Changing trend in bacterial etiology and antibiotic resistance in sepsis of intramural neonates at a tertiary care hospital.

BACKGROUND: Septicemia is an important cause of neonatal morbidity and mortality. However, organized data on causative organisms and their resistant pattern are scanty from developing countries. The changing trend in causative organisms and their antibiotic resistance is yet to be documented in India. The present study examines the trends in bacterial profile and antibiotic resistance of the organisms causing sepsis in hospitalized neonates.
MATERIALS AND METHODS: A retrospective laboratory-based analysis of blood cultures obtained from Neonatal Intensive Care Unit of a tertiary care hospital in New Delhi was done for the period of 1999-2014, divided into five phases.
RESULTS: A total of 4700 isolates were considered. Over time, Gram-negative organisms have replaced Gram-positives as frequent isolates. Initially, there was predominance of Klebsiella pneumoniae, then of Staphylococcus aureus which recently has been changed with coagulase negative-Staphylococcus and Acinetobacter. Growing resistance against the first and second line of drugs has been noted, including methicillin-resistant S. aureus and vancomycin-resistant Enterococcus.
CONCLUSION: The etiological profile of neonatal sepsis has changed tremendously in the past 15 years. High resistance against common drugs necessitates continued surveillance and review of empirical antibiotic policy for neonatal sepsis. These steps are important to effectively curtail the surge of further antibiotic resistance.

Introduction

Neonatal sepsis refers to systemic infection of the newborn, characterized by nonspecific symptoms, and documented by positive blood culture.[1] An estimated 1.6 million deaths occur due to neonatal infections worldwide, 40% of them being limited to developing countries.[2] A multicentric study from India found sepsis as one of the most common causes of mortality, contributing to 19% of all neonatal deaths. The incidence of neonatal sepsis in the country is 30/1000 live births.[3] Considering the fact that these children are more likely to have neurodevelopmental side effects, this topic deserves paramount significance.[4]

The spectrum of microbial etiology of neonatal sepsis varies from region to region and even varies in different hospitals of the same region. In addition, one organism or group of organisms may be replaced by others over a period of time.[5] For example, in developed countries, Escherichia coli and other Gram-negative organisms were the most common cause of neonatal sepsis in mid-1960s.[6] Thereafter, Group B Streptococcus and coagulase-negative Staphylococcus (CONS) species have been implicated frequently from 1970s.[78] Some scientists even documented the change in pattern of newborn sepsis over decades in a single study area.[9] In India, most of the studies so far concentrated on the prevalence of different organisms in neonatal sepsis. For example, Gram-negative bacteria till recently were reported to be the major cause of neonatal sepsis with predominance of Klebsiella pneumoniae although the proportion of cases due to Gram-positive bacteria, especially Staphylococcus aureus, has gradually increased over the last two decades.[210] A recent study from Mangalore suggested dominance of Gram-positive cocci in neonatal sepsis.[11] The shortcoming with the available researches is their cross-sectional nature. To our knowledge, no study from India has tried to document the secular trend of bacterial etiology in neonatal sepsis.

Widespread emergence of resistance to multiple and commonly used antibiotics in isolates is another challenge for determining appropriate empirical therapy.[3] In the absence of any national antibiotic policy, the practice at the present hospital for neonatal sepsis is cloxacillin and aminoglycoside. Ciprofloxacin was used for the treatment of extended-spectrum beta-lactamase (ESBL) producing isolates till carbapenem was introduced in 2004.

In this background, the present study aimed to know the trend in etiology of neonatal sepsis and their antibiotic susceptibility pattern over a period of 15 years in a tertiary care hospital.

Materials and Methods

Settings

The study was conducted at Department of Microbiology and Neonatal Division, Department of Pediatrics at a tertiary hospital in New Delhi. The hospital has a maternity center with a delivery rate of 18,000–20,000/year and 50 bedded Neonatal Intensive Care Unit (NICU) which admits only intramural newborns. Almost 20% of the infants delivered in our hospital require admission to the NICU. A laboratory-based retrospective analysis was conducted on blood cultures obtained from the NICU during the past 15 years divided in five phases: January, 1999 to December, 2000 (Phase I); January, 2004 to December, 2005 (Phase II); January, 2009 to December, 2010 (Phase III); January, 2011 to December, 2012 (Phase IV); and January, 2013 to December, 2014 (Phase V). More emphasis was given to recent period to include all the years within analysis.

Microbiological methods

Centers for Disease Control/National Healthcare Safety Network criteria were used for assessment of bloodstream infections.[12] We also considered maternal risk factors for sepsis such as maternal fever, premature rupture of membrane of more than 24 h duration, unclean vaginal examination before admission, assisted delivery by forceps, or ventouse application; clinical signs suggestive of sepsis in the neonates were included and remained constant throughout the study period. Samples were collected before starting antibiotic therapy routinely, following hospital policy.

Two milliliters of blood was collected after aseptic precaution, inoculated in 10 ml Brain Heart Infusion Broth, and incubated at 37°C for 48 h following which subculture was performed on sheep blood agar and MacConkey agar plates. After overnight incubation at 37°C, culture media were examined for growth, and organisms identified by standard microbiological techniques.[1314] Cultures yielding no growth at 48 h were further incubated for 7 days before reporting as sterile.[2] Growth of mixed bacterial flora or diphtheroids was considered as contamination. CONS was considered a pathogen only when isolated in paired cultures. All isolates were included in the analysis.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed on Mueller–Hinton agar media by standard disk diffusion method and interpreted as per Clinical and Laboratory Standard Institute (CLSI) guidelines.[15] Gram-negative bacteria were tested for the following antimicrobials (μg): amikacin (30), gentamicin (10), ciprofloxacin (5), cefotaxime (30), ceftazidime (30), piperacillin (100), cefoperazone/sulbactam (75/10), piperacillin/tazobactam (100/10), ertapenem (10), imipenem (10), and meropenem (10). Imipenem and meropenem which were introduced in the hospital formally in 2004 and ertapenem in 2009 were tested against isolates resistant to the third-generation cephalosporins. Staphylococci were tested for penicillin (10 U), gentamicin (10), ciprofloxacin (5), chloramphenicol (30), erythromycin (15), clindamycin (2), and vancomycin (30). Enterococci were additionally tested for high-level aminoglycoside resistance using high-concentration gentamicin (120) from 2004 onward. In addition, methicillin resistance in S. aureus (MRSA) was determined by oxacillin (1 μg) disk throughout the study period. However, from 2009, cefoxitin was also introduced to screen MRSA. The presence of ESBL was determined by double-disk diffusion method using ceftazidime/cefotaxime with and without clavulanic acid. ESBL production is indicated by an increase in zone size of more than 5 mm, with clavulanic acid.[15] Standard strains of E. coli ATCC 25,922, S. aureus ATCC 25,923, Pseudomonas aeruginosa ATCC 27,853, and K. pneumoniae ATCC 700,603 (ESBL positive) were used as control.

CLSI continues to recommend disk diffusion for detecting vancomycin-resistant S. aureus (VRSA) mediated by VAN-A gene. It recommends screening all isolates with zone diameter ≥7 for vancomycin-intermediate S. aureus (VISA), and all isolates of zone ≤6 mm are defined as VRSA. In our study, all isolates had zone diameter of ≥16 mm. We did not screen for VISA.[16]

WHONET 5.6 software (Boston, USA) was used for data entry and subsequent analysis. Repeat isolates were not included in analysis.

Results

Over five phases, a total of 4700 isolates were studied [Table 1]. In Phase I, Gram-negative organisms (70.6%) were the predominant isolates. However, in the subsequent phases, Gram-positive organisms took the upper hand. S. aureus was the most common organism among Gram-positive bacteria till the fourth phase (2011–2012), and K. pneumoniae was the predominant pathogen among the Gram-negative bacteria till the third phase (2009–2010). Recently, CONS has replaced S. aureus as the most common Gram-positive bacteria, and Acinetobacter baumannii claimed the top spot among Gram-negative infections. In the last phase (2013–2014), Acinetobacter maintained the top spot causing more than half cases of Gram-negative sepsis.

Table 1

Distribution of predominant Gram-positive and Gram-negative organisms isolated in Phase I through V

Organism	Phase I (1999-2000) 850 (%)	Phase II (2004-2005) 979 (%)	Phase III (2009-2010) 750 (%)	Phase IV (2011-2012) 1065 (%)	Phase V (2013-2014) 1057 (%)	P value for trend
Gram-positive	250 (29.4)	590 (60.2)	525 (70)	601 (56.4)	575 (54.3)	<0.0001
Staphylococcus aureus	88 (35.2)	418 (70.9)	375 (71.5)	340 (56.6)	193 (33.6)	<0.0001
Enterococci spp	55 (22)	89 (15)	90 (17.1)	50 (8.3)	64 (11.1)	<0.0001
CONS	52 (20.8)	74 (12.6)	48 (9.1)	159 (14.9)	284 (49.4)	0
Others*	55 (22)	9 (1.5)	12 (2.3)	52 (8.7)	34 (5.9)	0.0104
Gram-negative	600 (70.6)	389 (39.8)	225 (30)	464 (43.6)	472 (45.7)
Klebsiella pneumoniae	504 (84)	289 (74.3)	68 (30.2)	91 (19.6)	83 (17.6)	0
Escherichia coli	64 (10.7)	48 (12.3)	54 (24)	100 (21.6)	76 (16.1)	<0.0001
Acinetobacter baumannii	10 (1.7)	26 (6.7)	64 (28.5)	182 (39.2)	254 (53.8)	0
Others**	22 (3.6)	26 (6.7)	39 (17.3)	91 (19.6)	70 (14.8)	<0.0001

*Others include Group B, D, G Streptococci, and Streptococci viridans. **Others include Enterobacter spp., Citrobacter spp., Serratia spp., Proteus spp., Pseudomonas spp., Haemophilus spp., and Salmonella spp. CONS: Coagulase-negative Staphylococcus

β-lactam resistance was high in all the phases with approximate half of S. aureus being methicillin resistant. Vancomycin resistance was not observed till Phase II (2004–2005) after which, however, enterococci have been increasingly showing resistance to this drug.

Figure 1 shows the resistance pattern of E. coli during different phases of the study. A high degree of resistance to the first and second line of antibiotic was observed. Over the years, the organism has documented a gradual rise in the resistance against amikacin. However, netilmicin resistance in E. coli has been reducedfrom 50% (Phase I) to 23.6% (Phase V). Most susceptibility to carbapenems and β-lactam plus β-lactamase inhibitors has been noted.

Figure 1

Trend of antibiotic resistance for Escherichia coli over years

Figure 2 depicts how resistance pattern of K. pneumoniae changed over the years. Resistance against the third-generation cephalosporins was almost the same over this long period. Among K. pneumoniae and E. coli, resistance to ampicillin was high (approximately 90%) till Phase III, after which it was not tested (not shown). Among the aminoglycosides, resistance to amikacin in K. pneumoniae was high throughout the study period. Resistance to penems and β-lactamase inhibitors is gradually emerging in this organism.

Figure 2

Trend of antibiotic resistance for Klebsiella pneumoniae over years

Figure 3 shows resistance pattern of Acinetobacter. The strains showed an increase in resistance to almost all drugs. From the third phase (2009–2010), gradually increasing resistance to netilmicin and amikacin has been noted. The resistance to carbapenem and piperacillin plus tazobactam recorded a particular steep rise in the recent years (>80% against both the drugs). As of now, colistin and tigecycline remain the only drugs effective against it.

Figure 3

Trend of antibiotic resistance for Acinetobacter spp. over years

Among Gram-positives, resistance to vancomycin is gradually increasing in Enterococcus spp. Overall, 15.2% of the total number of samples was vancomycin resistance. Similarly, an overall rise has been noted in methicillin resistance among S. aureus over the years [Figure 4]. Over the years, 47.4% of the total number of samples was methicillin resistance.

Figure 4

Change in methicillin resistance in Staphylococcus aureus and vancomycin resistance in Enterococcus over years

Discussion

Despite recent advances in health care, morbidity and mortality due to neonatal sepsis remains a major cause of concern in both term and low birth weight neonates. In developing countries, reported rates of neonatal sepsis are 3–20 times higher than those reported from hospitalized infants in developed countries.[17] As neonatal sepsis may not have specific signs and symptoms, a delay in diagnosis and treatment contributes to higher morbidity and mortality. Sepsis-related mortality is preventable with early appropriate empiric antibiotic therapy, based on periodic epidemiological survey of local bacterial flora, and their antibiotic susceptibility pattern.

A review from developing countries shows that K. pneumoniae and S. aureus were the major pathogens of neonatal sepsis.[18] Our study documented clear dominance of CONS and Acinetobacter among Gram-positive and negative organisms, respectively. The finding suggests a paradigm shift in the etiology of neonatal sepsis in recent times. The shift is not only from Gram-negative to Gram-positive but also in individual organisms. The spectrum of pathogen in South-East Asia and Africa is quite different from that observed in neonates in developed regions, where Group B Streptococcus, E. coli, and CONS are the predominant pathogens.[19]

A change in resistance pattern has also been noted in the present study. The surge of MRSA has been noted carefully in the last one decade.[20] Resistance to methicillin to the extent of our study was reported earlier by Karthikeyan and Premkumar, but most of the previous Indian studies documented the resistance in the range of 25%–40%.[9202122] The use of glycopeptides and linezolid has been encouraged with restrictions for such cases. For Enterococci, our finding is in accordance with a previous study which reported vancomycin resistance in 20% cases.[23]

Among Gram-negative organisms, ESBL production has been noted in more than one-third cases. A study from Aligarh also documented similar pattern.[24] Plasmid-mediated resistance, resulting from such production, renders third- and fourth-generation cephalosporins inactive against Gram-negatives and considered notorious for neonatal infections, particularly in ICU setup. Addition of amikacin along with the third-generation cephalosporins could be a solution for Gram-negative organisms.[25]

Some change in antibiotic resistance could be attributed to frequency of the use of particular antibiotics. For example, widely used amikacin witnessed a steady resistance across study period. Similarly, netilmicin, used infrequently, saw a drop in resistance over the years. The latter case probably supports keeping high-end antibiotics in reserve and their judicious use.

During the later phases of the study (2009 onward), there was a significant increase in proportion of A. baumannii, with notable resistance pattern. Being frequently multidrug resistant, Acinetobacter survives and multiplies in hospital environment and may prove very difficult to be treated. As of now, it has not been isolated from maternal genital tract. In Thailand, it has already raised alarm by causing death in more than 50% patients infected with imipenem-resistant variant.[26] However, a study has documented susceptibility of Acinetobacter to ceftriaxone plus ethylenediaminetetraacetic acid plus sulbactam even when it is resistant to other antibiotics.[27]

The heavily contaminated environmental reservoirs of organism to which mother and child are exposed during labor, delivery, and postnatal care may serve as the probable source of infection. In recent years, Staphylococci are emerging as the most common cause of neonatal septicemia.[18] The main source of spread of infection is through the hands of health-care providers.[2]

The other reason for increase in Gram-positive organisms could be explained by emergence of ESBL producing Enterobacteriaceae in late 1990. Widespread use of broad-spectrum antibiotic for the treatment of ESBL producing organism is known to have impact on flora within the hospitals. High prevalence of MRSA and Enterococcus has been observed in areas where ESBL is endemic.[2829]

The WHO recommended the first-line antibiotics for neonatal sepsis are ampicillin and aminoglycosides for neonatal sepsis and both have shown alarming resistance in the pathogens isolated during the present study.[18] Increasing resistance to the third-generation cephalosporins seen in the study suggests widespread dissemination of ESBL producing K. pneumoniae and E. coli. As ESBL is plasmid mediated, these organisms are resistant to other antibiotics including aminoglycosides. It was earlier reported that 50% of the neonates with early onset Gram-negative septicemia were infected with ESBL producing Enterobacteriaceae.[30] In view of the high resistance to these antibiotics, one hospital policy in Delhi was to use fluoroquinolones for treatment of Gram-negative infections till β-lactam plus β-lactamase inhibitors and carbapenem were available for management in 2002 and 2006, respectively.[31] Based on review of 11,471 blood cultures from developing countries in South-East Asia, Zaidi et al. have recommended imipenem and amikacin regimen for initial treatment of suspected sepsis in hospitalized neonates but considering increase in proportion of Acinetobacter, rampant use of carbapenem group of drugs cannot be encouraged.[18]

Predominance of MRSA, emergence of vancomycin-resistant Enterococcus, and carbapenem-resistant Acinetobacter can also be attributed to widespread use of broad-spectrum antibiotics for ESBL producers and vancomycin for MRSA. The current scenario suggests that there is need for cohorting and isolation of neonates with multidrug-resistant organisms, strict adherence to hand hygiene practices, periodic surveillance of pathogens, and a need to strictly implement antibiotic policy to effectively curtail spread of these resistant organisms.

The study has certain limitations. Being not correlated with clinical features and lack of correlation with outcome are the areas where we could improvise. Still, putting an effort to document change in sepsis pattern and antibiotic resistance over a period of 15 years and large sample size are the strongest points the study has. Lack of written hospital antibiotic policy during earlier phases is another drawback.

Conclusion

The bacterial profile of neonatal sepsis in hospitalized neonates has changed significantly with increase in CONS and Acinetobacter. There is a need to study colonization pattern of maternal genital tract and neonatal gut and hospital environment to know the source of these infections. With high degree of resistance to commonly used antibiotics, there is an urgent need to review the use of empirical antibiotics currently recommended by the WHO for the management of neonatal sepsis. Low-cost bundled interventions for improved infection control are therefore urgently required.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.