Enumeration of Obesumbacterium proteus in brewery yeasts and characterization of isolated strains using Biolog GN microplates and protein fingerprinting

Of a range of media tested for enumeration of Obesumbacterium proteus in brewers' yeast, Universal Beer agar and Wallerstein Laboratories Differential medium were most effective. MacConkey agar (several types) and Membrane Lauryl Sulphate agar were least effective. Other media (Wort agar, YM agar) were of intermediate efficacy. Nine O. proteus strains from commercial yeast samples were characterized using the API 20E test kit, the Biolog GN microplate (BGNM) and by SDS-PAGE of their total soluble proteins. Both the BGNM and SDS-PAGE techniques allowed the strains to be differentiated from one another: the API 20E kit did not. All strains isolated from UK breweries belonged to O. proteus biogroup II. Four of these strains displayed a branching cell morphology not hitherto described in any member of the Enterobacteriaceae.     

Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.     

A comparative evaluation of five typing techniques for determining the diversity of fluorescent pseudomonads

Five typing methods were evaluated, utilising 63 strains of fluorescent pseudomonads, to assess their usefulness as tools to study the bacterial diversity within this complex group. The methods used were Biolog metabolic profiling, restriction fragment length polymorphism ribotyping, PCR ribotyping, and repetitive element sequence-based PCR (rep-PCR) utilising BOX and enterobacterial repetitive intergenic consensus (ERIC) primers. Cluster analysis of the results clearly demonstrated the considerable homogeneity of Pseudomonas aeruginosa isolates and, conversely, the heterogeneity within the other species, in particular P. putida and P. fluorescens, which need further taxonomic investigation. Biolog metabolic profiling enabled the best differentiation among the species. Rep-PCR proved to be highly discriminatory, more so than the other DNA fingerprinting techniques, demonstrating its suitability for the analysis of highly clonal isolates. RFLP ribotyping, PCR ribotyping, and rep-PCR produced specific clusters of P. aeruginosa isolates, which corresponded to their origins of isolation, hence we recommend these methods for intraspecific typing of bacteria.     

Characterization of the brewery spoilage bacterium Obesumbacterium proteus by automated ribotyping and development of PCR methods for its biotype 1

AIMS: To study the ability of automated ribotyping to characterize Obesumbacterium proteus and Hafnia alvei, to design primers and to evaluate standard end-point and real-time PCR for the detection of O. proteus biotype 1 in beer and in brewers's yeast-containing samples. METHODS AND RESULTS: Automated ribotyping was carried out using the standard method with EcoRI and PvuII. The digestions with both enzymes clearly differentiated O. proteus biotypes 1 and 2 and H. alvei. PCR primers were designed according to the 16S rRNA gene sequence of the O. proteus type strain. Two primer sets (Obs137-Obs558 and Obs137-Obs617) detected O. proteus biotype 1 and H. alvei but not O. proteus biotype 2 or other tested beer spoilage bacteria (40 species) in the end-point and real-time PCR, indicating their high specificity. The detection limit for O. proteus was 160-1600 CFU 100 ml(-1) beer in the end-point PCR reactions and < or =160 CFU 100 ml(-1) beer in the real-time PCR reactions. More cells (from 16 to 3200) were needed for detection in the presence of brewer's yeast cells. CONCLUSIONS: Automated ribotyping is a useful tool to characterize and identify O. proteus and H. alvei isolates. The designed primers are suitable for the rapid detection of O. proteus biotype 1 and H. alvei in brewery samples by PCR. Significance and IMPACT OF THE STUDY: Automated ribotyping and PCR could improve microbiological quality control in breweries by facilitating the detection, identification and tracing of spoilage bacteria.     

Detection of peptidoglycan and β-glucan with silkworm larvae plasma test

Abstract Discrimination of 70 Salmonella strains previously studied by ribotyping was realized by RAPD and ERIC-PCR analysis. RAPD results on the 56 S. typhimurium isolates did not closely match those of ribotyping. With ERIC-PCR, two fingerprints only were obtained. For the 14 S. enteritidis strains, a helpful discrimination was obtained with RAPD analysis, while ERIC-PCR resulted in a single fingerprint.     

Differentiation of marine luminous bacteria using commercial identification plates

Differentiation of marine luminous bacteria using Biolog GN plate combined with API 20e or the BBL Crystal ID plate inoculated with cell suspensions in artifical seawater was accomplished by comparison to type species using cluster analysis. Inoculum density affected the results from Biolog GN plates, but had less of an effect on the reactions obtained from API 20e strip or BBL Crystal ID plate. In a few cases, combination of the Biolog GN traits along with either the API 20e or Crystal ID traits was necessary to differentiate some marine luminous bacteria. © 1998 John Wiley & Sons, Ltd.     

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.     

Comparison of Parental and Transgenic Alfalfa Rhizosphere Bacterial Communities Using Biolog GN Metabolic Fingerprinting and Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

Abstract Rhizosphere bacterial communities of parental and two transgenic alfalfa (Medicago sativa L.) of isogenic background were compared based on metabolic fingerprinting using Biolog GN microplates and DNA fingerprinting of bacterial communities present in Biolog GN substrate wells by enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR). The two transgenic alfalfa expressed either bacterial (Bacillus licheniformis) genes for alpha-amylase or fungal (Phanerochaete chrysosporium) genes for Mn-dependent lignin peroxidase (Austin S, Bingham ET, Matthews DE, Shahan MN, Will J, Burgess RR, Euphytica 85:381–393). Cluster analysis and principal components analysis (PCA) of the Biolog GN metabolic fingerprints indicated consistent differences in substrate utilization between the parental and lignin peroxidase transgenic alfalfa rhizosphere bacterial communities. Cluster analysis of ERIC-PCR fingerprints of the bacterial communities in Biolog GN substrate wells revealed consistent differences in the types of bacteria (substrate-specific populations) enriched from the rhizospheres of each alfalfa genotype. Comparison of ERIC-PCR fingerprints of bacterial strains obtained from substrate wells to substrate community ERIC-PCR fingerprints suggested that a limited number of populations were responsible for substrate oxidation in these wells. Results of this study suggest that transgenic plant genotype may affect rhizosphere microorganisms and that the methodology used in this study may prove a useful approach for the comparison of bacterial communities. Received: 1 June 1998; Accepted: 20 October 1998     

Nitrate reduction and N-nitrosation by Obesumbacterium proteus

An isolate of Obesumbacterium proteus biogroup 2 was found to possess a formate-dependent nitrate and nitrite reductase system the activity of which was rapidly repressed upon exposure to oxygen. N-nitrosation of dimethylamine at pH 7·8 was characteristic of an enzyme-catalysed reaction and found to be dependent upon nitrite reductase activity.     

Molecular epidemiological tools for Salmonella Dublin typing

Abstract A total of 32 strains of Salmonella Dublin recovered from cattle were differentiated by electrophoretic typing of their esterases (zymotyping), restriction fragment length polymorphism of ribosomal DNA (ribotyping), arbitrarily primed PCR (AP-PCR) using five primers, PCR based on repetitive extragenic palindromic sequences (REP-PCR) and PCR based on enterobacterial repetitive intergenic consensus sequences (ERIC-PCR). ERIC-PCR and REP-PCR each gave one type, zymotyping gave three, AP-PCR gave five and ribotyping gave seven types. Combination of ribotyping and AP-PCR produced a total of 11 types, whereas 14 different types were obtained by all five methods. Thus a combination of several methods enhanced the discrimination of cattle-adapted strains among the genotypically homogeneous serovar Salmonella Dublin.     

Comparison of arbitrarily primed polymerase chain reaction and ribotyping for subtyping Actinobacillus actinomycetemcomitans

This study investigated the compatibility of arbitrarily primed polymerase chain reaction (AP-PCR) and ribotyping in the characterization of Actinobacillus actinomycetemcomitans , a major pathogen in the mixed anaerobic microflora of human periodontitis. AP-PCR was performed directly on lysed bacterial colonies using a random-sequence 10-base oligonucleotide primer. Ribotyping was carried out by using purified bacterial chromosomal DNA digested with BglI. DNA fragments were separated electrophoretically, blotted onto a nylon membrane and hybridized with the plasmid pKK3535 containing the rRNA operon of Escherichia coli. The two genetic methods were evaluated on isolates from single individuals and from family members. Twelve AP-PCR types and 47 ribotypes were distinguished among 76 A. actinomycetemcomitans isolates of different serotypes. AP-PCR typing and ribotyping gave compatible results in 18 of 20 comparisons. Although AP-PCR detected less genetic heterogeneity in A. actinomycetemcomitans than ribotyping, the rapid and relatively simple AP-PCR technique seems to be sufficiently discriminative to be used in large scale epidemiological studies which preclude the application of the more laborious ribotyping technique.     

New host plants of Erwinia amylovora in Bulgaria

Nine strains of Erwinia amylovora were isolated from new host plants in Bulgaria--chokeberry and strawberry. The strains were characterized morphologically and biochemically using the API 20E and BIOLOG system. It was established that they showed three different API 20E metabolic profiles, not found by previous studies of E. amylovora. All strains were identified as E. amylovora due to their metabolic fingerprint patterns obtained by the BIOLOG system. The identification was confirmed by PCR amplification of a specific region of plasmid pEA29 and genome ams-region. This study is the first characterization and identification of E. amylovora strains isolated from chokeberry and strawberry by the API 20E and BIOLOG system and by polymerase chain reaction.     

Sensitivity of Listeria monocytogenes to sanitizers used in the meat processing industry

Nineteen Listeria monocytogenes strains were characterized by automated ribotyping, pulsed-field gel electrophoresis, and plasmid profiling to determine the relationship between genotype and sanitizer resistance. Isolates within a ribogroup had a consistent sensitivity or resistance phenotype except for ribogroup C isolates. All isolates with resistance phenotypes harbored two plasmids. The sensitivity of L. monocytogenes strains to quaternary ammonium compounds (QACs) was correlated with sensitivity to sanitizers and antibiotics with other modes of action. All isolates tested contained the mdrL gene, which encodes an efflux pump that confers resistance to QACs and is both chromosome and plasmid borne.     

Identification of pediococci by ribotyping

Pediococci are among the most prevalent microbial contaminants in breweries and they can cause ropiness and the accumulation of high levels of diacetyl in beer. The accurate identification of pediococci is important, because different species do not possess equal spoilage potential. In this study, 18 Pediococcus strains, mainly of brewery origin, were first identified using phenotypical characterization (API 50 CHL and SDS-PAGE profiling), and then ribotyped using a RiboPrinterR System. Six Pediococcus type strains and three other Pediococcus strains were used as references. Ribotyping showed higher discriminative capacity than phenotypical identification methods. Strains could be identified to species level and in many cases, differentiated even at strain level using this genetic fingerprinting method. The identifications performed by ribotyping were confirmed by 16S rDNA sequencing of selected strains. Automated ribotyping was found to be a rapid and reliable method for identifying pediococci, but requires the construction of a comprehensive fingerprint library.     

Phenotypic diversity of Erwinia amylovora in Bulgaria

Fifty-one strains of Erwinia amylovora isolated from nine host plants in Bulgaria were characterized phenotypically and identified by the API 20E and BIOLOG system. The identification was confirmed by PCR amplification of a specific region of the plasmid pEA29 and the genome ams region. The phenotypic diversity of the strains was studied on the basis of their API 20E and BIOLOG metabolic profiles, as well as of their SDS-PAGE protein profile. Metabolic diversity among the strains was established, but no connection with the origin of the strains was revealed. The Bulgarian strains showed API 20E metabolic profiles not found in previous studies of E. amylovora. The strains formed a homogenous group on the basis of their protein profiles. All the strains were sensitive to the antibiotics streptomycin, tetracycline and oxytetracycline. This study was an initial step towards an investigation of the diversity and evolution in the Bulgarian population of E. amylovora, and it was the first characterization of E. amylovora strains isolated from different host plants in the period 1995-2005 in Bulgaria.     

Direct comparison of four bacterial source tracking methods and use of composite data sets

AIMS: Four bacterial source tracking (BST) methods, enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR), automated ribotyping using HindIII, Kirby-Bauer antibiotic resistance analysis (KB-ARA) and pulsed-field gel electrophoresis (PFGE) were directly compared using the same collection of Escherichia coli isolates. The data sets from each BST method and from composite methods were compared for library accuracy and their ability to identify water isolates. METHODS AND RESULTS: Potential sources of faecal pollution were identified by watershed sanitary surveys. Domestic sewage and faecal samples from pets, cattle, avian livestock, other nonavian livestock, avian wildlife and nonavian wildlife sources were collected for isolation of E. coli. A total of 2275 E. coli isolates from 813 source samples were screened using ERIC-PCR to exclude clones and to maximize library diversity, resulting in 883 isolates from 745 samples selected for the library. The selected isolates were further analysed using automated ribotyping with HindIII, KB-ARA and PFGE. A total of 555 E. coli isolates obtained from 412 water samples were analysed by the four BST methods. A composite data set of the four BST methods gave the highest rates of correct classification (RCCs) with the fewest unidentified isolates than any single method alone. RCCs for the four-method composite data set and a seven-way split of source classes ranged from 22% for avian livestock to 83% for domestic sewage. Two-method composite data sets were also found to be better than individual methods, having RCCs similar to the four-method composite and identification of the same major sources of faecal pollution. CONCLUSIONS: The use of BST composite data sets may be more beneficial than the use of single methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comprehensive comparisons using composite data from several BST methods. While the four-method approach provided the most desirable BST results, the use of two-method composite data sets may yield comparable BST results while providing for cost, labour and time savings.     

Comparison of API 20NE and Biolog GN identification systems assessed by techniques of multivariate analyses.

The increasing use of commercial multitest systems for identification of environmental bacteria creates the problem of how to compare the identification results obtained from different systems. The limited use of species designations in such comparisons is caused by low usage of environmental bacteria in the development of commercial identification schemes. Two multivariate statistical methods, the Mantel's test and the co-inertia analysis, were applied to analyze data derived from the Biolog GN and the API 20NE systems of identification for 50 environmental bacterial strains. We found these two methods to be useful for revealing the relationship between the two sets of numerical taxonomic traits. Both of these methods showed that the distances according to the Biolog GN results between the studied strains were related to those derived from the API 20NE results, despite the differences in the test sets of the two systems. In addition, the co-inertia analysis allowed us to visualise the relationships between classifications of strains derived from the two identification systems and, simultaneously, to estimate the contribution of particular tests to the differentiation of bacterial strains.     

Comparative evaluation of plasmid profiling and ribotyping in the analysis of Lactobacillus plantarum strain heterogeneity in silage

F. DUFFNER AND M. O'CONNELL. 1995. Seventy-two Lactobacillus plantarum isolates were recovered from six uninoculated grass silages for the purposes of firstly evaluating the usefulness of (1) restriction endonuclease digestion of total genomic DNA, (2) plasmid profiling and (3) ribotyping in Lact. plantarum strain differentiation and secondly, examining the strain heterogeneity in well preserved silage.
The three methods for differentiation were applied to 72 of the isolates and allowed at least 32 different strains to be identified. Twenty-five different plasmid profiles were detected (26 if the absence of plasmids is included as a profile). Ribotyping with Eco RI identified only 11 patterns among the silage isolates. A variety of restriction enzymes was screened to increase the sensitivity of ribotyping to detect strain differences and Bam HI was used successfully for this purpose, differentiating all of the strains tested.
Two dominant strains (I and II) were identified in one particular silage, comprising 47% and 17% respectively of the isolates, while strains III and V comprised 37% and 25% of the Lact. plantarum population isolated from another of the silages.     

Identification and genetic characterisation of orthopaedic Staphylococcus isolates collected in Italy by automated EcoRI ribotyping

The aim of this study was to evaluate the possibility to use automated EcoRI ribotyping to address, during the same analysis, both identification and genetic characterisation of 38 Staphylococcus aureus and 64 coagulase-negative staphylococci collected from surgical injuries. The ribotyping identification results confirmed those obtained using the API Staph system for 96% of the isolates. All strains were successfully genotyped and the ribotyping discriminatory power, calculated using the Simpson's index of discrimination, was very high for both groups of staphylococci tested. The same, as well as different biotypes, were identified among isolates with the identical ribotyping profile.