1. MED. MICROBI0L.-VOL.
0022-261 5/81/0467 0443 $02.00
14 (1981). 4 4 3 4 5 0
0 1981 The Pathological Society of Great Britain and Ireland
T H E ABILITY O F STRAINS O F KLEBSZELLA
A E R O G E N E S TO SURVIVE O N THE HANDS
E. MARYCOOKE, A.
s. EDMONDSON*
AND w.STARKEY
Department of Microbiology, University of Leeds, Leeds LS2 9NL
SUMMARY.The ability of Klebsiella aerogenes strains isolated from
outbreaks of infection, from sporadic infections and from the environment to survive on hands were compared. Considered as groups, the
outbreak strains survived best and the environmental strains least well.
The possible importance of these observations in relation to outbreaks
of klebsiella infection in hospitals and to heterogeneity of pathogenic
potential and transmissibility in Klebsiella aerogenes is discussed.
INTRODUCTION
Evidence has been obtained by several workers that Klebsiella aerogenes is
found on the hands of hospital staff and that transmission by hands may be an
important mechanism by which spread of this organism occurs in hospitals
(Salzman, Clark and Klemm, 1967; Casewell and Phillips, 1977; Shinebaum,
Cooke and Brayson, 1979).
Strains of K. aerogenes are found in a great variety of situations in the
environment, as causes of sporadic infections and associated with outbreaks of
epidemic spread. The present study was undertaken to determine whether
differences in the behaviour in hospitals of isolates with these different
associations might be correlated with differences in their ability to survive on
the skin.
MATERIALS
AND METHODS
Sources of the organisms
Ten strains of K. aerogenes from the environment, ten from sporadic infections and ten from
outbreaks of infection were examined. The sources of the organisms and their capsular types
are given in table I. All organisms were stored on Dorset egg slopes and none was serially
subcultured. Identification and serotyping were done as previously described by Edmondson
and Cooke (1979). One strain of Escherichia coli was used; this had been isolated from the
faeces of an infant with diarrhoea but did not belong to any of the recognised enteropathogenic
serotypes.
Details relating to outbreak ( 0 )strains. 01: the outbreak occurred during a short period
and involved 17 patients in a ward and an intensive-care unit. 0 2 : was isolated during a long
Received 2 Mar. 1981; accepted 16 Mar. 1981.
address: Johnson & Johnson, Microbiology Laboratories, Airebank, Gargrave, Skipton,
North Yorkshire.
* Present
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E. MARY COOKE, A . S. EDMONDSON AND W. STARKEY
TABLEI
Sources of strains
Strain
no.
Environmental strains
Source
Capsular
serotype
Flowers from florists
E 4
E 7
E 8
E 9
E 10
Ward environment, settle plate
sink
Hospital food: lettuce
Lake water
Flowers from ward
[
K26
K12/19
K27
K70
K14
K80
Clinical strains
Strain
Capsular
no.
Source serotype
C 1 Urine
c 2
c 3
C 4 Sputum
c 5
C 6 Blood
C 7 Sputum
C 8
c 9
c10
K2 1
K2
K64
K16
K27
K16
K38
K62
K16
K16
Outbreak strains*
Strain
Capsular
no.
serotype
0 1
0 2
0 3
0 4
0 5
0 6
0 7
0 8
0 9
010
K2 1
G23
K2 1
K9
K9
K21
K68
K43
K2
K39
* Details are given in the text.
and serious outbreak in a neurosurgical unit which has been described by Price and Sleigh
(1970). G23 may be a new serotype and was the predominant type present. 03: nine patients
were infected, after prostatectomy, by the same strain of K . aerogenes. The outbreak had some
of the characteristics of common-source infection but patient-to-patient transmission may have
occurred. 04: was isolated during a small outbreak lasting 2 months in three wards and
involved mainly urinary- tract infection. The organism was detected because it had multiple
antibiotic resistance. 0 5 : forty-seven patients mainly in one ward were involved in the outbreak
which caused urinary-tract infection and lasted for 9 months. 06: this strain was isolated during
a period of 2 months from five patients, four of whom were in an intensive-care department. 0 7 ,
08, 09, 010: all of these strains were isolated from a prolonged outbreak and all were
predominantly associated with urinary-tract infection. Strain 0 7 infected nine, 0 8 twelve, 0 9
ten, and 0 1 0 35 patients.
These strains and information about the outbreaks were kindly provided by Dr A. A. B.
Mitchell (Ol), Dr J. D. Sleigh (02), Dr N. Peel (03), Dr A. C. Maddocks (04), Dr G. A. J.
Ayliffe ( 0 5 ) , Mrs M. I. Carr (06) and Dr C. A. Hart ( 0 7 , 0 8 , 0 9 and 010). A more detailed
report of strains 01-05 and the outbreaks with which they were associated was given by
Edmondson (1978).
Contamination and sampling offingertips
The methods used were based on those of Lilly and Lowbury (1978). From an 18-h culture
of the organism in nutrient broth, 5 ml were centrifuged; the supernate was discarded and the
bacteria were resuspended in quarter-strength Ringer’s solution; 10 p1 of a tenfold dilution was
spread over an area of c. 1 cm2on one fingertip, allowed to dry for 30 s, and then sampled. The
same procedure was followed on two more fingers of the same volunteer, one being sampled at 5
and one at 10 min.
Fingers were sampled by rubbing for 45 s against sterile glass beads in 0 . 4 5 ~phosphate
buffer with 0.1% peptone and 0.1% Triton X-100. Organisms were counted by the method of
Miles, Misra and Irwin (1938). Klebsiellae were counted on MacConkey inositol carbenicillin
agar (Cooke et al., 1979a) and on nutrient agar, and E. coli on MacConkey agar containing
fusidic acid 1 pg/ml and on nutrient agar.
RESULTS
Each of the test strains of K . aerogenes classed as 10 environmental, 10
clinical and 10 outbreak strains was tested on each of the volunteers to give 120
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SURVIVAL OF KLEBSIELLAE O N HANDS
experiments in all and 40 experiments for each class of strain. In addition, one
strain of E. coli was tested on each volunteer.
The loss of viability during the immediate drying period was approximately
50% and there were no differences between strains from different sources.
The median survival times of the K . aerogenes strains at 5 and 10 rnin after
inoculation, the results for the E. coli strain, and a summary of the results are
shown in tables 11-IV. Table V shows the results of studies with one strain
examined on six occasions on one volunteer.
TABLE
I1
Percentage suruiual of Klebsiella aerogenes strains at 5 and 10 min after inoculation onto the
fingertips (median values of four experiments)
I
1
Environmental
strain
no.
E l
E 2
E 3
E 4
E 5
E6
E7
E 8
E9
El0
Median percentage
survival at
5 min
10 min
Clinical
strain
no.
0.38
0.05
0.04
0.03
0.69
0.00
0.07
0.10
0.00
0.00
0.02
0.12
0.00
0.00
0.00
0.00
0.00
c 1
c 2
c 3
c 4
c 5
C 6
c7
C 8
c 9
c10
0.00
0.05
0.02
Median percentage
survival at
5 min
10min
Outbreak
strain
no.
1.51
1.85
1.91
3.68
0.27
8-75
0.64
0.29
0.1 1
0.54
0.86
2.07
0.53
1-98
0.14
1.17
0-18
0.07
0.00
0.2 1
0 1
0 2
0 3
0 4
0 5
0 6
0 7
0 8
0 9
010
Median percentage
survival at
5 min
10 min
8-95
69.85
0.12
1.11
3.31
6.00
2.60
1.81
4.40
1.63
1.12
2.76
0.16
0.32
1.25
4.29
1.60
0.93
1.61
0-48
TABLE
I11
Survival of E. coli test strain on the hands of four subjects at 5 and 10 min*
Percentage
survival at
Subject
'I
C
D
5 min
10 min
0.00
0.00
0.02
0.51
0.00
0.00
0.04
0.00
I
* Tested once on each subject.
TABLE
IV
Survival of klebsiella strains at 5 and 10 min after inoculation onto fingertips
Median percentage
survival at
Test strains*
Environmental
Clinical
Outbreak
1
5 min
10 min
0.05
1.08
2.96
0.00
0.37
1-18
-
* In each case, 10 strains were used in 40 experiments.
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E. MARY COOKE, A . S. EDMONDSON AND W . STARKEY
TABLE
V
Results of six replicate experiments to determine survival of strain 0 2 on fingertips of subject 4
Experiment
no.
Percentage survival* at
5
10
20
40
60 min
1.73
1-39
26.20
2.74
3.46
2.58
0.83
0.34
5.71
0.80
0.73
1.60
0.89
0.48
0.04
2.01
0-22
0.09
1.76
0.28
0.02
1.81
0.44
0.18
0.50
0.05
2.62
0.36
0.23
2.58
* Each entry is the result of one experiment.
Although there were variations between strains, the outbreak strains as a
group survived for longer than the other strains. The environmental strains
survived least well. The strains were ranked in order of increasing percentage
survival for each individual. The strains giving the 10 best survival times at 5
min were noted for each series of 40 experiments with each volunteer; in this
analysis, the outbreak strains scored 26, the clinical strains 13, and the
environmental strains only 1. In a similar analysis for the best 10-minute
survivors, the scores were 23, 17 and 0 respectively. When the converse
analysis was done, strains surviving least well at 5 and 10 min produced scores
of 31 and 29 for environmental strains, 7 and 8 for clinical strains, and only 2
and 3 for outbreak strains. These data are summarised in figs. 1 and 2.
DISCUSSION
The role of hands in the spread of infections has aroused interest in several
respects. Transmission of salmonellae by hands may be responsible for the
protracted outbreaks of infection that sometimes affect hospitals; survival of
salmonellae has been investigated by Pether and Gilbert (1971) in relation to
the possibility of contamination of food. Disinfection of the hands of
surgeons and the value of different disinfectants in the prevention of
postoperative wound sepsis has been extensively studied (Lowbury and Lilly,
1960, 1973; Lowbury, Lilly and Ayliffe, 1974).
Recent severe outbreaks of klebsiella infection have focused attention on
the sources of this organism. Faecal carriage (Cooke et al., 1979a; Houang et
al., 1980),hand transmission (Salzman et al., 1967;Casewell and Phillips, 1977;
Shinebaum et al., 1979) and the ward environment (Cooke et al., 19793) may
all be important. Casewell and Phillips (1977) examined the survival on hands
of types K47 and K2 1, the commonest endemic strains in the unit they were
studying, and showed regular survival with tenfold or less reduction in count
during periods of up to 150 min.
The present investigation confirms that some strains of K . aerogenes
survive much better on the skin than did the strain of E. coli tested, and
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SURVIVAL OF KLEBSIELLAE O N HANDS
80
8
0
0
0
8
0
0
0
8
8
0
8
4
8
c
0
0
0
447
0
0
?
:
0
0
0
i
.t.
0
F
i.
0
:
0
0
8
r
0
0
880
8
t
0
;8
:
P
t.
8
0
0
i
0
0
0
0
0
0
0
00
0
0
0
0
0
0
m
0
0
0
0
Time fmin)
Results
with;
5
0.0
10
strains isolated from
outbreaks
OOO
5
m
oooa.0
10
strains isolated from
sporadic infections
10
strains isolated from
the environment
FIG.1 .-Percentage survival times of Klebsiella aerogenes isolates from different sources in experiments in
which they were inoculated onto fingertips and sampled at 5 and 10 min.
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E. MARY COOKE, A . S. EDMONDSON AND W . STARKEY
448
HlQH SURVIVAL
MODERAE SURVIVAL
LOW SURVIVAL
3025
!i
-
E
20-
E
15-
32
1050-
5
10
5
10
5
10
DURATION OF SURVIVAL
FIG.2.-Number of experiments in which strains of KlebsieIfa aerogenes isolated from different sources
showed high, moderate and low percentage survival 5 and 10 min after inoculation onto fingertips.
El = Strains from outbreaks; W =strains from sporadic infections; IID =strains from the environment.
demonstrates that strains of Klebsiella isolated from outbreaks survive longer
than do other strains.
Recent outbreaks of klebsiella infection have generally been associated
with gentamicin resistance of the organism and with a limited range of
serotypes (Curie et al., 1978; Casewell and Talsania, 1979). Edmondson et al.
(1980) obtained evidence that clinical strains differ in other ways from
environmental strains, particularly in their temperature growth-range. The
present investigation provides additional evidence of heterogeneity in K.
aerogenes. Previously these organisms have been distinguished only by their
biochemical properties, capsular serotype and antibiotic resistance. In some
other bacteria there is great variation in pathogenic properties between strains;
more investigation may reveal a similar heterogeneity in K. aerogenes.
The results shown in table V illustrate the experimental variation found.
The problems of counting bacteria have been discussed by Meynell and
Meynell(l970). In this type of investigation, in addition to two sets of counts,
there are the problems associated with the application of organisms to and
their removal from the skin. Similar variation, particularly between results
obtained with different subjects, was also found by Pether and Gilbert (1971).
However, in our study, the differences between strains from different sources
were marked (figs. 1 and 2). Because little is known about the infecting dose of
K. aerogenes in different situations, it is difficult to assess what numbers on the
hands may be important, but our findings indicate that transmission of viable
organisms between patients by the hands of staff in a hospital ward could
readily occur in a period of 5-10 min.
The outbreaks from which strains were investigated differed in severity and
duration. It is interesting that the longest and most severe outbreak (02) was
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SURVIVAL OF KLEBSIELLAE ON HANDS
449
associated with a strain with prolonged survival on skin. Outbreak 0 5 was
also severe and lasted for 9 months and again skin survival was good.
Outbreak 0 3 may have been a common-source outbreak and survival of this
organism was poor.
The reasons for the poor survival of environmental strains of K. aerogenes
compared with clinical and outbreak isolates are not obvious. Survival of
organisms on the skin is related to pH, humidity, the presence of antibacterial
substances and of competitive organisms. Gram-negative bacilli are generally
susceptible to drying and survive less well than gram-positive cocci. However,
there are differences in the survival of different gram-negative bacilli on the
skin, and some K. aerogenes strains survive better than E. coli strains. This
may account, in part, for their increasing importance as hospital pathogens.
More investigation of the heterogeneity of K. aerogenes strains in relation
to their pathogenicity and transmissibility may yield information that could be
useful in the control of hospital outbreaks of K. aerogenes infection.
Our thanks are due to Dr A. B. Mitchell, Dr J. D. Sleigh, Dr N. Peel. Dr A. C. Maddocks, Dr
G. A. J. Ayliffe, Mrs M. Carr and Dr C. A. Hart, who kindly provided the klebsiella outbreak
strains; to Mrs J. Siegerstetter (Department of Community Medicine and General Practice) for
advice on the analysis of results, and to Mr A. P. D. Wilcock and Mrs Mary E. Walker for
technical assistance. This work was supported in part by a project grant from the Medical
Research Council.
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