Neonatal sepsis in southwest of Iran; prevalence of microbial pathogens, risk factors, clinical manifestations and laboratory findings

Background: Neonatal sepsis is a serious worldwide problem causing significant rates of mortality and morbidity in newborns, especially in cases with delayed infection diagnosis and management. The present study aimed to evaluate the bacteriological profiles, antibiotic susceptibility patterns, risk factors, clinical manifestations, and laboratory findings in neonatal sepsis in southwest of Iran. Methods : In this descriptive-analytic study, 342 neonates with suspected sepsis admitted to the neonatal ward and NICU were included. Using standard protocols, blood samples were transported to the BACTEC blood culture system. Then, conventional biochemical tests were used for the identification of bacterial genera and species. The bacterial antimicrobial susceptibility patterns were determined using agar disk diffusion method according to the CLSI guidelines. Demographic data, clinical findings, risk factors, mortality rates, and laboratory parameters were collected for each patient. Results : Forty-three (12.6%) cases were culture-positive, among which CoNS, Staphylococcus aureus , Escherichia coli , Acinetobacter and Beta hemolytic streptococcus were the most prevalent. The prevalence of early-onset sepsis and late-onset sepsis were 53.5% and 46.5%, respectively. Significant differences between prematurity, very low birth weight, and invasive procedures were observed between neonates with and without sepsis. Teicoplanin and vancomycin were the most efficient antibiotics against Gram-positive bacteria, while amikacin was more efficient against Gram-negative bacteria. Conclusion : Risk factors such as prematurity, abnormal birth weight, anemia, leukopenia, prolonged hospitalization, and invasive processes and cesarean section, can increase the incidence of neonatal sepsis.


Introduction
Neonatal sepsis is a clinical syndrome referring to the systemic presence of microorganisms within the first 28 days of life. The undesirable effects of this clinical syndrome are due to the uncontrolled systemic inflammatory responses against infection, leading to organ dysfunction through several pathophysiological mechanisms. [1] The neonatal period is considered as the most vulnerable period of life owing to high susceptibility to infectious agents, immaturity of immune system, low levelor short term exposure to infectious agents, and low immunoglobulin production. [2] Neonatal sepsis is a serious complication leading to mortality and morbidity, especially among very low birth weight (VLBW, <1500 g) and preterm infants in Neonatal Intensive Care Units (NICUs). [3,4] Late complications of neonatal sepsis include impaired bone marrow function (neutropenia, thrombocytopenia, and anemia), renal failure, heart failure, neurological disorders, disseminated intravascular coagulation (DIC), and shock. [5,6] Depending on the neonatal age and timing of the presentation, sepsis has been classified into early-onset-sepsis (EOS) and late-onset-sepsis (LOS). Early-onset infections are usually acquired through mother-to-infant vertical transmission during the first week after birth and late-onset infections appear after first week of life due to interactions with the hospital environment or the community with nonspecific clinical symptoms, such as respiratory distress, temperature instability, seizure, cyanosis, tachypnea, apnea, lethargy, irritability, poor feeding, and tachycardia. [7] Maternal, neonatal, and environmental risk factors as well as the virulence of infectious organisms can lead to neonatal sepsis. [8] Various etiologic agents, including Gram-positive and Gram-negative bacteria, cause this syndrome. Organisms responsible for neonatal sepsis can vary depending on geographical region and the age of onset. [2,9] In addition, bacteriological profile of neonatal sepsis has changed over time in various areas due to lifestyle differences and antibiotic overuse, and subsequent alterations in antibiotic susceptibility patterns. [10,11] The most common organisms isolated from blood culture are Gram-positive bacteria, including coagulase-negative Staphylococcus (CoNS), Group B Streptococcus (GBS), Enterococus, Listeria monocytogenes, and Gram-negative bacteria, such as Escherichia coli, Enterobacter, Klebsiella, Citrobacter, Serratia, Acinetobacter,Pseudomonas. [11,12] Pro-inflammatorymediatorsare recruited following bacterial entry tothe bloodstream.
Bacterial virulence factors, such as lipopolysaccharide, lipoteichoic acid, peptidoglycan, enzymes, super-antigens, and toxins not only over-stimulate the immune system, but also lead to bacterial tolerance to the destructive and deadly effects of the immune system.
Non-specific symptoms of neonatal sepsis can challenge the diagnosis and treatment of this syndrome, especially in preterm very low birth weight neonates. [2] Prolonged and inappropriate antibiotic administrations can adversely affect the intestinal microbiota, especially in early days of life. Therefore, broad-spectrum antibiotics may easily replace a pathogen with a more dangerous one. [13][14][15] Given the alterations in microbial profile and antibiotic susceptibility patterns, identification of the most common bacteria isolated from neonatal sepsis as well as the appropriate antibiotic administration are highly important.
Little information is present regarding the epidemiology of bacterial profile and risk factors associated with neonatal sepsis in Iran. Hence, the present study aimed to evaluate the bacteriological profile, antibiotic susceptibility patterns, risk factors, clinical manifestations and laboratory findings in neonatal sepsis in southwest of Iran.

Sample collection
This descriptive-analytic study was conducted from June 2017 to April 2018 in neonatal ward and NICU of Imam Sajjad hospital in Yasuj, southwest of Iran. The study was conducted in accordance with the Declaration of Helsinki. This study has been approved by Yasuj University of Medical Sciences, Research Ethics Committee (IR.YUMS. REC.1395.190). Prior to sample collection, written informed consents were obtained from parents / guardians of each individual.
Under aseptic conditions, one ml blood was taken from each neonate before antibiotic administration and transferred to BACTEC pediatric bottles (BACTEC 9050; Becton Dickinson, MD, United States of America).
Demographic information, clinical manifestations, risk factors, and laboratory findings of each neonate were extracted from their medical records.
For bacterial identification, after alerting by BD system, bottle contents were sub-cultured on blood agar, chocolate agar, and McConkey agar media and incubated in both CO2 and non-CO2 conditions at 37ºC for 24 -48h. After Gram staining, biochemical tests, such as catalase, coagulase, Novobiocin sensitivity, bile solubility, optochin sensitivity, hippurate hydrolysis, CAMP, and PYR tests were performed for the identification of Gram-positive cocci.

Statistical analysis
Data were analyzed by Chi-square and odds ratio (OR) tests using SPSS software version 18.
A P value of < 0.05 was considered statistically significant.

The demographic characteristics
A total of 342 neonates suspected with sepsis were admitted to Imam Sajjad hospital in Yasuj, Iran, of which 207 were in the NICU and 135 were in the neonatal ward. Among 342 cases suspected with sepsis,43 neonates (12.6%) had positive blood cultures, of which 32 were in the NICU and 11 in the neonatal ward. The mean and median hospitalization duration were 13.4 ± 0.8 and 9 days, respectively. Among neonates with culture-positive samples, 28 (65.2%) were preterm and 24 (55.8%) had abnormal birth weight. Twenty-eight (65.2%) of these neonates were female and15 (34.8%) were male among which25 (58.2%)were born by cesarean section. Demographic characteristics and risk factors based on the wards and sepsis type are illustrated in Table 1.
The prevalence of EOS was 53.3 % (23:43) of which 60.8% were preterm and 43.5% had abnormal birth weight ( Table 1). The mean and median age of neonates with EOS were2.1 ± 0.3 and one day. In this group, 11 and 12neonates were born by cesarean section and vaginal delivery, respectively. The mean and median hospitalization duration in neonates with EOS was 14.5±3and 10.5 days, respectively. LOS accounted for 46.5 % (20:43)of sepsis episodes and was observed in 70% of preterm neonates and 70% of neonates with abnormal birth weight. The mean and median age of neonates with LOS were 27.5± 4.5and 20 days, respectively. In the LOS group, 14 neonates were born by cesarean section and sixneonates were born by vaginal delivery. The mean and median hospitalization duration in neonates with LOS were 26.7± 4.6and 17 days, respectively.

Mortality
Out of 342 cases suspected with sepsis, 33 (9.6%) died, of which seven cases (21.2%)had positive bacterial culture. The most common bacteria isolated from fatal sepsis was CoNS.

Risk Factors and Clinical Symptoms
The common risk factors for sepsis included prematurity (65.2% ;P = 0.02), cesarean section   (Table 7).

Laboratory parameters
The laboratory parameters studied in neonates with sepsis included CRP, white blood cells,  (Table 8).

DISCUSSION
Today, infectious diseases, especially sepsis, are one of the main causes of death in neonates.
Sepsis is a clinical syndrome due to an inflammatory systemic inflammatory response to infection which can lead to organ dysfunction through several pathophysiologic mechanisms. [19] The greatest challenge in combating high mortality due to sepsis is the lack of rapid, accurate, and effective detection as well as inappropriate treatment and improper discontinuation of treatment. [5,20] The present study was carried out for the first time in Yasuj, southwest of Iran. The prevalence of neonatal sepsis in the present study was 12.6% which is higher than similar previous studies by Mohammadi  and Atefi et al. (2016)showed a higher prevalence of sepsis in females compared to the males. [9,[25][26][27][28] In the present study, high prevalence of immature and very low birth weight neonates, which is one of the most important risk factors for sepsis, was attributed to the female sex, so they were more have chance for sepsis than males.  [8,25,27,[31][32][33] In the cesarean section, the neonate is exposed to environmental microbes, while in vaginal delivery, the neonate is exposed to normal vaginal flora. Moreover, neonates born by cesarean section have a higher risk of immune response disorders. [34] Similar to the studies by Pokhrel et al. neonates, neonates with abnormal birth weight and neonates who underwent invasive procedures were more likely to contract sepsis. [8,27,[45][46][47] In the studies by Tsai et al. (2015) and Hematyar et al. (2014),similar to the present study, an increased incidence of sepsis was observed in anemia neonates. [37,48] In the present study, neonates with anemia, polycythemia, and leukopenia were more likely to develop sepsis in comparison with those who had abnormal hemoglobin. Increased recruitment of leukocytes and neutrophils caused by bacterial antigens in the first 96 hours of the life in neonates is associated with hypoxia-ischemia, leading to abnormal neurodevelopment, as well as neutropenia, leukopenia, and anemia. [49] In this regard, oxygenation to the neonatal organs is impaired, leading to a suitable condition for bacterial growth and pathogenicity. [50] In this study, platelet and white blood cell counts were not associated with sepsis and the incidence of sepsis in neonates with leukopenia increased similarly to the study by Shah (2014), positive CRP was associated with bacterial presence in blood. [32,35,37,41,48] CRP depends on the type of delivery, age, type of microorganism causing sepsis, leukopenia, and type of sepsis. [34,51] In the present study, CRP in neonates with LOS was twice morethanneonates with EOS, indicatingthat the higher the age of the neonate, the more advanced and specific the immune system works. In the present study, there was a correlation between positive CRP and bacterial species, and this marker is greater in Gram-negative bacteria than in Gram-positive bacteria and fungi. Lipid A is a partial endotoxin of the cell wall in Gram-negative bacteria.
Its presence in blood leads to the release of inflammatory mediators such as CRP and TNF-α.
On the other hand, CoNS can reduce interaction with the host immune system by biofilm formation, and plays a significant role in reducing the host inflammatory responses. [52][53][54] Sepsis is the third most common cause of death among neonatesworldwide. 23  Gram-negative bacteria, with immunogenic cell wall components, can cause severe sepsis and a subsequent septic shock. In our study, all neonates who died following sepsis had respiratory distress due to the production of pro-inflammatory cytokines, excessive activation of the immune system, and severe damage to body organs caused by endotoxins and cell wall elements, especially in Gram-negative bacteria. Systemic inflammation and leukocyte recruitment due to LPS is associated with cerebral and myocardial infarction. [49]

Conclusion
More than half of the studied neonates were infected with skin normal microflora and hospital-residing bacteria. Most of these neonates were subjected to invasive procedures and were born via cesarean section. Risk factors such as prematurity, abnormal birth weight, anemia, leukopenia, prolonged hospitalization, and invasive procedures increase the chance of sepsis in newborns. Clinical symptoms are nonspecific and therefore are not considered as appropriate indicators of sepsis. In this study, all neonates with sepsis developed a different pattern of clinical symptoms. Increased antibiotic administration and increased reservoir of antibiotic resistance genes in bacteria can alleviate the immunity against microbial pathogens in newborns. In the present study, the most effective antibiotics for the experimental treatment of Gram-positive bacteria were teicoplanin, aminoglycosides, and fluoroquinolones. The most common and effective antibiotics for the treatment of Gramnegative organisms were beta-lactam antibiotics, such as piperacillin, beta-lactamase inhibitor, aminoglycosides, and ciprofloxacin.