Enhancing the scalability of Wolbachia-based vector-borne disease management: time and temperature limits for storage and transport of Wolbachia-infected Aedes aegypti eggs for field releases.

BACKGROUND: Introgression of the bacterial endosymbiont Wolbachia into Aedes aegypti populations is a biocontrol approach being used to reduce arbovirus transmission. This requires mass release of Wolbachia-infected mosquitoes. While releases have been conducted using a variety of techniques, egg releases, using water-soluble capsules containing mosquito eggs and larval food, offer an attractive method due to its potential to reduce onsite resource requirements. However, optimisation of this approach is required to ensure there is no detrimental impact on mosquito fitness and to promote successful Wolbachia introgression. METHODS: We determined the impact of storage time and temperature on wild-type (WT) and Wolbachia-infected (wMel or wAlbB strains) Ae. aegypti eggs. Eggs were stored inside capsules over 8 weeks at 18 °C or 22 °C and hatch rate, emergence rate and Wolbachia density were determined. We next examined egg quality and Wolbachia density after exposing eggs to 4-40 °C to determine how eggs may be impacted if exposed to extreme temperatures during shipment. RESULTS: Encapsulating eggs for 8 weeks did not negatively impact egg viability or resulting adult emergence and Wolbachia density compared to controls. When eggs were exposed to temperatures within 4-36 °C for 48 h, their viability and resulting adult Wolbachia density were maintained; however, both were significantly reduced when exposed to 40 °C. CONCLUSIONS: We describe the time and temperature limits for maintaining viability of Wolbachia-infected Ae. aegypti eggs when encapsulated or exposed to extreme temperatures. These findings could improve the efficiency of mass releases by providing transport and storage constraints to ensure only high-quality material is utilised during field releases.

Trash to Treasure: How Insect Protein and Waste Containers Can Improve the Environmental Footprint of Mosquito Egg Releases.

Release and subsequent establishment of Wolbachia-infected Aedes aegypti in native mosquito populations has successfully reduced mosquito-borne disease incidence. While this is promising, further development is required to ensure that this method is scalable and sustainable. Egg release is a beneficial technique that requires reduced onsite resources and increases community acceptance; however, its incidental ecological impacts must be considered to ensure sustainability. In this study, we tested a more environmentally friendly mosquito rearing and release approach through the encapsulation of diet and egg mixtures and the subsequent utilization of waste containers to hatch and release mosquitoes. An ecologically friendly diet mix was specifically developed and tested for use in capsules, and we demonstrated that using either cricket or black soldier fly meal as a substitute for beef liver powder had no adverse effects on fitness or Wolbachia density. We further encapsulated both the egg and diet mixes and demonstrated no loss in viability. To address the potential of increased waste generation through disposable mosquito release containers, we tested reusing commonly found waste containers (aluminum and tin cans, PET, and glass bottles) as an alternative, conducting a case study in Kiribati to assess the concept's cultural, political, and economic applicability. Our results showed that mosquito emergence and fitness was maintained with a variety of containers, including when tested in the field, compared to control containers, and that there are opportunities to implement this method in the Pacific Islands in a way that is culturally considerate and cost-effective.

Comparative Transcriptomic and Functional Assessments of Linezolid-Responsive Small RNA Genes in Staphylococcus aureus.

Staphylococcus aureus contains a repertoire of at least 50 and possibly 500 small RNAs (sRNAs). The functions of most sRNAs are not understood, although some are known to respond to environmental changes, including the presence of antibiotics. Here, in an effort to better understand the roles of sRNAs in the context of antibiotic exposure, we took a clinical methicillin-resistant S. aureus (MRSA) isolate and separately deleted eight sRNAs that were significantly upregulated in response to the last-line antibiotic linezolid as revealed by transcriptome sequencing (RNA-seq) comparisons. We also deleted an additional 10 sRNAs that were either highly expressed or previously found to respond to antibiotic exposure. There were no significant changes for any of the 18 mutants in a variety of phenotypic screens, including MIC screens, growth competition assays in the presence of linezolid, biofilm formation, and resistance to whole-blood killing. These data suggest sRNA functional redundancy, because despite their high expression levels upon antibiotic exposure, individual sRNA genes do not affect readily observable bacterial phenotypes. The sRNA transcriptional changes we measured during antibiotic exposure might also reflect sRNA "indifference," that is, a general stress response not specifically related to sRNA function. These data underscore the need for sensitive assays and new approaches to try and decipher the functions of sRNA genes in S. aureus IMPORTANCE Bacterial small RNAs (sRNAs) are RNA molecules that can have important regulatory roles across gene expression networks. There is a growing understanding of the scope and potential breadth of impact of sRNAs on global gene expression patterns in Staphylococcus aureus, a major human pathogen. Here, transcriptome comparisons were used to examine the roles of sRNA genes with a potential role in the response of S. aureus to antibiotic exposure. Although no measurable impact on key bacterial phenotypes was observed after deleting each of 18 sRNAs identified by these comparisons, this research is significant because it underscores the subtle modes of action of these sometimes abundant molecules within the bacterium.

RNA-seq analysis of virR and revR mutants of Clostridium perfringens.

BACKGROUND: Clostridium perfringens causes toxin-mediated diseases, including gas gangrene (clostridial myonecrosis) and food poisoning in humans. The production of the toxins implicated in gas gangrene, α-toxin and perfringolysin O, is regulated by the VirSR two-component regulatory system. In addition, RevR, an orphan response regulator, has been shown to affect virulence in the mouse myonecrosis model. RevR positively regulates the expression of genes that encode hydrolytic enzymes, including hyaluronidases and sialidases. RESULTS: To further characterize the VirSR and RevR regulatory networks, comparative transcriptomic analysis was carried out with strand-specific RNA-seq on C. perfringens strain JIR325 and its isogenic virR and revR regulatory mutants. Using the edgeR analysis package, 206 genes in the virR mutant and 67 genes in the revR mutant were found to be differentially expressed. Comparative analysis revealed that VirR acts as a global negative regulator, whilst RevR acts as a global positive regulator. Therefore, about 95 % of the differentially expressed genes were up-regulated in the virR mutant, whereas 81 % of the differentially expressed genes were down-regulated in the revR mutant. Importantly, we identified 23 genes that were regulated by both VirR and RevR, 18 of these genes, which included the sporulation-specific spoIVA, sigG and sigF genes, were regulated positively and negatively by RevR and VirR, respectively. Furthermore, analysis of the mapped RNA-seq reads visualized as depth of coverage plots showed that there were 93 previously unannotated transcripts in intergenic regions. These transcripts potentially encode small RNA molecules. CONCLUSION: In conclusion, using strand-specific RNA-seq analysis, this study has identified differentially expressed chromosomal and pCP13 native plasmid-encoded genes, antisense transcripts, and transcripts within intergenic regions that are controlled by the VirSR or RevR regulatory systems.

Hyperexpression of α-hemolysin explains enhanced virulence of sequence type 93 community-associated methicillin-resistant Staphylococcus aureus.

BACKGROUND: The community-associated methicillin-resistant S. aureus (CA-MRSA) ST93 clone is becoming dominant in Australia and is clinically highly virulent. In addition, sepsis and skin infection models demonstrate that ST93 CA-MRSA is the most virulent global clone of S. aureus tested to date. While the determinants of virulence have been studied in other clones of CA-MRSA, the basis for hypervirulence in ST93 CA-MRSA has not been defined. RESULTS: Here, using a geographically and temporally dispersed collection of ST93 isolates we demonstrate that the ST93 population hyperexpresses key CA-MRSA exotoxins, in particular α-hemolysin, in comparison to other global clones. Gene deletion and complementation studies, and virulence comparisons in a murine skin infection model, showed unequivocally that increased expression of α-hemolysin is the key staphylococcal virulence determinant for this clone. Genome sequencing and comparative genomics of strains with divergent exotoxin profiles demonstrated that, like other S. aureus clones, the quorum sensing agr system is the master regulator of toxin expression and virulence in ST93 CA-MRSA. However, we also identified a previously uncharacterized AraC/XylS family regulator (AryK) that potentiates toxin expression and virulence in S. aureus. CONCLUSIONS: These data demonstrate that hyperexpression of α-hemolysin mediates enhanced virulence in ST93 CA-MRSA, and additional control of exotoxin production, in particular α-hemolysin, mediated by regulatory systems other than agr have the potential to fine-tune virulence in CA-MRSA.

The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.

Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

Attachment and invasion of Neisseria meningitidis to host cells is related to surface hydrophobicity, bacterial cell size and capsule.

We compared exemplar strains from two hypervirulent clonal complexes, strain NMB-CDC from ST-8/11 cc and strain MC58 from ST-32/269 cc, in host cell attachment and invasion. Strain NMB-CDC attached to and invaded host cells at a significantly greater frequency than strain MC58. Type IV pili retained the primary role for initial attachment to host cells for both isolates regardless of pilin class and glycosylation pattern. In strain MC58, the serogroup B capsule was the major inhibitory determinant affecting both bacterial attachment to and invasion of host cells. Removal of terminal sialylation of lipooligosaccharide (LOS) in the presence of capsule did not influence rates of attachment or invasion for strain MC58. However, removal of either serogroup B capsule or LOS sialylation in strain NMB-CDC increased bacterial attachment to host cells to the same extent. Although the level of inhibition of attachment by capsule was different between these strains, the regulation of the capsule synthesis locus by the two-component response regulator MisR, and the level of surface capsule determined by flow cytometry were not significantly different. However, the diplococci of strain NMB-CDC were shown to have a 1.89-fold greater surface area than strain MC58 by flow cytometry. It was proposed that the increase in surface area without changing the amount of anchored glycolipid capsule in the outer membrane would result in a sparser capsule and increase surface hydrophobicity. Strain NMB-CDC was shown to be more hydrophobic than strain MC58 using hydrophobicity interaction chromatography and microbial adhesion-to-solvents assays. In conclusion, improved levels of adherence of strain NMB-CDC to cell lines was associated with increased bacterial cell surface and surface hydrophobicity. This study shows that there is diversity in bacterial cell surface area and surface hydrophobicity within N. meningitidis which influence steps in meningococcal pathogenesis.

Neisserial Correia repeat-enclosed elements do not influence the transcription of pil genes in Neisseria gonorrhoeae and Neisseria meningitidis.

Two human-specific neisserial pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, require the expression of type IV pili (tfp) for initial attachment to the host during infection. However, the mechanisms controlling the assembly and functionality of tfp are poorly understood. It is known that the gonococcal pilE gene, encoding the major subunit, is positively regulated by IHF, a multifunctional DNA binding protein. A neisserial specific repetitive DNA sequence, termed the Correia repeat-enclosed element (CREE) is situated upstream of three pil loci: pilHIJKX (pilH-X), pilGD, and pilF. CREEs have been shown to contain strong promoters, and some CREE variants contain a functional IHF binding site. CREEs might therefore be involved in the regulation of tfp biogenesis in pathogenic Neisseria. Site-directed and deletion mutagenesis on promoter::cat reporter constructs demonstrated that transcription of pilH-X and pilGD is from a σ(70) promoter and is independent of the CREE. The insertion of a CREE in the pilF promoter region in N. meningitidis generated a functional σ(70) promoter. However, there is also a functional promoter at this position in N. gonorrhoeae, where there is no CREE. These results suggest CREE insertion in these three pil loci does not influence transcription and that IHF does not coordinately regulate tfp biogenesis.

ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates.

Fluconazole (FLC) remains the antifungal drug of choice for non-life-threatening Candida infections, but drug-resistant strains have been isolated during long-term therapy with azoles. Drug efflux, mediated by plasma membrane transporters, is a major resistance mechanism, and clinically significant resistance in Candida albicans is accompanied by increased transcription of the genes CDR1 and CDR2, encoding plasma membrane ABC-type transporters Cdr1p and Cdr2p. The relative importance of each transporter protein for efflux-mediated resistance in C. albicans, however, is unknown; neither the relative amounts of each polypeptide in resistant isolates nor their contributions to efflux function have been determined. We have exploited the pump-specific properties of two antibody preparations, and specific pump inhibitors, to determine the relative expression and functions of Cdr1p and Cdr2p in 18 clinical C. albicans isolates. The antibodies and inhibitors were standardized using recombinant Saccharomyces cerevisiae strains that hyper-express either protein in a host strain with a reduced endogenous pump background. In all 18 C. albicans strains, including 13 strains with reduced FLC susceptibilities, Cdr1p was present in greater amounts (2- to 20-fold) than Cdr2p. Compounds that inhibited Cdr1p-mediated function, but had no effect on Cdr2p efflux activity, significantly decreased the resistance to FLC of seven representative C. albicans isolates, whereas three other compounds that inhibited both pumps did not cause increased chemosensitization of these strains to FLC. We conclude that Cdr1p expression makes a greater functional contribution than does Cdr2p to FLC resistance in C. albicans.