Relation between the puppy microbiome and the dam’s microbial flora

Early microbial colonization of the gastrointestinal tract plays a crucial role in the development and health of neonatal mammals. In the dog, current knowledge on microbiota acquisition and its influencing factors remains limited. This doctoral project investigated maternal microbial transfer and gut microbiota development in puppies, combining classical culture methods and nextgeneration sequencing (NGS) techniques, and integrating complementary studies on nutrition and antimicrobial resistance. The first phase involved a culture-based approach to study bacterial colonization in the meconium and feces of puppies, and its relationship with maternal microbial reservoirs. Samples were collected longitudinally from six dam–litter units (3 Lagotto Romagnolo and 3 Appenzeller Cattle Dogs) housed in the same kennel conditions. Bacterial isolates were obtained from the dams’ rectum, vagina, and mammary secretions, and from the puppies’ rectum at days 0, 2, 30, and 60. Meconium samples collected before colostrum intake and dam contact showed cultivable bacteria in 93.3% of cases. Enterococcus spp., Escherichia coli, and Staphylococcus pseudintermedius were the most commonly identified species. Overlap between dam and litter isolates increased with age, suggesting progressive maternal transmission. A total of 48 microbial overlaps were identified between dam and litter isolates, supporting vertical transmission, while cluster analysis showed that samples from the same litter became more similar over time. The second part employed 16S rRNA NGS to investigate bacterial DNA and to assess microbial diversity in the same biological sites of the previous experiment. The first NGS study focused on meconium samples collected at birth, demonstrating bacterial DNA in all of them, even before maternal contact and colostrum intake. Lower alpha diversity and unique clustering of meconium samples distinguished them from the dam’s rectal, vaginal, and mammary microbiota. However, shared taxa suggested vertical seeding during parturition. The second NGS study followed 24 puppies from 4 dam–litter units over the first month of life. A temporal shift was observed, from Proteobacteria dominance to increasing Firmicutes. Alpha diversity increased with age, and ordination plots showed family-specific clustering. Results supported rapid maturation of the gut microbiota and the central role of maternal microbial imprinting. Complementary studies evaluated the influence of maternal diet on neonatal growth. Pregnant dams were assigned to either a standard mixed-protein diet or a limited-ingredient diet (LID). Puppies from LID-fed dams had higher normalized birth weight but showed lower weight gain by day 60. These results suggest that both prenatal nutrition and postnatal microbial dynamics affect neonatal growth trajectories. A final collateral investigation compared antimicrobial resistance in rectal and perivulvar isolates of Escherichia coli and Staphylococcus pseudintermedius from 25 bitches housed in breeding kennels and 25 household ones. Resistance to doxycycline and cephalosporins was significantly higher in kennel dogs, with increased detection of ESBL-producing E. coli and methicillin-resistant S. pseudintermedius isolates. These findings highlight environmental and antimicrobial use-related risks in breeding settings. In conclusion, this dissertation confirms that puppies are colonized by bacteria before maternal care begins. The dam’s microbiota—especially vaginal and mammary—is a primary source of early microbial seeding. Microbial colonization follows individual and family-specific paths, even under uniform conditions. The potential risk of increased bacterial resistance in kennels suggests the need of promoting breeders’ awareness of the effects of antimicrobials use on bacterial populations homeostasis and on bacterial resistance profile.