ABSTRACT Context: The study helps to make antibiotic policy in neonatal septicemia.ObjectiveThe present study was undertaken to investigate the high incidence of multidrug resistant β lactamases producing Gram-negative bacilli causing neonatal septicemia along with their antimicrobial sensitivity pattern Design & SettingThe eighteen months prospective study was conducted from June 2011 to Dec 2012. The Blood samples from the suspected neonatal septicemias were processed in the Department of Microbiology SGRDIMSR Amritsar. After identification and antibiotic susceptibility testing, beta-lactamases were detected as per CLSI guidelines.ResultsIn 235 blood cultures 37.87% (89/235) positivity was seen. Out of them, 55.05% were Gram positive and 43.82% were Gram negative isolates while Candida was 1.12%.Among S. aureus 62.0% were MRSA while 37.93% were MSSA. CONS were 40.8%. Klebsiella (25.64%) was predominant gram negative isolate followed by Enterobacter (20.51%), Acinetobacter (17.94%), Pseudomonas (15.38%), E coli (12.8%) and Citrobacter (7.69%).69.23% were lactamases producer gram negative isolates. Maximum 80.0% was in Klebsiella followed by 71.42% Acinetobacter, 66.67% in Citrobacter and Pseudomonas spp 62.5% Enterobacter, and 60.0% in E coli. ESBL producers were maximum 45.74% followed by co-producers of MBL + AmpC 18.5%,ESBL+AmpC 14.8%, ESBL+MBL11.12%,AmpC and MBL 7.4%.ConclusionsThe varying microbiological pattern of neonatal septicaemia warrants the need for periodic review of neonatal sepsis as the knowledge of the pathogens and their antibiotic susceptibility would be a useful guide in the antibiotic therapy  This will also facilitate infection control interventions like hand hygiene, patient isolation and contact precautions.

Keywords: MDR , ESBL, AmpC, MBL, GNB.

 Introduction

World Health Organization (WHO) estimates about 5 million neonatal deaths in a year and 98% are in developing countries1Medical achievements of the last twenty years have increased the survival rate of neonates but these babies with low immunity, always need prolonged hospitalization which is a factor contributing to the high risk of post-infectious complications.2 The control over nosocomial infections has been a formidable challenge for a long period of time.3 Most common bacterial organisms responsible neonatal septicemias in developed countries includes Coagulase-negative staphylococcus and group B Streptococcus while in developing countries like Pakistan, India, Nigeria, Bangladesh, are E coli, Klebsiella,  Enterobacter etc.4 Multiresistant bacteria causing neonatal sepsis in developing countries are increasing and such multiresistance in Gram-negative bacteria may be associated with the production of extended-spectrum beta-lactamase (ESBL). Klebsiella and Enterobacter species are often reported in this context.5 There is not enough information from the Indian subcontinent regarding the prevalence of β-lactamases mediated resistance among gram negative bacteria in Neonatal septiceamis and hence the aim of the present study is to find the prevalence of β-lactamases mediated resistance which helps to formulate a policy of empirical therapy. Spectrum of multiresistant organisms causing neonatal sepsis changes over time and across geographical boundaries, thus periodic bacteriologic surveillance is a necessity to reduce neonatal sepsis.

Material and methods

A prospective study was conducted over a period of eighteen months (1st June 2011to 31st Dec 2012) at the Department of Microbiology, Sri Guru Ram Das Institute of Medical Sciences & Research Vallah, Amritsar. Blood samples were collected from 235 suspected cases of neonatal septicemia from NICU and processed by standard techniques. The organisms grown in pure culture were identified by standard microbiological techniques.6 Antimicrobial susceptibilities were detected by Kirby baur standard disk diffusion (SDD) method using various antimicrobial agents as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Quality control was achieved by using standard strain of Escherichia coli ATCC 25922.7 Gram-negative isolates found resistant to third-generation cephalosporin were further tested for beta lactamases production.  Each strain was screened for ESBL production against cefotaxime, ceftazidime & cefpodoxime. ESBL production in these strains was confirmed by using ceftazidime and ceftazidime-clav disc as per CLSI guidelines.8 Amp-C β-lactamases producers were screened by using reduced cefoxitin susceptibility and production was confirmed by modified three dimensional test. An indentation or a flattening of the zone of inhibition indicated AmpC production 8 Organisms showed resistance to imepenm and meropenem screened as Metallo- β- lactamase producers and Metallo- β- lactamase production was detected in these isolates by Meropenem – EDTA & Imipenam – EDTA disc potentiating test. Organism was considered MBL producer if the increase in inhibition zone with EDTA disk was ≥ 5 mm. 9  

Results

A total of 235 neonates with neonatal sepsis were included in the study. Blood samples were taken from all cases and 37.87% (89/235) positivity was seen. The predominant organisms responsible for septicemia were S.aureus, Klebsiella Enterobacter, Pseudomonas and Acinetobacter spp. Out of all 89 isolates 49 (55.05% ) were Gram positive and 39 ( 43.82%) were Gram negative isolates while Candida was 1 (1.12%) as shown in Fig 1.

Fig 2 shows the distribution of organisms grown on blood culture. It can be seen that the Klebsiella 10 (25.64%) was predominant gram negative isolate followed by Enterobacter 8 (20.51%), Acinetobacter 7 (17.94%), Pseudomonas 6 (15.38%), E coli 5 (12.8%) and Citrobacter 3 (7.69%).

Coagulase positive staphylococci were 59.13 % while Coagulase negative staphylococci were 40.81 % as shown in Fig 3. Among Coagulase positive staphylococci 18 (62.0%) were methicillin resistant (MRSA) and (37.93%) were methicillin sensitive (MSSA) as shown in Fig 4.

27 (69.23%) prevalence of various lactamases was seen in gram negative isolates. Maximum 80.0% prevalence was found in Klebsiella spp. followed by 71.42% in Acinetobacter, 66.67% in Citrobacter and Pseudomonas spp 62.5% in Enterobacter, and 60.0% in E coli as shown in Fig 5. ESBL producers were maximum 45.74% followed by co-producers of MBL+AmpC 18.5%, ESBL+AmpC 14.8%, ESBL+MBL11.12%, AmpC and MBL 7.4% .

Low sensitivity of third generation cephalosporin -Cefotaxime 11-25%, Ceftriaxone 10-18%, Cefpodoxime 13-40% was found due to high prevalence of beta-lactamases in gram negative organisms. The sensitivity of amikacin and levofloxacin showed sensitive trend with most of the organisms. Imepenem showed maximum sensitivity against various gram negative organisms. Third generation with inhibitor ie Pipra + tazobactam and Ceftazidime+clavunate also showed good sensitivity 52-76.92% and 44.82 %-67.30 % respectively as shown in Table 1. In case of Gram positive Vancomycin, Erythromycin, Linezolid, Tiecoplanin were found most effective as shown in Table 2.

Discussion

Neonatal septicaemia remains as the major cause of mortality and morbidity in spite of recent advances in the technology world wide. Neonatal sepsis is one of the most common factors contributed for admission to NICU in developing countries and is defined as invasive infection usually bacterial and documented by a positive blood culture. There has been a wide variation in the growth positivity obtained by blood culture over the years.10 In our study, blood culture positivity was 37.87% and predominant isolated organisms were Klebsiella spp and S.aureus which is similar to the report from NNPD 2000. 11This revealed Klebsiella spp and S.aureus the most frequent neonatal sepsis causative organism in India.

We obtained maximum 10 (25.64%) isolates of klebsiella spp among gram negative isolates and  most sensitive drugs were Imipenem (65.62%), Pipra+tazobactam (56.25%), Caftazidime+clavunate (53.12%), Levofloxacin(40.62%) and Amikacin (34.36%). With the emergence of multiresistant beta –lactamases  producing gram negative bacilli as the predominant pathogen in Neonatal sepsis 12 the third generation cephalosporins, which have been used extensively as a life saving first-line antibiotic are rendered useless.13 Present study suggests the use of higher generation cephalosoporin with beta lactamases inhibitor combination for empirical therapy.

We found 18 (62.0 %) MRSA among S aureus and sensitive drugs were Tiecoplanin (82.35%), Vancomycin (79.41 %), Linezolid (73.52 %), Amikacin (67.64%) and  Pipra + tazobactam (58.82%).

We found 69.23% prevalence of beta lactamases in gram negative organism which is similar to other studies14 but also contrary to some studies15 due to geographical variations. Failure to detect lactamses enzymes like Extended Spectrum ß-lactamases (ESBLs), AmpC ß-lactamases, Metallo ß-lactamases and their combination in same organism has contributed to therapeutic failures.16 In our region especially in Amritsar, ESBLs production is not routinely tested in most centers resulting the dissemination of ESBL-producing strains within and between hospitals and is  remain undetected for long periods. Similar to study conducted by Jain A and Mondal R.17 we found high 80% of lactamases production in Klebsiella  spp which may be due to inappropriate antibacterial use. Therefore, preventive antibiotics should be used as little as possible, while therapeutic antibiotics should be specific and used as short period of time as possible. In conditions wherein the use of antibiotics is necessary, rotating antibiotic regimens is suggested.

We found 40.81% of CONS infection which can be can be reduced by proper skin treatment before collection of blood samples, reducing the duration of invasive lines and proper hand hygiene, barrier nursing and environment monitoring. The combined approaches of rotational antibiotic therapy and educational programs may be beneficial in fighting against such types of infections. Considering antibiotic resistance in India, it is necessary to determine the prevalence of β-lactamases producers in a hospital so as to formulate a policy of empirical therapy in NICU.

Conclusion

The varying microbiological pattern of neonatal septicaemia warrants the need for periodic review of neonatal sepsis as the knowledge of the pathogens and their antibiotic susceptibility would be a useful guide in the antibiotic therapy  This will also facilitate infection control interventions like hand hygiene, patient isolation and contact precautions

Acknowledgments

We are thankful to the clinical department, SGRD Charitable Hospital for their cooperation in study.

References

  1. WHO. Perinatal mortality. Report No.: WHO/FRH/MSM/967. Geneva: WHO, 1996.
  2. Adams-Chapman I, Stoll BJ. Prevention of nosocomial infections in the neonatal intensive care unit. Curr Opin Pediatr, 2002; 14:157-64
  3. Goldmann DA, Freeman J, DurbinWA. Nosocomial infection and death in a neonatal intensive care unit. J Infect Dis1983; 147:635-41.
  4. Waheed M, Laeeq A, Maqbool S. The etiology of neonatal sepsis and patterns of antibiotic resistance. J Coll Physicians Surg Pak 2003; 13: 449-52.
  5.  Karunasekera KA, Pathirana D. A preliminary study on neonatal septicaemia in a tertiary referral hospital paediatric unit. Ceylon Med J 1999; 44:81–6.
  6. Collee JG, Miles RS, Watt B. Test for the identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie & MacCartney practical medical microbiology, 14th ed. Edinburgh: Churchill Livingstone; 1996.p.151-79.
  7. National Committee for Clinical Laboratory Standards (2000). Performance standards for antimicrobial susceptibility testing-Ninth information supplement: M100-S10. NCCLS.Wayne, A, USA.
  8.  Shahid S, Malik A, Agrawal M, Singhal S, The phenotypic detection of the extended spectrum and the AmpC β lactamases by a new spot inoculation method and a modified three dimensional extract test J AntimicrobialChemother  2004; 54:684-87.
  9.  Pitout JD, Gregson DB, Poirel L, McClure JA, Le P, Church DL, The detection of Pseudomonas aeruginosa which produced metallo- β lactamases in a large centralized laboratory J Clin Microbiol  2005; 43:3129-35.
  10. Mondal GP, Raghavan M, Bhat BV and Srinivasan S. Neonatal septicaemia among inborn and outborn babies in a referral hospital, Indian J Pediatr 1991;58:529-533
  11. Report of the National Neonatal Perinatal Database (National Neonatology Forum) 2000
    1. Jain A, Roy I, Gupta MK, Kumar M, Agarwal SK. Prevalence of extended-spectrum beta-lactamase-producing Gram-negative bacteria in septicaemic neonates in a tertiary care hospital. J Med Microbiol. 2003 May; 52(Pt5):421-5.
    2. Krishna BV, Patil AB, Chandrasekhar MR. Extended-spectrum beta-Lactamase producing Klebsiella pneumoniae in neonatal intensive care unit. Indian J Pediatr. 2007 Jul; 74(7):627-30.
    3. Sehgal R, Gaind R, Chellani H, Agarwal P. Extended-spectrum beta lactamase-producing gram-negative bacteria: clinical profile and outcome in a neonatal intensive care unit. Ann Trop Paediatr. 2007 Mar; 27(l):45-54.
    4. Kumar CS, Neelagund YF. Extended-spectrum of P-lactamase mediated resistance to third generation cephalosporins among klebsiella pneumoniae in neonatal septicemia. Indian Pediatr.2004 Jan; 41(l):97-9.
    5. Rodrigues C, Johi P, Jani SH, Alphonse M, Radhakrishnan R, Meheta A. Detection of ß-lactamases in nosocomial Gramnegative clinical isolates. Indian J Med Microbiol 2004; 22 (4): 247-250.
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TABLES LEGENDS

Table 1. SENSITIVITY AND RESISTANCE PERCENTAGE OF GRAM NEGATIVE ORGANISMS TO ANTIBIOTICS

Table 2. SENSITIVITY AND RESISTANCE PERCENTAGE OF GRAM POSITIVE ORGANISMS TO ANTIBIOTICS

FIGURES LEGENDS

Fig. 1. DISTRIBUTION OF CLINICAL ISOLATES

Fig. 2. DISTRIBUTION OF VARIOUS GRAM NEGATIVE ISOLATES

Fig. 3 DISTRIBUTION OF COAGULASE POSITIVE AND NEGATIVE STAPHYLOCOCCUS

Fig. 4. DISTRIBUTION OF METHICILLIN RESISTANT AND SENSITIVE STAPHYLOCOCCUS

Fig. 5. DISTRIBUTION OF BETA LACTAMASES PREVELANCE IN VARIOUS GRAM NEGATIVE  ISOLATES

Table 1

Antibiotic

E coli

Klebsiella

Pseudomonas

Citrobacter

Acinetobacter

Enterobacter

  S %

R%

S%

R%

S%

R%

S%

R%

S%

R%

S%

R%

Gentamicin 57.69 42.31 31.25 68.75 37.5 62.5 24.13 75.87 33.34 66.66 14.81 85.19
Amikacin 67.30 32.7 34.37 65.63 46.87 53.13 20.68 79.32 30.95 69.05 25.92 74.08
Ciprofloxacin 48.07 51.93 18.75 81.25 28.12 71.88 13.79 86.21 19.04 80.96 14.81 85.19
Levofloxacin 59.61 40.39 40.62 59.38 53.12 46.88 37.93 62.07 50.0 50.0 51.85 48.15
Netilmicin 51.92 48.08 28.12 71.88 43.75 56.25 17.24 82.76 30.80 69.20 25.92 74.08
Cefotaxime 15.38 84.62 21.87 78.13 25.0 75.0 13.79 86.21 16.67 83.33 11.12 88.88
Ceftraixone 17.30 82.70 15.62 84.38 15.62 84.38 10.34 89.66 14.28 85.72 14.23 85.77
Cefpodoxime 36.53 63.47 28.12 71.88 40.6 59.40 13.79 86.21 21.42 78.58 25.92 74.08
Cefoxitin 32.0 68.0 31.25 68.75 28.12 71.88 20.68 79.32 19.04 80.96 33.34 66.66
Imepenem 80.39 19.61 65.62 34.38 78.12 21.88 68.96 31.04 64.28 35.72 68.45 31.55
Pipra +Tazo 76.92 23.08 56.25 43.75 56.25 43.75 72.41 27.59 57.14 42.86 52.3 47.7
Cefta +Clav 67.30 32.70 53.12 46.88 53.12 46.88 44.82 55.8 50.0 50.0 46.87 53.13

 

Table 2

Antibiotic

S aureus

CONS

 

S %

R %

S %

R %
Cefotaxime

38.23

61.77

66.67

33.33
Gentamicin

51.54

48.46

73.34

26.66
Amikacin

67.64

32.36

92.85

7.15
Cefoxitin

29.41

70.59

66.67

33.33
Vancomycin

79.41

20.59

100.0

00
Erythromycin

58.83

41.17

86.67

13.33
Oxacillin

27.29

72.71

80.0

20.0
Clindamycin

54.23

45.77

86.67

13.33
Pipra+tazo

58.82

41.18

80.0

20
Linezolid

73.52

26.48

82.4

17.6
Tiecoplanin

82.35

17.65

100.0

00.0

Fig. 1

Fig. 2.

Fig. 3

Fig. 4

Fig. 5.

Microbiological Surveillance of Operation Theatre : Why… What…How …Where…Which…?” S. Poongodi @lakshmi, N. Palaniappan, M.Kannan, S.Nithya gomatheeswari
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