Transcriptomic signature of bacteria exposed to benzalkonium chloride.

The COVID-19 pandemic has highlighted our reliance on biocides, the increasing prevalence of resistance to biocides is a risk to public health. Bacterial exposure to the biocide, benzalkonium chloride (BAC), resulted in a unique transcriptomic profile, characterised by both a short and long-term response. Differential gene expression was observed in four main areas: motility, membrane composition, proteostasis, and the stress response. A metabolism shift to protect the proteome and the stress response were prioritised suggesting these are main resistance mechanisms. Whereas "well-established" mechanisms, such as biofilm formation, were not found to be differentially expressed after exposure to BAC.

The Hormetic Effect Observed for Benzalkonium Chloride and Didecyldimethylammonium Chloride in Serratia sp. HRI.

Hormesis, or the hormetic effect, is a dose- or concentration-dependent response characterised by growth stimulation at low concentrations and inhibition at high concentrations. The impact of sub-lethal levels of disinfectants on the growth of Serratia species is critical to understanding the increasing number of outbreaks caused by this pathogen in healthcare settings. Serratia sp. HRI and Serratia marcescens ATCC 13880 were cultivated in sub-lethal levels of benzalkonium chloride (BAC), Didecyldimethylammonium chloride (DDAC), and VirukillTM. The maximum specific growth rates, doubling times, and cell counts were compared. The results revealed significant increases in maximum specific growth rates and shorter doubling times for Serratia sp. HRI when cultivated in sub-lethal levels of BAC and DDAC. The significant stimulatory effect of sub-lethal levels of these disinfectants for Serratia sp. HRI represents the first time hormesis has been observed in a Gram-negative bacterium for any disinfectant. Furthermore, this study is the first to observe the hormetic effect after treatment with DDAC and the second study to date analysing the impact of sub-lethal levels of disinfectants on the growth of bacterial species.

Genomic Islands Identified in Highly Resistant Serratia sp. HRI: A Pathway to Discover New Disinfectant Resistance Elements.

Molecular insights into the mechanisms of resistance to disinfectants are severely limited, together with the roles of various mobile genetic elements. Genomic islands are a well-characterised molecular resistance element in antibiotic resistance, but it is unknown whether genomic islands play a role in disinfectant resistance. Through whole-genome sequencing and the bioinformatic analysis of Serratia sp. HRI, an isolate with high disinfectant resistance capabilities, nine resistance islands were predicted and annotated within the genome. Resistance genes active against several antimicrobials were annotated in these islands, most of which are multidrug efflux pumps belonging to the MFS, ABC and DMT efflux families. Antibiotic resistance islands containing genes encoding for multidrug resistance proteins ErmB (macrolide and erythromycin resistance) and biclomycin were also found. A metal fitness island harbouring 13 resistance and response genes to copper, silver, lead, cadmium, zinc, and mercury was identified. In the search for disinfectant resistance islands, two genomic islands were identified to harbour smr genes, notorious for conferring disinfectant resistance. This suggests that genomic islands are capable of conferring disinfectant resistance, a phenomenon that has not yet been observed in the study of biocide resistance and tolerance.

Heterologous expression and characterisation of a keratinase produced by Chryseobacterium carnipullorum.

Chryseobacterium carnipullorum 9_R23581T, isolated from raw chicken meat, was evaluated for its potential to degrade keratin found in feathers. The focus of this study was to heterologously express and characterise a keratinolytic enzyme produced by C. carnipullorum. Chryseobacterium carnipullorum secretes proteolytic enzymes that have feather degrading capabilities during its exponential growth phase. This study concluded that the most likely main component of the keratinolytic enzymes of C. carnipullorum was peptidase M64, a serine-endopeptidase with a molecular weight in crude form of 49.46 kDa. Primers were designed on the selected gene of interest, which was amplified from the genome of C. carnipullorum (accession number NZ-FRCD01000002.1). The gene coding for peptidase M64 was further cloned, propagated and expressed in E. coli BL21 [DE3] cells. Purification was by Immobilised Metal Affinity Chromatography (IMAC). The molecular weight of the keratinase was about 50 kDa after purification while its optimum temperature and pH were 50 °C and 8.5, respectively. The activity of this keratinase was inhibited by phenylmethylsulfonyl fluoride (PMSF) and it was enhanced by the presence of divalent metal ions such as Mg2+ and Ca2+. Enzyme activity was further assayed by application to chicken feathers and observed degradation was an indication of keratinolytic potential.

Genome Sequence of Resistant Serratia sp. Strain HRI, Isolated from a Bottle of Didecyldimethylammonium Chloride-Based Disinfectant.

Antimicrobial resistance is a significant issue, and it threatens the prevention and effective treatment of a range of bacterial infections. Here, we report the whole-genome sequence of the multidrug-resistant isolate Serratia sp. strain HRI. A hybrid assembly was created using sequences from a first (MiSeq) and second (PacBio) sequencing run. This work is imperative for understanding antimicrobial resistance and adds to the knowledge base for combating multidrug-resistant bacteria.

Molecular basis of bacterial disinfectant resistance.

Antibiotic resistance could accelerate humanity towards an already fast-approaching post-antibiotic era, where disinfectants and effective biosecurity measures will be critically important to control microbial diseases. Disinfectant resistance has the potential to change our way of life from compromising food security to threatening our medical health systems. Resistance to antimicrobial agents occurs through either intrinsic or acquired resistance mechanisms. Acquired resistance occurs through the efficient transfer of mobile genetic elements, which can carry single, or multiple resistance determinants. Drug resistance genes may form part of integrons, transposons and insertions sequences which are capable of intracellular transfer onto plasmids or gene cassettes. Thereafter, resistance plasmids and gene cassettes mobilize by self-transmission between bacteria, increasing the prevalence of drug resistance determinants in a bacterial population. An accumulation of drug resistance genes through these mechanisms gives rise to multidrug resistant (MDR) bacteria. The study of this mobility is integral to safeguard current antibiotics, disinfectants and other antimicrobials. Literature evidence, however, indicates that knowledge regarding disinfectant resistance is severly limited. Genome engineering such as the CRISPR-Cas system, has identified disinfectant resistance genes, and reversed resistance altogether in certain prokaryotes. Demonstrating that these techniques could prove invaluable in the combat against disinfectant resistance by uncovering the secrets of MDR bacteria.

Regulation of outer-membrane proteins (OMPs) A and F, during hlyF-induced outer-membrane vesicle (OMV) biosynthesis.

BACKGROUND: Gram-negative bacteria actively secrete outer membrane vesicles into the surrounding environment and these vesicles have been shown to play various physiological and protective roles such as carrying antibiotic-degrading enzymes and acting as decoys against host defences, therefore promoting the pathogenesis of the bacterium. It has been shown that avian pathogenic Escherichia coli species can increase vesicle biosynthesis through the acquisition of the hlyF gene but the effect this has on the cell by scavenging outer-membrane associated proteins (OmpA, OmpF) into the vesicles during vesicle release have not yet been investigated. RESULTS: Relative quantitative real-time PCR data obtained from hlyF expressing and non-expressing cells showed that during hlyF induction, ompF showed a nearly 2-fold down regulation relative to the non-expressing cells during the entire 24 hours, while ompA was expressed at the same level as the non-expressing cells during the first 8 hours of expression. At 24 hours post-hlyF expression, ompA was up-regulated 4-fold. CONCLUSIONS: The regulatory effects of the newly described outer-membrane vesicle biosynthesis-related gene, hlyF, on E. coli has not previously been investigated. As hlyF-induced vesicles contain OmpA and OmpF scavenged from the bacterial outer-membrane, potential regulatory effects on the host was investigated. An increase in ompA expression and an insignificant decrease in ompF expression was observed during hlyF induction demonstrating that hlyF-related biosynthesis is not related to decreased ompA expression, which is one of the potential mechanisms discussed in literature for biosynthesis. Outer-membrane vesicle biosynthesis during hlyF over-expression could potentially be accomplished through a different mechanism(s).

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.

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.

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

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.

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.

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-kß leading to the production of inflammatory cytokines. This work provides an informative outlay of immune response initiation upon infection with this pathogen.

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.

Transcriptional profiling of chicken immunity-related genes during infection with Avibacterium paragallinarum.

Avibacterium paragallinarum is the causative agent of Infectious Coryza (IC), which is an upper respiratory tract disease in chickens. The occurrence of outbreaks has emphasized the significance of the disease globally in the chicken industry. Studies have demonstrated that early immune responses are critical in defining the severity and physiological outcome of an infection. This prompted the need to investigate the regulation of immune functions by the number of genes that are expressed during the chickens' response to A. paragallinarum serovar C3 insult. This study consisted of 15 male leghorn birds that were scored into groups (score 1, 2, 3) according to severity of symptoms after they were challenged. Expression patterns of immunity-related genes were followed as symptoms progressed from a disease score of 1 to 3. The data proposed that initial pathogen recognition was either through Toll-like receptors 2 or 4. Unique expression patterns were observed such as the up-regulation of TLR7 which recognizes viral-like particles. This substantiated the presence of prophages reported in the genome of A. paragallinarum. Significant down-regulation of metabolic pathways was observed, which led us to hypothesize that the host may rely on an oxidative stress response as initial immune response. The data sheds light onto the mechanisms that govern the immune system towards infection and/or towards the initial response to infections with highly virulent A. paragallinarum.

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.