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Occurrence and fate of antibiotic resistant bacteria in sewage

4. Effects of sewage treatment on total numbers and percentages of resistant bacteria

The possibility that resistant bacteria occurring in sewage can reach natural aquatic habitats is correlated to their ability to survive sewage treatment. It is generally assumed that sewage treatment determines a marked reduction in the bacterial numbers, including the total numbers of resistant bacteria. However, some studies have documented higher percentages of multiple-resistant bacteria in treated sewage compared with raw sewage ^64,65, indicating that resistant and susceptible bacterial populations may not be equally affected by treatment.

In the second part of the project, we investigated the effects of tertiary sewage treatment on total numbers (section 4.1) and percentages (section 4.2) of resistant bacteria. The numbers of resistant bacteria in raw sewage, treated sewage and anaerobically digested sludge from two large-scale treatment plants (Avedøre Spillevandscenter I/S and Lynettefællesskabet I/S) were enumerated on media containing ampicillin, gentamicin, tetracycline or all three antibiotics (antibiotic selective method). Bacteriological counts were determined using media selective for two distinct bacterial populations, i.e. coliforms and acinetobacters. This afforded possible discrimination as to whether the effects of sewage treatment varied among different bacterial populations.

In addition, the levels of susceptibility to 14 antibiotics were determined in 442 Acinetobacter isolates obtained from culture on agar media without antibiotics (antibiotic non-selective method) and identified at the genus level. The use of two different methods for quantitative assessment of antibiotic resistance allowed us to compare results obtained by different methods. The description of the sampling sites and the methods used for sampling, bacteriological counts, bacterial isolation, identification, antibiotic susceptibility testing and statistical analysis are described in Chapter 2.

4.1 Effects on total numbers of resistant bacteria

At both plants under study, sewage treatment was associated with a marked reduction in the total numbers of resistant bacteria. The numbers of coliforms resistant to ampicillin, gentamicin and tetracycline were generally 100 to 1000 times lower in treated sewage compared with raw sewage (Figs. 4.1). The only exception was the first sampling time (August) at Lynetten plant, where the numbers of resistant coliforms in treated sewage were about 10 times lower compared with raw sewage. Similar results were seen with acinetobacters (see Paper 4).

Independent of the sample type, coliforms resistant to ampicillin occurred more frequently in comparison with bacteria resistant to gentamicin or tetracycline (Fig. 4.1). Bacteria resistant to all three antibiotics in raw sewage were relatively frequent with regard to both coliforms (10² to 10³ CFU/ml) and acinetobacters (10³ to 10⁴ CFU/ml). Such multiple-resistant bacteria were not detected in treated sewage from the Avedøre plant, and were seen only at low numbers (# 10² CFU/ml) in treated sewage from Lynetten.

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Figure 4.1.
Numbers of coliforms resistant to ampicillin (AMP-resistant), gentamicin (GEN-resistant), tetracycline (TET-resistant) or all three antibiotics (Multi-resistant) in raw and treated sewage from the two treatment plants under study.

Single- and multiple-resistant bacteria occurred in high numbers in digested sludge from both plants (Table 4.1), indicating that anaerobic digestion had little effect on the total numbers of resistant bacteria occurring in sludge. Differences were observed between the two plants, with sludge from Avedøre plant containing lower numbers of resistant bacteria compared with sludge from Lynetten plant. Since the total numbers of resistant bacteria occurring in raw sewage were similar at the two plants (Fig. 4.1), it appeared that the process of sludge treatment at the Avedøre plant reduced the numbers of resistant bacteria more efficiently than at Lynetten plant. However, at both plants digested sludge was further treated by incineration (850° C for 2 sec), which eliminates any form of bacterial life. Furthermore, the resulting ash was not used for agricultural purposes. Therefore, these results do not imply any potential risks for the dissemination of resistant bacteria in the environment through use of sludge in agriculture.

Table 4.1.
Average numbers (CFU/g) of coliforms and acinetobacters resistant to antibiotics occurring in anaerobically digested sludge.

  
The average percentages of acinetobacters and coliforms among total heterotrophic bacteria for each plant and sample type are shown in Table 4.2. Independent of the sample type, acinetobacters were more prevalent in the total heterotrophic bacterial population compared with coliforms. In the Lynetten plant, the prevalence of coliforms in treated sewage was significantly higher than in raw sewage (P<0.05). No other significant differences were observed in the prevalence of the two bacterial populations in different sample types (Table 4.2).

Table 4.2.
Average percentages of coliforms and acinetobacters among total culturable bacteria in raw sewage, treated sewage and anaerobically digested sludge.

*The percentage of coliforms in treated sewage was significantly higher than in raw sewage (P<0.05).

4.2 Effects on percentages of resistant bacteria

The relative numbers of antibiotic-resistant coliforms and acinetobacters were not significantly increased by sewage treatment (Table 4.3). On the contrary, at the Avedøre plant the percentage of ampicillin-resistant acinetobacters in treated sewage was significantly lower than in raw sewage (P<0.05). At the same plant, digested sludge contained significantly lower percentages of ampicillin-resistant acinetobacters (P<0.005), ampicillin-resistant coliforms (P<0.05) and gentamicin-resistant coliforms (P<0.05) compared with raw sewage, indicating that anaerobic digestion also had a positive effect on percentages of resistant bacteria.

Although these results clearly indicate that the relative numbers of single- and multiple-resistant bacteria were not increased by sewage treatment, it should be noted that strains demonstrating multiple-resistant to ampicillin, gentamicin and tetracycline, such as those detected in treated sewage from the Lynetten plant, are unlikely to occur naturally in the environment. Indeed, the occurrence of such multiple-resistant strains was not detected in seawater collected from Øresund that was analysed using the same media and antibiotic concentrations employed in this study (data not shown).

The average percentages of resistant bacteria occurring in raw sewage, treated sewage and digested sludge are shown in Table 4.3. Ampicillin resistance occurred more frequently than tetracycline and gentamicin resistance in both coliforms and acinetobacters. This could be due to the fact that many bacterial species are intrinsically resistant to penicillins in general but particularly to ampicillin. Coliforms were generally more resistant to ampicillin and less resistant to gentamicin and tetracycline compared with acinetobacters (Table 4.3).

Table 4.3.
Average percentages (%) of antibiotic-resistant coliforms and acinetobacters in raw sewage, treated sewage and digested sludge determined by counts on media countaining antibiotics.

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The results obtained by bacteriological counts were confirmed by antibiotic susceptibility testing of Acinetobacter isolates. Among the 14 antibiotics tested, statistically significant differences were rarely observed in the percentages of resistant isolates originating from different sample types (Table 4.4), indicating a limited effect of sewage treatment on percentages of resistant bacteria. Only the percentage of isolates resistant to nalidixic acid was significantly higher in treated sewage than in raw sewage (P<0.05). Furthermore, the increase in the percentage of nalidixic acid resistance in treated sewage was observed only at the Avedøre plant, indicating that this effect was not generally associated with sewage treatment per se, but more specific to the conditions occurring at this particular plant.

The highest overall percentages of Acinetobacter isolates resistant to antibiotics were observed for aztreonam (38.0%), cefoxitin (23.8%), chloramphenicol (18.6%), cefotaxime (10.2%), tetracycline (8.4%) and nalidixic acid (6.8%). Resistances to amikacin, imipenem and tobramycin were not detected. For the remaining antibiotics, the percentages of resistant isolates were less than 3% (Table 4.4). The percentage of ampicillin resistance among Acinetobacter isolates was very low (0.9%) in comparison with the percentages of ampicillin-resistant acinetobacters obtained by bacteriological counts on Baumann agar containing this antibiotic (see Table 4.3). This is probably because bacterial species other than Acinetobacter, including bacteria intrinsically resistant to penicillins (e.g. Pseudomonas), are able to grow on this medium ⁵⁴.

As described in section 2.2.2, in some cases antibiotic resistance was defined according to two different breakpoints, a breakpoint determined empirically based on the distribution of the inhibition zone diameters and a breakpoint used in clinical practice for definition of antibiotic resistance in clinical Acinetobacter isolates. Although the two breakpoints differed only by 1 to 3 mm, such a difference was seen to substantially influence the overall percentages of resistance to aztreonam and chloramphenicol (Table 4.4).

For chloramphenicol and tetracycline, the determination of the breakpoint also influenced the results of the statistical analysis (Table 4.4). According to one breakpoint value, isolates resistant to chloramphenicol occurred more frequently in digested sludge when compared with raw sewage at both the Avedøre plant (P<0.005) and the Lynetten plant (P<0.05), and no statistically significant differences were observed in the occurrence of tetracycline-resistant isolates between different sample types. On the other hand, the use of the second breakpoint value showed that the differences in the occurrence of chloramphenicol-resistant isolates between digested sludge and raw sewage were no longer statistically significant and only tetracycline-resistant isolates from Lynetten occurred more frequently in digested sludge compared with raw sewage (P<0.01).

Table 4.4.
Percentages (%) of antibiotic resistance among Acinetobacter isolates from raw sewage, treated sewage and digested sludge.

* Resistances to aztreonam, chloramphenicol and tetracycline were defined according to two different breakpoints.
^a The percentage of resistant isolates in treated sewage was significantly lower than in raw sewage (P<0.05).
^b The percentage of resistant isolates in digested sludge was significantly lower than in raw sewage (P<0.05).
^c The percentage of resistant isolates in treated sewage was significantly higher than in raw sewage (P<0.05).
^d The percentage of resistant isolates in digested sludge was significantly higher than in raw sewage (P<0.05).

The analysis of the distribution of the inhibition zone diameters showed an atypical distribution for aztreonam-resistance and chloramphenicol-resistance among Acinetobacter isolates, which made the distinction between resistant and susceptible populations quite arbitrary (Paper 4). This type of distribution indicates the occurrence of multiple and gradual levels of resistance to these compounds. A similar distribution was observed also for cefoxitin and cefotaxime, although changes in the selection of the breakpoint values for definition of resistance to these antibiotics did not significantly affect the results of the study.

It should be noted that Acinetobacter isolates obtained from the two plants showed markedly lower percentages of single and multiple antibiotic resistance in comparison with Acinetobacter isolates previously obtained from sewers receiving waste effluent from a pharmaceutical plant (see section 3.2). The fact that this pharmaceutical plant was located in the catchment area of the Avedøre plant suggests that the numbers of resistant bacteria were substantially reduced along the sewage system before sewage reached the treatment plant.

The results of our study are in contrast with some previous studies ^64,65, which indicated an increase in the percentage of multiple-resistant bacteria following sewage treatment. The divergence could be due to either factors affecting the efficiency of removal of resistant bacteria at different plants (e.g. initial composition of sewage, type of treatment, plant operation, etc.) or differences in the bacterial indicators and methods used for quantitative assessment of antibiotic resistance (e.g. type of medium, definition of the breakpoint value, etc.). Indeed, even in this study, slightly different results were obtained depending on the plant, the target bacterial population and the antibiotic under study, as well as on the method and the breakpoint values used to define antibiotic resistance.

4.3 Conclusions

Based on the analysis of samples obtained from the two large-scale sewage treatment plants during a period of six months, it can be concluded that sewage treatment substantially reduces the total numbers of resistant bacteria without increasing their relative numbers. In some cases, the relative numbers of resistant bacteria in treated sewage appeared even to be reduced in comparison with raw sewage.

Nevertheless, it was shown that in some cases low numbers of multiple-resistant bacteria survived sewage treatment and persisted in treated sewage. Consequently, the release of municipal sewage effluents into natural aquatic habitats appears to contribute to the spread of multiple antibiotic resistance in the indigenous aquatic microflora. Similarly, anaerobically digested sludge could contribute to the dissemination of multiple-resistant bacteria when applied directly to agricultural land without any further treatment. In countries such as Denmark, where further treatment is required for the use of sewage sludge as a fertilizer, it would be relevant to investigate the effects of post-treatment and storage of digested sludge on the occurrence of multiple-resistant bacteria.

The results of this investigation indicate that there is a need to understand how long multiple-resistant bacteria originating from municipal sewage effluents are able to survive after they are introduced in natural aquatic habitats. Furthermore, their ability to retain their resistance properties and transfer resistance genes to aquatic bacteria should also be elucidated. The results of our studies on survival and in vitro transfer of antibiotic resistance by multiple-resistant strains isolated from the municipal sewage effluent of the Lynetten plant are described in Chapter 5.

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