Etiology of early onset neonatal sepsis in neonatal intensive care unit

Abstract

INTRODUCTION:

This study was conducted to find out the bacterial causes of early onset neonatal sepsis and their susceptibility pattern for different classes of antibiotics in neonates admitted to neonatal intensive care unit (NICU) of Mansoura-Egypt.

METHODS:

A descriptive cross-sectional study was conducted. All admitted newborns to our study were infants at <72 hours of age with clinical features of sepsis or product of an in-house delivery at Mansoura University Hospital with risk factors for neonatal sepsis. Identification of Gram-negative isolates was confirmed by API 20E kits (bioMerieux). Antibiotic susceptibility was performed by Kirby-Bauer disc diffusion method. Bacteria resistant to ≥3 antimicrobial classes were counted as multidrug resistant.

RESULTS:

One hundred eighty eight babies were admitted to our study. Positive blood culture was reported in 34.6% of newborn infants with prevalence of 4.02/1,000 live births. The most common isolated bacteria in early onset neonatal sepsis were coagulase negative staphylococci followed by Klebsiella pneumoniae and Serratia marcescens. The Gram-positive bacteria showed high resistance to ampicillin 93.9% while all the isolates were susceptible to vancomycin. The isolated Gram-negative bacteria were highly resistant to ampicillin [96.9%], amoxicillin-clavulanic acid [90.6%], cefotaxime [84.4%] and ceftazidime [84.4%]. Best sensitivity among all isolates was observed to imipenem. Multidrug resistance was observed among 45.5% of the Gram-positive and 68.8% of Gram-negative isolates.

CONCLUSION:

Appropriate identification of the source of infection and initiation of an effective management can reduce both mortality and morbidity associated with neonatal sepsis. The challenge is to initiate immediate empirical antibiotic therapy according to a strictly implemented updated antibiotic policy based on an individualized community established antimicrobial sensitivity pattern of microorganisms causing early onset neonatal sepsis.

Keywords

EONS NICU Mansoura

1 Introduction

Sepsis is a leading cause of neonatal mortality all over the world, accounting for almost 40% of mortality rate globally. The neonatal death rate in Africa was found to be four folds or even higher than that in Europe [1 –4]. Neonatal sepsis is known to be classified into early or late onset sepsis. The early onset sepsis [EONS] is defined if manifestations appear within the first 72 hours of life while the late onset sepsis [LONS] is defined if manifestations appear from fourth days of life ahead till the end of neonatal period [5]. The causative organisms of EONS are commonly maternal in origin that could be transmitted to the newborn antenatally or during labor. However, the causative organisms of LONS may be acquired either from community or nosocomial if patients are hospitalized [6]. Recently, studies from developing countries have reported an alteration in the etiological patterns of neonatal sepsis without discrimination between both EONS and LONS [7 –9]. While in developed countries the etiological trend of EONS was kept almost unchanged. Therefore, there is an urgent need in developing countries to take urgent steps addressing the high infection rates and subsequent high mortality caused by neonatal sepsis. The increment prevalence of the multidrug-resistant organisms recently is considered as an alarming issue, making it a challenging task to initiate an immediate empirical antibiotic therapy without adding to such problem [10]. This necessitates implementation of a strict antibiotic policy based on locally updated sensitivity pattern of microorganisms causing EONS.

This study was conducted to find out the bacterial causes of EONS and their susceptibility pattern for different classes of antibiotics in neonates admitted to neonatal intensive care unit (NICU) of Mansoura-Egypt.

2 Methods

2.1 Study design

A descriptive cross-sectional study was conducted in Mansoura University children hospital-EGYPT, between November 2013 and January 2015 on newborn babies admitted to the NICU.

2.2 Participants

All admitted newborns to our study were infants at <72 hours of age with clinical features of septicemia or product of an in-house delivery at Mansoura University Hospital with risk factors for neonatal sepsis. A written consent was taken from pregnant women after full explanation of the research protocol. The institution research board of Faculty of Medicine – Mansoura University has approved the research protocol.

2.3 Data collection

All the antenatal maternal risk factors for septicemia were identified. The maternal clinical risk factors according to the unit policy to be considered as risk factors for neonatal sepsis included abnormal vaginal discharge prior to delivery, peri-partum fever [temperature ≥ 38°C recorded 72 hours before or after labor], offensive liquor, rupture of membranes for more than 18 hours, meconium-stained liquor, mothers suffering from abdominal tenderness or dysuria, antepartum hemorrhage and prolonged 2nd stage labor. The maternal laboratory risk factors included leukocytosis and positive high vaginal swab or urine cultures. Demographic and clinical data of the mothers including pregnancy, delivery, and perinatal history, were obtained from ward records of the mother if in-house delivery or from history if outside delivery. The clinical features for EONS included temperature instability such as hypothermia or fever, respiratory distress including tachypnea, feeding intolerance or lethargy.

2.4 Sampling procedure

A blood sample was collected by venous puncture from a peripheral vein from each neonate for C-reactive protein [CRP], complete blood count [CBC], and blood culture. Approximately 1 mL of baby’s blood was directly inoculated into a pediatric blood culture bottle and sent to the microbiology laboratory for subsequent processing before starting the empirical antibiotics. According to the policy, ampicillin and gentamicin were the empirical antibiotics given to all suspected cases of early neonatal sepsis.

2.5 Bacterial identification

The bottles were aerobically incubated for 7 days at 37°C. They were daily examined for signs of bacterial growth as hemolysis, turbidity or production of gas. Then they were examined after 24–48 hours of incubation for growth. Enriched and selective media such as blood, MacConkey and chocolate agar plates were used for subcultures. On daily bases for one week, subcultures were repeated before considering blood culture negative.

Identification of obtained isolates was made by regular microbiological techniques such as Gram staining, colony morphology and biochemical properties. Gram-negative isolates was confirmed by API 20E identification kits [bioMerieux] USA. Identification of staphylococcal and streptococcal species were confirmed by API^® Staph and API^® 20 Strep identification kits respectively [bioMerieux] USA.

Identification of antibiotic susceptibility for bacterial isolates was performed by Kirby-Bauer disc diffusion method using Mueller-Hinton agar [Oxoid] as recommended by CLSI, 2011 [11]. Oxacillin disc diffusion test was used to identify methicillin-resistant Staphylococcus aureus [MRSA]. Bacteria resistant to three or more antimicrobial classes were counted as multidrug resistant [MDR] [12].

2.6 Statistical analysis

Study data was initially captured into Microsoft Excel program then exported to Statistical Package for Social Sciences (SPSS, Version 15) for editing and statistical analysis. [Chicago, IL, USA].

3 Results

There were 6719 in-house deliveries during the study period. A total of 188 newborn infants were fulfilling the criteria for admission to our study, out of them 72 [38.3%] were born inside the hospital (inborn) and 116 [61.7%] were born in another hospital, home birth or other (outborn) as shown in Fig. 1. Among our participants, there were 95 males and 93 females. The criteria of the study subjects are shown in Table 1.

Fig.1

Flow diagram of participant recrutiment and blood culture results.

Table 1

Neonatal characteristics

Characteristics	Inborn n [%]	Outborn n [%]	All babies n [%]
Birth weight [kg]
<1.5	9 [12.5]	16 [13.8]	25 [13.3]
1.5 –<2.5	21[29.2]	36 [31.0]	57 [30.3]
>2.5	42[58.3]	64 [55.2]	106[56.4]
Total	72 [100.0]	116 [100.0]	188 [100.0]
Gestational age
<28 weeks	2 [2.8]	5 [4.3]	7 [3.7]
28–32 weeks	14 [19.4]	28 [24.1]	42 [22.3]
33–36 weeks	21 [29.2]	36 [31.0]	57 [30.3]
37–42 weeks	34 [47.2]	41 [35.3]	75[39.9]
>42 weeks	1 [1.4]	6 [5.2]	7 [3.7]
Total	72 [100.0]	116 [100.0]	188 [100.0]

3.1 Clinical manifestations

Overall, 162 [86.2%] of our subjects presented with one or more of clinical manifestations of sepsis, while 26 [13.8%] had risk factors only. Out of these clinical presentations the most common was respiratory distress [63.4%]; where temperature instability [28.7%] and lethargy [28.7%] were less common presented features among our participants (Table 2).

Table 2
Presenting clinical features

Clinical features Inborn [N = 72] [%] Outborn [N = 116] [%] Total [N = 188][%]

Temperature instability 19 [26.4] 39 [33.6] 58 [30.9]

Lethargy/reduced activity 16 [22.2] 42 [36.2] 58 [30.9]

Respiratory distress 48 [66.7] 80 [69.0] 128 [68.1]

Seizures 2 [2.8] 24 [20.7] 26 [13.8]

Apnea 5 [6.9] 15 [13.0] 20 [10.6]

Pallor 1 [1.4] 5 [4.3] 6 [3.2]

Jaundice 12 [16.7] 11 [9.5] 23 [12.2]

Cyanosis 6 [8.3] 12 [10.3] 18 [9.6]

Tachypnea 24 [33.3] 27 [23.3] 51 [27.1]

Bleeding diathesis 4 [5.6] 16 [13.8] 20 10.6]

Poor feeding 7 [9.7] 13 [11.2] 20 [10.6]

Abdominal distension 12 [16.7] 11 [9.5] 23 [12.2]

Regurgitation/vomiting 6 [8.3] 4 [3.4] 10 [5.3]

Periumbilical redness 2 [2.8] 2 [1.7] 4 [2.1]

Sclerema 0 [0.0] 8 [6.9] 8 [4.3]

Mottled skin 3 [4.2] 8 [6.9] 11 [5.9]

Prolonged capillary refill 0 [0.0] 1 [0.9] 1 [0.5]

Hepatomegaly 0 [0.0] 17 [14.7] 17 [9.0]

Clinical features	Inborn [N = 72] [%]	Outborn [N = 116] [%]	Total [N = 188][%]
Temperature instability	19 [26.4]	39 [33.6]	58 [30.9]
Lethargy/reduced activity	16 [22.2]	42 [36.2]	58 [30.9]
Respiratory distress	48 [66.7]	80 [69.0]	128 [68.1]
Seizures	2 [2.8]	24 [20.7]	26 [13.8]
Apnea	5 [6.9]	15 [13.0]	20 [10.6]
Pallor	1 [1.4]	5 [4.3]	6 [3.2]
Jaundice	12 [16.7]	11 [9.5]	23 [12.2]
Cyanosis	6 [8.3]	12 [10.3]	18 [9.6]
Tachypnea	24 [33.3]	27 [23.3]	51 [27.1]
Bleeding diathesis	4 [5.6]	16 [13.8]	20 10.6]
Poor feeding	7 [9.7]	13 [11.2]	20 [10.6]
Abdominal distension	12 [16.7]	11 [9.5]	23 [12.2]
Regurgitation/vomiting	6 [8.3]	4 [3.4]	10 [5.3]
Periumbilical redness	2 [2.8]	2 [1.7]	4 [2.1]
Sclerema	0 [0.0]	8 [6.9]	8 [4.3]
Mottled skin	3 [4.2]	8 [6.9]	11 [5.9]
Prolonged capillary refill	0 [0.0]	1 [0.9]	1 [0.5]
Hepatomegaly	0 [0.0]	17 [14.7]	17 [9.0]

3.2 Prevalence of culture positive EONS

Positive blood culture results were reported in 65 [34.6%] newborn infants, 27 of them were delivered inside the hospital resulting in definitive EONS prevalence of 4.02/1,000 live births. Out of the 27 neonates delivered inside the hospital with positive cultures, there were 21 neonate [77.8%] born to mothers had their routine antenatal care at health care centers [primary health care or private centers] and came only for delivery due to medical complications. Out of the 116 neonates delivered outside the hospital, 38 [32.8%] had positive blood cultures for EONS.

Among the 188 neonates admitted with suspected EONS, CRP level was positive [>6 mg/L] in 160 [85.1%] cases, while CBC abnormalities were documented in 14 [7.4%] in the form of leucopenia [WBC<5,000/mm3], in 47 [25%] as leukocytosis [WBC >20,000/mm³] and in 88 [46.8%] as thrombocytopenia [platelets<140,000/mm3].

Isolated Pathogens: Out of the 188 blood cultures, only 65 [34.6%] showed growth of different types of bacteria. The type and frequency of isolated pathogens are shown in Table 3. Gram-positive bacteria were responsible for 33 [50.8%] of culture-proven neonatal sepsis and the coagulase negative staphylococci were the most common isolated bacteria in EONS, followed by Klebsiella pneumoniae [K. pneumoniae] and Serratia marcescens [S. marcescens].

Table 3
Bacterial isolates in eons among inborn and outborn neonates

Bacterial isolates Inborn N = 27 n [%] Outborn N = 38 n [%] Total N = 65 n [%]

Gram-positive bacteria

Coagulase negative staphylococci 8 [29.6] 12 [31.6] 20 [30.8]

Enterococcus faecalis 2 [7.4] 3 [7.9] 5 [7.7]

MSSA 2 [7.4] 2 [7.9] 4 [6.2]

MRSA 1 [3.7] 1 [2.6] 2 [3.1]

Streptococcus pneumoniae 1 [3.7] 1 [2.6] 2 [3.1]

Gram-negative bacteria

Klebsiella pneumoniae 5 [18.5] 8 [21.0] 13 [20.0]

Serratia marcescens 3 [11.1] 5 [13.1] 8 [12.3]

Acinetobacter baumannii 3 [11.1] 3 [7.9] 6 [9.2]

Escherichia coli 2 [7.4] 3 [7.9] 5 [7.7]

Bacterial isolates	Inborn N = 27 n [%]	Outborn N = 38 n [%]	Total N = 65 n [%]
Gram-positive bacteria
Coagulase negative staphylococci	8 [29.6]	12 [31.6]	20 [30.8]
Enterococcus faecalis	2 [7.4]	3 [7.9]	5 [7.7]
MSSA	2 [7.4]	2 [7.9]	4 [6.2]
MRSA	1 [3.7]	1 [2.6]	2 [3.1]
Streptococcus pneumoniae	1 [3.7]	1 [2.6]	2 [3.1]
Gram-negative bacteria
Klebsiella pneumoniae	5 [18.5]	8 [21.0]	13 [20.0]
Serratia marcescens	3 [11.1]	5 [13.1]	8 [12.3]
Acinetobacter baumannii	3 [11.1]	3 [7.9]	6 [9.2]
Escherichia coli	2 [7.4]	3 [7.9]	5 [7.7]

MSSA: methicillin-sensitive Staphylococcus aureus.

Table 4

Resistance patterns of bacterial isolates

Antibiotic	Resistance of gram-positive bacteria N = 33 [%]	Resistance of gram-negative bacteria N = 32 [%]	Total resistance N = 65 [%]
Ampicillin	31 [93.9]	31 [96.9]	62 [95.4]
Amoxicillin/clavulanic acid	15 [45.5]	29 [90.6]	44 [67.7]
Cefotaxime	18 [54.5]	27 [84.4]	45 [69.2]
Ceftazidime	18 [54.5]	27 [84.4]	45 [69.2]
Imipenem	3 [9.1]	8 [25]	11 [16.9]
Vancomycin	0 [0]	NT	–
Gentamycin	16 [48.5]	15 [46.9]	31 [47.7]
Amikacin	10 [30.3]	12 [37.5]	22 [33.8]
Erythromycin	18 [54.5]	NT	–
Ciprofloxacin	10 [30.3]	11 [34.4]	21 [32.3]

NT: not tested.

The types of isolated bacteria from positive blood cultures in different gestational age groups and the place of delivery (whether inborn or outborn) were shown in Tables 5 and 6.

Table 5

Bacterial isolates in EONS among inborn neonates

Bacterial isolates	<28 weeks n = 2	28–32 weeks n = 8	33–36 weeks n = 6	37–40weeks n = 11	>40 weeks n = 0
Gram-positive bacteria
Coagulase negative staphylococci	1	3	2	2	0
Enterococcus faecalis	0	0	1	1	0
MSSA	0	0	1	1	0
MRSA	1	0	0		0
Streptococcus pneumoniae	0	0	0	1	0
Gram-negative bacteria
Klebsiella pneumoniae	0	2	1	2	0
Serratia marcescens	0	1	0	2	0
Acinetobacter baumannii	0	1	0	2	0
Escherichia coli	0	1	1	0	0

Table 6

Bacterial isolates in EONS among outborn neonates

Bacterial isolates	<28 weeks n = 3	28–32 weeks n = 9	33–36 weeks n = 14	37–40weeks n = 10	>40 weeks n = 2
Gram-positive bacteria
Coagulase negative staphylococci	1	3	5	2	1
Enterococcus faecalis	0	0	2	1	0
MSSA	0	1	1	0	0
MRSA	1	0	0	0	0
Streptococcus pneumoniae	0	0	1	0	0
Gram-negative bacteria
Klebsiella pneumoniae	1	1	3	2	1
Serratia marcescens	0	2	2	1	0
Acinetobacter baumannii	0	1	0	2	0
Escherichia coli	0	1	0	2	0

3.3 Antibiotic susceptibility pattern

The antimicrobial resistance of the isolated bacteria was illustrated in Table 4. Gram-positive bacteria showed high resistance to ampicillin [93.9%] and amikacin was found to be more effective on Gram-positive isolates than gentamicin. All the isolated Gram-positive bacteria were susceptible to vancomycin. On the other hand, the isolated Gram-negative bacteria were highly resistant to ampicillin [96.9%], amoxicillin-clavulanic acid [90.6%], cefotaxime [84.4%] and ceftazidime [84.4%]. Best sensitivity among all isolates was observed to imipenem. Quinolones [ciprofloxacin] in spite not being recommended for use in neonates and young children, but they may be used in culture-proven sepsis with bacteria resistant to other antibiotics.

Multidrug Resistance was observed in 37 isolates [56.9%] among of which 45.5% [15/33] were Gram-positive isolates while, among 68.8% [22/32] were Gram-negative isolates.

4 Discussion

The clinical signs and symptoms of neonatal sepsis are nonspecific and elusive, making its diagnosis a difficult task [13, 14]. The gold standard for definitive diagnosis of neonatal sepsis remains blood culture. The incidence of neonatal sepsis in developing countries is widely variable, ranging from 5.6/1,000 live births in Pakistan [15], 15.5/1,000 live births in subcontinent of India [16] and up to 55/1,000 live births in Nigeria [17]. This has been referred to many possible factors such as variability in socioeconomic standard, perinatal care, infection control practices and commitment to antibiotic protocols. The prevalence of EONS in developed countries is 0.9–3.3/1,000 live births that was mainly attributed to the strict implementation of infection control guidelines and avoidance of irrational use of antibiotics [18]. In our study, the prevalence of EONS among newborn infants was 4.02/1,000 live births that is considered similar to previous reports from Egypt and higher than that from developed countries.

In our study, the incidence of neonatal sepsis caused by Gram-positive cocci (predominantly CoNS) was slightly higher than that caused by Gram-negative bacteria. The extensive use of invasive devices that been used regularly for caring of immunologically immature neonates might be an attributive cause of CoNS bacteremia in NICU. This finding supports the previous reports that majority of cases of CoNS bacteremia in NICU, could be related with the use of venous catheters [19 –24]. On the contrary, some studies in developing counties have shown that Staphylococcus aureus was the predominant Gram-positive organism responsible for EONS [25] while Gram-negative bacteria was the predominant cause in other studies [26 –28].

Coagulase negative staphylococci were the most prevalent organism isolated from newborn infants either delivered inside or outside the hospital. Similarly, other reports from India showed that the commonest organism isolated among inborn infants was CoNS followed by K. pneumoniae. This observation may therefore refers to lack of adherence to infection control measures in Egypt similar to other developing countries and constitute a major risk factor for neonatal sepsis. K. pneumoniae was found to be the commonest Gram-negative pathogen responsible for EONS among newborn infants in our study. The predominance of K. pneumoniae was also reported in other studies from Egypt [26 –30] and other different countries [31, 32]. Therefore, it is substantially important to take this into consideration during constructing protocols for first-line empirical antibiotics.

Serratia marcescens was found to be the 2nd most common Gram-negative isolate in our study. This was consistent with a study from Bangladesh in which S. marcescens was the 2nd commonest Gram-negative bacteria to cause EONS [32]. However, other studies failed to detect S. marcescens among pathogens isolated from cases of neonatal sepsis [22]. In our study, we have reported that group B streptococci was not a causative pathogen of EONS similar to previous studies from other developing countries [18] and in contrary to reports from developed countries that consider group B streptococci a common pathogen for EONS [12, 13]. There is no clear explanation for such observation, but prior maternal antibiotic exposure can be blamed as a contributory factor. Further studies of maternal carriage of group B streptococci would be required to find out if the organism is truly not prevalent in the developing countries. The limited number of positive blood cultures in our study did not allow us to establish any relation involving different gestational age groups or the place of delivery (whether inborn or outborn) and types of isolated bacteria. Further studies with a larger number of positive blood culture may be required in the future to establish such relation.

Among the Gram-negative isolates, all K. pneumoniae, S. marcescens, and Acinetobacter baumannii isolates were found to be resistant to ampicillin, cefotaxime, amoxicillin-clavulanic acid, and ceftazidime. The resistance of all Klebsiella isolates to ampicillin was previously reported [33]. In our study, amikacin has shown a better efficacy against K. pneumoniae than gentamicin, in contrary to a study from Iran, where isolated K. pneumoniae were more resistant to amikacin than gentamicin, 46%, and 27% respectively [34]. This finding should be reflected on our policy to support the amikacin usage instead of gentamicin. Despite the high resistance of Acinetobacter isolates, 50% of these isolates were sensitive to imipenem.

In our study, all the isolated CoNS were resistant to ampicillin. Staphylococcus haemolyticus isolates showed the highest resistance [≥85%] among the other CoNS isolates to amoxicillin-clavulanic acid and other antimicrobials including gentamicin, cefotaxime, ceftazidime and erythromycin. These results are in agreement with previous studies [35, 36]. There was a variability of sensitivity of different CoNS isolates to amikacin, imipenem and ciprofloxacin. All staphylococcal isolates were sensitive to vancomycin as previously reported [37].

5 Conclusion

Disclosure

There was no funding secured for this study. All Authors have no conflict of interest to disclose. All procedures performed in this study were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from at least one of parents of participants included in the study. The study protocol has been approved by Faculty of Medicine - Mansoura University ethical committee.

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