Rough Colony Research Articles

Connections between Exoproteome Heterogeneity and Virulence in the Oral Pathogen Aggregatibacter actinomycetemcomitans.

ABSTRACTAggregatibacter actinomycetemcomitans is a Gram-negative bacterial pathogen associated with severe periodontitis and nonoral diseases. Clinical isolates of A. actinomycetemcomitans display a rough (R) colony phenotype with strong adherent properties. Upon prolonged culturing, nonadherent strains with a smooth (S) colony phenotype emerge. To date, most virulence studies on A. actinomycetemcomitans have been performed with S strains of A. actinomycetemcomitans, whereas the virulence of clinical R isolates has received relatively little attention. Since the extracellular proteome is the main bacterial reservoir of virulence factors, the present study was aimed at a comparative analysis of this subproteome fraction for a collection of R isolates and derivative S strains, in order to link particular proteins to the virulence of A. actinomycetemcomitans with serotype b. To assess the bacterial virulence, we applied different infection models based on larvae of the greater wax moth Galleria mellonella, a human salivary gland-derived epithelial cell line, and freshly isolated neutrophils from healthy human volunteers. A total number of 351 extracellular A. actinomycetemcomitans proteins was identified by mass spectrometry, with the S strains consistently showing more extracellular proteins than their parental R isolates. A total of 50 known extracellular virulence factors was identified, of which 15 were expressed by all investigated bacteria. Importantly, the comparison of differences in exoproteome composition and virulence highlights critical roles of 10 extracellular proteins in the different infection models. Together, our findings provide novel clues for understanding the virulence of A. actinomycetemcomitans and for development of potential preventive or therapeutic avenues to neutralize this important oral pathogen.IMPORTANCE Periodontitis is one of the most common inflammatory diseases worldwide, causing high morbidity and decreasing the quality of life of millions of people. The bacterial pathogen Aggregatibacter actinomycetemcomitans is strongly associated with aggressive forms of periodontitis. Moreover, it has been implicated in serious nonoral infections, including endocarditis and brain abscesses. Therefore, it is important to investigate how A. actinomycetemcomitans can cause disease. In the present study, we applied a mass spectrometry approach to make an inventory of the virulence factors secreted by different clinical A. actinomycetemcomitans isolates and derivative strains that emerged upon culturing. We subsequently correlated the secreted virulence factors to the pathogenicity of the investigated bacteria in different infection models. The results show that a limited number of extracellular virulence factors of A. actinomycetemcomitans have central roles in pathogenesis, indicating that they could be druggable targets to prevent or treat oral disease.

Identification of novel biofilm genes in avian pathogenic Escherichia coli by Tn5 transposon mutant library.

Avian pathogenic Escherichia coli (APEC) is the main pathogens that inflict the poultry industry. Biofilm as the pathogenic factors of APEC, which can enhance the anti-host immune system of APEC and improve its survival in the environment. In order to screen for new genes related to APEC biofilm. The APEC strain APEC81 was used to construct a mutant library by Tn5 insertion mutagenesis. Moreover the 28 mutant strains with severely weakened biofilm were successfully screened from 1500 mutant strains by crystal violet staining, in which 17 genes were obtained by high-efficiency thermal asymmetric interlaced PCR. The reported genes include 3 flagella genes (fliS, fliD, andfliR), 4 curli fimbriae genes (csgD, csgA, csgF, andcsgG) and 3 type 1 fimbriae genes (fimA, fimD, andfimC). The novel genes include 3 coenzyme genes (gltA, bglX, and mltF) and 4 putative protein genes (yehE, 07045, 11735, 11255). To investigate whether these 17 genes co-regulate the biofilm, the 17 identified genes were deleted from APEC strain APEC81. The results showed that except for the 11735 and 11255 genes, the deletion of 15 genes significantly reduced the biofilm formation ability of APEC81 (P < 0.05). The result of rdar (red, dry and rough) colony morphology showed that curli fimbriae genes (csgD, csgA, csgF, andcsgG) and other functional genes (fimC, glxK, yehE, 07045, and11255) affected the colony morphology. In particular, the hypothetical protein YehE had the greatest influence on the biofilm. It was predicted to have the same structure as the type 1 fimbria protein. When yehE was deleted, the fimE transcription was up-regulated, and the fimA and fimB transcription were down-regulated, resulting in a decrease in type 1 fimbriae. Hence, the yehE mutant significantly reduced the biofilm and the adhesion and invasion ability to cells (P < 0.05). This study identified 5 novel genes (gltA, bglX, mltF, yehE, and07045) related to biofilm formation and confirmed that yehE affects biofilm formation by type 1 fimbriae, which will benefit further study of the mechanism of biofilm regulation in APEC.

Mycobacterium senriense sp. nov., a slowly growing, non-scotochromogenic species, isolated from sputum of an elderly man.

A slowly growing mycobacteria, identified as strain TY59T, was isolated from sputum of an elderly man with pneumonia. Sequencing of the 16S rRNA gene indicated that this strain was similar to members of the Mycobacterium avium complex and closely related species. Strain TY59T has highest 16S rRNA gene sequence similarities to the type strains of Mycobacterium colombiense (99.80 % sequence similarity), Mycobacterium vulneris (99.74 %), Mycobacterium timonense (99.54 %), Mycobacterium avium subsp. avium (99.54 %) and Mycobacterium avium subsp. silvaticum (99.54 %). Analysis of the internal transcribed spacer (ITS) and DNA-directed RNA polymerase subunit beta (rpoB) sequences gave similar results to the 16S rRNA gene analysis. The closest species to strain TY59T were M. colombiense and M. vulneris with 97.90-98.25 % identity in ITS and 96.4-96.6 % in rpoB. The strain's 65 kDa heat shock protein (hsp65) gene was different from those of M. vulneris, M. colombiense and M. avium subsp. silvaticum with 72.4-74.2 % identity. Average nucleotide identity results showed a 93.4 % match to M. vulneris as the maximum value. Phenotypically, the non-chromogenicity, rough colonies, growth at 42 °C, negative results for nitrate reduction, β-glucosidase and Tween 80 hydrolysis, and positive results for catalase activity set this strain apart from closely related species. We propose that Mycobacterium senriense sp. nov. is a novel species of slowly growing mycobacteria. The type strain is TY59T (RIMD 1371001T=CIP 111917T).

Rough and smooth variants of Mycobacterium abscessus are differentially controlled by host immunity during chronic infection of adult zebrafish

Prevalence of Mycobacterium abscessus infections is increasing in patients with respiratory comorbidities. After initial colonisation, M. abscessus smooth colony (S) variants can undergo an irreversible genetic switch into highly inflammatory, rough colony (R) variants, often associated with a decline in pulmonary function. Here, we use an adult zebrafish model of chronic infection with R and S variants to study M. abscessus pathogenesis in the context of fully functioning host immunity. We show that infection with an R variant causes an inflammatory immune response that drives necrotic granuloma formation through host TNF signalling, mediated by the tnfa, tnfr1 and tnfr2 gene products. T cell-dependent immunity is stronger against the R variant early in infection, and regulatory T cells associate with R variant granulomas and limit bacterial growth. In comparison, an S variant proliferates to high burdens but appears to be controlled by TNF-dependent innate immunity early during infection, resulting in delayed granuloma formation. Thus, our work demonstrates the applicability of adult zebrafish to model persistent M. abscessus infection, and illustrates differences in the immunopathogenesis induced by R and S variants during granulomatous infection.

First Report of Pantoea dispersa Causing Brown Blotch Disease in Flammulina filiformis in China.

Flammulina filiformis (previously known as F. velutipes) is one of the most frequently cultivated and consumed edible mushrooms in China. In October 2020, brown blotch disease was observed on the pileus of F. filiformis at a mushroom factory in Ganzhou (25.74°N; 114.95°E), Jiangxi, China, with a disease incidence of approximately 6%. Symptoms initially appeared as small, irregular spots on the infected pileus, with color ranging from pale yellow to light brown. Such spots were enlarged and pitted at high relative humidity within several days, and finally caused malformation of the caps and yield reduction. To isolate the causal agent, the blotches on F. filiformis caps were homogenized and diluted with sterilized distilled water, and the resulting suspension (100 μl) was spread onto LB agar plates. After incubation at 28°C for 48 h, three colonial types were obtained: (i) yellow, convex, and smooth colonies, (ii) light yellowish, irregular, and rough colonies, and (iii) milky white, glistening, and smooth colonies. The first colonial type was predominant. A single colony of each type was randomly selected and streaked on fresh LB agar plates to obtain pure cultures namely PF1, PF2 and PF3 respectively. To test pathogenicity of the three isolates, young F. filiformis fruiting bodies (8- or 10-day-old primordium) grown in culture bottles were inoculated by spraying a bacterial suspension (108 CFU/ml, 3 ml per bottle on average), and cultivated in a mushroom house at 15±2°C and 95% relative humidity. The fruiting bodies sprayed with sterilized distilled water served as controls. The pathogenicity tests repeated three times, and at least five culture bottles were included in each experiment. Among the three types of bacteria, only strain PF1 induced symptoms similar to the original disease. The brown spots were observed on treated pileus 10 days after inoculation, and a fresh weight reduction of 30.9% per culture bottle was observed. In contrast, those fruiting bodies treated with water remained asymptomatic. Same yellow colonies were also re-isolated from the infected pileus, and identified by subsequent methods. The strain PF1 was gram negative, motile, and short rods. Biochemical analysis showed that the strains belonged to genus Pantoea (positive for citric acid, inositol, mannitol, methyl red test, and Voges-Proskauer test but negative for lysine, ornithine, phenylalanine, H2S, urease, D-melibiose, sorbitol, adonitol, and raffinose). Further PCR amplification and sequencing of four genes, 16S rRNA gene with primer 27F/1492R, fusA gene with primer fusA3/fusA4, gyrB gene with primer gyrBf1/gyrBr1, and rpoB genes with primer Vic3/Vic2 (Delétoile et al. 2009; Palemon et al. 2021), were performed to identify the species. A BLASTn showed 100%, 100%, 99.51%, and 99.26% homology, respectively, with those of P. dispersa (MT072166, CP045216, CP076369, MH015168). The four gene sequences were deposited in GenBank (accession numbers: MZ373179, MZ393661, MZ393662, MZ393663). A phylogenetic analysis based on the four concatenated genes also showed that the strain PF1 well clustered with the type strain of P. dispersa. This species has been reported to cause leaf blight in rice (Toh et al. 2019), soft rot in Agave angustifolia (Palemon et al. 2021), and bulb decay in onion (Chang et al. 2018). To the best of our knowledge, this is the first report of P. dispersa causing brown blotch diseases on cultivated F. filiformis, which was previously known to be caused by Pseudomonas tolaasii (Lee et al. 2002). Our results also indicate P. dispersa could induce malformation of pileus and lead to a severe yield loss if not controlled effectively. Therefore, it should be considered in future disease management of F. filiformis cultivation.

Bap-Independent Biofilm Formation in Staphylococcus xylosus.

The biofilm associated protein (Bap) is recognised as the essential component for biofilm formation in Staphylococcus aureus V329 and has been predicted as important for other species as well. Although Bap orthologs are also present in most S. xylosus strains, their contribution to biofilm formation has not yet been demonstrated. In this study, different experimental approaches were used to elucidate the effect of Bap on biofilm formation in S. xylosus and the motif structure of two biofilm-forming S. xylosus strains TMW 2.1023 and TMW 2.1523 was compared to Bap of S. aureus V329. We found that despite an identical structural arrangement into four regions, Bap from S. xylosus differs in key factors to Bap of S. aureus, i.e., isoelectric point of aggregation prone Region B, protein homology and type of repeats. Disruption of bap had no effect on aggregation behavior of selected S. xylosus strains and biofilm formation was unaffected (TMW 2.1023) or at best slightly reduced under neutral conditions (TMW 2.1523). Further, we could not observe any typical characteristics of a S. aureus Bap-positive phenotype such as functional impairment by calcium addition and rough colony morphology on congo red agar (CRA). A dominating role of Bap in cell aggregation and biofilm formation as reported mainly for S. aureus V329 was not observed. In contrast, this work demonstrates that functions of S. aureus Bap cannot easily be extrapolated to S. xylosus Bap, which appears as non-essential for biofilm formation in this species. We therefore suggest that biofilm formation in S. xylosus follows different and multifactorial mechanisms.

Elimination of PknL and MSMEG_4242 in Mycobacterium smegmatis alters the character of the outer cell envelope and selects for mutations in Lsr2

Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of pknL and adjacent regulator MSMEG_4242 in rough colony M. smegmatis mc2155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated lsr2 genes due to mutations and different IS1096 insertions. When complemented with lsr2, the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS1096 transposase TnpA and MSMEG_4727, which encodes a protein similar to PKS5. When MSMEG_4727 was deleted, smooth pknL/MSMEG_4242/lsr2 mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough delpknL/del4242 mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of lsr2 changes colony morphology from rough to smooth, augments sliding motility and increases expression of MSMEG_4727 and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and MSMEG_4242 regulate lipid components of the outer cell envelope and their absence selects for lsr2 inactivation. A regulatory, phosphorylation cascade model is proposed.

Secretome characterization of clinical isolates from the Mycobacterium abscessus complex provides insight into antigenic differences

BackgroundMycobacterium abscessus (MAB) is a widely disseminated pathogenic non-tuberculous mycobacterium (NTM). Like with the M. tuberculosis complex (MTBC), excreted / secreted (ES) proteins play an essential role for its virulence and survival inside the host. Here, we used a robust bioinformatics pipeline to predict the secretome of the M. abscessus ATCC 19977 reference strain and 15 clinical isolates belonging to all three MAB subspecies, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense.ResultsWe found that ~ 18% of the proteins encoded in the MAB genomes were predicted as secreted and that the three MAB subspecies shared > 85% of the predicted secretomes. MAB isolates with a rough (R) colony morphotype showed larger predicted secretomes than isolates with a smooth (S) morphotype. Additionally, proteins exclusive to the secretomes of MAB R variants had higher antigenic densities than those exclusive to S variants, independent of the subspecies. For all investigated isolates, ES proteins had a significantly higher antigenic density than non-ES proteins. We identified 337 MAB ES proteins with homologues in previously investigated M. tuberculosis secretomes. Among these, 222 have previous experimental support of secretion, and some proteins showed homology with protein drug targets reported in the DrugBank database. The predicted MAB secretomes showed a higher abundance of proteins related to quorum-sensing and Mce domains as compared to MTBC indicating the importance of these pathways for MAB pathogenicity and virulence. Comparison of the predicted secretome of M. abscessus ATCC 19977 with the list of essential genes revealed that 99 secreted proteins corresponded to essential proteins required for in vitro growth.ConclusionsThis study represents the first systematic prediction and in silico characterization of the MAB secretome. Our study demonstrates that bioinformatics strategies can help to broadly explore mycobacterial secretomes including those of clinical isolates and to tailor subsequent, complex and time-consuming experimental approaches accordingly. This approach can support systematic investigation exploring candidate proteins for new vaccines and diagnostic markers to distinguish between colonization and infection. All predicted secretomes were deposited in the Secret-AAR web-server (http://microbiomics.ibt.unam.mx/tools/aar/index.php).

Comparative genomics reveals an SNP potentially leading to phenotypic diversity of Salmonella enterica serovar Enteritidis.

An SNP is a spontaneous genetic change having a potential to modify the functions of the original genes and to lead to phenotypic diversity of bacteria in nature. In this study, a phylogenetic analysis of Salmonella enterica serovar Enteritidis, a major food-borne pathogen, showed that eight strains of S. Enteritidis isolated in South Korea, including FORC_075 and FORC_078, have almost identical genome sequences. Interestingly, however, the abilities of FORC_075 to form biofilms and red, dry and rough (RDAR) colonies were significantly impaired, resulting in phenotypic differences among the eight strains. Comparative genomic analyses revealed that one of the non-synonymous SNPs unique to FORC_075 has occurred in envZ, which encodes a sensor kinase of the EnvZ/OmpR two-component system. The SNP in envZ leads to an amino acid change from Pro248 (CCG) in other strains including FORC_078 to Leu248 (CTG) in FORC_075. Allelic exchange of envZ between FORC_075 and FORC_078 identified that the SNP in envZ is responsible for the impaired biofilm- and RDAR colony-forming abilities of S. Enteritidis. Biochemical analyses demonstrated that the SNP in envZ significantly increases the phosphorylated status of OmpR in S. Enteritidis and alters the expression of the OmpR regulon. Phenotypic analyses further identified that the SNP in envZ decreases motility of S. Enteritidis but increases its adhesion and invasion to both human epithelial cells and murine macrophage cells. In addition to an enhancement of infectivity to the host cells, survival under acid stress was also elevated by the SNP in envZ. Together, these results suggest that the natural occurrence of the SNP in envZ could contribute to phenotypic diversity of S. Enteritidis, possibly improving its fitness and pathogenesis.

Biofilm Formation by Multidrug-Resistant Serotypes of Salmonella Isolated from Fresh Products: Effects of Nutritional and Environmental Conditions

Salmonella serotypes can develop biofilms in fresh food products. This study focused on determining the antimicrobial resistance profile and the effects of different growth media and environmental conditions on biofilm formation by multidrug-resistant serotypes of Salmonella. All 49.4% of the Salmonella strains (five serotypes) were multidrug resistant. Assessment of the ability to form biofilms using the crystal violet staining method revealed that 95.6% of the strains of Salmonella were strong biofilm producers in 96-well polystyrene microtiter plates. Overall, 59.3% of the Salmonella strains showed the rdar (red dry and rough colony) morphotype, 2.1% pdar (pink dry and rough colony), 27.4% bdar (brown dry and rough colony) and 10.9% saw (smooth and white colony), at two temperatures (22 and 35 °C). Mono-species biofilms of Salmonella serotypes showed a mean cell density of 8.78 log10 CFU/cm2 ± 0.053 in TSBS (1/20 diluted TSB (tryptic soy broth) + 1% strawberry residues) and 8.43 log10 CFU/cm2 ± 0.050 in TSBA (1/20 diluted TSB + 1% avocado residues) on polypropylene type B (PP) (p &lt; 0.05). In addition, epifluorescence microscopy and scanning electron microscopy (SEM) enabled visualizing the bacteria and extracellular polymeric substances of biofilms on PP. Salmonella form biofilms depending on the serotype of the strains and the environmental conditions. Mono-species biofilms formed by Salmonella serotypes respond to nutrient limitation with the use of simplified culture media such as TSBA and TSBS.

Mycobacterium abscessus Strain Morphotype Determines Phage Susceptibility, the Repertoire of Therapeutically Useful Phages, and Phage Resistance.

Mycobacterium abscessus is an opportunistic pathogen whose treatment is confounded by widespread multidrug resistance. The therapeutic use of bacteriophages against Mycobacterium abscessus infections offers a potential alternative approach, although the spectrum of phage susceptibilities among M. abscessus isolates is not known. We determined the phage infection profiles of 82 M. abscessus recent clinical isolates and find that colony morphotype-rough or smooth-is a key indicator of phage susceptibility. None of the smooth strains are efficiently killed by any phages, whereas 80% of rough strains are infected and efficiently killed by at least one phage. The repertoire of phages available for potential therapy of rough morphotype infections includes those with relatively broad host ranges, host range mutants of Mycobacterium smegmatis phages, and lytically propagated viruses derived from integrated prophages. The rough colony morphotype results from indels in the glycopeptidolipid synthesis genes mps1 and mps2, negating reversion to smooth as a common route to phage resistance. Resistance is thus rare, and although mutations in polyketide synthesis, uvrD2, and rpoZ can confer resistance, these likely also impair survival in vivo The expanded therapeutic repertoire and the resistance profiles show that small cocktails or single phages could be suitable for controlling infections with rough strains.IMPORTANCEMycobacterium abscessus infections in cystic fibrosis patients are challenging to treat due to widespread antibiotic resistance. The therapeutic use of lytic bacteriophages presents a new potential strategy, but the great variation among clinical M. abscessus isolates demands determination of phage susceptibility prior to therapy. Elucidation of the variation in phage infection and factors determining it, expansion of the suite of therapeutic phage candidates, and a greater understanding of phage resistance mechanisms substantially advances the potential for broad implementation of new therapeutic options for M. abscessus infections.

Beyond phenotype: The genomic heterogeneity of co-infecting Mycobacterium abscessus smooth and rough colony variants in cystic fibrosis patients

Two unrelated cystic fibrosis patients were co-infected with Mycobacterium abscessus smooth and rough phenotypes. Smooth M. abscessus is proposed as the infecting form, and the subsequent loss of glycopeptidolipids in the host leads to a rough phenotype. Whole-genome sequencing (WGS) diagnosed two different M. abscessus strains in patient N°1 but only one strain in patient N°2. In patient N°1, rough isolate had novel mutations potentially involved in smooth-to-rough morphology changes. In patient N°2, four genes were present in only the smooth isolate. In addition, we obtained different susceptibility profiles in the four clinical isolates. We revealed a new paradigm describing a cystic fibrosis patient infected with two different clones, including a rough isolate, and identifying a rough M. abscessus clone that did not lose glycopeptidolipids. We propose WGS for the identification of heterogenic isolates and genetic determinants of antimicrobial resistance, which we believe will positively influence treatment prognosis.

Application of Rough Ant Colony Algorithm in Adolescent Psychology

With the rapid development of big data, big data research in the security protection industry has been increasingly regarded as a hot spot. This article mainly aims at solving the problem of predicting the tendency of juvenile delinquency based on the experimental data of juvenile blindly following psychological crime. To solve this problem, this paper proposes a rough ant colony classification algorithm, referred to as RoughAC, which first uses the concept of upper and lower approximate sets in rough sets to determine the degree of membership. In addition, in the ant colony algorithm, we use the membership value to update the pheromone. Experiments show that the algorithm can not only solve the premature convergence problem caused by stagnation near the local optimal solution but also solve the continuous domain and combinatorial optimization problems and achieve better classification results. Moreover, the algorithm has a good effect on predicting classification and can provide guidance for predicting the tendency of juvenile delinquency.

Serological detection of brucellosis among febrile, malaria-negative children and domesticated dogs in an urban African setting.

BackgroundChildhood brucellosis and malaria are co-endemic febrile illnesses in some sub-Saharan African countries. Malaria and brucellosis co-infection or brucellosis sole infections are often missed due to an over emphasis on malaria and the lack of appropriate diagnostic infrastructure. Brucellosis in dogs is usually overlooked and yet there is extensive contact between humans and their pets.ObjectiveThis study investigated brucellosis in children and dogs using a confirmatory serological testing series that screens for three Brucella sp.MethodsResidual blood samples from malaria smear-negative febrile children were collected and tested for Brucella sp and malaria parasite. During the same period, residual blood samples presented to a veterinary microbiology laboratory in the same area were tested for brucellosis using the same approach.ResultsA total of 105 human and 80 canine blood samples were tested for brucellosis antibodies. The seroprevalence of brucellosis was 22.86% (25/105) in children and 1.3% (1/80) in dogs using the Card, buffered acidified plate antigen, and standard plate agglutination tests but was 0% using the rivanol precipitation plate agglutination test.ConclusionGiven that brucellosis can be caused by both smooth and rough colony strains, there is a need to modify the current serological surveillance strategy (targeted at only Brucella abortus and other smooth colony Brucella strains) to figure out the relative contribution of rough colony Brucella strains (B. ovis and B. canis). Since Uganda is endemic for brucellosis there is a need to modify the brucellosis surveillance strategy.

First Report of Pre-Harvest Soft Rot of Peach Fruit (Prunus persica) Caused by Enterobacter mori in China.

Peach (Prunus persica L. Batsch) is one of the major fruit crops of China and a rich source of vital nutrients and fibers. In July 2019, a peach orchard was visited at Pinggu District, Beijing, China. The average temperature and relative humidity at the sampling site were 28±2 °C and 75±2 % respectively. In the orchard, unripe and near to ripe peach fruits cv. 'China Pearl' were observed with soft brown patches (3-4×2-3cm) on the surface. The samples (n=25) were collected based on typical symptoms. When the rotten part of the fruit was pressed, a liquid oozed out of the fruit emitting an unpleasant odor. The disease incidence of fruit soft rot was 22 %. The percent incidence was calculated based on total number of infected fruits divided by total number of fruits examined. Infected fruit tissues were excised into small pieces (5mm) and surface disinfested with 1% NaClO for 25s and rinsed twice with sterilized distilled water (SDW). These pieces (4-5 pieces per sample) were macerated with 1.5 ml SDW in a 2 ml tube. Later, 5 µl supernatant was streaked on Luria Bertani (LB) agar plates and incubated at 28 ºC for 3 days. Oozing liquid (50 µl) was also inoculated on 100ml LB broth and incubated for 24 h and 5 µl was streaked on LB agar medium and incubated at 28 ºC. Purified colonies were obtained by re-streaking 3 times. The isolate formed creamy to light yellowish, irregular, round, rough or smooth-looking colonies on LB medium and pink colonies on Eosin Methylene blue. The bacterium was rod shaped, measuring 0.8 to 1.4 μm in length and 0.2 to 0.5 μm in width (Fig 1) observed using a scanning electron microscope (Hitachi-SU8010). Morphological characteristics were similar to previously described characteristics of Enterobacter spp. (Zhu et al. 2011; Nishijima et al. 1987). Genomic DNA was extracted from all twenty-five isolates by using TIANamp Bacteria DNA Kit (Tiangen-Biotech, Beijing, China) according to the manufacturer's instructions. The 16S ribosomal RNA gene of 25 isolates was amplified by using universal primers (27F:5'-AGAGTTTGATCCTGGCTCAG-3',/1492R:5'-CTACGGCTACCTTGTTACGA-3'), and rpoB gene (F2: 5'-AACCAGTTCCGCGTTGGCCTGG-3', R2: 5'-CCTGAACAACACGCTCGGA-3') (Mollet et al., 1997). Sequences for EPT-1 were submitted to GenBank (accessions MN548761 (16S rRNA), MN594495 (rpoB)). Accessions MN548761 and MN594495 had 99.35 and 99.77% sequence identity with E. mori (GenBank accessions KF747680, GQ406571). Maximum Likelihood phylogenetic tree constructed (1000 replicates) using Mega X (Kumar et al. 2018) indicated that isolate EPT-1 clustered with E. mori sequences. To confirm the pathogenicity, medium-sized (n=60) surface disinfested ripe peach fruits cv. 'China Pearl' were wound inoculated with 5 µl suspension of EPT-1 (107cfu/ml) and the control fruit were treated with 5 µl sterilized water. The fruit were kept in sterilized plastic box and incubated at 28 ºC for 7 days at 70% relative humidity, and the pathogenicity test was repeated 3 times. Symptoms similar to the original fruit samples were observed on all inoculated fruit (Fig 2). The pathogen was re-isolated, the colonies obtained from the oozing liquid were similar to those of infected fruit tissues and identified as E. mori based on morphological and sequencing analysis. Previously, E. mori has been reported to cause bacterial wilt on Morus alba L. in China (Zhu et al. 2011). To our knowledge, this is the first report of E. mori causing soft rot of peaches in China.

Nutritional medium for differentiation of malignant anthrax

The purpose of the work is to develop a nutrient medium for differentiation of bacillus anthracis from soil aerobic bacilli. In order to achieve the set goal, we used the method of introduction into the environment of cultivation of microorganisms separated from animals and objects of external environment (water, soil, feed, air, scrapes from different surfaces suspected of contamination by their bacillus anthracis) of nutrient substrate - sucrose, used by bacteria of Bacillus genus for synthesis of levan - product of their metabolism, a sign absent in bacillus anthracis. This feature is essential for identification and differentiation of bacillus anthracis from closely related saprophytes. To identify and differentiate the bacillus anthracis, the microbes isolated from the external environment were cultivated in a nutrient medium consisting of meat-and-peptone agar (MPA) and stimulator (inductor) synthesis of levan sucrose in the amount of 10% to 100 ml of melted agar. The proposed nutrient media was prepared as follows. Meat-peptonic agar (500 ml) was melted at 100°C, 10 g of sucrose was added per 100 ml of medium and after the complete dissolution of sucrose, the nutrient medium was poured into Petri dishes and used for sowing the studied material for identification and differentiation of grown crops. The efficiency of the method has been tested in production experiments with positive evaluation. For this purpose, soil samples taken from the territory of old cattle cemeteries were fractionally sown on MPA and thermostatted for 16-18 hours at 37°C and examined crops for the presence of matte and rough (R-form) colonies.

Features of isolation of the anthrax pathogen depending on the type of nutrient medium

The purpose of the work is to develop a nutrient medium for differentiation of bacillus from soil aerobic bacilli. In order to achieve the set goal, we used the method of introduction into the environment of cultivation of microorganisms separated from animals and objects of external environment (water, soil, feed, air, scrapes from different surfaces suspected of contamination by their bacillus ) of nutrient substrate sucrose, used by bacteria of Bacillus genus for synthesis of product of their metabolism, a sign absent in bacillus . This feature is essential for identification and differentiation of bacillus from closely related saprophytes. To identify and differentiate the bacillus , the microbes isolated from the external environment were cultivated in a nutrient medium consisting of agar (MPA) and () synthesis of sucrose in the amount of 10% to 100 ml of melted agar. The proposed nutrient media was prepared as follows. agar (500 ml) was melted at 1000C, 10 g of sucrose was added per 100 ml of medium and after the complete dissolution of sucrose, the nutrient medium was poured into dishes and used for sowing the studied material for identification and differentiation of grown crops. The efficiency of the method has been tested in production experiments with positive evaluation. For this purpose, soil samples taken from the territory of old cattle cemeteries were fractionally sown on MPA and for 16-18 hours at 370C and examined crops for the presence of matte and rough (R-form) colonies.

The rough colony morphotype of Mycobacterium avium exhibits high virulence in human macrophages and mice

Introduction. The incidence of Mycobacterium avium complex (MAC) pulmonary disease (MAC PD), a refractory chronic respiratory tract infection, is increasing worldwide. MAC has three predominant colony morphotypes: smooth opaque (SmO), smooth transparent (SmT) and rough (Rg).Aim. To determine whether colony morphotypes can predict the prognosis of MAC PD, we evaluated the virulence of SmO, SmT and Rg in mice and in human macrophages.Methodology. We compared the characteristics of mice and human macrophages infected with the SmO, SmT, or Rg morphotypes of M. avium subsp. hominissuis 104. C57BL/6 mice and human macrophages derived from peripheral mononuclear cells were used in these experiments.Results. In comparison to SmO- or SmT-infected mice, Rg-infected mice revealed severe pathologically confirmed pneumonia, increased lung weight and increased lung bacterial burden. Rg-infected macrophages revealed significant cytotoxicity, increased bacterial burden, secretion of proinflammatory cytokines (TNF-α and IL-6) and chemokines (CCL5 and CCL3), and formation of cell clusters. Rg formed larger bacterial aggregates than SmO and SmT. Cytotoxicity, bacterial burden and secretion of IL-6, CCL5 and CCL3 were induced strongly by Rg infection, and were decreased by disaggregation of the bacteria.Conclusion. M. avium Rg, which is associated with bacterial aggregation, has the highest virulence among the predominant colony morphotypes.

The Effect of Pesticides on the Tomato Bacterial Speck Disease Pathogen Pseudomonas Syringae pv. Tomato

A significant part of the used pesticides does not reach the target organisms and, while remaining in the agrophytocenosis, influences all living organisms in it. Having a toxic and often mutagenic effect, pesticides induce morphological and physiological changes in the cells of microorganisms and are the cause of phenotypic heterogeneity of their populations. However, the effect of pesticides on phytopathogenic bacteria as non-target microorganisms remains out of the field of view for most researchers. However, the use of pesticides can lead to expansion of the diversity of existing phytopathogens and, as a consequence, complications of identification of the pathogens, loss of resistance by plants varieties, and increased harm from diseases caused by them. This study is focused on the effect of pesticides used in tomato plantations on the causative agent of bacterial speck of this crop—Pseudomonas syringae pv. tomato. The studies were carried out using the methods of classical microbiology. The mutagenic action of pesticides was recorded, taking into account the increase of the number of streptomycin resistance mutations in bacteria in the case of pesticide action. It is established that the fungicide aluminium phosethyl is characterised by a bacteriostatic effect on P. syringae pv. tomato. Deltamethrin insecticide does not affect the growth of P. syringae pv. tomato. However, there is an increase in the frequency of streptomycin resistance mutations in both studied strains of P. syringae pv. tomato after using deltamethrin. It is shown that the frequency of occurrence of R (rough colonies) forms of P. syringae pv. tomato IZ28 and IZ46 after using deltamethrin increased by 100 times when in comparison to the frequency of spontaneous morphological dissociation, or smooth-to-rough (S-R) mutation, of these bacteria. Therefore, aluminium phosethyl is characterised by moderate bacteriostatic action against P. syringae pv. tomato. Deltamethrin does not influence the growth of the pathogen of tomato speck but increases the frequency of formation of StrR mutants and R forms of phytopathogenic bacteria.

Rough Sets and Colonies of Artificial Ants for the Improvement of Training Sets

Improving training sets is an area of active research within l to Artificial Intelligence. In particular, it is of particular interest in supervised classification systems, where the quality of training data is crucial. This paper presents a new method for the improvement of training sets, based on approximate sets and artificial ant colonies. The experimental study carried out with international databases allows us to guarantee the quality of the new algorithm, which has a high efficiency.