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- W. Van Schaik (United Kingdom)
Ecology and Genomics of Antibiotic Production in Streptomyces
- G. P. Van Wezel (Netherlands)
Abstract
Abstract Body
Central in this talk is Streptomyces, a filamentous soil bacterium with a complex life cycle that reproduced via sporulation. Streptomycetes and other members of the Actinobacteria produce some two thirds of all known antibiotics and a range of other natural products and enzymes. The treasures that lie hidden in the actinomycete genomes may well be our final resource in the fight against the rapidly emerging multi-drug resistant pathogens. Many of the biosynthetic gene clusters (BGCs) for antibiotics are poorly expressed in the laboratory, while they are likely expressed in nature.
We harness host-microbe interactions to activate antibiotic production. Where BigPharma has routinely screened bacteria in isolation, in nature bacteria live in complex communities with other organisms, and these often competitive interactions elicit specific responses involving the production of natural products. We discover novel antibiotics by combining multi-omics approaches, such as genome mining, transcriptomics, proteomics and metabolomics.
We have discovered a range of chemical elicitors and growth conditions that allow the effective activation of silent BGCs for specialized metabolites. Application of our multi-omics platform identified several new antibiotics, including the Lugdunomycins, a class of compounds with new chemical scaffold, and a novel subfamily of lantibiotics.
Thus, understanding the ecological conditions under which antibiotic-producing streptomycetes live is a key factor in approaches to activate their production and discover novel bioactive molecules. This, combined with an efficient paired omics drug discovery platform, identified several novel bioactive molecules. Novel drug-discovery approaches and antibiotics we have discovered will be discussed.
EVALUATION OF A REAL-TIME PCR ARRAY TO DETECT MULTIPLE ANTIMICROBIAL RESISTANCE GENES IN VARIETY OF FOOD WITH SUBSEQUENT CONFIRMATION AND BACTERIAL IDENTIFICATION USING NANOPORE-SEQUENCING
- S. Naushad (Canada)
Abstract
Background and Aims
Microorganisms with antimicrobial resistance genes (ARGs) present in food is a public health concern worldwide. We evaluated a commercial real-time PCR array (Qiagen) that detects 87 clinically relevant ARGs for use in food for rapid and routine monitoring of ARGs.
Methods
Microbes from 63 food samples (meat=18, produce=(vegetables=21 and fruits=9) and dairy=15) collected in local retail in Canada were enriched (24 h at 37°C) using modified Schaedler media, followed by genomic DNA extraction, AMR genes detection, and identification of bacterial species by Nanopore MinION based metagenome sequencing.
Results
Among 63 food samples, 33 ARGs had at least one occurrence in all food groups with potential resistance against aminoglycoside, fluoroquinolone, tetracycline, macrolide and Class C beta-lactamase. Eight ARGs including erythromycin resistance genes were not detected. Some ARGs had unique distribution and were detected in one group of samples or one source of food, such as 4 genes including vanB (Vancomycin resistance) were detected in meat only, similarly 12 and 4 ARGs were detected in produce and dairy, respectively. Highest average number of ARGs were detected in meat (14 ARGs/sample), followed by produce (10 ARGs/sample) and dairy products (7 ARGs/sample). Nanopore sequencing confirmed the results of PCR and identified 23 bacterial genera in 20 representative samples.
Conclusions
In conclusion, we presented useful baseline data on ARGs presence in foods in Canada, and determined that this PCR array is a useful tool for routine ARG surveillance for food. MinION based metagenomics sequencing helps to identify bacterial species and establish link between AGRs and potential bacterial hosts.
QUANTIFYING CONCENTRATIONS OF AND RESISTANCE SELECTION BY ANTIMICROBIAL RESIDUES UNDER IN VIVO CONDITIONS IN BROILERS
- A. F. Swinkels (Netherlands)
Abstract
Background and Aims
Antimicrobial usage in livestock is one of the main drivers of antimicrobial resistance, especially due to the exposure of the gut microbiome to antimicrobials. Depending on their stability, some antimicrobials can persist in the farm environment after animal treatment. We examined if persistence of the antimicrobial is a factor in the selection for antimicrobial resistant bacteria in the gut microbiota of broilers, birds with coprophagic behaviour.
Methods
Four groups of broilers were divided in three subgroups (n=12). Groups were treated with amoxicillin (non-persistent), doxycycline or enrofloxacin (persistent), and an untreated control group. Faecal droppings and caecal material were collected at different time points after the treatment. Baseline measurement occurred before the treatment. Shotgun metagenomics was performed on the faecal material to determine the resistome. Phenotypic resistance analysis of E. coli isolates was conducted by plating on MacConkey agar with appropriate antimicrobials. Antimicrobials were extracted from the faecal samples and analysed by LC-MS/MS.
Results
We observed higher concentrations of persistent antimicrobials (doxycycline and enrofloxacin) over time after treatment than the non-persistent antimicrobial amoxicillin. Furthermore, doxycycline treatment resulted in a larger increase in the number of resistance genes over time than amoxicillin treatment. The group treated with enrofloxacin showed a slow but continuous increase of resistance genes after treatment. Phenotypically, the same difference was observed between the persistent and non-persistent antimicrobial treatment groups.
Conclusions
Persistent antimicrobials remain longer in the farm environment with a longer selection pressure. Therefore persistence of antimicrobials should be used in the assessment of priority classification of antimicrobials.
INVESTIGATING THE ROLE OF TRYPTOPHANASE IN E. COLI BIOFILMS
- C. Croft (United Kingdom)
Abstract
Background and Aims
Tryptophanase (TnaA), an enzyme present in many species of bacteria, is best known for the conversion of L-tryptophan to indole, pyruvate, and ammonia. However, TnaA can also metabolise other amino acids. We recently observed that a clumping phenotype seen during the exponential growth phase of a uropathogenic E. coli strain (ATCC® 25922™) was absent in a tnaA knock-out.
Methods
A tnaA knock-out, its derivative (ATCC® 25922™), and UTI clinical isolates were assayed for biofilm development in 96-well plates. Novel inhibitors of TnaA identified in our laboratory were then screened for their effects on biofilms.
Results
The clumping phenotype, which is characteristic of early-stage biofilms, was reduced by novel TnaA inhibitors. The compounds are of interest because they could lead to the use of TnaA inhibitors as a combination therapy with existing antibiotics to mitigate recurrent UTIs and biofilms.
Conclusions
We originally assumed that the absence of clumping in the mutant was due to the loss of indole production since previous studies indicated that indole influences biofilm development. Upon closer inspection, we found that the absence of L-tryptophan in growth medium had no effect on the phenotype, but the absence of L-arginine prevented clumping. These observations shed new light on the role of TnaA and suggest a route towards a more comprehensive understanding, and therefore treatment, of biofilm formation in uropathogenic E. coli.
COMPARATIVE ANALYSIS OF THE IMPACT OF DIFFERENT ANTIMICROBIALS ON CAMPYLOBACTER, SALMONELLA, AND E. COLI
- N. Nasser (United States of America)
Abstract
Background and Aims
The occurrence and persistence of foodborne pathogens on food products could be partially attributed to increases in resistance/tolerance to antimicrobials that are commonly used during food processing. Here, we assessed the impact of different antimicrobials, including organic acids, chlorine and peracetic acid (PAA), on different strains of Campylobacter, Salmonella and Shiga-toxin producing E. coli.
Methods
The minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined using a combination of the broth microdilution, kinetic growth, and colony counting assays. The antimicrobials were also tested against artificially-contaminated chicken meat and lettuce.
Results
The organic acids (formate, succinate, fumarate) had the lowest MICs (250-500mM) and MBCs (≥250mM) against Campylobacter. However, formate was superior to the other organic acids when considering solubility and Campylobacter control. The Salmonella and E. coli were notably less susceptible to the organic acids. Growth inhibition of all bacteria tested required high concentrations of chlorine (≥ 400ppm) while the inhibitory PAA concentrations varied considerably among bacterial species and strains (50- 200ppm). Furthermore, formate (1M) was insufficient in eliminating the bacteria on chicken breasts and lettuce. However, significant reductions in bacterial loads were noted at ≥ 200ppm PAA (1log CFU/g) and at ≥ 200ppm chlorine (0.5log CFU/g) for chicken samples. Lettuce decontamination showed a varying impact for PAA (50-200ppm) which depended on the bacterial species tested, with maximum reduction of 4.5 log CFU/g. Chlorine (≥ 100ppm) also significantly reduced bacterial counts (2log CUF/g) on lettuce.
Conclusions
It appears that certain bacterial strains can tolerate/resist antimicrobial concentrations that are commonly used during food production.