Conclusiones y Recomendaciones

Plasmids are able to transfer multiple resistance gene cassettes due to efficient capture by integrons. Integrons have evolved to capture and express gene cassettes. They were first discovered in clinical isolates but have since been found in a variety of environments including wastewater treatment plants, fish farms, on-farm biopurification systems, soil, manured soil, pig slurry, lakes poultry litters, estuaries and reed beds (Marathe et al., 2013, Agerso and Petersen, 2007, Dealtry et al., 2014, Ma et al., 2013, Tennstedt et al., 2005, Ghosh et al., 2009, Holmes et al., 2003, Byrne- Bailey et al., 2009, Tennstedt et al., 2003, Zhang et al., 2011, Du et al., 2014, Flach et al., 2015, Byrne-Bailey et al., 2011, Agerso and Sandvang, 2005, Heuer et al., 2012, Gaze et al., 2005, Nandi, 2004, Lu et al., 2015, L'Abee-Lund and Sorum, 2001, Ferreira da Silva et al., 2007). Integron structure allows for efficient insertion and expression of a gene cassette in to a genome without disruption of the genome making them highly favourable both in clinical and environmental conditions.

Integrons were first identified by Stokes and Hall in 1989 over 30 years after the first Japanese studies investigating plasmid-mediated transferable antibiotic resistance in 1950’s (Stokes and Hall, 1989, Ochiai, 1959). They consist of three key components: the integrase gene (intI1), a recombination site (attI) and a promoter (Pc) (Figure 1.5). All integrons will possess these three elements to enable the capture and expression of gene cassettes with minimal disruption to the genome (Labbate et al., 2009). They are able to capture genes from diverse backgrounds and hence act as genomic

diversity “hotspots” (Boucher et al., 2007, Hall and Collis, 1995). Over 130 different antibiotic resistance gene cassettes have been found on integrons providing resistance to most antibiotics used in the treatment of Gram-negative bacteria (Partridge et al., 2009, Centron and Roy, 2002, Falbo et al., 1999, Koeleman et al., 2001, L'Abee-Lund and Sorum, 2001, Maguire et al., 2001, Nordmann and Poirel, 2002, van Belkum et al., 2001). There are five known classes of integrons, class 1 through to 5 (Cambray et al., 2010). Classes 1, 2 and 3 are the most readily detected integrons with classes 4 and 5 having only been detected once (Hochhut et al., 2001). They are classified based on sequence homology of the integrase protein with 40- 58% identity (Mazel, 2006).

Figure 1.5 Structure of Class 1 integrons, capture by transposons and plasmid insertion.

The most commonly detected integron in both the clinic and environment is the class 1 integron (Deng et al., 2015). The suggestion that class 1 integrons may act as a marker for antibiotic resistance has been considered for over a decade, with the first

suggestions that antibiotic resistance in Enterobacteriaceae is strongly related to

integrons in 2003 (Leverstein-van Hall et al., 2003). The study conducted by Leverstein-van Hall et al. found the multidrug-resistance phenotype was attributed

to the presence of integrons within strains and that 100 % of strains possessing integrons expressed resistance to at least 1 antibiotic tested.

Recent work into class 1 integrons as a proxy for ARG has been carried out to investigate how efficient a marker it is. Amos et al. proposed a model to predict 3GC resistance prevalence based upon class 1 integron prevalence combined with environmental metadata (Amos et al., 2015). Furthermore, Gillings et al. suggested the class 1 integron-integrase gene may act as a marker for anthropogenic pollution based on the observation of high abundance of this gene in polluted environments in addition to many pathogenic and commensal bacteria in the humans and animals (Gillings et al., 2015, Goldstein et al., 2001, Stokes and Gillings, 2011, Amos et al., 2015, Berglund et al., 2015, Marathe et al., 2013, Gaze et al., 2011).

They also represent potential monitoring mechanisms due to their assembly, which has been influenced by human activities resulting in the accumulation of BRG and ARG on the same genetic element (Gillings et al., 2008). If the integrase gene is to be used as a marker for antibiotic resistance, care must be taken to ensure all integrons are detected. A study conducted by Dawes et al. found that of 79 isolates known to possess class 1 integrons, only 31 of them could be detected using standard PCR primers which typically amplify the 3’ region (Dawes et al., 2010).

Class 1 integrons are commonly associated with Gram negatives, but they have also been detected in Gram-positive bacteria. In 1998 a class 1 integron was found in Corynebacterium glutamicum. Interestingly this integron showed higher expression in this host than in E. coli suggesting an Gram-positives may be important reservoirs of ARG-carrying integrons (Xu et al., 2011). Other Gram-positive bacteria, in which

class 1 integrons have been found include Staphylococcus, Corynebacterium, and

Aerococcus (Nandi, 2004, Nesvera et al., 1998, Xu et al., 2007, Xu et al., 2008b, Xu et al., 2008a).

Class 1 integrons have been associated with many bacterial families in both commensal and pathogenic bacteria and have been found in a variety of

allowed efficient transfer from the environment in to the human food chain. Consequently this has led to a sub class of class 1 integrons that are found in human- dominated ecosystems (Gillings et al., 2015). This clinically related class 1 integron is characterised by a 3’ conserved region, a truncated qacEΔ1 and a sul1 gene. These components are believed to have arisen through capture of an environmental

betaproteobacterium integron containing qacE biocide resistance gene and

subsequent sul1 sulphonamide resistance gene which led to a truncated qacE which was captured by a Tn402 transposon which targets the res region of plasmids and hence allows the class 1 integron-transposon hybrid to transpose in to a wide range of plasmid and hence become high mobile (Minakhina et al., 1999).