Luria-Bertani Agar Medium Research Articles

First Report of Klebsiella variicola Causing Tomato Pith Necrosis in Yunnan Province, China.

Tomato (Solanum lycopersicum L.) is a key vegetable crop in China. In August 2023, an outbreak of bacterial pith necrosis in tomato occurred in Lufeng County, Yunnan Province, China, affecting over 40% of the tomato plants in a greenhouse. The stems of infected plants developed a waterlogged soft rot and the disease progressed, the lower leaves and lateral branches of infected plants gradually wilted and died. A longitudinal cut of the stem revealed hollow pith with brown vascular tissue. To isolate the pathogen, the plant surface was disinfested with 75% ethanol. Then, a piece of infected tissue from the base of the stem was excised and immersed in sterile water for 2 min. A small amount of liquid was streaked onto TTC (2,3,5-triphenyltetrazolium chloride) agar medium using an inoculation loop, and plates were incubated at 28℃ for 24 h. Colonies on the TTC plate were white, indicating that the pathogen was not Ralstonia solanacearum. Colonies grown on LB (Luria-Bertani) agar medium were randomly selected and subjected to preliminary pathogenicity tests. Based on the results, a colony named Kv4 was selected and purified through six subcultures in LB agar medium. Biochemical tests showed the strain utilized D-sorbitol, raffinose and citrate but not adonitol, and was positive for methyl red, D-glucose (acid), urea hydrolysis, lysine decarboxylase, and motility, and negative for phenylalanine deaminase, H2S production, indole production, and ornithine decarboxylase. These characteristics align with Klebsiella species (Garrity et al. 2007). To determine the species of strain Kv4, partial sequences of the 16S rDNA, phoE, leuS, and rpoB genes were amplified (Barrios-Camacho et al. 2019) and sequenced. Through BLASTn analysis, strain Kv4 sequences of 16S rDNA (OR888750) had 99.47% identity (1488/1496 bp), phoE (OR899599) had 98.69% (605/613 bp) identity, leuS (OR899598) had 99.07% identity (959/968 bp), and rpoB (OR899597) had 97.69% (633/648 bp) identity with Klebsiella variicola strain FF0907. Using the ClustalW algorithm in MEGA11 for nucleotide sequence alignment, phylogenetic trees were constructed with 16S and phoE, leuS, and rpoB via the neighbor-joining method, confirming strain Kv4 as K. variicola. To test pathogenicity, the roots of 25 'Moneymaker' tomato plants with four to five true leaves were wounded, then each plant inoculated with a 15 mL bacterial suspension (OD600=0.6) of strain Kv4, while the control plants received sterile water. Plants were incubated at 28℃ with a 16 h photoperiod. Experiments were done twice. At 15 days after inoculation (DAI), all plants inoculated with Kv4 showed yellowing, unevenly distributed small black necrotic spots on the leaf surface, and purple-brown soft rot at the stem base. By 18 DAI, there was a gradual transformation of the stem bases from green to purplish brown. At 21 DAI, 60% of the inoculated plants displayed brownish soft rot at the stem base. In contrast, the control plants remained symptom-free. The pathogen was re-isolated from the stem and identified as K. variicola via sequence analysis of 16S, phoE, leuS, and rpoB. In recent years, several new bacterial pith necrosis diseases were reported in tomato (Guo et al. 2023; Ivić et al. 2023). This is the first study documenting K. variicola causing bacterial pith necrosis in tomato. Once considered a benign plant endophyte, Sun et al. (2023) reported K. variicola causing banana sheath rot in Guangdong and Guangxi Provinces, China. Malik et al. (2023) reported that K. variicola caused leaf streak in sorghum in India. This report of bacterial pith necrosis in tomato caused by K. variicola strain Kv4 underscores the escalating threat posed by emerging pathogens to agricultural production. The emergence of K. variicola as a tomato pathogen complicates plant disease management strategies.

Essential oil of Lippia multiflora Moldenke Flowers Quenches Pseudomonas aeruginosa PAO1 Biofilm Formation and Motilities

Bacteria use different types of motilities in order to colonize and adapt to new environments. These motilities also play an important role in the formation of biofilm, allowing bacteria to develop resistance to antibiotics and host’s immune systems. The objective of this study was to evaluate the ability of essential oils of Lippia multiflora flowers to inhibit biofilm formation and motility in Pseudomonas aeruginosa PAO1. The capacity of essential oil of Lippia multiflora flowers to inhibit biofilm formation in Pseudomonas aeruginosa was evaluated spectrophotometrically by using the crystal violet method. The property of the essential oil to inhibit different types of motilities such as swimming, swarming and twitching was further evaluated by measuring the diameters of bacterial migration on liquid or semi-liquid Luria Bertani agar medium. The essential oil exhibited good anti-biofilm activity in Pseudomonas aeruginosa. At a concentration of 1%, essential oil presented a degree of biofilm inhibition similar to that of salicylic acid used as a reference (P > 0.05). In addition, the essential oil significantly inhibited swarming, twitching and swimming in Pseudomonas aeruginosa compared to the control (P < 0.001). The inhibition of biofilm formation as well as that of bacterial motility increases with the concentration of the essential oil. The essential oil of Lippia multiflora flowers possesses an anti-bacterial potential to fight against multi-resistant bacteria.

First Report of Pectobacterium aroidearum Causing Soft Rot of Pinellia ternata in China.

Pinelliae rhizoma is the dried tuber of Pinellia ternata (Thunb.) Breit., and has been used for thousand of years in traditional Chinese medicine as an antivomit, anticough, and analgesic (Ying et al. 2007). In September 2022, P. ternata planted in Bijie, Guizhou Province, showed severe soft rot symptoms with incidence of about 50%. The diseased plants showed water-soaked symptoms and produced a foul soft rot smell, and finally the whole plant collapsed. Lesions were first observed at the tip of a leaf or wound, and symptoms of the disease spread rapidly, with the entire plant collapsing and dying within a week. The tissue sections of six plants with typical symptoms from the diseased field were disinfected with 75% ethanol for 30 seconds and 0.3% NaClO for 3 minutes. The tissue sections were then washed with sterile water for three times. A small piece of tissue (5x5mm) was removed from the edge of the lesion and mashed in a 1.5 ml centrifuge tube containing 20 μl of sterile water. The tissue liquid was then diluted 100 times with prepared sterile water. The bacteria were streaked on LB (tryptone/yeast extract/NaCl) AGAR medium and cultured at 37°C for 48 h (Kravitz, 1962). Isolated colonies were streaked on Luria-Bertani (LB) AGAR medium to obtain single colonies for further identification. A total of 13 representative isolates were selected for PCR amplification using primers targeting the conserved region of the 16S rDNA gene, which were in turn analyzed via the BLASTn search engine on the NCBI website. The results of the analysis revealed that seven of the isolates were similar to P. aroidearum strain SCRI 109 (GenBank accession no. NR_159926), with strain BX13 exhibiting the highest similarity to P. aroidearum (99.93% similarity), and therefore, this strain was selected for further investigation. The strain BX13 was incubated on LB solid medium for 24 h at 37 °C, and the single colonies were creamy white, translucent and round, slightly elevated in the center, with smooth surfaces and neat edges (Figure S1 B1). Then,the Scanning Electron Microscope revealed that the thalli of strain BX13 were short rod-shaped and somewhat blunt round at both ends (Figure S1 B2). The steward genes (icdA, gapA, proA) of BX13 were amplified and sequenced for further identification. The sequences of the amplified fragments were all deposited in GenBank 16S rDNA (OQ874505,) icdA (OQ954122)，gapA (OQ954123), proA (OQ954124). Sequence analysis using the BLASTn program at the NCBI revealed gene icdA, gapA, and proA had 100% identity to P. aroidearum strain QJ002 (GenBank accession no. CP090597).. Meanwhile, a maximum likelihood phylogenetic tree was constructed based on multigene sequence analysis of BX13 16S rDNA and steward genes (gapA, icdA, proA) by MEGA X (Liang et al. 2022). Phylogenetic results also showed that BX13 and P. aroidearum strain QJ002 gathered in the same clade(Figure S2). Accordingly, the morphological and molecular characteristics of strain BX13 indicate that it is P. aroidearum. (Nabhan S., et al.2013,Xu et al. 2020). In order to confirm the pathogenicity of strain BX13, a bacterial suspension containing 107 CFU/ml (10 ml/ inoculation point) was injected into the base of a one-week-old P. ternata stems, control seedlings were inoculated with sterile water, inoculated and control seedlings (each of six plants) were kept in a growth chamber maintained at 26°C with a relative humidity range of 70% to 80%. Plants were watered as needed. After 3 days, the stem base of the plants inoculated with bacteria solution showed water-soaked necrosis and stems began to rot, while the plants inoculated with water did not show this symptom. The strains were then successfully re-isolated from the symptomatic P. ternata. Then the strain re-isolated was identified using the BLASTn program at the NCBI and found that it has the same 16S rDNA, icdA, gapA, and proA sequences as strain BX13, thus completing the Koch's postulates. To our knowledge, this is the first report of P. aroidearum causing P. ternata soft rot in China, which expands its known host range. Accordingly, this study provides essential information for the breeding of P. ternata resistant to bacterial soft rot and the development of control measures in China.

Ilmenite-derived titanic acid species: exploring their outstanding light-independent antibacterial activity.

The emergence of resistance in detrimental pathogenic bacteria towards well-recognized antibiotics has greatly impacted global medicine, consequently exploring potent antibacterial compounds is becoming a potential area of research. Although photocatalytic metal oxides have been extensively explored in this regard, their applicability is diminished due to the requirement of photon energy. Therefore, in our study, we explored the light-independent antibacterial effect of two unexplored titanium species, known as metatitanic acid (MTA) and potassium titanate, against Staphylococcus aureus, Escherichia coli, and Pseudomonas spp. using the disk diffusion method in Luria-Bertani agar medium, where the well-known antibiotic, gentamicin, was used as the positive control. These two titanium compounds were readily synthesized through a novel process which was originally developed for the extraction of TiO2 from ilmenite. The synthesized MTA was characterized using FT-IR, Raman spectroscopy, XRD, TGA, UV-visible spectroscopy, and SEM. According to our findings, both MTA and potassium titanate exhibited superior light-independent antibacterial properties, where for some concentrations, the effect was even greater than gentamicin. However, nano-TiO2 totally failed as an antibacterial compound against the tested three strains under dark conditions.

The cloning of ESAT-6, EspC and Rv2029c genes obtained from an Indonesian tuberculosis-infected patient

Early secreted antigenic target of 6-kDa protein (ESAT-6), a protein related to tuberculosis virulence (EspC) and latent antigen Rv2029c are important biomarkers for Mycobacterium tuberculosis prevention. Recent advances in subunit protein vaccines offer promising solutions to the inadequacies of existing BCG vaccines. In this study, these antigens were cloned into pET plasmid series to express the genes using the Escherichia coli expression system. Polymerase Chain Reaction was used to amplify the target genes from genome DNA isolated from Indonesia tuberculosis-infected patient’s sputum. A 309 bp of ESAT-6 fragment and a 337 bp of EspC fragment were inserted into pET-21b. Meanwhile, the 1038 bp of Rv2029c fragment was inserted into pET-32b. All constructs were transformed into Escherichia coli TOP10 and screened on Luria Bertani agar media containing ampicillin. The transformants were subjected to colony PCR using T7 universal primers followed by plasmid isolation to confirm the recombination. All genes were successfully cloned into the pET plasmid series. These results serve as a basis for further studies to express the recombinant protein as a booster vaccine.

Antimicrobial Activity Test of Medicinal Plant Extract Using Antimicrobial Disc and Filter Paper

Plant extracts contain secondary metabolites such as flavonoids and phenolic compounds with antibiotic activity. Antibiotic activity test can be done by using the Kirby-Bauer disc method which the antibiotic activity is indicated by the formation of a clear zone. This research was conducted using extracts of Moringa oleifera leaves and Opuntia cochenillifera cladode extracted using ethyl acetate as a solvent. Antibiotic activity test against Staphylococcus aureus bacteria was done in Mannitol Salt Agar and Luria Bertani Agar media. The Kirby-Bauer discs used are Antimicrobial Disc Oxoid® and Filter Paper. The results of this study show that the use of the Antimicrobial Disc Oxoid® and Filter Paper shows no statistical difference in clear zone results (t-value = 0,45; p-value = 0,655; with α = 95%). While the Clear Zone results on Mannitol Salt Agar gave better results than Luria Bertani Agar and were statistically significant (t-value = 2,46; p-value = 0,02; with α = 95%). These findings indicate that filter paper can be an inexpensive alternative for the antibiotic test with no significantly different result compared to commercial antibiotic disc. However, an antibiotic test against Staphylococcus aureus is better done on Mannitol Salt Agar (MSA) compared to Luria Bertani Agar (LBA).

Ginkgolic acid promotes inflammation and macrophage apoptosis via SUMOylation and NF-κB pathways in sepsis.

Excessive inflammation and increased apoptosis of macrophages contribute to organ damage and poor prognosis of sepsis. Ginkgolic acid (GA) is a natural constituent extracted from the leaves of Ginkgo biloba, that can regulate inflammation and apoptosis. The present study aims to investigate the potential effect of GA in treating sepsis and its possible mechanisms. Here, a classic septic mice model and a lipopolysaccharide (LPS)-induced RAW 264.7 inflammation model were established. Cytokines in serum and culture supernatant were detected by ELISA, and the mRNA levels of them were examined by PCR. Hematoxylin and eosin (H&E) staining was performed to determine histopathological changes in liver, lung and kidney. Bacterial burden in the blood, peritoneal lavage fluids (PLFs) and organs were observed on Luria-Bertani agar medium. Flow cytometry and western blotting was used to detect apoptosis and the expression level of apoptosis related molecules, respectively. Moreover, the levels of SUMOylation were detected by western blotting. The activity of NF-κB p65 was assessed by immunofluorescence staining and western blotting. The result showed that GA promoted inflammatory responses, reduced bacterial clearance, aggravated organ damage, and increased mortality in septic mice. GA increased apoptosis in peritoneal macrophages (PMs) and RAW 264.7 cells. Meanwhile, GA inhibited SUMOylation and increased the nuclear translocation of NF-κB p65 as well as its phosphorylation level. Collectively, GA promotes inflammation and macrophage apoptosis in sepsis, which may be mediated by inhibiting the SUMOylation process and increasing NF-κB p65 activity.

In vitro antibacterial activity of essential oils from Carum copticum and Ziziphora clinopodioides plants against the phytopathogen Pseudomonas syringae pv. syringae

The present study aimed to determine the in vitro antibacterial activity and the chemical composition of essential oils (EOs) from two medicinal plants (seeds of Carum copticum L. and leaves of Ziziphora clinopodioides Lam) against the bacterial plant pathogen Pseudomonas syringae pv. syringae. The sensitivity of P. syringae to the two types of EOs was investigated through three methods including disc diffusion, well diffusion, and vapor phase tests. Gas chromatography (GC) and gas chromatography attached to a mass spectrometer (GC-MS) were used to identify and quantify the essential oil compounds. The greatest growth inhibition zones for disc diffusion, well diffusion, and vapor tests were observed for the highest essential oil volume (50 µL) of Z. clinopodioides as 0.925 cm, 1.75 cm, and 1.45 cm, respectively. Concerning the EOs from C. copticum seeds, the diameters of growth inhibition zones were 0.775 cm, 1 cm, and 0 cm, respectively. Based on the obtained results, the highest antibacterial activity, with a growth inhibition zone of 1.75 cm, belonged to 50 µL EOs of Z. clinopodioides on Luria-Bertani (LB) agar medium. The minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) for the EOs of Z. clinopodioides were 8 µL mL−1 and 1 µL mL−1 in LB agar medium. The MBC and MIC values for EOs of C. copticum were 16 µL mL−1 and 1 µL mL−1, in LB agar medium, respectively. According to the chromatography results, 16 different compounds were identified in the EOs of C. copticum, of which thymol (54.6%) was the major constituent. Moreover, 27 different compounds were identified in the Z. clinopodioides EOs, of which pulegone (23%) and 1,8 cineole (20.3%) were the main essential oil constituents. The results demonstrated that the EOs of Z. clinopodioides plants had higher chemical diversity and displayed stronger antibacterial activities against P. syringe pv. syringae, compared to those of C. copticum.

Karakterisasi Resistensi dan Dekolorisasi Berbagai Pewarna Oleh Bakteri Indigen Indonesia Escherichia coli Strain CN5

The development of the textile industry in Indonesia is increasing the amount of dye waste produced. Copper is often a component of dyes, both of which harm aquatic ecosystems because they cannot be degraded. These problems can be overcome by bioremediation using bacteria isolated from polluted waters, called indigenous bacteria. It is hoped that indigenous bacteria can degrade textile waste and reduce copper toxicity in waters. This study aims to characterize, test resistance, and test the decolorization of the Indonesian indigenous bacterial isolate CN5 to dye and copper. There are 12 kinds of dyes used, namely: methylene blue, malachite green, congo red, mordant orange, reactive black, direct yellow, basic fuchsin, reactive orange, disperse orange, remasol red, wantex yellow, and wantex red. Resistance and decolorization tests on solid medium were carried out by growing bacterial cultures into luria bertani agar medium, each of which added a different type of dye. The dye concentrations tested were 100 ppm and 500 ppm. The ability to decolorize is known from the presence of a clear zone around the bacterial colony. The percentage of decolorization was tested using a spectrophotometer at a wavelength of 300-900 nanometers. Bacterial identification was carried out by 16S-rRNA sequencing. The results showed that CN5 isolates had a base similarity of 100% with Escherichia coli, so hereinafter referred to as E. coli strain CN5 could grow at 200 ppm and 500 ppm methylene blue, malachite green, congo red, mordant orange, reactive black, direct yellow, reactive orange, disperse orange, red remasol, yellow wantex, and red wantex but did not grow on fuchsin basic dye. Colonies of E. coli strain CN5 were only able to decolorize methylene blue with a concentration of 200 ppm and 500 ppm seen from the clear zone formed around the colony. The decolorization of methylene blue that occurred was 92.47%. The addition of copper reduced the decolorization ability to 75.59%. Based on the results of this study, it can be concluded that the E. coli strain CN5 has the potential to be used as a bioremediation agent for textile waste containing copper and methylene blue.Key words: dye; E. coli; copper; decolorization; resistant

Serratia nevei 9rpt1, a Potential Microorganism for Phosphorus Recovery

Aims: Phosphorus (P) is an essential element for life, finite and irreplaceable. Its constant exploration on a global scale has motivated frequent alerts regarding an eventual crisis due to the shortage of this nutrient. However, it is possible to recycle it and reintroduce it into the ecological cycle. One viable alternative is the microbial recovery of phosphate.
 Study design: This study is based on systematic bioprospection of bacteria in phosphate-deficient Amazon regions.
 Place and Duration of Study: Bacteria were isolated from black water samples, collected in the Rio Pretinho, located at Serra do Aracá, Barcelos, Amazonas, Brazil, from January to July 2019.
 Methodology: Microbial isolation was performed in Luria Bertani agar medium. For the genomic study, the isolate with the best performance in the phosphate uptake test was chosen. The WGS was carried out in a Illumina HiSeq 2500 System. The assembly of the draft genome was carried out with the SPAdes and the annotation by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).
 Results: Serratia nevei 9rpt1 recovers 90% of the phosphate available in the culture medium. Its draft genome comprises 5.4 MB, the GC content is 59.52% and 4,922 coding sequences were identified, among these, two pst operons: one complete, containing the five pst genes and one missing pstS, pstC and phoU genes.
 Conclusion: Serratia nevei 9rpt1, isolated from an Amazonian environment poor in phosphate, is very efficient to uptake this nutrient in a Pi starvation condition. The genomic findings revealed this strain has an additional high affinity Pi uptake pst system containing the ATP-binding protein PstB, the canonical permease PstA, a putative permease other than PstC, upstream of the PstA and two essential enzymes in the polyphosphate metabolism: polyphosphate kinase 1 and exopolyphosphatase.

Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia

Abstract. Fazal BZ, Budiman C, Amin Z, Ling CMW. 2022. Screening, isolation, and characterization of amylase-producing bacteria from Poring Hot Spring Sabah, Malaysia. Biodiversitas 23: 2807-2815. Thermostable ?-amylases are being used in a wide range of industries, including food, textiles, detergents, pharmaceuticals, and fine chemicals. A good source of thermostable ?-amylases in thermophilic bacteria is found in high-temperature habitats like hot springs. Hence, this study aimed to screen, isolate, and characterize amylase-producing bacteria (APB) from the Poring hot spring in Sabah. Sediment and water samples were collected from the hot springs, serially diluted, plated onto the Luria Bertani agar medium containing starch, and incubated at 60°C for 48 hours. The amylase-producing bacterium was identified by the halo formation around the colony after the agar medium was stained with Lugol's solution. Nine colonies were found to be able to form halo zones, with a creamy colony (A7 strain) producing the highest amylolytic index (4.24). Further characterization of the A7 strain showed that the isolate was a Gram-positive, rod-shaped bacterium, with a positive reaction upon oxidase and catalase tests. The 16S rRNA sequence showed that the A7 strain had 99.81% similarity with the Anoxybacillus flavithermus, and therefore identified as A. flavithermus A7 strain. Further, the growth curve analysis indicated that the A7 strain grew well at 60°C. The 3, 5-dinitrosalicyclic acid (DNS) assay showed the crude enzyme secreted by the A7 strain exhibited optimum amylase activity at 60°C with 8.6 x 10-2 U/ml. This is the first APB obtained from hot springs in Sabah and promising for further studies and applications.

Sphingobacterium faecale sp. nov., a 1-aminocyclopropane-1-carboxylate deaminase producing bacterium isolated from camel faeces.

An investigation of the diversity of 1-aminocyclopropane-1-carboxylate deaminase producing bacteria associated with camel faeces revealed the presence of a novel bacterial strain designated C459-1T. It was Gram-stain-negative, short-rod-shaped and non-motile. Strain C459-1T was observed to grow optimally at 35 °C, at pH 7.0 and in the presence of 0 % NaCl on Luria-Bertani agar medium. The cells were found to be positive for catalase and oxidase activities. The major fatty acids (>10 %) were identified as iso-C15 : 0, summed feature 3 (C16 : 1 ω6c and/or C16 : 1 ω7c) and iso-C17 : 0 3-OH. The predominant menaquinone was MK-7. The major polar lipids consisted of phosphatidylethanolamine, one sphingophospholipid, two unknown aminophospholipids, three unknown glycolipids and five unknown lipids. The genomic DNA G+C content was 40.3 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C459-1T was affiliated with the genus Sphingobacterium and had the highest sequence similarity to Sphingobacterium tabacisoli h337T (97.0 %) and Sphingobacterium paucimobilis HER1398T (95.6 %). The average nucleotide identity and digital DNA-DNA hybridization values between strain C459-1T and S. tabacisoli h337T were 83.8 and 33.8 %, respectively. Phenotypic characteristics including enzyme activities and carbon source utilization differentiated strain C459-1T from other Sphingobacterium species. Based on its phenotypic, chemotaxonomic and phylogenetic properties, strain C459-1T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium faecale sp. nov. is proposed, with strain is C459-1T (CGMCC 1.18716T=KCTC 82381T) as the type strain.

Optimizing Bioremediation: Elucidating Copper Accumulation Mechanisms of Acinetobacter sp. IrC2 Isolated From an Industrial Waste Treatment Center.

Acinetobacter sp. IrC2 is a copper-resistant bacterium isolated from an industrial waste treatment center in Rungkut, Surabaya. Copper-resistant bacteria are known to accumulate copper inside the cells as a mechanism to adapt to a copper-contaminated environment. Periplasmic and membrane proteins CopA and CopB have been known to incorporate copper as a mechanism of copper resistance. In the present study, protein profile changes in Acinetobacter sp. IrC2 following exposure to copper stress were analyzed to elucidate the copper resistance mechanism. Bacteria were grown in a Luria Bertani agar medium with and without CuSO4 supplementation. Intracellular copper ion accumulation was quantified using atomic absorption spectrophotometry. Changes in protein profile were assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results showed that 6 mM CuSO4 was toxic for Acinetobacter sp. IrC2, and as a response to this copper-stress condition, the lag phase was prolonged to 18 h. It was also found that the bacteria accumulated copper to a level of 508.01 mg/g of cells’ dry weight, marked by a change in colony color to green. The protein profile under copper stress was altered as evidenced by the appearance of five specific protein bands with molecular weights of 68.0, 60.5, 38.5, 24.0, and 20.5 kDa, suggesting the presence of CopA, multicopper oxidase (MCO), CopB, universal stress protein (Usp), and superoxide dismutase (SOD) and/or DNA-binding protein from starved cells, respectively. We proposed that the mechanism of bacterial resistance to copper involves CopA and CopB membrane proteins in binding Cu ions in the periplasm and excreting excess Cu ions as well as involving enzymes that play a role in the detoxification process, namely, SOD, MCO, and Usp to avoid cell damage under copper stress.

Highly Cr(vi)-tolerant Staphylococcus simulans assisting chromate evacuation from tannery effluent

Abstract Chromium(vi) contaminated sites have been targeted for studying highly chromate-resistant bacteria. From a total of 23 Cr(vi)-resistant bacteria isolated on Luria-Bertani agar medium supplemented with K2CrO4 (1,500 μg/mL), only one strain UT8 (Staphylococcus simulans) was able to tolerate high concentration of chromate, i.e., up to 200 mg/mL in agar medium from tannery effluent. In acetate minimal medium (AMM), it showed maximum tolerance of up to 2,500 μg/mL. Cr(vi) removal potential was 94.6% after 24 h (K2CrO4 1,500 μg/mL). Parametric conditions were optimized in AMM to attain maximum chromate removal. Exopolysaccharides extracted from bacterial cells exposed to chromate exhibited major absorption shifts from 2,500 to 500 cm−1 revealed by Fourier transform infrared spectroscopy. Energy-disperse X-ray spectroscopy further confirmed the adsorption of oxyanions to the bacterial cells. Surface topography of the Cr(vi) treated cells showed transformation into concave shape by scanning electron microscopy. The presence of resistance genes, i.e., chromate reductase (chrR) and class I integrase (intI1), further confirmed tolerance toward chromate. Microarray data analysis of transcriptional gene expression suggested upregulation of cys gene cluster under chromate exposure. Concisely, the present investigations revealed the potential of S. simulans to be an effective candidate for chromate reclamation of wastewater.

ISOLASI DAN KARAKTERISASI ISOLAT BAKTERI RESISTEN TEMBAGA DARI SUNGAI CISADANE

The water quality of the Cisadane River, located in the industrial factory area, it is contaminated with copper, it is necessary to treat sewage. Biological wastewater treatment using bacteria is a more economical and effective method of removing copper from the environment than chemical methods. This study aims to isolate, identify, determine the level of resistance, and analyze the ability of bacteria to accumulate copper. Isolation was carried out by the spread method in Luria Bertani Agar medium containing CuSO4. Bacterial isolates were identified based on analysis of the 16S rDNA gene. The resistance level is determined based on the Minimum Inhibitory Concentration (MIC) value. The accumulation ability of copper was determined using an atomic absorption spectrophotometer. Six bacteria resistant to copper were isolated and identified as Pantoea agglomerans strain IrCis1, Klebsiella grimontii strain IrCis3, Klebsiella penumoniae strain IrCis4, Shigella flexneri strain IrCis5, Enterobacter cloacae strain IrCis6, and Enterobacter cloacae strain IrCis9 with MIC of 7 mM - 9 mM. Pantoea agglomerans strains IrCis1, Klebsiella grimontii strains IrCis3, Klebsiella pneumoniae strains IrCis4 can accumulate copper of 1,19 mg, 1,39 mg, and 0,92 mg, respectively, per gram of dry cell weight.

First report of stalk bacterial soft rot of sugarcane caused by Dickeya zeae in China.

In late September 2019, seven stalks of about 1400 stalks of sugarcane cultivar Zhongzhe 1 exhibited soft rot symptoms in a trial plot in Beihai city, Guangxi province of China. Symptoms included scorched and collapsed leaves, maceration of stalks, and sour smelling exudates from the stalks (Supplementary Fig. S1). Severely diseased stalks had collapsed and were dead. Internal stalk fragments of 5 × 5 mm were collected at the junction of healthy and diseased tissue after surface-sterilization of stalks with 70% ethanol for one minute, and three times rinsing with sterile distilled water. Stalk fragments were placed on Luria-Bertani agar medium (1 % w/v tryptone, 0.5 % w/v yeast extract, 1 % w/v NaCl, 1 % w/v agar, pH7.0) and plates were put in an incubator at 30°C for 48h. Four types of bacterial colonies were obtained, and small and white colonies with irregular margins were the most dominant. A single colony of each type was diluted in sterile distilled water and aliquots of each suspension were streaked on fresh medium plates to obtain pure cultures. Ten eight-week-old stalks (11 th leaf stage) of sugarcane plants, which derived from cuttings of symptomless cultivar Zhongzhe 1, were inoculated by injection of 300 μl of bacterial suspension (3.5x108 CFU/ml) into the stalks. Another 10 stalks were injected with pure water and served as control. The inoculated plants were kept in a greenhouse at 25-37℃.Among the four types of bacteria, only strain BH9 induced symptoms that were identical to those of diseased canes observed in the field (Supplementary Fig. S1). Elongated water-soaked lesions were observed around the inoculation sites three days post inoculation. Five of the 10 BH9-inoculated plants had collapsed two days later. Water-soaked stalks had a sour smell similar to the filed diseased plants. Eight days post inoculation, all BH9-inoculated plants exhibited symptoms but control plants remained symptomless up to 30 days after inoculation. Uniform white colonies with irregular margins were isolated from the inoculated stalks that developed soft rot symptom, and these bacteria caused again stalk soft rot symptoms when inoculated to a new batch of 10 healthy plants. The 16S rRNA gene of strain BH9 was amplified by PCR with primer pair fD2/rP1 and the PCR amplicons from three independent colonies were sequenced. The sequences of the three amplicons were identical (Accession No. MT723897). BLAST alignments of the 16S rDNA sequence from BH9 strain with the GenBank database revealed that BH9 belonged to the genus Dickeya (98.5% identity between D. zeae BH9 and D. zeae EC1). Further PCR assays and sequencing of three genes, DNA polymerase III gamma subunit gene dnaX with primers dnaXf/dnaXr, DNA gyrase gene gyrB with primers gyrBf1/gyrBr1, and recombinase A gene recA with primers recAf/recAr, were performed to identify the species within the genus Dickeya (Zhang et al., 2014). BH9 sequences of these genes (Accession No. MT723898 to MT723900) had highest identity (97.5%, 97.6%, and 97.7%, respectively) with those from D. zeae EC1 (GenBank accession No. CP006929.1). To determine the evolutionary relationship of BH9 to other Dickeya species and strains, a phylogenetic analysis was performed using dnaX, gyrB, and recA sequences. As shown in Supplementary Fig. S2, BH9 clustered with D. zeae strains and formed a lineage distinguishable from other Dickeya species. Among the closest strains, D. zeae NCPPB3531 (Accession No. CM001980.1) was isolated from potato and D. zeae CSL RW192 (Accession No. CM001972.1) from river water (Pritchard et al., 2013). Consequently, strain BH9 was identified as D. zeae. This bacterial species has been reported to cause soft rot in rice (Pu et al., 2012), banana (Zhang et al., 2014), maize (Martinez-Cisneros et al., 2014), and clivia (Hu et al., 2018). To the best of our knowledge, this is the first report of a bacterial stalk rot caused by D. Zeae in sugarcane. In fact, low incidence of D. zeae-caused stalk soft rot was recently found in sugarcane fields in Fusui County, about 150 km north to Beihai. Given the potential threat of this disease to the local sugarcane industry, the mode of transmission, cultivar resistance, and measures to control the disease should be investigated.

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.

Potensi Mikroorganisme Resisten dari Pantai Timur Surabaya Sebagai Biosorben Seng

East coast of Surabaya is one of region in Indonesia which contaminated by heavy metals, such as zinc. Zinc is one of essential heavy metals for organisms but be dangerous for human if comsumed in high concentration. So, it is important to to solve the problem to reduce the concentration of zinc in east coast of Surabaya through bioremediation using microorganism. Zinc resistant microorganism could be isolated from zinc contaminated region and be used for bioremediation agent because some microorganism developed resistance mechanism to zinc by bioaccumulation or biosorption. The aims of this study are to isolate and characterize zinc resistant microorganism from east coast of Surabaya. The most resistant microorganism will be observed for the growth in medium supplemented with zinc and without zinc. The potency of the chosen isolate to biosorb of zinc was also analyzed. Water sample from east coast of Surabaya was serries dilluted and innoculated on Luria Bertani Agar medium using spread plated method. Microbial resistance to zinc was analyzed by grown the cell on medium containing various concentration of zinc to determine Minimum Inhibitory Concentration (MIC) value of each microbial isolate. Cell turbidity will be observed in the chosen isolate using spectrophotometer in wave length of 600 nm to get the growth curve. The potency of the chosen isolate to biosorb of zinc was analyzed using atomic absorption spectrophotometer. From this study, six bacteria and three yeast have been isolated from east coast of Surabaya, designated as PmbZn1, PmbZn2, PmbZn3, PmbZn4, PmbZn5, PmbZn6, PmbZn7, PmbZn8, dan PmbZn9. All isolates could grow on medium containing 8 mM ZnSO4. The most resistant bacterial was isolate PmbZn5 with the MIC of 9 mM ZnSO4. The potency of the isolate to biosorb of zinc was 94,79 and 93,57% in medium containing 4 mM and 7 mM ZnSO4, respectively.Key words: Bacteria, biosorb, resistant, yeast, zinc.

Poly(vinyl pyrrolidone)-mediated synthesis of silver nanowires decorated with silver nanospheres and their antimicrobial activity

Pathogenic infectious diseases, like bacterial infections, are one of the most prevalent types of diseases all over the world. Although antibiotics are commonly used and widely available drugs for the treatment of bacterial infections, bacteria show multiple drug resistance, have a tendency to get genetically mutated and become resistant to commonly employed antibiotic drugs. This makes the invention of novel drugs essential. One of the possible approaches is the use of nanomaterials for this purpose. The present study attempts to synthesize silver nanospheres and silver nanowires and compare their antibacterial activity. Silver nanospheres were synthesized by chemical reduction of silver nitrate in the presence of a suitable stabilizer. For the synthesis of silver nanowires, polyol method was employed. Silver nanowires neatly decorated with silver nanospheres were obtained. The antibacterial activity was estimated by separately determining the minimum inhibition concentration of the two nanostructures on Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli cultured on Luria Bertani agar media. The comparison shows that the antibacterial activity of silver nanospheres is better than that of silver nanowires which is attributed to its higher surface area and the difference in uptake mechanism by the bacterial cells.

Use of Maldi-Tof MS biosensor in microbial assessment of Brazilian kefir grains

ABSTRACT The aim of this study was to evaluate the use of Maldi-Tof MS biosensor in microbial assessment of Brazilian kefir grains. Maldi-Tof MS is a new methodology for the rapid diagnosis of microorganisms. A total of 358 microorganisms were isolated, 31 were yeasts and 327 were bacteria (divided into lactic and acetic bacteria). Microbial colonies were grown in Luria-Bertani agar medium and incubated at 35 °C for 18h and used in the identification of species by Maldi-Tof MS. The microbial population identified in Brazilian kefir grains was Lactobacillus paracasei, Saccharomyces cerevisiae, Lactobacillus plantarum, Acetobacter pasteurianus, and Acetobacter syzygii. This study demonstrated a rapid and accurate identification of the Brazilian kefir grains microorganisms using the Maldi-Tof MS biosensor. In conclusion, the Maldi-Tof MS technology can facilitate the microbiological control in a fermentation process using kefir grains as starter cultures.