Selective Blood Agar Research Articles

Microbiological Studies on Clostridium Perfringens Type (A)

Background:Clostridium perfringens, a rapid-growing pathogen known to secrete an arsenal of >20 virulent toxins, has been associated with intestinal diseases in both animals and humans throughout the past century. Gas gangrene is synonymous with myonecrosis and is a highly lethal infection of deep soft tissue, caused by Clostridium species, with Clostridium perfringens being the most common. Clostridium perfringens is one of the most common cause’s gas gangrene that is associated with broadspectrum antibiotic treatment. The objectives of the present article were focusing on the diagnosis of Clostridium perfringens from wound swabs from patients suffering from gas gangrene associated with antibiotics treatment and determining the best antibiotics that can inhibit the growth of Clostridium perfringens.Materials and methods: Four hundred isolates were collected from Fever Abbassia hospital in Cairo, Egypt. Clinical samples were collected from swab wounds of limbs. Samples were taken during the period of the first of Oct 2017 till the end of Oct 2018. From four hundred cases collected, Two hundred twenty-eight identified as gas gangrene (57%), One hundred twenty-eightidentified as Clostridium perfringens type(A) (56.1%) confirmed by biochemical tests (Gram stain, Catalase test, Motility test, Skim milk test, Urease test, Indole test, Nagler reaction.) and Multiplex PCR. In this study, we are used different media such as (Thioglycalate broth, Reinforced Clostridial Agar, Blood agar, TryptoseSulfiteCycloserine Agar, Robertson's cooked meat media). The antibiotic sensitivity against CL.perfringens is assayed by two methods, disc diffusion Method and Determination of (MIC) using E-Test by following the reference standards established by the Clinical and Laboratory Standards Institute ( CLSI) and British Society for Antimicrobial Chemotherapy (BSAC). The antimicrobial susceptibility test was performed for all of the isolates (128) by disc diffusion method using antibiotics (Chloramphenicol (30 μg /disc), Cefoxitin (10 μg /disc), Clindamycin (2 μg /disc), Metrondiazole (5 μg /disc), Tetracyclin (30 μg /disc), Rifampin (5 μg /disc), Penicillin (10 μg /disc) and Piperacillin (30 μg /disc) and by E- test (MIC) (0.016- 256µg/1ml) method using antibiotics (Chloramphenicol, Cefoxitin, Clindamycin, Penicillin G, Tetracycline and Piperacillin) on blood agar and incubate anaerobically in Anaerobic jar for 24 hrs. at 37 ˚C. As Rifampin and Metrondiazole breakpoints (E-Test) for Clostridium perfringenhave not yet been established by either EUCAST or CLSI.Results and conclusion: In this study, isolates Clostridium perfringens from selective media TSC Agar or Blood agar and doing biochemical tests, the antibiotics susceptibility test showing that disc diffusion method and MIC method give the same result. Susceptibility tests were done on (128) isolates of CL.perfringens by E-Test to determine MIC.Our results revealed that the percentage of sensitivity of CL.perfringens to( Chloramphenicol , Cefoxitin, Clindamycin, Rifampin, Penicillin G, Metrondiazole and Tetracyclin) are (53.9% , 53.1% , 52.3 % , 41.4 % , 35.9 % , 21.1 % , 14.1 % ) respectively . On the other hand, the data revealed that CL.perfringens was resistant to PRL ( 100%). Future advanced research should participate in the prevention of C. perfringens gas gangrene outbreaks

Individual hosts carry H. pylori isolates with different cagA features - motifs and copy number.

H. pylori strains with different genetic contents may infect different or an individual human host. Genetic diversity of cagA is thought to contribute to differences in H. pylori strains pathogenicity. In this study, diversity of cagA genotype, EPIYA motif and copy number was assessed in H. pylori single colonies isolated from individual patients. Gastric biopsies from 14H. pylori-positive dyspeptic patients were cultured on selective brucella blood agar and incubated at 37°C under microaerobic conditions. Four single colonies were obtained from each biopsy subculture on brucella blood agar under similar incubation condition. Presence of cagA and types of EPIYA motifs was determined by polymerase chain reaction (PCR) and cagA copy number by quantitative real-time (RT) PCR. Single colonies of 5 patients showed no variation in cagA genotype, EPIYA motif and copy number. Out of the remaining 9 patients, 1 patient showed presence or absence of cagA gene, 2 patients had mixed EPIYA motifs, 2 patients had different cagA copy number, 1 patient showed absence or presence of cagA and mixed motifs, 2 patients had cagA genes with different nucleotide sequences, 1 patient showed presence or absence of cagA and difference in cagA nucleotide sequence. Four isolates that contained multiple copies of cagA, carried EPIYA-ABC motif. Genetic diversity of cagA among single colonies isolated from individual patients represents evidence that gastric mucosa of every individual is colonized with a specific and heterogeneous population of H. pylori. Future studies on patients in different disease groups may elucidate the role of mixed populations of H. pylori in development of gastric diseases.

Characterization of Pasteurella multocida isolates recovered from the oral flora of cats

In this study, it was aimed to investigate the presence of Pasteurella multocida in the swab samples taken from the oral cavity of cats, and to determine the capsular type and antimicrobial susceptibility of the isolates. For this purpose, swab samples taken from 300 cats were inoculated onto Knight's selective enriched medium and blood agar for isolation of P. multocida. Followingafter capsular typing of the isolates by PCR, the susceptibilities of the isolates to ceftiofur, clindamycin, erythromycin, enrofloxacin, amoxicillin+clavulanic acid, trimethoprim/ sulfamethoxazole, tetracycline, and chloramphenicol were examined by the disk diffusion method. The relationships between oral colonization and various physiological and behavioral variables were evaluated, statistically. P. multocida was isolated from 48 (16%) of the samples and all strains were typed as capsular type A. While all of the isolates were resistant to clindamycin and susceptible to enrofloxacin, 8,33% tetracycline resistance was also remarkable. Multiple antibiotic resistance (MDR) was detected in 27% of the isolates. The colonization of P. multocida was found to be statistically significant in cats under the age of 1 and in cats living outdoors. The importance of antibiotic resistance observed in bacteria with a zoonotic character such as P. multocida should not be ignored since it poses a threat to public health. It was concluded that, in this study determination of tetracycline resistance, which has started to be reported in feline Pasterurella multocida isolates in recent years, and detection of multiple antibiotic resistance in 27% of isolates, was extremely important for public health.

2704. Molecular Technology to Detect Pneumococcal Colonization in Young Children Reveals Increased Prevalence of Vaccine Serotypes as Compared with Enhanced Culture Methods

BackgroundHuman challenge studies demonstrate enhanced sensitivity of molecular technology for identification of vaccine serotype pneumococcal (SP) carriage in PCV13 immunized adults. We hypothesized that PCV13 immunized children would similarly harbor vaccine serotypes in their nasopharynx (NP) that could only be identified by molecular technology.MethodsWe compared use of enhanced microbiologic culture vs. molecular technology to characterize SP colonization among NP swabs collected from 995 healthy or sick children <5 years old at Boston Medical Center from November 2015 to May 2017. NP specimens were broth enriched for 4 hours and cultured on selective blood agar. Specimens were evaluated for presence of SP using both routine microbiologic methods and RT–PCR. RT–PCR assays targeted the lytA, and piaB (SP membrane permease) genes, and 26 SP serotypes: all serotypes included in 13-valent pneumococcal conjugate vaccine and 13 prevalent non-vaccine serotypesResultsA total of 162 (16.3%) NP specimens were positive for SP via enhanced culture, and an additional 163 (16.3%) were SP positive via lytA+ RT–PCR molecular technology. Prevalence of SP carriage was equivalent in children aged 0<2 years and 2≤5 years, but greater in children with respiratory tract infections (RTI) compared with children without RTI (26.5% vs. 9.6% among culture+ specimens only; and 43.2% vs. 25.8% among combined culture+ and molecular+ specimens). Using enhanced culture only, vaccine serotypes (VST) were identified in 4 (1%) of 450 children <2 years and 14 (2.6%) of 545 children 2 ≤5 years; adding molecular positive specimens increased the prevalence of VST to 2.9% in children <2 years and 4.6% in children 2 ≤5 years (table). Serotypes 3 and 19A were the two most commonly identified VST.ConclusionCombining molecular technology with enhanced culture reveals an increased prevalence of vaccine serotype colonization in young children. The ability of sensitive molecular methods to detect vaccine serotypes in culture-negative specimens suggests low-density vaccine serotype carriage persists in a highly immunized pediatric population. The importance of culture negative but RTPCR positive carriage for transmission requires further evaluation. Disclosures All authors: No reported disclosures.

Enhanced Detection of Vaccine Type Colonization and Dual Serotype Carriage with Molecular Strategies for Identification of Streptococcus pneumoniae Colonization

BackgroundDetecting Streptococcus pneumoniae (SP) carriage in children via conventional microbiological methods lacks sensitivity as high density is required for routine culture and identification of dual serotype colonization is a technical challenge. To increase understanding of post vaccine nasopharyngeal (NP) carriage, specifically persistence of vaccine serotypes, strategies for molecular identification of SP have been developed. These methods most often rely on the identification of the SP autolysin gene lytA through non-quantitative PCR or semi-quantitative real-time PCR (RTPCR).MethodsWe collected NP swabs from 600 healthy or sick children <5 years old at Boston Medical Center from Nov 2015 – Mar 2016 and used enhanced microbiologic culture and molecular identification strategies to characterize SP colonization. NP specimens were broth enriched for 4 hours and cultured on selective blood agar; routine microbiologic methods were used to isolate and identify SP. A second aliquot of enriched broth was used for DNA isolation. RTPCR assays were performed targeting the lytA, piaB (SP membrane permease), cpsA (SP capsule operon) genes, and 21 SP serotypes: all serotypes included in 13-valent pneumococcal conjugate vaccine and 8 additional non-vaccine serotypes.ResultsIn our sample, 16% of specimens were SP positive via culture and 33% were RTPCR-positive (Cq≤35) for the lytA gene. Multiplex RTPCR assays with both the lytA and piaB genes yielded a positive result for 24% of samples. Further RTPCR confirmed that 99% of the lytA/piaB positive samples were positive for cpsA (a second marker for assay validation) but only 70% of the lytA positive, paiB negative samples were cpsA positive. Serogroup 19 was the most frequently isolated vaccine serogroup using both culture and RTPCR; molecular analysis identified 6% of specimens with concurrent carriage of more than one serotype.ConclusionCompared with enhanced culture, we found a 50% increase in SP detection using combined lytA and piaB multiplex RTPCR. Similarly, the proportion of children colonized with vaccine serotypes increased from 2% to 7%. This work is funded by an investigator initiated grant to BUMC from Pfizer.Disclosures K. M. Shea, Pfizer, Inc: Consultant and Grant Investigator, Consulting fee and Grant recipient; S. I. Pelton, Pfizer: Board Member and Grant Investigator, Consulting fee, Research grant and Speaker honorarium; Merck vaccines: Board Member, Consulting fee and Speaker honorarium; GSK: Board Member, Consulting fee and Speaker honorarium; Sequiris: Board Member, Consulting fee and Speaker honorarium

Antimicrobial Efficacy of Various Essential Oils at Varying Concentrations against Periopathogen Porphyromonas gingivalis.

Porphyromonas gingivalis (P.gingivalis) is a notorious perio-pathogen with the ability to evade host defense mechanism and invade into the periodontal tissues. Many antimicrobial agents have been tested that curb its growth, although these agents tend to produce side effects such as antibiotic resistance and opportunistic infections. Therefore search for naturally occurring anti-microbials with lesser side effects is the need of the hour. The aim of this study was to substantiate the antimicrobial activity of various essential oils; eucalyptus oil, chamomile oil, tea tree oil and turmeric oil against P. gingivalis. Pure cultures of P. gingivalis were grown on selective blood agar. Antimicrobial efficacy of various concentrations of essential oils (0%, 25%, 50% and 100%) was assessed via disc diffusion test. Zone of inhibition were measured around disc after 48 hours in millimeters. Zones of inhibition were directly proportional to the concentration of essential oils tested. At 100% concentration all the tested oils possess antimicrobial activity against P.gingivalis with eucalyptus oil being most effective followed by tea tree oil, chamomile oil and turmeric oil. All essential oils tested were effective against P.gingivalis. After testing for their clinical safety they could be developed into local agents to prevent and treat periodontitis.

Different distribution of Helicobacter pylori EPIYA- cagA motifs and dupA genes in the upper gastrointestinal diseases and correlation with clinical outcomes in iranian patients.

Our aim was to determine the EPIYA-cagA Phosphorylation sites and dupA gene in H. pylori isolates among patients with upper gastrointestinal diseases. Pathogenicity of the cagA-positive Helicobacter pylori is associated with EPIYA motifs and higher number of EPIYA-C segments is a risk factor of gastric cancer, while duodenal ulcer-promoting gene (dupA) is determined as a protective factor against gastric cancer. A total of 280 non-repeated gastric biopsies obtained from patients undergoing endoscopy from January 2013 till July 2013. Samples were cultured on selective horse blood agar and incubated in microaerophilic atmosphere. The isolated organisms were identified as H. pylori by Gram staining and positive oxidase, catalase, and urease tests. Various motif types of cagA and the prevalence of dupA were determined by PCR method. Out of 280 specimens, 128 (54.7%) isolated organisms were identified as H. pylori. Of 120 H. pylori isolates, 35.9% were dupA positive and 56.26% were cagA positive, while cagA with ABC and ABCC motifs were 55.5% and 44.5%, respectively. Fifty six percent of the isolates with the ABCC motif have had dupA genes. We also found a significant association between strains with genotypes of dupA-ABC and duodenal ulcer disease (p = 0.007). The results of this study showed that the prevalence of cagA-positive H. pylori in Shiraz was as high as in western countries and higher numbers of EPIYA-C segments were seen in gastric cancer patients. We may also use dupA as a prognostic and pathogenic marker for duodenal ulcer disease and cagA with the segment C for gastric cancer and gastric ulcer disease in this region.

Evaluation of Antimicrobial susceptibility pattern of Streptococcus mutans isolated from dental plaques to chlorhexidine, nanosil and common antibiotics

Streptococcus mutans is one of the most important leading causes of dental diseases worldwide and is considered as one of the main causative agent of dental caries. Increasing resistance of oral pathogens to conventional antibacterial agents has resulted to find alternative therapies to overcome resistance development problems. The aim of this study was to examine susceptibility of Streptococcus mutans isolates to some antibiotics, chlorhexidine and nanosil. The study subjects comprised of caries active individual volunteers attending the outpatient department of different dental college hospitals of Kerman. The saliva sample of 2-5 ml was collected from each individual in sterile capped bottles and were immediately transported to the laboratory and processed for the screening of S. mutans. The specific selective media, Blood agar and Tryptone yeast cysteine media agar were used for the screening and isolation of Streptococcus mutans and incubated anaerobically at 37?C for 48 hrs. Colonies of mutans streptococci were examined under a dissecting microscope and identified by their distinctive colony morphology and biochemical tests .Mutacin production of all isolates were investigated.The isolates were tested for susceptibility to some antibiotics (penicillin, gentamycin, vancomycin, cephalotin), chlorhexidine and nanosil mouthwashes by disc diffusion method. The results showed that the majority of mutacin and non mutacin-producing isolates were more sensitive to the nanosil than antibiotics and chlorhexidine. More extensive research on the use of mouthwashes containing silver nanoparticles is suggested.DOI: http://dx.doi.org/10.3126/ijls.v9i2.12040 International Journal of Life Sciences 9 (2) : 2015; 18-21

Isolation and Identification of Dermatophilus in Bovine from Andhra Pradesh

A total of 727 samples (148 cattle, 579 buffalo) collected from different districts of Andhra Pradesh during the period from June 2010 to May 2011were subjected for isolation of Dermatophilus by using modified Haalstra`s method. On Gram’s staining revealed Gram positive, branching and filamentous organisms and are less visible but characteristic tram-track appearance was clearly visible in Giemsa and Methylene blue staining. The isolated agents were motile and appeared as grayish-white, small, beta haemolytic colonies on selective polymyxin blood agar media. The organisms were positive for catalase, oxidase and citrate utilization tests and produced acid with out gas. Gelatin liquefaction test showed the virulence of the isolates. The isolated agents were found sensitive to Penicillin G, Doxycyclin hydrochloride, Ceftriaxone and resistant to Kanamycin and Chloromphenicol.The isolates were 100% sensitive to Pencillin G, Doxycycline and Ceftriaxone and can be used for the treatment of animals in Dermatophilosis.

Pasteurellaceae isolated from bighorn sheep (Ovis canadensis) from Idaho, Oregon, and Wyoming

To elucidate the species and biovariants of Pasteurellaceae isolated from clinically normal bighorn sheep (Ovis canadensis) or bighorn sheep with evidence of respiratory disease. 675 Pasteurellaceae isolates from 290 free-ranging bighorn sheep in Idaho, Oregon and Wyoming. Nasal and oropharyngeal swab specimens were inoculated onto selective and nonselective blood agar media. Representatives of each colony type were classified via a biovariant scheme. The association of respective β-hemolytic isolates with respiratory disease was evaluated via χ(2) analyses. Bacterial isolates belonged to 4 species: Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia (Pasteurella) trehalosi. Within the latter 3 species, 112 subspecies, biotypes, and biovariants were identified. Bibersteinia trehalosi 2 and B trehalosi 2B constituted 345 of 675 (51%) isolates. Most (597/618 [97%]) isolates from adult sheep were from clinically normal animals, whereas most (47/57 [82%]) isolates from lambs were from animals with evidence of respiratory disease. Twenty-two Pasteurellaceae biovariants were isolated from sheep with respiratory disease; 17 of these biovariants were also isolated from clinically normal sheep. The ability of isolates to cause β-hemolysis on blood agar was associated with respiratory disease in adult bighorn sheep (OR, 2.59; 95% confidence interval, 1.10 to 6.07). Bighorn lambs appeared more susceptible to respiratory disease caused by Pasteurellaceae than did adult sheep. β-Hemolytic Pasteurellaceae isolates were more likely to be associated with respiratory disease than were non-β-hemolytic isolates in adult sheep. Identification of Pasteurellaceae with the greatest pathogenic potential will require studies to estimate the risk of disease from specific biovariants.

PWE-166 Increasing VacA toxin activity alters Helicobacter pylori colonisation density and the nature of the acquired immune response in a mouse model of infection

IntroductionHelicobacter pylori (Hp) infection causes chronic asymptomatic gastritis, and may lead to peptic ulceration and gastric cancer. Hp strains expressing more active forms of the vacuolating cytotoxin VacA are more...

Comparison of BD GeneOhm real-time polymerase chain reaction with chromogenic and conventional culture methods for detection of group B Streptococcus in clinical samples

A total of 200 antenatal high vaginal swabs were screened for the presence of group B Streptococcus (GBS) using a conventional culture method (recommended by Centers for Disease Control and Prevention). Screening was also performed by using a new chromogenic agar, chromID Strepto B, and by using the BD GeneOhm StrepB real-time polymerase chain reaction (PCR), which was performed directly on swabs without enrichment. Using a combination of all methods, we detected GBS in 101 samples. A total of 82 samples (81.2%) were positive using PCR, and 83 samples (82.2%) were confirmed as positive by culture (any method). PCR was more sensitive for detection of GBS than direct culture using any method ( P < 0.0005). PCR was also more sensitive than any single enrichment method, but this difference was not statistically significant. With culture as a “gold standard”, the PCR method showed a sensitivity of 77.1% and a positive predictive value of 79.3%. Of the culture-positive samples, significantly, more GBSs were detected by direct plating on chromID Strepto B than on selective sheep blood agar (67.5% versus 57% respectively, P < 0.02). After selective enrichment, 92.8% of GBS were isolated on chromID Strepto B compared with 89.2% isolated on sheep blood agar.

DETECTION THE OCCURRENCE OF HELICOBACTER PYLORI AMONG DYSPEPTIC PATIENT AND THEIR SUSCEPTIBILITY TO ANTIBIOTIC AGENTS

Fifty dyspeptic patient include 31 female and 19 male were subjected to esophageal gastroduodenoscopy and gastric biopsy and gastric biopsy specimens were taken from antrum and body were applied for microbiological analysis which include : urease test bacterial culture using selective and non selective media and histological examination for detecting H. pylori, and studying their susceptibility to various to antimicrobial agents. Results showed that occurrence of H.pyloria was (100%) among gastric ulcer patient,71% among duodenal ulcer and these with normal endoscopic appearance, as well as, (70%) of gastric patient were found to be infected with H.pylori. Biopsy urease test were proved to be most sensitive method for detecting H.pylori infection when 76% gave positive result for presence of H.pyloria using it, followed by histological examination when 70% of gastric tissues showed the existence of H.pylori organisms. Results also showed that using selective media was batter than not selective media for primary isolation of bacteria, with a recovery rate 61% using brain heart infusion agar and 39% was recovery rate for non selective blood agar. When isolates of H.pylori were subjected to the sensitivity test against (8) antibiotic groups, result, showed that ciprofloxacin was the most effective antibiotic against the isolates when the sensitivity percentage 90%pencilin G,tetracycline, amoxicillin and metronidazal on other hand, were the last Effective antibiotic antibiotics when bacterial sensitivity range between 30% and 90%.

Comoarison of PCR and bacterial culture methods for diagnosis of dairy cattles subclinical submastitis caused by staphylocuccus aureus

To compare PCR and bacterial culture methods for diagnosis of subclinical mastitis caused by Staphylococcus aureus, 100 milk samples from cattle with subclinical mastitis and 20 samples from healthy cattle were collected and tested. The samples were cultured on selective blood agar and bacteria were identified by standard methods. DNA extracted from samples was subjected to PCR reaction with species specific primers and PCR products were analyzed by agarose gel electrophoresis. Based on the PCR results the prevalence of subclinical mastitis due to S. aureus was 25%. In the bacteriological culture of single milk sampling, S. aureus was isolated from the same samples being positive in PCR. A correlation of 100% was found between PCR and single milk sampling culture method by Mc Nemar test. All of the CMT negative samples were also negative in culture and PCR methods. The results of this study indicate that the PCR reaction is sensitive and specific for diagnosis of S. aureus in subclinical mastitis and can detect this pathogen in milk samples at species level in few hours.

Real-time PCR targeting the sip gene for detection of group B streptococcus colonization in pregnant women at delivery

Group B streptococcus (GBS) is an important aetiological agent of serious neonatal infections. A rapid and sensitive method for the detection of GBS colonization in pregnant women at delivery could make intrapartum screening for GBS possible. A real-time PCR method targeting the sip gene of GBS in pregnant women at delivery has been evaluated. The performance of the real-time PCR was compared with optimized GBS culture. Separate vaginal and rectal swabs were collected from women hospitalized at the delivery department at St Olavs Hospital, Trondheim, Norway, from January 15 through May 2005. The specimens were cultured on selective blood agar plates and in selective broth and examined by real-time PCR. Of samples from 251 women, 87 (34.7%) were GBS positive by culture and 86 (34.3%) were positive by PCR. Using GBS culture as the 'gold standard', the sensitivity of real-time PCR was 0.97 (95% confidence interval 0.90-0.99) and specificity was 0.99 (95% confidence interval 0.97-1.00). In two women the PCR was positive and the culture negative. Additional analysis using cylE PCR substantiates that these two women were true GBS carriers with negative GBS culture. The rate of GBS colonization was lower in vaginal specimens than in rectal specimens both by culture and PCR. The real-time PCR assay is fast, highly sensitive and specific for detecting GBS colonization in pregnant women at delivery, and has the potential for intrapartum detection of GBS colonization. Both vaginal and rectal samples are required to achieve highest possible detection rate.

Rapid detection and identification of Brachyspira aalborgi from rectal biopsies and faeces of a patient

This study reports for the first time the detection of Brachyspira aalborgi in faeces and rectal biopsies of a female suffering for 3–4 months of abdominal pain with long-standing mucosal diarrhoea, rectal bleeding and suspected carcinoma of the rectum. After pre-treatment of samples (faeces and biopsies) with a liquid medium (trypticase soy broth—TSB) containing foetal calf serum (FCS, 10%) and spectinomycin and rifampicin (TSB-SR) the first detection of B. aalborgi isolate HBS1 was observed after 48 h in the primary plates of selective blood agar modified medium (BAM) containing spectinomycin and rifampicin (BAM-SR), where growth zones were signalled by a small weakly beta-haemolytic halo. Attempts to subculture spirochaetes in agar media failed. The new HBS1 isolate was only propagated in TSB broth and at electron microscopy it showed 4 endoflagella inserted at each tapered end. The phenotypic characterization of HBS1 demonstrated absence of hippurate hydrolysis, indole production, α-galactosidase, α- and β-glucosidase activities in accordance with the B. aalborgi type strain. Rapid identification of B. aalborgi isolate HBS1 was performed directly from faeces and rectal biopsies and subsequently from pure cultures by a genetic method based on 16S DNA restriction fragment length polymorphism (RFLP)—polymerase chain reaction (PCR). The sequence of 16S DNA amplicon of the isolate HBS1 was found 99.2% corresponding to that of the B. aalborgi type strain. Our results encourage further investigations for the development of a suitable selective agar medium for the isolating and cultivating B. aalborgi from human specimens.

A novel method for isolation of Brachyspira (Serpulina) hyodysenteriae from pigs with swine dysentery in Italy

Brachyspira (Serpulina) hyodysenteriae was isolated from 10 of 11 pigs with clinically suspected swine dysentery in six herds in northern Italy. All strains were successfully isolated in the selective blood agar modified medium with spectinomycin and rifampin (BAM-SR) currently used in our laboratory to isolate B. (S.) pilosicoli of human origin, after pre-treatment of intestinal material with spectinomycin and rifampin in foetal calf serum. Isolates had phenotypic characteristics typical of B. (S.) hyodysenteriae.

Survival of Helicobacter pylori in ready-to-eat foods at 4°C

The survival of Helicobacter pylori (NCTC 11638) in various semiprocessed and fresh, ready-to-eat foods, and one raw chicken was studied at 4°C and under aerobic conditions by experimentally inoculating these with 10 4 CFU. Cells were concentrated by two centrifugation cycles followed by plating onto selective blood agar medium made from Wilkins-Chalgren agar supplemented with 5% whole horse blood, and 30 mg/l colistin methanesulfonate, 100 mg/l cycloheximide, 30 mg/l nalidixic acid, 30 mg/l trimethoprim, and 10 mg/l vancomycin. H. pylori was recovered from spiked pasteurized milk and tofu samples up to 5 days and from spiked leaf lettuce and raw chicken up to 2 days. H. pylori could not be recovered from yogurt after any length of storage time. H. pylori is unlikely to grow in foods; however, it may survive in low acid–high moisture environments under refrigeration and pose a possible risk for transmission of infection via foods.

Haemolysin-deficient variants of Streptococcus pyogenes and S. dysgalactiae subsp. equisimilis may be overlooked as aetiological agents of pharyngitis.

Variants of large colony beta-haemolytic Lancefield group A, C and G streptococci that are non-haemolytic or alpha-haemolytic on sheep blood agar have been detected in clinical specimens due to their enhanced haemolytic activity when grown on a new selective and differential blood agar medium containing colistin, nalidixic acid and pH 7.5-adjusted PIPES buffer (CNA-P). The large colony Lancefield group C and G isolates were identified as Streptococcus dysgalactiae subsp. equisimilis by API 20 Strep classification and 16S rDNA profiling. The haemolytic activity of these variants on various blood agar media, including CNA-P, was closely similar to that of known streptolysin S-defective mutants of S. pyogenes and was blocked by addition of cholesterol, a specific inhibitor of the streptolysin O family of haemolysins. As haemolysin variants could be detected in large numbers in cultures from patients with clinical symptoms of pharyngitis it is suggested that they may function as primary pathogens in such infections. The high frequency with which haemolysin variants were isolated from clinical specimens during a 3-month trial (3%, 13% and 10%, respectively, of group A, C and G streptococcal isolates) indicated that a substantial proportion of streptococcal infections may go undetected if only conventional sheep blood agar media are used in clinical laboratories for the detection of beta-haemolytic streptococci. As haemolysin variants have been implicated in the development of serious streptococcal sequelae, further investigation of the full extent of their contribution to streptococcal disease is indicated.

Houseflies are an unlikely reservoir or vector for Helicobacter pylori.

The route of transmission of Helicobacter pylori from individual to individual remains undefined. It has recently been reported that the domestic housefly, Musca domestica, when fed pure cultures of H. pylori, was able to harbor the organism in its midgut for up to 30 h (P. Grubel, S. Hoffman, F. K. Chong, N. A. Barstein, C. Mepani, and D. R. Cave, J. Clin. Microbiol. 35:1300-1303, 1997). Our investigation examined whether houseflies could acquire H. pylori from fresh human feces. Domestic houseflies (40 flies/group) were exposed for 24 h to feces from an H. pylori-positive volunteer, feces from an H. pylori-negative volunteer, or feces from an H. pylori-negative volunteer to which a known amount of viable H. pylori had been added. At various intervals, flies were sacrificed and the midguts were excised, homogenized, and plated in duplicate onto selective horse blood agar plates. All plates were incubated under microaerobic conditions at 37 degreesC for 14 days. Emergent colonies presumptive of H. pylori were picked and tested biochemically to confirm the identity as H. pylori. H. pylori was not recovered from houseflies fed human feces either naturally infected or artificially infected with H. pylori. These results suggest that the domestic housefly is not a vector for transmission or a reservoir for H. pylori infection.