Characterisation of ß-lactam resistance mediated by blaZ in staphylococci recovered from captive and free-ranging wallabies.

Staphylococci are commensal organisms of animals, but some species are opportunistic pathogens that are resistant to almost all antimicrobial agents in clinical use. Bacterial resistance to ß-lactam antimicrobial agents is widespread and has been investigated in species isolated from humans in addition to food production and companion animals. However, minimal progress has been made towards identifying reservoirs of ß-lactam-resistant staphylococci in wildlife. This study was aimed at investigating and characterising ß-lactamase resistance from staphylococci of wallaby origin. Staphylococci from free-ranging and captive wallabies were assessed for their phenotypic susceptibility to ß-lactam antimicrobial agents prior to sequence analysis of their blaZ and blaR1 genes. Deduced amino acid sequences were classified according to the Ambler molecular characterisation method, assigned a protein signature type and compared with sequences generated from previous studies involving isolates from humans, cattle and companion animals. All BlaZ sequences identified in this study were assignable to a pre-existing ß-lactamase class and protein signature type, including the more recently discovered protein signature type 12. Three major phylogenetic groups were resolved upon phylogenetic analysis against published BlaZ sequences. This study has found antibiotic-resistant staphylococci both in free-ranging and captive wallaby populations and these bacteria harbour blaZ variants that are different to those recovered from humans, cattle and companion animals. Further studies of staphylococci from non-traditional sources are required in order to enhance our knowledge of the epidemiology of antibiotic resistance genes.

A molecular survey of a captive wallaby population for periodontopathogens and the co-incidence of Fusobacterium necrophorum subspecies necrophorum with periodontal diseases.

Periodontal diseases (PD) are diseases of polymicrobial aetiology and constitute major health problems in captive macropods. Increasing knowledge of the causal pathogens is therefore crucial for effective management and prevention of these diseases. PCR survey and sequence analyses of potential periodontopathogens in captive wallaby populations revealed a co-incidence of the diseases with the detection of Fusobacterium necrophorum subsp. necrophorum (Fnn) and its encoded leukotoxin (lktA) gene. Sequence analyses showed that the outer membrane protein of Fnn in the GenBank database shared significant homology (99%) with the Fnn encoded haemagglutinin-related-protein gene fragment identified in this study. In addition, this report suggests the existence of a variant of Fnn with no detectable lktA gene and thus warrants further studies. In contrast to reports associating Porphyromonas gingivalis and F. nucleatum with PD, this study revealed that PD in macropods are associated with Porphyromonas gulae and Fnn and raises the question: is there a possible host pathogen co-evolution in the pathogenesis of PD in animals and humans? These findings contribute to the understanding of the aetiology of periodontal disease in macropods as well as opening up a new direction of research into the microbial interactions involved in the pathogenesis of PD in macropods.

"Cycliplex PCR" confirmation of Fusobacterium necrophorum isolates from captive wallabies: a rapid and accurate approach.

Isolation and identification of obligate anaerobic bacteria is labour intensive and time consuming. This has led to the increased application of molecular tools to circumvent part of this problem. We report here the development of a rapid, accurate and cost-effective method to isolate and identify Fusobacterium necrophorum species from South Australian wallaby populations using a supplemented medium (BHIRS) in conjunction with a "Cycliplex PCR" method which involves a stepwise-selective amplification of target PCR products. This report demonstrates the complementation of phenotypic characterization by PCR for accurate and fast identification of F. necrophorum isolates from wildlife origin.

Nutrient regime regulates complex transcriptional start site usage within a Pseudoalteromonas chitinase gene cluster.

The chitinase gene cluster of the marine bacterium Pseudoalteromonas sp. S91, chiABC, which produces the major chitinases of this sp., was transcribed as an operon and from each individual gene. chiA, chiB and chiC were found to possess multiple transcriptional start points (TSPs), the use of which was determined by the nutrient regime used for S91 growth. In minimal medium containing glutamate, chiA, chiB and chiC each used 3, 1 and 1 TSP, respectively. Upon the addition of the chitin monomer N-acetylglucosamine, the number of chiA TSPs was unaffected. However, chiB used an additional 4 TSPs, and chiC used four new TSPs excluding the TSP used in glutamate only. In addition, the cluster was transcribed as an operon from TSP A1 of chiA. All TSPs were potentially associated with either a sigma(70)- or sigma(54)-dependent promoter. Under the growth conditions used, no TSPs were detected for chiB or chiC in S91CX, a chiA transposon mutant. The transcription of the S91 chiABC gene cluster produced at least four polycistronic mRNAs. In addition, the occurrence of operon transcription of chiABC, and identification of an additional 12 putative TSPs within the gene cluster, gave an indication that each gene appeared to be transcribed from more than one promoter region upstream of each in-frame translation start codon. Questions arose regarding the reason for this complexity of transcription within the gene cluster, leading to a re-evaluation of the Chi protein domains. By bioinformatic review, ChiA, ChiB and ChiC were found to potentially possess additional putative domains.

Nitrogen regulates chitinase gene expression in a marine bacterium.

Ammonium concentration and nitrogen source regulate promoter activity and use for the transcription of chiA, the major chitinase gene of Pseudoalteromonas sp. S91 and S91CX, an S91 transposon lacZ fusion mutant. The activity of chiA was quantified by beta-galactosidase assay of S91CX cultures containing different ammonium concentrations (NH4+; 0, 9.5 or 191 mM) and with different nitrogen sources (N-acetylglucosamine (GlcNAc) or glutamate (glt)). S91 chiA expression was found to depend on both the NH4+ concentration and source of nitrogen in marine minimal medium (MMM). Pseudoalteromonas sp. S91 and S91CX can use either GlcNAc or glt as a sole source of carbon in MMM containing a standard concentration of 9.5 mM NH4+. Adding excess NH4+, 20 times the standard concentration, to MMM significantly reduced chiA activity below that found in the presence of either GlcNAc or glt. When no NH4+ was added to MMM, S91CX was also able to use either GlcNAc or glt as a source of nitrogen; under these conditions chiA activity was significantly increased. Under all conditions tested, GlcNAc induced chiA activity significantly more than glt. Regulation of bacterial chitinases by nitrogen has not been previously reported. Transcriptional start point analysis of S91 chiA, using 5'RACE (ligation-anchored PCR), showed that during growth in MMM supplemented with (1) maltose (solely a carbon source for S91), chiA transcription occurred from only one putative sigma(70)-dependent promoter; (2) the chitin monomer GlcNAc, transcription initiated from two putative sigma(54)-dependent promoters and (3) glt, transcription initiated from all three putative promoters.

Multiple genes involved in chitin degradation from the marine bacterium Pseudoalteromonas sp. strain S91.

A cluster of three closely linked chitinase genes organized in the order chiA, chiB and chiC, with the same transcriptional direction, and two unlinked genes, chiP and chiQ, involved in chitin degradation in Pseudoalteromnas sp. strain S91 were cloned, sequenced and characterized. The deduced amino acid sequences revealed that ChiA, ChiB and ChiC exhibited similarities to chitinases belonging to family 18 of the glycosyl hydrolases while ChiP and ChiQ belonged to family 20. ChiP and ChiQ showed different enzymic activities against fluorescent chitin analogues, but neither was able to degrade colloidal chitin. ChiA possessed chitinase activity but did not bind chitin; ChiB bound chitin but had no chitinase activity; ChiC possessed strong chitinase activity and also bound chitin. Production of ChiC in S91 appeared to be controlled by chiA expression, since insertion of a transposon into the ORF of chiA resulted in the loss of chitinase activity as well as loss of ChiC proteins in a chitinase-negative mutant. In Escherichia coli, ChiC appeared to be expressed from its own promoter.