neonatal sepsis continues to be an important cause of morbidity and mortality both in term and in low - birth - weight infants despite advances in healthcare . there are approximately 7 cases of neonatal sepsis per 1000 live births , increasing to 162 per 1000 live births in very low birth weight neonates . the incidence of the neonatal sepsis varies with the geographical area , the socio - economic structure and various customs and practices in the perinatal period . neonates are particularly vulnerable to infection , so any delay in the initiation of therapy or wrong choice of antibiotics is to be avoided[4 , 5 ] . diagnosing neonatal infection is challenging , since clinical signs and symptoms are often slight and nonspecific for a particular infection . therefore , in suspected sepsis , two or three days empirical antibiotic therapy should begin immediately after cultures have been obtained without awaiting the results . in line with this idea the recognition of the epidemiological and antimicrobial susceptibility pattern of common pathogens , and determination of the risk factors for neonatal sepsis mortality is extremely relevant in the clinical setting , since it contributes to the diagnostic reasoning and supports clinical decisions . the aim of the study was to determine the demographic characteristics of newborns diagnosed as late - onset sepsis and to evaluate their microbiological characteristics ( prevalence and antimicrobial susceptibility ) . to view the changes in years , we compared them with the data which we gained in former years . also , we investigated the clinical and microbiological characteristics of the survivors and non - survivors to describe the mortality risk factors due to neonatal sepsis . this study was carried out in the neonatal intensive care unit ( nicu ) and department of microbiology at tepecik educational and research hospital ( terh ) in izmir , turkey . terh is a 1000bed tertiary - care hospital in izmir , the biggest city in aegean region in turkey with a population of 3,400,000 according to the census 2000 . the nicu has a level iii nursery with 45 bed capacity . because there is not an obstetric service in our hospital , the prevalence of sepsis was calculated by dividing the number of newborns with sepsis by the total number of newborns admitted to the nicu . data collected from the medical records of each patient were recorded in standardized collection sheets and included demographic information , birth weight , gestational age , gender , outcome , and delivery data ( type of delivery , maturity ) . gestational age was classified into two distinct groups : 24 - 29 weeks and 30 weeks . birth weight categories were : 600 - 1499 g and 1500 g. the definition of sepsis was based on a positive blood culture in the presence of clinical signs and symptoms of infection ; tachypnea ( respiratory rate > 60 bpm ) plus either grunting/ retraction or desaturation , temperature instability ( < 36c or > 38c ) , heart rate > sd above normal for age ( 180 beats / min , 100 beats / min ) , lethargy / altered mental status , systolic pressure < 65 mm hg ( infants 1 month ) , capillary refill time > 3 sec , wbc count ( < 400010/l or > 3400010/l , immature/ total neutrophil ratio > 0.2 , cpr>10 mg / ml , il-6 or il-8 > 70 pg / ml ) was diagnosed septicemia in this study . late - onset infection was defined when positive blood cultures were obtained after first 72 hours of life . newborns that were younger than three days were excluded . when coagulase - negative staphylococci ( cons ) were isolated the definition of true infection was based on the presence of clinical symptommatology strongly suggestive of infection together with at least two blood cultures positive for cons strains , drawn on different occasions ; otherwise , isolated cons was defined as contamination . cultures positive for organisms that are generally considered to be contaminants including cornybacterium , propionibacterium , and diphtheroids were excluded from analysis . copy isolates , defined as the same organism isolated from multiple blood cultures of the same patient within 48 h and contaminant bacteria were excluded from this study . during the study period , standard initial empirical treatment protocols in our nicu included ampicillin and cefotaxime or teicoplanin and meropenem in serious clinical conditions . all blood cultures were collected from peripheral vein under proper aseptic precautions before starting any antibiotic therapy . blood culture samples were incubated in the bact / alert 3d ( biomerieux , france ) and isolates identified by standard laboratory techniques or automated system ( vitek2 , biomerieux , france ) , as required . eosin methylene blue and blood agars were used for sub - culture and incubated at 37 c aerobically . antibiotic susceptibility testing was performed by kirby - bauer disk diffusion method according to the clinical laboratory standard institute ( clsi ) criteria . extended - spectrum -lactamase ( esbl ) activity was detected by phenotypic confirmation with ceftazidime/ ceftazidime - clavulanate and cefotaxime/ cefotaxime - clavulanate disks recommended by the clsi . in this test , a 5 mm increase in the zone diameter for disks ceftazidime - clavulanate or cefotaxime - clavulanate versus ceftazidime or cefotaxime disks alone demonstrates that the strain has esbl activity . when susceptibility was intermediate , the bacteria were considered resistant . the comparison of categorical variables was performed by using chi - square test or fisher 's exact test . univariate analysis was done to identify the risk factors of mortality . to find out the overall effects of significant variables , the standard significance level , p<0.05 , was used and all tests of statistical significance were two - tailed . this study was carried out in the neonatal intensive care unit ( nicu ) and department of microbiology at tepecik educational and research hospital ( terh ) in izmir , turkey . terh is a 1000bed tertiary - care hospital in izmir , the biggest city in aegean region in turkey with a population of 3,400,000 according to the census 2000 . the nicu has a level iii nursery with 45 bed capacity . because there is not an obstetric service in our hospital , the prevalence of sepsis was calculated by dividing the number of newborns with sepsis by the total number of newborns admitted to the nicu . data collected from the medical records of each patient were recorded in standardized collection sheets and included demographic information , birth weight , gestational age , gender , outcome , and delivery data ( type of delivery , maturity ) . gestational age was classified into two distinct groups : 24 - 29 weeks and 30 weeks . birth weight categories were : 600 - 1499 g and 1500 g. the definition of sepsis was based on a positive blood culture in the presence of clinical signs and symptoms of infection ; tachypnea ( respiratory rate > 60 bpm ) plus either grunting/ retraction or desaturation , temperature instability ( < 36c or > 38c ) , heart rate > sd above normal for age ( 180 beats / min , 100 beats / min ) , lethargy / altered mental status , systolic pressure < 65 mm hg ( infants 1 month ) , capillary refill time > 3 sec , wbc count ( < 400010/l or > 3400010/l , immature/ total neutrophil ratio > 0.2 , cpr>10 mg / ml , il-6 or il-8 > 70 pg / ml ) was diagnosed septicemia in this study . late - onset infection was defined when positive blood cultures were obtained after first 72 hours of life . newborns that were younger than three days were excluded . when coagulase - negative staphylococci ( cons ) were isolated the definition of true infection was based on the presence of clinical symptommatology strongly suggestive of infection together with at least two blood cultures positive for cons strains , drawn on different occasions ; otherwise , isolated cons was defined as contamination . cultures positive for organisms that are generally considered to be contaminants including cornybacterium , propionibacterium , and diphtheroids were excluded from analysis . copy isolates , defined as the same organism isolated from multiple blood cultures of the same patient within 48 h and contaminant bacteria were excluded from this study . during the study period , standard initial empirical treatment protocols in our nicu included ampicillin and cefotaxime or teicoplanin and meropenem in serious clinical conditions . all blood cultures were collected from peripheral vein under proper aseptic precautions before starting any antibiotic therapy . blood culture samples were incubated in the bact / alert 3d ( biomerieux , france ) and isolates identified by standard laboratory techniques or automated system ( vitek2 , biomerieux , france ) , as required . eosin methylene blue and blood agars were used for sub - culture and incubated at 37 c aerobically . antibiotic susceptibility testing was performed by kirby - bauer disk diffusion method according to the clinical laboratory standard institute ( clsi ) criteria . extended - spectrum -lactamase ( esbl ) activity was detected by phenotypic confirmation with ceftazidime/ ceftazidime - clavulanate and cefotaxime/ cefotaxime - clavulanate disks recommended by the clsi . in this test , a 5 mm increase in the zone diameter for disks ceftazidime - clavulanate or cefotaxime - clavulanate versus ceftazidime or cefotaxime disks alone demonstrates that the strain has esbl activity . when susceptibility was intermediate , the bacteria were considered resistant . the comparison of categorical variables was performed by using chi - square test or fisher 's exact test . univariate analysis was done to identify the risk factors of mortality . to find out the overall effects of significant variables , the standard significance level , p<0.05 , was used and all tests of statistical significance were two - tailed . . 
  demographic characteristics of the study groups data are presented as meanstandard deviation microorganisms causing late - onset sepsis were significantly increased in years ( p<0.001 ) . as shown in table 2 , gram - positive microorganisms were more frequently isolated than gram - negative microorganisms . among gram - positive pathogens cons cons showed a significant increase from 2004 to 2008 ( 76 vs 115 , p=0.04 . distribution of pathogens of late - onset sepsis in 2004 and 2008 during the years 2004 to 2008 , the number of sepsis caused by gnb showed an increase of more than 3-fold ( p<0.001 ) . escherichia coli ( e. coli ) , together , were responsible for 77% and 67% of late - onset sepsis which was caused by gnb in 2004 and 2008 , respectively . the most important finding of our study is that a considerable proportion of pseudomonas aeruginosa isolates increased in recent years . while 21 non - fermentative bacteria were isolated from blood cultures in 2008 , only one non - fermentative pathogen ( p. aeruginosa ) isolate was responsible for late - onset sepsis in 2004 ( p<0.05 ) . table 3 shows the antimicrobial susceptibility of cons , staphylococcus aureus ( s. aureus ) , klebsiella spp . and e. coli that made up 79 - 85% of all isolates . gentamicin and trimethoprim - sulfamethoxazole ( sxt ) showed increased resistance rate for cons and s. aureus in years ( ns ) . there were no detected vancomycin and teicoplanin resistance to the gram - positive bacteria . antimicrobial resistance pattern of isolates in 2004 and 2008 , no ( % ) cons : coagulase - negative staphylococci ; s. aureus : staphylococcus aureus ; esbl : extended - spectrum -lactamase ; e. coli : escherichia coli 
  although gram - negative strains showed increase in resistance rate , there was no statistically significant difference between years . especially for e. coli , gentamicin and sxt resistance rate although number of p. aeruginosa isolates was very low , high resistance rates were detected in recent years ( ns ) . generally , there was a trend of increasing resistance rate of all isolates to tested antibiotics in 2008 , compared to those in 2004 . table 4 shows clinical characteristics of the survivors and non - survivors of the study groups . preterm neonates , smaller gestational age ( < 30 weeks ) and low birth weight ( < 1500 g ) were found significantly associated with mortality ( p<0.05 ) . the variables associated with mortality according to the univariate analyses were : birth weight ( p<0.001 ) , gestational age ( p<0.001 ) , and infected with klebsiella spp . logistic regression analysis showed birth weight ( p=0.000 , or : 1.001 , 95%ci : 1.0009 - 1.002 ) and infected with klebsiella spp . ( p=0.005 , or : 2.472 , 95%ci : 1.003 - 6.092 ) associated with high mortality rate in neonates . clinical characteristics of the survivors and non - survivors of the study group data are presented as meanstandard deviation/ ns : not significant distribution of 227 patients according to demographic variables and microbiologic characterization and occurrence of mortality in 2008 ns : not significant/ cons : coagulase - negative staphylococci ; s. aureus : staphylococcus aureus ; e. coli : escherichia coli ; esbl : extended - spectrum -lactamase ; gbs : group b streptococcus microorganisms causing late - onset sepsis were significantly increased in years ( p<0.001 ) . as shown in table 2 , gram - positive microorganisms were more frequently isolated than gram - negative microorganisms . among gram - positive pathogens cons was the leading cause of late - onset sepsis . cons showed a significant increase from 2004 to 2008 ( 76 vs 115 , p=0.04 . distribution of pathogens of late - onset sepsis in 2004 and 2008 during the years 2004 to 2008 , the number of sepsis caused by gnb showed an increase of more than 3-fold ( p<0.001 ) . escherichia coli ( e. coli ) , together , were responsible for 77% and 67% of late - onset sepsis which was caused by gnb in 2004 and 2008 , respectively . the most important finding of our study is that a considerable proportion of pseudomonas aeruginosa isolates increased in recent years . while 21 non - fermentative bacteria were isolated from blood cultures in 2008 , only one non - fermentative pathogen ( p. aeruginosa ) isolate was responsible for late - onset sepsis in 2004 ( p<0.05 ) . table 3 shows the antimicrobial susceptibility of cons , staphylococcus aureus ( s. aureus ) , klebsiella spp . and e. coli that made up 79 - 85% of all isolates . gentamicin and trimethoprim - sulfamethoxazole ( sxt ) showed increased resistance rate for cons and s. aureus in years ( ns ) . there were no detected vancomycin and teicoplanin resistance to the gram - positive bacteria . antimicrobial resistance pattern of isolates in 2004 and 2008 , no ( % ) cons : coagulase - negative staphylococci ; s. aureus : staphylococcus aureus ; esbl : extended - spectrum -lactamase ; e. coli : escherichia coli 
  although gram - negative strains showed increase in resistance rate , there was no statistically significant difference between years . especially for e. coli , gentamicin and sxt resistance rate although number of p. aeruginosa isolates was very low , high resistance rates were detected in recent years ( ns ) . generally , there was a trend of increasing resistance rate of all isolates to tested antibiotics in 2008 , compared to those in 2004 . table 4 shows clinical characteristics of the survivors and non - survivors of the study groups . preterm neonates , smaller gestational age ( < 30 weeks ) and low birth weight ( < 1500 g ) were found significantly associated with mortality ( p<0.05 ) . the variables associated with mortality according to the univariate analyses were : birth weight ( p<0.001 ) , gestational age ( p<0.001 ) , and infected with klebsiella spp . logistic regression analysis showed birth weight ( p=0.000 , or : 1.001 , 95%ci : 1.0009 - 1.002 ) and infected with klebsiella spp . ( p=0.005 , or : 2.472 , 95%ci : 1.003 - 6.092 ) associated with high mortality rate in neonates . clinical characteristics of the survivors and non - survivors of the study group data are presented as meanstandard deviation/ ns : not significant distribution of 227 patients according to demographic variables and microbiologic characterization and occurrence of mortality in 2008 ns : not significant/ cons : coagulase - negative staphylococci ; s. aureus : staphylococcus aureus ; e. coli : escherichia coli ; esbl : extended - spectrum -lactamase ; gbs : group b streptococcus early diagnosis and therapy are essential for the prevention of morbidity and mortality of neonatal sepsis in nicu . in the vast majority of cases , antimicrobial therapy must be initiated empirically . therefore , the accurate prediction of likely pathogens and antimicrobial resistance patterns is crucial for successful therapy . the distribution of the causative pathogens for sepsis showed that these infections are caused mainly by gram - positive microorganisms , mostly cons in our nicu . our results are in agreement with other studies that reported cons as the most common bacteria causing late - onset sepsis in neonates , accounting for more than 50% of cases[1 , 5 , 10 ] . these infections are often associated with use of indwelling vascular catheters or central lines[3 , 10 , 11 ] . hensey et al , described the rise in incidence as coincident with change in skin desinfection usage and general use of 3 generation cephalosporins to which the cons were resistant . also , cons are among the most common causes of blood culture contaminants , since they are the most common microorganisms found colonizing the skin and mucous membranes of neonates . although cons were the leading pathogen of sepsis in nicu , it remains difficult to determine which blood isolates of cons represent true infection and which are contaminants . the virulence factor of cons may well be a biofilm formation , which aids colonization of not only intravascular devices but also tissues , and may also allow the organism to persist in hospital environments . thus , cons strains must be tested for biofilm formation or its genetic markers and if proven so , the offending central lines must be removed[13 , 14 ] . although gram - positive bacteria dominate over gnb in all years , gnb increased in the recent years . in this study , 15% and 31% of isolates were related to gnb in 2004 and 2008 ( p<0.05 ) respectively , very similar to that found by others , with reports varying between 10%-67%[5 , 10 ] . although klebsiella spp . were the most common microorganisms among gram - negative germs , p. aeruginosa was significantly increased in our nicu in recent years ( p<0.05 ) . increased gnb rate was possibly related to many possibilities : primarily the widespread and injudicious use of antibiotics , also  selection pressure  on the esbl producing multi - resistant clones by the use of higher antibiotics , and catheter related bloodstream infections especially by  biofilm  forming organisms ( like p. aeruginosa , and s. epidermidis [ 2 , 15 ] ) . jain et al , found that gram - negative rods were responsible for the 67.7% of septicemic neonates and authors authors indicate that this high rate may be due to the selective pressure imposed by extensive use of antimicrobials in the intensive care unit . in our study , esbl was detected 14% and 33% for e. coli ; 30% and 44% for klebsiella spp . in 2004 and 2008 , respectively ( ns ) . in this study the resistance rate of -lactam antibiotics and aminoglycosides to gnb showed increase in years . there may be different explanations for increased resistance rate in our nicu ; our nicu serves very wide area in aegean and patients were usually premature , received more mechanical ventilation and were more exposed to antibiotic treatment , which in turn may lead to selection of resistant microbes . some data suggest that the recent introduction of intrapartum antibiotic prophylaxis programs , through which selected women are administered penicillin or ampicillin during labor to prevent vertically transmitted group b streptococcal disease , has increased the incidence and resistance of gnb sepsis in the newborn[2 , 16 ] . cordero et al , assessed the enteric gnb sepsis during 17-years period and showed that antepartum antibiotic prophylaxis may have increased antibiotic resistance in e. coli isolates . in our study , group b streptococcal infections showed increase in the last years ( ns ) , but we do not have any data on antibiotic prophylaxis during pregnancy . rapid treatment with antibiotics is essential for favorable outcome for neonatal sepsis . in the present study , staphylococcus and gram - negative strains were frequently found to be resistant to ampicillin , thus indicating that the use of these drugs alone may be ineffective . since staphylococci are mostly resistant to methicillin the most common gram - negative pathogens , k. pneumoniae and e. coli are resistant to most antibiotics but susceptible to amikacin and carbapenem . therefore , the appropriate empiric regimen seems to consist of teicoplanin and carbapenem until culture results arrive . mortality rate in neonatal sepsis has been reported 7 - 55% in many studies[10 , 17 ] . in our study , a number of antenatal and intrapartum factors have been associated with perinatal and neonatal mortality . although , neonatal sepsis is an important risk factor for mortality in itself , low birth weight , prematurity , and infection with klebsiella spp . basu et al and locatelli et al showed that shorter gestational age and low birth weight were independent predictors of survival . in the present study , cordero et al , also found high mortality rate related to k. pneumoniae and e. coli in neonates . multi - resistant gnb , including esbl producing bacteria are associated with increased mortality . in this study , lautenbach et al showed that patients with esbl - positive strains had significantly higher average hospital costs , longer median hospital stays after the onset of bacteremia , and higher mortality compared with control individuals . in addition , mortality risk factors ( major neonatal co - morbidities such as presence or absence of underlying congenital abnormalities , bronchopulmonary dysplasia , necrotizing enterocolitis , duration of mechanical ventilation , the need for invasive procedure , etc . ) were omitted . nevertheless , large - scale multicentric prospective studies are necessary to identify the changing patterns of the prevailing flora and mortality risk factors of neonatal sepsis . knowledge of prevailing strains and antibiotic sensitivity patterns in the region is essential to meet the challenge of neonatal sepsis with equal competence and opposing force . observed decline in susceptibility of pathogens to common antibiotics in this study calls for increased efforts to ensure monitoring , restricting and rationalizing the use of these antimicrobials . prevention measures should therefore be employed with an emphasis on proper hand hygiene and evaluation of other potential reservoirs as sources of bacterial acquisition and transmission . low birth weight and infection with klebsiella spp were found related to high mortality rate in our nicu . these results raise two important tasks , first , providing a better antenatal care to minimize low birth weight and second , importance of infection control practices .
objectiveneonatal sepsis is a common cause of morbidity and mortality among newborns in the developing world . 
 we have investigated the causative agents and their antimicrobial susceptibility of late - onset sepsis ( > 72 h post - delivery ) , and determined the possible association between various risk factors and the mortality due to neonatal sepsis in 2008 . to view the changes in years , we compared them with the data which we gained in 2004.methodsmedical records of all neonates with late - onset sepsis were reviewed for demographic characteristics ( birth weight , gestational age , gender , type of delivery , and mortality rate ) , positive cultures and risk factors of mortality.findingsone hundred and forty - seven and 227 neonates had been diagnosed as late - onset sepsis in 2004 and 2008 , respectively . 
 coagulase - negative staphylococcus was the most frequent microorganisms . 
 gram - negative bacilli , particularly pseudomonas aeruginosa showed a significant increase in years . 
 the mortality rate was 11.5% and 19% in 2004 and 2008 , respectively . 
 birth weight , gestational age , and infection with klebsiella spp . 
 isolates were found to have significant association with sepsis mortality in our neonatal intensive care unit ( nicu).conclusionthe present study emphasizes the importance of periodic surveys of sepsis encountered in particular neonatal setting to recognize the trend . increased gram - negative bacilli rate was possibly related to the widespread use of antibiotics in our nicu .