Small colony variants in Staphylococcus aureus and other species : antibiotic selection, antimicrobial susceptibility, and biofilm formation

Staphylococcus aureus is one of the leading causes of hospital acquired infections. The ability of S. aureus to acquire resistance to a diverse range of antimicrobial compounds, results in limited treatment options, particularly in methicillin-resistant S. aureus. A mechanism by which S. aureus develops reduced susceptibility to antimicrobials is through the formation of small colony variants (SCVs). Reduced antimicrobial susceptibility in S. aureus SCVs is not related to ‘classical’ mechanisms of resistance, but occurs as a direct result of the development of the SCV phenotype. S. aureus SCVs are frequently associated with defects in the bacterial electron transport chain and these defects are responsible for the characteristics associated with the SCV phenotype. This study aimed to investigate and characterise the selection of S. aureus SCVs in the presence of various antibiotics and also to examine their biofilm forming capabilities. Four members of the aminoglycoside family of antibiotics were shown to select for S. aureus SCVs. In addition, a broad range (X 0.25 MIC – X 4 MIC) of aminoglycoside concentrations were shown to select for S. aureus SCVs. Characterisation of these isolates revealed that differences in auxotrophy, biochemical profiles, carotenoid production, haemolysis, levels of intracellular ATP, mutation frequency and reversion rate were present. Members of the tetracycline family of antibiotics were also shown to select for S. aureus SCVs. Tetracycline selected S. aureus SCVs show attenuated catalase, coagulase and heamolysis activity and reduced production of extracellular DNase and lipase and reduced susceptibility to various antimicrobial agents. As SCVs have been linked to persistent and recurrent infections their ability to form biofilms was also investigated. A range of S. aureus SCVs isolated from various backgrounds were shown to form greater biofilms in comparison to parent strains, which was attributed to increased production of polysaccharide intracellular adhesin. In addition S. aureus SCV biofilms displayed a more pronounced reduction in antimicrobial susceptibility, which was attributed to a reduction in antimicrobial penetration through SCV biofilms. Limited discovery of novel antibiotics in recent years and the observation that S. aureus SCVs can be selected for by various antimicrobial compounds highlights the need for novel antimicrobial compounds. Accordingly, an investigation into the susceptibility of S. aureus to various plant compounds was undertaken. Both S. aureus SCVs and parent strains showed susceptibility to five plant antimicrobials tested, of which SCVs were more susceptible to cinnamon bark, green tea and oregano. Resistance to these plant antimicrobials could not be induced and synergistic relationships between certain plant antimicrobials and antibiotics were demonstrated. Finally, formation of SCVs in bacterial species other than S. aureus was examined. Gentamicin induced SCV selection in Escherichia coli, Pseudomonas aeruginosa and S. epidermidis as well as chloroamphenicol and ciprofloxacin in E. coli and tetracycline in S. epidermidis. SCVs from these bacterial species shared common characteristics associated with the SCV phenotype including altered growth and biochemical profiles, auxotrophy for compounds involved in electron transport, reduction in expression of virulence factors and reduced antimicrobial susceptibility. Additionally all SCVs showed an increased capacity to form biofilms. The ability of certain antibiotics to select for SCVs and their increased capacity to form biofilms suggest that SCV are an important adaptation to aid survival and persistence in times of stress. Reduced susceptibility to commonly used antibiotics in SCVs signifies that the development of new antimicrobial compounds is required. Harnessing naturally occurring plant antimicrobials and their synergistic relationship with antibiotics may offer a novel approach to treating antibiotic resistant infections whilst overcoming antibiotic selection for SCVs.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Small colony variants in Staphylococcus aureus and other species: antibiotic selection, antimicrobial susceptibility, and biofilm formation

Staphylococcus aureus is one of the leading causes of hospital acquired infections. The ability of S. aureus to acquire resistance to a diverse range of antimicrobial compounds, results in limited treatment options, particularly in methicillin-resistant S. aureus. A mechanism by which S. aureus develops reduced susceptibility to antimicrobials is through the formation of small colony variants (SCVs). Reduced antimicrobial susceptibility in S. aureus SCVs is not related to ‘classical’ mechanisms of resistance, but occurs as a direct result of the development of the SCV phenotype. S. aureus SCVs are frequently associated with defects in the bacterial electron transport chain and these defects are responsible for the characteristics associated with the SCV phenotype. This study aimed to investigate and characterise the selection of S. aureus SCVs in the presence of various antibiotics and also to examine their biofilm forming capabilities. Four members of the aminoglycoside family of antibiotics were shown to select for S. aureus SCVs. In addition, a broad range (X 0.25 MIC – X 4 MIC) of aminoglycoside concentrations were shown to select for S. aureus SCVs. Characterisation of these isolates revealed that differences in auxotrophy, biochemical profiles, carotenoid production, haemolysis, levels of intracellular ATP, mutation frequency and reversion rate were present. Members of the tetracycline family of antibiotics were also shown to select for S. aureus SCVs. Tetracycline selected S. aureus SCVs show attenuated catalase, coagulase and heamolysis activity and reduced production of extracellular DNase and lipase and reduced susceptibility to various antimicrobial agents. As SCVs have been linked to persistent and recurrent infections their ability to form biofilms was also investigated. A range of S. aureus SCVs isolated from various backgrounds were shown to form greater biofilms in comparison to parent strains, which was attributed to increased production of polysaccharide intracellular adhesin. In addition S. aureus SCV biofilms displayed a more pronounced reduction in antimicrobial susceptibility, which was attributed to a reduction in antimicrobial penetration through SCV biofilms. Limited discovery of novel antibiotics in recent years and the observation that S. aureus SCVs can be selected for by various antimicrobial compounds highlights the need for novel antimicrobial compounds. Accordingly, an investigation into the susceptibility of S. aureus to various plant compounds was undertaken. Both S. aureus SCVs and parent strains showed susceptibility to five plant antimicrobials tested, of which SCVs were more susceptible to cinnamon bark, green tea and oregano. Resistance to these plant antimicrobials could not be induced and synergistic relationships between certain plant antimicrobials and antibiotics were demonstrated. Finally, formation of SCVs in bacterial species other than S. aureus was examined. Gentamicin induced SCV selection in Escherichia coli, Pseudomonas aeruginosa and S. epidermidis as well as chloroamphenicol and ciprofloxacin in E. coli and tetracycline in S. epidermidis. SCVs from these bacterial species shared common characteristics associated with the SCV phenotype including altered growth and biochemical profiles, auxotrophy for compounds involved in electron transport, reduction in expression of virulence factors and reduced antimicrobial susceptibility. Additionally all SCVs showed an increased capacity to form biofilms. The ability of certain antibiotics to select for SCVs and their increased capacity to form biofilms suggest that SCV are an important adaptation to aid survival and persistence in times of stress. Reduced susceptibility to commonly used antibiotics in SCVs signifies that the development of new antimicrobial compounds is required. Harnessing naturally occurring plant antimicrobials and their synergistic relationship with antibiotics may offer a novel approach to treating antibiotic resistant infections whilst overcoming antibiotic selection for SCVs

Dual species dry surface biofilms; Bacillus species impact on Staphylococcus aureus survival and surface disinfection

Abstract: Aims: Dry surface biofilms (DSB) survive on environmental surfaces throughout hospitals, able to resist cleaning and disinfection interventions. This study aimed to produce a dual species DSB and explore the ability of commercially available wipe products to eliminate pathogens within a dual species DSB and prevent their transfer. Methods and Results: Staphylococcus aureus was grown with two different species of Bacillus on stainless steel discs, over 12 days using sequential hydration and dehydration phases. A modified version of ASTM 2967–15 was used to test six wipe products including one water control with the Fitaflex Wiperator. Staphylococcus aureus growth was inhibited when combined with Bacillus subtilis. Recovery of S. aureus on agar from a dual DSB was not always consistent. Our results did not provide evidence that Bacillus licheniformis protected S. aureus from wipe action. There was no significant difference of S. aureus elimination by antimicrobial wipes between single and dual species DSB. B. licheniformis was easily transferred by the wipe itself and to new surfaces both in a single and dual species DSB, whilst several wipe products inhibited the transfer of S. aureus from wipe. However, S. aureus direct transfer to new surfaces was not inhibited post‐wiping. Conclusions: Although we observed that the dual DSB did not confer protection of S. aureus, we demonstrated that environmental species can persist on surfaces after disinfection treatment. Industries should test DSB against future products and hospitals should consider carefully the products they choose. Significance and Impact of the Study: To our knowledge, this is the first study reporting on the production of a dual species DSB. Multispecies DSB have been identified throughout the world on hospital surfaces, but many studies focus on single species biofilms. This study has shown that DSB behave differently to hydrated biofilms

Rapid detection of emerging pathogens and loss of microbial diversity associated with severe lung disease in cystic fibrosis

Copyright © 2015, American Society for Microbiology. All Rights Reserved. Respiratory infection in cystic fibrosis (CF) is polymicrobial, but standard sputum microbiology does not account for the lung microbiome or detect changes in microbial diversity associated with disease. As a clinically applicable CF microbiome surveillance scheme, total sputum nucleic acids isolated by a standard high-throughput robotic method for accredited viral diagnosis were profiled for bacterial diversity using ribosomal intergenic spacer analysis (RISA) PCR. Conventional culture and RISA were performed on 200 paired sputum samples from 93 CF adults; pyrosequencing of the 16S rRNA gene was applied to 59 patients to systematically determine bacterial diversity. Compared to the microbiology data, RISA profiles clustered into two groups: the emerging nonfermenting Gram-negative organisms (eNFGN) and Pseudomonas groups. Patients who were culture positive for Burkholderia, Achromobacter, Stenotrophomonas, and Ralstonia clustered within the eNFGN group. Pseudomonas group RISA profiles were associated with Pseudomonas aeruginosa culture-positive patients. Sequence analysis confirmed the abundance of eNFGN genera and Pseudomonas within these respective groups. Low bacterial diversity was associated with severe lung disease (P < 0.001) and the presence of Burkholderia (P < 0.001). An absence of Streptococcus (P < 0.05) occurred in individuals with lung function in the lowest quartile. In summary, nucleic acids isolated from CF sputum can serve as a single template for both molecular virology and bacteriology, with a RISA PCR rapidly detecting the presence of dominant eNFGN pathogens or P. aeruginosa missed by culture (11% of cases). We provide guidance for how this straightforward CF microbiota profiling scheme may be adopted by clinical laboratories

The unexpected discovery of a novel low-oxygen-activated locus for the anoxic persistence of Burkholderia cenocepacia

Burkholderia cenocepacia is a Gram-negative aerobic bacterium that belongs to a group of opportunistic pathogens displaying diverse environmental and pathogenic lifestyles. B. cenocepacia is known for its ability to cause lung infections in people with cystic fibrosis and it possesses a large 8 Mb multireplicon genome encoding a wide array of pathogenicity and fitness genes. Transcriptomic profiling across nine growth conditions was performed to identify the global gene expression changes made when B. cenocepacia changes niches from an environmental lifestyle to infection. In comparison to exponential growth, the results demonstrated that B. cenocepacia changes expression of over one-quarter of its genome during conditions of growth arrest, stationary phase and surprisingly, under reduced oxygen concentrations (6% instead of 20.9% normal atmospheric conditions). Multiple virulence factors are upregulated during these growth arrest conditions. A unique discovery from the comparative expression analysis was the identification of a distinct, co-regulated 50-gene cluster that was significantly upregulated during growth under low oxygen conditions. This gene cluster was designated the low-oxygen-activated (lxa) locus and encodes six universal stress proteins and proteins predicted to be involved in metabolism, transport, electron transfer and regulation. Deletion of the lxa locus resulted in B. cenocepacia mutants with aerobic growth deficiencies in minimal medium and compromised viability after prolonged incubation in the absence of oxygen. In summary, transcriptomic profiling of B. cenocepacia revealed an unexpected ability of aerobic Burkholderia to persist in the absence of oxygen and identified the novel lxa locus as key determinant of this important ecophysiological trait