Clostridioides difficile from Fecally Contaminated Environmental Sources: Resistance and Genetic Relatedness from a Molecular Epidemiological Perspective.

Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3')-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven "hypervirulent" RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential.

Prevalence, Antimicrobial Resistance and Toxin-Encoding Genes of Clostridioides difficile from Environmental Sources Contaminated by Feces.

Clostridioides difficile (C. difficile) is the most common pathogen causing antibiotic-associated intestinal diseases in humans and some animal species, but it can also be present in various environments outside hospitals. Thus, the objective of this study was to investigate the presence and the characteristics of toxin-encoding genes and antimicrobial resistance of C. difficile isolates from different environmental sources. C. difficile was found in 32 out of 81 samples (39.50%) after selective enrichment of spore-forming bacteria and in 45 samples (55.56%) using a TaqMan-based qPCR assay. A total of 169 C. difficile isolates were recovered from those 32 C. difficile-positive environmental samples. The majority of environmental C. difficile isolates were toxigenic, with many (88.75%) positive for tcdA and tcdB. Seventy-four isolates (43.78%) were positive for binary toxins, cdtA and cdtB, and 19 isolates were non-toxigenic. All the environmental C. difficile isolates were susceptible to vancomycin and metronidazole, and most isolates were resistant to ciprofloxacin (66.86%) and clindamycin (46.15%), followed by moxifloxacin (13.02%) and tetracycline (4.73%). Seventy-five isolates (44.38%) showed resistance to at least two of the tested antimicrobials. C. difficile strains are commonly present in various environmental sources contaminated by feces and could be a potential source of community-associated C. difficile infections.

Prophage Carriage and Genetic Diversity within Environmental Isolates of Clostridioides difficile.

Clostridioides difficile is an important human pathogen causing antibiotic-associated diarrhoea worldwide. Besides using antibiotics for treatment, the interest in bacteriophages as an alternative therapeutic option has increased. Prophage abundance and genetic diversity are well-documented in clinical strains, but the carriage of prophages in environmental strains of C. difficile has not yet been explored. Thus, the prevalence and genetic diversity of integrated prophages in the genomes of 166 environmental C. difficile isolates were identified. In addition, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems were determined in the genomes of prophage regions. Predicted prophages and CRISPR-Cas systems were identified by using the PHASTER web server and CRISPRCasFinder, respectively. Phylogenetic relationships among predicated prophages were also constructed based on phage-related genes, terminase large (TerL) subunits and LysM. Among 372 intact prophages, the predominant prophages were phiCDHM1, phiCDHM19, phiMMP01, phiCD506, phiCD27, phiCD211, phiMMP03, and phiC2, followed by phiMMP02, phiCDKM9, phiCD6356, phiCDKM15, and phiCD505. Two newly discovered siphoviruses, phiSM101- and phivB_CpeS-CP51-like Clostridium phages, were identified in two C. difficile genomes. Most prophages were found in sequence types (STs) ST11, ST3, ST8, ST109, and ST2, followed by ST6, ST17, ST4, ST5, ST44, and ST58. An obvious correlation was found between prophage types and STs/ribotypes. Most predicated prophages carry CRISPR arrays. Some prophages carry several gene products, such as accessory gene regulator (Agr), putative spore protease, and abortive infection (Abi) systems. This study shows that prophage carriage, along with genetic diversity and their CRISPR arrays, may play a role in the biology, lifestyle, and fitness of their host strains.

Effect of magnetite addition on transcriptional profiles of syntrophic Bacteria and Archaea during anaerobic digestion of propionate in wastewater sludge.

Anaerobic digestion (AD) is an important technology for the effective conversion of waste and wastewater to methane. Here, syntrophic bacteria transfer molecular hydrogen (H2 ), formate, or directly supply electrons (direct interspecies electron transfer, DIET) to the methanogens. Evidence is accumulating that the methanation of short-chain fatty acids can be enhanced by the addition of conductive material to the anaerobic digester, which has often been attributed to the stimulation of DIET. Since little is known about the transcriptional response of a complex AD microbial community to the addition of conductive material, we added magnetite to propionate-fed laboratory-scale reactors that were inoculated with wastewater sludge. Compared to the control reactors, the magnetite-amended reactors showed improved methanation of propionate. A genome-centric metatranscriptomics approach identified the active SCFA-oxidizing bacteria that affiliated with Firmicutes, Desulfobacterota and Cloacimonadota. The transcriptional profiles revealed that the syntrophic bacteria transferred acetate, H2 and formate to acetoclastic and hydrogenotrophic methanogens, whereas transcription of potential determinants for DIET such as conductive pili and outer-membrane cytochromes did not significantly change with magnetite addition. Overall, changes in the transcriptional profiles of syntrophic Bacteria and Archaea in propionate-fed lab-scale reactors amended with magnetite refute a major role of DIET in the studied system.

Microbiome Analysis via OTU and ASV-Based Pipelines-A Comparative Interpretation of Ecological Data in WWTP Systems.

Linking community composition and ecosystem function via the cultivation-independent analysis of marker genes, e.g., the 16S rRNA gene, is a staple of microbial ecology and dependent disciplines. The certainty of results, independent of the bioinformatic handling, is imperative for any advances made within the field. In this work, thermophilic anaerobic co-digestion experimental data, together with primary and waste-activated sludge prokaryotic community data, were analyzed with two pipelines that apply different principles when dealing with technical, sequencing, and PCR biases. One pipeline (VSEARCH) employs clustering methods, generating individual operational taxonomic units (OTUs), while the other (DADA2) is based on sequencing error correction algorithms and generates exact amplicon sequence variants (ASVs). The outcomes of both pipelines were compared within the framework of ecological-driven data analysis. Both pipelines provided comparable results that would generally allow for the same interpretations. Yet, the two approaches also delivered community compositions that differed between 6.75% and 10.81% between pipelines. Inconsistencies were also observed linked to biologically driven variability in the samples, which affected the two pipelines differently. These pipeline-dependent differences in taxonomic assignment could lead to different conclusions and interfere with any downstream analysis made for such mis- or not-identified species, e.g., network analysis or predictions of their respective ecosystem service.

Mechanisms Driving Microbial Community Composition in Anaerobic Co-Digestion of Waste-Activated Sewage Sludge.

Anaerobic co-digestion (Co-AD) is used to increase the effectiveness of anaerobic digestion (AD) using local "wastes", adding economic and environmental benefits. Since system stability is of existential importance for the operation of wastewater treatment plants, thorough testing of potential co-substrates and their effects on the respective community and system performance is crucial for understanding and utilizing Co-AD to its best capacity. Food waste (FW) and canola lecithin (CL) were tested in mesophilic, lab-scale, semi-continuous reactors over a duration of 120 days with stepwise increased substrate addition. Key performance indicators (biogas, total/volatile solids, fatty acids) were monitored and combined with 16S-rRNA amplicon sequencing to assess the impact of co-substrate addition on reactor performance and microbial community composition (MCC). Additionally, the latter was then compared with natural shifts occurring in the wastewater treatment plant (WWTP, source) at the same time. An almost linear increase in biogas production with both co-substrates at an approximate 1:1 ratio with the organic loading rate (OLR) was observed. The MCCs in both experiments were mostly stable, but also prone to drift over time. The FW experiment MCC more closely resembled the original WWTP community and the observed shifts indicated high levels of functional redundancy. Exclusive to the CL co-substrate, a clear selection for a few operational taxonomic units (OTUs) was observed. There was little evidence for a persistent invasion and establishment of microorganisms from typical primary substrates into the stable resident community of the reactors, which is in line with earlier findings that suggested that the inoculum and history mostly define the MCC. However, external factors may still tip the scales in favor of a few r-strategists (e.g., Prolixibacter) in an environment that otherwise favors K-strategists, which may in fact also be recruited from the primary substrate (Trichococcus). In our study, specialization and diversity loss were also observed in response to the addition of the highly specialized CL, which in turn, may have adverse effects on the system's stability and reduced resilience and recovery.

Syntrophic acetate oxidation replaces acetoclastic methanogenesis during thermophilic digestion of biowaste.

BACKGROUND: Anaerobic digestion (AD) is a globally important technology for effective waste and wastewater management. In AD, microorganisms interact in a complex food web for the production of biogas. Here, acetoclastic methanogens and syntrophic acetate-oxidizing bacteria (SAOB) compete for acetate, a major intermediate in the mineralization of organic matter. Although evidence is emerging that syntrophic acetate oxidation is an important pathway for methane production, knowledge about the SAOB is still very limited. RESULTS: A metabolic reconstruction of metagenome-assembled genomes (MAGs) from a thermophilic solid state biowaste digester covered the basic functions of the biogas microbial community. Firmicutes was the most abundant phylum in the metagenome (53%) harboring species that take place in various functions ranging from the hydrolysis of polymers to syntrophic acetate oxidation. The Wood-Ljungdahl pathway for syntrophic acetate oxidation and corresponding genes for energy conservation were identified in a Dethiobacteraceae MAG that is phylogenetically related to known SAOB. 16S rRNA gene amplicon sequencing and enrichment cultivation consistently identified the uncultured Dethiobacteraceae together with Syntrophaceticus, Tepidanaerobacter, and unclassified Clostridia as members of a potential acetate-oxidizing core community in nine full-scare digesters, whereas acetoclastic methanogens were barely detected. CONCLUSIONS: Results presented here provide new insights into a remarkable anaerobic digestion ecosystem where acetate catabolism is mainly realized by Bacteria. Metagenomics and enrichment cultivation revealed a core community of diverse and novel uncultured acetate-oxidizing bacteria and point to a particular niche for them in dry fermentation of biowaste. Their genomic repertoire suggests metabolic plasticity besides the potential for syntrophic acetate oxidation. Video Abstract.

Candidatus Syntrophosphaera thermopropionivorans: a novel player in syntrophic propionate oxidation during anaerobic digestion.

Propionate is an important intermediate in the anaerobic mineralization of organic matter. In methanogenic environments, its degradation relies on syntrophic associations between syntrophic propionate-oxidizing bacteria (SPOB) and Archaea. However, only 10 isolated species have been identified as SPOB so far. We report syntrophic propionate oxidation in thermophilic enrichments of Candidatus Syntrophosphaera thermopropionivorans, a novel representative of the candidate phylum Cloacimonetes. In enrichment culture, methane was produced from propionate, while Ca. S. thermopropionivorans contributed 63% to total bacterial cells. The draft genome of Ca. S. thermopropionivorans encodes genes for propionate oxidation via methymalonyl-CoA. Phylogenetically, Ca. S. thermopropionivorans affiliates with the uncultured Cloacimonadaceae W5 and is more distantly related (86.4% 16S rRNA gene identity) to Ca. Cloacimonas acidaminovorans. Although Ca. S. thermopropionivorans was enriched from a thermophilic biogas reactor, Ca. Syntrophosphaera was in particular associated with mesophilic anaerobic digestion systems. 16S rRNA gene amplicon sequencng and a novel genus-specific quantitative PCR assay consistently identified Ca. Syntrophosphaera/Cloacimonadaceae W5 in 9 of 12 tested full-scale biogas reactors thereby outnumbering other SPOB such as Pelotomaculum, Smithella and Syntrophobacter. Taken together the ubiquity and abundance of Ca. Syntrophosphaera, those SPOB might be key players for syntrophic propionate metabolism that have been overlooked before.

Wet biowaste digestion: ADM1 model improvement by implementation of known genera and activity of propionate oxidizing bacteria.

Anaerobic digestion of biowaste not only reduces environmental burden but also plays an important role for sustainable energy supply. For process optimization simulation based on the Anaerobic Digestion Model No. 1 (ADM1) is commonly used. The ADM1 was extended to include the known three genera of propionate oxidizing bacteria (POB) and the two routes of propionate degradation (methyl-malonyl CoA and C6-dismutation pathway). Kinetic parameters for anaerobic propionate oxidation by single strains of the three propionate oxidizing genera were determined from defined tri-cultures of the POB with hydrogenotrophic and acetotrophic methanogens and implemented into ADM1. The such improved model ADM1xpro was evaluated with operational data from a full scale wet biowaste digestion plant. Predicted amounts of biogas and composition with ADM1xpro (2201 m³ d-1, 68.1 % CH4 and 31.9 % CO2) correlated well with full-scale process data (2171 m³ d-1, 67.5 % CH4 and 31.9 % CO2).

Concentration of facultative pathogenic bacteria and antibiotic resistance genes during sewage treatment and in receiving rivers.

Whereas the hygienic condition of drinking and bathing water by law must be monitored by culture-based methods, for quantification of microbes and antibiotic resistance in soil or the aquatic environment, often molecular genetic assays are used. For comparison of both methods, knowledge of their correlation is necessary. Therefore the population of total bacteria, Escherichia coli, enterococci and staphylococci during sewage treatment and in receiving river water was compared by agar plating and quantitative polymerase chain reaction (qPCR) assays. In parallel, all samples were investigated for clinically relevant antibiotic resistance genes. Whereas plating and qPCR data for total bacteria correlated well in sewage after primary treatment, qPCR data of river water indicated higher cell numbers for E. coli. It is unknown if these cells are 'only' not growing under standard conditions or if they are dead. Corresponding to the amount of non-culturable cells, the 'breakpoints' for monitoring water quality should be adapted. The abundances of clinically relevant antibiotic resistance genes in river water were in the same order of magnitude or even higher than in treated sewage. For estimation of the health risk it is important to investigate which species carry respective genes and whether these genes are disseminated via gene transfer.

Growth Behavior of E. coli, Enterococcus and Staphylococcus Species in the Presence and Absence of Sub-inhibitory Antibiotic Concentrations: Consequences for Interpretation of Culture-Based Data.

Culture-based approaches are used to monitor, e.g., drinking water or bathing water quality and to investigate species diversity and antibiotic resistance levels in environmental samples. For health risk assessment, it is important to know whether the growing cultures display the actual abundance of, e.g., clinically relevant antibiotic resistance phenotypes such as vancomycin-resistant Enterococcus faecium/Enterococcus faecalis (VRE) or methicillin-resistant Staphylococcus aureus. In addition, it is important to know whether sub-inhibitory antibiotic concentrations, which are present in surface waters, favor the growth of antibiotic-resistant strains. Therefore, clinically relevant bacteria were isolated from different water sources and the growth behavior of 58 Escherichia coli, 71 Enterococcus, and 120 Staphylococcus isolates, belonging to different species and revealing different antibiotic resistance patterns, was studied with respect to "environmental" antibiotic concentrations. The finding that VRE could only be detected after specific enrichment can be explained by their slow growth compared to non-resistant strains. Interpreting their absence in standardized culture-based methods as nonexistent might be a fallacy. Sub-inhibitory antibiotic concentrations that were detected in sewage and receiving river water did not specifically promote antibiotic-resistant strains. Generally, those antibiotics that influenced cell metabolism directly led to slightly reduced growth rates and less than maximal optical densities after 48 h of incubation.

Sensitivity of antibiotic resistant and antibiotic susceptible Escherichia coli, Enterococcus and Staphylococcus strains against ozone.

Tolerance of antibiotic susceptible and antibiotic resistant Escherichia coli, Enterococcus and Staphylococcus strains from clinical and wastewater samples against ozone was tested to investigate if ozone, a strong oxidant applied for advanced wastewater treatment, will affect the release of antibiotic resistant bacteria into the aquatic environment. For this purpose, the resistance pattern against antibiotics of the mentioned isolates and their survival after exposure to 4 mg/L ozone was determined. Antibiotic resistance (AR) of the isolates was not correlating with higher tolerance against ozone. Except for ampicillin resistant E. coli strains, which showed a trend towards increased resistance, E. coli strains that were also resistant against cotrimoxazol, ciprofloxacin or a combination of the three antibiotics were similarly or less resistant against ozone than antibiotic sensitive strains. Pigment-producing Enterococcus casseliflavus and Staphylococcus aureus seemed to be more resistant against ozone than non-pigmented species of these genera. Furthermore, aggregation or biofilm formation apparently protected bacteria in subsurface layers from inactivation by ozone. The relatively large variance of tolerance against ozone may indicate that resistance to ozone inactivation most probably depends on several factors, where AR, if at all, does not play a major role.

Pilot-scale chitin extraction from shrimp shell waste by deproteination and decalcification with bacterial enrichment cultures.

Extraction of chitin from mechanically pre-purified shrimp shells can be achieved by successive NaOH/HCl treatment, protease/HCl treatment or by environmentally friendly fermentation with proteolytic/lactic acid bacteria (LAB). For the last mentioned alternative, scale-up of shrimp shell chitin purification was investigated in 0.25 L (F1), 10 L (F2), and 300 L (F3) fermenters using an anaerobic, chitinase-deficient, proteolytic enrichment culture from ground meat for deproteination and a mixed culture of LAB from bio-yoghurt for decalcification. Protein removal in F1, F2, and F3 proceeded in parallel within 40 h at an efficiency of 89-91 %. Between 85 and 90 % of the calcit was removed from the shells by LAB in another 40 h in F1, F2, and F3. After deproteination of shrimp shells in F3, spent fermentation liquor was re-used for a next batch of 30-kg shrimp shells in F4 (300 L) which eliminated 85.5 % protein. The purity of the resulting chitin was comparable in F1, F2, F3, and F4. Viscosities of chitosan, obtained after chitin deacetylation and of chitin, prepared biologically or chemically in the laboratory, were much higher than those of commercially available chitin and chitosan.

Detection of the macrolide-lincosamide-streptogramin B resistance gene erm(44) and a novel erm(44) variant in staphylococci from aquatic environments.

Resistance to macrolides, lincosamides and streptogramin B antibiotics (MLSB) is not restricted to staphylococci from clinical samples but can also be present in staphylococci from the aquatic environment. Two coagulase-negative staphylococci-Staphylococcus xylosus and S. saprophyticus were obtained from sewage and receiving river water samples and were investigated for the genetic basis of inducible MLSB resistance by whole-genome sequencing. Two rRNA methylases encoded by erm(44) and a novel erm(44) variant were identified, which had only 84% amino acid identity. While fragments of phage DNA were found in the vicinity of the erm(44) gene of S. xylosus, no relics of mobile genetic elements were detected in the sequences flanking the erm(44) variant gene in the S. saprophyticus strain. The functionality of the erm genes was confirmed by cloning and transformation experiments. Based on the obtained sequences, specific PCR assays for both erm genes were developed and used to identify erm(44) in another 7 S. xylosus and 17 S. saprophyticus isolates from aquatic environments.

Staphylococcus argensis sp. nov., a novel staphylococcal species isolated from an aquatic environment.

A staphylocoagulase-negative, novobiocin-susceptible strain (M4S-6T) of a species of the genus Staphylococcus was isolated from the river Argen in Southern Germany. It was assigned to the genus Staphylococcus due to the presence of the fatty acids, ai-C15 : 0, i-C15 : 0, i-C17 : 0, ai-C17 : 0, and of menaquinone (MK-7) in the cytoplasmic membrane, which are typical of coagulase-negative staphylococci. The polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, an unknown phospholipid and an unknown glycolipid. Although the 16S gene sequence of strain M4S-6T revealed a 98% similarity with its closest relative, Staphylococcus pettenkoferi, it could be distinguished by several phenotypical and physiological markers. In contrast to S. pettenkoferi, M4S-6T was ornithine decarboxylase-positive, urease-negative and could use formiate and l-histidine as carbon-sources; nitrate was not reduced. Whereas S. pettenkoferi could grow with d(-)-mannitol, d-sorbitol, gluconic acid, l-proline, carboxymethylcellulose and lignosulfonate, M4S-6T was not able to grow with these substances. The results of 16S rRNA gene sequence analysis and of phenotypic testing indicated that M4S-6T was a representative of a novel species for which the name Staphylococcus argensis sp. nov., is proposed with the type strain M4S-6T (DSM 29875T = CIP 110904T).

Co-digestion of wheat and rye bread suspensions with source-sorted municipal biowaste.

Acidification of wheat bread (WBS), rye bread (RBS) and fresh biowaste suspensions (FBS), leading to lactate+acetate, lactate+acetate+n-buyrate, and acetate+propionate+n-butyrate, respectively, and biogas production as well as population dynamics were investigated. Co-fermentation of FBS (14 kg m(-3) d(-1) organic loading rate (OLR)) with WBS or RBS was stable up to an OLR of 22 kg m(-3) d(-1) and resulted in up to 3 times as much biogas. During co-fermentation at more than 20 kg m(-3) d(-1) OLR the total population increased more than 2-fold, but the originally low share of propionate-oxidizing bacteria significantly decreased. The proportion of methanogens also decreased. Whereas the proportion of Methanosarcinales to Methanomicrobiales in biowaste and biowaste+WBS remained constant, Methanosarcinales and in particular Methanosaeta spec. in the biowaste+RBS assay almost completely disappeared. Methanomicrobiales increased instead, indicating propionate oxidation via acetate cleavage to CO2 and hydrogen.

Microbial Community Shifts during Biogas Production from Biowaste and/or Propionate.

Propionate is the most delicate intermediate during anaerobic digestion as its degradation is thermodynamically unfavorable. To determine its maximum possible degradation rates during anaerobic digestion, a reactor was fed Monday to Friday with an organic loading rate (OLR) of 12/14 kg CODbiowaste·m-3·d-1 plus propionate up to a final OLR of 18 kg COD·m-3·d-1. No feed was supplied on weekends as it was the case in full-scale. To maintain permanently high propionate oxidizing activity (POA), a basic OLR of 3 kg CODpropionate·m-3·d-1 all week + 11 kg CODbiowaste·m-3·d-1 from Monday to Friday was supplied. Finally a reactor was operated with an OLR of 12 kg CODbiowaste·m-3·d-1 from Monday to Friday and 5 kg CODpropionate·m-3·d-1 from Friday night to Monday morning to maintain a constant gas production for permanent operation of a gas engine. The propionate degradation rates (PDRs) were determined for biowaste + propionate feeding. Decreasing PDRs during starvation were analyzed. The POA was higher after propionate supply than after biowaste feeding and decreased faster during starvation of a propionate-fed rather than a biowaste-fed inoculum. Shifts of the propionate-oxidizing and methanogenic community were determined.

Removal of total and antibiotic resistant bacteria in advanced wastewater treatment by ozonation in combination with different filtering techniques.

Elimination of bacteria by ozonation in combination with charcoal or slow sand filtration for advanced sewage treatment to improve the quality of treated sewage and to reduce the potential risk for human health of receiving surface waters was investigated in pilot scale at the sewage treatment plant Eriskirch, Baden-Wuerttemberg/Germany. To determine the elimination of sewage bacteria, inflowing and leaving wastewater of different treatment processes was analysed in a culture-based approach for its content of Escherichia coli, enterococci and staphylococci and their resistance against selected antibiotics over a period of 17 month. For enterococci, single species and their antibiotic resistances were identified. In comparison to the established flocculation filtration at Eriskirch, ozonation plus charcoal or sand filtration (pilot-scale) reduced the concentrations of total and antibiotic resistant E. coli, enterococci and staphylococci. However, antibiotic resistant E. coli and staphylococci apparently survived ozone treatment better than antibiotic sensitive strains. Neither vancomycin resistant enterococci nor methicillin resistant Staphylococcus aureus (MRSA) were detected. The decreased percentage of antibiotic resistant enterococci after ozonation may be explained by a different ozone sensitivity of species: Enterococcus faecium and Enterococcus faecalis, which determined the resistance-level, seemed to be more sensitive for ozone than other Enterococcus-species. Overall, ozonation followed by charcoal or sand filtration led to 0.8-1.1 log-units less total and antibiotic resistant E. coli, enterococci and staphylococci, as compared to the respective concentrations in treated sewage by only flocculation filtration. Thus, advanced wastewater treatment by ozonation plus charcoal or sand filtration after common sewage treatment is an effective tool for further elimination of microorganisms from sewage before discharge in surface waters.

Removal of micropollutants, facultative pathogenic and antibiotic resistant bacteria in a full-scale retention soil filter receiving combined sewer overflow.

Combined sewer systems collect surface runoff as well as wastewater of industrial and domestic origin. During periods of heavy rainfall the capacity of the sewer system is exceeded and the overflow is discharged into receiving waters without any treatment. Consequently, combined sewer overflow (CSO) is considered as a major source of water pollution. This study investigates the effectiveness of a retention soil filter (RSF) for the removal of micropollutants as well as facultative pathogenic and antibiotic resistant bacteria from CSO. The removal of organic group parameters like total organic carbon was excellent and the removal efficiency for micropollutants of the RSF and the wastewater treatment plant (WWTP), which treats wastewater of the same origin during dry and normal weather conditions, was comparable. Compounds of high environmental concern like estrogens or certain pharmaceuticals, e.g. diclofenac, were completely eliminated or removed to a high degree during RSF passage. RSF treatment also reduced the number of E. coli, enterococci and staphylococci by 2.7, 2.2 and 2.4 log-units (median values), respectively. Obviously, some Staphylococcus species can better adapt to the conditions of the RSF than others as a shift of the abundance of the different species was observed when comparing the diversity of staphylococci obtained from the RSF influent and effluent. RSF treatment also decreased the absolute number of antibiotic resistant bacteria. The percentage of antibiotic resistant E. coli and staphylococci isolates also decreased during passage of the RSF, whereas the percentage of resistant enterococci did not change. For E. coli ampicillin and for enterococci and staphylococci erythromycin determined the antibiotic resistance level. The results demonstrate that RSFs can be considered as an adequate treatment option for CSO. The performance for the removal of micropollutants is comparable with a medium sized WWTP with conventional activated sludge treatment. The number of facultative pathogenic and antibiotic resistant bacteria was considerably decreased during RSF passage. However, as RSF effluents still contained antibiotic resistance genes and traces of micropollutants; receiving waters may still be at risk from negative environmental impacts.

Resistance behaviour of inducible clindamycin-resistant staphylococci from clinical samples and aquatic environments.

In this study, the species diversity of staphylococci with inducible resistance to macrolides, lincosamides and streptogramin B (MLSB) isolated from clinical samples, sewage and river water was investigated. Inducible clindamycin resistance was tested using a D-test and macrodilution assays. Inducible cross-resistance (iMLSB phenotype) was examined by PCR of erm gene classes A, B, C, F, G, Q, T and 43. Although ermC was the most frequently detected resistance gene in iMLSB phenotypes of environmental staphylococci (61.2%), resistance genes encoding iMLSB were more diverse than in staphylococci from hospital samples. In 22.4% of iMLSB staphylococci from aquatic environments, none of the eight tested erm genes was found. Those isolates and erm43-expressing Staphylococcus lentus displayed low erythromycin MICs (3-16 µg ml(-1)) compared with ermC-positive environmental staphylococci (≥256 µg ml(-1)). In contrast to clinical isolates with clearly defined resistance behaviour, resistance patterns against MLSB and MICs for clindamycin of environmental isolates were more diverse. Although the abundance of iMLSB staphylococci in the aquatic environment was lower than in staphylococci from hospital samples, the diversity of resistance genes encoding this phenotype seemed to be higher. Oleandomycin is the best marker to correlate iMLSB phenotype and the respective erm gene. The phenotypical behaviour of environmental isolates may differ from the resistance pattern of clinical iMLSB staphylococci expressing ermA or ermC, and this should be considered for successful treatment of infections.