Research Article | Open Access
Saurabh Chhotalal Gamit , Rachana Dhirubhai Patel, Himani Bhardwaj Pandya and Sucheta Jitendra Lakhani
Department of Microbiology, Smt. BK Shah Medical Institute & Research Centre, Sumandeep Vidyapeeth Deemed to be University, Piparia – 391 760, Vadodara, Gujarat, India.
J Pure Appl Microbiol. 2022;16(1):638-642 | Article Number: 6930
https://doi.org/10.22207/JPAM.16.1.65 | © The Author(s). 2022
Received: 11/03/2021 | Accepted: 31/01/2022 | Published online: 25/02/2022
Issue online: March 2022
Abstract

Neonatal septicemia is a major cause of infection and mortality in neonates. Neonatal sepsis is a type of neonatal infection, particularly bacteremia, diagnosed by the presence of microorganisms in blood via microbiological culture tests during the first 4 weeks of birth. Neonatal sepsis is classified into early bacterial neonatal septicemia (less than 72 h) and late bacterial neonatal septicemia (>72 h–28 d) according to age. Isolation of septicemia-causing bacteria from the blood of neonates and investigation of their antibiotic susceptibility pattern for disease management. This study was a retrospective analysis of blood cultures from neonates admitted to the NICU with symptoms of septicemia from June to December, 2019. All samples were processed according to standard microbiological procedures using the BACTEC 9050 automated culture system. Results were reported on the basis of microbial appearance on the culture media and microscopic examination using the Gram staining method, and authentication was performed using standard biochemical reactions. Of the 228 patients, 32.89% were positive for blood culture. Early bacteremia was detected in 64% of the patients, and late bacteremia was detected in 36% of the patients. The most effective drugs against the gram-positive bacteria were linezolid, vancomycin, and ciprofloxacin and against the gram-negative bacteria were tigecycline, imipenem, and amikacin. Coagulase-negative staphylococci and gram-negative organisms such as Klebsiella, Acinetobacter, and Staphylococcus aureus were the major cause of neonatal septicemia in this study, and almost all the isolates were resistant to different antibiotics. Overall, antimicrobial drug resistance was observed in all the positive blood culture samples.

Keywords

Neonatal septicemia, blood culture, antibiotics, antibiotic susceptibility

Introduction

Neonatal septicemia is one of the most common causes of infection and mortality in neonates, due to which 30–50% of neonates die each year in developing nations. Various studies have suggested that bacteremia occurs in 20% of neonates, and approximately 1% die due to neonatal sepsis.1 According to the National Perinatal Database (NNPD) study, neonatal septicemia occurred in 30 live births out of 1000 in India during 2002–2003.2 The classification of neonatal sepsis is based on the age at infection initiation; according to this classification, it is categorized into early bacterial neonatal sepsis (less than 72 h) and late bacterial neonatal sepsis (>72 h–28 d). Early bacterial neonatal septicemia is diagnosed mostly at the time of fetal delivery and during the nursery period.4 There are various gram-positive and gram-negative bacteria that cause bacterial neonatal septicemia.5

Currently, the appearance of multiple antibiotic-resistant microorganisms poses challenges in the management of neonatal sepsis.6,7 In order to utilize the antimicrobial therapy effectively in septicemia patients to detect the prevalence of bacteria and the related antibiotic drugs, it is important to identify their sensitivity patterns. Early detection and antimicrobial treatment in sepsis are useful for reducing morbidity and mortality.8 This study was performed in an institute to determine the incidence rate and microbiological profile of neonatal sepsis along with the antibiotic susceptibility testing (AST) pattern of the isolates from neonates who were admitted to the NICU of Dhiraj Hospital, Pipariya, Vadodara.

Materials and Methods

This study was conducted in the central laboratory of the Department of Microbiology, Dhiraj Hospital, Vadodara (Gujarat) from June to December, 2019. The study evaluated the bacteriological profile and antibiotic susceptibility patterns of 228 neonates clinically suspected of septicemia, admitted to the NICU of Dhiraj Hospital, Piparia, Vadodara (Gujarat) from June to December, 2019.

Blood samples were collected from suspected neonates with septicemia under aseptic conditions after obtaining consent from their relatives. Then, 1–2 ml of the collected venous blood was inoculated in a blood culture bottle containing 40 ml sterile BD BACTEC™ Peds Plus™/F culture vials. All samples were processed according to standard microbiological procedures using the BACTEC 9050 automated culture system. If microbes are present in the collected blood specimen injected into the sterile bd bactec peds plus/f culture vials, CO2 is generated when the microbes break down the substance present in the vial. The rise in the fluorescence of the vial due to the higher amount of CO2 released by microbes was monitored using the BD BACTEC 9050 instrument. The positive blood cultures were subcultured on sheep blood agar and MacConkey agar and incubated at 37°C for 24 h.

Statistical analysis
Results were reported on the basis of the appearance of microbial growth on culture media and microscopic examination using the Gram staining method, and interpretation was performed using standard biochemical reaction tests. Several bacteria from the Enterobacteriaceae family have been identified using various biochemical reactions such as indole test, H2S production-based catalase test, citrate utilization test, hanging drop test, urease test, oxidase test, sugar fermentation test, and other tests. For gram-positive bacteria, coagulase, catalase, bacitracin, and optochin disk tests and other tests were performed. The correlation of the results was also performed using BioMerieux’s VITEK® 2 COMPACT (automated microbial identification system).

The antimicrobial susceptibility test was performed on Muller Hilton agar (MHA) using the Kirby-Bauer disc-diffusion antibiotic sensitivity test method in accordance with the Clinical Laboratory Standards Institutes (CLSI) guidelines.9 Following drugs were used for the disc-diffusion antibiotic sensitivity test: For gram-positive bacteria: penicillin (10 units), levofloxacin (5 μg), ciprofloxacin (5 μg), gentamicin (10 μg), vancomycin (5 μg), and linezolid (30 μg). For gram-negative organisms, ampicillin (10 μg), ciprofloxacin (5 μg), gentamicin (10 and 30 μg), co-trimoxazole (1.25 μg trimethoprim/23.75 μg sulfamethoxazole), amikacin (30 μg), cefepime (5 μg), ceftriaxone (30 μg), imipenem (10 μg), piperacillin/tazobactam (100/10 μg), and tigecycline (15 μg). The discs were obtained from HiMedia Laboratories (India).9

RESULTS

A total of 228 suspected neonatal blood samples were collected for blood culture during the study; among these, bacterial sepsis was confirmed in 32.89% (75 out of 228) patients. Of the 75 patients, 48 (64%) had early septicemia (EOS; <72 h of age) and 27 (36%) had late septicemia (LOS; >72 h of age).

In this study, 46 gram-positive and 29 gram-negative bacteria were isolated. Among these, coagulase-negative staphylococci (CONS) were detected in 46.67% (35 out of 75), gram-negative Klebsiella spp. in 17.33% (13 out of 75), Acinetobacter in 16% (12 out of 75), and Staphylococcus aureus in 14.67% of the patients (11 out of 75). The main causal bacteria for septicemia are listed in Table 1.

Table (1):
Main Causal bacteria for Neonatal septicemia.

Isolated bacteria Total (n = 75) EOS (n = 48) LOS (n = 27)
Gram-positive bacteria (n = 46)
1. CONS 35 (46.67%) 20 15
2. Staphylococcus aureus 11 (14.67%) 09 02
Gram-negative bacteria (n = 29)
1. Klebsiella spp. 13 (17.33%) 10 03
2. Acinetobacter spp. 12 (16%) 07 05
3. E. coli 03 (4%) 01 02
4. Pesudomonas 01(1.33%) 01

Table 2 suggests that the most sensitive antibiotics to gram-positive bacteria were vancomycin and linezolid, followed by ciprofloxacin and gentamicin. Almost all the gram-positive bacteria were resistant to penicillin.

Table (2):
Antibiotic susceptibility of gram-positive bacteria (n = 46).

Antibiotic
Resistant (%)
Sensitive (%)
Penicillin (P)
40 (87)
06 (13)
Levofloxacin (LE)
31 (67)
15 (33)
Gentamicin (GEN)
23 (50)
23 (50)
Ciprofloxacin (CIP)
22 (47)
  24 (52.1)
Vancomycin (VA)
46 (100)
Linezolid (LZ)
46 (100)

Table 3 suggests that the most sensitive antibiotics to gram-negative bacteria were tigecycline, imipenem, and piperacillin/tazobactam, followed by ciprofloxacin and amikacin. Almost all the gram-negative bacteria were resistant to ampicillin and co-trimoxazole.

Table (3):
Antibiotic susceptibility of gram-negative bacteria (n = 29).

Antibiotics
Resistant (%)
Sensitive (%)
Ampicillin (AMP)
28 (97)
01 (03)
Co-trimoxazole (COT)
20 (69)
09 (31)
Cefepime (CPM)
19 (65)
10 (35)
Ceftriaxone (CTR)
18 (62)
11 (38)
Gentamicin (GEN)
16 (55)
13 (45)
Amikacin (AK)
13 (45)
16 (55)
Ciprofloxacin (CIP)
07 (24)
22 (76)
Piperacillin/ tazobactam (PT)
06 (21)
23 (79)
Imipenem (IPM)
04 (14)
25 (86)
Tigecycline (TGC)
01 (03)
28 (97)
DISCUSSION

This study shows that gram-positive bacteria such as Klebsiella, Acinetobacter, CONS, and S. aureus are the major cause of neonatal septicemia, and most of them are resistant to multiple antibiotics that are routinely used.

Table (4):
Comparison of the prevalence of neonatal septicemia reported in different studies.

Parameter
This study
Muley et al.10
Kumar et al.11
Thakur et al.12
Prevalence
32.89%
26.6%
26.2%
42%

Table 4 shows that in the four studies, the prevalence of neonatal septicemia was did not differ considerably, which indicates that the prevalence of neonatal septicemia is high in developing countries such as India.

Table (5):
Comparison of the organisms isolated from blood culture of neonatal septicemia samples.

Organism
This study
Jyothi et al.13
Yadav et al.14
Roy et al.15
CONS
46.67%
27.5%
10.2%
16.6%
Klebsiella
17.33%
30.5%
15.3%
24.6%
Acinetobacter
16%
12.2%
11.9%
Staphylococcus
11%
10.6%
35.6%
14%

Table 5 shows that in most of the studies compared, neonatal septicemia was caused by Klebsiella, Acinetobacter, Coagulase-negative Staphylococcus (CONS), and Staphylococcus aureus. The most typical organism was Klebsiella.

Limitation
Although we aimed to include all patients during the indicated period, data were limited due to the prevalence of this disease. Furthermore, we found that the automated instruments failed to isolate a few organisms. In such patients, confirmation and identification via traditional methods are useful; however, we used only preliminary tests for such patients. More sensitive automation may be required in such patients.

Table (6):
Comparison of the antibiotic susceptibility pattern of blood culture isolates from neonatal septicemia samples.

Antibiotics      This study              Jyothi et al.13                 Muley et al.10
Gram-negative bacteria Resistant Sensitive Resistant Sensitive Resistant Sensitive
(%) (%) (%) (%) (%) (%)
Imipenem 03 97 07 93 00 100
Amikacin 45 55 48 52 35.3 64.7
Gentamicin 55 45 49 51 41.2 58.8
Ampicillin 97 03 97 03 70.5 29.5
Gram-positive bacteria Resistant (%) Sensitive (%) Resistant (%) Sensitive (%) Resistant (%) Sensitive (%)
Linezolid/Vancomycin 00 100 09 91 0 100
Ciprofloxacin 47 53 48 52 27.3 72.7
Gentamicin 55 45 60 40 44.5 55.5
Penicillin 87 13 90 10 72.7 27.3
CONCLUSION

Neonatal mortality and morbidity associated with bacterial neonatal septicemia can be attributed to a variety of gram-positive and gram-negative bacteria with different sensitivity patterns in different geographic areas and populations. We suggest that any suspected case of bacterial septicemia, particularly in NICU settings, should undergo bacterial culture and sensitivity profiling, and disease management should be performed according to the isolated and detected organism and its sensitivity to antibiotics.

Declarations

ACKNOWLEDGMENTS
The authors are thankful to the institution for granting permission to carry out the study and to the teaching staff of the Microbiology Department of SBKS MI & RC and Central Laboratory Dhiraj Hospital, Vadodara, India for their advice and support. They are grateful for the cooperation of the patients who participated in this study.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.

AUTHORS’ CONTRIBUTION
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

FUNDING
None.

ETHICS STATEMENT
 This study was approved by the Institute Ethics Committee, Sumandeep Vidhyapeeth University, Vadodara, India.

AVAILABILITY OF DATA
All datasets generated or analyzed during this study are included in the manuscript.

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