R. del Campo

Clonal dissemination of Enterococcus faecalis ST201 and Enterococcus faecium CC17–ST64 containing Tn5382–vanB2 among 16 hospitals in Chile

We report the clonal dissemination of ST201 Enterococcus faecalis carrying Tn5382-vanB2 and of CC17-ST64 Enterococcus faecium carrying Tn5382-vanB2-ISEnfa110 among 16 hospitals in four geographically distant regions in Chile. This is the first epidemiological characterization of vancomycin resistance in Chile, and also the first report of interhospital dissemination of enterococcal vanB2 in South America.

Comparative in vitro bacteriostatic and bactericidal activity of trovafloxacin, levofloxacin and moxifloxacin against clinical and environmental isolates of Legionella spp.

The susceptibility of 140 Legionella spp isolates (106 clinical and 34 environmental isolates) to trovafloxacin (TRFX), levofloxacin (LEVX), moxifloxacin (MOFX), ciprofloxacin (CIPX), ofloxacin (OFLX), erythromycin (ERY), azithromycin (AZI) and rifampicin (RIF) was studied using a standard microdilution method and buffered yeast extract broth (BYE) supplemented with 0.1% alpha-ketoglutarate. The post-antibiotic effects (PAEs) of the study drugs against 10 clinical isolates of Legionella pneumophila sg.1 were compared. The MIC inhibiting 90% of strains tested on BYEalpha broth were 0.008, 0.016, 0.016, 0.06, 0.125, 0.5, 0.5, and 0.004 mg/l for TRFX, LEVX, MOXX, CIP, OFLX, ERY, AZI, and RIF, respectively. The MBC/MIC ratios ranged from one to eight depending on the antibiotic tested: TRFX [1x-2 x MIC], LEVX, MOFX, CIPX and OFLX [1x-4 x MIC], RIF [2x-4 x MIC], ERY and AZI [2x-8 x MIC]. TRFX, RIF, LEVX, MOFX, CIPX, OFLX, ERY and AZI showed similar activity against Legionella species other than L. pneumophila. One-hour exposures to the study antimicrobial agents at a concentration of 4 x MIC resulted in PAEs as follows (average in hours): TRFX: 2.68 h; RIF: 2.63 h; CIPX: 2.62 h; MOFX: 2.56 h; LEVX: 2.41 h; OFLX: 2.25 h; AZI: 1.65 h; and ERY: 1.54 h. In conclusion, our in vitro data confirm that trovafloxacin, levofloxacin, moxifloxacin and rifampicin have excellent bacteriostatic and bactericidal activity against Legionella spp and show significant post-antibiotic effect.

Comparison of different methods for identification of species of the genus Raoultella: report of 11 cases of Raoultella causing bacteraemia and literature review

The genus Raoultella was excised from Klebsiella in 2001, but difficulties in its identification may have led to an underestimation of its incidence and uncertainty on its pathogenic role. Recently, clinical reports involving Raoultella have increased, probably through the introduction of mass-spectrometry in clinical microbiology laboratories and the development of accurate molecular techniques. We performed a retrospective analysis using our blood culture collection (2011-14) to identify Raoultella isolates that could have been erroneously reported as Klebsiella. PCR and gene sequencing of highly specific chromosomal class A β-lactamase genes was established as the reference method, and compared with 16S rRNA and rpoβ sequencing, as well as matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), MicroScan Walkaway system and API20E biochemical identification. MALDI-TOF and rpoβ correctly identified all Raoultella isolates, whereas 16S rRNA provided inconclusive results, and MicroScan and API20E failed to detect this genus. The analysis of the clinical characteristics of all Raoultella bacteraemia cases reported in the literature supports the role of Raoultella as an opportunistic pathogen that causes biliary tract infections in elderly patients who suffer from some kind of malignancy or have undergone an invasive procedure. Two salient conclusions are that Raoultella shows tropism for the biliary tract and so its identification could help clinicians to suspect underlying biliary tract disease when bacteraemia occurs. Concomitantly, as most phenotypic identification systems are not optimized for the identification of Raoultella, the use of MALDI-TOF or additional phenotypic tests is recommended for the reliable identification of this genus.

Characterization of the Mechanisms of Fluoroquinolone Resistance in Vancomycin-Resistant Enterococci of Different Origins

Abstract The mutations in gyrA and parC genes were analyzed in 22 vancomycin-resistant enterococci of different origins and species, which had varying susceptibility to ciprofloxacin (minimum inhibitory concentration, MIC: 0.5->256 mg/L). All vanA or vanB2-containing strains with ciprofloxacin MIC of >32 mg/L presented amino acid changes in GyrA protein (S83I, S83Y, S83R or S83IE87G) with/without changes in parC protein (S80I or S80R or S80L). Strains with lower ciprofloxacin MICs presented the GyrA and parC wild type. One vanA-containing Enterococcus durans strain with a ciprofloxacin MIC of 64 mg/L presented the S83i and S80i changes in GyrA and parC proteins, respectively. Two vanB2 Enterococcus faecium strains were typed by multi-locus-sequencetyping and both were ascribed to the CC17 clonal complex with two sequence-types (ST78 and ST17-like). All seven vancomycin-resistant and ciprofloxacin-resistant E. faecium strains showed ampicillin resistance (MIC 32-256 mg/L), identifying the following amino acid changes in PBP5 protein: Q461K, V462K, H470Q, M485A, N496K, A499T, E525D, N546T, A558T, G582S, K632Q, P642l, E629V and P667S, together with a serine insertion at position 466′. The 12 Enterococcus gallinarum and Enterococcus casseliflavus isolates included in the study exhibited an MIC for ciprofloxacin in the range 0.5-16 mg/L and no amino acid changes were identified in GyrA or parC proteins. Specific mutations in gyrA and parC genes are associated with fluoroquinolone resistance in E. faecium and E. durans of different origins.

The rise of ampicillin-resistant Enterococcus faecium high-risk clones as a frequent intestinal colonizer in oncohaematological neutropenic patients on levofloxacin prophylaxis: a risk for bacteraemia?

Levofloxacin extended prophylaxis (LEP), recommended in oncohaematological neutropenic patients to reduce infections, might select resistant bacteria in the intestine acting as a source of endogenous infection. In a prospective observational study we evaluated intestinal emergence and persistence of ampicillin-resistant Enterococcus faecium (AREfm), a marker of hospital adapted high-risk clones. AREfm was recovered from the faeces of 52 patients with prolonged neutropenia after chemotherapy, at admission (Basal), during LEP, and twice weekly until discharge (Pos-LEP). Antibiotic susceptibility, virulence traits and population structure (pulsed-field gel electrophoresis and multilocus sequence typing) were determined and compared with bacteraemic isolates. Gut enterococcal population was monitored using a quantitative PCR quantification approach. AREfm colonized 61.4% of patients (194/482 faecal samples). Sequential AREfm acquisition (25% Basal, 36.5% LEP, 50% Pos-LEP) and high persistent colonization rates (76.9-89.5%) associated with a decrease in clonal diversity were demonstrated. Isolates were clustered into 24 PFGE-patterns within 13 sequence types, 95.8% of them belonging to hospital-associated Bayesian analysis of population structure subgroups 2.1a and 3.3a. Levofloxacin resistance and high-level streptomycin resistance were a common trait of these high-risk clones. AREfm-ST117, the most persistent clone, was dominant (60.0% isolates, 32.6% patients). It presented esp gene and caused 18.2% of all bacteraemia episodes in 21% of patients previously colonized by this clone. In AREfm-colonized patients, intestinal enrichment in the E. faecium population with a decline in total bacterial load was observed. AREfm intestinal colonization increases during hospital stay and coincides with enterococci population enrichment in the gut. Dominance and intestinal persistence of the ST117 clone might increase the risk of bacteraemia.

Detection of phage-associated virulence/resistance genes ininduced prophages of Streptococcus pyogenes clinical isolates

Objectives: AmpC b-lactamases hyperproducing (ABLH) Enterobacteriaceae have been reported worldwide, but few data are available about their prevalence in human clinical specimens. We aimed to investigate the prevalence of ABLH (chromosomal or plasmid-borne) E. coli in the faecal flora of hospitalised patients. Material and Methods: 253 consecutive faecal samples from 17 patients collected between April and July 2006 were screened for the presence of ABLH E. coli. Samples were incubated overnight in BHI broth containing vancomycin (20mg/L) and cefoxitin (12mg/L) then subcultured onto CHROMagar Orientation agar (BD) plates on which a 30mg cefoxitin disk and a 30mg ceftazidim disk (I2A) were placed. E. coli colonies growing close to cefoxitin and/or ceftazidim disks were tested for antimicrobial susceptibility by disk diffusion. Strains with reduced susceptibility to ampicillin, amoxi-clavulanate, cefazolin and cefoxitin (compatible with a ABLH phenotype) were further characterised by AmpC disk test, isoelectric focusing gel (IEF), plasmid-borne ampC PCR and sequence analysis of the chromosomal ampC promoter regions. An ABLH was defined on the basis of molecular tests (presence of a plasmidic ampC gene, a mutated ampC promoter and/or corresponding band by IEF). Results: 23 E. coli strains (9% of the total faecal samples) isolated on the CHROMagar plates displayed a cephalosporinase resistance phenotype, but only 12 were confirmed as ABLH (prevalence of 6.7% of all patients); 11 strains were chromosomal ampC hyperproducers (prevalence of 6.2%) including 7 showing mutations at position −42 of the promoter region; only 1 strain harboured a plasmidic CMY-2 gene. The other 11 isolates were considered as putative penicillinase producers associated with porin deficiency. Two extended-spectrum b-lactamases (ESBL) producing E. coli strains (including one having both ampC and ESBL enzymes) were recovered by our screening method. Conclusion: Our screening technique appeared promising to detect ABLH E. coli. In our study, chromosomal ampC hyperproducers E. coli represented the major proportion (85%) of faecal carriage of ABLH E. coli whereas plasmid-mediated ampC was encountered in only 1 of the 12 ABLH isolates. Further studies of larger scale are needed to characterise their prevalence.