Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM).

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64- Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.

Emergence of Enterococcus gallinarum carrying vanA gene cluster displaying atypical phenotypes.

Motile enterococci such as Enterococcus gallinarum has the ability to acquire and transfer antibiotic resistance genes to other enterococci. Even though infections caused by E. gallinarum are rare, the discovery of this bacteria in food sources and in clinical environments is disturbing. Here, we report the isolation and identification of E. gallinarum from the wound of a hospital in-patient. The isolate was identified using 16S rDNA sequencing. Isolate 146 harboured the vanA and vanC1 gene clusters, was vancomycin-susceptible, and displayed resistance to ampicillin, penicillin, erythromycin and teicoplanin. This isolate also showed intermediate resistance to linezolid and sequencing of the 23S rRNA peptidyl transferase region did not unveil any known mutations associated to the conferment of linezolid resistance. The presence of vanA did not confer resistance to vancomycin. Structural analyses into the Tn1546 transposon carrying the vanA gene revealed distinct genetic variations in the vanS, vanY and vanS-vanH intergenic region that could be associated to the atypical antibiotic resistance phenotypes of isolate 146. Finding from this study are suggestive of the occurrence of interspecies horizontal gene transfer and that similarities in genotypic characteristic may not necessarily correlate with actual antibiotic resistance pattern of E. gallinarum.

A Report of Vancomycin-susceptible, Teicoplanin-resistant Enterococcus faecalis ST6 in Malaysia.

The ability to acquire antibiotic resistance and virulence has propelled Enterococcus faecalis to become a major nosocomial pathogen. In Malaysia, data on the antibiotic resistance determinants and virulence of Enterococcus circulating strains are still scarce. This study aimed to assess the genotype of an E. faecalis isolate initially identified as Streptococcus uberis, examine the antibiotic resistance genotypes, analyze the genetic variations within Tn1546 and investigate the presence of virulence genes. The E. faecalis isolate was genetically characterized using multilocus sequence typing (MLST). Minimum inhibitory concentrations to vancomycin and teicoplanin were determined. Antibiotic resistance and other virulence genes were amplified using nucleic acid polymerase chain reaction. Mapping of the Tn1546 transposon was performed and compared to the prototype sequences. The E. faecalis isolate was found to have a MLST profile corresponding to sequence type 6. The isolate was resistant to teicoplanin but susceptible to vancomycin. Its genome consisted the vanA and vanC1 genes. Novel genetic variations in the vanS, vanS-vanH intergenic region and vanY genes were present and six virulence genes were detected. The detection of the vanC1 gene, thought to be non-transferable, suggests the potential emergence of inter-species enterococcal vanC1 gene transfer. The peculiar antibiotics resistance phenotype of this E. faecalis isolate could be associated to the novel genetic variations found. This study highlights the presence of E. faecalis belonging to the high-risk clonal complex with multiple virulence factors in Malaysia.

A Report of Vancomycin-susceptible, Teicoplanin-resistant Enterococcus faecalis ST6 in Malaysia

The ability to acquire antibiotic resistance and virulence has propelled Enterococcus faecalis to become a major nosocomial pathogen. In Malaysia, data on the antibiotic resistance determinants and virulence of Enterococcus circulating strains are still scarce. This study aimed to assess the genotype of an E. faecalis isolate initially identified as Streptococcus uberis, examine the antibiotic resistance genotypes, analyze the genetic variations within Tn1546 and investigate the presence of virulence genes. The E. faecalis isolate was genetically characterized using multilocus sequence typing (MLST). Minimum inhibitory concentrations to vancomycin and teicoplanin were determined. Antibiotic resistance and other virulence genes were amplified using nucleic acid polymerase chain reaction. Mapping of the Tn1546 transposon was performed and compared to the prototype sequences. The E. faecalis isolate was found to have a MLST profile corresponding to sequence type 6. The isolate was resistant to teicoplanin but susceptible to vancomycin. Its genome consisted the vanA and vanC1 genes. Novel genetic variations in the vanS, vanS-vanH intergenic region and vanY genes were present and six virulence genes were detected. The detection of the vanC1 gene, thought to be non-transferable, suggests the potential emergence of inter-species enterococcal vanC1 gene transfer. The peculiar antibiotics resistance phenotype of this E. faecalis isolate could be associated to the novel genetic variations found. This study highlights the presence of E. faecalis belonging to the high-risk clonal complex with multiple virulence factors in Malaysia.

Emergence of Enterococcus gallinarum carrying vanA gene cluster displaying atypical phenotypes

Motile enterococci such as Enterococcus gallinarum has the ability to acquire and transfer antibiotic resistance genes to other enterococci. Even though infections caused by E. gallinarum are rare, the discovery of this bacteria in food sources and in clinical environments is disturbing. Here, we report the isolation and identification of E. gallinarum from the wound of a hospital in-patient. The isolate was identified using 16S rDNA sequencing. Isolate 146 harboured the vanA and vanC1 gene clusters, was vancomycin-susceptible, and displayed resistance to ampicillin, penicillin, erythromycin and teicoplanin. This isolate also showed intermediate resistance to linezolid and sequencing of the 23S rRNA peptidyl transferase region did not unveil any known mutations associated to the conferment of linezolid resistance. The presence of vanA did not confer resistance to vancomycin. Structural analyses into the Tn1546 transposon carrying the vanA gene revealed distinct genetic variations in the vanS, vanY and vanS-vanH intergenic region that could be associated to the atypical antibiotic resistance phenotypes of isolate 146. Finding from this study are suggestive of the occurrence of interspecies horizontal gene transfer and that similarities in genotypic characteristic may not necessarily correlate with actual antibiotic resistance pattern of E. gallinarum.

Multiplex sequencing of SARS-Cov-2 genome directly from clinical samples using the Ion Personal Genome Machine (PGM)

@#Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13 751-13 965 and 23 941-24 106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13 730 and 23 929, whereas the other isolates possess T at these positions. The genome nucleotide variations observed suggest that the naturally occurring variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS CoV 2 Research Panel. The possible impact of other genome nucleotide variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS CoV 2 Research Panel, hence, should be considered.