- Email: [email protected]
Phage-Typing of Klebsiella Strains from Cologne and Wroclaw
Zbl. Bakt. Hyg., I.Abt. Orig. A 250,296-306 (1981)
Department of Microbiology, Medical Academy, Wrodaw, Poland; Institute of Hygiene, University of Cologne (Director: Prof. Dr. GerhardPulverer) , Federal Republic of Germany
Phage-Typing of Klebsiella Strains from Cologne and Wroclaw Phagentypisierung von Klebsiella-Stammen aus Koln und aus Breslau ANNA PRZONDO HESSEK 1, GEORG PETERS, BARBARA BYCZYNSKA, and GERHARD PULVERER
Received April 6, 1981
Summary 276 strain of Klebsiella pneumoniae and Klebsiella oxytoca were phage-typed. 126 of these strains were isolated from clinical sources in the Institut of Hygiene in Cologne and 150 came from Wrodaw. The set of 19 phages used for phage-typing was chosen out of 46 Klebsiella specific bacteriophages. 80% of the isolates from Cologne were sensitive to the phages in comparison to 83% of the strains from the Wrodaw region. The phage sensitivity of the German strains differed from that of the Polish strains. 28 of the 67 phagetypes were seen in Cologne and 35 in Wrodaw, only 6 phage-types were observed in both regions. The frequency and the spectrum of the phage-types were different, too. Phagetypes denoted by the numbers 1, 17, 32, 36, 37, 59, 64 appeared to be most frequent (10 to 16 strains). No significant differences in the sensitivity to typing phages between strains of Klebsiella pneumoniae and Klebsiella oxytoca were demonstrated.
Zusammenfassung 276 Starnme von Klebsiella pneumoniae und Klebsiella oxytoca wurden phagentypisiert, 126 dicser Starnme wurden im Hygienc-Institut der Universitat Koln aus klinischem Material angeziichtet, 150 Starnme kamen aus Breslau. Der zur Lysotypie verwendete Satz von 19 Bakteriophagen wurde aus insgesarnt 46 Klebsiella-spezifischen Phagen aufgrund ihrer Aktivitat und ihres Wirkungsspektrums ausgewahlt, 80% der isolierten Kolner KlebsiellaStamme waren Iysotypisierbar im Vergleich zu 83% der Breslauer Stamrne. Allerdings differierte die Phagenempfindlichkeit der deutschen und der polnischen Starnme voneinander. Von insgesamt 67 beobachteten Phagtypen entfielen 28 auf Kolner Starnme und 35 auf Breslauer Klebsiellen, nur 6 Phagtypen kamen in beiden Regionen vor. Auch die Haufigkeit und das Spektrum der Phagtypen waren unterschiedlich. Am haufigsten kamen die folgen1
Sripendiarin der Alexander von Humboldt-Stiftung am Hygiene-Institut der Universitat
zu Koln.
Phage- Typing of Klebsiella Strains
297
den Phagtypen vor: 1, 17, 32, 36, 37, 59 und 64 (10 bis 16 Starnme). Keine signifikanten Unterschiede im Phagtypenspektrum der beiden Spezies Klebsiella pneumoniae und Klebsiella oxytoca wurden beobachtet.
Introduction Klebsiella bacteria may be responsible for severe diseases including pneumonia, lung abscess, urinary and alimentary tract infections and sepsis (2, 4, 6, 8, 13). Especially Klebsiella pneumoniae and Klebsiella oxytoca have often been associated with morbidity and mortality in hospitalized patients (4, 8). To solve the increasing problems of infections evoked by Klebsiella strains, more attention should be paid to the study of epidemiology of these organisms. An important method in epidemiological studies is phage-typing. To obtain a more detailed differentiation within bacterial species this method can be used together with serotyping, biotyping and typing by antibiograms (3,7, 12). This paper reports the results of Klebsiella phagetyping using a selected set of 19 specific bacteriophages.
Materials and Methods
1. Strains: 276 clinical isolates were identified as Klebsiella pneumoniae or Klebsiella oxytoca, 126 in the Institute of Hygiene in Cologne and 150 strains in the Department of Microbiology of the Medical Academy in Wrodaw. Clinical specimens from upper and lower respiratory tract, urinary tract or woundinfections were the source of these isolates. Species identification was based on the conventional biochemical tests. 2. Bacteriophages: The bacteriophages and their titers used for typing of Klebsiella are shown in Table 1. This list includes phages selected from the two collections of Klebsiella phages, those of Przondo Hessek (7) and Milch and Dedk (9). 3. Determination of sensitivity of Klebsiella to bacteriophages: Klebsiella strains were inoculated on solid medium of the following composition: agar 1.7% , bacteriologic peptone (Difco) 0.5%, meat extract 0.3%, lactose 0.1 %, bromcresol purple 0.025%, pH 7.6. After 24 h incubation at room temperature, single colonies were transferred to broth and incubated for 4 h at 37°C. A few slowly growing strains were incubated for 6 h. The number of viable bacterial cells in the 4 hand 6 h cultures was about lOs/m!' Two mililiters of the broth culture were poured out on a Petri plate, containing about 20 ml agar medium of the following composition: agar (Difco) 1.2%, bacteriologic peptone (Difco) 1%, NaCI 0.3%, Na2HP04 0.2%, pH 7.2. The inoculum was spread evenly on the surface of the agar. Excess of broth was removed. The inoculated plates were dried for 20 min at 37°C. Undiluted phage Iysates of the concentration given in Table 1 were placed on each plate immediately after drying. The phage suspensions were applied by means of Pasteur pipettes giving 10 III drops. The plates were dried for 20 min at room temperature and then incubated at 37°C for 6 h. Results were recorded after 6 h incubation. Confluent lysis and semiconfluent lysis were regarded as a positive reactions and designated as ( +), isolated plaques and negative reaction were designated as (-). All investigated strains were examined three times at one-month intervals.
Results A total of 276 Klebsiella strains was tested for sensitivity to 46 bacteriophages
(7,9). Out of these phages from the Polish and Hungarian collections, 19 most useful phages were selected: Phages KI: 1,2,3,8,9,12,13,14,16,19,21,27,30,34
298
A.Przondo Hessek, G.Peters, BiByczynska, and G.Pulverer Table 1. List of Klebsiella bacteriophages used for typing Tabelle 1. Liste der verwendeten Klebsiella-Bakteriophagen Phage designation
Klebsiella propagation strain
Bacteriophage titer"
KI 1 KI 2 KI 3 KI 8 KI 9 Kl12 KI13 Kl14 Kl16 Kl19 Kl21 Kl27 Kl30 Kl34 4B 42B 106 B 171 B 765 B
K 67/264/1 K 59/2212/52 K 70/167 K 37/8238 K 14 Kleb 1193 K 40/8588 K 68/265/1 L 174/K 3 K30/7824 K 58/636/52 K 2/B 5055 K44/7730 K 57/4425/51 K 30/7826 K4 K 71 K 196 K344 K997
108 10· 10· 10' 10· 108 108 10· 108 10' 108 108 108 108 10· 108 10' 10· 108
" pfu/ml (pfu = plaque forming units).
and Hungarian phages 4B, 42B, 106B, 171B, 765B. These phages typed 80% Klebsiella strains from Cologne and 83% isolates from Wrodaw. Sensitivity of the studied Klebsiella strains is presented in Table 2. Four of the 19 typing bacteriophages showed a markedly different lytic activity towards the two groups of strains. Phages Kl 1, K112, Kl21 lysed greater number of strains isolated in the Wrodaw region, whereas phage 171B lysed 62% of strains isolated in Cologne and only 4% of strains isolated in Wrodaw. The usefullnes of the remaining phages was practically identical in both groups. The results of phage-typing are sumarized in Table 3.67 phage-types were found, buth the frequency of the different types varied widely. 37 phage-types were represented only by single strains, 24 types by 2 to 9 strains. Most frequent were the following types: 1, 17,32,36,37,59,64. A distinct correlation between the place of the strains isolation and their sensitivity patterns was observed. Only 6 phagetypes, i.e. 1, 2, 21, 25, 26, 27 appeared among strains both from Cologne and Wrodaw. Of the remaining 61 types, 28 were found within strains isolated in Germany and 35 in Poland. No significant differences in the phage-sensitivity between Klebsiella pneumoniae and Klebsiella oxytoca strains were observed, the degree of sensitivity as well as the lytic patterns were similar. As a result, numerous phage-types consisted of strains of both species (Table 4). Table 5 summarizes the distribution of phage-types among various clinical sources. No particular phage-type was associated with a given source.
Phage-Typing of Klebsiella Strains
299
T able 2. Lytic activity of th e bacter ioph ages agai nst Klebsiella strains isolated in Cologne and in Wro daw Tabelle 2. Bakteri oph agen-Aktivitat gegeniiber Klebsiella-Stammen aus Koln und au s Breslau Bacterioph ages
Percent of sensitivity strai ns isolated in : Co logne Wrodaw
Kl :
19
21.4 42.0 38.8 2.3 34.1 4.9 1.5 11.1 2.3 24.6
21 27 30 34 4n 42n 106n 171B 765n
0.8 0.8 1.5 0.8 1.5 4.7 62.0 3.1
1 2 3 8 9 12 13 14 16
T otal num ber of strai ns :
o
126.0
36.0 50.0 39.3 8.0 32.0 18.0 2.0 3.3 2.6 34.6 18.0 0.6
o 2.6
o 4.0 8.0 4.0 2.6
150.0
Discussion 19 phages out of 46 Klebsiella phages known were selected for phage-typing of Klebsiella strains from clinical sources. This set comprises all bacteriophages used in 1978-79 for typin g Klebsiella strains isolated in the Wrodaw region (8) as well as 5 other phages: Kl 8, KI13, Kl 30, Kl 34 and 4n. Thi s group of phages appeared to be properly selected since 80% of the strains isolat ed in Cologne and 83% of those in Wrodaw were sensitive, the phage -bacter ia reactio ns were reproducible and 67 phage-types were defined. To pro vide repro ducibility of the phag e-typing resu lts, the inoculum of the bacterial strains has to be th e same and the lysate s used sho uld have identi cal and possibly high tite rs (Table 1). Due to the mucou s grow th of the major ity of Klebsiella strains, reactions with phages in th e form of singe plaques were not always repro ducible and cannot be tak en int o consider at ion (denoted as negative). If Klebsiella strains differ in respect to capsular antigen (K), serological typing used by some autho rs may be an efficient meth od (1, 10, 11). However, all th e techn ique s used for th e deter mination of Klebsiella capsu lar antige ns, i. e. swelling reactions, indi rect immunofluor escence and counter-current immunoelectrophoresis,
21 22 23
19 20
-
-
-
-
-
-
-
+ + + + + + + + + +
-
-
-
11 12 13 14 15 16 17
18
-
+ + +
+ + + + + + + + + + + + + +
+ + + + +
+ + + + + + + +
1 2 3 4 5 6 7 8 9 10
-
3
2
1
Phag e types
-
+
-
-
-
-
-
+
-
8
-
+ + + +
-
-
+ + + + + + + + + + + + +
9
-
+
-
-
+
12
-
-
-
-
-
-
-
+ +
-
-
-
-
-
-
+
-
-
-
-
+ +
14
13
+
-
-
-
+
-
-
+
-
-
-
+
-
+ +
-
-
+ +
-
-
+ + +
-
-
+ +
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
Bacteriophages 19 21 27 16
-
-
-
-
-
30
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
48
-
-
-
-
-
34
-
-
-
-
-
-
-
-
-
+ +
-
-
-
-
-
42n
-
+
-
+ +
-
+
-
+ +
-
-
+
+ + + + + + + +
+ + + + + +
-
-
-
+ + -
-
-
-
-
-
+ -
-
_.
-
-
-
106n 17111 765n
T able 3. Distribution of phage-types among the Klebsiella strains iso lated in Co logne and in Wrodaw Tabelle 3. Verteilung der Phagtypen bei Klebsiell a-Stdmmen a us Koln und aus Breslau
4 3 1
1 1 1 3 9 1 14 1 1 2
3 3 1 1 6 1 1 1 1
12
1
(5)
1 (13) 2 (5)
Origin a nd number of str ains Cologne Wrodaw
w
~
~
'"::l
r.> ....
....
<" t':>
c
0
c,
::3
J'
~
:;l-
'<
t:O n N '<
Y'
c:> ....
rc
'"::l
0
l'
'" '" rc
t':>
::r:
0
p...
:;l
0
~ -e .... N
0 0
51 52 53
41 42 43 44 45 46 47 48 49 50
31 32 33 34 35 36 37 38 39 40
-
-
-
-
+ + + + + + + + + + + + + + + + +
-
-
-
-
25
26 27 28 29 30
-
24
+ + + +
-
+ + + + + + + + + + + + + +
-
-
-
-
+ + + + + + +
-
+ + + + + + + + + + + +
-
-
-
-
-
+
-
-
-
-
-
-
-
-
+ + + + -
-
+ + + + + +
-
+ + +
-
+
-
-
+ +
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
+
+
-
-
-
+ + +
+
-
-
-
+
-
-
-
-
-
-
-
-
+ -
-
-
-
-
-
-
-
-
-
-
-
-
+
-
+ + + + +
+ + + + +
-
-
-
-
-
-
+
-
-
-
-
-
-
+ +
-
-
-
-
-
-
-
-
-
+ + + +
+ +
-
-
-
-
-
-
+
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
+ -
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
+
-
+ + +
-
+
-
-
-
-
-
-
-
-
-
-
-
-
+
+ + + + +
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+ 2
1 15
1
1 1 1
3
4
2 1 2
5 1
1
1 1
1
1
1 1 1
2 3
6
to
14
1 1
1
1 (5) (6) 1 (2) 3
"'::l
::r
0>
.....
w
0
~ .... 0> 5" 'J>
~
~
'J>
0-
'"n
2-
IJ'l
5"
'< >0
'" .:,
(7:l
Phage-Typing of Klebsiella Strains
303
Table 4. Frequency of occurrence of bacteriophage types in Klebsiella pneumoniae and Klebsiella oxytoca Tabelle 4. Haufigkeit der Phagtypen bei Stammen von Klebsiella pneumoniae und Klebsiella oxytoca Phage types
Number of strains Klebsiella pneumoniae ' oxytoca 2
Phage types
Number of strains Klebsiella pneumoniae 1 oxytoca 2
1 2 3 4 5 6 7 8 9 10 11
6 2 1 1 1 1 1 1
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67
1
7 3 2
5
1 1 1 1
12
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
1 1 8
2 1 1 8
6 1 1
2 1
4 3 1 2 5 5 2 1
1 1
1 1 6 1
9
Total number of strains:
1
138
2
5 6 2 2
1 9 4 4 2 1 1 1 1
1 1 1 1 2
1 3 1 2 1 2
1 1 1
2 1 10 1 1 4 5 12 3 7 3
()
87
are laborious (5). Moreover, anti K sera are not commercially available. Due to these difficulties, serotyping cannot be used as a common method and has to be replaced by phage-typing of Klebsiella strains without their previous serological classification. Lysotyping within previously defined antigenic types seems to be the most efficient and precise way of differentiation (12). The strains used in our ex-
-
61 62 63 64 65 66 67
-
-
-
-
-
-I-I-
-
-
+ +
-
3
-
-I-I-
-
-
-
8
-
-
-
-I-I-
+
-
-
-
-
-I-
-
-
13
-
-
-
-
-
12
-
-
9
-
+ -
-
+
-I-
-
-I-
+
-
-
-
-
-
-
-
-
-
-
-
-I-
21
-
-
+
-I-I-
-I-
19
-I-
-
+
-
-
-
16
14
Bacteriophages
-
-
-
-
27
Confluent lysis, opaque lysis, sernico nfluent lysis are designated as ( -I-) . Negative reacti on is designat ed as (-) Tota l number of strains is given in bra ckets.
T otal n umber of strains :
-
-
-I-I-I-
-
54 55 56 57 58 59 60
+
2
1
Phage typ es
-
-
-
-
-
-
-
-I-
-
-
+
-
-
-
-
-
34
-
30
-
-
-
-
-
-
-
-
-
-
-
-
-
-
42I1
4B
-
-I-
-
+
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
106B 171I1 765n
101
124
1 4 5 12 3 7 3
1 1 1 2 1 16 1
Ori gin and n umber of strains Col ogne Wrodaw
w
'"0
~
(1)
,.,
<"
r=
0
:::l
P-
J'
.,'"
'"
:;l-
-e
N
-e r,
I;l:I
?'
J"
~
,.,
(1)
;.."
o
.'"
(1)
~
(1)
::r:
o
P-
0 :::l
N
>,.,;.."
0 N
304
A.Przondo Hessek, G.Peters, BiByczyriska, and G.Pulverer
Table 5. Klebsiella phage types among various clinical isolates Tabelle 5. Verteilung der Phagtypen bei Klebsiella-Stammen aus unterschiedlichem kIinischem Material Phage type
1 2 3 4 5 6 7-8 9 10 11 12 13 14 15 16 17 18-19 20
21 22 23 24 25 26 27-30 31 32 33 34 35 36 37 38 39 40 41-43 44-47 48 49 50 51 52-53
Number of Klebsiella pneumoniae from Respira- Wound Urine tory tract
Number of Klebsiella oxytoca from Respira- Wound Urine tory tract
Total
5 1
5 3 2
2 1
2
4
13 5 3 1 1 6 2 1 1
1
1
1 1 2 1 1 1
1
1 1 1 7
1
1 1
1
1
7
1
1
1 2 2 2 1
2
2
2
1 2
5 3 1 2 5 6 5
1 2 3 4 5 7
2 1
2
1
2 1
1 5 6 2 2
1 9 4 4 2 1
1
1 3
1
1 15 1 1 1 14 10 6 2 3
1 3 1
3 4 1 5 1
1 3
2 3
3 4 2
1 1 3 9 1 14 2 2
Phage-Typing of Klebsiella Strains
1 2 3 16 36
1
54 55-56 57-58 59 W-67
3 10 36
Total
118
2 6
11
6
64
305
21
5
225
periments were not serologically typed. For the phage-typing numerous phages of various lytic range were tested, they were isolated from various sources, selected and propagated on serologically different Klebsiella strains, thus they achieved lytic activity confined to various capsular types (7). No differences were found in the sensitivity to typing phages between strains of Klebsiella pneumoniae and Klebsiella oxytoca. Out of 36 phage-types, represented by more than one strain, 16 were composed of Klebsiella strains of both species (Table 4). This phenomenon can be explained by the occurrence of the same serotypes in the above species (1). Marked disproportions in the number of strains from three different clinical sources did not permit the analysis of the participation of particular phage-types in different infections. However, our investigations suggest that strains belonging to the same phage-type may induce infections in the respiratory and urinary tracts as well as in the soft tissues (Table 5). Substantial differences in phage sensitivity patterns were observed in strains from Cologne and Wrodaw, only 6 phage-types were common. Similar results of differing Klebsiella phage-types in connection with the place of their isolation have been previously observed in Klebsiella rhinoscleromatis (10). Analogous data were observed when the results of Klebsiella serotyping were analysed (1, 2, 7, 11). Our investigations confirm in accordance with the data of RENNIE et al. (12) the usefulness of phage-typing for the epidemiological work on Klebsiella strains. References 1. Blanchette, E. A. and S. J. Rubin: Seroepidemiology of clinical isolates of Klebsiella in
Connecticut. J. Clin. Microbio!. 11 (1980) 474-478 2. Hill, H.R., C.E.Hunt, and J.M.Matsen: Nosocomial colonization with Klebsiella type 26, in a neonatal intensive-care unit, associated with an outbreak of sepsis, meningitis and necrotizing enterocolitis. J. Pediat. 85 (1974) 415-419 3. Meitert, T. and E. Meitert: Usfulness, applications and limitations of epidemiological typing methods to elucidate nosocomial infections and the spread of communicable diseases. In: T.Bergan and ].R.Norris: Methods in Microbiology, Vo!. 10. Academic Press, London-New York-San Francisco (1978) 4. Montgomerie,]. Z.: Epidemiology of Klebsiella and hospital-associated infections. Rev. infect. Dis. 1 (1979) 736-753 5. 0rskov, F. and I. 0rskov: Serotyping of Enterobacteriaceae, with special emphasis on K antigen determination. In: T.Bergan and J.R.Norris: Methods in Microbiology, Vo!. 11. Academic Press, London - New York - San Francisco (1978) 6. 0rskov, I.: Nosocomial infections with Klebsiella in lesion of the urinary tract. Acta path. microbio!. scand. 93, Supp!. (1952) 259-271
306
A.Przondo Hessek, G.Peters, B.Byczynska, and G.Pulverer
7. Przondo Hessek, A.: Bacteriophages of Klebsiella bacilli. Isolation properties and use
in typing. Arch. Immunol. Ther. Exp. 14 (1966) 413-435 8. Przondo Hessek, A. and B. Byczyl1ska: The properties of Klebsiella isolated from patients with respiratory infections. Med. dosw, Mikrobiol. 32 (1980) 31-38 9. Przondo Hessek, A. and S.Slopek: Typing of Klebsiella bacilli by means of the Klebsiella bacteriophages of the collection of Milch and Deah, Arch. Immunol. Ther. Exp.16
(1967) 578-582 10. Przondo Hessek, A., S. Slopek, and ]. Miodonska: Phage typing of Klebsiella rhinoscleromatis. Arch. Immunol. Ther. Exp. 16 (1968) 402-405 11. Reniie, R.P. and I.B.R.Duncan: Combined biochemical and serological typing of
clinical isolates of Klebsiella. Appl. Microbiol. 28 (1974) 534-539 12. Rennie, R.P., C.E.Nord, LiSioberg, and I.B.R.Duncan: Comparison of bacteriophage
typing, serotyping and biotyping as aids in epidemiological surveillance of Klebsiella infections. J. Clin. Microbiol. 8 (1979) 638-642 13. Terman, J.W., R.H.Alford, and R. E. Bryant: Hospital acquired Klebsiella bacteremia. Amer. J. med. Sci. 264 (1972) 191-196 Prof. Dr. Gerhard Pulverer, Hygiene-Institut der Universitat, Goldenfelsstr. 21, D-5000 Kaln 41, FRG Dr. Anna Przondo Hessek, Department of Microbiology, Medical Academy, ul. Chaiubinskiego 4,50-368 Wrodaw, Poland
Department of Microbiology, Medical Academy, Wrodaw, Poland; Institute of Hygiene, University of Cologne (Director: Prof. Dr. GerhardPulverer) , Federal Republic of Germany
Phage-Typing of Klebsiella Strains from Cologne and Wroclaw Phagentypisierung von Klebsiella-Stammen aus Koln und aus Breslau ANNA PRZONDO HESSEK 1, GEORG PETERS, BARBARA BYCZYNSKA, and GERHARD PULVERER
Received April 6, 1981
Summary 276 strain of Klebsiella pneumoniae and Klebsiella oxytoca were phage-typed. 126 of these strains were isolated from clinical sources in the Institut of Hygiene in Cologne and 150 came from Wrodaw. The set of 19 phages used for phage-typing was chosen out of 46 Klebsiella specific bacteriophages. 80% of the isolates from Cologne were sensitive to the phages in comparison to 83% of the strains from the Wrodaw region. The phage sensitivity of the German strains differed from that of the Polish strains. 28 of the 67 phagetypes were seen in Cologne and 35 in Wrodaw, only 6 phage-types were observed in both regions. The frequency and the spectrum of the phage-types were different, too. Phagetypes denoted by the numbers 1, 17, 32, 36, 37, 59, 64 appeared to be most frequent (10 to 16 strains). No significant differences in the sensitivity to typing phages between strains of Klebsiella pneumoniae and Klebsiella oxytoca were demonstrated.
Zusammenfassung 276 Starnme von Klebsiella pneumoniae und Klebsiella oxytoca wurden phagentypisiert, 126 dicser Starnme wurden im Hygienc-Institut der Universitat Koln aus klinischem Material angeziichtet, 150 Starnme kamen aus Breslau. Der zur Lysotypie verwendete Satz von 19 Bakteriophagen wurde aus insgesarnt 46 Klebsiella-spezifischen Phagen aufgrund ihrer Aktivitat und ihres Wirkungsspektrums ausgewahlt, 80% der isolierten Kolner KlebsiellaStamme waren Iysotypisierbar im Vergleich zu 83% der Breslauer Stamrne. Allerdings differierte die Phagenempfindlichkeit der deutschen und der polnischen Starnme voneinander. Von insgesamt 67 beobachteten Phagtypen entfielen 28 auf Kolner Starnme und 35 auf Breslauer Klebsiellen, nur 6 Phagtypen kamen in beiden Regionen vor. Auch die Haufigkeit und das Spektrum der Phagtypen waren unterschiedlich. Am haufigsten kamen die folgen1
Sripendiarin der Alexander von Humboldt-Stiftung am Hygiene-Institut der Universitat
zu Koln.
Phage- Typing of Klebsiella Strains
297
den Phagtypen vor: 1, 17, 32, 36, 37, 59 und 64 (10 bis 16 Starnme). Keine signifikanten Unterschiede im Phagtypenspektrum der beiden Spezies Klebsiella pneumoniae und Klebsiella oxytoca wurden beobachtet.
Introduction Klebsiella bacteria may be responsible for severe diseases including pneumonia, lung abscess, urinary and alimentary tract infections and sepsis (2, 4, 6, 8, 13). Especially Klebsiella pneumoniae and Klebsiella oxytoca have often been associated with morbidity and mortality in hospitalized patients (4, 8). To solve the increasing problems of infections evoked by Klebsiella strains, more attention should be paid to the study of epidemiology of these organisms. An important method in epidemiological studies is phage-typing. To obtain a more detailed differentiation within bacterial species this method can be used together with serotyping, biotyping and typing by antibiograms (3,7, 12). This paper reports the results of Klebsiella phagetyping using a selected set of 19 specific bacteriophages.
Materials and Methods
1. Strains: 276 clinical isolates were identified as Klebsiella pneumoniae or Klebsiella oxytoca, 126 in the Institute of Hygiene in Cologne and 150 strains in the Department of Microbiology of the Medical Academy in Wrodaw. Clinical specimens from upper and lower respiratory tract, urinary tract or woundinfections were the source of these isolates. Species identification was based on the conventional biochemical tests. 2. Bacteriophages: The bacteriophages and their titers used for typing of Klebsiella are shown in Table 1. This list includes phages selected from the two collections of Klebsiella phages, those of Przondo Hessek (7) and Milch and Dedk (9). 3. Determination of sensitivity of Klebsiella to bacteriophages: Klebsiella strains were inoculated on solid medium of the following composition: agar 1.7% , bacteriologic peptone (Difco) 0.5%, meat extract 0.3%, lactose 0.1 %, bromcresol purple 0.025%, pH 7.6. After 24 h incubation at room temperature, single colonies were transferred to broth and incubated for 4 h at 37°C. A few slowly growing strains were incubated for 6 h. The number of viable bacterial cells in the 4 hand 6 h cultures was about lOs/m!' Two mililiters of the broth culture were poured out on a Petri plate, containing about 20 ml agar medium of the following composition: agar (Difco) 1.2%, bacteriologic peptone (Difco) 1%, NaCI 0.3%, Na2HP04 0.2%, pH 7.2. The inoculum was spread evenly on the surface of the agar. Excess of broth was removed. The inoculated plates were dried for 20 min at 37°C. Undiluted phage Iysates of the concentration given in Table 1 were placed on each plate immediately after drying. The phage suspensions were applied by means of Pasteur pipettes giving 10 III drops. The plates were dried for 20 min at room temperature and then incubated at 37°C for 6 h. Results were recorded after 6 h incubation. Confluent lysis and semiconfluent lysis were regarded as a positive reactions and designated as ( +), isolated plaques and negative reaction were designated as (-). All investigated strains were examined three times at one-month intervals.
Results A total of 276 Klebsiella strains was tested for sensitivity to 46 bacteriophages
(7,9). Out of these phages from the Polish and Hungarian collections, 19 most useful phages were selected: Phages KI: 1,2,3,8,9,12,13,14,16,19,21,27,30,34
298
A.Przondo Hessek, G.Peters, BiByczynska, and G.Pulverer Table 1. List of Klebsiella bacteriophages used for typing Tabelle 1. Liste der verwendeten Klebsiella-Bakteriophagen Phage designation
Klebsiella propagation strain
Bacteriophage titer"
KI 1 KI 2 KI 3 KI 8 KI 9 Kl12 KI13 Kl14 Kl16 Kl19 Kl21 Kl27 Kl30 Kl34 4B 42B 106 B 171 B 765 B
K 67/264/1 K 59/2212/52 K 70/167 K 37/8238 K 14 Kleb 1193 K 40/8588 K 68/265/1 L 174/K 3 K30/7824 K 58/636/52 K 2/B 5055 K44/7730 K 57/4425/51 K 30/7826 K4 K 71 K 196 K344 K997
108 10· 10· 10' 10· 108 108 10· 108 10' 108 108 108 108 10· 108 10' 10· 108
" pfu/ml (pfu = plaque forming units).
and Hungarian phages 4B, 42B, 106B, 171B, 765B. These phages typed 80% Klebsiella strains from Cologne and 83% isolates from Wrodaw. Sensitivity of the studied Klebsiella strains is presented in Table 2. Four of the 19 typing bacteriophages showed a markedly different lytic activity towards the two groups of strains. Phages Kl 1, K112, Kl21 lysed greater number of strains isolated in the Wrodaw region, whereas phage 171B lysed 62% of strains isolated in Cologne and only 4% of strains isolated in Wrodaw. The usefullnes of the remaining phages was practically identical in both groups. The results of phage-typing are sumarized in Table 3.67 phage-types were found, buth the frequency of the different types varied widely. 37 phage-types were represented only by single strains, 24 types by 2 to 9 strains. Most frequent were the following types: 1, 17,32,36,37,59,64. A distinct correlation between the place of the strains isolation and their sensitivity patterns was observed. Only 6 phagetypes, i.e. 1, 2, 21, 25, 26, 27 appeared among strains both from Cologne and Wrodaw. Of the remaining 61 types, 28 were found within strains isolated in Germany and 35 in Poland. No significant differences in the phage-sensitivity between Klebsiella pneumoniae and Klebsiella oxytoca strains were observed, the degree of sensitivity as well as the lytic patterns were similar. As a result, numerous phage-types consisted of strains of both species (Table 4). Table 5 summarizes the distribution of phage-types among various clinical sources. No particular phage-type was associated with a given source.
Phage-Typing of Klebsiella Strains
299
T able 2. Lytic activity of th e bacter ioph ages agai nst Klebsiella strains isolated in Cologne and in Wro daw Tabelle 2. Bakteri oph agen-Aktivitat gegeniiber Klebsiella-Stammen aus Koln und au s Breslau Bacterioph ages
Percent of sensitivity strai ns isolated in : Co logne Wrodaw
Kl :
19
21.4 42.0 38.8 2.3 34.1 4.9 1.5 11.1 2.3 24.6
21 27 30 34 4n 42n 106n 171B 765n
0.8 0.8 1.5 0.8 1.5 4.7 62.0 3.1
1 2 3 8 9 12 13 14 16
T otal num ber of strai ns :
o
126.0
36.0 50.0 39.3 8.0 32.0 18.0 2.0 3.3 2.6 34.6 18.0 0.6
o 2.6
o 4.0 8.0 4.0 2.6
150.0
Discussion 19 phages out of 46 Klebsiella phages known were selected for phage-typing of Klebsiella strains from clinical sources. This set comprises all bacteriophages used in 1978-79 for typin g Klebsiella strains isolated in the Wrodaw region (8) as well as 5 other phages: Kl 8, KI13, Kl 30, Kl 34 and 4n. Thi s group of phages appeared to be properly selected since 80% of the strains isolat ed in Cologne and 83% of those in Wrodaw were sensitive, the phage -bacter ia reactio ns were reproducible and 67 phage-types were defined. To pro vide repro ducibility of the phag e-typing resu lts, the inoculum of the bacterial strains has to be th e same and the lysate s used sho uld have identi cal and possibly high tite rs (Table 1). Due to the mucou s grow th of the major ity of Klebsiella strains, reactions with phages in th e form of singe plaques were not always repro ducible and cannot be tak en int o consider at ion (denoted as negative). If Klebsiella strains differ in respect to capsular antigen (K), serological typing used by some autho rs may be an efficient meth od (1, 10, 11). However, all th e techn ique s used for th e deter mination of Klebsiella capsu lar antige ns, i. e. swelling reactions, indi rect immunofluor escence and counter-current immunoelectrophoresis,
21 22 23
19 20
-
-
-
-
-
-
-
+ + + + + + + + + +
-
-
-
11 12 13 14 15 16 17
18
-
+ + +
+ + + + + + + + + + + + + +
+ + + + +
+ + + + + + + +
1 2 3 4 5 6 7 8 9 10
-
3
2
1
Phag e types
-
+
-
-
-
-
-
+
-
8
-
+ + + +
-
-
+ + + + + + + + + + + + +
9
-
+
-
-
+
12
-
-
-
-
-
-
-
+ +
-
-
-
-
-
-
+
-
-
-
-
+ +
14
13
+
-
-
-
+
-
-
+
-
-
-
+
-
+ +
-
-
+ +
-
-
+ + +
-
-
+ +
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
Bacteriophages 19 21 27 16
-
-
-
-
-
30
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
48
-
-
-
-
-
34
-
-
-
-
-
-
-
-
-
+ +
-
-
-
-
-
42n
-
+
-
+ +
-
+
-
+ +
-
-
+
+ + + + + + + +
+ + + + + +
-
-
-
+ + -
-
-
-
-
-
+ -
-
_.
-
-
-
106n 17111 765n
T able 3. Distribution of phage-types among the Klebsiella strains iso lated in Co logne and in Wrodaw Tabelle 3. Verteilung der Phagtypen bei Klebsiell a-Stdmmen a us Koln und aus Breslau
4 3 1
1 1 1 3 9 1 14 1 1 2
3 3 1 1 6 1 1 1 1
12
1
(5)
1 (13) 2 (5)
Origin a nd number of str ains Cologne Wrodaw
w
~
~
'"::l
r.> ....
....
<" t':>
c
0
c,
::3
J'
~
:;l-
'<
t:O n N '<
Y'
c:> ....
rc
'"::l
0
l'
'" '" rc
t':>
::r:
0
p...
:;l
0
~ -e .... N
0 0
51 52 53
41 42 43 44 45 46 47 48 49 50
31 32 33 34 35 36 37 38 39 40
-
-
-
-
+ + + + + + + + + + + + + + + + +
-
-
-
-
25
26 27 28 29 30
-
24
+ + + +
-
+ + + + + + + + + + + + + +
-
-
-
-
+ + + + + + +
-
+ + + + + + + + + + + +
-
-
-
-
-
+
-
-
-
-
-
-
-
-
+ + + + -
-
+ + + + + +
-
+ + +
-
+
-
-
+ +
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
+
+
-
-
-
+ + +
+
-
-
-
+
-
-
-
-
-
-
-
-
+ -
-
-
-
-
-
-
-
-
-
-
-
-
+
-
+ + + + +
+ + + + +
-
-
-
-
-
-
+
-
-
-
-
-
-
+ +
-
-
-
-
-
-
-
-
-
+ + + +
+ +
-
-
-
-
-
-
+
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
+ -
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
+
-
+ + +
-
+
-
-
-
-
-
-
-
-
-
-
-
-
+
+ + + + +
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+ 2
1 15
1
1 1 1
3
4
2 1 2
5 1
1
1 1
1
1
1 1 1
2 3
6
to
14
1 1
1
1 (5) (6) 1 (2) 3
"'::l
::r
0>
.....
w
0
~ .... 0> 5" 'J>
~
~
'J>
0-
'"n
2-
IJ'l
5"
'< >0
'" .:,
(7:l
Phage-Typing of Klebsiella Strains
303
Table 4. Frequency of occurrence of bacteriophage types in Klebsiella pneumoniae and Klebsiella oxytoca Tabelle 4. Haufigkeit der Phagtypen bei Stammen von Klebsiella pneumoniae und Klebsiella oxytoca Phage types
Number of strains Klebsiella pneumoniae ' oxytoca 2
Phage types
Number of strains Klebsiella pneumoniae 1 oxytoca 2
1 2 3 4 5 6 7 8 9 10 11
6 2 1 1 1 1 1 1
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67
1
7 3 2
5
1 1 1 1
12
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
1 1 8
2 1 1 8
6 1 1
2 1
4 3 1 2 5 5 2 1
1 1
1 1 6 1
9
Total number of strains:
1
138
2
5 6 2 2
1 9 4 4 2 1 1 1 1
1 1 1 1 2
1 3 1 2 1 2
1 1 1
2 1 10 1 1 4 5 12 3 7 3
()
87
are laborious (5). Moreover, anti K sera are not commercially available. Due to these difficulties, serotyping cannot be used as a common method and has to be replaced by phage-typing of Klebsiella strains without their previous serological classification. Lysotyping within previously defined antigenic types seems to be the most efficient and precise way of differentiation (12). The strains used in our ex-
-
61 62 63 64 65 66 67
-
-
-
-
-
-I-I-
-
-
+ +
-
3
-
-I-I-
-
-
-
8
-
-
-
-I-I-
+
-
-
-
-
-I-
-
-
13
-
-
-
-
-
12
-
-
9
-
+ -
-
+
-I-
-
-I-
+
-
-
-
-
-
-
-
-
-
-
-
-I-
21
-
-
+
-I-I-
-I-
19
-I-
-
+
-
-
-
16
14
Bacteriophages
-
-
-
-
27
Confluent lysis, opaque lysis, sernico nfluent lysis are designated as ( -I-) . Negative reacti on is designat ed as (-) Tota l number of strains is given in bra ckets.
T otal n umber of strains :
-
-
-I-I-I-
-
54 55 56 57 58 59 60
+
2
1
Phage typ es
-
-
-
-
-
-
-
-I-
-
-
+
-
-
-
-
-
34
-
30
-
-
-
-
-
-
-
-
-
-
-
-
-
-
42I1
4B
-
-I-
-
+
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
106B 171I1 765n
101
124
1 4 5 12 3 7 3
1 1 1 2 1 16 1
Ori gin and n umber of strains Col ogne Wrodaw
w
'"0
~
(1)
,.,
<"
r=
0
:::l
P-
J'
.,'"
'"
:;l-
-e
N
-e r,
I;l:I
?'
J"
~
,.,
(1)
;.."
o
.'"
(1)
~
(1)
::r:
o
P-
0 :::l
N
>,.,;.."
0 N
304
A.Przondo Hessek, G.Peters, BiByczyriska, and G.Pulverer
Table 5. Klebsiella phage types among various clinical isolates Tabelle 5. Verteilung der Phagtypen bei Klebsiella-Stammen aus unterschiedlichem kIinischem Material Phage type
1 2 3 4 5 6 7-8 9 10 11 12 13 14 15 16 17 18-19 20
21 22 23 24 25 26 27-30 31 32 33 34 35 36 37 38 39 40 41-43 44-47 48 49 50 51 52-53
Number of Klebsiella pneumoniae from Respira- Wound Urine tory tract
Number of Klebsiella oxytoca from Respira- Wound Urine tory tract
Total
5 1
5 3 2
2 1
2
4
13 5 3 1 1 6 2 1 1
1
1
1 1 2 1 1 1
1
1 1 1 7
1
1 1
1
1
7
1
1
1 2 2 2 1
2
2
2
1 2
5 3 1 2 5 6 5
1 2 3 4 5 7
2 1
2
1
2 1
1 5 6 2 2
1 9 4 4 2 1
1
1 3
1
1 15 1 1 1 14 10 6 2 3
1 3 1
3 4 1 5 1
1 3
2 3
3 4 2
1 1 3 9 1 14 2 2
Phage-Typing of Klebsiella Strains
1 2 3 16 36
1
54 55-56 57-58 59 W-67
3 10 36
Total
118
2 6
11
6
64
305
21
5
225
periments were not serologically typed. For the phage-typing numerous phages of various lytic range were tested, they were isolated from various sources, selected and propagated on serologically different Klebsiella strains, thus they achieved lytic activity confined to various capsular types (7). No differences were found in the sensitivity to typing phages between strains of Klebsiella pneumoniae and Klebsiella oxytoca. Out of 36 phage-types, represented by more than one strain, 16 were composed of Klebsiella strains of both species (Table 4). This phenomenon can be explained by the occurrence of the same serotypes in the above species (1). Marked disproportions in the number of strains from three different clinical sources did not permit the analysis of the participation of particular phage-types in different infections. However, our investigations suggest that strains belonging to the same phage-type may induce infections in the respiratory and urinary tracts as well as in the soft tissues (Table 5). Substantial differences in phage sensitivity patterns were observed in strains from Cologne and Wrodaw, only 6 phage-types were common. Similar results of differing Klebsiella phage-types in connection with the place of their isolation have been previously observed in Klebsiella rhinoscleromatis (10). Analogous data were observed when the results of Klebsiella serotyping were analysed (1, 2, 7, 11). Our investigations confirm in accordance with the data of RENNIE et al. (12) the usefulness of phage-typing for the epidemiological work on Klebsiella strains. References 1. Blanchette, E. A. and S. J. Rubin: Seroepidemiology of clinical isolates of Klebsiella in
Connecticut. J. Clin. Microbio!. 11 (1980) 474-478 2. Hill, H.R., C.E.Hunt, and J.M.Matsen: Nosocomial colonization with Klebsiella type 26, in a neonatal intensive-care unit, associated with an outbreak of sepsis, meningitis and necrotizing enterocolitis. J. Pediat. 85 (1974) 415-419 3. Meitert, T. and E. Meitert: Usfulness, applications and limitations of epidemiological typing methods to elucidate nosocomial infections and the spread of communicable diseases. In: T.Bergan and ].R.Norris: Methods in Microbiology, Vo!. 10. Academic Press, London-New York-San Francisco (1978) 4. Montgomerie,]. Z.: Epidemiology of Klebsiella and hospital-associated infections. Rev. infect. Dis. 1 (1979) 736-753 5. 0rskov, F. and I. 0rskov: Serotyping of Enterobacteriaceae, with special emphasis on K antigen determination. In: T.Bergan and J.R.Norris: Methods in Microbiology, Vo!. 11. Academic Press, London - New York - San Francisco (1978) 6. 0rskov, I.: Nosocomial infections with Klebsiella in lesion of the urinary tract. Acta path. microbio!. scand. 93, Supp!. (1952) 259-271
306
A.Przondo Hessek, G.Peters, B.Byczynska, and G.Pulverer
7. Przondo Hessek, A.: Bacteriophages of Klebsiella bacilli. Isolation properties and use
in typing. Arch. Immunol. Ther. Exp. 14 (1966) 413-435 8. Przondo Hessek, A. and B. Byczyl1ska: The properties of Klebsiella isolated from patients with respiratory infections. Med. dosw, Mikrobiol. 32 (1980) 31-38 9. Przondo Hessek, A. and S.Slopek: Typing of Klebsiella bacilli by means of the Klebsiella bacteriophages of the collection of Milch and Deah, Arch. Immunol. Ther. Exp.16
(1967) 578-582 10. Przondo Hessek, A., S. Slopek, and ]. Miodonska: Phage typing of Klebsiella rhinoscleromatis. Arch. Immunol. Ther. Exp. 16 (1968) 402-405 11. Reniie, R.P. and I.B.R.Duncan: Combined biochemical and serological typing of
clinical isolates of Klebsiella. Appl. Microbiol. 28 (1974) 534-539 12. Rennie, R.P., C.E.Nord, LiSioberg, and I.B.R.Duncan: Comparison of bacteriophage
typing, serotyping and biotyping as aids in epidemiological surveillance of Klebsiella infections. J. Clin. Microbiol. 8 (1979) 638-642 13. Terman, J.W., R.H.Alford, and R. E. Bryant: Hospital acquired Klebsiella bacteremia. Amer. J. med. Sci. 264 (1972) 191-196 Prof. Dr. Gerhard Pulverer, Hygiene-Institut der Universitat, Goldenfelsstr. 21, D-5000 Kaln 41, FRG Dr. Anna Przondo Hessek, Department of Microbiology, Medical Academy, ul. Chaiubinskiego 4,50-368 Wrodaw, Poland