Microbiological investigation and clinical significance of Alloscardovia omnicolens in human samples

Breaking news about Alloscardovia omnicolens Conclusions 24th ECCMID 10-13 May 2014 Barcelona, Spain 1791 The genus Alloscardovia has been initially described by Huys & al. in 2007 [1]. It only comprises one species, Alloscardovia omnicolens. This species is a catalase negative, facultative-anaerobic, non-motile Gram-positive rod (Fig. 1) belonging to the Bifidobacteriaceae family, which is frequently misidentified or considered as a contaminant in clinical specimens. Fig.1: Gram staining of A. omnicolens Materials and methods Bacterial strains, antimicrobial susceptibility and clinical data A total of 16 clinical isolates of A. omnicolens collected from two different institutions (1 in France and 1 in Switzerland) were included. MICs of 16 antibiotics were determined using E-test strips on MuellerHinton agar plate supplemented with lysed horse blood (5%) and β-NAD (20 mg/L) according to the EUCAST recommendations. Following clinical data were obtained: age, gender, site of isolation, underlying pathologies and clinical presentation for each patient. Identification assays Identification was performed by conventional biochemical tests (API 32 A, bioMerieux), MALDI-TOF mass spectrometry technology (MicroFlex, Bruker Daltonics) according to manufacturer’s recommendations and confirmed by sequencing of the rrs (16S rRNA) gene . PCR assay and DNA sequencing Bacterial genomic DNA was extracted using the easyMAG® NucliSens extractor (bioMérieux) according to the protocol suggested by the manufacturer. PCR experiments for the detection of genes putatively involved in MLS resistance [erm(A), erm(B), erm(C), erm(F), erm(TR), erm(T), erm(X), msr(A) and mef(A)] were carried out as previsouly described [2]. Specific mutations in the 23S rRNA (rrl) gene were also detected. Sequencing of rrs gene and 23S rRNA encoding gene were performed with the Sanger sequencing method (GATC Biotech, Konstanz, Germany) Antimicrobials MIC (mg/L) MIC50 MIC90 Range Gentamicin 32 256 8 256 Amoxicillin 0.12 0.25 0.06 – 0,25 Amoxicillin-Clavulanic acid 0.25 0.38 0.12 – 0.5 Piperacillin-Tazobactam 0.25 0.5 0.06 – 0.5 Cotrimoxazole 0.03 0.12 0.002 – 0.12 Cefotaxim 0.25 0.38 0.12 – 0.5 Ceftazidim 0.5 0.75 0.12 – 1.5 Daptomycin 1 4 0.01 – 8 Ciprofloxacin 0.5 1 0.12 – 1 Levofloxacin 0.5 0.5 0.5 – 1 Erythromycin 0.01 0.12 0.01 >256 Clindamycin 0.01 0.01 0.01 >256 Quinupristin-Dalfopristin 0.5 1 0.5 – 2 Vancomycin 0.5 0.5 0.25 – 1 Nitrofurantoin 0.5 128 0.12 >512 Metronidazole >32 >32 >32 Results 1 Clinical data A total of 16 clinical isolates were isolated from different clinical specimens: urine (n=13), BAL (n=2) and seminal fluid (n=1). Main clinical characteristics were: • Mean age: 63 years (range, 32-94 years) • Sex ratio M/F: 0.9 • Underlying comorbidities: • elderly age (>65 years old) (n=8/16) • preexistent nephropathy (n=5/16) • Clinical presentation: • cystitis (n = 10/16) • pyelonephritis (n = 2/16) 2 Bacterial identification As previously described [3], biochemical tests did not provide reliable identification of the species, but all the bacterial strains were identified by MALDI-TOF (Table 1) 3 Antimicrobial susceptibility Like all members of the Bifidobacteriaceae family, A. omnicolens exhibited an intrinsic resistance to metronidazole and aminosides. Out of the 16 strains, all were susceptible to β-lactams (i.e. penicillins, cephalosporins and carbapenems), glycopeptides (i.e. vancomycin and teicoplanin), cotrimozaxole, fluoroquinolones (i.e. ciprofloxacin and levofloxacin) and other antibiotics commonly prescribed in the treatment of UTIs. Noteworthy, 31% of strains were resistant to nitrofurantoin, antimicrobial usually recommended in empirical treatment of uncomplicated cystitis. One strain, showed an acquired resistance to MLS. This resistance was screened by PCR and was explained by a single mutation (A2058G) in the 23S rRNA gene. Table 2: Antimicrobial susceptibility of 16 A. omnicolens clinical isolates •Alloscardovia omnicolens: score ≥ 2 n = 21 / 32* (66%) = species identification •Alloscardovia spp.: score 1.7 < x < 2 n = 10 / 32* (31%) = genus identification •1 nonsignificant identification score = 1.532 (3%) We report herein the first study on clinical significance, bacterial identification and antimicrobial susceptibility of A. omnicolens, a recentlydescribed human uropathogen. These data may be useful for clinical microbiologists and physicians for the diagnosis and the treatment of UTIs in order to determine the potential of emergence of this Gram-positive rod. 1) Huys, G., Vancanneyt, M., D’Haene, K., Falsen, E., Wauters, G., Vandamme, P., 2007. Alloscardovia omnicolens gen. nov., sp. nov., from human clinical samples. Int. J. Syst. Evol. Microbiol. 57, 1442–1446. 2) Isnard, C., Guérin, F., Dahyot, S., Cattoir, V., 2014. Molecular detection of genes coding for resistance to macrolides, lincosamides and streptogramins in Gram-positive cocci. Methods Mol Biol; In press. 3) Mahlen, S.D., Clarridge, J.E., 3rd, 2009. Site and clinical significance of Alloscardovia omnicolens and Bifidobacterium species isolated in the clinical laboratory. J. Clin. Microbiol. 47, 3289–3293. [email protected] A. omnicolens is likely a commensal of the human urogenital tract since it has been mainly isolated in urine samples from patients suffering from UTIs. Only a few case reports have been described so far, while no data on bacterial identification antimicrobial susceptibility of this emerging uropathogen are available. The aim of the study was to determine clinical significance, methods of identification and antimicrobial susceptibility of A. omnicolens in 16 clinical isolates. *Each strain was identified in duplicate (16 2)