Bacterial and fungal colonization of burn wounds

A prospective study of fungal and bacterial flora of burn wounds was carried out from February 2004 to February 2005 at the Burns Unit of Hospital Regional da Asa Norte, Brasília, Brazil. During the period of the study, 203 patients were treated at the Burns Unit. Wound swab cultures were assessed at weekly intervals for four weeks. Three hundred and fifty four sampling procedures (surface swabs) were performed from the burn wounds. The study revealed that bacterial colonization reached 86.6% within the first week. Although the gram-negative organisms, as a group, were more predominant, Staphylococcus aureus (28.4%) was the most prevalent organism in the first week. It was however surpassed by Pseudomonas aeruginosa form third week onwards. For S. aureus and P. aeruginosa vancomycin and polymyxin were found to be the most effective drugs. Most of the isolates showed high level resistance to antimicrobial agents. Fungi were found to colonize the burn wound late during the second week postburn, with a peak incidence during the third and fourth weeks. Species identification of fungi revealed that Candida tropicalis was the most predominant, followed by Candida parapsilosis. It is crucial for every burn institution to determine the specific pattern of burn wound microbial colonization, the time-related changes in the dominant flora, and the antimicrobial sensitivity profiles. This would enable early treatment of imminent septic episodes with proper empirical systemic antibiotics, without waiting for culture results, thus improving the overall infection-related morbidity and mortality.     

烧烫伤创面感染的小鼠模型构建

目的探讨一种简单、稳定的烧烫伤创面感染的小鼠模型构建方法,以便进行相关烧烫伤创面修复研究。方法取30只BALB/c小鼠,采用自制木质烫伤板,沸水浴法烫取直径8 mm的圆形创面,烫伤时间分别为5 s、10 s、15 s。伤后48 h,取创面组织进行HE染色观察,筛选最佳创面烫伤时间。另取72只小鼠制成深Ⅱ度烫伤创面,采用擦刮法分别接种20μL菌浓度为1×106、l×107、1×108CFU/mL金黄色葡萄球菌标准菌株ATCC 25923的菌液。接种细菌后72 h,取创面组织HE染色观察创面炎症反应情况,并测定3、7、14 d的皮肤菌负荷,筛选最佳的细菌接种浓度。最后,按最佳条件建模后,观察创面的完全愈合时间以及创面愈中、愈后的组织学变化,以确定此创面感染模型是否建立成功。结果组织学结果表明,10 s为深Ⅱ度创面的理想致伤时间,最佳接种菌浓度为l×108CFU/mL,此时期,14 d内菌负荷均高于l×105CFU/g。该模型的创面愈合时间(21±0.95 d)较正常创面愈合时间(15.92±0.34 d)明显延长(P<0.01),炎性反应明显,愈后不佳。结论烧烫伤创面感染的小鼠模型构建成功,可作为感染创面防治研究的实验动物模型。     

IFN-gamma regulated chemokine production determines the outcome of Staphylococcus aureus infection

Immunomodulatory therapy represents an attractive approach in treating multidrug-resistant infections. Developing this therapy necessitates a lucid understanding of host defense mechanisms. Neutrophils represent the first line of systemic defense during Staphylococcus aureus infections. However, recent research suggests that survival of S. aureus inside neutrophils may actually contribute to pathogenesis, indicating that neutrophil trafficking to the infection site must be tightly regulated to ensure efficient microbial clearance. We demonstrate that neutrophil-regulating T cells are activated during S. aureus infection and produce cytokines that control the local neutrophil response. S. aureus capsular polysaccharide activates T cell production of IFN-gamma in a novel MHC class II-dependent mechanism. During S. aureus surgical wound infection, the presence of IFN-gamma at the infection site depends upon alphabetaTCR+ cells and functions to regulate CXC chemokine production and neutrophil recruitment in vivo. We note that the reduced neutrophil response seen in IFN-gamma-/- mice during S. aureus infection is associated with reduced tissue bacterial burden. CXC chemokine administration to the infection site resulted in an increased survival of viable S. aureus inside neutrophils isolated from the wound. These data demonstrate that T cell-derived IFN-gamma generates a neutrophil-rich environment that can potentiate S. aureus pathogenesis by facilitating bacterial survival within the neutrophil. These findings suggest avenues for novel immunomodulatory approaches to control S. aureus infections.     

Host-pathogen interactions between the skin and Staphylococcus aureus

Staphylococcus aureus is responsible for the vast majority of bacterial skin infections in humans. The propensity for S. aureus to infect skin involves a balance between cutaneous immune defense mechanisms and virulence factors of the pathogen. The tissue architecture of the skin is different from other epithelia especially since it possesses a corneal layer, which is an important barrier that protects against the pathogenic microorganisms in the environment. The skin surface, epidermis, and dermis all contribute to host defense against S. aureus. Conversely, S. aureus utilizes various mechanisms to evade these host defenses to promote colonization and infection of the skin. This review will focus on host-pathogen interactions at the skin interface during the pathogenesis of S. aureus colonization and infection.     

Hemoglobin promotes Staphylococcus aureus nasal colonization

Staphylococcus aureus nasal colonization is an important risk factor for community and nosocomial infection. Despite the importance of S. aureus to human health, molecular mechanisms and host factors influencing nasal colonization are not well understood. To identify host factors contributing to nasal colonization, we collected human nasal secretions and analyzed their ability to promote S. aureus surface colonization. Some individuals produced secretions possessing the ability to significantly promote S. aureus surface colonization. Nasal secretions pretreated with protease no longer promoted S. aureus surface colonization, suggesting the involvement of protein factors. The major protein components of secretions were identified and subsequent analysis revealed that hemoglobin possessed the ability to promote S. aureus surface colonization. Immunoprecipitation of hemoglobin from nasal secretions resulted in reduced S. aureus surface colonization. Furthermore, exogenously added hemoglobin significantly decreased the inoculum necessary for nasal colonization in a rodent model. Finally, we found that hemoglobin prevented expression of the agr quorum sensing system and that aberrant constitutive expression of the agr effector molecule, RNAIII, resulted in reduced nasal colonization of S. aureus. Collectively our results suggest that the presence of hemoglobin in nasal secretions contributes to S. aureus nasal colonization.     

Role of Staphylococcus aureus catalase in niche competition against Streptococcus pneumoniae

Nasal colonization by Staphylococcus aureus is a major predisposing factor for subsequent infection. Recent reports of increased S. aureus colonization among children receiving pneumococcal vaccine implicate Streptococcus pneumoniae as an important competitor for the same niche. Since S. pneumoniae uses H2O2 to kill competing bacteria, we hypothesized that oxidant defense could play a significant role in promoting S. aureus colonization of the nasal mucosa. Using targeted mutagenesis, we showed that S. aureus expression of catalase contributes significantly to the survival of this pathogen in the presence of S. pneumoniae both in vitro and in a murine model of nasal cocolonization.     

Properties of Staphylococcus aureus in case of the unfavorable course of burn infection

Clinical and microbiological study of wound discharge from 35 patients demonstrated a relationship between biological properties of S. aureus, the causative agent of burn infection, and the course of the infected burn trauma. The prognostic importance of the antioxidant properties of these staphylococci was established: in cases of the unfavorable course of the burn process they showed essentially greater resistance to peroxinitrite and higher superoxide dismutase activity in comparison with the cultures isolated from patients with uncomplicated wound infection.     

In vivo efficacy of antimicrobe-impregnated saline-filled silicone implants

Bacterial colonization of mammary implants is a prelude to clinical infection and has been implicated in the etiology of capsular contracture. Antimicrobial impregnation of a variety of medical devices with the combination of minocycline and rifampin has recently emerged as a potentially effective method for preventing device colonization and device-related infection. The objective of this animal study was to examine in vivo the antimicrobial efficacy of minocycline/rifampin-impregnated, saline-filled silicone implants. A rabbit model of Staphylococcus aureus colonization and infection of subcutaneously placed implants was used. A total of 48 saline-filled silicone implants (24 antimicrobe-impregnated and 24 control unimpregnated implants) were suspended in a 106 colony-forming units/ml bacterial suspension of S. aureus for 30 minutes at room temperature, allowed to dry for 60 minutes, and then implanted subcutaneously in the back of 12 rabbits (two antimicrobe-impregnated and two control implants were placed in each rabbit). Rabbits were monitored daily, then killed either at 2 weeks (10 rabbits) or at 4 weeks (two rabbits) and cultured. The antimicrobe-impregnated implants were 12 times less likely to be colonized than control unimpregnated implants (two of 24 versus 23 of 24; p < 0.001), and they were a significantly less likely cause of implant-related infection (0 of 24 versus 22 of 24; p < 0.001) and implant-related abscess (0 of 24 versus 21 of 24; p < 0.001) than control implants. The minocycline/rifampin-impregnated implants routinely demonstrated zones of inhibition against S. aureus at the time of explantation. These results indicate that minocycline/rifampin-impregnated implants can significantly decrease the rate of bacterial colonization, implant-related infection, and implant-related abscess. Antimicrobe-impregnated implants also have the potential of reducing the likelihood of capsular contracture.     

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。     

Effects of burn wound excision on bacterial colonization and invasion

Rates of survival after thermal injury have improved in the past two decades, and rates of wound infections and sepsis have decreased during the same period. Early excision has been advocated as one of the major factors, but its safety and efficacy and the exact timing of burn excision are still under debate. It was hypothesized that acute burn wound excision (in the first 24 hours after burning) would be superior to conservative treatment and delayed excision in preventing bacterial colonization and invasion. Twenty consecutive patients with thermal injuries were studied. Twelve patients underwent acute burn wound excision, and eight patients underwent conservative treatment and delayed excision. The second group of patients received topical treatments in another facility and underwent delayed excision after transfer to our service, on postburn day 6. Quantitative bacteriological assessments of the excised wound and biopsy samples of the wound bed, obtained before autografting and/or homografting, were performed. The effects of time on bacterial counts, differences between superficial and deep biopsy samples, and the effects of early versus late debridement were studied. Patients admitted early exhibited bacterial counts of less than 10 bacteria per gram of tissue. Patients in this group did not experience infection or graft loss. Patients admitted late exhibited counts of more than 10 bacteria (p = 0.001, compared with early admission). Three patients in the late excision group experienced infection and graft loss (p < 0.05, compared with the early excision group). Burn wound excision significantly decreased bacterial colonization for all patients (p < 0.001). Greater bacterial colonization and higher rates of infection were correlated with topical treatment and late excision (p < 0.001). It is concluded that burn wound excision significantly reduces bacterial colonization. Patients who undergo topical treatment and delayed burn wound excision exhibit greater bacterial colonization and increased rates of infection. Acute burn wound excision should be considered for all full-thickness burns.     

Increased susceptibility to Staphylococcus aureus colonization of the skin of the NOA mouse: a potentially useful animal model for evaluating antiseptic effects.

Isolation of bacteria from wet skin lesions was attempted using Naruto Research Institute Otsuka Atrichia (NOA) mice, which develop such lesions spontaneously at a high rate. As a result, Staphylococcus aureus was demonstrated to have colonized the wet skin lesions at high density. In addition, the isolated S. aureus was found to be similar to the strain of S. aureus thought to colonize the eczematous lesions seen in humans with atopic dermatitis. Furthermore, a survey of the S. aureus colonization status of NOA mice with no wet skin lesions confirmed colonization at higher density than in HR-1 mice as control, indicating that the skin of the NOA mouse has the novel characteristic of increased susceptibility to S. aureus colonization. Thus, by using changes in S. aureus counts as an index, the NOA mouse can be expected to serve as a useful animal model for evaluating the effects of topical antiseptics. The antiseptic effects of an ointment and a lotion containing chlorhexidine gluconate were confirmed using this animal model.     

树鼩细菌感染模型的建立及抗菌药物的治疗效果评价(英文)

在抗微生物感染药物开发过程中, 动物模型是必不可少的。虽然目前已经用啮齿类动物建立了一些细菌感染动物模型, 但在小型灵长类动物中还很少见。这里首次报道两个树鼩细菌感染动物模型。第一种是在三度烫伤后的皮肤表面接种 5×106 CFU 的金黄色葡萄球菌构建的皮肤烫伤感染模型。这个数量的金黄色葡萄球菌可以造成 7 d 持续性感染, 并且在第 4天可以看到明显的炎症反应。第二种是用绿脓杆菌构建的涤纶补片感染模型, 接种 2×106 CFU 的绿脓杆菌同样可以引起持续 6 d 感染, 并在第三天在伤口处观察到大量的脓液。进一步用这两种模型评价头孢哌酮钠和左氧氟沙星的治疗效果。左氧氟沙星和头孢哌酮钠在皮肤烫伤感染模型中能分别将 100 mg 皮肤组织中的细菌降低到 4log10 和 5log10 CFU, 并且在涤纶补片植入感染模型中这两种抗生素都能显著地将感染的细菌降低了 4log10 CFU (P<0.05)。结果表明用金黄色葡萄球菌和绿脓杆菌成功构建了两个细菌感染的树鼩模型。此外, 树鼩对金黄色葡萄球菌和绿脓杆菌很敏感, 适合用于构建细菌感染动物模型和评价新的抗细菌感染药物的效果。     

Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes

Staphylococcus aureus is the most common cause of hospital-acquired infection. In healthy hosts outside of the health care setting, S. aureus is a frequent colonizer of the human nose but rarely causes severe invasive infection such as bacteremia, endocarditis, or osteomyelitis. To identify genes associated with community-acquired invasive isolates, regions of genomic variability, and the S. aureus population structure, we compared 61 community-acquired invasive isolates of S. aureus and 100 nasal carriage isolates from healthy donors using a microarray spotted with PCR products representing every gene from the seven S. aureus sequencing projects. The core genes common to all strains were identified, and 10 dominant lineages of S. aureus were clearly discriminated. Each lineage carried a unique combination of hundreds of "core variable" (CV) genes scattered throughout the chromosome, suggesting a common ancestor but early evolutionary divergence. Many CV genes are regulators of virulence genes or known or predicted to be expressed on the bacterial surface and to interact with the host during nasal colonization and infection. Within each lineage, isolates showed substantial variation in the carriage of mobile genetic elements and their associated virulence and resistance genes, indicating frequent horizontal transfer. However, we were unable to identify any association between lineage or gene and invasive isolates. We suggest that the S. aureus gene combinations necessary for invasive disease may also be necessary for nasal colonization and that community-acquired invasive disease is strongly dependent on host factors.     

抑菌生的研究总结报告

本文报告一种由枯草杆菌制成的生态制剂,并命名为抑菌生(Subtilobiogen)。该制剂对创、烧伤感染有治疗作用。经过安全试验、急性毒性试验、Ames试验和微核试验证明,该制剂是一种无害、无毒和有致突变作用的活菌制剂。抑菌生有膏剂、乳剂及粉剂3种剂型。抑菌生在试管和体内对金黄色葡萄球菌、绿脓杆菌和大肠杆菌均具有抑菌作用。临床观察证明,将抑菌生喷洒在创面上,对浅Ⅱ°度、深Ⅱ°度及混合型烧伤感染均具有明显疗效。实验组(181例)与对照组(174例)相比较,在统计学上具有显著性差异。抑菌生的作用机制,经过初步试验证明,与营养争夺和占位性保护有关,因为枯草杆菌的生长速度超过金黄色葡萄球菌、绿脓杆菌和大肠杆菌的生长速度。     

Gram Negative Wound Infection in Hospitalised Adult Burn Patients-Systematic Review and Metanalysis-

Background

Gram negative infection is a major determinant of morbidity and survival. Traditional teaching suggests that burn wound infections in different centres are caused by differing sets of causative organisms. This study established whether Gram-negative burn wound isolates associated to clinical wound infection differ between burn centres.

Methods

Studies investigating adult hospitalised patients (2000–2010) were critically appraised and qualified to a levels of evidence hierarchy. The contribution of bacterial pathogen type, and burn centre to the variance in standardised incidence of Gram-negative burn wound infection was analysed using two-way analysis of variance.

Primary Findings

Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanni, Enterobacter spp., Proteus spp. and Escherichia coli emerged as the commonest Gram-negative burn wound pathogens. Individual pathogens’ incidence did not differ significantly between burn centres (F (4, 20) = 1.1, p = 0.3797; r2 = 9.84).

Interpretation

Gram-negative infections predominate in burn surgery. This study is the first to establish that burn wound infections do not differ significantly between burn centres. It is the first study to report the pathogens responsible for the majority of Gram-negative infections in these patients. Whilst burn wound infection is not exclusive to these bacteria, it is hoped that reporting the presence of this group of common Gram-negative “target organisms” facilitate clinical practice and target research towards a defined clinical demand.     

Use of nasal mupirocin for Staphylococcus aureus: effect on nasal carriers and nosocomial infections

Staphylococcus aureus is the agent of community-acquired and nosocomial infections. Twenty to 35% of the population permanently carries it in the nose and oropharynx, and additional 50%, carries it intermittently. Topical calcium mupirocin is an antibacterial agent against Staphylococcus aureus recommended to eradicate nasal and hand colonization in patients and health care workers. The prevalence of nasal S. aureus was determined in patients undergoing cardiovascular surgery. In addition, the effect of mupirocine on the number of carriers and rate of nosocomial infections was evaluated. An experimental prospective study was undertaken with two groups of patients: one treated with mupirocin (n = 96), and the other without treatment (n = 95). Tests for presence of nasal S. aureus and nosocomial infections were conducted in all patients. A 34% prevalence of S. aureus carriers was found. A decrease of the prevalence was found in both treated (87%) and untreated patients (33%), but in significantly different proportions (p = 0.0002, RR = 0.22, 95%CI = 0.09-0.054). This result demonstrated the effectiveness of a mupirocin treatment program to decrease numbers of nasal carriers. With regard to nosocomial infection, S. aureus prevalence was 3.6%, occurring mostly in control patients (6 of 7). Total nosocomial infection prevalence was 17.3%, evenly distributed in treated and untreated patients. This suggested that mupirocin use did not decrease the number of nosocomial infections.     

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.     

An oxidation-sensing mechanism is used by the global regulator MgrA in Staphylococcus aureus

Staphylococcus aureus is a human pathogen responsible for most wound and hospital-acquired infections. The protein MgrA is both an important virulence determinant during infection and a regulator of antibiotic resistance in S. aureus. The crystal structure of the MgrA homodimer, solved at 2.86 A, indicates the presence of a unique cysteine residue located at the interface of the protein dimer. We discovered that this cysteine residue can be oxidized by various reactive oxygen species, such as hydrogen peroxide and organic hydroperoxide. Cysteine oxidation leads to dissociation of MgrA from DNA and initiation of signaling pathways that turn on antibiotic resistance in S. aureus. The oxidation-sensing mechanism is typically used by bacteria to counter challenges of reactive oxygen and nitrogen species. Our study reveals that in S. aureus, MgrA adopts a similar mechanism but uses it to globally regulate different defensive pathways.     

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.