Incidence of antibiotic resistance and sex pheromone response among enterococci isolated from clinical human samples and from municipal waste water

The incidence of resistance to various antibiotics as well as the capacity to elicit aggregation response to sex pheromones have been investigated in strains of Enterococcus faecalis isolated from clinical and municipal waste waters (MWW). While clinical isolates showed a high incidence of antibiotic resistance (87%) and sex pheromone response (33%), these traits appeared with a much lower frequency in MWW isolates (12% and 4% respectively). The simultaneous incidence of both traits was of 52% and 0% for clinical and MWW isolates, respectively. The capacity to elicit a positive pheromone response as well as antibiotic resistance traits seemed to be strongly correlated with the presence of gelatinase activity among clinical isolates. Among MWW isolates, only sex pheromone response seemed to correlate with the presence of gelatinase activity.     

Male orientation to trail sex pheromones in parasitoid wasps: does the spatial distribution of virgin females matter?

We studied male locomotory response to trails and patches of sex pheromone (left respectively by free-ranging females and females constrained to stay on a small area) in the two parasitoids Aphelinus asychis (Hymenoptera: Aphelinidae) and Trichogramma brassicae (Hymenoptera: Trichogrammatidae). Under the hypothesis that the spatial distribution of virgin females differs between these species (scattered among host plants in A. asychis, gregarious at emergence sites in T. brassicae), we predicted that male locomotory response to their sex pheromones should also differ: A. asychis males should follow pheromone trails on plants in order to encounter the females along these trails, whereas T. brassicae males should stay on pheromone patches, at emergence sites, and mate the females on these patches. Using an improved video-tracking system, we found that males of both species respond to conspecific sex pheromone trails and patches, but that the response does not differ much between species. Males released on marked substrates walked in a more convoluted pattern (i.e. higher path fractal dimension and higher number of crossings within tracks) than males released on unmarked substrates. On pheromone patches, males turned persistently in the same direction when leaving the patch, which explains a higher number of visits on marked patches than on unmarked patches, and possibly, higher track convolution on pheromone trails. Contrary to our hypothesis, male A. asychis did not follow female trails more accurately than male T. brassicae, and male T. brassicae did not stay longer on pheromone patches than male A. asychis. We argue that these discrepancies between our predictions and the observed responses originates from discrepancies between the assumed spatial distribution of virgin females and their actual distribution in the wild.     

Do Pheromone Binding Proteins Converge in Amino Acid Sequence When Pheromones Converge?

Convergence in amino acid sequences between proteins can be strong evidence for selection. Here, I look for evidence of convergence in the amino acid sequences of pheromone binding protein (PBP) in response to convergence in pheromones. PBPs are involved in sex pheromone reception by the antennae of male moths. In this role PBPs may selectively bind pheromone components and experience convergent selection in response to convergence in pheromone components. However, examination of the PBPs of the taxa that have converged upon the use of (E)- or (Z)-11-tetradecenyl acetate as their major pheromone component reveals little evidence for convergence in the PBPs identified from these taxa. A few sites show a pattern consistent with convergence or parallelism; however, it cannot be ruled out that these sites share the ancestral state. Two of these sites fall within the proposed binding region of PBPs. These results suggest that PBPs either have not converged in sequence or have converged at very few sites in response to convergence on the same pheromone component. Received: 29 July 1999 / Accepted: 8 November 1999     

EAG measurement of pheromone distribution in apple orchards treated for mating disruption of Cydia pomonella

Pheromone concentrations were measured in an apple orchard using an advanced EAG device featuring short-term recalibration of the antenna with two calibration standards, a lightweight remotely operated probe and automated measurement cycles. The effects of wind direction on pheromone density and distribution at the edge of the orchard were studied as well as the change of pheromone density with height from 1 to 6.5 m. Results show that pheromone can be found at heights of up to 6 m when moderate winds are present. Very low wind speeds tend to reduce the height of the pheromone cloud. Clean wind entering the orchard creates a transition zone up to 15 m wide where the pheromone is depleted. At the down-wind edge of the orchard, pheromone signals were recorded at distances of up to 60 m downwind from the treated zone at concentrations in the same order of magnitude as within the treated orchards. The consequences of these results concerning the design and interpretation of mating disruption field tests are discussed.     

Genomic and experimental evidence for a potential sexual cycle in the pathogenic thermal dimorphic fungus Penicillium marneffei

All meiotic genes (except HOP1) and genes encoding putative pheromone processing enzymes, pheromone receptors and pheromone response pathways proteins in Aspergillus fumigatus and Aspergillus nidulans and a putative MAT-1 alpha box mating-type gene were present in the Penicillium marneffei genome. A putative MAT-2 high-mobility group mating-type gene was amplified from a MAT-1 alpha box mating-type gene-negative P. marneffei strain. Among 37 P. marneffei patient strains, MAT-1 alpha box and MAT-2 high-mobility group mating-type genes were present in 23 and 14 isolates, respectively. We speculate that P. marneffei can potentially be a heterothallic fungus that does not switch mating type.     

Characterization of dominant cultivable lactobacilli and their antibiotic resistance profiles from faecal samples of weaning piglets

AIMS: To examine the lactic acid bacteria flora of weaning piglets, to define the distribution of different lactobacilli species in piglet faecal samples, and to determine the susceptibility phenotype to 11 antibiotic of different families. METHODS AND RESULTS: The faecal samples were taken from piglets with good herd status at 11 and 28 days after weaning. The Lactobacillus isolates (n = 129) from 78 animals housed in pairs in 39 pens were preliminarily identified by their morphology and biochemical characteristics. Partial 16S ribosomal DNA (16S rDNA) was used to identify the isolates to the species level, and RAPD (randomly amplified polymorphism DNA) profiles to differentiate Lactobacillus isolates to the strain level. Based on these studies, 67 strains were selected for antibiotic resistant tests. The most numerous Lactobacillus species found in the piglets was Lactobacillus reuteri (n = 43). Other lactobacilli were L. salivarius (n = 15), L. agilis (n = 4), L. johnsonii (n = 2), L. vaginalis (n = 1), L. mucosae (n = 1) and L. gallinarum (n = 1). All the strains were susceptible to chloramphenicol, ampicillin and gentamicin. Two L. salivarius isolates and two L. reuteri isolates were found to be multiresistant. CONCLUSIONS: This study indicates that the faecal Lactobacillus flora in piglets consists mainly of L. reuteri, L. salivarius and L. acidophilus group lactobacilli, and the distribution of lactobacilli is similar between individuals of the same age and with the same diet. Most of the Lactobacillus isolates tested were sensitive to the antibiotics used in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Valuable information on Lactobacillus species distribution and their antibiotic resistance profiles in piglets is obtained.     

EVOLUTION AND MITOCHONDRIAL DNA IN NEUROSPORA CRASSA

We studied mitochondrial DNA variability in 19 natural Neurospora crassa isolates and one wild-type isolate to examine evolution of these fungi and their mitochondrial DNA (mtDNA). We combined restriction endonuclease analysis of natural isolate mtDNA with DNA-DNA hybridization to cloned EcoR I fragments of a wild-type genome to discriminate between length mutations and site changes due to nucleotide substitution. Most variability was due to length mutations (insertions and deletions); genome size could vary 25% between pairs of isolates. Length-mutation distribution was not random, nor simply explained by the presence of coding versus noncoding regions. Restriction-site changes were few; the estimated amount of nucleotide substitution per nucleotide between the most divergent pair of isolates was 0.78%. Evolutionary relationships among isolates based on both types of mutations were compatible, and suggest that geographically distinct populations of mitochondrial DNA exist in the biological species, N. crassa. In contrast, no such correlation was shown by the previously determined distribution of nuclear heterokaryon incompatibility genes in the same isolates (Mylyk, 1975, 1976).     

Evidence that the hemolysin/bacteriocin phenotype of Enterococcus faecalis subsp. zymogenes can be determined by plasmids in different incompatibility groups as well as by the chromosome.

The hemolysin (Hly/Bac) determinant in strains of Enterococcus faecalis was found to be present on plasmids in different incompatibility groups (conferring different sex pheromone responses) as well as on the chromosome. Of 33 Hly/Bac plasmids identified in clinical isolates, the related pheromone for 30 was cAD1; the related pheromone for another two (pYI1 and pYI3) or one (pYI2) was cOB1 or cY12, respectively. The representative Hly/Bac plasmids pAD1, pYI1, pOB1, and pYI2, which responded to pheromones cAD1, cOB1, cOB1, and cYI2, respectively, were compatible with one another. As additions to the incompatibility group IncHly of pAD1, groups for pOB1, pYI1, and pYI2 were designated IncHlyII, IncHlyIII, and IncHlyIV, respectively. Eleven of the 30 plasmids conferring a response to cAD1 were very similar to pAD1 on the basis of their restriction endonuclease profiles. EcoRI fragment D, F, or H containing parts of the Hly/Bac gene(s) of pAD1 hybridized to similar EcoRI fragments from each of the other three representatives of incompatibility groups (i.e., pOB1, pYI1, and pYI2) and to homologous DNA representing the chromosome of the plasmid-free Hly/Bac strain YI6-1.     

Clonality and genetic variation in Amylostereum areolatum and A. chailletii from northern Europe

Genetic variation within and between vegetative compatibility groups (VCGs) of Amylostereum areolatum (Fr.) Boid. and Amylostereum chailletii (Pers.: Fr.) Boid. isolates was investigated. DNA fingerprints were made using the M13 core sequence as a primer. A total of 53 isolates of A. areolatum and 57 isolates of A. chailletii from Lithuania, Sweden, Denmark and Great Britain were studied. In all cases isolates belonging to the same VCG showed identical DNA banding patterns, suggesting that VCGs correspond to clones. In A. areolatum the vast majority of the isolates (spread by Sirex juvencus L.) were assigned to dispersive clones, that have wide geographical distribution (i.e. the same genotypes were detected in Lithuania, Sweden and Denmark), with low genetic variation between the different clones. By contrast, A. chailletii population structure was consistent with the spread of airborne basidiospores produced by outcrossing. Only a small fraction of A. chailletii isolates studied, could be assigned to dispersal clones with a local distribution, spread by Urocerus gigas L. Overall, M13 fingerprinting detected low genetic differentiation in both species in the samples we studied.     

The structure and interrelationship of fungal populations in native and cultivated soils

Mitochondrial DNA haplotypes have been characterized for 120 isolates of the asexual fungus Fusarium oxysporum. Sixty of these isolates were obtained from soil in a native grassland in the San Joaquin Valley of California, including 20 isolates from each of six different sampling locations. The same sampling strategy was used to obtain 60 additional isolates from an agricultural field of the same soil type directly adjacent to the native soil. Twenty-three different mitochondrial DNA haplotypes were identified among the 120 isolates, including 11 haplotypes represented by two or more isolates and 12 that were unique. The five most common mitochondrial DNA haplotypes accounted for 93 (78%) of the 120 isolates. Isolates representing each of these five mitochondrial DNA haplotypes were found both in the cultivated and in the native soil. Seventy-two per cent of the isolates found in the cultivated soil were associated with the same mitochondrial DNA haplotype as one or more isolates in the native soil. The remaining isolates in the cultivated soil were associated with comparatively rare mitochondrial DNA haplotypes, most of which showed a close relationship to one of the haplotypes found in the native soil. Hierarchial gene diversity analysis indicated that a significant proportion of the mitochondrial DNA haplotype diversity was attributable to differences between sampling sites in the native soil but not in the cultivated soil. This may reflect significant spatial structuring of genetic diversity in populations of F. oxysporum in a native soil. The proportion of mtDNA haplotype diversity attributable to differences between populations in the native and cultivated soils was not significant. This suggests that our entire collection, encompassing strains from both native and cultivated soils, is representative of a single population of F. oxysporum.     

香菇自然群体中个体间的空间分布及其遗传联系*

应用体细胞不亲和性反应、交配型因子分析和基因组ＤＮＡ的ＲＡＰＤ分析,研究了一个分布于方圆约１ｋｍ的６根倒木上的１８个香菇野生菌株间的遗传差异。结果表明,该群体大多数菌株间配对（８０．４％）体细胞不亲和,而同一倒木菌株间配对的体细胞亲和率达 ６２．５％。不同倒木菌株间未发现体细胞亲和的配对。该群体存在 １１个特异的Ａ因子和７个特异的Ｂ因子。同一倒木的菌株有的交配型因子相同,有的则不同。不同倒木的菌株大多数交配型因子不同,未发现交配型因子完全相同的菌株。ＲＡＰＤ分析显示,体细胞亲和的菌株,交配型因子完全相同的菌株,在基于ＤＮＡ相似系数的遗传相关聚类中,首先聚为小类。总起来看,在自然群体中,香菇个体间的遗传差异与其空间分布之间存在一定的联系,随着空间距离的增大,菌株间的异质性相应增高。     

蛾类昆虫性信息素受体及其作用机理

蛾类昆虫性信息素受体首先从烟芽夜蛾Heliothis virescens和家蚕Bombyx mori中鉴定出来, 到目前为止已经克隆得到了19种蛾类昆虫的几十种性信息素受体基因, 并且这些基因在系统发育树中聚成一个亚群。性信息素受体从蛾类蛹期开始表达, 主要表达在雄性触角的毛形感器中, 少部分受体在雌性触角、 雄性触角其他感器以及身体其他部位中也有表达。大部分蛾类性信息素受体的配体并不是单一的, 而是能够对多种性信息素组分有反应, 部分性信息素受体还能够识别性信息素以外的其他物质, 还有一部分性信息素受体的识别配体目前尚不清楚。另外发现在雌性蛾类触角中也存在一些嗅觉受体能够识别雄性分泌的性信息素。在蛾类性信息素受体与性信息素识别的过程中, 性信息素结合蛋白不仅能够特异性地运送配体到嗅觉神经元树状突上, 还能够提高性信息素与性信息素受体之间的结合效率。另外, OrCo类受体与性信息素受体共表达在嗅觉神经元中, 在蛾类性信息素受体与配体的识别过程中扮演了重要角色。但是蛾类信息素对神经元刺激的终止并非由性信息素受体控制, 而是由细胞中的气味降解酶等其他因子调控。蛾类性信息素受体研究中还有很多疑问需要解答, 其过程可能比我们想象的更为复杂。     

Patterns of response to sex pheromone by young and mature adult male cockroaches, Periplaneta americana

Male American cockroaches were isolated from females upon becoming adults and were exposed one single time to sex pheromone on days 1 to 11, 13, or 15 after adult ecdysis. The behavioural components of adult male sexual behaviour are rapid antennation, erect body posture, locomotion, running, wing raising, and abdominal extension. These components appear after the adult ecdysis in the same sequence as they appear in later adult life in response to different concentrations of sex pheromone. The appearance of these behavioural components is described using two theoretical concepts, threshold and a “sequencer”. It is proposed that the components are ordered sequentially before or during ecdysis by the same “sequencer” that organizes the responses to different concentrations of pheromone. The appearance of the components during development is due to decreasing thresholds as the adult male matures.     

Electrophysiological correlates of attraction in Trichoplusia ni

A quantitative comparison was made between pheromone-induced electroantennogram (EAG) potentials and the attraction of male cabbage loopers, Trichoplusia ni. For this comparison, duplicate pheromone dispensers were used in both assays. The slope of the function of evoked EAG potentials was the same as the slope of the function of percentage attraction to different amounts of pheromone, but the EAG was calculated to be about 3 × 10⁴ times less sensitive than the attraction response. Thus, the EAG of T. ni was not a reliable indicator of relative attraction to various batches of synthetic pheromone, and no difference in the evoked EAG was observed between an inhibitor of the behavioural response to the pheromone and the pheromone itself.     

Genetic mapping of sexual isolation between E and Z pheromone strains of the european corn Borer (Ostrinia nubilalis)

The E and Z pheromone strains of the European corn borer (ECB) provide an exceptional model system for examining the genetic basis of sexual isolation. Differences at two major genes account for variation in female pheromone production and male behavioral response, components of the pheromone communication system known to be important for mate recognition and mate choice. Strains of ECB are morphologically indistinguishable, and surveys of allozyme and DNA sequence variation have revealed significant allele frequency differences at only a single sex-linked locus, Tpi. Here we present a detailed genetic linkage map of ECB using AFLP and microsatellite markers and map the factors responsible for pheromone production (Pher) and male response (Resp). Our map covers 1697 cM and identifies all 31 linkage groups in ECB. Both Resp and Tpi map to the Z (sex) chromosome, but the distance between these markers (>20 cM) argues against the hypothesis that patterns of variation at Tpi are explained by tight linkage to this "speciation gene." However, we show, through analysis of marker density, that Tpi is located in a region of low recombination and suggest that a second Z-linked reproductive barrier could be responsible for the origin and/or persistence of differentiation at Tpi.     

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

Summary During conjugative transfer of sex pheromone plasmids ofEnterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3 .8 kb fragment of the sex pheromone plasmid pAD1 containing the structural genesea1 for surface exclusion protein and a small open reading frame (ORF) upstream ofsea1. Surface exclusion protein Seal was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene seal demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5 to that for aggregation substance.