High amounts of antibiotic resistance genes and Class I integrons are disseminated in rivers of the Mekong delta (Viet Nam) from the effluents of intensive shrimp farms

In the Mekong delta, intensive shrimp farming rely heavily on a wide panel of antibiotics (ABs) to prevent disease epidemics. Consequently, large amounts of antibiotic multi-resistant bacteria are emerging, threatening adjacent aquatic habitats used for irrigation and drinking water, and potentially impairing human health. In this study, quantification of AB resistance genes (ARGs) was carried out for two consecutive years along the Vam Co River (Long An Province, Viet Nam), a river receiving several effluent channels from intensive shrimp farms. qPCR analyses were conducted on resistance genes sul2 (sulfonamide), oxa1 (ß-lactams), ermB (erythromycin), as well as on intl1 (Class1 integrons) and on bacterial 16S rRNA genes. Results showed that resistance genes and Class 1 integrons were present in all river compartments (sediment and water). Both sul2 and oxa1 reached highest abundances. For instance, sul2 reached respectively a maximum of 5.9×104 copies per gram and 1.3×104 copies per mL of sediment and water. Ratio between 16S rRNA and oxa1 showed that up to 5.1% of all bacterial cells may harbor this resistance gene in water samples. Distribution patterns of both genes were constant along the course of the river, an indication of the generalized use of the related ABs in the shrimp cultures. Contrariwise, strong seasonal and local fluctuations were found for ermB, denoting a more limited and localized usage of the related AB. In conclusion, massive presence of ARGs along the river confirmed that both water and sediment are dissemination routes, stressing for the development of alternative farming practices.

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

Seasonal and spatial variability of antibiotic resistance genes and Class I integrons in the rivers of the Mekong delta, Vietnam

Florian Frédéric Vincent Breider, Emmanuelle Rohrbach, Pierre Rossi

Aquaculture activities are steadily expanding in Vietnam, covering an estimated 700,000 ha, with 89% of these culture ponds located in the Mekong Delta. Since 2009, large-scale bacterial epidemics have spread in response to this intensive cultivation. Antibiotics, even those considered as a last resort, have only partially mitigated this problem. In turn, the side effects of the massive use of these chemicals include the appearance of mobile genetic elements associated with antibiotic resistance genes (ARGs). The large-scale emergence of a diverse bacterial resistome, along with severe economic losses, has posed significant health risks to local residents. In this study, the seasonal and spatial distributions of the class I integrase (CL1) intl1 and the ARGs sul2 (sulfonamide), BLA-oxa1 (ß-lactams), and ermB (erythromycin) were quantified from water and sediment samples collected during two consecutive seasons along the Vam Co River and its tributary (Long An province, Vietnam). The results showed that CL1 was present in all river compartments, reaching 2.98×104 copies/mL and 1.07×106 copies/g of sediment, respectively. The highest relative copy abundances to the 16S rDNA gene were measured in water samples, with up to 3.02% for BLA-oxa1, followed by sul2 (1.16%) and ermB (0.46%). Strong seasonal (dry season vs. rainy season) and spatial patterns were recorded for all resistance genes. Higher amounts of ARGs in river water could be associated with higher antibiotic use during the rainy season. In contrast, higher amounts of ARGs were recorded in river sediments during the dry season, making this habitat a potential reservoir of transient genes. Finally, the observations made in this study allowed us to clarify the environmental and anthropogenic influences that may favor the dispersal and persistence of ARGS in this riverine ecosystem.

2021

Antibiotics and pesticides in water and sediments from intensive shrimp farms in Southern Vietnam: Preliminary results

Maria Inmaculada Carpinteiro Botana, Luiz Felippe De Alencastro, Nicolas Estoppey

Vietnam is one of the largest shrimps exporters in the world. Intensive shrimp farming uses a lot of insecticides to sanitize the shrimp ponds and antibiotics for the prevention of diseases. The aims of this study are to develop a passive sampling method for evaluation of six antibiotics and twelve pesticides in the surface water and determine the contamination of surface waters and sediments by these compounds in Can Gio district – Ho Chi Minh City and Long An province, two major intensive shrimp farming sites in Southern Vietnam. Passive samplers (Polar Organic chemical Integrative Samplers - POCIS) enable to calculate time-weighted average concentrations of the free dissolved fraction of pollutants and thus provide precious data about pollutant bioavailability. The results shown that, passive sampling with POCIS is quite effective for evaluation of antibiotics and pesticides in surface water. The antibiotics and pesticides were found in both surface waters and sediments from the shrimp ponds and the canals. The maximum aqueous concentrations of antibiotics and pesticides were found in the shrimp ponds, 2'800 ng/l and 250 ng/l respectively. In the sediments concentrations of antibiotics and pesticides were close to 1'000 ng/kg. A new project is in preparation to confirm these findings and to extend the investigation to others shrimp farms. Possible antibiotic resistance by natural bacterias will also be investigated.