Daniela Cirasola

Evaluation of reduced susceptibility to quaternary ammonium compounds and bisbiguanides in clinical isolates and laboratory-generated mutants of Staphylococcus aureus

ABSTRACT The MICs and minimum bactericidal concentrations (MBCs) for the biocides benzalkonium chloride and chlorhexidine were determined against 1,602 clinical isolates of Staphylococcus aureus. Both compounds showed unimodal MIC and MBC distributions (2 and 4 or 8 mg/liter, respectively) with no apparent subpopulation with reduced susceptibility. To investigate further, all isolates were screened for qac genes, and 39 of these also had the promoter region of the NorA multidrug-resistant (MDR) efflux pump sequenced. The presence of qacA, qacB, qacC, and qacG genes increased the mode MIC, but not MBC, to benzalkonium chloride, while only qacA and qacB increased the chlorhexidine mode MIC. Isolates with a wild-type norA promoter or mutations in the norA promoter had similar biocide MIC distributions; notably, not all clinical isolates with norA mutations were resistant to fluoroquinolones. In vitro efflux mutants could be readily selected with ethidium bromide and acriflavine. Multiple passages were necessary to select mutants with biocides, but these mutants showed phenotypes comparable to those of mutants selected by dyes. All mutants showed changes in the promoter region of norA, but these were distinct from this region of the clinical isolates. Still, none of the in vitro mutants displayed fitness defects in a killing assay in Galleria mellonella larvae. In conclusion, our data provide an in-depth comparative overview on efflux in S. aureus mutants and clinical isolates, showing also that plasmid-encoded efflux pumps did not affect bactericidal activity of biocides. In addition, current in vitro tests appear not to be suitable for predicting levels of resistance that are clinically relevant.

Cell surface hydrophobicity: a predictor of biofilm production in Candida isolates?

Biofilm-forming ability undoubtedly represents one of the major medical concerns in clinical practice, being a relevant pitfall for an efficient therapeutic intervention (Donlan & Costerton, 2002). In this scenario, invasive fungal infections, especially those involving Candida species, become an important problem in terms of early diagnosis and medical treatment. A large proportion of Candida infections are associated with biofilm formation, mainly on the surface of implanted medical devices (Douglas, 2003). Rapid methods for biofilm-forming ability evaluation in clinical isolates could thus improve the clinical outcome of the fungal infection. Biofilm formation is a step-by-step and finely regulated process in which the adhesion phase is a crucial event (Chandra et al., 2001). Hydrophobic interactions play a role in the adherence of micro-organisms to a wide variety of surfaces.

Experimental biofilm-related Candida infections

AIM We investigated the pathogenic role of biofilm and the therapeutic efficacy of anidulafungin in experimental infections of the wax moth Galleria mellonella by Candida albicans clinical strains. MATERIALS & METHODS On the basis of the in vitro propensity to form biofilm, five biofilm-producer (BP) and four nonproducer (NP) C. albicans clinical strains were used in this study. For each strain, we assessed the virulence by infecting G. mellonella larvae and observing survival. Anidulafungin was administered 2 h after yeast inoculum at 0.6 µg, according to the therapeutic dose recommended for humans. RESULTS Biofilm-forming ability highly influenced the larva-killing rate. A significant (p < 0.0001) survival decrease was observed in the BP group, with 80% of the infected larvae dying within 72 h. NP isolates did not reach the same killing rate, even at the end of experiments (216 h). Larval survival was enhanced (p < 0.0001) by anidulafungin administration in both groups. Survival rate at 72 h was similar in both groups (BP 78.5% and NP 87.5%); whereas there were still differences at the end of the experiments, with a higher survival in the NP group (75 vs 48%). CONCLUSION Our data confirm the pathogenic role of biofilm in C. albicans infections. Its importance was further enhanced by a lack of contribution from extracellular enzymes, detected in both NP and BP strains. In addition, we demonstrated anidulafungin efficacy in treating biofilm-related invasive candidiasis.

A histological procedure to study fungal infection in the wax moth Galleria mellonella

The invertebrate model Galleria mellonella is a widely used factitious host to study the microbial pathogenesis in vivo. However, a specific procedure for the recovery and the processing of the infected tissues, important for a better understanding of the host-pathogen interactions, has not been reported to our knowledge. In the present study we describe a new procedure of fixation and processing of larval tissue that allows studying the larval topographic anatomy and assessing the morphological changes due to the fungal infection. Lepidopteran larvae were infected with Candida albicans strains displaying various biofilm-forming abilities. The whole larvae were then examined for tissue changes by histological techniques. We show that comparing cutting planes, serial transversal sections of paraffin-embedded larva result in better accuracy and information recovering. Using this technique, it was possible to preserve the integrity of G. mellonella internal structures allowing the detailed analysis of morphological differences in different experimental groups (i.e., healthy vs infected larvae). We were also able to study strain-related differences in the pathogenesis of C. albicans by observing the immune response elicited and the invasiveness of two isolates within the larval tissues. In general, by processing the whole larva and optimizing routinely histochemical stainings, it is possible to visualize and analyse infected tissues. Various degrees of pathogenicity (strain- or inoculum-related), and the infection time course can be described in details. Moreover, the host immune response events can be followed throughout the infectious process leading to a comprehensive picture of the studied phenomenon.

Antifungal resistance does not necessarily affect Candida glabrata fitness

Abstract Although there has been an overall good coverage of Candida glabrata infections by the echinocandins, emergence of antifungal resistance during therapy has been reported. We investigated, by using an invertebrate host model, the fitness of sequential C. glabrata isolates with different echinocandins susceptibility patterns. The studied strains were isolated from a case of recurrent fungemia with a fatal outcome due to C. glabrata that developed cross-resistance to echinocandins during caspofungin therapy. The sequential strains isolated post-therapy showed a S663P mutation in the Fks2p hot spot 1. In vivo study in the invertebrate host Galleria mellonella did not suggest a fitness cost related to the acquired antifungal resistance, the three isolates displayed a similar rate of killing (P  =  0·54). We observed a clear correlation between emergence of antifungal resistance and persistence of the causal agent, probably aided by the unchanged fitness and unresponsiveness in vivo to the adopted therapy.

Methicillin-Resistant Staphylococcus aureus in Raw Milk: Prevalence, SCCmec Typing, Enterotoxin Characterization, and Antimicrobial Resistance Patterns.

Staphylococcus aureus is a known major cause of foodborne illnesses, and raw milk and dairy products are often contaminated by enterotoxigenic and antimicrobial-resistant S. aureus strains. In the present study, 35 S. aureus strains were isolated from 383 raw milk samples collected from various dairy herds in the province of Milan (northern Italy). The isolates were characterized based on their antimicrobial susceptibility patterns and the presence of genes encoding staphylococcal enterotoxins (sea, seb, sec, sed, and see). About half (45.7%) of the strains were enterotoxigenic, and 37.1% were resistant to at least one of the antimicrobial drugs tested. Seven (20%) of 35 isolates were identified as methicillin-resistant S. aureus (MRSA), and SCCmec typing performed with a multiplex PCR assay revealed the presence of gene cassettes IV and V, typical of community-acquired MRSA, and I and II, characteristic of health care-associated MRSA. The MRSA strains were evaluated for the presence of the Panton-Valentine leukocidin gene, but this gene was not found. The results of the present study revealed the presence of toxin-producing S. aureus and MRSA strains in raw milk. MRSA and enterotoxigenic S. aureus in dairy farms are an important risk factor for the spread of staphylococcal infections; therefore, further studies are needed to find strategies for monitoring and controlling the presence of S. aureus, especially MRSA, in dairy products.

Correlation between Candida albicans biofilm formation and invasion of the invertebrate host Galleria mellonella

AIM The aim of our study was to investigate whether biofilm production by Candida albicans clinical isolates could be a hallmark of virulence in vivo. MATERIALS & METHODS Twenty clinical isolates of C. albicans were examined via histological studies on larvae infected with various fungal doses (from 10(3) to 10(5) CFU/larva) of biofilm producer and nonproducer strains. RESULTS The poor prognostic role of infection due to a biofilm-producing isolate was confirmed by the Wald test (hazard ratio: 2.63; 95% CI: 2.03-3.41). Histological examinations at 24 h showed a strong innate immune response, with evidence of melanization for both infection groups. However, at 48 h, we found huge differences in filamentation and tissue invasion capability between biofilm nonproducing and producing isolates, the latter being highly organized into biofilm and invading the larval intestinal tract. Invasion corroborated survival data. CONCLUSION The histological results demonstrate that the production of biofilm could enhance the invasiveness of C. albicans.

Molecular Identification and Echinocandin Susceptibility of Candida parapsilosis Complex Bloodstream Isolates in Italy, 2007–2014

The Candida parapsilosis group encompasses three species: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Here, we describe the incidence and echinocandin susceptibility profile of bloodstream isolates of these three species collected from patients admitted to an Italian university hospital from 2007 to 2014. Molecular identification of cryptic species of the C. parapsilosis complex was performed using polymerase chain reaction amplification of the gene encoding secondary alcohol dehydrogenase, followed by digestion with the restriction enzyme BanI. Minimum inhibitory concentrations were determined using the broth microdilution method according to European Committee for Antimicrobial Susceptibility Testing (EUCAST EDef 7.2) and Clinical Laboratory Standards Institute (CLSI M27-A3) guidelines, and the results were compared with those obtained using the E-test and Sensititre methods. Of the 163 C. parapsilosis complex isolates, 136 (83.4%) were identified as C. parapsilosis, and 27 (16.6%) as C. orthopsilosis. The species-specific incidences were 2.9/10,000 admissions for C. parapsilosis and 0.6/10,000 admissions for C. orthopsilosis. No resistance to echinocandins was detected with any of the methods. The percent essential agreement (EA) between the EUCAST and E-test/Sensititre methods for anidulafungin, caspofungin, and micafungin susceptibility was, respectively, as follows: C. parapsilosis, 95.6/97.8, 98.5/88.2, and 93.4/96.3; C. orthopsilosis, 92.6/92.6, 96.3/77.8, and 63.0/66.7. The EA between the CLSI and E-test/Sensititre methods was, respectively, as follows: C. parapsilosis, 99.3/100, 98.5/89.0, and 96.3/98.5; C. orthopsilosis, 96.3/92.6, 100/81.5, and 92.6/88.9. Only minor discrepancies, ranging from 16.9% (C. parapsilosis) to 11.1% (C. orthopsilosis), were observed between the CLSI and E-test/Sensititre methods. In conclusion, this epidemiologic study shows a typical C. parapsilosis complex species distribution, no echinocandin resistance, and it reinforces the relevance of using commercially available microbiological methods to assess antifungal susceptibility. These data improve our knowledge of the national distribution of species of the psilosis group, as there are very few studies of these species in Italy.

Lack of Evidence for Reduced Fitness of Clinical Staphylococcus aureus Isolates with Reduced Susceptibility to Triclosan

phenotypes and virulence potential (3). Phenotypiccharacterization was carried out on one mutant (P10) that hadbeenpassaged10timesinatriclosangradient.MutantP10exhib-itedmultiplephenotypes,includingsmallcolonysize,slowplank-tonic growth, impaired biofilm formation, impaired hemolyticactivity, impaired coagulase activity, and impaired virulence, in amodel of

Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm

BackgroundThe human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents.Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections.ResultsAntifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25–64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4–16) for both planktonic and sessile cells.Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms.An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles.ConclusionsThe direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance.