This research demonstrates that the TRACA system may be used to capture plasmids from bacteria in the oral metagenome. The presence of plasmids that contain antibiotic resistance genes in the absence of antibiotic selection pressure suggests that it is possible that there is another advantage that these plasmids are offering their bacterial hosts in addition to antibiotic re- sistance. It is likely that such an advantage is conferred by one of the ORFs identified. These results demonstrate that there is plasmid involvement in the maintenance of a reservoir of antibiotic resistance genes in commensal bacteria. The plasmids captured from saliva samples during this study had a greater homology to the plasmids that were captured via TRACA from the plaque from periodontal patients than those that were captured from the gut metagenome, this is not surprising due to the proximal relationship between where the samples were gathered from.
The plasmid group pTRACA42 appears to be common within the oral envi- ronment, to date this group of plasmids has not been observed in any other
setting. The pTRACA42 group appears to be both common in healthy and infected individuals, to date the pTRACA42 group have not been shown to harbour any genes that confer resistance to antibiotics.
Chapter 4
Bacteria Isolated from Swabs of
Infected Wounds
4.1
Antibiotic Resistance in a Clinical Set-
ting
4.1.1
Rationale
This section of research investigates the presence of antibiotic resistance genes and genes that are associated with mobile genetic elements in samples that have been obtained from within a clinical setting. The focus of this study is on tetracycline, erythromycin and vancomycin resistance. Additional work was carried out on methicillin regarding the isolates that were identified as Staphylococcus aureus.
Transposons are modular, the modules that they contain can be interchanged and replaced with other modules, therefore as the modules within the trans- poson are acquired and/or lost, they can acquire resistance to additional antibiotics, potentially resulting in multiple resistance genes being located
upon one mobile element.
4.1.2
Bacteria in Wounds
Bacteria that compose the skin flora often colonise wounds. Limitations in what bacterial species are culturable in the laboratory have historically led to a misrepresentation of what species are present on the skin flora with S. aureus and S. epidermis thought to be the most dominant species due to their being relatively easy to culture. Analysis of 16S rRNA has revealed that the diversity of bacterial species that inhabit the skin is greater than originally thought (Cogen et al., 2008; Grice et al., 2008). Wounds support communities of both anaerobic and aerobic bacteria, these bacteria may ini- tiate infections or establish a less invasive relationship with the host (Cooper et al., 2009). The development of infection in wounds typically results from either an increased host susceptibility or an increase in bacterial load or vir- ulence (Cooper, 2005). Bacteria that frequently reside within the skin flora and their pathogenicity are shown in Table 4.1.
Should a break in the skin occur forming a wound, the bacteria belonging to the skin flora may enter the wound. The quantity of bacteria that enter the wound impacts upon infection and the healing rate of the wound, the effect of the microbial load on wound healing was first identified when healing in decubitus ulcers only progressed when the bacterial load was less than 106
CFU/ml of wound fluid (Bendy and Landman, 1964). As well as microbial load wound healing is also affected by the differences between the specific species present (Bowler et al., 2001). Species that have been reported to have the most detrimental effect on wound healing are S. aureus, P. aeuriginosa and β-haemolytic streptococci (Bowler et al., 2001; Danielsen et al., 1998).
Table 4.1: Frequently studied bacteria that reside as part of the skin flora and their pathogenicity.
Species Pathogenicity
Staphylococcus aureus Typically pathogenic Staphylococcus epidermis Occasionally pathogenic Staphylococcus warneri Occasionally pathogenic Streptococcus mitis Occasionally pathogenic Streptococcus pyogenes Typically pathogenic Propionibacterium acnes Occasionally pathogenic Pseudomonas aeuriginosa Occasionally pathogenic Acdenitobacter johnsonii Occasionally pathogenic Corynebacterium spp. Occasionally pathogenic
4.1.2.0.1 Acute vs Chronic Wounds The bacterial species that colonise wounds tends to differ based on the length of time that the wound has been present, the early colonisers tend to be Gram positive bacteria that are able to grow in aerobic conditions such as Staphylococcus and Streptococcus. As time progresses anaerobic bacteria also colonise the wound, these are typi- cally acquired from exogenous sources such as bathwater (Dow et al., 1999). Chronic wounds, such as ulcers provide a polymicrobial environment where the exchange of genetic information is able to take place between bacteria. This creates conditions whereby the transfer of a resistance gene to a new species is more favourable, for example the first two cases of S. aureus resis- tant to vancomycin were isolated from chronic wound patients (Howell-Jones et al., 2005). A recent study investigating the bacteria that are found in wounds observed that the most common species present is S. aureus, fol-
lowed by P. aeruginosa (Wong et al., 2015).
4.1.3
Overview of Study
136 bacterial isolates have been received from different anatomical sites (Ta- ble 4.4 and 4.5), the isolates that were suspected to belong to the strepto- coccal or staphylococcal species were initially screened for resistance to the antibiotics tetracycline and erythromycin at concentrations of 4µg/ml, those isolates that were resistant to at least one of these antibiotics were taken forward for further analysis. These isolates are presented in Table 4.6. The isolates that were suspected of being enterococci were screened for resistance to the antibiotic vancomycin at a concentration of 4µg/ml. Vancomycin re- sistant isolates were taken forward for further analysis, these isolates are also presented in Table 4.6.