Pathway analysis of Candida albicans survival and virulence determinants in a murine infection model

One potentially rich source of possible targets for antifungal therapy are those Candida albicans genes deemed essential for growth under the standard culture (i.e., in vitro) conditions; however, these genes are largely unexplored as drug targets because essential genes are not experimentally amenable to conventional gene deletion and virulence studies. Using tetracycline-regulatable promoter-based conditional mutants, we investigated a murine model of candidiasis in which repressing essential genes in the host was achieved. By adding doxycycline to the drinking water starting 3 days prior to (dox - 3D) or 2 days post (dox + 2D) infection, the phenotypic consequences of temporal gene inactivation were assessed by monitoring animal survival and fungal burden in prophylaxis and acute infection settings. Of 177 selected conditional shut-off strains tested, the virulence of 102 was blocked under both repressing conditions, suggesting that the corresponding genes are essential for growth and survival in a murine host across early and established infection periods. Among these genes were those previously identified as antifungal drug targets (i.e., FKS1, ERG1, and ERG11), verifying that this methodology can be used to validate potential new targets. We also identify genes either conditionally essential or dispensable for in vitro growth but required for survival and virulence, including those in late stage ergosterol synthesis, or early steps in fatty acid or riboflavin biosynthesis. This study evaluates the role of essential genes with respect to pathogen virulence in a large-scale, systems biology context, and provides a general method for gene target validation and for uncovering unexpected antimicrobial targets.     

Spontaneous mutations in the CsrRS two-component regulatory system of Streptococcus pyogenes result in enhanced virulence in a murine model of skin and soft tissue infection

CsrS/CsrR is a 2-component system in Streptococcus pyogenes that negatively regulates hyaluronic capsule and several exotoxins. To detect spontaneous mutations in csrRS, mucoid and large colony variants of M1 strain MGAS166 were isolated from experimental murine skin infections. By use of complementation with a csrRS(+) plasmid, relevant mutations were also detected in 7 of 12 human clinical isolates. The presence of spontaneous mutants in mouse infection was associated with larger, more necrotic lesions. Most spontaneous changes in CsrR resulted from single amino acid substitutions, whereas most csrS mutations were frameshift or nonsense mutations. In 2 instances, IS1548 insertions were found in csrS. Experimental inoculation of mixtures of wild-type (wt) and csrRS(-) bacteria yielded larger, more necrotic lesions than did either strain at twice the inoculum, which suggests that these variants may exhibit pathogenic synergy. Spontaneous emergence of csrRS(-) mutants in vivo enhances the virulence of wt bacteria and increases severity of murine skin infection.     

Comparison of virulence in community-associated methicillin-resistant Staphylococcus aureus pulsotypes USA300 and USA400 in a rat model of pneumonia

BACKGROUND: The predominant genetic background of community-associated methicillin-resistant Staphylococcus aureus has transitioned from USA400 to USA300 in most US communities. The explanation for this shift is unclear. We hypothesized that USA300 must be more pathogenic--specifically, that USA300 would have increased virulence when compared with USA400 in an animal model. METHODS: Rats were inoculated intratracheally with 1 of 6 S. aureus isolates from the USA300 and USA400 backgrounds. We assessed mortality, in vivo bacterial growth, and histopathology. We assessed the in vitro expression of capsule and of selected genes believed to be important in virulence in S. aureus, including agr, saeRS, sarA, alpha-toxin (hla), and Panton-Valentine leukocidin (pvl). RESULTS: USA300 isolates were more lethal, produced more severe pneumonia, and had higher in vivo bacterial density in the lung than did USA400 isolates. In vitro expression of agr, saeRS, sarA, hla, and pvl were greater in USA300 isolates. USA300 isolates were unencapsulated, whereas 2 of 3 USA400 isolates produced capsule. CONCLUSIONS: USA300 isolates were more virulent than USA400 isolates in a model of necrotizing pneumonia. The explanation for this is unclear, but it likely results from increased expression of S. aureus regulatory systems (e.g., agr, saeRS, and sarA) and the resultant upregulation of key virulence factors including alpha-toxin and PVL.     

Microbial virulence determinants and the pathogenesis of urinary tract infection

The most frequent and best-studied agent of urinary tract infection (UTI) is Escherichia coli, which serves as a useful model pathogen for understanding microbial virulence in relation to UTI pathogenesis. The E. coli strains that cause most UTIs and other extraintestinal E. coli infections represent a highly specialized subset of the total E. coli population. The enhanced virulence potential of such strains, which collectively are known as uropathogenic E. coli or extraintestinal pathogenic E. coli (ExPEC), is thought to be caused mainly by their multiple virulence factors. These virulence factors include diverse adhesins, siderophores, toxins, polysaccharide coatings, and other properties that assist the bacteria in avoiding or subverting host defenses, injuring or invading host cells and tissues, and stimulating a noxious inflammatory response. Although the true evolutionary basis for ExPEC is unknown, the virulence factors of ExPEC serve as useful epidemiologic markers and in the future may provide effective targets for anti-UTI interventions.     

Comparative clinical study of roxithromycin and josamycin for suppurative skin and soft tissue infections by a double-blind method

The clinical efficacy and safety of Roxithromycin (RU 28965, RU), a new macrolide preparation, were compared with those of Josamycin (JM) in superficial suppurative skin infections. The study was designed as double-blind controlled trial with daily dosages of 300 mg in RU group and 1200 mg in JM group. A total of 209 cases (RU:105; JM:104) was analyzed and the final global improvement rating was 82.9% in the RU group and 80.8% in the JM group; there was no significant difference between the two groups. Slight adverse reactions were observed in 3.6% (4 cases) of the RU group and in 4.6% (5 cases) of the JM group. In conclusion, RU at daily doses of 300 mg is as effective as JM at daily doses of 1200 mg in superficial suppurative skin infections.     

Diaporthe soft tissue infection in a heart transplant patient

Infections caused by Diaporthe species are very uncommon. We describe a heart transplant recipient 14 years post transplant who developed a soft tissue fungal infection due to a Diaporthe species that responded well to surgical excision and posaconazole therapy. The Aspergillus galactomannan index was markedly elevated, and returned to normal following treatment. Solid organ transplant patients remain at risk of infection long after transplantation and should be counseled about risk avoidance.     

Staphylococci in primary skin and soft tissue infections in a Swedish county

Patients with skin and soft tissue infections (SSTI) are frequently encountered in primary health care. The majority are uncomplicated and treated empirically by surgical incision and drainage and/or antibiotics. This strategy may risk delaying the detection of methicillin-resistant Staphylococcus aureus (MRSA), which, although still rare in Sweden, is increasingly being found in patients with SSTI. To avoid 'late detection' of MRSA, primary health care physicians in Kronoberg county, Sweden, were asked to perform a culture as soon as the patient's first visit. Samples from 175 patients with primary SSTI confirmed that S. aureus is the dominant pathogen. Two cases of MRSA were detected. Furthermore, isolates of S. aureus producing the Panton-Valentine leukocidin (PVL) toxin were more common among isolates from SSTI than among S. aureus from secondary infections. Finally, we confirmed the importance of the coagulase-negative staphylococcal species S. lugdunensis as a pathogen as it was isolated as the only pathogen in 10% of the skin and soft tissue samples.     

Microsphaeropsis arundinis skin and soft tissue infection in renal transplant recipients: three case reports and a review of the literature

Microsphaeropsis arundinis, a dematiaceous mold, is emerging as a cause of skin and soft tissue infection in immunocompromised hosts. Diagnosis is challenging because of the difficulty in identifying Microsphaeropsis species morphologically and few data are available to guide optimal management. We report 3 renal transplant recipients with M. arundinis soft tissue infection, where the etiological agent was diagnosed using DNA sequencing, and who were successfully treated with prolonged courses of extended‐spectrum triazole antifungal agents.     

Interaction between group A streptococci and the plasmin(ogen) system promotes virulence in a mouse skin infection model

Group A streptococci are capable of acquiring a surface-associated, unregulatable plasmin-like enzymatic activity when incubated in human plasma. The effect of this enzymatic activity on virulence of group A isolate CS101 was examined in a mouse skin infection model. Initial studies demonstrated enhanced virulence for bacteria preincubated in human plasma but not in plasminogen-depleted plasma. A direct correlation between surface-associated enzymatic activity and virulence was not observed; however, an association between virulence and the assembly of a surface-associated plasminogen activator that could activate mouse plasminogen was noted. This activity enhanced virulence in wild type but not in plg-/- plasminogen-deficient mice. These results support the hypothesis that acquisition of a surface-associated plasmin(ogen)-dependent enzymatic activity can contribute to the virulence of group A streptococcal invasive infections.     

Complicated skin and soft tissue infections in remote indigenous communities

The burden and consequences of skin infections for remote living indigenous people are high. While skin infections are recognised as an antecedent to conditions such as acute rheumatic fever in children, data are limited concerning skin infection complications such as cellulitis, abscesses and osteomyelitis in older children and adults. In a 1‐year retrospective audit of 439 patients presenting to two remote health clinics, 330/439 (75%) patients presented with a skin infection and 18 (4%) developed a complication.     

New drugs to treat skin and soft tissue infections

Due to increasing antimicrobial resistance, a pressing need exists for new antibiotics to treat skin and soft tissue infections. Several newer agents such as tigecycline, daptomycin, and linezolid have been important additions for the treatment of multidrug-resistant pathogens. New drugs in development such as dalbavancin and ceftobiprole will further enhance our ability to treat mixed infections and improve patient compliance. These promising new antimicrobials will likely grow in importance as resistant bacterial strains increase in community-acquired infections.     

Skin and soft tissue infection: necrotizing fasciitis

The incidence of necrotizing fasciitis has increased in the past decade as a result of the resurgence severe group A streptococcal infection. Mortality has remained unchanged over the past 60 years, supporting the notion that immune modulators, such as intravenous immunoglobulin, are required to alter the physiological process during the early stages of infection.     

Contribution of protein G-related alpha2-macroglobulin-binding protein to bacterial virulence in a mouse skin model of group A streptococcal infection

Protein G-related alpha(2)-macroglobulin-binding (GRAB) protein is a cell wall-attached determinant of group A streptococcus (GAS) that interacts with the human protease inhibitor alpha(2)-macroglobulin (alpha(2)-M). Of 86 clinical isolates tested, 23% could bind alpha(2)-M. However, all strains tested contained the grab gene. High levels of anti-GRAB antibodies were found in the serum of convalescent GAS-infected patients, a finding that indicates that this protein is expressed during the infection process. Among the alpha(2)-M-binding strains, 80% were skin isolates, and 20% were throat isolates, findings that suggest that the skin environment is a preferential site for expression of alpha(2)-M-binding activity. To test this possibility, we determined the role of GRAB in a mouse model of GAS skin infection. The wild-type strain KTL3, which interacts with alpha(2)-M, showed high virulence. The isogenic mutant of KTL3, MR4, devoid of surface-bound GRAB, was attenuated in virulence, compared with the wild-type strain. Thus, mice infected with MR4 survived longer, developed smaller skin lesions, and exhibited lower levels of bacterial dissemination than did those infected with KTL3. These results emphasize the role of GRAB as a virulence factor of GAS.