A comparison of culture and PCR to determine the prevalence of ampicillin-resistant bacteria in the faecal flora of general practice patients

John Heritagea,*, Nicola Ransomeb, Philip A. Chambersa and Mark H. Wilcoxa,b

a Division of Microbiology, University of Leeds, Leeds LS2 9JT; b Department of Microbiology, The General Infirmary at Leeds, Leeds, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Of 95 faecal specimens containing bacterial DNA amplified by PCR, 24% contained cultivable bacteria that were resistant to high-level ampicillin. When these samples were examined by PCR using primers to amplify the blaTEM gene, the number of positive samples identified increased significantly to 49 (52%). These results indicate that ampicillin resistance is common in the study population. Furthermore, the blaTEM gene is more common than indicated by the recovery of resistant bacteria in culture. This points to potential anomalies in the assessment of the prevalence of resistance when relying on recovery of resistant bacteria by culture.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Most of our knowledge of the ecology of antibiotic resistance genes comes from hospital-based studies that do not provide a wider picture of the occurrence of genes that confer resistance in bacterial pathogens. Hospital patients are discharged into the community taking their antimicrobial-resistant flora with them. Once back in the community they may act as reservoirs for resistance genes until host bacteria are eliminated from their microbial flora. Furthermore, as general practitioners issue the majority of prescrip- tions for antibiotics,1 this compounds the selective pressure for resistance genes in this setting. If we are to understand the dissemination of resistance determinants we must consider the degree of colonization by resistant bacteria of the majority of people who do not frequent hospitals.

Molecular biological techniques for detection of antimicrobial resistance have rarely been applied to the study of the distribution of resistance genes in the commensal flora.2 Reliance upon culture alone ignores the bacteria that cannot be grown in artificial culture, although such bacteria may act as a reservoir for resistance genes.

The ß-lactams are the most commonly prescribed antibiotics in the UK. TEM-type ß-lactamases, coded for by the blaTEM gene, are among the commonest mechanisms by which facultative anaerobic gut flora resist ß-lactam antibiotics.3 Furthermore, the blaTEM genes display a remarkable tendency to mutate to give enzymes with an extended spectrum of activity. Although mutants are most often found in hospitalized patients, they will be carried out of hospital environments when patients are discharged, adding to the load of resistance genes in the wider community. In this study, we have compared conventional aerobic culture for identifying resistant bacteria with a PCR-based method for detecting resistance genes to identify individuals in the community who harbour bowel flora in which blaTEM genes are present, both in bacteria that can be isolated by culture and in non-cultivable microbes.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Sample population

One hundred consecutive faecal samples submitted by GP patients to the microbiology laboratory of the Leeds General Infirmary were collected and stored for up to 5 days at 4°C until culture for routine bacterial enteric pathogens was complete. Once samples were known not to contain Salmonella, Shigella, Campylobacter or verotoxin-producing Escherichia coli, they were processed for this study.

Isolation of resistant bacteria

Approximately 100 mg of each faecal sample were emulsified in tryptone–soya broth (TSB) by vortexing for 1 min. Three 10-fold dilutions were made in TSB. A sample of 25 µL from each dilution was plated on to CLED agar (Oxoid, Basingstoke, UK) containing 128 mg/L ampicillin. This concentration was chosen to permit the selective culture of bacteria producing efficient ß-lactamases, such as those of the TEM family. Plates were incubated aerobically at 37°C overnight and then observed for growth. Specimens were considered culture positive if even a single colony could be grown on repeated subculture in the presence of ampicillin.

Extraction of bacterial DNA from faeces

DNA was extracted from faecal samples using the QIAamp DNA Stool Mini Kit according to the manufacturer's instructions (Qiagen Ltd, Crawley, West Sussex, UK) with the following modifications: the optimal higher temperature of 95°C was used in Step 3 and at Step 17, DNA was eluted twice in 100 µL of buffer AE, rather than once in 200 µL as described in the manufacturer's protocol.

PCR amplification of bacterial targets

To ensure that bacterial DNA from each sample was available for PCR amplification of the blaTEM gene, each sample was subjected to PCR amplification using universal bacterial primers4 5'-AGGAGGTGATCCAACCGCA-3' and 5'-AACTGGAGGAAGGTGGGGAT-3'. Each reaction was carried out in 25 µL, containing 15 pmol of each primer, buffer, 2.5 mM (final concentration 0.2 mM) dNTPs, 1.5 mM MgCl2, 1 U Promega Taq polymerase and 0.1 g/L BSA with 2.5 µL of the extracted template. The reaction comprised 40 cycles of 30 s at 95°C followed by 30 s at 55°C with a final extension step of 10 min. The PCR product is 370 bp.

PCR amplification of the blaTEM gene

To optimize the detection of blaTEM sequences, a nested PCR was employed using the primers of Mabilat et al.5 and Hibbert-Rogers et al.6 The sequence of the TemA primer is 5'-ATAAAATTCTTGAAGAC-3'; TemB is 5'-TTACCAATGCTTAATCA-3'; TemE is 5'-TCGTCGTTTGGTATGGC-3'; and TemH is 5'-AGGAAGAGTATGAGTAT-3'. Each reaction was carried out in 25 µL, containing 15 pmol of each of the four primers, buffer, 2.5 mM dNTPs, 3 mM MgCl2, 1 U Promega Taq polymerase and 0.1 g/L BSA with 2.5 µL of the extracted template. The reaction comprised 30 cycles at 94°C for 15 s, followed by 30 s at 52°C and 1 min at 72°C. The final extension was 5 min, yielding a product of 533 bp.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Of the 100 samples examined, there was found to be no bias in the gender of the donor ({chi}2 = 2.92, 1 df, P > 0.05) and although young women, aged 20–40 years, appeared to be somewhat over-represented, this was not statistically significant. Because of the nature of the source material, samples provided for the detection of enteric pathogens, this study does not seek to model the healthy general population. It does, however, illustrate levels of resistance in a population who are not currently hospitalized.

Five samples failed to amplify with the universal bacterial PCR primers, indicating that DNA extraction had failed. These were excluded from further analysis. Twenty-three specimens (24%) yielded high-level ampicillin-resistant colonies upon culture. Of these, 18 also yielded an amplimer when subjected to PCR to detect the blaTEM gene. The absence of an absolute correlation between a positive culture and the production of a blaTEM amplimer is not surprising, since although TEM-type ß-lactamases are one of the most common causes of ampicillin resistance in the facultative Gram-negative bowel flora, they are not the only mechanism conferring high-level ampicillin resistance.7 Four specimens showed evidence of an inoculum effect: confluent or semi-confluent growth when plated neat but no growth when dilutions were examined. One of these was positive when tested for blaTEM by PCR. It is probable that the blaTEM found in this sample may be harboured by the obligate anaerobic flora and thus not recoverable by culture.

Although 23 of the specimens yielded ampicillin-resistant bacteria, when the presence of blaTEM was sought using PCR, 49 specimens (52%) were found to harbour this gene. The FigureGo shows representative results of nested PCR for blaTEM. Of the PCR-positive samples, only 36% also yielded ampicillin-resistant colonies upon aerobic culture. Thus, 31 samples appeared to harbour blaTEM but did not yield ampicillin-resistant colonies on culture. The difference between culture and PCR for the detection of ampicillin resistance was highly significant ({chi}2 = 7.298, P < 0.001). The most probable location of blaTEM in these specimens is the obligate anaerobic flora of the bowel.



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Figure. Results of nested PCR amplification of the blaTEM gene. Lane 1, 100 bp ladder; lane 2, UB1780 carrying a single chromosomal copy of blaTEM; lane 3, UB5201, isogenic with UB1780 but lacking blaTEM; lanes 4–11, PCR amplifications using faecal samples as templates. Lane 8 shows no amplification.

 
The discrepancy between the results obtained from culture experiments and those derived from a molecular analysis reveal the dangers inherent in relying upon a single method to determine the size of a potential gene pool. Reliance on aerobic culture has significantly underestimated the gene pool. It is not surprising that PCR has detected the blaTEM gene in more samples than were found by conventional culture. This is a technique capable of detecting genes in bacteria that cannot be recovered by conventional culture. In this context it is important to note that up to 80% of existing microorganisms may remain uncharacterized because of a reliance on culture detection.8 It is interesting to note that in a community-based study in The Netherlands, ampicillin-resistant faecal Enterobacteriaceae were found in 30% of specimens,9 and use of antibiotics by patients was subsequently found to increase selection for resistant E. coli.10 Notably, 27% of faecal samples collected in the initial Dutch study yielded no growth on antibiotic-free plates after 24 h.9 It must be remembered, however, that PCR alone does not reflect accurately the presence of active blaTEM genes. In order to determine the activity of the blaTEM gene in a population, it will be necessary to assay for the presence of mRNA derived from the blaTEM gene. Further characterization of the PCR products is also required to determine whether the genes detected encode narrow-spectrum ß-lactamases or those with an altered spectrum of activity. Only when such methodologies are applied will we obtain an accurate view of the distribution of active resistance genes in the population.


    Notes
 
* Corresponding author. Tel: +44-113-233-5592; Fax: +44-113-233-5638; E-mail: j.heritage{at}leeds.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Harrison, P. F. & Lederberg, J., Eds (1998). Antimicrobial Resistance: Issues and Options. National Academy Press, Washington DC.

2 . Hawkey, P. M. (1986). Resistant bacteria in the normal human flora. Journal of Antimicrobial Chemotherapy 18, Suppl. C, 133–9.[ISI][Medline]

3 . Livermore, D. M. (1995). ß-lactamases in laboratory and clinical resistance. Clinical Microbiology Reviews 8, 557–84.[Abstract]

4 . Greisen, K., Loeffelholz, M., Purohit, A. & Leong, D. (1994). PCR primers and probes for the 16S ribosomal RNA gene of most species of pathogenic bacteria, including bacteria found in cerebrospinal fluid. Journal of Clinical Microbiology 32, 335–51.[Abstract]

5 . Mabilat, C., Goussard, S., Sougakoff, W., Spencer, R. C. & Courvalin, P. (1990). Direct sequencing of the amplified structural gene and promoter for the extended broad-spectrum ß-lactamase TEM-9 (RHH-1) of Klebsiella pneumoniae. Plasmid 23, 27–34.[ISI][Medline]

6 . Hibbert-Rogers, L. C., Heritage, J., Todd, N. & Hawkey, P. M. (1994). Convergent evolution of TEM-26, a ß-lactamase with extended-spectrum activity. Journal of Antimicrobial Chemotherapy 33, 707–20.[Abstract]

7 . Livermore, D. M. (1998). ß-lactamase-mediated resistance and opportunities for its control. Journal of Antimicrobial Chemotherapy 41, Suppl. D, 25–41.[Abstract/Free Full Text]

8 . Ward, D. M., Weller, R. & Bateson, M. M. (1990). 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345, 63–5.[ISI][Medline]

9 . London, N., Nijsten, R., van den Bogaard, A. & Stobberingh, E. (1993). Antibiotic resistance of faecal Enterobacteriaceae isolated from healthy volunteers, a 15-week follow-up study. Journal of Antimicrobial Chemotherapy 32, 83–91.[Abstract]

10 . London, N., Nijsten, R., Mertens, P., van den Bogaard, A. & Stobberingh, E. (1994). Effect of antibiotic therapy on the antibiotic resistance of faecal Escherichia coli in patients attending general practitioners. Journal of Antimicrobial Chemotherapy 34, 239–46.[Abstract]

Received 19 October 2000; returned 30 March 2001; revised 30 April 2001; accepted 17 May 2001