RESULTS: The genome size of LM115 and LM41 was 2,959,041 and 2,963,111 bp, respectively. These two strains shared approximately 90% homologous genes. Comparative genomics and phylogenomic analyses revealed that LM115 and LM41 were more closely related to the reference strains F2365 and EGD-e, respectively. Our virulence profiling indicated a total of 31 virulence genes shared by both analysed strains. These shared genes included those that encode for internalins and L. monocytogenes pathogenicity island 1 (LIPI-1). Both the Malaysian L. monocytogenes strains also harboured several genes associated with stress tolerance to counter the adverse conditions. Seven antibiotic and efflux pump related genes which may confer resistance against lincomycin, erythromycin, fosfomycin, quinolone, tetracycline, and penicillin, and macrolides were identified in the genomes of both strains.
CONCLUSIONS: Whole genome sequencing and comparative genomics analyses revealed two virulent L. monocytogenes strains isolated from ready-to-eat foods in Malaysia. The identification of strains with pathogenic, persistent, and antibiotic resistant potentials from minimally processed food warrant close attention from both healthcare and food industry.
METHODS: A total of 210 Aeromonas clinical isolates were investigated: 116 from Singapore General Hospital and 94 archived clinical isolates from University of Malaya Medical Center, Malaysia. The isolates were genetically identified based on the gcat gene screening and the partial sequences of the rpoD housekeeping gene. Genetic relatedness, distribution of 15 virulence genes and 4 beta-lactamase resistance genes, and susceptibility patterns to 11 antimicrobial agents were compared.
RESULTS: Of the 210 Aeromonas isolates, A. dhakensis-94 (45%) was the dominant species in Singapore and Malaysia. Species composition was similar and enterobacterial repetitive intergenic consensus-PCR did not show genetic relatedness between strains from the two countries. Of the 15 virulence genes, A. dhakensis and A. hydrophila harbored the most compared with other species. Different combinations of 9 virulence genes (exu, fla, lip, eno, alt, dam, hlyA, aexU, and ascV) were present in A. dhakensis, A. hydrophila, and A. veronii from both the countries. Distribution of virulence genes was species and anatomic site related. Majority (>80%) of the strains were susceptible to all antimicrobial agents tested, except amoxicillin and cephalothin. A. dhakensis strains from Malaysia significantly harbored the cphA gene compared with A. dhakensis from Singapore. Multidrug resistance was mostly detected in strains from peritoneal fluids of dialysis patients.
CONCLUSION: This study revealed A. dhakensis as the dominant species isolated in both geographic regions, and that it carried a high number of virulence genes. It also highlights the geographic-related differences of virulence gene distribution and antimicrobial resistance profiles of clinical Aeromonas strains from Singapore and Malaysia.
Methodology: The pangolin P. fungorum (pangolin Pf) genome has a genomic size of approximately 7.7 Mbps with N50 of 69,666 bps. Our study showed that pangolin Pf is a Paraburkholderia fungorum supported by evidence from the core genome SNP-based phylogenetic analysis and the ANI analysis. Functional analysis has shown that the presence of a considerably large number of genes related to stress response, virulence, disease, and defence. Interestingly, we identified different types of secretion systems in the genome of pangolin Pf, which are highly specialized and responsible for a bacterium's response to its environment and in physiological processes such as survival, adhesion, and adaptation. The pangolin Pf also shared some common virulence genes with the known pathogenic member of the Burkholderiales. These genes play important roles in adhesion, motility, and invasion.
Conclusion: This study may provide better insights into the functions, secretion systems and virulence of this pangolin-associated bacterial strain. The addition of this genome sequence is also important for future comparative analysis and functional work of P. fungorum.
Methods: The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones.
Results: Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing.
Conclusion: NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.