Max Aravena-Roman

Comparison of Diagnostic Laboratory Methods for Identification of Burkholderia pseudomallei

ABSTRACT Limited experience and a lack of validated diagnostic reagents make Burkholderia pseudomallei, the cause of melioidosis, difficult to recognize in the diagnostic microbiology laboratory. We compared three methods of confirming the identity of presumptive B. pseudomallei strains using a collection of Burkholderia species drawn from diverse geographic, clinical, and environmental sources. The 95 isolates studied included 71 B. pseudomallei and 3 B. thailandensis isolates. The API 20NE method identified only 37% of the B. pseudomallei isolates. The agglutinating antibody test identified 82% at first the attempt and 90% including results of a repeat test with previously negative isolates. Gas-liquid chromatography analysis of bacterial fatty acid methyl esters (GLC-FAME) identified 98% of the B. pseudomallei isolates. The agglutination test produced four false positive results, one B. cepacia, one B. multivorans, and two B. thailandensis. API produced three false positive results, one positive B. cepacia and two positive B. thailandensis. GLC-FAME analysis was positive for one B. cepacia isolate. On the basis of these results, the most robust B. pseudomallei discovery pathway combines the previously recommended isolate screening tests (Gram stain, oxidase test, gentamicin and polymyxin susceptibility) with monoclonal antibody agglutination on primary culture, followed by a repeat after 24 h incubation on agglutination-negative isolates and GLC-FAME analysis. Incorporation of PCR-based identification within this schema may improve percentages of recognition further but requires more detailed evaluation.

Antimicrobial Susceptibilities of Aeromonas Strains Isolated from Clinical and Environmental Sources to 26 Antimicrobial Agents

ABSTRACT We determined the susceptibilities of 144 clinical and 49 environmental Aeromonas strains representing 10 different species to 26 antimicrobial agents by the agar dilution method. No single species had a predominantly nonsusceptible phenotype. A multidrug nonsusceptible pattern was observed in three (2.1%) clinical strains and two (4.0%) strains recovered from diseased fish. Common clinical strains were more resistant than the corresponding environmental isolates, suggesting that resistance mechanisms may be acquired by environmental strains from clinical strains.

Aeromonas aquariorum is widely distributed in clinical and environmental specimens and can be misidentified as Aeromonas hydrophila

ABSTRACT Genotypic characterization of 215 Aeromonas strains (143 clinical, 52 environmental, and 20 reference strains) showed that Aeromonas aquariorum (60 strains, 30.4%) was the most frequently isolated species in clinical and water samples and could be misidentified as Aeromonas hydrophila by phenotypic methods.

Cellular Fatty Acid Profile Distinguishes Burkholderia pseudomallei from Avirulent Burkholderia thailandensis

ABSTRACT Burkholderia pseudomallei, the cause of melioidosis, can be distinguished from the closely related but nonpathogenic Burkholderia thailandensis by gas chromatography (GC) analysis of fatty acid derivatives. A 2-hydroxymyristic acid derivative (14:0 2OH) was present in 95% of B. pseudomallei isolates and no B. thailandensis isolates. GC mass spectrophotometry confirmed that 2-hydroxymyristic acid was present in B. pseudomallei. GC-fatty acid methyl ester analysis may be useful in distinguishing these two closely related species.

First Description of Curtobacterium spp. Isolated from Human Clinical Specimens

ABSTRACT During a 4-year period, five strains (three of which were doubtless clinically significant) of yellow- or orange-pigmented, oxidative, slowly acid-producing coryneform bacteria were recovered from human clinical specimens in two reference laboratories or referred to them. The strains were motile, catalase positive, nitrate reductase negative, and urease negative, but strongly hydrolyzed esculin. In all reference and clinical strains described in the present study, anteisopentadecanoic (C15:0ai) and anteisoheptadecanoic (C17:0ai) acids represented more than 75% of all cellular fatty acids except in one clinical strain and in Curtobacterium pusillum, in which both the unusual ω-cyclohexyl fatty acid (identified as C18:1ω7cis/ω9cis/ω12trans by the Sherlock system) represented more than 50% of all cellular fatty acids. In all clinical strains, ornithine was the diamino acid of the cell wall, the interpeptide bridge consisted of ornithine, and acetyl was the acyl type of the peptidoglycan. Therefore, the five clinical strains were unambiguously identified as Curtobacterium spp. Analyses of the complete 16S rRNA genes of the five clinical strains with homologies to the established Curtobacterium species ranging from 99.2 to 100% confirmed the identifications as Curtobacterium spp. Data on the antimicrobial susceptibility pattern of curtobacteria are reported, with macrolides and rifampin showing very low MICs for all strains tested. This report is the first on the isolation of Curtobacterium strains from human clinical specimens.

Polymerase chain reaction for the detection of toxigenic Corynebacterium diphtheriae.

&NA; Several conventional methods have been described for the detection of Corynebacterium diphtheriae toxin, including Elek immunodiffusion, tissue culture using VERO cells and guinea pig inoculation. All these methods have the disadvantage of being either slow to complete or technically demanding, particularly when performed infrequently. We examined 64 strains of C. diphtheriae by PCR and Elek immunodiffusion, and strains showing a positive result in either assay were inoculated into guinea pigs. Seven isolates were positive in both Elek and PCR assays and subsequently positive in guinea pig inoculation assay. One isolate was negative in Elek testing but positive in PCR assay and guinea pig inoculation. All other isolates were negative in both Elek and PCR assays. The PCR assay is rapid with cycling and detection complete within 3–4 hrs of receipt of strains. PCR has now become the routine method for detection of C. diphtheriae toxin in our laboratory.

First case of Francisella bacteraemia in Western Australia

Francisella species are Gram-negative, nonmotile, pleomorphic coccobacilli, facultative intracellular fastidious bacteria. We report the isolation of a Francisella-like species from a blood culture collected from a 44-year-old bacteraemic patient in Perth, Western Australia. The organism was identified to species level by 16S rRNA sequencing and by fatty acid methyl esters analysis. The strain genotypically resembled Francisella hispaniensis, a species previously isolated from human blood in Spain.

Phenotypic characteristics of human clinical and environmental Aeromonas in Western Australia

Aim: To determine the phenotypic characteristics of 199 Aeromonas strains comprising 146 clinical and 53 environmental isolates. Methods: Identification was based on a scheme consisting of 62 biochemical tests including two novel tests introduced as potential phenotypic markers. Results: One hundred and eighty-five strains (93%) were identified to species level while eight (4%) resembled members of the Aeromonas hydrophila complex and six (3%) could not be assigned to any taxon. There were no significant phenotypic differences between clinical and environmental strains of the three most commonly isolated species A. hydrophila, Aeromonas veronii subspecies sobria and Aeromonas caviae. The most frequently isolated species in human clinical material and environmental samples was A. hydrophila (54.8% and 45.3%, respectively). Conclusions: Phenotypical identification showed that A. hydrophila was the most frequently isolated Aeromonas from clinical and water samples. The introduction of novel tests did not improve the discriminatory power of the scheme and the lack of definitive phenotypical markers continues to hinder Aeromonas taxonomy.