A bacterial culture collection from stable flies and manure relevant to bovine mastitis

TL;DR

This paper introduces a collection of bacteria from stable flies and cow manure, which could help understand bovine mastitis and bacterial interactions.

Contribution

The novel contribution is a curated bacterial collection with genomic and antimicrobial data relevant to bovine mastitis.

Findings

Over 1,100 bacterial isolates were collected from stable flies and cow manure.

Genome assemblies and antimicrobial susceptibility data are available for some isolates.

Abstract

We present a collection of over 1,100 bacterial isolates from hematophagous Stomoxys flies and cow manure. Genome assemblies and antimicrobial susceptibility data are available for a subset of isolates. This resource supports studies of bacterial ecology, host-vector interactions, and potential roles in bovine mastitis.

Full text

ANNOUNCEMENT

Stable flies of the genus Stomoxys (Diptera: Muscidae) are common blood-feeding pests of dairy cows and other livestock. They have long been recognized as mechanical vectors of manure-borne bacterial pathogens and antimicrobial resistance genes (1–4). More recently, these flies have also been implicated in the transmission of mastitis-causing bacteria in dairy environments, particularly through their frequent contact with manure and contaminated surfaces (2–4). Despite these associations, the phylogenetic and functional diversity of their microbiota remain poorly characterized.

To address this gap, we present a bacterial culture collection derived from Stomoxys flies, along with the publicly available draft genome assemblies of bacteria isolated from these insects, as detailed in a companion article to this announcement (5). This resource enables computational and experimental investigations into fly-bacteria interactions and their implications for veterinary health, with particular relevance to the ecology and transmission of environmental mastitis pathogens.

Isolates were collected from flies, manure, and mastitic cows housed at two interconnected dairy farms in Wisconsin, USA. Stomoxys flies were captured using adhesive alsynite fiberglass traps (Olson Products, Medina, OH, USA), and manure samples were obtained from both scraper systems and cattle pens between July and September 2021. Flies were washed in PBS-T (1× PBS with 0.01% Tween 80), and exogenous bacteria were recovered from the wash solution following centrifugation. Endogenous bacteria were isolated from fly homogenates prepared by bead-beating pooled flies with 3 × 5 mm^2^ stainless steel beads. Field samples—including diluted feces, fly homogenates, and fly washes—were plated on various nutrient agars, including MacConkey, Sorbitol-MacConkey, Mannitol Salt, Brain Heart Infusion, Tryptic Soy, blood, and Eosin Methylene Blue (EMB). Strains from mastitic cows were obtained from archived samples maintained by the Wisconsin Veterinary Diagnostic Laboratory (Madison, Wisconsin, USA). Detailed sampling and isolation procedures are described in previous studies (2–5).

The collection comprises 909 fly-derived, 206 manure-derived, and 12 mastitic cow-derived taxonomically identified isolates, including clinically relevant taxa within the Bacillales, Enterobacterales, and Pseudomonadales (Table 1). Species-level identification is available for the 296 isolates with draft genome assemblies. The remaining isolates were identified via Sanger sequencing of the 16S rRNA gene, phenotypic methods (e.g., colony morphology on EMB agar, where Escherichia coli typically produces a metallic green sheen), or PCR amplification using genus-specific primers.

TABLE 1: Collection composition and sequencing overview

DNA extractions for strains selected for whole genome sequencing were performed using either the NucleoSpin Tissue kit (Macherey-Nagel, Nordrhein-Westfalen, Germany) for samples processed by SeqCoast Genomics (Portsmouth, NH, USA) or the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) for samples processed by the UW-Madison Biotechnology Center, following standard manufacturer protocols. Genomic DNA from 190 isolates sent to SeqCoast Genomics was prepared using the Illumina DNA Prep Tagmentation Kit and sequenced on the NextSeq2000 platform, generating 2 × 150 bp paired-end reads with an average of 1.3 million reads per sample. DNA from 106 isolates sent to the UW-Madison Biotechnology Center was prepared using the Celero EZ DNA-Seq Prep Kit (Tecan Genomics, Männedorf, Switzerland) and sequenced on the Illumina NovaSeq6000 platform, producing 2 × 150 bp paired-end reads with approximately 1 million reads per sample.

Sequence read trimming was performed using DRAGEN v3.10.12 for SeqCoast Genomics samples and Trimmomatic v0.39 (6) for UW-Madison samples. Draft genome assemblies were generated using SPAdes v3.15.5 and annotated with PROKKA v 1.13 using the “rfam” setting (7, 8). Quality control was conducted throughout the workflow. Sequencing data quality was assessed using FastQC v0.11.9, QUAST v5.2.0, CheckM v1.0.18, and BUSCO v 5.7.1 (9–12). Summary statistics for sequence quality metrics are provided in Table 1.

Isolates from the Staphylococcaceae and Enterobacteriaceae were screened for antimicrobial susceptibility using CLSI disk diffusion guidelines (4). Notably, the collection includes fly-derived strains of E. coli, Klebsiella pneumoniae, and Enterobacter spp. carrying extended-spectrum beta-lactamase (ESBL) genes conferring resistance to ceftiofur—a key antibiotic used to treat bovine mastitis and other infections on U.S. dairy farms (13). These findings underscore the potential role of flies in the environmental dissemination of mastitis-associated pathogens and resistance determinants.

We encourage the scientific community to use this collection to investigate host-microbe interactions, bacterial dispersal by muscid flies, and the environmental reservoirs of mastitis pathogens. Comparative genomic analyses have already revealed shared strains and plasmids between flies and manure (5). In addition, the collection supports colonization and related studies using field-derived strains, which often retain genotypic and phenotypic traits absent in domesticated laboratory strains—traits that are essential for capturing the complexity of natural microbial associations relevant to mastitis ecology.