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 ~  Abstract
 ~  Subjects and Methods
 ~  Results
 ~  Discussion
 ~  References

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ORIGINAL ARTICLE
Year : 2003  |  Volume : 21  |  Issue : 2  |  Page : 82-86
 

Nosocomial klebsiella infection in neonates in a tertiary care hospital: Protein profile by SDS-page and klebocin typing as epidemiological markers


Departments of Microbiology, JN Medical College, Aligarh Muslim University, Aligarh - 202002, UP, India

Correspondence Address:
Departments of Microbiology, JN Medical College, Aligarh Muslim University, Aligarh - 202002, UP, India

 ~ Abstract 

PURPOSE: To find out the prevalence of Klebsiella in hospital acquired neonatal infections in a tertiary care set up and to evaluate the role of klebocin typing and protein profile by SDS-PAGE in epidemiological typing of the isolates. METHODS: Hospital born neonates transferred to the neonatal unit after birth and available in the unit 48 hours later comprised the study group. Two hundred and three neonates were found eligible for inclusion in the study. Repeated blood cultures, other relevant clinical specimens and environmental samples were collected and identified according to the standard techniques. Isolated clinical and environmental Klebsiella pneumoniae strains were subjected to klebocin typing and protein profiling by SDS-PAGE at regular intervals. RESULTS: Multi drug resistant K. pneumoniae were the commonest organism isolated in 30 neonates leading to the incidence of Klebsiella nosocomial infection to be 14.7%. Klebocin typing of the K. pneumoniae isolates showed four patterns with type 312 being the commonest (43.4%). Whole cell protein analysis by SDS-PAGE of K. pneumoniae isolates revealed four types of banding pattern. Analysis of the typing method showed that the typeability and reproducibility of klebocin was 83.3% and 73.3% respectively whereas typeability and reproducibility of SDS-PAGE was 100%. CONCLUSIONS: Based on the present study it is concluded that SDS-PAGE typing method is better than klebocin typing in neonatal nosocomial infection. It is also suggested that protein profile by SDS-PAGE may be used as a tool for epidemiological typing of Klebsiella pneumoniae isolates in laboratories where genomic based molecular typing technique is not available.

How to cite this article:
Malik A, Hasani S E, Shahid M, Khan H M, Ahmad A J. Nosocomial klebsiella infection in neonates in a tertiary care hospital: Protein profile by SDS-page and klebocin typing as epidemiological markers. Indian J Med Microbiol 2003;21:82-6


How to cite this URL:
Malik A, Hasani S E, Shahid M, Khan H M, Ahmad A J. Nosocomial klebsiella infection in neonates in a tertiary care hospital: Protein profile by SDS-page and klebocin typing as epidemiological markers. Indian J Med Microbiol [serial online] 2003 [cited 2019 Jan 22];21:82-6. Available from: http://www.ijmm.org/text.asp?2003/21/2/82/7980


Neonatal nosocomial infections are an important cause of neonatal morbidity and mortality, however its reporting in India has been non-uniform.[1] This may be due to the lack of surveillance work and scanty studies on this aspect. The reported incidence of nosocomial sepsis in India ranges from 1.5-37%. Although any pathogen may be acquired by the neonates in the hospital, Klebsiella is lately emerging as an important cause of neonatal nosocomial infection.[2] Epidemiological typing is useful in determining the extent of an outbreak and in elucidating the sources and spread of infection.[3] Various methods including protein profile by SDS-PAGE and klebocin have been documented for epidemiological typing of Klebsiella isolates but their results have not been consistent, therefore the present study was planned to study the incidence of Klebsiella nosocomial neonatal infection in our hospital and also to find out the role of klebocin typing and protein profile in epidemiological typing of K. pneumoniae strains.

 ~ Subjects and Methods Top

The present study was carried out in the department of microbiology, and neonatal unit of department of paediatrics, JNMCH, Aligarh, during a period of one year. Hospital born neonates transferred to the neonatal unit after birth and available in the unit for atleast 48 hours comprised the study group. Nosocomial infection was considered to be present if onset of infection was found after 48 hours of birth. Neonates presenting with infection beyond 48 hours and under 7 days of life but with maternal predisposing factors such as premature rupture of membranes, maternal fever and foul vaginal discharge were not considered to be nosocomial in origin.
Repeated Blood cultures and various other relevant clinical specimens including CSF, pus, umbilical swab, conjunctival swab and tracheal aspirate were collected from the neonates in whom infection was suspected. Environmental samples such as air samples, swabs from sinks, incubators, resuscitation trolley, endotracheal suction tubes, catheter tips, ambu bags etc. were also collected from the neonatal unit. Culture and identification of the isolates was done as per standard methods.[4] Antimicrobial susceptibility of the isolates was performed according to the method of Bauer et al[5] and all the isolates were stocked and later subjected to klebocin typing[6] and SDS-PAGE.[7] Klebocin typing and SDS PAGE was performed thrice at intervals of 1 month. Klebocin typing strains were kindly provided by Govt. Medical College, Rohtak, Haryana. For SDS-PAGE, 8% acrylamide gel was used and protein extraction of the K. pneumoniae isolates was done according to the method of Alavandi et al.[8] The stained gels were examined for presence or absence of bands. Dice index of similarity was determined with each group.
Statistical analysis
Dice index of similarity: Dice index similarity coefficient[9] was calculated between the groups formed by the banding pattern in SDS-PAGE by using the formula: S (%)=2X x 100/ a+b where a is the total number of bands in lane 1, b is the total number of bands in lane 2 and x is the total number of similar band in both the lanes. Dendrogram was made by single linkage method (Jaccord's method) using the SPSS software (SPSS Inc., USA).
Typeability: The typeability[10] was determined by using the formula: T = Nt / N where Nt is the number of isolates assigned a type and N is the number of isolates tested.
Reproducibility: The reproducibility[10] was determined by using the formula: R = Nr / N, where Nr is the number of isolates assigned the same type on repeat testing and N is the number of isolates tested.

 ~ Results Top

During the study period of one year, 203 neonates were found eligible for inclusion in the study. Epidemiological features of the study group along with various risk factors are shown in [Table - 1]. Out of 203 neonates, 30 (14.7%) developed Klebsiella nosocomial infection, 19 being males and 11 females.
All the isolates were identified as Klebsiella pneumoniae. Out of the 30 isolates of K. pneumoniae, 25 (83.7%) were isolated from cases of septicaemia, 2 (6.6%) each from cases of pneumonia and umbilical sepsis and 1 (3.3%) from a skin abscess. Among the environmental cultures, four K. pneumoniae isolates were from catheter tips while 2 isolates were from air samples. The pattern of drug resistance of the K. pneumoniae isolates showed that 96.7% of the isolates were multi drug resistant. The commonest resistance pattern was the resistance to tetracycline, gentamicin and ampicillin. None of the isolates showed resistance to cefotaxime or ceftazidime.
Klebocin typing of the K. pneumoniae isolates showed four patterns. Type 312 was the commonest (43.4%) followed by type 334 (23.4%), type 112 (16.6%) while remaining 16.6% strains could not be typed. K. pneumoniae isolates from environmental cultures also showed klebocin type 312 [Table - 2].
Whole cell protein analysis by SDS-PAGE of 29 isolates of K. pneumoniae divided them into four groups on the basis of protein banding pattern including number of bands and difference in molecular weights. The bands ranged from 12-17 major prominent bands. In the present study, out of 29 isolates, six isolates belonged to group I, 15 isolates belonged to group II, three and five isolates belonged to group III and IV respectively. The protein banding pattern in K. pneumoniae isolates from environmental culture showed that they belonged to group II. Dendrogram showing the cluster analysis by SPSS software program is shown in [Figure:1].

The cumulative results of the various typing methods used for K. pneumoniae were analysed. It was observed that six isolates obtained during one period and five isolates obtained during another period showed same pattern by klebocin typing and protein profile by SDS-PAGE, while antibiogram pattern revealed all of them to be multidrug resistant. Klebocin typing and protein profile by SDS-PAGE of the environmental isolates of K. pneumoniae also showed the same pattern (klebocin type 312 and SDS-PAGE group II) as was observed in patient isolates. It may be concluded that these isolates represent probably the same source of infection and probably a single strain.
All the klebocin type 312 strains were resistant to tetracycline, gentamicin and ampicillin, 85% of the klebocin type 334 strains were resistant to tetracycline and 71% to gentamicin, 100% of type 112 strains showed resistance to gentamicin and ampicillin and 80% resistance to tetracycline, 100% of untypeable strains were resistant to tetracycline, gentamicin and ampicillin.
All the SDS-PAGE group I strains were resistant to tetracycline and 83.3% were resistant to gentamicin and ampicillin. All the strains of group II were resistant to tetracycline, gentamicin and ampicillin. Analysis of the antibiogram pattern of the klebocin types and SDS-PAGE groups shows that type 312 and group II are probably similar while the untypable (klebocin) strains and SDS-PAGE group I were probably similar.
Analysis of the various typing methods showed that the typeability and reproducibility of klebocin typing was 83.3% and 73.3%, respectively. The typeability and reproducibility of SDS-PAGE method was found to be 100% [Table - 3].

 ~ Discussion Top

Nosocomial infections are major contributors to hospital associated morbidity and mortality, prolonged hospital stay and higher cost of treatment.[11] There is a wide variation in the rates of neonatal nosocomial infections that have been reported. In the present study, the incidence of Klebsiella nosocomial infection was 14.7% which is comparable with the rates reported by other workers.[12]
Klebsiella is a major agent of neonatal nosocomial infections. It continues to be a nightmare for neonatologists, microbiologists and hospital administrators.[13] In the present study, multiresistant K. pneumoniae species were the commonest organism isolated. The results of the klebocin typing and SDS-PAGE showed that the major group formed by these typing methods corresponded with the environmental K. pneumoniae isolates, thus confirming the environmental strains to be the source of nosocomial infection.
Cluster analysis also revealed that the major group formed by SDS-PAGE and the environmental K. pneumoniae strains were the same. Hierarchical cluster analysis showed that the majority of strains forming the group II, III and IV were closely related at phylogenetic tree whereas the strains in group I were completely unrelated to other groups and possibly, they were acquired separately and from a different source. For hospital epidemiology, typing techniques that define clonal relationship between individual isolates are warranted in order to recognise nosocomial transmission. The technique used must not only be sensitive enough to identify outbreak strains but also be sufficiently reproducible and have good discriminatory power.[14] In past, klebocin typing has been used in epidemiological investigations of K. pneumoniae isolates.[15] Recently, molecular methods including whole cell protein analysis (WCPA) by SDS-PAGE,[16] ribotyping and randomly amplified polymorphic DNA analysis (RAPD)[17] have been employed for differentiation of multi resistant Klebsiella isolates. The discriminatory power of WCPA, ribotyping and RAPD have been reported to be better than bacteriocin typing including the klebocin typing.[18] Though ribotyping and RAPD appear to be reliable methods for distinguishing K. pneumoniae strains they are expensive, time consuming and require skilled technical staff. In contrast, SDS-PAGE appears to be relatively economical and does not require skilled staff.
In the present study, we found the typeability and reproducibility of the klebocin typing to be low, and our results clearly indicate SDS-PAGE method for typing Klebsiella species by the protein profile as a suitable and convenient method. It has excellent reproducibility and the ability to type all isolates. Similar findings have also been reported by Costast et al.[16]
A bacterial strain, growing in standardised conditions always produces the same set of proteins. SDS-PAGE is currently one of the most commonly used techniques for the characterisation and analysis of proteins and it has been used as a taxonomic tool for identification of various bacterial species.[19] Therefore, we suggest that SDS-PAGE may be used as a tool for epidemiological typing of Klebsiella isolates in the laboratories where the facility to perform genomic based molecular typing is not available. 

 ~ References Top

1.Pawa AK, Ramji S, Prakash K, Thirupuram S. Neonatal nosocomial infection. Profile and risk factors. Indian paediatrics 1997;34:297-302.  Back to cited text no. 1    
2.Gupta P, Murali P, Murali MV, Faridi MMA, Kaul PB, Ramachandran VC, Talwar V. Clinical profile of Klebsiella septicaemia in neonates. Indian J Paediatr 1993;60:565-572.  Back to cited text no. 2    
3.Parker MT. Hospital acquired infections: Guidelines to laboratory methods, WHO Regional publication European series No. 4 P.35, 1978, WHO, Copenhagen.  Back to cited text no. 3    
4.Mackie and McCartney, Practical Medical Microbiology, Collee JG, Fraser AG, Marmion BP, Simmons A. Eds. 14th Ed. (Churchill Livingstone, London) 1996.  Back to cited text no. 4    
5.Bauer AW, Kirby WMM. 1966 Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-496.  Back to cited text no. 5    
6.Arora DR, Chugh TD. Klebocin types of Klebsiella pneumoniae isolated from normal and diarrhoeal stool. Indian J Med Res 1981;72:856.  Back to cited text no. 6    
7.Laemmli UK. Cleavage of structural proteins during assembly of head of bacteriophage T[4]. Nature 1970;227:680-685.  Back to cited text no. 7    
8.Alavandi SV, Ananthan S, Pramod NP. Typing of Aeromonas isolates from children with diarrhoea and water samples by randomly amplified polymorphic DNA, polymerase chain reaction and whole cell protein finger printing. Indian J Med Res 2001;113:85-97.   Back to cited text no. 8    
9.Dice LR. Measurement of the amount of ecological association between species. Ecology 1945;26:297-302.  Back to cited text no. 9    
10.Strueleus MJ. Consensus guidelines for appropriate use and evaluation of microbiologic typing system. Clin Microbiol Infect 1996;2:1-9.  Back to cited text no. 10    
11.Burgner D, Dalton D, Hanton M, Wong M, Kakakics A, Isaacs D. Repeated prevalence surveys of paediatric hospital acquired infection, J Hosp Infect 1996;34:163-170.  Back to cited text no. 11    
12.Monga K, Fernandez A, Deodher L. Changing bacteriology in neonatal septicaemia Indian J Paediatr 1986;53:505-508.  Back to cited text no. 12    
13.Bhutta ZA, Naqvi SH, Muzaffar T, Neonatal Sepsis in Pakistan, Acta Paediatr Scand 1991;80:596-601.  Back to cited text no. 13    
14.Grundman H, Schneider C, Hartung D, Daschner FD, Pitt TL. Discriminatology power of three DNA based typing techniques for Pseudomonas aeruginosa J Clin Microbiol 1995;33:528-34.  Back to cited text no. 14    
15.Chugh TD, Walia SK, Uma Sabherwal. Bacteriocin sensitivity typing as an epidemiological marker of Klebsiella pneumoniae. Indian J Med Res 1980;71:195-202.  Back to cited text no. 15    
16.Costas M, Holmes B, Sloss LL. Comparison of SDS-PAGE protein patterns with other typing methods for investigating the epidemiology of K. aerogenes. Epidemiol Infect 1990;104:455-465.  Back to cited text no. 16    
17.Lhopital S, Bonalorsi S, Meis D, Brahimi N, Mathy S, Navarro J, Aigrain Y, Bingen E. Molecular markers for differentiation of multiresistant Klebsiella pneumoniae isolates in a Paediatric hospital. Infect Control Hosp Epidemiol 1997;18(11):743-8.  Back to cited text no. 17    
18.Khan FG, Rattan A, Khan IA, Kalia A. Study of Pseudomonas aeruginosa causing ventilator associated pneumonia. Indian J Med Res 1998;107:68-74.  Back to cited text no. 18    
19.Chung MCM, Polyacrylamide gel Electrophoresis, In: Genes and proteins. A laboratory manual of selected techniques in molecular biology. Jeyaseelan K. Chung MCM, Kon OL, ed, Bangkok, UNDP/WB/WHO Special Programme for Research and Training in Tropical Diseases, 1987:99-112.  Back to cited text no. 19    
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