Arachidonic Acid Metabolism Regulates Escherichia coli Penetration of the Blood-Brain Barrier

Escherichia coli K1 meningitis occurs following penetration of the blood-brain barrier, but the underlying mechanisms involved in E. coli penetration of the blood-brain barrier remain incompletely understood. We have previously shown that host cytosolic phospholipase A₂α (cPLA₂α) contributes to E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, but the underlying mechanisms remain unclear. cPLA₂α selectively liberates arachidonic acid from membrane phospholipids. Here, we provide the first direct evidence that host 5-lipoxygenase and lipoxygenase products of arachidonic acid, cysteinyl leukotrienes (LTs), contribute to E. coli K1 invasion of HBMEC and penetration into the brain, and their contributions involve protein kinase C alpha (PKCα). These findings demonstrate that arachidonic acid metabolism regulates E. coli penetration of the blood-brain barrier, and studies are needed to further elucidate the mechanisms involved with metabolic products of arachidonic acid for their contribution to E. coli invasion of the blood-brain barrier.The mortality and morbidity associated with neonatal Gram-negative bacillary meningitis have remained significant despite advances in antimicrobial chemotherapy and supportive care. Inadequate knowledge of the pathogenesis has contributed to this mortality and morbidity (18-20). Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis. Most cases of neonatal E. coli K1 meningitis develop as a result of hematogenous spread, but the underlying mechanisms involved in E. coli penetration of the blood-brain barrier remain incompletely understood (18-20).Several lines of evidence from experimental animal models as well as human cases of E. coli K1 meningitis indicate that E. coli penetrates into the brain initially in the cerebral vasculature (3, 21). We have developed the in vitro model of the blood-brain barrier by isolation and cultivation of human brain microvascular endothelial cells (HBMEC) (22, 34, 37). Upon cultivation on collagen-coated Transwell inserts, these HBMEC exhibit morphological and functional properties of tight junction formation and a polarized monolayer. These are shown by our demonstrations of tight junction proteins (such as ZO-1), adherens junction proteins (such as β-catenin), and their spatial separation, limited permeability to inulin (molecular mass, 4,000 Da), and development of high transendothelial electrical resistance (22, 34, 37). We have also developed the animal models of experimental hematogenous meningitis, which mimic the pathogenesis of E. coli meningitis in humans, e.g., hematogenous infection of the meninges (10, 11, 17, 21, 40, 41).Using these in vitro and in vivo models, we have shown that E. coli invasion of HBMEC is a prerequisite for penetration into the brain and requires specific microbial determinants (10, 11, 17-20, 40, 41). This was shown in animal models of experimental hematogenous E. coli meningitis; mutants of E. coli K1 deleted of the structures contributing to HBMEC invasion (e.g., Ibe proteins and CNF1) were significantly less able to penetrate into the brain than the parent strain despite having similar levels of bacteremia. We subsequently showed that these E. coli K1 determinants interact with their respective host receptors, involving host signaling molecules for efficient invasion of HBMEC (4, 15-20, 33), but the contributions of microbe-host interactions and host signaling molecules to E. coli K1 penetration of the blood-brain barrier remain incompletely understood.We have shown that host cytosolic phospholipase A₂α (cPLA₂α) contributes to E. coli K1 invasion of HBMEC (5), but the underlying mechanisms remain unclear. cPLA₂α selectively liberates arachidonic acid from the sn-2 position of membrane phospholipids (7) (Fig. ​(Fig.1),1), and we hypothesize that the contribution of cPLA₂α to E. coli K1 invasion of HBMEC is related to arachidonic acid metabolism. In the present study, we showed that 5-lipoxygenase (5-LO) and 5-LO-derived products of arachidonic acid, cysteinyl leukotrienes (LTs) (7, 29) (Fig. ​(Fig.1),1), contribute to E. coli K1 invasion of HBMEC, and their contributions occur via protein kinase C alpha (PKCα). More importantly, E. coli K1 penetration into the brain was inhibited by gene deletion of cPLA₂α and 5-LO as well as by the administration of cPLA₂α inhibitor and type 1 cysteinyl LT receptor (CysLT1) antagonist (Fig. ​(Fig.11).Open in a separate windowFIG. 1.A schematic diagram for cytosolic phospholipase A₂α (cPLA₂α), 5-lipoxygenase (5-LO), 5-LO-activating protein (FLAP), and leukotrienes (LTs), LTB4 and cysteinyl LTs (LTC4, LTD4, LTE4), that have been examined in this paper for their contributions to E. coli K1 invasion of the HBMEC monolayer and/or penetration into the brain. cPLA₂α selectively liberates arachidonic acid (A.A) from membrane phospholipids. Pharmacological inhibitors of cPLA₂α, 5-LO, and FLAP and antagonists of CysLT1 and BLT-1 are shown as arrows with ×.     

Prevention of Escherichia coli K1 Penetration of the Blood-Brain Barrier by Counteracting the Host Cell Receptor and Signaling Molecule Involved in E. coli Invasion of Human Brain Microvascular Endothelial Cells

Escherichia coli meningitis is an important cause of mortality and morbidity, and a key contributing factor is our incomplete understanding of the pathogenesis of E. coli meningitis. We have shown that E. coli penetration into the brain requires E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. E. coli invasion of HBMEC involves its interaction with HBMEC receptors, such as E. coli cytotoxic necrotizing factor 1 (CNF1) interaction with its receptor, the 67-kDa laminin receptor (67LR), and host signaling molecules including cytosolic phospholipase A₂α (cPLA₂α). In the present study, we showed that treatment with etoposide resulted in decreased expression of 67LR on HBMEC and inhibited E. coli invasion of HBMEC. Pharmacological inhibition of cysteinyl leukotrienes, lipoxygenated products of arachidonic acid released by cPLA₂α, using montelukast (an antagonist of the type 1 cysteinyl leukotriene receptor) also inhibited E. coli invasion of HBMEC. E. coli penetration into the brain was significantly decreased by etoposide as well as by montelukast, and a combination of etoposide and montelukast was significantly more effective in inhibiting E. coli K1 invasion of HBMEC than single agents alone. These findings demonstrate for the first time that counteracting the HBMEC receptor and signaling molecule involved in E. coli invasion of HBMEC provides a novel approach for prevention of E. coli penetration into the brain, the essential step required for development of E. coli meningitis.The mortality and morbidity associated with neonatal Gram-negative bacillary meningitis have remained significant despite advances in antimicrobial chemotherapy and supportive care. Inadequate knowledge of the pathogenesis has contributed to this mortality and morbidity (10-12). Escherichia coli is the most common Gram-negative organism that causes neonatal meningitis. Several lines of evidence from experimental animal models as well as human cases of E. coli meningitis indicate that E. coli penetrates into the brain initially in the cerebral vasculature (2, 13), but the underlying mechanisms contributing to E. coli penetration of the blood-brain barrier remain incompletely understood (10-12).We have developed an in vitro blood-brain barrier model by isolation and cultivation of human brain microvascular endothelial cells (HBMEC) (14, 20-22). Upon cultivation on collagen-coated Transwell inserts, the HBMEC exhibit morphological and functional properties of tight junction formation and a polarized monolayer. These properties are shown by our demonstrations of tight junction proteins (such as ZO-1), adherens junction proteins (such as β-catenin), and their spatial separation, limited permeability to propidium iodide (molecular mass, 668 Da) and inulin (molecular mass, 4,000 Da), and development of high transendothelial electrical resistance (14, 20-22). We have also developed an infant rat model of experimental hematogenous meningitis (8, 13). This animal model has important similarities to E. coli meningitis in humans, such as hematogenous infection of the meninges. Using these in vitro and in vivo models, we have shown that E. coli binding to and invasion of HBMEC are prerequisites for penetration into the brain (10-12).We have shown that E. coli K1 binding to and invasion of HBMEC require specific E. coli determinants (e.g., cytotoxic necrotizing factor 1 [CNF1] and OmpA), and these E. coli determinants contribute to HBMEC binding and invasion via interactions with their respective HBMEC receptors (10-12). For example, CNF1 contributes to E. coli K1 invasion of HBMEC via its interaction with 37-kDa laminin receptor precursor (37LRP)/67-kDa lamin receptor (67LR), while OmpA contributes to E. coli K1 binding to and invasion of HBMEC via its interaction with the HBMEC receptor, gp96 (3, 7, 9). We also showed that the E. coli determinants contributing to HBMEC binding and invasion exploit specific host signaling molecules for efficient invasion of HBMEC. For example, OmpA, NlpI, FliC, and IbeC (the E. coli structures contributing to HBMEC binding and invasion) are shown to exploit host cytosolic phospholipase A₂α (cPLA₂α) for E. coli invasion of HBMEC (4, 12, 25).We also showed that blockade of the HBMEC receptors and/or host signaling molecules was effective in preventing E. coli K1 invasion of HBMEC. For example, anti-67LR and -gp96 antibodies inhibited E. coli K1 invasion of HBMEC in a ligand-dependent manner (7, 9), and pharmacological inhibition of host cPLA₂α exhibited a dose-dependent inhibition of E. coli invasion of HBMEC (4). These findings suggest that inhibition of the HBMEC receptors and host signaling molecules involved in E. coli K1 invasion of HBMEC is likely to affect the ability of E. coli to penetrate into the brain.In screening drugs for their effects on the HBMEC receptors, we determined that etoposide (a topoisomerase inhibitor) decreased the expression of 67LR on HBMEC. cPLA₂α mediates agonist-induced release of arachidonic acid (6). We showed that the contribution of host cPLA₂α to E. coli invasion of HBMEC occurs via lipoxygenated products of arachidonic acid, cysteinyl leukotrienes (LTs), formed via LT biosynthetic pathways involving 5-lipoxygenase, and acting via the type 1 cysteinyl leukotriene receptor (CysLT1) (4, 12). More importantly, etoposide and montelukast (the CysLT1 antagonist) were additive in their prevention of E. coli K1 invasion of HBMEC and also efficient in preventing E. coli K1 penetration into the brain.     

Identification of Escherichia coli K1 antigen

We compared the use of bacteriophage sensitivity, seroagglutination with polyclonal antisera raised in rabbits or horses, seroagglutination with murine monoclonal antibody, and the serum agar precipitin technique for the detection of K1 capsular polysaccharide among clinical isolates of Escherichia coli obtained from blood stream infections. Some E. coli isolates failed to yield agreement among these tests, indicating that reliable detection of K1 antigen may require the use of multiple tests.     

Ferredoxin Is Involved in Secretion of Cytotoxic Necrotizing Factor 1 across the Cytoplasmic Membrane in Escherichia coli K1

We previously showed that cytotoxic necrotizing factor 1 (CNF1) contributes to Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) and interacts with the receptor on the surface of HBMEC. CNF1 is the cytoplasmic protein, and it remains incompletely understood how CNF1 is secreted across the inner and outer membranes in E. coli K1. In order to investigate the genetic determinants for secretion of CNF1 in E. coli K1, we performed Tn5 mutagenesis screening by applying β-lactamase as a reporter to monitor secretion of CNF1. We identified a Tn5 mutant that exhibited no β-lactamase activity in the culture supernatant and in which the mutated gene encodes a ferredoxin gene (fdx). In the fdx deletion mutant, there was no evidence of translocation of CNF1 into HBMEC. Western blot analysis of the fdx deletion mutant revealed that ferredoxin is involved in translocation of CNF1 across the cytoplasmic membrane. The fdx mutant exhibited significantly decreased invasion of HBMEC, similar to the decreased HBMEC invasion observed with the CNF1 mutant. The failures to secrete CNF1 and invade HBMEC of the fdx mutant were restored to the levels of the parent strain by complementation with fdx. These findings demonstrate for the first time that ferredoxin is involved in secretion of CNF1 across the inner membrane in meningitis-causing E. coli K1.Neonatal Escherichia coli meningitis is associated with high mortality and morbidity, and a major contributing factor is our incomplete knowledge on the pathogenesis of E. coli meningitis (15, 16). Most cases of neonatal E. coli meningitis develop as a result of hematogenous spread (8, 14), but it is incompletely understood how circulating bacteria cross the blood-brain barrier and cause meningitis.We have shown that cytotoxic necrotizing factor 1 (CNF1) contributes to E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) and penetration into the central nerve system (CNS) via the interaction with its receptor, 37 laminin receptor precursor (37LRP)/67 laminin receptor (67LR) (4, 12, 13). CNF1 is a cytoplasmic protein, and its secretion is a strategy utilized by meningitis-causing E. coli K1 to invade the blood-brain barrier (12). CNF1 is the paradigm of the RhoGTPase-activating bacterial toxins (2, 19). The CNF1 secretion pathway, however, remains incompletely understood. No typical signal peptide is found in the CNF1 sequence. A previous study by Kouokam et al. showed that CNF1 is tightly associated with outer membrane vesicles (18). Outer membrane vesicles from a number of bacterial species have been found to contain virulence factors, exhibit immunomodulatory effects, and adhere to and intoxicate host cells (20).In order to study the genetic determinants for secretion of CNF1 in meningitis-causing E. coli K1, we designed a Tn5 mutational screening strategy by applying TEM β-lactamase as the reporter. Using this approach, we identified a mutant which was defective in CNF1 secretion into HBMEC, and this mutant is characterized in this report.     

The Diffusion Permeability to Water of the Rat Blood-Brain Barrier

The diffusion permeability to water of the rat blood-brain-barrier (BBB) was studied. Preliminary data obtained with the Oldendorf tissue uptake method (Oldendorf 1970) in seizure experiments suggested that the transfer from blood to brain of labelled water is diffusion-limited. More definite evidence of such a limitation was obtained using the single injection technique of Crone (1963). ¹⁴C-labelled sucrose was used as intravascular reference substance and tritium-labelled water as test substance. The non-exchanging (transmitted) fraction, I-E = T, of labelled water during a single passage increased from 0.26 to 0.67 when the arterial carbon dioxide tension was changed from 15 to 85 mm Hg, a change increasing the cerebral blood flow about sixfold. This finding suggests that water does not pass the blood-brain barrier as freely as lipophilic gases.     

Involvement of IbeA in meningitic Escherichia coli K1-induced polymorphonuclear leukocyte transmigration across brain endothelial cells

Transmigration of neutrophil [polymorphonuclear neutrophil (PMN)] across the blood-brain barrier (BBB) is a critical event in the pathogenesis of bacterial meningitis. We have shown that IbeA is able to induce meningitic Escherichia coli invasion of brain microvascular endothelial cells (BMECs), which constitutes the BBB. In this report, we provide evidence that IbeA and its receptor, vimentin, play a key role in E. coli-induced PMN transmigration across BMEC. In vitro and in vivo studies indicated that the ibeA-deletion mutant ZD1 was significantly less active in stimulating PMN transmigration than the parent strain E44. ZD1 was fully complemented by the ibeA gene and its product. E. coli-induced PMN transmigration was markedly inhibited by withaferin A, a dual inhibitor of vimentin and proteasome. These cellular effects were significantly stimulated and blocked by overexpression of vimentin and its head domain deletion mutant in human BMEC, respectively. Our studies further demonstrated that IbeA-induced PMN migration was blocked by bortezomib, a proteasomal inhibitor and correlated with upregulation of endothelial ICAM-1 and CD44 expression through proteasomal regulation of NFκB activity. Taken together, our data suggested that IbeA and vimentin contribute to E. coli K1-stimulated PMN transendothelial migration that is correlated with upregulation of adhesion molecule expression at the BBB.     

Chemical characterization of Escherichia coli K 1 capsular polysaccharide

The exact chemical characterization of the prepared Escherichia coli K 1 capsule polysaccharide is necessary and a prerequisite for using this antigen as a screening antigen in the production of monoclonal anti-K 1-antibodies. The K 1-antigen, prepared by phenol-water-extraction, was analyzed by protein, RNA, and neuraminic acid determination. An addition, the antigen was subjected to elementary analysis, infrared- and 13C-NMR-spectroscopy, and gelchromatography. After testing the serological specificity, the K 1-antigen was identified as a high molecular polyneuraminic acid.     

Recombinant interleukin-1 stimulates clearance of Escherichia coli K1 bacteraemia

Enhancement of non-specific resistance to neonatal Escherichia coli K1 infection by interleukin-1 (IL-1) was analysed. Recombinant human IL-1 administered prophylactically to newborn LPS-non-responsive C3H/HeJ mice induced rapid clearance of E. coli 018:K1 bacteraemia. The effect was dose-dependent and was observed with mice treated immediately to 1 day before bacterial challenge, whereas treatment 2 days before challenge was ineffective. was ineffective. Clearance of intravenously injected radiolabelled 018:K1 E. coli suggested that IL-1 triggered defence mechanisms that contribute to bacterial sequestration and killing in the spleen and liver. Comparable increase in bacterial clearance occurred in naturally resistant LPS-responsive mice that had been subjected to transient E. coli K1 bacteraemia and showed increased resistance to reinfection. In the course of E. coli K1 bacteraemia a strong synthesis of acute phase reactants was observed in both susceptible and resistant mouse strains, which indicated that these proteins alone cannot confer natural resistance to E. coli K1. IL-1 induced a very rapid synthesis of acute phase proteins. The clearance of K1 E. coli when still viable in IL-1-treated animals suggested that acute phase proteins are not likely to be major mediators of the IL-1-enhanced non-specific resistance.     

The modification of phagocytosis by different K1 capsule preparations of Escherichia coli

The influence of two different types of Escherichia coli K1 capsule preparation on the phagosytosis of an E. coli laboratory strain (LN 28) by polymorphnuclear leucocytes (PMNL) is described. Capsule material of E. coli 0.18 and 0.83 was prepared a) from the culture supernatant and b) from a bacterial pellet fraction. When capsule material from the pellet was added to opsonin, before mixing the laboratory strain with the PMNL, it decreased the rate of phagocytosis, compared with untreated opsonin. The preparation from the supernatant of capsule showed no alteration of phagocytosic rate or capacity. These results are explained by the different chemical compositions of the preparations.     

Novel model to study virulence determinants of Escherichia coli K1

It is shown here for the first time that locusts can be used as a model to study Escherichia coli K1 pathogenesis. E. coli K-12 strain HB101 has very low pathogenicity to locusts and does not invade the locust brain, whereas the injection of 2 × 10⁶ E. coli K1 strain RS218 (O18:K1:H7) kills almost 100% of locusts within 72 h and invades the brain within 24 h of injection. Both mortality and invasion of the brain in locusts after injection of E. coli K1 require at least two of the known virulence determinants shown for mammals. Thus, deletion mutants that lack outer membrane protein A or cytotoxic necrotizing factor 1 have reduced abilities to kill locusts and to invade the locust brain compared to the parent E. coli K1. Interestingly, deletion mutants lacking FimH or the NeuDB gene cluster are still able to cause high mortality. It is argued that the likely existence of additional virulence determinants can be investigated in vivo by using this insect system.     

Simple technique for detecting K1 antigen of Escherichia coli.

The K1 antigen is a poor immunogen, its detection by serological means is difficult, and previously described methods using K1 specific bacteriophages require standardised suspensions of five different bacteriophages. A simple technique was developed which uses an unstandardised suspension of a single K1 specific bacteriophage applied with a 1 mm wire loop to bacteria streaked on to cystine lactose electrolyte deficient (CLED) medium. The technique correctly identified all 99 known K1 strains tested, including 14 strains negative with the serological method. Among 71 clinical isolates from urinary tract infections, the single bacteriophage method distinguished 30 K1 strains from 41 strains without this antigen. Suspensions of the bacteriophage were shown to remain fully active for at least two years when stored at 4 degrees C. It is concluded that this technique required so little in materials, time, or equipment that it could be routinely used in most laboratories.     

Survival and Implantation of Escherichia coli in the Intestinal Tract

Preliminary experiments established that a 0.5-ml inoculum that is introduced directly into the stomach of mice was cleared rapidly into the small intestine. Bicarbonate buffer, but not skim milk, protected such an inoculum from stomach acid until at least 90% of it had entered the small intestine. Passage and survival of various Escherichia coli strains through the mouse gut were tested by introducing a buffered bacterial inoculum directly into the stomach, together with the following two intestinal tracers: Cr(51)Cl(3) and spores of a thermophilic Bacillus sp. Quantitative recovery of excreted bacteria was accomplished by collecting the feces overnight in a refrigerated cage pan. The data show that wild-type E. coli strains and E. coli K-12 are excreted rapidly (98 to 100% within 18 h) in the feces without overall multiplication or death. E. coli varkappa1776 and DP50supF, i.e., strains certified for recombinant DNA experiments underwent rapid death in vivo, such that their excretion in the feces was reduced to approximately 1.1 and 4.7% of the inoculum, respectively. The acidity of the stomach had little bactericidal effect on the E. coli K-12 strain tested, but significantly reduced the survival of more acidsensitive bacteria (Vibrio cholerae) under these conditions. Long-term implantation of E. coli strains into continuous-flow cultures of mouse cecal flora or into conventional mice was difficult to accomplish. In contrast, when the E. coli strain was first inoculated into sterile continuous-flow cultures or into germfree mice, which were subsequently associated with conventional mouse cecal flora, the E. coli strains persisted in a large proportion of the animals at levels resembling E. coli populations in conventional mice. Metabolic adaptation contributed only partially to the success of an E. coli inoculum that was introduced first. A mathematical model is described which explains this phenomenon on the basis of competition for adhesion sites in which an advantage accrues to the bacterium which occupies those sites first. The mathematical model predicts that two or more bacterial strains that compete in the gut for the same limiting nutrient can coexist, if the metabolically less efficient strains have specific adhesion sites available. The specific rate constant of E. coli growth in monoassociated gnotobiotic mice was 2.0 h(-1), whereas the excretion rate in conventional animals was -0.23 h(-1). Consequently, limitation of growth must be regarded as the primary mechanism controlling bacterial populations in the large intestine. The beginnings of a general hypothesis of the ecology of the large intestine are proposed, in which the effects of the competitive metabolic interactions described earlier are modified by the effects of bacterial association with the intestinal wall.     

Six widespread bacterial clones among Escherichia coli K1 isolates.

Variable properties among Escherichia coli isolates include serotype, electrophoretic migration of major outer membrane proteins, metabolic properties, production of hemolysin or colicin or both, and plasmid content. These characteristics were compared in E. coli strains of capsular types K1, K5, K92, and K100 and in non-encapsulated isolates. The 234 bacterial strains from the United States and Europe which we studied had been isolated from healthy or diseased individuals recently or as long ago as 1941. Regardless of source, most O7:K1, O16:K1, and O75:K100 isolates could be assigned to three unique, serotype-specific groups, which were interpreted as representing three bacterial clones. Two bacterial (sub)clones each were discerned among the O18:K1 and O18:K5 isolates, and two further, distinct clones were discerned among the O1:K1 isolates. The implications of these results for epidemiological analyses and for virulence are discussed.     

Breast milk lymphocyte response to K1 antigen of Escherichia coli.

Comparison milk and blood lymphocyte blastogenic responses to the K1 antigen of Escherichia coli and lipopolysaccharide (LPS) from E. coli O127,B8 were examined in 16 postpartum women by [3H]thymidine uptake. Rabbit hemolysincoated sheep erythrocyte monolayers were used to deplete macrophages from milk lymphocyte preparations and to enrich for T lymphocytes in order to make milk preparations more comparable to blood preparations. Response was defined as a stimulation index of greater than or equal to 2.0. There was no evidence of selective response to K1 antigen by milk lymphocytes, since both blood and milk lymphocytes responded in four women and neither blood nor milk lymphocytes responded in nine. Milk lymphocytes alone responded to K1 in one woman, whereas blood lymphocytes alone responded in two women. Additional nonpaired milk or blood cultures were available from three women. None of these responded to K1 antigen. Corresponding lymphocyte cultures were stimulated with LPS. A positive K1 response was always accompanied by an LPS response, and the LPS response correlated with the K1 response in 17 of 19 women. Stool cultures examined with an antiserum agar showed no correlation between the presence of K1 E. coli in the stool and milk or blood lymphocyte response to K1 antigen. In the system used here, no selectivity of response of breast milk lymphocytes to K1 antigen was noted.     

Isolation of bacteriophages specific for the K1 polysaccharide antigen of Escherichia coli.

Five bacteriophage stocks were prepared after enrichment of a sewage sample using Escherichia coli 02:K1:H4 (strain U9/41). The bacteriophages were tested for their ability to lyse 224 strains of E. coli that had been tested for the presence of the K1 antigen by means of an antiserum-agar diffusion technique, using a meningococcus group B antiserum known to detect the E. coli K1 antigen. The standard test strains for E. coli K antigens 2 to 99 were used as control strains. Of the 101 strains found to possess the K1 antigen using the antiserum-agar technique, 93 were lysed by at least one of the bacteriophages, whereas 8 of the 123 strains apparently lacking K1 were lysed by one or more of the bacteriophages. None of the standard test strains for K antigens 2 to 99 was lysed by any of the bacteriophages. The eight strains thought to lack K1 but that were lysed by bacteriophage were re-examined by immunoelectrophoresis, using meningococcus group B antiserum; five of the eight strains gave a precipitin line corresponding to K1. The use of K1-specific bacteriophages offers an inexpensive and easy method for the identification of the K1 antigen.