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Biodiversity of bacterial ecosystems in traditional Egyptian Domiati cheese.

Bacterial biodiversity occurring in traditional Egyptian soft Domiati cheese was studied by PCR-temporal temperature gel electrophoresis (TTGE) and PCR-denaturing gradient gel electrophoresis (DGGE). Bands were identified using a reference species database (J.-C. Ogier et al., Appl. Environ. Microbiol. 70:5628-5643, 2004); de novo bands having nonidentified migration patterns were identified by DNA sequencing. Results reveal a novel bacterial profile and extensive bacterial biodiversity in Domiati cheeses, as reflected by the numerous bands present in TTGE and DGGE patterns. The dominant lactic acid bacteria (LAB) identified were as follows: Leuconostoc mesenteroides, Lactococcus garvieae, Aerococcus viridans, Lactobacillus versmoldensis, Pediococcus inopinatus, and Lactococcus lactis. Frequent non-LAB species included numerous coagulase-negative staphylococci, Vibrio spp., Kocuria rhizophila, Kocuria kristinae, Kocuria halotolerans, Arthrobacter spp./Brachybacterium tyrofermentans. This is the first time that the majority of these species has been identified in Domiati cheese. Nearly all the dominant and frequent bacterial species are salt tolerant, and several correspond to known marine bacteria. As Domiati cheese contains 5.4 to 9.5% NaCl, we suggest that these bacteria are likely to have an important role in the ripening process. This first systematic study of the microbial composition of Domiati cheeses reveals great biodiversity and evokes a role for marine bacteria in determining cheese type.

Biodiversity↗

Microbial community structure and dynamics of dark fire-cured tobacco fermentation.

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.

Bacteria↗

Classification of bacteria from commercial egg washers and washed and unwashed eggs.

A total of 432 bacterial isolates from washed and unwashed eggs, egg-washer surfaces, and washwaters from five egg-grading plants in Maryland and southeastern Pennsylvania were classified. Counts on equipment surfaces showed considerable variation from plant to plant, reflecting care used in cleaning. Unwashed eggs had a higher percentage of gram-positive cocci (71%), and isolates included Streptococcus faecalis, Aerococcus, and Escherichia coli, which were not isolated from equipment surfaces and washwaters. Equipment surfaces had a higher proportion of actinomycetes than unwashed eggs, and predominant gram-negative rods were Alcaligenes and Moraxella, which were not found on unwashed eggs. Flavobacterium and Alcaligenes have been implicated in shell egg rots, Staphylococcus aureus has been implicated in food poisoning, and organisms resembling micrococci and actinomycetes have been found in broken-out egg products.

Animals↗

Microbiological examination of sebeel water.

Water samples from clay storage jugs ("zeers") located in homes and at public watering stands ("sebeels") at streets, mosques, and schools were examined. Coliforms, fecal coliforms, and fecal streptococci were detected in 100, 69, 88, and 91.56% of the samples, respectively. The general microbiology of the water and some factors affecting microbial load were studied. The predominant bacterial genera of sebeel water were found to be Staphylococcus. Aerococcus, Micrococcus, Streptococcus, Bacillus, Listeria, Lactobacillus, and Arthrobacter. A simple modification of zeer construction was suggested to help improve sanitation.

Bacteria↗

Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. We isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Some clusters could be tentatively identified with the help of reference strains. Samples from each environment had a typical composition of streptococcus types. Enterococcus faecalis was present, but not as a dominating enterococcal species, in samples in which fecal contamination was probable. Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii had protein profiles that were difficult to distinguish from each other. These bacteria were found in a variety of samples. Enterococcus casseliflavus and Enterococcus gallinarum had identical protein profiles. On the basis of the maximum temperatures for growth and pigment production, isolates of this protein profile group common in forest industry wastewaters were identified as E. casseliflavus. Lactococcus lactis subsp. lactis was also found in this environment. Nearly all strains from pristine waters belonged to protein profile groups which could not be identified with the aid of known Aerococcus, Enterococcus, Lactococcus, or Streptococcus strains. The maximum temperatures for growth and the results of fatty acid analysis were in general agreement within each protein profile group.

Electrophoresis, Polyacrylamide Gel↗

Digestion of herring by indigenous bacteria in the minke whale forestomach.

Northeastern Atlantic minke whales (Balaenoptera acutorostrata) have a multichambered stomach system which includes a nonglandular forestomach resembling that of ruminants. Bacteria from the forestomachs of herring-eating whales were enumerated and isolated in an anaerobic rumen-like culture medium (M8W medium). The total viable population of anaerobic bacteria ranged from 73 x 10 to 145 x 10/ml of forestomach fluid (n = 4). Lactobacillus spp. (19.7%), Streptococcus spp. (35.9%), and Ruminococcus spp. (12.8%) were the most common of the bacterial strains (n = 117) isolated by use of M8W medium from the forestomach fluid population of two minke whales. Most of the isolates stained gram positive (93.2%), 62.4% were cocci, and all strains were strictly anaerobic. The population of lipolytic bacteria in one animal, enumerated by use of a selective lipid medium, constituted 89.7% of the viable population. The total viable population of anaerobic bacteria in freshly caught and homogenized herring (Clupea harengus) ranged from 56.7 to 95.0 cells per gram of homogenized prey (n = 3) when M8W medium was used. Pediococcus spp. (30.6%) and Aerococcus spp. (25.0%) were most common of the bacterial strains (n = 72) isolated from the homogenized herring. Most of the bacterial strains were gram positive (80.6%), and 70.8% were cocci. Unlike the forestomach bacterial population, as many as 61.1% of the strains from the herring were facultatively anaerobic. All bacterial strains isolated from the prey had phenotypic patterns different from those of strains isolated from the dominant bacterial population in the forestomach, indicating that the forestomach microbiota is indigenous. Scanning electron microscopic examinations revealed large numbers of bacteria, surrounded by a glycocalyx, attached to partly digested food particles in the forestomach. These data support the hypothesis that symbiotic microbial digestion occurs in the forestomach and that the bacteria are indigenous to minke whales.

Journal Article↗

Enzyme characteristics of beta-D-galactosidase- and beta-D-glucuronidase-positive bacteria and their interference in rapid methods for detection of waterborne coliforms and Escherichia coli.

Bacteria which were beta-D-galactosidase and beta-D-glucuronidase positive or expressed only one of these enzymes were isolated from environmental water samples. The enzymatic activity of these bacteria was measured in 25-min assays by using the fluorogenic substrates 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl-beta-D-glucuronide. The enzyme activity, enzyme induction, and enzyme temperature characteristics of target and nontarget bacteria in assays aimed at detecting coliform bacteria and Escherichia coli were investigated. The potential interference of false-positive bacteria was evaluated. Several of the beta-D-galactosidase-positive nontarget bacteria but none of the beta-D-glucuronidase-positive nontarget bacteria contained unstable enzyme at 44.5 degrees C. The activity of target bacteria was highly inducible. Nontarget bacteria were induced much less or were not induced by the inducers used. The results revealed large variations in the enzyme levels of different beta-D-galactosidase- and beta-D-glucuronidase-positive bacteria. The induced and noninduced beta-D-glucuronidase activities of Bacillus spp. and Aerococcus viridans were approximately the same as the activities of induced E. coli. Except for some isolates identified as Aeromonas spp., all of the induced and noninduced beta-D-galactosidase-positive, noncoliform isolates exhibited at least 2 log units less mean beta-D-galactosidase activity than induced E. coli. The noncoliform bacteria must be present in correspondingly higher concentrations than those of target bacteria to interfere in the rapid assay for detection of coliform bacteria.

Enterobacteriaceae↗

Cloning and analysis of the L-lactate utilization genes from Streptococcus iniae.

The presence of lactate oxidase was examined in eight Streptococcus species and some related species of bacteria. A clone (pGR002) was isolated from a genomic library of Streptococcus iniae generated in Escherichia coli, containing a DNA fragment spanning two genes designated lctO and lctP. We show that these genes are likely to be involved in the L-lactic acid aerobic metabolism of this organism. This DNA fragment has been sequenced and characterized. A comparison of the deduced amino acid sequence of LctP protein demonstrated that the protein had significant homology with the L-lactate permeases of other bacteria. The amino acid sequence of the LctO protein of S. iniae also showed a strong homology to L-lactate oxidase from Aerococcus viridans and some NAD-independent lactate dehydrogenases, all belonging to the family of flavin mononucleotide-dependent alpha-hydroxyacid-oxidizing enzymes. Biochemical assays of the gene products confirm the identity of the genes from the isolated DNA fragment and reveal a possible role for the lactate oxidase from S. iniae. This lactate oxidase is discussed in relation to the growth of the organism in response to carbon source availability.

Amino Acid Sequence↗

Development of bacterial contamination during production of yeast extracts.

Baker's yeast suspensions having bacterial populations of 10(6) and 10(8) CFU/ml were subjected to autolysis processes designed to obtain yeast extracts (YE). The bacterial contaminants added to the yeast cell suspensions were produced with spent broths obtained from a commercial yeast production plant and contained 59% cocci (Leuconostoc, Aerococcus, Lactococcus) as well as 41% bacilli (Bacillus). Autolyses were conducted at four different pH levels (4.0, 5.5, 7.0, and 8.5) and with two autolysis-promoting agents (ethyl acetate and chitosan). Processing parameters were more important than the initial bacterial population in the development of contaminating bacteria during manufacture of YE. Drops in the viable bacterial population after a 24-h autolysis were observed when pH was adjusted to 4.0 or when ethyl acetate was added. A significant interaction was found between the effects of pH and autolysis promoters on the bacterial population in YE, indicating that the activity of ethyl acetate, as opposed to that of chitosan, was not influenced by pH.

Acetates↗

Spherical Lactic Acid-producing Bacteria of Southern-grown Raw and Processed Vegetables.

The frequency and levels of population of the spherical lactic acid-producing bacteria were determined on raw and processed yellow summer and zucchini squash, a variety of greens, green beans, okra, southern peas, and butter and lima beans, and on fresh cucumbers and corn flowers. Six taxa occurred consistently: Leuconostoc mesenteroides, yellow-pigmented streptococci, Streptococcus faecium, Aerococcus viridans, and S. faecalis and S. faecalis var. liquefaciens. The same taxa occurred with the same order of frequency on processed, frozen vegetables, but with a marked decrease in the occurrence of S. faecalis var. liquefaciens. S. lactis, S. cremoris, S. equinus, S. bovis, and pediococci were isolated infrequently. No other member of the viridans group of the streptococci and no member of the pyogenic group was isolated. Approximately 88% of the cultures were identified. Total counts of the lactic-acid-producing bacteria rarely exceeded 10 per gram of sample, and there was a reduction by 90% during the second year of study, probably because of drought. Only one bacterial species was found on 40% of the raw and 34% of the processed vegetable samples. Two or more species or taxa were present on the remainder of 153 raw and 56 processed vegetable samples. A. viridans was present on squash, greens, okra, and southern peas, and its frequency of occurrence on vegetables suggests that plants are its natural habitat.

Journal Article↗

Serum bactericidal activity in the horseshoe crab, Limulus polyphemus.

Serum from the horseshoe crab, Limulus polyphemus, was examined for bactericidal activity against five species of bacteria. Greatest activity was found against Pseudomonas putida and Flavobacterium sp.; with the former, serum dilutions as high as 1:20 were capable of reducing viable counts by 50% within 2 h. Bactericidal activity of a significantly lesser magnitude was demonstrated against Serratia marcesencs and Salmonella minnesota. No killing was seen when the lobster pathogen Aerococcus viridans (formerly Gaffkya homari) was used. The cidal activity against Flavobacterium sp. remained relatively consistent for 6 months.

Animals↗

Effect of capsulation of opportunistic pathogenic bacteria on binding of the pattern recognition molecules mannan-binding lectin, L-ficolin, and H-ficolin.

Mannan-binding lectin (MBL), L-ficolin, and H-ficolin are pattern recognition molecules of the innate immune system. We investigated their ability to bind to different serotypes and noncapsulated variants of two gram-positive bacterial species, Streptococcus pneumoniae and Staphylococcus aureus. MBL did not bind to capsulated S. aureus or capsulated S. pneumoniae but did bind to a noncapsulated S. aureus variant (Wood). L-ficolin bound to some capsulated S. aureus serotypes (serotypes 1, 8, 9, 11, and 12) and capsulated S. pneumoniae serotypes (11A, 11D, and 11F) but not to noncapsulated strains. H-ficolin did not bind to any of the S. pneumoniae and S. aureus serotypes included in this study but did bind to one strain of Aerococcus viridans. The concentrations of the three proteins in 97 plasma samples were estimated. The median concentrations were 0.8 mug per ml for MBL, 3.3 mug per ml for L-ficolin, and 18.4 mug per ml for H-ficolin.

Bacterial Capsules↗

Evaluation of the Rapid Strep system for the identification of clinical isolates of Streptococcus species.

A total of 247 strains of streptococci isolated from humans were tested for identification in the Rapid Strep system. The identification rates and identification levels were different for each Streptococcus species. Our data indicate that the Rapid Strep system will identify nearly all the beta-hemolytic Streptococcus species if serological procedures are used in conjunction with the rapid physiological procedures. Of the group D streptococci, 98% of the enterococci and 95% of the non-enterococci were correctly identified. Of the commonly occurring viridans species, 85% were correctly identified, but only 10% of the less frequently occurring viridans species were identified. A total of 90% of the Streptococcus pneumoniae and 60% of the Aerococcus strains were correctly identified.

Humans↗

Comparative evaluation of the API 20S and AutoMicrobic gram-positive identification systems for non-beta-hemolytic streptococci and aerococci.

The API 20S system (Analytab Products, Plainview, N.Y.) and the AutoMicrobic Gram-Positive Identification system (GPI; Vitek Systems, Hazelwood, Mo.) were evaluated for their capacity to identify the non-beta-hemolytic streptococci and aerococci to the species level. The 20S system identified 86% (six of seven strains) of nonhemolytic group B streptococci, whereas 100% of the same group B streptococcal strains were correctly identified by the GPI system. With both systems 99% (134 of 135 strains) of four species of group D enterococcus strains and 92% (24 of 26 strains) of the Aerococcus spp. strains were identified. The 20S system identified 84% (41 of 49 strains) of three species of group D non-enterococcus strains. The GPI system identified 96% of the same group D non-enterococcus strains. The 20S system identified 84% (190 of 226 strains) of 10 species of viridans streptococci; however, supplemental conventional tests were required to identify 49% (110 of the 226 strains) of the viridans strains to the species level. The GPI system identified 79% of the same viridans streptococci without the need for supplemental tests. Both systems identified 84% (161 of 192 strains) of the seven most commonly occurring viridans Streptococcus spp. The 20S system identified 82% (75 of 92 strains) and the GPI system identified 84% (54 of 64 strains) of Streptococcus pneumoniae.

Bacteriological Techniques↗

Cross-reactions between alpha-streptococci and Omniserum, a polyvalent pneumococcal serum, demonstrated by direct immunofluorescence, immunoelectroosmophoresis, and latex agglutination.

In recent years several groups have used serological methods to demonstrate pneumococcal capsular antigens in sputum. In the present study 123 strains of alpha-hemolytic streptococci (including 97 strains from sputum or pharyngeal specimens) were tested for cross-reactions with a polyvalent antipneumococcal serum (Omniserum). Representatives of the following species were included: Streptococcus bovis, S. equinus, S. intermedius, S. lactis, S. milleri, S. mitis, S. mutans, S. sobrinus, S. salivarius, S. sanguis, S. suis, and Aerococcus viridans. Serological reactions were detected by direct immunofluorescence, immunoelectroosmophoresis, and latex agglutination. Fifteen (12%) of the strains gave positive reactions by all three methods. Positive reactions were also observed with another 32 strains (26%) with two of the methods, whereas 37 strains (30%) gave positive reactions by just one technique. Altogether 84 (68%) strains gave positive reactions with one or more of the methods. Latex agglutination gave positive reactions with 26 (21%) strains compared with 57 (46%) in immunofluorescence and 63 (51%) in immunoelectroosmophoresis. Absorption of the antiserum with one alpha-hemolytic strain reduced but did not entirely eliminate the cross-reactions with five tested strains. These findings indicate a potential risk of cross-reactions with polyvalent antipneumococcal serum in tests carried out on sputa or other specimens which may be contaminated with alpha-hemolytic streptococci.

Cross Reactions↗

Comparison of physiologic tests used to identify non-beta-hemolytic aerococci, enterococci, and streptococci.

Twenty-one reference strains and 88 clinical isolates of Aerococcus, Enterococcus, and Streptococcus species were tested for reactions in the Rapid Strep (RS) and modified conventional tests. We conclude that some but not all of the tests in the RS system could be used to substitute for conventional tests. RS tests for hydrolysis of arginine, esculin, L-pyrrolidonyl-naphthylamide, production of acetyl methyl carbinol (Voges-Proskauer), and fermentation of arabinose, lactose, mannitol, raffinose, and sorbitol were satisfactory substitutes for conventional tests. However, the RS tests for hydrolysis of hippurate and starch and fermentation of inulin were not satisfactory substitutes for conventional tests. We also conclude that, of the five test tube Voges-Proskauer tests, the Coblentz modification is the most discriminatory.

Bacteriological Techniques↗

Description and evaluation of the semiautomated 4-hour rapid ID 32 Strep method for identification of streptococci and members of related genera.

The rapid ID 32 Strep system (bioMérieux, La Balme les Grottes, France) is a new system which allows the identification in 4 h of most streptococci and members of related genera encountered in medical and veterinary bacteriology. Four hundred thirty-three isolates first identified by conventional methods and belonging to the genera Streptococcus, Lactococcus, Enterococcus, Aerococcus, Gemella, Leuconostoc, Erysipelothrix, Gardnerella, and Listeria were tested. Overall, rapid ID 32 Strep correctly identified 413 (95.3%) of the strains, with 109 (25.1%) requiring extra tests for complete identification. Sixteen strains (3.7%) were not identified, and 4 (1.0%) were misidentified. The rapid ID 32 Strep system is a suitable alternative for rapid identification of members of the genus Streptococcus and of related genera.

Bacterial Typing Techniques↗

Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin.

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.

Animals↗