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The occurrence and epidemiology of intestinal spirochaetes in humans in Western Australia

Brooke, Celia Josephine (2003) The occurrence and epidemiology of intestinal spirochaetes in humans in Western Australia. PhD thesis, Murdoch University.

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      The intestinal spirochaetes Brachyspira aalborgi and Brachyspira pilosicoli colonise the large intestine of humans, whilst B. pilosicoli also colonises a number of animal species. Both organisms have been detected in human intestinal spirochaetosis (HIS), a syndrome where the spirochaetes attach end-on to the colorectal epithelium, forming a characteristic false brush border. Colonisation by B. pilosicoli occurs at 30% in Australian Aborigines (Lee and Hampson, 1992), homosexual males (Trivett-Moore et al., 1998), human immunodeficiency virus (HIV)-positive individuals and individuals from developing countries (Barrett, 1990; Trott et al., 1997a). Studies on B. aalborgi have focussed mainly on its detection from Caucasians.

      Clinically, colonisation with Brachyspira spp. has been linked to gastrointestinal disorders, including chronic diarrhoea, abdominal pain and rectal bleeding. More seriously, invasive colitis and hepatitis associated with intestinal spirochaetes have been recorded, and B. pilosicoli has been isolated from the blood of nine patients. However, the pathogenic significance of Brachyspira spp. remains uncertain, due to an inconsistent clinical picture, carriage by healthy individuals and the absence of accurate spirochaete identification in many studies.

      Identification of the organisms by culture and microscopy has been difficult, due to morphological similarities and biochemical heterogeneity of the Brachyspira spp.. In addition, B. pilosicoli is the easier spirochaete to culture, and so has predominated in studies assessing faecal carriage. The recent development of molecular methods has allowed unequivocal speciation of the Brachyspira spp. Thus the main aim of this thesis was to examine the prevalence and epidemiology of B. pilosicoli and B. aalborgi in Australia, using this new methodology.

      Intestinal carriage of both species was examined in faecal specimens using culture, microscopy and two species-specific 16S rRNA PCRs. Carriage of Brachyspira spp. was determined in individuals from rural areas of Western Australia (WA) with gastrointestinal symptoms, (including 151 Aboriginal and 142 non-Aboriginal Australians), 227 migrants to Australia, and 90 healthy control individuals from Perth, Western Australia. When samples were assessed by culture, B. pilosicoli was isolated from the faeces of 10.6% of migrant and 9.9% of Aboriginal Australian individuals. B. pilosicoli carriage was significantly higher in Aboriginal children aged 2 - 4 years (P = 0.0027) and migrants of African, Middle Eastern or Asian origin (P = 0.0034). Carriage in each population was associated with the detection of a number of intestinal parasites. It was not clear whether an association existed between B. pilosicoli isolation and disease, as similar proportions were found in sick Aboriginal patients and presumably healthy migrant individuals. B. pilosicoli may have caused disease in Aboriginal children where other pathogens were not present, or may have contributed to disease with known or suspected pathogens. In particular, colonisation was significantly more frequent in individuals with chronic diarrhoea or failure to thrive (FTT) (P = 0.016), and was also increased in elderly patients. The association of B. pilosicoli with parasites may have been due to behavioural factors, or an as yet unknown relationship between the organisms. B. aalborgi was not isolated from the specimens of rural individuals or migrants, as culture media and conditions for this organism were not optimal, but was thought to have been the spirochaetes seen by microscopy in culture negative samples. As a result, a more appropriate isolation medium for B. aalborgi was developed, containing brain heart infusion agar with 10% bovine blood, spectinomycin and polymyxin B. One isolate of B. aalborgi was obtained on this medium, from a control individual with self-reported chronic diarrhoea. The colonial, phase contrast and electron microscopic morphologies of two cell types present in the sample were described, and partial 16S rRNA sequences obtained. However, even with the modified media, other samples in this population contained spirochaetes that could not be cultured.

      PCR conducted on DNA extracted by the Qiagen QIAmp column method was more suitable for determining the prevalence of Brachyspira spp. in these specimens. B. pilosicoli was detected in higher proportions by PCR, in 15.0% of migrants and 14.5% of Aboriginals, but again not in symptomatic or healthy non-Aboriginal Australians. Detection of B. pilosicoli by PCR changed the identification of risk factors only a little; detection of protozoa in migrants, and protozoa, G. intestinalis, H. nana in Aboriginals were no longer associated with B. pilosicoli detection. B. aalborgi DNA was detected in 7.9% of migrants, 6.9% of Aboriginals, 5.6% of non-Aboriginal patients, and 5.6% of controls. Approximately equal proportions were obtained from migrant individuals from African, Middle Eastern and Eastern European countries. Both spirochaete species were detected in migrants (5.6%) and Aboriginal Australians (4.1%), and carriage of one spirochaete was significantly associated with carriage of the other spirochaete in both populations (P <0.001). These data suggest that the two Brachyspira spp. have a different epidemiology, and that B. aalborgi may be distributed at a similar prevalence in many human populations.

      Carriage of B. aalborgi was associated with larger family size in migrants (P <0.001), and if a family member was positive, it was significantly more likely that other family members would be colonised (P = 0.008). Two colonised control individuals also came from the same family, all suggesting that transmission of B. aalborgi can occur between family members. Carriage of B. aalborgi was significantly higher in Aboriginal patients aged 2 - 18 y, and in non-Aboriginal patients aged >18 y and between 60 - 69 y. Detection was associated with various parasites in Aboriginals, but only protozoans in migrants (P = 0.014). Detection of B. aalborgi was associated with diarrhoea in non- Aboriginals (P = 0.048), with an absence of diarrhoea (P = 0.016) amongst Aboriginals, and with eosinophilia, weight loss, being underweight and suspected worms in this group also (P <0.001). Whether B. aalborgi was a cause of these symptoms, or was excreted as a result of the symptoms caused by some other source, was unknown. The pathogenic significance ofB. aalborgi also could not be resolved in this investigation, as similar proportions were detected in ill and healthy individuals, and detection was associated with different reasons for investigation in the two ill population groups. B. aalborgi could be a commensal in the human gastrointestinal tract, or may have the capacity to cause disease under certain circumstances, such as in individuals with poor immune status, or in a conducive gut microenvironment.

      PCR products were sequenced to attempt to differentiate between B. aalborgi strains. All except five strains were identical to the type strain, three had nucleotide changes that placed them in cluster 3 described by Pettersson et al. (2000), another strain shared similarity with cluster 2 or 3 strains and the final strain remained in cluster 1. More complete sequencing of this gene, or the use of another sequence based typing method, such as multi-locus sequence typing, would be useful to further investigate the molecular epidemiology of B. aalborgi carriage in human populations. A comparison between techniques for intestinal spirochaete detection was made.

      PCR was the most successful method for detecting spirochaetes in faeces, with a lower limit of detection of B. pilosicoli in seeded faeces at 1 x 10^6 organism/g and 1 x 105 organism/g for B. aalborgi. Microscopy was inadequate for identifying the spirochaetes, and lacked sensitivity, with only 1 x 10^7 - 1 x 10^5 organisms/g of seeded faeces visible in this way. Culture was adequate for B. pilosicoli, with isolation possible from seeded faeces containing 1 x 10^2 - 2 x 10^3 cfu/g. Culture was inadequate for B. aalborgi detection, despite isolation from seeded faeces at 2 x 10^4 cfu/g, and advances made to isolation media for this spirochaete in the course of this investigation.

      There were concerns that B. pilosicoli spirochaetaemia might be occurring in Western Australian populations with a high rate of intestinal carriage, but going undiagnosed. An evaluation of the blood culture systems commonly used in Australia indicated that the automated BACTEC system using the BACTEC plus Anaerobic/F bottle was best for the detection of B. pilosicoli in seeded blood. A protocol longer than the 5 d normally utilised by clinical laboratories was required to detect the organism when seeded at low concentrations. Both manual Hemoline and Septi-Chek media were as reliable as the BACTEC for B. pilosicoli growth, however detection in these systems required frequent subculture, and was thus time-consuming and laborious. The automated BacT/Alert system performed poorly both for detection and recovery of B. pilosicoli, and medium and algorithmic components were probable contributing factors. BACTEC Plus Anaerobic/F bottles inoculated with blood from 801 patients thought to be at risk for blood infection with B. pilosicoli were cultured for 14 d, or 21 d after transport from rural centres. No isolate was obtained from these individuals, but there were some problems inherent in the investigation. However, while bacteraemia with B. pilosicoli is clearly a rare medical event, it should be considered as a differential diagnosis in immunocompromised individuals with gastrointestinal symptoms, and possibly in groups with high rates of intestinal carriage of B. pilosicoli.

      Pulsed-field gel electrophoresis, utilising MluI and SmaI restriction enzymes, was used to examine the relationships between B. pilosicoli isolates collected from Aboriginal and migrant individuals, and some pre-existing strains. In migrants, isolates were distributed throughout the dendrogram created after analysis with MluI; however, the migrants had originated from 10 countries around the world. Two B. pilosicoli isolates from two individuals from the same family were closely related, indicating that exposure from a common source or transmission from one individual to another might have occurred. As no other isolate from migrants were related, even among members of other families, transmission between family members did not appear to be common.

      Strains from Australian Aboriginals were also distributed throughout the MluI dendrogram, but the majority (89.7%) were clustered in one of six clonal groups. Isolates were obtained from different locations and years of sampling, supporting previous observations that B. pilosicoli colonising Aboriginal people have an epidemic population structure (Trott et al., 1998). An explanation for the presence of clonal groups was not clear. Increased virulence of isolates may have contributed to the spread of B. pilosicoli clones in WA Aboriginals. All isolates from hospitalised individuals, and those obtained from samples where no other pathogen was detected were members of clonal groups, however, no isolate from healthy Aboriginals was included in the investigation. Resistance to one or more antimicrobial also may have favoured selection for and spread of clonal isolates in the Aboriginal population.

      The antimicrobial susceptibility of 139 B. pilosicoli isolates was obtained by a procedure modified from the NCCLS agar dilution method (NCCLS, 1990). Strains were generally sensitive to agents tested, but variably resistant to clindamycin and amoxicillin, depending on the source of strains. Over 50% of strains contained a beta-lactamase that was inhibited by clavulanic acid. Significantly higher proportions of isolates from Aboriginal Australians (P <0.001) contained a beta-lactamase than other isolates, while no isolate from PNG natives did (P <0.001), nor was any isolate from this population resistant to any other antimicrobial. Isolates from pigs and homosexuals demonstrated raised MICs for tetracycline and resistance to amoxicillin and clindamycin. All Aboriginal isolates in clonal groups contained a beta-lactamase, as did other isolates not classified within clonal groups. Further analysis of the virulence characteristics and antibiotic susceptibility of the isolates is required.

      Based on susceptibility results and success in previous studies, metronidazole was a potential choice for treatment of HIS, and to remove intestinal carriage of B. pilosicoli. For therapy in spirochaetaemia, intravenous ceftriaxone or Timentin would be preferred over penicillin antibiotics in view of the frequent occurrence of resistance. Carriage of spirochaetes is currently going unrecognised in Australian laboratories, as current protocols do not specifically target their detection, and current medical opinion remains ambivalent about their significance. Detection by any of culture, microscopy, PCR is not difficult, and could be implemented to examine faecal specimens during investigations of individuals with chronic diarrhoea of unknown aetiology, or in children who are failing to thrive.

      In conclusion, the work presented in this thesis examined a range of issues relating to the epidemiology of intestinal spirochaetes in humans. The prevalence of B. aalborgi and B. pilosicoli was determined in faeces by microscopy, culture and PCR. The spirochaetes were present in both healthy and ill individuals, at around 15% for B. pilosicoli and 6% for B. aalborgi. Each organism was associated with specific risk factors in the populations assessed. The prevalence of bacteraemia with B. pilosicoli was examined, but no episode was identified in the study. The molecular epidemiology of B. pilosicoli was analysed using PFGE. MIC data were determined for B. pilosicoli and based on these, therapies for infections with this organism were suggested. Similar studies on B. aalborgi are now required to provide a more complete understanding of the epidemiology of this organism in humans.

      Publication Type: Thesis (PhD)
      Supervisor: UNSPECIFIED
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