Voges-Proskauer Research Articles

Microbiological and Biochemical Contamination Analysis of Refilled Drinking-water in Abeli, Kendari, Southeast Sulawesi

BACKGROUND: The access to safe drinkingwater is increasingly difficult, especially in water catchment or coastal areas. Due to the difficulty, people in coastal areas tend to consume ready-to-use refilled drinking-water. However, the sanitation facilities on most drinking-water refill depots do not meet the requirements, hence really prone to microbial contamination. We conducted this study to determine the types of bacteria causing the contamination of refilled drinking-water from refill depots in Abeli, a coastal area in Kendari.METHODS: Samples were collected from all 6 drinkingwater refill depots in Abeli. Three-series fermentation tubes were used for the microbiological test, including the presumptive, confirmative and completed tests. The biochemical test was performed with indole test, methyl red test, Voges-Proskauer test, and citrate utilization test (IMViC) methods and the triple sugar iron agar (TSIA) test.RESULTS: Among 6 refilled drinking-water samples, we found the growth of coliform bacterial colonies in 3 samples, exhibiting various characteristics. Based on the characteristics, both microbiological and biochemical, the identified bacteria were Proteus sp., Escherichia coli and Klebsiella oxytoca.CONCLUSION: Bacteria that contaminated refilled drinking-water in Abeli were Proteus sp., E. coli and K. oxytoca. Continuous supervision is required to ensure the quality of water from drinking-water refill depots.KEYWORDS: bacteria, Proteus sp., Escherichia coli, Klebsiella oxytoca, coastal areas, refilled drinking water

Isolation, screening and characterization of plant growth promoting rhizobacteria from rhizospheric soils of selected pulses

Many beneficial bacteria that colonize plant roots and facilitate plant growth to alleviate the stress posed by several biotic and abiotic factors through direct and indirect mechanisms are known as plant growth promoting rhizobacteria (PGPR). These PGPRs are recognized as efficient biofertilizers, biocontrol agents and microbial inoculants. Looking at the potential benefits of PGPRs in present study efforts have been made to isolate, screen and characterize rhizobacteria from the soil samples collected from the root zone of various pulses. Out of seven strains, six strains were methyl red positive and Voges-Proskauer's test positive indicating their fermentation ability. All seven strains except AV-1 produced catalase enzyme indicating their potential as promising biocontrol agents. AV-4 and AV-7 strains were found to be phosphate solubilizers and strains AV-1, AV-2 and AV-7 were siderophore producers. Two strains AV-3 and AV-5 were heavy metal tolerant for Arsenic (29.48 mg/l), Barium (12.56 mg/l) and Nickel (5.926 mg/l). Looking at the various traits of all the rhizobacterial isolates AV-7 can be considered as a potential biofertilizer to support the growth and production of the legumes in the soil which is not polluted with As, Ba and Ni. This will help in supporting sustainable pulse production.

Detection of carbapenem resistant Klebsiella species isolated from clinical samples

The dissemination of carbapenem resistance among the family of Enterobacteriaceae is one of the most potent intimidation to an individual’s health nowadays. In this research, the foremost focus is on carbapenemase-producing Klebsiella species. Carbapenems are a genre of beta-lactam ring containing antibiotics which includes meropenem imipenem, ertapenem, biapenem, doripenem & panipenem. Carbapenems are highly effective antibiotics as they arrest the cell wall of bacteria so if the resistance against carbapenem emerges than it will subsequently lead to narrow therapeutic options. The foremost purpose of this research is the determination of carbapenem resistance among Klebsiella species. Fifty (n=50) gram-negative samples were collected from the Centralized Diagnostic Laboratory of Karachi, Pakistan. All the samples were sub-cultured on EMB (eosin methylene blue) agar to differentiate them from other Enterobacteriaceaes. MacConkey agar was used to check lactose fermentation. Gram staining and capsule staining for morphological identification, biochemical tests (Indole, Methyl red, Voges Proskauer, Citrate, (IMViC), Triple Sugar Iron (TSI)) were carried out to differentiate among Klebsiella species. Carbapenem resistance against Imipenem and Meropenem was identified by the disc diffusion method based on the Clinical Laboratory Standard Institute (CLSI) guidelines. Out of which 26 isolates were identified as Klebsiella spp. In these 26 isolates, 10 isolates were identified as Klebsiella pneumoniae. Further results showed that K. pneumoniae isolates were more resistant to Meropenem as compared to the Imipenem. All ten isolates of K. pneumoniae were found resistant to Meropenem while only two isolates of K. pneumoniae were resistant to Imipenem. This study was done to determine the carbapenem resistance among Klebsiella so that data can be provided which will help in rapid and appropriate treatment of patients so that in the future we can use alternative drugs or cocktail of drugs to treat infections caused by Klebsiella. Keywords: Beta-lactamases; Carbapenems; Emergence; Klebsiella pneumoniae; Resistance http://dx.doi.org/10.19045/bspab.2020.90175

Isolation and characteristics of multi-drug resistant Streptococcus porcinus from the vaginal secretions of sow with endometritis

BackgroundSow endometritis is a common disease in pig breeding farms after artificial insemination, which leads to gray-green vaginal secretions and decreased conception rates. It is important to perform an etiologic diagnosis for effective treatments and control of diseases. The aim of this study was to carry out a pathogenic detection in five specimens of vaginal secretions collected from sick pigs with endometritis, implement identification of the pathogens by phenotypic detection and 16 s rDNA sequence and phylogeny analysis, and determinate antibiotic susceptibility of the isolates.ResultsA Streptococcus strain was isolated and identified from all of the five specimens. The isolate was positive for Voges-Proskauer (V-P) and for the hydrolysis of arginine, esculin and myelin-associated glycoprotein (MAG). Acid formation was observed for sorbitol, mushroom sugar, sucrose, and glucose. The 16S rDNA sequence of the isolate possessed 99.93% similarity to that of Streptococcus porcinus. The phylogenetic analysis of 16S rDNA sequence showed that the isolate belonged to the same clade as the S. porcinus strains from humans, pigs, and other animals. The isolate exhibited multi-drug resistance to aminoglycosides, quinolones, macrolides and tetracyclines except being sensitive to some β- lactams such as penicillin G, cephalothin, cefazolin, cephradine and cefuroxime.ConclusionsA S. porcinus isolate with multi-drug resistance was identified from vaginal secretions of sows with endometritis in one pig breeding farm, which suggests that the sow endometritis was caused by S. porcinus infection during artificial insemination. This study indicates that sensitive antibiotics such as penicillin G or some cephalosporins could be used for treatment of the diseases. In addition, the study hints that bacterial multi-drug resistance is a tough problem for disease treatment in pig farms.

Corynebacterium suranareeae sp. nov., a glutamate producing bacterium isolated from soil and its complete genome-based analysis.

Strain N24T was isolated from soil contaminated with starling's feces collected from Roi-Et province, Thailand. Cells of N24T were Gram-stain-positive rods, aerobic and non-spore-forming. N24T was positive for catalase, urease, citrate utilization, nitrate reduction and Methyl Red (MR) test but negative for oxidase, casein, gelatin liquefaction, tyrosine, Voges-Proskauer (VP) reaction and starch hydrolysis. Meso-diaminopimelic acid, rhamnose, ribose, arabinose and galactose were detected in its whole-cell hydrolysates. The results of the 16S rRNA gene sequence analysis indicated that N24T represented a member of the genus Corynebacterium. N24T was closely related to Corynebacterium glutamicum ATCC 13032T, with 99.0 % 16S rRNA gene sequence similarity. According to results obtained using in silico DNA-DNA hybridization approaches, N24T showed highest DNA-DNA relatedness (27.6 %) and average nucleotide identity (84.1 %) to Corynebacterium glutamicum ATCC 13032T. The DNA G+C content of N24T was 51.8 mol% (genome based). The major cellular fatty acids of N24T were C16 : 0, and C18 : 1ω9c. N24T had the nine isoprenes unit, MK-9(H2) as the predominant menaquinone. The predominant polar lipids were phosphatidylglycerol, phosphatidylinositol and diphosphatidylglycerol. Mycolic acids were also present. According to the complete genome sequence data, strain N24T and C. glutamicum ATCC 13032T are close phylogenetic neighbours, but have different genome characteristics. On the basis of the results of the genotypic and genomic studies and phenotypic characteristics including chemotaxonomy, strain N24T should be classified as representing a novel species of the genus Corynebacterium, for which the name Corynebacterium suranareeae sp. nov. is proposed. The type strain is N24T (TBRC 5845T=NBRC 113465T).

Paenibacillus ottowii sp. nov. isolated from a fermentation system processing bovine manure

Strain MS2379T was isolated from a pasteurized solution sample from a predominantly anaerobic fermentation system processing bovine manure in Pilot Point, Texas. Phylogenetic analyses based on both 16S rRNA gene and rpoB gene sequences showed that MS2379T was most closely related to Paenibacillus polymyxa (DSM 36T), P. jamilae (DSM 13815T), and P. peoriae (DSM 8320T), yet DNA-DNA relatedness through DNA-DNA hybridization revealed only 22.6, 32.0 and 24.7 % relatedness to these three species respectively. Rod-shaped cells of strain MS2379T are Gram-stain variable with sub-terminal, ellipsoidal, deforming endospores. The peptidoglycan contains meso-diaminopimelic acid (mDAP) and the predominant fatty acids are anteiso-C15 : 0 (61.9 %) and anteiso-C17 : 0 (11.6 %), confirming that strain MS2379T has diagnostic features of other Paenibacillus species. The G+C content of MS2379T is 45.9 mol%. Fermentation of glucose yields acid and gas end-products. The polar lipids found were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and glycolipids, but also included some unidentified lipids, aminolipids, aminoglycolipid, and phosphatidylmethylethanolamine. The growth range of MS2379T was observed from 10-45 °C with optimal growth temperature at 30 °C. Growth was observed between pH 6-10 and up to 3 % NaCl. Unlike the most closely related Paenibacillus species, strain MS2379T was negative in the Voges-Proskauer reaction. Nucleic acid, chemotaxonomic and biochemical features support the distinctiveness of strain MS2379T. Thus, strain MS2379T represents a novel species of the genus Paenibacillus for which the name Paenibacillus ottowii sp. nov. is proposed with the type strain MS2379T (=DSM 107750T=ATCC TSD-165T).

Multidrug-resistant Escherichia coli and Salmonella spp. isolated from pigeons.

Background and Aim:Pigeon rearing has been gaining popularity for recent years. They are reared remarkably very close to the house of the owner. This activity, therefore, may pose potential threats for humans as well as other animals as pigeons may carry and spread different pathogens including drug-resistant bacteria. This study was conducted to explore the prevalence of Escherichia coli and Salmonella spp. as well as their antibiogram profile along with an association analysis.Materials and Methods:Forty swab samples were collected from 20 pigeons during the study. E. coli and Salmonella spp. were isolated and identified on various types of agars, including MacConkey, Eosin methylene blue, Brilliant green, and Salmonella-Shigella agar. Biochemical tests such as the carbohydrate fermentation test, the triple sugar iron agar slant reaction, the indole test, the methyl red test, the catalase test, as well as the Voges–Proskauer test were also performed. Besides, the presence of E. coli was further confirmed by polymerase chain reaction (PCR). Moreover, antimicrobial susceptibility testing of the isolates was performed against nine antibiotics from seven classes on the Mueller-Hinton agar based on the Kirby–Bauer disk diffusion method.Results:The overall prevalence of E. coli and Salmonella spp. was 52.5 and 27.5%, respectively. The prevalence of the pathogenic E. coli was 61.90%. The antibiogram profile of 21 E. coli as well as 11 Salmonella spp. revealed that all isolates, except one, were resistant to one to six antibiotics. Around 61.90%, 71.43%, 23.81%, 61.90%, 23.81%, 19.05%, and 52.38% of E. coli showed resistance against amoxicillin, ampicillin, azithromycin, erythromycin, nalidixic acid, gentamicin, and tetracycline, respectively. Furthermore, E. coli resistance was not observed in case of ciprofloxacin and levofloxacin. Similarly, around 36.36%, 27.27%, 27.27%, 45.45%, 81.82%, 100%, and 18.18% of the Salmonella spp. showed resistance against amoxicillin, ampicillin, azithromycin, erythromycin, nalidixic acid, tetracycline, and levofloxacin, respectively. However, all Salmonella spp. (100%) were found to show sensitivity against ciprofloxacin and gentamicin. Multidrug-resistant (MDR) E. coli (23.80%) and Salmonella spp. (54.54%) were also isolated. Furthermore, both positive (odds ratio [OR] >1) and negative (OR <1) drug resistance associations, with a higher frequency of positive associations, were found in E. coli. A significant positive association was observed between ampicillin and amoxicillin (OR: 81.67, 95% confidence interval: 2.73-2447.57, p=0.01).Conclusion:Pigeon carrying MDR E. coli and Salmonella spp. may contribute to the transmission and spread of these microorganisms. Therefore, strict hygienic measures should be taken during the farming of pigeons to decrease the potential transmission of E. coli and Salmonella spp. from pigeon to humans as well as other animals. So far, this is the first report of the PCR-based identification of pathogenic E. coli from pigeons in Bangladesh.

Isolation And Characterization Of Gut Bacterial Species Of Earthworm Lampito Mauritii

Lampito mauriti is an Anecic earthworm living in the topsoil and it is geophytophagous in nature.This earthworm is an important soil macro fauna as it has the dual role of an ‘ecosystem engineer’ due to the ability to build burrows as well as ‘keystone species’ in soil food webs because of its function in degradationof organic wastes. The present study investigates the gut of this earthworm to find the most predominant bacterium harbored inside. Gut contents were extracted and streaked on bacteriological media. The predominant type of colony was identified based on size, pigmentation, form, margin and elevation. Thendifferent colonies were isolated and streaked separately to get pure colonies. The bacteria was subjected to several biochemical tests like Motility test, Gram staining, Methyl Red test, Voges-Proskauer test, Indole production test, Carbohydrate Fermentation test, Catalase test and Starch Hydrolysis test in order to identify the species. The bacterial species identification was done using Bergey’s manual. The bacterial isolates such as Micrococcus spp, Veillonella spp, Bacillus spp and Streptococcus spp were identified.

First Report of Ewingella americana Causing Bacterial Shot Hole on Anoectochilus roxburghii in China

Anoectochilus roxburghii (Wall.) Lindl., known as jinxianlian in Chinese, is a member of the Orchidaceae family. Traditionally, it has been considered as one of the most valuable medicinal plants used for therapeutic treatments. During the period from September to November in 2017 and 2018, symptoms of bacterial shot hole disease were observed in ∼10% of A. roxburghii plants in a 30-ha area (23°88′N; 100°08′E) in Lincang County, Yunnan Province. Chlorosis is the first symptom, and then the leaves turn brown, followed by profuse brown necrotic spotting and scorching. Then, the leaves eventually dry up and become punched out. No symptoms are observed on the stem or root. To identify the possible pathogens, three diseased leaves were disinfected with tap water and then 70% ethanol for 30 s, followed by maceration in sterile distilled water for three times, and 20 µl of the suspension was plated onto nutrient agar (NA) medium and incubated at 28°C for 48 h in the dark. A predominant type of colony was purified on NA, and a representative isolate designated JXL-2 was used for further study. The cultures formed an oyster white lawn and were short rod shaped, with a cell size around 1.5 to 1.3 × 0.6 to 0.8 μm, and were periflagellate with slow motility. The biochemical tests showed that JXL-2 was gram negative, nonfluorescent, and positive for the Voges–Proskauer test, o-nitrophenyl-β-D-galactoside reaction, and esculin hydrolysis. But it does not produce indoles, ureases, H₂S, phenylalanine deaminase, amylase, or lysine decarboxylase, or ornithine decarboxylase. The 16S ribosomal RNA and DNA gyrase subunit B (gyrB) partial genes were amplified using the primers 27F/1492R and UP1/UP2, respectively (Marchesi et al. 1998; Liu et al. 2018) and sequenced (MK460249.1 and MK460250.1). These sequences had 99% nucleotide identity with Ewingella americana in GenBank (KF308476.1, NR 104925.1). Koch’s postulates were fulfilled on 10 healthy leaves of 4-month-old potted A. roxburghii plants in an incubator. Bacterial suspension (0.2 ml, 1.0 × 10⁸ CFU/ml) was extracted with a sterilized syringe and injected into each leaf of A. roxburghii. The same amount of sterilized water was inoculated to 10 more A. roxburghii leaves as a control. The experimental and control groups were incubated in an artificial climate incubator, at 25°C with 12-h light/dark and 85% relative humidity. Symptoms similar to those of diseased leaves in the field were observed in the JXL-2-inoculated group after 15 days, whereas the control group remained healthy. E. americana was consistently reisolated from the inoculated symptomatic leaves, which were identified through 16S rRNA and gyrB nucleotide sequence analysis. Finally, we confirmed the pathogen was E. americana based on morphological, biochemical, and molecular characteristics. It has been reported that the ecological niche or natural source of E. americana is unknown, but it has been isolated from animals, mushrooms, carrots, and even vacuum-packaged meat (Liu et al. 2018; Reyes et al. 2004). To our knowledge, this is the first report of E. americana causing bacterial shot hole disease on A. roxburghii in China and around the world. It would be a new and noteworthy finding that E. americana could cause diseases on plants.

Plant growth promoting and antifungal activity in endophytic Bacillus strains from pearl millet (Pennisetum glaucum).

Bacterial endophytes are well known inhabitants of living plant system and perform important assignments in maintaining plant growth and health. Currently, limited reports are available on the endophytes of pearl millet (Pennisetum glaucum) reflecting antagonistic and plant growth promoting (PGP) attributes. Therefore, the major objectives of current investigation were to identify antagonistic strains of endophytic Bacillus from pearl millet and further illustrate their PGP capabilities. In this study, 19 endophytic Bacillus strains (EPP5, EPP21, EPP30, EPP32, EPP35, EPP42, EPP49, EPP55, EPP62, EPP65, EPP70, EPP71, EPP74, EPP78, EPP83, EPP86, EPP93, EPP100, and EPP102) displaying antagonistic activity towards Rhizoctonia solani (RS), Sclerotium rolfsii (SR), and Fusarium solani (FS) were isolated from different sections (root, leaf, stem, and root) of pearl millet. Phenotypic (shape, colony, gram staining reaction, endospore formation, and motility) and biochemical features (catalase, oxidase, citrate, gelatinase, urease, Voges Proskauer's, methyl red, indole, and nitrate reduction), along with the similarly comparison of 16S rRNA gene sequence with type strains identified eight antagonistic endophyhtes as B. amyloliquefaciens (EPP35, EPP 42, EPP62, and EPP 102), Bacillus subtilis subsp. subtilis (EPP65), and Bacillus cereus (EPP5, EPP71, and EPP74). The production of indole acetic acid and siderophores was varied among the isolated endophytes. Besides displaying enzymatic activities, these isolates varied in solubilizing capabilities of phosphate, potassium, and zinc. The presence of three antimicrobial peptide genes (ituD, bmyC, and srfA) also confirmed their antifungal nature. Further, single treatment of three promising strains (EPP5, EPP62, and EPP65) offered protection ranging from 35.68 to 45.74% under greenhouse conditions. However, microbial consortium (EPP5+ EPP62 + EPP65) provided the highest protection (71.96%) against root rot and wilt infection with significant increase in plant biomass. Overall, the current study indicated that pearl millet plant harbors various species of endophytic Bacillus that possess excellent biocontrol and growth promotion activities.

Isolation and characterization of biofilm-mediated vibriosis-causing bacteria from Macrobrachium rosenbergiii-aquaculture

Vibriosis is one of the major problems in prawn aquaculture leading to a significant loss in yield. The objective of the study was to isolate and identify the etiological agent of vibriosis from the giant freshwater prawn (Macrobrachium rosenbergiii) aquaculture. The study was conducted by isolating the bacteria using selective media of thiosulfate-citrate-bile salts-sucrose (TCBS) agar followed by the morphological and biochemical characterizations, i.e., indole, methyl-red (MR), Voges-Proskauer (VP), citrate, urease, NaCl 0%, gelatinase and lysine decarboxylase tests. The isolated bacteria were further assayed for their standard growth-curve as well as the ability to form a biofilm on Congo-red agar (CRA). The results showed that the bacteria showed a yellow color in colony appearance and had negative Gram staining. On the biochemical assays, the isolates showed positive reactions on the indole, MR, VP, gelatine, and lysine decarboxylase; but had negative reactions on the citrate, urea, and NaCl 0% tests. The isolates were then identified as Vibrio alginolyticus and after further characterization, the isolates showed the ability to form a biofilm. This result signifies the urgency to overcome V-. alginolyticus as the causing agent of vibriosis that was able to form a biofilm on the giant river prawn aquaculture.

Pararhodobacter marinus sp. nov., a bacterium isolated from marine sediment in the East China Sea and emended description of the genus Pararhodobacter.

A Gram-negative, rod-shaped, non-motile and strictly aerobic bacterium, designated ZQ420T, was isolated from marine sediment sampled on Zhoushan Island located in the East China Sea. Strain ZQ420T was able to grow at 10-45 °C, 0-12.0 % (w/v) NaCl and pH 5.5-9.0. Catalase and oxidase activities, nitrate reduction, H2S production, hydrolysis of starch, casein, Tween 20, 40 and 80 were positive. Indole, methyl red, Voges-Proskauer test, hydrolysis of gelatin and Tween 60 were negative. The major cellular fatty acids were C18 : 1 ω7c, C16 : 0 and 11-methyl C18 : 1ω7c. Ubiquinone-10 (Q-10) was the only detected respiratory quinone. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified phospholipids, three unidentified glycolipids, two unidentified aminolipid and two unidentified lipids. The DNA G+C content was 64.8 mol%. According to 16S rRNA gene sequence similarities, strain ZQ420T shared 97.9, 96.3 and 96.3 % similarities to the following species with validated names Pararhodobacteraggregans D1-19T, Pseudo rhodobacter psychrotolerans PAMC27389T and Pseudo rhodobacter collinsensis 4-T-34T, respectively. While sharing lower sequence similarities (<96.0 %) to other type species. Phylogenetic analyses showed that strain ZQ420T and P. aggregans D1-19T formed an independent cluster in the phylogenetic trees. The average nucleotide identity value between strain ZQ420T and P. aggregans D1-19T was 79.1 %. The in silico DNA-DNA hybridization analysis revealed that strain ZQ420T shared 21.5 % DNA relatedness with P. aggregans D1-19T. On the basis of its phenotypic, chemotaxonomic and genotypic characteristics, strain ZQ420T is considered to represent a novel species in the genus Pararhodobacter, for which the name Pararhodobactermarinus sp. nov. is proposed. The type strain is ZQ420T (=KCTC 62579T=MCCC 1K03530T).

Incidence of Enteropathogenic Escherichia coli among Infants at Eleyele Comprehensive Health Centre, Ile-Ife, Osun State

Abstract Objectives This work was carried out to find the incidence of enteropathogenic Escherichia coli in children below 5 years of age who complained of having diarrhea at Eleyele Comprehensive Health Centre, Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife. The work was also done to determine the incidence in different age groups of both sexes. Methods All specimens were cultured as routinely done at the bacteriology laboratory in Obafemi Awolowo University Teaching Hospital for stool samples. Colonies of lactose fermenters were processed after overnight incubation at 37°C. Nonlactose fermenter colonies were discarded. Out of 200 colonies isolates on MacConkey agar, there were 150 lactose fermenters and these were all processed. The other 70, which were Gram-positive cocci, were discarded. These were subjected to biochemical examination such as treating them with sugars, for the production of acid and gas, indole production, motility with sugars, citrate utilization, Voges-Proskauer, and methyl red tests. Results Out of 150 strains examined, only 80 strains obeyed IMVic ++ -- reactions. These were serotyped to know the enteropathogenic strains. From the result obtained, 23 strains reacted positively with polyvalent antisera (polyvalent 2, 3, 4 antisera), of which 13 strains gave a positive reaction with corresponding monovalent antisera. There were five strains from polyvalent 2 antisera, five strains from polyvalent 3 antisera, and three strains from polyvalent 4 antisera. In close study of this work, total percentage of females with diarrhea was 44.5% while total percentage of males with diarrhea was 55.5%. Out of these 80, E coli was isolated, and from these, 13 enteropathogenic E strains were isolated (16.25%). Patient aged below 2 years gave nine strains (69.2%), aged 2 to 3 years gave two strains (15.4%), aged 3 to 4 years gave one strain (7.7%), and aged 4 to 5 years gave one strain (7.7%). Also, close examination of result revealed that male children had the greater percentage of enteropathogenic infection, with 8 strains out of the 13 strains serotyped (61.5%) and female children with 5 strains (38.5%). Conclusion E coli remains an important cause of infant diarrhea. Although epidemics of infant diarrhea caused by enteropathogenic E coli have almost disappeared from the developed countries, it is still very common in developing countries. The relative importance of enteropathogenic E coli as a cause of infant diarrhea needs to be reassessed and new diagnostic techniques will also help to simplify this task.

Identification of the Mechanisms of Acetoin Production Change in Genetically Altered Vibrio cholerae Strains O1 Biovar El Tor

Objective of the study was to determine the mechanisms of acetoin biosynthesis change in genetically altered El Tor V. cholerae strains in Voges-Proskauer test.Materials and methods. We used nine genetically altered V. cholerae O1 strains, biovar El Tor, imported in the territory of the Russian Federation and Ukraine between 1993–2011, and four typical strains isolated in 1970–1972. When assessing acetoin production in V. cholerae strains in Voges-Proskauer test, the strain V. cholerae 569B O1 serogroup, classical biovar served as negative control of the assay. Relative gene expression was studied using real-time RT-PCR. Protein model construction was performed by means of automated server SWISS – MODEL.Results and discussion. It has been demonstrated that diagnostically significant feature – VogesProskauer reaction, utilized for V. cholerae O1 biovar differentiation, is changed in all investigated genetically altered strains of cholera agent, isolated in different periods of the current seventh pandemic (66.7 % of the strains show weakly positive reaction, 33.3 % – negative one). The data obtained testify to the reduction or absence of acetoin production in the investigated strains. Analysis of four structural genes of als operon, as well as expression of regulatory genes alsR and аphA, responsible for acetoin biosynthesis, has revealed that changes in acetoin production in the genovariants stem from the deletion of a single nucleotide (T in the position 315) in the structural gene alsD, encoding acetolaktat decarboxylase, and also from high levels of negative acetoin biosynthesis regulator expression – аphA gene. Modeling of the spatial (3-D) structure of AlsD protein in the genovariant M1293 and the reference-strain N16961 has shown that AlsD protein of the genovariant is, indeed, considerably reduced. However, spontaneous decarboxylation is possible in the absence of acetolaktat decarboxylase, which phenotypically manifests itself in borderline positive Voges-Proskauer test.

Isolation, identification, and characterization of diesel-oil-degrading bacterial strains indigenous to Changqing oil field, China.

In the present study, 12 indigenous diesel-oil-degrading bacteria were isolated from the petroleum-contaminated soils of the Changqing oil field (Xi'an, China). Measurement of the diesel-oil degradation rates of these strains by the gravimetric method revealed that they ranged from 42% to 66% within 2 weeks. The highest degradation rates were observed from strains CQ8-1 (66%), CQ8-2 (62.6%), and CQ11 (59%), which were identified as Bacillus thuringiensis, Ochrobactrum anthropi, and Bordetella bronchialis, respectively, based on their 16S rDNA sequences. Moreover, the physiological and biochemical properties of these three strains were analyzed by Gram staining, catalase, oxidase, and Voges-Proskauer tests. Transmission electron microscopy showed that all three strains were rod shaped with flagella. Gas chromatography and mass spectrometric analyses indicated that medium- and long-chain n-alkanes in diesel oil (C11-C29) were degraded to different degrees by B. thuringiensis, O. anthropi, and B. bronchialis, and the degradation rates gradually decreased as the carbon numbers increased. Overall, the results of this study indicate strains CQ8-1, CQ8-2, and CQ11 might be useful for environmentally friendly and cost-effectivebioremediation of oil-contaminated soils.

Two-tailed fancy goldfish challenged with Bacteroides spp. isolated from shubunkin goldfish

This study aimed to isolate, morpho-biochemically characterize and identify the most common bacterium in diseased shubunkin goldfish, and challenge the two-tailed fancy goldfish using the identified bacterium. With the aid of Bergey’s Manual of Determinative Bacteriology, the bacterium was identified as Bacteroides spp. The bacterium was characterized as follows: Gram-negative, coccobacillus, facultative anaerobe, hemolytic, glucose and sucrose fermentative but not lactose and xylose, negative to catalase, Methyl red, Voges-Proskauer and hydrogen sulphide production tests, and positive to Simmon’s citrate utilization, liquefaction of gelatin, nitrate reduction, urea hydrolysis and indole production. Two-tailed goldfish challenged with Bacteroides spp. showed clinical signs such as gasping for air, irritability, sloughing of scale, lesion and watery internal organs. Keywords: Shubunkin goldfish, two-tailed goldfish, biochemical tests, Bacteroides

Edaphovirga cremea gen. nov., sp. nov., isolated from the rhizospheric soil of Codonopsis clematidea.

A Gram-negative, facultatively anaerobic, non-motile, nonspore- forming, coccoid or rod-shaped and creamy-pigmented bacterium, designated SYP-B2100T, was isolated from the rhizospheric soil of Codonopsis clematidea in the Xinjiang Uygur Autonomous Region, China. The optimal growth occurred at 28°C, pH 5.0, in the absence of NaCl. The cells tested positive in catalase and methyl red tests but negative in oxidase, urease, gelatinase, milk coagulation, and peptonisation, H2S production, nitrate reduction, and Voges-Proskauer tests. The major isoprenoid quinone was ubiquinone-8 (Q-8). The major cellular fatty acids were C16:0 and summed feature 8. The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. The 16S rRNA gene sequence of strain SYP-B2100T was the most similar to that of Rahnella inusitata DSM 30078T (96.9%) within the family Enterobacteriaceae. The genomic DNA G + C content of strain SYP-B2100T was 50.3 mol%. The combined data from the phylogenetic, morphological, physiological, biochemical, and chemotaxonomic analyses presented in this study support the conclusion that strain SYP-B2100T represents a novel species of a new genus, for which the name Edaphovirga cremea gen. nov., sp. nov. is proposed; the type strain is SYPB2100T (= CGMCC 1.5857T = DSM 105170T = KCTC 62024T).

Molecular detection of Vibrio cholerae from human stool collected from SK Hospital, Mymensingh, and their antibiogram.

Objective:Vibrio spp., particularly, Vibrio cholerae is a major etiology of diarrhea in humans worldwide. In this study, we isolated and identified V. cholerae from the human stool of suspected cases along with antibiogram.Materials and Methods:In total, 25 stool samples from cholera suspected patients were analyzed. Isolation and molecular detection of Vibrio species were performed based on staining, motility, cultural and biochemical characteristics followed by polymerase chain reaction (PCR) using groEL gene-specific primers.Results:Among the 25 samples, seven showed growth of yellow color colonies on Thiosulfate-Citrate-Bile salts-Sucrose agar plates. The isolates were Gram-negative, curved shaped, and motile. Biochemically, they were found positive for indole and Methyl Red tests and negative for Voges–Proskauer test. Out of the seven positive samples, only three isolates were confirmed as Vibrio spp. using genus-specific primers. Subsequently, these three isolates were confirmed as V. cholerae by PCR using V. cholerae groEL gene-specific primers. Antibiotic sensitivity test revealed these three isolates as highly sensitive to azithromycin, chloramphenicol, gentamicin, and norfloxacillin while resistant to streptomycin, tetracycline, and oxacillin.Conclusion:Vibrio cholerae were isolated from the stool of diarrheic human patients and confirmed by PCR targeting the groEL gene. The isolates were found resistant to streptomycin, tetracycline and oxacillin, and need further characterization to reveal the molecular basis of their origin and resistance.

Klebsiella huaxiensis sp. nov., recovered from human urine

A strain of a member of the genus Klebsiella, WCHKl090001, was recovered from a human urine sample in PR China in 2017. Phylogenetic analysis based on gyrA and rpoB housekeeping genes revealed that the strain was distinct from any previously described species of the genus Klebsiella though it was clustered with the Klebsiella oxytoca phylogroup, including Klebsiella grimontii, Klebsiella michiganensis and Klebsiella oxytoca. The whole-genome sequence of strain WCHKl090001 has an up to 87.18 % average nucleotide identity with those of type strains of all known species of the genus Klebsiella. In silico DNA-DNA hybridization (isDDH) values between strain WCHKl090001 and type strains of all known species of the genus Klebsiella ranged from 22.3 to 35.2 %. Strain WCHKl090001 could be distinguished from species of the Klebsiella oxytocaphylogroup by its negative Voges-Proskauer reaction. Genotypic and phenotypic characteristics from this study indicate that strain WCHKl090001 should be considered to represent a novel species of the genus Klebsiella, for which the name Klebsiellahuaxiensis sp. nov. is proposed. The type strain is WCHKl090001T (=GDMCC 1.1379T=CNCTC 7650T).

Characterization of a strain representing a new Enterobacter species, Enterobacter chengduensis sp. nov.

An Enterobacter strain, WCHECl-C4, was recovered from a human blood sample in China in 2015. Phylogenetic analysis based on multi-locus sequence analysis of the rpoB, gyrB, infB and atpD housekeeping genes revealed that the strain is distinct from any previously described species of the genus Enterobacter. The whole genome sequence of strain WCHECl-C4 has an 80.48-93.34% average nucleotide identity with those of type strains of all known Enterobacter species. In silico DNA-DNA hybridization (isDDH) values between strain WCHECl-C4 and type strains of all known Enterobacter species range from 23.2 to 52.4%. The major cellular fatty acids of strain WCHECl-C4 are C16:0, C17:0 cyclo and C18:1ω7c, which are in the range of Enterobacter species. The genomic DNA G + C content was 55.7mol%. Strain WCHECl-C4 can be distinguished from all known Enterobacter species by its ability to ferment D-sorbitol, L-rhamnose and melibiose but with a negative Voges-Proskauer reaction. Genotypic and phenotypic characteristics from this study indicate that strain WCHECl-C4 represents a novel species of the genus Enterobacter, for which the name Enterobacter chengduensis sp. nov. is proposed. The type strain is WCHECl-C4T (= GDMCC1.1216T = CCTCC AB 2017105T = KCTC 52993T).