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PubMed1984

Gas chromatography-mass spectrometry of mycolic acids as a tool in the identification of medically important coryneform bacteria.

The mycolic acid derivatives of 11 unidentified pathogenic coryneform bacteria were examined by TLC, GLC and GLC-mass spectrometry. The resulting mycolic acid profiles of the unidentified isolates were compared with those of type or reference strains of possibly related coryneform species, namely Corynebacterium bovis, C. diphtheriae, C. xerosis and Rhodococcus equi. It was apparent that most of the unidentified strains showed a distinctive mycolic acid profile, with predominant amounts of relatively high molecular weight mycolic acids (C32-C36) and a high degree of unsaturation, and could thus be distinguished from both C. bovis, which had exceptionally low molecular weight mycolic acids (C24-C30), and C. diphtheriae (C28-C34), which had large amounts of saturated mycolic acids. The mycolates of C. xerosis and R. equi (C28-C36) were generally similar to those of the unidentified coryneforms but their overall mycolic acid patterns were different from one another as well as the unidentified strains. The mycolic acid profiles exhibited by the pathogenic coryneforms examined here were very similar to one another but unlike that of any of the type or reference strains included in the study.

Actinomycetales↗
PubMed1997

Strategy for detection and identification of bacteria based on 16S rRNA genes in suspected cases of Whipple's disease.

The 16S ribosomal RNA (rRNA) gene of the phylogenetic subdivision containing gram-positive bacteria with a high G + C content was detected specifically in clinical specimens from patients suspected of having Whipple's disease. The primary structure of 16S rDNA amplified from clinical samples was determined by cloning and sequencing. Two sorts of sequences were identified: one corresponded exactly to the rRNA sequence of Tropheryma whippelii (GenBank accession no. M87484) while the other was related to that of members of the genus Corynebacterium. No sequence related to Mycobacterium spp. or Rhodococcus equi was observed. Exhaustive examination of negative specimens with broad-range eubacterial primers detected one sequence related to Enterobacteriaceae and another related to Enterococcus spp. To speed identification of T. whippelii, a nested amplification method was devised. A first amplification specific for the gram-positive bacteria subdivision was performed, followed by a second amplification with T. whippelii-specific primers. The amplified T. whippelii product was checked by digestion with AvaII, StuI, and PstI endonucleases. These techniques were applied to DNA extracted from seven intestinal biopsy samples, two cerebrospinal fluid samples and one articular fluid from patients suspected of having Whipple's disease. T. whippelii 16S rDNA was found in two of the biopsy samples, one of the cerebrospinal fluid samples and in the articular fluid.

Actinobacteria↗
PubMed2005

Novel mycolic acid-containing bacteria in the family Segniliparaceae fam. nov., including the genus Segniliparus gen. nov., with descriptions of Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov.

Four strains of novel, rapidly growing, acid-alcohol-fast-staining bacteria were characterized with a polyphasic approach. Isolates were received by the Centers for Disease Control and Prevention from domestic health department laboratories for reference testing as unidentifiable, clinical mycobacteria. Bacteria were rod-shaped and produced non-pigmented (white to beige), non-photochromogenic, smooth or wrinkled-rough colonies on Middlebrook 7H10 and 7H11 media at 33 degrees C. The smooth and wrinkled colony forms were representative of two species with 68.0 and 72.0 mol% DNA G+C content. The cell wall contained meso-diaminopimelic acid and mycolic acids. Species were characterized by cellular fatty acids of C10:0, C14:0, C16:1omega9t, C16:0, C18:1omega9c and 10-methyl C18:0 (tuberculostearic acid). HPLC analysis of mycolic acids produced a novel late-emerging, genus-specific mycolate pattern. TLC analysis demonstrated a novel alpha(+)-mycolate. Species were 98.9% similar by comparison of 16S rRNA gene sequences; however, the DNA-DNA association was <28 %. Phylogenetic analysis of 16S rRNA gene sequences demonstrated an association with Rhodococcus equi, although a DNA-DNA relatedness value of 2% did not support a close relationship. PCR analysis of a proposed, selected actinomycete-specific 439 bp fragment of the 65 kDa heat-shock protein was negative for three of the four isolates. The creation of Segniliparaceae fam. nov. is proposed to encompass the genus Segniliparus gen. nov., including two novel species, the type species Segniliparus rotundus sp. nov. and Segniliparus rugosus sp. nov., with the respective type strains CDC 1076(T) (=ATCC BAA-972(T)=CIP 108378(T)) and CDC 945(T) (=ATCC BAA-974(T)=CIP 108380(T)).

Actinomycetales↗
PubMed1985

Analysis of cellular fatty acids by gas chromatography as a tool in the identification of medically important coryneform bacteria.

The fatty acid methyl esters of nineteen unidentified pathogenic coryneform bacteria were analysed by gas-liquid chromatography and the resulting profiles were compared with those of type or reference strains of possibly related species, namely Caseobacter polymorphus, Corynebacterium bovis, C. diphtheriae, C. xerosis and Rhodococcus equi. All of the strains had distinct fatty acid profiles but most of them conformed to a general pattern, with high levels of octadecanoic acids and only trace amounts of 10-methyl octadecanoic acid (tuberculostearic acid). These profiles were very similar to those from C. diphtheriae and C. xerosis but could be differentiated from C. bovis, Cas. polymorphus, R. equi and two unidentified pathogenic strains which had significantly higher levels of tuberculostearic acid.

Actinomycetales↗
PubMed1993

Pharmacokinetics of single intravenous and single and multiple dose oral administration of rifampin in mares.

The disposition of rifampin in six healthy mares after single intravenous (i.v.) and oral (p.o.) doses and after seven oral doses of 10 mg/kg administered twice a day was investigated using a high performance liquid chromatographic (HPLC) method. Pharmacokinetic variables for rifampin determined using the HPLC method were comparable to variables reported from earlier studies utilizing a microbiological assay. Desascetylrifampin, a major metabolite of the parent compound, could not be detected in the serum but was detected at low concentrations in urine. Mean trough concentrations of rifampin increased from the first to the second dose of the multiple dose oral study and then remained unchanged through 72 h. At 84 h after the first dose (i.e. 12 h after the final dose) the rifampin concentration was significantly decreased (P = 0.001). The harmonic mean of the half-life of rifampin decreased significantly from 13.3 h after a single oral dose of 7.99 h after the seventh oral dose. The mean serum protein binding of rifampin over the concentration range of 2-20 micrograms/ml was 78%. Mean trough serum concentrations of unbound rifampin after multiple oral doses ranged from 0.67 micrograms/ml at 24 h to 0.40 micrograms/ml at 72 h. The mean unbound serum rifampin concentration at 84 h (i.e., 12 h after the final dose) was 0.30 micrograms/ml. Trough concentrations and the 84-h sample concentration of unbound rifampin exceeded the minimum inhibitory concentration for most gram positive bacterial isolates from horses reported in this study. All organisms with minimum inhibitory concentrations less than 0.125 micrograms/ml were considered susceptible. Based on the pharmacokinetics of rifampin after p.o. administration, we concur with the current dosage recommendation of 10 mg/kg twice a day by mouth. At this dose, most streptococci, Rhodococcus equi, and coagulase-positive staphylococci would be considered susceptible to rifampin.

Administration, Oral↗
PubMed1996

The incidence and consequences of failure of passive transfer of immunity on a thoroughbred breeding farm.

Circulating IgG concentration was determined between 12 and 24 hours after birth for 323 foals born on a Thoroughbred breeding farm over 3 consecutive years. The incidence of failure of passive transfer (FPT) of maternal immunoglobulins (foal circulating IgG concentration < 8 g/L) was found to be 9.6%. Foals born late in the season (October to December) were found to be at increased risk for the development of FPT. The degree of assistance required at parturition and the presence of a periparturient problem in the mare or foal also significantly influenced the subsequent incidence of FPT. Passive immune status significantly influenced the likelihood of foals developing septic illness (joint ill, septicaemia, pneumonia) in the first month of life, but had no significant effect on the development of diarrhoea or Rhodococcus equi pneumonia. The results of the current study support the value of routine monitoring of passive immune status and the early speculative treatment of foals considered to be at risk for the development of FPT.

Animals↗
PubMedDate not supplied

Bronchointerstitial pneumonia and respiratory distress in young horses: clinical, clinicopathologic, radiographic, and pathological findings in 23 cases (1984-1989).

Twenty-three foals, between 1 and 7 months old, with signs of acute respiratory distress, were examined at the Veterinary Medical Teaching Hospital (VMTH), University of California, Davis, between 1984 and 1989. Characteristic features included sudden onset of severe respiratory distress and tachypnea, cyanosis unresponsive to nasal oxygen, pyrexia, hypoxemia, hypercapneic respiratory acidosis, poor response to treatment, and histopathologic lesions of bronchiolitis and bronchointerstitial pneumonia. Seven of the 23 foals were normal before the onset of respiratory distress, 3 foals were found dead, and 13 foals were being treated for respiratory tract infections at the time of presentation. Laboratory data obtained for 13 horses showed increased plasma fibrinogen concentration (630.7 +/- 193 mg/dL), leukocytosis (18,607 +/- 7,784/microL), and neutrophilia (13,737 +/- 8,211/microL). Thoracic radiographs showed a diffuse increase in interstitial and bronchointerstitial pulmonary opacity and, in 5 foals, an alveolar pulmonary pattern of increased density was also seen. In 3 foals heavy interstitial infiltration proceeded to a coalescing nodular radiographic appearance. Microbiological culture of tracheobronchial aspirates (TBA) from 9 foals yielded bacterial growth, but no one bacterial species was consistently isolated. Microbiological culture of postmortem specimens of the lung from 6 foals yielded growth of bacteria that included Escherichia coli, Enterobacter spp., Proteus mirabilis, Klebsiella pneumoniae, Rhodococcus equi, or beta-hemolytic Streptococcus spp. Tracheobronchial aspirates from 4 foals and lung samples collected from a further 4 foals at necropsy yielded no bacterial growth. Cultures were not taken from two foals premortem or postmortem. Virologic examination of TBA, lung tissue, or pooled organ tissue from 12 foals was negative. Viral culture of TBA from 1 foal showed cytopathic effects and positive immunofluorescence for equine herpes virus type II (EHV-II). In addition to the 3 foals that were found dead, 11 foals died or were euthanatized. Pathologic lesions were limited to the lungs in 50% of the foals; the remainder also had bowel lesions suggestive of hypoxic injury. The predominant histopathologic pulmonary lesions included bronchiolitis, bronchiolar and alveolar epithelial hyperplasia, and necrosis. Many bronchioles were filled with mucoid and fibrinocellular exudate. The peribronchiolar interstitium and adjacent alveolar spaces were also infiltrated with inflammatory cells and contained proteinaceous edema fluid. Type II cell hyperplasia and hyaline membrane formation were observed in the majority of foals and in 2 foals alveolar multinucleate giant cells were also present.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

In-vitro susceptibility to antimicrobial drugs of bacterial isolates from horses in The Netherlands.

Minimum inhibitory concentrations (MICs) of 30 antimicrobial agents (including the hitherto unreported antimicrobial agents doxycycline, minocycline, vanomycin, 3 quinolones and 3 combinations of antimicrobial agents) for isolates of Salmonella spp. (20), Escherichia coli (17), Klebsiella spp. (8), Proteus spp. (7), Pseudomonas aeruginosa (7), Actinobacillus equuli (5), Rhodococcus equi (4), Streptococcus zooepidemicus (23), Streptococcus equisimilis (6), Streptococcus equi (4), coagulase-positive Staphylococcus spp. (20) and Taylorella equigenitalis (19) were determined using the agar dilution method. All isolates were of equine origin. MICs were compared with recommended MIC breakpoints. The results indicate that, for some of the pathogenic bacteria evaluated, susceptibility testing of isolates from the individual patient is essential to determine an appropriate antimicrobial treatment.

Animals↗
PubMed2006

In vitro activities of the lipopeptides palmitoyl (Pal)-Lys-Lys-NH(2) and Pal-Lys-Lys alone and in combination with antimicrobial agents against multiresistant gram-positive cocci.

The in vitro activities of the lipopeptides palmitoyl (Pal)-Lys-Lys-NH(2) and Pal-Lys-Lys against gram-positive cocci were investigated. Enterococci and streptococci demonstrated higher susceptibilities than staphylococci and Rhodococcus equi. A positive interaction was shown when the lipopeptides were combined with beta-lactams and vancomycin. These results suggest that lipopeptides are promising candidates for antimicrobial therapy for infections caused by gram-positive organisms.

Anti-Bacterial Agents↗
PubMed1998

In vitro susceptibilities of aerobic and facultative non-spore-forming gram-positive bacilli to HMR 3647 (RU 66647) and 14 other antimicrobials.

The comparative in vitro activity of the ketolide HMR 3647 (RU 66647) and those of structurally related macrolide-lincosamide-streptogramin compounds (erythromycin, roxithromycin, azithromycin, clarithromycin, josamycin, lincomycin, pristinamycin, and quinupristin-dalfopristin) as well as those of benzylpenicillin, doxycycline, vancomycin, teicoplanin, levofloxacin, and rifapentine against 247 aerobic and facultative non-spore-forming gram-positive bacilli were determined by an agar dilution method. The ketolide was active against most organisms tested except Corynebacterium striatum, coryneform CDC group 12, and Oerskovia spp. The frequency of resistance to erythromycin and other macrolides as well as that to lincomycin was high. Pristinamycin and, to a lesser extent, quinupristin-dalfopristin were very active, but resistance to these agents was present in some strains of Rhodococcus equi, Listeria spp., C. striatum, Erysipelothrix rhusiopathiae, and Oerskovia spp. HMR 3647 was very active against all erythromycin-sensitive and many erythromycin-nonsusceptible strains, especially Corynebacterium minutissimum, Corynebacterium pseudodiphtheriticum, Corynebacterium amycolatum, and Corynebacterium jeikeium. In vitro resistance to benzylpenicillin was common, but doxycycline, vancomycin, and teicoplanin were very active against most organisms tested except E. rhusiopathiae, against which glycopeptide antibiotics were not active. The in vitro activity of levofloxacin was remarkable, but resistance to this agent was common for C. amycolatum, Corynebacterium urealyticum, C. jeikeium, and Oerskovia spp. strains. Rifapentine was also very active in vitro against many organisms, but resistance to this agent was always present in E. rhusiopathiae and was very common in C. striatum and C. urealyticum.

Anti-Bacterial Agents↗
PubMed2003

Activities of ABT-773 against Listeria monocytogenes and coryneform bacteria of clinical interest.

The in vitro activities of ABT-773 were evaluated against 15 Listeria monocytogenes strains and 196 coryneform bacteria isolated from clinical samples. One hundred percent of the L. monocytogenes strains were inhibited by </=0.015 micro g of ABT-773/ml. MICs of ABT-773 ( micro g/ml) at which 50% of the isolates tested were inhibited (MIC(50)s) and MIC(90)s for other organisms were 0.125 and 0.5 (Corynebacterium amycolatum), 1 and >32 (Corynebacterium jeikeium), 0.03 and >32 (Corynebacterium minutissimum), >32 and >32 (Corynebacterium pseudodiphtheriticum and Corynebacterium urealyticum), 0.125 and >32 (Corynebacterium striatum), and 0.03 and 0.5 (Rhodococcus equi), respectively.

Anti-Bacterial Agents↗
PubMed2006

Discovery of natural atypical nonhemolytic Listeria seeligeri isolates.

We found seven Listeria isolates, initially identified as isolates with the Xyl(+) Rha(-) biotype of Listeria welshimeri by phenotypic tests, which exhibited discrepant genotypic properties in a well-validated Listeria species identification oligonucleotide microarray. The microarray gives results of these seven isolates being atypical hly-negative L. seeligeri isolates, not L. welshimeri isolates. The aberrant L. seeligeri isolates were d-xylose fermentation positive, l-rhamnose fermentation negative (Xyl(+) Rha(-)), and nonhemolytic on blood agar and in the CAMP test with both Staphylococcus aureus (S(-) reaction) and Rhodococcus equi (R(-) reaction). All genes of the prfA cluster of L. seeligeri, located in the prs-ldh region, including the orfA2, orfD, prfA, orfE, plcA, hly, orfK, mpl, actA, dplcB, plcB, orfH, orfX, orfI, orfP, orfB, and orfA genes, were checked by PCR and direct sequencing for evidence of their presence in the atypical isolates. The prs-prfA cluster-ldh region of the L. seeligeri isolates was approximately threefold shorter due to the loss of orfD, prfA, orfE, plcA, hly, orfK, mpl, actA, dplcB, plcB, orfH, orfX, and orfI. The genetic map order of the cluster genes of all the atypical L. seeligeri isolates was prs-orfA2-orfP-orfB-orfA-ldh, which was comparable to the similar region in L. welshimeri, with the exception of the presence of orfA2. DNA sequencing and phylogenetic analysis of 17 housekeeping genes indicated an L. seeligeri genomic background in all seven of the atypical hly-negative L. seeligeri isolates. Thus, the novel biotype of Xyl(+) Rha(-) Hly(-) L. seeligeri strains can only be distinguished from Xyl(+) Rha(-) L. welshimeri strains genotypically, not phenotypically. In contrast, the Rha(+) Xyl(+) biotype of L. welshimeri would not present an identification issue.

Animals↗
PubMed1987

Keratinolytic activity of cutaneous and oral bacteria.

A test was developed to measure the keratinolytic activity of cutaneous and oral bacteria. Keratin, labeled with fluorescein isothiocyanate, was used in a phosphate buffer (pH 7.2) with 1 mM dithiothreitol. The degradation of keratin was estimated by measuring the fluorescence of the degradation products in the supernatant of the reaction mixtures in a luminescence spectrometer. Several oral and cutaneous bacteria were investigated: Bacteroides gingivalis, Bacteroides intermedius, Treponema denticola, Actinomyces odontolyticus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus saprophyticus, Brevibacterium epidermidis, Brevibacterium lines, Corynebacterium glutamicum, Corynebacterium minutissimum, Corynebacterium ovis, and Rhodococcus equi. The dermatophyte Trichophyton rubrum was used as a control organism. The test offered a simple and quantitative method for the estimation of keratin degradation and enabled us to show keratinolytic activity in Trichophyton rubrum, S. epidermidis, S. haemolyticus, B. linens, B. epidermidis, Bacteroides gingivalis, and Treponema denticola. The keratinolytic activity was cell bound and heat sensitive. The presence of dithiothreitol stimulated the degradation of keratin to mainly high-molecular-weight products.

Bacteria↗
PubMed1989

Purification and characterization of two Listeria ivanovii cytolysins, a sphingomyelinase C and a thiol-activated toxin (ivanolysin O).

The strong bizonal hemolysis on blood agar and the positive CAMP reaction with Rhodococcus equi denotes the production of two different cytolytic factors by Listeria ivanovii. One was characterized as a thiol-activated (SH) cytolysin of 61 kilodaltons and was termed ivanolysin O (ILO) since data suggested that it is different from listeriolysin O, the SH-cytolysin produced by Listeria monocytogenes. The other is a 27-kilodalton hemolytic sphingomyelinase C that was found to be the cytolytic factor responsible for the halo of incomplete hemolysis synergistically enhanced by R. equi exosubstances. When thiol-disulfide exchange affinity chromatography and gel filtration were applied to the purification of ILO from concentrated L. ivanovii culture supernatants, the copurification of the two cytolysins was observed. This phenomenon seems to be due to the formation of intermolecular disulfide bonds between ILO and the sphingomyelinase, since the latter was found to contain free SH groups, not essential for the activity. These SH groups could react with the single cysteine residue characteristically present in the SH-cytolysins, forming a dimeric cytolytic complex. The purification of ILO was achieved by a further gel filtration with a reducing agent (dithiothreitol) in the eluent. A method for the purification of the sphingomyelinase based on selective sequestration of ILO from the L. ivanovii concentrated culture supernatant by the SH cytolysin target molecule cholesterol and thiol-disulfide affinity chromatography is described.

Bacterial Toxins↗
PubMed1982

Routine test for in vitro differentiation of pathogenic and apathogenic Listeria monocytogenes strains.

The exosubstance of Rhodococcus equi in a prepurified form strongly enhanced the hemolytic effect of certain strains of Listeria monocytogenes. The strains which produced positive synergic hemolysis with this exosubstance were also pathogenic for guinea pigs and white mice. The other strains, which remained nonhemolytic in the presence of the R. equi. exosubstance, were apathogenic for those animals. A routine test was devised for the in vitro determination of the pathogenicity of L. monocytogenes strains.

Animals↗
PubMed1986

Microplate technique to determine hemolytic activity for routine typing of Listeria strains.

Because the hemolysis produced by Listeria monocytogenes and Listeria seeligeri on blood agar is frequently difficult to interpret, we developed a microplate technique for the routine determination of hemolytic activity with erythrocyte suspensions. This microtechnique is a simple and reliable test for distinguishing clearly between hemolytic and nonhemolytic strains and could be used instead of the CAMP (Christie-Atkins-Munch-Petersen) test with Staphylococcus aureus in the routine typing of Listeria strains. Furthermore, our results suggest that the quantitation of the hemolytic activity of the Listeria strains, along with the D-xylose, L-rhamnose, and alpha-methyl-D-mannoside acidification tests, allows the differentiation of L. monocytogenes, L. seeligeri, and Listeria ivanovii. We also observed that the treatment of erythrocytes with crude exosubstances of rhodococcus equi, Pseudomonas fluorescens, Acinetobacter calcoaceticus, and S. aureus enhanced the hemolytic activity of all Listeria strains with this characteristic.

Acinetobacter↗
PubMed1992

High-performance liquid chromatography of corynomycolic acids as a tool in identification of Corynebacterium species and related organisms.

A high-performance liquid chromatography (HPLC) study of 307 strains of Corynebacterium species and related taxa revealed that strains classified as "Corynebacterium aquaticum"; "Corynebacterium asperum"; and Centers for Disease Control (CDC) groups 1, 2, A-3, A-4, A-5, B-1, B-3, E, F-2, and I-2 as well as some unidentified coryneforms do not contain any corynomycolic acids; therefore, they should not be included in the genus Corynebacterium. Such an HPLC method of identification permitted the correct assignment to the genus Rhodococcus of two unpigmented strains of coryneform bacteria whose mycolic acid profiles were comparable to those of Rhodococcus equi. Bacteria belonging to CDC groups ANF-1, ANF-3, F-1, G-1, G-2, and I-1, as well as some other Corynebacterium sp. strains, yielded corynomycolic acid HPLC patterns related to those of Corynebacterium species. Either similarities or differences were observed in the corynomycolic acid profiles of Corynebacterium species tested after culture on sheep blood agar and/or sheep blood agar supplemented with Tween 80, which demonstrated that identification at the species or group level is possible. However, Corynebacterium striatum and CDC group I-1 bacteria as well as CDC group G-1 and group G-2 bacteria had indistinguishable HPLC patterns. Conversely, some variations were observed within some species as Corynebacterium xerosis, C. striatum, and Corynebacterium minutissimum. The evaluation procedure of this HPLC method by mass spectrometry analysis of isolated eluted peaks revealed that analytical reverse-phase HPLC alone does not provide any structural information, since isomers with identical polarities coeluted as a single peak. Nevertheless, HPLC is a rapid and reliable method for identification of corynomycolic acid-containing bacteria in the clinical microbiological laboratory.

Chromatography, High Pressure Liquid↗
PubMed1994

Alpha-mannosidase: a rapid test for identification of Arcanobacterium haemolyticum.

A 4-h alpha-mannosidase test for identification of Arcanobacterium haemolyticum strains (n = 139) and differentiation of A. haemolyticum from Actinomyces pyogenes strains (n = 30) and other gram-positive rods was evaluated. Practically all A. haemolyticum strains (138 of 139) and the Listeria monocytogenes type strain were alpha-mannosidase positive, while all A. pyogenes strains and Corynebacterium (n = .25) strains as well as the Rhodococcus equi and Erysipelothrix rhusiopathiae type strains were negative. The rapid alpha-mannosidase test, in conjunction with a Gram stain and catalase and reverse CAMP tests, was useful in identification of A. haemolyticum and in differentiation of A. haemolyticum from A. pyogenes and Corynebacterium spp.

Actinomyces↗