Knowledge and Habits Towards Antibiotic Use and Resistance of Public University Students in Nisava Region – Southern Serbia

Antibiotic resistance has become a serious threat to global health. This study aimed to assess knowledge and habits surrounding the use of antibiotics and antibiotic resistance of university students residing in urban areas compared to those in rural areas of Nisava region, Serbia. Data was gathered using an online survey and tested for statistical differences using the Chi-squared test. A total of 380 students participated with a response rate of 94.7 per cent. Of this, 84.4 per cent of students correctly identified antibiotics as being effective against bacteria and distinguished well between antibiotics and other types of medicines. However, 31.4 per cent believe that antibiotics help with most diseases, not just bacteria-related illnesses. While only 12.5 per cent said they believed that therapy can be interrupted when the symptoms fade, a total of 45.8% admitted to premature treatment interruption. As many as 59.7 per cent reported having bought antibiotics without a prescription, and a significant portion of 62.5 per cent reported having taken antibiotics for travel emergencies. There was no statistically significant difference related to the domicile of the students (p>0.05). Students demonstrated relatively acceptable knowledge on antibiotics use and antibacterial resistance – which is not reflected in their practice of using antibiotics. Campaigns are needed to promote awareness on antibiotic resistance as students’ habits are not satisfactory.


Introduction
Paul Ehrlich, Nobel prize laureate in medicine in 1908, famously called antibiotics magic bullets: a completely safe medicine directly specific for the target (Valent et al., 2016: 118). Throughout the Second World War, antibiotics were advertised as able to cure any disease, and the British Medical Journal stated that ' penicillin is to other antiseptics what radium is to other metals' (Shama, 2015: 132). Such beliefs have led to inappropriate usage of antibiotics in recent decades. Inappropriate use practices include self-medication , missed purpose, short duration of treatment, inadequate dose, or stopping prematurely upon improvement of disease symptoms (Ocan et al., 2015). The percentage of self-medicating patients in Europe has grown in the past years from 5 per cent in 2009 to 7 per cent in 2016 (Lescure et al., 2018), ranging from 3 per cent in Northern Europe  to the largest rates in countries of South-Eastern Europe. This includes 28 per cent in Slovenia and 20.5 per cent in Croatia, neighbouring countries to Serbia where this study was conducted (Grigoryan et al., 2010).
Literature data cites that the public unawareness level regarding the ineffectiveness of antibiotics in viral diseases was as high as 57 per cent (Machowska and Lundborg, 2018: 27). Extensive use of antibiotics has been reported in cases of upper respiratory tract infections, despite that these infections are mostly viral in origin (Ochoa et al., 2000). In general, antibiotics cannot be used to treat viral infections as viruses feature no molecular targets attacked by antibiotics (Dworkin, 2003). Non-adherence to dosing regimens is also common, either prolonging the course or ending it prematurely as soon as the symptoms fade. The latter is especially dangerous as it may leave some more resistant pathogens behind that reintroduce the illness.

Antibacterial resistance: a global danger
Antibacterial resistance (ABR) is an ability of bacteria to develop resistance towards the drug employed (Aslam et al., 2018(Aslam et al., : 1645. It occurs when bacteria change in response to usage of antibiotics. While it does occur naturally, the highly increasing rate of ABR is linked to inappropriate usage of antibiotics (World Health Organization, 2020).
The bacterial population, and not the organism in which they reside, develops resistance over generations, with the fittest bacteria surviving. (Brookes-Howell et al., 2012). First cases were reported soon after the discovery of penicillin (Lee Ventola, 2015). Antibiotic resistance leads to higher medical costs, prolonged hospital stays and increased mortality (World Health Organization, 2020). An estimated 25,000 deaths in Europe and 23,000 in USA per year are linked to antibiotic-resistant bacteria (Laxminarayan et al., 2013(Laxminarayan et al., : 1060. One typically identifies four mechanisms that bacteria employ to protect themselves from antibiotics (Hawkey, 1998): (i) The resistant bacteria may prevent the antibiotic from reaching the sensitive target on the bacterium, or (ii) the sensitive target may be altered, preventing the antibiotic from inhibiting bacterial activity due to structural changes in the molecule. (iii) Bacteria may also produce an alternative target that is resistant to inhibition by the antibiotic, therefore protecting the initial sensitive site.
Finally, (iv) the antibiotic may be prevented from entering the cell or pumped out of the cell at a rate faster than the one at which it enters the cell. Lowering the effect of antibiotics means that the disease becomes more dangerous and more difficult to cure, representing great danger to public health (Pechère et al., 2007, Fair andTor, 2014).

Need for a new study
Inappropriate use of antibiotics, ultimately leading to ABR, is a problem in the broader region of the Balkans and South-Eastern Europe, including very high consumption rates reported in Romania, Greece, Montenegro and Turkey (World Health Organization, 2018: 28). Antibiotic consumption in Serbia was among the highest in Europe in 2015 (World Health Organization, 2018). A nationwide campaign in 2016 managed to reduce it, but still keeping it much higher than the European average (World Bank, 2018). Also, past studies have reported erroneous practices in Serbia. Serbian households often keep antibiotics in home pharmacies (Paut Kusturica et al., 2015: 115) and over-prescription by physicians has occurred even after the completion of the campaign, notably in cases of acute bronchitis (Petrovic et al., 2019: 687). In addition, Serbia is among few European countries not participating in ESAC-Net (European Surveillance of Antimicrobial Consumption Network) . This indicates a link between high antibiotic consumption and erroneous attitudes of the public, showing that further investigation of attitudes of the public is needed. Reinvention: an International Journal of Undergraduate Research 13:2 (2020) Aforementioned dangers of inappropriate use and the unfavourable situation in Serbia have motivated this study, whose primary goal was to investigate students' knowledge and habits regarding antibiotic usage and ABR in a public university in the Nisava region of Southern Serbia and compare them to available data from the immediate region and from other developing countries. A secondary goal was to inspect differences between answers of students coming from rural areas and those from urban regions, as different socio-economic statuses and different beliefs of communities more inclined to traditional medicine and curing methods have shown to influence the attitudes of patients towards drug use (Akici et al., 2017;Peng et al., 2018).
Based on previous studies, healthcare and socio-economic differences (Primary Healthcare Centre Nis, 2020), we expected that our sample would demonstrate doubts regarding knowledge, as well as erroneous usage and self-medication, with less appropriate attitudes present in the rural region.
It is important to assess the knowledge on the subject among the population of university students, whose opinion will become that of the most educated population of the country. This way, gaps in their knowledge can be identified, and antibiotic stewardship campaigns can be planned to contribute to the action of stopping antibiotic resistance. Such campaigns were successfully conducted in many countries, such as France, where knowledge improved from 2002 to 2007 (Sabuncu et al., 2009).
Past studies in Serbia mostly concentrated on habits of antibiotics usage in hospitals in other regions of the country and were written from a clinician's point of view. There is currently a paucity of data addressing public's attitudes. Therefore, this study on the knowledge and habits towards antibiotic use and resistance of public university students in Southern Serbia provides novel information and contributes to the global knowledge on the subject.

Materials and methods
This study concentrated on students at Nis University, the only university centre in the region. It has 20,455 undergraduate students (Janković, 2017). The sample size of 378 was calculated using Raosoft calculator, with a confidence interval of 95 per cent and a margin of error of 5 per cent.
We used a stratified sample, aiming to roughly equalise the number of students coming from urban and rural areas for direct comparison. The students included were undergraduates -excluding students of medicine and pharmacology from the Faculty of Medicine -as well as students of biology and chemistry from the Faculty of Natural Sciences. This was to avoid potential response bias as the above-mentioned courses were linked to the current topic.
This research was exempt from ethical committee evaluation as it only acquired opinions of the surveyed parties. Prior to completing the survey, all participants gave personal informed consent about processing of personal information and data use for the purpose of the research. There was no compensation nor reward offered to the participants.
Questions were designed in compliance with previous successful studies from Saudi Arabia (Zaidi et al., 2020), Italy (Napolitano et al.,2013), China (Zhu et al., 2016) and South Sudan (Sa'adatu Sunusi et al., 2019). The questions were altered to include commercial names of the drugs used in Serbia (such as paracetamol ). The questionnaire had 16 questions in Serbian language and was available online and in paper format. The questions are given in Table 1.
Subsequently, the results have been quantified and represented in percentile. The data was analysed on the level of whole group, as well as on the two groups of students coming from the surrounding rural area belonging to the Nisava region (Group A) and from the urban territory of city of Nis where university is located (Group B). For statistical difference, the common χ 2 -test for independence (Chi-squared test) was used, where value p<0.05 was considered significant.   Most students studied understand that antibiotics cannot be taken preventively (92.2 per cent) and that it is essential to complete the treatment in its entirety (87.5 per cent).
Such numbers have been common in many studies on university students (Kanneppady et al., 2019) and on the general population in Europe, like in Italy (Napolitano et al., 2013). Based on these answers only, it would seem that the students are aware of antibiotic resistance and that they consider that treatment should not be interrupted as soon as signs of relief are shown, as more resistant pathogens may survive and reintroduce the illness. This is, however, not reflected in other answers. Almost a third stated that antibiotics help with most diseases (31.4 per cent). This is quite the opposite of what we wanted to see, as many studies on causes of antibiotic resistance have stated overuse and misconceptions on healing power of antibiotics to be one of the principal causes of resistance development (Machowska and Lundborg, 2018 This section aimed to assess if the students adhere to the principles of rational usage and whether they put the knowledge they have into practice. The questions also aimed to discover whether self-medication was common as in past studies Grigoryan et al., 2010). The answers are summarised in Table 3.  We expected that the section on students' habits would show erroneous usage and selfmedication, with less appropriate attitudes present in the rural region. As expected, this section yielded less acceptable results. Ironically, it seems that students' knowledge is not well reflected in practice.
As many as 59.7 per cent of students admitted to acquiring antibiotics without a prescription, which is higher than reported by comparable studies in both Serbia and abroad. The only Serbian study on general public stated that 47.2 per cent of interviewees has admitted to self-medication , and in neighbouring Croatia, this number was even lower (37 per cent in 2001, and 41 per cent in 1977) (Aljinovic-Vucic et al., 2005: 76). This shows that, as expected, self-medication with antibiotics is common among surveyed students. Even after introducing stricter regulations in 2011, which forbid selling antibiotics over the counter, antibiotics have been available for purchase without prescription . doses, and as many as 62.5 per cent carry antibiotics with them for any emergencies when travelling. According to media reports, these are common practices in Serbia (Radivojevic, 2019). It is therefore evident that students frequently alter their dosages and do not adhere to their regimens. However, these numbers are better than ones reported in a large study in China, where 70.8 per cent reported interruption of the treatment after first signs of relief (Zhu et al., 2016: 81). In another Chinese study, 55.6 per cent reported taking doses irregularly (Pan et al., 2012).
On the positive side, a large portion of surveyed students would not use alcohol during antibiotic treatment (80.8 per cent). Past studies on Serbian students' attitudes towards alcohol demonstrated that drinking alcohol was a common practice (Kilibarda et al., 2013). In any case, in order to avoid errors that could occur when students who do not drink would answer 'No' as an answer to this question, the statement was given in a hypothetical form. The large percentage of students not taking alcohol during therapy could demonstrate their awareness of reactions of alcohol with some antibiotics and the overall effect of fatigue that alcohol has, as discussed in many official online guides regarding alcohol usage during treatments (Steckelberg, 2018). In our study, we reported a better percentage than the one reported in Tamil Nadu, India (61.4 per cent) (Arul Prakasam et al., 2011). Another satisfactory result is that 72.5 per cent of students stated they always read the instructions and checked the expiry date before usage, which is an important habit in handling any medicine. A similar number (70.8 per cent) was reported in China (Zhu et al., 2016: 81).
The most frequently chosen motive for taking antibiotics without a prescription was keeping leftovers at home (25.6 per cent), in line with the answer that they end their treatments prematurely. Our result is lower than those reported by some older studies in Serbia (46.5 per cent) (Paut Kusturica et al., 2015), possibly indicating a positive outcome of the 2015-16 campaign. Also, this number is significantly lower than the one reported in a similar Chinese study, where 63 per cent of those studied kept antibiotics at home (Peng et al., 2018), but very similar to a study on the general public from neighbouring Romania, a country with a similarly high DDD (31.57 vs. 28.5 in 2015) (World Health Organization, 2018: 28). This study reported 22.9 per cent of respondents using their leftovers from last treatments (Voidazan et al., 2019: 3385), indicating similar habits across the Balkans.
The second most chosen reason is having a medical professional in the household (24.4 per cent). Similar percentages of students trust their experience (18.6 per cent) and knowledge (16.4 per cent). The answers to this question confirm that students tend to trust their acquaintances, experience and knowledge acquired through high-school education, and indicate that they might consider that some diseases are not worth visiting their doctor about. This reinforces the statement that the opinions of close acquaintances have a high impact on consumer's attitude towards the drug (Akici et al., 2017).

Rural vs. urban: no statistically signi cant difference
A secondary objective of this study was to inspect differences in answers between students who live in an urban area and those who come from less developed (rural) regions. The urban area was defined as the immediate area of the City of Nis, which is the largest urban centre in the studied region and the third-largest city in the country, featuring a large clinical centre, a large primary healthcare centre and 13 municipalityspecific primary healthcare centres (Primary Healthcare Centre Nis, 2020). The less developed -rural area included the surrounding villages and settlements in Nisava region, which includes a significantly lower number of pharmacies, 12 primary healthcare centres and 27 ambulances with highly irregular working hours dispersed over 163,244 people, according to the 2011 census (Vukmirovic, 2015a: 464;Primary Healthcare Centre Nis, 2020). Students from the urban area benefit from a larger mean parental salary (Statistical Office of the Republic of Serbia, 2020), higher rate of employment in tertiary sector of economy and a higher number of inhabitants with high school or higher education (Vukmirovic, 2015b: 88-89). As such, we expected that urban-area students would score better than their counterparts from the rural region.
However, there was no statistical difference found between the two groups from Nisava region. This shows that awareness of the surveyed students is the same across the region where the university is located, which can be partially attributed to a high-school education system that equally covers antibiotics in most study courses. The official textbooks for biology that were being used at the time when the survey was conducted address the topic equally in the two main study courses available in Serbia. These include the language-humanities study course and the mathematics-natural sciences study course. The chapter on bacteria and viruses describes different types of bacteria, along with most common illnesses they provoke. It outlines the main guidelines of usage of antibiotics and briefly describes antibiotic resistance. While the part on rational usage of the drugs is not very extensive nor puts much emphasis on the global threats of ABR, it could provide students with some basic knowledge, regardless of places where their high schools were located. This also explains the relatively acceptable results obtained in the section on knowledge.
Additionally, a national campaign conducted in Serbia in 2015-16 managed to bring down the DDD per thousand inhabitants from 36.5 to 30.03 (World Bank, 2018). Despite the age of these students, the campaign could have altered the perception of usage of antibiotics and affected students' attitudes as well. These were the only sources of knowledge we identified that could have influenced these results, as there were no records of newer campaigns addressing antibiotic resistance and consumption.

Limitations and further research
One of the main limitations we encountered was the lack of data on current public health attitudes and practices in Serbia, including data on usage of antibiotics, incidence of infections, as well as availability of antibiotics and any public health strategies or campaigns being implemented. This is due to the scarce treatment of this subject in Serbia and poor pharmaceutical management. The country requires national guidelines on antibiotic use in the outpatient primary care . Some information regarding the subject is available in the Serbian language only and as such is inaccessible to the international audience.
This study on its own does not address knowledge on the topic of all young people in the region of similar age, rather it focuses on university students. Our other work refers to the knowledge of students of biomedical sciences and comparison to their colleagues from other fields. This is in line with many similar studies conducted around the world.
However, further studies are required to assess the situation in the entire age group, including young people not attending universities. Also, one needs to take into account that students' habits could have easily been influenced by their parents or other members of the family (Akici et al., 2017). Due to the young age of the surveyed sample, their opinions can be easily formed on practices of their elders. As such, further investigations on the general public are needed to assess this link and to design campaigns appropriately.

Conclusions and the future
Despite demonstrating a satisfactory level of knowledge, habits of students regarding usage of antibiotics are not as appropriate. Comparable studies conducted in the developing world and in Serbia have given similar results regarding habits. Students are aware of the antibiotics' mechanism of functioning and can distinguish them well from other types of commonly used NSAIDs. However, many have reported changing their regimens, interrupting them prematurely or taking doses irregularly, as well as buying antibiotics without a prescription. Such behaviour will aggravate the already unfavourable situation in Serbia. Rate of students who acquired antibiotics with no prescription is higher than those in countries with a similar DDD per thousand inhabitants rating.
Students' answers did not demonstrate awareness of the potential dangers and consequences of such actions. No difference was found in relation to the domicile of students, most probably due to common high-school education. After