Targeted next-generation sequencing assay to detect 3 Moraxella spp. directly from bovine ocular swabs.

Infectious bovine keratoconjunctivitis (IBK) is associated with 2 species of Moraxella: M. bovis and M. bovoculi. A third novel Moraxella spp., designated tentatively as M. oculi, has been identified from the eyes of cattle with and without pinkeye. These 3 Moraxella spp. can be found in various combinations within the same clinical sample, making speciation of this genus directly from a sample impossible with Sanger sequencing. Assessing Moraxella diversity found in IBK- and non-IBK-affected cattle eyes, independent of culture, may provide additional information about IBK by avoiding the selectivity bias of culturing. We developed a targeted NGS panel to detect and speciate these 3 Moraxella spp. directly from bovine ocular swabs. Our targeted panel amplifies bacterial essential genes and the 16S-23S ribosomal RNA intergenic spacer region (ITS) of the 3 Moraxella spp. and speciates based on these sequences. Our panel was able to differentiate the 3 species directly from DNA extracted from 13 swabs (6 from healthy animals, 7 from animals with IBK), and every swab except one (clinically healthy eye) had the 3 Moraxella spp. Targeted NGS with sequencing of Moraxella spp. housekeeping genes appears to be a suitable method for speciation of Moraxella directly from ocular swabs.

First report of the whole genome of Moraxella bovoculi genotype 1 from India and comparative genomics of Moraxella bovoculi to identify genotype-specific markers.

Moraxella bovoculi has been isolated frequently from cattle with Infectious bovine keratoconjunctivitis (IBK). Two diverse genotypes of M. bovoculi, 1 and 2 were identified based on whole genome sequence analysis. It is essential to discriminate between the two genotypes to frame prevention and control measures. The whole genome of M. bovoculi TN7 was sequenced and compared to other M. bovoculi strains available in the NCBI database. M. bovoculi TN7 was found to be genotype 1, had an RTX toxin operon and pilA gene that are the known virulence factors in related Moraxella sp., but lacked antimicrobial resistance genes. M. bovoculi was found to have an open pangenome with 4051 (75.31%) accessory genes, and the addition of each new genome adds 18 genes to the pangenome. Comparison of pilin protein amino acid sequences revealed three new sequence types. Furthermore, the presence of linx, nagL, swrC and mdtA genes was found to be genotype 1 specific, whereas hyaD, garR, gbsA, yhdG, gabT, iclR, higB2, hmuU, hmuT and hemS were found only in genotype 2. Polymerase Chain Reaction (PCR) primers were designed and evaluated on strain TN7 plus seven additional strains accessible to us that had not been whole genome sequenced. This initial evaluation of the designed primers for the linX and hyaD genes produced the expected banding patterns on PCR gels for genotypes 1 and 2, respectively, among the 8 strains. The genotype-specific genes identified in this study can be used as markers for accurate diagnosis of genotype 1 isolates and this can aid in the development of autogenous or other molecular vaccines for treatment of infectious bovine keratoconjunctivitis (IBK) in resource-limited research settings.

Isolation of Moraxella spp. from horses with conjunctivitis in Southern Brazil.

Infectious keratoconjunctivitis (IKC) is the most frequent ocular disease in livestock worldwide and is primarily caused by Moraxella bovis, M. ovis, and/or M. bovoculi. The economic impact of IKC is mainly due to ocular damage, which leads to weight loss, management difficulties, pain and discomfort, and cost of treatments. In horses, limited information is available on the association of Moraxella spp. with keratoconjunctivitis. The present report describes two cases of equine keratoconjunctivitis caused by members of the genus Moraxella. Both animals presented with lacrimation, conjunctivitis, photophobia, mucoid or purulent secretions, blepharitis, and conjunctival hyperemia. The diagnosis of IKC was based on the epidemiological and clinical findings; the etiological agent was identified through bacteriological (culture and biochemistry assays) and molecular testing (PCR and nucleotide sequencing). Our study reports the isolation of Moraxella bovoculi (SBP 88/19) and a putative new species/mutant of Moraxella (SBP 39/19) recovered from ocular secretions in horses. Thus, we suggest the inclusion of Moraxella spp. infection in the differential diagnosis of conjunctivitis in horses in Southern Brazil.

Moraxella osloensis infection among adults and children: A pediatric case and literature review.

Moraxella osloensis has been reported in the literature as a human pathogen, particularly among immunocompromised adults. In contrast to the adult population, most pediatric cases are among patients with no underlying immunological defect; however, no patient underwent further investigation and no data about the long-term follow-up are available. We report the case of a 2-month-old previously healthy girl infected with Moraxella osloensis. Here, we review case reports and case series of children and adults with Moraxella osloensis infection and compare them with our experience. On the basis of our findings, we recommend further investigations (immunological or other underlying diseases) when a child is found to be infected with these bacteria.

Bacteremia Caused by Moraxella Osloensis: a Fatal Case of an Immunocompromised Patient and Literature Review.

BACKGROUND: Moraxella osloensis rarely causes infection in humans, and most of the reported cases are not fatal. It is often difficult to identify M. osloensis using conventional biochemical methods. METHODS: Here, we report a bacteremia case caused by M. osloensis in a patient with advanced lung cancer who initially presented symptoms of fever. RESULTS: Blood culture revealed growth of a gram-negative bacterium, which was identified as M. osloensis through 16S rRNA gene sequencing and MALDI-TOF analyses. The patient could not recover from sepsis with empirical treatment. CONCLUSIONS: As M. osloensis can cause serious infections in immunocompromised patients, its prompt identification is important.

Relatedness of type IV pilin PilA amongst geographically diverse Moraxella bovoculi isolated from cattle with infectious bovine keratoconjunctivitis.

Introduction. Moraxella bovoculi is frequently isolated from the eyes of cattle with infectious bovine keratoconjunctivitis (IBK; pinkeye). As with M. bovis, which has been causally linked to IBK, M. bovoculi expresses an RTX (repeats in the structural toxin) cytotoxin that is related to M. bovis cytotoxin. Pilin, another pathogenic factor in M. bovis, is required for corneal attachment. Seven antigenically distinct pilin serogroups have been described in M. bovis.Hypothesis/Gap Statement. Multiple different serogroups exist amongst type IV pilin encoded by M. bovis, however, it is not known whether M. bovoculi exhibits a similar degree of diversity in type IV pilin that it encodes.Aim. This study was done to characterize a structural pilin (PilA) encoded by M. bovoculi isolated from cases of IBK to determine if diversity exists amongst PilA sequences.Methodology. Ninety-four isolates of M. bovoculi collected between 2002 and 2017 from 23 counties throughout California and from five counties in four other Western states were evaluated.Results. DNA sequencing and determination of deduced amino acid sequences revealed ten (designated groups A through J) unique PilA sequences that were ~96.1-99.3 % identical. Pilin groups A and C matched previously reported putative PilA sequences from M. bovoculi isolated from IBK-affected cattle in the USA (Virginia, Nebraska, and Kansas) and Asia (Kazakhstan). The ten pilin sequences identified were only ~74-76 % identical to deduced amino acid sequences of putative pilin proteins identified from the previously reported whole-genome sequences of M. bovoculi derived from deep nasopharyngeal swabs of IBK-asymptomatic cattle.Conclusions. Compared to the diversity reported between structural pilin proteins amongst different serogroups of M. bovis, M. bovoculi PilA from geographically diverse isolates derived from IBK-affected cattle are more conserved.

Unexpected and Sudden Death Due to Undiagnosed Moraxella catarrhalis Meningoencephalitis in a 40-day-old Infant: Case Report and Literature Review.

Acute bacterial meningitis in infants and newborns represents a medical emergency and a significant cause of mortality and morbidity worldwide. Moraxella catarrhalis has been considered a microorganism with low pathogenic potential, and only in exceptional cases has it been found to cause meningitis in infants and immunocompetent people. We will now document an unusual case of an unexpected and sudden death of a 40-day-old infant due to acute meningitis from M. catarrhalis, apparently asymptomatic and subsequently diagnosed by an autopsy. According to our knowledge this is the first case of unexpected infant death due to undiagnosed M. catarrhalis meningitis.The suggested case, as well as for the rarity of such a fatal event, should be considered a caution to pediatrics and neonatologists for M. catarrhalis can cause paucisymptomatic meningoencephalitis in infants which can be potentially fatal.From a forensic point of view, an autopsy accompanied by a multidisciplinary assessment is always necessary in cases of unexpected infant deaths to identify the causes.

Rapid differentiation of Moraxella bovoculi genotypes 1 and 2 using MALDI-TOF mass spectrometry profiles.

Moraxella bovoculi is the most frequently isolated bacteria from the eyes of cattle with Infectious Bovine Keratoconjunctivitis (IBK), also known as bovine pinkeye. Two distinct genotypes of M. bovoculi, genotype 1 and genotype 2, were characterized after whole genome sequencing showed a large degree of single nucleotide polymorphism (SNP) diversity within the species. To date, both genotypes have been isolated from the eyes of cattle without clinical signs of IBK while only genotype 1 strains have been isolated from the eyes of cattle with clinical signs of IBK. We used 38 known genotype 1 strains and 26 known genotype 2 strains to assess the ability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to accurately genotype M. bovoculi strains using mass spectrum biomarkers. Mass spectrum data was analyzed with ClinProTools 3.0 software and six models were developed that classify strain genotypes with accuracies ranging from 90.6% - 100%. Finally, using four of the most genotype-specific peaks that also exhibited high peak intensities from the six automated models, we developed a customized model (UNL assisted model) that had recognition capability, validation, and classification accuracies of 100% for genotype classification. Our results indicate that MALDI-TOF MS biomarkers can be used to accurately discriminate genotypes of M. bovoculi without the need for additional methods.

[MORAXELLA OSLOENSIS BACTEREMIA WITH PNEUMONIA: FIRST REPORTED CASE IN ISRAEL].

INTRODUCTION: Community acquired pneumonia (CAP), an acute infection of the pulmonary parenchyma acquired in the community, is generally treated in an outpatient setting and involves different etiological agents. In the adult community, the most common pathogen in the disease is Streptococcus pneumonia, though other multiple etiological agents (atypical) have been involved, including Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila. The genus Moraxella consists of aerobic, oxidase-positive gram-negative coccobacilli. Moraxella catarrhalis is known to be a common inhabitant of the upper respiratory tract and has been implicated as an etiologic agent in multiple diseases of the respiratory tract (but not limited to), such as bronchitis, pneumonia, otitis media, and sinusitis. The species Moraxella osloensis is a gram-negative opportunistic human pathogen, which has been found to cause several human diseases and infections such as meningitis, vaginitis, sinusitis, bacteremia, endocarditis, and septic arthritis. However, due to the subject's rarity, there is a paucity of information in the medical literature regarding its clinical significance, epidemiological data and appropriate therapy. We present the first case reported in Israel of Moraxella osloensis bacteremia in a patient with multiple co-morbidities including C. difficile infection (CDI) carrier state which presented with clinical symptoms (supported by radiological features) of community-acquired pneumonia. The patient was initially treated with empiric antibiotics including a 3rd generation cephalosporin and a macrolide that were substituted with IV Augmentin (Amoxicillin-Clavulanic acid) according to the organism's sensitivity tests. Our patient showed remarkable clinical and laboratory improvement with the therapy mentioned above.

Rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by MALDI-TOF MS.

Respiratory tract infections are a major health problem and indication for antimicrobial use in cattle and in humans. Currently, most antimicrobial treatments are initiated without microbiological results, holding the risk of inappropriate first intention treatment. The main reason for this empirical treatment is the long turnaround time between sampling and availability of identification and susceptibility results. Therefore the objective of the present study was to develop a rapid identification procedure for pathogenic respiratory bacteria in bronchoalveolar lavage fluid (BALf) samples from cattle by MALDI-TOF MS, omitting the cultivation step on agar plates to reduce the turnaround time between sampling and identification of pathogens. The effects of two different liquid growth media and various concentrations of bacitracin were determined to allow optimal growth of Pasteurellaceae and minimise contamination. The best procedure was validated on 100 clinical BALf samples from cattle with conventional bacterial culture as reference test. A correct identification was obtained in 73% of the samples, with 59.1% sensitivity (Se) (47.2-71.0%) and 100% specificity (Sp) (100-100%) after only 6 hours of incubation. For pure and dominant culture samples, the procedure was able to correctly identify 79.2% of the pathogens, with a sensitivity (Se) of 60.5% (45.0-76.1%) and specificity (Sp) of 100% (100-100%). In mixed culture samples, containing ≥2 clinically relevant pathogens, one pathogen could be correctly identified in 57% of the samples with 57.1% Se (38.8-75.5%) and 100% Sp (100-100%). In conclusion, MALDI-TOF MS is a promising tool for rapid pathogen identification in BALf. This new technique drastically reduces turnaround time and may be a valuable decision support tool to rationalize antimicrobial use.

Diagnosis and Treatment of Adults with Community-acquired Pneumonia. An Official Clinical Practice Guideline of the American Thoracic Society and Infectious Diseases Society of America.

Background: This document provides evidence-based clinical practice guidelines on the management of adult patients with community-acquired pneumonia.Methods: A multidisciplinary panel conducted pragmatic systematic reviews of the relevant research and applied Grading of Recommendations, Assessment, Development, and Evaluation methodology for clinical recommendations.Results: The panel addressed 16 specific areas for recommendations spanning questions of diagnostic testing, determination of site of care, selection of initial empiric antibiotic therapy, and subsequent management decisions. Although some recommendations remain unchanged from the 2007 guideline, the availability of results from new therapeutic trials and epidemiological investigations led to revised recommendations for empiric treatment strategies and additional management decisions.Conclusions: The panel formulated and provided the rationale for recommendations on selected diagnostic and treatment strategies for adult patients with community-acquired pneumonia.

Moraxella species: infectious microbes identified by use of time-of-flight mass spectrometry.

PURPOSE: To report the clinical manifestations, identification, antimicrobial susceptibilities, and treatment outcomes of ocular infections caused by Moraxella species. STUDY DESIGN: Retrospective study. PATIENTS AND METHODS: The medical records of all patients treated at the Departments of Ophthalmology of the Ogaki Municipal Hospital and the Gifu University Graduate School of Medicine for ocular infections caused by Moraxella species between January 2011 and June 2017 were examined. The stored Moraxella species isolated from ocular samples were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), molecular identification, and the biochemical properties. RESULTS: Sixteen eyes of 16 patients were treated for Moraxella ocular infections. The patients' median age was 72 years. A predisposing systemic or ocular condition was identified in 15 of the patients. Nine of the patients developed keratitis; four, conjunctivitis; and three, blebitis. M lacunata (6 eyes), M catarrhalis (6), M nonliquefaciens (3), and M osloensis (1) were identified by MALDI-TOF MS. All isolates were sensitive to levofloxacin, tobramycin, ceftazidime, ceftriaxone, and cefazolin. Twelve patients with keratitis or blebitis were treated with various topical antimicrobial combinations, and systemic antibiotics were used in 10 of the 12 patients. The mean time for the complete closure of the epithelial defects with keratitis was 24 days. The visual outcomes after treatment were favorable except in 1 keratitis patient who underwent enucleation. CONCLUSIONS: The use of duo-therapy with a combination of fluoroquinolone and cefmenoxime should be considered in cases nonresponsive to monotherapy, such as keratitis and bleb-associated infections. MALDI-TOF MS is useful for the identification of Moraxella to the species level.

Fulminant scleral abscess secondary to infected scleral buckle with Moraxella species.

We here report a case of scleral buckle infection with fulminant scleral abscess secondary to Moraxella species. A 54-year-old chronic alcoholic male with a history of retinal detachment repair, with scleral buckle 8 years prior, presented with complaints of severe pain, redness, and swelling in the right eye since 2 weeks. The patient was diagnosed with scleral buckle infection, the buckle was removed, and cultures revealed Moraxella species. The postoperative course included fulminant scleral abscess treated with dual antibiotic therapy that included ceftriaxone and moxifloxacin. All systemic antibiotics were discontinued after 3 weeks, retina remained attached, and no recurrence occurred over a 1-year follow-up. Moraxella, though commonly associated with bacterial keratitis, can also lead to buckle infection, especially in chronic alcoholic and immunocompromised patients. In buckle infection, infected buckle along with sutures should be immediately removed without damaging underlying compromised sclera. Lastly, culture and drug sensitivity play a very important role in buckle infections.

Peritonitis due to Moraxella osloensis: A case report and literature review.

A-26-year-old man was admitted to our hospital with diffuse abdominal pain, nausea, and vomiting. He had a history of malignant nephrosclerosis, for which he had been receiving peritoneal dialysis (PD) for the past 14 months. His PD effluent was cloudy and turbid (white blood cell count, 10,528/µL; neutrophils 95.2%). A Gram-negative coccobacillus was isolated from peritoneal fluid culture. However, the organism could not be identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) (Vitek MS, bioMérieux), but was identified as Moraxella osloensis by the 16S rRNA gene sequencing. He was successfully treated with intraperitoneal cefazolin therapy for 3 weeks without removing the intra-abdominal catheter. A literature review revealed three previous case reports all of which were diagnosed by MALDI Biotyper (Bruker Daltonics), suggesting that the identification of M. osloensis may vary depending on the type of MALDI-TOF MS system. In conclusion, we experienced a case of M. osloensis infection in a PD patient, which was successfully treated by antibiotic treatment, without removing the PD catheter.

Infectious endocarditis and vertebral osteomyelitis caused by Moraxella catarrhalis.

Moraxella catarrhalis frequently colonises the oropharynges of healthy individuals. Disease is usually limited to the oropharynx, upper airways and lower airways in patients with predisposing conditions. The pathogen rarely causes more invasive disease. We present the case of a 65-year-old woman with Crohn's disease on azathioprine, who was diagnosed with native valve M. catarrhalis endocarditis and vertebral osteomyelitis several weeks after an upper respiratory tract infection. She presented to hospital with 5 weeks of worsening malaise, nausea, relapsing fevers, weight loss, acute-on-chronic exacerbation of lower back pain and diffuse myalgia. Transoesophageal echocardiogram showed a 12 mm vegetation on her mitral valve, contrast-enhanced MRI was consistent with L4 osteomyelitis and blood cultures were persistently positive for M. catarrhalis She was initially treated with ceftriaxone 2 g intravenously daily, and although her symptoms initially resolved, she experienced a relapse of osteomyelitis with L3 extension a few weeks after treatment discontinuation.

Moraxella nonliquefaciens septic arthritis in a hematopoietic stem cell transplant patient a case report and review of the literature.

BACKGROUND: Septic arthritis is a common rheumatologic condition with myriad microbiological causative agents. Moraxella is one of the very rare causes of septic arthritis. We hereby present the third case of Moraxella nonliquefaciens septic arthritis and the first case in a hematopoietic stem cell transplant patient (HSCT) along with a brief review of the literature. METHODS: We used PubMed with google search engine to search the literature for reported cases of moraxella septic arthritis. RESULTS: Information on 19 other cases of moraxella infectious arthritis was found. M. catarrhalis was the most common species isolated. Only 2 reports on M. nonliquefaciens were found; the first one in a multiple myeloma patient and the second one in a diabetic patient on hemodialysis. Predisposing conditions included inflammatory arthritis, prosthetic joints, diabetes, Human Immunodeficiency Virus (HIV) infection, Hepatitis C, hemodialysis, esophageal cancer, valve replacements, alcoholism and Intravenous (IV) drug use. The age group of the reported cases ranged from 3 months to 78 years. CONCLUSION: Infectious arthritis with Moraxella spp. is a very rare entity which can occur in any age group and in the setting of various underlying medical conditions.