Synergic combinations of antimicrobial peptides (AMPs) against biofilms of methicillin-resistant Staphylococcus aureus (MRSA) on polystyrene and medical devices.

Abstract Objectives Antimicrobial research is being focused to look for more effective therapeutics against antibiotic-resistant infections caused by methicillin-resistantStaphylococcus aureus (MRSA). In this direction, antimicrobial peptides (AMP) appears a promising solution. The aim of the present study was to investigate the potential activity of Temporin A, Citropin 1.1, CA(1–7)M(2–9)NH2 and Pal-KGK-NH2 in synergic activity against MRSA biofilms developed on polystyrene surface (PSS) and central venous catheter (CVC). Methods The research was subdivided into distinct phases to assess the ability of AMPs to inhibit biofilm formation, to identify a possible synergy between AMPs, and to eradicate preformed biofilms on PSS and CVC using AMPs alone or in combination. Results The activity of the AMPs was particularly evident in the inhibition of biofilm formation on PSS and on CVC, while the eradication of preformed biofilms was more difficult and was reached only after 24 h of contact. The synergic activity of AMPs combinations, selected by their FICI, has led to an improvement in the performance of all the molecules in the removal of different biofilms. Conclusions Overall, AMPs could represent the next generation of antimicrobial agents for a prophylactic or therapeutic tool to control biofilm of antibiotic-resistant and/or biofilm-associated infections on different medical devices

Clinical and microbiological features of ceftolozane/tazobactam-resistant Pseudomonas aeruginosa isolates in a university hospital in central Italy

Objectives: Ceftolozane/tazobactam (C/T) is a novel cephalosporin and β-lactamase inhibitor combination with great activity against Pseudomonas aeruginosa. To assess P. aeruginosa susceptibility to C/T, a surveil- lance study was conducted from October 2018 to March 2019 at the University Hospital ‘Ospedali Riuniti’ in Ancona, Italy. Methods: Minimum inhibitory concentrations (MICs) to C/T were determined by Etest strip. Resistant iso- lates were characterized by phenotypic (broth microdilution antimicrobial susceptibility testing and mod- ified Carbapenem Inactivation Method [mCIM]) and genotypic (Polymerase Chain Reaction [PCR], Pulsed Field Gel Electrophoresis [PFGE], and whole-genome sequencing [WGS]) methods. Clinical variables of patients infected by C/T-resistant P. aeruginosa were collected from medical records. Results: Fifteen of 317 P. aeruginosa collected showed resistance to C/T (4.7%). Ten strains demonstrated carbapenemase activity by mCIM method, and PCR confirmed that eight strains harbored a blaVIM gene while the other two were positive for blaIMP. Additionally, three isolates carried acquired extended spec- trum β-lactamase genes (two isolates carried blaPER and one carried blaGES). Eight strains were strictly related by PFGE and WGS analysis confirmed that they belonged to sequence type (ST)111. The other STs found were ST175 (two isolates), ST235 (two isolates), ST70 (one isolate), ST621 (one isolate), and the new ST3354 (one isolate). Most patients had received previous antibiotic therapies, carried invasive devices, and experienced prolonged hospitalization. Conclusion: This study demonstrated the presence of C/T-resistant P. aeruginosa isolates in a regional hospital carrying a number of resistance mechanisms acquired by different high-risk clone

Characterization of a Multiresistance Plasmid Carrying the optrA and cfr Resistance Genes From an Enterococcus faecium Clinical Isolate

open13noEnterococcus faecium E35048, a bloodstream isolate from Italy, was the first strain where the oxazolidinone resistance gene optrA was detected outside China. The strain was also positive for the oxazolidinone resistance gene cfr. WGS analysis revealed that the two genes were linked (23.1 kb apart), being co-carried by a 41,816-bp plasmid that was named pE35048-oc. This plasmid also carried the macrolide resistance gene erm(B) and a backbone related to that of the well-known Enterococcus faecalis plasmid pRE25 (identity 96%, coverage 65%). The optrA gene context was original, optrA being part of a composite transposon, named Tn6628, which was integrated into the gene encoding for the ζ toxin protein (orf19 of pRE25). The cfr gene was flanked by two ISEnfa5 insertion sequences and the element was inserted into an lnu(E) gene. Both optrA and cfr contexts were excisable. pE35048-oc could not be transferred to enterococcal recipients by conjugation or transformation. A plasmid-cured derivative of E. faecium E35048 was obtained following growth at 42°C, and the complete loss of pE35048-oc was confirmed by WGS. pE35048-oc exhibited some similarity but also notable differences from pEF12-0805, a recently described enterococcal plasmid from human E. faecium also co-carrying optrA and cfr; conversely it was completely unrelated to other optrA- and cfr-carrying plasmids from Staphylococcus sciuri. The optrA-cfr linkage is a matter of concern since it could herald the possibility of a co-spread of the two genes, both involved in resistance to last resort agents such as the oxazolidinones.openMorroni, Gianluca; Brenciani, Andrea; Antonelli, Alberto; Maria D’Andrea, Marco; Di Pilato, Vincenzo; Fioriti, Simona; Mingoia, Marina; Vignaroli, Carla; Cirioni, Oscar; Biavasco, Francesca; Varaldo, Pietro E.; Rossolini, Gian Maria; Giovanetti, EleonoraMorroni, Gianluca; Brenciani, Andrea; Antonelli, Alberto; Maria D’Andrea, Marco; Di Pilato, Vincenzo; Fioriti, Simona; Mingoia, Marina; Vignaroli, Carla; Cirioni, Oscar; Biavasco, Francesca; Varaldo, Pietro E.; Rossolini, Gian Maria; Giovanetti, Eleonor

Resistenza agli oxazolidinoni in isolati clinici di Staphylococcus spp. e Enterococcus spp.

Nella lotta alle infezioni batteriche causate da Gram-positivi, gli oxazolidinoni (linezolid e tedizolid) rappresentano l’ultima classe di antibiotici ad essere stati sviluppati per l’utilizzo in clinica. La resistenza a questi farmaci è dovuta a mutazioni nel ribosoma (23S rRNA, proteine L3 e L4) e a geni acquisiti [cfr, cfr(B) e optrA]. In questo studio sono stati caratterizzati i meccanismi di resistenza agli oxazolidinoni in ceppi clinici di Staphylococcus spp. ed Enterococcus spp. È stato identificato un clone di Staphylococcus epidermidis, endemico negli Ospedali Riuniti di Ancona da 12 anni, che è resistente al linezolid a causa d’una mutazione del 23S rRNA. Sono stati caratterizzati due ceppi di S. epidermidis linezolid-resistenti provenienti dall’ospedale Careggi (Firenze): la resistenza era mediata da mutazioni sul 23S rRNA, sulle proteine L3 e dal gene cfr, localizzato su due nuovi plasmidi coniugativi multi-resistenti correlati. È stato caratterizzato un isolato di Staphylococcus aureus linezolid-resistente proveniente da Firenze, primo S. aureus cfr-positivo isolato in Italia: il cfr era localizzato nel cromosoma all’interno di un plasmide enterococcico linearizzato (pRE25-like), in una nuova struttura a mosaico portante anche i geni di resistenza erm(B) e fexB. È stato effettuato uno screening su enterococchi con MIC del linezolid ≥4 mg/L alla ricerca di determinanti di resistenza agli oxazolidinoni. Sono stati identificati due ceppi di Enterococcus faecium con ridotta sensibilità al linezolid, che portavano i geni cfr e optrA sullo stesso elemento, un plasmide pRE25-like. Infine è stato caratterizzato un ceppo di E. faecium pienamente resistente al linezolid a causa di una mutazione sul 23S rRNA. Pur se la percentuale di resistenza rimane molto bassa, la sorveglianza e l’uso consapevole degli oxazolidinoni sono necessari per preservare l’efficacia di questi antibiotici.In the fight against bacterial infections due to Gram-positive bacteria, oxazolidinones (linezolid and tedizolid) represent the latest class of antibiotics developed for clinical use. Oxazolidinone resistance is caused by mutations in the ribosome (23S rRNA, L3 and L4 proteins) and acquired genes [cfr, cfr(B) and optrA]. In this study oxazolidinone resistance mechanisms have been characterised in clinical strains of Staphylococcus spp. and Enterococcus spp. A clone of Staphylococcus epidermidis, resistant to linezolid due to mutations in 23S rRNA, has been recognised as being endemic in the Ancona Regional Hospital for 12 years. Two linezolid-resistant S. epidermidis isolates from Careggi Hospital (Florence) have been characterised: resistance was mediated by 23S rRNA mutations, L3 protein mutations, and the cfr gene, located on two new related multi-resistance plasmids. A linezolid-resistant Staphylococcus aureus strain from Florence, the first cfr-positive isolate in Italy, has been studied: cfr was located on the chromosome, within a linearized plasmid of enterococcal origin (pRE25-like), in a mosaic structure carrying also resistance genes erm(B) and fexB. A screening was conducted on enterococci showing a linezolid MIC ≥4 mg/L in order to look for oxazolidinones resistance determinants. Two Enteroccocus faecium with reduced linezolid susceptibility were identified which carried both cfr and optrA on the same genetic element, a pRE25-like plasmid. Finally, an E. faecium isolate fully resistant to linezolid has been characterised: resistance was mediated by mutations in rRNA 23S. Even if the incidence of linezolid resistance remains very low, surveillance and conscious use of oxazolidinones are essential to preserve their effectiveness

Characterization and Clonal Diffusion of Ceftaroline Non-Susceptible MRSA in Two Hospitals in Central Italy

Background: Ceftaroline represents a novel fifth-generation cephalosporin to treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA). Methods: Ceftaroline susceptibility of 239 MRSA isolates was assessed by disk diffusion and a MIC test strip following both EUCAST and CLSI guidelines. Non-susceptible isolates were epidemiologically characterized by pulsed-field gel electrophoresis, spa typing, and multilocus sequence typing, and further investigated by PCR and whole genome sequencing to detect penicillin-binding protein (PBP) mutations as well as antibiotic resistance and virulence genes. Results: Fourteen isolates out of two hundred and thirty-nine (5.8%) were non-susceptible to ceftaroline (MIC > 1 mg/L), with differences between the EUCAST and CLSI interpretations. The characterized isolates belonged to seven different pulsotypes and three different clones (ST228/CC5-t041-SCCmecI, ST22/CC22-t18014-SCCmecIV, and ST22/CC22-t022-SCCmecIV), confirming a clonal diffusion of ceftaroline non-susceptible strains. Mutations in PBPs involved PBP2a for ST228-t041-SCCmecI strains and all the other PBPs for ST22-t18014-SCCmecIV and ST22-t022-SCCmecIV clones. All isolates harbored antibiotic resistance and virulence genes with a clonal distribution. Conclusion: Our study demonstrated that ceftaroline non-susceptibile isolates belonged not only to ST228 strains (the most widespread clone in Italy) but also to ST22, confirming the increasing role of these clones in hospital infections

Resistenza agli oxazolidinoni in isolati clinici di Staphylococcus spp. e Enterococcus spp.

Nella lotta alle infezioni batteriche causate da Gram-positivi, gli oxazolidinoni (linezolid e tedizolid) rappresentano l’ultima classe di antibiotici ad essere stati sviluppati per l’utilizzo in clinica. La resistenza a questi farmaci è dovuta a mutazioni nel ribosoma (23S rRNA, proteine L3 e L4) e a geni acquisiti [cfr, cfr(B) e optrA]. In questo studio sono stati caratterizzati i meccanismi di resistenza agli oxazolidinoni in ceppi clinici di Staphylococcus spp. ed Enterococcus spp. È stato identificato un clone di Staphylococcus epidermidis, endemico negli Ospedali Riuniti di Ancona da 12 anni, che è resistente al linezolid a causa d’una mutazione del 23S rRNA. Sono stati caratterizzati due ceppi di S. epidermidis linezolid-resistenti provenienti dall’ospedale Careggi (Firenze): la resistenza era mediata da mutazioni sul 23S rRNA, sulle proteine L3 e dal gene cfr, localizzato su due nuovi plasmidi coniugativi multi-resistenti correlati. È stato caratterizzato un isolato di Staphylococcus aureus linezolid-resistente proveniente da Firenze, primo S. aureus cfr-positivo isolato in Italia: il cfr era localizzato nel cromosoma all’interno di un plasmide enterococcico linearizzato (pRE25-like), in una nuova struttura a mosaico portante anche i geni di resistenza erm(B) e fexB. È stato effettuato uno screening su enterococchi con MIC del linezolid ≥4 mg/L alla ricerca di determinanti di resistenza agli oxazolidinoni. Sono stati identificati due ceppi di Enterococcus faecium con ridotta sensibilità al linezolid, che portavano i geni cfr e optrA sullo stesso elemento, un plasmide pRE25-like. Infine è stato caratterizzato un ceppo di E. faecium pienamente resistente al linezolid a causa di una mutazione sul 23S rRNA. Pur se la percentuale di resistenza rimane molto bassa, la sorveglianza e l’uso consapevole degli oxazolidinoni sono necessari per preservare l’efficacia di questi antibiotici.In the fight against bacterial infections due to Gram-positive bacteria, oxazolidinones (linezolid and tedizolid) represent the latest class of antibiotics developed for clinical use. Oxazolidinone resistance is caused by mutations in the ribosome (23S rRNA, L3 and L4 proteins) and acquired genes [cfr, cfr(B) and optrA]. In this study oxazolidinone resistance mechanisms have been characterised in clinical strains of Staphylococcus spp. and Enterococcus spp. A clone of Staphylococcus epidermidis, resistant to linezolid due to mutations in 23S rRNA, has been recognised as being endemic in the Ancona Regional Hospital for 12 years. Two linezolid-resistant S. epidermidis isolates from Careggi Hospital (Florence) have been characterised: resistance was mediated by 23S rRNA mutations, L3 protein mutations, and the cfr gene, located on two new related multi-resistance plasmids. A linezolid-resistant Staphylococcus aureus strain from Florence, the first cfr-positive isolate in Italy, has been studied: cfr was located on the chromosome, within a linearized plasmid of enterococcal origin (pRE25-like), in a mosaic structure carrying also resistance genes erm(B) and fexB. A screening was conducted on enterococci showing a linezolid MIC ≥4 mg/L in order to look for oxazolidinones resistance determinants. Two Enteroccocus faecium with reduced linezolid susceptibility were identified which carried both cfr and optrA on the same genetic element, a pRE25-like plasmid. Finally, an E. faecium isolate fully resistant to linezolid has been characterised: resistance was mediated by mutations in rRNA 23S. Even if the incidence of linezolid resistance remains very low, surveillance and conscious use of oxazolidinones are essential to preserve their effectiveness

Antifungal Combinations in Dermatophytes

Dermatophytes are the most common cause of fungal infections worldwide, affecting millions of people annually. The emergence of resistance among dermatophytes along with the availability of antifungal susceptibility procedures suitable for testing antifungal agents against this group of fungi make the combinatorial approach particularly interesting to be investigated. Therefore, we reviewed the scientific literature concerning the antifungal combinations against dermatophytes. A literature search on the subject performed in PubMed yielded 68 publications: 37 articles referring to in vitro studies and 31 articles referring to case reports or clinical studies. In vitro studies involved over 400 clinical isolates of dermatophytes (69% Trichophyton spp., 29% Microsporum spp., and 2% Epidermophyton floccosum). Combinations included two antifungal agents or an antifungal agent plus another chemical compound including plant extracts or essential oils, calcineurin inhibitors, peptides, disinfectant agents, and others. In general, drug combinations yielded variable results spanning from synergism to indifference. Antagonism was rarely seen. In over 700 patients with documented dermatophyte infections, an antifungal combination approach could be evaluated. The most frequent combination included a systemic antifungal agent administered orally (i.e., terbinafine, griseofulvin, or azole—mainly itraconazole) plus a topical medication (i.e., azole, terbinafine, ciclopirox, amorolfine) for several weeks. Clinical results indicate that association of antifungal agents is effective, and it might be useful to accelerate the clinical and microbiological healing of a superficial infection. Antifungal combinations in dermatophytes have gained considerable scientific interest over the years and, in consideration of the interesting results available so far, it is desirable to continue the research in this field

Oxazolidinones: mechanisms of resistance and mobile genetic elements involved

The oxazolidinones (linezolid and tedizolid) are last-resort antimicrobial agents used for the treatment of severe infections in humans caused by MDR Gram-positive bacteria. They bind to the peptidyl transferase centre of the bacterial ribosome inhibiting protein synthesis. Even if the majority of Gram-positive bacteria remain susceptible to oxazolidinones, resistant isolates have been reported worldwide. Apart from mutations, affecting mostly the 23S rDNA genes and selected ribosomal proteins, acquisition of resistance genes (cfr and cfr-like, optrA and poxtA), often associated with mobile genetic elements [such as non-conjugative and conjugative plasmids, transposons, integrative and conjugative elements (ICEs), prophages and translocatable units], plays a critical role in oxazolidinone resistance. In this review, we briefly summarize the current knowledge on oxazolidinone resistance mechanisms and provide an overview on the diversity of the mobile genetic elements carrying oxazolidinone resistance genes in Gram-positive and Gram-negative bacteria

Control of host mitochondria by bacterial pathogens

Mitochondria control various processes that are integral to cellular and organismal homeostasis, including Ca2+ fluxes, bioenergetic metabolism, and cell death. Perhaps not surprisingly, multiple pathogenic bacteria have evolved strategies to subvert mitochondrial functions in support of their survival and dissemination. Here, we discuss nonimmunological pathogenic mechanisms that converge on the ability of bacteria to control the mitochondrial compartment of host cells