Bacterial cell wall synthesis; undecaprenyl pyrophosphate

In combination with colistin, bacitracin (64 μg/mL, 24 h) demonstrated antibacterial action against Staphylococcus aureus BA01611 [3]. Cell borders become hazy as bacitracin (64 μg/mL, 1 or 2 h) breaks down the cell surface and creates clusters of grape-like cells [3].

Zinc bacitracin improved performance and modified the intestinal microbiota of non-challenged chickens. Zinc bacitracin improved performance and modified the intestinal microbiota of Eimeria-challenged chickens. Zinc bacitracin and hops β-acids had similar positive effect on the feed efficiency of challenged broilers and changed the microbial profile [1].

The Time-Kill Assay[3]
The time-killing curve assays were performed in triplicate to study the effect of the combination of bacitracin and colistin on S. aureus BA01611 growth as previously described by Mun et al. (2013) with minor modifications (Mun et al., 2013). A single bacterial colony was added to 2 mL of the MHB and grown overnight at 37°C with shaking at 180 rpm. The overnight culture was diluted with pre-warmed MHB to obtain a starting inoculum of approximately 5 × 105 CFU/mL. The S. aureus BA01611 strain was exposed to colistin at the concentrations of 0 or 1/2 MIC (64 μg/mL) in the presence or absence of 1/2 MIC (64 μg/mL, except 8 μg/mL for S. aureus BA01511) bacitracin. Samples were taken at 0, 2, 4, 6, 8, 16, and 24 h, serially diluted, spread on drug-free plates, and incubated at 37°C for 24 h before counting the colonies. Each experiment was repeated three times.

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Scanning Electron Microscope (SEM)[3]
SEM were performed as described previously (You et al., 2013). S. aureus BA01611 cells were treated with 1/2 MIC (64 μg/mL) colistin and/or 1/2 MIC (64 μg/mL, except 8 μg/mL for S. aureus BA01511) bacitracin for 1 h and 2 h. Untreated controls were also prepared. The bacterial cells were collected via centrifugation at 10,000 × g and then the pellet formed was washed with PBS for three times. Fixation was done by suspending the bacterial cells into 0.25% of glutaraldehyde solution (in PBS, pH 7.0) and then incubated at room temperature for 1 h before collecting the fixed bacterial pellet. Dehydration of the bacterial cells was done by washing the pellets with ethanol at different concentrations up to 100%. After the critical-point drying, the bacterial cells were observed with a field-emission scanning electron microscopy (FE-SEM; FEI Inspect F50).

Susceptibility Screen[3]
The susceptibility screen assay was performed as described previously (Haaber et al., 2015). S. aureus strains were grown overnight and the cultures were adjusted to subsamples of 5 × 105 CFU/mL in the warm MH broth. Colistin sodium sulfate was added as an inducer at the concentrations of 1/2 MIC (64 μg/mL) for each strain. After 90 min at 37°C with shaking at 180 rpm, 10 μL aliquots of the cultures were spotted on MH agar plates containing bacitracin at a concentration of 1/2 MIC (64 μg/mL). The plates were incubated overnight at 37°C before checking the bacterial growth. Each experiment was repeated three times.

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Determination of the in vitro Effects of Combinations of bacitracin and Colistin[3]
The antimicrobial combination assays were conducted with bacitracin plus colistin by using the broth microdilution checkerboard technique (Mataraci and Dosler, 2012). The test was performed using 96-well microtiter plates containing colistin and bacitracin in twofold serial concentrations. Bacterial suspensions were prepared to yield a final inocula of ∼5 × 105 CFU/mL. Plates were read after overnight incubation at 37°C. Fractional Inhibitory Concentration (FIC) Index was calculated according to the formulas: FICbacitracin = MICbacitracin+colistin/MICbacitracin, FICcolistin = MICbacitracin+colistin/ MICcolistin, FIC Index = FICbacitracin+ FICcolistin. FIC Index values were interpreted according to Mun et al. (2013): synergy (FIC Index ≤ 0.5); partial synergy (FIC Index > 0.5 to ≤ 0.75); additivity (FIC Index > 0.75 to ≤ 1); no interaction (indifference) (FIC Index > 1 to ≤ 4) and antagonism (FIC Index > 4.0) (Mun et al., 2013). Each experiment was repeated three times.

The experimental treatments were: basal diet, unsupplemented negative control (NC); basal diet supplemented with 30 mg/kg of Bacitracin Zinc, positive control (PC); NC + challenge; PC + challenge; NC + 30 mg/kg of hop β-acids + challenge and NC + 240 mg/kg of hop β-acids + challenge. Hops β-acids were added in microencapsulated form. The microencapsulated product used was prepared to contain 30% of β-acids. Both additives, β-acids and antimicrobial, were introduced by replacing an inert substance (kaolin) in the basal diet. The diets were not supplemented with any anticoccidial agent.[1]

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Seventy-two F1 Nellore × Angus young bulls (368 ± 16.3 kg), were stratified by body weight (BW) and allotted into 18 pens (4 animals per pen). The trial was carried out as a completely randomized design with three treatments: monensin (MON), monensin + Bacitracin Zinc (MONZB) and monensin + virginiamycin (MONVM), resulting in 6 replicates per treatment, assuming pen as the experimental unit. The doses of monensin, Bacitracin Zinc and virginiamycin were 18.9, 6.62 and 18.9 mg/kg dry matter (DM) of the diet, respectively, and they are in the margin of industry recommendations (i.e. monensin is 10–30 mg/kg DM). According to Tedeschi and Gorocica-Buenfil (2018), virginiamycin would be effective when fed around 12–24 mg/kg DM. As our diet did not present high lipid content, the additives inclusion should be around 20 mg/kg DM for monensin and virginiamycin and around 7 mg/kg DM for bacitracin. We decided to use a lower dose recommended by the industry due to the combined use of the additives. These concentrations are also regularly used in commercial feedlots in Brazil (Pinto and Millen, 2018, Silvestre and Millen, 2021). Since there is no recent recommendation due to the lack of research regarding bacitracin utilization, the bacitracin dose was recommended by the company (Agroceres Multimix) that commercialize the molecule in Brazil and supported part of the present study.[2]

[1]. Hops β-acids and zinc bacitracin affect the performance and intestinal microbiota of broilers challenged with Eimeria acervulina and Eimeria tenella. Animal Feed Science and Technology, 2015, 207: 181-189.

[2]. Effect of the combined use of monensin with virginiamycin or bacitracin on beef cattle performance, liver gluconeogenesis, lipid metabolism and intramuscular fat content. Animal Feed Science and Technology, 2023, 304: 115735.

[3]. Colistin Induces S. aureus Susceptibility to Bacitracin. Front Microbiol. 2018 Nov 20;9:2805.

Bacitracin Zinc is the zinc salt form of bacitracin, a complex of cyclic polypeptide antibiotics, mainly bacitracin A, produced by spore-forming organisms belonging to the licheniformin group of the Bacillus subtilis with antibacterial activity. Bacitracin zinc binds to C55-isoprenyl pyrophosphate, a biphosphate lipid transport molecule that carries the building blocks of the peptidoglycan bacterial cell wall, thereby interfering with the enzymatic dephosphorylation of the C55-isoprenyl pyrophosphate and preventing peptidoglycan synthesis which leads to cell lysis.

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The objective of this study was to evaluate the effects of dietary hops β-acids on the productive performance and intestinal microbiota of broilers challenged with Eimeria acervulina and Eimeria tenella. 1440 broiler chicks were allocated in a randomized design with 6 treatments and 6 replicates of 40 birds/pen. The treatments were: basal diet, negative control (NC); basal diet supplemented with 30 mg/kg of zinc bacitracin, positive control (PC); NC + challenge; PC + challenge; NC + 30 mg/kg of hop β-acids + challenge; and NC + 240 mg/kg of hop β-acids + challenge. At 14 days of age, the birds were challenged with Eimeria acervulina and E. tenella. The performance was evaluated and the analysis of the intestinal microbiota was performed. The coccidiosis infection impaired (P = 0.001) the weight gain, feed intake and the feed conversion ratio in the first week after the challenge. In the second week after the challenge, the effects were less evident. The feed conversion ratio was better (P = 0.05) in the challenged birds, compared to the non-challenged, in the last two weeks of the experiment. Considering the overall experimental period, the weight gain and feed intake were higher in the PC compared to the NC, and among the challenged birds, the feed conversion ratio was better in those receiving zinc bacitracin and hops β-acids in the diet. Among the challenged birds, the zinc bacitracin and both levels of hops β-acids decreased (P = 0.009) Clostridiales amount when compared to the unsupplemented birds, in the small intestine at 21 days of age. In addition, the challenge decreased the Staphylococcus (P = 0.01) and Enterococcus (P = 0.001) amounts; however, both additives tended to increase Enterococcus amount (P = 0.09). At 35 days of age, the both levels of β-acids increased Clostridiales (P = 0.04) and decreased Lactobacillus (P = 0.008) in the small intestine, among the challenged birds. At this age, the challenge tended to increase (P = 0.07) Bacteroides amount in the ceca of broilers, and the higher level of hops β-acids tended to decrease (P = 0.09) its amount when compared to the lower level. In conclusion, the hops β-acids showed similar positive effects to those obtained with zinc bacitracin supplementation on the productive performance changed the intestinal microbiota of challenged birds and have potential to replace the antimicrobial performance enhancers in the diets of broilers.[1]

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The objective was to evaluate the combined use of monensin with virginiamycin or zinc bacitracin on performance, serum D-lactate, feeding behavior, carcass traits, and the expression of gluconeogenic and lipogenic genes over time in feedlot young bulls. Nellore × Angus (F1) young bulls (n = 72; BW 368 ± 16.3 kg) were used in a completely randomized design. Animals were stratified by BW and allotted into 18 pens with 4 animals each. Pens (6 pens per treatment) were randomly assigned to the following treatments: monensin (MON), monensin + zinc bacitracin (MONZB) and monensin + virginiamycin (MONVM). The doses of monensin, zinc bacitracin and virginiamycin were 18.9, 6.62 and 18.9 mg/kg DM of the diet, respectively. The diet consisted of 85% corn-based concentrate and 15% corn silage. The finishing phase was 109 days with the first 27 days for diet adaptation and the remaining 82 days for the finishing diet. Treatment additives did not affect body weight (BW), overall average daily gain (ADG), dry matter intake (DMI) or gain:feed (P ≥ 0.13). Animals fed MONVM had lesser (P = 0.03) intramuscular fat than those fed MON and MONZB. Serum D-lactate concentration was greater (P = 0.05) in MONVM animals than in MON and MONZB animals. In the liver, MONVM animals upregulated (P < 0.03) Pyruvate carboxylase (PC) and Lactate dehydrogenase A (LDHA) expression compared with MON and MONZB. The expression of Peroxisome proliferator-activated receptor gamma (PPARG) in LT tended to be decreased (P = 0.07) in MONVM animals than in MONZB. Animals fed MONVM downregulated (P = 0.03) Acetyl-CoA carboxylase alpha (ACACA) expression compared with those fed MON. In conclusion, these additives affect growth performance in different times during feedlot phase. The combined use of monensin and virginiamycin reduce lipogenesis and increase lipid oxidation, triggering lesser IMF deposition compared to monensin alone or combined with bacitracin.[2]

C66H103N17O16SZN

1488.101

1485.677

C, 53.34; H, 6.85; N, 16.02; O, 17.23; S, 2.16; Zn, 4.40

1405-89-6

White to off-white solid powder

250 ºC (dec.)

DFCFJNHVZXJGQP-UHFFFAOYSA-L

InChI=1S/C66H103N17O16S.Zn/c1-9-35(6)52(69)66-81-48(32-100-66)63(97)76-43(26-34(4)5)59(93)74-42(22-23-50(85)86)58(92)83-53(36(7)10-2)64(98)75-40-20-15-16-25-71-55(89)46(29-49(68)84)78-62(96)47(30-51(87)88)79-61(95)45(28-39-31-70-33-72-39)77-60(94)44(

4-[[2-[[2-(1-amino-2-methylbutyl)-4,5-dihydro-1,3-thiazole-4-carbonyl]amino]-4-methylpentanoyl]amino]-5-[[1-[[3-(2-amino-2-oxoethyl)-18-(3-aminopropyl)-12-benzyl-15-butan-2-yl-6-(carboxymethyl)-9-(4H-imidazol-4-ylmethyl)-2,5,8,11,14,17,20-heptaoxo-1,4,7,10,13,16,19-heptazacyclopentacos-21-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-5-oxopentanoic acid;zinc

Bacifarmin; Bacifarmin (zinc)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

1M HCl : 50 mg/mL (~33.65 mM)
H2O : ~1 mg/mL (~0.67 mM)
DMSO :< 1 mg/mL

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)