Cystine Lactose Electrolyte Deficient Research Articles

Laboratory Dilutions of Thioridaxine with Potential to Enhance Antibiotic Sensitivity in a Multidrug Resistant Escherichia Coli Uropathogen

This research effort seeks to use doses of thioridaxine to enhance antibiotic sensitivity in a MDR Escherichia coli strain. Five axenic (pure) strains of Escherichia coli coded EC1 to EC5 were obtained from five infected midstream urine samples among several other urine samples inoculated on sterile Cystine Lactose Electrolyte Deficient (CLED) agar with appropriate labeling in the Microbiology and Biotechnology Laboratory of Western Delta University, Oghara, Nigeria and stocked on sterile Nutrient agar slants at 4oC in a refrigerator. Slant cultures were sub-cultured aseptically on fresh sterile CLED agar plates and incubated aerobically at 37oC for 24hrs to confirm Escherichia coli strains. Gram staining, indole production, methyl red test, voges praskaeur and citrate utilization tests were done on the resulting colonies to further confirm the strains as E.coli. Antibiotic susceptibility test was done by agar disc diffusion method on all confirmed strains on sterile Mueller- Hinton agar plates before and after treatment with laboratory dilutions of thioridaxine. Only E.coli strain 2 (EC2) was multidrug resistant as it resisted 4(44.4%) of the antibiotics used which were cefuroxime, nalidixic acid, augmentin and tetracycline. Other strains resisted 1-2 antibiotics. The highest (15.6±20.6mm) and least (2.0±8.1mm) zones of inhibition by all five strains were recorded for ofloxacin and cefuroxime respectively. Whereas all five uropathogen strains resisted augmentin, they were sensitive to ciprofloxacin, ofloxacin (both being fluoroquinolones), gentamicin, chloramphenicol and nitrofurantoin. After treatment with 2000-2240ug/ml laboratory dilutions of thioridaxine, ≤50.0% loss of resistance was recorded for 2040ug/ml, 2160ug/ml and 2240ug/ml dilutions. Thioridaxine dilution of 2040ug/ml induced 250% and 90% resistance losses of EC2 to ciprofloxacin and nitrofurantoin respectively with an overall mean±S.E loss of 68.0±24.4%. Resistance losses of 112.5%, 130.0% and 100.0% to ciprofloxacin, nitrofurantoin and chloramphenicol respectively were recorded after 2160ug/ml treatment and 68.5±16.3% overall loss of resistance. Thioridaxine dilutions of 2240ug/ml induced 55.6±25.0% overall loss of resistance with a corresponding 50.0%, 50.0%, 58.1%, 70.0% and 50.0% resistance losses with gentamicin, ciprofloxacin, ofloxacin, nitrofurantoin and chloramphenicol respectively. Less than 50% resistance losses were recorded for 2000, 2080, 2120 and 2200ug/ml dilutions. Minimum inhibitory concentration of chloramphenicol was lowered by 2080ug/ml, 2160ug/ml and 2240ug/ml dilutions by two-fold (15ug), two-fold (15ug) and four-fold (7.5ug) respectively. The medical/ chemotherapeutic implications of these findings are discussed.

English

Drug resistant Escherichia coli persist in the intestinal flora of poultry birds, and these serve as route via which they can be transmitted directly to humans, thus contributing to the already growing crisis of antibiotic resistance. The purpose of this study was to determine the cloacal feacal carriage and occurrence of antibiotic resistant E. coli isolates from chicken fed with and without antibiotic supplemented feeds. Cloacal feacal swabs (n = 200) were aseptically obtained from two poultry farms in Abakaliki metropolis, Ebonyi state of Nigeria, and these were inoculated on MacConkey and cystine-lactose-electrolyte-deficient (CLED) media and incubated at 37°C for 18 to 24 h. Suspected colonies of E. coli growing on the agar media were subcultured, purified and further characterized using standard microbiology techniques. Antibiogram was investigated using the Kirby-Bauer disk diffusion method as per the clinical laboratory standards institute (CLSI) criteria. A total of 45 E. coli was isolated from the 200 cloacal feacal swab samples used for this study. Overall, 28% of E. coli were isolated from chicken fed with feed supplemented with antibiotics while only 17% of E. coli was isolated from chicken that received feed without antibiotics supplements. All the E. coli isolates showed varying rates of resistance and susceptibility to the tested antibiotics. Our results strongly reveal the occurrence of antibiotic resistant E. coli from chicken fed with and without antibiotic supplemented feeds. It is very critical that the continuous use of antibiotics in poultry production be strictly monitored, controlled and discouraged in order to contain the emergence and spread of antibiotic resistant bacteria through poultry production. Key words: Resistance, Escherichia coli, poultry, veterinary, Nigeria.

Prevalence and antibiotic susceptibility of uropathogens among patients attending University of Abuja Teaching Hospital, Gwagwalada, Abuja

A urinary tract infection (UTI) is a condition where one or more structures in the urinary tract become infected by the presence and growth of microorganisms that overcome the structures strong natural defenses. This study reports the examination of bacterial organisms implicated in urinary tract infections among 180 patients suspected to have UTI that attended University of Abuja Teaching Hospital (UATH), Gwagwalada, Abuja for a period of 3 months and the antibiotic susceptibility patterns of the bacterial isolates. First morning mid stream urine sample cultures were done by the calibrated loop technique delivering 0.002 ml of urine plated on Cystine Lactose Electrolyte Deficient (CLED) agar and blood agar medium for isolation of uropathogens which were further analyzed for drug susceptibility by disk diffusion method. Result showed that 104 (57.78%) patients had urinary tract infection. The uropathogens isolated were Escherichia coli as the most abundant (43.27%) followed by Staphylococcus aureus (35.57%), Klebsiella species (11.54%), Proteus species (5.77%) and Pseudomonas species (3.85%) as the least occurring. Invitro antibiotic susceptibility test revealed that the Gram negative bacteria were most sensitive to quinolones (ofloxacin and levofloxacin), gentamycin and nalixidic acid while the Gram positive isolates were sensitive to erythromycin, chloramphenicol and quinolones (ofloxacin and levofloxacin). The most effective drug in both cases was the quinolones–ofloxacin (78.32%) and levofloxacin (76.98%), while the least sensitivity pattern was observed with ampicillin (14.29%). Key words: Urinary tract, uropathogens, antibiotics susceptibility.

Urinary tract infection among symptomatic outpatients visiting a tertiary hospital based in midwestern Nigeria.

Microbial pathogens implicated in urinary tract infection and their antibiotic susceptibility patterns as prevalent in UTI symptomatic outpatients resident in Benin City, Nigeria was the focus of this study. One hundred (100) midstream urine samples were collected into sterile plastic universal bottles from outpatients who visited the University of Benin Teaching Hospital, Nigeria and who were tentatively diagnosed as manifesting symptoms of UTI. Patients were referred to the Medical Microbiology department by the consulting doctors. Significant bacterial counts and neutrophil (pus cells) counts were carried out on samples by standard methods. Positive samples for both counts were inoculated aseptically on sterile MacConkey agar, Cystine Lactose Electrolyte Deficient (CLED) agar and Sabouraud Dextrose agar plates and incubated appropriately. Microbial isolates were identified and antibiotic sensitivity testing was carried out on isolates by standard methods. Thirty nine (39.0%) and 61 (61.0%) samples recorded significant microbial growth and no growth respectively. Gram negative bacilli constituted 86.1% (of which enterobacteriaceae made up 49.9%) while gram positive cocci made up 13.9%. Strains of uropathogens isolated were Alcaligenes spp (19.4%), Klebsiella aerogenes (16.7%), Escherichia coli (13.9%), Staphylococcus aureus (13.9%), Candida albicans (11.1%), Proteus mirabilis (8.3%), Pseudomonas aeruginosa (5.5%), Enterobacter spp (5.5%) and Providencia spp (5.5%). Occurrence of UTI in male and female patients were 58.3% and 41.7% respectively of which UTI occurred highest in the 25-46, 15-54 and 27-54 age groups in that decreasing order. Alcaligenes spp occurred most in very old female patients. Candida albicans (the only fungal uropathogen) occurred in an 8day old male patient. Other isolates occurred in much older patients. A significantly high microscopic neutrophil count or pyuria was recorded from deposits of UTI positive patients (i.e. < 5/HPF). Eighteen (representing 50.5%) and 15 (47.8%) of total microbial strains isolated were sensitive to nitrofurantoin and ceftriaxone respectively. Antibiotic susceptibility profile also showed 13(41.6%), 13(41.6%), 13(41.6%) for ciprofloxacin, cefuroxime and ofloxacin respectively suggesting moderate sensitivity of the fluoroquinolones and second/third generation cephalosporins. Gentamicin, ampicillin and augmentin recorded over 70.0% resistance level each. A total of nineteen bacterial strains made of E. coli, Enterobacter spp, Proteus mirabilis, Providencia spp, Staph. aureus and Pseudomonas aeruginosa were multi drug resistant as they resisted 3, 3, 4, 4, 5 and 8 antibiotics respectively.

Studies on Drug Sensitivity and Bacterial Prevalence of UTI in Tribal Population of Paschim Medinipur, West Bengal, India

Background: There is paucity of information in regards to prevalence of bacteria in urinary tract infection (UTI) in tribal population. Objectives: This study was conducted to observe dominant organisms causing UTI in tribal community of Medinipur, West Bengal, India and evaluate their sensitivity to common antibiotics. Patients and Methods: A total number of 4,416 urine samples of clinically suspected UTI attending outpatient department and hospitalized tribal patients of West Medinipur zone were collected. Urine samples were inoculated on Blood agar, MacConkey’s agar, and cystine lactose electrolyte deficient (CLED) agar and incubated at 37oC for 24 hr and extended up to 48 hr in cases of negative growth. Antibiotic susceptibility testing against the most common causative bacteria was also performed. Results: A total number of 1,190 samples revealed positive bacterial growth. Seven types of dominant organisms were isolated as causative agents like Escherichia coli (63.44%), Klebsiella sp. (14.62%), Pseudomonas aeruginosa (4.53%), Proteus sp. (4.62%), other Gram negative bacteria (5.79%), Staphylococcus aureus and coagulase negative Streptococci (5.21%), and other Enterococcus sp. (1.76%). E.coli was the most common pathogen of UTI in tribal population with female susceptibility predominantly more than male patients. Among16 antibiotics, amikacin (14.30%) and meropenem (12.0%) were shown to be the most sensitive to and Tobramycin (0.66%) the least effective on E.coli. Conclusions: Females are more susceptible to UTI than males and the most effective drug (sensitive against isolated E. coli) is Amikacin. Therefore, culture and antimicrobial drug sensitivity testing are essential procedures for proper management of UTI in tribal population.

A Study on the Interrelationship between Renal Calculi, Hormonal Abnormalities and Urinary Tract Infections in Iraqi Patients

Objective: The main objective of the present study is to assess the interrelationship between renal calculi, selected ions, thyroid and parathyroid hormones and urinary tract infections. Methods: This study was carried out on 150 patients attended Tikrit Teaching Hospital from 2008 to 2009. Stones and serum ions were analyzed utilizing relevant Biolab Company kits (France). Thyroid hormones were determined using ELISA microwells kit (Accuubind, USA) Parathyroid hormone was estimated by active I-PTH ELISA (DSL, USA).Urine cultures were done utilizing cystine-lactose- electrolyte deficient (CLED) medium. Various isolated pathogens were conventionally identified. Results: Urine cultures revealed that 42% of the patients had urinary tract infections particularly with Enterobacteriaceae. 68% of the stones tested were calcium oxalate. Infective and noninfective stones were classified. Calcium ion was more elevated in patients examined. Hypothyroidism was prevalent but parathyroid hormone (PTH) was elevated among 14 patients only. Conclusions: The frequency of UTI was higher among urolithiasis patients and the common causative agents were Gram negative bacteria. Renal stones of calcium origin were predominant. Hormonal abnormalities were seen. 15 patients with hyperthyroidism revealed hypercalcemia and hypercalciuria.

Prevalence and Antibiotics Susceptibility of Uropathogens in Patients from a Rural Environment, Tamilnadu

Problem statement: Urinary Tract Infections (UTIs) in human, rural en vironment cause significant morbidity due to insanitary condition, lack of knowledge of personal hygiene, lack of patient's compliance and economic burden. In our st udy, the bacterial etiologies and the resistance patterns found in human with serious UTIs and selec ting optimal antimicrobial therapy. Approach: A total of 105 patients first morning mid stream urin e samples, culture was done by the calibrated loop technique delivering 0.001 mL of urine plated on Cy stine-Lactose-Electrolyte Deficient (CLED) agar, MacConkey agar and Blood agar medium (Hi Media, India) for isolation of uropathogens. All pathogens were analyzed for drug susceptibility by disk diffu sion method. Results: More than 50% of them were confirmed to have UTI bacteriological positive. Women and man especially in the age group of 20-29 had higher incidence of bacterial in fection. A total of 58 isolates seven different uropathogens were identified among the Escherichia coli (31%) was dominant pathogens followed by Citrobacter spp., (20%) and Pseudomonas aeruginosa (17.24%). In all uropathogens antibiotics susceptibility the more active agents were norfloxa cin (87.93%), ciprofloxacin (70.68%), gentamicin (70.68%), nalidixic acid (68.96%), nitrofurantoine (60.34%) and tetracycline (60.34%) and less active in ampicillin (8.62%), rifampicin (37.93%) and carb enicillin (41.37%). Conclusion: Among commonly used antimicrobial agents for the treatment of UTI, there is a trend towards increasing resistance to ampicillin and a persistently low res istance rate to norfloxacin, ciprofloxacin and gentamicin should be consider for treatment.

Prevalence of Urine Glucose-Associated Bacteriuria in Diabetics and Antibiotic Susceptibility Patterns of the Isolated Bacteria.

Objectives: This study aimed at determining the influence of urine glucose on prevalence of bacteriuria in diabetic patients and to carry out antibiotic susceptibility patterns testing on the isolated bacteria. Methods: This is cross-sectional study conducted at Diabetes Clinic-Muhimbili National Hospital, by collecting 133 midstream urine (MSU) specimens from 133 patients who were attending the clinic during the study period. The MSU specimens were then cultured on cystine lactose electrolyte deficient (CLED) agar medium using the standard wire loop technique. The isolated microorganisms were identified and microbial counts determined by using conventional methods. Urine glucose levels (UGLs) were determined using urinalysis reagent strips (Acon Laboratory Inc., USA). Results were interpreted and classified as negative, trace, positive (+), positive (++) or positive (+++). Antibiotic susceptibility patterns of the isolated bacteria were conducted using disk-diffusion method in accordance with National Committee on Clinical Laboratory Standards (NCCLS) guidelines. Checklists of clinical histories were also used for collection of complementary data on the patient weight and age. All the obtained data were coded and entered in computer and then analyzed using computer software SPSS version 10. Results: Majority of the patients 118(88.7%) manifested no significant bacterial growth (NSBG). Out of the patients who manifested NSBG, majority (73%) had no/negative urine glucose levels (UGLs). Escherichia coli were the most frequently isolated microorganism (18.8%), which were also the most predominantly isolated microorganisms from patients with negative urine glucose levels. Klebsiella spp and Pseudomonas aeruginosa were found in all patients with glycosuria. More females (12%) manifested higher UGLs in comparison to males' counterpart (7.5%), which coincided with the higher bacterial counts in female diabetics. All the isolated microorganisms were found to be susceptible to nalidixic acid (30µg). Nevertheless, all the microorganisms were resistant to ampicillins (10µg), flucloxacillin (10µg) and tetracycline (30µg) with exception of E. coli which was susceptible to the latter drug. Conclusion: The study revealed that E. coli was the most frequently isolated microorganisms (18.8%) and bacteriuria was relatively more prevalent in female patients (25%) compared to males (16%). Notwithstanding, majority of the patients (88.7%) manifested NSBG. Direct correlation between bacteriuria and UGLs was observed though its relation is still unclear. The antibiotic resistances identified make it necessary for antibiotic susceptibility testing to be carried out prior antibiotic prescription. Tanzania Medical Journal Vol. 23 (1) 2008: pp. 16-19

Etiology and prevalence of urinary tract infection in primary school children in Port Harcourt metropolis, Nigeria

A total of two hundred urine samples were collected from pupils aged 4-14 years from four different primary schools in Port Harcourt Metropolis. The urine samples were cultured on Cystine Lactose Electrolyte Deficient (CLED) medium using standardized wire-loop technique and incubated aerobically at 370C for 24h. Twenty-nine (14.5%) samples were found to have significant bacterial growth (>105 organisms). Twenty-two (75.86%) of which were from females and seven (24.14%) from males. Sixteen (55.17%) of the bacteriuric pupils belong to the 10-12 age group. Eschericia coli (48.28%) was the most frequently isolated organism. Others were Klebsiella aerogenes (34.48%) Streptococcus faecalis (10.34%), Proteus mirabilis (3.45%) and Staphylococcus aureus (3.45%). Gentamycin was found to be the most effective anti-microbial. . International Journal of Natural and Applied Sciences Vol. 2 (4) 2006: pp. 362-365

Evaluation of a new chromogenic medium, Uriselect 4, for the isolation and identification of urinary tract pathogens

Aims: To compare the performance of a new chromogenic medium, Uriselect 4, with cystine lactose electrolyte deficient (CLED) agar and an established chromogenic agar, CPS ID 2 medium, for detection...

Prevention of biofilm formation in dialysis water treatment systems

Biofilm formations in dialysis systems may be relevant because they continuously release bacterial compounds and are resistant against disinfection. The aim of the study was to compare the development of biofilm between a water treatment system based on a single reverse osmosis unit producing purified dialysate water [bacterial count, 350 colony-forming unit (CFU)/L] (center A) and a water treatment system based on double reverse osmosis and electric deionization, which is continuously disinfected with ultraviolet light and treated with ozone once a week (bacterial count, 1 CFU/L) (center B). During a period of 12 weeks, biofilm formation was studied in the tubing segment between the water piping and the dialysis module, using four dialysis monitors in each center. On a weekly basis, tubing samples of 5 cm length (N = 96) were taken under aseptic conditions and investigated for microbiologic contamination [cystine lactose electrolyte-deficient (CLED) Agar], endotoxin levels [limulus amoeben lysate (LAL) gel test, cutoff value, 0.0125 EU/mL], and biofilm formation [electron scanning microscopy (SEM)]. In center A, tube cultures were positive (>100 CFU/mL) in 16% of samples at 22 degrees C and 37 degrees C, compared to 3% of samples of center B (P < 0.05; chi-square). Endotoxin levels were positive in 76% of the tubing samples of center A and negative in all of the samples of center B (P < 0.05). Biofilm was present in 91.7% of the samples of center A (Fig. 1), and only present in one sample (taken after 9 weeks) of center B (P < 0.05) (Fig. 2). In center A, biofilm formation was already observed after 1 week. In contrast to a standard water treatment system producing purified water, the use of a system producing highly purified water, which is also treated with regular disinfection procedures, leads to a significant reduction in biofilm formation, bacterial growth, and endotoxin levels in a highly vulnerable part of a water treatment system.

Prevalence of Bacteria in Diabetics and Non-Diabetics

The study was carried out to determine the prevalence of bacteriuria in diabetic and non-diabetic patients attending the University of Port-Harcourt Teaching Hospital and the Diabetic Association of Nigeria, Rivers State Chapter. A total of 257 (132 diabetic and 125 non-diabetic) mid-stream urine samples were collected and cultured on cystine lactose electrolyte deficient (CLED) agar medium using the standard wire loop technique. Forty-nine (37.2%) of the non-diabetics had significant bacteriuria. Among diabetics 32 (65.31%) females and 17 (34.69%) males had significant bacteriuria while 15 (68.25%) females and 7 (31.75%) males in non-diabetics had significant bacteriuria .In both groups studied, the incidence of significant bacteriuria was highest in the age range of 18-39 years for both groups, 55.11% in the diabetics and 68.25% in non-diabetics. Escherichia coli and Staphylococcus aureus were the most frequently isolated bacteria in the two groups, statistical analysis showed a significant prevalence of bacteriuria in diabetics compared with non-diabetics (P Key words: Bacteriuria, Diabetics, Non-Diabetics, Age, Sex [Mary Slessor Jnl of Medicine Vol.3(1) 2003: 52-54]

Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR.

The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to colony blots from five randomly picked E. coli-like colonies from CLED (cystine lactose electrolyte deficient) agar plates inoculated with the feces. When these probes were applied to blots from whole stool cultures collected from the agar plates (sweep blot), the detection limit was 10(6) CFU/g of feces. PCR of the sweep material could detect toxin genes when the concentration of ETEC strains was 10(2) CFU/g of feces. Results obtained with stool specimens from 29 healthy control subjects were negative. Testing stool specimens from 50 patients confirmed the observation that the number of samples containing ETEC enterotoxin genes was higher when PCR of sweeps was used than when oligonucleotide DNA probe hybridization of either sweep blots or colony blots was used. Furthermore, PCR of sweeps is an easy and rapid method which does not require DNA extraction and purification from fecal specimens.

Simple technique for detecting K1 antigen of Escherichia coli.

The K1 antigen is a poor immunogen, its detection by serological means is difficult, and previously described methods using K1 specific bacteriophages require standardised suspensions of five different bacteriophages. A simple technique was developed which uses an unstandardised suspension of a single K1 specific bacteriophage applied with a 1 mm wire loop to bacteria streaked on to cystine lactose electrolyte deficient (CLED) medium. The technique correctly identified all 99 known K1 strains tested, including 14 strains negative with the serological method. Among 71 clinical isolates from urinary tract infections, the single bacteriophage method distinguished 30 K1 strains from 41 strains without this antigen. Suspensions of the bacteriophage were shown to remain fully active for at least two years when stored at 4 degrees C. It is concluded that this technique required so little in materials, time, or equipment that it could be routinely used in most laboratories.

Sterilization of MacConkey agar and CLED medium by γ-radiation

MacConkey agar and Cystine-Lactose-Electrolyte-Deficient (CLED) agar, media widely used in the bacteriological laboratory and recommended for the detection of urinary tract infections, were sterilized by γ-radiation at a dose of 1.5 Mrad. Both were modified and adapted to radiation sterilization by adding sodium thioglycollate as a radioprotectant, and by increasing their indicator content. The media performed well when tested with different Enterobacteria and other micro-organisms. Growth and change of indicator reaction were equal in irradiated and autoclaved culture media. Culture media were also evaluated after storage for one month at room temperature and at 4°C and compared well with freshly autoclaved media.