Research On The Relation Of Free-living Amoebae And Its Intracellular Bacterium

Free-living amoebae (FLA) are a diverse group of ubiquitous unicellular organism. It exists in nature either as a trophic amoeba, feeding on the bacteria in soil and water, or as a dormant cyst. The amoebae, including Acanthamoeoba spp, Balamuthia manrillaris, Naegleria fowleri, Sappinia diploidea, are capable of causing human disease. They have been recognized as ecologic agents of encephalitis, some of the infectious are opportunistic, occurring mainly in immunocompromised hosts (Acanthamoebae and Balamuthia encephalitides), while others are non-oppotunitic (Acanthamoeba keratitis, Naegleria meningoencephalitis, and cases of Balamuthia encephalitides occurring in immunocompromised humans).Most importantly, many bacteria were found to survive and grow within the amoeba. Thus free-living amoebae were crowned as"Trojan horses" of the microbial world. FLA have evolved along a line of phagocytic cells engulfing entire bacterial cells as nutrients. They participate in nutrient and energy turnover in nature, acting as predators controlling bacterial populations and altering prevalence of bacterial species in the environment. As some of the engulfed organisms may avoid phagosomal lysis and maintain their intracellular viability, occasional infections may result in the destruction of the host cell. If the defensive mechanisms are inefficient or impaired, the internalized bacteria may persist intracellular and co-adaptation to a symbiotic relationship may evolve. Organisms which can resist digestion after being uptaken by the amoebae are called the amoebae-resisting bacteria (ARB). Legionella was the very first pathogen demonstrated to be able to multiply and persist in free-living amoebae, which are common inhabitants of natural aquatic environments and water systems. Since then, several other intracellular bacterial pathogens were found to be able to infect free-living amoebae, including Legionella-like amoebal pathogenitic, Chlamydia pneumoniae, parachlamydiaceae, Afipia felis, Burkholderia cepacia, Burkholderia pseudomalleis, E coli 0157, Listeria monocytogenes, Mycobacterium avium, Mycobacterium bivis, Simkania negevensis, Francisella tularensis, Vibrio cholerae, Cryptococcus neoformans, and the great Minivirus. Hense, study on the intracellular bacteria within the amoebae became one of the focus in modern microbiological field.More and more datum show the FLA and its endosymbionts are associated with the nosocomial or community-acquired pneumonia of unknown etiology, Nosocomialpneumonia, a frequent complication associated with considerable illness and death, is the leading cause of death from nosocomial infections. Community-acquired pneumonia, which is also common, is associated with a case-fatality rate of up to 8.8%. Despite use of standard diagnostic methods, no microbial cause could be identified in 47% to 55% of CAP worldwide in adults and 20%-75% of nosocomial pneumonia. Legionella spp.and Listeria monocytogenes are common bacteria in the water supply, ventilating and air conditioning units systems. Amoeba may act as reservoirs and vectors in the environment. Since amoeba has a cosmopolitan, people are at the high risk of the exposure to the ARB Hense, we begin the research on the association of the amoebae and its endosymbionts. Objective? to investigate the symbiosis between Chlamydia spp and free-living amoebae;(2)to establish methods of amoebal co-culture for the isolation and identification the Chlamydia, (3)to isolate and identify of the amoeba and its endosymbionts in the communist and hospital acquatic environment;(Dto isolate and identify the agents of clinical respiratory diseases with the amoebal co-culture methods;?to investigate the interaction between the Vibrio cholera and free-living amoebae. MethodsThe main Methods in our research include:the amoebal co-culture; bacteria culture and Giemensa/Giemensez-staining; electron microscope technology; molecular technology and so on. Results1. When Co-cultured with the FLA, Chlamydia spp (parachlamydia Acanthamoeba bn9, Chlamydia psittaci CGI, Chlamydia trachomatis TE55) were found to be able to survive and grow within the Acanthamoeba. Entry by phagocytosis of elementary bodies revealed their infective nature, elementary bodies could be differentiated in the reticulate bodies, which could divide by binary fission and were able to invade the amoebal cytoplasm. The reticular bodies located mainly in the vacuoles and less frequently within the amoebal cytoplasm. In the vacuoles, the reticulate bodies could be condensed in elementary bodies, which are released after amoebal lysis or expelled within vesicles. The amoebal size increased with infection partially due to vacuole formation and/or dilatation. The infected trophozoites were able to encyst partly, and the elementary bodies were observed in the vacuoles with in the cysts. Parachlamydia acanthamoeba were able to obligate intracellular grow within amoebae likeAcanthamoeba castellanii, Acanthamoeba polyphaga and Nergleria spp. Multiplication of Parachlamdiae result in the lysis of the amoebae, and both of the elementary bodies and reticular bodies were observed out of amoebae. A third developmental stage —the crescent body was observed, and its presence inside the amoebae was associated mainly the prolonged incubation time. The Chlamydia psittaci (CGI) and Chlamydia trachomatis (TE55) could grow within the trophozoites and survive in the cysts, so there exist the symbiosis between the Acanthamoeba and the CGI and TE55. Reticulate bodies of TE55 divided in the cytoplasm, not in the vacuoles The amoebae infected by CGI and TE55 were able to grow in trophoziotes without leading trophozoites' lysis. So elementary bodies were mainly expelled out of the amoebae.To establish the amoebal co-culture methods for the isolation and identification of Chlamydiae by the technology of Gimensa/Gimensez-staining and electron microscopy. The chlamydia bodies could be identified with the Giemensa/Giemensez-staining, parachlamydia acantbamoeba show small coccoid in red (G/e/we/ttor-staining) or small coccoid in deep blue (G/e/we/wez-staining) within the trophozoites. Electron micrograph of amoebae can demonstrate the presence of bacteria at different developmental stages typical of the Chlamydiales. Beside the parachlamydiae, other family members of chalmydiae like Chlamydia psittaci, Chlamydia trachomatis could also be cultured with Amoebal co-culture methods.2 We have observed trophozoites growing on the non-nutrient agar injected with the filter and recovered 25(33.8%) strains from the cool water of 74 environmental samples. Among them, 21(35.6%) strains for the 59 hospital water samples while 4(26.7%) strains for hotel cool water. The positive of amoebae of hospital water samples is higher than that of hotel cool water. There was the significant differences of ratio between the cool water and tower water in hospital( 41.0% V 25%, x2= 4.89, p<0.027). No strains were recovered from the tower water while 4 strains were isolated in the cool water in hotels. The positive rate in cooling water was higher than that of tower water(40%V20.8%, x 2 = 7.784, p=0.005). For the month distribution is concerned, positive rate were up to 50% from Jun. to Aug. while it decreased to 25% and 16.7% on Sept and Oct. respectively.The recovered 25 strains were tested with PCR technology. All were positive for the amoeba common primers. 15 out of 25 strains were positive with special-genusAcanthamoeba primers and 2 were positive for special Neagleria primers. The remainders were negative for either of Acanthamoeba or Nergleria, belonged to other amoebae. The dominant amoebae were Acanthamoeba spp in the hospital cooling water. Nergleria spp were recovered from the cooling water in hotels while none in hospital cooling water. 30(54%) strains of Legionella were recovered from 59 water samples in hospital, most of which were LP1. Both of amoebae and Legionella spp. were recovered from 13 (21%) water samples.Products of PCR with primer Al of sample B09 were sequenced, about 518bp called PDlwere obtained The sequence was compared with those available in the GenBank database by using the blastn 2.2.2 program. The sequence of isolated blasted with that oi Acanthamoeba sp. with 95 % identified, suggesting that the isolate belong to the genus Acanthamoeba. Phylogenetic analysis showed it was close to the member of Acanthamoebae, representing a new species within the genus Acanthamoebae.3. After amoebal co-cultured, six (1.92%) out of 312 respiratory sputum samples were positive to the chlamydia 16s rDNA for PCR test. But Chlamydia-like organisms failed to be recovered. When sequenced, one product, called cc66, was shown to belonge to Simkania negevensis while others termed cc56, cc76, ccl89, cc235, cc289, were related to the Chlamydiae. Percentages of homology were estimated from the nucleotide differences on the 305 retain sites. The sequence of CC66 has 95% similarity with the sequence of Simkania negevensis, the sequences of others showed 80%-85% similarity with each other. Phylogenetic reconstructions based a 305bp fragment assigned cc66 to the Simkania negevensis lineage and others to Simkania lineage.Additionally, some Bacteria were observed within the trophozoites from the 25(8.01%) clinical samples when co-cultured with amoebae. After being further cultured in the media of blood and BCYE media. 5 strains were recovered and identified, One belonging to the Candidatus spp, three to Stenotrophomonas maltophilia and one to Serratia marcescens.4.The interaction between the Vibrio cholerae 0139 with Acanthamoeba spp and Naegleria spp. were investigated. For the Acanthamoebapolyphaga, V. cholerae 0139 were observed to survive and grow within the trophozoites, It was found to be located both in the vacuole of cytoplasm and interspaces of two-layer wall of the cysts and itcould be re-isolated from excysting of amoebae At 30°C, V. cholerae 0139 could survive more than 120 days when cocultured with amoebae while it survive less than 45days in the PAS. While the number of viable V. cholerae decreased and reached undetectable levels by 30-day's incubation at 4°C both for the research group and control group V. cholerae 0139 within the cysts could be re-isolated after excystment in medium PYG712 at 30°C.It can survive more than 75 days within cysts. However cysts of amoeba lysed and even died after incubating more than 90 days at 4°C .V. cholerae 0139 within the cyst could not be isolated again. These findings indicate V. cholerae 0/39 can grow and survive in Acanthamoeba polyphaga. Survival time of V. cholerae 0139 can be improved within the cysts of Acanthamoeba polyphaga in low temperature.As far as the Naegleria sp is concered, Viable V cholerae 0139 were observed within the amoebal vacuoles within 4 hours. The vacuoles were filled more bacteria after co-cultured longer. Electron microscopy showed that after being uptaken, V cholerae 0139 could be embraced with the thick smooth endoplasmic seficulum, which later would form the vacuole for 0139 to survive and multiply. The matured vacuoles would lead trophozoites lysed and then expelled out. The 0139 were also found to be located both in the vacuole of plasm and the inter space of two-layer wall. It could also be recovered from excysting of Naegleria flowri. But the 0139 could not isolated from both the study group(being co-cultured with amoebae) and the control group( without amoebae) after 30 days at 4°C, the inner bacteria within the cysts could not be isolated when cysts were excited at 30°C. These findings indicate a symbiotic relationship exists between V. cholerae 0139 and Naegleria spp. However, the survival time of V. cholerae 0139 cannot be improved within the cysts of Naegleriaflowri in low temperature.Additionally, one strain of Acanthamoeba, called JD6, was isolated and identified from a pond that once led to an outbreak of cholera. A Strain of V. cholerae 0139 isolated from the same pond was confirmed to survive and growth in the trophozoites of JD6, which suggested a symbiotic relationship exist between V. cholerae 0139 and JD6.Conclusion 1. Parachlamydia acanthamoeba were able to obligate intracellular grow within the free-living amoebae and a new stage, termed crescent bodies could be obverted. 2. Chlamydia spp (Chlamydiapsittaci, Chlamydia trachomatis) were able to...