Three-dimensional structures of enzymes useful for beta-lactam antibiotic production

Significant advances have been made in the structure-based engineering of enzymes useful for beta-lactam antibiotic production. Structure-based engineering of penicillin G acylase and cephalosporin acylase has resulted in improved enzymes for use in enzymatic production processes. The structures of many other enzymes that could be used in the production of beta-lactam antibiotics, such as enzymes from the beta-lactam biosynthetic pathway and beta-lactam antibiotic-converting enzymes, have been determined. The interest in these structures suggests that the future may see an even more extensive use of rationally engineered biocatalysts in antibiotic production than today.     

Recombinant microorganisms for industrial production of antibiotics

The enhancement of industrial antibiotic yield has been achieved through technological innovations and traditional strain improvement programs based on random mutation and screening. The development of recombinant DNA techniques and their application to antibiotic producing microorganisms has allowed yield increments and the design of biosynthetic pathways giving rise to new antibiotics. Genetic manipulations of the cephalosporin producing fungus Cephalosporium acremonium have included yield improvements, accomplished increasing biosynthetic gene dosage or enhancing oxygen uptake, and new biosynthetic capacities as 7-aminocephalosporanic acid (7-ACA) or penicillin G production. Similarly, in Penicillium chrysogenum, the industrial penicillin producing fungus, heterologous expression of cephalosporin biosynthetic genes has led to the biosynthesis of adipyl-7-aminodeacetoxycephalosporanic acid (adipyl-7-ADCA) and adipyl-7-ACA, compounds that can be transformed into the economically relevant 7-ADCA and 7-ACA intermediates. Escherichia coli expression of the genes encoding D-amino acid oxidase and cephalosporin acylase activities has simplified the bioconversion of cephalosporin C into 7-ACA, eliminating the use of organic solvents. The genetic manipulation of antibiotic producing actinomycetes has allowed productivity increments and the development of new hybrid antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 216-226, 1997.     

Applications of Biocatalysis to Biotechnology

β-lactam antibiotics (e.g. penicillins, cephalosporins) are of major clinical importance and contribute to over 40% of the total antibiotic market. These compounds are produced as secondary metabolites by certain actinomycetes and filamentous fungi (e.g. Penicillium, Aspergillus and Acremonium species). The industrial producer of penicillin is the fungus Penicillium chrysogenum. The enzymes of the penicillin biosynthetic pathway are well characterized and most of them are encoded by genes that are organized in a cluster in the genome. Remarkably, the penicillin biosynthetic pathway is compartmentalized: the initial steps of penicillin biosynthesis are catalyzed by cytosolic enzymes, whereas the two final steps involve peroxisomal enzymes. Here, we describe the biochemical properties of the enzymes of β-lactam biosynthesis in P. chrysogenum and the role of peroxisomes in this process. An overview is given     

Preparation, characterization and evaluation of biocomposite films containing chitosan and sago starch impregnated with silver nanoparticles

The positive attributes of excellent biocompatibility and biodegradability of biopolymers with versatile biological activities have provided ample opportunities for further development of functional biomaterials of high potential in various fields. The biopolymers used in this study, i.e. chitosan and sago starch are abundantly available in nature and can be used in various biomedical applications. In the present study, the composite films of chitosan (Ch) and sago starch (SG) impregnated with silver nanoparticles (AgNP) with and without antibiotic gentamicin (G) were prepared by solvent casting method. The films prepared were characterized for their physic-chemical properties using conventional methods. The results obtained showed that with the increase of chitosan content in the composite results in decrease in its water absorption capacity. The FTIR and SEM studies have shown the composite nature of the films prepared. Ch-SG-AgNP and Ch-SG-AgNP-G composites were used as wound dressing materials in experimental wounds of rats. The healing pattern of the wounds was evaluated by planimetric studies, macroscopic observations, biochemical studies and histopathological observations. The results have shown faster healing pattern in the wounds treated with Ch-SG-AgNP and Ch-SG-AgNP-G composites compared to untreated control. This study suggests that Ch-SG-AgNP film may be a potential candidate as a dressing material for wound healing applications.     

Wound fluid bacterial levels exceed tissue bacterial counts in controlled porcine partial-thickness burn infections

In the present study, an established controlled burn wound model was used to test the hypothesis that controlled surface contamination with is capable of generating a noninvasive method for the creation of a reproducible deep tissue burn wound infection. Using a liquid tight-wound chamber in Yorkshire pigs, partial-thickness burns were inoculated with saline-immersed for 24 hours. Noninoculated burns and unwounded skin immersed in normal saline served as controls. Bacterial cultures of wound fluid were performed daily, and tissue biopsies for bacteriological and histological evaluations were performed on days 1, 3, and 5. was only recovered from -inoculated wounds (tissue and fluid), whereas all controls contained endogenous only. The number of colony-forming units per gram of wound tissue did not correlate with the bacterial counts found in the overlying wound fluid for any wounds. Fluid counts were consistently higher than tissue counts by two logs. -inoculated wounds showed three times deeper tissue destruction than control wounds. Obtaining consistently deep tissue colonization without cross-contamination among wounds, this study introduces a noninvasive model for controlled burn wound infection suitable for future investigations regarding the efficacy of topical antibiotic wound treatment in experimental burns.     

Microbial isopenicillin N synthase genes: structure, function, diversity and evolution.

Clinically and economically, penicillins and cephalosporins are the most important class of the beta-lactam antibiotics. They are produced by a wide variety of microorganisms including numerous species of Streptomyces, some unicellular bacteria and several filamentous fungi. A key step common to their biosynthetic pathways is the conversion of a linear, cysteine-containing tripeptide to a bicyclic beta-lactam antibiotic by isopenicillin N synthase. Recent successes in the cloning and expression of isopenicillin N synthase genes now permit production of a plentiful supply of this enzyme, which may be used for structural and mechanistic studies, or for biotechnological applications in the creation of novel beta-lactam compounds from peptide analogues. New ideas concerning the evolution and prevalence of the penicillin and cephalosporin biosynthetic genes have emerged from studies of isopenicillin N synthase genes.     

The effect of selected factors on the course of wound healing after surgery for laryngeal neoplasms

Finding of the a etiologic factors and participation of bacteria flora in wound healing in laryngeal cancer treatment was the purpose of our study. Investigations were performed in 27 patients. Swabs were taken from the postoperative wounds. Detailed identifications of the bacteria flora and antibiotic susceptibility of bacteria were performed. Wound healing was estimated according to extension of the carcinoma, applied antibiotics, state of the oral cavity, the kind of bacteriological flora isolated after surgical treatment from postoperative wounds. It was found that wound healing depended on the extension of carcinoma, as well as, type of isolated bacteria and antibiotic therapy used. The proper healing of postoperative wounds was not dependent on the state of the oral cavity and the dentition. The main cause of postoperative complication of wounds was methicillin-resistant Staphylococcus aureus (MRSA).     

PYOGENIC INFECTIONS IN ORTHOPEDIC SURGERY—Prevention and Management

A problem that confronts surgeons in clinical practice is that a patient may acquire new infections while in the hospital. When such infections occur they are predominantly staphylococcal and these bacteria are often, but not always resistant to penicillin, streptomycin and the tetracycline antibiotics. They are often but neither completely nor uniformly sensitive to the newer or less frequently used antimicrobial agents.The extension of antibiotic usage from proven situations to “routine” prophylaxis has been a widespread practice. There are many reasons to discourage and to reexamine the validity and purpose, as well as the safety of this practice. We now have sufficient background and experience to revert from widespread and indiscriminate use to a practice of discriminate prophylactic therapy.In general, soft tissue lacerations and clean wounds do not require operation under an “antibiotic umbrella.” Similarly, elective orthopedic surgical procedures of soft tissues such as muscle biopsy, tenorrhaphy and muscle and tendon transplants as well as plastic surgical procedures can be safely performed without antibiotic therapy if technique is good and operation not prolonged. Operations of major magnitude on the motor-skeletal system, such as open fractures, internal fixation of fractures with bone grafts, and major operations of joints are indication for antibiotic therapy for impending infection postoperatively for five days. Reliance is mainly on antistaphylococcal drugs to which hospital organisms are predominantly sensitive. The two remaining indications for antibiotic therapy against impending infection are: (1) major crush injury—for example, to the thigh—and (2) the need for a patient with a healing fracture to have other surgical procedures such as tooth extraction or excision of an infected area which might predispose to transient bacteremia and embolic infection in bone or joint.     

Nanocrystalline silver dressings in wound management: a review

This paper describes the properties of nanocrystalline silver products (Acticoat) and their applications and examines available evidence supporting their use in wound management. Acticoat utilizes nanotechnology to release nanocrystalline silver crystals. Acticoat releases 30 times less silver cations than silversulfadiazine cream or 0.5% silver nitrate solution but more of the silver released (by Acticoat). Silver-impregnated slow-release dressings release minute concentrations of silver which are quickly bound up by the chloride in the wound exudate. While extrapolations from in vitro and animal studies are cautious, evidence from these studies suggests Acticoat is: effective against most common strains of wound pathogens; can be used as a protective covering over skin grafts; has a broader antibiotic spectrum activity; and is toxic to keratinocytes and fibroblasts. Animal studies suggest a role for nanocrystalline silver in altering wound inflammatory events and facilitation of the early phase of wound healing. Quality human clinical trials into nanocrystalline silver are few. However, evidence suggests using Acticoat in wound management is cost effective, reduces wound infection, decreases the frequency of dressing changes and pain levels, decreases matrix metalloproteinase activity, wound exudate and bioburden levels, and promotes wound healing in chronic wounds. Although there is no in vivo evidence to suggest nanocrystalline silver is toxic to human keratinocytes and fibroblasts, there is in vitro evidence to suggest so; thus these dressings should be used cautiously over epithelializing and proliferating wounds. Future clinical research, preferably randomized controlled trials into nanocrystalline silver technology, may provide clinicians a better understanding of its applications in wound management.     

14S,21R-dihydroxydocosahexaenoic acid remedies impaired healing and mesenchymal stem cell functions in diabetic wounds

Treatment of diabetes-impaired wound healing remains a major unresolved medical challenge. Here, we identified suppressed formation of a novel reparative lipid mediator 14S,21R-dihydroxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid (14S,21R-diHDHA) in cutaneous wounds of diabetic db/db mice. These results indicate that diabetes impedes the biosynthetic pathways of 14S,21R-diHDHA in skin wounds. Administration of exogenous 14S,21R-diHDHA to wounds in diabetic animals rescued healing and angiogenesis. When db/db mesenchymal stem cells (MSCs) were administered together with 14S,21R-diHDHA to wounds in diabetic animals, they coacted to accelerate wound re-epithelialization, granulation tissue formation, and synergistically improved vascularization. In the pivotal cellular processes of angiogenesis, 14S,21R-diHDHA enhanced VEGF release, vasculature formation, and migration of db/db dermal microvascular endothelial cells (DMVECs), as well as remedied paracrine angiogenic functions of db/db MSCs, including VEGF secretion and the promotion of DMVEC migration and vasculature formation. Our results show that 14S,21R-diHDHA activates the p38 MAPK pathway in wounds, db/db MSCs, and DMVECs. Overall, the impeded formation of 14S,21R-diHDHA described in this study suggests that diabetes could affect the generation of pro-healing lipid mediators in wound healing. By restoring wound healing and MSC functions, 14S,21R-diHDHA is a new lead for the development of better therapeutics used in treating wounds of diabetics.     

Organization of the gene cluster for biosynthesis of penicillin in Penicillium nalgiovense and antibiotic production in cured dry sausages

Several fungal isolates obtained from two cured meat products from Spain were identified as Penicillium nalgiovense by their morphological features and by DNA fingerprinting. All P. nalgiovense isolates showed antibiotic activity in agar diffusion assays, and their penicillin production in liquid complex medium ranged from 6 to 38 microgram. ml-1. We constructed a restriction map of the penicillin gene cluster of P. nalgiovense and found that the organization of the penicillin biosynthetic genes (pcbAB, pcbC, and penDE) is the same as in Penicillium chrysogenum and Aspergillus nidulans. The pcbAB gene is located in an orientation opposite that of the pcbC and penDE genes in all three species. Significant amounts of penicillin were found in situ in the casing and the outer layer of salami meat during early stages of the curing process, coinciding with fungal colonization, but no penicillin was detected in the cured salami. The antibiotic produced in situ was sensitive to penicillinase.     

Incidence of Staphylococcus aureus isolated from patients treated at the Clinical Center of Skopje, Macedonia, with special attention to MRSA

The distribution of 3497 Staphylococcus aureus strains according to methicillin resistance, specimens, departmental profession and antibiotic resistance patterns was analysed. The strains were cultured from the patients of the Clinical Center of Skopje, Macedonia, between 1 January 2002 and 31 December 2004. The majority of the isolates was obtained from suppurated wounds (28.5%), nares (21%), intratracheal tubes (13%) and blood cultures (11.8%). Overall 1100 (31.4%) of the isolates was methicillin-resistant with 1 microg oxacillin disc. Of these 35.5%, 30.5% and 10.4% were cultured from wounds, intratracheal tubes and blood samples, respectively. The prevalence of MRSA strains was 78.6%, 75%, 44.2% and 37.3% in specimens of ICU, Coma Center, General Surgery and Haematology patients. There were extremely big differences in the frequency of MRSA between departments with particular specialisation. The 2397 MSSA isolates belonged to practically one antibiotic resistance pattern characterised with penicillin resistance and susceptibility to other antistaphylococcal drugs. The 1100 MRSA isolates distributed to four antibiotic resistance patterns on the basis of their resistance to oxacillin, penicillin, amoxicillin+clavulanic acid, azithromycin, clindamycin, amikacin, gentamicin, ciprofloxacin, trimethoprim+sulphamethoxasole, vancomycin and teicoplanin. All the MRSA isolates were multidrug resistant but sensitive to glycopeptides.     

Application of wound dressing Molndal technique in clean and potentially contamined postoperative wounds--initial comparative study

Because of a possible delayed wound healing, critical colonization and infection of wounds present a problem for surgeons, particularly in patients with compromised immune system or in case where the wound is heavy contaminated or poorly perfused. Molndal technique of wound dressing has proven to be effective in prevention of infection. In our study we wanted to describe the benefits of the application of Molndal technique wound dressing compared to traditional wound dressing technique at potentially contaminated and clean postoperative wounds. We examined postoperative wound after radical excision of pilonidal sinus and after implantation of partial endoprosthesis in hip fracture. Molndal technique consisted of wound dressing with Aquacel Ag - Hydrofiber. Traditional technique was performed using gauze compresses and hypoallergic adhesives. We analyzed the results of 50 patients after radical excision of pilonidal sinus. 25 patients were treated by Molndal technique and 25 patients by the traditional technique of wound dressing. In the group treated by Molndal technique only 1 (4%) patient has revealed a wound infection, proven by positive microbiological examination and suppuration. In the traditional technique group 4 (16%) patients developed wound infection as inflammation and secretion as a sign of superficial infection. In the other group we analyzed the results of 50 patients after implantation of partial endoprosthesis after hip fracture. 20 patients were treated by Molndal technique and 30 patients by the traditional technique of wound dressing. In the group treated by Molndal technique no patient has revealed a wound infection (0%). In the traditional technique group 4 (13%) patients developed wound infection. All complication in both group were superficial incisional surgical infection (according to HPSC). There was no deep incisional surgical site infection or organ/space surgical site infection. Our results are clearly showing that Molndal technique is effective in preventing the postoperative wound infection.     

Our Clinical Experience using Cryopreserved Cadaveric Allograft for the Management of Severe Burns

Human cadaveric allograft is the most commonly used alternative wound closure material for excised burn wounds when limited donor sites or the overall patient condition does not permit immediate grafting with autologous skin. The Skin Bank in Singapore has supplied a total of 33,000 cm² of cryopreserved cadaveric allograft to the Burns Centre in Singapore for the early post-burn treatment of 17 severely burned patients with body surface area (BSA) burn wounds averaging 58% (range 33–90). An average of 13% (range 3–30%) cadaveric allograft was transplanted on 17 patients. Seven patients had recovered from their burn injuries and ten patients died of multiple complications related to their burn injuries. Our clinical observation has shown good adherence of cadaveric allograft when applied on clean and debrided wound bed after 4–7 days of post-operation. The availability of cadaveric allograft permits early excision and wound coverage before the patients' condition is further compromised. Our clinical experience using cryopreserved cadaveric allograft is proving to be indispensable in the management of patients with severe burns. This revised version was published online in July 2006 with corrections to the Cover Date.     

烧伤病房患者创面金葡菌分布及耐药性分析

目的分析烧伤病房患者不同创面金葡菌的分布及耐药性,为临床合理选用抗菌药提供依据。方法对2006年1月至2013年12月间中国人民解放军第八五医院烧伤病房患者创面分离出金葡菌,采用K—B纸片扩散法进行药物敏感试验。分析金葡菌的耐药性,并对难愈性创面、非难愈性创面的耐甲氧西林金葡菌（MRSA）与甲氧西林敏感金葡菌（MSSA）的耐药性进行对比分析。结果分离出金葡菌112株,其中难愈性创面有70株MRSA和17株MSSA来自难愈性创面,16株MRSA和9株MSSA来自非难愈性创面。金葡菌对青霉素、红霉素、克林霉素的耐药率较高（分别为94．64％、81．25％和74．11％）,对复方新诺明、呋喃妥因的耐药率较低（分别为16．07％和1．79％）,对万古霉素、利奈唑烷的耐药率为0。MRSA的耐药率高于MSSA。来源于难愈性创面与非难愈性创面的MRSA仅在对利福平的耐药率上有明显差异,而来源于两创面的MSSA的耐药率无明显差异。结论创面金葡菌中MRSA的构成比高,难愈性创面MRSA耐药严重,应积极防控创面MRSA感染和扩散。     

Photodynamic therapy for Staphylococcus aureus infected burn wounds in mice.

The rise of multiply antibiotic resistant bacteria has led to searches for novel antimicrobial therapies to treat infections. Photodynamic therapy (PDT) is a potential candidate; it uses the combination of a photosensitizer with visible light to produce reactive oxygen species that lead to cell death. We used PDT mediated by meso-mono-phenyl-tri(N-methyl-4-pyridyl)-porphyrin (PTMPP) to treat burn wounds in mice with established Staphylococcus aureus infections The third degree burn wounds were infected with bioluminescent S. aureus. PDT was applied after one day of bacterial growth by adding a 25% DMSO/500 microM PTMPP solution to the wound followed by illumination with red light and periodic imaging of the mice using a sensitive camera to detect the bioluminescence. More than 98% of the bacteria were eradicated after a light dose of 210 J cm(-2) in the presence of PTMPP. However, bacterial re-growth was observed. Light alone or PDT both delayed the wound healing. These data suggest that PDT has the potential to rapidly reduce the bacterial load in infected burns. The treatment needs to be optimized to reduce wound damage and prevent recurrence.     

Antibiotic Therapy of Staphylococcal Infections

The antibiotic treatment of staphylococcal infections remains a problem. Isolation of the organism and sensitivity testing are necessary in the choice of antibiotic. Penicillin G is the most effective penicillin against non-penicillinase-producing staphy-lococci; for the penicillinase producers there is very little to choose between the semisynthetic penicillins, methicillin, cloxacillin, nafcillin and oxacillin. For patients who are hypersensitive to penicillin, the bacteriostatic drugs (erythromycin, novobiocin, tetracycline, chloramphenicol, oleandomycin) are useful for mild infections, while for more severe illness the bactericidal drugs (vancomycin, ristocetin, kanamycin, bacitracin, neomycin) have been used successfully. Acute staphylococcal enterocolitis is probably best treated by a semisynthetic penicillin. Other antibiotics which have been found useful, with clinical trials, for staphylococcal infections are cephalosporin, fucidin, cephaloridine and lincomycin. The latter drug has been reported of value in the treatment of osteomyelitis. There is little justification for the prophylactic use of antibiotics to prevent staphylococcal infection. Surgical drainage is still an important adjunct in the treatment of many staphylococcal infections.     

Antibiotic sensitivity of Corynebacterium amycolatum

C. amycolatum strains belongs to opportunistic bacteria considered as etiological factors of hospital infections. It's usually handled as a human natural flora, so antibiotic sensitivity is not checked. There's a few reports relative to antibiotic sensitivity of C. amycolatum in the world literature. So, we decided to examine antibiotic sensitivity of isolated strains. The 70 of C. amycolatum strains isolated from clinical samples from patients hospitalised at Samodzielny Publiczny Szpital Kliniczny in Bydgoszcz were analysed. Antimicrobial susceptibility testing of the strains was performed by means of a disk diffusion method. 28.6% of analysed strains were susceptible to penicillin and 38.6% to ampicillin. Susceptibility to another 16 antibiotics was from 40.0% for ceftazidime to 64.3% for ceftriaxone. Penicillinase was not produced by analysed strains. We stated higher percentage of strains susceptible to combinations of penicillin with inhibitors than to penicillin and ampicillin. The most strains were susceptible to quinupristin-dealfopristin, linezolid and glycopeptide antibiotics but resistance to mupirocin. 35.7% analysed strains were multiresistance; there were resistance to beta-lactams (approximately 100%), lincosamides (96.0%), macrolides (92.0%) and quinolones (92.0%). Multiresistant strains were the most frequently isolated from wound swabs (60.0%) and mainly came from patients treated at the departments of general surgery (28.0%) and vascular surgery (16.0%).     

Determination of the rate-limiting step(s) in the biosynthetic pathways leading to penicillin and cephalosporin

Summary This paper is a review of strategies that have been used, or that could be used, to determine the rate-limiting step(s) in the biosynthetic pathways leading to penicillin or cephalosporin. Information is summarized from published material that involves studies with low-producing strains ofPenicillium chrysogenum andCephalosporium acremonium. We also summarize information derived from some high-producing production strains. Identification of the rate-limiting step(s) was of great interest to us as the first step in a rational program to further improve antibiotic titers of these highly developed strains. A number of approaches that could be used to elucidate the rate-limiting step(s) are described herein.