Increased Typability of Multiresistant Staphylococcus aureus by Reverse Phage Typing

Zent.bl. Bakteriol. 289, 37-46 (1999) © Urban & Fischer Verlag

Zentralblatt fUr

~~k tel'iolOSIf:

Increased Typability of Multiresistant Staphylococcus aureus by Reverse Phage Typing Miriam Ben-Yaakov 1, Zi lia Lazarovich l ,2, Nathan Boldur 1 *, and Ida Boldur 1, 2 I

Department of Microbiology, Assaf Harofeh Medica l Center, Zerifin

2

Department of Life Sciences, Bar IIan University, Ramat Gan, Israel

Received March 20, 1998 . Rev ision received August 14, 1998· Accepted September

24, 1998

Summary Sixty percent of Staphylococcus aureU$ isolates from patients in Israeli hospitals proved to be non typablc by the conventional phage typing method. Hear pretreatment improved typabiliry only to 54 % while reverse typing increased rypabiliry to 75 %. In genera l isolates typablc by conventional phages belonged to group V, H, III, I, or to mixed groups. While isolates typable only by reverse typing belonged to group III, II, the extended group III + or to mixed groups, but seldom to group I. Although most isolates were resistam to penicillin G, onl y onc half were resistant to other antibiotics as we ll . Whi le one third of these isolates could be typed by conventiona l phage typing, typability was significantly improved to over 80 %, by the usc of reverse typing as the additional typing method. Two ma in groups of oxacillin resistant isolates were identified. The partial resistant group consisting of isolates resistant to penicillin G and oxacill in with no or few ot her resistances. These iso lates were mostly typab le by conventional phage typing (group V) and dominated in the first study period (1989-1990) but were only seldom isolated in the second one ('1991 - 1991). The mu ltiresistant group consisted of iso lates resistant to penicillin G and oxaci llin accompanied by res istances to 3-5 other ant ibiotics (ch loramphenicol, c1indamycin, eryth romycin, gentam icin and tetracycline). These iso lates were mostly typablc by reverse typing (the extended group II I + lIla ) and showed no change in isolation frequencies during the entire study period. Reverse typing is proposed by us as a typ ing tool for rhese multiresistant S. aureus isolates.

lila.

IIPresent Address: Department of Ophthalmology Bronx-Lebanon, Hosp ital Center NY 1045 7 USA.

0934-8840/991289/ 1·037 $ 12.0010

38

M. Ben-Yaakov et al.

Introduction Staphylococcus aureus is among the most prominent pathogens in both community-acquired and nosocomial infections (3, 6, 7, 10, 12, 14-16). The emergence of significantly high percentages of non typable virulent S. aureus clinical isolates urged us to explore alternative typing methods. Special emphasis was put on clinical isolates resistant to a wide variety of antibiotics including oxacillin. These isolates are involved in development and spread of nosocomial infections, causing problems in their treatment and eradication. A variety of typing methods of S. aureus are suggested, including traditional as well as molecular typing methods (2, 3, 5-7,11-13,15,16). The aim of the present study was to evaluate the use of relative simple typing methods for multiresistant S. aureus isolates that can be performed in laboratories not equipped for molecular typing. The use of reverse phage typing, originally developed for coagulase negative stapylococci (4) proved helpful in increasing typability of our S. aureus isolates (8).

Materials and Methods Bacterial isolates: The study included 2392 S. aureus isolates from sporadic infections

collected during a period of 4 years (1989-1992) from blood, wounds, respiratory tracr and other organs of patients hospitalized at the'" Assaf Harofeh" Medical Center. All isolates were confirmed as S. aureus by the coagulase rest. We also included in one part of the study 714 S. aureU5 isolates from 6 additional hospitals from all over the country, sent to our laboratory fur phage typing during the same period. All isolates were first typed by the conventional phage typing method and only those remaining non typable were further tested by other phage typing methods. 1. Conventional phage typing (PT): The international set of phages used for direct

typing comprised 28 phages grouped as follows: group I: 29, 52, 52A, 79, 80. group II: 3, 3C, 55, 7l. group III: 6,42E, 47, 53, 54, 75, 77, 83A, 84, 85, alone or extended by the experimen-

tal phages 88, 89 and 90 (called by us - III') which are used to differentiate group III strains (17). group V: 94, 96, D", HK,. group M: 81,95. Isolates were typed both at Routine Test Dilution (RTD) (1) and at 100 RID. 2. Heat pretreatment: Isolates were phage typed by PT after growth in nutrient broth No.2 (Oxoid, Unipath Ltd., Basingstoke, Hampshire, England) at 48 'C for 4 h (5). 3. Reverse typing (RT): RT was carried out by adaptation of the De Saxe and Notley method described for coagulase negative staphylococci (4 ). Lysogenic phages were

induced by mitomycin C (0.5

~glml)

(Sigma, M-503, St. Louis, USA) and their lytic

spectra tested on the propagating strain battery used for routine typing phages.

Reverse typing of multiresistant S. aureus

39

Antibiotic Susceptibility Tests Susceptibilities were determined by the disk diffusion method on Mueller-Hinton agar plates (Oxoid), with an inoculum of 10 5 CFU/ml. The following antibiotics were tested: chloramphenicol (C) - 30 Ilg, clindamycin

(Cm)- 2 ~g, erythromycin (E) - 15 units, fusidic acid (F) - 10 ~g, gentamicin (Gm) 10 fig, mcthicillin (M) - 5 ltg, oxacillin (Ox)- 5 1% penicillin G (PG) - 6 ~g, rifampicin (R) - 30 f'g, tetracycline (Tc) - 30 f'g, vancomycin (V) - 30 ~g. Since methicillin disks are considered relatively unstable upon storage, oxacillin disks which are more stable and better standardized were used in parallel in the study but results are presented only for oxacillin resistance.

Statistics The significance of data was determined using the Chi Square Test.

A probability value (p) of < 0.05 was considered significant.

Results Methods used to increase typability of s. aureus isolates [n a preliminary study 469 out of 1189 S. aureus isolates were typable by PT. When isolates were heat treated prior to typing (Fig. 1) additional 173 isolates could be phage typed. The use of RT as the additional typing method increased typability to a total of 892 isolates (75 %). This method was therefore used as the additional typing method in further investigations.

PHAGE TYPING

/ + HEAT PRETREATMENT

rIll1

EJ

+ REVERSE TYPING

TYPABLE NON·TYPABLE

Fig.1. Phage typing of S. aureus: Isolates non typable by conventional phage typing were further typed using heat pretreatment and reverse typing.

40

M. Beo-Yaakov ef al.

Typability of S. au reus isolates from different hospitals A collection of 2392 S. aureus isolates (including the preliminary study) from our medical center (hospital 1) and 714 isolates from six other centers (hospitaI2-7) were phage typed (Table 1). Only isolates from hospital 3 showed a significantly different typing pattern (p < 0.05).

Typability of S. aureus isolates from different anatomical sites No difference in typing patterns and improvement of typability by RT, were found for isolates from blood or pus (Table 2). Isolates from other sites showed reduced typability by RT (p < 0.05 compared to blood isolates) leaving 31 % of these isolates NT (oon typable).

Table 1. Typability of S. aureus isolates by hospital Hospital

No. of Isolates

Phage typing No.

1 2 3 4 5 6 7

2392 429 115 75 47 27 22

962 185 89 30 26 4

Total

3107

1307

11

(%)

Reverse typing

Non typable

(% )

No.

(%)

(35) (33) (16j> (40) (30 ) (52) (50)

599 103

(40) (55) (15 )' (50)

831 141 18 30 14 14 11

(25) (24) (7j> (20) (IS) (33) (0)'

(42)

1059

(34)

741

(40) (43) (77)'

No.

8

IS 7 9 0

(24)

'p < 0.05 compared to hospital I (Chi Square test ).

Table 2. Typability of S. au reus isolates by site Isolates

No.

Phage ryping

Reverse typing

Non rypablc

from

Blood Pus

Other·

351 1313 728

No.

(%)

No.

(%)

No.

(% )

123 558 281

(35) (42 ) (38)

141 486 222

(40) (36) (31)1

87 287 225

(22)

.. Respiratory tract and genitals. I p < 0.05 compared to blood isolates (Chi Square tcst).

(25 ) (31 )

Reverse typing of multiresistant S. aureus

41

Phage typing groups Out 01962 S. aureus isolates typable by PT (Fig. 2), 205 belonged to group V (21 'Yo) , 198 co group If (21 'Yo), 129 belonged to group III (13 'Yo) , 100 to group I (10 %) and 54 to the extended group III + III' (6 %). The rest 276 PT typable isolates (others) belonged co mixed groups (29 'Yo). Out of the 831 isolates typable only by RT, 203 belonged to group III (24 %),172 co group n (21 %) and 144 co the extended group III + Ill' (17%), but seldom to group l. The other 307 RT typahle isolates belonged to other groups or mixed groups (3 7%).

Susceptibility of S. aI/reus isolates to antibiotics Information about susceptibility co 1.0 different anribiotics is presented for 1330 S. aureus isolates (Table 3). Sixty seven isolates were susceptible to all tested antibiotics. Resistance to PG alone was detected in 641 isolates (48 %), while the other 556 PG resistant isolates were resistant to other antibiotics as well. Two hundred and fifty six isolates (19 %) were resistant to Ox. The

rh~gl

"

Gumps

0"',"

Fig.2. Typabi lity and phage typing groups of S. aureli, isolates. Typing methods: conventional phage typing (PT) and teverse typi ng (RT). Non typable (NT): by both methods. Phage group' (international set): I, II, Ill, V, M , the extended group UJ + Ill' and

other combin:lti o ns (others ).

42

M.Ben-Yaakov et al.

Table 3. Susceptibility of 1330 S. au reus isolates to antibiotics and their phage typing patterns

Total

Resistance to

PC (aU) PG (only) Tc Ox

Non typable

(%)

No.

(%)

No.

(%)

No.

(%)

1197 641 282 256 245 230 187 131

479 267 85 105

(40) (41) (30)(41) (31)' (32)(24)(24)-

446 208 146 111 125 125 119 84

(37) (33) (52)(43) (51 )' (54)' (64)' (64)'

272 166 51 40 43 32 24 15 4 4

(23) (26) (18) (16) (18) (14) (13) (12)

12

(25)

38

(57)

F

12

R

10

none

67

(5)

C Cm Cm

Reverse typing

No.

(90) (48) (21) (19) (18) (17) (14) (10) (0.9) (0.8)

E

Phage typing

77 73 44 32 5 4

3 2 (18)

17

'p < 0.05 compared to PC (all) (Chi Square test).

frequency of resistance to each of the other antibiotics ranged from 10 to 21 % for em, Gm, e, E, and Tc, and from 0.8 to 0.9 % for, Rand F.

Typability of antibiotic resistant S. aureus isolates by phages Forty percent of the PG resistant isolates were typable by PT, 37 % by RT and 23 % remained NT (Table 3). Typability of isolates resistant to Ox, did not significantly differ. Isolates resistant to T c, E , e, Gm or em had significantly different typing rates. Only 24-32 % could be typed by PT (p < 0.05), while 51-64% could be typed by RT (p < 0.05) leaving only 12-18% of the isolates NT (Table 3).

Oxacillin resistance The 256 isolates resistant to Ox could be divided into two groups according to their resistance profile (Table 4). 1) The partial resistant group: consisting of 110 isolates resistant to PG, Ox and to :5 2 of the following antibiotics: C, Cm, E, Gm, T c. 2) The multiresistant group: consisting of 146 isolates resistant to PG, Ox and to ~ 3 of the above mentioned antibiotics. Seventy of the 110 partial resistant isolates were typable by PT (64%) including 47 isolates belonging to phage type group V (43 %). Seventeen (15 %) were typable by RT, leaving 23 isolates (21 %) NT.

110

146

partial

multi

A B

A B

71 75

93 17

isolation Total period! No.

1

0

0

26

0

9

64 6

No. I

0 0

8

0

II

5 14

1

III

0 1 1

42 5

V

2 4

1 0 1 7

11

IIl+III" others

Phage typing groups

I

multi - resistant to PG, Ox with 3-5 of C, em, E, Gm, T c. Isolation period: A: 1989- 1990, first two study years (698 isolates), B, 1991-1992, second two study years (632 isolares).

* Resistance profile: partial - resistant to PG, Ox with 0- 2 of C, Cm, E, Gm, Tc,

No.

Resistance profile *

Table 4. Phage typing patterns of oxacillin resistant S. aureus isolates

39

55

14 3

0

0

0 0

No. I

0 0

0

5

II

10 3

2

0

III

1 0

1 1

V

42 35

0 0

2 1

6 2

lII+IIIa others

Reverse typing grou ps

7 10

15 8

Non typable No.

10.

w

"' ...

"

~

~

"

;-0

fZ g

~

". "

g.

3

""~

~

'< -g.

~

~

~

< ~

'"

44

M. Ben-Yaakov et aJ.

Only 35 multiresistant isolates were typable by PT (24 %) whereas 94 isolates were typable by RT (64 %), including 77 belonging to the extended reverse phage type group III + III'. Only 17 (12 %) multiresistant isolates remained NT. In the first two study years (1989-1990) 164 out of 698 isolates (24%) were resistant to Ox, while during the second two srudy years (1991-1992) only 92 out of 632 isolates (15%) were Ox resistant (p <- 0.05). During the first period 93 isolates (57%) were partial resistant, while during the second period, only 17 (18 %) belonged to this group (p < 0.05). No significant change in isolation of multiresistant S. aureus during the complete study period was recorded (p = 0.37).

Discussion In this study we investigated the possibility to improve typability of S. au reus clinical isolates, by methods available in laboratories not equipped for molecular typing. Sixty percent of S. au reus isolates from sporadic infections in Israeli hospitals proved to be non typable by the conventional phage typing method. Variable typability rates are reported from different parts of the world (3, 7, 10, 15). Heat pretreatment - a procedure suggested for increasing susceptibility of S. aureus isolates to phages - has, in our hand, only minor contribution to improved typability, though found useful by other investigators (5, 15). When we used the reverse typing method - based on host ranges of phages carried by the clinical isolates under investigation (15, 16) - in combination with the conventional phage typing method, we were able to type 75 % of our isolates. Typability of S. aureus isolates from different Israeli hospitals is comparable, with only one exception (hospital 3). Resistance to PG could be detected in 90 % of our isolates, as expected (10, 14). Resistance to other antibiotics is present in one half of our isolates. Some are resistant to a single or few, while others are resistant to a variety of antibiotics. Almost 20 % of our isolates are oxacillin resistant and comptise a category of great interest since they may playa major role in nosocomial infections

(2-3,6-7,9,14,16). Oxacillin resistant isolates can be divided into two main groups according to their resistance profile and typability. A partial resistant group includes isolates resistant to PG and Ox accompanied by $ 2 other resistances and a multiresistant group including isolates resistant to PG and Ox together with 2e: 3 other resistances. Most of the partial resistant isolates can be typed by conventional phage typing, especially by phages of group V, a restriction enzyme determined group. Isolates typable by group V are hardly ever reported by others to be oxacillin resistant (R. R. Marples - personal communication). These partial resist-

Reverse typing of multiresistant S. aureus

45

ant isolates predominate in our first study period (1989-1990) but are seldom isolated in the second one (1991-1992). We arc unable to exclude the possibility that these isolates are really only borderline resistant to oxacillin (BORSA). Multiresistant isolates are mostly non typable by conventional phage typing but are typable by reverse typing, especially by the extended group III + III' . These multiresistant isolates can be isolated during the entire study period and due to the disappearance of the partial resistant isolates - in the second part of the study - the multiresistant isolates become the major oxacillin resistant group. The use of reverse typing suggested in this study for the typing of S. aureus, enables typing of these otherwise untypable oxacillin resistant isolates. The present study only investigated typability of S. aureus isolates. Further studies are required to test the discriminatory power of this method in order to compare its value as an epidemiological tool with other typing methods. Acknowledgements. We thank Dr. R. R. Marples and Dr. J. E Richardson, Staphylococcal laboratory, Epidemiological typing unit, London, for reviewing the manuscript. We thank L. Sarel for technical assistance, Mrs. S. Zarini and Mrs. A, Cranit Sharifi for secretarial help.

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M. Ben-Yaakov et al.

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13. Tenover, 1'. C, R. Arbeit, G. Archer, j. Biddle, S. Byrnes, G. Goering, G. Hancock, G. A. Hebert, B. Hill, R. Hoollis, W. R.jarvis, B. Kreiswirth, M. A. Eisner, j. Maslow, 1. K. McDougal, j. M. Miller, M. Mulligan, and M. A. Pfaller: Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureu5.]. Clin. Microbiol. 32 (1994) 407-415 14. Thornsberry, c.: The development of antimicrobial resistance in staphylococci. J. Antimicrob. Chcmother. 21 (supp.C) (19.88) 9-16 15. Vinde/, A. , C. Martin-Bouran, and }. A. Saez-Nieto: Characterization of non-typable strains of Staphylococcus aureus from cases of hospital infection, Epidemiol. Infect. 99 (1987) 191-200 16, Wilkinson, D. N., S. Andrews, and R. P. Stewart: Bacteriophages associated with multiresistant Staphylococcus aurells in Australia. J. Med. Microbiol. 23 (1987) 119-126 17. Westh , H., j. a.jarlov, H. Kiersem, and V. T Rosdahl: The disappearance of multiresistant Staphylococcus aureus in Danemark: Changes in strains of the 83A complex between 1969 and 1989. Clin.Infect. Dis. 14 (1992) 1186- 1194 Corresponding author: Dr. Miriam Ben- Yaakov, Department of Microbiology, Assaf

Harofeh Medical Center, Zerifin, 70300, Israel, Tel.: 972-8-9779874, Fax: 972-89779865