The early initiation of targeted antibiotic therapy in patients with bacteraemia and septic shock impacts favourably on outcomes. Rapid methods are therefore increasingly employed for bacterial identification directly from positive blood culture bottles, but with variable success. We evaluated the performance of the Gram Positive 12 multiplex tandem PCR (MT-PCR) assay (AusDiagnostics; catalogue no. 6202, version 07) containing targets for the identification of staphylococci including Staphylococcus aureus, streptococci including Streptococcus pneumoniae, enterococci including Enterococcus faecalis and Enterococcus faecium and their common antibiotic resistance genes (mecA, vanA, vanB). A total of 673 aerobic and anaerobic blood culture broths demonstrating Gram-positive cocci on microscopy were analysed in parallel with traditional phenotypic methods. Amplification of the internal control was inhibited in 79/673 (11.7 %) samples; however, MT-PCR identification was in concordance with phenotypic identification to the genus level in 96.6 % (537/556) of the remaining monomicrobial specimens and to the species level, where applicable, in 100 % (172/172) of samples. MT-PCR identification for 94.7 % (36/38) of polymicrobial samples matched traditional phenotypic identification. Meticillin and vancomycin susceptibility results determined by MT-PCR in blood culture broths demonstrated complete agreement with those determined by phenotypic methods in all 143 Staphylococcus aureus isolates and eight E. faecium isolates, respectively. Gram-positive pathogens and their key antibiotic resistance markers were reliably identified with the MT-PCR assay within 3 h of a positive blood culture result.

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Viral infections engender bacterial colonization in the host by causing alteration in the innate immune response to common bacterial pathogens. These alterations may lead to severe bacterial pneumonia during influenza. Bacterial co-infection is common in influenza pneumonia and increases morbidity and mortality. With the onset of the COVID-19 pandemic worldwide, research in viral diseases has intensified. With this study, it can be thought that bacterial colonization may also support viral activity in the human body. This study sought to investigate bacterial co-infection in respiratory specimens using samples from the Australian influenza epidemic of 2009 as a model for analysis. Furthermore, it was thought that this study could shed light on bacterial co-infection studies in patients with COVID-19, which caused the current pandemic. 200 clinical specimens were extracted for isolation of genomic DNA and directly tested in a multiplexed quantitative PCR. Multiplex-tandem PCR (MT-PCR) assay based on 16S rRNA consensus sequence of a family (Enterobacteriaceae species) and four genera (Staphylococcus spp., Streptococcus spp., Enterococcus spp., and Pseudomonas spp.) was performed. According to MT-PCR results, respiratory tract samples showed a very high rate of bacterial identification (163/200-81.5%), and Enterococci were commonly found as the dominant genus identified in influenza patients. In a total of 200 putative co-infection samples, Enterococcus spp. was apparently present in 139 samples (69.5%), being the dominant signal (any other signal was ≤10%) from 106 samples. Streptococcus spp. was next most commonly indicated with 137 positive results (68.5%), but was the dominant signal in 19 of 137. It was determined that bacterial co-infection was high in the samples taken from patients with severe influenza, and Enterococcus was the dominant bacteria in the upper respiratory tract of patients treated for severe pneumonia, while Streptococcus was more dominant in the lower respiratory tracts than the other species.

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