New Insights into the Bacterial Targets of Antimicrobial Blue Light.

Antimicrobial blue light (aBL) offers efficacy and safety in treating infections. However, the bacterial targets for aBL are still poorly understood and may be dependent on bacterial species. Here, we investigated the biological targets of bacterial killing by aBL (λ = 410 nm) on three pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Initially, we evaluated the killing kinetics of bacteria exposed to aBL and used this information to calculate the lethal doses (LD) responsible for killing 90 and 99.9% of bacteria. We also quantified endogenous porphyrins and assessed their spatial distribution. We then quantified and suppressed reactive oxygen species (ROS) production in bacteria to investigate their role in bacterial killing by aBL. We also assessed aBL-induced DNA damage, protein carbonylation, lipid peroxidation, and membrane permeability in bacteria. Our data showed that P. aeruginosa was more susceptible to aBL (LD99.9 = 54.7 J/cm2) relative to S. aureus (LD99.9 = 158.9 J/cm2) and E. coli (LD99.9 = 195 J/cm2). P. aeruginosa exhibited the highest concentration of endogenous porphyrins and level of ROS production relative to the other species. However, unlike other species, DNA degradation was not observed in P. aeruginosa. Sublethal doses of blue light (LD99.9). We conclude that the primary targets of aBL depend on the species, which are probably driven by variable antioxidant and DNA-repair mechanisms. IMPORTANCE Antimicrobial-drug development is facing increased scrutiny following the worldwide antibiotic crisis. Scientists across the world have recognized the urgent need for new antimicrobial therapies. In this sense, antimicrobial blue light (aBL) is a promising option due to its antimicrobial properties. Although aBL can damage different cell structures, the targets responsible for bacterial inactivation have still not been completely established and require further exploration. In our study, we conducted a thorough investigation to identify the possible aBL targets and gain insights into the bactericidal effects of aBL on three relevant pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. This research not only adds new content to blue light studies but opens new perspectives to antimicrobial applications.

Phylogenomic analysis of CTX-M-15-producing Enterobacter hormaechei belonging to the high-risk ST78 from animal infection: another successful One Health clone?

OBJECTIVES: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacter cloacae complex (ECC) members have been a leading cause of severe infections in hospital setting and have lately been recognized as important pathogens for animals. In this article, we report phylogenomic data of a multidrug-resistant and CTX-M-15-positive E. hormaechei belonging to ST78 isolated from a calf with omphalitis. METHODS: Genomic DNA was extracted and sequenced using the Illumina NextSeq platform. De novo assembly was performed by Unicycler and in silico prediction accomplished by curated bioinformatics tools. Single nucleotide polymorphism (SNP)-based comparative phylogenomic analysis was conducted by using publicly available ECC genomes belonging to ST78. RESULTS: The genome size was calculated at 3 8465 40 bp, comprising 4717 total genes, 3 rRNAs, 43 tRNAs, 7 ncRNAs, and 74 pseudogenes. The animal-associated E. hormaechei (ECBEZ strain) ST78 harboured the blaCTX-M-15 ESBL gene in addition to other critically important resistance genes conferring resistance to ß-lactams, aminoglycosides, fosfomycin, phenicol, quinolones, sulphonamides, tetracyclines, and trimethoprim. Phylogenetic analysis revealed that ECBEZ is closely related to human-isolated strains from Asian and African countries. CONCLUSION: Phylogenomic analysis of CTX-M-15-producing E. hormaechei from animal infection reveals that ST78 is a successful One Health clone among ECC members. Furthermore, data presented in this study reinforce the urgent need to monitor ESBL-producing ECC members in veterinary settings.

Methylene blue-mediated antimicrobial photodynamic therapy can be a novel non-antibiotic platform for bovine digital dermatitis.

BACKGROUND: Bovine digital dermatitis (BDD) is one of the most important diseases that effect dairy cows. Methylene blue-mediated antimicrobial photodynamic therapy (MB-APDT) emerges as a promising technique to treat superficial infections in bovines. METHODS: Twenty BDD lesions located at the skin horn transition of the claw of pelvic limbs of 16 cows were treated by MB-APDT, using a red LED cluster (λ = 660 nm, irradiance =60 mW/cm2, exposure time = 40 s) combined with topical application of MB at 0.01 %; or by topical application of OXY (500 mg in 20 % solution). Each lesion was treated twice with an interval of 14 days. Lesions were weekly evaluated until day 28 by clinical analysis and by histological examination on days 0 and 28. RESULTS: Both treatments led to a similar reduction of lesions area. At day 28, three lesions treated by OXY did not present completely recovery, whereas no lesions were observed in MB-APDT group. OXY resulted in a slight increase in type I and III collagen levels, while MB-APDT led to a significant increase in the total area of both collagen types. An abundant number of spirochetes were histologically observed in all lesions before treatments. On the 28th day, five lesions treated by OXY still presented a slight number of spirochetes, whereas in MB-APDT group no spirochetes were evidenced. CONCLUSION: Our findings suggest that MB-APDT is more effective than OXY and could be used in Veterinary practice to fight BDD.

Antimicrobial photodynamic therapy can be an effective adjuvant for surgical wound healing in cattle.

BACKGROUND: Rumenostomy is a useful procedure commonly performed in cattle for medical treatment of domestic ruminants with forestomach diseases. Methylene blue (MB)-mediated antimicrobial photodynamic therapy (APDT) has been broadly investigated to treat infected wounds. AIM: The aim of the study was to evaluate the effectiveness of MB-mediated APDT (MB-APDT) combined with chlorhexidine and zinc oxide ointment on wound healing process after rumenostomy. METHODS: Fourteen Nelore bulls were subjected to rumenostomy procedure. Animals were randomly divided into MB-APDT (MB associated with a red diode laser performed immediately after surgery and repeated on days 3, 5, 7 and 10) and control groups. Daily care included topical cleaning with chlorhexidine 2% followed by topical zinc oxide ointment. Animals were followed-up until the 28th day. RESULTS: Wounds presented a better post-surgical profile in MB-APDT group when it was compared with the control group. In MB-APDT group, it was also possible to observe less pain on palpation of wounds borders, less edema and inflammatory exudate. Additionally, animals from MB-APDT group were faster discharged from the cattle care facility. CONCLUSION: Our results support the use of MB-APDT for the post-surgical management of rumenostomy. This pilot study ratifies the use of APDT in cattle and also suggests that it could be performed for other surgical procedures as a complementary approach or an alternative for topical administration of antibiotics.

Antimicrobial blue light and photodynamic therapy inhibit clinically relevant ß-lactamases with extended-spectrum (ESBL) and carbapenemase activity.

INTRODUCTION: The production of ß-lactamases by Gram-negative bacteria is among the most important factors of resistance to antibiotics, which has contributed to therapeutic failures that currently threaten human and veterinary medicine worldwide. Antimicrobial photodynamic therapy and antimicrobial blue light have a broad-spectrum antibacterial activity against multidrug-resistant and hypervirulent pathogens. OBJECTIVE: To investigate the ability of antimicrobial blue light to inhibit the hydrolytic activity of clinically relevant ß-lactamase enzymes (i.e., KPC, IMP, OXA, CTX-M, and SHV), with further comparison of the inhibitory effects of antimicrobial blue light with methylene blue-mediated antimicrobial photodynamic therapy. METHODS: Blue LED light (λ = 410 ± 10 nm) alone or red LED light (λ = 660 ± 10 nm) in combination with methylene blue were used to inactivate, in vitro, suspensions of Klebsiella pneumoniae strains producing clinically important ß-lactamase enzymes assigned to the A, B and D Ambler molecular classes. Furthermore, ß-lactamase activity inhibition mediated by antimicrobial blue light and methylene blue-mediated antimicrobial photodynamic therapy was measured by using the chromogenic ß-lactam substrate nitrocefin. RESULTS: ß-lactamase activities were effectively inactivated by both visible light-based approaches. In this regard, antimicrobial blue light and methylene blue-antimicrobial photodynamic therapy led to a significant reduction in the hydrolysis of nitrocefin (81-98 %). CONCLUSION: Sublethal doses of antimicrobial blue light and methylene blue-mediated antimicrobial photodynamic therapy are equally effective to inhibit clinically significant ß-lactamases, including extended-spectrum ß-lactamases and carbapenemases.

Hypervirulent and hypermucoviscous strains of Klebsiella pneumoniae challenged by antimicrobial strategies using visible light.

INTRODUCTION: Infections caused by hypervirulent and/or hypermucoviscous Klebsiella pneumoniae (K. pneumoniae) strains are frequently reported worldwide. Since convergence of hypervirulence and drug-resistance emerged as a serious clinical problem, novel therapeutic strategies are worthy of investigation. In this regard, antimicrobial photodynamic therapy and blue light have proven to be effective against a broad-spectrum of clinically relevant pathogens but have never been tested for hypervirulent/hypermucoviscous strains. Thus, this study investigated the influence of hypermucoviscosity and hypervirulence over the photoinactivation efficacy of blue light alone or antimicrobial photodynamic therapy mediated by methylene blue and red light. METHODS: Five clinical isolates of K. pneumoniae were screened for hypermucoviscosity by string test and for hypervirulence by a Galleria mellonella model of systemic infection. Strains were then challenged by both photoinactivation methods performed in vitro. All tests also included a non-hypervirulent/hypermucoviscous control strain for comparison. RESULTS: All K. pneumoniae strains were effectively inactivated by both light-based antimicrobial strategies. Hypervirulent/hypermucoviscous strains exposed to photodynamic therapy presented rapid and consistent inactivation kinetics, while blue light led to slower and more variable inactivation kinetics. CONCLUSION: Hypermucoviscosity and hypervirulence does not confer tolerance in K. pneumoniae against photoinactivation. Antimicrobial photodynamic therapy represents an interesting alternative to treat localised infections because it is a fast procedure with high effectiveness. On the other hand, antimicrobial blue light could be used to decontaminate hospital environments since no photosensitiser administration is required and harmful effects of ultraviolet light are avoided. Therefore, visible light-based strategies present great potential for the development of safe and effective antimicrobial technologies against such aggressive pathogens.

Antimicrobial blue light inactivation of international clones of multidrug-resistant Escherichia coli ST10, ST131 and ST648.

BACKGROUND: International clones of multidrug-resistant Escherichia coli have been a leading cause of human and animal infections worldwide. Microbial inactivation by blue light has been proposed as an effective treatment for superficial infections and surface contamination. AIM: To evaluate the inactivation efficacy of blue light irradiation against high-risk multidrug-resistant strains of E. coli. METHODS: Blue LED light (λ = 410 nm) was used to inactivate in vitro suspensions of colistin- broad-spectrum cephalosporin-, or carbapenem-resistant E. coli strains belonging to sequence types (STs) ST10, ST131 and ST648, carrying mcr-1, blaCTX-M or blaKPC-2 genes. RESULTS: Our results showed that all E. coli strains were susceptible to blue light irradiation, independently of antibiotic resistance and virulence profiles. In addition, blue light irradiation induced a strain-specific and dose-dependent bacterial effect. CONCLUSION: Our results support use of blue light as a promising antimicrobial option against MDR pathogens, including high-risk clones of E. coli displaying resistance to polymyxins or broad-spectrum ß-lactam antibiotics.

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.

The normal electrocardiogram in the clinically healthy Saanen goats / Eletrocardiograma normal de cabras Saanen clinicamente sadias

The purpose of this study was to establish normal reference electrocardiographic (ECG) values for standard limb lead II in Saanen goats. For this, were used 19 healthy adult female Saanen goats. And for reference values for the ECG parameters, were used 95% confidence level. The overall P, Q, R, S and T durations (seconds) were 0.03±0.01, 0.02±0.005, 0.03±0.014, 0.03±0.011, 0.06±0.014 respectively, and the reference values were 0.01-0.04, 0.01-0.02, 0.01-0.06, 0.02-0.04 and 0.04-0.08 respectively. The amplitudes (milivolts) of these waveforms were 0.1±0.031, 0.06±0.023, 0.44±0.312, 0.39±0.434, 0.26±0.164 (T negative) and 0.15±0.071 (T positive), respectively. The reference values were 0.05-0.15, 0.05-0.1, 0.05-1.2, 0.05-1.1 and -0.2-0.7 respectively. The PR and QT interval, the QRS complex and the ST segment durations (seconds) were 0.08±0.018, 0.26±0.03, 0.05±0.008, 0.15±0.041 respectively. The reference values were 0.06-0.12, 0.2-0.32, 0.04-0.07 and 0.11-0.26, respectively. It was possible to observe differences in ECG of Saanen goats regarding the amplitude and duration of the constituents when compared to the results of other breeds. Therefore it is necessary to conduct further studies to allow comparisons, detect and diagnose cardiac arrhythmias and help the development of therapies.

O objetivo deste estudo foi de estabelecer os valores de referência para o eletrocardiograma normal de derivação II de cabras Saanen sadias. As durações totais (segundos) das amplitudes de P, Q, R, S e T foram 0.03±0.01, 0.02±0.005, 0.03±0.014, 0.03±0.011, 0.06±0.014, respectivamente, e os valores de referência foram 0.01-0.04, 0.01-0.02, 0.01-0.06, 0.02-0.04 e 0.04-0.08, respectivamente. As amplitudes (milivolts) dessas formas de onda foram 0.1±0.031, 0.06±0.023, 0.44±0.312, 0.39±0.434, 0.26±0.164 (T negativo) e 0.15±0.071 (T positivo), respectivamente. Os valores de referência foram 0.05-0.15, 0.05-0.1, 0.05-1.2, 0.05-1.1 e -0.2-0.7, respectivamente. As durações (segundos) dos intervalos PR e QT, do complexo QRS e do segmento ST foram 0.08±0.018, 0.26±0.03, 0.05±0.008, 0.15±0.041, respectivamente. Os valores de referência foram 0.06-0.12, 0.2-0.32, 0.04-0.07, e 0.11-0.26, respectivamente. Foi possível observar diferenças em relação à duração e amplitude dos constituintes do ECG quando comparados com os resultados de outras raças. Portanto, torna-se necessário a realização de mais estudos para se permitir comparações, detectar e diagnosticar arritmias cardíacas bem como auxiliar no desenvolvimento de terapias.

Prolapso vaginal e uterino em ovelhas / Uterine and vaginal prolapse in ewes

O presente trabalho objetivou realizar um estudo retrospectivo sobre os prolapsos vaginal e uterino em ovelhas atendidas no Serviço de Clínica de Bovinos e Pequenos Ruminantes (CBPR) da FMVZ/USP no período compreendido entre 2000 a 2010, no qual, foram atendidas 56 ovinos com problemas inerentes ao sistema reprodutivo, dessas, 25 apresentaram prolapso vaginal ou uterino (44,6%). O prolapso vaginal total foi o de maior frequência (72%). As ovelhas acometidas, em sua maioria, possuíam idade superior a quatro anos (64%), eram sem raça definida (44%) ou da raça Ile de France (40%). As manifestações clínicas observadas durante a maioria dos atendimentos foram: taquipnéia, taquicardia, mucosas oculares avermelhadas indicando estado de toxemia, decúbito esternal ou lateral, apatia e anorexia. O tratamento instituído para todos os casos foi a limpeza, desinfecção e reintrodução do órgão prolapsado. A sutura de Bühner foi feita em 84% dos casos e a histeropexia em um caso (4%). A evolução foi satisfatória em 80% dos casos atendidos, nos demais casos (20%) observou-se óbito da fêmea acometida. Do total de óbitos, os prolapsos vaginais foram responsáveis por 60% (3/5) e os prolapsos uterinos por 40% (2/5). A etiologia dos prolapsos não foi definida nos casos atendidos, sendo esses associados com o período pós-parto em sua maioria (56%), provavelmente associados com quadros de hipocalcemia, altas concentrações séricas de estrógeno e hipertonia uterina. Além disso, a predisposição genética não pode ser descartada.

This study aimed to conduct a retrospective study on vaginal and uterine prolapse in sheep seen at the Clinic and Surgery on Cattle and Small Ruminants (CBPR) at University of São Paulo, from 2000 to 2010. During this period, 56 sheep were treated with problems of the reproductive system. Of these, 25 ewes had vaginal or uterine prolapse (44.6%). The total vaginal prolapse was the most frequently (72%). The majority of sheep that was affected were 4 years old (64%); most were mixed breed (44%) and 40% were Ile de France. Main clinical signs were increased cardiac and respiratory rates, congested ocular mucosa, sternal or lateral recumbence, apathy and anorexia, suggesting toxemia. The treatment of all cases was the cleaning and disinfection of the prolapsed organ and its reintroduction. The Bühner suture was made in 84% of the cases. The uterus fixation was made in one case (4%). Recovered was observed in 80% of the cases and 20% of the patients died. Vaginal prolapse corresponded to 60% of the deaths and uterine prolapse to 40%. The etiology of the prolapses had not been defined, but most cases (56%) occurred during the postpartum period, probably associated with hypocalcaemia, high serum concentrations of estrogen or uterine hypertonia. Furthermore, a genetic predisposition on affected sheep cannot be discharged.