Klebocin typing of Klebsiella species isolated from nosocomial infection in intensive care unit.

RB Pal, JS Rebello, DD Banker, PK Jain 
 Sir Hurkisondas Nurrotumdas Medical Research Society, Mumbai.

Correspondence Address:
R B Pal
Sir Hurkisondas Nurrotumdas Medical Research Society, Mumbai.

Full Text

::   Introduction

Klebsiellae are an important cause of nosocomial infections, particularly in intensive care units[1],[2]. With this regard, the accurate identification of organisms is of cardinal importance in defining nosocomial spread among patients. For epidemiological purpose, phage typing, bacteriocin (klebocin) typing, serotyping and antibiogram are important tools. Some workers have reported that klebocin typing in combination with other methods such as biotyping and serotyping, is a precise epidemiological marker for Klebsiella infection[3],[4]. The present study was designed to evaluate efficiency of klebocin typing in combination with antibiogram as a tool for detection of source of infection.

::   Material and method

The present study was carried out from September 1990 to October 1992 in the Intensive Care Unit (ICU) of Sir H.N. Hospital, Mumbai. Skin, nasal and throat swabs from patients were immediately collected after admission to ICU to determine their flora. Appropriate clinical samples were also collected after the onset of infection. Swabs from the patient’s environment (i.e. ICU sites such as wall, floor, trolley, suction tubing, bed, and curtains) as well as from the staff members (i.e. skin, nasal and throat swabs) were collected monthly to study the flora acquired. All Klebsiella isolates were subjected to klebocin typing by spot-inoculation method as described by Chugh et al (1980)[5], with some modifications using six producer strains 153, 154, 155, 156, US-5 and US-6. The methodology used was as follows:

To each broth culture (about 0.7 ml) of producer strains, 3.3 ml trypticase soy broth (TSB) and 1 ml of mitomycin C (0.5 mcg/ml) was added and incubated at 37°C for 5 hours with intermittent shaking. The cells were killed by addition of 0.25 ml chloroform. The suspension was centrifuged at 3000 rpm for 10 mins in cold centrifuge. Supernatant containing respective klebocins was aliquoted in screw cap vials and stored at - 20°C. Test culture was swabbed over a nutrient agar plate and 12 µl of each klebocin was spot inoculated on marked sectors with a micropipette instead of 23 gauge tuberculin syringe (as described in original method). A positive control plate streaked with strain WC-50 was also put up with every set of experiment. The negative and positive reactions to 6 klebocins were recorded using a mnemonic system proposed by Farmer (1976)[6]. Antibiotic sensitivity of the strains was determined by Kirby-Bauer disc diffusion method[7]. The untypable strains i.e. those not inhibiting the growth of any of the indicator strains, were designated as “444”.

::   Results

Klebocin typing was performed on a total of 518 strains obtained from various sources. Twenty-eight klebocin types were observed [Table:I]. The overall typability of the strains was 71.62%. The untypable strains accounted for the remaining 28.37% of the total. The isolates obtained from ICU staff members showed highest typability i.e. 75.82% among the various sources. The most common mnemonic types found among ICU sites and ICU staff isolates were 111 & 211. Types 111, 211 and 312 were commonly observed among ICU patient’s flora. Among the clinical isolates the common mnemonic types were 111 and 112. Klebocin typing and antibiogram of clinical isolates was compared with those from other sources, isolated during the period of infection. In 23 (60.52%) cases, the mnemonic types as well as antibiogram of Klebsiella isolates from clinical specimens matched with those of ICU sites and/or staff. In 10 (26.31%) cases, the mnemonic types and antibiogram matched with those from the endogenous flora of the patients [Table:II].

::   Discussion

Epidemiological investigation of nosocomial infections is greatly facilitated by the use of a precise typing system for the organism involved. Our experience about the nosocomial infections in ICU suggests a high prevalence of Klebsiella infections accounting for 26.66% of total cases. Respiratory tract infections were most common followed by UTI, wound, septicaemia, miscellaneous sites and multiple site infections. Evaluation of klebocin typing in conjunction with antibiogram was done in present study for epidemiological investigation. Using the spot-inoculation method, we observed 71.62% typability, whereas Telang et al (1983)[8], Chugh et al (1980)[5] and Shanmugam et al (1983)[9] reported 92%, 80.3% and 93.83% typability respectively. Bauernfeind et al (1981)[4], Hall (1971)[10] and Chugh et al (1980)[5] have reported 83.3%, 77% and 52% typability respectively using streak-and-point method for klebocin typing. In view of the high induction of klebocins by mitomycin C, storage stability at - 20°C and simplicity of the high degree of technique, spot inoculation method is more appropriate typing method.

We observed that klebocin types 111 (50%), 211 (5.59%), 312 (3.08%) and 112 (2.31%) were the most common types in our study. According to Chugh et al (1980)[5] type “444” is considered as untypable and these strains accounted for 28.37% of total strains studied. Chugh et al (1980)[5] reported types 313, 113, 111 and 311 as the commonest klebocin types, whereas Shanmugam et al (1983)[9] found types 113, 114 and 314 as the most prevalent ones in their study. Probably there is variation in the prevalence of various klebocin types in different topographical areas and institutions. In a study on klebocin typing by Chakravarty et al (1986)[11], no correlation between antibiogram and klebocin types was observed. We found that by combination of klebocin types and antibiogram of clinical isolates, the source of infection could be traced in 86.84% cases. The sources of infection were ICU sites and/or staff in 60.52% cases, 26.31% cases as endogenous flora and rest were unknown. Hence, combination of klebocin typing and antibiogram methods suggests an important tool for epidemiological studies.

::   Acknowledgment

We wish to thank Dr.Uma Sabherwal, Professor of Microbiology, Rohtak Medical College for providing the klebocin producer strains and valuable guidance.

References