Elimination and conjugal transfer of their resistance in isolated gram negative bacteria from Aksu River (Kahramanmaras-Turkey).
Overuse of antibiotics also elevates the emergence of resistant bacteria. In fact, antibiotics exert a selection pressure on pathogenic bacteria as well as on bacteria belonging to the normal endogenous flora. Occurance of resistance in pathogens may reduce the effectiveness of previously useful antibiotics.
Antibiotics (?ncluding synthetic antimicrobials) are likely the most successful therapeutic agents developed by humans. With the introduction of antibiotics, it was thought that infections should disappear. But, bacteria have been able to evolve to become antibiotic resistant. Nowadays, a sufficient pathogen must be virulent, epidemic, and resistant to antibiotics. The great majority of antibiotics currently used for treating infections and the antibiotic resistance genes acquired by human pathogens each have an environmental origin. Antibiotic resistance is vastly disseminated in non-clinical environments, and antibiotic producers contain a large number of antibiotic resistance genes in their genomes that might finally be transferred to pathogenic bacteria .
There have been many surveys of the occurrence of antibiotic resistant E. coli in animals [26,34]. Most recently, Son et al  demonstrated that both resistance and entereotoxigenicity were transferred using bacterial conjugation experiments with antibiotic-resistant isolates of E. coli from human and poultry sources in Peninsular-Malaysia.
The use of antibiotics in medicine, veterinary practise and agriculture has aroused some concern about the incidence and spread of antibiotics resistance among bacterial populations. This is particulary true when transfer occurs in environments such as hospitals where the human population is at risk. In the USA as much as half of antibiotic production is destined for use in animal feed, although the significance of this in relation to the transfer of resistance to human bacterial pathogens has been questioned recently . As a result of the usage of antibiotics in medical or veterinary practise, selected for resistant bacteria, these bacteria have inevitably entered the natural environment .
It is a subject of research how much diversity exists in the antibiotic resistance patterns within natural populations of enteric bacteria, and how the levels of resistance have changed since the use of antibiotics became widespread. Increased introduction of antimicrobial agents into the environment via medical therapy, agriculture, and animal husbandry [11,15] has resulted in new selective pressure on bacterial population . This has exacerbated the problem of controling microbes in a disease setting and has caused a resurgence of bacterial diseases worldwide due to the acquisition and transfer of virulence factors and antibiotic resistance genes [30,50].
The wide spread use of antimicrobial agents has failed to eradicate microbial infections despite their benefits. Antibiotic resistant bacteria have been a source of ever-increasing therapeutic problem. Continued mismanaged selective pressure has contributed towards the emergence of multiple drug resistant bacteria and that has been regarded as an inevitable genetic response to antimicrobial therapy .
Kahramanmaras, is a developing city, located in the southern part of Turkey, with the 315,000 inhabitants. The economy of the Kahramanmaras mainly depends on the textile, yarn industry and agriculture. The Aksu River receives the wastewater discharge from the sewage of the city of Kahramanmaras.
An attempt was made to answer the first two questions by determining the elimination of their resistance in gram-negative bacteria isolated from five different wastewater discharge Aksu River, in Kahramanmaras (Turkey). Then resistance transfer was examined.
Materials and methods
Study Area and Sample Collection
The Aksu River (28.3 [m.sup.3]/s), located in southern Turkey. Five different sampling stations were chosen along the pollution gradient in the river, with first site upstream at Aksu river, a second site at Erkenez brook, a third site Oklu brook a fourth site at Karasu brook, a fifth site downstream at Aksu river.
Isolation and identification of Enterobacteriaceae and Pseudomonas spp.
Triplicate plates with EMB were inoculated with appropriated dilutions from the water samples. Representative colonies were purified an EMB-agar. Organisms which exhibited a yellow-green metallic sheen on EMB-agar were incubated at 37[degrees]C for 24 [+ or -] 4 h. Preliminary identification of strains obtained in pure culture was based on Gram staining, respiration fermentation tests, and biochemical tests (IMVIC tests) . Pseudomonas spp. was isolated by incubation at 42[degrees]C, 37[degrees]C and 20[degrees]C on Pseudomonas selective medium (Oxoid CM 457). Isolate was then tested at these temperatures for casein hydrolysis and pigment production on milk-agar with cetrimide  and for a positive oxidase reaction. Complete identification of Enterobacteriaceae was achieved by use of the tests in Bergey's Manual of Determinative Bacteriology [23,51].
Antibiotic Susceptibility Testing
The isolates were tested for sensitivity to eleven (11) antibiotics applied by an agar disk diffusion test . The eleven antibiotics used were (mg/disc) as follows: Meropenem (MER 10mg/mL), Cefoxitin (FOX 30mg/mL), Ceftazidime (CFZ 30mg/mL), Ceftriaxone (CRO 30mg/mL), Imipenem (IPM 10mg/mL), Sulbactam/Cefoperazone (CRP 30mg/mL), Cefazoline (CZ 30mg/mL), Cefotaxsime (CTX 30mg/mL), Aztreonam (ATM 30mg/mL), Ampicillin/Sulbactam (SAM 10mg/mL), Penicilline-G (KP 10 unite/mL).
Analysis of Data
The percentage resistance ([%.sup.r]) to 1,2,3.... n antibiotics as exclusive classes was calculated.
The production of beta-lactamase was determined with the Nitrocefin-Method [32,39].
Analysis of the phenotype of susceptibility to penicillins and cephalosporins can give a close estimate of the beta-lactamase type produced by Enterobacteriaceae . Beta-lactamase activity was revealed by adding one drop of the Nitrocefin (Oxoid SR112C) solution on to a clean glass slide. Using a sterile loop, pick one colony from the plate and emulsify into the Nitrocefin drop, positive results noted if the colour changes from yellow to red within 30 minutes. In the presence of beta-lactamase and the opening of the beta-lactam ring, conjugation of the dinitrostyryl group at position 3 with the dihydrothiazine ring increases, with a change in color from yellow to red . All the bacterial isolates were tested for the production of beta-lactamases.
Plasmid Elimination (Curing Experiments)
Strains of the beta-lactamase positive and to be done antibiogram were incubated in LB broth containing 150 mg/mL EtBr at 37[degrees]C for 24 h . Isolated strains were subjected to EtBr mediated plasmid (bearing resistance markers) elimination. Later incubated strains were plated on EMB-agar by line inoculation and incubated at 37[degrees]C for 24 h. 8, 15, or 20 pure colonies of each strain were examined to test lost antibiotic resistance by plate with antibiotic. But no cured strains were isolated at 37[degrees]C for 24 h cured strains were not found.
Conjugation (Mating Experiments)
The tranferabilities of resistance determinants of the selected strains were further investigated by conjugation experiment. Conjugation experiments were carried out by a broth-mating and filter-mating procedure with at least a or two antibiotic resistant strain and different species sensitive strain. Isolated Klebsiella spp. were used as donors for the possible transfer of resistance markers to a standard recipient E.coli AB 1621 strain.
Recipient strains and donor strains (E. coli and Klebsiella spp.) were grown for 24 h in LB broth or nutrient broth. Equal volumes (2 ml) of parental strains were mixed and incubated at 37[degrees]C [l]. Later different species sensitive strain as chosen on EMB agar. The selective antibiotic concentrations used for conjugation experiments were as antibiotic's MIC.
In the filter-mating, donor and recipient cells were inoculated onto same filter, incubated overnight, diluted and cultivated on the appropriate media, exp. EMB agar in order to correlate the effect of the transconjugant detection technique with the occurence at transconjugant (plate-mating) .
Results and discussion
In this study, a total of 67 strains belonging to 3 bacterial genera of the Enterobacteriaceae and Pseudomonas spp. in the water samples taken from the Aksu River were isolated .
1 of 45 E. coli strain was sensitive, 14 strains were resistant to 1 kind of antibiotic and 30 strains (66.6 %) showed resistance against two or any more antibiotics. While the resistance ratio of isolated E. coli was 93.3 % to KP, all the strains were sensitive against CTX and CRO. While 1 of 20 Klebsiella spp. strain was resistant to 1 kind of antibiotics, 19 strains (95 %) showed resistance against two or any more antibiotics (Table 1). In this connection, EtBr mediated curing was performed (see Table 3). Resultantly, some of the resistance markers were lost. E. coli ve Klebsiella spp., isolated at different sampling stations were chosen as curing, after antibiogram and beta-lactamase test were made (see Table 2).
Although plasmid elimination was observed (100 %) in CFZ, CRP, CZ and CRO for Klebsiella spp. isolated at sampling station 1, there was no elimination in ATM, SAM and KP (0.0 %). In this Klebsiella spp. plasmid elimination was successful in 6 antibiotics out of 9. For sampling station 2, plasmid elimination was 100 % in ATM, CFZ, SAM, FOX, CRP, CZ, CRO and 0.0 % in KP for Klebsiella spp.. Plasmid elimination was successful in 8 antibiotics out of 9. While plasmid elimination in CRP was completed (30 %) in E. coli sampled at sampling station 3, there was no elimination in CZ (0.0 %). In this E. coli plasmid elimination was successful in 5 antibiotics out of 6. For sampling station 3, plasmid elimination in IPM and FOX was completed (100 %) in E. coli, there was no elimination in KP (0.0 %). In this E.coli plasmid elimination was successful in 7 antibiotics out of 8. While plasmid elimination in SAM was completed (100 %) in Klebsiella spp. sampled at sampling station 5, there was no elimination in ATM, CFZ, CRP, CZ, KP and CRO (0.0 %). In this Klebsiella spp. R plasmid elimination was successful in 2 antibiotics out of 9 (see Table 3).
In this study elimination ratios at sampling stations 1 and 2 in Klebsiella spp. were high. The ratios in Klebsiella spp., taken at sampling station 5 were according to these results low. The E. coli samples taken at sampling stations 3 and 4 showed lower elimination ratios than those of Klebsiella spp. As a result of the elimination tests elimination ratios were likely to depend on antibiotic resistance in plasmid.
Plasmids with traits of both resistance and conjugation have been known . Plasmid conjugation is an important mechanism of disseminating drug resistance among bacterial populations. In the present studies, solid substrate (Millipore filter paper) and liquid substrate (Stonier Broth) mating experiments were undertaken (using isolated Klebsiella spp. as donor and E. coli AB 1621 as the recipient strain) (see Table 4).
E. coli ve Klebsiella spp., isolated at sampling station 1 were chosen as donor, after antibiogram and beta-lactamase test were made; Klebsiella spp. and E. coli collection culture sample AB1621 strains were chosen as receiver. In conjugation experiments between E. coli and Klebsiella spp. resistance transfer was carried out for SAM (45%), CZ (40%), for MER it was not realized resistance transfer. In conjugation experiments between Klebsiella spp. and E. coli collection culture sample AB 1621 resistance transfer was carried out for KP (100%), SAM (96%), ATM (96%), FOX (83%), CZ (83%) CRP (78%), CFZ (74%), CRO (65%). The conjugation was realized by processing plasmid isolated strains with receiver strains being sensitive to antibiotics in stonier broth (Table 5).
In the conjugation experiment using filter, where at the sampling station 1 isolated Klebsiella spp. was chosen as donor and E. coli collection culture sample (AB1621) was chosen as receiver, resistance transfer was observed for SAM (100[degrees]/x), KP (100%), ATM (92%), CRP (92%), CZ (46%), CFZ (42%), CRO (29%) and FOX (10%) (Table 6).
The quality of water is of vital importance to public. Efficient surveillance and check strategies are important for executing a high-quality management of this resource. Kahramanmaras province has been settled in the southern region of Turkey, and a unique region which contains textile plants, yarn industry and agriculture. And the wastewater discharge from the sewage of the city of Kahramanmaras flows in Aksu River. We observed that there was a high fecal coliforms counts . And now, this sudy is shown multiple resistance of bacteria against antibiotics and their transferable (Table 1).
The multiple resistance of bacteria against antibiotics in resulted from the fact that domestic and industrial waste were emitted into running water without clarifying. It has been reported that multiple resistance of bacteria against antibiotics was also found in other regions polluted by medical, urban, industrial and agricultural waste [3,18,37,41,47,51]. Antibiotic resistance is often determined by genetic information of plasmid origin. The correlation between antibiotic resistance and plasmid profile may indicate that the genetic information is plasmid-borne .
Elimination of plasmids by curing is an essential step in procedures used to investigate to be reservoir resistance plasmid or chromosom . Curing agent was emplayed EtBr in Enterobacteriaceae strains because in curing of antibiotic resistance plasmid was determined an effective chemical agent [33,38]. The location (chromosomal or extra chromosomal) of drug resistance determinants was confirmed by plasmid curing strategies . EtBr as curing agent used for resistance lost in this study. As a result, resistance elimination was observed in different rates for antibiotics (See Tablet and Table 3).
Mac Faddin  have reported that there were a lot of mechanisms to transfer resistant genes to other bacteria. Generally, resistance completely or partially depends on beta-lactamase . Therefore, beta-lactamase enzyme was investigated and this result, most of antibiotic resistant bacteria were found carrying beta-lactamase enzyme.
The members of Klebsiella spp. genus have also been linked to epidemics of diarrhoea, because some strains appear to have acquired plasmids from E. coli (that code for the heat labile and heat stable enterotoxins) . Resistance in microbial pathogens like E. coli to two or more classes of antibiotics is now commonplace in both veterinary [19,21,25]. and human medicine . Although antibiotic susceptibility may return with the discontinuance of antibiotic usage, antibiotic-resistant bacteria can still persist long after the removal of the selection pressure [8,3].
Previous studies have shown that gene transfer is a process by which bacterial populations substantialy increase their rates of evolution and adaptation [10,22,46]. Particularly, plasmid-located genes, which are transferred by conjugation from donor to recipient cells, can disseminate rapidly between even phylogenetically different bacterial groups [14,31,35]. and microbial communities in different spatial habitats [29,52]. The conjugation results are similar to those of earlier studies [1,22,44]. Comparative results of conjugations (Pre and Post Conjugation) see shown Table 4, Table 5 and Table 6).
Plasmid DNA mediated transfer of resistance determinants has earlier been reported as well . Such broad host-range transferable plasmids play an important role in the spread of antibiotic resistance. However, conjugation (physical mating) strategies do not allow the pushable plasmids which need the sex pili provided by an independent F-plasmid . In such circumstances, transformation may work well; infact, the race to develop agents to overcome the resistance mechanism is one that man may never win, but the resistance trends should be kept under check through intensive research leading to novel and alternative drugs therapies .
The R plasmids offer resistance to antibiotics and are transmissible from one cell to another by direct cell contact. Conjugation (direct in vivo gene transfer) is a convenient method of transferring drug resistant genetic determinants among intra and inter generic bacterial populations . It is well known that industrialization and man's activities have partially or totally turned our environment to dumping sites for waste materials. As a result, many water resources have been rendered unwholesome and hazardous to man and other living systems .
In this study, resistance transfer among bacteria was observed in liquid (Stonier broth) and solid surface (filter). The results of this study showed that water, polluted with industrial, domestic, and agricultural waste, caused bacterial resistance and leaded to reservoirs for spreading these kinds of resistances.
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Sevil Toroglu, Department of Biology, Faculty of Arts and Science, Kahramanmaras Sutcu Imam University, 46045-Kahramanmaras, Turkey. Tlf: +90 344 2191312 Fax: +90 344 2191042 E-mail: email@example.com
(1) Sevil Toroglu, (2) Sadik Dincer
(1) Science and Art Faculty Biology Department Sutcuimam, University Kahramanmaras Turkey. firstname.lastname@example.org
(2) Science and Art Faculty Biology Department Cukurova University Adana Turkey. email@example.com
Sevil Toroglu, Sadik Dincer, Elimination and Conjugal Transfer of Their Resistance In Isolated Gram Negative Bacteria from Aksu River (Kahramanmaras-Turkey), Adv. Environ. Biol., 2(3):124-131, 2008
Table 1: Antibiotic Resistance Distribution of Bacterial Genera Numbers of antibiotics E. coli Klebsiella Citrobacter spp. spp. Sensitive 1 0 0 1 Antibiotic 14 1 0 2 Antibiotics 6 1 0 3 Antibiotics 4 2 1 4 Antibiotics 4 1 0 5 Antibiotics 3 3 0 6 Antibiotics 5 1 0 7 Antibiotics 7 1 0 8 Antibiotics 1 3 0 9 Antibiotics 0 5 0 10 Antibiotics 0 2 0 11 Antibiotics 0 0 0 Total 45 20 1 Numbers of antibiotics Pseudomonas Total of % Resistance spp. Strains Sensitive 0 1 1 1 Antibiotic 0 15 22 2 Antibiotics 0 7 10 3 Antibiotics 0 7 10 4 Antibiotics 0 5 7 5 Antibiotics 0 6 9 6 Antibiotics 0 6 9 7 Antibiotics 0 8 12 8 Antibiotics 0 4 6 9 Antibiotics 1 6 9 10 Antibiotics 0 2 3 11 Antibiotics 0 0 0 Total 1 67 Table 2: Results in Antibiogram and Beta-lactam ase of Isolates on Pre-Curing SaS. No B.G. ATM CFZ CTX 1 Klebsiella spp + + - 2 Klebsiella spp + + - 3 E. coli + - - 4 E. coli + - - 5 Klebsiella spp + + - SaS. No B.G. SAM MER IPM 1 Klebsiella spp + - - 2 Klebsiella spp + - - 3 E. coli + - - 4 E. coli + - + 5 Klebsiella spp + - - SaS. No B.G. FOX CRP CZ 1 Klebsiella spp + + + 2 Klebsiella spp + + + 3 E. coli - + + 4 E. coli + + - 5 Klebsiella spp + + + SaS. No B.G. KP CRO N .CEF 1 Klebsiella spp + + + 2 Klebsiella spp + + + 3 E. coli + - + 4 E. coli + - + 5 Klebsiella spp + + + SaS.No: Sampling Station No; B.G.: Bacteria Genera; N .Cef: Nitrocefin; MER: Meropenem, FOX: Cefoxitin, CFZ: Ceftazidime, CRO: Ceftriaxone, IPM :Imipenem, CRP: Sulbactam /Cefoperazone, CZ: Cefazoline, CTX: Cefotaxsime, ATM : Aztreonam, SAM : Ampicillin/Sulbactam, KP: Penicilline-G. Table 3: Results in Plasmid Elimination Test (on Post-Curing) SaS. No B.G. ANTIBIOTICS 1 Klebsiella spp ATM A P P.E. 8 8 0% CRP A P P. E. 8 0 100% 2 Klebsiella spp ATM A P P.E. 20 0 100% CRP A P P. E. 20 0 100% 3 E. coli ATM A P P.E. 20 16 20% KP A P P.E. 20 17 15% 4 E. coli ATM A P P.E. 20 13 35% CRP A P P.E. 20 18 10% 5 Klebsiella spp ATM A P P.E. 15 15 0% CRP A P P.E. 15 15 0% SaS. No B.G. ANTIBIOTICS 1 Klebsiella spp CFZ A P P. E. 8 0 100% CZ A P P. E. 8 0 100% 2 Klebsiella spp CFZ A P P.E. 20 0 100% CZ A P P.E. 20 0 100% 3 E. coli SAM A P P.E. 20 15 25% 4 E. coli SAM A P P.E. 20 19 5% KP A P P.E. 20 20 0% 5 Klebsiella spp CFZ A P P.E. 15 15 0% CZ A P P.E. 15 15 0% SaS. No B.G. ANTIBIOTICS 1 Klebsiella spp SAM A P P. E. 8 8 0% KP A P P. E. 8 8 0% 2 Klebsiella spp SAM A P P.E. 20 0 100% KP A P P.E. 20 20 0% 3 E. coli CRP A P P.E. 20 14 30% 4 E. coli IPM A P P.E. 20 0 100% CZ A P P.E. 20 18 10% 5 Klebsiella spp SAM A P P.E. 15 0 100% KP A P P.E. 15 15 0% SaS. No B.G. ANTIBIOTICS 1 Klebsiella spp FOX A P P. E. 8 3 63% CRO A P P. E. 8 0 100% 2 Klebsiella spp FOX A P P. E. 20 0 100% CRO A P P.E. 20 0 100% 3 E. coli CZ A P P.E. 20 20 0% 4 E. coli FOX A P P.E. 20 0 100% 5 Klebsiella spp FOX A P P.E. 15 14 7% CRO A P P. E. 15 15 0% SaS.No: Sampling Station No B.G.: Bacteria Genera A: Affected P: Producing P.E.: Plasmid Elimination MER: Meropenem, FOX: Cefoxitin, CFZ: Ceftazidime, CRO : Ceftriaxone, IPM :Imipenem, CRP: Sulbactam /Cefoperazone, CZ: Cefazoline, CTX: Cefotaxsime, ATM : Aztreonam, SAM : Ampicillin/Sulbactam, KP: Penicilline-G. Table 4: Results in Antibiogram and Beta-lactam ase of Isolates on Pre-Conjugation SaS. No B.G. ATM CFZ CTX SAM 1 E. coli + - - + 5 Klebsiella spp + - - - 1 Klebsiella spp + + - + Cul E. coli (AB1621) - - - - SaS. No B.G. MER IPM FOX CRP 1 E. coli + - + + 5 Klebsiella spp - + + + 1 Klebsiella spp - - + + Cul E. coli (AB1621) - - - - SaS. No B.G. CZ KP CRO N .CEF 1 E. coli + + - + 5 Klebsiella spp - + - - 1 Klebsiella spp + + + + Cul E. coli (AB1621) - - - - SaS.No: Sampling Station No; B.G.: Bacteria Genera; N .Cef: Nitrocefin; Cul: Culture MER: Meropenem , FOX: Cefoxitin, CFZ: Ceftazidime, CRO: Ceftriaxone, IPM :Imipenem, CRP: Sulbactam /Cefoperazone, CZ: Cefazoline, CTX: Cefotaxsime, ATM : Aztreonam, SAM : Ampicillin/ Sulbactam, KP: Penicilline-G. Table 5: Conjugation Results in Stonier Broth Sampling Station 1 E. coli 6 Sampling Station 5 Klebsiella spp. SAM CZ MER A P R% A P R% A P R% 20 9 45 20 8 40 20 0 0 Sampling Station 1 Klebsiella spp.6 Collection culture Sample E. coli (AB1621) ATM CFZ A P R% A P R% 23 22 96 23 17 74 KP CRO A P R% A P R% 23 23 100 23 18 65 SAM FOX A P R% A P R% 23 22 96 23 19 83 CZ CRP A P R% A P R% 23 19 83 23 18 78 A: Affected P: Producing R% : Resistance Percentage; MER: M eropenem, FOX: Cefoxitin, CFZ: Ceftazidime, CRO : Ceftriaxone, IPM :Imipenem, CRP: Sulbactam /Cefoperazone, CZ: Cefazoline, CTX: Cefotaxsime, ATM: Aztreonam, SAM : Ampicillin/Sulbactam, KP: Penicilline-G. Table 6: Conjugation Results using filter Sampling Station 1 Klebsiella spp. 6 Collection culture Sample E. coli (AB1621) ATM CFZ SAM A P R% A P R% A P R% 24 22 92 24 10 42 24 24 100 FOX CRP A P R% A P R% 24 24 10 24 22 92 KP CRO CZ A P R% A P R% A P R% 24 24 100 24 7 29 24 11 46 A: Affected P: Producing R% : Resistance Percentage; MER: Meropenem, FOX: Cefoxitin, CFZ: Ceftazidime, CRO : Ceftriaxone, IPM :Imipenem, CRP: Sulbactam /Cefoperazone, CZ: Cefazoline, CTX: Cefotaxsime, ATM : Aztreonam, SAM : Am picillin/Sulbactam, KP: Penicilline-G.
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|Title Annotation:||Original Article|
|Author:||Toroglu, Sevil; Dincer, Sadik|
|Publication:||Advances in Environmental Biology|
|Date:||Sep 1, 2008|
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