Evaluation of differential media for the identification of Corynebacterium genitalium and Corynebacterium pseudogenitalium (group JK corynebacteria)

Tween purple agar containing 1% fructose (TFP agar) differentiated Corynebacterium genitalium from C. pseudogenitalium, which respectively formed colorless and yellow colonies after 72 h incubation at 37 degrees C aerobically or in 5-10% CO2 in air. Thus TFP agar is a differential medium. Corynebacteria-like colonies grown on nonspecific urethritis (NSU) chocolate agar from urogenital material were identified as C. genitalium, C. pseudogenitalium, or commensals when subcultured on TPF agar. TFP agar was unsuitable for their primary isolation since the commensals turned the medium yellow with 24 h incubation. Gentamicin cannot be employed as a selective agent in medium for the isolation of these corynebacteria. TFP agar containing 10 micrograms/mL entamicin inhibited most strains of C. pseudogenitalium and C. genitalium isolated from urogenital infections. It did not inhibit isolates of these corynebacteria from cancer patients or suppress the normal bacterial flora of the urogenital tract. Evidence that gentamicin-resistant strains are characteristic of nosocomial infections is presented.     

Deoxyribonucleic acids of Corynebacterium genitalium and Corynebacterium pseudogenitalium: their genome molecular weights, base ratios, and DNA relatedness with other corynebacteria involved in urinary tract infections

Chromosomal deoxyribonucleic acids of Corynebacterium genitalium and Corynebacterium pseudogenitalium were isolated and analysed spectrophotometrically. Their genome molecular weights ranged from 1.1 X 10(9) to 1.6 X 10(9). The guanine-plus-cytosine content of C. genitalium ranged from 60.0 to 63.3%, whereas that of C. pseudogenitalium ranged from 56.1 to 58.7%. Five strains of C. genitalium showed relatively low levels of DNA relatedness to each other ranging from 35 to 64%. In contrast, most strains of C. pseudogenitalium showed high levels of DNA relatedness to each other ranging from 71 to 89%. Selected strains of C. genitalium and C. pseudogenitalium showed low levels of DNA relatedness (49 to 60%) to other corynebacterial species involved in urinary tract infection. Data obtained in this study indicate that all strains of C. genitalium consist of genetically divergent organisms while the most strains of C. pseudogenitalium belong to a single species.     

The identification of nondiphtheritic bacteria of the genus Corynebacterium and the determination of their antibiotic sensitivity]

A total of 221 patients with nonspecific inflammatory processes were studied with a view to determine the occurrence and species composition of microorganisms of the genus Corynebacterium on the mucous membrane and in the secretions of their genitals. Different representatives of this genus were detected in 23.1 +/- 3.5% of patients with cervicitis and endometritis and in 48.7 +/- 5.7% of patients with prostatitis. Among the isolated bacteria of this genus C. pseudogenitalium, C. genitalium and C. xerosis, as well as Corynebacterium cells of group JK, occurred most frequently. The strains under study were found to have high antibiotic resistance. On the basis of their cultural features, growth rate, the degree of contamination of the genitals and sensitivity to antibiotics, bacteria of this genus were differentiated into macro- and microcoryneforms.     

Infection et fertilité —Corynebacterium seminale: point de vue du bactériologiste

The authors present two independent studies designed to identify corynebacteria isolated from the semen of patients consulting for infertility. Corynebacteria were identified by conventional biochemical and physiological tests and by determination of volatile fatty acids. In the first study based on 420 patients, the commonest species were Corynebacterium seminale (synonym C. glucuronolyticum) found in 7.4% of specimens, CDC group G (5%) and C. amycolatum (3.8%). Of the 92 semen specimens with more than 103 cfu/ml, 44 were positive for corynebacteria, including 15 C. seminale strains, whereas streptococci, staphylococci and enterobacteriacae were found in 23, 18 and 6 of the 420 specimens, respectively. The presence of C. seminale was more frequently associated with a high bacteria count than the other corynebacteria (p<0.02). In the second study, we compared the presence of corynebacteria in the semen of 1,902 patients with semen indices. C. seminale was present at levels greater than 103 cfu/ml in 2.7% of these specimens, while several other species of corynebacteria were detected in 5.3% of cases. Normal motility was found in only 25.4% of semen specimens with a high C. seminale count in contrast with 45% of specimens containing similar counts of other corynebacteria. These studies demonstrate that the isolation rates from human genital specimens and their clinical implications are different according to the species isolated. Microbiologists should be aware of the need to accurately identify these corynebacteria for further in vitro or in vivo studies on genital infections.     

Heavy metal effects on Proteus mirabilis Superoxide dismutase production

Abstract Levels of genomic DNA relatedness were determined using a SI nuclease procedure for reference bacteria identified as biotypes of Corynebacterium diphtheriae , biovars of Corynebacterium pseudotuberculosis , and ' Corynebacterium ulcerans '. These results showed that the three species are separate taxa at the genomospecies level whereas biotypes and biovars are closely related genomically within each species. Phylogenetic analyses of small-subunit rDNA sequences revealed that ' Corynebacterium ulcerans ' forms a tight cluster with Corynebacterium pseudotuberculosis within the robust branch that groups all Corynebacterium sequenced to date. Therefore, we propose that the species incertae sedis ' C. ulcerans ' should be conclusively recognized as a distinct species within the genus Corynebacterium with strain CCUG 2708 = NCTC 7910 as type strain. This species is characterized by urease production and fermentation of glycogen.     

Bacteriocins of phytopathogenic Corynebacterium species.

The majority (85% of all strains tested) of 12 phytopathogenic Corynebacterium species produced bacteriocin(s) on nutrient broth--yeast extract (NBY) medium. All C. nebraskense, C. michiganense, C. insidiosum, C. oortii, and C. iranicum strains produced bacteriocin(s). The optimal conditions for production of 23 distinct bacteriocins by eight species of Corynebacterium generally were 20 degrees C and 4 days of incubation on NBY or on modified Burkholder's agar that lacked peptone (MBAL). Production in liquid was marginal and not augmented by adding mitomycin C. Bacteriocins generally had little effect on other strains within a species but were inhibitory to other species. Most bacteriocins appeared to be bactericidal proteins resistant to heat (75 to 80 degrees C, 30 min) but sensitive to proteolytic enzymes. Some strains of C. nebraskense, C. michiganense, C. insidiosum, and C. flaccumfaciens produced two bacteriocins which were clearly differentiated by varying or testing one or more of the following: conditions for production, the indicator, heat stability, and susceptibility to proteolysis. Within certain limitations, a convenient and reproducible typing scheme was devised for strain and species differentiation of most phytopathogenic corynebacteria.     

Genetic Identification of Members of the Genus Corynebacterium at Genus and Species Levels with 16S rDNA-Targeted Probes

16S rRNA gene-targeted probes were designed for the identification of corynebacteria at the genus and species levels. The genus-specific probe hybridized all clinically important members of the genus Corynebacterium and could distinguish them from other coryneform bacteria and phylogenetically related high G + C% gram-positive bacteria, including Actinomyces, Rhodococcus, Gordona, Nocardia, Streptomyces, Brevibacterium and Mycobacterium. The species-specific probes for C. jeikeium and C. diphtheriae could differentiate these two species from other members of this genus. The probes were used to select corynebacteria among gram-positive clinical isolates which had been tentatively identified as corynebacteria by biochemical tests. We screened 59 strains with the genus-specific probe; 51 strains hybridized to the genus-specific probe, 8 did not. Of the 51 strains that hybridized to the genus-specific probe, 1 hybridized to the C. diphtheriae species probe and 13 hybridized to the C. jeikeium species probe. The 8 strains that did not hybridize to the genus probe were further characterized by analyzing cell wall diaminopimelic acid and partial 16S rRNA sequencing. The results indicated that these strains were distributed in the genera Arthrobacter and Brevibacterium.     

Characterization of Corynebacterium group JK by whole-cell protein patterns

A total of 102 strains received as Corynebacterium 'group JK' were characterized by SDS-PAGE of their whole-cell proteins. Numerical taxonomy based on the protein pattern absorbance profiles indicated that 91 of the strains formed a cluster. Seventy strains isolated in the UK were identified as group JK, indicating the increasing detection of this group as opportunistic pathogens. Fine differences between strain patterns were visible but it was not possible to associate these with any particular clinical source.     

Sensitivity of aerobic bacteria and Candida species to chlormidazole hydrochloride in vitro

Over 100 bacterial stains, including Candida sp., gram-positive cocci and some gram-negative bacilli, were tested. The majority of microorganisms was isolated from man. Only one bacterial strain (5 micrograms/mL) and 76% of Candida strains were sensitive to chlormidazole HCl (10 micrograms/mL). It may be assumed that most infections with Candida sp. will respond to therapy with drugs containing chlormidazole HCl. Relatively simple and inexpensive tube test may serve to evaluate microbial sensitivity to this agent, especially in case of isolates from patients.     

Genetic structure of Corynebacterium diphtheriae strains isolated in Russia during epidemics of various intensity

The genetic structure of C. dipthteriae toxigenic strains isolated in Russia during the period of more than 50 years was analysed. The use of the method of ribotyping made it possible to register 17 C. diphtheriae ribotypes. The study revealed that the genetic structure of C. diphtheriae population varied in the dynamics of the epidemic process: each epidemic cycle characterized by predominant spread of epidemic strains of definite biovars and ribotypes. Thus, C. diphtheriae strains of biovar gravis, ribotype M11, dominated in the 40-60 years and C. diphtheriae strains of biovar mitis, closely related ribotypes M1 and M1v, dominated in the 80 years. During the last epidemic rise of diphtheriae morbidity in the 90 s C. diphtheriae strains of biovar gravis, closely related ribotypes G1 and G4, dominated among circulating strains. The proportion of these ribotypes began to increase 3 years before the rise of morbidity. The data of microbiological monitoring are recommended for use in the prognostication of the development of the epidemic process of diphtheria infection.     

Biochemical properties of Corynebacterium amycolatum strains

C. amycolatum is the most commonly isolated nonlipophilic species of Corynebacterium from clinical samples. However, the lack of good commercial identification tests in microbiology laboratories causes some difficulties in C. amycolatum diagnostics. We decided to examine biochemical and enzymatic properties of isolated strains and analyze the occurrence of particular biochemical profiles (biotypes). Perhaps it would let improve the identification schemes. 70 strains of C. amycolatum were analyzed. The estimation of biochemical properties consisted of the results of API Coryne and API ZYM tests (bioMérieux), the ability of excreting of protease, esterase, lipase and lecithinase. Analyzed strains had various biochemical and enzymatic properties. Almost all strains fermented glucose (98.6%) and maltose (95.7%) and produced pyrasinamidase (94.3%). All strains produced alkaline phosphatase and phosphohydrolase, and 95.7%--acid phosphatase. Biotypes of particular strains were determined on the biochemical reactions included in the API Coryne tests. In the group of 70 strains 21 profiles were distinguished among which 3100325 biotype (35.7%) was dominant. The lipolysis was defined on Tween 20, Tween 40, Tween 60, Tween 80 medium and with the API ZYM test usage. All strains produced esterase-lipase (esterase C-8), 95.7% of strains-esterase C-4, and 21.4% lipase C-14. Among analyzed strains 18.6% hydrolyzed Tween 20, 14.3% Tween 60, and 1.4% Tween 40. None of these strains demonstrated lipase and lecithinase activity. Difficulties in concerning C. amycolatum as pathogens justify further investigations.     

A numerical taxonomic study of the Pseudomonas flora isolated from poultry meat

Pseudomonas strains were isolated from both fresh and cold-stored broiler skin. Phenotypically-based numerical taxonomic techniques were used to characterize the isolates and 36 reference strains. For this purpose, Biolog GN Microplates, API 20NE and a number of other biochemical tests were used. Jaccard clustering revealed the predominance of four major Pseudomonas groups: Ps. fragi, Ps. lundensis, strains belonging to Ps. fluorescens biovars and an unidentified group of strains displaying a high degree of similarity to Ps. fluorescens biovars. Within Ps. fluorescens, biovar A was best represented. The marked proteolytic character of members of Ps. fluorescens biovars A, B and C, as well as of members of the unidentified cluster, supports their possible role in the origin of organoleptic defects. In the Ps. lundensis cluster, a distinct group of Ps. lundensis-like species was found. Further genotypic studies should be carried out to clarify the taxonomic status of the Ps. lundensis-like strains and that of the unidentified group resembling Ps. fluorescens biovars A and B.     

Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.     

Specific gene probe for detection of biotyped and serotyped Listeria strains

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.     

Recognition of biovar C of Fusobacterium necrophorum (Flügge) Moore and Holdeman as Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex Prévot 1940)

The cellular morphology, colonial morphology, biochemical properties, DNA base compositions, and DNA-DNA homolgies of three biovars of Fusobacterium necrophorum were examined. Some differences were found among the three biovars in cellular morphology, colonial morphology, and biochemical properties. The guanine-plus-cytosine contents of DNAs from biovar C strains Fn521T (T = type strain), Fn522, and Fn520 were 30.4, 29.3, and 28.0 mol%, respectively, and the guanine-plus-cytosine contents of DNAs from strains VPI 2891 (biovar A) and VPI 6161 (biovar B) were 31.3 and 32.0 mol%, respectively. Labeled DNA from biovar C strain Fn521T exhibited 96 and 82% relatedness to DNAs from biovar C strains Fn522 and Fn520, respectively; however, it exhibited only about 10% relatedness to DNAs from strains of biovars A and B. Labeled DNAs from strains VPI 2891 and VPI 6161 exhibited more than 70% relatedness to each other, but about 6 to 20% relatedness to DNAs from biovar C strains. Therefore, Fusobacterium pseudonecrophorum sp. nov., nom. rev. (ex Prévot 1940) is proposed for Fusobacterium necrophorum biovar C. The type strain is strain Fn521 (= JCM 3722).     

The genome stability in Corynebacterium species due to lack of the recombinational repair system

Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales. Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently. We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species. This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria. This is the first report that the genome structures have been conserved in free-living bacteria such as C. efficiens and C. glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species. The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.     

Specific gene probe for detection of biotyped and serotyped Listeria strains.

A total of 284 strains of Listeria, including all known serovars and biovars together with Listeria grayi and Listeria murrayi, were biotyped and serotyped. Biotyping and serotyping could be done in 2 days. A gene probe encoding a delayed hypersensitivity factor (DTH) was used in the detection of pathogenic biotypes and serotypes of the tested strains. The gene was found in all 117 tested Listeria monocytogenes strains of serogroups 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4c, 4d, 4e, 4ab, and 7. It was also present in Listeria ivanovii. Of 78 L. monocytogenes strains of serogroup 4b, 77 strains contained the gene, whereas it was absent in all 10 tested L. monocytogenes strains of serogroup 4a. Furthermore, the gene was absent in Listeria seeligeri, L. grayi, L. murrayi, and L. innocua of serogroups 3c, 4b, and 6a and in L. welshimeri of serogroups 1/2b, 3b, 6a, and 6b. Since L. monocytogenes and L. ivanovii are the only two biotypes of the genus Listeria considered pathogens, the data obtained indicate that the DNA probe tested may be a useful tool in the detection of virulent Listeria isolates in clinical, environmental, and food samples.     

Species of Pseudomonas obtained at 7°C and 30°C during aerobic storage of lamb carcasses

A total of 268 strains of Pseudomonas isolated during storage life of lamb carcasses was identified to species level. One-hundred and thirteen strains obtained at 30°C were Ps.fragi (51), Ps. lundensis (17), Ps. fluorescens biovars I (10), III (9) and VI (1), Ps. putida biovar A (8 strains) and unidentified (17 strains). Species and biovars isolated at 7°C (155) were Ps. fragi (101), Ps. lundensis (32), Ps. fluorescens biovar I (6), Ps. putida biovar A (8) and unidentified (8). Numerical analysis (82% S _SM, UPGMA) of 'psychrotrophic' and 'mesophilic' strains resulted in the formation of nine and eight clusters respectively. The dendrograms obtained exhibited similar structures. Most of the strains of Ps. lundensis and Ps. fragi clustered together. Strains of this latter species also joined the type strain of Ps. testosteroni and appeared included with phenons containing the Ps. putida strains. There were clusters made up exclusively of strains assigned to one biovar or group ( Ps. fluorescens biovars I and II and unidentified). A high level of similarity was observed between clusters of Ps. fluorescens biovar I and those containing the Ps. fragi-Ps. lundensis complex (>74% S _SM) and Ps. lundensis (>80%). The recovery of pseudomonads seemed to be affected by the sampling day.     

Analysis of antibiotic susceptibility and extrachromosomal DNA content of Ruminococcus albus and Ruminococcus flavefaciens

Seventeen Ruminococcus albus and Ruminococcus flavefaciens strains have been screened for naturally occurring antibiotic resistance, as determined by zones of inhibition from antibiotic disks. These strains were also examined for extrachromosomal DNA content. All strains screened are resistant to low levels (10-200 micrograms/mL) of streptomycin. In contrast to the previously reported data, we have found that R. flavefaciens C-94 is now susceptible to both kanamycin and tetracycline. However, R. flavefaciens FD-1 is not susceptible to kanamycin (minimum inhibitory concentration (MIC) = 40 micrograms/mL). Furthermore, R. albus 8 is resistant to tetracycline (MIC = 40 micrograms/mL), and erythromycin (MIC = 100 micrograms/mL). Six freshly isolated strains showed resistance to tetracycline (35-70 micrograms/mL), and all tetracycline-resistant strains also showed resistance to minocycline. None of these Ruminococcus determinants share homology with the streptococcal tetL, tetM, or tetN determinants. All 17 strains were screened for extrachromosomal DNA content. Nine different techniques for the detection and isolation of extrachromosomal DNA were tested. However, owing to difficulties in demonstrating or isolating plasmid DNA, it has not been possible to determine if these antibiotic resistance genes are plasmid borne. Evidence is presented to suggest that the presence of oxygen may affect the quality of the DNA obtained from Ruminococcus.     

Species and biotype identification ofSerratia strains associated with insects

Forty-eightSerratia strains associated with insects were, identified to species level and biotyped according to recent taxonomic schemes. Each strain was submitted to 36 biochemical tests, including 23 carbon source utilization tests. Twenty-eight strains were assigned to eight biotypes ofSerratia marcescens: A1a, A2a, and A6a (pigmented biotypes: 18 strains); and A3a, A3b, A4a, A5, and TCT (nonpigmented biotypes: 10 strains). However, biotypes A8a, A8b, and A8c, which are frequently involved in nosocomial infections, were not found in insects. Ninetten strains were identified asS. liquefaciens (S. proteamaculans) biotypes C1a (12 strains), C1c (4 strains), C1d (2 strains), and one atypicalS. liquefaciens strain. Only one strain was identified asS. marinorubra (a nonchitinolytic species). The recent emergence ofSerratia in human pathology calls for a reevaluation of the idea of usingSerratia to biologically control insects.