Canine Isolates Research Articles

Molecular detection and characterization of Hepatozoon canis in stray dogs from Cuba.

Canine hepatozoonosis caused by Hepatozoon canis is a worldwide distributed tick-borne disease of domestic and wild canids that is transmitted by ingestion of Rhipicephalus sanguineus sensu lato (s.l.) ticks. The present study was aimed to determine the prevalence of Hepatozoon infections in 80 stray dogs from Havana Province in Cuba, and to confirm the species identity and phylogenetic relationships of the causative agent. Samples were screened by microscopical examination of thin blood smears for the presence of Hepatozoon spp. gamonts and by genus-specific SYBR green-based real-time PCR assay targeting the 18S rRNA gene. Direct microscopy examination revealed Hepatozoon gamonts in the peripheral blood of 8 dogs (10.0%; 95% CI: 4.80-18.0%), while 38 animals (47.5%; 95% CI: 36.8-58.4%) were PCR-positive, including all microscopically positive dogs. Hence, the agreement between the two detection methods was 'poor' (κ = 0.20). Hematological parameters did not differ significantly between PCR-positive and PCR-negative dogs (p > 0.05). The DNA sequences of the 18S rRNA gene of the Hepatozoon spp. from Cuban dogs showed a nucleotide identity >99% with those of 18S rRNA sequences of Hepatozoon canis isolates from Czech Republic, Brazil and Spain. Phylogenetic analysis revealed that obtained sequences clustered within the Hepatozoon canis clade, different from the Hepatozoon felis or Hepatozoon americanum clades. The present study represents the first molecular characterization of Hepatozoon canis in stray dogs within Cuba.

Identification of Streptococcus dysgalactiae using matrix-assisted laser desorption/ionization-time of flight mass spectrometry; refining the database for improved identification

Matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) has revolutionized bacterial identification. However, the phylogenetic resolution is still insufficient for discerning several β-haemolytic streptococcal species. We aimed to improve the diagnostic performance of MALDI-ToF through manual curation of the reference spectra in Brukers Compass Library DB-7854. Before intervention, only 133 out of 217 (62%) Streptococcus dysgalactiae isolates were successfully identified to the species level, 83 isolates were identified to the genus level as either S. dysgalactiae, S. pyogenes or S. canis, and one S. dysgalactiae isolate was wrongly identified as S. canis. All 109 S. canis isolates were successfully identified to the species level. Removal of three reference spectra from the database significantly improved the identification of S. dysgalactiae to 94%, without compromising identification of S. canis. This illustrates the advantage of refinement of the reference database in order to improve the analytic precision of MALDI-ToF.

Phenotype evaluation of human and canine isolates of Leishmania infantum

Human visceral leishmaniasis (VL) and canine leishmaniasis (CanL) in countries of South and Central America are caused by Leishmania infantum and has been endemic in Brazil for several years. The parasite biodiversity as well as the pharmacologic properties of drugs and the host species, are involved in the efficacy or inefficacy of leishmaniasis treatments. Although there are substantial number of reports describing the genetic characterization of the clinical field isolates of L. infantum,the phenotypic parameters have been less studied. In this study isolates from human and canine leishmaniasis (Hum1 and Can1) obtained in Campinas, São Paulo state, Brazil were identified as L. infantum. The Hum1 and Can1 isolates exhibited typical promastigote growth pattern. Regarding morphological features Can1 isolate differed in cell size. The infectivity in vitro of both isolatesis lower compared to the reference strain of L. infantum. Moreover, the in vivo infectivity of the three parasites is similar in Balb/c mice. The Hum1 isolate is more sensitive to leishmanial drugs (amphotericin B, miltefosine and glucantime) than the Can1 isolate when inside human macrophages, but not when inside canine macrophages. These findings indicated that L. infantum isolates differs in some phenotypic characteristics.

Intracellular Invasion Ability and Associated Microbiological Characteristics of Streptococcus canis in Isolates from Japan.

This study evaluated the cell invasion ability (CIA) of Streptococcus canis isolates, and clarified the relationship between high-frequency CIA and its microbiological features. Of the companion animal-origin isolates (n = 117) that were obtained in 2017, 40 isolates were randomly selected with the host information, with two human blood-origin isolates included. CIA was measured using human colon carcinoma epithelium and the hemolytic activity (HA) using sheep blood, along with S. canis M-like protein (SCM) allele typing, sequence type (ST) determination, and antimicrobial resistance (AMR) phenotyping/genotyping. CIA measurements revealed that 19 and 24 isolates had high- and low-frequencies, respectively. HA assessment revealed that 24 and 19 isolates were categorized as high- and low- level, respectively. No difference was observed in the high-/low-level HA between the high- /low-frequency CIA populations. A significant difference was found in the high-/low-frequency CIA between the SCM group I/II populations. Additionally, a significantly higher CIA was found in the SCM allele type 10/type 11 than in the others. A significant association was observed between high-frequency CIA and the ST21/ST41 populations. No difference was found in the high-/low-frequency CIA between the presence and absence of the AMR phenotype/genotype. These observations suggest a relationship between high-frequency CIA and its microbiological characteristics (SCM allele type 10/type 11 or ST21/ST41).

Genetic tools discriminate strains of Leishmania infantum isolated from humans and dogs in Sicily, Italy.

BackgroundLeishmaniasis is one of the most important vector-borne diseases and it represents a serious world health problem affecting millions of people. High levels of Leishmania infections, affecting both humans and animals, are recognized among Italian regions. Among these, Sicily has one of the highest prevalence of Leishmania infection.Methodology/Principal FindingsSeventy-eight Leishmania strains isolated from human and animal samples across Sicily, were analyzed for the polymorphic k26-gene and genotypes were assigned according to the size of the PCR products. A multilocus microsatellite typing (MLMT) approach based on the analysis of 11 independent loci was used to investigate populations structure and genetic diversity of the isolated strains. Six L. infantum reference strains were included in the analysis for comparison. Bayesian clustering analysis of microsatellite data showed that all the isolated strains clustered in two genetically distinct populations, corresponding to human and canine isolates respectively. A further subdivision was observed between the two main groups, giving a good correlation between human strains and their geographic origin, conversely canine population showed a great genetic variability diffused in the territory.Conclusions/SignificanceAmong the 78 Leishmania isolates, K26 analysis detected 71 samples (91%) as MON-1 zymodeme, confirming it as the predominant strain in Mediterranean area and 7 human samples (9%) as non-MON-1. MLMT gives important insights into the epidemiology of leishmaniases and allows characterization of different strains to a higher resolution than possible with zymodeme typing. Two main populations presented a strong correlation respect to the different hosts, exhibiting a co-circulation of two distinct populations of L. infantum. The population found in infected humans exhibited a correlation with geographic origin. These clusters could represent a geographically restricted population of strains with the same or related genotypes. This study can contribute to an understanding of Leishmania epidemiology, including the spread of reservoirs and sand fly vectors in the different foci of infection, characterizing parasites within the different hosts.

Canine sporotrichosis: polyphasic taxonomy and antifungal susceptibility profiles of Sporothrix species in an endemic area in Brazil.

Sporotrichosis, a mycosis caused by pathogenic species of the genus Sporothrix, affects diverse species of mammals. Until 2007, Sporothrix schenckii was considered the unique etiologic agent of sporotrichosis. Canine sporotrichosis is a poorly reported disease, and the majority of cases are from Rio de Janeiro, Brazil. There are scarce studies on the characterization of canine isolates of Sporothrix schenckii complex, as well as few antifungal susceptibility data available. The aim of this study was to characterize the clinical isolates of Sporothrix from dogs from Brazil at species level and evaluate their antifungal susceptibility profile. Polyphasic taxonomy was used to characterization at species level (morphological, phenotypical characteristics, and molecular identification). Antifungal susceptibility profiles (amphotericin B, itraconazole, ketoconazole, posaconazole, and terbinafine) were determined using the Clinical and Laboratory Standards Institute broth microdilution method (M38-A2). According to phenotypic identification and molecular analysis, 46 isolates included in this study were identified as S. brasiliensis and one as S. schenckii. Amphotericin B presented the highest minimum inhibitory concentration values, and the other drugs showed effective in vitro antifungal activity. This is the first report of S. schenckii in dogs from Brazil, since S. brasiliensis is the only species that has been described in canine isolates from Rio de Janeiro to date. Nevertheless, no differences were observed in the antifungal susceptibility profiles between the S. brasiliensis and S. schenckii isolates, and it is important to continuously study new canine clinical isolates from Rio de Janeiro, Brazil.

Sequence analysis of Salmonella enterica isolates obtained from shelter dogs throughout Texas.

Dogs are a potential source of zoonotic Salmonella transmission. We had previously estimated the prevalence of Salmonella shedding among shelter dogs throughout Texas using a repeated cross‐sectional study design. Our current objectives were to fully characterize the Salmonella isolates and to assess their relatedness, using whole‐genome sequencing. Antimicrobial resistance (AMR) genes were detected in 4/27 (15%) of the isolates. The fosfomycin resistance gene fosA7 was identified in two isolates; to our knowledge, there are no published reports of this gene in canine Salmonella isolates. The biocide resistance gene qacEdelta1, conferring resistance to quaternary ammonium compounds, was detected in an isolate that had four additional AMR genes. The most frequently identified serotypes were Newport (6/27, 22%) and Javiana (4/27, 15%), both of which were widespread among animal shelters. For these serotypes, there was evidence of both transmission of Salmonella within the shelter environment and separate introductions of Salmonella into a shelter. Several canine Salmonella isolates were closely related to human clinical isolates (four canine isolates within 10 SNPs and six more within 20 SNPs), suggesting a shared pathogen population. Educational outreach programmes targeting animal shelter workers would be useful for optimizing knowledge of Salmonella and other canine‐associated zoonotic pathogens.

Comparison of Streptococcus halichoeri isolates from canine and fur animal infections: biochemical patterns, molecular characteristics and genetic relatedness

BackgroundStreptococcus halichoeri infections have been reported in grey seals, a European badger, a Stellar sea lion and humans, but its presence in companion and fur animals is unknown. Since 2010, S. halichoeri-like bacteria (SHL) have been isolated from fur animals and dogs in Finland. Our aim was to retrospectively investigate laboratory records for SHL from canine and fur animal infections, characterize the isolates and compare their genetic relatedness in relation to three reference strains: CCUG 48324T, originating from a grey seal, and strains 67100 and 61265, originally isolated from humans.ResultsA total of 138 and 36 SHLs from canine and fur animal infections, respectively, were identified in the laboratory records. SHL was commonly associated with skin infections, but rarely as the only species. A set of 49 canine and 23 fur animal SHLs were further characterized. MALDI-TOF confirmed them as being S. halichoeri. The growth characteristics were consistent with the original findings, but isolates were catalase positive. In total, 17 distinct API 20 Strep patterns were recorded among all 75 isolates tested, of which pattern 5563100 was the most common (n = 30). Antimicrobial resistance to erythromycin and clindamycin was common in canine isolates, but rare in fur animal isolates. Three clusters were observed by PFGE, and 16S rRNA sequencing revealed 98.1–100% similarities with the human strains and 98.1–99.5% with the seal strain. A phylogenetic tree of concatenated 16S rRNA and rpoB revealed closely related isolates with two clades. Fifteen canine isolates were identical to the human strains based on concatenated 16S rRNA and rpoB sequencing.ConclusionsStreptococcus halichoeri appears to be quite a common bacterial species in the skin of dogs and fur animals. The clinical significance of S. halichoeri is uncertain, as it was rarely isolated as a monoculture. No apparent temporal or spatial clustering was detected, but isolates from different sources were genetically very similar. Because many canine isolates were genetically similar to the human reference strains, transmission between dogs and humans may be possible. WGS sequencing of strains from different sources is needed to further investigate the epidemiology and virulence of S. halichoeri.

PERFORMANCE OF ACCESSORY GENE REGULATOR IN VIRULENCE EXPRESSION AND BIOFILM FORMATION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS PSEUDINTERMEDIUS

Staphylococcus pseudintermedius-induced soft tissue infection is a typical biofilm-related infectious disease. Recently, the occurrence of methicillin-resistant S. pseudintermedius (MRSP) infections in humans has increased. In this study, the virulence of different accessory gene regulator (agr) types of MRSP and MRSP[Formula: see text]induced skin infection were investigated through in vitro studies and the murine pyoderma model, respectively. Forty-seven canine clinical MRSP isolates were collected from northern Taiwan and classified into four agr types, namely, type I (52%), type II (2%), type III (35%), and type IV (10%). The majority of the MRSP isolates belonged to either strong (49%) or moderate (42.5%) biofilm producers. The skin damages induced by type III 77, type III 79, and type IV n10 isolates were larger than those induced by type I 58 and type II n3 isolates, but not by a significant degree. The effects of cell adhesion, cell invasion and cell cytotoxicity tests for agr type III 77, type III 79, and type IV n10 isolates were stronger than those caused by other types of MRSP isolates. However, the biofilm formation ability of these MRSP isolates did not show association with their virulence differences for causing murine skin abscesses. The agr type III 77, type III 79, and type IV n10 isolates of MRSP from the clinical samples revealed increased bacterial virulence for causing pyoderma, which is worthy of further genomic studies.

New Therapeutic Candidates for the Treatment of Malassezia pachydermatis -Associated Infections

The opportunistic pathogen Malassezia pachydermatis causes bloodstream infections in preterm infants or individuals with immunodeficiency disorders and has been associated with a broad spectrum of diseases in animals such as seborrheic dermatitis, external otitis and fungemia. The current approaches to treat these infections are failing as a consequence of their adverse effects, changes in susceptibility and antifungal resistance. Thus, the identification of novel therapeutic targets against M. pachydermatis infections are highly relevant. Here, Gene Essentiality Analysis and Flux Variability Analysis was applied to a previously reported M. pachydermatis metabolic network to identify enzymes that, when absent, negatively affect biomass production. Three novel therapeutic targets (i.e., homoserine dehydrogenase (MpHSD), homocitrate synthase (MpHCS) and saccharopine dehydrogenase (MpSDH)) were identified that are absent in humans. Notably, L-lysine was shown to be an inhibitor of the enzymatic activity of MpHCS and MpSDH at concentrations of 1 mM and 75 mM, respectively, while L-threonine (1 mM) inhibited MpHSD. Interestingly, L- lysine was also shown to inhibit M. pachydermatis growth during in vitro assays with reference strains and canine isolates, while it had a negligible cytotoxic activity on HEKa cells. Together, our findings form the bases for the development of novel treatments against M. pachydermatis infections.

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.

Specific staphylococcal cassette chromosome mec (SCCmec) types and clonal complexes are associated with low-level amoxicillin/clavulanic acid and cefalotin resistance in methicillin-resistant Staphylococcus pseudintermedius.

Background Staphylococcus pseudintermedius is a common pathogen in dogs and methicillin resistance has emerged over recent decades. According to the current guidelines, S. pseudintermedius displaying oxacillin resistance should be reported as resistant to all β-lactams.ObjectivesTo identify possible associations between β-lactam resistance levels and clonal complexes (CCs) and/or staphylococcal cassette chromosome mec (SCCmec) types in methicillin-resistant S. pseudintermedius (MRSP).MethodsMICs of oxacillin, penicillin, ampicillin, amoxicillin/clavulanic acid and cefalotin were determined by broth microdilution for 86 clinical canine MRSP isolates from Denmark and the Netherlands. PCR and sequencing were used for SCCmec typing and MLST.ResultsIsolates belonged to CC71 (n = 36), CC258 (n = 33), CC45 (n = 11), CC68 (n = 1) and five singleton STs. SCCmecII-III was exclusively found in CC71 and SCCmecIV was significantly associated with CC258. SCCmecV and non-typeable SCCmec types occurred in 4 and 14 isolates, respectively. SCCmecIV was associated with lower MICs of oxacillin (<2 mg/L), ampicillin (<8 mg/L) and amoxicillin/clavulanic acid (<4 mg/L) and with susceptibility to cefalotin (<4 mg/L). All isolates harbouring SCCmecV were susceptible to cefalotin as well.ConclusionsSCCmec types were associated with different CCs and with either high- or low-level resistance to different β-lactams. The finding of amoxicillin/clavulanic acid (20%) and cefalotin (70%) in vitro susceptibility across all CCs might have clinical implications, since amoxicillin/clavulanic acid and first-generation cephalosporins are first-choice antibiotics for treatment of S. pseudintermedius infections. Pharmacokinetic/pharmacodynamic and clinical outcome studies are warranted to evaluate the in vivo efficacy of these β-lactams for treatment of MRSP infections.

Investigation of the molecular characteristics of Brucella isolates from Guangxi Province, China

BackgroundHuman brucellosis has become a severe public health problem in China’s Guangxi Province, and there has been higher prevalence of brucellosis in this region after 2010. Both multiple locus variable-number tandem repeat analysis (MLVA) and multilocus sequence typing (MLST) assay schedules were used to genotype isolates and determine relationships among isolates.ResultsA total of 40 isolates of Brucella were obtained from humans, pigs, and dogs from 1961 to 2016. There were at least three species of Brucella detected in Guangxi Province, Brucella melitensis, Brucella suis, and Brucella canis, with 16, 17, and 7 isolates, respectively. Of which B. suis biovar 3 was the predominant species resulting in pig brucellosis in the area examined before 2000s. Moreover, B. melitensis biovar 3 was found to be mainly responsible for human brucellosis during 2012–2016. All B. melitensis isolates in this study belonged to East Mediterranean lineage. MLVA-11 genotype 116 was the dominant genotype and represented 81.2% of the isolates. MLVA cluster analysis showed there to be 44% (7/16) brucellosis cases caused by B. melitensis with a profile of outbreak epidemic from 2012 to 2016. However, nearly 83.3% (20/24) of brucellosis cases resulting from both B. suis and B. canis showed no epidemiological links or sporadic characteristics. MLVA-16 analysis confirmed extensive genotype-sharing events between B. melitensis isolates from Guangxi and other northern provinces within China. These data revealed that there are potential epidemiology links among these strains. B. suis strains of this study showed a unique genetic lineage at the global level and may have existed historically in this area. However, present B. canis isolates were closely related to previously reported isolates in Korea, where they may have originated. MLST typing showed that the population structure of Brucella strains had changed considerably in this province; ST17 and ST21, two previously predominant populations appeared to have been replaced by recently emerging ST8 group.ConclusionsOur investigation data have inspired the hypothesis that Guangxi Province had been subject to an imported human brucellosis epidemic. Our data suggest that strains found in Northern regions of China are the principal source of infections in recent cases of human brucellosis in Guangxi Province. Comparative genomic analysis from more strains is necessary to confirm this hypothesis. This work will facilitate better understanding of the epidemiology and improve the effectiveness of control and prevention of brucellosis in this region.

Antibacterial effect of N-acetylcysteine in combination with antimicrobials on common canine otitis externa bacterial isolates.

Approved treatments for canine otitis externa are limited in variety and may contain ototoxic ingredients. With bacterial resistance an ongoing concern, it would be ideal if non-ototoxic agents combined with antibiotics resulted in a synergistic effect, requiring lower antibiotic concentrations to treat infections. Evidence of synergism and antagonism between N-acetylcysteine (NAC) and various antibiotic classes has been reported; the present research group was interested in examining these interactions. To determine if NAC, an otoprotective and antimicrobial compound, has synergistic activity when combined with enrofloxacin or gentamicin in vitro against bacterial isolates causing canine otitis externa. Twenty-two isolates from canine clinical cases of otitis externa were identified and tested, including seven Staphylococcus pseudintermedius, 12 Pseudomonas aeruginosa and three Corynebacterium spp. isolates. Each isolate was grown on blood agar for 24h and transferred to Mueller-Hinton broth (MHB), with a final concentration of 5×105 cfu/mL. Each well was inoculated with 50μL of bacterial suspension. N-acetylcysteine was diluted in MHB to a starting concentration of 160mg/mL. Enrofloxacin and gentamicin were diluted to 64μg/mL. Individual and checkerboard serial microdilution assays were performed in triplicate with negative controls for all isolates tested. Interactions observed for NAC and enrofloxacin were synergistic (4.5%), indifferent (77.3%) or antagonistic (18.2%). Interactions observed for NAC and gentamicin were synergistic (4.5%), indifferent (45.5%) or antagonistic (50%). Most interactions between NAC and enrofloxacin or gentamicin were indifferent or antagonistic at the concentrations tested in vitro.

Antibacterial activity of chemotherapeutic drugs against Escherichia coli and Staphylococcus pseudintermedius.

The ability of chemotherapeutic agents to affect the growth of common bacterial pathogens and the relationship between the effects of chemotherapeutics and antimicrobials is largely unknown. The purpose of this study was to describe the susceptibility of canine bacterial isolates to chemotherapeutic agents and to compare these results to their antimicrobial susceptibility. The effects of bleomycin, doxorubicin, cytarabine, cyclophosphamide, methotrexate, 5-fluorouracil and gemcitabine on the growth of 33 Staphylococcus pseudintermedius isolates and 32 Escherichia coli isolates from dogs was determined by agar dilution. In addition to MICs, the lowest drug concentration associated with a decreased colony size was recorded. Results were compared to the MICs of a panel of antimicrobial agents. Bleomycin consistently inhibited bacterial growth of S.pseudintermedius and E.coli. Doxorubicin inhibited S.pseudintermedius but not E.coli while the opposite was seen for gemcitabine. Reduction in colony size on exposure to 5-fluorouracil for both organisms, and methotrexate for S. pseudintermedius was seen. No observable effect of cyclophosphamide or cytarabine was observed. Associations between elevated MICs to chemotherapeutic drugs and antimicrobial resistance were not found. These results indicate that chemotherapeutic agents affect the growth of bacteria, but do not support a role in the selection of antimicrobial resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that chemotherapy drugs commonly used in veterinary oncology have an effect of the growth of canine isolates of Escherichia coli and Staphylococcus pseudintermedius. No associations between susceptibility to chemotherapeutic drugs and antibiotics were found, which does not support selection of antimicrobial resistance by chemotherapy drugs.

In vitro activities of 15 antifungal drugs against a large collection of clinical isolates of Microsporum canis.

Microsporum canis is a zoophilic species, found to be the most frequently isolated species in animals. M. canis causes sporadic outbreaks of infections in humans, such as the one that occurred in Canada, where more than 1000 human cases were detected over an 8-year period. Despite the medical importance of M. canis infections, there are limited in vitro data on the antifungal susceptibility to antifungal drugs, including new generation triazoles and imidazoles. The aim of the current study was to comprehensively evaluate the in vitro activity of new azoles and comparator drugs against a large panel of M. canis isolates using a microdilution assay. The in vitro susceptibility to novel triazoles and imidazoles was compared to that of other antifungal drugs using a large collection of M. canis clinical isolates (n=208) obtained from patients and animals with dermatophytosis in Iran, France and Turkey. All isolates exhibited high susceptibility to the majority of the tested antifungal agents. However, luliconazole, lanoconazole and efinaconazole, as well as econazole, demonstrated superior activity against all strains in comparis on with the other drugs. FDA-approved antifungal drugs, that is luliconazole, efinaconazole and lanoconazole, showed the highest antifungal activity and should be promising candidates for the treatment of dermatophytosis caused by M canis. However, their therapeutic effectiveness remains to be determined in clinical settings.

Biofilm production by pathogens associated with canine otitis externa, and the antibiofilm activity of ionophores and antimicrobial adjuvants.

Otitis externa (OE) is a frequently reported disorder in dogs associated with secondary infections by Staphylococcus, Pseudomonas and yeast pathogens. The presence of biofilms may play an important role in the resistance of otic pathogens to antimicrobial agents. Biofilm production of twenty Staphylococcus pseudintermedius and twenty Pseudomonas aeruginosa canine otic isolates was determined quantitatively using a microtiter plate assay, and each isolate was classified as a strong, moderate, weak or nonbiofilm producer. Minimum biofilm eradication concentration (MBEC) of two ionophores (narasin and monensin) and three adjuvants (N-acetylcysteine (NAC), Tris-EDTA and disodium EDTA) were investigated spectrophotometrically (OD570nm ) and quantitatively (CFU/ml) against selected Staphylococcus and Pseudomonas biofilm cultures. Concurrently, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of planktonic cultures were assessed. 16/20 of the S.pseudintermedius clinical isolates were weak biofilm producers. 19/20 P.aeruginosa clinical isolates produced biofilms and were distributed almost equally as weak, moderate and strong biofilm producers. While significant antibiofilm activity was observed, no MBEC was achieved with narasin or monensin. The MBEC for NAC ranged from 5,000-10,000µg/ml and from 20,000-80,000µg/ml against S.pseudintermedius and P.aeruginosa, respectively. Tris-EDTA eradicated P.aeruginosa biofilms at concentrations ranging from 6,000/1,900 to 12,000/3,800µg/ml. The MBEC was up to 16-fold and eightfold higher than the MIC/MBC of NAC and Tris-EDTA, respectively. Disodium EDTA reduced biofilm growth of both strains at concentrations of 470µg/ml and higher. It can be concluded that biofilm production is common in pathogens associated with canine OE. NAC and Tris-EDTA are effective antibiofilm agents in vitro that could be considered for the treatment of biofilm-associated OE in dogs.

PSI-25 development of local Kansas E. coli UTI antibiograms to improve antimicrobial stewardship in companion animal medicine

Abstract Urinary tract infections are common in dogs, but rare in cats. Antimicrobial susceptibility testing of urine isolates is required to determine optimal antimicrobial therapy, but empirical antimicrobial guidelines are needed while culture is pending or if testing is declined. Antibiograms are summaries of local susceptibility trends that assist clinicians in selecting empirical antimicrobial therapy. Antibiograms were developed from E. coli feline (N = 138) and canine (N = 640) urinary isolates submitted by the KSU Veterinary Health Center and private veterinary practices from 2013–2017. Results showed a high prevalence of resistance among feline isolates to amoxicillin-clavulanate (99.3%, S ≤ 0.25) and ampicillin (99.3%, S ≤ 0.25), but lower prevalence among canine isolates (amoxicillin/clavulanate 7.7% S ≤ 8), ampicillin 46.7%, S ≤ 8). Resistance to other antimicrobials was uncommon, with no antimicrobial agents demonstrating resistance above 6% in feline isolates, or above 14% in canine isolates. Canine isolates from private veterinary practices had increased resistance as compared with KSU isolates to orbifloxacin (9.5% vs 0.1%) and pradofloxaxin (9.4% vs 0.1%). Application of different breakpoints and differences in antibiotic exposures may explain the disparity of Beta-Lactam resistance between cats and dogs. Different use patterns between hospitals may explain the disparity between fluoroquinolone resistance. These findings emphasize the need for definitive urine culture and susceptibility testing in pets with UTIs, so that appropriate and effective antimicrobial therapy can be prescribed. Veterinary clinicians in Kansas can use these antibiogram results in addition to following stewardship guidelines to maximize successful UTI therapy for veterinary patients and minimize development of antimicrobial resistance for the One Health community.

Streptococcus canis Are a Single Population Infecting Multiple Animal Hosts Despite the Diversity of the Universally Present M-Like Protein SCM.

Streptococcus canis is an animal pathogen which occasionally causes infections in humans. The S. canis M-like protein (SCM) encoded by the scm gene, is its best characterized virulence factor but previous studies suggested it could be absent in a substantial fraction of isolates. We studied the distribution and variability of the scm gene in 188 S. canis isolates recovered from companion animals (n = 152), wild animal species (n = 20), and humans (n = 14). Multilocus sequence typing, including the first characterization of wildlife isolates, showed that the same lineages are present in all animal hosts, raising the possibility of extensive circulation between species. Whole-genome analysis revealed that emm-like genes found previously in S. canis correspond to divergent scm genes, indicating that what was previously believed to correspond to two genes is in fact the same scm locus. We designed primers allowing for the first time the successful amplification of the scm gene in all isolates. Analysis of the scm sequences identified 12 distinct types, which could be divided into two clusters: group I (76%, n = 142) and group II (24%, n = 46) sharing little sequence similarity. The predicted group I SCM showed extensive similarity with each other outside of the N-terminal hypervariable region and a conserved IgG binding domain. This domain was absent from group II SCM variants found in isolates previously thought to lack the scm gene, which also showed greater amino acid variability. Further studies are necessary to elucidate the possible host interacting partners of the group II SCM variants and their role in virulence.

Genetic Profiling and Comparison of Human and Animal Methicillin-Resistant Staphylococcus aureus (MRSA) Isolates from Serbia.

The aim of this study was to characterize a collection of methicillin-resistant Staphylococcus aureus (MRSA) isolates of human and animal origin from Serbia. In total, 36 MRSA isolates—30 obtained from humans and six from companion animals—were investigated by PCR for the presence of antibiotic and biocide resistance determinants and virulence genes (PVL—Panton–Valentine leukocidin, ETs—exfoliative toxins, TSST—toxic shock syndrome toxin, SEs—staphylococcal enterotoxins, and MSCRAMMs—microbial surface components recognizing adhesive matrix molecules and biofilm). Isolates were analyzed by staphylococcal cassette chromosome mec (SCCmec), spa, and dru typing, as well as by multiple locus variable number of tandem repeat analyses (MLVA), multilocus sequence typing (MLST), and subsequently, eBURST. The majority of human MRSA isolates were resistant to gentamicin, erythromycin, clindamycin, and ciprofloxacin. Different antibiotic resistance genes were detected: aac-aphD, ant(6′)-Ia, erm(A), erm(B), erm(C), tet(K), tet(M), fexA, and catpC221. All isolates were susceptible to teicoplanin and linezolid. SCCmec type III was prevalent in human isolates, while SCCmec elements in animals were mostly nontypeable. t037 was the predominant spa type in human and t242 in animal MRSA isolates. The prevalent dru type was dt11c in human and dt10a in animal MRSA isolates. MRSA isolates exhibited 27 different MLVA types. ST239 was predominant in human, while ST5 was prevalent in canine MRSA isolates. PVL was found in two, while tsst-1 was detected in three human isolates. Human-associated clones belonging to ST5, ST45, and ST239 MRSA clones were discovered in companion animals, which suggests anthropozoonotic transmission.