Columbia Agar Research Articles

Isolation of Arcanobacterium haemolyticum from patients with pharyngitis in Belgium.

During a 40 months period, 49 Arcanobacterium haemolyticum strains were isolated from 12,000 throat swabs of patients with pharyngitis originating from 5 belgian provinces. All strains were recovered on sheep blood Columbia agar plates, supplemented by nalidixic acid and colistin. In two thirds of the cases, pharyngitis was associated with a rash. Half of the cases occurred in the age group between 10 and 20 years and isolates were twice more frequent in females than in males. Streptococcus pyogenes was isolated 23.2 times more frequently than Arcanobacterium haemolyticum.

Acute Myositis Associated withHemophilus Parasuisin Primary SPF Sows

Hemophilus parasuis (HPS) infection is frequently associated with polyserositis and meningitis (Glasser’s disease). HPS infection is recognized as a serious infection in susceptible specific pathogen-free (SPF) hogs. Acute HPS septicemia 4 or pneumonia, without polyserositis, has also been reported. This report describes an acute fasciitis and myositis associated with HPS infection in primary SPF sows. Thirty SPF sows were inappetent, weak, and ataxic. Several of these sows had swollen heads and 7 sows died over a 48hour period. Rectal temperatures ranged from 105 to 108 F. The remaining 23 sows responded to antibiotic therapy and recovered fully, suggesting a bacterial infection. The sows were second parity and were ceasarian derived through an SPF laboratory. They had no previous disease problems. Two of the 7 sows were necropsied. One sow had extensive subcutaneous edema of the head region (Fig. 1). Lesions of septicemia, polyserositis, or pneumonia were not present. The other sow lacked significant gross lesions. The lung, liver, and meninges of both sows were cultured aerobically in 5% bovine blood agar, 5% bovine blood Columbia agar, MacConkey agar, and mannitol salt agar, and on a 5% ovine blood agar plate with a Staphylococcus epidermidis nurse streak incubated under 5% CO, atmosphere. No bacteria were isolated from the above organs of either sow. The masseter muscle from 1 sow was cultured on a bovine blood agar plate under anaerobic conditions as well as with a Staphylococcus epidermidis nurse streak under 5% CO, atmosphere. These procedures failed to identify any anaerobic bacteria. Tiny, smooth, grayish colored, translucent, nonhemolytic bacterial colonies satellited the Staphy-

Evaluation of the E test for quantitative antimicrobial susceptibility testing of Helicobacter pylori

The Progressive Diagnostics Manufacturers epsilometer test (E test; AB Biodisk, Solna, Sweden), a quantitative variant of the disk diffusion technique, was evaluated comparatively to an agar dilution method for the antimicrobial susceptibility testing of Helicobacter pylori. A collection of 79 H. pylori clinical strains, including isolates with known resistance to various antimicrobial agents, was tested against 12 different antimicrobial agents. All strains were tested on Columbia agar supplemented with 10% horse blood. Plates were incubated at 37 degrees C in microaerobic atmosphere (5% O2, 10% CO2), and readings were done after 3 days of incubation. In general, E test MICs were easy to interpret and the correlation between MICs by the agar dilution method and the E test was good, with 86 and 99.5% of results being within, respectively, 1 and 2 log2 dilution steps in a total of 936 tests. All strains of H. pylori with documented resistance to the tested agents were detected by the E test. Thus, the E test appears to be an easy and reliable method for determination of MICs of antibiotics for H. pylori, and it may offer an interesting alternative to MIC determination by the agar dilution technique.

Egg yolk emulsion agar, a new medium for the cultivation of Helicobacter pylori

We developed a new agar, egg yolk emulsion (EYE) agar, for cultivation of Helicobacter pylori. EYE agar contains Columbia agar base (Oxoid), 10% EYE (Oxoid), 1% IsoVitaleX (BBL), and 40 mg of Triphenyleteraxolium chloride (Sigma) per liter. We compared EYE agar with the following agars: (i) brain heart infusion agar-7% horse blood-1% IsoVitaleX (GDW agar; C. S. Goodwin, E. D. Blincow, J. R. Warren, T. E. Waters, C. R. Sanderson, and L. Easton, J. Clin. Pathol. 38:1127-1131, 1985), (ii) brain heart infusion agar-10% horse serum-0.2% charcoal-1% yeast extract-40 mg of triphenyltetrazolium chloride per liter (GLU agar; Y. Glupczynski, M. Labbe, and F. Thiabaumont, p. 3-6, in F. Megraud and H. Lamouliatte, ed., Gastroduodenal Pathology and Campylobacter pylori, 1989), (iii) Columbia agar with 7% lysed horse blood (D&M agar; J. C. Dent and C. A. M. McNulty, Eur. J. Clin. Microbiol. Infect. Dis. 7:555-558, 1988), and (iv) brain heart infusion agar-10% EYE-1% IsoVitaleX (BHIE agar). H. pylori CFU counts, expressed as average percentages of maximum growth, were as follows: EYE agar, 96; GDW agar. 76; BHIE agar, 57; D&M agar, 52; and GLU agar, 23. Colony counts for EYE agar were significantly higher than for GDW agar (P = 0.027), BHIE agar (P = 0.005), D&M agar (P = 0.0001), and GLU agar (P less than 0.0001). EYE agar also had higher CFU counts than two commercial chocolate media; the EYE agar count was 80%, versus 33% for BBL chocolate medium and 63% for Remel chocolate medium.

Phagocytosis of mastitis isolates of Staphylococcus aureus and expression of type 5 capsular polysaccharide are influenced by growth in the presence of milk

Phagocytosis by bovine polymorphonuclear granulocytes of seven capsular polysaccharide type 5 Staphylococcus aureus strains isolated from mastitis [corrected] was investigated by means of luminol-dependent chemiluminescence. Bacteria were grown on four different agar media (brain heart infusion, Columbia broth, modified staphylococcus medium 110, and skim milk) and were opsonized by normal bovine serum. When compared to growth on brain heart infusion agar, Columbia agar, and modified staphylococcus medium 110 agar, growth on skim milk agar rendered five of the strains more resistant to opsonization. The other two strains were resistant in all culture media used. Short periods of incubation in milk after growth on brain heart infusion agar did not augment resistance to phagocytosis, indicating that mere adsorption of milk components on bacteria was not responsible. The variability of the chemiluminescence response of polymorphonuclear leukocytes was pronounced among strains with each growth medium except milk. Growth on modified staphylococcus medium 110 and on milk agar favored the masking of teichoic acid, as shown by inagglutinability with rabbit antiserum. Interestingly, agglutination by a monoclonal antibody to capsular polysaccharide type 5 was optimal when bacteria were grown on skim milk agar. This suggests that capsular polysaccharide participated in the masking effect. These findings indicate that masking of the bacterial target of most of the naturally acquired opsonins present in normal bovine serum occurred when bacteria grew in the presence of milk, resulting in an increased resistance to phagocytosis by polymorphonuclear leukocytes.

An elective and selective isolation medium for Bifidobacterium spp.

A modified Columbia agar medium, containing 5 or 10 ml/l propionic acid and adjusted to pH 5.0, is described. The medium is both elective and selective for bifidobacteria.

Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp.

A selective and differential medium (PALCAM agar) was elaborated for the isolation and enumeration of Listeria monocytogenes. PALCOM is based on Columbia agar with 0.05% glucose made selective by the addition of 0.001% polymyxin B, 0.0005% acriflavin, 1.5% lithium chloride and 0.002% ceftazidime. The diagnostic traits were attained by the incorporation of (i) 0.08% aesculin and 0.05% ferric salt; and (ii) 1% mannitol plus 0.008% phenol red. PALCAM recovered testrains of L. monocytogenes and other Listeria spp quantitatively and suppressed most other bacteria of common occurence in fresh food. L. monocytogenes colonies were approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. The occasional Enterococcus or Staphylococcus strains developing on the medium gave rise to grey colonies with a brown-green halo or yellow colonies with a yellow halo. PALCAM was the preferred medium out of 13 tested Listeria selective agars in current use. A similar differential enrichment broth, L-PALCAMY was developed based on peptone yeast extract broth with 2.5% egg yolk emulsion. The diagnostic traits and inhibitors used in this medium were the same as in PALCAM agar, through in different concentrations. Growth rate and cellcrop of L. monocytogenes in L-PALCAMY were of the same order as in Columbia broth. The growth of the majority of other bacteria of common occurrence in fresh foods was inhibited. The medium recovered L. monocytogenes more effectively from severely contaminated food than other current enrichment media.

Direct isolation of atypical thermophilic Campylobacter species from human feces on selective agar medium.

Campylobacter upsaliensis is the name which has been proposed for a new group of thermophilic campylobacter strains which differ from C. jejuni and C. coli in having a negative or weak catalase reaction. Primary isolation of these strains from human feces has been achieved only by use of filtration techniques. We report here direct isolation of strains corresponding to C. upsaliensis from stools of six children. The strains were isolated on a newly described campylobacter-selective medium. The strains were oxidase positive, hippurate negative, nitrate positive, negative for H2S in triple sugar iron, and susceptible to cephalothin (30-micrograms disk) and nalidixic acid (30-micrograms disk), and they grew at 37 and 43 degrees C, but not at 25 degrees C. The selective medium used was a blood-free, charcoal-based medium consisting of Columbia agar base, activated charcoal, cefoperazone (32 micrograms/ml), vancomycin (20 micrograms/ml), and cycloheximide (100 micrograms/ml). The medium supported the growth of the weakly reacting or catalase-negative strains, with colony counts equivalent to those obtained on antibiotic-free horse blood agar. These strains could not be isolated directly from stool on Skirrow medium, and colony counts confirmed that this medium could not support a low inoculum of these organisms. The clinical significance of these strains is unknown. We conclude that C. upsaliensis can be isolated directly from stool by using a selective medium, without the need for filtration.

Rapid identification of Escherichia coli by Fluorocult media and positive indole reaction.

To assess the specificity and sensitivity of Fluorocult media for the identification of Escherichia coli, the beta-glucuronidase activities of 1,258 bacterial strains, as well as 20 strains of Candida spp., were investigated. Fluorescence of colonies combined with positive indole reaction resulted in specificities of 99.6 to 99.8%. Sensitivities were 59.1% (MacConkey agar), 69.9% (brolacin agar), 85.5% (Columbia agar), and 85.8% (ECD agar).

New selective medium for Pseudomonas aeruginosa with phenanthroline and 9-chloro-9-[4-(diethylamino)phenyl]-9,10-dihydro-10- phenylacridine hydrochloride (C-390).

A new selective medium (PC agar) for the isolation of Pseudomonas aeruginosa was developed, consisting of 30 micrograms of 9-chloro-9-[4-(diethylamino)phenyl]-9,10-dihydro-10-phenylacridine hydrochloride (C-390) per ml and 30 micrograms of phenanthroline per ml in Columbia agar. PC agar was superior to phenanthroline, C-390, acetamide, and cetrimide agars for the selective growth of P. aeruginosa.

An improved selective and diagnostic medium for isolation and counting of Listeria spp. in heavily contaminated foods

Columbia agar base containing 0·001% acriflavine, 1·5% lithium chloride, 0·25% phenyl ethanol, 0·05% aesculin. 0·05% ferrous salt, 1% mannitol, 2·5% egg yolk emulsion and 0·008% phenol red (ALPAMY), recovered Listeria monocytogenes and some strains of Listeria seeligeri quantitatively, but suppressed Listeria ivanovii and virtually all other bacteria common in fresh foods. When used with foods processed for safety, repair on non-selective buffered glucose tryptone soya peptone yeast extract catalase agar must precede the use of ALPAMY.

Búsqueda bacteriológica e histológica de Campylobacter pyloris (CP) en mucosa gástrica

Endoscopic was performed on 135 patients looking for Campylobacter pylons (CP) . The germ was found in 80.7%. Bacteria was associated to gastritis in 84.8%. Ninety five point five percent of patients were suffering active cronic gastritis. Patients with normal gastric mucosa had CP in 22.2%. The CP was cultured in Columbia agar with sheep's blood; histologicaly CP was seen with Warthin-Starry and Giemsa stains. Rapid Urease Test was performed too and it was correlated with other tests in 70%.

Inhibition of mutans streptococci by the hematin formed on blood-containing media by proteolytic enterococci

Proteolytic Enterococcus faecalis isolates produced strong inhibitory activity against mutans streptococci in a deferred antagonism test on Columbia agar base medium supplemented with 5% human blood. Hematin was responsible for this inhibitory effect and its formation by the enterococci was dependent upon incubation in the presence of hemoglobin under conditions favourable to protease production.

Inhibition of mutans streptococci by the hematin formed on blood-containing media by proteolytic enterococci

Proteolytic Enterococcus faecalis isolates produced strong inhibitory activity against mutans streptococci in a deferred antagonism test on Columbia agar base medium supplemented with 5% human blood. Hematin was responsible for this inhibitory effect and its formation by the enterococci was dependent upon incubation in the presence of hemoglobin under conditions favourable to protease production.

DEMONSTRATION IN AN ANIMAL MODEL OF A HEAT LABILE TOXIN PRODUCED BY C PYLORIDIS (CP)

The production of enterotoxins by various bacterial cell lines has been widely studed in a variety of models including both animal and tissue culture cells. A report of a mouse model for measurement of virulence has been published recently (McCardell, et al., J. Infect. Immun; 153:177, 1986). Similar results with CP, a gram negative, curved bacterium associated with chronic gastritis, have been obtained in our laboratory. Using a modification of this animal model to identify the presence of toxins, sterilely filtered cell lysates of CP obtained from a pediatric patient were injected ip into 6-8 week old CF-1 outbred female mice. CP was grown on Columbia agar with 5% sheep blood and incubated at 37°C, microaerophically for 4 days. Harvested cells were lysed and the particulate matter was removed by centrifugatipn. The supernatant was sterilely filtered. One ml of sterile filtratres at various protein concentrations were injected ip into mice. The mice were observed for 4 days for death. Control mice were injected with PBS that was used to wash uninoculated Columbia agar plates. Protein concentrations ranging from 1 to 5 mg were evaluated. Concentrations greater than 1.3 mg were uniformly toxic (90% mortality within 48 hours). No deaths were noted during the 4 day study period with protein concentrations below 1.3 mg/ml. Additional work has snown that the toxic factor was inactivated by trypsin, heat (100°C for 15 min), and acidification to pH 4.0. The toxic factor was precipitated with 80% NH4SO4 and is nondialyzable. Postmortem examination of the mice shows edema and distention of the duodenum with the remainder of the bowel appearing normal. The above work demonstrates that there is a toxic factor that is present in certain strains of CP. This factor is lethal to mice when given ip and is probably protein in nature. Whether this factor is responsible for the epithelial damage seen in patients with active CP associated gastritis remains to be determined. Further work will also determine if this toxic factor can be demonstrated on the various tissue culture models used for toxin assays.

Relationship of bacteriocin-like inhibitor production to the pigmentation and hemolytic activity of mutans streptococci

An inhibitor production typing (P-typing) scheme originally devised for hemolytic streptococci of Lancefield groups A-G has been successfully applied to 35 mutans streptococcus isolates recovered from plaque cultures of 60 Dunedin schoolchildren. Thirteen different P-type designations were identified. Although 11 (31%) of the isolates failed to produce detectable inhibitory activity on the conventional blood agar medium used for P-typing, four of these isolates were inhibitor-positive on Trypticase Soy agar supplemented with 2% yeast extract and 0.5% calcium carbonate (TSYCa). Four mutans strains displayed strong beta-hemolysis on Columbia agar base containing human blood when incubated in a 5% CO2 in air atmosphere. Three of these also produced weak beta-hemolysis on sheep blood-supplemented medium and were further distinctive in that they were the only inhibitor P-type 767 strains to be detected in the present study. Five mutans isolates were pigment producers and this property seemed to occur independently of both the beta-hemolytic activity and the P-type designation. Upon testing an additional collection of 18 mutans strains of various serotypes, only seven (39%) were inhibitor-positive. However, three of the four serotype c strains were inhibitor producers. Two strains of serotype d and one of serotype g were more hemolytic on sheep than on human blood agar medium. In general, it seems that the most common human mutans streptococci (serotype c strains) are more likely than are other mutans strains to produce bacteriocin-like inhibitory activity and to be hemolytic for human rather than sheep erythrocytes.

In vitro susceptibility of Capnocytophaga species to 29 antimicrobial agents.

A hemoglobin-supplemented medium composed of Columbia agar base supplemented with 1% hemoglobin and 1% Polyvitex was used to investigate the in vitro activity of 29 antimicrobial agents against Capnocytophaga species. Clindamycin was the most active agent, with all strains being inhibited by 0.06 microgram/ml or less. Amoxicillin-clavulanic acid and imipenem were the most active among the beta-lactam antibiotics (MIC for 90% of strains tested [MIC90], 0.50 microgram/ml); other very active drugs were BMY 28142, cefpirome, cefotaxime, ceftazidime, and ceftriaxone (MIC90, 0.06 to 0.50 micrograms/ml), although at least one strain showed resistance to each of these antibiotics (MIC, greater than or equal to 16 micrograms/ml). Ciprofloxacin was the most active among the quinolones, with all strains being inhibited by 0.50 microgram/ml. The MICs of the other four drugs ranged from 0.12 to 4 micrograms/ml. Ampicillin, penicillin G, ticarcillin, aztreonam, and temocillin were moderately active (MIC90, 1 to 8 micrograms/ml; MIC range, less than or equal to 0.03 to greater than 128 micrograms/ml). All strains were uniformly resistant to the aminoglycosides, polymyxin B, vancomycin, trimethoprim, and amphotericin B. Three strains produced beta-lactamase. No significant difference was found between the susceptibility of strains isolated from various sources or patients.

Role of proteinase in the formation of inhibitory levels of hematin by group A streptococcus cultures on blood-containing media.

Group A streptococci were tested for proteinase production and for the possible relationship of this production to the generation of bacteriocinlike inhibitor activity. Of 126 strains tested, 83% were positive for proteinase, and a similar distribution was found among strains isolated in association with rheumatic fever (89%) and nephritis (94%) and from uncomplicated acute infections (78%). Although application of an inhibitor production (P) typing scheme demonstrated a variety of P types, all of the proteinase-positive strains produced inhibitory activity and over 65% of these strains were P type 204. It was shown that hematin was responsible for this P type 204 activity and that it was produced only by actively proteolytic strains when grown on a hemoglobin-containing medium. Conditions optimizing proteinase production (anaerobic incubation at 37 degrees C on a test medium prepared from Columbia agar base [GIBCO Laboratories, Grand Island, N.Y.]) increased P type 204 activity. Interference with proteinase activity either by growth of the cultures at an alkaline pH or by incorporation of sub-growth inhibitory concentrations of either iodoacetic acid or lincomycin into the medium prevented production of P type 204 activity. Whether significant conversion of hemoglobin to hematin occurs in vivo and the possible implications of this conversion with regard to the pathogenesis of group A streptococcal infections remain to be determined.

Evaluation of a blood-free, charcoal-based, selective medium for the isolation of Campylobacter organisms from feces

A blood-free, charcoal-based selective medium (CSM) consisting of a Columbia agar base, activated charcoal (4 g/liter), hematin (0.032 g/liter), sodium pyruvate (0.1 g/liter), cefoperazone (32 mg/liter), vancomycin (20 mg/liter), and cycloheximide (100 mg/liter) supported the growth of Campylobacter jejuni and C. coli with colony counts equivalent to those obtained on antibiotic-free horse blood agar. CSM was compared to Skirrow medium (SKM) for the recovery of C. jejuni and C. coli from stools of patients with diarrhea, the media being incubated for 2 days under reduced oxygen tension at 43 degrees C. These campylobacters were isolated from 35 (2.9%) of 1,227 stools tested (29 on both media, 5 on CSM alone, and one on SKM alone). Whenever C. jejuni and C. coli were recovered, growth was pure on 29 CSM cultures (85%), but on only 11 SKM cultures (37%). Complete suppression of "contaminating" flora occurred in 704 CSM cultures (57%) compared with 426 SKM cultures (35%). CSM more effectively suppressed contaminating pseudomonads, gram-positive organisms, and yeasts than did SKM; both media failed to suppress members of the family Enterobacteriaceae in about a quarter of the samples. Studies on 20 representative Enterobacteriaceae contaminants showed that susceptibility to cefoperazone and growth on CSM were markedly dependent on inoculum size; 12 strains were inhibited by cefoperazone (32 mg/liter) at inoculum sizes of 5 X 10(2) and 5 X 10(4) but not 5 X 10(6) organisms, indicating that the frequency of contaminants on CSM could probably be reduced further by ensuring that stools were not inoculated too heavily on CSM. Our findings confirm that charcoal is an effective substitute for blood in media for growing campylobacters, and that CSM is a highly effective blood-free selective medium for isolating C. jejuni and C. coli from stools.

Selective Medium for Isolation of Saprophytic Coryneform Bacteria

Out of 64 test chemicals for which the toxicity was analysed on pure cultures of Gram-positive and Gram-negative bacteria, nalidixic acid, polymyxin B sulfate, colistin, methyl red, cefotaxim, amikacin, 2-chlorphenol, metronidazole and potassium tellurite were shown to perform best for the selective isolation of coryneform microorganisms; sodium azide, potassium thiocyanate and lithium chloride were not suitable. A new selective agar medium (NPC) for coryneform bacteria containing standard agar, plate count agar or columbia agar together with nalidixic acid (20 mg/l), polymyxin B sulfate (40,000 IU/l) and cycloheximide (100 mg/l) was tested with samples of milk, surface water, waste water and soil. The NPC agar was not inhibitory to micrococci, probably due to their close relatedness with coryneforms. Use of sugar-free nutrient agar (standard agar) restricted growth of Streptococcus faecalis. Pseudomonads, flavobacteria and chromobacteria, which often accounted for 1-5% of the NPC agar-resistant Gram-negative rods, could be distinguished from the Gram-positive bacteria due to differences in colony morphology, since on this medium colony shape is not masked by color reactions. The NPC medium was more effective in eliminating Gram-negative bacteria, bacilli, moulds and yeasts than beta-phenethyl alcohol media and potassium tellurite media.