Comparative profiling of agr locus, virulence, and biofilm-production genes of human and ovine non-aureus staphylococci.

BACKGROUND: In a collaboration between animal and human health care professionals, we assessed the genetic characteristics shared by non-aureus staphylococci (NAS) infecting humans and dairy ewes to investigate their relatedness in a region concentrating half of the total National sheep stock. We examined by PCR 125 ovine and 70 human NAS for biofilm production, pyrogenic toxins, adhesins, autolysins genes, and accessory gene regulator (agr) locus. The microtiter plate assay (MPA) was used for the phenotypic screening of biofilm production. Ovine NAS included S. epidermidis, S. chromogenes, S. haemolyticus, S. simulans, S. caprae, S. warneri, S. saprophyticus, S. intermedius, and S. muscae. Human NAS included S. haemolyticus, S. epidermidis, S. hominis, S. lugdunensis, S. capitis, S. warneri, S. xylosus, S. pasteuri, and S. saprophyticus subsp. bovis. RESULTS: Phenotypically, 41 (32.8%) ovine and 24 (34.3%) human isolates were characterized as biofilm producers. Of the ovine isolates, 12 were classified as biofilm-producing while the remaining 29 as weak biofilm-producing. All 24 human isolates were considered weak biofilm-producing. Few S. epidermidis isolates harbored the icaA/D genes coding for the polysaccharide intercellular adhesin (PIA), while the bhp, aap, and embp genes coding biofilm accumulation proteins were present in both non-producing and biofilm-producing isolates. Fifty-nine sheep NAS (all S. epidermidis, 1 S. chromogenes, and 1 S. haemolyticus) and 27 human NAS (all S. epidermidis and 1 S. warneri) were positive for the agr locus: agr-3se (57.8%) followed by agr-1se (36.8%) predominated in sheep, while agr-1se (65.4%), followed by agr-2se (34.6%) predominated in humans. Concerning virulence genes, 40, 39.2, 47.2%, 52.8, 80 and 43.2% of the sheep isolates carried atlE, aae, sdrF, sdrG, eno and epbS respectively, against 37.1, 42.8, 32.8, 60, 100 and 100% of human isolates. Enterotoxins and tsst were not detected. CONCLUSIONS: Considerable variation in biofilm formation ability was observed among NAS isolates from ovine and human samples. S. epidermidis was the best biofilm producer with the highest prevalence of adhesin-encoding genes.

A challenging complication following SARS-CoV-2 infection: a case of pulmonary mucormycosis.

Severe acute respiratory syndrome coronavirus 2 infection might induce a significant and sustained lymphopenia, increasing the risk of developing opportunistic infections. Mucormycosis is a rare but severe invasive fungal infection, mainly described in immunocompromised patients. The first case of a patient diagnosed with coronavirus disease (COVID-19) who developed a pulmonary mucormycosis with extensive cavitary lesions is here reported. This case highlights how this new coronavirus might impair the immune response, exposing patients to higher risk of developing opportunistic infections and leading to worse outcomes.

Molecular typing of XDR Acinetobacter baumannii strains in an Italian ICU.

OBJECTIVE: To investigate the antimicrobial susceptibility and clonal relationship of Acinetobacter baumannii strains isolated in an Italian ICU. DESIGN: Epidemiological, observational, retrospective, longitudinal study. SETTING AND PARTICIPANTS: The ICU of the University Hospital of Sassari, Italy. MAIN OUTCOME MEASURES: Pulsed Field Gel Electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST) were used to evaluate the genomic features of the isolated strains. RESULTS: Drug susceptibility testing for all isolated strains showed the same resistance pattern, characterized by resistance to the most important antibiotics, with the only exception of colistin. PFGE showed a very poor between-strain variability; three distinct clusters, 11, 4, and 1 isolates in size, were identified (Dice's coefficient: 92.11%). MLST showed that all isolated strains belonged to sequence type 2 (ST2). All isolates collected from the environment and the human samples were positive for the following genes: blaOXA-23, blaOXA-51-like, blaVIM-like, blaIMP-like, andISAba1; however, blaOXA-24-like, blaOXA-58-like, and blaNDM-like were not detected. CONCLUSIONS: The survey identified XDR strains belonging to the same cell clone, confirming the wide circulation and environmental persistence of this microorganism.

Occult Vancomycin-Resistant Enterococcus faecium ST117 Displaying a Highly Mutated vanB2 Operon.

Rare information is available on clinical Enterococcus faecium encountered in Sardinia, Italy. This study investigated the antimicrobial susceptibility profiles and genotypic characteristics of E. faecium isolated at the University Hospital of Sassari, Italy, using the Vitek2 system and PCR, MLST, or WGS. Vitek2 revealed two VanB-type vancomycin-resistant Enterococcus faecium (VREfm) isolates (MICs mg/L = 8 and ≥32) but failed to detect vancomycin resistance in one isolate (MIC mg/L ≤ 1) despite positive genotypic confirmation of vanB gene, which proved to be vancomycin resistant by additional phenotypic methods (MICs mg/L = 8). This vanB isolate was able to increase its vancomycin MIC after exposure to vancomycin, unlike the "classic" occult vanB-carrying E. faecium, becoming detectable by Vitek 2 (MICs mg/L ≥ 32). All three E. faecium had highly mutated vanB2 operons, as part of a chromosomally integrated Tn1549 transposon, with common missense mutations in VanH and VanB2 resistance proteins and specific missense mutations in the VanW accessory protein. There were additional missense mutations in VanS, VanH, and VanB proteins in the vanB2-carrying VREfm isolates compared to Vitek2. The molecular typing revealed a polyclonal hospital-associated E. faecium population from Clade A1, and that vanB2-VREfm, and nearly half of vancomycin-susceptible E. faecium (VSEfm) analyzed, belonged to ST117. Based on core genome-MLST, ST117 strains had different clonal types (CT), excluding nosocomial transmission of specific CT. Detecting vanB2-carrying VREfm isolates by Vitek2 may be problematic, and alternative methods are needed to prevent therapeutic failure and spread.

Microarray technology for yeast identification directly from positive blood cultures. A multicenter Italian experience.

The authors evaluated the performance of the MycArray™ Yeast ID (Myconostica Ltd, UK) assay in the identification of a total of 88 yeast isolates recovered in culture as compared to that obtained through routine methods. The turn-around time for species identification directly from cultures by the MycArray was 6 hours, much quicker than classical methods and all yeasts were correctly identified. In two cases a double identification including Saccharomyces cerevisiae was noted, but it was not confirmed by culture. The results show that MycArray Yeast ID can be a potential tool for rapid detection and identification of Candida species.

Evaluation of the peptide nucleic acid fluorescence in situ hybridisation technology for yeast identification directly from positive blood cultures: an Italian experience.

Fungaemia is an increasing nosocomial pathology. The 'gold standard' for detection of fungaemia is blood culture, but it is time-consuming and its sensitivity for early detection is low. On the other hand, yeasts present different antifungal sensitivity patterns to be quickly detected to allow an effective treatment. The aim of this study was to evaluate the diagnostic performances of PNA-FISH to directly identify yeasts from blood cultures and to compare results with those obtained by culture. A total of 176 blood cultures positive for yeasts at direct Gram stain and 24 negative blood cultures as control collected from 15 Italian hospitals, included in a network coordinated by the Medical Mycology Committee, Italian Society of Clinical Microbiology (AMCLI), were examined both by culture and PNA-FISH technology. Sensitivity of the PNA-FISH technique evaluated for five Candida species was 99.3% and specificity, 100%. Distinguishing which yeast is implicated in fungaemia and whether the infection is caused by multiple species are important for the selection of antifungal therapy. The PNA-FISH technique is a very useful approach because the test discriminates between groups of Candida species with different susceptibility pattern, particularly against azoles and echinocandins, with only a 90-minute turn-around time after the Gram-stain reading.

Multicenter Italian Study on "In Vitro Activities" of Isavuconazole, Voriconazole, Amphotericin B, and Caspofungin for Aspergillus Species: Comparison between SensititreTM YeastOneTM and MIC Test Strip.

In this study the activity of Isavuconazole, Voriconazole, Amphotericin B, and Caspofungin against 224 clinical isolates of Aspergillus spp. originating from seven Italian hospitals, was comparatively evaluated with two commercial antifungal susceptibility tests (AST): SensititreTM YeastOneTM (SYO) and MIC Test Strip. More attention was focused on Isavuconazole activity, given the new introduction of the drug in widely distributed antifungal susceptibilities methods in the clinical microbiology lab. The minimum inhibitory concentrations of antifungal drug that can inhibit the growth of pathogen by 90% (MIC90) for Isavuconazole detected by SYO were 0.5, 1, 0.25, and 2 µg/mL for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus niger, respectively, whilst they were 0.25, 0.25, 0.5, and 0.75 µg/mL by MIC Test Strip. Essential agreement between the two tested methods for Isavuconazole is 70% for all the species tested, 75.7% for A. fumigatus, 45.2% for A. flavus, 90.6% for A. terreus, and 40% for A. niger. Although the tested strains do not express any phenotypic resistance, MIC results were quite different if tested with microdilution broth or gradient agar method. This is the first Italian multicenter report on Isavuconazole MIC obtained employing the widely used SensititreTM Yeast OneTM (SYO) and MIC Test Strip on clinical isolates of Aspergillus.

Interlaboratory evaluation of VITEK2 system and Sensititre YeastOne® for antifungal susceptibility testing of yeasts isolated from blood cultures against four antifungal agents.

An interlaboratory evaluation (seven centers) of VITEK2 System and Sensititre YeastOne® was conducted to test the antifungal susceptibilities of yeasts. The MICs of amphotericin B, fluconazole, flucytosine, and voriconazole were determined for 70 isolates of Candida spp. Our results demonstrated a higher interlaboratory agreement of VITEK 2 System than Sensititre YeastOne©. A good concordance between the two methods was observed for amphotericin B, fluconazole, voriconazole and 5-fluorocytosine (from 81.4% to 88.6%). The study suggests the potential value of the VITEK2 System as a convenient alternative method for testing the susceptibility of yeasts. It also indicates the need for further optimization of MIC endpoint criteria to improve interlaboratory agreement.

Water distribution systems in Sardinian hospitals host invasive clonal lineages of the Fusarium oxysporum and Fusarium solani species complexes.

Several Fusarium species cause disease on human hosts, including commonly fatal infections in immunocompromised individuals. Recently, cases of hospitalized patients affected by fusaria were reported in the Tyrrhenian Island of Sardinia, Italy. To precisely characterize the Fusarium species and haplotypes present in hospitals of the region, a multilocus DNA sequence typing (MLST) approach was applied. Water distribution systems in four departments belonging to four Sardinian hospitals were sampled. Fusarium species and sequence types (STs) were identified using MLST based on sequences of the elongation factor 1-alpha (EF-1α) gene, the nuclear ribosomal DNA intergenic spacer region (IGS rDNA), and/or a portion of the second-largest subunit of RNA polymerase (RPB2) gene. The majority of isolates obtained from Sardinian hospitals (90.7%) were identified as representatives of the Fusarium oxysporum species complex (FOSC), followed by those of the F. solani species complex (FSSC) (8.2%), and F. dimerum (1.1% of all isolates). Ten STs were found among the FOSC and FSSC, with more than 60% of the isolates identified as either FOSC ST 33 or FSSC 1 (F. petroliphilum). More than half of the FOSC isolates obtained from the water systems in all four hospitals belonged to the worldwide distributed clonal lineage ST 33. This haplotype is the most prevalent among the FOSC in different countries, being responsible for the vast majority of cases of human fusariosis.

Molecular phylogenetic diversity of dermatologic and other human pathogenic fusarial isolates from hospitals in northern and central Italy.

Fifty-eight fusaria isolated from 50 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusarioses in northern and central Italy. Sequence typing revealed that the isolates were nearly evenly divided among the Fusarium solani species complex (FSSC; n = 18), the F. oxysporum species complex (FOSC; n = 20), and the Gibberella (Fusarium) fujikuroi species complex (GFSC; n = 20). The three-locus typing scheme used for members of the FSSC identified 18 novel STs distributed among six phylogenetically distinct species, yielding an index of discrimination of 1.0. Phylogenetic analysis of the FOSC two-locus data set identified nine STs, including four which were novel, and nine isolates of ST 33, the previously described widespread clonal lineage. With the inclusion of eight epidemiologically unrelated ST 33 isolates, the FOSC typing scheme scored a discrimination index of 0.787. The two-locus GFSC typing scheme, which was primarily designed to identify species, received the lowest discrimination index, with a score of 0.492. The GFSC scheme, however, was used to successfully identify 17 isolates as F. verticillioides, 2 as F. sacchari, and 1 as F. guttiforme. This is the first report that F. guttiforme causes a human mycotic infection, which was supported by detailed morphological analysis. In addition, the results of a pathogenicity experiment revealed that the human isolate of F. guttiforme was able to induce fusariosis of pineapple, heretofore its only known host.

Management of antifungal susceptibility testing in Italy: comparative results of 2 nationwide surveys (1999 and 2004) in 102 Italian hospitals.

The purpose of this study was to verify the standard procedures and minimum level of knowledge of Italian public laboratories involved in the management of antifungal susceptibility testing (AST). Two nationwide surveys were performed in 1999 and 2004. One hundred and two Italian hospitals located in 85 provincial capitals (82.5%) participated to these surveys. In 1999, 28 (27.5%) laboratories versus 16 (15.7%) in 2004 stated that they did not perform any susceptibility testing. Some discrepancies observed in the survey confirm that AST is difficult to be correctly managed, and that it can be performed only in very well-trained centers. The great variability of the results of MIC determination and clinical interpretation underlines the urgent need to improve knowledge about indications, method choice, and interpretative criteria for AST both for clinical microbiologists and clinicians.