Adonitol Fermentation Research Articles

Molecular and metabolic characterization of petroleum hydrocarbons degrading Bacillus cereus.

Hydrocarbon constituents of petroleum are persistent, bioaccumulated, and bio-magnified in living tissues, transported to longer distances, and exert hazardous effects on human health and the ecosystem. Bioaugmentation with microorganisms like bacteria is an emerging approach that can mitigate the toxins from environmental sources. The present study was initiated to target the petroleum-contaminated soil of gasoline stations situated in Lahore. Petroleum degrading bacteria were isolated by serial dilution method followed by growth analysis, biochemical and molecular characterization, removal efficiency estimation, metabolites extraction, and GC-MS of the metabolites. Molecular analysis identified the bacterium as Bacillus cereus, which exhibited maximum growth at 72 hours and removed 75% petroleum. Biochemical characterization via the Remel RapID™ ONE panel system showed positive results for arginine dehydrolase (ADH), ornithine decarboxylase (ODC), lysine decarboxylase (LDC), o-nitrophenyl-β-D-galactosidase (ONPG), p-nitrophenyl-β-D-glucosidase (βGLU), p-nitrophenyl-N-acetyl-β-D-glucosaminidase (NAG), malonate (MAL), adonitol fermentation (ADON), and tryptophane utilization (IND). GC-MS-based metabolic profiling identified alcohols (methyl alcohol, o-, p- and m-cresols, catechol, and 3-methyl catechol), aldehydes (methanone, acetaldehyde, and m-tolualdehyde), carboxylic acid (methanoic acid, cis,cis-muconic acid, cyclohexane carboxylic acid and benzoic acid), conjugate bases of carboxylic acids (benzoate, cis,cis-muconate, 4-hydroxybenzoate, and pyruvate) and cycloalkane (cyclohexene). It suggested the presence of methane, methylcyclohexane, toluene, xylene, and benzene degradation pathways in B. cereus.

First Detection of Carbapenem-Resistant Escherichia fergusonii Strains Harbouring Beta-Lactamase Genes from Clinical Samples.

Recently discovered extraintestinal Escherichia fergusonii obtained from non-clinical samples has exhibited the potential for acquiring multiple beta-lactamase genes, just like many extraintestinal Escherichia coli strains. Albeit, they are often omitted or classified as E. coli. This study aimed to, therefore, identify carbapenem-resistant extended-spectrum beta-lactamase (ESBL) producing E. fergusonii isolates from clinical samples, determine their evolutionary relatedness using 16S rRNA sequencing analysis and screen for beta-lactamase genes. A total of 135 septic wound samples were obtained from patients on referral at a General Hospital in Lagos, Nigeria. For the phenotypic identification of isolates from culture-positive samples, morphological, and physiological tests were carried out. Identities of the isolates harbouring beta-lactamase genes were assigned to their genus strains using the 16S rRNA sequencing. The Kirby Bauer disc diffusion technique and double-disc synergy test were used to screen isolates for multidrug resistance and ESBL production. Carbapenem-resistant ESBL producing isolates were screened for beta-lactamase genes in a polymerase chain reaction. Three E. fergusonii isolates (CR11, CR35 and CR49) were obtained during this study. E. fergusonii strains were motile, non-lactose and non-sorbitol fermenting but positive for cellobiose and adonitol fermentation. The I6S rRNA assigned the phenotypically identified isolates to E. fergusonii species. All three isolates were multidrug-resistant, carbapenem-resistant and ESBL producers. Isolates CR11 and CR35 harboured cefotaximase (CTX-M) and temoniera (TEM) beta-lactamase genes while CR49 harboured sulfhydryl variable (SHV) beta-lactamase gene. We herein report the detection of multiple beta-lactamase genes in carbapenem-resistant ESBL producing E. fergusonii from clinical samples.

Phylogenetic Groups Among Klebsiella pneumoniae Isolates from Brazil: Relationship with Antimicrobial Resistance and Origin

The objectives of this study were to determine the distribution of phylogenetic groups among Klebsiella pneumoniae isolates from Recife, Brazil and to assess the relationship between the groups and the isolation sites and resistance profile. Ninety four isolates of K. pneumoniae from hospital or community infections and from normal microbiota were analyzed by gyrA PCR-RFLP, antibiotic susceptibility, and adonitol fermentation. The results revealed the distinction of three phylogenetic groups, as it has also been reported in Europe, showing that these clusters are highly conserved within K. pneumoniae. Group KpI was dominantly represented by hospital and community isolates while groups KpII and KpIII displayed mainly normal microbiota isolates. The resistance to third generation cephalosporins, aztreonam, imipenem, amoxicillin/clavulanic acid, and streptomycin was only observed in KpI. The percentage of resistance was higher in KpI, followed by KpII and KpIII. The differences in the distribution of K. pneumoniae phylogenetic groups observed in this study suggest distinctive clinical and epidemiological characteristics among the three groups, which is important to understand the epidemiology of infections caused by this organism. This is the first study in Brazil on K. pneumoniae isolates from normal microbiota and community infections regarding the distribution of phylogenetic groups based on the gyrA gene.

Development of a rapid identification method for Klebsiella pneumoniae phylogenetic groups and analysis of 420 clinical isolates

A rapid method combining gyrA PCR-restriction fragment length polymorphism analysis, parC PCR and adonitol fermentation was developed to identify Klebsiella pneumoniae phylogenetic groups KpI, KpII and KpIII. Analysis of 420 clinical isolates from 26 hospitals showed that the three groups were widespread geographically. KpI comprised 80.3% of 305 isolates from blood and 82.2-97.2% of isolates from other clinical sources. KpIII was never found among isolates from urinary tract infections. KpI isolates from blood were generally less susceptible than KpIII isolates to the ten antimicrobial agents tested, with KpII being intermediate. The frequencies of ceftazidime resistance were 21.6% and 8.6% in KpI and KpIII isolates, respectively (p 0.01).

Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping.

The infra-specific phylogenetic diversity and genetic structure of both Klebsiella pneumoniae and Klebsiella oxytoca was investigated using a combination of randomly amplified polymorphic DNA (RAPD) analysis, sequencing of gyrA and parC genes, and automated ribotyping. After RAPD analysis with four independent primers of 120 clinical isolates collected from 22 European hospitals in 13 countries, K. pneumoniae isolates fell into three clusters and K. oxytoca isolates fell into two clusters, while Klebsiella planticola isolates formed a sixth cluster. Each cluster was geographically widespread. K. pneumoniae cluster I (KpI) accounted for 80% of the isolates of this species and included reference strains of the three subspecies K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis. Clusters KpII and KpIII were equally represented, as were the two K. oxytoca clusters. Individualization of each cluster was fully confirmed by phylogenetic analysis of gyrA and parC gene sequences. In addition, sequence data supported the evolutionary separation of K. pneumoniae from a phylogenetic group including K. oxytoca, Klebsiella terrigena, K. planticola and Klebsiella ornithinolytica. Automated ribotyping using Mlu I appeared suitable for identification of each Klebsiella cluster. The adonitol fermentation test was found to be useful for cluster identification in K. pneumoniae, since it was negative in all strains of clusters KpIII and in some KpII strains, but always positive in cluster KpI. The usefulness of gyrA and parC sequence data for population genetics and cluster identification in bacteria was demonstrated, even for the phylogenetic positioning of quinolone-resistant isolates.

In vitro comparison of bovine mastitis and fecal Escherichia coli isolates

In vitro methods were used to test the hypothesis that Escherichia coli from bovine mastitis are essentially no different from isolates from bovine feces. Fifty E. coli isolates from bovine mastitic milk, 50 from feces of mastitic cows and 50 from feces of healthy cows were compared with respect to biochemical properties and certain potential virulence factors. There were no significant differences among the groups in tests for biotype; production of colicins, colicin V, or Vero cell cytotocity; and growth in 90% gnotobiotic calf serum or 90% normal milk whey. Resistance to killing in 90% gnotobiotic calf serum varied from 66 to 84%. Most isolates grew in normal whey: the percentage in a group varied from 86 to 96. Mastitic milk isolates were significantly different from the fecal isolates in adonitol fermentation ( P⩽0.006), production of aerobactin ( P⩽0.026), and ability to grow in 90% mastitic whey ( P⩽0.00004). However, only 40% of mastitis E. coli fermented adonitol and only 20% produced aerobactin. Ninety-six percent of mastitic milk E. coli grew in mastitic whey, whereas 64% and 60%, respectively, of mastitic fecal and normal fecal isolates grew in this medium. It is concluded that none of the properties that were investigated constitute potential virulence factors or markers for ability to induce mastitis; the data are consistent with the hypothesis that mastitic E. coli are simply opportunistic pathogens.

Characteristics of CDC group 1 and group 1-like coryneform bacteria isolated from clinical specimens.

Fifteen strains of CDC group 1 coryneform and biochemically similar bacteria were isolated from clinical specimens. Of the 15 strains isolated, 11 were derived from abscesses and purulent lesions, mostly from the upper part of the body, and 3 were grown from blood cultures. Nine strains were associated with mixed anaerobic but no other aerobic flora. Seven strains exhibited the classical biochemical profile of CDC coryneform group 1; however, eight strains were unable to reduce nitrate and were called "group 1-like." Other reactions to differentiate CDC group 1 and group 1-like coryneform rods include alpha-hemolysis on human blood agar, fermentation of adonitol, and the presence of alkaline phosphatase. Fifteen strains showed marked CAMP reactions on different erythrocyte agars. Gas-liquid chromatography of volatile and nonvolatile fatty acids as well as cellular fatty acid patterns and the composition of cell wall components suggest that CDC group 1 and group 1-like coryneform bacteria do not belong to the genus Corynebacterium but possibly to the genus Actinomyces or Arcanobacterium. DNA-DNA hybridization studies revealed that group 1 and group 1-like strains represent different species.

Characterization of Vibrio metschnikovii and Vibrio gazogenes by DNA-DNA hybridization and phenotype.

Vibrio metschnikovii and Vibrio gazogenes are two new Vibrio species that have been little studied. Thirteen strains of V. metschnikovii were highly related to the type strain, NCTC 8443, by DNA-DNA hybridization. Relatedness values were 83 to 90% at 60 degrees C and 75 to 84% at the more stringent 75 degrees C. Divergence values ranged from 0.7 to 1.9. Strains of V. metschnikovii were oxidase negative and did not reduce nitrate to nitrite. The other phenotypic characteristics agreed with published data. Twenty-three strains of V. gazogenes were isolated from salt marshes and marshy areas on the coast of North and South Carolina. A new medium, marine agar supplemented with an additional 2.5% agar, reduced the problem of swarming by marine Vibrio species and enhanced the isolation of V. gazogenes and other organisms. By DNA-DNA hybridization, 22 of 23 strains were 76% or more related to the type strain of V. gazogenes, ATCC 29988. However, four DNA hybridization subgroups were defined on the basis of divergence values and/or phenotype. Strains of DNA group 1 were more highly related to each other, and this group contained the type strain and six other strains. Strains of DNA group 2 were more highly related to each other, and this group contained reference strain ATCC 43942 and 14 other strains. Strains of DNA group 1 did not ferment melibiose or D-sorbitol (one strain was sorbitol positive), but strains of DNA group 2 fermented both sugars. A revised phenotypic description of V. gazogenes based on 24 strains was written on the basis of reactions (within 2 days of incubation) at 25 degrees C in media supplemented with Na+, K+, and Mg2+. Positive results (100% positive unless indicated) included motility; gas production during fermentation (96% at 2 days, 100% at 3 to 7 days); growth in nutrient broth with the addition of 1% NaCl (88%), 2% NaCl, 3.5% NaCl, 6% NaCl, 8% NaCl, and 10% NaCl (92%); dry red or orange colonies on marine agar; and fermentation of L-arabinose, cellobiose, D-galactose (88%), D-glucose, lactose (88%), maltose, D-mannitol (96%), D-mannose, salicin, sucrose, trehalose, and D-xylose. Negative results included oxidase; nitrate reduction to nitrite (4% positive); indole production; lysine decarboxylase; ornithine decarboxylase; arginine dihydrolase; swarming; growth on TCBS agar; growth in nutrient broth with 0% NaCl, 0.1% NaCl, 0.2% NaCl, 0.3% NaCl, and 0.4% NaCl (8% positive); and fermentation of adonitol, D-arabitol, dulcitol, erythritol, D-galacturonate, i-inositol, alpha-methyl-D-glucoside, raffinose, and L-rhamnose. Variable results were found for the Voges-Proskauer reaction (62% positive), growth in nutrient broth plus 0.5% NaCl (29%) and 12% NaCL (42%), and fermentation of melibiose (71%) and D-sorbitol (71%).

In vitro and in vivo Characterization of Avian Escherichia coli. II. Factors Associated with Pathogenicity

The pathogenicity of 197 Escherichia coli isolates obtained from clinically affected commercially grown broiler chickens and normal hatchery chicks was assessed by inoculating day-old broilers intratracheally. The degree of pathogenicity (high, intermediate, low) was judged according to mortality and lesions occurring within 7 days following inoculation. Serotype, metabolic activity, motility, and in vitro antibiotic sensitivity of each isolate were evaluated and related to pathogenicity. Seventy-five of the isolates of high to intermediate pathogenicity belonged to serogroup O2, O78, or O35. In addition, 51 pathogenic E. coli isolates could not be serotyped, and several had multiple serotypes. Most isolates had similar metabolic activity, as determined by amino acid decarboxylation and carbohydrate fermentation, regardless of pathogenicity. An exception was the fermentation of adonitol, which occurred more frequently with the highly pathogenic strains. Motility and in vitro antibiotic sensitivity were not related to pathogenicity. An age-associated resistance to intratracheal E. coli administration occurred by 15 days of age in uncompromised birds. Relative susceptibility of birds older than 2 weeks to intratracheal and/or intravenous E. coli inoculation could be increased by prior exposure to pathogenic reovirus 1733, adenovirus 3167, or infectious bursal disease virus (IBDV). Birds infected with IBDV at 3 weeks failed to clear apathogenic and pathogenic E. coli from circulating blood.

Escherichia fergusonii and Enterobacter taylorae, two new species of Enterobacteriaceae isolated from clinical specimens.

Escherichia fergusonii (formerly known as Enteric Group 10) and Enterobacter taylorae (formerly known as Enteric Group 19) are proposed as new species in the family Enterobacteriaceae. By DNA hybridization (32P, 60 degrees C, hydroxyapatite), strains of E. fergusonii were 90 to 97% related to the type strain (holotype) ATCC 35469. They were most closely related to Escherichia coli and more distantly related to species in other genera. E. fergusonii strains are positive for indole production, methyl red, lysine decarboxylase, ornithine decarboxylase, and motility. They ferment D-glucose with gas production and also ferment adonitol, L-arabinose, L-rhamnose, maltose, D-xylose, trehalose, cellobiose, and D-arabitol. They are negative for Voges-Proskauer, citrate utilization (17% positive), urea hydrolysis, phenylalanine deamination, arginine dihydrolase, growth in KCN, and fermentation of lactose, sucrose, myo-inositol, D-sorbitol, raffinose, and alpha-methyl-D-glucoside. By DNA hybridization (32P, 60 degrees C, hydroxyapatite), strains of E. taylorae were 84 to 95% related to the type strain (holotype) ATCC 35317. Their nearest relative was E. cloacae, to which they were 61% related. Other named species were more distantly related. Strains of E. taylorae are positive for Voges-Proskauer, citrate utilization, arginine dihydrolase, ornithine decarboxylase, motility, growth in KCN medium, and malonate utilization. They ferment D-glucose with gas production and also ferment D-mannitol, L-arabinose, L-rhamnose, maltose, D-xylose, trehalose, and cellobiose. They are negative for indole production, methyl red, H2S production on triple sugar-iron agar, urea hydrolysis, phenylalanine deamination, lysine decarboxylase, gelatin hydrolysis, and fermentation of adonitol, i-inositol, D-sorbitol, and raffinose. Both new species occur in human clinical specimens. Two strains of E. fergusonii were isolated from blood. Five stains of E. taylorae were isolated from blood, and one was from spinal fluid. These blood and spinal fluid isolates suggest possible clinical significance, but this point requires further study.

PROBLEMS IN THE RECOVERY AND IDENTIFICATION OF ENTEROPATHOGENIC ESCHERICHIA COLI FROM FOODS

During a recent outbreak of gastroenteritis associated with serogroup 0124:B17 of Escherichia coli, various problems complicated recovery and identification of the pathogen. Standard methods for E. coli were of limited value because of atypical behavior of isolates. Two modified recovery procedures have been presented. For rapid lactose fermenters, pre-enrichment in MacConkey broth with subsequent transfer to lauryl sulfate tryptose broth and incubation at 44 C is recommended. For slow lactose fermenters, pre-enrichment in nutrient broth with subsequent transfer to Mossel's enteric enrichment broth and incubation at 41.5 C is tentatively proposed. Isolation agars include Levine's eosin methylene blue for lactose fermenters and MacConkey agar for non-lactose fermenters. The merits of a direct streak are considered. To facilitate rapid differentiation of E. coli from closely related Enterobacteriaceae within 3 days a modified Lundbeck procedure is offered. Isolates are first screened for H2S formation, indole production, arabinose fermentation, urease and ONPG-ase. Secondary characterization based on results of the indole and TSI reactions includes Voges-Proskauer test (22 C), lysine decarboxylase activity, KCN tolerance, and fermentation of adonitol, cellobiose, sorbitol, or glucose. Confirmation by gram-reaction nitrate reduction, and cytochrome oxidase activity is required to differentiate from members of other families. Critical factors of serological analysis are stressed. Prospects for future research are discussed.

On the Bacteria in Dairy Products

The frequency of appearance of the coliform group and enterococci in the natural butter and the properties of isolated coliform bacteria were examined. 15 samples of the marketed butter 9 samples of the laboratory preparations, 15 samples of the makers, total 48 butter samples, were examined. The frequency of appearance was examined by the multiplying bacteria method using the selective medium for their bacteria.Both modified Eijkman medium and E. C. medium were used for Escherichia coli type, Koser medium for Aerobacter aerogenes type, Koser medium added Konnyaku flake for Aerobacter group decomposing Konnyaku mannan and S. F. medium for Enterococci.Escherichia coli type was found in two of 24 samples. One of them in the butter dilutions of them in the butter dilutions of 10-1-10-3, and the other in 10-1-10-2.Aerobacter group, chiefly Aerobacter aerogenes type, were found even in the butter dilutions as much high as 10-4-10-5 in some instances, but ware detected only in dilutions of 10-2-10-3 in many butter products, Aerobacter group decomposing mannan, chiefly Aerobacter cloacae type, were detected in lower dilutions than in the case of Aerobacter aerogenes type, and many of them were detected in dilutions of 10-1-10-2.The frequency of appearance of Enterococci was the same as that of Aerobacter group decomposing mannan in dilutions of 10-1-10-2.Escherichia coli type was also isolated. In the Aerobacter group, Aerobacter aerogenes type and Aerobacter cloacae type were detected Aerobacter aerogenes type was classified as 3 types by the fermentation of adonitol and dulcitol. All Aerobacter group decomposing mannan were Aerobacter cloacae type.

The fermentation of polysaccharids by bacillus aerogenes

From recent work on the coli-like bacteria it is agreed that they may be divided into two subgroups which are differentiated by the Voges-Proskauer or methyl red reactions. These two groups are quite well correlated with habitat. The B. coli group (Voges-Proskauer negative ? methyl red positive) is characteristically of fecal origin, whereas the B. aerogenes-cloaca group (Voges-Proskauer positive ? methyl red negative) is relatively infrequent in feces, but predominates in the soil and on grains. B. aerogenes, although rarely found in the feces as voided, is a common inhabitant of the upper part of the intestinal tract of man. Under certain conditions it may constitute a considerable portion of the coli-like bacteria of a stool. It thus becomes of some practical significance to differentiate the B. aerogenes of intestinal origin from the B. aerogenes found in soil and on grain. Rogers pointed out that the fermentation of adonitol may be employed for this purpose with a high degree of reliability. It was thought that a study of the fermentation of the polysaccharids from different sources, particularly glycogens from vegetable and animal sources might possibly be of value in differentiating fecal from nonfecal types of B. aerogenes. Historical