Publications by authors named "Dafne Bongiorno"

29 Publications

  • Page 1 of 1

Post-Mortem Detection of SARS-CoV-2 RNA in Long-Buried Lung Samples.

Diagnostics (Basel) 2021 Jun 24;11(7). Epub 2021 Jun 24.

Department of Medical and Surgical Sciences and Advanced Technologies "G.F. Ingrassia", Institute of Legal Medicine, University of Catania, 95123 Catania, Italy.

The Coronavirus Disease 19 (COVID-19) pandemic has caused an unexpected death toll worldwide. Even though several guidelines for the management of infectious corpses have been proposed, the limited number of post-mortem analyses during the pandemic has led to inaccuracies in the counting of COVID-19 deaths and contributed to a lack of important information about the pathophysiology of the SARS-CoV-2 infection. Due to the impossibility of carrying out autopsies on all corpses, the scientific community has raised the question of whether confirmatory analyses could be performed on exhumed bodies after a long period of burial to assess the presence of SARS-CoV-2 RNA. Post-mortem lung samples were collected from 16 patients who died from COVID-19 infection and were buried for a long period of time. A custom RNA extraction protocol was developed to enhance extraction of viral RNA from degraded samples and highly sensitive molecular methods, including RT-qPCR and droplet digital PCR (ddPCR), were used to detect the presence of SARS-CoV-2 RNA. The custom extraction protocol developed allowed us to extract total RNA effectively from all lung samples collected. SARS-CoV-2 viral RNA was effectively detected in all samples by both RT-qPCR and ddPCR, regardless of the length of burial. ddPCR results confirmed the persistence of the virus in this anatomical niche and revealed high viral loads in some lung samples, suggesting active infection at the time of death. To the best of our knowledge, this is the first study to demonstrate the persistence of SARS-CoV-2 viral RNA in the lung even after a long post-mortem interval (up to 78 days). The extraction protocol herein described, and the highly sensitive molecular analyses performed, could represent the standard procedures for SARS-CoV-2 detection in degraded lung specimens. Finally, the innovative results obtained encourage post-mortem confirmatory analyses even after a long post-mortem interval.
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http://dx.doi.org/10.3390/diagnostics11071158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8304625PMC
June 2021

Combination of aztreonam, ceftazidime-avibactam and amikacin in the treatment of VIM-1 Pseudomonas aeruginosa ST235 osteomyelitis.

Int J Infect Dis 2021 Jul 4;108:510-512. Epub 2021 Jun 4.

Infectious Diseases Section, Department of Medicine and Surgery, University of Insubria, Varese, Italy.

We describe a challenging case of patient with metallo-beta-lactamase-producing Pseudomonas aeruginosa sternal osteomyelitis following aortic valve replacement with biological prosthesis. The strain exhibited a multidrug-resistance phenotype carrying the bla gene and belonged to the high-risk clone sequence type ST235. The patient was successfully treated with surgical debridement plus antibiotic therapy with ceftazidime/avibactam, aztreonam, and amikacin. Time-kill curves showed that this triple antibiotic combination at 1 × MIC was strongly synergic after 8 h, achieving 99.9% killing and maintaining this until 48 h.
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http://dx.doi.org/10.1016/j.ijid.2021.05.085DOI Listing
July 2021

Resistance to Echinocandins Complicates a Case of Bloodstream Infection: A Case Report.

J Fungi (Basel) 2021 May 21;7(6). Epub 2021 May 21.

U.O.C. Laboratory Analysis Unit, A.O.U. Policlinico-San Marco, 95123 Catania, Italy.

Invasive candidiasis is known to be one of the most common healthcare-associated complications and is caused by several species. First-line drugs, particularly echinocandins, are effective, but there are increasing reports of resistance to these molecules, though rarely related to . Even though the rate of echinocandins resistance remains low (<3%), sporadic cases are emerging. Here, we present a case of bloodstream infection by a pan-echinocandin-resistant affecting a critically ill patient, who died in an intensive care unit following therapeutic failure and multiple organ dysfunction syndrome. This case highlights the need to suspect pan-echinocandin resistance in patients with prolonged echinocandin exposure, particularly in the presence of urinary tract colonization. Our study shows the importance of sequencing to predict therapeutic failure in patients treated with echinocandins and persistent candidemia.
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http://dx.doi.org/10.3390/jof7060405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224343PMC
May 2021

Staphylococcus aureus ST228 and ST239 as models for expression studies of diverse markers during osteoblast infection and persistence.

Microbiologyopen 2021 03;10(2):e1178

Department of Biomedical and Biotechnological Sciences (BIOMETEC), Medical Molecular Microbiology and Antibiotic Resistance laboratory (MMARLab, University of Catania, Catania, Italy.

The ability of S. aureus to infect bone and osteoblasts is correlated with its incredible virulence armamentarium that can mediate the invasion/internalization process, cytotoxicity, membrane damage, and intracellular persistence. We comparatively analyzed the interaction, persistence, and modulation of expression of selected genes and cell viability in an ex vivo model using human MG-63 osteoblasts of two previously studied and well-characterized S. aureus clinical strains belonging to the ST239-SCCmecIII-t037 and ST228-SCCmecI-t041 clones at 3 h and 24 h post-infection (p.i). S. aureus ATCC12598 ST30-t076 was used as a control strain. Using imaging flow cytometry (IFC), we found that these strains invaded and persisted in MG-63 osteoblasts to different extents. The invasion was evaluated at 3 h p.i and persistence at 24 h p.i., in particular: ATCC12598 internalized in 70% and persisted in 50% of MG-63 cells; ST239-SCCmecIII internalized in 50% and persisted in 45% of MG-63 cells; and ST228-SCCmecI internalized in 30% and persisted in 20% of MG-63 cells. During the infection period, ST239-III exerted significant cytotoxic activity resulting from overexpression of hla and psmA and increased expression of the genes involved in adhesion, probably due to the release and re-entry of bacteria inside MG-63 cells at 24 h p.i. The lower invasiveness of ST228-I was also associated with non-cytotoxic activity inside osteoblasts. This clone was unable to activate sufficient cellular reaction and succumbed inside MG-63 cells. Our findings support the idea of considering new strategies, based on a translational approach-eukaryotic host-pathogen interaction (EHPI)-and to be applied on a large scale, to predict S. aureus /osteoblast interaction and treat bone infections. Such strategies rely on the study of the genetic and biochemical basis of both pathogen and host.
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http://dx.doi.org/10.1002/mbo3.1178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087985PMC
March 2021

Internalization in Osteoblast Cells: Mechanisms, Interactions and Biochemical Processes. What Did We Learn from Experimental Models?

Pathogens 2021 Feb 19;10(2). Epub 2021 Feb 19.

Medical Molecular Microbiology and Antibiotic Resistance Laboratory (MMARLab), Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, 95125 Catania, Italy.

Bacterial internalization is a strategy that non-intracellular microorganisms use to escape the host immune system and survive inside the human body. Among bacterial species, showed the ability to interact with and infect osteoblasts, causing osteomyelitis as well as bone and joint infection, while also becoming increasingly resistant to antibiotic therapy and a reservoir of bacteria that can make the infection difficult to cure. Despite being a serious issue in orthopedic surgery, little is known about the mechanisms that allow bacteria to enter and survive inside the osteoblasts, due to the lack of consistent experimental models. In this review, we describe the current knowledge about internalization mechanisms and various aspects of the interaction between bacteria and osteoblasts (e.g., best experimental conditions, bacteria-induced damages and immune system response), focusing on studies performed using the MG-63 osteoblastic cell line, the best traditional (2D) model for the study of this phenomenon to date. At the same time, as it has been widely demonstrated that 2D culture systems are not completely indicative of the dynamic environment in vivo, and more recent 3D models-representative of bone infection-have also been investigated.
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http://dx.doi.org/10.3390/pathogens10020239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922271PMC
February 2021

Different Modulatory Effects of Four Methicillin-Resistant Clones on MG-63 Osteoblast-Like Cells.

Biomolecules 2021 01 7;11(1). Epub 2021 Jan 7.

Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, 95125 Catania, Italy.

is a Gram-positive bacterium responsible for a variety of mild to life-threatening infections including bone infections such as osteomyelitis. This bacterium is able to invade and persist within non-professional phagocytic cells such as osteoblasts. In the present study, four different strains, namely, 2SA-ST239-III (ST239), 5SA-ST5-II (ST5), 10SA-ST228-I (ST228), and 14SA-ST22-IVh (ST22), were tested for their ability to modulate cell viability in MG-63 osteoblast-like cells following successful invasion and persistence. Methicillin-sensitive (MSSA) ATCC-12598-ST30 (ST30) was used as control strain. Despite being proven that ST30, ST239, and ST22 have a similar ability to internalize and persist in MG-63 osteoblast-like cells under our experimental conditions, we demonstrated that the observed decrease in cell viability was due to the different behavior of the considered strains, rather than the number of intracellular bacteria. We focused our attention on different biochemical cell functions related to inflammation, cell metabolism, and oxidative stress during osteoblast infections. We were able to show the following: (1) ST30 and ST239 were the only two clones able to persist and maintain their number in the hostile environment of the cell during the entire period of infection; (2) ST239 was the only clone able to significantly increase gene expression (3 and 24 h post-infection (p.i.)) and protein secretion (24 h p.i.) of both interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in MG-63 osteoblast-like cells; (3) the same clone determined a significant up-regulation of the transforming growth factorbeta 1 (TGF-β1) and of the metabolic marker glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNAs at 24 h p.i.; and (4) neither the MSSA nor the four methicillin-resistant (MRSA) strains induced oxidative stress phenomena in MG-63 cells, although a high degree of variability was observed for the different clones with regard to the expression pattern of nuclear factor E2-related factor 2 (Nrf2) and its downstream gene heme oxygenase 1 (HO-1) activation. Our results may pave the way for an approach to -induced damage, moving towards individualized therapeutic strategies that take into account the differences between MSSA and MRSA as well as the distinctive features of the different clones. This approach is based on a change of paradigm in antibiotic therapy involving a case-based use of molecules able to counteract pro-inflammatory cytokines activity such as selective cytokine signaling inhibitors (IL-6, TNF-α).
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http://dx.doi.org/10.3390/biom11010072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825699PMC
January 2021

In Vitro Activity of Dalbavancin against Refractory Multidrug-Resistant (MDR) Isolates.

Antibiotics (Basel) 2020 Dec 3;9(12). Epub 2020 Dec 3.

Department of Biomedical and Biotechnological Sciences (BIOMETEC)-Medical Molecular Microbiology and Antibiotic Resistance Laboratory (MMARLab), University of Catania, 95123 Catania, Italy.

The high prevalence of methicillin-resistant (MRSA) infections, always treated with vancomycin and daptomycin, has led to the emergence of vancomycin-intermediate (VISA), heteroresistant vancomycin-intermediate (hVISA) and daptomycin non-susceptible (DNS) . Even if glycopeptides and daptomycin remain the keystone for treatment of resistant , the need for alternative therapies that target MRSA has now become imperative. The in vitro antibacterial and bactericidal activity of dalbavancin was evaluated against clinically relevant showing raised antibiotic resistance levels, from methicillin-susceptible to Multidrug-Resistant (MDR) MRSA, including hVISA, DNS and rifampicin-resistant (RIF-R) strains. A total of 124 strains were tested for dalbavancin susceptibility, by the broth microdilution method. Two VISA and 2 hVISA reference strains, as well as a vancomycin-resistant (VRSA) reference strain and a methicillin-susceptible (MSSA) reference strain, were included as controls. Time-kill curves were assayed to assess bactericidal activity. Dalbavancin demonstrated excellent in vitro antibacterial and bactericidal activity against all resistance classes, including hVISA and DNS isolates. The RIF-R strains showed the highest percentage of isolates with non-susceptibility, reflecting the correlation between B mutations and VISA/hVISA emergence. Our observations suggest that dalbavancin can be considered as an effective alternative for the management of severe MRSA infections also sustained by refractory phenotypes.
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http://dx.doi.org/10.3390/antibiotics9120865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761838PMC
December 2020

Gold standard susceptibility testing of fosfomycin in Staphylococcus aureus and Enterobacterales using a new agar dilution panel®.

J Glob Antimicrob Resist 2020 12 27;23:334-337. Epub 2020 Oct 27.

Department of Biomedical and Biotechnological Sciences, section of Microbiology, University of Catania, Italy.

Objectives: Many clinical laboratories have difficulty in routinely performing in vitro fosfomycin susceptibility testing using the agar dilution (AD) method, considered to be the gold standard method. The objective of our work was to evaluate a rapid commercial fosfomycin agar dilution panel against clinical Staphylococcus aureus and Enterobacterales strains, in two different centres located in Italy and in the UK.

Methods: A total of 99 Enterobacterales (mostly Escherichia coli and Klebsiella pneumoniae) and 80 S. aureus clinical isolates was used to evaluate the commercial device, a 12-well panel containing fosfomycin incorporated into CA-MH agar supplemented with 25mg/L of glucose-6-phosphate (Liofilchem S.r.l., Roseto degli Abruzzi, Italy). Testing was performed in two centres (Italy and UK) and kit results were compared against the gold standard in-house AD MIC method.

Results: According to the EUCAST breakpoints, fosfomycin inhibited 61% of the S. aureus strains, and 76% of the Enterobacterales isolates tested by the AD reference method. There was a Categorical Agreement (CA) of 100% and an Essential Agreement (EA) of 91.25% for S. aureus; while the Enterobacterales strains showed a CA of 94% and an EA of 97%. No evaluation errors were observed among S. aureus, while 5% Major Error and 1% Very Major Error were observed for the Enterobacterales.

Conclusions: Our results confirmed the feasibility of determining fosfomycin susceptibility using a commercial AD panel as a routine substitution for the AD test. The few differences observed were only in strains with MICs around the breakpoint used.
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http://dx.doi.org/10.1016/j.jgar.2020.08.025DOI Listing
December 2020

Sensitivity assessment of droplet digital PCR for SARS-CoV-2 detection.

Int J Mol Med 2020 Sep 13;46(3):957-964. Epub 2020 Jul 13.

Department of Biomedical and Biotechnological Sciences, Section of Microbiology, University of Catania, I‑95123 Catania, Italy.

Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) is the gold standard method for the diagnosis of COVID‑19 infection. Due to pre‑analytical and technical limitations, samples with low viral load are often misdiagnosed as false‑negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT‑qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID‑19 were analyzed by droplet digital PCR (ddPCR) and RT‑qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)‑approved probe for the SARS‑CoV‑2 N gene were used. SYBR‑Green RT‑qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT‑qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT‑qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10‑fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT‑qPCR for the diagnosis of COVID‑19 infection in false‑negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID‑19 and the follow‑up of positive patients until complete remission.
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http://dx.doi.org/10.3892/ijmm.2020.4673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388836PMC
September 2020

Fluoroquinolone Metalloantibiotics: A Promising Approach against Methicillin-Resistant .

Int J Environ Res Public Health 2020 04 30;17(9). Epub 2020 Apr 30.

REQUIMTE-LAQV (Rede de Química e Tecnologia - Laboratório Associado para a Química Verde), Departamento de Química e Bioquímica da Faculdade de Ciências da Universidade do Porto, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal.

Fluoroquinolones (FQs) are antibiotics commonly used in clinical practice, although nowadays they are becoming ineffective due to the emergence of several mechanisms of resistance in most bacteria. The complexation of FQs with divalent metal ions and phenanthroline (phen) is a possible approach to circumvent antimicrobial resistance, since it forms very stable complexes known as metalloantibiotics. This work is aimed at determining the antimicrobial activity of metalloantibiotics of Cu(II)FQphen against a panel of multidrug‑resistant (MDR) clinical isolates and to clarify their mechanism of action. Minimum inhibitory concentrations (MICs) were determined against MDR isolates of and methicillin-resistant (MRSA). Metalloantibiotics showed improved antimicrobial activity against several clinical isolates, especially MRSA. Synergistic activity was evaluated in combination with ciprofloxacin and ampicillin by the disk diffusion and checkerboard methods. Synergistic and additive effects were shown against MRSA isolates. The mechanism of action was studied though enzymatic assays and atomic force microscopy (AFM) experiments. The results indicate a similar mechanism of action for FQs and metalloantibiotics. In summary, metalloantibiotics seem to be an effective alternative to pure FQs against MRSA. The results obtained in this work open the way to the screening of metalloantibiotics against other Gram‑positive bacteria.
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http://dx.doi.org/10.3390/ijerph17093127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246690PMC
April 2020

Detection of methicillin-resistant Staphylococcus aureus persistence in osteoblasts using imaging flow cytometry.

Microbiologyopen 2020 05 1;9(5):e1017. Epub 2020 Apr 1.

Department of Biomedical and Biotechnological Sciences (BIOMETEC), Medical Molecular Microbiology and Antibiotic Resistance Laboratory (MMARLab), University of Catania, Catania, Italy.

Methicillin-resistant S. aureus has been reported as the main pathogen involved in chronic infections, osteomyelitis, and prosthetic joint infections. The host/pathogen interaction is dynamic and requires several changes to promote bacterial survival. Here, we focused on the internalization and persistence behavior of well-characterized Staphylococcus aureus invasive strains belonging to the main ST-MRSA-SCCmec clones. To overcome the limitations of the cell culture method, we comparatively analyzed the ability of internalization within human MG-63 osteoblasts with imaging flow cytometry (IFC). After evaluation by cell culture assay, the MRSA clones in the study were all able to readily internalize at 3h postinfection, the persistence of intracellular bacteria was evaluated at 24h both by routine cell culture and IFC assay, after vancomycin-BODIPY staining. A statistical difference of persistence was found in ST5-SCCmecII (26.59%), ST228-SCCmecI (20.25%), ST8-SCCmecIV (19.52%), ST239-SCCmecIII (47.82%), and ST22-SCCmecIVh (50.55%) showing the same ability to internalize as ATCC12598 (51%), the invasive isolate used as control strain for invasion and persistence assays. We demonstrated that the intracellular persistence process depends on the total number of infected cells. Comparing our data obtained by IFC with those of the cell culture assay, we obtained greater reproducibility rates and a number of intracellular bacteria, with the advantage of analyzing live host cells. Moreover, with some limitations related to the lack of whole-genome sequencing analysis, we validated the different proclivities to persist in the main Italian HA-MRSA invasive isolates and our results highlighted the heterogeneity of the different clones to persist during cell infection.
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http://dx.doi.org/10.1002/mbo3.1017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221431PMC
May 2020

Genotypic analysis of Italian MRSA strains exhibiting low-level ceftaroline and ceftobiprole resistance.

Diagn Microbiol Infect Dis 2019 11 15;95(3):114852. Epub 2019 Jun 15.

Department of Biomedical and Biotechnological Sciences (BIOMETEC) - Medical Molecular Microbiology and Antibiotic Resistance laboratory (MMARLab), - University of Catania, Italy. Electronic address:

The aim of this study was to address the involvement of PBP mutations in the bactericidal activity to novel cephalosporins, alone and in combination with daptomycin, in not-related multidrug-resistant methicillin-resistant Staphylococcus aureus strains isolated during a nationwide Italian survey. MICs determination and time-killing assays were performed and mecA, pbp1, pbp2, pbp3, pbp4, and gdpP genes were sequenced. Ten strains showed low-level resistance to ceftaroline and ceftobiprole. PBP2a sequence analysis identified four different mutations (N146K; N204K; T235I; E239K) uniquely present in the non-penicillin-binding domain (nPBD). Epidemiologically, this resistance was associated with the most widespread MDR Italian clone ST228-SCCmecI-t001/t041, confirming its proclivity to accumulate mutations, and it is also associated to substitutions in the GdpP signaling protein, involved in the maintenance of di-AMP balance, recently associated with resistance to beta-lactams. Despite these mutations, both drugs retained their potent in vitro bactericidal activity and showed a synergistic effect towards difficult-to-treat isolates.
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http://dx.doi.org/10.1016/j.diagmicrobio.2019.06.004DOI Listing
November 2019

Emergence of two novel sequence types (3366 and 3367) NDM-1- and OXA-48-co-producing K. pneumoniae in Italy.

Eur J Clin Microbiol Infect Dis 2019 Sep 5;38(9):1687-1691. Epub 2019 Jun 5.

Department of Biomedical and Biotechnological Sciences, section of Microbiology, University of Catania, Via Santa Sofia n.97, 95123, Catania, Italy.

The aim of this study was to analyze the alarming spread of NDM-1- and OXA-48-co-producing Klebsiella pneumoniae clinical isolates, collected between October 2016 and January 2018 in a neonatal intensive care unit of the University Hospital, Catania, Italy, through whole genome sequencing. All confirmed carbapenem-resistant K. pneumoniae (CRKp) isolates were characterized pheno- and geno-typically, as well as by whole genome sequencing (WGS). A total of 13 CRKp isolates were identified from 13 patients. Pulsed-field gel electrophoresis (PFGE) was performed, and the multilocus sequence typing (MLST) scheme used was based on the gene sequence as published on the MLST Pasteur website. Core genome MLST (cgMLST) was also performed. All isolates co-carried bla and bla genes located on different plasmids belonging to the IncM/L and IncA/C2 groups, respectively. The 13 strains had identical PFGE profiles. MLST and cgMLST showed that K. pneumoniae was dominated by CRKp ST101 and two novel STs (ST3666 and ST3367), identified after submission to the MLST database for ST assignment. All isolates shared the same virulence factors such as type 3 fimbriae, genes for yersiniabactin biosynthesis, yersiniabactin receptor, and iron ABC transporter. They carried the wzi137 variant associated with the K17 serotype. To the best of our knowledge, this is the first report of two novel STs, 3366 and 3367, NDM-OXA-48-co-producing K. pneumoniae clinical isolates, in Italy.
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http://dx.doi.org/10.1007/s10096-019-03597-wDOI Listing
September 2019

Spread of Vancomycin-Resistant Enterococcus faecium Isolates Despite Validated Infection Control Measures in an Italian Hospital: Antibiotic Resistance and Genotypic Characterization of the Endemic Strain.

Microb Drug Resist 2018 Oct 26;24(8):1148-1155. Epub 2018 Jan 26.

1 Department of Life Sciences, University of Trieste , Trieste, Italy .

An alarming increase of vancomycin-resistant Enterococcus faecium (VREfm) isolates was detected in an Italian referral hospital subjected to policies of infection control validated by the Joint Commission International. Analysis of the population structure of 122 consecutive, nonreplicate VREfm isolates collected over an 18-month period identified a single major clone that spread around the whole hospital, rapidly establishing an endemic state. It belonged to sequence type (ST) 17 and showed a highly multidrug-resistant phenotype, being resistant to all antimicrobial classes for the carriage of several resistance determinants. Furthermore, some strains with decreased susceptibility to daptomycin were detected. Eighteen out of the 122 isolates did not group in the major clone. They showed a low spreading potential inside the hospital wards, even if most of them displayed a multidrug-resistant phenotype and belonged to a hospital-adapted lineage. Causes that led to the VREfm endemic state have not been fully elucidated. However, it is conceivable that the increase in systemic antibiotic consumption and the use of selective digestive tract decontamination, including vancomycin in critically ill patients during the period before 2014, may have played a role in the ST17 clone dissemination, but additional traits conferring high fitness in hospital environment cannot be excluded.
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http://dx.doi.org/10.1089/mdr.2017.0314DOI Listing
October 2018

Burden of Rifampicin- and Methicillin-Resistant Staphylococcus aureus in Italy.

Microb Drug Resist 2018 Jul/Aug;24(6):732-738. Epub 2017 Nov 29.

Medical Molecular Microbiology and Antibiotic Resistance Laboratory (MMAR Lab), Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania , Catania, Italy .

Rifampicin is one of the major drugs used on its own and also in combination to treat numerous infections sustained by methicillin-resistant Staphylococcus aureus (MRSA). In Italy, rifampicin resistance (RIF-R) is increasing in multidrug-resistant-MRSA isolates (16.4%), with respect to Europe (5.7%). In our study, the relationship between clones, rpoB mutations, and susceptibility profiles in 50 RIF-R MRSA isolated from hospitalized patients was evaluated. Antimicrobial susceptibility testing was performed by the broth microdilution method. Isolates were typed by MLST/SCCmec/spa-typing. The rpoB gene was analyzed by PCR and sequence analysis. RIF-R isolates were 60% heterogeneous vancomycin-intermediate S. aureus (hVISA) and 22% daptomycin nonsusceptible and belonged to the major MRSA clones: ST228-SCCmec I (44%), ST8-SCCmec IV (18%), ST239-SCCmec III (16%), ST5-SCCmec II (14%), and ST22-SCCmec IVh (4%). Thirteen diverse RpoB amino acid substitutions were identified. Half of the strains harbored the H481N substitution, conferring low-level resistance. Different single mutations at the equivalent locus (H481D; H481Y) or in other loci, and multiple mutations conferred high-level resistance. In conclusion, this study investigated the nature of RIF-R in Italy among RIF-R-MRSA strains, finding a prevalence of ST228, strongly associated with reduced susceptibility to glycopeptides (hVISA). The spread of RIF-R strains in clinical settings represents a serious threat, due to their complex resistance nature even to new anti-Gram-positive drugs, making these infections particularly difficult to treat.
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http://dx.doi.org/10.1089/mdr.2017.0299DOI Listing
October 2018

Rapid containment of nosocomial transmission of a rare community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) clone, responsible for the Staphylococcal Scalded Skin Syndrome (SSSS).

Ital J Pediatr 2017 Jan 6;43(1). Epub 2017 Jan 6.

MMARLab - Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, Via Santa Sofia 97, 95123, Catania, Italy.

Background: The aims of this study were to identify the source and the transmission pathway for a Staphylococcal Scalded Skin Syndrome (SSSS) outbreak in a maternity setting in Italy over 2 months, during 2014; to implement appropriate control measures in order to prevent the epidemic spread within the maternity ward; and to identify the Methicillin-Resistant Staphylococcus aureus (MRSA) epidemic clone.

Methods: Epidemiological and microbiological investigations, based on phenotyping and genotyping methods, were performed. All neonates involved in the outbreak underwent clinical and microbiological investigations to detect the cause of illness. Parents and healthcare workers were screened for Staphylococcus aureus to identify asymptomatic carriers.

Results: The SSSS outbreak was due to the cross-transmission of a rare clone of ST5-CA-MRSA-SCCmecV-spa type t311, exfoliative toxin A-producer, isolated from three neonates, one mother (from her nose and from dermatological lesions due to pre-existing hand eczema) and from a nurse (colonized in her nose by this microorganism). The epidemiological and microbiological investigation confirmed these as two potential carriers.

Conclusions: A rapid containment of these infections was obtained only after implementation of robust swabbing of mothers and healthcare workers. The use of molecular methodologies for typing was able to identify all carriers and to trace the transmission.
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http://dx.doi.org/10.1186/s13052-016-0323-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217574PMC
January 2017

Epidemiology of Staphylococcus aureus in Italy: First nationwide survey, 2012.

J Glob Antimicrob Resist 2015 Dec 29;3(4):247-254. Epub 2015 Jul 29.

Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, Catania, Italy.

A 3-month epidemiological study to determine the prevalence and antibiotic resistance of Staphylococcus aureus nosocomial infections was performed in 52 centres throughout Italy in 2012. A total of 21,873 pathogens were analysed. The prevalence of S. aureus among all nosocomial pathogens isolated in that period was 11.6% (n=2541), whilst the prevalence of methicillin-resistant S. aureus (MRSA) among the S. aureus was 35.8% (n=910). All tested antimicrobials demonstrated ≥92.2% susceptibility against methicillin-susceptible S. aureus, with the exception of clindamycin (89.7%) and erythromycin (84.2%). Among MRSA, percentages of resistance ranged from 12.6% to >39% for tetracycline, rifampicin, clindamycin and gentamicin; higher percentages were found for erythromycin (65.4%) and fluoroquinolones (72.3-85.8%). Overall, the glycopeptide minimum inhibitory concentration (MIC) distribution showed that 58.3% of strains possessed MICs of 1-2mg/L and few strains were linezolid- or daptomycin-resistant. Molecular characterisation was performed on 102 MRSA selected from Northern, Central and Southern regions. Five major clones were found: Italian/ST228-I (t001-t023-t041-t1686-t3217), 33.3%; USA500/ST8-IV (t008), 17.6%; E-MRSA15/ST22-IVh (t020-t025-t032-t223), 16.7%; USA100/ST5-II (t002-t653-t1349-t2164-t3217-t388), 14.7%; and Brazilian/ST239/241-III (t030-t037), 3.9%. Five PVL-positive CA-MRSA isolates, belonging to USA300 and minor clones, were also identified. In conclusion, this first nationwide surveillance study showed that in Italy, S. aureus infections accounted for 11.6% of all nosocomial infections; MRSA accounted for approximately one-third of the S. aureus isolates and these were multidrug-resistant organisms. Five major MRSA epidemic clones were observed and were inter-regionally distributed, with ST228-SCCmecI becoming predominant.
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http://dx.doi.org/10.1016/j.jgar.2015.06.006DOI Listing
December 2015

High Resolution Melting-Typing (HRMT) of methicillin-resistant Staphylococcus aureus (MRSA): The new frontier to replace multi-locus sequence typing (MLST) for epidemiological surveillance studies.

J Microbiol Methods 2015 Oct 5;117:136-8. Epub 2015 Aug 5.

Medical Molecular Microbiology and Antibiotic Resistance laboratory (MMAR Lab), Department of Biomedical and Biotechnological Sciences, University of Catania, Italy. Electronic address:

We report an implemented molecular-typing-method based on HRMA to detect SNPs within MLST loci, characterizing 100 clinical MRSA and 11 control strains, representative of Italian clones. The results provide solid evidence that HRMT could be a fast, cost-effective and reliable alternative to MLST, for MRSA molecular epidemiology.
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http://dx.doi.org/10.1016/j.mimet.2015.08.001DOI Listing
October 2015

Methicillin-resistant Staphylococcus aureus nasal colonization in a department of pediatrics: a cross-sectional study.

Ital J Pediatr 2014 Jan 10;40. Epub 2014 Jan 10.

Bambino Gesù Children's Hospital IRCCS, Rome, Italy.

Background: We describe methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage at admission in patients admitted to a Department of Pediatrics.

Methods: All patients received a nasal swab at admission. A questionnaire was administered and molecular genetics analyses were performed on all identified MRSA isolates.

Results: We enrolled 785 patients, affected with both acute and chronic diseases. MRSA nasal colonization prevalence was 1.15% (CI: 0.5607%-2.093%). Methicillin-sensitive Staphylococcus aureus (MSSA) nasal colonization prevalence at admission was 19.75% (CI 17.07%-22.64%). Only one MRSA isolate carried the SCCmec V variant; all other isolates carried the SCCmecIV variant. Five out of 9 MRSA-colonized patients had an underlying condition. Antibiotic therapy in the previous 6 months was a protective factor for both MRSA (OR 0,66; 95% CI: 0,46-0,96) and MSSA (OR 0,65; 95% CI: 0,45-0,97) colonization. A tendency to statistical significance was seen in the association between hospitalization in the 6 months prior to admission and MRSA colonization at admission (OR 4,92; 95% CI: 0,97-24,83). No patient was diagnosed with an S. aureus infection during hospitalization.

Conclusions: The majority of our MRSA colonizing isolates have community origins. Nevertheless, most MRSA-colonized patients had been hospitalized previously, suggesting that strains that circulate in the community also circulate in hospital settings. Further studies should elucidate the role of children with frequent contact with health care institutions in the circulation of antibiotic resistant strains between the hospital and the community.
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http://dx.doi.org/10.1186/1824-7288-40-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896672PMC
January 2014

Worrisome trend of new multiple mechanisms of linezolid resistance in staphylococcal clones diffused in Italy.

J Clin Microbiol 2013 Apr 23;51(4):1256-9. Epub 2013 Jan 23.

University of Catania, Catania, Italy.

In order to assess the frequency of clinically relevant linezolid-resistant staphylococcal isolates, and the role of linezolid in maintaining and coselecting multiple resistance mechanisms (cfr, 23S rRNA, L3/L4 mutations), a prospective Italian study was performed from 2010 to 2011 to confirm the diffusion of three major multidrug-resistant clones (ST2, ST5, ST23).
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http://dx.doi.org/10.1128/JCM.00098-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3666802PMC
April 2013

MRSA nasal colonization in children: prevalence meta-analysis, review of risk factors and molecular genetics.

Pediatr Infect Dis J 2013 May;32(5):479-85

Bambino Gesù Children's Hospital, Rome, Italy.

Background: We report a meta-analysis of methicillin-resistant Staphylococcus aureus (MRSA) nasal colonization prevalence in children and a review of the risk factors as well as molecular genetic characterization.

Methods: All relevant studies reporting prevalence data on MRSA nasal colonization in children published between January 2000 and August 2010 were retrieved from the MEDLINE database and analyzed.

Results: After screening 544 studies, 50 studies were included. We obtained an estimate of MRSA prevalence of 2.7% (95% confidence interval [CI]: 2.2-3.1); of 5.2% (95% CI: 3.1-7.3) in children with underlying conditions and of 2.3% (95% CI: 1.8-2.7) in healthy children; 5.4% (95% CI: 3.1-7.7) in children recruited in hospitals and 3% (95% CI: 2.4-3.6) in children recruited in the community. Staphylococcal cassette chromosome mec type IV is the most diffused cassette globally.

Conclusion: The hospital remains the environment where the microorganism circulates most. Children with underlying conditions could act as vectors of microorganisms between the hospital and the community. MRSA prevention strategies should be tailored to each specific institution, taking into account the nosocomial prevalence of MRSA nasal colonization and infections, and the prevalence of nasal colonization in the community that refers to the specific health care center.
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http://dx.doi.org/10.1097/INF.0b013e3182864e4cDOI Listing
May 2013

DNA methylase modifications and other linezolid resistance mutations in coagulase-negative staphylococci in Italy.

J Antimicrob Chemother 2010 Nov 18;65(11):2336-40. Epub 2010 Sep 18.

Department of Microbiology, University of Catania, Via Androne 81, 95124 Catania, Italy.

Background: Despite 10 years of clinical use, linezolid resistance in Staphylococcus aureus and coagulase-negative staphylococci (CoNS) is still a rare phenomenon. This study reports the mechanisms of resistance and strain types seen in clusters of linezolid-resistant CoNS from two different hospitals in Italy during the period 2008-09.

Methods: Genes associated with linezolid resistance were subjected to molecular analysis and isolates were characterized by PFGE macrorestriction analysis using SmaI.

Results: Thirty-three linezolid-resistant isolates of methicillin-resistant CoNS comprising Staphylococcus epidermidis (24), Staphylococcus hominis (5) and Staphylococcus simulans (4) were studied. The isolates showed varying levels of linezolid resistance. Almost all isolates for which linezolid MICs were 64 mg/L possessed point mutations in domain V of 23S rRNA, while isolates for which the MICs were 256 mg/L expressed methylase activity at position A2503 mediated by the cfr gene. Overall, the isolates showed reduced susceptibility to vancomycin (MICs 1-2 mg/L) and 11 of the 33 isolates showed no susceptibility to teicoplanin. These strains were also resistant to chloramphenicol (28 of 33), lincomycin (24 of 33), erythromycin (17 of 33) and quinupristin/dalfopristin (13 of 33). S. epidermidis isolates, showing mutations or methylase modifications, belonged to different PFGE profiles and to two different sequence types (ST2 and ST23), in which the cfr gene was carried on a plasmid of ∼50 kb.

Conclusions: Clinical CoNS strains with resistance to linezolid and other second-line antibiotics, as well as reduced susceptibility to glycopeptides, have emerged in Italy.
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http://dx.doi.org/10.1093/jac/dkq344DOI Listing
November 2010

Heteroresistance to glycopeptides in Italian meticillin-resistant Staphylococcus aureus (MRSA) isolates.

Int J Antimicrob Agents 2010 Nov 19;36(5):415-9. Epub 2010 Aug 19.

Department of Microbiology, University of Catania (I), Catania, Italy.

The prevalence and molecular characterisation of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) strains were determined in a large group of Italian strains isolated between 2005 and mid 2007. Amongst the 1284 strains isolated from documented infections in hospitalised patients (bloodstream infection, pneumonia, and skin and skin-structure infections), 139 S. aureus with vancomycin minimum inhibitory concentrations (MICs) between 1 mg/L and 2 mg/L were screened for the presence of hVISA using three different methods and were confirmed by population analysis profile (PAP). Thirty-six hVISA strains (25.9%) were detected. Amongst the three screening methods used, the macro Etest (MET) demonstrated 100% specificity and 75% sensitivity. hVISA strains were accessory gene regulator (agr) types I and II and belonged to the major nosocomial clones circulating in Italy (ST8, ST239, ST247 and ST228). All strains were susceptible to quinupristin/dalfopristin, linezolid, daptomycin, tigecycline and dalbavancin. In conclusion, we have demonstrated that hVISA isolates are common amongst MRSA isolates with MICs between 1 mg/L and 2 mg/L in Italy. MET, with its high sensitivity and specificity, should be used for early detection of hVISA, especially in patients with serious or prolonged infections sustained by MRSA. Finally, the most recent anti-Gram-positive drugs maintained their full spectrum of in vitro activity against these strains.
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http://dx.doi.org/10.1016/j.ijantimicag.2010.06.044DOI Listing
November 2010

Linezolid Resistance in Staphylococci.

Pharmaceuticals (Basel) 2010 Jun 24;3(7):1988-2006. Epub 2010 Jun 24.

Department of Microbiology, University of Catania, Via Androne 81, 95124 Catania, Italy.

Linezolid, the first oxazolidinone to be used clinically, is effective in the treatment of infections caused by various Gram-positive pathogens, including multidrug resistant enterococci and methicillin-resistant Staphylococus aureus. It has been used successfully for the treatment of patients with endocarditis and bacteraemia, osteomyelitis, joint infections and tuberculosis and it is often used for treatment of complicated infections when other therapies have failed. Linezolid resistance in Gram-positive cocci has been encountered clinically as well as in vitro, but it is still a rare phenomenon. The resistance to this antibiotic has been, until now, entirely associated with distinct nucleotide substitutions in domain V of the 23S rRNA genes. The number of mutated rRNA genes depends on the dose and duration of linezolid exposure and has been shown to influence the level of linezolid resistance. Mutations in associated ribosomal proteins also affect linezolid activity. A new phenicol and clindamycin resistance phenotype has recently been found to be caused by an RNA methyltransferase designated Cfr. This gene confers resistance to lincosamides, oxazolidinones, streptogramin A, phenicols and pleuromutilins, decrease the susceptibility of S. aureus to tylosin, to josamycin and spiramycin and thus differs from erm rRNA methylase genes. Research into new oxazolidinones with improved characteristics is ongoing. Data reported in patent applications demonstrated that some oxazolidinone derivatives, also with improved characteristics with respect to linezolid, are presently under study: at least three of them are in an advanced phase of development.
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http://dx.doi.org/10.3390/ph3071988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036669PMC
June 2010

Hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) in Italy.

Ann Clin Microbiol Antimicrob 2009 Jun 24;8:22. Epub 2009 Jun 24.

Department of Microbiology, University of Catania, Italy.

The aim of our study was to trace the dynamic changes of hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) lineages in Italy, comparing the genotypic backgrounds of contemporary isolates over a period of 17 years, with those of a sample of early MRSA strains from 1980. In total, 301 non-repetitive MRSA clinical isolates, recovered from 19 Italian hospitals between 1990 and 2007 were selected and analyzed for their antibiotic resistance, typed by PFGE and SCCmec, grouped into clonal-types and further characterized using Multi Locus Sequence Typing (MLST). A sample of fifteen early MRSA strains from 1980 was also used for comparison. The most interesting feature was the recent increase of ST228-MRSA-I (formerly the Italian clone; PFGE E) over the period 2000-2007 (57%), when compared to the period 1990-1999 (29%), and its stability to date, associated with a decrease of the highly epidemic ST247-MRSA-IA (formerly the Iberian clone; PFGE A), (23% from 1990 to 1999, 6% from 2000 to 2007). ST1-MRSA-I (1 out of 2 strains carrying ccrA2B2), ST8-MRSA-I (4 strains), ST15-MRSA-I (1 out of 4 carrying ccrA2B2) and ST30-MRSA-I (2 out of 5 carrying no ccrAB-types and ccrC) were the predominant earliest STs among the MRSA strains in 1980. A temporal shift in the susceptibility levels to glycopeptides was observed: strains with vancomycin MIC of >or= 2 mg/L increased from 19.4% to 35.5%. In conclusion, we describe the alternation of MRSA clones that occurred in hospitals from 1990 to 2007 and the increase of the glycopeptide MIC levels, reflecting a worldwide trend. We document the detection of ST1, ST8, ST15 and ST30 in the 1980 isolates; we hypothesize their possible latency and their appearance as the current CA-MRSA clones.
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http://dx.doi.org/10.1186/1476-0711-8-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708121PMC
June 2009

Pathotype and susceptibility profile of a community-acquired methicillin-resistant Staphylococcus aureus strain responsible for a case of severe pneumonia.

Diagn Microbiol Infect Dis 2009 Jan;63(1):100-4

Department of Microbiology, University of Catania, 95124 Catania (I), Italy.

Recent articles have described an increasing number of infections due to Panton-Valentine leukocidin PVL-positive community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) worldwide. We report a case of necrotizing pneumonia successfully treated with levofloxacin in Italy, sustained by a PVL-positive CA-MRSA, belonging to ST88 and carrying a staphylococcal chromosomal cassette mec type V. Further molecular characterization of isolates revealed that they were PVL positive, belonged to the agr IV allele, possessed a capsular type 8, and had an almost complete pathotype composed of leukocidins and numerous adhesins. In addition, the strains were strong biofilm producers.
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http://dx.doi.org/10.1016/j.diagmicrobio.2008.09.012DOI Listing
January 2009

In vitro activity of daptomycin against methicillin- and multi-resistant Staphylococcus haemolyticus invasive isolates carrying different mec complexes.

Diagn Microbiol Infect Dis 2008 Jun 7;61(2):227-31. Epub 2008 Mar 7.

Department of Microbiology, University of Catania, Catania, Italy.

Fifty methicillin and multi-resistant Staphylococcus haemolyticus (MRSH) strains, were tested against daptomycin and comparators. Daptomycin showed an overall MIC(90) value of 1 mg/L, similar to that of quinopristin/dalfopristin and lower than the values for vancomycin (2 mg/L), linezolid (2 mg/L) and teicoplanin (32 mg/L). Eighteen strains showed heteroresistant subpopulations with teicoplanin, whereas two strains were also heteroresistant to vancomycin and these sub-populations retained susceptibility to daptomycin. Our results show the good in vitro activity of daptomycin in MRSH strains possessing a multi-resistant phenotype. Clinical data on daptomycin efficacy are necessary to confirm our in vitro findings.
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http://dx.doi.org/10.1016/j.diagmicrobio.2008.01.019DOI Listing
June 2008

Characterization of a variant of the SCCmec element in a bloodstream isolate of Staphylococcus intermedius.

Microb Drug Resist 2007 ;13(1):7-10

Department of Microbiological and Gynaecological Science, University of Catania, Via Androne 81, 95124 Catania, Italy.

Here we report for the first time, a detailed characterization of a variant of the SCCmec element, in a methicillin-resistant Staphylococcus intermedius human isolate. S. intermedius is a coagulase-positive zoonotic microrganism, recently classified as a separate species. In routine clinical laboratory practice, the coagulase production is used as criterion of pathogenicity related to S. aureus, but S. intermedius is frequently misidentified-being mistaken for S. aureus-and consequently its real incidence underestimated. S. intermedius have been found only occasionally in human beings, and methicillin-resistance is very rare for this organism. Even if the genetic element responsible for methicillin-resistance--the mecA gene carried by diverse staphylococcal chromosomal cassettes--has been described in various staphylococcal species, the current literature doesn't report any case of S. intermedius isolate carrying SCCmec-like elements. Our study could be useful to explain the mechanism and routes of transfer of the chromosomal cassette carrying the mec complex, among staphylococci.
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http://dx.doi.org/10.1089/mdr.2006.9991DOI Listing
August 2007
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