Probable Number Method Research Articles

Nutritional and Microbial Quality of Locally Processed Plantain Flour

Flour is a fibre rich in carbohydrate but the preparation and packaging process are exposed to microbial contaminations and nutritional alterations. This study was therefore undertaken to evaluate the microbial and nutritional quality of packaged and unpackaged plantain flour. Cultural, microscopic, biochemical test and most probable number (MPN) methods were employed to determine the various microorganisms; while pproximate analysis was done to estimate inherent nutrients in the flour. Significant differences between packaging, nutritional and microbial qualities were determined using ANOVA or T-test, at P<0.05. Results show Heterotrophic bacterial count in packaged plantain flour was 4.52 ± 1.20 x 102 Cfu/g and unpackaged flour 6.01 ± 1.43 x 102 Cfu/g. Coliform bacterial count in plantain flour showed packaged 2.77 ± 2.01 x 102 Cfu/g and unpacked 3,45 ± 0.50 x 102 Cfu/g. The Pseudomonas count showed the packaged plantain flour count of 3.24 ± 0.60 x 102 Cfu/g and the unpackaged 4.31 ± 1.23 x 102 Cfu/g. The proximate analysis of the flour sample revealed an increase in nutritional contents. This study concludes that exposure of flour to the environment increases microbial content and therefore health and hygiene concerns.

ANALISIS KEBERADAAN BAKTERI ENTEROBACTER PADA AIR CUCIAN IKAN LAYANG (DECAPTERUS SPP) DI PASAR MARDIKA DAN PASAR TAGALAYA AMBON

Background: Fish sales generally do not prioritize sanitation aspects in Ambon city. This study aims to determine the presence of Enterobacter bacteria and the morphological characteristics of Enterobacter bacteria in the washing water of flying fish (Decapterus spp) at Mardika Market and Tagalaya Market Ambon. Wash water samples were taken at Mardika and Tagalaya Markets in Ambon. Methods: The analysis used the Most Probable Number (MPN) method and biochemical tests (methyl red and citrate tests). Results: The results showed that 6 samples of flying fish washing water at the mardika market and tagalaya market that had been tested were identified as Enterobacter bacteria and based on the morphological characteristics found in the washing water samples of flying fish were pink, smooth edges, wavy, curved, and also not sequential and the elevation was flat and raised. The methyl red and citrate tests were negatife and positive respectively. Conclusion: Flying fish washing water at the Mardika and Tagalaya Markets has been contaminated with Enterobacter bacteria.

Maximum likelihood estimators for colony-forming units.

Measuring the abundance of microbes in a sample is a common procedure with a long history, but best practices are not well-conserved across microbiological fields. Serial dilution methods are commonly used to dilute bacterial cultures to produce countable numbers of colonies, and from these counts, to infer bacterial concentrations measured in colony-forming units (CFUs). The most common methods to generate data for CFU point estimates involve plating bacteria on (or in) a solid growth medium and counting their resulting colonies or counting the number of tubes at a given dilution that have growth. Traditionally, these types of data have been analyzed separately using different analytic methods. Here, we build a direct correspondence between these approaches, which allows one to extend the use of the most probable number method from the liquid tubes experiments, for which it was developed, to the growth plates by viewing colony-sized patches of a plate as equivalent to individual tubes. We also discuss how to combine measurements taken at different dilutions, and we review several ways of analyzing colony counts, including the Poisson and truncated Poisson methods. We test all point estimate methods computationally using simulated data. For all methods, we discuss their relevant error bounds, assumptions, strengths, and weaknesses. We provide an online calculator for these estimators.Estimation of the number of microbes in a sample is an important problem with a long history. Yet common practices, such as combining results from different measurements, remain sub-optimal. We provide a comparison of methods for estimating abundance of microbes and detail a mapping between different methods, which allows to extend their range of applicability. This mapping enables higher precision estimates of colony-forming units (CFUs) using the same data already collected for traditional CFU estimation methods. Furthermore, we provide recommendations for how to combine measurements of colony counts taken across dilutions, correcting several misconceptions in the literature.

Groundwater and indoor air quality of urban village housing: study in Janturan, Warungboto, Yogyakarta

Water and air are important elements for life. The presence of pathogenic bacteria in the surrounding environment can cause health problems resulting from the low quality of water and air. The purpose of this study is to measure the quality of groundwater and air based on microbiological parameters in urban village housing. This research was conducted in a descriptive survey, and a purposive sampling technique was carried out. Groundwater and air samples were taken from six residents' houses located in the Janturan area. The water quality test used the most probable number (MPN) method and the air quality test used the agar filter method using a microbiological air sampler (MAS) to trap microbes. The results of the MPN test for total coliform bacteria from six samples of groundwater showed that three samples were positive for coliform bacteria which exceeded the threshold according to Indonesian standard<50 MPN/100 ml. Assay of <em>E. coli</em> bacteria showed that all samples of groundwater positively contained <em>E. coli</em> bacteria which exceeded the threshold of 0 MPN/100 ml. The results of indoor air quality measurements from 6 residents' houses showed that five houses have air quality did not meet the standard for indoor air germ numbers based on Indonesian standard, less than 700 CFU/m3 (colony forming units per metres cubic) and 1 house that got the number of airborne germs, in the normal range, namely 356 CFU/m3. It was concluded that the water quality and air quality in the urban village did not meet health requirements.

Application of miniaturized most probable number method for bacterial detection in water samples: detection of multi-drug-resistant Ralstonia insidiosa in drinking water.

The detection of bacterial contamination in drinking water is essential for monitoring the spread of foodborne diseases. We developed a simple, portable, and low-cost method of mini most probable number (mini MPN) to semi-enumerate bacterial suspension in water as a drinking water analogue. In this study, there is no significant difference between mini MPN and the standard method, technique plate count (TPC), at 10 and 100 CFU/ml Klebsiella pneumoniae suspension with a P-value of 0.28. For the ease-of-use aspect of this method, we tested several variables to prove it can be mass-applied in society. The usage of a sterile-plastic pipette, sample inoculation conducted in a biosafety cabinet (BSC), the usage of a 3-month storage medium, and incubation temperature conducted at room temperature compared to aseptic standard laboratory technique showed P-value > 0.05. In a trial for this method, we used commercialized drinking water for bacterial enumeration and characterization. We found multi-drug resistant (MDR) Ralstonia insidiosa which was resistant to at least four antimicrobial classes, including aminoglycosides, penicillins, cephalosporin, and carbapenem. Vitek 2 Compact was used for bacterial identification and antimicrobial susceptibility testing. A virulence test in Omphisa fuscidentalis larvae showed R. insidiosa strain D had a low virulence.

Ubiquity of methanogenic archaea in the trunk of coniferous and broadleaved tree species in a mountain forest.

Wetwood of living trees is a habitat of methanogenic archaea, but the ubiquity of methanogenic archaea in the trunk of various trees has not been revealed. The present study analysed methanogenic archaeal communities inside coniferous and broadleaved trees in a cold temperate mountain forest by culture-dependent or independent techniques. Heartwood and sapwood segments were obtained from the trunk of seven tree species, Cryptomeria japonica, Quercus crispula, Fraxinus mandshurica, Acer pictum, Aesculus turbinata, Magnolia obovata, and Populus tremula. Amplicon sequencing analysis of 16S rRNA genes showed that Methanobacteriaceae predominated the archaeal communities and Methanomassiliicoccaceae also inhabited some trees. Real-time PCR analysis detected methanogenic archaeal mcrA genes from all the tree species, with a maximum of 107copiesg-1 dry wood. Digital PCR analysis also detected mcrA genes derived from Methanobacterium spp. and Methanobrevibacter spp. from several samples, with a maximum of 105 and 104copiesg-1 dry wood. The enumeration by the most probable number method demonstrated the inhabitation of viable methanogenic archaea inside the trees; 106cellsg-1 dry wood was enumerated from a heartwood sample of C. japonica. Methanogenic archaea related to Methanobacterium beijingense were cultivated from a heartwood sample of Q. crispula and F. mandshurica. The present study demonstrated that the inside of various trees is a common habitat for methanogenic archaeal communities and a potential source of methane in forest ecosystems.

Pengaruh Penyimpanan Suhu Refrigerasi terhadap Karakteristik Fisik Organoleptik, Nilai pH, Cemaran Coliform dan Salmonella spp., Yoghurt Susu Kambing dengan Penambahan Tepung Bekatul Beras Merah

Yogurt is a fermented milk product made with the help of Lactic Acid Bacteria (LAB). Goat milk yogurt with the addition of red rice bran flour contains high antioxidants, making it very beneficial for health. This study aims to evaluate the effect of storage duration on the organoleptic physical characteristics, pH value, and contamination by Coliform and Salmonella spp. in goat milk yogurt with the addition of red rice bran flour. The study used 20 samples divided into five treatment groups based on storage duration (0, 1, 2, 3, and 4 weeks) at 4 ℃, with four repetitions. Each week, organoleptic physical characteristics were tested hedonically and hedonic quality assessed by 20 semi-trained panellists, pH measured with a digital pH meter, Coliform contamination tested using the Most Probable Number (MPN) method, and Salmonella spp. contamination tested using SSA media followed by TSIA media. Data analysis for organoleptic physical characteristics was conducted using the Kruskal-Wallis test followed by the Mann-Whitney test. Data analysis for pH and Coliform contamination was conducted using one-way ANOVA followed by the Tukey test. Data analysis for Salmonella spp. contamination was done descriptively. The results showed that the storage duration significantly affected the physical characteristics of appearance, taste, and aroma (p<0.05) but did not significantly affect colour (p>0.05). There were very significant differences in pH values and Coliform contamination (p<0.01), whereas Salmonella spp. contamination was not affected by the storage duration. The conclusion of this study is that goat milk yogurt with the addition of red rice bran flour should be consumed within three weeks based on organoleptic physical characteristics, pH value, and levels of Coliform and Salmonella spp. contamination.

Raman Spectroscopy Applied to Early Detection of Clostridium Infection in Milk.

Detecting Clostridium in milk presents a significant challenge for the dairy industry given that traditional methods are time-consuming and not specific for these bacteria. Microbiological techniques are expensive and require qualified personnel. Clostridium, in the form of spores, can withstand pasteurization and revert to its vegetative form during cheese aging. These gas-producing bacteria are known for their production of carbon dioxide and hydrogen, causing the formation of slits, cracks, and irregular eyes in hard and semi-hard cheeses. However, gas analysis in the vial headspace of appropriate culture can be exploited to specifically detect Clostridium presence, since the closest competing bacterial Bacilli produces only carbon dioxide. The aim of this paper is to present a Raman-spectroscopy-based instrument for a rapid, inexpensive identification of Clostridium in milk with a limit of detection of 29 spores/L. The proposed measurement procedure is analog to that routinely used, based on the most probable number method. The Raman-based instrument speeds up the detection of a vial's positivity. A test conducted with Clostridium spores demonstrated its effectiveness in almost halving the time needed for the measurement campaign compared to the traditional method.

Development and evaluation of a modified most probable number (MPN) method for enumerating rifampicin‐resistant Escherichia coli in agricultural, food, and environmental samples

AbstractLow level of rifampicin‐resistant E. coli is frequently used in food safety research as a model microorganism and is typically enumerated using traditional Most Probable Number (MPN) method. To simplify the process, we developed and validated a modified MPN method based on lactose peptone broth (LPB) containing rifampicin and bromocresol purple, the broth changes to a yellow color due to pH shifts induced by lactose fermentation from E. coli. E. coli TVS353 were prepared in following suspensions including healthy cells, injured cells, competitive bacteria with or without rifampicin resistance, and unknown natural microflora. E. coli TVS353 was quantified by traditional MPN method and our improved method. At a predicted level of 1 log CFU/mL, there was no significant difference in the MPN index of LPBR compared with TSB and TSBR in these suspensions (p > 0.05). Confirmation results generated from this new color change MPN method were analogous to traditional MPN methods.

Spatiotemporal distribution of thermophilic free-living amoebae in recreational waters: A 5-year survey in Guadeloupe (French West Indies)

Free-living amoebae (FLA) such as Acanthamoeba, Balamuthia mandrillaris, Naegleria fowleri and Sappinia pedata are naturally widespread in freshwater, causing rare but fatal and debilitating infections in humans. Although recent studies have shown an increase in infection rates, there is a paucity of epidemiological studies regarding the presence of these emerging pathogens in water. Herein, we studied the diversity and relative abundance of thermophilic FLA in different recreational baths in a tropical climate for 5 years.From 2018 to 2022, a total of 96 water samples were collected from 7 recreational baths (natural, tiled, regularly cleaned or not, and with temperatures ranging from 27 to 40 °C). DNA was extracted from FLA cultivated at 37 °C to detect thermophilic culturable FLA. Metabarcoding studies were conducted through FLA 18S rRNA gene amplicons sequencing; amplicon sequence variants (ASV) were extracted from each sample and taxonomy assigned against PR2 database using dada2 and phyloseq tools. We also searched for Naegleria sp. and N. fowleri using PCR targeting ITS and NFITS genes (respectively) and we quantified them using an optimized most probable number (MPN) method for FLA. Our results showed that differences in FLA diversity and abundance were observed amongst the 7 baths, but without a clear seasonal distribution. Naegleria, Vermamoeba and Stenamoeba were the most represented genera, while the genera Acanthamoeba and Vahlkampfia were mainly found in 2 baths. The MPN values for Naegleria sp. (NT/l) increased between 2018 and 2022, but the MPN values for N. fowleri (NF/l) seemed to decrease.Globally, our results showed that since we cannot establish a seasonal distribution of FLA, the regular presence of FLA (namely Naegleria and Acanthamoeba) in recreational waters can pose a potential threat in terms of neuroinfections as well as Acanthamoeba keratitis. It is thus imperious to perform the regular control of these baths as a preventive health measure.

ISOLATION OF Escherichia coli FROM VANAME SHRIMP (Litopenaeus vannamei) POND AND THE SENSITIVITY TOWARD ANTIBIOTICS

Vannamei shrimp (Litopenaeus vannamei) is a fishery commodity widely cultivated in various parts of Indonesia. The main problem often found in vannamei shrimp production is poor water quality during the rearing period. This study aims to isolate and identify E. coli contained in water, sediment, and meat of vannamei shrimp cultivated in shrimp ponds and examine its sensitivity to antibiotics. The research was conducted from March to May 2023 at the Technical Implementation Unit (UPT) Vannamei Shrimp Ponds of the Maritime Affairs and Fisheries Service, Tanjung Punak Village, Rupat Utara District, Bengkalis Regency, Riau. The survey method was used in this study. The Most Probable Number (MPN) method was employed for E. coli isolation to calculate bacterial density, and the Kirby-Bauer method was used to test bacterial sensitivity to antibiotics. Based on the identification of the six isolates carried out, it was found that only two isolates were positively identified as E. coli bacteria, namely SD C 3.1 and IP B 1.1, which were isolated from sediment and the Wastewater Treatment Plant (WWTP) pond. The diameter of the inhibition zone for sediment isolate RF01 against the antibiotic chloramphenicol was 18.75 mm, indicating intermediate sensitivity. On the other hand, WWTP isolates RF02 exhibited an inhibition zone of 17 mm for chloramphenicol, indicating medium sensitivity as well. However, it showed resistance to penicillin with a diameter of 6.5 mm and resistance to ampicillin with a diameter of 2 mm

Occurrence of sulfate-reducing bacteria in well water: identification of anaerobic sulfidogenic bacterial enrichment cultures.

Bacteriological studies of well water mainly focus on aerobic and facultative aerobic coliform bacteria. However, the presence of obligate anaerobic bacteria in well water, especially sulfate-reducing bacteria (SRB), possible causative agents of some diseases, is often ignored. In this study, the presence of SRB and coexisting anaerobic bacteria with SRB in sulfate-reducing enrichment cultures obtained from 10 well water samples in Istanbul was investigated. A nested polymerase chain reaction-denaturing gradient gel electrophoresis strategy was performed to characterize the bacterial community structure of the enrichments. The most probable number method was used to determine SRB number. Out of 10, SRB growth was observed in only one (10%) enrichment culture and the SRB number was low (<10 cells/mL). Community members were identified as Desulfolutivibrio sulfodismutans and Anaerosinus sp. The results show that SRB coexist with Anaerosinus sp., and this may indicate poor water quality, posing a risk to public health. Furthermore, Anaerosinus sp., found in the human intestinal tract, may be used as an alternative anaerobic fecal indicator. It is worth noting that the detection of bacteria using molecular analyzes following enrichment culture techniques can bring new perspectives to determine the possible origin and presence of alternative microbial indicators in aquatic environments.

Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of Escherichia coli, E. marmotae, and E. ruysiae in water samples

Escherichia coli are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental Escherichia species such as E. marmotae (formerly named cryptic clade V) or E. ruysiae (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex droplet digital PCR (ddPCR) assay targeting lipB (E. coli and E. ruysiae) and bglC (E. marmotae) was designed. The ddPCR performance was assessed using in silico analysis; genomic DNA from 40 local, international, and reference strains of target and non-target coliforms; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous quantitative PCR (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/μL and 0.005 pg/μl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/μL of genomic DNA. The ddPCR allowed differentiation and quantification of three Escherichia species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. In silico specificity testing underlined the possibility to further detect and distinguish Escherichia cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where E. marmotae and E. ruysiae potentially confound E. coli counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.

The relationship between healthy behavior and environmental sanitation with water quality in stunting occurrence work area of Sikabu Community Health Center Padang Pariaman

Stunting is a long-term nutritional deficiency from insufficient intake and recurrent infectious diseases. One of the factors that can influence stunting is the limited access to clean water. This research analyzes the relationship between healthy behavior and environmental sanitation with water quality in stunting occurrences. This is a quantitative descriptive study with a cross-sectional design. The population includes all mothers with stunted toddlers in the working area of Sikabu Community Health Center, Padang Pariaman Regency, totaling 49 individuals. The sampling technique employed was total sampling. Behavior and sanitation assessments were conducted using a questionnaire. Data collection took place from September 14 to 18, 2023. Microbiological parameters were evaluated using the Most Probable Number (MPN) method, while inorganic chemical parameters were assessed using spectrophotometry. Data processing involved Spearman rank statistical tests, where a p-value <0.05 was considered statistically significant. The research results indicate a significant relationship (p<0.05) between behavior and microbiological and inorganic chemical water quality parameters, with correlation coefficients of 0.745 and 0.604, respectively. There is a significant relationship (p<0.05) between sanitation and microbiological and inorganic chemical water quality parameters, with correlation coefficients of 0.658 and 0.520, respectively. This study concludes that there is a significant relationship between good behavior that has a positive impact on sanitation and drinking water quality on the level of stunting in the Sikabu Puskesmas working area of Padang Pariaman Regency with a correlation coefficient of (p<0.05).

Microorganisms in water for daily consumption on Luis Vargas Torres Island in the city of Esmeraldas

Introduction: microbiological analysis of water involves evaluating water quality in terms of identification and presence of pathogenic and non-pathogenic agents to ensure that the water is safe for daily human use and consumption. Objetive: to identify the microorganisms in the water for daily consumption in the families living on Luis Vargas Torres Island in the city and province of Esmeraldas. Method: a cross-sectional quantitative analytical study was carried out on 121 water samples from reservoirs and taps selected through simple random probability sampling using sterile Whirl-Pak® bags with sodium thiosulfate for sample collection. In the laboratory analysis, the membrane filtration technique with a uniform diameter of 0.45 μm was used to obtain the bacteria on the surface of the membrane filter, which were inoculated in differential culture media for fungi and bacteria and in all cases the count was performed by the Most Probable Number (MPN) method. The final data were consolidated in an Excel table for the final elaboration of frequency tables and graphs. Results: of the total samples analyzed, E. coli was identified in 20 %, Salmonella spp. 10 % and Shigella spp. 4 %; and total coliforms such as Citrobacter spp. and Klebsiella spp. present in 8 %, Enterobacter spp. in 6 % and Pseudomona spp. 2 % indicators of fecal contamination and fungi such as Mucor spp. represents 2 %, Penicilum spp. and Aspergillus fumigatus 3 % and Alternaria alternata and Absidia spp. 4 % and 5 % respectively. Conclusions: research on microbiological water contamination in unplanned urban communities reveals the presence of pathogens and drinking water quality indicators, such as fungi, total coliforms, and species-specific species such as E. coli, Salmonella spp., and Shigella spp., indicating fecal contamination. These findings highlight the urgent need for sanitation and hygiene interventions in this community to effectively manage water quality and health risks

Isolation of a Moderately Acidophilic Nitrobacter from a Nitrifying Community Supplied with Urea.

Nitrite-oxidizing bacteria (NOB), which perform the second step of aerobic nitrification, play an important role in soil. In the present study, we report a novel isolate from agricultural soil affiliated with the genus Nitrobacter and its physiological characteristics. We sampled the surface soil of a vegetable field and obtained mixed culture A31 using the most probable number (MPN) method with inorganic medium containing 0.75‍ ‍mM urea (pH 5.5). The dilution-extinction procedure on culture A31 led to the isolation of a strain that was designated as Nitrobacter sp. A67. The nxrB1 gene sequence of Nitrobacter sp. A67 (302 bp) was classified into Cluster 5, and the highest sequence identity was 96.10% with Nitrobacter sp. BS5/19. The NO2- oxidation activity of Nitrobacter sp. A67 was investigated at various pH. The optimum pH for NO2- oxidation was 5.8-6.4. This result indicates that Nitrobacter sp. A67 is a moderately acidophilic nitrite-oxidizing bacterium.

Phenol Biodegradation by Free and Immobilized Acinetobacter calcoaceticus K5 and Candida tropicalis YM2 Isolated from Persian Gulf

Phenols exhibit high solubility in water, leading to their potential to contaminate significant volumes of freshwater, wastewater, groundwater, oceans, and soil. The primary objective of this study is to isolate and identify bacterial and yeast isolates with optimal phenol degradation capabilities from Persian Gulf. Sea water and sediment samples were collected to isolate bacterial and yeast isolates, which were then evaluated for their ability to degrade phenol both in their single-cell form and when encapsulated in chitosan and alginate. The phenol-degrading strains were quantified using colony-forming unit (CFU) and most probable number (MPN) methods. The most promising microorganisms were selected and identified using phenotypic and polymerase chain reaction techniques. The selected isolates were immobilized in alginate and chitosan carriers, and their bioremediation potential was assessed at different phenol concentrations. The removal of phenol by the isolates was confirmed using Gas Chromatography. Through molecular techniques, Acinetobacter calcoaceticus K5 and Candida tropicalis YM2 were identified as the most efficient phenol-degrading strains. Acinetobacter calcoaceticus K5 exhibited the highest growth at phenol concentrations of 200 and 300 ppm, while Candida tropicalis YM2 showed optimal growth at a concentration of 700 pp. In case of Acinetobacter calcoaceticus, chitosan immobilization and a phenol concentration of 500 ppm proved to be the best conditions for phenol bioremediation. In the case of Candida tropicalis YM2, the highest percentage of phenol removal was achieved using the chitosan carrier at a concentration of 1000 ppm. Comparing the immobilized strains to their free counterparts, it was observed that the stabilized bacteria exhibited greater phenol removal efficiency, particularly at a concentration of 500 ppm, with a significant difference between the two conditions. This study demonstrates that the novel strains Acinetobacter calcoaceticus K5 and Candida tropicalis YM2 are efficient strains for the bioremediation of various phenol-contaminated seawater and this is the first report to show such rapid phenol degradation by these organisms isolated from sediment and seawater samples collected from Persian Gulf.

Impedance method application for number detection Escherichia coli in molluscs testing by official laboratory

Bivalve molluscan shellfish (BMS) have an important role in the transmission of some pathogens responsible for food-borne disease because they are filter-feeding animals capable of greatly concentrating certain pathogenic agents (bacteria, viruses, or parasites) present in the water column, like Escherichia coli. The reference method for E. coli testing in bivalves is the most probable number (MPN) method therefore, as this method has some disadvantages, alternative techniques of equivalent accuracy could be used without the drawbacks of the MPN method such as the direct impedance technique. This method is based on the principle that bacteria produce positively or negatively charged end products causing an impedance variation of the medium that can be used to measure their growth. The present study evaluated the efficiency of the direct impedance measuring technique through a series of laboratory tests performed on 6 kg of clams belonging to two different batches. The preliminary obtained data, compared with the MPN reference method and dd-PCR to validate the alternative method, show that the direct impedance technique has the potential to be used in place of TBX culture for confirming E. coli in MPN assays.

Genomic insights into Enterococcus faecium isolates from marine bivalves highlight One Health concerns and healthcare linkages.

Enterococci, especially Enterococcus faecium, are one of today's leading causes of multidrug-resistant infections in hospital settings. The marine environment may harbour enterococci, but its role as an evolutionary niche and as a vector for the spread of enterococci is sparsely investigated. Hence, by applying enterococci in bivalves as a sentinel tool, this study aimed to describe the prevalence of enterocooci along the Norwegian coast and in addition the phylogeny of E. faecium in particular. Enterococci in batch samples of marine bivalves, harvested from 86 different locations, were quantitatively examined by a culture-dependent most probable number (MPN) method. Isolates were identified by MALDI-TOF-MS prior to antimicrobial susceptibility testing by broth microdilution. In-detail analyses of a representative selection of E. faecium isolates (n=148) were done by Illumina whole-genome sequencing, and assembled genomes were compared to closed E. faecium genomes in the public databases and to genomes from commensal and clinical isolates from Norway. Diversity among E. faecium within the same batch sample of bivalves was also explored. Enterococci were detected in 287 of the 471 examined bivalve samples, but in low concentrations with a median value of <18 MPN /100 g. From positive samples, 479 isolates of enterococci were identified belonging to ten different species, where E. faecium (n=247), Enterococcus hirae (n=114) and Enterococcus faecalis (n=66) were most frequently found. Resistance towards one or more antimicrobial agents was observed in 197 isolates (41 %), none of the isolates showed acquired resistance to vancomycin or linezolid. Phylogenetic analyses revealed high diversity among the E. faecium isolates and showed that the marine niche is dominated by strains from the non-clinical setting belonging to clade A2 (n=85) and B (E. lactis) (n=60). Only three isolates belonged to the hospital-associated clade A1 (ST80 and ST117). Two of these clustered with one isolate from a hospitalized patient and one from a non-hospitalized person. This study demonstrated a high prevalence, but low concentrations of enterococci in bivalves, and low levels of antimicrobial resistance. E. faecium genomes showed high population diversity and that very few E. faecium isolates in bivalves may have arisen from the human healthcare system. A systematic surveillance of target micro-organisms applying methods examining multiple isolates from the same bivalve sample provides important data to assess the enterococcal phylogeny, antimicrobial resistance and the level of faecal pollution in the marine environment.

Isolation and Identification of Phenol-Degrading Bacteria from Iranian Soil and Leaf Samples.

By considering the importance and role of soil in the health of humanity, it is important to remove the presence of harmful compounds, such as phenol. In this study, four types of soil and leaf samples were collected from Kerman, Iran, and the amounts of heterotrophic and degradation bacteria were determined using the serial dilution and most probable number (MPN) methods. The amount of removed phenol was investigated using the Gibbs method with different concentrations of phenol. Then, an isolate with the highest percentage of phenol degradation was identified as the superior strain using 16 sRNA sequencing. The effects of the different factors, such as the carbon source (1% molasses and 1 g glucose), nitrogen source (0.1 g yeast extract), mixed culture, and time (14 and 28 days), on the biodegradation ability of the superior strain was investigated. A total of 18 bacterial strains were isolated from the samples. Isolate B3 had the highest rate (75%) of phenol degradation, at a concentration of 1000 ppm, meaning it was identified as the superior strain. The molecular analysis results identified this isolate as the Comamonas testosteroni strain F4. This bacterium can degrade 89% of the phenol at 30 °C, 180 rpm, and 800 ppm over 28 days. C. testosteroni did not show a favorable phenol degradation ability in the presence of the investigated carbon sources, while this ability was also reduced in mixed cultures. C. testosteroni bacterial strain isolated from soil samples of pistachio orchards in Kerman, Iran, has a favorable ability to biodegrade phenol.