Polymyxin resistance of Pseudomonas aeruginosa phoQ mutants is dependent on additional two-component regulatory systems.

Pseudomonas aeruginosa can develop resistance to polymyxin as a consequence of mutations in the PhoPQ regulatory system, mediated by covalent lipid A modification. Transposon mutagenesis of a polymyxin-resistant phoQ mutant defined 41 novel loci required for resistance, including two regulatory systems, ColRS and CprRS. Deletion of the colRS genes, individually or in tandem, abrogated the polymyxin resistance of a ΔphoQ mutant, as did individual or tandem deletion of cprRS. Individual deletion of colR or colS in a ΔphoQ mutant also suppressed 4-amino-L-arabinose addition to lipid A, consistent with the known role of this modification in polymyxin resistance. Surprisingly, tandem deletion of colRS or cprRS in the ΔphoQ mutant or individual deletion of cprR or cprS failed to suppress 4-amino-L-arabinose addition to lipid A, indicating that this modification alone is not sufficient for PhoPQ-mediated polymyxin resistance in P. aeruginosa. Episomal expression of colRS or cprRS in tandem or of cprR individually complemented the Pm resistance phenotype in the ΔphoQ mutant, while episomal expression of colR, colS, or cprS individually did not. Highly polymyxin-resistant phoQ mutants of P. aeruginosa isolated from polymyxin-treated cystic fibrosis patients harbored mutant alleles of colRS and cprS; when expressed in a ΔphoQ background, these mutant alleles enhanced polymyxin resistance. These results define ColRS and CprRS as two-component systems regulating polymyxin resistance in P. aeruginosa, indicate that addition of 4-amino-L-arabinose to lipid A is not the only PhoPQ-regulated biochemical mechanism required for resistance, and demonstrate that colRS and cprS mutations can contribute to high-level clinical resistance.

PmrB mutations promote polymyxin resistance of Pseudomonas aeruginosa isolated from colistin-treated cystic fibrosis patients.

Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of colistin (polymyxin E) resistance in laboratory strains and clinical isolates of this organism (MICs of 8 to 64 mg/liter). To explore the role of PmrAB in high-level clinical polymyxin resistance, P. aeruginosa isolates from chronically colistin-treated cystic fibrosis patients, most with colistin MICs of >512 mg/liter, were analyzed. These cystic fibrosis isolates contained probable gain-of-function pmrB alleles that conferred polymyxin resistance to strains with a wild-type or pmrAB deletion background. Double mutant pmrB alleles that contained mutations in both the periplasmic and dimerization-phosphotransferase domains markedly augmented polymyxin resistance. Expression of mutant pmrB alleles induced transcription from the promoter of the arnB operon and stimulated addition of 4-amino-l-arabinose to lipid A, consistent with the known role of this lipid A modification in polymyxin resistance. For some highly polymyxin-resistant clinical isolates, repeated passage without antibiotic selection pressure resulted in loss of resistance, suggesting that secondary suppressors occur at a relatively high frequency and account for the instability of this phenotype. These results indicate that pmrB gain-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.

PhoQ mutations promote lipid A modification and polymyxin resistance of Pseudomonas aeruginosa found in colistin-treated cystic fibrosis patients.

Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of polymyxin resistance (MICs of 8 to 64 mg/liter) in laboratory and clinical strains of this organism. To explore the role of PhoPQ in high-level clinical polymyxin resistance, P. aeruginosa strains with colistin MICs > 512 mg/liter that had been isolated from cystic fibrosis patients treated with inhaled colistin (polymyxin E) were analyzed. Probable loss-of-function phoQ alleles found in these cystic fibrosis strains conferred resistance to polymyxin. Partial and complete suppressor mutations in phoP were identified in some cystic fibrosis strains with resistance-conferring phoQ mutations, suggesting that additional loci can be involved in polymyxin resistance in P. aeruginosa. Disruption of chromosomal phoQ in the presence of an intact phoP allele stimulated 4-amino-l-arabinose addition to lipid A and induced transcription from the promoter of the pmrH (arnB) operon, consistent with the known role of this lipid A modification in polymyxin resistance. These results indicate that phoQ loss-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.

Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function.

Effect of omeprazole on oral iron replacement in patients with iron deficiency anemia.

Hypochlorhydric states such as atrophic gastritis and partial gastrectomy have long been known to cause iron deficiency anemia. However, studies to date have failed to show a similar association with omeprazole, a proton pump inhibitor that also produces achlorhydria. These studies, however, have primarily involved nonanemic, iron-replete individuals. The effect of the drug has not been studied in patients with established iron deficiency, and to our knowledge the patients presented here are the first of their kind to be reported. Our observations support the probability that the profound hypochlorhydria induced by omeprazole may indeed impair the optimal absorption of orally administered iron in iron-deficient individuals, precluding them from obtaining therapeutically adequate amounts to establish the positive balance necessary for the resolution of anemia and the replenishment of stores. The possible explanations for this phenomenon are also discussed.

Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL adjuvant.

Three immunodominant leishmanial antigens (TSA, LmSTI1 and LeIF) previously identified in the context of host response to infection in infected donors and BALB/c mice, as well as their ability to elicit at least partial protection against Leishmania major infection in the BALB/c mouse model, were selected for inclusion into a subunit based vaccine. This is based on the premise that an effective vaccine against leishmaniasis (a complex parasitic infection) would require a multivalent cocktail of several antigens containing a broader range of protective epitopes that would cover a wide range of MHC types in a heterogeneous population. For practical considerations of vaccine development, we report on the generation of a single recombinant polyprotein comprising the sequences of all three open reading frames genetically linked in tandem. The resulting molecule, Leish-111f, comprises an open reading frame that codes for a 111kDa polypeptide. Evaluation of the immunogenicity and protective efficacy of Leish-111f formulated with IL-12 revealed that the immune responses to the individual components were maintained and as well, rLeish-111f protected BALB/c mice against L. major infection to a magnitude equal or superior to those seen with any of the individual components of the vaccine construct or SLA, a soluble Leishmania lysate. But because rIL-12 is expensive and difficult to manufacture and its efficacy and safety as an adjuvant for human use is questionable, we screened for other adjuvants that could potentially substitute for IL-12. We report that monophosphoryl lipid A (MPL) plus squalene (MPL-SE) formulated with rLeish-111f elicited protective immunity against L. major infection. The demonstrated feasibility to manufacture a single recombinant vaccine comprising multiple protective open reading frames and the potential use of MPL-SE as a substitute for IL-12, takes us closer to the realization of an affordable and safe Leishmania vaccine.