The conventional and most accepted method of measuring the lytic activity of a phage against its bacterial host is the plaque assay. This method is laborious, time consuming and expensive, especially in high throughput analyses where multiple phage-bacterial interactions are required to be monitored simultaneously. It can also vary considerably with the experimenter and by the growth and plating conditions. Alternatively, the lytic activity can be measured indirectly by following the decrease in optical density of the bacterial cultures owing to lysis. Here we describe an automated, high throughput, indirect liquid lysis assay to evaluate phage growth using the OmniLogTM system. The OmniLogTM system uses redox chemistry, employing cell respiration as a universal reporter. During active growth of bacteria, cellular respiration reduces a tetrazolium dye and produces a color change that is measured in an automated fashion. On the other hand, successful phage infection and subsequent growth of the phage in its host bacterium results in reduced bacterial growth and respiration and a concomitant reduction in color. Here we show that microtiter plate wells inoculated with Bacillus anthracis and phage show decreased or no growth, compared with the wells containing bacteria only or phage resistant bacteria plus phage. Also, we show differences in the kinetics of bacterial growth and the timing of appearance of phage resistant bacteria in the presence of individual phages or a cocktail of B. anthracis specific phages. The results of these experiments indicate that the OmniLogTM system could be used reliably for indirectly measuring phage growth in high throughput host range and phage and antibiotics combination studies.
Resistance to ceftriaxone in is mainly conferred by mosaic alleles that encode penicillin-binding protein 2 (PBP2) variants with markedly lower rates of acylation by ceftriaxone. To assess the impact of these mosaic alleles on gonococcal fitness, we introduced the mosaic alleles from two ceftriaxone-resistant (Cro) clinical isolates (H041 and F89) into a Cro strain (FA19) by allelic exchange and showed that the resultant Cro mutants were significantly outcompeted by the Cro parent strain and in a murine infection model. Four Cro compensatory mutants of FA19 were isolated independently from mice that outcompeted the parent strain both and One of these compensatory mutants (LV41C) displayed a unique growth profile, with rapid log growth followed by a sharp plateau/gradual decline at stationary phase. Genome sequencing of LV41C revealed a mutation (G348D) in the gene encoding the bifunctional aconitate hydratase 2/2 methylisocitrate dehydratase. Introduction of the allele into FA19 conferred both a growth profile that phenocopied that of LV41C and a fitness advantage, although not as strongly as that exhibited by the original compensatory mutant, suggesting the existence of additional compensatory mutations. The mutant aconitase appears to be a functional knockout with lower activity and expression than wild-type aconitase. Transcriptome sequencing (RNA-seq) analysis of FA19 revealed a large set of upregulated genes involved in carbon and energy metabolism. We conclude that compensatory mutations can be selected in Cro gonococcal strains that increase metabolism to ameliorate their fitness deficit. The emergence of ceftriaxone-resistant (Cro) has led to the looming threat of untreatable gonorrhea. Whether Cro resistance is likely to spread can be predicted from studies that compare the relative fitnesses of susceptible and resistant strains that differ only in the gene that confers Cro resistance. We showed that mosaic alleles found in Cro clinical isolates are outcompeted by the Cro parent strain and but that compensatory mutations that allow ceftriaxone resistance to be maintained by increasing bacterial fitness are selected during mouse infection. One compensatory mutant that was studied in more detail had a mutation in , which encodes the aconitase that functions in the tricarboxylic acid (TCA) cycle. This study illustrates that compensatory mutations can be selected during infection, which we hypothesize may allow the spread of Cro resistance in nature. This study also provides novel insights into gonococcal metabolism and physiology.
Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin-resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upwards of 60 amino acid changes relative to wild-type PBP2, or by non-mosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates were identified that harbored a mosaic penA allele with an A501P mutation. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon-501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, A501S) were isolated that increased resistance and preserved essential transpeptidase function. To understand their structural implications, these mutations were introduced into the non-mosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was solved and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active-site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.
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