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High level of resistance to Aztreonam and Ticarcillin in Pseudomonas aeruginosa isolated from soil of different crops in Brazil
Science of the Total Environment 473–474 (2014) 155–158
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
High level of resistance to Aztreonam and Ticarcillin in Pseudomonas aeruginosa isolated from soil of different crops in Brazil André Pitondo-Silva, Vinicius Vicente Martins, Ana Flavia Tonelli Fernandes, Eliana Guedes Stehling ⁎ Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto, Brazil
H I G H L I G H T S • • • • •
Aztreonam and ticarcillin are antibiotics used against P. aeruginosa. Forty isolates from Brazilian soil were analyzed for antibiotic resistance. The vast majority of the isolates presented resistance for both antibiotics. Four isolates presented plasmids and eight isolates possess the class 1 integron. Environmental P. aeruginosa showed surprising resistance for both antibiotics.
a r t i c l e
i n f o
Article history: Received 29 August 2013 Received in revised form 28 November 2013 Accepted 5 December 2013 Available online 22 December 2013 Keywords: Pseudomonas aeruginosa Plasmids Antibiotic resistance Soil
a b s t r a c t Pseudomonas aeruginosa can be found in water, soil, plants and, human and animal fecal samples. It is an important nosocomial pathogenic agent characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance through some mechanisms, among them, by the acquisition of plasmids and integrons, which are mobile genetic elements. In this study, 40 isolates from Brazilian soil were analyzed for antibiotic resistance, presence of integrons and plasmidial profile. The results demonstrated that the vast majority of the isolates have shown resistance for aztreonam (92.5%, n = 37) and ticarcillin (85%, n = 34), four isolates presented plasmids and eight isolates possess the class 1 integron. These results demonstrated that environmental isolates of P. aeruginosa possess surprising antibiotic resistance profile to aztreonam and ticarcillin, two antimicrobial agents for clinical treatment of cystic fibrosis patients and other infections occurred by P. aeruginosa. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Pseudomonas aeruginosa is a bacterium distributed in nature and has received the definition of bacterium ubiquity. It has a great ability of survival strategies such as pigments and production of biofilm (De Vos et al., 1997) and is an organism that has as its main habitat water, inhabiting superficial and average depth regions of the limnetic zone, where it can survive for some months in the ambient temperature (Schwartz et al., 2006) and in various terrestrial environments, including agricultural soil (Deziel et al., 1996; Kaszab et al., 2011). Beyond water and soil, this microorganism can also be found in plants and in human and animal fecal samples (Dubois et al., 2001; Colinon et al., 2010). The incidence in human excrement of healthy adults is 12%, and in clinic and hospital employees, 10 colony forming units per gram of excrement can be isolated. In hospitals, P. aeruginosa represents a great factor of risk because of its capacity to survive on surfaces of inert materials, ventilation equipment, in detergent solutions ⁎ Corresponding author. Tel.: +55 16 3620 0285. E-mail address: [email protected] (E.G. Stehling). 0048-9697/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.12.021
and on the hands of healthcare professionals (Dubois et al., 2001), and can still be isolated as member of the normal flora of the skin and throat of healthy individuals (Shrivastava et al., 2004). P. aeruginosa is also an important nosocomial pathogenic agent (Jarvis and Martone, 1992) characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance (Poole and Srikumar, 2001; Wang et al., 2006; Kerr and Snelling, 2009) through some mechanisms, among them, by the acquisition of plasmids and integrons, which are mobile genetic elements that are potentially important in the dissemination of multiple resistance among gram-negative bacteria, especially in Pseudomonas (Li et al., 2006; Poole, 2011). Many studies have shown the great potential of clinical isolates of P. aeruginosa to acquire antimicrobial resistance, however, few studies report the resistance profile and its acquisition mechanisms in environmental isolates, especially those obtained from soil. Thus, the aim of this study was to determine the antibiotic resistance profile of P. aeruginosa isolated from soil of some regions of Brazil and to determine if this resistance profile was correlated with the presence of integrons and plasmids.
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2. Material and methods 2.1. Isolation and identification of P. aeruginosa Isolation and identification of P. aeruginosa were performed as described previously by Mukherjee et al. (2011). Soil samples were collected between February 2009 and April 2013, from plantation areas of different vegetation (beetroot, cabbage, cherry, chili, chrysanthemums, coffee, eucalyptus, maize, orange, papaya, peppers sorghum, soya and sugar cane), originating from different areas of Brazil, covering six different states and contemplating 3 of the 5 regions of the country. The samples were collected 5 cm below the land surface in sterile containers. One gram of each soil sample was dispersed in 9 mL of a 0.85% NaCl sterile solution and a series of dilutions from 10−2 to 10−10 were performed. A 0.2 mL aliquot of the appropriate dilution was spread aseptically onto Cetrimide Agar (Fluka, USA) and was incubated at 37 °C for 48–72 h. Identification of P. aeruginosa was performed by the following tests: oxidase production, pigmentation, growth at 42 °C and biochemical tests, e.g., carbohydrate fermentation (−), citrate assimilation (+), indol (− ), and lysine decarboxylase (−) (Murray et al., 1995). 2.2. DNA extraction Genomic DNA from the isolates identified as P. aeruginosa was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany) following the manufacturer's instructions, after overnight growth in LB medium. 2.3. Research of oprL gene by PCR (Polymerase Chain Reaction) Confirmation of P. aeruginosa specie was performed by PCR using specific primers to amplify the open reading frame of the oprL gene (De Vos et al., 1997). The primers were obtained from Invitrogen (Brazil) and had the following sequences: PAL1, 59-ATGGAAATGCTG AAATTCGGC-39 (a 21-mer corresponding to the beginning of the open reading frame of oprL), and PAL2, 59-CTTCTTCAGCTCGACGCGACG-39 (a 21-mer corresponding to the end of the open reading frame of oprL). The PCR reactions were performed as described by De Vos et al. (1997). PCR products were observed after agarose gel (1.8%) electrophoresis by staining with ethidium bromide. P. aeruginosa ATCC 27853 served as positive control. 2.4. Antimicrobial susceptibility testing Antimicrobial susceptibility testing was performed by disc diffusion on Mueller–Hinton agar (Oxoid) in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI, 2013). The following antimicrobial drug discs and their respective concentrations tested in this study were: amikacin (30 μg), aztreonam (30 μg), cefepime (30 μg), ceftazidime (30 μg), ciprofloxacin (5 μg), imipenem (10 μg), levofloxacin (5 μg), lomefloxacin (10 μg), meropenen (10 μg), norfloxacin (10 μg), ofloxacin (5 μg), piperacillin–tazobactam (100 μg–10 μg), polymyxin B (300 units), sulfametoxazol-trimetoprim (23.75 μg–1.25 μg), tetracycline (30 μg), ticarcillin-clavulanate-acid 7.5:1 (85 μg) and tobramycin (10 μg). P. aeruginosa ATCC 27853 served as quality control strains for antimicrobial susceptibility tests. Isolates were considered multidrug resistant (MDR) if they displayed simultaneous resistance to two or more antimicrobial drug classes.
30 min. The plasmidial bands were revealed and documented under UV light. Eight plasmids of E. coli V517 (Macrina et al., 1987) with molecular known masses (in megadaltons; 35.84, 4.82, 3.67, 3.39, 2.63, 2.03, 1.79 and 1.36) and four plasmids of E. coli 39R861 (98, 42, 24 and 4.6) were used as control of extractions and also to measure the molecular weight of the plasmids present in bacterial isolates of the study. The molecular weights of the plasmids were calculated by comparison with the migrating plasmids of known sizes present in the reference strains using the software BioNumerics software. 5.1 (Applied Maths, Belgium). 2.6. Detection of integrons PCR was used to detect the presence of integrase genes. Class 1, 2 and 3 integrase primers used and PCR conditions were carried out according to Fonseca et al. (2005), with modifications according described by Zanetti et al. (2013). 3. Results and discussion In this study, 40 bacterial isolates (designated S03, S05, S10 and the 37 remaining denominated S21 to S57) were obtained from soil cultivated with several types of crops in 15 different cities of Brazilian states. The analysis of the plasmidial profile has demonstrated that four isolates (S05, S10, S41 and S51) presented plasmids with 54, 82, 16 and 35 MDa, respectively. The research of class 1, 2 and 3 integrons was also performed and it was observed that eight isolates (S03, S05, S15, S30, S33, S34, S35 and S47) possess the int1 gene for the class 1 integron, which has a size of 1224 pb (data not shown). All eight isolates presented resistance to sulfametoxazole. None of the isolates presented class 2 and 3 integrons (Table 1). Antimicrobial susceptibility testing was carried out with these isolates of P. aeruginosa and 17 different antimicrobials have been tested. The results have demonstrated that the vast majority of the isolates have shown resistance to aztreonam (92.5%, n = 37) and ticarcillin (85%, n = 34) (Fig. 1). The three isolates that did not present resistance to aztreonam, S03, S05 and S57, were ticarcillin resistant, and isolate S05 also presented resistance to tetracycline. Among the six isolates sensitive to ticarcillin, all of them were resistant to aztreonam and just one, isolate S10, has presented additional resistance for polymixin. Moreover, isolate 41, which was resistant to aztreonam and ticarcillin, also presented resistance to meropenem. Some works have analyzed the antimicrobial resistance profile in environmental isolates of P. aeruginosa. In the work of Kaszab et al. (2011) it was observed that 11 P. aeruginosa isolates (30.5%) were resistant to at least two different classes of antimicrobials, especially thirdgeneration cephalosporins, wide spectrum penicillins, aminoglycosides, and carbapenems. The work of Deredjian et al. (2011) has shown that in soil-related samples, 14% of the isolates showed resistance to up to 5 antibiotics (ciprofloxacin, gentamicin, imipenem, ticarcillin and clavulanic acid, and tobramycin). Deredjian et al. (2011) and Kaszab et al. (2011) found resistance to cephalosporine, ticarcillin, tobramycin and imipenem
Table 1 Antimicrobial resistance and plasmidial profile found in P. aeruginosa isolated from soil of different crops. Antimicrobial resistance profile
Isolates
Plasmidial profile MW (MDa)
ATM TCC TCC + TET ATM + PMB ATM + TCC + MEM ATM + TCC
All isolates, except S03, S05, S57 All isolates, except S10, S27, S28, S31, S42, S53 S05 S10 S41 S51
– – 54 82 16 35
2.5. Plasmid DNA extraction and agarose gel electrophoresis Plasmid DNA extraction was performed by the alkaline lysis method as recommended by Takahashi and Nagano (1984). Plasmid samples were resuspended in TE buffer and were mixed with a 10x loading buffer containing RNase (2 μg mL−1) (Fermentas, Brazil). Electrophoresis was carried out on a horizontal apparatus as outlined by Sambrook et al. (1989). The gel was stained with ethidium bromide (0.5 μg mL−1) for
Aztreonam (ATM), ticarcillin-clavulanate (TCC), polymixin B (PMB), tetracycline (TET), meropenem (MER). Molecular weight (MW).
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157
100 90
Total resistance (%)
80 70 60 50 40 30 20 10 0 ATM
TCC
MEM
TET
PMB
Antibiotics tested which presented resistance. Fig. 1. Frequency of total resistance to tested antibiotics for 40 P. aeruginosa studied. Aztreonam (ATM), ticarcillin-clavulanate-acid (TCC), meropenem (MEM), tetracycline (TET) and polymyxin B (PMB).
and in the work of da Silva et al. (2008), the isolates showed resistance to chloramphenicol, gentamicin and trimethoprim-sulfamethoxazole. None of these cited works have researched or made some correlation between antimicrobial resistance and plasmids or integrons. Knapp et al. (2010) showed that soil microbes have become progressively more resistant to antibiotics over the last 60 years, even with more stringent rules on the use of antibiotics in medicine and agriculture. These authors have concluded that there is an increasing chance of finding organisms in nature that are resistant to antimicrobial therapy. Other authors (LaPara et al., 2011) have demonstrated that in sewage treatment many of the bacterial genes that cause antibiotic resistance may be released into effluent and are a source of contamination. According to Martínez et al. (2011), in places mostly affected by human activity, contact between clinical pathogens and environmental bacteria, in the presence of several antimicrobial selective pressures, is favorable for the exchange and dissemination of resistance genes; in ecosystems where antibiotic-producing organisms (some bacteria and different types of fungi) are present, the spread of resistance genes is also higher (D'Costa et al., 2006). Isolates from soil plantations are in contact with human activity and can be close to soil bacteria and fungi that produce antibiotics which harbor resistance elements for self-protection (Arenas-Lago et al., 2013; Cerqueira et al., 2011; Oliveira et al., 2012). These isolates serve as a reservoir of resistance determinants that can be mobilized into the microbial community (D'Costa et al., 2006). This may be the explanation for the number of resistance elements and multi-resistant strains of soil found in this work. The present results are different from other studies, especially with respect to resistance to aztreonam in 92.7% of the isolates and ticarcillin in 85%. Although no multidrug resistance bacteria were found, the results draw attention to the high level of resistance to two antimicrobials commonly used in clinical practice, especially in the treatment of patients with cystic fibrosis (Zobelt et al., 2010; Oermann et al., 2011; Prescott et al., 2011; Assael et al., 2013). Since this resistance profile to aztreonam and ticarcillin is not correlated with the presence of plasmids or integrons in these isolates, the results suggest that other antimicrobial resistance mechanisms are present, as possible changes in the efflux pump (Poole and Srikumar, 2001), in MexAB or MexXY systems, which is subsequently investigated.
However, three of the four isolates possessing plasmids (S05, S10 and S41) are resistant to tetracycline, polymyxin and meropenem respectively, and in these isolates such resistance can be related to the presence of these mobile elements. Isolate S51, which has just aztreonam and ticarcillin resistance and a plasmid with 35 MDa has been studied, and in a work not yet published, this plasmid was related to the degradation of a herbicide and is therefore characterized as a catabolic plasmid (unpublished results). In conclusion, the present results demonstrate the potential of environmental isolates of P. aeruginosa to acquire antibiotic resistance, whether by acquisition of plasmids, integrons or by chromosomal alteration. These results suggest that restrictions on the use of antibiotics in humans, animals and agriculture are necessary to improve the current situation. Similar results were reported by previous researches (Hower et al., 2013; Kronbauer et al., 2013; Martinello et al., 2014; Quispe et al., 2012; Ribeiro et al., 2013a,b; Sanchis et al., 2013; Silva et al., 2011, 2012). Conflict of interest We have no conflict of interest to declare. Acknowledgments This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 2009/16657-3). We also thank John Carpenter for the English revision. References Arenas-Lago D, Vega FA, Silva LFO, Andrade ML. Soil interaction and fractionation of added cadmium in some Galician soils. Microchem J 2013;110:681–90. Assael BM, Pressler T, Bilton D, Fayon M, Fischer R, Chiron R, et al. Inhaled aztreonam lysine vs. inhaled tobramycin in cystic fibrosis: a comparative efficacy trial. J Cyst Fibros 2013;12:130–40. Cerqueira B, Vega FA, Serra C, Silva LFO, Andrade ML. Time of flight secondary ion mass spectrometry and high-resolution transmission electron microscopy/energy dispersive spectroscopy: a preliminary study of the distribution of Cu2+ and Cu2+/Pb2+ on a Bt horizon surfaces. J Hazard Mater 2011;195:422–31. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. CLSI M100-S23Wayne, PA: Clinical and Laboratory Standards Institute; 2013.
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Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
High level of resistance to Aztreonam and Ticarcillin in Pseudomonas aeruginosa isolated from soil of different crops in Brazil André Pitondo-Silva, Vinicius Vicente Martins, Ana Flavia Tonelli Fernandes, Eliana Guedes Stehling ⁎ Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto, Brazil
H I G H L I G H T S • • • • •
Aztreonam and ticarcillin are antibiotics used against P. aeruginosa. Forty isolates from Brazilian soil were analyzed for antibiotic resistance. The vast majority of the isolates presented resistance for both antibiotics. Four isolates presented plasmids and eight isolates possess the class 1 integron. Environmental P. aeruginosa showed surprising resistance for both antibiotics.
a r t i c l e
i n f o
Article history: Received 29 August 2013 Received in revised form 28 November 2013 Accepted 5 December 2013 Available online 22 December 2013 Keywords: Pseudomonas aeruginosa Plasmids Antibiotic resistance Soil
a b s t r a c t Pseudomonas aeruginosa can be found in water, soil, plants and, human and animal fecal samples. It is an important nosocomial pathogenic agent characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance through some mechanisms, among them, by the acquisition of plasmids and integrons, which are mobile genetic elements. In this study, 40 isolates from Brazilian soil were analyzed for antibiotic resistance, presence of integrons and plasmidial profile. The results demonstrated that the vast majority of the isolates have shown resistance for aztreonam (92.5%, n = 37) and ticarcillin (85%, n = 34), four isolates presented plasmids and eight isolates possess the class 1 integron. These results demonstrated that environmental isolates of P. aeruginosa possess surprising antibiotic resistance profile to aztreonam and ticarcillin, two antimicrobial agents for clinical treatment of cystic fibrosis patients and other infections occurred by P. aeruginosa. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Pseudomonas aeruginosa is a bacterium distributed in nature and has received the definition of bacterium ubiquity. It has a great ability of survival strategies such as pigments and production of biofilm (De Vos et al., 1997) and is an organism that has as its main habitat water, inhabiting superficial and average depth regions of the limnetic zone, where it can survive for some months in the ambient temperature (Schwartz et al., 2006) and in various terrestrial environments, including agricultural soil (Deziel et al., 1996; Kaszab et al., 2011). Beyond water and soil, this microorganism can also be found in plants and in human and animal fecal samples (Dubois et al., 2001; Colinon et al., 2010). The incidence in human excrement of healthy adults is 12%, and in clinic and hospital employees, 10 colony forming units per gram of excrement can be isolated. In hospitals, P. aeruginosa represents a great factor of risk because of its capacity to survive on surfaces of inert materials, ventilation equipment, in detergent solutions ⁎ Corresponding author. Tel.: +55 16 3620 0285. E-mail address: [email protected] (E.G. Stehling). 0048-9697/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.12.021
and on the hands of healthcare professionals (Dubois et al., 2001), and can still be isolated as member of the normal flora of the skin and throat of healthy individuals (Shrivastava et al., 2004). P. aeruginosa is also an important nosocomial pathogenic agent (Jarvis and Martone, 1992) characterized by an intrinsic resistance to multiple antimicrobial agents and the ability to develop high-level (acquired) multidrug resistance (Poole and Srikumar, 2001; Wang et al., 2006; Kerr and Snelling, 2009) through some mechanisms, among them, by the acquisition of plasmids and integrons, which are mobile genetic elements that are potentially important in the dissemination of multiple resistance among gram-negative bacteria, especially in Pseudomonas (Li et al., 2006; Poole, 2011). Many studies have shown the great potential of clinical isolates of P. aeruginosa to acquire antimicrobial resistance, however, few studies report the resistance profile and its acquisition mechanisms in environmental isolates, especially those obtained from soil. Thus, the aim of this study was to determine the antibiotic resistance profile of P. aeruginosa isolated from soil of some regions of Brazil and to determine if this resistance profile was correlated with the presence of integrons and plasmids.
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2. Material and methods 2.1. Isolation and identification of P. aeruginosa Isolation and identification of P. aeruginosa were performed as described previously by Mukherjee et al. (2011). Soil samples were collected between February 2009 and April 2013, from plantation areas of different vegetation (beetroot, cabbage, cherry, chili, chrysanthemums, coffee, eucalyptus, maize, orange, papaya, peppers sorghum, soya and sugar cane), originating from different areas of Brazil, covering six different states and contemplating 3 of the 5 regions of the country. The samples were collected 5 cm below the land surface in sterile containers. One gram of each soil sample was dispersed in 9 mL of a 0.85% NaCl sterile solution and a series of dilutions from 10−2 to 10−10 were performed. A 0.2 mL aliquot of the appropriate dilution was spread aseptically onto Cetrimide Agar (Fluka, USA) and was incubated at 37 °C for 48–72 h. Identification of P. aeruginosa was performed by the following tests: oxidase production, pigmentation, growth at 42 °C and biochemical tests, e.g., carbohydrate fermentation (−), citrate assimilation (+), indol (− ), and lysine decarboxylase (−) (Murray et al., 1995). 2.2. DNA extraction Genomic DNA from the isolates identified as P. aeruginosa was extracted using the QIAamp DNA Mini Kit (QIAGEN, Germany) following the manufacturer's instructions, after overnight growth in LB medium. 2.3. Research of oprL gene by PCR (Polymerase Chain Reaction) Confirmation of P. aeruginosa specie was performed by PCR using specific primers to amplify the open reading frame of the oprL gene (De Vos et al., 1997). The primers were obtained from Invitrogen (Brazil) and had the following sequences: PAL1, 59-ATGGAAATGCTG AAATTCGGC-39 (a 21-mer corresponding to the beginning of the open reading frame of oprL), and PAL2, 59-CTTCTTCAGCTCGACGCGACG-39 (a 21-mer corresponding to the end of the open reading frame of oprL). The PCR reactions were performed as described by De Vos et al. (1997). PCR products were observed after agarose gel (1.8%) electrophoresis by staining with ethidium bromide. P. aeruginosa ATCC 27853 served as positive control. 2.4. Antimicrobial susceptibility testing Antimicrobial susceptibility testing was performed by disc diffusion on Mueller–Hinton agar (Oxoid) in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI, 2013). The following antimicrobial drug discs and their respective concentrations tested in this study were: amikacin (30 μg), aztreonam (30 μg), cefepime (30 μg), ceftazidime (30 μg), ciprofloxacin (5 μg), imipenem (10 μg), levofloxacin (5 μg), lomefloxacin (10 μg), meropenen (10 μg), norfloxacin (10 μg), ofloxacin (5 μg), piperacillin–tazobactam (100 μg–10 μg), polymyxin B (300 units), sulfametoxazol-trimetoprim (23.75 μg–1.25 μg), tetracycline (30 μg), ticarcillin-clavulanate-acid 7.5:1 (85 μg) and tobramycin (10 μg). P. aeruginosa ATCC 27853 served as quality control strains for antimicrobial susceptibility tests. Isolates were considered multidrug resistant (MDR) if they displayed simultaneous resistance to two or more antimicrobial drug classes.
30 min. The plasmidial bands were revealed and documented under UV light. Eight plasmids of E. coli V517 (Macrina et al., 1987) with molecular known masses (in megadaltons; 35.84, 4.82, 3.67, 3.39, 2.63, 2.03, 1.79 and 1.36) and four plasmids of E. coli 39R861 (98, 42, 24 and 4.6) were used as control of extractions and also to measure the molecular weight of the plasmids present in bacterial isolates of the study. The molecular weights of the plasmids were calculated by comparison with the migrating plasmids of known sizes present in the reference strains using the software BioNumerics software. 5.1 (Applied Maths, Belgium). 2.6. Detection of integrons PCR was used to detect the presence of integrase genes. Class 1, 2 and 3 integrase primers used and PCR conditions were carried out according to Fonseca et al. (2005), with modifications according described by Zanetti et al. (2013). 3. Results and discussion In this study, 40 bacterial isolates (designated S03, S05, S10 and the 37 remaining denominated S21 to S57) were obtained from soil cultivated with several types of crops in 15 different cities of Brazilian states. The analysis of the plasmidial profile has demonstrated that four isolates (S05, S10, S41 and S51) presented plasmids with 54, 82, 16 and 35 MDa, respectively. The research of class 1, 2 and 3 integrons was also performed and it was observed that eight isolates (S03, S05, S15, S30, S33, S34, S35 and S47) possess the int1 gene for the class 1 integron, which has a size of 1224 pb (data not shown). All eight isolates presented resistance to sulfametoxazole. None of the isolates presented class 2 and 3 integrons (Table 1). Antimicrobial susceptibility testing was carried out with these isolates of P. aeruginosa and 17 different antimicrobials have been tested. The results have demonstrated that the vast majority of the isolates have shown resistance to aztreonam (92.5%, n = 37) and ticarcillin (85%, n = 34) (Fig. 1). The three isolates that did not present resistance to aztreonam, S03, S05 and S57, were ticarcillin resistant, and isolate S05 also presented resistance to tetracycline. Among the six isolates sensitive to ticarcillin, all of them were resistant to aztreonam and just one, isolate S10, has presented additional resistance for polymixin. Moreover, isolate 41, which was resistant to aztreonam and ticarcillin, also presented resistance to meropenem. Some works have analyzed the antimicrobial resistance profile in environmental isolates of P. aeruginosa. In the work of Kaszab et al. (2011) it was observed that 11 P. aeruginosa isolates (30.5%) were resistant to at least two different classes of antimicrobials, especially thirdgeneration cephalosporins, wide spectrum penicillins, aminoglycosides, and carbapenems. The work of Deredjian et al. (2011) has shown that in soil-related samples, 14% of the isolates showed resistance to up to 5 antibiotics (ciprofloxacin, gentamicin, imipenem, ticarcillin and clavulanic acid, and tobramycin). Deredjian et al. (2011) and Kaszab et al. (2011) found resistance to cephalosporine, ticarcillin, tobramycin and imipenem
Table 1 Antimicrobial resistance and plasmidial profile found in P. aeruginosa isolated from soil of different crops. Antimicrobial resistance profile
Isolates
Plasmidial profile MW (MDa)
ATM TCC TCC + TET ATM + PMB ATM + TCC + MEM ATM + TCC
All isolates, except S03, S05, S57 All isolates, except S10, S27, S28, S31, S42, S53 S05 S10 S41 S51
– – 54 82 16 35
2.5. Plasmid DNA extraction and agarose gel electrophoresis Plasmid DNA extraction was performed by the alkaline lysis method as recommended by Takahashi and Nagano (1984). Plasmid samples were resuspended in TE buffer and were mixed with a 10x loading buffer containing RNase (2 μg mL−1) (Fermentas, Brazil). Electrophoresis was carried out on a horizontal apparatus as outlined by Sambrook et al. (1989). The gel was stained with ethidium bromide (0.5 μg mL−1) for
Aztreonam (ATM), ticarcillin-clavulanate (TCC), polymixin B (PMB), tetracycline (TET), meropenem (MER). Molecular weight (MW).
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157
100 90
Total resistance (%)
80 70 60 50 40 30 20 10 0 ATM
TCC
MEM
TET
PMB
Antibiotics tested which presented resistance. Fig. 1. Frequency of total resistance to tested antibiotics for 40 P. aeruginosa studied. Aztreonam (ATM), ticarcillin-clavulanate-acid (TCC), meropenem (MEM), tetracycline (TET) and polymyxin B (PMB).
and in the work of da Silva et al. (2008), the isolates showed resistance to chloramphenicol, gentamicin and trimethoprim-sulfamethoxazole. None of these cited works have researched or made some correlation between antimicrobial resistance and plasmids or integrons. Knapp et al. (2010) showed that soil microbes have become progressively more resistant to antibiotics over the last 60 years, even with more stringent rules on the use of antibiotics in medicine and agriculture. These authors have concluded that there is an increasing chance of finding organisms in nature that are resistant to antimicrobial therapy. Other authors (LaPara et al., 2011) have demonstrated that in sewage treatment many of the bacterial genes that cause antibiotic resistance may be released into effluent and are a source of contamination. According to Martínez et al. (2011), in places mostly affected by human activity, contact between clinical pathogens and environmental bacteria, in the presence of several antimicrobial selective pressures, is favorable for the exchange and dissemination of resistance genes; in ecosystems where antibiotic-producing organisms (some bacteria and different types of fungi) are present, the spread of resistance genes is also higher (D'Costa et al., 2006). Isolates from soil plantations are in contact with human activity and can be close to soil bacteria and fungi that produce antibiotics which harbor resistance elements for self-protection (Arenas-Lago et al., 2013; Cerqueira et al., 2011; Oliveira et al., 2012). These isolates serve as a reservoir of resistance determinants that can be mobilized into the microbial community (D'Costa et al., 2006). This may be the explanation for the number of resistance elements and multi-resistant strains of soil found in this work. The present results are different from other studies, especially with respect to resistance to aztreonam in 92.7% of the isolates and ticarcillin in 85%. Although no multidrug resistance bacteria were found, the results draw attention to the high level of resistance to two antimicrobials commonly used in clinical practice, especially in the treatment of patients with cystic fibrosis (Zobelt et al., 2010; Oermann et al., 2011; Prescott et al., 2011; Assael et al., 2013). Since this resistance profile to aztreonam and ticarcillin is not correlated with the presence of plasmids or integrons in these isolates, the results suggest that other antimicrobial resistance mechanisms are present, as possible changes in the efflux pump (Poole and Srikumar, 2001), in MexAB or MexXY systems, which is subsequently investigated.
However, three of the four isolates possessing plasmids (S05, S10 and S41) are resistant to tetracycline, polymyxin and meropenem respectively, and in these isolates such resistance can be related to the presence of these mobile elements. Isolate S51, which has just aztreonam and ticarcillin resistance and a plasmid with 35 MDa has been studied, and in a work not yet published, this plasmid was related to the degradation of a herbicide and is therefore characterized as a catabolic plasmid (unpublished results). In conclusion, the present results demonstrate the potential of environmental isolates of P. aeruginosa to acquire antibiotic resistance, whether by acquisition of plasmids, integrons or by chromosomal alteration. These results suggest that restrictions on the use of antibiotics in humans, animals and agriculture are necessary to improve the current situation. Similar results were reported by previous researches (Hower et al., 2013; Kronbauer et al., 2013; Martinello et al., 2014; Quispe et al., 2012; Ribeiro et al., 2013a,b; Sanchis et al., 2013; Silva et al., 2011, 2012). Conflict of interest We have no conflict of interest to declare. Acknowledgments This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 2009/16657-3). We also thank John Carpenter for the English revision. References Arenas-Lago D, Vega FA, Silva LFO, Andrade ML. Soil interaction and fractionation of added cadmium in some Galician soils. Microchem J 2013;110:681–90. Assael BM, Pressler T, Bilton D, Fayon M, Fischer R, Chiron R, et al. Inhaled aztreonam lysine vs. inhaled tobramycin in cystic fibrosis: a comparative efficacy trial. J Cyst Fibros 2013;12:130–40. Cerqueira B, Vega FA, Serra C, Silva LFO, Andrade ML. Time of flight secondary ion mass spectrometry and high-resolution transmission electron microscopy/energy dispersive spectroscopy: a preliminary study of the distribution of Cu2+ and Cu2+/Pb2+ on a Bt horizon surfaces. J Hazard Mater 2011;195:422–31. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. CLSI M100-S23Wayne, PA: Clinical and Laboratory Standards Institute; 2013.
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