Microbiological contamination and resistance genes in biofilms occurring during the drinking water treatment process

Science of the Total Environment 443 (2013) 932–938

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Microbiological contamination and resistance genes in biofilms occurring during the drinking water treatment process Anca Farkas a,⁎, 1, Anca Butiuc-Keul b, 1, Dorin Ciatarâş a, Călin Neamţu a, Cornelia Crăciunaş b, Dorina Podar b, Mihail Drăgan-Bularda b a b

Someş Water Company, 79 21 December 1989 Boulevard, 400604 Cluj-Napoca, Romania Babeş-Bolyai University, 1 Kogălniceanu Street, 400084 Cluj-Napoca, Romania

H I G H L I G H T S ► ► ► ►

Class 1 integrons were assessed in biofilms throughout drinking water production. intI1, qacEΔ1 and sul1 genes were present in bacteria in contact with water. The origin of antimicrobial resistance may be linked to anthropogenic disturbance. Microbiological contamination of drinking water sources is of high concern.

a r t i c l e

i n f o

Article history: Received 15 October 2012 Received in revised form 19 November 2012 Accepted 19 November 2012 Available online 14 December 2012 Keywords: Class 1 integrons intI1 qacEΔ1 sul1 Biopollution Risk assessment

a b s t r a c t Biofilms are the predominant mode of microbial growth in drinking water systems. A dynamic exchange of individuals occurs between the attached and planktonic populations, while lateral gene transfer mediates genetic exchange in these bacterial communities. Integrons are important vectors for the spread of antimicrobial resistance. The presence of class 1 integrons (intI1, qac and sul genes) was assessed in biofilms occurring throughout the drinking water treatment process. Isolates from general and specific culture media, covering a wide range of environmental bacteria, fecal indicators and opportunistic pathogens were tested. From 96 isolates tested, 9.37% were found to possess genetic determinants of putative antimicrobial resistance, and these occurred in both Gram-positive and Gram-negative bacteria. Class 1 integron integrase gene was present in 8.33% of bacteria, all positive for the qacEΔ1 gene. The sul1 gene was present in 3.12% of total isolates, representing 37.5% of the class 1 integron positive cells. The present study shows that biofilm communities in a drinking water treatment plant are a reservoir of class 1 integrons, mainly in bacteria that may be associated with microbiological contamination. Eight out of nine integron bearing strains (88.8%) were identified based on 16S rRNA gene sequencing as either enteric bacteria or species that may be connected to animal and anthropogenic disturbance. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The occurrence of resistance to antibiotics and disinfectants and its spreading into drinking water bacterial communities has particular significance, considering its impact on human-associated microbiota. Genetic elements responsible for dissemination of antimicrobial resistance are dynamically shared between a range of bacterial species found in soil and freshwater environments (Gillings et al., 2009a). Direct observations in natural and pathogenic ecosystems have shown that more than 99% of bacteria grow and function as members of metabolically integrated communities, in biofilms (Costerton, 2007). The ecological advantages of the biofilm mode of life include biodiversity, ⁎ Corresponding author. Tel.: +40 740 093 442; fax: +40 264 430 886. E-mail address: [email protected] (A. Farkas). 1 These authors (Anca Farkas and Anca Butiuc-Keul) made equal contribution in this work and are both equally considered as first authors. 0048-9697/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2012.11.068

gene pool and facilitated genetic exchange, protection against biocides and other types of stress in a high-density population (Flemming, 2009). Since the attached state is the main mode of bacterial organization and the planktonic state is a dispersal phase (Webb, 2007), drinking water associated biofilms represent potential reservoirs for water contamination (Wingender and Flemming, 2011). Microbial aquatic ecosystems, mainly those integrating the urban water cycle, represent important vehicles for the dissemination of human-associated microorganisms (Faria et al., 2009) and a source of transmission of antimicrobial resistant bacteria (Xi et al., 2009; Coleman, 2012; Figueira et al., 2012). Risk assessment of the spread of antimicrobial resistance in such environments and strategies of water quality improvement are needed in the context of this urgent health issue (Lupo et al., 2012). In drinking water systems, a dynamic exchange of individuals constantly occurs between the attached and planktonic communities, and horizontal gene transfer generates genetic diversity in bacterial

A. Farkas et al. / Science of the Total Environment 443 (2013) 932–938

populations. Integrons are a diverse family of mobile genetic elements capable of acquisition and expression of genes conferring antibiotic and biocide resistance (Nield et al., 2001), with a significant role in the evolution of bacterial resistance (Cambray et al., 2010). When integrated in genetic vectors such as plasmids and transposons, integrons, together with their associated mobile gene cassettes facilitate lateral gene transfer in bacteria (Márquez et al., 2008). Over 100 discrete integron classes have been characterized to date, about 10% of sequenced bacterial genomes carrying these elements (Boucher et al., 2007; Hardwick et al., 2008). Class 1 integrons, the most common integrons (Koczura et al., 2012), were previously described to contain a 5′-conserved segment encoding integrase gene (intI), a recombination site (attI) and a promoter region (Pc) (Hall and Collis, 1995). The 3′-conserved segment located downstream of integrated gene cassettes contains either an ancestral qacE gene, encoding resistance to quaternary ammonium compounds, or a combination of three genes, qacEΔ1 (a modified version of qacE), sul (encoding resistance to sulfonamides) and orf5 (an open reading frame with unknown function) (Rosser and Young, 1999). A site-specific recombination mechanism, mediated by enzymes called integrases, allows excision and insertion of genes located in mobile cassettes in tandem (Larouche and Roy, 2011). The abundance of gene cassettes and recombination processes represents a prerequisite for horizontal gene transfer and this system of integron/gene cassette system substantially influences bacterial genome evolution (Holmes et al., 2003). Multidrug resistance was observed to be strongly associated with the presence of class 1 integrons (Leverstein-van Hall et al., 2002; Vo et al., 2006; Márquez et al., 2008; Nemec et al., 2010). They were commonly found in bacterial strains in clinical context (Márquez et al., 2008; Gündoğdu et al., 2011) or in the human gastrointestinal tract (Randall et al., 2004; Labbate et al., 2008; Dawes et al., 2010). They are also prevalent in environmental bacteria, even in the absence of anthropogenic disturbance (Rosser and Young, 1999; Nield et al., 2001; Stokes et al., 2006; Hardwick et al., 2008; Gillings et al., 2008, 2009a,b; Xuejun and Weijin, 2010). When originating from environmental samples, integrons are usually chromosomal and do not carry antibiotic resistance cassettes, but often carry small multidrug resistance genes, belonging to the qac family (Gillings et al., 2009b). One of the hypotheses of antibiotic resistance rising in clinical context considers that the ancestral mobilization of integrons in transposons (e.g. Tn21, Tn402) allowed their transfer to pathogens and commensals (Stokes et al., 2006; Labbate et al., 2008; Dawes et al., 2010). The transposition is supposed to have been followed by deletions in the qacE gene, incorporation of the sul1 gene and partial deletions of the tni transposition module (Stokes et al., 2006; Gillings et al., 2008, 2009b). It has been shown previously that the class 1 integrons found in human pathogens are descended from chromosomal integrons of non-pathogenic soil and freshwater Betaproteobacteria. They are also present on the chromosomes of soil and freshwater Proteobacteria (Gillings et al., 2009a,b). Previous investigations of biofilms occurring during drinking water processing in Cluj revealed complex microbial consortia, containing highly active ecophysiological groups of bacteria (Farkas et al., in press) and harboring fecal indicators and opportunistic pathogens (Farkas et al., 2012). The present study investigates the potential of drinking water biofilms to accumulate resistance determinants, by verifying the presence of class 1 integrons. Integron integrase gene, genes conferring resistance to quaternary ammonium compounds (qacEΔ1 and qacE), the sulfonamide resistance gene (sul1) and transposition modules (tni) were targeted. Searching for the optimal method of gene recovery in the autochthonous microbiota, multiple primers were used against the conserved regions of class 1 integrons. Environmental bacteria but also fecal indicators and opportunistic pathogens were tested, in order to confirm the hypothesis that microbiological contamination by anthropogenic pressures links to antimicrobial resistance.

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2. Material and methods 2.1. Sampling and isolation Biofilm samples were collected from surfaces in contact with water in a drinking water treatment plant, from steel and concrete walls of a clarifier and from a sand filter. This facility (Fig. 1) is supplied from a succession of three dam reservoirs situated in the mountainous area of Cluj County, Romania (coordinates 46°44′N latitude and 23°22′E longitude). In order to capture bacterial diversity in the attached communities, isolates were obtained by spreading biofilm suspensions on general, selective and differential microbiological media. Agar plates with Yeast Extract Agar (Merck) and with R2A Agar (Merck) were inoculated for heterotrophic and for oligotrophic bacteria. Fecal indicators were isolated according to the standard methods used in drinking water quality assessment. Coliform bacteria were grown on Tergitol-7-TTC Agar (Merck), intestinal enterococci on Slanetz and Bartley Agar (Merck), Clostridium perfringens on m-CP Agar (Scharlau). Aeromonads were selected from Ryan's Agar (Oxoid), while pseudomonads were grown on Cetrimide Agar (Sartorius Biotech). Escherichia coli were oxidase-negative, indole producing and glucuronidase positive (ISO 9308-1/2009). Intestinal enterococci were able to produce aesculin hydrolysis (ISO 7899-2/2002). Colonies developed on m-CP Agar, turning magenta when exposed to ammonium hydroxide, were considered as presumptive C. perfringens (Council Directive 98/83/EEC). Dark green colonies developed on Ryan's Agar that were oxidase positive, able to ferment trehalose, indole producing and resistant to vibriostatic agent O129 were considered Aeromonas hydrophila (UK HPA W9 and US EPA 1605/2001). Presumptive Pseudomonas aeruginosa were fluorescent and oxidase positive (UK HPA W6/2005; SR EN ISO 16266/2008). In addition, several media used for cultivating diverse physiological groups of bacteria were inoculated with biofilm suspensions. Ammonifying bacteria grown in Peptone Broth supplemented with mineral salts and red phenol were detected by the addition of Nessler reagent. Denitrifying bacteria cultured in Allen Broth were able to produce gaseous nitrogen and nitrogen oxides. Peptone medium, as well as Oppenheimer and Gunkel Broth, Starkey Broth and Postgate Broth were used for the recovery of bacteria involved in sulfur cycling, based on their ability to produce hydrogen sulfide, to precipitate sulfide and iron salts or to generate sulfur deposits. Iron reducing bacteria were detected in Ottow's culture medium, by pink staining of the bivalent iron ions resulted from Fe 3+ reduction with α-α-dipiridil. Manganese bacteria were grown in Manganese Agar no. 2 modified, and stained with leucoberbelin blue (Farkas et al., in press). The presence of bacteria belonging to the mentioned ecophysiological groups was biochemically confirmed in liquid broths. Subsequently, pure colonies were isolated by spreading the liquid cultures on the same media supplemented with 2% agar. Culture media components and reagents were purchased from Merck, BD Difco, Sigma-Aldrich, Chimopar and Silal Trading. Colonies of different phenotypes were selected from each agar plate: heterotrophic bacteria (3), oligotrophic bacteria (9), ammonifying bacteria (8), denitrifying bacteria (6), sulfur reducing aerobes (6) and anaerobes (4), sulfur oxidizing bacteria (5), sulfate reducing bacteria (3), iron reducing bacteria (7) and manganese oxidizing bacteria (7), coliforms (16), intestinal enterococci (5), presumptive C. perfringens (3), A. hydrophila (5) and P. aeruginosa (9). Gram staining and phenotypic identification based on API kits (API 20E, API 20NE, API 20S and API 20A) and APIWEB platform (bioMérieux) were performed. 2.2. PCR screening Class 1 integron-specific genes were amplified from biofilm isolates by direct PCR technique. The colony PCR protocol was modified, using bacterial suspensions as templates. Pure cultures obtained from a single

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Fig. 1. Localization of the sampling site.

bacterial colony after 24 to 48 h incubation were suspended in sterile water to a concentration of approximately 10 6 CFU/ml (Crăciunaş et al., 2010). Anticipating the variability of integrons in local bacterial strains, a broad range of specific primers were tested. Eleven combinations of 20 primers were used in the screening of class 1 integrons, integronintegrase, genes encoding antimicrobial resistance and transposition module (Table 1, Table 1S). The schematic representation of class 1 integron and the approximate locations of regions found in the investigated bacteria are illustrated in Fig. 2. PCR reaction mix contained in 25 μl: 1.0 μl 10× PCR master mix Fermentas (containing reaction buffer, 0.05 U Taq Polymerase, 4 mM MgCl2, 0.4 mM concentration of each dNTP), 11 μl water, nuclease-free, 25 pmol each primer, and 2 μl bacterial suspension. PCRs were performed using a Gradient Palm-Cycler, Corbett Life Science thermocycler following customized programs: after the initial denaturation 94 °C for 5 min, cycles 2, 3 and 4 were 35-fold repeated and followed by a final extension at 72 °C for 5 min. Specific PCR programs were used for different primers as shown in Table 1. PCR products were separated in 1.5% w/v agarose gels in 1 × TAE buffer, stained with ethidium bromide 0.5 μg/ml.

2.3. 16S rRNA gene amplification Bacterial isolates that generated PCR products, presumably containing in their genomes integron-integrase, qac and sul genes, were identified using 16S rRNA gene sequence analysis. Universal bacterial primers, targeting the consensus region of the bacterial 16S rRNA gene

were used (Table 1, Table 1S). Analysis of a large fragment, of 1499 bp was performed to improve discrimination at the species level. 2.4. DNA sequencing The presence of genetic determinants was confirmed by amplicon sequence analysis. PCR products were purified using NucleoSpin gel and PCR clean-up kit (Macherey-Nagel), according to the manufacturer instructions. Purified PCR products were sequenced by Sanger method (3730XL, Applied Biosystems) at Macrogen Inc., Netherlands. Nucleotide sequences were edited by BioEdit and compared with the known sequences in GenBank, using BLASTN of the NCBI database (http:// www.ncbi.nlm.nih.gov/BLAST/). 3. Results and discussion 3.1. Detection of class 1 integrons and associated genes Four out of the 11 primer pairs tested revealed positive PCR results. Nine isolates (9.37%) were found to possess class 1 integron specific genes. Previous investigations estimated that environmental bacteria contain class 1 integrons in percents from 3.6% – as found in Gramnegative bacteria isolated from Tay Estuary, UK (Rosser and Young, 1999) to 7.98% cells – in a quaternary ammonium compound polluted environment in the UK (Gaze et al., 2005). In Australia, Stokes et al. (2006) detected 2 to 4% integron-positive bacteria in lake sediments, while Gillings et al. (2008) found 1 to 3% integron bearing cells in freshwater sediments, respectively 30% in biofilms collected from a groundwater treatment plant. Percents from 1 to 9% integron-positive cells

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Table 1 PCR design targeting class 1 integrons, gene cassettes, transposition module and 16S rRNA gene. Program no.

Primers

Target

Amplicon size (bp)

PCR cycles 2, 3, 4

References

P1

MRG284 MRG285

Class 1 integron

Variable 400–2000

Gillings et al. (2009b)

P2

MRG284 MRG286

Class 1 integron

Variable 400–2000

P3

HS915 HS916

intI1

≈350

P4

HS458 HS459

5′CS–3′CS intI 1

1300

P5

HS722 HS715

oriV + intI1

1320

P6

HS724 HS725

tniAB

520

P7

HS464 HS721

intI1 + IRi

1520

P8

intF intR

intI1

497

P9

qacEF qacER

qacE

363

P10

qacEF qacEΔ1R

qacEΔ1

363

P11

HS549 HS550

sul1

1100

P16S

16S–8F 16S–1493R

16S rRNA gene

1499

94 60 72 94 60 72 94 60 72 94 60 72 94 60 72 94 60 72 94 60 72 94 60 72 94 67 72 94 67 72 94 60 72 94 56 72

occurred in diverse environmental samples in the Sydney area (Gillings et al., 2009b). The presence of class 1 integrons was assessed with two pairs of primers, which target the whole integron, from attI1 site beyond the attC site of the last cassette in the array (Gillings et al., 2009b). PCR results were negative for all isolates. Fragments inside the intI1 integrase gene were targeted with five pairs of primers. Two of them showed positive results (Table 2), for eight isolates (8.33%). The most successful primer combination in intI1 gene assessment proved to be HS915/HS916, yielding amplicons in case of seven isolates (7.29%). Amplicons with the expected length were obtained by PCR with primers intF and intR, for three isolates (3.12%). PCR failed to detect integrase gene when using primer combinations HS458/HS459, HS722/HS715 and HS464/HS721. This may be due to the considerable sequence diversity of intI1 gene, specific for environmental isolates (Gillings et al., 2008). The presence of multidrug efflux qac cassettes was also investigated. Nine isolates (9.37%) contained the qacEΔ1 gene (Table 2), assessed with primers qacEF/qacEΔ1R. PCR amplifications failed to detect

°C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C °C

30 45 90 30 45 90 30 30 90 60 45 80 60 45 80 60 30 80 60 45 80 60 45 80 45 45 45 45 45 45 45 45 90 45 45 90

s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s

Márquez et al. (2008)

Holmes et al. (2003)

Stokes et al. (2006)

Chuanchuen et al. (2007)

Stokes et al. (2006)

Weisburg et al. (1991); Crăciunaş et al. (2010)

products of the expected length when assessing the presence of the qacE gene with primers qacEF/qacER. Genes encoding resistance to quaternary ammonium compounds (qacEΔ1) were found in all of the intI1 bearing cells. One bacterial strain was found to possess a qacEΔ1 gene in the absence of class 1 integronintegrase. The percents of cells containing qacEΔ1 genes are different from the results obtained in previous studies: Gillings et al. (2009a) indicated that between 1 and 9% of the cells in aquatic biofilms were intI1 positive and the proportion of cells carrying qac gene cassettes was approximately half of them. Another study, performed by Xuejun and Weijin (2010) showed that 45% of intI1 positive cells from environmental samples carry qac gene cassettes. A high incidence of 95% environmental bacteria bearing a qacE gene was reported in an environment polluted with quaternary ammonium compounds (Gaze et al., 2005). Individual qac cassettes occur in many different contexts, suggesting a dynamic process of cassette acquisition and rearrangement in these arrays. The widespread distribution of diverse qac cassettes hints at a general role for the efflux pumps in environmental bacteria (Gillings et al., 2009b). They are part of innate defense mechanisms of bacteria

Fig. 2. Schematic representation of class 1 integron and the positions of primers that revealed positive results in amplifications. oriV — origin of replication site; intI1 — integrase gene; qacEΔ1 — gene encoding resistance to quaternary ammonium compounds; sul1 — gene encoding resistance to sulfonamides; orf — open reading frame; tni module — transposition module; IRi, IRt — transposase binding sites; Pc — promoter for gene cassette-associated genes; attI1 — attachment site; attC — cassette-associated recombination site. Adapted after Stokes et al., 2006; Chuanchuen et al., 2007; Gillings et al., 2009a,b; Larouche and Roy, 2011.

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absence of PCR products with primer combinations MRG284/MRG285 or MRG284/MRG286, which target the chromosomal integrons, further investigations are needed in order to confirm the location of int1I genes.

Table 2 Bacterial isolates positive for class 1 integrons specific genes. Primer pairs/target genes Isolate no.

14 84 88 91 92 93 94 95 96

P3

P8

P10

P11

intI1

intI1

qacEΔ1

sul1

x x x

x x x x x x x x x

x x x x x x x x

x x x

against toxins in natural ecosystems (Gilbert and McBain, 2003). Multiple antibiotic resistant bacteria were found to be not necessarily more resistant to quaternary ammonium compounds than antibiotic sensitive strains, even though qacE or qacEΔ1 were present (Kücken et al., 2000). The combination of primers HS549/HS550 was tested for sulfonamide resistance gene sul1, revealing its presence (Table 2) in three isolates (3.12%), representing 37.5% of the intI1 bearing cells. Class 1 integrons associated with qacEΔ1 and sul1 genes have been commonly detected in clinical isolates and in polluted environments (Randall et al., 2004; Vo et al., 2006; Chuanchuen et al., 2007). sul1 was found to be the most prevalent sulfonamide resistance gene in agricultural soils in the UK, being present in 23% of bacteria, but only 8% of the sul1 positive isolates were found to possess class 1 integrons (Byrne-Bailey et al., 2009). Increased frequencies of multiresistant bacteria and resistance genes, including sul1 were detected in rivers, lakes, drinking water treatment plants and distribution systems, as well as in treated and untreated wastewater effluents in USA and Switzerland (Pruden et al., 2006; Xi et al., 2009; Czekalski et al., 2012). Drinking and wastewater treatment were found to reduce bacterial load, but the selection of extremely multiresistant strains and the accumulation of resistance genes were observed (Xi et al., 2009; Czekalski et al., 2012). All isolates were tested for qacEΔ1 and sul1 genes, even if intI1 was not detected. The presence of sul genes in the absence of class 1 integrons was recently demonstrated (Byrne-Bailey et al., 2009; Gündoğdu et al., 2011). The bacteria investigated in the present assessment proved to contain sul1 genes only incorporated in class 1 integrons. The horizontal transfer of integron-related resistance cassettes was suggested to be explained by the association of integrase and transposase genes (Dawes et al., 2010). We found no evidence that class 1 integron genes are linked with Tn402 transposition genes, when assessing the presence of the site of origin of replication (oriV) and transposition module (tni) with primers HS722/HS715 and HS724/HS725, respectively. This might suggest that either other types or hybrid transposons are involved (Labbate et al., 2008), or class 1 integrons are located in bacterial chromosomes (Stokes et al., 2006; Gillings et al., 2008). Since the

3.2. Identification of positive isolates From the 96 isolates further examined in PCR screening, 58 isolates (60.41%) were selected from agar plates targeting environmental bacteria, and 38 isolates (39.58%) were collected from culture media used for detection of fecal indicators and opportunistic pathogens. Nevertheless, the great majority of class 1 integron bearing cells proved to be bacteria with presumable animal or anthropogenic origin. Additional biochemical tests and phenotypic profile based on API kits confirmed the hypothesis that genetic elements triggering antibacterial resistance in drinking water environment may be linked to microbial contamination. Subsequent identification of positive isolates based on 16S rRNA gene revealed certain discrepancies at the species level (Table 3). Two special situations registered in case of isolates no. 88 and no. 95. Isolate no. 88 was collected from Lactose TTC agar, as a typical yellow colony, oxidase negative. It was assumed to belong to the coliform group. Gram staining showed Gram-positive cocci, unidentifiable with API 20STREP kit. Molecular identification showed high similarity with Staphylococcus vitulinus, species belonging to Staphylococcus sciuri group, a staphylococcal clade described to possess cytochrome c oxidase (Whitman et al., 2009). Isolate no. 95 was collected from Slanetz and Bartley Enterococcus selective agar, confirmed as Enterococcus by standard test of aesculin hydrolysis. Gram staining revealed Gram-positive cocci, but the strain was not identified by apiweb software, based on API 20STREP profile. Molecular identification showed high similarity with Staphylococcus warneri. Staphylococci occurring in drinking water have been previously recovered from different culture media, other than those designed for Staphylococcus growth, including from Lactose TTC agar and Bile aesculin agar (Faria et al., 2009). Results showed that only one out of nine isolates (11.11%), positive for resistance determinants belongs to environmental species, identified as Pseudomonas fragi. Eight isolates (88.8%) represent enteric species or may be linked to a human or animal origin. They were identified as Clostridium baratii, Enterococcus faecalis, Enterococcus saccharolyticus, Escherichia fergusonii, Klebsiella oxytoca, Klebsiella pneumoniae, S. vitulinus and S. warneri (Table 3). Molecular identification validated the hypothesis that most of the bacteria triggering antibacterial resistance in drinking water microbiota may be attributed to anthropogenic disturbance. The high frequency of class 1 and class 2 integrons found among Enterobacteriaceae isolated from wastewater treatment confirms that sewage is a reservoir of integron-embedded antibiotic resistance genes (Mokracka et al., 2012). Wastewater effluent was demonstrated to contribute to increased frequency of integron-positive E. coli isolates in water environment (Koczura et al., 2012). Microbiological contamination with fecal origin in Cluj drinking water sources and in biofilms formed throughout the

Table 3 Identification of class 1 integron-bearing isolates. Isolate no.

Biochemical testing according to specific reference methods

Phenotypic identification with API kits

14 84 88 91 92 93 94 95 96

Pseudomonas aeruginosa Clostridium perfringens Coliform bacteria Escherichia coli E. coli Coliform bacteria Intestinal enterococcus Intestinal enterococcus Intestinal enterococcus

Pseudomonas putida C. perfringens Unacceptable profile E. coli Raoultella ornithinolytica Klebsiella oxytoca Enterococcus faecalis Unacceptable profile Enterococcus faecium

API API API API API API API API API

20NE 20A 20STREP 20E 20E 20E 20STREP 20STREP 20STREP

16S rRNA gene identification 64.5% 97.7% – 91.8% 99.9% 98.6% 55.5% – 99%

Pseudomonas fragi Clostridium baratii Staphylococcus vitulinus Escherichia fergusonii Klebsiella oxytoca Klebsiella pneumoniae E. faecalis Staphylococcus warneri Enterococcus saccharolyticus

ATCC 4973 IP 2227 ATCC 51145 ATCC 35469 ATCC 13182 DSM 30104 JCM 5803 AW 25 ATCC 43076

99% 95% 99% 99% 99% 99% 99% 99% 98%

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drinking water treatment plant was previously demonstrated (Farkas et al., 2010, 2012). Human activities represent a selective pressure, increasing the frequency of lateral gene transfer and influencing bacterial evolution (Stokes and Gillings, 2011). The presence of integrons (Nandi et al., 2004) and the increasing antibiotic resistance in Gram-positive bacteria such as methicillinresistant Staphylococcus aureus (Guo et al., 2011; Xu et al., 2011) has recently become a great concern. The current study found that besides Gram-negative Enterobacteriaceae, Gram-positive bacteria are a reservoir of class 1 integrons in drinking water associated biofilms. Both Gram-negative (4 out of 9 isolates, representing 44.4%) and Grampositive bacteria (5 out of 9 isolates, representing 55.5%) were found to possess class 1 integrons. sul1 genes were present only in Grampositive isolates: E. faecalis, E. saccharolyticus and S. warneri. The association of qacEΔ1 and sul1 indicates that integrons contain the typical 3′-conserved region in these three strains. 3.3. Health significance of antimicrobial genes bearing isolates Based on the literature, the great majority of bacteria found by us to contain class 1 integrons might be fecal indicators or common members of enteric bacteria. Most of them are human pathogens (Garrity et al., 2005; Whitman et al., 2009) or have been previously isolated from intestinal microbiota (Rajilic-Stojanovic, 2007). It is the case of eight out of nine species: C. baratii, E. faecalis, E. saccharolyticus, E. fergusonii, K. oxytoca, K. pneumoniae, S. vitulinus and S. warneri. Even if here we cannot assert the commensal or environmental origin of these integron bearing isolates, it is significant that their pathogenicity in humans has been already recognized. P. fragi is the only environmental species identified as bearing resistance determinants in the present study. A psychrophilic, biofilmforming bacterium, it is responsible for food spoilage and generally absent from the normal gastrointestinal microbiota in humans (Wagner et al., 2008). P. fragi strains bearing class 1 integrons and tetracycline resistance genes were recently isolated from an oxytetracycline-polluted environment, in China (Li et al., 2010). Recent investigations warn of the increasing evidence of antimicrobial resistance determinants in human environment. sul1, sul2, sul3 and the combination qacEΔ1–sul1 genes associated with class 1 and class 2 integrons were detected in E. coli isolates from human blood cultures (Vinué et al., 2010). Our results indicate the association of intI1 with both qacEΔ1 and sul1 genes in all the isolates containing sulfonamide resistance genes (3.12% of total bacteria). E. faecalis, E. saccharolyticus and S. warneri isolates proved to contain intI1, qacEΔ1 and sul1 genes, results confirmed by sequencing analysis. C. baratii, E. fergusonii, K. oxytoca, K. pneumoniae and S. vitulinus isolates contain integron integrase and qacEΔ1 genes. The genome of P. fragi is bearing the qacEΔ1 gene, but the presence of class 1 integron integrase was not evident. These findings have general implications for public health, indicating aspects of concern in terms of the linkage between microbiological and genetic contamination in drinking water sources. Animal and human pollution in freshwater catchments poses a risk by introducing genetic elements responsible for bacterial resistance that may be perpetuated in environmental species, especially in biofilms. The gene pool potential in this environment should not be overlooked, although integron-bearing strains may not always express the antimicrobial phenotype (Roe et al., 2003). Assessing the significance of human carriage of antimicrobial resistant E. coli with contaminated drinking water, Coleman (2012) concluded that the intake of resistant bacteria was 40% higher for individuals using contaminated water than for subjects consuming uncontaminated water or drinking water containing sensitive E. coli strains. Besides the genetic mechanisms, factors promoting survival of bacteria in chlorinated water supplies include aggregation, attachment, starvation but also ineffective disinfection (LeChevallier et al., 1988).

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Biofilm insusceptibility to biocides is sometimes considered a tolerance due to a physiological adaptation, rather than to the presence of genetic determinants (Bridier et al., 2011). This assessment warns of biopollution hazards in biofilm consortia occurring throughout drinking water processing, in a facility providing drinking water to almost 0.7 million people. However, it is unlikely that the treatment process may have contributed to the selection of resistant variants. A primary disinfection with gaseous chlorine is applied with intermittence, quarterly. The responsible use of biocides, following a professional procedure is not discouraged in situations where there is a real benefit (Gilbert and McBain, 2003), as in case of drinking water purification. 4. Conclusions The present study reveals how novel resistance mechanisms and microbiological contaminants are penetrating the urban aquatic environments, and should be regarded as biopollutants. Class 1 integrons and related gene cassettes are present in drinking water biofilms, mainly in bacteria linked to anthropogenic disturbance. Eight out of nine species were identified as potential bacteria of concern: C. baratii, E. faecalis, E. saccharolyticus, E. fergusonii, K. oxytoca, K. pneumoniae, S. vitulinus and S. warneri. A single bacterial isolate, containing qacEΔ1 gene with no evidence for its association with class 1 integron was identified as an environmental species, P. fragi. The microbiological contamination with fecal origin in drinking water sources is of high concern. Animal and human pollution in freshwater catchments poses a risk by introducing genetic elements responsible for bacterial resistance that may be perpetuated in environmental species, especially in biofilms. Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.scitotenv.2012.11.068. These data include Google map of the most important areas described in this article. References Boucher Y, Labbate M, Koenig JE, Stokes HW. Integrons: mobilizable platforms that promote genetic diversity in bacteria. Trends Microbiol 2007;15:301–9. Bridier A, Briandet R, Thomas V, Dubois-Brissonet F. Resistance of bacterial biofilms to disinfectants: a review. Biofouling 2011;27:1017–32. Byrne-Bailey KG, Gaze WH, Kay P, Boxall A, Hawkey PM, Wellington EMH. Prevalence of sulphonamide resistance genes in bacterial isolates form manured agricultural soils and pig slurry in the United Kingdom. Antimicrob Agents Chemother 2009;53:696–702. Cambray G, Guerout AM, Mazel D. Integrons. Annu Rev Genet 2010;44:141–66. Chuanchuen R, Khemtong S, Padungtod P. Occurrence of qacE/qacEΔ1 genes and their correlation with class 1 integrons in Samonella enterica isolates from poultry and swine. Southeast Asian J Trop Med Public Health 2007;38:855–62. Coleman BL. The role of drinking water as a source of transmission of antimicrobialresistant E. coli. Epidemiol Infect 2012;140:633. Costerton JW. The biofilm primer. Berlin Heidelberg New York: Springer; 2007. Crăciunaş C, Butiuc-Keul A, Flonta M, Brad A, Sigarteu M. Application of molecular techniques to the study of Pseudomonas aeruginosa clinical isolate in Cluj-Napoca, Romania, 17. Annals of Oradea University, Biology Fascicle; 2010. p. 243–7. Czekalski N, Berthold T, Caucci S, Egli A, Bürgmann H. Increased levels of multiresistant bacteria and resistant genes after wastewater treatment and their dissemination into Lake Geneva, Switzerland. Front Microbiol 2012;3:106. Dawes FE, Kuzevski A, Bettelheim KA, Hornitzsky MA, Djordjevic SP, Walker MJ. Distribution of class 1 integrons with IS26-mediated deletions in their 3′-conserved segments in Escherichia coli of human and animal origin. PLoS One 2010;5:e12754. Faria C, Vaz-Moreira I, Serapicos E, Nunes OC, Manaia CM. Antibiotic resistance in coagulase negative staphylococci isolated from wastewater and drinking water. Sci Total Environ 2009;407:3876–82. Farkas A, Bocoş B, Ţigan Ş, Ciatarâş D, Drăgan-Bularda M, Carpa R. Surveillance of two dam reservoirs serving as drinking water sources in Cluj, Romania. In: Dimkic MA, editor. Balkans Regional Young Water Professionals Conference Proceedings. Belgrade: Jaroslav Černi Institute for the Development of Water Resources; 2010. p. 91–7. Farkas A, Drăgan-Bularda M, Ciatarâş D, Bocoş B, Ţigan Ş. Opportunistic pathogens and faecal indicators in drinking water associated biofilms in Cluj, Romania. J Water Health 2012;10:471–83.

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