Department of Physics, Chemistry and Biology (IFM)

Staphylococcus aureus is a gram-positive nosocomial pathogen carried by about 25-50% of the healthy population, primarily in the anterior nares (Grundmann et al., 2006, van Belkum2009, Hu et al., 1995, Kluytmans et al., 1997). S. aureus has been a major cause of human infection throughout history and still causes a high percentage of hospital infections. S. aureus is an opportunistic pathogen whereby carriers can be asymptomatic for a long period of time. S. aureus can cause a wide range of diseases in humans, from minor skin infections to wound, bone, joint and implant infections, septicemia, pneumonia, septic arthritis, toxic shock syndrome and other toxicoses, infections of the central nervous system, respiratory and urinary tracts, and bacteremia (DeLeo et al., 2009, Grundmann et al., 2006, Monecke et al., 2011, Moore and Lindsay, 2001, Oliveira et al., 2002). 

The prevalence of MRSA infection has been increasing since it was first discovered in 1961. Several northern European countries have a low level of MRSA infections (<5% of all S. aureus infections, but as low as <1% has been reported) whereas many other countries, including western and southern European countries, have a high proportion of MRSA infection rate (up to > 50% of all S. aureus infections) (Grundmann et al., 2006, Lindsay, 2010, Tiemersma et al., 2004).

Rosdahl and Knudsen (1991) describes a decline in MRSA infections in Denmark in 1984 while Kayser (1975) describes a decline in MRSA infections in Switzerland since 1972. Both also report that the level of MRSA infections have remained low since then. The reason for the decrease in MRSA infections is not entirely clear, but it is accredited to changes in prescription of streptomycin and tetracycline, early detection of resistant strains and taking health precautions (Rosdahl and Knudsen, 1991, Grundmann et al., 2006). Tiemersma et al. (2004) found an increase in MRSA proportions in several European countries 1999-2002 and Chaberny et al. (2005) report that the proportion of nosocomial MRSA infections increased on intensive care units in Germany between 1997 and 2003 from 8% to 30%. In Canada an increase in the rate of MRSA infection has been detected between 1995 and 2003 (Noskin et al., 2007). Also in large hospitals in USA, MRSA infections increased from 4% to 50% between the 1980s and 1990s (Oliveira et al., 2002).

It has been debated whether MRSA infections are more lethal than meticillin susceptible S. aureus (MSSA) infections. Cosgrove et al. (2003) pooled the results from 31 studies and found that the mortality rate of MRSA bacteremia is significantly higher than that of MSSA bacteremia, even after adjusting for comorbidities or severity of illness. 

S. aureus has shown to be highly adaptable and has acquired resistance against all classes of antibiotics by either mutation of an existing bacterial gene or by horizontal transfer of mobile genetic elements (MGE) from another bacterium (Grundmann et al., 2006, Lindsay, 2010, Moore and Lindsay, 2001). Selective advantages such as increased resistance to commonly prescribed antibiotics, adaptations to the host and increased virulence are beneficial traits of MGEs, however, for MGE to spread successfully they cannot cause a fitness cost to the bacterium (Lindsay, 2010). Already 1957, S. aureusresistant against penicillin, streptomycin, tetracycline and erythromycin was detected (Oliveira et al., 2002). Following the introduction of meticillin as a prescription antimicrobial in 1959/60, meticillin resistant S. aureus (MRSA) strains were discovered in 1961, resistant to penicillin, streptomycin, tetracycline, meticillin and occasionally erythromycin (Oliveira et al., 2002, Lindsay, 2010).  The meticillin resistance evolved by acquisition of the mecA gene (Matsuhashi et al., 1986, Cameron et al., 2011) and is clinically the most important acquired trait since it confers resistance to β-lactam antibiotics, the most commonly prescribed group of antibiotics (Grundmann et al., 2006).

Bengal bay

The Bengal Bay clone originates from the Bengal bay area and has been detected in both India and Bangladesh, and also in Malaysia and England but then with links to India and/or Bangladesh (Ellington et al., 2009). The Bengal Bay clone is highly virulent highlighted by its high level of antimicrobial resistance (for being a CA-MRSA) and its geographic spread.

CC398

The clonal complex (CC) 398 is prevalent among livestock-associated MRSA (LA-MRSA) and has been found to cause numerous cases of MRSA in humans (Köck et al., 2013, Cuny et al., 2010). Many of the colonised patients in the study of Köck et al. (2013) had had no contact with farm animals, suggesting that CC398 spreads between humans. This was confirmed by Wulf et al. (2008) which found nosocomial transmission in a MRSA CC398 outbreak. The first finding of CC398 was in 2005 and since then it has predominantly been observed in Belgium, Netherlands and Denmark but seems to be spreading to other countries (Lindsay, 2010). CC398 has been documented to cause human infections such as endocarditis, wound infections and pneumonia (Köck et al., 2010).

EMRSA-15

EMRSA-15 and EMRSA-16 have been frequently occurring in UK hospitals whereby during the 1990s they were responsible for 40% of the HA-MRSA infections and in 2002 they were the dominant type of MRSA in the UK (Knight, 2012, Moore and Lindsay, 2002). EMRSA-15 and EMRSA-16 have a unique accessory genome which might explain why they are experts in surviving, spreading and infecting humans nosocomially (Moore and Lindsay, 2002).

USA300

USA300 is an infectious group of CA-MRSA isolates with similar PFGE patterns causing severe disease mainly in the USA, but they have also disseminated to Europe, South America and Australia (Tenover et al, 2009). USA300 has replaced USA400 as the main clone causing epidemic MRSA infections in the USA due to its enhanced virulence (DeLeo et al., 2009).

Microbial virulence is a property of the microbe dependent on the interaction between the microbe and the host it is colonising. This confers a difficulty in defining the concept of virulence factors, since the microbe is not the only actor, as the host response also plays a part. Casadevall and Pirofski (2009) have developed a “damage-response framework” of microbial pathogenesis that takes the host-microbe interaction into account.  In this framework, a pathogen is “a microbe capable of causing host damage” and virulence is defined as “the relative capacity of a microbe to cause damage in a susceptible host” making a virulence factor “a microbial component that can damage a susceptible host”. This also conveys that the host and its response affects the importance of specific virulence factors, resistance genes and adhesion factors. A response to a virulence factor in one host is not necessarily the same in another host. When trying to assess the influence of virulence factors on a host, it is also important to keep in mind that virulence is not often caused by one virulence factor alone and that damage to a host can be caused both directly and indirectly by the microbe. Furthermore, the host immune responses can neutralise many virulence factors (Casadevall and Pirofski, 2009). It is therefore not straightforward to relate virulence factors to hypothesised virulence in humans using a model organism.

Model organisms play an important role in the study of disease. Even though they cannot model the full natural disease process, they can nevertheless provide approximations of human disease that let us understand disease patterns and determinants (Wiles et al., 2006).

Traditionally, mammalian models have been used for assessing the virulence of microbial pathogens. A typical mammalian model though requires not only educated personnel and substantial ethical concern, but it is also time-consuming and entails considerable costs for animal maintenance. As the innate immune system has shown to have a high similarity in vertebrates and insects (Kavanagh and Reeves, 2003, Ratcliffe, 1985, Salzet, 2001, Kimbrell and Beutler, 2001, García-Lara et al., 2004), and they use similar mechanisms for pathogen killing (García-Lara et al., 2004, Kavanagh and Reeves, 2003, Desbois and Coote, 2010, Scully and Bidochka, 2006), insects are increasingly used to evaluate microbial virulence that affects the innate immune system. Although the insect immune system is not as complex as the mammalian immune system, they share many similarities of the innate immune response (Kavanagh and Reeves, 2003, Salzet, 2001, Scully and Bidochka, 2006) which enables the use of insects as a model of the innate immune system, such as to quantify the virulence of pathogens.

The insect model is not likely to replace the mammalian model for studies of in vivo virulence. It will rather be used as a means to refine the experimentation of mammals and rule out compounds not likely to be efficient in mammals, and by that reduce mammalian experimentation.

The G. mellonella model has earlier been used to determine the virulence and identify novel virulence genes in S. aureus (Desbois and Coote, 2010, Peleg et al., 2009, Gao et al., 2010, Purves et al., 2010). A correlation have been established between the virulence of some microbes in mice and in the Wax Moth larvae Galleria mellonella (Jander et al.,2000, Mylonakis et al., 2005, Chua et al., 2011, Peleg et al., 2009, Salamitou et al., 2000). That demonstrates the potential of the G. mellonella as a model for in vivo evaluation of microbial virulence. In comparison to a mouse model, the G. mellonella model is less time-consuming, less expensive and requires less ethical concern. G. mellonella can also be used in large numbers, are easily injected due to their large size, are commercially available and has unique features for the study of human invasive infection, as they can be adapted to the human physiological temperature of 37°C.