Occurrence of ESBL-producing avian pathogenic Escherichia coli (APEC) isolates in spiced chicken meat in Goias, Brazil.

Some extraintestinal pathogenic Escherichia coli isolates (ExPEC), obtained from humans and chickens avian pathogenic E. coli (APEC), share similar virulence genes. Thus, products of avian origin can be a source of human infection. Moreover, these APEC isolates are resistant to antimicrobials and can spread in the environment through the chicken feces. Although the development of multidrug-resistant (MDR) microorganisms in poultry is on the rise, healthcare entities have raised concerns since MDRs can horizontally transfer resistance genes to other microorganisms and complicate the management of human infections by MDR APEC. The results of our study showed that of 80 investigated spiced chicken meat samples, 55% were contaminated with E. coli, of which 34% (15/44) contaminate with APEC. No diarrheagenic E. coli (DEC) pathotypes were found. Twenty-six isolates were MDR E. coli. Among the APEC isolates, 87% (13/15) produced extended-spectrum beta-lactamase (ESBL). The emergence of MDR/ESBL-producing APEC with zoonotic potential for humans is extremely worrying. Therefore, further studies are required to identify the prevalence of MDR/ESBL-producing APEC in the entire chicken production chain from creation, slaughter, processing, and butchery.

Sporotrichosis in dogs: epidemiological and clinical-therapeutic profile and the emergence of itraconazole-resistant isolates.

Sporotrichosis is a neglected and emerging mycosis caused by the traumatic implantation of Sporothrix propagules into the (sub)cutaneous tissues of humans and animals. We evaluated canine sporotrichosis's clinical-therapeutic, epidemiological profile, and in vitro susceptibility of isolates to itraconazole. The variables were evaluated by a chi-square test. A total of 69 dogs were infected with Sporothrix spp., and the molecular identification revealed an overwhelming occurrence of S. brasiliensis as the etiological agent. The epidemiological profile was male (56.5%), adults (4.9 ± 1.92 years old; 69.6%), and mongrels (53.6%). The clinical signs were 76.8%, ulcers, draining tracts, and nodules were predominant, mainly in the nasal region (82.2%). Dogs were diagnosed late with an evolution time of up to 3 months (34.8%). According to the prior therapeutic information, 52.2% received empirical therapy, 79.2% antibiotics, and had a 0.29 significantly greater chance of presenting lesion evolution time ˃ 3 months (P < .05; Odds Ratio [OR] 1/0.29). Additionally, 25 S. brasiliensis isolates recovered between 2006-2012 (n = 15; Minimal inhibitory concentration (MIC): 0.06-2 µg/ml) and 2013-2018 (n = 10; MIC: 2→16 µg/ml) were tested against itraconazole (ITZ). These findings highlighted the resistance to ITZ in clinical cases due to S. brasiliensis occurring after 2013, showing the temporal evolution of ITZ-resistance. We warn of the importance of accurate and early diagnosis in Sporothrix-affected areas, and we report the emergence of ITZ-resistant isolates in Southern Brazil.

Sporotrichosis is a fungal zoonosis. We investigated the clinical-therapeutic, epidemiological profile, and in vitro susceptibility of isolates to itraconazole (ITZ) in canine cases in Southern Brazil. Our study highlighted the emergence of ITZ-resistant Sporothrix brasiliensis and the main challenges for clinical control of this neglected disease.

Novel phage display-derived recombinant antibodies recognizing both MPT64 native and mutant (63-bp deletion) are promising tools for tuberculosis diagnosis.

Tuberculosis (TB) is one of the top 10 causes of death in humans worldwide. The most important causative agents of TB are bacteria from the Mycobacterium tuberculosis complex (MTC), although nontuberculous mycobacteria (NTM) can also cause similar infections. The ability to identify and differentiate MTC isolates from NTM is important for the selection of the correct antimicrobial therapy. Immunochromatographic assays with antibodies anti-MPT64 allow differentiation between MTC and NTM since the MPT64 protein is specific from MTC. However, studies reported false-negative results mainly due to mpt64 63-bp deletion. Considering this drawback, we selected seven human antibody fragments against MPT64 by phage display and produced them as scFv-Fc. Three antibodies reacted with rMPT64 mutant (63-bp deletion) protein and native MPT64 from M. tuberculosis H37Rv in ELISA and Western blot. These antibodies are new biological tools with the potential for the development of TB diagnosis helping to overcome limitations of the MPT64-based immunochromatographic tests currently available.

Origanum vulgare essential oil: antibacterial activities and synergistic effect with polymyxin B against multidrug-resistant Acinetobacter baumannii.

Antimicrobial resistance is increasing around the world and the search for effective treatment options, such as new antibiotics and combination therapy is urgently needed. The present study evaluates oregano essential oil (OEO) antibacterial activities against reference and multidrug-resistant clinical isolates of Acinetobacter baumannii (Ab-MDR). Additionally, the combination of the OEO and polymyxin B was evaluated against Ab-MDR. Ten clinical isolates were characterized at the species level through multiplex polymerase chain reaction (PCR) for the gyrB and blaOXA-51-like genes. The isolates were resistant to at least four different classes of antimicrobial agents, namely, aminoglycosides, cephems, carbapenems, and fluoroquinolones. All isolates were metallo-ß-lactamase (MßL) and carbapenemase producers. The major component of OEO was found to be carvacrol (71.0%) followed by ß-caryophyllene (4.0%), γ-terpinene (4.5%), p-cymene (3,5%), and thymol (3.0%). OEO showed antibacterial effect against all Ab-MDR tested, with minimum inhibitory concentrations (MIC) ranging from 1.75 to 3.50 mg mL-1. Flow cytometry demonstrated that the OEO causes destabilization and rupture of the bacterial cell membrane resulting in apoptosis of A. baumannii cells (p < 0.05). Synergic interaction between OEO and polymyxin B (FICI: 0.18 to 0.37) was observed, using a checkerboard assay. When combined, OEO presented until 16-fold reduction of the polymyxin B MIC. The results presented here indicate that the OEO used alone or in combination with polymyxin B in the treatment of Ab-MDR infections is promising. To the best of our knowledge, this is the first report of OEO and polymyxin B association against Ab-MDR clinical isolates.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin.

BACKGROUND: The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES: We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS: BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS: Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS: The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Isolation, prevalence, and risk factors for infection by shiga toxin-producing Escherichia coli (STEC) in dairy cattle.

Rectal swabs of 198 Holstein × Gir crossbred beef cattle from 34 milk farms in the central west of Brazil were analyzed from August 2010 to February 2011. Strains of shiga toxin-producing Escherichia coli (STEC) were isolated from 72.73% (144/198) of the animals, on over 97% of the surveyed properties. The molecular characterization indicated the most common toxin gene stx1 in 70.88% of the animals (202/285), followed by 18.95% (54/285) stx1/sxt2, and 10.18% (29/285) stx2. The presence of STEC in animals together with the probable risk factors based on a questionnaire was evaluated in the owners of the evaluated animals. Results showed that the animal category "calves" and production/technification scale "low" of the farm were related to high STEC prevalence in cattle. The season did not significantly affect the presence of STEC in cattle. The STEC strains are considered a major pathogen, causing severe and potentially lethal diseases in humans such as hemorrhagic colitis and hemolytic uremic syndrome. This high prevalence of STEC in dairy cattle poses a significant risk to public health, since these microorganisms can contaminate products intended for human consumption, e.g., water, raw and pasteurized milk, meat products, dairy products, and/or products of plant origin.

Production of antigens expressed in Nicotiana benthamiana plant and Escherichia coli for the SARS-CoV-2 IgG antibody detection by ELISA.

The recent COVID-19 pandemic disclosed a critical shortage of diagnostic kits worldwide, emphasizing the urgency of utilizing all resources available for the development and production of diagnostic tests. Different heterologous protein expression systems can be employed for antigen production. This study assessed novel SARS-CoV-2 proteins produced by a transient expression system in Nicotiana benthamiana utilizing an infectious clone vector based on pepper ringspot virus (PepRSV). These proteins included the truncated S1-N protein (spike protein N-terminus residues 12-316) and antigen N (nucleocapsid residues 37-402). Two other distinct SARS-CoV-2 antigens expressed in Escherichia coli were evaluated: QCoV9 chimeric antigen protein (spike protein residues 449-711 and nucleocapsid protein residues 160-406) and QCoV7 truncated antigen (nucleocapsid residues 37-402). ELISAs using the four antigens individually and the same panel of samples were performed for the detection of anti-SARS-CoV-2 IgG antibodies. Sensitivity was evaluated using 816 samples from 351 COVID-19 patients hospitalized between 5 and 65 days after symptoms onset; specificity was tested using 195 samples collected before 2018, from domiciliary contacts of leprosy patients. Our findings demonstrated consistent test sensitivity, ranging from 85 % to 88 % with specificity of 97.5 %, regardless of the SARS-CoV2 antigen and the expression system used for production. Our results highlight the potential of plant expression systems as useful alternative platforms to produce recombinant antigens and for the development of diagnostic tests, particularly in resource-constrained settings.

Immunogenicity of an inactivated vaccine for intravaginal application against bovine alphaherpesvirus type 5 (BoHV-5).

The present study was carried out to evaluate the intravaginal vaccine potential against bovine alphaherpesvirus type 5 (BoHV-5). Sixty three cows were divided into seven groups (n: 9) and inoculated intravaginally (VA) or intramuscularly (IM) with inactivated BoHV-5, associated with the recombinant B subunit of the heat-labile enterotoxin of E. coli (rLTB), 2-hydroxyethylcellulose (Drug Delivery System A - DDS-A) or Poloxamer 407 (Drug Delivery System B - DDS-B) as follows: G1 (DDS-A + BoHV-5 + rLTB), G2 (DDS-A + BoHV-5), G3 (DDS-B + BoHV-5 + rLTB), G4 (DDS-B + BoHV-5), G5 (BoHV-5 + rLTB), G6 (Negative control) e G7 (Positive control). The local and systemic humoral responses were measured by indirect ELISA (IgA and IgG) and serum neutralization tests, and the cellular response was measured by a quantitative direct ELISA (IL-2 and IFN-Gamma). The results showed the group inoculated by the IM route, G5, demonstrated the highest levels of IgG in the vaginal mucosa among the experimental groups (p < 0.05). In the groups tested with polymers (G1 and G3) in the vaginal mucosa, even higher levels of IgG were seen in comparison to the positive control (G7; p < 0.01). Higher levels of IgA were also noted in relation to the other groups (p < 0.05) on days 30, 60 and 90 post-inoculations. The groups G1 and G3 also provided higher titers of neutralizing antibodies (Log2) in relation to other treatments (p < 0.01) 90 days after inoculation. In the nasal mucosa, there was an increase in the levels of IgA and IgG with the use of vaccines from groups G1 and G3, in relation to the positive control, G7 (p < 0.05) at 60 and 90 days after the first inoculation. Moreover, neutralizing antibodies titers were detected at 60 and 90 days by serum neutralization. The inclusion of the evaluated polymers resulted in a superior response (p < 0.05) of immunoglobulins and IL-2 and IFN-Gamma in relation to the treatment using only rLTB (G5). This data demonstrates the capabilities of a vaccine with an intravaginal application in cattle to stimulate a local and systemic immune response.

Clostridium haemolyticum, a review of beta toxin and insights into the antigen design for vaccine development.

Phospholipases C (PLCs) represent an important group of lethal toxins produced by pathogenic bacteria of the Clostridium genus, including the beta toxin of C. haemolyticum. Bacillary hemoglobinuria in cattle and sheep is the main disease caused by this pathogen and its incidence can be reduced by annual vaccination of herds. Currently, widely used vaccines depend on cultivating the pathogen and obtaining high concentrations of the toxin, disadvantages that can be overcome with the use of recombinant vaccines. In the development of this new generation of immunizing agents, identifying and understanding the structural and immunological aspects of the antigen are crucial steps, but despite this, the beta toxin is poorly characterized. Fortunately, the time and resources required for these investigations can be reduced using immunoinformatics. To advance the development of recombinant vaccines, in addition to a brief review of the structural and immunological aspects of beta toxin, this work provides in silico mapping of immunodominant regions to guide future vaccinology studies against C. haemolyticum. A review of alternatives to overcome the limitations of beta toxin vaccines (conventional or recombinant) is also proposed.

Recombinant Vaccine Design Against Clostridium spp. Toxins Using Immunoinformatics Tools.

The emergence of recombinant DNA technology has led to the exploration of the use of the technology to develop novel vaccines. With a fundamental role in vaccines design, several immunoinformatics tools have been created to identify isolated epitopes that stimulate a specific immune response, contributing to effective vaccines development. In the past, vaccine development projects relied entirely on animal experimentation, a relatively expensive and time-consuming process. Currently, use of immunoinformatics tools play a vital role in the antigen analysis and refinement, allowing the identification of possible protective epitopes capable of stimulating convenient humoral or cellular immune responses, in addition to facilitating time and cost reduction of vaccine production. The vaccination aimed at bacterial species of Clostridium spp. has been considered a promising example of use of these approaches in recent years. Based on the literature search, it is possible to understand the best immunoinformatics software used by researchers that facilitate recombinant vaccine antigens design and development. This chapter presents an overview of how these tools are supporting the antigen engineering, aiming at increasing the efficiency of inducing protective immune response in animals.

Recombinant Escherichia coli Cell Lysates as a Low-Cost Alternative for Vaccines Against Veterinary Clostridial Diseases.

This chapter describes a practical, industry-friendly, and efficient vaccine protocol based on the use of Escherichia coli cell fractions (inclusion bodies or cell lysate supernatant) containing the recombinant antigen. This approach was characterized and evaluated in laboratory and farm animals by the seroneutralization assay in mice, thereby showing to be an excellent alternative to induce a protective immune response against clostridial diseases.

Clostridium spp. Toxins: A Practical Guide for Expression and Characterization in Escherichia coli.

Farm animals are frequently affected by a group of diseases with a rapid clinical course, caused by Clostridium spp. and immunization is essential to provide protection. However, the current manufacturing platform for these vaccines has disadvantages and the main alternative is the use of an expression system that uses Escherichia coli to obtain recombinant vaccine antigens. In this chapter we describe procedures for cloning, expression and characterization of recombinant toxins from Clostridium spp. produced in E. coli for veterinary vaccine applications.

Antifungal resistance on Sporothrix species: an overview.

INTRODUCTION: The treatment of human and animal sporotrichosis is often performed with antifungal agents; however, the emergence of antifungal-resistant strains of Sporothrix species has been reported. We aimed to discuss the ability of Sporothrix species in developing resistance to the conventional antifungals and mechanisms for this. METHODOLOGY: Published data on databases (PubMed, Science Direct, Google Scholar) were investigated using a combination of keywords from 2008 to 2019 by the StArt tool. RESULTS: The minimal inhibitory concentrations values based on the Clinical and Laboratory Standards Institute (CLSI) from eight references were classified according to the epidemiological cutoff values in wild-type or non-wild-type strains. In this way, non-wild-type S. schenckii and, mainly, S. brasiliensis isolates were recognized on itraconazole, amphotericin B, terbinafine, and voriconazole, which are strains that deserve more attention toward antifungal control, with a probable risk of mutation to antifungal resistance. Among the few reviewed studied on antifungal resistance, the melanin production capacity (DHN-melanin, L-DOPA melanin, and pyomelanin), the low genetic diversity due to the abnormal number of chromosomes, and the mutation in cytochrome P450 are some of the factors for developing resistance mechanism. CONCLUSIONS: The emergence of Sporothrix species with in vitro antifungal resistance was evidenced and the possible mechanisms for resistance development may be due to the melanin production capacity, genetic diversity and mutations in cytochrome P450. Further studies should be carried out targeting gene expression for the development of antifungal resistance on Sporothrix species in order to prospect new therapeutic targets for human and veterinary use.

Clostridium septicum: A review in the light of alpha-toxin and development of vaccines.

Clostridium septicum (CS) is a pathogen that can cause the death of animals in livestock worldwide through its main virulence factor, alpha-toxin (ATX). The aspects involved in diseases caused by ATX, such as economic impact, prevalence, and rapid clinical course, require that animals should be systematically immunized. This review provides an overview of CS in livestock farming and discusses current immunization methods. Currently, commercial vaccines available against CS involve the cultivation and inactivation of microorganisms and toxins using a time-consuming, expensive, and high biological risk-carrying production platform, and some have been reported to be ineffective. An alternative to this process is the recombinant DNA technology, although recombinant ATX obtained thus far is no longer efficient in stimulating protective antibody titers despite improvements in the production methods. On the other hand, immunized animals have highly favorable levels of survival when subjected to challenge tests, suggesting that high titers of circulating serum antibodies may not be representative of protection after immunization and that the non-immune cellular defenses associated with the particularities of the mechanism of action of ATX may be involved in the immune response of the host. To contribute to the future of global livestock farming through the development of more efficient recombinant vaccines, we suggest novel perspectives and strategies, such as the location of immunodominant epitopes, expression of relevant functional domains, and construction of chimeras, in the rational design of recombinant ATX.

Feline sporotrichosis: a case series of itraconazole-resistant Sporothrix brasiliensis infection.

The treatment of feline sporotrichosis is a challenge for veterinary clinicians since refractory cases may occur, due either to patient and/or to pharmacological management errors or due to the development of antifungal resistance. Thus, we aimed to describe the therapeutic history of feline cases infected by itraconazole-resistant Sporothrix brasiliensis in an endemic region of Southern Brazil. Medical records of cats attended at the Veterinary Clinic Hospital (Pelotas/RS, Brazil) between 2016 and 2017 were reviewed. Twelve cases of infection by S. brasiliensis with that showed high minimum inhibitory concentration (MIC) values (≥ 4 µg/mL) to itraconazole by M38-A2 of CLSI were selected. At the hospital consultation, disseminated (cats 1-l0, 12) and localized (cat 11) skin lesions remained in the cats, even after treatment with fluconazole, ketoconazole (02/12), and itraconazole (ITZ, 09/12) performed before this study. High doses (25-100 mg/kg/day) of ITZ for up to 4 months (03/12, cats 2, 6, 12) or over 12 months (05/12, cats 1, 5, 7, 8, 11) did not provide a clinical cure, except for the association of ITZ plus potassium iodide (01/12, cat 12) for 3 months, which proved useful in infections with itraconazole-resistant S. brasiliensis. However, the combined issues of abandonment of therapy by owners for financial reasons, difficulties surrounding therapy administration (03/12, cats 6, 11, 12), and the inappropriate choice of medication (01/12, cat 6), together reflect the reality of this endemic region, which greatly compromises clinical healing. This study highlighted the occurrence of refractory cases by itraconazole-resistant S. brasiliensis in cats from Southern Brazil, as well as the abandonment of treatment and therapeutic errors. We warn of the need for antifungal susceptibility tests to adapt therapeutic protocols in feline sporotrichosis.

Human sporotrichosis outbreak caused by Sporothrix brasiliensis in a veterinary hospital in Southern Brazil.

Sporotrichosis is a mycotic disease caused by Sporothrix spp., whose zoonotic transmission by sick cats is the main infection route in Brazil. The aim of the current study is to report a human sporotrichosis outbreak caused by zoonotic transmission from a feline, with emphasis on the importance of making differential diagnosis and of using personal protective equipment. A hospital team member presented injury in the arm after having handled a cat that had been hospitalized for being hit by a car. The animal presented skin lacerations, myiasis, and full tibial fracture - there were no other signs of skin lesions. Clinical samples were collected from both the human and the suspected cat, for mycological culture; results have shown Sporothrix sp. growth. A search was conducted to identify other hospital team members who also had contact with the animal. Other six individuals also had suspected lesions in their arms, hands and ocular area; they were all subjected to sample collection. Mycological results have also confirmed Sporothrix spp.; sequencing analysis has shown that all seven humans were infected with Sporothrix brasiliensis. Since Southern Brazil is endemic of this disease, it is worth emphasizing the importance of taking into consideration zoonotic risks at the time to provide emergency care to stray animals, mainly felines, as well as of using Personal Protective Equipment while handling them - regardless of whether they present, or not, typical clinical symptoms or history of the disease, given the potential zoonotic risk posed by Sporothrix brasiliensis.

Subcutaneous phaeohyphomycosis due to Aureobasidium pullulans infection in a dog

A case of subcutaneous phaeohyphomycosis in a dog with an ulcerative lesion on the right limb during a post-operative period of castration was described for the first time. The macroscopic and microscopic characteristics of the fungal colonies growth on the Sabouraud­dextrose agar were detailed. The fungus was identified as Aureobasidium pullulans on the basis of the phenotypic analysis, which was confirmed by sequencing of the internal transcribed spacers (ITS) region of rDNA. The patient might have acquired the infection through traumatic inoculation by environmental contact, along with the immunological condition during the stressful period of postoperative. The spontaneous remission of the lesion was observed in five weeks without antifungal treatment. This work highlights the importance of considering the pathogenic potential of this environmental fungus and the need of including it in the differential diagnosis of cutaneous lesions in dogs.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin

BACKGROUND The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.