In recent years, methicillin-resistant Staphylococcus aureus (MRSA) isolates in Spain have increased dramatically; in 1986 there were only 1.2% MRSA amongst all nosocomial Staphylococcus aureus (SA) isolates, by 1989 this percentage had risen to 44% in some hospital causing a very serious epidemic situation in the country. We have characterized these isolates by direct, reverse and Fisk phage typing and we have also looked for an additional local set of phages to help us to differentiate these strains. We have been able to differentiate an epidemic strain from other MRSA strains which cause sporadic hospital outbreaks, and we have also distinguished between some variants of the epidemic strain.This research has been supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISS), Ministerio de Sanidad y Consumo, Spain; No. 93/0144.S

Aparicio, Pilar

Boquete Blanco, Maria Teresa

Gomez, E

Martin de Nicolas, M

Saez-Nieto, Juan A

Trincado, Pilar

Vindel, Ana

Epidemiology and Infection

English

Saez-Nieto, Juan Antonio

REPISALUD

Epidemniol. Infect. (1994), 112, 299-306 299Copyright C) 1994 Cambridge University PressAn additional set of phages to characterize epidemic methicillin-resistant Staphylococcus aureus strains from Spain (1989-92)A. VINDEL, P. TRINCADO, E. GOMEZ, P. APARICIO, M. MARTIN DENICOLAS, T. BOQUETE AND JA. SAEZ NIETOLaboratorio de Referencia de fagotipia de S. aureus, Servicio de Bacteriologt'a,Centro Nacional de Microbiologt'a, Virologt'a e Inmunologt'a Sanitarias, Instituto deSalud Carlos III, 28220 Majadahonda, Madrid, Spain(Accepted 17 October 1993)SUMMARYIn recent years, methicillin-resistant Staphylococcus aureus (MRSA) isolates inSpain have increased dramatically; in 1986 there were only 1P2% MRSA amongstall nosocomial Staphylococcus aureus (SA) isolates, by 1989 this percentage hadrisen to 44% in some hospital causing a very serious epidemic situation in thecountry. We have characterized these isolates by direct, reverse and Fisk phagetyping and we have also looked for an additional local set of phages to help us todifferentiate these strains. We have been able to differentiate an epidemic strainfrom other MRSA strains which cause sporadic hospital outbreaks, and we havealso distinguished between some variants of the epidemic strain.INTRODUCTIONDuring the past 10 years, methicillin-resistant Staphylococcus aureus (MRSA)strains had been causing important hospital infections around the world [1]. InSpain the percentage of MRSA isolates was formerly very low (1 2 %) [2] exceptfor some local problems [3], but late in 1989 MRSA strains caused importantnosocomial outbreaks in three Spanish hospitals from two regions. During 1990these MRSA strains spread throughout the country affecting several hospitals [4].Since then, there has been a constant increase in the epidemic situation; and in1991-2 MRSA strains were isolated from 31 hospitals from ten different regions.In this paper we report the results of phage typing 3759 MRSA isolates, theselection of an additional set of phages extracted from a collection of 115 MRSAisolates from different hospitals during the 1989-91 period, and the patternsobtained after applying such phages and ten supplementary phages for MRSA [5]to a group of 1415 MRSA strains (1376 epidemic and 39 non-epidemic).MATERIALS AND METHODSIsolatesA total of 3759 MRSA isolates was received in our laboratory from 31 Spanishhospitals between October 1989 and December 1992.12-2A. VINDEL AND OTHERSPhage typingPhage typing was performed by the routine method [6]. Non-typable isolateswere also phage typed after heat treatment [7]. According to the results obtainedwith direct phage typing we selected 202 isolates to perform further studies.Reverse typingProphages were induced by mitomycin C [8] from 2639 isolates non-typable bydirect phage typing and from the 202 selected strains and these were matched withthe propagating strains of the International set of S. aureus phages.Fisk typing (Cross spotting)Induced supernates from each of all the 202 selected strains were also spotinoculated onto lawns of all the 202 strains [9].Antimicrobial susceptibility testingThe 202 isolates were examined using 15 antimicrobial drugs: penicillin,clarithromycin, clindamycin, rifampicin, fosfomycin, erythromycin, neomycin,fusidic acid, vancomycin, gentamicin, ciprofloxacin, cloxacillin, methicillin,bacitracin and mupirocin.Minimal Inhibitory Concentrations (MICs) were determined by the agardilution method [10]. The isolates were assigned to susceptibility in accordancewith NCCLS criteria [11].Selection of a local set of phagesTo get an additional set of local phages, 115 isolates were selected according totheir phage pattern and origin amongst the total number of MRSA isolatesreceived in our laboratory during the period 1989-91. Prophages were inducedfrom isolates with mitomycin C and they were spotted onto all parenteral strains.Eight phages were selected because of their high percentage of lytic capacity anddiscriminatory efficacy. These phages were titrated on several strains and the mostsensitive was selected to propagate them. These phages were designated withnumbers from 30 to 38 (except number 36). The source and origin of the lysogenicand propagating strains are shown in Table 1. They were propagated by the softagar overlay method [12] and their lytic spectra were determined (Table 2).Additional phagesA set of ten supplementary phages for MRSA [5] was kindly sent to ourlaboratory by Dr Marples (CPHL, Colindale, London). We propagated them bythe soft agar overlay method [12].A selection of 1415 MRSA isolates, including isolates from each year of thestudy and from all affected hospitals was phage typed with the supplementary andthe local phages for MRSA strains.RESULTSBy direct phage typing we could differentiate two groups among 3580 epidemicisolates: one group was lysed at RTD with a large number of group III phages(6/42E/47/54/75/77/84/85) and the other group was non typable but was typedafter heat treatment with phages from groups I and III (29/77/84) (Table 3).300Additional phages for MRSA 301Table 1. Origin of lysogenic and propagating MRSA strains and designation ofphagesLysogenic strains Propagating strainsA A ,s 5 DesignationNumber Source Origin Number Source Origin of phages8361 Blood Madrid 8789 Sputum Granada 304114 Wound Sevilla 9302 Sputum Barcelona 315715 Wound Sevilla 6594 Blood Madrid 328261 Urine Sevilla 543 Unknown Madrid 336596 Unknown Madrid 44 Blood Sevilla 348762 Skin Granada 48 Catheter Sevilla 35709 Blood Madrid 4231 Catheter Madrid 372504 Blood Madrid 1148 Exudate Madrid 38Table 2. Lytic spectra of local phagesPhages*Strain 30 31 32 33 34 35 37 3829 - 052 0 - 052A 1 1 3 1 3 180 1 4 1 2 1 0 12009 0 4 0 03A - 1 1 1 1 18719 042C 0 0 042E 0 047 5 4 5 5 5 5 5 553 0 - 0 2 3 4 454 0 4 3 2 3 3 4 475 0 0 2 4 0 4 277 0 3 4 3 4 4* , indicates no reaction; 0, indicates inhibition; 1-5, indicates relative strength of reaction.Table 3. Phage typing ofMRSA i8olates in Spain (1989-92)Phage-group NumberYear (method) Phage type (percentage)1989 I-III (HT) 29/77/84 147 (51 8)III (RTD) 6/42E/47/54/75/77/84/85 113 (48 2)1990 I-III (HT) 29/77/84 493 (63 1)III (RTD) 6/42E/47/54/75/77/84/85 288 (369)1991 I-III (HT)* 29/77/84 614 (793)III (RTD) 6/42E/47/54/75/77/84/85 109 (144)I-III (100 RTD) 29/42E/53/83A 51 (66)1992 I-III (HT) 29/77/84 1385 (71-2)III (RTD) 6/42E/47/54/75/77/84/85 431 (222)I-III (100 RTD) 29/42E/53/83A 113 (58)III (RTD) 47/53/83A/84 15 (0 8)* HT, Heat treatment.A. VINDEL AND OTHERS198919900 "IWI __W1Andalucia Aragon Cast-Leon Cataluna Madrid C. Valenciana_ Group III m Non-typableFig. 1. Evolutioni of two phage-group between epidemic strains (1989-92).3028010080604020012010080604020010080604020Additional phages for MRSATable 4. Phage typing of 1376 epidemic MRSA with additional MRSA phagesPhage-groupI-III IIINumber Number(percentage) (percentage)Supplementary MRSA phagesNT 979 (71 1) 97 (7)618/622/626/630 110 (8) 190 (139)Local MRSA phagesNT 43 (31) 4 (03)34 59 (4 3) 18 (1 3)33/34/37/38 293 (213) 27 (2)30/32/33/34/35/38/38 673 (489) 237 (172)32 8 (06) 1 (01)38 13 (0 9)The distribution of these two groups of strains around the country washeterogeneous. Figure 1 shows the distribution of these two phage-groups ofepidemic strains during the 1989-92 period in the most affected regions.There were 179 isolates corresponding to two different outbreaks not relatedwith the epidemic strains, one typed with phage group I-III (29/42E/53/83A)and the other was lysed by phages from group III (47/53/83A/84) (Table 3).Phage typing with additional phagesA selection of 1415 MRSA isolates was studied by applying two sets ofadditional phages. The relationship between the patterns obtained with both setson the epidemic strains are shown in Table 4.After applying the MRSA supplementary set at 100 RTD we could distinguishtwo groups among the epidemic strains and also we differentiated a group of non-epidemic strains.With the nine local phages obtained in our laboratory, we could differentiateclearly the non-epidemic from the epidemic strains and we also distinguished twomajor and two minor groups among the epidemic strains.The evolution of the majority patterns obtained with the additional phagesamong the epidemic strains is shown in Figure 2.All the epidemic strains tested, including both groups of non-typable andtypable isolates showed a very similar pattern by reverse phage typing. But wecould differentiate clearly the non-epidemic strains.With the cross spotting (Fisk typing) we could differentiate at least two maingroups among the epidemic strains. Again, the non-epidemic strains were clearlydifferent.Antibiotic susceptibilityAll epidemic strains of both phage groups were resistant to penicillin (MIC >32 mg/l), clarithromycin (MIC > 129 mg/l), clindamycin (MIC > 8 mg/l), fosfo-mycin (mean MIC 64 mg/l), neomycin (MIC 32 mg/l), gentamicin (MIC 64 mg/l),30330410080604020A. VINDEL AND OTHERS- NT + Pattern 180011989 1990 1991*-- NT -6- Pattern 1 x Pattern 2 -6 Pattern 31992Fig. 2. Evolution of the major patterns obtained with additional MRSA phages.bacitracin (mean MIC 25 mg/i), ciprofloxacin (MIC 16 mg/i), cloxacillin (MIC128 mg/i) and methicillin (MIC 256 mg/i). Susceptibility to rifampicin wasreduced (MIC 4 mg/i). They were uniformly susceptible to the other non-betalactam antibiotic tested.The two non-epidemic strains had different antibiotic susceptibility patternsbetween them and with respect to the epidemic strains. One was susceptible torifampicin (MIC 0-12 mg/i) and ciprofloxacin (MIC 0-12 mg/i) and the other wassusceptible to clindamycin (mean MIC 0-06 mg/i), rifampicin (MIC 0-12 mg/i) andciprofloxacin (MIC 0-5 mg/i).Additional phages for MRSA 305DISCUSSIONIn recent years the incidence of nosocomial infection due to MRSA has increaseddramatically in many Spanish hospitals all around the country. In a survey of 74hospitals, methicillin-resistant strains were 1-5% of the total S. aureus [2], in1990 that percentage has risen to 11-0% [13]. Some hospitals have reported anincidence of 20-25% of MRSA of S. aureus isolates in their hospitals [14].By phage typing we have been able to identify and differentiate betweenepidemic and sporadic strains involved in hospital outbreaks over the last 4 years.Also, phage typing has allowed us to analyse the evolution and dissemination ofthe strains that initially appeared in Madrid and Seville and further spread toother Spanish regions [15].The fact that amongst the epidemic strains we found two different patterns withvery similar epidemic behaviour and the similar patterns obtained by reversetyping lead us to think of a possible single clonal origin. To answer that questionwe performed a deeper study on a selection of isolates, choosing strains from allthe regions and both patterns. After doing the cross-spotting phage typing wecould differentiate several patterns, all of them occurring in both the major phagetyping patterns, which could indicate a divergent evolution of the epidemic strains[4].To investigate this problem further we applied a supplementary set of phagesmore specific for methicillin-resistant strains which were useful to identify a non-related outbreak and to differentiate two groups among the epidemic strains.Again, in both groups there were representative isolates from the two phagetyping patterns. We also looked for additional local phages to increase ourknowledge in relation to the evolution and differentiation of the epidemic isolates.With these local phages we found four main variants and we could discriminatethe non-epidemic strains. The utility of supplementary phages to distinguishMRSA strains had been shown in different studies [16, 17] and we think that itwould be useful to use an international supplementary set of phages to analyse theevolution of MRSA strains.It further remains to apply molecular markers to complete these studies and toobtain an extensive characterization of these epidemic strains.ACKNOWLEDGEMENTSThis research has been supported by a grant from the Fondo de InvestigacionesSanitarias de la Seguridad Social (FISS), Ministerio de Sanidad y Consumo, Spain;No. 93/0144.REFERENCES1. Grubb WB. Molecular epidemiology of methicillin-resistant S. aureus. In: Molecular biologyof the staphylococci. Novick R, ed. New York: VHC Publisher, 1990: 595-606.2. Bouza E, Martinez Beltran J. Estudio multicentrico sobre la prevalencia de estafilococos enEspafia (informe preliminar). Enf Infec Microbiol Clin 1988; 6: 68-79.3. Martin Alvarez R. S. aureus resistentes a la meticilina. Enf Infec Microbiol Clin 1986; 4:15-7.4. Aparicio P, Richardson J, Martin S, Vindel A, Marples RR, Cookson BD. An epidemicmethicillin-resistant strain of S. aureus in Spain. Epidemiol Infect 1992; 108: 287-98.306 A. VINDEL AND OTHERS5. Richardson JF, Chittasobhon N, Marples RR. Supplementary phages for the investigationof strains of methicillin-resistant S. aureus. J Med Microbiol 1988; 25: 67-74.6. Blair JE, Williams REO. Phage typing of staphylococci. Bull WHO 1961; 245: 771-84.7. Vindel A, Martin-Bourgon C, Saez-Nieto JA. Characterization of non-typable strains ofS. aureus from cases of hospital infection. Epidemiol Infect 1987; 99: 191-200.8. De Saxe MJ, Notley CM. Experiences with typing of coagulase-negative staphylococci andmicrococci. In: Phage typing of coagulase-negative staphylococci. Pulverer G, Hecko PB,Peters G, eds. Stuttgart: Fisher Verlag, 1979: 46-59.9. Fisk RT. Studies on staphylococci. II. Identification of S. aureus strains means ofbacteriophage. J Infect Dis 1942; 71: 161-5.10. National Committee for Clinical Laboratory Standards. Method for dilution antimicrobialsusceptibility tests for bacteria that grow aerobically, 2nd edn. Approved Standard, M7A2,NCCLS, Vilanova, Pa. 1990.11. National Committee for Clinical Laboratory Standards. Performance standards forantimicrobial susceptibility testing, Third Information Supplement. NCCLS DocumentM100-S3, Vilanova, Pa. 1991.12. Swanstrom M, Adams MH. Agar layer method for production of high titer phage stocks.Proc Soc Exp Biol Med 1951; 78: 372-5.13. Rodriguez Creixems M. Evolucion de la resistencia a antimicrobianos de Staphylococcusaislados en hospitales espafioles. Enf Infec Microbiol Clin 1992; 10 (suppl. 2): 24-9.14. Alarcon T, Richardson JF, Cookson DD, Lopez-Brea M. Resistance pattern and biologicalcharacterization of methicillin resistant S. aureus clinical isolates. Rev Esp Quimioter 1992;54: 307-11.15. Vindel A, Trincado P, Martin de Nicolas MM, Gomez E, Martin-Bourgon C, Saez-Nieto JA.Hospital infections in Spain. I. S. aureus. Epidemiol Infect 1993; 110: 533-41.16. Vickery AM, Beard-Pegler MA, Rountree PM. Strain differentiation in methicillin-resistantS. aureus. Pathology 1983; 15: 135-240.17. Beard-Pegler MA, Vickery AM. Lysogenicity of methicillin-resistant S. aureus. J MedMicrobiol 1985; 20: 147-55.

An additional set of phages to characterize epidemic methicillin-resistant Staphylococcus aureus strains from Spain (1989-92).

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An additional set of phages to characterize epidemic methicillin-resistant Staphylococcus aureus strains from Spain (1989-92).

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