Comparison of methods for the isolation of beta-hemolytic streptococci

Throat swabs from 334 patients, submitted to the clinical laboratory of the Institute of Infectious Diseases (Siena University), for isolation of beta-hemolytic streptococci, were inoculated and incubated aerobically and anaerobically. Beta-hemolytic streptococci were identified in 117 (35%) cases, including 77 group A streptococci. Group A streptococci were recovered significantly more often with anaerobic incubation than with aerobic incubation (77 versus 53 cases: P less than 0.01). Non-group A beta-hemolytic streptococci also were recovered significantly more often with anaerobic incubation (40 versus 22; P less than 0.01). We conclude that anaerobic incubation for throat cultures is superior to incubation in normal air for detection of beta-hemolytic streptococci.     

Comparative evaluation of medium and atmosphere of incubation for isolation of Streptococcus pyogenes

To evaluate the effects of medium and atmosphere of incubation for the isolation of group A (GA) streptococci from throat cultures, we compared 1098 throat swabs plated on each of three blood agar plates (BAP). Two plates contained trimethoprim-sulfamethoxazole (SXT) and one had no antibiotics. The antibiotic-free medium and one SXT plate were incubated anaerobically (AnO2) at 35 degrees C, whereas the other SXT plate was incubated in air (O2) at 35 degrees C. The BAP-AnO2 identified 85% of 201 GA compared with 96% for SXT-AnO2 and 92% for SXT-O2. GA were confirmed from only 29% of the beta-hemolytic colonies on BAP-AnO2 compared with 53% from SXT-AnO2 and 44% from SXT-O2. SXT, however, appeared to delay recognition of GA; since 94% of positives were found on day 1 with BAP-AnO2 versus 80% and 62% with SXT-AnO2 and SXT-O2, respectively. We conclude that BAP-SXT (AnO2 or O2) is more sensitive and more specific than BAP-AnO2, but that a second day of incubation is required for optimal recovery of GA on the SXT selective media studied.     

Clinical evaluation of the improved streptex method for grouping streptococci

The improved Streptex method for serogrouping streptococci incorporates a new extraction enzyme and a simplified procedure requiring no centrifugation. A total of 114 clinical isolates of beta-hemolytic streptococci were serogrouped from primary plates, isolation plates, and Todd-Hewitt broth cultures using this system. Results were compared to those of the heat extraction Lancefield precipitin method. An additional 33 stock culture isolates of related streptococcal species and 5 strains of Listeria were serogrouped to assess the specificity of the test. Agreement between the two methods was 82.5% with primary plates and 96.5% with both isolation plates and broth cultures. Four isolates from three different serogroups were nongroupable by the Lancefield method, but did agglutinate in specific Streptex antisera; therefore, the enzyme extraction procedure appeared more sensitive than the heat extraction method. Streptex accurately grouped five isolates of gamma-hemolytic group B streptococci, but failed to detect antigen in 33% of the group D streptococcal extracts tested. In addition, cross-reactions were observed with strains of alpha-hemolytic streptococci. Streptex produced fewer ambiguous results and required fewer repeat tests. When used with isolation plates or broth cultures, Streptex is both sensitive and specific for the grouping of beta-hemolytic streptococci of groups A, B, C, F, and G.     

Effect of Several Components of Anaerobic Incubation on Antibiotic Susceptibility Test Results

The factors influencing the in vitro activity of antibiotics during anaerobic incubation were studied by the disc method with a facultative organism, Escherichia coli. We observed the effects of incubation aerobically, anaerobically (Torbal jars), in a CO(2) incubator, and aerobically and anaerobically with all CO(2) removed. We also monitored pH changes during incubation and observed the effect of two different initial agar pH values (7.4 and 8.3). With aminoglycosides, zones were larger at pH 8.3 and, in each agar pH group, zones were decreased by incubation with increased CO(2) (anaerobically and CO(2) incubator). A fall in agar pH took place during the first 5 to 7 hr of incubation when increased CO(2) was present. Decreased aminoglycoside zones in the presence of increased CO(2) were due to fall in agar pH. Erythromycin showed the same zone size changes as the aminoglycosides. Chloramphenicol zones were somewhat smaller at the lower medium pH. Zones around tetracycline discs were largest after incubation anaerobically. Further aerobic (or CO(2)) incubation of plates after anaerobic incubation resulted in large "zones of relative inhibition" around the aminoglycoside discs. This suggests that these antibiotics had become more active after exposure to aerobic conditions. Our studies indicate that antibiotic susceptibility test results can be significantly altered by several components of anaerobic incubation including changes in agar pH and CO(2) concentration as well as anaerobiosis per se.     

Effect of anaerobic growth on quinolone lethality with Escherichia coli

Quinolone activity against Escherichia coli was examined during aerobic growth, aerobic treatment with chloramphenicol, and anaerobic growth. Nalidixic acid, norfloxacin, ciprofloxacin, and PD161144 were lethal for cultures growing aerobically, and the bacteriostatic activity of each quinolone was unaffected by anaerobic growth. However, lethal activity was distinct for each quinolone with cells treated aerobically with chloramphenicol or grown anaerobically. Nalidixic acid failed to kill cells under both conditions; norfloxacin killed cells when they were grown anaerobically but not when they were treated with chloramphenicol; ciprofloxacin killed cells under both conditions but required higher concentrations than those required with cells grown aerobically; and PD161144, a C-8-methoxy fluoroquinolone, was equally lethal under all conditions. Following pretreatment with nalidixic acid, a shift to anaerobic conditions or the addition of chloramphenicol rapidly blocked further cell death. Formation of quinolone-gyrase-DNA complexes, observed as a sodium dodecyl sulfate (SDS)-dependent drop in cell lysate viscosity, occurred during aerobic and anaerobic growth and in the presence and in the absence of chloramphenicol. However, lethal chromosome fragmentation, detected as a drop in viscosity in the absence of SDS, occurred with nalidixic acid treatment only under aerobic conditions in the absence of chloramphenicol. With PD161144, chromosome fragmentation was detected when the cells were grown aerobically and anaerobically and in the presence and in the absence of chloramphenicol. Thus, all quinolones tested appear to form reversible bacteriostatic complexes containing broken DNA during aerobic growth, during anaerobic growth, and when protein synthesis is blocked; however, the ability to fragment chromosomes and to rapidly kill cells under these conditions depends on quinolone structure.     

Recovery of group A and nongroup A beta-hemolytic streptococci from throat swab specimens.

The recovery of beta-hemolytic streptococci from throat swab specimens, from 1973 through 1975, was analyzed. Group A and nongroup A beta-hemolytic streptococci were recovered from 16.5% and 16.1%, respectively, of the specimens received. Although the recovery of group A streptococci fluctuated during the year, with the greatest incidence during the winter and spring months, the recovery of nongroup A streptococci was constant throughout the year. The proportion of group A to nongroup A streptococci was significantly greater in specimens collected during the winter and spring months than in those collected during the summer and fall months, and in specimens from pediatric patients than in those from adults.     

Detection of group A streptococci by aerobic culture and a new simplified immunoassay in three pediatric practices and a hospital laboratory

Duplicate throat swabs for detection of group A streptococci were collected in three pediatric offices from 1,035 patients with symptoms of pharyngitis. In the collecting office and in the hospital laboratory, one swab from each patient was first inoculated to sheep blood agar (incubated at 35 degrees C aerobically for 2 days) and then tested for group A streptococcal antigen by using the SMART enzyme immunoassay technique (New Horizons Diagnostics Corp) incubated for up to 24 hours. Group A streptococci were recovered in culture (from one or both swabs) and serologically identified from 444 (42.9%) of the patients. Pediatric offices numbers 1, 2, and 3 detected 84.4%, 84.6%, and 82.2%, respectively, of their patients who had positive cultures (in the office and/or laboratory) by using their own culture system and 82.6%, 71.1%, and 84.9%, respectively, of these same patients by using the SMART technique. In the laboratory, SMART test sensitivity and specificity were 71.4% and 98.7%, respectively, after 5 minutes of test incubation. However, SMART test sensitivity improved to 86.5% after overnight incubation of the immunoassay and to 91.3% if the data from one defective lot of seven SMART production lots studied were excluded. SMART test results which are negative after 5 minutes of incubation should therefore be confirmed both by reincubation of the antigen test up to 24 hours and by culture.     

Diagnostic value of a single Streptozyme test for screening streptococcal sore throat infection

A Streptozyme test for screening individuals whose throat was affected or colonized with beta-hemolytic streptococci was evaluated. A total of 90 children and 32 adults suffering from sore throat, and 85 children and 30 adults healthy control were compared. A significant Streptozyme titer of 800 (P < 0·03) was found among the majority of individuals with positive streptococcal throat cultures, including eight with non-group A streptococci. In contrast, most individuals without isolation of streptococci showed a Streptozyme titer ≤ 400. The results suggest that accurate differentiation of patients with streptococcal sore throat from those who are carriers cannot be made on the basis of a single Streptozyme test.     

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

The 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 < 0.005). The PVP of the former lot was 76.3%, whereas that of the latter was 95.6% (P < 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.     

Increased production of beta-lactamase under anaerobic conditions in some strains of Escherichia coli.

A simple biological assay to detect beta-lactamase activity exhibited by selected cultures of Escherichia coli was used to test enzyme production in cells incubated aerobically and anerobically. Anaerobic incubation resulted in increased size of zones of drug inactivation by some beta-lactamase-producing strains. The beta-lactamase activity of cell lysates was determined iodometrically for aerobically and anaerobically grown cells. The specific beta-lactamase activity for anaerobically grown cells was three to five times greater than for aerobically grown cells. Beta-lactamase production was determined to be constitutive in all strains and to be plasmid mediated, as demonstrated by transfer to E. coli K-12 by conjugation.     

Beta-hemolytic streptococcal pharyngitis: uncommon in infectious mononucleosis

Infectious mononucleosis (IM) and beta-hemolytic streptococcal pharyngitis may present similar clinical pictures and may occur concurrently; however, recent studies have differed sharply with older ones that cited high rates of concurrent disease. Our study of 100 consecutive new patients meeting strict criteria for a diagnosis of IM found only 4% with positive throat cultures for group A beta-hemolytic streptococci at the time of diagnosis. Use of antibiotics for presumed streptococcal pharyngitis in patients with IM is unjustified. Only patients with positive cultures should be treated.     

Group F streptococci in the pharynx: pathogens or innocent bystanders?

beta-Hemolytic streptococci (BHS) of Lancefield group F were isolated in moderate to heavy growth from throat cultures taken from 46 children and adolescents with symptomatic pharyngitis. In most instances, oxygen deprivation by means of an anaerobe jar was required for these beta-hemolytic organisms to grow. In relation to a comparison group of children with throat cultures positive for group A BHS, children with group F isolates were more likely to be adolescents and less likely to have fever and cervical adenopathy. It appears that group F BHS are not a major cause of nonepidemic pharyngitis in the pediatric age group. More precise determination of how commonly these organisms cause pharyngitis will require either comparison of isolation rates of group F BHS from the throats of both sick and well children, or further elucidation of the serologic response to these organisms so as to distinguish invasive infection from asymptomatic carriage.     

Lack of correlation between streptococcal Fc receptors and symptomatic pharyngitis

To determine whether bacterial Fc(gamma) surface receptors are associated with symptomatic streptococcal pharyngitis, throat cultures were obtained from 264 children (grouped according to symptoms of pharyngeal infection) and from 328 who were asymptomatic. Group A beta-hemolytic streptococci which were recovered were tested for the presence of Fc(gamma) surface receptors. There were no statistically significant differences in the incidence of Fc(gamma) surface receptors on strains from the various patient groups.     

Efficacy of penicillin versus cefdinir in eradication of group A streptococci and tonsillar flora

Core tonsillar cultures were obtained from 40 children with recurrent tonsillitis treated with either penicillin or cefdinir. Group A beta-hemolytic streptococci were isolated from 11 penicillin- and 3 cefdinir-treated (P < 0.001) patients. beta-Lactamase producers were recovered from 17 penicillin- and 3 cefdinir-treated (P < 0.01) patients. Inhibiting alpha-hemolytic streptococci were isolated less often from penicillin-treated patients than from cefdinir-treated patients.     

A CONTRIBUTION TO THE BACTERIOLOGY OF ACUTE ANTERIOR POLIOMYELITIS

Four different varieties of easily cultivated microorganisms have been cultured from the cerebrospinal fluid and tissues of cases of acute anterior poliomyelitis; namely, a streptococcus, a diplococcus, diphtheroids, and Gram-negative bacilli. It is not contended that they were all inherent in the tissues; a part were doubtless extraneous. The streptococci and diplococd may be considered as the most significant of the bacteria cultivated and are distinguishable from each other by biological tests. The streptococci grew both aerobically and anaerobically; under anaerobic conditions growth was slow, the cocci became small and round, and were more easily decolorized with alcohol in the Gram stain. They were not found in the anaerobic cultures of 106 cerebrospinal fluids; they were found in one of twenty anaerobic blood cultures and frequently in the cerebrum, cerebellum, pons and medulla, cord, tonsils, lungs, liver, kidneys, spleen, pancreas, thymus gland, suprarenal glands, and mesenteric glands of fatal cases. The diplococci are Gram-positive and, transplanted to solid media, yield luxuriant growths and a staphylococcus grouping. They grew aerobically and anaerobically, but more slowly under the latter condition, and the cocci became smaller and more rounded. Diplococci were found in the anaerobic cultures of 48 of 106 cerebrospinal fluids; also in the cerebrum, cerebellum) pons and medulla, cord, tonsils, lungs, liver, kidneys, spleen, pancreas, and mesenteric glands of fatal cases. The filtrates of emulsions of tissues containing streptococci and diplococci passed through fine Kitasato and Pasteur-Chamberland filters were sterile unless large amounts of filtrates were collected. The amount of filtrate collected and cultured is therefore of considerable importance in filtration experiments. The small forms of streptococci and diplococci in old anaerobic cultures are filterable with these filters, while young aerobic cultures containing large forms are not, unless large amounts of culture are filtered. Intracranial, intravenous, and intraperitoneal injection of these easily cultivated streptococci, diplococci, diphtheroids, and Gramnegative bacilli failed to produce paralysis in rabbits or monkeys. With two exceptions all the cultures were transplants from the original anaerobic ascites-broth-kidney cultures of cerebrospinal fluid and various tissues. Arthritis and meningitis were produced by the streptococci, but there were neither clinical iior histological evidences of true poliomyelitis. Occasional bacteriological studies since 1898 have shown that easily cultivated micrococci and bacilli may be present in the cerebrospinal fluid and tissues of the central nervous system of persons suffering with acute anterior poliomyelitis. The majority of bacteriologists have found the cerebrospinal fluid, blood, and nervous organs sterile. Opinions have varied in regard to the significance of the organisms and the micrococci in particular, but the consensus of opinion has been to the effect that they are secondary invaders and unable of themselves to produce poliomyelitis in the lower animals. After allowing for contaminations due to technical errors in securing specimens, the total number of observations indicates that easily cultivated micrococci occur sometimes in the brain and cord of persons suffering from epidemic poliomyelitis. Our studies have shown that they may be found not only in these locations, but also in the spleen, kidneys, suprarenal glands, and other organs. It is not known that they exert an influence in this disease, although they may possibly give rise to the production of antibodies) assuming their entrance not to be wholly agonal,as the cultures of streptococci are frequently of sufficient virulence to produce meningitis in rabbits and monkeys. Our experiments are in accord with those of other investigators who found that these microorganisms do not produce poliomyelitis in the lower animals, and are therefore in sharp contrast with the recent reports which would attribute an etiologic relationship of streptococi and allied organisms to that disease. At present this wide divergence of result cannot be accounted for, but it does not seem that it is possible for it to reside in any condition of the cultures employed by us as they were obtained from undoubted cases of epidemic poliomyelitis and inoculated in early generations. As regards these easily cultivatable microorganisms, we agree at present with those who regard them as secondary and probably terminal invaders rather than the actual etiologic agent of the disease.     

Throat culture in impending peritonsillar abscess

Throat cultures done immediately before the development of a peritonsillar abscess were positive for beta-hemolytic streptococci in only one of 12 cases. To prevent suppurative complications such as peritonsillar abscess, treatment with antibiotics may be indicated in selected severe cases of tonsillar infections despite negative throat cultures.     

Clindamycin in Treatment of Aspiration Pneumonia in Children

Twenty-eight patients with anaerobic pleuropulmonary infections were treated with clindamycin alone or clindamycin with gentamicin. Sixteen of the patients presented with pneumonitis, nine with necrotizing pneumonia, and three with lung abscesses. The average length of treatment was 13.8 days, and the duration of temperature after initiation of therapy was 3.1 days. The predominant isolates were anaerobic gram-positive cocci (23 isolates), Bacteroides melaninogenicus (14), Bacteroides fragilis (9), and Fusobacterium nucleatum (11). The most frequent aerobic isolates were alpha-hemolytic streptococci (12), Diplococcus pneumoniae (12), Pseudomonas aeruginosa (9), Klebsiella pneumoniae (7), group A beta-hemolytic streptococci (5), Staphylococcus aureus (9), and Escherichia coli (6). All patients responded to the therapy and were cured of the infection. There were no side effects observed from the administration of clindamycin. None of the patients developed any blood dyscrasia, liver damage, diarrhea, or colitis. Clindamycin appears to be effective in the treatment of mixed aerobic and anaerobic pleuropulmonary infections in children, alone or with an aminoglycoside when indicated.     

Bacillus Calmette-Guérin-activated murine macrophages kill syngeneic melanoma cells under strict anaerobic conditions

We have studied the spontaneous killing of B5(59) melanoma cells by Bacillus Calmette-Guerin (BCG)-elicited macrophages under strictly anaerobic conditions to investigate the role of oxygen in macrophage- mediated cytotoxicity. The number of melanoma cells capable of forming colonies after aerobic or anaerobic incubation with BCG-macrophages was used as the index of cytotoxicity. The BCG-macrophages killed melanoma cells regardless of the amount of oxygen present. The killing observed was proportional to the ratio of effector cells added; a ratio of 25:1 effector to target cells was required to achieve nearly 90% cytotoxicity both aerobically and anaerobically. This cytotoxicity was not dependent on a diffusible macrophage product nor on alteration of the medium by macrophages, since tumor cells incubated in the same culture medium, but not in contact with a mixed population of tumor cells and macrophages, were not killed. These results also indicated that macrophage-mediated cytotoxicity was dependent on macrophage-tumor cell contact. The mechanism responsible for the oxygen-independent cytotoxicity is unknown at present.     

Use of Available Dosage Forms of Cephalexin in Clinical Comparison with Phenoxymethyl Penicillin and Benzathine Penicillin in the Treatment of Streptococcal Pharyngitis in Children

The effectiveness of cephalexin, an oral cephalosporin using a dosage equivalent to available capsular dosage forms, was studied in relation to the effectiveness of phenoxymethyl penicillin and benzathine penicillin in the treatment of 128 patients with beta-hemolytic streptococcal pharyngitis, all but six of whom had group A streptococci isolated from throat cultures. Approximately one-half, 66 patients, received cephalexin for 10 days; 34 patients received phenoxymethyl penicillin for 10 days; and 28 patients had a single injection of benzathine penicillin. There were four treatment failures determined bacteriologically post-therapy, two in the cephalexin treatment group and one each in the oral penicillin and intramuscular penicillin groups. Similar cure rates of 96.7, 97.1, and 96.4% were computed for the respective treatment regimens. Whereas intramuscular benzathine penicillin remains the regimen of choice in most instances, cephalexin appeared to be as effective as oral penicillin in the elimination of group A streptococci from the pharynx when oral treatment was desired for streptococcal pharyngitis.     

Reassessment of the incubation time in a controlled clinical comparison of the BacT/Alert aerobic FAN bottle and standard anaerobic bottle used aerobically for the detection of bloodstream infections

This study assessed the minimum incubation time required to detect bloodstream infections during a controlled clinical comparison of the performance characteristics of the BacT/Alert aerobic FAN bottle and the standard anaerobic bottle used aerobically except on a selective basis. Blood was collected from adults with suspected bloodstream infections and inoculated into each bottle, which was monitored in the BacT/Alert Microbial Detection System. The anaerobic bottle was vented before incubation except when cultures were obtained from patients on the colorectal and gynecologic surgical and emergency services. Statistical analysis was limited to those culture sets in which each bottle was inoculated with ≥8 mL of blood and bacterial growth was considered to be clinically significant. A total of 682 positive cultures from 243 patients satisfied the inclusion criteria. Significantly more isolates of Staphylococcus aureus (p <0.001), S. epidermidis (p <0.001), other coagulase-negative staphylococci (p <0.001), Enterococcus spp. (p = 0.04), Escherichia coli (p = 0.03), all Enterobacteriaceae (p <0.001), Pseudomonas aeruginosa (p = 0.001), and Candida spp. (p <0.001) were detected by the aerobic FAN bottle. Significantly more septic episodes due to S. aureus, S. epidermidis, other coagulase-negative staphylococci, Enterobacteriaceae, P. aeruginosa, and Candida spp. were detected by the aerobic FAN bottle. Significantly more bacterial isolates were detected by the aerobic FAN whether or not antibiotics were being administered at the time of blood culture, whereas there were significantly fewer positive cultures in the vented standard anaerobic bottle when patients were receiving antimicrobial therapy than when they were not. All but 5% of positive cultures were detected within three days. Only six of the cultures requiring four or five days of incubation represented true misses, and only one of these six resulted in a change in therapy which, however, did not affect the patent’s outcome.