Enhancement of Campylobacter hepaticus culturing to facilitate downstream applications

Campylobacter hepaticus causes Spotty Liver Disease (SLD) in chickens. C. hepaticus is fastidious and slow-growing, presenting difficulties when growing this bacterium for the preparation of bacterin vaccines and experimental disease challenge trials. This study applied genomic analysis and in vitro experiments to develop an enhanced C. hepaticus liquid culture method. In silico analysis of the anabolic pathways encoded by C. hepaticus revealed that the bacterium is unable to biosynthesise l-cysteine, l-lysine and l-arginine. It was found that l-cysteine added to Brucella broth, significantly enhanced the growth of C. hepaticus, but l-lysine or l-arginine addition did not enhance growth. Brucella broth supplemented with l-cysteine (0.4 mM), l-glutamine (4 mM), and sodium pyruvate (10 mM) gave high-density growth of C. hepaticus and resulted in an almost tenfold increase in culture density compared to the growth in Brucella broth alone (log10 = 9.3 vs 8.4 CFU/mL). The type of culture flask used also significantly affected C. hepaticus culture density. An SLD challenge trial demonstrated that C. hepaticus grown in the enhanced culture conditions retained full virulence. The enhanced liquid culture method developed in this study enables the efficient production of bacterial biomass and therefore facilitates further studies of SLD biology and vaccine development.

Effectiveness of Chemical Sanitizers against Campylobacter jejuni–Containing Biofilms

Survival of Campylobacter jejuni in mixed-culture biofilms was determined after treatment with chemical sanitizers including chlorine, quaternary ammonia, peracetic acid (PAA), and a PAA/peroctanoic acid mixture (PAA/POA). Biofilm-producing bacteria (gram-positive rods, Y1 and W1) were isolated from chicken house nipple drinkers. A meat plant isolate (Pseudomonas sp.) was also included as a biofilm producer. Two-day-old biofilms grown on polyvinyl chloride (PVC) plastic coupons in R2A broth at 12°C were incubated with 106 CFU/ml C. jejuni for 6 h to allow attachment. The coupons were then rinsed and incubated in fresh media for an additional 24 h. C. jejuni–containing biofilms were detached by vortexing with glass beads in modified brucella broth, which was then enumerated for C. jejuni on selective/differential media. The presence of biofilm enhanced (P < 0.01) the attachment and survival of C. jejuni. After the 24-h incubation, only 20 CFU/cm2 of C. jejuni were recovered from the control without biofilms compared to 2,500 to 5,000 CFU/cm2 in samples with preexisting biofilms. The presence of biofilm microflora decreased (P < 0.01) the effectiveness of sanitizers against C. jejuni. Chlorine was the most effective sanitizer since it completely inactivated C. jejuni in the biofilms after treatment at 50 ppm for 45 s. C. jejuni in biofilms was susceptible to all sanitizers tested but was not completely inactivated by treatment with quaternary ammonia, PAA, or PAA/POA mixture at 50 and 200 ppm for 45 s.

An Animal Model of Gastric Ulcer Due to Bacterial Gastritis in Mice

Conventional female BalbC mice were inoculated with Gastrospirillum-like bacteria in mouse gastric homogenate or in 5.0-μm filtrate of gastric homogenate. The bacteria were originally isolated from cheetahs with gastritis. The mice were killed 6 months, 7 months, or 1 year after inoculation. All mice became infected with Gastrospirillum-like bacteria that were confined to the gastric mucosa. Control mice, given either sterile Brucella broth, 0.22-μm filtrate of infected gastric homogenate, or uninfected gastric homogenate did not become infected with bacteria. Lesions in infected mice included severe lymphoplasmacytic gastritis (26/26 infected mice), gastric epithelial hyperplasia (25/26 infected mice), and gastric ulceration (11/26 infected mice). Neutrophilic inflammatory cell infiltrates were inconsistent. None of the uninfected control mice had Gastrospirillum-like bacteria, gastritis, gastric epithelial hyperplasia, or gastric ulceration. These results implicate Gastrospirillum-like bacteria from cheetahs in the pathogenesis of gastric ulceration. This model will be useful in investigating the mechanisms of gastric ulceration associated with bacterial gastritis.

Diluents and the Enumeration of Stressed Campylobacter jejuni

Significant discrepancies in calculated counts of Campylobacter jejuni strains ATCC 33250 and 29428 were observed between undiluted samples and samples diluted in 0.1% peptone water when cultures had been incubated at 42°C under micro-aerobic conditions in brucella broth containing 2% NaCl. The influence of 0.1% peptone water, brucella broth and phosphate buffer as diluents for enumeration of C. jejuni stored at 6 and −18°C in brucella broth with 0.5 and 2% NaCl was studied. The three diluents were equally effective for the recovery of C. jejuni from refrigerated broth containing 0.5% NaCl. However, when the organism had been refrigerated in the broth with 2% NaCl or frozen in the broth with either level of salt, dilution with brucella broth produced significantly higher counts than dilution with 0.1% peptone water or phosphate buffer.

Evaluation of an Enrichment-Plating Procedure for the Recovery of Campylobacter jejuni from Turkey Eggs and Meat

A selective enrichment-plating procedure was tested for the recovery and enumeration of Campylobacter jejuni from turkey eggs and meat. Enrichment was in brucella broth with ferrous sulfate, sodium metabisulfite, sodium pyruvate and five antimicrobial agents. Plating was on brucella agar supplemented with equine blood and antimicrobial agents. Incubation of tubes and plates was at 42°C in an atmosphere of 5% O2:10% CO2:85% N2. C. jejuni could be recovered from the enrichment broth when calculated initial cell numbers per ml of broth were as low as 0.3 to 3.3

Occurrence and Survival of Campylobacter jejuni in Milk and Turkey1

An enrichment procedure was used to determine the presence of Campylobacter jejuni in milk and ground turkey. This procedure consisted of subculturing a sample in antibiotic-supplemented brucella broth incubated at 37°C for 24 h, transferred to fresh broth, incubated microaerophilically at 42°C for 8 h and plated on agar medium selective for C. jejuni for detection. C. jejuni was suspected in 9 of 50 samples of raw milk, but was not confirmed. The organism was not recovered from rectal swabs of cows. Storage of whole milk and ground turkey inoculated with C. jejuni at 4, 37 and 42°C resulted in decreases in C. jejuni counts in milk at 4 and 42°C; and increases in counts in ground turkey at 37 and 42°C. No survivors were detected when suspensions of the organism were exposed to 10 ppm chlorine for 30 s or three common commercial sanitizers used according to manufacturers' specifications.

Effect of Temperature and pH on the Survival of Campylobacter fetus1

Test strains of C. fetus subsp. jejuni and C. fetus subsp. intestinalis failed to survive heating in skimmilk at 60 C for 1 min. A few strains survived heating in skimmilk at 55 C for 1–3 min. D50C values for C. fetus subsp. jejuni and C. fetus subsp. intestinalis in skimmilk ranged from 1.3–4.5 and from 1.0–3.7, respectively. No survivors of C. fetus subsp. jejuni and C. fetus subsp. intestinalis were detected in beef roasts inoculated at levels of 106–107 viable cells per g when the final temperature in the center was 57 and 55 C, respectively. At an internal temperature of 50–53 C, survivors of C. fetus were detected in beef roasts. Storage of skimmilk, beef and ground beef inoculated with C. fetus at −20, 1, 10, 20, 30 or 40 C resulted in decreases in C. fetus count. Survival of C. fetus was best at 1 and 10 C. Rapid increases in C. fetus counts occurred at 37 C in Brucella broth adjusted to pH 6–8. At pH 5, no survivors were detected after 24 h. At pH 9, counts of C. fetus subsp. jejuni decreased rapidly while those of C. fetus subsp. intestinalis increased slightly.

Examination of Poultry Giblets, Raw Milk and Meat for Campylobacter fetus subsp. jejuni

An MPN procedure was used to determine the presence of Campylobacter fetus subsp. jejuni in poultry giblets. This procedure consists of (a) subculturing a sample in Brucella broth supplemented with 0.15% agar, 0.05% sodium pyruvate and the following antimicrobial agents per liter: vancomycin 10 mg, trimethoprim 5 mg, polymyxin B sulfate 2,500 IU, amphotericin B 2 mg and cephalothin 15 mg and (b) subsequent streaking of a loopful of Brucella broth held at 42 C for 48 h on plates of Brucella agar supplemented with 10% defibrinated horse blood and the concentrations of antimicrobial agents identified above. C. fetus subsp. jejuni was present in 85% of the chicken livers and in 89% of the chicken gizzards obtained immediately after evisceration. The organism was not recovered from samples treated with chlorinated water. C. fetus subsp. jejuni was not recovered from raw milk (bulk tank samples or individual cow samples) or from beef (infraspinatus or biceps femoris muscles).