Eosin Methylene Blue Research Articles

Effects of Bacteriocin Extracted from Lactobacillus plantarum on Treatment-Resistant Escherichia coli Bacteria Isolated from Humans and Livestock

Background: Lactic acid bacteria (LAB) are among the most common probiotics and play a significant role in promoting health within the human intestinal microbiota. Their protective functions in the intestine include producing antimicrobial compounds, regulating the balance of the intestinal microbiome, and more. Objectives: This study aims to investigate the antimicrobial effects of bacteriocin extracted from Lactobacillus plantarum (L. plantarum) on Escherichia coli (E. coli) strains that cause treatment-resistant urinary tract infections (UTIs) in humans and mastitis in livestock. Methods: Twenty E. coli isolates were obtained from patients with UTIs and cows with mastitis. After culturing the samples on Eosin-methylene blue, McConkey agar, and blood agar, the strains were identified using biochemical tests. Bacteriocin produced by L. plantarum ATCC8014 was extracted, and its concentration was measured using the Bradford method. The antimicrobial effect of the bacteriocin was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Additionally, the bacteriocin's antimicrobial activity was evaluated through three repetitions of disk diffusion and agar well diffusion methods. Results: The findings revealed that the MIC and MBC concentrations for all human and animal strains were 13.38 μg/mL and 26.76 μg/mL, respectively. Notably, two animal samples (No. 3 and No. 10) and one human sample (No. 6) showed lower MIC and MBC concentrations compared to the other samples and the standard E. coli sample. However, all strains were resistant to bacteriocin in the disk diffusion and agar well diffusion methods. Conclusions: The data indicate a high risk of increasing treatment-resistant E. coli strains in UTIs and livestock, which could pose a significant threat to public and animal health. Therefore, it is essential to further investigate the effects of different bacteriocins on these resistant strains, in conjunction with antibiotic therapies.

A-295 New Transport Media to Preserve Viable Parasites, Bacteria and Fungus

Abstract Background Patients with various infections not always show positive results in their cultures. Therefore, the purpose of this study is to show that a transport media is needed to preserve bacteria, parasites and fungi, which can help patients when collecting samples to determine their infections, the most common urine or feces. The microorganisms can be kept immediately in the transport media when they are taken by the patient and the first step is very important to keep them alive. The preanalytical step is essential for a good diagnostic and accurately determines their etiology. Methods The transport media that I am referring to, was designed by our laboratory. It is a tube with an enriched broth, it can be kept at room temperature for a year. To show that this transport media can be trusted, different samples were tested such as: Urine, semen, wounds, eye secretions and feces were tested at different temperatures: refrigerator 2°C, environment 18°C and in a “kangaroo” corporal temperature or an incubator at 37°C. Also, times were tested between the collection and transport to the laboratory to be proceeded. To have a good diagnostic the sample should be smeared onto the transport media and kept there at least two or more hours at normal corporal temperature. There is no problem to send the tube at room temperature to the laboratory. All samples were smeared for bacterias in MacConkey agar, EMB (Eosin Methylene Blue) agar, CLED (Cystine-Lactose-Electrolyte Deficient) agar with Andrade Indicator, Blood agar, Chocolate agar, XLD agar. For fungi in Potato or Sabouraud agar. The parasites were studied in saline solution and parasitological lugol. Results The samples most studied were 1600 samples of feces, 700 urines and 100 other samples, such as genital, eye secretions, wounds. The percentage of positive etiology with this transport media was 67.9% of fungi versus 73.5% without the transport media. The aerobic bacteria and helminths eggs or adults can be in this transport media for many days, there is no difference and Giardia lamblia trophozoites can be kept as long as seven hours. Conclusions Nowadays, there are various commercial transport media for each microorganism. The great advantage of our transport media is used for three etiologies: Bacteria, parasites and fungi, plus can be kept at room temperature for several days. It is very easy to used, the tube of the transport media can be placed in a “kangaroo” style to keep the samples at corporal temperature. It is helpful in case the patient lives far away or takes the sample at night. The transport media can keep the samples at 37°C at all time at corporal temperature. This new transport media is essential to obtain accurate positive cultures and can give a better diagnostic, since the microorganisms are alive. Knowing the etiology, the treatment is accurate and the patient´s health is always necessary.

Molecular characterization of blaTEM and blaCTX-M ESBLs genes producing Escherichia coli isolates from urinary tract infections (UTIs) in Al-Basrah province, Iraq

Urinary tract infections (UTIs) are primarily caused by Gram-negative Enterobacteriaceae bacteria, accounting for 80–90% of uropathogenic Escherichia coli infections. Multidrug-resistant (MDR) bacteria, such as extended-spectrum β-lactamases (ESBLS), pose a significant threat to global healthcare systems. The current study relied on 200 urine samples collected from patients suffering urinary tract infections (UTIs) in Al Sadr Teaching Hospital in Al-Basrah Province,Iraq from the 5th January to 22nd February. A result of cultivated 200 samples collected from urinary tract infection patients showed 71 (35.5%) positive samples on MacConkey agar, eosin methylene blue (EMB), and HiChromeTM E.coli agar, and bacterial growth was distributed to 47 (66.2%) Escherichia coli and 24 (33.8%) of Gram-negative species. The diagnostic gene 16S rDNA by PCR method showed that all 47 E. coli isolates had a molecular weight of around 585 bp at 100%. Furthermore, positive results were shown in 44 (93.6%) of the E.coli isolates in the current study that produced ESBLs by using the double-disc approximation method (DAM). While 3 (6.4%) isolates revealed negative results for ESBLs, whereas the13(27.7%) of the E.coli isolates gave positive results for ESBL. While 34(72.3%) E.coli isolates showed negative results for produced ESBLs by using the double-disc synergy test (DDST), there were significant differences (P<0.01) between positive and negative isolates in both methods used to detect ESBL-producing isolates. Extended-spectrum β-lactamses (ESBLs) genes were detected by using PCR to amplify the blaTEM and blaCTX-M genes in E.coli isolates. The amplified genes' bands were characterised approximately at (800 bp) for blaTEM and (754 bp) for blaCTX-M , the products were compared to the standard molecular DNA ladder at (2000 bp). According to the results, 47(100%) E.coli isolates gave positive results for the blaTEM gene. While blaCTX-M gene was shown, only 23(48.9%) E.coli isolates had positive results for the blaCTX-M gene.

Proximate Analysis and Occurrence of Antibiotic Resistant Escherichia coli in Street Vended Ready to Eat Edible Larvae of Rhynchophorus phoenicis in Port Harcourt, Nigeria

Rhynchophorus phoenicis, known as the African palm tree larva weevil or edible worm, is a popular edible insect in southern Nigeria and other African countries. These larvae, by-products of oil palm and palm trees, are consumed roasted or boiled and are known for their nutritional and medicinal properties. In Nigeria, they are widely sold along highways in states like Bayelsa, Delta, Edo, Imo, Rivers, and the upper Cross River basin. Preparation methods include consuming them raw, boiled, fried, smoked, or in stews and soups. Typically, the larvae are washed, skewered, sprinkled with salt and pepper, and grilled. They are juicy and have a taste reminiscent of Torzo or cow intestines. Despite their popularity, edible worms pose safety and health concerns due to contamination and the emergence of antimicrobial-resistant bacteria. The indiscriminate use of antimicrobials in animals and humans has led to the development of resistant microorganisms, including multidrug-resistant Escherichia coli, which has been increasingly reported in food poisoning outbreaks. There is limited data on the incidence and antibiotic resistance of Escherichia coli in ready-to-eat foods in Nigeria, prompting this study. The study aimed to determine the proximate composition, occurrence, and antibiotic-resistant pattern of Escherichia coli from edible larvae (Rhynchophorus phoenicis). Objectives included determining the proximate composition of the larvae, the occurrence of Escherichia coli in them, and their antibiotic susceptibility pattern. Fifty ready-to-eat food samples of edible larvae were randomly collected from hawkers. These samples, often partially exposed and poorly packaged, were transported to the microbiology laboratory for analysis. Sterilized glassware and media were used, including Nutrient Agar, Eosin Methylene Blue (EMB) Agar, and Mueller Hinton Agar. Samples were diluted using peptone water, and bacteriological determination involved homogenizing 25 grams of the sample in 225 ml of peptone water, followed by serial dilutions. Aliquots were inoculated on EMB Agar plates, and incubated at 37°C for 24 hours, and colonies were counted and recorded as colony-forming units per gram (cfu/g). Isolates were identified using standard microbiological methods and biochemical tests. Proximate analysis revealed moisture (35.49%), carbohydrate (6.01%), protein (20.73%), ash (1.25%), lipid (22.3%), and fiber (14.23%) content. Bacterial counts ranged from 8.1 x 10^7 to 6.34 x 10^8 cfu/g, with the highest count at the Checkpoint sample and the lowest at Elele-Alumini. Of the 27 isolates, 15 (55.56%) were confirmed as E. coli, with 18.52% of samples exceeding the acceptable microbiological limit (≥10^7 CFU/g). Antibiotic susceptibility tests showed all isolates resistant to augmentin, penicillin, cephalosporin, and ofloxacin, with 46.67% resistance to sulfamethoxazole and streptomycin.

Isolation and identification of Escherichia coli O157:H7 from houseflies (Musca domestica L) at cattle barns in Al-Qadisiyah Province, Iraq

Recent studies identified that Escherichia coli O157:H7 (ECO157H7) causes hemorrhagic colitis in humans, a rare spectrum of diarrheal illness. According to the findings of epidemiological investigations, cattle serve as the principal reservoirs for this bacterium. This study was conducted to understand the incidence of ECO157H7 in houseflies (HFs), a key disease vector. HFs (n = 40) were collected from cattle barns. The HFs were cultured on MacConkey (MC) agars, and any suspected colonies were grown on Eosin Methylene Blue (EMB) agar. EMB-based metallic sheen colonies were grown on specific media, such as sorbitol Chromagar (SCA) and cefixime tellurite sorbitol-MacConkey (CT–SMAC) agar. Sorbitol non-fermenting (SNF) isolates were subjected to a commercial latex agglutination kit to identify the existence of O157 group. The results recorded 31/40 (77.5%) E. coli isolates identified from HF samples. The findings revealed that only 18/31 (58%) were from the SNF isolates. The identities of the isolates were further examined by targeting the rfbO157 and fliCH7 genes, as genetic markers in a PCR method. The PCR results reported 10/18 (55.5%) and 12/18 (66.6%) isolates that carried the rfbO157 and fliCH7 genes, respectively. Our research revealed that HFs represent a harbor for the pathogenic E. coli. The ecology and way this bacterium spreads among animals and across the environment may depend heavily on the presence of ECO157H7 in agricultural environments.

Molecular detection of avian pathogenic Escherichia coli (APEC) in broiler meat from retail meat shop

Avian pathogenic Escherichia coli (APEC) is a major bacterial pathogen responsible for the most widespread form of colibacillosis, resulting in substantial economic losses within the poultry sector and posing a potential public health risk. From July to September 2021, our study investigated the antibiotic resistance pattern of Escherichia coli (E. coli) and the presence of virulence-associated genes (iucD, iutA, iss, and ompT) linked to APEC using 105 broiler meat samples comprising liver, thigh, and breast muscle, in Chitwan, Nepal. E. coli was isolated and identified by culturing samples on MacConkey's agar, Eosin-methylene blue (EMB) agar and performing different biochemical tests. Antibiotic resistance patterns of E. coli were determined by the Kirby-Bauer disc diffusion method. Following the isolation of E. coli, the molecular detection of APEC was performed using conventional polymerase chain reaction (PCR). Out of the 105 samples analyzed, 61 (58.1 %) tested positive for E. coli. In antibiotic susceptibility test (AST), gentamicin and tetracycline exhibited the highest resistance rates, with 90.2 % and 67.2 %, respectively and 29.5 % of the E. coli isolates displayed multidrug-drug resistance. Out of 61 confirmed E. coli isolates, iutA was detected in 47 (77.0 %) samples, iucD in 46 (75.4 %), iss in 53 (86.8 %), and ompT in 39 (63.9 %) samples. This study reports the occurrence of MDR E. coli in meat samples, together with virulence genes associated with APEC which poses a public health threat. Continuous surveillance is vital for monitoring APEC transmission within poultry farms, coupled with efforts to raise awareness of food safety among consumers of broiler meat.

Effect of Multiple Types of High-fat Diet Induction on Gut Microbiota Profile and Butyrate Levels on NAFLD Rat Model

Dysbiosis, marked by a decline in Firmicutes (such as Clostridium spp.) and a rise in Proteobacteria (such as Escherichia coli), is recognized as a pathway linked to the progression of non-alcoholic fatty liver disease (NAFLD). High-fat food may influence the development of NAFLD. This study investigates the effect of different high-fat diets on gut microbiota composition and butyrate levels in a rat model. Four groups of Rattus norvegicus strain Wistar (n=27) were fed specific diets for 12 weeks: normal diet (ND, n=7), high-fat diet (HFD, n=6), western diet (WD, n=7), and high-fat-high-fructose diet (HFHFD, n=7). Stools were cultured on Eosin Methylene Blue (EMB) for Escherichia coli and Forget Fredette Agar (FFA) for Clostridium spp. Colonies were counted using Total Plate Count (TPC), and butyrate levels were measured with gas chromatography. Liver histology was assessed with the NAFLD activity score (NAS). Gut microbiota were analyzed using Crosstab and Chi-Square tests, and butyrate levels with the Kruskal-Wallis test. Butyrate levels decreased in NAFLD-inducing diet groups (HFD, WD, HFHFD) compared to ND (p=0.021). Escherichia coli colony counts were significantly higher in NAFLD-inducing diet groups (p=0.048), while Clostridium spp. counts were higher in ND (p=0.001). Liver histological changes, particularly in the WD group, resembled NAFLD. These findings imply that different high-fat diets can alter the gut microbiome, potentially influencing NAFLD development.

Estimation of IL-8 and TNF-α Levels in Pediatric Diarrhea Patients Infected with Enterohemorrhagic E. coli O157:H7

Abstract Background: Verotoxins are bacterial virulence factors produced by E. coli O157:H7, transmitted by the fecal-oral route. Objectives: The aim of this article was to diagnose E. coli O157:H7 which causes diarrhea and sometimes develops into HUS, which considers pig health problems and estimates the levels of interleukin (IL)-8 and tumor necrosis factor (TNF)-α in the sera of pediatric patients infected with Enterohemorrhagic E. coli compared to the control group. Materials and Methods: Stool and blood samples were collected from 421 pediatric patients with diarrhea, ranging in age from birth to 13 years old, from March to October 2022. Samples were collected from Al Noor Teaching Hospital, Babylon Hospital for Pediatric and Gynecology, Hilla, Iraq. E. coli O157:H7 was cultured on eosin methylene blue (EMB) and Sorbitol MacConkey agar (SMA), confirmed by biochemical test and cultured on HiCrome E. coli O157:H7 selective medium which was an agar base supplemented with cefixime tellurite agar. Serum from 30 pediatric diarrhea patients infected with E. coli O157:H7 compared with 30 healthy children as control group used to determine serum levels of IL-8 and TNF-α by sandwich ELISA. Results: The results revealed that out of the total 421 samples used in this study, E. coli O157H:7, represented 7% (30 of 421) stool samples. This 30serum samples of infected children as well as 30 samples from healthy children subjected to the estimate serum level of IL-8 and TNF-α which record significant differences P ≤ 0.01 and P ≤ 0.05 to this cytokines in different age group; the mean of IL-8 level was 283.62 ± 17.8 pg/mL (7–9 years), and the mean of TNF-α was 208.62 ± 28.7 pg/mL (10–13 years) comparative with the control group of 80.58 ± 15.4pg/mL and 32.50 ± 7.5 pg/mL, respectively, and also result showed an increased mean level of IL-8 than TNF-α in the male comparative with female 195.19 ± 10.4 pg/mL and 159.05 ± 12.4 pg/mL, respectively, comparative with the control group. The result showed no significant differences in IL-8 and TNF-α between watery diarrhea (192.43 ± 24.3 pg/mL and 136.05 ± 20.4 pg/mL) and bloody diarrhea (189.02 ± 22.5 pg/mL and 123.80 ± 13.5 pg/mL), and also result showed significant increase of mean sera level of IL-8 than TNF-α in formula feeding children comparative with breastfeeding children (187.87 ± 19.5 pg/mL and 119.93 ± 17.4 pg/mL, respectively). Conclusion: The finding of this study suggested that increased levels of IL-8 and TNF-α are present in all age groups, in male comparative with female, and also in pediatric diarrhea feeding by formula than breastfeeding and no differences of this cytokine according to consistency of diarrhea. These results contribute to using the immune profile as a serological marker for diagnosing diarrhea caused by E. coli O157:H7 in comparison with the control group.

Improve The Microbiological Quality of Groundwater Around Bonoloyo Cemetery by Boiling

Public cemeteries are considered to be a potential source of groundwater pathogen contamination. Many settlements in Indonesia are located near cemeteries and rely on groundwater for consumption. However, there is no information available on whether the simple method of boiling can improve the microbiological quality of groundwater around cemeteries. Therefore, this experimental study aimed to determine the effectiveness of boiling in improving the microbiological quality of groundwater around Bonoloyo Cemetery. The study had two treatments: groundwater without boiling (control) and groundwater that had been boiled. Samples were collected from two points (S1 and S2) inside the cemetery and three points (S3, S4, and S5) outside the cemetery. The microbiological quality of groundwater was analyzed using the MPN method for each treatment. Samples that produced gas in positive tubes were inoculated on eosin methylene blue (EMB) media. The results of the study showed that S1, S2, S3, and S5 had total coliform levels that exceeded the normal threshold for water. Fecal coliform was found in S3 and S5. However, after boiling, there was an average decrease in total coliforms in samples S1, S3, S4, and S5. There was also no growth of E. coli in samples S3 and S5. The decrease in MPN values ​​in the samples indicates that boiling can improve the microbiological quality of groundwater at cemetery Bonoloyo Surakarta.

ANTIMICROBIAL EFFECT OF CLOVES (Syzygium aromaticum) ON Escherichia coli FROM FEMALE ADULTS URINE WITHIN ALUU COMMUNITY, RIVERS STATE, NIGERIA.

The study was aimed at determining the antimicrobial effect of clove on Escherichia coli isolated from the urine of female adults in Aluu community, Rivers State. Clean-void, mid-stream urine (MSU) specimens were collected from 30 female adults and 20 pregnant women residing in Aluu community, Port Harcourt Rivers state. The samples were culture on Eosin Methylene Blue (EMB) agar by streaking method. Biochemical test was carried out on the isolated bacteria using Bergey’s Manual of Bacteriological Identification. Antibiogram of the isolated bacteria were determined by Kirby-Bauer disc diffusion method. Antimicrobial activity of clove extract was determined by well-in-agar method. Among the 30 sample of the adult female, only 20% of the urine sample were positive for the presence of Escherichia coli while 80% were devoid of Escherichia coli. In the case of urine sample from pregnant women, Escherichia coli was isolated from 45% of the sample while 55% of the sample showed no presence of Escherichia coli. The result of the antibiotic sensitivity showed that 14% of Escherichia coli were resistant to ofloxacin, 33% resistant to perfloxacin, 60%, resistant to augmentin, 74% resistant to nitrofurantoin, 73%, resistant to ceftazidime, 93% resistant to Nalidixic acid and 33% were resistant to gentamin. The antimicrobial activity of cloves produced zone inhibition ranged from 11.50±0.71 to 17.50±0.71mm of all the tested isolated. The antimicrobial potential of clove in the treatment of urinary tract infection by Escherichia coli was also shown in this study, hence could be applied in the treatment of urinary tract infections.

Isolation and Diagnosis of Bacteria and Fungi from Some Areas of Tikrit and Some Villages

The purpose of the study was to evaluate the amount of air pollution caused by bacteria and fungi in specific areas of Tikrit, including the Al-Alam area, Al-Bu Ajil, Al-Buhyazaa, and the village of Al-Karaat. The study was conducted in November and January of 2021–2022, using petri dishes filled with nutrient agar (potato dextrose agar, or PDA) distributed throughout the study areas. The petri dish was left open for fifteen minutes before being closed and incubated with an incubator. After that, the dish was closed and the species was allowed to grow on the media, including isolates and differential media like Macconkey agar and Eosin Methylene Blue (EMB). For microscopical diagnosis, gram stain was applied to the bacterial samples, and fungal stain Lactophenol was used for fungus isolates. The findings of the current study showed that the proportion of Penicillium sp colonies colonized agricultural areas was twice as high, particularly in the Albu-Ajil area, where the dish G2.1 had more than 96 colonies, which was twice or three times more than the rest of the petri dishes in the study area for remote areas. This is because the area had ideal growth conditions in terms of temperature, humidity, and other factorsThe analysis revealed that the majority of bacteria were negative, particularly those belonging to the genus Klebsiella sp. which showed that it could grow on a Macconkey agar dish in Tikrit, the village of Al-Karaat, and Al-Alam. This suggests that it can grow in a variety of environments. However, in the Albu Hayaza area, no bacterial growth was found on the Macconkey agar dish, which was designated with the symbol 3.1 H. This could be because the environmental conditions at the time were insufficient for bacterial growth.

Isolation and Diagnosis of Bacteria and Fungi from Some Areas of Tikrit and Some Villages

The purpose of the study was to evaluate the amount of air pollution caused by bacteria and fungi in specific areas of Tikrit, including the Al-Alam area, Al-Bu Ajil, Al-Buhyazaa, and the village of Al-Karaat. The study was conducted in November and January of 2021–2022, using petri dishes filled with nutrient agar (potato dextrose agar, or PDA) distributed throughout the study areas. The petri dish was left open for fifteen minutes before being closed and incubated with an incubator. After that, the dish was closed and the species was allowed to grow on the media, including isolates and differential media like Macconkey agar and Eosin Methylene Blue (EMB). For microscopical diagnosis, gram stain was applied to the bacterial samples, and fungal stain Lactophenol was used for fungus isolates. The findings of the current study showed that the proportion of Penicillium sp colonies colonized agricultural areas was twice as high, particularly in the Albu-Ajil area, where the dish G2.1 had more than 96 colonies, which was twice or three times more than the rest of the petri dishes in the study area for remote areas. This is because the area had ideal growth conditions in terms of temperature, humidity, and other factorsThe analysis revealed that the majority of bacteria were negative, particularly those belonging to the genus Klebsiella sp. which showed that it could grow on a Macconkey agar dish in Tikrit, the village of Al-Karaat, and Al-Alam. This suggests that it can grow in a variety of environments. However, in the Albu Hayaza area, no bacterial growth was found on the Macconkey agar dish, which was designated with the symbol 3.1 H. This could be because the environmental conditions at the time were insufficient for bacterial growth.

PREVALENCE OF ESCHERICHIA COLI, COLIFORM AND NON-COLIFORM BACTERIA IN MUD BUFFALO IN RANGGAGATA AND SERAGE VILLAGES, SOUTHWEST PRAYA SUBDISTRICT, DISTRICT OF CENTRAL LOMBOK, WEST NUSA TENGGARA

Escherichia coli (E. coli) is a common bacterium that inhabits the intestines of humans and animals with hundreds of different strains, both harmful and harmless. One of the diseases that can be caused by E. coli is diarrhea. Diarrhea is a disease in the form of increased frequency of defecation, thinner stool consistency and increased intestinal peristalsis. This study aims to determine the number of E. coli, coliform and non-coliform bacteria in mud buffaloes in Ranggagata and Serage Villages, Southwest Praya Subdistrict, Central Lombok Regency, West Nusa Tenggara with samples used are fresh feces samples, a total of 14 samples. Sample cultivation was carried out on Eosin methyleneblue agar (EMBA) media then samples suspected of E. coli colonies were Gram stained, and biochemical tests on Triple Sugar Iron Agar (TSIA) media, Sulfide Indole Motility (SIM), Methyl red-Voges Proskauer (MR-VP) media, Simmons Citrate and catalase tests. The results showed the number of bacterial populations varied depending on the age of the buffalo with an average number of E. coli bacteria 162.143 ± 196.476, average coliform 272.000± 328.307 and average non-coliform 110.714 ± 201.855. Based on the results of the study it can be concluded that there is an increase in the number of E. coli, coliform and non-coliform as the age of buffalo increases, namely for E. coli is Y = 2.527+0.009x, coliform is Y = 2.525+0.05x and non-coliform bacteria is Y = 3.199+0.007x. Further tests need to be conducted related to other strains or species of E. coli, coliform and non-coliform bacteria in mud buffalo feces.

Oriental beech decline caused by Brenneria alni

AbstractHyrcanian forests comprise a long strip along the southern coast of the Caspian Sea in northern Iran. Oriental beech (Fagus orientalis) is one of the most valuable forest tree species in the Hyrcanian forests. Bark necrosis and bleeding spots of oriental beech were observed in Savadkuh forests in Mazandaran province during summer 2021. Ten samples were collected from symptomatic oriental beech trees. The bark canker symptoms included stem tissue necrosis, dark brown to black spots on the trunk, bleeding with dark exudates and dark lesions in the inner bark. Twenty‐six strains of culturable bacteria were isolated from the samples which in culture were predominantly round, convex, smooth with entire margins on nutrient agar and a metallic green sheen pigment on eosin‐methylene blue (EMB) agar. Fourteen strains induced necrosis and browning of the tissues around the inoculation sites on acorns after 2 weeks. Five representative strains also caused dieback in twigs of oriental beech 3 weeks after inoculation. These bacteria were Gram‐negative, catalase positive and oxidase negative. All strains produced colonies with a green metallic sheen on EMB agar. Strains were negative in tests for starch and gelatine hydrolysis or production of levan. A phylogenetic tree based on concatenated sequences of gyrB and infB genes, of representative strains well as in each of the single gene‐based phylogenetic trees, indicated that these strains clustered with Brenneria alni NCPPB 3934T with high bootstrap values. To the best of our knowledge, this is the first report of B. alni associated with bark necrosis and bleeding spots on oriental beech.

Spread of Multi-drug Resistant Aerosolised Salmonella sp and Escherichiacoli from Dumpsites in Built Environment of Abraka Town, Nigeria

Waste dumpsites are known to be hot spots for the development of multi-drug resistant (MDR) pathogenic microorganisms, which can be dispersed via aerosols. The dispersal of MDR Salmonella sp and E. coli in dumpsite-infested neighbourhoods of Abraka, a university town, was therefore investigated. Eosine Methylene Blue and Bismuth Sulphite agar plates were used to isolate airborne E. coli and Salmonella sp, respectively using settling plate technique. Air sampling was carried out at 3 neighbourhood dumpsites and at intervals of 50m up to 200m away from the dumpsites along the cardinal directions. The isolates were tested for antibiotics resistance using 10 antibiotics and those with multiple antibiotics resistance (MAR) index≥0.3 were considered MDR and subsequently subjected to plasmid curing, and re-tested for antibiotics resistance. The population of both organisms varied with cardinal directions and dumpsites, but generally declined as the distance from dumpsites increased (E. coli, 2.96-1.72 log cfu/m3 ; Salmonella sp, 2.65-0.00 cfu/m3 ). By overall assessment, mean MAR index for E. coli isolates was 0.39±0.01-0.64±0.04 and 0.37±0.02-0.68±0.03 for Salmonella sp isolates. MDR E. coli and Salmonella sp constituted 26.7-45.7 and 0.0-47.1% of the total population, respectively; and were encountered up to 200m away from the dumpsites at all directions although with decreasing numbers. There were significant reductions (P<0.05) in the MAR indexes of both organisms after curing thereby indicating plasmid involvement in the MDR. In conclusion, the aerosolised plasmid-encoded and chromosome-based genes in MDR organisms from dumpsites can be dispersed in neighbourhoods; and plasmids can spread antibiotics resistant genes by horizontal transfer and compound therapeutic options

Chemical characteristics and microbiological loads of harvested rainwater run-off from roof tops in South Eastern Nigeria

The study evaluated rainwater quality harvested from roof top catchments viz Asbestos, Aluminum, Corrugated and Harvey roof tops in southeastern, Nigeria. Chemical and microbiological species viz pH, Total Dissolved Solids(TDS), Electrical conductivity (EC), Nitrate(NO3−), Sulphate (SO42−), Chloride(Cl−), Total bacteria load(TBL), Total Coliform Count (TCC), Salmonella Shigella Agar (SSA), Thiocitrate Bile-salt Sucrose( TCBS), Eosin Methylene Blue (EMB) were investigated from samples of rainwater roof top run off during onset rain (April), peak rain (July) and cessation (October) rain events in 2022. Results of chemical parameters show significant variation p ≥ 0.05 among the catchments with increased mean concentration of pH(6.5), TDS(25.7 mg/l), EC(43.6μS/cm),NO3−(0.72 mg/l) SO42(4.73 mg/l), Cl(8.81) during onset rain more than the peak and cessation rain, with asbestos rainwater having the highest levels but below WHO/FMENV drinking water limit. Positive and negative correlations existed between the chemical parameters in temporal scale and originating from human activities with the temporal variability in decrease order: April ≥ October ≥ July. Microbial parameters in rainwater revealed mean TBL(6.6 × 104 cfu/ml), TCC(2.6 × 103 cfu/ml) and control (5.8 × 104 cfu/ml) with Aluminum top (8.2 × 104 cfu/ml) recording highest in onset rain more than the peak and cessation rain events. All of the microbial parameters were above the WHO standards which indicates a very high public health risk if consumed without treatment. Organisms found in rainwater from roof types are: Enterobacter sp., Staphylococcus aurous, Streptococcus sp., Bacillus Sp. and Escherichia coli. Based on the results, treatment of rainwater, operation, maintenance methods should be upheld for quality assurance of harvested rainwater.

ANALISIS CEMARAN BAKTERI Coliform DAN Escherichia coli DARI SAMPEL ES BATU PADA PEDAGANG MINUMAN KAKI LIMA DI SEKITAR PASAR CIRACAS JAKARTA TIMUR MENGGUNAKAN METODE MOST PROBABLE NUMBER (MPN)

Background: Water is a basic need of living things. Water is a liquid, when it freezes it turns into ice cubes. People often use ice cubes as a drink additive to give a fresh taste. The process of freezing ice cubes cannot destroy bacteria, many bacteria can live for a long time at low temperatures. This study aims to determine the contamination of Coliform and Escherichia coli bacteria in ice cubes sold by street vendors around Ciracas Market, East Jakarta. Methods: The MPN method is used to determine the range of Coliform bacteria in ice cube samples. E.coli contamination was detected using Eosine Methylene Blue Agar media. This study uses primary data by collecting samples and testing them in the microbiology laboratory. The sampling technique by purposive sampling. The samples used were ice cubes from street vendors around Ciracas Market is 8 samples. Results: 8 positive samples of Coliform bacteria and 5 positive samples of E.coli bacteria. In Sample 5 it has the highest value of 2900 MPN/100 mL. Sample 6 has the lowest value, namely 140 MPN/100 mL. Conclusion: The eight samples exceeded the contamination threshold allowed by Permenkes 416/Men.Kes/Per/IX/1990, that is 0/100 mL. In the EMBA media there were 5 positive samples of E.coli bacteria.

Comparison of CHROMagar Orientation Medium with Traditional Methods in Urinary Tract Infection Cultures.

Urinary tract infections (UTIs) are the most common infections in adults, and urine culture is the parameter that uses the most time and cost in microbiology laboratories. For this reason, the selection of fast and cost-effective methods in the evaluation of urine samples is one of the priority issues of microbiology laboratories. The aim of this study was to investigate the compatibility and cost-effectiveness of routinely used Blood Agar (BA), Eosin Methylene Blue (EMB) medium, and CHROMagar Orientation Medium (CO Medium) in the identification of microorganisms in urine samples. Consecutive urine samples (n: 700) sent to our laboratory were simultaneously inoculated onto BA/EMB media and CO medium. Urine samples were evaluated after 18 - 24 hours of incubation at 37℃ and the compatibility of the two methods was compared. The use of 104 Gram stains, 198 biochemical tests, and 9 identification kits was required with BA/EMB agar. When 104 colonies with single growth were evaluated, presumptive identification with CO medium was found to be 100% compatible with the VITEK 2 system. The most isolated 62 Escherichia coli (E. coli) colonies gave dark pink-red color and were found to be fully compatible with the VITEK 2 system. Compatibility of BA and EMB medium evaluations with VITEK 2 system; E. coli (n: 62), KES group (n: 26), Pseudomonas spp. (n: 6) and Proteus spp. For (n: 2), it was determined as 69.3%, 57.69%, 100%, and 100%, respectively. According to the results of our study, when BA/EMB and CO Medium methods were compared, 182 Euro (€) savings were achieved in 700 urine cultures with CO Medium. It was estimated that the amount of savings could be 15,600 € per year. CHROMagar Orientation Medium method can be used routinely with its advantages such as being cost-effective, reducing the workload, and not requiring additional operations. CHROMagar Orientation Medium can also be considered as an easily accessible method and opportunity that does not require infrastructure and trained personnel, especially for laboratories with low test capacity and having problems with the supply of com-mercial kits and automated systems.

Antimicrobial Resistance in Escherichia coli and other Coliform Bacteria Isolated from Bagmati River

Objectives: To explore the presence of fecal indicator bacteria and assess antibiotic resistance status in Bagmati river water. Methods: In a cross sectional study during a year 2020; a total of 180 water samples were collected from the Bagmati River’s upstream, midstream, and downstream sources. Maintaining reverse cold chain, the samples were transferred to the laboratory of central department of Microbiology, Tribhuvan University. Organisms were isolated on Eosin Methylene Blue (EMB) and Nutrient Agar. The organisms were further identified based on the biochemical properties and antibiotic susceptibility testing was performed following CLSI (2020) guidelines. Results: Of the 546 isolates, 209 (or 38%) were Escherichia coli. Other coliforms isolated were Enterobacter spp (2%), Citrobacter spp (37%), and Klebsiella spp (23%). Upstream source revealed least percentage 3% (7/209) of E. coli. All the recovered Citrobacter spp. were resistant and E. coli showed >99% resistance towards Tetracycline, Ampicillin and Amoxiclav antibiotics. Klebsiella spp. was 100% resistant towards Ampicillin and Amoxiclav antibiotics. The coliforms exhibited least resistance (10%) towards Chloramphenicol. Based on antibiotic resistance percentage pattern, E. coli showed 27% similarity to Citrobacter spp. Conclusion: Coliforms showed maximum resistance towards first line antibiotics prescribed in human infection. Immediate water safety plans should be instituted to improve the water quality.

Hatching Failure and Success of Hawksbill Sea Turtles (Eretmochelys imbricata) in Semi-Natural Nests Karimunjawa.

Hatching success and failure is influences by the interaction between biotic and abiotic factors during the incubation period. Non optimal nest is susceptible to bacterial contaminated eggs during incubation period and hatchling morphology. The purpose of the study is to understand the relationship between environmental conditions of semi natural nest with bacteria contaminant, hatchlings allometry and abnormality. The method used was quantitative descriptive which are nest temperature and humidity measurement, hatchlings allometry measurement, bacteria observation of gram negative with EMB (Eosin Methylene Blue) agar, TPC (Total Plate Count), bacteria characteristic morphology and hatchlings abnormality observation. The result showed that semi natural nest have high temperature (30˚C) and high humidity (70%) caused smaller hatchlings and negative allometry growth, hatchlings abnormality and bacterial contaminant. TPC showed that the number of bacteria unhatched eggs (1,42 x ) more than hatched eggs (1,03 x ). The bacteria colonies were suspected to be from Enterobacteriaceae family according to microscopic and macroscopic morphological characteristic. Macroscopically, the bacteria morphology is dominated by a round shape, concentric surface, flat edges, flat elevation and pink color, meanwhile microscopic form is bacilli. There is significant correlation between environmental conditions of semi natural nest with hatchlings growth, hatchlings abnormality and eggshells bacteria