In£uence of growth media on vancomycin resistance of Enterococcusisolates and correlation with resistance gene determinants

Rajesh Nayak a, Saeed A. Khan a;�, Robert H. Watson b, Carl E. Cerniglia a

a US Food and Drug Administration, National Center for Toxicological Research, Division of Microbiology, Je¡erson, AR 72079, USAb Brooks Air Force Base, San Antonio, TX 78235, USA

Received 1 March 2002; received in revised form 28 May 2002; accepted 10 June 2002

First published online 8 August 2002

Abstract

The effect of Mueller^Hinton (MH), MH+blood or brain heart infusion medium (agar or broth) on 13 Enterococcus isolates wasdetermined, when testing their antibiotic susceptibility. Disk diffusion and Vitek methods were used to determine vancomycin resistance,while broth dilution and E-test methods were used to measure the minimum inhibitory concentration. The data were correlated with thepresence of vancomycin resistance genes. A definite correlation pattern could not be established between the presence of van genes andvancomycin resistance in any plating medium, when tested by the disk diffusion assay. The broth dilution, irrespective of the platingmedium, and Vitek methods were more reliable than the E-test method in testing isolates with vanA or vanB genes. However, for vanC2/C3 genotypes, the E-test method, irrespective of the plating medium, tested better than the broth dilution assay. 7 2002 Federation ofEuropean Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Vancomycin-resistant enterococcus; Plating medium; van gene; Antibiotic susceptibility testing method

1. Introduction

Vancomycin-resistant enterococci (VRE) exhibit highlevels of resistance to penicillin, aminoglycosides, and oth-er glycopeptides [1], and can transmit antibiotic resistanceto other sensitive organisms and pose serious health prob-lems in humans [2]. Predominant genes such as vanA, B,and C confer vancomycin resistance in enterococci [3].Accurate and prompt detection of VRE and determina-

tion of their antimicrobial susceptibility pro¢les by clinicallaboratories are necessary to prevent and control nosoco-mial outbreaks allowing patients to be administered ap-propriate drugs. Several studies have been conducted tovalidate the accuracy of antimicrobial susceptibility testingof VRE [4,5]. The composition of plating/broth mediumand the growth conditions of enterococcal isolates mayin£uence the antibiotic susceptibility pro¢les and the mini-mum inhibitory concentration (MIC) values [6,7]. The ef-fect of plating medium, however, was not correlated with

the presence of vancomycin resistance genes in these stud-ies. Several automated antibiotic susceptibility tests havefailed to detect enterococci with di¡erent resistance genes[8]. Hence, selection of a single medium formulation anddetection method, eliciting a high degree of correlationwith the presence of vancomycin gene(s), is critical to fa-cilitate reproducibility of results among di¡erent clinicallaboratories. This study evaluates the e⁄cacy of Muel-ler^Hinton (MH), MH with 5% sheep blood (MHB), orbrain heart infusion (BHI) medium (broth or agar) on theantimicrobial susceptibilities of enterococcal isolates. SinceMH and BHI are the media recommended by the NationalCommittee for Clinical Laboratory Standards (NCCLS),we have compared the e¡ect of plating medium with refer-ence to MH agar/broth. This study also attempts to cor-relate the vancomycin resistance of the enterococcal iso-lates with the presence of vancomycin resistance genes.

2. Materials and methods

2.1. Enterococcal isolates

Thirteen isolates (nine clinical isolates from patients inSan Antonio, TX and Las Vegas, NV and four ATCC

0378-1097 / 02 / $22.00 7 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 8 8 5 - 6

* Corresponding author. Tel. : +1 (870) 543-7197;Fax: +1 (870) 543-7307.

E-mail address: skhan@nctr.fda.gov (S.A. Khan).

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www.fems-microbiology.org

reference strains) were evaluated for their antimicrobialsusceptibilities.

2.2. Antimicrobial susceptibility testing

Cultures were grown overnight on tryptic soy agar (Dif-co Laboratories) with 5% sheep blood at 35‡C. Each iso-late was suspended in tryptic soy broth and the turbiditywas adjusted to 0.5 MacFarland standard, correspondingto approximately 1.2U108 CFU ml31. About 200 Wl ofthis suspension was evenly spread on a plating mediumand tested for antibiotic susceptibility. To compare thee¡ects of using three di¡erent media on the antimicrobialsusceptibility test, each isolate was plated on MH, MHBor BHI agar (Difco Laboratories) plates. The followingantibiotic disks (BD Biosciences) were used on each agarplate: gentamicin, 120 Wg; streptomycin, 300 Wg; cipro-£oxacin, 5 Wg; o£oxacin, 5 Wg; tetracycline, 10 Wg; baci-tracin, 10 Wg; sulfamethoxazole/trimethoprim (SXT), 23.75/1.25 Wg; chloramphenicol, 30 Wg; kanamycin, 30 Wg; na-lidixic acid, 30 Wg; erythromycin, 15 Wg; and vancomycin,30 Wg. The plates were incubated at 35‡C for 24 h beforemeasuring the zone of inhibition to the nearest tenth of amillimeter. The zone diameter was measured in triplicate,and the measurements were interpreted as resistant or sen-sitive according to the manufacturer’s guidelines. Suscep-tibility of enterococcal isolates to vancomycin was alsotested on Gram-positive diagnostic susceptibility (GPS-108) cards (BioMe¤rieux Vitek) as per the manufacturer’sinstructions.

2.3. Determination of MIC

Isolates were tested for vancomycin (Sigma) MIC by thebroth dilution method [9]. Each isolate was cultured on anMH, MHB or BHI plate and incubated overnight at 35‡C.Cultures were suspended in the corresponding broth andthe cell suspension was used to determine the MICs (0.5^256 Wg ml31). The tubes were incubated at 35‡C and readfor the presence of turbidity (culture growth) with indirectlight after 24 h. The end point for measuring turbidity ofMHB broth was determined by a change in color from red(no culture growth) to purple (culture growth). Vancomy-cin MIC was also measured using the epsilometer test (E-test). Overnight cultures, grown on MH, MHB or BHIbroth, were plated on the corresponding agar plates, andthe MIC was measured using E-strips (AB BioDisk).

2.4. Polymerase chain reaction (PCR) detection of genesencoding vancomycin resistance

Individual and multiplex PCR were used to detect van-comycin resistance genes vanA (732 bp), B (457 bp), C1(822 bp) and C2/C3 (429 bp) in each test isolate, usingreference ATCC enterococcal strains as positive controls.Each reaction tube contained 5 Wl of bacterial lysate (5^

10 ng DNA), 7.5 Wl of a 4 WM mixture of the gene-speci¢cPCR primers [10] and 12.5 Wl of 2UPCR mixture contain-ing 100 mM KCl, 2^6 mM MgCl2, 20 mM Tris^HCl, pH8.3, 0.002% gelatin (w/v), and 0.5 Wl of Taq DNA poly-merase. The ¢nal concentration of Mg2þ used in PCRreactions was 1 mM during the ampli¢cation of individualgenes and 3 mM for multiplex PCR ampli¢cation of vangenes. The reaction mixture was ampli¢ed for 30 cycles ;each cycle consisted of 97‡C denaturation for 1 min, 50‡Cannealing for 55 s, and 72‡C extension for 1.5 min. Theinitial denaturation and the ¢nal extension step were ex-tended for 2 and 10 min, respectively. Samples were ana-lyzed by electrophoresis on 1.5% agarose gels.

3. Results and discussion

All 13 enterococcal isolates were resistant to multipleantibiotics, when tested by the NCCLS method [11] onMH agar plates (Table 1). The gentamicin, streptomycin,SXT, bacitracin, kanamycin, erythromycin and vancomy-cin resistance of enterococcal isolates varied with the testmedia, using the disk di¡usion assay (Tables 2 and 3).This alteration in the resistance/sensitivity may be dueeither to variation in the potency of each antibiotic or tothe solubility and di¡usion properties of antibiotics in dif-ferent media [12]. Antimicrobial SXT activities against en-terococcal isolates have been inconsistent due to varyingconcentration of thymidine, a major inhibitor of SXT, inthe growth medium [13]. MH medium contains relativelylow levels of thymidine [13], and has been recommendedby the NCCLS for routine antimicrobial susceptibilitytesting [11]. In our investigation, eight clinical and threeATCC enterococcal isolates were sensitive to SXT on MHand resistant on MHB or BHI plates (Table 2). This ob-servation suggests that either the blood supplement orBHI medium may contain thymidine and/or other un-known chemical(s) that inhibited SXT activity. The thymi-dine concentration in BHI medium was not available fromthe commercial manufacturer. Sheep blood supplementhas also been reported to cause indistinct zones or a ¢lmof growth within the zone of inhibition around SXT disks,further a¡ecting the antibiotic susceptibility [14].In an earlier study, Endtz and coworkers evaluated the

accuracy of eight available susceptibility testing methodson 50 vanA, 15 vanB, 50 vanC1 and 30 vanC2 VREs [5].VRE isolates containing the vanA gene were detected byall methods. However, isolates containing the vanB genewere not detected by Vitek GPS-TA and MicroScan rapid.E-test and the agar screen methods were able to detect allVRE isolates. The investigation by Endtz and coworkers[5], however, did not evaluate the e¡ect of plating mediumon the antibiotic susceptibility testing methods. In ourstudy, the e¡ectiveness of MH, MHB or BHI mediumwas evaluated on the vancomycin susceptibility using thedisk di¡usion, broth dilution and E-test methods, and the

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data were correlated with the presence of van genes.Although a good correlation was observed between vanAE. faecium isolates 26, 29 and 30, vanB E. faecalis isolate31 and vancomycin resistance on all three plating media,the disk di¡usion assay appeared unreliable for vancomy-cin susceptibility testing (Table 3). E. faecium isolates 5and ATCC 51559, containing the vanA gene, were sensi-tive on MHB and MH/BHI agar plates, respectively, while

vanB E. faecalis ATCC 51299 was sensitive on MH plate.Vancomycin resistance, as determined by the disk di¡u-sion assay, did not correlate with the presence of thevanC2/C3 genotypes (Table 3). These isolates were foundto be sensitive to vancomycin on all three plating media.Vancomycin resistance as determined by the Vitek GPS-108 method was consistent with the presence of vanA andvanB, but not with vanC2/C3 genotypes (Table 3). Both

Table 2E¡ect of plating medium on antibiotic susceptibility pro¢le of selective enterococcal isolates by the disk di¡usion assay

Antibiotic Isolate ID Species Antibiotic testing mediaa

MH MHB BHI

Gentamicin (GM) 26 faecium S R SStreptomycin (S) 26 faecium R S R

51559 faecium R R SSulfamethoxazole/trimethoprim (SXT) 2 faecalis S S R

5 faecium S R R26 faecium S R R27 faecalis S S R28 faecalis S S R29 faecium S R R30 faecium S R S31 faecalis S S R29219 faecalis S S R51299 faecalis S S R25788 casseli£avus S S R

Bacitracin (B) 31 faecalis R S S25788 casseli£avus R R S

Kanamycin (K) 2 faecalis R S R27 faecalis R S R29212 faecalis R S R

Erythromycin (E) 2 faecalis R R S51559 faecium R R S

aBD Biosciences interpretive standards based on zone diameter (mm): GM: R=6, Sv 10; S: R= 6, Sv 10; SXT: R9 10, Sv 16; B: R9 8, Sv 13;K: R9 13; E: R9 13, I = 14^22, Sv 23.R= resistant; S = sensitive.

Table 1Antibiotic susceptibility pro¢le of enterococci in MH agar by the disk di¡usion assay

Isolate Species Source Antibiotics testeda

GM S CIP OFX SXT TE B C K NA E VA

2 faecalis SA S S S S S S S S R R R S5 faecium LV S S R R S R S S R R I R12 faecium SA S S R R R S S S R R R S26 faecium LV S R R R S R S S R R R R27 faecalis SA S S I S S R R I R R R S28 faecalis SA S R S S S R R S R R R S29 faecium LV S R R R S R S S R R R R30 faecium LV S R R R S R S S R R R R31 faecalis SA R R S S S S R R R R R R29212 faecalis ATCC S S I S S R S S R R I S51559 faecium ATCC R R R R R S S S R R R S51299 faecalis ATCC R R S S S S S R R R R S25788 casseli£avus ATCC S S I R S S R S R R I S

aBD Biosciences interpretive standards based on zone diameter (mm): GM: R=6, Sv 10; S: R=6, Sv 10; CIP: R9 15, I = 16^20, Sv 21; OFX:R9 12, Sv 16; SXT: R9 10, Sv 16; TE: R9 14, Sv 19; B: R9 8, Sv 13; C: R9 12, I = 13^17, Sv 18; K: R9 13; NA: R9 13; E: R9 13, I = 14^22, Sv 23; VA: R9 14, Sv 17.SA=San Antonio; LV=Las Vegas; GM=gentamicin; S= streptomycin; CIP= cipro£oxacin; OFX=o£oxacin; SXT= sulfamethoxazole/trimethoprim;TE= tetracycline; B= bacitracin; C= chloramphenicol ; K=kanamycin; NA=nalidixic acid; E= erythromycin; VA=vancomycin; S= sensitive ; I = inter-mediate resistance; R= resistant.

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disk di¡usion assay and Vitek methods proved equallyreliable in testing vancomycin-sensitive isolates.The presence of the vanA gene in Enterococcus spp. is

associated with high vancomycin resistance (MIC=64^s 1000 Wg ml31), while vanB (MIC=4^1024 Wg ml31)and vanC (MIC=2^32 Wg ml31) are associated with me-dium to low levels of vancomycin resistance, respectively[3]. In general, a good correlation was observed betweenthe presence of vanA and vanB genes in Enterococcus iso-lates and the MIC values for all three plating media by thebroth dilution and E-test methods (Table 4). However, thereason for the unexpectedly lower MIC values (1^1.8 Wgml31) observed for vanA E. faecium ATCC 51559 in allthree plating media by the E-test method remains unclear.Broth dilution, irrespective of the plating medium, ap-peared to be a better choice than the E-test methodwhen testing vanA genotypes. For vanB genotypes, MHor BHI proved more reliable than MHB in testing vanco-

mycin MIC by the E-test method; MHB medium elicitedlow MIC values (2.5^3 Wg ml31). In the case of isolateswith vanC2/C3 genotypes, the E-test method, irrespectiveof the plating medium, appeared more reliable than thebroth dilution method (Table 4). E. faecalis isolate 2, con-taining the vanC2/C3 gene, showed MIC values of 6 0.5and 64 Wg ml31 in MH and BHI broths, respectively.These values were not within the typical range of 2^32 Wgml31 reported for vanC genotypes [3].In summary, the Vitek and broth dilution methods, ir-

respective of the plating medium, proved most reliable intesting vanA and B genotypes. However, the E-test meth-od, irrespective of the plating medium, was a better choicefor testing vanC genotypes. A combination of Vitek, brothdilution and E-test methods predisposes the choice of aplating medium for determining vancomycin resistance inenterococcal isolates. A companion study addressing thein£uence of plating medium on SXT resistance and its

Table 3E¡ect of plating medium on vancomycin resistance of enterococci by disk di¡usion and Vitek methods

van gene Species Isolate Vancomycin resistancea Vancomycin resistance pro¢leby Vitek GPS-108

MH MHB BHI

A faecium 5 R S R RA faecium 26 R R R RA faecium 29 R R R RA faecium 30 R R R RA faecium 51559 S R S RB faecalis 31 R R R RB faecalis 51299 S R R RC2/C3 faecalis 2 S S S SC2/C3 casseli£avus 25788 S S S Snd faecium 12 S S S Snd faecalis 27 S S S Snd faecalis 28 S S S Snd faecalis 29212 S S S S

aBD Biosciences interpretive standards based on zone diameter (mm): R9 14, Sv 17.R= resistant; S = sensitive; nd= not detected.

Table 4E¡ect of plating medium on the MIC of vancomycin by broth dilution and E-test methods

van gene Serotype Isolate Broth dilution (Wg ml31) E-test (Wg ml31)

MH MHB BHI MH MHB BHI

A faecium 5 128 128 s 256 s 256 s 256 48.0A faecium 26 256 s 256 s 256 s 256 s 256 s 256A faecium 29 256 256 s 256 s 256 s 256 96.0A faecium 30 128 256 s 256 s 256 s 256 s 256A faecium 51559 256 s 256 s 128 1.5 1.0 1.8B faecalis 31 32 8 64 6.0 2.5 16.0B faecalis 51299 16 16 16 6.0 3.0 16.0C2/C3 faecalis 2 6 0.5 2 64 2.0 2.5 2.0C2/C3 casseli£avus 25788 2 2 8 4.0 3.0 5.5nd faecium 12 6 0.5 1 16 1.5 1.5 1.8nd faecalis 27 6 0.5 1 16 1.0 1.5 2.0nd faecalis 28 6 0.5 1 16 1.5 1.3 2.0nd faecalis 29212 2 2 4 1.0 3.0 4.0

nd=not detected.

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correlation with the presence of gene(s) conferring SXTresistance in enterococci is warranted.

Acknowledgements

The work was supported in part by the postgraduateresearch program at the National Center for ToxicologicalResearch (NCTR) and administered by the Oak RidgeInstitute for Science and Education through an inter-agency agreement between the U.S. Department of Energyand U.S. Food and Drug Administration. We thank Drs.John Sutherland and Doug Wagner at NCTR for theirvaluable comments in preparing the manuscript.

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