Assessment of the Potential Risks Associated with the Proposed Use of Composted Waste from the Production of Bacitracin as a Soil Additive

Bacitracin is a hexapeptide antibiotic, with a substituted thiazolidine nucleus, produced by some strains of B. licheniformis. It is mainly active against Grampositive bacteria, although many differences in susceptibility exist among the bacterial species. Alpharma A.S. Norway has produced bacitracin for use in human medicine since 1954. Until 1998, the fermentation waste from the production of bacitracin was added to animal feed in some European countries, including Norway, to promote growth of pigs and domestic fowl. In 1998, fermentation waste containing bacitracin as a food additive was banned by the EU to reduce the risk of developing bacitracin-resistant bacteria in animals, and the subsequent possible transfer of such bacteria to humans via the food chain. Use of fermentation waste containing bacitracin as a feed additive has not been officially banned in Norway, but it is no longer used for this purpose. Alpharma is therefore actively seeking alternative uses for their production waste. As the waste material is rich in nutrients, the company proposes that it could be developed as a soil additive by fermenting it with chipped bark and lime. The Norwegian Food Safety Authority (Mattilsynet) commissioned the Panel on Biological Hazards of the Norwegian Scientific Committee for Food Safety (Vitenskapskomitéen for mattrygghet) to develop a risk assessment regarding the use of composted waste material from Alpharma’s production of bacitracin, as a soil additive. In response, an ad hoc Working Group of experts was appointed with the mandate to draft a risk assessment which should include the following elements: assessment of risk to human health and/or the environment in relation to residual content of bacitracin in the finished soil additive product and assessment of the risk in relation to dissemination of the production strain and antimicrobial resistance genes. The Panel on Biological Hazards concludes that the risks to human health and the environment posed by residual bacitracin present in the finished product are minimal. Furthermore, as Bacillus licheniformis is considered essentially non-pathogenic, occurring rarely as an opportunistic pathogen, the risk posed by this bacterium to human health or the environment is very low. It is reasonable to assume that during the early composting process horizontal transfer of bacitracin and erythromycin resistance genes, from the B. licheniformis producer strain to environmental bacteria, will exceed background levels. However, this is considered to represent a low risk to human health and the environment.

Metabolic engineering of L-cysteine supply modules for enhanced production of bacitracin in Bacillus licheniformis / 生物工程学报

Bacitracin is a broad-spectrum antibiotics mainly produced by Bacillus, and is used as veterinary medicine in the fields of livestock and poultry breeding. Insufficient supply of precursor amino acids might be an important factor that hinders high-level microbial production of bacitracin. We investigated the effect of strengthening L-cysteine supply on bacitracin production by an industrial bacitracin producer, Bacillus licheniformis DW2. Overexpression of cysK encoding L-cysteine synthase led to a 9.17% increase of the bacitracin titer. Moreover, overexpression of cysE encoding L-serine acetyltransferase and cysP encoding thiosulfate/sulfate intracellular transporter increased the bacitracin titers by 7.23% and 8.52%, respectively. Moreover, overexpression of a putative cystine importer TcyP led to a 29.19% increase of intracellular L-cysteine, and bacitracin titer was increased by 7.79%. Subsequently, the strong promoter PbacA was used to replace the promoters of genes cysP, cysE and tcyP in strain DW2::ysK, respectively. The resulted strain CYS4 (DW2::cysK-PbacA-(cysP)-PbacA(cysE)- PbacA(tcyP) produced 910.02 U/mL bacitracin, which was 21.10% higher than that of the original strain DW2 (747.71 U/mL). Together with the experiments in 3 L fermenters, this research demonstrated that enhancing intracellular L-cysteine supply is an effective strategy to increase bacitracin production of B. licheniformis.

Probiotic and synbiotic in broiler diet: performance and Enterobacteriaceae / [Probiótico e simbiótico em ração para frangos: desempenho e Enterobacteriaceae]

The objective of this study was to evaluate the effects of probiotics and synbiotics on the performance and Enterobacteriaceae count of broiler chickens. A total of 640 one-day-old male broiler chicks were distributed in a completely randomized design with four treatments and eight replicates with 20 birds each. The treatments were: ration with performance enhancer (zinc bacitracin; positive control); ration without performance enhancer and probiotic/synbiotic (negative control); ration with probiotics; and ration with synbiotics. At 35 days, five birds from each treatment were euthanized and intestinal contents were harvested for determining the Enterobacteriaceae count. The performance data and average colony-forming units (CFUs) transformed as log CFU/g were subjected to analysis of variance and Tukey's test. The effects of probiotics and synbiotics were observed in the initial phase, with supplemented birds exhibiting comparable weight gain to those supplemented with bacitracin. No effect of the treatment on broiler performance was observed after 42 days. The enterobacterial count was comparable among all experimental treatments. Supplementation with probiotics and synbiotics did not compromise the performance of broilers and did not alter the Enterobacteriaceae count.(AU)

Objetivou-se avaliar o efeito do probiótico e do simbiótico sobre o desempenho e a contagem de Enterobacteriaceae em frangos. Foram utilizados 640 pintos de corte, machos, de um dia de idade, distribuídos em delineamento inteiramente ao acaso, com quatro tratamentos, oito repetições com 20 aves cada. Os tratamentos foram: ração com melhorador de desempenho (bacitracina de zinco) (controle positivo); ração sem melhorador de desempenho e sem probiótico/simbiótico (controle negativo); ração com probiótico e ração com simbiótico. Aos 35 dias, cinco aves por tratamento foram eutanasiadas para retirada de conteúdo intestinal e determinação de Enterobacteriaceae. As médias das unidades formadoras de colônias, transformadas em log/UFC/g, e de desempenho foram submetidas à análise de variância e comparadas pelo teste Tukey. Foi observado efeito do probiótico e do simbiótico na fase inicial, sendo que aves apresentaram os mesmos resultados de ganho de peso e de peso corporal que o grupo de aves alimentado com bacitracina. Aos 42 dias, não houve efeito dos tratamentos sobre o desempenho. Aves que não receberam nenhum aditivo não apresentaram maior contagem de enterobactérias, sendo semelhantes aos demais tratamentos. A adição do probiótico e do simbiótico não compromete o desempenho dos frangos e não altera a contagem de Enterobacteriaceae.(AU)

Enhanced production of bacitracin via energy metabolism engineering in Bacillus licheniformis DW2 / 生物工程学报

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.

Comparative studies on the efficacy of lincomycin and bacitracin for the control of necrotic enteritis in broiler chickens

Background: The present study was conducted to evaluate the efficacy of lincomycin and/or bacitracin for control of experimentally-induced Clostridium perfringens (CP) infection in broiler chickens.Methods: A total of 100 one-day-old Cobb-mixed chicks were divided into five groups (A, B, C, D and E, each of 20 bird). At the 15th day of age, all birds (except group A) were inoculated orally with CP broth culture (109 CFU/mL). Two days later, drugs were orally administered once daily for five consecutive days as follow; Group A and B were left untreated. Group C, D, and E were treated with lincomycin (0.5 g/l), bacitracin (100 mg/l), lincomycin and bacitracin, respectively. The efficacy of used drugs was estimated based on clinical symptoms, body weight, weight gain, feed conversion rate. Hematobiochemical changes were also determined.Results: Necrotic enteritis in broiler chickens induced a significant decrease in body weight, weight gain, erythrocytic count, hemoglobin content, PCV %, serum proteins, catalase, and superoxide dismutase. Additionally, a marked decrease in serum lipids was obtained. Furthermore, a significant increase in feed conversion rate, leukocytic count, phagocytic activity, phagocytic index, serum total globulin, ? globulin and malondialdehyde coupled with a marked increase in ? and ? globulins were determined. Medication of infected broilers with lincomycin and/or bacitracin improved clinical signs and reduced mortality rate to 8, 6 and 2%, respectively, as well as restored the performance and hematobiochemical alterations.Conclusions: a combination of lincomycin and bacitracin was of considerable value for the control of necrotic enteritis in broiler chickens.

Molecular epidemiological characteristics of Streptococcus pyogenes causing scarlet fever and angina in children / 中华微生物学和免疫学杂志

Objective@#To investigate the antimicrobial resistance, macrolide-resistance genes, virulence genes and emm types in Streptococcus pyogenes isolates.@*Methods@#A total of 247 oropharyngeal swab specimens were collected from pediatric outpatients (aged 2-11 years) who were clinically diagnosed as scarlet fever in Children′s Hospital of Fudan University from January to December, 2018. These specimens were timely sent to the Microbiology Laboratory for isolation and identification of Streptococcus pyogenes strains were isolate after culturing and identified with bacitracin susceptibility test. Moreover, the diameter of bacitracin inhibition zone was measured by vernier caliper. Their susceptibility to seven antibiotics, including erythromycin, clarithromycin, clindamycin, ampicillin, ceftriaxone, norfloxacin and chloramphenicol, were measured using KB method. Macrolide-resistance genes (mefA, ermA, ermB and Tn916 transposon) and virulence genes (speA, speB, speC, speG, speH, speI, speJ and speK) were detected by PCR. Amplification and sequencing of emm gene were conducted according to the protocol in the website of Centre for Disease Control and Prevention (CDC).@*Results@#A total of 86 strains of Streptococcus pyogenes were isolated from the 247 specimens. Their resistance rates to erythromycin, clarithromycin and clindamycin were 89.5%, 95.3% and 96.5%, respectively. However, these isolates showed high susceptibility to ampicillin (100.0%), ceftriaxone (100.0%), norfloxacin (90.7%) and chloramphenicol (95.3%). The positive rates of mefA, ermA, ermB and Tn916 genes were 20.9%, 24.4%, 98.8% and 97.7%. There was significant difference in the mefA-carrying rates between patients with scarlet fever and angina. The positive rates of virulence genes of speA, speB, speC, speG, speH, speI, speJ, speK, speL and speM were 8.1%, 100.0%, 95.3%, 100.0%, 80.2%, 90.7%, 10.5%, 100.0%, 5.8% and 5.8%. Seven emm types were identified and the predominant types were emm12.0 (75.6%), emm12.19 (9.3%) and emm1.0 (8.1%). The diameter of bacitracin inhibition zone was smaller in isolates of emm1.0 type than in emm12.0 type strains. The profile of virulence genes varied in the strains of different emm types. All types of strains carried speB, speG and speK genes. The isolates of emm1.0 type carried speA virulence gene, while speB, speC, speG, speH, speI and speK genes were more often identified in emm12.0 type isolates.@*Conclusions@#This study showed that emm types were associated with the profile of virulence genes and the diameter of bacitracin inhibition zone. It was recommended that the diameter of bacitracin inhibition zone should be measured in bacitracin inhibition susceptibility test apart from only observing the formation of inhibition zone.

Analysis of the Related Substances and Content of Effective Bacitracin by HPLC Combined with Component Preparation / 中国药学杂志

OBJECTIVE: To develop a suitable HPLC method combined with component preparation to analyze the related substances and effective contents in bacitracin.METHODS: The separation was performed on Agilent HPLC system with a Diamonsil Plus C18 column (4.6 mm×250 mm, 5 μm) using 50 mmol•L-1 ammonium formate (pH adjusted to 4.0 with formic acid) as mobile phase A and acetonitrile as mobile phase B at a flow rate of 1 mL•min-1 under gradient elution. The detection wavelength was set at 254 nm. The HPLC method was carried out by adjusting the pH of ammonium formate solution, the ratio of mobile phase and the solvent. Furthermore, the specificity, linearity, precision, repeatability, stability and durability were studied. The known components like bacitracin A, B1/B2/B3, C1/C2/C3 and F were collected by preparative HPLC according to the HPLC method for bacitracin in USP 40. They were positioned in ammonium formate-acetonitrile HPLC system.RESULTS: The established HPLC method was verified to be suitable for analyzing the related substances and effective contents in bacitracin. Baseline separation between the known components and adjacent impurities was achieved, especially the B1/B2. The elution positions of the known component were clearly defined. The contents of the known components in different batches of bacitracin were compared. More detailed sample information was provided in this research.CONCLUSION: The HPLC method can simultaneously analyze the related substances and effective contents in bacitracin, and is suitable for the quality control of the product.

Enhanced production of bacitracin by knocking out of amino acid permease gene yhdG in Bacillus licheniformis DW2 / 生物工程学报

Bacitracin is a broad-spectrum polypeptide antibiotic, which is formed by 11 amino acids residues. Precursor amino acids supply might be the limit factor during bacitracin fermentation. First, our results demonstrated that increasing Ile and Leu supplies were regarded as the efficient strategies for the enhanced titer of bacitracin. Then, the amino acid permease YhdG, which was identified as the BCAA permease, was deleted and overexpressed in DW2, respectively. Our results showed that knocking out of permease YhdG could improve bacitracin production remarkablely. The bacitracin titer of the yhdG deficient strain DW2ΔyhdG reached 917.35 U/mL by flask fermentation, increased by 11% compared with that of DW2. In addition, the bacitracin titer was decreased by 25% in the YhdG overexpressed strain. Meanwhile, the intracellular concentrations of BCAA were higher than DW2 during the biosynthesis of bacitracin. The above results suggested that the permease YhdG might act as an exporter for branched chain amino acids in B. licheniformis DW2. Taken together, the increasing intracellular concentrations of branched chain amino acids by deleting amino acid permease YhdG could improve bacitracin titer. This study provided a new strategy for high-level production of bacitracin.

Analysis of bacitracin and its related substances by liquid chromatography tandem mass spectrometry / 药物分析学报

A suitable liquid chromatography quadrupole time-of-flight mass spectrometric (LC–Q-TOF–MS) method was developed for separation and characterization of related substances in bacitracin test drug. The separation was performed on LiChrospher RP-18 column using methanol as mobile phase A and 0.2% ammonium acetate buffer solution as mobile phase B in gradient elution. A total of 12 related substances were detected through high resolution mass spectrometric determination in a positive electrospray ionization mode. They were identified as co-existing active components and degradation products of bacitracin through the analysis and elucidation of both the protonated parents and the product ions of all the related substances and their fragmentation pathways were also proposed.

Bacitracin Inhibits the Migration of U87-MG Glioma Cells via Interferences of the Integrin Outside-in Signaling Pathway

OBJECTIVE: Protein disulfide isomerase (PDI) acts as a chaperone on the cell surface, and it has been reported that PDI is associated with the tumor cell migration and invasion. The aims of this study are to investigate the anti-migration effect of bacitracin, which is an inhibitor of PDI, and the associated factor in this process. METHODS: U87-MG glioma cells were treated with bacitracin in 1.25, 2.5, 3.75, and 5.0 mM concentrations. Western blot with caspase-3 was applied to evaluate the cytotoxicity of bacitracin. Adhesion, morphology, migration assays, and organotypic brain-slice culture were performed to evaluate the effect of bacitracin to the tumor cell. Western blot, PCR, and gelatin zymography were performed to investigate the associated factors. Thirty glioma tissues were collected following immunohistochemistry and Western blot. RESULTS: Bacitracin showed a cytotoxicity in 3rd (p<0.05) and 4th (p<0.001) days, in 5.0 Mm concentration. The cell adhesion significantly decreased and the cells became a round shape after treated with bacitracin. The migration ability, the expression of phosphorylated focal adhesion kinase (p-FAK) and matrix metalloproteinase-2 (MMP-2) decreased in a bacitracin dose- and time-dependent manner. The U87-MG cells exhibited low-invasiveness in the 2.5 mM, compared with the untreated in organotypic brain-slice culture. PDI was expressed in the tumor margin, and significantly increased with histological glioma grades (p<0.001). CONCLUSION: Bacitracin, as a functional inhibitor of PDI, decreased the phosphorylated FAK and the secreted MMP-2, which are the downstream of integrin and play a major role in cell migration and invasion, might become one of the feasible therapeutic strategies for glioblastoma.

Identification of Non-Aggregatibacter actinomycetemcomitans Bacteria Grown on the Tryptic soy-Serum-Bacitracin-Vancomycin Medium

The aim of this study was to identify the non-Aggregatibacter actinomycetemcomitans bacteria grown on the tryptic soy-serum-bacitracin-vancomycin (TSBV) medium, an A. actinomycetemcomitans selective medium. A total of 82 unidentified bacterial isolates from the oral cavities of a Korean population were kindly provide by the Korean Collection for Oral Microbiology. All the clinical isolates were grown on TSBV medium and bacterial DNA purified from each isolate was subjected to PCR with universal primers specific for bacterial 16S rRNA genes (16S rDNAs) sequence. The each bacterial 16S rDNA was amplified by PCR and the nucleotide sequences of it was determined by the dideoxynucleotide chain termination method. They were identified by 16S rDNA sequence comparison method at the specie-level. The data showed that Neisseria spp. (42 strains), Fusobacterium spp. (10 strains), Capnocytophaga spp. (8 strains), Propionibacterium acnes (5 strains), Aggregatibacter aprophilus (4 strains), Campylobacter spp. (5 strains), Veillonella dispar (3 strains), Streptococcus sp. (1 strain), Haemophilus parainfluenzae (1 strain), Leptotrichia wadei (1 strain), Morococcus sp./Neisseria sp. (1 strain), and Staphylococcus sp. (1 strain) were identified. These results could be used to develop a new A. actinomycetemcomitans-selective medium which is more effective than the TSBV medium in future studies.

Systematic mutagenesis method for enhanced production of bacitracin by Bacillus licheniformis mutant strain UV-MN-HN-6

The purpose of the current study was intended to obtain the enhanced production of bacitracin by Bacillus licheniformis through random mutagenesis and optimization of various parameters. Several isolates of Bacillus licheniformis were isolated from local habitat and isolate designated as GP-35 produced maximum bacitracin production (14±0.72 IU ml-1). Bacitracin production of Bacillus licheniformis GP-35 was increased to 23±0.69 IU ml-1 after treatment with ultraviolet (UV) radiations. Similarly, treatment of vegetative cells of GP-35 with chemicals like N-methyl N'-nitro N-nitroso guanidine (MNNG) and Nitrous acid (HNO2) increased the bacitracin production to a level of 31±1.35 IU ml-1 and 27±0.89 IU ml-1 respectively. Treatment of isolate GP-35 with combined effect of UV and chemical treatment yield significantly higher titers of bacitracin with maximum bacitracin production of 41.6±0.92 IU ml-1. Production of bacitracin was further enhanced (59.1±1.35 IU ml-1) by optimization of different parameters like phosphate sources, organic acids as well as temperature and pH. An increase of 4.22 fold in the production of bacitracin after mutagenesis and optimization of various parameters was achieved in comparison to wild type. Mutant strain was highly stable and produced consistent yield of bacitracin even after 15 generations. On the basis of kinetic variables, notably Yp/s (IU/g substrate), Yp/x (IU/g cells), Yx/s (g/g), Yp/s, mutant strain B. licheniformis UV-MN-HN-6 was found to be a hyperproducer of bacitracin.

Validación del método analítico para la cuantificación de bacitracina / Validation of the analytical method to quantify the bacitracine

Se desarrolló y validó un método analítico para la determinación cuantitativa de bacitracina zinc al 15 por ciento y bacitracina metilen disalicilato al 11 por ciento, por el método de cilindro en placa (difusión en agar), con el fin de ser usado en el control de calidad de las materias primas y productos farmacéuticos. Se evaluaron los parámetros de especificidad, selectividad, linealidad del sistema, y del método, exactitud, límite de cuantificación y precisión. Mediante el diseño experimental y la evaluación estadística de los resultados, se demostró que el método analítico es específico, selectivo, lineal, preciso (CV< 5 por ciento) y exacto (sesgo< 3 por ciento, Gexp< Gtab y t exp< t tab) en el intervalo de las concentraciones estudiadas. El límite de cuantificación y de detección fue de 0,02 y 0,005 UI/mL respectivamente. Las características de desempeño analítico cumplen con los requisitos para la aplicación analítica propuesta.

An analytical method was developed and validated for quantitative determination of 15 percent zinc bacitracine and 11 percent disalicylate methylene-bacitracine by the plate-cylinder method (agar diffusion) to be used in quality control of raw products and pharmaceutical products. Specificity, selectivity, system and method linearity, accuracy, quantification and precision parameters were assessed. By the experimental design and the statistic evaluation of results, it was demonstrated that the analytical method is specific, selective, linear, precise (CV< 5 percent) and exact (bias < 3 percent, Gexp< Gtab< t exp< t tab) during the study concentrations. The quantification and detection limit was of 0.02 and 0.005 Ul/mL, respectively. The analytical performance characteristics fulfill the requirement for the proposal analytical implementation.

Comparison of Dio-Bacit, Bacitracin-Trimethoprim/ Sulphamethoxazole and Latex Agglutination in the Diagnosis of Group A Beta-Hemolytic Streptococci

Not only is Group A beta-hemolytic Streptococcus (GAS) the most frequent cause of bacterial pharyngitis, it is also the culprit in various skin and systemic infections, acute rheumatic fever, post streptococcal glomerulonephritis, and other disorders and complications. A new, ready-to-use media, Dio-Bacit, in a two section plate containing 5% sheep blood agar on one side and sheep blood agar with bacitracin (2microgram/ml) on the other was compared for its efficiency in identifying GAS with bacitracin and bacitracin + sulphamethaxazole / trimethoprim disk tests applied after isolation of beta-hemolytic colonies. We also used the latex-agglutination test as the gold standard method for differentiating GAS from streptococci belonging to other groups. Compared with the latex-agglutination test, we found the sensitivity and specificity of the Dio-Bacit method to be 92.0% and 96.9%, respectively. Dio-Bacit plates provide an easy and very useful way to identify GAS within one day, saving time, labor, and money for routine diagnostic microbiology laboratories.

Bacitracin A: Structure-activity relationship.

The single imidazole nucleus of L-histidine residue in bacitracin-A seems to be important for the anti-bacterial activity of the molecule, since iodination, carboxymethylation and coupling of diazobenzene sulphonic acid to the histidine residue in the antibiotic caused 90-94% loss of antibacterial activity of the antibiotic. In contrast, the bacitracin sulphone and sulphoxide derivatives are as active as the parent antibiotic.

Chemical modification of bacitracin A and the biological activity of modified bacitracins.

The single side chain amino group of the D-ornithine residue in bacitracin seems to be important for the antibacterial activity of the molecule, since, acetylation, formylation, carbamylation and deamination of the antibiotic caused 90-92% loss of antibacterial activity. In contrast, nearly 80-91% of the antibacterial activity of the parent antibiotic was retained after the esterification, amide formation and acid-chloride formation of the a - and Υ –carboxyl groups of D-asparagine and D-glutamic acid residues of the antibiotic, respectively.

Antifungal activity of bacitracin and its interaction with metals.

Bacitracin was more growth-inhibitory to Neurospora crassa on a minimal magnesium medium than on a normal magnesium-medium. Both magnesium and manganese were able to counteract the growth inhibition. The antifungal activity of bacitracin was potentiated by zinc. Potassium could not counteract the growth inhibition by this antibiotic. The mycelial magnesium levels were low in bacitracin-inhibited cultures.