The vector potential of houseflies (Musca domestica) for Klebsiella spp. was investigated. Klebsiella spp. (mostly Klebsiella pneumoniae) were isolated from.
Epidemiol Infect. (1992), 109, 143-147 Printed in Great Britain
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Vector potential of hospital houseflies with special reference to Klebsiella species R. FOTEDAR, U. BANERJEE, J. C. SAMANTRAY AND SHIRNIWAS Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India
(Accepted 10 February 1992) SUMMARY The vector potential of houseflies (Musca domestica) for Klebsiella spp. was investigated. Klebsiella spp. (mostly Klebsiella pneumoniae) were isolated from 36-7 % of hospital flies and 28 1 % of infected wounds of patients. Antibiograms of Klebsiella spp. showed that 82 0 % of isolates from hospital flies and 96 3 % from infected wounds were resistant to four or more commonly used antimicrobials. In contrast, from the control group, only 877% klebsiella isolates showed similar antimicrobial resistance pattern (P < 0 001). Similar strains of Klebsiella spp. were encountered among patients and hospital houseflies. The results indicate that flies in the hospital environs are potential vectors of hospital resistant strains of Klebsiella spp. INTRODUCTION
Houseflies (Musca domestica) have been considered important agents in the dissemination of numerous infectious diseases [1]. The behavioural characteristics of M. domestica and its variants, ensure its contact with food and detritus of man and his animals and provides opportunity to disseminate pathogenic microorganisms [2]. Klebsiella spp. are one of the major pathogens responsible for nosocomial infections. They are known to be responsible for about 10% of Gram negative infections in hospitals [3]. Being opportunistic pathogens they are capable of producing infections of the respiratory tract and urinary tract, as well as post operative wound infections [4]. In recent years the incidence of nosocomial infections due to drug resistant Klebsiella species has increased [4, 5]. Isolation of various bacteria including Klebsiella spp. has been reported from hospital houseflies by various investigators [2, 6, 7]. The role of houseflies in epidemics like cholera., gastroenteritis, etc. is also well established [2, 8]. So far, there is no report of isolation of drug resistant Klebsiella spp. from houseflies collected from hospital wards and patients in the same ward. A prospective study was therefore initiated to ascertain the role of houseflies in transmission/carriage of drug resistant strains of klebsiellas in a hospital environment.
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R. FOTEDAR
AND OTHERS
MATERIALS AND AIETHODS
Collection and identification of houseflies Two hundred and forty live houseflies were caught, 120 from the surgery ward of the All India Institute of Medical Sciences Hospital (hospital group) and 120 from a high income group residential area situated 5 km from the hospital (control group), during the same period of study. Simultaneously, wound swabs from 96 patients with post operative infection, were also collected. The flies were collected from the immediate environment of the patients (beds, cupboards, benches, stools, and bed covers etc.) as well as from the floor of the dressing area of the ward. The control flies were collected from the kitchens and store rooms of the residential areas. Each housefly was caught in a sterilized fly net and immediately transferred to a sterile test tube (one fly/tube) inflicting minimal injury during the process. The flies were quickly transported to the laboratory, anaesthetized by keeping them at 0 °C for 5 min. Identification was made by examining the fly under a low power microscope, following standard taxonomic keys [9]. Isolation of Klebsiella spp. from external surfaces of houseflies Each housefly was transferred to a single sterile test tube containing 2 ml of sterile normal saline (0-85%) and shaken thoroughly for 2 min. A fixed volume of these washings were then cultured on to MacConkey agar (MA) plates (primary isolations) and also inoculated into thioglycollate broth (TGB) and incubated overnight at 37 'C. Subcultures were made from TGB on to MacConkey agar plates, incubated overnight at 37 'C. Colonies of Klebsiella spp. recovered on the MacConkey agar were identified according to Cruickshank [10]. Briefly, lactose fermenting colonies were identified by their macroscopic morphology, Gram's reaction and biochemical reactions.
Isolation of Klebsiella spp. from gut of houseflies After external washing, the flies were washed in 70 % ethyl alcohol for 5 mim to decontaminate external surfaces and dried. The fly was then washed with sterile normal saline to remove traces of alcohol and the gut dissected out aseptically. The gut was then transferred to a sterile pestle and mortar and emulsified in 2 0 ml of sterile normal saline. The resulting macerate was processed, as described above.
Isolation from patients WVound swabs from 96 patients with wound infection were collected, using sterile cotton swabs. transported to the laboratory and processed within 1 h. The swabs were inoculated on MacConkey agar plates and into TGB and subcultured on MacConkey agar plates. Culture plates for primary and secondary sub-cultures were incubated overnight at 37 °C. The lactose fermenting mucoid colonies (resembling Klebsiella spp.) were identified by their macroscopic morphology, Gram's reaction, biochemical tests and other specific characteristics. Antibiograms Antimicrobial sensitivity testing was done on all the strains of Klebsiella spp. isolated, using a modified Stokes disk diffusion method using MacConkey's agar
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Table 1. Percentage biotypes of Klebsiella isolates Houseflies Patients (0) Hospital ("O) (N = 27) (N = 50)
Species
K. K. K. K.
pneurnoniae oxytoca ozanae
rhintoscleromnatis
85-1 74 7-4 0
90 8 0 2
Control (0) (N = 46) 84 7 152 0 0
Table 2. Correlation by antimicrobial resistance Antibiotics ,
Source
Infected -wounds Houseflies (hospital) Houseflies (control)
A~~
&
Three or less No
%o
1 9 42
Fotur or more No
0
:37
26
181 913
41 4
96:3 82.0* 8.7*
Statistically* significant (P < 0 001).
with Mitomvein-C [11]. The following antibiotics were tested: amikacin (30 jtg), streptomyein (100 ,ug), gentamicin (10 ,ug), tetracycline (25 ,ug), erythromycin (15 ,ag), chloromycetin (25,ag), sisomycin (10,tg), cephalexin (30 ,ug), ampicillin (10 1ag).
T'yping Typing of all the isolates was done by broth extraction method using Mitomycin-C [12]. Briefly the klebocine producers strains (153, 154, 155, 156, 157. 158. 159 Fkt3. Fkt4. and GH824) were supplied by Dr Ayliffe. Summerfield Hospital, Birmingham, UK. One local producer strain LPS (AIIMS 77) was added to the original set of nine producer strains. The nutrient agar plates with Mitomycin C (0 5 mcg/ml) were evenly seeded with 4 h growth of the test strain in peptone water. Klebocines were applied to the seeded plates with a phage applicator. The plates were incubated at 32 °C for 16-18 h. The results were read and reported according to the modified Aljumali's mnemonic coding [13]. RESULTS
All the houseflies (hospital and control) were identified as Musca domestica. Klebsiella spp. were isolated from 36 7 0/o (44/120) of hospital flies, 31P7 % (38/12O) of the control flies, either externally or internally. Klebsiella spp. were isolated from 27 (284t %) of the infected wounds of the 96 swabs collected from patients having post-operative wound infection. 6 2
R. FOTEDAR AND OTHERS 146 On biotyping four species of Klebsiella (K. pneumoniae, K. oxytoca, K. ozanae, K. rhinoscleromatis, were identified from all the three sources. The majority of Klebsiella spp. isolates from patients and flies were K. pneurnoniae (Table 1). Antibiograms of the klebsiella isolates from patients and hospital flies were resistant to the commonly used hospital antibiotics, e.g. streptomycin, tetracycline, cephalexin, chloromycetin, erythromycin and ampicillin. Eighty-two percent of the klebsiella isolates from hospital houseflies and 96K3 % isolates from infected wounds were resistant to four or more commonly used antimicrobials (Table 2) in contrast to only 8-7 % from control houseflies (P) < 0-001). Sixty-three percent of klebsiella isolates from wounds and 48 % isolates from test houseflies had similar antibiograms. On klebocine typing, few similar klebocine types were obtained from the three sources. However, no statistically significant difference was seen among these klebocine types obtained from patients and houseflies (test and control). D)TSCUSSION .Musca domestica was the only fly species found in the hospital ward and the residential areas, other species could have been found if the collections had been made from more diverse sources. The occurrence of ill. domestica in hospitals has been reported by many workers [6, 7, 14]. The carriage rate of Klebsiella spp. in this study was 36-7 % in hospital houseflies and 31-7 % in control houseflies. Carriage rates of Klebsiella spp. from flies collected from hospital environment, has not previously been reported [2, 6]. In this study drug resistant Klebsiella spp. have been isolated from the gut as well as from external surfaces of the hospital houseflies. This is of importance since houseflies can be more than simple mechanical vectors. Bacteria in the alimentary canal can remain viable for several days or weeks and be expelled later. A higher percentage of klebsiella isolates from the hospital houseflies and patients showed multiple drug resistance to four or more antimicrobials, than in the controls (P < 0O001). This suggests that the drug-resistant strains of Klebsiella spp. carried by patients, are acquired by the flies. Klebsiella spp. isolated from control houseflies showed very little resistance to antimicrobials, indicating that these strains were entirely different and probably of environmental origin. Klebocine typing was unable to reveal any significant difference among the klebsiella strains isolated from the three sources, and may not be a useful discriminatory epidemiological tool in such type of studies.
1. 2.
3. 4.
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