Detection by polymerase chain reaction of Clostridium perfringens producing epsilon toxin in faeces and in gastrointestinal contents of goats

F.A. UZAL, J.J. PLUMB, L.L. BLACKALL, D. O'BOYLE AND W.R. KELLY. 1996. A polymerase chain reaction (PCR) was used to identify the gene-encoding epsilon toxin production in Clostridium perfringens types B and D in faeces and in gastrointestinal contents of goats. The samples were cultured in thioglycollate broth and centrifuged. The upper layer of the pellet was used as a template for PCR, obviating the need for DNA extraction. This technique specifically differentiated Cl. perfringens types B and D from Cl. perfringens types A and C and from Escherichia coli . When used to identify Cl. perfringens type D in samples artificially spiked with the micro-organism, the PCR detected as few as 1.4 × 10² cfu g⁻¹ of sample. Gastrointestinal contents and faeces were collected from 20 goats at slaughter and processed by PCR. Several positive results were obtained from the first five goats that were slaughtered and sampled a few days after their arrival at the abattoir, but only a few samples gave positive results during the following weeks, after the goats had been fed a concentrated ration containing monensin. A possible role of this drug in control of enterotoxaemia is suggested.     

PCR detection of Clostridium perfringens type D in formalin-fixed, paraffin-embedded tissues of goats and sheep

A polymerase chain reaction (PCR) was used to identify the genes encoding the alpha, epsilon and beta toxins of Clostridium perfringens in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep. When pure cultures of Cl. perfringens types B and D were used as control templates in the PCR, products of the following sizes were observed on the agarose gel: 247 bp (alpha primers), 1025 bp (beta primers) and 403 bp (epsilon primers). When used to identify Cl. perfringens type D in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep, the PCR technique resulted in the detection of this micro-organism in 11 out of 13 samples known to be infected with Cl. perfringens. No false positive results were obtained when 13 culturally negative samples were analysed by the PCR technique.     

Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens

A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10² bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans.
The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.     

Characterization of glycerol kinase and NAD-independent glycerol-3-phosphate dehydrogenase from Pediococcus pentosaceus N5p

The polymerase chain reaction (PCR) has the potential to detect low levels of the human pathogen Escherichia coli O157 : H7 in bovine faeces. To improve the utility of PCR for this application, several methods for preparing template DNA from bovine faeces, both directly and after non-selective enrichment, were tested. These were boiling, enzyme treatment, enzyme treatment plus phenol-chloroform extraction, and enzyme treatment plus phenol-chloroform extraction plus Geneclean^® purification. Of these, the boiling method was the most consistent and had a sensitivity of approximately 3 cfu g⁻¹ faeces, with an assay time of less than 32 h. The boiling method was also combined with immunomagnetic separation (IMS) to detect E. coli O157 : H7 in less than 8 h, but with a sensitivity of approximately 10³ cfu g⁻¹ faeces. These methods can be used to prepare template for PCR screening of bovine faeces using any appropriate PCR primers.     

Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques

A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l⁻¹ cefoperazone. Both MF culture and the PCR assay detected 10⁵ colony-forming units (cfu) g⁻¹ faeces. Selective agar culture of some strains could detect as few as 10³ cfu g⁻¹ faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus.     

In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics

Minimal inhibitory concentration (MIC) determinations were carried out with seven growth-enhancing antibiotics against 95 Clostridium perfringens field isolates obtained during 1991 and 1992 from poultry, pigs and calves. All were resistant to 64 μg ml⁻¹ of the bambermycin antibiotic, flavomycin (flavophospholipol) and susceptible to avoparcin (MIC₉₀ 0.25 μg ml⁻¹), avilamycin (MIC₉₀ 0.5 μg ml⁻¹) and salinomycin (MIC₉₀≤ 0.12 μg ml⁻¹). Acquired resistance against bacitracin was detected in some isolates from poultry and bovines and resistance to tylosin and virginiamycin in some strains from all species investigated. Overall, the prevalence of resistance was comparable to the low levels recorded in 1979 in Cl. perfringens isolates from the same animal host species.     

The use of PCR to monitor the population abundance of six human intestinal bacterial species in an in vitro semicontinuous culture system

Abstract Six PCR primer sets complementary to the 16S rDNAs (rRNA genes) were developed and shown to be specific for the following anaerobic bacteria: Clostridium clostridiiforme, C. perfringens, C. leptum, Bacteroides vulgatus, B. distasonis , and B. thetaiotaomicron , respectively. These primers were used for PCR to detect and monitor the bacteria in a semicontinuous culture system designed to mimic intestinal microflora in the human gastrointestinal tract. Except for C. perfringens , the five species of Bacteroides and Clostridia present in the in vitro culture system were detected by the PCR, and the titers varied from 10⁻² to 10⁻⁶ dilutions. The role of azo dye reduction by these bacterial species in the system was examined and discussed.     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

Microbiological composition of raw milk from selected farms in the Camembert region of Normandy

Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens , yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas , coliforms, Escherichia coli , enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml⁻¹), lactococci (760 cfu ml⁻¹) and Micrococcaceae (720 cfu ml⁻¹) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts <100 cfu ml⁻¹. Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml⁻¹. About 80% of supplies had ≤10 E. coli cfu ml⁻¹ and all samples had 1 Cl. perfringens cfu ml⁻¹. Two of the tested milks were positive for salmonellas (2·9%), four were positive for Listeria monocytogenes (5·8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1·4%).     

Growth-inhibitory effects of Galla Rhois-derived tannins on intestinal bacteria

The growth-inhibitory activity of Galla Rhois-derived materials towards 17 intestinal bacteria was evaluated using an impregnated paper disc method. The biologically active components of Galla Rhois were characterized as the tannins methyl gallate (MG) and gallic acid (GA) by spectral analysis. The growth responses varied with bacterial strain tested. In the test using 10 mg disc⁻¹, MG and GA produced a clear inhibitory effect on harmful bacteria such as Clostridium perfringens , Cl. paraputrificum , Eubacterium limosum , Bacteroides fragilis , Staphylococcus aureus and Escherichia coli . Methyl gallate showed no growth-inhibitory activity towards Bifidobacterium adolescentis or B. longum whereas the growth of B. bifidum , B. breve , B. infantis , B. animalis , B. thermophilum , Lactobacillus acidophilus , Lact. plantarum and Streptococcus faecalis was slightly affected. However, GA did not adversely affect the growth of the bifidobacteria and lactobacilli. At 5 mg disc⁻¹, MG significantly inhibited the growth of Cl. perfringens and Cl. paraputrificum but did not affect the growth of the bifidobacteria and lactobacilli. At 1 mg disc⁻¹, MG greatly inhibited the growth of Cl. perfringens alone. These results may be an indication of at least one of the pharmacological actions of Galla Rhois.     

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non- E. coli bacteria. Its detection limit was 10²–10³ cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10⁰ cells 100 ml⁻¹ of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

Development and use of 16S rRNA gene targeted PCR primers for the identification of Escherichia coli cells in water

The primary sequences of the V₃ and V₆ regions of the 16S rRNA gene of pathogenic and non-pathogenic strains of Escherichia coli were determined and compared with those obtained for a number of reference strains which belong to the family Enterobacteriaceae. Three oligonucleotide primers 16E1, 16E2 and 16E3 were designed and used in the polymerase chain reaction to identify specifically all E. coli isolates. When 16E1, 16E2 and 16E3 were used as primers for the identification of E. coli cells present in tap, underground and pond waters, as low as 1 cfu 100 ml⁻¹ of water could be detected if an 8 h pre-culture step was performed prior to the PCR reaction.     

Note: Occurrence and persistence of culturable clostridial spores on the leaves of horticultural plants

Clostridial spores were found in numbers from less than 1 to over 50 colony-forming units cm^-2 on mature leaves of 19 species of horticultural plants under commercial cultivation in five localities in Apulia (SE Italy). Of 1828 clostridial isolates, 87% were identified phenotypically and ascribed to Clostridium pasteurianum, Cl. sporogenes, Cl. butyricum, Cl. roseum, Cl. perfringens, Cl. felsineum and Cl. acetobutylicum , in decreasing order of frequency. When spore suspensions of Cl. pasteurianum, Cl. perfringens, Cl. roseum and Cl. sporogenes were inoculated onto the leaves of basil, leaf-beet, lettuce, rocket-salad, spinach and tomato in the greenhouse, spore counts at first invariably declined shortly after inoculation, then rose again significantly for Cl. pasteurianum and Cl. perfringens on basil and for Cl. sporogenes on tomato in summer.     

Design and evaluation of malolactic enzyme gene targeted primers for rapid identification and detection of Oenococcus oeni in wine

Rapid identification and detection of Oenococcus oeni was achieved by species-specific PCR. Two primers flanking a 1025 bp region of the O. oeni gene encoding the malolactic enzyme were designed. The expected DNA amplificate was obtained only when purified DNA from O. oeni was used. The identity of PCR product was confirmed by nested PCR and restriction analysis. Within 8 h, 10³ cfu ml⁻¹ of oenococci were detected in fermenting grape must containing 10⁷ yeast cells, whereas the detection limit in wine was 10⁴ cfu ml⁻¹. The rapidity and reliability of the PCR procedure established suggests that the method may be profitably applied in winery laboratories for quality control.     

Development and evaluation of polymerase chain reaction tests as an aid to diagnosis of swine dysentery and intestinal spirochaetosis

Polymerase chain reaction (PCR) tests were established for detection of Serpulina hyodysenteriae , the agent of swine dysentery, and S. pilosicoli , the agent of intestinal spirochaetosis. Both reactions were specific when tested with DNA from 107 strains of various intestinal spirochaetes. For diagnostic use, faeces were plated to selective medium, and diatomaceous earth extraction used to obtain DNA prior to PCR. This procedure detected 10³–10⁴ cells of either organism seeded into 0·2 g of faeces. When applied to 63 samples from pigs of eight piggeries naturally infected with either S. hyodysenteriae or S. pilosicoli , both PCRs were specific, more rapid, and detected more positive samples than did routine faecal culture and isolation.     

Isolation and identification of Burkholderia pseudomallei from soil using selective culture techniques and the polymerase chain reaction

An environmental soil survey to detect Burkholderia pseudomallei was performed during the dry and wet seasons in Darwin, Northern Territory, Australia. Soil was sampled at regular intervals during a 15-month period at different depths from areas which were representative of the local, soil environment. Selective culture techniques using Ashdown's and Galimand and Dodin's methods and the polymerase chain reaction (PCR) using specific 16S rRNA primers were used to detect and identify the organism and determine its distribution within the soil stratum over the change in seasons. Results showed that Ashdown's method gave higher isolation rates in the dry season, and Galimand and Dodin's method gave higher isolation rates during the wet season. PCR of the soil enrichment proved to be a more sensitive method than culture and was also a useful confirmatory test in determining the identification of isolates where biochemical tests gave inconsistent results. The PCR primers were specific and able to detect 10¹ cfu g^-1 soil and 10⁴ cfu g^-1 of soil using Ashdown's enrichment broth and Galimand and Dodin's broth, respectively. Overall the isolation of B. pseudomallei was greatest during the dry season and at the higher and lower soil depths, which is contradictory to epidemiological evidence that melioidosis occurs primarily during the wet season among patients exposed to contaminated surface soil and water.     

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg I⁻¹), amphotericin (10 mg I⁻¹) and teicoplanin (4 mg I⁻¹) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0·65 μm filters and a 150-fold reduction using the 0·45 μm filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10⁴ cfu ml⁻¹). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0·65 μm) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².     

New selective media for isolation of clostridia from faecal specimens

Two selective media, novobiocin-colistin agar (NCA) and colistin-crystal violet agar (CCA), were developed for isolating clostridia from human and animal faeces. The basal medium was modified Eggerth-Gagnon agar. The NCA medium contains novobiocin (8 μg ml^-1) and colistin (8 μg ml^-1) and the CCA medium contains colistin (10 μg ml^-1) and crystal violet (10 μg ml^-1). Nine faecal specimens were cultured. Clostridia isolated on these media were similar to those on non-selective media, and higher than those isolated after heat treatment. However, more clostridial species were isolated on the new selective media compared with the non-selective medium. These selective agars were particularly useful for enumerating and isolating clostridia from human faeces.