What contributions have the authors mentioned in the paper "Factors influencing antibiotic resistance burden in municipal wastewater treatment plants" ?

Manaia et al. this paper compared three municipal wastewater treatment plants differing on the dimensions and bio-treatment processes for the loads of amoxicillin-, tetracycline-, and ciprofloxacinresistant heterotrophic bacteria, enterobacteria, and enterococci in the raw inflow and in the treated effluents.

What was the effect of plant TF on bacterial removal?

In fact, plant TF presented very low bacterial removal rates,although contributed to significant reductions on antibiotic resistance percentages.

Why is the variability in plant TF due to the low hydraulic residence time?

In turn, the variability reported in plant TF may be due to both the low hydraulic residence time and the fact that in percolating bioreactors the detachment of biomass aggregates is often observed leading to the heterogeneity observed.

Why was the resistance in the wastewater treated wastewater lower than in the other groups?

In spite of this, and probably due to the heterogeneity of bacteria growing on PCA, antibiotic resistance in heterotrophs from raw wastewater was lower than in the other groups examined (Table 3).

(Open Access) Factors influencing antibiotic resistance burden in municipal wastewater treatment plants (2010) | Ana Novo

Q: What is the main reason for the spread of antibiotic resistance in wastewater treatment plants?

Human sewage comprises both antibioticresistant bacteria and antibiotic residues, a mixture that under favorable conditions, of high nutrient content and close contact between bacteria, may promote antibiotic resistance dissemination (Martínez 2009).

Factors influencing antibiotic resistance burden in municipal

wastewater treatment plants

Ana Novo

Célia M. Manaia

Abstract Municipal wastewater treatment plants are

recognized reservoirs of antibiotic-r esistant bacteria.

Three municipal wastewater treatment plants differing on the

dimensions and bio-treatment processes were compared for

the loads of amoxicillin-, tetracycline-, and ciprofloxacin-

resistant heterotrophic bacteria, enterobacteria, and enterococ-

ci in the raw inflow and in the treated effluents. The sewage

received by each plant, in average, corresponded to 85,000

inhabitant equivalents (IE), including pretreated industrial

effluents (≤30%) in plant activated sludge, 105,000 IE,

including pretreated hospital effluents (≤15%) in plant

trickling filter, and 2,000 IE, exclusively of domestic sewage,

in plant submerged aerated filter. The presence of pretreated

industrial effluents or of pretreated hospital sewage in the raw

inflow did not imply significantly higher densities (per

milliliter or per IE) of antibiotic-resistant bacteria in the raw

wastewater. Longer hydraulic residence periods (24 h)

corresponded to higher bacterial removal rates than shorter

periods (12 and 9 h), although such efficiency did not imply

significant average decreases in the antibiotic resistance

prevalence of the treated effluent. The bacterial loads in the

treated ef fluent could be ranked according to the treatment

efficiency, suggesting that the characteristics of the raw inflow

may have less relevance on the quality of the treated

wastewater than other aspects, such as the inflow volume, the

type of biological treatment, or the hydraulic residence time.

Keywords Wastewater treatment

Antibiotic resistance

Amoxicillin

Tetracycline

Ciprofloxacin

Introduction

Several studies on antimicrobial resistance in municipal

wastewaters have contributed to include wastewater treatment

plants among the leading reservoirs of antibiotic-resistant

bacteria in the environment (Gallert et al. 2005; Ferreira da

Silva et al. 2006, 2007; Goñi-Urriza et al. 2000;Baqueroet

al. 2008;Kümmerer2009a, b; Martínez 2009; Servais and

Passerat 2009). Human sewage comprises both antibiotic-

resistant bacteria and antibiotic residues, a mixture that under

favorable conditions, of high nutrient content and close

contact between bacteria, may promote antibiotic resistance

dissemination (Martínez 2009).

The loads of antibiotic-resistant bacteria in the raw and

treated wastewater of municipal treatment plants suffer sharp

variations from day to day, hampering clear-cut conclusions

on the influence of wastewater treatment on the diminution or

increase of antibiotic resistance prevalence (Guardabassi et al.

2002; Servais and Passerat 2009;Manaiaetal.2010). It has

been referred that the input of antibiotic-resistant bacteria in

the environment represents a major source of antibiotic

resistance dissemination (Kümmerer 2009a;Martínez2009).

Considering this argument, high antibiotic-resistant bacteria

loads in the raw inflow would be decisive for the spreading

of antibiotic resistance by wastewater treatment plants. In

summary, three major driving forces contributing for

antibiotic resistance dissemination by wastewater treatment

plants can be equated: (a) the presence of antibiotic-resistant

bacteria in the raw inflow, with a possible dose-effect; (b) the

variable efficiency of the wastewater treatment process on

the removal of bacteria, namely, those harboring antibiotic

resistance determinants; and (c) the potential increase or

decrease of antibiotic resistance after wastewater treatment,

which may differ among different bacterial and antibiotic

resistance groups.

A. Novo

C. M. Manaia (*)

CBQF, Escola Superior de Biotecnologia,

Universidade Católica Portuguesa,

4200-072 Porto, Portugal

e-mail: cmmanaia@esb.ucp.pt

In respect to (a), the higher the load of antibiotic-resistant

bacteria in the raw wastewater, the more intense can be the

burden of antibiotic resistance in the discharged effluent. For

example, hospital wastewaters, which are supposed to contain

higher loads of antibiotic-resistant organisms, represent

relevant suppliers of resistance into the environment (e.g.,

Guardabassi et al. 2002; Blanch et al. 2003; Reinthaler et al.

2003; Baquero et al. 2008; Servais and Passerat 2009). In

general, it can be hypothesized that municipal wastewater

treatment plants receiving higher loads of antibiotic-resistant

bacteria, for example, hospital-derived effluents, will have

higher environmental burden in terms of antibiotic resistance

spreading. This hypothesis was addressed in the current

study. The efficiency of the wastewater treatment process on

the removal of bacteria, referred to above (b), depends on

several factors, namely, on the type of biological treatment

and on the hydraulic residence time, as longer periods may

favor antibiotic resistance genes exchange, or on the volume

of wastewater treated per day (Tchobanoglous et al. 2003).

Plants in which these aspects differed were compared in the

current study in order to infer the possible implication on

antibiotic resistance removal.

Concerning (c), it is known that wastewater treatment

may impose rearrangements in bacterial populations and

thus, antibiotic-resistant bacteria belonging to different

taxonomic groups may be selected/ eliminated differently

during wastewater treatment (Vilanova et al. 2002; Forster

et al. 2003; Tchobanoglous et al. 2003). On the other hand,

the potential effect of selective pressure imposed by

different antibiotics may vary according to its properties

(as solubility, adsorption, degradability, etc.) and, thus,

within a same group of bacteria, organisms tolerant to one

drug may behave differently, during wastewater treatment,

than others that are tolerant to another antimicrobial agent.

In summary, the antibiotic resistance burden in the treated

effluent is not necessarily the same for different antimicro-

bial drugs and for different bacterial groups. This issue was

addressed in the current study.

To test the hypotheses referred to above, three municipal

wastewater treatment plants that use different biological

treatments (activated sludge (AS), trickling filter (TF), and

submerged aerated filter (SAF); Table 1) were compared.

Three resistance phenotypes against amoxicillin, tetracy-

cline, and ciprofloxacin were selected—the first two were

chosen bec ause t he y are am on g th e most pr es cr ibed ,

namely, in Portugal (Observatório Nacional de Saúde

2002), and the third because we observed that wastewater

treatment could lead to an increase in resistance rates

(Ferreira da Silva et al. 2006, 2007; Manaia et al. 2010).

Residues of these antibiotics and others belonging to the

same families (beta-lactams, tetracyclines, and fluoroqui-

nolones) have been widely quantified in waste- and surface

waters (Kim and Aga 2007; Kümmerer 2009b). Given the

relevance that inflow bacteria may have on the spreading of

antibiotic resistance, we selected three bacterial groups

clearly associated with humans, with high environmental

fitness and co mprising very well known vectors of

antibiotic resistance (www.earss.rivm.nl). Specifically, we

intended to (1) assess the influence of the plant size,

estimated on the bases of inhabitant equivalents and of the

reception of pretreated hospital or industrial effluents on the

load of antibiotic-resistant organisms reaching a municipal

wastewater treatment plant; (2) estimate if, within the same

plant, bacteria resistant to different antibiotics (or belonging

to different groups) were removed at similar rates; and (3)

compare the removal of antibiotic-resistant bacteria and

assess if the loads of these bacteria in the treated outflow

are directly proportional to those in the raw wastewater and/

or bacterial removal rates.

Materials and methods

Wastewater treatment plants and sampling

This study involved three municipal wastewater treat-

ment plants with different dimensions and type of

biological treatment (Table 1). In all the studied plants,

the influent sewage undergoes a preliminary treatment to

remove voluminous solids, but only plants AS and TF

have a primary settling tank to remove the settleable

solids. In plant AS, the settled sewage is biologi cally

treated through an A S process. In the plants TF and SAF,

the biological treatment occurs in fixed film reactors,

constituted by a bed of a highly permeable matrix

supporting a mixed population of microorganisms, which

formaslimelayer.ThefilmreactorsareaTF(inplantTF)

and a subm erged aerated filter, constituting approximately

50% of the volume of the biological tank (in plant SAF).

The treated wastewater discharged from the secondary

settling tank of the three plants enters a natural water-

course without previous d isinfection. Plants AS and TF

are located in different towns, where services represent the

major activity, with industry occupying, respectively,

15.3% and 11.2% of the total urban area. Plant SAF is

located in a touristic village, near the Atlantic coast, with

9.0% of urban area dedicated to industry and 6.5% to

tourism (http://www.ine.pt).

Twenty-four-hour composite samples of the influent of the

biological treatment tank, designated here as raw wastewater,

and of final treated effluent (treated wastewater) were

collected in glass sterile bottles (1 L), transported refrigerated

to the lab, and analyzed within 12 h. Samples were collected at

independent sampling campaigns. In plants AS and TF, six

samples were collected monthly, respectively, from February

to May 2008 and from March to April of 2009, and from

March to May of 2008 and from April to June of 2009. In

plant SAF, four samples were collected monthly from

February and May of 2008.

Enumeration of total cultivable and antibiotic-resistant

bacteria

Bacteriological analyses were performed using the

membrane filtration method as described before (Ferreira

da Silva et al. 2006; Manaia et al. 2010). Heterotrophic

bacteria, enterobacteria, and enterococci were enumerated,

respectively, on plate count agar (PCA, Pronadisa), m-

fecal coliforms (m-FC, Difco), and on m-enterococcus

agar (m-Ent, Difco). The respective antibiotic-resistant

subpopulations were enumerated on the same media

supplemented with the following antibiotic concentrations:

32 mg/L amoxicillin, 16 mg/L tetracycline, or 4 mg/L

ciprofloxacin. These antibiotic concentrations were deter-

mined in previous studies, as adequate to recover

antibiotic-resistant bacteria (Watkinson et al. 2007), and

we assume that bacteria growing in these media are

resistant to the respective antibiotic, independently of the

clinical resistance definition. Membranes, t hrough which

were filtered volumes of 1– 10 mL of sample or of the

adequate serial dilution, were placed onto the culture media

and incubated for 24 h at 30 °C (total heterotrophs) or 37 °C

(enterobacteria), or for 48 h at 37 °C (enterococci). All

analyses were made in triplicate. After the incubation

period, the number of colony forming units (CFU) was

registered on the basis of filtering membranes containing

between ten and 80 colonies. Values of CFU per milliliter

obtained for each of the 12 culture media constituted the

basis for the estimates indicated below.

Data analysis

The values of inhabitant equivalents were estimated for the

time-period comprehending the sampling campaigns, on the

basis of the definition of the Council Directive 91/271/EEC

of 21 May 1991 concerning urban wastewater treatment, as

equated below:

Inhabitant equivalent ¼

BOD5 g=LðÞdaily flow L=dayðÞ

60 g=dayðÞ

Antibiotic resistance percentage was estimated for each

antibiotic in the raw and treated wastewater of each

sampling as:

% Resistance ¼

CFU=mLðÞmedium with antibiotic

CFU=mLðÞmedium without antibiotic

 100:

The bacterial removal rate was estimated for each bacterial

group: heterotrophs, enterobacteria, and enterococci, con-

sidering in each sampling campaign, the ratio between the

Table 1 Operational characteristics of wastewater treatment plants examined in this study

WWTP AS TF SAF

Type of sewage Domestic (∼70%) and

pretreated industrial (∼30%)

Domestic (∼85%) and

pretreated hospital (∼15%)

Domestic

Biological treatment Activated sludge Trickling filter Submerged

aerated filter

Average daily flow (m

/day) 20,000 32,500 900

Hydraulic residence time (h) 12 9 24

Agglomeration population 100,000 150,000 8,700

Inhabitant equivalent 85,000 105,000 2,000

Range of COD in WW (mgO

/L)

Raw 553–604 425–462 107–800

Treated 67–124 120–143 92–187

Range of BOD

in WW (mgO

/L)

Raw 167–400 247–312 26–600

Treated 16–35 38–40 4–26

Heavy metals

in WW

Arsenic (As)

(µg/L)

Raw 1.8–2.4 2.2–2.7 2.7–3.5

Treated 1.3–1.8 1.5–1.7 1.9–2.4

Mercury (Hg)

(µg/L)

Raw <0.10–0.46 <0.10–0.18 <0.10–0.15

Treated <0.10–0.15 <0.10–0.17 <0.10

Site of WWTP

discharge

Water stream River, through a

2–3 km drain

Water stream, 500 m

from the sea

ND not determined

Data from WWTP

Other metals quantified were above limit of quantification (LOQ): Cd<0.05 mg/L; Pb<0.10 mg/L; Cr<0.05 mg/L

CFU per milliliter observed, respectively, on PCA, on m-

FC, or on m-Ent in treated and in raw wastewater:

% Removal rate ¼ 1 

CFU=mLðÞin treated wastewater

CFU=mLðÞin raw wastew ater



 100

The loads of total and antibiotic-resistant bacteria expressed

as CFU per day and per inhabitant equivalent were

calculated for each sampling campaign as:

CFU

dayðÞ

Inhab: Equiv:

CFU

mLðÞ10

 daily flow L

dayðÞ



Inhabitant equivalents

Data on CFU per milliliter on each medium, resistance

percentage in the raw and in the treated wastewater , bacterial

removal rate of total and antibiotic-resistant bacteria, and

resistant CFU per day per inhabitant equivalent were compared

in and between the three plants through the analysis of variance

and the post hoc test of Tukey (SPSS 16.0 for Windows).

Results

Raw wastewater: effect of the type of inflow and population

served size

In this study, we examined three municipal wastewater

treatment plants (Table 1), which are dimensioned to serve

populations with different sizes. Our results suggest that

neither the size of the agglomeration population nor of the

inhabitant equivalents affects the density (CFU per milliliter)

of heterotrophs and enterococci, antibiotic-resistant or not,

found in the raw inflow, as the respective counts (CFU per

milliliter) were in the same order of magnitude in the three

plants. In contrast, enterobacteria presented significantly

lower counts in plant TF than in the other plants (p<0.001;

Table 2). The abundance of bacteria expressed as the counts

of CFU per day per inhabitant equivalent gave a slightly

different picture of the characteristics of the raw inflow

(Fig. 1). In this respect, the most relevant difference was that

in general, plant SAF received significantly more heterotro-

phic bacteria and enterococci (p≤0.001). Among the three

plants examined, TF receives pretreated hospital effluents in

a percentage that ranges 15% of the total. It could be

hypothesized that these pretreated effluents could contain

higher loads of antibiotic-resistant bacteria and thus produce

a noticeable effect on the quality of the raw inflow received

in this plant. Nevertheless, no significantly higher loads of

antibiotic-resistant bacteria were observed in the raw inflow

of this plant (TF), when compared with others. In spite of

this, the comparison of the prevalence of antibiotic resistance

(Table 3) leads to a different conclusion, as tetracycline

resistance was observed to be more prevalent among

heterotrophs and enterobacteria of the raw inflow of the TF

plant (p≤0.05). Similarly, ciprofloxacin resistance was more

prevalent among enterococci of the influent of this plant (p<

0.001). In summa ry, it is possible to conclude that

although the numbers of antibiotic-resistant bacteria are

not significantly higher in the TF plant, the received

wastewater may contain higher percentages of bacteria able

to grow in the presence of tetracycline or ciprofloxacin.

Comparative analysis of bacterial removal rates

The bacterial removal rates were compared for the three plants

(Fig. 2), yielding average reductions of about 1.3 log units of

the CFU per milliliter in plant AS, of 0.6 in plant TF, and of

2.1 in plant SAF. As these values express, the plant SAF

presented the highest efficiency, yielding higher removal

bacterial rates (p<0.001) with ranging values of 98.9–99.7%

for every bacterial groups analyzed (heterotrophs, enter-

obacteria, and enterococci, total and antibiotic resistant). In

plant AS, remo val ra tes rang ed 87.3– 98.3%, and no

significant differences regarding the removal of total and

antibiotic-resistant heterotrophs and enterobacteria (p<0.1).

In contrast, in this same plant, amoxicillin-resistant entero-

cocci were removed with less efficiency than total or

ciprofloxacin- or tetracycline-resistant enterococci (p<

0.001). When compared with others, plant TF presented

significantly lower bacterial removal rates, ranging 53.9–

85.1% (p<0.001) and also higher heterogeneity on the

removal of the different bacterial groups examined. In this

plant, tetracycline-resistant organisms were among the

groups which were removed more extensively that included

also total and amoxicillin-resistant organisms (p<0.05).

Ciprofloxacin-resistant enterobacteria and enterococci were

amongst the antibiotic resistance groups with lower removal

rates, mainly when compared with tetracycline (p<0.05).

When, within the same plant, the removal rates were

compared for different bacterial groups (heterotrophs, enter-

obacteria, and enterococci), it was observed that wastewater

treatment may have different implications depending on the

antibiotic resistance group under analysis (Table 4

). In plant

AS, amoxicillin- and tetracycline-resistant enterobacteria

were rem oved more efficiently than amoxicillin- or

tetracycline-resistant heterotrophs (p≤0.005). In this plant,

amoxicillin-resistant enterococci were also removed at lower

rates than the other bacterial groups (p<0.001). In plant TF,

total and tetracycline-resistant enterococci were removed

more extensively than the other bacterial groups (p<0.001;

p<0.05, respectively). Plant SAF, which presented the

highest removal rates, removed total enterobacteria and

enterococci more efficiently than heterotrophs (p<0.001).

However, this difference was not observed for ciprofloxacin-

resistant enterococci, which were not removed with signif-

icantly lower rates than heterotrophs (p≤0.001). In summary,

the plant with longer hydraulic residence time (SAF)

presented higher removal rates and homogeneity, i.e., different

bacterial groups and resistance phenotypes were removed

similarly, whereas the opposite was observed for shorter

periods of treatment (plant TF). In general, in plant TF,

facultative anaerobes of fecal origin, as the enterobacteria and

enterococci, were removed more efficiently than total hetero-

trophs (Fig. 2 and Table 4).

Treated wastewater: effect of the plant size and bacterial

removal rates

The values of CFU per milliliter discharged differed

significantly among the three plants examined is this study

Table 2 Bacterial density (CFU per milliliter) of the different bacterial taxonomic and antibiotic resistance groups in the raw inflow and in the

treated effluent

Bacteria group counts (CFU/mL) Resistance group WWTP Raw wastewater Treated wastewater

Range Mean Range Mean

Heterotrophs Total AS 8.7×10

–5.5×10

2.9×10

a 6.0×10

–3.3×10

1.4×10

TF 1.0×10

–3.4×10

2.1×10

a 5.3×10

–2.6×10

9.9×10

SAF 2.1×10

–4.8×10

3.0×10

a 1.4×10

–3.7×10

2.3×10

AML AS 2.3×10

–1.2×10

6.4×10

a 5.8×10

–7.7×10

3.3×10

TF 2.0×10

–8.7×10

4.2×10

a 6.1×10

–6.2×10

2.3×10

SAF 3.7×10

–7.9×10

6.0×10

a 2.8×10

–7.8×10

5.0×10

TET AS 1.7×10

–4.5×10

3.0×10

a 4.0×10

–5.0×10

2.3×10

TF 2.0×10

–1.1×10

5.3×10

a 6.7×10

–1.8×10

1.0×10

SAF 3.2×10

–8.0×10

5.1×10

a 1.6×10

–5.0×10

2.8×10

CIP AS 1.8×10

–9.7×10

5.7×10

a 9.3×10

–7.4×10

3.9×10

TF 2.3×10

–1.5×10

5.3×10

a 6.3×10

–2.1×10

1.2×10

SAF 3.8×10

–1.2×10

7.3×10

a 3.2×10

–9.0×10

5.8×10

Enterobacteria Total AS 8.2×10

–3.4×10

1.7×10

b 2.0×10

–2.3×10

7.1×10

TF 3.1×10

–1.3×10

6.4×10

a 1.1×10

–2.6×10

2.0×10

SAF 8.4×10

–1.8×10

1.5×10

b 1.7×10

–9.5×10

5.1×10

AML AS 4.5×10

–2.0×10

9.9×10

b 3.7×10

–5.1×10

1.7×10

TF 2.3×10

–6.8×10

4.2×10

a 6.6×10

–1.2×10

8.9×10

SAF 6.4×10

–1.3×10

8.6×10

b 1.1×10

–3.3×10

2.3×10

TET AS 2.4×10

–1.5×10

6.5×10

a 5.0×10

–3.3×10

1.8×10

TF 1.2×10

–1.8×10

6.2×10

a 4.5×10

–8.6×10

5.8×10

SAF 2.9×10

–6.8×10

4.4×10

a 6.9×10

–1.9×10

1.2×10

CIP AS 5.6×10

–7.5×10

3.1×10

b 3.0×10

–2.4×10

1.0×10

TF 1.9×10

–1.8×10

8.5×10

a 1.3×10

–3.1×10

1.9×10

SAF 7.3×10

–4.4×10

2.5×10

b 5.6×10

–2.3×10

1.0×10

Enterococci Total AS 8.3×10

–2.6×10

1.9×10

a 5.3×10

–2.3×10

1.0×10

TF 7.4×10

–2.9×10

1.6×10

a 1.5×10

–6.1×10

3.5×10

SAF 1.3×10

–1.7×10

1.6×10

a 2.7×10

–5.8×10

4.3×10

AML AS 5.3×10

–1.2×10

8.4×10

a 5.3×10

–1.8×10

8.8×10

TF 1.6×10

–8.8×10

6.3×10

a 1.3×10

–4.3×10

2.0×10

SAF <1.0×10

<1.0×10

TET AS 2.1×10

–3.6×10

3.2×10

a 1.1×10

–2.7×10

2.0×10

TF 1.9×10

–5.5×10

3.4×10

a 6.8×10

–1.1×10

4.7×10

SAF 2.0×10

–4.5×10

3.4×10

a 3.8×10

–1.2×10

1.0×10

CIP AS 1.6×10

–5.7×10

3.1×10

b 1.4×10

–3.7×10

2.1×10

TF 1.5×10

–1.7×10

7.4×10

b 3.1×10

–5.5×10

2.2×10

SAF 2.5×10

–3.2×10

1.5×10

a <1.0×10

–3.4×10

<1.0×10

a, b, and c homogeneous subsets on the basis of Tukey test

Factors influencing antibiotic resistance burden in municipal wastewater treatment plants

Figures

Citations

Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review.

Antibiotic-Resistance Genes in Waste Water.

Correlation of tetracycline and sulfonamide antibiotics with corresponding resistance genes and resistant bacteria in a conventional municipal wastewater treatment plant

Antibiotic resistance, antimicrobial residues and bacterial community composition in urban wastewater.

Wastewater reuse in irrigation: a microbiological perspective on implications in soil fertility and human and environmental health.

References

Wastewater engineering : treatment and reuse

Antibiotics in the aquatic environment - A review - Part II

Antibiotics and antibiotic resistance in water environments

Environmental pollution by antibiotics and by antibiotic resistance determinants

Resistance in Gram-Negative Bacteria: Enterobacteriaceae

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Frequently Asked Questions (8)

Q1. What contributions have the authors mentioned in the paper "Factors influencing antibiotic resistance burden in municipal wastewater treatment plants" ?

Q2. What type of bacteria were enumerated on plate count agar?

Q3. What was the effect of plant TF on bacterial removal?

Q4. What is the main reason for the spread of antibiotic resistance in wastewater treatment plants?

Q5. What is the effect of the secondary settling tank on the wastewater?

Q6. Why is the variability in plant TF due to the low hydraulic residence time?

Q7. What is the main argument for the spread of antibiotic resistance in wastewater treatment plants?

Q8. Why was the resistance in the wastewater treated wastewater lower than in the other groups?