Semantide- and chemotaxonomy-based analyses of some problematic phenotypic clusters of slowly growing mycobacteria, a cooperative study of the International Working Group on Mycobacterial Taxonomy.

During previous cooperative numerical taxonomic studies of slowly growing mycobacteria, the International Working Group on Mycobacterial Taxonomy described a number of strains whose taxonomic status was ambiguous. A new study of DNA, RNA, and proteins from 66 of these organisms was performed to correlate their properties with phenotypic clustering behavior; the results of this study permitted 51 of the strains studied to be assigned to known species. The methods used to characterize the semantides included nucleotide sequencing and assessment of levels of semantide relatedness by affinity binding techniques, including whole DNA-DNA hybridization, probe hybridization, and antibody binding. There was good overall agreement between the phenotypic and chemotaxonomic clusters and the groups of organisms identified by semantide analyses. Our results supported the conclusion that we should continue to rely on polyphasic taxonomy to provide satisfactory systematic resolution of members of the genus Mycobacterium. We identified no single 16S rRNA interstrain nucleotide sequence difference value that unequivocally defined species boundaries. DNA-DNA hybridization remains the gold standard, but common resources are needed to permit DNA-DNA hybridization analyses to be made available to laboratories that are not prepared to use this technology. One of the large novel clusters which we studied corresponds to the recently described species Mycobacterium interjectum, a pathogen that resembles the nonpathogen Mycobacterium gordonae phenotypically. We also identified strains that appear to represent ribovars of Mycobacterium intracellulare which do not react with the commercial diagnostic probes that are currently used for identification of this species. Other branches or clusters consisted of too few strains to permit a decision about their taxonomic status to be made.

Fourth report of the cooperative, open-ended study of slowly growing mycobacteria by the International Working Group on Mycobacterial Taxonomy.

The open-ended study of the International Working Group on Mycobacterial Taxonomy is an ongoing project to characterize slowly growing strains of mycobacteria that do not belong to well-established or thoroughly characterized species. In this fourth report we describe two numerical taxonomic clusters that represent subspecies or biovars of Mycobacterium simiae, one cluster that encompasses the erstwhile type strain of the presently invalid species "Mycobacterium paraffinicum," one cluster that is phenotypically very similar to Mycobacterium avium and Mycobacterium intracellulare but may be a separate genospecies, one cluster that appears to be phenotypically distinct from M. avium but reacts with a nucleic acid probe specific for M. avium, and three tentatively defined clusters in proximity to a cluster that encompasses the type strain of Mycobacterium malmoense. Of special practical interest is the fact that one of the latter three clusters is composed of clinically significant scotochromogenic bacteria that can be misidentified as the nonpathogenic organism Mycobacterium gordonae if insufficient biochemical tests are performed.

Draft results of a workshop to develop guidelines for studies involving microbial incidence or populations in the oral cavity.

The following five outlines are the results to date of the Workshop held in Rockville, Maryland, in January, 1990. The topics considered in these outlines are: (1) validation of immunological and/or nucleic acid identification probes, (2) cross-calibration of methods and/or laboratories for multi-laboratory cooperative studies, (3) choosing methods for identifying or describing microbial populations appropriate to the scientific question asked, (4) microbial ecology methods (e.g., population dynamics) for the oral cavity studies, and (5) epidemiological methods (e.g., incidence, risk factor analysis) for oral microbial studies. Each topic was considered by two independent groups of participants and later rationalized into one. These outlines are meant to be working outlines for evolution of a set of guidelines to advise on designing studies with microbial incidence and/or population components. We are publishing this preliminary version to elicit comment and criticism from people who did not attend the Workshop. (Attendance at the Workshop was necessarily limited by both space and funds). Some of the topic outlines have been condensed to save Journal space. The full document is available on request. The next stage will be an open forum to gather and discuss further amplification of the "Guidelines", planned for April 17, 1991, Acapulco, Mexico, in conjunction with the IADR/AADR Meeting. Written comments and requests for further information should be sent to the Workshop organizer (MIK) at the above address.(ABSTRACT TRUNCATED AT 250 WORDS)

The CODATA/IUIS Hybridoma Data Bank: development of a hybrid system to handle complex data relationships.

System design for the Hybridoma Data Bank, a database of comprehensive information on immunoreagents for use by scientists in diverse disciplines, is described. Unique problems include: use of nomenclature from diverse fields that is neither static nor standard; the need for two representations of the database--textual for readability and numeric for complex search capabilities, analysis and data compression; and a method of translating between the two representations of the database.

Changes in antimicrobial resistance in fecal bacteria associated with pig transit and holding times at slaughter plants.

Fecal coliforms, fecal streptococci, and antimicrobial resistance (AMR) associated with various pig transit and holding times were investigated at slaughter plants. Changes in the relative abundance of two biotypes of Streptococcus faecium were associated with transit and holding of pigs, although approximately 20% of the isolates were unidentified. The greatest variety of coliforms was isolated from porcine feces after short transit (2 h) or holding (3 h) times and was qualitatively similar to those from pigs on farms. Isolates from pigs with longer average transit or holding times were almost all Escherichia coli (four biotypes). Streptococcal resistance to most antimicrobial agents was significantly greater (P less than 0.05) in isolates from live pigs at slaughter plants than in those from pigs at farms and was apparent after a short transit time (2 h). Streptococci from pigs held an average of 15 h were less resistant to most antimicrobial agents than those from pigs held 3 or 43 h. When compared with short transit times, moderate transit times (6 h) were associated with significantly decreased (P less than 0.05) coliform resistance and decreased resistance transfer but a greater diversity of AMR patterns. Holding pigs overnight (14 h) was associated with lowered coliform resistance to several antimicrobial agents, compared with the resistance of isolates from pigs held 3 or 39 h. A substantial increase (18 to 48%) in the ability to transfer streptomycin resistance was demonstrated in coliforms from pigs held 39 h, when compared with those from pigs held 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary chlortetracycline on the antimicrobial resistance of broiler faecal Streptococcaceae.

A breeder flock and a control group of progeny birds were fed antimicrobial-free rations; a second group of progeny received rations supplemented with 50 g chlortetracycline (Ctc)/ton. Effects of dietary Ctc on the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Antimicrobial resistance (AMR) pattern diversity and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1321 (97%) of 1360 isolates into eight species or biotypes. The largest cluster (n = 659, 48%) was a biotype of Streptococcus faecalis. Three clusters were biotypes of Streptococcus faecium and contained 580 isolates (42%). The isolates were susceptible to ampicillin and almost uniformly resistant to methicillin, neomycin, streptomycin, sulfadiazine and tetracycline. There were 54 and 47 different AMR patterns, including 0 to 11 and 1 to 11 resistance determinants, in isolates from control and Ctc-fed birds, respectively. Modal AMR patterns for Strep. faecalis and one biotype of Strep. faecium were very similar for all three groups of birds. However, modal patterns in a second biotype of Strep. faecium varied considerably for all three groups. Interpretation of AMR pattern diversities were equivocal among biotypes from both progeny groups. The variable distribution of isolates, proportions of resistant strains, modal patterns and diversity indices among the progeny were probably due to their exposure to different environmental sources of bacteria.

Effects of dietary chlortetracycline on the antimicrobial resistance of porcine faecal streptococcaceae.

Breeding pigs and one-half of their progeny were fed antimicrobial-free rations; the other half of the progeny received rations supplemented with 100 g of chlortetracycline (Ctc)/ton. Effects of dietary Ctc with respect to the distribution of species and biotypes of faecal Gram-positive cocci and their relative resistance to 12 antimicrobial agents were studied. Diversity of antimicrobial resistance (AMR) patterns and modal AMR patterns were determined for bacterial species common to all three groups. Numerical taxonomic analysis placed 1140 of 1150 isolates (99%) into 10 groups. Three of these were biotypes of Streptococcus faecium and contained the largest number of isolates (n = 934, 81%). Streptococcus faecalis, Strep. morbillorum, Pediococcus halophilus and Gemella haemolysans also were isolated. Generally, the proportion of tetracycline-resistant strains for a species or biotype was greater from pigs fed Ctc, although differences were not significant (P greater than 0.05). There was a significant difference (P less than 0.05) among all the groups for the percentage of penicillin-resistant strains in a biotype of Strep. faecium. Overall, 57 and 43 different AMR patterns, including 2 to 11 and 1 to 11 resistance determinants, were demonstrated in isolates from control pigs and pigs fed Ctc, respectively. Modal AMR patterns in species and biotypes were the same from both progeny groups, except for Strep. faecium. AMR pattern diversity was decreased for strains from pigs fed Ctc. Similar proportions of resistant strains from each group of progeny pigs were accompanied by decreased AMR pattern diversity in strains from pigs fed Ctc. These results indicated a change in distribution of AMR phenotypical patterns, rather than a change in overall frequency of individual resistant phenotypes.

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia.

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of conventional and miniaturized biochemical techniques for identification of animal streptococcal isolates.

Human clinical streptococcal isolates can be identified rapidly by means of commercially available miniaturized biochemical systems, in contrast to animal and environmental isolates which may require extensive characterization using conventional methods. Streptococcal isolates (n=548) from fresh animal feces of cattle, swine, and broiler chickens were tested by means of conventional biochemical and physiological techniques, and also with a miniaturized technique in which conventional formulations were dispensed in 0.1 ml volume into microtiter plates. Agreement of the positive feature frequencies of the two methods were compared. Results from the tolerance tests in the two methods were generally in good agreement. However, the miniaturized method tended to give false negative results in some carbohydrate fermentation tests. Agreement between the 2 methods ranged from 100% for bile esculin tests to 71% for raffinose fermentation. Cluster analysis of the conventional method data indicated that there were 11 biochemically related groups of isolates, 2 of which were identified asStreptococcus faecalis, andS. morbillorum. Half of the isolates biochemically resembledS. faecium. Errors of miniaturized tests occurred mainly in certain tests and in certain biochemically related clusters of isolates. The data indicate that further investigation of experimental conditions such as medium formulation and inoculum size could lead to a successful miniaturized technique for testing animal streptococcal isolates.

Diagnostic probability matrix for identification of slowly growing mycobacteria in clinical laboratories.

A probability matrix is presented for identification of slowly growing mycobacteria that are likely to be encountered in clinical laboratories. The matrix includes 23 features that are useful for identifying members of 14 species or species complexes. The computer program identifies strains as a function of the ID (identification) score, which measures the discrimination among possible alternative identifications, and the R (ratio) score, which measures the degree of fit to the most likely taxa. It is not necessary to employ all 23 tests when initiating an identification; the program will suggest additional tests to perform when a partial data set fails to yield a definitive identification. Two independent sets of cultures comprising a total of 1,212 strains were used to test the matrix. Correct diagnoses were based on clustering behavior in numerical taxonomic analysis with larger numbers of features. The probable efficiencies with the two sets were 94.2 and 83.4%, respectively, and the accuracy of the definitive identifications for both sets exceeded 95%. A discussion is presented of situations when it may be appropriate to override an R score that has caused the rejection of an identification and to thereby enhance the efficiency.

Characterization of some groups of gram-negative nonfermentative bacteria by the carbon source alkalinization technique.

A total of 541 gram-negative nonfermentative bacterial strains comprising 26 species and unclassified groups were characterized by routine diagnostic and carbon substrate alkalinization techniques. These microorganisms were tested for the ability to cause alkalinization of a basal medium on a total of 217 substrates. We found that 58 carbon substrates had some discriminatory potential. We also performed 30 routine diagnostic tests. The results of these studies were evaluated by numerical taxonomy techniques. A cluster analysis of the results by the Jaccard coefficient method identified 30 clusters at the 45% level. We identified 39 tests that separated most of the groups. Groups of similar organisms or organisms that were difficult to identify were analyzed, and tests that were differential were identified. Because of variability within the clusters, further studies utilizing deoxyribonucleic acid-deoxyribonucleic acid homologies should be undertaken.

Method for coding data on protozoan strains for computers.

Traditionally, observations on the nature of protozoa have been published in periodicals or books, or remain buried in research notebooks. The retrieval and processing of information on a particular species or strain are dependent solely upon individual investigators. Although various modern methods have been applied to the study of protozoa, no attempt has been made to develop a system with which information on protozoan strains can be stored, retrieved easily, and processed for various analyses by computer technology. Based upon an existing system for encoding data on bacterial strains, a complementary system applicable to protozoan strains was developed and is described herein.

Analysis of the exudate produced by Streptococcus mutans SL-1 colonies of sucrose-containing agar media.

The watery exudate produced by Streptococcus mutans SL-1 colonies on sucrose-containing agar media was found to contain about 7% (wt/vol) of a water-soluble, branched dextran, 4% sucrose, and smaller (less than 1%) amounts of fructose, Folin-phenol-positive material, and lactic acid.

Growth of Dictyostelium discoideum in the presence of antibiotics.

Cultures of the amoeboid slime mold, Dictyostelium discoideum, were grown in the presence of various antibiotics and antimetabolites. Results indicate that many antibiotics may be used in amoeba cultures to help minimize contamination (for example: polymyxin B, kanamycin, tetracycline, neomycin, oxacillin, ampicillin, cephalothin, and erythromycin). A few potent antibiotics were found to be unsuitable (colistin, nystatin, amphotericin B, actinomycin, and cycloheximide); the mechanisms of the inhibitions observed were not investigated further.

Distribution of enzymes forming polysaccharide from sucrose and the composition of extracellular polysaccharide synthesized by Streptococcus mutans.

The distribution of polysaccharide-forming activity from sucrose was investigated in cultures of three strains of Streptococcus mutans by using an assay which conveniently determines total polysaccharide. The enzymatic activity for polysaccharide formation from sucrose is almost exclusively extracellular. The ratio of the fructan to glucan in the polysaccharide differs among the three strains investigated. The enzymatic activity for the formation of polysaccharide from sucrose has been shown to be bound to the cell-free polymer itself.