Occurrence and ecological determinants of the contamination of floodplain wetlands with Klebsiella pneumoniae and pathogenic or antibiotic-resistant Escherichia coli

The survival and multiplication of human pathogenic and antibiotic-resistant bacteria in ecosystems is of increasing concern but has been little explored. Wetlands can be contaminated by water fluxes from rivers and may present environmental conditions leading to bacterial survival and multiplication. To test this hypothesis, we sampled 16 wetlands located along three rivers of the Jura Massif, France. The bacterial contamination of the wetland and river waters was measured monthly over a one-year cycle together with the water physico-chemical characteristics. We assessed the abundance of three pathogenic species: Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. The concentrations of E. coli producing extended-spectrum beta-lactamase (ESBL E. coli) or belonging to the phylogenetic group B2 (E. coli B2-more pathogenic) were also measured. We found that rivers carried total E. coli, ESBL E. coli, and K. pneumoniae to wetlands. ESBL E. coli poorly survived in wetlands, whereas total E. coli and K. pneumoniae possibly met favourable physico-chemical conditions for survival and multiplication in these habitats. K. pneumoniae peaked in summer in warm and shallow wetlands. Total E. coli and E. coli B2 potentially reached wetlands through sources other than rivers (hillslope groundwater or leaching from contaminated fields).

Sources, Spreading and Fate of Antibiotic Resistance Genes and Resistant Bacteria in Vidy Bay, Lake Geneva, Switzerland

Nadine Czekalski

Increasing numbers of multiresistant bacterial pathogens cause severe health problems on a global scale. Large quantities of bacteria and their resistance genes are discharged into natural environments via human and animal waste. Not much is known so far about the fate and further spread of resistance determinants in the environment. There is evidence that aquatic environments may function as important compartments for preserving and exchanging antibiotic resistance genes (ARG) from sewage and drinking water. Lake Geneva is the largest fresh water reservoir of Western Europe and supplies the surrounding cities. At the same time these cities discharge sewage into the lake. Lausanne is the largest city in the lake’s catchment and discharges the highest amount of treated, as well as in case of heavy rain events, partly untreated waste waters. Also two rivers, Chambering and Flown, transport untreated sewage (bypass), in case of storm events. Only 3.4 km from the sewage discharge point lake water is pumped for drinking water preparation for Lausanne. Many studies have reported on contamination of the bay with heavy metals, inorganic nutrients, organic carbon and fecal indicator bacteria due to the sewage discharge. The present thesis aimed on investigating the sources, spreading and fate of antibiotic resistant bacteria and antibiotic resistance genes in the Vida bay, Lake Geneva. In the first part of this dissertation, a screening for antibiotic- and specifically multi-resistant bacteria was carried out as one of the first studies in a Swiss waste water stream and the receiving fresh water lake. For this purpose, a combined set of culture based (viable bacterial counts on nutrient media supplemented with antibiotics) and molecular tools (detection and quantitation of antibiotic resistance genes from environmental DNA-extracts using quantitative real-time PCR) was applied. Raw hospital and communal sewage, as well as partly treated/mixed sewage, which is discharged into the lake via an effluent pipe 700 m off shore, were taken into account. Furthermore lake water samples nearby the end of the waste water discharge pipe and the drinking water pump as well as close to the two river mouths were analyzed. Finally, the role of Lausannes WTP was evaluated for its potential to remove resistant and multiresistant bacteria, as well ARG. Results show highest levels of multiresistant bacteria and ARG in both hospital and communal sewage. Incidence was found that the WTP is a selective environment for multiresistant bacteria and resistance genes. Moreover, lake water and sediments nearby the end of the WTP discharge pipe and the two river mouths exhibited significantly higher levels of resistant determinants as compared to samples from the drinking water uptake. If the level of antibiotic resistance close to the drinking water uptake represents the natural background level of the lake or whether it is impacted by the WTP plume remained open during this first survey. The spatial distribution and persistence of ARG discharged via waste water into lake systems has been evaluated for the first time in the second part of this thesis. 22 sediment cores were sampled along two major transects (leading either into the deep lake or along the south-western shore line to the drinking water uptake) and in the vicinity of the WTP discharge pipe in the Vidy bay. A subset of samples was also analyzed for contamination with a “conventional” pollutant of sewage (mercury), for which a similar transport behavior (particle bound) as for bacteria and their resistance genes is assumed. The microbial community composition in all sediment as well as in waste water samples was analyzed using ARISA. Strong pollution with both ARG and mercury was detected in close proximity of the WTP discharge pipe and both contaminants significantly decreased along the transect into the deep lake. In general however, the spatial contamination of mercury and ARG was only moderately correlated. While the mercury contaminated sediment surface area tended to have a pollution tail in south eastern direction, ARG showed only a slow decrease along the south western transect. These findings imply differences in transport and fate of mercury and ARG in the bay, or additional sources of ARG contamination. Analysis of the microbial diversity in the samples support these hypotheses. This study revealed feasibility of qPCR as a tool to semi-quantitatively track spatial ARG contamination in fresh water lake sediments. Not much is known yet on the antibiotic resistance background level of fresh water lakes and how this may be influenced by anthropogenic activities in the surrounding catchment. Water samples from 21 Swiss lakes have been collected and were analyzed for the prevalence of different ARG (2 sulfonamide resistance genes sul1 & sul2, 3 tetracycline resistance genes tetB, tetM, tetW and one plasmid mediated fluoroquinolone resistance gene qnrA). In order to document the abundance of these genes in the bacterial populations, they were normalized to bacterial 16S rRNA gene copy numbers. The lakes differ in size, geographical position, land use pattern, and trophical status. The composition of the microbial community of all lakes was analyzed using ARISA. Sulfonamide resistance genes were detected in all investigated lakes (0.2-4%, sul1 and 0.01-0.3%, sul2), with highest levels in Lake Baldegger (21%). Lake Baldegger also contained all of the three tetracycline resistance genes, which were found in only 4 other lakes. None of the lakes was positive for qnrA. Important anthropogenic catchment activities linked to sul gene prevalence could not be identified based on our data and may require further analysis and an additional variables to complete our data set. However, our study demonstrates contamination of Swiss lakes with sulfonamide resistance genes and the potential of fresh water lakes preserve these genes, even remote from direct sources of pollution.

EPFL2013

Pilus production in Acinetobacter baumannii is growth phase dependent and essential for natural transformation

Melanie Blokesch, Nina Vesel

Acinetobacter baumannii is a severe threat to human health as a frequently multidrug-resistant hospital-acquired pathogen. Part of the danger from this bacterium comes from its genome plasticity and ability to evolve quickly by taking up and recombining external DNA into its own genome in a process called natural competence for transformation. This mode of horizontal gene transfer is one of the major ways pathogens can acquire new antimicrobial resistances and toxic traits. Because these processes in A. baumannii are not well studied, we herein characterized new aspects of natural transformability in this species that include the species’ competence window. We uncovered a strong correlation with a growth–phase-dependent synthesis of a type IV pilus (TFP), which constitutes the central part of competence-induced DNA-uptake machinery. We used bacterial genetics and microscopy to demonstrate that the TFP is essential for the natural transformability and surface motility of A. baumannii, whereas pilus-unrelated proteins of the DNA-uptake complex do not impact the motility phenotype. Furthermore, TFP biogenesis and assembly is subject to input from two regulatory systems that are homologous to Pseudomonas aeruginosa, namely the PilSR two-component system and the Pil-Chp chemosensory system. We demonstrated that these systems not only impact the piliation status of cells but also their ability to take up DNA for transformation. Importantly, we report on discrepancies between TFP biogenesis and natural transformability within the same genus by comparing A. baumannii to data reported for A. baylyi, the latter of which served for decades as a model for natural competence.

2021

Microtissue models for studying the dynamics of host-pathogen interactions in the urinary bladder

Kunal Sharma

Urinary tract infections (UTIs) are amongst the most common bacterial infections and are the second-most frequent reason for antibiotic prescriptions. Moreover, in about 25% of all treated cases, recurrence of infection occurs. Uropathogenic Escherichia coli (UPEC) is the most common causative agent of UTIs. Much of our current understanding about UTIs has come from mouse models of UTI which have highlighted the intracellular lifestyle of this pathogen in the forms of intracellular bacterial communities (IBCs) as the prominent cause for bacterial persistence. Recurrent infections are thought to be caused by bacterial fluxing from persistent IBCs in the umbrella cell layer of the epithelia in the bladder. Although in vitro model systems of UTIs have been developed to study acute phases of infection including the formation of IBC and their dispersal, they suffer from certain shortcomings. For example, these models lack vasculature and the possibility to impose mechanical stresses, such as those experienced by the cells during bladder filling and voiding. Moreover, the design of these in vitro systems makes it difficult to study long-term UPEC persistence when exposed to antibiotic or neutrophil-mediated stresses. In the last two decades, two bioengineering approaches have emerged to generate functional and physiological tissues: organoid and organ-chip systems. Differentiating cells self-organize in a hydrogel and enable the formation of organoids, which mimic various aspects of organ physiology and function in a three-dimensional structure. Organ-chip systems rely upon co-culturing pre-differentiated cells across a synthetic membrane in a defined geometry. The organ-chip systems allow dynamic control over environmental (nutritive) state and over the mechanical stresses experienced by the emulated tissue. In this thesis, I showcase the development of model systems to study UPEC pathogenesis using both these approaches. In the first part of this thesis, we developed a bladder organoid model of UPEC infection that recapitulates the stratified bladder architecture within a volume suitable for high-resolution live-cell imaging. Bacteria injected into the organoid lumen rapidly enter umbrella cells and proliferate to form IBCs. Unexpectedly, we identified populations of individual âsolitaryâ bacteria that form independently of IBCs and which penetrate deeper layers of the organoid wall, and evade killing by antibiotics and neutrophils. Volumetric electron microscopy revealed that these solitary bacteria may be intracellular or pericellular (sandwiched between uroepithelial cells) and are rod-shaped and flagellated, unlike coccoid-shaped and unflagellated bacteria within IBCs. Bacterial fluxing from IBCs is therefore not an essential requirement for the establishment of persistent bacterial populations in the bladder wall. The dynamic responses of IBCs to host stresses and antibiotic therapy are difficult to assess in situ. In another study, we focussed on examining the lifecycle and persistence of IBCs during early stages of UTIs. We developed a human bladder-chip model wherein superficial umbrella cells and bladder microvascular endothelial cells are co-cultured under flow in urine and nutritive media respectively. Bladder filling and voiding was mimicked mechanically by application and release of linear strain. Using time-lapse microscopy, we show that rapid recruitment of neutrophils from the vascular channel