Skirrow Agar Research Articles

Placentitis and abortion caused by a multidrug resistant strain of Campylobacter fetus subspecies fetus in a sheep in Uruguay

Campylobacter fetusfetus (Cff) is a major infectious cause of abortion in sheep worldwide, and an opportunistic human pathogen. Information on Cff as an ovine abortifacient in South America is limited. We describe a case of abortion caused by a multidrug resistant strain of Cff in a sheep in Uruguay. In August 2017, 3/57 pregnant ewes (5.3%) aborted whithin one week. Histopathologic examination of the placenta of an aborted ewe revealed severe neutrophilic and fibrinonecrotizing placentitis with vasculitis and thrombosis of the chorionic arterioles. Cff was isolated on microaerobic culture in Skirrow agar, and further confirmed by 16S rDNA PCR amplification and sequencing, and endpoint and real time PCR assays. Antimicrobial sensitivity testing revealed resistance to tetracyclines, nalidixic acid, telithromycin and clindamycin. Other abortifacients were not detected. Further studies are necessary to determine the geographic distribution, ecology, epidemiology, economic impact, and antimicrobial resistance of Cff in sheep flocks in Uruguay.

Clinical sensitivity and specificity of a real-time PCR assay for Campylobacter fetus subsp venerealis in preputial samples from bulls.

To determine clinical sensitivity and specificity of a quantitative real-time PCR (qRT-PCR) assay for Campylobacter fetus subsp venerealis (Cfv) in preputial samples of bulls. 313 beef bulls. Preputial samples were collected from 300 virgin bulls and 13 Cfv-infected bulls. Specificity of the qRT-PCR assay, determined on the basis of results for samples collected from virgin bulls, was compared with specificity of bacteriologic culture performed with transport enrichment medium (TEM). Sensitivity of the qRT-PCR assay, determined on the basis of results for multiple samples collected at weekly intervals from infected bulls, was compared with sensitivity of the direct fluorescent antibody test (DFAT), bacteriologic culture, and bacteriologic culture with TEM. Specificity was 85% for the qRT-PCR assay and 100% for bacteriologic culture; results were significantly different. Mean sensitivity was 85.4% for the qRT-PCR assay, 82.3% for direct culture in blood agar, 72.1% for the DFAT, 32.7% for direct culture in Skirrow agar, 30% for bacteriologic culture with TEM and blood agar, and 38.1% for bacteriologic culture with TEM and Skirrow agar. Differences in sensitivity among tests varied with ambient outdoor temperature. Repeated sampling significantly increased sensitivity of the qRT-PCR assay. Use of the qRT-PCR assay as a screening test on direct preputial samples had comparable sensitivity to bacteriologic culture, and repeated sampling improved sensitivity. Although improved performance of the qRT-PCR assay, compared with direct bacteriologic culture, was dependent on temperature, transport times that allow direct culture are unlikely under field conditions. The qRT-PCR assay would provide a fast and sensitive screening method for Cfv in bulls.

RELATIONSHIP BETWEEN QUANTITATIVE 13C BREATH TESTING AND QUANTITATIVE H. PYLORI CULTURE BY ENDOSCOPY FOR ITS USEFULNESS AS AN EARLY ASSAY OF ANTIBIOTIC EFFECT

Purpose: To quantify, compare, and correlate non-invasive and invasive diagnostic assays for assessing degrees of H. pylori infection for use in antibiotic development. Methods: Volunteers (N = 152, 100%) were screened for circulating IgG antibodies to H pylori using the QuickVue H. pylori gII test and, if positive (N = 42,27.6%), further screened with13 C urease breath test (UBT) (N = 20, 13.2%). UBT data were obtained at baseline, 15, 30, 45, and 60 min. post po administration of 13C; fifteen patients were positive (9.9%). Urease activity was calculated as umolesof 13CO2 formed/minute at time points (15, 30,45, 60 min). Urease activity was highest at 30 min., consistent with previously published reports. Volunteers with positive results for both screening tests underwent endoscopy (N = 4, 2.6%). Six biopsy samples were obtained, two each from the lesser and greater curvatures of the antrum (GA, LA, 2 cm and 4 cm from pylorus), and two samples from the greater curvature of the corpus (GC, 3 and 5 cmproximal to angulus). Within 2 hours, biopsy samples were homogenized, serially diluted and plated on on Skirrow's agar for H. pylori colony forming units (cfu). H. pylori colonies were counted on Day 3 after plating and cfu/biopsy site was calculated. Correlation analysis was used to relate cfu obtained per biopsy site to urease activity. Results: We report here the preliminary findings of this study which demonstrate good correlation (r2 = 0.93, p = 0.023) between log cfu at biopsy location GA-4cm and 30 minute urease activity rate. However, no correlation with the 30 minute urease activity rate was found with other biopsy sites. Conclusions: A correlation was observed between log cfu and urease activity at 30 minutes on UBT at one of six biopsy sites in this limited sample. We speculate that either inadequate sample size or uneven distribution of H. pylori infection may explain the lack of correlation at other sites. This may be useful in antibiotic therapy development by providing an early indicator of bacterial kill, which could guide the dosing and sequence of component administration in combination therapies. This warrants further study.

Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures.

Usually, bacteria are cryopreserved for short-term storage at low and ultra-low temperatures. There are no reports as to whether Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures as compared with other intestinal bacteria. A study was done on seven H. pylori strains and other intestinal bacteria to compare different temperatures for storage of organisms in saline solution. Seven H. pylori strains, specifically American Type Culture Collection (ATCC) strains 43504 and TN2GF4, and five strains isolated from the present patients were grown on a modified Skirrow's agar for H. pylori. Escherichia coli and Bacteroides distasonis, both representing isolates from the present patients, were grown on trypticase soy blood agar for E. coli, and EG agar for B. distasonis. Culture was for 4-5 days under microaerobic, aerobic and anaerobic conditions at 37 degrees C. Cells were harvested by scraping growth from the solid medium and into sterile saline. The cells were adjusted to concentrations of 109 viable cells/mL in saline and preserved at 4 degrees C, -20 degrees C, or -80 degrees C for 3 weeks before reculture under microaerobic, aerobic and anaerobic conditions at 37 degrees C for 7 days. After incubation, morphologically distinct colonies were counted, isolated, and identified by standard bacteriologic techniques. The H. pylori were morphologically analyzed by electronic microscopy before and after preservation. Mongolian gerbils were inoculated with the cryopreserved H. pylori to evaluate the bacterial infectivity. Six of the seven H. pylori strains failed to culture after being preserved at 4 degrees C, -20 degrees C, or -80 degrees C. Only ATCC 43504 could be cultured after freezing at -80 degrees C. The number of H. pylori ATCC 43504 before preservation was 9.0 +/- 0.5 (log10 no. organisms/mL) and decreased to 5.7 +/- 0.6 after preservation. Morphologically, all H. pylori except ATCC 43504 strains transformed from a bacillary to a coccoid form after preservation. In addition, none of the H. pylori strains could infect Mongolian gerbils after preservation. Escherichia coli and B. distasonis were recovered. Titers before and after 4 degrees C, -20 degrees C, and -80 degrees C, respectively, were 9.1 +/- 0.2, 8.9 +/- 0.5, 8.6 +/- 0.3, and 8.7 +/- 0.3 for E. coli and 9.1 +/- 0.4, 8.7 +/- 0.6, 8.6 +/- 0.5, and 8.8 +/- 0.3 for B. distasonis. Helicobacter pylori is a fragile bacteria for storage at low and ultra-low temperatures in comparison with other intestinal bacteria.

MODIFIED POLYMERASE CHAIN REACTION AND SELECTIVE MEDIA METHODS FOR DETECTION OF HELICOBACTER PYLORI IN PORK LOIN CHOPS1

Abstract Selective media, and a modified polymerase chain reaction (PCR) were evaluated for use in the detection and enumeration of Helicobacter pylori in pork loin chops. The chops were treated with tap water, or H. pylori‐ATCC 43504, packaged in polyvinyl chloride (PVC) film (7 days), or vacuum packaged (24 days), stored at 2C, and analyzed for survival of H. pylori. Modifying the amplification cycles and annealing temperature of the PCR resulted in detection of H. Pylori on inoculated chops. Helicobacter pylori survived for 5, and 10 days on the PVC, and vacuum packaged chops, respectively. Skirrow agar was the most effective medium, when compared to blood agar, and egg yolk emulsion agar.

Incidence of thermotolerantCampylobacterin foods assessed by NF ISO 10272 standard: results of a two-year study

Fifteen hundred food samples were analysed for thermotolerantCampylobacteraccording to the NF ISO 10272 standard during a two-year investigation period using Preston and Park & Sanders methods and three isolation media (Karmali, Butzler and Skirrow agar). The samples, consisting of 660 oysters(Crassostrea gigas), 400 ready-to-eat vegetables, 300 poultry products and 140 raw milk cheeses, were positive 5, 2, 12 and 0 times respectively. All isolates were identified asCampylobacter jejunisubsp.jejuniexcept oneC. coli.Biotype and serotype determination revealed a large diversity among the strains, although biotypes I (59%) and II (18%) were predominant. Serotyping (Lior scheme) showed that 85% of the strains were among the 10 most common Campylobacter serogroups isolated in human campylobacteriosis. Park & Sanders enrichment and isolation on Karmali agar appeared to be the most efficient combination for detecting thermotolerant Campylobacter in the food samples tested.

Resuscitation from the viable but nonculturable state of Helicobacter pylori

It is well known that the change of state from the culturable to not culturable on pathogenic organisms could easily occurred. These states of bacteria were so called viable but nonculturable (VNC). The coccoid form of H. pylori is seems to be in this state. The possibility of resuscitation of H. pylori was examined in vitro. The coccoid form bacteria was treated with Ammonium Sulfate and heat shock before culture, and cultured in Brucella broth containing, sodium pyruvate, laked human erythrocyte and serum. The growth of the rod spiral form was found in the coccoid form bacteria, and further the bacteria could grow on blood agar and Skirrow agar as well as the original strain. It was strongly suggested that the resuscitation from VNC state of cells to culturable form consists of two processes, stimulation and supplemented of appropriate nutrition.

Gastric epithelial cells stimulate Helicobacter pylori growth.

Helicobacter pylori infects only human gastric epithelium, causes gastritis, and is strongly associated with gastroduodenal ulceration and gastric cancer. Colonization of the stomach with H. pylori is accompanied in the acute stage by an increased number of neutrophils in the lamina propria, indicative of gastric inflammation. It is interesting that H. pylori colonizes specifically human gastric-type epithelial cells. We studied whether the presence of gastric epithelial cells influenced H. pylori growth. H. pylori (NCTC 11637) was cultured on Skirrow agar with 7% horse blood. Kato-III cells, a human gastric cancer cell line, were cultured with RPMI 1640 plus 10% fetal bovine serum (FBS). Kato-III cells (10(5)/ml) were cultured with/ without H. pylori (10(8) cfu/ml) with RPMI 1640 + 1% FBS for 3 days. The number of Kato-III cells was counted with a hemacytometer. H. pylori with/without Kato-III cells was cultured with RPMI 1640 + 10% FBS for 2 hours, and plated on Skirrow agar. After 3 days we counted the number of H. pylori colonies. To detect the H. pylori colonies, we used a colony hybridization method. DNA of colonies was transferred to positively charged nylon membrane and hybridized by PCR with Hpl (5'-CTG-GAG-AGA-CTA-AGC-CCT-CC-3') and Hp2 (5'-ATT-ACT-GAC-GCT-GAT-TGT-GC-3')-amplified primers. We previously reported that the number of Kato-III cells was significantly decreased by co-incubation with H. pylori. The number of H. pylori colonies was significantly increased by coincubation with Kato-III cells. We conclude that the presence of human gastric epithelial cells is important for the growth of H. pylori.

Helicobacter pylori in dental plaque and gastric mucosa

Objectives The aim of this study was to investigate the presence of Helicobacter pylori in both dental plaque and gastricmucus. Study design Dental scaling hand instruments were used to collect supragingival and subgingival dental plaque from 81 dentate participants. Denture plaque was obtained from the fitting surfaces of dentures from 41 edentulous patients. Gastric mucus from gastric mucosa of antrum and body of stomach were collected from all 122 participants (92% white) with a soft gastroscopic brush. These samples were dispersed in modified urea broth and normal saline solution before being inoculated onto selective Skirrow's agar and incubated in a microerophilic atmosphere for culturing H. pylori. Results Dental plaque from all dentate participants was negative for H. pylori culture. Only one 80-year-old edentulous patient had positive H. pylori culture in both gastric mucus and denture plaque. Conclusions Although dental plaque has a mixed flora that may act as a reservior for gastric reinfection, dental plaque could not be implicated as the major reservoir of H. pylori for gastric reinfection.

Comparison of Selective Enrichment Broths for Isolation of Campylobacter jejuni/coli from Freshly Deboned Market Chicken

The efficiency of Brucella broth supplemented with vancomycin-trimethoprim-polymyxin B (VTP) at the same concentration as Skirrow agar and bacitracin-colistin-cephalothincycloheximide-novobiocin (BCN) at the same concentration as Butzler agar was evaluated in the recovery of Campylobacter jejuni/coli from fresh retail chicken. Ninety-two samples were acquired from retail markets in Guadalajara, Mexico, and analyzed by enrichment in VTP and BCN broths, each streaked on Skirrow and Butzler agars. Thirty-three (36%) of the samples were positive by any route of analysis. The recovery of Campylobacter (19.6% in VTP alone, 25.0% in BCN, and 35.9% in VTP+BCN) was enhanced by simultaneously using two instead of only one enrichment broth, especially when VTP broth was used (p<0.05). There was an elevated rate of samples containing Campylobacter which gave false-negative results on VTP (45.2% of false-negatives) and on BCN (30.3%). While isolation on Butzler agar showed a similar distribution between samples derived from VTP or BCN broths, Skirrow agar was effective only when the inoculum was derived from BCN broth. Our results indicated that vancomycin-trimethoprim-polymyxin B mixture was not a suitable agent for use in an enrichment-plating procedure to recover Campylobacter from chicken, and that by simultaneously using two enrichment broths the ability to recover Campylobacter was enhanced significantly.

Campylobacter pylori in Japan: bacteriological feature and prevalence in healthy subjects and patients with gastroduodenal disorders.

The presence of Campylobacter pylori was investigated in biopsy specimens obtained during gastrofiberscopy from 103 consecutive patients prospectively. Patients included 25 with gastric ulcer, 4 with duodenal ulcer, 5 with coexisting gastroduodenal ulcer, 31 with gastroduodenal ulcer with gastritis, 27 with gastritis, 3 with gastric polyps and 8 with gastric cancer. Results were compared with 20 healthy control subjects who were endoscopically normal. Two specimens each were taken from 3 sites in the stomach. One part was used for a histological study to examine the presence of the organisms. The other part was cultured using Skirrow's agar microaerophilically. Conventional microflora and C. pylori were examined in gastric contents of some cases. Bacteriological features of isolated strains of C. pylori were identical to the NCTC strain. C. pylori was the most dominant organism in gastric contents at any pH level. Detection rates of C. pylori by bacteriological culture were 96% in gastric ulcer, 100% in duodenal ulcer, 80% in coexisting gastroduodenal ulcer, 84% in gastroduodenal ulcer with gastritis, 70% in gastritis, 100% in gastric polyps and 100% in gastric cancer, and the percentages recognized by histological studies were 81, 100, 100, 84, 71, 67, and 57%, respectively. The values in healthy controls were 55% by histological and bacteriological methods (P less than 0.001 compared with overall ulcer patients). These results supported the close association between C. pylori and gastroduodenal diseases.

Comparison of rapid urease tests, staining techniques, and growth on different solid media for detection of Campylobacter pylori

Thirty-nine single antral biopsies (phase 1) and 99 sets of six antral biopsies (phase 2) were collected from 132 patients, and 87 (63%) yielded positive cultures for Campylobacter pylori. Of several primary media tested in phase 1, tryptic soy agar and Skirrow agar, each supplemented with 10% whole sheep blood, supported relatively good growth of C. pylori. In phase 2, four of the six biopsies in each set were tested with different urease systems. Selective urea agar for rapid identification was the most sensitive (39 of 63 [62%] at 1 h) and specific (100%); however, the difference between this system and the CLOtest was not statistically significant. The remaining two biopsies, one transported in saline and the other transported in a supplemented tryptic soy broth, were ground separately and inoculated onto tryptic soy agar and Skirrow agar, each supplemented with 10% whole sheep blood. In selected instances, 10% horse serum or 10% horse serum and 5 mM urea or 1% cholesterol were also added to the media. Smears stained with a modified Gram stain or acridine orange detected 68% of 63 culture-positive biopsies; no false-positive results were reported. Skirrow agar supported better growth of C. pylori than did tryptic soy agar; the nonselective medium was also overgrown with contaminants in 25 to 30% of the positive cultures. Based on colony size, Skirrow agar supplemented with 10% whole sheep blood, 10% horse serum, and 1% cholesterol supported optimal growth of C. pylori. Fresh media supported better growth than did prepoured commercial media (P less than or equal to 0.004). Saline was a convenient and satisfactory transport medium for antral biopsies.

Comparison of media and filtration procedures for qualitative recovery of thermotolerant Campylobacter spp. from naturally contaminated surface water

Different media and filtration procedures were compared for efficiency in recovering thermotolerant campylobacters from three naturally contaminated water sources representing different turbidities and levels of contamination. Three selective enrichment broths were evaluated. Regardless of the kind of broth employed, enrichment did not significantly enhance recovery over that produced by direct plating onto both Skirrow's agar and colistin-amphotericin-keflin (CAK) agar. However, enrichment was clearly advantageous when Skirrow's agar was used as the sole plating medium. Of the four filtration procedures compared, the highest recovery was obtained with 0.45 μm Millipore membrane filters. Subsequent filtration through a 0.22 μm membrane did not increase the yield. Of the three plating media evaluated, the best overall recovery was obtained with Preston agar, followed by CAK agar, whereas Skirrow's medium was inferior. For all three agar media, incubation for 48 h greatly increased the yield as compared with 24 h incubation. We could not demonstrate any statistically significant differences between the plating media as regards their relative efficiency in isolating different Campylobacter species.

Campylobacter jejuni and Campylobacter coli as surface contaminants of fresh and frozen poultry carcasses

Campylobacter jejuni and Campylobacter coli were isolated as surface contaminants from 216 (31.3%) of 691 freshly eviscerated poultry carcasses which were examined just prior to packaging in a highly automatized poultry processing plant. The highest overall contamination rate was recorded among turkey carcasses (56.7%), followed by hens (48.2%), and broiler chickens (13.8%). A total of 245 carcasses were re-examined after they had been processed in the plant and kept frozen (−25°C) for 1 to 15 weeks. The results indicate that campylobacters may survive frozen storage for at least four weeks. Selective enrichment proved essential for isolation from frozen carcasses; the procedure employed enabled a 108.3% increase in isolation rate, compared with direct plating. Colistin-amphotericin-keflin agar was superior to Skirrow's agar for isolation from poultry cascasses. The most common biotype was C. jejuni biotype 1 (82.0%), followed by C. coli (16.9%), and C jejuni biotype 2 (1.1%). It was possible to serotype 129 (72.9%) of 177 campylobacters by means of heat-stable antigens. The most common serotype was LAU 2 which comprised 21.5% of all isolates, followed by LAU 1 with 13.6%. Six additional serotypes occurred regularly (3.4 to 6.2%), while nine serotypes were rare. The serotypes found to be most prevalent in poultry (LAU 1 and LAU 2) were the same ones as recovered most frequently from Norwegian patients.

Isolation of Campylobacter fetus subsp. jejuni from human fecal specimens by incubation at 35 and 42 degrees C.

Campylobacter strains were isolated from human stool specimens that were cultured on modified Skirrow agar and incubated at 35 and 42 degrees C. Incubation at 42 degrees C resulted in more isolates, increased colony numbers, and earlier isolation than was found at 35 degrees C.

Studies on sporadic Campylobacter enteritis

Three-year study (April 1979 to May 1982) was carried out on Campylobacter enteritis in sporadic cases of diarrhea. Stool cultures taken from 1114 patients with acute diarrhea resulted in 13.6% and 8.6% isolation of Campylobacter in children and adults, respectively. The frequency of isolation of Campylobacter was superior in numbers to that of other enteropathogens among the same 1114 patients with diarrhea. Stool cultures were also taken from 6815 asymptomatic subjects including pregnant women and infants, and only 1% of the cultures were positive for Campylobacter.The age distribution of 111 patients with Campylobacter enteritis indicated an excess of infection in children: the excess, however, may mean that children withdiarrhea are more likely to be taken to hospital and that sampled are adults. Males were infected more often than females.The symptoms included diarrhea, abdominal pain, and fever which were not specific in Campylobacter enteritis.It was noticed that occurrence of gross or occult blood in stool specimens and in the microscopy of stools presence of neutrophiles and red cells and campylobacters showing specific morphology and motility yielded valuable information for the diagnosis of Campylobacter enteritis. Apparent rising antibody titers of sera from convalescent patients and a long term positive stool cultures (2 to 7 weeks) were also confirmend as important features of the enteritis.Biochemical characteristics and enzyme profiles of human isolates of Campylobacter were studied. Although insignificant differences of some enzymatic activities were observed among the isolates, all but 2 were determined to be Campylobacter jejuni.The two hippurate negative isolates were likely to be C. coli.Two selective media, Skirrow's agar and Butzler's agar modified by adding 40u./ml of colistin, for the isolation of Campylobacter were compared using 48 positive stool specimens. It was appeared that the modified Butzler's agar was superior to the former in selectivity and isolation of C. jejuni and C. coli.