Genotypic characterization of Streptococcus didelphis causative of fatal infection in white-eared opossums.

Streptococcus didelphis was once reported as related to severe infections in opossums. Thus, we present the first comprehensive whole-genome characterization of clinical S. didelphis strains isolated from white-eared opossums (Didelphis albiventris). Long-read whole-genome sequencing was performed using the MinION platform, which allowed the prediction of several genomic features. We observed that S. didelphis genomes harbor a cluster for streptolysin biosynthesis and a conserved genomic island with genes involved in transcriptional regulation (arlR) and transmembrane transport (bcrA). Antimicrobial resistance genes for several drug classes were found, including beta-lactam, which is the main antimicrobial class used in Streptococcus spp. infections; however, no phenotypical resistance was observed. In addition, we predicted the presence of 33 virulence factors in the analyzed genomes. High phylogenetic similarity was observed between clinical and reference strains, yet no clonality was suggested. We also proposed dnaN, gki, pros, and xpt as housekeeping candidates to be used in S. didelphis sequence typing. This is the first whole-genome characterization of S. didelphis, whose data provide important insights into its pathogenicity.

Development of a long-read next generation sequencing workflow for improved characterization of fastidious respiratory mycoplasmas.

Mycoplasma cynos and Mycoplasma felis are often associated with canine and feline infectious respiratory disease in dogs and cats, respectively. Mycoplasmas have a reduced genome and dearth of many biosynthetic pathways, making them dependent on rich medium for growth. Due to this fastidious nature, mycoplasmas have been historically underdiagnosed. The aim of this study was to develop a cost-effective and accurate sequencing workflow for genotypic characterization of clinical isolates of M. cynos and M. felis using a rapid long-read sequencing platform. We explored the following critical aspects of bacterial whole genome sequencing, including: (i) five solid and liquid-based culture approaches based on a specialized media formulation for Mycoplasma culture, (ii) three DNA extraction methods modified for long-read sequencing purposes, and (iii) two de novo assembly platforms, Flye and Canu, as key components of a bioinformatics pipeline. DNA extraction method 1, a solid-phase and column-based kit with enzymatic lysis, provided the best DNA quality and concentration followed by high coverage and sequencing contiguity. This was obtained with a culture volume of 45 ml in modified Hayflick's broth incubated for 48 h. DNA extracted directly from colonies on agar or from small broth volumes (6 ml) did not meet the criteria required for long-read sequencing. Overall, Flye generated more contiguous assemblies than the Canu assembler and was more time efficient. This 4-5 day sample-to-sequence workflow provides the scientific and clinical communities with a more comprehensive tool than laborious conventional methods for complete genomic characterization of M. cynos and M. felis clinical isolates.

High rates of multidrug resistance in bacteria associated with small animal otitis: A study of cumulative microbiological culture and antimicrobial susceptibility.

The etiology of otitis in dogs and cats is multifactorial and complex, involving bacterial and fungal pathogens. As empiric antimicrobial prescription is a common practice when treating such cases, antimicrobial resistance may represent a complicating factor. The aim of this study was to describe microbiological features and susceptibility profiles of pathogens associated with 142 cases of external otitis, comprising 138 dogs and 4 cats.. The specimens were processed to identify bacterial and fungal etiologies following standard microbiological methods. Antimicrobial susceptibility was determined in vitro against 15 antibiotics and 3 antifungals. Further, Staphylococcus spp. isolates were screened for the detection of ß-lactamase enzymes using cefinase paper discs. Pseudomonas spp. and isolates from Enterobacteriaceae family were screened for colistin (Polymyxin E) resistance and for the mcr-1-mediated colistin resistance gene by PCR. The presence of mixed cultures of Enterobacteriaceae, Pseudomonas spp. and Staphylococcus spp., and co-infections with Malassezia spp., emphasizes the polymicrobial etiology of external otitis in small animals. Emerging rates of multidrug resistance observed in almost 50% of the isolates may alert for a near future of challenging veterinary cases unresponsive to first-line antimicrobials. In addition, these results highlight a potential public health concern of multidrug resistant bacteria, given the proximity of pets and their owners. This study addressed central aspects of external otitis, providing microbiologists and clinicians updated information on the etiology and treatment of challenging cases of multidrug resistant bacteria. It also provides priceless surveillance value in monitoring resistant bacteria in small animals.

Genomic and Pathologic Findings for Prototheca cutis Infection in Cat.

Severe nasal Prototheca cutis infection was diagnosed postmortem for an immunocompetent cat with respiratory signs. Pathologic examination and whole-genome sequencing identified this species of algae, and susceptibility testing determined antimicrobial resistance patterns. P. cutis infection should be a differential diagnosis for soft tissue infections of mammals.

Ocular and Lacrimal Gland Lesions in Naturally Occurring Rabies of Domestic and Wild Mammals.

Investigations describing the ocular and lacrimal gland lesions associated with rabies are sparse. Here we characterize the pathological changes and distribution of rabies viral antigen in the eye, optic nerve, and lacrimal gland of 18 rabies cases from different mammalian species. Histology and immunohistochemistry for rabies virus, CD3, CD20, and Iba1 were performed on tissue sections of eye, optic nerve, and lacrimal gland. Polymerase chain reaction (PCR) for rabies was performed on all cases, including 7 formalin-fixed, paraffin-embedded (FFPE) and 11 frozen tissue samples of eye and lacrimal gland. Pathological changes in the eye consisted of retinal necrosis (12/18 cases) with occasional viral inclusions within ganglion cells (8/12 cases). Immunohistochemically, viral antigen was detected within the nerve fiber layer, ganglion cells, and inner plexiform layer in all 12 cases with retinal lesions and in 2 cases with no retinal lesions, as well as optic nerve (6/18 cases) and lacrimal gland epithelium (3/18 cases). CD3+ T lymphocytes were present in the retina (11/18 cases), optic nerve (2/18 cases), and lacrimal gland (11/18 cases). No CD20+ B lymphocytes or Iba1+ macrophages were detected. PCR for rabies virus was positive in 9 of 11 frozen samples but in only 2 of 7 FFPE samples. Five samples that were negative for rabies by PCR were positive by immunohistochemistry, and 2 samples were negative by both tests. These results provide evidence that rabies virus infection extends to the eye, likely via the ocular nerve, and that the lacrimal gland might be a source of viral infection.

Laboratory diagnostics, phylogenetic analysis and clinical outcome of a subcutaneous Mycoleptodiscus indicus infection in an immunocompetent cat.

BACKGROUND: Mycoleptodiscus indicus is a dematiaceous hyphomycete fungus found on plant leaves. It has been rarely reported as a cause of human or animal disease, possibly because it is difficult to culture and identify from clinical specimens. Infections are presumably acquired by traumatic implantation. CASE PRESENTATION: An 8-year-old non-immunosuppressed cat from Georgia, USA, presented with a left front leg swelling without lameness. Cytology from a fine needle aspirate revealed pyogranulomatous inflammation with both cytoplasmic and extracellular fungal elements. There were septate hyphae with irregularly sized segments, non-staining uneven walls, and rounded yeast-like forms from which longer hyphae arose in a hub-and-spoke pattern. A mold was isolated on agar from a fine needle aspirate collected 1 week later and identified as M. indicus by morphology, DNA sequencing and phylogenetic analysis. The cat recovered completely and uneventfully with antifungal treatment. CONCLUSIONS: We report a previously undescribed presentation of M. indicus causing a subcutaneous infection in a cat with successful antifungal treatment. In this study we highlight the potential of M. indicus to infect immunocompetent animals, and the veterinary medical community should be aware of its unusual but characteristic clinical, microbiological and cytologic presentation.

Lymphoplasmacytic Meningoencephalitis and Neuronal Necrosis Associated With Parvoviral Infection in Cats.

Neurologic manifestations other than cerebellar hypoplasia are rarely associated with feline panleukopenia virus (FPV) infection in cats. Here the authors describe lymphoplasmacytic meningoencephalitis and neuronal necrosis in 2 cats autopsied after exhibiting ataxia and nystagmus. Gross changes consisted of cerebellar herniation through the foramen magnum, with flattening of cerebrocortical gyri and narrowing of sulci. Histologically, lymphoplasmacytic meningoencephalitis, extensive neuronal necrosis, and neuroaxonal degeneration with digestion chambers were present in the telencephalon and brain stem in both cats. Frozen brain tissue of both cats was positive for parvoviral antigen via fluorescent antibody testing, and formalin-fixed, paraffin-embedded tissue sections of brain were immunoreactive for parvovirus antigen and positive for parvoviral DNA on in situ hybridization. Frozen brain tissue from 1 case was positive for parvovirus NS1 and VP2 genes using conventional polymerase chain reaction, and subsequent DNA sequencing and phylogenetic analysis revealed that the viral strain was a FPV. Reverse transcription quantitative polymerase chain reaction on formalin-fixed, paraffin-embedded brain tissue revealed high levels of parvovirus in both cases, supporting an acute and active viral infection. Although rare, FPV infection should be considered in cases of lymphoplasmacytic meningoencephalitis and neuronal necrosis in cats.

Complete genome sequences of Mycoplasma cynos and Mycoplasma felis isolated from dogs and cats with infectious respiratory disease.

Mycoplasma cynos is a primary agent of pneumonia in dogs, and Mycoplasma felis is associated with upper respiratory tract disease in cats. We present complete genome sequences of 26 isolates from clinically affected dogs and cats. These genome sequences will facilitate new molecular and epidemiological analyses.

Predicting host species susceptibility to influenza viruses and coronaviruses using genome data and machine learning: a scoping review.

Introduction: Predicting which species are susceptible to viruses (i.e., host range) is important for understanding and developing effective strategies to control viral outbreaks in both humans and animals. The use of machine learning and bioinformatic approaches to predict viral hosts has been expanded with advancements in in-silico techniques. We conducted a scoping review to identify the breadth of machine learning methods applied to influenza and coronavirus genome data for the identification of susceptible host species. Methods: The protocol for this scoping review is available at https://hdl.handle.net/10214/26112. Five online databases were searched, and 1,217 citations, published between January 2000 and May 2022, were obtained, and screened in duplicate for English language and in-silico research, covering the use of machine learning to identify susceptible species to viruses. Results: Fifty-three relevant publications were identified for data charting. The breadth of research was extensive including 32 different machine learning algorithms used in combination with 29 different feature selection methods and 43 different genome data input formats. There were 20 different methods used by authors to assess accuracy. Authors mostly used influenza viruses (n = 31/53 publications, 58.5%), however, more recent publications focused on coronaviruses and other viruses in combination with influenza viruses (n = 22/53, 41.5%). The susceptible animal groups authors most used were humans (n = 57/77 analyses, 74.0%), avian (n = 35/77 45.4%), and swine (n = 28/77, 36.4%). In total, 53 different hosts were used and, in most publications, data from multiple hosts was used. Discussion: The main gaps in research were a lack of standardized reporting of methodology and the use of broad host categories for classification. Overall, approaches to viral host identification using machine learning were diverse and extensive.

Utilizing machine learning and hemagglutinin sequences to identify likely hosts of influenza H3Nx viruses.

Influenza is a disease that represents both a public health and agricultural risk with pandemic potential. Among the subtypes of influenza A virus, H3 influenza virus can infect many avian and mammalian species and is therefore a virus of interest to human and veterinary public health. The primary goal of this study was to train and validate classifiers for the identification of the most likely host species using the hemagglutinin gene segment of H3 viruses. A five-step process was implemented, which included training four machine learning classifiers, testing the classifiers on the validation dataset, and further exploration of the best-performing model on three additional datasets. The gradient boosting machine classifier showed the highest host-classification accuracy with a 98.0 % (95 % CI [97.01, 98.73]) correct classification rate on an independent validation dataset. The classifications were further analyzed using the predicted probability score which highlighted sequences of particular interest. These sequences were both correctly and incorrectly classified sequences that showed considerable predicted probability for multiple hosts. This showed the potential of using these classifiers for rapid sequence classification and highlighting sequences of interest. Additionally, the classifiers were tested on a separate swine dataset composed of H3N2 sequences from 1998 to 2003 from the United States of America, and a separate canine dataset composed of canine H3N2 sequences of avian origin. These two datasets were utilized to look at the applications of predicted probability and host convergence over time. Lastly, the classifiers were used on an independent dataset of environmental sequences to explore the host identification of environmental sequences. The results of these classifiers show the potential for machine learning to be used as a host identification technique for viruses of unknown origin on a species-specific level.

Epidemiologic investigation and genetic characterization of canine respiratory coronavirus in the Southeastern United States.

Canine respiratory coronavirus (CRCoV) is one of the main causative agents of canine infectious respiratory disease (CIRD), an illness whose epidemiology is poorly understood. We assessed the prevalence, risk factors, and genetic characterization of CRCoV in privately owned dogs in the Southeastern United States. We PCR-screened 189 nasal swabs from dogs with and without CIRD clinical signs for 9 CIRD-related pathogens, including CRCoV; 14% of dogs, all diagnosed with CIRD, were positive for CRCoV, with a significantly higher rate of cases in younger dogs and during warmer weather. Notably, the presence of CRCoV, alone or in coinfection with other CIRD pathogens, was statistically associated with a worse prognosis. We estimated a CRCoV seroprevalence of 23.7% retrospectively from 540 serum samples, with no statistical association to dog age, sex, or season, but with a significantly higher presence in urban counties. Additionally, the genomes of 6 CRCoVs were obtained from positive samples using an in-house developed targeted amplicon-based approach specific to CRCoV. Subsequent phylogeny clustered their genomes in 2 distinct genomic groups, with most isolates sharing a higher similarity with CRCoVs from Sweden and only 1 more closely related to CRCoVs from Asia. We provide new insights into CIRD and CRCoV epidemiology in the Southeastern United States and further support the association of CRCoV with more severe cases of CIRD. Additionally, we developed and successfully tested a new amplicon-based approach for whole-genome sequencing of CRCoV that can be used to further investigate the genetic diversity within CRCoVs.

Feline respiratory disease complex: insights into the role of viral and bacterial co-infections.

Feline respiratory disease complex (FRDC) is a highly prevalent syndrome in cats that often result in fatal outcomes. FRDC etiology is complex, and often, multiple viral and bacterial pathogens are simultaneously associated with disease causation. There is limited information about the role of co-infections in pathogenesis and the current prevalence of pathogens in North America. We aimed to conduct a study using technical advances in molecular diagnosis and statistical modeling analysis to elucidate the occurrence of pathogens and how co-infections affect disease severity. We attained information from three diagnostic laboratories in North America regarding the occurrence of Bordetella bronchiseptica, Chlamydia felis, Mycoplasma, Felid alphaherpesvirus 1 (FeHV-1), feline calicivirus (FCV), and influenza A, along with age, seasonality, sex, and clinical signs. We also evaluated the role of co-infections in disease severity. These pathogens were also investigated in clinically normal cats (control). The most detected pathogens were Mycoplasma, FCV, and FeHV-1. Most pathogens were detected in the control group, highlighting the challenge of interpreting positive testing results. Co-infections of Mycoplasma and FCV, as well as Mycoplasma and FeHV-1, were important predictors of disease severity. Age, sex, and season had a minor impact on pathogen occurrence. This study provides new insights into FRDC and underlines the relevance of diagnostic panels to screen for a range of pathogens, providing knowledge for timely diagnosis and therapeutic interventions.

High-throughput rapid amplicon sequencing for multilocus sequence typing of Mycoplasma ovipneumoniae from archived clinical DNA samples.

Introduction: Spillover events of Mycoplasma ovipneumoniae have devastating effects on the wild sheep populations. Multilocus sequence typing (MLST) is used to monitor spillover events and the spread of M. ovipneumoniae between the sheep populations. Most studies involving the typing of M. ovipneumoniae have used Sanger sequencing. However, this technology is time-consuming, expensive, and is not well suited to efficient batch sample processing. Methods: Our study aimed to develop and validate an MLST workflow for typing of M. ovipneumoniae using Nanopore Rapid Barcoding sequencing and multiplex polymerase chain reaction (PCR). We compare the workflow with Nanopore Native Barcoding library preparation and Illumina MiSeq amplicon protocols to determine the most accurate and cost-effective method for sequencing multiplex amplicons. A multiplex PCR was optimized for four housekeeping genes of M. ovipneumoniae using archived DNA samples (N = 68) from nasal swabs. Results: Sequences recovered from Nanopore Rapid Barcoding correctly identified all MLST types with the shortest total workflow time and lowest cost per sample when compared with Nanopore Native Barcoding and Illumina MiSeq methods. Discussion: Our proposed workflow is a convenient and effective method for strain typing of M. ovipneumoniae and can be applied to other bacterial MLST schemes. The workflow is suitable for diagnostic settings, where reduced hands-on time, cost, and multiplexing capabilities are important.

Nontuberculous Mycobacteria in Horses: A Narrative Review.

Nontuberculous mycobacteria (NTM) infections are increasing in human and veterinary medicine. Although horses were initially thought to be resistant to NTM infection, reports of horses suffering from gastrointestinal, respiratory, and reproductive diseases associated with NTM have increased in the last few decades. The aim of this literature review is to summarize the mycobacteria species found in horses, describe clinical manifestations, diagnostic and treatment approaches, and public health concerns of NTM infection in horses. Clinical manifestations of NTM in horses include pulmonary disease, lymphadenitis, soft tissue, bone infections, and disseminated disease. NTM are also linked to granulomatous enteritis, placentitis, and abortions. Currently, diagnostic methods for NTM are limited and include acid-fast microscopy, bacterial cultures, species-specific PCR assays, and gene sequencing. In humans, NTM treatment guidelines are available, but their application appears inadequate and inconsistent. In horses, treatment guidelines for NTM infections are not available. NTM are a serious public health threat as 70% of people with untreated acquired immunodeficiency syndrome (AIDS) have a chronic pulmonary disease caused by NTM. Thus, it is essential that we gain a better understanding of NTM infections in horses and their zoonotic potential.

Review of methods for detection and characterization of non-tuberculous mycobacteria in aquatic organisms.

Mycobacteriosis is an emerging and often lethal disease of aquatic organisms caused by several non-tuberculous mycobacteria (NTM) species. Early diagnosis of mycobacteriosis in aquaculture and aquatic settings is critical; however, clinical diagnoses and laboratory detection are challenging, and the available literature is scarce. In an attempt to fill the gap, here we review the most relevant approaches to detect and characterize mycobacteria in clinical specimens of aquatic organisms. Emphasis is given to recent advances in molecular methods used to differentiate NTM species spanning from targeted gene sequencing to next-generation sequencing. Further, given that there are major gaps in our understanding of the prevalence of the different NTM species, partially because of their distinct requirements for in vitro growth, we also reviewed the most relevant NTM species reported to cause disease in aquatic organisms and their specific in vitro growth conditions. We also highlight that traditional bacterial culture continues to be relevant for NTM identification, particularly in non-automated laboratories. However, for NTM species discrimination, a high level of accuracy can be achieved with MALDI-TOF MS and molecular approaches, especially targeted gene sequencing applied from clinical specimens or from pure NTM isolates.

Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA.

Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community.

Detection of asinine gammaherpesviruses in association with pulmonary fibrosis in free-ranging donkeys.

A mortality event among recently captured feral donkeys (Equus asinus) occurred in south-central Utah in 2016. The deaths were sporadic, and clinical signs were indicative of respiratory disease, likely associated with an infectious etiology. Ten of 13 donkeys autopsied had moderate-to-severe interstitial fibrosing pneumonia, and one had pyogranulomatous pneumonia. Consensus PCRs directed toward the DNA polymerase and DNA packaging terminase subunit 1 for herpesviruses were performed followed by sequencing of the PCR amplicons and phylogenetic analysis. Asinine herpesvirus 4 (AsHV4) and 5 (AsHV5) were consistently identified in lung tissues of affected donkeys. No other herpesviruses were identified, and herpesviral DNA was not detected in lung tissues of 2 donkeys without evidence of respiratory disease. The detection of asinine gammaherpesviruses may have been associated with the lesions described. AsHV4 and AsHV5 have been reported in previous studies as novel gammaherpesviruses based on sequences obtained from donkeys with interstitial pneumonia and marked syncytial cell formation. Our findings suggest that the association of asinine gammaherpesviruses with respiratory conditions in equids deserves further attention.

Mortality in Common (Sterna hirundo) and Sandwich (Thalasseus sandvicensis) Terns Associated with Bisgaard Taxon 40 Infection on Marco Island, Florida, USA.

Widely distributed aquatic species such as terns are highly dependent on, and can serve as indicators of, the global health of marine and other aquatic environments. Documented mass mortality events in terns have been associated with anthropogenic, weather-related and, less commonly, infectious causes. This study describes a multispecies mortality event associated with brevetoxicosis and Bisgaard taxon 40-induced sepsis involving common (Sterna hirundo) and sandwich (Thalasseus sandvicensis) terns off the southwest coast of Florida, USA, in November and December 2018. During an approximately 6-8-week period, a large number of birds were found dead or displayed weakness, ataxia or other neurological signs. Many were admitted to a wildlife hospital for evaluation, but most died or were euthanized due to poor prognosis. Necropsy of 12 birds revealed minimal or non-specific gross lesions. Initial toxicology screening of tissues for brevetoxins revealed levels that could be consistent with brevetoxicosis. However, histology revealed multiorgan inflammation and necrosis associated with a gram-negative bacillus. A bacterium isolated on aerobic culture of liver and heart tissues was unidentifiable in the MALDI-TOF database. Subsequently, 16 S rRNA gene sequencing revealed that the isolate shared 99.33% homology with Bisgaard taxon 40 from the Pasteurellaceae family. While the source of the bacterium and potential association with brevetoxin exposure are unclear, histopathology suggests that the bacterium was the proximate cause of clinical signs and mortality in all birds examined as well as the scale of the mortality event. This report highlights the need to conduct detailed investigations into wildlife mortality events and expands on the current, limited knowledge of the effects of novel Pasteurellaceae bacteria on avian health.

Comparative Genomics Analyses Support the Reclassification of Bisgaard Taxon 40 as Mergibacter gen. nov., With Mergibacter septicus sp. nov. as Type Species: Novel Insights Into the Phylogeny and Virulence Factors of a Pasteurellaceae Family Member Associated With Mortality Events in Seabirds.

The Pasteurellaceae family has been associated with fatal diseases in numerous avian species. Several new taxa within this family, including Bisgaard taxon 40, have been recently described in wild birds, but their genomic characteristics and pathogenicity are not well understood. We isolated Bisgaard taxon 40 from four species of seabirds, including one sampled during a mass, multi-species mortality event in Florida, United States. Here, we present a comprehensive phenotypic and genetic characterization of Bisgaard taxon 40 and comparative genomic analysis with reference strains from the Pasteurellaceae family, aiming at determining its phylogenetic position, antimicrobial susceptibility profile, and identifying putative virulence factors. In silico multilocus sequence-based and whole-genome-based phylogenetic analysis clustered all Bisgaard taxon 40 strains together on a distinct branch separated from the other members of the Pasteurellaceae family, indicating that Bisgaard taxon 40 could represent a new genus. These findings were further supported by protein similarity analyses using the concatenation of 31 conserved proteins and other taxonomic approaches such as the percentage of conserved protein test. Additionally, several putative virulence factors were identified, including those associated with adhesion (capsule, ompA, ompH) and colonization (exbD, fur, galU, galE, lpxA, lpxC, and kdsA) of the host and a cytolethal distending toxin (cdt), which may have played a role in disease development leading to the mortality event. Considerably low minimum inhibitory concentrations (MICs) were found for all the drugs tested, in concordance with the absence of antimicrobial resistance genes in these genomes. The novel findings of this study highlight genomic and phenotypic characteristics of this bacterium, providing insights into genome evolution and pathogenicity. We propose a reclassification of these organisms within the Pasteurellaceae family, designated as Mergibacter gen. nov., with Mergibacter septicus sp. nov. as the type species. The type strain is Mergibacter septicus A25201T (=DSM 112696).

Antimicrobial resistance patterns of Acinetobacter spp. of animal origin reveal high rate of multidrug resistance.

Antimicrobial resistance has been declared by the World Health Organization as one of the biggest threats to public health and Acinetobacter baumannii is a notable example. A. baumannii is an important human nosocomial pathogen, being along with other multidrug resistant (MDR) bacteria, one of the biggest public health concerns worldwide. In Veterinary Medicine, resistance patterns of Acinetobacter species other than A. baumanii are unclear, and the scarce information available is limited and fragmented. We applied a statistical modeling approach to investigate the occurrence, clinical relevance and antimicrobial resistant phenotypes of Acinetobacter spp. originated from animals. Seven Acinetobacter species were identified in clinical specimens of more than 15 different domestic, zoo and exotic animal species. We found a high rate of MDR A. baumannii of canine origin with some of these isolates originating from serious systemic or wound infections, which highlights their potential pathogenic profile and spread in the human environment. Data also revealed different antimicrobial resistance patterns of animal-origin Acinetobacter species, emphasizing the necessity to implement specific antimicrobial susceptibility recommendations for animal isolates as there are no such clinical breakpoints currently in place. This study provides substantial advancing in our understanding of Acinetobacter spp. in animal clinical specimens, and highlights the role of animals in the dynamics of multidrug resistance in bacteria. The data presented here is a valuable source of information for further establishment of clinical breakpoints for susceptibility testing of animal-associated Acinetobacter isolates.