|Year : 2010 | Volume
| Issue : 4 | Page : 380-384
Nosocomial outbreak of septicaemia in neonatal intensive care unit due to extended spectrum β-lactamase producing Klebsiella pneumoniae showing multiple mechanisms of drug resistance
V Rastogi1, PS Nirwan1, S Jain2, A Kapil3
1 Department of Microbiology, J.L.N. M.C & Asso. Hospitals, Ajmer - 305 001, India
2 Department of Paediatrics, J.L.N. M.C & Asso. Hospitals, Ajmer - 305 001, India
3 Department of Microbiology, AIIMS, New Delhi - 110 029, India
|Date of Submission||17-May-2010|
|Date of Acceptance||10-Aug-2010|
|Date of Web Publication||20-Oct-2010|
Department of Microbiology, J.L.N. M.C & Asso. Hospitals, Ajmer - 305 001
A total of 14 phenotypically similar clinical isolates of Klebsiella pneumoniae, resistant to multiple drugs including cefotaxime and ceftazidime, were isolated from blood of neonates admitted to neonatal intensive care unit (NICU) within a short span of 10 days. Alarmed at the possibility of occurrence of outbreak, a thorough investigation was done. Microbiological sampling of the NICU and labour room (LR) environment yielded 12 K. pneumoniae isolates. The presence of extended spectrum β-lactamase (ESBL) in the clinical and environmental strains was detected by double-disk synergy test (DDST), CLSI phenotypic confirmatory disk diffusion test (PCDDT) and E-test ESBL strips. Amp-C screen (disk) test was done to determine Amp-C β-lactamase production. 100% clinical strains, 57% NICU strains and 80% LR strains were ESBL positive. 57% clinical, 43% NICU and 20% LR strains were Amp-C screen positive. Polymerase chain reaction (PCR) of representative ESBL positive (10 clinical and 5 environmental) strains showed CTX gene and TEM and/or SHV gene in all. K. pneumoniae showing multiple mechanisms of drug resistance was responsible for the outbreak.
Keywords: Extended spectrum β-lactamase, Klebsiella pneumoniae, neonatal septicaemia, outbreak
|How to cite this article:|
Rastogi V, Nirwan P S, Jain S, Kapil A. Nosocomial outbreak of septicaemia in neonatal intensive care unit due to extended spectrum β-lactamase producing Klebsiella pneumoniae showing multiple mechanisms of drug resistance. Indian J Med Microbiol 2010;28:380-4
|How to cite this URL:|
Rastogi V, Nirwan P S, Jain S, Kapil A. Nosocomial outbreak of septicaemia in neonatal intensive care unit due to extended spectrum β-lactamase producing Klebsiella pneumoniae showing multiple mechanisms of drug resistance. Indian J Med Microbiol [serial online] 2010 [cited 2014 Sep 18];28:380-4. Available from: http://www.ijmm.org/text.asp?2010/28/4/380/71834
| ~ Introduction|| |
Neonatal septicaemia is difficult to diagnose clinically as it presents with non-specific and usually subtle signs and symptoms. Nevertheless, septicaemia remains a significant cause of morbidity and mortality in the newborns, more so in the developing countries. Extended spectrum β-lactamase (ESBL) producing strains of Klebsiella pneumoniae (K. pneumoniae) have emerged as important agents in the list of nosocomially acquired neonatal pathogens.  Plasmid mediated resistance to the third generation cephalosporins and the ease by which resistant plasmids are able to transfer from one genus to another further complicates the control of these resistant organisms and is the major cause of various outbreaks of nosocomial infections.  We report here the characteristics of a nosocomial outbreak of septicaemia in the neonatal intensive care unit (NICU) of our hospital.
| ~ Patients and Methods|| |
In August 2005, within a period of 1 week, six strains of phenotypically similar K. pneumoniae subspecies aerogenes were isolated from the blood of neonates admitted in the NICU of our hospital. This clustering prompted us to evaluate the emergence of an outbreak in the NICU. Diagnosis of neonatal septicaemia was based on clinical features and a positive blood culture.  By the time of suspicion of an outbreak, we had already isolated 14 similar strains of K. pneumoniae from 14 neonates. To identify the source of outbreak, a microbiological survey of NICU and labour room (LR) environment was done. A total of 23 samples were collected from NICU including linen, humidifier, incubator, cradle, refrigerator, feeding trolley, water tap, suction fluid, umbilical cord stump of neonates and hands of health care workers (HCW). Thirteen samples were collected from LR including linen, antiseptic solution in which scissor (for cord cutting) was kept, water tap, suction tube, steriliser and hands of HCW. All the 36 environmental samples were collected in trypticase soy Broth (TSB) and subcultured after 18 hours incubation onto MacConkey agar and Blood agar. The organism grown was identified up to subspecies level as per standard protocol. Only strains of K. pneumoniae subspecies aerogenes isolated from environmental sources were followed further, as all clinical isolates were also K. pneumoniae subspecies aerogenes.
Thus, a total of 26 (14 neonatal + 7 environmental from NICU + 5 environmental from LR) strains were subjected to antibiotic susceptibility testing with ampicillin (A), amikacin (Ak), amoxyclav (Ac), ciprofloxacin (Cf), ofloxacin (Of), gentamicn (G), netilmicin (Nt), ceftazidime (Ca), cefotaxime (Ce), ceftriaxone (Ci), cefoperazone (Cs), cefoxitin (Cn), Imipenem (I) (Himedia labs, Mumbai, India) and cefoperazone-sulbactam (Cfs, Pfizer India Ltd, Jaipur, India.) by modified Kirby-Bauer disk diffusion technique as per CLSI criteria. 
All 26 isolates were also tested for ESBL production by modified double-disk synergy test (DDST), phenotypic confirmatory disk diffusion test (PCDDT) and E-test Ce-Cec ESBL strips (AB biodisk, Solna, Sweden). In addition, minimum inhibitory concentration (MIC) for ceffazidime was determined using E-test Ca MIC Strip (Hi-Media labs). Amp-C disk test was also done to screen for Amp-C β-lactamase production. Multiplex polymerase chain reaction (PCR) for detection of TEM, SHV and CTX genes was carried out on representative strains (n = 15) from neonates (n = 10) and environment (n = 5; 3 from NICU, 2 from LR) at Department of Microbiology, AIIMS, New Delhi.
| ~ Results|| |
During the outbreak, 14 out of 23 (60.9%) blood cultures of neonates received were positive for K. pneumoniae subspecies aerogenes. Preterm deliveries(70%, ≤37 weeks) with low birth weight (70%, ≤2500 g) of the neonates was the most important predisposing factor observed. Others were low Apgar score (50%), birth asphyxia (35%), prolonged rupture of membranes (21%) and twin pregnancy (7%). The main presenting symptom was respiratory distress associated with diminished spontaneous activity and feeding intolerance. Disseminated intravascular coagulation (DIC) was seen in three, whereas jaundice and peritonitis was seen in one neonate each as complication. Empiric treatment of the first six neonates included a combination of cefotaxime and amikacin. With this, condition of four neonates worsened and they succumbed to the infection. Subsequent to culture and sensitivity, the therapy was switched over to imipenem. Ciprofloxacin in combination with amikacin or cefoperazone-sulbactam was given in two cases (due to non-affordability and/or poor compliance) based on antibiogram. With this, 6 out of 10 (60%) neonates could be saved. The mortality rate was 57% (8 out of 14).
All the 14 clinical strains of K. pneumoniae tested positive for ESBL production by PCDDT. The results of E-test and DDST are shown in [Table 1]. Phantom zones in E-test were seen in 60% of the strains, signifying ESBL production. All strains were cefoxitin resistant (100%) and eight strains (57%) were Amp-C β-lactamase screen positive. All strains were uniformly susceptible to imipenem. Resistance to multiple antibiotics was seen, viz., Ak (78.5%), Cf (28.5%), Of (28.5%), Cfs (42.8%). 100% resistance was seen with rest of the antibiotics tested. MIC for ceftazidime was >120 μg/ml in all the strains.
|Table 1: Characteristic features of the representative clinical and environmental strains of the outbreak |
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K. pneumoniae subspecies aerogenes, similar to the clinical strains of the outbreak, was isolated from the disinfectant solution, suction fluid, water tap and hand of HCW of the LR as also from humidifier, water tap, incubator and hands of HCW at NICU. However, only the strains isolated from the disinfectant solution of LR, humidifier, tap and hands of HCW at NICU were found to be ESBL and /or Amp-C β-lactamase producers, showing an antibiogram similar to that of the outbreak strain. MIC for ceftazidime in these strains also was >120 μg/ml. All the clinical strains and the representative strains from suspected environmental sources were found to possess TEM and /or SHV gene [Figure 1].and universal presence of CTX gene [Figure 2].
|Figure 1: TEM-SHV PCR results of samples. Fifteen strains of K. pneumoniae were tested. Lanes 2 and 3: strains positive for SHV only; lanes 4, 5, 7-16: strains were positive for both TEM and SHV; lane 6: strain positive for TEM only; lane 17: K. pneumoniae positive control (SHV); lane 1: negative control (with no template added)|
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|Figure 2: PCR results of CTX gene. Lane M: 100 bp molecular weight marker; lanes 1-9: K. pneumoniae clinical isolates; lane 10: negative control (no template DNA added)|
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| ~ Discussion|| |
K. pneumoniae is an important hospital acquired pathogen with the potential of causing severe morbidity and mortality in paediatric patients.  Several outbreaks of infection caused by multidrug resistant K. pneumoniae have been widely reported.  The information regarding the probable source of ESBL positive K. pneumoniae in a patient is important for infection control, especially in an intensive care setting as NICU. This organism colonises the bowel and skin and is probably transmitted via hands of medical personnel. ,
Our study reveals the humidifier solution, water tap, hands of HCW at NICU and the disinfectant solution (in which umbilical cord cutting scissors was kept) from LR as the probable sources of the outbreak strain. The most probable source of the outbreak, however, seems to be the disinfectant solution. Once the cord was cut and the neonate transferred to NICU, the strain became established there and spread through environmental dissemination and via hands of HCW (in the absence of stringent infection control practices) aided by its virulence and transfer of resistance plasmids. Similar observations in neonatal infections due to Klebsiella in the nursery have been reported in 1993. 
Neonatal septicaemia due to ESBL producing K. pneumoniae was reported to produce 25% mortality.  Eighty percent mortality was reported during an ESBL outbreak in NICU of JIPMER, Pondicherry.  In our case, the mortality rate was 57%. Prompt intimation of culture and sensitivity results to the treating neonatologist influenced subsequent treatment regime with imipenem. This resulted in saving 6 (42.8%) of the neonates. However, the mortality of two neonates could have been averted if imipenem were timely instituted. Our hospital does not provide imipenem as a part of hospital supply. We have, however, recommended the same to the authorities for future use. Carbapenems are important antibiotics for treatment of nosocomial infections  and all the outbreak strains were uniformly susceptible to this drug. (However, in recent times, since 2009, we have been witnessing emergence of imipenem resistant strains in our setting, though only rarely, in Klebsiella.) Hence, carbapenem is the drug of choice for ESBL and/or Amp-C producing K. pneumoniae in our setting.
All the 14 clinical strains (100%) were ESBL positive by PCDDT and 13 (92.8%) were positive in E-test ESBL strip method [Table 1]. However, only 11 strains (78.5%) were ESBL positive by modified DDST method. Thus, PCDDT is seen to be a highly sensitive, specific and feasible method for ESBL detection in a routine lab, as has been confirmed by the CLSI.  One strain [C 4 in [Table 1] was ESBL negative by DDST and non determinable in E-test ESBL test. However, this strain was Amp-C positive, cefoxitin resistant and showed the presence of CTX, TEM and SHV0 genes in PCR. This strain probably elaborates inhibitor resistant TEM β-lactamases (IRTs) and Amp-C β lactamases in addition to ESBLs (which was not detectable probably due to loss of outer membrane proteins). Amp-C β-lactamase production was seen in 8 (57%) strains although this needs to be confirmed further. Since all strains were cefoxitin resistant, the outbreak strain seems to possess multiple mechanisms of drug resistance,  including loss of outer membrane porins (OMPs). Hence, it is difficult to comment whether cefoxitin resistance was due to Amp-C production or loss of OMPs.
ESBL and Amp-C production in the strains of the present outbreak was 100% and 57% (clinical strains), 57% and 43% (environmental strains from NICU) and 80% and 20% (environmental strains from LR), respectively. Out of the 26 strains of K. pneumoniae characterised, 22 (84.6%) were cefotaxime resistant, whereas only 17 (65.3%) were ceftazidime resistant. The molecular analysis also showed the universal presence of CTX gene [Figure 2] in the 15 representative strains tested by PCR, explaining the higher degree of cefotaxime resistance due to the presence of cefotaximases that preferentially hydrolyse cefotaxime. Similar findings were reported by Kumar et al. from Hyderabad.  However, further studies are warranted to establish the presence of cefotaximases and to know their types.
Hospital colonisation by ESBL producing bacteria is usually a complex phenomenon involving different mechanisms including dissemination of several epidemic strains, plasmids and resistant genes.  This was truly evidenced in this outbreak as ESBL producing K. pneumoniae present in our setting were found to produce multiple β-lactamases. Similar findings were reported by Rodrigues et al. from Mumbai. 
The following measures were taken, which helped in successfully controlling the outbreak:
- switch over of therapy to imipenem based on antibiogram results;
- isolation and cohorting of neonates having ESBL and/or Amp-C positive K. pneumoniae infection;
- shifting of NICU to new prefumigated room and fumigation of the existing NICU in an attempt to ward off K. pneumoniae from all possible environmental sources;
- use of common scissors for cord cutting was abandoned and disposable surgical blade was arranged for every delivery;
- stringent infection control measures were reinforced and the NICU staff was counselled for the same (this, though the simplest, was nevertheless the most effective);
- mobilisation of adequate NICU staff and resources for care of neonates; and
- review of hospital antibiotic policy and avoiding irrational overuse of cephalosporins and other β-lactams.
The last two measures were the most difficult task to accomplish and maintain. I am sure the medical fraternity unanimously agrees to that!
Klebsiella strains producing ESBLs, Amp-c β-lactamases and other β-lactamases, possibly IRTs, are ubiquitously present in our hospital environment. This can lead to serious septicaemic outbreaks in the neonates and possibly other patients with predisposing factors. Infection control measures need to be strictly strengthened in our setting lest we witness still more devastating outbreaks. A continued and dedicated multidisciplinary effort is therefore inexpendable.
| ~ Acknowledgement|| |
We are thankful to Dr. Prabha Lal of AIIMS, New Delhi, for carrying out molecular typing of the strains.
| ~ References|| |
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[Figure 1], [Figure 2]
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