Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.Mikrobien ja säilöntäaineiden määrälle elintarvikkeissa on luotu ja asetettu raja-arvoja, jotta voidaan varmistaa kuluttajien turvallisuus. Erityisesti ruokamyrkytysepidemioissa on oleellista, että taudinaiheuttajat voidaan havaita ja tunnistaa mahdollisimman nopeasti ja tarkasti. Tämän lisäksi päivittäisessä mikrobien ja säilöntäaineiden seurannassa ja kontrolloinnissa käytettävien ilmaisutekniikoiden ja metodologioiden tulee olla helppokäyttöisiä. Tässä väitöskirjassa tutkittiin vaihtoehtoisia nopeampia mittausmenetelmiä bakteerien tunnistamiseen DNA-sormenjälkien avulla. Työssä kehitettiin virtaussytometriin perustuva menetelmä vaihtoehdoksi pulssikenttägeelielektroforeesille ( the golden method ). DNA-palojen koon mittaaminen erittäin herkällä virtaussytometrillä mahdollisti lajien ja kantojen erottamisen toistettavalla ja vertailtavissa olevalla tavalla pulssikenttägeelielektroforeesiin nähden. Uusi menetelmä oli satoja kertoja nopeampi ja 200,000 kertaa herkempi kuin vertailumenetelmä. Tämän lisäksi kehitettiin myös toinen DNA-sormenjälkitunnistusmenetelmä. Menetelmässä bakteerin DNA:ta pilkotaan yhdellä entsyymillä ja syntyneitä DNA-paloja monistetaan erityisillä alukkeilla, jolloin syntyy kullekin bakteerille tunnusomainen sormenjälkikuvio. Tämä menetelmä mahdollisti patogeenibakteerien: Bacilli, Staphylococci, Yersinia, ja Escherichia coli sukujen, lajien ja kantojen erottamisen. Uudella menetelmällä sormenjälkikuviot olivat myös yksinkertaisempia ja helpompia analysoida kuin perinteisellä menetelmällä, jossa käytetään kahta entsyymiä. Nisiiniä (E234) käytetään yleisesti säilöntäaineena erilaisissa elintarvikkeissa ja erityisesti maitotuotteissa ympäri maailmaa. Nisiinin määrittämiseen on olemassa erilaisia mittausmenetelmiä, mutta ne eivät ole yleensä tarpeeksi herkkiä, spesifisiä ja nopeita. Tässä tutkimuksessa kehitettiin testi, jossa näytteen sisältämä nisiini osoitetaan reportterigeenin avulla. Testissä käytettiin Lactococcus lactis kanta, jolla on kromosomissa kaksikomponenttisignaalijärjestelmä NisRK ja plasmidissa nisA-promoottori gfpuv reportterigeenin edessä. Nisiini aktivoi nisA-promoottorin NisRK-komponentin välituksellä, mikä saa aikaan reportterigeenin ja vihreän fluoresoivan proteiinin (GFPuv) ilmentymisen, joka voidaan mitata. Testi oli erittäin herkkä ja sillä pystyttiin havaitsemaan jopa 10 pg/ml nisiinipitoisuus kasvatusliemessä. Tämän lisäksi se oli sovellettavissa mittauksiin erilaisista elintarvikkeista, kuten maidosta, salaattikastikkeista, prosessoiduista juustoista, nestemäisistä kananmunista ja säilyketomaatista. Alkoholipitoisuus, alhainen pH-taso ja orgaanisen aineen pitoisuus antavat viinille hyvät antimikrobiset ominaisuudet, minkä vuoksi viiniä pidetään yleisesti mikrobien suhteen turvallisena elintarvikkeena. Tämän väitöskirjan yhtenä tavoitteena oli tutkia asiakkaiden palauttamia viinejä, joiden oletettiin olleen syynä ruokamyrkytysoireisiin. Osittaisen 16S rRNA geenisekvenssin analysoinnin, ribotyypityksen ja sian siittiöiden liikkuvuuskokeen avulla yhdestä viinistä löydettiin ja tunnistettiin Bacillus simplex BAC91 bakteeri, joka tuotti siittiösolujen mitokondrioihin vaikuttavaa myrkkyä. Viinin antimikrobinen aktiviteetti mitattiin B. simplex BAC91, B. cereus ATCC 14579 (tyyppikanta) ja kereulidi-myrkkyä tuottavan B. cereus F4810/72 bakteerien vegetatiivisilla soluilla ja itiöillä. Vaikka B. simplex BAC91 bakteerin vegetatiiviset solut ja itiöt olivat herkkiä viinin antimikrobisille ominaisuuksille, B. cereus tyyppikanta ATCC 14579 ja B. cereus F4810/72 -bakteereiden itiöt pysyivät elossa ainakin neljän kuukauden ajan. Tulosten perusteella Bacillus spp. ja erityisesti niiden itiöt voivat olla mahdollinen riski viinin kuluttajille

A Novel Linear Plasmid Mediates Flagellar Variation in Salmonella Typhi

Unlike the majority of Salmonella enterica serovars, Salmonella Typhi (S. Typhi), the etiological agent of human typhoid, is monophasic. S. Typhi normally harbours only the phase 1 flagellin gene (fliC), which encodes the H:d antigen. However, some S. Typhi strains found in Indonesia express an additional flagellin antigen termed H:z66. Molecular analysis of H:z66+ S. Typhi revealed that the H:z66 flagellin structural gene (fljBz66) is encoded on a linear plasmid that we have named pBSSB1. The DNA sequence of pBSSB1 was determined to be just over 27 kbp, and was predicted to encode 33 coding sequences. To our knowledge, pBSSB1 is the first non-bacteriophage–related linear plasmid to be described in the Enterobacteriaceae

A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium

Subtyping below the serovar level is essential for surveillance and outbreak detection and investigation of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant 1,4,[5],12:i:- (S. 1,4,[5],12:i:-), frequent causes of foodborne infections. In an attempt to overcome the intrinsic shortcomings of currently used subtyping techniques, a multiplex oligonucleotide ligation-PCR (MOL-PCR) assay was developed which combines different types of molecular markers in a high throughput microsphere suspension array. The 52 molecular markers include prophage genes, amplified fragment length polymorphism (AFLP) elements, Salmonella genomic island 1 (SGI1), allantoinase gene allB, MLVA locus STTR10, antibiotic resistance genes, single nucleotide polymorphisms (SNPs) and phase 2 flagellar gene fljB. The in vitro stability of these markers was confirmed in a serial passage experiment. The validation of the MOL-PCR assay for subtyping of S. Typhimurium and S. 1,4,[5],12:i:- on 519 isolates shows that the method is rapid, reproducible, flexible, accessible, easy to use and relatively inexpensive. Additionally, a 100 % typeability and a discriminatory power equivalent to that of phage typing were observed, and epidemiological concordance was assessed on isolates of 2 different outbreaks. Furthermore, a data analysis method is provided so that the MOL-PCR assay allows for objective, computerised data analysis and data interpretation of which the results can be easily exchanged between different laboratories in an international surveillance network

Amplified fragment length polymorphism analysis in strain typing and identification of Listeria and Clostridium species

DNA-pohjaiset genotyypitysmenetelmät ovat nykyään tärkeitä työkaluja bakteerien tunnistamisessa ja sukulaisuussuhteiden selvittämisessä. Eri genotyypitysmenetelmien soveltuvuudessa eri bakteerilajeille sekä erilaisiin tutkimustarkoituksiin on kuitenkin eroja. Tässä väitöstutkimuksessa selvitettiin amplified fragment length polymorphism (AFLP) –menetelmän soveltuvuutta elintarvikevälitteisten tautia-aiheuttavien bakteerien Listeria monocytogeneksen, Clostridium botulinumin ja Clostridium perfringensin geneettiseen tyypittämiseen. Väitöstutkimuksessa kehitettiin AFLP-menetelmästä hyvin erotteleva sovellutus L. monocytogenes, C. botulinum ja C. perfringens kantojen karakterisointiin. Menetelmän todettiin olevan hyvin toistettava, nopea sekä helppokäyttöinen. Tutkimuksessa todettiin myös, että AFLP-menetelmää voidaan käyttää apuna eri listeria- ja klostridi-lajien tunnistamisessa. Menetelmästä on hyötyä etenkin klostridi-lajien diagnostiikassa, sillä nykyisin käytössä olevat diagnostiset menetelmät ovat osin puutteellisia. Kehitetyn AFLP sovellutuksen avulla luodut AFLP-tyypitystulokset voidaan kerätä tietokantoihin, joita voidaan hyödyntää mm. epidemiologisissa tutkimuksissa, elintarviketeollisuuden kontaminaatioreittien selvittämisessä, ruokamyrkytysepidemia selvityksissä sekä listeria- ja klostridi-lajien tunnistamisessa. Väitöstyössä tutkittiin AFLP-menetelmän avulla L. monocytogeneksen kontaminaatioreittejä eineksiä valmistavassa elintarvikelaitoksessa kahdeksan vuoden aikana. Siivousrutiinien, valmistettavan tuotteen ja osastoinnin todettiin vaikuttavan tuotantoympäristön kontaminaatiotilanteeseen kuumennettuja eineksiä valmistavilla osastoilla. Osastolla, jolla valmistettiin eineksiä, joiden valmistusprosessiin ei kuulu kuumennuskäsittelyä, raaka-aineiden todettiin saastuttavan valmiita tuotteita. Siten raaka-aineiden laatuun tulisi kiinnittää erityistä huomiota kun valmistetaan eineksiä, joita ei kuumennuskäsitellä. Lisäksi tutkimuksessa osoitettiin, että tuotantolinjan rakenteelliset muutokset voivat edesauttaa elintarvikelaitosta pääsemään eroon listeriakontaminaatiosta. Tutkimuksessa selvitettiin myös elintarvikkeiden tuotantoympäristössä pitkäkestoisen (persistoivan) kontaminaation aiheuttavien sekä satunnaisesti (sporadisesti) esiintyvien L. monocytogenes –kantojen välisiä eroja AFLP- ja pulssikenttäelektroforeesi-menetelmillä. Vaikka persistoivien kantojen todettiin eroavan sporadisista kannoista, ei persistoivilla kannoilla todettu olevan yhteistä geneettistä alkuperää.In epidemiological studies, techniques that effectively discriminate between individual bacterial strains are essential. Recent developments in molecular techniques necessitate an ongoing need to tailor new genotyping methods for optimal characterization of different bacterial species and to evaluate their performance and suitability for research purposes. In this thesis amplified fragment length polymorphism (AFLP) analysis was tailored for optimal characterization of Listeria monocytogenes and Clostridium botulinum. The suitability of the developed AFLP protocol to type L. monocytogenes, C. botulinum and Clostridium perfringens at strain level was evaluated. AFLP proved to be a highly reproducible, easy-to-use, relatively fast and highly discriminative approach. When AFLP was applied to L. monocytogenes strains, its discriminatory power was shown to equal that of PFGE, which is considered the current gold standard for molecular fingerprinting of L. monocytogenes. These features make AFLP analysis a useful alternative to other genotyping methods in, for example, outbreak investigations and contamination route studies. Since phenotypic identification of Clostridium isolates is laborious, the suitability of AFLP for genomic species identification was assessed. The AFLP technique was applied to 129 strains representing 24 different Clostridium species. AFLP differentiated all species tested, except for Clostridium ramosum and Clostridium limosum. AFLP also differentiated between six different Listeria species. If AFLP profiles of well-defined strains are collected in identification libraries, the database can be a valuable additional tool for identification of Clostridium and Listeria species. Due to high throughput of samples, AFLP proved to be especially suitable for screening large numbers of isolates. AFLP was also used to trace contamination routes of L. monocytogenes in a chilled food processing plant producing ready-to-eat and ready-to-reheat meals during an 8-year period. Cleaning routines, product type and degree of compartmentalization seemed to have an influence on the contamination status in compartments that produced cooked meals. In addition, raw materials were shown to cause contamination of uncooked meals. Thus, special attention should be paid to quality control of raw ingredients when uncooked ready-to-eat meals are produced. This work also demonstrated that structural adjustments of a production line may facilitate the eradication of L. monocytogenes from the food processing environment. AFLP and PFGE analysis of sporadic L. monocytogenes strains and strains that cause persistent plant contamination revealed that persistent strains differ from sporadic strains. However, no specific evolutionary lineage of persistent strains was observed

Multiple-Locus Variable Number Tandem Repeat Analysis of Staphylococcus Aureus: Comparison with Pulsed-Field Gel Electrophoresis and spa-Typing

(MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

<p>Abstract</p> <p>Background</p> <p>Fluorescence <it>in situ </it>hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100 kb, careful probe selection and characterization are of paramount importance.</p> <p>Results</p> <p>We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific ~6 kb plasmid onto an unusually small, ~55 kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-κB2 locus.</p> <p>Conclusion</p> <p>The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.</p

Characterisation of <i>Burkholderia cepacia</i> from clinical and environmental origins.

Burkholderia cepacia isolates were obtained from the sputum of cystic fibrosis patients or isolated from the environment. The isolates were characterised phenotypically by determining antibiotic resistance profiles and their ability to macerate onion tissue, and genetically by PCR ribotyping and macro-restriction analysis (genome fingerprinting). The replicon (chromosomal) organisation was determined by pulsed field gel electrophoresis (PFGE) and the presence of plasmids was also investigated. Plasmid transfer by conjugation was also investigated. A high degree of genetic and phenotypic variation was found both within and between clinical and environmental populations of B. cepacia. Environmental isolates were generally less resistant to antibiotics but showed greater ability to macerate onion tissue. Genetic characterisation showed little evidence of acquisition of B. cepacia from the environment by CF patients, but revealed strong evidence supporting person-to-person transmission of B. cepacia in the Cardiff CF centre and other European centres. Two or more chromosomes were found in 93 % of B. cepacia isolates tested, and were also found in other closely related species suggesting such organisation may be common in P-2 proteobacteria. Plasmids, often in excess of 100 kb, were found to be harboured in 52 % of isolates, though were more commonly found in CF isolates (65 % of isolates) than environmental isolates (23 % of isolates). Plasmid transfer by conjugation was demonstrated into, between and from B. cepacia strains. Evidence of plasmids encoding antibiotic resistance was also found

Construction, Characterization, and Preliminary BAC-End Sequence Analysis of a Bacterial Artificial Chromosome Library of the Tea Plant (Camellia sinensis)

We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%–87.24%), followed by DNA retrotransposons (11.16%–11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers