Extraction and Fractionation of Bioactives from Dipsacus fullonum L. Leaves and Evaluation of Their Anti-Borrelia Activity

Lyme disease (LD) is a tick-borne bacterial disease that is caused by Borrelia burgdorferi. Although acute LD is treated with antibiotics, it can develop into relapsing chronic form caused by latent forms of B. burgdorferi. This leads to the search for phytochemicals against resistant LD. Therefore, this study aimed to evaluate the activity of Dipsacus fullonum L. leaves extract (DE) and its fractions against stationary phase B. burgdorferi in vitro. DE showed high activity against stationary phase B. burgdorferi (residual viability 19.8 ± 4.7%); however, it exhibited a noticeable cytotoxicity on NIH cells (viability 20.2 ± 5.2%). The iridoid-glycoside fraction showed a remarkable anti-Borrelia effect and reduced cytotoxicity. The iridoid-glycoside fraction was, therefore, further purified and showed to contain two main bioactives—sylvestrosides III and IV, that showed a considerable anti-Borrelia activity being the least toxic to murine fibroblast NIH/3T3 cells. Moreover, the concentration of sylvestrosides was about 15% of DE, endorsing the feasibility of purification of the compounds from D. fullonum L. leaves.

Bacterial biofilms predominate in both acute and chronic human lung infections

BackgroundA basic paradigm of human infection is that acute bacterial disease is caused by fast growing planktonic bacteria while chronic infections are caused by slow-growing, aggregated bacteria, a phenomenon known as a biofilm. For lung infections, this paradigm has been thought to be supported by observations of how bacteria proliferate in well-established growth media in the laboratory—the gold standard of microbiology.ObjectiveTo investigate the bacterial architecture in sputum from patients with acute and chronic lung infections.MethodsAdvanced imaging technology was used for quantification and direct comparison of infection types on fresh sputum samples, thereby directly testing the acute versus chronic paradigm.ResultsIn this study, we compared the bacterial lifestyle (planktonic or biofilm), growth rate and inflammatory response of bacteria in freshly collected sputum (n=43) from patient groups presenting with acute or chronic lung infections. We found that both acute and chronic lung infections are dominated by biofilms (aggregates of bacteria within an extracellular matrix), although planktonic cells were observed in both sample types. Bacteria grew faster in sputum from acute infections, but these fast-growing bacteria were enriched in biofilms similar to the architecture thought to be reserved for chronic infections. Cellular inflammation in the lungs was also similar across patient groups, but systemic inflammatory markers were only elevated in acute infections.ConclusionsOur findings indicate that the current paradigm of equating planktonic with acute and biofilm with chronic infection needs to be revisited as the difference lies primarily in metabolic rates, not bacterial architecture.

Anti-Quorum Sensing Effects of Some Medicinal and Aromatic Plant Extracts on Xanthomonas axonopodis pv. Phaseoli

Xanthomonas axonopodis pv. phaseoli (Xap) is known as one of the most important seed-borne destructive pathogens on beans worldwide. Nowadays, Xap is considered to acquire resistance to antibiotics and synthetic bactericides which concerns the scientific world for its future management. This has made the use of plant extracts, the best alternative in the control of plant disease pathogens by inhibiting the quorum sensing (QS) mediated virulence factors. This research was designed to investigate the antibacterial activities and the anti-QS effects of the 14 different aromatic and medicinal plants against QS-mediated virulence factors of Xap. The results revealed that Syzgium aromaticum showed the largest inhibition zone diameter and strongest antimicrobial (antibacterial) effect among the 14 plant extracts followed by Thymus vulgaris and Coriandrum sativum. Similarly, the lowest swarming, swimming, and twitching motility values were measured from the Syzgium aromaticum application followed by Coriandrum sativum, Thymus vulgaris, Brassica nigra, Lepidium sativum, and Ruta chalepenis. These results indicated that S. aromaticum, C. sativum, T. vulgaris, B. nigra, L. sativum, and R. chalepenis will be a potential candidates as anti-quorum sensing agents in preventing common bacterial disease of beans caused by Xap. Compounds derived from aromatic and medicinal plants have demonstrated successful control of diseases in crops and the use of these substances provides a valuable tool to the growers around the world for diseases management in organic production.

Cloning and Expression of PirA gene of Vibrio parahaemolyticus Strain K5 Causing Acute Hepatopancreatic Necrosis Disease in Whiteleg Shrimp in E. coli Host Cell

Background: Acute hepatopancreatic necrosis disease (AHPND), is a bacterial disease of whiteleg shrimp, which has a high mortality rate (100%) and incurs economic losses. Our objective was to identify the genes which lead to cell and organ damage and investigate bioproducts to prevent and treat. Methods: Litopenaeus vannamei shrimp in Thua Thien Hue province, Vietnam were collected from an infected pond and analysed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence and paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant protein was purified and determined the PirAvp binding ratio, cloning and sequencing of PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. Results: PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/IPTG/X-Gal medium. PCR showing a band of about 347 bp, matching the size of PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that PirA gene has a length of 336 bp and similar to PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected a highest protein concentration (1,586.54 µg/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.

Identification of bacterial blight resistance genes in rice landraces from Yunnan Province, China

Bacterial blight (BB), a serious bacterial disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo) affects rice growth and yield. Yunnan Province is regarded as a center of rice diversity in China and indeed the world, and has abundant rice landrace resources, which may offer prospective candidate donors in rice improvement and breeding. In this study, a set of 200 rice landraces were evaluated to determine their resistance to 10 pathogenic Xoo strains resistance by the leaf-clipping method. The results indicated that the tested rice landraces had different resistance levels against different Xoo strains. Multiple comparisons showed that the Xoo strain PXO99 was virulent to the tested rice landraces. Sixty-six rice landraces conferred resistance against at least one Xoo strain. These resistant rice landraces screened were then performed the presence of 14 cloned BB resistance genes by closely linked molecular markers and designed specific primers. The results showed that none of these resistant accessions contained xa13, Xa21, Xa27, and Xa45(t) homologous fragments, while 9, 24, 4, 7, 9, 15, 1, 5, 4 and 27 accessions contained Xa1, Xa2/Xa31(t), Xa14, Xa3/Xa26, Xa4, xa5, Xa7, Xa10, Xa23 and xa25 homologous fragments, respectively. Sequence analysis further revealed that nucleotide variations around functional nucleotide polymorphisms region were observed within these accessions containing the Xa1, Xa2/Xa31(t), Xa14, Xa3/Xa26, Xa4, xa5, Xa10, Xa23 and xa25 homologous fragments. These results along with phenotypic resistance spectrum supported that these accessions carried nine resistance homologous genes. Only one accession (Qishanggu_Wenshan) carried the Xa7 resistance gene. We also found that some resistant rice landraces, especially Xilandigu_Baoshan, and Laoyaling_Lincang without the above resistance genes, which mediated broad spectrum resistance to multiple Xoo strains, were identified as potential sources for breeding rice lines resistance to BB.

Nanoparticles in inhibiting Pantoea ananatis and to control maize white spot

ABSTRACT: Maize white spot (MWS) caused by Pantoea ananatis is one main maize leaf diseases, and nanoparticles (NPs) are an innovative approach for bacterial disease control. This research evaluated the toxicity of pure NPs and doped NPs with different elements in inhibiting bacterial growth and to control MWS. Pure NPs and ZnO NPs doped with silver (Ag), gold (Au), copper (Cu), iron (Fe), manganese (Mn), and nickel (Ni) at different concentrations were used to determine the toxicity for P. ananatis in vitro, evaluating the bacterial growth inhibition zone. To assess the control of MWS, in the preventive application, maize plants were sprayed with NPs of ZnO:0.1Cu, ZnO:0.05Fe, ZnO:0.2Mn and ZnO:0.7Ni at 10, 5 or 2.5 mg mL-1, and after 3 days, the plants were inoculated with bacterial suspension. To assess the curative application, plants were inoculated with the bacteria, and 3 days later sprayed with the NPs. The disease severity was assessed and the area under the disease-progress curve (AUDPC) was calculated. The doped ZnO NPs with different elements, and at different concentrations inhibited bacterial growth in vitro. NPs of ZnO:0.1Cu and ZnO:0.2Mn at 5 or 2.5 mg mL-1, in both applications reduced the severity of MWS, showing potential for use in the disease management.

Occurrence of Antibiotic-Resistant Staphylococcus spp. in Orange Orchards in Thailand

Background: The widespread indiscriminate application of antibiotics to food crops to control plant disease represents a potential human health risk. In this study, the presence of antibiotic-resistant staphylococci associated with workers and orange orchard environments was determined. A total of 20 orchards (orange and other fruits) were enrolled in the study. Trees in the orange orchards were treated with ampicillin on a pre-determined schedule. Environmental samples (n = 60) included soil, water, and oranges; 152 hand and nasal samples were collected from 76 healthy workers. Antibiotic susceptibility profiles were determined for all staphylococcal isolates. Results: This investigation revealed that of the total Staphylococcus spp. recovered from the orange orchard, 30% (3/10) were resistant to erythromycin, 20% (2/10) were resistant to ampicillin, and 20% (2/10) resistant to both erythromycin and ampicillin. Conclusion: The application of antibiotics to orange trees in open production environments to halt the spread of bacterial disease presents risks to the environment and creates health concerns for Thai farmers using those agents. ARB on crops such as oranges may enter the global food supply and adversely affect public health.

T6SS Accessory Proteins, Including DUF2169 Domain-Containing Protein and Pentapeptide Repeats Protein, Contribute to Bacterial Virulence in T6SS Group_5 of Burkholderia glumae BGR1

Burkholderia glumae are bacteria pathogenic to rice plants that cause a disease called bacterial panicle blight (BPB) in rice panicles. BPB, induced by B. glumae, causes enormous economic losses to the rice agricultural industry. B. glumae also causes bacterial disease in other crops because it has various virulence factors, such as toxins, proteases, lipases, extracellular polysaccharides, bacterial motility, and bacterial secretion systems. In particular, B. glumae BGR1 harbors type VI secretion system (T6SS) with functionally distinct roles: the prokaryotic targeting system and the eukaryotic targeting system. The functional activity of T6SS requires 13 core components and T6SS accessory proteins, such as adapters containing DUF2169, DUF4123, and DUF1795 domains. There are two genes, bglu_1g23320 and bglu_2g07420, encoding the DUF2169 domain-containing protein in the genome of B. glumae BGR1. bglu_2g07420 belongs to the gene cluster of T6SS group_5 in B. glumae BGR1, whereas bglu_1g23320 does not belong to any T6SS gene cluster in B. glumae BGR1. T6SS group_5 of B. glumae BGR1 is involved in bacterial virulence in rice plants. The DUF2169 domain-containing protein with a single domain can function by itself; however, Δu1g23320 showed no attenuated virulence in rice plants. In contrast, Δu2g07420DUF2169 and Δu2g07420PPR did exhibit attenuated virulence in rice plants. These results suggest that the pentapeptide repeats region of the C-terminal additional domain, as well as the DUF2169 domain, is required for complete functioning of the DUF2169 domain-containing protein encoded by bglu_2g07420. bglu_2g07410, which encodes the pentapeptide repeats protein, composed of only the pentapeptide repeats region, is located downstream of bglu_2g07420. Δu2g07410 also shows attenuated virulence in rice plants. This finding suggests that the pentapeptide repeats protein, encoded by bglu_2g07410, is involved in bacterial virulence. This study is the first report that the DUF2169 domain-containing protein and pentapeptide repeats protein are involved in bacterial virulence to the rice plants as T6SS accessory proteins, encoded in the gene cluster of the T6SS group_5.

AVIAN ESCHERICHIA COLI INFECTION IN MEAT-TYPE CHICKEN: PREVALENCE AND PATHOLOGY

Avian colibacillosis is considered the major bacterial disease in the poultry industry worldwide. This study was conducted to determine prevalence and pathology by avian pathogenic Escherichia coli (APEC) in broiler chicken. Various organs of affected broilers were checked for the E. coli infection. One hundred and forty commercial poultry farms were divided into seven age groups (A, B, C, D, E, F, and G). Group A (1st week), B (2nd week), C (3rd week), D (4th week), E (5th week), F (6th week), G (7th to 9th week). Investigations were conducted from day 1 until the marketing of birds based on clinical findings. A total of 2491 (23.71% of sick birds) dead birds were observed positive for E. coli infection. Three forms of infection were observed i.e. omphalitis, colisepticaemia, and colibacillosis at the rate of 1.55, 17 and 56.65%, respectively. Omphalitis was confirmed by microbial culture and revealed in only groups A and B with 76.24 and 23.76 respectively. While, Colisepticaemia was observed at the rate of 3.11, 6.74, 20.18, 29.63, 19.16, and 22.18 in groups B, C, D, E, F, and G respectively. Whereas Colibacillosis was noticed as 0.075, 3.35, 5.99, 19.20, 26.70, 16.11 and 28.55% in groups A, B, C, D, E, F, and G respectively. Petechiation of heart, kidney, and gizzard besides hemorrhagic enteritis, hemorrhagic tracheitis, and necrotic foci on the liver and caseous exudates in air sacs were the prime gross lesions noticed in colibacillosis. Microscopically, no lesion was observed in serosa, muscularis externa, and submucosa. However, loss of epithelial tissue and breaches in the mucosal layer of the small intestine were observed. Furthermore, characteristic pink and grayish colonies were observed in E. coli cultures on McConkey’s and Blood agar respectively.