Susceptibility of Burkholderia cepacia and other pathogens of importance in cystic fibrosis to u.v. light

To investigate the potential usefulness of u.v. germicidal irradiation (UVGI) in preventing the spread of Burkholderia cepacia, an important pathogen in cystic fibrosis (CF), the in-vitro susceptibility of B. cepacia to UVGI was determined. Five strains were exposed to UVGI from a 7.2-W source. Burkholderia cepacia was less susceptible to UVGI than other important CF-related pathogens, namely Staphylococcus aureus and Pseudomonas aeruginosa, but was more susceptible than Stenotrophomonas maltophilia. No strain of B. cepacia survived longer than an 8 s exposure to UVGI, with doses required to achieve 1 log reduction in bacterial numbers ranging from 28.3 to 57.5 J m(-2).     

Antibacterial activity of different honeys against pathogenic bacteria

To study the antimicrobial activity of honey, 60 samples of various botanical origin were evaluated for their antimicrobial activities against 16 clinical pathogens and their respective reference strains. The microbiological quality of honeys and the antibiotic susceptibility of the various isolates were also examined. The bioassay applied for determining the antimicrobial effect employs the well-agar diffusion method and the estimation of minimum active dilution which produces a 1 mm diameter inhibition zone. All honey samples, despite their origin (coniferous, citrus, thyme or polyfloral), showed antibacterial activity against the pathogenic and their respective reference strains at variable levels. Coniferous and thyme honeys showed the highest activity with an average minimum dilution of 17.4 and 19.2% (w/v) followed by citrus and polyfloral honeys with 20.8 and 23.8% respectively. Clinical isolates of Staphylococcus aureus subsp. aureus, Escherichia coli, Salmonella enterica subsp. Enterica, Streptococcus pyogenes, Bacillus cereus and Bacillus subtilis were proven to be up to 60% more resistant than their equal reference strains thus emphasizing the variability in the antibacterial effect of honey and the need for further research.     

土壤中产脂肪酶洋葱伯克霍尔德菌的分离与鉴定

洋葱伯克霍尔德菌(Burkholderia cepacia)在生物防治、生物降解等农业领域有着广泛的应用,它产生的脂肪酶则在有机合成、精细化工等领域潜力巨大。采用改良的TB-T平板筛选法从土壤中初步筛选出300株洋葱伯克霍尔德菌,然后用脂肪酶活性检测平板对300株菌进行筛选,最终获得6株脂肪酶产量高的菌,通过发酵发现6株菌均有较好的产脂肪酶能力。随后通过16S rDNA比对的方法将6株全部鉴定为B.cepacia。在此基础上,采用HaeⅢ-recA RFLP和基因种特异性PCR对6株菌进行了基因种鉴定,结果表明JWT16、G63YL、WJ158和JWT137属于Burkholderia cenocepacia菌,JWP9属于Burkhold-eria vietnamiensis,JWT267则属于Burkholderia multivorans。     

The sensitivity to honey of Gram-positive cocci of clinical significance isolated from wounds

AIMS: To determine the sensitivity to honey of Gram-positive cocci of clinical significance in wounds and demonstrate that inhibition is not exclusively due to osmotic effects. METHODS AND RESULTS: Eighteen strains of methicillin-resistant Staphylococcus aureus and seven strains of vancomycin-sensitive enterococci were isolated from infected wounds and 20 strains of vancomycin-resistant enterococci were isolated from hospital environmental surfaces. Using an agar incorporation technique to determine the minimum inhibitory concentration (MIC), their sensitivity to two natural honeys of median levels of antibacterial activity was established and compared with an artificial honey solution. For all of the strains tested, the MIC values against manuka and pasture honey were below 10% (v/v), but concentrations of artificial honey at least three times higher were required to achieve equivalent inhibition in vitro. Comparison of the MIC values of antibiotic-sensitive strains with their respective antibiotic-resistant strains demonstrated no marked differences in their susceptibilities to honey. CONCLUSIONS: The inhibition of bacteria by honey is not exclusively due to osmolarity. For the Gram-positive cocci tested, antibiotic-sensitive and -resistant strains showed similar sensitivity to honey. SIGNIFICANCE AND IMPACT OF THE STUDY: A possible role for honey in the treatment of wounds colonized by antibiotic-resistant bacteria is indicated.     

PCR-based identification and characterization of Burkholderia cepacia complex bacteria from clinical and environmental sources

AIMS: To study the genotypic identification and characterization of the 119 Burkholderia cepacia complex (Bcc) strains recovered from clinical and environmental sources in Japan and Thailand. METHODS AND RESULTS: Based on the results of analysis by 16S rDNA RFLP generated after digestion with DdeI, the Bcc strains were differentiated into two patterns: pattern 1 (including Burkholderia vietnamiensis) and pattern 2 (including B. cepacia genomovar I, Burkholderia cenocepacia and Burkholderia stabilis). All strains belonged to pattern 2 except for one strain. In the RFLP analysis of the recA gene using HaeIII, strains were separated into eight patterns designated as A, D, E, G, H, I, J and K, of which pattern K was new. Burkholderia cepacia epidemic strain marker (BCESM) encoded by esmR [corrected] and the pyrrolnitrin biosynthetic locus encoded by prnC were present in 22 strains (18%) and 88 strains (74%) from all sources, respectively. All esmR-positive [corrected] strains belonged to B. cenocepacia, whereas most prnC-positive strains belonged to B. cepacia genomovar I. CONCLUSIONS: Strains derived from clinical sources were assigned to B. cepacia genomovar I, B. cenocepacia, B. stabilis and B. vietnamiensis. The majority of Bcc strains from environmental sources (77 of a total 95 strains) belonged to B. cepacia genomovar I, whereas the rest belonged to B. cenocepacia. On the basis of genomovar-specific PCR and prnC RFLP analysis, strains belonging to recA pattern K were identified as B. cepacia genomovar I. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the genotypic identification of a collection of the Bcc strains from Japan and Thailand. RFLP analysis of the prnC gene promises to be a useful method for differentiating Burkholderia pyrrocinia from B. cepacia genomovar I strains.     

Surface characteristics of Pseudomonas cepacia

Two major surface characteristics of Pseudomonas cepacia were examined in this study: reactivity with lectins and hydrophobicity. The results indicated that the surfaces of P. cepacia strains are heterogeneous with regard to the distribution of lectin receptors. Only lima bean agglutinin was found to strongly agglutinate all strains. The strains were also heterogeneous with regard to hydrophobicity as determined by adhesion to hexadecane. The degree of hydrophobicity, however, was not significantly altered when selected strains were mixed with either fibronectin or bovine serum albumin. In addition, the strains exhibited no apparent affinities for buccal epithelial cells and gave no evidence for an ability to haemagglutinate human red cells.     

从植物根际定向批量筛选广谱有机溶剂耐受性脂肪酶产生菌

洋葱伯克霍尔德菌脂肪酶是一类具有重要工业应用价值的优良脂肪酶之一。根据已公布的洋葱伯克霍尔德菌基因组信息, 在传统的洋葱伯克霍尔德菌选择性培养基中添加适量的氨苄青霉素和卡那霉素, 从植物根际的土壤中筛选洋葱伯克霍尔德菌。对获得的单菌落再用含罗丹明B指示剂的产脂肪酶定性检测平板检测, 从4个根际土壤中筛选到35株产脂肪酶的洋葱伯克霍尔德菌, 阳性率达到65%。其中15株对体积浓度为10%的苯、己烷和正庚烷同时具有耐受性。用recA基因分子鉴定上述15株菌种, 全部属于洋葱伯克霍尔德菌菌群。     

洋葱伯克霍尔德菌致恶性肿瘤患者医院获得性感染的耐药性分析

目的分析我院恶性肿瘤患者医院获得性感染洋葱伯克霍尔德菌的临床特点及对常用抗菌药物的耐药性,为临床合理治疗提供依据。方法回顾性分析2011年1月至2013年12月,从我院恶性肿瘤感染患者送检的细菌培养标本;细菌鉴定用美国BD公司phoenix-100全自动细菌鉴定药敏系统,药敏试验采用纸片法,同时使用WHONET 5.6软件对相关资料进行统计。结果从检测部位分析主要分布在下呼吸道（74.1%）,其次为血液（9.4%）;药敏试验表明139株洋葱伯克霍尔德菌对米诺环素、氯霉素、美罗培南、头孢他啶和头孢哌酮/舒巴坦仍较敏感,可作为临床治疗洋葱伯克霍尔德菌感染的首选药物,其余15种抗菌药物的耐药率高达30.0%~100%。结论洋葱伯克霍尔德菌在恶性肿瘤患者中的耐药现象非常严重,临床应引起高度关注,及早进行微生物学检测,并根据药敏试验结果合理选用抗菌药物。     

Insight into probiotic properties of lactic acid bacterial endosymbionts of Apis mellifera L. derived from the Polish apiary

Taking into account that fructophilic lactic acid bacteria (FLAB) can play an important role in the health of honey bees and can be used as probiotics, phenotypic properties of probiotic interest of Lactobacillus kunkeei (12 strains) and Fructobacillus fructossus bacteria (2 strains), isolated from Apis mellifera gastrointestinal tract, have been studied. We have evaluated survival of tested FLAB in honey bee gut, their susceptibility to antibiotics (ampicillin, erythromycin, tylosin), cell surface hydrophobicity, auto-aggregation ability, co-aggregation with model pathogenic bacteria, biofilm formation capacity, and effect of studied FLAB, added to sucrose syrup bee diet, on longevity of honey bees. The tested FLAB exhibited good gastrointestinal tract tolerance and high antibiotic susceptibility, which are important criteria in the screening of probiotic candidates. It was also found that all FLAB studied have high cell surface hydrophobicity and fulfil next selection criterion for their use as probiotics. Symbionts of A. mellifera showed also auto- and co-aggregation capacities regarded as valuable features for biofilm formation and inhibition of pathogens adhesion to the bee gut cells. Biofilm-development ability is a desired characteristic of probiotic lactic acid bacteria. As indicated by quantitative crystal violet-stained microplate assay and confocal laser scanning microscopy imaging, all studied A. mellifera gut isolates exhibit a biofilm positive phenotype. Moreover, it was also documented, on honey bees kept in cages, that supplementation of A. mellifera sucrose diet with FLAB decreases mortality and improves significantly longevity of honey bees. Presented research showed that A. mellifera FLAB symbionts are good candidates for application as probiotics.     

A quorum-quenching approach to investigate the conservation of quorum-sensing-regulated functions within the Burkholderia cepacia complex

Taxonomic studies of the past few years have shown that the Burkholderia cepacia complex, a heterogeneous group of B. cepacia-like organisms, consists of at least nine species. B. cepacia complex strains are ubiquitously distributed in nature and have been used for biocontrol, bioremediation, and plant growth promotion purposes. At the same time, B. cepacia complex strains have emerged as important opportunistic pathogens of humans, particularly those with cystic fibrosis. All B. cepacia complex species investigated thus far use quorum-sensing (QS) systems that rely on N-acylhomoserine lactone (AHL) signal molecules to express certain functions, including the production of extracellular proteases, swarming motility, biofilm formation, and pathogenicity, in a population-density-dependent manner. In this study we constructed a broad-host-range plasmid that allowed the heterologous expression of the Bacillus sp. strain 240B1 AiiA lactonase, which hydrolyzes the lactone ring of various AHL signal molecules, in all described B. cepacia complex species. We show that expression of AiiA abolished or greatly reduced the accumulation of AHL molecules in the culture supernatants of all tested B. cepacia complex strains. Phenotypic characterization of wild-type and transgenic strains revealed that protease production, swarming motility, biofilm formation, and Caenorhabditis elegans killing efficiency was regulated by AHL in the large majority of strains investigated.     

Differential interaction of bacterial species from the Burkholderia cepacia complex with human airway epithelial cells

To increase knowledge of the pathogenic potential of the Burkholderia cepacia complex (BCC), we investigated the effects of reference strains of the nine BCC species on human bronchial epithelial cells in vitro. B. multivorans exhibited the highest rates of adherence to and internalization by host cells. Two out of three clinical isolates recovered from cystic fibrosis patients confirmed the B. multivorans high adhesiveness. All four B. multivorans isolates exhibited an aggregated pattern of adherence but any of them expressed cable pili. When bacteria were centrifuged onto cell cultures to circumvent their poor adhesiveness, B. pyrrocinia exhibited the highest internalization rate, followed by B. multivorans. The percentages of apoptotic cells in cultures infected with B. cepacia, B. multivorans, B. cenocepacia (subgroups IIIA and IIIB), B. stabilis and B. vietnamiensis were significantly higher than in control non-infected cultures. All nine BCC species triggered a similar release of the inflammatory cytokine IL-8, that was not reduced by cell treatment with cytochalasin D. Hence, our data demonstrate, for the first time, that all BCC species exhibit a similar ability to induce the expression of host immune mediators whereas they differ on their ability to adhere to, invade and kill airway epithelial cells.     

In vitro effect of some Indian honeys on Staphylococcus aureus from wounds

Staphylococcus aureus is the most frequently isolated pathogen from wounds with multiple resistances to antibiotics. Honey has been demonstrated and reported to be effective antibacterial agent on Gram positive and Gram negative organisms. Hence, the present study was conducted to evaluate the in vitro antibacterial effect of Indian honeys on Staphylococcus aureus obtained from wounds. A total of 123 Staphylococcus aureus isolates along with ATCC 25923 were categorized as sensitive, multi drug resistant (MDR) and non-MDR strains. Out of total nine Indian honeys (three each of unifloral, multifloral and branded marketed honey) used, three unifloral and three multifloral honey samples showed antibacterial activity against all the organisms tested by Agar diffusion method but not the branded marketed honeys. The MIC values of all honey samples for all studied Staphylococcus aureus isolates ranged between 5-15% (v/v). Unifloral honey samples showed higher antibacterial activity than multifloral honey. The single sample of Jambhul honey showed the highest activity. Thus, Indian honeys were found to be effective for their antimicrobial activity on sensitive, non-MDR, MDR and ATCC strains of S. aureus.     

重症监护病房洋葱伯克霍尔德菌医院感染的特征及耐药性分析

目的了解重症监护病房（ICU）洋葱伯克霍尔德菌引起医院感染的特征及耐药情况,为临床治疗及控制该菌的暴发流行提供实验依据。方法常规方法对我院2003年1月至2007年10月ICU的病人的各种临床标本进行分离培养,细菌鉴定及药敏试验采用全自动微生物鉴定仪VITEK-2进行。结果引起ICU医院感染的洋葱伯克霍尔德菌共有99例,感染以肺部感染为主,对临床常用的多种抗菌药物表现交叉耐药,对头孢匹肟、亚胺培南、哌拉西林、阿米卡星、庆大霉素的敏感率较差在50．0％以下;对环丙沙星、左氧氟沙星、头孢他啶、氨曲南、哌拉西林／他唑巴坦、美诺培南和复方新诺明的敏感率较高,分别为82．8％、87．9％、91．9％、72．7％、55．6％、62．6％和100．0％。结论引起ICU医院感染的洋葱伯克霍尔德菌具有多重耐药性,临床治疗时应根据药敏结果选用抗菌药物。     

Genotypic and phenotypic relationships in Burkholderia cepacia isolated from cystic fibrosis patients and the environment

Twenty-one strains of Burkholderia cepacia isolated from the environment, and 21 clinical strains isolated principally from sputum of cystic fibrosis (CF) patients, were characterized genotypically by macrorestriction analysis (genome fingerprinting) and PCR ribotyping, and phenotypically by susceptibility to antibiotics and the ability to macerate onion tissue. The plasmid content of the strains was also investigated. Environmental isolates showed a high degree of genetic variability, all strains differing from both one another and the CF isolates. The CF isolates were less variable, with common strains found in patients attending three geographically distinct CF centres. Phenotypic variation was found both within and between CF and environmental strains. Generally, CF isolates displayed higher levels of antibiotic resistance, while the ability to macerate onion tissue was more prevalent amongst environmental isolates. Plasmids were more frequently found in CF isolates, but were of similar size in both groups of strains. Such variability is not surprising in view of the existence of multiple genomovars within the B. cepacia complex.     

Antibacterial activity of honey from stingless honeybees (Hymenoptera; Apidae; Meliponinae).

The aim of the study was to examine antibacterial activity of the honey of stingless honeybees (Meliponinae). An agar well diffusion assay demonstrated that many honey samples of stingless honeybees inhibited the growth of test strains of Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa; moreover, they exhibited non-peroxide antibacterial activity against those strains. This is the first time that non-peroxide antimicrobial activity of honey from a number of species of stingless honeybees has been demonstrated. These antibacterial activities appear to be powerful, even when compared to those of"manuka honey" from Apinae honeybees.     

Multiple chromosomes in Burkholderia cepacia and B. gladioli and their distribution in clinical and environmental strains of B. cepacia

Burkholderia cepacia is found in soils and waters, it can be used in biocontrol and bioremediation but is also a human pathogen. It is not yet clear what differentiates pathogenic from non-pathogenic strains of the organism. In this study the multiple replicon structure was investigated in 28 strains of B. cepacia by pulsed field gel electrophoresis. All strains examined, whether of clinical, environmental or plant pathogenic origin, were found to have two, three or four large (> 500 kbp) replicons. Many strains also contained small replicons. Clinical strains were more likely to have three or four large replicons than non-clinical strains. Multiple replicon structure was also demonstrated in B. gladioli and Alcaligenes eutrophus.     

Antibacterial potential of some Saudi honeys from Asir region against selected pathogenic bacteria

Honey is a nutrient rich natural product and has been utilized as traditional and complementary medicine since ancient times. In this study, antibacterial activity of Sider (Ziziphus spina-christi), Dharm (Lavandula dentata), and Majra (Hypoestes forskaolii) honey samples collected from Asir region of Saudi Arabia was in vitro evaluated at 80% and 50% w/v concentrations against five pathogenic bacteria i.e. Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Shigella flexneri, and Staphylococcus epidermidis. Well diffusion assays to measure the average zone of inhibition (ZOI) and minimum inhibitory concentration (MIC) values were employed in the experiments. All the tested honey samples showed antibacterial activity in a dose-dependent manner. Sider and Dharm exhibited a good antibacterial activity at high concentrations while, Majra honey of Apis mellifera jemenitica and of Apis florea showed comparatively low antibacterial activity. The average MIC values of Sider, Dhram from Rijal Alma, Dharm from Al-Souda, Majra (A.m. jemenitica), and Majra (A. florea) honey against all tested bacteria were 22%, 16%, 18%, 32%, and 28% (v/v) respectively. Dharm and Sider honeys showed better antibacterial activity than Majra honey. Saudi honey can be considered as a promising future antimicrobial agent and should be further investigated as an alternative candidate in the management of resistant bacterial pathogens.     

Diversity of Burkholderia isolates from woodland rhizosphere environments

AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.     

Bacteriocin typing of Pseudomonas cepacia strains isolated from patients and the rhizosphere of plants

The work deals with the bacteriocin typing of 34 P. cepacia strains isolated from different sources with respect to both the capacity of synthesizing bactericins and sensitivity to them. The standard set of strains comprizing 8 P. cepacia bacteriocin-sensitive strains and 6 highly active cepaciacin producer strains was used. 24 P. cepacia strains belonged to 11 different S-types, 20 strains synthetized cepaciacins of new types.     

Cloning, expression, and regulation of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase genes.

The genes for the alpha and beta subunits of the enzyme protocatechuate 3,4-dioxygenase (EC 1.13.11.3) were cloned from the Pseudomonas cepacia DBO1 chromosome on a 9.5-kilobase-pair PstI fragment into the broad-host-range cloning vector pRO2317. The resultant clone was able to complement protocatechuate 3,4-dioxugenase mutations in P. cepacia, Pseudomonas aeruginosa, and Pseudomonas putida. Expression studies showed that the genes were constitutively expressed and subject to catabolite repression in the heterologous host. Since the cloned genes exhibited normal induction patterns when present in P. cepacia DBO1, it was concluded that induction was subject to negative control. Regulatory studies with P. cepacia wild-type and mutant strains showed that protocatechuate 3,4-dioxygenase is induced either by protocatechuate or by beta-carboxymuconate. Further studies of P. cepacia DBO1 showed that p-hydroxybenzoate hydroxylase (EC 1.14.13.2), the preceding enzyme in the pathway, is induced by p-hydroxybenzoate and that beta-carboxymuconate lactonizing enzyme, which catalyzes the reaction following protocatechuate 3,4-dioxygenase, is induced by both p-hydroxybenzoate and beta-ketoadipate.