Pseudintermedius Isolates Research Articles

Diversity and Risk Factors Associated with Multidrug and Methicillin-Resistant Staphylococci Isolated from Cats Admitted to a Veterinary Clinic in Eastern Province, Saudi Arabia.

Understanding the distribution, antimicrobial resistance (AMR), and risk factors associated with multidrug-resistant (MDR) and methicillin-resistant staphylococci (MRS) isolated from cats admitted to veterinary clinics may decrease the risk of MDR and MRS transmission to humans and other cats. As such, the objectives of this study were to investigate the diversity in Staphylococcus spp. recovered from different anatomical locations in healthy and diseased cats and to determine the occurrence of MDR and MRS spp. as well as possible risk factors associated with colonization in these cats. Five swabs were collected from the anus, skin, ear canal, conjunctival sac, and nares of each cat (209 healthy and 191 diseased) admitted to a veterinary clinic in Eastern Province, Saudi Arabia, between January and December 2018. Prior to sample collection, cat owners completed a questionnaire collecting information on cat demographics, health status, management, and antimicrobial usage. In total, 179 Staphylococcus isolates were recovered from healthy (n = 71) and diseased (n = 108) cats, including 94 (52.5%) coagulase-positive staphylococci (CoPS), and 85 (47.5%) coagulase-negative staphylococci (CoNS). Five Staphylococcus spp. were identified, namely, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus felis, Staphylococcus capitis, and Staphylococcus saprophyticus. Staphylococcus isolates were most commonly resistant to penicillin (56.4%) and ciprofloxacin (25.7%); however, no isolate was resistant to clindamycin. Thirty (16.8%) Staphylococcus spp. (24 S. aureus and 6 S. pseudintermedius) isolates were MDR, with resistance to up to six different antibiotic classes. Only 17 (9.5%) Staphylococcus spp. (15 methicillin-resistant S. aureus and 2 methicillin-resistant S. pseudintermedius) harbored the mecA gene. Risk factor analysis showed that cats with a history of antibiotic therapy, those raised mainly indoors with a child, and those who visit a veterinary clinic for treatment were at higher risk of MDR and MRS colonization. In conclusion, MDR and MRS were common in healthy and diseased cats in Saudi Arabia. Thus, an effective antimicrobial stewardship program and further studies using a One Health approach are required to investigate the role of cats as vectors for AMR transmission to humans.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017–2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Whole genome sequencing of coagulase positive staphylococci from a dog-and-owner screening survey

BackgroundStaphylococcus aureus and S. pseudintermedius are the two most common coagulase positive staphylococci (CPS). S. aureus is more prevalent among humans, whereas S. pseudintermedius is more commonly isolated from dogs, however, both can cause various community and hospital acquired diseases in humans.MethodsIn the current study we screened 102 dogs and 84 owners in Hungary. We tested the antibiotic susceptibility of the strains and in order to get a better picture of the clonal relationship of the strains, we used pulsed-field gel electrophoresis. In addition, three pairs of isolates with identical PFGE patterns were whole genome sequenced, MLST and spa types were established.ResultsCarriage rate of S. aureus was 23.8% in humans and 4.9% in dogs and two cases of co-carriage were found among dogs and owners. S. pseudintermedius carriage rate was 2.4% and 34.3%, respectively, with only one co-carriage. The isolates were generally rather susceptible to the tested antibiotics, but high tetracycline resistance of S. pseudintermedius strains was noted. The co-carried isolates shared almost the same resistance genes (including tet(K), bla(Z), norA, mepR, lmrS, fosB) and virulence gene pattern. Apart from the common staphylococcal enzymes and cytotoxins, we found enterotoxins and exfoliative toxins as well. The two S. aureus pairs belonged to ST45-t630, ST45-t671 and ST15-t084, ST15-t084, respectively. The co-carried S. pseudintermedius isolates shared the same housekeeping gene alleles determining a novel sequence type ST1685.ConclusionsBased on the genomic data, dog-owner co-carried strains displayed only insignificant differences therefore provided evidence for potential human-to-dog and dog-to-human transmission.

MOLECULAR AND BIOCHEMICAL CHARACTERIZATION OF STAPHYLOCOCCUS PSEUDINTERMEDIUS FROM CANINE PYODERMA IN SHIVAMOGGA REGION OF KARNATAKA

Shivamogga. Exudate/pus/lesion swabs were collected from clinical cases of canine pyoderma (n=126) and subjected to isolation and identification of bacterial isolates by phenotypic methods. The bacteriological processing of the samples resulted in the recovery of 95 staphylococcal isolates and 18 other bacterial isolates. On culture, staphylococci were the most predominantly (n=95, 75.39%) isolated organisms. The PCR was employed as molecular method in this study for the detection of species of staphylococcal isolates by targeting nuc gene and it was also used for the detection of virulence gene and antibiotic resistance gene in staphylococcal isolates by targeting siet gene and mecA gene, respectively, by using primers published earlier. One of the S. pseudintermedius isolates which confirmed by PCR and sequencing of partial nuc gene was used as positive reference strain for further screening of isolates by PCR. Based on nuc gene-based PCR, out of 95 staphylococcal isolates obtained, 82 (86.1%)of the isolates were found belonging to S. pseudintermedius. And out of 82 S. pseudintermedius isolates, siet gene was detected in 69 (86.1%) isolates. S. pseudintermedius was found to be predominant bacterial pathogen responsible for pyoderma in dogs.

SEQUENCE ANALYSIS OF siet gene IN STAPHYLOCOCCUS PSEUDINTERMEDIUS ISOLATEDFROM CANINE PYODERMA

Astudy was conducted on isolation, identification and characterization of staphylococcal organisms associated with the canine pyoderma. The bacteriological processing of the samples resulted in the recovery of 95 staphylococcal isolates and 18 other bacterial isolates. On culture, staphylococci were the most predominantly (n=95, 75.39%) isolated organisms. Based on nucgene-based PCR, out of 95 staphylococcal isolates obtained, 82 (86.1%) of the isolates were found belonging to S. pseudintermedius. And out of 82 S. pseudintermedius isolates, siet gene was detected in 69 (86.1%) isolates. S. pseudintermedius was found to be predominant bacterial pathogen responsible for pyoderma in dogs. Two PCR products which were amplified for nuc gene and one each of siet and mec A gene were sent for commercial sequencing and the sequence alignment revealed 99.23 per cent, 99.14 per cent, 98.98 per cent and 100 per cent, similarities with the corresponding genes of S. pseudintermedius respectively.

Frequency of Staphylococcus pseudintermedius in Canine Skin Infections and Antibiotic Resistance Profiles of the Recovered Isolates

In this study, 61 Staphylococcus pseudintermedius strains were isolated from 77 dogs with skin infections. Antimicrobial resistance to commonly used antibiotics was evaluated by disc diffusion method. The presence of the blaZ and mecA genes responsible respectively for penicillin and methicillin resistance were investigated by PCR. The S. pseudintermedius isolates were highly resistant to tetracycline (47.5%) and penicillin (40.9%) while all isolates were susceptible to amikacin (100%). All 18 methicillin-resistant S. pseudintermedius (MRSP) strains were positive for the mecA gene while the 25 S. pseudintermedius strain isolates with the blaZ gene were also resistant to penicillin phenotypically. Of the 18 MRSP isolates, 14 S. pseudintermedius strains were resistant to three or more antibiotics, indicating that these strains showed multiple drug resistance (MDR). This report confirms previous findings that S. pseudintermedius is the most frequently isolated bacteria from dog skin infections. It can also be concluded that amikacin is a useful agent for treating S. pseudintermedius infections since all the S. pseudintermedius strains tested in this study were susceptible to amikacin.

Lytic Activity of Polyvalent Staphylococcal Bacteriophage PhiSA012 and Its Endolysin Lys-PhiSA012 Against Antibiotic-Resistant Staphylococcal Clinical Isolates From Canine Skin Infection Sites.

The spread of antibiotic-resistant bacteria (ARB) in human and veterinary medicine is of global concern. Notably, the emergence of methicillin-resistant Staphylococcus pseudintermedius has become a serious problem. In this context, bacteriophages and their lytic enzymes, endolysins, have received considerable attention as therapeutics for infectious diseases in place of antibiotics. The aim of the present study was to investigate the antibiotic-resistance patterns of staphylococcal species isolated from canine skin at a primary care animal hospital in Tokyo, Japan and evaluate the lytic activity of the staphylococcal bacteriophage phiSA012 and its endolysin Lys-phiSA012 against isolated antibiotic-resistant staphylococcal strains. Forty clinical staphylococcal samples were isolated from infection sites of dogs (20 from skin and 20 from the external ear canal). Susceptibility to antimicrobial agents was determined by a disk diffusion method. The host range of phiSA012 was determined by using a spot test against staphylococcal isolates. Against staphylococcal isolates that showed resistance toward five classes or more of antimicrobials, the lytic activity of phiSA012 and Lys-phiSA012 was evaluated using a turbidity reduction assay. Twenty-three S. pseudintermedius, 16 Staphylococcus schleiferi, and 1 Staphylococcus intermedius were detected from canine skin and ear infections, and results revealed 43.5% methicillin resistance in S. pseudintermedius and 31.3% in S. schleiferi. In addition, the prevalence multidrug resistance (MDR) S. pseudintermedius was 65.2%. PhiSA012 could infect all staphylococcal isolates by spot testing, but showed little lytic activity by turbidity reduction assay against MDR S. pseudintermedius isolates. On the other hand, Lys-phiSA012 showed lytic activity and reduced significantly the number of staphylococcal colony-forming units. These results demonstrated that ARB issues underlying in small animal hospital and proposed substitutes for antibiotics. Lys-phiSA012 has broader lytic activity than phiSA012 against staphylococcal isolates; therefore, Lys-phiSA012 is a more potential candidate therapeutic agent for several staphylococcal infections including that of canine skin.

Population genomics of Staphylococcus pseudintermedius in companion animals in the United States

Staphylococcus pseudintermedius is a commensal bacterium and a major opportunistic pathogen of dogs. The emergence of methicillin-resistant S. pseudintermedius (MRSP) is also becoming a serious concern. We carried out a population genomics study of 130 clinical S. pseudintermedius isolates from dogs and cats in the New England region of the United States. Results revealed the co-circulation of phylogenetically diverse lineages that have access to a large pool of accessory genes. Many MRSP and multidrug-resistant clones have emerged through multiple independent, horizontal acquisition of resistance determinants and frequent genetic exchange that disseminate DNA to the broader population. When compared to a Texas population, we found evidence of clonal expansion of MRSP lineages that have disseminated over large distances. These findings provide unprecedented insight into the diversification of a common cutaneous colonizer of man’s oldest companion animal and the widespread circulation of multiple high-risk resistant clones.

Isolation and Molecular Characterization of Methicillin-Resistant Staphylococcus pseudintermedius in dogs

Isolation and Molecular Characterization of Methicillin-Resistant Staphylococcus pseudintermedius (MRSP) in dogs were carried out. A total of 25 ear swabs were collected aseptically from both diseased (otitis externa) and apparently healthy dogs (for vaccination) presented to teaching Veterinary Clinical Complex, Rajiv Gandhi Institute of Veterinary Education and Research (RIVER), Puducherry. On analysis of 25 ear swab samples, a total of 14 (56%) Staphylococcus pseudintermedius (S. pseudintermedius) isolates were recovered, 6 (24%) isolates from healthy, and 8 (32%) isolates from diseased dogs. Among the 14 S. pseudintermedius isolates, eight isolates for mecA, which is 21.40% (3/14), and 35.70% (5/14) of MRSP isolates were detected in healthy and diseased dogs, respectively. The present study highlighted the presence of MRSP in dogs in Puducherry region.

Molecular and phenotypic characterization of methicillin-resistant Staphylococcus pseudintermedius from ocular surfaces of dogs and cats suffering from ophthalmological diseases

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) have recently emerged as a major therapeutic challenge in small animal medicine because of their antimicrobial multidrug resistance and their role as nosocomial pathogens. This study focused on the prevalence, molecular characteristics and antimicrobial resistance pheno- and genotypes of MRSP isolated from conjunctival swabs of dogs and cats. Conjunctival swabs were collected from 72 dogs and 24 cats suffering from conjunctivitis/blepharitis, keratitis or uveitis and screened for the presence of MRSP. S. pseudintermedius was isolated from 38 (39.6 %) of all samples. Three (7.9 %) S. pseudintermedius isolates were confirmed as MRSP. They harboured the mecA gene and originated from dogs. One MRSP isolate was from a case of uveitis while the other two MRSP isolates originated from cases of conjunctivitis/blepharitis. All MRSP isolates were subjected to broth microdilution and whole genome sequencing (WGS). Resistance and virulence genes, multilocus sequence (MLS), spa, dru and SCCmec types were deduced from WGS data. Two of the three MRSP isolates, IMT360/16 and IMT515/16, shared the same MLS type (ST71), spa type (t02), dru type (dt9a), SCCmec type (II-III), and indistinguishable multidrug resistance pheno- and genotypes, including resistance to β-lactams (blaZ, mecA), erythromycin and clindamycin (erm(B)), streptomycin (aphA3), gentamicin (aacA-aphD), enrofloxacin (mutations in grlA and gyrA), tetracycline (tet(K)), and trimethoprim (dfrG)/sulfamethoxazole. The third isolate, IMT1670/16, differed in all those characteristics (MLST (ST1403), dru type (dt10h), SCCmec type (IVg), except the spa type (t02). In addition, isolate IMT1670/16 carried a different tetracycline resistance gene (tet(M)) and was susceptible to erythromycin and clindamycin.

Investigation of the in vitro antimicrobial activity of triclosan-coated suture material on bacteria commonly isolated from wounds in dogs.

To investigate in vitro effects of triclosan coating of suture materials on the growth of clinically relevant bacteria isolated from wounds in dogs. 6 types of suture material and 10 isolates each of methicillin-susceptible Staphylococcus pseudintermedius, methicillin-resistant S pseudintermedius, Escherichia coli, and AmpC β-lactamase and extended-spectrum β-lactamase-producing E coli from clinical wound infections. Isolates were cultured on Mueller-Hinton agar with 3 types of triclosan-coated suture, uncoated counterparts of the same suture types, and positive and negative controls. Zones of inhibition (ZOIs) were measured after overnight incubation. Sustained antimicrobial activity assays were performed with susceptible isolates. The ZOI measurements and durations of sustained antimicrobial activity were compared among suture types and isolates by statistical methods. Suture surface characteristics and bacterial adherence were evaluated qualitatively with scanning electron microscopy. ZOIs were generated only by triclosan-coated materials; triclosan-coated suture had sustained antimicrobial activity (inhibition) for 3 to 29 days against all tested pathogens. The ZOIs around triclosan-coated suture were significantly greater for S pseudintermedius isolates than for E coli isolates. Bacterial adherence to uncoated polyglactin-910 was greatest, followed by triclosan-coated polyglactin-910, and then uncoated monofilament sutures, with least adherence to coated monofilament sutures. Surface characteristics of suture materials may be as important or more important than triclosan coating for microbial inhibition; however, triclosan coating appeared to affect bacterial adherence for multifilament sutures. Triclosan-coated, particularly monofilament, sutures inhibited pathogens commonly isolated from wounds of dogs, including multidrug-resistant bacteria. Further studies are required to assess clinical efficacy of triclosan-coated suture materials in vivo.

Profiles of Enterotoxin Genes and Antimicrobial Resistance in <i>Staphylococcus pseudintermedius</i> Strains Isolated from Livestock and Companion Animals

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and is recognized as a zoonotic pathogen causing public health concern. Although canine-associated S. pseudintermedius has mainly been recognized for its antimicrobial resistance and ability to cause skin infections in dogs, information on antimicrobial resistance profiles and enterotoxigenicity of S. pseudintermedius in livestock is very limited. In this study, we investigated the prevalence of 18 different staphylococcal enterotoxin (SE) genes and toxic shock syndrome toxin gene (tst-1) in S. pseudintermedius strains isolated from dogs, pigs, and beef cattle. Moreover, antimicrobial resistance profiles of the strains were determined along with the presence of mecA and SCCmec types. Except for one bovine isolate, all S. pseudintermedius isolates from dogs and pigs were resistant to multiple drugs (≥ 4 different drugs). Four out of six canine isolates were methicillin resistant and carried SCCmec type V. In addition, 11 different SE genes (seb, sec, see, seg, sei, sej, sel, seo, sep, seq, and seu) and tst-1 were identified in S. pseudintermedius isolates from dogs, pigs, and beef cattle. Most S. pseudintermedius isolates (83%) harbored multiple SE genes, and sel (42%) and sep (42%) were most frequently detected in the isolates. Our results suggested that S. pseudintermedius isolates from livestock and companion animals may serve as a reservoir for SE genes and antimicrobial resistance.

Diagnostic accuracy of phenotypic assays for determining antimicrobial resistance status in Staphylococcus pseudintermedius isolates from canine clinical cases

This study evaluated the diagnostic test accuracy of disc diffusion relative to broth-microdilution for clinical Staphylococcus pseudintermedius isolated from dogs in Australia (n = 614). Accuracy of disc diffusion and broth-microdilution for oxacillin relative to mecA real-time PCR was also assessed. Each isolate had paired minimum inhibitory concentration and zone diameter values for ten antimicrobial agents. Data was dichotomised using Clinical and Laboratory Standards Institute susceptible and resistant clinical breakpoints. Test accuracy was reported using relative diagnostic sensitivity (RSe), specificity (RSp), likelihood ratio pairs, diagnostic odds ratio, and area-under-the receiver-operating characteristic (ROC AUC) analysis. Disc diffusion was found to have high test accuracy for most antimicrobials (ROC AUC range: 0.96 – 0.99) except rifampicin (ROC AUC = 0.80). The RSp of disc diffusion was high for all antimicrobials (range, 97.1%–100%). However, RSe was considerably variable (range, 35.7%–98.8%), particularly for amoxicillin-clavulanic acid (51.5%, 95% CI, 38.9%, 64.0%), cefoxitin (35.7%, 95% CI, 12.8%, 64.9%), and cephalothin (43.6%, 95% CI, 27.8%, 60.4%). When disc diffusion and broth-microdilution were compared to mecA real-time PCR, the overall accuracy of both assays was similar (ROC AUC, 0.99 respectively). However, the RSe for broth-microdilution (96.1%, 95% CI, 88.9%, 99.2%) was significantly higher than for disc diffusion (86.8%, 95% CI, 77.1%, 93.5%) (McNemars mid-p value 0.01). Overall, these findings demonstrate that for most antimicrobials, disc diffusion performed according to CLSI guidelines can be used to differentiate clinical S. pseudintermedius isolates that might otherwise be assessed by broth-microdilution, provided consideration is given to the performance estimates reported here.

EUCAST disc diffusion criteria for the detection of mecA-Mediated β-lactam resistance in Staphylococcus pseudintermedius: oxacillin versus cefoxitin

ObjectivesUntil recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated β-lactam resistance in Staphylococcus pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius. MethodsWe included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-μg cefoxitin and 1-μg oxacillin discs from three manufacturers and Mueller–Hinton agar from two manufacturers. ResultsCefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6–20 mm and 19–30 mm. For cefoxitin 16% (95% CI 14.8–18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6–2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) <20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively. ConclusionsThis investigation confirms that the 1-μg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-μg cefoxitin disc. For a 1-μg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter <20 mm are resistant to all β-lactam antibiotics except those with activity against methicillin-resistant staphylococci.

Pangenomic analysis of Staphylococcus pseudintermedius to better understand antimicrobial resistance profiles

Although Staphylococcus pseudintermedius are commensal bacteria of dogs, they are also opportunistic pathogens, being the primary cause of canine skin and ear infections. Recently, more attention has been given to this species, due to the emergence of antimicrobial resistance. Further to this a methicillin resistant S. pseuintermedius was isolated from a human and was subsequently found to be multidrug resistant. To better understand the frequency of carriage of antimicrobial resistance, as well as the presence of multidrug resistance, we examined the pangenome of over 200 S. pseudintermedius isolates from across the globe. Focussing on methicillin resistance we were able to identify staphylococcal cassette chromosome mec (SCCmec) types unique to specific Bayesian Analysis of Population Structure (BAPS) groups, including groups which carried mec resistance genes independent of a known SCCmec element. In addition, we identified an SCCmec element, within isolates from North America, that shares 99 % nucleotide identity with a recently described non-typeable SCCmec element carried by Staphylococcus aureus isolated from a human in the Netherlands. Beyond methicillin resistance we found over 50 % of those strains analysed were putatively multidrug resistant (resistant to 3 or more antimicrobial drug classes). This highlights the diverse resistance determinants present in animal staphylococci and the importance of monitoring S. pseudintermedius.

Staphylococcus pseudintermedius septicemia in puppies after elective cesarean section: confirmed transmission via dam\u2019s milk

BackgroundIn humans, transmission of bacteria causing fatal sepsis in the neonates through mother’s milk has been reported. In dogs, it is believed that bacteria from canine milk are not the primary cause of neonatal infections. Staphylococcus pseudintermedius is colonizing the skin and mucocutaneous junctions in adult dogs and can act as an opportunistic pathogen. This bacterium was previously isolated from the canine milk and, although, its transmission from the dam’s milk to the newborn puppies causing a neonatal sepsis was suggested, this hypothesis has not been confirmed.Case presentationA 4.5-year-old healthy Boston terrier dam had an elective cesarean section, delivering five normal puppies and one dead runt. Next day, two puppies developed pustules on their legs and around the muzzle. After two more days, strings of blood were noticed in the stool of the biggest puppy that suddenly died later that night. The same day, blood became visible in the feces of all other puppies. Necropsy of the dead puppy revealed a distended abdomen, catarrhal gastroenteritis with lymphadenopathy, dark red and slightly firm lung, mild dilatation of the right heart chamber and congestion of the liver, spleen, pancreas and meninges. The thoracic cavity contained white-yellow slightly opaque exudate, and there was transudate in the abdominal cavity. Histopathology revealed an acute interstitial pneumonia and multifocal myocardial necrosis with mineralization. Bacteriology of the internal organs, body cavity effusions of the dead puppy and dam’s milk revealed a diffuse growth of S. pseudintermedius in pure culture. Whole genome sequencing (WGS) revealed that all isolates belonged to the sequence type 241 and differed in 2–5 single nucleotide polymorphisms; thus, the epidemiological link between the outbreak-associated isolates was confirmed.ConclusionsThis is the first report of a confirmed transmission of S. pseudintermedius through dam’s milk causing a neonatal sepsis in a puppy after an elective cesarean section. The epidemiological link between S. pseudintermedius isolates obtained from dam’s milk and internal organs of the affected puppy was confirmed by WGS. Our findings indicate that milk of healthy dams can serve as a reservoir of bacteria that can cause fatal sepsis in the newborn puppies.

High frequency of coagulase-positive staphylococci carriage in healthy wild boar with detection of MRSA of lineage ST398-t011.

The objective of this study was to determine the frequency and diversity of coagulase-positive staphylococci (CoPS) in nasal samples of healthy wild boar, to study their resistance phenotypes/genotypes and to check the occurrence of the MRSA-ST398. Nasal samples of 371 wild boars were collected in Spain for staphylococci and MRSA recovery. Staphylococci identification was performed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). The susceptibility to 11 antimicrobials was tested by disc-diffusion and the presence of resistance genes by PCR. Molecular typing and virulence factors determination were carried out by PCR and sequencing. The rate of CoPS carriage (Staphylococcus aureus, Staphylococcus hyicus and Staphylococcus pseudintermedius) in wild boar was of 17.8% (13.7%, 2.7% and 1.6%, respectively). Susceptibility to all tested antimicrobials was shown in 74.5% of S. aureus and one strain was MRSA [lineage ST398-t011-agrI, carrying blaZ, mecA, tet(M) and tet(K) genes]. A total of 22 spa-types and 17 STs were detected among S. aureus, including: ST398/CC398 (n=1), ST2328-ST133/CC133 (n=20), ST425/CC425 (n=7), ST5/CC5 (n=5), ST1/CC1 (n=3), ST130/CC130 (n=2) and ST88/CC88 (n=1). Two spa-types (t02, t15) and four STs (ST455, ST796, ST797, ST798) were detected among the six S. pseudintermedius isolates recovered, and all of them carried the lukF/S-I and siet virulence genes. All S. hyicus isolates were susceptible to antimicrobials tested.

Characterization of a leukocidin identified in Staphylococcus pseudintermedius.

Bacterial infections from Staphylococcus pseudintermedius are the most common cause of skin infections (pyoderma) affecting dogs. Two component pore-forming leukocidins are a family of potent toxins secreted by staphylococci and consist of S (slow) and F (fast) components. They impair the innate immune system, the first line of defense against these pathogens. Seven different leukocidins have been characterized in Staphylococcus aureus, some of which are host and cell specific. Through genome sequencing and analysis of the S. pseudintermedius secretome using liquid chromatography mass spectrometry we identified two proteins, named “LukS-I” and “LukF-I”, encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. Phylogenetic analysis of LukS-I components in comparison to the rest of the leukocidin family showed that LukS-I was most closely related to S. intermedius LukS-I, S. aureus LukE and LukP, whereas LukF-I was most similar to S. intermedius LukF-I S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermedius LukS-I and LukF-I on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-Luk-I produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of Luk-I as a promising component in a vaccine against canine S. pseudintermedius infections.

Comparative genomics of phenotypic antimicrobial resistances in methicillin-resistant Staphylococcus pseudintermedius of canine origin

Staphylococcus pseudintermedius is an important pathogen in dogs. Since 2004, methicillin- resistant S. pseudintermedius (MRSP) isolates, often multidrug resistant, have been observed in dogs in the Netherlands. This study aims to link the observed resistance phenotypes in canine MRSP to genotypic antimicrobial resistance markers, and to study the phylogeny of MRSP by genomic comparisons.The genomes of fifty clinical isolates of MRSP from dogs from the Netherlands were sequenced. The resistance genes were identified, and for twenty one different antimicrobials their presence and sequence were associated with the resistance phenotypes. In case of observed discrepancies, the genes were aligned with reference genes. Of the phenotypic resistances, 98.3% could be explained by the presence of an associated resistance gene or point mutation. Discrepancies were mainly resistance genes present in susceptible isolates; 43.8% (7/16) were explained by an insertion, deletion or mutation in the gene. In relation with the resistance gene presence or absence, a single-nucleotide polymorphism (SNP) based phylogeny was constructed to define the population dynamics. The resistance gene content differed according to clonal complex, from very conserved (CC45), to partly conserved (CC71) to highly diverse (CC258) resistance gene patterns.In conclusion, this study shows that the antimicrobial genotype from whole genome sequencing is highly predictive of the resistance phenotype in MRSP. Interestingly, the observed clonal complexes of MRSP isolates were linked with resistance gene patterns

Minimum inhibitory concentration and killing properties of rifampicin against canine Staphylococcus pseudintermedius isolates from dogs in the southeast USA.

Meticillin-resistant (MR) staphylococcal pyoderma in dogs has led to increased use of alternate antibiotics such as rifampicin (RFP). However, little information exists regarding its pharmacodynamics in MR Staphylococcus pseudintermedius. To determine the minimum inhibitory concentration (MIC) and killing properties of RFP for canine Staphylococcus pseudintermedius isolates. The MIC of RFP was determined using the ETEST® for 50 meticillin-susceptible (MS) and 50 MR S.pseudintermedius isolates collected from dogs. From these isolates, two MS isolates (RFP MIC of 0.003 and 0.008μg/mL, respectively) and two MR isolates (RFP MIC of 0.003 and 0.012μg/mL, respectively) were subjected to time-kill studies. Mueller-Hinton broth was supplemented with RFP at 0, 0.5, 1, 2, 4, 8, 16 and 32 times the MIC for 0, 2, 4, 10, 16 and 24h. The number of viable colony forming units in each sample was determined using a commercial luciferase assay kit. The MIC50 and MIC90 were the same for MS and MR isolates, at 0.004μg/mL and 0.008μg/mL, respectively. Rifampicin kill curves were not indicative of concentration-dependency, suggesting time-dependent activity. Two isolates (MS 0.003 and 0.008μg/mL) exhibited bacteriostatic activity, whereas two others (MR 0.003 and 0.012μg/mL) exhibited bactericidal activity. This study demonstrated that MS and MR S.pseudintermedius isolates were equally susceptible to rifampicin and that dosing intervals should be designed for time-dependent efficacy. These data can support pharmacokinetic studies of RFP in dogs with susceptible infections caused by S.pseudintermedius.