Antibiotic Production by Anaerobic Bacteria

Soils from aerobic and anaerobic sources were investigated for the possible presence of bacteria which produce antibiotics under anaerobic conditions of growth. The screening techniques devised for this study yielded 157 soil bacteria which, during anaerobic growth, produced antibiotic activity against aerobic test bacteria.

Studies on choice of media, presence of oxygen, and changes in antibiotic activity during growth indicated that representative strains of these bacteria produced mixtures of antibiotics. The activity was heat labile.

     

Effects of glutamate, glucose, phosphate, and alkali metal ions on cephamycin C production by Nocardia lactamdurans in defined media

A High cephamycin C producing strain of Nocardia lactam-durans was used to study cell growth and antibiotics production in defined media. Batch fermentations in shake flasks and stirred tanks showed that antibiotic production occurred during cell growth and the production rate rapidly decline as the growth slowed. Glutamate served as a primary substrate during this phase. Later, ammonia was utilized along with a remainder of the glucose. Rapid antibiotic production occurred in this phase. Increased glutamate promoted higher growth, a rise in ammonium ion concentration, and a marked reduction in antibiotic titers. An increase of the glucose concentration along with the glutamate concentration balanced to the medium; no ammonium ion rise occurred and a peak specific antibiotic titer comparable to the control medium was obtained. In a phosphate-limited medium, cell growth equivalent to the control medium and increased antibiotic titers were obtained. In these experiments, adjustment of Na(+) and K(+) ion concentration equal to that in the control medium was found to be important. Based on carbon and nitrogen balances, the activity of the key nitrogen metabolism enzymes, and the published literature, a two-stage model of regulation is suggested.     

Multiple antibiotics produced by Pseudomonas fluorescens HV37a and their differential regulation by glucose

Pseudomonas fluorescens HV37a inhibited growth of the fungus Pythium ultimum on potato dextrose agar (PDA). An antibiotic activity produced under these conditions was fractionated and partially characterized. Extracts prepared from the PDA on which HV37a was grown revealed a single peak of antibiotic activity on thin-layer chromatograms. Similar extracts were prepared from mutants of HV37a. Their analysis indicated that the antibiotic observed in thin-layer chromatograms was responsible for fungal inhibition observed on PDA. The production of the PDA antibiotic required the presence of glucose, whereas two other antibiotic activities were produced only on potato agar without added glucose. Two mutants (denoted AfuIa and AfuIb) previously characterized as deficient in fungal inhibition on PDA showed altered regulation of the production of all three antibiotics in response to glucose. These mutants were also deficient in glucose dehydrogenase. Mutants isolated as deficient in glucose dehydrogenase were also deficient in fungal inhibition and were grouped into two classes on the basis of complementation analysis with an AfuI cosmid. Glucose regulation of antibiotic biosynthesis therefore involves at least two components and requires glucose dehydrogenase.     

Positive selection of antibiotic-producing soil isolates.

Stepwise discriminant analysis was used to identify the most powerful selective substrates which could be used to formulate media capable of enriching for antibiotic-producing soil isolates. This was achieved by characterizing a collection of 74 soil bacteria, including eubacteria and actinomycetes, according to their ability to produce antibacterial antibiotics and their growth responses to 43 physiological and nutritional tests. The characters which were selective for actinomycetes relative to eubacteria included growth on proline (1%, w/v) and humic acid (0.1%) as sole sources of both carbon and nitrogen, growth on nitrate as a nitrogen source, and growth at pH 7.7-8.0. Growth on proline (1%) and humic acid (0.1%) as sole carbon/nitrogen sources, growth on asparagine as a nitrogen source, and growth in the presence of vitamins were among the characteristics which allowed antibiotic-producing actinomycetes to be differentiated from non-antibiotic-producing strains. Several simple isolation media which incorporated the selective substrates identified by discriminant analysis succeeded in increasing the proportion of actinomycetes isolated from soil samples. Furthermore, the percentage of isolates capable of antibiotic production was considerably increased.     

Modified antibiotics-methylated ampicillin and ethylated ampicillin-inhibit growth of ampicillin-resistant strain of bacteria

Bacterial resistance to antibiotics is a significant problem in health facilities and results in higher costs for health care and increased fatalities due to infection. The work presented here suggests that antibiotic molecular structure can be altered in a selected manner, which will revive the bacterial growth inhibiting capability. A bacterial strain PKK3535(DH1), which is resistant to the antibiotic ampicillin, was found to be highly growth inhibited by these altered forms of ampicillin when tested in tissue culture. The level of growth inhibition of bacterial strain PKK3535(DHI) was greater than 50%, for both molecular variants of ampicillin that were investigated. The bacteria strain used for testing was a clinical isolate obtained from the University Hospital of the University of Nebraska, Omaha. These two antibiotic variants were methylated ampicillin and ethylated ampicillin. The synthetic procedure for generating these variants is presented as well as the molecular structure. The methylated and ethylated ampicillin were found to be stable at 0 degrees C for many weeks, were somewhat less soluble than normal ampicillin, but dissolved in LB plate media. The resistant bacteria strain was plated onto LB media with altered ampicillin and profound inhibition of bacteria growth was seen within the first 24 hours of incubation. These molecular variants of ampicillin provide evidence of a means to combat the proliferation of resistant bacterial strains. The molecular alteration of antibiotics may provide a suitable means to study and combat the appearance of antibiotic-resistant bacteria.     

Effect of different grades of agar-agar on the nature of the test microbe growth inhibition zones in controlling antibiotic activity by means of agar diffusion]

The effect of various agar grades on the size and margin character of the inhibition growth zones in assay of antibiotic activity by the agar diffusion method was studied. It was shown that not all the agar grades could be used in antibiotic activity assay. Depending on the agar type the size of the inhibition growth zones produced by the same antibiotic concentration significantly varied. The variations in the size of the inhibition growth zones depended on the agar ability to bind antibiotics and were mainly defined by the agar purity. The agars with low content of nitrogen admixtures bound the antibiotics to a low extent. The commerical grades of the agars of the South-Sea and Korsakov Plants, the experimental grade of the TINRO agar with additional purification, as well as the agars imported from Argentina and France proved to be most useful for determination of the antibiotic activity by the agar diffusion method.     

Slow-growing heterokaryons as potential intermediates in supernumerary chromosome transfer between biotypes of Colletotrichum gloeosporioides

The vegetative transfer of a supernumerary chromosome between two biotypes (A and B) of Colletotrichum gloeosporioides pathogenic on Stylosanthes spp. has been previously demonstrated. The mechanism of transfer is unknown, and transient heterokaryosis has been implicated as an intermediary step, but inter-biotype heterokaryons have not so far been isolated. Conidia of a hygromycin-resistant strain of biotype A and a phleomycin-resistant strain of biotype B were mixed, co-cultured and plated on media containing both antibiotics and extremely slow-growing colonies resistant to both antibiotics isolated using repeated hyphal tip sub-culturing. Mononucleate conidia derived from these colonies were unable to germinate on media containing both antibiotics, but were able to germinate on media containing only one antibiotic, with hygromycin-resistant colonies predominating, indicating that unbalanced heterokaryons had formed. The heterokaryons had highly impaired growth rates suggesting some incompatibility. DNA marker analysis confirmed their heterokaryon status. These results demonstrate that unfit inter-biotype heterokaryons can form and potentially provide an intermediate step for supernumerary chromosomal exchange.     

Changes in microorganism sensitivity to antibiotics as affected by divalent metal ions

Ca+ and Mg+ in nutrient media significantly influence the results of antibiotic sensitivity determination in microorganisms. The data of the study are indicative of a necessity for the media standardization with respect to the content of bivalent metal ions. It is recommended that agar-agar manufactured by the South Sea Factory (tafuinsky) be used for preparation of nutrient media for determination of microbial sensitivity to antibiotics and Hottinger meat pancreatic digest as the nitrogen source providing the content of 120 mg per cent of amine nitrogen in the medium.     

Induced tolerance of Sclerotium cepivorum to antibiotics in the presence of onion exudates

The onion white rot pathogen Sclerotium cepivorum was cultured on agar media containing 2% malt extract and one of the antifungal antibiotics, endomycin, griseofulvin, venturicidin and cycloheximide at concentrations that reduced but did not prevent growth of mycelium. When onion seeds or agar discs impregnated with diffusates from onion bulbs were placed on the antibiotic media, radial growth of the fungus was greatly increased, and there was a profuse development of aerial mycelium. Gaseous diffusates from onion tissue and from impregnated agar discs were also effective. On the antibiotic media, tomato, cabbage and radish seeds did not stimulate the growth of S. cepivorum and the onion exudates did not stimulate the growth of four other fungi. This and other evidence is considered to show that the stimulation of growth of S. cepivorum was not caused by any direct effect on the antibiotics but by a tolerance of the fungus to them, which was specifically induced by an exudate from its host plant. The phenomenon may be related to the reported reversal by onion extracts of the inhibitory effects of soil mycostasis on germination of sclerotia of the fungus.     

Effects of reduced oxygen on growth and antibiotic production inStreptomyces clavuligerus

Summary The effects of reduced oxygen on growth and antibiotic production were studied in batch cultures ofStreptomyces clavuligerus in defined media. Antibiotic levels were unaffected by reduced oxygen for the first 50 hours of fermentation. After growth ceased, antibiotic concentrations dropped by a factor of about three under reduced oxygen, while the antibiotic concentration was stable when air was used. This suggests that enzymes for hydrolyzing antibiotics may be regulated by the aeration conditions.     

Simplified liquid nutrient media for controlling antibiotic activity, the spectrum of their antibacterial action and the sensitivity of microorganisms]

Beaf-peptone broth and some of its modifications, one of which is a simple and in expensive one to a leser extent binding to antibiotics, such as penicillin, oxytetracycline and streptomycin and providing sufficient growth of the test microbes were used to determine the antibiotic activity with the methods of serial dilutions. The simple modification was recommended for practical use. The MIC of the antibiotics in the above simple medium was less than that in the control. The results of the antibiotic activity determination on both media coincided.     

Sigma B对单核细胞增生李斯特菌耐受抗生素作用的影响

单核细胞增生李斯特菌是重要的革兰阳性食源性致病菌。近年来的报道显示出该菌耐受抗生素的能力有不断增强的趋势,为了探讨其耐药机制,对Sigma B（σB,李斯特菌中应对环境胁迫的主要调控因子）在抗生素耐受性中的作用进行了初步研究。检测和比较单核细胞增生李斯特菌标准菌株EGDe和其σB缺失突变菌株EGDeΔsigB对盘尼西林青霉素、氨苄西林青霉素、利福平、硫酸庆大霉素、四环素盐酸和红霉素6种抗生素的最小抑菌浓度（MIC）;在测定的MIC的基础上,利用MTT（噻唑蓝活体染色法）法比较EGDe和EGDeΔsigB在1×MIC、2×MIC和8×MIC的氨苄西林青霉素、红霉素和利福平3种抗生素中的生长活性。EGDe对盘尼西林青霉素（0.16μg/mL）、四环素盐酸（0.25μg/mL）和硫酸庆大霉素（0.5μg/mL）的MIC高于EGDeΔsigB（分别为0.08、0.125和0.125μg/mL）;而对氨苄西林青霉素、红霉素和利福平的MIC 2种菌株没有差别,分别为0.19、0.125和0.032μg/mL;与EGDe相比,EGDeΔsigB在氨苄西林青霉素、红霉素和利福平培养基中的生长活性较差,对抗生素的抑制更为敏感,而且随着这3种抗生素浓度的增加,其抑制程度也随之增强。Sigma B在单核细胞增生李斯特菌对抗生素的耐受中起到重要调节作用。     

Nutritional requirements ofLysobacter lactamgenus for the production of cephabacins

Summary To optimize the fermentation medium for the production of new cephem compounds, cephabacins, by an eubacteriaLysobacter lactamgenus IFO 14,288, the effects of medium components on cephabacin production were investigated. Supplementation of glucose as a sole carbon source in liquid media was the best for the antibiotic production as well as for the cell growth. Casamino acid was the best nitrogen source for antibiotic biosynthesis and cell growth among nitrogen sources tested, and this strain could utilize sulfate or thiosulfate as a sulfur source. No significant effects of growth factors (vitamins) on the antibiotic production and cell growth were observed, but ferrous, magnesium and nickel ions slightly enhanced the cephabacin production.     

Antibiotic production in a spatially structured environment

Ecological factors influencing the effects of antibiotic production were explored experimentally and theoretically. A spatially structured model was used to model the dynamics of antibiotic-producing and nonproducing bacteria in which growth of the nonproducers was reduced by neighbouring antibiotic producers. Various factors affecting spatial interactions between the bacteria were examined for their impact on antibiotic producers. Spatial clustering had a positive impact on the effect of antibiotic production, as measured by the decline in growth of the nonproducing strain, while increasing the initial density of the nonproducing strain had a negative impact. Experiments examined the growth of antibiotic-producing Streptomyces species and a nonproducing, antibiotic-sensitive strain of Bacillus subtilis that were coinoculated on surface media. There was an effect of the Streptomyces on Bacillus growth in some experiments but not in others. In light of the predictions from the model, unintentional clustering of cells is a more likely explanation for this finding than different initial Bacillus densities. The importance of spatial structure seen in this study is consistent with a terrestrial rather than an aquatic distribution of antibiotic-producing bacteria, and may have implications in the search for novel antibiotics.     

Impairment of ribosomal subunit synthesis in aminoglycoside-treated ribonuclease mutants of Escherichia coli

The bacterial ribosome is an important target for many antimicrobial agents. Aminoglycoside antibiotics bind to both 30S and 50S ribosomal subunits, inhibiting translation and subunit formation. During ribosomal subunit biogenesis, ribonucleases (RNases) play an important role in rRNA processing. E. coli cells deficient for specific processing RNases are predicted to have an increased sensitivity to neomycin and paromomycin. Four RNase mutant strains showed an increased growth sensitivity to both aminoglycoside antibiotics. E. coli strains deficient for the rRNA processing enzymes RNase III, RNase E, RNase G or RNase PH showed significantly reduced subunit amounts after antibiotic treatment. A substantial increase in a 16S RNA precursor molecule was observed as well. Ribosomal RNA turnover was stimulated, and an enhancement of 16S and 23S rRNA fragmentation was detected in E. coli cells deficient for these enzymes. This work indicates that bacterial RNases may be novel antimicrobial targets.     

Pantoea agglomerans strain EH318 produces two antibiotics that inhibit Erwinia amylovora in vitro

Pantoea agglomerans (synonym: Erwinia herbicola) strain Eh318 produces through antibiosis a complex zone of inhibited growth in an overlay seeded with Erwinia amylovora, the causal agent of fire blight. This zone is caused by two antibiotics, named pantocin A and B. Using a genomic library of Eh318, two cosmids, pCPP702 and pCPP704, were identified that conferred on Escherichia coli the ability to inhibit growth of E. amylovora. The two cosmids conferred different antibiotic activities on E. coli DH5alpha and had distinct restriction enzyme profiles. A smaller, antibiotic-conferring DNA segment from each cosmid was cloned. Each subclone was characterized and mutagenized with transposons to generate clones that were deficient in conferring pantocin A and B production, respectively. Mutated subclones were introduced into Eh318 to create three antibiotic-defective marker exchange mutants: strain Eh421 (pantocin A deficient); strain Eh439 (pantocin B deficient), and Eh440 (deficient in both pantocins). Cross-hybridization results, restriction maps, and spectrum-of-activity data using the subclones and marker exchange mutants, supported the presence of two distinct antibiotics, pantocin A and pantocin B, whose biosynthetic genes were present in pCPP702 and pCPP704, respectively. The structure of pantocin A is unknown, whereas that of pantocin B has been determined as (R)-N-[((S)-2-amino-propanoylamino)-methyl]-2-methanesulfonyl-s uccina mic acid. The two pantocins mainly affect other enteric bacteria, based on limited testing.     

Genetic analysis of absB, a Streptomyces coelicolor locus involved in global antibiotic regulation.

The filamentous soil bacterium Streptomyces coelicolor is known to produce four antibiotics which are genetically and structurally distinct. An extensive search for antibiotic regulatory mutants led to the discovery of absB mutants, which are antibiotic deficient but sporulation proficient. Genetic analysis of the absB mutants has resulted in definition of the absB locus at 5 o'clock on the genetic map. Multiple cloned copies of the actII-ORF4 gene, an activator of synthesis of the antibiotic actinorhodin, restore actinorhodin biosynthetic capability to the absB mutants. These results are interpreted to mean that the failure of absB mutants to produce antibiotics results from decreased expression of the antibiotic genes. The absB gene is proposed to be involved in global regulation of antibiotic synthesis.     

Diversity among Rhizobia Effective with Robinia pseudoacacia L.

The diversity of rhizobia that form symbioses with roots of black locust (Robinia pseudoacacia L.), an economically important leguminous tree species, was examined by inoculating seedling root zones with samples of soil collected from the United States, Canada, and China. Bacteria were isolated from nodules, subcultured, and verified to be rhizobia. The 186 isolates varied significantly in their resistance to antibiotics and NaCl, their growth on different carbohydrates, and their effect on the pH of culture media. Most isolates showed intermediate antibiotic resistance, the capacity to use numerous carbohydrates, and a neutral to acid pH response. Isolates had greater similarity within sampling locations than among sampling locations. The isolates were grouped by using numerical taxonomy techniques, and representative strains of 37 groups were selected. The mean generation times of these isolates ranged from 3 to 9 h, and the protein profile of each of the 37 isolates was unique. Nitrogen fixation, total nitrogen accumulation, and plant growth varied significantly among black locust seedlings inoculated with the representative isolates. We conclude that great variation exists among Rhizobium spp. that nodulate black locust, and selection of strains for efficiency of the symbiotic association appears possible.     

Development of an accelerated method of determining the antibiotic sensitivity of Cl. perfringens type A]

An express method for determination of antibiotic sensitivity in the strains of Cl. perfringens of type A using Soviet dry nutrient media and antibiotics is proposed. The criteria for estimation of the level of the antibiotic sensitivity of the causative agent of gas gangrene in short periods on the basis of comparison of the data of the antibiotic agar diffusion procedure and the antibiotic MIC were worked out. Twelve antibiotics and 45 collection strains of Cl. perfringens of type A were used in the experiment. The antibiotic agar diffusion method with the use of the nutrient media, microbial load and cultivation conditions developed by the authors is recommended for tentative determination of the antibiotic sensitivity in Cl. perfringens of type A for 4 hours. The use of the agar diffusion method and determination of the antibiotic MIC provided complete estimation of tha antibiotic sensitivity of Cl. perfringens of type A within not more than 24 hours.