ORIGINAL ARTICLE. P.L. Ho 1, R.C. Wong 2, K.H. Chow 1 and T.L. Que 2. Abstract

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Distribution of integron-associated trimethoprim sulfamethoxazole resistance determinants among Escherichia coli from humans and food-producing animals P.L. Ho 1, R.C. Wong 2, K.H. Chow 1 and T.L. Que 2 1 Division of Infectious Diseases, Department of Microbiology and Centre of Infection, The University of Hong Kong, Hong Kong, China 2 Tuen Mun Hospital, Hospital Authority, Hong Kong, China Keywords antimicrobial resistance, E. coli, gene cassettes, integrons. Correspondence Pak-Leung Ho, Division of Infectious Diseases, Department of Microbiology and Centre of Infection, The University of Hong Kong, Queen Mary hospital, Pokfulam Road, Pokfulam, Hong Kong SAR, China. plho@hkucc.hku.hk : received 23 May 2009, revised and accepted 8 August 2009 doi: /j x x Abstract Aims: To compare the distribution of integrons and trimethoprim sulfamethoxazole resistance genes among Escherichia coli isolates from humans and foodproducing animals. Methods and Results: A collection of 174 multidrug-resistant E. coli isolates obtained from faecal samples of food-producing animals (n = 64) and humans (n = 59), and patients with urinary tract infections (n = 51) in Hong Kong during were studied. The strains were analysed for their phylogenetic groups, the presence of sul genes (sul1 and sul2), integrons (intl1 and intl2) and class 1 integron-associated dfr cassette genes by PCR, restriction enzyme analysis and sequencing. Integrons were identified in 110 (63Æ2%) isolates. The prevalence of integrons was significantly different according to the specimen sources (animal faecal 84Æ4%, human faecal 67Æ8% and human urinary 31Æ4%) and phylogenetic groups (B1 80Æ8%, A 77Æ6%, D 54Æ1% and B2 11Æ5%). Faecal isolates (both human and animal) are more likely to belong to group A and B1. In contrast, most urinary isolates were either groups B2 and D. Among dfr containing isolates, dfra1 and dfra12 were almost exclusively found in strains of phylogenetic groups A and B1; and were present in animal and human faecal isolates. In contrast, dfra17 was found in both faecal and urinary isolates and comprised strains from all phylogenetic groups. The sul1 and sul2 genes were equally prevalent among the isolates irrespective of the specimen source and phylogenetic group status. Pulsed-field gel electrophoresis analysis of isolates with identical cassette genes showed that they were genetically diverse. Conclusions: More animal faecal isolates carry class 1 integrons than human faecal and human urinary isolates, and the distribution of phylogenetic groups is common across animal and human faecal isolates but different from human urinary isolates. Significance and Impact of the Study: Commensal isolates from food-producing animals are an important reservoir for integrons carrying antibiotic resistance genes. Introduction Integrons play major roles in the spread of antimicrobial resistance in Gram-negative bacteria by virtue of their ability to capture multiple resistance genes as cassettes and their localization on transposons and conjugative plasmids (Mazel 2006). In Escherichia coli, trimethoprim sulfamethoxazole resistance often correlates with the Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009)

2 presence of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes in integrons (Huovinen et al. 1995; White et al. 2001). Currently, at least 20 different DHFR gene (dfr) sequences have been reported (Huovinen et al. 1995). The dfr genes have most often been reported as cassettes in class 1 integrons and in association with a wide range of other resistance genes. In contrast, very few dfr genes occurred within class II integrons because the integrase gene is nonfunctional (Mazel 2006). Currently, only three different types of DHPS genes (sul1, sul2 and sul3) encoding sulfonamides (i.e. sulfamethoxazole) resistance are recognized. The sul1 gene is usually found in class 1 integron in the 3 end, whilst sul2 is usually located on small plasmids belonging to the IncQ family or another type represented by pbp1 (van Treeck et al. 1981; Huovinen et al. 1995). Both sul1 and sul2 are highly prevalent, whilst the third type sul3 has only been occasionally detected (Blahna et al. 2006). According to the World Health Organization, it is estimated that half of the world s antibiotic production is used in the animal industries (World Health Organization 2002). Antibiotics are added to feed for food animals, either for mass treatment of infections or for growth promotion. However, this is not the case in all countries worldwide because antibiotics in animal feed as growth promoters are banned by countries in the European Union since 2006 (Hammerum and Heuer 2009). In animals, there is clear evidence that antibiotics select the resistant bacteria in the animals themselves (Aarestrup et al. 2008). Bacteria and resistant genes can pass between humans and animals (Hammerum and Heuer 2009). Furthermore, commensal E. coli of animal origin may act as a donor of antibiotic resistance genes for pathogenic E. coli (Leverstein-van Hall et al. 2002). For decades, it is recognized that administration of antibiotics to animals may be followed by the establishment of antibiotic-resistant strains of E. coli in the human gut flora (Aarestrup et al. 2008), and this has been postulated to cause urinary tract infection () (Shooter et al. 1970). Recently, a relationship between avian pathogenic E. coli and human urinary tract pathogenic strains has been reported (Rodriguez-Siek et al. 2005). Among clinical isolates of E. coli from Europe and Canada, it has been suggested that horizontal transfer of resistance genes may be playing a larger role than clonal expansion in the increase in trimethoprim sulfamethoxazole resistance (Blahna et al. 2006). However, very few studies have investigated the relationship between trimethoprim sulfamethoxazole resistance genes among animal and human isolates (Kang et al. 2005). Here, we assess the extent to which identical integron cassette arrays were carried by E. coli isolates obtained from humans and food-producing animals in Hong Kong. Materials and methods Bacterial strains and susceptibility testing A total of 174 isolates were obtained from three sources in (Duan et al. 2006; Ho et al. 2007b, 2008). These included urinary isolates obtained from outpatients with urinary tract infection (n = 51), isolates from faeces of food-producing animals (cattle, poultry and pigs) (n = 64) and isolates obtained from faeces of university students (age range, years; n = 20) or paediatric outpatients (n = 39). Most of the food-producing animals from which the isolates were obtained, were imported to Hong Kong from the mainland China. The isolates were obtained as part of antimicrobial resistance surveillance in humans and animals. Hence, these bacterial populations provide an opportunity to assess the antimicrobial resistance genes in food animals and humans in the same geographical region. A subset of ciprofloxacin-resistant isolates was chosen for investigation. The isolates were also resistant to ampicillin, trimethoprim sulfamethoxazole and or gentamicin. We decided to focus on the ciprofloxacin-resistant isolates because it has been shown that animal and human isolates showed greater similarity to each other than their susceptible counterparts (Johnson et al. 2006). Antimicrobial susceptibility of the isolates was determined by the disc diffusion method, and results were interpreted according to the CLSI (Clinical and Laboratory Standards Institute 2007). The following antibiotics (disc content) were tested: amikacin (30 lg), ampicillin (10 lg), chloramphenicol, ciprofloxacin (5 lg), gentamicin (10 lg), sulfonamides (300 lg), tetracycline (30 lg), trimethoprim (5 lg) and trimethoprim sulfamethoxazole (1Æ25 lg 23Æ75 lg). Control strains (E. coli ATCC and Staphylococcus aureus ATCC 25923) were included on each day of testing. Molecular studies Primers used are listed in Table 1. To determine the regions with identical content, the PCR products of the gene cassette regions were digested separately with restriction enzymes, ApoI and BsiE1 (New England Biolabs Ltd, Hertfordshire, UK). Those with identical restriction fragment patterns were assigned to the same cassette array. The content of the gene cassette region was determined by bidirectional sequencing. One to two isolates for each unique restriction patterns were sequenced. Multiplex PCR was used to assign the E. coli isolates to the four phylogenetic groups (A, B1, B2 and D) using previously described primers (Clermont et al. 2000) according to a modified protocol (Ho et al. 2007a). The epidemiological 628 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009)

3 Table 1 Primers used in this study Primer name Sequence (5 3 ) Target References intl1f GGG TCA AGG ATC TGG ATT TC Class 1 integron This study intl1b GTT CTA CGG CAC GTT TGA AG intl2f GCA AAT GAA GTG CAA CGC Class 2 integron (Reyes et al. 2003) intl2b ACA CGC TTG CTA ACG ATG sul1-f CGG CGT GGG CTA CCT GAA CG sul1 (Kerrn et al. 2002) sul1-r GCC GAT CGC GTG AAG TTC CG sul2-f GCG CTC AAG GCA GAT GGC ATT sul2 (Kerrn et al. 2002) sul2-r GCG TTT GAT ACC GGC ACC CGT 5 -CS GGC ATC CAA GCA GCA AG Gene cassette region (Sunde 2005) 3 -CS AAG CAG ACT TGA CCT GA relatedness of the strains was studied by pulsed-field gel electrophoresis (PFGE). XbaI (Amersham Pharmacia Biotech, Little Chalfont, UK) was used for digestion of the genomic DNA and patterns were analysed with Gelcompar II (Applied Maths, Sint-Martens-Latem, Belgium). Dendrogram was created by means of the Dice coefficient and the unweighted pair-group mean arithmetic (UP- MAG) method. A similarity coefficient of >85% was selected to define clonal relationship (Ho et al. 2005). Statistical analysis Chi-square or Fisher exact tests were used for the comparison of categorical variables. A P-value of <0Æ05 was considered to be statistically significant. For the purpose of analysis, both intermediate and resistant were grouped together as resistant. A statistical package (spss 16Æ0; SPSS Hong Kong Ltd, Hong Kong, China) was used for all analysis. Table 2 Antimicrobial resistance for the 174 Escherichia coli isolates included in this study % Resistant Animal isolates (n = 64) Human isolates (n = 110) Both (n = 174) Amikacin 14 3Æ6 7Æ5 Ampicillin 95Æ3 98Æ1 97Æ1 Chloramphenicol 79Æ7 56Æ3 64Æ9 Ciprofloxacin Gentamicin 59Æ4 61Æ8 60Æ9 Sulfonamides 93Æ8 91Æ8 92Æ5 Tetracycline 98Æ4 91Æ8 94Æ3 Trimethoprim 92Æ Æ8 Trimethoprim 90Æ6 91Æ8 91Æ4 sulfamethoxazole Multidrug resistance 4 6 drugs 46Æ8 62Æ7 56Æ9 7 8 drugs 48Æ4 31Æ8 37Æ9 Results The antimicrobial susceptibility of the isolates was summarized in Table 2. The majority (92Æ5%, ) of the isolates were resistant to five or more antibiotics. Among the 174 isolates, three isolates were resistant to trimethoprim alone, five were resistant to sulfonamides alone, 155 were resistant to both trimethoprim and sulfonamides, and 159 were resistant to the combination trimethoprim sulfamethoxazole. Overall, the intl genes were identified in 110 (63Æ2%) of the 174 isolates. The intl1 gene alone was found in 107 isolates, the intl2 gene alone in one isolate, and both genes were found in two isolates. Chi-square analyses showed that there was significant difference in the intl-positive rates according to sources, Table 3 Prevalence of integrons in 174 Escherichia coli isolates from humans and animals Factor % intl-positive (No. positive subtotal) Source* Cattle 81Æ8 (9 11) Poultry 80 (20 25) s 89Æ3 (25 28) Faecal human 67Æ8 (40 59) Urinary tract infection 31Æ4 (16 51) Phylogenetic group* A 77Æ6 (66 85) B1 80Æ8 (21 26) B2 11Æ5 (3 26) D 54Æ1 (20 37) Trimethoprim sulfamethoxazole Resistant 66Æ7 ( ) Sensitive 26Æ7 (4 15) *P < 0Æ001 for comparisons of the prevalence of integrons among the isolate sources and phylogenetic groups. P = 0Æ002 for comparison of the prevalence of integrons among trimethoprim sulfamethoxazole-resistant vs trimethoprim sulfamethoxazole-sensitive isolates. Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009)

4 % intl-positive (No. positive subtotal) Table 4 Prevalence of integrons according to the number of drug resistance Animal isolates Human isolates Both P-value No. of drug resistance* Æ3 (1 3) 0 (0 6) 11Æ1 (1 9) 0Æ (24 30) 47Æ8 (33 69) 57Æ6 (57 99) 0Æ Æ5 (29 31) 65Æ7 (23 35) 78Æ8 (52 66) 0Æ006 Total 84Æ4 (54 64) 50.9 (56 110) 63Æ2 ( ) <0Æ001 Fisher exact or chi-square comparison of % intl-positive among animal isolates vs human isolates. *According to the number of drug resistance for eight drugs including ampicillin, amikacin, zchloramphenicol, ciprofloxacin, gentamicin, sulfonamides, tetracycline and trimethoprim. phylogenetic groups and resistance to trimethoprim sulfamethoxazole (Table 3). Two of the three isolates resistant to trimethoprim alone were intl-positive, and three of the five isolates resistant to sulfonamides alone were intl-positive. Faecal isolates (both human and animal) are more likely to belong to group A and B1 (60 64 and for the faecal isolates from animal and human, respectively). In contrast, most urinary isolates were either groups B2 (19 51) or D (23 59). Furthermore, the frequency of intl genes in the isolates increased positively with the number of drug resistance (Table 4). One of nine isolates resistant to 1 3 drugs was intl-positive. This increased to 57Æ6% and 78Æ8% among those with resistance to 4 6 and 7 8 drugs, respectively (P =0Æ001 for comparisons between isolate groups with resistance to 1 3, 4 6 and 7 8 drugs). The frequencies of integrons were higher among animal isolates than the human isolates. The cassette arrays for the 105 trimethoprim sulfamethoxazole-resistant isolates with class 1 integron were investigated further. Amplicons with sizes of 1, 1Æ3, 1Æ6, 1Æ7, 1Æ8, 2 or 3 kb was obtained in 54 (51Æ4%) isolates (Table 5). A single band was obtained in 50 isolates. Two bands were obtained in four isolates. Eleven cassettes encoding resistance to trimethoprim (dfr), aminoglycosides, rifampicin and chloramphenicol were found in nine different arrays, of which six were shared by animal and human isolates. The most prevalent aminoglycoside resistance genes were those encoding resistance to streptomycin (aada1, aada2, aada5 and aada23). Those encoding gentamicin and amikacin resistance were aadb and aaca4, respectively. No resistance genes encoding resistance to ampicillin and tetracycline were found in the cassette arrays. Three array patterns with dfr genes (dfra1-aada1, dfra12-aada2 and ) and three other array patterns without dfr genes (arr-3, aada1 and aada23) were shared by isolates from human and animal sources. At least one dfr allele was found in 41Æ9% (44 105) of the isolates [44Æ2% (23 52) for faecal animal isolates, 36Æ8% Table 5 Class 1 integron-associated resistance genes for 54 trimethoprim sulfamethoxazole-resistant Escherichia coli Source of isolates Phylogenetic group PCR product size (kb) Cassette arrays* n Animal faecal Human faecal -human A B1 B2 D 1Æ Æ3 dfraa Æ6 dfra1-aada Æ6 and 1 dfra1-aada1 and aada Æ8 dfra12-aada Æ6 and 1Æ7 dfra1-aada1 and aada aada aadb-aada Æ3 arr aaca4-cmla Undefined Total , urinary tract infection. *Gene cassette arrays found inside the variable region of class 1 integrons. The resistance genes encoded resistance to trimethoprim (dihydrofolate reductase, alleles dfra1, dfra12 and dfra17), aminoglycosides (aminoglycoside adenyltransferases [ANT(3 )-I], aada1, aada2, aada5, aada23; aminoglycoside acetyltransferase [AAC(6 )-Ib], aaca4; and aminoglycoside adenylytransferase [ANT(2 )-I], aadb), rifampin (arr-3) and chloramphenicol (cmla1). 630 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009)

5 (14 38) for faecal human isolates and 46Æ6% (7 15) for urinary isolates]. The most common allele was dfra17 (56Æ8%, 25 44), followed by dfra1 (40Æ9%, 18 44) and dfra12 (9Æ1%, 4 44). Allele dfra1 was most common in animal isolates, whilst dfra17 was most common in human isolates. This was compared with detection of at least one sul gene in 91Æ4% (96 105) of the isolates. Of these, 28Æ6% (30 105) had sul1 alone, 27Æ6% (29 105) had sul2 alone and 35Æ2% (37 105) had both sul1 and sul2. Unlike the dfr alleles, the two sul genes exhibited similar distribution among isolates from animal and human sources. Phylogenetic group analysis showed that all except one of the isolates with dfra1 and dfra12 were of groups A and B1. In contrast, dfra17 allele was found in isolates of all four phylogenetic groups. All three dfr alleles (A1, A12 and A17) were found among faecal isolates from animal and human. PFGE analysis for the subset of human and animal isolates with identical cassette arrays showed they were not clonally related (Fig. 1). Discussion The present study demonstrated the presence of identical integron-associated dfr genes and cassette arrays among E. coli isolates obtained from humans and food-producing animals. All three integron-associated dfr alleles were present in animal and human faecal isolates, but dfra17 was the only allele found in urinary isolates. One potential 85% similarity m m Strain Source Cassette array 6A4 aaca4 [AAC(6 )-lb]-cmla1 Pi241 Pi B4 Pn268 N39 G6 Pi131 N37 9A3 Poultry aada23 dfra1-aada1 dfra1- aada1 dfra12-aada2 dfra1-aada1 & G1 B1 aada1 1F6 5F6 O68 9C5 ch02 ch37 N29-2 Pi222 11C9 O42-2 ch36 Pi010 Cattle Poultry Poultry Cattle Poultry dfra1-aada1 dfra1-aada1 dfra1- aada1 dfra1-aada1 Pi012 4D5 dfra1-aada1 & aadb-aada1 Pi055-2 B6 arr-3 Figure 1 Dendrogram showing the genetic relatedness of 28 Escherichia coli isolates obtained from food-producing animals and humans with identical cassette arrays (unweighted pair-group mean arithmetic, Dice, dotted vertical line indicated 85% similarity) (Ho et al. 2007a). Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009)

6 explanation is that horizontal transfer of integron-associated genes may occur more readily for the commensal commensal pair than the commensal urinary pair. Our findings of higher prevalence of integrons among groups A and B1 isolates (77Æ6 80Æ8%) than among groups B2 and D isolates (11Æ5 54Æ1%) lend support to this hypothesis. In a study of faecal E. coli isolates from healthy subjects, class 1 integrons likewise were reported to occur more frequently among E. coli strains in non-b2 groups than in B2 group, although the difference was statistically insignificant (Skurnik et al. 2005). Geographical and temporal variations in dfr alleles among E. coli isolates from different host and specimen sources are recognized to exist (Huovinen et al. 1995; Yu et al. 2004; Blahna et al. 2006). The distribution of dfr genes among our E. coli isolates was similar to that reported by some investigators (Yu et al. 2004; Kang et al. 2005). Among isolates obtained in Korea during , it was reported that dfra1 and dfra12 were predominate alleles among intestinal E. coli from food animals, whilst dfra17 was the major allele among urinary E. coli from humans (Yu et al. 2004; Kang et al. 2005). In another investigation of E. coli isolates obtained from meat and meat products in Norway during , dfra1 and dfra12 together account for 88% of all dfr genes found within class 1 integrons (Sunde 2005). In a multi-centre surveillance, dfra1 and dfra17 were found in 45% and 35%, respectively, of the trimethoprim sulfamethoxazoleresistant urinary E. coli isolates obtained from Europe and Canada (Blahna et al. 2006). However, these previous studies did not assess the dfr allelic distribution in relation to the major phylogenetic groups (Sunde and Sorum 1999; Kang et al. 2005; Blahna et al. 2006). Our PFGE analysis showed that clonal expansion played little role in the dissemination of trimethoprim sulfamethoxazole resistance between food-producing animal and human isolates. These findings are in accord with previous studies (Kang et al. 2005). In the United States, community expansion of the so-called clonal group A (CgA) strains with an integron-associated gene cassette was found to be important in the increase in trimethoprim sulfamethoxazole resistance among uropathogenic E. coli (Solberg et al. 2006). Ramchandani et al. (2005) reported one bovine isolate with 94% PFGE similarity to a human CgA strain, but no information was available as to whether it was a commensal or clinical isolate. Because our urinary isolates with gene cassette belonged to different phylogenetic groups and exhibited diverse PFGE patterns, it would appear that horizontal gene transfer is playing more important roles than clonal expansion in the increase in trimethoprim sulfamethoxazole resistance in our locality (Ho et al. 2007b). In future study, it would be interesting to analyse whether the human and animal isolates share other transferable characteristics, such as plasmid profiles and virulence traits (Johnson et al. 2008). This study has limitations. Because most of our isolates were resistant to five or more drugs, the relatively low proportion (51Æ4%) of identifiable gene cassette content may be explained by the inability of PCR to amplify integrons with three or more cassettes (>3 kb). Secondly, the sample sizes for E. coli isolates with dfr genes were relatively small. Thus, some caution is necessary in interpretation of the dfr allele distribution. Given that integron-associated dfr genes are often encoded on transferable genetic elements, dissemination of similar transposons or plasmids among the animal and human isolates cannot be excluded. In conclusion, identical gene cassette arrays encoding several dfr alleles and other resistance genes were found to be carried by E. coli isolates obtained from animal faeces, human faeces and or those causing in outpatients. The findings suggest that food-producing animals are an important reservoir for integrons carrying antimicrobial resistance genes. Future experimental studies to assess the efficiency of integron transfer using different donor and recipient E. coli combinations would be of value to improve our understanding of how resistance genes may flow across strains in the major phylogenetic groups. Acknowledgement Part of the work was submitted by River C. Wong to the University of Hong Kong in partial fulfillment of the requirement for Master in Philosophy. Funding The work is supported by research grants from RGC (HKU M) and the Research Fund for the Control of Infectious Diseases (RFCID) of the Health, Welfare and Food Bureau of the Hong Kong SAR Government. Transparency declarations The authors have nothing to declare. References Aarestrup, F.M., Wegener, H.C. and Collignon, P. (2008) Resistance in bacteria of the food chain: epidemiology and control strategies. Expert Rev Anti Infect Ther 6, Blahna, M.T., Zalewski, C.A., Reuer, J., Kahlmeter, G., Foxman, B. and Marrs, C.F. (2006) The role of horizontal gene transfer 632 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009)

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