Aerobic and anaerobic growth modes and expression of type 1 fimbriae in Salmonella

Pathophysiology 14 (2007) 61–69

Aerobic and anaerobic growth modes and expression of type 1 fimbriae in Salmonella Elias Hakalehto a,e,∗ , Jouni Pesola b,c , Lauri Heitto d , Ale N¨arv¨anen a , Anneli Heitto e b

a Department of Chemistry, University of Kuopio, P.O.B. 1627, FI-70211 Kuopio, Finland Institute of Clinical Medicine, Paediatrics, University of Kuopio, P.O.B. 1627, FI-70211 Kuopio, Finland c Children’s Hospital, Kuopio University Hospital, P.O.B. 1777, FI-70211 Kuopio, Finland d Environmental Research of Savo-Karjala, Yritt¨ aj¨antie 24, FI-70150 Kuopio, Finland e Finnoflag Ltd., P.O.B. 262, FI-70101 Kuopio, Finland

Received 4 January 2007; accepted 5 January 2007

Abstract The aim of this study was to clarify the growth rates of facultatively anaerobic Salmonella enterica serovar Enteritidis strain in aerobic and anaerobic conditions and the expression of type 1 fimbriae in relation to the growth phases. The cultivation was carried out in a Portable Microbe Enrichment Unit (PMEU) where in same conditions one can grow the cells in parallel by modifying, e.g. aerobiosis only. The results obtained show that although the anaerobic metabolism is generally believed to be a slower producer of biomass or metabolites, in these circumstances S. enterica serovar Enteritidis strain gave comparable growth rates in anaerobiosis with nitrogenation as in aerobic cultures with constant aeration. Fimbrial antigens were produced in the beginning of logarithmic phase of the growth cycle both in the aerobic and anaerobic conditions. The fimbria remained in the presence of oxygen. This capability is possibly used for the intrusion of oxygen containing tissues of host body by the invading pathogens. In conclusion S. enterica serovar Enteritidis strain suspensions grow equally well in constant nitrogenation and aeration, and fimbria were produced in both conditions, during the early logarithmic phase but they prevailed in the presence of aeration. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Salmonella; Fimbriae; Enrichment; Growth; Aerobiosis; Anaerobiosis; Facultative anaerobes; Antibodies; Pathogenesis; Gastrointestinal tract

1. Introduction In microbial physiology and human pathophysiology especially interesting groups of prokaryotes are facultatively anaerobic bacteria [1]. They include enteric pathogens, which can use both aerobic and anaerobic modes of growth. The members of the genus Salmonellae exhibit a vast amount of different immunological reactivities, forming a multitude of serovars [2]. It has been generally accepted that the aerobic mode of growth and metabolism is much more effective than the anaerobic one [1]. This may not necessarily be true when the same conditions are used as the value of the key metabolite ATP is the same not depending how it is made if enough energy substrates are availabe and harmful metabolites are ∗

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removed with proper gas flows, which is made possible, e.g. in the Portable Microbe Enrichment Unit (PMEU) [3]. The members of the genus Salmonella have genes coding at least 12 different fimbrial types, including in the S. enterica serovar Enteritidis SEF 14, 17, 18, and 21, long polar fimbriae (lpf) and plasmid-encoded fimbriae (pef), many of which have been associated to the bacterial virulence [4]. The SEF 21 fimbriae is corresponding to the type 1 fimbriae of S. enterica serovar Typhimurium [5,6]. Usually it is assumed that fimbrial expression can be provoked during the anaerobic growth mode, but this is possibly relevant only for the first phases of the growth. When moving ahead in the host gastrointestinal tract, the invading salmonellas face after acidic stomach favourable pH and nutritional conditions in the small intestine [7,8] and form colonies, eventually biofilms on host cells in mucosal membranes [9]. Salmonellas rapidly invade the gut epithelium in

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humans. They possess several surface structures, fimbriae, for attachment. The most intensively studied fimbrial structures are the enterobacterial type 1 fimbriae, which mediate the mannose-sensitive binding of the bacterial cells to the target cells [6]. Type 1 fimbriae are straigth rod-like structures of about 1 ␮m in length and 7 nm thick. The fimbrins are synthesized at the cytoplasmic membrane and directed to periplasmic space, where chaperones readily bind to them [10]. The chaperones liberate the fimbrin molecules and add them to growing fimbrial filament. The fimbrial assembly occurs about 3 min after the subunit synthesis. The reservoir of the synthesized fimbrins is limited in number [11]. The fimbrial assembly is suggested to occur without protein synthesis. In static cultures the fimbriation has been suggested to offer a selective advantage for these cells by allowing them to a better access of oxygen due to cell mesh on the broth surface [12]. Salmonella strains in the intestine, after initial attachment, invade the body by breaking through the host membrane structures [13]. This invasion is genetically activated by the hilA, which is independently regulated by multiple factors [14]. The type 1 fimbriae are also important mediators of bacterial adhesion and invasion of Escherichia coli and other enteric bacteria [15]. The role of type 1 fimbriae in the pathogenesis of E. coli has recently been summarised [16]. Similar structures have also been found in other bacteria, such as Pseudomonas aeruginosa [17]. Depending on physiological or environmental conditions bacteria can exist in fimbriated and nonfimbriated states. The phase variation is under transcriptional control [18,19]. In earlier studies we have examined the expression of type 1 fimbriae on Salmonella cells by using antibodies to the fimbrin protein with enzyme immuno assay and transmission electron microscopy [20,21]. Peak immunoreactivity occurred after 3 h cultivation at 42 ◦ C in shaken cultures. In electron micrographs, the highest numbers of fimbriae occurred at the time of peak immunoreactivity, which suggests that fimbrins were assembled during a short period of the early exponential growth phase. Antifimbrial antibodies can be produced both against the terminal and the middle part sequences of the fimbrial proteins [20]. Synthetic fibril-forming peptides derived from E. coli fimbriae have been described [22] as also for bacterial flagella [23]. These antibodies produced against the fimbrin peptides usually cross-react not only with different Salmonella strains but also with some related enteric species. This remarkable cross-reactivity of Salmonella type 1 fimbrial antibodies produced with synthetic peptides is an undesired property according to conventional ideas in diagnostics. However, it could be exploited in specifying antibodies with an adequately broad spectrum for the detection of different serotypes [20]. If the antibodies produced with the synthetic peptides were not adequately species-specific, selectivity in the immunoanalysis could be achieved by controlling the enrichment conditions prior to the analysis [24]. In E. coli, the synthesis of mRNA coding the type 1 fimbriae takes place in the lag and early logarithmic phases

[11]. In Salmonella sp., the expression of type 1 fimbrial antigens occurs also in an early growth phase, which indicates that these appendages are assembled quickly after the arrival of the cells to the correct growth temperature and rich nutritional conditions [20]. This quick “mobilization” of fimbriae facilitates the attachment of enterobacteria to the small intestinal epithelia soon after the cells have passed the acidic conditions of the stomach. Therefore, the understanding of fimbrial kinetics is crucial also for the understanding the pathophysiology of bacterial invasions. Unfortunately present knowledge is still too limited. The aims of the present study have been to clarify the anaerobic and aerobic growths and type 1 fimbrin antigen production during the growth by benefitting the conditions in the PMEU to increase understanding about the pathophysiological capacity of Salmonellae and other facultatively anaerobic pathogenic bacteria.

2. Materials and methods 2.1. Bacterial strains and culture conditions The strain Salmonella serovar Enteriditis phagetype 4 (IHS 59813) and the Salmonella serovar Typhimurium phagetype 1 (IHS 59929) were stored at 37 ◦ C in TYG medium (5% tryptone, 2.5% yeast extract, 1% glucose) and seeded every 2 weeks throughout the study. Cultivation for the EIA was started with 3–4 days old starter cultures by inoculating 5% of the cultures into fresh RVS medium (Rappaport–Vassiliadis soya peptone broth, Oxoid, UK). The cultures were shaken in Erlenmeyer flasks (100 ml each) at 20 and 42 ◦ C or in peptone water at 37 ◦ C for inoculating the PMEU for Transia experiments, see below. Samples were stored at 4 ◦ C before coating to microtitration plates, up to 8 h after cultivation at 42 ◦ C and up to 24 h after cultivation at 20 ◦ C. For the plate cultivation samples were diluted to 10−2 –10−8 with 0.9% NaCl, added to XLDplates (xylose–lysine–deoxycholate) and incubated at 37 ◦ C for 24 h. The number of colonies were counted and colony forming units (cfu)/ml were calculated for every time point. 2.2. Enrichment under different gas flows in the PMEU The cultivation syringes were positioned into a Portable Microbe Enrichment Unit (PMEU) (Finnoflag Oy, Kuopio and Siilinj¨arvi, Finland). This equipment has been designed for enhanced cultivation of microbes under aseptic gas flow in adjusted temperatures (Fig. 1) for use in monitoring in the hospitals [3]. It has also been applied in the screening of water hygiene [25]. In the present experimentation, the PMEU unit was set to the enrichment temperature of 35–40 ◦ C. The air flow was adjusted to 0–100% of the pump capacity in the aerobic cultivations. During the anaerobic cultures, the gas stream was visually adjusted using the valves of the pressurized gas bottles.

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sis were taken before starting the cultivation and after 2.5, 4, 5.5 and 7 h of its onset. Samples for TYG-agar plates were collected in the beginning and after 4 and 7 h, and the plates were cultivated for 24 h at 40 ◦ C. In another experiment the samples were cultivated using 10 different combinations of inoculum age and the gas combination (see Table 1). All syringes were inoculated with Salmonella enterica serovar Enteritidis strain IHS 59813. Temperature was programmed to 35 ◦ C for the first 2 h, after that to 40 ◦ C. Samples for TYG-agar plates were collected in the beginning and after 3 and 7 h, and the plates were cultivated for 24 h at 37 ◦ C. 2.3. Synthetic fimbrin peptide Fig. 1. Schematic presentation of PMEU (Portable Microbe Enrichment Unit).

The samples were taken during the incubation periods by taking the injection syringes from the PMEU equipment and using them for transferring 1 ml of the cultivation broth into the first tubes of a dilution series. After adequate dilutions 100 ␮l of the chosen dilutes were then applied to the TYG (Tryptone, Yeast extract, Glucose) agar plates for enumeration. Salmonella enterica serovar Enteritidis strain IHS 59813 was grown in TYG broth overnight (18 h) at room temperature prior to inoculating the TYG broth in PMEU cultivation syringes with an inoculum concentration of 104 –105 CFU/ml. The cultivation was first carried out in four syringes: Syringe 1. Aerobic cultivation in PMEU. Air flow 100% of the pump capacity. Temperature 37 ◦ C for the first 2 h, after that 40 ◦ C; Syringe 2. As syringe 1 but switched to anaerobic cultivation after 3 h of cultivation (100% nitrogen); Syringe 3. Anaerobic cultivation in PMEU with 100% nitrogen. Temperature settings as in syringes 1 and 2. Switched to aerobic cultivation at 40 ◦ C. Samples for dot blot analy-

The amino acid sequence for the synthetic peptide was derived from Salmonella enterica serovar Typhi type 1 fimbrin [26]. The selected sequence ASFTAIGDTTAQVPFSIV shares 52.9% identity in 17 aa with corresponding polypeptide of E. coli fimbrin type 1 [27] (sequence similarity and homology program Fasta3, EMBL). A peptide was synthesized as multiple-antigen peptide (MAP) with four branches using Millipore’s PerSeptive 9050 Plus automated peptide synthesizer and Fmoc synthesis strategy. Fmoc-Lys(Fmoc)-OH comprised the backbone of the branched structure. The branched peptide was used for immunization without conjugation to carriers. Rabbits were subcutaneously immunized with 500 ␮g of MAP-peptide in Freund’s complete adjuvant. Boosters (500 ␮g) were injected in Freund’s incomplete adjuvant every 2 weeks, for 5 months. 2.4. Enzyme immunoassay (EIA) The reactivity of the fimbrial anti-peptide antibodies with whole cells of Salmonella enterica serovar Typhimurium was tested with conventional indirect EIA [28]. Microtitration

Table 1 Growth conditions in different cultivation of Salmonella enterica serovar Enteritidis in PMEU Experiment

Innoculum age

Gas mixture

A1 A2

4 days 4 days

A3 A4

4 days 4 days

B1 B2

1 day (24 h) 1 day (24 h)

B3 B4

1 day (24 h) 1 day (24 h)

C2

4.5 h, preincubation with air flow 10% at +35 ◦ C. 4.5 h, preincubation with gas mixture (N2 85%, CO2 10%, O2 5%) at +35 ◦ C.

Aerobic, air flow 60% from the beginning Aerobic, air flow 0% for the first hour, then gradual increasing, after 3 h airflow the same as in syringe A1 (60%) Anaerobic, with 100% nitrogen Anaerobic, nitrogen flow 0% for the first hour, then gradual increasing, after 3 h flow the same as in syringe A3 (60%). Aerobic, air flow 60% from the beginning Aerobic, air flow 0% for the first hour, then gradual increasing, after 3 h airflow the same as in syringe A1 (60%) Anaerobic, with 100% nitrogen Anaerobic, nitrogen flow 0% for the first hour, then gradual increasing, after 3 h nitrogen (100%) flow the same as in syringe A3 (60%). Aerobic, air flow 0% for the first half an hour, then gradual increasing, after 3 h airflow the same as in syringe A2 (60%) Anaerobic, nitrogen flow 0% for the first half an hour, then gradual increasing, after 3 h nitrogen (100%) flow the same as in syringe A4 (60%).

C4

Temparature was +35 ◦ C for the first 2 h, after that +40 ◦ C.

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plates (MicroTest III, Becton & Dickinson) were first pretreated with 0.5% glutaraldehyde. The bacterial coating of the microtiter wells was adapted to the same level in different samples, 1:2 dilution of 0 time point as reference. Microtitration wells were coated by bacterial cells (1–2 × 106 cfu), incubated overnight at 4 ◦ C and tested with rabbit anti-peptide antibodies (1:100). Bound antibodies were detected with alkaline phosphatase conjugated anti-rabbit IgG and visualised with para-nitrophenyl. The reactivity of the fimbrial anti-peptide antibodies with purified fimbriae was tested with recombinant S. enterica serovar Typhimurium type 1 fimbriae isolated from E. coli (pISF101) [26], and with E. coli type 1 fimbriae purified from LE392/pRPO-1 strain [29]. The EIA for purified fimbria was carried out on Maxisorp Nunc-immuno plate coated with isolated fimbriae in TBS. The plates were not pretreated and fimbriae were exponentially titrated from 10 ␮g/ml. Otherwise, the plates were treated as in the EIAprocedure for whole cells. Anti-peptide antibodies reacted in EIA both with recombinant S. enterica serovar Typhimurium type 1 fimbriae and with E. coli type 1 fimbriae purified from LE392/pRPO-1 strain. The detection limit of the assay varied between 1 and 5 ␮g/ml. The sequence similarity was high enough to produce antibodies cross-reacting with these two type 1 fimbrial variants. Therefore, the produced antibodies could be considered as anti-type 1 fimbrin antibodies.

Tween in TBS). Then the strips were incubated in alkaline phosphatase—conjugated goat anti-rabbit IgG (Zymed, San Francisco, USA) (dilution 1:1000) for 1 h, washed as above for two times, and one time in the Afos buffer and stained with NTB and BCIP reagent solutions (Bio-Rad Laboratories, Hercules, California, USA) (in Afos buffer).

3. Results 3.1. PMEU cultures in different gas flow arrangements The growth curve of the PMEU experiment with different gas flow arrangements is illustrated in Fig. 2 and the corresponding dot blot results are seen in Fig. 3. The early anaerobic and aerobic growths were equally fast in the simultaneous suspensions gassed in PMEU either with nitrogen or air. Using four different gas compositions and gas distribution strategies for Salmonella enterica serovar Enteritidis indicated that the growth started quickly in the anaerobic conditions, but equal growth rates were rapidly achieved by the aerobic cultures. In 7 h from the onset of cultures in late exponential or early stationary phase, same cell densities were measured for the anaerobic cultures (AN), and the cultures which were started aerobically and switched to anaerobiosis,

2.5. Transia gold immunoassay Salmonella enterica serovar Enteritidis strain IHS 59813 cultivated as described above was used for inoculating the PMEU cultivation syringes (with a concentration of 100 000 cells/ml). The cultivation in the PMEU took place at 41.5 ◦ C using sterile air flow, and the samples were taken at 0, 4, 7 and 9 h after the onset of cultivation. The Transia experiment was carried out as instructed in the product manual of the test system (Diffchamb Ab, V¨astra Fr¨olunda, Sweden). For the enumeration of the cells, the Dry Cult TPC slides of Orion Diagnostica Oy (Espoo, Finland) were inoculated with the enrichment broth and incubated at 41.5 ◦ C. 2.6. Dot blot immunoassay Samples were collected directly from the cultivation syringes of the PMEU equipment to the nitrocellulose strips, and the intensity of the antibody–antigen reactions were tested with a simple dot-blot analysis as follows. After drying the transferred spots of about 2 ␮l, the strips were dried and refrigerated for about a week. Then the strips were moved to BSA-TBS solution in a shaker for an hour in order to block the unspecific binding of antibodies to the antigens during the consequent steps. The anti-type 1 fimbrin antibody H463 was used as the primary antibody (dilution 1:70 in TBS buffer). After 1 h incubation in the antibody the strips were washed three times for 10 min (in 1.5% milk powder/0.5%

Fig. 2. Growth curves of Salmonella enterica serovar Enteritidis. AE = aerobic growth mode; AE + AN = aerobic growth mode for 3 h, then anaerobiosis; AN + AE = anaerobic growth conditions for 3 h, then aerobiosis; AN = anaerobic growth mode.

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3.2. Dot blot analysis

Fig. 3. Dot blot of the PMEU culture experiment, blotted against antitype 1 fimbrin-peptide antibody as the primary antibody (see Section 2). AE = aerobic growth mode; AE + AN = aerobic growth mode for 3 h, then anaerobiosis; AN + AE = anaerobic growth conditions for 3 h, then aerobiosis; AN = anaerobic growth mode.

or vice versa (AE + AN, AN + AE) the aerobic (AE) growth yield being somewhat higher (Fig. 2). In anaerobic cultivation (AN) the type 1 fimbrial antigen expression levels peaked at 5.5 h in the dot blot decreasing thereafter (Fig. 3). This finding was consistent with our earlier results, which indicated that the fimbrial expression levels were rapidly coming down after they had reached the peak values [20]. On the contrary, according to the present results, the fimbrial expression in the well aerated PMEU cultures remained at high level. In the second experiment the same Salmonella strain was cultivated using different gas compositions and distribution strategies. The results indicated that the growth rates were comparable both in aerobic and anaerobic gassings during early periods of growth. The corresponding growth curves are presented in the Fig. 4 and the growth rates in Table 2. The growth started in similar speed but higher growth yields were finally achieved by the aerobic cultures probably due to the quicker deprivation of exploitable carbon substrates in the anaerobic fermentation. Table 2 Specific growth rates of the PMEU cultures as K (K = Ln (N2/N1)/(t2 − t1), where N1 and N2 are CFU’s (colony forming units)/ml at the beginning (t1) and in the end (t2) of logarithmic growth phase) when testing different types of inoculum and gas arrangements 0–3 h

3–7 h

A1 A2 A3 A4

1.8 1.1 0.3 0.8

1.2 1.6 1.0 0.6

B1 B2 B3 B4

1.9 1.9 1.1 1.1

1.0 0.9 0.4 0.9

C1 C3

0.9 1.0

1.6 0.8

See Table 1 for explanations of different test conditions. Aerobic and anaerobic growth rates closely correspond to each other during 0-3 h in A2 and A4; and in C1 and C3. During 3-7 h the similarity in growth rates was found in A1 and A3; and in B2 and B4.

Four different PMEU cultures that were originally inoculated with identical inocula were studied with respect to their type 1 fimbrial antigen expression in different gas conditions. The results are shown in the Fig. 3. According to these results the fimbriae are produced in the beginning of logarithmic phase in the growth cycle both in the aerobic and anaerobic conditions. In the latter ones this production seemingly ceased after forming a peak in the 5.5 h sample. This finding is compatible with our earlier observations [7], according to which also the electron microscopic evidence indicated that the fimbrial structures were seen on the Salmonella cells after 3.5 h of cultivation in a static culture in a test tube, whereas on the cells from the 7 h old cultures possessed hardly any fimbriae. The fimbrial expression was diminishing already during the late logarithmic phase. According to the present findings it seemed evident that in the presence of adequate amounts of oxygen in the PMEU the bacterial cells continued to have type 1 fimbriae on their surfaces. 3.3. Transia gold immunoassay The PMEU cultures that were analyzed with the Transia method yielded a clear and rapid increase both in the cell number and according to the immunoreaction (Fig. 5). In a simultaneous cultivation of a corresponding inoculum in a test tube the growth yield (the final cell concentration was about 1–10% of the yield in the PMEU unit (results not shown here). It is noteworthy that the antibodies used in this test were not produced with fimbrial antigens but with other surface antigens (LPS). This result proves that the cultivation in the PMEU unit rapidly increased the various bacterial antigens and together with the population growth during the earliest growth phases. However, the fimbrial antigens are expressed faster than the LPS antigens, for example. In the present experiment the detection limit level of the immunoassay was achieved in about 7 h. When the inoculum was somewhat smaller, 10–100 cells/ml or 100–1000 cells/ml, that level was met after 8 or 10 h from the onset of the cultivation, respectively (results not shown).

4. Discussion Having the focus on the early phases of the microbe cultivation in the enhanced enrichment equipment (PMEU) produced new information on: (1) rapid speed of the early phase anaerobic growth; (2) instant expression of type 1 fimbriae in the early logarhitmic phase, and (3) continuous expression of the type 1 fimbriae under oxygen flow. The reasons for assumptions that the aerobic growth mode would generally be more effective than the anaerobic fermentation are based on the fact that every glucose mole yields anaerobically only two moles of ATP with the substrate level

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Fig. 4. Growth curves of Salmonella enterica serovar Enteritidis. See Table 1 for explanations.

phosphorylation in homolactic fermentation versus about 38 moles of ATP in aerobic respiration of the same substrate [2]. However, according to our findings with the PMEU, this difference is not seen in maximal growth rate of the population, i.e. both metabolic systems apparently provide equal energy for the growth in gassed suspensions in the logarithmic phase. In the PMEU, different gases are directed to the liquid growth medium [24]. This allows not only an effective distribution of gases but also effective diffusion of the nutri-

ents in the medium as well as removal of the metabolites. Therefore, the speed of reactions is not that much restricted by diffusion limitation as in the case of static cultures or in production fluids [30,31]. In the gastrointestinal tract the incoming salmonellas have to overcome several defense mechanisms of the body, such as defensins, mucosal immunoglobulin A, HCl, bile salts, and pancreatic enzymes as well as cationic antimicrobial peptides of the epithelial cell surfaces in the low-oxygen,

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Fig. 5. Transia immunoassay in connection with PMEU enrichment experiment with Salmonella enterica serovar Enteritidis. In this assay the antibodies were produced against LPS (lipopolysaccharide) antigens. The rise in immunoreactivities followed the population growth, whereas in the assays described in Sections 3.1 and 3.2 with type 1 fimbrial antibodies it preceded the growth in the early phases.

hyper-osmotic environment of the small intestine [8,32]. In that environment the bacterial cells are well able to propagate according to the present results, if fermentable foods are present, with their anaerobic mode of metabolism. Salmonella pathogenesis could be a kind of escape from the above-mentioned hostile defenses, and also a way to avoid the competition in the large intestine with better facilitated bacterial rivals in there by intruding deeper into the body system, where the salmonellas avoid the competion and are better equipped for meeting the confrontation of the host defenses. Once attached the gut epithelium, the cells which prevail in anaerobiosis (in the conditions inside the gut) soon loose their fimbriae [20], whereas those cells which intrude through the mucosal barrier get in touch with oxygen provision, where they compete for the oxygen with host cells [33] switching their metabolism into the aerobic mode. In this light, the present finding that in the aerobic conditions the salmonellas are not ceasing the type 1 fimbrial expression seems logical. These fimbrial structures are still needed for the attachment and support better survival of the pathogens using the host oxygen reservoirs. It is possible that this aerobic activity also lowers the formation of bacteriocidic oxygen radicals by the host cells as the bacteria consume also oxygen for their own metabolism [34]. In these conditions more Salmonella cells are able to invade the host cells [35]. Gram-negative bacteria have been shown to respond with the formation of so called sigma factors to the environmental stresses. These factors are controlled by rpoS regulon in many bacteria [36]. In Salmonella enterica the inactivation of rpoS and other stress factor genes, namely PhoPQ and

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OmpR/EnvZ resulted in attenuated virulence [32]. The need for the stress factor functions for pathogenecity is a sign of the general hostility of the intestinal environment for Salmonella as well as the defensive barriers on host mucosal membranes. There exist somewhat different methods for intrusion in S. enterica serovars Typhi and Typhimurium [35]. The former has been shown to use unique fimbrial facilities, such as the type IVB fimbriae for intrusion [37]. The existence of the fimbrial structures could also show way like war flags to new recruits in a battle for the newly formed bacterial cells, which are thus anchored and directed through the breakthrough as like in the surface net of cells in a static Salmonella culture [12]. Then the growth could take place principally to the direction of increased oxygen content inside the body due to blood circulation. The serovar Typhimurium enters the murine membranes by penetrating and destroying the specialized epithelial M cells in the small intestine [13]. If the oxygen concentration turns low, the fimbrial structures are removed and their synthesis is ceased, and consequently the newly born cells are liberated from the gut wall in order to infect new intact intestinal surfaces. This phenomenon could explain also the findings of Hakalehto et al. [20] that in cultures originally grown at 20 ◦ C where the type 1 fimbriae are not necessarily expressed even though the temperatures were elevated to the physiological levels and fresh medium was added. In these cultures the active growth had deprived most oxygen and probably continued to do so in the shaken flask cultures also in the new conditions. Most likely cells on the liquid surface possessed more fimbriae, as discovered by Old and Duguid [12]. On the contrary the inlet of oxygen gases into the medium in the PMEU unit could form a different situation where the majority of cells could keep up their type 1 fimbriae. In our earlier studies, the maximum reactivity of antipeptide fimbrial antibodies with bacterial cells cultivated at 42 ◦ C was reached at 3 h, after which the reactivity rapidly decreased in shaken flask cultures [20]. At 20 ◦ C there was already immunoreactivity at the starting point and the maximum reactivity was reached at 9 h. There was essentially no EIA reactivity after 9 h. These results suggest that the fimbrins are transferred out of the bacterial cell only during a short period of the growth cycle. In the light of the present findings this may have caused by the deprivation of oxygen and nutrients from the medium. In the electron microscopy a clear pattern of fimbriation was observed at 3.5 h of culture at 42 ◦ C whereas at the starting point and at 7 h only few fimbriae could be seen [20]. Most cells were clearly smaller after 5 h of cultivation, as compared with the same cells after 3–4 h of growth in the RVS medium. Intestinal acid formation may have an overall contribution on the composition of the bacterial flora in the gut, which consequently affects the immunoreactions [38]. An interesting hypothesis has been proposed that the rectal mucosa-associated flora could reflect the situation with respect to inflammatory bowel disease [39]. In the light of this finding the results of the present study, which indicate differ-

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ent antigen formation of facultative salmonellas in aerobic and anaerobic conditions could give a clue for understanding the build up of some of the body’s immunodefenses. The rectal area is the major site where the gut-colonizing or invading bacteria meet increasingly aerobic conditions. For example, the potential production of type 1 fimbrial antigens could then, according to the present results, be more continuous there than in the case of conditions where the oxygen has been almost totally deprived. Thus, the facultatively anaerobic bacteria meet the challenges of host immune system differently in the rectum than in the anaerobic conditions of the inner intestines. Therefore, the rectal area could turn out to be an important location for building up the immune defenses of the body, and the interest on immunological studies on the rectal microflora could consequently increase. This hypothesis may also give new ideas for the studies of the pathogenesis of colitis ulcerosa that is originated in the rectal part of colon. The differing conditions in different parts of the intestines may thus contribute on the inflammatory diseases of the gastrointestinal tract. The bacteria use sophisticated macromolecular machineries such as chaperone-usher pathway for the type 1 fimbrial assembly [16] or type III secretion systems for molecular communication between the colonizing bacterial flora and host body [40,41]. The relations of the corresponding cell surface processes with the levels of molecular oxygen in the surroundings should also be studied as well as the bacterial switching of the facultative culture from aerobic growth mode into the anaerobic one or vice versa. The different growth modes could cause, besides the use of distinct metabolic pathways, also different functional roles of anaerobic and aerobic cultures in the invasion of host bodies. Acknowledgements We thank Dr. Anja Siitonen (National Public Health Institute of Finland, Laboratory of Enteric Pathogens, Helsinki, Finland) for giving us the bacterial strains, and Professor Timo K. Korhonen (Department of Biosciences, University of Helsinki, Finland) for providing us with the isolated fimbrin proteins. We also thank Professor Jukka Finne (Department of Medical Biochemistry, University of Turku, Finland) for suggestions to some parts of the manuscript. References [1] R.Y. Stanier, J.L. Ingraham, E.A. Adelberg, General Microbiology, fourth edition, The Macmillan press Ltd., 1976. [2] F.C. Neidhardt, J.L. Ingraham, M. Schaechter, Physiology of the Bacterial Cell, a Molecular Approach, Sinauer Associates, Inc., Sunderland, Massachusetts, 1990. [3] E. Hakalehto, Semmelweis’ present day follow-up: updating bacterial sampling and enrichment in clinical hygiene, Pathophysiology 13 (2006) 257–267. [4] T.A. Cogan, T.J. Humphrey, The rise and fall of Salmonella Enteritidis in the UK, J. Appl. Microbiol. 94 (2003) 114s–119s.

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