柑橘衰退病毒柚类分离株的分子鉴定

应用限制性片段长度多态性(RFLP)、双向逆转录-聚合酶链式反应(BD-PCR)、序列分析等技术对我国部分地区柑橘衰退病毒(Citrus tristeza virus,CTV)柚类分离株进行了鉴定分析。结果表明:柚类以受相对单一的CTV株系侵染为主;柚类上的优势流行株系属于p25/HinfⅠRFLP 6组群(占79.4%)和p23/BD-PCRⅢ组群(占84.1%);在对柚类CTV分离株S051~S058及国外CTV分离株PB61、T30、T36、T385、SY568、VT、NUAGA的p23、p25基因序列分析中,S051与弱毒株PB61的同源性最高;S052与弱毒株T30、T385的同源性最高;S053、S054、S055、S056、S057、S058与其它CTV分离株的同源关系均较远,并在系统发生树上形成了独立的分枝。     

Phenotypic and Molecular Identification of <Emphasis Type="Italic">Sporothrix</Emphasis> Isolates from an Epidemic Area of Sporotrichosis in Brazil

Sporotrichosis has significantly increased in Brazil in the last decade, particularly in the state of Rio de Janeiro, with the occurrence of an epidemic related to zoonotic transmission from cats to humans. Recently, four new phylogenetic species were incorporated into the Sporothrix species complex based on the phenotypic and molecular characteristics, and a new species name (Sporothrix brasiliensis) was proposed for some of the Sporothrix isolates from this epidemic. This study describes the characterization of 246 isolates obtained from patients attending the Laboratory of Infectious Dermatology, IPEC-FIOCRUZ, between 1998 and 2008, together with one environmental sample. Two hundred and six of the isolates (83.4%) were characterized as S. brasiliensis, 15 (6.0%) as S. schenckii, and one (0.5%) as S. mexicana. Twenty-five isolates (10.1%) could not be identified according to their phenotype and were classified as Sporothrix spp. The calmodulin gene was sequenced to confirm the identity of these isolates. The molecular analysis demonstrated that 24 of the isolates were S. brasiliensis, with the remainder being a S. globosa isolate. The isolate characterized phenotypically as S. mexicana was clustered on the S. schenckii clade. The correlation between molecular data and phenotypic characteristics described in this study is fundamental to the identification of the Sporothrix complex.     

Development of Genomic SSR Markers and Molecular Characterization of Magnaporthe oryzae Isolates from Wheat in Brazil

Magnaporthe oryzae, the causal agent of wheat blast, was characterized on a molecular level with 38 newly isolated genomic SSR loci. Among the 31 wheat isolates analyzed, 15 polymorphic loci were detected, with an average of 1.7 alleles per locus, 28.9% of them being highly or reasonably informative. The number of polymorphic loci was higher in isolates from Londrina in the Brazilian state of Paraná and Coromandel in Minas Gerais compared with Goiânia in Goiás and São Borja in Rio Grande do Sul. The rice isolate was clearly different from the wheat isolates, and the size difference in polymorphic SSR loci between one isolate from wheat and one isolate from rice was associated with the number of repeats. Some isolates collected from different states and in different years demonstrated similarities of 100%. The markers developed here are useful for the genetic analysis of M. oryzae isolated from wheat, and isolates representing the variability detected in the field can be used to search for better wheat blast resistance.     

Molecular Characterization of Clostridium difficile Isolates from Human Subjects and the Environment

Clostridium difficile is a spore-forming, gram-positive, anaerobic bacillus that can cause C. difficile infection (CDI). However, only a few studies on the prevalence and antibiotic resistance of C. difficile in healthy individuals in China have been reported. We employed a spore enrichment culture to screen for C. difficile in the stool samples of 3699 healthy Chinese individuals who were divided into 4 groups: infants younger than 2 years of age and living at home with their parents; children aged 1 to 8 years of age and attending three different kindergarten schools; community-dwelling healthy adult aged 23–60 years old; and healthcare workers aged 28–80 years old. The C. difficile isolates were analyzed for the presence of toxin genes and typed by PCR ribotyping and multilocus sequence typing (MLST). The minimum inhibitory concentration of 8 antimicrobial agents was determined for all of the isolates using the agar dilution method. The intestinal carriage rate in the healthy children was 13.6% and ranged from 0% to 21% depending on age. The carriage rates in the 1654 community-dwelling healthy adults and 348 healthcare workers were 5.5% and 6.3%, respectively. Among the isolates, 226 were toxigenic (225 tcdA+/tcdB+ and 1 tcdA+/tcdB+ ctdA+/ctdB+). Twenty-four ribotypes were found, with the dominant type accounting for 29.7% of the isolates. The toxigenic isolates were typed into 27 MLST genotypes. All of the strains were susceptible to vancomycin, metronidazole, fidaxomicin, and rifaximin. High resistance to levofloxacin and ciprofloxacin at rates of 39.8% and 98.3%, respectively, were observed. ST37 isolates were more resistant to levofloxacin than the other STs. The PCR ribotypes and sequence types from the healthy populations were similar to those from the adult patients.     

Distribution of Virulence Factors and Molecular Fingerprinting of Aeromonas Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-Human Transmission

A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.Aeromonas species cause both intestinal and extraintestinal infections (25, 33, 78), and the latter include septicemia, cellulitis, wound infections, urinary tract infections, hepatobiliary tract infections, soft tissue infections, and, occasionally, meningitis and peritonitis (25, 30, 78). In immunocompromised children, these pathogens can cause even more severe forms of infections, such as hemolytic-uremic syndrome (HUS) and necrotizing fasciitis (3, 23), although detailed studies are needed to establish such associations. Worldwide, the rate of isolation of Aeromonas from diarrheic stools has been reported to be as high as 10.8%, compared to 2.1% for healthy controls (25, 37, 78). An increased rate of isolation of Aeromonas species was reported in flood water samples during Hurricane Katrina in New Orleans (58), and skin and soft tissue infections caused by Aeromonas species were among the most common infections in the survivors of the 2004 tsunami in southern Thailand (28). In particular, Aeromonas salmonicida causes fish infections that result in huge economical losses in the fishing industry (6, 22). The ability of aeromonads, as well as other bacteria, to survive in chlorinated water when they are in biofilms and their resistance to multiple antibiotics are major public health concerns (46).Aeromonas-related gastroenteritis remains somewhat controversial (24, 36). There have been a number of well-described cases and a few documented outbreaks, but whether all aeromonad fecal isolates from symptomatic persons are the actual causes of diarrheal disease is still questionable. One theory for this conundrum was posed in 2000 by two of us, who suggested that only specific subsets of Aeromonas strains within and between species are actually pathogenic for humans (38). This highlights the importance of developing accurate biotyping, molecular fingerprinting, and virulence factor analysis methods for differentiating environmental and clinical aeromonads from one another and for comparing them (38).Of the 19 currently recognized Aeromonas species, A. hydrophila, A. caviae, and A. veronii biovar sobria are the most common species known to cause the majority of human infections, and they account for more than 85% of all clinical isolates (34). The pathogenesis of Aeromonas infections is multifactorial, as aeromonads produce a wide variety of virulence factors, including hemolysins, cytotonic and cytotoxic enterotoxins, proteases, lipases, leucocidins, endotoxin, adhesions, and an S layer, that act in concert to cause disease in the host (12-14, 50, 51). The cytotoxic enterotoxin Act, which has some similarities to aerolysin (31), is one of the most significant virulence factors in diarrheal isolate SSU of A. hydrophila and was first characterized in our laboratory (12). Act is secreted by the type II secretion system (T2SS) and has hemolytic, cytotoxic, and enterotoxic activities (12). In addition, our laboratory recently sequenced and characterized two other secretion systems, T3SS and T6SS, that were found to contribute to the virulence of A. hydrophila SSU (66, 67, 72). We recently characterized an effector of the T3SS, which was designated AexU, and found that it was associated with ADP ribosylation of host cell proteins, a rounded phenotype in HeLa cells, inhibition of phagocytosis, induction of apoptosis, and mouse mortality (66, 67). In recent studies, we also investigated the role of two T6SS-associated effectors, the valine-glycine repeat G (VgrG) family of proteins and hemolysin-coregulated protein (Hcp), in the virulence of A. hydrophila (71, 72). We demonstrated that VgrG1 of A. hydrophila had actin-ADP ribosylation activity that induced host cell cytotoxicity (71). Based on the model for T6SS, the VgrG1 protein must assemble with the highly homologous VgrG2 and VgrG3 proteins to form a cell-puncturing device to deliver effector proteins into the host cells (59). We also obtained evidence that expression of the hcp gene in HeLa cells led to their apoptosis, and animals immunized with recombinant Hcp were protected from subsequent challenge with a lethal dose of wild-type A. hydrophila SSU (72).In addition, cytotonic enterotoxins (e.g., Alt [heat labile] and Ast [heat stable]) were identified in a diarrheal A. hydrophila SSU isolate (14, 63) that induced fluid secretion in the ligated small intestinal loops of animals (47). More recently, we identified some additional virulence factor-encoding and regulatory genes, such as the enolase, hlyA (hemolysin), gidA (glucose-inhibited division A), vacB (virulence-associated protein B), dam (DNA adenine methyltransferase), and tagA (ToxR-regulated lipoprotein) genes, which modulated the virulence of A. hydrophila SSU (19-21, 57, 62, 64). The production of such a wide array of virulence factors by Aeromonas species is indicative of their potential to cause severe diseases in humans. These virulence factor-encoding genes might be differentially expressed in Aeromonas species depending on the environmental conditions, such as water or the human host.A cell-to-cell signaling system, known as quorum sensing (QS), might play an important role in sensing physiological conditions and helping bacteria express the virulence genes at an appropriate time under the appropriate conditions. Thus far, at least three QS circuits have been identified in Gram-negative bacteria, and they were designated LuxRI (autoinducer 1 [AI-1]), LuxS (AI-2), and AI-3 (epinephrine/norepinephrine). All of these QS systems were detected in our SSU clinical strain of A. hydrophila, and we recently demonstrated that N-acyl homoserine lactone (AHL) (AI-1) and AI-2-mediated QS controlled the virulence of A. hydrophila SSU (40, 43). Further, we observed decreased production of N-acyl homoserine lactones when we deleted two major virulence factor-encoding genes, the act gene and the gene encoding an outer membrane protein (aopB), an important component of the T3SS (65), from A. hydrophila SSU. Likewise, we observed that N-acyl homoserine lactone production was also modulated by regulatory genes, such as dam and gidA, in A. hydrophila SSU (18). Thus, differential expression of genes might also be an important factor in the pathogenesis of Aeromonas species.The presence of any virulence gene in strains of Aeromonas isolated from water should be carefully scrutinized, as such genes could be expressed better in a human host, which could lead to devastating outcomes. In addition, it is possible that in the environment certain Aeromonas clones may predominate and cause human diseases more frequently than other clones. Thus, it is important to determine the clonal variation of a range of Aeromonas species isolated from various sources and identify predominant clones by a polyphasic approach that includes biochemical phenotyping, virulence marker detection, and molecular fingerprinting techniques.In the present study, we compared 199 Aeromonas isolates, 146 of which were from water sources and 53 of which were from human patients with diarrhea in the Unites States. In addition, 28 reference and classical strains that were obtained from various culture collections and/or were isolated from specimens obtained in diverse geographical areas of the world, including water and clinical specimens, were also characterized. All isolates were biochemically identified to the phenospecies group level, examined for the presence of a set of 11 virulence factors by using DNA colony blot hybridization, and fingerprinted by using pulsed-field gel electrophoresis (PFGE). Some of the virulence factors selected, including T6SS effectors, were also examined by using functional assays. Our data provide the first suggestive evidence of water-to-human transmission, i.e., of successful colonization and infection by particular strains of certain Aeromonas species.     

Molecular Identification and Antifungal Susceptibilities of Black Aspergillus Isolates from Otomycosis Cases in Hungary

Otomycosis, also known as fungal otitis externa, has been used to describe a fungal infection of the external auditory canal, but sometimes involving the middle ear. Many fungal species have been identified as infectious agents in otomycosis, with Aspergillus and Candida species being the most common. Among aspergilli, Aspergillus niger is the most commonly described species in the literature. In this study, 14 black Aspergillus strains were analyzed, which were isolated from otomycosis cases in Hungary between 2010 and 2011. These strains were identified as A. niger according to conventional morphological methods. Species identification was based on sequencing of part of the calmodulin gene. Our results indicate that instead of A. niger, A. awamori and A. tubingensis are the predominant species that cause ear infections in Southern Hungary. Antifungal susceptibility tests were carried out against four antifungal drugs: amphotericin B, itraconazole, ketoconazole and terbinafine. All isolates were found to exhibit low in vitro MIC values to amphotericin B, terbinafine and itraconazole. However, the examined isolates exhibited high in vitro MIC values to ketoconazole.     

Detection of Achromobacter xylosoxidans in Hospital,Domestic, and Outdoor Environmental Samples and Comparison with Human Clinical Isolates

Achromobacter xylosoxidans is an aerobic nonfermentative Gram-negative rod considered an important emerging pathogen among cystic fibrosis (CF) patients worldwide and among immunocompromised patients. This increased prevalence remains unexplained, and to date no environmental reservoir has been identified. The aim of this study was to identify potential reservoirs of A. xylosoxidans in hospital, domestic, and outdoor environments and to compare the isolates with clinical ones. From 2011 to 2012, 339 samples were collected in Dijon''s university hospital, in healthy volunteers'' homes in the Dijon area, and in the outdoor environment in Burgundy (soil, water, mud, and plants). We designed a protocol to detect A. xylosoxidans in environmental samples based on a selective medium: MCXVAA (MacConkey agar supplemented with xylose, vancomycin, aztreonam, and amphotericin B). Susceptibility testing, genotypic analysis by pulsed-field gel electrophoresis, and bla_OXA-114 sequencing were performed on the isolates. A total of 50 strains of A. xylosoxidans were detected in hospital (33 isolates), domestic (9 isolates), and outdoor (8 isolates) samples, mainly in hand washing sinks, showers, and water. Most of them were resistant to ciprofloxacin (49 strains). Genotypic analysis and bla_OXA-114 sequencing revealed a wide diversity among the isolates, with 35 pulsotypes and 18 variants of oxacillinases. Interestingly, 10 isolates from hospital environment were clonally related to clinical isolates previously recovered from hospitalized patients, and one domestic isolate was identical to one recovered from a CF patient. These results indicate that A. xylosoxidans is commonly distributed in various environments and therefore that CF patients or immunocompromised patients are surrounded by these reservoirs.     

Molecular Structure and Transferability of Tn1546-Like Elements in Enterococcus faecium Isolates from Clinical, Sewage, and Surface Water Samples in Iran

The molecular structure and transferability of Tn1546 in 143 vancomycin-resistant Enterococcus faecium (VREF) isolates obtained from patients (n = 49), surface water (n = 28), and urban and hospital sewage (n = 66) in Tehran, Iran, were investigated. Molecular characterization of Tn1546 elements in vanA VREF was performed using a combination of restriction fragment length polymorphism analysis and DNA sequencing of the internal PCR fragments of vanA transposons. Long-PCR amplification showed that the molecular size of Tn1546 elements varied from 10.8 to 12.8 kb. The molecular analysis of Tn1546 showed that 45 isolates (31.5%) harbored a deletion/mutation upstream from nucleotide 170. No horizontal transfer of Tn1546 was observed following filter-mating conjugation with these isolates. Nevertheless, the rates of transferability for other isolates were 10⁻⁵ to 10⁻⁶ per donor. Insertion sequences IS1216V and IS1542 were present in 103 (72%) and 138 (96.5%) of the isolates, respectively. The molecular analysis of Tn1546 elements resulted in three genomic organizations. The genomic organization lineage 1 was dominated by the isolates from clinical samples (3.4%), lineage 2 was dominated mostly by sewage isolates (24.5%), and lineage 3 contained isolates obtained from all sources (72.1%). The genetic diversity determined using pulsed-field gel electrophoresis (PFGE) revealed a single E. faecium clone, designated 44, which was common to the samples obtained from clinical specimens and hospital and municipal sewage. Furthermore, the results suggest that lineage 3 Tn1546 was highly disseminated among our enterococcal isolates in different PFGE patterns.     

Molecular Identification and Antifungal Susceptibility of Clinical Isolates of Sporothrix schenckii Complex in Medellin,Colombia

Background

Sporotrichosis is a subcutaneous mycosis that affects humans and other animals. Infection prevails in tropical and subtropical countries. Until a few years ago, it was considered that two varieties of Sporothrix schenckii caused this mycosis, but by applying molecular taxonomic markers, it has been demonstrated that there are several cryptic species within S. schenckii complex which varies in susceptibility, virulence, and geographic distribution.

Objective

This study aimed to identify the clinical isolates of Sporothrix spp. from patients with sporotrichosis in Medellin, Colombia, using two markers and to evaluate the in vitro susceptibility to itraconazole.

Methods

Thirty-four clinical isolates of Sporothrix spp. from Colombia, three from Mexico, and one from Guatemala were identified through sequencing of the noncoding region ITS-1?+?5.8SDNAr?+?ITS-2 and of the fragment containing exons 3 and 4 of the β-tubulin gene. Clinical isolate sequences were compared with GenBank reference sequences using the BLASTN tool, and then, phylogenetic analysis was performed. Besides, the in vitro susceptibility to itraconazole was evaluated by determining the minimum inhibitory concentrations according to the CLSI M38-A2 method.

Results

Clinical isolates were identified by morphology as Sporothrix spp. Using the molecular markers, ITS and β-tubulin, isolates were identified as S. schenckii sensu stricto (25) and Sporothrix globosa (13). Susceptibility to itraconazole was variable among clinical isolates.

Conclusion

This is the first scientific publication that identifies species that cause sporotrichosis in Colombia, along with the antifungal susceptibility to itraconazole.

     

Human Salmonella Clinical Isolates Distinct from Those of Animal Origin

The global trend toward intensive livestock production has led to significant public health risks and industry-associated losses due to an increased incidence of disease and contamination of livestock-derived food products. A potential factor contributing to these health concerns is the prospect that selective pressure within a particular host may give rise to bacterial strain variants that exhibit enhanced fitness in the present host relative to that in the parental host from which the strain was derived. Here, we assessed 184 Salmonella enterica human and animal clinical isolates for their virulence capacities in mice and for the presence of the Salmonella virulence plasmid encoding the SpvB actin cytotoxin required for systemic survival and Pef fimbriae, implicated in adherence to the murine intestinal epithelium. All (21 of 21) serovar Typhimurium clinical isolates derived from animals were virulent in mice, whereas many (16 of 41) serovar Typhimurium isolates derived from human salmonellosis patients lacked this capacity. Additionally, many (10 of 29) serovar Typhimurium isolates derived from gastroenteritis patients did not possess the Salmonella virulence plasmid, in contrast to all animal and human bacteremia isolates tested. Lastly, among serovar Typhimurium isolates that harbored the Salmonella virulence plasmid, 6 of 31 derived from human salmonellosis patients were avirulent in mice, which is in contrast to the virulent phenotype exhibited by all the animal isolates examined. These studies suggest that Salmonella isolates derived from human salmonellosis patients are distinct from those of animal origin. The characterization of these bacterial strain variants may provide insight into their relative pathogenicities as well as into the development of treatment and prophylactic strategies for salmonellosis.     

草原珍稀食用菌蒙古口蘑菌种的分子鉴定

目的:蒙古口蘑是一种著名的野生食用菌.为了辨别蒙古口蘑与其相似的草原蘑菇种及分离菌株的真伪.方法:采用ITS序列分析方法对从内蒙古地区采集的子实体和纯培养菌株进行分子鉴定.结果:在20个供试材料中,从呼伦贝尔草原采集的4个子实体(来源于不同地区)中有2个属于蒙古口蘑,从子实体分离得到的纯培养物中有2个为蒙古口蘑菌株;在呼伦贝尔地区购买的蘑菇干品和冷冻品都属于蒙古口蘑;从锡林郭勒草原的不同地区采集分离得到的10株纯培养物中有6株为蒙古口蘑菌株.结论:在呼伦贝尔地区和锡盟地区都有蒙古口蘑存在,而且从不同地区采集的蒙古口蘑亲缘关系比较近.     

Molecular Identification of Staphylococcus aureus in Airway Samples from Children with Cystic Fibrosis

Background

Staphylococcus aureus is a common and significant pathogen in cystic fibrosis. We sought to determine if quantitative PCR (qPCR) and 16S rRNA gene sequencing could provide a rapid, culture-independent approach to the identification of S. aureus airway infections.

Methods

We examined the sensitivity and specificity of two qPCR assays, targeting the femA and 16S rRNA gene, using culture as the gold standard. In addition, 16S rRNA gene sequencing to identify S. aureus directly from airway samples was evaluated. DNA extraction was performed with and without prior enzymatic digestion.

Results

87 samples [42 oropharyngeal (OP) and 45 expectorated sputum (ES)] were analyzed. 59 samples (68%) cultured positive for S. aureus. Using standard extraction techniques, sequencing had the highest sensitivity for S. aureus detection (85%), followed by FemA qPCR (52%) and 16SrRNA qPCR (34%). For all assays, sensitivity was higher from ES samples compared to OP swabs. Specificity of the qPCR assays was 100%, but 21.4% for sequencing due to detection of S. aureus in low relative abundance from culture negative samples. Enzymatic digestion increased the sensitivity of qPCR assays, particularly for OP swabs.

Conclusion

Sequencing had a high sensitivity for S. aureus, but low specificity. While femA qPCR had higher sensitivity than 16S qPCR for detection of S. aureus, neither assay was as sensitive as sequencing. The significance of S. aureus detection with low relative abundance by sequencing in culture-negative specimens is not clear.     

Molecular Characterization and Identification of Biocontrol Isolates of Trichoderma spp.

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as “Trichoderma harzianum” Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.     

诺卡氏菌酰胺酶基因的分子克隆及在大肠杆菌中表达的初步研究

酰胺酶是一种重要的工业酶。利用生物信息学手段,在和已知酰胺酶基因序列分析比对的基础上,首次从Ncordiasp.YS-2002中成功地克隆得到酰胺酶基因ami,并对其基因序列及氨基酸序列的性质进行了分析。结果表明,所得酰胺酶基因ami片段大小共为1446bp,由启动子区、阅读框和回文结构终止区三部分构成。序列分析和进化树分析表明,Ncordiasp.YS-2002酰胺酶是一种比较特殊的酰胺酶,不含大多数酰胺酶共同具有的保守区序列。进一步将酰胺酶基因连接到pET-28a( )上,转入大肠杆菌BL21(DE3)中筛选获得重组菌株PEAB。酶活测定结果表明重组菌具有酰胺酶酶活,但较低,其原因可能是因为大量表达的产物主要以包涵体的形式存在。     

Host-range Characterization of Two Pratylenchus coffeae Isolates from Brazil

Two isolates of Pratylenchus coffeae were collected from coffee roots (in Marília, São Paulo State, Brazil) and Aglaonema (in Rio de Janeiro City, Rio de Janeiro State, Brazil) and maintained in the laboratory on alfalfa callus. Twenty-four plants were tested in the greenhouse to characterize the host preference of these isolates. The host ranges of the isolates differed from each other and, interestingly, coffee, banana, and citrus were not among the better hosts of either isolate. Rather, sorghum, maize, rice, millet, okra, melon, eggplant, and lettuce were the best hosts of the Marília isolate. Poor hosts included French marigold, Rangpur lime, banana, sesame, peanut, sunflower, cotton, French bean, onion, and small onion. The best hosts of the Rio de Janeiro isolate were sesame, soybean, sorghum, castor oil plant, watermelon, squash, eggplant, and melon; the poorest hosts were French marigold, coffee, Rangpur lime, banana, sunflower, peanut, maize, millet, French bean, cotton, onion, sweet pepper, lettuce, okra, and small onion. These isolates have important molecular and morphological differences, suggesting host preference is linked to these characteristics.     

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.     

Molecular Identification and In Vitro Antifungal Susceptibility of Aspergillus Isolates Recovered from Otomycosis Patients in Western China

Mycopathologia - Aspergillus species are the most common causative agents involved in otomycosis. In this study, 45 Aspergillus isolates were obtained from patients with otomycosis in western China...