Sheep Blood Agar Plates Research Articles

Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli

The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis. In vitro , the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete. Our laboratory has been investigating the pathobiology of T. denticola, and has demonstrated the production of several hemolysins by T. denticola. In this report two hemolysin genes from T. denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T. denticola on sheep blood agar plates. Physical maps of the insert fragments were not identical. Southern blot analyses suggested some degree of homology in the nucleotide sequence. Maxicell analyses of [35S]-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide. Biochemical characterization of the T. denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA. Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids. Iron partially inhibited the hemolytic activities. Addition of 2-2′ bipyridyl moderately enhanced the activities, possibly by iron limitation. These results suggest the isolation of an identical hemolysin gene from T. denticola strains TD-4 and GM-1.

Detection of penicillin and extended-spectrum cephalosporin resistance among Streptococcus pneumoniae clinical isolates by use of the E test

Increasing penicillin resistance and the initial recognition of resistance to extended-spectrum cephalosporins among Streptococcus pneumoniae isolates have placed greater emphasis on accurate methods for susceptibility testing of clinical isolates. This study has evaluated the use of the E test (AB Biodisk NA, Piscataway, N.J.) for the detection of penicillin and cefotaxime resistance among 147 pneumococcal clinical isolates in three geographically separate laboratories. These included 42 penicillin-resistant (MIC, > or = 2 micrograms/ml) and 14 cefotaxime-resistant (defined here as an MIC of > or = 2 micrograms/ml) isolates. E test strips were applied to the surface of Mueller-Hinton sheep blood agar plates and incubated at 35 degrees C in 5% CO2 for 20 to 24 h. E test MICs were compared with MICs determined with lysed horse blood-supplemented Mueller-Hinton broth in a microdilution format as recommended by the National Committee for Clinical Laboratory Standards. Penicillin MICs agreed within one log2 dilution for 136 of 147 (92.5%) isolates, and cefotaxime MICs agreed within one log2 dilution for 142 of 147 (96.6%) isolates. No very major or major interpretive errors occurred with either penicillin or cefotaxime E test MIC results. There were 9.5 and 5.4% minor interpretive category errors with penicillin and cefotaxime E test MICs, respectively. These data indicate that the E test represents a convenient and reliable method for the detection of penicillin or cephalosporin resistance in pneumococci.

Comparison of an immunochromatographic method for rapid identification of group A streptococcal antigen with culture method

To compare the sensitivity and specificity of Concise Strep A (Hybritech, San Diego, Calif), an immunochromatographic group A streptococcal rapid antigen detection system, with a two-plate culture method for the diagnosis of streptococcal pharyngitis, and to evaluate the need for routine back-up culture when this rapid test is used. Throat cultures were obtained from 351 children with acute pharyngitis by duplicate rayon-tipped swabs held in parallel and vigorously rubbed against both tonsils and the posterior pharyngeal wall. One swab was tested for group A streptococcal antigen by a registered licensed laboratory technologist in the pediatrician's office. The other swab was streaked over each of two sheep blood agar plates, one of which was enhanced with trimethoprim in combination with sulfamethoxazole. The plain sheep blood agar plate was then incubated in a candle-extinguish jar. The enhanced agar plate was placed in a gas-pack anaerobic jar. Both plates were incubated for up to 48 hours at 35 degrees C. A six-person group pediatric practice. Three hundred fifty-one children. The Concise Strep A antigen detection test produced 129 positive results. Only six of the 129 were not confirmed by culture method. There were four false-negative rapid streptococcal antigen detection test results, all of which were found after a single overnight incubation. The sensitivity for the Concise Strep A test was 96.9% and the specificity was 97.4%. The plain 5% sheep blood agar plate (without trimethoprim and sulfamethoxazole), which was incubated in a candle-extinguish jar, identified 123 (97%) of the 127 positive throat cultures. The second 24-hour incubation and use of trimethoprim and sulfamethoxazole agar were not rewarding for this study. Concise Strep A, a polyclonal antibody test, in conjunction with a color immunochromatographic assay for soluble streptococcal carbohydrate antigen A appears to be accurate, sensitive, and specific when throat swabs are carefully obtained and when qualified, licensed laboratory technologists perform the procedure. Further studies should be done to confirm our findings, especially when nurses or office staff perform the rapid test procedure in the office setting. If our findings are confirmed, the use of back-up cultures for negative rapid test results obtained using Concise Strep A would be unnecessary.

Cloning of two distinct hemolysin genes from Porphyromonas (Bacteroides) gingivalis in Escherichia coli

Hemolysin is considered a potent virulence factor in a large number of Gram-positive and Gram-negative bacterial pathogens. The hemolysin produced by the oral pathogen Porphyromonas gingivalis functions to provide the cell with its required heme-containing molecules for growth in the periodontal pocket. Two distinct P. gingivalis genes, each of which confers a hemolytic phenotype in Escherichia coli, were isolated by screening genomic DNA libraries of P. gingivalis on sheep blood agar plates. The results obtained from physical maps and Southern blots indicated a considerable degree of divergence in the nucleotide sequences of these two genes. Maxicell analyses of the recombinant plasmids in E. coli suggested that plasmid pPGH5 encoded a polypeptide of molecular weight 48 kDa, while an 18-kDa polypeptide was obtained with pPGH1 and pPGH7. When E. coli harboring these hemolysin genes was subjected to iron starvation, the levels of hemolysin activity increased. Biochemical characterization of hemolytic activities indicated that the activity of both hemolysins was inhibited by Mg 2+ and Ca 2+; but not by EDTA. Elevated levels of hemolytic activity were obtained from the E. coli recombinant strains in the presence of glutathione, DTT and 2-mercaptoethanol. Cholesterol inhibited the activity.

Coccidioides immitis Mastitis in a Mare

Mastitis in the mare is an uncommon problem. Most instances are recognized during lactation; however, mastitis can occur at any time. Equine mastitis is almost invariably bacterial, with a variety of bacterial species involved. Infections are usually unilateral and occur via the teat canal, often as a result of trauma or insect feeding. The perception of pain on palpation and edema are additional, common findings. This report describes an unusual case of mycotic mastitis in a mare. The causative agent was Coccidioides immitis, and the mastitis was the result of a disseminated coccidioidal infection. In February 1992, a 15-year-old Thoroughbred mare was examined because of a swollen left mammary gland of 1 week duration. The owner had treated the mare with an intramammary infusion of ampicillin for 5 days with no effect. The mare was subsequently treated with an intramammary infusion of cloxacillin for 5 days, which resulted in only slight improvement. Physical examination revealed that the left mammary gland was swollen, firm, and apparently painful to palpation. There was a moderate degree of edema in tissue surrounding the gland. Fluid expressed from the gland was clear, gelatinous, and yellow and was intermixed with strands of cloudy material. The mare was 7 months pregnant and had been losing weight until it was moved to a new pasture and the plane of nutrition improved. Change in available feed had resulted in a noticeable weight gain, No other physical abnormalities were detected. Fluid was collected from the affected gland and submitted to the California Veterinary Diagnostic Laboratory for microbiologic evaluation. The fluid was inoculated onto 5% sheep blood agar and MacConkey agar plates and incubated at 37 C in 7.5% CO2. No growth was detected after 48 hours; however, after incubation for an additional 48 hours small numbers of weakly hemolytic 2-3-mm glabrous colonies were apparent. Following incubation at 25 C for an additional 48 hours, the colonies developed white aerial mycelium at the apex. A lactophenol cotton blue wet mount preparation revealed septate, hyaline hyphae with occasional branching and rare areas of arthroconidia with alternating empty cells. Macroscopic and microscopic morphology were suggestive of C. immitis. Examination of a second sample of the mastitic fluid following 10% KOH digestion revealed rare doublewalled spherules with endospores. A serum sample was submitted for coccidioidal serology. The immunodiffusion titer for immunoglobulin G, which corresponds to complement fixation antibody, was 1:64, suggesting a disseminated coccidioidal infection. Passage of the isolate in mice demonstrated conversion of arthroconidia to endosporulating spherules and confirmed that the isolate was C. immitis. The mare was maintained without additional treatment for the duration of her pregnancy and foaled without difficulty approximately 4 months after initial presentation. At this time, the left mammary gland was still firm to palpation but had decreased in size. With some difficulty, fluid similar to that collected previously could be expressed from the gland. The right mammary gland appeared unaffected and milk from the gland was of normal consistency. The mare appeared to have a moderate degree of weight loss but otherwise appeared clinically normal. The foal was removed from the mare without being allowed to suckle and a blood sample was collected from the foal for coccidioidal serology. Because of the likelihood that the infection in the mare was disseminated and the poor potential for successful treatment, the owners elected to have the mare euthanized. The foal was fostered on to another lactating mare. Necropsy of the mare revealed that the left mammary gland was shrunken and hard (Fig. 1). Yellow caseous nodules 3-8 mm in diameter were scattered throughout the gland. No lesions were noted in the right gland, which was distended with milk. There were multiple 4-15-mm nodules scattered throughout the lungs. When sectioned, the nodules had dry, caseous centers. Although not notably enlarged, the mediastinal and bronchial lymph nodes were firm, and the normal parenchyma was replaced with dry caseous material. There

Interactive Effect of Temperature, Atmosphere and Storage Time on the Probability of Colony Formation on Blood Agar by Four Listeria Species

The probability (P) of one Listeria cell and the cells needed to initiate colony formation on sheep blood agar plates as affected by atmospheric conditions (AC), storage temperature (T), time (t), and Listeria species was evaluated. The factorial design experiments included Listeria monocytogenes (2 strains), Listeria seeligeri, Listeria ivanovii, and Listeria innocua, as test organisms, storage of the plates at 4, 8, 20, and 30°C under air (A), modified atmosphere (MA) of 5% O2, + 10% CO2 + 85% N2, 100% CO2 (CO2), vacuum (V), and candle jar (CJ) for 7, 14, 21, 42, and 56 d. Statistical analysis indicated the significant effect of AC (p<0.0004), T (p<0.0001), t (p<0.0001) but not of species (p>0.71). None of the interactions with atmospheric conditions were significant (AC × species, AC × t, AC × T, all with p>0.46). Pairwise comparison of the P's for each of the AC's indicated that 100% CO2 was significantly more inhibitory to growth initiation than any other AC (p<0.004). No difference among the other AC's was shown. The effect of CO2 on delaying growth of Listeria was enhanced with decreasing storage T. Thus, under CO2, ≤6 cells of L. monocytogenes formed a colony within 7 d at 20°C and 42 d at 4°C. L. ivanovii was the most sensitive to CO2 and required 4.7 × 104 cells to form a colony after 42 d at 4°C. CO2 (100%) extended the lag phase at ≤8°C and decreased the rate of growth of the test organisms at 4°C but not at higher temperatures.

Reassessment of Microbiology Protocol for Cerebrospinal Fluid Specimens

Over a 1-year period, 874 cerebrospinal fluid (CSF) specimens from 105 patients were examined by gramstained smear, culture, and white blood cell count. With these parameters, the efficacy of currently recommended strategies for the detection of organisms in CSF specimens was evaluated. We conclude that all CSF specimens should be cultured on a sheep blood agar plate, a chocolate agar plate, and one tube of thioglycolate broth without indicator regardless of cell count. The sensitivity, specificity, and positive and negative predictive values for a gram-stained smear were 61.9%, 98.9%, 92.9%, and 91.9%, respectively; these same values for white blood cell counts greater than 10/mm3 were 72.0%, 86.4%, 62.1%, and 90.0%, respectively. Gram-positive cocci, particularly staphylococci, were recovered frequently from both truepositive cultures and contaminated cultures. Recovery of the same organism from all media inoculated helped distinguish true-positive from contaminated cultures.

Clostridium orbiscindens sp. nov., a Human Intestinal Bacterium Capable of Cleaving the Flavonoid C-Ring

Clostridium orbiscindens sp. nov. is an obligate anaerobe that is capable of cleaving the C-3-C-4 bond of the natural anticarcinogen quercetin. The metabolic products, 3,4-dihydroxyphenylacetic acid and presumably phlorglucinol, are not known to possess anticarcinogen properties. This organism was isolated from human feces. On sheep blood agar plates C. orbiscindens forms minute, irregular, convex, gray or white, shiny, smooth, nonhemolytic colonies. It is beta-hemolytic on rabbit blood agar. The motile peritrichous rods are gram variable. Subpolar spores are common. Cultures are resistant to 80 degrees C for 10 min. Capsules are absent. This asaccharolytic organism does not metabolize esculin, urea, meat, gelatin, casein, or nitrate. The G + C content is 56 to 57 mol%. DNA hybridization experiments did not reveal relatedness to phenotypically similar Clostridium strains. Strain 265 (= ATCC 49531) is the type strain.

Phospholipase C and haemolytic activities of Clostridium perfringens alpha‐toxin cloned in Escherichia coli: sequence and homology with a Baciilus cereus phospholipase C

The Clostridium perfringens alpha-toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli. The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined. The cloned cpa gene product, which is exported to the periplasm in E. coli, possesses both phospholipase C and haemolytic activities. Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep-blood agar plates. Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region. The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45,481 D. This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription-translation system (Mr 46,000), but larger than the cpa gene product detected in E. coli minicells, E. coli whole cells or in C. perfringens cells (Mr 43,000), suggesting post-translational processing. The 28 N-terminal residues of the deduced alpha-toxin sequence possess the consensus features of a signal peptide and may be removed during secretion. The deduced alpha-toxin sequence shares significant structural homology with the phosphatidylcholine-preferring phospholipase C of Bacillus cereus.

Synergistic hemolytic reactions between staphylococci and Micrococcus lylae.

The primary culture of a clinical specimen obtained from a dog with an acute squamous eczema revealed three different bacterial species which demonstrated synergistic hemolytic activities on sheep blood agar plates. The three cultures were identified as beta-hemolytic Staphylococcus intermedius, as a coagulase-negative staphylococcal species, producing a delta-like hemolysin and as non-hemolytic Micrococcus lylae. The coagulase-negative staphylococcal species as well as M. lylae produced synergistically with beta-hemolytic S. intermedius zones of complete hemolysis. The occurrence of three different synergistically active bacterial species from one clinical specimen might be of clinical significance.

Performance of the tandem ICON strep A® enzyme immunoassay system for detection of group a streptococci from oropharyngeal swabs

AbstractThe Tandem ICON Strep A® (TISA Hybri‐tech, Inc.) was compared to culture for detection of group A streptococci, using throat swabs obtained from patients with symptoms of pharyngitis. Each swab was first inoculated to a 5% sheep blood agar plate and then tested for streptococcal antigen using the TISA. Cultures were incubated anaerobically at 35°C for 36 to 48 hours unless positive following overnight incubation. Group A streptococci were recovered in culture and serologically identified from 163 (21.0%) of the 776 swabs processed. The sensitivity and specificity of the TISA were 76.7% and 96.4%, respectively, whereas the predictive values of positive (PVP) and negative (PVN) results were 85.0% and 94.0%, respectively. However, TISA specificity ranged from 93.6% in one of 3 lots tested to 99.1% in another (P &lt; 0.005). The PVP of the former lot was 76.3%, whereas that of the latter was 95.6% (P &lt; 0.005). The relative lack of sensitivity and lot‐to‐lot variation observed with the TISA in this study indicate that the product is not a suitable alternative to a competently performed throat culture.

Performance of an enzyme immunoassay test and anaerobic culture for detection of group A streptococci in a pediatric practice versus a hospital laboratory

The ability of pediatricians and hospital laboratory personnel to detect group A streptococci in patients with suspected streptococcal pharyngitis was evaluated using the TestPack Strep A and anaerobic culture. Duplicate throat specimens (for similar processing by both the pediatricians and laboratory technologists) were simultaneously collected on rayon-tipped swabs from patients with symptoms of pharyngitis. Each swab was first inoculated to a 5% sheep blood agar plate, then tested for group A streptococcus antigen using the TestPack Strep A according to the manufacturer's instructions. Cultures were incubated anaerobically at 35 degrees C for 2 nights unless positive after 1 night. Group A streptococci were identified using specific antisera. Pediatric office or laboratory cultures from 112 (31.3%) of the 358 patients contained group A streptococci. Of the patients with positive cultures, 96 (85.7%) and 107 (95.5%) were detected by the pediatricians and laboratory, respectively. Respective findings with the TestPack Strep A by the pediatricians and laboratory were sensitivity 68.8% and 74.8%, specificity 94.3% and 95.6%, predictive value of a positive result 81.5% and 87.9%, and predictive value of a negative result 89.2% and 89.9%. Anaerobic culture was significantly more sensitive than the TestPack Strep A for detection of group A streptococci by both the pediatricians (P less than 0.005) and laboratory personnel (P less than 0.05).

Detection of Group A Streptococcus

Eight hundred thirty-seven consecutive throat cultures from pediatric patients were cultured comparatively on conventional sheep blood agar and selective agar containing 1.25 micrograms/ml trimethoprim and 23.75 micrograms/ml sulfamethoxazole. In two sequential studies the sheep blood agar-sulfamethoxazole-trimethoprim plates were significantly (P less than 0.01) more sensitive for Group A Streptococcus (Strep-A) detection than conventional sheep blood agar plates or Todd-Hewitt broth using the fluorescent antibody-antigen detection technique. The selective plates inhibited normal upper respiratory flora but not Strep-A. An incubation time of 18 hours was significantly better than one of 4 hours for the detection of Strep-A antigen in cultures incubated in Todd-Hewitt broth alone or Todd-Hewitt-sulfamethoxazole-trimethoprim broth, resulting in a 24% increase in positives. These studies demonstrate that Strep-A detection in throat swab specimens, by either culture or antigen detection, will be influenced by the use of selective antibiotics and incubation time.

Multiply resistant Streptococcus mitis isolated from conjunctival exudate of newborns.

Streptococcus mitis resistant to most of the commonly used antimicrobial agents but sensitive to cefotaxime, clindamycin, vancomycin and rifampicin was isolated from conjunctival exudate of newborns. The isolate had group C streptococcal antigen, was beta-haemolytic on horse but alpha-haemolytic on sheep blood agar plates and might therefore be misidentified as beta-haemolytic group C streptococci.

Detection of Group A Streptococci From Throat Cultures: Comparison of Enzyme Fluorescence, Latex Agglutination, and Routine Culture

Two rapid commercial methods were compared with conventional throat cultures for their effectiveness in the detection of group A streptococci. Three throat swabs were obtained simultaneously from 213 pediatric patients and subsequently tested by culture on a 5% sheep blood agar plate, Culturette Brand 10-Minute Group A Strep ID (Marion Scientific), and StrepAFIuor (BioSpec). A total of 110 specimens contained group A streptococci confirmed by conventional grouping methods. Culturette detected all 110 specimens containing group A streptococci, but StrepAFIuor detected only 22. False-positive reactions were obtained with two specimens tested by Culturette and with nine specimens tested by StrepAFIuor. The Culturette system appears to offer a rapid and accurate alternative to culture for the detection of group A streptococcal antigen. The current StrepAFIuor system does not have the sensitivity necessary to be recommended as an acceptable alternative to culture for the detection of group A streptococci

Human Fc(gamma) receptors for differentiation in throat cultures of group C "Streptococcus equisimilis" and group C "Streptococcus milleri".

The biochemical characteristics and the presence of human Fc(gamma) receptors of 52 throat isolates of group C beta-hemolytic streptococci were examined. Among these isolates, 38 were identified as "Streptococcus milleri" and 14 were identified as "Streptococcus equisimilis." The differentiation of group C "S. equisimilis" from "S. milleri" with identical group antigens was easy to perform by the measurement of the size of the hemolytic zone on a sheep blood agar plate in an anaerobic atmosphere and by biochemical tests (Voges-Proskauer test). A clear-cut criterion for differentiation was noted among these isolates, i.e., the presence of Fc(gamma) receptors. "S. equisimilis," which are generally associated with pharyngitis, possess human Fc(gamma) receptors, while "S. milleri", which are generally isolated from healthy persons, have no such receptors.

Eubacterium desmolans sp. nov., a Steroid Desmolase-Producing Species from Cat Fecal Flora

Eubacterium desmolans sp. nov., an obligate anaerobe with desmolytic activity, was isolated from cat feces. Desmolase, which was previously observed only in a Clostridium species isolated from human fecal flora, cleaves the carbon-carbon bond of 17-hydroxylated corticoids between C-17 and C-20, thereby converting these compounds to androstenes (C19 steroids). In primary cultures on sheep blood agar plates, E. desmolans forms nonhemolytic, circular to irregular, convex, shiny, entire, white to colorless colonies that are barely visible to the unaided eye. The cells are plump, short rods that are 0.8 to 1.1 μm wide by 1.7 to 2.3 μm long and have capsules and flagella. They occur singly or in short chains and are gram positive. Inositol is the sole carbohydrate fermented. Indole is produced. The major fermentation products in inositol-enriched media are acetic and butyric acids. The type strain is strain ATCC 43058.

Distribution of bacteria and yeasts within the 10-ml Isolator during the processing of seeded blood samples.

Forty-five organisms consisting of stock cultures and clinical isolates of bacteria and yeast were separately inoculated into outdated blood bank blood to achieve a concentration of approximately 100 CFU/ml. Blood with each organism was introduced into groups of four Isolators (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.), which were then processed according to the Isostat instructions of the manufacturer. The supernatant, sediment, and wash (material removed from the surface of the slanted stopper after sediment removal) were inoculated onto 5% sheep blood agar plates. Cultures were incubated aerobically (5 to 10% CO2) at 35 degrees C for 48 to 72 h. From the 180 Isolators, the mean recovery was 6% (range, 0 to 48%) for the supernatant, 87% (range, 47 to 98%) for the sediment, and 8% (range, 3 to 23%) for the wash. Neither variation among technologists nor intentional misalignment of additional Isolators in the centrifuge could explain all of the losses of microorganisms from the sediment. The manual nature of the Isolator procedure, which led to the loss of significant amounts of organisms from the sediment, may help to explain false-negative Isolator results obtained from blood of patients, particularly when small numbers of pathogens are present.

Clostridium scindens sp. nov., a Human Intestinal Bacterium with Desmolytic Activity on Corticoids

Clostridium scindens sp. nov., an obligate anaerobe with desmolytic activity, was isolated from human fecal flora. The desmolase, not associated previously with any specific intestinal microorganism, cleaves the carbon-carbon bond of 17-hydroxylated corticoids at C17-C20, thereby converting them to androstans (C19 steroids). In primary cultures on sheep blood agar plates, C. scindens forms minute, nonhemolytic colonies. The gram-positive rods (0.5 to 0.7 by 1 to 2.5 μm) are slightly curved. The rare oval terminal spores (0.8 to 2.0 μm in diameter) are extremely difficult to demonstrate in Gram-stained smears. More than 40% of the cells are fimbriated. Neither a capsule nor flagella are present. d-Fructose, d-glucose, lactose, d-mannose, d-ribose, and d-xylose are fermented. The major fermentation products are acetic acid, ethanol, and hydrogen. The type strain is ATCC 35704 (Bokkenheuser strain 19).

723 PERIANAL STREPTOCOCCAL CELLULITIS

Group A beta hemolytic streptococci can cause perianal cellulitis. First described by Amrert et al, (AJDC 112:546, 1966), this infection receives little attention in subsequent literature. This study describes 14 patients with perianal streptococcal cellulitis evaluated from 1975 to 1984. Characteristically, the infection caused painful defecation and constipation with an intensely erythematous, well-demarcated perianal rash and blood-streaked stools. The average age of patients was 3.9 years (range 1-10 yrs.); the male:female ratio was 3.7:1. Seven of the 14 children had rectal bleeding, 5 had anal fissures, 6 had constipation. The mean duration of symptoms before diagnosis was 6.2 months (range 1-12 months). Often, previous evaluation included multiple diagnostic tests and local therapies. Mis-diagnoses included simple anal fissure, inflammatory bowel disease, psychogenic stool holding, psoriasis and moniliasis. We established the diagnosis in all patients by culture of affected perianal skin and plating on 5% sheep-blood agar plates. Treatment with oral penicillin resulted in rapid resolution of the rash and disappearance of all complaints. Recrudescence of infection was not uncommon, necessitating a repeat course of oral antibiotics. On followup examination no patient had underlying gastrointestinal or systemic disease.