Antimicrobial Resistance Profile of Bacteria Isolated from Canine and Feline Samples at the Preventive Veterinary Laboratory of the Federal University of Rio Grande do Sul (UFRGS) - Brazil

Background: Antimicrobial resistance (AMR) is a major global health threat. In small animals such as dogs and cats, antimicrobials are most commonly prescribed for skin and genitourinary diseases; therefore, the AMR of bacteria involved in these infections should be monitored. In addition, the results of antimicrobial susceptibility testing (AST) may be interpreted as a local epidemiological history of AMR. The Preventive Veterinary Medicine Laboratory (PVML) received clinical samples from dogs and cats for bacterial isolation and AST. Thus, this study aimed to assess the AMR of bacteria isolated from the samples of dogs and cats received at the Preventive Veterinary Medicine Laboratory (PVML). Materials, Methods & Results: Data from bacteriological examinations performed at the PVML of the Universidade Federal do Rio Grande do Sul (UFRGS) during 5 years were analyzed. Skin and ear canal samples were inoculated in 5% sheep blood agar, and urine samples were streaked on CHROMagar™ orientation. After incubation at 36±1°C for up to 72 h, identification and AST were performed according to routine protocols. Of 1,534 samples submitted to the PVML, 1,086 (70.8%) were collected from dogs and  29.2% from feline patients. Otological swabs (n = 533, 49.1%) were the most frequent samples from dogs, while cat urine samples (n = 384, 84.8%) predominated by far. Considering the canine samples, no bacterial growth (NBG) was observed in 443 (40.8%) samples, while only one colony type was noted in 516 (47.5%) samples. Gram-positive bacteria (n = 298) were more frequent than gram-negative bacteria (n = 77) in the skin. In urine samples, gram-negative bacteria (n = 94) were isolated more frequently than gram-positive bacteria (n = 47). In feline samples, a high number of NBG (n = 308, 68%) was observed. Gram-positive (n = 22) was predominant in comparison to gram-negative bacteria (n = 9) in cultures from the ear and skin swabs. Enterococcus spp. and Escherichia coli were the most frequently identified bacteria in urine samples. Among the Staphylococcus sp. strains of any origin, AMR frequency varied from 4.22% (amikacin) to 50.70% (sulfa/trimethoprim). Enterococcus spp. showed AMR frequencies from 12.5% (amoxicillin/clavulanic acid) to 62.06% (enrofloxacin). Among the gram-negative genera, E. coli presented AMR frequencies from 10.20% (gentamicin) to 60.0% (neomycin). The frequency of AMR was stable over time, and a profile of much higher resistance to fluoroquinolones in comparison to beta-lactams was observed. Discussion: The recurrence of skin and urinary infections implies the need for frequent treatment with antibiotics, which exerts selection pressure for resistance and multidrug resistance. In this study, the frequency of multidrug resistance was low, and the resistance to the tested antimicrobials showed high variation. However, a trend of high resistance to the fluoroquinolone group was observed in contrast to the low resistance to beta-lactams. This trend was consistent among the isolated bacteria, regardless of the type of sample or origin. The overprescription of fluoroquinolones in small animal practices has been widely documented in several countries. However, this class of antimicrobials, is highly prioritized for the treatment of infections in humans. Therefore, the selection of resistant strains has gained special emphasis, especially when considering the possibility of the transmission of resistant bacteria between pets and humans. In summary, the results of bacteriological tests conducted at the PVML-Universidade Federal do Rio Grande do Sul confirmed that ubiquitous bacteria predominate in clinical samples of dogs and cats. The high frequency of resistance to the fluoroquinolone group, while a predominance of susceptible strains in the first-choice drugs such as amoxicillin/clavulanic acid, may indicate excessive and empirical use of the second-choice drugs in clinical practice. Keywords: otitis, dermatitis, urinary infection, dogs, cats, antibiotic resistance, AMR

Antimicrobial resistance profile of bacteria isolated from canine and feline samples at the Preventive Veterinary Laboratory of the Federal University of Rio Grande do Sul (UFRGS) – Brazil

Background: Antimicrobial resistance (AMR) is a major global health threat. In small animals such as dogs and cats, antimicrobials are most commonly prescribed for skin and genitourinary diseases; therefore, the AMR of bacteria involved in these infections should be monitored. In addition, the results of antimicrobial susceptibility testing (AST) may be interpreted as a local epidemiological history of AMR. The Preventive Veterinary Medicine Laboratory (PVML) received clinical samples from dogs and cats for bacterial isolation and AST. Thus, this study aimed to assess the AMR of bacteria isolated from the samples of dogs and cats received at the Preventive Veterinary Medicine Laboratory (PVML). Materials, Methods & Results: Data from bacteriological examinations performed at the PVML of the Universidade Federal do Rio Grande do Sul (UFRGS) during 5 years were analyzed. Skin and ear canal samples were inoculated in 5% sheep blood agar, and urine samples were streaked on CHROMagar™ orientation. After incubation at 36±1°C for up to 72 h, identification and AST were performed according to routine protocols. Of 1,534 samples submitted to the PVML, 1,086 (70.8%) were collected from dogs and 29.2% from feline patients. Otological swabs (n = 533, 49.1%) were the most frequent samples from dogs, while cat urine samples (n = 384, 84.8%) predominated by far. Considering the canine samples, no bacterial growth (NBG) was observed in 443 (40.8%) samples, while only one colony type was noted in 516 (47.5%) samples. Gram-positive bacteria (n = 298) were more frequent than gram-negative bacteria (n = 77) in the skin. In urine samples, gram-negative bacteria (n = 94) were isolated more frequently than gram-positive bacteria (n = 47). In feline samples, a high number of NBG (n = 308, 68%) was observed. Gram-positive (n = 22) was predominant in comparison to gram-negative bacteria (n = 9) in cultures from the ear and skin swabs. Enterococcus spp. and Escherichia coli were the most frequently identified bacteria in urine samples. Among the Staphylococcus sp. strains of any origin, AMR frequency varied from 4.22% (amikacin) to 50.70% (sulfa/trimethoprim). Enterococcus spp. showed AMR frequencies from 12.5% (amoxicillin/clavulanic acid) to 62.06% (enrofloxacin). Among the gram-negative genera, E. coli presented AMR frequencies from 10.20% (gentamicin) to 60.0% (neomycin). The frequency of AMR was stable over time, and a profile of much higher resistance to fluoroquinolones in comparison to beta-lactams was observed. Discussion: The recurrence of skin and urinary infections implies the need for frequent treatment with antibiotics, which exerts selection pressure for resistance and multidrug resistance. In this study, the frequency of multidrug resistance was low, and the resistance to the tested antimicrobials showed high variation. However, a trend of high resistance to the fluoroquinolone group was observed in contrast to the low resistance to beta-lactams. This trend was consistent among the isolated bacteria, regardless of the type of sample or origin. The overprescription of fluoroquinolones in small animal practices has been widely documented in several countries. However, this class of antimicrobials, is highly prioritized for the treatment of infections in humans. Therefore, the selection of resistant strains has gained special emphasis, especially when considering the possibility of the transmission of resistant bacteria between pets and humans. In summary, the results of bacteriological tests conducted at the PVML-Universidade Federal do Rio Grande do Sul confirmed that ubiquitous bacteria predominate in clinical samples of dogs and cats. The high frequency of resistance to the fluoroquinolone group, while a predominance of susceptible strains in the first-choice drugs such as amoxicillin/clavulanic acid, may indicate excessive and empirical use of the second-choice drugs in clinical practice

In vitro antibacterial activity of tigecycline against clinical isolates of Linezolid-Intermediate (LIE) and Linezolid-Resistant Enterococci (LRE) by time-kill assay

Introduction: Enterococci have become the third major leading cause of nosocomial bacteraemia, an infection which is significantly associated with the risk of developing infective endocarditis. Linezolid provides high rates of clinical cure and microbiologic success in complicated infections due to Enterococcus spp. However, several instances of emergence of resistance during linezolid treatment have been reported. The aim of this study was evaluate the activity of tigecycline against Linezolid-Intermediate (LIE) and Linezolid-Resistant Enterococcus faecalis (LRE) by the time-kill assay.Methods: Five isolates of LRE and two isolates of LIE were used in this study. MICs were determined by broth dilution following the CLSI (2014) guidelines. Time-kill assay was employed to access the in vitro response profile of tigecycline.Results: All seven of the isolates presented MIC of 0.125μg/mL. Tigecycline activity was individually evaluated and in three of the five isolates of LRE it presented bactericidal. Against the other isolates, tigecycline showed bacteriostatic activity. The tigecycline activity was measured according to CLSI criteria.Conclusions: Tigecycline presented both bacteriostatic and bactericidal activity against tested isolates, result not yet described in previous studies. Time and concentrations above MIC were key factors to achieving bactericidal effect. MIC and the tested concentration below it resulted in bacteriostatical effect to enterococci, corroborating previous data

Enhancement of antistaphylococcal activities of six antimicrobials against sasG-negative methicillin-susceptible Staphylococcus aureus: an in vitro biofilm model

AbstractThis study was designed to evaluate antimicrobial activities against methicillin-susceptible Staphylococcus aureus in both sessile and planktonic forms and to detect genes associated with this biofilm phenotype. Minimal biofilm inhibition and eradication concentrations (MBIC and MBEC, respectively) were determined by an in vitro biofilm model, and icaA, atlA, and sasG genes were detected by polymerase chain reaction. Vancomycin and tigecycline presented better biofilm inhibitory activity (MBIC range: 4–8 μg/mL) (P ≤ 0.05) and lower MBEC/MIC ratios (P ≤ 0.001) than other antimicrobials. All isolates harbored icaA and atlA, whereas sasG was present only in strong biofilm formers (P ≤ 0.05). Interestingly, antimicrobial activities against sasG− weak biofilm formers were significantly higher than those against sasG+ strong biofilm formers (P ≤ 0.05), demonstrating that number of cells in a biofilm matrix affected the antimicrobial activity, which was also variable, and might be associated with specific genetic determinants. To our knowledge, this was the first study reporting the presence of sasG in clinical isolates of S. aureus in South America

In vitro antibacterial activity of tigecycline against clinical isolates of Linezolid-Intermediate (LIE) and Linezolid-Resistant Enterococci (LRE) by time-kill assay

Introduction: Enterococci have become the third major leading cause of nosocomial bacteraemia, an infection which is significantly associated with the risk of developing infective endocarditis. Linezolid provides high rates of clinical cure and microbiologic success in complicated infections due to Enterococcus spp. However, several instances of emergence of resistance during linezolid treatment have been reported. The aim of this study was evaluate the activity of tigecycline against Linezolid-Intermediate (LIE) and Linezolid-Resistant Enterococcus faecalis (LRE) by the time-kill assay. Methods: Five isolates of LRE and two isolates of LIE were used in this study. MICs were determined by broth dilution following the CLSI (2014) guidelines. Time-kill assay was employed to access the in vitro response profile of tigecycline. Results: All seven of the isolates presented MIC of 0.125μg/mL. Tigecycline activity was individually evaluated and in three of the five isolates of LRE it presented bactericidal. Against the other isolates, tigecycline showed bacteriostatic activity. The tigecycline activity was measured according to CLSI criteria. Conclusions: Tigecycline presented both bacteriostatic and bactericidal activity against tested isolates, result not yet described in previous studies. Time and concentrations above MIC were key factors to achieving bactericidal effect. MIC and the tested concentration below it resulted in bacteriostatical effect to enterococci, corroborating previous data

Molecular characteristics of vancomycin-susceptible Staphylococcus aureus could help to predict treatment failure due to reduced vancomycin susceptibility

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of healthcare-associated and community-acquired infections and with its advancement, vancomycin became the main therapeutic option. However, its indiscriminate 2 use favored the emergence of MRSA with reduced susceptibility to vancomycin, commonly associated with vancomycin treatment failure, persistent bacteremia, prolonged hospitalization and adverse clinical outcome. This study evaluated the occurrence of MRSA with reduced vancomycin susceptibility and determined some molecular characteristics in comparison with vancomycin-susceptible MRSA (VS-MRSA). Methods: Determination of antimicrobial susceptibility profile, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for vancomycin, vancomycin-tolerance, SCCmec and agr typing were performed in a total of 177 MRSA. Thereafter, they were screened for hVISA by BHIA-3V and BHIA-6V and confirmed with population analysis profile - area under the curve method (PAPAUC). Results: VT-MRSA and hVISA phenotypes were found in 13.6% and 5.1% of clinical isolates of MRSA, respectively, and the presence of hVISA was statistically significant among VT-MRSA isolates (

In-Depth Genomic Characterization of a Meropenem-nonsusceptible Pseudomonas otitidis Strain Contaminating Chicken Carcass

Background: The indiscriminate use of antibiotics in food-animal production has a major impact on public health, particularly in terms of contributing to the emergence and dissemination of antimicrobial resistant bacteria in the food-animal production chain. Although Pseudomonas species are recognized as important spoilage organisms in foodstuff, they are also known as opportunistic pathogens associated with hospital-acquired infections. Furthermore, Pseudomonas can play a role as potential reservoirs of antimicrobial resistance genes, which may be horizontally transferred to other bacteria. Considering that cephalosporins (3rd and higher generations) and carbapenems are critically important beta-lactam antimicrobials in human medicine, this study reports the occurrence and genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain recovered from a chicken carcass in Brazil.Materials, Methods & Results: During the years 2018-2019, 72 frozen chicken carcasses were purchased on the retail market from different regions in Brazil. Aliquots from individual carcass rinses were screened for Extended Spectrum Beta-lactamase (ESBL)-producing bacteria in MacConkey agar supplemented with 1mg.L-1 cefotaxime. Phenotypically resistant isolates were further tested for resistance to other antimicrobials and confirmed as ESBL-producers by means of disk-diffusion method using Müller-Hinton agar. Only one meropenen-nonsusceptible isolate was detected and submitted to whole genome sequencing (WGS) in Illumina Miseq. The strain was identified as Pseudomonas otitidis by local alignment of the 16S rRNA sequence using BLASTn and confirmed by Average Nucleotide Identity (ANI) analysis using JspeciesWS database. Genes encoding for antimicrobial resistance were detected by means of Resfinder and Comprehensive Antibiotic Resistance Database (CARD) databases. The phenotypic non-susceptibility to meropenen was attributed to the gene blaPOM-1. A total of 192 different genes encoding for quorum sensing system, antiphagocytosis, iron uptake, efflux pump, endotoxin and toxin, adherence, and secretion systems were detected by means of Virulence Factor Database (VFDB). Pseudomonas otitidis-pan genome was built using Roary-rapid large-scale prokaryote pan genome analysis using the present strain (K_25) and other two P. otitidis genomes (PAM-1, DSM 17224) publicly available at the NCBI. The core genome analysis of the two human strains resulted in similar percentages.Discussion: Carbapenems are critically important drugs for human health and bacterial strains resistant to these antimicrobials pose a public health problem. The blaPOM-1 gene harbored by the Pseudomonas otitidis K_25 strain encodes a metallo-beta-lactamase (MBL) conferring resistance to carbapenems. Pseudomonas otitidis was the first confirmed pathogenic Pseudomonas species expressing MBL constitutively in the absence of inducible beta-lactamase genes. Furthermore, the several virulence genes associated with the capacity of the P. otitidis K_25 to colonize, evade the immune system and cause lesions in the human host confirm this strain as a potential opportunistic pathogen contaminating foodstuff. These reinforce the need to address antimicrobial resistance in a One Health perspective, in which resistant bacteria and resistance determinants circulate among environment, animals and humans

In-depth genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain contaminating chicken carcass

Background: The indiscriminate use of antibiotics in food-animal production has a major impact on public health, particularly in terms of contributing to the emergence and dissemination of antimicrobial resistant bacteria in the food-animal production chain. Although Pseudomonas species are recognized as important spoilage organisms in foodstuff, they are also known as opportunistic pathogens associated with hospital-acquired infections. Furthermore, Pseudomonas can play a role as potential reservoirs of antimicrobial resistance genes, which may be horizontally transferred to other bacteria. Considering that cephalosporins (3rd and higher generations) and carbapenems are critically important beta-lactam antimicrobials in human medicine, this study reports the occurrence and genomic characterization of a meropenem-nonsusceptible Pseudomonas otitidis strain recovered from a chicken carcass in Brazil. Materials, Methods & Results: During the years 2018-2019, 72 frozen chicken carcasses were purchased on the retail market from different regions in Brazil. Aliquots from individual carcass rinses were screened for Extended Spectrum Beta-lactamase (ESBL)-producing bacteria in MacConkey agar supplemented with 1mg.L-1 cefotaxime. Phenotypically resistant isolates were further tested for resistance to other antimicrobials and confirmed as ESBL-producers by means of disk-diffusion method using Müller-Hinton agar. Only one meropenen-nonsusceptible isolate was detected and submitted to whole genome sequencing (WGS) in Illumina Miseq. The strain was identified as Pseudomonas otitidis by local alignment of the 16S rRNA sequence using BLASTn and confirmed by Average Nucleotide Identity (ANI) analysis using JspeciesWS database. Genes encoding for antimicrobial resistance were detected by means of Resfinder and Comprehensive Antibiotic Resistance Database (CARD) databases. The phenotypic non-susceptibility to meropenen was attributed to the gene blaPOM-1. A total of 192 different genes encoding for quorum sensing system, antiphagocytosis, iron uptake, efflux pump, endotoxin and toxin, adherence, and secretion systems were detected by means of Virulence Factor Database (VFDB). Pseudomonas otitidis-pan genome was built using Roary-rapid large-scale prokaryote pan genome analysis using the present strain (K_25) and other two P. otitidis genomes (PAM-1, DSM 17224) publicly available at the NCBI. The core genome analysis of the two human strains resulted in similar percentages. Discussion: Carbapenems are critically important drugs for human health and bacterial strains resistant to these antimicrobials pose a public health problem. The blaPOM-1 gene harbored by the Pseudomonas otitidis K_25 strain encodes a metallo-beta-lactamase (MBL) conferring resistance to carbapenems. Pseudomonas otitidis was the first confirmed pathogenic Pseudomonas species expressing MBL constitutively in the absence of inducible beta-lactamase genes. Furthermore, the several virulence genes associated with the capacity of the P. otitidis K_25 to colonize, evade the immune system and cause lesions in the human host confirm this strain as a potential opportunistic pathogen contaminating foodstuff. These reinforce the need to address antimicrobial resistance in a One Health perspective, in which resistant bacteria and resistance determinants circulate among environment, animals and humans

Características moleculares de Staphylococcus aureus suscetível à vancomicina poderia ajudar a prever falhas no tratamento devido à reduzida suscetibilidade à vancomicina

Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of healthcare-associated and community-acquired infections and with its advancement, vancomycin became the main therapeutic option. However, its indiscriminate 2 use favored the emergence of MRSA with reduced susceptibility to vancomycin, commonly associated with vancomycin treatment failure, persistent bacteremia, prolonged hospitalization and adverse clinical outcome. This study evaluated the occurrence of MRSA with reduced vancomycin susceptibility and determined some molecular characteristics in comparison with vancomycin-susceptible MRSA (VS-MRSA). Methods: Determination of antimicrobial susceptibility profile, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for vancomycin, vancomycin-tolerance, SCCmec and agr typing were performed in a total of 177 MRSA. Thereafter, they were screened for hVISA by BHIA-3V and BHIA-6V and confirmed with population analysis profile - area under the curve method (PAPAUC). Results: VT-MRSA and hVISA phenotypes were found in 13.6% and 5.1% of clinical isolates of MRSA, respectively, and the presence of hVISA was statistically significant among VT-MRSA isolates (pJustificación y objetivos: Staphylococcus aureus resistente a la meticilina (MRSA) es una de las causas más frecuentes de infecciones relacionadas con la asistencia sanitaria y comunitarias, y con su avance, a la vancomicina se ha convertido en la principal opción terapéutica. Sin embargo, su uso indiscriminado favoreció el surgimiento de MRSA con reducida susceptibilidad a la vancomicina, comúnmente asociados con fallas en el tratamiento, bacteriemia persistente, hospitalización prolongada y resultados clínicos adversos. Este estudio evaluó la ocurrencia de MRSA con reducida susceptibilidad a la vancomicina y determinó algunas características moleculares en comparación con MRSA susceptible a la vancomicina (VS-MRSA). Métodos: Determinación del perfil de susceptibilidad a los antimicrobianos, la concentración inhibitoria mínima (CIM) y la concentración bactericida mínima (CBM) para vancomicina, tolerancia a la vancomicina, tipificación del SCCmec y agr se realizaron en un total de 177 MRSA. Resultados: Los fenotipos VT-MRSA y hVISA se encontraron en el 13,6% y el 5,1% de los aislados clínicos de MRSA, respectivamente, y la presencia de hVISA fue estadísticamente significativa entre los aislados de VT-MRSA (pJustificativa e Objetivos: Staphylococcus aureus resistente à meticilina (MRSA) é uma das causas mais frequentes de infecções relacionadas à assistência à saúde e comunitárias, e com seu avanço, a vancomicina tornou-se a principal opção terapêutica. Entretanto, o seu uso indiscriminado favoreceu o surgimento de MRSA com reduzida suscetibilidade à vancomicina, comumente associados com falhas no tratamento, bacteremia persistente, hospitalização prolongada e desfechos clínicos adversos. Este estudo avaliou a ocorrência de MRSA com reduzida suscetibilidade à vancomicina e determinou algumas características moleculares em comparação com MRSA suscetível à vancomicina (VS-MRSA). Métodos: Determinação do perfil de suscetibilidade aos antimicrobianos, a concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) para vancomicina, tolerância à vancomicina, tipagem do SCCmec e agr foram realizadas em um total de 177 MRSA. Posteriormente, foram triados para hVISA por BHIA-3V e BHIA-6V e confirmados com a Análise do Perfil Populacional - Área Abaixo da Curva (PAP-AUC). Resultados: Os fenótipos VT-MRSA e hVISA foram encontrados em 13,6% e 5,1% dos isolados clínicos de MRSA, respectivamente, e a presença de hVISA foi estatisticamente significativa entre os isolados de VT-MRSA (