Abstract: Bacterial sepsis is one of the most common causes of significant mortality and morbidity in neonates. The researchers analyzed bacterial isolates and their antibiotic susceptibilities for cases of septicemia in a Neonatal Intensive Care Unit (NICU) of a teaching hospital in Tehran, Iran. During a 36 months period, the incidence of bacteremia and the causing organisms and mortality of sepsis as well as antibiotic susceptibility were investigated. Neonatal Late-Onset Sepsis (LOS) was defined as clinical signs suggestive of infection with a positive Blood Culture (B/C) after 72 h of birth. About 909 neonates were admitted to the NICU. A total of 9.13% of neonates (83/909) had at least one positive B/C after 72 h of birth. The vast majority (56.6%) of sepsis were caused by Gram-negative organism. Gram-positive pathogens accounted for 41% infections. The most common cause of late-onset sepsis was Klebsiela p. (31%) and followed by Staph aureus (18.1%). In this study, the researchers have observed that the old empiric therapy with cephalothine plus Amikacin for suspected late-onset sepsis seems ineffective. Now, however may be the best choice regimen is the combination vancomycin plus amikacin and the vancomycin plus imipenem for the severe ill patients.

INTRODUCTION

Sepsis is considered to comprise a spectrum of disorders that result from infection by bacteria, viruses and fungi ora parasites or the toxic products of these microorganisms. Bacteremia, viremia, fungemia and parasitemia refer to bloodstream invasion that may be associated with fever but no other signs or symptoms of circulatory compromise or end-organ malperfusion or dysfunction (Stoll, 2004; Palazzi et al., 2006). Neonatal sepsis may be categorized as early or late onset.

About 85% of newborns with early-onset infection present within 24 h, 5% at 24-48 h and a smaller percentage of patients present between 48 h and 6 days of life (Palazzi et al., 2006; Edwards, 2006). Late-onset sepsis syndrome occurs from 3rd- 90th days of life and is acquired from the caregiving environment.

Organisms that have been implicated in causing late-onset sepsis syndrome include Coagulase-Negative Staphylococci (CONS), Staphylococcus aureus, E. coli, Klebsiella, Pseudomonas, Enterobacter candida, Group-B Streptococcus (GBS), Serratia, Acinetobacter and anaerobes. The infant's skin, respiratory tract, conjunctivae, gastrointestinal tract and umbilicus may become colonized from the environment, leading to the possibility of late-onset sepsis from invasive microorganisms (Palazzi et al., 2006). Surveillance of late-onset neonatal sepsis is required to monitor the quality of Neonatal Intensive Care Unit (NICU) related care (Cloherty et al., 2004).

Despite major advances in NICU, sepsis continues to be an important cause of morbidity/mortality among neonates. A gradual change in the spectrum and organisms responsible for neonatal sepsis has been recognized. Constant surveillance is important to guide empirical antibiotic therapy. The pathogens associated with neonatal sepsis are known to vary geographically. Antibiotic regimens for treatment or prevention of these infections have been associated with variable (Stoll, 2004; Palazzi et al., 2006; Edwards, 2006; Cloherty et al., 2004; Gomella and Cunnigham, 2004). In order to understand, the epidemiology of late-onset sepsis in the NICU of Ali-Asghar Hospital, a teaching center, the researchers retrospectively collected data on bacteremia to analyze its microbiology and determine the antibiotic susceptibilities of the causative organisms.

MATERIALS AND METHODS

Ali-Asghar Hospital NICU is a level III referral nursery in Iran University of Medical Sciences, Tehran, Iran. This NICU is one of the greatest tertiary-teaching level (III) in Iran. It has 6 units with >40 neonatal beds capacity and one neonatal surgery unit (6 beds).

Study population: From 2004/5 -2007/5 all neonates with age >72 H admitted to the NICU of Ali-Asghar Hospital were included in this study. This study is based on results of blood culture in neonatal late-onset sepsis. Empirical antibiotic regimen for suspected LOS was changed on the basis of organism antimicrobial susceptibility. Neonatal LOS was defined as clinical signs suggestive of infection with a positive Blood Culture (B/C) after 72 h of birth. That similar with definition of LOS at the Ohio State University Medical Center (Cordero et al., 1999) and NICU of (VGH-Taipei) in Taiwan (Lee et al., 2004).

Data collection: Data collection for neonate with positive B/C included: gender, birth weight and gestational age, the pathogenic bacteria of positive B/C and their antibiotic susceptibilities. B/C were performed routinely on all neonate with clinical sepsis.

RESULTS AND DISCUSSION

About 909 neonates were admitted to the NICU. A total of 9.13% of neonates (83/909) had at least one positive B/C after 72 h of birth. About 60% of the infants were male. The proportion late-onset sepsis in neonates with normal-birth weight (45/81) (>2500 g) and low-birth weight (26/81) (<2500 g) and very low-birth weigh (12/81) (<1500 g) was (54.32, 30.86, 14.82%), respectively. Very low-birth weight infants average rate is lower in this study because usually more complicated term cases refereed to the NICU as referral center. GNB was higher in very low birth-weight than other groups.

The vast majority (56.6%) of septic cases were caused by gram-negative organism. Klebsiela p. were the most common pathogens (31.3%) of all infections, 55.31% (26/47) of gram-negative infections. Other gram-negative organisms included Enterobacter, Acinetobacter, E. coli, Pseudomonase (Fig. 1). Gram-positive pathogens accounted for 41% infections. Staphylococcus aureus, CONS, other Streptococcus sp. and Enterococus were the most frequent gram-positive pathogens. Fungal organisms were responsible for 2.4% of sepsis. The most common cause of late-onset sepsis was Klebsiela sp. (31%) and followed by Staph aureus (18.1%).

Fig. 1:	The incidence of neonatal LOS by frequency of bacteria

Overall 27(32.5%) of the 83 infants with sepsis died. About 15/30 neonates (50%) in the group of neonates surgery were died. The highest mortality rate was due to Klebsiela pneumonia in the group of neonates surgery. The most common cause of mortality in this group might be sepsis or complications of surgery.

Antibiotic susceptibilities: The antibiotic susceptibilities of gram-positive cocci shown in Fig. 2. GPC was resistant to most of the antibiotics including cephalothin, Cloxacillin, Ampicillin, Cefotaxime. Strains resistant to vancomycin and imipenem were not found in the gram-positive pathogens. All cultured CONS and S. aureus organism were vancomycin-sensitive. S. aureus was resistant to cephalothin (87.5%), cefotaxim (70%), cloxacilin (66.67%), ampicillin (50%).

The antibiotic susceptibilities of gram-negative bacilli shown in Fig. 3. GNB was resistant to most of the antibiotics including cefotaxim, ceftazidim, gentamicin and amikacin. GNB had high sensitivity to imipenem (95%) and only one strains of E. coli was resistant to imipenem. Klebsiela sp. was resistant to cefotaxim (95%), ceftazidim (93.75%), gentamicin (87%) and amikacin (80%). All cultured Klebsiela sp. was imipenem-sensitive.

Sepsis is a severe problem for neonates. The most common nosocomial infection among neonates is blood stream infections. It is associated with marked morbidity and mortality and should be a major focus of surveillance and prevention efforts in high-risk nurseries. Positive B/C together with clinical signs of infection makes the diagnosis of blood stream infection more accurate. The reserchers have found the LOS rate of 9.13% in the center.

The overall incidence of bacteremia in NICU have ranged from 1.9-30.4% (Lee et al., 2004; Fok et al., 1998; Jogn et al.,1982). Some other investigators in different centers of NICU in Iran had shown similar results (Samaee, 1998; Movahedion et al., 2006). In the NICU the case fatality rate due to sepsis was 32.5%. Among them, >50% mortality was taken place in the group of neonates after surgery. The predominant causative organisms in the study were gram-negative organism (56.6%).

Fig. 2:	The antibiotic susceptibilities of gram-negatieve cocci in neonatal LOS

Fig. 3:	The antibiotic suscepitbilties of gram-negative bacilli in neonatal LOS

Overall, there was a greater distribution of gram-negative bacilli than gram positive infections. This data consistence with the finding of other NICU centers in Iran (Samaee, 1998; Masheof, 1999). While gram-positive cocci including coagulase-negative staphylococci is the most common isolated organisms in late-onset sepsis in NICU at other countries (Palazzi et al., 2006; Lee et al., 2004; Kapoor et al., 2005; Yalaz et al., 2006; Haque et al., 1990; Rubin et al., 2002). It seems that incidence of CONS infection is increasing as important pathogen in late-onset sepsis in the NICU. It is may be because of different approach of the neonatologists about the definition, interpretation and management of suspected late-onset sepsis, particularly those caused by CONS positive blood culture in neonatal period.

Therefore, it should be cautiously interpreted CONS positive blood culture as possibility of contamination. At the present study, Klebsiela sp. was the most common bacteria (31.3%) followed by Staph aureus (18.1%) and CONS (10.7%). Overall the data that Klebsieal sp. is one of the three common bacteria is consistent with other reports in different center in Iran with previous study in this center at 1992 (Samaee, 1998; Movahedion et al., 2006; Masheof, 1999) and with the report from hospital in Dehli, India (Yalaz et al., 2006). A study from Ohio shows that the number of bacteremia due to Klebsiella sp. and Enterobacter were more prominent and more often than the previous years (Cordero et al., 1999). In this study, most of GNB cases were highly resistant to aminoglycosides (gentamicin and amikacin) and 3rd generation cephalosphorins (cefotaxim, ceftazidm) while sensitive to the imipenem. Since 1983, productions of Extended Spectrum Beta-Lactamase (ESBL) have been found in many GNB, such as Klebsiela sp., E. coli, Enterobacteriacea (Paterson et al., 2004). The occurrence of multi-drug resistant in our NICU and recently prominence of Klebsiela sp. might result from these mechanism that was against most 3rd generation cephalosporins in the routine usage.

However, further analysis should be done for confirmation. Unfortunately, we observed increase resistant of Klebsiela sp. to Amikacin (80%), gentamicin (87%) and cefotaxim (95%) that’s is similar with other study in Iran (Masheof, 1999). While cordero in Ohio reported that 100% Klebsiela sp. were sensitive to gentamicin and cefotaxim and the combination of (vancomycin and gentamicin) for suspected LOS been successful (100% sensitivity).

In this study, GPB were highly resistance to cephalothin (90%), cloxacillin (75%) and cefotaxim (77%). All of them were susceptible to vancomycin and imipenem. While, Rashidy et al. (2003) in Sanandage, Iran reported that GPB resistance to vancomycin was 8.6% (Makhoul et al., 2005).

The data have shown that fungemia was the lowest incidence (2.4%) which is unlike high incidence of fungemia in Turkey (18%) and Tel-Aviv University so that in both studies antifungal was added to the routine empirical antibiotic regimen for LOS, especially in VLBW (Haque et al., 1990; Makhoul et al., 2001, 2005). In the NICU, prophylactics antibiotics were prescribed for suspected late-onset sepsis with Cephalothin plus amikacin as the empiric therapy, according to previous studies (>10 years ago). In this study, the researchers have observed that the old empiric therapy with cephalothine plus amikacin for suspected late-onset sepsis seems ineffective. Now, therefore may be the best choice regimen is the combination of vancomycin plus amikacin and the vancomycin plus imipenem for the severe ill patients.

CONCLUSION

Studies of bacterial etiology have implications for presumptive antibiotic therapy of bacterial infection in neonates. Now, the significance of antibiotic resistance is recognized to be a global unsolved problem and the emergence of antibiotic-resistant pathogens is particularly alarming in developing countries. The some reason of this problem seems due to the widespread availability of antibiotics and inappropriate use of it at outpatients, uncontrolled antibiotics programs, overuse of antibiotics at inpatients, especially lack of establish protocols to prohibit the unnecessary administration of antibiotics in neonates whose culture are negative.

ACKNOWLEDGEMENTS

The researchers thank Dr. Mosavinasab for statistical analysis. The researchers also thank Mrs. Miersaiedy and the staff of the microbiology Section of Laboratory department in Ali-Asghar-Children Hospital for their continued interest in the preparation of these data.