Lysed Horse Blood Research Articles

Development of a blood-free Campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance.

The capacity of six basal media to support the growth of thermophilic campylobacters was tested. The most successful was Nutrient Broth No. 2 (Oxoid) solidified with New Zealand agar but it gave at best only a 9% recovery rate. Various blood products, iron compounds, detoxifying agents, reducing agents, growth stimulants and an antimetabolite were added to the selected basal medium and counts of inoculated organisms were compared with counts on basal medium containing 5% lysed horse blood. Of 22 supplements tried only blood, Fildes' peptic digest of blood, heamatin, iron salts, charcoal, sodium metabisulphite and sodium pyruvate greatly improved the basal medium. The ability of these supplements used singly and in combinations to facilitate aerotolerance of campylobacters was investigated. Two aspects of aerotolerance were tested; (a) the ability of the supplements to sustain the viability of campylobacters seeded onto culture plates left on the bench for up to 6 h before microaerobic incubation; and (b) the ability of the supplements to facilitate the growth of campylobacters at increasing oxygen tension (6, 10 and 17% oxygen). A combination of 0.4% charcoal, 0.025% ferrous sulphate and 0.025% sodium pyruvate was found to be as effective as blood in both tests.

Trimethoprim activity in media selective for Campylobacter jejuni.

The activity of trimethoprim (TMP) in two selective media used for isolation of Campylobacter jejuni was evaluated. The two selective media, Campy-BAP and Skirrow medium, contain TMP in addition to other antimicrobial agents. The minimal inhibitory concentrations of TMP in blood agar base (basal agar for Skirrow medium) or brucella agar (basal agar for Campy-BAP) for three sensitive control organisms were compared with those in Mueller-Hinton agar, which contains low levels of thymidine. TMP was inactive in both blood agar base and brucella agar, even when lysed horse blood or thymidine phosphorylase was added. TMP had activity when used in combination with the other antimicrobial agents normally included in Skirrow medium and Campy-BAP, probably indicating synergism between TMP and one or more of the other antimicrobial agents. Sheep blood could be substituted for lysed horse blood in Skirrow medium without compromising the activity of TMP.

Recovery of Campylobacter jejuni and Campylobacter coli from inoculated foods by selective enrichment

A direct enrichment procedure was developed to selectively recover small numbers of Campylobacter jejuni, C. coli, and nalidixic acid-resistant thermophilic Campylobacter from foods. The procedure includes an enrichment medium composed of brucella broth, 7% lysed horse blood, 0.3% sodium succinate, 0.01% cysteine hydrochloride, vancomycin (15 micrograms/ml), trimethoprim (5 micrograms/ml), polymyxin B (20 IU/ml), and cycloheximide (50 micrograms/ml) that is inoculated with 10 or 25 g of food and incubated with agitation under microaerophilic conditions at 42 degrees C for 16 to 18 h. After incubation, the medium is plated directly onto Campy-BAP agar plates (M. J. Blaser et al., Ann. Intern. Med. 91:179-185, 1979), and resulting colonies that resemble Campylobacter are identified by conventional tests. The foods evaluated included raw milk, hamburger, and chicken skin which had aerobic plate counts of 10(5) to 10(9) bacteria/g. The procedure was effective in recovering as few as 0.1 cell of Campylobacter per g of food. Of the 50 isolates of Campylobacter evaluated, all were recovered from raw milk and hamburger at a level of 1 to 4 cells/g, and 41 and 40 isolaes were recovered from the hamburger and milk, respectively, at 0.1 to 0.4 cell/g. The enrichment was least effective for recovering campylobacters from chicken skin, as 7 and 26 of 50 isolates were not recovered at 1 to 4 and 0.1 to 0.4 cell/g, respectively. This new procedure is more rapid, direct, and effective than other enrichment or direct plating procedures for recovering small numbers of campylobacters from foods.

The in vitro resistance of canine staphylococci to trimethoprim and sulphamethoxazole alone and in combination.

The in vitro resistance of 40 strains of Staphylococcus aureus isolated from dogs to trimethoprim, sulphamethoxazole and co-trimoxazole (SXT) was assessed using the disc diffusion technique on Diagnostic Sensitivity Test Agar (DSTA), DSTA with 5% lysed horse blood (DSTA + B), IsoSensitest Agar (ISTA) and Wellcotest Sensitivity Test Agar (WSTA). When sensitivity was based on a zone width of greater than or equal to 4 mm all 40 strains were sensitive to SXT on DSTA + B and ISTA, 39 were sensitive on WSTA while only 31 (77.5%) were sensitive on DSTA. A comparison of the results obtained for each strain for trimethoprim and sulphamethoxazole with those obtained with the Oxford strain of S. aureus and using a scheme for interpretation recommended by Garrod & O'Grady (1971) indicated that the proportion of strains classed as sensitive for therapeutic purposes was 5%, 55%, 47.5% and 15% respectively on DSTA, DSTA + B, ISTA and WSTA.

Counts on viableBacteroides fragilis:a modification of the microdroplet technique

A modification of Sharpe's microdroplet counting technique is presented which permits viable counting of Bacteroides fragilis. Lysed horse blood was added to 0.1 soy digest agar droplets and 2% was found to be the optimal concentration which combined adequate intradroplet colony size with acceptable visibility on the counting screen of the Colworth Droplette machine.

Susceptibility tests for sulfamethoxazole-trimethoprim by a broth microdilution procedure

Most media used for susceptibility testing contain sulfonamide inhibitors that make them unacceptable for testing sulfonamides. The major substance that inhibits sulfonamides has been identified as thymidine, and recent efforts to remove it from Mueller-Hinton medium have made it possible to perform susceptibility tests with sulfamethoxazole-trimethoprim by broth microdilution. Some lots of Mueller-Hinton broth are thymidine free, but some contain small amounts of thymidine and require the addition of thymidine phosphorylase or lysed horse blood which contains thymidine phosphorylase. Some lots may contain too much thymidine so that its activity cannot be reversed by adding the recommended amont of thymidine phosphorylase. Therefore the suitability of a medium must be determined by testing with control strains. Some organism, for example enterococci, cannot be tested in thymidine phosphorylase-treated media because thymine works in the same way thymidine does to inhibit the action of sulfamethoxazole-trimethoprim.

Synergy between sulphonamide and trimethoprim in the presence of pus.

Synergy between sulphadiazine and trimethoprim against Escherichia coli has been demonstrated in culture media not containing lysed horse blood despite the presence of pus or a pus extract which inhibited the action of each drug separately. Synergy may therefore be important where pus is present in vivo.

腸, パラチフスに対するSulfamethoxazole-Trimethoprim合剤の臨床的および細菌学的検討

Twenty three cases of patients of typhoid and paratyphoid were treated with sulfamethoxazoletrimethoprim (SMX-TMP) combination product (SMX 400mg and TMP 80mg per tablet) to assess its clinical and bacteriological efficacy. The drug sensitivity of isolated strains of Salmonellae was also studied.Nineteen cases of manifested patients with enteric feven and 4 carriers of Salmonella typhi were admitted to the department of communicable diseases, Kyoto City Hospital during the period from Sept. 1975 to Aug. 1976.In all patients in acute stage (2 children, 17 adults), SMX-TMP was administered in dosis of 10mg trimethoprim per kilogram body weight daily in two divided doses with maximum of 640mg daily before complete defervescence, whereupon the dosis was reduced to maintenance dosis in a half cases. SMX-TMP therapy was terminated about 10 days after defervescence.Among 19 cases of typhoid fever and paratyphoid fever at acute stage, 18 responsed to the therapy, the mean period to defervescence being 4.1 days.Relapse of typhoid fever occurred in a patient 18 days after cessation of the drug therapy. The relapse was successfully controlled with chloramphenicol. In 4 cases, trasient redischarge of the organism in stool occurred after the therapy.The therapy proved effective in removing the organism in 3 of 4 carriers of Salmonella typhi. Two cases were cured after the operation upon cholelithiasis after the clinical course of unsuccessful elimination of the causatives.Strains isolated from patients, consisting of 20 strains of Salmonella typhi, 3 strains of Salmonella paratyphi, were tested in vitro to assess their susceptibility to SMX, TMP and SMX-TMP combination (mixed at a ratio 20: 1), respectivelyThe MIC of SMX, TMP and SMX-TMP combination in Mueller-Hinton agar with 7.5% lysed horse blood for Salmonella typhi ranged from 12.5μg/ml to 6.25μg/ml, 0.1-0.05μg/ml and 0.78-0.2μg/ml, respectively. Three strains of Salmonella paratyphi were inhibited by 25μg/ml of SMX, by 0.39-0.1μg/ml of TMP and by 1.56-0.78μg/ml of SMX-TM, respectively.No adverse effects such as gastrointestinal disturbance, skin eruption and hematological disorder were noted throughout the series.

Influences of media and inocula on the in vitro susceptibility of Haemophilus influenzae to co-trimoxazole, ampicillin, penicillin, and chloramphenicol.

The effects of inocula and media on the activities of ampicillin, penicillin, chloramphenicol and co-trimoxazole against Haemophilus influenzae were examined in vitro. Two inocula and four media were tested by the disk diffusion, broth dilution, and agar dilution methods. Chloramphenicol activity versus H. influenzae was least affected by changes in inocula and media, whereas co-trimoxazole was most susceptible to these effects. Filde's and Levinthal's agar dilution tests were most satisfactory for ampicillin. Penicillin was less active on Levinthal's than on Filde's agar. Both ampicillin and penicillin were less active when tested against the higher inoculum. Co-trimoxazole was most active (<1% H. influenzae was resistant) when tested at an inoculum of 10(6) colony-forming units/ml on diagnostic susceptibility test agar with 5% lysed horse blood added. The majority of H. influenzae appeared resistant to co-trimoxazole with increases in the test inocula and/or when tested on brain heart infusion with Filde's, Levinthal's or "low-thymidine" Mueller-Hinton medium.

Chronic herpes simplex ocular infection in rabbits.

Rich media support the growth of bacteria in the presence of concentrations of sulfonamides and diaminopyrimidines that are highly inhibitory when the organisms are grown on minimal media. Many such rich media can be made more suitable for susceptibility testing by the incorporation of lysed horse blood. Harper and Cawston characterized the active substance, Harper-Cawston factor (HCF), and later studies indicated it to be a protein. It has now been identified as thymidine phosphorylase. The identification follows from the identical purification pattern of HCF and thymidine phosphorylase activities from horse blood to a high degree of purity. Blood of goats, sheep, oxen, geese, chickens, cows, dogs, rats, and humans had neither biological activity. The identification of HCF as thymidine phosphorylase is consistent with the earlier findings of Koch and Burchall (1971) that most of the interfering effects of rich media could be accounted for by their thymidine contents, and that thymidine is much more active in this respect than is thymine.

Sensitivity of Streptococcus pyogenes to sulphamethoxazole, trimethoprim, and cotrimoxazole.

When tested on Wellcotest (sensitivity test agar, Wellcome) supplemented with lysed horse blood, 59 clinical isolates of beta-haemolytic streptococci, belonging to several serological groups, are all sensitive to sulphamethoxazole, to trimethoprim, and to cotrimoxazole. The results obtained on diagnostic sensitivity test agar (Oxoid) containing lysed horse blood are comparable. When Mueller Hinton agar (Difco) is used, some of these strains are called resistant: this is especially true in the case of sulphamethoxazole.Furthermore, on Mueller Hinton agar, even supplemented with lysed blood, discrepancies between diameters of inhibition zone and MIC values exist for the three drugs tested, strains with a low MIC being considered resistant by the disc method. Such disagreements are not observed with non-streptococcal control strains.

Antagonism between penicillin and chloramphenicol in experimental pneumococcal meningitis.

Trimethoprim and sulfamethoxazole were tested alone and in combination against 227 recently isolated Shigella strains. Variations in medium constituents and inoculum size were used to determine the optimal testing conditions. The plate dilution method with addition of 5% lysed horse blood to the susceptibility test medium and an inoculum size of 10(2) organisms was found to provide satisfactory results. All 227 strains were inhibited by low concentrations of trimethoprim, and all were susceptible to the combination of 0.06 mug of trimethoprim per ml and 1.25 mug of sulfamethoxazole per ml. Sixteen percent of these strains were resistant to ampicillin, 33% to tetracycline, 15% to chloramphenicol, and 27% to cephalothin. Based on these in vitro observations, trimethoprim and sulfamethoxazole appear worth evaluating for treatment of shigellosis due to multiply antibiotic-resistant strains.

The susceptibility of Serratia marcescens to sulfamethoxazole, trimethoprim, and trimethoprim-sulfamethoxazole.

The sensitivities of strains of Serratia marcescens isolated from clinical specimens, to trimethoprim (TMP), sulfamethoxazole (SMZ), and to a combination of the two drugs (trimethoprim-sulfamethoxazole) were examined. All strains were sensitive to TMP (MICs, 0.1-1 ELg/ml) with varying degrees of sensitivity to SMZ (MICs, 1.0 tg/ml). Potentiation of antibacterial activity of the two drugs together was indicated by the larger clear zones of inhibition around the combination disk and by results of checkerboard titrations of combinations of the two drugs. Small colonies were present in many of the zones around the TMP and SMZ disks on Mueller-Hinton agar and sensitivity test agar (Wellcome), with and without 5% lysed horse blood.

Some aspects of trimethoprim-sulphamethoxazole

Results of routine sensitivity testing using discs on Oxoid DST Agar with added lysed horse blood were surveyed, and experiments simulating <i>in vivo</i> conditions in urine performed to assess bactericidal activity. A comparison of <i>in vitro</i> sensitivity studies of urinary pathogens in 1969-1970, before the advent of trimethoprim-sulphamethoxazole (T-S) therapy, with a similar period 2 yr later showed no increase in the number of resistant strains, with the possible exception of Proteus. Claims that all strains of a particular organism were initially sensitive to the combination are usually based on totally insufficient data. Organisms from wound swabs (apart from Pseudomonas), and from 'pus swabs show a similar high percentage of sensitive strains as is found in urinary organisms. Strains of staphylococci are almost universally sensitive and enterobacteriaceae isolated from sources other than urine or faeces have a similar proportion of sensitive strains as is in those from urine. More general use of T-S for soft tissue infections is justified. The development of strains resistant to T-S has been less than that often experienced with other antibacterials after a similar period of widespread clinical use. Soluble sulphonamides should not in general be used except in combination with trimethoprim, since the exhibition of sulphonamides alone readily leads to the emergence of resistant strains, and an increase in the number of such strains might lessen the effectiveness of the more important sulphonamide-trimethoprim combination. These experiments simulating <i>in vivo</i> conditions confirm that T-S is bactericidal, at any rate in urine.

Stimulation of growth on a deficient medium of a Klebsiella aerogenes species by sulphonamide, para-amino benzoic acid, nitrofurantoin, and some other substances.

A strain of Klebsiella aerogenes is described which was unable to grow from light inocula on 5% lysed horse blood diagnostic sensitivity test (DST) agar. It was able to grow on DST agar without added horse cells and MacConkey and nutrient agar. Growth on a deficient medium was promoted by sulphonamides and nitrofurantoin as well as by para-amino benzoic acid, para-amino salicylic acid, procaine hydrochloride, thymine, and uracil.