黑大蒜提取物联合抗生素对金黄色葡萄球菌和肠埃希菌的体外抗菌作用研究

评价黑大蒜提取物分别与头孢唑林或庆大霉素联合应用,对金黄色葡萄球菌和大肠埃希菌的体外抗菌效应。采用液体稀释法分别测定黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的最低抑菌浓度（MIC）。采用棋盘法设计,微量肉汤稀释法测定黑大蒜提取物联合头孢唑林或庆大霉素对金黄色葡萄球菌和大肠埃希菌的MIC,并计算部分抑菌浓度（FIC指数）。测定黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的时间-杀菌曲线。黑大蒜提取物对金黄色葡萄球菌的MIC为256μg/mL,黑大蒜提取物对大肠埃希菌的MIC为256μg/mL。时间-杀菌曲线结果显示黑大蒜提取物对金黄色葡萄球菌和大肠埃希菌的抑菌作用呈现较强的浓度依赖性。黑大蒜提取物联合头孢唑林后对金黄色葡萄球菌的FIC指数为0.75;黑大蒜提取物联合庆大霉素后对大肠埃希菌的FIC指数为0.5。黑大蒜提取物与头孢唑林或庆大霉素联合用药,可明显降低抗生素对金黄色葡萄球菌和大肠埃希菌的MIC,表现为相加和协同效应。     

几种中药单体和抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性研究

考察没食子酸等6种中药单体和诺氟沙星等3种抗生素对嗜水气单胞菌及温和气单胞菌的体外抑菌活性及其联合抑菌作用。通过琼脂扩散法测定受试物的抑菌作用,用微量二倍稀释法测定受试物最小抑菌浓度（MIC）和最小杀菌浓度（MBC）,用棋盘法考察受试物的联合抑菌作用。6种中药单体中没食子酸和槲皮素对嗜水气单胞菌及温和气单胞菌抑制作用较强,抑菌圈均达到20 mm以上,没食子酸对嗜水气单胞菌及温和气单胞菌的MIC和MBC均为250 g/mL;槲皮素对嗜水气单胞菌的MIC和MBC均为500 g/mL,对温和气单胞菌的MIC和MBC分别为250和500 g/mL;而大黄素甲醚等4种中药单体无明显的抑菌作用。嗜水气单胞菌及温和气单胞菌对诺氟沙星、恩诺沙星、氟苯尼考等抗生素均极敏感。在联合药敏试验中,没食子酸与恩诺沙星或氟苯尼考、槲皮素与氟苯尼考联合用药对嗜水气单胞菌具有相加抑菌作用;没食子酸与恩诺沙星联合用药对温和气单胞菌具有协同抑菌作用,没食子酸与诺氟沙星或氟苯尼考、槲皮素与氟苯尼考联合用药对温和气单胞菌具有相加抑菌作用。没食子酸和槲皮素对嗜水气单胞菌及温和气单胞菌具有显著的抑制作用,二者与恩诺沙星、诺氟沙星、氟苯尼考等抗生素联合应用具有相加或协同作用,有助于降低抗生素的用量及残留。       

Synthesis and anti Methicillin resistant Staphylococcus aureus activity of substituted chalcones alone and in combination with non-beta-lactam antibiotics

A total of 30 chalcone analogues was synthesized via a base catalyzed Claisen Schmidt condensation and screened for their in vitro antibacterial activity against Methicillin-sensitive Staphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) alone or in combination with non beta-lactam antibiotics namely ciprofloxacin, chloramphenicol, erythromycin, vancomycin, doxycycline and gentamicin. In the checkerboard technique, fractional inhibitory concentration indices (FICI) show that the following combinations like ciprofloxacin with 25 (4'-bromo-2-hydroxychalcone); doxycycline with 21 (4-hydroxychalcone); doxycycline with 25; and doxycycline with 4 (2',2-dihydroxychalcone) were synergistic against MRSA. In term SAR study, the relationship between chalcone structure and their antibacterial activity against S. aureus and synergy with tested antibiotics were discussed. Possible mechanisms for antibacterial activity of chalcones alone as well as the synergistic effect in combinations were proposed by molecular modeling studies, respectively. Combinations of chalcones with conventional antibiotics could be an effective alternative in the treatment of infection caused by MRSA.     

Antibacterial potential of Malaysian ethnomedicinal plants against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA)

This study aimed to evaluate the antibacterial activities of 61 plant extracts from 49 Malaysian ethnomedicinal plants and to investigate the interaction of the active plant extracts in combination with synthetic antibiotics against the MSSA and MRSA strains. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the plant extracts were determined using a microdilution method against MSSA and MRSA strains. The interaction between active plant extracts and the antibiotics was assessed using the checkerboard method. The total fractional inhibitory concentration (∑FIC) indices from the combination were calculated to determine the nature of the interaction. Out of the 61 plant extracts tested against the MSSA strain, 7 plant extracts (̴ 11%) showed MIC values of less than 200 μg/mL, 17 extracts (̴ 28%) showed MIC between 200 and 800 µg/mL and seed extracts of Areca catechu showed MBC values of 400 μg/mL. The seed extract of A. catechu showed MIC and MBC of 400 μg/mL against the MRSA strains while leaf extract of Cocos nucifera showed MIC of 400 μg/mL against MRSA NCTC 12493. When the active plant extracts (MIC ≤ 200 µg/mL for MSSA, and ≤ 400 µg/mL for MRSA) were tested in combination with vancomycin and ciprofloxacin, they showed no interaction against both MSSA and MRSA with ∑FIC between 1.06 and 2.03. These findings provide a preliminary overview of the anti-MSSA and anti-MRSA properties of Malaysian ethnobotanical plants to combat Staphylococcal infections. Further research is needed to establish an antibacterial profile of the tested plant extracts.     

A combination effect of epigallocatechin gallate, a major compound of green tea catechins, with antibiotics on Helicobacter pylori growth in vitro

Since green tea catechins are known to have antimicrobial activity against a variety of microorganisms, their possible effects on Helicobacter pylori in combination with antibiotics were examined. Fifty-six clinical isolates of H. pylori, including 19 isolates highly resistant to metronidazole (MTZ) and/or clarithromycin (CLR), were used to determine in vitro sensitivity to tea catechins. The MIC90 of both epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) was 100 microg/ml. However, other tea catechins tested did not show any anti-H. pylori activity. Highly antibiotic-resistant clinical isolates showed a similar sensitivity to both EGCg and ECg. The kinetic study of antibacterial activity in liquid cultures revealed a relatively slow but strong activity on the growth of H. pylori. In combination with sub-MIC of amoxicillin (AMX), the antibacterial activity of AMX was significantly enhanced by the presence of EGCg. To estimate the general combination effect between EGCg and other antibiotics, such as MTZ and CLR, on the antibacterial activity against clinical isolates, the fraction inhibitory concentration (FIC) was determined by checkerboard study. The FIC indexes showed additive effects between EGCg and antibiotics tested. These results indicatethat EGCg may be a valuable therapeutic agent against H. pylori infection.     

Synergistic effect between dieckol from <Emphasis Type="Italic">Ecklonia stolonifera</Emphasis> and β-lactams against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We have been attempting for some time to discover a compound evidencing antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The dieckol isolated from Ecklonia stolonifera has been shown to exhibit antibacterial activity against methicillin-susceptible S. aureus (MSSA) and MRSA. The minimum inhibitory concentrations (MICs) of dieckol were determined in a range of 32 to 64 μg/mL against standard MSSA and MRSA strains. Furthermore, dieckol clearly reversed the high-level ampicillin and penicillin resistance of MRSA. The MICs of ampicillin against two standard strains of MRSA were dramatically reduced from 512 to 0.5 μg/mL in combination with 1/4 MIC of dieckol (16 μg/mL). The fractional inhibitory concentration (FIC) indices of ampicillin and penicillin were measured from 0.066 to 0.266 in combination with 8 or 16 μg/mL of dieckol against all tested MRSA strains, thereby suggesting that dieckol-ampicillin or dieckol-penicillin combinations exert a synergistic effect against MRSA. The results of this study indicate that dieckol, administered in combination with β-lactams, may prove effective in the treatment of MRSA infections. Our finding may also contribute to the development of an alternative phytotherapeutic anti-MRSA agent.     

A Combination Effect of Epigallocatechin Gallate,a Major Compound of Green Tea Catechins,with Antibiotics on Helicobacter pylori Growth In Vitro

Since green tea catechins are known to have antimicrobial activity against a variety of microorganisms, their possible effects on Helicobacter pylori in combination with antibiotics were examined. Fifty-six clinical isolates of H. pylori, including 19 isolates highly resistant to metronidazole (MTZ) and/or clarithromycin (CLR), were used to determine in vitro sensitivity to tea catechins. The MIC₉₀ of both epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) was 100 Î&frac;g/ml. However, other tea catechins tested did not show any anti-H. pylori activity. Highly antibiotic-resistant clinical isolates showed a similar sensitivity to both EGCg and ECg. The kinetic study of antibacterial activity in liquid cultures revealed a relatively slow but strong activity on the growth of H. pylori. In combination with sub-MIC of amoxicillin (AMX), the antibacterial activity of AMX was significantly enhanced by the presence of EGCg. To estimate the general combination effect between EGCg and other antibiotics, such as MTZ and CLR, on the antibacterial activity against clinical isolates, the fraction inhibitory concentration (FIC) was determined by checkerboard study. The FIC indexes showed additive effects between EGCg and antibiotics tested. These results indicate that EGCg may be a valuable therapeutic agent against H. pylori infection.Received: 2 September 2002 / Accepted: 12 November 2002     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Expression of 4kD scorpion defensin and its in vitro synergistic activity with conventional antibiotics

The 4kD scorpion defensin (SD) is a potent disulfide-linked peptide. In this study, we expressed it in methylotrophic yeast Pichia pastoris and purified it using Ni–NTA His Bind Resin. We investigated its in vitro antibacterial activity and effect in combination with several conventional antibiotics. We first examined its antibacterial activity towards several Gram-positive and Gram-negative bacteria. Then we used the broth microdilution method to test drugs alone and in combination and used the fractional inhibitory concentration (FIC index) to classify the drug interactions. Our study showed the expressed SD peptide has antibacterial activity against Salmonella typhimurium, E. coli and S. aureus etc. Synergy or additive interaction was observed between SD and Norfloxacin, Polymyxin B and Ampicillin. Cell growth tests showed that combination of SD and Norfloxacin can improve their activity against bacteria. This result maybe permit lower using of the conventional antibiotic agents more effectively and safely.     

Susceptibility to antibiotics of Ureaplasma urealiticum strains isolated at the primorye region]

Susceptibility to antibiotics of Ureaplasma urealyticum strains isolated in Primorye Region and its changes were evaluated. Among the sexually transmited diseases urogenital ureaplasmosis has the leading position in combination with gonorrhoea, trichomoniasis and infections caused by opportunistic pathogens. The spectrum of investigated antibacterial agents included gentamycin, clarithromycin, roxythromycin, azithromycin, doxycycline, three fluoroquinolones of II generation and for cephalosporins of the I and III generations. The most potent activity against U. urealyticum was demonstrated for pefloxacin (67 per cent of susceptible strains), ofloxacin (63 per cent) and roxythmycin (54 per cent). It is concluded that at Primorye Region the drugs of first choice for the urogenital infections therapy are fluoroquinolones.     

Characterization of antimicrobial resistance among Escherichia coli isolates from chickens in China between 2001 and 2006

Escherichia coli is a common commensal bacterium and is regarded as a good indicator organism for antimicrobial resistance for a wide range of bacteria in the community and on farms. Antimicrobial resistance of E. coli isolated from chickens from 49 farms in China between 2001 and 2006 was studied. A total of 536 E. coli isolates were collected, and minimal inhibitory concentrations (MICs) of eight antimicrobials were determined by the broth microdilution method. Isolates exhibited high levels of resistance to ampicillin (80.2%), doxycycline (75.0%) and enrofloxacin (67.5%). Relatively lower resistance rates to cephalothin (32.8%), cefazolin (17.0%) and amikacin (6.5%) were observed. Strains were comparatively susceptible to colistin (MIC(50)=1 mug mL(-1)). A marked increase in isolates with elevated MICs for florfenicol was observed over the study period. Therefore, five resistance genes leading to the dissemination of phenicol resistance in the isolates (n=113) with florfenicol MICs>/=32 mug mL(-1) were analyzed. The gene floR was the most prevalent resistance gene and was detected in 92% of the 113 isolates, followed by the cmlA (53%), catA1 (23%) and catA2 (10%) genes. catA3 was not detected in these isolates. Eight isolates with florfenicol MICs=32 mug mL(-1) and one with MIC=64 mug mL(-1) were negative for the floR gene.     

Interaction of aminoglycosides and the other antibiotic on selected bacterial strains

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.     

Synthesis, antibacterial activities and molecular docking studies of Schiff bases derived from N-(2/4-benzaldehyde-amino) phenyl-N'-phenyl-thiourea

A series of novel Schiff base derivatives have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of FabH. These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Compounds with potent antibacterial activities were tested for their E. coli FabH inhibitory activity. Compound 3v showed the most potent antibacterial activity with MIC of 1.56-6.25 μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC(50) of 4.3 μM. Docking simulation was performed to position compound 3v into the E. coli FabH active site to determine the probable binding conformation.     

Activity and synergistic interactions of stilbenes and antibiotic combinations against bacteria in vitro

The synergistic antibacterial activity of two stilbenes [3,4??,5-trihydroxystilbene (1) and 3,5-dihydroxy-4-isopropylstilbene(2)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) in combination with ciprofloxacin and cefotaxime was investigated. The activity of the stilbenes and standard antibiotics on bacteria were determined using the macrodilution method. The minimum inhibitory concentration and minimum bactericidal concentration of the stilbenes was compared with that of the standard antibiotics. The antibacterial activities of stilbenes against pathogenic bacteria were assessed using the checkerboard and time-kill methods to evaluate the synergistic effects of stilbenes in treatment with ciprofloxacin or cefotaxime. The results of the present study showed that the combination effects of both stilbenes with ciprofloxacin were synergistic (FIC index <0.5). Whereas stilbene 2 with cefotaxime recorded additive. Furthermore, time-kill study showed that the growth of the tested bacteria was completely attenuated with 12?C24?h of treatment with 50:50 ratios of stilbenes and antibiotics. These results suggest that stilbenes combined with antibiotics may be microbiologically beneficial and not antagonistic. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and stilbenes). The in vitro synergistic activity of stilbenes and antibiotics against bacteria is reported here for the first time.     

Synthesis, molecular modeling and biological evaluation of β-ketoacyl-acyl carrier protein synthase III (FabH) as novel antibacterial agents

A series of novel cinnamic acid secnidazole ester derivatives have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of FabH. These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus. Compounds with potent antibacterial activities were tested for their E. coli FabH inhibitory activity. Compound 3n showed the most potent antibacterial activity with MIC of 1.56-6.25 μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC?? of 2.5 μM. Docking simulation was performed to position compound 3n into the E. coli FabH active site to determine the probable binding conformation.     

In-Vitro Activity of Doxycycline and β-Lactam Combinations Against Different Strains of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis>

Although doxycycline is active against Burkholderia pseudomallei and has been used in the eradication stage of melioidosis therapy, it is not regularly used during the initial intensive phase. In order to assess its potential use in intensive phase therapy, we investigated in vitro pharmacodynamic activity of doxycycline and β-lactams alone and in combination against four Malaysian strains of B. pseudomallei. Using a checkerboard assay, the combinations of doxycycline and imipenem, doxycycline and ceftazidime, and doxycycline and amoxicillin–clavulanate tested against four strains showed indifferent effects with summation fractional inhibitory concentration values ranging from 0.62 to 2.12. Time-kill experiments also indicated that the combinations of doxycycline/β-lactam antibiotics against four tested strains did not fulfil synergy criteria, in which all combinations showed indifferent effects with ? 1.36 to 1.26-log CFU/mL compared to the most active monotherapy regimen in each combination. No re-growth of bacteria was detected after the early killing in doxycycline/β-lactam combination regimens compared to β-lactam monotherapy regimens, in which 9 out of 10 were associated with re-growth of bacteria. As no synergistic activity was observed, this in vitro study showed that doxycycline offers no additional benefit to be used in combination with β-lactams in the intensive phase of therapy.     

In Vitro Activity of Sodium New Houttuyfonate Alone and in Combination with Oxacillin or Netilmicin against Methicillin-Resistant Staphylococcus aureus

Background

Staphylococcus aureus can cause severe infections, including bacteremia and sepsis. The spread of methicillin-resistant Staphylococcus aureus (MRSA) highlights the need for novel treatment options. Sodium new houttuyfonate (SNH) is an analogue of houttuynin, the main antibacterial ingredient of Houttuynia cordata Thunb. The aim of this study was to evaluate in vitro activity of SNH and its potential for synergy with antibiotics against hospital-associated MRSA.

Methodology

A total of 103 MRSA clinical isolates recovered in two hospitals in Beijing were evaluated for susceptibility to SNH, oxacillin, cephalothin, meropenem, vancomycin, levofloxacin, minocycline, netilmicin, and trimethoprim/sulfamethoxazole by broth microdilution. Ten isolates were evaluated for potential for synergy between SNH and the antibiotics above by checkerboard assay. Time-kill analysis was performed in three isolates to characterize the kill kinetics of SNH alone and in combination with the antibiotics that engendered synergy in checkerboard assays. Besides, two reference strains were included in all assays.

Principal Findings

SNH inhibited all test strains with minimum inhibitory concentrations (MICs) ranging from 16 to 64 µg/mL in susceptibility tests, and displayed inhibition to bacterial growth in concentration-dependent manner in time-kill analysis. In synergy studies, the combinations of SNH-oxacillin, SNH-cephalothin, SNH-meropenem and SNH-netilmicin showed synergistic effects against 12 MRSA strains with median fractional inhibitory concentration (FIC) indices of 0.38, 0.38, 0.25 and 0.38 in checkerboard assays. In time-kill analysis, SNH at 1/2 MIC in combination with oxacillin at 1/128 to 1/64 MIC or netilmicin at 1/8 to 1/2 MIC decreased the viable colonies by ≥2log₁₀ CFU/mL.

Conclusions/Significance

SNH demonstrated in vitro antibacterial activity against 103 hospital-associated MRSA isolates. Combinations of sub-MIC levels of SNH and oxacillin or netilmicin significantly improved the in vitro antibacterial activity against MRSA compared with either drug alone. The SNH-based combinations showed promise in combating MRSA.     

六种中药及其复方对鳗鲡致病性气单胞菌的体外抑制作用

针对病鳗内脏中分离的5 株致病性气单胞菌, 采用琼脂稀释法测定五倍子、石榴皮、大黄、虎杖、黄芩及黄连各味中药的最低抑菌浓度(MIC)和最低杀菌浓度(MBC); 再根据棋盘法设计15 种双联用药方和4 种三联用药方, 同样检测各组合配方的抑菌作用。实验结果表明, 6 味中药对养殖鳗鲡5 株致病性气单胞菌均有不同程度的抗菌效果, 其中五倍子的抑菌作用最强, 其次是大黄和石榴皮, 而黄连的抑菌效果最差; 15 种双联用药方较各味中药单用的抑菌活性绝大多数出现增强, 抑菌浓度至少减低39%, FICFIC≤0.5 表现显著增强抗菌活性的协同比例占23.3%; 4 种三联用药方对5 株致病性气单胞菌均具有显著的协同抑制效应, 复方中单味中药的抑菌浓度可以降低80%以上; 而双联用HC14 对4 株致病菌出现FIC≥2 的降低彼此抗菌活性的相互拮抗现象。由此说明合理运用不同中药的联用配伍, 不仅可提高单味中药的抗菌疗效, 而且大大减少了单一中药在实际养殖生产中的给药浓度, 降低药物在环境中的残留量, 防止残留药物造成环境污染, 并且降低用药成本, 提高水产养殖业的经济和社会效益。研究为中药复方防治细菌性鱼病提供科学理论参考。       

Development of Tyrocidine A analogues with improved antibacterial activity

The development of new antibacterial therapeutic agents capable of halting microbial resistance is a chief pursuit in clinical medicine. Classes of antibiotics that target and destroy bacterial membranes are attractive due to the decreased likelihood that bacteria will be able to generate resistance to this mechanism. The amphipathic cyclic decapeptide, Tyrocidine A, is a model for this class of antibiotics. Tyrocidine A is composed of a hydrophobic and a hydrophilic face, allowing for insertion into bacterial membranes, creating porous channels and destroying membrane integrity. We have used a combination of molecular modeling and solid phase synthesis to prepare Tyrocidine A and analogues 1-8. The minimum inhibitory concentrations (MICs) of these compounds were determined for a host of gram positive species and E. coli as a representative gram negative bacterium. Analogues 2 and 5 demonstrated moderate 2- to 8-fold increases in antibacterial activity over the parent Tyrocidine A for a variety of pathogenic microbes (best MICs for E. coli 32 microg/mL and 2 microg/mL for most gram positives). Examination of the structure- activity relationship between the analogues demonstrated a preference for increased amphipathicity but did not show a clear preference for increasing hydrophilicity versus hydrophobicity in improving antibacterial activity. Of note, movement of positively charged lysine residues or neutral pentafluorophenyl residues to different positions within the cyclopeptide ring system demonstrated improvements in antibacterial activity.     

Inactivation of chloramphenicol and florfenicol by a novel chloramphenicol hydrolase

Chloramphenicol and florfenicol are broad-spectrum antibiotics. Although the bacterial resistance mechanisms to these antibiotics have been well documented, hydrolysis of these antibiotics has not been reported in detail. This study reports the hydrolysis of these two antibiotics by a specific hydrolase that is encoded by a gene identified from a soil metagenome. Hydrolysis of chloramphenicol has been recognized in cell extracts of Escherichia coli expressing a chloramphenicol acetate esterase gene, estDL136. A hydrolysate of chloramphenicol was identified as p-nitrophenylserinol by liquid chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. The hydrolysis of these antibiotics suggested a promiscuous amidase activity of EstDL136. When estDL136 was expressed in E. coli, EstDL136 conferred resistance to both chloramphenicol and florfenicol on E. coli, due to their inactivation. In addition, E. coli carrying estDL136 deactivated florfenicol faster than it deactivated chloramphenicol, suggesting that EstDL136 hydrolyzes florfenicol more efficiently than it hydrolyzes chloramphenicol. The nucleotide sequences flanking estDL136 encode proteins such as amidohydrolase, dehydrogenase/reductase, major facilitator transporter, esterase, and oxidase. The most closely related genes are found in the bacterial family Sphingomonadaceae, which contains many bioremediation-related strains. Whether the gene cluster with estDL136 in E. coli is involved in further chloramphenicol degradation was not clear in this study. While acetyltransferases for chloramphenicol resistance and drug exporters for chloramphenicol or florfenicol resistance are often detected in numerous microbes, this is the first report of enzymatic hydrolysis of florfenicol resulting in inactivation of the antibiotic.