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Environmental waters and blaNDM-1 in Belgrade, Serbia: Endemicity questioned
Science of the Total Environment 511 (2015) 393–398
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Environmental waters and blaNDM-1 in Belgrade, Serbia: Endemicity questioned K. Novovic a, B. Filipic a,b, K. Veljovic a, J. Begovic a, N. Mirkovic c, B. Jovcic a,d,⁎ a
University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P.O. Box 23, 11010 Belgrade, Serbia University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia University of Belgrade, Faculty of Agriculture, Nemanjina 6, Belgrade, Serbia d University of Belgrade, Faculty of Biology, Studentski trg 16, 11000 Belgrade, Serbia b c
H I G H L I G H T S • • • • •
Serbia was designated as the endemic for blaNDM-1, a significant public health threat Contamination of urban rivers, lake and springheads with NDM-1 producing strains was tested We propose that NDM-1 is a transplant and that Serbia is not endemic region for NDM-1. Special emphasis was on prevalence of resistance in the most popular Belgrade lake. We report presence of strains producing SHV, DHA-1 and CMY-2 β-lactamases.
a r t i c l e
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Article history: Received 7 October 2014 Received in revised form 8 November 2014 Accepted 22 December 2014 Available online xxxx Editor: C.E.W. Steinberg Keywords: Antibiotic resistance Environment New Delhi metallo-beta-lactamase (NDM-1)
a b s t r a c t New Delhi metallo-beta-lactamase-1 (NDM-1) will soon become the most commonly isolated and distributed metallo-beta-lactamase worldwide due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative pathogens. NDM-positive bacteria pose a significant public health threat in the Indian subcontinent and the Balkans, which have been designated as endemic regions. Our study was focused on urban rivers, a lake and springheads as a potential source of NDM-1-producing strains in Serbia, but also as a source of other metallo-beta-lactamases and extended-spectrum beta-lactamase (ESBL) producing bacteria. A total of 69 beta-lactam resistant isolates, belonging to 12 bacterial genera, were collected from 8 out of 10 different locations in Belgrade, of which the most were from a popular recreational site, Ada Ciganlija Lake. Phenotypic tests revealed 7 (10.14%) ESBL-producing isolates and 39 (56.52%) isolates resistant to imipenem, of which 32 were positive for metallo-beta-lactamase (MBL) production. PCR and sequencing revealed the presence of genetic determinants for SHV (3 isolates), DHA-1 (1 isolate) and CMY-2 (1 isolate) beta-lactamases. However, we did not detect any NDM-1-producing strains (previously described cases of NDM-1 from Serbia were limited to Belgrade), so we propose that Serbian NDM-1 is in fact a transplant and a nosocomial, rather than an environmental, issue and that Serbia is not an endemic region for NDM-1. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Antibiotics, besides their application for improving human health, have been used for preventing and treating animal and plant infections and for promoting growth in animal farming. These applications have
⁎ Corresponding author at: University of Belgrade, Faculty of Biology, Studentski trg 16, 11000 Belgrade, Serbia. E-mail address: [email protected] (B. Jovcic).
http://dx.doi.org/10.1016/j.scitotenv.2014.12.072 0048-9697/© 2014 Elsevier B.V. All rights reserved.
caused antibiotics to be released and abundant in natural ecosystems (Cabello, 2006). Significant public use or misuse of antibiotics, caused by several social factors and low awareness of antibiotic resistance, is predominantly present in countries with high levels of antimicrobial resistance. Due to all of these factors antibiotic resistant bacteria have been found widely in aquatic environments (Baquero et al., 2008; Xi et al., 2009). The occurrence of resistant bacteria in a water environment could be the result of anthropogenic activity or natural processes (many acquired resistance mechanisms have originated in producers of antibiotics) (Martinez, 2008). Nevertheless, anthropogenic and/or naturally occurring resistant bacteria in water environments
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pose a threat to human health. Moreover, these bacteria may transfer their antibiotic resistance genes to other pathogens through horizontal gene transfer. Therefore the contamination of water and the environment with multidrug-resistant Gram-negative bacteria is one of the main routes for the spread of antibiotic resistance and is therefore a crucial area for control. Beta-lactam antibiotics, including the sub-groups of penicillins, cephalosporins and carbapenems, are among the most valuable antibiotics for human use owing to their comparatively high effectiveness, low cost, ease of delivery and minimal side effects. Bacterial resistance against beta-lactam antibiotics is increasing at a significant rate and has become a common problem in primary care medicine. There are several mechanisms of antimicrobial resistance to betalactam antibiotics, one of which is the production of beta-lactamases (Wilke et al., 2005). Acquired resistance to beta-lactams is mainly mediated by extendedspectrum beta-lactamases (ESBLs) that confer bacterial resistance to all beta-lactams except carbapenems and cephamycins (Wilke et al., 2005). The increase of ESBL prevalence at the beginning of the 21st century in Europe has been one of the most significant phenomena in antimicrobial resistance (Barlow et al., 2008). ESBLs are generally acquired by horizontal gene transfer (e.g., TEM or SHV, plasmid-encoded CTX-M) or are mobilized from environmental bacteria (e.g., progenitors of CTX-M from Kluyvera spp.). Interestingly a shift in the distribution of different ESBLs has occurred in Europe, which is the result of a significant increase of CTX-M enzymes over TEM and SHV variants (Coque et al., 2008). Besides the ESBLs, of great importance are carbapenemases which confer resistance to carbapenems that are potent and broadspectrum beta-lactam antibiotics traditionally reserved for the treatment of the most serious infections (El-Gamal and Oh, 2010). The emergence and rapid worldwide spread of carbapenemase-producing pathogens contribute significantly to the morbidity and mortality of patients. Carbapenem-resistant Gram-negative bacilli remain an alarming threat as few antimicrobial agents are reliably effective and very few are expected to be available in the near future (Patel and Bonomo, 2013). Among the plethora of resistance mechanisms against carbapenems, production of metallo-beta-lactamases (MBLs) is especially significant. The clinical significance of MBLs is further magnified by their ability to hydrolyze all beta-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. New Delhi metallo-beta-lactamase-1 (NDM-1) will soon become the most commonly isolated and distributed metallo-beta-lactamase worldwide due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative pathogens (Patel and Bonomo, 2013). Initial reports frequently demonstrated an epidemiologic link to the Indian subcontinent where these MBLs are endemic (Kumarasamy et al., 2010), and among others to the Balkans as another area of endemicity for NDM-1 (Struelens et al., 2010; Jovcic et al., 2011; Mirovic et al., 2012). Previous environmental studies done by Walsh et al. (2011) revealed the prevalence of NDM-1-positive bacterial strains in drinking water and waste seepage samples in New Delhi, India. That study suggested widespread environmental contamination in New Delhi and is in correlation with the fact that not all of the patients with blaNDM-1-positive bacteria were treated in hospitals there. Thus, rather than being a nosocomial problem, NDM-1-producing bacteria seem to be circulating in the community and to be imported into hospitals with admitted patients (Walsh et al., 2011). Therefore, the aim of our study was to determine whether the environment is a source of NDM-1-producing bacteria. The study was focused on urban rivers, a lake and springheads in the Belgrade area, since all the documented cases of Serbian NDM-1 infections have been located in Belgrade. The results of this study lead us to doubt that Serbia, among other Balkan countries, is an endemic area for NDM. In addition, testing for the presence of bacteria producing ESBL and MBL other than NDM-1 was performed in order to determine the role of the environment in the dissemination of these types of resistance.
2. Materials and methods 2.1. Sampling and isolation of beta-lactam-resistant bacteria Water samples were collected at ten sites (rivers, a lake and springheads) in the area of Belgrade, Serbia, from June to September 2013. The locations were: the Danube River—Ada Huja (GPS coordinates: N 44°49′ 29.854″, E 20°31′53.187″), the Danube River—Dorcol (GPS coordinates: N 44°49′49.276″, E 20°27′46.651″), the Sava River (GPS coordinates: N 44°47′44.934″, E 20°23′57.149″), Ada Ciganlija Lake I (GPS coordinates: N 44°47′10.451″, E 20°24′20.584″), Ada Ciganlija Lake II (GPS coordinates: N 44°47′1.381″, E 20°23′37.841″), Ada Ciganlija Lake III (GPS coordinates: N 44°46′37.389″, E 20°22′33.499″), Hajducka Cesma Spring (GPS coordinates: N 44°45′57.479″, E 20°26′8.914″), the Topcider River (GPS coordinates: N 44°46′55.73″, E 20°26′14.678″), Sakinac Spring (GPS coordinates: N 44°46′55.73″, E 20°26′14.678″), and Brace Jerkovic Spring (GPS coordinates: N 44°45′56.451″, E 20°29′27.228″). Water samples from the Danube River (Ada Huja and Dorcol sites), the Sava River, Hajducka Cesma Spring, Brace Jerkovic Spring, Sakinac Spring and the Topcider River were sampled in August 2013, while samples from Ada Ciganlija Lake were taken in June, August and September. Ada Ciganlija Lake is 4.2 kilometer long and 200 meter wide on average, with a depth of between 4 and 6 m. At its southwestern embankment the lake is connected by a water-permeable barrier to Taloznik Lake, a smaller, fenced-in lake, which is part of Belgrade's water supply system. Water constantly filters from Taloznik Lake into Ada Ciganlija Lake, while pumps at the opposite end of the lake artificially ensure its flow. Water samples were collected in sterile bottles from 0.5 m below the water's surface. Each water sample (500 ml) was concentrated using a sterile 0.45 μm membrane filter (Sarstedt, USA). The filter was then placed into Luria Bertani (LB) broth supplemented with 100 μg/ml ampicillin and 10 μg/ml cycloheximide and incubated for 24 h at 37 °C with aeration for enrichment. Different dilutions of enrichment cultures were inoculated onto LB agar plates with 100 μg/ml ampicillin and 10 μg/ml cycloheximide for selection of beta-lactam-resistant bacteria and incubated for 24 h at 37 °C. Colonies with different morphologies and colorations were then selected for further analysis. 2.2. Molecular identification of isolated bacteria The total genomic DNA of each selected bacteria was isolated according to the method described by Hopwood et al. (1985). The PCR fragments of 16S rRNA genes were obtained using primers UNI 16SF (5-GAG AGT TTG ATC CTG GC-3) and UNI 16SR (5-AGG AGG TGA TCC AGC CG-3) (Jovcic et al., 2009). The PCR products were purified with a GeneJET PCR Purification Kit (Thermo Scientific, Lithuania) and subjected to sequencing by the Macrogen DNA sequencing service (Macrogen Inc., Netherlands). The resulting sequences were aligned against known sequences by using Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were submitted to the European Nucleotide Archive (http://www.ebi.ac. uk/ena/data/view/LN558584-LN558652, accession nos.: LN558584– LN558652). 2.3. Detection of ESBL and MBL producing isolates ESBL-producing activity was first screened with a double disc synergy test assay (DDST) and subjected to a phenotypic confirmatory test as previously described (Tsering et al., 2009). Only isolates that were positive for ESBL production in confirmatory tests were taken into account for molecular analysis. The detection of MBL producers was carried out by disc diffusion tests with imipenem (10 μg) and imipenem in combination with EDTA (10/930 μg) (Yong et al., 2002). Isolates selected within this test were further subjected to molecular analysis.
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2.4. Determination of ESBL and MBL genes The presence of ESBL, AmpC type and MBL genes in isolates selected on basis of phenotypic tests was examined by the PCR method. A set of primers specific to genes TEM, SHV, CTX-M, GES-7, DHA-1, CMY-1, and CMY-2, and to genes NDM-1, SPM-1, IMP, VIM-2, SIM, and GIM were used for the detection of ESBL, AmpC type and MBL genes, respectively (Table 1). The PCR products were purified with a GeneJET PCR Purification Kit (Thermo Scientific, Lithuania), sequenced by the Macrogen DNA sequencing service (Macrogen Inc., Netherlands) and aligned against known sequences by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast. cgi). The sequences were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena/data/view/LN558653-LN558657, accession nos.: LN558653–LN558657). 2.5. Antimicrobial susceptibility testing ESBL positive isolates detected by the disc diffusion method were tested against four antimicrobial agents by microdilution testing in cation-adjusted Mueller-Hinton broth by following The European Committee on Antimicrobial Susceptibility Testing (Version 4.0, 2014) criteria and using their susceptibility and resistance breakpoints. Resistance was tested against piperacillin, piperacillin–tazobactam, ceftazidime and aztreonam (ranging from 0 to 256 μg/ml). Cell density was monitored by OD620 measurements after 24 h of incubation at 37 °C in a microtiter plate reader (Multiscan RC, Labsystems, UK). Experiments were done in triplicate. The concentrations of antimicrobial agents which inhibited the growth rate by 50% (IC50) were calculated by using Microsoft Excel software. IC50 values were determined from an individual curve equation specific for each sample. The IC50s reported are the results of three independent experiments. 3. Results 3.1. Identification and distribution of ampicillin resistant bacteria Water sampling was performed at 10 different locations in the Belgrade area in order to examine the presence and distribution of ESBL and MBL producing bacteria, and to determine if New Delhi
Table 1 List of primers used for detection of ESBL and MBL genes.
ESBL
MBL
CMY-2R
5-GTTTTCTCCTGAACGTGGCTGGC-3
Primer TEMF TEMR SHVF SHVR CTX-MF CTX-MR GES-7 F GES-7R DHA-1F DHA-1R CMY-1F CMY-1R CMY-2F NDMfullF NDMfullR SPM-1F SPM-1R IMPF IMPR VIM-2F VIM-2R SIMF SIMR GIMF GIMR
Sequence 5-ATAAAATTCTTGAAGACGAAA-3 5-GACAGTTACCAATGCTTAATCA-3 5-GGGTTATTCTTATTTGTCGC-3 5-TTAGCGTTGCCAGTGCTC-3 5-AAAAATGATTGAAAGGTGGTTGT-3 5-TTACAGCCCTTCGGCGATGA-3 5-ATCTTGAGAAGCTAGAGCGCG-3 5-GTTTCCGATCAGCCACCTCT-3 5-GTCGCGGCGGTGGTGGAC-3 5-CCGCACCCAGCACACCTGT-3 5-GCTGCTCAAGGAGCACAGGATCCCG-3 5-GGCACATTGACATAGGTGTGGTGCATG-3 5-ACTGGCCAGAACTGACAGGCAAA-3 5-ATGGAATTGCCCAATATTATG-3 5-TCAGCGCAGCTTGTCGGCC-3 5-CTGCTTGGATTCATGGGC-3 5-CCTTTTCCGCGACCTTGA-3 5-GAAGGYGTTTATGTTCAT-3 5-GTAMGTTTCAAGAGTGAT-3 5-GTTTGGTCGCATATCGCA-3 5-AATGCGCAGCACCAGGAT-3 5-TACAAGGGATTCGGCATC-3 5-TAATGGCCTGTTCCCATG-3 5-TCGACACACCTTGGTCTG-3 5-AACTTCCAACTTTGCCAT-3
Reference Teo et al. (2012) Teo et al. (2012)
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metallo-beta-lactamase is present in the surface waters of Belgrade. Locations were selected due to their exposure to different anthropogenic impacts from industrial and domestic origins. Among other sites, Ada Ciganlija Lake was studied in more detail since it has been known as the most popular recreational area during the summer season, with up to 150,000 swimmers daily. Therefore, sampling from Ada was carried out three times: before, during and after the swimming season (June, August and September). In order to fulfill our goals we used culture-dependent and molecular techniques to investigate the presence and the prevalence of extended-spectrum beta-lactamase- and carbapenemase-producing Gram-negative bacteria in water samples from the Belgrade area. A total of 69 ampicillin-resistant isolates were collected from 8 out of the 10 analyzed locations (42 from three sites at Ada Ciganlija Lake, 10 from the Sava River, 6 from the Danube River at Ada Huja, 6 from the Topcider River, 3 from the Danube River at Dorcol and 2 isolates from Hajducka Cesma spring) whereas at two springs (Brace Jerkovic and Sakinac) beta-lactam resistant isolates were not detected. Among the 69 isolates, molecular identification revealed the presence of species belonging to 12 different bacterial genera, including 13 isolates of Pseudomonas otitidis, 9 Klebsiella pneumoniae, 9 Proteus mirabilis, 8 Pseudomonas aeruginosa, 5 Morganella morganii, 5 Escherichia coli, 4 Aeromonas veronii, 3 Providencia sp., 3 Enterobacter sp., 3 Comamonas aquatica, 2 Stenotrophomonas maltophilia, 2 Proteus vulgaris, 1 Providencia stuartii, 1 Comamonas testosteroni and 1 Acinetobacter baumannii isolate (Fig. 1). All identified isolates were Gram negative rods and most were members of the Enterobacteriaceae family (53.62%), while others belonged to Pseudomonadaceae (30.43%), Aeromonadaceae (5.8%), Comamonadaceae (5.8%), Xanthomonadaceae (2.9%) and Moraxellaceae (1.45%) family. Since Ada Ciganlija Lake is one of the most popular natural swimming locations in Belgrade, water samples were collected three times in order to reveal if there is a correlation between periods of the season and the prevalence of beta-lactam-resistant bacteria. Also, we wanted to determine anthropogenic influence on the presence of beta-lactamresistant bacteria. In June, at the beginning of the season, 11 different ampicillin-resistant isolates were obtained, while in August, during the peak of the swimming season, the number of isolates was expectedly the largest, in total 25. At the end of the season, in September, 6 isolates were obtained from the sampled water (Fig. 2). In addition, the distribution of bacterial species in June, August and September varied (Fig. 2). The most common species in June was Klebsiella pneumonia (4 / 11, 36.36%), while in August P. otitidis (11 / 25, 44%) and P. aeruginosa (6 / 25, 24%) dominated. In September, K. pneumoniae, P. otitidis and P. aeruginosa were not detected and the isolates mostly belonged to species from the Enterobacteriaceae family (E. coli, M. morganii and Enterobacter sp.) (Fig. 2).
Teo et al. (2012)
3.2. Phenotypic and genotypic analysis of ESBL and MBL producers
Teo et al. (2012)
Phenotypic confirmation tests for detecting ESBL producers, done by the disc diffusion method, revealed 7 (10.14%) ESBL-producing isolates. Four of the ESBL-producing isolates were from the Ada Ciganlija Lake III site, of which three were sampled in August (one isolate of K. pneumoniae and two isolates of S. maltophilia), while the fourth was isolated in September (M. morganii). Three of the ESBLproducing candidates were isolated from rivers, two from the Sava River (C. aquatica isolates) and one from the Danube River at Dorcol (P. stuartii). These 7 ESBL-producing candidates were further analyzed by the PCR method with primers specific for TEM, SHV, CTX-M, GES-7, DHA-1 and for AmpC-type beta-lactamases CMY-1 and CMY-2 genes. The genetic basis of beta-lactamase production was determined for five of the seven isolates. Among those are all four phenotypically selected ESBL producers from Ada Ciganlija Lake III. The SHV gene was found in K. pneumonia and two S. maltophilia (isolated in August), while the presence of DHA-1 gene was determined for M. morganii
Teo et al. (2012) Teo et al. (2012) Teo et al. (2012) Teo et al. (2012) Ellington et (2007) Ellington et (2007) Ellington et (2007) Ellington et (2007) Ellington et (2007)
al. al. al. al. al.
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Fig. 1. Prevalence of different beta-lactam resistant species isolated from rivers, lake and springheads in Belgrade, Serbia.
(isolated in September). The fifth ESBL-producing candidate was P. stuartii, isolated from the Danube River, for which we confirmed that carries CMY-2 gene, but neither one of the analyzed ESBL genes was found. The molecular basis of ESBL production was not determined for C. aquatica isolated from the Sava River. Phenotypic tests revealed a total of 39 (56.52%) isolates resistant to imipenem, of which 32 were positive for MBL production. These isolates were predominantly selected from Ada Ciganlija Lake (n = 20), of which 8 were isolated in June, 9 in August and 3 in September. In the June isolates the same number of candidates was isolated from Ada Ciganlija Lake sites I and II (3 isolates) and 2 were selected from Ada Ciganlija Lake III, while in August 4 of the isolates were from Ada Ciganlija Lake I, 2 from Ada Ciganlija Lake II and 3 from the Ada Ciganlija Lake III sampling site. Out of 3 MBL-positive bacteria isolated in September, 1 was from Ada Ciganlija Lake I and 2 were from the Ada Ciganlija Lake II site. Twelve MBL-producing isolates
Fig. 2. Number and distribution of beta-lactam resistant isolates before (June), during (August) and after (September) the swimming season at Lake Ada Ciganlija in Belgrade, Serbia.
were selected from samples taken from rivers: 2 from the Danube River at Ada Huja, 6 from the Sava River and 4 from the Topcider River. Molecular analyses failed to detect the genetic basis of MBL production when primers specific for NDM-1, SPM-1, IMP, VIM-2, SIM and GIM genes were used. 3.3. Antimicrobial susceptibility profiles Antimicrobial susceptibility profiles were done for all 7 ESBLproducing candidates obtained by phenotypic confirmation tests. The selected isolates showed variable antibiotic resistance patterns (Table 2). All strains except two S. maltophilia isolates and M. morganii were sensitive to piperacillin in combination with the beta-lactamase inhibitor tazobactam. 4. Discussion The enzyme NDM-1 was designated as a global threat considering its rapid worldwide dissemination and association with other determinants of resistance to antibiotics. Not long after the first European cases were described, the Balkan countries (including Serbia) were highlighted, besides the Indian subcontinent, as an NDM-1 endemic region (Struelens et al., 2010). Monitoring of public water supplies in India revealed significant environmental contamination with NDM-1producing strains (Walsh et al., 2011). The results of a survey performed by Walsh et al. (2011) were striking, since blaNDM-1 was detected in 4% of drinking water samples and 30% of seepage samples. This survey clearly indicated the environment as a source of NDM-1producing strains in India. Although it was shown for India that NDM1-producing strains were abundant in the environment, this type of study was never performed in Serbia, a region designated as endemic. This is of importance because Struelens et al. (2010) stated that of 55 European patients (until 2010), 31 had visited the Indian subcontinent and five had visited Balkan countries, leading to the conclusion of endemicity in the Balkans. Endemicity of blaNDM-1 in the Balkans has been the subject of many discussions, but without environmental data all were just hypothetical. One of the hypotheses that had been proposed by
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Table 2 IC50 of ESBL producing isolates from Belgrade environmental waters, for selected antibiotics. Isolate
Isolation site
Type of ESBL
Piperacillin
Piperacillin/tazobactam
Ceftazidime
Aztreonam
K. pneumoniae AAIII-1 S. maltophilia AAIII-2 S. maltophilia AAIII-3 P. stuartii DD3 M. morganii ASIII-2 C. aquatica RS3 C. aquatica RS8
Ada Ciganlija Lake III Ada Ciganlija Lake III Ada Ciganlija Lake III The Danube River - Dorcol Ada Ciganlija Lake III The Sava River The Sava River
SHV SHV SHV CMY-2 DHA-1 ND ND
14.88 ± 0.28 24.69 ± 1.04 N256 60.16 ± 0.64 16 ± 3.14 47.15 ± 1.93 24.53 ± 0.56
b4 12.16 ± 2.26 28.56 ± 4.86 b4 16 ± 2.26 b4 b4
b4 40.96 ± 4.07 35.68 ± 0.68 98.24 ± 25.8 38.34 ± 9.12 17.44 ± 0.68 90.24 ± 0.45
26.13 ± 3.42 186.88 ± 45.45 44.96 ± 0.48 b4 56.32 ± 2.36 48.1 ± 0.98 34.4 ± 1.13
ND — not determined.
Liveromore et al. (2011) to explain a Balkan association for blaNDM-1 was that the blaNDM-1 gene was recruited to plasmids from an external origin, probably an environmental organism without clinical relevance. A similar phenomenon was previously reported for the blaCTX-M genes, which code for cephalosporin-hydrolyzing extended-spectrum betalactamases that have escaped repeatedly from Kluyvera spp. (Woodford et al., 2009). Alternatively, Liveromore et al. (2011) hypothesized that the NDM-1 enzyme, which was already circulating in India, could have been imported from the subcontinent to the Balkans. The second hypothesis was also highlighted by Johnson and Woodford (2013) and they supported it by means of documenting the travel of patients from the Balkans to Pakistan for commercial kidney transplants that were associated with frequent infections. These types of findings lead to the assumption that such medical tourism could have introduced NDM to the Balkans. Having in mind that we did not detect any NDM-1-producing strains in the environmental waters of Belgrade area we could speculate that Serbian NDM-1 in fact is a transplant and that Serbia is not an endemic region for NDM-1. ESBL-producing strains, which are recognized as a threat by clinicians, were also one of the focal points of our study. The number of studies investigating potential environmental reservoirs of ESBL-producing strains is still very limited (Dhanji et al., 2010; Girlich et al., 2011; Tacao et al., 2012; Zurfluh et al., 2013). In addition, data from Balkan Peninsula rivers are unavailable. These studies are of great importance since rivers and lakes are considered putative reservoirs of resistant bacteria, and because they represent collectors of surface waters containing materials from different origins that could be contaminated. Having that in mind, we have done an extensive study of the presence and prevalence of ESBL and AmpC-type beta-lactamase producing bacteria in the surface waters of Belgrade. Among the results we would like to emphasize that SHV-producing bacteria were found only in Ada Ciganlija Lake, the most popular swimming recreation lake, in August when there is the largest number of visitors. Since we didn't find SHV-producing strains in Ada Ciganlija Lake in June or September we could speculate that these strains were only transient in this lake and that their source is anthropogenic. Our finding of SHVproducing K. pneumoniae is in correlation with previously published results (Babini and Livermore, 2000), however finding SHV-producing S. maltophilia is striking, since this bacteria is considered an emerging nosocomial pathogen. Additionally, as phenotypic detection of ESBLs in non-fermenters is complicated, ESBLs are often underestimated and underreported in these strains and therefore S. maltophilia may be a hidden reservoir for such ESBLs (al Naiemi et al., 2006). The presence of CMY-2-producing P. stuartii in the Danube River at Dorcol is also of importance, since P. stuartii is an opportunistic pathogen which is essentially responsible for urinary tract infections (Tumbarello et al., 2004). Besides ESBL production, we analyzed the presence of MBL producing bacteria (other than NDM-1) as well. Our results revealed the low specificity and positive predictive value of conventional phenotypic methods for detecting MBL producers among non-imipenemsusceptible strains. However, this phenomenon has previously been described for P. aeruginosa strains (Samuelsen et al., 2008).
4.1. Conclusions New Delhi metallo-beta-lactamase-producing strains weren't present in the analyzed environmental waters, which strongly reinforces the need for a revision of the designation of NDM-1 endemic regions. However, beta-lactam resistant Gram-negative bacteria are present in the environment and anthropogenic factors seem to have a crucial role in the dissemination of resistance to antibiotics in Belgrade, Serbia. This assumption is supported by several findings: (i) the highest number of beta-lactam resistant Gram-negative bacteria was collected from Ada Ciganlija Lake which is, if we compare it to other analyzed sites, most exposed to anthropogenic influence, (ii) out of 42 betalactam resistant isolates from Ada Ciganlija Lake 25 were collected in August, when the highest number of visitors and swimmers are present at the site, and (iii) out of 7 phenotypically confirmed ESBL producing strains, 4 were from Ada Ciganlija Lake, of which 3 were collected in August. Therefore, this study emphasizes the importance of cohesive research related to the spread of resistance to antibiotics which must include both clinical and environmental data in order to draw the final conclusions. Furthermore, this study reinforces the importance of continued study and monitoring of antimicrobial resistance to combat the growing infections caused by ESBL- and MBL-producing bacteria.
Acknowledgments This work was supported by grant no. 173019 from the Ministry of Education, Science and Technological Development of the Republic of Serbia.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Environmental waters and blaNDM-1 in Belgrade, Serbia: Endemicity questioned K. Novovic a, B. Filipic a,b, K. Veljovic a, J. Begovic a, N. Mirkovic c, B. Jovcic a,d,⁎ a
University of Belgrade, Institute of Molecular Genetics and Genetic Engineering, Vojvode Stepe 444a, P.O. Box 23, 11010 Belgrade, Serbia University of Belgrade, Faculty of Pharmacy, Vojvode Stepe 450, 11221 Belgrade, Serbia University of Belgrade, Faculty of Agriculture, Nemanjina 6, Belgrade, Serbia d University of Belgrade, Faculty of Biology, Studentski trg 16, 11000 Belgrade, Serbia b c
H I G H L I G H T S • • • • •
Serbia was designated as the endemic for blaNDM-1, a significant public health threat Contamination of urban rivers, lake and springheads with NDM-1 producing strains was tested We propose that NDM-1 is a transplant and that Serbia is not endemic region for NDM-1. Special emphasis was on prevalence of resistance in the most popular Belgrade lake. We report presence of strains producing SHV, DHA-1 and CMY-2 β-lactamases.
a r t i c l e
i n f o
Article history: Received 7 October 2014 Received in revised form 8 November 2014 Accepted 22 December 2014 Available online xxxx Editor: C.E.W. Steinberg Keywords: Antibiotic resistance Environment New Delhi metallo-beta-lactamase (NDM-1)
a b s t r a c t New Delhi metallo-beta-lactamase-1 (NDM-1) will soon become the most commonly isolated and distributed metallo-beta-lactamase worldwide due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative pathogens. NDM-positive bacteria pose a significant public health threat in the Indian subcontinent and the Balkans, which have been designated as endemic regions. Our study was focused on urban rivers, a lake and springheads as a potential source of NDM-1-producing strains in Serbia, but also as a source of other metallo-beta-lactamases and extended-spectrum beta-lactamase (ESBL) producing bacteria. A total of 69 beta-lactam resistant isolates, belonging to 12 bacterial genera, were collected from 8 out of 10 different locations in Belgrade, of which the most were from a popular recreational site, Ada Ciganlija Lake. Phenotypic tests revealed 7 (10.14%) ESBL-producing isolates and 39 (56.52%) isolates resistant to imipenem, of which 32 were positive for metallo-beta-lactamase (MBL) production. PCR and sequencing revealed the presence of genetic determinants for SHV (3 isolates), DHA-1 (1 isolate) and CMY-2 (1 isolate) beta-lactamases. However, we did not detect any NDM-1-producing strains (previously described cases of NDM-1 from Serbia were limited to Belgrade), so we propose that Serbian NDM-1 is in fact a transplant and a nosocomial, rather than an environmental, issue and that Serbia is not an endemic region for NDM-1. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Antibiotics, besides their application for improving human health, have been used for preventing and treating animal and plant infections and for promoting growth in animal farming. These applications have
⁎ Corresponding author at: University of Belgrade, Faculty of Biology, Studentski trg 16, 11000 Belgrade, Serbia. E-mail address: [email protected] (B. Jovcic).
http://dx.doi.org/10.1016/j.scitotenv.2014.12.072 0048-9697/© 2014 Elsevier B.V. All rights reserved.
caused antibiotics to be released and abundant in natural ecosystems (Cabello, 2006). Significant public use or misuse of antibiotics, caused by several social factors and low awareness of antibiotic resistance, is predominantly present in countries with high levels of antimicrobial resistance. Due to all of these factors antibiotic resistant bacteria have been found widely in aquatic environments (Baquero et al., 2008; Xi et al., 2009). The occurrence of resistant bacteria in a water environment could be the result of anthropogenic activity or natural processes (many acquired resistance mechanisms have originated in producers of antibiotics) (Martinez, 2008). Nevertheless, anthropogenic and/or naturally occurring resistant bacteria in water environments
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pose a threat to human health. Moreover, these bacteria may transfer their antibiotic resistance genes to other pathogens through horizontal gene transfer. Therefore the contamination of water and the environment with multidrug-resistant Gram-negative bacteria is one of the main routes for the spread of antibiotic resistance and is therefore a crucial area for control. Beta-lactam antibiotics, including the sub-groups of penicillins, cephalosporins and carbapenems, are among the most valuable antibiotics for human use owing to their comparatively high effectiveness, low cost, ease of delivery and minimal side effects. Bacterial resistance against beta-lactam antibiotics is increasing at a significant rate and has become a common problem in primary care medicine. There are several mechanisms of antimicrobial resistance to betalactam antibiotics, one of which is the production of beta-lactamases (Wilke et al., 2005). Acquired resistance to beta-lactams is mainly mediated by extendedspectrum beta-lactamases (ESBLs) that confer bacterial resistance to all beta-lactams except carbapenems and cephamycins (Wilke et al., 2005). The increase of ESBL prevalence at the beginning of the 21st century in Europe has been one of the most significant phenomena in antimicrobial resistance (Barlow et al., 2008). ESBLs are generally acquired by horizontal gene transfer (e.g., TEM or SHV, plasmid-encoded CTX-M) or are mobilized from environmental bacteria (e.g., progenitors of CTX-M from Kluyvera spp.). Interestingly a shift in the distribution of different ESBLs has occurred in Europe, which is the result of a significant increase of CTX-M enzymes over TEM and SHV variants (Coque et al., 2008). Besides the ESBLs, of great importance are carbapenemases which confer resistance to carbapenems that are potent and broadspectrum beta-lactam antibiotics traditionally reserved for the treatment of the most serious infections (El-Gamal and Oh, 2010). The emergence and rapid worldwide spread of carbapenemase-producing pathogens contribute significantly to the morbidity and mortality of patients. Carbapenem-resistant Gram-negative bacilli remain an alarming threat as few antimicrobial agents are reliably effective and very few are expected to be available in the near future (Patel and Bonomo, 2013). Among the plethora of resistance mechanisms against carbapenems, production of metallo-beta-lactamases (MBLs) is especially significant. The clinical significance of MBLs is further magnified by their ability to hydrolyze all beta-lactams and by the fact that there is currently no clinical inhibitor, nor is there likely to be for the foreseeable future. New Delhi metallo-beta-lactamase-1 (NDM-1) will soon become the most commonly isolated and distributed metallo-beta-lactamase worldwide due to its rapid international dissemination and its ability to be expressed by numerous Gram-negative pathogens (Patel and Bonomo, 2013). Initial reports frequently demonstrated an epidemiologic link to the Indian subcontinent where these MBLs are endemic (Kumarasamy et al., 2010), and among others to the Balkans as another area of endemicity for NDM-1 (Struelens et al., 2010; Jovcic et al., 2011; Mirovic et al., 2012). Previous environmental studies done by Walsh et al. (2011) revealed the prevalence of NDM-1-positive bacterial strains in drinking water and waste seepage samples in New Delhi, India. That study suggested widespread environmental contamination in New Delhi and is in correlation with the fact that not all of the patients with blaNDM-1-positive bacteria were treated in hospitals there. Thus, rather than being a nosocomial problem, NDM-1-producing bacteria seem to be circulating in the community and to be imported into hospitals with admitted patients (Walsh et al., 2011). Therefore, the aim of our study was to determine whether the environment is a source of NDM-1-producing bacteria. The study was focused on urban rivers, a lake and springheads in the Belgrade area, since all the documented cases of Serbian NDM-1 infections have been located in Belgrade. The results of this study lead us to doubt that Serbia, among other Balkan countries, is an endemic area for NDM. In addition, testing for the presence of bacteria producing ESBL and MBL other than NDM-1 was performed in order to determine the role of the environment in the dissemination of these types of resistance.
2. Materials and methods 2.1. Sampling and isolation of beta-lactam-resistant bacteria Water samples were collected at ten sites (rivers, a lake and springheads) in the area of Belgrade, Serbia, from June to September 2013. The locations were: the Danube River—Ada Huja (GPS coordinates: N 44°49′ 29.854″, E 20°31′53.187″), the Danube River—Dorcol (GPS coordinates: N 44°49′49.276″, E 20°27′46.651″), the Sava River (GPS coordinates: N 44°47′44.934″, E 20°23′57.149″), Ada Ciganlija Lake I (GPS coordinates: N 44°47′10.451″, E 20°24′20.584″), Ada Ciganlija Lake II (GPS coordinates: N 44°47′1.381″, E 20°23′37.841″), Ada Ciganlija Lake III (GPS coordinates: N 44°46′37.389″, E 20°22′33.499″), Hajducka Cesma Spring (GPS coordinates: N 44°45′57.479″, E 20°26′8.914″), the Topcider River (GPS coordinates: N 44°46′55.73″, E 20°26′14.678″), Sakinac Spring (GPS coordinates: N 44°46′55.73″, E 20°26′14.678″), and Brace Jerkovic Spring (GPS coordinates: N 44°45′56.451″, E 20°29′27.228″). Water samples from the Danube River (Ada Huja and Dorcol sites), the Sava River, Hajducka Cesma Spring, Brace Jerkovic Spring, Sakinac Spring and the Topcider River were sampled in August 2013, while samples from Ada Ciganlija Lake were taken in June, August and September. Ada Ciganlija Lake is 4.2 kilometer long and 200 meter wide on average, with a depth of between 4 and 6 m. At its southwestern embankment the lake is connected by a water-permeable barrier to Taloznik Lake, a smaller, fenced-in lake, which is part of Belgrade's water supply system. Water constantly filters from Taloznik Lake into Ada Ciganlija Lake, while pumps at the opposite end of the lake artificially ensure its flow. Water samples were collected in sterile bottles from 0.5 m below the water's surface. Each water sample (500 ml) was concentrated using a sterile 0.45 μm membrane filter (Sarstedt, USA). The filter was then placed into Luria Bertani (LB) broth supplemented with 100 μg/ml ampicillin and 10 μg/ml cycloheximide and incubated for 24 h at 37 °C with aeration for enrichment. Different dilutions of enrichment cultures were inoculated onto LB agar plates with 100 μg/ml ampicillin and 10 μg/ml cycloheximide for selection of beta-lactam-resistant bacteria and incubated for 24 h at 37 °C. Colonies with different morphologies and colorations were then selected for further analysis. 2.2. Molecular identification of isolated bacteria The total genomic DNA of each selected bacteria was isolated according to the method described by Hopwood et al. (1985). The PCR fragments of 16S rRNA genes were obtained using primers UNI 16SF (5-GAG AGT TTG ATC CTG GC-3) and UNI 16SR (5-AGG AGG TGA TCC AGC CG-3) (Jovcic et al., 2009). The PCR products were purified with a GeneJET PCR Purification Kit (Thermo Scientific, Lithuania) and subjected to sequencing by the Macrogen DNA sequencing service (Macrogen Inc., Netherlands). The resulting sequences were aligned against known sequences by using Basic Local Alignment Search Tool (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were submitted to the European Nucleotide Archive (http://www.ebi.ac. uk/ena/data/view/LN558584-LN558652, accession nos.: LN558584– LN558652). 2.3. Detection of ESBL and MBL producing isolates ESBL-producing activity was first screened with a double disc synergy test assay (DDST) and subjected to a phenotypic confirmatory test as previously described (Tsering et al., 2009). Only isolates that were positive for ESBL production in confirmatory tests were taken into account for molecular analysis. The detection of MBL producers was carried out by disc diffusion tests with imipenem (10 μg) and imipenem in combination with EDTA (10/930 μg) (Yong et al., 2002). Isolates selected within this test were further subjected to molecular analysis.
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2.4. Determination of ESBL and MBL genes The presence of ESBL, AmpC type and MBL genes in isolates selected on basis of phenotypic tests was examined by the PCR method. A set of primers specific to genes TEM, SHV, CTX-M, GES-7, DHA-1, CMY-1, and CMY-2, and to genes NDM-1, SPM-1, IMP, VIM-2, SIM, and GIM were used for the detection of ESBL, AmpC type and MBL genes, respectively (Table 1). The PCR products were purified with a GeneJET PCR Purification Kit (Thermo Scientific, Lithuania), sequenced by the Macrogen DNA sequencing service (Macrogen Inc., Netherlands) and aligned against known sequences by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast. cgi). The sequences were submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/ena/data/view/LN558653-LN558657, accession nos.: LN558653–LN558657). 2.5. Antimicrobial susceptibility testing ESBL positive isolates detected by the disc diffusion method were tested against four antimicrobial agents by microdilution testing in cation-adjusted Mueller-Hinton broth by following The European Committee on Antimicrobial Susceptibility Testing (Version 4.0, 2014) criteria and using their susceptibility and resistance breakpoints. Resistance was tested against piperacillin, piperacillin–tazobactam, ceftazidime and aztreonam (ranging from 0 to 256 μg/ml). Cell density was monitored by OD620 measurements after 24 h of incubation at 37 °C in a microtiter plate reader (Multiscan RC, Labsystems, UK). Experiments were done in triplicate. The concentrations of antimicrobial agents which inhibited the growth rate by 50% (IC50) were calculated by using Microsoft Excel software. IC50 values were determined from an individual curve equation specific for each sample. The IC50s reported are the results of three independent experiments. 3. Results 3.1. Identification and distribution of ampicillin resistant bacteria Water sampling was performed at 10 different locations in the Belgrade area in order to examine the presence and distribution of ESBL and MBL producing bacteria, and to determine if New Delhi
Table 1 List of primers used for detection of ESBL and MBL genes.
ESBL
MBL
CMY-2R
5-GTTTTCTCCTGAACGTGGCTGGC-3
Primer TEMF TEMR SHVF SHVR CTX-MF CTX-MR GES-7 F GES-7R DHA-1F DHA-1R CMY-1F CMY-1R CMY-2F NDMfullF NDMfullR SPM-1F SPM-1R IMPF IMPR VIM-2F VIM-2R SIMF SIMR GIMF GIMR
Sequence 5-ATAAAATTCTTGAAGACGAAA-3 5-GACAGTTACCAATGCTTAATCA-3 5-GGGTTATTCTTATTTGTCGC-3 5-TTAGCGTTGCCAGTGCTC-3 5-AAAAATGATTGAAAGGTGGTTGT-3 5-TTACAGCCCTTCGGCGATGA-3 5-ATCTTGAGAAGCTAGAGCGCG-3 5-GTTTCCGATCAGCCACCTCT-3 5-GTCGCGGCGGTGGTGGAC-3 5-CCGCACCCAGCACACCTGT-3 5-GCTGCTCAAGGAGCACAGGATCCCG-3 5-GGCACATTGACATAGGTGTGGTGCATG-3 5-ACTGGCCAGAACTGACAGGCAAA-3 5-ATGGAATTGCCCAATATTATG-3 5-TCAGCGCAGCTTGTCGGCC-3 5-CTGCTTGGATTCATGGGC-3 5-CCTTTTCCGCGACCTTGA-3 5-GAAGGYGTTTATGTTCAT-3 5-GTAMGTTTCAAGAGTGAT-3 5-GTTTGGTCGCATATCGCA-3 5-AATGCGCAGCACCAGGAT-3 5-TACAAGGGATTCGGCATC-3 5-TAATGGCCTGTTCCCATG-3 5-TCGACACACCTTGGTCTG-3 5-AACTTCCAACTTTGCCAT-3
Reference Teo et al. (2012) Teo et al. (2012)
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metallo-beta-lactamase is present in the surface waters of Belgrade. Locations were selected due to their exposure to different anthropogenic impacts from industrial and domestic origins. Among other sites, Ada Ciganlija Lake was studied in more detail since it has been known as the most popular recreational area during the summer season, with up to 150,000 swimmers daily. Therefore, sampling from Ada was carried out three times: before, during and after the swimming season (June, August and September). In order to fulfill our goals we used culture-dependent and molecular techniques to investigate the presence and the prevalence of extended-spectrum beta-lactamase- and carbapenemase-producing Gram-negative bacteria in water samples from the Belgrade area. A total of 69 ampicillin-resistant isolates were collected from 8 out of the 10 analyzed locations (42 from three sites at Ada Ciganlija Lake, 10 from the Sava River, 6 from the Danube River at Ada Huja, 6 from the Topcider River, 3 from the Danube River at Dorcol and 2 isolates from Hajducka Cesma spring) whereas at two springs (Brace Jerkovic and Sakinac) beta-lactam resistant isolates were not detected. Among the 69 isolates, molecular identification revealed the presence of species belonging to 12 different bacterial genera, including 13 isolates of Pseudomonas otitidis, 9 Klebsiella pneumoniae, 9 Proteus mirabilis, 8 Pseudomonas aeruginosa, 5 Morganella morganii, 5 Escherichia coli, 4 Aeromonas veronii, 3 Providencia sp., 3 Enterobacter sp., 3 Comamonas aquatica, 2 Stenotrophomonas maltophilia, 2 Proteus vulgaris, 1 Providencia stuartii, 1 Comamonas testosteroni and 1 Acinetobacter baumannii isolate (Fig. 1). All identified isolates were Gram negative rods and most were members of the Enterobacteriaceae family (53.62%), while others belonged to Pseudomonadaceae (30.43%), Aeromonadaceae (5.8%), Comamonadaceae (5.8%), Xanthomonadaceae (2.9%) and Moraxellaceae (1.45%) family. Since Ada Ciganlija Lake is one of the most popular natural swimming locations in Belgrade, water samples were collected three times in order to reveal if there is a correlation between periods of the season and the prevalence of beta-lactam-resistant bacteria. Also, we wanted to determine anthropogenic influence on the presence of beta-lactamresistant bacteria. In June, at the beginning of the season, 11 different ampicillin-resistant isolates were obtained, while in August, during the peak of the swimming season, the number of isolates was expectedly the largest, in total 25. At the end of the season, in September, 6 isolates were obtained from the sampled water (Fig. 2). In addition, the distribution of bacterial species in June, August and September varied (Fig. 2). The most common species in June was Klebsiella pneumonia (4 / 11, 36.36%), while in August P. otitidis (11 / 25, 44%) and P. aeruginosa (6 / 25, 24%) dominated. In September, K. pneumoniae, P. otitidis and P. aeruginosa were not detected and the isolates mostly belonged to species from the Enterobacteriaceae family (E. coli, M. morganii and Enterobacter sp.) (Fig. 2).
Teo et al. (2012)
3.2. Phenotypic and genotypic analysis of ESBL and MBL producers
Teo et al. (2012)
Phenotypic confirmation tests for detecting ESBL producers, done by the disc diffusion method, revealed 7 (10.14%) ESBL-producing isolates. Four of the ESBL-producing isolates were from the Ada Ciganlija Lake III site, of which three were sampled in August (one isolate of K. pneumoniae and two isolates of S. maltophilia), while the fourth was isolated in September (M. morganii). Three of the ESBLproducing candidates were isolated from rivers, two from the Sava River (C. aquatica isolates) and one from the Danube River at Dorcol (P. stuartii). These 7 ESBL-producing candidates were further analyzed by the PCR method with primers specific for TEM, SHV, CTX-M, GES-7, DHA-1 and for AmpC-type beta-lactamases CMY-1 and CMY-2 genes. The genetic basis of beta-lactamase production was determined for five of the seven isolates. Among those are all four phenotypically selected ESBL producers from Ada Ciganlija Lake III. The SHV gene was found in K. pneumonia and two S. maltophilia (isolated in August), while the presence of DHA-1 gene was determined for M. morganii
Teo et al. (2012) Teo et al. (2012) Teo et al. (2012) Teo et al. (2012) Ellington et (2007) Ellington et (2007) Ellington et (2007) Ellington et (2007) Ellington et (2007)
al. al. al. al. al.
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Fig. 1. Prevalence of different beta-lactam resistant species isolated from rivers, lake and springheads in Belgrade, Serbia.
(isolated in September). The fifth ESBL-producing candidate was P. stuartii, isolated from the Danube River, for which we confirmed that carries CMY-2 gene, but neither one of the analyzed ESBL genes was found. The molecular basis of ESBL production was not determined for C. aquatica isolated from the Sava River. Phenotypic tests revealed a total of 39 (56.52%) isolates resistant to imipenem, of which 32 were positive for MBL production. These isolates were predominantly selected from Ada Ciganlija Lake (n = 20), of which 8 were isolated in June, 9 in August and 3 in September. In the June isolates the same number of candidates was isolated from Ada Ciganlija Lake sites I and II (3 isolates) and 2 were selected from Ada Ciganlija Lake III, while in August 4 of the isolates were from Ada Ciganlija Lake I, 2 from Ada Ciganlija Lake II and 3 from the Ada Ciganlija Lake III sampling site. Out of 3 MBL-positive bacteria isolated in September, 1 was from Ada Ciganlija Lake I and 2 were from the Ada Ciganlija Lake II site. Twelve MBL-producing isolates
Fig. 2. Number and distribution of beta-lactam resistant isolates before (June), during (August) and after (September) the swimming season at Lake Ada Ciganlija in Belgrade, Serbia.
were selected from samples taken from rivers: 2 from the Danube River at Ada Huja, 6 from the Sava River and 4 from the Topcider River. Molecular analyses failed to detect the genetic basis of MBL production when primers specific for NDM-1, SPM-1, IMP, VIM-2, SIM and GIM genes were used. 3.3. Antimicrobial susceptibility profiles Antimicrobial susceptibility profiles were done for all 7 ESBLproducing candidates obtained by phenotypic confirmation tests. The selected isolates showed variable antibiotic resistance patterns (Table 2). All strains except two S. maltophilia isolates and M. morganii were sensitive to piperacillin in combination with the beta-lactamase inhibitor tazobactam. 4. Discussion The enzyme NDM-1 was designated as a global threat considering its rapid worldwide dissemination and association with other determinants of resistance to antibiotics. Not long after the first European cases were described, the Balkan countries (including Serbia) were highlighted, besides the Indian subcontinent, as an NDM-1 endemic region (Struelens et al., 2010). Monitoring of public water supplies in India revealed significant environmental contamination with NDM-1producing strains (Walsh et al., 2011). The results of a survey performed by Walsh et al. (2011) were striking, since blaNDM-1 was detected in 4% of drinking water samples and 30% of seepage samples. This survey clearly indicated the environment as a source of NDM-1producing strains in India. Although it was shown for India that NDM1-producing strains were abundant in the environment, this type of study was never performed in Serbia, a region designated as endemic. This is of importance because Struelens et al. (2010) stated that of 55 European patients (until 2010), 31 had visited the Indian subcontinent and five had visited Balkan countries, leading to the conclusion of endemicity in the Balkans. Endemicity of blaNDM-1 in the Balkans has been the subject of many discussions, but without environmental data all were just hypothetical. One of the hypotheses that had been proposed by
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Table 2 IC50 of ESBL producing isolates from Belgrade environmental waters, for selected antibiotics. Isolate
Isolation site
Type of ESBL
Piperacillin
Piperacillin/tazobactam
Ceftazidime
Aztreonam
K. pneumoniae AAIII-1 S. maltophilia AAIII-2 S. maltophilia AAIII-3 P. stuartii DD3 M. morganii ASIII-2 C. aquatica RS3 C. aquatica RS8
Ada Ciganlija Lake III Ada Ciganlija Lake III Ada Ciganlija Lake III The Danube River - Dorcol Ada Ciganlija Lake III The Sava River The Sava River
SHV SHV SHV CMY-2 DHA-1 ND ND
14.88 ± 0.28 24.69 ± 1.04 N256 60.16 ± 0.64 16 ± 3.14 47.15 ± 1.93 24.53 ± 0.56
b4 12.16 ± 2.26 28.56 ± 4.86 b4 16 ± 2.26 b4 b4
b4 40.96 ± 4.07 35.68 ± 0.68 98.24 ± 25.8 38.34 ± 9.12 17.44 ± 0.68 90.24 ± 0.45
26.13 ± 3.42 186.88 ± 45.45 44.96 ± 0.48 b4 56.32 ± 2.36 48.1 ± 0.98 34.4 ± 1.13
ND — not determined.
Liveromore et al. (2011) to explain a Balkan association for blaNDM-1 was that the blaNDM-1 gene was recruited to plasmids from an external origin, probably an environmental organism without clinical relevance. A similar phenomenon was previously reported for the blaCTX-M genes, which code for cephalosporin-hydrolyzing extended-spectrum betalactamases that have escaped repeatedly from Kluyvera spp. (Woodford et al., 2009). Alternatively, Liveromore et al. (2011) hypothesized that the NDM-1 enzyme, which was already circulating in India, could have been imported from the subcontinent to the Balkans. The second hypothesis was also highlighted by Johnson and Woodford (2013) and they supported it by means of documenting the travel of patients from the Balkans to Pakistan for commercial kidney transplants that were associated with frequent infections. These types of findings lead to the assumption that such medical tourism could have introduced NDM to the Balkans. Having in mind that we did not detect any NDM-1-producing strains in the environmental waters of Belgrade area we could speculate that Serbian NDM-1 in fact is a transplant and that Serbia is not an endemic region for NDM-1. ESBL-producing strains, which are recognized as a threat by clinicians, were also one of the focal points of our study. The number of studies investigating potential environmental reservoirs of ESBL-producing strains is still very limited (Dhanji et al., 2010; Girlich et al., 2011; Tacao et al., 2012; Zurfluh et al., 2013). In addition, data from Balkan Peninsula rivers are unavailable. These studies are of great importance since rivers and lakes are considered putative reservoirs of resistant bacteria, and because they represent collectors of surface waters containing materials from different origins that could be contaminated. Having that in mind, we have done an extensive study of the presence and prevalence of ESBL and AmpC-type beta-lactamase producing bacteria in the surface waters of Belgrade. Among the results we would like to emphasize that SHV-producing bacteria were found only in Ada Ciganlija Lake, the most popular swimming recreation lake, in August when there is the largest number of visitors. Since we didn't find SHV-producing strains in Ada Ciganlija Lake in June or September we could speculate that these strains were only transient in this lake and that their source is anthropogenic. Our finding of SHVproducing K. pneumoniae is in correlation with previously published results (Babini and Livermore, 2000), however finding SHV-producing S. maltophilia is striking, since this bacteria is considered an emerging nosocomial pathogen. Additionally, as phenotypic detection of ESBLs in non-fermenters is complicated, ESBLs are often underestimated and underreported in these strains and therefore S. maltophilia may be a hidden reservoir for such ESBLs (al Naiemi et al., 2006). The presence of CMY-2-producing P. stuartii in the Danube River at Dorcol is also of importance, since P. stuartii is an opportunistic pathogen which is essentially responsible for urinary tract infections (Tumbarello et al., 2004). Besides ESBL production, we analyzed the presence of MBL producing bacteria (other than NDM-1) as well. Our results revealed the low specificity and positive predictive value of conventional phenotypic methods for detecting MBL producers among non-imipenemsusceptible strains. However, this phenomenon has previously been described for P. aeruginosa strains (Samuelsen et al., 2008).
4.1. Conclusions New Delhi metallo-beta-lactamase-producing strains weren't present in the analyzed environmental waters, which strongly reinforces the need for a revision of the designation of NDM-1 endemic regions. However, beta-lactam resistant Gram-negative bacteria are present in the environment and anthropogenic factors seem to have a crucial role in the dissemination of resistance to antibiotics in Belgrade, Serbia. This assumption is supported by several findings: (i) the highest number of beta-lactam resistant Gram-negative bacteria was collected from Ada Ciganlija Lake which is, if we compare it to other analyzed sites, most exposed to anthropogenic influence, (ii) out of 42 betalactam resistant isolates from Ada Ciganlija Lake 25 were collected in August, when the highest number of visitors and swimmers are present at the site, and (iii) out of 7 phenotypically confirmed ESBL producing strains, 4 were from Ada Ciganlija Lake, of which 3 were collected in August. Therefore, this study emphasizes the importance of cohesive research related to the spread of resistance to antibiotics which must include both clinical and environmental data in order to draw the final conclusions. Furthermore, this study reinforces the importance of continued study and monitoring of antimicrobial resistance to combat the growing infections caused by ESBL- and MBL-producing bacteria.
Acknowledgments This work was supported by grant no. 173019 from the Ministry of Education, Science and Technological Development of the Republic of Serbia.
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