Choosing the right animal model to study device- associated infections

Biofilms and implant-associated infections

3.3 Choosing the right animal model to study device- associated infections

Since the 1960s different experimental infection models have been utilized to under- stand pathogenesis and to find the best drug therapy and prophylaxis for biofilm infections. An animal model for implant- associated infections has to reproduce the common characteristics of human diseases, such as the ease of implant infection without spontaneous healing (Zimmerli, 1999). However, there is no gold standard animal model for device- associated infections and different models using total joint replacement, skeletal implant, and soft tissue implants have been described. Those models use different animal species (mouse, rat, guinea pig, rabbit, cat, dog, chick and sheep) and each of them offers advantages and disadvantages that needs to be taken into consideration. In the following, we discuss what may be taken into account concerning the different animal models, animal species, and examined microbial pathogen in implant- associated infections.

3.3.1 Considerations of the microbial pathogen to be tested The microbial species should be chosen to reflect what is seen clinically. Common pathogens in implant- associated infection models are Staphylococcus aureus , Staphylococcus epidermidis , Streptococcus pyogenes , Escherichia coli and Pseudomonas aeruginosa . In addition to the choice of bacterial species, choosing the appropriate pathogenic strain is one of the most crucial steps when investigat- ing implant- associated infections because biofilm formation is strain dependent (Christensen et al. , 2007; O’Neill et al. , 2007; Smeltzer et al. , 1997). For example Smeltzer and colleagues found that an inoculum of 2 × 10 ³ CFU of S. aureus strain UAMS–1 was able to create an infection in devascularized bone whereas the heavily encapsulated S. aureus strain Smith caused reduced rates of infection despite using a higher inoculum (Smeltzer et al. , 1997). Therefore, the capacity of a bacterial spe- cies to form a biofilm has to be carefully investigated before using it for an implant- associated infection model. Alternatively, a clinical strain from a proven biofilm infection can be chosen. However, most published studies have used standard strains (e.g American Type Culture Collection, ATCC) because most of these strains are well characterized with results allowing direct comparison to other studies using the same strain.

After choosing the appropriate bacterial species and strain, several factors in the animal model, such as the timeline of a spontaneous infection, lack of clearance of the infection, lethality or minimal dose of injection, or ability to cause haemato genous or local infection of an implant, must be considered (Zimmerli et al. , 1982). An important question is how many bacteria are needed to cause an implant- associated infection. The inoculum of bacterial species varies between 10 ² to 10 ⁸ CFU depend- ent on the animal model and bacterial species (Zimmerli et al. , 1982; Blomgren, 1981; An and Friedman, 1998). In order to standardize the model, the ability to cause infection, the severity and duration of chronic infection has to be determined

50 Biomaterials and Medical Device-associated Infections

with each new strain. Bacterial challenge to produce an implant- associated infection can be performed by either (1) pre- colonization of bacteria on an implant in vitro before implantation, (2) direct inoculation of the bacterial suspension at the implant site, or (3) infection of the bacteria into the bloodstream (Saleh Mghir et al. , 1998;

An and Friedman, 1998). For haematogenous induction of an implant infection with S. aureus , the best time for bacterial challenge is five days postoperatively as shown in a rabbit tibia bone plate model (Johansson et al. , 1999). A challenge at the day of surgery can be overwhelming and cause high mortality. Also when given the bacterial inoculum locally, the quantity of bacteria given should be carefully determined in order not to overwhelm the host immune system. For this purpose it is usually recommended to determine the 50% lethal dose (LD ₅₀ ) for the strain used (Frimodt-Moller et al. , 1999).

3.3.2 Animal model selection

An optimal animal model is characterized by having a reasonable expense, high infection success, ease of infection initiation, low mortality rate of the animal species, and a close approximation to human pathological properties and immunological pat- terns of the disease. But there is no gold standard animal model in implant- associated infections. Depending on the type of the implant, the pathogenesis (haematogenous, intraoperative or postoperative acquired), antibiotic resistance profile of the patho- gen, and the goal of the study (testing prophylaxis, short or long antibiotic treatment, diagnostic procedures) the animal model has to be carefully selected. In the follow- ing text and in Table 3.1 , we describe the most common implant- associated infection models and distinguish between implant models localized in bone, soft- tissue, perito- neal, bloodstream or urinary tract.

3.3.2.1 Subcutaneous models

One of the first subcutaneous models to study the pathogenesis of a foreign body infec- tion was developed by Zimmerli and colleagues. They utilized implanted tissue cages (perforated Teflon cylinders) placed bilaterally in the flank of guinea pigs (Zimmerli et al. , 1982). Following blunt dissection of the subcutaneous compartment, the tissue cages were sterilely implanted, and the host was allowed to heal for two weeks before actual or mock infection by bacterial injection. These tissue cages allowed the estab- lishment of infection with as few as 100 S. aureus bacteria while those guinea pigs without the cages fail to develop abscesses with an injection of as much as 10 ⁸ bacteria.

The imported-associated infection did not spread to any other organs (kidney, lung, liver, spleen) mimicking the localized nature of certain implant infections in humans (Zimmerli, 1999). The tissue cages create a fluid filled dead space allowing later aspi- ration for microbial counts, assays on infiltrating immune cells, and pharmacokinetic assays. Using this model, Zimmerli and co- workers demonstrated decreased opsonisa- tion of S. aureus after approximately 20 hours of infection (Zimmerli et al. , 1982).

Later modifications of this model expanded the range of animals used and included mice (Dayer et al. , 1987), calves (Bengtsson et al. , 1991, 1992), ponies (Voermans

In vivo infection studies 51

Table 3.1

Common implant- associated infection models – an overview

Model Reference Animal species Pathogen Notes Subcutaneous models Tissue cage implant Zimmerli et al. , 1982 Guinea pig S. aureus Wood 46 Chronic infection of implant dead space allowing tests on tissue cage fluid Widmer et al. , 1991 Guinea pig Salmonella dublin Widmer et al. , 1991 Guinea pig E. coli Kristian et al. , 2003 Mouse (C57BL/6, wildtype, TLR2 -/-) S. aureus , 113 and isogenic dlt(-) strain Tissue cages containing 8-sinter glass beads Fluckiger et al. , 2005 Mouse S. epidermidis 1457, S. aureus RN6390, S. aureus Newman Tissue cages containing catheter pieces Voermans et al. , 2006 Pony S. aureus Tissue cages implanted subcutaneously in the pony’s neck Nair et al. , 2008 Rat (Sprague-Dawley) Actinomyces radicidentis Furustrand Tafin et al. , 2011 Guinea pig (male albino) Enterococcus faecalis Furustrand et al. , 2012 Guinea pig Propionibacterium acnes Subcutaneous catheter model Christensen et al. , 2007 Christensen et al. , 1983 Mouse (Swiss albino) S. epidermidis Sterile plastic catheters implanted into mouse flanks, allowed to heal, and infected by injection Roehrborn et al. , 1995 Mouse (CF–1) S. aureus Van Wijngaerden et al. , 1999 Rat S. aureus, S. epidermidis Polyurethane catheter implants infected with a low dose of bacteria immediately prior to implantation Ricicova et al. , 2010 Rat Candida albicans Pre- colonized polyurethane catheters implanted into immunosuppressed hosts (Continued overleaf )

52 Biomaterials and Medical Device-associated Infections

Table 3.1

Continued

Central venous catheter models Central venous catheter Rupp et al. , 1999b Rat S. epidermidis Silastic lumen- within-lumen catheter was surgically placed in the right jugular vein and tunneled subcutaneously to the rat’s shoulder where is held immobilized by a catheter restraint jacket Rupp et al. , 2001 Rat E. faecium Cirioni et al. , 2006 Rat S. aureus Kadurugamuwa et al. , 2003 Mouse S. aureus, P. aeruginosa Precolonized catheter Schinabeck et al. , 2004 Rabbit C. albicans Fernandez-Hidalgo et al. , 2010 Rabbit S. aureus Totally implantable venous access port model

Chauhan et al. , 2012a Rat E. coli, S. aureus, S. epidermidis, P. aeruginosa , Urinary Tract Models Zinc disc vesicular implant

Satoh et al. , 1984 Rat Proteus mirabilis Zinc discs are implanted in the animal’s bladed and infected transvesicularly with P. mirabilis Rabbit catheter model Morck et al. , 1993 Rabbit E. coli Closed urinary catheter drainage system Glass bead renal infection model Haraoka et al. , 1995 Rat (Sprague-Dawley) E. coli Pre- colonized glass beads are implanted in the bladder followed by clamping of the urethra

Model Reference Animal species Pathogen Notes

In vivo infection studies 53

Non-surgical urinary catheter model

Kurosaka et al. , 2001 Rat P. aeruginosa Spiral polyethylene tube placed transurethrally into the bladder followed by inoculation with bacteria Kadurugamuwa et al. , 2005 Mouse P. aeruginosa, P. mirabilis Urinary stent model Fung et al. , 2003 Rabbit P. aeruginosa Curled portion of double pig- tailed ureteric stents were colonized with bacteria then inserted transuretherally into the bladder of the animals Cirioni et al. , 2007 Rat S. aureus Stents were implanted surgically into the bladder of rats Orthopaedic device models Femoral implant model Petty et al. , 1985 Dog Staphylococcus epidermidis, Staphylococcus aureus , and Escherichia coli

Instilliation of bacteria in the femoral canal followed by insertion of an implant (different materials) Tibial pin model Li et al. , 2008 Mouse S. aureus Xen29 Pre- colonized stainless steel insect pin inserted transcortically through the tibia Prabhakara et al. , 2011a, 2011b Mouse S. aureus M2 Crane et al. , 2009 Mouse Acinetobacter baumanii Femur K-wire model Niska et al. , 2012a Mouse S. aureus K-wire implant inserted into the femur intramedullary cavity with projection into the knee joint space, infectious inoculum applied to knee joint space Infected tibial non-union Alt et al. , 2011 Rat S. aureus Intramedullary device used to stabilize osteotomy site and directly inoculated with bacteria (Continued overleaf )

54 Biomaterials and Medical Device-associated Infections

External vertebral fixation

Holt et al. , 2011 Rat Environmental bacteria Vetebrae exposed and a 1.8 mm hole drilled through the vertebrae, into which a 2 mm titanium pin is screwed until flush with the surface of the vertebrae Intraperitoneal models Silastic subdermal catheter implant model

Buret et al. , 1991 Rabbit P. aeruginosa Precolonized catheter material implanted into rabbit peritoneal cavity and infection sustained past day 42 Gallimore et al. , 1988 Mouse S. epidermidis Carsenti-Etesse et al. , 1992b Mouse (Swiss albino) S. aureus, S. epidermidis Christensen et al. , 2007 Mouse (BALB/c and NMRI) P. aeruginosa and quorum- sensing mutant strain

Table 3.1

Continued

Model Reference Animal species Pathogen Notes

In vivo infection studies 55

et al. , 2006), rats (Schaad et al. , 1994), rabbits (Bamberger et al. , 1995) and dogs (Gruet et al. , 1997). In addition to the use of the model to study infections with S. aureus (John et al. , 2009; Trampuz et al. , 2007; Baldoni et al. , 2009), it has been used to study infec- tions with Actinomyces radicidentis (Nair et al. , 2008), S. epidermidis (Widmer et al. , 1990b), E. coli (Widmer et al. , 1991), Salmonella dublin (Widmer et al. , 1990a), P.

acnes (Furustrand et al. , 2012) and E. faecalis (Furustrand Tafin et al. , 2011). The tis- sue cage model imitates the clinical situation of extravascular devices such as cardiac or neurosurgical devices batteries, subcutaneous catheters, shunts or breast implants. It allows the testing of different aspects of pathogenesis of implant- associated infections, including haematogenous infections of implants, biocompatibility of different bioma- terial and efficacy of prophylaxis and treatment (Zimmerli, 1993). Using this model, it was demonstrated that a significant cure rate could be provided against S. aureus infection when animals were given rifampin in combination with a second antibiotic to prevent emergence of rifampin resistance (Baldoni et al. , 2009; John et al. , 2009;

Zimmerli et al. , 1994). This success also translated into clinical success when tested, albeit in clinical implant- associated bone infections (Zimmerli et al. , 1998).

3.3.2.2 Orthopedic device models

Animal models of osteomyelitis frequently use sclerosing agents such as sodium morrhuate or the creation of devascularized bone to allow the establishment of a chronic infection (Smeltzer et al. , 1997; Rissing et al. , 1985; Cremieux and Carbon, 1997; Mader and Shirtliff, 1999). However, by implanting orthopaedic hardware such as screws, pins and wire the infection can be maintained without artificially creating dead host bone (Andriole et al. , 1973). The pin associated- osteomyelitis model by (Li et al. , 2008) consists of a biofilm- coated, 0.25 mm insect pin (stain- less steel) inserted trans- cortically into the tibia of a mouse. The leg is shaved and decontaminated prior to the surgery, after which the skin is sutured close preventing contamination of the pin. This model allows for quantitative observation of microbial growth kinetics using longitudinal in vivo bioluminescent imaging of luxA-E trans- formed S. aureus (Xen29) for chronic infections of up to three months (Funao et al. , 2012; Li et al. , 2008). This model has also been used to determine the host immune response to implant- associated osteomyelitis in S. aureus infection, to demonstrate the efficacy of immune modulation at clearing such an infection (Prabhakara et al. , 2011a, 2011b; Varrone et al. , 2011), and to test colistin- impregnated beads’ ability to treat Acinetobacter baumanii implant- associated osteomyelitis (Crane et al. , 2009).

Other models of orthopedic implant- associated infections include a femur implant model using Kirschner-wire in mice (Niska et al. , 2012a, 2012b), and implant-related infected non-unions of the tubia in rats (Alt et al. , 2011). Holt and colleagues devel- oped a model of external vertebral fixation in the rat and used it to demonstrate the microbicidal effect of nitric oxide- releasing coatings for fixation pins (Holt et al. , 2011). Several large- animal models using long- bone implants in goats and sheep have been demonstrated and are particularly useful for the ability to use human- size implants and the ability to test weight- loading of the limbs with infected implants (Hill et al. , 2002; Curtis et al. , 1995; Perry et al. , 2010).

56 Biomaterials and Medical Device-associated Infections

3.3.2.3 Intraperitoneal models

Intraperitoneal (IP) models of implant infection utilizing biofilm- coated silastic cath- eter material implanted into the peritoneal cavity of rabbits or mice were developed to study early (days–weeks) as well as chronic (up to 6 months) infections using the bacterial species S. epidermidis (Gallimore et al. , 1988, 1991). Buret and co- workers studied the morphology, ultrastructure, and microbiology of the colonized implant infected with P. aeruginosa (Buret et al. , 1991). This model was used in extensive trials to study the efficacy of vancomycin (Gagnon et al. , 1993), cefaman- dole, cefuroxime (Carsenti-Etesse et al. , 1992a), pefloxacin (Carsenti-Etesse et al. , 1992b), and ciprofloxacin (Owusu-Ababio et al. , 1995) to treat implant- associated infections. However, these trials only demonstrated the ability of the antibiotics to reduce bacterial burdens but rarely provide implant sterilization when infected with S. epidermidis (Carsenti-Etesse et al. , 1992a), S. aureus (Carsenti-Etesse et al. , 1992a, 1992b; Espersen et al. , 1993, 1994), or P. aeruginosa (Owusu-Ababio et al. , 1995). IP models have also been used to study the effect of novel catheter coatings on bacterial colonization in vivo (Kim et al. , 2001).

More recently this model has been used to demonstrate that quorum- sensing defi- cient or inhibited P. aeruginosa are cleared from implants at a faster rate compared to wild- type bacteria suggesting a role for quorum sensing inhibitors in chemotherapy of biofilms (Christensen et al. , 2007).

3.3.2.4 Central venous catheter models

Next to prosthetic valve endocarditis and vascular graft infections, there are many experimental models to imitate catheter- associated blood stream infections (CABSI).

The ‘Rupp’ model (Rupp et al. , 1999a) was developed and used to evaluate the efficacy of a range of antibiotics and anti- microbial peptides in CABSI infections (Cirioni et al. , 2006). This model was adapted for mice (Kadurugamuwa et al. , 2003;

Kokai-Kun et al. , 2009) and rabbits (Fernandez-Hidalgo et al. , 2010; Schinabeck et al. , 2004) for the study of S. aureus , S. epidermidis , P. aeruginosa , and the fungal pathogen Candida albicans .

A recent model published by Chauhan and colleagues has taken the established CABSI model one step further with the placement of totally implantable venous access ports (TIVAPs) replicating chronic infections of such devices in immunocom- petent and immunosuppressed rats (Chauhan et al. , 2012a). To study biofilm forma- tion, the model demonstrated colonization of the TIVAP via haematogenous seeding as well as metastasis of the infection from the TIVAP to organs. This model was also used to demonstrate the efficacy of treating S. aureus , S. epidermidis , E. coli or P.

aeruginosa infected TIVAPs using a gentamicin plus EDTA antibiotic- lock therapy clearing the infection (Chauhan et al. , 2012b).

3.3.2.5 Urinary tract catheter models

Initial models of urinary tract infections induced struvite urolithiasis by implant- ing zinc discs in the bladder of rats (Satoh et al. , 1984; Buret et al. , 1991; Olson

In vivo infection studies 57

et al. , 1989) while another model implanted glass beads into the bladder to cause renal infections (Haraoka et al. , 1995). A rabbit urinary catheter model was later developed and used to demonstrate the ability of different antibiotics to partially eliminate the bacteria that cause catheter- associated urinary tract infections (CAUTIs) from urinary catheters (Morck et al. , 1993). This model was followed by a transurethral catheterized model in mice (Kadurugamuwa et al. , 2005), rats (Kurosaka et al. , 2001), and rabbits (Fung et al. , 2003). The mouse model in particular has recently been used to model E. faecalis CAUTI and demonstrated the importance of endocarditis and biofilm- associated pili (Ebp) as a virulence factor (Nielsen et al. , 2012)

3.3.3 Most suitable animal species for the model

An ideal species for a model would have a low cost for purchase and housing a well- documented genetic and immunological profile that is comparable with humans, easy and convenient handling (size of the animal for handling but also for appropriate size of implant), and the possibility to observe numerous animals over a short time (Schimandle and Boden, 1994).

Theoretically, to imitate the human situation, it is better to use large animals, such as sheep, goats and dogs, which in general tolerate surgical interventions better (Auer et al. , 2007; An and Friedman, 1998). The shortcomings of using large animals include the need for a large housing space and high costs. Smaller animals such as rats, mice, rabbits and guinea pigs might be housed and fed more easily, thereby allowing for a larger number of animals, a more rapid endpoint, and more statisti- cally powerful results to be obtained (Auer et al. , 2007; Calabro et al. , 2013; An and Friedman, 1998).

When testing potential antimicrobial agents in animal models, it should be recognized that the agent will interact with the selected animal and have variable clearance rates, local or systemic toxicity, or disruption of the host microbial flora.

Therefore, potential side effects in humans can be over- or underestimated (Morris, 1995), so that the selection of the most suitable animal species for the experiment is a crucial factor for a proper experiment. Proper pharmacokinetic (PK) studies must be performed with each new agent to ensure that the antibiotic concentrations seen in the animal match that proposed for the clinical studies. These PK studies should be performed on both serum and tissue samples from single dose and repeated dose regimens at multiple time points following administration in order to get an accurate representation of the antibiotic concentrations seen in the host over time (Mader and Shirtliff, 1999).

The most commonly used small animals in implant- associated infections are mice (Christensen et al. , 2007; Gallimore et al. , 1991), rats (Schaad et al. , 1994), rab- bits (Mader and Shirtliff, 1999; Brady et al. , 2011), or guinea pigs (Zimmerli et al. , 1982). No single model is a perfect representation of its human disease counterpart (An et al. , 2003) so that for some hypotheses, more than one species has to be tested.

In the following and in Table 3.2 , pros and cons of the different animal species are summarized.

58 Biomaterials and Medical Device-associated Infections

Table 3.2

Pros and cons of the most commonly used small animal species in implant- associated infection models

Animal species

Advantage Disadvantage

Mice Small, low cost for housing compared to rabbits

Short life span, proclivity for reproduction

Known genetic background Minimal expense for purchase and maintenance

Very low susceptibility to bacterial Inoculum

Spontaneous healing in BALB/c mice are reported

Rats Tolerate beta lactams

Available from many commercial and private sources

Good adaptable to novel environments Well understood and characterized anatomically, physiologically, and genetically

Economical and easy to handle

Very low susceptibility to bacterial inoculum and need of high minimal infecting dose of bacteria

Spontaneous healing of infection is possible

Rabbits Easy to handle for surgery and postoperative observe

Reproducible infection profile Tolerance of implantation of prosthesis

Small light and fragile bone structure for implants

Very vulnerable to antibiotic side effects (diarrhoea, ileus) Vulnerable to post- implantation fractures due to a fragile weight- bearing femur

Needs surgical expertise to conduct surgery.

Guinea pigs Close similarity to human device- associated infections Very low minimal infecting dose (100 CFU S. aureus )

Tractable disposition and size Readily available, relatively inexpensive and easy to maintain.

Do not tolerate beta lactams (weight loss, diarrhoea) Relatively high susceptibility to infection

Mice. Mice have been the mainstay of in vivo models since they are small, easily housed, genetically tractable and usually inexpensive. In addition, there is a litany of commercially available reagents to study and/or to modify their histological, immu- nological and pathological responses to disease. Due to their short gestation and life span, mice allow rapid breeding of a large number of animals and, consequently, allowing for the feasibility of multiple studies in a relatively short period.