Survey of molecular determinants in Gram-positive cocci isolated from hospital settings in Argentina

Background: In order to study the resistance mechanisms to aminoglycosides, tetracyclines and erythromycin, we investigated the genetic determinants on 85 Streptococcus spp., Staphylococcus spp., and Enterococcus spp. isolates collected from 46 hospitals of Argentina over a two-year period. Methodology: The MICs to amikacin, gentamicin, kanamycin, and streptomycin, tetracycline and erythromycin were determined by the standard broth dilution method according to CLSI recommendations. Detection of resistance genes to the antibiotic tested was assessed by the PCR standard technique whereas the clonal relationships of each species was performed by PFGE. Results: Major heterogeneity was detected in aminoglycoside and erythromycin resistances. Indeed, 37.6% of the isolates harbored the aac(6’)-aph(2’’) genes; 27% harbored the aph(3’)-IIIa and ant(6)-Ia genes along with the aac(6’)-aph(2’’) gene; 7% carried the ant(4')-Ia gene; and 71% harbored one or more of the erm(A), erm(B), erm(TR), mef(A), mef(E) and msr(A) genes. The tetracycline resistance was determined by the tet(M) gene and was found in 23 isolates that were resistant to this antibiotic. Spreading of tet(M) by the Tn916-like transposon was not a frequent event since the integrase of this element was detected only in 3 Streptococcus spp. isolates. Instead, a 370 bp fragment was detected that corresponded to a region of the CW459-like element integrase in 10 of 11 methicillin-resistant Staphylococcus aureus and in 3 group G Streptococcus isolates, a finding that implies a novel mechanism for tetracycline resistance spreading. Conclusion: This study demonstrates the wide spreading of resistance mechanisms in our nosocomial cocci population and underscores the importance of continuous and efficient epidemiological surveillance.


Introduction
The incidence of infections caused by multidrug-resistant Gram-positive organisms has been increasing over the last 20 years as a major clinical problem.Troublesome pathogens in our country include the following: Staphylococcus aureus isolates resistant to methicillin, macrolides, lincosamides and aminoglycosides; coagulasenegative staphylococci resistant to beta-lactams, aminoglycosides, macrolides, lincosamides and glycopeptides; Streptococcus pyogenes and groups B, C, and G Streptococcus resistant to macrolides; Streptococcus pneumoniae resistant to beta-lactams and macrolides; viridans group streptococci resistant to beta-lactams and aminoglycosides; enterococci resistant to vancomycin, teicoplanin and highly resistant to penicillins and aminoglycosides.The great variability of germs involved requires the performance of a continuous survey of the prevalent microorganisms and their susceptibility.
A number of tet determinants were described that generate antibiotic efflux or ribosomal protection in Gram-positive cocci such as tet(K), tet(L), tet(M), tet(O), tet(T), tet(U), tet(S), tet (W) and tet38 (19).The tet(M) gene is widely spread and is usually found in mobile elements such as the Tn916-Tn1545 family of transposons (16), Tn5397 (18) and the newly described Tn2009 and Tn2010, (6) where the tet(M) gene is cotransferred with macrolide -resistance genes mef(A) and msrD or with erm(B) respectively.Other elements described to carry tet(M) were Tn5801 in Staphylococcus aureus, and the conjugative element CW459 in Clostridium spp.(8,18).According to the authors, the nucleotide sequences of Tn916, Tn5801 and CW459 are related and the encoded integrases of Tn5801 and CW459 are identical; however, they are different from that of Tn916 with an amino acid identity less than 19% (8).
The aim of this study was to establish which genes were involved in the resistance pattern exhibited by 85 Gram-positive cocci isolates of the Staphylococcus, Enterococcus and Streptococcus genera collected from nosocomial patients with invasive diseases over a two-year period.We performed a molecular study that included 46 hospitals from 16 cities of Argentina where we analyzed the possible source of antimicrobial resistance determinants found in these isolates.This study also describes the spreading of tetracycline resistance by transposable elements between staphylococci and group G streptococci.

Bacterial strains
Eighty-five bacterial and nine coagulase-negative staphylococci (CoNS)] and eleven vancomycinresistant Enterococcus faecium (VRE) were investigated.The isolates were epidemiologically unrelated and collected from different hospitals at distant locations in Argentina.The isolates were obtained over a twenty-month period between 1999 and 2000.The sources of infections ranged from pharingitis to bacteremia (Table 2).Culture conditions.Bacteria were grown on Columbia agar plates (Merck, Darmstadt, Germany) supplemented with 5% v/v of sheep blood and incubated for 24 hours at 37°C in the presence or absence of a 5% CO2 atmosphere.

Susceptibility test
The MICs to amikacin, gentamicin, kanamycin, streptomycin, tetracycline and erythromycin (Roemmers) were determined by standard broth dilution method according to CLSI recommendations for each genera studied using Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213 as controls (5).Briefly, the broth dilution method was performed: 2-fold serial dilutions of the antibiotic were made in Mueller Hinton broth which was inoculated with a standardized number of bacteria and incubated for 18 to 20 hours at 37 0 C. The lowest concentration (highest dilution) of antibiotic preventing appearance of turbidity was considered to be the minimal inhibitory concentration (MIC).
The PCR products were sequenced using the ABI3730XL system (Macrogen, Korea).

Transposon detection
The mobile elements Tn4001 (product size: 1,500 bp), Tn5405, the integrases of "Tn916-like" and "CW459-like" elements and the insertion sequences IS256, IS1181, IS1182, IS257 and IS1216 were investigated by PCR.Table 1 lists all primers that were used, as well as annealing temperatures and PCR product sizes.The protocol included a denaturation step of 5 minutes at 95 0 C followed by 30 amplification cycles at different annealing temperatures (Table 1), 1 or 2 minutes at 72 0 C, and final extension of 10 minutes at 72 0 C. Sequence analysis of the amplification products was performed by automated sequencing.

Genotyping
Genotyping was assessed for each genera with SmaI PFGE by a standard protocol using the CHEF DR-III system (Biorad®, Hercules, California).Percent similarity was estimated by the Simple Matching coefficient, and the matrix was clustered by the unweight pair group method (UPMGA).In this study an 80% similarity level was considered, corresponding to differences between 9 to 16 bands.The analysis was performed previously described (9).
Erythromycin resistance was observed in isolate GGS 01 and in 11 GAS with MIC > 4 µg/ml.It was also detected in 11 MRSA, 4 CoNS and in 11 VRE (MIC>8 µg/ml).

Antimicrobial resistance determinants
The aminoglycoside-resistance genes aac(6')aph(2''), aph(3')-IIIa and ant(6)-Ia were found in one GGS isolate (GGS 01) as well as in all MRSA, in seven CoNS and in all VRE isolates.In isolates GCS 02 and GBS 34, the aac(6')-aph(2'') was the only gene detected whereas in six of the nine CoNS the ant(4')-Ia was also detected.The tetracycline-minocycline-resistant isolates (n=23) harbored the tet(M) gene.Among erythromycin resistance determinants, erm(TR) was found in one GGS isolate (GGS 01), in three GAS (GAS 03, 07 and 16) and in one CoNS (CoNS 03).The sequencing of the amplicon showed 100% homology with sequence AF002716 from Streptococcus pyogenes.The erm(A) was detected in ten MRSA; erm(B) was observed in eleven VRE; and erm(M) was found in one CoNS (CoNS 02).We did not observe the presence of erm(C) in our population.The mef(A) gene was present in nine GAS, in one CoNS (CoNS 03) and in one VRE (VRE 10).The mef(E) gene was detected in nine MRSA, and msr(A) was found in two CoNS (CoNS 06 and 07).

Transposon detection
In one GGS (GGS 01), in one GCS (GCS 02), in seven CoNS, and in the eleven MRSA isolates, the combination of both aac primers with the IS256U primer rendered a 1,500 bp amplification product, whereas in ten of the eleven VRE only the combination aac6R/IS256U rendered a product of the same size.In one VRE isolate (VRE 9) no amplification product was detected with any of the two combinations.Sequence analysis revealed that the aac(6')-aph(2'') gene was flanked by two IS256 in those isolates in which both combinations rendered an amplification product; meanwhile, only one IS256 downstream of the aac(6')-aph(2'') gene was present in VRE isolates (01 to 08, 10 and 11).
Tn5405 was detected in all the MRSA isolates.Sequence analysis of the amplification products of 3.6 kb (the region between IS1182 and ant(6)-Ia), 5.7 kb (the region between IS1182 and aph3'-IIIa), 2.2 kb (the region between ant(6)-Ia and aph3'-IIIa) and 2.5 kb (the region between aph3'-IIIa and orfz) revealed that these fragments were identical to portions of the Tn5405 structure in MRSA.In one GGS (GGS 01) and in the eleven VRE isolates, sequence analysis of the 2.2 kb amplification product demonstrated concomitant presence of aph(3')-IIIa, sat4 and ant(6)-Ia.
The integrase of the "Tn916-like" element was found in two GGS (GGS 10 and 11) and in one GCS (isolate GCS 15), whereas the integrase of the "CW459-like" or "Tn5801-like" elements was detected in three GGS (GGS 21, 22 and 24) and in 10 of the 11 MRSA; both integrases were demonstrated by PCR and blast sequence analysis.Insertion sequences IS256 and IS1216 were found in one GGS isolate (GGS 01), one GCS (isolate GCS 02), one GBS (isolate GBS 34) and in the eleven VRE.In Staphylococcus spp., IS256 was detected in seven CoNS and eleven MRSA whereas IS1182 and IS1181 and IS257 were detected only in the isolates.

Isolates clonal relationships
PFGE analysis of the 23 SGG, six of which were multiresistant isolates, showed twelve band patterns.These 23 isolates were grouped in ten clones or "clusters"; similarity was 80% (I, II, III, IV, V, VI, VII, VIII, IX and X).The number of band differences among the clusters was between seven and ten with three or four bands appearing in the same cluster.The ten SGC were grouped in four patterns, two of which were resistant to tetracycline and the clusters were grouped in four clones at the 80% similarity.The difference of bands among the clusters was between five and ten with four bands appearing in the same cluster (data not shown).
The eleven SGA isolates were resistant to erythromycin and we analysed the spreading of such resistance.Three of them harboured the erm(TR) gene (SGA 3, SGA 7 y SGA 16) and nine possessed the mef(A) gene (SGA 4, SGA 5, SGA 6, SGA 8, SGA 9, SGA 12, SGA 16, SGA 17 y SGA 18).The PFGE analysis revealed the presence of seven-band patterns among the resistant isolates, which were grouped in seven clones or clusters (A-G).Six different bands were detected among the clones (data not shown).
PFGE analysis of the CoNS isolates revealed the presence of seven-band patterns that were grouped in four clones according to the phylogenetic evaluation (A, B, C and D).The difference in the number of bands among the clones ranged from nine to sixteen, and within the same cluster between one and five.Within the three isolate clone A cluster, the difference between the isolates was the acquisition of various resistance markers.The isolate CoNS 4 did not harbor any of the markers tested (clone A subtype 1), whereas the other two isolates (subtypes 2 and 3) acquired the mecA, aac(6´)-aph(2´´), ermTR and mef(A) genes (Table 2).The acquisition of mecA and aac(6´)-aph(2´´) differentiated the subtypes 1 from the subtypes 2 in cluster D (isolates CoNS 8 and 9).Among the MRSA isolates, we observed seven PFGE patterns that were grouped in four clones at the 80% similarity.The difference in the number of bands among the clones ranged between eight and twelve bands, and within the same clone between two and five bands.In the MRSA population the difference among the clusters was determined by the presence or absence of macrolides resistance genes mef(E) or ermA (Table 2).The VRE population showed five PFGE patterns that were grouped in three clusters (A, B and C).The difference among the clusters was of four to six bands and within the same cluster between one and three bands.In this group, one isolate belonging to cluster A (VRE 9) differed from the other members by the acquisition of tet(M) gene and one isolate classified by cluster B (VRE 10) harbored the mef(A) gene that was not observed in the other isolates.

Discussion
The high level of aminoglycoside resistance was due to the presence of several aminoglycoside-modifying enzymes such as aac(6')-aph(2''), aph(3')-IIIa and ant( 6)-Ia harbored in the same strain in our bacterial population.The spreading of aac(6')-aph(2'') was due to Tn4001 present in all resistant isolates in three forms: i) the α-form, which has 2 IS256 flanking the aac(6')-aph(2'') gene in opposite directions, as it was observed in one GGS isolate (GGS 01) and in MRSA; ii) the Tn4001-truncated type I-like element, observed in ten of eleven VRE isolates, in which the IS256 on the right-hand flanking region was absent; or iii) the Tn4001truncated type III-like element observed in one VRE (isolate 09), in which both IS256 were missing (2).This isolate (VRE 09) was the only one that harbored tet(M), a finding that may explain the presence of a truncated Tn4001.
The gene cluster aph(3')-IIIa, ant(6)-Ia and sat4 (which encodes the resistance to streptothricin) was detected in staphylococci mostly integrated into the Tn5405 transposable element (7).One hypothesis to explain why these three genes are regularly associated could be the presence of a common promoter (7).The genes aph(3´)-IIIa, ant(6)-Ia and sat4 were amplified separately to confirm their presence in the genome of the isolates.As expected, this gene cluster was found inserted in Tn5405 in all the MRSA isolates investigated in this study, whereas IS1182, orfX, orfY and orfZ (elements that are part of Tn5405) were not found in either multiresistant GGS isolate 01 or any of the Enterococcus faecium isolates investigated.This observation agrees with the results of a report by Werner et al. (23), who described that 29.8% of the Enterococcus faecium isolates investigated had the 5' end of Tn5405 deleted, and the cluster ant( 6)-Ia-sat4-aph(3')-IIIa linked to erm(B).In our VRE population, we observed the presence of erm(B) and the cluster ant( 6)-Ia-sat4-aph(3')-IIIa, suggesting a common structure that harbored them.Probably, in a recombination event, Tn5405 could have lost the IS1182, a fact that may explain the finding of the aph(3')-IIIa, sat4 and ant( 6)-Ia genes together.Six of the eleven VRE isolates and one GGS (isolate 01) were recovered from the same nosocomial setting, suggesting that an interspecies transfer may have occurred among them.
With regard to tetracycline resistance, we found the tet(M) gene in tetracycline and minocycline-resistant isolates.From all known conjugative transposons carrying tet(M), we designed primers to amplify the integrases of Tn916 and CW459 since these are the regions where major differences were found (18).We observed that spreading of tet(M) by the Tn916like transposon was not a frequent event since we only detected the integrase of this element in three Streptococcus spp.isolates.Instead, we amplified a 370 bp fragment that corresponded to a region of the CW459-like element integrase in 10 of 11 MRSA and in 3 GGS isolates, a finding that could suggest a novel mechanism for tetracycline resistance spreading.Ito et al. (8) reported similar findings and described a new element called Tn5801 in MRSA.The Tn5801 integrase is identical to that of the element CW459 from Clostridium spp., which permits the speculation that the same conjugative transposon harboring tet(M) is transmissible between these two genera (8).It can also be hypothesized that the integrase that we detected by PCR might be analogous to the one found in Tn5801 and that this element was spread into streptococci in a yet undisclosed manner.Further studies are required to determine which mobile element is responsible for spreading of this transposon in our cocci population and to establish whether there are more strains that harbor this mobile element.The importance of this knowledge is that this putative mobile element seems to be linked to the primary mechanism of tet(M) spreading in our country and that we have also observed it in group G streptococci.Different authors have reported the presence of the tet(M) gene within a transposable element in S. pneumoniae, (6) streptococci group viridans and Gemella species being the latter reservoirs of resistance genes that can be transferred to S .pneumoniae(3).To our knowledge this is the first study where tet(M) is associated with an intTn gene in group G streptococci which suggests the presence of elements similar to those of the Tn916 family in this species.
From the two erythromycin-resistance mechanisms described in the bacterial population under scrutiny, the one mediated by methylases was the most frequently found among Grampositive cocci (27 isolates), although antibiotic efflux (mef(A)) was the most prevalent mechanism in Streptococcus spp.(GAS) isolates.We observed that those isolates that harbor an erm gene, no matter what type of MLS B phenotype they expressed, also hold a tet gene such as tet(M).This finding suggests that these genes could be harbored by a common transposable element such as Tn2009 (6,19).
The antiseptic resistance was due to qacE delta1 gene in some isolates (Table 2).The reason for searching this particular gene was the hypothesis that as it is widely spread in Gramnegative bacteria, it could have been transmitted to Gram-positive cocci.In fact Kazama et al. (11) found qacEdelta1 in Staphylococcus spp.and Enterococcus spp.(11) suggesting an integron-like structure in Gram positive cocci.
From the presence of multiple resistance in our cocci population, we hypothesize the existence of a more complex genetic structure.We searched IS1216 that flanks the Tn5385 element described in Enterococcus faecalis (17).This element confers resistance to several compounds due to the insertion of different transposons.The IS1216like element was detected in Streptococcus spp.and in VRE whereas we did not observe its presence in Staphylococcus spp.
In brief, we found a marked heterogeneity in the cocci population under scrutiny since isolates in one genus exhibited the same resistance determinants but belonged in different pulsotype clusters.An exception was the VRE and the CoNS populations, which contained few clones with a highly prevalent one.In conclusion, the appearance of isolates of different genera from different nosocomial settings with the same resistance determinants and transposable elements demonstrates significant horizontal spreading of resistance to aminoglycosides, tetracycline and erythromycin in hospitals in Argentina.These results underscore the importance of planning control strategies in order to limit the spreading of mechanisms of multiple resistance which could have a deleterious impact on human therapy.

Table 1 .
Primers used in this study.

Table 2 .
Isolates analyzed in this study.