In this report, we used a cell-free system with reagent H2O2 and HOCl to examine the intrinsic resistance of prevalent CF and Non-CF respiratory pathogens to the oxidants. We found that the in vitro HOCl-resistance profile (PsA > SA > BC > EC > KP) best fits the infection profile observed clinically in CF lungs; that is, the most HOCl-resistant bacteria such as PsA and SA are the most frequent pathogens in CF patients. This finding implies that differential HOCl resistance across microbial species may allow for persistence of some infections over others by subversion of the host innate immunity and supports our previous finding that CF neutrophils with a compromised HOCl production may not be able to clear the most resistant organisms effectively [12, 13]. From a microbiological point of view, PsA and SA, the relatively more resistant strains to HOCl, would be more likely to survive and be selected for, if the host neutrophils were deficient in their ability to make HOCl. Burns and coworkers did a longitudinal study on young children with CF and found that 97% of the children are colonized with PsA . The early isolates tend to be nonmucoid and antibiotic-sensitive. However, if the initial infection is not effectively eradicated by the host defense, which could happen, for example, if HOCl or other oxidant production was suboptimal, then the bacteria which escape the initial host defenses will grow and spread within the lung, establishing a long-term chronic colonization. Subsequently, environmental pressure in the lung such as antibiotic application selects for the mucoid PsA phenotype. Increased PsA density in the lower respiratory tract and development of antibiotic-resistant mucoid biofilms causes chronic airway inflammation and deteriorating lung function [19–22]. SA has long been recognized to be among the first organisms to colonize the airways of CF patients . Colonization with SA occurs within the first few months of life, and persistent variants of this organism may arise due to a selective pressure from long-term antibiotic treatment in CF patients . However, SA infection does not usually persist or progress to chronic disease. We would like to point out that our current study only tested bacteria in log-phase growth. Such an experimental design was intended to study the nonmucoid form which is assumed by the bacteria during the early CF infections. It is important to recognize that only after initial bacterial colonization is established, can chronic persistent infections ensue in CF lungs.
Neutrophils are highly specialized for bacterial killing especially in the case of extracellular infections. The cells employ at least two microbicidal mechanisms to execute this function: one is oxidant-mediated and another is non-oxidant-mediated. Pseudomonas bacteria possess tough polysaccharide capsules, which are resistant to nonoxidant killing mechanisms, such as protease and hydrolase digestion . This feature determines the requirement of oxidant-killing mechanisms for complete eradication. The importance of neutrophils in defending Pseudomonas infection is reflected by significant increase in infection rate in neutropenic patients . Winterbourn and colleagues modeled the reactions of oxidant production in neutrophil phagosomes. They calculated that superoxide is produced at a rate of ~312 mM/min and HOCl 134 mM per minute . In this current study, the maximal concentration of H2O2 used was 5 mM and HOCl 0.07 mM. A recent report documented that bleaching of GFP expressed in SA is seen at concentrations of 0.05-0.1 mM HOCl which correlated well with killing of SA by this oxidant , suggesting that similar concentrations of HOCl were likely achieved in vivo. The mathematical model proposed by Winterbourn and colleagues predicts that such levels can be reached within seconds after activation of the NADPH oxidase . Thus, we believe that the selected concentrations of H2O2 and HOCl in our studies are well within the scope of the achievable oxidant levels in neutrophils.
Precise mechanisms of oxidant-mediated bacterial killing are not fully defined. Early studies using EC as a model organism indicated a correlation between EC envelope permeabilization and bacterial inactivation by HOCl; however, only low-molecular weight compounds became freely permeable while the cell maintained its barrier function to proteins . Albrich et al. (1986) tested the small-molecule permeability theory in EC by measuring the transport of H+ ion and glycerol and reported that the intercellular movements of these molecules were only marginally affected . Their conclusion was that HOCI inactivation of bacteria does not occur by loss of membrane structural integrity, which contradicts the previous report. In the current study, we demonstrated that membrane integrity is affected by H2O2 and HOCl, but the effect is organism-specific (Figures 2 and 3). Statistically, permeability of BC and EC caused by H2O2 and HOCl did correlate with loss of viability while permeability of KP with only H2O2 exposure correlated with loss of viability. It is notable that permeability and CFU viability were statistically independent of each other for PsA and SA, the two most prevalent CF pathogens, in both H2O2 and HOCl exposures.
EC and PsA have been shown to recover from reduced adenylate energy charge, when subsequently supplied with nutrients which facilitate ATP hydrolase activity of the F1F0 complex of the bacterial ATP synthase . After treatment with bactericidal doses of HOCl, however, adenylate energy charge is unrecoverable and further ATP production is abolished . These findings suggest that a potent oxidant-induced killing mechanism may cause destruction of ATP production by specific oxidation of the F1F0 ATP synthase . We measured the ATP concentration in bacterial suspensions after exposure to H2O2 and HOCl exposure and compared the degree of ATP reduction to the degree of loss of CFU viability at various oxidant concentrations (Figure 4). Of the organisms tested, all except PsA demonstrated significant decline in ATP production which correlated with loss of CFU viability; ATP production in PsA declined significantly up to 5 mM but did not correlated with decline in CFU viability. These data present evidence that H2O2 affects ATP production in bacteria suggesting that there are H2O2-sensitive sites in the bacterial ATP production machinery or that H2O2 assault disrupts pathways of energy production.
The profile of abolished ATP production with HOCl treatment was different from that of H2O2 in that HOCl-induced loss of ATP production correlated significantly with the loss of CFU viability in PsA, BC, and EC, while these two parameters were statistically independent in SA and KP (Figure 5). Interestingly, ATP production in KP was unaffected by HOCl concentrations up to 0.1 mM, a dose exceeding that required for complete eradication of the entire samples at the cellular densities used herein. Given the results obtained in SA and KP, it can be inferred that loss of CFU viability is not completely dependent on disruption of ATP production. In light of these results, further studies are required to elucidate the specific mechanisms of oxidant-induced bactericidal activity against different bacterial species.