Comparative evaluation of enrichment and selective culture strategies for detecting Salmonella from cockroaches

Cockroaches, including the German cockroach (Blattella germanica) can harbor and disseminate enteric human pathogens, such as Salmonella spp. Detecting Salmonella in field-collected cockroach samples is important for monitoring public health risk but is complicated by the low abundance of the pathogen relative to the rich and complex cockroach microbiota. Studies using different culture methods to detect Salmonella in cockroaches have reported disparate results, emphasizing the need for sensitive, accurate strategies to recover and identify Salmonella from infected cockroaches. Here, we evaluated the efficacy of several Salmonella enrichment and selective detection strategies- Rappaport-Vassiliadis (RV) broth, Tetrathionate (TT) broth, and a sequential TT followed by RV enrichment, combined with selective plating on Brilliant Green (BG) or Xylose Lysine Tergitol 4 (XLT-4) agar. Efficacy was assessed on cockroach samples collected one to seven days after experimental infection with a laboratory reference strain of Salmonella. The relative abundance of Salmonella in each enrichment broth was determined by 16S rRNA gene amplicon sequencing, and colony morphologies on selective plates were visually compared. Overall, RV broth, TT broth, and sequential TT-RV broth strategies were equally effective at enriching Salmonella from infected cockroach samples. However, selective plating on XLT-4 was more effective at differentiating Salmonella from commensal Pseudomonas and Serratia spp. that were also enriched in RV and TT broth and produced similar colony phenotypes to Salmonella on BG agar. These results enhance methodological capacity for Salmonella surveillance in cockroaches and highlight the importance of considering the potential for cockroach commensals to produce false positives, interfering with accurate pathogen detection.