Antibiotic susceptibility Patterns against Upper Respiratory Tract Pathogens in Tertiary Care Hospitals of Lahore, Pakistan CURRENT STATUS: UNDER REVIEW

Backgrounds In Pakistan, despite the surge in antibiotic consumption scanty of literature evidences exist regarding antibiotic susceptibility patterns in URTIs. Thus, we conducted the study to evaluate the antibiotic susceptibility patterns in URTIs. Methods A cross-sectional study was conducted by collecting 259 culture samples from tertiary care hospitals of Lahore, Pakistan. Using simple random sampling, culture reports of even numbered patient were included as per study inclusion criteria. Results Two hundred sixty samples were processed out of which only 144 (55.4%) samples yielded growth, i-e., 42.7% males and 49% females. In males, the resistance was high against ceftazidime (19.3%), ampicillin (13.1%), ciprofloxacin (11.6%) and gentamicin (10.8%), while in female samples it was significant against cefuroxime (6.9%), ampicillin (5.4%), tobramycin (4.6%) and ciprofloxacin (6.5%). Besides, males were sensitive against imepenem (25.8%), amikacin (22%), ceftazidime (19.3%), ciprofloxacin (19.3%) and piptaz (16.2%), while females were sensitive against amikacin (9.2%), imepenem (9.2%) and ciprofloxacin (6.9%). Overall, carbepenems, imipenem (35%) and meropenem (30.8%), were found highly sensitive followed by aminoglycosides (amikacin; 31.9%), quinolones (ciprofloxacin; 26.3%), piptaz (19.2%) among penicillin and cephalosporin, while resistant was maximum against cephalosporin (ceftazidime; 25.4%) followed by penicillin (ampicillin, 18.5%) and quinolones. Conclusion These data suggested that P. and , most

carbapenem and aminoglycoside class of antibiotics. Thus, health care professionals, policy makers and drug regulators must devise policies to overcome the menace of growing antimicrobial resistance.

Background
Upper respiratory tract infection (URTI) represents a persistent health issue among all the age groups, and is considered as the most common reason of consultation and hospitalization, thus imposes enormous burden on society [1]. The most common bacterial causes of RTIs include, Streptococcus, Klebsiella, Pseudomonas, Staphylococcus and Haemophilus influenza [2] nevertheless, the causative pathogens are not identified in almost 50% of cases [3]. Recently, it is estimated that the global antibiotic consumption, expressed in defined daily doses (DDDs), increased from 21.1 to 34.8 billion DDDs -an increase of 65% from 2000 to 2015 [4]. This increase in global antibiotic consumption was primarily driven by increased consumption in low middle-income countries (LMICs), including Pakistan. In this context, between 2000 and 2015, the highest surge in antibiotic consumption was observed among LMICs, i-e., 103% in India, 79% in China and 65% in Pakistan [4].
Antibiotics are prescribed more frequently in URTIs, but irrational and abundant use of antibiotics increase the chances of resistance among different species and effect the cost of total treatment [5]. A recent study from USA suggested that 51% of patients with acute URTIs were prescribed antibiotics although out of them 20% didn't require antibiotics [6], [7]. Antibiotic resistance not only results in severe infections leading to increase mortality but can also contribute towards undue financial burden [8], [9]. A study from Kenya reported that approximately 90% of E.
coli at Kenyan Hospital were found resistant to fluoroquinolones due to production of beta lactamases [10]. In UK, 25,000 patient die every year due to hospital acquired infections caused by multi drug resistant microorganisms [11]. In developing countries URTIs are more frequently reported at primary care centers and are of great concern due to almost non-existing standard prescribing/treatment guidelines, or if available, a very poor compliance by the prescribers that probably have an impact on patient's finances along with increase chances of developing resistance against antibiotics [12]. According to one estimate, Streptococcus pneumoniaand Moraxella catarrhalis are found in 54% and 72% of children, respectively, in first year of their lives [13], while 44% children between 2-4 years of age exhibited colonies of Haemophilus influenza [14]. The percentage of culture sensitivities ranges between 60%-95% [12], which sometimes falls below 31% depending upon the technique and personal expertise [15].
In Pakistan, most of the physicians start antibiotic therapy assuming that culture would be positive rather than performing culture sensitivity test and treat patient empirically, yet not according to standard criteria [16]. Irrational and empirical prescribing pattern reduce the rate of effectiveness and patient compliance as well.
Thus, inappropriate prescribing pattern of antibiotics is directly proportional to development of resistance in microorganisms, which in turn increases cost of therapy. A study from Pakistan, published in 1989, demonstrated that 97% of the isolated strains of Streptococcus pneumoniae from children's blood with acute lower respiratory tract infection were resistant to at least one antimicrobial drug, while, 62% exhibited decreased susceptibility to co-trimoxazole, 39% were resistant to chloramphenicol and 31% were fully resistant (Mastro 1991 Inclusion criteria: The samples from patients above 18 and below 74 years of age with confirmed diagnosis of URTIs (Runny nose, tonsillitis, pharyngitis, sinusitis, otitis media, cough, sore throat or common cold), irrespective of gender, ethnicity, financial, employment status and disease duration and willing to participate were included in the study.
Exclusion criteria: All samples from patients below 18 and above 74 years of age having unconfirmed diagnosis, multiple infections and not williing to participate were excluded from study.

Data Collection
Data was collected by employing comprehensive instrument of measure designed after extensive literature review [24,25], [26], [27].The questionnaire was sent to subject expert/academician for content validation, thereafter their expert opinion was incorporated to make the questionnaire more simple and objective driven. The reliability of the questionnaire was evaluated with Cronbach's alpha (0.78) using SPSS version 22. Face validation of the questionnaire was done by conducting a pilot study by collecting data of 20 samples and additional information gathered during data collection was incorporated in the final data collection form. The data obtained during the pilot study was not included in the final analysis. The field administrator docuemnted all the necessary parameters by evaluationg laboratory reports of the enrolled subjects. The questionnaire was outlined into the following sections; basic demographic, specimen type, organisim type & name and drug culture sensitivity pattern.
After documentation of the necessary information, the samples were collected with the help of sterile culture swab stick from the throat, pharynx and nasal cavity by rubbing deeply. Samples were shifted to laboratory immediately after collection for culture sensetivity analysis [28].

Data Analysis
Data were analyzed using SPSS (IBM, version 22), unless otherwise stated.
Descriptive analysis was performed to estimate the percentages and frequencies.
Differences in dicrete and continous variables were estimated using studetn t-test.
An alpha value of less than 0.05 was considred statistically significant.

Gender-wise prevalence of bacterial isolates
Gender wise prevalence of bacterial samples are shown in Figure S1 & Table 1. Out of total culture samples, n = 259, 169 were of males (65.4%) and 90 were of females (34.6%). Only 42.7% males samples exhibited growth compared to 49% in female samples (Fig. 1). Thus, out of 259 selected culture samples, only 61.5% test reports had bacterial growth while 38.5% reports had no growth (Table 1).
Among aminoglycosides, amikacin was tested sensitive in 22% male culture samples compared to other classes of antibiotics followed by gentamicin (17.%) and tobramycin (10.4%), while for the same drugs, 9.6%, 6.9% and 1.9 female culture samples were tested sensitive to amikacin, gentamicin and tobramycin, respectively (Table S1).
Among fluoroquinolones, ciprofloxacin was found sensitive in 19.3% male culture samples followed by levofloxacin (14.6%). Similar sensitivity pattern was observed in females, i-e., 6.9% for ciprofloxacin and 6.2% for levofloxacin. Furthermore, 11.6% male and 6.5% female culture samples were found resistant to ciprofloxacin. However, mild susceptibility patterns were observed for sulphonamides, macrolides, lincosamides and oxazolidiones (Table S1).

Discussion
Upper respiratory tract infection (URTIs) are amongst the most common and diverse group of infections in humans worldwide with prevalence rate 22% to 25% [29]. It is estimated that almost 38.5% cultures of URTIs have negative bacterial growth, indicating that such infections may be of viral origin as evident by previous report [30]. Data from the present study suggested that more than 60% cultures exhibited bacterial growth with S. aureus, MRSA and MSSA as most common gram-positive isolates, while P. aeruginosa, Klebsiella and E. coli were the most frequent gramnegative isolates in both males and females. Additionally, antibiogram showed that P. aeruginosa, Klebsiella and E. coli were most sensitive to carbapenem, while Klebsiella and P. aeruginosa exhibited highest resistance to cephalosporin. The frequency of resistance was highest among males for penicillin, cephalosporins, aminoglycosides and fluoroquinolones in comparison to females. Overall, carbepenems were found highly sensitive followed by aminoglycosides, quinolones, piptaz among penicillin and cephalosporins, while resistant was maximum against cephalosporin followed by penicillin and quinolones.
Several lines of literature evidences suggested that the most prevalent pathogens of URTIs include S. pneumonia, S. aureus, P. aeruginosa, E.coli, K. pneumonia and H.
influenza [31], [32]. Similar bacterial pathogens have been implicated in URTIs by studies from Pakistan with minor variations in frequency distribution of bacterial pathogens [33], [34]. Similar to these reports, we also observed that P. aeruginosa, Klebsiella, E. coli, and S. aureus were among the most common bacterial isolates in subjects having URTIs. There is scanty of literature evidences regarding gender specific consumption and laboratory antibiogram in Pakistani population with URTIs.
Literature evidences suggest that gender base differences exist in the incidence and severity of respiratory tract infections [35], [36]-more common in males compared to females. We also observed that the clinical enrollments of males were greater in number compared to females. However, in Pakistan, it is highly likely that these differences might also be due to higher social interaction of males in comparison to females, thus males probably have higher propensity to be infected.
Nevertheless, there is scanty of literature evidences, from Pakistan or from any other country, regarding gender differences in antibiotic susceptibility patterns in URTIs. We observed that imipenem, meropenem, amikacin, ciprofloxacin, gentamicin and piptaz were the most sensitive antibiotics in males, while amikacin, imepenem, ciprofloxacin and gentamicin were the most sensitive antibiotics in females with URTIs. On the other hand, maximum resistance in males was observed for ceftazidime, ceftriaxone and ampicillin, whereas in females, it was more frequent for cefuroxime, ciprofloxacin, ceftazidime and ampicillin. Thus, subtle gender wise differences exist regarding sensitivity and resistance to antibiotics, yet with a few commonalties. These data also suggested that cephalosporin class of antibiotics, second and third generation, are more frequently utilized in both males and females in URTIs, but probably their un-necessary use might resulted in considerable

Limitations of the study
There are several limitations of this study. The cross-sectional design of the study does not allow us to observe the susceptibility patterns over a period. The only access to culture reports resulted in non-availability of patients to crosscheck the information so have to rely on the information given on the reports with lots of missing information that needs to be excluded from the study. It is pertinent to mention that we have to reply on written diagnosis and were unable to counter check with the clinician. A very few studies were available from Pakistan for a direct comparison of susceptibility patterns with our findings.

Conclusion
In conclusion, our data suggested that gram -ve bacteria, P. aeruginosa, Klebsiella and E.coli were among the top bacterial isolates in URTIs, in both males and females. The very same bacteria exhibited significant resistance against penicillin, cephalposporins and ciprofloxacin, while imipenem, meropenem, amikacin and piptaz exhibited highest sensitivity against these bacteria. I was further concluded that males exhibited much higher resistance against penicillin, cephalosporin, aminoglycosides and quinolones in comparison to females. Thus, education and training programs must be introduced for clinicians to rationalize antibiotic therapy choices, dosing and duration. Likewise, educational and counselling sessions must be in place for the patients to cope with self-prescribing and timely completion of antibiotic therapy to avoid and curb anti-microbial resistance and irrational use,

Funding
This study received no finds from any institution or department, governmental or private.

Availability of data and materials
The data sets used in the study can be provided upon request to the corresponding author.

Consent for Publication
Consent for publication was obtained from the hospital administration and the participants.

Competing interest
Authors declared that they have no conflicts of interest [8].Avorn J, Harvey K, Soumerai SB, Herxheimer A, Plumridge R, Bardelay G: Information and education as determinants of antibiotic use: report of Task Force 5.