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PubMed2004

The microflora of fermented nixtamalized corn.

Nixtamalization is a traditional process that improves the nutritional quality of corn. To provide a means of utilizing the nutritional benefits of nixtamalized corn and improve product acceptability, lactic acid fermentation was applied. The objective of the study was to study the microbial profile and establish the important lactobacilli of fermenting nixtamalized corn dough. Two batches of cleaned whole corn were subjected to the process of nixtamalization, using two concentrations of lime (0.5 or 1.0%), milled, made into a dough (50% moisture) and fermented spontaneously for 72 h. A control sample was prepared without alkaline treatment. pH and titratable acidity of the dough were measured. Aerobic mesophiles, lactic acid bacteria, yeasts and molds were enumerated on Plate Count Agar (PCA), deMan, Rogossa and Sharpe (MRS) Agar and Malt Extract Agar (MEA), respectively. The identity of lactobacilli present was established at the species level using API 50 CHL. The pH of all the fermenting systems decreased with fermentation time with concomitant increase in titratable acidity. Lactic acid bacteria in numbers of 1.6 x 10(9), 2.3 x 10(9) and 1.8 x 10(9) cfu/g, respectively yeasts and molds, and numbers of 8.0 x 10(7), 5.0 x 10(5) and 1.7 x 10(5) cfu/g, respectively were observed in the control and the two nixtamalized (0.5% and 1.0% lime) samples after 48 h of fermentation. Lactobacilli identified in the fermenting nixtamalized corn dough were Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus cellobiosus as well as Pediococcus spp. The study demonstrates that nixtamalized corn though alkaline in nature can be subjected to spontaneous fermentation to produce a sour product.

Colony Count, Microbial↗
PubMed2003

Beer spoilage bacteria and hop resistance.

For brewing industry, beer spoilage bacteria have been problematic for centuries. They include some lactic acid bacteria such as Lactobacillus brevis, Lactobacillus lindneri and Pediococcus damnosus, and some Gram-negative bacteria such as Pectinatus cerevisiiphilus, Pectinatus frisingensis and Megasphaera cerevisiae. They can spoil beer by turbidity, acidity and the production of unfavorable smell such as diacetyl or hydrogen sulfide. For the microbiological control, many advanced biotechnological techniques such as immunoassay and polymerase chain reaction (PCR) have been applied in place of the conventional and time-consuming method of incubation on culture media. Subsequently, a method is needed to determine whether the detected bacterium is capable of growing in beer or not. In lactic acid bacteria, hop resistance is crucial for their ability to grow in beer. Hop compounds, mainly iso-alpha-acids in beer, have antibacterial activity against Gram-positive bacteria. They act as ionophores which dissipate the pH gradient across the cytoplasmic membrane and reduce the proton motive force (pmf). Consequently, the pmf-dependent nutrient uptake is hampered, resulting in cell death. The hop-resistance mechanisms in lactic acid bacteria have been investigated. HorA was found to excrete hop compounds in an ATP-dependent manner from the cell membrane to outer medium. Additionally, increased proton pumping by the membrane bound H(+)-ATPase contributes to hop resistance. To energize such ATP-dependent transporters hop-resistant cells contain larger ATP pools than hop-sensitive cells. Furthermore, a pmf-dependent hop transporter was recently presented. Understanding the hop-resistance mechanisms has enabled the development of rapid methods to discriminate beer spoilage strains from nonspoilers. The horA-PCR method has been applied for bacterial control in breweries. Also, a discrimination method was developed based on ATP pool measurement in lactobacillus cells. However, some potential hop-resistant strains cannot grow in beer unless they have first been exposed to subinhibitory concentration of hop compounds. The beer spoilage ability of Pectinatus spp. and M. cerevisiae has been poorly studied. Since all the strains have been reported to be capable of beer spoiling, species identification is sufficient for the breweries. However, with the current trend of beer flavor (lower alcohol and bitterness), there is the potential risk that not yet reported bacteria will contribute to beer spoilage. Investigation of the beer spoilage ability of especially Gram-negative bacteria may be useful to reduce this risk.

ATP-Binding Cassette Transporters↗
PubMed1999

Characterization of lactic acid bacteria isolated from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation.

Lactic acid bacteria (LAB) isolated from raw materials (fish, rice, garlic and banana leaves) and processed som-fak (a Thai low-salt fermented fish product) were characterized by API 50-CH and other phenotypic criteria. Lactococcus lactis subsp. lactis and Leuconostoc citreum were specifically associated with fish fillet and minced fish, Lactobacillus paracasei subsp. paracasei with boiled rice and Weisella confusa with garlic mix and banana leaves. In addition, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus pentosaceus were isolated from raw materials. A succession of aciduric, homofermentative lactobacillus species, dominated by Lb. plantarum/pentosus, was found during fermentation. In total, 9% of the strains fermented starch and 19% fermented garlic, the two main carbohydrate components in som-fak. The ability to ferment garlic was paralleled by a capacity to ferment inulin. An increased percentage of garlic fermenting strains was found during fermentation of som-fak, from 8% at day 1 to 40% at day 5. No starch fermenting strains were isolated during fermentation. Three mixed LAB cultures, composed of either starch fermenting Lc. lactis subsp. lactis and Lb. paracasei subsp. paracasei, or garlic fermenting Lb. plantarum and Pd. pentosaceus, or a combination of these strains were inoculated into laboratory prepared som-fak with or without garlic. In som-fak without garlic, pH was above 4.8 after three days, irrespective of addition of mixed LAB cultures. The starch fermenting LAB were unable to ferment som-fak and sensory spoilage occurred after three days. Fermentation with the combined mix of starch and garlic fermenting strains led to production of 2.5% acid and a decrease in pH to 4.5 in two days. The fermentation was slightly slower with the garlic fermenting strains alone. This is the first report describing the role of garlic as carbohydrate source for LAB in fermented fish products.

Animals↗
PubMed1999

Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST 31 isolated from sourdough.

Lactobacillus plantarum ST31 isolated from sourdough produced an antimicrobial substance inhibiting other strains of the genera Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Bacillus and some foodborne pathogens including Staphylococcus aureus. This antimicrobial substance was inactivated by proteolytic enzymes. Consequently, it was characterized as a bacteriocin and was designated plantaricin ST31. This bacteriocin was stable in the pH range 3-8 and it was not affected by amylolytic enzymes. Production of plantaricin was pH and temperature dependent, and maximum yields were obtained in MRS broth cultures maintained at pH 6 and incubated at 30 degrees C in the exponential phase to the early stationary growth phase of the producer organism. This bacteriocin was purified by using consecutive ammonium sulfate and reversed-phase chromatography. It is a peptide of 20 amino acid residues with a mass of 2755+/-0.3 Da, as determined by electrospray mass spectrometry. The sequence of Plantaricin ST31 showed no similarity to those of other bacteriocins. Plantaricin ST31 production appeared to be chromosomally encoded.

Adsorption↗
PubMed2000

A rapid PCR based method to distinguish between Lactococcus and Enterococcus.

Phenotypic characterisation of Lactococcus and Enterococcus species remains unreliable as strains of both genera have been isolated which do not conform to the traditional criteria for separation of these genera. A bank of 131 isolates was phenotypically characterised by three methods: (a) traditional broth tests, (b) API Rapid ID 32 Strep and (c) BBL Crystal ID kits. Differences in genus designation between commercial kits were evident for 12 strains (9%), while 7 strains (5%) remained unidentified by either kit. Published 16S rRNA sequences were aligned and used to design genus-specific primers which, when used in separate PCR reactions, were capable of distinguishing all type strains of Lactococcus and Enterococcus. These primers did not react with known species of Streptococcus, Pediococcus, Lactobacillus, Leuconostoc or Tetragenococcus. Isolates which could not be identified by phenotype were assigned to either genus on the basis of the gene primers.

DNA Primers↗
PubMed1998

Diversity of lactic acid bacteria associated with ducks.

The diversity of lactic acid bacteria occurring in the crop and intestinal content of ducks of different ages (5 to 34 days) was evaluated using the RAPD-PCR technique and comparative sequence analysis of 16S rRNA. Cluster analysis derived from isolates grown on Rogosa agar used for the enrichment of lactic acid bacteria, revealed at least twenty six different groups of organisms representing species, subspecies and strains belonging to Streptococcus, Enterococcus, Pediococcus, Weissella and Lactobacillus. Homofermentative and heterofermentative lactobacilli were identified, belonging to the phylogenetically defined Lb. acidophilus, Lb. salivarius and Lb. reuteri groups.

Age Factors↗
PubMed1996

In vitro activity of quinupristin/dalfopristin (RP 59500) against a large collection of infrequently isolated or tested species.

Quinupristin/dalfopristin (RP 59500, Synercid) is a parenteral streptogramin combination antimicrobial that possesses a synergistic and often bactericidal action against many Grampositive species. In this study, a collection of 1270 uncommonly isolated or tested strains were evaluated for susceptibility to quinupristin/dalfopristin using agar dilution minimum inhibitory concentration (MIC) methods described in the National Committe for Clinical Laboratory Standards. The greatest antimicrobial activity observed for quinupristin/dalfopristin was against staphylococci, streptococci, the pathogenic neisseria, Legionella spp., Lactobacillus spp., and Peptostreptococcus spp. (MIC90 range, 0.5-2 micrograms/ml). Marginal activity (MIC90s, 4 to 8 micrograms/ml) was identified for the rarer enterococci, Leuconostoc spp., Pediococcus spp., and Streptococcus bovis. Against Haemophilus parainfluenzae, Bacteroides thetaiotaomicron, Fusobacterium spp., and Prevotella spp., the streptogramin was inactive. Although no susceptible breakpoint has been approved for quinupristin/dalfopristin, three possible breakpoints (< or = 1, < or = 2, or < or = 4 micrograms/mL) were evaluated. Acceptance of the lower breakpoints (< or = 1 or < or = 2 micrograms/mL) would limit quinupristin/dalfopristin use to staphylococci, streptococci, gonococci, meningococci, and Legionella spp. These results markedly expand the understanding of the usable spectrum of quinupristin/dalfopristin.

Bacteria↗
PubMed1997

Antimicrobial characteristics of quinupristin/dalfopristin (Synercid at 30:70 ratio) compared to alternative ratios for in vitro testing.

Quinupristin/dalfopristin is a new streptogramin combination that occurs at a natural ratio and formulation of 30:70. Rapid metabolism of the dalfopristin component to RP 12536 in vivo puts in question the validity of in vitro test of spectrum with the parent combination. In studies of quinupristin with both dalfopristin and RP 12536, a wide range of ratios (30:70, 50:50, 70:30) were tested by reference MIC and MBC tests. No significant potency differences were observed between combination ratios or metabolic components when testing 256 bacterial strains. Quinupristin/dalfopristin or quinupristin/RP 12536 remained active, by bactericidal action against many staphylococci and Streptococcus ssp. Enterococcus faecium strains were susceptible (MIC90, 2 micrograms/ml; static effect only) to the streptogramin, but E. faecalis, Pasteurella multocida, Pediococcus ssp., Haemophilus influenzae, and Bacteroides fragilis were generally less susceptible (MIC90, > or = 8 micrograms/ml). The log phase inoculum was preferred for MBC and kill-curve tests with this combination. The 30:70 ratio in vitro susceptibility test of quinupristin/dalfopristin as used to date, seems to predict the potency and spectrum of this streptogramin accurately and all clinically important in vivo ratios of the injectable form or its major metabolites. Quinupristin/dalfopristin should be further investigated for clinical use against emerging resistant Gram-positive infections, especially penicillin-resistant streptococci and glycopeptide-resistant E. faecium that exhibit susceptibility in this investigation.

Anti-Bacterial Agents↗
PubMed2004

Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes.

Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation.

Journal Article↗
PubMed2004

Effects of indigenous starter cultures on the microbial and physicochemical characteristics of Urutan, a Balinese fermented sausage.

Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25 degrees C at night to 50 degrees C during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P. acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.

Journal Article↗
PubMed2004

Diversity of lactic acid bacteria isolated from AOC Salers cheese.

The objective of this work was to describe the diversity of lactic acid bacteria in traditional raw milk Salers cheeses at the species and strain levels. The characterization of 381 strains isolated during ripening and various strain collections was investigated using physiological analysis and molecular techniques: Rep-PCR, species and genus specific amplifications and the sequence analysis of 16S rDNA for strain typing and taxonomic identification. The strains belonged to Lactobacillus plantarum, Lactobacillus paracasei, Lactococcus lactis, Lactococcus garviae, Enterococcus faecalis, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Streptococcus salivarius, Streptococcus millieri, Streptococcus macedonicus and Pediococcus pentosaceus. A wide phenotypic and genomic heterogeneity was observed within the different species (Lactobacillus plantarum, Lactobacillus paracasei and Leuconostoc mesenteroides) according to the origin and the time of ripening. The natural microflora was different from strain collection and each method must be combined to identify and characterize natural microflora. This study revealed the low selectivity of selective media used for the isolation of different groups of lactic acid bacteria except the Facultatively Heterofermentative lactobacilli medium selecting mesophile lactobacilli and SB medium selective for Enterococcus. The study reveals, for the first time, the microbial lactic acid bacteria community of Salers cheese and its diversity. A better knowledge of microbial flora will be useful to improve understanding of sensory quality of cheeses.

Cheese↗
PubMed2001

Detection and typing of vancomycin-resistance genes of enterococci from clinical and nosocomial surveillance specimens by multiplex PCR.

Ninety-three clinical isolates of vancomycin-resistant enterococci (VRE) collected from nine hospitals in Taiwan were examined for the presence of vanA, vanB, vanC1, or vanC2/vanC3 genes by a multiplex PCR. Forty-seven of these VRE isolates were vanA positive, 1 contained both vanC1 and vanA, 40 harboured vanB, 2 were vanC1, and 3 were identified to be vanC2/vanC3. Twenty-four vanA isolates were sensitive to teicoplanin and thus did not have a typical VanA phenotype. Five isolates with the VanC phenotype harboured vanB. None of the 40 clinically isolated vancomycin-susceptible E. faecium or E. faecalis and the vancomycin-resistant Leuconostoc and Pediococcus isolates were positive for any of the van genes. While performing nosocomial surveillance, VRE were isolated from 47 of 467 rectal swabs by culture. Compared with the conventional culture method, the sensitivity and specificity of the multiplex PCR for detecting and identifying vancomycin-resistance genes in enterococci directly from culture-positive broth were 97.9% and 100%, respectively. The results suggest that genotypic characterization of vancomycin-resistance is necessary for all clinical VRE isolates and that the multiplex PCR assay can be an alternative method for this purpose.

Bacterial Proteins↗
PubMed1977

Antifolate studies. Activities of 40 potential antimalarial compounds against sensitive and chlorguanide triazine resistant strains of folate-requiring bacteria and Escherichia coli.

As part of the search for new antimalarial drugs, a screening program was developed using sensitive and chlorguanide triazine (CGT, cycloguanil) resistant strains of the folate-requiring bacteria, Streptococcus faecium durans, Lactobacillus casei, and Pediococcus cerevisiae. The activities of 40 compounds have been studied against these strains and Escherichia coli. Observations have been made on the points of 50% growth inhibition, the fold increase of resistance shown to each compound by the resistant strains as compared with the parent sensitive strains, and the reversal of growth inhibition by folic acid with S. faecium and L. casei by folinic acid with P. cerevisiae and by p-aminobenzoic acid with E. coli. Comparisons have been made of the activities of the test compounds with those of the standard antimalarial antifoltes, CGT and pyrimethamine (PM), and the antibacterial results have been compared with the activities of the compounds against Plasmodium berghei infections in the mouse and against human malaria infections where data are available. Of the 17 compounds reversed by folates, five had patterns of activity similar to CGT and PM in that they were most active against S. faecium and nine compounds exhibited a different pattern, being highly active against all four test bacteria. This suggests that these latter compounds either have different pharmacokinetic properties or have additional modes of action. The three CGT-resistant organisms responded to antifolates in different ways. S. faecium (R) and P. cerevisiae (R) strains were cross resistant to 4,6-diaminotriazines, 2,4-diaminopyrimidines, 2,4-diaminoquinazolines, and active 2,4-diaminopteridines. L. casei (R) was cross resistant to the triazines but was collaterally sensitive to all the other antifolates. Most of the compounds not reversed by folates were much less inhibitory for the test organisms; they were most active against L. casei. In general, their growth inhibitory concentrations varied less for the four test organisms and the responses of the sensitive and CGTR strains were similar. However, there was some cross resistance to five compounds and some collateral sensitivity to five others. Comparison of the bacteriological data with the activities of the compounds against Plasmodium berghei in the mouse showed little correlation between the two test systems; each appears to provide independent and useful information.

Animals↗
PubMed1998

Fermentation of yam: microbiology and sensory evaluation of cooked fermented yam tissues.

The microflora of white yam (Dioscorea rotundata L.) slices fermented anaerobically in 1.5 percent brine for five days at room temperature were studied. The hydrolysis of the carbohydrate and the subsequent conversion of sugars and minerals by the fermenting microbes contributed much to the increased microbial load especially within the first 72 hours of fermentation. The organisms implicated in the fermentation include the species of Pediococcus, Lactobacillus and Pseudomonas, Bacillus subtilis and two other Gram negative coccal cells, yet to be identified. The decrease in microbial counts at the latter stage of fermentation was attributed to the high total acidity of the medium, which was about 2.67 times the initial value of 0.027% lactic acid. Meanwhile, the lactic acid bacteria increased continuously throughout the period of fermentation.

Cooking↗
PubMed1999

Production of African breadfruit (Treculia africana) and soybean (Glycine max) seed based food formulations, 2: Effects of germination and fermentation on microbiological and physical properties.

The effects of germination (G) and naturally fermented (F) on the bacterial flora, viscosities and moisture sorption isotherms of soybean (S) and African breadfruit (B) seed based food products were investigated. Bacillus, Enterobacter, Enterobacteriaceae, Proteus, Serratia and Staphylococcus species dominated in the nonfermented products. Lactobacillus, Leuconostoc, Pediococcus and yeast species dominated in the fermented products whose gruels also inhibited growth of coagulase positive Staphylococcus aureus in challenge tests. Germination and fermentation resulted in significant (p < 0.05) decreases in cooked paste viscosities which is advantageous in increasing nutrient density. The monolayer moisture contents (g H2O/g solid) and surface areas for monolayer adsorption (m2/g solid) derived from BET model were 0.0422 and 148.1 (GFSB); 0.0428 and 150.4 (NGFSB); 0.0436 and 153.3 (NGNFSB); 0.0531 and 186.6 (GNFSB), respectively.

Absorption↗
PubMed2004

Intra-specific variation of Lactobacillus plantarum and Lactobacillus pentosus in sensitivity towards various bacteriocins.

Fifty-two strains belonging to the Lactobacillus plantarum species group were identified and typed. They represented 32 clones of Lactobacillus plantarum and 7 clones of Lactobacillus pentosus. Sensitivity of all strains towards bacteriocins of four different producer strains was investigated using a deferred inhibition test (DIT). Substantial intra-specific variation in sensitivity of clones was observed towards bacteriocinogenic lactic acid bacteria producing nisin ( Lactococcus lactis ATCC 11454) or pediocin PA-1 ( Pediococcus acidilactici PAC-1.0), while none of the strains were sensitive towards the two remaining bacteriocin producers. The minimum inhibitory concentration (MIC) of nisin towards selected strains confirmed the DIT results. No correlation between the susceptibility of fourteen selected strains towards nisin and an array of antibiotics was found. The present study indicates that the variation in bacteriocin-sensitivity within target species might be a potential limitation for the application of bacteriocins as biopreservatives.

Anti-Bacterial Agents↗
PubMed1997

Detection and partial characterization of a bacteriocin produced by Carnobacterium piscicola 213.

BLIS 213, is a bacteriocin-like inhibitory substance produced by Carnobacterium piscicola 213. It is active against Carnobacterium, Enterococcus and Listeria spp. No activity was observed against tested Lactobacillus, Lactococcus, Leuconostoc and Pediococcus strains, nor against Gram-negative bacteria. The BLIS 213 activity was inactivated by several proteolytic enzymes. It was heat resistant (121 degrees C for 20 min), and stable over a pH range of 2-8. Activity was determined by a dilution micromethod; it was increased after SDS treatment. A mutant strain which lacks bacteriocin production was isolated and designated as Carnobacterium piscicola 213a. It had the same phenotypic and biochemical properties as the parent strain, and was not sensitive to bacteriocin activity. The apparent molecular weight of the bacteriocin in the crude extract was greater than 10 kDa. It was about 6 kDa after SDS-PAGE of a partially purified bacteriocin by adsorption on producer cells. The isoelectric point of the BLIS 213 was around 9.3.

Bacteriocins↗
PubMed1997

Detection and speciation of bacteria through PCR using universal major cold-shock protein primer oligomers.

The detection of bacteria using PCR is a well-established diagnostic technique. However, conventional PCR requires the use of DNA primer oligomers that are specific to the target organism and, as a consequence, a sample can only be tested for the presence of that specific target. A significant advantage would be to probe a sample for the presence of any bacteria, followed by identification. To achieve this it is necessary to identify a DNA sequence common to all bacteria. Here we demonstrate that such a sequence may be that encoding the major cold-shock proteins. Using two universal PCR primer oligomers from conserved regions of these gene homologues, we have amplified a 200 base-pair DNA sequence from more than 30 diverse Gram-positive and Gram-negative bacteria, including representatives from the genera Aeromonas, Bacillus, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Listeria, Pediococcus, Photobacterium, Proteus, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Sequence analysis of the amplified products confirmed a high level of DNA homology. Significantly, however, there are sufficient nucleotide variations to allow the unique allocation of each amplified sequence to its parental bacterium.

Amino Acid Sequence↗