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ABSTRACTWe have found an avian pathogenic Escherichia coli(APEC) plasmid, pAPEC-O2-ColV, which contains many of the genes associated with APEC virulence and also shows similarity in content to a plasmid and pathogenicity island of human uropathogenic E. coli(UPEC). To test the possible role of this plasmid in virulence, it was transferred by conjugation along with a large R plasmid, pAPEC-O2-R, into a commensal avian E. colistrain. The transconjugant was compared to recipient strain NC, UPEC strain HE300, and donor strain APEC O2 using various assays, including lethality for chicken embryos, growth in human urine, and ability to cause urinary tract infection in mice. The transconjugant killed significantly more chicken embryos than did the recipient. In human urine, APEC O2 grew at a rate equivalent to that of UPEC strain HE300, and the transconjugant showed significantly increased growth compared to the recipient. The transconjugant also significantly outcompeted the recipient in colonization of the murine kidney. These findings suggest that APEC plasmids, such as pAPEC-O2-ColV, contribute to the pathogenesis of avian colibacillosis. Moreover, since avian E. coliand their plasmids may be transmitted to humans, evaluation of APEC plasmids as possible reservoirs of urovirulence genes for human UPEC may be warranted.

ABSTRACTVirulence factors of pathogenic Escherichia colibelonging to a recently emerged and disseminated clonal group associated with urinary tract infection (UTI), provisionally designated clonal group A (CGA), have not been experimentally investigated. We used a mouse model of ascending UTI with CGA member strain UCB34 in order to identify genes of CGA that contribute to UTI. ihawas identified to be expressed by strain UCB34 in the mouse kidney using selective capture of transcribed sequences. ihafrom strain UCB34 demonstrated a siderophore receptor phenotype when cloned in a catecholate siderophore receptor-negative E. coliK-12 strain, as shown by growth promotion experiments and uptake of 55Fe complexed to enterobactin or its linear 2, 3-dihydroxybenzoylserine (DHBS) siderophore derivatives. Siderophore-mediated growth promotion by Iha was TonB dependent. Growth and iron uptake were more marked with linear DHBS derivatives than with purified enterobactin. The reported phenotype of adherence to epithelial cells conferred by expressing ihafrom a multicopy cloning vector in a poorly adherent E. coliK-12 host strain was confirmed to be specific to iha, in comparison with other siderophore receptor genes. ihaexpression was regulated by the ferric uptake regulator Fur and by iron availability, as shown by real-time reverse transcriptase PCR. In a competitive infection experiment using the mouse UTI model, wild-type strain UCB34 significantly outcompeted an isogenic ihanull mutant. Iha thus represents a Fur-regulated catecholate siderophore receptor that, uniquely, exhibits an adherence-enhancing phenotype and is the first described urovirulence factor identified in a CGA strain.

ABSTRACTTo survive within the host urinary tract, Escherichia colistrains that cause urinary tract infection (UTI) presumably must overcome powerful oxidant stresses, including the oxygen-dependent killing mechanisms of neutrophils. Accordingly, we assessed the global oxygen stress regulator OxyR of Escherichia colias a possible virulence factor in UTI by determining the impact of oxyRinactivation on experimental urovirulence in CBA/J and C57BL (both wild-type and p47phox−/−) mice. The oxyRand oxySgenes of wild-type E. colistrain Ec1a (O1:K1:H7) were replaced with a kanamycin resistance cassette to produce an oxyRSmutant. During in vitro growth in broth or human urine, the oxyRSmutant exhibited the same log-phase growth rate (broth) and plateau density (broth and urine) as Ec1a, despite its prolonged lag phase (broth) or initial decrease in concentration (urine). The mutant, and oxyRSmutants of other wild-type ExPEC strains, exhibited significantly increased in vitro susceptibility to inhibition by H2O2, which, like the altered growth kinetics observed with oxyRSinactivation, were reversed by restoration of oxyRon a multiple-copy-number plasmid. In CBA/J mice, Ec1a significantly outcompeted its oxyRSmutant (by >1 log10) in urine, bladder, and kidney cultures harvested 48 h after perurethral inoculation of mice, whereas an oxyR-complemented mutant exhibited equal or greater colonizing ability than that of the parent. Although C57BL mice were less susceptible to experimental UTI than CBA/J mice, wild-type and p47phox−/−C57BL mice were similarly susceptible, and the oxyRmutant of Ec1a was similarly attenuated in C57BL mice, regardless of the p47phoxgenotype, as in CBA/J mice. Within the E. coliReference collection, 94% of strains were positive for oxyR. These findings fulfill the second and third of Koch's molecular postulates for oxyRas a candidate virulence-facilitating factor in E. coliand indicate that oxyRis a broadly prevalent potential target for future preventive interventions against UTI due to E. coli. They also suggest that neutrophil phagocyte oxidase is not critical for defense against E. coliUTI and that the major oxidative stresses against which OxyR protects E. coliwithin the host milieu are not phagocyte derived.

ABSTRACTThe role of the Escherichia coliiron-regulated gene homologue adhesin (Iha) in the pathogenesis of urinary tract infections (UTIs) is unknown. We performed a series of complementary analyses to confirm or refute the hypothesis that Iha is a virulence factor in uropathogenic E. coli. Fecal E. coliisolates exhibited significantly lower prevalences of iha(range, 14 to 22%) than did clinical isolates from cases of pediatric cystitis or pyelonephritis, adult pyelonephritis or urosepsis, or bacteremia (range, 38 to 74%). Recombinant Iha from E. colipyelonephritis isolate CFT073 conferred upon nonadherent E. coliORN172 the ability to adhere to cultured T-24 human uroepithelial cells. In a well-established mouse model of ascending UTI, CFT073 and its derivative UPEC76 (a pap[P fimbriae] mutant version of strain CFT073) each significantly outcompeted their respective ihadeletion mutants in CBA/J mice 48 h after bladder challenge (P< 0.03 for urine, both kidneys, and bladders of both constructs, except for bladders of mice challenged with UPEC76 and its deletion mutant, where P= 0.11). These data suggest that IhaCFT073is a virulence factor and might be a target for anti-UTI interventions.

ABSTRACTIsogenic Enterococcus faecalisstrains that differ in their expression of aggregation substance (AS) and its cognate receptor, enterococcal binding substance (EBS), were compared for urovirulence in mice. Strain OG1SSp/pCF500 (inducible AS+, constitutive EBS+) failed to outcompete isogenic derivative INY3000 (AS−EBS−) in the urine, bladders, or kidneys of mice harvested at 48 h postinoculation. Neither mouse nor human urine induced AS expression by OG1SSp/pCF500. Recombinant strain OG1SSp/pINY1801 (constitutive AS+, EBS+) exhibited plasmid segregation that was as extensive in vivo as in vitro. These data suggest that AS and EBS do not contribute to upper or lower urinary tract colonization by E. faecalisand that growth in urine does not induce AS expression by strains carrying plasmids in the pCF10 family.

ABSTRACTWe studied cytokine proteins and mRNAs in mice with two forms ofToxoplasma gondiipneumonia resulting from reactivation of infection. In the first form, mice were infected with T. gondii, developed and recovered from systemic disease, and then developed pneumonia 3 weeks later. As pulmonary inflammation developed, levels of cytokine mRNAs for gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-4, and IL-10 increased in bronchoalveolar lavage (BAL) cells or lung tissue, and the level of IFN-γ protein increased in BAL fluid. The second form of pneumonia occurred as a complication of primary cytomegalovirus (CMV) disease in mice with dormant T. gondiiinfection. During CMV disease, IL-2 mRNA levels decreased in lung tissue, IL-10 protein levels increased in lung tissue, and IL-10 protein levels increased in BAL fluid. As the mice recovered from CMV disease, T. gondiiinfection was reactivated in the lungs and was manifested as T. gondiipneumonia. During CMV-inducedT. gondiipneumonia, IFN-γ, IL-2, IL-4, and IL-10 mRNA levels increased in BAL cells or lung tissue, and both IFN-γ and IL-2 protein levels increased in BAL fluid. We concluded that both forms of T. gondiipneumonia are accompanied by increases in both type 1 T-helper and type 2 T-helper cytokine levels in lungs. The mechanism of CMV-induced reactivation ofT. gondiiinfection in lungs may involve local decreases in IL-2 levels and/or increases in IL-10 levels.