Spectrum and Antimicrobial Susceptibility Pattern of Micro-Organisms Associated With Neonatal Sepsis in a Hospital in Karachi, Pakistan

Background Neonatal sepsis is one of the most common causes of neonatal mortality and morbidity, particularly in developing countries. Its causative bacteria and their respective sensitivity patterns are different in each hospital and region. The objective of this study was to determine the causative bacteria and their antibiotics sensitivity patterns at the neonatal unit. Methods This prospective study was carried out at the Neonatology Unit of Kharadar General Hospital (KGH) from January 2017 to Jun 2019. A total of 162 neonates with suspected sepsis and positive blood cultures were included in the study. Blood culture was done by standard microbiological techniques (BACTEC Method). Continuous data were presented as mean and standard deviation, while categorical data were presented in frequency and percentages. Result Out of a total of 162 neonates with blood culture positive neonatal sepsis, males were 106 (65.4%). Gram-positive and Gram-negative bacteria were found with a frequency of 83 (51.5%) and 79 (48.5%), respectively. Staphylococcus aureus and Pseudomonas were the commonest isolates in 50.5% and 25.7% of cases, respectively. The Gram-positive organism was mostly sensitive to amikacin and vancomycin whereas the Gram-negative was mostly sensitive to amikacin, imipenem, meropenem, and ciprofloxacin. Conclusion Staphylococcus aureus was the most common bacteria isolated. For the sepsis, the causative bacteria and antibiotics sensitivity pattern changes over a period of time. Continued surveillance is required to help reduce morbidity and mortality through developing institution-based guidelines.


Introduction
Neonatal sepsis is defined as bacteremia and clinical symptoms caused by micro-organisms and their toxic products [1]. It can be either early-onset sepsis which usually presents within the first 72 hours of life or late-onset sepsis, which presents at 72 hours after birth up to 28 days of life [2].
The incidence of neonatal sepsis varies from 7.1 to 38 per 1000 live births in Asia, 6.5 to 23 per 1000 live births in Africa, 3.5 to 8.9 per 1000 live births in South America, and six to nine per 1000 live births in the United States [3]. Sepsis is a significant cause of morbidity and mortality in newborns where it contributes up to 13%-15% of deaths in developed countries, 30%-50% deaths in developing countries, and 28% deaths in Pakistan [4][5][6]. Clinical manifestation of neonatal sepsis is diverse which includes the maternal feeling of unwell baby, lethargy, vomiting, variability in temperature, respiratory rate and heart rate, jaundice, hepatomegaly, cyanosis, apnea, and abdominal distention [7]. In developing countries, Gram-negative organisms like Escherichia coli and Klebsiella have been found to be the leading cause of neonatal infections followed by Gram-positive organisms including Staphylococcus aureus, Group B Streptococcus (GBS), and Staphylococcus epidermidis [8][9][10]. In contrary to this, in developed countries, Group B Streptococcus, followed by Escherichia coli, Staphylococcus aureus, Coagulase-negative staphylococcus (CoNS), Listeria monocytogenes, Klebsiella spp., Enterococcus spp., and Pseudomonas aeruginosa are causative organisms of neonatal sepsis [11][12][13].
Neonatal sepsis is one of the commonest reasons for admission in the neonatology unit. As culture and sensitivity pattern varies at different places and duration, the WHO recommends determination of local culture and sensitivity pattern for starting antibiotics. In our setup, we lack the susceptibility spectrum of microorganisms responsible for neonatal sepsis, which is essential to make a choice of appropriate antibiotics in affected neonates. The results of this study will not only help to determine the causative organisms of sepsis in our population but also promote the judicious use of empirical antibiotics. This will result in shorter hospital stay of neonates, reduction in adverse effects of drugs, and cost-effective management of sepsis.

Materials And Methods
This prospective cross-sectional study was conducted from 1st January 2017 to 30th June 2019 in the neonatology unit, Kharadar General Hospital (KGH), Karachi. The study was initiated after obtaining permission from the IRB. Informed consent was obtained from the parents or guardians. A total of 162 neonates of either gender admitted in the neonatal unit through paediatric OPD/emergency room who presented with sepsis and their blood cultures were found to be positive were included in the study. Detailed history and examination were carried out in all patients presenting with sepsis. The neonates with incomplete data on proforma were excluded from the study. About 5 cc (minimum 1 cc) of venous blood samples were withdrawn and, directly injected into BACTEC® PEDS Plus (Becton Dickinson, Towson, MD) culture media bottle after using aseptic technique. The sample was transported to the laboratory for processing blood cultures. In the microbiology section of KGH lab, the BACTEC bottle was loaded in BACTEC 9050 automated blood culture instrument, based on fluorescent technology with continuous, unattended testing of cultures. When positive vials were identified, the lab technologist took out a specific bottle from the BACTEC instrument for gram staining as well as subculturing on specific media by inoculating the culture broth on blood, chocolate and MacConkey agar plates. These plates were then incubated in an incubator at 37°C in chocolate plate also under a candle jar for 24 hours. On the next day, the culture plate was read for their colony characteristic and the further biochemical test was run for the identification of specific microorganisms, as well as antibiotic susceptibility test was performed by disk diffusion technique. As per protocol, the negative sample remained the instrument for five days and then the report declared as negative. Results were analyzed using SPSS version 20 (IBM Corp., Armonk, NY). The qualitative variable was presented as mean and standard deviation, while quantitative variables presented in frequency and percentages. A Chi-square test was used to compare the variables. P-value ≤0.05 was considered statistically significant.

Results
Between January 2015 and June 2017, 1960 neonates were admitted to the neonatology unit with a diagnosis of sepsis or developed sepsis during the hospital stay. A total of 162 out of 1960 neonates had a positive blood culture. A total of 106 (65.4%) were male and 56 (34.5%) were female babies making a male: female ratio 1.89:1. The mean age of admission was 140 ± 131 hours. Mean birth weight was 2.44 + 0.71 kg (0.8-4.5 kg). Gram-positive bacteria were more frequent than Gram-negative bacteria with a frequency of 83 (51.5%) and 79 (48.5%), respectively. Among Gram-positive isolates, Staphylococcus aureus (50.1%) was the commonest organism isolated followed by Enterococci spp. (1%), while in Gram-negative isolates, Pseudomonas spp. was most common (25.7%) followed by Klebsiella (10.5%) (Figure 1).

FIGURE 1: Distribution of micro-organisms involved in neonatal sepsis.
The bacteria responsible for the sepsis showed a variable pattern of antibiotics sensitivity. The results of antibiotic sensitivity, the pattern of Gram-positive bacteria were as shown in Table 1  The results of antibiotic sensitivity, the pattern of Gram-negative bacteria were as shown in Table 2.

Discussion
Neonatal sepsis is considered to be an important cause of neonatal morbidity and mortality [14]. Developing countries share 99% of the estimated 4 million neonatal deaths [15]. Out of 4 million one-fourth die due to neonatal sepsis [16][17]. The timely diagnosis of neonatal sepsis plays an important role in determining the outcome of the babies. Blood culture is a gold standard test for sepsis [18].
Hubacek et al. have shown that the polymorphism in genes for lysosomal binding protein is responsible for the immunological difference between males and females [19]. Marriott et al. observed that cell surface receptors like TLR 4 trigger a different response in males than females in terms of inflammatory cytokines and acute-phase reactants thus making males more prone to sepsis [20]. Our study was conducted on 162 neonates with confirmed sepsis on blood culture. Male babies were 106 (65.4%) and female babies were 56 (34.5%) with a ratio of 1.89 to 1, which is consistent with several studies [14,[21][22].
In the present study, however, 54.6% of isolates were  [17,21,29,30]. The other Gram-positive organisms to be isolated were Enterococci. A similar pattern of the predominance of Gram-positive organisms has been shown in studies conducted by Shrestha et al. [27]. In these studies, hospital-acquired sepsis was included.
Pseudomonas was the most common Gram-negative organism. These results are consistent with Awoniyi et al. [25]. In contrary to this, Shrestha et al. found Klebsiella pneumonia the most common Gram-negative organism [27]. Muhammad [21,26].
Staphylococcus aureus was most sensitive to amikacin and vancomycin as 85% and 88.67%, respectively. Similar results were also observed in the studies done by Shaw et al. and Najeeb et al. [21,30].
Regarding the sensitivity pattern of Pseudomonas spp., following drugs showed sensitivity in decreasing pattern as amikacin, imipenem, ciprofloxacin, meropenem, and piperacillin. A study conducted by Awoniyi et al. found Pseudomonas spp. was the most sensitive to amikacin [25]. These results coincide with our study. Pseudomonas spp. sepsis was sensitive to imipenem in 77.7% in our study which is consistent with a study done by Shrestha et al. [27]. The study performed by Shrestha et al. found that Pseudomonas showed sensitivity to ciprofloxacin (77%) which is consistent with our result [27].
Kayange et al. found Klebsiella was highly sensitive to meropenem which coincides with the results of our study [28]. In a study conducted by Najeeb et al., 93% of Klebsiella was sensitive to imipenem, 73% is sensitive to amikacin and 74% to ciprofloxin [30]. These results are in conformity with our study.
Amikacin, imipenem, meropenem, and ciprofloxacin showed a high sensitivity of 100%, 100%, 100%, and 57% in the case of E. coli. The study by Muhammad et al. found almost similar sensitivity pattern [14].

Conclusions
Staphylococcus aureus and Pseudomonas are the most common organisms causing neonatal sepsis. Grampositive bacteria have sensitivity to vancomycin and amikacin, while Gram-negative bacteria have sensitivity to amikacin, imipenem, meropenem, and ciprofloxacin. In our country, common pathogens responsible for neonatal sepsis change over the period of time and from region to region. Improved and revised local data on the pattern of causative bacteria and antibiotics sensitivity patterns are required to help reduce morbidity and mortality through the development of institution-based guidelines.

Additional Information Disclosures
Human subjects: All authors have confirmed that this study did not involve human participants or tissue. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.