Charlotte E. Boucher

Bacterial resistance to Quaternary Ammonium Compounds (QAC) disinfectants.

Control of bacterial diseases has, for many years, been dependent on the use of antibiotics. Due to the high levels of efficacy of antibiotics in the past other disease control options have, to a large extent, been neglected. Mankind is now facing an increasing problem with antibiotic resistance. In an effort to retain some antibiotics for human use, there are moves afoot to limit or even ban the use of antibiotics in animal production. The use of antibiotics as growth promoters have been banned in the European Union and the USA. The potential ban on the use of antibiotics to treat diseases in production animals creates a dilemma for man-suffer significant problem with bacterial infection or suffer from a severe shortage of food! There are other options for the control of bacterial diseases. These include vaccine development, bacteriophage therapy, and improved biosecurity. Vaccine development against bacterial pathogens, particularly opportunistic pathogens, is often very challenging, as in many cases the molecular basis of the virulence is not always clearly understood. This is particularly true for Escherichia coli. Biosecurity (disinfection) has been a highly neglected area in disease control. With the ever-increasing problems with antibiotic resistance-the focus should return to improvements in biosecurity. As with antibiotics, bacteria also have mechanisms for resistance to disinfectants. To ensure that we do not replace one set of problems (increasing antibiotic resistance) with another (increasing resistance to disinfectants) we need to fully understand the modes of action of disinfectants and how the bacteria develop resistance to these disinfectants. Molecular studies have been undertaken to relate the presence of QAC resistance genes in bacteria to their levels of sensitivity to different generations of QAC-based products. The mode of action of QAC on bacteria has been studied using NanoSAM technology, where it was revealed that the QAC causes disruption of the bacterial cell wall and leaking of the cytoplasm out of the cells. Our main focus is on the control of bacterial and viral diseases in the poultry industry in a post-antibiotic era, but the principles remain similar for disease control in any veterinary field as well as in human medicine.

Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s.

Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria −42; fungi −19; plant −28; animal −22; plant and animal −1 and common P450 family −1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study’s results offer new understanding of the dynamic structural nature of P450s.

Bacteriophages as Potential Treatment Option for Antibiotic Resistant Bacteria

The world is facing an ever-increasing problem with antibiotic resistant bacteria and we are rapidly heading for a post-antibiotic era. There is an urgent need to investigate alterative treatment options while there are still a few antibiotics left. Bacteriophages are viruses that specifically target bacteria. Before the development of antibiotics, some efforts were made to use bacteriophages as a treatment option, but most of this research stopped soon after the discovery of antibiotics. There are two different replication options which bacteriophages employ. These are the lytic and lysogenic life cycles. Both these life cycles have potential as treatment options. There are various advantages and disadvantages to the use of bacteriophages as treatment options. The main advantage is the specificity of bacteriophages and treatments can be designed to specifically target pathogenic bacteria while not negatively affecting the normal microbiota. There are various advantages to this. However, the high level of specificity also creates potential problems, the main being the requirement of highly specific diagnostic procedures. Another potential problem with phage therapy includes the development of immunity and limitations with the registration of phage therapy options. The latter is driving research toward the expression of phage genes which break the bacterial cell wall, which could then be used as a treatment option. Various aspects of phage therapy have been investigated in studies undertaken by our research group. We have investigated specificity of phages to various avian pathogenic E. coli isolates. Furthermore, the exciting NanoSAM technology has been employed to investigate bacteriophage replication and aspects of this will be discussed.

Pathogenic Gram-positive cocci in South African rainbow trout, Oncorhynchus mykiss (Walbaum)

Fish cultured in aquaculture systems are continuously exposed to pathogens present in the water, soil, air or in the fish (Rottmann, Francis-Floyd & Durborow 1992). The fish are weakened by stress conditions including increased fish density and poor water quality, injury during handling, inadequate nutrition, poor sanitation and increased water temperatures. During the last decade, Gram-positive cocci have become important pathogens causing disease in fish (Eldar & Ghittino 1999). Diseases have been reported from Japan (Hoshina, Sanu & Marimoto 1958), Singapore (Foo, Ho & Lam 1985), Australia (Carson, Gudkovs & Austin 1993), Israel (Eldar, Frealier, Asanta, Varner, Lawhon & Bercovier 1995), Italy (Ghittino & Praero 1992), Spain (Toranzo, Curtin, Romalde, Nunez & Barja 1995; Domenech, Fernandez-Garayzabal, Pasqual, Garcia, Cutuli, Moreno, Collins & Dominguez 1996), France (Michel, Nougayrède, Eldar, Sochon & De Kinkelin 1997), South Africa (Bragg & Broere 1986) and the United States (Perera, Johnson, Collins & Lewis 1994). This includes species from streptococci, lactococci and vagococci (Eldar & Ghittino 1999). In South Africa, a Streptococcus infection caused large numbers of deaths in rainbow trout, Oncorhynchus mykiss (Walbaum), (Bragg & Broere 1986) in the former Eastern Transvaal area. Clinical signs seen in the fish included extreme exophthalmus, often with haemorrhages in the eye chamber, and often resulting in the rupture of one or both eyes. Darkening of the skin pigmentation also occurred. Internal examination showed enlargement of the spleen, and a haemorrhagic intestine filled with a yellowish fluid, while both the stomach and intestine contained food. Only adult fish were affected and the largest, fast growing fish showed the disease first in most cases. These Streptococcus isolates could, however, not be classified (Bragg & Broere 1986). A number of the isolates from the study done by Bragg & Broere (1986) which were preserved by freeze drying were used in the current study. The aims were to identify some of these isolates using phenotypic tests as well as 16S rRNA sequencing. The twelve isolates used in this study are given in Table 1. The freeze-dried isolates were revived in brain heart infusion (BHI) broth (Biolab C114) and incubated under anaerobic conditions at 25 C for 3 days using an anaerobic jar with a gasgenerating kit (Oxoid BR0038). Anaerobic conditions enhanced the growth of the organisms. The purity of the cultures was confirmed by Gram staining. For the phenotypic tests, growth from a 24-h BHI agar slant culture was standardized in 5 mL sterile 1 N phosphate buffer to a density comparable to a McFarland 2 standard (Difco 0691326). Journal of Fish Diseases 2011, 34, 483–487 doi:10.1111/j.1365-2761.2011.01259.x

The Potential Use of Bacteriophage Therapy as a Treatment Option in a Post-Antibiotic Era

The impending postantibiotic era creates an urgent requirement for alternative treatments of infectious diseases in humans and animals. Bacteriophages are viruses that infect and kill bacteria. The application of bacteriophages as a treatment option was investigated before the development of antibiotics. However, the initial success of antibiotic therapy soon shifted the focus from bacteriophage research. The revitalization of phage therapy has received increased global attention since the appearance of multidrug-resistant bacteria. Bacteriophages replicate via either the lytic or lysogenic cycle. While both life cycles have potential applications in bacteriophage therapy, the lytic cycle seems most suited to antibacterial therapy. The most striking advantage of bacteriophage therapy is the high degree of host specificity exhibited by these viruses, which enables the formulation of tailored treatments that kill only pathogenic bacteria. However, the high specificity of such treatments requires highly accurate diagnostic procedures in order to succeed. Other restrictions of bacteriophage therapy, such as limitations with the registration of phage therapy options, may possibly be overcome by the expression and engineering of phage lytic enzymes, which break the bacterial cell wall. The problem of bacterial immunity to phage infection also cannot be ignored, although it is more solvable than resistance to antibiotics. Considering the available information, phage therapy holds promise as an alternative treatment option, although the road ahead is not without obstacles.

Selection of Avibacterium paragallinarum Page serovar B strains for an infectious coryza vaccine

Infectious coryza is an important respiratory disease of chickens around the world and is caused by Avibacterium paragallinarum. Among the three Page serovars currently recognized for this bacterium, serovar B is a major circulating serovar in China nowadays. The cross-protection ability of the Page serovar B reference strain (0222) and five local isolates was evaluated by a vaccination-challenge trial in SPF chickens. The clinical signs seen in control birds challenged by strain 0222 and isolate HB 01 were significantly different, with isolate HB 01 giving more severe clinical signs. In terms of cross-protection, the protection in the groups vaccinated with isolate HB 01 and BJ 02 was significantly higher than that in the groups vaccinated with 0222 and the other three isolates. In addition, an experimental oil adjuvant trivalent vaccine, containing field isolate HB 01 antigen, was compared for immune efficacy with two commercial trivalent infectious coryza vaccines containing internationally recognized serovar B strains. The experimental oil adjuvant trivalent vaccine elicited best protection (80%) among the three trivalent vaccines. In conclusion, the oil adjuvant vaccine, containing field isolate HB 01 may be a better choice in control of current serovar B Av. paragallinarum outbreaks in China under current circumstances.

Detection of virulence factors of South African Lactococcus garvieae isolated from rainbow trout, Oncorhynchus mykiss (Walbaum)

Lactococcus garvieae is a Gram-positive bacterium that causes mortalities in freshwater and marine fish worldwide and therefore results in severe economic losses in the aquaculture industry. Apart from the apparent integral role of the exopolysaccharide (EPS) capsule in pathogenesis, factors associated with virulence of this bacterium are poorly understood. However, recent studies have indicated that the ability of L. garvieae to cause disease does not depend on the presence of the EPS capsule. Lack of knowledge of virulence factors, pathogenesis and serology of L. garvieae is an impediment to the development of effective typing methods and control measures. This study, therefore, aimed to detect the presence of EPS capsules and other putative virulence factors in South African L. garvieae fish pathogenic isolates and a non-virulent isolate, and to identify possible candidates for subunit vaccine development. No indication of the presence of the EPS capsule was detected by negative staining or amplification of the EPS biosynthesis gene cluster in the virulent isolates or the avirulent strain, discrediting the notion that the EPS capsule is the sole determinant of virulence. However, a set of putative virulence factor genes was detected in all isolates, and candidates for subunit vaccine development (enolase, lactate dehydrogenase phosphoenolpyruvate-protein phosphotransferase) were identified by identification of extracellular proteins of virulent strains.

Evaluation of the ERIC-PCR as a probable method to differentiate Avibacterium paragallinarum serovars

ABSTRACT Infectious coryza, an upper respiratory tract disease in chickens, caused by Avibacterium paragallinarum, leads to huge economic losses. The disease is controlled through vaccination; but vaccination efficacy is dependent on correct identification of the infecting serovar, as limited cross-protection is reported amongst some serovars. Current identification methods include the heamagglutination inhibition test, which is demanding and could be subjective. To overcome this, molecular typing methods proposed are the Multiplex polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism-PCR, but low reproducibility is reported. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR has been suggested for molecular groupings of various bacterial species. This study focuses on evaluating the ERIC-PCR as a probable method to differentiate between different Av. paragallinarum serovars by grouping with reference isolates, based on clonal relations. The ERIC-PCR was performed on 12 reference isolates and 41 field isolates originating from South Africa and South America. The data indicate that the ERIC-PCR is not ideal for the differentiation or for molecular typing of Av. paragallinarum serovars, as no correlation is drawn upon comparison of banding patterns of field isolates and reference strains. However, the results do indicate isolates from the same origin sharing unique banding patterns, indicating potential clonal relationship; but when compared to the reference isolates dominant in the specific area, no correlation could be drawn. Furthermore, although the ERIC-PCR serves a purpose in epidemiological studies, it has proved to have little application in differentiating amongst serovars of Av. paragallinarum and to group untyped field strains with known reference strains.

Regulation of chicken immunity-related genes and host response profiles against Avibacterium paragallinarum pathogen challenge

Infectious coryza (IC) is a well-recognised and commonly encountered upper respiratory tract disease in chickens. The aim of this study was to monitor aspects of the immune response of chickens infected with Avibacterium paragallinarum. Gene expression profiling of 30 genes was carried out for 11 chicken nasal area samples belonging to four groups, including one non-infected control group. For this purpose, 30 biomarker transcripts were selected for comparative gene expression analysis and were analysed by real-time PCR using TaqMan(®) assays. The biomarkers included three reference genes. The reference genes were used to normalise the results in a relative quantification approach. The gene expression changes of the 27 biomarker transcripts (genes of interest) were quantified between all treated groups in six pair-wise comparisons. It was concluded from the data that immune response initiation is via TLR4, which leads to a Th2 dominant type response. Furthermore, TLR4 results in signalling via the MyD88-dependent pathway, resulting in early onset of NF-kβ leading to the production of inflammatory cytokines. This work provides an informative outlay of immune response initiation upon infection with this pathogen.