Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES: To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS: WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS: mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS: Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales.

Mur ligase F as a new target for the flavonoids quercitrin, myricetin, and (-)-epicatechin.

MurC, D, E, and F are ATP-dependent ligases involved in the stepwise assembly of the tetrapeptide stem of forming peptidoglycan. As highly conserved targets found exclusively in bacterial cells, they are of significant interest for antibacterial drug discovery. In this study, we employed a computer-aided molecular design approach to identify potential inhibitors of MurF. A biochemical inhibition assay was conducted, screening twenty-four flavonoids and related compounds against MurC-F, resulting in the identification of quercitrin, myricetin, and (-)-epicatechin as MurF inhibitors with IC50 values of 143 µM, 139 µM, and 92 µM, respectively. Notably, (-)-epicatechin demonstrated mixed type inhibition with ATP and uncompetitive inhibition with D-Ala-D-Ala dipeptide and UM3DAP substrates. Furthermore, in silico analysis using Sitemap and subsequent docking analysis using Glide revealed two plausible binding sites for (-)-epicatechin. The study also investigated the crucial structural features required for activity, with a particular focus on the substitution pattern and hydroxyl group positions, which were found to be important for the activity. The study highlights the significance of computational approaches in targeting essential enzymes involved in bacterial peptidoglycan synthesis.

Diminishing hERG inhibitory activity of aminopiperidine-naphthyridine linked NBTI antibacterials by structural and physicochemical optimizations.

Novel bacterial topoisomerase inhibitors (NBTIs) are an important new class of antibacterials targeting bacterial type II topoisomerases (DNA gyrase and topoisomerase IV). Notwithstanding their potent antibacterial activity, they suffer from a detrimental class-related hERG blockage. In this study, we designed and synthesized an optimized library of NBTIs comprising different linker moieties that exhibit reduced hERG inhibition and retain inhibitory potencies on DNA gyrase and topoisomerase IV of Staphylococcus aureus and Escherichia coli, respectively, as well as potent antibacterial activities. Substitution of the linker's tertiary amine with polar groups outcome in diminished hERG inhibition. Compound 17 expresses nanomolar enzyme inhibitory potency and antibacterial activity against both Gram-positive and Gram-negative bacteria as well as reduced hERG inhibition relative to our previously published NBTI analogs. Here, we point to some important NBTI's structural features that influence their hERG inhibitory activity.

Mur ligases inhibitors with azastilbene scaffold: Expanding the structure-activity relationship.

Antibiotic resistance represents one of the biggest public health challenges in the last few years. Mur ligases (MurC-MurF) are involved in the synthesis of UDP-N-acetylmuramyl-pentapeptide, the main building block of bacterial peptidoglycan polymer. They are essential for the survival of bacteria and therefore important antibacterial targets. We report herein the synthesis and structure-activity relationships of Mur ligases inhibitors with an azastilbene scaffold. Several compounds showed promising inhibitory potencies against multiple ligases and one compound also possessed moderate antibacterial activity. These results represent a solid ground for further development and optimization of structurally novel antimicrobial agents to combat the rising bacterial resistance.

Clinical and histologic tissue responses of skin incisions closed with glycomer 631 and lactomer 9-1 using a subcuticular technique in pigs.

BACKGROUND: Glycomer 631 and lactomer 9-1 are absorbable suture materials indicated for soft tissue approximation in non-infected wounds. Pigs are often used as surgical models in translational research; however, reports of tissue reactions to both suture materials in pigs are limited. The aim of this study was to assess clinical and histologic responses of skin incisions closed with a subcuticular technique using glycomer 631 and lactomer 9-1 in pigs. RESULTS: Skin incisions on 17 pigs were closed with glycomer 631 and lactomer 9-1, and a clinical reactive score (CRS) including erythema, swelling, discharge and dehiscence was calculated on postoperative days 7 and 14. Subcuticular tissue reaction was assessed histologically on postoperative day 14 (the presence of extravascular neutrophils, macrophages, multinucleated giant cells, lymphocytes, fibroblasts, bacterial colonies and the overall severity of the inflammatory response to the suture material), and the cumulative score of the variables was calculated as an aggregate tissue irritation score (ATIS). Tissue samples were examined for suture extrusion and evaluated microbiologically. The clinical reactive score did not differ between the suture materials. Only one ATIS variable, namely the overall severity of the inflammatory response, was lower (p = 0.029) when glycomer 631 was used. Suture extrusion was found in 10/17 of the incisions closed by glycomer 631 and in 7/13 of the incisions closed by lactomer 9-1. Trueperella pyogenes was isolated from the skin and from the area of tissue reaction in six pigs. CONCLUSIONS: No difference in CRS between the suture materials was observed, and thus both materials may be used for the subcuticular technique in pigs. Glycomer 631 induced less tissue reaction only in terms of the overall severity of the inflammatory response. Suture extrusion was observed in more than 50% of incisions regardless of the suture material, possibly due to a large amount of suture material in the wound. Trueperella pyogenes was the only pathogen isolated from the tissue surrounding the suture material.

Staphylococcus pseudintermedius septicemia in puppies after elective cesarean section: confirmed transmission via dam's milk.

BACKGROUND: In 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 PRESENTATION: A 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. CONCLUSIONS: This 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.

Determination of N-acylhomoserine lactones of Pseudomonas aeruginosa in clinical samples from dogs with otitis externa.

BACKGROUND: Bacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules. In Gram-negative bacteria, like Pseudomonas aeruginosa, these signaling molecules are N-acylhomoserine lactones (AHLs). P. aeruginosa is a common cause of inflammation of the ear canal (otitis externa) in dogs. It employs quorum sensing to coordinate the expression of host tissue-damaging factors, which are largely responsible for its virulence. The treatment of P. aeruginosa-associated otitis is challenging due to a high intrinsic resistance of P. aeruginosa to several antibiotics. Attenuation of quorum sensing signals to inhibit bacterial virulence is a novel strategy for the treatment of resistant bacterial pathogens, including P. aeruginosa. Therefore, it is important to recognize and define quorum sensing signal molecules in clinical samples. To date, there are no reports on determination of AHLs in the veterinary clinical samples. The purpose of this study was to validate an analytical procedure for determination of the concentration of AHLs in the ear rinses from dogs with P. aeruginosa-associated otitis externa. Samples were obtained with rinsing the ear canals with physiological saline solution. For validation, samples from healthy dogs were spiked with none or different known amounts of the selected AHLs. With the validated procedure, AHLs were analyzed in the samples taken in weekly intervals from two dogs, receiving a standard treatment for P. aeruginosa-associated otitis externa. RESULTS: Validation proved that the procedure enables quantification of AHLs in non-clinical and clinical samples. In addition, a time dependent reduction of AHL concentration was detected for the treated dogs. CONCLUSIONS: Our results indicate that liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is superior in detecting AHLs compared to other chromatographic techniques. This is the first report on determination of AHLs in the clinical samples of veterinary importance. The analytical procedure described in this paper is capable of supporting novel antimicrobial strategies, which target quorum sensing.

A novel metagenomic approach uncovers phage genes as markers for increased disinfectant tolerance in mixed Listeria monocytogenes communities.

Listeria monocytogenes is an important human pathogen with a high mortality rate. Consumption of contaminated ready-to-eat food is the main mode of transmission to humans. Disinfectant-tolerant L. monocytogenes have emerged, which are believed to have increased persistence potential. Elucidating the mechanisms of L. monocytogenes disinfectant tolerance has been the focus of previous studies using pure cultures. A limitation of such approach is the difficulty to identify strains with reduced susceptibility due to inter-strain variation and the need to screen large numbers of strains and genes. In this study, we applied a novel metagenomic approach to detect genes associated with disinfectant tolerance in mixed L. monocytogenes planktonic communities. Two communities, consisting of 71 and 80 isolates each, were treated with the food industry disinfectants benzalkonium chloride (BC, 1.75 mg/L) or peracetic acid (PAA, 38 mg/L). The communities were subjected to metagenomic sequencing and differences in individual gene abundances between biocide-free control communities and biocide-treated communities were determined. A significant increase in the abundance of Listeria phage-associated genes was observed in both communities after treatment, suggesting that prophage carriage could lead to an increased disinfectant tolerance in mixed L. monocytogenes planktonic communities. In contrast, a significant decrease in the abundance of a high-copy emrC-harbouring plasmid pLmN12-0935 was observed in both communities after treatment. In PAA-treated community, a putative ABC transporter previously found to be necessary for L. monocytogenes resistance to antimicrobial agents and virulence, was among the genes with the highest weight for differentiating treated from control samples. The undertaken metagenomic approach in this study can be applied to identify genes associated with increased tolerance to other antimicrobials in mixed bacterial communities.

Report on overcoming the poor quality of Apai pulsotypes with a short review on PFGE for listeria monocytogenes.

Since listeriosis, caused by Listeria monocytogenes, is one of the important concerns of public health in Europe related to foodborne zoonoses, an efficient protocol for isolate typing is necessary when performing epidemiological studies. Three standardized PFGE protocols available for L. monocytogenes were briefly reviewed. Since observing a poor-quality of ApaI pulsotypes in our laboratory, enzymes from three different manufacturers were compared. The obtained pulsotypes showed that restriction digestion with ApaI from New England BioLabs should be complemented with a subsequent overnight incubation of PFGE plugs in TE buffer for better performance.

Exploring Alternative Pathways to Target Bacterial Type II Topoisomerases Using NBTI Antibacterials: Beyond Halogen-Bonding Interactions.

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of antibacterial agents that target bacterial type II topoisomerases (DNA gyrase and topoisomerase IV). Our recently disclosed crystal structure of an NBTI ligand in complex with DNA gyrase and DNA revealed that the halogen atom in the para position of the phenyl right hand side (RHS) moiety is able to establish strong symmetrical bifurcated halogen bonds with the enzyme; these are responsible for the excellent enzyme inhibitory potency and antibacterial activity of these NBTIs. To further assess the possibility of any alternative interactions (e.g., hydrogen-bonding and/or hydrophobic interactions), we introduced various non-halogen groups at the p-position of the phenyl RHS moiety. Considering the hydrophobic nature of amino acid residues delineating the NBTI's binding pocket in bacterial topoisomerases, we demonstrated that designed NBTIs cannot establish any hydrogen-bonding interactions with the enzyme; hydrophobic interactions are feasible in all respects, while halogen-bonding interactions are apparently the most preferred.

Amide containing NBTI antibacterials with reduced hERG inhibition, retained antimicrobial activity against gram-positive bacteria and in vivo efficacy.

Novel bacterial topoisomerase inhibitors (NBTIs) are new promising antimicrobials for the treatment of multidrug-resistant bacterial infections. In recent years, many new NBTIs have been discovered, however most of them struggle with the same issue - the balance between antibacterial activity and hERG-related toxicity. We started a new campaign by optimizing the previous series of NBTIs, followed by the design and synthesis of a new, amide-containing focused NBTI library to reduce hERG inhibition and maintain antibacterial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). This optimization strategy yielded the lead compound 12 that exhibits potent antibacterial activity against Gram-positive bacteria, reduced hERG inhibition, no cardiotoxicity in zebrafish model, and a favorable in vivo efficacy in a neutropenic murine thigh infection model of MRSA infection.

Coagulase-negative staphylococci from non-mastitic bovine mammary gland: characterization of Staphylococcus chromogenes and Staphylococcus haemolyticus by antibiotic susceptibility testing and pulsed-field gel electrophoresis.

During routine microbiological examination of milk samples from dairy cows without clinical signs of mastitis, quarter milk samples of 231 dairy cows from 12 herds were investigated for the presence of coagulase-negative staphylococci (CNS). The isolates were identified on the basis of colony morphology, Gram staining, catalase and coagulase test and the commercial kit, API Staph. CNS was detected in 29% (67/231) of the cows. A total of seven CNS species were identified with the most prevalent being Staphylococcus (Staph.) chromogenes (30%) and Staph. haemolyticus (28·8%), followed by Staph. simulans (11·2%), Staph. xylosus (11·2%), Staph. epidermidis (7·5%), Staph. hyicus (6·3%) and Staph. sciuri (5%). The predominant species, Staph. chromogenes and Staph. haemolyticus, were further characterized by antibiotic susceptibility testing using the agar disc diffusion method (Kirby-Bauer) and by pulsed-field gel electrophoresis (PFGE). Considerable resistance to ampicillin and penicillin was observed in both species. Isolates with identical or highly similar PFGE profiles were detected at the herd level despite a marked heterogeneity seen for both species. On the basis of somatic cell count, absence of clinical signs of inflammation and heterogeneity of genotypes, we assume that CNS isolated in this study could not be considered as important causative agents of the bovine mammary gland inflammation.

Lactic acid bacteria and bifidobacteria deliberately introduced into the agro-food chain do not significantly increase the antimicrobial resistance gene pool.

Lactic acid bacteria (LAB) and bifidobacteria may serve as reservoirs of antimicrobial resistance, but the risk posed by strains intentionally introduced into the agro-food chain has not yet been thoroughly investigated. The aim of our study was to evaluate whether probiotics, starter and protective cultures, and feed additives represent a risk to human health. In addition to commercial strains of LAB and bifidobacteria, isolates from human milk or colostrum, intestinal mucosa or feces, and fermented products were analyzed. Phenotypic susceptibility data of 474 strains showed that antimicrobial resistance was more common in intestinal isolates than in commercial strains. Antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) were characterized in the whole genome sequences of 1114 strains using comparative genomics. Intrinsic ARGs were abundant in enterococci, bifidobacteria, and lactococci but were considered non-risky due to the absence of MGEs. The results revealed that 13.8% of commercial strains contained acquired ARGs, most frequently for tetracycline. We associated 75.5% of the acquired ARGs with known or novel MGEs, and their potential for transmission was assessed by examining metagenomic sequences. We confirmed that ARGs and MGEs were not as abundant or diverse in commercial strains as in human intestinal isolates or isolates from human milk, suggesting that strains intentionally introduced into the agro-food chain do not pose a significant threat. However, attention should be paid especially to individual probiotic strains containing elements that have been shown to have high potential for transferability in the gut microbiota.Abbreviations: ARG, antimicrobial resistance gene; ICE, integrative and conjugative element; IME, integrative and mobilizable element; LAB, lactic acid bacteria; MDR, multidrug resistance; MIC, minimum inhibitory concentration; MGE, mobile genetic element; TRRPP, tetracycline-resistant ribosomal protection protein; WGS, whole genome sequences.

Antimicrobial Resistance and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus from Two Pig Farms: Longitudinal Study of LA-MRSA.

Pigs were identified as the most important reservoir of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), mostly belonging to the emergent zoonotic clonal complex (CC) 398. Here, we investigated the presence of MRSA in sows and piglets over a period of several months in two pig farms (intensive farm A and family-run farm B). Isolates underwent antimicrobial susceptibility testing, PCR characterization and spa typing. We collected 280 samples, namely 206 nasal swabs from pigs and 74 environmental samples from pig housings at 12 consecutive time points. A total of 120/161 (74.5%) and 75/119 (63.0%) samples were MRSA-positive in farms A and B, respectively. All isolates harbored mecA but lacked mecC and PVL-encoding genes. The identified spa types (t571, t034, t1250 and t898 in farm A, t1451 and t011 in farm B) were indicative of CC398. Antimicrobial resistance patterns (all multidrug resistant in farm A, 57.2% in farm B) depended on the farm, suggesting the impact of farm size and management practices on the prevalence and characteristics of MRSA. Due to the intermittent colonization of pigs and the high contamination of their immediate environment, MRSA status should be determined at the farm level when considering preventive measures or animal trade between farms.

Covalent inhibitors of bacterial peptidoglycan biosynthesis enzyme MurA with chloroacetamide warhead.

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first committed step in the cytoplasmic part of peptidoglycan biosynthesis and is a validated target enzyme for antibacterial drug discovery; the inhibitor fosfomycin has been used clinically for decades. Like fosfomycin, most MurA inhibitors are small heterocyclic compounds that inhibit the enzyme by forming a covalent bond with the active site cysteine. The reactive chloroacetamide group was selected from a series of suitable electrophilic thiol-reactive warheads. The predominantly one-step synthesis led to the construction of the final library of 47 fragment-sized chloroacetamide compounds. Several new E. coli MurA inhibitors were identified, with the most potent compound having an IC50 value in the low micromolar range. The electrophilic reactivity of all chloroacetamide fragments in our library was evaluated by a high-throughput spectrophotometric assay using the reduced Ellman reagent as a surrogate for the cysteine thiol. LC-MS/MS experiments confirmed the covalent binding of the most potent inhibitor to Cys115 of the digested MurA enzyme. The covalent binding was further investigated by a biochemical time-dependent assay and a dilution assay, which confirmed the irreversible and time-dependent mode of action. The efficacy of chloroacetamide derivatives against MurA does not correlate with their thiol reactivity, making the active fragments valuable starting points for fragment-based development of new antibacterial agents targeting MurA.

Dermatophyte Trichophyton mentagrophytes Produces Cysteine Protease Inhibitor.

The protein inhibitor of cysteine proteases was isolated from an important zoophilic dermatophyte species Trichophyton mentagrophytes (T. mentagrophytes) and partially characterized. The isolation process involved affinity chromatography, followed by ion-exchange chromatography and reverse phase high performance liquid chromatography. The fungal inhibitor appears to exist in a high (24 kDa) and low (12 kDa) molecular mass form. It inhibits proteolytic activity of papain, cathepsins B and L but not of cathepsin H or trypsin. Results of immunoblotting procedures indicate that sera of T. mentagrophytes infected rabbits contain antibodies against higher molecular mass forms of the inhibitor. Since no sequence homology has been found between partial protein sequences of T. mentagrophytes inhibitor and other known cysteine protease inhibitors so far, we can speculate that this inhibitor has some structurally unique characteristics. The T. mentagrophytes inhibitor shares some biochemical similarities (molecular mass, high and low molecular mass forms, inhibitory profiles) with clitocypin from Clitocybe nebularis and macrocypins from Macrolepiota procera.

Impact of Type of Parturition on Colostrum Microbiota Composition and Puppy Survival.

The objective of our study was to determine whether the type of parturition affects the microbiota of the colostrum and the growth and survival of the puppies. Seventy-nine newborn puppies were divided into three groups regarding the type of parturition: vaginal delivery (VD), elective caesarean section (EL-CS), and emergency caesarean section (EM-CS). After the birth of the puppies, swabs of meconium were collected from the puppies and colostrum was obtained from the dam. Many aerobic and anaerobic bacteria were isolated and identified by mass spectrometry (MALDI-TOF MS). The colostrum microbiota of VD and EL-CS puppies contained a significantly higher abundance of bacteria belonging to the genera Staphylococcus, Kocuria and Enterococcus compared with EM-CS colostrum samples. The composition of the meconium microbiota of the puppies present at birth was similar to the colostrum microbiota of their mothers. It was also found that puppies without a meconium microbiota at birth gained weight more slowly compared with puppies with a meconium microbiota at birth. The type of parturition influenced the bacterial composition of the microbiota in the colostrum. Future studies are necessary to further define the significance of the observed differences in microbiota composition between EM-CS compared with EL-CS and VD colostrum microbiota.

Genomic insights into the emergence and spread of methicillin-resistant Staphylococcus pseudintermedius in veterinary clinics.

Staphylococcus pseudintermedius is a common cause of skin and soft tissue infections in dogs but can also cause infections in cats and humans. The frequency of methicillin-resistant S. pseudintermedius (MRSP) strains is increasing worldwide. Here, we obtained 43 MRSP isolates from dogs (n = 41), one cat (n = 1) and the small animal clinic environment (n = 1) in Slovenia from the period 2008-2018, which underwent whole-genome sequencing (WGS) and antimicrobial susceptibility testing. Five sequence types (STs) were identified, with ST71 (32/43) and ST551 (8/43) being the predominant. In Slovenia, ST551 was first detected in 2016, whereas a decrease in the frequency of ST71 was observed after 2015. All isolates were multidrug-resistant and most antimicrobial-resistant phenotypes could be linked to acquisition of the corresponding resistance genes or gene mutations. Core-genome multilocus sequence typing (cgMLST) revealed several potential MRSP transmission routes: (i) between two veterinary clinics by a single MRSP-positive dog, (ii) between the environment of a veterinary clinic and a dog, and (iii) between a canine and a feline patient through the contaminated environment of a veterinary clinic. Of the six dogs that were additionally sampled from 14 days to five months after the initial sampling, each harbored the same MRSP strain, suggesting a limited within-host diversity of MRSP in symptomatic dogs. The present results highlight the importance of MRSP-positive dogs in the spread of veterinary care-associated MRSP infections and call for the implementation of strict control measures to reduce MRSP contamination in veterinary clinic environments originating from animal-contact surfaces.

How Accurate Are Veterinary Clinicians Employing Flexicult Vet for Identification and Antimicrobial Susceptibility Testing of Urinary Bacteria?

Antibiotics are frequently used for treating urinary tract infections (UTI) in dogs and cats. UTI often requires time-consuming and expensive antimicrobial susceptibility testing (AST). Alternatively, clinicians can employ Flexicult Vet, an affordable chromogenic agar with added antibiotics for in-clinic AST. We investigated how well veterinary microbiologists and clinicians, without any prior experience, employ Flexicult Vet for the identification and AST of the most common canine and feline urinary pathogenic bacteria. We prepared 47 monoculture plates containing 10 bacterial species. The test's mean accuracy was 75.1% for bacteria identification (84.6% and 68.7% for microbiologists and clinicians, respectively) and 79.2% for AST (80.7% and 78.2%). All evaluators employed Flexicult Vet with the accuracies over 90% for the distinctively colored bacteria like Escherichia coli (red), Enterococcus faecalis (turquoise), and Proteus spp. (pale brown). However, the evaluators' experience proved important in recognizing lightly colored bacteria like Staphylococcus pseudintermedius (accuracies of 82.6% and 40.3%). Misidentifications of E. faecium additionally worsened AST performance since bacterial intrinsic resistance could not be considered. Finally, only 33.3% (3/9) of methicillin-resistant S. pseudintermedius (MRSP) were correctly detected. To conclude, Flexicult Vet proved reliable for certain urinary pathogens. In contrast, light-colored bacteria (e.g., Staphylococcus), often misidentified, require a standard AST.

Feline Otitis Externa Caused by Methicillin-Resistant Staphylococcus aureus with Mixed Hemolytic Phenotype and Overview of Possible Genetic Backgrounds.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infections in humans, but its importance in small animal practice is increasing. Here, we present a case of feline otitis externa (OE) caused by MRSA; both hemolytic and nonhemolytic variants with a stable phenotype were recovered from the external auditory canal after infection was detected by routine otoscopy. One isolate per variant underwent antimicrobial susceptibility testing (AST) by broth microdilution method, conventional spa typing and whole-genome sequencing (WGS). The results showed that both variants were genetically related and were of sequence type (ST) 1327, SCCmec type IV and spa type t005. AST and WGS showed that both isolates were resistant to ß-lactams and sensitive to all tested non-ß-lactam antibiotics. Both isolates were pvl-negative, but encoded several other virulence genes (aur, hlgABC, sak, scn, seg, sei, sem, sen, seo and seu). Genetic background of the mixed hemolytic phenotype was not identified; no differences in the agr locus or other regulatory regions were detected. Three single-nucleotide polymorphisms were identified but could not be associated with hemolysis. This well-documented case of MRSA infection in companion animals adds to the reports of MRSA infections with a mixed hemolytic phenotype.