Resistance of Enterococci to Ampicillin and ...

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Resistance of Enterococci to Ampicillin and Glycopeptide Antibiotics in Italy Roberta Fontana, Marco Ligozzi, Annarita Mazzariol, Gianluca Veneri, the Italian Surveillance Group for Antimicrobial Resistance,* and Giuseppe Cornaglia

From the Istituto di Microbiologia, Universita` degli Studi di Verona, Verona, Italy

We report the results of the monitoring of resistance to ampicillin, vancomycin, and teicoplanin in enterococci by an Italian network of clinical microbiology laboratories. A total of 16,226 strains were analyzed; 9,169 of these strains were Enterococcus faecalis, 913 were Enterococcus faecium, and 6,144 were Enterococcus species. The average rate of resistance to ampicillin was 1.9% (range, 0 – 6.5%) for E. faecalis and 70% (range, 33.3% – 98.7%) for E. faecium; the average rate of resistance to vancomycin was 1.1% (range, 0.1% – 2%) for E. faecalis and 8.5% (range, 0 – 36.1%) for E. faecium; and the average rate of resistance to teicoplanin was 0.8% (range, 0 – 2.4%) for E. faecalis and 8.7% (range, 0 – 36.1%) for E. faecium.

Grant support: These studies were partially supported by grants from the Ministry of the University and Scientific and Technological Research (40% and 60% funding) and from the National Research Council (96.03074.CT04). * Group members are listed at the end of the text. Reprints or correspondence: Dr. Roberta Fontana, Istituto di Microbiologia, Strada Le Grazie, 8, 37134 Verona, Italy. Clinical Infectious Diseases 1998;27(Suppl 1):S84–6 q 1998 by the Infectious Diseases Society of America. All rights reserved. 1058–4838/98/2702–0012$03.00

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Enterococci were identified to the species level by performing conventional biochemical and immunologic tests or by use of commercially available systems such as the API and ATB systems (bioMe´rieux, Rome), the Vitek system (bioMe´rieux), or the Sceptor system (Becton Dickinson, Milan, Italy). Five of the 20 laboratories did not routinely identify all enterococcal isolates to the species level but reserved differentiation between Enterococcus faecalis and E. faecium for serious infections. The susceptibility tests were performed either by use of the disk diffusion method (with automated reading of the halo diameter sizes by bioVideobact [biokit, Barcelona, Spain]) or by use of an automated microdilution test (ATB and Vitek or Sceptor). Manual tests were performed according to the National Committee for Clinical Laboratory Standards (NCCLS)

Table 1. Italian clinical microbiology laboratories involved in a study of resistant enterococci. Center

Location

Ospedali Riuniti Istituto di Microbiologia, Universita` di Brescia Azienda Ospedaliera di Careggi Istituto Ortopedico Toscano Ospedale Generale Provinciale ‘‘San Giuseppe’’ Ospedale Civile Pio Albergo Trivulzio Ospedale Fatebenefratelli Ospedale San Gerardo Dipartimento di Igiene e Microbiologia Istituto di Microbiologia, Universita` di Perugia Ospedale Civile Istituto di Microbiologia, Universita` di Parma Spedali Riuniti Ist. di Microbiologia, Universita` Cattolica del Sacro Cuore Azienda Ospedaliera di Bormio e Sondalo Istituto di Microbiologia, Universita` di Sassari Istituto di Igiene, Universita` di Sassari Ospedale San Bortolo Istituto di Microbiologia, Universita` di Verona

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Bergamo Brescia Florence (1) Florence (2) Empoli Gorizia Milan (1) Milan (2) Monza Palermo Perugia Pordenone Parma Pistoia Rome Sondalo Sassari (1) Sassari (2) Vicenza Verona

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Enterococcal strains that exhibit in vitro resistance to penicillins and glycopeptide antibiotics began to be considered a clinical problem at the end of the 1980s. Two mechanisms have been identified as being responsible for resistance to penicillins: one is based on overproduction of a low-affinity penicillin-binding protein (PBP), which is a normal component of the PBP pattern of these bacteria, while the other is based on the production of a b-lactamase [1, 2]. An increase in resistance to b-lactams is more frequently reported for Enterococcus faecium: these strains do not produce b-lactamase, and the high MICs of penicillin are the extreme examples of the intrinsic resistance common to E. faecium and associated with low-affinity PBPs [3]. Acquisition of glycopeptide resistance was first described in enterococci in 1988 and appeared to be of two types: the VanA type, characterized by high-level resistance to vancomycin and cross-resistance to teicoplanin, and the VanB type, characterized by low-level resistance to vancomycin alone [4]. Since 1993, a national network of clinical microbiology laboratories (the Italian Surveillance Group for Antimicrobial Resistance) has been involved in monitoring resistances to antimicrobial agents in bacteria isolated from human pathological specimens in Italy. This network consists of ú30 clinical laboratories and is managed by the Institute of Microbiology of the University of Verona. Twenty of these laboratories monitored the resistances of enterococci to antimicrobial agents (table 1). The strains were isolated from clinical specimens during the period from 1993 through the first quarter of 1995. The concern caused by emergence of ampicillin and glycopeptide resistances prompted us to analyze the data collected with the aim of determining the incidence of these resistances.

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b-Lactam and Glycopeptide Resistance in Enterococci

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Table 2. Resistances to ampicillin, vancomycin, and teicoplanin of enterococcal strains isolated in 20 Italian centers from 1993 to 1995. Resistance (%) Test method

Ampicillin

Vancomycin

Teicoplanin

Enterococcus faecalis

Brescia (1,678) Florence 1 (1,383) Florence 2 (134) Empoli (138) Milan 1 (721) Milan 2 (83) Monza (1,375) Palermo (294) Pordenone (75) Pistoia (478) Rome (525) Sassari (109)† Vicenza (396) Verona (1,780)

Vitek Vitek Vitek Sceptor Kirby Bauer ATB system Sceptor Kirby Bauer Manual dilution Sceptor More than one ATB system Kirby Bauer More than one

Enterococcus faecium

Brescia (150) Florence 1 (75) Milan 1 (79) Monza (193) Palermo (118) Pistoia (15) Rome (139) Vicenza (38) Verona (106)

Vitek Vitek Kirby Bauer Sceptor Kirby Bauer Sceptor More than one Kirby Bauer More than one

Enterococcus species

Bergamo (1,991) Gorizia (95) Parma (3,219) Perugia (250) Sondrio (589)

KB ATB system More than one Sceptor Kirby Bauer

3.0 0.9 1.5 1.4 3.0 0 1.1 2.7 1.9 0.4 1.1 2.7 6.5 0.8 (1.9)‡ 77.5 76.0 98.7 79.8 57.6 33.3 67.6 73.7 66.0 (70.0)‡ 10.2 2.6 7.8 4.4 5.9 (6.2)‡

2.0 1.0 1.5 0.7 0.1 3.6 0.2 NA 0 0.6 0.9 1.8 1.7 0.2 (1.1)‡ 10.0 2.6 3.8 2.6 NA 0 12.3 36.1 0.9 (8.5)‡ 0.2 3.1 NA 1.6 NA (1.6)‡

1.0 NT NT 0 0.1 2.4 0.1 NA NT 0.4 0.6 1.8 1.7 0.2 (0.8)‡ 10.0 NT 3.8 2.6 NA 0 8.6 36.1 0.9 (8.8)‡ 0.2 3.1 NA 1.6 NA (1.6)‡

Species

NOTE. NA Å data not available; NT Å not tested. * Centers with õ10 isolates are not reported. † The results from the two centers in Sassari were pooled. ‡ Mean percentages.

performance standards [5], and the automated test procedures were carried out as recommended by the respective manufacturers. The test results, including those embedded in instruments’ software, were assigned to the different interpretive categories according to the NCCLS documents in force up to late 1995 [5]. No center included intermediate endpoints in the resistance category. The critical zones for resistance in the disk diffusion tests were 16 mm for ampicillin (10-mg disk content), 14 mm for vancomycin (30 mg), and 10 mm for teicoplanin (30 mg). The equivalent MIC breakpoints were 16 mg/mL for ampicillin, 32 mg/mL for vancomycin, and 32 mg/mL for teicoplanin. Antimicrobial susceptibility data were collected with use of the MyMic software package, as previously described [6]. Briefly, the data stemmed from each automated reader device via data acquisition interfaces created for the purpose by the

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respective manufacturers, and the data were subsequently translated from each individual proprietary format into a common Xbase format and transmitted to the reference center on magnetic supports or via telecommunications systems. A total of 16,226 strains were analyzed. Of these, 9,169 were identified as E. faecalis, 913 as E. faecium, and 6,144 generically as Enterococcus species (table 2). The overall rate of isolation of E. faecium, as compared with that for E. faecalis, was 9.5% in agreement with data reported in the literature indicating a range of 10% – 20%. Only one center was above the upper limit (Palermo, 28.6%), and four were below the lower limit (Florence 1, 5.1%; Pistoia, 3%; Vicenza, 8.7%; Verona, 5.6%). As shown in table 2, the average rate of resistance to ampicillin was 1.9% (range, 0 – 6.5%) for E. faecalis and 70% (range, 33.3% – 98.7%) for E. faecium; the average rate of resistance

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Center (total no. of isolates)*

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Fontana et al.

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which evaluated a large number of hospitals over an extensive geographic area, revealed that vancomycin-resistant strains were isolated in nearly all centers but that the rate was significant in only a few centers, mostly in oncology-hematology units. Acknowledgments

The authors thank Becton Dickinson Italia S.p.A., Biokit S.r.l. Italia, and bioMe´rieux Italia S.p.A. for interfacing their instruments with the MyMic software. This paper is dedicated to Giuseppe Satta, who conceived and promoted the Italian Surveillance Group for Antimicrobial Resistance.

Members of the Italian Surveillance Group for Antimicrobial Resistance

A. Goglio, Bergamo; A. Gallina and P. Troupioti, Sondalo; A. Turano and G. Ravizzola, Brescia; B. Maranini and R. Cioni, Empoli; P. Nicoletti, R. Degli Innocenti, and P. Pecile, Florence; F. Komauli, Gorizia; S. Frugoni and A. Berlusconi, Milan; D. Ottomano and M. Saudelli, Milan; R. Rescaldani and S. Bramati, Monza; A. Giammanco and A. Chiarini, Palermo; C. Chezzi, W. Magliani, and M. G. Menozzi, Parma; G. Sbaraglia, Perugia; R. Rossetti, Pistoia; G. Santini and A. Callegaro, Pordenone; G. Fadda and T. Spanu, Rome; P. Cappuccinelli, A. Maida, B. N. Are, S. Sanna, and E. Muresu, Sassari; and M. Scagnelli, Vicenza. Data manager: G. Cornaglia.

References 1. Fontana R. Leading articles: penicillin-binding proteins and the intrinsic resistance to beta-lactams in gram-positive cocci. J Antimicrob Chemother 1985; 16:412 – 6. 2. Murray BE. The life and times of the enterococcus. Clin Microbiol Rev 1990; 3:46 – 65. 3. Fontana R, Aldegheri M, Ligozzi M, Lopez H, Sucari A, Satta G. Overproduction of a low affinity penicillin-binding protein and high level ampicillin resistance in Enterococcus faecium. Antimicrob Agents Chemother 1994; 38:1980 – 3. 4. Woodford N, Johnson AP, Morrison D, Speller DCE. Current perspective on glycopeptide resistance. Clin Microbiol Rev 1995; 8:585 – 615. 5. National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing: sixth informational supplement. NCCLS document M100-S6. Villanova, Pennsylvania: National Committee for Clinical Laboratory Standards, 1995. 6. Cornaglia G, Satta G. MyMic: computerised listing of antimicrobials with optimal activity and pharmacokinetic properties for individual infections. Binary 1993; 5:159 – 64. 7. Tenover FC, Tokars J, Swenson J, Paul S, Spitalny K, Jarvis W. Ability of clinical laboratories to detect antimicrobial agent – resistant enterococci. J Clin Microbiol 1993; 31:1695 – 9. 8. Manso E, De Sio G, Biavasco F, Varaldo PE. Vancomycin-resistant enterococci. Lancet 1993; 342:616 – 7.

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to vancomycin was 1.1% (range, 0.1% – 2%) for E. faecalis and 8.5% (range, 0 – 36.1%) for E. faecium; and the average rate of resistance to teicoplanin was 0.8% (range, 0 – 2.4%) for E. faecalis and 8.7% (range, 0 – 36.1%) for E. faecium. The ampicillin resistance rate found in most of the centers fell within the average reported for both E. faecalis and E. faecium in other countries [2]. Exceptions were Vicenza, which reported resistance rate to ampicillin of ú5% for E. faecalis, and Milan 1, where nearly all E. faecium isolates were resistant to ampicillin. Such variations in resistance to ampicillin might be explained by epidemiological factors. Most centers that tested vancomycin reported isolation of strains resistant to this antibiotic, even if at a very low rate. We were aware of the dubious reliability of the data from some of the automated systems in detecting the VanB phenotype [7], and we considered the resistance rates we obtained in our study as mainly due to strains with the VanA phenotype. Centers that reported high resistance rates were Brescia, Rome, and Vicenza (14.3%, 12.2%, and 42.1%, respectively). The high rate of resistance to glycopeptides found in Vicenza was due to an outbreak of bacteremia (June 1993 through October 1994) caused by vancomycin-resistant E. faecium of the VanA type, which occurred in the center’s hematology unit. In the same center, resistant strains were also isolated sporadically from patients in other units and from the stools of nonbacteremic hematology patients. Epidemiological analysis of this outbreak is in progress, and preliminary results indicate a multiclonal origin. Sixty-four percent of vancomycin-resistant strains isolated in Rome from March 1993 to July 1993 were recovered from patients in the hematology unit and sporadically from patients in other units in 1994. Of the four vancomycin-resistant strains that accounted for 14.3% of all E. faecium isolates recovered in Brescia, two were recovered in May 1995 and June 1995 in the infectious disease unit, one was recovered in the transplantation unit, and one was recovered from an outpatient. In conclusion, analysis of the rate of vancomycin resistance in Italy indicates that the phenomenon is nationwide, without reaching significant levels in the majority of the centers contributing to the surveillance network. Two, or possibly three, ‘‘hot spots’’ have been identified and are the Vicenza, Brescia, and Rome centers. Before our study, only one outbreak caused by vancomycin-resistant enterococci had been reported in Italy. It occurred from December 1992 to May 1993 at the regional hospital of Ancona and was caused by a single clone of E. faecalis that infected inpatients in the neurological intensive care unit [8]. Most of the concern about vancomycin-resistant enterococci is generated by studies that evaluated only one hospital or a few medical centers in a limited geographic area. Our study,

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