m-Endo Agar Research Articles

Isolation and Identification of Coliform Bacteria and Multidrug-Resistant Escherichia coli from Water Intended for Drug Compounding in Community Pharmacies in Jordan.

(1) Background: Water is necessary for the preparation of some medicines found in pharmacies where the local water source does not meet the required purity. This study aimed to investigate the presence of coliform contamination in water used for drug reconstitution in community pharmacies in Jordan. (2) Methods: Two water samples from 50 randomly selected community pharmacies representing all Jordanian governorates were filtered and then cultured in plate count agars to determine total microbial count, and in m-Endo Agar Les and Eosin Methylene Blue (EMB) agar to cultivate Escherichia coli (E. coli). The presence of E. coli was further characterized with gram stains, biochemical tests, and Polymerase chain reaction (PCR). Antibiotic susceptibility of isolated E. coli was tested against a variety of standard antibiotics. (3) Results: Community pharmacies used droppers filled with water from coolers (62%), bottled water (20%), boiled tap water (16%) and tap water (2%). The majority of the sampled water contained coliform bacteria (88%), and E. coli was isolated from 26% of all samples. Statistical analysis showed no significant difference in the percentage of contaminated water samples based on its source location. Nonetheless, the results showed a tendency for higher proportions of contamination in droppers filled from boiled tap water (37.5%; SE: 17.1), followed by water from water coolers (25.8%; SE: 7.9), and then from bottled water (20%; SE: 12.7). All of the isolated E. coli were sensitive to gentamycin, ciprofloxacin and levofloxacin. The susceptibility of the isolates to ceftazidime, doxycycline, tetracycline, azithromycin and amoxicillin/clavulanic acid were 92%, 61%, 46%, 23% and 15%, respectively. (4) Conclusions: This study confirms the widespread presence of multidrug-resistant bacteria in water intended for reconstituting drugs in local pharmacies. These findings expose an alarming situation that needs special attention by the acting pharmacists and competent authorities. Higher levels of personal hygiene in the pharmacies coupled with regular inspection of water quality may reduce the risk of microbial contamination in compounded products, especially multidrug-resistant strains of E. coli and other index microorganisms.

Microbial Analysis of Drinking Water from Randomly Selected Boreholes and Shallow Wells around Hargeisa, Somaliland

Background: Shallow wells and boreholes are vital sources of potable water in Hargeisa. This water can be polluted by runoff, in particular during the rainy season, causing outbreaks of waterborne infections. Objectives: This research aimed at evaluating the microbial quality of shallow wells and boreholes water around Hargeisa, Somaliland. Methods: The total coliform and Escherichia coli count were done by using the membrane filtration method. Overall, 100 ml of each water sample was filtered via a 0.45 μm membrane filter, and then the filters were put on m-Endo agar plates that were incubated at 37°C for 24 to 48 hours. Results: The mean value of total coliform counts for the boreholes and shallow wells ranged from 1.288 × 103 to 8.8 × 103 CFU/100ml, while the mean value of total E. coli counts also ranged from 3.5 × 102 to 4.429 × 103 CFU/100ml. Results from this study have demonstrated that all water sources (Arabsiyo, Dararweyne, Darasalaam, Dabaraqas, and Jaleelo) don’t comply with the WHO guideline for drinking water. Results from the analysis of water samples of 28 wells demonstrated a significant correlation between total coliform and E. coli counts (P = 0.01). Therefore, this water is not fit for human consumption unless it is treated. Conclusion: This study has demonstrated that all results of both mean values of total coliform and E. coli counts from groundwater of selected shallow wells and boreholes were beyond WHO standards, so water from Arabsiyo, Jaleelo, Dabaraqas, Dararweyne, and Darasalaam requires treatment before human consumption.

Impact of wave action and rainfall on incidence and antibiotic resistance of total coliforms in Southern California beaches.

Coliforms are important bacterial contamination indicators in recreational waters. Little is known about the antibiotic resistance of coliforms from Southern California beaches. This study examined the numbers of coliforms as well as the incidence of antibiotic-resistant coliforms in beaches with restricted and non-restricted wave action by sampling from the shores of both types of beaches following dry and wet weather. Total coliforms were selected by membrane filtration onto mEndo agar and then enumerated. Randomly selected isolates from each location were screened for resistance to nine classes of antibiotics by disk diffusion, and the multiple antibiotic resistance (MAR) index was calculated. Numbers of total coliforms were significantly higher following rain compared to dry weather. Total coliform numbers were not significantly elevated at non-restricted wave action sites. Restricted wave action sites had a 78.5% increase in MAR index following wet weather compared to dry weather. Resistance to ampicillin was observed in almost 50% of isolates and was not significantly impacted by wave action or weather. Minimum inhibitory concentration testing revealed that many isolates were highly resistant to ampicillin. This study is the first to report on the antibiotic resistance of coliforms found in Southern California beaches and highlights the prevalence of ampicillin resistance.

Inactivation with UV-radiation and internalization assessment of coliforms and Escherichia coli in aquaponically grown lettuce

Escherichia coli O157:H7 can internalize in produce. Therefore, a food safety risk exists for lettuce grown using mixed water sources such as aquaponics, which combines aquaculture with hydroponics. Objectives were to determine antimicrobial efficacy of UV-radiation on reduction of E. coli/coliform in a pilot-scale demonstration aquaponic system and assessment for possibility of E. coli/coliform internalization in aquaponically-grown lettuce. Water from aquaculture and hydroponic was sampled for 6 weeks prior to lettuce harvest. Water and lettuce samples were spread-plated on 3-M petrifilm™ EC (E. coli/coliform) and on m-Endo agar. The average bacterial counts in aquaculture raceways were approximately 102 CFU/ml and 104 CFU/L on 3-M petrifilm and m-Endo agar, respectively. Bacterial counts did not (p > 0.05) increase in the aquaculture raceways or hydroponic greenhouse, suggesting that neither fish nor plants contributed to bacterial growth. The coliform bacteria detected in the aquaponic system reflected their normal presence in the environment where the aquaponic system was located. UV treatment reduced (p < 0.05) bacterial counts by approximately 1.5 and 3.0-log on 3-M petrifilm and m-Endo agar, respectively. Internalized coliforms or E. coli were not detected in lettuce. This study demonstrates the effectiveness of coliform inactivation by UV-radiation in a pilot-scale real-life aquaponic system.

Bacteriological and Physicochemical Analysis of Drinking Water in Tokha, Kathmandu, Nepal

Background: This study reports the comparative studies and microbial risk assessment of different water samples used for drinking water. The results obtained were compared with WHO and EPA standards for drinking and recreational water.Methods: Physicochemical and bacteriological analysis of water samples were carried out from source, taps, well and stone spouts used for drinking purpose in Tokha (Saraswati and Chandeswari Village Development Committee). Total viable count was carried out by pour plate technique. Total coli form and fecal coli form were performed by membrane filtration technique. The results obtained were compared with World Health Organization (WHO), National Agency for Food and Drug Administration and Control (NAFDAC) and Nepal Standard of Drinking Water Quality (NSDWQ) standards for drinking water.Results: The pH, total hardness, chloride, nitrate and arsenic content of samples were found within permissible guideline value however well sample was found to exceed Nepal standard values for calcium hardness and ammonia content. The total viable counts for all the water samples were high exceeding the limit for water (1.0×102 cfu/ ml). All the water samples were found to contain coli forms and fecal organisms in numbers greater than the required WHO/FAO standards for water. The fecal coli form colonies on M-endo agar plate ranged between 143 and 152 and total coli form from 110 to 248 per 100 ml water also exceed the standard limit for water. The Isolated organisms were identified to be E.coli, Klebsiella spp. And Citrobacter spp.MED PhoenixVolume (1), Issue (1) July 2016, page: 15-18

A NEW MEDIUM FOR SCREENING OF GRAM NEGATIVE BACTERIA IN PORTABLE WATER

A series of laboratory experiments were performed to screen the presence of gram negative bacteria (coliform group) in drinking water to determine its suitability for human consumption and in clinical, pathological, and ophthalmology purpose. A new medium (Makka medium) with methylene blue as an indicator which inhibit Gram positive bacteria was developed, evaluated and used in the present study. Composition of this medium doesn't contain lactose found as in m-Endo agar medium. Generally lactose is used by the bacteria during fermentation process but in Makka medium fermentation occurred in the absence of lactose which was recorded as the change in the color of the medium due to pH drift and Dipotassium phosphate dibasic serves as buffer and enhance organism's growth (coliform group). In comparison with the commercially available synthetic media, Makka medium is unique in composition, economical, safe and easy to use for detection of contamination of drinking water with coliforms.

Laboratory testing protocol to identify critical factors in bacterial compliance monitoring

This research focused on providing guidelines for water utilities on the collection and handling of routine bacteriological samples and in developing scientifically-based approaches in selecting the most representative sampling locations. A laboratory-scale pilot distribution system was designed comprising two parallel loops, one using unlined cast-iron pipe and one using PVC pipe. Each loop contained six sampling ports, including (1) a distribution main dead end faucet, (2) one long (5.5 m; 18 feet) and (3) one short (0.3 m; 1 foot) household copper service line with threaded hose-bibb taps, (4) one hose-bibb with welded faucet, (5) one dedicated sampling port (modeled after a manufacturer's specifications) and (6) one laboratory-style (PVC) stop-cock sampling port. Residual chlorine concentrations were maintained at 0, 0.5, 1.5 and 2.0 mg/L stages during the course of the experiment. Bacterial samples were collected from the different sampling ports and assayed by membrane filtration and/or spread plate. Nutrient and R2A agars were used for heterotrophic plate counts (HPC), m-Endo agar for total coliform (TC) counts and Chromocult agar for injured bacterial analyses. Several methods of sample collection were tested using various combinations of flushing and tap disinfection, including "first flush" (no flushing, without tap disinfection), flushing only, tap disinfection only (using alcohol or hypochlorite solution) and flushing coupled with tap disinfection. The results indicated that the bacterial counts in samples drawn from dead ends were not significantly different from counts in samples from the other sample port configurations. First flush samples consistently produced the highest bacterial count results. Bacterial counts in samples from the long household copper service line were typically three orders of magnitude higher than in samples from the other sample ports. Thus, there is evidence that long copper household service connections may be unsuitable sample tap configurations for collecting samples intended to represent microbial quality in the distribution system.

Microbial quality of domestic and imported brands of bottled water in Trinidad

A cross-sectional study was conducted to determine the microbial quality of domestic and imported brands of bottled water available in Trinidad, purchased from six geographical regions in Trinidad, and representing the whole island. A sample size of 344 bottles of water was determined by using a precision rate of 2% and a Type 1 error of 5%. The membrane filter technique was used with cultures grown on m-Endo agar and m-FC agar for total coliforms and thermotolerant coliforms, respectively. Aerobic plate count (APC) was determined on nutrient agar; Pseudomonas aeruginosa was detected on MacConkey agar, Escherichia coli was isolated on eosin methylene blue (EMB) and Salmonella spp. was assayed by using standard methods. Of the 344 water samples tested, 262 (76.2%) and 82 (23.8%) were domestic and imported brands, respectively. Eighteen (5.2%) of the 344 samples contained coliforms with a mean count of 0.88±6.38 coliforms per 100 ml, while 5 (1.5%) samples contained E. coli. The prevalence of total coliforms in domestic brands of bottled water was 6.9% (18 of 262) as compared with 0.0% (0 of 82) detected in imported brands. The difference was statistically significant ( p=0.004). Similarly, the prevalence of aerobic bacteria in domestic brands of bottled water (33.6%) was significantly higher ( p=0.001) than was found in imported brands (14.8%). Twenty-six (7.6%) of the total samples of water contained Pseudomonas species, but all were negative for thermotolerant coliforms and Salmonella spp. It was concluded that based on the recommended zero tolerance for coliforms in potable water, 5% of bottled water sold in Trinidad could be considered unfit for human consumption.

Reduction of Indicator Organisms During Biological Solubilization of Metals: A Pilot Study

Laboratory and pilot scale studies on the biological solubilization of metals from undigested raw sludge were carried out using elemental sulphur as the energy source. Metals (Cu, Zn, Ni and Cd), pH, sulfate and indicator bacteria [total coliforms (TC) faecal coliforms (FC) and faecal streptococci (FS)] were used to evaluate the system effectiveness. This paper does not report the detailed design and operational data of the pilot plant (available elsewhere [1]) but focuses on the biological tests carried out at the Main Wastewater Treatment Plant in Toronto from July to December, 1997. Since the indicator bacteria are injured from exposure to low pH and potentially toxic metal concentrations, improved alternative methods for their enumeration and their ability to repair acid-metal induced injury in a resuscitation medium (CASO tryptone soya broth) were also investigated. Resuscitation was not effective in repairing cells injured during bacterial leaching. The MPN technique (using lauryl tryptose broth) for enumerating indicator bacteria (total coliforms) gave higher counts and was therefore superior to the spread plate technique (using m-Endo agar) in recovering bacteria from acidic leached sludge (biosolids). However, the coliform bacterial counts from raw sludge were similar by the two methods. This study indicated that the biological solubilization process could significantly reduce the pathogenic indicators. Concentrations of TC, FC and FS in the leached biosolids from the solubilization tank were lower than concentrations in the raw sludge by 4 to 6 orders of magnitude and the finished product (biosolids) met U.S.EPA requirements for pathogen and metal concentrations for Class A biosolids to be used on agricultural land.

Microbiological Quality of Drinking Water in South-Western Greece

A study was carried out in order to estimate the presence of enteric and non-enteric indicators in the water distribution systems of Western Greece and to evaluate different methods for culture and isolation of coliforms in that region, under several incubation conditions and using different media. According to the different media and techniques used, the numbers of water samples found unsafe for consumption represented 21, 17 and 10% of the total, when mT7 agar, M-Endo agar LES (Membrane Filtration technique) and Most Probable Number (MPN) techniques were used, respectively. In one third of the samples oxidase positive microorganisms were present, which were almost eliminated by using anaerobic incubation of the media. Faecal streptococci were found only in 5% of the samples tested. The MPN technique proved to be significantly less efficient in recovering the coliform colonies than the Membrane Filtration (MF) technique (x2 = 125.758 162.422). However, a larger mean number of colonies per sample developed on mT7 agar, indicating that the latter medium should be used in our region for the detection of total coliforms.

Interlaboratory evaluation of MI agar and the US Environmental Protection Agency-Approved Membrane Filter Method for the recovery of total coliforms and Escherichia coli from drinking water

A new membrane filter (MF) medium, MI agar, recently validated for use in recovering chlorine-damaged total coliforms (TC) and Escherichia coli from drinking water, was compared to the US Environmental Protection Agency (EPA)-approved MF method (mEndo agar and nutrient agar supplemented with 4-methylumbelliferyl-β- d-glucuronide) in a collaborative study. Six wastewater-spiked Cincinnati tap water samples, containing three different concentrations of E. coli (≤ 10, 11–30, and >30 E. coli/100 ml), were analyzed concurrently by 19 geographically dispersed laboratories. Results showed that although the overall recoveries of TC and E. coli with MI agar were 12% and 26% greater, respectively, than those of the approved method, the differences were not statistically significant (α=0.05). However, the overall recovery of background/non-coliforms by the new method, 5% of the mEndo agar recovery, was significantly lower ( P=0.0009). Since the results of this study show MI agar to be equal to or better than the current method, it meets the criteria for routine compliance monitoring of drinking water.

Investigation of injury of coliforms after chlorination

The extent of coliform injury after chlorination and the relationship between regrowth of injured coliforms and assimilable organic carbon concentrations (AOC) were studied. Seeded (Escherichia coli E25), sterilized tap water (AOC concentration adjusted to 400mg C/l) was incubated for a period of 3 weeks (25-28°C) after chlorination (0.5 and 1mg/l). Bacteria were enumerated using m-Endo agar LES and m-T7 agar. Chlorination resulted in a 3 log reduction in coliform counts within one minute. No viable colonies were found for 27% of the water samples enumerated with m-Endo agar LES. Injured coliforms were enumerated from all the water samples except one and were present in higher numbers than non-injured coliforms in the majority of the water samples. During the second and third weeks of incubation, only injured bacteria were observed in chlorinated samples. Additional AOC contributed little to the survival and regrowth of chlorinated bacteria.

Investigation of injury of coliforms after chlorination

The extent of coliform injury after chlorination and the relationship between regrowth of injured coliforms and assimilable organic carbon concentrations (AOC) were studied Seeded (Escherichia coli E25), sterilized tap water (AOC concentration adjusted to 400mg C/l) was incubated for a period of 3 weeks (25–28°C) after chlorination (0.5 and lmg/l). Bacteria were enumerated using m-Endo agar LES and m-T7 agar. Chlorination resulted in a 3 log reduction in coliform counts within one minute. No viable colonies were found for 27% of the water samples enumerated with m-Endo agar LES. Injured coliforms were enumerated from all the water samples except one and were present in higher numbers than non-injured coliforms in the majority of the water samples. During the second and third weeks of incubation, only injured bacteria were observed in chlorinated samples. Additional AOC contributed little to the survival and regrowth of chlorinated bacteria.

New medium for the simultaneous detection of total coliforms and Escherichia coli in water

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.

Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies.

A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies. mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive. In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium. Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal. Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.

Ammonia-induced cell envelope injury in Escherichia coli and Enterobacter aerogenes

Ammonia-induced cell envelope injury was examined in pure cultures of Escherichia coli and Enterobacter aerogenes. Cell injury, as determined by the ratio of colony-forming units on m-T7 agar to colony-forming units on m-Endo agar, increased with exposure to increasing concentrations of ammonia. Cell envelopes appeared to be the site of injury as indicated by increasing susceptibility to lysozyme with increasing ammonia concentration. Cells exposed to ammonia also exhibited more cellular leakage than control cells. Leakage from cells exposed to ammonia included proteins, and all leaked substances increased in concentration as ammonia concentrations increased. The concentration of 2-keto-3-deoxyoctonate (KDO) in the outer membrane of E. coli increased with ammonia exposure, while KDO concentration in the outer membrane of E. aerogenes decreased. The results suggest that exposure of E. coli cells to high concentrations of ammonia disrupts the outer membrane and lipopolysaccharide-associated proteins, while E. aerogenes cells are affected through the disruption of bonds between KDO and the outer membrane.

New screening test to determine the acceptability of 0.45-micron membrane filters for analysis of water

During routine membrane filter (MF) quality control testing, irregularities such as partial or complete inhibition of microbial growth at grid lines, abnormal spreading of colonies, growth in or along the grid lines, nonwetting areas, poor colony sheen and metallic sheen on the MF surface with mEndo agar, brittleness, decreased recovery, and severe wrinkling were observed with several lots of filters. To study these effects and to develop a more sensitive screening test for MF quality, we compared five different MFs with various types and degrees of defects by using five stock coliform cultures and five different media. Results showed that the Enterobacter aerogenes-tryptic soy agar test system detected more MF defects than any other combination did and was superior to the Escherichia coli-mFC agar American Society for Testing and Materials method for grid line inhibition. Filtered natural samples grown on the same media showed the same effects as were observed with the pure cultures. Poor colony sheen and sheen on the MF surface were best detected with Enterobacter aerogenes on mEndo agar. The use of tryptic soy agar and mEndo agar with this organism permitted the maximum detection of MF irregularities. Of the 142 MF lots tested by this method, 30% were acceptable, 10% were marginally acceptable, and 61% were unacceptable. This method provides a valuable screening test for determining the acceptability of 0.45-microm-pore-size MFs used for coliform and heterotroph analysis and may also be useful in conjunction with other methods.

Ammonia-induced injury in pure cultures and natural populations of coliform bacteria

Ammonia-induced injury was investigated in pure cultures of Escherichia coli and Enterobacter aerogenes, and in natural coliform populations obtained from the oligotrophic Luxapallila and the eutrophic Sunflower Rivers in northern Mississippi. Pure cultures were affected by ammonia exposure as indicated by changes in the injury ratio (IR) of CFU on m-T7 agar/CFU on m-Endo agar. Ammonia concentrations between 0 and 20 (mg NH3-N/1) had little or no effect and concentrations between 40 and 80 caused the greatest injury. Natural coliform populations from the oligotrophic river were more prone to ammonia-induced injury than those from the eutrophic river. The results stress the need for the routine use of m-T7 media and the enumeration of injured cells when using the membrane filter procedure to ascertain domestic water quality.

Ammonia-induced injury in pure cultures and natural populations of coliform bacteria.

Ammonia-induced injury was investigated in pure cultures of Escherichia coli and Enterobacter aerogenes, and in natural coliform populations obtained from the oligotrophic Luxapallila and the eutrophic Sunflower Rivers in northern Mississippi. Pure cultures were affected by ammonia exposure as indicated by changes in the injury ratio (IR) of CFU on m-T7 agar/CFU on m-Endo agar. Ammonia concentrations between 0 and 20 (mg NH3-N/1) had little or no effect and concentrations between 40 and 80 caused the greatest injury. Natural coliform populations from the oligotrophic river were more prone to ammonia-induced injury than those from the eutrophic river. The results stress the need for the routine use of m-T7 media and the enumeration of injured cells when using the membrane filter procedure to ascertain domestic water quality.

Injured coliforms in drinking water.

Coliforms were enumerated by using m-Endo agar LES and m-T7 agar in 102 routine samples of drinking water from three New England community water systems to investigate the occurrence and significance of injured coliforms. Samples included water collected immediately after conventional treatment, during the backwash cycle, at various points in the distribution system, and 1 week after the break and subsequent repair of a distribution main. Injured coliforms in these samples averaged greater than 95%. m-T7 agar yielded 8- to 38-fold more coliforms than did m-Endo agar LES. The geometric mean of coliforms recovered by m-Endo agar LES was less than 1 confirmed coliform per 100 ml, although m-T7 agar yielded 5.7 to 67.5 confirmed coliforms per 100 ml. In addition, the majority of these samples giving positive results on m-T7 agar produced no detectable counts on m-Endo agar LES. These findings indicated that coliforms were injured and largely undetected by use of accepted analytical media in the systems examined.