ANTIBIOTIC SUSCEPTIBILITY PROFILE OF KLEBSIELLA PNEUMONIAE STRAINS ISOLATED FROM DRINKING AND SURFACE WATER

Присуство Klebsiella pneumoniae (K. pneumoniae) у пијаћим и површинским водама представља индикатор фекалне контаминације хуманог и животињског порекла. K. pneumoniae има способност да стекне и пренесе гене резистенције. Изолати из животне средине могу представљати резервоар ових гена који се могу пренети и на друге бактеријске врсте. Циљ рада је изолација, идентификација и тестирање антибиотске осетљивости K. pneumoniae из пијаћих и површинских вода. Проспективна студија (спроведена од 1. октобра до 31. децембра 2015. године) испитала је 1276 узорака воде. Узорци су обрађени мембран-филтерском методом. За идентификацију K. pneumoniae коришћени су стандардни биохемијски тестови. Осетљивост на антибиотике је тестирана диск-дифузионом методом. K. pneumoniae је пронађена у 2,98% (38/1276) узорака, oд којих су 42,11% (16/38) били узорци површинских, а 57,89% (22/38) узорци пијаће воде. Teстирани изолати су били резистентни на ампицилин, док резиABSTRACT


ABSTRACT
Presence of Klebsiella pneumoniae (K. pneumoniae) in drinking and surface water indicates fecal contamination of human or animal origin. K. pneumoniae has the ability to acquire and transfer resistance genes. Isolates found in the environment may represent a reservoir of these genes, which can be transmitted among different bacterial species. The aim of the study was identification and testing susceptibility of K. pneumoniae to antibiotics after isolation from drinking and surface water. Prospective study (conducted from October the 1 st 2015 to December the 31 st 2015) included 1276 samples of drinking and surface water. The samples were processed by membranefilter technique. Standard biochemical tests were used for identification of K. pneumoniae. Antimicrobial susceptibility was determined by diskdiffusion method. K. pneumoniae was found in 2.98% (38/1276) of samples, from both surface 42.11% (16/38) and drinking water 57.89% (22/38). Tested strains were resistant to ampicillin, but resistance to other antibiotics was not confirmed. K. pneumoniae could be found in drinking

INTRODUCTION
Microbiologically safe drinking water is essential for human health 1 and its availability is a major concern of global community 2,3 . Contamination by different pathogens indicates an inadequate treatment, which is the reason why drinking water undergoes through strict microbiological control of hygienic quality 4 .
Surface waters, such as rivers, often contain large number of pathogenic microorganisms whose presence is mostly caused by human activity 5 . Being common place for waste products of industries, hospitals and pharmaceutical companies, these surface waters are favorable environment for proliferation of fecal coliform bacteria 6,7 .
Detection of coliform bacteria in water (Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Serratia spp.) indicates that there was fecal contamination of human or animal origin 8 . Although these microorganisms represent the portion of physiological flora of lower intestinal tract and are primarily not considered to be pathogenic, they can still cause different diseases 4 .

Изолација бактерија
Као метода за доказивање укупних и фекалних колиформних бактерија коришћена је мембран-филтер техника. Апарат за филтрацију (Sartonius Membranfilter, Немачка) је ка-cies to expand rapidly inside hospitals, but also in natural surroundings such as an aqueous environment, and its potential resistance to natural and synthetic antimicrobial drugs, classifies K. pneumoniae as highly dangerous microorganism, particularly when it comes to vulnerable groups such as immunocompromised patients 16 . The aim of this study was to investigate presence of K. pneumoniae in samples of surface and drinking water as well as to test sensitivity of isolates to antimicrobial drugs.

Sampling of water
A prospective study that included 1276 samples collected in a period of three months (from 01 October 2015 until 31 December 2015) was conducted. Samples of surface waters were collected from several rivers and from several different places established as sampling points for sanitary testings. In cases of some rivers several samples were taken from different parts of the same riverbed, with larger number of samples in cases of bigger rivers. Samples of drinking waters were taken from several sources (central and local water supplying network and public water supplying objects -wells, capped faucet and open springs) in order to cover all possible water supplying sources. Further on, drinking water samples were classified according to the treatment method before its use. All of the processed samples in this paper were chosen randomly from a larger group of samples used for routine analyses.
The samples were collected in sterile sealed bottles using standard technique in accordance with ISO 5667-1, ISO 5667-2 and ISO 5667-3 17 . All bottles were transported in a refrigerator at 4°C within 2 to 4 hours of sampling. The testing was conducted in the Department for sanitary bacteriology of the Center for Hygiene in Institute of Public Health of Vojvodina, Novi Sad.
the water filtration all parts of the apparatus were sterilized.
Volume of the water samples was 100 ml. In order to perform faster and more efficient filtration vacuum pump was used. Upon completion of filtration the membrane was aseptically removed and placed in the Petri dish. Слика 1. Апарат за филтрацију са узорцима воде и припремљеним хранљивим подлогама Figure 1. An apparatus for filtration of water samples with prepared agar plates

Isolation of Klebsiella pneumoniae
For examination of total coliform bacteria, endo agar was incubated at 37° C for 24 hours, whereas for the fecal coliform bacteria MacConkey agar was incubated at 44° C for 24 hours. MacConkey agar and endo agar are substrates on which Klebsiella pneumoniae grows in single large lactose-positive colonies of up to 3 mm in diameter. The colonies are convex in M shape.

Biochemical identification of Klebsiella pneumoniae
Biochemical tests used to identify K. pneumoniae included: fermentation test of dextrose and lactose, ability to utilize citrates, test of urea decomposition, the reaction of methyl red, test of hydrogen sulphide production, motility test and test of indole production from tryptophan.
Presence of K. pneumoniae has been confirmed in 16 samples of surface waters (originating from 6 different rivers). In Table 2 the samples positive on presence of K. pneumoniae are presented, depending on the river where the sample was taken (Table 2).
K. pneumoniae was also found in the samples of drinking water. Drinking waters were classified depending on whether the water was purified/unpurified and chlorinated/unchlorinated. In the Table 3 samples positive on the presence of K. pneumoniae are shown with reference to the mode of water treatment (Table 3).
In all tested K. pneumoniae isolates resistance to ampicillin was found. Resistance to other tested antibiotics was not proved. K. pneumoniae isolates were sensitive to penicillins with betalactamase inhibitors, cephalosporins, carbapenems, aminoglycosides, fluorinated quinolones and folate antagonists (Table 4).

DISSCUSION
Detection of K. pneumoniae in the water is an indicator of fecal contamination. If it is isolated from a sample of drinking water, it can indicate poor treatment of water in terms of purification and chlorination. Certain strains show significant resistances to chlorination which is mediated through a number of mechanisms, such as change of bacterial cell lipid membrane which can consequently bring into question the adequacy of chlorination as a method for treatment of drinking water 16 . As it was expected, the highest number of positive drinking water samples belonged to the group of unpurified and unchlorinated waters. However, in our examination, 2 of the drinking water samples, that were positive on K. pneumoniae, were previously purified and chlorinated and 1 sample was from unpurified and chlorinated water, which raises doubts about the effectiveness of this method. The newest approach to effective inactivation of persistent pathogens in water is oxidation with titanium dioxide 16,19 .
Resistance genes are ubiquitous and they date from ancient times 14 . The bacteria that inhabit the aquatic environment and are dating millions of years before the antibiotic era, carry certain genes which are responsible for development of resistance towards some of the antibiotics essential for modern therapy 21,22 . Several studies showed that some bacteria, which are naturally found in water, can produce antibiotic substances as secondary metabolites which may persist in aquatic environment and come into contact with the bacteria present in that specific ecosystem. Bacteria can acquire resistance to these metabolites and this can be an opportunity for cross-resistance with important groups of antibiotics used in modern medicine 12,23 . Many studies have confirmed presence of resistant isolates of K. pneumoniae, especially in hospital drainage pipes. Based on epidemiological studies it is believed that these Klebsiella spp. are originating from the external environment, from where they also received these resistance genes 10,24 .
Use of antibiotics in the meat industry has become one of the main targets for combat against antibiotic resistance due to the enormous quantity of drugs used on a yearly basis. In Denmark during 2010, 71% of all antibiotics were used for treatment of animals. Consumption of such meat can unnecessarily expose an indivudal to large amount of antibiotics. Besides the fact that they impair physiological intestinal microflora, they can be transferred to local environment through fecal waste water 14 .
In our study, resistant strains were not found in the tested samples gained from different rivers (Danube, Tisa, Begej, Nera, Tamiš, Moravica) or in the samples of drinking water and only resistance to ampicillin was determined with statistical significance. This resistance is intrinsic and mediated via chromosomal beta-lactamases 10 . There are indications that the results would be different if the study was repeated at some other time of the year because the composition of the surface waters is very variable and depends on the numerous factors such as the dynamics of disposal, rainfall, flooding, temperature and others 5 . The mere presence of K. pneumoniae in water creates opportunity for these strains to acquire a certain genes of resistance 7,27-31 . Skariyachan and coworkers 7 examined bacteriological quality of the river Kaveri in India and found that 93.51% of strains were multiresistant and Klebsiella spp. strains, which were examined in this study, showed resistance to as many as 26 different antibiotics. Strains from Dambovita River in Romania have shown resistance to 16 antibiotics and 60% of them were multiresistant 27 . In Turkey, in the river Seihan, isolated strains showed resistance to ampicillin, streptomycin, cefazolin and cotrimoxazole 28 . In river Mhlathuze in South Africa, the detected strains were resistant to 10 different antibiotics 29 . Comparison with studies in our површинских вода у доступној литератури.
Consumption of hygienically inadequate water or contact with contaminated surface waters can lead to colonization of intestinal tract with pathogenic, resistant bacterial strains in completely healthy individuals who have never been hospitalized or taken antibiotics 14 . Infections with resistant strains are becoming an increasing therapeutic problem, which can lead to increased morbidity, mortality and costs of treatment 30 .
Bacterial genome is large and hypervariable, so it is difficult to define exact place and time of the creation and transfer of resistance genes. It is necessary to understand the importance of the aquatic environment in the development and evolution of resistance genes. Moreover it is mandatory to reduce usage of antibiotics in human and veterinary medicine. However, reducing only the selective pressure of antibiotics will not lead to reduction of resistance if the resistant strains are constantly appearing from different sources. That is why constant control and microbiological testing of samples from the water environment must be performed. It is important to understand that resistance evolution is not local, but international problem, in which the main focus is on travelling and global food market 14 . Overzealous control is the key to overcome this problem. Therefore the World Health Organization on April the 7 th , 2011 in Geneva decided to launch the project "No action today -No cure tomorrow". This initiative aims to reduce the use of antibiotics in hospitals, community and in the meat industy 14,31 .
This paper highlights the importance of monitoring the bacteria resistance in aqueous media. This monitoring should be continuous and it requires great effort by many branches of science and industry. However, it is clear that only synergistic efforts of different sectors can bring notable changes in this field.
Our study pointed to the presence of K. pneumoniae both in drinking and surface waters. This opportunistic pathogen is detected despite the implementation of appropriate measures for its elimination. This indicates the importance of constant microbiological control, but also raises the question of the appropriateness of sanitary measures. Despite the fact that the isolates were of expected sensitivity and without acquired resistance, ideal results would show absolute absence апсолутно одсуство сојева K. pneumoniae у пијаћим и површинским водама. Само присуство овог микроорганизма у води отвара могућност да она у сваком моменту стекне гене резистенције различитим механизмима, као и да исте пренесе другим бактеријским врстама.
of strains of K. pneumoniae from drinking and surface waters. The mere presence of this microorganism in the water creates an opportunity of acquiring resistance genes at any moment by different mechanisms and further transfer of the genes to other bacterial species.