Epidemiology of Pseudomonas cichorii,the Cause of Lettuce Midrib Rot

Bacterial midrib rot, caused by Pseudomonas cichorii, has become a serious threat to the production of greenhouse butterhead lettuce (Lactuca sativa L. var. capitata) in Belgium. Currently, there are no strategies for controlling this pathogen. Therefore, greenhouse experiments were conducted to obtain more knowledge about the epidemiology of P. cichorii on butterhead lettuce. Greenhouse butterhead lettuce becomes susceptible to lettuce midrib rot infections at head formation, and a single overhead irrigation with water containing 10² CFU/ml P. cichorii was sufficient to cause disease. The use of surface drip irrigation instead of overhead sprinkler irrigation significantly reduced midrib rot incidence in the greenhouse. P. cichorii isolates can be divided into subgroups based on BOX‐PCR genomic fingerprinting, with isolates belonging to subgroup C1 and C2 being more virulent than those of (or related to) subgroup C3. P. cichorii infections with distinct symptoms comparable to midrib rot have also been observed on field‐grown crisphead lettuce in California and Japan which, respectively, are referred to as ‘varnish spot’ or ‘tar’. We showed that symptom expression is strongly influenced by the lettuce cultivar group, irrespective of the P. cichorii isolate, resulting in varnish spot/tar on crisphead lettuce and midrib rot on butterhead or cutting group lettuce.     

Pseudomonas cichorii as the causal agent of midrib rot,an emerging disease of greenhouse-grown butterhead lettuce in Flanders

Bacterial midrib rot of greenhouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) is an emerging disease in Flanders (Belgium) and fluorescent pseudomonads are suspected to play an important role in the disease. Isolations from infected lettuces, collected from 14 commercial greenhouses in Flanders, yielded 149 isolates that were characterized polyphasically, which included morphological characteristics, pigmentation, pathogenicity tests by both injection and spraying of lettuce, LOPAT characteristics, FAME analysis, BOX-PCR fingerprinting, 16S rRNA and rpoB gene sequencing, as well as DNA–DNA hybridization. Ninety-eight isolates (66%) exhibited a fluorescent pigmentation and were associated with the genus Pseudomonas. Fifty-five of them induced an HR⁺ (hypersensitive reaction in tobacco leaves) response. The other 43 fluorescent isolates were most probably saprophytic bacteria and about half of them were able to cause rot on potato tuber slices. BOX-PCR genomic fingerprinting was used to assess the genetic diversity of the Pseudomonas midrib rot isolates. The delineated BOX-PCR patterns matched quite well with Pseudomonas morphotypes defined on the basis of colony appearance and variation in fluorescent pigmentation. 16S rRNA and rpoB gene sequence analyses allowed most of the fluorescent isolates to be allocated to Pseudomonas, and they belonged to either the Pseudomonas fluorescens group, Pseudomonas putida group, or the Pseudomonas cichorii/syringae group. In particular, the isolates allocated to this latter group constituted the vast majority of HR⁺ isolates and were identified as P. cichorii by DNA–DNA hybridization. They were demonstrated by spray-inoculation tests on greenhouse-grown lettuce to induce the midrib rot disease and could be re-isolated from lesions of inoculated plants. Four HR⁺ non-fluorescent isolates associated with one sample that showed an atypical midrib rot were identified as Dickeya sp.     

Molecular Identification and Biofilm-Forming Ability of Culturable Aquatic Bacteria In Microbial Biofilms Formed in Drinking Water Distribution Networks

Drinking water distribution networks are known to harbor microbial biofilms. The aim of the present work is to (i) identify the culturable bacteria presented in the drinking-water distribution network, (ii) investigate the ability of isolated bacteria to form biofilm under some environmental stress conditions and some eliminating or removing treatments. To achieve it, 57 strains were isolated from biofilm (43 isolates) and water samples (14 isolates) collected from five stations in drinking-water distribution network in Taif city, Kingdom of Saudi Arabia (KSA). Partial sequences of 16S rRNA gene in the 57 isolates ensured the presence of only 22 different strains in biofilm samples. Among these strains, only 14 strains were also detected in water samples. Gram-negative Aeromonas hydrophila was the most occurred bacterium in the microbial biofilm obtained from the purified-water storage tanks followed by Gram-negative Pseudomonas sp. Gram-positive Bacillus subtilis was the most occurred bacterium in the microbial biofilm collected from the ends of the distribution pipes. Among the 22 isolated strains, 13 strains were strong biofilm producers at 30 and 37°C. The effects of environmental stresses including nutrient starvation (diluted TSB, 20:1), heating (100°C for 10 min), UV-treatment (240 nm for 10 min) and dynamic incubation (150 rpm min^?1) on the formation of biofilm were also investigated. These conditions affected the biofilm formation ability of the isolated strains at different levels. Nutrient starvation enhanced biofilm formation by most of the isolates. Among some biofilm deforming treatments, SDS and trypsin had considerable effects on preventing biofilm formation by most of the isolated strains. In conclusion, the results of the present work indicated that not all biofilm strains released from biofilm to the drinking water. Also, not all biofilm strains were able to form biofilm. Most of isolated bacteria had ability to form biofilm at suboptimum temperature of growth. These results may provide basic information on formation of microbial biofilms and overcome the problem of deteriorating of water quality in the drinking-water distribution networks.     

Oxidative stress response and virulence factors in <Emphasis Type="Italic">Candida glabrata</Emphasis> clinical isolates

We determined the susceptibility to oxidative stress and assessed the four virulence factors of the 38 Candida glabrata clinical isolates originating from two teaching hospitals in Slovakia. All the isolates were susceptible to hydrogen peroxide, diamide, and 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT) inducing an increased formation of reactive oxygen species in fungal cells. The mean relative cell surface hydrophobicity (CSH) of isolates was 21.9, ranging from 1.92 to 56.96. All isolates showed biofilm formation. A high biofilm formation was observed among 60.5% of isolates. Positive correlations were observed between biofilm formation and moderate values of CSHs. The 76.3% and 84.2% of isolates displayed varying degrees of proteinase and phospholipase activity, respectively. These results demonstrate a differential distribution of factors contributing to virulence of C. glabrata clinical isolates and point to their significance in pathogenesis that would be targeted by novel antifungals.     

Moderate Prevalence of Antimicrobial Resistance in Escherichia coli Isolates from Lettuce,Irrigation Water,and Soil

Fresh produce is known to carry nonpathogenic epiphytic microorganisms. During agricultural production and harvesting, leafy greens can become contaminated with antibiotic-resistant pathogens or commensals from animal and human sources. As lettuce does not undergo any inactivation or preservation treatment during processing, consumers may be exposed directly to all of the (resistant) bacteria present. In this study, we investigated whether lettuce or its production environment (irrigation water, soil) is able to act as a vector or reservoir of antimicrobial-resistant Escherichia coli. Over a 1-year period, eight lettuce farms were visited multiple times and 738 samples, including lettuce seedlings (leaves and soil), soil, irrigation water, and lettuce leaves were collected. From these samples, 473 isolates of Escherichia coli were obtained and tested for resistance to 14 antimicrobials. Fifty-four isolates (11.4%) were resistant to one or more antimicrobials. The highest resistance rate was observed for ampicillin (7%), followed by cephalothin, amoxicillin-clavulanic acid, tetracycline, trimethoprim, and streptomycin, with resistance rates between 4.4 and 3.6%. No resistance to amikacin, ciprofloxacin, gentamicin, or kanamycin was observed. One isolate was resistant to cefotaxime. Among the multiresistant isolates (n = 37), ampicillin and cephalothin showed the highest resistance rates, at 76 and 52%, respectively. E. coli isolates from lettuce showed higher resistance rates than E. coli isolates obtained from soil or irrigation water samples. When the presence of resistance in E. coli isolates from lettuce production sites and their resistance patterns were compared with the profiles of animal-derived E. coli strains, they were found to be the most comparable with what is found in the cattle reservoir. This may suggest that cattle are a potential reservoir of antimicrobial-resistant E. coli strains in plant primary production.     

Evaluation of Adherence,Hydrophobicity, Aggregation,and Biofilm Development of <Emphasis Type="Italic">Flavobacterium johnsoniae</Emphasis>-Like Isolates

Flavobacterium spp. isolates have been identified in diverse biofilm structures, but the mechanism of adherence has not been elucidated. The absence of conventional biofilm-associated structures such as fimbriae, pili, and flagella suggest that surface hydrophobicity, and/or autoaggregation and coaggregation may play an important role in adherence and biofilm formation. The biofilm-forming capacity of 29 Flavobacterium johnsoniae-like isolates obtained from South African aquaculture systems was assessed using microtiter plate assays. The role of hydrophobicity [salting aggregation test (SAT) and bacterial adherence to hydrocarbons (BATH) assays], autoaggregation, and coaggregation on biofilm formation by Flavobacterium spp. was also investigated, while biofilm structure was examined using flow cells and microscopy. All isolates displayed a hydrophilic nature, but showed varying levels of adherence in microtiter assays. Significant negative correlations were observed between adherence and biofilm-forming capacity in nutrient-poor medium at 26°C and BATH hydrophobicity and motility, respectively. Isolates displayed strain-to-strain variation in their autoaggregation indices and their abilities to coaggregate with various Gram-negative and Gram-positive organisms. Microcolony and/or biofilm development were observed microscopically, and flavobacterial isolates displayed stronger biofilm structures and interaction with a Vibrio spp. isolate than with an Aeromonas hydrophila isolate. The role of extracellular polysaccharides and specific outer membrane proteins will have to be examined to reveal mechanisms of adherence and coaggregation employed by biofilm-forming F. johnsoniae-like strains.     

Probiotic Lactobacilli Interfere with <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilm Formation In Vitro

In clinical studies, probiotic bacteria have decreased the counts of salivary mutans streptococci (MS). We compared the effects of probiotic Lactobacillus strains on the biofilm formation of Streptococcus mutans. The bacterial strains used included four S. mutans strains (reference strains NCTC 10449 and Ingbritt and clinical isolates 2366 and 195) and probiotic strains Lactobacillus rhamnosus GG, L. plantarum 299v, and L. reuteri strains PTA 5289 and SD2112. The ability of MS to adhere and grow on a glass surface, reflecting biofilm formation, was studied in the presence of the lactobacilli (LB). The effect of LB culture supernatants on the viability of the MS was studied as well. All of the LB inhibited the biofilm formation of the clinical isolates of MS (P < 0.001). The biofilm formation of the reference strains of MS was also inhibited by the LB, but L. plantarum and L. reuteri PTA 5289 showed a weaker inhibition when compared to L. reuteri SD2112 and L. rhamnosus GG. Viable S. mutans cells could be detected in the biofilms and culture media only when the experiments were performed with the L. reuteri strains. The L. reuteri strains were less efficient in killing the MS also in the tests performed with the culture supernatants. The pHs of the supernatants of L. reuteri were higher compared to those of L. rhamnosus GG and L. plantarum; P < 0.001. In conclusion, our results demonstrated that four commonly used probiotics interfered with S. mutans biofilm formation in vitro, and that the antimicrobial activity against S. mutans was pH-dependent.     

Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene among biofilm forming clinical isolates of Acinetobacter spp

Background

Quorum sensing is a term that describes an environmental sensing system that allows bacteria to monitor their own population density which contributes significantly to the size and development of the biofilm. Many gram negative bacteria use N-acyl-homoserine lactones as quorum sensing signal molecules. In this study, we sought to find out if the biofilm formation among clinical isolates of Acinetobacter spp. is under the control of autoinducing quorum sensing molecules.

Methodology/Principal Findings

Biofilm formation among clinical isolates of Acinetobacter spp. was assessed and the production of signal molecules were detected with Chromobacterium violaceum CV026 biosensor system. Characterisation of autoinducers was carried out by mass spectrometric analysis. We have also reported the identification of an autoinducer synthase gene, abaΙ among the isolates that produce quorum sensing signal molecules and have reported that the mutation in the abaI gene influences their biofilm forming capabilities. Using a microtitre-plate assay it was shown that 60% of the 50 Acinetobacter spp. isolates significantly formed biofilms. Further detection with the biosensor strain showed that some of these isolates produced long chain signal molecules. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C₁₂. The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown.

Conclusions/Significance

These data are of great significance as the signal molecules aid in biofilm formation which in turn confer various properties of pathogenicity to the clinical isolates including drug resistance. The use of quorum sensing signal blockers to attenuate bacterial pathogenicity is therefore highly attractive, particularly with respect to the emergence of multi antibiotic resistant bacteria.     

Pseudomonads associated with midrib rot and soft rot of butterhead lettuce and endive

During the past ten years, bacterial soft rot and midrib rot of glasshouse-grown butterhead lettuce (Lactuca sativa L. var. capitata) and field-grown endive (Cichorium endivia L.) has become increasingly common in the region of Flanders, Belgium. Severe losses and reduced market quality caused by bacterial rot represent an important economical threat for the production sector. Symptoms of midrib rot are a brownish rot along the midrib of one or more inner leaves, often accompanied by soft rot of the leaf blade. Twenty-five symptomatic lettuce and endive samples were collected from commercial growers at different locations in Flanders. Isolations of dominant bacterial colony types on dilution plates from macerated diseased tissue extracts yielded 282 isolates. All isolates were characterized by colony morphology and fluorescence on pseudomonas agar F medium, oxidase reaction, and soft rot ability on detached chicory leaves. Whole-cell fatty acid methyl esters profile analyses identified the majority of isolates (85%) as belonging to the Gammaproteobacteria, which included members of the family Enterobacteriaceae (14%) and of the genera Pseudomonas (73%), Stenotrophomonas (9%), and Acinetobacter (3%). Predominant bacteria were a diverse group of fluorescent Pseudomonas species. They were further differentiated based on the non-host hypersensitive reaction on tobacco and the ability to rot potato slices into 4 phenotypic groups: HR-/P- (57 isolates), HR-/P+ (54 isolates), HR+/P (16 isolates) and HR+/P+ (35 isolates). Artificial inoculation of suspensions of HR-, pectolytic fluorescent pseudomonads in the leaf midrib of lettuce plants produced various symptoms of soft rot, but they did not readily cause symptoms upon spray inoculation. Fluorescent pseudomonads with phenotype HR+ were consistently isolated from typical dark midrib rot symptoms, and selected isolates reproduced the typical midrib rot symptoms when spray-inoculated onto healthy lettuce plants.     

Screening of bacteria for the suppression of Botrytis cinerea and Rhizoctonia solani on lettuce (Lactuca sativa) using leaf disc bioassays

Two leaf disc bioassays were developed for screening bacteria as putative biological control agents of Botrytis cinerea and Rhizoctonia solani on lettuce. Aerobic spore and non‐spore forming bacteria were isolated from the phylloplane, rhizoplane and rhizosphere of symptom‐free lettuce plants grown in the presence and absence of chitin or composted bark soil amendments. Bacteria, previously isolated from other plants, were also included in the primary screen initially against B. cinerea. One hundred and twenty‐seven of 700 isolates reduced botrytis rotting of lettuce leaves by more than 50% in the primary screen. Following a secondary screen against B. cinerea, the lead 50 isolates were also tested for suppression of R. solani infection. Four isolates significantly reduced both botrytis and rhizoctonia leaf rotting. Eleven and five isolates gave control of botrytis and rhizoctonia, respectively, equal to that given by the standard fungicides Rovral WP (iprodione) and Basilex (tolclofos methyl). The two most effective isolates against B. cinerea and R. solani were both identified as Bacillus subtilis. Use of soil amendments did not increase the proportion of efficacious isolates recovered. Effective isolates were originally recovered from roots of oilseed rape and lettuce leaves. In general, it was found that bacteria which controlled one disease effectively did not control the second disease nearly as well. The bioassay protocols developed in this study were used successfully in screening a large number of bacterial isolates in a short time.     

c-di-AMP调控细菌生物被膜的形成

细菌生物被膜是细菌持续性致病的重要机制。研究细菌生物被膜的形成和发展可为顽固性细菌感染防治提供新的思路与策略。环二腺苷酸c-di-AMP(Cyclic diadenosine monophosphate)是继c-di-GMP之后在细菌中新发现的一种核苷酸第二信使分子。研究发现,c-di-AMP参与调节细菌多种生理功能,包括细菌生长代谢、生物被膜形成、细胞壁的合成以及细菌毒力因子等。本文综述了c-di-AMP参与调控细菌生物被膜形成的不同方式及其分子机制。鉴于c-di-AMP在调控细菌生物被膜中的重要性,其可作为抗细菌生物被膜感染新药研发的潜在靶点。     

The Activities of Adhesion and Biofilm Formation by <Emphasis Type="Italic">Candida tropicalis</Emphasis> Clinical Isolates Display Significant Correlation with Its Multilocus Sequence Typing

Adhesion and biofilm formation, which can occur on abiotic and biotic surfaces, are key components in Candida pathogenicity. The aims of this study were to infer about the C. tropicalis clinical isolates ability to adhere and form biofilm on abiotic and biotic surfaces and to correlate that with the multilocus sequence typing and other virulence factors. Adhesion and biofilm formation were measured in 68 C. tropicalis isolates from 3 hospitals in China on abiotic (polystyrene) and biotic (human urinary bladder epithelial cell) surfaces by crystal violet assay and 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide reduction assay. In our study, almost all C. tropicalis isolates could adhere and produce biofilm on abiotic and biotic surfaces in a strain-dependent manner. The isolates from blood showed relatively lower adhesion and biofilm capacity on polystyrene surface, but had strong secreted aspartyl proteinase activity. Moreover, significant differences were found among MLST groups for adhesion and biofilm capacity. C. tropicalis in multilocus sequence typing group5 and group6 showed high adhesion and biofilm, while isolates in group1 exhibited low adhesion and biofilm formation. Overall, it is important to note that C. tropicalis isolates adhere to and produce biofilm on abiotic and biotic surfaces with strain specificity. These data will play an important role in subsequent research on the pathogenesis of C. tropicalis.     

Overexpressed recombinant quorum quenching lactonase reduces the virulence,motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min⁻¹ for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48–75.9%), and rhamnolipids (52.7–63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.

     

Staphylococcus epidermidis in Orthopedic Device Infections: The Role of Bacterial Internalization in Human Osteoblasts and Biofilm Formation

Background

Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms.

Materials and Methods

Orthopedic device infection S. epidermidis strains (n = 15) were compared to nasal carriage isolates (n = 22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method.

Results

No difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/−631 and 347+/−431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups.

Conclusion

Osteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.     

sarA negatively regulates Staphylococcus epidermidis biofilm formation by modulating expression of 1 MDa extracellular matrix binding protein and autolysis‐dependent release of eDNA

Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant‐associated infections. Nonetheless, large proportions of invasive Staphylococcus epidermidis isolates fail to form a biofilm in vitro. We here tested the hypothesis that this apparent paradox is related to the existence of superimposed regulatory systems suppressing a multicellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm‐negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA, chief regulator of staphylococcal virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via overexpression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed to biofilm formation in mutant 1585ΔsarA. Increased eDNA amounts indirectly resulted from upregulation of metalloprotease SepA, leading to boosted processing of autolysin AtlE, in turn inducing augmented autolysis and release of eDNA. Hence, this study identifies sarA as a negative regulator of Embp‐ and eDNA‐dependent biofilm formation. Given the importance of SarA as a positive regulator of polysaccharide mediated cell aggregation, the regulator enables S. epidermidis to switch between mechanisms of biofilm formation, ensuring S. epidermidis adaptation to hostile environments.     

Endophytic fungi occurring in fennel, lettuce, chicory, and celery — commercial crops in southern Italy

The occurrence of endophytic fungi in fennel, lettuce, chicory, and celery crops was investigated in southern Italy. A total of 186 symptomless plants was randomly collected and sampled at the stage of commercial ripeness. Fungal species of Acremonium, Alternaria, Fusarium, and Plectosporium were detected in all four crops; Plectosporium tabacinum was the most common in all crop species and surveyed sites. The effect of eight endophytic isolates (five belonging to Plectosporium tabacinum and three to three species of Acremonium) inoculated on lettuce plants grown in gnotobiosis was assessed by recording plant height, root length and dry weight, collar diameter, root necrosis, and leaf yellowing. P. tabacinum and three species of Acremonium, inoculated on gnotobiotically grown lettuce plants, showed pathogenic activity that varied with the fungal isolate. Lettuce plants inoculated with the isolates Ak of Acremonium kiliense, Ac of Acremonium cucurbitacearum, and P35 of P. tabacinum showed an increased root growth, compared to the non-inoculated control. The high frequency of P. tabacinum isolation recorded in lettuce plants collected in Bari and Metaponto, and in fennel plants from Foggia agricultural districts, suggests a relationship not only between a crop species and P. tabacinum, but also between the occurrence of the endophyte and the crop rotation history of the soil.     

Biofilm formation by Mycobacterium avium isolates originating from humans, swine and birds

Background  

Mycobacterium avium includes the subspecies avium, silvaticum, paratuberculosis and hominissuis, and M. avium subspecies has been isolated from various environments all over the world including from biofilms in water distribution systems. The aim of this study was to examine isolates of M. avium subsp. avium and M. avium subsp. hominissuis of different origin for biofilm formation and to look for correlations between biofilm formation and RFLP-types, and to standardise the method to test for biofilm formation. In order to determine the best screening method, a panel of 14 isolates of M. avium subsp. avium and M. avium subsp. hominissuis, were tested for their ability to form biofilm in microtiter plates under different conditions. Subsequently, 83 additional isolates from humans, swine and birds were tested for biofilm formation. The isolates were tested for the presence of selected genes involved in the synthesis of glycopeptidolipids (GPLs) in the cell wall of M. avium, which is believed to be important for biofilm formation. Colony morphology and hsp65 sequvar were also determined.     

鲍曼不动杆菌碳青霉烯耐药性与生物膜形成及O-甘露糖蛋白相关性研究

鲍曼不动杆菌(Acinetobacter baumannii)是引起医院感染的常见致病菌,该细菌不仅容易产生耐药性,而且在人体及无生命物质表面易形成生物膜,临床治疗较为棘手。从临床分离24株鲍曼不动杆菌,药物敏感试验观察这些分离株对常用抗菌药物的敏感性,针对耐碳青霉烯鲍曼不动杆菌,检测是否含有耐药基因碳青霉烯酶基因OXA-23,采用结晶紫染色法观察耐药性与生物膜形成的相关性,并用刀豆蛋白凝集素结合试验及质谱分析耐药性与O-甘露糖蛋白的相关性。结果显示鲍曼不动杆菌耐药性与生物膜形成呈正相关,某些O-甘露糖蛋白表达有利于细菌获得耐药性。     

Investigation of N-acyl homoserine lactone (AHL) molecule production in Gram-negative bacteria isolated from cooling tower water and biofilm samples

In this study, 99 Gram-negative rod bacteria were isolated from cooling tower water, and biofilm samples were examined for cell-to-cell signaling systems, N-acyl homoserine lactone (AHL) signal molecule types, and biofilm formation capacity. Four of 39 (10 %) strains isolated from water samples and 14 of 60 (23 %) strains isolated from biofilm samples were found to be producing a variety of AHL signal molecules. It was determined that the AHL signal molecule production ability and the biofilm formation capacity of sessile bacteria is higher than planktonic bacteria, and there was a statistically significant difference between the AHL signal molecule production of these two groups (p?<?0.05). In addition, it was found that bacteria belonging to the same species isolated from cooling tower water and biofilm samples produced different types of AHL signal molecules and that there were different types of AHL signal molecules in an AHL extract of bacteria. In the present study, it was observed that different isolates of the same strains did not produce the same AHLs or did not produce AHL molecules, and bacteria known as AHL producers did not produce AHL. These findings suggest that detection of signal molecules in bacteria isolated from cooling towers may contribute to prevention of biofilm formation, elimination of communication among bacteria in water systems, and blockage of quorum-sensing controlled virulence of these bacteria.     

The Impact of spgM,rpfF, rmlA Gene Distribution on Biofilm Formation in Stenotrophomonas maltophilia

Background

Stenotrophomonas maltophilia is emerging as one of the most frequently found bacteria in chronic pulmonary infection. Biofilm is increasingly recognized as a contributing factor to disease pathogenesis. In the present study, a total of 37 isolates of S. maltophilia obtained from chronic pulmonary infection patients were evaluated to the relationship between biofilm production and the relative genes expression.

Methods

The clonal relatedness of isolates was determined by pulse-field gel electrophoresis. Biofilm formation assays were performed by crystal violet assay, and confirmed by Electron microscopy analysis and CLSM analysis. PCR was employed to learn gene distribution and expression.

Results

Twenty-four pulsotypes were designated for 37 S. maltophilia isolates, and these 24 pulsotypes exhibited various levels of biofilm production, 8 strong biofilm-producing S. maltophilia strains with OD492 value above 0.6, 14 middle biofilm-producing strains with OD492 average value of 0.4 and 2 weak biofilm-producing strains with OD492 average value of 0.19. CLSM analysis showed that the isolates from the early stage of chronic infection enable to form more highly structured and multilayered biofim than those in the late stage. The prevalence of spgM, rmlA, and rpfF genes was 83.3%, 87.5%, and 50.0% in 24 S. maltophilia strains, respectively, and the presence of rmlA, spgM or rpfF had a close relationship with biofilm formation but did not significantly affect the mean amount of biofilm. Significant mutations of spgM and rmlA were found in both strong and weak biofilm-producing strains.

Conclusion

Mutations in spgM and rmlA may be relevant to biofilm formation in the clinical isolates of S. maltophilia.