Genetic and biochemical analyses of a eukaryotic-like phospholipase D of Pseudomonas aeruginosa suggest horizontal acquisition and a role for persistence in a chronic pulmonary infection model

Phospholipases D (PLDs) are virtually ubiquitous in eukaryotic organisms; however, they are relatively uncommon in prokaryotes. In this report, we demonstrate that the environmentally acquired, opportunistic pathogen Pseudomonas aeruginosa expresses PLD activity. A gene designated pldA was identified in the genomic database of P. aeruginosa PAO1 encoding a protein with significant homology to eukaryotic PLDs, but not to any prokaryotic PLDs. PldA is most homologous to PLDs from mammals and yeast. The pldA gene was cloned and shown to express an approximately 116 kDa protein with calcium-regulated PLD activity that is localized to the periplasm. Interestingly, not all strains of P. aeruginosa carry pldA. When present, pldA is always linked to an open reading frame (ORF), ORF4, and a gene (vgrA1) encoding a protein homologous to Vgr from Escherichia coli. Vgr proteins contain regularly repeated dipeptide motifs (valine-glycine repeats). In E. coli, genes encoding Vgr are associated with multicopy genetic elements designated Rhs (rearrangement hot-spots). P. aeruginosa PAO1 has 10 vgr homologues dispersed throughout its genome, but the copy number of these genetic elements varies considerably in different strains. Neither vgrA1 nor ORF4 is present in strains lacking pldA. Furthermore, sequences flanking vgrA1, pldA and ORF4 in the P. aeruginosa strains examined are highly conserved, suggesting a specific site of insertion. These and other data suggest that vgrA1, pldA and ORF4 constitute an approximately 7 kb mobile genetic element and that pldA was acquired horizontally, perhaps from a eukaryotic organism. Competition studies between a PldA knock-out mutant and the parental wild-type strain indicate that PldA contributes to the ability of P. aeruginosa PAO1 to persist in a chronic pulmonary infection model in rats.     

Positive role of peptidoglycan breaks in lactococcal biofilm formation

Bacterial attachment to solid matrices depends on adhesive molecules present on the cell surface. Here we establish a positive correlation between peptidoglycan (PG) breaks, rather than particular molecules, and biofilm-forming capacity in the Gram-positive bacterium Lactococcus lactis. The L. lactis acmA strain, which is defective in PG hydrolase, adhered less efficiently than the wild-type (wt) strain to different solid surfaces and was unable to form biofilms. These phenotypes were abolished by addition of lysozyme, a PG hydrolytic enzyme. Thus, the presence of PG breaks introduced by PG hydrolase, and not the AcmA protein itself, appears to be responsible for biofilm formation. Two different genetic screens confirmed the importance of PG breaks in L. lactis biofilm formation. Using the chain-forming ability of the acmA strain as a phenotypic indicator of PG integrity, we selected for insertional mutants generating short chains. Five independent mutants were all mapped to ponA, which encodes the PG synthesis enzyme PBP1A. Double acmA ponA mutants displayed increased adhesion and biofilm-forming capacity. Direct selection for strains with increased biofilm-forming capacity resulted in the isolation of another five mutations in ponA. Based on these results, we conclude that PG breaks are important for both adhesion and biofilm formation in L. lactis.     

Examination of the genetic variability among biofilm-forming Candida albicans clinical isolates

Biofilms are microbial communities encased in a self-produced polymeric matrix and represent a common mode of microbial growth. Candida albicans is able to colonize the surface of catheters, prostheses, and epithelia, forming biofilms that are highly resistant to antimicrobial drugs. The objective of this study was the genotypic characterization of biofilm-forming C. albicans clinical isolates using RAPD (Random Amplified Polymorphic DNA). We have studied 25 clinical isolates of C. albicans from oral cavities, blood, skin, nail, stool, oesophagus biopsy and vaginal fluids from patients suffering from candidiasis. For each strain biofilm formation was analysed by measuring the ability to adhere to and grow on polystyrene plastic surfaces using XTT [2,3-bis(2-methoxi-4nitro-5sulfophenil)-2H tetrazolium-5carboxanilide] reduction assay. The similarity coefficients generated by RAPD using four different primers varied from 49 to 91%, indicating a high degree of genetic variability between the clinical isolates. The dendrogram clustered the isolates in four related groups, all groups included strains with very different abilities to form biofilms. The isolates with similar genotypes often showed very different biofilm formation abilities. Strains were grouped into clusters independently of their clinical sources. Our results suggested that a direct correlation does not exist between the biofilm-forming ability of natural populations of C. albicans and the genotype as determined by RAPD.     

Determination of beta-lactamase types of clinical strains of bacteria using a modified microiodometric method

The substrate profiles and sensitivity to dicloxacillin inhibition were studied in the enzymes of the clinical strains of Pseudomonas aeruginosa and the transconjugants of E. coli carrying the plasmids discovered earlier in P. aeruginosa. The study was performed with a modified microiodometric method for determination of the activity of beta-lactamases. According to the M. Richmond classification of beta-lactamases the enzymes detected in P. aeruginosa strains 4529, 5290 and 9902 may correspond to the 5th class, the enzymes of P. aeruginosa strain 8208 to the 2nd class and the beta-lactamases of the E. coli transconjugants to the 3rd class. Two different beta-lactamases were detected in P. aeruginosa strain 10294.     

上海市某三甲医院幽门螺杆菌耐药性和毒力与其生物膜形成能力的相关性研究

为探讨复旦大学附属华东医院(以下简称本院)分离培养的幽门螺杆菌(Helicobacter pylori,H. pylori)的耐药性、毒力和感染特征与其生物膜形成能力的相关性,收集2014年12月-2015年6月于本院消化内镜中心的胃活检组织标本及相应临床病例资料,分离培养获得幽门螺杆菌,分析菌株的耐药性、毒力基因型、临床病例特征。结果显示,从胃活检组织样本中共分离培养28株幽门螺杆菌,对左氧氟沙星(levofloxacin,LEV)、甲硝唑(metronidazole,MTZ)和克拉霉素(clarithromycin,CLA)的耐药率分别为32%、75%和11%,未发现阿莫西林(amoxicillin,AMX)耐药。单一药物耐药17株(17/28,61%),双重耐药10株(10/28,36%)。毒力基因cagA、oipA和vacAs1检出率为100%,未检出vacAs2。基因型vacAs1m1占39%(11/28),vacAs1m2占61%(17/28);iceA1占54%(15/28),iceA2占21%(6/28),iceA1A2占25%(7/28);dupA^＋占36%(10/28)。28株菌株均能形成生物膜,但能力不尽相同。单因素及独立样本t检验分析显示,45～59岁、iceA1^＋dupA^－基因型和甲硝唑敏感菌株形成生物膜的能力较强。结果提示,本院分离的幽门螺杆菌对甲硝唑耐药率最高,双重耐药不容忽视。菌株主要毒力基因型为cagA、oipA、vacAs1m2。幽门螺杆菌的生物膜形成能力与患者年龄有关,45～59岁组较强;携带毒力基因iceA1的菌株生物膜形成能力强;dupA基因型及甲硝唑耐药与菌株生物膜形成呈负相关。     

一株工业腐败物中魏氏柠檬酸杆菌的分离鉴定及产生物膜特性分析

【目的】分离和鉴定工业腐败物中高产细菌生物膜菌株,并明确该菌的部分产膜特性。【方法】通过微孔板结晶紫染色法对分离的菌株进行产膜能力评价,根据菌落形态、生理生化特性和16S rRNA序列的系统进化树分析进行菌株鉴定;同时利用扫描电子显微镜(SEM)和结晶紫染色法分别研究材料及温度对该菌产膜特性和能力的影响。【结果】筛选出一株高产细菌生物膜菌株,经鉴定该菌为魏氏柠檬酸杆菌;其在玻璃、不锈钢和聚氯乙烯(PVC)材料表面均能形成生物膜;温度条件显著影响产膜能力,在30°C时,菌株在PVC材料表面形成生物膜能力最强。【结论】工业腐败物中含有高产细菌生物膜菌株,并且产膜受附着物和温度影响。     

Biofilm formation by asymptomatic and virulent urinary tract infectious Escherichia coli strains

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. In contrast to uropathogenic E. coli (UPEC) that cause symptomatic urinary tract infection, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the biofilm-forming capacity on abiotic surfaces of groups of ABU strains and UPEC strains in human urine. We found that there is a strong bias; ABU strains were significantly better biofilm formers than UPEC strains. Our data suggest that biofilm formation in urinary tract infectious E. coli seems to be associated with ABU strains and appears to be an important strategy used by these strains for persistence in this high-flow environment.     

Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa.

Mucoid strains of Pseudomonas aeruginosa isolated from the sputum of cystic fibrosis patients produce copious quantities of an exopolysaccharide known as alginic acid. Since clinical isolates of the mucoid variants are unstable with respect to alginate synthesis and revert spontaneously to the more typical nonmucoid phenotype, it has been difficult to isolate individual structural gene mutants defective in alginate synthesis. The cloning of the genes controlling alginate synthesis has been facilitated by the isolation of a stable alginate-producing strain, 8830. The stable mucoid strain was mutagenized with ethyl methanesulfonate to obtain various mutants defective in alginate biosynthesis. Several nonmucoid (Alg-) mutants were isolated. A mucoid P. aeruginosa gene library was then constructed, using a cosmid cloning vector. DNA isolated from the stable mucoid strain 8830 was partially digested with the restriction endonuclease HindIII and ligated to the HindIII site of the broad host range cosmid vector, pCP13. After packaging in lambda particles, the recombinant DNA was introduced via transfection into Escherichia coli AC80. The clone bank was mated (en masse) from E. coli into various P. aeruginosa 8830 nonmucoid mutants with the help of pRK2013, which provided donor functions in trans, and tetracycline-resistant exconjugants were screened for the ability to form mucoid colonies. Three recombinant plasmids, pAD1, pAD2, and pAD3, containing DNA inserts of 20, 9.5, and 6.2 kilobases, respectively, were isolated based on their ability to restore alginate synthesis in various strain 8830 nonmucoid (Alg-) mutants. Mutants have been assigned to at least four complementation groups, based on complementation by pAD1, pAD2, or pAD3 or by none of them. Introduction of pAD1 into the spontaneous nonmucoid strain 8822, as well as into other nonmucoid laboratory strains of P. aeruginosa such as PAO and SB1, was found to slowly induce alginate synthesis. This alginate-inducing ability was found to reside on a 7.5-kilobase EcoRI fragment that complemented the alg-22 mutation of strain 8852. The pAD1 chromosomal insert which complements the alg-22 mutation was subsequently mapped at ca. 19 min of the P. aeruginosa PAO chromosome.     

Isolation and characterization of rhamnolipid-producing bacterial strains from a biodiesel facility

Novel strains of rhamnolipid-producing bacteria were isolated from soils at a biodiesel facility on the basis of their ability to grow on glycerol as a sole carbon source. Strains were identified as Acinetobacter calcoaceticus , Enterobacter asburiae , Enterobacter hormaechei , Pantoea stewartii , and Pseudomonas aeruginosa . The strains of the former five species were found to produce rhamnolipids in quantities the same as, or similar to, coisolated strains of P. aeruginosa . Measurements of surface tension revealed that that emulsifying properties of these strains were similar to levels displayed by rhamnolipids produced by P. aeruginosa . Results of matrix-assisted laser desorption/ionization time-of-flight MS analyses revealed that the predominant compounds made by all strains were C₁₀–C₁₀ mono- and dirhamnolipids. Notably, E. hormaechei and one strain of A. calcoaceticus produced rhamnolipids in amounts similar to the pseudomonads. As all strains examined were from the same taxonomic class of Proteobacteria , further examination of this group may reveal many additional species not previously known to produce rhamnolipids in addition to novel strains of species currently known to produce rhamnolipids.     

Structure of Pseudomonas aeruginosa populations analyzed by single nucleotide polymorphism and pulsed-field gel electrophoresis genotyping

Pseudomonas aeruginosa has a wide ecological distribution that includes natural habitats and clinical settings. To analyze the population structure and distribution of P. aeruginosa, a collection of 111 isolates of diverse habitats and geographical origin, most of which contained a genome with a different SpeI macrorestriction profile, was typed by restriction fragment length polymorphism based on 14 single nucleotide polymorphisms (SNPs) located at seven conserved loci of the core genome (oriC, oprL, fliC, alkB2, citS, oprI, and ampC). The combination of these SNPs plus the type of fliC present (a or b) allowed the assignment of a genetic fingerprint to each strain, thus providing a simple tool for the discrimination of P. aeruginosa strains. Thirteen of the 91 identified SNP genotypes were found in two or more strains. In several cases, strains sharing their SNP genotype had different SpeI macrorestriction profiles. The highly virulent CHA strain shared its SNP genotype with other strains that had different SpeI genotypes and which had been isolated from nonclinical habitats. The reference strain PAO1 also shared its SNP genotype with other strains that had different SpeI genotypes. The P. aeruginosa chromosome contains a conserved core genome and variable amounts of accessory DNA segments (genomic islands and islets) that can be horizontally transferred among strains. The fact that some SNP genotypes were overrepresented in the P. aeruginosa population studied and that several strains sharing an SNP genotype had different SpeI macrorestriction profiles supports the idea that changes occur at a higher rate in the accessory DNA segments than in the conserved core genome.     

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum.     

Vero cell toxins in Escherichia coli and related bacteria: transfer by phage and conjugation and toxic action in laboratory animals, chickens and pigs

Sixty-eight of 519 strains of Escherichia coli and six of 10 strains of Pseudomonas aeruginosa produced toxins acting on Vero cells (VT+); all of 63 Salmonella, Shigella, Klebsiella, Enterobacter and Proteus strains were VT-. Most of the VT+ E. coli strains were from weaned pigs suffering from oedema disease and/or diarrhoea and belonged to serogroups O141:K85,88, O141:K85, O138:K81, and O139:K82; six VT+ E. coli strains were from diarrhoeic human babies, four of serogroup O26 and two of serogroup O128. The VT genes in two of the O26 strains and in the O128 strains were located in the genome of the phages with which they were lysogenized. One O141:K85,88 pig E. coli strain transferred its VT genes, probably by conjugation, to E. coli K12. The VTs of the human E. coli strains, the pig E. coli strains and the P. aeruginosa strains were antigenically different from each other; unlike the others, the P. aeruginosa VT was heat-resistant. Cell-free preparations of cultures of E. coli K12 to which the VT genes of the four human E. coli strains had been transferred caused fluid accumulation in ligated segments of rabbit intestine. Inoculated intravenously, they were lethal for mice and rabbits; similar preparations of E. coli K12 to which the VT genes of the pig E. coli strain had been transferred produced a disease in pigs that clinically and pathologically resembled oedema disease.     

The ability to entrain to long photoperiods differs between 3 Drosophila melanogaster wild-type strains and is modified by twilight simulation

The ability to adapt to different environmental conditions including seasonal changes is a key feature of the circadian clock. Here, we compared the ability of 3 Drosophila melanogaster wild-type strains to adapt rhythmic activity to long photoperiods simulated in the laboratory. Fruit flies are predominantly crepuscular with activity bouts in the morning (M) and evening (E). The M peak follows dawn and the E peak follows dusk when the photoperiod is extended. We show that this ability is restricted to a certain extension of the phase angle between M and E peaks, such that the E peak does not delay beyond a certain phase under long days. We demonstrate that this ability is significantly improved by simulated twilight and that it depends additionally on the genetic background and the ambient temperature. At 20 °C, the laboratory strain CantonS had the most flexible phase angle between M and E peaks, a Northern wild-type strain had an intermediate one, and a Southern wild-type strain had the lowest flexibility. Furthermore, we found that the 3 strains differed in clock light sensitivity, with the CantonS and the Northern strains more light sensitive than the Southern strain. These results are generally in accord with the recently discovered polymorphisms in the timeless gene (tim) that affect clock light sensitivity.     

Grazing rates on toxic and non-toxic strains of cyanobacteria by Hypophthalmichthys molitrix and Oreochromis niloticus

The tilapia Oreochromis niloticus and the silver carp Hypophthalmichthys molitrix were exposed to toxic and non-toxic strains of the cyanobacterium Microcystis aeruginosa in order to determine if cells of the toxic strain were ingested and, if not, by what mechanism they were excluded. Enumeration of cyanobacterial particles before and after exposure to fish showed that there were no significant differences (P<0.05) at the end of the trial between the toxic treatment and the control consisting of toxic M. aeruginosa with no fish. Fish exposed to the non-toxic strain increased opercular beat rate, elevating the volumes of water and food material passed over the gills whereas those that were held in the toxic strain did not. Of the cyanobacterial toxins (microcystins) presented to the fish, most were in the cyanobacterial cells, toxin levels in the water being below the level of detectability (<250 ng l⁻¹), The ability of the fish to differentiate between toxic and non-toxic cyanobacterial strains may thus be determined by very low levels of extracellular microcystins or/and other features which distinguish toxic from non-toxic M. aeruginosa strains, such as cell surface components.     

铜绿假单胞菌对碳青霉烯类药物的耐药机制研究

目的 探讨铜绿假单胞菌对碳青霉烯类药物的耐药机制.方法 收集2008年11月至2009年4月我院临床分离的铜绿假单胞菌31株,根据药敏结果分为碳青霉烯类耐药组(21株)和碳青霉烯类敏感组(10株).另设1株标准株ATCC 27853,用亚胺培南-EDTA(乙二胺四乙酸)抑制试验检测菌株是否产生金屑酶,采用PCR法检测各菌株的外膜孔道蛋白oprD2基因,探讨铜绿假单胞菌对碳青霉烯类抗生素耐药机制.结果 21株耐药株有7株产生金属酶;21株耐药株经oprD2基因扩增,15株阴性,6株阳性,10株敏感株全部阳性.统计学检验结果表明,碳青霉烯类耐药组与敏感组oprD2基因阳性率的差异有极显著性(P＜0.01).结论 oprD2基因缺失和金属酶是本院铜绿假单胞菌对碳青霉烯类抗生素耐药的重要机制.     

Expression of foreign LpxA acyltransferases in Neisseria meningitidis results in modified lipid A with reduced toxicity and retained adjuvant activity

A major problem in the development of vaccines against Gram-negative bacteria is the endotoxic -activity of lipopolysaccharide (LPS), which is determined by its lipid A moiety. Nevertheless, LPS would be an interesting vaccine component because of its immune-stimulating properties. In the present study, we have changed the fatty acid composition of Neisseria meningitidis LPS by replacing the lpxA gene of strain H44/76 with the Escherichia coli or Pseudomonas aeruginosa homologue. The majority of the O-linked 3-OH C12 in N. meningitidis lipid A was replaced by 3-OH C14 (strain HA01E) and 3-OH C10 (strain HA25P) respectively. Both strains, but most notably strain HA01E, had reduced amounts of LPS compared with the wild-type strain. In addition, growth was severely impaired for HA01E. The major outer membrane proteins were expressed normally. Outer membrane complexes of both strains normalized on their LPS content showed a 10-fold reduction in their ability to induce tumour necrosis factor (TNF)-alpha. Immunogenicity studies in BALB/c mice revealed that the adjuvant activity of the LPS was not affected. Thus, the replacement of the O-linked fatty acids in meningococcal lipid A results in immunogenic outer membranes with reduced endotoxic activity, more suitable for use in outer membrane vesicle vaccines.     

Burkholderia spp. alter Pseudomonas aeruginosa physiology through iron sequestration

Pseudomonas aeruginosa and members of the Burkholderia cepacia complex often coexist in both the soil and the lungs of cystic fibrosis patients. To gain an understanding of how these different species affect each other's physiology when coexisting, we performed a screen to identify P. aeruginosa genes that are induced in the presence of Burkholderia: A random gene fusion library was constructed in P. aeruginosa PA14 by using a transposon containing a promoterless lacZ gene. Fusion strains were screened for their ability to be induced in the presence of Burkholderia strains in a cross-streak assay. Three fusion strains were induced specifically by Burkholderia species; all three had transposon insertions in genes known to be iron regulated. One of these fusion strains, containing a transposon insertion in gene PA4467, was used to characterize the inducing activity from Burkholderia: Biochemical and genetic evidence demonstrate that ornibactin, a siderophore produced by nearly all B. cepacia strains, can induce P. aeruginosa PA4467. Significantly, PA4467 is induced early in coculture with an ornibactin-producing but not an ornibactin-deficient B. cepacia strain, indicating that ornibactin can be produced by B. cepacia and detected by P. aeruginosa when the two species coexist.     

Comparison of Listeria monocytogenes Exoproteomes from Biofilm and Planktonic State: Lmo2504, a Protein Associated with Biofilms

The food-borne pathogen Listeria monocytogenes is the causative agent of the severe human and animal disease listeriosis. The persistence of this bacterium in food processing environments is mainly attributed to its ability to form biofilms. The search for proteins associated with biofilm formation is an issue of great interest, with most studies targeting the whole bacterial proteome. Nevertheless, exoproteins constitute an important class of molecules participating in various physiological processes, such as cell signaling, pathogenesis, and matrix remodeling. The aim of this work was to quantify differences in protein abundance between exoproteomes from a biofilm and from the planktonic state. For this, two field strains previously evaluated to be good biofilm producers (3119 and J311) were used, and a procedure for the recovery of biofilm exoproteins was optimized. Proteins were resolved by two-dimensional difference gel electrophoresis and identified by electrospray ionization-tandem mass spectrometry. One of the proteins identified in higher abundance in the biofilm exoproteomes of both strains was the putative cell wall binding protein Lmo2504. A mutant strain with deletion of the gene for Lmo2504 was produced (3119Δlmo2504), and its biofilm-forming ability was compared to that of the wild type using the crystal violet and the ruthenium red assays as well as scanning electron microscopy. The results confirmed the involvement of Lmo2504 in biofilm formation, as strain 3119Δlmo2504 showed a significantly (P < 0.05) lower biofilm-forming ability than the wild type. The identification of additional exoproteins associated with biofilm formation may lead to new strategies for controlling this pathogen in food processing facilities.     

Scanning electron microscopic study of uropathogen adherence to a plastic surface

We used a polyethylene surface to study the adherence of various urinary pathogens to a representative inert surface. The bacteria were suspended in filter-sterilized urine during this adhesion study, and differential adhesion was clearly demonstrated. Pseudomonas aeruginosa adhered most avidly and formed large microcolonies that were surrounded by an extensive amorphous matrix. Staphylococcus saprophyticus also formed microcolonies on the surface of the plastic droppers. In general, piliated strains of Escherichia coli adhered less avidly than the other organisms, but more avidly than nonpiliated strains; however, one piliated strain of E. coli adhered very poorly and behaved like a nonpiliated strain.     

Scanning electron microscopic study of uropathogen adherence to a plastic surface.

We used a polyethylene surface to study the adherence of various urinary pathogens to a representative inert surface. The bacteria were suspended in filter-sterilized urine during this adhesion study, and differential adhesion was clearly demonstrated. Pseudomonas aeruginosa adhered most avidly and formed large microcolonies that were surrounded by an extensive amorphous matrix. Staphylococcus saprophyticus also formed microcolonies on the surface of the plastic droppers. In general, piliated strains of Escherichia coli adhered less avidly than the other organisms, but more avidly than nonpiliated strains; however, one piliated strain of E. coli adhered very poorly and behaved like a nonpiliated strain.