Whole genome sequencing and de novo assembly of Staphylococcus pseudintermedius: a pangenome approach to unravelling pathogenesis of canine pyoderma.

BACKGROUND: Staphylococcus pseudintermedius is the main aetiological agent of canine pyoderma. Whole genome sequencing is the most comprehensive way of obtaining relevant genomic information about micro-organisms. HYPOTHESIS/OBJECTIVES: Oxford Nanopore technology enables quality sequencing and de novo assembly of the whole genome of S. pseudintermedius. Whole genome analysis of S. pseudintermedius may help to better understand the pathogenesis of canine pyodermas. METHODS AND MATERIALS: Twenty-two strains of S. pseudintermedius isolated from the skin of five healthy dogs and 33 strains isolated from skin of 33 dogs with pyoderma were analysed. DNA was extracted and sequenced using Oxford Nanopore MinION, a new technology that delivers longer reads in a hand-held device. The pangenome was analysed and visualised with Anvi'o 6.1. RESULTS: Nanopore technology allowed the sequencing and de novo assembly of the genomes of 55 S. pseudintermedius strains isolated from healthy dogs and from dogs with pyoderma. The average genome size of S. pseudintermedius was 2.62 Mbp, with 48% being core genome. Pyoderma isolates contained a higher number of antimicrobial resistance genes, yet the total number of virulence factors genes did not change between isolates from healthy dogs and from dogs with pyoderma. Genomes of meticillin-resistant S. pseudintermedius (MRSP) strains were larger than those of meticillin-susceptible (MSSP) strains (2.80 Mbp versus 2.59 Mbp), as a consequence of a greater presence of antimicrobial resistance genes, phages and prophages. CONCLUSIONS AND CLINICAL IMPORTANCE: This technique allows much more precise and easier characterisation of canine S. pseudintermedius populations and may lead to a better understanding of the pathogenesis of canine pyodermas.

Extraintestinal Pathogenic Escherichia coli: Virulence Factors and Antibiotic Resistance.

The One Health approach emphasizes the importance of antimicrobial resistance (AMR) as a major concern both in public health and in food animal production systems. As a general classification, E. coli can be distinguished based on the ability to cause infection of the gastrointestinal system (IPEC) or outside of it (ExPEC). Among the different pathogens, E. coli are becoming of great importance, and it has been suggested that ExPEC may harbor resistance genes that may be transferred to pathogenic or opportunistic bacteria. ExPEC strains are versatile bacteria that can cause urinary tract, bloodstream, prostate, and other infections at non-intestinal sites. In this context of rapidly increasing multidrug-resistance worldwide and a diminishingly effective antimicrobial arsenal to tackle resistant strains. ExPEC infections are now a serious public health threat worldwide. However, the clinical and economic impact of these infections and their optimal management are challenging, and consequently, there is an increasing awareness of the importance of ExPECs amongst healthcare professionals and the general public alike. This review aims to describe pathotype characteristics of ExPEC to increase our knowledge of these bacteria and, consequently, to increase our chances to control them and reduce the risk for AMR, following a One Health approach.

In Vitro Antibacterial Activity of Biological-Derived Silver Nanoparticles: Preliminary Data.

Silver nanoparticles (AgNPs) are promising alternatives to antibiotics. The aims of this study were to produce AgNPs using two biological methods and determine their antibacterial activity against Pseudomonas aeruginosa and Staphylococcus pseudintermedius. AgNPs were biosynthesized from an infusion of Curcuma longa (turmeric) and the culture supernatant of E. coli. Characterization was achieved by ultraviolet-visible spectroscopy and by Transmission Electron Microscopy (TEM). The antibacterial properties of NPs from C. longa (ClAgNPs) and E. coli (EcAgNPs), alone and in combination with carbenicillin and ampicillin, were investigated through the Kirby-Bauer disk diffusion assay and the minimum inhibitory concentration (MIC). Dimensions of NPs ranged from 11.107 ± 2.705 nm (ClAgNPs) to 27.282 ± 2.68 nm (EcAgNPs). Kirby-Bauer and MIC assays showed great antibacterial abilities for both NPs alone and in combination with antibiotics. EcAgNPs alone showed the most powerful antibacterial activities, resulting in MIC values ranging from 0.438 ± 0.18 µM (P. aeruginosa) to 3.75 ± 3.65 µM (S. pseudintermedius) compared to those of ClAgNPs: 71.8 ± 0 µM (P. aeruginosa) and 143.7 ± 0 µM (S. pseudintermedius). The antibiofilm abilities were strain-dependent, but no statistical differences were found between the two NPs. These results suggest the antibacterial potential of AgNPs for the treatment of infectious diseases.

Investigation on Antibiotic-Resistance, Biofilm Formation and Virulence Factors in Multi Drug Resistant and Non Multi Drug Resistant Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is a commensal bacterium frequently isolated from canine skin and recognized as a zoonotic agent especially for dog-owners. This study focused on (a) the antibiotic-resistance phenotypes; (b) the ability to produce biofilm (slime); and (c) the dissemination of virulence factors in S. pseudintermedius strains. Seventy-three S. pseudintermedius strains were screened for antibiotic-resistance against 22 different molecules by means of Kirby-Bauer assay. The ability to produce biofilm was investigated using the microtiter plate assay (MtP) and the amplification of icaA and icaD genes. Virulence factors such as cytotoxins (lukI), enterotoxins (seC), and exfoliative toxins (siet, expA, and expB) were evaluated. The antibiotic-resistance profiles revealed 42/73 (57%) multi-drug resistant (MDR) strains and 31/73 (43%) not-MDR. All the MDR strains and 8/31 (27%) of not-MDR resulted in biofilm producers. Leukotoxin LukI was found in 70/73 (96%) of the isolates. Moreover, the enterotoxin gene seC was detected in 47/73 (64%) of the strains. All the isolates carried the siet gene, whereas expA and expB were found in 3/73 (4%) and 5/73 (7%), respectively. In conclusion, S. pseudintermedius should be considered a potential zoonotic and human agent able to carry different virulence determinants and capable of producing biofilm which facilitates horizontal gene transfer.

High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

A survey on bacterial involvement in neonatal mortality in dogs.

Bacterial infections represent the second cause of neonatal morbidity and mortality in dogs, so the present study aimed to investigate the bacterial involvement in canine neonatal mortality and to evaluate the antibiotic susceptibility of the isolated bacteria. Fifty-one newborn purebred puppies, born dead or dead within 28 days of age, belonging to 36 different litters, were enrolled and the following procedures were performed on their fresh dead bodies: necropsy, collection of swabs by liver, kidney, lung, small bowel, and possible thoracic and/ or abdominal effusion, for both bacteriological examination and antimicrobial susceptibility testing, and collection of samples by the same organs for histology. About 47% of total swabs were positive at bacteriology (pure bacterial culture or bacterial association). In 65% of the newborn puppies the mortality could be attributed to a bacterial infection. Although the high multidrug resistance, the most effective antimicrobials were third generation cephalosporins and fluorquinolones. In case of neonatal mortality, bacterial culture and antimicrobial susceptibility testing become essential for a targeted therapy in surviving littermates and for the management of following pregnancies in bitches with recurrent neonatal loss.