Investigation of Conditions for Capture of Live Legionella pneumophila with Polyclonal and Recombinant Antibodies

Since Legionella pneumophila has caused punctual epidemics through various water systems, the need for a biosensor for fast and accurate detection of pathogenic bacteria in industrial and environmental water has increased. In this report, we evaluated conditions for the capture of live L. pneumophila on a surface by polyclonal antibodies (pAb) and recombinant antibodies (recAb) targeting the bacterial lipopolysaccharide. Using immunoassay and PCR quantification, we demonstrated that, when exposed to live L. pneumophila in PBS or in a mixture containing other non-target bacteria, recAb captured one third fewer L. pneumophila than pAb, but with a 40% lower standard deviation, even when using the same batch of pAb. The presence of other bacteria did not interfere with capture nor increase background by either Ab. Increased reproducibility, as manifested by low standard deviation, is a characteristic that is coveted for biosensing. Hence, the recAb provided a better choice for immune adhesion in biosensors even though it was slightly less sensitive than pAb. Polyclonal or recombinant antibodies can specifically capture large targets such as whole bacteria, and this opens the door to multiple biosensor approaches where any of the components of the bacteria can then be measured for detection or characterisation.


Introduction
Legionella pneumophila is a gram-negative bacterium that is ubiquitous in water sources, environmental or man-made [1]. Sixteen serogroups of L. pneumophila are defined by the various components of the surface, but principally due to the variability of the lipopolysaccharide (LPS) [2]. In the environment, L. pneumophila is able to survive, despite its nutritional dependence on cysteine and iron, by having a high resilience in water [2,3] and by infecting protozoa, especially amoeba [4]. When engulfed by amoeba or human macrophages, they prevent lysosome fusion after phagocytosis and use nutriments of the host to divide and eventually cause host cell death [5]. This ability to survive intracellularly makes them pathogenic for humans, with different levels of pathogenicity depending on the individual's

Bacterial Culture
In preliminary experiments, we occasionally observed overgrowth with strains resembling L. pneumophila, so we employed a strain-producing GFP to provide a double guarantee of the identity of the bacteria tested as follows: it does not grow on BCYE plates without L-Cys, and colonies appear green on BCYE plates overlaid with chloramphenicol, which induces GFP production. L. pneumophila ssp 5 was also confirmed by its lack of growth on BCYE plates without L-Cys. L. pneumophila was plated and grown on BCYE agar plates coated with chloramphenicol for 4-5 days at 37 • C. An isolated colony was inoculated in Legionella enrichment broth (L.ER) supplemented with iron-pyrophosphate and L-cysteine and incubated for 24-48 h at 37 • C with shaking (~100 rpm). E. coli, B. subtilis and S. aureus were cultivated on LB plates overnight at 37 • C (O/N, 37 • C) then an isolated colony was inoculated into LB broth and grown (O/N, 37 • C) with shaking (~100 rpm). Exponential phase cultures of L. pneumophila were used as their outer membranes varied only slightly from viable but non-culturable L. pneumophila found in nutriment lacking environments [2], such as CTW. They were confirmed by having neutral pH [29] and no brown colour [30] (see Appendix A.1). The same protocol was applied to P. aeruginosa, but at a temperature of 25 • C. The bacterial concentrations were quantified by optical density measurements at 600 nm (OD 600 nm ). OD 600 nm of 0.1 was 6 × 10 7 CFU/mL for L. pneumophila, 2.5 × 10 8 CFU/mL for both E. coli and S. aureus [31], and 2.5 × 10 7 CFU/mL for both B. subtilis [32] and P. aeruginosa [33], respectively. All bacterial suspensions were prepared in PBS.

Immuno-Capture of Live Bacteria on 96 Well Plates
As a proxy for capture of bacteria with Ab on GaAs plates or other biosensor surfaces, L. pneumophila was captured with Ab on 96 well Costar plates. Ab solutions at 100 µg/mL in PBS were incubated in the plate wells for 1 h at room temperature (RT). Following adsorption, the following two blocking steps were carried out: first with BSA 2% in PBS and then with ethanolamine (pH 8, 1 M) in DI water. Both blocking solutions (200 µL/well) were incubated in the wells for 1 h at RT. After blocking, the wells were washed 3 times with PBS. For bacterial capture, 100 µL of L. pneumophila suspensions were added to each well (1 h, RT). Finally, 3 successive washes were made with PBS + Tween 0.05% (PBST). The effect of 0.05% Tween in PBS on the viability of L. pneumophila was evaluated (see Appendix A.2). To test the impact of Tween on reducing non-specific and antibody-specific capture, experiments were performed with or without Tween 0.05% in the bacterial washing step. All described incubations were performed with shaking (~500 rpm). For background level wells, the step of adding Ab solution was replaced by adding PBS instead, while all other steps remain unchanged.

Effect of Antibody Pre-Sonication on Bacterial Capture
In order to test the effect of sonication on possible rAb aggregation, 100 µL aliquots of Ab were pre-sonicated in a sonication bath at 4 • C in glass tubes for various times to reduce possible Ab aggregation before adding the Ab solution to the wells. This step was performed in a walk-in cold room at 4 • C. The ensuing blocking and washing steps remained the same as described in the previous section.

Direct DNA Extraction
An adaptation of the EtNa DNA extraction [34] protocol was chosen for direct DNA extraction of different microorganisms from Costar plates because of the limited material requirements and minimal loss of DNA. The DNA suspension solution from the EtNa protocol (Triton X-100 1%, Tween 20 0.05%, Tris-HCL 50 mM, EDTA 0.1 mM, pH 8) was used to detach bacteria captured on the plate wells. After adding 200 µL of the DNA extraction solution to the wells, the plate was heated to 85 • C for 30 min in a water bath, then cooled on ice for 5 min, quickly centrifuged and placed at −15 • C O/N. After thawing, the samples were aliquoted into microtubes and used for PCR.

Polymerase Chain Reaction (qPCR)
A 2 µL sample of the direct DNA extract was added to 8 µL of amplification solution in a LightCycler PCR plate as follows: 0.25 µM MIP forward and reverse primers, 0.15 µM MIP probe, (and/or 0.1 µM 16S rDNA forward and reverse primers, 0.075 µM 16S rDNA probe), 5 µL of 2X Taqman master mix (produced by the protein and purification service of the University of Sherbrooke, Sherbrooke, QC, Canada), completed to 8 µL with Mol Grade H 2 O. MIP gene primers and probes were employed for specific detection of L. pneumophila [35], as shown in Table 1. For universal amplification, 16S ribosomal DNA (16S rDNA) primers and probes, adapted from the CDC protocol [36], were added, as shown in Table 1. PCR program (activation: 95 • C-15 s; 50 amplification cycle: 95 • C-15 s, 60 • C-1 min) was optimized for the primers and probes. After amplification, the LightCycler programme determined the cycle threshold (Ct) of detection.

Bacteria Quantification Curve
Triplicate 100 µL volumes containing concentrations from 10 3 to 10 8 CFU/mL of bacteria were added to a Costar plate in 1 log 10 increments to obtain 6 concentrations of L. pneumophila. The addition of 100 µL of 2X concentrated DNA extraction solution to the wells resulted in concentrations from 5 × 10 2 to 5 × 10 7 CFU/mL of L. pneumophila. DNA extraction of the bacteria added to the Costar plate was carried out as described above and amplified by qPCR. Quantification curve wells were included in each experiment to assess the variability of extraction and amplification of positive controls and to obtain

Calibration Curve
Following DNA extraction of L. pneumophila from suspensions with concentrations ranging from 5 × 10 2 to 5 × 10 7 CFU/mL, qPCR amplification was carried out, and Ct values were determined with two sets of primers and probes [37]. Standard curves generated by linear regression revealed an inverse linear correlation between Ct and the concentration of L. pneumophila ( Figure 1). Lower concentrations were not tested as the Ct for 5 × 10 2 CFU or less showed a plateau, indicating that the detection limit of the system had been reached. This is not surprising as, theoretically, there would be only one bacterium in a 2 µ L aliquot from a bacterial solution with 5 × 10 2 CFU/mL, so lower concentrations of bacteria might be expected to generate negative qPCR results. The difference in Ct values between the two curves for the same bacterial concentration was due to the presence of three copies of the 16S rDNA sequence [38] but only a single copy of the MIP gene per Legionella bacteria [39,40]. The calibration curve was used to estimate the number of immobilised bacteria in the plate wells, with the MIP gene curve used for L. pneumophila and the 16S gene curve for L. pneumophila and other bacteria. Each capture test included calibration curve wells that also Lower concentrations were not tested as the Ct for 5 × 10 2 CFU or less showed a plateau, indicating that the detection limit of the system had been reached. This is not surprising as, theoretically, there would be only one bacterium in a 2 µL aliquot from a bacterial solution with 5 × 10 2 CFU/mL, so lower concentrations of bacteria might be expected to generate negative qPCR results. The difference in Ct values between the two curves for the same bacterial concentration was due to the presence of three copies of the 16S rDNA sequence [38] but only a single copy of the MIP gene per Legionella bacteria [39,40]. The calibration curve was used to estimate the number of immobilised bacteria in the plate wells, with the MIP gene curve used for L. pneumophila and the 16S gene curve for L. pneumophila and other bacteria. Each capture test included calibration curve wells that also served as positive controls for the experiment.

Optimal Bacterial Concentration for Incubation in Ab-Coated Wells
We had previously observed that the optimal concentration of the solution of Ab to be deposited on GaAs or microplate surfaces was 100 µg/mL [41]. Hence, we used this concentration to evaluate the saturation level of the two Ab used to capture L. pneumophila.
To estimate the saturation level of an Ab-coated surface, Ab-coated wells were exposed to various concentrations of bacteria. It was presumed that in order to compare the ability of pAb and recAb to capture bacteria, it would be necessary to use bacterial concentrations that did not saturate the surface, as results would be expected to plateau with saturating concentrations and variation in conditions might not affect the values. The highest concentration tested was 10 9 CFU/mL, and it provided the highest bacterial capture, as shown in Figure 2. When suspensions at 10 9 CFU/mL were added to wells coated with Ab, 2.2 × 10 6 CFU/mL (0.22% of the input bacteria) were captured with pAb and 1.5 × 10 6 CFU/mL (0.15%) with recAb, whereas without Ab, a non-specific capture of 3.6 × 10 4 CFU/mL (0.0035%) was observed. Exposure to suspensions at lower concentrations resulted in specific capture of 4.1 × 10 5 ± 2.2 × 10 5 (0.41%) CFU/mL by pAb and 2.8 × 10 5 ± 1.1 × 10 5 (0.28%) CFU/mL by recAb at 10 8 CFU/mL. The attachment of 3.0 × 10 4 ± 1.7 × 10 3 (0.30%) CFU/mL by pAb and 1.5 × 10 4 ± 9.0 × 10 3 (0.15%) CFU/mL by recAb were obtained by exposure to 10 7 CFU/mL of L. pneumophila. Non-specific attachment of 5.8 × 10 3 ± 4.8 × 10 3 (0.0057%) and 1.0 × 10 3 ± 1.6 × 10 2 (0.01%) CFU/mL were obtained with exposure of 10 8 CFU/mL and 10 7 CFU/mL, respectively. The exposure to suspensions at lower concentrations (10 8 CFU/mL and 10 7 CFU/mL) resulted in lower bacterial retention than the 10 9 CFU/mL, but similar capture percentages, indicating that capture was not less efficient. Hence, the 10 8 and 10 7 CFU/mL concentrations did not saturate the surface, and we do not have evidence that even 10 9 CFU/mL saturated the surface. The percentage of capture on wells without Ab increased slightly with a decrease in the exposure concentration. This could be attributed to the saturation of non-specific capture sites on the wells with increasing concentrations of bacteria.
These results confirmed the use of 10 8 or 10 7 CFU/mL as exposure concentrations for experimentation as they did not saturate the surface. This would allow us to evaluate the variation in capture by Ab under different conditions. They also maintained values for non-specific detection of >10 3 CFU/mL, which is within the range of the calibration curve.

Effect of Pre-Sonication of Antibodies on Bacterial Capture
Since only 0.1-1% of the total bacteria in the suspension were captured by Ab attached to the wells, we hypothesized that we lost some of the Ab capacity due to the presence of Ab aggregates. To reduce potential Ab aggregation, the Ab solutions were sonicated before adsorption to the wells to enhance bacterial capture. Figure 3 shows that different durations of sonication for both recAb and pAb did not significantly improve bacterial capture. sonicated before adsorption to the wells to enhance bacterial capture. Figure 3 shows that different durations of sonication for both recAb and pAb did not significantly improve bacterial capture. This result was expected for pAb due to its good solubility as it is produced by an organism for activity in solution [42]. Since the added scFv fragment may interfere with the solubility of recAb and cause self-aggregation [42], we checked this solution for aggregation. However, we did not observe that sonication improved capture efficiency, so we concluded that it was ineffective for breaking up aggregates or that aggregation did not pose a problem under our conditions.

Specificity of Polyclonal and Recombinant Antibodies
To evaluate Ab specificity, wells coated with pAb or recAb were exposed to different non-target bacteria (E. coli, S. aureus, B. subtilis, and P. aeruginosa). L. pneumophila ssp 5 was also used to test the selectivity towards other Legionella serogroups. Results based on the amplification of a universal 16S RNA showed that the average value of the capture of nontarget bacteria to wells with pAb or recAb did not vary significantly from the background adhesion to the No-Ab wells, as shown in Figure 4, but the higher non-specific attachment of B. subtilis and P. aeruginosa to wells without Ab. This result was expected for pAb due to its good solubility as it is produced by an organism for activity in solution [42]. Since the added scFv fragment may interfere with the solubility of recAb and cause self-aggregation [42], we checked this solution for aggregation. However, we did not observe that sonication improved capture efficiency, so we concluded that it was ineffective for breaking up aggregates or that aggregation did not pose a problem under our conditions.

Specificity of Polyclonal and Recombinant Antibodies
To evaluate Ab specificity, wells coated with pAb or recAb were exposed to different non-target bacteria (E. coli, S. aureus, B. subtilis, and P. aeruginosa). L. pneumophila ssp 5 was also used to test the selectivity towards other Legionella serogroups. Results based on the amplification of a universal 16S RNA showed that the average value of the capture of nontarget bacteria to wells with pAb or recAb did not vary significantly from the background adhesion to the No-Ab wells, as shown in Figure 4, but the higher non-specific attachment of B. subtilis and P. aeruginosa to wells without Ab. This indicated that Ab wells did not capture non-target bacteria significantly. However, L. pneumophila ssp5 did show significant capture with recAb (2.0 × 10 3 CFU/mL), albeit nearly 10 times less than the capture of L. pneumophila ssp1 (1.5 × 10 4 CFU/mL).
It was surprising that pAb did not capture more L. pneumophila ssp 5. Indeed, pAb is directed towards multiple components of L. pneumophila ssp 1, as indicated by the manufacturer, Virostat, who reported some cross-reactivity of this Ab with other serogroups of L. pneumophila [43,44]. However, this reactivity may not be towards components that allow the capture of L. pneumophila. LPS, or serogroup-specific LPS epitopes, may be the preferred target that allows the capture of these bacteria in the current experiments. When it was formulated, recAb was chosen to detect the LPS of ssp 1, and indeed, it was found highly specific for human isolates of serogroup 1 of L. pneumophila and did not detect an environmental serogroup 1, nor a serogroup 6 strain [22].
For further evaluation of specificity, mixtures of L. pneumophila at a concentration of 10 7 CFU/mL were added together with competitor bacteria to evaluate the interference of other bacteria present in the solution with the adhesion of L. pneumophila to the surface of the wells as follows: E. coli; S. aureus; L. pneumophila sg5 at a concentration of 10 7 CFU/mL (ratio 1:1) and B. subtilis; P. aeruginosa at a concentration of 10 6 CFU/mL (ratio 10:1). Both sets of primers were used on all sample wells to evaluate the total number of bacteria captured on the surface vs. the number of Legionella. The results indicated that the presence of other bacteria did not interfere significantly with the attachment of L. pneumophila to the surface ( Figure 5A,B, black bars). This indicated that Ab wells did not capture non-target bacteria significantly. However, L. pneumophila ssp 5 did show significant capture with recAb (2.0 × 10 3 CFU/mL), albeit nearly 10 times less than the capture of L. pneumophila ssp 1 (1.5 × 10 4 CFU/mL).
It was surprising that pAb did not capture more L. pneumophila ssp 5. Indeed, pAb is directed towards multiple components of L. pneumophila ssp 1, as indicated by the manufacturer, Virostat, who reported some cross-reactivity of this Ab with other serogroups of L. pneumophila [43,44]. However, this reactivity may not be towards components that allow the capture of L. pneumophila. LPS, or serogroup-specific LPS epitopes, may be the preferred target that allows the capture of these bacteria in the current experiments. When it was formulated, recAb was chosen to detect the LPS of ssp 1, and indeed, it was found highly specific for human isolates of serogroup 1 of L. pneumophila and did not detect an environmental serogroup 1, nor a serogroup 6 strain [22].
For further evaluation of specificity, mixtures of L. pneumophila at a concentration of 10 7 CFU/mL were added together with competitor bacteria to evaluate the interference of other bacteria present in the solution with the adhesion of L. pneumophila to the surface of the wells as follows: E. coli; S. aureus; L. pneumophila sg5 at a concentration of 10 7 CFU/mL (ratio 1:1) and B. subtilis; P. aeruginosa at a concentration of 10 6 CFU/mL (ratio 10:1). Both sets of primers were used on all sample wells to evaluate the total number of bacteria captured on the surface vs. the number of Legionella. The results indicated that the presence of other bacteria did not interfere significantly with the attachment of L. pneumophila to the surface ( Figure 5A,B, black bars).  It was observed that other bacteria did not adhere to the surface in the presence of attached L. pneumophila, as there was no increase in the 16S rDNA (grey columns) compared to the MIP (black columns) when other bacteria were added. The highest rate of nonspecific attachment in wells not coated with Ab was observed with P. aeruginosa as follows: 1.2 × 10 4 CFU/mL ( Figure 5C), but this high non-specific attachment did not affect capture levels of L. pneumophila with Ab even when 16S primers and probe were used for evaluation ( Figure 5A,B). This observation was also noted with all other bacteria mixtures. This result suggested that the Ab-L. pneumophila complex absorbed to the surface had acted as a blocking agent, preventing further binding of the non-target bacteria.
Considering specificity, both recAb and pAb showed excellent results when exposed to a variety of bacteria, making them both adequate choices for biosensor use.

Evaluation of Sensitivity and Reproducibility of Polyclonal and Recombinant Antibodies
From the various experiments (Figures 2-5), both pAb and recAb showed comparable sensitivity under the same stringent conditions. pAb showed slightly higher numbers of captured bacteria compared to recAb. A compilation of capture levels and the standard deviation obtained for L. pneumophila captured with each Ab and quantified with 16S rDNA primers (from Figure 5) is presented in Figure 6.
Biosensors 2022, 12, x FOR PEER REVIEW 11 of 17 It was observed that other bacteria did not adhere to the surface in the presence of attached L. pneumophila, as there was no increase in the 16S rDNA (grey columns) compared to the MIP (black columns) when other bacteria were added. The highest rate of non-specific attachment in wells not coated with Ab was observed with P. aeruginosa as follows: 1.2 × 10 4 CFU/mL ( Figure 5C), but this high non-specific attachment did not affect capture levels of L. pneumophila with Ab even when 16S primers and probe were used for evaluation ( Figure 5A,B). This observation was also noted with all other bacteria mixtures. This result suggested that the Ab-L. pneumophila complex absorbed to the surface had acted as a blocking agent, preventing further binding of the non-target bacteria.
Considering specificity, both recAb and pAb showed excellent results when exposed to a variety of bacteria, making them both adequate choices for biosensor use.

Evaluation of Sensitivity and Reproducibility of Polyclonal and Recombinant Antibodies
From the various experiments (Figures 2-5), both pAb and recAb showed comparable sensitivity under the same stringent conditions. pAb showed slightly higher numbers of captured bacteria compared to recAb. A compilation of capture levels and the standard deviation obtained for L. pneumophila captured with each Ab and quantified with 16S rDNA primers (from Figure 5) is presented in Figure 6. These data indicate that pAb captured 4.32 × 10 5 ± 2.72 × 10 5 CFU/mL of live L. pneumophila, whereas recAb captured 2.99 × 10 5 ± 1.64 × 10 5 CFU/mL.
A slightly lower (70%) capture efficiency was observed for recAb compared to pAb; however, a slightly lower (60%) standard deviation was also observed for recAb compared to pAb, indicating that recAb captured L. pneumophila more consistently and more reproducibly than pAb. Several factors could contribute to these differences. RecAb is directed towards a single epitope of the L. pneumophila ssp1 LPS, whereas pAb is probably composed of antibodies directed towards multiple epitopes of the LPS and perhaps other proteins as well. These other epitopes are not necessary for efficient capture but might Figure 6. Compilation of number of L. pneumophila captured in wells coated with pAb (left) and recAb (right) exposed to 10 7 CFU/mL L. pneumophila from Figure 5. Data were quantified with qPCR by employing 16S rDNA universal primers. Error bars are shown to indicate standard deviation.
A slightly lower (70%) capture efficiency was observed for recAb compared to pAb; however, a slightly lower (60%) standard deviation was also observed for recAb compared to pAb, indicating that recAb captured L. pneumophila more consistently and more reproducibly than pAb. Several factors could contribute to these differences. RecAb is directed towards a single epitope of the L. pneumophila ssp 1 LPS, whereas pAb is probably composed of antibodies directed towards multiple epitopes of the LPS and perhaps other proteins as well. These other epitopes are not necessary for efficient capture but might contribute slightly to the strength of attachment to the target. On the other hand, reaction with other epitopes did not result in the capture of serogroup 5 L. pneumophila by pAb, perhaps because they are not as solidly anchored in the cell wall or membrane, or not as exposed as the LPS epitope targeted by recAb. It is interesting to note that recAb provided efficient capture, demonstrating that single epitopes of LPS can be a useful target for capturing bacteria and can be employed in biosensors.
It is to be expected that there will be variation between batches of pAb since they are produced in rabbits or other mammals where individual variation is observed. Since the production of a recAb does not require synthesis by a living vertebrate organism, it should be highly reproducible and provide identical specificity. The increased variation of results that we observed with pAb could not be attributed to batch-to-batch dissimilarity as we employed only a single batch for all of our tests. We hypothesize that it might be due to variation associated with the multiple epitopes targeted. We could further hypothesise that the reproducibility of pAb would have been even lower with multiple batches, considering reports in the literature that there are batch-to-batch differences from one animal to another for pAb [45].
It is generally considered that polyclonal antibodies will have higher apparent binding capacities than monoclonal or recombinant antibodies due to the variety of epitopes targeted compared with the single epitope target of a monoclonal or recombinant antibody [23][24][25]. The recAb that we employed was almost as efficient as pAb, so it is no doubt directed towards a dominant epitope that allows efficient capture. The increased reproducibility that we observed with recAb is no doubt a considerable advantage over pAb for use in biosensors, especially considering that its capture efficiency was almost as high as that of pAb. In the future, the use of multiclonal antibodies, a mixture of monoclonal recombinant antibodies with a completely defined composition, further promises to provide detection reagents that combine the advantages of both recombinant and polyclonal antibodies [45].

Conclusions
With the ubiquity of L. pneumophila in water sources, the development of an effective biosensor is imperative to improve prospective and preventive action toward outbreaks. We report two important steps towards attaining this objective. A new recombinant antibody targeting the LPS of L. pneumophila spp1 has been developed [28], which we have evaluated in the development of biosensors for the capture of whole L. pneumophila. We have also developed a stringent washing protocol that maintains the viability of L. pneumophila. Polyclonal and recombinant antibodies have been shown to specifically capture large targets, such as whole bacteria, and this opens the door to multiple biosensor approaches where any of the components of the bacteria can be measured for detection or characterisation, such as we have used with PCR detection of bacterial DNA sequences.
Through the advancement of biotechnology, reproducible production processes have been developed to manufacture recAb in order to forgo the use of animal and pAb production with their inherent variation and animal welfare concerns. Although they target only single epitopes, we have demonstrated that recAb are capable of capturing intact target bacteria. The capture of L. pneumophila at different concentrations by recAb and pAb in different environments showed comparable sensitivity and specificity. However, the lower standard deviations, indicating a more reproducible capture, favour recAb, even compared with a unique batch of pAb. For biosensors, precision is crucial in order to obtain constant, reproducible, and stable results for repeated tests.
As these results are in favour of the use of recombinant antibodies under pristine laboratory conditions, it is still necessary to confirm these results with cooling tower water or other environmental water samples using surfaces of different biosensors, such as those based on gold surfaces or on digital photocorrosion of GaAs/AlGaAs nanoheterostructures. After 72 h of culture, Legionella entered the stationary phase, and the medium started browning due to the production of homogentisic acid (HGA) [30]. HGA is produced by Legionella on quorum sensing at high bacterial concentrations and is used to inhibit the growth of other bacterial species. However, in aerobic environments, HGA is oxidised and polymerised into HGA-melanine, causing a browning of the surrounding environment. The browning of the medium is thus a visual indicator that the culture has entered the stationary phase and should not be used for experiments where exponential phase growth is required. Using yeast extract as a nutrient source and at an optimal pH of 6.8 for Legionella, the cultures became more alkaline with growth, unlike most other bacteria, which usually acidify the medium [29]. After 5 days in the stationary phase, the pH did not decrease further, as Legionella was still intact and viable despite the lack of nutrients. If Legionella had died and lysed, the cytosol content would have been liberated and this would probably have changed the pH. After the stationary phase, it would be expected that Legionella enters a latency phase where the bacteria stop dividing and move towards To investigate the effect of different detergents used in washing solutions on the viability of L. pneumophila and to ensure that there was no lysis of the target bacteria during the washing process, different detergent solutions containing Tween-20, Triton X-100 or SDS were tested at different concentrations. For SDS and Triton, 0.02; 0.04; 0.06; 0.08; 0.1; 0.2; 2 and 4% solutions in PBS were prepared, whereas for Tween, the tested concentrations were: 0.1; 0.2; 0.5; 0.1; 1.5; 2 and 4%. A volume of 200 µL of each detergent solution was mixed with 200 µL of L. pneumophila suspension at 10 3 CFU/mL was prepared in PBS from an exponential L.ER broth. After 5 min of exposure, 100 µL of the bacteria-detergent solution was spread on each of three BCYE agar plates. Two control tests were also carried out: (1) a solution of PBS without detergent was used as a positive control, and (2) a bacterial suspension in 1% bleach was used as a negative control. After 72-h of incubation, colonies were counted, and the results were normalised with respect to the positive control.
Detergent was added to washing solutions to ensure an efficient washing of the plates and to avoid the non-specific attachment of bacteria. To ensure that bacteria had not been degraded by detergents and that only bacterial outer membrane shells were still bound to the surface by Ab, we evaluated the toxicity of detergents, assuming that if the bacteria were still viable, they would be intact, whereas if dead, they might have ruptured. Exposing Legionella to different detergents showed that 0.05% and even 0.1% Tween-20 did not affect its viability, as shown in Figure A2.
was mixed with 200 µ L of L. pneumophila suspension at 10 3 CFU/mL was prepared in PBS from an exponential L.ER broth. After 5 min of exposure, 100 µ L of the bacteria-detergent solution was spread on each of three BCYE agar plates. Two control tests were also carried out: (1) a solution of PBS without detergent was used as a positive control, and (2) a bacterial suspension in 1% bleach was used as a negative control. After 72-h of incubation, colonies were counted, and the results were normalised with respect to the positive control.
Detergent was added to washing solutions to ensure an efficient washing of the plates and to avoid the non-specific attachment of bacteria. To ensure that bacteria had not been degraded by detergents and that only bacterial outer membrane shells were still bound to the surface by Ab, we evaluated the toxicity of detergents, assuming that if the bacteria were still viable, they would be intact, whereas if dead, they might have ruptured. Exposing Legionella to different detergents showed that 0.05% and even 0.1% Tween-20 did not affect its viability, as shown in Figure A2. This result demonstrates that 0.05% Tween-20 is not toxic at these concentrations and could be used to attempt to reduce the non-specific adhesion of bacteria. However, the use of SDS and triton X-100 at concentrations ranging from 0.01% to 2% resulted in the complete inactivation of L. pneumophila (not shown). The observation of more than 100% viability for between 0.05 and 0.1% concentrations of Tween-20 could be due to the reduction of bacterial aggregation due to mixing and the appearance of more individual bacterial colonies on the plates.