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.     

Complete Genome Sequence of the Electricity-Producing “Thermincola potens” Strain JR

“Thermincola potens” strain JR is one of the first Gram-positive dissimilatory metal-reducing bacteria (DMRB) for which there is a complete genome sequence. Consistent with the physiology of this organism, preliminary annotation revealed an abundance of multiheme c-type cytochromes that are putatively associated with the periplasm and cell surface in a Gram-positive bacterium. Here we report the complete genome sequence of strain JR.“Thermincola potens” strain JR, a Gram-positive anaerobe isolated from a thermophilic microbial fuel cell (MFC), constituted a dominant member of the current-producing bacterial community (10). Strain JR is a Thermincola species in the phylum Firmicutes belonging to the family Peptococcaceae in the order Clostridiales. It shares 99% 16S rRNA gene sequence identity with the two known members of the Thermincola genus, T. carboxdophilia and T. ferriacetica (8, 12). This strain coupled acetate oxidation to reduction of the insoluble electron acceptors MFC anodes and hydrous ferric oxide (HFO) (10). Strain JR is also capable of growth with CO as the sole electron donor and carbon source.This member of the Firmicutes is the first MFC isolate and Thermincola species to have its genome sequenced and is one of only a few bacteria in the Peptococcaceae to have its genome sequenced (5, 11). Genomic analysis will aid elucidation of electron transfer mechanisms by strain JR, contributing to the knowledge of extracellular respiration by Gram-positive bacteria. By comparing these mechanisms to those in Gram-negative organisms, the conserved and disparate aspects of this seminal metabolism can be identified. This will include analysis of the c-type cytochrome gene makeup of the genome, especially the increased number of proteins with double heme (CXXCH) motifs and multiple heme binding domains compared to the nearest phylogenetic neighbors with sequenced genomes (4, 6, 7). c-type cytochromes are essential for the reduction of insoluble electron acceptors by model Gram-negative bacteria, such as Geobacter or Shewanella species (3, 9); however, their role in Gram-positive mineral respiration is still unknown.Joint Genome Institute (JGI) sequencing used a combination of 454 and Illumina techniques with 27× coverage. All library construction and sequencing techniques are available at http://www.jgi.doe.gov/. Illumina reads were assembled into 121 contigs using Velvet 0.7.1.18 (13) and shredded into 1-kb pseudoreads (with 100-bp overlap). The pseudoreads were incorporated into a hybrid 454/Illumina assembly using the parallel Phrap assembler (CodonCode Corporation, Dedham, MA) (1, 2). Misassemblies were corrected with Dupfinisher (C. S. Han and P. Chain, presented at the 2006 International Conference on Bioinformatics and Computational Biology). Gene modeling was performed using Prodigal (http://prodigal.ornl.gov/), and resulting protein translations were assigned by comparisons to Pfam, KEGG, and COGs databases using BLASTP or HMMER. The complete genome was a single circular chromosome of approximately 3,036,819 bp with an average G+C content of 45.9%. A total of 2,963 protein-encoding genes were predicted, and 393 (6.9%) had no similarity to public database sequences.     

Complete Genome Sequence of Klebsiella oxytoca E718, a New Delhi Metallo-β-Lactamase-1-Producing Nosocomial Strain

We report the complete genome sequence of Klebsiella oxytoca E718, a New Delhi metallo-β-lactamase-1 (NDM-1)-producing strain isolated from a renal transplant patient. The genome contains a 6,097,032-bp chromosome and two multidrug resistance plasmids with sizes of 324,906 bp and 110,781 bp.     

Complete Genome Sequence of φHSIC, a Pseudotemperate Marine Phage of Listonella pelagia

The genome for the marine pseudotemperate member of the Siphoviridae HSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. HSIC enters into a pseudolysogenic relationship with its host, Listonella pelagia, characterized by sigmoidal growth curves producing >10⁹ cells/ml and >10¹¹ phage/ml. The genome (37,966 bp; G+C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a β subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of Salmonella phage P22 and Shigella phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), HSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.     

Complete Genome Sequence of Serratia plymuthica Bacteriophage ?MAM1

A virulent bacteriophage (ϕMAM1) that infects Serratia plymuthica was isolated from the natural environment and characterized. Genomic sequence analysis revealed a circular double-stranded DNA sequence of 157,834 bp, encoding 198 proteins and 3 tRNAs. The ϕMAM1 genome shows high homology to previously reported ViI-like enterobacterial bacteriophage genomes.     

Cloning and Sequence Analysis of Genome from the Inner Mongolia Strain of the Endogenous Betaretroviruses （enJSRV）

In order to amplify the complete genome of enJSRV from the strain of Inner Mongolia （enJSRV-NM）, we used enJSRV-specific and JSRV-specific DNA probes in dot blot hybridization. Seven pairs of primers were designed based on Genbank sequences. Seven fragments were obtained by PCR and were cloned into the PMD19-T vectors. The recombinant plasmids were sequenced and analyzed. The results showed that the genome was 7 942 bp in length and contained four overlapping open reading frames corresponding to the gag, pro, pol and env genes as well as an additional open reading frame （orf-x） that overlaps the 3＇ end of the pol gene. The nucleotide acid sequences of the enJSRV-NM loci were compared with the sequences of South Africa enJS56A1 strain （Accession No.AF153615） and USA JSRV21 strain （Accession No. AF105220）. The nucleotide acid identities were 99.2% and 92.3% respectively. Two zinc fingers were found in the NC region in the predicted amino acid sequence. However, the YXXM motif, which is a reliable molecular marker for the infectious exogenous virus, was not found in the TM region. It was found that the enJSRV-NM region was 90%-98% identical at the amino acid level to its exogenous infectious counterparts in most of the retroviral genome. This is the first nucleotide sequence of enJSRV reported in P.R China. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the clinical diagnosis of OPA.     

Alarming Proportions of Methicillin-Resistant Staphylococcus aureus (MRSA) in Wound Samples from Companion Animals,Germany 2010–2012

Staphylococcus (S.) aureus is an important cause of wound infections in companion animals, and infections with methicillin-resistant S. aureus (MRSA) are of particular concern due to limited treatment options and their zoonotic potential. However, comparable epidemiological data on MRSA infections in dogs, cats and horses is scarce, also limiting the knowledge about possible links to MRSA isolates from human populations. To gain more knowledge about the occurrence and genotypic variation of MRSA among wound swabs of companion animal origin in Germany we performed a survey (2010–2012) including 5,229 samples from 1,170 veterinary practices. S. aureus was identified in 201 (5.8%) canine, 140 (12.2%) feline and 138 (22.8%) equine swabs from a total of 3,479 canine, 1,146 feline and 604 equine wounds, respectively. High MRSA rates were identified with 62.7%, 46.4% and 41.3% in S. aureus of canine, feline and equine origin, respectively. Further genotyping including spa typing and multilocus sequence typing (MLST) revealed a comparable distribution of spa types among canine and feline MRSA with CC22 (47.6%; 49.2%) and CC5 (30.2%; 29.2%) as predominant lineages followed by CC398 (13.5%; 7.7%) and CC8 (4.0%; 9.2%). In contrast, the majority of equine MRSA belonged to CC398 (87.7%). Our data highlight the importance of S. aureus and MRSA as a cause of wound infections, particularly in cats and horses in Germany. While “human-associated” MRSA lineages were most common in dogs and cats, a remarkable number of CC398-MRSA was detected in horses, indicating a replacement of CC8-MRSA as the predominant lineage within horses in Germany. These data enforce further longitudinal epidemiological approaches to examine the diversity and temporal relatedness of MRSA populations in humans and animals to assess probable sources of MRSA infections. This would enable a sound risk assessment and establishment of intervention strategies to limit the additional spread of MRSA.     

Comparative Evaluation of Five Culture Media with Triplex PCR Assay for Detection of Methicillin-Resistant Staphylococcus aureus

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) is responsible for nosocomial and community-acquired infections. Hence, rapid and accurate laboratory diagnosis of MRSA is a vital constituent of control measures. The present study evaluated five different methods for the identification of MRSA. A total of 207 S. aureus clinical isolates that consisted of 89 MRSA and 118 methicillin-susceptible S. aureus (MSSA) strains confirmed by PCR were tested. MRSA strains were evaluated by five different methods: chromogenic MRSA agar (CMRSA), oxacillin resistance screening agar base (ORSAB), mannitol salt oxacillin agar (MSO), mannitol salt cefoxitin agar with two different concentrations of cefoxitin [4 μg/ml (MSC-4) and 6 μg/ml (MSC-6)]. The results of the different methods were compared to mecA PCR as the gold standard. MSC-6 showed only six false-positive MRSA in comparison with PCR. The sensitivities and specificities of MSC-6, MSC-4, MSO-4, ORSAB, and CMRSA were as follows: 98.9/94.9%, 100/83.1%, 89.9/87.3%, 97.8/96.6%, and 95.5/94.9%, respectively. In comparison with PCR, it was found that both MSC-6 and ORSAB were relatively the least expensive screening tests (0.70 and0.70 and 1.00, respectively). In conclusion, all methods were comparable, but MSC-6 was the least expensive medium for MRSA screening.     

Channel-Forming Abilities of Spontaneously Occurring α-Toxin Fragments from Staphylococcus aureus

Pore formation by four spontaneously occurring α-toxin fragments from Staphylococcus aureus were investigated on liposome and erythrocyte membranes. All the isolated fragments bound to the different types of membranes and formed transmembrane channels in egg-phosphatidyl glycerol vesicles. Fragments of amino acids (aa) 9–293 (32 kD) and aa 13–293 (31 kD) formed heptamers, similar to the intact toxin, while the aa 72–293 (26 kD) fragment formed heptamers, octamers, and nonamers, as judged by gel electrophoresis of the liposomes. All isolated fragments induced release of chloride ions from large unilamellar vesicles. Channel formation was promoted by acidic pH and negatively charged lipid head groups. Also, the fragments’ hemolytic activity was strongly decreased under neutral conditions but could be partially restored by acidification of the medium. We paid special attention to the 26-kD fragment, which, despite the loss of about one-fourth of the N-terminal part of α-toxin, did form transmembrane channels in liposomes. In light of the available data on channel formation by α-toxin, our results suggest that proteolytic degradation might be better tolerated than previously reported. Channel opening could be inhibited and open channels could be closed by zinc in the medium. Channel closure could be reversed by addition of EDTA. In contrast, digestion at the C terminus led to premature oligomerization and resulted in species with strongly diminished activity and dependent on protonation.     

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...     

Properties of a Novel PBP2A Protein Homolog from Staphylococcus aureus Strain LGA251 and Its Contribution to the ��-Lactam-resistant Phenotype

Methicillin-resistant Staphylococcus aureus (MRSA) strains show strain-to-strain variation in resistance level, in genetic background, and also in the structure of the chromosomal cassette (SCCmec) that carries the resistance gene mecA. In contrast, strain-to-strain variation in the sequence of the mecA determinant was found to be much more limited among MRSA isolates examined so far. The first exception to this came with the recent identification of MRSA strain LGA251, which carries a new homolog of this gene together with regulatory elements mecI/mecR that also have novel, highly divergent structures. After cloning and purification in Escherichia coli, PBP2A_LGA, the protein product of the new mecA homolog, showed aberrant mobility in SDS-PAGE, structural instability and loss of activity at 37 °C, and a higher relative affinity for oxacillin as compared with cefoxitin. The mecA homolog free of its regulatory elements was cloned into a plasmid and introduced into the background of the β-lactam-susceptible S. aureus strain COL-S. In this background, the mecA homolog expressed a high-level resistance to cefoxitin (MIC = 400 μg/ml) and a somewhat lower resistance to oxacillin (minimal inhibitory concentration = 200 μg/ml). Similar to PBP2A, the protein homolog PBP2A_LGA was able to replace the essential function of the S. aureus PBP2 for growth. In contrast to PBP2A, PBP2A_LGA did not depend on the transglycosylase activity of the native PBP2 for expression of high level resistance to oxacillin, suggesting that the PBP2A homolog may preferentially cooperate with a monofunctional transglycosylase as the alternative source of transglycosylase activity.     

Crystal Structures of Penicillin-Binding Protein 3 (PBP3) from Methicillin-Resistant Staphylococcus aureus in the Apo and Cefotaxime‐Bound Forms

Staphylococcus aureus is a widespread Gram‐positive opportunistic pathogen, and a methicillin‐resistant form (MRSA) is particularly difficult to treat clinically. We have solved two crystal structures of penicillin‐binding protein (PBP) 3 (PBP3) from MRSA, the apo form and a complex with the β-lactam antibiotic cefotaxime, and used electrospray mass spectrometry to measure its sensitivity to a variety of penicillin derivatives. PBP3 is a class B PBP, possessing an N-terminal non-penicillin‐binding domain, sometimes called a dimerization domain, and a C-terminal transpeptidase domain. The model shows a different orientation of its two domains compared to earlier models of other class B PBPs and a novel, larger N-domain. Consistent with the nomenclature of “dimerization domain”, the N-terminal region forms an apparently tight interaction with a neighboring molecule related by a 2-fold symmetry axis in the crystal structure. This dimer form is predicted to be highly stable in solution by the PISA server, but mass spectrometry and analytical ultracentrifugation provide unequivocal evidence that the protein is a monomer in solution.     

Combinations of β-Lactam or Aminoglycoside Antibiotics with Plectasin Are Synergistic against Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus

Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101) and MRSA (n = 115). Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI), plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin) displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin) in 87–89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA infections. We demonstrated that plectasin strongly rejuvenates the therapeutic potencies of existing antibiotics in vitro and in vivo. This is a novel strategy that can have major clinical implications in our fight against bacterial infections.     

Bacteriolytic enzymes from Staphylococcus aureus. Specificity of action of endo-β-N-acetylglucosaminidase

The bacteriolytic enzyme with an isoelectric point of 9.5 that is produced by all strains of Staphylococcus aureus investigated was purified from strain M18 (Wadström & Hisatsune, 1970). This enzyme released reducing groups from cell walls of Micrococcus lysodeikticus and was thus shown to be a bacteriolytic hexosaminidase. Although dinitrophenylation and acid hydrolysis of cell walls hydrolysed by a partially purified enzyme gave DNP-alanine and DNP-glycine from staphylococcal peptidoglycan, which indicated the presence of a peptidase and probably also an N-acetylmuramyl-l-alanine amidase, hydrolysis of cell walls by the extensively purified enzyme did not give any DNP-amino acids. The enzyme digest was purified by Amberlite CG-120 and Sephadex G-10 chromatography. Reduction by sodium borohydride of the disaccharide obtained was followed by acid hydrolysis and paper chromatography. Glucosamine completely disappeared after this treatment and a new spot identical with glucosaminitol appeared. The muramic acid spot remained unchanged. The purified enzyme was found to be devoid of exo-β-N-acetylglucosaminidase activity. These results are compatible with the action of a bacteriolytic endo-β-N-acetylglucosaminidase. It is also proposed that this enzyme is probably identical with the staphylococcal lysozyme. The mode of action of this has not previously been investigated.     

Complete Amino Acid Sequence of Crystalline (α–Glucosidase from Aspergillus niger

Rhodanine (1), rhodanine-3-acetic acid (2), methylrhodanine (3), and aminorhodanine (4) inhibited the growth of plants. Among them, 4 was the strongest inhibitor of the roots of all the tested plants. On the other hand, N-acetylaminorhodanine (5) and N-benzoylaminorhodanine (6) greatly decreased the inhibitory activity. The results suggest that the free amino group at N-3 of 4 is essential to the greater inhibitory activity of rhodanine derivatives. The plant-growth inhibition of 1–6 is related to the chlorophyll content of the plant treated with them and their acute toxicities in mice.     

Lytic effects of melittin and δ-haemolysin from Staphylococcus aureus on vesicles of dipalmitoylphosphatidylcholine

The effects of the lytic peptides, melittin and δ-haemolysin, are compared in vesicles of gel-phase dipalmitoylphosphatidylcholine (DPPC), using calcein as trapped marker. At low concentration, both toxins cause vesicles to lose contents in 5 mM phosphate buffer near neutral pH, with melittin being the more active. As phosphate concentration is increased, the kinetics of melittin-induced leakage change from a slow, sustained loss to a rapid ‘burst’ of leakage when melittin is present mainly as tetramer in solution, under conditions where it is reported to lose haemolytic activity towards erythrocytes. At low phosphate concentration, the leakage induced by δ-haemolysin is preceded by a lag phase, though fluorescence measurements show that binding of toxin is rapid. At higher phosphate concentration, the toxin binds rapidly to vesicles, but causes no leakage of entrapped calcein. Steady-state fluorescence spectra show no obvious differences in tryptophan emission for δ-haemolysin bound to lipid in high- or low-phosphate buffer. Spin-label fluorescence-quenching studies show that the single tryptophan residue of δ-haemolysin is buried within the lipid bilayer at all phosphate concentrations used. In gel-phase DPPC, δ-haemolysin shows no tendency to cause vesicle aggregation over several hours, as judged by light scattering, though a slow non-linear effect is seen above the lipid phase transition temperature. These effects are contrasted with those of melittin under similar conditions.