# Biofilms: Novel Strategies Based on Antimicrobial Peptides
## 5 In Vivo Models for Biofilms  
To broaden in vitro findings, it is important to enforce in vivo mimicking conditions. Models of infection in vivo and pathogenesis are a continuous interaction between the host and microbes and between microbes themselves [67]. These interactions are complex and dynamic and make it difficult to study them in in vitro models. To validate results in vitro, that could be further translated into higher organisms or clinical trials, it is necessary to use the right in vivo model [68]. It is very difficult to reproduce the host environment in in vitro models and for this reason there is a need to use multiple in vivo models ranging from non-mammalian to mammalian models, mainly rodents [69]. Because of ethical concerns, the use of in vivo models is now submitted to legal regulations, as it is necessary to consider not only medical benefits but also animal welfare [70]. This partly explains why researchers are still using in vitro and in vivo non-mammalian models. Non-mammalian models show advantages such as rapid development and cheap experiments to perform. Moreover, the genome of most of these organisms have been sequenced, and it is possible to do genetics both on the bacteria and the host. However, these non-mammalian models may have limitations such as the growth temperature of pathogens and are not suitable for studying chronic infections. In the past years, non-mammalian models like _Drosophila melanogaster_, among invertebrates, and _Danio rerio_, among vertebrates, have been adapted to study host-microbe interactions and immune system responses related to colonization of the intestine by biofilms [69]. Zebrafish are an emergent model to study human disease. They have a highly developed innate cellular immune systems that make them a perfect model to study infectious diseases. Currently, zebrafish are used as a model for imaging and genetics to investigate the molecular and cellular foundations of host-microbe relationships for the in vivo biofilm studies. Microbiome studies in zebrafish hosts have concentrated on embryonic and larval stages, when the advantages of this model are maximized. It is fundamental to understand the interactions between microbe and host to know the host developmental level when microbes colonize it and know the effects on host ontogeny [71]. [PERSON] et al. [72] have produced a model of bacterial pathogenesis injecting _Streptococcus iniae_ into zebrafish dorsal muscles. Zebrafish are also susceptible to _Streptococcus_ pyogenes, the human pathogen. They used genetics and mutagenesis to obtain several attenuated mutants in zebrafish. The success of the combination Streptococcus-zebrafish makes it a great model for the analysis of infectious disease. [PERSON] et al. [73] colonized germ-free zebrafish with _P. aeruginosa_ strains containing mutated genes for motility obtaining the attenuation of the host innate immune responses. They show the utility of germ-free zebrafish in determining the comportment and localization of bacteria in vertebrate intestine and identified the bacterial genes involved on host/microbial interactions. The protective mechanism against pathogens of probiotic or commensal bacteria in germ-free animal models, remain poorly understood. [PERSON] et al. [74] pre-colonized zebrafish larvae with 37 probiotic bacterial strains to screen the survival upon _Edwardsiella itcaluri_ infection identifying 3 strong protective strains, including _Vibrio parahaemolyticus_ and two _E. coli_ strains. They favor the emergence of probiotic bacteria in zebrafish larvae. The trait of tuberculosis is the formation of granulomas, [PERSON] et al. [75] identified new mycobacterial factors involved in the granuloma formation. They utilized the zebrafish embryo-Mycobacterium marinum model to selected _M. marinum_ mutants that did not induce granuloma formation. One of the mutants was found to be defective in the espL gene, which is located in the ESX-1 cluster.