Detection and genomic characterization of a multidrug-resistant Salmonella Newport co-harbouring blaCMY-2, qnrB19 and mcr-9 from the diarrheic faeces of a foal.

OBJECTIVES: This study reports the genomic characterization of the multidrug resistant Salmonella Newport strain 195_20 recovered from the diarrheic faeces of a foal in Brazil and co-harbouring the mcr-9, blaCMY-2 and qnrB19 antibiotic resistance genes. METHODS: Bacterial isolate positive for mobile colistin resistance gene (mcr-9) was submitted to antimicrobial susceptibility testing by disk diffusion and broth microdilution for colistin and polymyxin B. The isolate was submitted to whole genome sequencing by Illumina technology and Nanopore Sequencing. Conjugation assays, plasmid sizes determined by S1-PFGE and plasmid content were investigated by hybrid assembly after MinIon long reads sequencing. RESULTS: Isolate 195_20 was identified as sequence type ST45, resistant to penicillin and cephalosporins (ampicillin, ceftazidime, ceftriaxone and cefotaxime), aminoglycosides (streptomycin and gentamicin), phenicol (chloramphenicol), quinolones and fluoroquinolones (nalidixic acid, ciprofloxacin, and pefloxacin), folate pathway antagonists (sulfonamides and trimethoprim-sulfamethoxazole), and tetracycline. A transferable IncHI2/IncHI2A plasmid sized ca. 262kb was found to carry the mcr-9 gene in a module consisting of IS903-mcr-9-wbuC-IS26. In addition, an 174kb IncC and a 48kb IncN plasmid were also identified in the 195_20 isolate, carrying blaCMY-2 and qnrB19, respectively. CONCLUSIONS: Not surprisingly, isolate 195_20 was susceptible to polymyxins, possibly due to absence of qseBC regulatory operon. Presence of mobile colistin resistance (mcr-9), third-generation cephalosporins (blaCMY-2) and quinolone (qnrB19) resistance determinants in zoonotic pathogens from animals in close contact with humans alerts for the possible route of transmission between these different reservoirs.

Overview of antimicrobial resistance and virulence factors in Salmonella spp. isolated in the last two decades from chicken in Brazil.

Foodborne infections caused by Salmonella have been linked to a variety of poultry products. The aim of this study was to compare the molecular profile of virulence genes considering different serotypes of Salmonella, isolates were from chicken breast sampled during the last two decades (1999 to 2010 and 2011 to 2018). The resistance to antimicrobials was also evaluated, establishing a comparative epidemiological parameter on the pathogenic potential on this bacterium over time. We tested 238 Salmonella isolates, and 18 different serotypes were observed. These being S. Enteritidis (42.3%, 58/137) and S. Ohio (28.3%, 36/137), the most frequent in the first decade; and S. Heidelberg (25.7%, 26/101) and S. Typhimurium (21.8%, 22/101), in the second. We found four (1.68%) multidrug resistant isolates from the first decade and 28 (11.76%) in the second. All extended spectrum beta-lactamase (ESBL) positive isolates belonged to the S. Heidelberg serotype, and were also detected in the second decade. Considering the nine different antimicrobial classes tested, an increase in the number of resistant isolates was observed over time: from five classes with resistant isolates in the first decade to eight classes in the second, with cefotaxime being the antimicrobial with the highest number of resistant isolates in both decades. All isolates (100%) presented the invA, sitC and tolC genes. In sequence, the most frequent genes were flgL (99.6%), sopB (98.3%), flgK (97.9%), fljB (96.6%), sipA (94.9%), sipB (88.6%), sifA (86.4%), sipD (66.1%), ssaR (51.3%), sopD (37.3%) and spvB (34.3%) was the least frequent; and 13 isolates showing all 14 virulence genes investigated. The ability of these isolates to resist certain antimicrobials, and to express genes encoding virulence factors, reinforce their marked pathogenic potential; while the possibility to trigger diseases in humans through the food chain is a serious public health threat through.

Genomic characterization of a multi-drug resistant, CTX-M-65-producing clinical isolate of Salmonella Infantis isolated in Brazil.

A multi-drug resistant, CTX-M-65 producing Salmonella Infantis was identified from a patient in Brazil. Whole genome sequencing followed by hybrid assembly (short and long reads) indicated the presence of blaCTX-M-65 in a pESI-like megaplasmid in this ST32 isolate and phylogenetic analysis showed high similarity with IncFIB S. Infantis isolates from food and poultry in the USA.

Padronização de técnica molecular para a detecção de salmonella spp em carne moída in natura / Standardization of molecular technique for the detection of salmonella spp in fresh ground meat

A Salmonella spp. é um dos principais agentes etiológicos incriminados nos surtos de doenças transmitidas por alimentos (DTAs) ocorridos no Brasil entre 2009 a 2018. A carne moída apresenta maior risco de contaminação microbiológica quando comparada aos demais cortes cárneos, podendo carrear micro-organismos patogênicos, representando um sério risco à saúde pública. O diagnóstico microbiológico (teste padrão ouro) para a bactéria é realizado em casos de surtos, porém o resultado conclusivo demora cerca de 7 dias, o que retarda as medidas de vigilância em saúde por parte das vigilâncias epidemiológica e sanitária. Devido ao longo tempo demandado para o diagnóstico conclusivo pela microbiologia, cresce a necessidade de desenvolver técnicas laboratoriais mais eficientes, capazes de agilizar a identificação correta das salmoneloses em alimentos. Com isso, este estudo objetivou desenvolver e padronizar uma reação em cadeia da polimerase (PCR) convencional para detectar Salmonella spp. em carne moída bovina. Para atingir tal objetivo, 4 amostras de carne moída in natura foram contaminadas experimentalmente com diluições seriadas da bactéria a partir da escala 0,5 de McFarland e após processo de descongelamento dessas amostras, o sedimento do filtrado cárneo resultante foi submetido a PCR para análise do limite de detecção da técnica padronizada. Em 75% das amostras a técnica foi capaz de detectar uma concentração de 1x106 UFC/mL. Não houve êxito na validação da técnica, uma vez que amostras positivas e negativas para a Salmonella spp., confirmadas pela microbiologia, foram negativas quando submetidas ao protocolo padronizado, demonstrando que, neste estudo, o padrão ouro foi mais sensível do que o PCR, sendo necessários ajustes para que a técnica seja uma ferramenta viável e possa ser empregada rotineiramente.

Efficacy of cefquinome and a combination of cloxacillin and ampicillin for treatment of dairy cows with Streptococcus agalactiae subclinical mastitis.

A randomized clinical trial was conducted to assess efficacy of intramammary cloxacillin and ampicillin (CLOXIMM), intramammary cefquinome (CEFIMM), and intramuscular cefquinome (CEFIM) to treat Streptococcus agalactiae intramammary infections (Trial 1). Subsequently, two treatment groups were extended to assess whether CLOXIMM was not inferior to CEFIMM (Trial 2). Nine farms were included in the study. Milk samples were collected from all quarters of all lactating cows for microbiological identification of S. agalactiae. Positive cows were randomly allocated into four groups: CLOXIMM, CEFIMM, CEFIM, or negative control (CONTROL). Study outcomes were bacteriological cure at 14 (CURE14), 21 (CURE21), and 14 and 21 (CURE1421) days after treatment onset, and somatic cell count. Logistic regression was used to estimate the odds of cure between each treatment and CONTROL. Non-inferiority analysis was performed considering a one-sided 95% confidence interval (CI) and non-inferiority margins (Δ) of 0.10, 0.15, 0.20, and 0.25. Adjusted S. agalactiae bacteriological cure for CLOXIMM, CEFIMM, CEFIM, and CONTROL was 86, 98, 55, and 25% at day 14; 82, 93, 52, and 0% at day 21; and 82, 92, 40, and 0% at days 14 and 21, respectively. Treatment with CLOXIMM and CEFIMM resulted in greater bacteriological cure rates, as compared with CEFIM or CONTROL, which does not justify the use of CEFIM in S. agalactiae eradication programs. The CURE14 difference between CEFIMM and CLOXIMM was of 12.1 percentage points (95% CI: 0.056-0.184). CLOXIMM was considered not inferior to CEFIMM for Δ = 0.20 or 0.25 and inconclusive for Δ = 0.10 or 0.15. Thus, it should be pondered by veterinarians whether an expected 12.1 (5.6-18.4) percentage points increase in cure rate would justify the use of a fourth-generation cephalosporin, as opposed to a combination of traditional IMM drugs (cloxacillin and ampicillin) to treat S. agalactiae subclinical mastitis.