Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.

We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC(fm), is transcribed as an operon, that its putative major pilus subunit, EbpC(fm) (also called pilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC(fm) operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC(fm) expression and eliminated EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), was not affected. Importantly, the ebpABC(fm) deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC(fm) in trans, which also restored cell surface expression of EbpC(fm) and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC(fm) locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.

Importance of the ebp (endocarditis- and biofilm-associated pilus) locus in the pathogenesis of Enterococcus faecalis ascending urinary tract infection.

BACKGROUND: We recently demonstrated that the ubiquitous Enterococcus faecalis ebp (endocarditis- and biofilm-associated pilus) operon is important for biofilm formation and experimental endocarditis. Here, we assess its role in murine urinary tract infection (UTI) by use of wild-type E. faecalis OG1RF and its nonpiliated, ebpA allelic replacement mutant (TX5475). METHODS: OG1RF and TX5475 were administered transurethrally either at an ~1 : 1 ratio (competition assay) or individually (monoinfection). Kidney pairs and urinary bladders were cultured 48 h after infection. These strains were also tested in a peritonitis model. RESULTS: No differences were observed in the peritonitis model. In mixed UTIs, OG1RF significantly outnumbered TX5475 in kidneys (P=.0033) and bladders (P< or =.0001). More OG1RF colony-forming units were also recovered from the kidneys of monoinfected mice at the 4 inocula tested (P=.015 to P=.049), and 50% infective doses of OG1RF for kidneys and bladder (9.1x10(1) and 3.5x10(3) cfu, respectively) were 2-3 log(10) lower than those of TX5475. Increased tropism for the kidney relative to the bladder was observed for both OG1RF and TX5475. CONCLUSION: The ebp locus, part of the core genome of E. faecalis, contributes to infection in an ascending UTI model and is the first such enterococcal locus shown to be important in this site.

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).

Characterization of Staphylococcus aureus adhesins important for respiratory tract infection

Staphylococcus aureus is a leading cause of lower respiratory tract infections in both adult and pediatric populations. In the past two decades, reports have described emergent incidence of severe necrotizing pneumonia in previously healthy individuals, frequently caused by antibiotic resistant strains. Additionally, S. aureus remains the most common cause of ventilator-associated pneumonia, contributing morbidity and mortality in intensive care units. As treatment of infection is made more difficult by the resistance to multiple antibiotics including vancomycin, there is a pressing need for novel strategies to prevent and treat S. aureus infections. Targeting essential mechanisms that promote infection such as adhesion, colonization, invasion, evasion of immune system and signaling may lead to inhibition of pathogenic surge. Staphylococcal adhesins of the MSCRAMM family (microbial surface components recognizing adherent matrix molecules) represent viable targets for such investigations. Understanding the molecular mechanism of binding is the first step toward the development of such therapies. Analysis of bacterial strains isolated from patients with staphylococcal pneumonia show increased expression of protein A, SdrD, SdrC and ClfB, cell surface proteins members of the MSCRAMM family. In this study the interaction of these MSCRAMMs with candidate ligands has been examined. We found that SdrD mediates S. aureus adherence to the lung epithelial cell line A549. Consistently, bacteria expressing SdrD have increased persistence in the lungs of infected mice after bronchoalveolar lavage in comparison with bacteria lacking this protein. Inhibition studies revealed that bacterial attachment can be abolished using neutralizing antibodies against SdrD. Using phage display, neurexin β isoforms were identified as SdrC binding partners. Previous reports postulated that MSCRAMMS bind their ligands by a 'dock, lock and latch' mechanism of interaction. Our data suggested that ClfB, an MSCRAMM responsible for nasal colonization, binds cytokeratin 10 by a 'dock and lock' variant of this model, in which the 'latching' event is not necessary. In summary, we have characterized aspects of molecular interaction between several MSCRAMMS and host components. We hope that continued delineation of these interactions will lead to identification of novel therapeutic targets or preventive strategies against S. aureus infections. ^

Effects of breastfeeding and naturally acquired immunity on infection with {\it Giardia lamblia}: A study in a cohort of Mexican infants

Giardia lamblia is one of the most common causes of gastrointestinal tract infection among young children worldwide. Yet host protection against this parasite and the effect of infection with Giardia on infant growth are poorly understood. It was hypothesized that among young children, protection against infection with Giardia is afforded by breastfeeding and previous infection with the parasite and further, that infection with Giardia decreases growth velocity. From 4/88 to 4/90, 197 infants in a poor area of Mexico City were followed from 0 to 18 months of age, with stool specimens, symptoms and feeding status data collected weekly. A total of 6,031 stool specimens were tested for Giardia antigen by enzyme-linked immunosorbent assay. There were 1.0 Giardia infections per child-year; 25% were symptomatic and 54% lasted more than 1 month; 94 infants had 1, and 33 had 2 or more infections. Breastfeeding status was coded and analyzed for each child-week of follow up. 91% of study infants were breastfed from birth, 57% at 6 months and 38% at 12 months of age. Rate ratios for non-breastfeeding adjusted for confounding factors were calculated from stratified analyses and the Cox proportional hazards model. Not breastfeeding was a significant risk factor for first infection with Giardia vs. any breastfeeding (adjusted RR = 1.8; 1.1, 2.8) at all ages; a dose response was demonstrated by degree of breastfeeding. The adjusted rate ratio for non-breastfeeding vs. partial breastfeeding was 1.6 (1.03, 2.6) and for non-breastfeeding vs. complete breastfeeding was 4.7 (1.4, 15.9). Among Giardia infected infants, breastfeeding did not protect against diarrheal symptoms or shorten the duration of carriage. First and repeat infections with Giardia did not differ in duration or the percent symptomatic. The analysis of growth and Giardia infection was inconclusive but suggested that a history of Giardia infection might be associated with decreased weight velocity, while an immediate chronic infection might be associated with increased weight velocity. In summary, these data indicate that breastfeeding protects infants against infection with Giardia; provide no evidence of protection against repeat infections resulting from a prior infection and suggest but do not establish that a history of Giardia infection might be associated with decreased growth in young children. ^

Bicarbonate enhances expression of the endocarditis and biofilm associated pilus locus, ebpR-ebpABC, in Enterococcus faecalis.

BACKGROUND: We previously identified ebpR, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the Enterococcus faecalis endocarditis and biofilm associated pilus operon, ebpABC. Although ebpR is not absolutely essential for ebpABC expression (100-fold reduction), its deletion led to phenotypes similar to those of an ebpABC mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated ebpABC mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the ebpR-ebpABC locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the ebpR-ebpABC locus. RESULTS: In this study, we examined the effect of CO2/HCO3(-), pH, and the Fsr system on the ebpR-ebpABC locus expression. The presence of 5% CO2/0.1 M HCO3(-) increased ebpR-ebpABC expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the ebpR-ebpABC locus expression appears independent of the effects of CO2(-) bicarbonate. Furthermore, by using an ebpA::lacZ fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO2, induced ebpA expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, P < 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with ebpA mRNA levels under the conditions tested. CONCLUSIONS: This study reports that the ebp locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, E. faecalis joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO2.

The association between uterus shape and development of chronic conditions in a retrospective corhort of diethylstilbestrol-exposed women in Minnesota and Texas

Diethylstilbestrol (DES) exposed women are well known to be at increased risk of gynecologic cancers and infertility. Infertility may result from DES associated abnormalities in the shape of women's uteri, yet little research has addressed the effect of uterine abnormalities on risk of infertility and reproductive tract infection. Changes in uterine shape may also influence the risk of autoimmune disease and women's subsequent mental health. A sample of consenting women exposed in utero to hormone who were recruited into the DESAD project, underwent hysterosalpingogram (HSG) from 1978 to 1984. These women also completed a comprehensive health questionnaire in 1994 which included women's self-reports of chronic conditions. HSG data were used to categorize uterine shape abnormalities as arcuate shape, hypoplastic, wide lower segment, and constricted. Women were recruited from two of the four DESAD study sites in Houston (Baylor) and Minnesota (Mayo). All women were DES-exposed. Adjusted relative risk estimates were calculated comparing the range of abnormal uterine shaped to women with normal shaped uteri for each of the four outcomes: infertility, reproductive tract infection, autoimmune disease and depressive symptoms. Only the arcuate shape (n=80) was associated with a higher risk of infertility (relative risk [RR]= 1.53, 95% CI = 1.09, 2.15) as well as reproductive tract infection (RR= 1.74, 95% CI = 1.11, 2.73). In conclusion, DES-associated arcuate shaped uteri appeared to be associated with the higher risk of a reproductive tract infection and infertility while no other abnormal uterine shapes were associated with these two outcomes.^

Health status of Saudi women, the case of Yanbu al-Sinaiya

Detailed data on the health status of Saudi women are lacking. This cross sectional study attempts to provide a comprehensive description of the health status of Saudi females between the ages of 15-45 residing in Yanbu Al-Siniyah. The purpose is to assess women's needs for health services. The health status indicators are chronic tracer conditions, reported symptoms and multidimensional functioning levels. The generic functioning instrument of the Medical Outcome Study was used to estimate physical, social, and role functioning; degree of pain and health perceptions. The information was obtained by interviewing subjects and abstracting facts from their medical records. The results show functioning scores are in the "well health" range for physical, social, role and pain. Crowding and education have an equal or stronger effect of reducing functioning levels than the diagnosed tracer conditions. The highest prevalence conditions having a definite functional impact and diagnosed adequately in primary care are anemia, urinary tract infection, hemorrhoids, rheumatoid arthritis, caries and gingivitis. Reported symptoms strongly reducing function levels in this study are dyspnea, heart pain, incontinence, eye and skin problems, and joint ache. The impact of the reliability and validity of the measures used and other limitations of the results are discussed. Finally, some policy implications and suggestions for future study are presented. ^

Bacteremia and mortality within 30 days of catheter-associated bacteriuria

Background: The distinction between catheter-associated asymptomatic bacteriuria (CAABU) and catheter-associated urinary tract infection (CAUTI) has only recently been widely appreciated. Our aims were to describe the relationship between CAUTI/CAABU and subsequent bacteremia and to investigate whether CAUTI/CAABU and antimicrobial use was associated with either bacteremia or mortality within 30 days. ^ Methods: Our study design was retrospective cohort. Patients with a urinary catheter and a positive urine culture between October 2010 and June 2011 at a large tertiary care facility were included. A multivariable model for analysis was constructed which controlled for age, race, Charlson co-morbidity score, catheter type and duration, category of organism,antimicrobials and classification of the catheter-associated bacteriuria as CAUTI or CAABU. ^ Results: Data from 444 catheter associated urine culture episodes in 308 unique patients were included in the analysis. Overall mortality was 21.1% (61 of 308 patients) within 30 days. Among the 444 urine culture episodes, 402 (90.5%) of these episodes were associated with antibiotic use. 52 (11.7%) of episodes were associated with bacteremia, but only 3 episodes of bacteremia (0.7% of 444 CAB episodes) were caused by an organism from the urinary tract. One of these episodes was CAABU and the other 2 were CAUTI. Bacteremia within 30 days was associated with having CAUTI rather than CAABU and having an indwelling urinary catheter rather than a condom catheter. The variables which were found to be significant for mortality within 30 days were a higher Charlson co-morbidity score and the presence of Candida in the urine culture. Use of antimicrobial agents to treat the bacteriuria was not associated with an increase or decrease in either bacteremia or mortality. ^ Conclusions: Our findings call into question the practice of giving antimicrobial agents to treat bacteriuria in an inpatient population with nearly universal antimicrobial use. A better practice may be targeted treatment of bacteriuria in patients with risk factors predictive of bacteremia and mortality.^

Respiratory syncytial virus infection in hematopoietic stem cell transplant patients

Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections in immunocompromised patients such as children less than 2 years, premature infants with congenital heart disease and chronic lung disease, elderly patients and patients who have undergone hematopoietic stem cell transplant (HSCT). HSCT patients are at high risk of RSV infection, at increased risk of developing pneumonia, and RSV-related mortality. Immunodeficiency can be a major risk factor for severe infection & mortality. Therapy of RSV infection with Ribavirin, Palivizumab and Immunoglobulin has shown to reduce the risk of progression to LRI and mortality, especially if initiated early in the disease. Data on RSV infection in HSCT patients is limited, especially at various levels of immunodeficiency. 323 RSV infections in HSCT patients have been identified between 1/1995 and 8/2009 at University of Texas M D Anderson Cancer Center (UTMDACC). In this proposed study, we attempted to analyze a de-identified database of these cases and describe the epidemiologic characteristics of RSV infection in HSCT patients, the course of the infection, rate of development of pneumonia and RSV-related mortality in HSCT patients at UTMDACC.^ Key words: RSV infections, HSCT patients ^