Using a human colonoid-derived monolayer to study bacteriophage translocation.

Bacteriophages (phages) are estimated to be the most abundant microorganisms on Earth. Their presence in human blood suggests that they can translocate from non-sterile sites such as the gastrointestinal tract where they are concentrated. To examine phage translocation ex vivo, we adapted a primary colonoid monolayer model possessing cell diversity and architecture, and a thick layer of mucus akin to the colonic environment in vivo. We show that the colonoid monolayer is superior to the Caco-2 cell-line model, possessing intact and organized tight junctions and generating a physiologically relevant mucus layer. We showed, using two different phages, that translocation across the colonoid monolayer was largely absent in differentiated monolayers that express mucus, unlike Caco-2 cultures that expressed little to no mucus. By stimulating mucus production or removing mucus, we further demonstrated the importance of colonic mucus in preventing phage translocation. Finally, we used etiological drivers of gut permeability (alcohol, fat, and inflammatory cytokines) to measure their effects on phage translocation, demonstrating that all three stimuli have the capacity to amplify phage translocation. These findings suggest that phage translocation does occur in vivo but may be largely dependent on colonic mucus, an important insight to consider in future phage applications.

Australian Group on Antimicrobial Resistance (AGAR) Australian Gram-negative Surveillance Outcome Program (GnSOP).

Abstract: The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric gram-negative pathogens. The 2021 survey was the ninth year to focus on bloodstream infections caused by Enterobacterales, and the seventh year where Pseudomonas aeruginosa and Acinetobacter species were included. The 2021 survey tested 8,947 isolates, comprising Enterobacterales (8,104; 90.6%), P. aeruginosa (745; 8.3%) and Acinetobacter species (98; 1.1%), using commercial automated methods. The results were analysed using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2022). Of the key resistances, resistance to the third-generation cephalosporin ceftriaxone was found in 12.5%/12.5% (CLSI/EUCAST criteria) of Escherichia coli and in 6.1%/6.1% of Klebsiella pneumoniae. Resistance rates to ciprofloxacin were 12.3%/12.3% for E. coli; 7.2%/7.2% for K. pneumoniae; 5.4%/5.4% for Enterobacter cloacae complex; and 3.7%/8.0% for P. aeruginosa. Resistance rates to piperacillin-tazobactam were 2.8%/6.5%; 2.9%/9.9%; 18.4%/28.1%; and 6.9%/12.8% for the same four species, respectively. Seventeen Enterobacterales isolates from 17 patients were shown to harbour a carbapenemase gene: 12 blaIMP-4; two blaNDM-7; one blaNDM-1; one blaOXA-181; and one blaKPC-2. No transmissible carbapenemase genes were detected among P. aeruginosa or Acinetobacter isolates in the 2021 survey.

Australian Group on Antimicrobial Resistance (AGAR) Australian Gram-negative Sepsis Outcome Programme (GnSOP) Annual Report 2020.

Abstract: The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric gram-negative pathogens. The 2020 survey was the eighth year to focus on bloodstream infections caused by Enterobacterales, and the sixth year in which Pseudomonas aeruginosa and Acinetobacter species were included. Eight thousand seven hundred and fifty-two isolates, comprising Enterobacterales (7,871, 89.9%), P. aeruginosa (771, 8.8%) and Acinetobacter species (110, 1.3%), were tested using commercial automated methods. The results were analysed using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2021). Of the key resistances, resistance to the third-generation cephalosporin ceftriaxone was found in 13.5%/13.5% (CLSI/EUCAST criteria) of Escherichia coli and 8.7%/8.7% of Klebsiella pneumoniae. Resistance rates to ciprofloxacin were 16.1%/16.1% for E. coli; 9.9%/9.9% for K. pneumoniae; 5.8%/5.8% for Enterobacter cloacae complex; and 4.5%/8.1% for P. aeruginosa. Resistance rates to piperacillin-tazobactam were 2.5%/6.6%; 3.9%/12.5%; 16.9%/26.3%; and 5.5%/14.4% for the same four species respectively. Thirty-two isolates from 32 patients were shown to harbour at least one carbapenemase gene: 19 blaIMP-4, three blaGES-5, two blaNDM-1, two blaNDM-5, two blaOXA-48, two blaOXA-181, one blaIMI-1, and one blaOXA-23+NDM-1.

Australian Group on Antimicrobial Resistance (AGAR) Australian Gram-negative Sepsis Outcome Programme (GNSOP) Annual Report 2019.

The Australian Group on Antimicrobial Resistance (AGAR) performs regular period-prevalence studies to monitor changes in antimicrobial resistance in selected enteric gram-negative pathogens. The 2018 survey was the sixth year to focus on bloodstream infections, and included Enterobacterales, Pseudomonas aeruginosa and Acinetobacter species. Eight thousand eight hundred and fifty-seven isolates, comprising Enterobacterales (7,983; 90.1%), P. aeruginosa (764; 8.6%) and Acinetobacter species (110; 1.2%), were tested using commercial automated methods. The results were analysed using Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints (January 2020). Of the key resistances, resistance to the third-generation cephalosporin ceftriaxone was found in 13.3%/13.3% (CLSI/EUCAST criteria) of Escherichia coli and 8.4%/8.4% of Klebsiella pneumoniae. Resistance rates to ciprofloxacin were 16.0%/16.0% for E. coli, 10.2%/10.2% for K. pneumoniae complex, 5.9%/5.9% for Enterobacter cloacae complex, and 4.1%/9.3% for P. aeruginosa. Resistance rates to piperacillin-tazobactam were 3.2%/5.7%, 4.7%/8.5%, 14.8%/21.4%, and 6.9%/12.5% for the same four species/complex respectively. Twenty-nine isolates from 29 patients were shown to harbour a carbapenemase gene: 15 blaIMP-4, five blaOXA-181, four blaOXA-23 (one with blaOXA-58 also), three blaNDM-4/5, one blaGES-5, and one blaIMP-1.

Characterizing the role of porin mutations in susceptibility of beta lactamase producing Klebsiella pneumoniae isolates to ceftaroline and ceftaroline-avibactam.

OBJECTIVES: Evaluate the role of porins in the susceptibility of Klebsiella pneumoniae to ceftaroline and ceftaroline-avibactam. METHODS: Susceptibility to ceftaroline and ceftaroline-avibactam was tested by broth microdilution method in Klebsiella pneumoniae isolates (n = 65), including isogenic mutants (n = 30) and clinical isolates (n = 35), with different outer membrane porin defects in the presence or absence of beta lactamases. RESULTS: Ceftaroline exhibited excellent activity against all the isogenic porin mutants with a MIC range of 0.125-0.25 µg/ml. Ceftaroline showed limited activity in the presence of extended spectrum ß-lactamase enzymes in isogenic mutant constructs as expected but regained effectiveness in combination with avibactam against these isolates except those carrying metallo-carbapenemase (IMP-4) with an MIC range of 0.25->32 µg/ml. Ceftaroline-avibactam was able to inhibit 86% of the clinical isolates (n = 35) of Klebsiella pneumoniae carrying porin defects and multiple beta lactamases with only four isolates showing raised MICs against the combination (MIC range 0.125-4 µg/ml). One clinical isolate with IMP-4 carbapenemase had an MIC value of >32 µg/ml. CONCLUSION: Outer membrane porins play a key role in the transport of ceftaroline inKlebsiella pneumoniae but it remains effective in isolates with altered permeability due to common porin mutations. The addition of avibactam substantially enhances the potency of ceftaroline providing an effective remedy to the problem of omnipresent beta lactamases in these bacteria.