Phenotypic and molecular characterization of three clinical isolates of Mycobacterium interjectum

Introduction of molecular biology-based technology into an Australian mycobacterial reference laboratory has resulted in the identification of three isolates of Mycobacterium interjectum in the past 12 months. Conventional phenotypic methods failed to identify the species of these isolates, and high-performance liquid chromatography found that only one of the three isolates had a mycolic acid pattern similar to that of the type strain. In contrast, all three isolates were rapidly identified as M. interjectum by 16S rRNA gene sequence analysis. Two isolates were recovered from the lymph nodes of children with cervical lymphadenitis, confirming the pathogenicity of this organism. However, the third isolate was obtained from the sputum of an elderly male with chronic lung disease without evidence of clinical or radiological progression, suggesting that isolation of M. interjectum should not imply disease. With the increasing use of molecular biology-based technology in mycobacterial laboratories, M. interjectum may be recognized more frequently as a pathogen or commensal organism.     

A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".     

Etiological agents of mycobacterioses in pet birds between 1986 and 1995

Between May 1986 and June 1995, mycobacteriosis was diagnosed by histology and microscopy in 204 (3.8%) of 5,345 necropsied pet birds. The predominant macroscopic changes were enlargement of the liver and spleen and thickening of intestinal walls. Attempts to cultivate mycobacteria were made in 110 cases. Acid-fact bacilli grew in 66 specimens (60%) only. In 18 cases we failed to obtain subcultures. Therefore, species identification could be performed for only 48 isolates. Identification was carried out by conventional biochemical tests as well as by PCR-mediated sequencing of the 16S rRNA gene. The majority of the isolates were Mycobacterium genavense (34 isolates), followed by M. avium complex (8), M. fortuitum (2), M. tuberculosis (2), M. gordonae (1), and M. nonchromogenicum (1). The significance of M. genavense as a zoonotic agent remains to be determined.     

A probability matrix for the identification of campylobacters, helicobacters and allied taxa

A probabilistic identification matrix for campylobacteria, comprising 76 phenotypic characters and 37 taxa, is described. The accuracy and integrity of the matrix was evaluated using established computer-assisted methods. Certain taxa (for example, Campylobacter concisus and Camp. gracilis) demonstrated significant phenotypic diversity; previous data corroborated these findings. Differentiation between a few pairs of taxa proved difficult, although discriminatory characteristics were noted in each of these cases. The results indicate that most campylobacteria can be identified accurately and objectively with phenotypic tests when probabilistic methods of data assessment are employed.     

Molecular identification and epidemiology of animal mycoplasmas

Molecular genetic techniques show a high potential for rapid and accurate identification of Mycoplasma species isolated from animals. An important field of application for such methods is the differentiation of species and/or subspecies which are phenotypically closely related, but which show significant differences in epidemiological impact. This need is particularly important for the mycoplasmas of the "mycoides group", which are phenotypically and phylogenetically very closely related. Molecular typing techniques based on 16S rRNA genes give straightforward phylogenetic answers on the species level. For more refined methods of subtyping at the subspecies level, the use of defined genes characteristic of certain Mycoplasma species or clusters is recommended. Genetic fingerprinting, especially insertion sequence typing has proved to be a valuable tool for subtyping and strain identification in particular of vaccine strains and for epidemiological investigations.     

Branched-DNA assay for detection of the mecA gene in oxacillin-resistant and oxacillin-sensitive staphylococci

The identification of methicillin-resistant staphylococcus isolates in the clinical laboratory has typically been performed by using methods that detect phenotypic expression of resistance determinants. However, these methods may be difficult to interpret and some isolates do not express resistance until selective pressure is administered. Assays that detect genetic determinants are not subject to these limitations and have been effective in distinguishing isolates that are capable of expressing the resistance phenotype. In this study, a novel branched-DNA (bDNA) hybridization assay was used to test for the mecA gene in 416 clinical staphylococcal isolates. The results were compared with those obtained by a PCR-based assay and oxacillin disk diffusion. For 155 Staphylococcus aureus and 261 coagulase-negative Staphylococcus isolates, the bDNA assay and PCR results were 100% concordant. Among the S. aureus isolates, 20 were MecA+ and 135 were MecA-. For the coagulase-negative staphylococci, 150 were MecA+ and 111 were MecA-. The results from the genotypic detection methods were compared with those obtained by oxacillin disk diffusion. No discrepancies were detected among the S. aureus isolates; however, 10 coagulase-negative isolates were MecA+ but oxacillin sensitive and 1 isolate was MecA- but oxacillin resistant. Oxacillin resistance was induced in 6 of the 10 MecA+ isolates previously classified as oxacillin sensitive. These results suggest that the bDNA method described here is a sensitive and efficient method for detection of methicillin resistance in staphylococci and that genetic detection methods may be useful for detection of potential methicillin resistance in the clinical laboratory.     

Facklamia ignava sp. nov., isolated from human clinical specimens

Two strains of a hitherto-undescribed gram-positive, catalase-negative coccus isolated from human sources were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains are genealogically identical and constitute a new line close to, but distinct from, Facklamia hominis. The unknown bacterium was readily distinguished from F. hominis by biochemical tests and electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Facklamia ignava sp. nov. The type strain of Facklamia ignava is CCUG 37419.     

Description of Gemella sanguinis sp. nov., isolated from human clinical specimens

Six strains of a hitherto undescribed gram-positive, catalase-negative, facultatively anaerobic coccus isolated from human sources were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains were genealogically identical and constitute a new subline within the genus Gemella. The unknown bacterium was readily distinguished from Gemella haemolysans, Gemella bergeriae, and Gemella morbillorum by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Gemella sanguinis sp. nov. The type strain is CCUG 37820(T).     

Characterization of glycopeptide-resistant enterococci from a Swiss hospital

Between August 1994 and September 1996, 28 glycopeptide-resistant enterococci (GRE) were isolated from 8 infected patients and 11 intestinal carriers hospitalized at the University Hospital of Geneva. Identification to the species was made by both phenotypic (API 20 STREP and Rapid ID 32 STREP systems, and Vitek Gram Positive Identification Card) and genotypic methods using a multiplex PCR assay developed also for the determination of the genotype of glycopeptide resistance (vanA, vanB, vanC1, and vanC2-C3 genes). Fifteen isolates were identified as Enterococcus faecium, 8 as E. gallinarum, 4 as E. faecalis, and 1 as E. hirae. All of the phenotypic identification methods failed to differentiate some isolates of E. gallinarum from E. faecium, or vice versa. Both vanA (n = 18) and vanB (n = 4) glycopeptide resistance genotypes were found. For the first time, the vanB determinant was found in two isolates of E. gallinarum. Two patients were colonized by two different species containing the vanA gene and one by two different species containing the vanB gene. All vanA isolates were highly resistant to both vancomycin and teicoplanin except for three isolates which were susceptible to teicoplanin. Molecular typing by pulsed-field gel electrophoresis showed identical or similar patterns among E. faecium isolates with the vanA gene in five patients for whom the epidemiological link could not be always elucidated. This study emphasizes the necessity of utilizing both phenotypic and genotypic methods to characterize GRE.     

Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by amplification of the 16S-23S ribosomal DNA spacer

Differentiation between Mycobacterium tuberculosis and M. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains of Mycobacterium. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates of M. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers of M. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.     

rpoB sequence analysis as a novel basis for bacterial identification

Comparison of the sequences of conserved genes, most commonly those encoding 16S rRNA, is used for bacterial genotypic identification. Among some taxa, such as the Enterobacteriaceae, variation within this gene does not allow confident species identification. We investigated the usefulness of RNA polymerase beta-subunit encoding gene (rpoB) sequences as an alternative tool for universal bacterial genotypic identification. We generated a database of partial rpoB for 14 Enterobacteriaceae species and then assessed the intra- and interspecies divergence between the rpoB and the 16S rRNA genes by pairwise comparisons. We found that levels of divergence between the rpoB sequences of different strains were markedly higher than those between their 16S rRNA genes. This higher discriminatory power was further confirmed by assigning 20 blindly selected clinical isolates to the correct enteric species on the basis of rpoB sequence comparison. Comparison of rpoB sequences from Enterobacteriaceae was also used as the basis for their phylogenetic analysis and demonstrated the genus Klebsiella to be polyphyletic. The trees obtained with rpoB were more compatible with the currently accepted classification of Enterobacteriaceae than those obtained with 16S rRNA. These data indicate that rpoB is a powerful identification tool, which may be useful for universal bacterial identification.     

Increased dopamine turnover in the putamen after MPTP treatment in common marmosets

Nocardia species are ubiquitous in the environment and may be found in the soil. They are generally responsible for sporadic pulmonary diseases acquired by inhalation of spores, with secondary localizations in the central nervous system and subcutaneous tissues. There is no absolute evidence for person to person transmission. Presumptive outbreaks of nocardiosis were observed in immunocompromised patients, more frequently in kidney transplant patients than in cardiac transplant patients. Nocardia spp., being present in dust particles, closure and disinfection of the transplantation unit with formaldehyde arrested the sequence of cases of nocardiosis. The original sources of the Nocardia sp. remain doubtful. Other possible sources of contamination are other patients, medical staff and the hospital environment. The first studies of Nocardia spp. typing were based on the detection of extracellular antigens, on the susceptibility of actinomycete strains to killer yeasts, and on the biochemical profiles with fluorogenic substrate. The use of molecular typing techniques have given very promising results. Analysis of plasmid profiles is an interesting way to compare the identity of isolates, although the reliability of this method depends of the presence of plasmids in the isolates. Other typing methods, including analysis of restriction length fragment polymorphism of total DNA, ribosomal DNA fingerprinting, require further investigations to evaluate their discriminating power or to be easily interpretable, whereas a random amplified polymorphic DNA (RAPD) assay was successful for epidemiological purposes. Progress in epidemiological analysis of cases of nocardiosis will be consistent when an improved diagnosis of this infection (molecular and serological diagnosis) will be available, when the genetic diversity of Nocardia spp. isolates will be better known, and when molecular typing, that hold promise in complementing investigations of outbreak of these infections, will be systematically performed when an abnormal increase of cases of nocardiosis in a population with risk factors is observed.     

PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples

Three sets of primers were designed for PCR detection and differentiation of Campylobacter jejuni and Campylobacter coli. The first PCR assay was designed to coidentify C. jejuni and C. coli based on their 16S rRNA gene sequences. The second PCR assay, based on the hippuricase gene sequence, identified all tested reference strains of C. jejuni and also strains of that species which lack detectable hippuricase activity. The third PCR assay, based on the sequence of a cloned (putative) aspartokinase gene and the downstream open reading frame, identified all tested reference strains of C. coli. The assays will find immediate application in the rapid identification to species level of isolates. The assays combine with a protocol for purification of total DNA from fecal samples to allow reproducible PCR identification of campylobacters directly from stools. Of 20 clinical samples from which campylobacters had been cultured, we detected C. jejuni in 17, C. coli in 2, and coinfection of C. jejuni and Campylobacter hyointestinalis in 1. These results were concordant with culture and phenotypic identification to species level. Strain typing by PCR-restriction fragment length polymorphism of the flagellin (flaA) gene detected identical flaA types in fecal DNA and the corresponding campylobacter isolate. Twenty-five Campylobacter-negative stool samples gave no reaction with the PCR assays. These PCR assays can rapidly define the occurrence, species incidence, and flaA genotypes of enteropathogenic campylobacters.     

Comparison of the API Candida system with the AUXACOLOR system for identification of common yeast pathogens

Two commercial systems for the identification of yeasts were evaluated by using 159 clinical isolates that had also been identified by conventional biochemical and morphological methods. The API Candida system correctly identified 146 isolates (91.8%), and the AUXACOLOR system correctly identified 145 isolates (91.2%). However, of the 146 isolates identified by the API Candida system, 23 required supplemental biochemical tests or morphological assessment to obtain the correct identification. The AUXACOLOR system gave no identification in 13 cases (8.2%), while the API Candida system gave an unreadable profile in only one case. Incorrect identifications were more common with the API Candida system (12 isolates; 7.5%) than with the AUXACOLOR system (1 isolate; 0.6%).     

Chronic gypsum fertiliser ingestion as a significant contributor to a multifactorial cattle mortality

A number of high-resolution molecular typing systems have been developed in recent years. Their availability raises the new issues of selecting the method (s) best suited for a particular purpose and interpreting and communicating typing results. Most of the currently available methods are comparative only: they allow testing of a sample of isolates for delineation of those closely related from those markedly different in genomic backgrounds. This approach is adequate for outbreak investigation, allowing determination of clonal spread in a microenvironment and identification of the source of infection. Comparative methods with sufficient resolution for most pathogens include restriction fragment-length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and arbitrarily primed and randomly amplified polymorphic DNA-polymerase chain reaction (PCR) analysis. For surveillance systems, monitoring clonal spread and prevalence in populations over extended periods of time requires library typing systems. These must be standardized, must have a high throughput, and must use a uniform nomenclature. Promising or validated methods include serotyping, insertion sequence fingerprinting, ribotyping, PFGE, amplified fragment-length polymorphism (AFLP), infrequent-restriction-site amplification PCR, interrepetitive element PCR typing (rep-PCR) and PCR-RFLP of polymorphic loci. PCR methods generating arrays of size-specific amplicons (AFLP, rep-PCR) can be more reproducibly analyzed by using denaturing polyacrylamide gel or capillary electrophoresis with automated laser detection. Binary probe typing systems appear optimal and should be enhanced further through use of DNA chip technology. In these systems, amplification of polymorphic regions is followed by solid-phase hybridization with a reference panel of sequence-variant specific probes. The resulting binary type results allow determination of reproducible, numeric profiles. However, interpretation and nomenclature of typing results for large-scale surveillance purposes still require a better understanding of population structure and microevolution of most microbial pathogens.     

A functional role for osteopontin in experimental crescentic glomerulonephritis in the rat

Fifty two isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and geographical locations, were heterogeneous in terms of molecular and phenotypic characteristics, and represented taxa which could not be accommodated by the current classification of four subspecies. Generally, there was incongruence between the molecular (PCR, RAPD and ribotyping) and phenotypic methods in terms of cluster membership. By PCR, 6 groups were described of which Group 1 encompassed 12 isolates including the type strain of A. salmonicida subsp. smithia. Group 2 accommodated 23 isolates including the reference cultures of subspecies achromogenes and masoucida. The named culture of Haemophilus piscium was recovered in Group 6. By ribotyping and RAPD, the reference cultures were recovered in separate groups. All methods pointed to the uniqueness of subspecies smithia. Most isolates contained 2-6 plasmids, of 2.3 to 150 kb in length. Nevertheless, all isolates possessed certain key characteristics, including Gram-negativity, and the absence of motility.     

Comparison of different DNA fingerprinting techniques for molecular typing of Bartonella henselae isolates

Seventeen isolates of Bartonella henselae from the region of Freiburg, Germany, obtained from blood cultures of domestic cats, were examined for their genetic heterogeneity. On the basis of different DNA fingerprinting methods, including pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, repetitive extragenic palindromic (REP) PCR, and arbitrarily primed (AP)-PCR, three different variants were identified among the isolates (variants I to III). Variant I included 6 strains, variant II included 10 strains, and variant III included only one strain. By all methods used, the isolates could be clearly distinguished from the type strain, Houston-1, which was designated variant IV. A previously published type-specific amplification of 16S rDNA differentiated two types of the B. henselae isolates (16S rRNA types 1 and 2). The majority of the isolates (16 of 17), including all variants I and II, were 16S rRNA type 2. Only one isolate (variant III) and the Houston-1 strain (variant IV) comprised the 16S rRNA type 1. Comparison of the 16S rDNA sequences from one representative strain from each of the three variants (I to III) confirmed the results obtained by 16S rRNA type-specific PCR. The sequences from variant I and variant II were identical, whereas the sequence of variant III differed in three positions. All methods applied in this study allowed subtyping of the isolates. PFGE and ERIC-PCR provided the highest discriminatory potential for subtyping B. henselae strains, whereas AP-PCR with the M13 primer showed a very clear differentiation between the four variants. Our results suggest that the genetic heterogeneity of B. henselae strains is high. The methods applied were found useful for typing B. henselae isolates, providing tools for epidemiological and clinical follow-up studies.     

The ethics of death-hastening or death-causing palliative analgesic administration to the terminally ill

The Phene Plate (PhP) system is a commercially available typing system based on the measurements of kinetics of selected biochemical reactions of bacteria grown in liquid medium in 96-well microplates. The system uses numerical analysis to identify biochemical phenotypes among the tested strains. In the present study, a set of 16 discriminatory tests were used to differentiate 117 strains of Vibrio cholerae O1 from MExico and Bangladesh. The stability of PhP types of 16 isolates under different storage temperatures and after repeated subcultures were also evaluated. The PhP system had a reproducibility of 95%. Storage either at +4 degrees C or -70 degrees C, did not affect the reactions of the isolates, whereas 4 strains (25%) stored at room temperature and 5 strains (31%) subjected to 30 consecutive subcultures, exhibited minor changes in their biochemical reactions. Endemic isolates of V. cholerae O1 from Bangladesh were more diverse (diversity index = 0.84 to 0.93) than epidemic isolates from Mexico (diversity index = 0.73). Using a collection of 33 heterogeneous isolates of classical biotype of vibrios, PhP typing and ribotyping were compared. PhP typing discriminated more types (n = 23) than ribotyping (n = 5), whereas a combination of both yielded 27 types. The PhP system appears to be a simple, reliable and highly discriminating method for typing of V. cholerae, and may prove especially useful as a first screening method in epidemiological studies of V. cholerae.     

Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis

A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp. RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolates of N. otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.