Genomic features and comparative analysis of a multidrug-resistant Acinetobacter bereziniae strain infecting an animal: a novel emerging one health pathogen?

Acinetobacter bereziniae has recently gained medical notoriety due to its emergence as a multidrug resistance and healthcare-associated pathogen. In this study, we report the whole-genome characterization of an A. bereziniae strain (A321) recovered from an infected semiaquatic turtle, as well as a comparative analysis of A. bereziniae strains circulating at the human-animal-environment interface. Strain A321 displayed a multidrug resistance profile to medically important antimicrobials, which was supported by a wide resistome. The novel Tn5393m transposon and a qnrB19-bearing ColE1-like plasmid were identified in A321 strain. Novel OXA-229-like ß-lactamases were detected and expression of OXA-931 demonstrated a 2-64-fold increase in the minimum inhibitory concentration for ß-lactam agents. Comparative genomic analysis revealed that most A. bereziniae strains did not carry any antimicrobial resistance genes (ARGs); however, some strains from China, Brazil, and India harbored six or more ARGs. Furthermore, A. bereziniae strains harbored conserved virulence genes. These results add valuable information regarding the spread of ARGs and mobile genetic elements that could be shared not only between A. bereziniae but also by other bacteria of clinical interest. This study also demonstrates that A. bereziniae can spill over from anthropogenic sources into natural environments and subsequently be transmitted to non-human hosts, making this a potential One Health bacteria that require close surveillance.

Genomic features of mecA-positive methicillin-resistant Mammaliicoccus sciuri causing fatal infections in pets admitted to a veterinary intensive care unit.

Methicillin-resistant staphylococci have become leading cause of infectious diseases in humans and animals, being categorized as high priority pathogens by the World Health Organization. Although methicillin-resistant Staphylococcus sciuri (recently moved to Mammaliicoccus sciuri) has been widely reported in companion animals, there is scarce information regarding their clinical impact and genomic features. Herein, we reported the occurrence and genomic characteristics of methicillin-resistant M. sciuri recovered from fatal infections in pets admitted to an intensive care unit of a veterinary hospital, in Brazil. Two M. sciuri strains were isolated from bronchoalveolar lavage samples collected from dog (strain SS01) and cat (strain SS02) presenting with sepsis and acute respiratory distress syndrome. Both isolates displayed a multidrug-resistant profile, whereas whole-genome sequencing analysis confirmed the presence of the mecA gene, along to genetic determinant conferring resistance to macrolides, streptogramins, aminoglycosides, and trimethoprim. For both strains, the mec and crr gene complex shared high identity (≥97%) with analogue sequences from a M. sciuri isolated from a human wound infection, in the Czech Republic. Strains were assigned to the sequence type ST52 and the novel ST74. Phylogenomic analysis revealed a broad host range association of these strains with several hosts and sources, including humans, animals, food, and the environment through different years and geographic locations. Our findings demonstrate that infections caused by mecA-positive M. sciuri strains can be a serious threat for veterinary intensive care patients and the medical staff, with additional implications for One Health approaches.

The influence of sample processing time on the performance of Microsporum canis cultures in cats.

BACKGROUND: Fungal culture is widely used as a diagnostic tool for detecting dermatophytosis. However, the presence of fungal contaminants can influence the culture's performance and compromise the diagnosis. OBJECTIVE: To verify whether the sample processing time can affect the performance of fungal culture for the diagnosis of Microsporum canis infection in cats. ANIMALS: Forty Persian cats. METHODS AND MATERIALS: Hair and scale samples were collected by combing the coat using a 5 × 5 cm sterile polyester carpet. The carpets were assigned randomly to four groups based on time point of processing samples after collection (i.e. used for culture on a selective agar medium for dermatophytes): Group 1: 8 h (n = 10); Group 2: 24 h (n = 10); Group 3: 48 h (n = 10); and Group 4: 72 h (n = 10). Cultures were compared regarding the degree of fungal invasion by either M. canis or nondermatophytic contaminant moulds (NDM). RESULTS: Processing samples after 24 h of storage resulted in increased isolation rates of NDM and decreased isolation rates of M. canis. Samples processed after 48 h and 72 h presented more than half of the plates with a high degree of fungal contamination (i.e. NDM occupying ≥50% of the total fungal mass). However, samples processed after 8 h and 24 h presented a lower degree (P < 0.05) of NDM plate invasion and higher recovery rates of M. canis when compared to samples processed after 48 h and 72 h. CONCLUSIONS AND CLINICAL IMPORTANCE: Delayed processing time is closely associated with the overgrowth of contaminants and with lower recovery rates of M. canis.

Rapid spread of critical priority carbapenemase-producing pathogens in companion animals: a One Health challenge for a post-pandemic world.

The COVID-19 pandemic has increased relationships and interactions between human and companion animals, supported by widespread social distancing and isolation measures. Additionally, the COVID-19 pandemic has led to an exponential growth in antibiotic and biocide use worldwide, possibly inducing further pressure, contributing to the selection of antibiotic-resistant bacteria, including WHO critical priority pathogens. While data from global surveillance studies reveal a linear trend of increasing carbapenem resistance among Gram-negative pathogens from companion animals, the acquisition of carbapenemase-producing Enterobacterales through direct contact with colonized hosts and contaminated veterinary hospital environments has been documented. This article highlights the rapid spread of WHO critical priority carbapenemase-producing pathogens in companion animals, which is a One Health challenge for a post-pandemic world.

Genomic insights of Klebsiella pneumoniae isolated from a native Amazonian fish reveal wide resistome against heavy metals, disinfectants, and clinically relevant antibiotics.

A multidrug-resistant CTX-M-15-producing Klebsiella pneumoniae (KpP1 strain) was isolated from a native Amazonian fish (Brachyplatystoma filamentosum) at the Brazilian Amazon. The strain was identified by MALDI-TOF. The genome was extracted, purified and a Nextera DNA Flex library was prepared and sequenced by Illumina platform. The sequenced genome was de novo assembled using Unicycler and in silico prediction accomplished by curated bioinformatics tools. The size of the genome is 5.6 Mb with 5715 genes. Whole-genome sequencing analysis revealed the presence of wide resistome, with genes conferring resistance to clinically relevant antibiotics, heavy metals and disinfectants. The KpP1 strain was assigned to the sequence type ST3827, KL111 (wzi113) and O3b locus. Native freshwater fish sold in wet markets of the Amazonian region could be an important vehicle for transmission of multidrug-resistant bacteria to humans. This study may give genomic insights on the spread of critical-priority WHO pathogens in a One Health context.

The use of ultrasound to assist epidural injection in obese dogs.

OBJECTIVE: To evaluate the use of ultrasound for identifying the site for needle puncture and to determine the depth to the epidural space in obese dogs. STUDY DESIGN: Prospective study in dogs undergoing elective orthopedic surgery. ANIMALS: A group of seven obese Labrador male dogs aged 6.93 ± 2.56 years and weighing 46.5 ± 4.1 kg (mean ± standard deviation). METHODS: The anesthetic protocol for these dogs included epidural anesthesia. With the dogs anesthetized and positioned in sternal recumbency with the pelvic limbs flexed forward, ultrasound imaging was used to locate the lumbosacral intervertebral space. Intersection of dorsal and transverse lines about the probe identified the point of needle insertion. A 17 gauge, 8.9 cm Tuohy needle was inserted perpendicularly through the skin and advanced to the lumbosacral intervertebral space. The number of puncture attempts was recorded and needle depth was compared with skin to ligamentum flavum distance. RESULTS: Epidural injection was performed in all dogs at the first attempt of needle insertion. The distance from skin to epidural space was 5.95 ± 0.62 cm measured by ultrasound and 5.89 ± 0.64 cm measured with the Tuohy needle. These measurements were not different (p = 0.26). A highly significant correlation coefficient of 0.966 between measurement techniques was obtained (p < 0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Ultrasound imaging identified the point of needle insertion for lumbosacral epidural injection in seven obese dogs. The results indicate that ultrasound can be used to locate the lumbosacral intervertebral space and identify an appropriate point for needle insertion to perform epidural injection.

Effective treatment and decolonization of a dog infected with carbapenemase (VIM-2)-producing Pseudomonas aeruginosa using probiotic and photodynamic therapies.

BACKGROUND: Carbapenem-resistant bacterial infections are a critical problem in veterinary medicine with limited treatment options. OBJECTIVE: To describe effective probiotic and photodynamic therapy of a dog with gut colonization and ear infection caused by a hospital-associated lineage of carbapenemase (VIM-2)-producing Pseudomonas aeruginosa. ANIMALS: A 5-year-old Lhasa apso dog presented with otitis externa. METHODS AND MATERIALS: Unilateral otitis externa caused by carbapenem-resistant P. aeruginosa was treated with antimicrobial photodynamic therapy (aPDT) using methylene blue as photosensitizer [wavelength 660 nm, fluence 140 J/cm2 , 8 J and 80 s per point (six equidistant points), 100 mW, spot size 0.028 cm2 and fluence rate 3.5 W/cm2 ]. The isolated bacterial strain also was tested for susceptibility to in vitro aPDT where the survival fraction was quantified by colony forming unit counts after exposure to increasing light doses. For decolonization, probiotic supplements were orally administered (once daily) for 14 days. Effectiveness of probiotics and photodynamic therapy was evaluated by clinical and microbiological culture assays. RESULTS: Complete resolution of clinical signs was achieved by Day 7 after aPDT. Samples collected immediately and after seven and 14 days following aPDT were negative for VIM-2-producing P. aeruginosa. Oral and rectal swabs collected on days 7, 14 and 21 after probiotic therapy, confirmed effective gastrointestinal decolonization. CONCLUSIONS AND CLINICAL IMPORTANCE: Combined use of aPDT and probiotics could be a promising therapeutic strategy for treatment of superficial infections produced by carbapenem-resistant bacteria, while avoiding recurrent infection due to intestinal bacterial carriage of these multidrug-resistant pathogens.

Zooanthroponotic Transmission of Drug-Resistant Pseudomonas aeruginosa, Brazil.

We recovered VIM-2 carbapenemase-producing Pseudomonas aeruginosa isolates from an infected dog, its owner, and the domestic environment. Genomic investigation revealed household transmission of the high-risk hospital clone sequence type 233 in the human-animal-environment interface. Results suggest zooanthroponotic transmission of VIM-2-producing P. aeruginosa in the household following the patient's hospital discharge.

Colistin-Resistant mcr-1-Positive Escherichia coli on Public Beaches, an Infectious Threat Emerging in Recreational Waters.

The emergence and rapid spread of colistin-resistant Escherichia coli carrying the mcr-1 gene have generated an urgent need to strengthen surveillance. We performed a meticulous investigation of strains of this sort, which resulted in the identification of international clones of E. coli carrying IncX4-plasmid-mediated mcr-1 and blaCTX-M genes in recreational waters of public urban beaches in cities with high tourist turnover, highlighting a new environmental reservoir.

Preliminary study of a teaching model for ultrasound-guided peripheral nerve blockade and effects on the learning curve in veterinary anesthesia residents.

OBJECTIVE: To evaluate the use of an experimental colloid model for teaching veterinary anesthesia residents ultrasound-guided technique for nerve blockade. STUDY DESIGN: Prospective, blinded and randomized. METHODS: Colloid models were constructed for practice in ultrasound-guided needle location. Nine veterinary anesthesia residents with no prior experience of ultrasound-guided technique for nerve blocks were randomly divided into three groups. Each group received theoretical orientation. Two groups were assigned to practical training using the experimental model: group 1 (G1) received 2 hours of training and group 2 (G2) received 1 hour of training prior to testing with specific tasks. Group 3 (G3) received no practical training. During testing, the time required for task completion (e.g., display of structures and positioning a needle) and the number of failures were recorded. RESULTS: The average times to completion of the tasks and the number of technical failures were: G1, 47 seconds and 1 failure; G2, 68 seconds and 2 failures; G3, 187 seconds and 7 failures. CONCLUSIONS AND CLINICAL RELEVANCE: In residents with no prior experience of ultrasound-guided needle placement, using an experimental colloid model and a longer training period was associated with increased accuracy and decreased time to task completion. Based on the results of this study, training with an experimental model can be recommended to improve the speed and accuracy of needle manipulation using ultrasound in clinicians with no prior experience of ultrasound-guided technique.

Genome-based diagnostic of MDR Escherichia coli ONT:H19 ST10955 causing human gastrointestinal infection.

This study focuses on the genomic characterization of a multidrug-resistant Escherichia coli strain responsible for a severe gastrointestinal infection in a 33-year-old male. The patient initially received sulfamethoxazole/trimethoprim treatment, which proved ineffective. Fecal culture confirmed the presence of E. coli displaying a MDR profile to ampicillin, nalidixic acid, ciprofloxacin, sulfamethoxazole, trimethoprim, and tetracycline. Serotyping identified the strain as ONT:H19. Virulence analysis indicated a highly virulent profile with numerous virulence markers. Plasmid analysis uncovered various plasmids carrying both antimicrobial resistance and virulence genes. MLST assigned the strain to ST10955. Phylogenomic analysis revealed similarity to an older Brazilian isolate, suggesting the persistence of a common lineage with evolving antimicrobial resistance. This report highlights the first identification of a multidrug-resistant ST10955 E. coli strain with a wide resistome and virulence potential, emphasizing the importance of ongoing surveillance of E. coli strains due to their potential for severe infections, resistance development, and virulence.

Hybrid genome assembly of colistin-resistant mcr-1.5-producing Escherichia coli ST354 reveals phylogenomic pattern associated with urinary tract infections in Brazil.

BACKGROUND: The rapid and global spread of Escherichia coli carrying mcr-type genes at the human-animal-environmental interface has become a serious global public health problem. OBJECTIVE: To perform a genomic investigation of a colistin-resistant E. coli strain (14005RM) causing urinary tract infection, using a hybrid de novo assembly of Illumina/Nanopore sequence data, presenting phylogenomic insights into the relationship with mcr-1-positive strains circulating at the human-animal-environmental interface, in Brazil. METHODS: Genomic DNA was sequenced using both the Illumina NexSeq and Nanopore MinION platforms. De novo hybrid assembly was performed by Unicycler. Genomic data were assessed by in silico prediction and bioinformatic tools. RESULTS: The genome assembly size was 5 333 039 bp. The mcr-1.5-positive E. coli strain 14005RM belongs to the sequence type ST354 and presented a broad resistome (antibiotics, heavy metals, disinfectants, and glyphosate) and virulome. The mcr-1.5 gene was carried by an IncI2 plasmid (p14005RM, sizing 65,458 kb). Full genome SNP-based phylogenetic analysis reveals that mcr-1.5-producing E. coli strain 14005RM is highly related (> 98% identity) to colistin-resistant mcr-1.1-positive ST354 lineages associated with urinary tract infections in Brazil since 2015. CONCLUSION: Mobile colistin resistance within the Brazilian One Health microbiosphere is mediated by mcr gene variants propagated by IncX4, IncHI2, and IncI2 plasmids, circulating among global clones of E. coli.

Successful expansion of hospital-associated clone of vanA-positive vancomycin-resistant Enterococcus faecalis ST9 to an anthropogenically polluted mangrove in Brazil.

Mangrove ecosystems are hotspots of biodiversity, but have been threatened by anthropogenic activities. Vancomycin-resistant enterococci (VRE) are nosocomial bacteria classified as high priority by the World Health Organization (WHO). Herein, we describe the identification and genomic characteristics of a vancomycin-resistant Enterococcus faecalis strain isolated from a highly impacted mangrove ecosystem of the northeastern Brazilian, in 2021. Genomic analysis confirmed the existence of the transposon Tn1546-vanA and clinically relevant antimicrobial resistance genes, such as streptogramins, tetracycline, phenicols, and fluoroquinolones. Virulome analysis identified several genes associated to adherence, immune modulation, biofilm, and exoenzymes production. The UFSEfl strain was assigned to sequence type (ST9), whereas phylogenomic analysis with publicly available genomes from a worldwide confirmed clonal relatedness with a hospital-associated Brazilian clone. Our findings highlight the successful expansion of hospital-associated VRE in a mangrove area and shed light on the need for strengthening genomic surveillance of WHO priority pathogens in these vital ecosystems.

Genomic Features of an MDR Escherichia coli ST5506 Harboring an IncHI2/In229/blaCTX-M-2 Array Isolated from a Migratory Black Skimmer.

Migratory birds have contributed to the dissemination of multidrug-resistant (MDR) bacteria across the continents. A CTX-M-2-producing Escherichia coli was isolated from a black skimmer (Rynchops niger) in Southeast Brazil. The whole genome was sequenced using the Illumina NextSeq platform and de novo assembled by CLC. Bioinformatic analyses were carried out using tools from the Center for Genomic Epidemiology. The genome size was estimated at 4.9 Mb, with 4790 coding sequences. A wide resistome was detected, with genes encoding resistance to several clinically significant antimicrobials, heavy metals, and biocides. The blaCTX-M-2 gene was inserted in an In229 class 1 integron inside a ∆TnAs3 transposon located in an IncHI2/ST2 plasmid. The strain was assigned to ST5506, CH type fumC19/fimH32, serotype O8:K87, and phylogroup B1. Virulence genes associated with survival in acid conditions, increased serum survival, and adherence were also identified. These data highlight the role of migratory seabirds as reservoirs and carriers of antimicrobial resistance determinants and can help to elucidate the antimicrobial resistance dynamics under a One Health perspective.

Genomic scan of a healthcare-associated NDM-1-producing Citrobacter freundii ST18 isolated from a green sea turtle impacted by plastic pollution.

BACKGROUND: Carbapenemase-producing Citrobacter freundii has been reported as a leading cause of healthcare-associated infections. Particularly, C. freundii belonging to the sequence type (ST) 18 is considered to be an emerging nosocomial clone. OBJECTIVES: To report the genomic background and phylogenomic analysis of a multidrug-resistant NDM-1-producing C. freundii ST18 (strain CF135931) isolated from an endangered green sea turtle affected by plastic pollution in Brazil. METHODS: Genomic DNA was extracted and sequenced using the Illumina NextSeq platform. De novo assembly was performed by CLC Workbench, and in silico analysis accomplished by bioinformatics tools. For phylogenomic analysis, publicly available C. freundii (txid:546) genome assemblies were retrieved from the NCBI database. RESULTS: The genome size was calculated at 5 290 351 bp, comprising 5263 total genes, 4 rRNAs, 77 tRNAs, 11ncRNAs, and 176 pseudogenes. The strain belonged to C. freundii ST18, whereas resistome analysis predicted genes encoding resistance to ß-lactams (blaNDM-1, blaOXA-1, blaCMY-117, and blaTEM-1C), aminoglycosides (aph(3'')-Ib, aadA16, aph(3')-VI, aac(6')-Ib-cr, and aph(6)-Id), quinolones (aac(6')-Ib-cr), macrolides (mph(A) and erm(B)), sulphonamides (sul1 and sul2), tetracyclines (tetA and tetD), and trimethoprim (dfrA27). The phylogenomic analysis revealed that CF135931 strain is closely related to international human-associated ST18 clones producing NDM-1. CONCLUSION: Genomic surveillance efforts are necessary for robust monitoring of the emergence of drug-resistant strains and WHO critical priority pathogens within a One Health framework. In this regard, this draft genome and associated data can improve understanding of dissemination dynamics of nosocomial clones of carbapenemase-producing C. freundii beyond hospital walls. In fact, the emergence of NDM-1-producing C. freundii of global ST18 in wildlife deserves considerable attention.