Epidemic Escherichia coli ST131 and Enterococcus faecium ST17 in

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Epidemic Escherichia coli ST131 and Enterococcus faecium ST17 in Coastal Marine Sediments from an Italian Beach C. Vignaroli,*,† G. M. Luna,‡ S. Pasquaroli,† A. Di Cesare,† R. Petruzzella,† P. Paroncini,† and F. Biavasco† †

Department of Life and Environmental Sciences, Polytechnic University of Marche, via Brecce Bianche, 60131 Ancona, Italy Institute of Marine Sciences−National Research Council (ISMAR−CNR), Castello 2737/f −Arsenale Tesa 104, 30122 Venezia, Italy

‡

S Supporting Information *

ABSTRACT: Fecal indicator bacteria (FIB) are used worldwide to assess water quality in coastal environments, but little is known about their genetic diversity and pathogenicity. This study examines the prevalence, antimicrobial resistance, virulence, and genetic diversity of FIB isolated from marine sediments from a central Adriatic seaside resort. FIB, recovered from 6 out of 7 sites, were significantly more abundant at sampling stations 300 m offshore than close to the shore. Escherichia coli accounted for 34.5% of fecal coliforms, and Enterococcus faecalis accounted for 32% of enterococci. Most isolates (27% of E. coli and 22% of enterococci) were recovered from the sediments that had the highest organic content. Multidrug-resistant E. coli (31%) and enterococci (22%) were found at nearly all sites, whereas 34.5% of E. coli and 28% of enterococci harboring multiple virulence factors were recovered from just two sites. Pulsed-field gel electrophoresis typing showed wide genetic diversity among isolates. Human epidemic clones (E. coli ST131 and Enterococcus faecium ST17) were identified for the first time by multilocus sequence typing in an area where bathing had not been prohibited. These clones were from sites far removed from riverine inputs, suggesting a wide diffusion of pathogenic FIB in the coastal environment and a high public health risk.

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INTRODUCTION The main sources of microbiological contamination of coastal marine waters, which have the potential to affect water quality and cause swimming bans, are riverine waters, terrestrial runoff, the residues of animal farming, and wastewater effluents.1,2 According to European Directive 2006/7/EC,3 which regulates the matter, the quality of coastal water, including recreational beach water, is monitored by analyzing water samples for Escherichia coli and enterococci, whose abundance in the gastrointestinal tract of humans and animals has led to their widespread use as fecal indicator bacteria (FIB).3,4 Recent studies have demonstrated that freshwater and marine sediments can be major reservoirs of FIB,5−8 including virulent and multidrug-resistant (MDR) strains, which can become naturalized to secondary habitats.2,9,10 Several studies have shown that sediment resuspension can give rise to elevated FIB concentrations in overlying seawater even more than runoff from surrounding land;11 nonetheless, regulatory standards for recreational coastal water quality have failed to adopt FIB evaluation in sediment both in Europe3 and in the United States.4 Investigations conducted during the last decades suggest that FIB can survive, or even regrow, in coastal marine sediments (ref 2 and references therein). Survival in such habitats depends on a variety of environmental factors, including biotic © 2013 American Chemical Society

interactions with benthic organisms, physical-chemical conditions, and availability of organic matter.5,12,13 The acquisition of virulence traits and antimicrobial resistance genes can also transform these bacteria into pathogens highly adapted to humans and animals.14−16 Phylogenetic analysis of pathogenic E. coli strains has demonstrated a correlation between strain phylogroup and the type of infection. Strains of phylogroups A and B1 are generally responsible for intestinal diseases, whereas B2 and D strains are associated with extraintestinal infections such as meningitis, bacteremia, and urinary tract infections.17,18 Enterococci are recognized as opportunistic pathogens and are responsible for nosocomial infections in patients with underlying disease; the species most frequently involved are Enterococcus faecalis and Enterococcus faecium.15,16,19 Acquired virulence factors increase the pathogenicity of both E. coli and enterococci by enhancing host tissue colonization and invasion as well as evasion of host immune responses. Treating bacterial infections is becoming increasingly difficult20,21 because of the spread of antimicrobial resistance, which contributes to the pathogenic potential of micro-organisms and to the evolution Received: Revised: Accepted: Published: 13772

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Figure 1. Study area and sampling stations. The satellite image was acquired from Google Maps (http://maps.google.it/; image provider: Immagini ©2013 Cnes/Spot Image, DigitalGlobe, European Space Imaging).

N, longitude 13°38.908′ E to latitude 43°23.367′ N, longitude 13°41.340′ E. The sites were selected to represent various gradients of putative microbiological contamination in relation to the town and the river mouths. The sea was calm at the time of sampling, and the weather had been good on the days prior to sampling. Each site included two sampling stations, one close to the shore (30−50 m, depth 2 m) and the other further offshore (maximum 300 m, depth 5 m), designated SHO and OFF, respectively. According to data from Marche’s environmental protection agency (http://www.arpa.marche.it/), which collected water samples on the same day as we did, all seven sampling sites lie in areas where water quality complied with bacterial water standards;3 nonetheless, the water quality at sites 14 and 15 (about 500 m north and 500 m south of the estuary of the river Potenza), respectively, was lower than at the other sites. Superficial sediment samples were collected at each station using a Van Veen grab sampler (30 L capacity) in three replicates, as previously described.13 The main environmental parameters of the water column (i.e., temperature, salinity, oxygen content, and pH), which are likely to exert an influence on bacterial assemblages in the superficial sediment layers (the upper 0−2 cm) investigated in this study, were measured as described in a previous study.13 For culture-based analyses, sediment samples were first mixed, and three aliquots (ca. 20 g) per station, each obtained from a different grab deployment, were also homogenized by mixing, kept at 5−10 °C, and analyzed within 6 h of sampling. Chloroplastic pigment equivalents (the sum of chlorophyll a and phaeopigments) and biopolymeric organic carbon, selected as indices of available organic resources for bacterial metabolism,29 were analyzed as described.29 For these biochemical tests, sediment samples were homogenized by mixing and stored at −20 °C until analysis (performed within 2 weeks of sampling).

of MDR strains unresponsive to antibacterial drugs, particularly in hospital settings. Besides being indices of fecal pollution, virulent/MDR FIB found in marine environments may thus also pose a direct threat to human health.1,22,23 Despite mounting evidence of the occurrence and distribution of E. coli and enterococci in coastal marine waters, usually with higher concentrations in sediments than in the overlaying water column,7,41 few studies have characterized pathogenic FIB to date,24,25 and information on the virulence and antibiotic resistance properties or the genetic diversity of FIB isolates from marine environments, particularly sediment, is still limited.26,27 We have previously reported the presence of virulent and antimicrobial-resistant E. coli in coastal marine sediments from the central Adriatic Sea13,28 and of MDR enterococci in sediment from a mariculture plant in southern Italy.8 The present study was devised to explore whether marine sediments at a recreational beach harbored FIB that could represent a risk for bathers. FIB frequency and distribution as well as the physical-chemical and trophic characteristics of sediment were determined to gain insights into the potential impact of contaminated sediments on water quality and human health. FIB isolates were also characterized for their virulence and antimicrobial resistance properties, genetic diversity, and relationship with known pathogenic clones.

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MATERIALS AND METHODS Site Description and Sediment Sampling. The study was carried out in July 2009 at an Adriatic beach resort lying between the estuaries of two small rivers (Musone and Potenza) that carry a significant load of organic nutrients and pollutants from industrial and farming activities (Figure 1). Seven sampling sites, lying 12−64 km north of those analyzed in a previous study by our group,13 were selected along a 9 km stretch of beach in front of the town, from latitude 43°27.785′ 13773

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Isolation, Quantification, and Identification of FIB. Culturable FIB were isolated from sediment samples, as described in previous papers,8,13 and quantified using membrane filtration. Triplicate 5 g aliquots of wet sediment were suspended in 20 mL of sterile seawater, shaken, and sonicated in an ice bath.13 Aliquots (1 mL) of undiluted and 10-fold serial dilutions of the resulting seawater were filtered (pore size 0.22 μm), and the filters were placed on selective agar plates. Fecal Coliform (FC) (BBL, Becton Dickinson & Co., Sparks, MD) and Slanetz Bartley (SB) (Oxoid, Basingstoke, UK) agar plates were used for enumeration of presumptive fecal coliforms and enterococci, respectively; mENDO (BBL, Becton Dickinson) agar plates were used to quantify total coliforms. FC agar plates were incubated at 44.5 °C, and SB and mENDO agar plates, at 37 °C for 24−48 h. Counts were expressed as colony forming units (CFU) per gram of dry sediment. Colonies grown on FC agar were first streaked on MacConkey agar (Oxoid) and then identified using the API 20E system (BioMérieux, Marcy l′Etoile, France). E. coli phylogenetic groups were determined by multiplex PCR, as described by Clermont et al.30 Presumptive enterococci (i.e., colonies selected from SB agar and grown in 6.5% NaCl at 42 °C) were screened by PCR to determine at first whether they belong to the genus Enterococcus and then for identification on the species level.8,31 Enterococcus mundtii and Enterococcus durans were identified by amplification of 16S−23S rDNA intergenic regions (ITS-PCR) as previously described for E. coli.13 E. faecium BM4147, E. faecalis ATCC51299, E. casseliflavus ATCC14432, E. gallinarum AIB39, E. durans PF1 V, and E. mundtii CM1T (previously described31,32) were used as control strains. Antimicrobial Susceptibility of FIB. Susceptibility of enterococci to tetracycline (30 μg), vancomycin (30 μg), erythromycin (15 μg), gentamicin (120 μg), synercid (15 μg), linezolid (30 μg), chloramphenicol (30 μg), and ciprofloxacin (5 μg) and susceptibility of E. coli isolates to ampicillin (10 μg), cefotaxime (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), tetracycline (30 μg), chloramphenicol (30 μg), nalidixic acid (30 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), and streptomycin (10 μg) was assessed by the disk-diffusion method and broth microdilution test according to CLSI recommendations for testing Enterobacteriaceae and Enterococcus spp.33 E. coli strains resistant to β-lactams were also evaluated for extended spectrum β-lactamase (ESBL) production using CLSI screening and confirmatory tests.33 Antibiotic disks were purchased from Oxoid and BBL, Becton Dickinson, and antibiotic powders, from Sigma-Aldrich (Saint Louis, MO). Phenotypic Characteristics of Enterococci. Gelatinase and hemolysis production by Enterococcus strains were assessed in gelatin infusion broth containing 40 mg mL−1 of gelatin (Bio-Rad Laboratories, Richmond, CA) and in blood agar base (Oxoid) supplemented with fresh horse blood (5%), respectively. The biofilm production test was performed as described by Elhadidy and Elsayyad34 with some modifications. Briefly, bacterial cells were grown overnight in tryptic soy broth (TSB) (Oxoid) supplemented with 1% glucose (TSBG) at 37 °C and diluted to an OD650 of 0.1. 96-well polystyrene microtiter plates (Falcon, Becton Dickinson Labware) were inoculated with 0.2 mL of these suspensions, incubated at 37 °C, washed with PBS, dried at 60 °C for 1 h, and stained with 0.2 mL of Hucker’s Crystal Violet (CV) solution (10% crystal violet in 20% ethanol

containing 1% ammonium oxalate) for 10 min. CV was aspirated, and wells were washed with sterile water. CV was extracted from adhering bacterial cells by ethyl alcohol/acetone (80:20 v/v), and the OD690 was measured using a microplate reader (Thermo Electron Corporation, Madison, WI). Each assay was performed in triplicate. Strains were classified as nonproducer (OD ≤ ODc), weak producer (ODc < OD ≤ 4 × ODc), or strong producer (OD > 4 × ODc).35 The OD cutoff (ODc) was defined as 3 standard deviations above the mean OD of the negative control represented by uninoculated wells containing TSBG.35 E. faecalis ATCC 29212, a strong biofilm producer, was the positive control. PCR Detection of Virulence and Antibiotic Resistance Genes. E. coli isolates were tested for the virulence genes found more often in intestinal and/or extraintestinal pathogenic E. coli, which encode toxins (stx1, stx2, EAST1, and hlyA), an adhesin (papG), an intimin (eaeA), iron acquisition systems ( f yuA and iroN), and a transmembrane protein involved in neonatal meningitis (ibeA), using previously described PCR protocols.13,28 Enterococci were analyzed for four Enterococcusassociated virulence determinants, esp, efa, cylB, and gelE, encoding the enterococcal surface protein, E. faecalis antigen A, a cytolysin, and a gelatinase, respectively. Primers and PCR protocols were those described by Biavasco et al.36 and Moraes et al.37 Genes associated with resistance to ampicillin (blaTEM, blaSHV, and blaCTX‑M), tetracycline (tet(A), tet(B), tet(C), tet(D), tet(E), and tet(G)), thrimethoprim/sulfamethoxazole (df rA1), sulfonamides (sul1, sul2, and sul3), and streptomycin (strA, strB, aadA1, and ant(3″)) in E. coli isolates were sought by PCR as described earlier.28 Tetracycline resistance genes tet(M), tet(L), tet(O), and tet(K), erythromycin resistance genes erm(A), erm(B), mef, and msr, and the gentamicin resistance gene aac(6′)-Ie-aph(2″)-Ia in enterococci were sought as previously described.8,32 FIB Typing by Pulsed-Field Gel Electrophoresis (PFGE). Genomic DNA was extracted after embedding a standardized bacterial suspension (in 10 mM Tris-HCl pH 8.0, 1 M NaCl, 10 mM EDTA, pH 8.0) in low-melting 1.6% agarose, according to standard protocols (ref 38 and http:// www.pulsenetinternational.org). Restriction digestion of DNA in agarose plugs was performed using 30 U of SmaI or 50 U of XbaI (both from Fermentas Life Sciences, M-Medical, Cornaredo, Italy) for enterococcal and E. coli strains, respectively. DNA fragments were separated using a CHEFMAPPER apparatus (Bio-Rad) with the pulse time increasing from 1 to 20 s (Enterococcus spp.) or from 5 to 50 s (E. coli) for 20 h at 14 °C and 200 V (6 V/cm). Low Range PFG Marker (New England BioLabs, Ipswich, MA) was used as a molecular weight marker. PFGE pattern similarity was determined as previously described.36 Multilocus Sequence Typing (MLST). MDR strains (four E. coli, two E. faecium, and two E. faecalis) were subjected to MLST according to the guidelines of the relevant MLST Web sites (http://www.mlst.net and http://mlst.ucc.ie) as described in previous studies.28,36 Statistical Analysis. Sediments were assessed for the abundance of total coliforms, fecal coliforms and enterococci using one-way analysis of variance (ANOVA). Differences were considered significant at P values 102 CFU g−1), where a lower FIB concentration was documented in the overlying water, likely because of the absence of resuspension phenomena on the day preceding sampling or because of sunlight inactivation. Site #15-OFF provided the majority of isolates (27% of E. coli and 22% of enterococci); this is probably related to a higher content in organic substrates compared with the other stations because of its proximity to tourist facilities, the estuary of the river Potenza, which is impacted by urban and agricultural activities, and possibly to marine currents favoring the settling out of organic matter and FIB. A considerable fraction of enterococci (28%) and a smaller percentage of E. coli (10%) isolates were also recovered from station #16-OFF even though the environmental parameters there did not differ from those of the other stations. A greater concentration of FIB in coastal sediments than in the overlying waters has already been described,39,41 suggesting that besides being a reservoir of FIB, or even because of this, sediments may also provide a more stable indicator of long-term fecal

Figure 5. Frequency (%) of E. coli and Enterococcus isolates carrying the selected resistance (A) and virulence (B, C) genes.

Genetic Diversity of FIB. E. coli and Enterococcus isolates were subjected to PFGE to assess their genetic diversity and to MLST to identify known pathogenic clones. The similarities among E. coli isolates are illustrated in Figure S2 (Supporting Information), and those among Enterococcus isolates (E. faecalis, E. faecium, E. casseliflavus, and E. mundtii), in Figure S3 (Supporting Information). Both sets of isolates were characterized by marked genetic diversity, with no prevalence of any clones or relatedness among isolates from the same site/station. However, despite being recovered from different sites and stations, most (73%) E. coli strains of phylogroup A formed a cluster characterized by 83−95% similarity (Figure S2). Similarity among enterococci ranged from 50 to 85% for E. faecalis and E. faecium, from 40 to 70% for E. casseliflavus, and from 20 to 70% for E. mundtii (Figure S3). Eight MDR and/or multivirulent isolates (four E. coli and four enterococci) were tested for ST determination. All four E. coli strains (14E2, 13777

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isolates.54,55 Both isolates were also resistant to gentamicin and showed β-hemolysis activity, and one (ENT15E25) carried aac(6′)Ie-aph(2″)Ia and cylB. The clone was recovered at site #15, the nearest to the estuary of river Potenza, suggesting contamination of coastal water and sediments with animal feces through river inputs. The present study documents virulent epidemic clones of FIB in marine sediments from an area where the overlying seawater complied with regulatory standards for recreational coastal water quality. The clones were recovered from sites not affected by riverine inputs, suggesting a very wide spread of pathogenic genotypes in the coastal marine environment. The presence of a FIB reservoir in marine coastal sediments compared to overlaying water involves an increased risk of exposure to pathogenic bacteria after possible resuspension phenomena induced by recreational activities (e.g., bathing) or natural events (e.g., currents and tidal or wind-driven waves).1,11 In the study area, large amounts of sediment can be resuspended by bottom fishing, for example, hydraulic dredging to harvest the bivalve Chamelea gallina.56 These data not only reinforce previous evidence13,28 suggesting the need for screening marine sediments for FIB, especially during the bathing season and in potentially contaminated areas, but also highlight the importance of identifying epidemic clones associated with greater public health risks when evaluating the microbiological safety of recreational environments. Further research, including epidemiological and numerical modeling studies, on the impact of resuspension of benthic pathogens on water quality at bathing beaches and on the main exposure routes would be essential to assess the real risk for human health.

contamination. Little information is available about the enterococcal species recovered most frequently from marine sediments.26,41 In the present work, E. faecalis was the predominant species followed by E. casseliflavus, E. faecium, and E. mundtii. These species have already been described in seawater and sediment,26,41,42 in human and animals feces,2,15 and in food of animal origin;15,43 E. faecalis has also been described in the feces of wildlife and marine mammals and seagulls.44However, no single Enterococcus species is a reliable indicator of the host fecal source, and their origin is difficult to establish. The esp protein plays an important role in the pathogenicity of enterococci, being involved in host tissue adhesion and biofilm formation.15,16 Because it is detected more frequently in clinical isolates than in environmental or animal strains, it has been suggested for use as a marker of human fecal contamination.45,46 However, in our isolates, the presence of the esp gene did not correlate with strong biofilm production nor did it help to assess the source of contamination, as only 4% of isolates were positive for it. Whereas MDR E. coli and enterococci (31% and 18%, respectively) were recovered from six of the seven sites sampled, isolates harboring multiple virulence factors (34.5% and 28%, respectively) were found, especially at sites #15 and #16. Multidrug resistance was predominantly observed among group A E. coli and among E. faecium isolates, whereas multivirulence was mostly associated with E. coli group B2 and with E. faecalis. These findings agree with previous data from studies of marine sediment.8,28 PFGE typing of the 29 E. coli and 50 enterococcal strains documented a broad genetic diversity and the absence of predominant clones either within or among sites/stations. MLST evidenced epidemic and widely disseminated clones of both E. coli (ST131) and E. faecium (ST17 and ST18) at different sites and depths. E. coli ST131, first identified in 2008, is a worldwide pandemic clone causing predominantly community-onset urinary tract infections. It has been described as an antimicrobial-resistant, virulent, CTX-M family βlactamase-producing strain isolated from human infection in multiple countries in Europe, North America, and Asia47 as well as from farm and wild animals and from food.48−50 The present study is the first report of its detection in marine sediments. Our ST131 isolate (E. coli 14E2) carries the ibeA gene and shows multidrug resistance (to β-lactams, tetracycline, and nalidixic acid); it is also a CTX-M ESBL-producing strain, as was reported for ST131 isolates from humans and poultry.49 The E. faecium genetic lineages ST17 and ST18, both belonging to CC17, are the clonal complex associated with the majority of hospital outbreaks and clinical infections and are also isolated from food and wastewater.51,52 Interestingly, although most strains were recovered from sites #15 and #16, such epidemic STs were isolated from sites #13, #14, and #17, which are not affected by river inputs, suggesting a different source of contamination for these sediments. For instance, E. coli ST131 has recently been isolated from seagull feces collected at two beaches in Portugal50 and E. faecium CC17 from wild animals.53 Our findings firmly establish marine sediment as a further reservoir of pathogenic strains and epidemic clones and suggest that it may well be involved in their dissemination in multiple countries and in different settings via contaminated waters, seafood, and wild birds. Two E. faecalis isolates (ENT15E25 and ENT15i31) were assigned to ST16, a gentamicin-resistant clone recently isolated from human infections and closely related to poultry and swine

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ASSOCIATED CONTENT

* Supporting Information S

Proportion of E. coli and enterococci isolates resistant to each antimicrobial agent tested; similarity, phylogroup allocation (for E. coli), and phenotypic traits of E. coli and Enterococcus spp. isolates analyzed in the study; and environmental and trophic variables of sediment samples from the 14 stations. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39 71 220 4638. Fax: +39 71 220 4638. E-mail: c. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Antonio Pusceddu and Dr. Silvia Bianchelli (Polytechnic University of Marche) for providing sediment samples and data on the environmental and trophic variables.

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REFERENCES

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