Examining the genetic variation of reference microbial cultures used within food and environmental laboratories using fluorescent amplified fragment length polymorphism analysis

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to genetically fingerprint 'working culture control strains' used by accredited food microbiology laboratories. A working culture control strain is defined as a subculture from a strain initially obtained from an authenticated source [such as the National Collection of Type Cultures (NCTC)] that is maintained for use with routine testing within the laboratory. Working culture control strains from eight food examination laboratories, representing four bacterial species, were analysed by FAFLP; these were Salmonella Nottingham, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus. The resultant FAFLP profiles of the eight working culture control strains for each of these species were compared against the appropriate freeze-dried ampoules obtained directly from NCTC. FAFLP results demonstrated that within 50% of working cultures analysed, several laboratories were routinely using working cultures that were genetically different from the original reference NCTC strains. This study highlights the need for laboratories to review the protocols used to process and maintain control strains and working cultures, with a potential view to utilize single-use quality control materials.     

LENTICULE discs provide a homogenous format for external quality assessment samples: a comparison with freeze-dried samples for shellfish microbiology

AIMS: The aim was to compare the variability in Escherichia coli enumeration data and detection of Salmonella spp. between four samples of LENTICULE discs and freeze-dried samples for the Health Protection Agency's External Quality Assessment (EQA) scheme for shellfish microbiology. METHODS AND RESULTS: Four samples of known but undisclosed microbiological content were dispatched in both freeze-dried and LENTICULE disc formats to 57 participating laboratories in 20 countries. Participants examined samples using their routine methods for the most probable number (MPN) of E. coli per 100 g and the presence/absence of Salmonella spp. There was no significant difference between the Food and Environmental Proficiency Testing Unit and participating laboratories for E. coli and Salmonella spp. results. There were significantly less outlying results using the LENTICULE discs than freeze-dried sample format and equivalent or less variance for the former for E. coli MPN. There was no significant difference between LENTICULE discs and freeze-dried samples for the presence/absence of Salmonella spp. CONCLUSIONS: Overall the results indicated that there was equivalent or less variance in results for the LENTICULE discs than for freeze-dried samples, therefore LENTICULE discs are a homogenous and stable matrix for EQA samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides validation data for the replacement of freeze-dried samples by LENTICULE discs for the Health Protection Agency Shellfish EQA Scheme.     

The stability of micro-organisms preserved in LENTICULE discs, demonstrated by repeat sample distributions of the EQUAL Scheme for Indicator Organisms (water microbiology)

Aims:  To demonstrate the stability of microorganisms preserved in LENTICULE discs, employed by the EQUAL Scheme for Indicator Organisms.
Methods and Results:  Five sample batches containing several indicator organism parameters were each despatched in two separate distributions at intervals of up to 24 months apart. A comparison of participants' median counts and bar charts of their cumulated results was then made.
Conclusions:  The results demonstrated that LENTICULE discs provided a remarkably stable preparation format for the maintenance of viable organisms in reproducible numbers.
Significance and Impact of the Study:  Complex mixtures of organisms can be maintained in stable numbers for extended periods of time at a freezer temperature of −20°C and satisfactorily transported without the requirement for cold chain conditions.     

A new method for liquid nitrogen storage of phototrophic bacteria under anaerobic conditions

A simple, effective and economical method for the long-term preservation of bacteria in liquid nitrogen under anaerobic conditions is described. As a case example anaerobic photosynthetic bacteria were successfully preserved. Gas tight small screw-cap glass ampoules with butyl rubber septa were used for freezing the specimen anaerobically. During experimental manipulations no anaerobic chamber or glove boxes were required. All teste cultures yielded high recoveries after repeated thawing and during storage. After freezing, survival recoveries of Rhodospirillaceae range from 70–100%, whereas with strict anaerobic strains of Chlorobiaceae and Chromatiaceae a maximum loss of 1–2 log₁₀ counts was observed. No further loss in viability occurred after 1–2 years of storage.The small size of the ampoules and the use of single ampoule for 15–20 repeated retrievals proved economical with respect to storage space and costs.The system is compact and suitable for the preservation of anaerobic phototropic bacteria and other fragile anaerobic microorganisms.     

Preservation of laboratory strains of Toxoplasma gondii in vitro

A procedure to obtain viable stabilates of virulent laboratory strains of Toxoplasma gondii with a prolonged storage life is described. Viable endozoites recovered from the sediment of mouse exudate or tissue cultures (LEP, HeLa) are suspended in Eagle's MEM medium supplemented with 10% calf serum and 10% dimethylsulfoxide and sealed into glass ampoules of 1-2 ml in volume. The ampoules are placed in an apparatus for gradual cell freezing, frozen to --35 degrees C at a rate of --1 degree C/min, and stored in liquid nitrogen. Reinoculation experiments on mice given the suspension intraperitoneally confirmed that such Toxoplasma gondii strains retain viability for at least 4 years. This in vitro preservation technique is compared with the analogous T. gondii preservation procedures described in the literature.     

Assessment of the stability of cell-surface components of microorganisms by MALDI-TOF-MS following preservation on lenticule discs

Strains representing the species Campylobacter coli, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enterica, and Staphylococcus aureus were randomly selected to assess the consistency of cells preserved on lenticule discs to those archived in traditional freeze-dried ampoules. Each matched pair was cultured using identical conditions and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) to profile the surface-associated molecules of the cells. In addition, the cytosolic/membrane-bound proteins of C. coli and S. aureus strains were further analysed by surface-enhanced laser desorption/ionization time-of-flight MS. The mass spectral profiles in all cases showed a high degree of concordance between cells preserved by both methods and suggest that the properties of cells preserved on lenticule disc are consistent with those archived by the traditional method of freeze-drying.     

Amplified fragment length polymorphisms reveal genetic differentiation among strains of Xanthomonas albilineans

Xanthomonas albilineans, the causative agent of leaf scald disease (LSD), colonizes the vascular system of sugarcane (Saccharum spp. hybrids). In this study X. albilineans strains from 28 countries were differentiated by using two methods of amplified fragment length polymorphism (AFLP). In the manual procedure, AFLP reactions were performed on 57 X. albilineans strains and after selective amplification using radiolabelled primers, the resulting products were separated using polyacrylamide gel electrophoresis. The autoradiographs were analyzed using GelCompar version 4.1 software (Applied Maths) to construct dendograms from similarity matrices. Fluorescent AFLP (FAFLP) was also employed on 52 X. albilineans strains using three fluorescently labelled primer combinations (automated AFLP). The FAFLP data was converted to a binary format using the Genemapper Software 3.7 (Applied Biosystems). Thereafter, dendograms were generated using the NTSYSpc. Software (USA). Distinct AFLP profiles were produced for the majority of the strains and were found to be useful in differentiating X. albilineans strains from various geographical locations. Fingerprints unique to each strain were reproducibly obtained and may be used to create a database for use in the identification of the various X. albilineans strains. It can be also concluded from the results obtained that the FAFLP has considerable technical advantages compared with the manual AFLP and also that the FAFLP is more sensitive than AFLP using radiolabelled primers in differentiating X. albilineans.     

Long-Term Preservation of Fungus Cultures with Liquid Nitrogen Refrigeration

A preservation technique was tested on 162 strains of culturally fastidious fungi sensitive to lyophilization, representing five classes. The results indicated that liquid nitrogen storage of frozen specimens may be used as an alternative to lyophilization for long-term preservation of stock cultures of fungi. The fungus was frozen in 10% (v/v) glycerol-water menstruum in heat-sealed ampoules. The cooling from ambient temperatures to -35 C was controlled at a rate of approximately 1 C per minute. Further cooling to the storage temperature of -165 to -196 C was uncontrolled and took place at an accelerated rate. Frozen ampoules were thawed in a water bath at 38 to 40 C. Viable and unmutated cultures were developed from reactivated specimens after storage for as long as 5 years.     

Intraspecific genotypic characterization of Lactobacillus rhamnosus strains intended for probiotic use and isolates of human origin

A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.     

Intraspecific Genotypic Characterization of Lactobacillus rhamnosus Strains Intended for Probiotic Use and Isolates of Human Origin

A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.     

The effect of culture preservation techniques on patulin and citrinin production by Penicillium expansum Link

AIMS: To study the influence of culture preservation methods and culture conditions on the production of the mycotoxins patulin and citrinin by Penicillium expansum. METHODS AND RESULTS: Ten strains of Penicillium expansum were preserved using subculture and maintenance at 4 degrees C, mineral oil, drying on silica gel and freeze-drying. Patulin and citrinin production was assessed on yeast extract sucrose agar (YES) and grape juice agar (GJ), using TLC before and after 0.5, 2-3, 6 and 12 months preservation. Citrinin was detected in all cultures for all preservation techniques on YES. The patulin profiles obtained differed with strain and culture media used. CONCLUSIONS: Citrinin production seems to be a stable character for the tested strains. There is a tendency for patulin detection with time apparently more consistent for silica gel storage and freeze-drying, especially when the strains are grown on GJ. SIGNIFICANCE AND IMPACT OF THE STUDY: Variability in the profiles of the mycotoxins tested seems to be more strain-specific than dependent on the preservation technique used.     

AmpliBASE MT: a Mycobacterium tuberculosis diversity knowledgebase

AmpliBASE MT is an online databank of high-resolution DNA fingerprints representing fluorescent amplified fragment length polymorphism (FAFLP) profiles or amplitypes developed for the Mycobacterium tuberculosis complex strains from 48 different countries. AmpliBASE MT is based on a relational database management system that is hyperlinked to visualize genotyping results in the form of DNA fingerprint images for individual strains. A flexible search system based on systematic comparisons of fragment sizes in base pairs allows inter-laboratory comparison of FAFLP profiles. Besides this, the database also displays previously published data on IS6110 profiles, spoligotypes, MIRU-VNTRs and large sequence polymorphisms along with the FAFLP records that will give the overall comparisons. Being the first of its kind, AmpliBASE MT is expected to be a very helpful tool in strengthening the concept of 'geographic genomics' and will be very helpful to molecular epidemiologists and those interested in diagnostic development for tuberculosis.     

A new freeze-drying method for the preservation of nitrogen-fixing and other fragile bacteria

A simple effective and compact freeze-drying method involving skim milk 20% (w/v) and glutamate 5% or meso-inositol 5% or honey 10% or raffinose 5% for the long-term preservation of bacteria is described. As a case example more than 160 strains representing 36 species of nitrogen-fixing bacteria, 11 species of chemolithorutotrophic bacteria and five species of Aquaspirillum were successfully preserved. All tested strains proved viable and showed about 10–100% survival after freeze-drying and during 2–3 years of storage at +9°C. In such lyophilized cultures no loss in plasmids or other desirable characters was observed. The method is also suitable for the preservation of other fragile and difficult microorganisms as several other strains including bacteria with introduced plasmids could equally survive well and retained plasmids after lyophilization with this method.     

Variation in Pathogenicity and Virulence of Strains of Xanthomonas campestris pv. glycines, the Incitant of Bacterial Pustule of Soybean

Standardized methods were developed to determine the pathogenicity and the degree of virulence of Xanthomonas campestris pv. glycines (Xcg) as well as the reaction of soybean plants in the greenhouse. A glass atomizer is described which allowed uniform inoculation without damaging the leaves. Optimum bacterial concentration was determined as 6 × 10⁶ CFU/ml for the pathogenicity test and 1.3 × 10⁵ CFU/ml for the virulence test. A total of 64 isolates were tested. Forty-five strains were designated as pathogenic, six of which were considered highly virulent. It was shown, for the first time, that large differences in the virulence of Xcg strains exist. All the highly virulent strains of Xcg were fresh isolates from diseased soybean leaves collected in Thailand. On the other hand, all the “old” cultures from bacterial collections possessed a low or very low virulence. Decrease of virulence of the pathogen did not occur very fast, however, that is: not within 2 years when stored on YDC-agar slants. Therefore, the bacteria may be kept on slants at 15 °C for short time storage, but the strains should be preserved permanently as lyophilized cultures.     

Survival of microorganisms after drying and storage

Bacteria, yeasts and fungi suspended in a dextran solution were added to ampoules containing strips of filter paper which were dried without vacuum conditions. The ampoules were sealed and stored in the dark at room temperature. Viability counts were made of the original suspension immediately after drying and after storage periods of 3–48 months. Although bacterial cultures of many genera did not show much resistance against dry conditions, bacteria of 13 other genera had survived well or moderately after 4 years of storage. Most of the dried yeast cultures had survived after this period. Of the 16 fungal genera tested, species of 6 genera exhibited growth after 4 years. Results of this study were compared with those of two other preservation methods by which the same microorganisms were used.     

Comparison of culture media and chairside assays for enumerating mutans streptococci

AIM: This study compared several traditional culture-based media and chairside cultural assays for ability to recover mutans streptococci (MS) from pure cultures and from saliva samples. METHODS AND RESULTS: When pure cultures were used with traditional culture-based media, mitis-salivarius bacitracin (MSB) agar demonstrated less support for bacterial recovery than trypticase-yeast extract-cysteine sucrose-bacitracin (TYCSB) agar and the modified medium of Ritz (HLR-S). One species of MS, Streptococcus ferus (c), was not recovered on MSB medium. Chairside cultural tests displayed considerable disparity between tests in recovering bacteria from pure cultures. On the glass adherence assay (Mucount), S. ferus was not detected and Streptococcus criceti was not detected on the dipslide assay (Cariescreen SM) or on the plastic adherence assay (Dentocult SM Strip mutans). The frequency of isolation of pure strains of bacteria other than MS was common. From saliva samples, the frequency of isolation of MS on HLR-S and TYCSB media and the glass adherence assay was 91-97%. The frequency of isolation on MSB medium and on the dip-slide and plastic adherence assays was significantly decreased (37, 47 and 69%, respectively). Recovery scores varied considerably among the culture methods studied and tended to be highest on the HLR-S medium and on the glass adherence assay. CONCLUSIONS: Growth and recovery profiles of pure bacterial cultures and of saliva samples for the MS varied according to different media. SIGNIFICANCE AND IMPACT OF THE STUDY: Caution should be exercised in comparing results between studies that employ different cultural methods for MS enumeration.     

Filamentous growth ofPseudomonas aeruginosa

Summary The growth of two strains ofPseudomonas aeruginosa in stirred batch cultures was monitored by optical density, DNA concentration, and acridine orange direct cell count measurements. Growth of adherent bacteria in pure culture was also observed on suspended glass discs by light and scanning electron microscopy. Strain MUCOID produced significant numbers of filamentous cells in broth culture and in the adherent population, while strain PAO 381 did not produce elongated cells. Filamentous growth of MUCOID could be prevented by the addition of 5 × 10^–2 M Mg²⁺. However, the addition of 0.66 mM EDTA caused an increased proportion of the population (>50%) of MUCOID cells to become filamentous in broth culture. The results are discussed and related to theories regarding bacterial plasticity, and filamentation of normally bacillary cells.     

Constraints on the Preservation of Ferriferous Microfossils

The taphonomy of ancient microbial communities is understood via rare windows of microbes that are successfully entombed and preserved in the fossil record. Laboratory investigations using live bacterial cultures, combined with observations on microfossils preserved in ferriferous oolitic beds formed around 161 million years ago, were used to constrain conditions that are conducive to preservation by mineralization. Lab experiments confirmed redox conditions in conjunction with bacterial Fe metabolism as the critical parameter. When Fe was mobilized during the reductive dissolution of ferric hydroxide in laboratory studies, bacteria did not sorb sufficient amounts of ferriferous phases to result in casts. By contrast, iron-oxidizing bacteria sorbed sufficient ferric hydroxide to entomb the cells, although there was variation in Fe oxide sorption within a single culture. The comparison of information from lab and field investigations opens an avenue to infer processes of preservation by mineralization in a geological time frame.     

Fluorescent amplified-fragment length polymorphism analysis of the MRSA epidemic

Genotypes and putative genetic relationships were characterised for epidemic methicillin-resistant Staphylococcus aureus (EMRSA) strains and isolates from England and Wales, using a high resolution DNA fingerprinting technique, fluorescent amplified-fragment length polymorphism (FAFLP). Each of the phage types of EMRSA had a distinct FAFLP profile. The technique revealed clusters of strains and isolates, and could distinguish isolates belonging to the same phage type. FAFLP provides a new approach to the epidemiological study and control of MRSA.