Impact of Exposure of Methicillin-Resistant Staphylococcus aureus to Polyhexanide In Vitro and In Vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) resistant to decolonization agents such as mupirocin and chlorhexidine increases the need for development of alternative decolonization molecules. The absence of reported severe adverse reactions and bacterial resistance to polyhexanide makes it an excellent choice as a topical antiseptic. In the present study, we evaluated the in vitro and in vivo capacity to generate strains with reduced polyhexanide susceptibility and cross-resistance with chlorhexidine and/or antibiotics currently used in clinic. Here we report the in vitro emergence of reduced susceptibility to polyhexanide by prolonged stepwise exposure to low concentrations in broth culture. Reduced susceptibility to polyhexanide was associated with genomic changes in the mprF and purR genes and with concomitant decreased susceptibility to daptomycin and other cell wall-active antibiotics. However, the in vitro emergence of reduced susceptibility to polyhexanide did not result in cross-resistance to chlorhexidine. During in vivo polyhexanide clinical decolonization treatment, neither reduced polyhexanide susceptibility nor chlorhexidine cross-resistance was observed. Together, these observations suggest that polyhexanide could be used safely for decolonization of carriers of chlorhexidine-resistant S. aureus strains; they also highlight the need for careful use of polyhexanide at low antiseptic concentrations.

Antimicrobial activity of ceftaroline against methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 2013-2014 at the Geneva University Hospitals.

Ceftaroline is a broad-spectrum antibiotic with activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. Ceftaroline susceptibility of an MRSA set archived between 1994 and 2003 in the Geneva University Hospitals detected a high percentage (66 %) of ceftaroline resistance in clonotypes ST228 and ST247 and correlated with mutations in PBP2a. The ceftaroline mechanism of action is based on the inhibition of PBP2a; thus, the identification of PBP2a mutations of recently circulating clonotypes in our institution was investigated. We analyzed ceftaroline susceptibility in MRSA isolates (2013 and 2014) and established that resistant strains correlated with PBP2a mutations and specific clonotypes. Ninety-six MRSA strains were analyzed from independent patients and were isolated from blood cultures (23 %), deep infections (38.5 %), and superficial (skin or wound) infections (38.5 %). This sample showed a ceftaroline minimum inhibitory concentration (MIC) range between 0.25 and 2 µg/ml and disk diameters ranging from 10 to 30 mm, with a majority of strains showing diameters ≥20 mm. Based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, 76 % (73/96) of isolates showed susceptibility to ceftaroline. Nevertheless, we still observed 24 % (23/96) of resistant isolates (MIC = 2 µg/ml). All resistant isolates were assigned to clonotype ST228 and carried the N146K mutation in PBP2a. Only two ST228 isolates showed ceftaroline susceptibility. The decreasing percentage of ceftaroline-resistant isolates in our hospital can be explained by the decline of ST228 clonotype circulating in our hospital since 2008. We present evidence that ceftaroline is active against recent MRSA strains from our hospital; however, the presence of PBP2a variants in particular clonotypes may affect ceftaroline efficacy.

Randomized, placebo-controlled, double-blind clinical trial to evaluate the efficacy of polyhexanide for topical decolonization of MRSA carriers.

OBJECTIVES: The objective of this study was to evaluate the efficacy of polyhexanide (Prontoderm(®)) in eliminating MRSA carriage. METHODS: In a 1900 bed teaching hospital, MRSA-colonized patients were randomized into a double-blind, placebo-controlled superiority trial between January 2011 and July 2014. Patients were treated with either polyhexanide or placebo applied to the anterior nares (thrice daily) and skin (once daily) for 10 days. The primary outcome was MRSA decolonization at day 28 (D28) after the end of treatment assessed by ITT responder and PP analyses (microbiological follow-up ± 7 days and topical treatment ≥ 5 days). Secondary outcomes included safety, emergence of resistance and MRSA genotype changes. Registered trial number ISRCTN02288276. RESULTS: Of 2590 patients screened, 146 (polyhexanide group, 71; placebo group, 75) were included. ITT analysis showed that 24/71 (33.8%) patients in the polyhexanide group versus 22/75 (29.3%) in the placebo group were MRSA-free at D28 (risk difference, 4.5%; 95% CI, -10.6% to 19.5%; P = 0.56). PP analysis confirmed the results with 19/53 (35.8%) decolonized polyhexanide-treated patients versus 17/56 (30.4%) in the placebo arm (risk difference, 5.5%; 95% CI, -12.2% to 23%; P = 0.54). Nine serious adverse events occurred in the polyhexanide group versus 12 in the placebo group; none was attributable to study medication. Emergence of polyhexanide resistance or cross-resistance between polyhexanide and chlorhexidine was not observed. No case of exogenous recolonization by a genotypically different MRSA strain was documented. CONCLUSIONS: This study suggests that under real-life conditions, a single polyhexanide decolonization course is not effective in eradicating MRSA carriage.

Tracing the human movements of three thousand years ago by volcanic grinding tools in the Final Bronze Age settlement of Monte Croce Guardia (Arcevia-Marche Region, central Italy).

Volcanic rocks were among the most sought-after materials to produce grinding tools in antiquity because lavas lithologies, either mafic or felsic, ensured good wear resistance and grinding capacity with respect to many other kinds of rocks. The interest in findings made of vesciculated lavas, referable to parts of querns, mortars, and/or pestles of the Final Bronze Age site of Monte Croce Guardia (Arcevia) lies in the fact that this settlement was built upon limestones belonging to the sedimentary sequence of the Marche-Umbria Apennines (central Italy) and far away from potential raw materials of volcanic rocks. A petrologic study of 23 grinding tool fragments clearly indicates a provenance from the volcanic provinces of central Italy: Latium and Tuscany Regions. Few leucite tephrites (5) and one leucite phonolite lavas have a clear magmatic affinity with the high-K series of the Roman Volcanic Province (Latium) whereas the most abundant volcanic lithotype (17 samples) is represented by shoshonites (K-series) whose thin section texture, modal mineralogy and major-trace elements contents closely match with the shoshonite lavas from the Radicofani volcanic centre in the Tuscan Magmatic Province. At Radicofani (a volcanic neck in the eastern sector of Tuscany) a Final Bronze Age site coeval to that of Arcevia is present and a potential pathway corridor from that site towards Arcevia (air-line distance of ca. 115 km) is dotted with many settlements of the same age. Through analytical algorithms based on the slope and the different human-dependent cost-functions which can be applied to determine non-isotropic accumulated cost surface, least-cost paths and least-cost corridors, the best route from Radicofani to Monte Croce Guardia, approximately 140 km long, was simulated, with a walking time of 25-30 h, possibly using pack animals and wheel chariots. Three thousand years ago the Apennine Mountains did not thus constitute a barrier for human movements. This study also shed light on some other possible patterns of interactions between Final Bronze Age communities of central Italy through the present-day regions of Tuscany, Umbria and Marche, aimed towards the best performance of strategic economic activities at that time such as that of the transformation of cereals, and accompanied to cultural and social reasons.

Prevalence of isolates with reduced glycopeptide susceptibility in orthopedic device-related infections due to methicillin-resistant Staphylococcus aureus.

We evaluated, by an improved susceptibility testing method, the prevalence and significance of low-level glycopeptide resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolates, which belonged to a previously described, retrospective cohort of patients treated for orthopedic device-related infections (ODRI) at the Geneva University Hospital between 2000 and 2008. Fifty-seven individual or multiple isolates were retrieved from 41 ODRI patients for glycopeptide susceptibility and clonality studies, including 20 patients with prosthetic joint (PJ) and 21 with osteosynthesis (OS) MRSA infections. Low-level glycopeptide resistance was detected by elevated teicoplanin or/and vancomycin minimum inhibitory concentrations (MICs ≥ 4 mg/L), as determined by a previously validated combination of macrodilution and agar dilution assays of improved sensitivity. MRSA isolates with elevated teicoplanin MICs were detected in 20/41 (49 %) ODRI patients at the onset or during the course of glycopeptide therapy, namely, in 10 of 20 patients with PJ and 10 of 21 patients with OS infections. Only one isolate developed a concomitant increase in vancomycin MIC during therapy. 13/20 (65 %) glycopeptide-intermediate S. aureus (GISA)-infected patients, including 7/10 (70 %) with PJ and 6/10 (60 %) with OS, experienced treatment failure. In contrast, therapy failed in only 5/21 (24 %) ODRI patients with non-GISA isolates (p = 0.012), including 2/10 (20 %) with PJ and 3/11 (27 %) with OS infections. The emergence of low-level teicoplanin resistance could not be explained by teicoplanin administration, since only four patients received teicoplanin. The evaluation of low-level teicoplanin resistance may improve the detection of GISA isolates. Further studies are warranted to evaluate the impact of low-level teicoplanin resistance on the outcome of glycopeptide therapy.

Geoarchaeological Evidence of Middle-Age Tsunamis at Stromboli and Consequences for the Tsunami Hazard in the Southern Tyrrhenian Sea.

Large-scale landslides at volcanic islands are one of the most dangerous geological phenomena, able to generate tsunamis whose effects can propagate far from the source. However, related deposits are scarcely preserved on-land in the geologic records, and are often difficult to be interpreted. Here we show the discovery of three unprecedented well-preserved tsunami deposits related to repeated flank collapses of the volcanic island of Stromboli (Southern Italy) occurred during the Late Middle Ages. Based on carbon datings, on stratigraphic, volcanological and archaeological evidence, we link the oldest, highest-magnitude investigated tsunami to the following rapid abandonment of the island which was inhabited at that time, contrary than previously thought. The destructive power of this event is also possibly related to a huge marine storm that devastated the ports of Naples in 1343 (200 km north of Stromboli) described by the famous writer Petrarch. The portrayed devastation can be potentially attributed to the arrival of multiple tsunami waves generated by a major landslide in Stromboli island, confirming the hypothetical hazard of these phenomena at a regional scale.

Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation.

OBJECTIVE: To investigate the potential roles of PBPs, efflux pumps and slow drug influx for imipenem heteroresistance in nontypeable Haemophilus influenzae (NTHi). METHODS: Fifty-nine NTHi clinical isolates examined in this study were collected at Geneva University Hospitals between 2009 and 2014. Alterations in PBPs were investigated by gene sequencing. To evaluate the affinities of the PBPs to imipenem, steady-state concentration-response experiments were carried out using imipenem in a competition assay with Bocillin-FL. The effect of the carbonyl cyanide m-chlorophenylhydrazone (CCCP) on imipenem susceptibility was assessed using broth dilution and viable cell counting. Using whole-genome sequencing, we explored the potential roles of outer membrane protein P2 (OmpP2), LytM proteins and the dcw gene cluster in imipenem heteroresistance. RESULTS: All 46 imipenem-heteroresistant isolates (IMIhR) harboured amino acid substitutions in the ftsI gene, which encodes PBP3, corresponding to 25 different mutation patterns that varied from the ftsI gene mutation patterns found in imipenem-susceptible isolates. Among all PBPs, the highest affinity to imipenem was documented for PBP3 (IC50, 0.004 µg/mL). Different amino acid substitutions and insertions were noted in OmpP2, suggesting a relationship with imipenem heteroresistance. The IMIhR isolates were affected by CCCP differently and displayed a higher percentage of killing by imipenem in CCCP-treated cells at concentrations ranging between 0.5 and 8 µg/mL. CONCLUSIONS: The present study provides robust evidence indicating that in combination with the altered PBP3, the slowed drug influx and its enhanced efflux due to the loss of regulation led to the development of imipenem heteroresistance in NTHi.

Delayed evoked potentials in children exposed to methylmercury from seafood.

Methylmercury poisoning may cause constriction of visual fields and deafness, especially if exposure occurs prenatally. However, the risks associated with exposure from contaminated seafood is unclear. We examined 149 children attending first grade in a Madeiran fishing community. As maternal dietary habits were relatively unchanged, current maternal hair concentrations were used as indicator of the child's prenatal exposure to methylmercury (geometric average, 9.64 microg/g [48.2 nmol/g]). After adjustment for age and sex, the mean (+/-SD) latency of peak III of the brainstem auditory evoked potentials at 40 Hz was increased by 0.128+/-0.047 ms when maternal hair-mercury concentrations exceeded 10 microg/g (50 nmol/g) (p for association, 0.002), and the increase of the N145 pattern-reversal visual evoked potential latency at 15 minutes of arc was 3.16+/-1.57 ms (p for association, 0.002). No such relationships were seen with the child's own hair-mercury concentration, and other clinical examinations revealed no mercury-associated deficits. Neurophysiological evidence of adverse effects on brain function are relatively independent of confounders, and should be considered in the risk assessment of this seafood pollutant.

Organochlorine contamination in bird's eggs from the Danube Delta.

In this study we report the levels of organochlorine compounds in eggs of aquatic birds from the Danube Delta, a major European wetland. The eggs were collected in 1997 and belonged to the following species: the mallard (Anas platyrhynchos), the greylag goose (Anser anser), the mute swan (Cygnus olor), the coot (Fulica atra), the glossy ibis (Plegadis falcinellus), the spoonbill (Platalea leucorodia), the little egret (Egretta garzetta), the night heron (Nycticorax nycticorax), the grey heron (Ardea cinerea), the great white egret (Egretta alba), the red-necked grebe (Podiceps griseus), the Dalmatian pelican (Pelecanus crispus), the Pygmy cormorant (Phalacrocorax pygmaeus) and the common cormorant (Phalacrocorax carbo). Dichlorodiphenyltrichloroethane (DDT) levels were higher in eggs of the little egret, the great white egret, the cormorant and the Pygmy cormorant with respect to the other species (48,399, 13,613, 12,400 and 10,417 ng/g dry wt., respectively). Hexachlorobenzene (HCB) levels were lower than 1393 ng/g dry wt. in all species while polychlorinated biphenyl (PCB) concentrations in the Pygmy cormorant (2565 ng/g dry wt.) were higher than in the other species. The toxicity evaluation was based on 2,3,7,8-TCDD toxic equivalent factors (TEF) and non-ortho PCB congeners contributed much more than mono-ortho PCBs in most of species. A further aim of this study was to evaluate the possible differences of organochlorine levels in bird eggs collected in the same area in 1982 and in 1997; generally speaking the levels detected in the latter period were lower than those detected in the earlier one.

Heavy metals in organisms from the northern Tyrrhenian sea.

The concentrations of zinc, manganese, copper, cadmium, lead and mercury have been determined in soft tissues of four marine organisms (Mytilus galloprovincialis Lmk., Nephrops norvegicus L., Mullus barbatus L., Engraulis encrasicolus L.) collected seasonally from the winter of 1976 to the spring of 1980 in various areas of the Tyrrhenian Sea (western Mediterranean). In all four species levels of all metals, except mercury in all areas are alike while mercury levels vary and, furthermore, are higher than in specimens from other areas of the Mediterranean and also from other seas. The possible natural origin of the mercury from cinnabar (HgS) ore as well as the suitability of the four species as bioindicators is discussed.

Trace elements and chlorinated hydrocarbons in eggs of Pelecanus crispus, a world endangered bird species nesting at Lake Mikri Prespa, North-western Greece.

Eggs of the Dalmatian pelican, Pelecanus crispus, collected from 1984 to 1986 at Lake Mikri Prespa, north-western Greece, contained residues of trace elements and PCBs at low concentrations, and of DDE at rather high concentrations. DDE is negatively related to eggshell thickness. Eggshell thickness was reduced by 12-20% in comparison with the pre-1947 (before DDT use) eggshell thickness. However, this decrease did not affect the reproductive success of this species. The main fish species eaten by the Dalmatian pelican at Lake Mikri Prespa were analyzed for pollutants. All the fish contained low concentrations of residues. The contribution of the diet while the birds are on the wintering grounds is unknown. Recommendations for a monitoring programme of pollutants in eggs of the Dalmatian pelican, a world-endangered species, are proposed.

Interspecies differences in mixed function oxidase activity in birds: Relationship between feeding habits, detoxication activities and organochlorine accumulation.

The relationship between feeding habits and interspecies differences in the detoxication ability of the mixed function oxidase (MFO) system was investigated in birds. The role of MFO (particularly aldrin epoxidase activity) in the detoxication/bioaccumulation of organochlorines was also investigated. Euriphagic (yellow-legged herring gull (Larus cachinnans), black-headed gull (Larus ridibundus), jackdaw (Corvus monedula), magpie (Pica pica) and stenophagic (cormorant (Phalacrocorax carbo), coot (Fulica atra) and sparrow (Passer italiae)) species were collected in northern and central Italy. The following liver microsomal monooxygenase activities were measured: aldrin epoxidase, ethoxyresorufin dealkylation (EROD), benzyloxyresorufin dealkylation (BROD) and pentoxyresorufin dealkylation (PROD). NADPH-and NADH-cytochrome c reductase (NAD(P)H-CYT-CRED) and NADH-ferricyanide reductase (NADH-FERRIRED) activities were also measured in the liver microsomal fraction. Glutathione concentration (GSH) was measured in the liver cytosolic fraction. As a marker of liver damage gamma-glutamyl transpeptidase (GGTP) activity was assayed in the serum. The residues of organochlorines (HCB, pp'DDTs and PCBs) were determined in muscle samples. Omnivorous species, particularly the yellow-legged herring gull, had the highest aldrin epoxidase activities. In the yellow-legged herring gull the activity was approximately twice as high as in the jackdaw (p < 0.05), black-headed gull (p < 0.05) and coot (p < 0.05). Values five times lower were detected in the specialist fish-eater, the cormorant (p < 0.001). The lowest values of aldrin epoxidase activity were detected in the sparrow. Feeding habits were found to be related to evolutionary interspecies differences in MFO activity. A significant statistical correlation (r = 0.656) was found between the 'omnivore index' and MFO detoxication activity expressed as aldrin epoxidation. Organochlorines, and particularly PCBs, were higher in cormorants and yellow-legged herring gulls from a polluted lagoon than in the other species analysed (black-headed gull p < 0.01; sparrow p < 0.001; jackdaw p < 0.01; coot p < 0.001). Levels of pp'DDE were much higher in the cormorant and sparrow than in the other birds. Knowledge of the different species-specific detoxication abilities was found to be a useful tool for the identification of species potentially at risk in environments with high pollution by chlorinated hydrocarbons, or in general contaminants metabolized by the MFO system.

The contamination of birds with organic pollutants in the Lake Baikal region.

Lake Baikal is considered to be the largest reservoir of fresh natural water in the world. Nevertheless industrial enterprises on its banks as well as river effluents contaminate this unique basin. In the present study birds' eggs (15 species) collected in the Baikal region (Selenga river estuary) have been analysed. Quantitative determination of more than 40 individual organic pollutants (polycyclic aromatic hydrocarbons, phenols, organochlorine compounds) has been carried out using GC-MS as an analytical tool. The results obtained demonstrated a wide range of toxicant concentrations (2-3 orders of magnitude) for various species. Very high levels of polycyclic aromatic hydrocarbons have been detected in the eggs of Anas platyrynchos (mallard), Tringa stagnatilis (marsh sandpiper) and Podiceps auritus (slavonian grebe). These particular species also have the highest levels of other toxicants. Taking into account high rate of metabolism of certain of these compounds in birds, it has been proposed that the major route of transfer into higher trophic levels is via water and aquatic invertebrates.

Recalcitrant organochlorine compounds in captive bottlenose dolphins (Tursiops truncatus): biomagnification or bioaccumulation?

Organochlorine residues were measured in the diet, blood, faeces and exhaled air of captive bottlenose dolphins (Tursiops truncatus) to evaluate the absorption efficiency from food and the eliminatory potential via faeces and exhaled air. Differences between air-breathing and water-breathing animals feeding on similar prey are briefly discussed. It is concluded that high concentrations of recalcitrant organochlorines currently found in marine mammals feeding on fish are essentially due to the lack of branchial elimination and not to their predator status.

Safety and immunogenicity of two influenza virus subunit vaccines, with or without MF59 adjuvant, administered to human immunodeficiency virus type 1-seropositive and -seronegative adults.

The objective of this study was to evaluate and compare both the safety and tolerability and the humoral and cell-mediated immune responses for two influenza virus subunit vaccines, one with MF59 adjuvant (Fluad) and one without an adjuvant (Agrippal), in healthy and in human immunodeficiency virus type 1 (HIV-1)-infected adult individuals. To achieve this aim, an open, randomized, comparative clinical trial was performed during the 2005-2006 season. A total of 256 subjects were enrolled to receive one dose of vaccine intramuscularly. Blood samples were taken at the time of vaccination and at 1 and 3 months postvaccination. A good humoral antibody response was detected for both vaccines, meeting all the criteria of the Committee for Medical Products for Human Use. After Beyer's correction for prevaccination status, Fluad exhibited better immunogenicity than Agrippal, as shown from the analysis of the geometric mean titers, with significant differences for some virus strains; however, no definitive conclusions on the clinical significance of such results can be drawn, because the method used to estimate antibody response is currently nonstandard for influenza virus vaccines. Significant induction of an antigen-specific CD4+ T-lymphocyte proliferative response was detected at all time points after immunization, for both the vaccines, among HIV-1-seronegative subjects. This was different from what was observed for HIV-1-infected individuals. In this group, significance was not reached at 30 days postvaccination (T30) for those immunized with Agrippal. Also when data were compared between treatment groups, a clear difference in the response at T30 was observed in favor of Fluad (P = 0.0002). The safety profiles of both vaccines were excellent. For HIV-1-infected individuals, no significant changes either in viremia or in the CD4+ cell count were observed at any time point. The results showed good safety and immunogenicity for both vaccines under study for both uninfected and HIV-1-infected adults, confirming current recommendations for immunization of this high-risk category.

Trends in mercury in hair of Greenlandic polar bears (Ursus maritimus) during 1892-2001.

Mercury concentrations in hair from 397 Greenland polar bears (Ursusmaritimus) sampled between 1892 and 2001 were analyzed for temporal trends. In East Greenland the concentrations showed a significant (p < 0.0001, n = 27) increase of 3.1%/year in the period 1892-1973. In Northwest Greenland, a similar (p < 0.0001, n = 69) increase of 2.1%/year was found, which continued until 1991, when the most recent samples were obtained. In East Greenland, a significant (p = 0.009, n = 322) decrease of 0.8%/year was observed after 1973. Two Northwest Greenland samples from 1300 A.D. had a mean value of 0.52 mg/kg of dry weight, which can be considered as a baseline level. The Hg concentration during 1985-1991 from Northwest Greenland (mean value of 7.45 mg/kg of dry weight) was more than 14-fold higher than the assumed baseline level from 1300 A.D. from the same region (i.e., about 93% anthropogenic). Although a decrease was found in East Greenland after 1973, the concentration is still ca. 11-fold higher than the baseline level (i.e., about 90% anthropogenic).