可注射性硫酸钙在免椎体松质骨中成骨性能的研究

目的 观察可注射性硫酸钙在兔椎体松质骨中的成骨降解性能.方法 18只成年新西兰兔随机分为A、B两组,每组9只.其中A组向兔椎体松质骨注入硫酸钙骨水泥（calcium sulfate cement,CSC）;B组作为空白对照组不植入材料.在不同时间点（2,6和12周）采用大体观察、X片影像学、组织学等方法观察和评估CSC植入后在椎体松质骨内的成骨和降解情况.结果 术后兔进食,活动正常,标本周围软组织无炎症反应.CSC在植入2周见少量新生骨,6周植入物部分吸收,见大量新生骨,12周植入物全部吸收,并有编织骨形成,成骨量明显高于对照组（P＜0.05）.结论 可注射性硫酸钙有良好的生物相容性,植入椎体后成骨作用明显,新生骨与植入材料的吸收降解同步,是一种良好的骨移植材料.     

Sonication of Explanted Cardiac Rhythm Management Devices for the Diagnosis of Pocket Infections and Asymptomatic Bacterial Colonization

Background: Correct diagnosis of the causative organism is critical for the treatment of pacemaker and defibrillator pocket infections. No gold standard for this exists, although swab and tissue cultures are frequently used. The purpose of this study was to determine the value of ultrasonication of explanted generators in the diagnosis of pocket infections and asymptomatic bacterial colonization. Methods: Samples were prospectively collected during pacemaker and defibrillator generator extractions for elective replacements, upgrades, or pocket infections. The devices were placed in an ultrasonicator for 5 minutes and the fluid sent for culture, along with swab and tissue cultures. Results: Eighty‐two patients with pacemakers (n = 46) or defibrillators (n = 36) underwent generator explantation, 66 of these for elective reasons and 16 for pocket infection. In patients with pocket infection, 15 (94%) received a definitive bacterial diagnosis using a combination of all three‐culture modalities. Cultures were positive in 15 sonicated fluid, 13 tissue, and 11 swab samples, with Staphylococcus aureus and other skin flora commonly seen. In asymptomatic patients, 14 (21%) had positive cultures. Cultures were positive in 11 sonicated fluid, eight tissue, and two swab samples. Skin flora was commonly seen, but three of the sonicated fluid cultures grew gram‐negative rods. No patients with asymptomatic colonization developed clinical infection during the follow‐up period. Conclusions: Ultrasonication is an inexpensive and simple technique that improves the bacteriologic diagnosis of device pocket infections. It also identifies a significant proportion of patients with asymptomatic colonization, although this is not a marker of future pocket infection. (PACE 2011; 34:143–149)     

Prevention of Biofilm Colonization by Gram-Negative Bacteria on Minocycline-Rifampin-Impregnated Catheters Sequentially Coated with Chlorhexidine

Resistant Gram-negative bacteria are increasing central-line-associated bloodstream infection threats. To better combat this, chlorhexidine (CHX) was added to minocycline-rifampin (M/R) catheters. The in vitro antimicrobial activity of CHX-M/R catheters against multidrug resistant, Gram-negative Acinetobacter baumannii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia was tested. M/R and CHX-silver sulfadiazine (CHX/SS) catheters were used as comparators. The novel CHX-M/R catheters were significantly more effective (P < 0.0001) than CHX/SS or M/R catheters in preventing biofilm colonization and showed better antimicrobial durability.     

A Review on Basic Biology of Bacterial Biofilm Infections and Their Treatments by Nanotechnology-Based Approaches

Biofilms are responsible for causing 80% of human infections including chronic infections like-cystic fibrosis, endocarditis and osteomyelitis. The growing ability of the biofilm to resist most of the available antibiotics has caused a serious threat to different life forms. Plenty of research work has already been reported, and some are ongoing to combat this serious health issue worldwide. Recent developments in nanotechnology have given a great boost in dealing biofilm infections. The unique size-dependent properties for antibacterial and antibiofilm activities provide the nanoparticles better options to eradicate biofilms. Here, the authors have discussed the basic biology of bacterial biofilm and their impact on human health. In addition, different nanotechnology-based strategies to overcome serious health issues caused by biofilm infections have been highlighted.

     

Impact of Bacterial Biofilm Formation on In Vitro and In Vivo Activities of Antibiotics

The impact of bacterial adherence on antibiotic activity was analyzed with two isogenic strains of Staphylococcus epidermidis that differ in the features of their in vitro biofilm formation. The eradication of bacteria adhering to glass beads by amikacin, levofloxacin, rifampin, or teicoplanin was studied in an animal model and in a pharmacokinetically matched in vitro model. The features of S. epidermidis RP62A that allowed it to grow on surfaces in multiple layers promoted phenotypic resistance to antibiotic treatment, whereas strain M7 failed to accumulate, despite initial adherence on surfaces and growth in suspension similar to those for RP62A. Biofilms of S. epidermidis M7 were better eradicated than those of strain RP62A in vitro (46 versus 31%; P < 0.05) as well as in the animal model (39 versus 9%; P < 0.01).     

硫酸镁与葡萄糖酸钙联合防治奥沙利铂神经毒性临床观察

目的:观察硫酸镁与葡萄糖酸钙联合防治奥沙利铂神经毒性的近期疗效。方法:采用FOFLOX 4方案化疗的结直肠癌患者81例,随机分为A、B两组。A组予硫酸镁+葡萄糖酸钙+化疗。B组单纯化疗。结果:可评价病例81例,A组41例,神经毒性1级7例,2级4例,3级0例,总发生率26.8%。B组40例,神经毒性1级9例,2级10例,3级4例,总发生率57.5%。两组差异有统计学意义(P<0.01)。结论:硫酸镁与葡萄糖酸钙联用能降低奥沙利铂神经毒性发生。     

细菌生物被膜的防治进展

细菌生物被膜（bacterialbiofilm,BF）是指多个细菌不可逆地黏附于机体或物体表面,被自身细胞分泌的基质所包裹,能表达一套与浮游细菌不同的基因,即一个新的表型。它是细菌的三维聚集体,在许多生物医学材料相关性感染和慢性感染疾病中起着重要作用,所致疾病迁延不愈,是医学工作者面临的巨大挑战。因此,如何防止其形成和有效清除已形成的生物被膜,在临床实践中至关重要。现对细菌生物被膜的防治综述如下。     

Derivatives of the Mouse Cathelicidin-Related Antimicrobial Peptide (CRAMP) Inhibit Fungal and Bacterial Biofilm Formation

We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 μM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 μM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.     

In Vitro Elution of Daptomycin by a Synthetic Crystallic Semihydrate Form of Calcium Sulfate,Stimulan

A synthetic crystallic semihydrate form of calcium sulfate, Stimulan, was evaluated as a biodegradable carrier for the daily in vitro elution of daptomycin. Daptomycin and Stimulan were admixed at a ratio of 95:5. Elution lasted for 28 days. Eluted concentrations peaked on days 1 and 11, when the mean values were 1,320.1 and 949.2 μg/ml, respectively. The lowest eluted concentration was detected on day 28. These results support the application of the system described in experimental models of osteomyelitis.Chronic osteomyelitis is an infection difficult to treat due both to multidrug resistance of common pathogens and to poor penetration of antibiotics into bone (16). Carriers for local delivery of antimicrobials have been developed, attempting to provide locally high concentrations of antibiotics (5). Some newly developed biodegradable carriers have been shown to be very potent for the eradication of experimental osteomyelitis (6, 7). The semihydrate form of calcium sulfate (CaSO₄), commonly known as plaster of Paris, may be applied as a biodegradable system for local drug delivery. It has been used for decades to fill bone cavities resulting from disease, trauma, or surgery (11). It was recently shown to be potent in vitro for the release of vancomycin, teicoplanin, gentamicin, and clindamycin (15). The antimicrobial applied in such an elution system should be active against the most commonly involved pathogens of chronic bone infections, namely, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (CoNS) (5). Daptomycin, which is a novel lipopeptide antimicrobial with excellent in vitro activity against these isolates (4), may be a candidate for application in a system for local drug delivery.Stimulan (Biocomposites, Keele Science Park, Staffordshire, United Kingdom) is a synthetic biocompatible bone graft material made of calcium sulfate. It is completely reabsorbed and replaced with new bone. It is synthesized at 100% purity with no traces of potentially toxic impurities that have been associated with naturally occurring mineral sources of calcium sulfate. It has been recently shown by our group that moxifloxacin and fusidic acid may be eluted at high concentrations in vitro by using Stimulan as a carrier (11). The purpose of the present study was to develop an in vitro system for daptomycin elution by using Stimulan as a delivery system.Stimulan was mixed with daptomycin (Novartis, Basel, Switzerland) at a ratio 95:5 to a total weight of 3 g. This was put into sterile vials (160 by 100 mm) and left at room temperature for 15 to 30 min for solidification. Five similar vials were prepared. One milliliter of Mueller-Hinton broth (Trec Diagnostic Systems, West Sussex, United Kingdom) was added over the free surface of each mixture and replaced every 24 h. The vials were incubated at 37°C. On each consecutive day, the eluent was removed, transferred to a sterile plastic tube, and replaced with 1 ml of broth. That procedure was repeated until the optical degradation of the prepared mixture. Samples were stored at −70°C until analysis.Concentrations of daptomycin were estimated in duplicate by a modification of the method already described (3). Briefly, 300 μl of sample was mixed with 20 μl of 99% methanol and centrifuged for 10 min at 5,000 rpm and 4°C. Twenty microliters of the supernatant was injected into a high-pressure liquid chromatography system (1100 series; Agilent, Waldbronn, Germany) with the following elution characteristics: a Zorbax Eclipse XDB-C₈ (4.6 by 150 mm, 5 μm) separating column (Agilent Technologies) warmed at 37°C, a mobile phase of 32.6% acetonitrile and 67.4% 0.5% ammonium phosphate buffer at a flow rate of 1.5 ml/min, and UV detection at 214 nm. The retention time of daptomycin was 3.3 min, and it was estimated as micrograms per milliliter by a standard curve created with known concentrations of daptomycin. The lower detection limit was 6.25 μg/ml, and the interday coefficient of variation of the assay was 4.7%. Results were expressed as means ± standard errors. The area under curve (AUC) for each vial was determined by the linear trapezoidal rule.Elution of daptomycin lasted 28 days. Then the prepared mixture was destroyed. Eluted concentrations peaked on days 1 and 11, when the mean concentrations were 1,320.1 and 949.2 μg/ml, respectively. The lowest eluted concentration was detected on day 28, and the mean value was 233.9 μg/ml (Fig. ​(Fig.1).1). This gradual step-down rate of release over the 28 days was consistent for all five replicates. The mean ± standard deviation AUC of daptomycin elution over this 28-day period was 14,542.7 ± 1,925.9 μg · day/ml. Short peaks of daptomycin release were apparent on days 5 and 11. These may be related to the properties of Stimulan.Open in a separate windowFIG. 1.Elution of daptomycin by Stimulan.Daptomycin is a new lipopeptide that has been recently licensed for the management of skin and soft-tissue infections (13). It has excellent intrinsic activity against MRSA and methicillin-resistant CoNS but also vancomycin-resistant enterococci (10). The MICs for 90% of the strains of MRSA, methicillin-resistant CoNS, and vancomycin-resistant enterococcal isolates tested are reported to be 0.78, 0.44, and 0.5 μg/ml, respectively (1, 4). Although the eluted daptomycin concentrations obtained with the system described here are much greater than the above-mentioned MICs for 90% of the strains tested, results should be interpreted with caution, since the kinetics of release apply to an in vitro system and not to in vivo conditions. It should, however, be underscored that eluted levels are considerably greater than the concentrations that are required to eliminate the growth of small-colony variants of S. aureus that are often implicated in chronic bone infections (1).The estimated AUC of elution may be considered indirect evidence of the total amount of drug released (2). The AUC for daptomycin by the elution system presented here is much greater than the AUC required for the management of an experimental thigh infection with MRSA in mice (9).Calcium sulfate has been applied as a carrier for daptomycin in two previous studies. The drug was admixed with CaSO₄ in pellet form. The total time of drug elution was 28 days, but the eluted concentrations were 10- to 100-fold lower than those achieved here (12). Even at a low rate of elution, the drug amounts released inhibited the growth of 10⁴ CFU of two different locally instilled isolates, one of S. aureus and another of S. epidermidis (14).Although the effectiveness of daptomycin has not been evaluated in any prospective randomized clinical trial for the therapy of staphylococcal osteomyelitis, retrospective data favor its application for the management of such infections (8). These favorable clinical prospects, along with the successful in vitro elution of daptomycin from Stimulan reported in the present study, support the application of the biodegradable system described here in experimental models of osteomyelitis.     

Perioperative and Modifiable Risk Factors for Periprosthetic Joint Infections (PJI) and Recommended Guidelines

Purpose of Review

Periprosthetic joint infection (PJI) remains a complication that is associated with high morbidity as well as high healthcare expenses. The purpose of this review is to examine patient and perioperative modifiable risk factors that can be altered to help improve rates of PJI.

Recent Findings

Evidence-based review of literature shows that improved control of post-operative glycemia, appropriate management of obesity, malnutrition, metabolic syndrome, preoperative anemia, and smoking cessation can help minimize risk of PJI. Additionally, use of Staphylococcus aureus screening, preoperative evaluation of vitamin D levels, screening for urinary tract infection, and examination of dental hygiene can help with improving rates of PJI; similarly, appropriate management of perioperative variables such as limiting operating room traffic, appropriate timing, and selection of prophylactic antibiotics and surgical site preparation can help to decrease rates of PJI.

Summary

In summary, PJI is a morbid complication of total joint arthroplasty. Surgeons should be vigilant of modifiable risk factors that can be improved upon to help minimize the risk of PJI.

     

Bacteriophage Cocktail for the Prevention of Biofilm Formation by Pseudomonas aeruginosa on Catheters in an In Vitro Model System

Microorganisms develop biofilms on indwelling medical devices and are associated with device-related infections, resulting in substantial morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated catheters with Pseudomonas aeruginosa bacteriophages on biofilm formation by P. aeruginosa in an in vitro model. Hydrogel-coated catheters were exposed to a 10 log₁₀ PFU ml⁻¹ lysate of P. aeruginosa phage M4 for 2 h at 37°C prior to bacterial inoculation. The mean viable biofilm count on untreated catheters was 6.87 log₁₀ CFU cm⁻² after 24 h. The pretreatment of catheters with phage reduced this value to 4.03 log₁₀ CFU cm⁻² (P < 0.001). Phage treatment immediately following bacterial inoculation also reduced biofilm viable counts (4.37 log₁₀ CFU cm⁻² reduction; P < 0.001). The regrowth of biofilms on phage-treated catheters occurred between 24 and 48 h, but supplemental treatment with phage at 24 h significantly reduced biofilm regrowth (P < 0.001). Biofilm isolates resistant to phage M4 were recovered from catheters pretreated with phage. The phage susceptibility profiles of these isolates were used to guide the development of a five-phage cocktail from a larger library of P. aeruginosa phages. The pretreatment of catheters with this cocktail reduced the 48-h mean biofilm cell density by 99.9% (from 7.13 to 4.13 log₁₀ CFU cm⁻²; P < 0.001), but fewer biofilm isolates were resistant to these phages. These results suggest the potential of applying phages, especially phage cocktails, to the surfaces of indwelling medical devices for mitigating biofilm formation by clinically relevant bacteria.Indwelling medical devices of various kinds may become colonized with microorganisms, resulting in the formation of microbial biofilms (16). Biofilm-associated organisms are tolerant to antimicrobial agents, can evade the host immune system, and can act as a nidus for infection (16). As a result, device-related infections, such as catheter-associated bloodstream infections, cause substantial morbidity and mortality among specific patient populations (9). Attributable mortality rates for healthcare-associated bloodstream infections have been estimated to be 25% (44).A number of novel strategies have been proposed to more effectively prevent and control device-associated biofilms, either by minimizing microbial attachment to device surfaces or by targeting the biofilm after it has developed. One such strategy is to use bacteriophages (phages) (17). Phages have been used for the treatment of infectious diseases in plants (26), animals (6), and humans (33, 39, 43). The use of phages to control biofilms has potential for several reasons. Phages can replicate at the site of an infection, thereby increasing in numbers where they are most required. During the lytic replication cycle, the infection of a bacterial host cell by a single phage virion will result in the production of dozens or hundreds of progeny phage, depending on the particular phage and host strains. Some phages also have been shown to produce enzymes that degrade the extracellular polymeric substance (EPS) matrix of a biofilm (23, 25). Doolittle et al. (19) showed that progeny phage will propagate radially through a biofilm. At least in theory, a single phage dose should be capable of treating a biofilm infection as progeny phage infect adjacent cells and degrade the biofilm matrix.Curtin and Donlan (13) demonstrated that a phage that is active against Staphylococcus epidermidis could be incorporated into a hydrogel coating on a catheter and significantly reduce biofilm formation by this organism in an in vitro model system. Based on those studies with S. epidermidis, we have investigated whether phages specific for Pseudomonas aeruginosa also can reduce biofilm formation by this organism in a similar in vitro model. Catheters were treated with a single phage or a combination of phages prior to, immediately following, or 24 h after inoculation with the test P. aeruginosa culture, and the effects of the phage treatments on biofilm formation and maintenance were characterized.(Portions of this paper were presented as poster no. A-011 at the 2007 American Society for Microbiology General Meeting in Toronto, Canada [20a].)     

In Vivo Protection Provided by a Synthetic New Alpha-Galactosyl Ceramide Analog against Bacterial and Viral Infections in Murine Models

Alpha-galactosyl ceramide (α-GalCer) has been known to bind to the CD1d receptor on dendritic cells and activate invariant natural killer T (iNKT) cells, which subsequently secrete T-helper-cell 1 (Th1) and Th2 cytokines, which correlate with anti-infection activity and the prevention of autoimmune diseases, respectively. α-GalCer elicits the secretion of these two cytokines nonselectively, and thus, its effectiveness is limited by the opposing effects of the Th1 and Th2 cytokines. Reported here is the synthesis of a new α-GalCer analog (compound C34), based on the structure of CD1d, with a 4-(4-fluorophenoxy) phenyl undecanoyl modification of the N-acyl moiety of α-GalCer. Using several murine bacterial and viral infection models, we demonstrated that C34 has superior antibacterial and antiviral activities in comparison with those of several other Th1-selective glycolipids and that it is most effective by administering it to mice in a prophylactic manner before or shortly after infection.Natural killer T (NKT) cells contribute to a variety of immunological processes through the recognition of NKT cell receptors by lipid and glycolipid antigens presented by CD1d molecules (2, 35). CD1d molecules are major histocompatibility complex class I-like proteins and are expressed by most monocytes, macrophages, dendritic cells, and B cells as well as by nonlymphoid cells. CD1d presents glycolipid antigens to CD1d-restricted T cells (or NKT cells), which are implicated in the host innate defense system through the production of T-helper-cell 1 (Th1) and Th2 types of cytokines, such as gamma interferon (IFN-γ) and interleukin-4 (IL-4), respectively (32, 39). The production of Th1 cytokines is shown to correlate with the antitumor, antibacterial, and antiviral effects of glycolipids, while the ability to induce the Th2 cytokines is thought to correlate with the amelioration of certain autoimmune diseases, such as type 1 diabetes (9, 10, 28).Among the variety of ligands that bind to CD1d, the most well studied one is alpha-galactosyl ceramide (α-GalCer) (7, 17, 30), which is a synthetic, structurally optimized compound of the natural product lead identified from the marine sponge (Agelas mauritianus) as agelasphin-9b (24). The synthetic analog (2S,3S,4R)-1-O-(α-d-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol was made as KRN7000 at Kirin Brewery, Japan (23), and has generally been referred to as α-GalCer. Mice treated with α-GalCer were shown to be protected against a variety of infections (13, 15, 16, 22, 33). However, the effectiveness of α-GalCer is limited by the opposing effects of the Th1 and Th2 cytokines induced by this glycolipid. In order to develop selective Th1 or Th2 activators, many α-GalCer analogs were synthesized (1, 3, 5, 8, 12, 14, 21, 34, 36), and their immune-modulating activities were shown to be related to the affinity of binding to CD1d (8). Recent studies using a CD1d array established that the secretion of IFN-γ and IL-4 by NKT cells is determined by the binding of α-GalCer analogs to CD1d (21), and a higher affinity for CD1d would shift the cytokine release profile toward a stronger Th1 response (3, 21).This report described the synthesis of the 4-(4-fluorophenoxy)phenyl undecanoyl derivative of α-GalCer as a selective Th1 activator. This analog was designed on the basis of the structure of CD1d, where the N-acyl group of the glycolipid interacts most favorably with the lipid binding moiety of CD1d (36). Its efficacies for protection against both bacterial and viral infections were compared with those of α-GalCer and several other analogs using murine models of infection. Our study found that among the synthetic α-GalCer analogs tested, compound C34 is the most effective in offering protection against all of the bacterial and viral infection models tested, especially when it is given in a prophylactic manner or shortly after infection.     

重症监护病房获得性细菌感染临床调查分析

目的探讨重症监护病房(ICU)感染的易感因素、病原菌流行趋势及防治措施。方法采用回顾性调查研究的方法对我院重症监护病房的286例患者进行分析。结果所研究病例中发生医院感染率为19.80%,次感染率26.92%,其中以下呼吸道感染最多,占58.20%;其次为血液感染,表现为菌血症,占20.80%。引起感染的病原菌主要以革兰阴性菌(G-)为主,占49.20%,其中以铜绿假单胞菌(33.98%)为多;其次是革兰阳性菌(32.16%);再次为真菌(18.64%)。在所发生感染的病例中,大部分以脑出血、脑梗死、大脑裂伤和脑挫伤为主。诱发因素包括患者基础病情危重、进行气管切开手术(气道开放)、长期大量广谱抗生素和激素的应用、住院时间较长等。结论重症监护病房的医院感染发生率高,成为危害患者生命的重要原因,积极采取相关措施,综合治理,减少发生率,改善患者的预后。     

Insights into Cave Architecture and the Role of Bacterial Biofilm

Caves offer a stable and protected environment from harsh and changing outside conditions. They lend living proof of the presence of minute life forms that delve deep within the earth’s crust where the possibility of life seems impossible. Devoid of all light sources and lacking the most common source of energy supplied through photosynthesis, the mysterious microbial kingdom in caves are consequently dependent upon alternative sources of energy derived from the surrounding atmosphere, minerals and rocks. There are a number of features that can be observed within a cave that may serve as evidence of microbial activity, for example, formation of biofilms comprised of multiple layers of microbial communities held together by protective gel-like polymers which form complex structures. Different bacterial biofilms can develop on the walls of the cave which can be visually distinguished by their colorations. Moreover, the pH generated by the metabolism of bacterial biofilm on the cave environment can lead to precipitation or dissolution of minerals in caves. Caves also offer an excellent scenario for studying biomineralization processes. The findings on the association of bacteria with secondary minerals as mentioned in this review will help to expand the existing knowledge in geomicrobiology and specifically on the influence of microorganisms in the formation of cave deposits. This paper reviews the current state of knowledge of biospeleology of caves and the associated bacterial biofilms. Recommendations for future research are mentioned to encourage a drift from qualitative studies to more experimental studies.