Evaluation of selective and enrichment media for isolation of glycopeptide-resistant enterococci from faecal specimens

Derek F. J. Brown* and Enid Walpole

Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge CB2 2QW, UK

Received 11 April 2002; returned 27 August 2002; revised 22 October 2002; accepted 4 November 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Vancomycin-resistant enterococcus enrichment broth (VEB) and vancomycin-resistant enterococcus selective agar with vancomycin 6 mg/L (VSA) are novel azide–aesculin agar-based media that contain meropenem as an additional selective agent. The media were compared with enterococcosel broth (EB) and enterococcosel agar with vancomycin 6 mg/L (EA) for the isolation of glycopeptide-resistant enterococci (GRE) from routine faecal screening specimens.

Methods: Two hundred and eighteen routine faecal screening specimens from patients at Addenbrooke’s Hospital were examined. The majority were from patients on haematology wards (155) or the intensive therapy unit (ITU) (21). Specimens were inoculated on to VSA and EA directly, and after enrichment in VEB and EB, respectively.

Results: One hundred and twenty-eight GRE isolates were recovered from 93 (43%) specimens with enterococci carrying vanA or vanB genes. There were no statistically significant differences between media (specimens positive; numbers of GRE isolates) on direct plating on VSA (87; 104) or EA (86; 97) or following 24 h enrichment in VEB (89; 103) or EB (86; 98). There was no significant advantage to enrichment compared with direct plating. Incubation of enrichment broth cultures for only 6 h appeared detrimental. Enterococci with vanC were isolated significantly less frequently from VEB and VSA than from EB and EA. Growth of organisms other than GRE was more common on VSA than on EA.

Conclusions: VEB and VSA were at least as effective as EB and EA for the recovery of GRE from faecal screening specimens, but substantially more non-GRE grew on VSA than on EA. Enrichment culture offered no significant advantages over direct plating.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Over the past 10 years there has been a rapid increase in the incidence of infection and colonization of patients with glycopeptide-resistant enterococci (GRE).1 Enterococci are intrinsically resistant to many antimicrobial agents and the emergence of strains resistant to commonly used agents, including ampicillin, aminoglycosides and vancomycin, has serious implications for immunosuppressed and debilitated patients, who are at particular risk of infection. Enterococci are found in the gastrointestinal tract, and many infections are attributed to endogenous sources, although cross-infection with GRE has been shown to occur.2 Colonization rates may be high on intensive therapy unit (ITU),3,4 renal,5 oncology68 and transplant9 wards. Some patients carry multiple strains of GRE10 and indistinguishable strains are isolated from different patients, suggesting cross-infection between patients on the same wards.11,12

Faecal colonization has been found to precede infection1316 and colonized patients may provide a reservoir for GRE, which may cross-colonize and cause infection in other patients. Once GRE have been isolated in a hospital, routine surveillance cultures of high-risk patients at weekly intervals have been recommended to allow early identification of colonized patients,13,16 and screening specimens should be cultured to identify colonized patients during outbreaks of infection with GRE.2,13

Selective media containing vancomycin have been recommended for surveillance cultures for GRE.13 However, a range of different media has been used, including 10% horse blood agar with aztreonam and amphotericin,17 colistin nalidixic acid agar,18 Mueller–Hinton agar with polymyxin and streptomycin,4 kanamycin aesculin azide agar,19 5% horse blood agar with neomycin,20,21 bile aesculin azide agar,22,23 campylobacter blood agar with clindamycin,19 cefalexin aztreonam arabinose agar24 and colistin nalidixic acid aesculin azide agar.25 There is no consensus on which medium should be used or the concentration of vancomycin to be included in the medium. Concentrations of vancomycin from 4 to 64 mg/L have been used, but 4 or 6 mg/L has been recommended to facilitate the detection of low-level vancomycin resistance.26,27 Enrichment in broth followed by plating on vancomycin-selective agar has been found to be more sensitive than direct screening on selective agar media containing vancomycin;28 however, enrichment in broth has the disadvantage that the procedure requires an extra day for incubation of the broth.

PCR methods of screening for GRE in faeces have the advantage that results can be available within a few hours of receipt of the sample.29,30 However, extraction of DNA is necessary to remove inhibitory substances in the sample, and the method may not be cost-effective when dealing with large numbers of specimens or when the prevalence is low.29 In these situations, the use of PCR methods on enrichment broths or colonies on selective medium may be appropriate.29,30 If typing of isolates for epidemiological purposes is required, culture will also be necessary.

There are no widely accepted standards for evaluation of selective media for use in screening for GRE. However, enterococcosel agar (EA) and broth (EB), which are bile-aesculin-azide formulations, have been used in several studies.28,31,32 Oxoid vancomycin-resistant enterococcus enrichment broth (VEB) and selective agar (VSA) are novel in that they are aesculin-azide media with meropenem as an additional selective agent. The objectives of this study were to assess the performance of VEB and VSA in comparison with EB and EA for the isolation of vancomycin-resistant enterococci (VRE) from faecal screening specimens. In addition, 6 h enrichment was investigated to determine whether the extra day required for broth enrichment could be avoided without loss of sensitivity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Specimens

Two hundred and eighteen routine faecal screening specimens from patients at Addenbrooke’s Hospital were included. The majority were from patients on haematology wards (155) or the ITU (21).

Media

The following media were used in screening for GRE. VEB was VRE broth (Oxoid, Basingstoke, UK) containing meropenem 2 mg/L (meropenem supplement; Oxoid). VSA was VSA agar (Oxoid) containing meropenem 1 mg/L and vancomycin 6 mg/L (meropenem and vancomycin supplements; Oxoid). The antibiotic supplements in VEB and VSA were as defined by the manufacturer. EB (Becton Dickinson, Oxford, UK) was unsupplemented and EA (Becton Dickinson) was supplemented with vancomycin 6 mg/L (Sigma, Poole, UK).

Screening methods

Specimens were inoculated directly on to VSA and EA media by transferring faeces on a sterile swab and spreading with a loop. Pea-sized portions of faeces were also transferred to 10 mL volumes of VEB and EB, vortexed and incubated at 37°C. After 6 and 24 h incubation the VEB was subcultured to VRE selective agar. After 24 h incubation the EB was subcultured to EA. All plates were incubated in air at 37°C and examined for colonies after 24 and 48 h. On both agar media enterococci produce brown colonies with black/brown zones around them. Different colony types, including colonies not typical of enterococci, were subcultured to blood agar plates from each selective plate. At least four separate colonies were cultured to increase the chances of detecting mixtures of organisms. Subcultures were incubated in air at 37°C for 24 h. Isolates were tentatively identified as enterococci on the basis of colony appearance, Gram’s stain and a positive test for pyrrolidonylarylamidase activity. Identity and van gene status were confirmed by a PCR method33,34 and antimicrobial susceptibility was tested by disc diffusion.35 Differences in the performance of media were tested statistically using the {chi}2 method.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolation of GRE with vanA or vanB

From the 218 specimens, GRE with vanA or vanB genotypes were isolated from 93 (43%) (Table 1). Individual isolation protocols detected GRE in between 86 (direct plating on EA, enrichment for 6 h in VEB followed by plating on VSA, and enrichment in EB followed by plating on EA) and 89 specimens (enrichment for 24 h in VEB followed by plating on VSA) but there were no statistically significant differences among the treatments.


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Table 1.  Isolation of glycopeptide-resistant enterococci with vanA and vanB resistance from 218 faecal specimens
 
More than one GRE with vanA or vanB genotypes was isolated from several specimens and in total 128 isolates with different identification or van gene status were recovered (Table 1). Variation in susceptibility to agents other than vancomycin and teicoplanin was common with isolates from the same specimen (data not shown). However, for the purposes of this analysis, isolates were considered distinct if they were different species and/or had different van gene types. Enterococcus faecium (102 isolates) was isolated more frequently than Enterococcus faecalis (25 isolates). The vanB genotype (59 isolates) was more common than vanA (43 isolates) in E. faecium, but no isolates of E. faecalis with vanB resistance were detected. There were two isolates of Enterococcus gallinarum with vanA, one being isolated on all media and one only on EA following enrichment. Individual isolation protocols detected between 97 (direct plating on EA) and 104 (direct plating on VSE) GRE, but there were no statistically significant differences among the treatments.

Enrichment in broth did not significantly increase the isolation rates with either VEB or EB. It was notable that the density of growth obtained on plates after 6 h incubation in VEB was frequently less than that obtained on direct plating or after 24 h enrichment. Where more than one GRE was isolated, the predominant strain was not uncommonly different on direct plating and after enrichment. The majority of isolates of GRE grew in large numbers on all media.

On all media, almost all GRE were detected after incubation of plates for 24 h. However, the colonies of GRE were generally larger on VSA than on EA, in that, after incubation for 24 h, colonies on VSA agar were generally at least 0.5 mm in diameter, whereas colonies on EA were usually <0.5 mm in diameter. On both the media, the brown/black colour greatly assisted in indicating GRE colonies, but, particularly after incubation of plates for 24 h, colonies on the same plate could differ in appearance from very pale brown to black. One isolate of E. faecalis on VSA inoculated directly and following enrichment for 6 or 24 h did not give colonies typical of enterococci, in that they did not show any brown or black pigment and would not have been subcultured as VRE in the routine situation.

Isolation of vanC enterococci

E. gallinarum and Enterococcus casseliflavus with vanC resistance were detected in eight and nine specimens, respectively (Table 2). They were detected significantly less frequently (P < 0.05) on VSA following enrichment than on EA following enrichment, and significantly less frequently (P < 0.05) on direct plating on VSA than on EA following enrichment.


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Table 2.  Positive isolates of enterococci other than vanA and vanB from 218 faecal specimens
 
Isolation of vancomycin-susceptible enterococci

Vancomycin-susceptible enterococci, mostly E. faecalis, were isolated from 36 specimens (Table 2). Differences between media were not statistically significant (P > 0.05). One isolate of E. faecalis on Oxoid VRE agar inoculated following enrichment for 6 h and on enterococcosel agar inoculated directly did not give colonies typical of enterococci, in that they did not show any brown or black pigment.

Isolation of organisms other than enterococci

Isolates that were not enterococci were not fully identified, but were grouped on the basis of colony morphology and Gram’s stain (Table 3). They were also grouped on the basis of whether the colonies on the selective media appeared to be similar to those of enterococci or clearly different.


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Table 3.  Organisms other than enterococci isolated from 218 faecal specimens
 
Organisms other than GRE mostly grew with smaller numbers of colonies than VRE and were frequently not detected until plates had been incubated for 48 h. Gram-positive bacilli grew significantly more frequently following direct plating on VSA than direct plating on EA (P < 0.01), enrichment in VEB for 6 or 24 h followed by plating on VSA (P < 0.05), direct plating on EA (P < 0.01) or enrichment in EB followed by plating on EA (P < 0.001). Gram-positive bacilli grew significantly more frequently on VSA following enrichment in VEB than on EA following enrichment in EB (P < 0.05). Colonies of Gram-positive bacilli were frequently, but not always, smaller than GRE colonies, but they very frequently had the characteristic appearance of enterococci. A few staphylococci grew on the VSA in particular (Table 3). In appearance of the colonies, about half were indistinguishable from GRE. A few other Gram-positive cocci, which were not enterococci, grew on both media. In appearance, they were mostly indistinguishable from GRE. With Gram-positive cocci other than enterococci, differences between media were not statistically significant (P > 0.05). Yeasts were isolated significantly more frequently on VSA, without enrichment (P < 0.01), following enrichment for 6 h (P < 0.01) or enrichment for 24 h (P < 0.001), than on EA with or without enrichment. However, colonies were usually atypical of enterococci.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Most of the specimens examined in this study were from patients on haematology wards and the ITU. Patients on these wards are known to have high GRE colonization rates, and the high overall positivity rate (43%) is in line with that of previous publications.39 Multiple isolates of GRE from the same specimen were not uncommon, in that 128 isolates of enterococci with vanA or vanB were found in the 93 positive specimens. Carriage of multiple strains of GRE has been reported previously,10,36 but different colony morphologies were generally not obvious on selective plates with either medium in this study. Multiple strains were commonly detected because different strains grew on different media, or different strains grew on direct plating and enrichment. Some were detected through routine selection of four colonies for identification and susceptibility testing. In routine screening for GRE in faecal specimens, multiple screening procedures are unlikely to be used, and although this might be ameliorated to some extent by picking multiple colonies, some false-negative reports appear to be inevitable.

There were more isolates of GRE (vanA and vanB) on VSA, both with and without enrichment, than on EA with and without enrichment, but differences were not statistically significant. Enterococci with vanC include E. gallinarum and E. casseliflavus, which were isolated less frequently from VSA than from EA, particularly following enrichment in VSB, which eliminated all enterococci with vanC except one strain also carrying the vanA gene. As the concentrations of vancomycin in VSA and EA were the same, the difference in isolation rates of E. gallinarum and E. casseliflavus, which are intrinsically low-level resistant to vancomycin, must be related to other differences between the media. Enterococci with vanC can cause significant infections,37 but this is rare and they have not been implicated in nosocomial outbreaks.38 In most situations when screening for GRE it might therefore be considered an advantage if enterococci with only the vanC glycopeptide resistance genes did not grow on GRE screening media. This would avoid unnecessary work involved in identification and susceptibility testing of isolates that are unlikely to cause infections or require infection control precautions.

Enrichment in VSB for 24 h did not significantly increase the rate of isolation of GRE on VSA, although there was a marginal increase in the number of specimens positive for GRE; and enrichment in EB did not increase the rate of isolation of GRE on EA. These results conflict with a study by Ieven et al.,28 which indicated that broth enrichment significantly increases isolation of GRE when screening faeces. The difference may relate to several differences in the studies. First, vancomycin was added to EB in the study by Ieven et al.28 but not in ours. The benefits of adding vancomycin to enrichment media have not been established and may depend on the particular combination of enrichment broth and selective agar. Klare et al.39,40 have reported isolation of some GRE only after enrichment in a medium without vancomycin. Secondly, the study by Ieven et al.28 included enterococci with vanC, which accounted for 56% of their isolates. Thirdly, there were differences in the patient populations studied, in that Ieven et al.’s study28 included patients from all hospital wards, whereas ours concentrated on patients known to be at high risk of colonization with GRE. Patients in these high-risk groups may be colonized with larger numbers of GRE than other patients, in which case the benefit of a more sensitive enrichment method would be reduced. Fourthly, the study by Ieven et al.28 included rectal swabs if faecal specimens were not available, and the numbers of GRE in rectal swabs may be lower than in faecal samples, which again might favour a more sensitive enrichment method. Detection of GRE carriers colonized with low numbers of GRE may be important in control of epidemic strains of GRE, so it would be useful to extend the present work to other patient groups and to rectal or peri-anal swab samples, as these are included in some GRE screening protocols because swab samples are easier to obtain.

The use of enrichment broth adds 24–48 h to the time taken for screening, which is undesirable if screening is undertaken for infection control purposes. In order to investigate whether enrichment for a short period offered any benefit over direct plating, VEB cultures were subcultured after incubation for 6 h. However, there were slightly fewer isolates of GRE when VEB was subcultured after 6 h, and the density of growth was frequently less than that obtained with direct plating or 24 h enrichment. It is likely that 6 h is insufficient time for recovery and growth of VRE following the initial dilution of organisms in broth.

Growth of organisms other than GRE on GRE selective media is undesirable because it leads to additional work in checking isolates, and even if colonies are not typical of GRE in appearance they can obscure GRE colonies or make it difficult to subculture colonies for confirmation. A few vancomycin-susceptible enterococci grew on both media. Increasing the concentration of vancomycin in the media would increase specificity, but isolation of GRE with low-level resistance is likely to be compromised and concentrations of vancomycin >6 mg/L are not recommended.26,27 Non-GRE colonies were isolated more frequently on VSA, particularly on direct plating. While many of these organisms could be distinguished because they did not give typical colonies on the aesculin-based media, significant numbers were not distinguishable from GRE solely on colony appearance. Non-GRE colonies commonly grew more slowly than GRE, with colonies not clearly visible until 48 h incubation, but this was also the case with some GRE, so the colonies could not be discounted.

In conclusion, VSA and VEB were as effective as EA and EB for the isolation of GRE from faecal specimens. The media differed in that VSA and VEB were more effective in preventing growth of enterococci with vanC, whereas EA and EB were generally more effective at preventing growth of other organisms. Enrichment was not shown significantly to increase isolation of GRE in the largely high-risk population studied, and enrichment for only 6 h may be deleterious.


    Acknowledgements
 
This study was supported by a grant from Oxoid Ltd.


    Footnotes
 
* Corresponding author. Tel: +44-1223-257020; Fax: +44-1223-242775; E-mail: dfjb2{at}cam.ac.uk Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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