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Sludge as a potential important source of antibiotic resistance genes in both the bacterial and bacteriophage fractions William Calero-Caceres, Anna Melgarejo, Claudia Stoll, Marta Colomer Lluch, Francisco Lucena Gutierrez, Juan Jofre , and Maite Muniesa Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es501851s • Publication Date (Web): 29 May 2014 Downloaded from http://pubs.acs.org on June 6, 2014

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Environmental Science & Technology

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Sludge as a potential important source of

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antibiotic resistance genes in both the

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bacterial and bacteriophage fractions

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William Calero-Cáceres,† Ana Melgarejo,† Marta Colomer-Lluch,† Claudia Stoll,

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Francisco Lucena,† Juan Jofre,† and Maite Muniesa, †,*

‡

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†

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E-08028 Barcelona. Spain

9

‡

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Department of Microbiology. University of Barcelona. Diagonal 643. Annex. Floor 0.

DVGW-Technologiezentrum Wasser (TZW), Karlsruher Street 84, D-76139 Karlsruhe,

Germany.

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*corresponding author

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Maite Muniesa

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Phone: +34934039386

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Fax: +34934039047

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e-mail: [email protected]

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Keywords: sludges, antibiotic resistance, bacteriophages, beta-lactamase, sulfonamides

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Author Contributions

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The manuscript was written through contributions of all authors. All authors have given

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approval to the final version of the manuscript.

1 Calero-Cáceres et al., 2014

ACS Paragon Plus Environment


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ABSTRACT

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The emergence and prevalence of antibiotic resistance genes (ARGs) in the

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environment is a serious global health concern. ARGs found in bacteria can become

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mobilized in bacteriophage particles in the environment. Sludge derived from secondary

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treatment in wastewater treatment plants (WWTPs) constitutes a concentrated pool of

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bacteria and phages that are removed during the treatment process. This study evaluates

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the prevalence of ARGs in the bacterial and phage fractions of anaerobic digested

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sludge; five ARGs (blaTEM, blaCTX-M, qnrA, qnrS and sul1) are quantified by qPCR.

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Comparison between the wastewater and sludge revealed a shift in the prevalence of

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ARGs (blaTEM and sul1 became more prevalent in sludge), suggesting there is a change

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in the bacterial and phage populations from wastewater to those selected during the

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secondary treatment and the later anaerobic mesophilic digestion of the sludge. ARGs

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densities were higher in the bacterial than in the phage fraction, with high densities in

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both fractions; particularly for blaTEM and sul1 (5 and 8 log10 gene copies (GC)/g

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respectively in bacterial DNA; 5.5 and 4.4 log10 GC/g respectively in phage DNA).

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These results question the potential agricultural uses of treated sludge, as it could

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contribute to the spread of ARGs in the environment and have an impact on the

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bacterial communities of the receiving ecosystem.

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INTRODUCTION

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Microorganisms that cause infections are becoming resistant to the most common

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antibiotic treatments. Thus, antibiotic resistance has become a serious and growing

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problem in contemporary medicine and has emerged as one of the primary public health

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concerns of the 21st century.1,2 An estimated 25,000 people die every year in Europe

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directly from antibiotic-resistant bacterial infections, while many more die from other

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conditions that are complicated by an antibiotic-resistant infection.3

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Antibiotic resistance occurs as a natural phenomenon but can be enhanced by human

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activities; in medicine, the major causes of such enhancement are incorrect diagnosis,

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and inappropriate prescription of antibiotics or their improper use by patients. The

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spread of antibiotic resistance can also be attributed to certain practices in the

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pharmaceutical industry or to the non-therapeutic use of antibiotics in animals intended

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for human consumption.

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Despite the urgent need for a global system to monitor antibiotic resistance and the

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health and economic burden it causes, information concerning the expansion of

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resistance in the environment is still remarkably limited. Different environments (bodies

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of water or soils) are suitable locations for the emergence of new bacterial resistance,

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including those that can cause infection in both humans and animals.4,5 Several studies

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suggest that antibiotic resistance occurs in nature and has an ancient origin which is not

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always linked to the use of antibiotics. However, the presence of antibiotics in the

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environment, even at subinhibitory concentrations, could have favoured the selection of

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some types of resistance,6 as well as the dissemination of resistant clones.

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In addition, aquatic environments (including surface water and groundwater) provide

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ideal settings for the horizontal exchange of mobile genetic elements (MGEs) that

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encode antibiotic resistance.7-9 The elements that mobilize antibiotic resistance genes

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(ARGs) can be insertion sequences, transposons, intragenic chromosomal elements,

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plasmids, pathogenicity islands, chromosome cassettes or, as recently reported,

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bacteriophage particles.10-12

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The horizontal transfer occurring anywhere at random and the assembly of genetically

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mobile platforms is more likely to occur in environments where there is a selective

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pressure, such as human and animal biomes or wastewater,13,14 where phages and

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bacteria are abundant but there is antibiotic exposure. Wastewater treatment plants

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(WWTPs) are therefore a plentiful source of ARGs from which they spread into the

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environment.11,15-17 Activated sludge derived from secondary treatment at WWTPs

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represents a concentrated pool of bacteria, and phages that infect these bacteria, which

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have been removed during the treatment process. A common treatment of activated

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sludge includes anaerobic digestion; this further transforms organic matter into biogas,

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reduces the final amount of sludge solids for disposal and destroys most of the

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pathogens present in the sludge.18,19 The mechanisms by which biological processes in

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WWTPs influence the development/selection of resistant bacteria and ARGs transfer are

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still poorly understood. The potential agricultural use of WWTP sludge may result not

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only in the transfer of resistant bacteria into the environment, but also in the

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dissemination of ARGs.

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To gain insight into these questions, the present study evaluates the abundance of five

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ARGs in the bacterial and bacteriophage DNA fractions of sludge obtained following

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anaerobic digestion in an urban WWTP.

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MATERIALS AND METHODS

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Samples.- Thirty raw urban wastewater samples were collected weekly between autumn

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2012 and spring 2013 from the influent of a WWTP in the Barcelona metropolitan area

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which serves approximately 500,000 people. The same plant was the source of 30

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wastewater sludge samples collected from the digestor. Sludge samples comprised a

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mixture of raw primary sludge (about two-thirds) and secondary sludge (about one-

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third) that was thickened and subjected to anaerobic mesophilic (35°C) digestion for 20-

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25 days. Dry weight of sludges was 25 % in average.

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All the samples were collected in sterile containers, transported to the laboratory at

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5ºC±2ºC within two hours of collection and processed immediately for bacterial counts

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and further experiments.

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Bacterial strains and media.- Escherichia coli strain C600 containing the pGEM

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vector with the blaTEM gene was used as a control for blaTEM. E. coli strains isolated

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from the urban wastewater during this study were used as controls for blaCTX-M genes

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carrying types CTX-M. The clinical E. coli strain 266 was used as a control for qnrA,

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the environmental Enterobacter cloacae strain 565 was used as a control for qnrS and

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E. coli J53 R38820 was used as a control for sul1. E. coli WG5 (ATCC 700078) was

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used as a host for the evaluation of somatic coliphages.21 5 Calero-Cáceres et al., 2014



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Luria-Bertani (LB) agar or broth were used for routine bacterial propagation to prepare

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the standards for qPCR assays. For the detection of heterotrophic bacteria, Tryptic Soy

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Agar (TSA) media or Chromocult® Coliform Agar (Merck, Darmstadt, Germany) were

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used. Media were supplemented with: 32 mg/L of ampicillin (AMP); 25 mg/L of

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nalidixic acid (NAL); or 350 mg/L of sulfamethoxazole (SUL), all from Sigma-Aldrich

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(Steinheim, Germany). These antibiotic concentrations were chosen based on previous

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studies that analyse environmental resistant bacteria.22,23

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Microbiological parameters.- The extent of faecal contamination in the wastewater

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and sludge samples was established by counting total heterotrophic bacteria and E. coli

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as bacterial indicators and somatic coliphages as viral indicators. The latter were

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included to indicate the levels of virulent bacteriophages in the samples.21 This method

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allows detection of many virulent infectious phages from a faecal sample with a single

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host strain (E. coli WG5) through the double agar layer plaque assay.

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For bacterial assays, serial decimal dilutions (1/100, 1/1000 and 1/10000) of the

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wastewater samples were filtered through 0.45-µm-pore-diameter membrane filters (47

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mm, white gridded, EZ-Pak® Membrane Filters, Millipore, Bedford, MA). The

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membranes were placed upside up on TSA for heterotrophic bacteria and Chromocult®

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Coliform Agar for E. coli, and incubated at 37ºC for 18 h. To evaluate somatic

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coliphages, serial decimal dilutions of the wastewater samples were filtered through low

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protein-binding 0.22-µm-pore-size membrane filters (Millex-GP, Millipore, Bedford,



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MA) and assayed by the double agar method, as described in the standard ISO

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methods.21 Samples were analysed in triplicate.

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For the analysis of microbiological parameters in sludge, 5 g of sludge sample was

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homogenized 1/10 (w:v) with sterile phosphate saline buffer (PBS) and suspended by

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magnetic stirring at room temperature for 30 min. This suspension was used to prepare

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10-fold dilutions as described in the US EPA standard procedures.24

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To evaluate the presence of bacteria resistant to antibiotics, samples were processed as

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described above and incubated in TSA or Chromocult® coliform agar for 2 hours at

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37ºC. The membranes were then transferred to TSA or Chromocult® coliform agar

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containing the respective antibiotics and incubated at 37ºC for a further 18 h.

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PCR procedures

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Standard PCR procedures.- Conventional PCRs were performed for ARGs (Table 1).

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All the PCRs were performed with a GeneAmp PCR system 2700 (Applied Biosystems,

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Barcelona, Spain). Five µL of each PCR product was analysed by agarose (0.8%) gel

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electrophoresis and bands were viewed using ethidium bromide staining. When the

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amplicons were used for cloning, PCR products were purified using a PCR Purification

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Kit (Qiagen Inc., Valencia, CA).

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qPCR procedures.- For the quantification of each ARG in phage DNA, TaqMan qPCR

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assays were performed using standards prepared as described in the next section.




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Conditions for each qPCR assay were as described previously.11,25 Limits of detection

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were calculated by performing 8-10 replicates of each dilution of the standard for each

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gene, in three independent qPCR reactions and by triplicate. We considered as limit of

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detection the higher threshold cycle (Ct) that showed positive amplification in all

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replicates. Calculating the gene copies (GC) for each Ct value, the limit of detection of

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each qPCR assay was 7.6 GC/µL for blaTEM, 8.4 gene copies GC/µL for blaCTX-M-1, 3.1

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GC/µL for qnrA and 8.3 gene copies GC/µL for qnrS.

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A new real-time qPCR assay for sul1 was developed to detect the most common

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sulphonamide resistance gene in the DNA of the bacterial and bacteriophage fractions

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of the samples.

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sul1 primers and a probe set were designed using the Primer Express 3.0 software

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(Applied Biosystems); primers and probes were selected to be used in a standardized

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TaqMan amplification protocol. Their specificity was determined via sequence

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alignments using 21 sequences of sul1 available in NCBI nucleotide database. A FAM-

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labelled fluorogenic probe was commercially synthesized by Applied Biosystems. The

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sul1 probe was minor-groove binding (MGB) with an FAM reporter (FAM: 6-

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carboxyfluorescein) and an NFQ quencher (non-fluorescent quencher). The sul1 qPCR

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assay showed a limit of detection of 5.9 GC/µL (a threshold cycle of 35).

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Preparation of standard curves.- For the generation of standards for all qPCR assays, a

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plasmid construct was used containing the insert of each ARG amplified by

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conventional PCR (Table 1). Each fragment was purified using Pure LINK Quick Gel

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Extraction and a PCR Purification Combo Kit (Invitrogen Carlsbad, CA) and was 8 Calero-Cáceres et al., 2014


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cloned in a pGEM-T Easy vector following the manufacturer’s instructions (pGEM®-T-

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Easy Vector, Promega, Barcelona, Spain). The construct was transformed by

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electroporation into E. coli DH5α electrocompetent cells. The cells were electroporated

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at 2.5 kV, 25 F capacitance and 200 Ω resistance in a BTX ECM 600 Electroporation

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System (Harvard Apparatus, Inc. Holliston, MA). Colonies containing the vector were

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screened by conventional PCR to evaluate the presence of the insert in the vector and its

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orientation, and the amplimers were sequenced. For sul1, plasmid R388 obtained from

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E. coli J53 was used. The vector containing the insert was purified from the positive

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colonies using the Qiagen Plasmid Midi purification kit (Qiagen Inc., Valencia, CA)

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and its concentration was quantified using a NanoDrop ND-1000 spectrophotometer

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(NanoDrop Technologies. Thermo Scientific. Wilmington. DE). The reaction product

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was linearized by digestion with XmnI restriction endonuclease (Promega Co., Madison,

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USA). The restricted product was purified and quantified for use in the qPCR assays.

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The GC/µL of the stock prepared for each gene was calculated and the standard curve

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for qPCR was calculated as described previously.11 Three replicates of each dilution

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were added to each qPCR reaction.

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All the qPCR assays were performed under standard conditions, as indicated by the

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manufacturer, in a Step One Real-Time PCR System (Applied Biosystems). The assays

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were tested for cross-reactions with the respective susceptible strains. They were

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amplified in a 20 µL reaction mixture with the TaqMan Environmental Real-Time PCR

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Master Mix 2.0 (Applied Biosystems). The reaction contained 9 µL of the DNA sample

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or quantified plasmid DNA. All the samples were assayed in duplicate, as were the 9 Calero-Cáceres et al., 2014



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standards, and positive and negative controls (described below). The GC was defined as

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the average of the duplicate data obtained.

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To screen for PCR inhibition, dilutions of the standard were spiked with environmental

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DNA and the experimental difference was compared to the true copies of the target

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genes in the standards. No inhibition of PCR by environmental DNA extracts was

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detected.

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Extraction of bacterial DNA

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To extract bacterial DNA from the wastewater samples, 50 mL of each sample was

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filtered through 0.45-µm-pore-diameter membrane filters (47 mm, white gridded, EZ-

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Pak® Membrane Filters (Millipore, Bedford, MA). The bacterial content of the

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membranes was suspended in Luria Bertani broth (LB). The samples were centrifuged

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at a 3000xg for 10 minutes. The pellet was suspended in 200 µL of LB and bacterial

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DNA was obtained using the QIAamp® DNA blood Mini Kit (Qiagen Inc., Valencia,

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CA) following the manufacturer’s instructions.

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For DNA extraction from sludge, 0.25 g of sample was processed using the PowerSoil®

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DNA isolation kit (MO BIO® Laboratories, Inc. Carlsbad, CA) following the

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manufacturer’s instructions.

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Purification of phage particles

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To purify the bacteriophage fraction of the water samples, a previous described method

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was used with minor modifications.11 Briefly, 50 mL of each sample was centrifuged at



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3000xg for 10 min, passed through low protein-binding 0.22-µm-pore-size membrane

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filters (Millex-GP, Millipore, Bedford, MA) and then 100-fold concentrated at 3000xg

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by means of protein concentrators (100 kDa Amicon Ultra centrifugal filter units,

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Millipore, Bedford, MA), following the manufacturer’s instructions.

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To purify the bacteriophage fractions from the sludge, the following two protocols were

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assayed.

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Organic flocculation based on the protocol proposed by the US EPA for elution of

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enteroviruses.24 Briefly, liquid raw sludge was conditioned by the addition of AlCl3 to a

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final concentration of 0.0005 mol/L, and thoroughly mixed by shaking and

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centrifugation. From this point on, the pellet was eluted with 10% beef extract and

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viruses were concentrated by organic flocculation according to Katzenelson et al. 26

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Homogenization, as described previously by Lasobras et al.,27 was performed here with

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minor modifications. The samples were mixed in a 1:10 (w/v) ratio with PBS pH=7.4

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and homogenized by magnetic stirring for 30 min at room temperature. The suspension

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was then centrifuged at 10,000xg for 30 min at 4°C. The supernatant was subsequently

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filtered through low protein-binding 0.22-µm-pore-size membrane filters (Millex-GP,

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Millipore, Bedford, MA) and then 100-fold concentrated at 3000xg by means of protein

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concentrators as described above.

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The concentrated phage fraction from wastewater and sludge samples was recovered

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and treated with chloroform to rule out the presence of possible vesicles containing

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DNA. DNase treatment (100 units/mL of the phage lysate) was performed to eliminate




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any free DNA that might be present in the samples outside the phage particles. After

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that, DNase was heat inactivated at 80ºC for 10 minutes.

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Controls

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Negative controls included controls of the DNA extraction with sterile water. In

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addition, to exclude non-packaged DNA, the protocol for DNA extraction from the

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phage fraction of the samples was always accompanied by several controls. To rule out

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the possibility of contamination with free DNA, an aliquot of the sample taken after

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DNase treatment and before desencapsidation was processed. At this stage, the samples

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were used as a template for conventional PCR of eubacterial 16S rDNA and for the

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qPCR assay of each ARG. Both amplifications should be negative to confirm that

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DNase has removed all non-packaged DNA from the samples.

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To verify the DNase treatment, the samples were spiked with serial decimal dilutions of

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the standards (DNA concentrations from 222-2.2x10-2 ng/µl, containing from 109-105

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GC/µl) and then treated with DNase followed by the heat inactivation of the enzyme

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and amplified.

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To confirm that the DNase was correctly inactivated by the heat treatment and that no

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degradation of the qPCR primers or probe occurred, the samples were spiked with serial

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decimal dilutions of the standard after the DNase heat inactivation and then amplified.

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Extraction of DNA from phage particles

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Phage DNA was extracted from the purified phage particles as previously described.11

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The concentration and purity of the extracted phage DNA were determined using the 12 Calero-Cáceres et al., 2014


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NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Thermo Scientifics,

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Wilmington, DE).

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Sequencing.- The ARGs amplified from the strains used as controls and the amplimers

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used to prepare the qPCR standards were sequenced with an ABI PRISM Big Dye 3.1

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Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems), following the

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manufacturer’s instructions and with the primers shown in Table 1. All sequences were

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performed at least in duplicate.

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Statistical analysis.- Data were compiled and statistical tests performed using the

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Statistical Package for Social Science software (SPSS). One-way analysis of variance

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(ANOVA) was used to evaluate the differences between the two methods used to

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extract phages from sludge and to compare the occurrence of ARGs detected in DNA

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from the bacterial and phage fractions. Evaluations were based on a 5% significance

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level in both cases: a P value of

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