Severe cases of Buruli ulcer (infection with Mycobacterium ulcerans) in common ringtail possums in Victoria adversely affect animal welfare.

Buruli ulcer is a chronic ulcerative disease of the skin and subcutaneous tissues caused by infection with Mycobacterium ulcerans. Although Australian possums are known to be susceptible to Buruli ulcer, many aspects of the disease in possums, including welfare impacts, remain largely unreported. Severe clinical Buruli ulcer was identified in four common ringtail possums (Pseudocheirus peregrinus) from Melbourne, Victoria. All four possums were euthanased due to the presence of deep ulcerative lesions on paws, with extensive tissue necrosis that exposed bones and tendons in three cases. Histologically, there was severe ulcerative necrotising pyogranulomatous dermatitis, panniculitis and myositis, with intralesional acid-fast bacteria. M. ulcerans was detected by real-time PCR in all swabs, tissues and faeces collected from all four cases. Buruli ulcer may be an important and under-recognised cause of poor possum welfare in endemic areas. The physical impacts of the severe cutaneous lesions, especially those extending to underlying bones and joints, would have directly impaired the mobility of these possums, affecting navigation of their natural environments and expression of natural behaviours including foraging and socialising. Systemic distribution of M. ulcerans throughout all major internal organs, as observed here, may further impact the health and fitness of infected possums. Faecal shedding of M. ulcerans in all four cases supports the role of possums as zoonotic reservoirs. Further research is needed to investigate the epidemiology, pathogenesis and welfare impacts of Buruli ulcer in possums and to inform the design of interventions that may protect their health and welfare.

Vancomycin-intermediate Staphylococcus aureus isolates are attenuated for virulence when compared with susceptible progenitors.

OBJECTIVES: Vancomycin-intermediate Staphylococcus aureus (VISA) is associated with genetic changes that may also impact upon pathogenicity. In the current study, we compared the virulence of clinical VISA strains with their isogenic vancomycin-susceptible progenitors (VSSA). METHODS: Production of the critical virulence protein, α toxin, was assessed using Western blot analysis and was correlated to agr activity using a bioluminescent agr-reporter. Cytotoxicity and intracellular persistence were compared ex vivo for VSSA and VISA within non-professional phagocytes (NPP). Virulence and host immune responses were further explored in vivo using a murine model of bacteraemia. RESULTS: VISA isolates produced up to 20-fold less α toxin compared with VSSA, and this was corroborated by either loss of agr activity due to agr mutation, or altered agr activity in the absence of mutation. VISA were less cytotoxic towards NPP and were associated with enhanced intracellular persistence, suggesting that NPP may act as a reservoir for VISA. Infection with VSSA strains produced higher mortality in a murine bacteraemia model (≥90% 7-day mortality) compared with infection with VISA isolates (20% to 50%, p <0.001). Mice infected with VISA produced a dampened immune response (4.6-fold reduction in interleukin-6, p <0.001) and persistent organ bacterial growth was observed for VISA strains out to 7 days. CONCLUSIONS: These findings highlight the remarkable adaptability of S. aureus, whereby, in addition to having reduced antibiotic susceptibility, VISA alter the expression of pathogenic factors to circumvent the host immune response to favour persistent infection over acute virulence.

Possible healthcare-associated transmission as a cause of secondary infection and population structure of Staphylococcus aureus isolates from two wound treatment centres in Ghana.

We have previously shown that secondary infections of Buruli ulcer wounds were frequently caused by Staphylococcus aureus. To gain understanding into possible routes of secondary infection, we characterized S. aureus isolates from patient lesions and surrounding environments across two Ghanaian health centres. One hundred and one S. aureus isolates were isolated from wounds (n = 93, 92.1%) and the hospital environment (n = 8, 7.9%) and characterized by the spa gene, mecA and the Panton-Valentine leucocidin toxin followed by spa sequencing and whole genome sequencing of a subset of 49 isolates. Spa typing and sequencing of the spa gene from 91 isolates identified 29 different spa types with t355 (ST152), t186 (ST88), and t346 dominating. Although many distinct strains were isolated from both health centres, genotype clustering was identified within centres. In addition, we identified a cluster consisting of isolates from a healthcare worker, patients dressed that same day and forceps used for dressing, pointing to possible healthcare-associated transmission. These clusters were confirmed by phylogenomic analysis. Twenty-four (22.8%) isolates were identified as methicillin-resistant S. aureus and lukFS genes encoding Panton-Valentine leucocidin were identified in 67 (63.8%) of the isolates. Phenotype screening showed widespread resistance to tetracycline, erythromycin, rifampicin, amikacin and streptomycin. Genomics confirmed the widespread presence of antibiotic resistance genes to ß-lactams, chloramphenicol, trimethoprim, quinolone, streptomycin and tetracycline. Our findings indicate that the healthcare environment probably contributes to the superinfection of Buruli ulcer wounds and calls for improved training in wound management and infection control techniques.

Enterococcal vanB resistance locus in anaerobic bacteria in human faeces.

While developing a rapid method to detect carriers of vancomycin-resistant enterococci (VRE), we found the vanB gene by PCR in 13 of 50 human faecal specimens that did not contain culturable VRE. Passaging under antibiotic selection allowed us to isolate two species of anaerobic bacteria that were vanB PCR positive, vancomycin resistant, and teicoplanin sensitive. Sequence analysis of the 16S rRNA genes showed that one isolate resembled Eggerthella lenta (98% identity), and the other Clostridium innocuum (92% identity). Southern hybridisation and nucleotide sequencing showed a vanB locus homologous to that in VRE. We propose that vanB resistance in enterococci might arise from gene transfer in the human bowel.

Comparative genetic analysis of Mycobacterium ulcerans and Mycobacterium marinum reveals evidence of recent divergence.

Previous studies of the 16S rRNA genes from Mycobacterium ulcerans and Mycobacterium marinum have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18 M. ulcerans strains and 22 M. marinum strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of M. ulcerans that have identified distinct genotypes within a geographical region. Single isolates of both M. ulcerans and M. marinum that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for M. ulcerans and M. marinum, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS2404 and IS2606, have recently been reported in M. ulcerans, and significantly, these elements are not present in M. marinum. Hybridization of the AseI restriction fragments from M. ulcerans with IS2404 and IS2606 indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that M. ulcerans has recently diverged from M. marinum by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of M. tuberculosis, where species diversity is being driven mainly by the activity of mobile DNA elements.

Identification of Mycobacterium ulcerans in the environment from regions in Southeast Australia in which it is endemic with sequence capture-PCR.

We recently described the use of PCR to identify the environmental source of Mycobacterium ulcerans during an outbreak of ulcerative disease that occurred in a localized region of southeast Australia. The PCR used was based on amplification of the M. ulcerans-specific insertion sequence, IS2404. In this study we developed a new test that is a substantial improvement over the original PCR method in terms of sensitivity, reliability, and ease of use. In the new method magnetic bead sequence capture-PCR is used to detect two M. ulcerans sequences (IS2404 and IS2606) and total mycobacterial 16S ribosomal DNA. We used sequence capture-PCR to test water and plant material collected over a 12-month period during 1998 and 1999 from sites near the centers of two distinct foci of M. ulcerans infections. A golf course irrigation system in one area and a small shallow lake in another area repeatedly were PCR positive for M. ulcerans. Nearby sites and sites unrelated to the endemic areas were negative. Based on the PCR data, a most-probable-number method was used to estimate the concentration of M. ulcerans cells in positive samples from both regions. This procedure resulted in average concentrations of 0.5 cell per 100 ml of water and 40 cells per 100 g of detritus. Loss of the PCR signal coincided with a decrease in ulcerative disease in each area. These results provide further evidence that M. ulcerans may be transmitted from a point environmental source and demonstrate the utility of magnetic bead sequence capture-PCR for identification of nonculturable microbial pathogens in the environment.

A simple PCR method for rapid genotype analysis of Mycobacterium ulcerans.

Two high-copy-number insertion sequences, IS2404 and IS2606, were recently identified in Mycobacterium ulcerans and were shown by Southern hybridization to possess restriction fragment length polymorphism between strains from different geographic origins. We have designed a simple genotyping method that captures these differences by PCR amplification of the region between adjacent copies of IS2404 and IS2606. We have called this system 2426 PCR. The method is rapid, reproducible, sensitive, and specific for M. ulcerans, and it has confirmed previous studies suggesting a clonal population structure of M. ulcerans within a geographic region. M. ulcerans isolates from Australia, Papua New Guinea, Malaysia, Surinam, Mexico, Japan, China, and several countries in Africa were easily differentiated based on an array of 4 to 14 PCR products ranging in size from 200 to 900 bp. Numerical analysis of the banding patterns suggested a close evolutionary link between M. ulcerans isolates from Africa and southeast Asia. The application of 2426 PCR to total DNA, extracted directly from M. ulcerans-infected tissue specimens without culture, demonstrated the sensitivity and specificity of this method and confirmed for the first time that both animal and human isolates from areas of endemicity in southeast Australia have the same genotype.

Identification and characterization of IS2404 and IS2606: two distinct repeated sequences for detection of Mycobacterium ulcerans by PCR.

Molecular analysis of Mycobacterium ulcerans has revealed two new insertion sequences (ISs), IS2404 and IS2606. IS2404 was identified by complete sequencing of a previously described repetitive DNA segment from M. ulcerans. This element is 1,274 bp long, contains 12-bp inverted repeats and a single open reading frame (ORF) potentially encoding a protein of 327 amino acids (aa), and apparently generates 7-bp direct repeats upon transposition. Amino acid similarity was found between the putative transposase and those encoded by ISs in other bacterial sequences from Aeromonas salmonicida (AsIs1), Escherichia coli (H repeat element), Vibrio cholerae (VcIS1), and Porphyromonas gingivalis (PGIS2). The second IS, IS2606, was discovered by sequence analysis of a HaeIII fragment of M. ulcerans genomic DNA containing a repetitive sequence. This element is 1,404 bp long, with 12-bp inverted repeats and a single ORF potentially encoding a protein of 445 aa. Database searches revealed a high degree of amino acid identity (70%) with the putative transposase of IS1554 from M. tuberculosis. Significant amino acid identity (40%) was also observed with transposases from several other microorganisms, including Rhizobium meliloti (ISRm3), Burkholderia cepacia (IS1356), Corynebacterium diphtheriae, and Yersinia pestis. PCR screening of DNA from 45 other species of mycobacteria with primers for IS2404 confirm that this element is found only in M. ulcerans. However, by PCR, IS2606 was also found in Mycobacterium lentiflavum, another slow-growing member of the genus Mycobacterium that is apparently genetically distinct from M. ulcerans. Testing the sensitivity of PCR based on IS2404 and IS2606 primers demonstrated the ability to detect 0.1 and 1 M. ulcerans genome equivalents, respectively. The ability to detect small numbers of cells by using two gene targets will be particularly useful for analyzing environmental samples, where there may be low concentrations of M. ulcerans among large numbers of other environmental mycobacteria.

Sensitive and rapid detection of viable Giardia cysts and Cryptosporidium parvum oocysts in large-volume water samples with wound fiberglass cartridge filters and reverse transcription-PCR.

We recently described a reverse transcription-PCR (RT-PCR) for detecting low numbers of viable Cryptosporidium parvum oocysts spiked into clarified environmental water concentrates. We have now modified the assay for direct analysis of primary sample concentrates with simultaneous detection of viable C. parvum oocysts, Giardia cysts, and a novel type of internal positive control (IPC). The IPC was designed to assess both efficiency of mRNA isolation and potential RT-PCR inhibition. Sensitivity testing showed that low numbers of organisms, in the range of a single viable cyst and oocyst, could be detected when spiked into 100-microliter packed pellet volumes of concentrates from creek and river water samples. The RT-PCR was compared with an immunofluorescence (IF) assay by analyzing 29 nonspiked environmental water samples. Sample volumes of 20 to 1,500 liters were concentrated with a wound fiberglass cartridge filter. Frequency of detection for viable Giardia cysts increased from 24% by IF microscopy to 69% by RT-PCR. Viable C. parvum oocysts were detected only once by RT-PCR (3%) in contrast to detection of viable Cryptosporidium spp. in four samples by IF microscopy (14%), suggesting that Cryptosporidium species other than C. parvum were present in the water. This combination of the large-volume sampling method with RT-PCR represents a significant advance in terms of protozoan pathogen monitoring and in the wider application of PCR technology to this field of microbiology.

Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR.

[This corrects the article on p. 1743 in vol. 64, PMID: 9572946.].

Detection of Mycobacterium ulcerans in environmental samples during an outbreak of ulcerative disease.

Mycobacterium ulcerans is an environmental bacterium which causes chronic skin ulcers. Despite significant epidemiological evidence to suggest that water is the source of infection, the organism has never been identified in the environment. Environmental water samples were collected from a small town in which an outbreak of 29 cases had occurred in a 3-year period. These were examined by mycobacterial culture and PCR amplification. Similar to previous studies, M. ulcerans was not cultured from the water samples. However, five samples were positive for M. ulcerans by PCR. These samples were collected from a swamp and a golf course irrigation system within the outbreak area. This is the first time that M. ulcerans has been demonstrated to be present in the environment and supports the postulated epidemiology of disease due to this organism.

Detection of a Single Viable Cryptosporidium parvum Oocyst in Environmental Water Concentrates by Reverse Transcription-PCR.

Volume 62, no. 9, p. 3386, column 1, last line: "(specific gravity, 2.10) and centrifuging the mixture at 2,000 x g for 10 min" should read "(specific gravity, 1.10) and centrifuging the mixture at 1,050 x g for 10 min." Page 3387, column 1, line 16: "25 cycles at 90(deg)C for 5 min" should read "25 cycles at 90(deg)C for 5 s." [This corrects the article on p. 3385 in vol. 62.].

Detection of a single viable Cryptosporidium parvum oocyst in environmental water concentrates by reverse transcription-PCR.

Current methods for detection of Cryptosporidium parvum oocysts in water are time-consuming and difficult. We have developed a reverse transcription (RT)-PCR which can detect the presence of a single viable oocyst spiked into concentrated environmental water samples. The test is based on the detection of mRNA from a C. parvum heat shock protein (hsp). The synthesis of hsp was induced by a short 45 degrees C incubation followed by oocyst lysis by a freeze-thaw process. Hsp70 mRNA, produced only from viable oocysts, was then isolated by hybridization to oligo(dT)25-coated magnetic beads. Detection was achieved by RT-PCR amplification of a 590-bp region of hsp70 mRNA specific for C. parvum. To test the method, samples of reticulated, reservoir, bore, and river water were concentrated by chemical flocculation and Percoll-sucrose gradient centrifugation and then spiked with dilutions of oocysts. In all four of the water types examined, the detection of single oocysts was possible by RT-PCR combined with Southern hybridization. RT-PCR products were not obtained from formalin-inactivated oocysts. An RNA internal positive control fragment was synthesized that was included with each reaction to guard against RT-PCR false-negative results that may be caused by the presence of inhibitory substances. However, when the magnetic beads were used to extract and concentrate mRNA, no inhibition was observed. The technique is versatile, straightforward, and rapid (1 day) and provides a sensitive and economic means of screening concentrated water samples for the presence of C. parvum.

Detection of pathogenic Yersinia enterocolitica in environmental waters by PCR.

The isolation of pathogenic strains of Yersinia enterocolitica from food and water samples by culture is time-consuming and unreliable. A two-step PCR procedure has been developed which, after a period of bacterial enrichment, can detect and confirm the presence of pathogenic Y. enterocolitica within a single day. This PCR method works effectively for a range of environmental water types, including reticulated waters, reservoirs and creeks. A survey of environmental waters in Victoria, Australia, showed that the PCR method detected pathogenic Y. enterocolitica in water sampled from four separate sites (two creeks and two reservoirs). Repeat samplings of the two reservoirs yielded PCR-positive results on all but one occasion. Culture analysis of the same samples detected pathogenic Y. enterocolitica in only one sample, indicating that the PCR can detect pathogenic Y. enterocolitica which are undetectable by culture. Results from this study confirm that potentially pathogenic strains of Y. enterocolitica can exist in environmental waters.