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].

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In broilers infected with Eimeria, dietary supplementation with 30 mg/kg of Bacitracin Zinc improved the feed conversion ratio from days 35 to 42 post-infection (i.e., the final week of the experiment).
Throughout the entire experimental period (days 1–42), the feed conversion ratio was improved in infected birds supplemented with either Bacitracin Zinc or hop beta-acids compared with the infected control group without supplementation.[2]
In uninfected broilers, dietary supplementation with Bacitracin Zinc increased body weight gain and feed intake during the final week of the experiment (days 35–42) as well as over the entire experimental period (days 1–42) compared with the negative control group.[2]
In infected birds, dietary supplementation with Bacitracin Zinc (30 mg/kg) reduced the relative abundance of Clostridiales in the small intestinal contents at 7 days post-infection (21 days of age).[2]
At 21 days post-infection (35 days of age), dietary Bacitracin Zinc showed a trend toward increasing the relative abundance of Clostridiales in the small intestine and reduced the relative abundance of Lactobacillales.[2]
At 21 days post-infection (35 days of age), dietary Bacitracin Zinc decreased the relative abundance of Staphylococcus and Streptococcus in the small intestine of infected broilers.[2]
At 21 days post-infection (35 days of age), the number of Campylobacter (although detected at low levels) in the cecum of infected broilers supplemented with Bacitracin Zinc was numerically lower than that in the infected control group without supplementation.[2]

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]

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The experiment used 1440 one-day-old male Ross 308 broilers, randomly assigned to 36 pens (40 birds per pen, 6 replicates per treatment). The experimental design included a negative control group (basal diet), a positive control group (basal diet + 30 mg/kg Bacitracin Zinc), an infected control group (basal diet + coccidial challenge), and an infected supplemented group (basal diet + 30 mg/kg Bacitracin Zinc + coccidial challenge). Bacitracin Zinc was added by replacing the inert material (kaolin) in the basal diet. Birds had free access to pelleted feed and water. On day 14 of age, except for the negative and positive control groups, all birds were inoculated via oral gavage with a mixed suspension of live sporulated oocysts of Eimeria acervulina and Eimeria tenella (2×10^5 and 5×10^4 oocysts per bird, respectively). Performance indicators (body weight gain, feed intake, feed conversion ratio, survival rate) were measured weekly on a per-pen basis. On days 21 and 35 of age, three birds per pen close to the average body weight were selected, euthanized without fasting by cervical dislocation, and gastrointestinal tract contents (small intestine and cecum) were collected for microbiota analysis. [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 cyclic polypeptide antibiotic complex whose main component is bacitracin A. It is produced by spore-forming bacteria of the Bacillus subtilis genus and possesses antibacterial activity. Bacitracin zinc binds to C55-isoprene pyrophosphate, a bisphospholipid transport molecule responsible for transporting building blocks of peptidoglycan from the bacterial cell wall. This interferes with the enzymatic dephosphorylation of C55-isoprene pyrophosphate, preventing peptidoglycan synthesis and ultimately leading to cell lysis. This study aimed to evaluate the effects of dietary supplementation with hop β-acid on the production performance and gut microbiota of broilers infected with Eimeria acervulina and Eimeria tenella. A randomized design was used, with 1440 broiler chicks randomly assigned to 6 treatment groups, with 6 replicates per treatment group and 40 chicks per replicate. The treatment groups included: a basal diet group (negative control group, NC); a basal diet supplemented with 30 mg/kg bacitracin zinc group (positive control group, PC); NC + challenge group; PC + challenge group; NC + 30 mg/kg hop β-acid + challenge group; and NC + 240 mg/kg hop β-acid + challenge group. At 14 days of age, chicks were challenged with Eimeria acervulina and E. tenella. After the experiment, the growth performance of the chicks was assessed and the gut microbiota was analyzed. Results showed that in the first week after challenge, coccidiosis infection significantly reduced weight gain, feed intake, and feed conversion ratio in chicks (P = 0.001). These effects weakened in the second week after challenge. In the last two weeks of the experiment, the feed conversion ratio of the challenged groups was significantly higher than that of the unchallenged groups (P = 0.05). Throughout the experiment, the weight gain and feed intake of chickens in the challenged group (PC) were higher than those in the unchallenged group (NC). Within the challenged group, chickens supplemented with bacitracin zinc and hop β-acid had higher feed conversion ratios. In the challenged group, the number of Clostridium species in the small intestine was significantly lower at 21 days of age compared to chickens without supplementation (P = 0.009). Furthermore, challenge also reduced the number of Staphylococcus (P = 0.01) and Enterococcus (P = 0.001); however, both supplements tended to increase the number of Enterococcus (P = 0.09). At 35 days of age, both levels of β-acid increased the number of Clostridium species in the small intestine of challenged chickens (P = 0.04) and decreased the number of Lactobacillus species (P = 0.008). At this age, challenge treatment tended to increase the number of Bacteroides in the cecum of broilers (P = 0.07), while high levels of hop β-acid tended to decrease the number of Bacteroides compared with low levels of hop β-acid (P = 0.09). In summary, the positive effects of hop β-acid on production performance are similar to those of bacitracin zinc supplementation, which can alter the gut microbiota of infected poultry and may replace antimicrobial performance enhancers in broiler diets. [1]

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This study aimed to evaluate the effects of monensin combined with virginiamycin or bacitracin zinc on the production performance, serum D-lactic acid levels, feeding behavior, carcass traits, and gluconeogenesis and adipogenesis gene expression in young bulls in a fattening farm. This study used a completely randomized design and selected 72 Nellol × Angus (F1) young bulls (weight 368±16.3 kg). The animals were stratified according to weight and assigned to 18 pens with 4 animals in each pen. Six pens (each treatment group) were randomly assigned to the following treatment groups: monensin (MON), monensin + bacitracin zinc (MONZB), and monensin + virginiamycin (MONVM). The addition levels of monensin, bacitracin zinc, and virginiamycin were 18.9, 6.62, and 18.9 mg/kg dry matter (DM), respectively. The diet consisted of 85% corn concentrate and 15% corn silage. The fattening period was 109 days, with the first 27 days being the dietary adaptation period and the following 82 days being the fattening period. The additives had no significant effect on body weight (BW), daily gain (ADG), dry matter intake (DMI), or feed conversion ratio (P ≥ 0.13). Compared with the MON and MONZB groups, the MONVM group had significantly lower intramuscular fat content (P = 0.03). Compared with the MON and MONZB groups, the MONVM group had higher serum D-lactic acid concentrations (P = 0.05). In the liver, compared with the MON and MONZB groups, the expression of pyruvate carboxylase (PC) and lactate dehydrogenase A (LDHA) was upregulated in the MONVM group (P < 0.03). Compared with the MONZB group, the expression of peroxisome proliferator-activated receptor γ (PPARG) in the liver of the MONVM group tended to be reduced (P = 0.07). Compared with animals fed MON, the expression of acetyl-CoA carboxylase α (ACACA) was downregulated in animals fed MONVM (P = 0.03). In summary, these additives affected growth performance at different stages of the fattening process. Monensin combined with virginiamycin reduced lipogenesis and increased lipid oxidation, and reduced intramuscular fat deposition compared with monensin alone or in combination 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.)