Key Words

neonatal septicemia, Acinetobacter , Carbapenems , Vancomycin

Introduction

Neonatal sepsis or sepsis neonatorum refers to systemic infection of the newborn. It is characterized by a constellation of a nonspecific symptomatology in association with bacteremia. Prompt recognition, appropriate antimicrobial therapy and judicious supportive care are the key determinants of positive outcome in this serious pediatric emergency. In developing countries, sepsis including meningitis, respiratory infections, diarrhea, and neonatal tetanus is the commonest cause of mortality responsible for 30-50 per cent of 5 million total neonatal deaths each year. It is estimated that almost 20 per cent of all neonates develop infection and approximately 1 per cent die of the serious systemic infections. Not surprisingly, sepsis is the commonest admitting diagnosis among neonates at referral facilities.

The detection of microorganisms in a patient’s blood has a great diagnostic and prognostic significance. Blood cultures provide essential information for the evaluation of a variety of diseases like endocarditis, pneumonia, and pyrexia of unknown origin and particularly, in patients with suspected sepsis. Many infections in the neonatal and paediatric age group can only be established on the basis of etiological agent recovered from blood. A positive blood culture does not necessarily confirm infection, since contamination of blood can occur. The recovery of organisms traditionally considered as pathogens pose no problems in interpretation. However, recovery of organisms such as coagulase negative staphylococci (CONS), Corynebacterium or Candida spp. is often difficult to interpret. Additional information like the density of bacteremia, number of positive cultures, duration of incubation of the broth to obtain a positive culture, presence of risk factors or an underlying disease, is required in order to determine whether infection is truly present 2

In 16 major hospitals in our country, the incidence of neonatal sepsis among intramural babies was 38 per 1000 live births as per the report of National Neonatal Perinatal Database (NNPD) 3 With this background the current study was undertaken to determine the bacteriological profile of neonatal septicemia in rural Nagpur.

Materials and Methods

Lata Mangeshkar Hospital is a 500 bedded multispeciality and tertiary care rural hospital. The present cross sectional study was conducted in the Department of Microbiology from October 2005 to July 2007.

We have a 08 bedded Neonatal Intensive Care Unit (NICU). After obtaining the necessary permission from Institutional Ethics Committee (IEC), we started this study. Neonatal septicemia was divided into two types depending on whether the onset was during the first 72 hours of life or later: 3

  • Early Onset Septicemia (EOS) occurring within 72 hours of birth
  • Late Onset Septicemia (LOS) occurring after 72 hours of birth.

All neonates of either sex clinically suspected of septicemia admitted in NICU were included. Blood

Cultures were carried out as per standard protocol 4,5,6. Under proper aseptic conditions, 1ml of venous blood was inoculated in 10 ml of sterile 1% Glucose broth. This was further incubated for 24 hours at 35-37 degree Celsius. Blind subcultures were done on 5% Sheep Blood Agar and MacConkey’s agar after 24 hours, 48 hours, 72 hours, 5th day and final on 7th day as per standard protocol. Just before subculture a Gram’s stain of the broth containing blood sample was performed and result reported telephonically to the Paediatrician for necessary action. A provisional report was issued after every subculture and if after 7th day, no growth was obtained, the sample was reported as negative. The various organisms were identified on the basis of colony morphology and standard biochemical tests. Antimicrobial susceptibility testing was performed by KirbyBauer disc diffusion susceptibility method on Mueller Hinton Agar7 , performed in accordance to CLSI (formerly NCCLS) guidelines for Gram positive and Gram negative isolates. Gram negative isolates were subjected to testing for Extended Spectrum Beta Lactamases (ESBL) production 8 .

Results

TABLE 1: Distribution of isolates in Early Onset Septicemia (EOS).

TABLE 2: Distribution of isolates in Late Onset Septicemia (LOS).

In EOS, K.pneumoniae and S.aureus were predominantly isolated (TABLE 1). In LOS, S.epidermidis, Acinetobacter spp. and P.aeruginosa were most commonly seen (TABLE 2). Over all, Gram negative bacteria formed the major part of isolates at 61.3%. Enterobacteriaceae formed the most prevalent group at 38.5%.

For the Enterobactericeae group, Imipenem showed 100% efficacy followed by Ciprofloxacin and Levofloxacin at 66% sensitivity for each.

Table 3:Enterobacteriaceae (n=27) isolated from 70 Blood Culture positive samples were tested for the following antimicrobials and their Sensitivity pattern is given in

For Acinetobacter spp. And P.aeruginosa, again Imipenem (100% sensitivity) followed by Cefepime (50% sensitivity). Antimicrobials least effective were Aztreonam (6.2%), Ceftazidime (12.5%) and Gentamicin (12.5%) For Staphylococcus spp., Linezolid, Vancomycin and Pristinomycin and Oxacillin were 100% effective followed by Clindamycin (82.6% sensitivity) and Penicillin with 78.2% sensitivity (TABLE 4). No MRSA was isolated in our study.

TABLE 4: Sensitivity pattern of Staphylococcus spp. (n=23)

SBL production was tested in K.pneumoniae and E.coli. Out of 12 K.pneumoniae isolated, 7 showed production of ESBL. Out of 6 E.coli isolated, 2 showed production of ESBL.

Discussion

In the present study, blood culture positivity in neonatal septicemia cases was 26.9%, whereas in 73.1% of cases there was no growth. There has been a wide variation in the growth positivity obtained by blood culture over the years 9,10,11,12,13 A higher isolation rate of 52.63% was reported by Murty et al probably due to a low sample size (n=22).

In our study, Early Onset Septicemia (EOS) was seen in 81.5% cases of neonatal septicemia which was also seen by Movahedian AH et al14 . We obtained K.pneumoniae and S.aureus as most common cause of EOS. S.epidermidis was isolated in maximum cases of LOS as also seen by Berger A et al and Kilani RA. Sanghvi et al15,16,17 also reported that CoNs was the most common cause of LOS like in our study.

Chugh K et al have reported 68.8% cases as EOS and 31.2% cases as LOS18. Here too number of EOS>LOS, although in their study, they considered the duration of EOS as <7 days and LOS as >7days. But in both varieties of septicemia, K.pneumoniae remained the most common isolate obtained from blood culture.

Kuruvilla KA et al have reported a lower rate of EOS (24%) than LOS (76%)19. This could be due to the fact that they defined EOS as <48 hours and LOS as >48 hours.

In our study, we obtained a maximum growth of Gram negative bacteria (61.3%). K.pneumoniae (17.1%) was the most common organism grown in this study and others 20, 21

We isolated 14.3% isolates of S.aureus in the present study. Our results are also matched by reports of Narang A et al.

We also obtained 14.3% isolates of Acinetobacter spp. in our study. Similar result is also reported by Mondal et al, Narang A et al, Agnihotri N et al, Arora U and Jaitwani J22 .

We obtained 18.6% Coagulase negative Staphylococci (CoNS) in the present study which are similar to results reported by Jain A et al (16.6%) and Movahedian et al who obtained 20.7% CoNS

Hammerberg O et al stated that if the venepuncture site had been carefully cleansed, the growth of CoNS in blood culture of specimens of premature neonates indicated bacteremia rather than skin contamination in vast majority of cases23 . This view is also shared by Favre B et al who concluded their study reporting that CoNS bacteremia harbor a significant mortality and a single positive blood culture in the presence of signs of sepsis should be considered as clinically relevant24 .

There were 27 isolates in the Enterobacteriaceae group; the commonest isolate was K.pneumoniae 12 (17.1%), followed by Enterobacter spp. 08 (11.4%), E.coli 06 (8.6%) and Citrobacter spp. 01 (1.4%). (Figure-1)

K.pneumoniae isolates were maximally sensitive to Imipenem (100%), Ciprofloxacin and Levofloxacin (66.6% each). Most resistant drugs were Aztreonam (100%), Cefoperazone (91.6%), Piperacillin (91.6%), Cefpodoxime, Ampicillin and Gentamicin (83% each). Enterobacter spp. isolates were most sensitive to Imipenem (100%) and Cefepime (50%) while high resistance was observed for Ampicillin, gentamicin and Ceftazidime (100% each) followed by Amikacin, Amoxicillin-clavulanic acid, Netilmycin and Cefpodoxime (87.5% each)

We obtained 06 (8.6%) isolates of E.coli in our study. The most sensitive drugs were Imipenem (100%), Ciprofloxacin, Levofloxacin and Ceftazidime (100% each). The most resistant drugs were Cefotaxime (100%), Ceftriaxone (100%), Gentamicin, Amikacin and Netilmycin (83.3% each).

We isolated 06 (8.6%) P.aeruginosa in the present study. These were sensitive to Imipenem (100%) and Netilmycin (66%). Resistance was observed for Gentamicin, Ceftazidime, Aztreonam (100% each) and Cefotaxime (83.3%). (TABLE 5)

Jain Ahad reported 50% resistance for Gentamicin and Cefotaxime in their study on neonatal sepsis25 . In their study, Movahedian AH et al stated that Pseudomonas and Klebsiella spp. showed a high degree of resistance to commonly used antimicrobials (Ampicillin & Gentamicin) as well as 3GCs which are also seen in the present study.

Maximum sensitivity (100%) was seen for Vancomycin, Linezolid, Pristinomycin and oxacillin. For S.hemolyticus, 100% sensitivity was seen for Vancomycin, Linezolid, Pristinomycin and oxacillin, Gentamicin and penicillin.

In the present study, 10 (14.3%) Acinetobacter spp. were isolated. It was 100% sensitive to Imipenem; while high resistance was seen for Aztreonam and Netilmycin (90% each).

There were 23 isolates of Staphylococcus spp; with S.aureus 10 (14.3%), S.epidermidis 09 (12.9%) and S.hemolyticus 04 (5.7%) in our study.

For S.aureus, 100% sensitivity was seen for Vancomycin, Linezolid, Pristinomycin and oxacillin. Azithromycin and erythromycin showed 90% each. For S.epidermidis, maximum (88.8%) resistance was seen for Azithromycin and Erythromycin

Ceftazidime, Gentamicin, Piperacillin, Amikacin, Cefotaxime and ceftriaxone showed 80% resistance each. (TABLE 5) isolated 3 (4.3%) isolates of C.albicans in our study. However, we have not done antifungal susceptibility testing on them.

ESBL production was shown by 58.3% of K.pneumoniae isolates, followed by 50% E.coli isolates. Various authors have given different observations of ESBL production in their studies on neonatal septicemia. Kim YK et al reported ESBL production by 52.9% of K.pneumoniae and 17.9% of E.coli isolates26 . Jain A et al however have reported a high ESBL production by Klebsiella spp (86.6%), Enterobacter spp (73.4%) & E.coli strains. The high percentage of ESBL producing Klebsiella spp may be due to the selective pressure imposed by extensive use of antimicrobials. Intensive care unit, in which antibiotic use is heaviest and the potential for patient-to-patient transmission of organisms is greatest, is an important factor for ESBL production26

TABLE 5: Sensitivity Pattern of Acinetobacter & Pseudomonas spp. (n=16)

Conclusion

Gram negative bacteria showed high resistance to first line drugs like Gentamicin, Ampicillin and to drugs like Aztreonam and Ceftazidime. Most of them had good sensitivity to Ciprofloxacin and Levofloxacin while Imipenem is still the best for infections with multidrug resistant gram negative bacilli. Gram positive bacteria responded very well to Vancomycin. Reserve drugs like Linezolid and Pristinomycin have not yet developed resistance. Multidrug resistant K.pneumoniae and E.coli are major pathogens in neonatal septicemia.