Changing Pattern of Bacteriological Profile, Antimicrobial Resistance and Mortality in Neonatal Sepsis in a Developing Country: A Retrospective Study

Introduction: Neonatal sepsis is one of the major contributors of mortality and morbidity among neonates. Irrational and overuse of antibiotics have led to an increase in antimicrobial resistance. This study was undertaken to investigate the bacteriological profile, antimicrobial resistance and predictors of mortality among blood culture-positive cases of neonatal sepsis . Methods: Demographic and bacteriological data were collected from electronic and manual case records. Automated BACTEC 9050 system using Peds Plus Vial was used for blood culture. Multidrug resistance was defined as a resistance to any three of five antibiotic classes like aminoglycoside, carbapenem, extended spectrum cephalosporins, fluoroquinolones and piperacillin. Results: Among 7180 admitted neonates, 433 (6.03%) were blood culture positive with early onset sepsis (EOS) in 50.1% of cases. Gram negative bacteria was the causative organism in 371 (85.7%) babies with klebsiella being the commonest pathogen (43.6%). The pathogen mix of early onset and late onset sepsis was similar and 90% of gram negative isolates were resistant to penicillin group. Multi drug resistance (MDR) was found in 51.2% of the gram negative organisms. EOS (Odds ratio 1.99; 95% confidence interval, 1.29-3.05) and MDR (Odds ratio 2.07; 95% confidence interval, 1.77-4.12) were independently associated with neonatal death due to sepsis. Conclusions: Gram-negative pathogens, specifically klebsiella accounted for a huge burden of neonatal sepsis. EOS and MDR were found to be independent predictors of death due to such sepsis. This study calls for multicentric studies on early onset neonatal infection and its relationship with pathogenic maternal flora.


INTRODUCTION
Neonatal sepsis remains the major cause of mortality and morbidity among neonates worldwide despite great advances in neonatal care. In India, the incidence of neonatal sepsis is 30/1000 live births. 1,2 Neonatal sepsis alone contributes to 19% of all neonatal deaths in India which increases to 50% in case of culture-proven sepsis. 3 Overuse of empirical broad spectrum antibiotics has increased the incidence of multi drug resistance (MDR). 4 In a recent multi-center Indian cohort study from North India, 5 gram -negative organisms contributed to two third of isolates that showed a high degree of antimicrobial resistance to antibiotics like extended spectrum cephalosporins and carbapenems. Almost similar picture has been drawn in another retrospective study from Delhi, India. 6 In India, a country with diverse demographics and heterogeneous level of neonatal care, bacterial isolates at NICUs and their antimicrobial sensitivity may differ in relation to different places and population and also with secular change. However, there is paucity of long term collaborative national data for bacteriological isolates of neonatal sepsis. This study was undertaken to investigate the distribution of bacteriological isolates and antimicrobial susceptibility patterns and mortality among blood culture positive cases of neonatal sepsis from a level III NICU of a large tertiary-care hospital in eastern India from 2014-2019. In reference to the earlier reports of neonatal sepsis from the same NICU in previous decade, 7,8 this study will also pave the way to determine the trend of bacterial isolates of neonatal sepsis and their antibiotic resistance over the years, which is yet to be documented in India.

METHODS
Demographic and bacteriological data were collected from electronic and manual case records. Automated BACTEC 9050 system using Peds Plus Vial was used for blood culture. Multidrug resistance was defined as a resistance to any three of five antibiotic classes like aminoglycoside, carbapenem, extended spectrum cephalosporins, fluoroquinolones and piperacillin.

RESULTS
During the study period, a total of 7180 babies were admitted in the NICU of which 1977 babies were treated for suspected neonatal sepsis. Blood culture was positive in 661 samples. Excluding fungal growth and growth of contaminants, a total of 455 blood culture samples were positive in 433 babies (6.03%) who were eligible for the study. The culture positivity rate of suspected sepsis was 21.9% (433 among 1977 babies). Table 1 showed the baseline characteristics of the study participants. Proportion of inborn and out born babies was almost equal among culture positive babies. EOS occurred in 50.1% (217) of the study infants.

Early onset versus late onset sepsis
There were equal share of EOS and LOS (table 2). Klebsiella pneumoniae was predominant in both early and late infection. However, the incidence of staphylococcus aureus sepsis was higher in LOS than EOS (17.1 % versus 8.3%, p < 0.005).

Mortality proportion and case fatality of different organisms
The CFR was 35.3% (153) among culture positive babies and mortality was significantly more in EOS than LOS (43.8 % versus 26.6%, p = 0.01) (Tables 1 and 2). Klebsiella pneumoniae caused highest number of deaths (80, 52.3%). Gram positive p a t h o g e n s ( S t a p h y l o c o c c u s a u r e u s a n d enterococcus spp.) were responsible for only five (3.3 %) deaths. Klebsiella pneumoniae and escherichia coli sepsis carried almost equal high CFR of 42 to 46% (Table 2). Overall the CFR was significantly higher in gram negative sepsis than gram positive sepsis (39.9% vs. 8.1%, p < 0.05).
Multi drug resistance MDR in cases of gram negative organism is depicted in table 2. Individually klebsiella pneumoniae topped the list of MDR organisms with 93 (48.9%) cases but the proportion of MDR was highest in acinetobacter spp (71.1%) followed by escherichia coli (57%), klebsiella pneumoniae (49.4%) and pseudomonas spp (37.3%) and the overall MDR proportion was 51.2% among gram negative bacteria. The CFR of MDR positive sepsis was 46.3% (88 / 190).

The trend of bacteriological isolates
The trend of blood culture positive sepsis cases over the years is shown in table 4. Klebsiella pneumoniae was the commonest isolate in early years (2014 -2017) but was outnumbered by escherichia coli and pseudomonas aeruginosa in subsequent years (2018 -2019). The proportion of MDR showed similar trend over the years (45% -60%).

Mortality risk factors
Prematurity, LBW, infection with MDR organisms and early onset sepsis were significantly associated with increased risk of mortality as found in univariate analysis (Table 5). In multiple logistic regression modelling, only EOS (OR 1.99, 95 % CI 1.29 -3.05) and MDR infection (OR 2.07 , 95% CI 1.77 -4.12) were found as independent predictors of morality in culture positive sepsis.

DISCUSSION
Our study has presented the epidemiology of neonatal sepsis and its causative bacterial organisms from a single center NICU of Eastern India dealing with more than 7000 admitted newborn over half a decade.  Two earlier reports from the same unit during 2007 to 2014 had also shown acinetobacter spp as the most common causal organism even before the result of the DeNis study was published. 5,7,8 The present study represents the subsequent era of the same unit from 2014-2019. The authors of the previous reports found that more resistant nonfermenting gram-negative bacteria (NFGNB) dominated the causative organisms of neonatal sepsis including certain rare organisms. Now in the same unit, acinetobacter spp (12%) has been outnumbered by klebsiella pneumoniae (43.6%) and escherichia coli (14.5%). The present-day incidence of acinetobacter spp and pseudomonas spp (NFGNB) sepsis is less (12 -13%) than the previous report from the same NICU (23.6%) in 2014. 8 One organism or a group of organisms may be replaced over a period of time in critical areas like NICU which was also reflected in a previous study by Roy et al. 2 This could be the result of continuous quality control initiatives for prevention of infection and implementation of antimicrobial stewardship program adopted since 2012 with a target of zero tolerance to sepsis.
Incidence of EOS and LOS were almost equal in the present study, which is in sharp contrast with the reports published from other Asian countries, 19,20 as well as those from developed countries where most infection are late onset in NICU. 21 Our data is somewhat consistent with the recent report published from Delhi where EOS was more than LOS and the pathogen mix of EOS and LOS was almost similar with the predominance of gram negative organism. 6 In a large referral unit like ours the predominance of in-utero transfer of sick pregnant mothers and heavy load of high-risk delivery increases the likelihood of perinatal and early onset infection among NICU babies.
Resistance to reserve drugs like colistin and carbapenems were found in 16-35% cases. Third generation cephalosporins have already been ineffective with documented resistance in 75 -80% of cases. In view of resistance to more than 90% of gram negative isolates and 85 -90% of gram positive isolates; ampicillin as the first line antibiotic needs revision. In the present study, the incidence of MDR was already 50% among gram negative organisms. The observation was quite similar to a few reports published from North India. 14,16,22 Hence judicious use of empirical antibiotics based on local isolates and antibiotic sensitivity pattern should be made mandatory in neonatal units. Based on a review of 11,471 blood cultures from developing countries in South-East Asia, Zaidi et al 23 have recommended imipenem and amikacin regimen for initial treatment of suspected sepsis. However, considering an increase in the proportion of acinetobacter spp, use of carbapenem group of drugs also needs restriction as the antibacterial armamentarium is extremely limited at present.
The case fatality was higher in case of gram negative organisms and even higher in MDR strains. High load of antimicrobial resistance may be the cause behind this. Contrary to previous reports, 5,7,8 the isolation of more resistant and fatal acinetobacter spp was less in our study. The CFR of culture positive sepsis in developed countries is higher in case of LOS. 21 But in the present study death was significantly higher in EOS like which was similar to other reports from developing   NT countries also mentioned a significant role of similar maternal risk factors in developing EOS. 24,25 This burden of gram negative organism has increased as a result of very early horizontal transmission from delivery rooms and NICUs 23,26 or vertical transmission from the maternal genital tract colonized with these pathogens. 25,27 Hence infection control in delivery room and resuscitation area, eradication of maternal pathogenic flora and restriction on the indiscriminate use of antibiotics in expectant mothers might be the only sustainable measures to prevent this disaster.
Our study has explored the pertinent issue of neonatal sepsis in the resource limited set up. We have conducted our research in one of the largest NICUs of India with more than 7000 newborns over a half decade. This study has a bigger implication in Indian scenario where presently the neonatal care is going through the phase of rapid expansion with many level-II sick newborn care units. Despite being a large study, it does have limitations of being retrospective and single centric study. Similarly, we also have to acknowledge that molecular mechanism of resistance especially e x t e n d e d -s p e c t r u m b e t a -l a c t a m a s e a n d carbapenemases were not tested. This study calls for further multicentric, prospective studies on the burden of EOS and its relationship with of pathogenic maternal flora in view of significant case fatality observed in EOS.

CONCLUSIONS
Gram negative pathogens and more specifically klebsiella pneumonia was the predominant organism of neonatal sepsis. EOS and MDR infection have been found to be two independent predictors of mortality in neonatal culture proven sepsis. There is an urgent need for revision of commonly used combination of ampicillin and gentamycin as empirical antibiotic therapy in view of a very high ampicillin resistance.