Extended period of selection for antimicrobial resistance due to recirculation of persistent antimicrobials in broilers.

OBJECTIVES: Antimicrobials can select for antimicrobial-resistant bacteria. After treatment the active compound is excreted through urine and faeces. As some antimicrobials are chemically stable, recirculation of subinhibitory concentrations of antimicrobials may occur due to coprophagic behaviour of animals such as chickens. METHODS: The persistence of three antimicrobials over time and their potential effects on antimicrobial resistance were determined in four groups of broilers. Groups were left untreated (control) or were treated with amoxicillin (unstable), doxycycline or enrofloxacin (stable). Antimicrobials were extracted from the faecal samples and were measured by LC-MS/MS. We determined the resistome genotypically using shotgun metagenomics and phenotypically by using Escherichia coli as indicator microorganism. RESULTS: Up to 37 days after treatment, doxycycline and enrofloxacin had concentrations in faeces equal to or higher than the minimal selective concentration (MSC), in contrast to the amoxicillin treatment. The amoxicillin treatment showed a significant difference (P ≤ 0.01 and P ≤ 0.0001) in the genotypic resistance only directly after treatment. On the other hand, the doxycycline treatment showed approximately 52% increase in phenotypic resistance and a significant difference (P ≤ 0.05 and P ≤ 0.0001) in genotypic resistance throughout the trial. Furthermore, enrofloxacin treatment resulted in a complete non-WT E. coli population but the quantity of resistance genes was similar to the control group, likely because resistance is mediated by point mutations. CONCLUSIONS: Based on our findings, we suggest that persistence of antimicrobials should be taken into consideration in the assessment of priority classification of antimicrobials in livestock.

Genetic Characterization of Mutations Related to Conidiophore Stalk Length Development in Aspergillus niger Laboratory Strain N402.

Aspergillus niger is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the A. niger N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of A. niger, the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of A. niger strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed cspA and cspB) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 (cspA) was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the A. nidulans UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 (cspB) was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.

Spatial structure increases the benefits of antibiotic production in Streptomyces.

Bacteria in the soil compete for limited resources. One of the ways they might do this is by producing antibiotics, but the metabolic costs of antibiotics and their low concentrations have caused uncertainty about the ecological role of these products for the bacteria that produce them. Here, we examine the benefits of streptomycin production by the filamentous bacterium Streptomyces griseus. We first provide evidence that streptomycin production enables S. griseus to kill and invade the susceptible species, S. coelicolor, but not a streptomycin-resistant mutant of this species. Next, we show that the benefits of streptomycin production are density dependent, because production scales positively with cell number, and frequency dependent, with a threshold of invasion of S. griseus at around 1%. Finally, using serial transfer experiments where spatial structure is either maintained or destroyed, we show that spatial structure reduces the threshold frequency of invasion by more than 100-fold, indicating that antibiotic production can permit invasion from extreme rarity. Our results show that streptomycin is both an offensive and defensive weapon that facilitates invasion into occupied habitats and also protects against invasion by competitors. They also indicate that the benefits of antibiotic production rely on ecological interactions occurring at small local scales.