Susceptibility testing of Danish isolates of Capnocytophaga and CDC group DF-2 bacteria

Twelve Capnocytophaga and seven DF-2 strains were tested for their susceptibility to 14 antimicrobial agents using an agar dilution and an agar diffusion method. Twenty-three other antibiotics were evaluated using the diffusion test only. All strains were fully susceptible to penicillin, ampicillin, cefuroxime, cefotaxime, erythromycin, clindamycin, chloramphenicol, doxycycline, rifamycin and ofloxacin using both methods. Clindamycin, rifamycin and cefotaxime were most active. Using agar dilution some strains were susceptible to gentamicin, but agar diffusion showed total resistance. One Capnocytophaga strain was susceptible and another moderately susceptible to metronidazole, other strains were resistant. The agar diffusion test showed that both Capnocytophaga and DF-2 were resistant to most other aminoglycosides, to fosfomycin, polymyxin and trimethoprim. All strains of both taxa were fully susceptible to piperacillin, cefoxitin, imipenem and fusidic acid and showed different susceptibilities to the other agents. Susceptibility testing by means of agar diffusion using an enriched chocolate agar and 5% CO2 atmosphere could be used to test Capnocytophaga and DF-2 strains and gives sufficient accuracy for routine use, when revised inhibition zone breakpoints are employed.     

Characterization of CDC group DF-3 by cellular fatty acid analysis.

Fourteen strains of Centers for Disease Control group DF-3 bacteria were examined for cellular fatty acid composition to evaluate their chemical relatedness to known bacterial species and groups. The fatty acids were liberated from whole cells by base hydrolysis, methylated, and analyzed by capillary gas-liquid chromatography. All group DF-3 strains possessed a distinct fatty acid profile which was characterized by large amounts (24%) of 12-methyltetradecanoate (a-C15:0), moderate amounts of saturated iso-branched-chain acids (i-C14:0 and i-C15:0), and small to moderate amounts of both branched- and straight-chain hydroxy acids (3-OH C15:0, i-3-OH C16:0, 3-OH C16:0, and i-3-OH C17:0). This fatty acid profile was unique as compared with the profiles of other bacteria we have previously tested but was most similar to the profiles of Capnocytophaga species.     

Clinical and microbiological observations on CDC group DF-3, a gram-negative coccobacillus.

Sequential stool cultures submitted for routine culture were screened for the presence of CDC group DF-3. Of 690 specimens, 11 (1.6%) yielded moderate to heavy growth of DF-3. Information on the 11 patients from whom these specimens were obtained showed that 4 had a history of prolonged diarrheal disease that resolved after specific therapy to eradicate DF-3, while for the other 7 patients no clear role could be established. Microbiological characterization of the stool isolates and 10 CDC strains of DF-3 suggested the presence of two subtypes within the group. Antibiotic susceptibility studies showed DF-3 to be relatively resistant to a wide variety of antibiotics.     

Characteristics of CDC group 3 and group 5 coryneform bacteria isolated from clinical specimens and assignment to the genus Dermabacter.

Over a 1-year period, 11 isolates (including 5 from blood cultures) of the recently described CDC group 3 and group 5 coryneform bacteria were derived from clinical specimens and compared with reference strains. Biochemical characteristics indicated a very close relationship between CDC group 3 and group 5 coryneform bacteria. The ability of CDC group 3 and the inability of CDC group 5 coryneform bacteria to ferment xylose were the only reactions that were different for the two taxa. Chemotaxonomic features of the two groups included the presence of meso-diaminopimelic acid, a lack of mycolic acids, and the presence of predominantly branched cellular fatty acids, a combination found among gram-positive rods only in Brevibacterium spp., Brachybacterium faecium, and Dermabacter hominis. 16S rRNA gene sequence analysis revealed that CDC group 3 and group 5 coryneform bacteria are members of the genus Dermabacter, which to date has been isolated exclusively from human skin.     

Chemical and cultural characterization of CDC group WO-1, a weakly oxidative gram-negative group of organisms isolated from clinical sources.

Ninety-six strains of weakly oxidative gram-negative rods isolated primarily from clinical specimens form a distinct group that has been designated Centers for Disease Control (CDC) group WO-1 (WO stands for weak oxidizer). The phenotypic characteristics of CDC group WO-1 were most similar to those of Comamonas acidovorans, Pseudomonas mallei, and CDC pink coccoid group III. The WO-1 group can be differentiated from C. acidovorans by the oxidation of glucose (often weak and sometimes delayed), motility by means of one or two polar flagella, and, when positive, the complete reduction of nitrate and nitrite. Motility and usually the failure to produce arginine dihydrolase distinguish this group from P. mallei. The WO-1 strains differ from the pink coccoid group III by the absence of pink growth pigment, the lack of predominantly coccoid cellular morphology, and usually the inability to produce acid from xylose. The cellular fatty acid compositions of 29 group WO-1 strains were characterized by large amounts of C16:0 and C16:1w7c; smaller amounts of C18:1w7c, C14:0, C12:0, and 3-OH-C10:0; and trace to small amounts of C15:1w6 and C17:0 acids. The fatty acid profile of WO-1, compared with the profiles of other bacteria we have tested previously, was most similar to the profiles of two phenotypically different organisms, Comamonas terrigena (a nonoxidative, multipolar gram-negative rod) and Chromobacterium violaceum (a fermentative gram-negative rod). Ubiquinone-8 was the major quinone in the five WO-1 strains examined. Eighty-five percent of the WO-1 strains were isolated from human specimens. Thirty-three percent were from blood, and 10% were from cerebrospinal fluid.     

Phenotypic and genetic characterization of clinical isolates of CDC coryneform group A-3: proposal of a new species of Cellulomonas, Cellulomonas denverensis sp. nov

CDC coryneform group A-3 bacteria are rare human pathogens. In this study, six group A-3 isolates (two from blood, one from cerebrospinal fluid, and one each from homograft valve, lip wound, and pilonidal cyst) were compared to the type strains of phenotypically related organisms, Cellulomonas fimi, Cellulomonas hominis, Oerskovia turbata, and Sanguibacter suarezii, and characterized by phenotypic, chemotaxonomic, and genotypic studies. DNA-DNA reassociation analysis identified two genomic groups, and phylogenetic analysis of the 16S rRNA gene sequence identified the taxonomic positions of these groups to genus level. Two groups were defined, and both were more closely related to Cellulomonas species: one group of three strains, for which we propose the new species Cellulomonas denverensis sp. nov., with the type strain W6929 (ATCC BAA-788(T) or DSM 15764(T)), was related to C. hominis ATCC 51964(T) (98.5% 16S rRNA gene sequence similarity), and the second group of three strains was related to C. hominis ATCC 51964(T) (99.8 to 99.9% 16S rRNA gene sequence similarity). The definition of this new Cellulomonas species and the confirmation of three strains as C. hominis serve to further clarify the complex taxonomy of CDC coryneform group A-3 bacteria and will assist in our understanding of the epidemiology and clinical significance of these microorganisms.     

Use of chemotaxonomy as an aid to differentiate among Capnocytophaga species, CDC group DF-3, and aerotolerant strains of Leptotrichia buccalis.

Four strains of fastidious gram-negative rods, thought to be Capnocytophaga species (formerly CDC group DF-1 or Bacteroides ochraceus) or CDC group DF-3 on the basis of conventional phenotypic criteria, were also analyzed for cellular fatty acid (CFA) composition. It was found that the CFA compositions of these strains were qualitatively incorrect for those taxa. Subsequently, it was determined that all four bacteria were in fact aerotolerant strains of Leptotrichia buccalis, based on biochemical reactions, CFA composition, and lactic acid as the major end product of glucose fermentation. It is recommended that, in addition to conventional cultural and biochemical criteria, all strains of Capnocytophaga or CDC group DF-3 should also be tested for metabolic end products of fermentation and CFA composition as essential adjuncts for identification.     

Primary identification of Microbacterium spp. encountered in clinical specimens as CDC coryneform group A-4 and A-5 bacteria.

Over nearly two decades, 13 yellow- or orange-pigmented, fermentative gram-positive rods belonging to the genus Microbacterium were encountered in clinical specimens. All 13 strains, 10 of which came from blood cultures, were initially identified as CDC coryneform group A-4 and A-5 bacteria according to the scheme of Hollis and Weaver for the identification of gram-positive rods. The clinical isolates were compared with the type strains of the six species constituting the genus Microbacterium as well as with three Microbacterium strains isolated from hospital environments. By biochemical methods only 5 of 13 clinical isolates could be identified to species level. Peptidoglycan analysis proved to be a valuable tool for differentiation between Microbacterium spp. and related genera, whereas cellular fatty acid analysis did not allow species identification within the genus Microbacterium. The 22 Microbacterium strains studied were, in general, susceptible to antimicrobial agents used in the treatment of infections caused by gram-positive rods. This report is the first one concerning the isolation of Microbacterium strains from clinical specimens. The sources as well as the mode of transmission remain to be established.     

Peritonitis associated with a CDC group EO-3 organism.

A 63-year-old female with chronic renal failure on continuous ambulatory peritoneal dialysis developed chronic peritonitis. A CDC group EO-3 organism was isolated from the peritoneal dialysis fluid on five occasions over a period of 4 months. This is the first reported isolation of this organism in which it is associated with a patient on continuous ambulatory peritoneal dialysis.     

Identification and characterization of clinical isolates of members of the Staphylococcus sciuri group

A total of 28 staphylococcal isolates from human clinical specimens belonging to the Staphylococcus sciuri group were identified and characterized. The API Staph and ID32 STAPH correctly identified S. sciuri and S. lentus but not S. vitulinus strains. Identification to the subspecies level was possible only by a PCR-based method.     

Comparable endotoxic properties of lipopolysaccharides are manifest in diverse clinical isolates of gram-negative bacteria

In general there is a poor correlation between serum lipopolysaccharide (LPS; the biologically active constituent of endotoxin) levels and mortality in septic patients. The objective of this study was to determine if chemical, structural, or biological differences among LPS from different clinical isolates of gram-negative bacteria might explain this discrepancy. LPS preparations were made using the hot phenol-water extraction method from eight clinical isolates of gram-negative bacteria. As a percentage of the total weight of the LPS, the phosphate content ranged from 3.0 to 13.8% (average, 6.7 +/- 3.6%), and the 2-keto-3-deoxyoctonate content ranged from 1.9 to 27.4% (average, 8.9 +/- 8.5%). These values were not dissimilar to those obtained for a reference endotoxin. In a standard measure of LPS activity, the Limulus amoebocyte lysate assay, there was approximately a twofold difference between the least and most active preparations. The two preparations with the greatest difference in their ability to elicit the secretion of tumor necrosis factor alpha from a mouse peritoneal macrophage cell line were similar in lethality when administered to mice sensitized to the effects of LPS by D(+)-galactosamine. These relatively minor differences in LPS activity seem unlikely to explain the generally observed discrepancy between serum endotoxin levels and mortality in patients with gram-negative sepsis.     

Achromobacter species (CDC group Vd): morphological and biochemical characterization.

Twenty-three isolates of Achromobacter species (CDC group Vd) were examined morphologically and biochemically. Gram stains revealed gram-variable bacilli frequently curved or hooked at one pole and often coryneform in shape and arrangement. Electron microscopy revealed the presence of extracellular material in polar accumulations and demonstrated the polar flagella arrangement seen by light microscopy to be lateral. Two colony types were produced; one was minute and watery at 24 h (35 degrees C) progressing to large, mucoid colonies at 48 h, and the other type was shiny, glistening, opaque but nonmucoid. All isolates grew on MacConkey agar and produced catalase, oxidase, and urease. Most grew on salmonella-shigella agar, reduced nitrate to nitrite and gas, hydrolyzed esculin, deaminated phenylalanine (2 to 4 days) and produced H2S in triple sugar iron agar (4 to 12 days). Oxidation of carbohydrates was weak, delayed, and limited to glucose and xylose. Two isolates also oxidized maltose, mannitol, and sucrose. The ability of miniaturized "nonfermenter" kits to identify Achromobacter species was tested. The Minitek (Baltimore Biological Laboratory, Cockeysville, Md.) and N/F (Corning, Roslyn, N.Y.) systems, respectively, identified 21 and 19 of the 23 isolates, whereas the Oxi/Ferm (Roche, Nutley, N.J.) identified 13 and the API 20E (Analytab Products, Plainview, N.Y.) identified only 3.     

Phenotypic characteristics, fatty acid composition, and isoprenoid quinone content of CDC group IIg bacteria.

Eleven strains of eugonic, nonoxidative, gram-negative rods isolated from clinical specimens formed a distinct group that was designated CDC group IIg. Five of the 11 isolates were from wounds. The phenotypic characteristics of CDC group IIg were most similar to those of Weeksella species, with the major difference being that CDC group IIg strains grew on MacConkey agar in 1 to 2 days, did not hydrolyze gelatin, and did not produce urease. All 11 strains of CDC group IIg possessed a distinct fatty acid profile that was characterized by large amounts (19 to 29%) of 18:1 omega 7c, 16:0, and 16:1 omega 7c, moderate amounts (6 to 10%) of 3-OH-14:0 and 14:0, and smaller amounts (1 to 2%) of 18:2, 18:0, and 3-OH-16:0. This fatty acid profile differs from those of Weeksella species by the absence of branched-chain fatty acids. CDC group IIg contains ubiquinone-8, as opposed to menaquinone-6 in Weeksella species. The isolates were susceptible to a variety of antimicrobial agents, including the aminoglycosides, tetracyclines, quinolones, sulfonamides, and polymyxin B.     

Antimicrobial susceptibility pattern of clinical isolates of non-fermentative bacteria

152 nonfermentative bacteria were isolated from a total number of 965 clinical samples processed routinely in the laboratory of Microbiology Department, M.K.C.G Medical College in South Orissa accounting to a prevalence rate of 15.75%. Pseudomonas spp. (both pigmented and non-pigmented strains) were isolated in maximum percentage (73.6%) followed by Acinetobacter spp. (19.7%) and Alkaligenes faecalis (4.6%). Rarely encountered species were Eikenella corrodens (1.3%) and Stenotrophomonas maltophila (0.6%). Pus from various sites was the major source (116; 76%). 81% of all isolates were sensitive to amikacin and 74% to ofloxacin. Sensitivity to cefotaxime, ciprofloxacin, tobramycin, gentamicin and netlimycin ranged from 53% to 68%. Least effective drugs were carbenicillin and ceftriaxone (48% each).     

Protease production by clinical isolates of type III group B streptococci.

Six strains of serotype III group B streptococci isolated from confirmed cases of neonatal disease were examined for their ability to produce proteolytic enzymes. Three neuraminidase-producing strains and three non-neuraminidase-producing strains were employed in this study. Protease production was examined in 1,000-fold concentrated filtrates of stationary-phase cells with an insoluble substrate derived from horse hide powder labeled covalently with Remazol brilliant blue. Protease activity was not detected in any cultural supernatant fluids until they were fractionated on Sephadex G-100. After fractionation, the neuraminidase-producing strains were shown to elaborate approximately sixfold more protease than the non-neuraminidase-producing strains. The finding that clinical isolates of group B streptococci that elaborated high levels of neuraminidase also produced elevated levels of extracellular protease may indicate that the production of several different factors may determine the virulence of these organisms.     

Development of reference systems for automatic identification of clinical isolates of bacteria.

The reported work describes experiments on automatic identification based on a material of 636 clinical isolates of bacteria. It is shown that three different and mutually independent automatic identification principles produce almost identical identification conclusions. It is also shown that an automatic procedure which continuously corrects the basic classification gives results which are independent of the classification background. Automatic correction logics develop conventionally-based, as well as numerically-based initial reference systems toward almost similar solutions. This may indicate that automated identification methods based on numerical classifications possess general validity.     

Capacity of Helicobacter pylori to generate ionic gradients at low pH is similar to that of bacteria which grow under strongly acidic conditions.

Helicobacter pylori colonized the highly acidic human gastric mucosa. At pH 3.0 to 7.0, this bacterium maintained a nearly neutral internal pH. Its membrane potential changed reciprocally with the pH gradient so that a relatively constant proton motive force was maintained. Possible, the capacity to maintain an appropriate transmembrane ionic gradient at a low pH contributes to the pathogenic propensities of this bacterium.     

Molecular characterization of resistance to rifampicin in clinical isolates of Neisseria meningitidis

Among 3904 meningococcal isolates collected between October 2002 and June 2007 by the French Meningococcal Reference Centre, eight (0.20%) were resistant to rifampicin (Rif-R; MIC >1 mg/L) and 27 (0.69%) were intermediate-resistant to rifampicin (Rif-I; MICs between 0.38 mg/L and 1 mg/L) according to the E-test method. The MICs determined by agar dilution were lower, eliminating the E-test intermediate category. All Rif-R isolates had mutations in the rpoB gene, resulting in substitutions at or near amino acid position 552, which were absent in non-resistant isolates. These data suggest that a rifampicin clinical breakpoint of 1.0 mg/L should be adopted for Neisseria meningitidis .     

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.