Curr Microbiol DOI 10.1007/s00284-014-0587-7

Toxin Production and Antibiotic Resistances in Escherichia coli Isolated from Bathing Areas Along the Coastline of the Oslo Fjord Colin Charnock • Anne-Lise Nordlie Bjarne Hjeltnes



Received: 27 September 2013 / Accepted: 1 March 2014 Ó Springer Science+Business Media New York 2014

Abstract The presence of enterovirulent and/or antibiotic resistant strains of Escherichia coli in recreational bathing waters would represent a clear health issue. In total, 144 E. coli isolated from 26 beaches along the inner Oslo fjord were examined for virulence determinants and resistance to clinically important antibiotics. No isolates possessed the genetic determinants associated with enterotoxigenic strains and none showed the prototypic sorbitol negative, O157:H7 phenotype. A small number (*1 %) produced alpha-hemolysin. Occurrences and patterns of antibiotic resistances were similar to those of E. coli isolated previously from environmental samples. In total, 6 % of the strains showed one or more clinically relevant resistances and 1.4 % were multi-drug resistant. Microarray analyses suggested that the resistance determinants were generally associated with mobile genetic elements. Resistant strains were not clonally related, and were, furthermore not concentrated at one or a few beach sites. This suggests that these strains are entering the waters at a low rate but in a widespread manner. The study demonstrates that resistant E. coli are present in coastal bathing waters where they can come into contact with bathers, and that the resistance determinants are potentially transferable. Some of the resistances registered in the study are to important antibiotics used in human medicine such as fluoroquinolones. The spread of antibiotic resistant genes, from the clinical C. Charnock (&)  A.-L. Nordlie  B. Hjeltnes Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, St. Olavs Plass, Oslo, Norway e-mail: [email protected] A.-L. Nordlie e-mail: [email protected] B. Hjeltnes e-mail: [email protected]

setting to the environment, has clear implications with respect to the current management of bacterial infections and the long term value of antimicrobial therapy. The present study is the first of its kind in Norway.

Introduction The presence of Escherichia coli is the most widely used indicator of fecal contamination of drinking and recreational waters. The current EU directive on bathing water quality [14] uses this indicator as follows: if, in the set of bathing water quality data for the last assessment period, the percentile values for microbiological enumerations are equal to or better than 250 cfu E. coli/100 ml, coastal bathing waters are considered excellent with regard to this parameter. However, some E. coli are themselves pathogenic through a variety of mechanisms (e.g., toxin production) [21, 26, 28]. Furthermore, E. coli are probably the Gram-negative bacteria most commonly found in connection with infections in humans. E. coli that can cause gastro-intestinal disease can be transmitted through contaminated food and water. These include Shiga toxin-producing E. coli (STEC) and Enterotoxigeneic E. coli (ETEC). Enterohaemorrhagic E. coli (EHEC) constitutes a subset of serotypes of STEC that has been firmly associated with bloody diarrhea and hemolytic uraemic syndrome (HUS) in industrialised countries [17]. Reported rates of STEC/EHEC infections in Europe are increasing steadily. The prototypic EHEC bacterium E. coli O157:H7 is the most prevalent EHEC isolate. In the identification of this pathogen, phenotypic features such as its lack of sorbitol fermentation are often used to differentiate it from other E. coli [16 and references therein] Since 2010 almost half of the reported cases of infection were related to serogroup

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O157 [12]. There are also several recorded incidences of infection with serotype O157:H7 implicating contaminated bathing waters [32] and references therein. However, of particular public health interest was the recent disease outbreak in Germany traceable to serotype O104:H4 [12]. ETEC is the name given to a group of E. coli that can produce heat-stable (ST) and/or heat-labile (LT) toxins which stimulate the lining of the intestines, causing them to secrete excessive fluid producing diarrhea. The toxins and the diseases produced by ETEC are not related to E. coli O157:H7. It is likely that the burden of antibiotic-resistant bacteria will soon shift toward an increasing prevalence of antibiotic-resistant Gram-negative bacteria [11]. Resistance to antimicrobial agents by members of the Enterobacteriaceae (including E. coli) is of growing concern. Most notable is resistance to the newer, third-generation cephalosporins that have been associated with treatment failures both in hospital and community-acquired infections [11, 31]. The average proportion of the third-generation cephalosporin-resistant isolates among E. coli is rising steadily in the European Union, Iceland, and Norway and reached 8 % in 2007 [11]. There are four major functional groups of beta-lactamases [8]: penicillinases, extended-spectrum b-lactamases (ESBLs), carbapenemases, and AmpC-type cephalosporinases [30]. ESBLs are a widespread problem in a range of species including E. coli [1, 30]. The spread of antibiotic resistant microorganisms is widely recognized, but data about their sources, presence, and significance in marine environments (e.g., coastal bathing waters) are limited. A study of enterovirulent E. coli isolated from Adriatic marine sediments showed that 35 % were resistant to a single antibiotic and 14 % were resistant to 3 or more agents. Resistances to tetracycline, ampicillin, and trimethoprim-sulphamethoxazole were the most common. Class 1 or 2 integrons were also detected in the majority of multi-resistant strains [39]. Similar results were found for public beach waters in Quebec [38]. In the period covered, 28–38 % of beaches sampled showed the presence of resistant E. coli, with tetracycline, ampicillin, and sulphamethoxazole being the most common resistances. Neither study reports the presence of ESBLs. The referenced studies suggest that marine coastal areas are suitable environments for the survival of enterovirulent and antimicrobial-resistant E. coli potentially capable of contributing to resistance spread. A detailed discussion of antibiotic resistance genes and their mobile genetic elements is beyond the scope of the present work. The reader is referred to the recent review by Zhang et al. [41] which deals comprehensively with antibiotic resistance mechanisms and especially those associated with aquatic environments. No consensus has yet been reached on the definition and use of terms such as

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‘‘multidrug-resistant’’ (MDR) microbes. Recently, a panel of international experts has proposed standardized definitions to be used by laboratories [25]. These include specific recommendations for the Enterobacteriaceae. In brief, MDR would be used for strains showing resistance or borderline resistance to one or more agents from 3 or more of the named antimicrobial categories. This system is adopted below. The goal of the present study is to investigate the prevalence of enterotoxigenic and/or resistant E. coli in bathing waters along the Oslo fjord. It seeks to provide an indication of the spread of clinically important E. coli into an environment where they come into close contact with humans. The presence of antibiotic resistance determinants in coastal waters might contribute to the spread and evolution of antimicrobial resistances, especially if the genes are carried on mobile genetic elements. Antimicrobial resistant bacteria ingested during bathing activities could potentially transfer their resistance determinants to the intestinal flora increasing the potential for further dissemination. Such an investigation has to our knowledge not previously been undertaken in Norway. Tools used include microarray analysis to identify resistance determinants. The data are compared with published values for E. coli from humans, animals, and marine environments in Norway and abroad.

Materials and Methods Locations/Sampling Samples were taken at 26 beach locations on both sides of the Oslo fjord in the period April 16th–June 20th, 2012. Samples were taken aseptically 1–2 m from the shore by sinking sterile 2L bottles beneath the surface. Bottles were kept cool in a refrigerator bag prior to analysis. Analyses were generally performed the same day and within a maximum of 24 h. Samples (100 and 500 ml) were filtered through Millipore HA filters (0.45 lm) and the filters were placed onto MacConkey agar without salt (LAB M, Heywood, UK). In addition, 1 ml sample was plated directly. Thus, a total volume of 601 ml was analyzed. Plates were allowed to stand for a recovery period of 1 h at room temperature before incubation at 44 ± 1 °C for 18–24 h. Dark pink/red colonies were counted and all (or at least 10 if C10 colonies) were inoculated (after subculture where necessary) onto CHROMagar orientationTM (BD, USA) and incubated at 37 ± 1 °C for 18–24 h. Dark pink/red colonies were recorded as presumptive E. coli. Confirmation of colonies as E. coli was based on the following combination of results: a positive indole test ? purple coloration on BRILLIANCE E. coli/coliform agar (Oxoid,

C. Charnock et al.: Toxin Production and Antibiotic Resistances in Escherichia coli

Basingstoke, UK) ? brown/black pigment on UricultÒ Trio E. coli medium (ORION diagnostica, Finland). These media were incubated at 37 ± 1 °C for 18–24 h before interpretation. ATCC 25922 (E. coli) and ATCC 13076 Salmonella enterica subsp. enterica serovar Enteritidis were included for quality control of the selective media.

Straw-colored colonies were scored as sorbitol negative, and pink colonies as sorbitol positive. An E. coli O157:H7 (see above) and ATCC 25922 (sorbitol positive) were used for quality control purposes. Detection of E. coli O157 Serogroup Among SorbitolNegative Strains

Hemolysin Production Isolates were inoculated onto sheeps blood agar (g/l): peptones, 20; NaCl, 5; CaCl2, 1.1; agar 11 and 5 % sheep blood erythrocytes). Blood was obtained as 50 % solution (Labor Dr. Merk, GmbH) and erythrocytes were washed 39 in phosphate-buffered saline prior to use. Plates were incubated at 37 ± 1 °C and observed for hemolysis after 3–6 h (indicating only a-hemolysin, HlyA) and after 18– 24 h incubation (indicating all types of hemolysins: HlyA; enterohemolysin, HlyE) [3]. A clinical O157:H7 isolate (a kind gift from Mette Lundstrøm Dahl, Vestfold Hospital, Norway), ATCC 25922 and ATCC 25923 strains were included as controls. Multiplex PCR Assay for the Detection of ETEC LT and ST-Genes The method used was basically that described by [33] for the detection of heat-stable toxins (STh and STp) and the heat-labile toxin (LT). Primer sequences are given in the original publication. The expected product sizes were STh (120 bp), STp (166 bp), and LT (273 bp). E. coli DSM 10973 reported to produce both ST and LT toxins was used as a positive control organism (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ, GmbH). ATCC 25922 was the negative control. The procedure was as follows: the master mix contained 1 9 colorless TaqÒ Flexi Buffer (Promega, Madison); 1.5 mM MgCl2 (Promega), 0.2 mM DNTPs (Sigma-Aldrich, St. Louis, MO, USA), 1.25 U GoTaqÒ Hot start polymerase (Promega) and primer pairs (obtained from Eurofins mwg operon, GmbH) separately or together at 0.2 lM of each. Various templates (a pinprick of colony material; 10–100 ng DNA from the clear supernatant of boiled colonies; pure DNA used for microarray analyses described below) were tested. PCR conditions were as follows: 1 cycle of 5 min at 94 °C, followed by 35 cycles of 45 s at 94 °C, 45 s at 53 °C, and 45 s at 72 °C, and a finishing 5 min. incubation at 72 °C. The PCR products were analyzed by electrophoresis in 3 % agarose gels. Testing for the Sorbitol-Negative Phenotype Escherichia coli were inoculated onto Sorbitol MacConkey agar (Oxoid) and incubated at 35 ± 0.5 °C for 18–24 h.

Isolated sorbitol negative E. coli, E. coli O157:H7, ATCC 25922, and a number of the sorbitol positive isolates (as controls) were tested for the O157 antigen using the DrySpot E. coli seroscreen latex agglutination test (Oxoid) exactly as described by the manufacturer. Multiplex PCR for the Detection of E. coli O157:H7 Strains agglutinated by O157 antibody (DrySpot) were tested for genetic determinants of the E. coli O157:H7 serotype basically as previously described [5]. A multiplex PCR using simultaneously 5 primer sets targeting genes associated with the O157 serotype was used: Rfb (O-antigen synthesis gene), expected product size = 292 bp; FLICh7 (encoding the H7 flagellin), expected product size = 625 bp, Int (Intimin that mediates the intimate adherence of the organism to host cells), expected product size = 368 bp; SLT-1 (Shiga-like toxins I), expected product size = 210 and SLT-II (Shiga-like toxins II), expected product size = 484 bp). The 100 ll PCR mix contained 0.2 mM DNTPS; 2.5 mM MgCl2; 2.5 U Taq polymerase; 0.075 lM of each Int primer; 0.1 lM of each FLIC primer, 0.2 lM of each SLT primer and 0.1 lM of each Rfb primer. The template was *100 ng cleaned DNA per 100 ll reaction mix, or 5 ll of boiled cell preparations (1 colony was boiled for 10 min. in 0.1 ml H2O. The sample was centrifuged for 2 min. at 12,0009g and 5 ll supernatant was extracted). PCR conditions were: an initial denaturation of 95 °C for 60 s followed by 30 cycles of 94 °C/ 30 s; 59 °C/60 s; 72 °C/60 s, and a final elongation of 72 °C/7 min. Positive (E. coli O157:H7 described above) and negative (ATCC 25922) controls were included. Antibiotic Resistance Testing The disk diffusion method with the specifications and breakpoints given in version 2.0 of the European Committee on Antimicrobial Susceptibility Testing, EUCAST [13] recommendations (available on-line: http://www. eucast.org) was used. In some few instances (see below), results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints given in the MO2-A11 document [9]. Susceptibility testing was done on Mueller–Hinton agar (Oxoid) using a McFarland 0.5 (measured spectrophotometrically) inoculum made

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from cells grown at 37 ± 1 °C for 18–24 h on tryptonesoya agar (Oxoid). Several colonies were used for inoculum production. After applying antibiotic disks (Oxoid), plates were incubated at 35 ± 1 °C for 16–20 h prior to reading of zone diameters. E. coli ATCC 35218 (b-lactamase producing) and E. coli ATCC 25922 were used for control purposes. The following antibiotics and concentrations were tested. The text in brackets indicates the standard abbreviation for each antibiotic and the disk content in lg: Mecillinam (MEC, 10), Piperacillin (PIP, 30), Ampicillin (AMP, 10), Ticarcillin (TIC, 75), Amoxicillin-Clavulanate (AMC, 30), Cefotaxime (CTX, 5), Ceftazidime (CAZ, 10), Doripenem (DOR, 10), Ciprofloxacin (CIP, 5), Amikacin (AMK, 30), Gentamicin (GEN, 30), Chloramphenicol (CHL, 30), Nitrofurantoin (NIT, 100), Trimethoprim-sulphamethoxazole (SXT, 25), Aztreonam (ATM, 30), Nalidixic acid* (NAL, 30), Tetracycline* (TET, 30) Sulphamethoxazole (SMZ, 100)**. Trimethoprim (TMP, 5) was tested only against strains showing clearly reduced zones around the trimethoprim-sulphamethoxazole disk, in order to distinguish the effects of the two agents. Streptomycin (STR, 10)* was used in instances where strains were sensitive to amikacin and/or gentamicin but showed an underlying aminoglycoside resistant determinant in microarray tests. *No Eucast breakpoints; the CLSI breakpoints [8] were used. **No breakpoints available. Screening for ESBL Production Two tests were performed to screen for ESBL production. Strains were inoculated onto the chromogenic BrillianceTM ESBL Agar (Oxoid, Basingstoke UK) containing cefpodoxime as selective agent. Plates were incubated and interpreted according to the manufacturer’s instructions. In brief, blue colonies were taken as confirmatory of ESBL production. The absence of growth was taken to indicate nonproduction of ESBLs. In addition, the presence of an ESBL based on synergy between clavulanic acid and cefpodoxim was investigated using double-combination disks (Cefpodoxime/clavulanic 10/1 lg; Cefpodoxime 10 lg). A commercially available test system (Combination Kit, OXOID, DD0029) was employed for this purpose. The tested strain was interpreted as producing an ESBL if there was an increase in zone size of C5 mm between the combination disks compared to that of the cephalosporin alone. Testing for Additional Aminoglycoside Resistance Determinants The AMR-ve system has probes for N-acetyltransferases, O-adenylyltransferases, and phosphotransferases (Str). The assay was supplemented by testing for additional resistance

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determinants. In brief, the genetic determinants of the resistance-associated 16S rRNA methylases RmtA, RmtB, and ArmA were amplified exactly as previously described [40]. Both whole cells and purified DNA preparations were used as template. The presence/absence of amplicons was shown by agarose gel electrophoresis. AMR-ve Microarray A DNA-based assay (IDENTIBAC AMR-ve Genotyping kit Version 05; CLONDIAG/ALERE, GmbH) developed and validated for the parallel detection of 47 resistance and integrase genes in E. coli and Salmonella [1 and references therein] was used to elucidate resistance mechanisms. Basic details of the method are given in the original publication and the kit protocol (available at www.identibac.com). In brief: E. coli were grown for 18–24 h at 37 °C on TSA (Oxoid). Genomic DNA was isolated from a 1-mm loopful of cells using the DNeasyÒ Blood and Tissue set (Qiagen, Venlo, the Netherlands) with the following modifications. Cells were suspended in the kit’s ATL solution (0.18 ml). Thereafter, 20 ll proteinase K (kit) and 200 ll AL solution (kit) were added. Incubation for 45 min at 55 °C in a thermoshaker (Quantifoil, GmbH) set at 550 rpm followed. After incubation, 4 ll RNase A (100 mg/ml; Qiagen, pnr 19101) was added to the mix, which was further incubated at room temperature for 5 min before DNA-isolation was completed using the kit protocol for animal tissues. DNA was eluted from columns with molecular biology grade water. The DNA concentration and purity were determined using a Nanodrop (ThermoScientific, USA), and the DNA integrity and the absence of intact RNA were checked by agarose electrophoresis. About 750 ng DNA was made up in 5 ll ultrapure molecular biology grade water (Sigma Aldrich). PCR, biotin labeling, hybridization to arrays, and washing and development of spots were performed exactly as described in the AME-ve protocol. Signal intensities were read within 10– 15 min of the final step (addition of buffer D1). Arrays were aligned in the reader where required by manual setting of array reference marker spots as described in the ATR03 2.0 installation and user guide. Results were exported, and the mean signal value for three replicate spots per probe was determined manually. Probes with intensity value C0.4 were considered positive while those \0.3 were considered negative. Values between 0.3 and\0.4 were considered ambiguous. Repetitive sequence-derived PCR-profiling (rep-PCR) using the Box A1R primer for investigating clonal structure among sorbitol-negative and antibioticresistant isolates The procedure was performed basically as described by Johnson et al. [20], with the following modifications. The

C. Charnock et al.: Toxin Production and Antibiotic Resistances in Escherichia coli Table 1 Information on beaches where samples were taken and the numbers of E. coli found at each Beach

Salient details of beach areaa

Weather conditions

Water (°C)

Number of E. coli (601 ml)b

Important E. coli: designated numbers in main text followed by either: AR-antibiotic resistant or SN-sorbitol negative

1

Residential area

Recent rain

15

150b

92-SN

2

Summer homes

Recent rain

17

0

3

Summer homes

Recent rain

17

1

4

Residential area

Slight rain

15

0

5

Summer homes

Slight rain

15

66b

6

Summer homes

No recent rain

16

7

107-AR, 110-AR

7

Residential area

No recent rain

15

36

114-AR, 116-SN

8

Summer homes

No recent rain

17

13

118-SN

9

Residential area

No recent rain

10

102

136-AR

Moored boats 93-AR

Moored boats 10

Summer homes. Drain for surface waterb

Slight rain

8

2

11

Summer homes

Slight rain

8

0

12 13

Residential area Island

Slight rain Recent rain

9 8

0 29

Drain for surface water 14

Island (no homes/cottages in proximity)

Recent rain

8

0

15

Summer homes. Sewage treatment plant 1 km distant

Slight rain

7

5

12-AR

16

Residential area

Slight rain

7

1

14-AR

17

Summer homes

Recent rain

7

2

18

Residential area (high density)

Slight rain

11

11

19

Island

No recent rain

11

0

Residential area Drain for surface water 20 21

Residential area Residential area

No recent rain Recent rain

11 14

49 36

22

Island

No recent rain

18

42

48-SN

No recent rain

18

114

58-SN

Residential area 23

Island Residential area

24

Summer homes

Recent rain

15

10

83-AR

25

Residential area

Recent rain

15

4

87-AR

Recent rain

14

8

Moored boats 26

Residential area Moored boats

a

Unless otherwise stated there were no known or visible discharge points commonly associated with fecal material (e.g., drains, rivers, agriculture, bird flocks) in close proximity

b

500 ml sample gave too many colonies to count. The results is calculated based on numbers of confirmed E. coli from 1 and 100 ml samples

template was *50 ng cleaned DNA used for microarray analyses (see below). The ReadyMixTM Taq PCR reaction mix (Sigma-Aldrich) was used in 50 ll volumes containing the Box A1R primer (CTACGGCAAGGCGACGCT GACG) at a final concentration of 2 lM. After PCR, 5 ll of the product was run using a 0.59 TAE buffer system

with 1.5 % molecular biology grade agarose gels containing 0.5 lg/ml ethidium bromide. Gels were run at 70 V at 4 °C for approximately 12–15 h until best separation of the bands, as determined by trial runs, was obtained. Molecular weight standards (Invitrogen 1 kb ? DNA ladder) were run flanking the profile. Gels were visualized using the Gel

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DocTMXR ? System and the gel picture was stored as tiffiles for analysis. Gel analysis and construction of a phylogenetic tree based on band matching was performed using the Quantity-1 (Bio-Rad) software package. In brief, lanes in the gel were defined manually and the start and end points for lane scanning were positioned (using corner anchor points). Background was removed using the ‘‘all lanes at same level’’ default values, and thereafter bands were detected automatically. Common and unique bands in the profiles were detected and digitized using the softwares’ automatic band matching function. Thereafter, phylogenetic similarity trees were constructed based on calculated Dice coefficients of correlation and using the unweighted pair group method with arithmetic averages clustering (UWPGMA).

Results and Discussion Locations/Sampling The Oslo fjord is extensively used for recreational purposes, including swimming and wind surfing, and there are many small beaches along both coasts. The main aim of the present study was to investigate the proportion of E. coli in beach areas showing properties of particular health concern: i.e., enteroenterovirulent E. coli (EHEC: O157/H7; ETEC) and strains with clinically significant and potentially transferable resistance determinants. In total, 26 beaches spanning both sides of the inner fjord region were investigated, and E. coli were enumerated in and isolated from 601 ml of sample at each location. The standard EUrecommendations when enumerating E. coli are that where possible, samples are to be taken 30 cm below the water’s surface and in water that is at least 1 m deep [14]. Due to low accessibility to deep water ([1 m) at some few sites, this recommendation could not always be strictly followed. Notwithstanding, none of the samples contained more than the 10 % of the 250 cfu E. coli/100 ml EU median mark for excellent quality (see Introduction). Table 1 provides an overview of the numbers of E. coli and gives also salient details on water temperature, rainfall, and beach conditions. At 5 beaches no E. coli were found. Although some beaches were in close proximity to residential areas, there were, with the exception of drains depositing excess surface water at 2 sites, no obvious sources of fecal contamination at any site. An important factor was probably the temperature of the water. In the sampling period, the average water temperature measured was (13 ± 3.9, n = 26 beaches), which is much lower than the species’ optimum growth temperature. Strains showing antibiotic resistances/negative sorbitol reaction (discussed below) were few in number and not concentrated at one or a few

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beach sites. This suggests that these strains are entering the waters at a low rate but in a widespread manner. A total of 144 E. coli were selected as described in Materials and Methods for further characterization. Testing for ETEC and EHEC Genetic Determinants and Related Traits None of the 144 isolates tested positive for any of the underlying genetic determinants of St and Lt production typical of ETEC. Multiplex PCR showed that the control strain DSM 10973 had the genetic determinants for Sth and Lt production, but not Stp. Strains can be positive for both Sth and Stp [6] though this seems to be the exception. The prototypic and most prevalent EHEC E. coli O157:H7 is generally sorbitol-negative [16 and references therein]. Of the 144 isolates, 5 (48, 58, 92, 116, 118) were sorbitol negative. These plus controls were screened using the dry-spot agglutination test for the presence of the O157 antigen. The control EHEC strain gave strong agglutination. In addition, definite but weaker agglutination was also seen with isolate 48. All other strains were clearly negative. The results of testing were investigated further using a multiplex PCR which both identifies E. coli O157:H7 strain-related antigens and pathogenesis associated genes. The control strain produced all 5 expected PCR products (results not shown), whereas no PCR products were obtained with isolate 48. Thus, the E. coli O157:H7 biotype was not found among the 144 isolates. The reason for the weak agglutination of strain 48 is not known, but might represent a degree of cross-reaction with a similar Oantigen. Most O157 STEC and many non-O157 STEC produce HlyE. This is distinguished from HlyA produced by other E. coli by slower hemolytic action and by its inhibition in vitro by serum. Only the control E. coli O157:H7 strain produced a thin zone of complete hemolysis after 24 h typical of HlyE activity. Thus, none of the 144 isolates were HlyE-producers. The control ATCC 25922 (a clinical isolate), and isolates 20, and 117 gave hemolysis after 3 h which became a large zone of complete hemolysis after 24 h, probably indicating production of HlyA. Invasive E. coli frequently produce factors such as HlyA which enhance strain virulence [26]. According to Burgos and Beutin [7], HlyA is frequently associated with human uropathogenic E. coli and with ETEC, STEC, and EPEC strains that cause diarrhea and edema in animals. As the HlyA producing strains in the present study had neither determinants for toxin production nor showed antibiotic resistances, they were not investigated further. In summary, none of the 144 bathing water isolates belonged to the major groups of enterovirulent E. coli which were assayed for, although two strains had the

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virulence-enhancing HlyA phenotype. As few studies of marine environments exist for comparison, the significance of the results are difficult to gauge at the present time. In a study of 109 E. coli isolated from marine sediments along the Adriatic coast, more than 50 % of the strains chosen for further testing were shown to harbor virulence genes [24]. However, unlike the present work only specific phylotypes (i.e., those commonly associated with virulence) were investigated, creating a bias in the study. As in the present work, no St-determinants were found in any strain. In a larger study of almost 25,000 E. coli from Californian beach waters, no ETEC were detected, whereas as many as 10 % of E. coli were classified as enteropathogenic [19]. Although the results from the present study indicate that the coastal waters of the Oslo fjord may not represent a significant reservoir of enterovirulent E. coli, additional studies where a wider range of virulence determinants and phenotypes are tested for (e.g., microarray analyses) might reveal other pathogenic strains. For example, non-O157 EHEC and sorbitol-fermenting O157 (which now account for[10 % of cases of HUS in Germany) could be included in future studies [16]. In addition testing of E. coli present in fjord sediments is also desirable. Antibiotic Resistance Testing (Disk Diffusion) and Additional Screening for ESBL Production All 144 isolates and controls were tested against a panel of clinically relevant antibiotics for which breakpoints have been established for the Enterobacteriaceae. About 94 % of the isolates were sensitive to all the agents tested. Approximately 6 % (9 isolates) showed resistance to one or more antibiotic. Of these, 6 isolates were resistant to the quinolone nalidixic acid. Isolates 107 and 114 were only resistant to nalidixic acid. The next most common resistances were to penicillins, tetracycline and trimethoprim (all 2 isolates). With respect to the penicillins, the following salient points can be made: all strains were sensitive to mecillinam whereas strains 12, 110, 136 were resistant to piperacillin, ampicillin, and ticarcillin. All strains with the exception of 12 and 136 (borderline) were sensitive to amoxicillin-clavalunate, and no ESBLs were present (see also below). A recent study of Norwegian clinical E. coli isolates (human blood cultures), showed the presence of ESBLs and generally a much higher incidence of resistant strains: 40 % were resistant to ampicillin, 16 % were resistant to nalidixic acid, and 3.3 % were ESBL producers [29]. All strains were sensitive to carbapenems (here Doripenem), monobactams (Aztreonam) and the third-generation cephalosporins (cefotaxime, ceftazidime) suggesting that none were ESBL producers [8, 31, 36]. The absence of ESBLs was further supported by the microarray data given

below. Notwithstanding, given the importance of this class of enzymes, cellular activity of ESBLs was assayed for. No strains grew on BrillianceTM ESBL Agar (Oxoid, Basingstoke UK) containing cefpodoxime. Furthermore, there was no synergy between the effects of cefpodoxime and clavulanic acid. i.e., zones of equal size were produced by cefpodoxime alone and in combination with clavulanic acid. Both results strongly suggest the absence of ESBL activity. With respect to the fluoroquinolones (here, ciprofloxacin), isolate 14 alone showed resistance. With respect to aminoglycosides (amikacin, gentamicin), none of the isolates showed resistance to amikacin, whereas isolates 14 and 83 were resistant to gentamicin. Isolates 14 and 87 were resistant to tetracycline. Of the miscellaneous agents tested (chloramphenicol, nitrofurantoin, trimethoprim-sulfamethoxazole), isolate 83 was resistant to chloramphenicol and trimethoprim-sulfamethoxazole, but no isolates showed resistance to nitrofurantoin. Isolates were also tested against sulfamethoxazole (100 lg) for which no breakpoints seem to exist. Isolate 83, grew into the disk indicating obvious resistance. Isolates 14 and 93 showed smaller zones around STX than other non-resistant isolates. These were susceptible to sulphamethoxazole, and subsequently shown to be resistant to trimethoprim. Isolate 14 was MDR: it showed resistance to ciprofloxacin, gentamicin and tetracycline representing 3 of the categories of agents in the proposed guidelines [25], as well as additional resistances to trimethoprim and nalidixic acid. Isolate 83 was also MDR by virtue of its resistance to gentamicin, chloramphenicol, and trimethoprim-sulfamethoxazole. The results of the disk diffusion assays for resistant isolates are summarized in the last line of Table 2. With one exception the resistant isolates came from different beaches, indicating an even spread of the resistance phenotypes as opposed to a possible single focal point for the origin of resistant strains (Table 1). In order to learn more about the underlying resistance mechanisms, and if they might be associated with mobile genetic elements, the isolates showing clinically important resistances were subjected to microarray analysis using the AMR-ve system. IDENTIBAC AMR-ve Genotyping and PCR-Based Detection of Additional Resistance Determinants Table 2 shows the results of microarray analysis of antibiotic resistant strains. Results for the control probes, which confirmed that the tested strains were E. coli, are not shown. The highest number of resistance genes (not counting integrons) was 7 (strain 87). By way of comparison, in a study of 50 clinical isolates using the same array the highest recorded number was 15 [1]. This finding is not

123

C. Charnock et al.: Toxin Production and Antibiotic Resistances in Escherichia coli Table 2 Microarray detected resistance and integrase determinants in control strains and bathing water isolates Gene

Strain 12

14

83

prob_ant2Ia_1 (aadB gene cassette)

P

prob_aadA1_1 (aadA aad1 aminoglycoside)

P

87

93

110

136

P

prob_aadA2_1 (aadA2 aadA2a aadA2b aadA2c, aadA3 aadA8 aminoglycoside)

P

prob_floR_11 (chloramphenicol/ florfenicol)

P

prob_sul2_11 (sulfonamide)

P

prob_sul1_11 (sulfonamide) prob_act1_11 (class 1 integrase)

P

prob_catB3_11 (catB3/B4 cloramphenicol acetyl transferase) Prob_tem1_1 (all blaTEM genes betalactam)

P P

P P

P

P P

P

P

Prob_cmlA1_11 (cnlA cmlA1 cmlA4 cmlA5 cmlA6 cmlA7 chloramphenicol exporter)

P P

Prob _tetB_11 (tetracycline)

P

Prob_dfrA7_12 (trimethoprim)

P

probdfrA17_11 (trimethoprim)

P

P

probdfrA19_1 (trimethoprim)

P

A

P

Prob_qnrB_11 (quinolone)

P

Prob_qnrB_12 (quinolone)

P

Prob_qnrS_11 (quinolone)

A

Prob_qnr_12 (qnrA quinolone)

A

Prob_strA_611 (aminoglycoside)

P

Prob_strB_611 (aminoglycoside) Prob_dfrA7_11 (trimethoprim)

P

Measured resistances

ATCCa 35218

P

P AMC, PIP, AMP, TIC

CIP,NAL, GEN,TET, TMP

NAL, GEN, CHL, SMZ, SXT

NAL, TET, STR

NAL, TMP

PIP, AMP, TIC

PIP, AMP, TIC

PIP, AMP, TIC, CHL, SMZ, STR

P positive result, A ambiguous result; see ‘‘materials and methods’’ section a

ATCC25922 (not shown in table) was also included. This strain was sensitive to all antibiotics tested, and no resistance determinants were found

unexpected, given the selective pressure for resistance development in the clinical setting. In the present study, blaTEM genes, which are generally found on plasmids [1, 31], were the most common resistance determinants and underpin the observed resistances to the penicillins (Table 2). The TEM b-lactamase is reported to be the most common mechanism of ampicillin resistance in E. coli, and it has also previously been detected in E. coli from a range of environments [35 and references therein]. However, in line with susceptibility testing no strains gave hybridization with the ESBL determinants included in the array. Class 1 integrons have been described as the main mobilizers of antibiotic resistance genes in enteric bacteria. In E. coli

123

class 1 integrons are also generally associated with human clinical isolates [10]. Several isolates, including both MDR strains (14, 83), tested positive for class 1 integrons suggesting a potential for resistance gene mobility. Similar results have been previously reported for MDR E. coli from marine sediments [39]. Some important observations with regard to the resistance profiles and genotypes of individual strains are as follows: The MDR E. coli 83 was resistant to chloramphenicol, a trait seen only rarely in other marine isolates [38, 39]. Furthermore two distinct chloramphenicol resistance mechanisms, an acetyl transferase and an efflux pump, are indicated (Table 2). In addition to a determinant

C. Charnock et al.: Toxin Production and Antibiotic Resistances in Escherichia coli

for a class 1 integron, hybridization to a gene associated with a DNA-cassette conferring aminoglycoside and chloramphenicol resistance in Salmonella was demonstrated. Taken together, these results might suggest evolution of the strain under selective pressure from chloramphenicol. This strain’s resistance to gentamicin can be explained by the presence of an aminoglycoside 300 adenyltransferase gene, whereas resistance to sulphametoxazole is attributable to the sul1 dihydropteroate synthase which is common in E. coli [e.g., 38]. The MDR E. coli 14 was the only isolate showing resistance to ciprofloxacin. However, no corresponding resistance genotype was found. This perhaps suggests a mutation-based mechanism. The main mechanism of resistance to quinolones is accumulated mutations in the genes coding for the target bacterial enzymes. Resistance to nalidixic acid is also known to increase the risk for the development of fluoroquinolone resistance, as nalidixic resistant strains can become resistant to fluoroquinolones by additional mutations [37, 41]. E. coli 14 was resistant to gentamicin (growing almost completely into the disk), and also here no resistance genetic determinant was found. By contrast, sensitivity to amikacin was shown. The isolate was accordingly tested for sensitivity to streptomycin. Based on the CLSI guidelines, the strain was recorded as sensitive. Thus, the results indicate a resistance mechanism with a high degree of specificity for gentamicin. The AMR-ve array is able to detect genes for N-acetyltransferases, O-adenylyltransferases, O-phosphotransferases (str). As these were not found, we assayed for a series of 16S rRNA methylase also known to confer resistance to gentamicin [40]. No determinants for methylases were found, and thus at present the resistance mechanism for gentamicin in this strain remains unknown. E. coli 87 was resistant to nalidixic acid. However, unlike other isolates showing quinolone resistance, hybridization with the qnrB and qnrS resistance genes, which are plasmid mediated, was obtained. Qnr determinants are believed to bind to and protect DNA gyrase and/ or topoisomerase IV from inhibition [1]. Good hybridization signals were obtained with the aminoglycoside resistance genes (strA and B). These are aminoglycoside phosphotransferases which are associated with streptomycin resistance [37]. As the strain was sensitive to both amikacin and gentamicin, it was further tested for streptomycin resistance and this was found. The results from the AMR-ve were generally in good accord with those from the disk diffusion assays (Table 2). Furthermore, a number of the resistance determinants identified (including those found in the MDR strains) are strongly associated with mobile genetic elements and thus a potential for horizontal transfer to pathogenic strains. Further support for this comes from the demonstration of class 1 integrons in several strains.

Repetitive sequence-derived PCR-profiling using the Box A1R primer for investigating clonal structure among sorbitol-negative and antibiotic-resistant isolates Isolates suggested by one or more test to be of potential human health significance (i.e., sorbitol negative and especially antibiotic resistant E. coli) were examined for clonal relationships using BOX A1R repetitive-element sequence based PCR (Fig. 1a). The technique has been widely used to examine the genetic diversity and to track the spread of E. coli [15, 18, 22, 27] including environmental isolates [16, 25]. Although less amenable to standardization than for example multilocus sequence typing (MLST), rep-PCR is simple and rapid and has been shown to have similar and in some cases greater discriminatory power than MLST when applied to E. coli and other species [e.g., 2, 15, 22]. Johnson et al. [20] have considered Box A1R fingerprints to represent unique isolates (from the same host) when the DNA similarity coefficients were \90 %. If applicable to the present study, all of the isolates are clearly unique (Fig. 1b). The most similar (65–75 %) isolate pairs were 110/136, 12/93 and 14/83, which are pairs consisting of antibiotic resistance strains. However, there are no clusters of either antibiotic strains or sorbitol-negative strains, indicating that these traits in the present study are not traceable to clonal relationships. This is in line with the observation that the sorbitol negative strains and almost all of the antibiotic resistant strains originated from different beaches (Table 1). An absence of clonal-relatedness might be the expected result given that the strains presented with unique resistance profiles (Table 2). However, although a correlation between clonal relatedness and resistance characteristics is often demonstrated (due to the spread of resistant clones), this is not invariably so. Genotyping techniques, including both pulsed-field electrophoresis and MLST have shown both temporal and spatial examples of variation in the resistance profile of well-defined clones [4, 34]. This can potentially be explained by the loss or gain of mobile resistance determinants by horizontal gene transfer, in response to changes in selective pressure such as the introduction of new antibiotics. Fingerprinting techniques such as Fig. 1a can thus also provide reference material for future studies. However, the number of strains analyzed is too small to draw conclusions on population structure, and based on the BOX A1R analysis, it can only be concluded that no two isolates represent the same or highly similar clones.

Conclusion Our data suggest that coastal marine waters may be a suitable environment for the survival of antimicrobialresistant E. coli strains. E. coli is generally considered a

123

C. Charnock et al.: Toxin Production and Antibiotic Resistances in Escherichia coli Fig. 1 Box A1R repetitive sequence-derived PCR profile (a) and corresponding cluster analysis (b) of antibiotic resistant (12, 14, 83, 87, 93, 110, 136, ATCC 35218), sorbitol negative (48, 58, 92, 116, 118, O157:H7), ATCC 25922 and DSM 10973 E. coli

a bp 12000

5000

2000 1650

1000 850

650 500 400 300 200 48

Standard

O157: H7

58

92

116

118

83 110

DSM 10973

112 14

87

93 136

Standard ATCC ATCC 35218 25922

b 48 O157:H7

118 116 ATCC25922 ATCC35218

136 110 DSM10973

92 93 12 58 87 14 83

transient member of the natural microbiota. However, it has recently been suggested that E. coli that persist outside the host can become naturalized members of native bacterial populations, including aquatic communities [39 and references therein]. If this applies to the present strains, the potential for their spread in the environment would be greatly increased. The number and types of resistances found were similar to those of E. coli isolated from a range of environmental samples. In a study of Norwegian cervids,

123

*7 % of E. coli were resistant to one or more antibiotics, chiefly streptomycin, sulfamethoxazole, and tetracycline [23]. E. coli isolated from marine sediments in the Adriatic showed a higher percentage of MDR (14 %). However, as in the present study, no ESBLs were detected and resistances to tetracycline, ampicillin and sulphmethoxazoletrimethoprim were widespread [39]. The significance of the present and previous studies is that resistances to antibiotics important to human medicine are present and

C. Charnock et al.: Toxin Production and Antibiotic Resistances in Escherichia coli

persistent in a variety of nonclinical environments, presumably also in the absence of selective pressure. Although the E. coli found in the present study may not cause disease, they come into contact with humans engaging in water activities, and can act as a reservoir for resistance genes that could be transmitted to bacteria of the intestinal flora, as well as to pathogenic bacteria. This is a threat to the current management of bacterial infections and the long term value of microbial agents. The study, which is the first of its kind in Norway, can provide a benchmark for later studies. Expected climate changes and changes in precipitation can bring about increases in water flows, and this could lead to increased contamination in future years. Acknowledgments The authors would like to thank Bjørg C. Haldorsen (Reference Centre for Detection of Antimicrobial Resistance, University Hospital of North-Norway, Tromso, Norway) for helpful comments on the manuscript.

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Toxin production and antibiotic resistances in Escherichia coli isolated from bathing areas along the coastline of the Oslo fjord.

The presence of enterovirulent and/or antibiotic resistant strains of Escherichia coli in recreational bathing waters would represent a clear health i...
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