Nitrogen concentrations in watersheds increased with numbers of poultry barns in and around the watershed. • Cephalosporin-resistant E. coli in streams was positively correlated with the estimated poultry manure exposure. • Studying small watersheds has the potential to reveal sources of antibiotic resistant bacteria and nutrient pollution.
Rapid molecular typing methods are important tools in surveillance and outbreak investigations of human Salmonella infections. Here we described the development of a three-genes PCR-RFLP typing method for the differentiation of Salmonella species, subspecies and serovars using the Agilent 2100 Bioanalyzer. The fliC, gnd, and mutS genes were PCR-amplified in 160 Salmonella strains representing the two Salmonella species, six subspecies, and 41 different serovars of S. enterica subspecies enterica. PCR products were individually cut with two different restriction enzymes and the resulting 930 restriction patterns were collected using the Agilent 2100 Bioanalyzer followed by cluster analysis. Both species of Salmonella were differentiated by conventional PCR. All of S. bongori tested were gnd PCR negative due to a mismatch at the 3′-end in one the PCR primers. Salmonella subspecies were differentiated into third-teen homogeneous groups representing each of the six subspecies by cluster analysis of restriction patterns generated from the mutS gene cut with AciI. S. enterica subspecies enterica serovars were further differentiated by the combination of the three target genes and five out the six sets of restriction patterns with a discriminatory power of 0.9725 by cluster analysis. The combined RFLP results of five sets of restriction patterns allowed us to assign each of the 160 strains to one of 128 restriction types. During inoculation studies we were able to identify S. Saintpaul and Typhimurium from 24 h pre-enrichment samples using the described method. The use of fliC, gnd, and mutS PCR-RFLP with the Agilent 2100 Bioanalyzer can provide an accessible and automated alternative method for differentiation of Salmonella pathogens.
RNA viruses can exchange genetic material during coinfection, an interaction that creates novel strains with implications for viral evolution and public health. Influenza A viral genetic exchange occurs when genome segments from distinct strains reassort in coinfection. Predicting potential reassortment between influenza strains is a longstanding goal. Experimental coinfection studies have shed light on factors that limit or promote reassortment. However, determining the reassortment potential between diverse Influenza A strains has remained an elusive goal. To fill this gap, we developed a high throughput genotyping approach to quantify reassortment among a diverse panel of human influenza virus strains, encompassing 41 years of epidemics, multiple geographic locations, and both circulating human subtypes A/H1N1 and A/H3N2. We found that the reassortment rate (proportion of reassortants) is an emergent property of a pair of strains where strain identity is a predictor of the reassortment rate. We show little evidence that antigenic subtype drives reassortment as intersubtype (H1N1xH3N2) and intrasubtype reassortment rates were, on average, similar. Instead, our data suggest that certain strains bias the reassortment rate up or down, independently of the coinfecting partner. We also observe that viral productivity is an emergent property of coinfections and that it is not correlated to reassortment rate, thus affecting the total number of reassortant progeny produced. Assortment of individual segments among progeny, and pairwise segment combinations within progeny, were not random and generally favored homologous combinations. This outcome was not related to strain similarity or shared subtype. Reassortment rate was closely correlated to both the proportion of unique genotypes and the proportion of progeny with heterologous pairwise segment combinations. We provide experimental evidence that viral genetic exchange is potentially an individual social trait subject to natural selection, which implies the propensity for reassortment is not evenly shared among strains. This study highlights the need for research incorporating diverse strains to discover the traits that shift the reassortment potential as we work towards the goal of predicting influenza virus evolution resulting from segment exchange.
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