Arg1 from Cryptococcus neoformans lacks PI3 kinase activity and conveys virulence roles via its IP3-4 kinase activity.

Inositol tris/tetrakis phosphate kinases (IP3-4K) in the human fungal priority pathogens, Cryptococcus neoformans (CnArg1) and Candida albicans (CaIpk2), convey numerous virulence functions, yet it is not known whether the IP3-4K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) CnArg1 strain (dkArg1). We verified that, although dkARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP3-4K activity. Using recombinant CnArg1 and CaIpk2, we confirmed that, unlike the IP3-4K homologs in humans and Saccharomyces cerevisiae, CnArg1 and CaIpk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP3 conversion is blocked in the dkArg1 and ARG1 deletion (Cnarg1Δ) strains and that 1-IP7 and a recently discovered isomer (4/6-IP7) are made by wild-type C. neoformans. Importantly, the dkArg1 and Cnarg1Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP3-4K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since CnArg1 is dispensable for growth on different nitrogen sources. IP3-4K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.IMPORTANCEThe World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP3-4K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans, and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP3-4K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP3-4K catalytic activity of CnArg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion, reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP3-4K catalytic site, as a new antifungal drug class.

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.

Pre-Treatment with Bromelain Prevents Intestinal Dysbiosis in Pigs with Post-Weaning Diarrhea, without Increasing Antimicrobial Resistance in Escherichia coli.

Pigs are especially vulnerable to intestinal pathogens and dysbiosis in the first two weeks after weaning. Infection with enterotoxigenic strains of Escherichia coli (ETEC) in combination with poor nutrition and hygiene can lead to diarrhea, poor growth and increased mortality. While neomycin and zinc oxide can prevent post-weaning diarrhea (PWD), their broad-spectrum activity also kills commensal microbiota and can lead to the emergence of heavy metal and antimicrobial resistance. Bromelain prevents attachment of F4 ETEC to intestinal enterocytes by cleaving the host receptor. In controlled environmental facilities, weaned pigs treated with either therapeutic levels of neomycin sulfate, zinc oxide, bromelain or non-treated were monitored for diarrhea, weight gain, feed intake, feed efficiency, excretion of F4 ETEC, changes to their intestinal microbiomes and antimicrobial resistance in E. coli. The treatment effects were evaluated at weaning, during two weeks of treatment and for three weeks after treatments ceased. Minimal clinical signs of PWD were observed, except in zinc-treated pigs post treatment. Intestinal dysbiosis was observed in response to diarrhea and in pigs treated with both neomycin and zinc. Antimicrobial resistance increased in commensal E. coli isolated from neomycin- and zinc-treated pigs. In contrast, bromelain controlled PWD and prevented intestinal dysbiosis without inducing antimicrobial resistance.

Dysregulating PHO Signaling via the CDK Machinery Differentially Impacts Energy Metabolism, Calcineurin Signaling, and Virulence in Cryptococcus neoformans.

Fungal pathogens uniquely regulate phosphate homeostasis via the cyclin-dependent kinase (CDK) signaling machinery of the phosphate acquisition (PHO) pathway (Pho85 kinase-Pho80 cyclin-CDK inhibitor Pho81), providing drug-targeting opportunities. Here, we investigate the impact of a PHO pathway activation-defective Cryptococcus neoformans mutant (pho81Δ) and a constitutively activated PHO pathway mutant (pho80Δ) on fungal virulence. Irrespective of phosphate availability, the PHO pathway was derepressed in pho80Δ with all phosphate acquisition pathways upregulated and much of the excess phosphate stored as polyphosphate (polyP). Elevated phosphate in pho80Δ coincided with elevated metal ions, metal stress sensitivity, and a muted calcineurin response, all of which were ameliorated by phosphate depletion. In contrast, metal ion homeostasis was largely unaffected in the pho81Δ mutant, and Pi, polyP, ATP, and energy metabolism were reduced, even under phosphate-replete conditions. A similar decline in polyP and ATP suggests that polyP supplies phosphate for energy production even when phosphate is available. Using calcineurin reporter strains in the wild-type, pho80Δ, and pho81Δ background, we also demonstrate that phosphate deprivation stimulates calcineurin activation, most likely by increasing the bioavailability of calcium. Finally, we show that blocking, as opposed to permanently activating, the PHO pathway reduced fungal virulence in mouse infection models to a greater extent and that this is most likely attributable to depleted phosphate stores and ATP, and compromised cellular bioenergetics, irrespective of phosphate availability. IMPORTANCE Invasive fungal diseases cause more than 1.5 million deaths per year, with an estimated 181,000 of these deaths attributable to Cryptococcal meningitis. Despite the high mortality, treatment options are limited. In contrast to humans, fungal cells maintain phosphate homeostasis via a CDK complex, providing drug-targeting opportunities. To investigate which CDK components are the best targets for potential antifungal therapy, we used strains with a constitutively active (pho80Δ) and an activation-defective (pho81Δ) PHO pathway, to investigate the impact of dysregulated phosphate homeostasis on cellular function and virulence. Our studies suggest that inhibiting the function of Pho81, which has no human homologue, would have the most detrimental impact on fungal growth in the host due to depletion of phosphate stores and ATP, irrespective of phosphate availability in the host.

Population Structure and Genomic Characteristics of Australian Erysipelothrix rhusiopathiae Reveals Unobserved Diversity in the Australian Pig Industry.

Erysipelothrix rhusiopathiae is a bacterial pathogen that is the causative agent of erysipelas in a variety of animals, including swine, emus, turkeys, muskox, caribou, moose, and humans. This study aims to investigate the population structure and genomic features of Australian isolates of E. rhusiopathiae in the Australian pig industry and compare them to the broader scope of isolates worldwide. A total of 178 isolates (154 Australian, seven vaccine isolates, six international isolates, and 11 of unknown origin) in this study were screened against an MLST scheme and publicly available reference isolates, identifying 59 new alleles, with isolates separating into two main single locus variant groups. Investigation with BLASTn revealed the presence of the spaA gene in 171 (96%) of the isolates, with three main groups of SpaA protein sequences observed amongst the isolates. Novel SpaA protein sequences, categorised here as group 3 sequences, consisted of two sequence types forming separate clades to groups 1 and 2, with amino acid variants at positions 195 (D/A), 303 (G/E) and 323(P/L). In addition to the newly identified groups, five new variant positions were identified, 124 (S/N), 307 (Q/R), 323 (P/L), 379 (M/I), and 400 (V/I). Resistance screening identified genes related to lincomycin, streptomycin, erythromycin, and tetracycline resistance. Of the 29 isolates carrying these resistance genes, 82% belonged to SpaA group 2-N101S (n = 22) or 2-N101S-I257L (n = 2). In addition, 79% (n = 23) of these 29 isolates belonged to MLST group ST 5. Our results illustrate that Australia appears to have a unique diversity of E. rhusiopathiae isolates in pig production industries within the wider global context of isolates.

RTA1 Is Involved in Resistance to 7-Aminocholesterol and Secretion of Fungal Proteins in Cryptococcus neoformans.

Cryptococcus neoformans (Cn) is a pathogenic yeast that is the leading cause of fungal meningitis in immunocompromised patients. Various Cn virulence factors, such as the enzyme laccase and its product melanin, phospholipase, and capsular polysaccharide have been identified. During a screen of knockout mutants, the gene resistance to aminocholesterol 1 (RTA1) was identified, the function of which is currently unknown in Cn. Rta1 homologs in S. cerevisiae belong to a lipid-translocating exporter family of fungal proteins with transmembrane regions and confer resistance to the antimicrobial agent 7-aminocholesterol when overexpressed. To determine the role of RTA1 in Cn, the knock-out (rta1Δ) and reconstituted (rta1Δ+RTA1) strains were created and phenotypically tested. RTA1 was involved in resistance to 7-aminocholesterol, and also in exocyst complex component 3 (Sec6)-mediated secretion of urease, laccase, and the major capsule component, glucuronoxylomannan (GXM), which coincided with significantly smaller capsules in the rta1Δ and rta1Δ+RTA1 strains compared to the wild-type H99 strain. Furthermore, RTA1 expression was reduced in a secretory 14 mutant (sec14Δ) and increased in an RNAi Sec6 mutant. Transmission electron microscopy demonstrated vesicle accumulation inside the rta1Δ strain, predominantly near the cell membrane. Given that Rta1 is likely to be a transmembrane protein located at the plasma membrane, these data suggest that Rta1 may be involved in both secretion of various fungal virulence factors and resistance to 7-aminocholesterol in Cn.

Co-Occurrence of Multidrug Resistant Klebsiella pneumoniae Pathogenic Clones of Human Relevance in an Equine Pneumonia Case.

The global epidemiology of multidrug resistant Klebsiella pneumoniae, a serious threat to both animal and human health, is dominated by the spread of pathogenic clones, each separately evolving via acquisition of transferable antibiotic resistance or niche-specific virulence determinants. In horses, K. pneumoniae infection can lead to severe respiratory illness. Here, we characterized multiple isolates recovered from bronchial aspirates of a mare with pneumonia refractory to antibiotics. First, we used a combination of standard microbiology, bacteriophage cross-susceptibility and antibiotic resistance testing to profile the infecting K. pneumoniae population. The genomes of isolates with distinct fingerprints (pulsed-field gel electrophoresis) and unique combined bacteriophage/antibiotic profiles were then further analyzed using whole-genome sequencing. Adhesion to human epithelial cells and biofilm production were also measured as virulence indicators. Although it is commonly expected for one clone to dominate an infection episode, we identified five coexisting multidrug resistant K. pneumoniae sharing the same niche. One was a novel sequence type (ST4656), while the other four were all members of emerging human pathogenic clonal groups (ST307, ST628, ST893 and ST392). These isolates did not display significant differences from one another in terms of virulence or resistance and differed only in plasmid content from isolates implicated in severe human infections, with equal potential to prolong duration and severity of infection when sharing the same niche. This study highlights the importance of more precise surveillance and detection measures to uncover bacterial heterogeneity, reminding us that the "single clone" concept is not an absolute in invasive bacterial infections. IMPORTANCE Multidrug resistant Klebsiella pneumoniae are agents of life-threatening infections in animals and humans, with several multidrug resistant clones causing outbreaks of disease worldwide. It is generally accepted that only one clone will be dominant in an infection episode. In this study, we investigated K. pneumoniae isolates from a horse with severe pneumonia and demonstrated co-occurrence of multiple sequence types previously identified as emerging human pathogens. The equine isolates are not significantly different from one another in terms of virulence or resistance, with equal potential to prolong duration and severity of infection, and are indistinguishable from isolates recovered from humans, except for plasmid content. Our study highlights how the "one dominant clone" concept is not an absolute in severe infection, illustrating the need for improved diagnostics to track heterogeneity of infection, and reinforces the importance of cross-monitoring of environmental and human reservoirs of multidrug resistant pathogens.

Inositol polyphosphate-protein interactions: Implications for microbial pathogenicity.

Inositol polyphosphates (IPs) and inositol pyrophosphates (PP-IPs) regulate diverse cellular processes in eukaryotic cells. IPs and PP-IPs are highly negatively charged and exert their biological effects by interacting with specific protein targets. Studies performed predominantly in mammalian cells and model yeasts have shown that IPs and PP-IPs modulate target function through allosteric regulation, by promoting intra- and intermolecular stabilization and, in the case of PP-IPs, by donating a phosphate from their pyrophosphate (PP) group to the target protein. Technological advances in genetics have extended studies of IP function to microbial pathogens and demonstrated that disrupting PP-IP biosynthesis and PP-IP-protein interaction has a profound impact on pathogenicity. This review summarises the complexity of IP-mediated regulation in eukaryotes, including microbial pathogens. It also highlights examples of poor conservation of IP-protein interaction outcome despite the presence of conserved IP-binding domains in eukaryotic proteomes.

Effects of Antibiotic Treatment with Piperacillin/Tazobactam versus Ceftriaxone on the Composition of the Murine Gut Microbiota.

Effective antimicrobial stewardship requires a better understanding of the impact of different antibiotics on the gut microflora. Studies with humans are confounded by large interindividual variability and difficulty in identifying control cohorts. However, controlled murine models can provide valuable information. In this study, we examined the impact of a penicillin-like antibiotic (piperacillin-tazobactam [TZP]) or a third-generation cephalosporin (ceftriaxone [CRO]) on the murine gut microbiota by analysis of changes in fecal microbiome composition by 16S rRNA amplicon sequencing and standard microbiology. Resistance to colonization by multidrug-resistant Escherichia coli sequence type 131 (ST131) and Klebsiella pneumoniae ST258 was also tested. Changes in microbiome composition and a significant (P < 0.05) decrease in diversity occurred in all treated mice, but dysbiosis was more marked and prolonged after CRO exposure, with a persistent rise in ProteobacteriaEnterobacteriaceae blooms occurred in all antibiotic-treated mice, but for TZP, unlike CRO, these were significant only under direct antibiotic pressure. At the height of dysbiosis after antibiotic termination, the murine gut was highly susceptible to colonization with both multidrug-resistant enterobacterial pathogens. Cohabitation of treated mice with untreated individuals had a notable mitigating effect on dysbiosis of treated guts. The administration of a third-generation cephalosporin caused a more severe imbalance in the murine fecal microflora than that caused by a penicillin/ß-lactam inhibitor combination with comparable activity against medically important virulent bacteria. At the height of dysbiosis, both antibiotic treatments equally led to microbial instability associated with loss of resistance to gut colonization by antibiotic-resistant pathogens.

Fine capsule variation affects bacteriophage susceptibility in Klebsiella pneumoniae ST258.

Multidrug resistant (MDR) carbapenemase-producing (CP) Klebsiella pneumoniae, belonging to clonal group CG258, is capable of causing severe disease in humans and is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as therapeutic alternatives to antibiotics. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the genomes of 18 Kumoniae target strains and tested them against novel lytic bacteriophages (n = 65). The genomes of K pneumoniae carrying blaNDM and blaKPC were sequenced and CG258 isolates selected for bacteriophage susceptibility testing. The local K pneumoniae CG258 population was dominated by sequence type ST258 clade 1 (86%) with variations in capsular locus (cps) and prophage content. CG258-specific bacteriophages primarily targeted the capsule, but successful infection is also likely blocked in some by immunity conferred by existing prophages. Five tailed bacteriophages against K pneumoniae ST258 clade 1 were selected for further characterization. Our findings show that effective control of K pneumoniae CG258 with bacteriophage will require mixes of diverse lytic viruses targeting relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic bacteriophage candidates against this serious pathogen.

Phage therapy for severe bacterial infections: a narrative review.

Bacteriophage (phage) therapy is re-emerging a century after it began. Activity against antibiotic-resistant pathogens and a lack of serious side effects make phage therapy an attractive treatment option in refractory bacterial infections. Phages are highly specific for their bacterial targets, but the relationship between in vitro activity and in vivo efficacy remains to be rigorously evaluated. Pharmacokinetic and pharmacodynamic principles of phage therapy are generally based on the classic predator-prey relationship, but numerous other factors contribute to phage clearance and optimal dosing strategies remain unclear. Combinations of fully characterised, exclusively lytic phages prepared under good manufacturing practice are limited in their availability. Safety has been demonstrated but randomised controlled trials are needed to evaluate efficacy.

Diversity of P1 phage-like elements in multidrug resistant Escherichia coli.

The spread of multidrug resistance via mobile genetic elements is a major clinical and veterinary concern. Pathogenic Escherichia coli harbour antibiotic resistance and virulence genes mainly on plasmids, but also bacteriophages and hybrid phage-like plasmids. In this study, the genomes of three E. coli phage-like plasmids, pJIE250-3 from a human E. coli clinical isolate, pSvP1 from a porcine ETEC O157 isolate, and pTZ20_1P from a porcine commensal E. coli, were sequenced (PacBio RSII), annotated and compared. All three elements are coliphage P1 variants, each with unique adaptations. pJIE250-3 is a P1-derivative that has lost lytic functions and contains no accessory genes. In pTZ20_1P and pSvP1, a core P1-like genome is associated with insertion sequence-mediated acquisition of plasmid modules encoding multidrug resistance and virulence, respectively. The transfer ability of pTZ20_1P, carrying antibiotic resistance markers, was also tested and, although this element was not able to transfer by conjugation, it was able to lysogenize a commensal E. coli strain with consequent transfer of resistance. The incidence of P1-like plasmids (~7%) in our E. coli collections correlated well with that in public databases. This study highlights the need to investigate the contribution of phage-like plasmids to the successful spread of antibiotic resistant pathotypes.

An unusual culprit: Klebsiella pneumoniae causing septicaemia outbreaks in neonatal pigs?

This study investigated the cause of recent outbreaks of septicaemia in neonatal pigs in Australia (Victoria and Queensland). Septicaemia in neonatal pigs is commonly caused by enterotoxigenic E. coli, extraintestinal pathogenic E. coli and beta-haemolytic streptococci. Infrequent causes of septicaemia are Actinobacillus suis and Citrobacter freundii. Therefore, it was quite unexpected when Klebsiella pneumoniae was isolated in predominant growth from multiple organs of pigs dying of septicaemia from six days of age. Two cases in Victoria were associated with multilocus sequence type 25 (ST25), whereas the cases on a single farm in Queensland were associated with two different sequence types (ST278 and ST1978). Similar outbreaks of septicaemia have also occurred in England, but all were associated with K. pneumoniae ST25. The K. pneumoniae sequence types did not cluster phylogenetically into groups of isolates from the same geographical location or into groups which caused either septicaemia or pneumonia. Antibiotic resistance also varied between isolates and showed neomycin resistance in Queensland. These results suggest that several sequence types of K. pneumoniae are involved in causing outbreaks of septicaemia in neonatal pigs, in addition to its previously recognised role in pneumonia and mastitis.

A Comparison of Diets Supplemented with a Feed Additive Containing Organic Acids, Cinnamaldehyde and a Permeabilizing Complex, or Zinc Oxide, on Post-Weaning Diarrhoea, Selected Bacterial Populations, Blood Measures and Performance in Weaned Pigs Experimentally Infected with Enterotoxigenic E. coli.

The effects of feeding a diet supplemented with zinc oxide (ZnO) or a blend of organic acids, cinnamaldehyde and a permeabilizing complex (OACP) on post-weaning diarrhoea (PWD) and performance in pigs infected with enterotoxigenic E. coli (ETEC) were examined. Additionally, changes in selected bacterial populations and blood measures were assessed. A total of 72 pigs weaned at 22 d of age and weighing 7.2 ± 1.02 kg (mean ± SEM) was used. Treatments were: base diet (no antimicrobial compounds); base diet + 3 g ZnO/kg; base diet + 1.5 g OACP/kg. Dietary treatments started on the day of weaning and were fed ad libitum for 3 weeks. All pigs were infected with an F4 ETEC on d 4, 5 and 6 after weaning. The incidence of PWD was lower in pigs fed ZnO ( p = 0.026). Overall, pigs fed ZnO grew faster ( p = 0.013) and ate more ( p = 0.004) than the base diet-fed pigs, with OACP-fed pigs performing the same ( p > 0.05) as both the ZnO- and base diet-fed pigs. Feed conversion ratio was similar for all diets ( p > 0.05). The percentage of E. coli with F4 fimbriae was affected a day by treatment interaction ( p = 0.037), with more E. coli with F4 fimbriae found in pigs fed ZnO on d 11 ( p = 0.011) compared to base diet-fed pigs. Only significant time effects ( p < 0.05) occurred for blood measures. Under the conditions of this study, inclusion of OACP gave statistically similar production responses to pigs fed ZnO, however pigs fed ZnO had less PWD compared to OACP- and the base diet-fed pigs.