6^th World Congress and Exhibition on Antibiotics and Antibiotic Resistance June 03-04, 2019 London, UK

Jian-Ming Huang is currently studying in National Cheng Kung University. His major project is to study the protein expression in the parasitic protozoan Acanthamoeba. He also developed antibody to identify a rapid and sensitive antibody-based assay and investigate molecular and characteristics of the M28_ AAP. A total of 2 papers were published by him based on his work.

Background: Acanthamoeba keratitis (AK) is a serious disease caused by pathogenic Acanthamoeba through wounds of cornea; by and large, most high-risk patients catching AK wear contact lens generally over a long period of time. However, microscopic examination and polymerase chain reaction following the culture that they are traditional diagnostic tests for pathogenic Acanthamoeba need long time to confirm AK. Hence, we purpose to develop a rapid and sensitive antibody-based assay to improve the diagnostic procedure. Objective: Comparing with proteomic analysis of extracellular secreted proteins expressed by two pathogenic Acanthamoeba castellanii clinical isolates and a non-pathogenic ATCC strain, we will evaluate the efficiency of Acanthamoeba M28 aminopeptidase (M28_AAP) as a diagnostic target for developing a rapid and sensitive antibodybased assay and investigate molecular and characteristics of the M28_AAP. Methods: The M28_AAP antibody was produced to detect Acanthamoeba isolates through an antibody-based diagnostic test such as a western blot or an enzyme-linked immunosorbent assay (ELISA). Results: We have produced successfully M28_AAP antibody to detect Acanthamoeba isolates through a western blot, an immunofluorescence assay (IFA) and an ELISA. Furthermore, the complement can be degraded by M28_AAP in the human innate immunity. We believe that M28_AAP will be the opportunity to develop a rapid diagnostic marker and newer target to decrease the AK patient cases. To evaluate the sensitivity and specificity of the Acanthamoeba M28_AAP antibody, we will plan to develop rapid diagnostic tests such as test strips on clinical diagnosis.

Pin-Ju, Ko is a second-year master’s student now, who is majored in parasitology. She has focused on drug metabolism, especially cytochrome P450 enzymes. Base on her research which may provide a better understanding of CYPs in Acanthamoeba but also provide insight into the development of treatment for AK.

Acanthamoeba is a free-living parasite present in rivers, lakes, seawater and soils that can cause severe infection, such as Granulomatous Amoebic Encephalitis (GAE) and Amoebic Keratitis (AK). Two main stages can be recognized in the life cycle of Acanthamoeba castellanii: the trophozoite and the cyst. Since the transformation process from a trophozoite into a cyst, also called encystation, plays an important role in resisting extreme physical and chemical conditions for Acanthamoeba cell survival. Therefore, we tried to figure out the difference between these two stages by collecting the trophozoites and encysting samples for transcriptomic high-throughput surveys. According to the transcriptome analysis, we found out several cytochrome P450 and antibiotic biosynthesis related genes expressed higher in the cyst form. Cytochromes P450 (CYPs) are proteins of the superfamily which is necessary for the detoxification of foreign chemicals and the metabolism of drugs which might be an essential process to against potential toxicity from the environment that Acanthamoeba uptake. Since previous studies have shown that CYPs play a role in drug metabolism and therefore we will try to use different expression vectors to express CYPs in both prokaryotes and eukaryotes and followed by drugs treatment to investigate the effects of CYPs. To sum up, these findings may not only provide us with a better understanding of CYPs in Acanthamoeba but also provide insight into the development of treatment for AK.

Shahneela Mazhar is a final year PhD student, pursuing a joint research project between Teagasc Food Research Centre and Univ ersity College Cork. Her project is based on in-depth understanding of the organism Macrococcus, in terms of its f antibiotic resistance acquisition and the pathogenic potential. To - date she has developed a screening method to isolate and differentiate species of Macrococcus from closely related Staphylococcus species from diverse sources.

Members of the Macrococcus genus are disseminated in nature as animal commensals that are evolutionarily closely related to the species Staphylococcus. Recent reports have identified a plasmid-encoded methicillin resistance determinant, mecB, in Staphylococcus aureus of macrococcal origin, obtained from an elderly cardiology patient. Other reports have identified a novel mecD gene in Macrococcus caseolyticus strains isolated from bovine and canine sources. The mecD gene confers resistance to all classes of β-lactams including anti-MRSA cephalosporins. Thus, there is an increasing body of evidence emerging recognizing macrococci as reservoirs of antibiotic resistance determinants. However, due to the lack of availability of genomic data for species of the Macrococcus genus, information on the genome-wide distribution of antibiotic resistance determinants across Macrococcus species is limited. In this study, we used a whole genome sequencing approach to characterize and identify antibiotic resistance genes present in a collection of the type strains of six Macrococcus species; Macrococcus bovicus ATCC 51825T, Macrococcus carouselicus ATCC 51828T, Macrococcus equipercicus ATCC 51831T, Macrococcus brunensis CCM4811T, Macrococcus hajekii CCM4809T Macrococcus lamae CCM4815T as well as in a collection of 14 strains from the Teagasc DPC culture collection belonging to Macrococcus caseolyticus, Macrococcus canis, Macrococcus bohemicus, and Macrococcus goetzii isolated from bovine tongue and milk samples. In silico analysis of the resistome using resistance gene identifier (RGI) identified the methicillin resistance mecD gene in M. bohemicus strain DPC7215. This gene displayed 100% sequence identity to that of M. caseolyticus strain IMD0819. Further analysis with Islanviewer4 revealed that this mecD gene complex is harbored on a genomic island within the chromosome. Interestingly, the complete sequence of a plasmid found in S. aureus pS0385-2, (GenBank: AM990994.1) carrying streptomycin resistance, was also found to be integrated in the M. bohemicus DPC7152 genome. All other sequenced genomes illustrated partial identity to various other resistance determinants including tetracycline, aminoglycosides and glycopeptides. Conclusion & Significance: The results demonstrate that antibiotic resistance, particularly the mecD determinant, may be acquired from closely related species by horizontal gene transfer, and that M. bohemicus DPC7152 may serve as a reservoir of antibiotic resistance genes. Given that the integrated pS0385-2 appears to be transferrable between species.