Panton–Valentine Leukocidin Enhances the Severity of Community-Associated Methicillin-Resistant Staphylococcus aureus Rabbit Osteomyelitis

Background

Extensive spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the United States, and the concomitant increase in severe invasive staphylococcal infections, including osteomyelitis, in healthy children, has led to renewed interest in Panton-Valentine leukocidin (PVL). However, the pathogenetic role of PVL in staphylococcal infections remains controversial, possibly because it depends on the site of infection.

Methodology/Principal Findings

We compared the course of experimental rabbit osteomyelitis due to the PVL-positive CA-MRSA strain USA 300 (LAC) and its PVL-negative isogenic derivative (LACΔpvl), using a low and a high inoculum (8×10⁵ and 4×10⁸ CFU). With the low inoculum, bone infection was less frequent on day 7 (D7) and day 28 (D28) with LACΔpvl than with LAC (respectively 12/19 and 18/19 animals, p = 0.042). With the high inoculum of both strains, all the animals were infected on D7 and the infection persisted on D28 in almost every case. However, tibial bacterial counts and the serum CRP concentration fell significantly between D7 and D28 with LACΔpvl but not with LAC. Respectively 67% and 60% of LAC-infected rabbits had bone deformation and muscle/joint involvement on D7, compared to 0% and 7% of LACΔpvl-infected rabbits (p = 0.001 and p = 0.005 respectively). Between D0 and D28, the anti-PVL antibody titer increased significantly only with the high inoculum of LAC.

Conclusions/Significance

PVL appears to play a role in the persistence and rapid local extension of rabbit osteomyelitis, in keeping with the greater severity of human bone infections due to PVL-positive S. aureus. The possible therapeutic implications of these findings are discussed.     

Analysis of the potential evapotranspiration demands in the Czech Republic between 1961–1990

Dynamics of the evapotranspirational demands in the Czech Republic within three decades from 1961 to 1990 has been studied. The determination of the levels of influence of the respective natural components depends on the regionalized modelling techniques. The project of regionalized modelling is theoretically based on the potential evapotranspiration values (ET ₀) calculated by FAO methodology (eqs. 1, 2) and the series of temperature and rainfall observations obtained by climatologic stations during a 30-year period from 1961 to 1990 and its relation to the absolute altimetric. The DMR-2 military digital elevation model of the Czech Republic relief consisting of a regular network of points with their altitudes specified in meters was used for the purpose of regionalized modelling. One step of the network in the S-42 coordinate system with the Gauss conforms cylindrical projection is equal to 100 meters; this implies that the smallest area for which the data can be processed is 1 hectare. The digital relief model can be linked to both direct (temperature, precipitation) and derived (evapotranspiration) quantities, which is one of the many novelties of regionalized modelling. The climatic data used in regionalized modelling records daily measurements were obtained by 85 climatologic and rainfall-monitoring stations from 1961 to 1990. Our results showed an appreciable decrease of the most drying area (ratio P/ET₀ up to 0.755) in the last decade 1981–90; half as less amounts compared with the previous decade 1971–80 (about 500,000 hectares). On the other hand, an apparent increase (more than 500,000 hectares in comparison with the previous decade 1971–80) of the wettest area (ratio P/ET₀ over 1.508) through the last decade was observed. Both first decades 1961–70 and 1971–80 look similar. The project mentioned in this article has made it possible to create models for the different time intervals which have showed higher reliability for heterogeneous application.     

Trends in stage-specific population-based survival of cancer patients in the Czech Republic in the period 2000–2008

BackgroundThe objective of this study was to assess trends in overall and in stage-specific 5-year relative survival rates of the Czech cancer patients between periods 2000–2004 and 2005–2008.MethodsAll Czech cancer patients diagnosed between 1995 and 2008 were included in the analysis. Period analysis was employed to calculate 5-year relative survival for 21 cancers.ResultsSignificant improvements in crude 5-year relative survival for 14 of 21 assessed types of cancer, including the most frequent diagnoses, such as, colorectal, prostate, breast, lung, kidney, pancreatic, and bladder cancer and melanoma, were identified. Moreover, in case of colorectal, lung, and prostate cancer, improvement in stage-specific 5-year relative survival was confirmed as statistically significant for all clinical stages. No diagnosis showed significant decrease in the 5-year relative survival. However, the 5-year relative survival remained poor in patients with metastatic cancers at diagnosis, particularly in case of liver, pancreatic, lung, and oesophageal cancer.ConclusionsThe cancer-specific outcomes in the Czech Republic are improving. Nevertheless, despite the overall significant improvement in 5-year relative survival of most of the cancer diagnoses, the high proportion of patients primarily diagnosed with metastatic cancer still represents a substantial challenge for prevention and early detection.     

Decreased impacts of the 2003 heat waves on mortality in the Czech Republic: an improved response?

The paper examines impacts on mortality of heat waves in 2003, the hottest summer on record in the Czech Republic, and compares them with previous similar events. While most summer heat waves over the period since 1986 were associated with significantly elevated mortality, this was not the case for three out of the four heat waves in 2003. The relatively weak mortality response was particularly noteworthy for the most severe heat wave which occurred in the first 10 days of August 2003 and resulted in enormous excess mortality in some western European countries. A mortality displacement effect and short-term adaptation to heat contributed to the reduced mortality impacts of the heat waves that followed after previous relatively warm periods. However, the decreased mortality response of the 2003 heat waves compared to previous heat waves in the 1990s is also likely to have arisen from positive health-care and other socio-economic changes in the post-communist central European region over the past decade, as well as a better public awareness of heat-related risks due to enhanced media coverage and regular biometeorological forecast and warnings.     

Use of human buildings by Eurasian badgers in the Moravskoslezské Beskydy Mountains,Czech Republic

An inspection of human buildings used by Eurasian badgersMeles meles (Linnaeus, 1758) in 28 sites in the Moravskoslezské Beskydy Mountains, Czech Republic, was carried out in 2001. The buildings inhabited or visited by badgers were as follows: wooden barns (18 cases), masonry buildings used for residential purposes (4), abandoned buildings (1), wooden sheds (2), wooden beehouses (2) and a non-residential part of a house (1). In three sites, female badgers with their cubs inhabited buildings. Badgers use the buildings more frequently in winter than in summer. Use of human buildings and the occurrence of badgers in setts in the wild in these mountains was observed in detail on a study area of 950 ha around the village of Lubno. In total, 12 setts were discovered. Eight of them were in the wild: two setts were located closer than 50 m, five between 100 m and 300 m, and one 700 m from human buildings. In four sites badgers inhabited human buildings.     

Late Pleistocene–early Holocene transition recorded in the sediments of a former shallow lake in the Czech Republic

Quaternary limnic sediments from Velanská Cesta, a former lake in the Czech Republic, were investigated to document the response of an aquatic environment to global climatic shifts during the late Pleistocene–early Holocene transition. The obtained multi-proxy record, including subfossil diatoms, diatom-inferred variables (index of trophic state, halobic index, TP, and pH), plant macrofossils, and geochemical data, was compared with the δ¹⁸O GISP2 curve. Diatom accumulation zones and subzones established on the basis of a ConSLink cluster analysis corresponded well with the boundaries of the Bölling, Older Dryas, Alleröd, Younger Dryas, and Preboreal periods. The diatom-inferred trophic state and salinity data showed increased values during the colder epochs, which were supported by the findings of low-temperature macrofossil indicators in the corresponding samples. The results of this study document a long-term forcing of a shallow lacustrine ecosystem in central Europe by climatic events of a global scale.     

The Emergence and Spread of Multiple Livestock-Associated Clonal Complex 398 Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains among Animals and Humans in the Republic of Ireland, 2010–2014

Clonal complex (CC) 398 methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) are associated with carriage and infection among animals and humans but only a single case of CC398 MRSA has been reported in the Republic of Ireland (ROI). The present study investigated the molecular epidemiology of CC398 MRSA (n = 22) and MSSA (n = 10) from animals and humans in the ROI from 2010–2014. Isolates underwent antimicrobial susceptibility testing, spa typing, DNA microarray profiling and PCR for CC398-associated resistance genes. All MRSA underwent SCCmec IV or V subtyping. Four distinct CC398-MRSA incidents were identified from (i) a man in a nursing home (spa type t011-SCCmec IVa, immune evasion complex (IEC) negative), (ii) a horse and veterinarian who had recently travelled to Belgium (t011-IVa, IEC positive), (iii) pigs (n = 9) and farm workers (n = 9) on two farms, one which had been restocked with German gilts and the other which was a finisher farm (t034-V_T, IEC negative, 3/9 pigs; t011- V_T, IEC negative, 6/9 pigs & 9/9 farm workers), and (iv) a child who had worked on a pig farm in the UK (t034-V_T, IEC negative). Isolates also carried different combinations of multiple resistance genes including erm(A), erm(B), tet(K), tet(M) & tet(L), fexA, spc, dfrG, dfrK aacA-aphD and aadD further highlighting the presence of multiple CC398-MRSA strains. CC398 MSSA were recovered from pigs (n = 8) and humans (n = 2). CC398 MSSA transmission was identified among pigs but zoonotic transmission was not detected with animal and human isolates exhibiting clade-specific traits. This study highlights the importation and zoonotic spread of CC398 MRSA in the ROI and the spread of CC398 MSSA among pigs. Increased surveillance is warranted to prevent further CC398 MRSA importation and spread in a country that was considered CC398 MRSA free.     

Long-term changes in numbers and distribution of wintering waterbirds in the Czech Republic, 1966–2008

Capsule?Of 26 species of wintering waterbirds, 18 showed an increase in numbers, five showed a decrease and two showed no change.

Aim?To assess long-term trends in the numbers and distribution of the 26 most abundant wintering waterbird species in the Czech Republic.

Methods?We used International Waterbird Census data from between 48 and 639 wetland sites which had been counted annually in the Czech Republic from 1966 to 2008. From these data long-term changes in numbers and distributions were determined. Log-linear Poisson regression analysis was used to estimate missing data using trim software. The distribution of each species was described as the ratio of the number of sites occupied by that species to the total number of sites investigated.

Results?Increasing trends were found for 18 species, five species were found to be declining, one species was stable and two species were found with uncertain trends. Wintering distributions (the ratio of sites occupied by a given species to the total number of sites counted) increased in 16 species and decreased in two species, broadly correlated with the species changes in numbers.

Conclusion?In most species changes in numbers as well as changes in distribution followed the Western Palearctic population trends. Those species which increased were mainly piscivores and included geese, ducks and gulls. Scarcer species also exhibited an increase in numbers. The changes in numbers (both positive and negative) were more frequent among species associated with running water, whereas species which showed uncertain trends were more frequently recorded on standing water, which is more affected by variable weather conditions.     

The Peptidoglycan Hydrolase of Staphylococcus aureus Bacteriophage ?11 Plays a Structural Role in the Viral Particle

The role of virion-associated peptidoglycan hydrolases (VAPGHs) in the phage infection cycle is not clear. gp49, the VAPGH from Staphylococcus aureus phage ϕ11, is not essential for phage growth but stabilizes the viral particles. ϕ11Δ49 phages showed a reduced burst size and delayed host lysis. Complementation of gp49 with HydH5 from bacteriophage vB_SauS-phiIPLA88 restored the wild-type phenotype.     

Dissection of events in the resistance to β-lactam antibiotics mediated by the protein BlaR1 from Staphylococcus aureus

A heterologous expression system was used to evaluate activation of BlaR1, a sensor/signal transducer protein of Staphylococcus aureus with a central role in resistance to β-lactam antibiotics. In the absence of other S. aureus proteins that might respond to antibiotics and participate in signal transduction events, we documented that BlaR1 fragmentation is autolytic, that it occurs in the absence of antibiotics, and that BlaR1 directly degrades BlaI, the gene repressor of the system. Furthermore, we disclosed that this proteolytic activity is metal ion-dependent and that it is not modulated directly by acylation of the sensor domain by β-lactam antibiotics.     

Occurrence of the toxigenic fungi (producers of aflatoxins and ochratoxin A) in foodstuffs in the Czech Republic 1999–2000

The occurrence of toxigenic fungi producing aflatoxins and ochratoxin A in foodstuffs was studied in the Czech Republic. Twenty five commodities were collected at twelve collection places in the Czech Republic (300 food samples). The presence of potentially toxigenicAspergillus flavus was observed in 28% of the sampled foods (black pepper, caraway seeds, fruit tea, black tea, oat flakes, fine flour, rolled oat flakes and semolina) in the year 1999, and in 25% of the sampled foods (black pepper, black tea, fine flour) in the year 2000.A tamarii (aflatoxins producer) was found in 3 black pepper samples (25%) in both years. Aflatoxins were detected in black pepper and caraway seed samples in the year 1999 and in sweet red pepper in the year 2000.A parasiticus andA nomius were not isolated. Aspergillus section Nigri (potential producer of ochratoxin A) was detected in some foodstuffs. Ochratoxin A was detected in raisins.Penicillium verrucosum andA ochraceus were not isolated from foodstuffs.     

The N-Terminal Domain of the Repressor of Staphylococcus aureus Phage Φ11 Possesses an Unusual Dimerization Ability and DNA Binding Affinity

Bacteriophage Φ11 uses Staphylococcus aureus as its host and, like lambdoid phages, harbors three homologous operators in between its two divergently oriented repressor genes. None of the repressors of Φ11, however, showed binding to all three operators, even at high concentrations. To understand why the DNA binding mechanism of Φ11 repressors does not match that of lambdoid phage repressors, we studied the N-terminal domain of the Φ11 lysogenic repressor, as it harbors a putative helix-turn-helix motif. Our data revealed that the secondary and tertiary structures of the N-terminal domain were different from those of the full-length repressor. Nonetheless, the N-terminal domain was able to dimerize and bind to the operators similar to the intact repressor. In addition, the operator base specificity, binding stoichiometry, and binding mechanism of this domain were nearly identical to those of the whole repressor. The binding affinities of the repressor and its N-terminal domain were reduced to a similar extent when the temperature was increased to 42°C. Both proteins also adequately dislodged a RNA polymerase from a Φ11 DNA fragment carrying two operators and a promoter. Unlike the intact repressor, the binding of the N-terminal domain to two adjacent operator sites was not cooperative in nature. Taken together, we suggest that the dimerization and DNA binding abilities of the N-terminal domain of the Φ11 repressor are distinct from those of the DNA binding domains of other phage repressors.     

The Galactose-Binding Lectin Isolated from Vatairea macrocarpa Seeds Enhances the Effect of Antibiotics Against Staphylococcus aureus–Resistant Strain

Probiotics and Antimicrobial Proteins - The use of natural products together with standard antimicrobial drugs has recently received more attention as a strategy to combat infectious diseases...     

Effect of rotational and continuous grazing on vegetation of an upland grassland in the Jizerské hory Mts., Czech Republic

The effect of different grazing regimes on pasture vegetation was studied during the intensive grazing of heifers in the Jizerské hory mountains during 1993–1997. The vegetation was monitored in 3 pairs of permanent 1×1 m plots using a continuous grid of nine 0.33 × 0.33 m subplots. We applied continuous stocking and rotational grazing. Vegetation varied as a result of time and differences between treatments. Several prostrate dicotyledonous species (Trifolium repens, Taraxacum sp.,Bellis perennis andLeontodon autumnalis) increased under continuous stocking. This treatment also promoted the growth of the perennial grassLolium perenne, which was able to cope with frequent defoliation. Tall grasses sensitive to frequent defoliation (Poa trivialis, Holcus mollis, Alopecurus pratensis, Dactylis glomerata andElytrigia repens) were more abundant in rotationally grazed paddocks. Species diversity was not significantly influenced by the different grazing systems. The decrease in the potential sward height under continuous stocking revealed the replacing of tall dominants by lower species. Our results indicate that different grazing systems alter the composition and structure of grassland vegetation. Defining the intensity of grazing under continuous stocking or rotational grazing is complex due to the different stocking rates and the heights of sward during a grazing season. Information about pasture management should therefore involve not only grazing intensity but also the grazing system used.     

Inhibition of Pore Formation by Blocking the Assembly of Staphylococcus aureus α-Hemolysin Through a Novel Peptide Inhibitor: an In Silco Approach

Staphylococcus aureus self-assembling α-hemolysin heptamer is an acute virulence factor that determines the severity of S. aureus infections. Hence, inhibiting the heptamer formation is of considerable interest. However, both natural and chemical inhibitors reported so far has difficulties related to toxicity, bioavailability, and solubility, which necessitate in identifying some alternatives. Hence, in this study, potential peptides for α-hemolysin inhibition was developed using in silico based approach. Haddock server was used to understand the residues involved in complex formation. Based on the key residues involved in the interaction, 20 peptides were designed and docked with the α-hemolysin monomer (Chain A). Further, the best scored Chain A-peptide complex was chosen and docked with Chain B to identify the ability of dimer formation in the presence of designed peptide. The stability of the Chain A–B dimer, Chain A-peptide and Chain A-peptide-Chain B complex was studied by performing molecular dynamic simulation over 3,000 ps. The peptide IYGSKANRQTDK was found to be binding efficiently with Chain A of α-hemolysin with highest binding energy and also revealed that the designed peptide disturbed the dimer formation, which provided useful information in developing promising lead for inhibiting α-hemolysin assembly in the future.     

Expression of δ-toxin by Staphylococcus aureus mediates escape from phago-endosomes of human epithelial and endothelial cells in the presence of β-toxin

Staphylococcus aureus is able to invade non-professional phagocytes by interaction of staphylococcal adhesins with extracellular proteins of mammalian cells and eventually resides in acidified phago-endosomes. Some staphylococcal strains have been shown to subsequently escape from this compartment. A functional agr quorum-sensing system is needed for phagosomal escape. However, the nature of this agr dependency as well as the toxins involved in disruption of the phagosomal membrane are unknown. Using a novel technique to detect vesicular escape of S. aureus, we identified staphylococcal virulence factors involved in phagosomal escape. Here we show that a synergistic activity of the cytolytic peptide, staphylococcal δ-toxin and the sphingomyelinase β-toxin enable the phagosomal escape of staphylococci in human epithelial as well as in endothelial cells. The agr dependency of this process can be directly explained by the location of the structural gene for δ-toxin within the agr effector RNAIII.