Donavon J. Hess

Comparative virulence of Candida albicans yeast and filamentous forms in orally and intravenously inoculated mice.

ObjectiveCandida albicans, a dimorphic fungus that switches from yeast to filamentous forms, is a major cause of complicating systemic infection in intensive care patients. The aim of this study was to compare the pathogenic potential of C. albicans yeast and filamentous forms. DesignSeparate groups of mice were inoculated either intravenously or orally with C. albicans CAF2 (wild type), HLC54 (yeast forms defective in filament formation), or BCa2-10 (constitutively filamentous). Mice were killed 1, 7, 14, and 21 days after intravenous C. albicans and kidneys and liver were quantitatively cultured; cohort groups were observed for mortality. Mice were pretreated with antibiotics for 3 days before oral inoculation with C. albicans, and killed 3 days later with dexamethasone administered for the latter 3 days; at sacrifice, the mesenteric lymph nodes and kidneys were cultured to monitor extraintestinal dissemination of C. albicans. SettingUniversity teaching hospital research laboratory. SubjectsFemale, Swiss Webster, adult mice. Measurements and Main ResultsIn intravenously inoculated mice, mortality was highest with wild-type C. albicans CAF2 (92%), intermediate with HLC54 (56%), and not detected with constitutively filamentous BCa2-10 (0%); BCa2-10 was cleared from the kidney and liver, but CAF2 and HLC54 were recovered at approximately 105–7/g kidney and 104–5/g liver. There was only occasional mortality in orally inoculated mice and the numbers of cecal C. albicans CAF2 and HLC54 were similarly high (approximately 10/g), whereas numbers of cecal BCa2-10 were at least 100-fold lower. Extraintestinal dissemination was greatest with HLC54, intermediate with CAF2, and undetectable with BCa2-10. ConclusionsOf the three C. albicans strains studied, wild-type CAF2 was most virulent in intravenously inoculated mice and HLC54 (defective in filament formation) was most virulent in orally inoculated mice. The constitutively filamentous BCa2-10 was avirulent in both models, suggesting that filamentous forms by themselves might not be critically important for C. albicans virulence.

Intracellular survival of Staphylococcus aureus within cultured enterocytes

BACKGROUND Little is known about the mechanisms involved in bacterial translocation from the intestinal lumen to extraintestinal sites. Because Staphylococcus aureus can colonize the intestinal tract, and because the intestinal tract is a reservoir for antibiotic resistant S. aureus, experiments were designed to clarify the interactions of S. aureus with cultured intestinal epithelial cells, and assays included measurements of bacterial internalization, enterocyte apoptosis, and epithelial barrier function. METHODS AND RESULTS Mature, confluent enterocytes were incubated 1 h with S. aureus, and the gentamicin protection assay was used to quantify intracellular bacterial survival at various time intervals up to 120 h later. Enterocyte apoptosis was assessed using Annexin V, and the permeability of confluent enterocyte cultures was measured by transepithelial electrical resistance and by transmigration of Escherichia coli across confluent enterocytes.S. aureus was internalized by cultured enterocytes and remained viable for up to 120 h within both HT-29 and Caco-2 enterocytes. S. aureus intracellular survival was associated with enterocyte apoptosis and with decreased transepithelial electrical resistance across confluent Caco-2 enterocytes. S. aureus intracellular survival over time was also associated with increased E. coli transmigration across confluent Caco-2, but not HT-29, enterocytes. CONCLUSIONS S. aureus appeared to survive within cultured enterocytes for prolonged time periods, up to several days. Survival of S. aureus within host eukaryotic cells, such as enterocytes, might facilitate persistence of S. aureus in infected tissue despite appropriate antibiotic therapy.

When patients choose: comparison of Nuss, Ravitch, and Leonard procedures for primary repair of pectus excavatum

BACKGROUND/PURPOSE Pectus excavatum is a common chest wall deformity, and several procedures have been developed for its correction. We allow patients to choose among Leonard, Nuss, and Ravitch procedures. This study aimed to determine which procedure most patients select and the resultant outcomes. METHODS Charts were reviewed of all pectus excavatum repairs performed for 4 years by a practice covering a university-based childrens hospital. Procedure choice, operative time, length of stay, analgesia, fees, and complications were recorded. RESULTS The Ravitch procedure was chosen by 60.9% of our patients, Leonard procedure by 23.9%, and Nuss procedure by 15.2%. Operative times were not significantly different among the groups. The mean length of stay was 2.2 days (Ravitch), 1.5 days (Leonard), and 3.9 days (Nuss) (P < .005). Epidural analgesia/patient-controlled analgesia pump requirements were 50% (Ravitch), 5% (Leonard), and 100% (Nuss). The mean charges were

Ability of the heparan sulfate proteoglycan syndecan-1 to participate in bacterial translocation across the intestinal epithelial barrier.

27,414 (Ravitch),

Importance of Colonization Site in the Current Epidemic of Staphylococcal Skin Abscesses

18,094 (Leonard), and

Role of heparan sulfate in interactions of Listeria monocytogenes with enterocytes

43,749 (Nuss) (P < .05). The overall complication rate was 16.3%. The complications among each group were as follows: Ravitch, 14.3%; Leonard, 9.1%; and Nuss, 35.7%. CONCLUSIONS We allow patients to choose among Leonard, Ravitch, and Nuss procedures for repair of pectus excavatum. Most select the Ravitch procedure. Length of stay, fees, analgesic needs, and complication rate were highest among patients in the Nuss group; all of these variables were lowest in the Leonard group.

Bacterial contamination of surgical suture resembles a biofilm.

Although hundreds of microbial species reside in the human intestinal tract, comparatively few (e.g., Escherichia coli and other enterobacteria, Enterococcus faecalis, etc.) are typically associated with systemic infection in postsurgical, shock, and trauma patients. Syndecan-1 is the predominant cell surface heparan sulfate proteoglycan expressed on epithelia, and there is substantial evidence that heparan sulfate participates in interactions of a variety of frankly pathogenic microbes with mammalian cells. To investigate the role of syndecan-1 in interactions of enteric flora with intestinal epithelium, bacteria that might use the enterocyte as a portal of entry for systemic infection (including E. faecalis, E. coli, and other enterobacteria, and several species of staphylococci and streptococci) were studied for their abilities to interact with syndecan-1. Streptococcus bovis, S. agalactiae, S. pyogenes, Staphylococcus aureus, and S. epidermidis showed increased adherence to ARH-77 cells transfected to express syndecan-1. Heparin, a heparan sulfate analog, inhibited internalization of S. bovis, S. agalactiae, S. pyogenes, and S. aureus by HT-29 enterocytes (prominent syndecan-1 expression), but not Caco-2 enterocytes (relatively low syndecan-1 expression). Data from experiments with Chinese hamster ovary cells with altered glycosaminoglycan expression indicated that heparan sulfate and chondroitin sulfate (glycosaminoglycans on the syndecan-1 ectodomain) participated in bacterial interactions with mammalian cells. Thus, although E. faecalis, E. coli, and other gram-negative enterobacteria did not appear to interact with syndecan-1, this heparan sulfate proteoglycan may mediate enterocyte interactions with some staphylococci and streptococci that are known to cause systemic infections in specific populations of high-risk, immunosuppressed, postsurgical, and trauma patients.

Heparan sulfate proteoglycans mediate Staphylococcus aureus interactions with intestinal epithelium

OBJECTIVE: The goal was to compare rectal and nasal Staphylococcus aureus colonization rates and S aureus pulsed-field types (PFTs) for children with S aureus skin and soft-tissue abscesses and normal control subjects. METHODS: Sixty consecutive children with S aureus skin and soft-tissue abscesses that required surgical drainage and 90 control subjects were enrolled. Cultures of the nares and rectum were taken in both groups. S aureus isolates from all sites were characterized through multiple-locus, variable-number, tandem-repeat analysis, pulsed-field gel electrophoresis, staphylococcal cassette chromosome mec typing for methicillin-resistant S aureus isolates, and determination of the presence of Panton-Valentine leukocidin genes. RESULTS: S aureus was detected significantly more often in the rectum of children with abscesses (47%) compared with those in the control group (1%; P = .0001). Rates of nasal colonization with S aureus were equivalent for children with abscesses (27%) and control subjects (20%; P = .33). S aureus recovered from the rectum was identical to S aureus in the abscess in 88% of cases, compared with 75% of nasal isolates. PFT USA300, staphylococcal cassette chromosome mec type IV, and Panton-Valentine leukocidin genes were significantly increased in the S aureus isolates from children with abscesses compared with those from control subjects. CONCLUSIONS: Skin and soft-tissue abscesses in the current epidemic of community-associated staphylococcal disease are strongly associated with rectal colonization by PFT USA300. Nasal colonization in children does not seem to be a risk factor.

Adherence of yeast and filamentous forms of Candida albicans to cultured enterocytes

Abstract. Heparan sulfate is known to participate in binding a wide variety of microbes to mammalian cells, but few studies have focused on the enterocyte. Normal human colonic and small intestinal enterocytes, and cultured HT-29 (but not Caco-2) enterocytes, reacted prominently with antibodies specific for heparan sulfate and for the core protein of syndecan-1 (a heparan sulfate proteoglycan). The heparan sulfate analog, heparin, inhibited interactions of Listeria monocytogenes (adherence and internalization) with HT-29, but not Caco-2, enterocytes. Internalization of L. monocytogenes by HT-29 enterocytes was inhibited by heparan sulfate and to a lesser extent by chondroitin sulfate, but not by the non-sulfated glycosaminoglycan hyaluronic acid. Compared to plasmid control ARH-77 cells, adherence of L. monocytogenes, was increased using ARH-77 cells transfected with syndecan-1 cDNA. Heparin binding protein(s) on L. monocytogenes were confirmed using biotinylated heparin. To determine if these in vitro observations might have in vivo relevance, L. monocytogenes was preincubated with heparin and then orally inoculated into mice. Compared to L. monocytogenes not pretreated with heparin, L. monocytogenes pretreated with heparin was associated with decreased extraintestinal dissemination to the mesenteric lymph nodes and liver of orally inoculated mice. Thus, heparan sulfate (possibly as the heparan sulfate proteoglycan syndecan-1) appears to participate in interactions of L. monocytogenes with enterocytes.

Gentamicin promotes staphylococcus aureus biofilms on silk suture

BACKGROUND Although much attention is currently directed to studying microbial biofilms on a variety of surfaces, few studies are designed to study bacterial growth on surgical suture. The purpose of this study was to compare the kinetic development of Staphylococcus aureus and Enterococcus faecalis on five surgical suture materials and to clarify factors that might influence this growth. METHODS Pure cultures of S. aureus and E. faecalis were incubated with five types of suture for four days using either tissue culture medium or a bacterial growth medium. Suture-associated bacteria were quantified daily. In selected experiments, the bacterial growth medium was supplemented with heparin, a substance known to promote S. aureus biofilm formation. The ultrastructure of S. aureus biofilm developing on braided suture was studied with scanning electron microscopy. RESULTS Staphylococcus aureus and E. faecalis were recovered in greater numbers (typically p < 0.01) from braided than from monofilament suture, and the numbers of bacteria were greater (often p < 0.01) on sutures incubated in bacterial growth medium rather than tissue culture medium. Addition of heparin 1,000 U/mL to silk or braided polyglactin 910 suture incubated three days with S. aureus resulted in greater numbers of bacteria on day one but not on subsequent days. Scanning electron microscopy showed a maturing S. aureus biofilm that developed from small clusters of cells among amorphous material and fibrillar elements to larger clusters of cells that appeared covered by more consolidated extracellular material. CONCLUSIONS Bacterial growth was favored on braided vs. monofilament suture, and heparin enhanced bacterial adherence after day one, but not at subsequent times. Staphylococcus aureus adhered to suture material and formed a structure consistent with a bacterial biofilm.