Tratándose del segundo y del tercer ejercicio, además de los requisitos previstos en las fracciones anteriores, que el porcentaje de las cuentas y documentos por cobrar derivados

Marcellin et al. (2010) reported that Phosphomycin concentrations as low as 0.5 ng/mL were lethal to the S. zooepidemicus strain of interest in their study. This indicates extremely potent antimicrobial activity which is more typically in the µg/mL range. Phosphomycin MICs for other gram positive species such as methicillin susceptible S. aureus strains were found to range 0.5-32 µg/mL with methicillin resistant strains being as high as 128 µg/mL and in the same study overall from the 590 common bacterial species tested the Phosphomyin MICs varied 0.25-512 µg/mL (Lu et al., 2011). Due to the variation in MIC’s observed between strains the effects of Phosphomycin on growth of the strains of interest in this study were investigated as it was postulated that they may not be as low as claimed by Marcellin et al. (2010). Phosphomycin is transported into the cell via two uptake systems, the first is the L-α-glycerophosphate uptake system and the second is the glucose-6-phosphate (G6P) uptake system (Zoeiby,

Sanschagrin and Levesque, 2003). Due to this, when testing the

susceptibility of Phosphomycin the media is often supplemented with G6P in order to enhance the antimicrobial activity (Lu et al., 2011;

Michalopoulos, Livaditis and Gougoutas, 2011; Falagas et al., 2016). However for the purpose of this study it was not necessary to do this as the ultimate aim was to investigate the possibility of introducing

Phosphomycin into the current fermentation process for HA production in our strains of interest. From the results it can be concluded that the

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visually observed MIC for all four strains is 15 µg/mL in BHI media, a large contrast to the reported lethal concentration of 0.5 ng/mL observed for S. zooepidemicus ATCC 35246. No EUCAST breakpoint data for

Streptococcus groups A, B, C and G are available as these organisms are

not recommended targets for Phosphomycin treatment, however it has been claimed MIC values for Phosphomycin of ≤ 64 µg/mL are considered susceptible (Michalopoulos, Livaditis and Gougoutas, 2011). Accepting this criteria it can be concluded that all four strains of interest are

susceptible to Phosphomycin in BHI media. Growth curves are not a EUCAST requirement for susceptibility testing however they were performed in order to provide a range of concentrations which did not inhibit growth for the purpose of this project. Due to the previously reported low lethal concentration of Phosphomycin on S. zooepidemicus MIC assays were originally set up with a maximum concentration of 0.5 µg/mL Phosphomycin however no effect on growth was observed so the maximum concentration was increased to 1, 3, 5 and 10 µg/mL before arriving at the concentration of 15 µg/mL (Data not shown). In addition, MIC’s of Ampicillin were determined for each strain as a control.

Breakpoint data from EUCAST declare that MIC’s for Ampicillin in group A, B, C and G Streptococcus of ≤ 0.25 µg/mL indicate susceptibility. Inhibition of growth was observed manually at 0.0156 µg/mL for SE40327 and SER17037, 0.0625 µg/mL for SEE20561 and 0.0313 µg/mL for SEZ20727 indicating that all strains were susceptible to Ampicillin in BHI (Data not shown). As previously mentioned the aim was to establish a range of below inhibitory concentrations of antibiotic to investigate further.

4.4.2. Capsule production in the presence of Phosphomycin

It has previously been reported that N-acetylglucosamine (NAG) is the limiting precursor with regards to HA molecular weight (Chen et al., 2009; Chen et al., 2014). As Phosphomycin specifically targets MurA which directly competes with HasA for NAG, it was hypothesised that

supplementing the media with a non-inhibitory concentration of the antibiotic would result in an increase in HA production and or molecular weight as it would increase NAG availability. The highest concentration of Phosphomycin which did not result in visible inhibition of growth in the

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strains was 7.5 µg/mL and so this was chosen as a concentration for analysis of HA production with Phosphomycin. With regards to

SER17037 the large error bars observed for 7.5 µg/mL were considered an indication of variation between biological replicates, possibly due to the MIC being nearer to 7.5 than 15 µg/mL, however as turbidity was

manually observed in all replicates this concentration was not considered inhibitory by EUCAST guidelines and so was tested in SER17037

regardless of the variation observed in the growth curve. Since an aim was to assess the highest Phosphomycin concentration possible without

inflicting a marked change to the growth of the strains, a concentration of 2 µg/mL was selected in addition. This was chosen after analysing the growth curve data the concentration of 1.875 µg/mL was considered to be the highest concentration reached before the curves differed markedly compared to the control curve. Bacteria cultures were set up and grown as previously described (2.2.5) in order to ensure results would be comparable to non-antibiotic supplemented growth.

From the results of the HPLC analyses this was not the case however with both capsule producing strains displaying significantly reduced HA

concentrations compared to when cultured in BHI media alone. In addition to the reduced concentration, although the HPLC was not set up to measure molecular weight the slightly increased retention time may indicate that the HA molecular weight is also reduced compared to that produced in BHI media alone.

The reduced HA production in the presence of Phosphomycin was an unexpected result. It is possible that this observation is the result of the emergence of a tolerance strategy in these strains. Mechanisms of resistance to Phosphomycin include mutational changes in either MurA which lowers the affinity to the antibiotic or in the uptake systems which reduces entry of Phosphomycin into the bacterial cells. Additionally, increasing MurA expression is another means of developing resistance (Falagas et al., 2016; Silver, 2017). It was previously reported that over expression of either of the copies of murA lead to a reduction in HA molecular weight (Marcellin, Chen and Nielsen, 2010) and so it is possible

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that to counteract the presence of Phosphomycin in the media, expression of murA is increased resulting in reduced HA production.