when an endpoint of no growth was used with either agar. The agar disk elution method offers a practical method for testing of most antibacterial agents against these mycobacterial species.

Virulence conversion of Legionella pneumophila serogroup 1 by passage in guinea pigs and embryonated eggs.

When virulent Legionella pneumophila is passaged on supplemented Mueller-Hinton agar, it remains virulent for guinea pigs and embryonated hen eggs for two passages. However, by the fifth passage the cultures become avirulent for guinea pigs. Flagella were not produced by L. pneumophila on the first passage on supplemented Mueller-Hinton agar. In contrast, 12 passages on charcoal-yeast extract agar did not result in the reduction of virulence or the loss of flagella of L. pneumophila. Growth in supplemented yeast extract broth or on Norit-A-filtered supplemented yeast extract agar also did not result in a reduction of the virulence of L. pneumophila. However, L. pneumophila did not produce flagella when grown on these two media. Thus, it appears that the production of flagella is not required for the virulence of L. pneumophila when administered by the intraperitoneal route of infection. A virulent flagellated form of L. pneumophila was recovered by passing an avirulent form six times in guinea pigs. When avirulent L. pneumophila was passaged 12 times in embryonated eggs, a nonflagellated form of the bacterium was recovered which had an increased virulence for guinea pigs and embryonated eggs. However, virulent forms were not recovered by passage of avirulent forms on commonly used laboratory media. These results support the suggestion that a suitable host is required for the selection of the virulent form of L. pneumophila from avirulent cultures.

Aromatic substrate specificity of browning by cultures of the Legionnaires' disease bacterium.

When the Legionnaires' disease (LD) bacterium is grown on supplemented Mueller-Hinton agar, brown pigmentation of the medium occurs. Since this browning may result from tyrosinase-mediated formation of melanin, we supplemented yeast-extract agar with various aromatic precursors of melanin and inoculated it with eight strains of the LD bacterium. Browning occurred with growth of each LD strain of the bacterium on yeast-extract agar enriched with 2.5 mmol/L of L-phenylalanine or L-tyrosine but not without such enrichment. Equimolar D-phenylalanine or D-tryosine in yeast-extract agar did not enhance browning. The LD bacterium may possess L-phenylalanine hydroxylase activity, but it does not use D-aromatic amino acids effectively in pigment production.

Exotoxin activity associated with the Legionnaires disease bacterium.

Hemolysis occurred around growth of the Legionnaires disease bacterium on supplemented Mueller-Hinton agar containing sterile defibrinated blood from each of five mammalian species. Hemolysis was most pronounced with guinea pig or rabbit blood, was less intense with horse or sheep blood, and was slight with blood from a human donor. Sterile filtrates of allantoic fluid from embryonated hen's eggs that had been infected with this organism displayed hemolytic activity in a radial hemolysis assay with guinea pig cells in agar. Growth of the Legionnaires disease bacterium on F-G agar with 5% hen's egg yolk was surrounded by a zone of clearing and more circumscribed zones of iridescence and increased opacity on and in the medium. Attempts to detect activity analogous to that of Escherichia coli heat-labile or heat-stable enterotoxin in allantoic fluid from infected eggs or in cultures of the Legionnaires disease bacterium were not successful.

Effect of supplementall-tyrosine on pigment production in cultures of the Legionnaires' disease bacterium

The etiologic agent of Legionnaires' disease grows on certain agar media. Cultures of this organism on supplemented Mueller-Hinton agar are characterized by the appearance of brown pigment in and around areas of bacterial growth. The major peptone source in Mueller-Hinton agar is an acid hydrolysate of casein. Legionnaires' disease bacterium also grows on a medium in which the peptone source is 0.25% yeast extract, but no pigment is produced. If the yeast extract agar is enriched withl-tyrosine, pigment formation can occur. Pigmentation of cultures of Legionnaires' disease bacterium may be mediated by a phenolo-monooxygenase, or tyrosinase.

Standardized Disk-diffusion Susceptibility Test forHaemophilus influenzae

The emergence of ampicillin-resistant strains of Haemophilus influenzae has emphasized the need for an improved practical method for routine susceptibility testing of clinical isolates. We have previously described a simplified medium for quantitative dilution susceptibility testing that is composed of Mueller-Hinton medium plus Supplement C (Difco). In the present study, paired broth-dilution and disk-diffusion susceptibility tests with ampicillin and chloramphenicol were performed on 100 strains of Haemophilus (95 H. influenzae and five H. parainfluenzae), including 30 strains with previously documented ampicillin resistance. Disk-diffusion tests were performed in exactly the same manner as the standardized Kirby-Bauer procedure used for less fastidious organisms, except that supplemented Mueller-Hinton agar plates were incubated in an increased-CO2 atmosphere. Using this method, ampicillin-susceptible strains of Haemophilus produced zone diameters of 22 mm or more, while ampicillin-resistant strains produced zones of 18 mm or less. All strains were chloramphenicol-susceptible and produced zone diameters of 30 mm or more. This method would allow routine disk-diffusion testing of isolates of H. influenzae by hospital diagnostic laboratories, using a clear medium that closely resembles unsupplemented Mueller-Hinton agar.