4) Discussion

4.1 Urban waste water treatment and AMR

The major known sources and transmission routes for AMR are through health and food applications. Pathways to the environment through urban waste water treatment largely arise from people excreting resistant bacteria themselves, or taking medicine and excreting some of the active ingredient, which may allow bacteria in the environment to develop resistance.

Gene transfer can take place in urban waste water treatment plants, though the rate and extent may be dependent on a range of factors, including the type of treatment applied. The longer the effluent is stored, the more opportunity there may be for AMR to be generated. This creates challenges: for instance, while disinfection is used to reduce the bacterial load, the waiting time between secondary treatment and disinfection can allow resistant bacteria to grow.

  • Andrea Roskosch (invited by Caroline Whalley) 01 Mar 2019 14:17:45

    Exposure to heavy metals or desinfectants can e. g. induce bacterial adaptions resulting in reduced susceptibility to antibiotics.

    Wales, A.D. and Davies, R.H. 2015. Co-selection of resistance to antibiotics, biocides and heavy metals, and its relevance to foodborne pathogens. Antibiotics 4: 567-604.

  • Bertrand Vallet (invited by Caroline Whalley) 22 Mar 2019 12:59:13

    I would not focus on the effluent but on the sludge. The hydraulic retention time in a WWTP is in general around 24h when the retention time of sludge is from 4 to 12 days in the water tretament steps and even longer for the sludge treatment steps.

    "This creates challenges: for instance, while disinfection is used to reduce the bacterial load, the waiting time between secondary treatment and disinfection can allow resistant bacteria to grow."

    It does not seem very significant to consider this case. The hydraulic retention time between those 2 treatment steps is rather short (min). 

    • Célia Manaia (invited by Caroline Whalley) 28 Mar 2019 11:16:15

       I agree

       The longer the hydarlic residence time during wastewater treatment the more opportunity there may be for AMR to be generated. Also, disinfection used to reduce the bacterial load, may lead to bacteria regrowth. 

       

      I would not focus on the effluent but on the sludge. The hydraulic retention time in a WWTP is in general around 24h when the retention time of sludge is from 4 to 12 days in the water tretament steps and even longer for the sludge treatment steps.

      "This creates challenges: for instance, while disinfection is used to reduce the bacterial load, the waiting time between secondary treatment and disinfection can allow resistant bacteria to grow."

      It does not seem very significant to consider this case. The hydraulic retention time between those 2 treatment steps is rather short (min). 

       

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Disinfection can affect different genes in different ways (Fig [C]). Particularly in the context of urban waste water, it should be noted that the presence of other substances, such as some metals, can co-select for resistance [EDITING NOTE - why does this matter? super-resistant BUGS OR SOMETHING ELSE?].

  • Bertrand Vallet (invited by Caroline Whalley) 22 Mar 2019 13:04:42

    It is important to understand better how the resistance can be enhanced in the UWWTP if it is. Effluent composition veries a lot and identifying co-selection factors may help to also target the measures to be eventually put in place.

  • Célia Manaia (invited by Caroline Whalley) 28 Mar 2019 11:24:38

    A method to assess disinfection efficacy could be based on the quantification of ARGs (Fig [C]). Some genes can even be used as proxy to assess water qaulity in terms of antibiotic resistance load - examples of these are the intI1 integrase gene, or the sul1 gene.

  • Célia Manaia (invited by Caroline Whalley) 28 Mar 2019 11:25:42

    This figure was originaly shown to demonstrate regrowth after disinfection.

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Fig [C] Number of antibiotic resistant genes following disinfection treatment of urban waste water effluents – ozonation and UV.

Source: Sousa et al, J. Hazard Mater. 2017 5;323(Pt A):434-441.

LOQ = below limit of quantification.

Selected genes 16S rRNA gene (marker for bacteria), the intl 1 gene (maker for class 1 integrons, common in Gram-negative bacteria), the blaTEM gene (marker for beta-lactamase resistant Gram-negative bacteria) and the antibiotic resistant genes qnrS and sul1.

  • Dominique Monnet (invited by Caroline Whalley) 26 Mar 2019 14:40:30

    "... the intl1 gene (marker..."

    "and the antibiotic resistance genes qnrS and sul1"

    Note: resistance genes always iin italics (but not other words), plus corrected typo.
    Also applies to the X-axis legends of Fig [C].

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A study in the Netherlands considered 100 UWWTPs (a third of all Dutch UWWTPs) and loadings in the context of well-characterised understanding of water across the country[1]. UWWT reduced the number of bacteria 100-1000 times and was significantly correlated with E. coli. Most of the differences in removal efficiency can be explained by rainfall, where increased rainfall can flush more waste into sewers but can also dilute the influent.

Some work suggests that urban waste water from hospitals represents a potential hotspot for AMR. However, the study showed that usually hospital effluents were not a very significant proportion of influent concentrations (below 10%), although some showed antibiotic concentrations over the minimum inhibitory concentration.

  • Bertrand Vallet (invited by Caroline Whalley) 22 Mar 2019 13:06:58

    The differences in removal efficiency is important but it is even more important to stress that any treatment, even not enforced by disinfection, is better than direct release of effluent into the environment (Combine Sewer Overflows)

  • Célia Manaia (invited by Caroline Whalley) 28 Mar 2019 11:31:17

    However, the study showed that usually hospital effluents were not a very significant proportion of influent concentrations (below 10%), although some showed antibiotic concentrations over the minimum inhibitory concentration. However, besides the load of antibiotics released by hospitals, also the fact that they are important sources of emerging antibiotis resistance genes and bacteria, often multidrug resistant, should be taken into consideration.

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In a study considering treated effluents from UWWTPs in six countries (China, Estonia, Finland, Portugal, Spain and USA), Manaia et al. (2016) found that the numbers of antibiotic resistant bacteria and antibiotic resistant genes released each day varied between plants with a range of 6 orders of magnitude (1012-1018)[2]. While this is still a large number, significant reduction of the loads released was considered an adequate strategy to mitigate antibiotic resistance.

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4.2 Pathways and exposure in the environment

There is limited information on the pathways for and significance of AMR in the environment to reach humans. Because genes can be so readily transferred between microorganisms, there may be several stages involved in any pathway.

A reservoir of AMR may exist in microorganisms that rarely reach humans, but these may interact with “carriers” which readily acquire and transfer genes. These carriers can interact with “vectors” which are widespread, human associated bacteria which can pass on genes to pathogens which have high capacity to infect and cause disease. The physical routes back into humans or animals, then, are through ingestion of contaminated (raw) food and drinking water; physical contact between people/animals, or from surfaces in public areas, recreation (water, sand) or health care. The remaining way is through inhalation, where sanitation workers would be most at risk of inhaling droplets, or populations downwind of intensive farms which have been shown to be more likely to be colonised by animal associated AMR bacteria. Fig [D] provides examples of these different pathways.

Fig [D] Possible routes of transmission of AMR back into humans.

Source: Manaia, Trends Microbiol. 2017 25(3):173-181.

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A key knowledge gap is the significance of environmental exposure of AMR to humans, relative to health and food pathways. However this issue is complicated by the fact that relatively rare gene transfer events from environmental bacteria may lead to pandemic spread of AMR in human associated bacteria. The threshold for environmental levels becoming of concern in terms of acute exposure risk needs to be assessed, although studies such as that by Leonard et al., (2018)[3] suggest that environmental exposure and transmission does occur. Better understanding of the drivers for resistance propagation and persistence is required, and we need to identify which resistance determinants should be of most concern.

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Escherichia coli (E. coli) are widely used as indicators of fecal pollution of water. Extended spectrum beta-lactase (ESBL)-producing E. coli are representative of enzymes produced by bacteria to destroy antibiotics and are one way that bacteria develop resistance. Certain groups of people have greater exposure to such resistant bacteria, principally those swimming in fresh or coastal waters e.g. surfers[4].

  • Dominique Monnet (invited by Caroline Whalley) 26 Mar 2019 14:49:29
    • "Extended-spectrum beta-lactamase" (not "Extended spectrum beta-lactase")
    • We have evidence for swimming in coastal waters (Leonard et al. 2015, and several other references). But not sure that we have evidence for swimming in fresh waters. This must be checked.

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4.3 Monitoring

Robust monitoring procedures are key to ensuring the reliability and comparability of results between different workers and sites. The relative novelty of AMR in the environment means broadly applicable monitoring methods and strategies applicable have yet to be agreed.

Clarity of the monitoring purpose is essential. For AMR in the environment, there are different possibilities. To protect human and domestic animal health, monitoring could assess prevalence of the level of resistance, the use of antibiotics, or the transmission or evolution of resistance, for example. For environmental protection, some measure of ecological effect would be more appropriate. Selection of suitable sites follows the purpose. Monitoring programmes also need to consider costs, method robustness and skills required to implement the methods.

  • Célia Manaia (invited by Caroline Whalley) 28 Mar 2019 11:37:08

    Definition and harmonization of the monitoring purpose is essential. ..... Wastewater AMR monitoring should be framed within the One-Health perspective - humans, animals and the environment. Therefore, a dialogue among different players dealing with AMR issues is required.

  • Thomas Berendonk (invited by Caroline Whalley) 08 Apr 2019 17:20:05

    However, the establishment of a monitoring system for the aquatic and terrestrial environment is most urgent as it exists already for humans and animals, but not for the avorementioned environments. Also monitoring techiques for the these environemnts may differ significantly due to technical reasons to the exsisting monitoring systems and a method bridge needs to be established. Monitoring data for the environment is paramount as at the moment there is no data for orientation - currently we cannot say how much AMR has increased in the envirtonment and if no monitoring data will be setup we will not be able to tell in the future either.

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[1] rivm.nl/bibliotheek/rapporten/2017-0058

[2] Manaia et al., 2016. Applied Microbiology and Biotechnology. 100:1543–1557

[3] https://doi.org/10.1016/j.envint.2017.11.003

[4] Leonard et al, 2017. DOI: 10.1016/j.envint.2015.02.013

  • Dominique Monnet (invited by Caroline Whalley) 26 Mar 2019 14:56:03

    Leonard et al. 2015.

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