PCR-based detection of DNA from the human pathogen Blastomyces dermatitidis from natural soil samples.

Blastomyces dermatitidis is the dimorphic fungal agent of blastomycosis, a disease that primarily affects humans and dogs. The clinical appearance of this mycosis is well characterized, but there is still little known about its environmental niche, having been isolated from nature only 21 times. We have developed a PCR-based assay to detect B. dermatitidis from soil samples using primers specific to a portion of the promoter region of the BAD1 virulence gene. An internal standard control, pTJV2, was constructed to validate the results from soil samples. Amplification of this control indicated adequate removal of ambient soil inhibitors. The PCR detection limits for the control plasmid and B. dermatitidis genomic DNA were 0.1 and 500 femtograms, respectively. No PCR cross-reactivity was observed against bacteria, actinomycetes, and 13 other fungi that were genetically related or found in the same geographic areas. In spiked soil samples, this method was sensitive to 304 copies of pTJV2 DNA and 8,450 live B. dermatitidis yeast cells. Three of eight natural soil samples from a dog kennel near Lexington, KY in which dogs suffered from blastomycosis were positive using the described method, demonstrating its utility in detecting B. dermatitidis in its natural surroundings.

Differential bacterial survival, replication, and apoptosis-inducing ability of Salmonella serovars within human and murine macrophages.

Salmonella serovars are associated with human diseases that range from mild gastroenteritis to host-disseminated enteric fever. Human infections by Salmonella enterica serovar Typhi can lead to typhoid fever, but this serovar does not typically cause disease in mice or other animals. In contrast, S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, which are usually linked to localized gastroenteritis in humans and some animal species, elicit a systemic infection in mice. To better understand these observations, multiple strains of each of several chosen serovars of Salmonella were tested for the ability in the nonopsonized state to enter, survive, and replicate within human macrophage cells (U937 and elutriated primary cells) compared with murine macrophage cells (J774A.1 and primary peritoneal cells); in addition, death of the infected macrophages was monitored. The serovar Typhimurium strains all demonstrated enhanced survival within J774A.1 cells and murine peritoneal macrophages, compared with the significant, almost 100-fold declines in viable counts noted for serovar Typhi strains. Viable counts for serovar Enteritidis either matched the level of serovar Typhi (J774A. 1 macrophages) or were comparable to counts for serovar Typhimurium (murine peritoneal macrophages). Apoptosis was significantly higher in J774A.1 cells infected with serovar Typhimurium strain LT2 compared to serovar Typhi strain Ty2. On the other hand, serovar Typhi survived at a level up to 100-fold higher in elutriated human macrophages and 2- to 3-fold higher in U937 cells compared to the serovar Typhimurium and Enteritidis strains tested. Despite the differential multiplication of serovar Typhi during infection of U937 cells, serovar Typhi caused significantly less apoptosis than infections with serovar Typhimurium. These observations indicate variability in intramacrophage survival and host cytotoxicity among the various serovars and are the first to show differences in the apoptotic response of distinct Salmonella serovars residing in human macrophage cells. These studies suggest that nonopsonized serovar Typhimurium enters, multiplies within, and causes considerable, acute death of macrophages, leading to a highly virulent infection in mice (resulting in death within 14 days). In striking contrast, nonopsonized serovar Typhi survives silently and chronically within human macrophages, causing little cell death, which allows for intrahost dissemination and typhoid fever (low host mortality). The type of disease associated with any particular serovar of Salmonella is linked to the ability of that serovar both to persist within and to elicit damage in a specific host's macrophage cells.

Impact of the high-affinity proline permease gene (putP) on the virulence of Staphylococcus aureus in experimental endocarditis.

Staphylococcus aureus causes a wide variety of invasive human infections. However, delineation of the genes which are essential for the in vivo survival of this pathogen has not been accomplished to date. Using signature tag mutagenesis techniques and large mutant pool screens, previous investigators identified several major gene classes as candidate essential gene loci for in vivo survival; these include genes for amino acid transporters, oligopeptide transporters, and lantibiotic synthesis (W. R. Schwan, S. N. Coulter, E. Y. W. Ng, M. H. Langhorne, H. D. Ritchie, L. L. Brody, S. Westbrock-Wadman, A. S. Bayer, K. R. Folger, and C. K. Stover, Infect. Immun. 66:567-572, 1998). In this study, we directly compared the virulence of four such isogenic signature tag mutants with that of the parental strain (RN6390) by using a prototypical model of invasive S. aureus infection, experimental endocarditis (IE). The oligonucleotide signature tag (OST) mutant with insertional inactivation of the gene (putP) which encodes the high-affinity transporter for proline uptake exhibited significantly reduced virulence in the IE model across three challenge inocula (10(4) to 10(6) CFU) in terms of achievable intravegetation densities (P, <0.05). The negative impact of putP inactivation on in vivo survival in the IE model was confirmed by simultaneous challenge with the original putP mutant and the parental strain as well as by challenge with a putP mutant in which this genetic inactivation was transduced into a distinct parental strain (S6C). In contrast, inactivation of loci encoding an oligopeptide transporter, a purine repressor, and lantibiotic biosynthesis had no substantial impact on the capacity of OST mutants to survive within IE vegetations. Thus, genes encoding the uptake of essential amino acids may well represent novel targets for new drug development. These data also confirm the utility of the OST technique as an important screening methodology for identifying candidate genes as requisite loci for the in vivo survival of S. aureus.

Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments.

The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.

Physical limitations on Salmonella typhi entry into cultured human intestinal epithelial cells.

Kinetic studies of Salmonella typhi invasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation on S. typhi entry at MOIs of >/=40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously for S. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally contained S. typhi cells by a macropinocytic process, which enhanced the entry of simultaneously added Escherichia coli HB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of approximately 400, maximal S. typhi entry is virtually completed within 30 to 35 min. Monolayers pretreated with S. typhi for 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains of S. typhi or S. typhimurium, but E. coli HB101(pRI203) expressing the cloned Yersinia inv gene was not reduced in entry. In invasion inhibition assays, anti-beta1 integrin antibodies markedly reduced E. coli HB101(pRI203) invasion efficiency but did not reduce S. typhi entry. Collectively, these data provide direct physical and visual evidence which indicates that S. typhi organisms are internalized at a limited number (i.e., two to four) of sites on host cells. S. typhi and S. typhimurium likely share INT407 cell entry receptors which do not appear to be members of the beta1 integrin superfamily.

Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models.

Staphylococcus aureus is an important pathogen of humans and other animals, causing bacteremia, abscesses, endocarditis, and other infectious syndromes. A signature-tagged mutagenesis (STM) system was adapted for use in studying the genes required for in vivo survival of S. aureus. An STM library was ultimately created in S. aureus RN6390, with Tn917 being used to create the transposon mutations. Pools of S. aureus RN6390 mutants were screened in mouse abscess, bacteremia, and wound infection models for growth attenuation after in vivo passage. One of the mutants that was identified displayed marked attenuation following large-pool screening in all three animal models, which was confirmed in bacteremia and endocarditis models of infection with a smaller pool of mutants. Sequence analysis of the entire open reading frame showed a 99% identity to the high-affinity proline permease (putP) gene characterized in another strain of S. aureus. In wound and murine abscess infection models, the putP mutant was approximately 10-fold more attenuated than was wild-type strain RN6390. Another S. aureus strain transduced with the putP mutation also displayed an attenuated phenotype after passage in the wound model. A [3H]proline uptake assay showed that less proline was specifically transported into the putP mutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in the S. aureus high-affinity proline permease suggests that proline scavenging by the bacteria is important for in vivo growth and proliferation and that analogs of proline may serve as potential antistaphylococcal therapeutic agents.

Uptake of pathogenic intracellular bacteria into human and murine macrophages downregulates the eukaryotic 26S protease complex ATPase gene.

A differential PCR technique detected the transcriptional downregulation of the mss1 (mammalian suppressor of svg1) gene in murine J774A.1 macrophages following uptake of Salmonella typhimurium. This downregulation was also noted after entry of virulent strains of Listeria monocytogenes and Shigella flexneri, two other facultative intracellular bacterial species. In contrast, uptake of nonpathogenic Escherichia coli HB101, an aroA mutant of S. typhimurium, an invasion plasmid antigen B (ipaB) mutant of S. flexneri, hemolysin (hly) and positive-regulatory factor (prfA) mutants of L. monocytogenes, or latex beads produced mss1 expression levels similar to that of uninfected macrophages. Transcriptional downregulation of mss1 was also shown to occur in S. typhimurium-infected human U937 cells, albeit to an extent less than that in murine J774A.1 cells. In addition to a lower abundance of mss1 transcripts, we also demonstrate for the first time that less MSS1 protein was detected in intracellular-bacterium-infected cells (beginning about 1 h after entry of the pathogenic intracellular bacteria) than in noninfected cells. Some strains with specific mutations in characterized genes, such as an ipaB mutant strain of S. flexneri and an hly mutant strain of L. monocytogenes, did not elicit this lower level of expression of MSS1 protein. The decrease in MSS1 within infected macrophages resulted in an accumulation of ubiquitinated proteins, substrates for MSS1. Since MSS1 comprises the ATPase part of the 26S protease that degrades ubiquitinated proteins, we hypothesize that downregulation of the mss1 gene by intracellular bacterial entry may help subvert the host cell's normal defensive response to internalized bacteria, allowing the intracellular bacteria to survive.

Transcriptional downregulation of a mouse Golgi mannosidase gene following Salmonella typhimurium uptake into murine macrophages.

Uptake of Salmonella typhimurium into murine macrophage cells caused transcriptional downregulation of a mRNA population in eukaryotic cells after 2 h of infection. The cloned cDNA that was identified showed extensive homology with a murine alpha 1,2-mannosidase gene involved in N-oligosaccharide processing. These findings suggest a disruption of eukaryotic protein processing after phagocytosis of S. typhimurium by the murine macrophage cells.

Detection and characterization by differential PCR of host eukaryotic cell genes differentially transcribed following uptake of intracellular bacteria.

Host eukaryotic cell genes that are differentially transcribed after phagocytosis of various pathogenic and nonpathogenic bacterial cells were identified by a differential PCR (DPCR) system. This DPCR procedure favors detection and isolation of host genes affected at the transcriptional level by selecting for poly(A) tails but differs substantially from reverse transcription-PCR. Several unidentified macrophage gene fragments from genes that were either transcriptionally activated or downregulated following uptake of Listeria monocytogenes into J774 mouse macrophage cells were initially defined by this DPCR procedure. Because of the sensitivity of the DPCR technique, all of the genes exhibited less than a 10-fold difference in transcription compared with noninfected cells as measured by limiting-dilution PCR. One of the gene fragments has a very high level of homology with a mitogen-activated protein kinase phosphatase (MKP-1), whereas the other affected fragments showed no homologies to known gene sequences. In addition, one of the gene fragments (WS30-B2/1) was specifically downregulated after L. monocytogenes uptake and another gene was repressed by uptake of either Shigella flexneri or L. monocytogenes, while transcription of the genes represented by fragment WS13-B9/9, and to some extent MKP-1, was activated following general phagocytosis (i.e., following uptake of any species of bacterium tested). Further characterization of the affected genes was conducted by using mutants of L. monocytogenes. A hemolysin-negative mutant of L. monocytogenes failed to elicit transcriptional regulation of gene fragment WS10-B4/14 or WS30-B2/1, and it elicited only minimal regulation of MKP-1, suggesting that escape from the phagosome may be required to initiate these responses. Furthermore, mutants with mutations in mpl and actA, two genes whose gene products are involved in actin polymerization and intrahost spread, also did not induce regulation of WS10-B4/14. These results demonstrate that (i) DPCR can identify specific host cell genes which are differentially transcribed after infection with certain microorganisms and (ii) some of these genes may be new or may never before have been linked to interactions between hosts and pathogens.

Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua.

Listeria monocytogenes is a facultative intracellular organism that is capable of replicating within macrophage and macrophage-like cells. The species secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) encoded by the plcA gene. A plcA gene from L. monocytogenes was cloned downstream of a gram-positive promoter in the plasmid pWS2-2. To determine what effect plcA would have on intracellular survival when introduced into Listeria innocua, a species that does not growth intracellularly or contain plcA, transformation with the recombinant pWS2-2 plasmid was performed. Phospholipase C activity in Listeria innocua/pWS2-2 was confirmed on a brain heart infusion-phosphatidylinositol agar plate, whereas wild-type L. innocua did not produce PI-PLC activity. Intracellular growth of L. innocua/pWS2-2 was subsequently measured in the macrophage-like cell line J774 by Giemsa staining and viable count determinations at specific time points following infection. The J774 cells infected with wild-type L. innocua showed a falling viable count through 8 h postinfection. Although J774 cells infected with L. innocua/pWS2-2 also initially displayed reduced viable counts, the viable count rose after 6 h postinfection and increased further at 8 h postinfection before a subsequent decline again at 16 h postinfection. Giemsa staining revealed fewer than 6 bacteria in individual macrophage cells at 2 h postinfection, and yet approximately 15% of the J774 cells had 6 to 12 bacteria localized to one area of the macrophage cell after 6 h; moreover, electron micrographs showed that the L. innocua/pWS2-2 cells were replicating inside the phagosome of the host cell. Furthermore, Thoria Sol labeling demonstrated that lysosomes had fused with these phagosomes, and acridine orange staining revealed that the compartments were acidified. These results demonstrate that L. innocua cells transformed with the plasmid-borne plcA gene, and expressing functional PI-PLC, are able to grow intracellularly in what appear to be phagolysosomes, although between 3 and 6 h is needed for this to manifest itself. Intracellular growth specifically in L. innocua may be a secondary function associated with the plcA gene product. The addition of this one gene, plcA, to a species of Listeria that in the wild-type state does not replicate intracellularly apparently can now allow some of the bacteria to transiently multiply inside the phagosomes of host macrophage cells.

Host cell responses to Listeria monocytogenes infection include differential transcription of host stress genes involved in signal transduction.

We examined the effect of Listeria monocytogenes infection of J774 macrophage-like mouse cells on induction of several stress genes, including genes for heat shock proteins (HSPs) and a protein-tyrosine phosphatase (PTP), to understand the host response in various steps of the bacterial invasion process. Exposure to wild-type L. monocytogenes strain EGD elicited an early induction of HSP70 mRNA with a corresponding early appearance of HSP70 protein. Cytochalasin D pretreatment prevented the induction of HSP70 mRNA in L. monocytogenes-infected macrophages. After a 2-hr infection with L. monocytogenes, PTP and to a lesser extent HSP90 mRNA levels were elevated. A listeriolysin-negative mutant of L. monocytogenes strain EGD and a noninvasive species of Listeria, Listeria innocua, did not induce PTP or HSP90 mRNA in infected macrophages. Mutations in other virulence genes did not affect transcription of PTP or HSP90. Expression of HSP60 mRNA remained constant over the time course studied in wild-type or mutant strains. These results suggest that phagocytosis of L. monocytogenes triggers transcription of HSP70 mRNA in macrophages; however, escape from the phagosome appears to be necessary for induction of PTP and HSP90 mRNA. Since both PTP and HSP90 may have links with signal transduction pathways in eukaryotic cells, the induction of these mRNAs suggests a role for L. monocytogenes in influencing the signal transduction routes of the host cell.

Analysis of the fimB promoter region involved in type 1 pilus phase variation in Escherichia coli.

Many strains of Escherichia coli express type 1 pili which undergo phase variation during growth in broth, but become uniformly nonpiliated when passaged on agar media. In a previous analysis of these agar phase locked strains, we demonstrated that fimB and fimE, which mediate the site-specific DNA rearrangement involved in phase variation, are differentially transcribed under the two growth conditions. In this study the fimB promoter region was sequenced and characterized in agar phase locked strain J96. Primer extension analysis of the fimB gene identified three putative transcription initiation sites. Transcription starting from two of the sites (P1 and P2) would produce an mRNA that approximates the transcript size of fimB previously detected by Northern blot hybridizations. Gel mobility shift studies revealed the presence of promoter binding activity in broth-grown cell extracts, but not in extracts from agar-grown cells, that reacted specifically with DNA fragments upstream of the P1 and P2 promoter regions. This putative binding protein may be involved in the regulation of fimB, perhaps by acting as a transcriptional activator.

Growth conditions mediate differential transcription of fim genes involved in phase variation of type 1 pili.

Type 1 pili in Escherichia coli undergo phase variation in which individual cells in a population reversibly switch between piliated (Pil+) and nonpiliated (Pil-) states. The switching process is mediated by an invertible DNA fragment which contains the promoter for fimA, the gene encoding the major structural subunit of type 1 pili. Although type 1 pili randomly phase vary in broth cultures, many clinical isolates of E. coli do not express type 1 pili when cultured on agar media. We investigated the role of the invertible element and the upstream genes, fimB and fimE, in the agar-mediated suppression of pili in an agar-negative clinical isolate, strain 149. Southern hybridization and polymerase chain reaction analyses of the fimA promoter region in broth-grown 149 cells indicated that the invertible element was present in orientations corresponding to both Pil+ and Pil- phenotypes. In contrast, only one orientation of the invertible element, corresponding to the Pil- phenotype, was observed in strain 149 cells cultured on agar. A second clinical isolate, strain 2-7, which expresses type 1 pili on agar was also examined; the invertible element was found in both the Pil+ and Pil- orientations during growth of this strain on agar as well as in broth. The introduction of the fim gene cluster from strain J96 on a multicopy plasmid into agar-negative strain 149 resulted in the production of both J96 and 149 pili during growth on agar. Experiments with subclones of the J96 genes indicated that the presence of an intact fimB gene allowed strain 149 pili to be produced on agar. Differences in pilus production between agar and broth cultures appear to be the result of differential transcription of fimB and fimE under the two growth conditions. In contrast, the pattern of expression of these genes in agar phase-variable strain 2-7 did not differ between broth- and agar-grown cells.

Bacteria as solid phase in a concentration fluorescence immunoassay analysis of antibodies to surface antigens.

A modified solid-phase fluorescence immunoassay was developed using bacterial cells as the solid phase to screen antibodies produced against surface antigens from a clinical isolate of Escherichia coli, strain 1-149. The bacterial solid phase was used to analyze both polyclonal and monoclonal antibodies. The bacterial concentration fluorescence immunoassay (BCFIA) showed up to 50-fold greater sensitivity in bacterial cell detection as compared to ELISA (enzyme-linked immunosorbent assay). Moreover, BCFIA was considerably faster than ELISA with uniform reproducibility. This paper demonstrates the utility of using bacteria and their surface antigens as solid-phase matrices for antibody characterization in a FIA.

In vivo expression and variation of Escherichia coli type 1 and P pili in the urine of adults with acute urinary tract infections.

In vivo expression of pili by Escherichia coli in the urine of 41 adults with lower urinary tract infections was analyzed by immunostaining with polyclonal antiserum to type 1 and P pili. Type 1 pili were detected in 31 of 41 urine specimens, while P pili were detected in 6 of 18 specimens. The piliation status of bacterial populations in urine was heterogeneous, varying from predominantly piliated to a mixture of piliated and nonpiliated cells. Bacteria frequently adhered to exfoliated uroepithelial cells and leukocytes in urine. Expression of pili in vivo did not always correlate with the hemagglutination phenotype after growth in vitro. Strains isolated from different sites in the urogenital tract of two individuals showed phenotypic variation in the state of piliation. The results demonstrate that E. coli type 1 and P pili are expressed and are subject to variation in vivo during acute urinary tract infections in adults.

Role of Escherichia coli adhesins in urethral colonization of catheterized patients.

Escherichia coli is a major cause of catheter-associated urinary tract infection and frequently colonizes the urethra prior to invading the urinary tract. Bacterial pili, filamentous protein cell surface-associated appendages, have been shown to mediate colonization of epithelial cells. Pili associated adhesins can be detected in vitro by their ability to mediate bacterial hemagglutination of erythrocytes. We have assessed the role of bacterial adhesins in supporting urethral colonization by determining the hemagglutination reactions of 56 E. coli isolates from the urethra of patients with indwelling urethral catheters. The adhesin detected most frequently was the type 1 mannose-sensitive hemagglutinin (43%), and 43% of isolates failed to hemagglutinate guinea pig or human erythrocytes. E. coli hemagglutinins were no more common on urethral isolates from patients that were persistently colonized (E. coli present 70% of the time catheterized), than from those that were transiently colonized (E. coli present 30% of the time catheterized). Analysis of the HA reactions and DNA plasmid profiles of multiple isolates from persistently colonized patients suggested that the E. coli strain colonizing the urethra changed over time. The data suggest that bacterial colonization of the urethra is mediated in part by adhesins and changes over time.

Relationship of type 1 pilus expression in Escherichia coli to ascending urinary tract infections in mice.

The role of type 1 pili and P adhesins during the in vivo growth of Escherichia coli inoculated into the urethras of BALB/c mice was studied. Strains which produced type 1 pili when grown in broth but lost this trait when grown on agar (regulated variants) were tested. Broth-grown organisms colonized the bladder of every animal tested, with counts of 10(3) to 10(4) viable organisms recovered from bladder homogenates. Agar-grown organisms gave lower rates of infection and the number of viable organisms recovered from bladders was significantly reduced. The degree of inoculum piliation influenced bladder colonization in a direct way: as piliation increased, the number of bacteria recovered from bladders also increased. After intraurethral inoculation, all of the bladders and 44% of the kidneys were colonized on day 1, and by day 5, 94% of the bladders and 16% of the kidneys were positive. Hemagglutination titers remained high for the bladder isolates, but the organisms colonizing the kidneys became significantly less piliated with time. Bacteriuria was unrelated to bladder or renal colonization. Strains that demonstrated random phase variation of type 1 pili during growth on agar produced similar colonizations of the urinary tract with broth- and agar-grown inocula. Strains that produced only P adhesins were less effective in colonizing the urinary tract than were type 1 piliated organisms. Other strains which did not produce pili only minimally colonized the bladder. The results suggest that type 1 pili play an essential role in ascending infections of the urinary tract.

Regulation of production of type 1 pili among urinary tract isolates of Escherichia coli.

The piliation and hemagglutination properties of 54 consecutive Escherichia coli isolates from women with recurrent urinary tract infections were studied. Mannose-sensitive hemagglutination (MSHA) of guinea pig erythrocytes, characteristic of type 1-piliated bacteria, was produced by 75% of the isolates, 32% produced mannose-insensitive hemagglutination, and 14% produced no hemagglutination reaction. The production of type 1 pili was examined in those strains that produced MSHA only. Studies with antiserum prepared against purified pili suggested that at least three subtypes of type 1 hemagglutinins were represented among the isolates. All of the type 1-piliated isolates produced MSHA after serial subculture in static broth. After growth on agar, selected type 1-piliated isolates were subdivided into two groups. Many strains apparently suppressed piliation during growth on agar (regulated variants); all colonies became MSHA negative and were composed of nonpiliated cells as shown by electron microscopy. The loss of the MSHA phenotype often occurred after a single overnight passage on agar, and any remaining hemagglutinin was gradually lost with one to three additional passages. Seven strains, however, retained a significant hemagglutination titer after multiple subcultures on agar, and they produced colonies consisting of a mixed population of piliated and nonpiliated cells. These strains were apparently able to oscillate between states of pilus expression and nonexpression during growth on agar (random phase variants). When nonpiliated cells isolated from the mixed, random variant population were plated on agar, they gave rise to hemagglutination-positive colonies that consisted of both piliated and nonpiliated cells. The distinction between random variants and regulated variants was also observed in shaking broth cultures inoculated with nonpiliated cells. The random variants produced MSHA-positive cultures composed of piliated and nonpiliated cells, whereas the regulated strains remained nonpiliated. The results indicate that type 1 pili are a predominant adhesin of uropathogenic E. coli and that during growth on agar only about one-fourth of the type 1-piliated isolates regulate pilus expression by random phase variation.