Neutrophil Response to Nontypable Haemophilus influenzae in Respiratory Infections

Sputa from patients with respiratory infections by nontypable Haemophilus influenzae (H. influenzae) were investigated by electron microscopy. The cell wall of H. influenzae appeared wavy and nonwavy. In the cell wall the peptidoglycan layer was ill-defined. These patients had adequate IgG response in the serum against H. influenzae. However neither capsule nor fimbriae were found. Different stages of phagocytosis and destruction of the bacteria by polymorphonuclear neutrophils (PMN) were observed. PMNs were also found to phagocytose the debris. Evidences were found that the debris is formed mainly by the destruction of polymorphonuclear neutrophil. Extracellular lysosomes were also observed, which may have a role in destruction of both bacteria and host tissue. It was concluded that nontypable H. influenzae are nonfimbriated and noncapsulated during infection. Debris are the end product of PMN destruction, and phagocytosis of debris by PMNs has a role in the pathogenesis of chronic respiratory diseases.     

Expression of Fimbriae and Host Response in Branhamella catarrhalis Respiratory Infections

Sputum during the acute exacerbation of chronic respiratory diseases were observed under the electron microscope, to determine the in vivo expression of surface structures of Branhamella catarrhalis (B. catarrhalis), the polymorphonuclear neutrophil (PMN) response to B. catarrhalis infections, and the composition of sputum. It was found that during infection fimbriae are expressed in B. catarrhalis. However, there were sparsely to densely fimbriated bacteria in each sputum sample. The length of the fimbriae were from 50 to 76 nm. In the sparsely fimbriated B. catarrhalis, external to the cell wall, a thin, granular, electron-dense layer was observed. Due to the presence of fimbriae, this layer was not seen in densely fimbriated B. catarrhalis. Blebs were also found in B. catarrhalis. PMNs were found to phagocytose both B. catarrhalis and debris. Evidence was found that debris were formed mainly by the destruction of PMNs. Bacteria as well as debris were phagocytosed by PMNs.     

Airway Interleukin-8 in Elderly Patients with Bacterial Lower Respiratory Tract Infections

We investigated the interleukin-8 (IL-8) levels and neutrophil numbers in the sputum of 9 elderly patients with lower respiratory tract infections, including Pseudomonas aeruginosa infection, before and after treatment with various antimicrobial agents. The IL-8 levels in sputum supernatants and the neutrophil numbers in sputum smears from 9 patients decreased significantly after the elimination of the causative respiratory pathogens. We also demonstrated that human recombinant IL-8 at a range of 6.25-25 ng/ml significantly enhanced opsonophagocytic killing of P. aeruginosa immunotype-1 strain by human neutrophils in the presence of a serotype-specific anti-lipopolysaccharide monoclonal antibody and fresh normal human serum. These data suggest that the level of IL-8 production in the airways of patients with lower respiratory tract infections is dependent on bacterial densities, and indicate the important role of IL-8 not only in neutrophil migration but also in opsonophagocytic killing of bacteria in the lower respiratory tract.     

Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.     

Capsule Influences the Deposition of Critical Complement C3 Levels Required for the Killing of Burkholderia pseudomallei via NADPH-Oxidase Induction by Human Neutrophils

Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD₅₀ via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo.     

Role of Mutation in Pseudomonas aeruginosa Biofilm Development

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.     

Quorum‐sensing regulator RhlR but not its autoinducer RhlI enables Pseudomonas to evade opsonization

When Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum‐sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI‐independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester‐containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4‐mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild‐type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense.     

Virulence of Pseudomonas aeruginosa Cells Surviving Photodynamic Treatment with Toluidine Blue

We report results of a study carried out to investigate photodynamic effect of Touildine blue (TBO) on virulent factors secreted by Pseudomonas aeruginosa. Viability of MCF-7 cells incubated with culture supernatants of bacteria photosensitized with TBO (experimental) was found to be higher than that for MCF-7 cells incubated with culture supernatants of bacterial cells treated either with TBO in dark (control II) or not receiving any treatment (neither TBO nor light (control I)). Furthermore, whereas MCF-7 cells incubated with supernatants of bacteria, control I and II, showed significant morphological alterations, no such changes were observed in MCF-7 cells incubated with supernatant of experimental cells. The activities of phospholipase C and proteases, virulent factors of P. aeruginosa, were also found to decrease on photosensitization. These results suggest that the potency of virulent factors is reduced in cells surviving photodynamic treatment with TBO and this may have implication in treatment of infections caused by P. aeruginosa.     

Phagocytosis and nodule formation by hemocytes of Manduca sexta larvae following injection of Pseudomonas aeruginosa

The time course of clearance of an injected dose of 10⁶ CFU ml^?1 hemolymph of Pseudomonas aeruginosa ATCC 9027 in larvae of the tobacco hornworm, Manduca sexta, has been examined in detail. The clearance process has been subdivided into three stages during which the rates of reduction in concentration of circulating viable bacteria were clearly different. Contributions of hemocyte reactions to bacterial clearance were examined during stages I and II. During stage I (0–2 hr postinoculation (PI), nodule formation produced a dramatic reduction in circulating bacteria by entrapping over 90% of the injected dose in the first 30 min. Phagocytosis of bacteria by circulating hemocytes and subsequent intracellular digestion contributed significantly to reductions in circulating bacteria during stage II (2–8 hr PI). Viable cells of the virulent P. aeruginosa P11-1 were trapped in nodules as efficiently as the less virulent 9027 during the first 30 min after injection into M. sexta. Bacteria of strain P11-1 were also phagocytosed by hemocytes during stage II, however, phagocytosed bacteria were observed less frequently in P11-1-treated insects and intracellular digestion of these bacteria was only rarely observed. The increased virulence of P11-1 in larvae of M. sexta may be due to less efficient phagocytosis by circulating hemocytes and to insensitivity of this strain to killing reactions in nodules and following phagocytosis.     

Antibacterial efficacy of lytic <Emphasis Type="Italic">Pseudomonas</Emphasis> bacteriophage in normal and neutropenic mice models

Recently, lytic bacteriophages (phages) have been focused on treating bacterial infectious diseases. We investigated the protective efficacy of a novel Pseudomonas aeruginosa phage, PA1Ø, in normal and neutropenic mice. A lethal dose of P. aeruginosa PAO1 was administered via the intraperitoneal route and a single dose of PA1Ø with different multiplicities of infection (MOI) was treated into infected mice. Immunocompetent mice infected with P. aeruginosa PAO1 were successfully protected by PA1Ø of 1 MOI, 10 MOI or 100 MOI with 80% to 100% survival rate. No viable bacteria were found in organ samples after 48 h of the phage treatment. Phage clearing patterns were different in the presence or absence of host bacteria but PA1Ø disappeared from all organs after 72 h except spleen in the presence of host bacteria. On the contrary, PA1Ø treatment could not protect neutropenic mice infected with P. aeruginosa PAO1 even though could extend their lives for a short time. In in vitro phage-neutrophil bactericidal test, a stronger bactericidal effect was observed in phage-neutrophil co-treatment than in phage single treatment without neutrophils, suggesting phage-neutrophil co-work is essential for the efficient killing of bacteria in the mouse model. In conclusion, PA1Ø can be possibly utilized in future phage therapy endeavors since it exhibited strong protective effects against virulent P. aeruginosa infection.     

Adherence of clinical isolates ofPseudomonas aeruginosa to hamster trachael epithelium in vitro

The adherence to hamster tracheal epithelium, of mucoid and nonmucoid clinical isolates ofPseudomonas aeruginosa from cystic fibrosis patients, was studied using tracheal organ cultures. Tracheal cultures were infected with 10⁷ colony-forming units per ml of either mucoid or nonmucoid clinical isolates ofP aeruginosa. The tracheal explants were rinsed at various time intervals to remove nonadherent bacteria, fixed, and prepared for transmission-and scanning-electron microscopy. Mucoid isolates were seen adhering to the ciliated epithelium as early as 4 h after initiation of infection, whereas nonmucoid isolates were only observed adhering at 6 to 8 h after infection. Mucoid organisms were found as clusters of bacteria embedded in an extensive extracellular matrix. The nonmucoid isolates were generally found as single organisms with no evidence of an extracellular matrix. These results suggest that the prevalence of mucoid isolates ofP. aeruginosa in cystic fibrosis may be due to adherent properties of the mucoid organism.     

Flagellin Treatment Prevents Increased Susceptibility to Systemic Bacterial Infection after Injury by Inhibiting Anti-Inflammatory IL-10+ IL-12- Neutrophil Polarization

Severe trauma renders patients susceptible to infection. In sepsis, defective bacterial clearance has been linked to specific deviations in the innate immune response. We hypothesized that innate immune modulations observed during sepsis also contribute to increased bacterial susceptibility after severe trauma. A well-established murine model of burn injury, used to replicate infection following trauma, showed that wound inoculation with P. aeruginosa quickly spreads systemically. The systemic IL-10/IL-12 axis was skewed after burn injury with infection as indicated by a significant elevation in serum IL-10 and polarization of neutrophils into an anti-inflammatory (“N2”; IL-10⁺ IL-12⁻) phenotype. Infection with an attenuated P. aeruginosa strain (ΔCyaB) was cleared better than the wildtype strain and was associated with an increased pro-inflammatory neutrophil (“N1”; IL-10⁻IL-12⁺) response in burn mice. This suggests that neutrophil polarization influences bacterial clearance after burn injury. Administration of a TLR5 agonist, flagellin, after burn injury restored the neutrophil response towards a N1 phenotype resulting in an increased clearance of wildtype P. aeruginosa after wound inoculation. This study details specific alterations in innate cell populations after burn injury that contribute to increased susceptibility to bacterial infection. In addition, for the first time, it identifies neutrophil polarization as a therapeutic target for the reversal of bacterial susceptibility after injury.     

Compromised host defense on Pseudomonas aeruginosa biofilms: characterization of neutrophil and biofilm interactions

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H(2)O(2) accumulation was transient, reaching a maximal measured value of 1 micro M. Following contact, stimulated degranulation was 20-40% (myeloperoxidase, beta-glucuronidase) and 40-80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H(2)O(2). Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.     

A tracheal culture model of respiratory tract infection withPseudomonas Aeruginosa

Summary The pathogenesis ofPseudomonas aeruginosa for the respiratory tract has been examined using hamster tracheal organ cultures. Tracheal rings prepared from male Syrian hamsters, strain LSH/LAK, were infected withP. aeruginosa for 4 h and processed at 4-h intervals for 24 h for examination by light- and electron microscopy. Tissue destruction was observed within 8 h after infection with 10⁸ colony-forming units (cfu)/ml and within 12 h after infection with 10⁴ or 10⁶ cfu/ml. Ciliated cells that contained abnormal subcellular organelles were expelled from the epithelium. By 20 h the epithelial borders were composed primarily of nonciliated cells. Transmission- and scanning electron microscopy revealed details of the cellular destruction and attachment ofP. aeruginosa to the ciliated epithelium.Pseudomonas aeruginosa causes a rapid destruction of the epithelium of hamster trachea in cultures. Hamster tracheal organ cultures have been shown to be useful in studying the pathogenesis ofP. aeruginosa for the respiratory tract. This work was supported by Grants G-430B and G-431B from the Cystic Fibrosis Foundation.     

Bacteria can form interconnected microcolonies when a self-excreted product reduces their surface motility: evidence from individual-based model simulations

Recent experimental observations of Pseudomonas aeruginosa, a model bacterium in biofilm research, reveal that, under specific growth conditions, bacterial cells form patterns of interconnected microcolonies. In the present work, we use an individual-based model to assess the involvement of bacteria motility and self-produced extracellular substance in the formation of these patterns. In our simulations, the pattern of interconnected microcolonies appears only when bacteria motility is reduced by excreted extracellular macromolecules. Immotile bacteria form isolated microcolonies and constantly motile bacteria form flat biofilms. Based on experimental data and computer simulations, we suggest a mechanism that could be responsible for these interconnected microcolonies.     

Phagocytosis by carp granulocytes; in vivo and in vitro observations

The phagocytic ability of carp (Cyprinus carpio L.) granulocytes was evaluated in vivo and in vitro. In suspensions of head kidney cells, neutrophil granulocytes incorporated both latex beads and coccidian merozoites. In intestinal tissues from carp with a Goussia carpelli infection, all granulocyte cell types (neutrophils and cells of the basophilic-eosinophilic complex) phagocytosed cell detritus and coccidian developmental stages, mainly merozoites.     

A New Hypothalamic Polypeptide Is a Regulator of Myelopoiesis

The effect of hypothalamic proline-rich polypeptide (PRP) against Pseudomonas aeruginosa infection in BALB/c mice with leukopenia was investigated. Mice were treated with cyclophosphamide (CPA) and were then injected with PRP 24 h after CPA treatment. The lethal doses of P. aeruginosa were injected to mice when the number of peripheral blood leukocytes reached a nadir on day 5 after CPA administration. The administration of PRP significantly increased the survival of infected mice, and had a pronounced protective effect during the period of development of the infection. The number of bacteria in internal organs of PRP-treated mice was significantly lower than that in control mice. In PRP-treated mice, the neutrophil levels in peripheral blood started to increase 7 days after CPA administration and were consistently higher, and they were more mature than those in controls. Our results may indicate the ability of PRP to stimulate recovery of myelopoiesis and enhance mature neutrophil function.     

Immunological and biological relationship among flagellin of <Emphasis Type="Italic">Pseudomonas aeruginosa,Burkholderia cepacia</Emphasis>, and <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.     

Does Streptococcus mitis,a neonatal oropharyngeal bacterium,influence the pathogenicity of Pseudomonas aeruginosa?

Microorganisms in a biofilm might promote or suppress each other. We previously found that Pseudomonas aeruginosa (P. aeruginosa) and the normal colonized bacteria in the oropharynx, Streptococcus mitis (S. mitis), were the most common bacteria in the biofilm found on newborns' endotracheal tubes. Here, we found that S. mitis enhanced the adhesion and biofilm formation of P. aeruginosa. Furthermore, it alleviated the immune response induced by P. aeruginosa. These findings remind us that we should not ignore the role of traditionally viewed non-pathogenic bacteria in biofilms and provide new insights into exploring bacterial interaction mechanisms in biofilm related infections.