Infectious diseases are becoming increasingly important. Humans and animals transfer pathogens and antibiotics resistant bacteria into the environment. They can multiply in water, in food or even in technical facilities and are thus a danger to human health. Pathogens should be avoided in concentrations that can lead to diseases. For this reason, limits are set for drinking water, defined food, but also for technical plants. This is where analytics comes into play. Quantitative results must be achieved as soon as possible. Using the example of Legionella in technical aquatic systems like evaporative recooling systems, it is currently clear that we can not comprehensively assess hygiene in technical systems solely with culture-based methods. Culture-independent measurement methods are based on molecular biological screening methods [1] or immunoassays [2]. These rapid measurement techniques are important in quantifying pathogenic contamination. In addition, the identification of a pathogen and its state of activity (infectious, living, dormant, or dead) can lead to a better risk analysis for technical installations [3]. The entry of antibiotics and antibiotic-resistant bacteria in sewage treatment plant and their elimination up to the fourth purification stage can be examined with molecular methods. The emergence or reduction of antibiotic resistance genes of pathogenic bacteria in a microbiological community is still completely unclarified. New analytical methods that address the analysis and handling of single bacteria cells are important [4]. Additionally, metagenomics methods have to be improved in combination with bioinformatics [5]. In conclusion, Applied Microbiology is a field of research that is developing rapidly, because on the one hand, highly modern analytical instruments are used and on the other hand, new methods are developed with analytical principles, in order to be able to evaluate cells independently in a timely manner. The specialist committee "Pathogens and Antibiotic-Resistant Bacteria" of the Water Chemical Society has set itself the goal of assessing and establishing new diagnostic detection methods in respect of the gold standard cultivation with regard to various pathogens in the water cycle. Here, we will give an overview of new technologies for the quantification and characterization of pathogens and antibiotic resistant bacteria and we will give first examples for applications.

Infectious diseases are becoming increasingly important. Humans and animals transfer pathogens and antibiotics resistant bacteria into the environment. They can multiply in water, in food or even in technical facilities and are thus a danger to human health. Pathogens should be avoided in concentrations that can lead to diseases. For this reason, limits are set for drinking water, defined food, but also for technical plants. This is where analytics comes into play. Quantitative results must be achieved as soon as possible. Using the example of Legionella in technical aquatic systems like evaporative recooling systems, it is currently clear that we can not comprehensively assess hygiene in technical systems solely with culture-based methods. Culture-independent measurement methods are based on molecular biological screening methods [1] or immunoassays [2]. These rapid measurement techniques are important in quantifying pathogenic contamination. In addition, the identification of a pathogen and its state of activity (infectious, living, dormant, or dead) can lead to a better risk analysis for technical installations [3]. The entry of antibiotics and antibiotic-resistant bacteria in sewage treatment plant and their elimination up to the fourth purification stage can be examined with molecular methods. The emergence or reduction of antibiotic resistance genes of pathogenic bacteria in a microbiological community is still completely unclarified. New analytical methods that address the analysis and handling of single bacteria cells are important [4]. Additionally, metagenomics methods have to be improved in combination with bioinformatics [5]. In conclusion, Applied Microbiology is a field of research that is developing rapidly, because on the one hand, highly modern analytical instruments are used and on the other hand, new methods are developed with analytical principles, in order to be able to evaluate cells independently in a timely manner. The specialist committee "Pathogens and Antibiotic-Resistant Bacteria" of the Water Chemical Society has set itself the goal of assessing and establishing new diagnostic detection methods in respect of the gold standard cultivation with regard to various pathogens in the water cycle. Here, we will give an overview of new technologies for the quantification and characterization of pathogens and antibiotic resistant bacteria and we will give first examples for applications.

Infectious diseases are becoming increasingly important. Humans and animals transfer pathogens and antibiotics resistant bacteria into the environment. They can multiply in water, in food or even in technical facilities and are thus a danger to human health. Pathogens should be avoided in concentrations that can lead to diseases. For this reason, limits are set for drinking water, defined food, but also for technical plants. This is where analytics comes into play. Quantitative results must be achieved as soon as possible. Using the example of Legionella in technical aquatic systems like evaporative recooling systems, it is currently clear that we can not comprehensively assess hygiene in technical systems solely with culture-based methods. Culture-independent measurement methods are based on molecular biological screening methods [1] or immunoassays [2]. These rapid measurement techniques are important in quantifying pathogenic contamination. In addition, the identification of a pathogen and its state of activity (infectious, living, dormant, or dead) can lead to a better risk analysis for technical installations [3]. The entry of antibiotics and antibiotic-resistant bacteria in sewage treatment plant and their elimination up to the fourth purification stage can be examined with molecular methods. The emergence or reduction of antibiotic resistance genes of pathogenic bacteria in a microbiological community is still completely unclarified. New analytical methods that address the analysis and handling of single bacteria cells are important [4]. Additionally, metagenomics methods have to be improved in combination with bioinformatics [5]. In conclusion, Applied Microbiology is a field of research that is developing rapidly, because on the one hand, highly modern analytical instruments are used and on the other hand, new methods are developed with analytical principles, in order to be able to evaluate cells independently in a timely manner. The specialist committee "Pathogens and Antibiotic-Resistant Bacteria" of the Water Chemical Society has set itself the goal of assessing and establishing new diagnostic detection methods in respect of the gold standard cultivation with regard to various pathogens in the water cycle. Here, we will give an overview of new technologies for the quantification and characterization of pathogens and antibiotic resistant bacteria and we will give first examples for applications.

Microbiological water quality can be assessed by standardized cultivation-based enumeration of fecal indicator bacteria (FIB). During the last two decades, molecular detection of general or host-associated genetic bacterial fecal markers has been increasingly complementing FIB methodology [e.g. 1,2] . Polymerase chain reaction (PCR) has become the favorite tool for enumeration. However, qPCR methodology requires well equipped laboratory infrastructure and specialized personal. Recently, isothermal amplification methods (ISOAMP) has been suggested as possible alternatives to PCR, with potential for high-throughput screening or rapid detection. In this presentation a brief overview will be given on recent ISOAMP developments for molecular fecal pollution diagnostics from our group [3-5]. Future visions on the application of ISOAMP methods in water quality testing will be discussed.

Microbiological water quality can be assessed by standardized cultivation-based enumeration of fecal indicator bacteria (FIB). During the last two decades, molecular detection of general or host-associated genetic bacterial fecal markers has been increasingly complementing FIB methodology [e.g. 1,2] . Polymerase chain reaction (PCR) has become the favorite tool for enumeration. However, qPCR methodology requires well equipped laboratory infrastructure and specialized personal. Recently, isothermal amplification methods (ISOAMP) has been suggested as possible alternatives to PCR, with potential for high-throughput screening or rapid detection. In this presentation a brief overview will be given on recent ISOAMP developments for molecular fecal pollution diagnostics from our group [3-5]. Future visions on the application of ISOAMP methods in water quality testing will be discussed.

Microbiological water quality can be assessed by standardized cultivation-based enumeration of fecal indicator bacteria (FIB). During the last two decades, molecular detection of general or host-associated genetic bacterial fecal markers has been increasingly complementing FIB methodology [e.g. 1,2] . Polymerase chain reaction (PCR) has become the favorite tool for enumeration. However, qPCR methodology requires well equipped laboratory infrastructure and specialized personal. Recently, isothermal amplification methods (ISOAMP) has been suggested as possible alternatives to PCR, with potential for high-throughput screening or rapid detection. In this presentation a brief overview will be given on recent ISOAMP developments for molecular fecal pollution diagnostics from our group [3-5]. Future visions on the application of ISOAMP methods in water quality testing will be discussed.

Antibiotic resistance (AR) is a phenomenon that was first recognised short after the antibiotic´s discovery by A. Fleming in 1928. From an ecological point of view, it is a natural mechanism of species competition and evolution. But today, AR is associated to human pathogens. Emerging species cover multiple resistances against different antibiotic classes which makes therapy critical and therefore, AR are declared to be a serious threat to modern medicine. [1] Antibiotic resistant bacteria enter the environment by different pathways, mainly wastewater and manure, and can find their way back to other humans. Acquired resistance genes are often located on mobile elements like plasmids and can be transferred from one bacterium to another via horizontal gene transfer. In combination with globalisation and mobility, thus lead to resistance dissemination. Antibiotics entering the environment are also seen as a reason for AR spread. [2] For wastewater, an association between single antibiotics and multi resistant bacteria in samples was identified. [3] There are various possibilities to detect AR, e.g. cultivation of bacteria followed by testing of growth inhibition, direct selective cultivation of resistant bacteria only, gene analysis within cultivated isolates or gene analysis within mixed bacterial populations of environmental samples. Depending on the species, genes or analysis methods selected, study results differ significantly. This makes a comparison between a statistical meta-analysis of studies difficult, not only between countries. This lecture aims to overview chances and limitations of popular analysis methods. It planned to comes forward with a proposal of a focused parameter combination for problem-based, target-oriented and nationwide standardized monitoring to enable future reliable trend analysis of the environmental AR status, based on recommendations of the BMBFfunded collaborative research project ‘HyReKA’ [grant number FKZ 02WRS1377].

Antibiotic resistance (AR) is a phenomenon that was first recognised short after the antibiotic´s discovery by A. Fleming in 1928. From an ecological point of view, it is a natural mechanism of species competition and evolution. But today, AR is associated to human pathogens. Emerging species cover multiple resistances against different antibiotic classes which makes therapy critical and therefore, AR are declared to be a serious threat to modern medicine. [1] Antibiotic resistant bacteria enter the environment by different pathways, mainly wastewater and manure, and can find their way back to other humans. Acquired resistance genes are often located on mobile elements like plasmids and can be transferred from one bacterium to another via horizontal gene transfer. In combination with globalisation and mobility, thus lead to resistance dissemination. Antibiotics entering the environment are also seen as a reason for AR spread. [2] For wastewater, an association between single antibiotics and multi resistant bacteria in samples was identified. [3] There are various possibilities to detect AR, e.g. cultivation of bacteria followed by testing of growth inhibition, direct selective cultivation of resistant bacteria only, gene analysis within cultivated isolates or gene analysis within mixed bacterial populations of environmental samples. Depending on the species, genes or analysis methods selected, study results differ significantly. This makes a comparison between a statistical meta-analysis of studies difficult, not only between countries. This lecture aims to overview chances and limitations of popular analysis methods. It planned to comes forward with a proposal of a focused parameter combination for problem-based, target-oriented and nationwide standardized monitoring to enable future reliable trend analysis of the environmental AR status, based on recommendations of the BMBFfunded collaborative research project ‘HyReKA’ [grant number FKZ 02WRS1377].

Antibiotic resistance (AR) is a phenomenon that was first recognised short after the antibiotic´s discovery by A. Fleming in 1928. From an ecological point of view, it is a natural mechanism of species competition and evolution. But today, AR is associated to human pathogens. Emerging species cover multiple resistances against different antibiotic classes which makes therapy critical and therefore, AR are declared to be a serious threat to modern medicine. [1] Antibiotic resistant bacteria enter the environment by different pathways, mainly wastewater and manure, and can find their way back to other humans. Acquired resistance genes are often located on mobile elements like plasmids and can be transferred from one bacterium to another via horizontal gene transfer. In combination with globalisation and mobility, thus lead to resistance dissemination. Antibiotics entering the environment are also seen as a reason for AR spread. [2] For wastewater, an association between single antibiotics and multi resistant bacteria in samples was identified. [3] There are various possibilities to detect AR, e.g. cultivation of bacteria followed by testing of growth inhibition, direct selective cultivation of resistant bacteria only, gene analysis within cultivated isolates or gene analysis within mixed bacterial populations of environmental samples. Depending on the species, genes or analysis methods selected, study results differ significantly. This makes a comparison between a statistical meta-analysis of studies difficult, not only between countries. This lecture aims to overview chances and limitations of popular analysis methods. It planned to comes forward with a proposal of a focused parameter combination for problem-based, target-oriented and nationwide standardized monitoring to enable future reliable trend analysis of the environmental AR status, based on recommendations of the BMBFfunded collaborative research project ‘HyReKA’ [grant number FKZ 02WRS1377].

E. coli, coliform bacteria and enterococci are widely used indicators for the determination of the hygienic quality of drinking water. A positive detection of E. coli indicates a fecal contamination and consequently a potential risk of pathogens. In contrast, coliform bacteria and enterococci do not exclusively originate from feces, but have also been isolated from environmental sources like plants, invertebrates or sediments. If indicator organisms are detected in drinking water systems, the reasons or sources for these contaminations are often unknown. Traditionally, fecal bacteria were identified with classic biochemical methods. In the last years, molecular biological approaches were increasingly used to identify bacteria by genetics or proteins and not by biochemical properties. These fast and specific methods are helpful to identify the sources of fecal contaminations in drinking water. The identification is crucial for further decisions and measures like boiling advices or disinfection measures. DNA-Sequencing is the most common method for the identification of bacteria. The sequence of the 16S-rRNA of an unknown bacterial isolate is compared to the sequences of other known species in a database. For closely related bacteria like Enterobacter, the sequencing of a single gene is not accurate enough for a correct identification. Therefore, additional genes have to be sequenced and compared. MALDI-TOF MS is an identification method based on bacterial protein spectra und is routinely used for the identification of clinically relevant bacteria. With less than one hour of sample preparation and analysis time, MALDI-TOF MS has a huge time advantage compared to other methods. Combined with high precision and sensitivity as well as the simple operative method, MALDI-TOF MS has a high potential in the drinking water sector. Comparisons of MALDI-TOF MS and traditional sequencing indicate that MALDI-TOF MS is in principle a suitable method for the identification of indicator bacteria from water [1-3]. Due to the fastness, MALDI-TOF MS has the potential to become the routine identification method also in the drinking water sector. Yet, the current database is not sufficient to correctly identify all relevant strains within the Enterobacteriaceae and enterococci, as the database is still dominated by clinically relevant bacteria and many environmental isolates are missing. Financial support by the German Ministry of Education and Research (BMBF) and the German Technical and Scientific Association for Gas and Water (DVGW) is gratefully acknowledged (02WGW1460 (BMBF);W201823 (DVGW)).

E. coli, coliform bacteria and enterococci are widely used indicators for the determination of the hygienic quality of drinking water. A positive detection of E. coli indicates a fecal contamination and consequently a potential risk of pathogens. In contrast, coliform bacteria and enterococci do not exclusively originate from feces, but have also been isolated from environmental sources like plants, invertebrates or sediments. If indicator organisms are detected in drinking water systems, the reasons or sources for these contaminations are often unknown. Traditionally, fecal bacteria were identified with classic biochemical methods. In the last years, molecular biological approaches were increasingly used to identify bacteria by genetics or proteins and not by biochemical properties. These fast and specific methods are helpful to identify the sources of fecal contaminations in drinking water. The identification is crucial for further decisions and measures like boiling advices or disinfection measures. DNA-Sequencing is the most common method for the identification of bacteria. The sequence of the 16S-rRNA of an unknown bacterial isolate is compared to the sequences of other known species in a database. For closely related bacteria like Enterobacter, the sequencing of a single gene is not accurate enough for a correct identification. Therefore, additional genes have to be sequenced and compared. MALDI-TOF MS is an identification method based on bacterial protein spectra und is routinely used for the identification of clinically relevant bacteria. With less than one hour of sample preparation and analysis time, MALDI-TOF MS has a huge time advantage compared to other methods. Combined with high precision and sensitivity as well as the simple operative method, MALDI-TOF MS has a high potential in the drinking water sector. Comparisons of MALDI-TOF MS and traditional sequencing indicate that MALDI-TOF MS is in principle a suitable method for the identification of indicator bacteria from water [1-3]. Due to the fastness, MALDI-TOF MS has the potential to become the routine identification method also in the drinking water sector. Yet, the current database is not sufficient to correctly identify all relevant strains within the Enterobacteriaceae and enterococci, as the database is still dominated by clinically relevant bacteria and many environmental isolates are missing. Financial support by the German Ministry of Education and Research (BMBF) and the German Technical and Scientific Association for Gas and Water (DVGW) is gratefully acknowledged (02WGW1460 (BMBF);W201823 (DVGW)).

E. coli, coliform bacteria and enterococci are widely used indicators for the determination of the hygienic quality of drinking water. A positive detection of E. coli indicates a fecal contamination and consequently a potential risk of pathogens. In contrast, coliform bacteria and enterococci do not exclusively originate from feces, but have also been isolated from environmental sources like plants, invertebrates or sediments. If indicator organisms are detected in drinking water systems, the reasons or sources for these contaminations are often unknown. Traditionally, fecal bacteria were identified with classic biochemical methods. In the last years, molecular biological approaches were increasingly used to identify bacteria by genetics or proteins and not by biochemical properties. These fast and specific methods are helpful to identify the sources of fecal contaminations in drinking water. The identification is crucial for further decisions and measures like boiling advices or disinfection measures. DNA-Sequencing is the most common method for the identification of bacteria. The sequence of the 16S-rRNA of an unknown bacterial isolate is compared to the sequences of other known species in a database. For closely related bacteria like Enterobacter, the sequencing of a single gene is not accurate enough for a correct identification. Therefore, additional genes have to be sequenced and compared. MALDI-TOF MS is an identification method based on bacterial protein spectra und is routinely used for the identification of clinically relevant bacteria. With less than one hour of sample preparation and analysis time, MALDI-TOF MS has a huge time advantage compared to other methods. Combined with high precision and sensitivity as well as the simple operative method, MALDI-TOF MS has a high potential in the drinking water sector. Comparisons of MALDI-TOF MS and traditional sequencing indicate that MALDI-TOF MS is in principle a suitable method for the identification of indicator bacteria from water [1-3]. Due to the fastness, MALDI-TOF MS has the potential to become the routine identification method also in the drinking water sector. Yet, the current database is not sufficient to correctly identify all relevant strains within the Enterobacteriaceae and enterococci, as the database is still dominated by clinically relevant bacteria and many environmental isolates are missing. Financial support by the German Ministry of Education and Research (BMBF) and the German Technical and Scientific Association for Gas and Water (DVGW) is gratefully acknowledged (02WGW1460 (BMBF);W201823 (DVGW)).

Before reaching the consumer, potable water passes through a non-sterile drinking water distribution system (DWDS) comprising of pipes and valves of different material and ages. While upstream water treatment processes (i.e. filtration and disinfection) are carefully controlled and monitored according to regulations and guidelines in order to ensure water safety, no similar monitoring of the distribution network exists. With their installation underground, consortia of microorganisms and extracellular polymeric substances referred to as Biofilms form on the surface of the pipes and valves. Biofilms have been shown to harbour microbial communities associated with corrosion of the pipe material, breakdown of residual disinfectants like monochloramine into nutrients, and supporting the growth of opportunistic waterborne pathogens. They have also been described as potentially enabling the horizontal transfer of antimicrobial resistance genes between different bacteria species [1, 2]. The installation of 14 physico-chemical sensor nodes in two operational campuses in Singapore allowed us to generate a full-scale DWDS testbed, thus continuously monitoring Biofilm community dynamics under realistic large-scale conditions. Regular high-resolution flow cytometrical measurements are currently conducted throughout the testbeds together culture-based and molecular techniques to evaluate the composition of the microbial community in both bulk water and Biofilm. In a next step, the algorithm-based Phenoflow, an approach coupling Flow Cytometry with NGS, will be utilized to recognize and identify deviations from a “healthy microbial state” in the DWDS. This will allow ensuring appropriate water quality for the consumer and minimizing the risk of corrosion in critical potable water infrastructure [3].

Before reaching the consumer, potable water passes through a non-sterile drinking water distribution system (DWDS) comprising of pipes and valves of different material and ages. While upstream water treatment processes (i.e. filtration and disinfection) are carefully controlled and monitored according to regulations and guidelines in order to ensure water safety, no similar monitoring of the distribution network exists. With their installation underground, consortia of microorganisms and extracellular polymeric substances referred to as Biofilms form on the surface of the pipes and valves. Biofilms have been shown to harbour microbial communities associated with corrosion of the pipe material, breakdown of residual disinfectants like monochloramine into nutrients, and supporting the growth of opportunistic waterborne pathogens. They have also been described as potentially enabling the horizontal transfer of antimicrobial resistance genes between different bacteria species [1, 2]. The installation of 14 physico-chemical sensor nodes in two operational campuses in Singapore allowed us to generate a full-scale DWDS testbed, thus continuously monitoring Biofilm community dynamics under realistic large-scale conditions. Regular high-resolution flow cytometrical measurements are currently conducted throughout the testbeds together culture-based and molecular techniques to evaluate the composition of the microbial community in both bulk water and Biofilm. In a next step, the algorithm-based Phenoflow, an approach coupling Flow Cytometry with NGS, will be utilized to recognize and identify deviations from a “healthy microbial state” in the DWDS. This will allow ensuring appropriate water quality for the consumer and minimizing the risk of corrosion in critical potable water infrastructure [3].

Before reaching the consumer, potable water passes through a non-sterile drinking water distribution system (DWDS) comprising of pipes and valves of different material and ages. While upstream water treatment processes (i.e. filtration and disinfection) are carefully controlled and monitored according to regulations and guidelines in order to ensure water safety, no similar monitoring of the distribution network exists. With their installation underground, consortia of microorganisms and extracellular polymeric substances referred to as Biofilms form on the surface of the pipes and valves. Biofilms have been shown to harbour microbial communities associated with corrosion of the pipe material, breakdown of residual disinfectants like monochloramine into nutrients, and supporting the growth of opportunistic waterborne pathogens. They have also been described as potentially enabling the horizontal transfer of antimicrobial resistance genes between different bacteria species [1, 2]. The installation of 14 physico-chemical sensor nodes in two operational campuses in Singapore allowed us to generate a full-scale DWDS testbed, thus continuously monitoring Biofilm community dynamics under realistic large-scale conditions. Regular high-resolution flow cytometrical measurements are currently conducted throughout the testbeds together culture-based and molecular techniques to evaluate the composition of the microbial community in both bulk water and Biofilm. In a next step, the algorithm-based Phenoflow, an approach coupling Flow Cytometry with NGS, will be utilized to recognize and identify deviations from a “healthy microbial state” in the DWDS. This will allow ensuring appropriate water quality for the consumer and minimizing the risk of corrosion in critical potable water infrastructure [3].