Age-related variations in the in vitro bactericidal activity of human sera against Pseudomonas aeruginosa

Introduction

Pseudomonas aeruginosa is a Gram-negative bacterium causing an array of infections in animals and humans. In humans, it is considered as a common opportunistic pathogen causing a variety of diseases in general population. It is a part of normal human microflora; however, it becomes more harmful when an individual becomes immu­nocompromised [1]. It is believed to be one of the most frequent pathogens responsible for nosocomial infection in the blood, lungs, urinary tract and wounds. In addition, it may cause community-acquired infections outside hospitals including pneumonia, skin rashes, external ear canal infections, eye and heart valve infections [2]. For the effective treatment of P. aeruginosa infection, an appropriate empirical antimicrobial therapy is important [3, 4]. However, P. aeruginosa poses a great healthcare challenge due to its antibiotic resistance, which is increasing with the passage of time, rendering the commonly used antibiotics ineffective against this bacterium [5–7].
The innate immune system plays a vital role in defense against P. aeruginosa invasions [8]. Among different effectors of the innate immunity, the complement system plays a critical role in the clearance of P. aeruginosa infection [9–14]. Complement, a collective term used for more than thirty serum proteins, is an integral part of the innate immune system. In addition to its role in innate immunity, it also acts as a bridge in regulating different functions of adaptive immunity. The major biological functions of the complement system include enhanced inflammation and phagocytosis along with direct cell lysis by the formation of MAC on the surface of pathogens [15].
The bactericidal effect of serum is an essential innate immune mechanism of the host that provides protection against harmful bacteria. The protective capacity of antibody and complement proteins in the serum is referred to as complement-mediated bactericidal activity or serum bactericidal activity and is determined via an in vitro technique known as serum bactericidal assay (SBA). Several studies have investigated the bactericidal activity of serum against different pathogens [16–20]. It is widely known that the in vitro killing of bacteria by serum is mainly complement-driven [16, 21, 22].
A number of recent studies have established the fact that complement-mediated bactericidal activity helps in the protection of the human body against disease-causing organisms, including P. aeruginosa [8, 12, 13]. In vitro studies, using healthy human sera, reflected the significance of both alternative and classical pathways in the complement-mediated killing of serum-sensitive strains of P. aeruginosa [23–26]. On the other hand, sera of complement-deficient patients demonstrated weak killing activity of P. aeruginosa [27–29], which further supports the fact that serum bactericidal activity is mostly complement-driven. Several murine model studies of P. aeruginosa infection have described the important contributions of the classical and alternative pathways in complement-mediated phagocytosis of P. aeruginosa in lung infections [13, 30, 31]. Similarly, the importance of the lectin pathway has been reported in burn wounds infection of P. aeruginosa a rodent model, describing the promising role of mannose-binding lectin (MBL) in recognizing and eradicating this bacterium [32]. In addition, MBL deficiency has also been linked with the earlier infection of P. aeruginosa and increased rate of death in patients with cystic fibrosis [33]. Although, previous in vitro and in vivo studies have clearly established an association of complement deficiencies with the killing of P. aeruginosa, the association of age with the serum killing of P. aeruginosa has not been described. Age-dependent variations in serum killing of P. aeruginosa have thus been investigated in the present study to describe the association between age and serum bactericidal activity against P. aeruginosa.

Material and methods

Subjects

Subjects of the current study were healthy individuals with no prior history of P. aeruginosa infection. Subjects were categorized into three groups, children, adults and elderly. Twenty subjects from each group were investigated for serum bactericidal activity of P. aeruginosa. Subjects between 0–10 were placed in children, 11–40 were placed in adults and 41 and above were placed in elderly. A detailed description of age groups in presented in Table 1. All the procedures were performed in the Microbiology Laboratory of the Pakistan Institute of Medical Sciences (PIMS), Islamabad.

Bacterial strain

Pseudomonas aeruginosa (ATCC® 51679™) was used in this study. Before using the strain in the assay, bacterial cultures were identified via Gram-staining, oxidase and catalase test as Gram-negative, oxidase and catalase positive.

Stock culture preparation

To prepare a stock culture, one colony of P. aeruginosa from the MacConkey agar plate was inoculated into 10 ml of nutrient broth and incubated overnight at 37°C. Next day, 15% of glycerol was mixed with bacterial growth in log phase and stored at –80°C as 300 µl aliquots for future use.

Preparation of sera

After receiving written consent, blood samples were collected from 60 healthy subjects, 20 each from three selected age groups. 10 ml of blood was drawn by venipuncture and transferred to polypropylene tubes (Becton Dickinson). To prevent the complement activation, tubes were immediately transferred to the ice. Serum was separated after 1–3 hours of incubation on ice by centrifuging the blood for 7 minutes at 7000 rpm. A portion of each sample was heat inactivated at 56^oC for half an hour to inactivate the complement system. Serum was stored in a –80^oC freezer until used in experiments. Ethical approval for the collection of blood was provided by the ethical committee of PIMS, Islamabad.

Serum bactericidal assay

An aliquot of bacteria was centrifuged at 10,000 rpm for 2 minutes and the pellet was washed by resuspending in 300 ml of Hanks’ Balanced Salt Solution (HBSS) (Sigma-Aldrich). After centrifugation, the bacterial pellet was subsequently diluted in HBSS to achieve a bacterial count of 1 × 108 CFU/ml. The SBA for each serum sample was carried out in two Eppendorf tubes, one for normal human serum and the other for heat inactivated serum as a negative control. The total volume of each tube was 1 ml, which contained 10% of serum and 1 × 108 CFU/ml of bacteria suspended in HBSS solution. Both tubes were incubated for two hours at 37^oC in a rotary mixer. During incubation, 20 ml of the sample was separated from each tube at time points 0, 30, 60, 90 and 120 minutes and diluted in saline at different concentrations. Selected time points were chosen to evaluate the progress/response time of sera from selected age groups in killing P. aeruginosa during the experiment. 20 ml of each dilution was spread onto MacConkey agar plates and incubated overnight. Colonies were counted next day to determine the CFU/ml of each sample.

Statistical analysis

Data were analyzed by using GraphPad Prism, Version 6 (GraphPad Software). The significance of differences in means of different groups was determined by using Student’s unpaired t-test. Correlation between age and killing activity was analyzed by the third order polynomial (cubic), drawing an interpolated standard curve. The differences were considered significant when a p value of < 0.05 was obtained.

Results

Bactericidal activity of fresh human sera and heat-inactivated sera

Bactericidal activity of sera, collected from three selected age groups, was investigated in this study. Two types of sera from each individual were examined; the normal human serum and the heat inactivated serum in which the complement was deactivated and used as a negative control. Both the fresh and heat inactivated sera collected from children were completely compromised in their ability to kill P. aeruginosa (Fig. 1A). In adults, fresh sera exhibited a significantly higher level of killing of P. aeruginosa as compared to heat-inactivated sera with p values of < 0.0001, 0.0008 and < 0.0001 at time points 60, 90 and 120 min during incubation (Fig. 1B). Similarly, fresh sera from elderly also showed a significantly higher level of killing as compared to heat-inactivated sera with p values of 0.0003, 0.0067 and < 0.0001 at time points 60, 90 and 120 min, respectively (Fig. 1C).

Comparison of bactericidal activity of normal human sera of different age groups

When a comparison in killing of P. aeruginosa by fresh serum collected from three age groups was made, it was evident that adult sera were highly efficient in the killing of P. aeruginosa with substantially decreased mean CFU/ml at time points 90 and 120 minutes, whereas children sera did not show any decrease (Fig. 2). Bacterial killing of adult sera was significantly higher as compared to children with p values of < 0.0001, 0.002 and < 0.0001 at time points 60, 90 and 120 min, respectively. On the other hand, elderly sera also exhibited a significantly higher level of bactericidal activity as compared to children after 60, 90 and 120 min with p values of < 0.0001, 0.0036 and < 0.0001, respectively. Although, the killing of elderly sera was decreased to a certain degree as compared to adults, statistical analysis did not affirm any significant differences between these two age groups (Fig. 2).

Correlation between age and Pseudomonas aeruginosa killing

Analysis of all 60 volunteers showed a significant correlation between age and bactericidal killing of P. aeruginosa. A scattered graph of all samples revealed that all the sera of children under 10 years did not kill bacteria, and the bacterial count remained similar even after the incubation of 120 min. When the age exceeded ten, sera revealed some killing with the increasing age. Sera from individuals between 18 and 45 were highly efficient in killing P. aeruginosa, a majority of which completely killed bacteria by time point 120. When age exceeded 45 years, the bulk of sera showed a decline in the killing. Only three sera from this group completely killed bacteria, whereas rest exhibited reduced killing. As obvious from scatter graphs that the overall trend in the bacterial killing was an initial increase from children to adults and a subsequent decrease from adults to elderly. Therefore, to determine the correlation between age and P. aeruginosa killing, data were analyzed by the third order polynomial (cubic) after drawing an interpolated standard curve. Results suggest a significant correlation between age and bactericidal killing with coefficient of determination values of 0.34, 0.27 and 0.58 and p values of < 0.0001, < 0.001 and < 0.0001 at time points 60, 90 and 120 min, respectively (Fig. 3).

Discussion

The serum is an unfavorable environment for invading pathogens due to its bactericidal properties mediated by a system of more than 35 proteins collectively known as the complement system. This phenomenon has been widely known since the late 19th century. Bactericidal effects of serum can be demonstrated in vitro against bacteria, especially Gram-negative pathogens [34–36]. Various studies have documented the bactericidal activity of serum against P. aeruginosa to investigate different aspects of host interaction with P. aeruginosa [23, 26, 37, 38]. Here we described the age-related differences in the serum-killing of P. aeruginosa. Subjects of the study were categorized into three age groups; children, adults and elderly.
The initial evaluation of fresh normal human serum with heat-inactivated serum revealed that all the heat inactivated sera from selected age groups were unable to kill P. aeruginosa with no decrease in CFU/ml at all during two-hour incubation. It is a known fact that complement proteins are heat labile, which are deteriorated at high temperatures [39, 40]. On the other hand, fresh sera from adults and elderly individuals showed a significantly higher bactericidal effect against P. aeruginosa. Therefore, the absence of bactericidal activity in heat inactivated sera, in line with a higher level of killing demonstrated by fresh human sera, further supports the evidence that bactericidal activity of serum is complement-driven as reported previously by several other authors [16, 22, 41].
Interestingly, fresh sera from children were fully compromised in their ability to kill P. aeruginosa with no noticeable differences as compared to heat-inactivated children sera. This is in agreement with a previously reported study documenting a less efficient bactericidal activity exhibited by neonatal sera against several Gram-negative bacteria as compared to normal adult sera [42]. The major contributor of serum bactericidal killing in the absence of blood cell types is the formation of MAC (C5b6789) [21, 43]. Children and neonates have a maturational deficiency of several complement factors [44]. However, the most relevant deficiency that may contribute to the lack of bactericidal activity of children is the C9 and C8 deficiency as described previously, with C9 present in the lowest relative concentration as compared to adults [45, 46]. Although, C8 and C9 might be important in forming MAC, the role of recognition molecules cannot be ignored due to the fact that the complement cannot be activated in the absence of these molecules, and other complement factors become irrelevant if the complement is not activated by these molecules. Different studies have reported lower levels of complement recognition molecules in neonates and children, which are below normal adult values. These include C1q [47], MBL [48–50], M-ficolin and L-ficolin [50, 51]. Hence, the diminished serum concentrations of MAC components C8 and C9, in parallel with lower concentrations of the recognition molecules for complement activation may explain the inefficiency of children sera in killing P. aeruginosa. However, there is a need to precisely investigate the role of these molecules in fighting Pseudomonas infection in order to identify the components of the complement system; deficiency of which significantly alters the bactericidal activity in children and neonates against P. aeruginosa.
Although, having no significant differences statistically, elderly sera were partially compromised in bactericidal activity against P. aeruginosa as compared to fresh adult sera. Most of the serum samples from adults completely killed bacteria by 120 min after incubation. In contrast, only three serum samples from individuals > 45 years totally cleared bacteria by the time point 120 min, whereas the rest of sera from this group revealed partial killing. This is a clear indication that with the age exceeding 45 years, bactericidal activity declines (Fig. 3), which may be due to age-related regression of the complement system. With aging, the immune system undergoes several alterations and remodeling at organic and cellular levels, eventually predisposing elderly to an increased risk of several infections [52, 53]. As a result of these age-related changes, mortality rates in elderly patients are three times higher as compared to adults [54]. Although, the majority of studies have largely focused on defects in the immune system arising with aging, like compromised phagocytosis and cytokine responses [53, 55], age-related dysregulations in the complement system have been largely ignored in humans. However, some animal studies conducted in mice are in agreement with our findings. Hazlett et al. reported compromised phagocytosis response in aged mice, arising due to defects in complement response against P. aeruginosa [30]. Similarly, other studies also revealed altered immune responses in aged mice against P. aeruginosa [56, 57]. Since, the compromised function of the complement system affects several other biological functions like opsonophagocytosis and inflammation, defects in the phagocytosis and other cellular responses in aged mice as described previously, indirectly supports the finding of our study.
In conclusion, in vitro findings of our study demonstrate the variations in the bactericidal activity of serum by establishing that children sera show a complete failure, whereas elderly sera display a partial decrease in the killing of P. aeruginosa. This indicates that components of the innate immune system, especially the complement system, do not work with full potential in these two age groups as compared to adult sera. Hence, our findings open a window for future research in investigating these components in relation to P. aeruginosa infections with an emphasis on those proteins, which are essential in providing defense against P. aeruginosa, and are not fully functional in children or partially compromised in elderly. If these innate immune components are successfully identified, they can be reconstituted in children and elderly to compensate the compromised immunity against P. aeruginosa, or even boost the innate immune response in adults. Immune therapy strategies like these may be a substitute for antibiotic therapy, which is becoming ineffective with the passage of time due to increasing resistance of this pathogen against commonly used antibiotics.

Acknowledgments

We thank Dr. Rubeena Khan, Head, Department of Microbiology, Pakistan Institute of Medical Science, Islamabad for helping in sera collection and providing lab facilities. We are also grateful to Dr. Azka and Shafiq Ahmed for their kind support during this study.

The authors declare no conflict of interests.

References

1. Ryan KJ, Ray CG, Sherris JC (2004): Sherris medical microbio­logy: an introduction to infectious diseases. 4th ed. New York: McGraw-Hill.
2. Diekema DJ, Pfaller MA, Jones RN, et al. (1999): Survey of bloodstream infections due to gram-negative bacilli: frequency of occurrence and antimicrobial susceptibility of isolates collected in the United States, Canada, and Latin America for the SENTRY Antimicrobial Surveillance Program, 1997. Clin Infect Dis 29: 595-607.
3. Garnacho-Montero J, Garcia-Garmendia JL, Barrero-Almodovar A, et al. (2003): Impact of adequate empirical antibiotic therapy on the outcome of patients admitted to the intensive care unit with sepsis. Crit Care Med 31: 2742-2751.
4. Ibrahim EH, Sherman G, Ward S, et al. (2000): The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting. Chest 118: 146-155.
5. Owens RC, Lautenbach E (2008): Antimicrobial resistance: problem pathogens and clinical countermeasures, New York: Informa Healthcare.
6. Gellatly SL, Hancock RE (2013): Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 67: 159-173.
7. Chastre J (2008): Evolving problems with resistant pathogens. Clin Microbiol Infect 14 Suppl 3: 3-14.
8. Lavoie EG, Wangdi T, Kazmierczak BI (2011): Innate immune responses to Pseudomonas aeruginosa infection. Microbes Infect 13: 1133-1145.
9. Mishra M, Ressler A, Schlesinger LS, et al. (2015): Identification of OprF as a complement component C3 binding acceptor molecule on the surface of Pseudomonas aeruginosa. Infect Immun 83: 3006-3014.
10. Zhang J, Koh J, Lu J, et al. (2009): Local inflammation induces complement crosstalk which amplifies the antimicrobial response. PLoS Pathog 5: e1000282.
11. Rhein LM, Perkins M, Gerard NP, et al. (2008): FcgammaRIII is protective against Pseudomonas aeruginosa pneumonia. Am J Respir Cell Mol Biol 38: 401-406.
12. Mueller-Ortiz SL, Drouin SM, Wetsel RA (2004): The alternative activation pathway and complement component C3 are critical for a protective immune response against Pseudomonas aeruginosa in a murine model of pneumonia. Infect Immun 72: 2899-2906.
13. Younger JG, Shankar-Sinha S, Mickiewicz M, et al. (2003): Murine complement interactions with Pseudomonas aeruginosa and their consequences during pneumonia. Am J Respir Cell Mol Biol 29: 432-438.
14. Hopken UE, Lu B, Gerard NP, et al. (1996): The C5a chemoattractant receptor mediates mucosal defence to infection. Nature 383: 86-89.
15. Trouw LA, Daha MR (2011): Role of complement in innate immunity and host defense. Immunol Lett 138: 35-37.
16. Desar IM, Van Deuren M, Sprong T, et al. (2009): Serum bactericidal activity against Helicobacter pylori in patients with hypogammaglobulinaemia. Clin Exp Immunol 156: 434-439.
17. Kwil I, Kazmierczak D, Rozalski A (2013): Swarming growth and resistance of Proteus penneri and Proteus vulgaris strains to normal human serum. Adv Clin Exp Med 22: 165-175.
18. Jang MS, Sahastrabuddhe S, Yun CH, et al. (2016): Serum bactericidal assay for the evaluation of typhoid vaccine using a semi-automated colony-counting method. Microb Pathog 97: 19-26.
19. Futoma-Koloch B, Bugla-Ploskonska G (2009): The efficiency of the bactericidal action of serum raised by complement and lysozyme against bacteria which avoid the immunological response of higher organisms. Postepy Hig Med Dosw (Online) 63: 471-484.
20. Goh YS, Maclennan CA (2013): Invasive African nontyphoidal Salmonella requires high levels of complement for cell-free antibody-dependent killing. J Immunol Methods 387: 121-129.
21. O’shaughnessy CM, Cunningham AF, Maclennan CA (2012): The stability of complement-mediated bactericidal activity in human serum against Salmonella. PLoS One 7: e49147.
22. Mcintosh ED, Broker M, Wassil J, et al. (2015): Serum bactericidal antibody assays – the role of complement in infection and immunity. Vaccine 33: 4414-4421.
23. Meshulam T, Verbrugh H, Verhoef J (1982): Serum-induced lysis of Pseudomonas aeruginosa. Eur J Clin Microbiol 1: 1-6.
24. Meshulam T, Verbrugh HA, Verhoef J (1982): Opsonization and phagocytosis of mucoid and non-mucoid Pseudomonas aeruginosa strains. Eur J Clin Microbiol 1: 112-117.
25. Baltimore RS, Shedd DG (1983): The role of complement in the opsonization of mucoid and non-mucoid strains of Pseudomonas aeruginosa. Pediatr Res 17: 952-958.
26. Nakae T, Tanaka J, Nakano A, et al. (2008): Complement-mediated bactericidal effect of antibodies in human intravenous preparation against multi-drug resistant Pseudomonas aeruginosa. Jpn J Antibiot 61: 379-387.
27. Jankowski S, Grzybek-Hryncewicz K, Kopec W, et al. (1988): Bactericidal activity of human sera with a physiologic or genetic defect of the complement system. Acta Microbiol Pol 37: 309-315.
28. Peterson PK, Kim Y, Schmeling D, et al. (1978): Complement-mediated phagocytosis of Pseudomonas aeruginosa. J Lab Clin Med 92: 883-894.
29. Offredo-Hemmer C, Berche P, Veron M (1983): A complement-sensitive mutant of Pseudomonas aeruginosa. Ann Micro­biol (Paris) 134A: 281-294.
30. Hazlett LD, Masinick-Mcclellan SA, Barrett RP (1999): Complement defects in aged mice compromise phagocytosis of Pseudomonas aeruginosa. Curr Eye Res 19: 26-32.
31. Mueller-Ortiz SL, Hollmann TJ, Haviland DL, et al. (2006): Ablation of the complement C3a anaphylatoxin receptor causes enhanced killing of Pseudomonas aeruginosa in a mouse model of pneumonia. Am J Physiol Lung Cell Mol Physiol 291: L157-165.
32. Moller-Kristensen M, Ip WK, Shi L, et al. (2006): Deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with Pseudomonas aeruginosa. J Immunol 176: 1769-1775.
33. Chalmers JD, Fleming GB, Hill AT, et al. (2011): Impact of mannose-binding lectin insufficiency on the course of cystic fibrosis: A review and meta-analysis. Glycobiology 21: 271-282.
34. Taylor PW (1983): Bactericidal and bacteriolytic activity of serum against gram-negative bacteria. Microbiol Rev 47: 46-83.
35. Leddy JP, Steigbigel RT (1979): Complement, serum bactericidal activity, and disseminated gram-negative infection. Ann Intern Med 90: 984-985.
36. Olling S (1977): Sensitivity of gram-negative bacilli to the serum bactericidal activity: a marker of the host-parasite relationship in acute and persisting infections. Scand J Infect Dis Suppl: 1-40.
37. Vitkauskiene A, Scheuss S, Sakalauskas R, et al. (2005): Pseudomonas aeruginosa strains from nosocomial pneumonia are more serum resistant than P. aeruginosa strains from noninfectious respiratory colonization processes. Infection 33: 356-361.
38. Kays MB, White RL, Friedrich LV (2001): Effect of serum from different patient populations on the serum bactericidal test. J Antimicrob Chemother 48: 417-420.
39. Noguchi H, Bronfenbrenner J (1911): Effects of Mechanical Agitation and of Temperature Upon Complement. J Exp Med 13: 229-233.
40. Pohl AW, Rutstein DD (1944): The Deterioration of Complement Activity in Normal Human Serum. J Clin Invest 23: 177-180.
41. Siggins MK, Cunningham AF, Marshall JL, et al. (2011): Absent bactericidal activity of mouse serum against invasive African nontyphoidal Salmonella results from impaired complement function but not a lack of antibody. J Immunol 186: 2365-2371.
42. Jankowski S (1995): The role of complement and antibodies in the impaired bactericidal activity of neonatal sera against gram-negative bacteria. Acta Microbiol Pol 44: 5-14.
43. Gondwe EN, Molyneux ME, Goodall M, et al. (2010): Importance of antibody and complement for oxidative burst and killing of invasive nontyphoidal Salmonella by blood cells in Africans. Proceedings of the National Academy of Sciences of the United States of America 107: 3070-3075.
44. Mcgreal EP, Hearne K, Spiller OB (2012): Off to a slow start: under-development of the complement system in term newborns is more substantial following premature birth. Immunobiology 217: 176-186.
45. Hogasen AK, Overlie I, Hansen TW, et al. (2000): The analysis of the complement activation product SC5 b-9 is applicable in neonates in spite of their profound C9 deficiency. J Perinat Med 28: 39-48.
46. Lassiter HA, Watson SW, Seifring ML, et al. (1992): Complement factor 9 deficiency in serum of human neonates. J Infect Dis 166: 53-57.
47. Davis CA, Vallota EH, Forristal J (1979): Serum complement levels in infancy: age related changes. Pediatr Res 13: 1043-1046.
48. De Benedetti F, Auriti C, D’urbano LE, et al. (2007): Low serum levels of mannose binding lectin are a risk factor for neonatal sepsis. Pediatr Res 61: 325-328.
49. Hilgendorff A, Schmidt R, Bohnert A, et al. (2005): Host defence lectins in preterm neonates. Acta Paediatr 94: 794-799.
50. Sallenbach S, Thiel S, Aebi C, et al. (2011): Serum concentrations of lectin-pathway components in healthy neonates, children and adults: mannan-binding lectin (MBL), M-, L-, and H-ficolin, and MBL-associated serine protease-2 (MASP-2). Pediatr Allergy Immunol 22: 424-430.
51. Swierzko AS, Atkinson AP, Cedzynski M, et al. (2009): Two factors of the lectin pathway of complement, l-ficolin and mannan-binding lectin, and their associations with prematurity, low birthweight and infections in a large cohort of Polish neonates. Mol Immunol 46: 551-558.
52. Weiskopf D, Weinberger B, Grubeck-Loebenstein B (2009): The aging of the immune system. Transpl Int 22: 1041-1050.
53. Simon AK, Hollander GA, Mcmichael A (2015): Evolution of the immune system in humans from infancy to old age. Proc Biol Sci 282: 20143085.
54. Yoshikawa TT (2000): Epidemiology and unique aspects of aging and infectious diseases. Clin Infect Dis 30: 931-933.
55. Polignano A, Tortorella C, Venezia A, et al. (1994): Age-associated changes of neutrophil responsiveness in a human healthy elderly population. Cytobios 80: 145-153.
56. Hazlett LD, Rosen DD, Berk RS (1978): Age-related susceptibility to Pseudomonas aeruginosa ocular infections in mice. Infect Immun 20: 25-29.
57. Berk RS, Iglewski BH, Hazlett LD (1981): Age-related susceptibility of mice to ocular challenge with Pseudomonas aeruginosa exotoxin A. Infect Immun 33: 90-94.