Distribution of mef(A) in Gram-Positive Bacteria from Healthy Portuguese Children

Abstract

We screened 615 gram-positive isolates from 150 healthy children for the presence of the erm(A), erm(B), erm(C), erm(F), and mef(A) genes. The mef(A) genes were found in 20 (9%) of the macrolide-resistant isolates, including Enterococcus spp., Staphylococcus spp., and Streptococcus spp. Sixteen of the 19 gram-positive isolates tested carried the other seven open reading frames (ORFs) described in Tn1207.1, a genetic element carrying mef(A) recently described in Streptococcus pneumoniae. The three Staphylococcus spp. did not carry orf1 to orf3. A gram-negative Acinetobacter junii isolate also carried the other seven ORFs described in Tn1207.1. A Staphylococcus aureus isolate, a Streptococcus intermedius isolate, a Streptococcus sp. isolate, and an Enterococcus sp. isolate had their mef(A) genes completely sequenced and showed 100% identity at the DNA and amino acid levels with the mef(A) gene from S. pneumoniae.

The normal flora is thought to act as a reservoir for many bacterial antimicrobial resistance genes, including those that confer macrolide resistance (12). In 1999, there were 20 different rRNA methylases described in the literature, which coded for macrolide-lincosamide-streptogramin B resistance, and 24 efflux and inactivating genes, which coded for one or more of the macrolide-lincosamide-streptogramin B complex of antimicrobials (14). However, relatively few of these 44 genes are found in the majority of macrolide-resistant gram-positive bacteria (1, 2, 13). Resistance to macrolides in the absence of resistance to lincosamides and streptogramin B has been associated with the presence of the mef(A) gene in Streptococcus pneumoniae (17, 18). The mef(A) gene has become more common than erm(B) in macrolide-resistant S. pneumoniae isolates from North America (7, 15). We have shown that the mef(A) gene is present in macrolide-resistant oral Streptococcus spp. and Enterococcus spp. isolated in Seattle, Wash., and Micrococcus luteus and Corynebacterium spp. isolated in the United Kingdom (5), as well as in gram-negative Acinetobacter junii and Neisseria gonorrhoeae (6). All of these species have been able to conjugally transfer the mef(A) genes to a variety of recipients. Recently, two genetic elements, Tn1207.1 (16) and mega (3), have been characterized from macrolide-resistant S. pneumoniae. A highly related gene has been sequenced from Streptococcus pyogenes, while related genes have been identified in Lancefield group C and G streptococci from Finland (4).

In this study, we examined randomly selected gram-positive isolates collected from healthy Portuguese children for the presence of the common macrolide resistance genes, erm(A), erm(B), erm(C), erm(F), and mef(A). Representative mef(A) genes were sequenced, and the presence of the other seven open reading frames (ORFs) from Tn1207.1 was investigated.

(The data in Table 2 were presented in part at the First Annual Symposium on Resistant Gram-Positive Infections in San Antonio, Tex., 3 to 5 Dec. 2000.)

TABLE 2.

Distribution of macrolide resistance genes found in oral and urine isolates

Organism	no. of urine isolates	No. of isolates with gene(s)	No. of oral isolates	No. of isolates with gene(s)
Enterococcus spp.	38
		34 erm(B)
		1 erm(C)
		1 erm(B), erm(C)
		1 erm(B), erm(F)
		1 mef(A)
E. faecalis	2	1 erm(B)
		1 erm(B), mef(A)
S. aureus	11	4 erm(B)	6	1 erm(B)
		4 erm(C)		1 erm(C)
		2 erm(F)		1 erm(B)
		1 erm(C), erm(F), mef(A)		1 erm(B), erm(C)
				1 erm(C), erm(F)
				1 erm(B), erm(C), erm(F)
Staphylococcus spp.	52	1 erm(A)	9
		15 erm(B)
		12 erm(C)		6 erm(C)
		11 erm(F)		3 erm(F)
		7 erm(B), erm(C)
		3 erm(B), erm(F)
		1 erm(B), erm(C), erm(F)
		2 mef(A)
Staphylococcus epidermidis	3	1 erm(A)
		2 erm(B)
S. haemolyticus	2	1 erm(C)
		1 mef(A)
Staphylococcus hominis	1	1 erm(B)
Staphylococcus saprophyticus	2	1 erm(B)
		1 erm(B), erm(C)
S. intermedius			6	1 erm(C)
				3 erm(F)
				1 erm(F), mef(A)
				1 mef(A), erm(B), erm(C), erm(F)
Streptococcus spp.			90	1 erm(A)
				53 erm(B)
				13 erm(C)
				7 erm(F)
				1 erm(B), erm(C)
				2 erm(B), erm(F)
				1 erm(C), erm(F)
				11 mef(A)
				1 mef(A), erm(C)
Total	111		111

MATERIALS AND METHODS

Bacterial isolates.

A total of 615 randomly chosen isolates were included in the study: 392 oral and 223 urine gram-positive isolates collected from 150 healthy children enrolled in a randomized study of amalgam versus composite fillings. The children were 73 girls and 77 boys; 81% were Caucasian, 17% were of African descent, and 2% identified themselves as Asian or Pacific Islander. From the children's records, we found that during the collection period, five or six children per year received some type of medication from doctors. This included both antibiotics and nonantibiotics. The oral isolates were isolated from samples collected from the gingiva and buccal mucosa using the BBL CultureSwab Plus Transport System (Becton Dickinson, Sparks, Md.), while the urine isolates were collected from cultured urine. Individual colonies were identified using standard biochemical protocols (9) and frozen at −70°C until needed. Two Corynebacterium sp. isolates, one Corynebacterium jeikeium isolate, one A. junii isolate, and two S. pneumoniae isolates were tested for the presence of the seven different orf genes from Tn1207.1. These six isolates had previously been shown to carry the mef(A) gene (5-7).

Determination of antibiotic resistance phenotype.

Susceptibilities were determined by disk diffusion assay, following NCCLS protocols, using the control organisms Staphylococcus aureus ATCC 25923 and S. pneumoniae ATCC 49619 (10).

Identification of resistance genes.

For the initial testing, we used bacterial dot blots and radiolabeled internal oligonucleotide probes to screen the isolates for the presence of erm(A), erm(B), erm(C), erm(F), and/or mef(A) genes, as previously described (8). Positive and negative controls were included in each assay. The results were confirmed using PCR assays with hybridization of the PCR products as previously described (1, 2, 8). Representative isolates carrying the mef(A) gene were examined by DNA-DNA hybridization using seven radiolabeled oligonucleotide probes for the presence of the other seven ORFs described in Tn1207.1 (Table 1).

TABLE 1.

Oligonucleotide probes and primers used to identify the mef and orf genes

Oligonucleotide	Sequence (5′-3′)	Genea	Reference
ORF1	TGA TGA AGA GGA AAA TTA G	orf1	This study
ORF2	GCA TCA GGA ACA TCA ATC	orf2	This study
ORF3	GGT ATT GTT CAG GTA GGT C	orf3	This study
MEFF	ATG GAA AAA TAC AAC AAT TGG AAA C	mef(A)	This study
MEFRa	TTA TTT TAA ATC TAA TTT TCT TAA	mef(A)	This study
MF4	ACC GAT TCT ATC AGC AAA G	mef(A)	6
MF4AR	TTC TTT GCT GAT AAA ATC GGT GT	mef(A)	This study
MF6	GGA CCT GCC ATT GGT GTG	mef(A)	This study
MF6R	GCA CAC CAA TGG CAG GTC C	mef(A)	This study
MF7	ATG CAG ACC AAA AGC CAC AT	Upstream from mef(A)	6
ORF5	CAT CTG GGT GAA CTT GCC	orf5	This study
ORF5FRev	GGC AAG TTC ACC CAG ATG	orf5	This study
ORF6	CAT GTT GGA GTA GCG GTA G	orf6	This study
ORF7	GAA ATC TTT GGT CAG ACT TGG	orf7	This study
ORF8	CCC TCC AAT CCA CCA GCG	orf8	This study

^aTaken from Tn1207.1. All the primers except MEFF, MEFR, and MF7 are within the genes listed. MEFF is the first 25 bp of the mef(A) gene; MEFR contains the last 6 bp of the mef(A) gene and 15 bp downstream of the gene.

PCR assays.

The PCR assay for the erm(F) gene was done as previously described (1, 2). The PCR assay for the mef(A) gene used MF4 and MF6, both internal to the ends of the gene as previously described (5, 6). For sequencing the mef(A) gene, PCR with primers MEFF and MEFR, which are at the ends of the mef(A) gene, were used. The assay used 30- to 100-ng genomic DNA as a template, 2 U of Taq polymerase in a thermal cycler from Perkin-Elmer Cetus (Norwalk, Conn.), 200 μM deoxynucleoside triphosphates, 1× PCR buffer (1.5 mM MgCl₂), and 100 ng of each primer. The reactions consisted of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and elongation at 72°C for 2 min for 35 cycles. To finish sequencing the beginning of the gene, we used the MF7 primer, which is 102 bp upstream of the start of the mef(A) gene in Tn1207.1, and we used the Orf5FRev primer, which is 1,240 bp downstream of the mef(A) gene in Tn1207.1 and internal to the orf5 gene, to sequence the end of the mef(A) gene. We used ORF5FRev and MF4AR primers for one PCR assay. The reaction mixture included 30 to 100 ng of genomic DNA as a template, 2 U of Taq polymerase (Perkin-Elmer Cetus), 200 μM deoxynucleoside triphosphates, 1× PCR buffer (1.5 mM MgCl₂), and 100 ng of each primer. The reactions were carried out by denaturing at 96°C for 1 min, annealing at 42°C for 1 min, and elongation at 72°C for 2 min for 35 cycles. The PCR using the MF7 and MF6 primers used 30 to 100 ng of genomic DNA as a template, 2 U of Taq polymerase (Perkin-Elmer Cetus), 200 μM deoxynucleoside triphosphates, 1× PCR buffer (1.5 mM MgCl₂), and 100 ng of each primer. The reaction mixtures were denatured at 96°C for 1 min, annealed at 54°C for 1 min, and elongated at 72°C for 2 min for 35 cycles. The PCR products were dried, resuspended in 1/10 volume of sterile water, and separated on a 1.5% agarose gel with 0.5× TBE running buffer. The bands were visualized by ethidium bromide staining. Positive and negative controls were run with each assay. All primers are listed in Table 1.

Sequencing.

We sequenced the PCR products from selected strains as previously described (2, 8, 11). The complete mef(A) genes were sequenced and compared to the previously sequenced mef(A) genes from S. pneumoniae (U83667) and S. pyogenes (U70055) and to AF227520 from Tn1207.1 and AF376746 from the mega element. The mef(A) sequences AF227520 and U70055 have 100% DNA and amino acid identity with each other and 91% DNA and amino acid identity with U83667 and AF376746. The mef(A) sequences from U83667 and AF376746 have 100% DNA and amino acid identity with each other. The PCR product for erm(F) was sequenced and compared to the GenBank sequence accession no. M1712. The sequences were compared using the Biological Information Resource software at the University of Washington.

Mating procedure.

Matings were done with Enterococcus faecalis JH2-2 as the recipient and mef(A)-positive isolates, including S. aureus 5, Streptococcus intermedius 424, Enterococcus sp. strain 130, and E. faecalis 2, as the donors. Matings were done as previously described (1, 2, 5, 6, 8, 13). We also used the S. aureus 5 and S. intermedius 424 donors with Kingella denitrificans 87.023461 and Neisseria mucosa CTM1.1 as recipients as previously described (6).

RESULTS

Detection of the five macrolide genes.

We found that 375 (61%) of the isolates were susceptible to macrolides and did not hybridize with the five oligonucleotide probes used. This included 278 of the 392 (71%) oral isolates and 97 of the 223 (44%) urine isolates examined. Susceptible isolates from both the oral and urine sites included enterococci, coagulase-negative staphylococci, and S. aureus, while the oral isolates also included Streptococcus spp., S. intermedius, Streptococcus mutans, and Streptococcus salivarius (data not shown). Of the remaining isolates, 223 (36.3%) hybridized with one or more of the five genes used (Table 2).

The mef(A) gene was found in 20 isolates, including Enterococcus sp. (1 isolate), E. faecalis (1 isolate), S. aureus (1 isolate), Staphylococcus haemolyticus (1 isolate), Staphylococcus spp. (2 isolates), S. intermedius (2 isolate), and Streptococcus spp. (12 isolates), either alone (16 isolates) or with erm genes (4 isolates) (Table 2). Five of the isolates were urine isolates, and 15 were from oral samples; the oral Streptococcus spp. were the most prevalent with 11 isolates (Table 2). To verify the presence of the mef(A) genes, all 20 isolates were tested with the mef PCR assay. Each isolate gave PCR products of the appropriate size which hybridized with an internal probe (data not shown).

The mef(A) sequences were determined from the isolates S. aureus 5, S. intermedius 424, Streptococcus sp. strain 6, and Enterococcus sp. strain 130. These sequences had 100% DNA and amino acid identity with the U83667 and AF376746 sequences from GenBank and 91% identity with the AF227520 and U70055 sequences from GenBank (data not shown).

S. aureus 5, S. intermedius 424, Enterococcus sp. strain 130, and E. faecalis 2 were used as donors in mating experiments with the recipient E. faecalis JH2-2. All isolates were able to transfer the mef(A) gene to JH2-2 at frequencies ranging from 2.9 × 10⁻⁵ to 6.2 × 10⁻⁸ per recipient (data not shown). Similar frequencies were found when S. aureus 5 and S. intermedius 424 donors were mated with K. denitrificans 87.023461 or N. mucosa CTM1.1 as recipients.

Of the remaining 213 isolates, 1 enterococcus hybridized with the erm(F) probe. The PCR sequence had 99% base pair identity with the erm(F) GenBank sequence M1712 (data not shown). The erm(A) gene was found in three isolates: two staphylococci and one streptococcus. The erm(B), erm(C), and erm(F) genes were commonly found in the different genera examined, with 90% carrying a single determinant (Table 2). Of the 240 erythromycin-resistant isolates, 18 (7.5%) isolates (15 urine and 3 oral) were macrolide resistant but did not hybridize with the five probes used. Of these, 13 were staphylococci, which have been known to carry other macrolide resistance genes, including erm(Y) and msr(A) (14).

Detection of the seven orf genes from Tn1207.1.

Recently Santagati and colleagues (16) described a genetic element (Tn1207.1) carrying the mef(A) gene and seven other ORFs in an S. pneumoniae isolate that was not able to transfer the mef(A) gene by conjugation. However, more recently, a new genetic element has been described (Tn1207.3) which carried Tn1207.1, and this element has been shown to be conjugative (M. Santagati, F. Iannelli, C. Messina, M. R. Ogginoi, S. Stefani, and G. Pozzi, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2014, 2001). The mef(A) gene is orf4 in the Tn1207.1 element. All six of these strains carried orf1 to orf8 (Table 3). We also tested S. intermedius (two isolates), Streptococcus spp. (seven isolates), E. faecalis (one isolate), Enterococcus sp. (one isolate), and three species of Staphylococcus (Table 3) with the seven other orf probes. The nine streptococcal and two enterococcal isolates hybridized with probes specific for each of the seven orf probes. In contrast, the three staphylococcal isolates did not hybridize with orf1, orf2, or orf3 probes (Table 3) but did hybridize with orf5 to orf7 probes (Table 3).

TABLE 3.

Presence of the ORF regions from Tn1270.1 in representative isolates

Isolate	Strain no.	Presence of genea
		orf1	orf2	orf3	mef(A)d	orf5	orf6	orf7	orf8
Gram positive
Corynebacteria sp.b	274	+	+	+	+	+	+	+	+
Corynebacteria sp.b	214	+	+	+	+	+	+	+	+
C. jeikeiumb	388	+	+	+	+	+	+	+	+
S. intermediuse	424	+	+	+	+	+	+	+	+
S. intermedius	357	+	+	+	+	+	+	+	+
Streptococcus sp.e	6	+	+	+	+	+	+	+	+
Streptococcus sp.	14	+	+	+	+	+	+	+	+
Streptococcus sp.	18	+	+	+	+	+	+	+	+
Streptococcus sp.	281	+	+	+	+	+	+	+	+
Streptococcus sp.	65	+	+	+	+	+	+	+	+
Streptococcus sp.	68	+	+	+	+	+	+	+	+
Streptococcus sp.	215	+	+	+	+	+	+	+	+
S. pneumoniaeb	163	+	+	+	+	+	+	+	+
S. pneumoniaeb	970147	+	+	+	+	+	+	+	+
Enterococcus sp.e	130	+	+	+	+	+	+	+	+
E. faecalis	2	+	+	+	+	+	+	+	+
S. haemolyticus	6	−	−	−	+	+	+	+	+
Staphylococcus saprophyticus	167	−	−	−	+	+	+	+	+
S. aureuse	5	−	−	−	+	+	+	+	+
Gram negative
A. juniic	329	+	+	+	+	+	+	+	+

^a+, present; −, absent.

^bDescribed in reference 5.

^cDescribed in reference 6.

^dmef(A) is orf4 in Tn1270.1.

^emef(A) sequenced.

Nucleotide sequence accession numbers.

The GenBank accession numbers are AY064721 for the S. aureus 5 isolate, AY064722 for the S. intermedius 424 isolate, AY071835 for the Streptococcus sp. strain 6 isolate, and AY071836 for the Enterococcus sp. strain 130 isolate.

DISCUSSION

A collection of 615 normal-flora gram-positive isolates, taken from healthy children, were screened for susceptiblity to macrolides and for the presence of the five most common macrolide resistance genes. Only five or six children in the study received any doctor-approved medication during the collection period. Thus, the number of children who had exposure to antibiotics was low; however, 29% of the oral and 56% of the urine isolates were macrolide resistant. The urine isolates were primarily staphylococci and enterococci, while the oral isolates were predominately streptococci. This could explain why the urine isolates were twice as likely as the oral isolates to be macrolide resistant. In addition, these isolates were commonly multidrug resistant, with enterococci being the most commonly multidrug-resistant genus. Many of the macrolide resistance genes were associated with conjugative elements and thus could act as reservoirs of these genes for pathogenic bacteria.

The mef(A) gene was found in 9% of the macrolide-resistant isolates. This is the first description of the mef(A) gene in the genus Staphylococcus. The complete mef(A) gene was sequenced for the first time from S. aureus, Streptococcus sp., S. intermedius, and Enterococcus sp. isolates. All four of these sequences were identical to each other at the DNA and amino acid levels and identical with the GenBank U83667 and AF376746 sequences. Seventeen of the isolates in Table 3 hybridized with orf1 to orf8 from Tn1207.1, and seven of the isolates have been shown here or previously (5, 6) to transfer the mef(A) gene to related and/or unrelated recipients. Whether these strains carry transposons similar to Tn1207.3, a recently described conjugal element from S. pyogenes (Santagati et al., 41st ICAAC), needs to be investigated. The three Staphylococcus spp. tested hybridized with orf5 to orf8 but not with the orf1, orf2, and orf3 probes, suggesting that these strains may have transposons related to the recently described S. pneumoniae mega element (3). One of the most interesting findings was that the gram-negative A. junii isolate hybridized with all seven orf genes, suggesting that it carries a transposon related to Tn1207.1 from gram-positive isolates. This is the first indication that this family of transposons may be transferred and maintained in some gram-negative species. The presence of the mef(A) gene in other gram-negative bacteria from the Portuguese collection is being determined.