Molecular epidemiology of coagulase-negative Staphylococcus species isolated at different lactation stages from dairy cattle in the United States

Abstract

Background: Coagulase negative Staphylococcus (CNS) species are currently the most prevalent intra-mammary pathogens causing subclinical mastitis and occasional clinical mastitis or persistent infection in lactating dairy cattle. More than 10 CNS species have been identified, but they are generally managed as one group on most dairies in the United States. However, improved management decisions and treatment outcomes may be achieved with better understanding of the prevalent species, pathogenicity and strain diversity within and across dairies.

Methodology: A total of 604 CNS isolates were cultured from milk samples collected during a dry-cow treatment clinical trial conducted on 6 dairy herds in 4 states in the US. All the study cows were randomized to receive 1 of the 3 different intra-mammary antimicrobial infusions (Quatermaster, Spectramast DC or ToMorrow Dry Cow) at dry-off. Milk samples were collected at dry-off, calving (0-6 days in milk, DIM), post-calving (7-13 DIM) and at mastitis events within the first 100 DIM. The CNS isolates were identified to species level by partial sequencing of the rpoβ gene, and genetic relatedness within species was investigated by phylogenetic analysis of the pulse-field gel electrophoresis profiles of the isolates.

Results: The major CNS species identified were S. chromogenes (48.3%), S. haemolyticus (17.9%), S. simulans and S. epidermidis (each at 6.5%). Other CNS species identified at lower frequencies included S. hominis, S. auricularis, S. sciuri, S. spp KS-SP, S. capitis, S. cohnii, S. warneri, S. pasteuri, S. xylosus, S. hyicus, S. equorum, S. microti, S. rostri, S. gallinarum, S. saprophyticus and S. succinus. Phylogenetic analyses of the major species types demonstrated an association between genetic relatedness and epidemiological distributions of S. chromogenes, S. simulans, S. haemolyticus and S. auricularis. Additionally, identical strains of S. chromogenes and S. simulans were isolated from the same udder quarter of several cows at consecutive sample stages. The rest of the minor species had no deducible genetic-epidemiological link.

Discussion: The observed association between genetic and epidemiological distributions indicated animal-adapted nature of four CNS species, suggesting possible host-adapted and environmental transmission of these species. Multi-stage isolation of the same udder quarter strain was evidence for chronic intra-mammary infection.

Conclusion: The different CNS species and strains circulating on US dairy herds were genetically diverse. Four species identified were likely udder-adapted pathogens, 2 of which caused persistent infection. Our findings are important in guiding the design of effective mastitis control strategies.

Keywords: Coagulase negative Staphylococcus (CNS); Epidemiology; Genetic relatedness; Mastitis pathogens; Phylogeny; Pulse-field gel electrophoresis (PFGE).

Figure 1. Genetic relatedness of coagulase negative Staphylococcus spp. (CNS) isolated from US dairies.

The unweighted pair group method with arithmetic mean (UPGMA) phylogenetic genetic trees were constructed based on PFGE banding patterns. Similarity scores are shown at the tree branches. (A) Staphylococcus epidermidis strains clustered into two clades (A and B) and three distinct isolates. The isolates showed wide genetically diversity and heterogenous distribution between the five dairies. (B) Staphylococcus auricularis strains had least genetic diversity among the isolates. The isolates clustered into 1 clade, except for a single isolate from a California dairy. The rest of the isolates were from Midwestern dairies. Within the clade, most of the isolates had close genetic relationship (>80% similarity) and two isolate pairs were identical (100% similarity). (C) Staphylococcus hominis strains had wide genetic diversity and clustered into 5 clades (A, B, C, D and E) and a single distinct isolate. Clade A had two closely related isolates from WI and a possibly related isolate from IA. Clade B contained identical isolates from two MN and IA. Clade C had three identical isolates from IA. Clade D was the most diverse with isolates from all the states (CA, IA, MN and WI). Clade E contained identical isolates from IA and the rest of the isolates were from WI. (D) Staphylococcus sciuri. S. sciuri clustered into 1 clade and 3 distinct isolates. The group had wide genetic diversity and none of the isolates were closely related. Dairies: CA1, California 1; CA2, California2; IA, Iowa; MN, Minnesota; WI or WI1, Wisconsin1. Udder quarters: RF, Right front; LF, Left front; RR, Right rear; LR, Left rear.

Figure 2. Genetic relatedness of coagulase-negative Staphylococcus spp. (CNS) isolated from US dairies.

The unweighted pair group method with arithmetic mean (UPGMA) phylogenetic trees were constructed based on PFGE banding patterns. (A) Staphylococcus simulans. All the S. simulans strains were isolated from Midwestern dairies and had close genetic relationships, constituting one clade and a two distinct strains from IA. Six cows had indistinguishable isolated from the same udder quarter at calving (S2) and 5 days post calving (S3) (black boxes). The S2 and S3 isolates from Cow 1712 and 5132 had close genetic relationship, but the S1 (dry-off) and S2 isolates from Cow ID 9012 were genetically distant (red boxes). (B) Staphylococcus cohnii strains clustered into a single clade and one distinct isolate. Two isolates from IA and WI1 were genetically indistinguishable. (C) Staphylococcus capitis strains had wide genetic diversity with genetically distinct strains, except two isolates from MN and CA2 that showed possible genetic relationship. Dairies: CA2, California 2; IA, Iowa; MN, Minnesota; WI or WI1, Wisconsin 1. Udder quarter: RF, Right front; LF, Left front; RR, Right rear; LR, Left rear. Similarity scores are shown at the tree branches.

Figure 3. Phylogenetic cluster analysis of Staphylococcus haemolyticus strains isolated from US dairies.

The multidimensional network was created by agglomeration scheme from unweighted pair group method with arithmetic means (UPGMA) dendrogram based on PFGE banding pattern. The S. haemolyticus isolates formed six major heterogenous clusters (1–6) comprised of strains from the different dairies. The branch lengths labels indicate the genetic distance and isolates with zero distance are clustered together at the end node. Dairies: CA1, California 1; CA2, California 2; IA, Iowa; MN, Minnesota; WI1, Wisconsin 1.

Figure 4. Phylogenetic cluster analysis of Staphylococcus chromogenes strains isolated from US dairies.

The multidimensional network was created by agglomeration scheme from unweighted pair group method with arithmetic means (UPGMA) dendrogram based on PFGE banding pattern. The isolates formed 10 major heterogenous clusters (numbered 1–10) that encompassed strains from the different dairies. Clusters 9 and 10 had the most number of isolates with close genetic distances or genetically identical isolates. The branch labels indicate genetic distance and isolates with zero distance are clustered together at the node. Dairies: CA1, California 1; CA2, California 2; IA, Iowa; MN, Minnesota; WI1, Wisconsin 1.

Figure 5. Persistence of Staphylococcus chromogenes intramammary infection in lactating cows on US dairies.

The phylogenetic tree for the repeat isolate strains (n = 48) was created using unweighted pair group method with arithmetic means (UPGMA) method, based on PFGE band patterns. The strains were isolated from quarter milk samples at dry off (S1), calving (S2), 5 days post calving (S3) and at any clinical mastitis event (CM) within 100 days in milk (DIM). Eight cows (black boxes) had genetically indistinguishable strains isolated from the same udder quarter at two different stages (three cows had S1/S2 each, one cow had a S1/S3 isolate pair, and three cows S2/S3 isolates each, and one cow had 2 S2/3 isolates from different quarters). Two cows each had a pair of genetically indistinguishable strains isolated from different quarters at the same sample stage (blue boxes). Twelve cows had related strains isolated from 1 or 2 udder quarters at the same stage or different stages (red boxes and connecting red lines). Only one cow had CM isolate that was closely related to S1 isolate and possibly related to S3 isolate of the same quarter. Dairies: CA1, California 1; CA2, California 2; MN, Minnesota; WI1, Wisconsin 1. Udder quarters: LF, Left Front; RF, Right Front; RR, Right Rear; LR, Left Rear.