Cultural, Biochemical, and Immunological Properties of Mima, Herellea, and Flavobacterium Species

From study of cultural and biochemical characteristics of 40 strains of Herellea, Mima, or Flavobacterium species, a proposed schema for identification was developed. The reactions observed by agglutination, gel diffusion, and immunofluorescence suggest antigenic heterogeneity of this group of organisms.     

Radiation Treatment of Foods: I. Radurization of Fresh Eviscerated Poultry1

Radurization processing of fresh eviscerated poultry has been microbiologically studied. The recommended dose of 0.5 Mrads extended the shelf life at 5 C by approximately 14 days. This treatment also effected a 10 and 11 log reduction in the number of viable Salmonella species and Staphylococcus aureus strains, respectively. Recycling these organisms at sublethal doses of irradiation resulted in strains possessing increased irradiation resistance. Shifts in the microbial ecology after irradiation and storage resulted, in some instances, in the isolation of organisms tentatively identified as Moraxella sp. and Herellea vaginicola.     

Herellea (Acinetobacter) and Pseudomonas ovalis (P. putida) from Frozen Foods

Seventeen strains of Herellea vaginicola (Acinetobacter antitratus) and 8 of Pseudomonas ovalis (P. putida), isolated from 23 (6.3%) of 364 samples of frozen, foil-pack foods, were identified and characterized morphologically and biochemically. Herellea was isolated from 17 foods (4.7%), P. ovalis from 6 (1.6%). No Mima were found. The food samples included precooked frozen meats, precooked and uncooked frozen vegetables, and uncooked frozen desserts. The bacteria were detected in the food with a procedure used generally for the detection of salmonellae. The pseudomonad simulated the characteristics of Herellea on Sellers differential agar, except for the fact that it fluoresced. From consideration of the habitat and pathogenicity of Herellea and Mima, it is concluded that, although the presence of these bacteria may not be desirable, their significance in food remains unanswered.     

Nucleic Acid Enzyme Studies of Nonfermentative Gram-Negative Bacteria Using Thin-Layer Chromatography

A rapid and easy to perform procedure for determining the nucleic acid enzyme reactions of intact bacterial cells was developed. Overnight organism growth on triple sugar agar was tested for nucleoside phosphotransferase, nucleosidase, and nucleotidase activity. Reaction products were detected by means of thin-layer chromatography and fluorescence. Characteristic patterns were seen with certain strains of nonfermentative gram-negative bacteria, which indicated that these tests could aid in classification and identification of E0-1 group, Pseudomonas multivorans, P. cepacia, P. maltophilia, certain other Pseudomonas species, and Herellea vaginicola.     

Aminoglycoside-modifying enzymes in clinical isolates ofAcinetobacter calcoaceticus

Aminoglycoside-modifying enzymes were identified in clinical strains ofAcinetobacter calcoaceticus subsp.anitratus (Herellea vaginicola) isolated between 1971 and 1979. Resistance to kanamycin, neomycin, and lividomycin was explained by an aminoglycoside-3′-phosphotransferase enzyme. The resistance of gentamicin was due to an aminoglycoside 3N-acetyltransferase type I enzyme which had not previously been described in this species. Several strains producing both enzymes showed resistance to kanamycin, gentamicin, lividomycin, neomycin, and sisomicin and susceptibility to tobramycin and amikacin.     

Response of the Mimeae to some Physical and Chemical Agents

Isolates of Mima polymorpha and Herellea vaginicola obtained from retail food products were investigated for their ability to hydrolyze proteins, starch, and lipids as well as for their ability to initiate growth under varying conditions of pH, sodium chloride concentration, and temperature. None of the isolates hydrolyzed starch and a few hydrolyzed proteins, whereas most were actively lipolytic. Members of each genus grew over the range 6 to 42 C; in general, H. vaginicola was more tolerant to high concentrations of sodium chloride and to acid conditions than was M. polymorpha.     

Identification of the Mima-Herellea Group of Organisms and Selected Members of the Genus Neisseria

To determine a rapid and reliable protocol for the differentiation of Mima polymorpha, M. polymorpha var. oxidans, Herellea, Bacterium antitratum, Neisseria gonorrheae, and other related members of the genus Neisseria, reference cultures were examined on a variety of microbiological media. The media were selected because of the typical morphological and biochemical characteristics exhibited by the test organisms and were those media which would be readily available in a microbiology facility. After compiling data obtained from 21,714 observations, an inclusive protocol is presented which has proven to be quite adequate for the identification of these particular gram-negative bacteria.     

Differentiation of Klebsiella-Enterobacter (Aerobacter)-Serratia by biochemical tests and antibiotic susceptibility

Studies were undertaken for the differentiation of members of the Klebsiella-Enterobacter (Aerobacter)-Serratia division by biochemical tests and antibiotic susceptibility. A total of 67 cultures were tested. Strain identification was readily accomplished with the use of motility tests and arabinose fermentation. In addition, a practical schema, based on sensitivity pattern, proved valuable in the classification of the different strains. Most (if not all) Klebsiella strains were susceptible to cephalothin, and all were sensitive to colistin. Enterobacter strains were resistant to cephalothin but susceptible to colistin. In contrast to those other members of the group, all Serratia strains were resistant to both cephalothin and colistin. The combination of a limited number of biochemical reactions and single-disc sensitivity tests appears to be a logical approach for the tentative identification of Klebsiella-Enterobacter (Aerobacter)-Serratia strains.     

Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

Non-tuberculous mycobacteria I: one year clinical isolates identification in Tertiary Hospital Aids Reference Center,Rio de Janeiro,Brazil, in pre highly active antiretroviral therapy era

The aim of this study was to determine the prevalence of non-tuberculous mycobacteria (NTM) isolates at University Hospital, Reference Center for Aids in Rio de Janeiro, Brazil, during one year. We used standard biochemical tests for species identification and IS1245 PCR amplification was applied as a Mycobacterium avium specific identification marker. Four hundred and four specimens from 233 patients yielded acid-fast bacilli growth. M. tuberculosis was identified in 85% of the patients and NTM in 15%. NTM disseminated infection was a common event correlated with human immunodeficiency virus (HIV) infected patients and only in HIV negative patients the source of NTM was non sterile site. M. avium complex (MAC) was biochemically identified in 57.8% (49/83) of NTM isolates, most of them from sterile sites (75.5%), and in 94% (46/49) the IS 1245 marker specific for M. avium was present. Twenty NTM strains showed a MAC biochemical pattern with the exception of a urease-positive (99% of MAC are urease-negative), however IS1245 was detected in 96% of the strains leading to their identification as M. avium. In this group differences in NTM source was not significant. The second most frequently isolated NTM was identified as M. scrofulaceum (7.2%), followed by M. terrae (3.6%), M. gordonae (2.4%), M. chelonae (1.2%), M. fortuitum (1.2%) and one strain which could not be identified. All were IS1245 negative except for one strain identified as M. scrofulaceum. It is interesting to note that non-sterile sites were the major source of these isolates (92.8%). Our finding indicated that M. avium is still the major atypical species among in the MAC isolates recovered from Brazilian Aids patients without highty active antiretroviral therapy schema. Some discrepancies were seen between the identification methods and further investigations must be done to better characterize NTM isolates using other phenotypic and genotypic methods.     

Numerical taxonomy and laboratory identification of Actinomyces and Arachnia and some related bacteria.

A numerical taxonomic study was made on 49 facultative anaerobic Gram-positive filamentous and/or diphtheroidal organisms isolated from dental plaques, carious dentin and faeces, together with 63 reference strains belonging to the genera Actinomyces, Arachnia, Bifidobacterium, Actinobacterium, Propionibacterium, Eubacterium and Lactobacillus. They were examined for 90 unit characters covering a wide range of tests and properties. The data were subjected to computer analysis in which the simple matching coefficient (SSM) and the similarity index (SJ) were calculated, and the results of single linkage techniques and an unweighted average linkage cluster analysis technique were compared. The strains fell into six major groups (phena). The Actinomyces strains were recovered in two phena; the first contained Actinomyces israelii and the other facultative anaerobic Actinomyces, including subclusters equal to taxospecies of A. odontolyticus and A. viscosus/A. naeslundii, while the other phenon corresponded to the genera Arachnia, Actinobacterium, Bifidobacterium and Propionibacterium. The groups of Arachnia and Actinobacterium each contained one species, representing taxospecies of Arachnia propionica and Actinobacterium meyerii. Taxonomic criteria, both constant and discriminative, were selected to form a diagnostic table useful for laboratory identification of this group of organisms. Immunofluorescence supported the numerical data.     

Random amplified ribosomal DNA restriction analysis for rapid identification of thermophilic Actinomycete-like bacteria involved in hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a pulmonary disease characterised by inflammation that can be caused by, amongst other substances, a subset of 4 thermophilic mycelial bacteria: Saccharopolyspora rectivirgula, Saccharomonospora viridis, Thermoactinomyces sacchari, and Thermoactinomyces vulgaris. Air sampling analyses in highly contaminated environments are often performed to evaluate exposure to these species which are difficult and fastidious to identify by conventional techniques. The aim of this study was to use amplified ribosomal DNA restriction analysis (ARDRA) to develop a method of identification for those thermophilic organisms that would be more rapid and simple. Strains of these 4 species were obtained from the American type culture collection (ATCC) and were characterized using biochemical tests and ARDRA patterns obtained on their partial-lenght amplified 16S rDNAs. To validate this approach, ARDRA with two restriction enzymes, TaqI and HhaI, was applied to 49 thermophilic actinomycete-like strains from environmental samples (sawmills). The results obtained show that combining some cultural characteristics and biochemical tests, such as xanthine or hypoxanthine decomposition, growth in the presence of NaCl, lysozyme or novobiocin, and spore resistance over 100 degrees C provide a rough identification and selection of the genera of interest. Consequently, target species could be confirmed by digestion of partial-lenght 16S rDNA with the use of Taql and HhaI restriction enzymes that gave specific restriction patterns. ARDRA analyses on the 49 environmental actinomycete-like organisms revealed the presence of 8 Saccharopolyspora rectivirgula, 2 Saccharomonospora viridis, and 15 Thermoactinomyces vulgaris strains, the other strains had restriction patterns different than those of the species of interest. Results of the present study will be applicable to other potential HP environments such as dairy barns, peat bogs and compost plants.     

The description of strains of anaerobic 'corroding' organisms isolated from soft tissue infections in cats and dogs

Thirty-eight strains of Gram negative anaerobic 'corroding' rods were isolated from subcutaneous abscesses and pyothorax in cats and dogs. These organisms all exhibited the colony morphology and biochemical properties of organisms described as Bacteroides ureolyticus (Jackson & Goodman 1978) except that they did not hydrolyse urea and they had a guanine plus cytosine ratio of 39–44 mol%. As described for Bact. ureolyticus , 11 of the strains were non-motile. The remaining 27 strains were motile and exhibited a single polar flagellum when examined by negative staining with phosphotungstic acid in an electron microscope. The motile organisms resembled the so-called 'anaerobic vibrios' or Vibrio succinogenes except that these organisms are curved do not liquefy gelatin and are thought by some to have a G + C ratio of 49 mol%. The non-motile strains fitted no group described previously.     

Microarray-based genetic identification of beneficial organisms as a new tool for quality control of laboratory cultures

The use of beneficial organisms to help control pests and pathogens in field and greenhouse crops is constantly increasing. Insects and mites are commonly used as beneficial organisms and, nowadays, rearing companies have to produce them in large quantities. Because of the peculiarities of laboratory culture conditions, the quality of lab-reared organisms generally degrades over time. To maintain high fitness levels, cultures are refreshed with field specimens at regular intervals. However, this bears the risk of contaminating laboratory cultures with species or strains other than the intended natural enemy. To ensure that the correct species is produced and also to facilitate surveys after field release, we have developed a diagnostic microarray for identification of beneficial species. Probes have been designed from the different haplotypes of a fragment of the mitochondrial cytochrome oxidase I (COI) gene of each species. Hybridization of labeled PCR amplicons of COI on the microarray chip allows precise identification of 28 economically relevant arthropod species.     

Sequence Analysis of 96 Genomic Regions Identifies Distinct Evolutionary Lineages within CC156, the Largest Streptococcus pneumoniae Clonal Complex in the MLST Database

Multi-Locus Sequence Typing (MLST) of Streptococcus pneumoniae is based on the sequence of seven housekeeping gene fragments. The analysis of MLST allelic profiles by eBURST allows the grouping of genetically related strains into Clonal Complexes (CCs) including those genotypes with a common descent from a predicted ancestor. However, the increasing use of MLST to characterize S. pneumoniae strains has led to the identification of a large number of new Sequence Types (STs) causing the merger of formerly distinct lineages into larger CCs. An example of this is the CC156, displaying a high level of complexity and including strains with allelic profiles differing in all seven of the MLST loci, capsular type and the presence of the Pilus Islet-1 (PI-1). Detailed analysis of the CC156 indicates that the identification of new STs, such as ST4945, induced the merging of formerly distinct clonal complexes. In order to discriminate the strain diversity within CC156, a recently developed typing schema, 96-MLST, was used to analyse 66 strains representative of 41 different STs. Analysis of allelic profiles by hierarchical clustering and a minimum spanning tree identified ten genetically distinct evolutionary lineages. Similar results were obtained by phylogenetic analysis on the concatenated sequences with different methods. The identified lineages are homogenous in capsular type and PI-1 presence. ST4945 strains were unequivocally assigned to one of the lineages. In conclusion, the identification of new STs through an exhaustive analysis of pneumococcal strains from various laboratories has highlighted that potentially unrelated subgroups can be grouped into a single CC by eBURST. The analysis of additional loci, such as those included in the 96-MLST schema, will be necessary to accurately discriminate the clonal evolution of the pneumococcal population.     

Production of long-chain alcohols by yeasts

Fourteen yeast strains from six genera were analysed for the presence of long-chain alcohols. Six strains from three genera contained long-chain alcohols, highest levels being found in Candida albicans. The alcohols were identified and determined by TLC, GLC and GLC-MS. The major long-chain alcohols synthesized by these organisms were saturated, primary alcohols with C14, C16 or C18 chain length. Unsaturated long-chain alcohols were not detected. In all strains that produced long-chain alcohols, the relative proportions were C16 greater than C18 greater than C14. Long-chain alcohol contents were higher in organisms from anaerobically, as compared with aerobically, grown cultures reaching about 650 micrograms (g dry wt organisms)-1 in stationary-phase cultures of C. albicans. In cultures of C. albicans, synthesis of long-chain alcohols occurred only after the end of exponential growth. The alcohols were predominantly present as free alcohols. The fatty-acyl chain-length profile of the triacylglycerol and to a lesser extent the sterol/wax ester fractions from C. albicans reflected that of the long-chain alcohols produced by this yeast.     

Phylogeny of Proteobacteria and Bacteroidetes from oxic habitats of a tidal flat ecosystem

Bacteria of the phyla Proteobacteria and Bacteroidetes are known to be the most prominent heterotrophic organisms in marine surface waters. In order to investigate the occurrence of these phyla in a coastal environment, the tidal flat ecosystem German Wadden Sea, we analyzed a clone library of PCR-amplified and sequenced 16S rRNA gene fragments and isolated 46 new strains affiliated with these phyla from the water column with various polymers and complex media as substrates. The phylogenetic affiliation of these strains was analyzed on the basis of sequenced 16S rRNA gene fragments. Subsequently, a comprehensive phylogenetic analysis of Proteobacteria and Bacteroidetes including available sequences from oxic habitats of earlier studies of this ecosystem was performed. Sequences of the earlier studies were derived from isolation approaches and from denaturing gradient gel electrophoresis (DGGE) analyses of environmental samples and high dilution steps of MPN (most probable number) cultures. The majority of the 265 sequences included in this analysis affiliated with alpha-Proteobacteria (45.3%), gamma-Proteobacteria (31.7%), and Bacteroidetes (16.2%). Almost 7% belong to the delta-Proteobacteria and several of these clones affiliated with the Myxococcales, a group comprising obligate aerobic organisms. Within the alpha- and gamma-Proteobacteria specific clusters were identified including isolates from high dilution steps of dilution cultures and/or clones from the clone library or DGGE gels, implying a high abundance of some of these organisms. Within the gamma-Proteobacteria a new cluster is proposed, which consists of marine surface-attached organisms. This SAMMIC (Surface Attached Marine MICrobes) cluster comprises only uncultured phylotypes and exhibits a global distribution. Overall, the analysis indicates that Proteobacteria and Bacteroidetes of the Wadden Sea have a surprisingly high diversity, presumably a result of the signature of this ecosystem as a melting pot at the land-sea interface and comprising a great habitat variety.     

Numerical taxonomy of gram-negative, nonmotile, nonfermentative bacteria isolated during chilled storage of lamb carcasses.

A numerical taxonomic study using 75 characters was performed with 132 strains of gram-negative, nonmotile, nonfermentative bacteria selected on the basis of lack of motility and Gram reaction among 1,200 cultures isolated during aerobic storage of lamb carcasses. At the 80% similarity level (SSM), eight clusters were formed. Strains in clusters 1 to 6 could be identified as members of the family Moraxellaceae and, more specifically, as members of the Psychrobacter-[Moraxella] phenylpyruvica subgroup. Of these strains, clusters 1 and 2 (88 strains) were identified as [Moraxella] phenylpyruvica and cluster 3 (15 strains) was identified as Psychrobacter immobilis. Clusters 4, 5, and 6 were not identifiable with any species. Clusters 7 and 8 consisted of 14 strains considered nonmotile variants of Pseudomonas fragi. The highest separation indices corresponded to acid production from certain carbohydrates (melibiose, L-arabinose, and cellobiose). Although strains of Psychrobacter-Moraxella clusters were relatively frequently identified at the completion of slaughter, very few cultures were detected on spoiled carcasses. It appears, therefore, that this group of organisms has only low spoilage potential.     

Numerical taxonomy of gram-negative, nonmotile, nonfermentative bacteria isolated during chilled storage of lamb carcasses.

A numerical taxonomic study using 75 characters was performed with 132 strains of gram-negative, nonmotile, nonfermentative bacteria selected on the basis of lack of motility and Gram reaction among 1,200 cultures isolated during aerobic storage of lamb carcasses. At the 80% similarity level (SSM), eight clusters were formed. Strains in clusters 1 to 6 could be identified as members of the family Moraxellaceae and, more specifically, as members of the Psychrobacter-[Moraxella] phenylpyruvica subgroup. Of these strains, clusters 1 and 2 (88 strains) were identified as [Moraxella] phenylpyruvica and cluster 3 (15 strains) was identified as Psychrobacter immobilis. Clusters 4, 5, and 6 were not identifiable with any species. Clusters 7 and 8 consisted of 14 strains considered nonmotile variants of Pseudomonas fragi. The highest separation indices corresponded to acid production from certain carbohydrates (melibiose, L-arabinose, and cellobiose). Although strains of Psychrobacter-Moraxella clusters were relatively frequently identified at the completion of slaughter, very few cultures were detected on spoiled carcasses. It appears, therefore, that this group of organisms has only low spoilage potential.