Multicenter Study on Spreading of the tet(M) Gene in Tetracycline-Resistant Streptococcus Group G and C Isolates in Argentina

Abstract

A prospective multicenter study on invasive infections caused by beta-hemolytic streptococci was performed over 6 months and involved 42 centers from 16 cities in Argentina. Among 33 isolates recovered, 9 group G Streptococcus isolates (39.1%) and 2 group C Streptococcus isolates (20%) exhibited resistance to tetracycline and harbored the tet(M) gene. Genealogical analysis revealed that tetracycline resistance has a polyclonal origin in Argentina.

Group G beta-hemolytic streptococci (GGBHS) and group C beta-hemolytic streptococci (GCBHS) form a heterogeneous collection of microorganisms, which can be classified as organisms that form large (diameter, >0.5 mm) or small (diameter, <0.5 mm) colonies. Unlike other beta-hemolytic streptococci, GGBHS and GCBHS can be isolated from either human or animal specimens (4). Infections caused by GCBHS and GGBHS include pharyngitis, skin and soft tissue infections, septicemia, endocarditis, bacteremia, osteomyelitis, and septic arthritis, among others (8).

Although treatment for invasive streptococcal infections involves combination of a β-lactam with an aminoglycoside, tetracyclines have been widely used as the second option. The emergence of tetracycline resistance in several genera of clinical relevance, however, has limited its use. Recently, tetracycline resistance accompanied by high-level resistance to aminoglycosides and other antibiotics has been reported in GGBHS from France (7) and Argentina (P. E. Jeric, B. E. Orman, S. Arduino, M. Dictar, M. T. Verón, P. Vidal, H. Lopardo, C. Lopreto, D. O. Sordelli, and D. Centrón, Abstr. 100th Gen. Meet. Am. Soc. Microbiol. 2000, abstr. C-287, p. 197, 2000). Three tetracycline resistance mechanisms have been described: drug inactivation, efflux by a proton antiporter system, and ribosomal protection (3, 6, 16). Moreover, six kinds of tetracycline resistance determinants that encode efflux or ribosomal protection have been found in Staphylococcus, Enterococcus, and Streptococcus: tet(M), tet(O), tet(K), tet(L), tet(T), and tet(U) (9).

The present study was aimed at the assessment of the incidence of tetracycline resistance among GGBHS and GCBHS in the main medical centers of Argentina. The mechanisms of resistance involved and the degree of clonal diversity were established in order to analyze the potential spread of the predominant genotypes.

Between December 1998 and May 1999, 23 GGBHS and 10 GCBHS were isolated from 42 centers in 16 cities in Argentina. Isolates were grown on brain heart infusion agar supplemented with 5% sheep blood and were incubated for 24 h at 37°C under a CO₂ atmosphere. The MICs of tetracycline, minocycline, and gentamicin were established by broth dilution, according to NCCLS guidelines (10), using Enterococcus faecalis ATCC 29212 as the control strain. Genomic DNAs were extracted as described by Pitcher et al. (11). The presence of the tet(M), tet(O), tet(K), and tet(L) genes was determined by a standard PCR technique with primers described elsewhere (2) or with primers designed for the present study with OLIGO software. The PCR for tet(M) (397 bp) detection was carried out by denaturation over 10 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 46°C, and 1 min at 72°C and a final extension at 72°C for 10 min. For tet(O) (515 bp) detection, primers TETOF (5′-AACTTAGGCATTCTGGCTCAC-3′) and TETOR (5′-TCCCACTGTTCCATATCGTCA-3′) were used and the annealing temperature was changed to 55°C. For tet(K) (172 bp) and tet(L) (993 bp), detection, the protocols consisted of denaturation for 1 min at 94°C, followed by 40 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C, with a final elongation step of 10 min at 72°C. The primers used were TETKU (5′-TCCTGGAACCATGAGTGT-3′) and TETKL (5′-AGATAATCCGCCCATAAC-3′) for detection of tet(K) and TETLU (5′-TGAACGTCTCATTACCTG-3′) and TETLL (5′-ACGAAAGCCCACCTAAAA-3′) for detection of tet(L). PCR was also used to investigate gentamicin resistance mechanisms (18). Clonal characterization of the 33 isolates was assessed by pulsed-field gel electrophoresis (PFGE) with the SmaI enzyme by a standard protocol with the CHEF DR-III system (Bio-Rad, Hercules, Calif.), as described previously (1, 15). Resistant isolates were compared with susceptible ones in order to search for similarities among isolates. Percent similarity was estimated with the simple matching coefficient (14), and the matrix was clustered by the unweighted pair group method (13). In this study, an 80% similarity level was considered, with this level of similarity corresponding to differences in seven bands by PFGE with SmaI, as reported previously (5, 17).

Nine isolates of GGBHS (39.1%) and two isolates of GCBHS (20%) exhibited resistance to tetracycline and minocycline according to their MICs (MICs, 32 to 64 μg/ml) (Table 1). One isolate of GGBHS and one isolate of GCBHS were also resistant to gentamicin (MICs, >1,024 μg/ml) (data not shown), and one isolate of GCBHS showed the macrolide-lincosamide-streptogramin B resistance phenotype. The tet(M) gene was present in all 11 tetracycline-resistant isolates, and the isolates with high-level gentamicin resistance harbored the aac(6′)-aph(2′′) gene (data not shown). PFGE analysis of 23 isolates of GGBHS yielded 12 different fingerprints. Six of them corresponded to the nine tetracycline-resistant isolates (Fig. 1). Cluster analysis of the 23 isolates revealed 10 distinct clones. Intercluster variations in the numbers of bands ranged from 7 to 10, whereas variations in the numbers of bands within a cluster ranged from 3 to 4 (Fig. 2). Analysis of 10 isolates of GCBHS by PFGE revealed four different banding patterns, two of which corresponded to those for tetracycline-resistant isolates (Fig. 1). Cluster analysis revealed four different clusters, with intercluster variations in the number of bands ranging from 5 to 10. The number of band differences among isolates within the same cluster was four (Fig. 3).

TABLE 1.

Isolates of GGBHS and CGBHS analyzed

Tetracycline susceptibility and isolate no.a	City of origin	MIC (μg/ml)		Lancefield group	PFGE typeb
		Tetra- cycline	Mino- cycline
Resistant
01	Buenos Aires	64	32	G	VII
02	La Plata	64	32	C	C
10	Buenos Aires	64	32	G	I
11	Buenos Aires	32	32	G	I
15	Buenos Aires	32	32	C	D
19	Neuquén	48	32	G	II
20	Rosario	64	32	G	II
21	Tandil	32	32	G	IIIa
22	Tandil	32	32	G	IIIa
24	Rosario	48	32	G	IVb
26	Buenos Aires	48	32	G	V
Susceptible
13	Buenos Aires	0.5		G	X
14	Buenos Aires	0.5		G	IVa
23	Buenos Aires	2		G	IIIb
25	Buenos Aires	2		G	VI
27	Buenos Aires	0.5		G	VIII
28	Buenos Aires	0.5		C	A2
29	Buenos Aires	0.5	G	IX
30	Buenos Aires	0.5		C	B
32	Buenos Aires	0.5		C	B
33	Buenos Aires	0.5		C	A1

^aIsolates 01 and 02 were also resistant to gentamicin (MIC, >1,024 μg/ml) and harbored the aac(6′)-aph(2′′) gene. Note that all the tetracycline-resistant isolates harbored the tet(M) gene.

^bDifferent clones were identified with Roman numerals in GGBHS and with capital letters in CGBHS.

FIG. 1.

PFGE patterns after SmaI digestion of genomic DNAs of GGBHS and GCBHS. Lanes 1 to 15, GGBHS; lanes 16 to 21, GCBHS. Lanes 1 to 9, tetracycline-resistant isolates 10, 11, 19, 20, 21, 22, 24, 26, and 01, respectively; lanes 10 to 15, tetracycline-susceptible isolates 23, 13, 14, 27, 29, and 25, respectively; lanes 16 and 17, tetracycline-resistant isolates 02 and 15, respectively; lanes 18 to 21, tetracycline-susceptible isolates 33, 28, 30, and 32, respectively; lane L, bacteriophage lambda ladder. The numbers on the right are in base pairs. The PFGE types are shown in Table 1.

FIG. 2.

Dendrogram showing the 10 different clusters of GGBHS. The roman numerals represent the PFGE types, and the letters represent the subtypes (for more information, refer to Table 1). The arrow indicates the 80% similarity level used as the breakpoint to differentiate the clusters.

FIG. 3.

Dendrogram showing the four different clusters of GCBHS. The letters A, B, C, and D represent the PFGE types. As shown, tetracycline-resistant isolates are indicated with the letters C and D (for more information, see Table 1). The arrow indicates the 80% similarity level used as the breakpoint to differentiate the clusters.

Until the present, GGBHS and GCBHS have had relatively minor effects on human health because of their low prevalences and high degree of susceptibility to antibiotics. The present study shows that during the period under scrutiny, resistance to tetracycline was frequently found, especially among GGBHS. Although six types of tetracycline resistance determinants were described in gram-positive cocci, the tet(M) gene seems to be the most prevalent in our population of streptococci. An explanation for this predominance could be that in most cases tet(M) is carried by conjugative transposons such as Tn916 or by composite structures such as Tn3701. The finding of two isolates that also harbor gentamicin resistance determinants would support the hypothesis of a composite structure.

PFGE analysis revealed that in our GGBHS population, cluster 1 included two isolates, one tetracycline-resistant isolate (isolate 24) and one tetracycline-susceptible isolate (isolate 14), which were identified as clone IV (Fig. 2) and which were recovered from distant locations. Their PFGE patterns differed by only three bands, which suggested that the occurrence of a single genetic event such as the acquisition of a transposon may have been responsible for the new PFGE banding pattern observed. A similar situation was detected with cluster 7 isolates (Table 1, clone III). Conversely, isolates grouped in cluster 5 (clone II) were recovered from cities 590 mi apart (Table 1). The finding of a single clone in these cities could suggest that the spreading of such resistance might have occurred by a similar mechanism. Further studies are needed to establish the means of tet(M) transfer among these isolates.

In conclusion, our study showed that tetracycline resistance in our populations of GGBHS and GCBHS was mediated by the tet(M) gene and that the spreading of such resistance has a polyclonal origin in Argentina. The availability of new derivatives, such as those of the glycylcycline family, may extend the usefulness of tetracyclines in human therapy (12).