| Abstract: | ![]()
Parachlamydia acanthamoebae is an obligately intracellular bacterium that infects free-living amoebae and is a potential human pathogen in hospital-acquired pneumonia. We examined whether the presence of P. acanthamoebae is related to the presence of Acanthamoeba in an actual hospital environment and assessed the in vitro survival of P. acanthamoebae. Ninety smear samples were collected between November 2007 and March 2008 (trial 1, n = 52) and between October 2008 and February 2009 (trial 2, n = 38) from the floor (dry conditions, n = 56) and sink outlets (moist conditions, n = 34) of a hospital. The prevalences of P. acanthamoebae DNA in the first and second trials were 64.3% and 76%, respectively. The prevalences of Acanthamoeba DNA in the first and second trials were 48% and 63.1%, respectively. A statistical correlation between the prevalence of P. acanthamoebae and that of Acanthamoeba was found (trial 1, P = 0.011; trial 2, P = 0.022), and that correlation increased when samples from just the dry area (floor smear samples, P = 0.002) were analyzed but decreased when samples from a moist area were analyzed (P = 0.273). The in vitro experiment showed that, without Acanthamoeba, P. acanthamoebae could not survive in dry conditions for 3 days at 30°C or 15 days at 15°C. Thus, both organisms were coincidentally found in an actual hospital environment, with the presence of Acanthamoeba having a significant effect on the long-term survival of P. acanthamoebae, suggesting that this potential human pathogen could spread through a hospital environment via Acanthamoeba.Chlamydiae, which are obligate intracellular bacterial pathogens, have been reclassified as the order Chlamydiales, which includes four families: Chlamydiaceae, Parachlamydiaceae, Waddliaceae, and Simkaniaceae (5). The family Chlamydiaceae is well known to have a broad range of distribution in animals and humans and to be the causative agents of human diseases (4, 20, 24, 31, 36). This family includes two major human pathogens: Chlamydophila pneumoniae, a causal agent of common respiratory infection and also suspected of being involved in some chronic diseases, such as asthma and atherosclerosis (6), and Chlamydia trachomatis, responsible for sexually transmitted disease and preventable blindness (33).Parachlamydiaceae, Waddliaceae, and Simkaniaceae have recently been recognized as chlamydiae that exhibit a wide range of distribution in the natural environment, such as in rivers and in soil (9). All these species can grow and survive dependently within the free-living amoeba Acanthamoeba, the most abundant genus of amoeba (1, 9, 11, 12). Parachlamydia acanthamoebae and Simkania negevensis have been associated with lower respiratory tract infections (3, 27), and Waddlia chondrophila, which was originally isolated from an aborted bovine fetus, is considered a potential abortogenic agent (9). There is accumulating evidence supporting the pathogenic role of P. acanthamoebae in humans (3, 9, 13). Several studies have reported that parachlamydial DNA was detected by PCR in mononuclear cells of sputa and in bronchoalveolar lavage samples from a patient with bronchitis (10). Other studies have suggested that P. acanthamoebae may cause inhalation pneumonia and be responsible for hospital-acquired pneumonia in HIV-infected patients and organ transplant recipients receiving immunosuppressive therapy (7, 8, 14). Thus, P. acanthamoebae, presumably spreading through amoebae, is emerging as a potential etiological agent of hospital-acquired pneumonia. However, the overlap between the distribution of P. acanthamoebae and that of Acanthamoeba in hospitals and the survival of the bacteria in harsh conditions without Acanthamoeba remain unknown. We examined whether the prevalence of P. acanthamoebae correlates with that of Acanthamoeba in a hospital in Sapporo, Japan. We also examined the in vitro survival of P. acanthamoebae and its requirement for Acanthamoeba.P. acanthamoebae Bn9 (ATCC VR-1476) was purchased from the American Type Culture Collection. The bacteria were propagated in an amoeba cell culture system according to methods described previously (17). The numbers of infective progeny were determined according to the procedure described below. Free-living amoebae, Acanthamoeba castellanii C3 (ATCC 50739), were purchased from the American Type Culture Collection. Amoebae were maintained in PYG broth (0.75% [wt/vol] peptone, 0.75% [wt/vol] yeast extract, and 1.5% [wt/vol] glucose) at 30°C (35). The numbers of infective progeny of P. acanthamoebae were determined by a procedure known as the amoeba infectious unit (AIU) assay using coculture with amoebae as described previously (28).Ninety smear samples were obtained from the floor (n = 56) and sink outlets (n = 34) of a hospital (Hokkaido University Hospital) containing approximately 900 beds (number of outpatients, approximately 3,000 per day; number of hospital patients, approximately 1,000 per day) in a 12-story building located in the central area of Sapporo, Japan, from November 2007 to March 2008 (trial 1, n = 52) and from October 2008 to February 2009 (trial 2, n = 38). The samples were collected by wiping an approximately 1-m2 area on the floor or sink outlet with sterilized gauze (approximately 25 cm2) moistened with Page''s amoeba saline (PAS) (30). Each piece of gauze was then vortexed for 60 s in 20 ml sterilized PAS containing 0.05% (vol/vol) Tween 80, and the suspension was then centrifuged at 2,100 × g for 20 min. Pellets were resuspended in 200 μl PAS and then used for P. acanthamoebae culture and DNA extraction.One-half of the resuspended smear pellet (100 μl) was used for P. acanthamoebae culture. Culture detection was performed by a method based on amoeba lysis described previously (18). In brief, serially diluted sample solution was added to 100 μl of amoeba suspension containing 1 × 105 A. castellanii C3 cells in 1 well of a 96-well microplate and incubated for up to 10 days at 30°C in a normal atmosphere. The microplate was read daily to determine the highest dilution of bacteria that led to amoeba lysis.The DNA extraction was performed using the UltraClean soil DNA extraction kit (MBL, Carlsbad, CA) according to the manufacturer''s instructions. The primers used in PCR amplification were as follows: PacI (5′-GAG GTG AAG CAA ATC CCA AA-3′) and Pac2 (5′-CTC CTT GCG GTT AAG TCA GC-3′) for amplification of P. acanthamoebae 16S rRNA (191 bp), JDP1 (5′-GGC CCA GAT CGT TTA CCG TGA A-3′) and JDP2 (5′-TCT CAC AAG CTG CTA GGG AGT CA-3′) for amplification of 18S rRNA from Acanthamoeba species (423 to 551 bp) (34), and Bac11 (5′-GAG GAA GGT GGG GAT GAC GT-3′) and Bac12 (5′-AGG CCC GGG AAC GTA TTC AC-3′) for amplification of bacterial 16S rRNA excluding the order Chlamydiales (216 bp) (38). The primers for amplification of P. acanthamoebae 16S rRNA were designed based on GenBank cDNA sequences (accession number NR026357) using the program Primer 3 (http://frodo.wi.mit.edu/primer3/input.htm). To overcome inhibition of PCR amplification by humic acid, bovine serum albumin (BSA) was added to each reaction mixture according to methods described previously (23, 39). The quality of extracted DNA was confirmed by PCR amplification using universal primers that target bacterial 16S rRNA, which is conserved across a broad spectrum of bacteria. All smear samples (n = 90) yielded PCR amplicons of the expected size and were then used in specific P. acanthamoebae and Acanthamoeba PCRs. Search results returned by the BLAST program showed that the primers used for each PCR were specific for P. acanthamoebae and Acanthamoeba detection. The amount of template DNA used in each PCR was 2 μl. The total reaction volume was 25 μl and consisted of 200 μM each deoxynucleoside triphosphate (dNTP), 10 μM BSA, 1× commercial reaction buffer, and 0.625 U Taq DNA polymerase (New England Biolabs, Herts, United Kingdom). The thermal cycling profile involved an initial denaturation step at 94°C for 10 min; 35 cycles, each consisting of 30 s of denaturation at 94°C and 30 s of annealing at 60°C for P. acanthamoebae 16S rRNA, 60°C for Acanthamoeba species 18S rRNA, and 52°C for bacterial 16S rRNA; and a 45-s extension at 72°C. The amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. The presence of amplified target genes in randomly selected positive specimens was confirmed by direct oligonucleotide sequencing of the PCR products (Macrogen, Seoul, South Korea). As a quality control for each PCR, diluted DNA extracted from the P. acanthamoebae Bn9 or A. castellanii C3 strain was used in each amplification. As a negative control, DNA-free water (Sigma) was also used in amplification. To prevent contamination, the preparation of the PCR mixture was performed in a separate room. Alignment analysis and the construction of a phylogenetic tree for P. acanthamoebae amplicons (n = 4) with previously reported Parachlamydiaceae sequences were performed using Genetyx-Mac software (version 10.1) and the neighbor-joining method in MEGA software (version 4). The detection limits of the PCR for P. acanthamoebae 16S rRNA and Acanthamoeba 18S rRNA were examined by using DNA extracted from the sterilized gauze moistened with PAS that had been spiked with defined numbers of P. acanthamoebae AIU and Acanthamoeba cells, respectively. The detection limits of the PCR targeted to P. acanthamoebae and Acanthamoeba were 102 AIU and 10 cells, respectively.The procedure for monitoring bacterial viability was as follows. A bacterial solution of 100 μl containing approximately 107 to 108 AIU prepared with PAS was placed into wells of a 24-well plate with (moist conditions) or without (dry conditions) 900 μl PAS, and the plates incubated for up to 28 days at 15°C or 30°C in a normal atmosphere. At 0, 3, 7, 15, and 28 days after inoculation, the supernatant in each well was collected and centrifuged at 3,500 × g for 30 min. In the case of the samples for moist conditions, supernatants were directly transferred to a centrifuge tube. For the dry condition samples, the wells were washed with 900 μl PAS and this was then transferred to a centrifuge tube. The resulting bacterial pellet was resuspended in 100 μl PAS. The numbers of infective progeny as a marker of bacterial viability in the solution were determined by the AIU assay as described above. The bacterial membrane integrity as a possible indicator for bacterial viability was also confirmed with fluorescence microscopy by using a Live/Dead reduced biohazard viability/cytotoxicity kit (Molecular Probes, Eugene, OR), according to the manufacturer''s instructions.The correlation between the frequency of P. acanthamoebae and that of Acanthamoeba spp. was analyzed by Fisher''s exact test. The influence of floor level on the prevalence of both organisms was also analyzed by a two-way analysis of variance (ANOVA) test. Comparison of bacterial numbers in the in vitro experiment was assessed by an unpaired t test. A P value of less than 0.05 was considered significant. |
