Rimtas Dargis

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis of Gram-Positive, Catalase-Negative Cocci Not Belonging to the Streptococcus or Enterococcus Genus and Benefits of Database Extension

ABSTRACT Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry with a Bruker Daltonics microflex LT system was applied to 90 well-characterized catalase-negative, Gram-positive cocci not belonging to the streptococci or enterococci. Biotyper version 2.0.43.1 software was used singly or in combination with a database extension generated in this study with 51 collection strains from 16 genera. Most strains were identified by using both databases individually, and some were identified only by applying the combined database. Thus, the methodology is very useful and the generated database extension was helpful.

Mass spectrometry: Pneumococcal meningitis verified and Brucella species identified in less than half an hour

Abstract Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently being introduced for the rapid and accurate identification of bacteria. We describe 2 MALDI-TOF MS identification cases – 1 directly on spinal fluid and 1 on grown bacteria. Rapidly obtained results had great value for the continued treatment and for the elucidation of exposure.

Identification of Clinically Relevant Nonhemolytic Streptococci on the Basis of Sequence Analysis of 16S-23S Intergenic Spacer Region and Partial gdh Gene

ABSTRACT Nonhemolytic streptococci (NHS) cause serious infections, such as endocarditis and septicemia. Many conventional phenotypic methods are insufficient for the identification of bacteria in this group to the species level. Genetic analysis has revealed that single-gene analysis is insufficient for the identification of all species in this group of bacteria. The aim of the present study was to establish a method based on sequence analysis of the 16S-23S intergenic spacer (ITS) region and the partial gdh gene to identify clinical relevant NHS to the species level. Sequence analysis of the ITS region was performed with 57 NHS reference or clinical strains. Satisfactory identification to the species level was achieved for 14/19 NHS species included in this study on the basis of sequence analysis of the ITS region. Streptococcus salivarius and Streptococcus vestibularis obtained the expected taxon as the best taxon match, but there was a short maximum score distance to the next best match (distance, <10). Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae could not be unambiguously discriminated by sequence analysis of the ITS region, as was also proven by phylogenetic analysis. These five species could be identified to the group level only by ITS sequence analysis. Partial gdh sequence analysis was applied to the 11 S. oralis strains, the 11 S. mitis strains, and the 17 S. pneumoniae strains. All except one strain achieved a satisfactory identification to the species level. A phylogenetic algorithm based on the analysis of partial gdh gene sequences revealed three distinct clusters. We suggest that sequence analysis of the combination of the ITS region and the partial gdh gene can be used in the reference laboratory for the species-level identification of NHS.

Six cases of Aerococcus sanguinicola infection: Clinical relevance and bacterial identification

Aerococcus sanguinicola is a Gram-positive coccus first described in 2001. Infections in humans are rare but the use of 16S rRNA gene sequencing and improved phenotypic methods has facilitated the identification of A. sanguinicola. We report here 6 cases of A. sanguinicola bacteraemia, 2 of which were associated with infective endocarditis. Most patients were elderly (median age 70 y) and had underlying neurological disorders including dementia, cerebral degeneration, and myelomeningocele. The primary focus of infection was the urinary tract in 3 cases and the gallbladder in 1; no focus was detected in 2 cases. Long-term prognosis was poor reflecting the frailty of the patients. All strains were susceptible to penicillin, ampicillin, cefuroxime, vancomycin, erythromycin, and rifampicin. The optimal treatment of infection with A. sanguinicola has yet to be determined.

Imported brucellosis in Denmark: Molecular identification and multiple-locus variable number tandem repeat analysis (MLVA) genotyping of the bacteria

Abstract A polymerase chain reaction was used to identify Brucella species isolated from humans in Denmark. Consecutive analysis of referred bacteria and re-examination of historical isolates identified all as Brucella melitensis. Multiple-locus variable number tandem repeat analysis (MLVA) placed the isolates in the previously defined ‘East Mediterranean’ B. melitensis group.

Cardiobacterium valvarum infective endocarditis and phenotypic/molecular characterization of 11 Cardiobacterium species strains

Cardiobacterium valvarum is a newly recognized human pathogen related to infective endocarditis. Cardiobacterium species are, however, only rarely the aetiology of infective endocarditis. An infective endocarditis case is presented and, additionally, phenotypic and phylogenetic comparison of a further 10 collection strains, representing the two species within the genus, was performed. C. valvarum was isolated from the blood and DNA was present in valvular tissue (partial 16S rRNA gene analysis) from a 64-year-old man with infective endocarditis of the mitral valve, rupture of chordae and prolapse of pulmonary valves in addition to a fluttering excrescence. A mechanical mitral valve and neochordae were inserted successfully. Phenotypically, the two species within the genus Cardiobacterium resemble each other greatly. When using the Vitek 2 Neisseria-Haemophilus identification card, the reaction for phenylphosphonate was positive for all Cardiobacterium hominis strains, but negative for all C. valvarum strains, thereby separating the two species. The two species made up two separate clusters by phylogenetic examination using 16S rRNA gene sequence analysis.

Routine ribosomal PCR and DNA sequencing for detection and identification of bacteria

Detection and identification of bacteria by PCR and DNA sequencing from clinical sample material has been introduced as a diagnostic routine analysis during the last 5-10 years. Assays analyzing ribosomal genes have been found to be particularly useful. The technique has identified unusual bacteria as well as well-known bacteria in unusual infectious foci. Thereby, it has proven its value both in diagnosing infections in individual patients and as a tool to establish the pathogenic potential of bacteria not previously associated with disease. To be of clinical relevance, results from ribosomal PCR and DNA sequencing must be obtained fast and at acceptable costs. Processing of a high number of samples by individual laboratories can ensure both speed and low price. By continued technical development and further investigations of its usefulness in various clinical settings ribosomal DNA sequencing will most probably become as common a part of clinical bacteriology as culture is today.

Detection of Burkholderia pseudomallei by SYBR Green Real Time PCR

Development of rapid and sensitive techniques for detection of B. pseudomallei is an important aim for public health. Here we report a B. pseudomallei specific SYBR Green-based real time PCR targeting a species specific toxin gene. The assay is able to detect 1-2 genome equivalents of B. pseudomallei.

PCR and DNA sequencing in establishing the aetiology of bacterial infections in children

The results of partial polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene and subsequent DNA sequencing of clinical samples from children are described. In 13 out of 62 samples, DNA from bacteria likely to be the cause of infection was identified. In the vast majority (11/13) of samples with significant pathogen culture the specimen had been negative. Antibiotics had been given in all cases except for three prior to sampling. PCR and subsequent DNA sequencing is a valuable supplementary tool for establishing the cause of bacterial infections in children when culture is negative.

Report of the First Human Case of Caulobacter sp. Infection

ABSTRACT A Caulobacter sp. isolate was recovered from the dialysis fluid of a patient undergoing peritoneal dialysis. Bacterial identification included electron microscopy and 16S rDNA sequencing. To our knowledge, this is the first report of human Caulobacter infection. Special growth requirements suggest that Caulobacter spp. may be overlooked in the clinical microbiology laboratory.