Screening Fecal Enterococci from Greek Healthy Infants for Susceptibility to Antimicrobial Agents

Abstract

Enterococci are among the first lactic acid bacteria to colonize the neonatal gastrointestinal tract, but they are also characterized as significant nosocomial pathogens. The aim of this study was to investigate the incidence of antibiotic resistance in enterococci isolated from neonates' gut microbiota as well as the presence of genetic determinants encoding for certain antibiotic resistance traits. A total of 263 fecal samples derived from 97 infants were collected on day 4, 30, and 90 after delivery. Enterococcus faecalis was the most frequently identified species (54.6%) followed by E. faecium, while E. casseliflavus/E. flavescens and E. gallinarum were also traced. The isolates were examined for their resistance to 12 antibiotics. Rifampicin resistance was the highest observed (53.2%), followed by resistance to tetracycline (42.0%), erythromycin (35.7%), and vancomycin (11.2%). Multiresistant strains were highly prevalent. Only intrinsic vancomycin resistance (vanC1 and vanC2/C3) was traced. The ermB gene was detected in 49 out of 96 erythromycin-resistant isolates, while tet genes were detected in 51 out of 113 tetracycline-resistant strains, with tet(L) being the most frequently observed. In conclusion, antibiotic-resistant enterococci are already established in the fecal microbiota of healthy neonates, from the first days of an infant's life.

Introduction

Enterococci are gram-positive, facultative anaerobic bacteria, members of the commensal microbiota of the intestinal tract of both animals and humans. In fact, they are among the first lactic acid bacteria to colonize the neonatal gastrointestinal tract.¹⁵ On the other hand, enterococci are also opportunistic pathogens; despite their low virulence, studies have documented that they are very significant nosocomial pathogens.²³ The intestinal microbiota is a potential source of pathogens, causing nosocomial infections, including bacteraemia, endocarditis, urinary tract infections, and contamination associated with surgery. The occurrence of large number of resistant bacteria within this flora is highly undesirable due to the risk of obstructing the effect of antibiotic treatment.²⁷

Enterococci express intrinsic resistance against cephalosporins and semisynthetic penicillins and acquired resistance against aminopenicillins and glycopeptides,^5,30 probably due to the use of broad-spectrum antibiotics (cephalosporins) or multiantibiotic regimens.²²

Enterococci acquire resistance to antimicrobial agents through the transfer of plasmids and transposons, chromosomal exchange, or mutation.²⁹ The resistance to vancomycin, tetracycline, and erythromycin is encoded by genetic determinants usually harbored in transferable genetic material.⁴⁰ To date, eight types of vancomycin resistance have been described in enterococci (vanA, vanB, vanC1, vanC2/C3, vanD, vanE, vanG, and vanL), each of them associated with a different ligase gene, resulting in a decrease or inhibition of the glycopeptide action. vanC-type resistance is an intrinsic, nontransferable property of Enterococcus gallinarum and E. casseliflavus/flavescens, related to the chromosomal presence of the species-specific genes vanC1 and vanC2/C3, respectively. All other types of vancomycin resistance are acquired and transferable characteristics, with vanA being the most clinically important.^25,43 Erythromycin resistance in enterococci is mainly associated with the presence of ermB gene on the conjugative transposon Tn917, which inhibits erythromycin binding by methylation of 23S rRNA.^21,25 Resistance to tetracycline in Enterococcus spp. can be mediated by energy-dependent efflux of tetracycline [tet(K) and tet(L) genes], by ribosomal protection from the action of tetracycline [tet(M), tet(O) and tet(S) genes] and by unknown mechanisms [tet(U) gene].⁷ In certain bacteria, tet(M) and tet(S) are harbored in conjugative transposons, implying a high potential for genetic exchange, especially in environments with high bacterial concentrations such as the human intestine. The tet(S) gene is mainly found in some important food-related bacteria, such as lactobacilli and enterococci.¹⁷

Exchange of genetic material between prokaryotes in the intestinal environment is of particular interest, since antibiotic resistance genes transferred by the ingested food may spread to other bacteria. The present study examines the incidence of antibiotic-resistant enterococci and the presence of selected resistance genes isolated from the fecal microbiota of Greek healthy, full-term infants, under no apparent antimicrobial selective pressure.

Materials and Methods

Subjects of research

Fecal specimens were collected from newborn infants born at the Department of Neonatology of the General-Maternity District Hospital Helena Venizelou in Athens, Greece, after written informed consent of their parents. The infants were healthy, full-term, Greek (of both sexes), born with no congenital abnormalities and delivered after uncomplicated pregnancy. The mothers did not receive antimicrobial agents within the month that preceded the delivery, neither did the babies during the study. The specimens were collected from 97 infants at day 4, 30, and 90 after infants' delivery.

Sample collection and bacterial isolates

Specimens were collected rapidly into sterile plastic containers and transferred under anaerobic conditions (GENbag anaer, 45534 Biomérieux^® SA) to a laboratory for microbiological analysis.

Approximately 1 g of the specimen was weighted and diluted in 9-ml prereduced peptone physiological saline, containing 0.1% bacteriological peptone (Oxoid) and 0.85% NaCl.³⁵ M17 agar (LabM Limited)³⁹ was used for the cultivation and isolation of cocci under aerobic conditions at 37°C for 48 hr.

Identification of bacterial isolates

All the Gram-positive, catalase-negative cocci isolated from M17 agar (LabM Limited), were examined for: (1) gas production in de Man, Rogosa and Sharpe broth cultures (Oxoid), (2) growth in the M17 broth (Oxoid) at 10°C and 45°C, and (3) growth in the 6.5% NaCl M17 broth (Oxoid) at 45°C, after 48 hr of incubation under aerobic conditions. Enterococci typically do not produce gas and they are able to grow at 10°C, at 45°C and in high NaCl concentration. Identification to the species level for Enterococcus spp. was performed by a multiplex polymerase chain reaction (PCR) methodology, as it is described ahead.

The isolates not identified by the multiplex PCR assay were further analyzed by partial amplification of the 16S rDNA gene with the universal primers p0 and p6^10,12 and sequencing of PCR products at Macrogen, Inc.

Susceptibility testing

The isolates were subjected to agar disc diffusion antimicrobial susceptibility testing in Mueller Hinton agar (Oxoid), according to the Clinical and Laboratory Standards Institute (CLSI).^8,9 Twelve different antimicrobial agents were applied in dried filter disc form with the following potencies (BBL™ Sensi-Disc™ Antimicrobial Susceptibility Test Discs, Becton Dickinson): ampicillin 10 μg, vancomycin 30 μg, bacitracin 10 U, tetracycline 30 μg, erythromycin 15 μg, streptomycin 300 μg (high level resistance), gentamycin 120 μg (high level resistance), ciprofloxacin 5 μg, rifampicin 5 μg, ofloxacin 5 μg, norfloxacin 10 μg, and nitrofurantoin 300 μg. Isolates were characterized as susceptible, intermediate, or resistant according to CLSI zone diameter interpretive standards.

Detection of antibiotic resistance genes

DNA extraction

DNA extraction was performed in bacterial cell suspensions, as reported in literature,²⁰ using a density equivalent to a McFarland standard of 5. Identification of the isolates and detection of vancomycin resistance (van) genes was performed by using multiplex PCR. The vancomycin-susceptible strains were subcultured to M17 agar (LabM Limited) and vancomycin-resistant isolates to M17 agar containing 6 mg/L vancomycin (Abbott Laboratories Hellas S.A.) (37°C for 48 hr).

For molecular detection of tet genes and ermB determinant, tetracycline- resistant cocci were subcultured to brain heart infusion agar (LabM Limited) with 8 mg/L tetracycline (Sigma-Aldrich Chemie GmbH) and erythromycin-resistant isolates to brain heart infusion agar (LabM Limited) with 5 mg/L erythromycin (Sigma-Aldrich Chemie GmbH), respectively.

Multiplex PCR for Enterococcus spp. identification and detection of vancomycin resistance (van) genes

A multiplex PCR assay was performed for the detection of four glycopeptide resistance genotypes (vanA, vanB, vanC1, vanC2/C3) and the identification to the species level of E. faecalis, E. faecium, E. gallinarum, and E. casseliflavus/E. flavescens, as previously described and improved.^14,24 This assay also includes partial amplification of 16S rRNA (rrs) as an internal PCR control to improve reliability.²⁴ The reference strains used were E. faecalis 208 (vanA gene), E. faecium VRE3 (vanA gene), E. faecalis 140 (vanB gene), and E. faecium VRE2 (vanB gene), as kindly provided by Prof. E. Skoulika, as well as two strains from home strain collection (E. gallinarum [vanC1 gene] and E. casseliflavus/E. flavescens [van C2/C3 gene]).

Detection of tetracycline resistance (tet) genes and erythromycin resistance (ermB) gene

A multiplex PCR analysis was performed for the detection of the tet(K), (L), (M), (O), and (S) tetracycline resistance determinants, as previously described,³¹ with some modifications; a set of primers recommended by Doherty et al. for the detection of the tet(M) gene¹³ was used. Reference strains for tetracycline resistance detection were Escherichia coli HB101/pAT102 [tet(K) gene], E. coli JM83/pAT103 [tet(L) gene], E. coli HB101/pAT183 [tet(M) gene], E. coli JM83/pAT51 [tet(O) gene], and Listeria monocytogenes BM4210/pIP811 [tet(S) gene] (CRBIP, Institut Pasteur, France). The presence of the ermB gene (class IV) in erythromycin-resistant isolates was determined by a PCR methodology based on the sequence of the transposon Tn917,²¹ and E. faecalis JH2-2 (Tn1545)=BM4137 was used as reference strain (CRBIP, Institut Pasteur, France).

Ethics

The study has been approved by the Bioethics Committee of Harokopio University.

Statistics

Comparisons of antibiotic resistance prevalence and presence of resistance determinants were performed by the Chi-square test. The statistical analysis was performed by the software program SPSS^® for Windows Release 11.5 and p<0.05 was considered to be statistically significant.

Results

Two hundred sixty three fecal specimens derived from 97 infants were examined. In the case of 14 infants, only the first sample (day 4) was collected. Enterococci or Streptococci were isolated from all the sampling periods (days 4, 30, and 90).

Identification of bacterial isolates

E. faecalis was the most frequently identified species, since it represented 54.6% of the 269 Gram-positive, catalase-negative cocci and it predominated on day 30 and 90 (Table 1). It was followed by E. faecium, which was more frequently detected on the fourth day. E. casseliflavus/E. flavescens and E. gallinarum were isolated in lower proportions.

Table 1.

Identification of Isolates at 4 (n=79), 30 (n=86), and 90 (n=104) days of age

Sampling	Enterococcus faecalis	Enterococcus faecium	Enterococcus casseliflavus/E. flavescens	Enterococcus gallinarum	Enterococcus hirae	Streptococcus spp.
4 days	24	45	2	—	1	7^a
30 days	67	5	9	1	—	4^b
90 days	56	18	16	7	—	7^c
Total strains	147	68	27	8	1	18

Values are expressed as numbers of strains.

Streptococcus vestibularis (one strain), Str. salivarius (five strains), and Str. pasterianus (one strain).

Str. pasterianus.

Str. pasterianus (four strains), Str. mitis 2 (one strain), Str. salivarius (one strain), and Str. equinus (one strain).

Resistance phenotypes

Rifampicin resistance was the highest observed (53.2%). Furthermore, 42.0% of the isolates were resistant to tetracycline, 35.7% were resistant to erythromycin, and 11.2% were resistant against vancomycin. Resistance to nucleic acid synthesis inhibitors (ciprofloxacin, rifampicin, ofloxacin, norfloxacin, and nitrofurantoin) was also significant. Resistance to ampicillin (6.0%), bacitracin (5.2%), gentamycin (2.2%), and high-level resistance to streptomycin (5.6%) were relatively limited.

As it is presented in Table 2, resistance to tetracycline (p=0.000) was significantly increased on day 30 and 90 in comparison to day 4, but maintained rather stable within the last two sampling periods. Resistance to nitrofurantoin and erythromycin was higher on day 4 compared to day 30 and 90, but it did not significantly change from day 30 to 90. Resistance to ciprofloxacin demonstrated fluctuations as it decreased (p=0.015) from day 4 to 30 and then increased (p=0.013) from day 30 to 90. Resistance to ofloxacin decreased at day 30 compared to day 4 (p=0.041) and resistance to norfloxacin was significantly increased at day 90 compared to day 30 (p=0.009). No other significant changes in the susceptibility profiles of isolates were detected during sampling. Thirty-six strains (13.4%) (12 E. faecalis, 9 E. casseliflavus/E. flavescens, 7 E. faecium, 4 Streptococcus saliv.salivarius, 2 E. gallinarum, 1 E. hirae and 1 Str. mitis 2) expressed no resistance to the tested antibiotics. Furthermore, one strain Str. vestibularis, one Str. Salivarius, and one E. hirrae isolated at day 4, one strain of E. faecium isolated at day 30 and finally, one E. faecium and one E. casseliflavus/E. flavescens isolated at day 90 were susceptible to all antimicrobial agents.

Table 2.

Antibiotic Susceptibility Profile of the 269 Cocci Isolates at 4 (n=79), 30 (n=86), and 90 (n=104) Days of Age by Disk-Diffusion Technique

	Susceptibility profile
	Susceptible			Intermediate			Resistant
Antibiotics	4 days	30 days	90 days	4 days	30 days	90 days	4 days	30 days	90 days
Ampicillin	73 (92.4)	84 (97.7)	95 (91.3)	1 (1.3)	—	—	5 (6.3)	2 (2.3)	9 (8.7)
Vancomycin	60 (75.9)	52 (60.5)	67 (64.4)	11 (13.9)	26 (30.2)	23 (22.1)	8 (10.1)	8 (9.3)	14 (13.5)
Bacitracin	32 (40.5)	47 (54.7)	52 (50.0)	44 (55.7)	36 (41.9)	44 (42.3)	3 (3.8)	3 (3.5)	8 (7.7)
Tetracycline	61 (77.2)	43 (50.0)	46 (44.2)	—	—	6 (5.8)	18 (22.8)	43 (50.0)^a	52 (50.0)^a
Erythromycin	10 (12.7)	9 (10.5)	21 (20.2)	28 (35.4)	54 (62.8)	51 (49.0)	41 (51.9)	23 (26.7)^a	32 (30.8)^a
Streptomycin	76 (26.2)	75 (87.2)	94 (90.4)	—	4 (4.7)	5 (4.8)	3 (3.8)	7 (8.1)	5 (4.8)
Gentamycin	75 (94.9)	83 (96.5)	103 (99.0)	1 (1.3)	1 (1.2)	—	3 (3.8)	2 (2.3)	1 (1.0)
Ciprofloxacin	25 (31.6)	30 (34.9)	33 (31.7)	37 (46.8)	49 (57.0)	49 (47.1)	17 (21.5)	7 (8.1)^a,b	22 (21.2)
Rifampicin	22 (27.8)	26 (30.2)	34 (32.7)	9 (11.4)	19 (22.1)	16 (15.4)	48 (60.8)	41 (47.7)	54 (52.0)
Ofloxacin	36 (45.6)	52 (60.5)	49 (47.1)	28 (35.4)	27 (31.4)	40 (38.5)	15 (19.0)	7 (8.1)^a,b	15 (14.4)
Norfloxacin	36 (45.6)	34 (39.5)	31 (29.8)	27 (34.2)	40 (46.5)	42 (40.4)	16 (20.3)	12 (14.0)^b	31 (29.8)
Nitrofurantoin	43 (54.4)	73 (84.9)	91 (87.5)	19 (24.1)	8 (9.3)	4 (3.8)	17 (21.5)	5 (5.8)^a	9 (8.7)^a

Statistically different compared to day 4 (p<0.05).

Statistically different compared to day 90 (p<0.05).

Values are expressed as number of isolates; numbers in parentheses are susceptibility rates (%).

Distribution of resistant isolates according to identification data and sampling period is presented in Table 3. Vancomycin resistance was mostly observed in E. faecalis, E. gallinarum, and E. casseliflavus/E. flavescens isolates. None of the E. faecium isolates was characterized as vancomycin-resistant. Erythromycin and tetracycline resistances were rather distributed among species, with their occurrence being particularly high in E. faecium isolates at day 4 (64.4%) and day 90 (55.6%), respectively. Among inhibitors of nucleic acid synthesis, rifampicin was associated with the highest resistance levels, reaching 71.1% of E. faecium isolates on day 4, 58.9% of E. faecalis, and 56.3% of E. casseliflavus/E. flavescens stains 90 days after infants' delivery. Ampicillin resistance was mainly associated with E. faecium species and gentamycin resistance with E. faecalis, whereas streptomycin and bacitracin resistance exhibited a wider distribution.

Table 3.

Resistant Cocci Strains at 4, 30, and 90 Days of Age by Disk-Diffusion Technique

	E. faecalis			E. faecium			E. casseliflavus/E. flavescens			E. gallinarum			E. hirae	Streptococcus spp.
Antibiotics	4 days	30 days	90 days	4 days	30 days	90 days	4 days	30 days	90 days	4 days	30 days	90 days	4 days	4 days	30 days	90 days
Ampicillin	0 (0.0)	0 (0.0)	1 (1.8)	5 (11.1)	2 (40.0)	8 (44.4)	0 (0.0)	0 (0.0)	0 (0.0)	—	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Vancomycin	7 (29.2)	7 (10.4)	11 (19.6)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (12.5)	—	1 (100.0)	1 (14.3)	0 (0.0)	1 (14.3)	0 (0.0)	0 (0.0)
Bacitracin	1 (42.0)	1 (1.5)	2 (3.6)	2 (4.4)	1 (20.0)	2 (11.1)	0 (0.0)	0 (0.0)	1 (6.3)	—	1 (100.0)	3 (42.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Tetracycline	13 (54.2)	35 (52.2)	34 (60.7)	3 (6.7)	1 (20.0)	10 (55.6)	1 (50.0)	2 (22.2)	2 (12.5)	—	1 (100.0)	1 (14.3)	0 (0.0)	1 (14.3)	4 (100.0)	5 (71.4)
Erythromycin	9 (37.5)	18 (26.9)	15 (26.8)	29 (64.4)	1 (20.0)	10 (55.6)	1 (50.0)	1 (11.1)	3 (18.8)	—	1 (100.0)	1 (14.3)	0 (0.0)	2 (28.6)	2 (50.0)	3 (42.3)
Streptomycin	2 (8.3)	4 (6.0)	3 (5.4)	1 (2.2)	1 (20.0)	2 (11.1)	0 (0.0)	0 (0.0)	0 (0.0)	—	1 (100.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (25.0)	0 (0.0)
Gentamycin	3 (12.5)	2 (3.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (5.6)	0 (0.0)	0 (0.0)	0 (0.0)	—	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ciprofloxacin	6 (25.0)	1 (1.5)	7 (12.5)	10 (22.2)	3 (60.0)	8 (44.4)	0 (0.0)	2 (22.2)	4 (25.0)	—	1 (100.0)	2 (28.6)	0 (0.0)	1 (14.3)	0 (0.0)	1 (14.3)
Rifampicin	16 (66.7)	36 (53.7)	33 (58.9)	32 (71.1)	2 (40.0)	11 (61.1)	0 (0.0)	3 (33.3)	9 (56.3)	—	0 (0.0)	1 (14.3)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Ofloxacin	5 (28.8)	1 (1.5)	7 (12.5)	10 (22.2)	2 (40.0)	5 (27.8)	0 (0.0)	3 (33.3)	3 (18.8)	—	1 (100.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
Norfloxacin	6 (25.0)	6 (9.0)	12 (21.4)	9 (20.0)	1 (20.0)	7 (38.9)	0 (0.0)	3 (33.3)	5 (31.3)	—	1 (100.0)	3 (42.3)	0 (0.0)	1 (14.3)	1 (25.0)	4 (57.1)
Nitrofurantoin	6 (25.0)	4 (6.0)	5 (8.9)	11 (24.4)	0 (0.0)	3 (16.7)	0 (0.0)	0 (0.0)	1 (6.3)	—	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (25.0)	0 (0.0)

Values are expressed as numbers of strains; numbers in parentheses are resistance rates (%).

Multiresistant strains (≥3 antibiotics) were highly prevalent in our study, representing 36.7%, 27.9%, and 39.4% of cocci isolates on day 4, 30, and 90, respectively. Multiresistance was detected in all enterococci and streptococci species and its patterns were highly heterogeneous. Most striking multiresistance cases were those of an E. faecalis and an E. faecium strain from day 4, both presenting seven different antimicrobial resistances, of an E. faecalis and an E. gallinarum strain from day 30, with seven and eight different antibiotic resistances, respectively, and of an E. faecalis and two E. faecium isolates from day 90, presenting resistances against eight and nine different antibiotic agents, respectively. Furthermore, an E. faecium isolate from day 90 and one E. faecalis isolate from day 30 exerted resistance to erythromycin, tetracycline, and to high levels of both aminoglycosides (gentamycin, streptomycin).

Molecular detection of resistance genes

Thirty-five fecal samples (13.3% of the samples) were found positive for vancomycin resistance. Only intrinsic vancomycin resistance (vanC1 and vanC2/C3) was reported among tested isolates (7.3% of vancomycin-resistant enterococci [VRE]). None of the 25 vancomycin-resistant E. faecalis strains or the Str. salivarius isolate carried vanA or vanB genes. Only two strains of E. gallinarum and two strains of E. casseliflavus/E. flavescens (carriers of the vanC1 and vanC2/C3 gene, respectively), expressed a vancomycin-resistant phenotype.

The results of the molecular-based detection of the ermB gene are presented in Table 4, pointing out a high prevalence of the ermB gene in the case of E. faecalis. Eleven strains from day 4, 17 from day 30 and 21 from day 90 were characterized as ermB-positive. Presence of the ermB gene was increased among erythromycin-resistant isolates from day 4 to 30 (73.9% vs. 26.8%, p=0.000), as well as to day 90 (65.6% vs. 26.8%, p=0.000).

Table 4.

Molecular-Based Detection of the ermB Gene in Erythromycin-Resistant Isolates

ermB gene	E. faecalis	E. faecium	E. casseliflavus/E. flavescens	E. gallinarum	Streptococcus spp.	Total
ND	8	30	4	1	4	47
Yes	34	10	1	1	3	49
Total	42	40	5	2	7	96

All values are expressed as numbers of isolates.

ND, not detected.

Tet genes were detected in 51 out of 113 tetracycline-resistant strains (7, 18, and 26 strains on days 4, 30, and 90, respectively), with tet(L) determinant being the most frequently detected (Table 5). The tet(K) gene was not detected.

Table 5.

Molecular-Based Detection of tet Genes in Tetracycline-Resistant Isolates

tet genes	E. faecalis	E. faecium	E. casseliflavus/E. flavescens	E. gallinarum	Streptococcus spp.	Total
ND	52	1	4	2	3	62
tet (M)	10	—	—	—	—	10
tet (L)	17	13	2	—	6	38
tet (S)	2	—	—	—	—	2
tet (O)	—	—	—	—	1	1
Total	81	14	6	2	10	113

All values are expressed as numbers of isolates.

The detection of tet genes among tetracycline-resistant isolates was increased from 38.9% (day 4) to 41.9% (day 30) and finally to 50% (day 50), without reaching statistical significance.

Copresence of tet(L) and ermB genes was observed in one E. faecalis isolate on day 4, in six strains on day 30, and in three strains on day 90. In E. faecium isolates, copresence of tet(L) and ermB genes was also observed in one strain on day 4, one on day 30, and in seven strains on day 90. The high level resistant to aminoglycosides E. faecium strain was also a carrier of ermB and tet(L) genetic determinants. Two E. faecalis isolates on day 90 carried simultaneously tet(S) and ermB genes, and two E. faecalis isolates on day 30 carried ermB and tet(M) genes. All the aforementioned strains were multiresistant (three to nine antibiotics). In Streptococcus spp. group, ermB and tet(L) genes were detected separately, only in Str. pasterianus isolates. One Str. pasterianus isolate carried tet(O) and ermB genes simultaneously.

Discussion

This study examines the presence of antibiotic resistance in fecal samples of healthy newborn infants. Little information in available on the frequency of carriage of antibiotic-resistant bacteria in healthy children in the community outside a childcare setting. In our study, fecal specimens were taken from healthy infants just before their departure from the neonatal care unit (4 days after delivery) and, respectively, at 1 and 3 months postpartum. Data from the initial sampling period (day 4) provide information about acquisition of fecal microflora during the first days of an infant's life. At birth, the newborn's gastrointestinal tract is almost sterile, but it is rapidly colonized by facultative anaerobes such as Enterobacteriaceae.¹⁵ Mother, health-care workers, and hospital environment could serve as the microbial reservoirs for this colonization.²⁸ The subsequent samples provide information about the infantile fecal microflora after babies are taken to their home.

The presence of antibiotic-resistant microbes poses a major problem in both hospital and community. An important feature contributing to the dissemination of antibiotic resistance is the ability of the resistant genes to shift into other bacteria by a variety of genetic means. Selective antibiotic pressure is another variable in the epidemiology of antibiotic-resistant pathogens.²⁶

In our study, vancomycin resistance was highly traced among enterococci, with a fecal recovery level of 15.6% using a non-selective enrichment medium. Also, none of the E. faecium isolates was characterized as vancomycin resistant and only intrinsic resistance (vanC-type) was reported. vanC-resistant enterococci have been occasionally found as part of the normal fecal microbiota,^16,41 with no reported incidences of nosocomial outbreaks.²⁵

It has been reported that the oral administration of glycopeptides strongly selects for the intestinal carriage of VRE in healthy Belgians subjects.⁴² Yüce et al.⁴⁴ analyzed rectal swab specimens from 110 neonates hospitalized in a neonatal intensive care unit. In hospitalized patients, 7.3% of isolates were characterized as VRE, while no VRE were detected in healthy neonates.⁴⁴ Furthermore, a 3-year recruitment of 1,820 patients in a neonatal intensive care unit in the United States detected a prevalence of VRE ranging from 2.0% to 4.0%. All but two VRE recovered were characterized as E. faecium; the rest two were characterized as E. gallinarum.³⁷ In another study in Germany, 274 infants were checked for their enterococcal colonization and none of the enterococcoccal isolates was vancomycin resistant.¹⁸

A great number of our isolates (35.7%) were found resistant to erythromycin with over the half of them carrying the ermB gene. It is worth noticing that 30.2% of the resistant strains were characterized as E. faecium and were isolated on the first sampling period (4 days after delivery). In addition, 69.4% of the E. faecalis strains were carriers of ermB genes. The use of macrolides in the animal husbandry as growth promoters could likely reinforce the high ratio of resistant strains.^1,3 Our results are in accordance with other studies based on children or adults.^1,2,33 In Lebanon, the investigation of enterococci isolates from patients' specimens pointed out high rates of erythromycin-resistant enterococci (54.0%).⁴⁷ Clinical isolates of enterococci from Cuba carried the ermB gene at a rate of 72.7%.³⁴ The absence of the ermB gene in some resistant isolates suggests that other yet to be elucidated unknown resistance mechanisms could possibly exist in the community, as it has already been demonstrated in a study from Portugal where 95 fecal samples from healthy humans were tested.³²

In this study, 42.0% of the enterococci strains were resistant to tetracycline, while 45.1% of the positive strains were carrying tet genes. This is consistent with previous studies reporting high levels of tetracycline-resistant enterococci.^1,2,33,38 The previously mentioned study from Lebanon indicated even a higher rate of resistant strains (64.0%), with their majority being characterized as E. faecalis isolates.⁴⁷

Detection levels of tet genes among resistant isolates in our study were lower than those reported in other reports.^1,2,33 Furthermore, tet(K) was not detected in our study. Likewise, Aarestrup et al. did not observe the tet(K) gene among enterococci isolates of human and animal origin.¹ In our study, tet(L) was the most commonly detected tetracycline-resistance gene, followed by the tet(M) gene. This is in contrast with other studies, which indicated tet(M) as the most frequently reported tetracycline resistance gene in enterococci.^1,2,6,33 The tet genes were mainly identified in E. faecalis isolates. The tet(S) gene was found in two E. faecalis strains, which both harbored the ermB gene and one expressed high level resistance to streptomycin. Plasmid pIP811, which is associated with the tet(S) gene, also confers resistance to chloramphenicol, erythromycin, and streptomycin and the tet(S) gene is reported to be exchanged between Enterococcus and Listeria spp. in nature.⁶ The tet(O) gene was found in a Str. pasterianus strain that carried also the ermB gene. In streptococci and enterococci, the tet(O) gene is rarely encountered and is identified in large, structurally related plasmids.⁴⁶ Gueimonde et al. studied the presence of tet genes in the fecal specimens of babies and their mothers.¹⁷ Their study demonstrated that tet genes are normally present in the human gut microbiota of both healthy adults and breast-fed infants. The most widely distributed gene was tet(M), as it was detected in all subjects. The tet(O) gene was present in all the mothers' samples, while only 35.0% of the infants harbored this gene. The tet(S) gene was not detected.¹⁷ In another case study, De Vries et al. used a metagenomic approach to determine the diversity of micro-organisms conferring tetracycline resistance in the gut of a healthy mother-infant pair 1 month after childbirth, and to investigate the potential for horizontal transfer and maternal transmission of tet genes.¹¹ The similarities found between mother and infant resistant clones suggest that the infant's antibiotic resistance genetic determinant arose through a process involving intraspecific genetic exchange.¹¹

A high rate of enterococci was found resistant to nucleic acid synthesis inhibitors (ciprofloxacin, rifampicin, ofloxacin, norfloxacin, and nitrofurantoin). Most of the resistant strains belonged to E. faecalis species. Ciprofloxacin resistance in healthy humans was previously estimated to reach 12.0%–13.0% among fecal enterococci,^3,33 with higher level prevalence being reported in clinical isolates.^45,47 In our study, norfloxacin resistance was higher, while rifampicin resistance was more limited compared to previous data from clinical enterococci.⁴⁷

In accordance with other studies, ampicillin resistance was associated mainly to E. faecium isolates,^2,47 but it was overall estimated in higher levels in our research compared to others.^2,33,47 On the contrary, Kaçmaz and Aksoy detected higher ampicillin resistance levels compared to our results.²³ Butler indicated that the dissemination of ampicillin-resistant E. faecium is dramatic.⁴ In Spain, ampicillin resistance was reported in 17.0% of bloodstream isolates of E. faecium in 1991, 53.0% in 1995, and 75.0% in 2002. Conversely, in E. faecalis species, this type of aminopenicillin resistance remained rather rare merely due to misidentification of E. faecium isolates. Furthermore, resistance levels of gentamycin and streptomycin were lower in our investigation compared to previous studies.^1,2,23,33,47

The incidence of multiresistant cocci in our study was high, in contrast with previously reported lower rates.^1,33 In the recent study of Hufnagel et al., 57.0% of the enterococci isolated from the meconium and the body of infants were multiresistant, without a trace of any vancomycin-resistant isolate.¹⁸ Also, half of the enterococcal clinical isolates from Cuba were resistant to three or more antimicrobial agents, including aminoglycosides, erythromycin, tetracycline, chloramphenicol, and ciprofloxacin.³⁴ Huys et al. mentioned that a significant proportion of tetracycline-resistant isolates exhibited coresistance to erythromycin and/or chloramphenicol.¹⁹ This observation strengthens the hypothesis that the selection of tetracycline-resistant genotypes may provide a suitable molecular basis for the further selection of multiple resistances.

The enterococcal populations of the human intestinal tract are probably influenced by many factors. Aarestrup et al.¹ proposed that ingestion of enterococci of animal origin through food is one of these factors. Similar combinations of resistance traits to antimicrobial agents were observed among isolates of both human and animal origin, and the same resistance genes were detected. These findings indicate that genes encoding resistance can be transferred from human to animal reservoirs and vice versa.¹

In conclusion, our study tried to enlighten the spread of resistant enterococci and streptococci in the infantile gut microbiota. The presence of genes encoding antibiotic resistance in the fecal microbiota since the first days of life enhances the aspect that the human gut may serve as a reservoir of antibiotic resistance traits.³⁶

Footnotes

Acknowledgments

We would like to thank Professor E. Scoulika (Department of Clinical Bacteriology, Faculty of Medicine, University of Crete, Heraklion, Greece) for kindly providing us the VRE reference strains.

Author Disclosure Statement

The authors have nothing to declare.

References

Aarestrup

F.M.

, Agerso

, Gerner-Smidt

, Madsen

, Jensen

L.B.

2000. Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagn. Microbiol. Infect. Dis., 37:127–137.

Barreto

Â.

, Guimarães

, Radhouani

, Araújo

, Gonçalves

, Gaspar

, Rodrigues

, Igrejas

, Poeta

2009. Detection of antibiotic resistant E. coli and Enterococcus spp. in stool of healthy growing children in Portugal. J. Basic Microbiol., 49:503–512.

Bruinsma

, Hutchinson

J.M.

, van den Bogaard

A.E.

, Giammarellou

, Degener

, Stobberingh

E.E.

2003. Influence of population density on antibiotic resistance. J. Antimicrob. Chemother., 51:385–390.

Butler

K.M.

2006. Enterococcal infection in children. Semin. Pediatr. Infect. Dis., 17:128–139.

Cetinkaya

, Falk

, Mayhall

C.G.

2000. Vancomycin-resistant enterococci. Clin. Microbiol. Rev., 13:686–707.

Charpentier

, Gerbaud

, Courvalin

1994. Presence of the Listeria tetracycline resistance gene tet(S) in Enterococcus faecalis. Antimicrob. Agents Chemother., 38:2330–2335.

Chopra

, Roberts

2001. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev., 65:232–260.

Clinical, Laboratory Standards Institute. 2009. Approved Standards M2-A10. Performance Standards for Susceptibility Tests, 10th. CLSI: Wayne, PA.

Clinical and Laboratory Standards Institute. 2011. M100-S21 (M2). Disk Diffusion Supplemental Tables. CLSI: Wayne, PA.

10.

De Bellis

, Valerio

, Sisto

, Lonigro

S.L.

, Lavermicocca

2010. Probiotic table olives: microbial populations adhering on olive surface in fermentation sets inoculated with the probiotic strain Lactobacillus paracasei IMPC2.1 in an industrial plant. Int. J. Food Microbiol., 140:6–13.

11.

De Vries

L.E.

, Vallés

, Agersø

, Vaishampayan

P.A.

, Garcia-Montaner

, Kuehl

J.V.

, Christensen

, Barlow

, Francino

P.M.

2011. The gut as reservoir of antibiotic resistance: microbial diversity of tetracycline resistance in mother and infant. PLoS One, 6:e21644

12.

Di Cello

, Bevivino

, Chiarini

, Fani

, Paffetti

, Tabacchioni

, Dalmastri

1997. Biodiversity of a Burkholderia cepacia population isolated from maize rhizosphere at different plant growth stages. Appl. Environ. Microbiol., 63:4485–4493.

13.

Doherty

, Trzcinski

, Pickerill

, Zawadzki

, Dowson

C.G.

2000. Genetic diversity of the tet(M) gene in tetracycline-resistant clonal lineages of Streptococcus pneumoniae. Antimicrob. Agents Chemother., 44:2979–2984.

14.

Elsayed

, Hamilton

, Boyd

, Mulvey

2001. Improved primer design for multiplex PCR analysis of vancomycin-resistant Enterococcus spp. J. Clin. Microbiol., 39:2367–2368.

15.

Fanaro

, Chierici

, Guerrini

, Vigi

2003. Intestinal microflora in early infancy: composition and development. Acta Paediatr. Suppl 91:48–55.

16.

Gambarotto

, Ploy

M.-C.

, Turlure

, Grèlaud

, Martin

, Bordessoule

, Denis

2000. Prevalence of vancomycin-resistant enterococci in fecal samples from hospitalized patients and nonhospitalized controls in a cattle-rearing area of France. J. Clin. Microbiol., 38:620–624.

17.

Gueimonde

, Salminen

, Isolauri

2006. Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota. FEMS Immunol. Med. Microbiol., 48:21–25.

18.

Hufnagel

, Liese

, Loescher

, Kunze

, Proempeler

, Berner

, Krueger

2007. Enterococcal colonization of infants in a neonatal intensive care unit: associated predictors, risk factors and seasonal patterns. BMC Infect. Dis., 7:107.

19.

Huys

, D'Haene

, Collard

J.M.

, Swings

2004. Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food. Appl. Environ. Microbiol., 70:1555–1562.

20.

Jayaratne

, Rutherford

1999. Detection of clinically relevant genotypes of vancomycin-resistant enterococci in nosocomial surveillance specimens by PCR. J. Clin. Microbiol., 37:2090–2092.

21.

Jensen

L.B.

, Frimodt-Møller

, Aarestrup

F.M.

1999. Presence of erm gene classes in Gram-positive bacteria of animal and human origin in Denmark. FEMS Microbiol. Lett., 170:151–158.

22.

Jones

R.N.

1985. Gram-positive superinfections following beta-lactam chemotherapy: the significance of enterococcus. Infection, 13:S81–S88.

23.

Kaçmaz

, Aksoy

2005. Antimicrobial resistance of enterococci in Turkey. Int. J. Antimicrob. Agents, 25:535–538.

24.

Kariyama

, Mitsuhata

, Chow

J.W.

, Clewell

D.B.

, Kumon

2000. Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. J. Clin. Microbiol., 38:3092–3095.

25.

Klare

, Konstabel

, Badstübner

, Werner

, Witte

2003. Occurrence and spread of antibiotic resistances in Enterococcus faecium. Int. J. Food Microbiol., 88:269–290.

26.

Levy

2002. The 2000 Garrod lecture. Factors impacting on the problem of antibiotic resistance. J. Antimicrob. Chemother., 49:25–30.

27.

Licht

, Wilcks

2006. Conjugative gene transfer in the gastrointestinal environment. Adv. Appl. Microbiol., 58:77–95.

28.

Marques

T.M.

, Wall

, Ross

P.R.

, Fitzgerald

G.F.

, Ryan

A.C.

, Stanton

2010. Programming infant gut microbiota: influence of dietary and environmental factors. Curr. Opin. Biotechnol., 21:149–156.

29.

Mundy

L.M.

, Sahm

D.F.

, Gilmore

2000. Relationships between enterococcal virulence and antimicrobial resistance. Clin. Microbiol. Rev., 13:513–522.

30.

Murray

B.E.

1998. Diversity among multidrug-resistant enterococci. Emerg. Infect. Dis., 4:37–47.

31.

L.-K.

, Martin

, Alfa

, Mulvey

2001. Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell Probes, 15:209–215.

32.

Novais

, Coque

T.M.

, Sousa

J.C.

, Peixe

L.V.

2006. Antimicrobial resistance among faecal enterococci from healthy individuals in Portugal. Clin. Microbiol. Infect., 12:1131–1134.

33.

Poeta

, Costa

, Rodrigues

, Torres

2006. Antimicrobial resistance and the mechanisms implicated in faecal enterococci from healthy humans, poultry and pets in Portugal. Int. J. Antimicrob. Agents, 27:131–137.

34.

Quiñones-Pérez

, Goñi

, Rubio

M.C.

, Baquero

, Gómez-Lus

, Del Campo

2006. Genetic relatedness and antimicrobial resistance determinants among clinical isolates of enterococci from Cuba. Clin. Microbiol. Infect., 12:793–797.

35.

Roy

2001. Media for the isolation and enumeration of bifidobacteria in dairy products. Int. J. Food Microbiol., 69:167–182.

36.

Salyers

, Gupta

, Wang

2004. Human intestinal bacteria as reservoirs for antibiotics resistant genes. Trends Microbiol., 12:412–416.

37.

Singh

, Léger

M.M.

, Campbell

, Short

, Campos

J.M.

2005. Control of vancomycin-resistant enterococci in the neonatal intensive care unit. Infect. Control Hosp. Epidemiol., 26:646–649.

38.

Solheim

, Aakra

, Snipen

L.G.

, Brede

D.A.

, Nes

I.F.

2009. Comparative genomics of Enterococcus faecalis from healthy Norwegian infants. BMC Genomics, 10:194.

39.

Terzaghi

B.E.

, Sandine

W.E.

1975. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol., 29:807–813.

40.

Teuber

, Meile

, Schwarz

1999. Acquired antibiotic resistance in lactic acid bacteria from food. Antonie Van Leeuwenhoek, 76:115–137.

41.

Toye

, Shymanski

, Bobrowska

, Woods

, Ramotar

1997. Clinical and epidemiological significance of enterococci intrinsically resistant to vancomycin (possessing the vanC genotype) J. Clin. Microbiol., 35:3166–3170.

42.

Van der Auwera

, Pensart

, Korten

, Murray

B.E.

, Leclercq

1996. Influence of oral glycopeptides on the fecal flora of human volunteers: selection of higly glycopeptide-resistant enterococci. J. Infect. Dis., 173:1129–1136.

43.

Werner

, Coque

T.M.

, Hammerum

A.M.

, Hope

, Hryniewicz

, Johnson

, Klare

, Kristinsson

K.G.

, Leclercq

, Lester

C.H.

, Lillie

, Novais

, Olsson-Liljequist

, Peixe

L.V.

, Sadowy

, Simonsen

G.S.

, Top

, Vuopio-Varkila

, Willems

R.J.

, Witte

, Woodford

2008. Emergence and spread of vancomycin resistance among enterococci in Europe. Euro Surveill., 13:19046.

44.

Yüce

, Karaman

, Gülay

, Yulug

2001. Vancomycin-resistant enterococci in neonates. Scand. J. Infect. Dis., 33:803–805.

45.

Zhanel

G.G.

, Hoban

D.J.

, Karlowsky

J.A.

2001. Nitrofurantoin is active against vancomycin-resistant enterococci. Antimicrob. Agents Chemother., 45:324–326.

46.

Zilhao

, Papadopoulou

, Courvalin

1988. Occurence of the Campylobacter resistance gene tetO in Enterococcus and Streptococcus spp. Antimicrob. Agents Chemother., 32:1793–1796.

47.

Zouain

M.G.

, Araj

G.F.

2001. Antimicrobial resistance of Enterococci in Lebanon. Int. J. Antimicrob. Agents, 17:209–213.