Reactive oxygen species-mediated immunity against bacterial infection in the gut of cat fleas (Ctenocephalides felis).

Fleas (Order Siphonaptera) transmit numerous bacterial pathogens that cause severe human diseases (e.g., cat scratch disease, flea-borne spotted fever, murine typhus, plague). Because initial entry of these infectious agents occurs while blood feeding, the immune response in the flea gut is considered to be the first line of defense against invading microbes. However, relatively few studies have identified the flea immune molecules that effectively resist or limit infection in the gut. In other hematophagous insects, an immediate immune response to imbibed pathogens is the generation of reactive oxygen species (ROS). In this study, we utilized cat fleas (Ctenocephalides felis) to investigate whether oral infection with a well-known insect bacterial pathogen (Serratia marcescens) induces ROS synthesis in the flea gut, and whether production of ROS provides a defense mechanism against microbial colonization. Specifically, we treated fleas with an antioxidant to limit the number of free radicals in the digestive tract prior to infection, and then measured the following: S. marcescens infection loads, hydrogen peroxide (ROS) levels, and mRNA abundance of ROS signaling pathway genes. Overall, our data shows that ROS levels increase in response to infection in the flea gut, and that this increase helps to strengthen the flea immune response through the microbicidal activity of ROS.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems.

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

The maternal transfer of bacteria can mediate trans-generational immune priming in insects.

Parents invest in their offspring by preparing them for defense against pathogens and parasites that only the parents have encountered, a phenomenon known as trans-generational immune priming. We investigated the underlying mechanism using the established lepidopteran model host Galleria mellonella. When larvae were fed with non-pathogenic bacteria, or the entomopathogenic species Pseudomonas entomophila and Serratia entomophila, the activity of lysozyme and phenoloxidase increased in the hemolymph, and immunity-related genes encoding antibacterial proteins such as gloverin were induced. Remarkably, the ingestion of bacteria by female larvae resulted in the differential expression of immunity-related genes in the eggs subsequently laid by the same females, providing evidence for trans-generational immune priming in G. mellonella. To determine the fate of these ingested microbes, the larval diet was supplemented with bacteria carrying a fluorescent label. We observed these bacteria crossing the midgut epithelium, their entrapment within nodules in the hemocoel, their accumulation within the ovary, and ultimately their deposition in the eggs. Therefore, we propose that trans-generational immune priming in Lepidoptera can be mediated by the maternal transfer of bacteria or bacterial fragments to the developing eggs.

Parasitic suppression of feeding in the tobacco hornworm, Manduca sexta: parallels with feeding depression after an immune challenge.

The parasitic wasp, Cotesia congregata, suppresses feeding in its host Manduca sexta. Feeding suppression in the host coincides with the emergence of the wasps through the host's cuticle. During wasp emergence, host hemocyte number declined, suggesting that the host mounts a wound/immune response against the exiting parasitoids and/or resulting tissue damage. Eliciting a different type of immune response by injecting heat-killed Serratia marcescens also resulted in a decline in feeding and a reduction in hemocyte number. Both the emerging wasps and the bacteria induced an increase in hemolymph octopamine concentration and a decrease in foregut peristalsis in M. sexta. The emerging parasitoids produced the largest changes. The source of the additional octopamine appeared to be the host in both cases. S. marcescens was found to contain no detectable amounts of octopamine. The parasitoids had insufficient octopamine to account for the amount found in host hemolymph and they did not secrete octopamine in vitro. One cause for the high concentration of octopamine in parasitized M. sexta was that octopamine was removed from the hemolymph approximately 23 times more slowly after the wasps emerged than prior to wasp emergence. The striking similarity between the effects of parasitoids and bacteria on M. sexta feeding, hemocyte number, hemolymph octopamine concentration, and foregut peristalsis supports the possibility that the immune/wound reaction induced by the emerging wasps could play a role in the suppression of host feeding. These results also support the hypothesis that M. sexta exhibit an immune-activated anorexia.

Antigenic crossreactivity and lipopolysaccharide neutralization properties of bovine immunoglobulin G.

We investigated a possible mechanism by which immunization against core and lipid A determinants of lipopolysaccharide reduced clinical cases of mastitis and symptoms commonly associated with heterologous Gram-negative IMI. The IgG fraction of sera from cows immunized with either Escherichia coli J5 bacterin, E. coli J5 lipopolysaccharide conjugate vaccine, or unimmunized controls was purified by precipitation with caprylic acid and ammonium sulfate. The degree of IgG crossreactivity with Gram-negative bacteria that were isolated from clinical quarters was greater than that with Gram-positive isolates of Staphylococcus aureus. The highest magnitude of crossreactivity was against smooth strain E. coli isolates, followed by heterologous species of Enterobacter, Serratia, and Klebsiella isolates. Serum IgG from cows immunized with conjugate was highly crossreactive to E. coli J5, E. coli O111:B4, Serratia marcescens, Klebsiella pneumoniae, and Salmonella typhimurium lipopolysaccharides. The magnitude of antibody crossreactivity with lipopolysaccharides coincided with the ability of IgG to suppress the mitogenic effect of lipopolysaccharides on bovine lymphocytes.

Structural and serological characterisation of an O-specific polysaccharide from Serratia plymuthica.

The surface polysaccharides of a strain of Serratia plymuthica were characterised and shown to consist of a linear, acidic galactoglucomannan as well as a major and a minor neutral galactan. Immunoblotting results demonstrated cross-reactions between this strain and others with similar galactans (S. marcescens O16 and O20, Klebsiella O1, and Pasteurella haemolytica T4 and T10).

Human natural anti-Gal IgG regulates alternative complement pathway activation on bacterial surfaces.

One percent of circulating IgG in humans recognizes galactose alpha 1,3 galactose residues (anti-Gal) and is synthesized in response to stimulation by enteric bacteria. In this study, we found that the prevalence of binding of anti-Gal to blood isolates is significantly higher than its binding to normal stool isolates. When anti-Gal bound onto the lipopolysaccharide of a representative blood isolate, Serratia marcescens #21, it blocked its alternative complement pathway (ACP) lysis and made the organism serum resistant. In contrast, when anti-Gal bound to the capsular polysaccharide of a serum sensitive Serratia, #7, it increased ACP killing of this strain. The mechanism of blockade of ACP lysis by anti-Gal did not involve a decrease in the number of C3 molecules deposited onto Serratia #21 or an inhibition of the binding of C3b to its LPS, nor did it change the iC3b and C3d degradation products of bound C3b or prevent membrane attack complex formation on this organism. Our findings suggest that the effect of anti-Gal on immune lysis is dependent on the bacterial outer membrane structure to which it binds. We postulate that anti-Gal may play a role in the survival of selected Enterobacteriacae in Gram-negative sepsis by blocking ACP-mediated lysis of such bacteria by the nonimmune host, and that this effect depends on where anti-Gal finds its epitope on the bacterial outer membrane.

Comparative biotyping, bacteriocin typing, and serogrouping (O-antigens) of Serratia liquefaciens.

A total of 83 clinical isolates of Serratia liquefaciens from 81 patients were biotyped, bacteriocin typed (with group A phage tail bacteriocins from S. marcescens), and serogrouped. Biotyping afforded least discrimination, because 71 of the 83 isolates (85.5%) comprised 2 biotypes; the remainder were of 5 different biotypes. Bacteriocin typing differentiated 70 of the 83 isolates (84.3%) into 20 types. Polyclonal rabbit anti-O immune sera identified 16 O-antigens, and all 83 isolates could be serogrouped.

Serotyping of Serratia liquefaciens: H-antigens.

Polyclonal anti-H rabbit immune sera differentiated 12 flagellar (H) antigens among 79 motile of 83 clinical isolates of Serratia liquefaciens from 81 patients. Seven of the anti-H S. liquefaciens sera cross-reacted with H-antigens of S. marcescens, specifically anti-S. liquefaciens H6, H7, H8, H9, H10, H11, and H12 with S. marcescens H-antigens H7, H8, H12, H14, H5, H3, and H17, respectively. These cross-reactions were reciprocal.

The comparative in-vitro activity of norfloxacin, ciprofloxacin, enoxacin and nalidixic acid against 423 strains of gram-negative rods and staphylococci isolated from infected hospitalised patients.

The in-vitro activities of four quinolone carboxylic acids against 423 clinical isolates of Gram-negative rods and staphylococci from infected hospitalised patients were compared. The antibiotics included nalidixic acid and the newer compounds, norfloxacin (MK-0366), ciprofloxacin (Bay 09867) and enoxacin (AT 2266 or CI919). Norfloxacin showed slightly more activity than enoxacin, but both agents had markedly greater potencies and broader antibacterial spectrums than nalidixic acid. Ciprofloxacin was the most active quinolone tested against both gentamicin-susceptible and gentamicin-resistant stains, having an MIC90 equal or less than 1 mg/l for all species studied.

Induction of germ-line anti-alpha 1-3 dextran antibody responses in mice by members of the Enterobacteriaceae family.

A large panel of enteric organisms was screened for agglutination with a panel of lambda monoclonal antibodies of different heavy chain isotypes specific for alpha 1-3 dextran (DEX). Two strains were initially isolated that were bound by most of the anti-DEX antibodies. One organism, Enterobacter cloacae strain MK7, which was characterized in detail, induced a typical lambda anti-DEX response in Igh-Ca mice that had a fine idiotope profile comparable with that induced by purified B1355S dextran containing alpha 1-3 glucosidic linkages (alpha 1-3-DEX). The determinant on the bacterial surface was shown by binding inhibition with nigerotriose to contain alpha 1-3 linkages. Hyperimmunization with these organisms of normal, athymic (nu/nu), or germ-free mice induced large amounts of IgM antibodies but very little IgG. This is the first description of an organism isolated from the normal gut flora of mice that can be shown directly to be bound by alpha 1-3-DEX antibodies and to induce the typical germ-line response of the DEX family of antibodies.

Multiple fimbrial haemagglutinins in Serratia species.

Fifty-six strains from nine Serratia species, grown under a variety of cultural conditions, were examined in rocked-tile tests for the presence of haemagglutinins (HAs) and with the electron microscope for fimbriae. All strains were haemagglutinating; most (71%) formed two or three of the different kinds of HA detected. These were: (i) a mannose-sensitive HA (MS-HA); (ii) a mannose-resistant, klebsiella-like HA (MR/K-HA); (iii) two mannose-resistant, proteus-like HAs (MR/P-HA), one of which showed broad-spectrum HA activity against different species of erythrocytes and the other narrow-spectrum HA activity. Five types of fimbriae associated with the different HAs were observed. This description of the fimbrial HAs in nine species of Serratia is the most comprehensive so far produced and should provide a basis for future studies directed towards elucidating the ecological role of the adhesins in in-vivo colonization by serratiae as commensals or pathogens.

New fimbrial hemagglutinin in Serratia species.

Strains of Serratia marcescens, Serratia liquefaciens, Serratia marinorubra, and Serratia plymuthica produced one or more of the following hemagglutinins (HAs): mannose-sensitive HA and mannose-resistant K-HA (MR/K-HA) and P-HA (MR/P-HA) (J. P. Duguid and D. C. Old, in E. H. Beachey (ed.), Bacterial adherence, vol. 6., p. 185-217, 1980). Most strains (82%) were multiply hemagglutinating. The properties of the three HAs are described. Each HA was associated with a distinct type of fimbria: mannose-sensitive HA with type 1 fimbriae. MR/K-HA with type 3 fimbriae, and MR/P-HA with a new type of thin fimbriae provisionally called MR/P fimbriae. This is the first report of the production of MR/P-HA and MR/P fimbriae by Serratia species. The range of Serratia HAs, which may reflect in vivo colonization potential, is more complex than previously reported.

A serologic response in human infection with Enterobacteriaceae.

Sera from patients infected with Escherichia coli, Proteus, Klebsiella, and Serratia were studied for precipitins to ultrasonic extracts of these organisms in gel-diffusion plates. Sera from 66 per cent of these patients contained precipitins when initially tested. Twenty-four per cent of sera tested in the first week after onset of infection contained precipitins, but this rose to 78 per cent by the third week. Cross-reactions of sera with Pseudomonas antigens were unusual, but were common with other enterobacterial antigen extracts. However, higher titers were usually present to homologous as compared to heterologus antigens. Sera from seven patients contained precipitins to a common enterobacterial antigen. Precipitins to E. coli, Proteus, Klebsiella, and Serratia were detected in only a small proportion of control sera.

Serologic relationship of fimbriae among Enterobacteriaceae.

The existence of serological relationship of fimbriae among serotypes of the same genus and among genera of Enterobacteriaceae was determined by agglutination and absorption procedures. It was found that fimbriae of different serotypes of the same genus and among the genera a) Escherichia, Shigella and Klebsiella, and b) Salmonella, Arizona and Citrobacter were more or less antigentically related. No relationship was found among fimbriae of the Edwardsiella, Enterobacter, Hafnia, Serratia, Proteus and Providencia. It was also found that in Klebsiella, MS and MR fimbriae differed antigenically. On the basis of the results obtained nine antigentically distinct types of fimbriae were distinguished.

Effect of cyclophosphamide on the immune response to Pseudomonas aeruginosa in mice.

Natural resistance in mice to Pseudomonas aeruginosa was decreased 10-fold with a single dose of 300 mg of cyclophosphamide (CY) per kg intraperitoneally. Mice were resistant to infection when immunized actively with Pseudomonas vaccine or passively with Pseudomonas immune serum before receiving CY. Syngeneic spleen, thymus and/or bone marrow cells were transfused into CY-treated recipient mice. Protective anti-Pseudomonas antibody was elicited in the recipient mice when they were vaccinated 1 day after receiving normal spleen cells and challenged 8 days after vaccination. When 1.6 X 10(7) normal thymus and bone marrow cells were infused before vaccination, 69% of the recipients of both cell preparations responded serologically compared with 15 and 27% of those receiving either thymus or bone marrow cells, respectively. CY-treated thymus or bone marrow cell recipients were resistant to Pseudomonas infection when 6 X 10(7) of either cell population was transfused.

Effects of IgM and IgG antibody in patients with bacteremia due to gram-negative bacilli.

Earlier studies, which indicated that high titers of O-specific antibody to the patient's infecting organism in acute-phase serum specimens were not associated with a decrease in the frequency of subsequent shock and death in bacteremia due to gram-negative bacilli, were reexamined for evaluation of the protective activity of specific IgG and IgM antibody. Titers of hemagglutination antibody and levels of IgM, determined by indirect immunofluorescent staining of the patient's infecting organism, as well as hemagglutination titers after reduction of serum with 2-mercaptoethanol and IgG levels, correlated closely (P less than 0.001). High titers of IgG antibody to the patient's infecting organism in acute-phase specimens were associated with a significant reduction in the frequency of shock and death in bacteremia. In contrast, high titers of IgG antibody were not associated with a diminution in the frequency of shock and death. The previously demonstrated protective activity of antibody to an antigen, Re lipopolysaccharide, shared by most gram-negative bacilli was reconfirmed and shown to be independent of the protective activity of O-specific IgG antibody.