The continuous evolution of β-lactamases resulting in bacterial resistance to β-lactam antibiotics is a major concern in public health, and yet the underlying molecular basis or the pattern of such evolution is largely unknown. We investigated the mechanics of the substrate fspectrum expansion of the class A β-lactamase using PenA of Burkholderia thailandensis as a model. By analyzing 516 mutated enzymes that acquired the ceftazidime-hydrolyzing activity, we found twelve positions with single amino acid substitutions (altogether twenty-nine different substitutions), co-localized at the active-site pocket area. The ceftazidime MIC (minimum inhibitory concentration) levels and the relative frequency in the occurrence of substitutions did not correlate well with each other, and the latter appeared be largely influenced by the intrinsic mutational biases present in bacteria. Simulation studies suggested that all substitutions caused a congruent effect, expanding the space in a conserved structure called the omega loop, which in turn increased flexibility at the active site. A second phase of selection, in which the mutants were placed under increased antibiotic pressure, did not result in a second mutation in the coding region, but a mutation that increased gene expression arose in the promoter. This result suggests that the twelve amino acid positions and their specific substitutions in PenA may represent a comprehensive repertoire of the enzyme’s adaptability to a new substrate. These mapped substitutions represent a comprehensive set of general mechanical paths to substrate spectrum expansion in class A β-lactamases that all share a functional evolutionary mechanism using common conserved residues.

We identified and isolated a novel Hendra virus (HeV) variant not detected by routine testing from a horse in Queensland, Australia, that died from acute illness with signs consistent with HeV infection. Using whole-genome sequencing and phylogenetic analysis, we determined the variant had ≈83% nt identity with prototypic HeV. In silico and in vitro comparisons of the receptor-binding protein with prototypic HeV support that the human monoclonal antibody m102.4 used for postexposure prophylaxis and current equine vaccine will be effective against this variant. An updated quantitative PCR developed for routine surveillance resulted in subsequent case detection. Genetic sequence consistency with virus detected in grey-headed flying foxes suggests the variant circulates at least among this species. Studies are needed to determine infection kinetics, pathogenicity, reservoir-species associations, viral-host coevolution, and spillover dynamics for this virus. Surveillance and biosecurity practices should be updated to acknowledge HeV spillover risk across all regions frequented by flying foxes.

PurposeRobust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness.MethodsWe conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 (IFI27) in COVID-19 patients.ResultsWe show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression in the lower respiratory tract is associated with the presence of a high viral load. We further demonstrate that the systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 infection. For clinical outcome prediction (e.g., respiratory failure), IFI27 expression displays a high sensitivity (0.95) and specificity (0.83), outperforming other known predictors of COVID-19 outcomes. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 influenza virus infection, IFI27-like genes were highly upregulated in the blood samples of severely infected patients.ConclusionThese data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus.

bWe describe a deletion mutation in a class A ␤-lactamase, PenA, of Burkholderia thailandensis that extended the substrate spectrum of the enzyme to include ceftazidime. Glu168del was located in a functional domain called the omega loop causing expansion of the space in the loop, which in turn increased flexibility at the active site. This deletion mutation represents a rare but significant alternative mechanical path to substrate spectrum extension in PenA besides more common substitution mutations. Burkholderia pseudomallei is the etiological agent of septicemic melioidosis, which is endemic in Southeast Asia and northeastern Australia (4). Burkholderia mallei, the cause of glanders, is a species derived from a clone of B. pseudomallei (20). Bcc, which is a complex composed of more than 10 Burkholderia species, including Burkholderia cepacia, Burkholderia cenocepacia, and Burkholderia multivorans, is a group of nosocomial pathogens that cause respiratory and systemic infections in patients with cystic fibrosis or chronic granulomatous disease and in other immunocompromised patients (10). The antibiotic regimen used to treat infections with these bacteria generally includes ceftazidime (10,26 (15,17,18,22). Similarly, two orthologs of PenA, PenB2 and PenB3, with seven and two amino acid alterations, respectively, were shown to be associated with ceftazidime resistance in clinical isolates of B. cenocepacia (13).Here we report a new mutation that extends to ceftazidime the substrate spectrum of PenA of B. thailandensis (3) (BTH_II1450 of B. thailandensis strain E264), which is closely related to PenA enzymes of the pathogenic species B. pseudomallei, B. mallei, and Bcc (13,17,22). The resistant isolate arose against a high level of ceftazidime (5 g/ml) in LB medium. The MIC of ceftazidime for this isolate was 48 g/ml, which was significantly higher than the 1.75 g/ml of the wild type ( Fig. 1). By PCR amplifying penA from its genomic DNA using primers penA-F (5=-CGTCAATCCGATG CAGTACC-3=) and penA-R (5=-GCCGTTATCGCACCTTTAT C-3=) and sequencing the amplicon in both directions using a 3730XL DNA analyzer (Applied Biosystems, Foster City, CA), we found a three-base deletion mutation in the coding region of penA (Fig. 1). To confirm that this penA gene with the mutation was responsible for the ceftazidime resistance that developed in the mutants, we inactivated the penA gene by replacing a region spanning 196 bp in the middle of the coding region with a Tet r cassette, which was obtained from broad-host-range vector pRK415K (9). The penA-null mutant was verified by PCR using primers penA_LF (5=-AACAGATCGCCGAGATGG-3=) and penA_LR (5=-GCGAACGTTGCCCGATAC-3=), which hybridize to the genomic regions outside penA. The mutant strain with inactivated penA-Glu168del (that is, with ⌬penA-Glu168del) lost resistance to ceftazidime, whose MIC was comparable to that for the wild-type strain with ⌬penA-WT (Fig. 1). The MICs were measured by the Etest (8) in accordance with the manufacturer's instructions (AB Biodisk, Solna, Sweden...

Expansion or shrinkage of existing tandem repeats (TRs) associated with various biological processes has been actively studied in both prokaryotic and eukaryotic genomes, while their origin and biological implications remain mostly unknown. Here we describe various duplications (de novo TRs) that occurred in the coding region of a β-lactamase gene, where a conserved structure called the omega loop is encoded. These duplications that occurred under selection using ceftazidime conferred substrate spectrum extension to include the antibiotic. Under selective pressure with one of the original substrates (amoxicillin), a high level of reversion occurred in the mutant β-lactamase genes completing a cycle back to the original substrate spectrum. The de novo TRs coupled with reversion makes a genetic toggling mechanism enabling reversible switching between the two phases of the substrate spectrum of β-lactamases. This toggle exemplifies the effective adaptation of de novo TRs for enhanced bacterial survival. We found pairs of direct repeats that mediated the DNA duplication (TR formation). In addition, we found different duos of sequences that mediated the DNA duplication. These novel elements—that we named SCSs (same-strand complementary sequences)—were also found associated with β-lactamase TR mutations from clinical isolates. Both direct repeats and SCSs had a high correlation with TRs in diverse bacterial genomes throughout the major phylogenetic lineages, suggesting that they comprise a fundamental mechanism shaping the bacterial evolution.