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JAC Advance Access originally published online on June 25, 2008
Journal of Antimicrobial Chemotherapy 2008 62(4):703-708; doi:10.1093/jac/dkn263
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Colonization dynamics of ampicillin-resistant Escherichia coli in the infantile colonic microbiota

Nahid Karami*, Charles Hannoun, Ingegerd Adlerberth and Agnes E. Wold

Department of Clinical Bacteriology and Virology, University of Gothenburg, Gothenburg, Sweden

* Corresponding author. Tel: +46-31-3424636; Fax: +46-31-3424729; E-mail: nahid.karami{at}microbio.gu.se

Received 3 March 2008; returned 16 April 2008; revised 28 May 2008; accepted 1 June 2008


   Abstract
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Objectives: To compare the colonization dynamics of ampicillin-resistant and ampicillin-susceptible Escherichia coli strains in the infantile intestinal microbiota.

Methods: We followed 128 infants over the first year of life with regular quantitative faecal cultures and recordings of antibiotic treatment. E. coli strains were quantified, and their resistance pattern and carriage of β-lactamase genes (TEM, SHV and OXA), phylogenetic group (A, B1, B2 or D), virulence gene profile (fimA, papC, sfaD/E, kfiC neuB, hlyA and iutA) and time of persistence in the microbiota were determined.

Results: Twelve percent (n = 32) of the E. coli strains were resistant to ampicillin, as they carried the blaTEM (84%) or blaSHV genes. Ampicillin-resistant strains belonged mostly to phylogenetic group D and carried pap genes (P = 0.023) significantly more often than ampicillin-susceptible strains due to a strong association between carriage of pap and blaSHV. In 31 of 32 cases, colonization by ampicillin-resistant strains occurred in infants not previously treated with β-lactam antibiotics. Ampicillin-resistant strains were equally capable as susceptible ones of persisting in the intestinal microbiota and did not have lower faecal population counts. Genes encoding β-lactamases were in most cases retained during the entire colonization period.

Conclusions: The results suggest that ampicillin-resistant E. coli strains are not hampered in their colonizing capacity, and β-lactamase genes, therefore, may only slowly be eliminated from the commensal E. coli strain pool.

Keywords: E. coli , β-lactamases , virulence factors , persistence , phylogeny


   Introduction
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Escherichia coli belong to the normal microbiota of the intestines of humans and animals,1 but may also cause urinary tract infection and neonatal septicaemia/meningitis.2–4 Strains causing such extraintestinal infections carry an array of virulence genes, including P fimbriae, aerobactin, capsules, haemolysin and aerobactin.5 Virulent strains mostly belong to the phylogenetic B2 and D sublineages, whereas strains of A and B1 sublineages generally have low extraintestinal pathogenicity.6,7

The capacity to persist in the normal microbiota varies strikingly among E. coli strains. Some strains, termed resident, may colonize an individual for months or years.8–10 Others, so-called transient strains, persist in the microbiota only for a short period and may not, in a strict sense, colonize. Interestingly, the same traits that confer extraintestinal pathogenicity are partly identical to those conferring the capacity to persist in the commensal microbiota. Thus, colonic resident strains carry the genes for P fimbriae,11–13 haemolysin and aerobactin12,14,15 more often than do transient strains. They also have O and K antigens of types similar to those in uropathogenic E. coli strains13,14 and belong to the B2 sublineage.16,17

Microbial resistance to antibiotics is a growing problem. The intestinal microbiota may provide an important reservoir for antibiotic-resistant bacteria, but the influence of antibiotic resistance gene carriage in such complex bacterial communities has received little attention. Due to the metabolic cost of reproducing antimicrobial resistance elements, susceptible strains would in theory be at advantage in direct competition between strains. In the absence of selective pressure from antibiotics, colonization dynamics of resistant and susceptible strains should favour elimination of resistance genes or make resistant strains less capable of persisting in the complex microbiota of the gut, where competition for nutrients is fierce. However, as we previously reported, tetracycline-resistant E. coli strains persist equally well as susceptible strains in the intestinal microbiota of infants who have never been exposed to this antibiotic.18 The persistence of resistant strains may be attributed to a genetic linkage between resistance and traits beneficial for persistence, or adaptive mutations may compensate for the cost of carriage resistance elements.19,20

The most widely prescribed antibiotics among children are the β-lactam agents, in particular, penicillin V and amoxicillin,21 which act by interfering with cell wall synthesis. Penicillin V has good activity against Gram-positive bacteria and Gram-negative cocci, whereas ampicillin/amoxicillin is also active against certain Gram-negative bacteria, including E. coli. However, since ampicillin was put on the market, a significant population of E. coli has acquired β-lactamases, i.e. enzymes that hydrolyse the β-lactam ring and thus inactivate ampicillin.22 A range of β-lactamases are produced by Gram-negative bacteria, the most common β-lactamases being TEM-1, TEM-2 and SHV-1, which confer resistance to ampicillin/amoxicillin and early generation cephalosporins.23,24 Expression of β-lactamase is regulated by promoter genes with varying efficiency. For blaTEM genes, both weak (P3) and strong (P4 and Pa/Pb) promoters have been identified,25 the difference deriving from single nucleotide mutations. Overproduction of β-lactamases due to a strong promoter decreases sensitivity to the β-lactamase inhibitor, clavulanic acid.26

The present study describes the colonization dynamics of ampicillin-resistant and -susceptible E. coli strains in the gut microbiota of 128 Swedish infants followed over their first year of life. E. coli were isolated and quantified in regular stool samples and characterized regarding antibiotic resistance, carriage of virulence genes, phylogenetic group identity and time of persistence in the intestinal microbiota. As the antibiotic consumption of each child was registered, the influence of antibiotic treatment on the establishment of β-lactam-resistant strains could be investigated.


   Materials and methods
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E. coli strains

A collection of 272 commensal intestinal E. coli strains was studied. They were derived from 128 infants born in 1998–2001 in Göteborg, Sweden. The infants participated in ALLERGYFLORA, a prospective birth-cohort study designed to investigate the relationship between intestinal colonization pattern in infancy and later allergy development.27 The children's intake of antibiotics was registered by the parents in a diary. The records were checked by a study nurse who interviewed the parents by telephone at 6 and 12 months. Informed consent was obtained from the parents, and the study was approved by the Medical Ethics Committee of Gothenburg University.

Rectal swabs were obtained at 3 days of age and cultured semi-quantitatively for facultative bacteria.27 Faecal samples were obtained at 1, 2, 4 and 8 weeks and at 6 and 12 months of age. They were transported under anaerobic conditions and cultured quantitatively within 24 h by plating serial dilutions on a range of selective and non-selective media, followed by incubation under aerobic and anaerobic conditions.27 Enterobacteria were isolated on Drigalski agar, on which colonies differing even slightly in morphology were identified. Each morphotype was separately enumerated, Gram-stained, subcultured, speciated using an API 20E biotyping system (bioMérieux, Marcy-I’Étoile, France) and assayed by random amplified polymorphic DNA (RAPD) for strain identity.15 All E. coli isolates deriving from one infant (an average of seven isolates per infant) were assayed together on the same gel. RAPD profiles with identical major bands and differing in not more than three minor bands were considered as representing the same strain.28

Virulence genotyping and phylotyping

Each strain was analysed for carriage of genes for type 1 (fimA), P (papC) and S (sfaD/E) fimbriae, Dr haemagglutinin (draA), K5 (kfiC) and K1 (neuB) capsules, haemolysin (hlyA) and aerobactin (iutA). A multiplex PCR was used for this purpose, as described in detail elsewhere.15

Each strain was assigned to one of the phylogenetic groups A, B1, B2 or D using PCR as described previously.29 Data regarding virulence gene carriage and phylogenetic origin of E. coli strains have previously been published for the cohort.16,18

Susceptibility testing

All E. coli strains (a total of 272 strains identified among 950 isolates deriving from 128 infants) were tested for antibiotic resistance using the agar disc diffusion method as outlined by the Swedish Reference Group for Antibiotics.21 If the infant received antibiotics during the first year of life, all isolates of each strain from this infant were tested (30 infants, 55 strains and 125 isolates). Furthermore, if the first isolate of an E. coli strain colonizing an infant was resistant to ampicillin, all isolates of this and all other strains appearing in that infant were tested (20 infants, 46 strains and 109 isolates). In the remaining cases (78 infants, 171 strains and 716 isolates), only the first isolate of each strain was tested.

The following 14 antibiotic-containing discs (AB Biodisk, Solna, Sweden) were applied to Iso-Sensitest agar: ampicillin, cefoxitin, cefuroxime, mecillinam, cefadroxil, ceftazidime, chloramphenicol, gentamicin, tobramycin, nitrofurantoin, nalidixic acid, norfloxacin, tetracycline and trimethoprim. Strains with clearance zones of <11 mm around the ampicillin disc were further analysed by Etest to determine the MIC value of ampicillin, as outlined by the SRGA.21 Ampicillin-resistant strains (MIC > 8 mg/L) were further tested for susceptibility to amoxicillin in combination with clavulanic acid by the disc diffusion method. E. coli ATCC 25922 (CCUG 17620) was used as a fully susceptible control strain.

Molecular detection of TEM, SHV and OXA β-lactamase genes

Ampicillin-resistant E. coli strains were assessed for carriage of the ampicillin resistance genes blaTEM, blaSHV and blaOXA using PCR. In brief, one bacterial colony was picked from an agar culture and suspended in HotStarTaq Master Mix (Qiagen GmbH, Hilden, Germany). First, a PCR was performed for the amplification of the entire blaTEM gene using primers and conditions described previously.30,31 The following primers for blaTEM were used: TEM-F (5'-TTCTTGAAGACGAAAGGGC-3') and TEM-R (5'-ACGCTCAGTGGAACGAAAAC-3'). The size of the entire blaTEM amplicons was 1150 bp. A duplex PCR was performed for the amplification of the blaSHV and blaOXA genes using primers and conditions described previously.32 The following primers were used: for blaSHV, primers SHV-F (5'-AGGATTGACTGCCTTTTTG-3') and SHV-R (5'-ATTTGCTGATTTCGCTCG-3'); and for blaOXA, primers OXA-F (5'-ATATCTCTACTGTTGCATCTCC-3') and OXA-R (5'-AAACCCTTCAAACCATCC-3'). The sizes of the blaSHV and blaOXA amplicons were 392 and 619 bp, respectively. The strains carrying known bla genes were used as positive controls (CCUG 30600 for blaTEM, CCUG 45421 for blaSHV and CCUG 52541 for blaOXA, all from the Culture Collection of the University of Gothenburg, CCUG, Sweden).

The blaTEM-positive PCR products were purified with magnetic beads and sequenced using the Big Dye Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems). Three forward primers (TEM-F, TEM-M1 and TEM-M2) and two reverse primers (TEM-R and TEM-end-R3) were used to obtain sequences covering both strands of the entire blaTEM gene.31,33 Sequence-PCR products were purified, analysed (3730 DNA Sequencer, Applied Biosystems) and aligned (Sequencher 4.2.2 software, Gene Codes Corporation, MI, USA). The nucleotide sequences obtained were compared with previously described sequences obtained from the GenBank database.

Statistics

Frequencies were compared using Fisher's exact test. The time of persistence in the bowel flora of susceptible and resistant strains was compared using the Mann–Whitney test. Population counts of susceptible and resistant strains colonizing the same individual were compared using the Wilcoxon signed rank test.


   Results and discussion
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 Abstract
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Prevalence and mechanism of ampicillin resistance among commensal E. coli strains

Of the E. coli strains isolated from the commensal infantile microbiota, 12% (32/272) were phenotypically resistant to ampicillin. They all had MICs exceeding 256 mg/L. They were all susceptible to the cephalosporins tested: cefadroxil, cefoxitin, cefuroxime and ceftazidime. The prevalence of ampicillin resistance is very moderate compared with the 59% ampicillin resistance found among E. coli from Spanish children,34 but still higher than the 2% ampicillin resistance found among commensal E. coli sampled from Swedish schoolgirls in the 1970s.35

Ampicillin resistance occurred either as a single trait (n = 15) or in combination with resistance to trimethoprim (n = 8), tetracycline (n = 3), tetracycline and trimethoprim (n = 3), chloramphenicol and trimethoprim (n = 1), chloramphenicol, tetracycline and trimethoprim (n = 1), or chloramphenicol, nalidixic acid, tetracycline and trimethoprim (n = 1).

Ampicillin-resistant strains were analysed with respect to carriage of blaTEM, blaSHV and blaOXA genes by PCR. All ampicillin-resistant strains carried at least one gene belonging to these groups. The blaTEM-1 genes were most common, found in 84% (27/32) of the resistant strains, one of which also carried the blaOXA genes. The rest (n = 5) had blaSHV genes. Strains carrying blaTEM-1 were more often resistant to other antibiotics (63%) than ampicillin-susceptible strains (12%, P = 0.0017) or blaSHV-positive strains (0%, P = 0.01).

The promoter region was sequenced in all 27 blaTEM amplicons. The weak P3 promoter was found in 24 and the strong Pa/Pb promoter in 3 of these sequences. The occurrence of the stronger promoter coincided with a decreased susceptibility to amoxicillin/clavulanic acid in these three strains, as previously reported.36

Ampicillin resistance in relation to phylogenetic group

Resistance to ampicillin was analysed in relation to phylogenetic group (A, B1, B2 or D). Strains belonging to phylogenetic group D were more often phenotypically ampicillin-resistant (26% versus 9%, P = 0.01) and more often carried the blaTEM-1 genes (23% versus 8%, P = 0.002) compared with strains of the other phylogenetic groups. Strains belonging to phylogenetic group B1 were significantly less often ampicillin-resistant (0% versus 13%, P = 0.03) than those belonging to the other phylogenetic groups. Strains belonging to phylogenetic group A or B2 did not differ significantly in ampicillin resistance, compared with the other phylogenetic groups (group A, 13% versus 18%, P = 0.37; group B2, 10% versus 14%, P = 0.45).

Four strains carrying blaSHV genes belonged to phylogenetic group B2, whereas only one belonged to group D. The SHV type of β-lactamase has previously been associated with phylogenetic group B2 E. coli.37

Resistance to ampicillin and carriage of virulence genes

We examined whether ampicillin-resistant and -susceptible strains differed in their carriage of genes encoding a range of virulence factors. The results are shown in Figure 1. Strains with phenotypic resistance to ampicillin carried the P-fimbrial gene papC (47% versus 27%, P = 0.023) significantly more often than ampicillin-susceptible strains. This association was due to an accumulation of virulence genes among blaSHV-positive strains. All strains with the blaSHV genotype carried the same combination of virulence genes, namely fimA, papC, sfaD/E and hlyA, the last three genes being significantly more common among blaSHV-positive strains than among ampicillin-susceptible strains (P = 0.0015 for papC and sfaD/E and P = 0.0006 for hlyA) or among blaTEM-positive strains (P = 0.015 for papC, P = 0.0013 for sfaD/E and P = 0.0006 for hlyA). Virulence genes papC, sfaD/E and hlyA are linked to the capacity for long-term persistence.11–13,15 blaTEM genes more often had the aerobactin gene iutA than strains susceptible to ampicillin (46% versus 26%, P = 0.039, Figure 1). Aerobactin is a virulence factor that is also associated with enhanced capacity to persist in the gut microbiota.15


Figure 1
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  		Figure 1. Virulence gene carriage among ampicillin-resistant (n = 32) and ampicillin-susceptible (n = 240) E. coli strains from the microbiota of 128 Swedish infants. Ampicillin-resistant strains were further divided into blaTEM-positive (n = 27) and blaSHV-positive strains (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

 
Impact of antibiotic treatment on E. coli colonization dynamics

We examined whether colonization by ampicillin-resistant strains occurred in relation to antibiotic treatment. In all, 30 infants were treated with antibiotics at least once during the first year of life. Fourteen of these received amoxicillin or cephalosporin antibiotics that could select for β-lactamase-producing E. coli. Figure 2 shows the E. coli strain colonization pattern during the first year of life in relation to antibiotic treatment in these 14 infants. In only one case could a temporal relation between β-lactam treatment and colonization by β-lactamase-producing E. coli be detected: infant no. 12 was colonized by an ampicillin-resistant strain at 12 months of age after having received amoxicillin shortly after 6 months of age. However, 12% (11/92) of E. coli strains picked up by non-treated infants between 6 and 12 months of age were also ampicillin-resistant, indicating that the acquisition of a resistant strain in 1/14 treated infants could occur by chance without relation to treatment. In all, 31 of 32 ampicillin-resistant strains were found in infants who had not previously been treated with β-lactam antibiotics that could select for such resistance. Thus, it appeared as if β-lactam-resistant E. coli are part of the common pool of circulating strains and may establish, unimpeded by their resistance, in the commensal microbiota of non-treated hosts. In one infant (no. 5), an E. coli strain that was initially susceptible to ampicillin acquired resistance after treatment with ampicillin due to transfer of a resistant plasmid from another E. coli strain that co-inhabited the gut flora of that infant (Figure 2). This event has been described in detail elsewhere.38


Figure 2
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  		Figure 2. E. coli colonization pattern of the 14 infants who were treated with amoxicillin or a cephalosporin at least once during their first year of life. Each E. coli strain colonizing an infant is represented by a symbol. Filled symbols indicate strains resistant to ampicillin (filled circles) or ampicillin and trimethoprim (filled upside-down triangles). Open symbols indicate strains susceptible to ampicillin; different symbols indicating individual strains colonizing an infant. A horizontal line connecting the strain symbols indicates persistence of a strain in the intestinal microbiota. A vertical line marks treatment with antibiotics: a solid line representing amoxicillin/ampicillin or a cephalosporin, and a dashed line representing treatment with other antibiotics. The antibiotics given are specified on the right-hand side of the diagram as follows: AMP, ampicillin; AMX, amoxicillin; PEN, penicillins (PcV or PcG); TMP, trimethoprim; CTB, ceftibuten; NIT, nitrofurantoin; CAZ, ceftazidime; TOB, tobramycin; ERY, erythromycin.

 
Faecal population levels of ampicillin-resistant E. coli strains

The stool population counts of each strain were determined on each culture occasion, which permitted us to compare the population levels of ampicillin-resistant and -susceptible strains. Because the population levels of facultative bacteria, including E. coli, decrease with time as the infantile gut microbiota become increasingly complex,27 the population levels should be compared in infants of the same age. Figure 3(a) shows the faecal population levels of ampicillin-resistant and ampicillin-susceptible strains at different time points in infants who never received antibiotics during their first year of life. At no time point were the faecal population counts of ampicillin-resistant strains lower than those of ampicillin-susceptible strains in this cross-sectional analysis.


Figure 3
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  		Figure 3. (a) Faecal population counts (log 10 values) of all ampicillin-resistant (filled circles) and ampicillin-susceptible (open circles) E. coli strains at each time point in infants not receiving any antibiotics during their first year of age. The numbers within parentheses show the number of ampicillin-resistant strains at each sampling occasion. (b) Faecal population counts of ampicillin-resistant and ampicillin-susceptible strains colonizing simultaneously in eight infants. In one infant, one ampicillin-resistant strain and three ampicillin-susceptible strains were present simultaneously; the population counts of the susceptible strains were averaged before comparison with those of the resistant strain.

 
To investigate a situation with direct competition between resistant and susceptible E. coli strains, we examined the eight infants who simultaneously harboured an ampicillin-resistant strain and at least one fully susceptible strain in the same stool sample. The population counts were slightly lower for resistant than for susceptible strains (average 107.40 versus 107.79), but the difference was not significant in paired analysis (P = 0.10, Figure 3b). These results show that ampicillin-resistant genes have a low fitness cost on E. coli strains and that they are able to compete favourably in the gut flora.

Impact of ampicillin resistance on capacity to persist in the intestinal microbiota

Ampicillin-resistant and -susceptible strains were examined with respect to their capacity to persist in the microbiota. Strains that were isolated at least twice in faecal samples collected at least 3 weeks apart were classified as resident, whereas strains whose colonization period was shorter than a month were classified as transient. Strains appearing only once in samples spaced >1 month in time could neither be grouped as resident nor as transient and were not included in the analysis. Among ampicillin-resistant strains, 82% were resident and 18% transient. Among blaTEM-positive strains, 87% (13/15) were resident, and among the five blaSHV-positive strains, one strain was resident and one transient, while the three remaining strains could not be classified. Among ampicillin-susceptible strains, 81% were resident.

The average time of persistence was 34±15 (SD) weeks for the 13 resident strains that were resistant to ampicillin and 29±17 weeks for the 82 resident strains that were susceptible (P = 0.35). Thus, ampicillin-resistant strains were equally able to persist in the infantile microbiota as ampicillin-susceptible strains.

To exclude the possibility that the good colonizing capacity of ampicillin-resistant strains was the result of selection by antibiotic treatment, we excluded all infants ever treated with antibiotics from the analysis. Among strains that colonized infants never treated with antibiotics, 91% (10 of 11) of the ampicillin-resistant and 85% (63 of 74) of the ampicillin-susceptible strains were resident in the microbiota. The average time of persistence was 29±15 weeks for the ampicillin-resistant resident strains and 26±17 weeks for the susceptible strains (P = 0.42). Thus, even in the absence of any antibiotic, ampicillin-resistant strains persisted equally well as fully susceptible strains in the microbiota.

Stability of ampicillin resistance

We examined the stability of the resistance phenotype in the 13 ampicillin-resistant E. coli strains that persisted in the microbiota of an infant for at least 3 weeks. Four of the 13 infants harbouring such a strain received β-lactam antibiotics. Eleven of the strains kept their resistance genes during the entire colonization period (the average time of persistence being 36±15 weeks). Resistance to β-lactam antibiotics tended to be a fairly stable trait. However, two strains, both from infants who received no antibiotics, lost their blaTEM genes. The strain identity of these isolates as determined by RAPD was confirmed by PFGE and virulence gene profiles. The strains also lost resistance to tetracycline and trimethoprim simultaneously with the loss of blaTEM, suggesting the loss of a multiple drug resistance plasmid.18 As described previously in a paper reporting on the effect of tetracycline resistance genes on colonization dynamics,18 the loss of the resistance phenotype was associated with increased population levels in one case and decreased population levels in the other.

Conclusions

Our results suggest that carriage of β-lactamase genes does not impact negatively on survival of E. coli in the gut microbiota because: (i) resistant strains are freely established in children not treated with antibiotics; (ii) resistant strains persist equally well in the microbiota as susceptible ones and attain approximately equal population levels; and (iii) resistance genes were, in most cases, retained during long-time colonization in the absence of selective pressure. The good colonizing ability could be due to the carriage, in ampicillin-resistant strains, of virulence factor genes that promote persistence in the colonic milieu. These findings suggest that resistance genes may not be easily eliminated from the commensal strain pool.


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The study was supported by grants from the Swedish Medical Research Council (no. K98-06X-12612-01A), the Swedish Strategic Programme for the Rational Use of Antimicrobial Agents and Surveillance of Resistance, the Capio Research Foundation, the Magnus Bergvall Foundation, the Wilhelm and Martina Lundgren Foundation, the May Flower Foundation and the Swedish Freemasons Foundation.


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None to declare.


   References
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