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Original Article Serum Bactericidal Effects on Common Clinical Bacterial Isolates in Thais Suwanna Trakulsombon, M.Sc. Nalinee Aswapokee, M.D. Somwang Danchaivijitr, M.D. Abstract Serum from healthy human is known to have bactericidal effect on some bacteria such as Enterobacteriaceae. This effect may act, in concert with phagocytes and complement to pre vent bacteremia from those species. We studied serum bactericidal effect on clinically important bacteria. Pooled sera were obtained from Thai healthy donors during June to September 1987. These subjects were free from diarrheal disease for 6 months before sera were taken. Standard time killing curves were done using 80%, 40% serum and controls with Mueller-Hinton broth and heated serum. Bacterial isolates wereS. aureus (4 strains),E. coli (5),K. pneumonioe (4),S. typhi (8), nontyphi Salmonella (16) and Shigella species (10) with 105 CFU/ml at the beginning. It was found that sera from normal Thai subjects exerted bactericidal effect upon Salmonella typhi and Shigella species but not for S. aureus, E. coli and K. pneumoniae. This study suggests that specific antibodies to Salmonella and Shigella may readily present in healthy Thai subjects, and that this may reflect frequent exposure. It might be suggested that there is protective antibody in adult sera and vaccination may be unnecessary in this group. 1lHfnlfl')..lI'YlU~()1I <l..... IIJ' <l <l ..... 1IJ.1 <l IIJ 'V ...... UflYlnUYl19 IUYlIIUflIYl'Yll-3fl'l')..lfl ' i " , fO ... , "I " qYlfi'lftl-3fll<jCJ.tll'IHl . t'nnon \lI'1::fli.H1llUH~, llail rl'rnInfl, tlll'td'-3 ~111'lfUl~Y11 "" A ( (':'A 1llm"1l1fl1qJtf!lY~J, flm:::U't'lYl£Jtfll~mtflJJl"1l'VHJ1Ul~. 1. <:>. ~ A A Q..o :lJVi1dYl£J1~£J:lJ'V\~~. n~ -JbYl't'l"1 10700 v.q, nJi17'i (mnf'll'JJfJIUl::lJ7mUf/fl'IIW 2532; 6: 12'21 I . G" ... ~I.o:::I c:I I ... 4J _I qq <\1 ' oo!f, q c:5 q 1 .at. 1 ' lullYlYl11UVll1tl1JJ'Utl-3fllluflYlJJqYlfi llm1'J.l11'1ftl lIUflYl I'W Ul-3'11llVl, 1'IIll l'lftl llfl~JJ Entero bacteriaceae. qYli111mHhi.llul~tl~tll;)~~1I1~tli1tl.:Ulllfln~VlI~tl 111fl1::lltll~tlVl;)lfll~tll't1~~ijll. IVIU v h i ' !l"-., .... '" .. .1 '" '" I "I .... tlltlU P agocyte 1Ii.l:: comp ement 'lflU. fllU::Hl;)U;)-3IV1'Yllm1flfl1o:l1u1::tlYlfiillVlm1'J.l11'1ftlIlUflYlI1U I t • l1ii1rlty'Yll'lflailfl111ih"JJflll'YlUtJfl~. . ~ I • ilfJJ111'1f'llflmlfl1mUlI pooled sera '\ltl-3flll'YlUUfl~1ll'lil-3l~tllliJOll1Ulli\'1l~tlll«llU1Ull'Vtfl. 2530. .q 4" 1 4, mu 1I 6IVltlllfltlllJJ1U1;)lflli.ltlVl. ~'Yl';)lflfl11U;;)lfl1 ~tlVl flllUfl~lm;,il'uiiu1d~tl;);)nd 1'1 " 1llfll1flfllol1 .. 1'lfl1i time-kill curve VllUtl1JJ~tlUi.l~ 'j/ 80 lla~ 40, ~<:I <:I v 9/ lli.l~ 1'li'Mueller-Hinton broth lli.l::ftfJJilOflfl11JJftlll 56''11. IUlIl1i.l1 30 1I111 lUll Y1"lfl1UflJJflOl illVl. l~tl~111fll1ftfl1o:l1'vi'llnS. aureus (4 stra~ns), E. coli (5), K. pneumoniae (4), S. typhi ;10;, non typhi S?lmonella (16) II a:: Sh!gella 10). , -1..q<:l..q1 VI <:I v VlUl1tl11l'lltl-3flllIYlU~flYlJJqYlfi <:I 4, 'iI spe~ies IVlUliJJYl'lUl~mhllll1I05CFU IJJi.l. ,"'t • llm1'J.lll'lftl Salmonella typhi Ui.l:: Shigella specIes. ~ ,4, 't11tlJJHi.llltlUJJ1flYlfll'lftl S. aureus, ~. coli 1Ii.l:: K. pneumoniae. _I..q, VI <:I oC!t, .q c" 4, ~ .q e:t c' ~..;. , _~ Q..I .q ~ '" 'iI ou tl-3flllfln VlVlI 'lftl IVI. 'iI <:I 1"'" '" :'. lIfi11 JJ'lf1Y1 Ui.l::tll;)lull 1 'j/..q it v 4':; 1 0 'j/ fJ afln flfl1l111 t)l;)'Yll 't1 Y16-3Vl;)n 01111 tl-3 lllU U1Ufll1 ;1 VI lfleUlI i1tl-3flll h flIVlUli1Vl1::,.,j'Yl~uvl6Uvl1't1u. Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 12 '''I' <:I llYlIJJJJHi.l i1~1l1lHi.lfl111;)Ulllli.1V1'1 't1I't11111 flllIl1UlJflYlll1;)::JJlltlllVlUtlVlUYlflI'lftl Salmonella lli.l:: Shigella tlyual 11t)1IY1UtlVlUmVl1~I'lftl'll-3i.11 J Jn .q flUftmn Vol. 6 No.1 Jan. - Mar. 1989 J Infect Dis Antimicrob Agents INTRODUCTION The ability to combat invasion into human body by microorganisms involves two mechanisms. The first is a specific immunity and the second is a so called natural or non-specific resistance which does not occur for every invading microorganisms. 1-4 These two host defense mechanisms virtually present in healthy hosts. However, they may vary among in dividuals depending on several known and unidenti The variation of host immunity in fied factors. human may occur through known factors such as age, sex, genetic, pregnancy, environment and nutri tion. 3 ,4 One of these nonspecific mechanism occur in serum of healthy human, that is bactericidal activity against some bacteria. This immunity may act among various defenses, in concert with phagocytes and complement to prevent bacteremia from those species. 5 This bactericidal activity is mainly against some gram-negative bacteria 6 - 9 as described by Buchner in 1889. This effect is abolished when serum has been incubated for 30 minutes at 56°C. Gram-positive bacteria generally are less sensitive than gram-negative bacteria to this killing effect. lO - 13 Sera from Thai people, however, are not known, to the best of our knowledge, to have this effect to any of these bacteria. We, therefore, study serum bacterici dal effect of Thai adult due to the interest that such effect in area where prevalence of bacterial infections are high may be different from elsewhere. It is also known that the environment, nutrition, hygiene and sanitation, behavior of Thais differ from those in de veloped nation, and that these factors might render the serum bactericidal activity to be different from the published information. MATERIALS AND METHODS Pooled nonnal human serum (PNHS). Pooled serum from 144 healthy donors, aged between 20-45 years, were obtained from blood bank, Siriraj Hospi tal, Mahidol University, Bangkok, Thailand, during June to September 1987. The individuals whose blood were taken did not suffer from diarrheal disease for 6 months prior to this study. PNHS was made into aliquot and was stored in -70°C before use. The Widal agglutination test of this pooled serum was negative for both 0 and H antigens of Sal monella typhi and also Salmonella para A and para B. Controls were PNHS heated at 56°C for 30 minutes and Mueller-Hinton broth. Bacteria. Bacterial strains were recent clinical isolates from patients hospitalized at Siriraj Hospital. Some strains were obtained from National Institute of Health, Thailand. These were Staphylococcus 13 aures (5 isolates), Escherichia coli (5; 2 from blood 2 from urine, 1 from pus), Klebsiella pneumoniae (4), Salmonella paratyphi (8), S. typhimurium (4), S. choleraesuis (4), S. typhi (10), Shigella dysenteriae (2), S. flexneri (8), S. boydii (3) and S. sonnei (2). Most isolates were from blood. Only Shigella species and some S. typhi were from stool and lung. These bacteria were grown in trypticase soy broth, and then adjusted to 10 6 CFU/ml with Mueller-Hinton broth as initial inoculum size. Serum bactericidal test. The serum bactericidal activity of PNHS against various bacteria were per formed by modifying plate count method of Reantree and Rantg 14 and Olling and et al. 18 The test for bactericidal activity of serum used throughout this study was set up as follows: The 0.1 ml of 10 6 CFU Iml culture broth was added into 80% and 40% PNHS (0.8 or 0.4 ml of PNHS with 0.1 or 0.2 mlof Mueller-Hinton broth respectively). These were done in steriled 13 x 100 mm. glass tubes and incubated at 37°C for 24 hours, at time interval of 1, 1/2 , 1, 2, 4, 6 and 24 hours. A standard plate counting was per formed for each test. The viable counts expressed as log CFU Iml, with the minimum detection scale of 10 2 CFU/ml. The percentage of bactericidal activity was cal culated as previously described 19 as follows: % bactericidal activity = 100-100 x number CFU of experimental tubes number of CFU of control tubes RESULTS The activity of serum bactericidal. The activity of PNHS bactericidal against tested bacteria are shown by standard time-kill curves presented in Figures 1-9. The viable bacteria counts are presented as log CFU Iml at each period of serum exposure. The percentage of bactericidal activity of S. au reus, E. coli and K. pneumoniae; S. typhi and S. paratyphi; non-typhi Salmonella; and Shigella species were cal culated and reported in tables 1-5. Growth rate of bacteria in serum. All tested or ganisms had normal growth rates when incubated with heated serum or Mueller-Hinton broth. After 2-4 hours of incubation the growth rate of all tested bacteria in heated serum (Figures 1-9) were slightly lower than that incubated in broth. The growth rates of bacteria in fresh serum were slower than those in heated serum. This effect was seen after 4-6 hours of serum exposure from some strains of E. coli, all tested strains of K. pneumoniae, non-typhi Salmonella and Shigella group B. The difference in growth rate is clearly seen in S. typhi, S. paratyphi and Shigella species with the exception of Shigella group B. 14 n"" 6 "'1J~1Jn""l ::'I II J< .. 2532 JJ.fI.-J.J.fI. The serum bactericidal activity among genus and bacterial strains. There were significant dif ferences in bactericidal activity by PNHS among the genera of gram-negative bacilli. PNHS exert strong bactericidal activity against some in genus Salmonella and genus Shigella, while activity against Escherichia and Klebsiella was less remarkable. No major difference in bactericidal activity was demonstrated among clinical isolates of each species, in which patterns of bactericidal activity was quite similar. Serum resistance. Some species of clinical iso lated bacteria such as S. au reus, K. pneumoniae, E. coli, non-typhi Salmonella (S. typhimurium and S. choleraesuis) and Shigella group B. (Figure 1-3, 6-7 and 9) were relatively resistant to killing by PNHS. This could be seen from the distribution of percent age of serum bactericidal activity against these or ganisms in Tables 1, 4 and 5. It might be noted that sera from normal Thai subjects had minimal bacteri - - 80% serum ++++-i 40% seru m ....... control (heated serum) --- -- control (Mueller-Hinton broth) O'------.---.-----.--I~ 24 6 4 2 Time (hours) Fig. 1 Bactericidal activity of normal human serum against S. aureus. 10 ,, 10 / --E ~ u. c..J e / 9 9 / / 8 / / / r- ------/ 8 / / 7 / 7 1E298 MUCI 6 / / / / 6 / C> 0 5 5 "iij > .~ JJ 4 3 2 - - 80% serum ++++-l 40% serum .... control (heated serum) ----- control (Mueller-Hinton broth) 4 3 2 1 0 2 E -~ u. c..J e 4 f24 0 10 9 9 8 8 7 7 6 'f---, 2 10 4 6 24 6 C> 0 6 IE 299 Pus I 5 IE 300 H/C I 5 "iij > .~ ::::I (I) 4 4 3 3 2 2 1 1 0 'f-----, 2 4 Time (hours) 6 24 r---. 0 2 4 Time (hours) Fig. 2 Bactericidal activity of normal human serum against E. coli. 6 24 Vol. 6 No.1 Jan. - Mar. 1989 J Infect Dis Antimicrob Agents 10 10 9 9 15 ,~ / / / / / / E -~ LL. B --E 7 ~ LL. e e 6 Cl Cl 0 c;; > .~ 0 5 c;; IK132H/CI 4 > .~ ,If ,/ 7 6 ,/ , / / / 5 Sal group B 1024 4 H/C ::::l ::::l en B U U / - - BO%serum +-+++ 40% serum ...... control (heated serum) -- - - control (Mueller-Hinton broth) 3 2 1 0 'j- 2 4 6 en 3 2 1 - - BO%serum +-+++ 40% serum ..... control (heated serum) - - - - control (Mueller-Hinton broth) 0 2 24 Fig. 3 Bactericidal activity of normal human serum against K. pneumoniae. ,. 9 -- B e 6 E ~ LL. u 7 Cl g c;; > .~ / 5 !' , , , ,, " . .- " .- / 9 B 7 6 5 H/C 4 4 - - BO%serum +-+++ 40% serum ...... control (heated serum) -- - - control (Mueller-Hinton broth) 3 1 '/------------. Yo 1 4 2 6 24 Time (hours) , 10 I I I B I I I 7 6 I I ,, ,If Cl o Sal Para A 236 5 4 3 2 1 - - BO%serum +-+++ 40% serum ..... control (heated serum) -- - - control (Mueller-Hinton broth) 0 2 4 f- 6 24 Fig. 7 Bactericidal activity of normal human serum against S. choleraesuis (C). I I e 2 I 9 LL. 3 Time (hours) Fig. 4 Bactericidal activity of normal human serum against S. typhi U Fig. 6 Bactericidal activity of normal human serum against S. typhimurium (B) 10 / 0 E 24 / Sal group 0 202 2 ~ r------. / ,,~' ::::l en " ,~ / 6 Time (hours) Time (hours) 10 4 H/C - - BO%serum +-+++ 40% serum ...... control (heated serum) -- - - control (Mueller-Hinton broth) OL-.~---,-----.-----, 2 4 6 Time (hours) Fig. 5 Bactericidal activity of normal human serum against S. paratyphi (A) cidal or bacteriostatic activity against these organisms only in the first 4 hours of serum exposure. After 6 hours of incubation, complete killing against these organisms was not found. These organisms, moreo ver, were able to regrow to the stationary phase, and after 24 hours of incubation, the sera exerted no acti vity at all. Serum sensitive. The results reported in Tables 2, 3 and 5 show that fresh PNHS from Thai donors are able to reduce the viability of S. typhi, S. paraty phi and Shigella species with the exception of Shigella group B. The rate of bactericidal of all strains of S. typhi., S. paratyphi and Shigella species group A, C and D in this study were very rapid, i.e. only 2-4 hours were needed for complete killing. The serum bactericidal activity among different sites of infection. A comparison of the sensitivity to 16 :'In "" ,6 . ,''lUvun"'' 1 I I .; u.n.-un. 2532 10 10 9 B 5 4 - BO%serum 40% serum ..... control (he.ted serum) t+++ ~ jL--~~ 4 . , 10 9 __c_on_t~~o_1 _(M_U_el...,'e_r'--.Hi~:roth) 24 10 ,I ,/ B I I I . I I I l I / I I I I .......... ---_.~ 5 4 1 1 OL-~..-,--~-..-,---I'f-- o L--..---.----,----.--I,f---, 4 4 24 Time (hours) 24 Time (hours) Fig. 8 Bactericidal activity of normal serum against Shigella spp. 10 10 , 9 // B ] ,, ,./ ~ u.. c..> o , / '" .g ~ .~ 4 4 en IShig B 335 stool I o L---.-..-,--,--~,...----/J--, 4 , 10 ,/ , , ,, 24 10 I - - BO%serum t+++ 40% serum ..... control (heated seruml control (Mueller·Hinton broth) -~,--~---I/f---, 4 Time (hours) 4 24 Time (hours) Fig. 9 Bactericidal activity of normal human serum against Shigella B. Vol. 6 No.1 Jan. - Mar. 1989 J Infect Dis An timicrob Agents 17 Table 1 Distribution of Serum Bactericidal Activity Against S. aureus, E. coli and K. pneumoniae. Organisms Tests· Yz 1 Percent Antibactericidal Activityt 2 4 6 24 S. aureus 41 H/C I 300 300 33.3 300 60 0 33.3 14.3 II 42 H/C I 43 H/C II I 65 H/C II I II 95 0 70 55 92 84 98 66.6 99.95 37.5 99.50 0 99.98 94.28 95.0 95.0 0 0 0 0 0 0 0 0 E coli 298 MUC I II 314 MUC I II 299 PUS I 300 H/C II I II 301 MUC I II 100 14.3 99.99 99.97 76.66 76.66 0 50 99.1 0 70.0 92.5 100 100 99.6 99.5 80 90 99.11 99.1 93 93 90 90 99.97 99.97 0 0 0 0 0 0 99.0 97.5 94.0 70.0 60.0 60.0 99.5 99.5 99.99 99.99 99.99 99.99 99.99 99.99 97.0 97.0 99.99 99.99 95.0 95.0 99.99 99.99 99.07 99.07 0 0 0 0 0 0 0 0 0 0 0 0 0 0 K. pneumoniae 132 H/C I 133 H/C II I II 138 H/C I 145 H/C II I II *1 is nonnal pooled serum (80%) compared with control broth. II is nonnal pooled serum (80%) compared with control heat-inactivated serum. tserum exposured time (hours). H/C is hemoculture MUC is midstream urine culture CFU/ml of test %Antibactericidal = 100 - 100 x - - - - ' ' - - - - CFU/ml of control serum of E. coli, isolated from different sites of in fection was performed. E. coli strains isolated from blood or pus were more resistant to the bactericidal effect of normal human serum than that from urine, as illustrated strain E 300 HIC, E 299 PUS, E 314 MUC and E 298 MUC in Figure 2. No significant dif ference was found in serum sensitivity of S. typhi which isolated from different sources, such as blood, stool and sputum (Table 2). DISCUSSION The bactericidal action of pooled serum from 144 healthy donors from Blood Bank Unit, Siriraj Hospital, Mahidol University, aged between 20-45 years, against various isolated bacteria commonly en countered in clinical situation in Thais has been studies. Since the serum from individual donor had a similar bactericidal action to pooled serum (data from ::"n"" 6""1J~1Jn"" 1 JJ. fl. -JJ.<\ fl. 2532 u 18 Table 2 Distribution of Serum Bactericidal Activity Against S. typhi Organisms Tests· Percent Antibactericidal Activityt 2 4 6 Yz I 99.5 99.0 99.90 99.90 99.9 99.9 90.0 90.0 99.5 99.5 97.5 97.5 99.0 99.0 99.0 98.0 99.5 99.66 98.5 99.5 99.9 99.5 100 100 100 100 99.82 99.7 99.82 99.55 99.82 99.77 100 100 99.9 99.1 99.88 99.9 99.94 99.95 24 Salmonella gr. D 169 H/C I II 202 H/C I II 100 H/C I II 45 bronchitis I II 48 pneumonitis I II 190 stool I II 211 stool I II 54 H/C I II 178 H/C I II 215 H/C I II 100 100 99.95 99.77 100 100 100 100 100 100 ~ 100 100 100 100 100 100 *1 is normal pooled serum (80%) compared with control broth. II is normal pooled serum (80%) compared with control heat-inactivated serum. tSerum exposured time (hours) Table 3 Distribution of Serum Bactericidal Activity Against S. paratyphi Organisms Tests· I Percent Antibactericidal Activityt 2 4 6 Salmonella gr. A 232 H/C I 99.85 99.82 100 100 100 100 100 100 II 233 H/C I II 236 H/C I II 57 H/C I II 58 H/C I II 59 H/C I II 60H/C I II 1 H/C I II 100 100 99.7 99.4 100 100 99.97 99.97 *1 is normal pooled serum (80%) compared with control broth. II is normal pooled serum (80%) compared with control heat-inactivated serum. tSerum exposured time (hours) 100 100 100 100 99.88 99.88 100 100 24 J Infect Dis Antimicrob Agents 19 Vol. 6 No.1 Jan. - Mar. 1989 the preliminary study) and we studied different first two hours. There was no difference in bacteri strains of the bacteria rather than sera from different cidal effect of serum at concentrations of 40 and 80% against serum susceptible strains and serum resistant individuals. The bactericidal action of serum is demonstrable strains. The rapid bactericidal effects of even low over a wide range of serum concentrations, although concentration of sera occurred with some clinically the rate of killing is known to generally increase with important bacteria, that is S. typhi, S. paratyphi, and increasing serum concentration. For the study of Shigella species (with the exception of some strains of serum bactericidal activity in relation to infection, Shigella group B), is interesting. This may be an in the majority of investigators have employed systems direct evidence that Thai people possess specific containing relatively high concentration of serum to antibody to these bacteria when reaching adult ensure that killing is not limited by the availability age group, or the bactericidal activity can be attri of essential components of bactericidal system: thus, buted to complement because this effects was abo concentrations in the 20 to 80% range have been suc lished by heated at 56°C. cessfully utilized in a number of studies. 6 ,20-24 From After 2-4 hours of incubation the growth rates these reasons, serum bactericidal activity of PNHS of all tested bacteria in heated serum were slightly was studied in concentration of 40 and 80%. lower than that incubated in broth. From these re Serum bactericidal activity is one of the most sults, we can conclude that there are some growth in important host defense mechanism for prevention of hibition factors in heated serum or there are some systemic invasion of serum sensitive bacteria. The bactericidal antibodies in PNHS of Thais, in addition ability of serum to inhibit the growth of bacteria is to the bactericidal effect of the complement. The PNHS from healthy Thai donors is able to repeatedly shown to be highest within the first 2 hours of serum exposure. 9 ,14,17,25,26 The results in our kill S. typhi, S. paratyphi and Shigella group A, C studies confirmed those studies, in which complete and D. Contradictory finding were presented by killing of sensitive bacteria by sera occurred in the other, 29,30 which revealed that their serum was not Table 4 Distribution of Serum Bactericidal Activity Against S. typhimurium and S. cholerasuis. Organisms Tests* Y2 1 Percent Antibactericidal Activityt 2 4 6 24 Salmonella gr B 1024 H/C I 2977 H/C II I 99.33 -100 85 II 40 3999 H/C I 2162 H/C II I 2681 H/C II I 95 66.66 90.00 -100 50 25 99.99 90.0 99.99 98.33 99.96 99.50 99.92 98.66 99.99 87.5 66.66 -100.00 97.5 50.0 90.0 50 70 90 99.95 40.0 99.99 -60 99.99 -75.0 99.95 80.0 II Salmonella gr C 4910 (18) H/C 0 -100 I 4907 (6) H/C II I 4907 (5) H/C II I 4910 (2) H/C II I II * I is normal pooled serum (80%) compared with control broth. II is normal pooled serum (80%) compared with control heat-inactivated serum. tSerum exposured time (hours) 80.0 -33.3 99.99 98.66 99.99 99.99 99.99 99.92 99.99 99.5 99.90 99.9 99.99 99.2 99.99 -60 99.99 99.85 99.99 95.71 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 lJ'<n 6 o~ <i <i n7il77 hfl~~1 iJiJlUl::lJ7ri'7lJ iJililw , iwmll JJ. fl. - 1/. fl. 2532 Table 5 Distributionof Serum Bactericidal Activity Against Shigella spp. Organisms Tests· Y2 1 Percent Antibactericidal Activityt 2 6 4 24 Shigella gr. A 3034 stool I 0 0 90.0 90.0 99.95 99.90 100.0 100.0 50.0 50.0 900.0 566.6 50.0 33.33 0 100.0 70.0 70.0 60.0 33.33 50.0 50.0 70.0 0 66.66 66.66 80.0 66.6 93.33 33.33 95.0 300.0 99.94 99.85 99.0 92.5 99.4 50.0 99.40 50.0 98.5 85.0 99.11 20.0 99.99 40.0 99.99 70.0 100 100 99.99 87.5 99.99 50 99.99 42.86 50.0 50.0 99.0 98.0 100 100 80.0 80.0 99.77 99.77 99.33 99.0 100 100 100 100 90.0 90.0 50.0 50.0 99.0 99.0 90.0 90.0 99.99 99.96 99.33 99.20 II Shigella gr. A 4421 stool I II 335 stool I II 337 stool I II 4453 stool I II 3068 stool I II 51 stool I II 45 stool I II 46 stool I II 70.0 70.0 0 0 50.0 33.33 0 0 50.0 50.0 0 0 50.0 50.0 0 0 99.!77 99.93 99.93 99.99 10.0 99.91 98.71 99.99 0 0 0 0 0 0 0 0 99.99 75.0 99.99 33.33 99.95 44.44 0 0 0 0 0 0 99.99 99.99 100 100 Shigella gr. C 4240 stool I II 21 stool I II 4131 stool I II Shigella gr. D 3947 stool I II 3945 stool I II able to reduce the viability of these organisms. Roan tree 14 found that the Shigella and Klebsiella groups showed a higher percentage of serum sensitive strains than do the others, whearas there is a higher per centage of serum resistant strains of Salmonella, using serum from the American healthy donors or patients. This contradictory finding may come from the dif ference in the characteristic of nutrition, environ ment, hygienic, sanitation and behavior among the Thais and the other races. The most important fac tors may be the differences in prevalence of these or ganisms in the environment and the rate of exposure 100 100 99.99 99.99 to them. The PNHS of Thais is unable to reduce the viabi lity of S. aureus, K. pneumoniae and some strain of E. coli. The lack of this bactericidal activity is simi lar to that reported from the sera from Westerns healthy donors. 8 - 11 ,14 The serum bactericidal activities among the groups of Salmonella spp. and Shigella spp. are signi ficant different. PNHS was unable to kill Salmonella group Band C and Shigella group B, whereas it exert ed high activity against Salmonella group A and D, Shigella group A, C and D. The mechanism for these J Infect Dis Antimicrob Agents phenomena is not known, but may be due to the dif ference in some antigenic determinant or the dif ference of some biochemical groups or structure in some compositions on the cell surface of individuals species or genus. 29 It was found that sera from Thai subjects exert ed bactericidal effect upon Salmonella typhi and Shigella species but not for S. au reus, E. coli and K. pneumoniae. This study suggests that specific antibo dies to Salmonella and Shigella may readily present in healthy subjects in Thailand. It may indicate previous apparent or subclinical infection with S. typhi or Shigella infections. It is known that humoral and cellular immunity to S. typhi in those who live or do not live in endemic area of typhoid infection are different. n ,28 Previous infection with other bacteria that share common antigenicity with Salmonella or Shigella 27 may be the cause of this finding. From this study, two hypotheses are generated. First, the rates of Salmonella and Shigella exposure in normal healthy adults in Thailand are relatively higher than that of Western countries, and that, this may be pro tective immunity against these organisms. Second, the protective immunity to Salmonella in the blood may reflects the unnecessary vaccination policy, especially in adults. It is also the explanation for observations which were made for many decades that, in Thailand, there are rare cases of enteric fever in the middle age group of the Thai people and that the differential diagnosis of febrile illness in adults aged over 40 who suffer from enteric fever will come out with low probability. 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