Usefulness of Cellular Analysis of Bronchoalveolar Lavage Fluid for Predicting the Etiology of Pneumonia in Critically Ill Patients

Background

The usefulness of bronchoalveolar lavage (BAL) fluid cellular analysis in pneumonia has not been adequately evaluated. This study investigated the ability of cellular analysis of BAL fluid to differentially diagnose bacterial pneumonia from viral pneumonia in adult patients who are admitted to intensive care unit.

Methods

BAL fluid cellular analysis was evaluated in 47 adult patients who underwent bronchoscopic BAL following less than 24 hours of antimicrobial agent exposure. The abilities of BAL fluid total white blood cell (WBC) counts and differential cell counts to differentiate between bacterial and viral pneumonia were evaluated using receiver operating characteristic (ROC) curve analysis.

Results

Bacterial pneumonia (n = 24) and viral pneumonia (n = 23) were frequently associated with neutrophilic pleocytosis in BAL fluid. BAL fluid median total WBC count (2,815/µL vs. 300/µL, P<0.001) and percentage of neutrophils (80.5% vs. 54.0%, P = 0.02) were significantly higher in the bacterial pneumonia group than in the viral pneumonia group. In ROC curve analysis, BAL fluid total WBC count showed the best discrimination, with an area under the curve of 0.855 (95% CI, 0.750–0.960). BAL fluid total WBC count ≥510/µL had a sensitivity of 83.3%, specificity of 78.3%, positive likelihood ratio (PLR) of 3.83, and negative likelihood ratio (NLR) of 0.21. When analyzed in combination with serum procalcitonin or C-reactive protein, sensitivity was 95.8%, specificity was 95.7%, PLR was 8.63, and NLR was 0.07. BAL fluid total WBC count ≥510/µL was an independent predictor of bacterial pneumonia with an adjusted odds ratio of 13.5 in multiple logistic regression analysis.

Conclusions

Cellular analysis of BAL fluid can aid early differential diagnosis of bacterial pneumonia from viral pneumonia in critically ill patients.     

Etiology of pneumonia in children in the absence of pneumococcal and antihaemophilus vaccines

Childhood pneumonia represents an important pathology, a cause of morbidity and mortality worldwide. Our study aims to determine etiology of pneumonia in hospitalized children using several laboratory methods. We performed a prospective study that enrolled 560 children age 1 up to 18 years old all diagnosed with pneumonia by clinical and radiological features. We applied various laboratory methods (serologic, bacteriologic: bronchial aspirate, sputum, pleural effusion and blood culture) in order to identify a pathogen agent that caused pneumonia. Statistics used Statistical Package for Social Science. An etiology was established in 68.92% of all cases included in the study, as follows: in 33.93% viral etiology, in 25.13% we identified Streptococcus pneumoniae, in 20.2% Mycoplasma pneumoniae, Klebsiella pneumoniae in 8.29%, Staphylococcus aureus in 7.51%, Haemophilus influenzae in 4.92%. Mixed bacterial and viral infection was identified in 4.40% of all cases. A potential causative agent of childhood pneumonia was determined in most cases, S. pneumoniae being the main agent involved in community acquired childhood pneumonia in our country.     

Procalcitonin and C-reactive protein for invasive bacterial pneumonia diagnosis among children in Mozambique, a malaria-endemic area

Background

Pneumonia is the major cause of mortality and morbidity in children worldwide. Procalcitonin (PCT) and C-reactive protein (CRP) are used in developed countries to differentiate between viral and bacterial causes of pneumonia. Validity of these markers needs to be further explored in Africa.

Methodology and Principal Findings

We assessed the utility of PCT and CRP to differentiate viral from invasive bacterial pneumonia in children <5 years hospitalized with clinical severe pneumonia (CSP) in rural Mozambique, a malaria-endemic area with high HIV prevalence. Prognostic capacity of these markers was also evaluated. Out of 835 children with CSP, 87 fulfilled definition of viral pneumonia and 89 of invasive bacterial pneumonia. In absence of malaria parasites, levels of PCT and CRP were lower in the viral group when compared to the invasive bacterial one (PCT: median = 0.21 versus 8.31 ng/ml, p<0.001; CRP: 18.3 vs. 185.35 mg/l, p<0.001). However, in presence of malaria parasites distribution between clinical groups overlapped (PCT: median = 23.1 vs. 21.75 ng/ml, p = 0.825; CRP: median = 96.8 vs. 217.4 mg/l, p = 0.052). None of the two markers could predict mortality.

Conclusions

Presence of malaria parasites should be taken into consideration, either for clinical or epidemiological purposes, if using PCT or CRP to differentiate viral from invasive bacterial pneumonia in malaria-endemic areas.     

The Diagnostic and Prognostic Accuracy of Five Markers of Serious Bacterial Infection in Malawian Children with Signs of Severe Infection

Background

Early recognition and prompt and appropriate antibiotic treatment can significantly reduce mortality from serious bacterial infections (SBI). The aim of this study was to evaluate the utility of five markers of infection: C-reactive protein (CRP), procalcitonin (PCT), soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), CD163 and high mobility group box-1 (HMGB1), as markers of SBI in severely ill Malawian children.

Methodology and Principal Findings

Children presenting with a signs of meningitis (n = 282) or pneumonia (n = 95), were prospectively recruited. Plasma samples were taken on admission for CRP, PCT, sTREM-1 CD163 and HMGB1 and the performance characteristics of each test to diagnose SBI and to predict mortality were determined. Of 377 children, 279 (74%) had SBI and 83 (22%) died. Plasma CRP, PCT, CD163 and HMGB1 and were higher in HIV-infected children than in HIV-uninfected children (p<0.01). In HIV-infected children, CRP and PCT were higher in children with SBI compared to those with no detectable bacterial infection (p<0.0005), and PCT and CD163 were higher in non-survivors (p = 0.001, p = 0.05 respectively). In HIV-uninfected children, CRP and PCT were also higher in children with SBI compared to those with no detectable bacterial infection (p<0.0005), and CD163 was higher in non-survivors (p = 0.05). The best predictors of SBI were CRP and PCT, and areas under the curve (AUCs) were 0.81 (95% CI 0.73–0.89) and 0.86 (95% CI 0.79–0.92) respectively. The best marker for predicting death was PCT, AUC 0.61 (95% CI 0.50–0.71).

Conclusions

Admission PCT and CRP are useful markers of invasive bacterial infection in severely ill African children. The study of these markers using rapid tests in a less selected cohort would be important in this setting.     

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses.     

Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia

Secondary bacterial pneumonia frequently claimed the lives of victims during the devastating 1918 influenza A virus pandemic. Little is known about the viral factors contributing to the lethality of the 1918 pandemic. Here we show that expression of the viral accessory protein PB1-F2 enhances inflammation during primary viral infection of mice and increases both the frequency and severity of secondary bacterial pneumonia. The priming effect of PB1-F2 on bacterial pneumonia could be recapitulated in mice by intranasal delivery of a synthetic peptide derived from the C-terminal portion of the PB1-F2. Relative to its isogenic parent, an influenza virus engineered to express a PB1-F2 with coding changes matching the 1918 pandemic strain was more virulent in mice, induced more pulmonary immunopathology, and led to more severe secondary bacterial pneumonia. These findings help explain both the unparalleled virulence of the 1918 strain and the high incidence of fatal pneumonia during the pandemic.     

病毒性肺炎患者外周血干扰素刺激基因15 mRNA表达特征的研究

通过分析病毒性肺炎患者外周血干扰素刺激基因15(interferon stimulated gene 15,ISG15)mRNA在感染状态下的特征性表达,探究ISG15与病毒性肺炎发病机制及疾病活动性之间的可能联系,为探索特异性呼吸道病毒感染的宿主生物标志物提供依据.本研究共收集157例社区获得性肺炎(communit...     

Clinical Characteristics and Outcomes in Hospitalized Patients with Respiratory Viral Co-Infection during the 2009 H1N1 Influenza Pandemic

Background

The clinical consequences of co-infection with two or more respiratory viruses are poorly understood. We sought to determine if co-infection with pandemic 2009–2010 influenza A H1N1 (pH1N1) and another respiratory virus was associated with worse clinical outcomes.

Methods

A retrospective cohort study was performed of all hospitalized patients with a positive respiratory viral panel (RVP) for two or more viruses within 72 hours of admission at our institution from October 2009 to December 2009. We compared patients infected with one respiratory virus to those with respiratory viral co-infection.

Results

We identified 617 inpatients with a positive RVP sample with a single virus and 49 inpatients with a positive RVP sample for two viruses (i.e. co-infection). Co-infected patients were significantly younger, more often had fever/chills, tachypnea, and they more often demonstrated interstitial opacities suggestive of viral pneumonia on the presenting chest radiograph (OR 7.5, 95% CI 3.4–16.5). The likelihood of death, length of stay, and requirement for intensive care unit level of care were similar in both groups, but patients with any respiratory virus co-infection were more likely to experience complications, particularly treatment for a secondary bacterial pneumonia (OR 6.8, 95% CI 3.3–14.2). Patients co-infected with pH1N1 and another respiratory virus were more likely to present with chest radiograph changes suggestive of a viral pneumonia, compared to mono-infection with pH1N1 (OR 16.9, 95% CI 4.5–62.7). By logistic regression using mono-infection with non-PH1N1 viruses as the reference group, co-infection with pH1N1 was the strongest independent predictor of treatment for a secondary bacterial pneumonia (OR 17.8, 95% CI 6.7–47.1).

Conclusion

Patients with viral co-infection, particularly with pH1N1, were more likely to have chest radiograph features compatible with a viral pneumonia and complications during their hospital course, particularly treatment for secondary bacterial pneumonia. Despite this, co-infection was not associated with ICU admission.     

280例呼吸道感染儿童呼吸道病毒病原学检出情况及流行规律分析

目的:了解呼吸道感染儿童呼吸道病毒病原学检出情况及其流行规律,为儿童呼吸道感染的预防、诊断及治疗提供病原学依据。方法:选取2016年1月-2017年12月期间中国人民解放军中部战区总医院收治的280例呼吸道感染患儿为研究对象,分析患儿呼吸道分泌物中呼吸道病毒的检出情况,并分析呼吸道感染儿童呼吸道病毒感染与年龄、季节、疾病类型的关系。结果:280例呼吸道感染患儿中共检出98份阳性标本,阳性率为35.00%,其中有2份标本中检出2种病毒感染,混合感染阳性率为0.71%;在所有病毒类型中,呼吸道合胞病毒(RSV)病毒感染阳性率最高。1岁患儿的病毒感染阳性率最高,与其他年龄段病毒感染阳性率比较差异有统计学意义(P0.05)。呼吸道感染患儿春季、冬季的病毒感染阳性率明显高于夏季、秋季(P0.05)。不同呼吸道感染疾病类型患儿病毒感染阳性率比较差异有统计学意义(P0.05),以喘息性肺炎、毛细支气管炎、肺炎患儿病毒感染阳性率较高。结论:RSV是呼吸道感染儿童呼吸道病毒感染的主要致病病原体,1岁的婴幼儿较易感染,春季、冬季为其高发季节,且以肺炎、毛细支气管炎、喘息性肺炎患儿的病毒感染阳性率较高。     

An Opportunistic Bacterial Pathogen, Pseudomonas alcaligenes , May Limit the Perpetuation of Oryctes Virus, a Biocontrol Agent of Oryctes rhinoceros L.

Pseudomonas alcaligenes was detected at a high concentration (10 9 -10 10 cells mL -1 ) in the haemolymph of some dead Oryctes rhinoceros grubs collected from its breeding sites in the three southern districts, viz. Alleppey, Quilon and Kottayam of Kerala State, India. In a laboratory colony maintained for production of Oryctes virus, an important biocontrol agent of this major coconut pest, approximately 52% of the grubs succumbed to septicaemia with similar symptoms. The bacterium was found to be a component of the gut microflora of healthy grubs. Occurrence of the viral infection naturally or when induced in the laboratory in the O. rhinoceros grubs, appeared to be one of the biotic stress factors for P.alcaligenes to become an opportunistic pathogen. A preponderance of this bacterial infection in field populations during the periods when natural viral infection in grubs was above average, agrees with this observation. This finding becomes significant as infection by the opportunistic bacterial pathogen, P. alcaligenes , reduces the production of Oryctes virus inoculum in nature and limits the field-perpetuation of this viral biocontrol agent.     

Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner

Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and Streptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.     

血清降钙素原与传统感染检测指标对细菌性血流感染的临床诊断价值比较

目的比较血清降钙素原(PCT)和传统感染检测指标在细菌性血流感染中的诊断价值。方法选取我院2018年1月至11月59例血培养显示细菌感染阳性患者为血培养阳性组,选取同期63例血培养阴性患者为血培养阴性组,分析两组患者白细胞(WBC)、中性粒细胞(NEU)计数和血清PCT水平,比较PCT、WBC和NEU对细菌性血流感染的诊断阳性率和ROC曲线下面积。结果两组患者WBC、NEU计数和血清PCT水平差异均有统计学意义(均P<0.05),PCT对细菌性血流感染的诊断阳性率和ROC曲线下面积显著大于WBC和NEU(均P<0.05)。PCT诊断细菌性血流感染的灵敏度为96.6%,特异度为95.2%。结论血清PCT对细菌性血流感染具有较高的诊断价值。     

闽南地区5 869例儿童社区获得性肺炎患者 病原学特点分析

目的通过对闽南地区社区获得性肺炎(CAP)患儿病原特点进行分析,为CAP早期预警、预防和早期经验治疗提供指导和依据。方法收集我院2015年1月至2017年5月5 869例CAP患儿静脉血、呼吸道分泌物及痰标本分别进行肺炎支原体(MP)、呼吸道病毒检测以及细菌培养和鉴定。结果 5 869例CAP患儿中明确病原体感染4 931例,其中单纯细菌感染3 054例,单纯病毒感染966例,单纯MP感染620例,混合感染291例,未检出病原体938例。细菌性病原主要以肺炎链球菌、流感嗜血杆菌和卡他莫拉菌为主,在各季节检出率不同且男性多于女性,主要在低龄儿童中检出。病毒感染者主要分布于低龄儿童,夏季检出率最高,病原以呼吸道合胞病毒(RSV)为主,占68.22%。该类患者在性别上比较差异无统计学意义。MP感染者中女性多于男性,主要分布于大龄儿童,冬季检出率最高。结论本地区CAP患儿病原检出率由高到低分别为细菌、病毒和MP。细菌和MP检出率在患儿中具有性别差异,各种病原在不同年龄段儿童及季节中检出率不同。     

住院儿童肺炎发病情况及病毒病原学分析

目的:统计我院1-7岁住院儿童肺炎发病情况并进行病毒病原学分析。方法:收集2015年1月到2017年12月河北省人民医院1-7岁住院儿童8532例的临床资料,统计不同年龄段的肺炎患儿、重症肺炎患儿的发病情况及不同季节肺炎分布特点,统计不同病毒病原体住院肺炎儿童入院时的症状或体征情况,记录住院肺炎儿童并发症发生情况。结果:8532例住院儿童中,肺炎患儿2476例。1岁患儿肺炎、重症肺炎的发病率均最高,分别为54.67%(703/1286)、1.14%(8/703);7岁患儿肺炎发病率最低,为12.52%(126/1006);6岁和7岁重症肺炎发病率均为0.00%。冬季肺炎发病率最高,为33.38%(724/2169),春季、夏季、秋季发病率相当。病毒病原学显示,至少1种病毒检测阳性的有2061例,阳性率为83.24%。腺病毒和偏肺病毒阳性患儿出现发热、呼吸急促和呼吸困难的比例高于其他病毒感染;各种病毒阳性病例中,咳嗽、咳痰、流涕等呼吸道症状出现的频率相当。住院肺炎患儿中出现并发症以呼吸衰竭为主,占比为21.16%,其次是心力衰竭,占比为14.82%,脓毒血症的发生率为8.72%,其他并发症发生率均较低。结论:2015-2017年我院1-7岁住院儿童肺炎和重症肺炎的发生率以1岁最高,且随着年龄的增长发病率呈下降趋势,冬季最多见,病毒病原体中以腺病毒和偏肺病毒感染导致的发热、呼吸急促和呼吸困难症状较多,并发症以呼吸衰竭为主。     

急性呼吸道感染患儿白细胞计数、内毒素、C 反应蛋白水平变化及 临床意义

目的:探讨急性呼吸道感染患儿白细胞(WBC)计数、血清内毒素(ET)、C反应蛋白(CRP)水平变化及临床应用价值。方法:对急性呼吸道感染组106例患儿(细菌感染组62例、病毒感染组44例)进行WBC计数、血清ET、CRP水平等指标进行检测,并与正常对照组的40例健康儿童进行比较分析。结果:急性呼吸道感染组患儿WBC计数、血清ET、CRP水平均显著高于正常对照组,差异具有统计学意义(P<0.05或P<0.01);血清ET、CRP水平对诊断脓毒症的敏感性差异无显著性(P>0.05),均显著高于WBC计数,差异具有统计学意义(P<0.05);WBC计数特异性显著高于ET、CRP水平,ET水平特异性显著低于CRP,差异均具有统计学意义(P<0.05);细菌感染组患儿血清ET、CRP水平显著高于病毒感染组,差异具有统计学意义(P<0.05),而两组间WBC计数差异不显著(P>0.05)。结论:测定WBC计数、ET、CRP对急性呼吸道感染患儿具有一定的诊断价值。     

Histopathological Features and Viral Antigen Distribution in the Lung of Fatal Patients with <Emphasis Type="Italic">Enterovirus</Emphasis> 71 Infection

Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease (HFMD) in children and might be accompanied by severe neurological complications. It has become one of the most important pathogens of central nervous system infection. To explore the causes of lung injury by EV71, the distribution of EV71 receptors, SCARB2 and PSGL-1, in human lung tissues was examined. Our results revealed that SCARB2 was positively distributed in the bronchial and bronchiolar epithelial cells, alveolar cells and macrophages, while PSGL-1 was positively scattered in bronchial and bronchiolar epithelial cells and macrophages, and negatively distributed in alveolar cells. The pathological changes of fatal lung with EV71 infection demonstrated intrapulmonary bronchitis and bronchiolitis, diffuse or focal infiltration of inflammatory cells, such as T cells and B cells in the wall and surrounding tissues, widened alveolar septum, capillaries in the septum with highly dilated and congested, and infiltrated inflammatory cells, showing different degrees of protein edema with fibrin exudation in the alveolar cavity, as well as obvious hyaline membrane formation in some alveolar cavities. The EV71 antigen in lung tissues was detected, and the viral antigen was positive in lung bronchial and bronchiolar epithelial cells, and positively scattered in the alveolar cells and macrophages. Therefore, in addition to the complications of central nervous system injury, the lung remains the main target organ for virus attack in severe EV71 infected patients. Lung injury was mainly caused by neurogenic damage and/or direct invasion of the virus into the lungs in critically serious children, and the lesions were mainly pulmonary edema and interstitial pneumonia.     

IL-10 is an important mediator of the enhanced susceptibility to pneumococcal pneumonia after influenza infection

Secondary pneumococcal pneumonia is a serious complication during and shortly after influenza infection. We established a mouse model to study postinfluenza pneumococcal pneumonia and evaluated the role of IL-10 in host defense against Streptococcus pneumoniae after recovery from influenza infection. C57BL/6 mice were intranasally inoculated with 10 median tissue culture infective doses of influenza A (A/PR/8/34) or PBS (control) on day 0. By day 14 mice had regained their normal body weight and had cleared influenza virus from the lungs, as determined by real-time quantitative PCR. On day 14 after viral infection, mice received 10(4) CFU of S. pneumoniae (serotype 3) intranasally. Mice recovered from influenza infection were highly susceptible to subsequent pneumococcal pneumonia, as reflected by a 100% lethality on day 3 after bacterial infection, whereas control mice showed 17% lethality on day 3 and 83% lethality on day 6 after pneumococcal infection. Furthermore, 1000-fold higher bacterial counts at 48 h after infection with S. pneumoniae and, particularly, 50-fold higher pulmonary levels of IL-10 were observed in influenza-recovered mice than in control mice. Treatment with an anti-IL-10 mAb 1 h before bacterial inoculation resulted in reduced bacterial outgrowth and markedly reduced lethality during secondary bacterial pneumonia compared with those in IgG1 control mice. In conclusion, mild self-limiting influenza A infection renders normal immunocompetent mice highly susceptible to pneumococcal pneumonia. This increased susceptibility to secondary bacterial pneumonia is at least in part caused by excessive IL-10 production and reduced neutrophil function in the lungs.     

学龄前儿童细菌性腹泻的病原学特征及PCT、CD64和sTREM-1测定的临床意义

目的:分析学龄前儿童细菌性腹泻的病原学特征及血清中降钙素原(PCT)、中性粒细胞分化群64(CD64)和髓样细胞触发受体-1(s TREM-1)检测的临床意义。方法:选择2017年1月～2018年3月南方医科大学附属东莞市人民医院收治的214例学龄前感染性腹泻患儿为研究对象,包括细菌性腹泻(细菌组,n=101)和病毒性腹泻(病毒组,n=113),以同期入院行儿童保健的113例健康学龄前儿童为对照组。统计细菌组患儿的病原菌种类,比较两组腹泻患儿与对照组儿童血清PCT、CD64和s TREM-1水平的差异、三种血清学指标的阳性率及三种指标对细菌性腹泻患儿的诊断效能。结果:101例细菌性腹泻患儿粪便培养的细菌菌株中,共培养菌株213株,大肠埃希菌、志贺菌属和沙门菌属阳性率最高,分别占41.78%、23.01%和15.96%;细菌组的血清PCT、CD64和s TREM-1水平及阳性率均高于病毒组和对照组,且病毒组的s TREM-1水平高于对照组(P0.05);三种指标的灵敏度、特异度、阳性预测值和阴性预测值比较,差异无统计学意义(P0.05)。结论:学龄前儿童细菌性腹泻的致病菌主要为革兰阴性菌,PCT、CD64和s TREM-1均可作为判断细菌性腹泻的早期敏感指标。     

Towards a Better Understanding of the Use of Probiotics for Preventing Chytridiomycosis in Panamanian Golden Frogs

Populations of native Panamanian golden frogs (Atelopus zeteki) have collapsed due to a recent chytridiomycosis epidemic. Reintroduction efforts from captive assurance colonies are unlikely to be successful without the development of methods to control chytridiomycosis in the wild. In an effort to develop a protective treatment regimen, we treated golden frogs with Janthinobacterium lividum, a skin bacterium that has been used to experimentally prevent chytridiomycosis in North American amphibians. Although J. lividum appeared to colonize A. zeteki skin temporarily, it did not prevent or delay mortality in A. zeteki exposed to Batrachochytrium dendrobatidis, the causative agent of chytridiomycosis. After introduction of J. lividum, average bacterial cell counts reached a peak of 1.7 × 10(6) cells per frog ~2 weeks after treatment but declined steadily after that. When J. lividum numbers declined to ~2.8 × 10(5) cells per frog, B. dendrobatidis infection intensity increased to greater than 13,000 zoospore equivalents per frog. At this point, frogs began to die of chytridiomycosis. Future research will concentrate on isolating and testing antifungal bacterial species from Panama that may be more compatible with Atelopus skin.     

Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin.