Exploring Diversity and Origins of Streptomycin-Resistant Erwinia amylovora Isolates in New York Through CRISPR Spacer Arrays

Abstract

Streptomycin is the most effective and widely used chemical control in the eastern United States for blossom blight of apple caused by Erwinia amylovora; however, resistance to this antibiotic has been a concern in New York since 2002. From 2011 to 2014, statewide collections of E. amylovora were conducted resulting in the isolation of streptomycin-resistant (SmR) E. amylovora from several commercial orchards. Further genetic analysis of isolates was necessary to understand the origins and the diversity of these bacteria. Clustered regularly interspaced short palindromic repeat (CRISPR) spacer sequencing was employed to explore the diversity and possible origins of New York SmR E. amylovora isolates. The spacer array CR1, CR2, and CR3 regions of 27 SmR E. amylovora isolates and 76 streptomycin-sensitive (SmS) E. amylovora isolates were amplified and subsequently sequenced, revealing 19 distinct CRISPR spacer profiles for New York isolates. The majority of SmR E. amylovora isolates had the same CRISPR profile as SmR E. amylovora isolates discovered in 2002. This may infer that eradication efforts in 2002 failed and the bacterial populations continued to spread throughout the state. Several CRISPR profiles for SmR E. amylovora were identical to SmS E. amylovora collected from the same orchards, leading to the hypothesis that resistance may be developing within New York. Profiles not unique to New York were identical to many isolates from the Midwestern, eastern, and western United States, implying that streptomycin resistance may be due to the introduction of SmR E. amylovora from other regions of the United States. The increased understanding as to how SmR E. amylovora isolates are introduced, evolve, or have become established afforded by CRISPR profiling has been useful for disease management and restricting the movement of streptomycin resistance in New York.