SENSITIVITY AND RESISTANCE PATTERN OF ANTIMICROBIAL AGENTS USED IN CASES OF NEONATAL SEPSIS AT A TERTIARY CARE CENTRE IN WESTERN INDIA

SENSITIVITY AND RESISTANCE PATTERN OF ANTIMICROBIAL AGENTS USED IN CASES OF NEONATAL SEPSIS AT A TERTIARY CARE CENTRE IN WESTERN INDIA

Anand J. Amin ^*1, Prakash P. Malam ², Pratik D. Asari ¹, Urja R. Patel ¹ and Archana B. Behl ¹

Department of Pharmacology ¹, Medical College Baroda, Vadodara, Gujarat, India

Department of Pharmacology ², Government Medical College, Surat, Gujarat, India

ABSTRACT: Purpose: Neonatal sepsis, a major killer among neonate, necessitates urgent implementation of empirical therapy and later specific antimicrobial therapy against causative microorganism. Empirical therapy relies upon data obtained from previous studies. We conducted this study to evaluate the blood culture, sensitivity and resistance pattern of various antimicrobial agents in neonatal sepsis at Neonatal Intensive Care Unit at a tertiary care hospital in western part of India, which will show us the changing pattern of etiological organism and antimicrobials needed to curb them. Methods: We conducted a prospective cross sectional study over a period of six month duration in Neonatal Intensive Care Unit at tertiary care hospital.  We collected and analyzed the blood culture reports and sensitivity pattern of antimicrobials used. Results: We enrolled 163 patients of neonatal sepsis. Paediatricians sent 163 blood samples for culture of which 62 % culture were positive. Out of 101 positive cultures 70 % were Gram negative organisms. Most frequently encountered organisms were Klebsiella Pneumonia followed by Acinobacter species while least common was Coagulase Negative Staphylococci. Conclusion: Our world is facing a huge issue of development of resistance to antimicrobial agents by microorganisms. Irrational prescribing habits of physicians are leading to increasing morbidity, mortality and treatment costs. To prevent antimicrobial resistance, regular educational awareness programs should be conducted in hospitals at a regular basis. Also comparative studies in the same hospital over years will help us to generate proper antimicrobial policies ultimately leading to rationale drug therapy

Neonate, Sepsis, Culture, Antibacterial drug resistance

INTRODUCTION: Neonatal  sepsis  is  defined  as  “a  clinical  syndrome  of  bacteraemia  with  systemic  signs  and  symptoms  of  infection  in  the  first  four  weeks  of  life”.  When  pathogenic  bacteria  gain  access  into  the  blood  stream,  they  may  cause  overwhelming  infection  without  much  localization  (septicaemia)  or  may  get  predominantly  localized  to  the  lung  (pneumonia)  or  the  meninges  (meningitis) ¹.

Various  systemic  infections  of  the  newborn  such  as  septicaemia,  meningitis,  pneumonia,  arthritis,  osteomyelitis,  and  urinary  tract  infections  are  considered  as  part  of  neonatal  sepsis  but  not  superficial  infection  like  conjunctivitis  or  oral  thrush.

Neonatal sepsis is divided into two types based on the time of onset of the symptoms i.e. early onset sepsis (EOS) if sepsis presents within first 72 hours of life or late onset sepsis (LOS) if after 72 hours of life ².

Clinically neonatal sepsis may present as hypo/hyperthermia, lethargy, poor cry, refusal of feeding, poor perfusion, hypotonia, absent neonatal reflex, respiratory distress, brady/tachycardia etc.

Sepsis is the commonest cause of neonatal mortality which is responsible for about 30-50% of the total neonatal deaths in developing countries ^{3, 4}. It is estimated that up to 20% of neonates develop sepsis and approximately 1% die of sepsis related causes ⁴. World Health Organisation estimates that of the four million neonatal deaths all over the world every year, over 35% are due to infection in the neonatal period⁵.

In India the incidence of neonatal sepsis according to the National Neonatal Perinatal Database is 30 per 1000 live births ⁶. The database comprising 18 tertiary care neonatal units across India found sepsis to be one of the commonest causes of neonatal mortality contributing to 19% of all neonatal deaths. Septicemia was the commonest clinical category with an incidence of 23 per 1000 live births while the incidence of meningitis was reported to be 3 per 1000 live births. Klebsiella Pneumoniae was the most frequently isolated pathogen in intramural setting (32.5%) and extramural setting (27%), followed by Staphylococcus Aureus (in intramural-13.6% and in extramural -15%) and Pseudomonas in extramural setting (13%). To treat neonatal sepsis, we need immediate targeted antimicrobial treatment directed against the causative organism and based upon their sensitivity pattern. To identify causative agents, microbial culture is considered as the gold standard and antimicrobial resistance pattern is of utmost importance for a definitive and prompt cure.

However it has been observed that the etiological agents and their sensitivity towards antimicrobial agents keep on changing over a period of time, so for optimum management of neonatal sepsis we need data of all these important variables. This study will provide us important data regarding culture and sensitivity patterns of causative organisms which will help us to intensify or modify the treatment policies if necessary. Also this study was conducted in a tertiary care hospital in western India; it may help for conducting large multi-centric studies in future.

MATERIALS AND METHODS:

Study design: A prospective cross sectional observational and single centre study was conducted for 6 months in neonatal intensive care unit (NICU) at a tertiary care hospital located in western part of India. We included patients from both extramural and intramural divisions.

The microbiology laboratory of the hospital is accredited by NABL (National Accreditation Board for Testing and Calibration Laboratory) which is an autonomous authority under Government of India. Various microbiological cultures and testing were done as per standard guidelines.

 Sample size and study population:

We included 163 neonates (zero to four weeks) admitted to NICU, who were suspected/ diagnosed cases of neonatal sepsis and in whom blood cultures were done.

 Data collection:

Period of data collection:

We collected data starting from 1^st April 2013 to 31^st September 2013.

 Inclusion criteria:

• All patients (zero to four weeks of age) of suspected/diagnosed neonatal sepsis of either sex admitted to NICU after 1^st April 2013.

• Only the patients in whom blood cultures were done.

• Only the patients whose parents gave informed consent were enrolled in this study.

 Exclusion criteria:

If parents of the septic neonates were not willing to participate in the study.

 Informed consent:

We took informed consent from all the parents of the neonates having suspected/ diagnosed as neonatal sepsis before enrolment.

 Study method:

We took prior permissions from the Head and Professor of concerned departments of the concerned hospital.

We obtained necessary ethical permission was from institutional ethics committee. We visited the NICU and Microbiology department daily to confirm that patients were selected and enrolled according to inclusion and exclusion criteria.

 Blood collection method:

Two blood samples were collected aseptically from patients for routine blood culture. The vein puncture site was disinfected with 70% alcohol and 2% tincture of iodine before collecting approximately 2 ml of blood for culture.

 Blood culture:

Approximately 2 ml of collected blood was added in glucose broth maintaining sample broth ratio of approximately 1:8 to 1:10. After this culture bottle was sent to microbiology laboratory as soon as possible and kept for initial incubation at 35-37 ⁰C for 6-12 hours. Then one loop full of sample was inoculated on Mac-Conkey agar and Nutrient/blood agar each and were incubated at 35⁰C, then observed for growth after 24 hours. If growth was seen then various tests like gram staining and microscopic examination, citrate, urease, catalase, coagulase, bile solubility etc. were done to identify and confirm the species of microorganism.

 Antimicrobial resistance:

Few of the colonies were inoculated in peptone water and kept for incubation at 37⁰C for six hours, which later was subjected to antimicrobial sensitivity tests. Modified Kirbey-Bauer method was used (Clinical and Laboratory Standards Institute). Depending upon microorganism (Gram positive and negative), various antimicrobial agents were checked for the sensitivity pattern. We collected data from indoor case papers as well as records from microbiology department and recorded on a prestructured case reporting form (CRF).

Data analysis:

We entered data in Microsoft excel sheet 2007 and tabulated later.

 RESULTS:

In this study, we considered 220 patients and depending upon inclusion criteria, enrolled 163 patients having neonatal sepsis and admitted to Neonatal Intensive Care Unit (NICU), at a tertiary care hospital in western part of India. Blood cultures of these 163 patients were sent for microbial examination. Out of total 163 microbial cultures done, we found 101 (62%) cultures to be positive which were subjected to antimicrobial sensitivity check.

In current study, out of the 101 positive results, we identified gram positive organisms in 30 isolates (30%) and gram negative organisms in 71 isolates (70 %). As shown in Table 1, most frequently found organism responsible for neonatal sepsis was Klebsiella Pneumoniae in 28 cultures (28%), followed by Acinobacter species in 23 cultures (23 %), while least frequently found organisms were Pseudomonas in 8 culture (8%) and Coagulase Negative Staphylocci (CONS) in 4 cultures (4%).

TABLE 1: FREQUENCY OF ORGANISMS ISOLATED BY CULTURE

Results displayed in Table 2 shows that the most effective antimicrobial against Staphylococcus aureus were vancomycin and ceftazidime.

TABLE 2: SENSITIVITY PATTERNS OF STAPHYLOCOCCUS AUREUS

 Results showed in the Table 3 states that against CONS organism gentamycin, vancomycin and levofloxacin were most effective while ceftriaxone was not effective.

TABLE 3: SENSITIVITY PATTERNS OF COAGULASE NEGATIVE STAPHYLOCOCCI

As per the results in Table 4, we can say that against Klebsiella organism meropenem, piperacillin + tazobactam combination, levofloxacin and imepenem were most effective while cefepime and cefotaxime were least effective.

TABLE 4: SENSITIVITY PATTERNS OF KLEBSIELLA SPECIES

Results displayed in Table 5 shows that against Acinobacter organism piperacillin + tazobactum combination, levofloxacin and meropenem were most effective.

TABLE 5: SENSITIVITY PATTERNS OF ACINOBACTER SPECIES

Results have been displayed in Table 6, these show that against E. coli organism piperacillin + tazobactum combination, meropenem, aztreonam and imepenem were effective while ceftriaxon, gentamycin, cefepime and levofloxacin were not effective.

TABLE 6: SENSITIVITY PATTERNS OF ESCHERICHIA COLI

As per the results showed in the Table 7, we can say that against Pseudomonas organism piperacillin + tazobactum combination, amikacin, gentamycin, and meropenem, were most effective while imepenem was least effective.

TABLE 7: SENSITIVITY PATTERNS OF PSEUDOMONAS

The results displaying sensitivity pattern of Enterococci have been shown in Table 8. Results showed that against Enterococci organism linezolide and vancomycin were most effective while gentamycin, erythromycin, ciprofloxacin and ceftazidime were not effective.

TABLE 8: SENSITIVITY PATTERNS OF ENTEROCOCCI

DISCUSSION: Septicaemia is one the major causes of morbidity and mortality in the neonatal period, and it often has a rapid and fulminant course. The database comprising 18 tertiary care neonatal units across India found sepsis to be one of the commonest causes of neonatal mortality contributing to 19% of all neonatal deaths ⁶.

In neonatal sepsis, culture and sensitivity reports are of utmost importance. We observed that in our study total 163 microbial culture and sensitivity tests were done, of which 62 % culture were found to be positive. This is higher than that reported by Shreshtha S et al and Shahian et al in which the blood culture positivity rate was 44% and 43% respectively ^7-8. In our hospital culture and sensitivity reports are done on the day of admission and empirical antimicrobial therapy is started with Cefotaxime and Amikacin combination. Culture is repeated later, if clinical improvement is not seen despite of empirical antimicrobial therapy. So many times undue culture tests are avoided, yielding higher rate of positive culture observed in our study.

In this study gram positive organisms were identified in 30 % cultures and gram negative organisms were seen in 70 % of cultures.  Study conducted by Rahman showed that in their study 70 % cases of neonatal sepsis were caused by gram negative organisms ¹⁰. The results in our study reflects what have been earlier established that in developing countries gram negative organisms are causative organisms in majority of cases of neonatal sepsis ⁴.

We observed that most frequently found organism was Klebsiella Pneumoniae, followed by Acinobacter species and Staphylococcus Aureus. Least frequently found organisms were Pseudomonas and Coagulase Negative Staphylocci (CONS).

Aletayeb et al reported Klebsiella Pneumoniae, E.coli and Acinobacter as the major organisms, which is in accordance with our study ¹¹. Klebsiella Pneumoniae, Staphylococcus Aureus and CONS were the predominant organisms in the study done by Shrestha S et al and Jyothi P et al ^{7, 12}. Pseudomonas Aeroginosa was the predominant organism for neonatal sepsis followed by Klebsiella and Acinobacter in the study done by Bhat R et al ¹³. Shahian et al reported CONS, E. coli and Staphylococcous as the major organisms which is in contrast to observations in our study ⁹.

Staphylococcus Aureus was the predominant organism for neonatal sepsis followed by Klebsiella and Escherischia Coli in the study done by Mhada TV(2012) et al ⁸. Shahian et al and Dias E et al reported CONS as the major organisms for neonatal sepsis^{9 and 14}. The similar or contradictory findings found in above mentioned studies reflect differential patterns of causative organisms in neonatal sepsis at different geographical areas.

The results in our study shows that for Staphylococcus Aureus infection most effective antimicrobial given in the mentioned study were vancomycin and ceftazidime as all isolates checked were found to sensitive to them. This was in accordance with the study by Shrestha, which showed that vancomycin was effective in 100 % isolates⁷. However, in study conducted by Kayagne  vancomycin was effective against 14 % strains only while study by Shahian showed that ceftazidime was effective only in 22% ^{15, 9}.

In our study according to sensitivity results we noted showed that against CONS organism gentamycin, vancomycin and levofloxacin were most effective in all cultures while ceftriaxone was not effective in any. These results were comparable with study done by Shrestha ⁷. In some other studies done by Shahian ⁹ and Dias ¹⁴, results showed that strains were sensitive to gentamycin only in 50 % and 63 % cultures respectively ^{9, 14}.

Our study also showed that  Klebsiella organism were sensitive to  meropenem in 100 % cultures, levofloxacin in 83 %  and imepenem in 77 % of cultures while cefepime and cefotaxime were not effective at all. Similar results were observed in studies done by Aletayeb and Shrestha who showed 90 % and 100 % sensitivity towards imepenam, respectively ^{11, 7}. In contrast to our study, Kayagne showed only 2 % sensitivity to meropenem and Shahian showed only 33 % sensitivity to imepenem^{15, 9}.

As per our study Acinobacter organism were most sensitive to piperacillin + tazobactum combination in 93 % of cultures, levofloxacin in 90 % of cultures and meropenem in 80 % of cultures. These results were comparable to study done by Shrestha showing 100% sensitivity to meropenem⁷.

In our study results showed that against E. coli organism piperacillin + tazobactum combination, meropenem, aztreonam and imepenem were effective in 100 %, 100 %, 80 % and 80 % of positive cultures respectively  while ceftriaxon, gentamycin, cefepime and levofloxacin were not effective.  Other studies to be compared are, by Shrestha showing 100 % sensitivity to imepenem but 100 % resistance aztreonam, Shahian showing 67 % and 33 % sensitivity to imepenem and gentamycin respectively, Mhada showing 43 % sensitivity to gentamycin, Kayagne showing 0% and 68 % sensitivity to meropenem and gentamycin^{7, 9, 8, 15}.

We observed that against Pseudomonas organism piperacillin + tazobactum combination, amikacin, gentamycin, and meropenem, were found to be effective in 100 %, 100 %, 100 % and 83 % of cultures respectively   while imepenem was effective only in 17 % of cultures. Study by Rehman showed gentamycin was effective only against 21 % strains ¹⁰. Shrestha showed 100 % sensitivity to imepenem but only 11 % and 0 % sensitivity to gentamycin and amikacin respectively⁷. Mhada showed that amikacin and gentamycin were not effective ⁸.

This study showed that against Enterococci organism linezolide and vancomycin were effective in all cultures while gentamycin, erythromycin, ciprofloxacin and ceftazidime were not effective. Similar results were shown by Shahian and Shrestha ^{9, 7}.

The differences found in sensitivity patterns in neonatal sepsis mentioned in above studies shows that susceptibility of various microorganisms differs at different places and different time period.

Limitations of current study were shorter duration of study and study was conducted in single centre only. Ideally blood samples for culture should be repeated after 3-4 days and antimicrobial therapy should be changed as per the report, however as the hospital is a limited resource hospital and to minimize the cost of the treatment, blood cultures were repeated only in case the empirical antimicrobial therapy was not effective.

 CONCLUSION: Various studies indicate gradual increase in the emergence of antibiotics resistance organisms. Factors which play role in the development of resistance include no uniformity in the usage of antibiotics, indiscriminate use, availability of antibiotics etc. Also microbial pattern and their sensitivity towards antimicrobials vary depending on the study group and the hospital setup. So trend nowadays is towards comparative studies in the same hospital over years, which will help us to generate proper antimicrobial policies for various infective conditions. This may ultimately lead us towards rationale drug therapy.

 ACKNOWLEDGEMENTS: We are very much thankful to the Head of the department of concerned departments for giving us permission for the study. We are also helpful to the healthcare staff of NICU (Neonatal Intensive Care Unit) and microbiology laboratory for their support. No financial support was taken for this study.

 CONFLICT OF INTEREST: We, the authors of this article declare that we have no conflict of interest of any means.

 REFERENCES:

1. NNF Teaching Aids: Newborn care. (newbornwhocc. org/ pdf/ teaching - aids/ neonatal sepsis. pdf)
2. Singh M, Narang A, Bhakoo ON: Predictive perinatal score in the diagnosis of neonatal sepsis. J Trop Pediatr 1994 Dec; 40:365-8.
3. Bang AT, Bang RA, Bactule SB, Reddy HM, Deshmukh MD: Effect of home-based neonatal care and management of sepsis on neonatal mortality: field trial in rural India. Lancet 1999; 354:1955-61.
4. Stoll BJ: The global impact of neonatal infection. Clinics in Perinatology 1997; 24:1-21.
5. Joy E Lawn, Katarzyna Wilczynska-Ketende, Simon N Cousens: Estimating the causes of 4 million neonatal deaths in the year 2000. International Journal of Epidemiology 2006; 35:706-718. DOI:10.1093/ije/dyl043
6. Report of the National Neonatal Perinatal Database (National Neonatology Forum) 2002-2003.
7. Shrestha S, Shrestha NC, Dongol Singh S, Shrestha RPB, Kayestha S, Shrestha M, et al: Bacterial Isolates and its Antibiotic Susceptibility Pattern in NICU. Kathmandu University Medical Journal 2013; 41:66-70.
8. Mhada TV, Fredrick F, Matee MI, Massawe A: Neonatal sepsis at Muhimbili National Hospital,Dar es Salaam, Tanzania; aetiology, antimicrobial sensitivity pattern and clinical outcome. BMC Public Health 2012; 12:904.
9. Shahian M, Pishva N, Kalani M: Bacterial Etiology and Antibiotic Sensitivity Patterns of Early-Late Onset Neonatal Sepsis among Newborns in Shiraz, Iran 2004-2007. Iran Journal of Medical Science 2010 December; 35:293-8.
10. Rahman S, Hameed A, Roghani MT, Ullah Z: Multidrug resistant neonatal sepsis in Peshawar, Pakistan. Arch Dis Child Fetal Neonatal Ed 2002; 87:F52–4.
11. Aletayeb SM, Khosravi AD, Dehdashtian M, Kompani F, Mortazavi SM, Aramesh MR: Identification of bacterial agents and antimicrobial susceptibility of neonatal sepsis: A 54-month study in a tertiary care hospital. African Journal of Microbiology Research 2011; 5:528-31.
12. Jyothi P, Basavaraj MC, Basavraj P: Bacteriological profile of neonatal septicemia and antibiotic susceptibility pattern of the isolates. Journal of Natural Science Biology and Medicine 2013; 4:306-9.
13. Bhat R, Lewis LE, Vandana KE: Bacterial isolates of early-onset neonatal sepsis and their antibiotic susceptibility pattern between1998 and 2004: an audit from a center in India. Italian Journal of Pediatrics 2011; 37:512-17. Doi: 10.1186/1824-7288-37-32.
14. Dias E, Vigneshwaran P: The Bacterial Profile of Neonatal Septicaemia In A Rural Hospital In South India. Journal of Clinical and Diagnostic Research. 2010 December; 4:3327-30.
15. Kayange N, Kamugisha E, Mwizamholya DL, Jeremiah S, Mshana SE: Preditors of positive blood culture and deaths among neonates with suspected neonatal sepsis in a tertiary hospital, Mwanza- Tanzania. BMC Pediatrics 2010; 10:45-8.
    

    ---

    How to cite this article:

    Amin AJ, Malam PP, Asari PD, Patel UR and Behl AB: Sensitivity and Resistance Pattern of Antimicrobial Agents Used In Cases of Neonatal Sepsis at a Tertiary Care Centre in Western India. Int J Pharm Sci Res 2016; 7(7): 3060-67.doi: 10.13040/IJPSR.0975-8232.7(7).3060-67

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---