DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract total bacterial DNA from environmental samples based on the sodium hydroxide lysis of cells with or without capture by magnetic beads for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in as little as 10 min at room temperature with dilution, or the DNA can be further purified using silica-coated paramagnetic beads. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrated that an impinger and portable pump can efficiently capture bacteria from the air in poultry facilities for rapid NaOH extraction to quantify total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well suited for routine, high-throughput screening and for metagenomic analyses for specific pathogens, even in resource-limited situations.

DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract genomic DNA from environmental samples based on sodium hydroxide lysis of cells with or without capture by magnetic beads, for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in 10 minutes at room temperature. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrate that an impinger and portable pump can efficiently capture bacteria in poultry facilities for rapid DNA extraction for quantification of total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well-suited for routine, high throughput screening, and for metagenomic analyses for specific pathogens, even in resource-limited situations.

S. agnetis has been previously associated with subclinical or clinically mild cases of mastitis in dairy cattle and is one of several Staphylococcal species that have been isolated from the bone and blood of lame broilers. We were the first to report that S. agnetis could be obtained frequently from bacterial chondronecrosis with osteomyelitis (BCO) lesions of lame broilers. Further, we showed that a particular isolate of S. agnetis, chicken isolate 908, can induce lameness in over 50% of exposed chickens, far exceeding normal BCO incidences in broiler operations. We have previously reported the assembly and annotation of the genome of isolate 908. To better understand the relationship between dairy cattle and broiler isolates, we assembled 11 additional genomes for S. agnetis isolates, including an additional chicken BCO strain, and ten isolates from milk, mammary gland secretions or udder skin, from the collection at the University of Missouri. To trace phylogenetic relationships, we constructed phylogenetic trees based on multi-locus sequence typing, and Genome-to-Genome Distance Comparisons. Chicken isolate 908 clustered with two of the cattle isolates along with three isolates from chickens in Denmark and an isolate of S. agnetis we isolated from a BCO lesion on a commercial broiler farm in Arkansas. We used a number of BLAST tools to compare the chicken isolates to those from cattle and identified 98 coding sequences distinguishing isolate 908 from the cattle isolates. None of the identified genes explain the differences in host or tissue tropism. These analyses are critical to understanding how Staphylococci colonize and infect different hosts and potentially how they can transition to alternative niches (bone vs dermis).Importance: Staphylococcus agnetis has been recently recognized as associated with disease in dairy cattle and meat type chickens. The infections appear to be limited in cattle and systemic in broilers. This report details the molecular relationships between cattle and chicken isolates in order to understand how this recently recognized species infects different hosts with different disease manifestations. The data show the chicken and cattle isolates are very closely related but the chicken isolates all cluster together suggesting a single jump from cattle to chickens.