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Clostridioides difficileinfection (CDI) is considered as an urgent threat to the publicby CDC, 2019.It causes life-threatening diarrhea and pseudomembranous colitis,mostly in those taking antibiotics or at the end of their antibiotic course.It is also notifiedas hospital-associated pathogensbecause one-third of the CDIhas occurredinthe health care center. Norepinephrine (NE) is a stress-associated neuroendocrine hormone released upon sympathetic stimulation to mediate stress.Gut walls are highly innervated by the sympathetic nervous system. During stress, elevated level of NE released in the GI tractcould influence bacterial overgrowth & translocation. It isalready known for its role in modulating the behavior of several bacterial pathogens suchas Staphylococcus, Escherichia coli, Salmonella, and Vibrio cholera. This study aims to evaluate the effect of NE treatment on the growth and virulence of C. difficile.Here, we studied the effect of NE on six different C. difficilestrains isolated from humans. To understandthe influence on growth, bacterial culture was treated (+/-)NE (5μM & 50 μM)during their log phase and recorded the density of the cell each time period for constructing the growth curve. In addition, after NE treatment, bacterial cells were taken for further analysis. For investigating the impact of NE on the virulence genes expression, a qPCR reaction was performed along with -RT / noRT control reactions for assessingthe RNA sample free from genomic DNA contamination. In the case of growth,higher growth was observed in VPI 10463at 6 hourtime pointonly,and in strain,NR 49277 significantly stimulated after 6 hoursand continued till 8 hours after treatmentwith50μM NE. In strain NR 49282, decreasedgrowth was observed at7-hourtime pointsafter 50 μM NEtreatment.But, there was no difference in cell density between control & 5μM NE treated bacterial culture in all strains. Toxingenes(tcdA&tcdB)and flagellin gene(fliC),were upregulated in NR 49290, NR 49277 & VPI 10463strains in both concentrations of NE and down-regulated in NR 49282.In strain NR 32888, toxin genes were downregulated while treated with 5μM NEbut upregulated after 50μM NEtreatment, though fliC was downregulated in both concentrations. In strain NR 32891, tcdAwas downregulated,but tcdB& fliCwere upregulatedafter NE treatmentin both concentrations. Increased expression in pilin gene,pilA1in strain NR 49277, NR 49290, VPI 10463& NR 32891 in both concentrationswas observed. In addition, pilA3in NR 49277, VPI 10463& NR 32891 and PilA5in NR 49277 & NR 49290 showed an upregulation pattern while treated with both concentrations. Modulating this response, it is possible to reduce the pathogenicity of C. difficileduring medical care & antibiotic use.

Mass spectrometry (MALDI-TOF) has become one of the most common tools for identifying bacteria in diagnostic laboratory specimens. However, identification of unknown organisms is only possible if the protein profile exists in the database. Although the database is extensive there are some organisms that continue to provide challenges in identification. Our laboratory has experienced a challenge in identifying Gram positive organisms, particularly from equine uterine samples. The goal of this study was to identify these bacteria, whose protein profiles were not well represented in the Bruker database. Forty-eight isolates from equine uterine samples submitted to the Wyoming State Veterinary Laboratory (WSVL) between June 2018 to September 2020 were maintained frozen. All were identified as Gram-positive rods, with low or no MALDI scores to profiles in the existing database. The isolates were sub-cultured, MSP profiles were created for our local MALDI database, and DNA extracted for whole genome sequencing. The MSP profiles for each isolate were used to create a dendrogram, and a minimum of one isolate from each branch was targeted for whole genome sequencing. We hypothesized that unidentified Gram-positive rods would be in the same genus of bacteria, however preliminary sequencing results indicate that there are at least three distinct genera. These include Cutibacterium, Corynebacterium, and Arcanobacterium. All are now associated with MSP profiles in our expanded database and will aid in more efficient identification of Gram-positive rods in future WSVL diagnostic samples.

Diagnosing the underlying cause of diarrhea and enterocolitis in horses is difficult. One bacterium that is associated with enterocolitis in foals is the anaerobic bacterium, Clostridium difficile. This bacterium is difficult to culture, thus other diagnostic methods must be used. A real-time PCR assay for the Toxin A and Toxin B genes of Clostridium difficile was developed and validated for more efficient diagnosis of this bacterium from clinical samples. Four sets of primers (two for Toxin A and two for Toxin B) were evaluated. Toxin A Set 5 and Toxin B Set 2 were determined to be most effective at detection the respective toxin genes. We used Sanger sequencing to confirm that the amplicon was the correct gene target, and then optimized the PCR protocol for maximum sensitivity and efficiency. PCR parameters included annealing time and temperature, primer concentration, and probe concentration. We used different fluorophores on the probes, to be able to multiplex this PCR reaction; FAM for Toxin A, HEX for Toxin B and Cy5 for the internal control. However when a multiplex reaction was attempted, there was interference. Thus the final assay includes a separate reactions for each toxin gene. A serial dilution of a positive C. diff ATCC control (BAA-1870DQ Quantitative Genomic DNA from Clostridium difficile Strain 4118) was used to test the efficiencies which were determined to be 91.3% for Toxin A and 97.6% for Toxin B. Validation was performed using thirty negative sample controls, while positive sample validation will be ongoing.

Brucellosis is an economically important cause of abortions in livestock and wildlife worldwide caused by the facultative intracellular pathogen Brucella abortus. One understudied molecular mechanism of in-vivo survival used by B. abortus is adherence to target cells by Type-V (auto-transporting) proteins. BruAb1_1988 and BruAb1_1989 are two such proteins that have been identified within a Cluster of Orthologous Groups (COGs) of proteins expressed in-vivo during infection. Attempts to express BruAb1_1989 have been unsuccessful. A bioinformatics approach, including a 3-D structural analysis, has predicted that it is likely a multimeric inner membrane protein composed of primarily two (folded) ��-helical domains. BruAb1_1988 has been expressed through traditional cloning methods, in two segments separating the passenger and ��-barrel domains. Western blots conducted on the domains using NVSL Bovine check serum for B. abortus haven't yielded any positive results. This result isn't entirely unexpected, as the gene for BruAb1_1988 should produce a truncated product in B. abortus. The ortholog in B. melitensis however is expected to produce a functional protein. Because of this future characterization will continue, including further characterization of the ��-barrel domain because of its potential use in identifying other in-vivo expressed autotransporters. Further understanding of the structural and functional nature of these and other Brucella Type-V proteins will potentially lead to their use in a next-generation diagnostic assay that separates B. abortus and B. melitensis, another clinically relevant species contributing to Brucellosis outside of the United States.

Bovine in the Greater Yellowstone Area (GYA) of the United States are consistently exposed to Brucella abortus. This bacterium has been known to cause abortions in cattle resulting in an economic loss to the owner and operator. The current veterinary vaccine, B. abortus RB51, is inadequate in giving broad host range protection against infection in domestic livestock. A complete understanding of how the vaccine strain, RB51 when given in a single dose, affects the immune response of cattle, Bos taurus. We propose to study the effects of multiple doses of RB51 in cattle. This study focuses on the methods needed to continue this research and move toward quantifying the results of vaccination of cattle against the Brucella abortus. This is a presentation of the methods developed to aid in that study.

A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Physiology, Anatomy and Microbiology, College of Science, Health and Engineering, La Trobe University, Victoria, Australia.

Lecture given by Doctor Shao-Cong Sun, Gladstone Institute of Virology and Immunology, University of California, USA at the Waite Campus, University of Adelaide, 15.12.1993This lecture is part of the Campus Seminars and Distinguished Lecturer Series, Waite Campus, University of Adelaide, 1991 – 1997. https://figshare.com/projects/Campus_Seminars_and_Distinguished_Lecturer_Series_Waite_Campus_University_of_Adelaide/17756

Summary A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide-based enzyme-linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence-associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11-14. The peptide corresponds to the N-terminal B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut-off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA-specific IgGb antibodies against N-terminal B-cell epitope of the VapA protein rather than IgG antibodies. The VapA-IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6-week-old foals showed that the VapA-IgGb ELISA provided an increasing trend (P = 0.0572) in sensitivity of 82.4% in comparison with the VapA-IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P = 0.357) as analysed by the McNemar test. These results indicated that detection of VapA-specific IgGb antibodies may be a better predictor of R. equi disease in foals.

1. SUMMARY Dogs and cats have become an integral part of modern society in the developed world and as such attention is given to their care and welfare. At some stage of their lives many cats and dogs suffer from skin and other superficial staphylococcal infections such as pyoderma and otitis externa and are therefore treated with antibiotics. This practice has led to the emergence of resistant staphylococci in these animals. Antimicrobial susceptibility studies in companion animals have revealed that staphylococci isolated from cats and dogs exhibit similar resistance patterns to human staphylococci and that resistance development is influenced by the frequency of use of certain antibiotics. This review examines the antibiotic resistance patterns of the most frequently isolated staphylococci from cats and dogs and the mechanisms underlying resistance. The possible transfer of methicillin-resistant staphylococci from cats and dogs to humans has been identified as a potential public health issue. Typing tools used for epidemiological differentiation of pathogenic and nonpathogenic staphylococci and for species identification studies may help to elucidate the importance or otherwise of this problem.

The aim of this study was to evaluate the usefulness of the previously identified B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein of Rhodococcus equi and its association with R. equi pneumonia. A modified peptide designated PN11-14 corresponding to the epitope was recognized by all sera from experimentally infected foals with virulent R. equi ATCC103+ containing the virulence plasmid but not by its plasmid-cured derivative ATCC103- strain. Marked levels of VapA-specific immunoglobulin (Ig)G were detected in all sera from the ATCC103+ infected foals at 2 weeks after the infection. One control animal had high titres as determined by the peptide enzyme-linked immunosorbent assay (ELISA), indicating the ELISA may not absolutely differentiate between foals with R. equi pneumonia and healthy exposed foals in farms where the prevalence of disease is high. However, numbers of animals used were small. Further evaluation of the peptide ELISA with field samples is necessary to determine whether the assay is diagnostically useful. This study showed that levels of passive transfer of maternal IgG antibodies to the epitope in newborn foals could be measured. Interestingly, the maternally derived antibodies were found to significantly (P

McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli , enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli , enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.

Sewage and its microbiology, treatment and disposal are important to the topic of Antarctic wildlife health because disposal of untreated sewage effluent into the Antarctic marine environment is both allowed and commonplace. Human sewage contains enteric bacteria as normal flora, and has the potential to contain parasites, bacteria and viruses which may prove pathogenic to Antarctic wildlife. Treatment can reduce levels of micro-organisms in sewage effluent, but is not a requirement of the Environmental Protocol to the Antarctic Treaty (the Madrid Protocol). In contrast, the deliberate release of non-native organisms for any other reason is prohibited. Hence, disposal of sewage effluent to the marine environment is the only activity routinely undertaken in Antarctica knowing that it will likely result in the release of large numbers of potentially non-native species. When the Madrid Protocol was negotiated, the decision to allow release of untreated sewage effluent was considered the only pragmatic option, as a prohibition would have been costly, and may not have been achievable by many Antarctic operators. In addition, at that time the potential for transmission of pathogens to wildlife from sewage was not emphasised as a significant potential risk. Since then, the transmission of disease-causing agents between species is more widely recognised and it is now timely to consider the risks of continued discharge of sewage effluent in Antarctica and whether there are practical alternatives.

Bacteriophages are viruses that are able to infect and kill bacterial cells. This capability indicates that they may have the potential to become new antibacterial agents, as an alternative to antibiotics. Yet, while bacteriophages are relatively new to Western researchers, they have been extensively used in the former Eastern Block and its successors, including use in human clinical medicine. Western research on this topic is far more limited. Bacteriophage products have recently been licensed in the USA for control of two different bacteria: Listeria monocytogenes and Escherichia coli O157:H7. Neither of these products is, however, approved for direct administration to humans or animals. The use of bacteriophages to treat bacterial infections in humans or animals is a much more complex issue, but there has been a considerable amount of research into this field, and results are quite promising. Bacteriophage therapy and prevention of bacterial pathogens has been assessed in numerous species, and animals are also used as models for human disease. The behaviour of bacteriophages is, however, not as predictable in vivo as it is in artificial laboratory models. There is also concern regarding possible undesirable outcomes of bacteriophage therapy, such as the development of bacterial resistance to bacteriophages, and even the possibility of transfer of virulence or resistance genes, via bacteriophages, into other bacteria. Therefore, extensive research is still required into the behaviour of bacteriophages in vivo , but research to date shows considerable promise.

A total of 129 Pseudomonas spp. and 90 Aeromonas spp. were isolated from nine rainbow trout (Oncorhynchus mykiss) farms in Australia. All the isolates were tested for sensitivity to 15 antibiotics and the multiresistant strains were tested for sensitivity to seven heavy metals. Minimal inhibitory concentrations (MICs) were determined by the agar dilution method. In Pseudomonas spp., resistance to amoxicillin, cefalothin, ceftiofur, ticarcillin, chloramphenicol, florfenicol, streptomycin, nitrofurantoin and trimethoprim was widespread, whereas resistance to cefotaxime and oxolinic acid was less common and only single isolates were resistant to tetracycline and sulfamethoxazole; all isolates were sensitive to ciprofloxacin and gentamicin. In Aeromonas spp., resistance to amoxicillin and cefalothin was widespread, resistance to ticarcillin, tetracycline and streptomycin was common, whilst resistance to ceftiofur, florfenicol and sulfamethoxazole was less common. Single isolates were resistant to chloramphenicol, nitrofurantoin and trimethoprim, and all isolates were sensitive to cefotaxime, oxolinic acid, ciprofloxacin and gentamicin. Multiple resistance was also observed. Most isolates were tolerant to different concentrations of various heavy metals, as evidenced by their MICs ranging from 6.25 microg/mL to >3200 microg/mL. These results confirm our previous findings that bacteria resistant to antibiotics are present in fish and sediments from aquaculture in Australia. In addition, we have found resistance to heavy metals in fish and sediment isolates. Much of the antibiotic resistance detected is likely to be intrinsic, although resistance to oxytetracycline, streptomycin and sulfonamides suggests either contamination from run-off from farms or perhaps off-label use of antibiotics in a situation where no antibiotics are licensed for use in aquaculture.

Staphylococci are part of the normal microflora of humans and animals and some are potential pathogens that have become resistant to almost all known antibiotics. Despite the widespread reports of penicillin resistance in cat and dog staphylococci, the mechanism underlying penicillin resistance has not been examined. This study was aimed at investigating the molecular basis of resistance to penicillin in cat and dog staphylococcal isolates that showed phenotypic resistance to beta-lactam antibiotics. An 861 bp fragment of the structural blaZ gene which codes for beta-lactamase production in staphylococci was amplified by polymerase chain reaction (PCR) and the products were sequenced. Sequenced fragments were analysed by protein signature typing and sequences were compared to published blaZ sequences of human and bovine staphylococcal strains held in a public database. Four known protein signature types (1, 3, 5 and 6) and one new type (12) were identified in this study. When sequences were compared with published blaZ sequences, gene phylogenetic analysis revealed three major groups. The four variants of beta-lactamases types (A, B, C and D) belonged to each major group except for types A and D which were both in group II. These findings confirm that the blaZ gene is responsible for beta-lactamase production leading to subsequent resistance to beta-lactam antibiotics in feline and canine staphylococci and that the gene shows similar diversity and relatedness as found with blaZ sequences obtained from human and bovine staphylococci.

Aims: To carry out a preliminary assessment of the occurrence of resistance to antimicrobials in bacteria that has been isolated from a variety of aquaculture species and environments in Australia. Method and Results: A total of 100 Gram-negative (Vibrio spp. and Aeromonas spp. predominantly) and four Gram-positive bacteria isolated from farmed fish, crustaceans and water from crab larval rearing tanks were obtained from diagnostic laboratories from different parts of Australia. All the isolates were tested for sensitivity to 19 antibiotics and Minimal Inhibitory Concentrations were determined by the agar dilution method. Plasmid DNA was isolated by the alkali lysis method. Resistance to ampicillin, amoxycillin, cephalexin and erythromycin was widespread; resistance to oxytetracycline, tetracycline, nalidixic acid and sulfonamides was common but resistance to chloramphenicol, florfenicol, ceftiofur, cephalothin, cefoperazone, oxolinic acid, gentamicin, kanamycin and trimethoprim was less common. All strains were susceptible to ciprofloxacin. Multiple resistance was also observed and 74·4% of resistant isolates had between one and ten plasmids with sizes ranging 2–51 kbp. Conclusions: No antibiotics are registered for use in aquaculture in Australia but these results suggest that there has been significant off-label use. Significance and impact of study: Transfer of antibiotic resistant bacteria to humans via the food chain is a significant health concern. In comparison with studies on terrestrial food producing animals, there are fewer studies on antibiotic resistance in bacteria from aquaculture enterprises and this study provides further support to the view that there is the risk of transfer of resistant bacteria to humans from consumption of aquaculture products. From the Australian perspective, although there are no products registered for use in aquaculture, antimicrobial resistance is present in isolates from aquaculture and aquaculture environments.

Rhodococcus equi is a significant pathogen in foals predominantly causing a pyogranulomatous bronchopneumonia. Many vaccine candidates have been tested for the prevention of R. equi disease in foals. However, none of these have been developed for widespread commercial use. Previous studies have shown that a Th1 immune response is imperative for the protection of foals against R. equi disease. In this study a DNA and a protein vaccine based upon the well-characterised R. equi virulence-associated protein VapA were developed. The vaccines were tested in the BALB/c murine model and the results showed that both vaccine candidates elicited a Th1-type response in the host. Upon coadministration of an IL-12 expression plasmid with the DNA vaccine, an increase in the Th1 response was observed. However, when mice were challenged with 1.5 x 10(7) virulent R. equi ATCC 33701 none of the vaccinated mice showed protection apart from the mice immunised with live R. equi. These results indicate that despite their immunogenicity the VapA-based DNA and recombinant protein vaccines developed in this study were unable to prevent bacterial replication following a high-dose systemic challenge with virulent R. equi in the BALB/c model.

Land application of animal waste can result in bacterial contamination of shallow groundwater and/or waterways. Using 500-mm-diameter barrel lysimeters, we investigated the potential for bacterial indicators to leach through 4 New Zealand soils treated with dairy shed effluent (DSE). DSE was applied to soil lysimeters containing poorly drained Gley Soils (Te Kowhai and Netherton) and well-drained Allophanic (Waihou) and Pumice Soils (Atiamuri) at 50 mm/h, a typical field application rate used by farmers. Simulated rainfall was applied continuously at a rate of 5–10 mm/h, and leachate collected at a depth of 700 or 750 mm was analysed for faecal coliforms, Escherichia coli, and faecal enterococci. Bacterial indicators of faecal contamination readily moved through the Te Kowhai and Netherton soils, but not the Waihou and Atiamuri soils. Differential microbial movement was attributed to differences in soil structure. The poorly drained soils have coarse subsoil structures with macropores, favouring bypass flow. In contrast, the welldrained soils have a finer, more uniformly porous soil structure that minimised bypass flow and allowed matrix flow.