Article pubs.acs.org/est
Prevalence of Antibiotic Resistance Genes in the Bacterial Flora of Integrated Fish Farming Environments of Pakistan and Tanzania Syed Q. A. Shah,*,† Duncan J. Colquhoun,‡ Hamisi L. Nikuli,§ and Henning Sørum† †
Department of Food Safety & Infection Biology, Norwegian School of Veterinary Science, P.O. Box 8146 Dep, Oslo N-0033, Norway ‡ Norwegian Veterinary Institute, Ullevålsveien 68, P.O. Box 750 Sentrum, N-0106 Oslo, Norway § Ministry of Livestock Development and Fisheries, P.O. Box 9152, Dar es Salaam, Tanzania S Supporting Information *
ABSTRACT: The use of a wide variety of antimicrobials in human and veterinary medicine, including aquaculture, has led to the emergence of antibiotic resistant pathogens. In the present study, bacteria from water, sediments, and fish were collected from fish farms in Pakistan and Tanzania with no recorded history of antibiotic use. The isolates were screened for the presence of resistance genes against various antimicrobials used in aquaculture and animal husbandry. Resistant isolates selected by disk diffusion and genotyped by Southern hybridization were further screened by polymerase chain reaction (PCR) and amplicon sequencing. The prominent resistance genes identified encoded tetracycline [tetA(A) and tetA(G)], trimethoprim [df rA1, df rA5, df rA7, df rA12, and df rA15], amoxicillin [blaTEM], streptomycin [strA-strB], chloramphenicol [cat-1], and erythromycin resistance [mefA]. The int1 gene was found in more than 30% of the bacterial isolates in association with gene cassettes. MAR indices ranged from 0.2 to 1. The blaNDM‑1 gene was not identified in ertapenem resistant isolates. It is hypothesized that integrated fish farming practices utilizing domestic farm and poultry waste along with antibiotic residues from animal husbandry may have contributed to a pool of resistance genes in the aquaculture systems studied.
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INTRODUCTION Although infectious fish diseases undoubtedly occurred prior to the origin of aquaculture practices, intensive fish farming creates various stressors that increase the susceptibility of fish to various pathogens.1 Aquatic fish pathogenic bacteria transmit more easily by water from fish to fish than terrestrial pathogenic bacteria transfer by air.2 Antimicrobials are the most important therapeutic agents used in control of infectious bacterial diseases and have saved millions of human lives since their introduction in the 1940s.3 The use of a wide variety of antimicrobials in human and veterinary medicine, agriculture, and aquaculture has led to the emergence of antibiotic resistant pathogens.3−5 Antimicrobials used in aquaculture are introduced mainly as medicated feed or immersion baths.2 As a result, water, sediments, and surrounding biological systems are often directly and indirectly exposed to the antimicrobials used.2,4 Persistence and spread of antibiotic resistance genes (ARGs) have been reported not only in pathogenic bacteria but also among environmental bacteria.3,6 To date, although the majority of antibiotic resistance (AR) studies focus on pathogenic bacteria, there is an increasing awareness that environmental bacteria comprise an important reservoir of drug resistance genes. It is evident from various reports that environmental bacteria, even in the absence of selective © 2012 American Chemical Society
antibiotic pressure, possess many diverse ARGs identical to those circulating in pathogenic microbiota in clinical environments.2−4 Countries such as Pakistan and Tanzania represent nations which may establish intensive aquaculture in the near future. Pakistan has great potential for aquaculture, especially in inland waters. Indian major carps and Chinese carps are cultivated on a commercial scale. Although fish farming is expanding, the industry remains at an early developmental stage (http://www. punjabfisheries.gov.pk/pdf/manual.pdf). Similarly, in Tanzania, although fish farming started in 1949,7 it is currently limited to small scale pond culture of native Tilapia species Pakistan and Tanzania have no recorded history of antibiotic use in aquaculture. Although information regarding the use of antimicrobials and AR in the natural environment is available for many European, South- and North-American, and East Asian countries, very little is known of South-East Asia and Africa.8 The aim of the present study was, therefore, to survey the presence of ARGs in aquatic and sedimental environmental bacteria in aquaculture sites on the Indian subcontinent and in Received: Revised: Accepted: Published: 8672
May 10, 2012 July 18, 2012 July 23, 2012 July 23, 2012 dx.doi.org/10.1021/es3018607 | Environ. Sci. Technol. 2012, 46, 8672−8679
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from swine, poultry, and cattle.7 Integrated fish farming (IFF) practices are common, especially in the Eastern regions, and polyculture of fish and duck is popular. Organic waste from cattle and poultry production is applied to fish ponds, 2 −7 times weekly at around 1000 kg/ha/annum (Personal communication). No antimicrobials are used in aquaculture, although different types of antimicrobials and growth promoters are used in poultry and animal husbandry10,11 (Table 6S). Antibiotic Susceptibility Testing. Antibiotic susceptibility testing was performed by the disk diffusion method on MüllerHinton (MH) agar (Oxoid Ltd., Basingstoke, U.K.) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.12 Inocula were prepared by making a direct saline suspension of isolated colonies selected from an overnight agar plate (a non-selective LA medium). All isolates were tested with tetracycline (80 μg), trimethoprim (5.2 μg), sulfamethoxazole (240 μg), amoxicillin (30 μg), oxolinic acid (10 μg), streptomycin (100 μg), chloramphenicol (60 μg), florfenicol (30 μg), and erythromycin (78 μg). Amoxicillin resistant isolates were further tested with ertapenem (10 μg) (NeoSensitabs Rosco, Taastrup, Denmark). Inhibition zone diameters (IZD) were measured to the nearest full millimeter (Table 1). There are no specific established IZD/MIC
East Africa, prior to a potential increase in intensive aquaculture and use of antimicrobials in these regions.
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MATERIALS AND METHODS Collection of Bacterial Samples. Bacterial isolates were collected in September to October 2009 from carp farms from the Multan and Lahore districts of the Punjab province, Pakistan, including fish hatcheries (Multan; 30°157744′N, 71°443909′E, Lahore; 31°585153′N, 74°458981′E) operated by the Punjab Fisheries Department. Personal communications were made with owners of private fish farms to obtain relevant information regarding use of antimicrobials, organic waste, and fish feed. In Tanzania, isolates were collected from tilapia farms from August to September 2008 in the Morogoro rural (7°023672′S, 37°340920′E, 6°075187′S, 37°373722′E, 6°500632′S, 37°414212′E) and Mvomero (6°234376′S, 38°692045′E) in Eastern Tanzania, and Masasi (10°400358′S, 38°555169′E, 11°060000′S, 38°390000′E) and Songea (10°404759′S, 35°283163′E, 10°492114′S, 35°322649′E) in Southern Tanzania. Bacterial samples were collected from water, sediments, and clinically healthy and diseased fish. Water samples were collected at a depth of approximately 5 cm subsurface from three different sites within each pond, and were subsequently pooled in a single sample. Sediment samples were collected with a sterile plastic pipet at a depth of ≥2 cm from the same sites and were pooled together as a single sample. Each sediment sample of approximately 0.1 g was mixed with 0.9 mL of sterilized saline solution. Samples were serially diluted up to 10-fold for inoculum preparation. Samples from fish were collected by gently rotating a sterilized cotton swab in the fish vent. The cotton swab was then agitated in sterilized saline to release the contents. All samples were stored on ice under transportation to the laboratory. A total of 253 bacterial isolates were collected, which included 127 from Pakistan (water, 50; sediments, 52; healthy fish, 25) and 126 from Tanzania (water, 42; sediments, 39; healthy fish, 34; diseased fish, 11). The samples were inoculated on Luria−Bertani agar (LA) plates and grown overnight at 30 °C. Manuring of the Fish Ponds. In Punjab, fresh organic waste, which is a mixture of both cattle and poultry waste, is applied approximately 10,000 to 12,000 kg/ha to newly constructed ponds (http://www.punjabfisheries.gov.pk/pdf/ manual.pdf). Fish fry are normally transferred from hatching ponds to rearing ponds in March/April when application of mixed organic waste is increased (totally 20−25,000 kg/ha/ annum in 7−10 installments between March and August) to boost production of zooplankton and phytoplankton. On average, farms use 30,000 to 40,000 kg organic waste ha/ annum,9 although the amount varies according to location, nature of the soil, and water pH (Personal communications). According to the official Web site of the Department of Fisheries Punjab, Pakistan (http://www.punjabfisheries.gov.pk/ pdf/manual.pdf), oxytetracycline and terramycin medicated feed is recommended for the treatment of abdominal dropsy and chloromycetin bath for treatment of the fin rot diseases of culture carps. However, during sampling, no fish farmer reported use of antimicrobials in their fish farm. Different types of antimicrobials and growth promoters are widely used in poultry and livestock farming (Table 5S of the Supporting Information). In Tanzania, fish feed includes maize bran, wheat bran, vegetables, kitchen waste, natural grasses, and animal manure
Table 1. Numbers and Percentages of Phenotypical Resistance Bacteria Isolated from Aquaculture Facilities of Pakistan and Tanzaniaa Pakistan antimicrobial tetracycline
Nb 55
trimethoprim
113
sulfonamides
70
amoxicillin
91
oxolinic acid
75
streptomycin
46
chloramphenicol
60
florfenicol
67
erythromycin
96
PRc 43.3 [26.9−60.1] 89 [77.1−98.6] 55.1 [38.3−71.7] 71.6 [55.8−86.2] 59 [42.3−75.3] 36.2 [20.5−52.8] 47.2 [30.6−64.1] 52.7 [35.9−69.4] 75.6 [60.3−89.3]
Tanzania Nb 62 86 84 89 87 58 59 81 91
PR 49.2 [32.4−66.1] 68.3 [52−83.4] 66.6 [50.3−82.1] 70.6 [54.6−85.4] 69 [52.9−84.1] 46 [29.3−63] 46.8 [30.1−63.7] 64.2 [47.7−80] 72.2 [56.4−86.7]
p-value 0.34 ≪0.01d 0.05 0.85 0.09 0.11 0.94 0.06 0.54
a
Results expressed in percentage with 95% confidence interval. bN, number of phenotypically resistant isolates. cPR, percentages of phenotypic resistance determined by disk diffusion. dSignificant difference.
breakpoints for susceptible and resistant environmental bacteria. However, using the guidelines of Kronvall13 and Smith et al.,14 we selected an IZD ≤ 25−30 mm as a breakpoint on the basis of our own data (Figures 1S and 2S). To verify the significance level of IZD, 95% confidence intervals of expected neighboring IZD values were constructed using the theory of simple binomial sequences.15 Determination of Multiple Antibiotic Resistance (MAR) Index. The MAR index values were calculated for selected isolates. The MAR index for a single isolate is “a/b”, where “a” represents the number of antimicrobials to which the particular isolate was resistant and “b” represents the number of 8673
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Table 2. Number and Percentages of Resistance Genes in the Aquaculture Facilities of Pakistan and Tanzania as Detected by PCRa Pakistan antimicrobial
geneb
Na
Tanzania frequencya
tetracycline trimethoprim
sulfonamides β-lactam streptomycin chloramphenicol erythromycin
df rA1 df rA5 df rA12 df rA15 sul1 sul2 blaTEM strA-B cat-1
42 18 58 1 8 13 33 8 5
37.1 15.9 51.3 0.8 11.4 18.5 36.2 17.3 8.3
geneb
Na
frequencya
tetA(A) tetA(G) df rA1 df rA7 df rA12
14 11 44 5 45
22.5 17.7 51.1 5.8 52.3
sul1 sul2 blaTEM strA-B cat-1 mefA
31 21 15 15 12 8
36.9 25 16.8 25.8 20.3 8.7
a Percentage of phenotypically resistant strains. N, number of positive isolates bDifferent screened resistance genes which were not detected in the present study are as follows. Tetracycline: tetA(B), tetA(C), tetA(D), tetA(E), tetA(H), and tetA(31). Trimethoprim: dfrA3, 5, 8, 9, 10, 14, 17, 24, 26, 2d. Sulfonamides: sul3. β-lactamase; blaSHv, blaOXA, blaNDM‑1. Streptomycin: aadA1, aadA2. Chloramphenicol/florfenicol: cmlA1, f lo. Erythromycin: ermB. Quinolones: qnrA, B, S1.
antimicrobials to which the isolate was exposed. 16 As antimicrobials are not used directly in the studied aquaculture facilities (the presumed source of antimicrobials being the livestock waste), we assumed that the nine antimicrobials tested represent the antimicrobials to which the isolates had been exposed in the present study. Colony Blotting and Southern Hybridization. Bacteria were transferred onto Hybond-N+ Nylon filters (Amersham Biosciences, U.K.) placed on LA plates from freshly grown LA plates and incubated overnight at 30 °C. DNA was released by treating the filters with 10% SDS, neutralizing solution (0.5 M Tris-Cl (pH 7.2), 1 M NaCl), and denaturing solution, 2× SSPE (3 M NaCl, 0.2 M NaH2PO4, 0.02 M EDTA) for 5 min each. The DNA was fixed to the nylon-filters by UV light. Probes specific for selected ARGs were oligolabeled in vitro [α-32P]dCTP (Hartmann Analytic, Braunschweig, Germany) using Ready-to-go DNA beads (GE Healthcare, Little Chalfont, U.K.) according to the manufacturer’s instructions. All membranes were prehybridized in 2× SSC, 0.1% SDS, 5× Denhardt’s solution for 2 h at 65 °C prior to hybridizations in 2× SSC, 0.1% SDS, 5× Denhardt’s solution, and 10% dextran sulfate for 18 h at 65 °C. The membranes were washed twice at 65 °C in 5× SSC, 0.1% SDS for 30 min, and once for 15 min.17 The membranes were exposed to X-ray film (Kodak XR, Rochester, NY) for 3−7 days at −80 °C depending on the radioactive probe signal strength. Films were developed in a dark room using G150 developer and G354 fixer solutions (Agfa Laboratories, Mortsel, Belgium) according to the manufacturer’s instructions. Polymerase Chain Reaction Amplifications and Gene Sequencing. All isolates showing phenotypical resistance and positive hybridization with probes were screened for the presence of various ARGs (Tables 2 and 3) with polymerase chain reaction (PCR). Positive and negative controls were used for each resistance gene tested (Tables 1S−3S). The oligonucleotide sequences of the primers used for the synthesis of probes and afterward for screening of ARGs with their annealing temperature and amplicon sizes are listed in Tables 1S−3S. Total DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Sollentuna, Sweden) according to the manufacturer’s instructions and stored at −20 °C. All the
isolates with class 1 integrons were screened for the presence of potential gene cassettes (GCs) with primers L1, L2, and R1 as described by Maguire et al.18 All PCR products were sequenced for the identity of the GCs. Screening of GCs for class 2 integrons was carried out with primer pairs Ti−F and Ti−B as described by Su et al.19 Because of an expected low frequency of int2, and the link with df rA1, we screened 14 dfr positive isolates from Pakistan and 12 from Tanzania for the occurrence of the int2 gene. The region between sul2 and tnpa is regarded as a hot spot for insertion of resistance genes due to the presence of an integrative transposase.20 The occurrence of such gene inserts was screened for by using the primer pair S4 and S16. Phenotypical characteristics and the amplified 16S rDNA gene sequences were used for identification of selected isolates. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Duesseldorf, Germany) and sequenced by GATC Biotech AG, Konstanz, Germany. Confirmation of the identity of sequences was carried out by NCBI BLASTn. Conjugation Experiments. In order to measure the extent of horizontal DNA transfer, 25 multiresistant environmental isolates, 12 from Tanzania and 13 from Pakistan, were selected for conjugation experiments. Conjugation was carried out as described elsewhere.21 However, Escherichia coli DH5α and HB101 were used as recipients according to the resistance profile of the donor, and bacteria were allowed to conjugate at 30 °C. Presumptive transconjugants were selected on MH plates with 10 μg/mL tetracycline, 50 μg/mL streptomycin, 100 μg/mL sulfonamide, 150 μg/mL ampicillin, 25 μg/mL nalidixic acid, and 50 μg/mL trimethoprim according to the donor and recipient resistance properties. The identity of transconjugants was confirmed with biochemical tests specific for E. coli. Selected transconjugants were further screened for the presence of potential resistance genes by PCR and amplicon sequencing. Aeromonas salmonicida ss salmonicida [NVI-2402/89 (718)] with promiscuous IncU plasmid pRAS1 was used as a positive control donor. Statistical Analysis. The results are expressed in percentages with 95% confidence intervals (Tables 1). Confidence intervals were constructed using the theory of simple binomial sequences.15 Differences between groups were considered significant if the p-value of the χ2 test was found less 8674
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Figure 1. Percentages of single and multiple antimicrobial resistances among the Pakistani and Tanzanian isolates (S, susceptible; A, antimicrobial/s; PK, Pakistan; TNZ, Tanzania).
(53%) Pakistani sul2 positive isolates also possessed the strAstrB genes. The existence of cassettes between sul2 and tnpa was screened for. We found sul2 and the transposase of ISVS3 in four Pakistani isolates without any inserted cassette. Screening of the 14 df r positive isolates from Pakistan and 12 from Tanzania for the occurrence of the int2 gene yielded int2 among eight Pakistani isolates and two Tanzanian isolates. None incorporated df rA1 as a potential GC. int3 was not found in any of the tested isolates. Among the trimethoprim resistant isolates, df rA1 and df rA12 carriers were most frequently identified. In Pakistani isolates, these genes were copresent in 25 isolates. df rA1, df rA5, and df rA12 were copresent in 14 while a single sediment isolate contained df rA1, df rA5, df rA12, and dfrA15. Only dfrA15 was found as a GC in a class 1 integron in this isolate. Most of the Tanzanian isolates contained both dfrA1 and dfrA12. No Pakistani or Tanzanian isolates contained df rA3, 7, 8, 9, 10, 14, 17, 24, 26, or df r2df (Table 2). Among amoxicillin resistant isolates, blaTEM was the most frequently identified resistance determinant, while blaSHV and blaOXA were not found. Amoxicillin resistant isolates were further tested for ertapenem (10 μg) resistance, and the resistant isolates were then screened for blaNDM‑1, but with negative results. Among streptomycin resistant isolates, all Pakistani strA-strB containing isolates also contained sul2 (with the exception of a single isolate). sul2 could not be identified in Tanzanian strA-strB positive isolates. Isolates with strA-strB genes were also screened for the RSF1010 plasmid, but it was
or equal to a significance level of 5%. Comparison of groups with regard to categorical variables was performed by using Contingency Table Analysis.15
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RESULTS Phenotypical Resistance, MAR Indices, and Resistant Bacterial Species Found in Fish Farming Environments of Pakistan and Tanzania. Prevalences of phenotypical resistance as determined by disk diffusion are listed in Table 1. Of the 253 isolates included in the present study, only 2% (6/ 253) were phenotypically susceptible and 10% (26/253) were resistant to all the nine tested antimicrobials. All remaining isolates demonstrated from one to eight drug resistance phenotypes (Figure 1). MAR indices of 0.4 to 1 were identified for Pakistani isolates and 0.2 to 1 for Tanzanian isolates (Table 3). The spectrum of multiresistant bacterial species identified by phenotypical investigation and partial 16S rDNA gene sequencing, and having three or more drug resistance genes, is listed in Tables 3 and 4S. Detection of Resistance Genes, Integrons, and Gene Cassettes in Bacterial Isolates from Pakistan and Tanzania. Identified ARGs are listed in Table 2. Tetracycline resistance genes, tetA(A) and tetA(G), were found only in Tanzanian isolates. With the exception of a single isolate, all tetA(G) positive isolates also possessed tetA(A). All sul1 containing isolates also possessed intl1. Pakistani isolates harboring intl1 incorporated df rA15 as a GC while Tanzanian isolates incorporated dfrA7. Eleven (52%) Tanzanian and seven 8675
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Table 3. Antimicrobial Resistance Genes and Gene Cassettes Present in Multiple Antimicrobial Resistant Bacteria Isolated from Pakistan and Tanzaniaa isolate Acinetobacter baumannii LS110 Acinetobacter baumannii LW90 Aeromonas hydrophila LF81 Alcaligenes sp. MS34 Alcaligenes sp. MS31 Enterobacter cloacae MF68 Enterobacter cloacae LF86 Pseudomonas aeruginosa MW11B Pseudomonas aeruginosa MS53 Pseudomonas f luorescens MW17B Pseudomonas sp. MW20B Enterococcus casselif lavus MF69 Aeromonas sp. T30 Acinetobacter sp. T4 Exiguobacterium spp. T14 Kurthia gibsonii T31 Empedobacter brevis T96 Klebsiella oxytoca T106 Proteus sp. T117 Proteus vulgaris T17 Proteus sp. T50 Proteus sp. T51 Proteus sp. T59 Planococcus sp. T16 Pseudomonas sp .T7 Pseudomonas f luorescens T19 Pseudomonas sp. T29 Pseudomonas f luorescens T103 Pseudomonas f luorescens T34 Providencia alcalifaciens T27 Vagococcus f luvialis T99
region
Tet
MAR index
Sul sul1
int1
cassette between Intl1and sulI
+
dfrA15
cassette between tnpAand Sul.II
Strept
Trim
Amox
Chl, Flor
0.6
PK
0.7
PK
0.5
sul2
PK
0.4
sul2
ISCR Element
PK
1
sul2
ISCR Element
PK
0.8
sul2
+
PK
0.5
sul1
+
df rA1,12
PK
0.7
sul1, sul2
+
df rA1,5,12
PK
0.8
sul1, sul2
+
PK
0.7
sul1
+
df rA5,12
PK
0.8
sul1, sul2
+
df rA12
blaTEM
PK
0.8
df rA1,5,12
blaTEM
cat-1
TNZ TNZ TNZ
0.8 0.6 0.5
df rA1,12 df rA1,12 df rA1,12
blaTEM blaTEM
cat-1
TNZ
0.2
TNZ
0.6
sul1
+
TNZ
0.4
sul1, sul2
+
1 0.8 1 0.8 0.7 0.3 1 1
sul1, sul1, sul1, sul1, sul1,
sul2 sul2 sul2 sul2 sul2
+ + + + +
sul1 sul1, sul2
+ +
1
sul1
+
TNZ TNZ TNZ TNZ TNZ
A, G A A, G A
TNZ TNZ
A, G
TNZ
+
df rA1 strA-strB
strA-strB
strA-strB
+
df rA1,12
df rA1,12 blaTEM
df rA12
mefA
df rA1,12 df rA1,12
blaTEM blaTEM
dfrA7 strA-strB strA-strB strA-strB strA-strB
dfrA7 dfrA7 dfrA7
strA-strB
df rA1,7,12 df rA1,12 DfrA7 DfrA7 df rA1,7,12 df r1,12 df rA1,12 df rA1,12
mefA blaTEM blaTEM blaTEM
cat-1 cat-1 cat-1
blaTEM blaTEM
cat-1
mefA
cat-1
TNZ
A, G
0.8
sul1, sul2
+
strA-strB
TNZ
A, G
0.7
sul1, sul2
+
strA-strB
TNZ
blaTEM
strA-strB
+ sul1
Eryth
df rA1,5,12,15
PK
df rA1,12
mefA cat-1
0.5 df r1,12
0.7
a
Pk, Pakistan; TNZ, Tanzania; Tet, tetracycline; Trim, trimethoprim; Sul, sulfonamide; Amox, amoxicillin; Strept, streptomycin; Chl, chloramphenicol; Flor, florfenicol; Eryth, erythromycin.
Detection of Conjugation in the Pakistani and Tanzanian Isolates. Two transconjugants were found, with one donor from Pakistan and one from Tanzania. Both transconjugants harbored the expected tetA(A) and dfrA1 genes. No transconjugants appeared from isolates with sulfonamide, and amoxicillin resistance determinants.
not found. Florfenicol/chloramphenicol resistant isolates possessed only the cat-1 gene. f lo and cmlA1 were not detected. Plasmid mediated quinolone (oxolinic acid) resistance (qnrA, qnrB, and qnrS1) was not found. Of erythromycin resistant isolates, mefA was found only in Tanzanian isolates, while ermB was not detected (Table 2). 8676
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more than 70−80% of described trimethoprim resistance.35,36 We found co-localization of df r genes in both studied areas. Although co-occurrence of multiple df r genes together is not common,36 it has been reported in European and Korean studies.35,37 Although use of sulfonamides has decreased worldwide, they remain in common use in developing Asian and African countries due to their low cost. We found GCs (Tanzania, df rA7; Pakistan, dfrA15) in class 1 integrons but none in class 2 integrons, although df rA1 was most commonly identified. It may be that df rA1 is omitted from the original df rA−sat1aadA1 arrangement.19 The sul2−ISVS3 region is considered as a hotspot for the insertion of genes due to the presence of an integrative transposase.20 We found sul2 and transposase of ISVS3 in four Pakistani isolates without any gene insert. The sul2−ISVS3 region has been reported in different types of bacterial taxa (Accession No. AJ289135.1),20 both freshwater and marine, but to the best of our knowledge, no gene insert has yet been reported. Resistance to streptomycin was comparatively infrequent in bacteria isolated from the two study areas, but the presence of strA-strB linked with sul2 conferring resistance against streptomycin and sulfonamides was a trend in Pakistani isolates. Linkage of strA-strB genes with sul2 is a characteristic feature of the RSF1010 plasmid.38 We screened the strA-strB carrying isolates for RSF1010, but the plasmid was not found. The absence of RSF1010 may indicate co-selection and/or cross-selection of streptomycin resistance determinants21,22,24 or the presence of some other plasmid (p9123, Accession No. AY360321.1; pV1678, Accession No. EF 090911.1; pQ1-1, Accession No. HM 371192.1; PETEC-6, Accession No. gb CP000798.1). As many of the resistance genes are inserted as GCs in different types of integrons in resistance plasmids (Rplasmids),23 we expected a considerable transfer of resistant determinants from environmental isolates to E. coli, but only two transconjugants were identified. Acinetobacter baumannii from Pakistan and Pseudomonas f luorescens from Tanzania were able to transfer tetA(A) and dfrA1 genes. This indicates that the frequency of R-plasmid transfer is lower in environmental bacteria than in clinical bacteria. The proportion of class 1 integrons in environmental bacteria is relatively low compared with clinical isolates, but this estimation may be biased as 0.2 are regarded as an indicator of anthropogenic contamination. MAR index values
