Sheep Blood Agar Plates Research Articles

Streptococcus pneumoniae Sheds Syndecan-1 Ectodomains through ZmpC, a Metalloproteinase Virulence Factor

Several microbial pathogens stimulate the ectodomain shedding of host cell surface proteins to promote their pathogenesis. We reported previously that Pseudomonas aeruginosa and Staphylococcus aureus activate the ectodomain shedding of syndecan-1 and that syndecan-1 shedding promotes P. aeruginosa pathogenesis in mouse models of lung and burned skin infections. However, it remains to be determined whether activation of syndecan-1 shedding is a virulence mechanism broadly used by pathogens. Here we show that Streptococcus pneumoniae stimulates syndecan-1 shedding in cell culture-based assays. S. pneumoniae-induced syndecan-1 shedding was repressed by peptide hydroxamate inhibitors of metalloproteinases but not by inhibitors of intracellular signaling pathways previously found to be essential for syndecan-1 shedding caused by P. aeruginosa, S. aureus, or other shedding agonists. A 170-kDa protein fraction with a peptide hydroxamate-sensitive shedding activity was purified by ammonium sulfate precipitation, DEAE chromatography, and size exclusion chromatography. Mass spectrometry analyses revealed that the 170-kDa fraction is composed of ZmpB and ZmpC, two metalloproteinase virulence factors of S. pneumoniae. Both the purified 170-kDa ZmpB/ZmpC fraction and unfractionated S. pneumoniae culture supernatant generated syndecan-1 ectodomains that are smaller than those released by endogenous shedding. Further, a mutant S. pneumoniae strain deficient in zmpC, but not zmpB, lost its capacity to stimulate syndecan-1 shedding. These data demonstrate that S. pneumoniae directly sheds syndecan-1 ectodomains through the action of ZmpC.

血液培養からの<I>Helicobacter cinaedi</I>及びその類縁菌の分離培養と簡便同定法に関する検討

A spiral Gram-negative bacterium, Helicobacter cinaedi, has been isolated from clinical specimens of immunodeficient patients in Japan. We determined the isolation procedure and features using practical phenotypic tests for H. cinaedi isolated from blood culture. We applied 28 clinically isolated strains from 5 different metropolitan institutions, whose species was reconfirmed in technical support at another expert veterinary medicine institute in identifying H. cinaedi. We also used 6 strains of type and reference. Positive signals from aerobic bottles in automated blood culture systems, BACTEC, were observed 4-9 days after culture was started. A thin spread colony was found 3-4 days after the start of subcultures consisting of growth on 5% sheep blood agar plates with immediate incubation under wet and microaerophilic conditions at 35 degrees C. In phenotypic tests, all specimens of H. cinaedi were shown to be positive in nitrate reduction and to be negative in both indoxyl acetate hydrolysis and urease activity, underlining the usefulness of these 3 phenotypes. Tests of alkaline phosphatase hydrolysis, growth at different temperatures, and growth with 1% glycin provided additional information when referencing standard results of identification. We took speculative profiling data of H. cinaedi identification with a simple phenotypic kit, Api campy, indicating its usefulness in testing. These observations suggest combined methods of previously available tests (nitrate reduction, indoxyl acetate hydrolysis, and urease activity) with Api campy may be useful in differentiating H. cinaedi from H. cinaedi-like microorganisms when observing the characteristic thin spread colony within subcultures under microaerophilic conditions.

Eikenella Corrodens Cervical Spinal Epidural Abscess Induced by a Fish Bone

Cervical spinal epidural abscess, caused by fish bone injury and a secondary infection by Eikenella Corrodens which is part of the normal flora, has not been reported. A 72-yr-old man came to the hospital with pain in his posterior neck and both shoulders for 2 months. He also was experiencing weakness on his right side for 3 days. A fish bone had been stuck in his throat for about 2 months. Neurological examination revealed right hemiparesis, hypesthesia on the left extremities and neck stiffness. Laboratory findings showed an elevated ESR/CRP and leukocytosis, and magnetic resonance imaging revealed a retropharyngeal abscess and cervical myelitis. The patient was treated with emergency surgical decompression and antibiotics. A fish bone was removed from the C3-C4 intervertebral disc space. In the culture of chocolate blood agar and 5% sheep blood agar plate, E. corrodens was detected as a causative organism.

In vitro evaluation of three bacterial culture systems for the recovery of Escherichia coli from equine blood

To evaluate the effectiveness of a commercial conventional blood culture system (BCS), a commercial resin-containing BCS, and a commercial lysis-centrifugation-based BCS for the recovery of Escherichia coli from equine blood samples inoculated with that organism. Samples of blood obtained from a clinically normal horse that were inoculated with E coli. Blood samples were aseptically collected and inoculated with an E coli specimen (50 CFUs/mL) that had been previously isolated from a foal with sepsis. Subsequently, samples were spiked with gentamicin at a concentration of 30 mug/mL, and 10 mL of each mixture was inoculated into 1 bottle or tube of each BCS. Samples were processed and incubated according to the manufacturer's guidelines and inoculated onto 5% sheep blood agar plates. Plated samples were examined macroscopically at regular intervals for as long as 72 hours. Detection of E coli and time to detection were recorded for each medium. Detection frequency of E coli was significantly greater by use of the resin-containing BCS (14/23 bottles) than that achieved by use of the conventional BCS (7/23 bottles) or the lysis-centrifugation-based BCS (0/10 tubes). Mean detection time (6 hours after plating) did not differ between the BCS with conventional medium and the BCS with resincontaining medium. Results suggest that a BCS with resin-containing medium may provide clinical benefit in the successful recovery of E coli from the blood of foals with sepsis that have been previously administered gentamicin.

Calf Respiratory Disease and Pen Microenvironments in Naturally Ventilated Calf Barns in Winter

Relationships between air quality, a variety of environmental risk factors, and calf respiratory health were studied in 13 naturally ventilated calf barns during winter. A minimum of 12 preweaned calves were randomly selected and scored for the presence of respiratory disease in each barn. An air sampling device was used to determine airborne bacteria colony-forming units per cubic meter (cfu/m3) of air in calf pens and central alleys within the barns. Airborne bacteria samples were collected on sheep blood agar (BAP) and eosin methylene blue (EMB) agar plates. Temperature and relative humidity were recorded in each calf pen, the barn alley, and outside the barn. Samples of bedding were collected in each pen and DM was measured. Pen bedding type and a calf nesting score (degree to which the calves could nestle into the bedding) was assigned to each barn. Calf numbers, barn and pen dimensions, ridge, eave, and curtain openings, and exterior wind speed and direction were determined and used to estimate building ventilation rates. Factors that were significantly associated with a reduced prevalence of respiratory disease were reduced pen bacterial counts (log10 cfu/m3) on BAP, presence of a solid barrier between each calf pen, and increased ability to nest. Individual calf pen bacterial counts were significantly different from barn alley bacterial counts on both BAP and EMB. Significant factors associated with reduced calf pen bacterial counts on BAP were increasing pen area, increasing number of open planes of the calf pen, decreasing pen temperature, and wood-particle bedding. Significant factors associated with reduced alley bacterial counts on BAP were increased ventilation changes per hour, increased barn volume per kilogram of calf, reduced pen bacterial counts, and barn type.

First Isolation of Dysgonomonas mossii from Intestinal Juice of a Patient with Pancreatic Cancer

Dysgonomonas species were first designated in 2000. However, clinical infections due to this microorganism have rarely been described. Our aim was to present the first isolation of Dysgonomonas mossii from intestinal juice of a patient with pancreatic cancer. Predominantly appearing grayish-white colonies grown on chocolate and sheep blood agar plates were characterized morphologically by Gram stain, biochemically by automated instrument using Vitek II ID-GNB card together with commercially available kit systems, ID-Test HN-20 and API rapid ID 32A32A, and genetically by sequencing the 16S rRNA gene of the organism using a Taq DyeDeoxy Terminator Cycle Sequencing and a model 3100 DNA sequencer instrument. The isolate was further characterized by antimicrobial susceptibility using MicroFast 4J Panels and additional biochemical and physiological properties. The isolate was finally identified as D. mossii from the findings of the morphological, cultural, and biochemical properties together with the comparative sequence of the 16S rRNA genes. The isolate was highly susceptible to many antibiotics but resistant to penicillins and cephems. As D. mossii was rarely encountered in the clinical microbiology laboratory, it may be misidentified as an X-factor-dependent Haemophilus species due to its negative result for the porphyrin test. Accumulation of the case reports with the isolation of this species is expected to elucidate the infections due to D. mossii. The presence of D. mossii caused no significant clinical infection despite repeated isolations, as the patient had no conspicuous abdominal complaints. However, our report is a noteworthy and useful piece of information.

Influences of Long‐Term Antibiotic Administration on Peyer's Patch Lymphocytes and Mucosal Immunoglobulin A Levels in a Mouse Model

Long-term antibiotic administration is sometimes necessary to control bacterial infections during the perioperative period. However, antibiotic administration may alter gut bacterial flora, possibly impairing gut mucosal immunity. We hypothesized that 1 week of subcutaneous (SC) antibiotic injections would affect Peyer's patch (PP) lymphocyte numbers and phenotypes, as well as mucosal immunoglobulin A (IgA) levels. Sixty-one male Institute of Cancer Research mice were randomized to CMZ (cefmetazole 100 mg/kg, administered SC twice a day), IPM (imipenem/cilastatin 50 mg/kg x 2), and control (saline 0.1 mL x 2) groups. After 7 days of treatment, the mice were killed and their small intestines removed. Bacterial numbers in the small intestine were determined using sheep blood agar plates under aerobic conditions (n = 21). PP lymphocytes were isolated to determine cell numbers and phenotypes (CD4, CD8, alphabetaTCR, gammadeltaTCR, B220; n = 40). IgA levels in the small intestinal and bronchoalveolar washings were also measured with ELISA. Antibiotic administration decreased both bacterial number and the PP cell yield compared with the control group. There were no significant differences in either phenotype percentages or IgA levels at any mucosal sites among the 3 groups. Long-term antibiotic treatment reduces PP cell numbers while decreasing bacterial numbers in the small intestine. It may be important to recognize changes in gut mucosal immunity during long-term antibiotic administration.

Streptobacillus moniliformisEndocarditis

To the Editor: Streptobacillus moniliformis is a facultatively anaerobic, pleomorphic, gram-variable bacillus often seen in chains and as long unbranched filaments. It is found in the nasopharynx and oropharynx of wild and laboratory rats. Human infections result either from rodent bites (rat bite fever) or contaminated milk or other foods (Haverhill fever). The most common manifestations of infection are arthralgia, fever, and rash; endocarditis occurs as a rare complication (1). We report a case of S. moniliformis endocarditis in India in a patient with congenital heart disease. An 18-year-old man was admitted to the Department of Cardiology at the Government General Hospital in Chennai, India, in November 2005, with a fever of 2 months' duration with cough, epistaxis, palpitations, and persistent joint pain. His medical history indicated congenital heart disease with a ventricular septal defect. On physical examination, his blood pressure was 100/70 mm Hg, pulse rate was 100 beats/min, and temperature was 38.5°C. Laboratory tests showed a leukocyte count of 7,600/μL, a platelet count of 127,000/μL, and an erythrocyte sedimentation rate of 70 mm/h. An electrocardiogram showed normal sinus rhythm. A transthoracic echocardiogram demonstrated a ventricular septal defect and vegetations on the septal leaflet of the tricuspid valve. Three blood cultures were prepared, and treatment with antimicrobial drugs (intravenous penicillin G, 3 × 106 U every 6 h, and gentamicin, 50 mg every 8 h for 4 weeks) was initiated. The blood cultures were incubated at 37°C in an atmosphere of 5%–10% CO2. Characteristic white, downy, crumblike granules were observed on the surface of the erythrocytes in all 3 cultures within 18–24 h of incubation. Characteristic puff balls were seen after 48 h of incubation. Gram-stained smears showed gram-negative bacilli in long chains. Cultures were subcultured onto 5% sheep blood agar plates and MacConkey agar plates. The plates were incubated at 37°C in an atmosphere of 5%–10% CO2. After 18–24 h of incubation, growth was seen on the sheep blood agar plates. Colonies were 1–2 mm in diameter, gray, smooth, and butyrous. A Gram stain of these colonies identified gram-variable, pleomorphic coccobacilli that were negative for catalase, oxidase, urease, and citrate, and did not produce indole or reduce nitrate. Antimicrobial susceptibility testing was performed by using the Kirby-Bauer disk diffusion method according to recommendations of the National Committee for Clinical Laboratory Standards (2). The isolate was sensitive to penicillin G, ceftriaxone, cephalexin, amoxicillin, gentamicin, and erythromycin. The patient responded well to treatment and became afebrile within 48 h after initiation of therapy. Treatment with antimicrobial drugs was continued for 4 weeks. The blood cultures were negative when repeated after 2 weeks. The patient had an uneventful recovery and was discharged from the hospital. Rat bite fever is a zoonosis caused by either Streptobacillus moniliformis or Spirillum minus (1,3). S. moniliformis is found in the nasopharynx of small rodents, especially rats. Rats that are carriers have no symptoms but can effectively transmit the infection by bite or through infected body fluids such as urine. This patient had a history of living in a rat-infested area, and admitted having been bitten by a rat several months before the onset of symptoms. However, we considered it unlikely that disease contracted by a rat bite would take months to be manifested. Thus, it is more likely that he contracted the infection from food or water contaminated with rat excreta. Endocarditis is a rare complication of S. moniliformis infection, and cardiac valvular abnormalities have been reported in ≈50% of cases (4). This patient, however, had only a small ventricular septal defect. This is the first report of S. moniliformis endocarditis from India.

The Role of Decay-accelerating Factor as a Receptor for Helicobacter pylori and a Mediator of Gastric Inflammation

Persistent gastritis induced by Helicobacter pylori is the strongest known risk factor for peptic ulcer disease and distal gastric adenocarcinoma, a process for which adherence of H. pylori to gastric epithelial cells is critical. Decay-accelerating factor (DAF), a protein that protects epithelial cells from complement-mediated lysis, also functions as a receptor for several microbial pathogens. In this study, we investigated whether H. pylori utilizes DAF as a receptor and the role of DAF within H. pylori-infected gastric mucosa. In vitro studies showed that H. pylori adhered avidly to Chinese hamster ovary cells expressing human DAF but not to vector controls. In H. pylori, disruption of the virulence factors vacA, cagA, and cagE did not alter adherence, but deletion of DAF complement control protein (CCP) domains 1-4 or the heavily O-glycosylated serine-threonine-rich COOH-terminal domain reduced binding. In cultured gastric epithelial cells, H. pylori induced transcriptional up-regulation of DAF, and genetic deficiency of DAF attenuated the development of inflammation among H. pylori-infected mice. These results indicate that DAF may regulate H. pylori-epithelial cell interactions relevant to pathogenesis.

Multilaboratory Evaluation of Disk Diffusion Antimicrobial Susceptibility Testing of Neisseria meningitidis Isolates

In 2005, the Clinical and Laboratory Standards Institute published MIC interpretive criteria for 13 antimicrobial agents used for either therapy or prophylaxis of Neisseria meningitidis infections. The MIC method includes the use of lysed horse blood-supplemented Mueller-Hinton broth with incubation in 5% CO2 for 20 to 24 h. Since some clinical laboratories might prefer the option of disk diffusion testing for infrequently encountered isolates a multicenter collaborative study was conducted to evaluate the reproducibility of a disk diffusion method for testing isolates of N. meningitidis. Interpretive criteria were developed for 12 antimicrobial agents. Four laboratories tested a common collection of 50 meningococcal strains and then tested 25 unique isolates per laboratory. Isolates were tested using Mueller-Hinton sheep blood agar plates incubated for 20 to 24 h in 5% CO2; they were also tested by the reference broth microdilution method in parallel. Pooling of the MIC and disk diffusion data from the common and unique isolates provided a sufficient sample size to develop susceptible, intermediate, and resistant zone diameter interpretive criteria using the error rate-bounded method for the following agents: chloramphenicol, trimethoprim-sulfamethoxazole, ciprofloxacin, and rifampin. Due to the lack of resistant strains at the present time, "susceptible only" interpretive criteria were proposed for cefotaxime, ceftriaxone, meropenem, azithromycin, and minocycline. The numbers of minor interpretive errors with penicillin and ampicillin disk tests were unacceptably high and precluded recommended testing of those agents by the disk method. However, amdinocillin, an agent that preferentially binds to the altered penicillin binding protein responsible for diminished penicillin susceptibility, has potential utility as a surrogate screening reagent for ampicillin resistance. A disk diffusion breakpoint was derived for nalidixic acid to serve as a surrogate marker for gyrase A mutations associated with diminished fluoroquinolone susceptibility. Disk diffusion testing with meningococci can be performed in a reproducible manner with several antimicrobial agents and represents a practical and cost-effective option for testing sporadic clinical isolates or for surveillance purposes by resource-limited laboratories.

The impact of incubating the throat culture for 72 h on the diagnosis of group A beta-hemolytic streptococci

Objective The accurate detection and treatment of group A beta-hemolytic streptococcal pharyngitis is important to prevent the potential sequels. Throat culture remains the gold standard for the diagnosis of streptococcal pharyngitis. Many authors recommended 48 h incubation time for the isolation of group A Streptococcus (GAS). In this study the diagnostic efficacy of prolonging the incubation time of throat cultures to 72 h for isolation of GAS was researched. Methods Throat swab specimens were inoculated to 5% sheep blood agar plate and incubated at 37 °C, and evaluated at the 24th hour. GAS negative plates were incubated again for another 24 h and read at the 48th hour. Negative plates at 48 h were incubated again, read at the 72nd hour, and positive results of all measurements were evaluated. Results A total of 367 cultures were examined. At the 24th hour evaluation 34 (9.3%) cultures were positive for GAS. Evaluation of 333 cultures that were found to be negative in the first 24th hour evaluation revealed 44 (13.6%) positive cultures at 48th hour evaluation. Out of 289 cultures that were negative during the 48th hour evaluation, an additional 13 (4.7%) cultures were found to be positive during the 72nd hour evaluation. The agreement between 24th–48th and 24th and 72nd were both moderate (kappa 0.54 and 0. 46, respectively), and between 48th and 72nd hour was very good (kappa: 0.90). Conclusion Throat cultures that are negative for GAS at 24 h should be incubated for second day. We conclude that, to confirm the throat cultures GAS negative, results of throat cultures need to be incubated for 72 h, since 72nd hour evaluations revealed additional 5% positive results.

Antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus epidermidis biofilms isolated from infected total hip arthroplasty cases

BackgroundIt is difficult to treat implant-related infections because bacteria form biofilms on implants. Biofilms protect the organisms from the host’s immune system and prevent penetration of antimicrobial agents. We investigated the susceptibility of six strains isolated from infected total hip arthroplasty (THA) cases to six antimicrobial agents. MethodsSix Staphylococcus aureus and S. epidermidis strains isolated from infected THA cases and S. aureus ACTT 25923, positive strains of biofilms, were used in this study. Biofilms were developed on 96-well titer plates. After 7days the nutrient solution was aspirated, and wells were rinsed three times with sterile water. A series of doubling dilutions of each antimicrobial agent (16, 32, 64, 128, 256, 512, and 1024μg/ml) in trypticase soy broth were added to the wells. The plates were incubated at 37°C. After 24h, the contents were aspirated and wells were rinsed three times with sterile water. The surfaces of wells were vigorously scraped with a sterile swab and applied to sheep blood agar plates at 37°C in an aerobic chamber for 24h. The minimum bactericidal concentrations (MBCs) were determined as the lowest concentration of antibiotic resulting in 100% reduction of viable cells compared to the antibiotic-free control. Biofilms were produced on stainless steel washers, and the time-course changes were observed by scanning electron microscopy (SEM). ResultsPlanktonic bacteria were sensitive to the antimicrobial agents, but biofilm bacteria were markedly resistant. SEM revealed that biofilms grew larger with time. ConclusionsThe MBCs of antimicrobial agents for biofilms bacteria were higher than those of planktonic bacteria. Therefore, it is suggested that implant-related infection is difficult to treat with antimicrobial agents alone.

A serum-free medium for colony growth and hyaluronic acid production by Streptococcus zooepidemicus NJUST01

A hyaluronic acid (HA)-producing strain, Streptococcus zooepidemicus NJUST01, can grow in a serum-free agar medium, with starch as exclusive carbon source, but not glucose, sucrose, dextrine, xylose, or lactose. In this starch medium, the strain NJUST01 reproduced successively at 37 degrees C for 60 generations, with no obvious variation on morphology and physiology, but colonies of the strain after 60th generation could not produce a clear hemolytic zone on sheep blood agar plates. Hyaluronic acid production by the strain NJUST01 was analyzed relative to the starch medium. Employing a multifactor cross experiment, an optimum medium revealed for hyaluronic acid production was composed of 5% starch, 0.3% glucose, 0.5% peptone, 0.15% MgSO4, and 2.0% K2HPO4. The amount of HA 6.7 g/l was obtained in batch fermentation on a rotary shaker at 37 degrees C, 220 rpm for 36 h.

Production and Validation of the Use of Gamma Phage for Identification of Bacillus anthracis

Gamma phage specifically lyses vegetative cells of Bacillus anthracis and serves as part of the basis for identification of isolates from agar cultures. We report our study to standardize gamma phage production and preparation and to validate the assay for routine use. Unstable phage preparations were largely reduced through propagation of phage on blood agar cultures of the avirulent B. anthracis strain CDC684 and were adequately stable for extended storage beyond 1 to 2 years at 4 degrees C, provided that the preparation initially gave rise to clearly discernible plaques (macroplaques, 5 to 10 mm in diameter) on dilution at 1:8 or greater during potency testing with the Sterne strain or its equivalent. The primary intent of the assay was to test nonhemolytic, ground-glass-appearing bacterial B. anthracis-like colonies arising from culture of clinical or nonclinical samples on 5% sheep blood agar. Specifically, the assay was designed to show clear or primarily clear circular zones of lysis on bacterial lawns at the site of gamma phage inoculation after incubation at 35 degrees C +/- 2 degrees C for 20 h. When tested with 51 B. anthracis strains and 49 similar non-B. anthracis Bacillus species, the analytical specificity was >95%, a value that is intentionally low because our study design included two rare nonsusceptible B. anthracis strains as well as a rare susceptible non-B. anthracis strain, B. cereus ATCC 4342. Repeatability, day-to-day precision, and analyst-to-analyst precision were superior. The assay was rugged to variations among phage lots, phage concentration, amounts of bacterial inoculum, and incubation times as short as 6 to 8 h. System suitability evaluation showed improved robustness when bacterial lawns were tested with high- and low-density inoculum using the first and second quadrants of a serial four-quadrant streak on 5% sheep blood agar plates.

Identification and classification of all potential hemolysin encoding genes and their products from Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai1

To identify and classify all potential hemolysin candidates of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai. All of the potential hemolysin encoding genes were characterized in silico. These genes were cloned and expressed in Escherichia coli. The hemolytic activities of the expressed proteins were assayed observing the hemolysis on sheep blood agar plates. Sphingomyelinase activities of the hemolysin candidates were measured by thin-layer chromatography (TLC) and HPLC for sphingomyelin-hydrolysis. Expression and secretion of the hemolysins in L interrogans were studied by reverse transcription polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assays. The hemolytic activities of hemolysin candidates (LA0327, LA0378, LA1027, LA1029, LA1650, LA3050, LA3937, LA4004) from L interrogans strain Lai were confirmed. They were further divided into two groups, sphingomyelinase hemolysins and non-sphingomyelinase hemolysins, based on their ability to hydrolyze sphingomyelin. Most of these hemolysins were actually expressed in living L interrogans and some of them were secreted into the environment. This study establishes an essential and complete basis for further studying the contribution of hemolysins to the pathogenesis of L interrogans.

In Vitro Bacterial Adherence to Teicoplanin and Calcium Sulfate-Soaked Bone Cement

The aim of this study was to assess In Vitro the improvement in release kinetics for teicoplanin and the inhibition of bacterial adhesion on calcium sulfate-soaked PMMA discs. Calcium sulfate has been used in vivo and shown to be biocompatible, and prevention of bacterial adhesion may be expected with calcium sulfatesoaked polymethylmethacrylate (PMMA). Discs were made by adding teicoplanin and calcium sulfate in powder form to PMMA powder.The antibiotic concentration eluted from PMMA discs was assayed by agar diffusion assay. Nonadherent bacteria were removed by washing and adherent bacteria were detached by sonication. The suspension including nonadherent bacteria was seeded on sheep blood agar plate and incubated for 24 h at 37°C for the growth of microorganisms.The teicoplanin released from discs containing calcium sulfate was higher than that released from discs which had not been soaked with calcium sulfate. The count of bacteria adhering to the calcium sulfate-soaked discs was lower than that from the discs without calcium sulfate.In conclusion, the addition of calcium sulfate to teicoplanin-loaded PMMA bone cement may provide local antibiotic concentrations higher than MIC values due to increased antibiotic release. Furthermore, calcium sulfate was found to be effective in reducing bacterial adherence to treated discs.

Prevalência da colonização pelo estreptococo do grupo B em gestantes atendidas em ambulatório de pré-natal

PURPOSE: to determine the prevalence of group B Streptococcus (GBS) in pregnant women in the third trimester of pregnancy and explore the factors potentially associated with colonization. METHODS: a sample of 273 pregnant women in the third trimester of pregnancy, from the prenatal care center in Southern Brasil, was investigated. Vaginal and anorectal samples were collected and innoculated in Todd-Hewitt selective broth supplemented with 10 µg/mL colistin and 15 µg/mL nalidixic acid and afterwards cultured on defibrinated sheep blood agar plates. All suspected colonies were submitted to the agglutination test for detection of the specific group B antigen. The Camp test was used for GBS identification in non-hemolytic varieties. Demographic, socioeconomic, reproductive, and clinico-obstetric data were also analyzed. Prevalence ratio (PR) was used as risk measurement. Confidence interval was considered significant at the level of 95% (a=0.05). RESULTS: prevalence of Streptococcus (GBS) colonization amounted to 21.6% (59), 9.9% (27) of pregnant women showing positivity in both sites; vaginal site colonization was found in 6.95% (19) of the women and 4.75% (13) of the samples showed positivity only in the anal site. GBS prevalence was slightly higher in pregnant women under 20 years, in those with less schooling and in primiparae and was twice as high among those who did not reported spontaneous abortion, but with no statistical significance. No difference was found in GBS prevalence according to the history of sexually transmittted diseases and tabagism. When analyzed together, the factors detected as potentially associated with colonization by GBS were: primiparae over 30 years (PR=1.55) and women with more than one sexual partner and increased frequency of sexual activity (p<0.05) (55,6 vs 20.5%; p<0.05). No difference regarding prevalence was found to exist in relation to the history of sexually transmitted diseases, previous spontaneous abortion and tabagism. CONCLUSION: these results confirm the need for routine collection for GBS culture from both sites (vaginal and anal) in all pregnant women in the third trimester of pregnancy.

Beta-haemolytic streptococci in school children 5-15 years of age with an emphasis on rheumatic fever, in the tri-island state of Grenada

The objective of this study is to determine the prevalence of beta-haemolytic streptococci in the pharynx and antibodies to beta-haemolytic streptococci in school children 5-15 years of age in the tri-island state of Grenada Blood samples and throat swabs were obtained from 1388 school children, aged 5-15 years old attending randomly selected schools in each parish of the tri-island state of Grenada. Serum samples were subjected to antistreptolysin o testing (ASOT) while throat swabs were cultured on sheep blood agar plates. The prevalence of positive throat swab was 15.4% (95% CI = 13.4%, 17.2%). The rate was highest in St Patrick (21.8%) and lowest in Carriacou (5.7%). The prevalence of antibodies was 38.6% (95% CI = 37.6%, 42.8%). It was highest in St Patrick (54.4%) and lowest in Petit Martinique (26.8%). In St Patrick, males were significantly more likely than females to have a positive ASOT (p = 0.0084). In St George's, males were significantly more likely than females to have a positive throat culture (p = 0.0172). Thirty-four per cent of the positive cultures were type A, 10% were type C and 56% type G. The data illustrate that there is a high prevalence of beta-haemolytic streptococci in school children in certain parishes in Grenada. Public health measures should address prevention and control of beta3-haemolytic streptococcal infection in order to prevent the possible sequelae of this disease.

Biological characterization of Aeromonas spp. isolated from the environment.

Cytotoxic enterotoxin (Act) is a key virulence factor in the pathogenesis of infections caused by Aeromonas spp. The cytotoxic enterotoxin gene (act) was detected in 32 out of 69 environmental isolates of Aeromonas spp. by hybridization with the act gene probe. To evaluate the pathogenic potential of the act gene probe-positive isolates, 32 act gene probe-positive and 31 randomly selected act gene probe-negative isolates were tested for enterotoxicity in a suckling mice assay (SMA), for haemolytic activity on sheep blood agar plates, for the presence of CAMP-like factors, and for cytotoxicity in a Vero cell line. The act gene probe-positive isolates significantly differed from the toxin gene probe-negative ones with respect to enterotoxicity in the SMA (P=0.009) and haemolytic activity (P=0.005). The CAMP-haemolysin phenotype was significantly associated with the rabbit ileal loop assay (P= 0.08), Vero cell assay (P= 0.064), and haemolysin production under the microaerophilic conditions (P= 0.056) of the act gene probe-positive isolates of Aeromonas spp. These data indicated the role of Act in the pathogenesis of Aeromonas infections and that the enterotoxic potential of Aeromonas spp. could be assessed by simply performing a CAMP-haemolysin assay.

ENTEROCOCCAL ISOLATES FROM RAW MILK AND DAIRY PRODUCTS IN RIYADH REGION AND THEIR SUSCEPTIBILITY TO COMMON ANTIBIOTICS

A total of 100 samples of raw milk, various cheeses, labnah, yogurt, and egett were collected from appropriate suppliers and markets in Riyadh region. Bacteriological analysis for typing of enterococci and other lactic Acid Bacteria (LAB) was carried out by plating appropriate dilutions of each sample on sheep blood agar and Edwards blood agar plates. After overnight aerobic incubation at 37°, the presumptive identification was done by colony morphology, cultural characteristics, Gram-stain and catalase production. Final identification to the genera and species level of the total 125 bacterial isolates was completed by API-20 strips as well as Lancefield-serogrouping. Results revealed that Enterococcus faecium (88 isolates) accounts of 70% of total bacterial isolates, while Enterococcus faecalis (26 isolates) accounts of up to 21% and other LAB constituted about 9% of total recovered isolates. The later isolates comprises 3, 3, and 5 isolates of Enterococcus gallinarum, Enterococcus durans, and Aerococcus viridans respectively. The results revealed that nature of sample, its pH, and salinity clearly affect the incidence and number of recovered bacterial isolates. Thus as pH rises towards neutrality, with no salt or low salinity, E. faecalis and other LAB were recovered more frequently, and vice versa. In contrast, E. faecium was routinely isolated from most of the examined samples regardless of their pH range and salinity- content, reflecting its ubiquitous nature and its tolerance to drastic environmental conditions, thereby facilitating person to person transmission. The dominance or persistence of enterococci in examined samples is most probably attributed to their wide range of growth temperatures, their tolerance to heat, salt and acid. In addition, the MIC of each of the tested 120 isolates was determined by serial dilution in Muller Hinton sheep blood agar against 9 antibiotics. All isolates were sensitive to ampicilin with the exception of one E. faecalis strain that showed an MIC of 4 ug/ml. While Erythromycin (EM) exhibited also a good activity with an MIC50/MIC90 of 1/1, 1/4, 2/8 and 4/4 ug/ml. for E. gallinarum, E. faecalis, E. faecium, and E. durans or Aerococcus viridans isolates respectively. Whereas all isolates were resistant to cefoxitin and about 50% were also resistant to Chloramphenicol (CM), Tetracycline (TC), or Trimethoprim / Sulfamethoxazole (SXT). Thus at the breakpoint of MIC (>16 ug/ml.) (TC) resistance rate for E. faecalis was 16% and for E. faecium and E. gallinarum was 35% and 100% respectively. Whereas that for vancomycin (VM) the figures were 44%, 19% and 100% respectively. It is concluded that the examined samples may constitute a potential source for the dissemination of antibiotic resistant determinants to human.