Daptomycin Liposomes Exhibit Enhanced Activity against Staphylococci Biofilms Compared to Free Drug.

The purpose of the present study was to investigate the anti-staphylococcal activity of liposomal daptomycin against four biofilm-producing S. aureus and S. epidermidis clinical strains, three of which are methicillin-resistant. Neutral and negatively charged daptomycin-loaded liposomes were prepared using three methods, namely, thin-film hydration (TFH), a dehydration-rehydration vesicle (DRV) method, and microfluidic mixing (MM); moreover, they were characterized for drug encapsulation (EE%), size distribution, zeta-potential, vesicle stability, drug release, and drug integrity. Interestingly, whilst drug loading in THF and DRV nanosized (by extrusion) vesicles was around 30-35, very low loading (~4%) was possible in MM vesicles, requiring further explanatory investigations. Liposomal encapsulation protected daptomycin from degradation and preserved its bioactivity. Biofilm mass (crystal violet, CV), biofilm viability (MTT), and growth curve (GC) assays evaluated the antimicrobial activity of neutral and negatively charged daptomycin-liposomes towards planktonic bacteria and biofilms. Neutral liposomes exhibited dramatically enhanced inhibition of bacterial growth (compared to the free drug) for all species studied, while negatively charged liposomes were totally inactive. Biofilm prevention and treatment studies revealed high antibiofilm activity of liposomal daptomycin. Neutral liposomes were more active for prevention and negative charge ones for treating established biofilms. Planktonic bacteria as well as the matured biofilms of low daptomycin-susceptible, methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis (MRSE) strains were almost completely eradicated by liposomal-daptomycin, indicating the need for their further exploration as antimicrobial therapeutics.

Clonal dissemination and resistance genes among Stenotrophomonas maltophilia in a Greek University Hospital during a four-year period.

Treatment of Stenotrophomonas maltophilia infections comprises of sulfamethoxazole/tripethoprim (SXT) or fluoroquinolones. We investigated antimicrobial resistance, presence of resistance genes (sul1, smqnr) and clonal dissemination in S. maltophilia from a university hospital. Among 62 isolates, 45 (73%) represented infection. Two isolates (3%) were resistant to SXT and three (5%) to levofloxacin. Twenty-nine isolates (47%), including two out of three levofloxacin-resistant, carried smqnr. Resistance of S. maltophilia was low and was not associated with sul1 or smqnr carriage. Although high degree of genetic diversity was identified (29 pulsotypes), 22/62 (35.5%) strains were classified into four clones; clone b was associated with bacteraemias.

Persistent Coagulase-Negative Staphylococcal Bacteremia in Neonates: Clinical, Microbiological Characteristics and Changes within a Decade.

Atypical outbreaks of persistent coagulase-negative staphylococci (CoNS) bacteremias, defined as three or more consecutive positive blood cultures with the same CoNS species, at least 48 h apart, have been reported in neonatal intensive-care units (NICUs). Our aim was to describe the profile of these cases in our NICU over a two-year period with the objective of assessing possible changes within a decade. Demographics, clinical and microbiological data were recorded for all CoNS bacteremias in our tertiary NICU during 2016-2017 and compared with the results of the same study in 2006-2007. Fifty-six cases of CoNS sepsis were recorded. Fourteen (25%) of them were persistent. There were no significant differences in demographic and clinical characteristics between cases with persistent vs. non-persistent bacteremia. Staphylococcus epidermidis was the most common species. In logistic regression analysis, biofilm production (ß = 2.464, p = 0.04) was the most significant determinant for the development of persistent CoNS bacteremia. Our isolates were less likely to produce biofilm and carry ica operon as compared to those of 2006-2007. The cases of persistent CoNS sepsis have decreased within a decade, which could be attributed to the implementation of intensive infection control practices. Biofilm production remains the most important risk factor.

Predominance of community-associated, methicillin-susceptible Staphylococcus aureus infections among hospitalized children and adolescents.

Introduction. Staphylococcus aureus infections cause significant morbidity and mortality in children and adolescents.Gap Statement. There is limited data on the characteristics of S. aureus infections requiring hospitalization in childhood.Aim.To investigate the molecular epidemiology and antibiotic resistance of S. aureus clinical isolates from children and adolescents.Methodology.All S. aureus isolates recovered from patients aged <18 years, admitted to a referral hospital, with culture-proven invasive or non-invasive infections during the 4 year period 2015 to 2018 were analysed for antimicrobial resistance, virulence genes, PFGE and multilocus sequence typing (MLST). Cases were assigned to community-associated, community-onset healthcare-associated or hospital-associated infections based on epidemiological case definitions.Results.Among 139 S. aureus infections, 88.5 % (123/139) were caused by methicillin-susceptible isolates (MSSA) and 73.4 % (102/139) were classified as community-associated infections. tst and lukS/lukF-PV genes were more common among MRSA as compared to MSSA isolates (tst, p 0.04; lukS/lukF-PV, p 0.007). Invasive disease was noted in 22/139 patients (15.8 %). Staphylococcal scalded skin syndrome caused by fusidic-resistant MSSA increased over time (22.8 % in 2017-2018 vs 8.3 % in 2015-2016, OR 3.24; 95 % CI 1.10-8.36; P 0.03). By PFGE genotyping, 22 pulsotypes were identified. A total of five sequence types (STs) were identified among 58 isolates analysed by MLST. More than one third of MSSA isolates (40/123, 32.5 %) and 13/23 (56.5 %) of SSSS isolates belonged to pulsotype 1, classified as sequence type 121 (ST121). MRSA isolates were equally distributed to pulsotypes A (ST30), B (ST239), C (ST80), H (ST225). ST121 isolates carried fnbA (40/40), eta/etb genes (29/40), exhibited high resistance to fusidic acid and were increasingly resistant to mupirocin.Conclusion.In our population, community-associated MSSA was the predominant cause of S. aureus infections characterized by polyclonality, increasing resistance to fusidic acid and mupirocin. PFGE type 1 ST121 clone, harboured exfoliative toxin genes and was associated with rising trends of SSSS.

Moxifloxacin Liposomes: Effect of Liposome Preparation Method on Physicochemical Properties and Antimicrobial Activity against Staphylococcus epidermidis.

The aim of this study was the development of optimal sustained-release moxifloxacin (MOX)-loaded liposomes as intraocular therapeutics of endophthalmitis. Two methods were compared for the preparation of MOX liposomes; the dehydration-rehydration (DRV) method and the active loading method (AL). Numerous lipid-membrane compositions were studied to determine the potential effect on MOX loading and retention in liposomes. MOX and phospholipid contents were measured by HPLC and a colorimetric assay for phospholipids, respectively. Vesicle size distribution and surface charge were measured by DLS, and morphology was evaluated by cryo-TEM. The AL method conferred liposomes with higher MOX encapsulation compared to the DRV method for all the lipid compositions used. Cryo-TEM showed that both liposome types had round vesicular structure and size around 100-150 nm, while a granular texture was evident in the entrapped aqueous compartments of most AL liposomes, but substantially less in DRV liposomes; X-ray diffraction analysis demonstrated slight crystallinity in AL liposomes, especially the ones with highest MOX encapsulation. AL liposomes retained MOX for significantly longer time periods compared to DRVs. Lipid composition did not affect MOX release from DRV liposomes but significantly altered drug loading/release in AL liposomes. Interestingly, AL liposomes demonstrated substantially higher antimicrobial potential towards S. epidermidis growth and biofilm susceptibility compared to corresponding DRV liposomes, indicating the importance of MOX retention in liposomes on their activity. In conclusion, the liposome preparation method/type determines the rate of MOX release from liposomes and modulates their antimicrobial potential, a finding that deserves further in vitro and in vivo exploitation.

Resveratrol loaded in cationic glucosylated liposomes to treat Staphylococcus epidermidis infections.

Glucosylated liposomes composed of the natural saturated phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Chol) and a cationic amphiphile featuring a glucosyl moiety (GL4), have been developed for delivering the antimicrobial trans-Resveratrol (RSV) to S. epidermidis, characterized by carbohydrate-specific adhesins able to recognize glucose. The cationic derivative of cholesterol, DC-Chol, was also included in liposome formulations, alone or in combination with GL4, in order to explore the role of both cationic charge and sugar moiety in the interaction of liposomes with bacterial cells. RSV was included inside glucosylated cationic liposomes by the thin film method, coupled with either extrusion or sonication; liposome mean diameter, polydispersity index, surface charge, RSV entrapment efficiency and concentration have been measured by DLS, electrophoretic mobility, and HPLC. The antimicrobial activity of RSV-loaded liposomes was evaluated by monitoring the bacterial growth curves of two cell lines of Staphylococcus epidermidis, a slime positive strain (i.e. a strain able to form a biofilm) and a slime negative one. Results point out that, when the glucosylamphiphile GL4 is included in the formulation, only the extrusion protocol allows obtaining monodisperse liposomes with high RSV entrapment efficiency. The mean diameters of empty and resveratrol-loaded liposomes are all around 120-140 nm and size distribution are narrow, except for samples including GL4 at 5 molar percentage. Here the higher polydispersity index may be the indication of the occurrence of a restructuring phenomenon. The microbiological tests put in evidence a different response of the two bacterial cell lines to liposome treatments, in fact, the slime negative bacterial cells, that are not able to produce the extracellular polymeric substances, are more susceptible to the cationic charge of the liposomes and to the detergent effect of GL4. The most interesting results concern DPPC/Chol/GL4 liposomes on the slime positive strain: this formulation, non-toxic in itself, displays an enhanced antibacterial efficacy with respect to free RSV, killing bacteria even at concentration tenfold under the MIC.

Methicillin-resistant Staphylococcus aureus transmission and hospital-acquired bacteremia in a neonatal intensive care unit in Greece.

BACKGROUND: Staphylococcus aureus is a common pathogen causing hospital acquired infections (HAIs) in neonates. In this study, the epidemiology of methicillin-resistant S. aureus (MRSA) colonization and infections in a 30-bed, level III university-affiliated neonatal intensive care unit (NICU) located in a children's hospital was retrospectively investigated for the period 2014-2018. METHODS: Genes encoding Panton-Valentine Leukocidin (lukS/lukF-PV, PVL), toxic shock syndrome toxin (tst), exfoliative toxins (eta, etb), and the resistance genes mecA, mecC and fusB, were defined in 46 representative strains by PCRs. Relatedness of strains was assessed by MLST. RESULTS: Of 1538 neonates, 77 (5%) had a positive culture for MRSA (23/77 were NICU-acquired and 54/77 imported cases). Four MRSA bacteremias occurred. Most isolates were multi-resistant. One major clone was identified, ST225, among 40 tested neonatal strains (23/40, 58%). Of these, 14/23 were imported from the same maternity hospital (MH). Another clone, ST217, was predominant (4/6) among health care workers (HCWs), found colonized. Four isolates classified as ST80 were PVL-positive. Additional four strains carried tst (10%), belonging to ST30 and ST225 (two strains each), and two etb. The implicated MH was notified for the problem, decolonization treatment was successfully performed in HCWs and neonates. Strengthening of infection control measures with emphasis on hand hygiene was applied. CONCLUSIONS: Uncovering reservoirs for on-going MRSA transmission in NICUs has proved challenging. Well known nosocomial MRSA clones are being constantly introduced and transmitted via MHs and HCWs. Effective infection prevention and control requires constant vigilance.

In Vitro Anti-Biofilm Activity of Bacteriophage K (ATCC 19685-B1) and Daptomycin against Staphylococci.

The purpose of the present study was to investigate anti-staphylococcal activity of daptomycin and bacteriophage K, alone or in combination, against biofilm-producers and non-producers S. aureus and S. epidermidis strains, under biofilm forming and cells' proliferation conditions. Daptomycin and bacteriophage K (ATCC 19685B1), in different concentrations, were tested against 10 Staphylococcus aureus and 10 S. epidermidis, characterized by phenotypes and genotypes. The quantitative microtiter plate (crystal violet, CV), methylthiazoltetrazolium (MTT), and growth curve (GC) assays were performed. No statistically significant difference was found between species, whereas daptomycin alone performed better using medium and high concentrations of the drug and bacteriophage K was more active against strains with higher susceptibility, by CV and MTT assays. Best results were achieved using both agents combined in high concentrations. Bacteriophage K was effective within 3.8 and 2.4 h, depending on the concentration used, by the GC assay. Combination of daptomycin with bacteriophage K was more effective against staphylococci, depending on the concentrations used and strains' susceptibility. Further studies are needed to evaluate if this approach might be a choice for prevention or therapy of biofilm-associated infections.

In vitro activity of dalbavancin and other anti-staphylococcal agents against infecting isolates of methicillin-resistant coagulase-negative staphylococci.

Introduction. Dalbavancin was approved in Europe in 2015 for skin and soft tissue infections.Hypothesis/Gap Statement. Data on methicillin-resistant coagulase-negative staphylococci (MR-CNS) dalbavancin susceptibility are scarce.Aim. To assess the susceptibility of MR-CNS to dalbavancin and other anti-staphylococcal agents.Methodology. A total of 443 MR-CNS clinical isolates from patients hospitalized in a Greek university hospital during a 2.5-year period (January 2018 to June 2020) were included. The MICs for vancomycin, teicoplanin, linezolid and daptomycin were investigated by Etest and the MIC for dalbavancin was determined according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines in 196 isolates. The consumption of the aforementioned antimicrobials was calculated.Results. In total, 51 isolates were resistant to teicoplanin (11.5 %) and 211 (47.6 %) to linezolid; all were susceptible to vancomycin and daptomycin. Among 196 isolates tested, 32 (16.3 %) were resistant to dalbavancin. A significant increase of MIC during the study period was found for vancomycin, teicoplanin and daptomycin, while a decrease in linezolid's MIC was observed. Dalbavancin's MIC remained stable. No difference in consumption was observed among the studied anti-staphylococcal agents.Conclusion. An increase of vancomycin, teicoplanin and daptomycin MICs among MR-CNS was observed, whereas 47.6 % of isolates were non-susceptible to linezolid. Dalbavancin retains excellent potency against MR-CNS, even in the presence of non-susceptibility to other anti-staphylococcal antibiotics.

Risk factors for isolation of fluconazole and echinocandin non-susceptible Candida species in critically ill patients.

Introduction. Resistance rates to azoles and echinocandins of Candida spp. increased over the last decade.Hypothesis/Gap Statement. Widespread use of antifungals could lead to development and dissemination of resistant Candida spp.Aim. To identify risk factors for isolation of Candida spp. non-susceptible to either fluconazole or echinocandins.Methodology. All patients hospitalized in the Intensive Care Unit (ICU) of the University General Hospital of Patras, Greece with Candida spp. isolated from clinical specimens during a ten-year period (2010-19) were included. Candida isolates were identified using Vitek-2 YST card. Consumption of antifungals was calculated.Results. During the study period, 253 isolates were included. C. non-albicans predominated (64.4 %) with C. parapsilosis being the most commonly isolated (42.3 %) followed by C. glabrata (nomenclatural change to Nakaseomyces glabrataa; 8.7 %) and C. tropicalis (11.9 %). Among all isolates, 45.8 and 28.5 % were non-susceptible and resistant to fluconazole, respectively. Concerning echinocandins, 8.7 % of isolates were non-susceptible to at least one echinocandin (anidulafungin or micafungin) and 3.1 % resistant. Multivariate analysis revealed that hospitalization during 2015-19, as compared to 2010-14, isolate being non-albicans or non-susceptible to at least one echinocandin was associated with isolation of fluconazole non-susceptible isolate. Administration of echinocandin, isolate being C. glabrata or C. tropicalis, or Candida spp. non-susceptible to fluconazole were independently associated with isolation of Candida spp. non-susceptible to at least one echinocandin. Fluconazole's administration decreased during the study period, whereas liposomal-amphotericin B's and echinoncandins' administration remained stable.Conclusion. Fluconazole's non-susceptibility increased during the study period, despite the decrease of its administration. Although echinocandins' administration remained stable, non-susceptibility among Candida spp. increased.

Emergence of a mupirocin-resistant, methicillin-susceptible Staphylococcus aureus clone associated with skin and soft tissue infections in Greece.

BACKGROUND: Staphylococcus aureus causes various infections, including skin and soft tissue infections (SSTIs). In this study, methicillin-susceptible S. aureus (MSSA) from SSTIs among patients in three tertiary-care hospitals in Greece were studied in terms of antimicrobial resistance, clonal distribution, toxin and adhesin genes carriage. RESULTS: During a five-year period (2014-2018), 6145 S. aureus were recovered from 13,244 patients with SSTIs and tested for antimicrobial susceptibility. MSSA were 4806 (78.21 %) including 1484 isolates with mupirocin minimum inhibitory concentration (MIC) > 64 mg/L (30.88 %). Two hundred and sixty representative mupirocin-resistant MSSA were analyzed for genes encoding Panton-Valentine leukocidin (PVL, lukS/lukF-PV), exfoliative toxins (eta, etb), adhesin FnbA (fnbA) and resistance genes mupA (high-level resistance to mupirocin), fusB (fusidic acid), aminoglycosides' modifying enzymes, ermA, ermC and msrA (macrolides/lincosamides) by PCRs. Strains were classified into clones by PFGE and MLST. All mupirocin-resistant MSSA were penicillin-resistant; 92.7 % expressed resistance to fusidic acid and 88.9 % to tobramycin. All 260 molecularly analyzed isolates were mupA-positive; all fusidic acid-resistant (241/260) carried fusB whereas, the tobramycin-resistant ones (230), ant(4')-Ia. The majority carried eta (93.85 %), etb (98.08 %) and fnbA (88.85 %). PFGE typing revealed a mostly unvarying population; 260 MSSA were grouped into three types. One major eta/etb-positive clone comprising of 258/260 strains (99.2 %), PFGE type 1, was classified as ST121, including nine strains co-carrying PVL. Another PVL-positive strain was identified as ST1, and one toxins-negative as ST21. CONCLUSIONS: A mupirocin-resistant MSSA clone, ST121, carrying resistance, exfoliative toxins and adhesin genes, was spread and predominated in SSTIs from patients in Greece during the five-year studied period.

Nanobiosystems for Antimicrobial Drug-Resistant Infections.

The worldwide increased bacterial resistance toward antimicrobial therapeutics has led investigators to search for new therapeutic options. Some of the options currently exploited to treat drug-resistant infections include drug-associated nanosystems. Additionally, the use of bacteriophages alone or in combination with drugs has been recently revisited; some studies utilizing nanosystems for bacteriophage delivery have been already reported. In this review article, we focus on nine pathogens that are the leading antimicrobial drug-resistant organisms, causing difficult-to-treat infections. For each organism, the bacteriophages and nanosystems developed or used in the last 20 years as potential treatments of pathogen-related infections are discussed. Summarizing conclusions and future perspectives related with the potential of such nano-antimicrobials for the treatment of persistent infections are finally highlighted.

External validation of INCREMENT-CPE score in a retrospective cohort of carbapenemase-producing Klebsiella pneumoniae bloodstream infections in critically ill patients.

OBJECTIVES: Our aim was to validate the INCREMENT-CPE score (ICS) in patients hospitalized in the intensive care unit (ICU) with bacteraemia due to carbapenemase-producing Klebsiella pneumoniae (CP-Kp). METHODS: The study was conducted in the ICU of the University General Hospital of Patras, Greece, during a 10-year period (2010-2019). Patients with monomicrobial bacteraemia due to CP-Kp were included. Primary outcome was 14-day mortality. MICs of meropenem, tigecycline, fosfomycin and ceftazidime/avibactam were determined by Etest, whereas for colistin the broth microdilution method was applied. PCR for blaKPC, blaVIM, blaNDM and blaOXA genes was used. RESULTS: Among 384 CP-Kp bacteraemias, most were primary (166, 43.2%) followed by catheter-related (143, 37.2%). Most isolates carried blaKPC (318, 82.8%). Fourteen-day mortality was 26.3% (101 patients). ICS score was 11.1 ± 4.2. An ICS ≥10 showed a sensitivity of 98.0% and a negative predictive value of 98.7%. The area under the curve of ICS (0.800) was comparable to those of the Pitt bacteraemia score (0.799), the Simplified Acute Physiology Score II (SAPS II) (0.797) and the Sequential Organ Failure Assessment score (SOFA) (0.815). CONCLUSIONS: ICS showed predictive efficacy similar to that of the SAPS II, SOFA and Pitt bacteraemia scores.

Mortality of Pandrug-Resistant Klebsiella pneumoniae Bloodstream Infections in Critically Ill Patients: A Retrospective Cohort of 115 Episodes.

BACKGROUND: The increased frequency of bacteraemias caused by pandrug-resistant Klebsiella pneumoniae (PDR-Kp) has significant implications. The aim of the present study was to identify predictors associated with mortality of PDR-Kp bacteraemias. METHODS: Patients with monomicrobial bacteraemia due to PDR-Kp were included. K. pneumoniae was considered PDR if it showed resistance to all available groups of antibiotics. Primary outcome was 30-day mortality. Minimum inhibitory concentrations (MICs) of meropenem, tigecycline, fosfomycin, and ceftazidime/avibactam were determined by Etest, whereas for colistin, the broth microdilution method was applied. bla KPC, bla VIM, bla NDM, and bla OXA genes were detected by PCR. RESULTS: Among 115 PDR-Kp bacteraemias, the majority of infections were primary bacteraemias (53; 46.1%), followed by catheter-related (35; 30.4%). All isolates were resistant to tested antimicrobials. bla KPC was the most prevalent carbapenemase gene (98 isolates; 85.2%). Thirty-day mortality was 39.1%; among 51 patients with septic shock, 30-day mortality was 54.9%. Multivariate analysis identified the development of septic shock, Charlson comorbidity index, and bacteraemia other than primary or catheter-related as independent predictors of mortality, while a combination of at least three antimicrobials was identified as an independent predictor of survival. CONCLUSIONS: Mortality of PDR-Kp bloodstream infections was high. Administration of at least three antimicrobials might be beneficial for infections in critically ill patients caused by such pathogens.

Impact of Tigecycline's MIC in the Outcome of Critically Ill Patients with Carbapenemase-Producing Klebsiella pneumoniae Bacteraemia Treated with Tigecycline Monotherapy-Validation of 2019's EUCAST Proposed Breakpoint Changes.

BACKGROUND: Tigecycline is a therapeutic option for carbapenemase-producing Klebsiella pneumoniae (CP-Kp). Our aim was to evaluate the impact of the tigecycline's minimum inhibitory concentration (MIC) in the outcome of patients with CP-Kp bacteraemia treated with tigecycline monotherapy. METHODS: Patients with monomicrobial bacteraemia due to CP-Kp that received appropriate targeted monotherapy or no appropriate treatment were included. Primary outcome was 30-day mortality. MICs of meropenem, tigecycline, and ceftazidime/avibactam were determined by Etest, whereas for colistin, the broth microdilution method was applied. PCR for blaKPC, blaVIM, blaNDM, and blaOXA genes was applied. RESULTS: Among 302 CP-Kp bacteraemias, 32 isolates (10.6%) showed MICs of tigecycline ≤ 0.5 mg/L, whereas 177 (58.6%) showed MICs that were 0.75-2 mg/L. Colistin and aminoglycoside susceptibility was observed in 43.0% and 23.8% of isolates, respectively. The majority of isolates carried blaKPC (249; 82.5%), followed by blaVIM (26; 8.6%), both blaKPC and blaVIM (16; 5.3%), and blaNDM (11; 3.6%). Fifteen patients with tigecycline MIC ≤ 0.5 mg/L and 55 with MIC 0.75-2 mg/L were treated with tigecycline monotherapy; 30-day mortality was 20.0% and 50.9%, respectively (p = 0.042). Mortality of 150 patients that received other antimicrobials was 24.7%; among 82 patients that received no appropriate treatment, mortality was 39.0%. No difference in 30-day mortality was observed between patients that received tigecycline (MIC ≤ 0.5 mg/L) or other antimicrobials. CONCLUSION: Tigecycline monotherapy was as efficacious as other antimicrobials in the treatment of bloodstream infections due to CP-Kp isolates with a tigecycline's MIC ≤ 0.5 mg/L.

Evolution and Population Dynamics of Clonal Complex 152 Community-Associated Methicillin-Resistant Staphylococcus aureus.

Since the late 1990s, changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) were recognized with the emergence of community-associated MRSA (CA-MRSA). CA-MRSA belonging to clonal complex 152 (CC152), carrying the small staphylococcal cassette chromosome mec (SCCmec) type V and encoding the Panton-Valentine leukocidin (PVL), has been observed in Europe. The aim of this study was to investigate its origin, evolution, and dissemination. Whole-genome sequencing was performed on a global collection of 149 CC152 isolates spanning 20 years (93 methicillin-susceptible S. aureus [MSSA] and 56 MRSA isolates). Core genome phylogeny, Bayesian inference, in silico resistance analyses, and genomic characterization were applied. Phylogenetic analysis revealed two major distinct clades, one dominated by MSSA and the other populated only by MRSA. The MSSA isolates were predominately from sub-Saharan Africa, whereas MRSA was almost exclusively from Europe. The European MRSA isolates all harbored an SCCmec type V (5C2&5) element, whereas other SCCmec elements were sporadically detected in MRSA from the otherwise MSSA-dominated clade, including SCCmec types IV (2B), V (5C2), and XIII (9A). In total, 93% of the studied CC152 isolates were PVL positive. Bayesian coalescent inference suggests an emergence of the European CC152-MRSA in the 1990s, while the CC152 lineage dates back to the 1970s. The CA-MRSA CC152 clone mimics the European CC80 CA-MRSA lineage by its emergence from a PVL-positive MSSA ancestor from North Africa or Europe. The CC152 lineage has acquired SCCmec several times, but acquisition of SCCmec type V (5C2&5) seems associated with expansion of MRSA CC152 in Europe.IMPORTANCE Understanding the evolution of CA-MRSA is important in light of the increasing importance of this reservoir in the dissemination of MRSA. Here, we highlight the story of the CA-MRSA CC152 lineage using whole-genome sequencing on an international collection of CC152. We show that the evolution of this lineage is novel and that antibiotic usage may have the potential to select for the phage-encoded Panton-Valentine leukocidin. The diversity of the strains correlated highly to geography, with higher level of resistance observed among the European MRSA isolates. The mobility of the SCCmec element is mandatory for the emergence of novel MRSA lineages, and we show here distinct acquisitions, one of which is linked to the successful clone found throughout Europe today.

Inactivation of mgrB gene regulator and resistance to colistin is becoming endemic in carbapenem-resistant Klebsiella pneumoniae in Greece: A nationwide study from 2014 to 2017.

INTRODUCTION: In Greece, the spread of carbapenem-resistant Enterobacteriaceae in humans has led to the reintroduction of colistin as a therapeutic agent. Unfortunately, colistin resistance with different mechanisms has emerged. The present work aims to determine the prevalence of carbapenem and colistin resistance and the corresponding mechanisms in Klebsiella pneumoniae clinical isolates from Greece. METHODS: From 2014 to 2017, 288 carbapenem-resistant K. pneumoniae clinical strains were gathered from a collection of 973 isolates from eight different hospitals in Greece. Antibiotic susceptibility testing was performed using three different methods. Screening of carbapenem and colistin resistance genes was conducted using polymerase chain reaction (PCR) amplification and sequencing. RESULTS: Among the 288 (29.6 %) carbapenem-resistant isolates, 213 (73.9%) were colistin-resistant (minimum inhibitory concentration [MIC] >2 mg/L). The KPC type was the most common carbapenemase gene (116; 40.3%), followed by VIM (41; 14.2%), NDM (33; 11.5%) and OXA-48 (22; 7.6%). Moreover, 44 (15.3%) strains co-produced two types of carbapenemases. No mcr genes were detected for colistin resistance but mutations in chromosomal genes were found. These included inactivation of the mgrB gene for 148 (69.5%) strains, including insertion sequences for 94 (44.1%), nonsense mutations for 4 (1.9%) and missense mutations for 24 (11.3%). Moreover, PCR amplification of mgrB gene was negative for 26 (12.2%) strains. Finally, 65 (30.5%) colistin-resistant strains exhibited a wild-type mgrB, the mechanisms of which remain to be elucidated. CONCLUSION: This study shows that K. pneumoniae clinical strains in Greece are resistant to both carbapenems and colistin and this is endemic and is likely chromosomally encoded.

Molecular characteristics and predictors of mortality among Gram-positive bacteria isolated from bloodstream infections in critically ill patients during a 5-year period (2012-2016).

To identify the molecular characteristics of Gram-positive cocci isolated from blood cultures and clinical outcome among critically ill patients. This retrospective study was conducted in the general intensive care unit of the University General Hospital of Patras, Greece, during a 5-year period (2012-2016). All adult patients with a Gram-positive BSI were included. PCR was applied to identify mecA gene (staphylococci); vanA, vanB, and vanC genes (enterococci). Linezolid-resistant S. epidermidis, MRSA, and VRE were further typed by multilocus sequence typing. Mutations in region V of 23S rDNA and ribosomal protein L4were investigated by PCR and sequencing analysis. The presence of the cfr gene was tested by PCR. In total, 141 Gram-positive BSIs were included. Coagulase-negative staphylococci predominated (n = 69; 65 methicillin-resistant, 23 linezolid-resistant carrying both C2534T and T2504A mutations and belonging to the ST22 clone), followed by enterococci (n = 46; 11 vancomycin-resistant carrying vanA gene, classified into four clones), S. aureus (n = 22; 10 methicillin-resistant, classified into three clones) and streptococci (n = 4). The most common type of infection was catheter-related (66; 46.8%), followed by primary BSI (28; 19.9%). Overall 14-day fatality was 24.8%. Multivariate analysis revealed septic shock as independent predictor of fatality, while appropriate empiric antimicrobial treatment and catheter-related BSI were identified as a predictor of good prognosis. Even though most of Gram-positive cocci were multidrug-resistant, fatality rate was low, associated with catheter-related BSIs. Among CNS, LR isolates represented one-third of BSIs due to the dissemination of ST22 S. epidermidis propagated by utilization of linezolid.

Fatality of Staphylococcus aureus infections in a Greek university hospital: role of inappropriate empiric treatment, methicillin resistance, and toxin genes' presence.

The aim of the present study was to identify predictors of fatality among patients with S. aureus infections requiring hospitalization. Cases hospitalized with S. aureus infections at the University General Hospital of Patras, Greece, during a 4-year period (2013-2016) were studied. mecA, lukS/lukF-PV (Panton-Valentine leukocidin, PVL), tst (toxic shock syndrome toxin), fnbA (fibronectin-binding protein A), eta, and etb (epidermolytic toxins) genes' carriage was detected by PCR in 149 selected patients. Among 464 patients, 346 were included (118 with missing data). Primary bacteremia predominated (44.2%), followed by lower respiratory tract infections (13.6%), deep seated infections (9.8%), osteoarticular (9.5%), and catheter-related bloodstream infections (6.1%). Methicillin-resistant S. aureus (MRSA) represented 33.8% of infections and were less likely to receive appropriate empiric treatment (79.5% versus 97.4%; P < 0.001). Thirty-day fatality was 14.5%. Multivariate analysis revealed that development of septic shock, Charlson Comorbidity Index, lower respiratory tract infection, bacteremia (primary or secondary), MRSA, and CRP was significantly associated with fatality. Appropriate empiric treatment was a predictor of good prognosis. Thirty-two out of 149 S. aureus (21.5%) carried lukS/lukF-PV genes, whereas, 14 (9.4%), 133 (78.7%), four (2.7%), and one (0.7%) carried tst, fnbA, eta, and etb genes, respectively. No difference was found among toxin genes' presence and mortality. PVL was significantly more frequently found among MRSA as compared to MSSA (45.1% versus 9.2%; P < 0.001). MRSA represented one third of the infections requiring hospitalization and were independently associated with fatality, probably since were more likely to receive inappropriate antibiotic treatment as compared to MSSA.

In vitro activity of ceftazidime/avibactam against isolates of carbapenem-non-susceptible Enterobacteriaceae collected during the INFORM global surveillance programme (2015-17).

OBJECTIVES: To report data for ceftazidime/avibactam and comparators against meropenem-non-susceptible Enterobacteriaceae collected globally (excluding centres in the USA) from 2015 to 2017 as part of the International Network For Optimal Resistance Monitoring (INFORM) surveillance programme. METHODS: MICs and susceptibility were determined using EUCAST broth microdilution methodology and EUCAST breakpoints. Isolates were screened to detect genes encoding ß-lactamases using multiplex PCR assays. MBL-positive isolates were those in which one or more of the IMP, VIM and/or NDM genes were detected. RESULTS: A total of 1460 meropenem-non-susceptible isolates were collected and, of the agents on the panel, susceptibility was highest to ceftazidime/avibactam, colistin and tigecycline [73.0%, 77.0% (1081/1403) and 78.1%, respectively]. Ceftazidime/avibactam was not active against MBL-positive isolates (n=367); these isolates showed the highest rates of susceptibility to colistin (92.1%, 303/329), tigecycline (71.9%) and amikacin (46.6%). A total of 394 isolates were resistant to ceftazidime/avibactam and, of the 369 isolates that were screened, 98.4% were found to carry a gene encoding an MBL enzyme. Among isolates that were identified as carbapenemase positive and MBL negative (n=910), susceptibility was highest to ceftazidime/avibactam (99.8%). Susceptibility was also highest to ceftazidime/avibactam among isolates that were carbapenemase negative and MBL negative (94/98, 95.9%). CONCLUSIONS: These data highlight the need for continued surveillance of antimicrobial activity as well as the need for new antimicrobials to treat infections caused by meropenem-non-susceptible Enterobacteriaceae, for which the options are extremely limited.