BACTERIAL VIRULENCE IN URINARY TRACT INFECTION

URINARY TRACT INFECTIONS

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BACTERIAL VIRULENCE IN URINARY TRACT INFECTION Catharina Svanborg, MD, PhD, and Gabriela Godaly

Bacteriuria is associated with acute disease conditions of varying severity.48, Consequently, the definition of bacterial virulence for the urinary tract depends on the end-point chosen. Acute pyelonephritis is characterized by fever, flank pain, and general malaise. Laboratory findings include pyuria (leukocytes in the urine), elevated acute phase reactants in serum (C reactive protein [CRP], erythrocyte sedimentation rate [ESR]), elevated levels of cytokines in serum and urine, and later increased levels of antibacterial IgA antibodies in urine and serum. Cystitis is characterized by dysuria, urgency, frequency of urination, and sometimes suprapubic pain. Acute cystitis should not be accompanied by acute phase reactants or cytokines in the serum, however, there is pyuria, and IL-6 and IL-8 levels in urine may be elevated. Asymptomatic bacteriuria (ABU) is commonly detected at screening because it is not accompanied by any of the symptoms seen for acute pyelonephritis and acute cystitis. The laboratory findings vary. The patients may have low level cytokine responses and leukocytes in urine, or they may have no host response to infection.", 6y THE PATHOGENESIS OF UTI: AN OVERVIEW

Urinary tract infections (UTIs) are caused by a variety of gram-negative bacteria that ascend into the urinary tract and establish bacteriuria often at levels greater than or equal to lo" bacteria/mL of urine. Escherickia coli dominate as the causative agent in all patient groups. The second most common agent is Staphylococcus saprophyticus, accounting for about 10% to 30% of the infections in young adult females depending on the season. Other members of the Enterobacteriaceae, such as Klebsiella sp, Proteus sp, Enterobacter, and Psuedornonas sp From the Section of Clinical Immunology, Department of Medical Microbiology, Lund University, Lund, Sweden

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are a common cause of UTI in patients with reduced resistance to infection because of a variety of complicating disease conditions. The pathogenesis of UTI starts when a uropathogenic E. coli clone becomes established in the host at a site outside the urinary tract (often the large intestine).’” 32, 9y Colonization at this site is facilitated, in part, by the same bacterial components that make the bacteria virulent for the urinary tract. The bacteria then spread to the urinary tract and establish bacteriuria; this phase is counteracted by the urine flow, by secreted receptor analogues that trap some fimbriated bacteria, and by bactericidal molecules in the urine and mucosa. Uropathogenic clones overcome these defenses and target the mucosa in the kidneys, the bladder, or other parts of the urinary tract in which they elicit an inflammatory response. The bacteria stimulate epithelial and other cells to produce cytokines and proinflammatory factors?,”, ?K The systemic spread of cytokines, such as interleukin-6 (IL-6), may lead to fever and the activation of the acute phase response. The chemotactic cytokines, like interleukin-8 (IL-8), recruit polymorphonuclear granulocytes (PMNs) to the mucosal surface, and in parallel bacteriuria is cleared. A specific immune response to the infection follows thereafter. In about 30% of patients with acute pyelonephritis, bacteria invade through the mucosa into the bloodstream and cause bacteremia. The magnitude and localization of this inflammatory response determines many features of UTLZZ, Patients with acute pyelonephritis have inflammation of the renal pelvis and kidneys combined with generalized inflammation (fever, CRP, leukocytes). The inflammatory response influences the outcome of infection in terms of bacterial clearance from the kidneys.*,IZ1, I z 6 Although the exact mechanisms are poorly understood, it is known that defects of acute inflammation render animals hypersusceptible to experimental UTI. Imbalance of inflammation causes defective elimination of microbial components from the tissues and probably contributes to the development of renal scarring and other chronic changes in the urinary tract.*,33 The pathogenesis of acute cystitis is less well understood. Patients with acute cystitis often have inflammation restricted to the lower urinary tract.62The spread to the urinary tract and the establishment of bacteriuria presumably occurs by similar mechanisms as in pyelonephritis. There are, however, no bacterial parameters that identify “cystitogenic” E. coli clones or distinguish them from strains that cause acute pyelonephritis, although hemolysin, type 1 fimbriae, and the prsGIqhtype of P fimbriae have been proposed to occur more often in acute cystitis than in other E. coli strains.”, 34, The authors believe that the clinical syndrome of acute cystitis reflects other host response mechanisms than acute pyelonephritis and that cystitis prone individuals have a local accumulation of special inflammatory cells (e.g., mast cells) in the bladder mucosa. Patients with ABU may show signs of local inflammation in the urinary tract, but the magnitude is not sufficient to make them symptomatic.56, 71 ABU may either be the consequence of bacterial attenuation by the host or a primary condition in which bacteria of low virulence stably colonize the urinary tract without activating a sufficient host response to cause symptoms. The bacterial factors that explain this colonization, the host defects permitting it to occur, and the basis for the unresponsiveness of the colonized patients are unknown. THE INFLAMMATORY RESPONSE TO UTI The molecular mechanisms that regulate the inflammatory response have only recently become the focus of study.ZZ-Z4 The information about the bacterial

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properties involved in this process, the nature of the inflammatory mediators, and the functional consequences of the response is quite limited. It is clear, however, that bacteria directly activate cells in the urinary tract to produce inflammatory mediators that, in turn, activate the inflammatory cascades. Cytokines are among the early mediators of this response.36 UTI is accompanied by a cytokine response in the infected host. This was first demonstrated in mice with experimental UTIZ4and in patients who had been colonized with E. coli in the urinary 37 Cytokine responses subsequently have been shown to occur in children and adults with different forms of UTI.", 39,5y, y7, 12* IL-6 was found in the urine of children with febrile UTI but not in children with fever of another origin or in children with ABU." The highest IL-6 urine and serum levels were detected in children with reflux and renal scarring1' In adults, IL-6 was detected in the urine of patients with acute pyelonephritis and ABU.37 UTI also leads to the induction of the chemokine IL-8. Patients deliberately colonized with E. coli in the urinary tract secreted IL-8 into the urine within hours of colonization but serum IL-8 was not d e t e ~ t e d .In ~ studies of acute pyelonephritis or ABU, IL-8 levels were elevated in the urine of patients with p y ~ r i aIt. ~was ~ also shown in this study that urinary IL-8 levels were elevated in most patients with UTI regardless of the bacterial species causing infection. Studies conducted in children with febrile UTI or ABU showed that the frequency of IL-8 responders was higher in children with febrile UTI than in children with ABU.IZThe apparent correlation between bacteriuria and pyuria initiated discussion of the usefulness of urine IL-8 measurements to separate "relevant" from "irrelevant" bacteriuria. Cellular Origin of Urinary Cytokines

The uninfected urinary tract mucosa is dominated by epithelial cells that secrete cytokines when suitably stimulated. Epithelial cell lines and nontransformed cells from the human urinary tract produce cytokines when exposed to bacteria. Uropathogenic E. coli stimulate de novo synthesis of interleukin-la (ILl a ) ,interleukin-1P (IL-lP), IL-6, and IL-8 but not tumor necrosis factor a (TNFa) as deduced from the cytokine specific mRNA. There is an increase in intracellular IL-la, IL-6, and IL-8 as detected by immunofluorescence. IL-6 and IL-8 are secreted by the cells but not IL-la, IL-16, and TNFa.?,4, 38 On the basis of these observations, the authors have proposed that epithelial cells are a source of cytokines during the early stages of infection and that the repertoire and magnitude of the epithelial cytokine response influence the subsequent activation of mucosal inflammation and immunity. The epithelial cytokine response is influenced by the properties of the uropathogenic bacteria that activate the cells (see later discussion). Adherence through type 1 and P fimbriae enhances the response." 4", 41, '16 Lipopolysaccharide (LPS) is likely to play a role for cytokine activation, although not in the same manner as for macrophages. LPS alone is a poor stimulant of epithelial cell cytokine responses.'", 4(3,112 This may be because epithelial cells lack CD-14 or other LPS receptor molecules required for the binding of LPS and the activation of cells."" The influx of PMNs, T cells, and other inflammatory cells occurs subsequent to the primary interaction of bacteria with the mucosa. These cells may be activated by bacteria, or by epithelial cytokines. For example, bacteria stimulate PMNs to secrete IL-8 and lymphocytes to secrete immunoregulatory cytokines

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that modify the epithelial cell responses to bacteria (see later discussion). Inflammatory cytokines, such as IL-1 and TNF, induce urinary tract epithelial cell IL-6 and IL-8 secretion, and immunoregulatory cytokines modify bacterially induced cytokine responses. Thus, the production of cytokines by influxing cells may influence epithelial cytokine responses. Epithelial cell cytokines may in turn affect lymphocyte activities. This type of communication between mucosal cells forms the basis of a mucosal cytokine network. Mechanisms of Neutrophil Influx into the Urinary Tract

Pyuria is one of the classical signs of UTI. The mechanisms of neutrophil recruitment to the urinary tract and the interaction of PMNs with cells at the mucosa have only recently been examined. The influx of neutrophils during UTI involves the generation of a chemotactic gradient from the urinary tract mucosa, adherence of neutrophils to the endothelial vessel wall, their extravasation into the lamina propria, and movement to the epithelial barrier. Finally, neutrophils cross the urinary tract epithelium into the urine. Epithelial cells were recently shown to participate in neutrophil recruitment in two ways: (1) by secreting neutrophil chemoattractants (IL-8): 31, lz6 and (2) by expression of adhesion molecules involved in neutrophil transmigration (ICAM-1).6 Role of Inflammation for the Resistance Against UTI

Previous studies of host resistance to UTI have focused on specific immunity and the development of UTI vaccines. Inflammation was proposed to be detrimental to the integrity of the mucosal barriers. These concepts are contradicated by studies on inflammation and resistance to experimental UTI in murine models. The authors found that C3H/HeJ and C57BL/lOScCr mice are more susceptible to experimental UTI than their normal counterparts C3H/HeN and C57BL/ 6J.5,3, lZh The susceptible mice are LPS hyporesponders because of an alteration of the Ips locus on chromosome IV. Mice with this defect were more susceptible to UTI and had significantly lower neutrophil and cytokine responses to experimental UTI than LPS responder mice. The neutrophil influx in the LPS responder mice coincided with the clearance of infection. These observations suggest that the inflammatory response was essential for clearance of bacteria from the urinary tract. Furthermore, the treatment of mice and rats with anti-inflammatory agents severely reduced the clearance of infection, and treatment with antineutrophil serum led to a 1000-fold increase of bacteria in the kidneys.”, 84 At present, it is not known how bacteria are killed and cleared from the urinary tract. Many cellular (PMNs, macrophages) or secreted components (defensins, NO) of the inflammatory response are likely to contribute in this process. VIRULENCE FACTORS OF UROPATHOGENIC E. COD

The E. coli strains that cause acute pyelonephritis are a selected subset of the fecal E. coli flora. E. coli causing extraintestinal infections were first noted to have increased lethality for mice and to produce hemolysin more frequently than other fecal ~trains.5~ With the advent of serotyping, the extraintestinal isolates were shown to express a limited number of O K H antigen corn bin at ion^.^^, 8o Subsequently, these clones have been shown to possess an

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array of virulence factors that contribute to different stages in pathogenesis; they include adherence factors (P, type 1, S, Dr fimbriae); toxins (LPS, hemolysin); 11*, aerobactin; invasion factors, and serum

Bacterial Adherence

Uropathogenic E. coli colonize several mucosal sites in the UTI prone host.”, 117 Adherence mechanisms are thought to influence tissue targeting establishment and persistence. The influence of adherence mechanisms on colonization of uropathogens has been studied in the large intestine, the vaginal/introital and preputial areas, and in the urinary tract per se. Attachment is also a way for pathogens to attack the host mucosal tissues.22,3h, 74 Attachment results in the activation of receptor-bearing target cells in the urinary tract mucosa, kidney tissue, and in the systemic compartment. Attachment is regulated through specific interactions between bacterial surface components (adhesins) and host cell receptors.hsThe adhesins of uropathogenic E. coli exist either as filamentous surface organelles, termed pili or fimbriae or as nonfilamentous proteins in the outer membrane.lS,2h, 2K The covariation of fimbriation and adherence was noted earlylZnand purified fimbriae were shown to retain the binding properties of whole fimbriated bacteria.60,R6, 117 The fimbriae-associated adhesins of uropathogenic E. coli are lectins (carboh5 hydrate binding proteins) that recognize a variety of host cell glycoconj~gates.~~ Uropathogenic E. coli lectins associated with P fimbriae recognize epitopes within the globoseries of g l y c ~ l i p i d sh4,. ~6h,~7h~ Lectins associated with S fimbriae recognize glycoconjugates with sialic acid residues, and type 1 fimbriae bind mannose residues on the Tamm-Horsfall protein, secretory IgA, and fibronec&IS,

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P Fimbriae

The symbol P was chosen for two reasons: (1) P fimbriated E. coli are a frequent cause of acute pyelonephritis, and ( 2 ) the globoseries of glycolipids are as receptors for P fimbriae and antigens in the P blood group system. Three main approaches were taken to demonstrate that the receptor activity of epithelial cells resided in the globoseries of glycolipids: (1) Attachment was inhibited by pretreatment of E. coli with those glycolipids; (2) the glycolipids were shown to act as receptors when coated to an inert surface or a cell that lacked receptor-active glycolipids; and (3) attachment was P blood group dependent. Cells from individuals of blood group p, who lack the globoseries of glycolipids, did not interact with P fimbriated E. ~ ~ l i . ~ ~ , ~ ~ , ~ ~ The organization of the fimbrial organelle has been elucidated through studies of the chromosomal DNA sequences that encode these structure^.^^, 73 The P fimbriae are encoded by the pap chromosomal gene cluster. papA encodes the major fimbrilin subunits,ln and papE, papF, and papG sequences encode the adhesin complex.18,42, 55, l3I1 The adhesin complex papE-papG is located at the tip of the f i m b ~ i a e . ~papF ~ , ’ ~is the adaptor-initiator for the tip structure incorporating the subunits papE and papGjS papC has been proposed to form a pore through which the pilus is assembled.”, x8 papD acts as the chaperone through the h’ periplasmic space of the preassembly complexes of the various papH terminates fimbrial assembly and helps anchor the fimbriaeA6,lz7 The papJ

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gene product has been suggested to facilitate the assembly of papA subunits into the pilus s t r u c t ~ r e . The ' ~ ~ papK gene product has been suggested to be a pilinlike protein located at the pilus tip.Iz7papB and pap1 encode regulatory proteins involved in papA transcripti~n.~~, The receptor activity of different members of the globoseries of glycolipids varies with the P fimbrial subtype.", 76. 7K, 'I5The P fimbriae may be described as a family of adhesins that recognize the globoseries of glycolipids as receptors but differ in the preferred receptor epitope. The G adhesins of the papGIA,type dominate in the virulent E. coli strains that cause acute pyelonephritis.z'),5" Their adhesins recognize most members of the globoseries of glycolipids but appear to prefer globotetraosylceramide.4y~ '15 The G adhesins of the ~ Y s G type , ~ are less prevalent among clinical isolates and show no clear-cut association with disease, except possibly acute cystitis."" These adhesins prefer a receptor epitope composed of an N-acetyl-galactosamine a-linked to a globoseries core.55,76, '15 Although these adhesins also recognize globotetraosylceramide on thin-layer chromatogram plates, their binding to uroepithelial cells requires the Forssmann or globo-A glycolipids.", 77

P Fimbriae and the Severity of UTI Early studies compared the in vitro adherence to uroepithelial cells between E. coli strains from patients with different forms of UTI.lZSAttaching strains were found in about 80% of patients with acute pyelonephritis, 40% to 50% with acute cystitis, and about 20% of strains from ABU or the fecal flora of healthy carriers.lz3Subsequent to the identification of P fimbriae as the main adherence factors, the expression of P fimbriae was analyzed in different clinical isolates. P fimbriated E. coli are prevalent in patients with acute pyelonephritis, especially in children and women without predisposing disorders of the urinary tract21,05. 1U4, 113 but not in men. In contrast, P fimbriae are expressed by a minority of ABU isolates and acute cystitis strains form an intermediary group.21,9x P fimbriated E. coli dominate as a cause of urosepsis. Johnson et a1 found that up to 100% of the blood isolates from patients with uncomplicated urosepsis were P fimbriated.sl In contrast, only 5oyOof patients with urosepsis and complicating factors were infected with P fimbriated E. coli. These observations have been confirmed and extended by several gr0ups.9~ Probes specific for different regions of the pap gene cluster have been developed and used for molecular e p i d e m i ~ l o g y .Hybridization ~~ studies showed that pap positive strains were frequent in acute pyelonephritis.y,zy, y8-1uu Southern blot analysis showed that the pyelonephritis strains often carried two or three copies of the pap gene cluster. Multiple copies of pap were significantly less frequent among strains from other diagnostic groups. This suggested that pyelonephritis strains may vary the quantity or quality of P fimbrial expression during infection.gK The hybridization studies also showed that the pap DNA sequences occurred more frequently in ABU strains than in fecal isolates from healthy carriers?, zy, y4, y5, yR-lnO About 60% of the ABU strains were pap positive compared to 20% or less of the fecal strains. This was in contrast to earlier phenotypic studies that showed P fimbriae were expressed by less than 20% of ABU strains and fecal The hybridization studies, therefore, suggested that the pap DNA strains.12",IZ5 sequences characterize E. coli strains that cause UTI, regardless of the type of UTLYR, lo" It may be speculated that the ABU strains express P fimbriae during

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the early stages of infection but down-regulate the expression to persist in the urinary tract. Probes that are highly specific for the three known G adhesin classes (papGIAZ,p~pG,,~, prsGJ9J have provided additional i n f ~ r m a t i o n The . ~ ~ papGI, DNA sequences were found to be rare; the papG,,: probes did not hybridize probe hybridized with most acute pyelowith other clinical isolates. The papGrA2 nephritis isolates and blood isolates from patients with s e p t i ~ e m i aThe . ~ ~ prsG,,, probe hybridized with 36% of acute cystitis strains, but prsGIw,homologous sequences were less frequent in acute pyelonephritis (15%) and ABU strains (14%).su The G adhesin specific probes, therefore, seem to resolve relevant subgroups among P fimbriated E. coli. Functions of P Fimbriae in Pathogenesis Colonization of the Large Intestine

Receptors for P and type 1 fimbriae are expressed on human colonic epithelial cell lines and on cells derived from surgical specimens.’”, 133 P or type 1 fimbriae E. coli attach to these cells in vitro. In a prospective study of the fecal flora in UTI prone children, P fimbriated E. cob were shown to become resident and to persist longer in the large intestine than other E. coli Spread to the Urinary Tract

The tendency of E. coli in the fecal flora to cause UTI was compared between Most of the children with strains with or without the pap DNA UTI carried both pap positive and pap negative strains in their fecal flora. The pap positive E. coli strains were subsequently recovered from the urinary tract of the children with UTI. Pap negative fecal strains were only recovered from the urinary tract of children in whom no pup positive fecal strain was detected. Consequently, pap negative strains were not unable to colonize the urinary tract but were outcompeted by pap positive strains when present in the fecal flora.” Persistence in the Urinary Tract

The role of P fimbriae for the persistence of E. coli in the urinary tract remains unclear. Studies in animal models have shown that P fimbriated E. coli persist in kidneys and bladders better than isogenic strains lacking the pap DNA sequences.”, 9”, The observations in patients with UTI have provided conflicting evidence. Persisting bacteriuria is mainly found in patients with ABU. If left untreated, Their strains these patients may carry the same E. coli strain for several rarely express P fimbriae (less than 20%) suggesting that P fimbriae are not required for persistence of bacteria in the urinary tract.35,y8, lZ2,124 Colonization studies in humans with bladder disorders confirmed this hypothesis. Patients were infected with a nonvirulent E. coli strain lacking adherence factors and with transformants of this strain expressing P fimbriae (196) or The P fimbriated and type 1 fimbriated isogens were eliminated type 1firnbrk~e.~ more rapidly from the human urinary tract than the nonfimbriated clinical isolate. Elimination of bacteriuria coincided with an inflammatory response. These observations suggested that the persistence of E. coli in patients with voiding disorders did not require bacterial adherence but that adherence triggered a host response that eliminated the adhering nonvirulent strains.

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P fimbriae Enhance the Inflammatory Response in the Urinary Tract The role of bacterial adherence for the cytokine response was investigated by comparing recombinant E. coli strains expressing different fimbriae.74In addition, isolated P and S fimbriae with and without the receptor binding domain were used as stimulants. Adhering bacteria and adhesin-positive P fimbriae stimulated cells to secrete significantly more IL-6 than nonadhering bacteria or adhesin-negative P fimb~-iae.~, 4, 4", 'Ih Treatment of uroepithelial cell lines with PDMP (an inhibitor of ceramide glycosylation) reduced receptor expression, bacterial attachment, and cytokine activation.11hThe transmembrane signaling pathway was recently shown to involve the release of ceramide by cell membrane glycolipids.41 Type 1 Fimbriae

Type 1 or mannose-sensitive (MS) fimbrial adhesins recognize mannosecontaining receptors. Their binding is blocked by solutions of D-mannose or amethyl-D-rnanno~ide.~~~~*~ xy Receptors for type 1 fimbriae are present on a variety of cells from many ~pecies.~' Type 1 fimbriae bind mannose epitopes on secreted glycoproteins such as the Tamm-Horsefall protein and secretory IgA.", 132 When these substances coat uroepithelial cells, they may provide receptor epitopes for bacterial surface colonization. When secreted, they may eliminate type 1 fimbriated E. coli strains and prevent colonization or infection.9h The type 1 fimbriae are encoded by the pi1 or firrz gene cluster including nine genes that encode structural and regulatory proteins.4z,58 fimA encodes the fimbrial subunit protein and can be expressed independent of the firnH-encoded adhesin protein.RsThefimA andfirnH gene products must be present on the cells to confer the adhesive phenotype. Functions of Type 1 Fimbriae in Pathogenesis

The fim DNA sequences encoding type 1 fimbriae occur in most clinical isolates, virulent and avirulent. Consequently, there has been little evidence from epidemiologic studies of an association between type 1 fimbriae and the severity of In experimental UTI models, type 1 fimbriae have been proposed to aid in the persistence of E. coli in the urinary tract.x,31, 14, ' O K The addition of the FimH receptor analogue (a-methyl-D-mannoside) to an inoculum of type 1 fimbriated E. coli significantly reduced bacteriuria in mice.x Similarly, anti-type 1 fimbrial antibodies prevented pyelonephritis in a rat model of E. coli induced UTI.lDK A type 1 negative mutant of a wild type uropathogenic strain produced by chemical mutagenesis with nitrosoguanidine survived in significantly lower numbers in the mouse bladder than the type 1-positive parent.34 In contrast, bacterial survival in the human urinary tract was reduced after transformation of a wild type E. coli ABU strain with a plasmid encoding the fim sequence~.~ The authors recently reexamined the role of type 1 fimbriae for E. coli virulence in the urinary tract.'O In a clinical study, the authors observed a type 1 fimbriae-related difference in the severity of infection. Out of 88 children with acute pyelonephritis, 14 carried E. coli of serotype 01:K1:H7.K2The Ol:Kl:H7 E. coli strains were members of the same clone and were P fimbriated but differed in their expression of type 1 fimbriae. The children infected with type 1 positive

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01:Kl:H7 strains were more severely ill than children infected with type 1 negative 01:Kl:H7 isolates or other E. coli, suggesting that it was the expression of type 1 fimbriae by the E. coli 01:Kl:H7 isolates that led to the increased severity of infection in the children. The role of type 1 fimbrial expression for the virulence of the E . coli 01:Kl:H7 clone was further examined in a mouse UTI Type 1-positive isolates showed increased persistence in the kidneys and bladders of the mice and induced a greater inflammatory response than type 1 negative isolates. A role for type 1 mediated adhesion in the virulence of the E. coli 01:Kl:H7 isolate was confirmed with the construction of a fimH null mutant of a type 1 positive E. coli 01:Kl:H7 isolate?" This mutant survived in significantly lower numbers in mouse kidneys and bladders and induced a significantly lower neutrophil influx compared with the fimH-positive parent. The results demonstrate that type 1 fimbriae contribute to virulence in the urinary tract when expressed in the background of a fully virulent uropathogen. LIPOPOLYSACCHARIDE The lipopolysaccharides of gram-negative bacteria consist of a repeating polysaccharide, a core region, and the Lipid A that anchors the molecule in the bacterial outer membrane.'"z The lipopolysaccharide defines the outer surface of the bacteria; loss of the polysaccharide increases the hydrophobicity, with rough colony morphology and autoagglutinability in buffers. In contrast to many smooth bacteria, the rough mutants are susceptible to complement-mediated lysis. Clinical isolates of E. coli show a variable LPS phenotype with intermediate stages between the smooth and rough phenotypes. The lipid A moiety has toxic, inflammatogenic, and immunomodulatory properties. Many symptoms of gram-negative septicemia can be reproduced by administration of isolated lipopolysaccharide or lipid A. LPS binds to certain host cells bearing the CD-14 receptor and elicits an inflammatory response.'o' At present, it is not clear to what extent the LPS induced host response in the urinary tract can explain the symptoms and signs of UTI. The acute stage of symptomatic urinary tract infection may be caused, in part, by LPS presented by attaching bacteria to the mucosal surface.7s Certain 0:K:H serotypes (01KlH7 and OlKlH - , 04K12H1 or 04K12H -, 06K2HN, 07KlH-, 016KlH6, 018K5H-, and 075K5H5 or 075K5H-) are over-represented among E. coli that cause acute pyelonephritis compared with asymptomatic bacteriuria. This has been demonstrated in pediatric and adult populations. The eight 0 antigens 01, 0 2 , 04, 06, 07, 016, 018, and 075 comprise up to 80% of pyelonephritic E. coli. This association is thought to depend on 0 antigen-associated, clonally expressed virulence factors.', 17, loo CAPSULAR POLYSACCHARIDE The polysaccharide capsules are classical virulence factors for bacteria, such as Streptococcus pneumoniae, Haemopkilus influenzae, and Ncisseria meningitidis, thought to act by restricting the access of complement and phagocytes. The same mechanisms have been assumed to apply to uropathogenic E. coli. Encapsulated mutants have a significant advantage in that they resist phagocytosis in vitro and persist in the kidneys in vivo compared with capsule-deficient mutants. This difference is seen in C3H/HeN mice in which bacteria activate an inflammatory

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response but not in C3H/HeJ mice refractory to endotoxin. This supports the contention that the capsule increases the resistance of E. coli to the effectors of inflammation. The K1, K2, K5, K12 and K13 or K51 antigens are overrepresented among acute pyelonephritis strains.* HEMOLYSINS

The hemolysins are cytotoxic proteins detected by their ability to lyse erythrocytes. Hemolysins can be the products of chromosomal genes or encoded by plasmids. The chromosomal localization of the hemolysin genes seems to be prevalent in E. coli causing extraintestinal infections, whereas E. coli strains from veterinary sources carry the hemolysin genes on large plasmids. The chromosomal or extrachromosomal hemolysin genes appear to have a high degree of homology. In E. coli 06:K15, the hemolysin gene was located on a 50 Kb fragment adjacent to the genes encoding sialic acid-specific fimbriae and serum resistance. The hemolysin is toxic for a wide range of cells in addition to erythrocytes, for example, human polymorphonuclear leukocytes, and monocytes in vitro. The E. coli a-hemolysin can also injure isolated renal tubular cells. Hemolysin has been shown to contribute to virulence in intraperitoneal infection models. It remains, however, uncertain at what stage during the ascending UTI process the hemolysin is active. The presence of hemolysin does not enhance bacterial persistence in kidneys and bladders but may aid in causing the mucosal damage that is required for invasive disease. Hemolysin production is overrepresented in the pyelonephritic clones, but the frequency of hemolysin production varies between different geographic areas. In isolates collected during the 1970s in Sweden, hemolytic activity occurred in less than 50% of the pyelonephritic clones. In other European collections and in a more recent collection of febrile UTI isolates from infants with their first episode of acute pyelonephritis, the frequency of hemolytic strains was increased above 50%. Concurrent expression of hemolytic activity and Galoll-4GalP-adhesins proposed from a limited survey of 12 isolates mainly belonging to hemolytic serogroups cannot be taken as epidemiologic evidence for a decisive role of hemolysin in acute pyelonephritis. Rather, the frequency of hemolysin seems to be secondary to the distribution of clones in each population. Thus, adults with acute pyelonephritis had 40% hemolytic E. coli, pregnant women 42%, and patients with acute pyelonephritis and complications such as diabetes mellitus or urinary tract abnormality had 29% hemolytic E . coli. In the same geographic area, children with febrile UTI had 63% hemolytic strains compared with 27?0 in infants with screening bacteriuria. Epidemiologic studies have revealed an association between acute pyelonephritis caused by hemolysin producing E. coli and low blood hernoglobulin levels. Before antibiotics, therapy resistant anemia was observed in a fraction of children with acute pyelonephritis. Hemolysin may have a direct hemolytic effect on erythrocytes also in vivo, resulting in low hemoglobin levels. AEROBACTIN

E. coli needs iron for aerobic metabolism and multiplication. The ability to compete for free iron has been identified as an important virulence factor in *References 1, 17, 30, 52, 54, 66, 68, 81, 91, 105, 114, 119, 123, 125, and 129.

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several infection models outside of the urinary tract. Two mechanisms of iron uptake in E. coli have been identified-the hydroxamate type of siderophore aerobactin or the catechol type siderophore enterochelin. The quantitative contribution of iron-binding, or the stage in the pathogenetic process in which this is active, has not been identified. Isolates from patients with septicemia or UTI have an increased frequency of aerobactin production. Acute pyelonephritis isolates had a higher incidence of aerobactin production than ABU isolates. THE STRUCTURE OF E. CULl POPULATIONS

The analysis of virulence requires information about the stability of bacterial populations. In bacteria, two principally different processes are responsible for the introduction into a population of the new variants, which are a prerequisite for evolutionary changes: mutations or immigration of genes from another population followed by recombination. Recombination allows mendelial evolution with extensive exchange of DNA."" A clonal population structure results when each clonal predecessor gives rise to genetically identical or highly similar progeny. Clonal populations thus persist as a series of independent lineages or clones.67,92. Ill6 Although recombination can be induced under laboratory conditions, natural populations of E. coli are clonal in structure with a low rate of recombination. The first evidence for clonality among E. coli populations was based on serotyping and biotyping. Sjostedt observed a nonrandom coappearance of certain E. coli surface antigen.'"' Orskov et a1 extended this analysis to demonstrate that the three serotype antigens 0 (lipopolysaccharide), K (capsular polysaccharide), and H (flagellar antigen) were nonrandomly associated and that certain biotypes were stably expressed by isolates bearing certain serotype markers.q3Estimates of the rate of phage adsorption and conjugative plasmid transfer showed that even with substantial rates of mobilization of chromosomal genes, the rate of gene replacement by recombination would not be expected to exceed the rate of gene modification by mutation.h3,h7 Analysis by multilocus enzyme electrophoresis showed the occurrence of analogous genotypes in isolates from unrelated hosts. THE CLONE CONCEPT AND VIRULENCE

In theory, the ability to cause infection might be determined by a single characteristic, for example, a toxin or an invasion factor. The acquisition of such a factor, for example, by recombination, would then make E. coli strains virulent. The clonal population structure of E. coli bacteria suggests that such recombinational events are rare. Rather, virulence in the urinary tract seems to be additive. The clones that frequently cause acute pyelonephritis differ from others in that they possess the sum of properties required to colonize the intestine, spread to and ascendingly infect the urinary tract, and induce symptoms in the host.'h,7y Because the clones differ by multiple traits, epidemiologic associations with disease have to be considered as indicators rather than as evidence for the role of specific bacterial properties in pathogenesis. Studies in infection models that compare isogenic strains differing in specific virulence factors are crucial to fulfill the "molecular Koch postulates." Such studies have been performed for

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some virulence factors of uropathogenic E. coli (I' fimbriae, type 1 fimbriae), but more extensive information is required.

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