Antimicrobial activity of two antitumour agents and ribonucleotide reductase inhibitors, pyridine-2-carboxaldehyde thiosemicarbazone and the acetate form of its copper(II) chelate

Some copper chelates have potent antitumour activity, and in some cases also the free ligands have activity in vivo. Yet, little is known about their antimicrobial properties. Copper(II) chelates of the thiosemicarbazones of a-N-heterocyclic carboxaldehydes constitute one important group of such agents, also their ligands having marked antitumour activity. Both the ligands and chelates inhibit ribonucleotide reductase. Some ligands have been or are under clinical trials as antineoplastic agents. I report here a study on the antimicrobial properties of the prototype compounds of this group, pyridine-2-carboxaldehyde thiosemicarbazone and its copper(II) chelate. They were tested against nine microbes, including bacteria (Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus lactis), yeasts (Candida albicans and Saccharomyces cerevisiae) and one mold (Aspergillus niger). Two clinical isolates of Bacillus sp. and one reference strain were also studied. Both the ligand and the chelate had marked activity. The ligand displayed considerable activity against all bacteria except for S. lactis, and its activity against E. coli and P. aeruginosa was that high that practical applications might be considued. It was highly active against A. niger and moderately active against C. albicans. The chelate was highly active against S. epidermidis and S. cerevisiae. Both compounds inhibited the clinical isolates markedly. Since some related ligands have been or are in clinical trials on humans or are entering them, their route to clinical use, also as antimicrobials, might be much more straightforward than that of substances, whose toxicity in humans is wholly unexplored.     

铜绿假单胞菌体外抑菌活性研究

目的 观察铜绿假单胞菌抗菌物质对鲍曼不动杆菌等细菌的体外抑菌效果.方法 用交叉条带实验方法检测了铜绿假单胞菌对鲍曼不动杆菌、耐甲氧西林表皮葡萄球菌和粪肠球菌的体外抑制活性.结果 铜绿假单胞菌对鲍曼不动杆菌、耐甲氧西林表皮葡萄球菌和粪肠球菌体外抑菌活性良好,10株铜绿假单胞菌中,有8株对鲍曼不动杆菌的抑制率均达到了100％.另外有8株对耐甲氧西林表皮葡萄球菌的抑菌率均为100％;有6株对粪肠球菌的抑菌率为100％.结论 铜绿假单胞菌对上述3种致病菌具有较强的抗菌活性,具有开发前景.     

连翘苷和黄芩苷对表皮葡萄球菌生物膜抑制作用的研究

目的通过中药有效成分连翘苷和黄芩苷分别对表皮葡萄球菌生物膜抑制作用的研究,为表皮葡萄球菌生物膜引起的相关感染提供新的治疗途径。方法体外构建表皮葡萄球菌生物膜,XTT减低法评价连翘苷、黄芩苷对表皮葡萄球菌初始黏附及生物膜内细菌代谢的影响,显微镜下观察用药后表皮葡萄球菌生物膜形态和结构改变。结果连翘苷和黄芩苷对表皮葡萄球菌生物膜的早期黏附均无抑制作用;连翘苷对表皮葡萄球菌生物膜菌的SMIC50为31.25μg/ml,而黄芩苷对表皮葡萄球菌生物膜菌的代谢无影响;在显微镜下观察,连翘苷使部分表皮葡萄球菌被膜的形态发生改变,而黄芩苷对其形态影响不显著。结论连翘苷对表皮葡萄球菌生物膜的初始黏附阶段无抑制作用,对生物膜菌的代谢和生物膜形态均有显著影响;黄芩苷对表皮葡萄球菌生物膜无显著作用。     

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.     

Bacterial interference with skin colonization in germfree hairless mice

Bacterial colonization of the digestive tract and the skin was studied over a 3-week period in a group of 10 germfree HRS mice using Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa. Sequential utilization of two strains allowed us to carry out six assays and to show the presence of interference phenomena during colonization of the skin. When P. aeruginosa was given after challenge with S. aureus or S. epidermidis, it did not colonize the skin. If the first challenge was done with P. aeruginosa, this bacteria was eliminated within 10 days by S. aureus and S. epidermidis on the skin, but it succeeded in colonizing the digestive tract. When the first challenge was done with S. aureus, colonization of the skin and the digestive tract with S. epidermidis was prevented, whereas these two species were found in association when S. aureus was given in second place. None of the in vitro assays (mixed culture, bacteriocin production, adherence inhibition, antimicrobial activity) could explain the in vivo observations.     

Antimicrobial activity of the marine alkaloids haminol and pulo'upone and related compounds

The marine alkaloids haminol A, haminol B and pulo'upone as well as 17 related compounds (twelve 2-substituted pyridine derivatives, four 3-substituted ones and one analogue of the bicyclic terminus of pulo'upone) were tested for antimicrobial activity against a panel of six microbes (Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, Candida albicans and Saccharomyces cerevisiae) using the paper disc agar diffusion method. Six compounds were tested also against the mold Aspergillus niger. Some of the compounds displayed noteworthy antimicrobial activity, only one congener being completely devoid of activity. Nearly all compounds had activity against B. cereus and S. epidermidis. The growth of E. coli, C albicans and S. cerevisiae was also distinctly inhibited by many compounds. In contrast, most compounds were inactive or had minimal activity against P. aeruginosa. Interestingly, most of the compounds tested against the opportunistic pathogen A. niger were active, one of them having noteworthy inhibitory potency.     

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.     

Antimicrobial activity of some Salvia species essential oils from Iran

The aerial parts of Salvia multicaulis, S. sclarea and S. verticillata were collected at full flowering stage. The essential oils were isolated by hydrodistillation and analyzed by combination of capillary GC and GC-MS. The in vitro antimicrobial activity of the essential oils were studied against eight Gram-positive and Gram-negative bacteria (Bacillus subtilis, Bacillus pumulis, Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and three fungi (Candida albicans, Saccharomyces cerevisiae and Aspergillus niger). The results of antibacterial activity tests of the essential oils according to the disc diffusion method and MIC values indicated that all the samples have moderate to high inhibitory activity against the tested bacteria except for P. aeruginosa which was totally resistant. In contrast to antibacterial activity, the oils exhibited no or slight antifungal property, in which only the oil of S. multicaulis showed weak activity against two tested yeasts, C. albicans and S. cerevisiae.     

Effect of ovotransferrin, protamine sulfate and EDTA combination on biofilm formation by catheter-associated bacteria

AIMS: To determine the effect of a composition comprising ovotransferrin (OT), protamine sulfate (PS) and ethylenediaminetetraacetic acid (EDTA) on biofilm formation by catheter-associated bacteria. METHODS AND RESULTS: The in vitro activity of OT, PS and EDTA alone and in combinations against biofilm formation by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis and Staphylococcus epidermidis was investigated. All the three compounds either alone or in combinations failed to inhibit the growth completely at the concentrations tested. However, the subinhibitory concentrations of three compounds in a composition showed synergistic inhibitory effect on biofilm formation by K. pneumoniae, Ps. aeruginosa and S. epidermidis. Furthermore, 79-95% reduction in Ps. aeruginosa and S. epidermidis biofilm formation was observed in a clear vinyl urinary catheter treated with the composition. CONCLUSION: The subinhibitory concentrations of OT, PS and EDTA in a composition were effective in reducing biofilm formation by catheter-associated bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that a synergistic composition-comprising non-antibiotic generally regarded as safe (GRAS) compounds such as OT, PS and EDTA may be used in the prevention of catheter-related infections.     

Antibiofilm activity of an exopolysaccharide from marine bacterium Vibrio sp. QY101

Bacterial exopolysaccharides have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, Escherichia coli group II capsular polysaccharide was characterized to exert broad-spectrum biofilm inhibition activity. In this study, we firstly reported that a bacterial exopolysaccharide (A101) not only inhibits biofilm formation of many bacteria but also disrupts established biofilm of some strains. A101 with an average molecular weight of up to 546 KDa, was isolated and purified from the culture supernatant of the marine bacterium Vibrio sp. QY101 by ethanol precipitation, iron-exchange chromatography and gel filtration chromatography. High performance liquid chromatography traces of the hydrolyzed polysaccharides showed that A101 is primarily consisted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. A101 was demonstrated to inhibit biofilm formation by a wide range of Gram-negative and Gram-positive bacteria without antibacterial activity. Furthermore, A101 displayed a significant disruption on the established biofilm produced by Pseudomonas aeruginosa, but not by Staphylococcus aureus. Importantly, A101 increased the aminoglycosides antibiotics' capability of killing P. aeruginosa biofilm. Cell primary attachment to surfaces and intercellular aggregates assays suggested that A101 inhibited cell aggregates of both P. aeruginosa and S. aureus, while the cell-surface interactions inhibition only occurred in S. aureus, and the pre-formed cell aggregates dispersion induced by A101 only occurred in P. aeruginosa. Taken together, these data identify the antibiofilm activity of A101, which may make it potential in the design of new therapeutic strategies for bacterial biofilm-associated infections and limiting biofilm formation on medical indwelling devices. The found of A101 antibiofilm activity may also promote a new recognition about the functions of bacterial exopolysaccharides.     

Role of ClpP in biofilm formation and virulence of Staphylococcus epidermidis

Infections caused by the leading nosocomial pathogen Staphylococcus epidermidis are characterized by biofilm formation on implanted medical devices. However, the molecular basis of biofilm formation and its regulation are not completely understood. Here, we describe an important role of the ClpP protease in biofilm development and virulence of S. epidermidis. We constructed an isogenic clpP mutant strain of a biofilm-forming clinical isolate of S. epidermidis. The mutant strain showed decreased biofilm formation in vitro and reduced virulence in a rat model of biofilm-associated infection. Biofilm forming ability of the mutant strain could be restored by expressing clpP on a plasmid, but not when a catalytically inactive allele of clpP gene was introduced. These observations indicate that the peptidase function of ClpP determines its role in biofilm formation. Experimental data in this work also suggested that clpP influenced initial attachment of bacteria on the plastic surface, the first step of biofilm formation. Furthermore, clpP was found to be regulated by the quorum-sensing agr, suggesting that part of the previously described influence of agr on the initial attachment to plastic surfaces may be mediated by clpP.     

Antibacterial and bactericidal activities of Japanese green tea

We found that extracts of Japanese green tea leaves inhibited the growth of various bacteria causing diarrheal diseases. All tea samples tested showed antibacterial activity against Staphylococcus aureus, S. epidermidis, Vibrio cholerae O1, V. cholerae non O1. V. parahaemolyticus, V. mimicus, Campylobacter jejuni and Plesiomonas shigelloides. None of the tea samples had any effect on the growth of V. fluvialis, Aeromonas sobria, A. hydrophila, Pseudomonas aeruginosa, Salmonella enteritidis, enteroinvasive Escherichia coli, enterohemorrhagic E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Enterobacter cloacae or Yersinia enterocolitica. Salmonella and Shigella showed susceptibilities different depending on the kind of Japanese green tea. Japanese green tea showed also bactericidal activity over S. aureus, V. parahaemolyticus and even enteropathogenic E. coli which was not sensitive when tested by cup method. The bactericidal activity was shown even at the drinking concentration in daily life.     

Viper metalloproteinase (Agkistrodon halys pallas) with antimicrobial activity against multi-drug resistant human pathogens

Metalloproteinases are abundant enzymes in crotalidae and viperidae snake venoms. Snake venom metalloproteinases (SVMPs) comprise a family of zinc-dependent enzymes, which display many different biological activities. A 23.1 kDa protein was isolated from Agkistrodon halys (pallas, Chinese viper) snake venom. The toxin is a single chain polypeptide with a molecular weight of 23146.61 and an N-terminal sequence (MIQVLLVTICLAVFPYQGSSIILES) relatively similar to that of other metalloprotein-like proteases isolated from the snake venoms of the Viperidae family. The antibacterial effect of Agkistrodon halys metalloproteinase (AHM) on Burkholderia pseudomallei (strains TES and KHW), Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacterium) was studied at a concentration 120 microM. Interestingly, we found that the metalloproteinase exhibited antibacterial properties and was more active against S. aureus, P. vulgaris, P. mirabilis and multi-drug resistant B. pseudomallei (strain KHW) bacteria. AHM variants with high bacteriostatic activity (MIC 1.875-60 microM) also tended to be less cytotoxic against U-937 human monocytic cells up to 1 mM concentrations. These results suggest that this metalloprotein exerts its antimicrobial effect by altering membrane packing and inhibiting mechanosensitive targets.     

Electrospun zwitterionic poly(sulfobetaine methacrylate) for nonadherent, superabsorbent, and antimicrobial wound dressing applications

Zwitterionic poly(sulfobetaine methacrylate) (PSBMA) has been well studied for its superhydrophilic and ultralow biofouling properties, making it a promising material for superabsorbent and nonadherent wound dressings. Electrospinning provides multiple desirable features for wound dressings, including high absorptivity due to high surface-area-to-volume ratio, high gas permeation, and conformability to contour of the wound bed. The goal of this work is to develop a fibrous membrane of PSBMA via electrospinning and evaluate its properties related to wound dressing applications. Being superhydrophilic, PSBMA fibers fabricated by a conventional electrospinning method would readily dissolve in water, whereas if cross-linker is added, the formation of hydrogel would prevent electrospinning. A three-step polymerization-electrospinning-photo-cross-linking process was developed in this work to fabricate the cross-linked electrospun PSBMA fibrous membrane. Such electrospun membrane was stable in water and exhibited high water absorption of 353% (w/w), whereas the PSBMA hydrogel only absorbed 81% water. The electrospun membrane showed strong resistance to protein adsorption and cell attachment. Bacterial adhesion studies using Gram negative P. aeruginosa and Gram positive S. epidermidis showed that the PSBMA electrospun membrane was also highly resistant to bacterial adhesion. The Ag(+)-impregnated electrospun PSBMA membrane was shown microbicidal, against both S. epidermidis and P. aeruginosa. Such electrospun PSBMA membrane is ideal for a novel type of nonadherent, superabsorbent, and antimicrobial wound dressing. The superior water absorption aids in fluid removal from highly exudating wounds while keeping the wound hydrated to support healing. Because of the resistance to protein, cell, and bacterial adhesion, the dressing removal will neither cause patients' pain nor disturb the newly formed tissues. The dressing also prevents the attachment of environmental bacteria and offers broad-spectrum antimicrobial activity. It is the first work to develop the water-stable electrospun PSBMA membrane, which has great potential for wound dressing and other applications.     

Antipseudomonal Activity of α-Sulfoaminopenicillins

A series of penicillins characterized by the presence of a sulfoamino or a modified sulfoamino group in the side chain was subjected to in vitro antimicrobial screening tests. Although the most potent members of the series were less active than benzylpenicillin against gram-positive bacteria and comparably active against most gram-negative bacteria, they were, on the average, 8 to 16 times more effective against strains of Pseudomonas aeruginosa. In other comparative laboratory tests against P. aeruginosa, these compounds were about as active as carbenicillin and four to eight times more active than ampicillin. An examination of structure-activity relationships indicated that maximal potency was obtained with penicillins having an alpha-(aromatic or heteroaromatic)-alpha-sulfoaminoacetamido side chain. The compound with an alpha-phenyl group was comparable in activity to those having an alpha-(2- or 3-thienyl) group, whereas any modification in position or structure of the alpha-sulfoamino group reduced activity. Results of studies with a cell-free P. aeruginosa beta-lactamase suggest that the marked inhibitory effects of alpha-sulfoamino penicillins for P. aeruginosa can be attributed, at least in part, to their high degree of resistance to this enzyme. Some derivatives, however, had weak antipseudomonal activity, despite possessing a high degree of beta-lactamase resistance.     

Antimicrobial properties of substituted salicylaldehydes and related compounds

A systematic survey of the antimicrobial properties of substituted salicylaldehydes and some related aromatic aldehydes is reported. A total of 23 different compounds, each at four different concentrations, were studied using a panel of seven microbes (Aspergillus niger, Bacillus cereus, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Saccharomyces cerevisiae and Staphylococcus epidermidis) and employing the paper disc agar diffusion method. Several aldehydes, most notably halogenated, nitro-substituted and hydroxylated salicylaldehydes, displayed highly potent activity against the microbes studied, giving inhibitory zones up to 49 mm in diameter (paper disc diameter 6 mm), while unsubstituted benzaldehyde and salicylaldehyde had minimal activity. Further, 4,6-dimethoxysalicylaldehyde had considerable activity against C albicans and slight activity against S. cerevisiae, while displaying minimal activity against bacteria. Also two aromatic dialdehydes had interesting activity. In general, P. aeruginosa was the least sensitive microbe, a result that is in line with observations from a large screening project, in which this microbe was the one against which the least number of active substances was found. Interestingly, the structure-activity relationships of the aldehydes studied were clearly different for different microbes. Many of the aldehydes tested had such high antimicrobial activity that they are noteworthy candidates for practical applications as well as interesting lead compounds for the development of novel antimicrobial drugs and disinfectants. The structure-activity relationships are discussed in detail. For high activity, substituents are required in benzaldehyde as well as in its 2-hydroxy derivative salicylaldehyde. One hydroxy group alone (at the 2-position) is not enough, but further hydroxylation may produce high activity. The effects of substituents are in some cases dramatic. Halogenation, hydroxylation and nitro substitution may produce highly active compounds, but the effects are not easily predicted nor can they be extrapolated from one microbe to another.     

Competitive adherence as a mechanism of bacterial interference

To determine whether competition among bacteria for specific attachment sites on host cells can explain bacterial interference, Staphylococcus aureus strain 502A was tested in turn against two different nasal coryneforms, a strain of Pseudomonas aeruginosa, and a virulent strain of S. aureus, all in the presence of nasal mucosal cells. Particularly examined was the influence of sequence in which bacteria were presented to the nasal cells in comparison with initial mixtures and individual suspensions. Results paralleled those observed in clinical prophylaxis: the bacterium first to adhere to the epithelial cells was able, under uniform conditions, to interfere with the colonization of subsequently added bacteria. Secondary adherence was not eliminated but substantially reduced, and was probably related to steric blockage by the initial colonizer and its particular ability to dissociate from the host cell.     

Modulation of antibiotic resistance in bacterial pathogens by oleanolic acid and ursolic acid

Antibiotic resistance among bacterial pathogens is a serious problem for human and veterinary medicine, which necessitates the development of novel therapeutics and antimicrobial strategies. Some plant-derived compounds, e.g. pentacyclic triterpenoids such as oleanolic acid (OA) and ursolic acid (UA), have potential as a new class of antibacterial agents as they are active against many bacterial species, both Gram-positive and Gram-negative, and specifically target the cell envelope. The aim of the present study was to investigate the influence of OA and UA on the susceptibility of four bacterial pathogens (Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus and Staphylococcus epidermidis) to the β-lactam antibiotics ampicillin (Ap) and oxacillin (Ox). Antimicrobial assays were conducted with bacteria growing in liquid suspension cultures (planktonic cells) or as biofilms. Using FICI value estimation and the time-kill method it was demonstrated that in some combinations, the tested compounds acted in synergy to lower the susceptibility of S. aureus, S. epidermidis and L. monocytogenes to ampicillin and oxacillin, but no synergy was observed for P. aeruginosa. These results indicate that OA and UA may be useful when administered in combination with β-lactam antibiotics to combat bacterial infections caused by some Gram-positive pathogens.     

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.     

Bacteriolysis by immobilized enzymes.

Bacteriolytic enzymes produced by Achromobacter lunatus were immobilized in collagen membrane. Intact bacteria such as Pseudomonas solanacearum, Xanthomonas oryzae, Staphylococcus aureus, and Pseudomonas aeruginosa were lyzed with the bacteriolytic enzyme-collagen membrane. Relative activity of the bacteriolytic enzyme-collagen membrane against Pseu. solanacearum was about 2% of that of native bacteriolytic enzymes. No difference in the optimum pH was observed between immobilized enzymes and native enzymes. The bacteriolytic enzymes in the collagen membrane were stable against sodium chloride which was an inhibitor of the native bacteriolytic enzymes. Xanthomonas oryzae and Pseu. aeruginosa were continuously lyzed by a reactor containing the rolled bacteriolytic enzyme-collagen membrane.