Citrate Utilization Research Articles

Isolation and Antimicrobial Susceptibility Patterns of Klebsiella Spp. from Hund Pumped Sources Drinking Water in Kola Tembien, Central Tigray, Northern Ethiopia

Klebsiella Spp. is a common environmental human and animal associated microorganism that has become a major cause of nosocomial infections worldwide. The objective of the study was to isolate Klebsiella Spp. and determine the resistance patterns of the organism. Seventy-five hand pump fitted boreholes were selected randomly. Water samples were collected aseptically. Biochemical tests were performed on colonies from macConkey agar No.3 (Oxoid.LTD) for final identification of the isolates. Isolates from all culture positive water samples were identified based on the standard cultural and morphological characteristics in couple with indole production, H2S production, gas production, citrate utilization, motility test, lysine decarboxylation, lysine deamination and carbohydrate utilization tests. Antimicrobial susceptibility test isolates were done against Amikacin, Ampicillin, Ceftriaxone, Chloramphenicol, Ciprofloxacin, Tetracycline and Gentamicin using Kirby-Bauer Disk Diffusion. Among the total of water sampled from hand pump fitted boreholes, five water (6.6%) samples were positive for Klebsiella spp. and Among the five isolated of Klebsiella spp. from water sampled, 2 of them were resistance to Ampicillin and one was resistance to Tetracycline. This study showed that there were Klebsiella spp. from ground water which were resistance to Ampicillin and Tetracycline. Since community acquired antibacterial resistance of Klebsiella spp . has a great public health importance, researchers should give attention like hospital acquired one. Keywords : Klebsiella spp. antimicrobial resistance DOI : 10.7176/JHMN/59-02

Profiling marine bacterial dioxygenase gene involved in Polycyclic Aromatic Hydrocarbon degradation

Abstract. Mishra M, Das S. 2017. Profiling marine bacterial dioxygenase gene involved in Polycyclic Aromatic Hydrocarbon degradation. Ocean Life 1: 32-40. The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely reported. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, little is known regarding the diversity of microorganism involved in PAH degradation in the environment. The aim of this study was characterization of the gene for enzymes involve in PAHs Degradation, and to make a bank collection of strains for further screening research. Pure bacterial cultures were sampled from a highly enriched consortium for biodegradation analysis. Bacterial strains capable of degrading pyrene and anthracene were isolated from Paradeep estuary (Odisha, India) water sample by selective enrichment. Five strains of pyrene and anthracene degrading pure cultures were collected, named as MP-1, MP-4, MP-9, MP-14, MP-18. Isolates were characterized by gram staining, utilization of citrate, sugar fermentation, swimming and swarming motility and antibiotic sensitivity test. In seven days isolates MP-1,MP-4,MP-9,MP-14,MP-18 shown to degrade 58.4%, 42.1%, 29.1%, 31.7%, 31.8% of pyrene at a concentration 100 mg/l and 56.3%, 46%, 44.6%, 30.4%, 48.2% of anthracene at a concentration100mg/l. Polymerase chain reaction (PCR) with PAH-specific primers successfully amplifies a dioxygenase gene in MP-4. The presence of dioxygenase gene may lead to unraveling the underlying mechanism on how bacteria develop the abilities to degrade high-molecular-weight PAH. The understanding to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful way for both assessing bioremediation potential in the environment and the discovery of novel dioxygenase genes.

First Report of Clavibacter michiganensis subsp. michiganensis Infecting Potato in Russia

Yellow-pigmented clavibacteria were isolated from diseased potato plants and tubers from five regions of Russia between 2011 and 2017. Severe outbreaks of the disease were recorded in the central and northwest parts of the Russian Federation. Plants exhibited yellowing and necrosis of the leaf and wilting of whole leaves and plants, and brown veins around potato tuber eyes were visible on cross sections. Vascular tissue was removed from discolored nodes, crushed in sterile 10 mM phosphate buffer, and dilution plated on yeast extract dextrose calcium carbonate agar. Creamy yellow colonies developed from the extracts after 7 days incubation at 26°C. Isolates from each diseased potato plant (35 isolates deposited to the State Collection of Phytopathogenic Microorganisms at Russian Research Institute of Phytopathology, Bolshie Vyazemy) were identified as Clavibacter michiganensis subsp. michiganensis (Cmm) using taxon-specific polymerase chain reaction (PCR) with primers CMM5/CMM6 that gave expected product of 614 bp (Dreier et al. 1995). Additionally, isolates were positive for Cmm and negative for C. michiganensis subsp. sepedonicus using a commercial ELISA test (Agdia Biofords, France). Bacteria were gram positive aerobic rods with biochemical traits uniform and similar to the type strain Cmm NCPPB2979T (Schaad et al. 2001). All isolates were positive for esculin hydrolysis, gelatin liquefaction, H₂S production from peptone, utilization of citrate and succinate, and acid from d(+)mannose, and negative for starch hydrolysis, casein hydrolysis, methyl red reaction, acid from inulin, mannitol, d(+)melezitose and d(–)sorbitol, and utilization of acetate, formate, lactate, propionate, and ribose. Sequencing of the 16S rRNA (Weisburg et al. 1991) and gyrB gene (Zaluga et al. 2011) was performed for all 35 isolates. 16S rRNA sequencing (GenBank accession nos. MH035728.1 to MH035762.1) showed a >99% relatedness to Cmm type strain NCPPB2979ᵀ. gyrB gene analysis grouped the new potato-isolated strains (GenBank accession nos. MH042254 to MH042288) with Cmm strains from tomato obtained from the same regions. Pathogenicity of four typical isolates was tested on tomato (cv. Dubok) and potato (cv. Lion Heart) plants using suspensions of about 10⁸ cells/ml in sterile tap water. Four to six plantlets with two to four fully developed leaves were inoculated by stem infiltration (Zaluga et al. 2013) with 10 µl of bacterial suspension and were placed in a growth chamber at 20 to 25°C. The type strain of Cmm from tomato was used as a pathogenic control. The first symptoms appeared 14 days after inoculation and progressed slowly. In both tomato and potato plantlets, the four Cmm isolates displayed chlorosis of leaf margins, mottling and yellowing between veins, wilting or necrosis of individual leaf parts, and finally wilting of whole leaves. Each isolate was recovered from infected test plants and reidentified as Cmm by PCR with primers CMM5/CMM6. No symptoms were observed on negative control plants inoculated with sterile water and on potato inoculated with the Cmm type strain. 16S rRNA gene (GenBank accession nos. MH035728.1 to MH035762.1) showed a >99% relatedness to Cmm type strain NCPPB2979ᵀ. This is the first report of Cmm affecting potato in Russia.

Aerococcus urinae and Globicatella sanguinis Persist in Polymicrobial Urethral Catheter Biofilms Examined in Longitudinal Profiles at the Proteomic Level.

Aerococcus urinae (Au) and Globicatella sanguinis (Gs) are gram-positive bacteria belonging to the family Aerococcaceae and colonize the human immunocompromised and catheterized urinary tract. We identified both pathogens in polymicrobial urethral catheter biofilms (CBs) with a combination of 16S rDNA sequencing, proteomic analyses, and microbial cultures. Longitudinal sampling of biofilms from serially replaced catheters revealed that each species persisted in the urinary tract of a patient in cohabitation with 1 or more gram-negative uropathogens. The Gs and Au proteomes revealed active glycolytic, heterolactic fermentation, and peptide catabolic energy metabolism pathways in an anaerobic milieu. A few phosphotransferase system (PTS)–based sugar uptake and oligopeptide ABC transport systems were highly expressed, indicating adaptations to the supply of nutrients in urine and from exfoliating squamous epithelial and urothelial cells. Differences in the Au vs Gs metabolisms pertained to citrate lyase and utilization and storage of glycogen (evident only in Gs proteomes) and to the enzyme Xfp that degrades d-xylulose-5′-phosphate and the biosynthetic pathways for 2 protein cofactors, pyridoxal 6′-phosphate and 4′-phosphopantothenate (expressed only in Au proteomes). A predicted ZnuA-like transition metal ion uptake system was identified for Gs while Au expressed 2 LPXTG-anchored surface proteins, one of which had a predicted pilin D adhesion motif. While these proteins may contribute to fitness and virulence in the human host, it cannot be ruled out that Au and Gs fill a niche in polymicrobial biofilms without being the direct cause of injury in urothelial tissues.

Isolation and Characterization of an Entophytic Ethanol Resistant Bacterium from Sap of <i>Saccharum officinarum</i> for Efficient Fermentation

Bio fuel is one of the emerging industries around the world due to arise of crisis in petroleum fuel. Fermentation is a cost effective and eco-friendly process in production of bio-fuel. One major problem in microbial ethanol fermentation is the low resistance of conventional microorganisms to the high ethanol concentrations, which ultimately lead to decrease in the efficiency of the process. In the present investigation, an ethanol resistant bacterium was isolated from sap of Saccharum officinarum (sugar cane). The optimal cultural conditions such as, temperature, incubation period, and microbiological characteristics, morphological characteristics, biochemical characteristics, ethanol tolerance, sugar tolerance were investigated. Isolated microorganism was tolerated to 18% (V/V) of ethanol concentration in the medium and 35% (V/V) glucose concentration in the medium. Biochemical characteristics have revealed as Gram negative, non-motile, negative for Indole test, Methyl Red test, Voges- Proskauer`s test, Citrate Utilization test, and Urease test and positive results for Oxidase test. Sucrose, Glucose, Fructose, Maltose, Dextrose, Arabinose, Raffinose, Lactose, and Sachcharose can be utilized by this particular bacterium. It is a significant feature in effective fermentation. The fermentation process was carried out in glucose medium under optimum conditions; temperature 30°C, and incubated for 72 hours. Maximum ethanol production was recorded as 12.8±0.4% (V/V). Methanol was not detected throughout the fermentation process. This bacterium is especially useful in bio-fuel production due to high ethanol tolerance of this microorganism; it can be used to enhance the fermentation process over conventional microorganisms. Investigations are currently conducted on establishing the identity of the bacterium.

Characterization of Ralstonia solanacearum (Smith) Race1, Causing Bacterial Wilt of Brinjal

Ralstonia solanacearum (Smith) Yabuuchi et al. is one of the most severe pathogen of Solanaceous crops with a wide host range. Ralstonia solanacearum race 1 is responsible for causing bacterial wilt in brinjal. The pathogenic strains were isolated from wilted brinjal plants and isolated on triphenyl tetrazolium chloride (TTC) medium. The strains were confirmed by proving pathogenicity on brinjal plants. Phenotypic characterization revealed that out of five media tested TTC medium was an excellent source for growth and extracellular polysaccaride production (EPS) of R. solanacearum. Upon biochemical analysis the R. solanacearum strains showed positive reaction for catalase, KOH, citrate utilization and acid production while negative results were observed for Gram staining, starch hydrolysis and fluorescent pigment production. The biovar of the strain was identified by utilization of disaccharides (cellobiose, maltose and lactose) and sugar alcohols (dulcitol, mannitol and sorbitol). The strains of R. solanacearum were confirmed to belong to Race 1 based on symptom production on different hosts and hypersensitive response on tobacco plant. Therefore, it is confirmed that R. solanacearum race 1 causing bacterial wilt of brinjal belongs to biovar III. PCR amplification of ITS region of the strains of R. solanacearum was done and sequenced [MH481663 (ERY1) and MH481664 (VBN1)].

Identification of Four Thermophilic Geobacillus Isolates from Hammam Pharaon, Sinai, Egypt

The present study was conducted to identify thermophilic bacteria from local habitats and to test their ability for producing thermostable α-Amylase, cellulase, lipase and protease in order to pave the way for their use in industrial processes. Out of 29 isolates initially obtained mainly from Hammam Pharaon, four potential isolates were selected based on thermo-tolerance ability and enzyme activity. The four isolates were characterized using phenotypic and 16S rRNA sequencing which recognized the isolates as Geobacillus. All four isolates were found to be variable for indole test, methyl red and rod shaped morphology. However, they were found to be variable for citrate utilization, catalase test starch hydrolysis, gelatin hydrolysis, casein hydrolysis, and Tween 80 hydrolysis. These bacteria grow at temperatures ranging from 50 to 90°C and the pH ranged from 6 to 9. This study added a lot to our knowledge about the thermophilic bacteria inhabiting Hammam Pharaon and their characters that would open the door for their use in biotechnological applications..

Characterization of rhizobia from root nodule and rhizosphere of Vicia faba in Algeria

Nitrogen fixation resulting from mutual symbiosis of rhizobia and cultivated legume plants is therefore critical to food security as it directly affects agricultural production. Biological Nitrogen Fixation (BNF) can be an important factor in sustainable agriculture. A collection of 20 isolates from fresh Nodules of the legume plant Vicia faba was isolated from five soil of Algeria. The soil from each region has undergone physical-chemical analysis : (granulometry, total carbon, organic matter, pH, and electrical conductivity, determination of available phosphorus and determination of total nitrogen). A media viz., Glucose-Peptone Agar (GPA), Congo red, Yeast Mannitol Agar (YMA) containing 2% NaCl were employed to make presumptive decisions on the recognition and classification of the isolated bacterial strains. All the isolates were found with poor absorption of dye Congo red and little or no growth on the media of GPA and without altering the pH. Almost all of the isolates exhibit good growth on 2% NaCl, poor growth on GPA, thus confirming the rhizobia. After biochemical tests like catalase test and citrate utilization test isolates were confirmed. The presence of rhizobia on root nodules of leguminous plant.

Innovation in an E. coli evolution experiment is contingent on maintaining adaptive potential until competition subsides.

Key innovations are disruptive evolutionary events that enable a species to escape constraints and rapidly diversify. After 15 years of the Lenski long-term evolution experiment with Escherichia coli, cells in one of the twelve populations evolved the ability to utilize citrate, an abundant but previously untapped carbon source in the environment. Descendants of these cells became dominant in the population and subsequently diversified as a consequence of invading this vacant niche. Mutations responsible for the appearance of rudimentary citrate utilization and for refining this ability have been characterized. However, the complete nature of the genetic and/or ecological events that set the stage for this key innovation is unknown. In particular, it is unclear why it took so long for citrate utilization to evolve and why it still has evolved in only one of the twelve E. coli populations after 30 years of the Lenski experiment. In this study, we recapitulated the initial mutation needed to evolve citrate utilization in strains isolated from throughout the first 31,500 generations of the history of this population. We found that there was already a slight fitness benefit for this mutation in the original ancestor of the evolution experiment and in other early isolates. However, evolution of citrate utilization was blocked at this point due to competition with other mutations that improved fitness in the original niche. Subsequently, an anti-potentiated genetic background evolved in which it was deleterious to evolve rudimentary citrate utilization. Only later, after further mutations accumulated that restored the benefit of this first-step mutation and the overall rate of adaptation in the population slowed, was citrate utilization likely to evolve. Thus, intense competition and the types of mutations that it favors can lead to short-sighted evolutionary trajectories that hide a stepping stone needed to access a key innovation from many future generations.

Safety assessment and functional properties of four enterococci strains isolated from regional Argentinean cheese

The members of the Enterococcus genus are widely distributed in nature. Its strains have been extensively reported to be present in plant surfaces, soil, water and food. In an attempt to assess their potential application in food industry, four Enterococcus faecium group-strains recently isolated from Argentinean regional cheese products were evaluated using a combination of whole genome analyses and in vivo assays. In order to identify these microorganisms at species level, in silico analyses using their newly reported sequences were conducted. The average nucleotide identity (ANI), in silico DNA-DNA hybridization, and phylogenomic trees constructed using core genome data allowed IQ110, GM70 and GM75 strains to be classified as E. faecium while IQ23 strain was identified as E. durans. Besides their common origin, the strains showed differences in their genetic structure and mobile genetic element content. Furthermore, it was possible to determine the absence or presence of specific features related to growth in milk, cheese ripening, probiotic capability and gut adaptation including sugar, amino acid, and peptides utilization, flavor compound production, bile salt tolerance as well as biogenic amine production. Remarkably, all strains encoded for peptide permeases, maltose utilization, bile salt tolerance, diacetyl and tyramine production genes. On the other hand, some variability was observed regarding citrate and lactose utilization, esterase, and cell wall-associated proteinase. In addition, while strains were predicted to be non-human pathogens by the in silico inspection of pathogenicity and virulence factors, only the GM70 strain proved to be non-virulent in Galleria mellonella model. In conclusion, we propose that, in order to improve the rational selection of strains for industrial applications, a holistic approach involving a comparative genomic analysis of positive and negative features as well as in vivo evaluation of virulence behavior should be performed.

Rhizobium sp.CCNWYC119: a single strain highly effective as biofertilizer for three different peas (Pigeon pea, Sweet pea and Chick pea)

Rhizobium spp. was isolated from root nodules of Pigeon pea (Cajanus cajan L.), Sweet pea (Lathyrus sativus L.), Chick pea (Cicer arietinum L.). The isolates ware rod shaped, aerobic, gram negative, motile and non-spore forming. Isolates were positive to Catalase, Citrate utilization, Urea hydrolysis, Congored, Nitrification, Oxidase, Triple sugar iron and MacConkey agar test. The isolates can ferment all nine sugars. Then, the isolates identified as Rhizobium spp. depending on above results were subjected to 16S rRNA sequencing for further confirmation and identification. Surprisingly, the isolates were same strain or member of same cluster of Rhizobium and identified as Rhizobium sp.CCNWYC119 strain based on 16S rRNA sequence (98% similarity). Then, different parameters of soil quality enrichment and plant growth viz. plant height; weight of pods and seeds; number, fresh and dry weight of nodules were studied to test the efficacy of the isolate as biofertilizer. Here, inoculant of Rhizobium sp. isolated from Pigeon pea was used as biofertilizer. The results showed the significant increase of nodulation, enrichment of soil of rhizosphere, plant growth and yield for all three types of inoculated peas as compared with non-inoculated control peas indicating that the isolated strain could be used as a common efficient biofertilizer for Pigeon pea, Sweet pea and Chick pea. It was also found that the isolate grew optimally at temperature 28°C and pH 7.0. Moreover, the isolate was sensitive to the higher concentration of NaCl (>1%) and to antibiotics- Mecillinam, Ciprofloxacin, Cotrimoxazole, Pefloxacin, Ceftazidime and Tetracycline.

EFFECTS OF STATIC MAGNETIC FIELD ON Escherichia coli MUTATION

The effects of a dipolar static magnetic field of strength 400, 800, 1200 and 1600 Gauss were prepared locally, on the ultra-structure of E. coli type 1 bacterium cells have been studied. Equal volumes of liquid culture media were exposed to the magnetic field for different periods, the three most effective periods, namely: 24 hrs, 48 hrs and 72 hrs were chosen for all our experimental studies. After API kit test of treated E. coli culture media as control group. Results indicated that exposure of the microorganisms to the demonstrated magnetic field caused pronounced changes in the Arginine dihydrolase (ADH), Citrate utilization (CIT) and Gelatinase (GEL) were observed on the cell growth. Besides, changes in the morphology of the E. coli colonies were observed after exposure period and subculture on MacConkey agar. Furthermore, the bacterial growth subculture tested for morphological and biological activity, the results suggested that a mutation occurred in bacterial cells.

Sphingobacterium bovisgrunnientis sp. nov., isolated from yak milk.

A novel Gram-negative, rod shaped, non-motile bacterium, designated strain YK2T, was isolated from yak milk from Leh, India. The strain was positive for oxidase- and catalase-activities and negative for starch hydrolysis, nitrate reduction, citrate utilization, urease, lysine decarboxylase and ornithine decarboxylase activities. The predominant fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, iso-C17 : 1ω9c and C16 : 1ω7c and/or C16 : 1ω6c and/or iso-C15 : 0 2-OH (summed feature 3). The major polar lipids were phosphatidylethanolamine, one unidentified aminophospholipid and six unidentified lipids. The DNA G+C content of the strain was 38.9 mol%. The 16S rRNA gene sequence analysis indicated that strain YK2T was a member of the genus Sphingobacterium and closely related to Sphingobacterium alimentarium and Sphingobacterium composti with pair-wise sequence similarity of 98.3 and 97.9 %, respectively. The sequence similarity to other members of the genus Sphingobacterium was between 92.6 to 96.3 %. Phylogenetic analysis showed that strain YK2T clustered with Sphingobacterium alimentarium and together clustered with Sphingobacterium composti. DNA-DNA hybridization of strain YK2T with Sphingobacterium alimentarium WCC 4521T and Sphingobacterium composti T5-12T showed a relatedness of only 38 and 54 %, respectively. Based on the phenotypic characteristics and on phylogenetic inference, it appears that strain YK2T represents a novel species of the genus Sphingobacterium, for which the name Sphingobacterium bovisgrunnientis sp. nov. is proposed. The type strain of Sphingobacterium bovisgrunnientis sp. nov. is YK2T (=MTCC 12631T=KCTC 52685T=JCM 31951T).

Bacteriological Quality of Ready-to-Eat Meat (Smoked and <i>Kilishi</i>) Sold in Nigerian Markets

Nine different samples of ready to-eat meat comprising of three samples of smoked meat bought from hawkers in Ihiala market and three samples bought from hawkers in Danboyi market, Jos. Three samples of kilishi meat were purchased from West Mile market, Jos. These were analyzed to access the public health implications of eating vended ready-to-eat meat. Standard microbiological methods were used for isolation. Morphological and biochemical analysis were carried out on discrete colonies using pure cultures to establish their identities. The biochemical tests utilized include: catalase, oxidase, coagulase, citrate utilization, and urease tests. The result of this study revealed that a total of four Genera were isolated, characterized and identified. The isolates include: Pseudomonas, Staphylococcus, Bacillus and Proteus species. Meat from all three sources were contaminated with heterotrophic bacteria. Results revealed that ready-to-eat kilishi and smoked meat studied were grossly contaminated with bacteria. Aseptic precautions should be employed in the processing, packaging and preservation of ready-to-eat meat to avoid outbreak of diseases.

Probiotic Potential and Antibiotic Resistance of Lactic Acid Bacteria Isolated from Curd in Sri Lanka

Lactic acid bacteria (LAB) are organisms beneficial to the human beings as probiotics which the organism can remain viable under stress conditions in the gastrointestinal tract.The aim of this study was to characterize and evaluate probiotic properties, and antibiotic resistance of thirteen isolates of lactic acid bacteria obtained from curd. In order to characterize the lactic acid bacteria isolates from curd; Gram’s staining, endospore staining, motility, catalase reaction, indole production, methyl red test, Voges-Proskauer test, citrate utilization, oxidase activity, urease activity, H2S production, arginine hydrolysis, growth at different temperatures, gas production from glucose and fermentation pattern of different carbohydrates were performed. In order to screen the probiotic potential of the isolates; tolerance to pH, bile, and phenol, survival in the presence of simulated gastric and pancreatic juices were examined. Antibiotic resistance of selected isolates was evaluated using 12 antibiotics. Results of biochemical tests and physiochemical tests revealed that all thirteen isolates (ITI_CD001, ITI_CD 002, ITI_CD 003, ITI_CD 004, ITI_CD 005,ITI_CD006, ITI_CD 009, ITI_CD 010, ITI_CD 011, ITI_CD 012 ITI_CD013, ITI_CD 014, andITI_CD 015) are belonged to the genus Lactobacillus. Species were identified as Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus delbrukeii lactis. Most of the isolates were able to tolerate and grow well under low pH conditions (1.5 - 3.0), high pH (9.0), extremes of bile up to 0.5% and phenol up to 0.6%.The isolates were able to growin the presence of simulated gastric and pancreatic juices too. Results for the antibiotic resistance varied among isolates. Isolates ITI_CD004, ITI_CD 009, ITI_CD 010, ITI_CD 011, ITI_CD 012 and ITI_CD 015 were identified as higher probiotic isolates. The study revealed that the tested LAB isolates possess desirable probiotic properties. Hence, these isolates can be used to produce foods high in probiotics.

Pseudomonas fluvialis sp. nov., a novel member of the genus Pseudomonas isolated from the river Ganges, India.

A bacterial strain, designated ASS-1T, was isolated and identified from a sediment sample of the river Ganges, Allahabad, India. The strain was Gram-stain-negative, formed straw-yellow pigmented colonies, was strictly aerobic, motile with a single polar flagellum, and positive for oxidase and catalase. The major fatty acids were C16 : 1ω7c/ 16 : 1 C16 : 1ω6c, C18 : 1ω7c and C16 : 0. Sequence analysis based on the 16S rRNA gene revealed that strain ASS-1T showed high similarity to Pseudomonas guguanensis CC-G9AT (98.2 %), Pseudomonas alcaligenes ATCC 14909T (98.2 %), Pseudomonas oleovorans DSM 1045T (98.1 %), Pseudomonas indolxydans IPL-1T (98.1 %) and Pseudomonas toyotomiensis HT-3T (98.0 %). Analysis of its rpoB and rpoD housekeeping genes confirmed its phylogenetic affiliation and showed identities lower than 93 % with respect to the closest relatives. Phylogenetic analysis based on the 16S rRNA, rpoB, rpoD genes and the whole genome assigned it to the genus Pseudomonas. The results of digital DNA-DNA hybridization based on the genome-to-genome distance calculator and average nucleotide identity revealed low genome relatedness to its close phylogenetic neighbours (below the recommended thresholds of 70 and 95 %, respectively, for species delineation). Strain ASS-1T also differed from the related strains by some phenotypic characteristics, i.e. growth at pH 5.0 and 42 °C, starch and casein hydrolysis, and citrate utilization. Therefore, based on data obtained from phenotypic and genotypic analysis, it is evident that strain ASS-1T should be regarded as a novel species within the genus Pseudomonas, for which the name Pseudomonasfluvialis sp. nov. is proposed. The type strain is ASS-1T (=KCTC 52437T=CCM 8778T).

First Report of Pink Seed of Lucerne Caused by Erwinia rhapontici in China

Erwinia rhapontici, causal agent of pink seed of pea and other crops, was found in seed samples from commercial lucerne (Medicago sativa) seeds imported from Canada in 2002 and 2003. The seed samples were stored at 5oC and 55% moisture content at the Quality Supervision and Inspection Centre for Forage and Turf Grass Seeds, Agricultural Ministry, Lanzhou, China. In germination tests of July 2015, we observed seeds with brown lesions on the seed coat, and pale pinkish brown to bright pink discoloration throughout the seed coat. Isolations from 10 surface-sterilized pink seeds resulted in bacterial cultures that produced a water-soluble pink pigment on tryptone soy agar (TSA). All isolates were gram-negative, motile, facultative anaerobic rods with mucoid colonies and produced a pink pigment on TSA. They were positive for citrate utilization, catalase, methyl red, Voges-Proskauer, and acid production from: N-acetyl glucosamine, l-arabinose, amygdalin, arbutin, cellobiose, esculin (hydrolysis), d-fructose, d...

Prevalance of Extended Spectrum Beta Lactamases (ESBL) Producing Escherichia Coli Isolated From Clinical Samples at Tertiary Care Hospital Peshawar

Escherichia coli is Gram negative, facultative and non sporulating rod shaped bacteria. It is commonly inhabitant of the gastrointestinal tract of humans and animals. E. coli cause diseases like urinary tract infection cholecystitis, cholangitis and traveler’s diarrhoea and the UTI which is more prevalent worldwide. E. coli cause complication like Hemolytic Uremic Syndrome which leads to renal failure, Thrombotic Thrombocytopenic Purpura, septicemia and peritonitis. Extend spectrum beta lactamase enzyme produce by E. coli which capable of hydrolyzing first and third generation cephalosporin, and is inhibited by beta lactamase inhibitor. A total of 150 clinical samples (blood, urine, wound swab, body fluids) were collected from Post Graduate Lady Reading Hospital Peshawar. Different media used were Nutrient agar, MacConky agar and Cysteine, Lactose and Electrolyte-Deficient agar. E. coligive pink colonies on MacConky agar because it is lactose fermenter. For further confirmation different biochemical tests were performed like triple sugar iron, Indole, and citrate utilization tests. The antibiotics susceptibility and resistivity was checked by disk diffusion method and different antibiotics were used. For ESBL detection combined disk method was performed. In the clinical samples the percentage of Gram positive bacteria in blood was (20%), urine (14.2%), wound swab (83.3%), and body fluids (8%), and the Gram negative in urine was (80%), blood (7%), wound swab (10%), and body fluids (0%). E. coli was more prevalent in urine which was 25(35.71%) and ESBL producing E. coli was 5(20%). The ESBL producing E. coli was resistant to ciprofloxacin (100%), amikacin (40%), amoxicillin+clavulanic acid (40%), levofloxacin (80%), tazobactum+pipracilline (20%), gentamycin (100%), trimethoprim (60%), cefotaxime (100%) and meropenem (0%). Sensitivity toward levofloxacin (20%), tazobactum+pipracilline (80%), gentamycin (0%), trimethoprim (40%), cefotaxime (0%) and meropenem (100%), ciprofloxacin (0%), amikacin (60%), amoxicillin+clavulanic acid (60%). The most effective antibiotic against ESBL producing E. coli was Meropenem while least effective antibiotics against ESBL producing E. coli were Gentamycin and Ciprofloxacin.

Citrate, low pH and amino acid limitation induce citrate utilization in Lactococcus lactis biovar diacetylactis.

SummaryIn Lactococcus lactis subsp. lactis biovar diacetylactis, citrate transport is facilitated by the plasmid‐encoded citrate permease (CitP). In this work, we analysed regulation of citrate utilization by pH, nutrient limitation and the presence of citrate at four different levels: (i) plasmid copy number, (ii) citP transcription, (iii) citP mRNA processing and (iv) citrate utilization capacity. Citrate was supplied as cosubstrate together with lactose. The citP gene is known to be induced in cells grown at low pH. However, we demonstrated that transcription of citP was even higher in the presence of citrate (3.8‐fold compared with 2.0‐fold). The effect of citrate has been overlooked by other researchers because they determined the effect of citrate using M17 medium, which already contains 0.80 ± 0.07 mM citrate. The plasmid copy number increased in cells grown under amino acid limitation (1.6‐fold) and/or at low pH (1.4‐fold). No significant differences in citP mRNA processing were found. Citrate utilization rates increased from approximately 1 to 65 μmol min−1 gDW−1 from lowest to highest citP expression. Acetoin formation increased during growth in an acidic environment due to induction of the acetoin pathway. Quantification of the relative contributions allowed us to construct a model for regulation of citrate utilization in L. lactis biovar diacetylactis. This knowledge will help to select conditions to improve flavour formation from citrate.

Screening and Identification of Cellulase- Producing Bacteria Isolated From Rumen of Camel in Sokoto Main Abattoir

Cellulose is the most abundant polymers on the earth and was extensively used for many industrial purposes. The aim of the study was screening and isolation of cellulase producing bacteria from rumen of camel in slaughter house of Sokoto. A numbers of rumen content samples were collected from four different camels for analysis. Rumen cellulase producing bacteria were Isolated and characterized in this experiment following serial dilution up to six fold and inoculated into Nutrient agar. Biochemical test such citrate utilization, indole, catalase, urease, methyl red, vogues proskauer, motility, and sugar fermentation, starch hydrolysis, hydrogen sulphide production (H2S) from triple sugar iron (TSI) agar and sugar fermentation were adopt in this study. Screening of the isolates was performed in an enriched media containing Carboxymethyl cellulose (CMC) flooded with Congo red and washed with NaCl. The result revealed the colonial characterization such as shape, color, size, elevation margin, opacity, and gram reaction were determined for each of the isolates. A total of seven isolates were identified, six were identified as gram positive while one as gram negative. Bergy’s manual of determinative bacteriology confirmed the isolates to be genus of Bacillus spp. The mean total count of bacteria are 2.5 x 106 cfu/g, 2.8 x 106 cfu/g, 3.5 x 106 cfu/g, 3.6 x 106 cfu/g, 2.7 x 106 cfu/g, 3.5 x 106 cfu/g and 3.0 x 106 cfu/g. Five out of seven show CMCase activity after flooding with Congo red and are considered as RC3, RC4, RC5, RC6, and RC7 while two isolates RC1 and RC2 lack ability to produce cellulase RC7 show highest ability to produce cellulase with highest diameter of 6.0 mm. It has concluded that rumen content of camel is good source of carbon sole and cellulase producing bacteria.