Gram-negative bacillary infections

Gram-negative bacillary infections

Gram-Negative Bacillary Infections Pathogenic and PathophysiologicCorrelates RICHARD J. DUMA, M.D., Ph.D. Richmond, Virginia Fromthe Departmentof In...

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Gram-Negative Bacillary Infections Pathogenic and PathophysiologicCorrelates

RICHARD J. DUMA, M.D., Ph.D. Richmond, Virginia

Fromthe Departmentof Internal Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia. Requestsfor reprints should be addressedto Dr. RichardJ. Duma, Division of Infectious Diseases, Box 49, Medical College of Virginia, Richmond, Virginia 23298-0001. 154

Gram-negative bacillary infections continue to be extremely important. Escherichia coil is the single most frequently encountered pathogen, followed by organisms belonging to the tribe KlebsiellaEnterobacter-Serratia and Proteus-Providencia. Pseudomonas aeruginosa, although it receives considerable (perhaps excessive) attention, is found relatively less frequently, occurring principally in the hospitalized patient who is immunocompromised. Many factors, both host and microbial, are responsible for invasiveness, virulence, and pathogenicity of gram-negative bacilli, but their relative roles, importance, and the pathophysiologic reactions they trigger are yet to be precisely defined. Certain aspects of many (but certainly not all)of the pathogenic correlates considered important in gram-negative bacillary infections, such as microbial flora, local barrierS, surface and serum antibodies, complement, cell-mediated immunity, slime production, capsules, pill, endotoxin, cell wall components, extracellular products, and inoculum size are discussed herein. Points at which preventive or therapeutic strategies might be developed are offered. The benefits of antibiotics in managing susceptible gram-negative bacillary infections appear to be plateauing. If further advances are to be made in the therapy of these infections, new approaches to rapidly identifying the responsible etiologic agent and a better understanding of the factors responsible for invasiveness, virulence, and pathogenicity are needed. The numbers of families, genera, and species of gram-negative bacilli that are of major medical concern to contemporary practicing physicians in the United States are finite (Table I). Studies of bacteremia, both in university [1,2] and in nonuniversity hospitals [3], show that gram-negative bacilli and their infections are predominantly of nosocomial origin, especially those due to Pseudomonas and certain other nonfermentative bacilli [2]. In all such studies, the most frequently encountered gram-negative bacillus without exception is Escherichia coil, usually accounting for 35 to 40 percent of infections. In descending order of frequency, bacilli belonging to the tribe Klebsiella-Enterobacter-Serratia are next, followed by the Proteus-Providencia group and P. aeruginosa. With the exception of gram-negative bacilli whose immediate source is extrinsic to the hospital [4], other fermentative and nonfermentative gram-negative bacilli generally account for less than 5 percent of nosocomial isolates, Patient populations infected with gram-negative bacilli are generally at the "extremes" of life. Their immunologic systems and host defenses are either poorly developed or compromised, and a variety of underlying diseases, whether inherited, congenital, or degenerative, are present. The

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mortality from gram-negative bacillary infection varies considerably, depending on the age of the patient, the site and severity of infection, the responsible microorganism(s), and perhaps most importantly, as pointed out by McCabe and Jackson [5], the severity and prognosis of the underlying disease. When bacteremia occurs, the mortality from gram-negative bacillary infections may range from 23 percent for community-acquired E. coil to 73 percent for hospital-acquired P. aeruginosa [1]. Nosocomial infections are the major source of current gram-negative bacillary infections in this country; and results from the National Nosocomial Infection Study, conducted in 1979 by the Centers for Disease Control [6], warrant attention (Table II). According to the report, E. coil is the single most common species of gram-negative bacilli infecting hospitalized patients: Among all organisms studied, it accounts for 18.3 percent of 416 pathogens per 10,000 patient discharges. Klebsiella-Enterobacter-Serratia, Proteus-Providencia, and P. aeruginosa clearly dominate the remainder of gram-negative bacilli encountered. The major site of gram-negative bacillary infections cited in the National Nosocomial Infection Study report is the urinary tract, accounting for more isolates than all other sites combined. Nevertheless, wounds (especially burns), respiratory and intra-abdominal sepsis also account for a considerable number of isolates. Although the studies of gram-negative bacteremia mentioned above [1 3] generally support the observations of the Centers for Disease Controll a greater percentage of isolates (particularly of Pseudomonas) result from lower respiratory tract infections than reported by the National Nosocomial Infection Study report.

TABLE II

TABLE I

Major Gram-Negative Bacillary Pathogens Producing Infections in the United States

Enterobacteriaceae Escherichia coil (38 percent) Klebsiella-Enterobacter-Serratia Proteus-Morganeila--Providencia Citrobacter Salmonella-Shigella* Pseudomonadaceae Pseudomonas aeruginosa (10-15 percent) Other species, Pseudomonascepacia, Pseudomonas maltophilia Other nonfermentative bacilli Acinetobacter calcoaceticus Alcaligenes faecalis Bacteroidaceaet *Usually community-acquired, occurs in epidemics, and produces easily recognizablesyndromes. tprobably not comparable in virulence to other organisms in table because they do not contain a biologically potent endotoxin. A summation of isolates of Enterobacteriaceae encountered by clinical investigators studying the use of imipenem/cilastatin in a wide variety of gram-negative bacillary infections (Dr. Gary B. Calandra, Merck Sharp and Dohme Research Laboratories) showed the same relative frequencies of Enterobacteriaceae as outlined in Table I. Escherichia coil, Klebsiella-Enterobacter-Serratia, and Proteus-Providencia accounted for 99 percent of the Enterobacteriaceae isolated from 466 patients (Table Ill). Thus, although the predominant species of gram-nega ~ tive bacilli may vary from hospital to hospital, data suggest that physicians can predict with considerable preciSion which gram-negative bacilli they may expect to encounter. Since the outcome of a serious gram-negative bacillary

1979 National Nosocomial Infection Study of the Rate* and Relative Frequency t of Gram-Negative Bacillary Pathogens

Gram-Negative Bacilli

Primary Bacteremia

Surgical Wounds

Lower Respiratory Tract

Urinary Tract

Skin

Other

All Sites

2.7 13.9 4.4 49.4 1.9 (14.6) (13.4) (6.8) (31.9) (7.2) Klebsiella-Enterobacter-Serratia 4.4 11.4 13.7 23.9 2.6 (23.8) (11.0) (21.3) (15.5) (9.8) Proteus-Providencia 0.4 6.3 3.3 14.3 1.3 (2.2) (6.1) (5.1) (9.2) (4,9) Pseudomonas aeruginosa 0.9 6.1 6.1 17.6 1.6 (4.9) (5.9) (9.5) (11.4) (6.0) Other Pseudomonas species 0.3 0.6 0.9 1.9 0.2 (1.6) (0.6) (1.4) (1.2) (0.8) Bacteroides fragilis 0.5 3.3 0.1 0 0.2 (2.7) (3.2) (0.1) 0 (0.8) Totals 9.2 41.6 28.5 107 7.8 (50) (40) (44) (69) (29) All pathogenst 18.5 103.5 64.3 154.6 26.5 *Number of isolates reported per 10,000 patients discharged; up to four isolates may be reported per infection. tNumber in parentheses represents relative frequency expressed as percent of all isolates from each site. tlncludes gram-positive organisms and fungi.

3.9 (8.0) 3.9 (8.0) t .8 (3.7) 2.7 (5.6) 0.3 (0.7) 0.6 (1.2) 13.2 (27) 48.6

76.2 (18.3) 59.9 (14.4) 27.4 (6.6) 35 (8.4) 4.2 (i .0) 4.7 (1.1) 207 (48) 416

Escherichia coil

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TABLE III

Enterobacteriaceae Encountered in Imipenem/Cilastatin Clinical Studies

Organism

Number of Patients(percent)

Escherichia coli Klebsieila-EnterobacterSerratia Proteus-Providencia Citrobacter Others (Hafnia, Salmonella, Yersinia) Total

173 (37) 173 (37) 97 (21) 19 (4) 4 (1) 466 (100)

infection may depend on an initial, correct, presumptive diagnosis and on early institution of an appropriate antibiotic, it is prudent in this age of statistically dominated thinking that practicing physiciads confronted with selecting rational antibiotic programs pay attention to these facts. PATHOGENESIS AND PATHOPHYSIOLOGY

To improve the management of gram-negative bacillary infections, an appreciation of the factors and mechanisms responsible for, or associated with, invasion, virulence, and pathogenicity of gram-negative bacilli is helpful. Although many such factors exist, they may be conveniently divided into host and microbial (Table IV); however, their relative roles and importance in sepsis are not easy to separate or define. Studies that examine these factors utilize animal species other than human or highly artificial animal models; or the factor is studied as an isolated event. Thus, results obtained may be of questionable, extrapolative clinical value. On the other hand, studies conducted in humans are often retrospective and their clinical meaning may be just as difficult to interpret.

TABLE IV

Factors Ass6ciated with Invasiveness, Virulence, and Pathogenicity of GramNegative Bacilli

Host Microbial flora (interference) Local barriers Surface antibody Serum antibody Complement Phagocytes (polymorphonuclear cells, macrophages, and reticuloendothelial system) Cell-mediated immunity Microbial Slime glycocalyx Capsule Pili Cell wall components (~.g;, protein) Endotoxi n (tipopolysaccharide)

Extracellular products Inoculum size

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An understanding of the pathophysiologic mechanism(s) and precise molecular events triggered by gramnegative bacilli are even more abstruse, principally because the proposed mechanism(s) are difficult to examine, having been Studied as isolated events. Shock complicating gram-negative bacteremia is particularly confounding, and only in recent years has a widespread appreciation for its diverse pathophysiologic etiologies and dynamic nature emerged [7]. The same can be said for adult respiratory distress syndrome, which is more often associated with bacterial sepsis, particularly gramnegative, than with any other infection [8]. A discussion of all of the factors responsible for, or associated with, the occurrence and progression of gi'amnegative infections, as well as such topics as foreign bodies and antimicrobial resistance, is beyond the scope of this text. However, several prominent host and microbial factors are worth reviewing, since real possibilities exist for their neutralization or control (or the reactions they precipitate), thus ~.betting the prevention and therapeutic interdiction of the infections they produce. HOST FACTORS Microbial Flora. With the advent of antimicrobial agents came the early realization that alterations in normal body flora promptly result from their use [9]. Depending on the effectiveness and spectrum of the agents employed, indigenous floral changes and subsequent colonization by other microorganisms emerge, usually resistant to the antibiotic(s) employed. Often, such colonizing organisms are gram-negative bacilli, usually multiply resistant to a broad array of antibiotics. Not only is the protective barrier of microbial "interference" erased [10], but also the host may be exposed to heavy colonization by drug-resistant organisms for which little or no immunologic experience existS. The result is often local or systemic invasion with considerable morbidity and mortality. Thus, any antimicrobial agent that permits preservation of the normal flora is an asset. Such preserv~.tion might be via the possession of a narrow antimicrobial spectrum or through poor localization resulting in low antimicrobial concentrations to body Surface cavities, so as not to affect indigenous microflora. In terms of prevention, discriminate use of antimicrobials is essential and, as suggested in a recent editorial by Sanford [11], antibiotic programs directed against gram-negative bacilli might be streamlined to contain one rather than multiple antibiotics. Considerable help in reaching such a goal may be secured if the progress of clinical microbiology is able to keep pace with the accelerated development of therapeutic agents. Greater emphasis on rapid techniques for specific identification of bacteria or infectious metabolic by-products in clinical specimens is necessary, e.g., through antigen detection, gas liquid chromatography, or gene probing [i2,13].

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Contemporary medicine, or what has been referred to as the "blessings of modern medicine," is witnessing the emergence of a plethora of devices, techniques, and therapeutic modalities, which are used to bridge or to alter local host defense barriers. Indwelling urinary and intravenous catheters and tracheostomy tubes are all valuable to today's practicing physician. However, they are also excellent examples of how to bypass highly effective host barriers. Such devices are notorious, not only for their frequent use and abuse [14], but also for the risks they impose [15-17]. Anything that can be done to reduce their use surely will decrease the incidence of gram-negative sepsis. Burn wound sepsis is another frustrating problem, especially in patients with second- and third-degree burns involving more than 50 percent of the body surface. In burn patients, most septic complications and death from gram-negative bacilli result from septicemia and an inability to quickly replace skin barriers. However, replacement of burned skin by recently developed artificial integument [18] or by laboratory cultured skin tissue [19] offers considerable promise. A n t i b o d y . The role and relative importance of secretory surface antibody in protecting against gram-negative bacillary infections are unknown; however, it is well appreciated that certain enteric gram-negative pathogens are more likely to colonize and produce serious infections in the absence of gut secretory IgA than in its presence [20]. In addition, commonly invasive bacteria, such as meningococci, pneumococci, and Hemophilus influenzae often possess proteases specific for IgA, as compared to noninvasive bacteria which frequently colonize the upper respiratory tract and do not produce such proteases [21]. Some gram-negative bacilli that frequent the urinary tract, as well as respiratory and alimentary tracts, may also produce this type of enzyme [22]. The mechanism by which IgA protects against bacterial invasion is not precisely known, but blocking bacterial adherence via binding to bacterial surface sites, which ordinarily bind to glycoprotein cell surface receptors, is a probable mechanism. In addition, IgA may neutralize toxic or lytic enzymes elaborated by various organisms. The protective and therapeutic values of humoral antibodies against gram-negative bacilli are more difficult to assess than are antibodies against polysaccharide-encapsulated organisms, such as pneumococci, meningococci, or H. influenzae. Nevertheless, substantial evidence indicates that humoral antibodies directed against the somatic polysaccharide O antigen of cell walls of gram-negative bacilli may protect the host from the mortality associated with these organisms [23-25]. In addition to the diversity of somatic antigens possessed by gram-negative bacilli (at least 164 known Ogroup antigens for E. coil), another reason why the success of humoral immunity against gram-negative bacilli is Local Barriers.

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not as effective as against pneumococci, meningococci, or H. influenzae may be the number of surface immunologic structures possessed by most gram-negative bacilli. These structures, located external to the somatic O antigen, are the H or flagella antigen (which has antiphagocytic activity), pili or fimbriae, capsular envelopes (especially with E. coli and Klebsiella), and a glycocalyx or slime layer. Since polysaccharide O antigens offer opsonizing sites for circulating antibody, their "cloaking" by other surface structures may reduce the efficiency of phagocytosis. This is suggested by the increase in virulence of meningococci which possess pili as compared to those that do not [26]. (The current assumption is that enhanced virulence of piliated strains is entirely due to attachment.) More recently, increasing attention has been paid to outer membrane "core" glycolipid antigens (endotoxin), which are common to all Enterobacteriaceae and which are located deep within the membranes of bacilli. (The endotoxin of P. aeruginosa possesses a chemically different lipid A structure.) Patients who possess high antibody titer~ to such antigens at the beginning of gram-negative bacteremia appear to be significantly protected against shock and death [24,27]. Efforts to develop prote~;tive vaccines [25] or antiserums that may be passively infused are meeting with limiting but encouraging success. For example, Young et al [28] immunoprophylaxed patients with cancer in a randomized, controlled, prospective study with a heptovalent vaccine directed against P. aeruginosa and showed a significant reduction in Pseudomonasassociated deaths; but because of severe neutropenia in many patients, the vaccine did not protect against bacteremic deaths. On the other hand, Ziegler et al [29] gave human antiserum, prepared from healthy men vaccinated with heat-killed E. coli J 5 - - a rough mutant that contains only core antigen and no oligosaccharide side chains, to patients with gram-negative bacillary septicemia. In this randomized, controlled study, survival was highest among recipients of the J5 antiserum, and the effect was most evident in patients with shock. In the future, monoclonal antibodies may offer considerable promise in improving the effectiveness of passive immunotherapy [30]. The continued pursuit of immunotherapy is fueled by the knowledge that the clinical effects of endotoxin (shock, disseminated intravascular coagulation, complement activation, leukopenia, etc.) cannot be eliminated, or necessarily affected, by antimicrobials. In fact, antimicrobial therapy may contribute to the release of significant amounts of endotoxin when bacteria are killed [31,32], thus possibly abetting morbidity and mortality. C o m p l e m e n t . The value of complement in gram-negative bacillary infections is also difficult to assess. The present weight of evidence suggests that it may be more detrimental than useful because a considerable number of pathophysiologic changes may result from its activation.

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Besides, most invasive gram-negative bacilli are resistant to complement-mediated bacteriolysis [33], ordinarily a valuable first line of host defense against encapsulated organisms such as meningococci. Constituents of the cell walls of bacteria activate both the classic and alternative complement pathways [34]; however, it is the alternative pathway that is important early in 'infection, particularly in bacteremia in the nonimmune host, because the alternative path may be activated in the absence of specific antibodies. C3a and C5a serve as anaphylatoxins, and probably contribute to the cascade of reactions eventuating in clinical shock. Although C5a is an extremely potent chemo-attractant for neutrophils and macrophages [35], it also induces granuIocytes to aggregate, forming leukocytic emboli, which may contribute to the pathogenesis of adult respiratory distress syndrome [36], which occasionally complicates gram-negative bacteremia. The argument in favor of complement usefulness is made principally on the basis of studies involving encapsulated coccal organisms. C3b is thought to enhance both ant!body attachment to the bacteria and ingestion by the phagocyte. C3b also promotes B cell lymphokine and antibody production [35], whereas C3e may induce granulocytosis [37]. C1 through C6 may neutralize endotoxin in vitro and protect animals in vivo [35]. Nevertheless, it is interesting that abnormally high numbers of serious or recurrent infections due to gram-negative bacilli are not frequently associated with complement-deficient conditions (particularly of the late components) as has been reported for Neisseria. In the future, it may appear that agents that may control specific reactions triggered by the complement cascade may be valuable therapeutic adjuncts to prevent the sequelae or complications of gramnegative bacillary infections, such as adult respiratory distress syndrome and shock. P h a g o c y t e s . Polymorphonuclear leukocytes are one of the most important cells capable of ingesting and killing gram-negative bacilli. As early as 1966, in a study in humans with acute leukemia, Bodey et al [38] clearly established that the occurrence of infections was related to the level of circulating granulocytes and, furthermore, the critical level at which infection was likely to occur appeared to be a leukocyte count of about 1,500/mm3. The best indicator of the risk of infection is the total granulocyte count, and this risk increases with increasing duration of granulocytopenia. Migrating neutrophils play an extremely important role in localizing or confining invading bacteria. When bacteria invade, circulating granulocytes adhere to capillary endothelium, diapedese into surrounding infected tissues, recognize bacterial or other chemical attractants, migrate toward the insult, and then ingest and kill the bacteria they encounter. Some inherited diseases in which neutrophils are reduced or are malfunctioning and which are eventu-

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ally complicated by gram-negative bacterial infections are Chediak-Higashi syndrome and chronic granulomatous disease of childhood. Whether or not neutrophil malfunction results from infection per se is still debatable. Several investigators [39,40] report various neutrophilic abnormalities appearing after or during infection, whereas others [41] note stimulatory effects. In a recent study in granulocytic function versus autologous infecting strains, Weinstein [25] was unable to demonstrate an intrinsic killing abnormality during the acute phase of gram-negative bacillary bacteremia in patients without leukopenia. When differences in neutrophilic activity between control subjects and infected patients occured, the differences appeared to be confined to plasma factors (type-specific antibody) and not to granulocytes. The reticuloendothelial system may be the most important line of defense against bloodstream invasion and the persistence of bacteremia. Thirty years ago, Bennett and Beeson [42] demonstrated that bacterial counts in the bloodstream of humans with bacteremia due to endocarditis decrease remarkably after passage of the blood through the liver. In subsequent animal studies, such removed bacteria were found in the macrophages of the liver and spleen. Mononuclear phagocytes of the reticuloendothelial system are derived from bone marrow, circulate in the bloodstream for about three days, then migrate to organs such as the liver, spleen, and lung, where they establish themselves as fixed tissue macrophages [34]. The role of these cells includes recognizing, ingesting, and killing microorganisms; processing antigens for T lymphocytes; and inducing B cell antibody synthesis. Diseases or conditions that lead to destruction or removal of the liver (e.g., cirrhosis) or spleen (e.g., splenectomy) may be associated with severe, high-grade bacteremias, which often result in death. Primarily, the liver removes bacteria that are well opsonized and which have activated complement, whereas the spleen removes bacteria which are poorly opsonized [43]. Cell-Mediated Immunity. The importance of thymusderived T lymphocytes in defense against invading gramnegative bacilli is indirect and appears to be exerted principally through T cell induction of B cells to proliferate and produce antibody. A fine balance of T cell suppressor and helper cells is necessary, or decreased antibody synthesis may result [44]. In the future, useful agents that might alter or stimulate the various limbs of the immune system to reduce the frequency of, or prevent, gram-negative sepsis may yet be foundl For example, one such agent levamisole significantly decreased sepsis as defined by bacteremia and visceral abscesses in patients with decreased host defenses (anergic surgical patients) when compared with control subjects [45]. Such studies await confirmation, but the

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thought of preventing or controlling sepsis in patients at high-risk by manipulating their immune systems is intriguing. MICROBIAL FACTORS

Exopolysaccharide slime may be produced by many species of gram-negative, as well as gram-positive, bacteria [46]. Such exopolysaccharides are highly viscous and may be comprised of several different sugars, usually polymers of hexoses (D-glucose, D-galactose, and D-mannose) or methylpentoses (fucose and rhamnose), and N-acetylated aminosugars. Acetate and formyl residues are also found within them and, in plant pathogens such as Pseudomonas, levan and dextran may be present as homopolysaccharides. The slime glycocalyx is hydrophilic, carries a net negative charge, and stains poorly. Most exopolysaccharide synthesizing bacteria are aerobes or facultative anaerobes, and unlimited availability of oxygen appears to be associated with increased slime production. Excess carbohydrate substrates or calcium ions also serve to stimulate slime production, and deficiencies of nitrogen, phosphorous, or sulfur increase it. Most exopolysaccharides are produced during exponential growth, and if its production is eliminated, in vitro growth and viability of the bacteria do not seem to be affected. The major virulent or pathogenic roles of the slime glycocalyx appear to be enhanced adherence [47] and microcolony formation and resistance to phagocytosis [48]. The slime of P. aeruginosa markedly impairs human neutrophilic in vitro motility, endocytosis, and phagosome formation, but without alteration of viability [49]. In addition, the biofilm formed by secreted exopolysaccharides may permit microcolonies of gram-negative bacilli to establish themselves firmly and to preferentially adhere to biomaterials of prosthetic devices or intravenous catheters. In a study by Sensakovic and Bartell [50], in which purified slime from P. aeruginosa was injected intraperitoneally into mice, the response observed was identical to that seen in lethal infections initiated with viable bacteria-toxicity, leukopenia, and death occurred. In addition, active and passive immunization against slime prevented these responses, and protection was type-specific. Of further interest, in comparison to the lipopolysaccharide endotoxin of P. aeruginosa, slime was two to three times more toxic. The importance, particularly in vivo, of glycocalyx slime in preventing antimicrobial agents from entering the bacterial cell is unclear; however, the negative charge of slime polysaccharides could impede the penetration or activity of certain antibiotics at the bacterial cell surface level [51]. This has been suggested as a problem in the management of pulmonary infections due to slime-producing P. aeruginosa [52]. Also, the importance of slime glycocalyx interfering with the binding of antibody and Slime Glycocalyx.

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complement is unclear, but the possibility of such an effect is distinct. In the future, therapeutic agents or antibiotics that interfere with slime production or block its action (by binding to it or its receptors) may offer innovative adjuncts to current chemotherapeutic approaches. Long-term, refractory urinary tract, respiratory tract, skin, or gastrointestinal tract infections may be extremely fruitful areas in which to consider such an approach. Capsule, One of the most well=known and well-studied virulence factors of bacteria is the antiphagocytic capsule. For example, the K1 capsular antigen may be found in 85 percent of E. coil responsible for neonatal meningitis [53]. The K1 capsular polysaccharide is an alpha2, 9-1inked N-actyl neuraminic acid homopolymer that is structurally identical to the capsule of N. meningitidis group B. Both the chemical composition and size of the capsule are important in resisting phagocytosis. Opsonization of bacteria requires that ligands of the microbial cell wall bind to granulocyte membrane receptors. Usually, such ligands are either the complement cleavage product C3b or the Fc gamma of antibody. Encapsulated bacteria frequently fail to activate the alternate complement pathway. Bacterial capsules bind C3b but, because of their chemical structure, they inhibit C3b from forming C3/C5 convertase [54]. However, if specific anti-capsular antibody is present, complement will fix via the classic path, and both C3b and Fc ligands will bind to the capsule and thence to the surface of the neutrophil. In experiments using encapsulated K ÷ E. coil that bind Concanavilin A, Horwitz and Silverstein [55] demonstrated that although encapsulated E. coil bind to neutrophils, they are not ingested; however, unencapsulated mutants are. Apparently, reactive moieties on the microbial cell surface are sufficiently masked to prevent phagocytosis. Nevertheless, antibodies directed against capsular antigens--supplied intrinsically via active vaccination or extrinsically via passive infusion--may be protective or therapeutic, respectively. Also, encapsulated strains that are highly virulent for newborns and infants may not be so for adults. The reasons for this are obscure and may not be explained entirely by the host-possessing protective antibodies. For example, Pitt [56] in a study of 137 cases of E. coil bacteremia in adults, found only 16 (12 percent) infected by K1 capsular strains and in none of these did death occur, whereas in a comparable group of patients with non-K1 infections the mortality was 26 percent. Pitt speculated that the reason(s) for this might include the presence of either a tissue neuraminidase, which alters the antiphagocytic K1 capsule, or a tissue receptor for K1 antigen, which is unmasked in the newborn. Pill (Fimbriae). One major event that is usually required before colonization or invasion by gram-negative bacilli is attachment to a body cell surface. Most often, the cell surface is oropharyngeal, gastrointestinal, respiratory, or uro-

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epithelial. Attachment is mediated by specific bacterial surface components, and most often (but not always) by fimbriae. Fimbriae are protruding microtubular protein structures that serve as colonizing factors for bacteria. Two major enterotoxigenic E. coli fimbrial antigens have been identified: colonization factor antigen land colonization factor antigen II [57]. Both are plasmid-determined, with ger~es residing on ent plasmids, and both are hostspecific. Neither has ever been identified on a strain of non-enterotoxigenic E. coli. The nature of attachment of enteropathogenic E. coli is unclear. Most uropathogenic E. coli serotypes produce fimbriae or P pili that result in attachment of the bacteria to uroepithelial cells [58-60]. The same fimbriae are present on the same serotypes, whether they represent isolates from urine, blood, or cerebrospinal fluid [57]. Colonization factor antigen fimbriae react specifically with receptors on host target cells. Recombinant plasmids containing all the genetic information necessary for expressing P pili may be present in uropathogenic E. coll. Nonadherent bacteria receiving such plasmids acquire adherence properties associated with P pili [61]. Adherence due to P pili may also be demonstrated for other Enterobacteriacae and for organisms isolated from extraintestinal sites other than the urinary tract. Most of these isolates contain gene sequences homologous to those found in the pap operon of uropathogenic E. coli which encodes for expression of P pili [62]. In contradistinction to colonization factor antigen fimbriae, E. coli also possess common fimbriae, which serve as a universal attachment factor for receptors containing mannose. These receptors exist on many cell types and in glycoproteins of mucous secretions [63]. However, they play a negative role in virulence, because the "mucous escalator" clearance mechanism of the respiratorY, gastrointestinal, and urinary tracts removes such bacteria more readily and the mannosyl receptors of phagocytes ingest them more efficiently. Thus, since attachment appears to be a prerequisite for bacterial invasion in most instances of natural infections, prevention or disruption of attachment offers a potential mechanism for the future management of infections. Antibodies against pili from non-mucoid strains of P. aeruginosa inhibit adherence of such bacteria to injured tracheal epithelium [64]. In the future, vaccines against pili may prove effective. Also, since attachment in many instances is a surface phenomenon, its prevention or erasure might be accomplished with topical or intraluminal agents. Cell Wall Components. An excellent example Of another nonpolysaccharide cell wall component playing an important role in virulence and perhaps in potential vaccine protection is the outer membrane 1H protein of H. influenzae. In studies of secondary cases of H. influenzae type B infections in children attending day care centers, the data of Barenkamp et al [65] suggest that protein sub-

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type 1H strains predominate, as compared to single, sporadic primary infections in which a variety of protein subtypes are found. Although vaccines against H. influenzae capsular type B polysaccharide are effective, they are not completely effective, especially in infants less than one year old, in whom polysaccharide antigens lack immunogenicity. Newer vaccines are combining protein subtype antigens (particularly 1H) with the polysaccharide. Minimal work has been done on the clinical significance or virulence of peptidoglycan in gram-negative bacilli. However, since it is present in all bacteria, is poorly biodegraded, may activate complement, and has endotoxin-like activities, its role in infection needs to be studied more fully. When studied in coccal infections, peptidoglycans are responsible for purpura in pneumococcal infections [66] and may contribute to fallopian tube damage in gonococcal infections [67]. Endotoxin. Much has been written about the somatic O antigen of gram-negative bacilli. At least 164 O groups exist [25,68]. The O-specific polysaccharide is characteristic of the smooth form of Enterobacteriaceae. Mutation in the synthesis of O-specific polysaccharides (which are side chains of the lipopolysaccharides of gram-negative bacilli) results in rough variants that give rough specificity to the core polysaccharides. Rough mutants are more readily phagocytosed than smooth forms, and thus are less virulent. Small variations in O polysaccharide composition appear to influence appreciably the virulence of gram-negative bacilli [68]. Nevertheless, at-the moment, only a few O serogroups are prevalent in the human gut and they are responsible for a large percentage of infections (O1, 02, 04, 06, 07, 08, 09, O l l , O18, O21, 022, 025, 045, 062, 075, O81, and 083). In an ongoing study of heatstable opsonizing antibodies directed against O antigens obtained from the blood samples of patients, which are culture-positive for gram-negative bacilli, O antibodies appear to protect against death [25]. Virtually all naturally occurring facultative and aerobic gram-negative bacilli possess heat-stable lipopolysaccharides with endotoxic activity in their cell walls. The toxin was first recognized by Pfeiffer [69] in culture filtrates of Vibrio cholera which were examined during the late logphase and named endotoxin to distinguish it from the exotoxin secreted by the bacterial cell during the growth phase [69]. Overwhelming evidence strongly implicates lipid A as the moiety of the lipopolysaccharide complex that possesses endotoxic activity [70]. Studies of many species of gram-negative bacilli reveal that the backbone of lipid A for each is similar, consisting of betal_6-1inked diglucosamines [71]. This backbone contains both esterand amide-linked pyrophosphates and fatty acids (principally 3-OH myristic). Aside from some immunostimulatory or immunogenic properties (probably related to protein bound lipids) that

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may or may not be of value to the host [70], endotoxin or lipid A may activate both complement systems or the Hageman factor, thus triggering a disseminated consumptive coagulopathy or the many factors responsible for, or associated with, the pulmonary and hemodynamic difficulties seen in gram-negative shock. Circulating free endotoxin appears to have a greater effect on the host than does the circulating cell-bound endotoxin. The studies of Demonty and De Graeve [72] show that filterable endotoxin liberated from serum-lysed E. coil is lethal when injected into mice sensitized to endotoxin by actinomycin D, whereas filtrates of E. coli incubated with heat-inactivated serum, reducing the concentration of free endotoxin, are tolerated by sensitized mice. The recent work of Shenep and Morgan [32] extends these findings, showing that rabbits infected with E. coli and treated with antibiotics have a 10- to 2,000-fold increase in plasma levels of free endotoxin as compared with placebo-treated animals with the same infection. The therapeutic implications of these and other studies suggest that if endotoxin can be neutralized, altered, or its release from bacteria inhibited, then the clinical outcome of gram-negative sepsis may be improved. Indeed, investigators are convinced that the outcome of gram-negative bacteremia treated with antibiotics and accompanied by shock can be improved by passive administration of antibodies directed against the core lipopolysaccharide [29]. Although the mechanism by which improvement occurs is unknown, it is presumed to be a result of neutralization of circulating liberated endotoxin. Rather than directly neutralizing released endotoxin, altering or blocking the end-organ receptor or the cascade of events triggered by endotoxin may prove useful. Although the use of corticosteroids in the treatment of septic shock is still controversial, massive doses appear beneficial in the early stages of shock [73]. This may occur via inhibition of complement-induced neutrophil aggregation, endothelial-cell cytotoxicity, pulmonary-capillary leakage, and/or endorphin release. Additionally, a variety of other agents is being examined, such as inhibitors of cyclooxygenase or thromboxane synthetase and naloxone, an endorphin antagonist [74]. Potentially, endotoxin itself may be altered to modify or eliminate its biologic effect. For example, Morrison and Kline [75] demonstrated a major role for the potysaccharide of lipopolysaccharide in activating the alternate complement pathway. Apparently, the polysaccharide stabilizes the binding of activated C3 and prevents its inactivation by C3blNA and beta 1H. Wilson et al [76] demonstrated that by using a variety of different lipopolysaccharide preparations, they could observe marked differences in the complement-dependent oxidative burst of neutrophils. E x t r a c e l l u l a r Products. With the exception of enterotoxin (which will not be discussed herein), comparatively

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little is known of the pathogenic importance of many bacterial exoproteases and other exocellular products in human infections due to gram-negative bacilli. Even less is known about immunity to them and whether or not (or to what degree) such immunity is protective. Yet, it is clear that this very important area may hold major clues to understanding the properties of invasion, virulence, and pathogenicity of gram-negative bacilli. Exotoxin A is a heat-labile, single chain polypeptide (molecular weight of 71,500) produced by approximately 90 percent of clinical isolates of P. aeruginosa [77]. No other bacteria produce it. As little as 2.5/~g/kg is lethal to a mouse and, therefore, is about 10,000 times more active than endotoxin. In subhuman primates it acts much like diphtheria toxin, inhibiting protein synthesis, decreasing cardiac output, and producing hypotension, shock, clotting abnormalities and hepatocellular necrosis. Nevertheless, since infections from non-exotoxin A-producing strains of Pseudomonas are not unusual, the significance of exotoxin A in human infections is unclear. Passively transferred antibodies against exotoxin A block the chemical aberrations it produces, limits bacterial invasion, and increases survival of infected animals. Antibodies against exotoxin A can be detected in human infections due to P. aeruginosa [78], and high titers of these antibodies, if present early in bacteremia, appear to enhance survival [79]. Recently, a toxoid produced from exotoxin A became available [80]. In mice and rabbits, it induces high titers of antibodies that neutralize mouse lethality, cytotoxicity, and ADP-ribosyl transferase activity. Elaboration of elastase, particularly by Pseudomonas, is believed to be another important factor in pathogenicity and virulence. In studies of mice infected with P. aeruginosa, strains containing elastase produced a significantly higher percentage of pneumonias than strains that did not [81]. In addition, elastase appears to inhibit luminol-enhanced myeloperoxidase-mediated chemiluminescence [82], suggesting that it interferes with intracellular phagocytic killing. A variety of other enzymes and extracellular products has been identified and merits concern--coliagenase, lecithinase, phospholipase, hemolysin, leukocidase, hydrolase, protease, hyaluronidase, urease, iron chelators, to name a few. Many of these substances are probably plasmid-mediated and capable of being transfer'red between closely related species [83]. Thus, future agents that might interfere with plasmid expression or cure bacteria of plasmids may well control or eliminate certain infections. I n o c u l u m Size. Suffice it to say that in virtually every infection there is a direct correlation between inoculum size and invasiveness and virulence, and thus, indirectly, in pathogenicity as well. The ingestion of typhoid bacilli by vaccinated volunteers in field trials is one of many excellent examples of the importance of this phenomenon in human infections [84].

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It is well known that certain body sites--for example, the subarachnoid space of the central nervous s y s t e m - possess extremely poor or inefficient host defenses, which enable invading bacteria to reach counts as high as 106-9 per ml of cerebrospinal fluid, resulting not only in considerable toxicity and severe disease, but also in an infection that may be extremely difficult to eradicate [85]. The susceptibility of large bacterial inoculums (106-9 or more per ml), particularly to beta-lactam derivatives, may be much less than that of small inoculums (103-5 per ml). An important advantage of the newly developed antibiotic imipenem, as compared with other beta-lactams, is the apparent lack of an "inoculum effect," especially against gram-negative bacilli such as Pseudomonas [86]. In this respect it resembles the aminoglycosides. COMMENTS

Today, the species of gram-negative bacilli infecting humans may be predicted with reasonable accuracy. Escherichia coil heads the list, followed by the tribe KlebsiellaEnterobacter-SerraUa and Proteus-Providencia. Except in special institutes and in certain situations, P. aeruginosa is encountered relatively less frequently. These well-documented facts provide the physician with a basis for rational antibiotic selection, especially when confronted with the initial therapy of a putative gram-negative bacillary infection in which the etiologic agent has not been identified. Currently, a wide variety of potent, broad-spectrum anti-

microbial agents is available to treat gram-negative infections, and most are highly efficacious against susceptible bacteria. Nevertheless, non-toxic agents highly active against Pseudomonas species and many nonfermenting gram-negative bacilli are still lacking, and this void needs to be filled. Imipenem/cilastatin, the new carbapenem discussed in this Symposium issue, may help in this regard. However, the major problem that confronts physicians when managing gram-negative sepsis is not a scarcity of effective antibiotics. Rather, the problem is varied and consists of an inability to promptly define the etiologic agent(s) and its susceptibility and to manage or quickly reverse the pathogenic and pathophysiologic reactions that the responsible microbes precipitate. A review of such microbes and their pathogenic correlates in terms of host responsiveness reveals that a large number of independent and interdependent factors exist, which may ignite a variety of poorly understood reactions. It appears that these reactions account for most of the currently observed morbidity and mortality. Continuous development of potent antibiotics is essential and emerging antimicrobial resistance is of vital concern. However, the benefits of antibiotics in successfully managing gram-negative bacillary infections appear to be plateauing. Thus, if significant improvements are to be made in the therapy of such infections, then physicians will need not only an in-depth working knowledge of the molecular events resulting from host-microbe interactions, but also methods of preventing or interrupting them.

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19. Gallico CG, O'Connor NE, Compton CC, Kehinde O, Green H: Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984; 311: 448451. 20. McNabb PC, Tomasi TB: Host defense mechanism at mucosal surfaces. Ann Rev Microbiol 1981; 35: 477-496. 21. Kornfeld SJ, Plaut AG: Secretory immunity and the bacterial IgA proteases. Rev Infect Dis 1981; 3: 521-534. 22. Milazzo FH, Delisle GJ: Immunoglobulin A proteases in gramnegative bacteria isolated from human urinary tract infections. Infect Immun 1984; 43: 11-13. 23. Roantree RJ: Salmonella O antigens and virulence. Ann Rev Microbiol 1967; 21: 443-466. 24. Zinner SH, McCabe WR: Effects of IgM and IgG antibody in patients with bacteremia due to gram-negative bacilli. J Infect Dis 1976; 133:37-45 . 25. Young LS, Meyer RD, Weinstein RJ, Anderson ET: Gram-negative rod bacteremia: microbiologic, immunologic, and therapeutic considerations. Ann Intern Med 1977; 86: 456-471. 26. Stephens DS, McGee ZA: Attachment of Neisseria meningitidis to human mucosal surfaces: influence of pill and type of receptor cell. J Infect Dis 1981; 143: 525-532. 27. McCabe WR, Kreger BE, Johns M: Type-specific and cross-reactive antibodies in gram-negative bacteremia. N Engl J Med 1972; 287: 261-267. 28. Young LS, Meyer RD, Armstrong D: Pseudomonas aeruginosa vaccine in cancer patients. Ann Intern Med 1983; 79: 518527. 29. Ziegler EJ, McCutchan JA, Fierer J, et al: Treatment of gramnegative bacterernia and shock with human antiserum to a mutant Escherichia coli. N Engl J Med 1982; 307: 12251230. 30. Sawada S, Suzuki M, Kawamura T, Fujinaga S, Masuho Y, Tomibe K: Protection against infection with Pseudomonas aeruginosa by passive transfer of monoclonal antibodies to lipopolysaccharides and outer membrane proteins. J Infect Dis 1984; 150: 570-576. 31. Spink WW, Braude AI, Castaneda MR, Goytia RS: Aureomycin therapy in human brucellosis due to Brucella melitensis. JAMA 1948; 138:1145-1148. 32. Shenep JL, Morgan KA: Kinetics of endotoxin release during antibiotic therapy for experimental gram-negative bacterial sepsis. J Infect Dis 1984; 150: 380-388. 33. Young LS: Role of antibody in infection due to Pseudomonas aeruginosa. J Infect Dis 1974; 130(suppl): 111-118. 34. Cates KL: Host factors in bacteremia. Am J Med 1983; 75: 1925 35. Johnston RB Jr, Stroud RM: Complement and host defense against infection. J Pediatr 1977; 90: 169-179. 36. Jacob HS, Craddock PR, Hammerschmidt DE, Moldow CF: Complement-induced granulocyte aggregation. N Engl J Med 1980; 302: 789-794. 37. Schultz DR: Complement in host defense. Scand J Infect Dis 1980; 24(suppl): 22-29. 38. Bodey GP, Buckley M, Sathe YS, Freireich EJ: Quantitative relationships between circulating leukocytes and infection in patients with acute leukemia. Ann Intern Med 1966; 64: 328340. 39. McCall CE, DeChatelet LR, Cooper MR, et al: Human toxic neutrophils. III. Metabolic characteristics. J Infect Dis 1973; 127: 26-33. 40. Solberg CO, Helium KB: Neutrophil granulocyte function in bacterial infections. Lancet 1972; Ih 727-730. 41. Stossel TP: Evaluation of opsonic and leukocyte function with a spectrophotometric test in patients with infection and with phagocytic disorders. Blood 1973; 42: 121-130. 42. Bennett IL Jr, Beeson PB: Bacteremia: a consideration of some experimental and clinical aspects. Yale J Biol Meal 1954; 26: 241-262. 43. Krivit W: Overwhelming postsplenectomy infection. Am J Hema-

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65. Barenkamp SJ, Granoff DM, Munson RS Jr: Outer-membrane protein subtypes of Haemophilus influenzae type b and spread of disease in day care centers. J Infect Dis 1981 ; 144: 210-217. 66. Chetty C, Kreger A: Role of autolysin in generating pneumococcal purpura-producing principle. Infect Immun 1981 ; 31 : 339344. 67. Rosenthal RS, Sinhara RK, Peterson BH, et al: Chemical and biological properties of gonococcal peptidoglycan. In: D~;nielsson D, Norwalk S, eds. Genetics and immunobiology of pathogenic Neisseria. Umea, Sweden: University of Umea Press, 1980; 7-11. 68. M&kel& PH, Valtonen VV, Valtonen M: Role of O antigen (LPS) factors in virulence of Salmonella. J Infect Dis 1973; 128(suppl): 81-85. 69. Pfeiffer R: Untersuchungen eber das cholera gift. Zeitschrift Hygiene Infectionshrankheiten 1892; 11: 393-412. 70. Morrison DC: Bacterial endotoxin and pathogenesis. Rev Infect Dis 1983; 5(suppl 4): 733-747. 71. Hase S, Rietschel ET: Isolation and analysis of the lipid A backbone: lipid A structure of lipopolysaccharides from various bacterial groups. Eur J Biochem 1976; 63: 101-107. 72. Demonty J, De Graeve J: Release of endotoxic lipopolysaccharide by sensitive strains of Escherichia coli submitted to the bactericidal action of human serum. Med Microbiol Immunol 1982; 170: 265-277. 73. Sprung CL, Caralis PV, Marcial EH, et al: The effects of highdose corticosteroids in patients with septic shock. A prospective, controlled study. N Engl J Med 1984; 311:1138-1143. 74. Sheagren JN: Septic shock and corticosteroids. N Engl J Med 1981 ; 305: 456-457. 75. Morrison DC, Kline LF: Activation of the classical and properdin pathway of complement by bacterial lipopolysaccharides (LPS). J Immunol 1977; 118: 362-368. 76. Wilson ME, Jones DP, Munkenbeck P, Morrison DC: Serum dependent and independent effects of bacterial lipopolysac-

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