Infect
Infect. mice, we found that, compared to normal rabbit sera, antisera to Ata significantly reduced the levels of ATCC 17978 and two MDR strains in the lungs of infected mice. The ability of Ata to engender anti-adhesive, bactericidal, opsonophagocytic, and protective antibodies validates its potential use as an antigenic target against MDR infections. INTRODUCTION is a strictly aerobic, Gram-negative coccobacillus that is now a major cause of nosocomial infections worldwide, particularly in patients who are critically ill, those requiring mechanical ventilation in the intensive care units (ICUs), and also among injured soldiers returning from the Iraq and Afghanistan conflicts (9, 24, 35). Many isolates manifest high levels of resistance (36) Rabbit Polyclonal to Cytochrome P450 7B1 to antibiotics such as ampicillin-sulbactam (6), carbapenems (28, 29), aminoglycosides (10, 26), tetracyclines (15, 17), and quinolones (8, 16). Of particular Amodiaquine dihydrochloride dihydrate concern is the emergence of strains resistant to all commonly used antibiotics, including colistin (13, 30), polymyxin B (27), or tigecycline (25). Furthermore, displays an extraordinary tolerance to desiccation and disinfectants, which contributes to its long-term persistence in the hospital environment and the occurrence of outbreaks of infection (1, 5, 18, 21). The mortality attributable to infection can range from 7.8 to 43%, with higher levels in patients admitted to ICUs than those admitted to wards (11, 12). Multiple studies have shown that pneumonia increases the length of ICU stay by several days (34, 41). With virtually no new antimicrobial agents active against in the pharmaceutical pipeline, the need to understand the biology of that could lead to the rational design of new drugs and/or vaccines is more urgent than ever. Previous work carried out by our group has shown that synthesizes the surface polysaccharide poly-biofilm formation (7). Moreover, we demonstrated that PNAG is a target for opsonic and protective antibodies in two models of infection: pneumonia and bacteremia in immunocompetent mice (3). We have recently identified, in addition to PNAG, another surface component of cells to collagen type IV, as well as for the survival of in a murine model of infection (2). PCR analysis of 75 clinical strains of obtained from various geographical locations and types of infections revealed that 44/75 (58.6%) of the strains were positive for by PCR, 43 of which (56.3%) produced variable but mostly high levels of surface Ata, as detected by flow cytometry (2). In the present study we evaluated the functional properties of antibodies raised to recombinant Ata protein and specifically tested their anti-adhesive, opsonophagocytic, and bactericidal activity and their protective efficacy in murine models of pneumonia in both immunocompetent and neutropenic mice. MATERIALS AND METHODS Ethics statement. The present study was carried out in accordance with the recommendations in the of the National Institutes of Health (NIH). All animal protocols were reviewed and approved by the Harvard Medical Area Standing Committee on Animals IACUC, which has Animal Welfare Assurance of Compliance number A3431-01 on file with the Office of Laboratory Animal Welfare of the U.S. Public Health Service. Studies involving human subjects were approved by the Partners Health Care System Institutional Review Board (IRB). All subjects donating blood provided written informed consent to participate in the studies. Bacterial strains and growth conditions. The bacterial strains used in the present study are listed in Table 1. Unless otherwise stated, the wild-type strains and the ATCC 17978mutant strain were grown to an optical density at 650 nm (OD650) of 0.025 in lysogeny broth (LB) broth to maximize Ata production. The complemented strain, ATCC 17978straincomplemented with the gene in pBAD18kan-Ori2S8MDR clinical isolate2S11MDR clinical isolate2I38Clinical isolate2S25MDR clinical isolate2 Open in a separate window Anti-adhesive properties of antisera to Ata. Microtiter tissue culture plates were coated with collagen type IV, blocked, and washed as described Amodiaquine dihydrochloride dihydrate previously Amodiaquine dihydrochloride dihydrate (2). To test the ability of rabbit antibodies to Ata, prepared as described elsewhere (2), to block the binding of strains to collagen type IV, wild-type ATCC 17978 and the 17978and 17978cells was determined by serial dilution and plating. To test the ability of antibodies to Ata to block the binding of to numerous ECM/BM parts, including collagen types I, III, IV, and V and laminin, wild-type was preincubated with either antibody to Ata or NRS at dilutions 1:100, 1:1,000, 1:10,000, and 1:100,000, and the remainder of the experiment carried as explained above for screening the anti-adhesive properties of antibody to Ata against collagen IV. Opsonophagocytic assay. The opsonophagocytic killing assay (OPKA) integrated four basic parts: target bacteria, polymorphonuclear cells (PMNs), test sera, and a match source. The prospective bacterial strains (ATCC 17978, ATCC 17978complemented with the gene cloned into the pBAD18kan-ori vector, ATCC 17978ATCC 17978to remove antibodies not directed to the prospective antigen. The match source (human being sera) was.