Evaluation of phenotypic and PCR-based approaches for routine analysis of Bacillus cereus group foodborne isolates

Identification of Bacillus cereus sensu stricto is a challenge for the food industry since it is being increasingly reported as implicated in many foodborne outbreaks. So far no conclusive microbiological or biochemical traits have been described for their specific differentiation. Here a polyphasic approach aiming at identification of new isolates is presented. It was conducted on a total of 75 strains, 59 Bacillus cereus group (29 reference strains and 30 food and environmental isolates) and 16 other Bacillus species. It includes biochemical traits (API 50CH and API 20E) and genetic profiles: PCR amplification of the internal spacer region (ISR) between 23S and 16S rRNA genes (ISR-PCR), randomly amplified polymorphic DNA (RAPD-PCR) with three universal primers (M13, T3, and T7), and PCR amplification using specific primers directed to genes encoding hemolysin BL (hbl), cytotoxin K (cytK) and cereulide (ces). As expected, PCR-enterotoxin profiles revealed the toxigenic potential of strains within the B. cereus group irrespective of the species. Cluster analysis combining the three RAPD fingerprints (RAPD-M13, RAPD-T3 and RAPD-T7) allowed almost a complete separation of strains within the B. cereus group. As a result, the ISR-PCR profile is proposed for the rapid assignation of isolates to B. cereus group with the advantage over the API profile of being a specific and culture-independent technique. Following, differentiation at species level can be obtained by RAPD profiles analysis.     

Isolation of endospore-forming bacilli toxic to Culiseta longiareolata (Diptera: Culicidae) in Jordan

Ten of 80 endospore-forming bacilli, isolated from various habitats of Jordan, were found to be highly toxic to the 4th instar larvae of Culiseta longiareolata (Macquart). The bacilli were identified into the following species and strains: Bacillus sphaericus (H6), B. sphaericus (H9a, 9b), B. cereus Frankland and Frankland, B. brevis Migula and B. megaterium Bary. Bacillus cereus comprised 50% of the isolates. The toxic concentrations of these isolates against C. longiareolata ranged between 1.2 x 10(7) and 1.1 x 10(9) viable spores ml-1.     

Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.     

Evaluation of the Biolog system for the identification of Bacillus anthracis

The potential of the Biolog system for the identification of Bacillus anthracis was evaluated. In-house generated databases allowed the correct identification of 19 of 20 isolates of B. anthracis within 24 h. Five strains of the closely related B. cereus/thuringiensis group were misidentified as B. anthracis. For this reason the test could only serve as a primary screen with further testing being required to confirm identity. In addition 20% of all the strains of bacilli examined during the study gave unreadable reaction profiles due to false-positive reactions.     

Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains

PCR-RFLP analysis of the vrrA gene and cerAB gene was used to investigate the genomic diversity in 21 strains of Bacillus anthracis and 28 strains of Bacillus cereus, and was compared with results obtained by ribotyping and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) analysis. VrrA-typing divided the B. anthracis into four groups. Except for one Pasteur vaccine strain, the vrrA PCR-RFLP profiles of the B. anthracis were separated into three groups, which were different from those of the B. cereus strains. Ribotyping separated the B. anthracis isolates into seven ribotypes, and a common fragment of an approximately 850 bp band from the ERIC-PCR fingerprints separated most B. anthracis strains into two groups. VrrA/cerAB PCR-RFLP, ribotyping and ERIC-PCR generated 18, 22 and 23 types, respectively, from B. cereus strains. The results suggest that a combination of all three methods provides a high resolution typing method for B. anthracis and B. cereus. Compared with ribotyping and ERIC-PCR, PCR-RFLP is simple to perform and has potential as a rapid method for typing and discriminating B. anthracis strains from other B. cereus group bacteria.     

Identification and characterization of toxigenic Bacillus cereus isolates responsible for two food-poisoning outbreaks

The epidemiology of Bacillus cereus strains responsible for food poisoning is scantly known, mostly because the genotypic and toxigenic properties of the B. cereus strains isolated during food-poisoning outbreaks have been never catalogued. The occurrence of two simultaneous food-poisoning outbreaks gave us the opportunity to wonder whether (i) the identity of individual strains isolated from clinical, environmental, and food samples could be established by random amplified polymorphic DNA (RAPD)-PCR and multiplex RAPD-PCR, and (ii) the toxigenic potential of the isolates could be determined by testing their ability to secrete hemolysin BL, phosphatidylcholine-specific phospholipase C, and cereulide, as well as by determining the presence of the genes encoding enterotoxins NHE, T, and FM/S, cytotoxin K, sphingomyelinase, and phosphatidylinositol-specific phospholipase C. This is the first report demonstrating that the combination of several phenotypic and genotypic traits provides a powerful tool for tracing the source of infection of toxigenic B. cereus strains relevant for epidemiological survey.     

Detection of Hippurate Hydrolase among Bacillus Species by Thin Layer Chromatography and other Methods

S ummary . The ability of 111 isolates of 33 species of Bacillus to hydrolyze hippurate into benzoic acid and glycine was tested. All, other than the strains classified as B. badius, B. cereus var. mycoides, B. cereus var. thuringiensis, B. macquariensis, B. medusa, B. pacificus, B. psychrosaccharolyticus and some B. macerans and B. polymyxa strains, were positive by thin layer chromatography (TLC). Of the 4 methods compared (hippurate agar, the sulphuric acid method, precipitation with ferric chloride and TLC), only the TLC procedure allowed a rapid and definite detection of the hydrolytic product, benzoic acid. The use of hippurate hydrolase as an additional diagnostic aid in the differentiation of aerobic spore-forming bacilli is discussed. Other characters were used to compare some recently described Bacillus spp. The combination B. cereus subsp. medusa n. subsp. is proposed.     

Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis

The three species of the group 1 bacilli, Bacillus anthracis, B. cereus, and B. thuringiensis, are genetically very closely related. All inhabit soil habitats but exhibit different phenotypes. B. anthracis is the causative agent of anthrax and is phylogenetically monomorphic, while B. cereus and B. thuringiensis are genetically more diverse. An amplified fragment length polymorphism analysis described here demonstrates genetic diversity among a collection of non-anthrax-causing Bacillus species, some of which show significant similarity to B. anthracis. Suppression subtractive hybridization was then used to characterize the genomic differences that distinguish three of the non-anthrax-causing bacilli from B. anthracis Ames. Ninety-three DNA sequences that were present in B. anthracis but absent from the non-anthrax-causing Bacillus genomes were isolated. Furthermore, 28 of these sequences were not found in a collection of 10 non-anthrax-causing Bacillus species but were present in all members of a representative collection of B. anthracis strains. These sequences map to distinct loci on the B. anthracis genome and can be assayed simultaneously in multiplex PCR assays for rapid and highly specific DNA-based detection of B. anthracis.     

Screening of plasmids in the bacteria of the genus Bacillus with antilysozyme activity

In the study of the microbiocenosis of the distal section of the patients' large intestine Bacillus strains with antilysozyme activity (ALA) were isolated. In B. cereus strain 26 with pronounced expression of antilysozyme factor the plasmid sized approximately 100 kb was detected. The transformation of the isolated plasmid in cells of B. cereus non-plasmid strain IP5832 the localization of genes encoding ALA and resistance to kanamycin was determined. The production of ALA factor in the recombinant clone of B. cereus strain IP5832 corresponded to the clinical isolate of B. cereus 26. The replicon of the detected plasmid could be used for the determination of the coding sequence of the antilysozyme sign of bacilli. Genetic determinants of antilysozyme factors and kanamycin resistance may be used for the construction of vector systems of cloning in bacilli.     

Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis.

A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacillus cereus and B. subtilis. From 12 out of 15 strains of B. cereus, plasmids could be isolated. Most of the B. cereus strains contained two or more plasmids. Their molecular weights ranged from 1.6 X 10(6) to 105 X 10(6). Bacteriocin production could be attributed to a 45 X 10(6)-dalton plasmid (pBC7) from B. cereus DSM 336, and tetracycline resistance to a 2.8 X 10(6) plasmid (pBC16) from B. cereus GP7. Two streptomycin-resistant strains of B. subtilis harbored plasmids of 5.2 X 10(6) and 9 X 10(6), respectively, which were, however, not correlated with the antibiotic resistance. The plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis, where it is stably maintained.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.     

多重PCR技术检测微生物肥料中巨大芽孢杆菌和蜡样群芽孢杆菌的研究与应用

巨大芽孢杆菌是微生物肥料生产中的常用菌种, 与之形态上相似的蜡样群芽孢杆菌(蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌)则是产品中常见的污染菌, 传统方法区分两者费时费力, 有必要建立检测这两类芽孢杆菌的PCR方法。本文利用已登录的spoOA基因序列分别设计和筛选了上述两个种(群)的特异引物, 并建立了多重PCR检测技术。使用该方法对巨大芽孢杆菌、蜡样群芽孢杆菌和其他芽孢菌共3属13种24株标准菌株的基因组DNA进行扩增, 以检验其特异性。结果显示, 巨大芽孢杆菌、蜡样群芽孢杆菌基因组DNA分别产生大小不同的唯一产物, 其他芽孢杆菌均为阴性。该多重PCR检测方法的灵敏度经测定为105 CFU/mL。同时对10株待测菌株和8个微生物肥料产品进行检测, 其鉴定结果与常规鉴定结果一致。以上结果表明, 本文建立的多重PCR方法具有较高的特异性和灵敏度, 可快速、准确鉴定巨大芽孢杆菌和蜡样群芽孢杆菌, 在微生物肥料检测方面有良好的实用前景。     

Chitinolytic activity of phylogenetically diverse Bacillus cereus sensu lato from natural environments

Bacillus cereus s.l., Gram-positive endospore-forming bacilli, persist ubiquitously in different natural habitats and play various ecological roles. Nevertheless, although chitin is one of the most abundant polymer on Earth, the study of the ability of B. cereus s.l. to hydrolyze this polymer were limited to individual B. cereus and B. thuringiensis strains only. Thus, to fill this gap in this research we focused on (i) the linkage between the capability to chitin degradation and the phylogenetic relatedness of B. cereus s.l. strains, and (ii) the genetic background of chitinolytic properties of these bacilli. Our results showed that chitin degradation is common among the B. cereus group members, yet strains clustered into particular phylogenetic groups differ in their chitinolytic capacity. Separate clustering of chitinolytic and non-chitinolytic strains in the phylogenetic tree indicates the ecotypic structure of these isolates. Two proteins belonging to subfamily A (ChiA) and subfamily B (ChiB) of the glycoside hydrolase GH18 family exhibited simultaneous chitobiosidase and endochitinase activities, and are responsible for chitin utilization by environmental B. cereus s.l. isolates.     

PCR assay of the groEL gene for detection and differentiation of Bacillus cereus group cells

Strains of species in the Bacillus cereus group are potentially enterotoxic. Thus, the detection of all B. cereus group strains is important. As 16S ribosomal DNA sequence analysis cannot adequately differentiate species of the B. cereus group, we explored the potential of the groEL gene as a phylogenetic marker. A phylogenetic analysis of the groEL sequences of 78 B. cereus group strains revealed that the B. cereus group strains were split into two major clusters, one including six B. mycoides and one B. pseudomycoides (cluster II) and the other including two B. mycoides and the rest of the B. cereus group strains (cluster I). Cluster I was further differentiated into two subclusters, Ia and Ib. The sodA gene sequences of representative strains from different clusters were also compared. The phylogenetic tree constructed from the sodA sequences showed substantial similarity to the tree constructed from the groEL sequences. Based on the groEL sequences, a PCR assay for detection and identification of B. cereus group strains was developed. Subsequent restriction fragment length polymorphism (RFLP) analysis verified the PCR amplicons and the differentiation of the B. cereus group strains. RFLP with MboI was identical for all the B. cereus group strains analyzed, while RFLP with MfeI or PstI classified all B. cereus and B. thuringiensis strains into two groups. All cluster II B. mycoides and B. pseudomycoides strains could be discriminated from other B. cereus group bacteria by restriction analysis with TspRI.     

The infection of bacilli by Mu cts phage integrated into the plasmid

The infection of Bacillus thuringiensis, B. cereus, B. mesentericus and B. polymyxa strains with temperate E. coli bacteriophage Mu cts62 integrated into plasmid RP4 under conditions of conjugative transfer is shown possible. The investigated strains of bacilli are not able to produce intact phage particles but they acquire the thermosensitive property determined by the phage genome. Gel electrophoresis and blot hybridization of DNA have confirmed the transfer of Mu cts62 genome or a part of it in the investigated strains of bacilli.     

A new rapid and sensitive detection method for cereulide-producing Bacillus cereus using a cycleave real-time PCR

Aims:  To develop a rapid and sensitive detection method for cereulide-producing Bacillus cereus using a real-time PCR based on the sequence of the cereulide synthesis gene.
Methods and Results:  A total of 56 cereulide-producing B. cereus and 15 cereulide-negative strains were tested. We designed specific primers and probes for the detection of cereulide-producing B. cereus . The new cycleave real-time PCR assay gave positive detections for all of 56 cereulide-producing B. cereus strains, whereas all other strains including 10 systemic infectious disease strains were negative. No cross-reaction was observed and the internal control showed positive for all samples.
Conclusions:  The performance of the assay was highly reproducible and specific for cereulide-producing B. cereus . The positive detection was obtained within only 2 h for cereulide-producing strains. The detection limit of this assay was evaluated as 10⁴ CFU g⁻¹ food sample. The assay also confirmed that strains from systemic infectious cases were cereulide-negative.
Significance and Impact of the Study:  This assay is applicable for contaminated foods as well as specimens from infectious disease cases. We recommend this assay for routine examination of suspected B. cereus food poisonings.     

Comparison of ribotyping and randomly amplified polymorphic DNA PCR for characterization of Vibrio vulnificus.

A total of 85 isolates of Vibrio vulnificus were characterized by ribotyping with a probe complementary to 16S and 23S rRNA of Escherichia coli and by randomly amplified polymorphic DNA-PCR (RAPD-PCR) with a 10-mer oligonucleotide primer. The RAPD-PCR results were scanned, and the images were analyzed with a computer program. Ribotype membranes were evaluated visually. Both the ribotyping and the RAPD-PCR results showed that the collection of strains was genetically very heterogeneous. Ribotyping enabled us to differentiate U.S. and Danish strains and V. vulnificus biotypes 1 and 2, while the RAPD-PCR technique was not able to correlate isolates with sources or to differentiate the two biotypes, suggesting that ribotyping is useful for typing V. vulnificus strains whereas RAPD-PCR profiles may subdivide ribotypes. Two Danish clinical biotype 2 strains isolated from fishermen who contracted the infection cleaning eels belonged to the same ribotype as three eel strains (biotype 2), providing further evidence that V. vulnificus biotype 2 is an opportunistic pathogen for humans. One isolate (biotype 2) from Danish coastal waters also showed the same ribotype as the eel strains. This is, to our knowledge, the first time the isolation of V. vulnificus biotype 2 from coastal waters has been described.     

Phylogenetic analysis of Bacillus cereus isolates from severe systemic infections using multilocus sequence typing scheme

Bacillus cereus strains from cases of severe or lethal systemic infections, including respiratory symptoms cases, were analyzed using multilocus sequence typing scheme of B. cereus MLST database. The isolates were evenly distributed between the two main clades, and 60% of them had allele profiles new to the database. Half of the collection's strains clustered in a lineage neighboring Bacillus anthracis phylogenetic origin. Strains from lethal cases with respiratory symptoms were allocated in both main clades. This is the first report of strains causing respiratory symptoms to be identified as genetically distant from B. anthracis. The phylogenetic location of the presented here strains was compared with all previously submitted to the database isolates from systemic infections, and were found to appear in the same clusters where clinical isolates from other studies had been assigned. It seems that the pathogenic strains are forming clusters on the phylogenetic tree.     

Identification of heterofermentative lactobacilli isolated from pig faeces by numerical analysis of total soluble cell protein patterns and RAPD-PCR

AIMS: The study deals with a number of heterofermentative Lactobacillus strains isolated from pig faeces and their identification. METHODS AND RESULTS: SDS-PAGE of total soluble cell proteins and RAPD-PCR profiles were used to identify the strains isolated from pig faeces. Protein profiles obtained with SDS-PAGE revealed that 15 strains clustered at r >or= 0.78 with Lactobacillus buchneri and nine strains at r >or= 0.77 with two reference strains of Lactobacillus reuteri. The identity of the strains was confirmed with RAPD-PCR. CONCLUSIONS: Numerical analysis of protein profiles and RAPD-PCR proved valuable in the differentiation of Lactobacillus spp. isolated from pig faeces. SIGNIFICANCE: This is the first report on the association of Lact. buchneri with pig faeces.     

Genotyping and toxigenic potential of Bacillus subtilis and Bacillus pumilus strains occurring in industrial and artisanal cured sausages

Artisanal and industrial sausages were analyzed for their aerobic, heat-resistant microflora to assess whether new emerging pathogens could be present among Bacillus strains naturally contaminating cured meat products. Sixty-four isolates were characterized by randomly amplified polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (fAFLP). The biotypes, identified by partial 16S rRNA gene sequence analysis, belonged to Bacillus subtilis, Bacillus pumilus, and Bacillus amyloliquefaciens species. Both RAPD-PCR and fAFLP analyses demonstrated that a high genetic heterogeneity is present in the B. subtilis group even in strains harvested from the same source, making it possible to isolate 56 different biotypes. Moreover, fAFLP analysis made it possible to distinguish B. subtilis from B. pumilus strains. The strains were characterized for their toxigenic potential by molecular, physiological, and immunological techniques. Specific PCR analyses revealed the absence of DNA sequences related to HBL, BcET, NHE, and entFM Bacillus cereus enterotoxins and the enzymes sphingomyelinase Sph and phospholipase PI-PLC in all strains; also, the immunological analyses showed that Bacillus strains did not react with NHE- and HBL-specific antibodies. However, some isolates were found to be positive for hemolytic and lecithinase activity. The absence of toxigenic potential in Bacillus strains from the sausages analyzed indicates that these products can be considered safe under the processing conditions they were produced; however, great care should be taken when the ripening time is shortened, particularly in the case of traditional sausages, which could contain high amounts of Bacillus strains and possibly some B. cereus cells.