Brain Heart Infusion Blood Agar Research Articles

Aerosols Generated during Endodontic Treatment: A Special Concern during the Coronavirus Disease 2019 Pandemic

IntroductionThe aims of this study were to investigate aerosolized microorganisms generated during endodontic emergencies and nonsurgical root canal therapy (NSRCT), to assess the spread of airborne microbes, and to verify the spatial distribution of airborne microbial spread. MethodsA total of 45 endodontic procedures were sampled, including full pulpotomy (n = 15), pulpectomy (n = 15), and NSRCT (n = 15). Samples were collected during room resting and after treatment. The passive air sampling technique using settle plates was applied. Agar plates were set at different locations in the operatory. The colony-forming unit (CFU) was counted in brain-heart infusion blood agar plates. A set of agar plates containing selective chromogenic culture media was used for the isolation and presumptive identification of target microorganisms. Fungi were investigated using Sabouraud dextrose agar. ResultsPulpotomy generated the lowest mean CFU count (P < .05). There was no difference between the mean CFU counts found in pulpectomy and NSRCT (P > .05). A higher mean CFU count was found close to the patient's mouth (0.5 m) than at a 2-m distance in pulpectomy and NSRCT (P < .05). There was no difference between the mean CFU count found in front of the patient's mouth versus diagonal in pulpectomy and NSRCT (P > .05). Staphylococcus aureus (22/45, 48.8%) was the most frequent bacteria species. Longer treatment times were associated with higher CFU counts. ConclusionsOur findings indicated that pulpotomy generates less aerosolized microorganisms than pulpectomy and NSRCT. The proximity to the patient's mouth and the treatment duration were implicated in the level of contamination.

BartonellaSpecies in Invasive Rats and Indigenous Rodents from Uganda

The presence of bartonellae in invasive rats (Rattus rattus) and indigenous rodents (Arvicanthis niloticus and Cricetomys gambianus) from two districts in Uganda, Arua and Zombo, was examined by PCR detection and culture. Blood from a total of 228 R. rattus, 31 A. niloticus, and 5 C. gambianus was screened using genus-specific primers targeting the 16S-23S intergenic spacer region. Furthermore, rodent blood was plated on brain heart infusion blood agar, and isolates were verified as Bartonella species using citrate synthase gene- (gltA) specific primers. One hundred and four fleas recovered from R. rattus were also tested for the presence of Bartonella species using the same gltA primer set. An overall prevalence of 1.3% (three of 228) was obtained in R. rattus, whereas 61.3% of 31 A. niloticus and 60% of five C. gambianus were positive for the presence of Bartonella species. Genotypes related to Bartonella elizabethae, a known zoonotic pathogen, were detected in three R. rattus and one C. gambianus. Bartonella strains, similar to bacteria detected in indigenous rodents from other African countries, were isolated from the blood of A. niloticus. Bartonellae, similar to bacteria initially cultured from Ornithodorus sonrai (soft tick) from Senegal, were found in two C. gambianus. Interestingly, bartonellae detected in fleas from invasive rats were similar to bacteria identified in indigenous rodents and not their rat hosts, with an overall prevalence of 6.7%. These results suggest that if fleas are competent vectors of these bartonellae, humans residing in these two districts of Uganda are potentially at greater risk for exposure to Bartonella species from native rodents than from invasive rats. The low prevalence of bartonellae in R. rattus was quite surprising, in contrast, to the detection of these organisms in a large percentage of Rattus species from other geographical areas. A possible reason for this disparity is discussed.

Characterization of tRNA-dependent Peptide Bond Formation by MurM in the Synthesis of Streptococcus pneumoniae Peptidoglycan

MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.

Evaluation of an extended blood culture protocol to isolate fastidious organisms from patients with AIDS.

Recent reports of fastidious pathogens suggest the need for special blood cultures for immunocompromised patients. Blood cultures from 45 human immunodeficiency virus (HIV)-infected patients with unexplained fever (> or = 38.0 degrees C) and CD4 counts of < 125 cells per mm3 were collected into a vacuum tube with sodium polyanetholsulfonate, an Isolator tube, and BACTEC aerobic and anaerobic bottles. Blood from the sodium polyanethosulfonate tube was inoculated into BACTEC 13A bottles, which were read weekly for 16 weeks. Isolator sediment was divided among eight agar media, including four sheep blood agar media: chocolate agar, brain heart infusion blood agar, heart infusion blood agar, and brucella blood agar. Other agar plates included Sabouraud's, buffered charcoal-yeast extract, Middlebrook 7H11 (M7H11) with hemoglobin, and M7H11 with mycobactin J. Incubation conditions included air and CO2-enriched aerobic, microaerophilic, and anaerobic atmospheres. Aerobic BACTEC broths received an acridine orange stain on day 8 and were subcultured at 2, 4, and 8 weeks. Anaerobic BACTEC bottles were subcultured at 4 weeks. All solid media, including subcultures, were incubated for 8 weeks, providing a total of 16 weeks of incubation for each specimen. Clinically significant isolates included eight Mycobacterium avium complex isolates and one each of Bartonella henselae, Bartonella quintana, Shigella flexneri, Klebsiella oxytoca, and Cryptococcus neoformans. All isolates were detected with commercially available media and, with the exception of Bartonella spp., were recovered within incubation times routinely used in most clinical laboratories.

Effect of culture media and incubation temperature on growth of selected strains of Francisella tularensis.

The rate and amount of growth of 4 field isolates and reference strain ATCC 6223 of Francisella tularensis were evaluated on isolation media with 2 different agar bases and with different supplements and incubated at 25 C, 35 C, and 42 C. Biochemical reactions on conventional differential media with and without cysteine were evaluated. Two of the field isolates and the reference strain were F. tularensis subspecies tularensis (formerly biovar tularensis or Type A), and 2 isolates were subspecies holarctica (formerly subspecies palaearctica or Type B). Bacto cystine heart blood agar supplemented with 1% hemoglobin, glucose cystine heart blood agar, and brain-heart infusion blood agar supported good growth of all 4 field strains, with the most luxuriant growth occurring on Bacto cystine heart blood agar with hemoglobin. Heart infusion blood agar and trypticase soy blood agar supported growth of the field isolates, although growth was diminished and delayed. Strain 6223 was distinctly fastidious and failed to grow on heart infusion or trypticase soy blood agars. Growth of strain 6223 was best on Bacto cystine heart blood agar with hemoglobin. The agar base did not affect growth unless the supplements became limiting, in which case Bacto agar base generally supported growth better than BiTek agar base. Incubation at 35 C was optimum for all 5 strains. Growth at 42 C was slow, with the greatest decrease in the rate and amount of growth occurring with field isolates of F. tularensis subspecies tularensis. Strain 6223 did not grow at 25 C, and the 4 field isolates grew slowly at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

A semiquantitative enzyme method for identifying Fusobacterium necrophorum biovars A and B.

The present study describes a semiquantitative enzyme method for distinguishing among the pathogenic biovars of Fusobacterium necrophorum. Since 1891 when McFaydean first described the organism as the etiologic agent of a bovine and ovine hepatic necrosis, numerous investigators have made significant contributions to the characterization and nomenclature of the bacterium. In 1963, Fievez classified the 3 Sphaerophorus species as one species, S. necrophorus, with biovars A, AB, B, and C. Beerens et a1. in 1971 similarly characterized the 3 Sphaerophorus species as follows: S. necrophorus was hemolytic, had a hemagglutinin, and was pathogenic for mice; S. funduliformis was hemolytic, had no hemagglutmm, and demonstrated only slight pathogenicity for mice; and S. pseudonecrophorus was not hemolytic, had no hemagglutinin, and was not pathogenic for mice. Biovars A and AB as described by Fievez corresponded to S. necrophorus, biovar B corresponded to S. funduliformis, and biovar C corresponded to S. pseudonecrophorus. In 1970, the International Committee on Nomenclature of Bacteria Taxonomic Subcommittee for Gram-Negative Anaerobic Rods recommended that the genus Sphaerophorus be included in the genus Fusobacterium. 3 The present classification of F. necrophorum, as described in 1984 in the first edition of Bergey’s Manual of Systematic Bacteriology 9 is based on biochemical characteristics, whereas the biovars of F. necrophorum, which have differences in cellular and colony morphology, are identified by their biological characteristics. Forty-two strains of F. necrophorum that had been previously isolated from bovine foot rot, bovine hepatic abscesses, 5 bovine ruminal contents and ovine hepatic abscesses were biotyped (Table 1) on the basis of direct hemagglutination (DHA) of chicken erythrocytes, sedimentation in broth culture, colony characteristics on enriched brain-heart infusion blood agar (BHIBA), and cellular morphology observed on gram-stained slides. The DHA tests were conducted as previously described. Twenty strains were identified as biovar A; 21 as biovar B; and 1 as biovar AB, which has morphologic characteristics of both biovars A and B and falls between these biovars on the basis of DHA and sedimentation pattern. The 42 strains were evaluated for their enzyme activities on 19 substrates using a commercial semiquantitative enzyme system as described by the manufacturer. Briefly, colonies from a BHIBA plate that had been incubated anaerobically in an atmosphere of 10% carbon dioxide, 10% hydrogen, and 80% nitrogen for 48 hours were suspended in

Transformation of Corynebacterium pseudotuberculosis by electroporation

Summary Corynebacterium pseudotuberculosis was transformed by electroporation, using pNG2, an erythromycin-resistaiice plasmid from C diphtheriae. Corynebacterium pseudotuberculosis cultivated in brain-heart infusion broth was washed 3 times with water, and resuspended to a final concentration of about 5 × 1013 colony-forming units/ml. An electroporator constructed in our laboratory incorporated an electrode with 0.8-mm interelectrode gap, using disposable spectrophotometer cuvettes as containers for electroporation. The pNG2 was prepared in Escherichia coli and 4 to 16 μg of pNG2 dna was mixed with 400-μl amounts of cell suspension in prechilled cuvettes. After incubation on ice for 5 to 10 minutes, the mixture was electroporated at field strengths of up to 18 kV/cm, mixed with 1.5 ml of brain-heart infusion broth, and incubated at 37 C for 2 hours with agitation. Aliquots were then plated on brain-heart infusion blood agar with 15 μg of erythromycin/ml. Corynebacterium pseudotuberculosis was transformed at a maximal efficiency of ≈ 4 × 104 transformants/μg of pNG2 dna. Most total transformants and most transformants per microgram of pNG2 were generated at a field strength of 18 kV/cm. When the concentration of pNG2 dna was varied, the average total number of transformants increased through a concentration of 30 μg/ml, but the efficiency of transformation was highest at the lowest dna concentration. Transformants contained unmodified pNG2.

Evaluation of enrichment, storage, and age of blood agar medium in relation to its ability to support growth of anaerobic bacteria

By measuring the colony size of a variety of anaerobic bacteria isolated from clinical specimens, an evaluation was made of the benefits derived from the addition of several enrichments to blood agar medium commonly used for the growth of anaerobes. Similar methods were used to study the effects of various storage conditions and age of the medium. The results were compared with those obtained on freshly prepared and enriched blood agar plates as well as commercially available blood agar plates. Freshly prepared and enriched blood agar was found to give substantially larger colonies than could be grown on commercially obtained blood agar plates when both were inoculated and incubated under identical conditions. Storage of plating media under CO2 for periods of up to 72 h had only a minor effect on the growth of the anaerobic bacteria studied, but longer periods of storage under CO2 resulted in a less efficient plating medium. Nonenriched brain heart infusion (BHI) was found to be a better basal medium than Trypticase soy agar (TSA) medium. Colony size on fully enriched BHI blood agar plates was greater than nonenriched BHI greater than nonenriched TSA greater than commercially prepared nonenriched TSA plates. The data suggest that freshness of the plates may be as important as using rich media.

Cultural and serologic studies on nonfatal histoplasmosis in mice, hamsters, and guinea pigs.

The definitive diagnosis of human histoplasmosis usually depends on the isolation of the etiologic agent Histoplasma capsulatum. However, in mild cases, it is often not possible to isolate the organism from or to demonstrate it in blood, gastric washings, tissue biopsies, sputum, or other available specimens. Accordingly, numerous methods have been employed in attempts to improve the chance of isolation of H. capsulatum from human patients. In addition, considerable research has been conducted on experimentally infected animals to determine the best methods for the demonstration of the etiologic agent. DeMonbreun (1934) and Parsons (1942) first demonstrated that mice could be infected by injection of mycelial or yeastlike forms of the fungus. Subsequently, Tager and Liebow (1942) found that four mice 3 to 4 weeks of age, inoculated intraperitoneally a mycelial suspension, succumbed to the infection 100 to 150 days later. Levy (1945) inoculated 145 6- to 8-week-old mice intravenously a mixture of the yeastlike and mycelial forms of H. capsulatum grown for 7 days on blood agar. Subsequently, positive cultures were obtained from the livers and spleens of all the mice. The size of the inoculum and the nature of the medium used for isolation were not mentioned. Howell (1948) reported brain-heart infusion blood agar to be of value for Received for publication May 11, 1953. The author acknowledges the technical assistance of Mr. J. C. Cory. demonstration of the fungus in the spleens of guinea pigs infected intraperitoneally with graded of the yeastlike phase. However, he did not correlate his cultural results direct microscopic observation of the fungus in the tissues, nor did he mention the actual numbers of organisms used. Kotcher et al (1951) inoculated 42 white mice intraperitoneally a saline suspension of ground elements of the mycelial phase, and showed (a) that H. capsulatum could be recovered by culture more readily than it could be identified by direct microscopic examination of stained tissue impressions, (b) that brain-heart infusion blood agar and Sabouraud's dextrose agar yielded the greatest number of positive cultures, and (c) that the highest percentage of isolations was obtained from spleens cultured on Sabouraud's dextrose agar. None of the earlier investigators inoculated laboratory animals accurately determined numbers of yeastlike cells. Similarly, no experiments have been recorded in which nonfatal histoplasmosis in small laboratory animals was studied the use of serological and cultural methods. Accordingly, experiments were conducted to obtain information on (a) the response of mice, hamsters, and guinea pigs to inoculation graded doses of yeastlike cells, (b) the possible enhancing effect of mucin in producing detectable histoplasmosis, (c) the presence of antibodies in animals inoculated small numbers of yeastlike cells, and (d) the relative efficiencies of cultural and serological