Isolation,structural characterization,and properties of mattacin (polymyxin M), a cyclic peptide antibiotic produced by Paenibacillus kobensis M

Mattacin is a nonribosomally synthesized, decapeptide antibiotic produced by Paenibacillus kobensis M. The producing strain was isolated from a soil/manure sample and identified using 16 S rRNA sequence homology along with chemical and morphological characterization. An efficient production and isolation procedure was developed to afford pure mattacin. Structure elucidation using a combination of chemical degradation, multidimensional NMR studies (COSY, HMBC, HMQC, ROESY), and mass spectrometric (MALDI MS/MS) analyses showed that mattacin is identical to polymyxin M, an uncommon antibiotic reported previously in certain Bacillus species by Russian investigators. Mattacin (polymyxin M) is cyclic and possesses an amide linkage between the C-terminal threonine and the side chain amino group of the diaminobutyric acid residue at position 4. It contains an (S)-6-methyloctanoic acid moiety attached as an amide at the N-terminal amino group, one D-leucine, six L-alpha,gamma-diaminobutyric acid, and three L-threonine residues. Transfer NOE experiments on the conformational preferences of mattacin when bound to lipid A and microcalorimetry studies on binding to lipopolysaccharide showed that its behavior was very similar to that observed in previous studies of polymyxin B (a commercial antibiotic), suggesting an identical mechanism of action. It was capable of inhibiting the growth of a wide variety of Gram-positive and Gram-negative bacteria, including several human and plant pathogens with activity comparable with purified polymyxin B. The biosynthesis of mattacin was also examined briefly using transpositional mutagenesis by which 10 production mutants were obtained, revealing a set of genes involved in production.     

Isolation of polymer-degrading bacteria and characterization of the hindgut bacterial community from the detritus-feeding larvae of Tipula abdominalis (Diptera: Tipulidae)

The Tipula abdominalis larval hindgut microbial community presumably facilitates digestion of the lignocellulosic diet. The microbial community was investigated through characterization of bacterial isolates and analysis of 16S rRNA gene clone libraries. This initial study revealed novel bacteria and provides a framework for future studies of this symbiosis.     

Identification and characterization of psychrotolerant sporeformers associated with fluid milk production and processing

Psychrotolerant spore-forming bacteria represent a major challenge to the goal of extending the shelf life of pasteurized dairy products. The objective of this study was to identify prominent phylogenetic groups of dairy-associated aerobic sporeformers and to characterize representative isolates for phenotypes relevant to growth in milk. Analysis of sequence data for a 632-nucleotide fragment of rpoB showed that 1,288 dairy-associated isolates (obtained from raw and pasteurized milk and from dairy farm environments) clustered into two major divisions representing (i) the genus Paenibacillus (737 isolates, including the species Paenibacillus odorifer, Paenibacillus graminis, and Paenibacillus amylolyticus sensu lato) and (ii) Bacillus (n = 467) (e.g., Bacillus licheniformis sensu lato, Bacillus pumilus, Bacillus weihenstephanensis) and genera formerly classified as Bacillus (n = 84) (e.g., Viridibacillus spp.). When isolates representing the most common rpoB allelic types (ATs) were tested for growth in skim milk broth at 6°C, 6/9 Paenibacillus isolates, but only 2/8 isolates representing Bacillus subtypes, grew >5 log CFU/ml over 21 days. In addition, 38/40 Paenibacillus isolates but only 3/47 Bacillus isolates tested were positive for β-galactosidase activity (including some isolates representing Bacillus licheniformis sensu lato, a common dairy-associated clade). Our study confirms that Paenibacillus spp. are the predominant psychrotolerant sporeformers in fluid milk and provides 16S rRNA gene and rpoB subtype data and phenotypic characteristics facilitating the identification of aerobic spore-forming spoilage organisms of concern. These data will be critical for the development of detection methods and control strategies that will reduce the introduction of psychrotolerant sporeformers and extend the shelf life of dairy products.     

Development of dilipid polymyxins: Investigation on the effect of hydrophobicity through its fatty acyl component

Continuous development of new antibacterial agents is necessary to counter the problem of antimicrobial resistance. Polymyxins are considered as drugs of last resort to combat multidrug-resistant Gram-negative pathogens. Structural optimization of polymyxins requires an in-depth understanding of its structure and how it relates to its antibacterial activity. Herein, the effect of hydrophobicity was explored by adding a secondary fatty acyl component of varying length onto the polymyxin structure at the amine side-chain of l-diaminobutyric acid at position 1, resulting to the development of dilipid polymyxins. The incorporation of an additional lipid was found to confer polymyxin activity against Gram-positive bacteria, to which polymyxins are inherently inactive against. The dilipid polymyxins showed selective antibacterial activity against Pseudomonas aeruginosa. Moreover, dilipid polymyxin 1 that consists of four carbon-long aliphatic lipids displayed the ability to enhance the antibacterial potency of other antibiotics in combination against P. aeruginosa, resembling the adjuvant activity of the well-known outer membrane permeabilizer polymyxin B nonapeptide (PMBN). Interestingly, our data revealed that dilipid polymyxin 1 and PMBN are substrates for the MexAB-OprM efflux system, and therefore are affected by efflux. In contrast, dilipid polymyxin analogs that consist of longer lipids and colistin were not affected by efflux, suggesting that the lipid component of polymyxin plays an important role in resisting active efflux. Our work described herein provides an understanding to the polymyxin structure that may be used to usher the development of enhanced polymyxin analogs.     

Structure–activity studies on polymyxin derivatives carrying three positive charges only reveal a new class of compounds with strong antibacterial activity

Recent years have brought in an increased interest to develop improved polymyxins. The currently used polymyxins, i.e. polymyxin B and colistin (polymyxin E) are pentacationic lipopeptides that possess a cyclic heptapeptide part with three positive charges, a linear “panhandle” part with two positive charges, and a fatty acyl tail. Unfortunately, their clinical use is shadowed by their notable nephrotoxicity. We have previously developed a polymyxin derivative NAB739 which lacks the positive charges in the linear part. This derivative is better tolerated than polymyxin B in cynomolgus monkeys and is, in contrast to polymyxin B, excreted into urine in monkeys and rats. Here we have conducted further structure-activity relationship (SAR) studies on 17 derivatives with three positive charges only. We discovered a remarkably antibacterial class, as exemplified by NAB815, that carries two positive charges only in the cyclic part.     

一株类芽孢杆菌的分离鉴定及其抗革兰氏阴性菌活性测定

【背景】随着碳青霉烯类和多粘菌素类可转移耐药基因的发现及扩散,多重耐药革兰氏阴性细菌感染更加难以治疗。【目的】筛选有效拮抗革兰氏阴性菌的菌株,为新型抗生素的发掘奠定基础。【方法】利用胰蛋白胨大豆琼脂培养基筛选土壤源细菌,通过16S rRNA基因序列鉴定其种属;通过全基因组测序,antiSMASH比对分析菌株产抗生素潜能,双层琼脂平板法验证其抗菌活性;通过甲醇萃取其次级代谢产物,高效液相色谱串联质谱(HPLC-MS/MS)进行次级代谢产物分析。【结果】从北京周边土壤样品中分离到一株类芽孢杆菌CAU136 (Paenibacillus pabuli CAU136),经过生物信息学分析和antiSMASH比对,表明该菌株有较强合成次级代谢产物的潜能,双层琼脂平板法验证其能抑制多株革兰氏阴性菌生长,HPLC-MS/MS检测结果显示其可能分泌多粘菌素E。【结论】类芽孢杆菌CAU136可能分泌多粘菌素E,能有效拮抗革兰氏阴性菌。     

Investigation of antibiotic and antibacterial agent cross-resistance in target bacteria from homes of antibacterial product users and nonusers

AIM: To describe the relationship between antibiotic and antibacterial resistance in environmental and clinical bacteria from home environments across geographical locations, relative to the use or nonuse of antibacterial products, with a focus on target organisms recognized as potential human pathogens. METHODS AND RESULTS: In a randomized study, environmental and clinical samples were collected from the homes of antibacterial product users (n=30) and nonusers (n=30) for the isolation of target bacteria for antibiotic and antibacterial testing in three geographical areas (in USA and UK). Isolates were tested for antibiotic susceptibility, with selected antibiotic-resistant and antibiotic-susceptible isolates tested against four common antibacterial agents (triclosan, para-chloro-meta-xylenol, pine oil and quaternary ammonium compound). Prequalified users and nonusers at each location were randomly selected after meeting exclusionary criteria. Of 1238 isolates, more target bacteria were recovered from nonuser than user homes. Of Staphylococcus aureus isolates (n=33), none showed resistance to oxacillin or vancomycin; for Enterococcus sp. (n=149), none were resistant to ampicillin or vancomycin; and for Klebsiella pneumoniae (n=54)and Escherichia coli (n=24), none were resistant to third generation cephalosporins. Antibiotic resistance to one or more of the standard test panel drugs for Gram-positive and Gram-negative target bacteria was comparable between nonuser and user homes for both environmental and clinical isolates [e.g. resistance of environmental coagulase-negative (CN) Staphylococcus sp. was 73.8% (124/168) from nonuser homes and 73.0% (111/152) from user homes, and Enterobacteriaceae other than E. coli, 75.9% (186/245) from nonuser homes compared with 78.0% from user homes]. Of 524 Gram-negatives tested against preferred/alternative drugs, 97.1% (509/524) were susceptible to all antibiotics, across both groups. Isolates of S. aureus, Enterococcus sp. and CN Staphylococcus sp. susceptible to all preferred treatment drugs showed comparable antibacterial minimum inhibitory concentration (MIC) results between nonuser and user home isolates. For Gram-positives resistant to one or more preferred drugs, greatest resistance to antibacterial active ingredients was found in the nonuser group. For Gram-negatives, the antibacterial MIC data were comparable for isolates that were fully susceptible and resistant to one or more preferred/alternative treatment antibiotics. CONCLUSIONS: The results showed a lack of antibiotic and antibacterial agent cross-resistance in target bacteria from the homes of antibacterial product users and nonusers, as well as increased prevalence of potential pathogens in nonuser homes. SIGNIFICANCE AND IMPACT OF THE STUDY: It refutes widely publicized, yet unsupported, hypotheses that use of antibacterial products facilitates the development of antibiotic resistance in bacteria from the home environment.     

Synthesis and characterization of the colistin peptide polymyxin E1 and related antimicrobial peptides.

Two strategies were developed to synthesize the acylated cyclic peptides know as polymyxins. Synthesis of polymyxin E1 and several analogs enabled us to evaluate the minimum inhibitory concentration of individual compounds against Gram-negative bacteria. In this study we also report the first identification of two component peptides in the complex polymyxin fermentation product colistin, a Thr2Ser isoform and an acyl group isomer. Both of these peptides, as well as a known component peptide, Leu7Ile, were similar to polymyxin E1 in potency, suggesting that conservative mutations in the colistin family are functionally inconsequential. In contrast, the acyclic analogs of all of these peptides were inactive, indicating that the characteristic lariat structure of the polymyxins is necessary for antimicrobial activity.     

Draft Genome Sequence of Paenibacillus polymyxa OSY-DF, Which Coproduces a Lantibiotic, Paenibacillin, and Polymyxin E1

Paenibacillus polymyxa OSY-DF is a Gram-positive rod-shaped bacterium isolated from a fermented vegetable food. This bacterial strain displays potent antimicrobial activities against Gram-positive and Gram-negative pathogenic bacteria, attributed to the production of the lantibiotic paenibacillin and the colistin peptide polymyxin E1. Here we report the draft genome sequence of Paenibacillus polymyxa OSY-DF.     

Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin

A new bacterial strain, displaying potent antimicrobial properties against gram-negative and gram-positive pathogenic bacteria, was isolated from food. Based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as Paenibacillus polymyxa and it was designated as strain OSY-DF. The antimicrobials produced by this strain were isolated from the fermentation broth and subsequently analyzed by liquid chromatography-mass spectrometry. Two antimicrobials were found: a known antibiotic, polymyxin E1, which is active against gram-negative bacteria, and an unknown 2,983-Da compound showing activity against gram-positive bacteria. The latter was purified to homogeneity, and its antimicrobial potency and proteinaceous nature were confirmed. The antimicrobial peptide, designated paenibacillin, is active against a broad range of food-borne pathogenic and spoilage bacteria, including Bacillus spp., Clostridium sporogenes, Lactobacillus spp., Lactococcus lactis, Leuconostoc mesenteroides, Listeria spp., Pediococcus cerevisiae, Staphylococcus aureus, and Streptococcus agalactiae. Furthermore, it possesses the physico-chemical properties of an ideal antimicrobial agent in terms of water solubility, thermal resistance, and stability against acid/alkali (pH 2.0 to 9.0) treatment. Edman degradation, mass spectroscopy, and nuclear magnetic resonance were used to sequence native and chemically modified paenibacillin. While details of the tentative sequence need to be elucidated in future work, the peptide was unequivocally characterized as a novel lantibiotic, with a high degree of posttranslational modifications. The coproduction of polymyxin E1 and a lantibiotic is a finding that has not been reported earlier. The new strain and associated peptide are potentially useful in food and medical applications.     

Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products

Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.     

Susceptibility to Polymyxin B of Penicillin G-induced Proteus mirabilis L Forms and Spheroplasts

A polymyxin B-resistant strain of Proteus mirabilis was converted into L forms and spheroplasts in the presence of penicillin G. This treatment caused a 400-fold increase in polymyxin B susceptibility. The acquired susceptibility was in the range of the natural susceptibility reported for susceptible gram-negative bacteria ( approximately 1 mug/ml). The high susceptibility to polymyxin B was lost as soon as the spheroplasts and L forms were allowed to reconvert into the bacillary form in penicillin-free media. This behavior is strong evidence that the natural resistance of Proteus strains to polymyxins is due to the impermeability of the outer cell wall structures to these antibiotic substances.     

Use of rpoB gene analysis for identification of nitrogen-fixing Paenibacillus species as an alternative to the 16S rRNA gene

AIM: To avoid the limitations of 16S rRNA-based phylogenetic analysis for Paenibacillus species, the usefulness of the RNA polymerase beta-subunit encoding gene (rpoB) was investigated as an alternative to the 16S rRNA gene for taxonomic studies. METHODS AND RESULTS: Partial rpoB sequences were generated for the type strains of eight nitrogen-fixing Paenibacillus species. The presence of only one copy of rpoB in the genome of P. graminis strain RSA19(T) was demonstrated by denaturing gradient gel electrophoresis and hybridization assays. A comparative analysis of the sequences of the 16S rRNA and rpoB genes was performed and the eight species showed between 91.6-99.1% (16S rRNA) and 77.9-97.3% (rpoB) similarity, allowing a more accurate discrimination between the different species using the rpoB gene. Finally, 24 isolates from the rhizosphere of different cultivars of maize previously identified as Paenibacillus spp. were assigned correctly to one of the nitrogen-fixing species. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study indicate that rpoB is a powerful identification tool, which can be used for the correct discrimination of the nitrogen-fixing species of agricultural and industrial importance within the genus Paenibacillus.     

Polymyxin E Induces Rapid Paenibacillus polymyxa Death by Damaging Cell Membrane while Ca2+ Can Protect Cells from Damage

Polymyxin E, produced by Paenibacillus polymyxa, is an important antibiotic normally against Gram-negative pathogens. In this study, we found that polymyxin E can kill its producer P. polymyxa, a Gram-positive bacterium, by disrupting its cell membrane. Membrane damage was clearly revealed by detecting the leakage of intracellular molecules. The observation using scanning electron microscopy also supported that polymyxin E can destroy the cell membrane and cause an extensive cell surface alteration. On the other hand, divalent cations can give protection against polymyxin E. Compared with Mg²⁺, Ca²⁺ can more effectively alleviate polymyxin E-induced damage to the cell membrane, thus remarkably increasing the P. polymyxa survival. Our findings would shed light on a not yet described bactericidal mechanism of polymyxin E against Gram-positive bacteria and more importantly the nature of limited fermentation output of polymyxin E from P. polymyxa.     

Diversity and characterization of antagonistic bacteria from tropical estuarine habitats of Cochin, India for fish health management

Mortalities due to pathogenic bacteria are a major problem in aquaculture, especially in larval rearing systems. Use of antibiotics to overcome this problem is not an option any more due to the increasing antibiotic resistance among pathogens. The present study aims to understand the diversity of bacteria with antagonistic properties in the tropical estuarine habitats of Cochin, located along the southwest coast of India, and to use them as an alternative to antibiotics in aquaculture. Among the 4,870 isolates screened, approximately 1 % showed significant antibacterial activity against six common aquaculture pathogens belonging to the genera Aeromonas and Vibrio. The antagonistic bacteria were identified as Bacillus (81 %) and Pseudomonas (19 %) using biochemical and 16S rRNA gene sequence homology. The isolates showing stable and higher levels of antibacterial activity were subjected to enzymatic expression profile, antibiotic resistance pattern and abiotic stress tolerance assays. As a result, five Pseudomonas spp. and four Bacillus spp., were identified as promising antagonistic isolates that could be exploited as probionts or microbial products (MP's), to control bacterial diseases in aquaculture rearing systems.     

Effect of polymyxins on Bacillus polymyxa sporogenesis

Bacillus polymyxa var. Ross. producing polymyxin M and Bacillus polymyxa 153 producing polymyxin B form spores during submerged cultivation when the rate of biosynthesis of antibiotic peptides is low and when the production of antibiotics is over. However, sporogenesis is stimulated if polymyxins are added at the early stage of cultural growth. Inhibition of the synthesis of antibiotics suppresses the formation of spores. Substances other than polymyxins do not exhibit such a specific effect on sporogenesis. The fact that the culture requires endogenous polymyxins which are most effective in the period prior to the appearance of spores in the culture suggests the regulatory action of these peptides at the stage between vegetative growth and spore formation in Bacillus polymyxa.     

An integrated high throughput strategy to screen mutants of Paenibacillus polymyxa with high polymyxin E-productivity

An integrated high-throughput screening (HTS) strategy was developed to screen large numbers of polymyxin E-producing mutants of Paenibacillus polymyxa. Various types of mutants were transferred onto the surfaces of solidified agar in 96-well microtiter plates, and then inoculated to 96-deep-well microtiter plates for micro-cultivation. The culture conditions were optimized for the production of polymyxin E. The supernatants from the micro-culture plates were transferred to 96-well bioassay microtiter plates containing Escherichia coli JM109 for high-throughput bioassay. By using this high-throughput screening (HTS) procedure, one best producer P. polymyxa PE 5.808 was identified from a large NTG mutated library with about 5,000 isolates. The volumetric productivity of polymyxin E of P. polymyxa PE 5.808 was 1,200 μg/ml in shake flasks, about 140% improvement compared with that of the wild type strain.     

Evaluation of the VITEK2 BCL card for identification of Bacillus species and other aerobic endosporeformers

Aims:  To evaluate the performance of the VITEK2 Bacillus identification card (BCL) for the identification of aerobic endospore-forming bacteria, using fresh isolates and reference strains.
Methods and Results:  One hundred and nine industrial, environmental and clinical isolates were tested using the BCL card. The card contained 46 substrates for measuring carbon source utilization, enzymatic activities, inhibition by 6·5% NaCl and resistance to the antibiotics kanamycin, oleandomycin and polymyxin B. Identifications were made after 14 h incubation, using a database allowing identification of 42 species of the genera Aneurinibacillus , Bacillus , Brevibacillus , Geobacillus , Paenibacillus and Virgibacillus . The reference identities of all isolates were authenticated by phenotypic methods, with 16S rRNA gene sequencing used to resolve discrepancies.
Conclusions:  One hundred and one strains (93%) were identified correctly to species level, seven strains (6%) were incorrectly identified, and one strain (1%) remained unidentified.
Significance and Impact of the Study:  The VITEK2 BCL card provides a major advance in the reliable identification of Bacillus species and members of related genera.     

Isolation and Identification of a Paenibacillus polymyxa Strain That Coproduces a Novel Lantibiotic and Polymyxin

A new bacterial strain, displaying potent antimicrobial properties against gram-negative and gram-positive pathogenic bacteria, was isolated from food. Based on its phenotypical and biochemical properties as well as its 16S rRNA gene sequence, the bacterium was identified as Paenibacillus polymyxa and it was designated as strain OSY-DF. The antimicrobials produced by this strain were isolated from the fermentation broth and subsequently analyzed by liquid chromatography-mass spectrometry. Two antimicrobials were found: a known antibiotic, polymyxin E1, which is active against gram-negative bacteria, and an unknown 2,983-Da compound showing activity against gram-positive bacteria. The latter was purified to homogeneity, and its antimicrobial potency and proteinaceous nature were confirmed. The antimicrobial peptide, designated paenibacillin, is active against a broad range of food-borne pathogenic and spoilage bacteria, including Bacillus spp., Clostridium sporogenes, Lactobacillus spp., Lactococcus lactis, Leuconostoc mesenteroides, Listeria spp., Pediococcus cerevisiae, Staphylococcus aureus, and Streptococcus agalactiae. Furthermore, it possesses the physico-chemical properties of an ideal antimicrobial agent in terms of water solubility, thermal resistance, and stability against acid/alkali (pH 2.0 to 9.0) treatment. Edman degradation, mass spectroscopy, and nuclear magnetic resonance were used to sequence native and chemically modified paenibacillin. While details of the tentative sequence need to be elucidated in future work, the peptide was unequivocally characterized as a novel lantibiotic, with a high degree of posttranslational modifications. The coproduction of polymyxin E1 and a lantibiotic is a finding that has not been reported earlier. The new strain and associated peptide are potentially useful in food and medical applications.     

草鱼肠道纤维素降解细菌的分离与鉴定

为更好地弄清草鱼（Ctenopharyngodon idella）肠道纤维素降解细菌的种类,采用羧甲基纤维素（CMC）作为唯一碳源的选择性培养基,分别从草鱼肠道内容物和肠道黏膜中分离到了40株产纤维素酶细菌。16S rRNA基因序列的分析结果显示,大多数产纤维素酶细菌为气单胞菌属（Aeromonas）的种类,其次为肠杆菌属（Enterobacter）的细菌以及未经分离纯培养的细菌（Uncultured bacterium）。进一步研究细菌产纤维素酶能力发现,纤维素酶活性显著性高于其他菌株的分别是A. veronii MC2、A. veronii BC6、肠杆菌科（Enterobacteriaceae）中一种未经分离纯培养的细菌BM3（Uncultured bacterium BM3）和A. jandaei HC9。草鱼肠道中简答气单胞菌（A. jandaei）、类志贺邻单胞菌（Plesiomonas shigelloides）、阴沟肠杆菌（E. cloacae）以及产气肠杆菌（E. aerogenes）是被作为产纤维素酶细菌的首次报道。