Molecular Characterisation and Profile of Extended Spectrum Beta Lactamase Producing Enterobacteriaceae Isolates Causing Neonatal Sepsis at a Tertiary Care Center

Introduction: Emergence of extended spectrum beta-lactamase producing strains of gram-negative bacteria is increasing and it affects outcome of neonatal sepsis. Present study was done to assess the prevalence, antibiogram and molecular characterisation of ESBL producing enterobacteriaceae organisms in neonatal sepsis. Methods: A cross-sectional study was conducted at a tertiary care centre in Agra from January 2016 to June 2017. Total 700 patients satisfying inclusion and exclusion criteria were enrolled. Workup for sepsis screen, blood culture, antibiotic susceptibility, disk approximation test for detection of ESBL producing organism and polymerase chain reaction were performed. Results: Out of 700 patients, blood culture was positive in 238 (34%) cases. Among all the blood-culture isolates, Gram positive, Gram negative and candida species were (54%), (39%) and (7%) respectively. Among gram negative isolates, Klebsiella (16%), E. coli (10%), Pseudomonas (6%), and Burkholderia (5%) were isolated. Prevalence of ESBL producing Enterobacteriaceae isolates was 42.42%. Among ESBL producers 13 were E. Coli and 15 were Klebsiella. ESBL producing bacilli were more common in males (p = 0.015) and out born patients (p = 0.042). CNS symptoms were the commonest manifestations (35.71%). All ESBL producing Klebsiella and 92.3% of E. coli were resistant to co-amoxyclav. All the ESBL producing Klebsiella and E. coli were sensitive to imipenem and colistin. CTXm gene was the commonest gene present in ESBL producing E. coli (61.54%) and Klebsiella (26.67%). Conclusions: ESBL producing Klebsiella and E. coli were resistant to most of the commonly used antibiotics but they were sensitive to carbapenems and colistin. CTXm gene was the commonest gene, associated with ESBL producing enterobacteriaceae.


INTRODUCTION
Neonatal sepsis is a clinical syndrome characterised by signs and symptoms of infection with or without accompanying bacteraemia in the first month of life. It encompasses septicaemia, meningitis, pneumonia, arthritis, osteomyelitis and urinary tract infection. Septicaemia is one of the most important causes of neonatal mortality and morbidity. Incidence of septicaemia varies between countries, but developing countries had reported higher incidence in comparison of developed nations. 1 In India also many studies had reported septicaemia as a leading cause of neonatal mortality and morbidity. 2 Both Gram-positive as well as Gramnegative bacteria and sometimes fungal infections are responsible for neonatal sepsis. 3 Because of the changing pattern of antibiotic use, the spectrum of organisms that cause neonatal sepsis change over times and vary regionally. 4 Gram negative bacteria form important group of neonatal sepsis worldwide. Among Gram negative bacilli, the two most important bacterial pathogens causing neonatal sepsis in developed countries are Klebisella pneumoniae and E. Coli. 5,6 Sepsis with extended-spectrum beta-lactamase (ESBL) producing bacteria is a major problem worldwide. 7 ESBLs are plasmid-mediated enzymes that hydrolyse broad-spectrum beta-lactams. 8 More than 100 ESBL variants from different types are known, commonest being SHV, TEM, OXA and CTX-M. While TEM-and SHV-type ESBLs have developed from their ancestors SHV-1 and TEM-1/-2 by point mutations, the origin of the other enzymes is less clear. 9 Molecular characterisation of ESBL from neonatal sepsis cases at Safdarjung Hospital, Delhi had shown CTX-M-15 as the most prevalent type, with other types being TEM-1, SHV-1, SHV-28, SHV-11 and SHV-12. 10 The present study was planned to know the prevalence and molecular characterisation of extended spectrum beta lactamase producing Enterobacteriaceae in newborns admitted in NICU with neonatal sepsis in our institute.

METHODS
A cross-sectional, prospective study was carried out among patients admitted with sepsis in NICU in Sarojini Naidu Medical College, Agra, India. All neonates with clinical features suggestive of sepsis like alteration in the established feeding behaviour, tachypnea, chest retraction, grunting, excessive or high pitched cry, seizure, blank look or bulging anterior fontanel, lethargy, hypothermia, fever, diarrhoea, vomiting and abdominal distension, sclerema, episodes of apneic spells or gasping were included in study during January 2016 to June 2017. Neonates who were already on antibiotics or those who developed signs of sepsis within 72 hours of discontinuation of antibiotics, neonates with major congenital anomalies, severe birth asphyxia were excluded. After taking written and informed consent from parents, a semi-structured questionnaire was filled that consisted of basic socio demographic profile. Sepsis screening was carried out in all the newborns. Blood samples were also collected for blood culture and antibiotic sensitivity. The isolates of culture were further analysed for identification of gram negative Enterobacteriaceae and ESBL production. Molecular characterisation of ESBL producers was analysed by PCR.
Neonates with two or more of the following were considered positive sepsis screen -Absolute neutrophil count < 1800 cell/mm 3 , total leucocyte count < 5000/mm 3 , ratio of immature to total neutrophil count > 0.2, micro ESR > 15 mm and CRP > 10 mg/l. Blood cultures that flagged positive were subjected to Gram staining and sub cultured on appropriate medium. Mac-Conkey agar and 5% sheep blood agar was used for sub culturing Gramn e g a t i v e a n d G r a m -p o s i t i v e o rg a n i s m s , respectively. Blood culture bottles incubated in the system for up to seven days.
Antibiotic susceptibility testing was done by Kirby Bauer Disk Diffusion method on Mueller Hinton Agar as per Clinical and Laboratory Standard Institute guidelines (CLSI). Escherichia coli ATCC 25922 was used as control. Samples showing an inhibition zone size of ≤ 22 mm with ceftazidime and ≤ 27 mm with cefotaxime were considered as potential ESBL producers and were further investigated for confirmation. 11 Isolates that s h o w e d r e s i s t a n c e t o t h i r d g e n e r a t i o n cephalosporins were screened to detect ESBL production. A modified double disk synergy (Disk Approximation Test) was carried out on resistant isolates. The ESBL producing isolates were further processed for genetic characterisation. Genetic material isolation was done using miniprep (reference) method. 11 Three sets of primers were used to amplify internal region of the TEM, SHV and CTX-M genes. PCR amplification was performed in 25 µl reaction mixture. A negative (no template) control and a positive control containing a DNA mixture of three control strains (SHV-1, TEM-2 and CTX-M betalactamase producers) were included. DNA was amplified with the BIO-RAD C1000 TM thermal cycler using 3 cycles. The PCR products were analysed on 2% Agarose gel with 1U Tris-borate-EDTA buffer. The results were confirmed for CTXm, SHV and TEM gene by appearance of band near 544, 928 and 837 base pair marker ladder respectively. Data were presented as frequency and percentage, Chi square test was used to find out association between study groups and p value < 0.05 was considered as statistically significant.
Present study was approved by Ethical research committee of the institute.

RESULTS
Out of 700 cases, sepsis screen was positive in 52.5% (n = 368) cases and blood culture was positive in 34% (n = 238) cases ( Figure 1). 60.1% (421) patients were males, 63.9% (447) were preterm, 65% (465) were LBW (< 2500 grams), 47.9% (335) patients were of lower class, 37.3% (261) were of lower middle class and 73.4% (514) were out born. There was statistically significant association of blood culture positivity with sepsis screen, onset of sepsis and place of delivery (p < 0.05) (  Figure 2). As shown in figure 3, all CTXm ESBL producing E. coli and Klebsiella pneumoniae were resistant to amoxyclav and sensitive to imipenem and colistin. Similar pattern was also seen with TEM positive E coli and Klebsiella pneumoniae isolates and SHV positive Klebsiella pneumoniae isolates, however all the isolates were sensitive to meropenem.

DISCUSSION
Antibiotic resistance is one of the major concerns in the management of neonatal sepsis and an important confounding factor is the development of ESBL producing organisms. 4    patients were of lower socio-economic class, as in the study of Ahmed et al. 15 Most common presenting symptoms were refusal to feed and lethargy, followed by CNS and respiratory manifestation and these findings were similar to several other studies. 14,16 The results of blood culture in our study were comparable to various other studies. In present study blood culture was positive more in outborn babies (p = 0.047), neonates with early onset sepsis (p = 0.041) and in neonates with positive sepsis screen (p < 0.001). 17,18 Among culture positive cases gram-positive bacteria were most common isolates. In this group Staphylococcus was most common organism followed by Streptococcus and Enterococcus. Similar results were seen in the study done by Kartikeyanet al. 19 An ESBL phenotype was recognized in 42.42% isolates in our study which was similar to the study done by Tofteland et al. 20 We found that among Gram negative isolates Klebsiella and E. coli were isolated most just like in other studies. 5  isolates followed by TEM and SHV gene. CTXm gene was associated with resistance to most of the first line cephalosporins and amikacin and fluroquinolones and sensitive to imipenem, meropenem and pipercillin -tazobactam. 10,24 This fact is also supported in our study. Several studies have reported that carbapenems along with β lactam / β lactamase inhibitors combination could be used as empirical therapy for suspicion of ESBL producing bacteria. 25,26 Although we tried to elaborate the organisms implicated in neonatal sepsis, our study is relatively small and single centric. We also could not identify gene responsible for ESBL production in three cases of ESBL producing E. coli and nine cases of ESBL producing Klebsiella because of resource constraints. Despite these limitations, we are hopeful that our research light help to shed more light on the topic of neonatal sepsis in resource limited set up like ours.

CONCLUSIONS
Among ESBL producers, E. coli and Klebsiella were the commonest organisms isolated and were more prevalent in outborns and male babies. These isolates were resistant to commonly used antibiotics like penicillin, cephalosporines, fluoroquinolons and aminoglycosides and were sensitive to carbapenems and colistin. CTXm gene was most common gene associated with ESBL producing E. coli and Klebsiella.