Evgeny A. Idelevich

Effects of Antibiotics on Shiga Toxin 2 Production and Bacteriophage Induction by Epidemic Escherichia coli O104:H4 Strain

ABSTRACT The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic-uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated for some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction of stx2-harboring bacteriophages, stx2 transcription, and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increased stx2-harboring phage induction and Stx2 production in outbreak isolates (P values of <0.001 to <0.05), while fosfomycin, gentamicin, and kanamycin insignificantly influenced them (P > 0.1) and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased them (P ≤ 0.05). Ciprofloxacin and chloramphenicol significantly upregulated and downregulated stx2 transcription, respectively (P < 0.01); the other antibiotics had insignificant effects (P > 0.1). Meropenem, azithromycin, and rifaximin, which were used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither induced stx2-harboring phages nor increased stx2 transcription or Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options for patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.

Rapid identification of microorganisms from positive blood cultures by MALDI‐TOF mass spectrometry subsequent to very short‐term incubation on solid medium

Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure.

Human MRSA Isolates with Novel Genetic Homolog, Germany

To the Editor: Methicillin-resistant Staphylococcus aureus (MRSA) represents a major cause of hospital-, community- and livestock-acquired infections that are increasingly difficult to manage (1–3). Detection and identification of MRSA by culture and nucleic acid–based methods is challenged by heterogeneous penicillin-binding protein 2a (PBP2a) expression and variability of the staphylococcal cassette chromosome (SCCmec) elements. Recently, a new SCCmec element (XI) carried in bovine and human isolates was described (4,5). This SCCmec element contains a novel mecA homolog, designated mecALGA251, that is not detectable by usual mecA-specific PCR approaches and PBP2a agglutination tests. Garcia-Alvarez et al. reported this novel mecA homolog exhibited 70% identity at DNA level to the mecA gene, and suggested these strains were transmitted from livestock to humans (4).

In Vitro Activity against Staphylococcus aureus of a Novel Antimicrobial Agent, PRF-119, a Recombinant Chimeric Bacteriophage Endolysin

ABSTRACT Antistaphylococcal activity of the novel chimeric endolysin PRF-119 was evaluated with the microdilution method. The MIC50 and MIC90 of 398 methicillin-susceptible Staphylococcus aureus isolates were 0.098 μg/ml and 0.391 μg/ml, respectively (range, 0.024 to 0.780 μg/ml). Both the MIC50 and MIC90 values of 776 methicillin-resistant S. aureus isolates were 0.391 μg/ml (range, 0.024 to 1.563 μg/ml). All 192 clinical isolates of coagulase-negative staphylococci exhibited MIC values of >50 μg/ml. In conclusion, PRF-119 exhibited very good activity specifically against S. aureus.

Rapid identification and susceptibility testing of Candida spp. from positive blood cultures by combination of direct MALDI-TOF mass spectrometry and direct inoculation of Vitek 2.

Fungaemia is associated with high mortality rates and early appropriate antifungal therapy is essential for patient management. However, classical diagnostic workflow takes up to several days due to the slow growth of yeasts. Therefore, an approach for direct species identification and direct antifungal susceptibility testing (AFST) without prior time-consuming sub-culturing of yeasts from positive blood cultures (BCs) is urgently needed. Yeast cell pellets prepared using Sepsityper kit were used for direct identification by MALDI-TOF mass spectrometry (MS) and for direct inoculation of Vitek 2 AST-YS07 card for AFST. For comparison, MALDI-TOF MS and Vitek 2 testing were performed from yeast subculture. A total of twenty four positive BCs including twelve C. glabrata, nine C. albicans, two C. dubliniensis and one C. krusei isolate were processed. Applying modified thresholds for species identification (score ≥1.5 with two identical consecutive propositions), 62.5% of BCs were identified by direct MALDI-TOF MS. AFST results were generated for 72.7% of BCs directly tested by Vitek 2 and for 100% of standardized suspensions from 24 h cultures. Thus, AFST comparison was possible for 70 isolate-antifungal combinations. Essential agreement (minimum inhibitory concentration difference ≤1 double dilution step) was 88.6%. Very major errors (VMEs) (false-susceptibility), major errors (false-resistance) and minor errors (false categorization involving intermediate result) amounted to 33.3% (of resistant isolates), 1.9% (of susceptible isolates) and 1.4% providing 90.0% categorical agreement. All VMEs were due to fluconazole or voriconazole. This direct method saved on average 23.5 h for identification and 15.1 h for AFST, compared to routine procedures. However, performance for azole susceptibility testing was suboptimal and testing from subculture remains indispensable to validate the direct finding.

Acceleration of Antimicrobial Susceptibility Testing of Positive Blood Cultures by Inoculation of Vitek 2 Cards with Briefly Incubated Solid Medium Cultures

ABSTRACT Briefly incubated agar cultures from positive blood cultures were used for antimicrobial susceptibility testing (AST) by Vitek 2. The cultivation time until inoculation was 3.8 h for Gram-positive cocci and 2.4 h for Gram-negative rods. The error rates were low, providing early and reliable AST without additional time or cost expenditure.

Comparative in vitro activity of finafloxacin against staphylococci displaying normal and small colony variant phenotypes

OBJECTIVES Staphylococcal small colony variants (SCVs) are associated with chronic and relapsing infections and their intracellular location may shield them from host defences and antibiotics. Finafloxacin is a novel fluoroquinolone that exhibits optimal activity at slightly acidic conditions where the activity of other marketed fluoroquinolones decreases. Here, the in vitro activity of finafloxacin against clinical strain pairs consisting of an SCV and its clonally identical parental strain displaying the normal phenotype (NP) was compared with those of other fluoroquinolones at standard and low pH. METHODS In vitro activities of finafloxacin, ciprofloxacin, levofloxacin and moxifloxacin were tested against 28 methicillin-susceptible Staphylococcus aureus (MSSA) and three methicillin-resistant S. aureus (MRSA) SCV-NP strain pairs. Additionally, two S. aureus mutants (ΔhemB and ΔthyA) displaying the SCV phenotype and their wild-type strains as well as four SCV-NP pairs of coagulase-negative staphylococcal (CoNS) strains were included. MIC(50,) MIC(90) and MIC ranges were calculated based on MIC determination by Etest(®) at pH 5.8 and pH 7.2. RESULTS Under acidic conditions, finafloxacin demonstrated superior activity against MSSA, MRSA and CoNS regardless of the phenotype. At neutral conditions, the activity against MSSA was as follows: moxifloxacin > finafloxacin > levofloxacin > ciprofloxacin. In comparison with methicillin-susceptible NP isolates, ciprofloxacin was less active against their corresponding SCVs. For other fluoroquinolones, there was no marked difference in activity against SCVs compared with NPs. CONCLUSIONS Particularly in acidic body compartments, finafloxacin appears to be a promising new antibiotic for the treatment of persistent staphylococcal infections, including those caused by SCVs.

Increased Prevalence and Resistance of Important Pathogens Recovered from Respiratory Specimens of Cystic Fibrosis Patients During a Decade.

Background: The study objective was to identify changes of prevalence and resistance of important pathogens in specimens of cystic fibrosis (CF) patients within a decade. Methods: Samples of 94 patients, who attended 2 CF centers from 2001 to 2011 were retrospectively analyzed. Results: Staphylococcus aureus was the most prevalent organism (74.5% in 2011) with an increase of methicillin-resistant S. aureus in patients (0% vs. 9.6%, n = 9). Resistance of S. aureus to gentamicin decreased (41.8% vs. 21%; P < 0.001), whereas resistance to rifampicin and trimethoprim/sulfamethoxazole (P < 0.05) increased significantly with a trend to increased resistance to clindamycin and erythromycin (P = 0.063). Methicillin-resistant S. aureus isolates belonged to 6 spa types (t003, t008, t011, t034, t045, t548). There was a significant increase of Pseudomonas aeruginosa prevalence (63.8% in 2011 vs. 46.8% in 2001, P = 0.019). Resistance of P. aeruginosa increased significantly to imipenem, gentamicin, amikacin, tobramycin, ciprofloxacin and fosfomycin, whereas resistance to piperacillin–tazobactam, meropenem and aztreonam decreased. Significantly fewer Stenotrophomonas maltophilia isolates were susceptible to all the analyzed antibiotics (trimethoprim/sulfamethoxazole, ciprofloxacin and colistin) in 2011 compared with 2001 (13.5% vs. 42.1%; P = 0.023), whereas the resistance to colistin increased significantly (11.1% vs. 62.2%; P < 0.001). Burkholderia cepacia complex and nontuberculous mycobacteria were not detected in 2001 but in 2011 in 7.4% (n = 9) and 7.4% (n = 9) of patients, respectively. B. cepacia complex isolates belonged to 8 multilocus sequence types. Conclusions: Our retrospective analysis revealed an increase of important CF-related pathogens, the emergence of new pathogens and a substantial increase of multidrug-resistant CF-specific isolates. Our findings are of importance to clinicians for the alertness of local epidemiology, which may be useful for prevention and treatment strategies.

Plasmid-Encoded Transferable mecB-Mediated Methicillin Resistance in Staphylococcus aureus

During cefoxitin-based nasal screening, phenotypically categorized methicillin-resistant Staphylococcus aureus (MRSA) was isolated and tested negative for the presence of the mecA and mecC genes as well as for the SCCmec-orfX junction region. The isolate was found to carry a mecB gene previously described for Macrococcus caseolyticus but not for staphylococcal species. The gene is flanked by β-lactam regulatory genes similar to mecR, mecI, and blaZ and is part of an 84.6-kb multidrug-resistance plasmid that harbors genes encoding additional resistances to aminoglycosides (aacA-aphD, aphA, and aadK) as well as macrolides (ermB) and tetracyclines (tetS). This further plasmidborne β-lactam resistance mechanism harbors the putative risk of acceleration or reacceleration of MRSA spread, resulting in broad ineffectiveness of β-lactams as a main therapeutic application against staphylococcal infections.

Pacemaker lead infection and related bacteraemia caused by normal and small colony variant phenotypes of Bacillus licheniformis.

Here, we report what we believe to be the first case of bacteraemia with small colony variants of Bacillus licheniformis related to a pacemaker lead infection by B. licheniformis displaying the normal phenotype. Arbitrarily primed PCR analysis showed a clonal strain. The infection was cured after the removal of the infected device.