The enrichment and early detection of Streptococcus pyogenes in skin lesions, using colistin, oxolinic acid, Todd Hewitt broth and latex agglutination

An improved selective broth, colistin, oxolinic acid, Todd Hewitt broth (COTHB) for the isolation of Streptococcus pyogenes and other streptococci from swabs is described. Staphylococci, coryneforms and Gram negative organisms are inhibited in COTHB inoculated with swabs from skin lesions. Streptococcus pyogenes could be detected by a fluorescent antibody method and by the Streptex and Phadebact streptococcal grouping methods after 6 and 24 h incubation. Other streptococci of groups B, C and G, were also detected. Streptex was the most sensitive and specific method for the detection of streptococci of groups A, B, C and G in this broth. The method detected 78% of these streptococci in COTHB after 6 h incubation and 93% after 24 h incubation. Of the Strep. pyogenes isolated, 82% were detected in the broth by the Streptex method.     

The enrichment and early detection of Streptococcus pyogenes in skin lesions, using colistin, oxolinic acid, Todd Hewitt broth and latex agglutination

An improved selective broth, colistin, oxolinic acid, Todd Hewitt broth (COTHB) for the isolation of Streptococcus pyogenes and other streptococci from swabs is described. Staphylococci, coryneforms and Gram negative organisms are inhibited in COTHB inoculated with swabs from skin lesions. Streptococcus pyogenes could be detected by a fluorescent antibody method and by the Streptex and Phadebact streptococcal grouping methods after 6 and 24 h incubation. Other streptococci of groups B, C and G, were also detected. Streptex was the most sensitive and specific method for the detection of streptococci of groups A, B, C, and G in this broth. The method detected 78% of these streptococci in COTHB after 6 h incubation and 93% after 24 h incubation. Of the Strep. pyogenes isolated, 82% were detected in the broth by the Streptex method.     

Evaluation of a new presumptive medium for group D streptococci.

A new medium designated as D streptococcus-enterococcus broth was formulated and evaluated for the enrichment and isolation of strains of serological group D streptococci. This medium was made by modifying Todd-Hewitt broth. Most-probable-number multiple-tube and membrane filter techniques were employed to estimate the numbers of enterococci in known cultures, wastewater, and other samples. Preliminary most-probable-number counts with this medium were as much as 3 logs higher than those counts obtained from four other media with which it was compared. The methodology for using this medium to estimate the numbers of group D streptococci in water is discussed.     

Evaluation of a new presumptive medium for group D streptococci.

A new medium designated as D streptococcus-enterococcus broth was formulated and evaluated for the enrichment and isolation of strains of serological group D streptococci. This medium was made by modifying Todd-Hewitt broth. Most-probable-number multiple-tube and membrane filter techniques were employed to estimate the numbers of enterococci in known cultures, wastewater, and other samples. Preliminary most-probable-number counts with this medium were as much as 3 logs higher than those counts obtained from four other media with which it was compared. The methodology for using this medium to estimate the numbers of group D streptococci in water is discussed.     

Selective Broth Medium for Isolation of Group B Streptococci

A selective medium containing Todd-Hewitt broth, sheep blood, nalidixic acid, and gentamicin sulfate was found to enhance significantly the isolation of group B streptococci from vaginal cultures. Preparation of the medium, which is stable for up to 4 weeks at 4 C, is simple and inexpensive. Use of such a medium should facilitate identification of vaginal colonization with group B streptococci.     

Detection of Group A Streptococci in Throat Swabs by Direct Fluorometry

A direct fluorometric test for the rapid detection of group A streptococci from throat swabs was compared with the microscopic fluorescent-antibody test. Formalinized throat swab cultures (490) were examined by the two methods, and the results agreed on 84% of the specimens. In another comparison, 15-hr broth cultures of 103 freshly taken throat swabs were tested by both methods. Of the specimens tested, 101 (98%) were either positive or negative by both methods. In all cases, the latter results correlated with the demonstration of presence or absence of group A streptococci in the specimens by cultural isolation and precipitin grouping tests. It may be feasible to use the direct fluorometric test in a diagnostic laboratory as described or possibly to adapt it for automatic processing of throat swab cultures.     

Demonstration of a Bactericidal Substance Against β-Hemolytic Streptococci in Supernatant Fluids of Staphylococcal Cultures

Staphylococcal skin isolates belonging to phage type 71 were found to produce a bactericidal substance against some streptococci, pneumococci, and corynebacteria. Fifteen strains of group A streptococci belonging to 13 different M types, group C streptococci, and group D streptococci were uniformly inhibited on solid media and in broth by membrane-filtered supernatant fluids of the staphylococcal broth cultures. Inhibition of group G streptococci and other staphyloccoci was variable, and no inhibition of group B streptococci or of a variety of gram-negative rods was demonstrable. A quantitative variation observed to exist among susceptible organisms was a function of the inoculum size of the inhibited strains. The bactericidal substance could be detected best from 24 to 48 hr after inoculation of the staphylococci in tryptic soy broth or in a dialysate of tryptic soy broth. Little or no bactericidal activity was noted when the organisms were grown in several other liquid media. The bactericidal substance was nondialyzable and could be precipitated with ammonium sulfate. It was heat-stable and its activity was not altered within a pH range of 4.0 to 8.5. Pronase and three times crystallized trypsin totally abolished its activity. The concentrated ammonium sulfate precipitate could be fractionated on a Sephadex G-100 column into several peaks, with the bactericidal activity localized to a single peak.     

Comparison of Selective Media for Isolation of Presumptive Group D Streptococci from Human Feces

Pfizer Selective Enterococcus (PSE) agar, a medium containing bile, sodium azide, and esculin, was evaluated for its sensitivity and selectivity for detection and enumeration of presumptive group D streptococci in human feces. SF broth and SF broth plus agar (1.5%), representing selective media in common use, were studied simultaneously. Presumptive group D streptococci were recovered on PSE agar from the feces of all 25 subjects. No growth was observed in 8% of specimens in SF broth. No gram-negative organisms were recovered in any medium. PSE agar has the advantages of selecting out Streptococcus bovis, earlier appearance of distinctive reactions, and lack of requirement for special incubation temperature.     

THE RATES OF GROWTH OF SOME THERMODURIC BACTERIA IN PURE CULTURE AND THEIR EFFECTS ON TESTS FOR THE KEEPING QUALITY OF MILK

SUMMARY: The growth rates of eleven representative thermoduric bacteria, comprising 3 aerobic spore formers, 3 streptococci, 1 Corynebacterium lacticum and 4 micrococci, have been determined in glucose broth and sterile pasteurized milk at 37·5°, 26° and 15°. The spore formers and streptococci were generally not affected by the presence of inhibitory factors in pasteurized milk. When multiplication of micrococci and C. lacticum occurred in milk this was only after a lag period. One micrococcus showed an unusual series of growth phases in glucose broth at 37·5°, possibly due to the appearance of mutants or to adaptation of the organism to growth at that temperature. This was not observed in pasteurized milk. C. lacticum died off when incubated in glucose broth at 37·5°.
None of the keeping quality tests was more effective than any other in detecting these organisms in milk. The micrococci and C. lacticum had little effect on the keeping quality of pasteurized milk within the period of 'commercial life'. Some of the spore formers and streptococci showed marked differences in the end-points with the clot-on-boiling and the alcohol precipitation tests.     

Predominant Catalase-negative Soil Bacteria. I. Streptococcal Population Indigenous to Soil

Streptococci related to Streptococcus sanguis were shown to represent a major segment of the bacterial flora of certain soils. Upon initial isolation, these streptococci were quite pleomorphic and displayed morphological characteristics common to several other genera of bacteria, but after prolonged cultivation on laboratory media the isolates demonstrated a typical streptococcal morphology. It was proposed that a requirement for correction of the control mechanisms affecting growth rate and cell wall synthesis in part might explain the difficulty encountered in isolation of these organisms from soil and their initial pleomorphism and cultural instability. The possible role of these soil organisms as etiological agents of disease and the occurrence of other groups of streptococci in soil were discussed.     

Extracellular biopolymeric flocculants. Recent trends and biotechnological importance

Many microorganisms secrete extracellular biopolymeric flocculants (EBFs) in the culture broth. This work reviews the development of EBF research and applications. Aspects discussed include a comparison of the chemical and biological flocculating agents, isolation of EBF-producing microorganisms, culture conditions, mechanisms of flocculation, the chemical structure of EBFs, and the role of physicochemical factors in the flocculating activity.     

Biosurfactants from thermophilic dairy streptococci and their potential role in the fouling control of heat exchanger plates

Recent work on biosurfactant release by thermophilic dairy streptococci is reviewed. There is a suggestion thatStreptococcus thermophilus isolates may release biosurfactants that stimulate detachment of already-adhering cells and leave an anti-adhesive coating on a substratum. A previously published rapid screening method is described for the identification of biosurfactant-releasing microorganisms, and growth medium supplements to enhance biosurfactant release by thermophilic dairy streptococci are reported. New experimental work described includes the isolation and purification of biosurfactants from dairy isolates by thin layer chromatography. Many compounds isolated were extremely surface-active and reduced the water surface tension to values around 30 mJ m^–2 at a concentration of 10 mg ml^–1. Most importantly, the thin layer chromatograms of various isolates resembled each other, and an adsorbed purified compound from one isolate retarded the deposition to glass of another isolate by a factor of two. Provided our findings implicate that these biosurfactants could also be adsorbed to heat exchanger plates in pasteurizers and thereby retard colonization by thermophilic streptococci, these compounds may have major economic implications. Further work is required, however.     

A Growth Initiation Factor for Host-Independent Derivatives of Bdellovibrio bacteriovorus

Host-independent (H-I) derivatives of Bdellovibrio bacteriovorus 109 Davis could not be isolated when concentrated suspensions of host-dependent (H-D) cultures, washed free of spent medium, were plated on host-free media. However, H-I colonies did appear when spent broth was incorporated into the isolation medium, indicating the presence of a factor in the spent medium essential for the growth of H-I cells. This growth factor (GIF) was also present in cell-free extracts of Escherichia coli and a variety of other microorganisms including H-D and H-I derivatives of strain 109 Davis. GIF was heat stable, non-dialyzable, and present in both soluble and particulate fractions of extracts. Heating of extracts at 70 C for 10 min resulted in 10- to 40-fold stimulation in GIF activity, and evidence for a heat-labile inhibitor was obtained. Colonies appearing on host-free medium in these experiments were shown to be those of typical H-I derivatives by isolation and subsequent host-independent cultivation of these organisms. GIF was a conditional requirement dependent on age and size of inoculum for all H-I derivatives characterized. Although GIF stimulated the growth of washed exponential phase cells transferred to fresh medium, it was not essential for growth. However, it was essential for the initiation of growth of washed stationary phase cells from small inocula transferred to fresh medium. It is proposed that GIF is required to initiate growth of metabolically quiescent cells.     

Periurethral aerobic microflora of pregnant and non-pregnant women.

Seventy-two pregnant and 88 non-pregnant women were examined to see whether the periurethral region had been colonised with group B streptococci (Streptococcus agalactiae), enterococci, and Gram-negative rods belonging to the Enterobacteriaeceae. A semi-quantitative method was used for periurethral sampling, and paired urethral swabs were also collected to compare the isolation rates of group B streptococci from the two sites and with the two sampling methods. A higher isolation rate was found with periurethral sampling. Most specimens showed no or scanty growth of Gram-negative rods. Pregnancy was often associated with heavy growth of enterococci. Sampling performed during menstruation and while oral contraceptives were being used produced high isolation rates of group B streptococci. These results seem to suggest that the periurethral area might protect against genital colonisation with group B streptococci as it does against urinary tract infection and that hormonal factors influence the carriage of these organisms.     

A new simple method of curing plasmids in lactic streptococci (Streptococcus cremoris; Streptococcus lactis, plasmids)

A simple method for curing plasmid DNA from lactic streptococci is described. When strains of lactic streptococci are grown overnight at 32 degrees C in an unbuffered medium (M17-) and held at the same temperature for an extended period (96 h), the acid environment induces loss of plasmid DNA of different sizes. If the process of growth in M17- broth followed by extended incubation at 32 degrees C is repeated, most of the plasmids are lost, as revealed by gel electrophoretic profiles of DNA. The method is simple and efficient in curing plasmids of lactic streptococci without use of any mutagenic chemical.     

Comparison of media for the isolation of Enterobacter sakazakii

Enterobacter sakazakii is associated with neonatal infections and is occasionally present at low levels (<1 CFU/g) in powdered infant formula milk (IFM). It has been previously reported that some E. sakazakii strains do not grow in standard media for Enterobacteriaceae and coliform bacteria; therefore, a reliable method is needed for recovery of the organism. Three E. sakazakii enrichment broths-Enterobacteriaceae enrichment broth (EE), E. sakazakii selective broth (ESSB), and modified lauryl sulfate broth (mLST)-were compared with a novel broth designed for maximum recovery of E. sakazakii, E. sakazakii enrichment broth (ESE). One hundred seventy-seven strains (100%) grew in ESE, whereas between 2 and 6% of strains did not grow in EE, mLST, or ESSB. E. sakazakii possesses alpha-glucosidase activity, and a number of selective, chromogenic agars for E. sakazakii isolation based on this enzyme have been developed. E. sakazakii isolation agar produced fewer false-positive colonies than did Druggan-Forsythe-Iversen agar. However, the latter supported the growth of more E. sakazakii strains. It was also determined that 2% of E. sakazakii strains did not produce yellow pigmentation on tryptone soya agar at 25 degrees C, a characteristic frequently cited in the identification of E. sakazakii. The recovery of desiccated E. sakazakii (0.2 to 2000 CFU/25 g) from powdered IFM in the presence of a competing flora was determined with various enrichment broths and differential selective media. Current media designed for the isolation and presumptive identification of E. sakazakii do not support the growth of all currently known E. sakazakii phenotypes; therefore, improvements in the proposed methods are desirable.     

THE SELECTIVE ACTION OF THALLOUS ACETATE FOR LACTOBACILLI

SUMMARY: The effect of thallous acetate has been examined on the growth of 23 strains of lactobacilli representing 13 different species. All strains grew satisfactorily in both broth and agar media containing 0.1% of thallous acetate. Of the 18 other organisms also examined the growth of the streptococci was unsuppressed, staphylococci, a micrococcus, B. licheniformis and Pr. mirabilis were partially inhibited and strains of Bact. coli, B. cereus, Chr. prodigiosum and Ps. fluorescens were almost completely inhibited by that concentration. By its use a valuable selective medium for isolating lactobacilli from natural habitats of mixed flora was obtained, other organisms, with the exception of streptococci, being almost completely eliminated.     

Comparison of Several Laboratory Media for Presumptive Identification of Enterococci and Group D Streptococci

Bile-esculin (Difco), modified bile-esculin (Difco), selective enterococcus (Pfizer Co.), and eosin-methylene blue agar media were evaluated for accuracy in identifying group D streptococci. The regular and modified bile-esculin media performed equally well, but the selective enterococcus and eosin-methylene blue agars did not accurately differentiate the group D from non-group D streptococci. A modified 6.5% NaCl broth was compared with unmodified 6.5% NaCl broth and Streptococcus faecalis (SF; Difco) broth for accuracy in differentiating enterococci from non-enterococci. The modified and unmodified broths worked equally well in the salt tolerance test, but the lot-to-lot variability of SF broth made this medium unusable as an indicator for enterococci. With all seven media, the number of strains giving positive tests decreased when the tests were incubated at 45 C as compared with 35 C, and the number of strains giving negative tests increased. Thus, the number of false-positive identifications decreased, but the number of false-negative identifications increased. Variability in the susceptibility of group D non-enterococcal streptococci to oxacillin and methicillin sensitivity disks limited the usefulness of these tests for presumptive identification of either enterococci or group D streptococci.     

Comparison of some enrichment broths and growth media for the isolation of thermophilic campylobacters from surface water samples

Three different enrichment broths and two selective growth media were compared for isolating thermophilic campylobacters by combined membrane filtration and enrichment techniques from surface waters of different physical, chemical and bacteriological characteristics. Fifty-two strains of campylobacters were isolated from total of 1668 cultures. The various broth/medium combinations did not affect the dominance of C. jejuni over C. coli (total 49 C. jejuni and three C. coli). The most efficient combinations of enrichment broth and growth media were either Oosterom broth/blood-free charcoal-cefoperazone-deoxycholate agar (CCDA) medium or blood-free charcoal-cefoperazone-deoxycholate (CCD) broth/CCDA medium. Modified Preston broth (sheep blood instead of horse blood) with either of the growth media gave significantly lower yields although it suppressed efficiently the growth of contaminants. Skirrow medium had lower selectivity than CCDA medium and gave slightly lower isolation rate. Enrichment time (24 or 48 h) did not affect the isolation frequency of campylobacters but longer enrichment time increased the growth of contaminants. Prefiltration through membranes of pore sizes 5.0 and 1.2 microns decreased the growth of contaminants. However, these membranes retain campylobacters and must be cultured to avoid underestimation. From more polluted waters campylobacters were isolated most frequently with CCD broth and CCDA medium.     

Selectivity of Mitis Salivarius agar and a new selective medium for oral streptococci in dogs

An evaluation on the applicability of Mitis Salivarius agar (MS) medium, commonly used for the detection of oral streptococci in human and animals, to dog specimens and the development of a new selective medium for isolating streptococci from the canine oral cavity are described. Oral samples from dogs were cultured on MS medium under anaerobic conditions. The predominant facultative anaerobic bacteria on MS plates were gram-negative rods. Selectivity of streptococci on MS medium was 21.2%. A new selective medium, designated MS-CAN-AE, was developed for the isolation of streptococci from the canine oral cavity. The average growth recovery of laboratory and clinically isolated strains of streptococci on MS-CAN-AE medium was 84.1% of that on MS medium. Gram-positive rods and gram-negative rods and cocci rarely grew on the MS-CAN-AE. The selectivity of MS-CAN-AE was 95.0% for clinical samples. MS-CAN-AE medium will be helpful for investigations of streptococci in the canine oral cavity.