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There has been an explosion of research into the microorganisms present within drinking water distribution systems (DWDS). However, previous studies have focused mainly on the taxonomic composition and little is known about the actual genes composing the metagenomes of DWDS and their function or whether such information could be used for genetic profiling and monitoring processes taking place in DWDS. We use here for the first time whole metagenome shotgun sequencing to characterise microbial communities from both biofilm and bulk water samples from operational, chlorinated DWDS. Gene content analysis revealed habitat-specific (biofilm vs. water) differences in terms of organisms as well as gene functions, suggesting adaptation to specific environments. In addition, several resistance mechanisms were identified preferentially within biofilms, including genes associated with the prevention and repair of disinfectant radical-induced damage and antibiotic resistance. This research highlights the potential of such information to help protect drinking water quality and safety in the future following further research and wider application.
We have used whole metagenome sequencing to assess the taxonomic affiliation and functional potential of chlorinated drinking water distribution systems (DWDS). This study illustrates how high throughput sequencing of environmental samples can inform on essential processes occurring in DWDS. This information can help on the development of faster and cost-effective monitoring strategies to indicate infrastructure or treatment failures and have the potential to be used in water risk assessment to protect water quality and safety.
The importance of microbial communities within DWDS is widely recognised, and recent research has illuminated the diversity and complexity of these communities.1–3 Microorganisms in DWDS, particularly those inhabiting biofilms, govern key processes including; transformation of metals involved in corrosion,4–6 degradation of disinfectant residual,7,8 changes in water organoleptic characteristics,9 discolouration,10 sheltering pathogens11 and production of toxins and virulence factors.12,13 However, there is virtually no understanding of how microorganisms work individually or as a community, and the impacts on the quality and safety of the drinking water or on the performance of the ageing pipe infrastructure. Prokaryotes are considered as simple organisms at genetic level when compared with eukaryotes, but they do have complex mechanisms to regulate gene expression. Prokaryotes have genomes that contain thousands of different genes which encode proteins with roles in different processes such as metabolism, defense mechanisms against other organisms, maintenance of cell structure, etc. All these genes are not expressed all the time and there are various ways of gene regulation to control which genes are expressed and at what levels.14 The aim of this study was to obtain a genetic fingerprint (gene presence) of microbial habitats in DWDS (biofilm vs. water) as a first step to understand genetic diversity. This initial knowledge will guide further steps in understanding specific mechanisms that can affect water infrastructure and quality such as biofilm formation, discolouration, corrosion, etc. Thus, our approach consists in understanding first what is there (all genes) to then focus further studies and monitoring strategies in specific functions and regulatory processes. To date, microbial ecological and metabolic interactions in drinking water-related systems have been studied using culturing methods, with a combination of two or three isolated microorganisms and under controlled laboratory conditions.15,16 The information presented by these laboratory studies is limited because: i) the laboratory growth conditions used are far from those experienced in real DWDS, and ii) interactions between a few selected microorganisms do not reflect highly diverse communities such as those present in biofilms. Currently, cultivation-independent methods based on targeted sequencing of specific genetic markers, such as the 16S rRNA gene, have provided a greater understanding of the bacterial diversity in DWDS17–20 and can be used to establish microbial networks and co-occurrence pattern.21 However, they do not yield information about prokaryotes and eukaryotes interactions and on the functional or genetic components of microbial communities. A step further in the use of molecular methods to understand DWDS is shotgun metagenomics, the use of DNA-based whole metagenomic analyses can define both taxonomy and potential function (presence of gene pools and expression when sequencing RNA) of complex microbial communities, providing greater insights into the processes and pathways of whole communities and their ecology.22 The whole metagenome sequencing analysis presented here includes the study of eukaryotes, organisms that despite their proven presence in these systems23 have been long ignored in most microbial studies in DWDS, with the exception of amoebas24,25 and the protozoa Cryptosporidium.26 In order to understand dynamics in DWDS and how they work as an ecosystem, we need to consider all interactions including those between eukaryotes and prokaryotes which are important for the maintenance of ecological processes.
Shotgun sequencing has been used in microbial ecology to identify environment-specific genes and interpreting environments27 and to study functional adaptation of planktonic prokaryotes in oceans.28 Environmental metagenome shotgun sequencing can provide new information on rapid diagnostic and monitoring tools29 that can be applied to analyse failures in DWDS, thus useful for public health protection. This research was conducted to assess the potential of whole metagenome sequencing to genetically characterise the ecology of DWDS, in particular aimed at uncovering habitat-specific genetic profiles that can provide further insights into the life styles of organisms living in DWDS. This information can be then used to inform next-generation management strategies that focus on the promotion of “friendly microorganisms” that outcompete pathogenic or undesirable ones. The proposed new monitoring strategies have the potential to be more sustainable, strengthening infrastructure performance while minimizing costly interventions by water utilities.
We studied two operational DWDS by sampling two different sites located in the Southwest of UK one of them supplied by surface water and another one by groundwater. The groundwater site is supplied with a mixture of water from boreholes and treated by marginal chlorination using sodium hypochlorite solution as disinfection residual. The surface water site is supplied with water from local springs, river abstraction and treated by coagulation with aluminium sulphate, flocculation and removal of floc particles by dissolved air flotation. Subsequently the water is filtered using sand filters and granular activated carbon in order to absorb and remove organics. Chlorine is used for disinfection and as a residual in the system. Biofilm sampling devices (Fig. 1) replacing 1 m high density polyethylene (HDPE) pipe in the system were installed at these two sites as described in Douterelo et al.3 These devices have coupons inserted that allowed for analysing compositional and functional characteristics of biofilms developed for a year under realistic conditions. Importantly, these coupons fit flush with the curved inner pipe surface thus maintaining boundary hydraulic conditions, such as shear stress for mobilising force and turbulence for exchange with the bulk water.30,31 Water that supplied both the ground and the surface water site were used to characterise planktonic communities and functional patterns under two different water supplies.
Fig. 1 A) Biofilm sampling devices installed in two chlorinated distribution systems in the south of the UK. The devices have 10 coupons (B) that were inserted in the system and left biofilm develop for a whole year, after this period coupons were collected for biofilm analysis (C).
On the day of coupon collection, after one year of biofilm development in situ, samples of the bulk water that supplied the systems were collected using designated containers for physico-chemical and microbiological characterization via sampling taps located immediately upstream of the devices.
Temperature and pH were measured in situ using a Hanna portable meter and probe HI 991003. All the other parameters were obtained by analysis of discrete water samples by an UK-accredited drinking water laboratory. Flow was measured by magnetic flow meters upstream of the biofilm. Coupons were installed and biofilm left to develop for a period of one year (2014–2015).
DNA was isolated from 24 samples, 6-bulk water and 6 biofilm samples at each site. To obtain DNA from biofilm samples, the whole area of the coupons was brushed and suspended in phosphate buffered saline (PBS), and then biofilm suspensions were concentrated by filtration in 0.22 μm nitrocellulose membrane (Millipore, Corp.). Bulk water samples (6 replicates of 3 L per site) were filtered through 0.22 μm membrane filters for subsequent DNA analysis. Biofilm and bulk water samples were then preserved in the dark at −80 °C until DNA was extracted. For the extractions we used a method based on proteinase K digestion followed by a standard chloroform/isoamyl alcohol method.32 The quantity and purity of the extracted DNA were assessed using Nanodrop ND-1000 spectrophotometer (NanoDrop, Wilmington, USA). In order to obtain enough DNA for whole metagenome sequencing it was necessary to pool DNA from the 6 coupons and the 6 water samples at each site, giving a total of 4 samples. However, because low DNA concentration of the pooled samples (<2 ng ul−1), whole genome amplification was carried out by the sequencing facility Research & Testing Laboratories. After amplification, DNA from the pooled biofilm samples from the surface water supply system was not of enough quantity and quality to proceed with whole metagenome sequencing. Thus, 2 water pooled samples (surface vs. ground) and 1 biofilm final pooled sample (groundwater) were used to obtain sequencing libraries and a description of the gene content of each habitat.
Sequencing libraries of the three pooled samples were prepared by Research and Testing Laboratory (Texas, USA) using a Kapa HyperPlus Single-Index Adapter Kit with TruSeq adapters (Illumina, San Diego, CA, USA), with sample-specific multiplex adaptor according to the manufacturer's instructions. The libraries were sequenced using a single lane in a HiSeq2500 System (Illumina).
MG-RAST Metagenomics Analysis Server v.4.0 (http://metagenomics.anl.gov)33 was used for shotgun metagenomics sequence analysis. We followed the standard steps: pre-processing and quality control, feature identification, feature annotation and profile generation. MG-RAST quality control included the removal of artificial replicates according to the method of Gomez-Alvarez et al.34 and removal of low quality sequences using a modified Dynamic trim35 for sequences with 5 bp below a 15 Phred score 8–23% of the reads were retained after this step (Table 1). Taxonomic profiling was carried out using searches against the SEED Subsystems database, which contains all publicly available genome sequences36 with maximum E-value of 10–5, minimum identity of 60%, and minimum alignment length of 15 amino acids.37 To identify genes and their functions, the reads were annotated using the clusters of orthologous groups of proteins (COGs) database,38 using the same parameters for the searches.
The flow in the groundwater site was low (<5 ML s−1) during the biofilm developmental period when compared with the surface supplied site which fluctuated between 2.5 to 12.5 ML s−1 (data not shown). Table 2 shows the results from the analysis of water samples collected on the day of coupon sampling, after one year of biofilm development. Results from the physico-chemical analysis showed that conductivity and the levels of the nitrous compounds were higher in the groundwater samples compared to the surface water ones. The other analysed parameters were similar at both sites. Very low levels of metals (≤0.01 mg l−1) and turbidity (0.11–≤0.1 NTU) were measured at both sites. Free chlorine concentrations were higher at the surface water site (0.62 mg l−1) than at the groundwater site (0.32 mg l−1).
The structural composition of biofilm and bulk water samples was characterised at a domain level (Fig. 2). Bacteria were clearly dominating the composition of all the studied samples, with a relative abundance of >85% in all samples. However, eukaryotes (4–5%) and viruses (5–6%) were slightly more represented in the structure of the planktonic communities than in biofilms. Among the source water type, the patterns were similar for both type of water, only archaea were slightly more abundant (0.3%) in surface water samples.
Fig. 2 Taxonomic analysis at domain level of water and biofilm samples.
Fig. 3 shows the eukaryotic profiles from biofilm and water samples at the groundwater site. Biofilms harboured a relatively high abundance of Chordata (28%), Ascomycota (23%), Streptophyta (15%) and Arthropoda (15%) (Fig. 3A). However, in bulk water diversity was dominated by fungal Ascomycota taxa representing 70% of all the sequences analysed, followed by Chordata (13%) and Streptophyta (3%) (Fig. 3B). Although found in relatively high abundance, animal and plant taxa are likely to be external contributions captured from the water sources. However, fungi are known to be the main eukaryotic components of biofilms in DWDS32,39 and were highly abundant in our samples.
Fig. 3 Taxonomic analysis of eukaryotes in the ground water supplied systems A) biofilm and B) water.
Fungal diversity (Fig. 4) was mostly composed by the phylum Ascomycota followed by Basidiomycota and Chytridiomicota. Within Ascomycota, the relative abundance of the genus Gibberella was high in both biofilm (37%) and bulk water (ground = 32% and surface = 24%). Schizosaccaromyces was highly abundant in biofilms (26%) whilst other fungal genera samples such as Neurospora (ground = 19% and surface = 13%) and Magnaporthe (ground = 12% and surface = 6%) were mainly present in bulk water.
Fig. 4 Most abundant fungi phylum and genera in the analysed samples in ground (G) and surface (S) supplied systems.
Regarding bacteria, the most abundant phylum in the two habitats were Bacteroidetes, Firmicutes and Proteobacteria (Fig. 5). Within biofilms Bacterioidetes was clearly dominating the composition of the community, with high relative abundance of genera such as Chitinophaga (19%) and Pedobacter (7%). In bulk water samples, Firmicutes was the main phylum represented in the samples, mainly the genera Staphylococus (11–13%) and Bacillus, which clearly dominated the composition of the surface water samples (25%).
Fig. 5 Most abundant bacteria phylum and genera in the analysed samples in ground (G) and surface (S) supplied systems.
Fig. 6 shows a heatmap with the relative abundance of annotated genes in the whole metagenome of each of the sequenced samples. MG-RAST analyses of biofilm and bulk water samples showed similitudes in the relative abundance of the most relevant level 1 (highest COGs functional level) functional categories, which are grouped in 4 categories; 1) cellular processes, 2) information storage and processing, 3) metabolism and 4) poorly characterized. When the genetic profile of biofilm and water samples for the groundwater site was characterised, the most relatively abundant level 1 functional categories were metabolism (biofilm average 50% and water 46%), followed by information storage and processing (biofilm and water average 18%) and cellular process and signaling (biofilm average 19% and water 20%). The fingerprint for both types of waters was similar.
Fig. 6 Heatmap of the relative abundance of annotated genes in the whole metagenome of each of the sequenced samples. COGs functional levels 1 and 2.
Within biofilm and analysing the genetic data at functional level 2, most of the genes involved in metabolism were related to aminoacid transport and metabolism (12%), with genes related to the expression of peptidases (enzymes that hydrolase proteins) in particular dipeptidyl aminopeptidases/acylaminoacyl-peptidases. Within metabolism, most genes were also related with energy production (9%) and the most abundant gene in this category was the aerobic-type carbon monoxide dehydrogenase. Carbon monoxide dehydrogenase is an enzyme that plays an important role in the carbon cycle and is involved in the metabolism of methanogenic, aerobic carboxidotrophic, acetogenic, sulfate reducing, and hydrogenogenic bacteria. Regarding the functional category of information storage and processing, most of the reads (8%) matched genes involved in translation, ribosomal structure and biogenesis and in particular the Asp-tRNAAsn/Glu-tRNAGln amidotransferase gene.
Table 3 shows the results for the specific functional annotated categories related to stress and resistance mechanisms and biofilm formation in samples from the groundwater supplied site. Different gene functions related to cell resistance and protection mechanisms were observed in biofilm and bulk water samples. Specific examples included extracellular polymeric matrix production and degradation (biofilm formation), glutathione protection, SoxRS system, the OyxR system and the RpoS regulated gene (all involved in cell protection), and antibiotic resistance. As it was expected, extracellular polymeric substances (EPS) production and degradation genes were almost exclusively identified in biofilms. The most abundant genes in the samples related to biofilm formation were sialic acid synthesis (0.09%) and dTDP-rhamanose synthesis (0.06%). Both of these genes are related to the synthesis of carbohydrates which enhances biofilm formation and influences motility and pathogenicity of bacteria. Gene categories related to cellular protection mechanisms were predominantly present in biofilms, such as catalase (0.13%) within the OxyR system and superoxide dismutase (0.14%) and aconitase A (0.28%) in the SoxRS system. Antibiotic related resistance genes were clearly more abundant in biofilms, represented mainly by beta-lactamase resistance genes (0.21%).
Genes related to multidrug efflux pump and transport mechanisms were similarly represented in both habitats (biofilm and bulk water), with multidrug efflux transport genes (biofilm = 0.37% and water = 0.54%) and cation/multidrug efflux pump (biofilm = 0.68% and water = 0.77%). Within the potential degradation genes, arylsulfatase A and related enzymes were highly represented in biofilms (0.34%) and these enzymes are related to the decomposition of organic matter and aromatic compounds. Beta-glucosidase activity related to the breakdown of carbohydrates was equally represented in both habitats (biofilm = 0.13% and water = 0.15%).
Our results show that not only prokaryotes are relevant in DWDS. Other taxonomic domains, including viruses, eukaryote and archaea were identified in the analysed samples, mostly in the bulk water samples at both sites. Eukaryotes are part of the ecological network of these engineered systems and consequently they can affect the quality and safety of the water. There are few studies based on eukaryotes in DWDS and those have been mainly focused in the parasitic protozoan Cryptosporidium and Giardia, or in planktonic amoeba studies24,40 and to some extent fungi, mainly Ascomycota.41,42 One of the few drinking water studies that reports the mixed presence of different eukaryotes in water from a desalination plant is that of Belila et al.39 The authors reported that uncultured fungi were the major group of eukaryotes, followed by Chordata and Arthropoda. We can confirm the taxonomic identification of different type of eukaryotes in chlorinated DWDS (water and biofilm); nematodes, amphipods, insect and copepods larvae or most likely fragments of those, these can overcome water treatment and enter in DWDS. Of particular interest in this study is the presence of a high percentage of Chordata, Arthropoda and Nematoda in the analysed biofilm samples that is different from bulk water samples mainly dominated by the fungal group Ascomycota. It must be noted that the identification of metazoan DNA does not necessarily implies that the actual organisms are in the samples. The identification of such organisms could be explained by the presence of free DNA released from animals or plants into the original source water. Alternatively, there may be potential sources of living organisms and biomass in DWDS other than the original treated water. Various interactions and sources through the DWDS, including service reservoirs used for breaks and repairs, ingress due to transients, back siphoning, or cross connections can influence and enrich biofilm communities.43 Thus, external organic matter contributions may be a key source of nutrients for biofilm. If that was the case, a potential good mechanism to control their development in DWDS would be to limit the entry of these sources at treatment work level. Similarly, monitoring for specific members of eukaryotic communities can offer an alternative way of assessing external organic matter entries in DWDS.
The importance of eukaryote–prokaryote relationships has been established in other environments and these interactions can be either beneficial or disadvantageous. There are cross-kingdom cell-to-cell signalling mechanisms that involve small molecules, mainly hormones, that are produced by eukaryotes and hormone-like chemicals produced by bacteria.44 Interactions between algae and bacteria are common in roots of higher plants for example, where microalgae serve as firm matrix to help create biofilms.45 Similarly, fungi hyphae can serve as building blocks and/or provide biotic support for the establishment and colonization of surfaces by bacteria. Cross-kingdom interactions such as those of bacterial-fungal associations are involved also in contact, adhesion and colonization of surfaces, thus in the process of mixed species biofilm formation.46 This study suggests that the contribution of fungi, particularly Ascomycota, to the microbial ecology of real DWDS is important, as has been previously reported in previous studies.41 Ascomycota form spores to propagate and the small size of these together with their resistant to disinfection can explain why they were the main representatives of eukaryotes in water samples. Bioremediation studies have proved the potential of non-ligninolytic fungi such as Ascomycota to degrade chlorinated and polycyclic aromatic hydrocarbons.47 Besides fungal–bacterial interactions, other cross-kingdom interactions reported in DWDS are predation of bacteria and viruses by amoeba or protozoa.48 The main concern related to the presence of amoeba and protozoa in DWDS, is the association of these with the transport of pathogens such as Legionella or viruses.49 Thus, based on this and our previous results in the same DWDS,41 it can be concluded that bacteria are the main inhabitants of these ecosystems, this can indicate that interactions between bacteria with many other domains are probably non beneficial for these organisms or that bacteria are the main organisms with the necessary mechanisms to thrive in this type of environment.
Knowing the mechanisms of biofilm formation will open the door to predicting the consequences of environmental changes and to engineering them to fulfil particular functions in DWDS. For instance, by directing biofilm formation we could favour “friendly” microbial communities that produce infrastructure-protective EPSs or neutralise pathogens.
Consequently, we suggest that by understanding of the metabolic use of a group of prevalent carbohydrates, such as sialic acids, it may be possible to partially identify factors controlling bacterial colonization of pipe surfaces and the probability of biofilm formation thus helping to predict it.
Following on our discussion on biofilm formation and the central role of bacteria on this, we observed that species belonging to the genus Pseudomonas were more abundant in biofilms. This is not surprising, since the involvement of these microorganisms in the initial steps of biofilm formation has been previously observed in this habitat.3 Our study proposes the potential use of other bacterial groups as alternative bioindicators, of biofilm formation such as Bacteroidetes. Lin et al.52 studying bacterial communities in a drinking water treatment plant proposed Bacterioidetes as a rapid indicator of faecal pollution since its presence can indicate failures in the system earlier than other commonly used faecal indicators such as E. coli. As has been indicated before, such taxonomic information is of practical relevance to applications such as design of new bioindicators. These two are examples of how the information obtained at genetic level can help to improve risk assessment, monitoring and management strategies.
Most genes in biofilms were related to the metabolism of amino acids and carbohydrates. This suggests that microbial communities in biofilms play a key role in carbon and nitrogen cycling through for example organic matter decomposition. Previous observation that biofilms may be self-sufficient in the generation of biomass brings into question the use of nutrient management strategies in DWDS.53 Therefore, rather than intervening at the water treatment works to control the incoming water quality, it might be better or also necessary to invest in network interventions, such as cleaning strategies. However, as highlighted by Husband et al.10 such operations are not a one off, biofilms and associated discolouration risk will re-accumulate/grow, thus an ongoing maintenance strategy is required.
Regarding the functional category of information storage and processing, most of the reads matched genes involved in translation, ribosomal structure and biogenesis and in particular the Asp-tRNAAsn/Glu-tRNAGln amidotransferase gene. This enzyme is fundamental for protein biosynthesis and is involved in the process of RNA translation into proteins. Our observations at a genetic level are complementary to previous studies at a structural level (i.e. physical structure) in DWDS biofilm. Fish et al.,54 showed by performing experimental tests in a chlorinated DWDS, that the main structural components of biofilms were proteins and carbohydrates. The authors demonstrated that the ratio of these two components varied with the flow rate in the system and was associated to the strength/mobilisation of biofilm and hence discolouration risk. Since the production of these structural components is regulated at a genetic level, we can suggest that hydraulic regimes might trigger or suppress the expression and level of expression of genes involved in EPS production such as those characterised in this study. These genetic changes can be also attributed to an evolutionary selection process, as long as the selective pressure (i.e. hydraulic regime) is kept long enough in the system. Future studies focused on linking genetic information and expression of these genes (e.g. amidotransferase gene), can certainly yield better insights into the mechanisms of biofilm formation and on how to improve biofilm control strategies in DWDS.
This study has characterised genes related to microbial resistance and cellular protection, that aid biofilm to be successful in DWDS, whilst affecting water quality and safety. Microbial cells can encounter reactive oxygen species (ROS), which are toxic due to their ability to damage molecules such as DNA, proteins, and lipids.55 Prokaryotes use defence mechanisms based on antioxidant scavenging enzymes, such as superoxide dismutase (SOD), peroxiredoxin, and catalase, to protect cells from ROS damage.56 In most gram-negative bacteria, the main defence systems induced under oxidative stress are: i) the OxyR system (responds to hydrogen peroxide); ii) the SoxRS system (respond to redox-active compound) and ii) several genes regulated by RpoS.57 Oxidative stress can stimulate OxyR and induce the transcription of several antioxidants, including glutaredoxin and thioredoxin reductases. Several genes related to the SoxRS system were found in this study, included superoxidase dismutase, acotinase A and glucose-6-phosphate isomerase. Another protection mechanisms characterised in this study is based on the activity of the enzyme glutathione reductase. Glutathione reductase reduces glutathione disulphide to the sulfhydryl form glutathione (GSH), which plays a key role in maintaining the reducing environment of the cells and resisting oxidative stress.58 GSH genes have been previously associated with enhanced resistance to chlorine by bacteria.59 Chao et al.,60 using drinking water biofilms grown on annular reactors suggested that oligotrophic conditions and chlorination can stimulate GSH synthesis, supporting previous observations on the limited effect of chlorine residual in controlling microbial growth and biofilm formation in DWDS.32 Gomez-Alvarez et al.,61 analysing drinking water receiving different disinfection treatments found that sequences related to glutathione reductase (gorA) and thioredoxin reductase (trxB) were highly present in water treated with chlorine and suggested that the gorA reductase serves as a repair mechanism against damage caused by oxygen radicals. Several studies have been able to associate the response of certain microorganism with specific genetic functions related to chlorine resistance. For example, Gomez-Alvarez et al.,61 in the study mentioned above observed that Gram-negative bacteria related to the families Caulobacteraceae and Rhodobacteraceae were responsible of encoding gorA reductase genes. Similarly, Chao et al.,60 noted that bacteria belonging to Alphaproteobacteria and Sphingomonaceae, which are resistance to chlorination, commonly carry GSH related genes. These are examples of how the specific association between a process (i.e. chlorine/chloramine resistance) and a certain type of microorganism can help to determine the type of treatment needed in DWDS and equally the inefficiency of it. This study succeed at characterising genes involved in processes used by microorganisms to respond to oxidative and chemical stress in water and biofilm, suggesting that both habitats experience oxidative stress. Although, the presence of these genes was higher in biofilms, indicating once again the protective habitat that biofilms offer to its inhabitants, and the limited effect that chlorine impose to control their development in DWDS.
This study reported high relative abundance of genes related to β-lactamase resistance, a common antibiotic, supporting the idea that attached communities on pipes have greater protection to environmental stressors. It has been documented that chlorination increases resistance to antibiotics62,63 and that the higher abundance of chlorine-resistant bacteria (e.g. Pseudomonas and Acidovorax) can contribute to the net increase in antibiotic resistance genes.64 However, it must be noted that antibiotic production and resistance are processes expected to be found in natural communities as a result of the competition between microorganisms.65 Distinguishing between human-driven and natural processes will require identifying and analysing the concentration of the antibiotics in bulk water in future studies. Genes that confer resistance can be transferred from one bacterium to another by mobile genetic elements, and they can be used by bacteria as a natural defence mechanism to avoid competition.66 Further research is also needed to disentangle the role of particular microbial species in biofilms in relation to the exchange of resistance mechanisms, as well as which factors favour the occurrence of such mechanisms.
This is the first ‘proof of concept’ and documented evidence of a whole metagenome sequencing study carried out in operational DWDS. As a consequence, we identified a number of procedural challenges and concerns while carrying out this study. One of such challenges is the difficulty to access biofilm samples in real DWDS, which in this case, this resulted in the need to pool samples prior whole metagenome sequencing. The amount of DNA recommended for whole metagenome sequencing using Illumina is 50–100 ng.67 Our coupons design had yielded enough DNA for our previous 16S-based analyses. However, it did not allow to obtain the larger amounts of DNA required for biological replication and whole metagenome sequencing. Future studies should utilise improved protocols that ensure that sufficient biofilm and bulk water is sampled to enable replicate results. Larger biofilm samples could be obtained by scratching a larger area of the internal pipe surface. However, the coupon design to enable this is difficult, as it would also have to follow the pipe curvature to maintain critical boundary effects that include shear stress and nutrient transfer. The use of removable pipe sections would be more feasible, but there are various complexities that would need to be addressed to ensure lack of contamination during removal and to replacement effects if multiple time points were to be studied. Another potential sampling strategy is to flushing sections of pipes by creating excess hydraulic forces and hence mobilise biofilm into the bulk water. However, it is well documented that this does not completely remove the attached biofilm and only the weakly adhered material would be sampled.68 Obtaining large water samples is relatively easier. For example, instead of filtering by vertical pressure, tangential flow filtration69 could be used that allows for the concentration of a higher volumes of water and consequently higher concentration of cells for subsequent DNA extraction.
Another challenge associated with the low amounts of DNA we isolated is that they are particularly sensitive to any contamination, especially when whole metagenome amplification is used. Contamination controls are used for NGS as a matter of routine, but additional controls when collecting samples and processing them in the laboratory might be advisable. For example, although we were careful in our procedures, we cannot completely rule out that the presence of metazoan DNA in our samples is not due to contamination instead of the most likely interpretation that it is DNA derived from organisms present in the original water source.
Despite the operational issues found, this study has shown for the first time that the use of whole metagenome shotgun sequencing is a promising powerful tool to better understanding the biological processes taking place in DWDS. Future steps will likely involve the selection of genetic markers as indicators of specific processes in DWDS, for example; corrosion, biofilm growth and virulence factors and ultimately test their use for monitoring purposes in real drinking water networks. With this aim, it will be necessary to establish a baseline of the presence (i.e. relative abundance) of these genes under optimal operation conditions. Once the baseline is established and known, significant changes in the relative abundance of these marker genes can indicate when a failure in the system occurs. This will yield data to inform a new risk assessment framework for DWDS that the water utilities can use to better manage these systems. Moreover, we have shown that it is possible to obtain additional information about the genes and their function, which could potentially be used to develop new and more sophisticated monitoring strategies. Thanks to this study, the follow-up research on microbial communities on DWDS will have a more solid methodological background. This will allow future studies to focus on linking genetic taxonomic information and functional analysis of genes, and on how to use our knowledge of the mechanisms of biofilm formation and bacterial resistance for improving the biofilm control strategies in DWDS.
We have used whole metagenome shotgun metagenomics to assess the taxonomic affiliation and functional potential of chlorinated DWDS for the first time. The information obtained in this study not only reinforces previous observations that DWDS hold a diverse community of prokaryotes and eukaryotes, but also provides new insight on the environmental genetic pool and the function of the genes involved. In particular, we have detected, mainly in biofilms, genes related to biofilm formation as well as mechanisms of resistance and damage-repair to external stressors such as chlorine and antibiotics. This study illustrates how high throughput sequencing of genetic data can inform on essential processes occurring in DWDS. This information could inform and help on the development of new indicators of infrastructure or treatment failures and has the potential to be used to protect and promote water quality and safety.
The sequencing data used for this project was deposited and is openly accessible in the MG-RAST server under project number ID MGP80824.
The research reported here was supported by the UK Engineering and Physical Sciences Research Council (EPSRC). EPSRC-LWEC Challenge Fellowship EP/N02950X/1, the Challenging Engineering Grant EP/G029946/1 and the EPSRC Platform Grant Funding EP/1029346/1. Victor Soria Carrasco is supported by a Leverhulme Trust Early Career Fellowship. We would like to recognise and thank Wessex Water Services Ltd for sampling sites, field work and physico-chemical sample analysis.
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Early and complete disclosure of discovery will limit discovery motions and help lead to the early and just resolution of criminal cases; in turn, saving tax dollars and reducing the risk of wrongful convictions.
As you all know, the United States Supreme Court in Riley v. California (134 S. Ct. 2473) held the "search incident to arrest" rules to not allow law enforcement to view and obtain every arrestee's cellular phone data. The Electronic Communication Privacy Act appears to be a step in the right direction.
When convicted, the court may require a waiver of digital privacy that is part and parcel to a 4th Waiver. This may be illegal as it circumvents the new digital privacy laws (i.e., the Electronics Communication Privacy Act.) Plus, a blanket waiver of passwords and cell phones, computers and the like lack necessary nexus, particularity, probable cause required by law. This waiver is a minefield.
AB 370 is the Legislature's most recent attempt to alleviate some of the arcane, overbroad, and harsh controlled substance laws. Now, the possession and transportation of most controlled substances (including marijuana and magic mushrooms) in "personal use" amounts is just a crime of simple possession. This law amends Health and Safety Code, Sections 11360, 11379.5, and 11391.
The newly created Business and Professions Code, Section 2525 makes it a misdemeanor for physicians to make cannabis recommendations, then refer patients to a facility in which the doctor has a financial interest.
Also, the new Department of Consumer Affairs' "Bureau of Medical Marijuana" was created.
California residents struggling with unpaid traffic fines or bail will be pleased to hear that eligibility for "amnesty" has been expanded under AB 405 (amending Penal Code, § 1214.1 and Vehicle Code, § 42008.8).
Under prior law, an amnesty program was available for those who had unpaid fines or bail for Vehicle Code infractions that was initially due on or before January 1, 2013. Payments under the prior law were accepted between October 1, 2015 through March 31, 2017. The program allowed those with unpaid bail or fines to apply to have the fines reduced or and to request permission to get their driver's license back. Reductions are available at 50%, or 80% for those on public assistance. This could reduce fines, which frequently exceed $1,000, to a mere $300, providing substantial relief for indigent populations.
The Legislature extended the program so it is now available to those who have not made payments after September 30, 2015.
The California Legislature also adopted a more relaxed and reasonable approach under the so-called "traffic amnesty" rules. Previously, the court could impose an additional $300 civil assessment against those who fail to appear or who fail to make timely payments on a fine installment plan, effective just 10 days after the court mails a warning letter. Perhaps most troubling, the prior law required payment of the civil assessment before the defendant can schedule his/her next court appearance.
Now, the penalty becomes effective 20 days after mailing the notice, and payment of bail, fines, penalties, fees or the civil assessment is not required before the court can vacate the assessment when the person appears. Payment of the civil assessment is no longer required to schedule a court appearance.
California residents are now automatically registered to vote whenever they get a driver's license or state identification card under AB 1465.
California codified the long-existing First Amendment dictate that citizens are allowed to photograph and video record public interactions with law enforcement. New Penal Code Sections 148 and 69 make it clear that recording police interactions is not a crime and is not, in and of itself, probable cause to arrest.
11.Body Cams and Discovery requests before Preliminary hearing.
This law will facilitate enforcement of pre-preliminary hearing discovery motion orders, as Due Process requires disclosure independent of criminal statutes. This is true so long as there is a mere reasonable probability said body cam evidence effects probable cause, the charge or allegations Bridgeforth v. Superior Court (2013); Brady v. Maryland (1963) 373 US 83. Said procedure helps set up a motion to suppress evidence (under Penal Code Section 1538.5) at the preliminary hearing. Magallan v. Superior Court (2011) 192 Cal App 4th 1444.
Early production of relevant discovery will facilitate prompt and equitable resolution of criminal cases.
I hope this information assists you in your practice as you find justice for your clients.


text:
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Mr. Martinez, proceeding pro se, appeals the district court's dismissal of his civil rights action. The district court dismissed Mr. Martinez's amended complaint in part as legally frivolous, under Neitzke v. Williams, 490 U.S. 319, 324 (1989), in part as barred by the rule in Heck v. Humphrey, 512 U.S. 477, 487 (1994) (stating that when judgment for a plaintiff in a § 1983 suit "would necessarily imply the invalidity of his conviction or sentence, . . . the complaint must be dismissed unless the plaintiff can demonstrate that the conviction or sentence has already been invalidated,") and in part for failure to exhaust state remedies.
Mr. Martinez's claims are difficult to understand. We construe his complaint liberally because Mr. Martinez is proceeding pro se, but we cannot be his advocate. See Haines v. Kerner, 404 U.S. 519, 520-21 (1972).
Mr. Martinez appears to challenge the validity of two state court convictions. Mr. Martinez seeks monetary damages and expungement of the convictions. As to his claims for monetary relief, we agree with the district court that Mr. Martinez's claims are barred under Heck and should be dismissed without prejudice.
To the extent Mr. Martinez is only seeking expungement of his criminal convictions, a § 1983 action is not the proper vehicle. The Supreme Court has held that "habeas corpus is the exclusive remedy for a state prisoner who challenges the fact or duration of his confinement and seeks immediate or speedier release, even though such a claim may come within the literal terms of § 1983." Heck, 512 U.S. at 481, 522; Carson v. Johnson, 112 F.3d 818, 820 (10th Cir. 1997) ( stating that "[g]enerally, § 1983 suits are the proper vehicle to attack unconstitutional conditions of confinement and prison procedures"). Thus the district court properly concluded that, because there is no evidence Mr. Martinez has exhausted his state court remedies, or that no adequate state remedies are available or effective to protect Mr. Martinez's rights, his claims seeking expungement of his criminal convictions should also be dismissed.
Mr. Martinez's remaining claims list a litany of challenges to the conditions of his confinement, including chronic headaches; restricted document preparation materials; being forced drinking tap rather than bottled water; having his clothing, bedding, toothbrush, and similar articles handled with unclean hands; continuing headaches and neck aches; stress from fear of being transferred and suffering from restricted privileges; and being forced to eat unpalatable and unwholesome food. From a review of Mr. Martinez's contentions and the entire record on appeal, we conclude that the district court properly dismissed these claims as well.
We therefore AFFIRM the dismissal of Mr. Martinez's complaint for substantially the reasons set forth in the district court's orders dated January 12, 2000.


FDA Warning Letter to Curatronic Ltd.
Refer to GEN0901250 when replying to this letter.
The Food and Drug Administration (FDA) has learned that your firm is marketing the BioMove 3000, BioMove 5000, Curatron 2000 HT, Curatron 2000 XP, Curatron 2000PC, and Curatron Ultra-Power 3-D Pulsed Electro-Magnetic Field Therapy (PEMF) in the United States without marketing clearance or approval, in violation of the Federal Food, Drug and Cosmetic Act (the Act). Under section 201(h) of the Act, 21 U.S.C. § 321(h), these products are devices, because they are intended for use in the diagnosis of disease or other conditions or in cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the body. As explained below, these devices are being marketed without the required clearance or approval in violation of the Act.
"The BioMove 3000 system . . . works by detecting the EMG signals still left in muscles . . . . This method teaches the brain to re-develop spontaneous and voluntary muscle control."
"The BioMove 3000 is a home device which will work in almost all standard stroke cases, except when the remaining voluntary movement of the stroke survivor is close to zero. If the patient still has voluntary movement e.g. a toe or finger, the BioMove home system is the right choice."
"The BioMove 5000 is not only the best Stroke Rehabilitation system in the world but also the easiest stroke therapy device for use by the stroke survivor."
"The BioMove 5000 is equally well suited for muscle rehabilitation therapy after Spinal Cord Injury [SCI] and Cerebral Palsy (CP)."
"If the stroke survivor hardly has any movement left, the BioMove 5000 Pro device is the right choice as it is able to measure very weak EMG signals with its superior EMG detector and signal processing system."
During the review of the 510(k) for the BioMove 5000 device, your firm was advised that the setting called "spinal cord injury" was not an indication under the proposed indications for use or cleared under the predicate device and that data to support the safe and effective use for specific ailments was required. In an email correspondence dated April 15, 2008, your firm agreed and revised by renaming the settings to reflect the gain levels for the device.
The promotion or labeling of these devices for use in the rehabilitation of spinal cord injury and cerebral palsy and the statements made regarding the devices' ability to teach the brain to re-develop represent new intended uses that require a premarket notification submission under section 510(k) of the Act, 21 U.S.C. § 360(k), per 21 CFR 807.81(a)(3). Additionally, comparative claims regarding the device being the best and easiest stroke rehabilitation system in the world requires clinical data and a new 510(k) submission.
The FDA has also learned that your firm is marketing the Curatron 2000HT, Curatron 2000XP, Curatron 2000PC, and the Curatron Ultra-Power 3-D PEMF devices in the United States without marketing clearance or approval, in violation of the Act.
"a safe and non-invasive way to effectively treat acute and chronic pain as well as rheumatic disorders including arthritis, fibromyalgia, and osteoporosis."
"PEMF promotes healing of soft tissue injuries, inflammatory joints, delayed and non-union fractures, and improves circulation and cellular metabolism."
“Lack of sleep often goes hand in hand with chronic pain and may result in a vicious circle of pain preventing sleep while sleep deprivation results in worsening pain. The Curatron PEMF systems address both these issues at once by simultaneously reducing pain and improving sleep."
The Curatron systems are bone-growth stimulators that incorporate pulsed electromagnetic field technology, resulting in them being classified as Class III devices that require PMA approval. Therefore the BioMove 3000, BioMove 5000, Curatron 2000 HT, Curatron 2000 XP, Curatron 2000PC, and Curatron Ultra-Power 3-D PEMF devices are adulterated under section 501(f)(1)(B) of the Act, 21 U.S.C. § 351(f)(1)(B), because your firm does not have an approved application for premarket approval (PMA) in effect pursuant to section 515(a) of the Act, 21 U.S.C. § 360(e)(a), or an approved application for an investigational device exemption under section 520(b) of the Act, 21 U.S.C. § 360j(g). The devices are also misbranded under section 502(o) of the Act, 21 U.S.C. § 352(o), because your firm did not notify the agency of its intent to introduce the devices into commercial distribution, as required by section 510(k) of the Act, 21 U.S.C. § 360(k). For a device requiring premarket approval, the notification required by section 510(k) of the Act, 21 U.S.C. § 360(k), is deemed satisfied when a PMA is pending before the agency, 21 CFR 807.81(b). .
The kind of information that your firm needs to submit in order to obtain approval or clearance for its device is described on the Internet at http://www.fda.gov/cdrh/devadvice/3122.html. The FDA will evaluate the information that your firm submits and decide whether the products may be legally marketed.
The Office of Compliance requests that Curatronic Ltd. immediately cease the dissemination of promotional materials for the BioMove 3000, BioMove 5000, Curatron 2000 HT, Curatron 2000 XP, Curatron 2000PC, and Curatron Ultra-Power 3-D PEMF devices the same as or similar to those described above. The Office of Compliance also requests that Curatronic Ltd. immediately cease distribution of the Curatron 2000HT, Curatron 2000XP, Curatron 2000PC, and Curatron Ultra-Power 3-D PEMF until clearance or approval is obtained.
You should take prompt action to correct the violations addressed in this letter. Failure to promptly correct these violations may result in regulatory action, which may include detaining your devices without physical examination upon entry into the United States until the corrections are completed. U.S. federal agencies may be advised of the issuance of Warning Letters about devices so that they may take this information into account when considering the award of contracts.
Please notify this office in writing within fifteen business days from the date you receive this letter of the specific steps that your firm has taken to correct the noted violations, as well as an explanation of how your firm plans to prevent these violations, or similar violations, from occurring again. Include documentation of the corrections and/or corrective actions (including any systemic corrective actions) that your firm has taken. If your firm's planned corrections and/or corrective actions will occur over time, please include a timetable for implementation of those activities. If corrections and/or corrective actions cannot be completed within fifteen business days, state the reason for the delay and the time within which these activities will be completed. Your firm's response should be comprehensive and address all violations included in this Warning Letter.
Please refer to the Unique Identification Number GEN0901250 when replying. If you have any questions about the contents of this letter, please contact Ms. Stifano at (301) 796-3517.
Finally, you should know that this letter is not intended to be an all-inclusive list of the violations at your firm’s facility. It is your firm’s responsibility to ensure compliance with applicable laws and regulations administered by FDA. Your firm should investigate and determine the causes of the violations, and take prompt actions to correct the violations and bring the products into compliance.
This page was posted on January 18, 2013.


HASSAN KHOKHAR, individually, J. BRAUNINGER, individually, JAMES WAYNE WHITMIRE, individually, Officers of the City of Atlanta Police Department, Defendants-Appellants.
Before JULIE CARNES, EDMONDSON, Circuit Judges, and WILLIAMS, [*] District Judge.
This action arises from Plaintiff Austin Gates's arrest for violating Georgia's mask statute, O.C.G.A. § 16-11-38, during a protest in downtown Atlanta on November 26, 2014. Plaintiff alleges that he was arrested without probable cause in violation of the Fourth Amendment, and that this flawed arrest also violated the First Amendment and various state laws. In this appeal, we consider his claims against three City of Atlanta police officers who were involved in the arrest: defendants Khokhar, Brauninger, and Whitmire (collectively "Defendants"). As to these individual officers, and based on this arrest, Plaintiff has asserted federal claims, pursuant to 42 U.S.C. § 1983, as well as state law claims for assault and battery, invasion of privacy, unlawful detention, and malicious prosecution. Defendants filed a motion to dismiss Plaintiff's § 1983 and state law claims on the grounds of qualified immunity and official immunity. The district court, however, denied their motion, and they now appeal.
Having carefully reviewed the record, and after hearing oral argument, we conclude that Defendants are entitled to qualified immunity on Plaintiff's § 1983 claims and to official immunity on Plaintiff's state law claims. Accordingly, we REVERSE the district court's order denying the motion to dismiss and REMAND the case for further proceedings consistent with this opinion.
We assume the following facts to be true for purposes of this appeal. On November 26, 2014, Plaintiff participated in a march in downtown Atlanta to protest a grand jury's decision in a police-shooting case in Ferguson, Missouri. During the protest, Plaintiff was given a "V for Vendetta" mask by another protestor. As the image attached to the complaint shows, the mask is a stylized image of the Guy Fawkes character from the movie "V for Vendetta." It is designed to cover the entire face. According to Plaintiff, the mask has become popular among people protesting against politicians, banks, and financial institutions. Plaintiff acknowledges that he and other protesters wore the "V for Vendetta" masks during this Ferguson protest in Atlanta. Plaintiff alleges that he wore the mask both to "express himself and his disagreement with the Ferguson, Missouri grand jury's decision, " and to maintain his anonymity during the protest. Plaintiff claims he never intended to threaten or intimidate anyone by wearing the mask.
At some point during the protest, Defendant Whitmire ordered the protesters to remove their masks. Plaintiff acknowledges that Whitmire warned the protesters multiple times over a loudspeaker that any person wearing a mask during the protest would be arrested. Plaintiff, however, claims he did not hear the warning. Whitmire subsequently issued an order over the radio for the police to arrest anyone who was wearing a mask.
According to Plaintiff, after Whitmire issued the order to arrest protesters wearing masks, a "swarm" of officers dressed in riot gear, including Defendant Khokhar, pushed their way into the protesting crowd. Plaintiff alleges that Khokhar grabbed Plaintiff by the shoulder, pulled him by the strap of his backpack, and arrested him. When Plaintiff asked what he had done and why he was being arrested, Khokhar did not immediately respond. After conferring with other officers, Khokhar "handcuffed [Plaintiff] with plastic cuffs" and "shoved [him] into [a] police car." Khokhar told Plaintiff that he was being arrested for wearing a mask.
I [Officer Khokhar] observed [Plaintiff] wear a "V for Vendetta" mask. [Plaintiff] was actively participating in a protest. The protest had been warned on the loud speakers multiple times that anyone wearing a mask will be arrested. This information was relayed by Unit 15 over the radio that anyone wearing a mask should be arrested. [Plaintiff] still had his mask on. [Plaintiff] was arrested for wearing a mask.
Defendant Brauninger, Khokhar's supervising officer, reviewed and authorized the offense report.
Based on the charges asserted against him in the offense report, Plaintiff, along with other arrestees from the protest, was booked, searched, and photographed at the precinct. After several hours of waiting at the precinct, Plaintiff was taken to the Fulton County jail. Once he arrived at the jail, Plaintiff was able to make a phone call and ultimately post bail.
Plaintiff filed a complaint about his arrest with the City of Atlanta Office of Professional Standards. The City determined that Plaintiff's arrest was "justified, lawful, and proper" and exonerated all of the officers who were involved in it. As noted, Plaintiff thereafter sued the City of Atlanta and the individual officers, asserting § 1983 claims and state law claims. The individual officers moved to dismiss Plaintiff's § 1983 claims on the ground of qualified immunity and his state law claims on the ground of official immunity. The district court denied the motion.
We review the denial of a Rule 12(b)(6) motion to dismiss on qualified or official immunity grounds de novo, applying the same standard as the district court. See Bailey v. Wheeler, 843 F.3d 473, 480 (11th Cir. 2016). When ruling on a motion to dismiss, we "accept the facts alleged in the complaint as true, drawing all reasonable inferences in the plaintiff's favor." Keating v. City of Miami, 598 F.3d 753, 762 (11th Cir. 2010). To avoid dismissal, the "complaint must contain sufficient factual matter . . . to state a claim to relief that is plausible on its face." Ashcroft v. Iqbal, 556 U.S. 662, 678 (2009) (internal quotation marks omitted). A complaint is plausible on its face when it contains sufficient facts to support a reasonable inference that the defendant is liable for the misconduct alleged. Id.
Defendants argue that they are entitled to qualified immunity on Plaintiff's federal constitutional claims asserted under § 1983. "Qualified immunity protects government officials performing discretionary functions from suits in their individual capacities unless their conduct violates clearly established statutory or constitutional rights of which a reasonable person would have known." Dalrymple v. Reno, 334 F.3d 991, 994 (11th Cir. 2003) (internal quotation marks omitted). "When properly applied, [qualified immunity] protects 'all but the plainly incompetent or those who knowingly violate the law.'" Ashcroft v. al-Kidd, 563 U.S. 731, 743 (2011).
To be clearly established, a right must be well-established enough "that every reasonable official would have understood that what he is doing violates that right." Reichle v. Howards, 566 U.S. 658, 664 (2012) (internal quotation marks omitted and alteration adopted). In other words, "existing precedent must have placed the statutory or constitutional question beyond debate" and thus given the official fair warning that his conduct violated the law. Id. (emphasis added); Coffin v. Brandau, 642 F.3d 999, 1013 (11th Cir. 2011) (en banc) ("The critical inquiry is whether the law provided [Defendant officers] with 'fair warning' that their conduct violated the Fourth Amendment.").
Fair warning is most commonly provided by materially similar precedent from the Supreme Court, this Court, or the highest state court in which the case arose. See Terrell v. Smith, 668 F.3d 1244, 1255 (11th Cir. 2012). However, a judicial precedent with identical facts is not essential for the law to be clearly established. Youmans v. Gagnon, 626 F.3d 557, 563 (11th Cir. 2010). Authoritative judicial decisions may "establish broad principles of law" that are clearly applicable to the conduct at issue. Griffin Indus., Inc. v. Irvin, 496 F.3d 1189, 1209 (11th Cir. 2007). And occasionally, albeit not very often, it may be obvious from "explicit statutory or constitutional statements" that conduct is unconstitutional. Id. at 1208-09. In all of these circumstances, qualified immunity will be denied only if the preexisting law by case law or otherwise "make[s] it obvious that the defendant's acts violated the plaintiff's rights in the specific set of circumstances at issue." Youmans, 626 F.3d at 563.
A defendant who asserts qualified immunity has the initial burden of showing he was acting within the scope of his discretionary authority when he took the allegedly unconstitutional action. See Bennett v. Hendrix, 423 F.3d 1247, 1250 (11th Cir. 2005). Assuming the defendant makes the required showing, the burden shifts to the plaintiff to establish that qualified immunity is not appropriate by showing that (1) the facts alleged make out a violation of a constitutional right and (2) the constitutional right at issue was clearly established at the time of the alleged misconduct. See Perez v. Suszczynski, 809 F.3d 1213, 1218 (11th Cir. 2016). Plaintiff does not dispute that Defendants were acting in their discretionary authority when they arrested him on November 26, 2014. The burden thus lies with Plaintiff to show that his arrest violated a constitutional right and that the right was clearly established at the time of the arrest. See id. Plaintiff cannot satisfy either prong of this analysis.
The district court implicitly agreed that Defendants had probable cause to arrest Plaintiff based on the elements of the mask law as set out in the statute. The court, however, noted that the Georgia Supreme Court has also imposed a mens rea element onto the statute, requiring that the wearer of the mask know or reasonably should know that his actions give rise to a reasonable apprehension of intimidation, threats, or impending violence. The district court further added that Plaintiff had alleged that he never intended to threaten, intimidate, or cause the apprehension of violence by his mask-wearing. Given this protestation by Plaintiff in his complaint, the district court concluded that the defendant officers lacked even arguable probable cause to arrest Plaintiff for violating the mask statute.
Addressing whether existing precedent gave Defendants fair notice that an arrest under these circumstances would be unlawful, the district court stated, "The Eleventh Circuit has concluded that it is 'clearly established that an arrest without probable cause to believe a crime has been committed violates the Fourth Amendment.' Von Stein v. Brescher, 904 F.2d 572, 579 (11th Cir. 1990)." (emphasis in district court order) (alteration accepted). The court concluded that Defendants were therefore on notice that their arrest in this case was unlawful. We disagree with the district court's analysis.


UNITED STATES of America, Appellee, v. Olsen KEY, also known as Babe, Defendant-Appellant.
No. 07-3528-cr.United States Court of Appeals, Second Circuit.
UPON DUE CONSIDERATION of this appeal from a judgment of conviction entered in the United States District Court for the Eastern District of New York, it is hereby ORDERED, ADJUDGED, AND DECREED that the judgment of conviction is AFFIRMED.
Lawrence D. Gerzog, New York, N.Y., for Appellant.
Judith Philips, Assistant United States Attorney (Jo Ann M. Navickas, on the brief), for Benton J. Campbell, United States Attorney for the Eastern District of New York, Brooklyn, N.Y., for Appellee.
Defendant-appellant Olsen Key appeals from a judgment of conviction entered on August 15, 2007 in the United States District Court for the Eastern District of New York (Korman J.), convicting him, upon his plea of guilty, of conspiracy to possess with intent to distribute cocaine in violation of 21 U.S.C. § 846, and aiding and abetting the use of a firearm in relation to a crime of violence, in violation of 18 U.S.C. § 924(c)(1). On appeal, Key challenges only the sufficiency of the factual basis for his plea to aiding and abetting the § 924(c)(1) violation. We assume the parties’ familiarity with the underlying facts and procedural history of the case.
on or about December 20, 1994, and lending them his car that morning. It is undisputed that the Mathis brothers subsequently used the guns and Key’s car to rob and murder two other drug-dealers. Although Key knew that the Mathis brothers “were going to carry guns either for a drug deal or  . . . there was a drug dealer they were going to rob,” he says he was unaware of any plans to commit specific acts of robbery or murder.
Fed.R.Crim.P. 11(b)(3)(“Before entering judgment on a guilty plea, the court must determine that there is a factual basis for the plea.”). We review for abuse of discretion a district court’s decision that a defendant’s factual admissions support conviction on the charge to which he has pleaded guilty United States v. Gonzalez, 420 F.3d 111, 120 (2d Cir. 2005) (citations omitted).
Section 924(c)(1) prohibits the use or carrying of a firearm “during and in relation to any crime of violence or drug trafficking crime.” We have held that to be convicted of aiding and abetting a § 924(c)(1) violation, a defendant must be shown “to have known of the underlying crime, to have had an interest in furthering it, and consciously to have assisted others in the use or carrying of weapons in the underlying crime.” United States v. Persico, 164 F.3d 796, 802 (2d Cir. 1999) (citing United States v. Pipola, 83 F.3d 556, 562-65 (2d Cir. 1996)). Key may not have known the identity of the Mathis brothers’ particular victims, but in light of his acknowledged participation in a narcotics conspiracy (particularly the regular salary he was paid by the Mathis brothers), and his knowledge that the Mathis brothers would use the guns that he stored for a robbery, it was no abuse of discretion for the district court to find that there was a sufficient factual basis for Key’s plea. Moreover, it is too late for Key to raise a potential defense of duress. A district court “has no duty under Rule 11 to anticipate or detect, and then rule out, defenses such as necessity.” United States v. Smith, 160 F.3d 117, 123 (2d Cir. 1998).
Finally, in a series of letters to the Court which can be construed as motions, Key requests replacement of his counsel, Lawrence Gerzog, and to be allowed to make new arguments that his counsel has not raised. These motions are all denied. Prior to oral argument, on November 3, 2008, we granted Gerzog’s motion for leave for Key to file a pro se supplemental brief. No brief was forthcoming. Gerzog is already Key’s fourth counsel appointed pursuant to the Criminal Justice Act, and Key’s chief ground for seeking new counsel is Gerzog’s refusal to make arguments that we granted Key leave to make for himself. Key writes that Gerzog never told him that we granted leave to file a supplemental pro se brief, but even if that were so, it would have no bearing on the merits of this appeal.


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International Baccalaureate (IB) music provides students with a greater knowledge and appreciation of music in both western and international traditions. Students develop performance skills and complete an independent research project. Topics covered include: music theory, ear-training, aural skills, music history, world music and analysis. Students also prepare for a Senior Recital in their senior year.
Senior Band is a credit course offered to Grades 10 to 12 instrumental music students. The course is taught on Mondays and Thursdays before school. The band performs in various concerts, festivals and community events. Each year, the group travels on tour to a provincial, national or international location. Past tours have visited Banff, Chicago, Los Angeles, Austria, Russia and Italy.
Visual Art 10 is offered as the culminating year in the International Baccalaureate (IB) Middle Years Programme (MYP). The course focuses on the culture of creativity which is designed to engage students in trans-disciplinary thinking along with using imaginary ability in pursuing thoughtful, provocative and unique art projects. Art 20 transitions into Art 30 through review of technical drawing, conceptual thinking and multiple mixed media projects. Students have increasing responsibility for developing both technical skills and personal “voice” in assignment driven work. In the final term, Art 30 students produce independent work which they have researched and developed up to their independent term. Art 20 and 30 are inquiry based courses.
IB Theatre is offered to students in STS. It is considered the “study of” theatre.
Students are introduced to units on movement and improvisation in Drama 10. In Drama 20, students expand their skill set into acting with scripts through units on script analysis, voice, character, playwriting and motivations. To finish the Alberta Drama Program of Studies, students delve deeper into the acting portion of theatre but also cover directing and marketing. Throughout their Senior School experience, students are given the opportunity to learn about technical theatre across all units.
Grade 10 students are introduced to film studies both through analysis of critically acclaimed motion pictures and through creating their own films. Students break down what makes a film through exercises in framing, camera angle and transitions. They will make three mini films throughout the semester including a static film, music video and final film. Students utilize Hi-Def cameras and iMovie on iMacs to complete their projects.
The STS Senior Choir builds on the strong musical techniques developed in all STS choral programs. This ensemble rehearses before school on Friday mornings and participation is open to all interested singers. The Senior Choir performs at school concerts, assemblies, and festivals.
The pit band is a smaller group of advanced players that accompanies performers in the annual musical. It typically includes wind and brass players, and keyboard, electric guitar, electric bass and drums.
Each fall senior school students are able to participate in the fall production. This usually takes the form of a full scale, Broadway musical. Past productions have included classical shows such as Guys and Dolls and My Fair Lady and contemporary musicals such as Shrek the Musical and Grease. The fall production is open to any student in the senior school. Rehearsals run after school and the performance traditionally goes up the last week of November. Auditions are held in the spring prior to the show opening.
Senior Jazz Band is a co-curricular group that meets once a week before school for rehearsals. The group focuses on big band repertoire with emphasis on ensemble performance, jazz styles, and improvisation. They perform at school events, concerts, and festivals.
Senior Jazz combo is a small jazz chamber group of advanced musicians that meets every two weeks for rehearsals before school. The group focuses on combo playing, jazz standards, and improvisation. They perform at school events, concerts, and community events.
Senior Band is a credit course offered for Grade 10, 11 and 12 instrumental music students. The course is taught on Monday and Thursdays before school. The band performs in various concerts, festivals and community events. Each year, the group travels on tour to a provincial, national or international location. Past tours have visited Banff, Chicago, Los Angeles, Austria and Russia.
STS competes annually in the Zone 5 High School One Act Festival. The One Act Festival contains plays that are under 60 minutes in length and can range in genre from solo pieces to full musical productions. Each year a winner is selected to move on to the Provincial Showcase in Red Deer where students not only perform but engage in workshops lead by theatre professionals.
Recently offered, students use their talents and skills in making a variety of art projects to support Round Square fund-raising initiatives. Students experience the joy of new art processes, the positive self-esteem of skillful production and the pride in using their abilities in the service of others. Workshop offered per term. This workshop is open to students in Grades 8 to 12.
This is an annual event held each spring featuring a fantastic evening of Jazz Music presented by all band students and the Spring Art Show showcasing art work from Grades 7 to 11.
The Graduation Show is an annual event opening each May. All work from Art 30 students over the year, including the Independent Term, is on display. This show is produced and designed entirely by the graduating class.

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