Matthew E. Wand

Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis

BackgroundBurkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella) infection models.ResultsB. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD) values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis.ConclusionsWe have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.

Mechanisms of increased resistance to chlorhexidine and cross-resistance to colistin following exposure of Klebsiella pneumoniae clinical isolates to chlorhexidine

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen that is often difficult to treat due to its multidrug resistance (MDR). We have previously shown that K. pneumoniae strains are able to “adapt” (become more resistant) to the widely used bisbiguanide antiseptic chlorhexidine. Here, we investigated the mechanisms responsible for and the phenotypic consequences of chlorhexidine adaptation, with particular reference to antibiotic cross-resistance. In five of six strains, adaptation to chlorhexidine also led to resistance to the last-resort antibiotic colistin. Here, we show that chlorhexidine adaptation is associated with mutations in the two-component regulator phoPQ and a putative Tet repressor gene (smvR) adjacent to the major facilitator superfamily (MFS) efflux pump gene, smvA. Upregulation of smvA (10- to 27-fold) was confirmed in smvR mutant strains, and this effect and the associated phenotype were suppressed when a wild-type copy of smvR was introduced on plasmid pACYC. Upregulation of phoPQ (5- to 15-fold) and phoPQ-regulated genes, pmrD (6- to 19-fold) and pmrK (18- to 64-fold), was confirmed in phoPQ mutant strains. In contrast, adaptation of K. pneumoniae to colistin did not result in increased chlorhexidine resistance despite the presence of mutations in phoQ and elevated phoPQ, pmrD, and pmrK transcript levels. Insertion of a plasmid containing phoPQ from chlorhexidine-adapted strains into wild-type K. pneumoniae resulted in elevated expression levels of phoPQ, pmrD, and pmrK and increased resistance to colistin, but not chlorhexidine. The potential risk of colistin resistance emerging in K. pneumoniae as a consequence of exposure to chlorhexidine has important clinical implications for infection prevention procedures.

Acinetobacter baumannii virulence is enhanced in Galleria mellonella following biofilm adaptation.

The opportunistic nosocomial pathogen Acinetobacter baumannii is responsible for a growing number of infections; however, few of its potential virulence factors have been identified, and how this organism causes infection remains largely unknown. Bacterial biofilms are often an important component in infection and persistence but there is no conclusive evidence to link biofilm formation with virulence and severity of infection in Acinetobacter. To investigate this link, several clinical isolates were assessed in biofilm culture models and were tested for virulence in the insect model Galleria mellonella. In both systems, the profiles showed significant differences between strains, but no correlation was observed between virulence and the ability to form biofilms. In contrast, A. baumannii cells from a biofilm produced higher mortality rates than an equivalent number of planktonic cells. Relative to planktonic cells, A. baumannii biofilm cultures also showed reduced sensitivity to antibiotics normally used in the treatment of A. baumannii, especially colistin. This model, therefore, provides a suitable system to investigate the link between biofilm growth and various factors influencing virulence during A. baumannii infection.

Superoxide dismutase C is required for intracellular survival and virulence of Burkholderia pseudomallei.

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a life-threatening disease of humans. Within host cells, superoxide is an important mediator of pathogen killing. In this study, we have identified the B. pseudomallei K96243 sodC gene, shown that it has superoxide dismutase activity, and constructed an allelic deletion mutant of this gene. Compared with the wild-type, the mutant was more sensitive to killing by extracellular superoxide, but not to superoxide generated intracellularly. The sodC mutant showed a markedly decreased survival in J774A.1 mouse macrophages, and reduced numbers of bacteria were recovered from human polymorphonuclear neutrophils (PMNs) when compared with the wild-type. The numbers of wild-type or mutant bacteria recovered from human diabetic neutrophils were significantly lower than from normal human neutrophils. The sodC mutant was attenuated in BALB/c mice. Our results indicate that SodC plays a key role in the virulence of B. pseudomallei, but that diabetics are not more susceptible to infection because of a reduced ability of PMNs to kill by superoxide.

Retention of virulence following adaptation to colistin in Acinetobacter baumannii reflects the mechanism of resistance

OBJECTIVES Colistin resistance in Acinetobacter baumannii has been associated with loss of virulence and a negative impact on isolate selection. In this study, exposure of clinical isolates to suboptimal concentrations of colistin was used to explore the capacity to develop resistance by diverse mechanisms, and whether acquired resistance always reduces fitness and virulence. METHODS Twelve colistin-susceptible clinical A. baumannii isolates were exposed to a sub-MIC concentration of colistin over 6 weeks with weekly increases in concentration. Stable resistance was then phenotypically investigated with respect to antibiotic/biocide resistance, virulence in Galleria mellonella and growth rate. Putative mechanisms of resistance were identified by targeted sequencing of known resistance loci. RESULTS Eight A. baumannii isolates acquired resistance to colistin within 1 week with MICs ranging from 2 to >512 mg/L. By 6 weeks 11 isolates were resistant to colistin; this was linked to the development of mutations in pmr or lpx genes. Strains that developed mutations in lpxACD showed a loss of virulence and increased susceptibility to several antibiotics/disinfectants tested. Two of the colistin-resistant strains with mutations in pmrB retained similar virulence levels to their respective parental strains in G. mellonella. CONCLUSIONS Acquisition of colistin resistance does not always lead to a loss of virulence, especially when this is linked to mutations in pmrB. This underlines the importance of understanding the mechanism of colistin resistance as well as the phenotype.

Complex interactions of Klebsiella pneumoniae with the host immune system in a Galleria mellonella infection model.

Worldwide, Klebsiella pneumoniae is an increasingly problematic opportunistic pathogen, with the emergence of carbapenem-resistant isolates of special importance. The mechanisms of virulence are poorly understood, and the current study utilized the invertebrate model Galleria mellonella to investigate facets of the virulence process. A range of UK clinical isolates and reference strains was assessed in Galleria by measuring survival as an end point. The clinical strains showed a range of virulence, with the majority of strains (68 %) causing greater than 50 % mortality at a challenge dose of 1×10(5) c.f.u. Three additional intermediate read-outs were developed to allow the mechanisms of virulence of Klebsiella to be dissected further. The release of lactate dehydrogenase as a marker of cell damage was the best predictor of virulence. Melanization as a marker of the insect innate immune system and ability to proliferate within Galleria as a marker of immune evasion also broadly correlated with survival but with some notable exceptions. No direct correlation was observed between virulence and either K1 or other defined capsular types, the carriage of defined virulence factors or particular functional phenotypes. Overall, the study showed that Galleria can provide significant insights into the mechanisms of virulence, and that this can be applied to the study of opportunistic human pathogens.

Evaluation of the effectiveness of hydrogen-peroxide-based disinfectants on biofilms formed by Gram-negative pathogens

BACKGROUND Hydrogen peroxide (H2O2)-based disinfectants are widely used in a number of different healthcare settings to control bacterial colonization and contamination, and reduce the risk of cross-infection. Efficacy tests of these formulations are performed on planktonic cultures, although it is well known that biofilms are the dominant form of bacterial contamination and more difficult to eradicate. AIM To determine if the biofilms of three different Gram-negative pathogens associated with multi-drug-resistant phenotypes can be eradicated effectively using different H2O2-based disinfectants. METHODS Planktonic cultures and single-species 24-h biofilms of seven strains of Acinetobacter spp., seven strains of Klebsiella pneumoniae and seven strains of Pseudomonas aeruginosa, including clinical isolates, were exposed to working concentrations of H2O2 and H2O2-based formulations for 1 min to 24h. Survival was monitored. FINDINGS The levels of susceptibility of planktonic cultures to unformulated and formulated H2O2 were similar in all organisms and strains tested, with minimum inhibitory concentrations ranging from 0.5 to 20mM H2O2. However, biofilms showed up to 266-fold less sensitivity to H2O2 and its formulations. The level of reduced susceptibility correlated with the strains propensity to form biofilm, and differed between species. The two formulations with additional acidic active ingredients performed better at short exposure times, whereas ethanol-containing products required longer exposure times to be effective. CONCLUSION Biofilms of a significant number of clinical isolates of multi-drug-resistant nosocomial pathogens are not susceptible to working concentrations of several H2O2-based disinfectants. This may compromise the ability to control these pathogens with such products.

Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity

ABSTRACT Burkholderia pseudomallei is a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created a relA spoT double mutant in B. pseudomallei strain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotype in vitro and in vivo. The B. pseudomallei ΔrelA ΔspoT mutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in the Galleria mellonella insect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ΔrelA ΔspoT mutant resulted in partial protection against infection with wild-type B. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation in B. pseudomallei, and the ΔrelA ΔspoT mutant may be a promising live-attenuated vaccine candidate.

Evaluation of antibiotic efficacy against infections caused by planktonic or biofilm cultures of Pseudomonas aeruginosa and Klebsiella pneumoniae in Galleria mellonella

The lack of novel antibiotics for more than a decade has placed increased pressure on existing therapies to combat the emergence of multidrug-resistant (MDR) bacterial pathogens. This study evaluated the Galleria mellonella insect model in determining the efficacy of available antibiotics against planktonic and biofilm infections of MDR Pseudomonas aeruginosa and Klebsiella pneumoniae strains in comparison with in vitro minimum inhibitory concentration (MIC) determination. In general, in vitro analysis agreed with the G. mellonella studies, and susceptibility in Galleria identified different drug resistance mechanisms. However, the carbapenems tested appeared to perform better in vivo than in vitro, with meropenem and imipenem able to clear K. pneumoniae and P. aeruginosa infections with strains that had bla(NDM-1) and bla(VIM) carbapenemases. This study also established an implant model in G. mellonella to allow testing of antibiotic efficacy against biofilm-derived infections. A reduction in antibiotic efficacy of amikacin against K. pneumoniae and P. aeruginosa biofilms was observed compared with a planktonic infection. Ciprofloxacin was found to be less effective at clearing a P. aeruginosa biofilm infection compared with a planktonic infection, but no statistical difference was seen between K. pneumoniae biofilm and planktonic infections treated with this antibiotic (P>0.05). This study provides important information regarding the suitability of Galleria as a model for antibiotic efficacy testing both against planktonic and biofilm-derived MDR infections.

An oxytocin-dependent social interaction between larvae and adult C. elegans.

Oxytocin has a conserved role in regulating animal social behaviour including parental-offspring interactions. Recently an oxytocin-like neuropeptide, nematocin, and its cognate receptors have been identified in the nematode Caenorhabditis elegans. We provide evidence for a pheromone signal produced by C. elegans larvae that modifies the behaviour of adult animals in an oxytocin-dependent manner increasing their probability of leaving a food patch which the larvae are populating. This increase is positively correlated to the size of the larval population but cannot be explained by food depletion nor is it modulated by biogenic amines, which suggest it is not an aversive behaviour. Moreover, the food-leaving behaviour is conspecific and pheromone dependent: C. elegans adults respond more strongly to C. elegans larvae compared to other nematode species and this effect is absent in C. elegans daf-22 larvae which are pheromone deficient. Neurotransmitter receptors previously implicated in C. elegans foraging decisions NPR-1 and TYRA-3, for NPY-like neuropeptides and tyramine respectively, do not appear to be involved in oxytocin-dependent adult food-leaving. We conclude oxytocin signals within a novel neural circuit that regulates parental-offspring social behaviour in C. elegans and that this provides evidence for evolutionary conservation of molecular components of a parental decision making behaviour.