Claudia Barberis

Evaluation of the Andromas Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Aerobically Growing Gram-Positive Bacilli

ABSTRACT Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Comparison of the Bruker MALDI-TOF Mass Spectrometry System and Conventional Phenotypic Methods for Identification of Gram-Positive Rods

In recent years, MALDI-TOF Mass Spectrometry (MS) method has emerged as a promising and a reliable tool for bacteria identification. In this study we compared Bruker MALDI-TOF MS and conventional phenotypic methods to identify a collection of 333 Gram-positive clinical isolates comprising 22 genera and 60 species. 16S rRNA sequencing was the reference molecular technique, and rpoB gene sequecing was used as a secondary gene target when 16Sr RNA did not allow species identification of Corynebacterium spp. We also investigate if score cut-offs values of ≥1,5 and ≥1,7 were accurate for genus and species-level identification using the Bruker system. Identification at species level was obtained for 92,49% of Gram-positive rods by MALDI-TOF MS compared to 85,89% by phenotypic method. Our data validates the score ≥1,5 for genus level and ≥1,7 for species-level identification in a large and diverse collection of Gram-positive rods. The present study has proved the accuracy of MALDI-TOF MS as an identification method in Gram-positive rods compared to currently used methods in routine laboratories.

Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry for species identification of Nonfermenting Gram-Negative Bacilli

Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) to identify 396 Nonfermenting Gram-Negative Bacilli clinical isolates was evaluated in comparison with conventional phenotypic tests and/or molecular methods. MALDI-TOF MS identified to species level 256 isolates and to genus or complex level 112 isolates. It identified 29 genera including uncommon species.

Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans

The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-H. pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.

Isolation of Kerstersia gyiorum from a Patient with Cholesteatomatous Chronic Otitis Media

ABSTRACT We describe the first case of a Kerstersia gyiorum strain isolated from a patient with cholesteatomatous chronic otitis media. We emphasize the isolation of members of the family Alcaligenaceae in serious infections and unusual sites and the importance of polyphasic identification addressing the definitive identification.

Recurrent Urinary Infection with Bifidobacterium scardovii

ABSTRACT Bifidobacterium species are difficult to identify and may be underreported or not recovered by many laboratories because of their slow growth. We emphasize the importance of the Gram stain in urine samples and the addition of enriched media and enhanced atmosphere over time for urine cultures with pyuria. This is the first report of a Bifidobacterium scardovii recurrent urinary infection in an elderly woman.

First Report of an Extensively Drug-Resistant VIM-2 Metallo-β-Lactamase-Producing Brevundimonas diminuta Clinical Isolate

ABSTRACT In the literature, only three Brevundimonas diminuta environmental isolates carrying metallo-β-lactamase genes were recently published. However, so far, no B. diminuta clinical isolates carrying these carbapenem resistance genes have been described. Here we report the first VIM-2 metallo-β-lactamase-producing B. diminuta clinical isolate obtained from an immunocompromised patient.

Actinobaculum schaalii causing urinary tract infections: report of four cases from Argentina

Actinobaculum schaalii may be a more common urinary tract pathogen than previously described. Here we report four cases of A. schaalii UTIs and we also propose a simple identification scheme to be used in the conventional microbiology laboratory based on standard biochemical tests.

Impact of heteroresistance to colistin in meningitis caused by Acinetobacter baumannii

We read with interest the paper by Li and colleagues concerning colistin hetero-resistance in Acinetobacter baumannii clinical isolates. The incidence of colistin-heteroresistance is still not well known due to its difficult detection. Moreover, its clinical impact has only been discussed in our previous report and in a communication of David. In a recent review of patients with meningitis due to A. baumannii treated with colistin, heteroresistant isolates have not been described. The aim of the present work is to report our experience regarding the clinical impact of colistin-heteroresistance in post neurosurgical meningitis caused by A. baumannii. Seven isolates were recovered from two cases (patients 1 and 2) of A. baumannii colistin heteroresistant in post neurosurgical meningitis were detected in the intensive care unit of Hospital de Clinicas Jose de San Martin of Buenos Aires city during the period 2004e2009. The selection intra-treatment with colistin of A. baumannii colistinresistant was analyzed. Strains were identified by phenotypic tests and genomospecies was determined by ARDRA (Amplified ribosomal DNA restriction analysis). Minimal inhibitory concentrations (MICs) to colistin (against the resistant subpopulations and the original strains) and the other antimicrobials (ampicillinesulbactam, ceftriaxone, ceftazidima, imipenem, meropenem, amikacin, gentamicin and levofloxacin) were performed by dilution method in cation-adjusted MuellerHinton agar and interpreted according to the Clinical and Laboratory Standards Institute. Pulsed-field electrophoresis (PFGE) was performed with ApaI as described previously. Population analysis profiles (PAPs) were determined according to a previous report. Timeekill studies were performed twice and thus, results were analyzed by using mean colony count values from the duplicate plates for each isolate, with the following concentrations: colistin sulfate: 4,8,12 and 80 MIC and rifampicin 4 mg/ ml. This study was performed with the colistin susceptible heteroresistant isolates from each patient (Ab86 and

Antimicrobial susceptibility of clinical isolates of Actinomyces and related genera reveals an unusual clindamycin resistance among Actinomyces urogenitalis strains

OBJECTIVES Patterns of antimicrobial susceptibility in Actinomyces and related genera are very limited in the literature. Data of predominant susceptibility profiles could contribute to the establishment of an accurate empirical treatment. METHODS A total of 113 isolates from clinical samples were included in this study. Each isolate was identified using phenotypic methods and MALDI-TOF/MS. When discrepancies were observed, 16S rRNA gene sequencing was performed. The minimum inhibitory concentrations (MICs) of nine antimicrobial agents (penicillin, ceftriaxone, linezolid, tetracycline, clindamycin, erythromycin, ciprofloxacin, levofloxacin and vancomycin) were tested against the species Actinotignum schaalii (n=23), Actinomyces turicensis (n=18), Actinomyces europaeus (n=13), Actinomyces naeslundii/Actinomyces viscosus group (n=12), Actinomyces urogenitalis (n=11), Actinomyces radingae (n=11), Actinomyces neuii (n=9), Actinomyces odontolyticus (n=8), Bifidobacterium scardovii (n=3), Actinomyces graevenitzii (n=2), Alloscardovia omnicolens (n=2) and Varibaculum cambriense (n=1). RESULTS All of the isolates were susceptible to penicillin, ceftriaxone, vancomycin and linezolid. Almost all of the A. urogenitalis isolates (8/11) were resistant to clindamycin and showed susceptibility to erythromycin, suggesting an L-phenotype, however no determinants of clindamycin resistance (lnu and lsa genes) were detected by PCR. High MIC values to quinolones were observed in 54/113 isolates (47.8%). All of the A. urogenitalis isolates were highly resistant to ciprofloxacin and levofloxacin. CONCLUSIONS These data highlight the importance of ongoing surveillance to provide relevant information for empirical management of infections caused by these organisms.