Virulence Genes and Genotypic Associations in Nasal Carriage,Community-Associated Methicillin-Susceptible and Methicillin-Resistant USA400 Staphylococcus aureus Isolates

It is not well understood why strains of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), a major cause of skin and soft tissue infections, became successful so quickly, overtaking the place of methicillin-sensitive S. aureus (MSSA) in many communities. To evaluate the genetic basis of differences in their virulence traits, 293 S. aureus isolates consisting of three cohorts, genotypically defined clinical CA-MRSA (n = 77), clinical MSSA (n = 103), and nasal carriage MSSA (n = 113), collected over a 19-year period in two Midwestern states in the United States, were (i) extensively genotyped and (ii) screened for 40 known virulence genes which included those for enterotoxins, leukocidins, hemolysins, and surface proteins and several newly identified putative toxin genes from the USA400 lineage of CA-MRSA. Genotypically, nasal carriage and clinical MSSA isolates were much more diverse than was the CA-MRSA group, which was found to be of USA400 lineage only. Virulence gene profiles of the three groups showed that CA-MRSA strains harbored significantly higher percentages (≥95%; P value, <0.05) of the sea, sec, sec4, seg2, seh, sek, sel, sel2, ear, ssl1, lpl10, lukSF-PV, lukD, lukE, and clfA genes than did the carriage and the clinical MSSA group (range, 0% to 58%). Genes of the enterotoxin gene cluster, seg, sei, sem, sen, and seo, were present in the clinical and carriage isolates but not in the CA-MRSA group. These results suggest that the presence of additional virulence factors in USA400 CA-MRSA strains compared to the nasal carriage and clinical MSSA strains probably contributed to their enhanced virulence.Staphylococcus aureus both is a benign commensal and common pathogen in humans and is responsible for a variety of infections, ranging from superficial skin and soft tissue infections to bacteremia, endocarditis, and osteomyelitis (33). Based on its susceptibility to beta-lactams, S. aureus is commonly described as methicillin-susceptible S. aureus (MSSA) or methicillin-resistant S. aureus (MRSA) (15). Infections due to health care-associated MRSA (HA-MRSA) have been a problem since the 1970s; however, starting in the 1990s, new strains of MRSA, referred to as community-associated MRSA (CA-MRSA), appeared in community dwellers and were genetically different from HA-MRSA strains (7). Until recently, individuals who presented with infections due to CA-MRSA typically have had none of the established risk factors associated with HA-MRSA, such as recent hospitalization, surgery, dialysis, long-term care residence, or indwelling medical devices. Lately however, CA-MRSA strains have been reported from both the community and the health care settings (25, 45). Genotyping tools such as pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa typing have helped in distinguishing the genotypes of CA-MRSA strains from those of other S. aureus strains (2, 14, 26). In PFGE, SmaI-restricted S. aureus genomes are compared to determine their genetic relatedness and also compared against the reference USA genotypes (USA100, USA200, etc., up to USA1200) as described by the Centers for Disease Control and Prevention (CDC) (34, 53). Of these, CA-MRSA isolates mostly belong to USA300 and USA400 clones and in some cases USA1000 and USA1100 clones as well. HA-MRSA isolates generally belong to USA100, USA200, and USA500 (34, 35). One of the two major clones of CA-MRSA, USA400, recognized in the early 1990s and initially referred to as the MW2 clone, was the predominant CA-MRSA clone that initially circulated in the midwestern United States in the 1990s (8, 17, 40, 52). The second and more recent CA-MRSA clone, USA300, was first recognized in 2000 and has since spread throughout the world (54). More than a thousand MLST allelic profiles for S. aureus have been identified so far, of which CA-MRSA strains are primarily represented by sequence type 1 (ST1) (USA400) and ST8 (encompasses USA300 and USA500). Of the several thousand spa types in the Ridom database (http://spaserver.ridom.de/), the most predominant CA-MRSA spa types are t008 and t128.CA-MRSA strains, besides their distinct PFGE, ST, and spa profiles, almost ubiquitously possess the Panton-Valentine leukocidin (PVL or lukSF-PV) genes, in contrast to only 1% to 5% of S. aureus strains overall having these genes (33, 50, 57). The PVL toxin has been implicated in many skin and soft tissue infections and lethal necrotizing pneumonia, but the exact role of PVL during S. aureus infections remains controversial (10, 28, 31, 58). The genome sequence of the CA-MRSA strain MW2 of the USA400 lineage showed the presence of several additional putative toxin genes (e.g., ear, sec4, sel2, seg2, ssl1 [set16], and lpl10) compared with other S. aureus strains that had been sequenced (1). Some of these toxin genes share homology with classic staphylococcal enterotoxin genes that encode pyrogenic exotoxins (49) typically produced during the post-exponential phase of growth, and the genes encoding these exotoxins are most often carried on plasmids, bacteriophages, or pathogenicity islands. The classic staphylococcal enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, and seo) are commonly found in strains of S. aureus (12, 23, 39, 42, 43, 46, 62). Pyrogenic exotoxin genes are common in S. aureus, and as many as 73% of S. aureus isolates carry at least one of the genes encoding a classic pyrogenic exotoxin; however, the distribution among various clonal types differs (3). Strains of USA400 CA-MRSA typically have been shown to possess the sea, sec, seh, and sek enterotoxin genes, whereas HA-MRSA strains usually carry the sed, seg, sei, sej, sem, sen, and seo enterotoxin genes (40). Currently, the distribution of the newly identified putative toxin genes (seg2, sel2, sec4, ssl1, and lpl10) (1) from the MW2 strain has not been reported from among CA-MRSA strains in general or from clinical and nasal carriage MSSA strains. Since CA-MRSA isolates are able to cause disease in humans without predisposing risk factors and have spread rapidly in communities, these strains may possess a greater number of toxin genes than do the other strains of S. aureus.The aim of this study was to compare the genotypes of clinical CA-MRSA USA400, clinical MSSA, and colonizing nasal carriage MSSA isolates and determine the frequency and distribution of the classic enterotoxin genes as well as the new putative toxin genes in them. Our results showed that MSSA strains were much more diverse in their genotypes than were CA-MRSA USA400 strains. In addition, CA-MRSA USA400 strains possessed a distinct array of toxin genes compared to MSSA strains. These data may provide insight into the success of CA-MRSA USA400 and its ability to cause severe disease in previously healthy people.     

Association of Campylobacter pylori with induced expression of class II transplantation antigens on gastric epithelial cells

Campylobacter pylori was identified with immunoperoxidase staining and a mouse monoclonal antibody directed against C. pylori in gastric biopsy specimens from 24 patients with gastritis. C. pylori was not found in gastric biopsy specimens from six subjects with histologically normal mucosa. The monoclonal antibody, which was reactive with a surface protein of approximately 20 kilodaltons, was found to be specific for C. pylori, and the immunoperoxidase staining proved to be more sensitive and rapid than culture in detecting the organism. In the tissue specimens where C. pylori was detected with the monoclonal antibody, there was a strong expression of class II transplantation antigens on the epithelial cells and an increased number of T lymphocytes. These findings indicate that C. pylori may initiate local immune responses.     

Phylogenetic analysis of the spirochetes Borrelia parkeri and Borrelia turicatae and the potential for tick-borne relapsing fever in Florida

Isolates of Borrelia turicatae, Borrelia parkeri, and the Florida canine borrelia (FCB) were examined to further phylogenetically characterize the identities of these spirochetes in the United States. DNA sequences of four chromosomal loci (the 16S rRNA gene, flaB, gyrB, and glpQ) were determined for eight isolates of B. turicatae and six isolates of B. parkeri, which grouped the spirochetes into two distinct but closely related taxa (>98% sequence identity) separate from Borrelia hermsii. The FCB was clearly separated with the group identified as B. turicatae, confirming this bacterium as a relapsing fever spirochete. Therefore, the potential for tick-borne relapsing fever in humans and other animals exists in Florida and future efforts are needed to determine the enzootic hosts and distribution of this spirochete in the southeastern United States. Analysis of plasmids demonstrated both linear and circular forms in B. turicatae but only linear plasmids in B. parkeri, which should be of interest to investigators concerned with plasmid diversity and evolution within this group of spirochetes.     

Activity and inhibition resistance of a phospholipase-resistant synthetic surfactant in rat lungs

This study investigates the activity and inhibition resistance in excised rat lungs of a novel synthetic surfactant containing the phospholipase-resistant diether phosphonolipid DEPN-8 plus 1.5% bovine surfactant protein (SP)-B/C compared to calf lung surfactant extract (CLSE). DEPN-8 + 1.5% SP-B/C surpassed CLSE in normalizing surfactant-deficient pressure-volume (P-V) deflation mechanics in lavaged excised lungs in the presence of phospholipase A(2) (PLA(2)) or C18:1 lyso-phosphatidylcholine (LPC). DEPN-8 + 1.5% SP-B/C had activity equal to CLSE in normalizing P-V mechanics in the absence of inhibitors or in the presence of serum albumin. These physiologic activity findings were directly consistent with surface activity measurements on the pulsating bubble surfactometer. In the absence of inhibitors, DEPN-8 + 1.5% SP-B/C and CLSE rapidly reached minimum surface tensions < 1 mN/m (0.5 and 2.5 mg surfactant phospholipid/ml). DEPN-8 + 1.5% SP-B/C maintained its high surface activity in the presence of PLA(2), while the surface activity of CLSE was significantly inhibited by exposure to this enzyme. DEPN-8 + 1.5% SP-B/C also had greater surface activity than CLSE in the presence of LPC, and the two surfactants had equivalent surface activity in the presence of albumin. DEPN-8 + 1.5% SP-B/C also had slightly greater surface activity than CLSE when exposed to peroxynitrite in pulsating bubble studies. These results support the potential of developing highly active and inhibition-resistant synthetic exogenous surfactants containing DEPN-8 + apoprotein/peptide constituents for use in treating direct pulmonary forms of clinical acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS).     

Analysis of supercoiled circular plasmids in infectious and non-infectious Borrelia burgdorferi

Linear plasmids are widely distributed in isolates of Borrelia burgdorferi, but the prevalence of supercoiled circular (SC) plasmids has not been clearly established. Enriching for SC plasmids on ethidium bromide density-gradients revealed that a low passage of strain Sh-2-82 of B. burgdorferi has at least six SC plasmids (8.4, 8.8, 20, 26, 29 and 51 kb). In addition, several of the SC plasmids were observed by electron microscopy to form concatenated structures. Because of the high incidence of coupled SC plasmids and the effect of such structures on plasmid mobility, we suggest that they could be mistaken for a single DNA species having a high molecular weight when total DNA from B. burgdorferi is examined by agarose-gel electrophoresis. Of the six SC plasmids in strain Sh-2-82, four could still be detected after two years of continuous in vitro passaging in BSK II medium. The two SC plasmids lost after a maximum of 20 passages, pBBC1 (8.4 kb) and pBBC2 (8.8 kb), shared a high degree of DNA sequence similarity, suggesting that their apparent instability may be a unique feature of these closely related plasmids. Two higher passaged variants of strain Sh-2-82, P20 and P202, were shown by hybridization to lack pBBC1 and pBBC2 sequences and were unable to infect the white-footed mouse, Peromyscus leucopus. However, six of 14 unrelated isolates that infected this rodent and Syrian hamsters also lacked pBBC1 and pBBC2 sequences. None of five unrelated but highly passaged non-infectious isolates carried either pBBC1 or pBBC2 sequences. These data indicate that pBBC1 and pBBC2 do not encode constitutively expressed proteins required for infectivity in our experimental system. Nevertheless, eight of the 13 infectious strains examined did carry pBBC1 and/or pBBC2, indicating that these two closely related plasmids can be detected in a wide range of unrelated isolates of B. burgdorferi.     

Polarisation impendance of pacemaker electrodes:in vitro studies simulating practical operation

The electrical properties of pacemaker electrodes were studiedin vitro under conditions prevailing in practical pacemaker operation. Emphasis was laid on a clear distinction between the changing modes of the pacemaker action. During sensing, the electrode can be represented by an a.c. series polarisation resistance and capacitance, generally accepted for biological electrodes obeying linearity rules. During stimulation, the electrode operates in the non-linear region. A nearly constant-voltage, short, rectangular pulse applied directly to the electrode-heart system, causes the electrode voltage and current to respond as a transient exponential, characterised approximately by a single time constant. This response allows modelling of the d.c. equivalent circuit of the electrode, in the form of a polarisation capacitance with a small resistance in series, shunted by a parallel resistance. Formulae were derived for calculation of these elements. The response of the electrode-heart system to a single stimulus was tested as a function of the amplitude and duration of the applied pulse. Also, the effect of repetitive stimulations was checked at a normal pacing rate. A nearly constant-voltage pacing source, as compared with a constant-current one, appears to be advantageous for preservation of the longevity of the electrode.     

Activation of RhoA,B,C by Yersinia Cytotoxic Necrotizing Factor (CNFy) Induces Apoptosis in LNCaP Prostate Cancer Cells

Prostate cancer is the most common malignancy, accounting for about 25% of all incident cases among men in industrialized countries. The human androgen-dependent prostate cancer cell line LNCaP, which is derived from a metastatic lesion of human prostatic adenocarcinoma, is frequently used to study prostate cancer associated signaling pathways in vitro. Recently it was described that Rho GTPase activation in these cells leads to apoptotic responses. We used the bacterial toxins CNFy and CNF1, which specifically and directly activate Rho GTPases by deamidation of a single glutamine. We asked whether these Rho activators could induce apoptosis in LNCaP cells. Our results indicate that RhoA activation, induced by CNFy, does lead to intrinsic apoptosis of the cells. Analysis of the underlying signaling pathway reveals that apoptosis induction requires the activity of Rho kinase (ROCK) and myosin activation, an apoptotic pathway previously identified in cancer stem cells.     

Detection of Lassa Virus, Mali

To determine whether Lassa virus was circulating in southern Mali, we tested samples from small mammals from 3 villages, including Soromba, where in 2009 a British citizen probably contracted a lethal Lassa virus infection. We report the isolation and genetic characterization of Lassa virus from an area previously unknown for Lassa fever.