Melioidosis, an infectious disease caused by Pseudomonas pseudomallei is a significant health problem in Thailand. This disease has been reported since 1912, but there are still many questions remaining. Among those questions are the role of glycocalyx in the chronicity and recrudescence of the infection. The present communication reports a study on the mode of growth of P.pseudomallei in culture media and in the lung tissue of infected humans and animals. The resistance of P. pseudomallei biofilms formed in vitro using the Modified Robbins Device (MRD) to ceftazidime (CTZ) and cotrimoxazole (SXT) was also studied. The experimental infection in animal was carried on in male guinea pigs. The interaction of guinea pig alveolar macrophages (AMs) with P.pseudomallei was examined. The viable count of P.pseudomallei and the immune complex levels were determined in guinea pigs lungs, spleen, kidney and blood at various time intervals after intratracheal inoculation with P.pseudomallei. Specific antibody to P.pseudomallei was determined using indirect fluorescent antibody (IFA) technique as well as the specific antigen (P.pseudomallei) in lungs, spleen and kidney. The role of circulating immune complex (CIC) in melioidosis was examined by detection of the CIC and specific antibodies to P.pseudomallei in sera of six patients who had either localized or disseminated melioidosis. CIC was detected by Raji-RIA and specific antibodies to P.pseudomallei were detected by IFA technique. The results showed that in culture media, P.pseudomallei cells grow in microcolonies within large amounts of intercellular fibrous glycocalyx material. In vivo study of humans and animals showed the bacterial cells growing in the lung tissue were surrounded by radially arranged fibres that constitute a very well-defined glycocalyx structure. In the infected areas of the animal lung tissue, bacterial cells could be seen with glycocalyx-enclosed microcolonies that displaced host cell components. P.pseudomallei biofilm cells were resistant to CTZ and SXT. A large number of these biofilm cells remained viable at 12 hours and negligible number of cells were killed after 24 hours except in the biofilm treated with 200 times the MIC of CTZ. When 10(4)-10(6) CFU/GP of either planktonic or agar-protected P.pseudomallei cells were introduced into normal guinea pigs the animals always developed an acute infection and all of them died within 3 days. No animal was found to clear the infection even those challenged with an inoculum of 10 CFU/GP of either agar-protected or unprotected planktonic cells. However, with the smaller inoculum, the guinea pig developed less acute infections. Although the dissemination of bacterial cells from the site of entry to other organs was detected in sacrificed animals, the organisms were rarely detected in the blood of the animals with chronic symptom. Blood obtained from animals that died from either the acute or exarcerbation of chronic disease always contained large numbers of bacteria, suggesting that septicemia is associated with fatal illness in guinea pigs which is similar to the picture seen in human melioidosis. By TEM the bacteria were seen. within virtually every host cell type involved in the inflammation of lung tissue, such as neutrophils, monocytes and macrophages. Many host cells showed signs of damage and the presence of bacteria in unusual locations, such as the nuclei of polymorphonuclear phagocytes, indicating that effective phagocytosis was not occurring. The interaction of guinea pig alveolar macrophage (AMs) with P.pseudomallei showed that P.pseudomallei was phagocytosed by AMs but was resistant to killing and digestion, resulting in intracellular multiplication detected both in vitro and in vivo. The multiplication of intracellular bacteria began 6 hours after incubation. There is a marked decrease in viability of AMs infected with viable P.pseudomallei or sterile culture filtrate of this organism, after incubating for 21 and 24 hours respectively. Moreover, the superoxide released from the P.pseudomallei infected AMs was significantly decreased. In the first week following the inoculation of <10 CFU/GP the organism multiplied in the lungs reaching the highest count of 10(5)-10(8) CFU/lungs resulting in the spread of P.pseudomallei to various organs i.e. spleen (102108 CFU/spleen) and kidney (10(2)-10(5) CFU/kidney). Antibody became detectable in the second week as did the immune complex and antigen which was found in lungs, kidney and spleen. The viable count meanwhile became lower or non culturable in certain animals. By the third week the viable count of P.pseudomallei became negative in the kidney and spleen in the majority of animals, only a few animals still had 10(3)-10(4) CFU in lungs. Immune complexes were constantly found in the lungs, kidney and spleen except in one animal. In the fourth week of infection, the organism was cleared from every organs studied, whereas the IFA of P.pseudomallei in those organs still remained positive. In an experimental model of guinea pig that had recovered from previous infection and which was subsequently subjected to long term treatment with dexamethasone for 3-4 weeks, the cryptic infection became apparent confirming the recrudescent nature of melioidosis as observed in human melioidosis. CIC was positive in all disseminated melioidosis patients, except in one kidney transplanted patient with disseminated melioidosis who was receiving an immune suppressive agent. No CIC were detected from two localized melioidosis patients. The result from this study can explain some aspects of the pathogenesis of melioidosis. The production of glycocalyx and the formation of microcolonies in lung tissue will protect the organism from various hostile conditions in the host environment such as

the phagocytes and the antimicrobial agents used for therapy. These conditions will allow the organism to persist silently in the host and to cause chronic infection. The success of P. pseudomallei as an intracellular parasite is another mechanism that prevents animals still had 10(3)-10(4) CFU in lungs. Immune complexes were constantly found in the lungs, kidney and spleen except in one animal. In the fourth week of infection, the organism was cleared from every organs studied, whereas the IFA of P.pseudomallei in those organs still remained positive. In an experimental model of guinea pig that had recovered from previous infection and which was subsequently subjected to long term treatment with dexamethasone for 3-4 weeks, the cryptic infection became apparent confirming the recrudescent nature of melioidosis as observed in human melioidosis. CIC was positive in all disseminated melioidosis patients, except in one kidney transplanted patient with disseminated melioidosis who was receiving an immune suppressive agent. No CIC were detected from two localized melioidosis patients. The result from this study can explain some aspects of the pathogenesis of melioidosis. The production of glycocalyx and the formation of microcolonies in lung tissue will protect the organism from various hostile conditions in the host environment such as

the phagocytes and the antimicrobial agents used for therapy. These conditions will allow the organism to persist silently in the host and to cause chronic infection. The success of P. pseudomallei as an intracellular parasite is another mechanism that prevents the bacteria from being eliminated by the host defense mechanisms resulted in chronic manifestation or long latent period of infection. The AMs also transmitted the intracellular bacteria to various organs which create the multiorgan involvement and the difficulty of eradication of the infection. The guinea pig model also demonstrates the ability of P.pseudomallei to remain in visceral organs. Any condition capable of impairing host immunity would lead to the reactivation of the organism resulting in the relapse of infection. Mimicking the human pattern, the animal disease caused by P.pseudomallei has both acute and chronic courses depending partially on the inoculum size and the type of bacteria whether unprotected or protected by an envelop such as agar which may represent microcolonies. The relationship between septicemic melioidosis and the high mortality rate may be partially explained by tissue injury due to IC which causes renal failure and septic shock at the terminal stages of the disease.