Inhibition of Macrophage Migration Inhibitory Factor Ameliorates Ocular Pseudomonas aeruginosa-Induced Keratitis

Pseudomonas aeruginosa causes severe sight-threatening corneal infections, with the inflammatory response to the pathogen being the major factor resulting in damage to the cornea that leads to loss of visual acuity. We found that mice deficient for macrophage migration inhibitory factor (MIF), a key regulator of inflammation, had significantly reduced consequences from acute P. aeruginosa keratitis. This improvement in the outcome was manifested as improved bacterial clearance, decreased neutrophil infiltration, and decreased inflammatory responses when P. aeruginosa-infected MIF knock out (KO) mice were compared to infected wild-type mice. Recombinant MIF applied to infected corneas restored the susceptibility of MIF deficient mice to P. aeruginosa-induced disease, demonstrating that MIF is necessary and sufficient to cause significant pathology at this immune privileged site. A MIF inhibitor administered during P. aeruginosa-induced infection ameliorated the disease-associated pathology. MIF regulated epithelial cell responses to infection by enhancing synthesis of proinflammatory mediators in response to P. aeruginosa infection and by promoting bacterial invasion of corneal epithelial cells, a correlate of virulence in the keratitis model. Our results uncover a host factor that elevates inflammation and propagates bacterial cellular invasion, and further suggest that inhibition of MIF during infection may have a beneficial therapeutic effect.     

Resistance of Pseudomonas aeruginosa to isothiazolone

This investigation was to determine whether Pseudomonas aeruginosa could acquire resistance to the bactericide isothiazolone, and what the nature of such a resistance mechanism would be. The Pseudomonas was cultured in nutrient-limited broth in the presence of sub-inhibitory concentrations of isothiazolone (a mixture of 1.15% 5-chloro- N -methylisothiazolone (CMIT) and 0.35% N -methylisothiazolone (MIT)). Three cultures tested in parallel adapted gradually during exposure for 15 d from an initial minimum inhibitory concentration (MIC) of 300 μl 1^-1 to 607 μl ^-1. The three parallel cultures adapted at similar rates, so the adaptation was not ascribed to mutation but to a specific mechanism. Resistant cells did not produce any extracellular isothiazolone-quenching compounds nor undergo detectable alterations in their lipopolysaccharide layer. In wild cells, a 35 kDa outer membrane protein (protein T) was detectable, whereas resistant cells lacked this protein. Production of protein T was suppressed within 24 h of exposure to isothiazolone. It was still suppressed after 72 h of growth in isothiazolone-free medium. It is proposed that Ps. aeruginosa acquires resistance to isothiazolone by a process of adaptation where the outer membrane protein T is suppressed.     

Resistance of Pseudomonas aeruginosa to gentamicin

The resistance to gentamicin 4 μg./ml. of 250 Pseudomonas aeruginosa isolates was measured by a proportion method. Twenty-eight (11.2%) of the cultures fell into the most resistant group, in whose populations between 10 and 100% of the organisms were resistant. A relatively high percentage of urinary isolates and a comparatively low percentage of isolates from respiratory sources occurred in this group. Three of the 28 were resistant to carbenicillin 150 μg./ml. and 6 of 18 tested were as resistant to gentamicin 8 μg./ml. as they were to 4 μg./ml. The distribution of Ps. aeruginosa isolates between the different grades of resistance did not change significantly during the 10 months in which the survey was performed.     

Lung Microbiota Changes Associated with Chronic Pseudomonas aeruginosa Lung Infection and the Impact of Intravenous Colistimethate Sodium

Background

Exacerbations associated with chronic lung infection with Pseudomonas aeruginosa are a major contributor to morbidity, mortality and premature death in cystic fibrosis. Such exacerbations are treated with antibiotics, which generally lead to an improvement in lung function and reduced sputum P. aeruginosa density. This potentially suggests a role for the latter in the pathogenesis of exacerbations. However, other data suggesting that changes in P. aeruginosa sputum culture status may not reliably predict an improvement in clinical status, and data indicating no significant changes in either total bacterial counts or in P. aeruginosa numbers in sputum samples collected prior to pulmonary exacerbation sheds doubt on this assumption. We used our recently developed lung segmental model of chronic Pseudomonas infection in sheep to investigate the lung microbiota changes associated with chronic P. aeruginosa lung infection and the impact of systemic therapy with colistimethate sodium (CMS).

Methodology/Principal Findings

We collected protected specimen brush (PSB) samples from sheep (n = 8) both prior to and 14 days after establishment of chronic local lung infection with P aeruginosa. Samples were taken from both directly infected lung segments (direct) and segments spatially remote to such sites (remote). Four sheep were treated with daily intravenous injections of CMS between days 7 and 14, and four were treated with a placebo. Necropsy examination at d14 confirmed the presence of chronic local lung infection and lung pathology in every direct lung segment.The predominant orders in lung microbiota communities before infection were Bacillales, Actinomycetales and Clostridiales. While lung microbiota samples were more likely to share similarities with other samples derived from the same lung, considerable within- and between-animal heterogeneity could be appreciated.Pseudomonadales joined the aforementioned list of predominant orders in lung microbiota communities after infection. Whilst treatment with CMS appeared to have little impact on microbial community composition after infection, or the change undergone by communities in reaching that state, when Gram negative organisms (excluding Pseudomonadales) were considered together as a group there was a significant decrease in their relative proportion that was only observed in the sheep treated with CMS. With only one exception the reduction was seen in both direct and remote lung segments. This reduction, coupled with generally increasing or stable levels of Pseudomonadales, meant that the proportion of the latter relative to total Gram negative bacteria increased in all bar one direct and one remote lung segment.

Conclusions/Significance

The proportional increase in Pseudomonadales relative to other Gram negative bacteria in the lungs of sheep treated with systemic CMS highlights the potential for such therapies to inadvertently select or create a niche for bacteria seeding from a persistent source of chronic infection.     

环丙沙星耐药的铜绿假单胞菌药敏情况分析

目的了解对环丙沙星耐药的铜绿假单胞菌的药敏情况。方法细菌的鉴定及药敏试验,均采用法国生物梅里埃公司的VITEK2微生物鉴定与药敏系统,选择经VITEK2测定耐环丙沙星的铜绿假单胞菌(耐药株)68株及对环丙沙星敏感的铜绿假单胞菌(敏感株)67株,统计分析两者对亚胺培南、阿米卡星、庆大霉素、头孢他啶、哌拉西林和哌拉西林他唑巴坦等几种临床上常用抗生素的药敏情况。结果耐药株对以上6种抗生素的敏感率分别为亚胺培南706%,阿米卡星206%,庆大霉素191%,头孢他啶279%,哌拉西林632%,哌拉西林他唑巴坦721%。而敏感株对6种抗生素的敏感率则分别为925%、328%、687%、657%、881%与896%,明显高于耐药株,经χ2检验,除阿米卡星005相似文献   

Resistance to Antibiotics of Clinical Relevance in the Fecal Microbiota of Mexican Wildlife

There are a growing number of reports of antibiotic resistance (ATBR) in bacteria living in wildlife. This is a cause for concern as ATBR in wildlife represents a potential public health threat. However, little is known about the factors that might determine the presence, abundance and dispersion of ATBR bacteria in wildlife. Here, we used culture and molecular methods to assess ATBR in bacteria in fecal samples from howler monkeys (Alouatta palliata), spider monkeys (Ateles geoffroyi), tapirs (Tapirus bairdii) and felids (jaguars, Panthera onca; pumas, Puma concolor; jaguarundis, Puma yagouaroundi; and ocelots, Leopardus pardalis) living freely in two regions of the Mexican state of Veracruz under different degrees of human influence. Overall, our study shows that ATBR is commonplace in bacteria isolated from wildlife in southeast Mexico. Most of the resistances were towards old and naturally occurring antibiotics, but we also observed resistances of potential clinical significance. We found that proximity to humans positively affected the presence of ATBR and that ATBR was higher in terrestrial than arboreal species. We also found evidence suggesting different terrestrial and aerial routes for the transmission of ATBR between humans and wildlife. The prevalence and potential ATBR transfer mechanisms between humans and wildlife observed in this study highlight the need for further studies to identify the factors that might determine ATBR presence, abundance and distribution.     

Resistance to Mercuric Chloride in Pseudomonas K-62

Mercuric ion-resistant mechanism in Pseudomonas K-62 was investigated by isolating a mercuric ion-sensitive mutant strain (H-2 strain) from a resistant wild-type strain (P strain) and comparing the physiological properties of these two strains. Growth curves obtained in mercuric ion-containing medium suggested that P strain constitutively has a resistant mechanism. The capacity of P and H-2 strains to decrease the concentration of mercuric ions in the medium was found to be almost equal; thus, the presence of a resistant mechanism was expected other than MMR-enzyme, which is an inducible system. From the characteristic property of the inhibitory effect of mercuric ions on respiration of the two strains, a resistant mechanism, a permeability barrier mechanism, was proposed. The barrier mechanism in P strain traps mercuric ions with high selectivity to prevent the penetration of ions to the intracellular space where the functional units are located. Finally, the resistance mechanism to mercuric ions in the bacterium was discussed including the MMR-enzyme system and the barrier mechanism.     

铜绿假单胞菌对11种抗生素的耐药性差异

目的探讨不同标本分离铜绿假单胞菌对11种抗生素耐药性的差异,指导临床合理使用抗生素。方法按常规方法培养分离后,经VITEK-AMS60或VITEK-II自动微生物鉴定分析仪的鉴定,用Kirty-Bauer法做药物敏感试验。结果尿液与血液之间,以及尿液与伤口分泌物之间,分离株均对所检测的11种抗生素的耐药性差异有非常显著性（P〈0.01）,尿液与穿刺液分离菌对除亚胺培南和美洛培南外的9种抗生素的耐药性差异有非常显著性（P〈0.01）,血液与穿刺液、血液与伤口分泌物和穿刺液与伤口分泌物间,除穿刺液与伤口分泌物分离菌对美洛培南的耐药性差异有显著性（P〈0.05）外,其他差异均无显著性（P〉0.05）。结论不同标本来源铜绿假单胞菌对哌拉西林等11种抗生素存在耐药性差异。     

Impact of Nisin-Activated Packaging on Microbiota of Beef Burgers during Storage

Beef burgers were stored at 4°C in a vacuum in nisin-activated antimicrobial packaging. Microbial ecology analyses were performed on samples collected between days 0 and 21 of storage to discover the population diversity. Two batches were analyzed using RNA-based denaturing gradient gel electrophoresis (DGGE) and pyrosequencing. The active packaging retarded the growth of the total viable bacteria and lactic acid bacteria. Culture-independent analysis by pyrosequencing of RNA extracted directly from meat showed that Photobacterium phosphoreum, Lactococcus piscium, Lactobacillus sakei, and Leuconostoc carnosum were the major operational taxonomic units (OTUs) shared between control and treated samples. Beta diversity analysis of the 16S rRNA sequence data and RNA-DGGE showed a clear separation between two batches based on the microbiota. Control samples from batch B showed a significant high abundance of some taxa sensitive to nisin, such as Kocuria rhizophila, Staphylococcus xylosus, Leuconostoc carnosum, and Carnobacterium divergens, compared to control samples from batch A. However, only from batch B was it possible to find a significant difference between controls and treated samples during storage due to the active packaging. Predicted metagenomes confirmed differences between the two batches and indicated that the use of nisin-based antimicrobial packaging can determine a reduction in the abundance of specific metabolic pathways related to spoilage. The present study aimed to assess the viable bacterial communities in beef burgers stored in nisin-based antimicrobial packaging, and it highlights the efficacy of this strategy to prolong beef burger shelf life.     

Survival and Impact of Genetically Engineered Pseudomonas putida Harboring Mercury Resistance Gene in Aquatic Microcosms

The survival of wild-type and genetically engineered Pseudomonas putida PpY101 that contained a recombinant plasmid pSR134 conferring mercury resistance were monitored in aquatic microcosms. We used lake, river, and spring water samples. The density of genetically engineered and wild-type P. putida decreased rapidly within 5 days (population change rate k -0.87 ~ -1.00 day^?1), then moderately after 5 to 28 days (-0.10~, -0.14 day^?1). The population change rates of genetically engineered and wild-type P. putida were not significantly different. We studied the important factors affecting the survival of genetically engineered and wild-type P. putida introduced in aquatic microcosms. Visible light exerted an adverse effect on the survival of the two strains. The densities of genetically engineered and wild-type P. putida were almost constant until 7 days after inoculation in natural water filtered with a 0.45-µm membrane filter, or treated with cycloheximide to inhibit the growth of protozoa. These results suggested that protozoan predation was one of the most important factors for the survival of two strains. We examined the impact of the addition of genetically engineered and wild-type P. putida on indigenous bacteria and protozoa. Inoculation of genetically engineered or wild-type P. putida had no apparent effect on the density of indigenous bacteria. The density of protozoa increased in microcosms inoculated with genetically engineered or wild-type P. putida at 3 days after inoculation, but after 5 to 21 days, the density of protozoa decreased to the same level as the control microcosms.     

R Factor-Mediated Resistance to Aminoglycoside Antibiotics in Pseudomonas aeruginosa

Conjugal transferability of drug resistance was examined, in eleven Pseudomonas aeruginosa strains which were isolated in Frankfurt. Four R factors were demonstrated from three strains using P. aeruginosa as recipients but they were nontransferable to Escherichia coli K12. Two R factors, i.e., R_ms146 and R_ms147, mediated resistances to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), kanamycin (KM), lividomycin (LV), gentamicin C complex (GM) and 3′,4′-dideoxykanamycin B (DKB). They mediated the formation of aminoglycoside-inactivating enzymes, i.e., SM phosphotransferase, SM adenylyltransferase, KM and LV phosphotransferase 1, and GM and DKB 6′-N-acetyltransferase. TC resistance conferred by these R factors was due to impermeability of the drug. P. aeruginosa Ps 142 carried two kinds of R factor in one cell, R_ms148 (SM) and R_ms149 (SM·SA·GM·CPC) (CPC, carbenicillin). R_ms148 (SM) was transferable at a high frequency of 10^–1 and mediated the formation of SM phosphotransferase. R_ms149 mediated the formation of drug-inactivating enzymes, i.e., GM 3-N-acetyltransferase and β-lactamase, but did not inactivate SM. SM resistance was probably due to impermeability of the drug.     

Resistance of Pseudomonas pseudomallei to Normal Human Serum Bactericidal Action

The effect of human normal serum (HNS) on Pseudomonas pseudomallei was determined. It is apparent from our data that the organism is resistant to the normal serum bactericidal mechanism. Ancillary experiments to confirm this serum-resistant property of P. pseudomallei were done by examining the effects of growth phase conditions of the bacteria (i.e., logarithmic and stationary phases) and different buffered systems used as diluent in our bactericidal assay. Results obtained showed similar degree of resistance to serum bactericidal killing by 5 strains of the organisms tested. The possible survival advantage of serum-resistant property to P. pseudomallei as bacterial pathogens known to invade the blood stream is discussed.     

Resistance of Pseudomonas aeruginosa to amphoteric and quaternary ammonium biocides

Pseudomonas aeruginosa was able to grow in high levels of an amphoteric and a quaternary ammonium compound following repeated subculturing in increasing concentrations of the biocides. Resistance was acquired and lost gradually. Adaptation to both biocides resulted in cross resistance to biguanides but whereas quaternary adapted cells were resistant to a range of quaternary ammonium compounds, the amphoteric adapted organisms were not. Amphoteric adapted cells had increased hydrophobicity and exhibited ultrastructural modifications which suggested that the outer membrane might be involved in resistance. Both amphoteric and quaternary ammonium adapted organisms showed changes in their fatty acid profiles consistent with outer membrane modification but the changes were different in each case. The mechanisms involved in biocide resistance are discussed.     

Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair

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Impact of Selection for Digestive Efficiency on Microbiota Composition in the Chicken

Objectives

Feed efficiency and its digestive component, digestive efficiency, are key factors in the environmental impact and economic output of poultry production. The interaction between the host and intestinal microbiota has a crucial role in the determination of the ability of the bird to digest its food and to the birds’ feed efficiency. We therefore investigated the phenotypic and genetic relationships between birds’ efficiency and the composition of the cecal microbiota in a F2 cross between broiler lines divergently selected for their high or low digestive efficiency.

Methods

Analyses were performed on 144 birds with extreme feed efficiency values at 3 weeks, with feed conversion values of 1.41±0.05 and 2.02±0.04 in the efficient and non-efficient groups, respectively. The total numbers of Lactobacillus, L. salivarius, L. crispatus, C. coccoides, C. leptum and E. coli per gram of cecal content were measured.

Results

The two groups mainly differed in larger counts of Lactobacillus, L. salivarius and E. coli in less efficient birds. The equilibrium between bacterial groups was also affected, efficient birds showing higher C. leptum, C. coccoides and L. salivarius to E. coli ratios. The heritability of the composition of microbiota was also estimated and L. crispatus, C. leptum, and C. coccoides to E. coli ratios were moderately but significantly heritable (0.16 to 0.24). The coefficient of fecal digestive use of dry matter was genetically and positively correlated with L. crispatus, C. leptum, C. coccoides (0.50 to 0.76) and negatively with E. coli (-0.66). Lipid digestibility was negatively correlated with E. coli (-0.64), and AMEn positively correlated with C. coccoides and with the C. coccoides to Lactobacillus ratio (0.48 to 0.64). We also detected 14 Quantitative Trait Loci (QTL) for microbiota on the host genome, mostly on C. leptum and Lactobacillus. The QTL for C. leptum on GGA6 was close to genome-wide significance. This region mainly includes genes involved in anti-inflammatory responses and in the motility of the gastrointestinal tract.