Christensen's Urea Agar Research Articles

An observational study on emerging antimicrobial resistance pattern in urinary tract Infection in Nepalese children

AIMS Evidence-based management and local antibiotic susceptibility pattern provides evidence to guide the development of urinary tract infection (UTI) treatments protocols decreasing the economic burden significantly. We aimed to investigate the aetiology and resistance pattern of bacterial uropathogens to commonly prescribed oral antibiotics (beta lactamase and fluoroquinolones) causing UTI and to recommend the most appropriate antibiotics.
 MATERIALS AND METHODS This study is cross sectional, retrospective study. We evaluated causative agents and antimicrobial resistance in urine, culture positive samples collected from July 2019 to June 2021 in a single hospital in Kathmandu, Nepal. To obtain urine samples, a midstream clean-catch method used in children who were toilet trained and transurethral catheterization performed in non-toilet-trained children. Urine samples were sent to the laboratory where they were inoculated using a 4 mm calibre loop on CLED (Cysteine Lactose Electrolyte Deficient) agar plate, and incubated at 37 °C for 18–24 h. Conventional methods (colony morphology, Gram stain) were opted. Different biochemical tests – catalase test, coagulase test and inoculation in Bile Esculin Agar was done for Gram-positive organisms, while Triple Sugar Indole (TSI) agar, Sulphide indole motility (SIM) agar, Simon's citrate agar and Christensen's urea agar were used for identification of Gram-negative bacilli. Significant growth was evaluated as ≥105 colony forming units (CFU)/ml of urine. Kirby-Bauer disc diffusion method was used to perform in vitro antimicrobial susceptibility tests in Mueller-Hinton agar plates. Total 13 drugs were tested for sensitivity pattern. To analyse resistance to antibiotics for different ages, subjects were divided into four age groups: Group I, 2 months - 1 year; Group II, 1 year - 5 year; Group III, 5 year - 10 year; Group IV, 10 year - 15 year.
 RESULTS Among 970 samples sent, a total of 230 positive urine cultures were identified, of which 116 (50.4%) were from girls and 114 (49.6%) were from boys. The most common age group was 2 months to 1 year (49.1%). The most common causative agent was Escherichia coli (49.1%) followed by Enterococcus faecalis (14.3%) and Klebsiella pneumoniae (11.3%). The overall resistance to Nalidixic acid (66%), Ceftriaxone (54.8%) Cefotaxime (48.3%) Ciprofloxacin (47.9%) and Co-trimoxazole (46.9%) was significant. The least resistance was for Chloramphenicol, Nitrofurantoin, and Norfloxacin was 9.5 %, 31.5 %, and 38.3 % respectively. Chloramphenicol (90.5%) was the most active agent against E. coli and Klebsiella, whereas Linezolid (92.7%) and cloxacillin (64.9%) was most active against Enterococcus and Staphylococcus species.
 CONCLUSION Escherichia coli was the most common causative agent of urinary tract infection in children. Nalidixic acid, Ceftriaxone, Cefotaxime, Ciprofloxacin and Co-trimoxazole had the highest resistance rates against urinary tract pathogens in our centre. For oral empirical antibiotic therapy, Chloramphenicol is the most appropriate choice for Escherichia coli and Klebsiella strains and Linezolid for Enterococcus species.

A quantitative, high-throughput urease activity assay for comparison and rapid screening of ureolytic bacteria

Urease is a dinickel enzyme commonly found in numerous organisms that catalyses the hydrolysis of urea into ammonia and carbon dioxide. The microbially induced carbonate precipitation (MICP) process mediated by urease-producing bacteria (UPB) can be used for many applications including, environmental bioremediation, soil improvement, healing of cracks in concrete, and sealing of rock joints. Despite the importance of urease and UPB in various applications, a quantitative, high-throughput assay for the comparison of urease activity in UPB and rapid screening of UPB from diverse environments is lacking. Herein, we reported a quantitative, 96-well plate assay for urease activity based on the Christensen's urea agar test. Using this assay, we compared urease activity of six bacterial strains (E. coli BL21, P. putida KT2440, P. aeruginosa PAO1, S. oneidensis MR-1, S. pasteurii DSM 33, and B. megaterium DSM 319) and showed that S. pasteurii DSM 33 exhibited the highest urease activity. We then applied this assay to quantify the inhibitory effect of calcium on urease activity of S. pasteurii DSM 33. No significant inhibition was observed in the presence of calcium at concentrations below 10 mM, while the urease activity decreased rapidly at higher concentrations. At a concentration higher than 200 mM, calcium completely inhibited urease activity under the tested conditions. We further applied this assay to screen for highly active UPB from a wastewater enrichment and identified a strain of S. pasteurii exhibiting a substantially higher urease activity than DSM 33. Taken together, we established a 96-well plate-based quantitative, high-throughput urease activity assay that can be used for comparison and rapid screening of UPB. As UPB and urease activity are of interest to environmental, civil, and medical researchers and practitioners, we envisage wide applications of the assay reported in this study.

First report of urease activity in the novel systemic fungal pathogen Emergomyces africanus: a comparison with the neurotrope Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogen responsible for the AIDS-defining illness, cryptococcal meningitis. During the disease process, entry of cryptococcal cells into the brain is facilitated by virulence factors that include urease enzyme activity. A novel species of an Emmonsia-like fungus, recently named Emergomyces africanus, was identified as a cause of disseminated mycosis in HIV-infected persons in South Africa. However, in contrast to C. neoformans, the enzymes produced by this fungus, some of which may be involved in pathogenesis, have not been described. Using a clinical isolate of C. neoformans as a reference, the study aim was to confirm, characterise and quantify urease activity in E. africanus clinical isolates. Urease activity was tested using Christensen's urea agar, after which the presence of a urease gene in the genome of E. africanus was confirmed using gene sequence analysis. Subsequent evaluation of colorimetric enzyme assay data, using Michaelis-Menten enzyme kinetics, revealed similarities between the substrate affinity of the urease enzyme produced by E. africanus (Km ca. 26.0 mM) and that of C. neoformans (Km ca. 20.6 mM). However, the addition of 2.5 g/l urea to the culture medium stimulated urease activity of E. africanus, whereas nutrient limitation notably increased cryptococcal urease activity.

전통 누룩으로부터 호산성 Urease 생산 효모의 분리 및 특성

Two hundred and twenty three yeast strains were randomly isolated from Korean traditional . Among them, six urease producing yeast strains (designated JJA, JJB, JJ22, SHA, SHC and SH10) were selected on the Christensen urea agar plates. They showed the same pattern in the PCR-RFLP analysis of the ITS I-5.8S-ITS II region digested with III and 1 restriction endonucleases. Its DNA sequences showed 100% (strains SHA, SHC and SH10) and 99.8% (strains JJA, JJB and JJ22) identity with those of type strain ATCC 24210. Phylogenetic analysis resulted in that all the strains were closely related to . Two representative strains, JJ22 and SH10, showing the highest urease activities were selected for further characterization. Their morphological, physiological and biochemical characteristics were also the same as . Therefore, both the two strains were identified as . They could grow at a wide range of temperature between as well as pH between 2.0 and 10.0. However, a higher level urease activity were obtained at acidic pH than that at alkalic pH. The maximal level of urease activity was obtained at (strain SH10) or (strain JJ22) and in a liquid medium adjusted to the initial pH 5.0.

A Single-Tube Screen forSalmonellaandShigella

Salmonella and Shigella species are routinely sought in stool specimens submitted for culture. It is a common practice to screen lactose-negative colonies by using triple sugar iron agar, lysine iron agar, and Christensen urea agar to determine if further identification is necessary. We designed and evaluated a novel combination of media, which are layered in a single tube, for screening isolates suspected to possibly represent Salmonella or Shigella. We tested this media combination with 106 Salmonella, 56 Shigella, and 56 other gram-negative bacilli. All Salmonella and Shigella isolates tested were appropriately characterized as possible Salmonella or Shigella by using an algorithm developed for use with this media combination. Similarly, 53 (95%) of 56 other gram-negative bacilli were appropriately screened as non -Salmonella and non -Shigella isolates. This unique media combination provides the most important biochemical reactions needed to screen for Salmonella and Shigella in a single-tube format, which decreases labor by two thirds (ie, 1 tube is inoculated vs 3).

Diagnostic value of morphological, physiological and biochemical tests in distinguishingTrichophyton rubrumfromTrichophyton mentagrophytescomplex

The two most frequently encountered dermatophyte etiologic agents of glabrous skin and nail dermatophytoses are Trichophyton rubrum and T. mentagrophytes. This study was aimed to discuss the efficacy of morphological, physiological and biochemical diagnostic tests commonly used in the identification of T. rubrum and members of the T. mentagrophytes complex. In this study, we evaluated; hydrolysis of urea in broth and on urea agar slants and Petri plates incubated at 22 degrees C, 28 degrees C and 37 degrees C, in vitro hair perforation (blond child, sheep and goat hair), pigment production on cornmeal dextrose agar (CMDA) and bromcresol purple-milk solids-glucose agar (BCP-MS-G), Tween opacity, sorbitol assimilation, and salt tolerance. Additionally, the production of micro- and macroconidia was investigated by using brain heart infusion agar (BHIA), Christensen's urea agar in Petri plates (UPA), CMDA, Lowenstein-Jensen agar (LJA), malt extract agar, oatmeal agar, Oxoid chromogenic Candida agar, and potato dextrose agar. All cultures were incubated at 28 degrees C, and conidial production was compared on days 5, 10 and 15. It was found that the urea hydrolysis test yielded more rapid and significant results when urea medium was prepared in Petri plates and incubated at 28 degrees C (P<0.01). LJA supported the highest production of microconidia after 15 days (P<0.001). Additionally, it was found that T. rubrum strains produced red pigment on CMDA (P<0.01) and BCP-MS-G, while strains of the T. mentagrophytes species complex did not. A special algorithm containing the various test procedures employed in these studies is presented which was found to be useful in the differentiation of T. rubrum strains from T. mentagrophytes complex. Our results revealed that UPA, CMDA, BCP-MS-G, LJA, and BHIA may be used as common mycological agars in routine practice.

Histoplasma capsulatum variety duboisii isolated in Japan from an HIV-infected Ugandan patient.

A strain of Histoplasma capsulatum var. duboisii (deposited as IFM 50954 in Chiba University) was isolated from the cerebrospinal fluid of a female Ugandan patient infected with HIV. The isolate had in vitro urease activity on Christensen's urea agar slants, although the common belief is that H. capsulatum var. duboisii is urease negative, and is, considered one of the characteristic markers that distinguishes the three varieties of H. capsulatum. Forty H. capsulatum var. capsulatum, five H. capsulatum var. duboisii, and five H. capsulatum var. farciminosum isolates were evaluated for urease activity on Christensen's urea agar slants and for other qualitative and quantitative urease assays of activity. All 50 isolates of H. capsulatum used in this study were positive for urease activity, suggesting that urease activity may be universal characteristic of H. capsulatum. We also compared the urease activity and pathogenicity of seven H. capsulatum isolates that convert into yeast-form cells. Although isolate IFM 50954 showed moderate virulence in mice and moderate urease activity among tested H. capsulatum isolates, there was no correlation between level of urease activity and pathogenicity. In addition, scanning electron microscopy revealed that some microconidia of isolate IFM 50954 formed "double-cell" configurations that were attached to each other by narrow bases.

The isolation ofCryptococcus neoformans from pigeon droppings and serotyping of naturally and clinically sourced isolates in China

This is the first report on the isolation of Cryptococcus neoformans from pigeon droppings in China and their serotypes. C. neoformans colonies which produced brown colonies on caffeic acid-cornmeal agar were found in Twenty-five out of thirty-six samples of pigeon droppings. Fifty-one colonies randomly picked from the positive samples were identified as C. neoformans by a commercially available kit for carbon source assimilation test and Christensen's urea agar. Forty (78%) out of the 51 strains were serotyped as A and 11 (22%) as AD. At the same time, seventeen out of nineteen clinical isolates were serotyped as A and 2 as B. There are three findings in our results. One is that only C. neoformans var. neoformans strains could be isolated from pigeon droppings, although the variety gattii strains were found in the clinical isolates obtained in the same geographic site in China. The second is that serotype A strains were most frequently seen in natural and clinical materials in the southeast part of China, and serotype AD strains were isolated in pigeon droppings but not in clinical materials. The third is that the coexistence of serotype A and AD cells of C. neoformans strains in same samples of pigeon droppings were observed.

On the false positive urease activity ofYarrowia lipolytica

The ability of Yarrowia lipolytica to produce ammonia from urea was found variable on some media. The colour change of the indicator in Christensen's urea agar was not due to the urease activity of this species but was a non-specific alkalization reaction. Rapid urea broth was reliable giving no false positive results. It was found that Y. lipolytica is a urease negative yeast species.

Microbiological, physical and chemical studies of Livestock-farm water in Taiwan

The properties of drinking water from 42 hog farms, including pH, color, turbidity, total soluble solids, concentrations of ammonium nitrogen, nitrate nitrogen, nitrite nitrogen, chlorine, fluorine, sulphate, calcium, copper, zinc, iron, manganese and chromium were investigated. The total number of coliforms, fecal coliforms, fecal streptococci and standard plate counts of both drinking water and waste water were also studied. It was found that all the waste water and some of the drinking water was polluted. The major pollutant was hog feces. The culture counts of aerobic Gram-negative bacilli of fresh feces and fecal polluted water on one hog farm were examined in detail by using selective media. There were 364 strains of pure cultures isolated from MacConkey agar. The biochemical characteristics of the isolates on the triple sugar iron agar, Simmon's citrate agar, Christensen's urea agar, sulfide-indole-motility medium, Voges-Proskauer broth, Moller's ornithine decarboxylase medium, etc., were investigated. Microbial identification was carried out according to a computer-coding system based on the characteristics shown in the above media. The identified species were Escherichia coli (71%), Enterobacter agglomerans (6%), Enterobacter cloacae, Citrobacter freundii, Citrobacter diversus, Citrobacter amalonaticus, Aeromonas hydrophila, Arizona hinshawii, Vibrio sp., Hafnia alvei (Enterobacter hafniae), Chromobacterium violaceum, Yersinia enterocolitica, Plesiomonas shigelloides, Serratia liquefaciens, Pectobacterium sp., Proteus mirabilis, Edwardsiella tarda, Vibrio vulnificus, Provindencia rettgeri, Provindencia alcalifaciens, Proteus vulgaris, Klebsiella ozaenae, Klebsiella sp., Shigella sp., Morganella morganii, Serratia marcescens and Vibrio (EF-6) group F. The significance of these isolates for public health is discussed.

Simple spot test for rapid detection of urease activity.

A spot test for the detection of urease activity was developed and evaluated with 761 strains of gram-negative bacteria. The test was compared with the conventional Christensen urea agar slants and urease test on the Vitek Enterobacteriaceae card (Vitek Systems, Inc., St. Ana, Mo.). Of the 348 strains of the Proteus-Providencia-Morganella group that were urease positive, 327 (94%) yielded positive results within 1 min, and all strains yielded positive results within 2 min. All these organisms also gave a positive urease reaction on the Vitek Enterobacteriaceae card test within 5 h and on the Christensen urea agar slants in 4 to 48 h. All the bacteria that did not hydrolyze urea by these two tests also remained negative by the spot test.

Rapid method for detection, identification, and susceptibility testing of enteric pathogens.

Three hundred and seven colonies believed to be enteric pathogens were selected from primary plates of MacConkey, xylose desoxycholate, or salmonella-shigella agar for inoculation to lactose-sucrose broth, urea-41 motility medium, modified Andrade glucose broth with inverted Durham tube, pregrowth broth, triple sugar iron agar, lysine iron agar (LIA), and Christensen urea agar. The rapid screen consisted of interpreting the lactose-sucrose, urea-41 motility, and modified Andrade glucose broth gas reactions after 4 to 6 h at 35 degrees C. These rapid screening biochemicals plus LIA were incubated for 24 h if further interpretation was required. Reference biochemicals (triple sugar iron, LIA, and Christensen urea agars) were interpreted at 24 h. Of 307 isolates, 49 (16%) were reported as negative for enteric pathogens after 4 to 6 h because their biochemical profiles were not compatible with those for enteric pathogens. A total of 87 (28.3%) isolates produced biochemical profiles at 4 to 6 h that were presumptive for enteric pathogens. The 87 presumptive pathogens were inoculated into the AutoMicrobic system Gram-Negative General Susceptibility Card and the AutoMicrobic system Enterobacteriaceae-Plus Biochemical Card (AMS-EBC+) after 4 to 6 h of growth in pregrowth broth. Of these isolates, 63 were confirmed to be enteric pathogens, of which 61 (96.8%) were correctly identified by the AMS-EBC+. One isolate was identified as Shigella dysenteriae by AMS-EBC+ but confirmed as Shigella flexneri biotype 6 by a reference laboratory. The other isolate was identified as Arizona hinshawii by AMS-EBC+ but was confirmed as Salmonella enteritidis. Of the 307 isolates, 166 (54.1%) required further interpretation of the rapid screening biochemicals plus LIA at 24 h; 5 of these were detected as enteric pathogens. The same 68 enteric pathogens were detected by both the rapid method and the reference method. The results from the general susceptibility card agreed with agar diffusion results at 99.2%. One Salmonella enteritidis and four Shigella spp. showed minor discrepancies with tetracycline. No very major or major discrepancies were observed.

An abbreviated scheme for identification of Yersinia enterocolitica isolated from food enrichments on CIN (cefsulodin-irgasan-novobiocin) agar.

An abbreviated procedure for the biochemical identification of Yersinia enterocolitica isolated from food enrichments on CIN (cefsulodin-irgasan-novobiocin) agar was investigated. A total of 170 colonies resembling Y. enterocolitica in colonial morphology and appearance on CIN agar were selected for identification using API strips. Ninety-three of these isolates were examined with the PathoTec ornithine decarboxylase, Voges-Proskauer, and urease test strips. The PathoTec urease strip alone was adequate for identification of all isolates of Y. enterocolitica. Christensen's urea agar was applied to the remaining 77 isolates and found less specific in the 1 isolate of Enterobacter agglomerans was urease positive along with 10 isolates of Y. enterocolitica. CIN agar is a highly specific medium for isolation of Y. enterocolitica, requiring only Kligler iron agar and urea slants for confirmation of presumptive colonies.

Utilization of standard laboratory methods in the laboratory diagnosis of problem dermatophytes.

The identification of certain dermatophytes may be simplified by using biochemical tests such as urease, nutritional requirements (with commercially available media), and the in-vitro hair penetration test. No study that combines these tests in a diagnostic scheme for identification of the common dermatophytes has been published. one to 20 isolates each of 29 species of dermatophytes (one Epidermophyton floccosum, 10 Microsporum species, and 18 Trichophyton species) were used. They were grown on Christensen's urea agar and the seven nutritional media for the differentiation of the Trichophyton species; the in-vitro hair penetration test was performed in duplicate. Patterns were developed that have been tested and proven to be useful for more than 22 months. In addition, the Microsporum species were all grown on polished rice, and color and sporulation were recorded. All dermatophytes in this study were grown on either Mycosel agar or 2% malt extract agar, and on modified potato dextrose agar and modified Sabouraud agar. Macroscopic and microscopic examinations were made to determine qualitatively the amounts of growth and sporulation on each medium.

Evaluation of a multitest system for rapid identification of Salmonella and Shigella.

The ability of Micro-ID, a multitest system for rapid (four hour) identification of Enterobacteriaceae, to identify Salmonella and Shigella was evaluated. Micro-ID, API 20E and a battery of tubed media consisting of triple sugar-iron agar, Christensen's urea agar, and Moeller's lysine decarboxylase medium were used to study 516 lactose nonfermenting strains of Enterobacteriaceae isolated from 500 consecutive pediatric stool specimens. Fifty-six of the isolates were Salmonella, and 21 were Shigella. Micro-ID correctly identified all isolates of Shigella and all but one isolate of Salmonella, whereas the conventional screening media failed to detect seven isolates of Salmonella and two isolates of Shigella. The false-positive rates were 1% and 20% for Micro-ID and the conventional battery, respectively. Use of Micro-ID as a substitute for conventional screening media for lactose nonfermenting stool isolates provides reliable presumptive idenfication of Salmonella and Shigella within four hours.

Vibrio parahaemolyticus: suspicion of presence based on aberrant biochemical and morphological features.

Vibrio parahaemolyticus was isolated from a stool specimen of a patient who developed gastroenteritis after ingestion of crab meat. Recognition and identification of this halophilic microorganism was facilitated by the microscopic observation of a darting, vibrant motility in condensate derived from Kligler iron agar and the bizarre morphological aberrations noted in the condensate obtained from Christensen urea agar. Sodium chloride supplementation (1.1%) of biochemical test media revealed the halophilic nature and fermentative capability of the isolate and abolished the aberrent morphology observed in unsupplemented Christensen medium.

Rapid urea broth test for yeasts

A rapid, miniaturized, urea broth test useful for detecting urease activity of yeasts was compared to Christensen urea agar. All urease-producing yeasts tested were positive on both media; however, 60% were reactive in the urea R broth within 30 min, and the remainder were reactive within 4 h. This urea multiwell test may be useful as a rapid screening method for detecting urease-producing yeasts recovered from clinical specimens and as an adjunct test with other rapid methods of yeast identification.

Rapid method for detection of urea hydrolysis by yeasts

A method is described for the rapid detection of urea hydrolysis by yeasts, using the Berthelot color reaction. The results could be determined within 30 to 50 min with this method, compared with 8 to 72 h usually required with Christensen urea agar.

Urease Activity of Enterobacteriaceae: Which Medium to Choose

Detection and intensity of urease activity in enterobacteriaceae greatly varies as a function of the media or techniques used, or both. A comparative investigation on several solid and liquid media led us to the following conclusions. (i) Detection of Proteus spp. can be adequately performed with the highly selective solid medium described by Cook (1948), as well as with the different liquid media described (Stuart standard and rapid media; Elek medium). (ii) Detection of Klebsiella should be based upon urease production on solid media with low buffer capacity (Christensen, 1946). (iii) For the identification of Yersinia, either the solid Christensen urea agar or the rapid Elek technique give optimal results.

Urease-positive oral viridans streptococci.

Products released from the hydrolysis of urea, as well as other metabolites, are considered to be histotoxic; therefore, they cause inflammation to tissues, as in periodontal diseases (R. J. GIBBONS, Int. Dent. J., 14:407-10, 1964). Some bacteria indigenous to the oral cavity are known to produce urease, but this property is generally unknown among the streptococci. In a study of oral micrococci, streptococci, and fecal streptococci, seven of 20 Streptococcus mitis-like species and 15 of 16 micrococci were found to be urease-positive (R. F. SMITH and H. L. BODILY, IADR Preprinted Abstract No. 239, 1967). A study was conducted to confirm this report and determine the frequency of urease-positive streptococci. Unstimulated saliva, taken at random from healthy adults, was conventionally diluted or directly streaked on mitis-salivarius and mannitol salt agars* to isolate streptococci and micrococci, respectively. Gram stains and the catalase test were used to aid separations. Purity was checked by restreaking each medium with isolated strains. Routine identification was conducted by standard procedures.t Urease activity was measured by inoculating Christensen's urea agar slants,T which were incubated at 37'C. for 24 hours (Table). Urease was found among the micrococci (81 percent) and in a small number of streptococci (14 percent). Each group was easily isolated by generally accepted diagnostic tests for identification.