Microbiology Research

Sinisa Vidovic completed his PhD program in 2008 at the University of Saskatchewan, Canada and Postdoctoral trainings at the Medical College, University of Ottawa and the Vaccine and Infectious Disease Organization, University of Saskatchewan. Presently, he works as an Assistant Professor at the Department of Veterinary and Biomedical Sciences, University of Minnesota, USA. He has published over 20 peer-reviewed articles.

Fluoroquinolones (FQs), broad-spectrum bactericides, are the antibiotics of choice for treatment of invasive, lifethreatening infections caused by Salmonella enterica. In this study, we aimed to identify and characterize non-synonymous single nucleotide polymorphisms (nsSNPs) mutations in the gyrA, gyrB, parC and parE genes, responsible for FQ-resistance in four resistant (MIC 8-16 μg/mL) and four highly resistant (MIC>128 μg/mL) S. enterica serovar Enteritidis strains, respectively. Initially, we used four clinical FQ-susceptible (MIC 0.06–0.12 μg/mL) S. Enteritidis strains (A5-S, A7-S, A21-S, and A33-S) and selected four spontaneous FQ-resistant (A5-R, A7- R, A21-R, and A33-R) and four FQ-highly resistant (A5-HR, A7- HR, A21-HR, and A33-HR) S. Enteritidis strains via a stepwise assay using ciprofloxacin as a selective agent. Among both, the resistant and highly resistant strains, no nsSNP were found in the gyrB and parE. In contrast, all the resistant strains had a substitution of S (Ser) for Y (Tyr) and F (Phe) at position 83 of the GyrA. The highly resistant strains together with A5-R strain acquired a second substitution of D (Asp) to G (Gly) at position 87 of the GyrA. For the ParC enzyme, we found a substitution of G for D at position 78 in the A7-HR strain and S for R (Arg) and I (Ile) at position 80 in the A5-R and A5-HR, respectively. By performing a cross-resistance assay using the resistant and highly resistant strains, we found that exposure of S. Enteritidis to FQ selects for additional resistance to cefoxitin, amikacin, chloramphenicol, and ceftiofur, antibiotics that do not belong to FQ class of antibiotics. Our study showed that amino acid substitution at position 83 of the GyrA is crucial for the acquisition of FQ-resistance, while substitution at position 87 of the same enzyme provides a high-level FQ-resistance. In addition, we showed that FQ exposure selects for the crossresistance to some beta-lactam (cefoxitin and ceftiofur), aminoglycoside (amikacin) and chloramphenicol classes of antibiotics.

Maurizio Manera has completed his PhD in Veterinary Pathology at Bologna University and MSc in Environmental Science at L’Aquila University. He is an Assistant Professor in Image Analysis in Histopathology and in Biomarkers in Environmental Monitoring. He has published 74 papers in indexed reputed journals and has been serving as an Editorial Board Member of reputed journals.

Liver texture was comparatively assessed in 20 specimens of carp after experimental exposure to two PFOA dosages (10 exposed to 200 ng l-1, 10 exposed to 2 mg l-1) for 56 days and in 10 unexposed (Ctr) specimens. Grayscale differential box counting (mass fractal dimension, DM and lacunarity, λ [Lac]; FracLac plugin in ImageJ package; Fig. 1) was adopted on images previously assessed for texture by means of MaZda software. Linear Discriminant Analysis on grayscale box counting data failed to discriminate with confidence between treated and untreated fish (40% of cases correctly classified: sensitivity of 69.0%, specificity of 52.6%) with particular regard to low dosage group (only 10% of Ctr cases correctly classified). Nevertheless, referring to the cumulative Redundancy Analysis ordination space (Fig. 2), the more the liver PFOA content augmented, the more the number of cells positive to Proliferating Cell Nuclear Antigen (PCNA), the liver mass, DM and λ (hence gray level pattern complexity) increased and Sum Entropy (hence the disorder of a vector from the gray level cooccurrence matrix) decreased. To date the exact biological implication of such correlations is not known, with lacunarity increase possibly due to hepatocyte degeneration (increased vacuolisation), though it surely deserves further investigation in order to use grayscale box counting with confidence in biomarker evaluation in course of environmental monitoring.

Soumya Palliyil is the Principal Scientist and Facility Manager of the Scottish

Biologics Facility (SBF), a biologic drug discovery unit which provides antibody and peptide hit generation services to academia and industry. She completed her PhD within Professor Andy Porter’s Antibody Engineering Group, University of Aberdeen and Wyeth/Pfizer Inc Protein Therapeutics Lab, Aberdeen. Utilizing the antibody engineering experience and expertise accumulated over the years, she manages a wide range of antibody based projects in the SBF including the development of antibody therapeutic candidates in areas such as cardiovascular, neurodegenerative and infectious diseases, several antibody-fragment based diagnostics in bacterial and fungal infections and in vivo imaging candidates for heart disease and fibrotic conditions of the liver, pancreas, etc. She is a recipient of the prestigious Commonwealth Scholarship and has also completed the Royal Society of Edinburgh Commercialization Fellowship.

Introduction: The human pathogen Pseudomonas aeruginosa uses a cell-density based intercellular communication system called quorum sensing (QS) to regulate virulence gene expression and pathogenic behavior within the host. In this study, immunomodulation of QS molecules by monoclonal antibodies (mAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect these compounds in bodily fluids as a possible first clue to an undiagnosed Gramnegative infection.

Methods: Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric mAbs to homoserine lactones (HSLs) was developed. Lead clones were tested in elastase and nematode slow killing assays to evaluate their quorum quenching activities and also for their ability to detect QS compounds in human urine. For survival studies in mice, lead mAbs and comparator antibiotic drugs at 10 mg/kg or vehicle-only control were co-administered with P. aeruginosa PA058 by the intranasal route with a second

treatment dose 4 h post infection. Bacterial burden in the lung tissue 24h post infection and survival up to seven days was monitored.

Results: Specific binding of lead mAbs to QS molecules has been demonstrated by a series of in vitro immunoassays. In the nematode slow killing assay, the survival rates of Caenorhabditis elegans increased from 15% to 60%. In a nonneutropenic lung model of mice infected with P. aeruginosa

PA058, mAb monotherapy demonstrated significant efficacy, prolonging survival up to 83%. HSL mAbs also retained functional recognition of its antigen in the presence of urine with very little reduction in sensitivity observed (IC50 value 4 nM in PBS vs. 10 nM in urine).

Conclusions: Antibodies are an attractive method for controlling bacterial virulence by block quorum sensing signaling as these ‘antipathogenic’ drugs are less likely to develop resistance in bacteria compared to conventional antibiotics. An immunoassay-based diagnostic system exploiting the high sensitivity of anti-QS MAbs could be developed to detect the presence of specific markers of infection (homoserine lactones, quinolones) in bodily fluids such as blood and urine.

Pathology branch is well known unorganized sector and 60 to 70 % medical treatment is dependant on it. So to meet growing demand and bring together inhouse pathologist and other consultants digitalization is very important. As we see evolution of pathology after inventing microscopy to complete digital platform we can able to connect them all. We manage remote units at five levels to ensure highest accuracy and fastest reports. Through technology now it is possilbe to reach to rural and suburban areas where good heathcare facilities

not available.

Swee Hua Erin Lim has completed her PhD in Medical Biotechnology from the Universiti Putra Malaysia. She is an Assistant Professor at the Health Sciences Division, Abu Dhabi Women’s College, Higher Colleges of Technology, in addition to being an Adjunct Associate Professor at the Perdana University- Royal College of Surgeons in Ireland and Perdana University-School of Data Sciences. She has published a book chapter, more than 15 papers in reputed journals and presented in many international conferences. She has also been serving as a Reviewer of reputable ISI indexed journals.

Nature-based tourism has become the fastest growing economic sector in Malaysia. Many ecotourism sites allow

for close human-animal interaction (HAI), lacking educated supervision and awareness of potential health risks. Nonhuman primates (NHPs) are of particular interest in this HAI having served as reservoir for more than 70 zoonotic diseases to humans. Ecotourism sites encourage direct contact with the NHPs, which increases the risk of cross-species transmission of infections. It has been recently discovered that methicillin resistant Staphylococcus aureus (MRSA), once thought to be clinically-originated, is community-acquired from non-clinical settings. This may apply not only to MRSA but also to other antimicrobial resistant (AMR) bacteria such as Enterococcus sp

and Escherichia coli. This study was carried out to investigate if the NHP-human interaction poses a risk in transmitting zoonotic pathogens based on microbiological data and to establish if a relationship exists between AMR carriage and host gut microbiome (MDR) bacteria in faecal samples of NHPs (Macaca fascicularis and Trachypithecus cristatus) and humans (Homo sapiens, HS). Methicillin-Resistant S. aureus (MRSA) and Vancomycin-Resistant Enterococcus (VRE) were isolated in different agar mediums and characterized by antimicrobial susceptibility using the disk diffusion method. The most resistant and least resistant faecal samples were sent for 16S rRNA sequencing to identify microbiota that might underpin antibiotic resistance. Principle Component Analysis (PCA) was used to group samples based on antibiotic profile, resistant bacteria and host species microbiome. From our study, humans were found to harbor higher levels of AMR suggesting a higher possibility of arthropozoonosis. Higher load of AMR was correlated to a less diverse gut microbiome and vice versa. Despite the initial focus of ecotourism intended for conservation, AMR spread can be minimised if proper regulatory guidelines are set via awareness programmes to staff, tourists and locals in addition to incorporation of these information into brochures and marketing materials.

Krzysztof Olesiejuk is a Medical Student from Medical University of Lodz, Poland. Having already taken part in research on three continents, he is engaged in scientific fields such as immunology, cancer biology and multi-drug-resistance of bacteria. Apart from this, his interests include wound healing processes and neural interfaces.

Dendritic cells (DC) are critical immune modulators. In the presented work, the author investigated the roles of genes, which might be involved in the development of classical DC (cDCs) and plasmacytoid DC (pDCs), using an immortalized hematopoietic stem and progenitor cell (iHSPC) line. The iHSPCs were first subjected to transduction with a lentivirus carrying shRNA in order to knockdown the following genes: IRF7, ETS1, PHF17 and Zfp719. The stable knockdown iHSPC cell lines were then cultured in vitro for 5 days with Flt3 ligand, a cytokine required for DC development, and analyzed with flow cytometry. The flow cytometry results and cell counts showed a strong impact of each of the genes on the survival and development of cDCs and pDCs, compared to shLacZ, a knockdown control. Normally highly expressed in pDCs, the genes proved to be crucial for their development – with the strongest effect observed for IRF7 knockdown. These results give a new insight into the DC biology and shows possible footholds for influencing their functions. This approach may prove useful in designing vaccines, fighting infections and paving the way for new strategies for immunotherapy in cancer.