Tetracycline and trimethoprim/sulfamethoxazole at clinical laboratory: can they help to characterize Staphylococcus aureus carrying different SCCmec types?

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen, which presents a penicillin-binding protein 2a (PBP2a) encoded by the mecA gene that is located in a mobile genetic element called staphylococcal cassette chromosome (SCCmec). The analysis of SCCmec has revealed eight different allotypes, which are designated as types I-VIII. Worldwide, most hospital-derived isolates belong to types I, II, and III; most community-derived isolates belong to type IV¹. In Brazil, isolates carrying types III and IV have been more frequently found in hospitals^2-4.

Recently, type IV MRSA isolates emerged in hospitals, and this change in hospital epidemiology had impact on the therapeutic choices for infections related to these strains and their control³. Thus, in this study, we analyzed MRSA isolates from inpatients to verify the possibility to use trimethoprim/sulfamethoxazole (TMP/STX) and/or tetracycline at the clinical laboratory to predict the SCCmec type on MRSA isolates.

One hundred and forty isolates of MRSA, obtained from patients in four hospitals in Rio de Janeiro City between 2004 and 2007, were randomly selected. These clinical strains were isolated from different sites: nares (57%), blood (8.5%), prosthesis secretion (7.8%), wound (6.4%), bone (4.3%), and others (16%). Pulsed field gel electrophoresis (PFGE) analysis of genomic DNA that was performed previously revealed many restriction profiles (68) clustered in 18 different genotypes, demonstrating a high genomic diversity among the isolates.

The genomic DNA was extracted with guanidinium thyocianate⁵, and the SCCmec typing was performed by multiplex PCR⁶. Control strains used in the multiplex PCR were Mu50 (SCCmec type II), HU25 (SCCmec type III), and HU39 (SCCmec type IV)⁷.

The susceptibility to 13 antimicrobials (ciprofloxacin, clindamycin, chloramphenicol, erythromycin, gentamicin, linezolid, mupirocin, rifampin, teicoplanin, TMP/STX, tetracycline, tigecycline, and vancomycin) was determined using the disk diffusion method⁸. The minimum inhibitory concentration (MIC) determination for oxacillin, TMP/STX, and tetracycline (Sigma Chemical Company, St Louis, MO, USA) was performed using the broth microdilution method⁹, which utilized Mueller Hinton broth (Difco Laboratories, Detroit, Michigan, USA) that does not contain sulfonamide inhibitors. Bacterial resistance was considered when the MIC values were ≥ 4, ≥ 4/76, and ≥ 16µg/mL for oxacillin, TMP/STX, and tetracycline, respectively¹⁰. ATCC 29213 and ATCC 33591 strains were used as methicillin susceptibility and resistance controls, respectively.

Correlation analyses were carried out using Fischer’s exact test. The p-values < 0.05 were considered statistically significant.

We found three SCCmec types among 140 MRSA isolates (II, n = 12; III, n = 63; and IV, n = 65). When type III isolates were compared with type IV isolates, the susceptibility of the latter was statistically higher for all antibiotics (p < 0.05), except for mupirocin (p = 0.06) and teicoplanin (p = 0.68) ( Table 1 ). All type III and 83% of type IV isolates were susceptible to mupirocin, but half (6/12) of type II isolates were resistant to this drug. Teicoplanin resistance was observed for a few isolates from type II (8%), III (5%), and IV (8%). All type III isolates were resistant to tetracycline and TMP/STX, while all type II and IV isolates were susceptible to both drugs, except for one type IV isolate that was resistant to TMP/STX.

As more than 98% of the isolates from type IV were susceptible to TMP/STX and tetracycline disks as compared to that of type III isolates, all strains were submitted to the broth microdilution test to determine the MIC for these drugs, confirm the results, and verify if they could be used as susceptibility markers. For TMP/STX, the values of MIC ranged from 16/304 to ≥ 32/608µg/mL among type III isolates, having MIC₉₀ ≥ 32/608µg/mL. However, for those isolates that harbored SCCmec IV, the MIC₉₀ was 0.25/4.75µg/mL, similar to that found among type II isolates. Only one type IV isolate showed MIC = 32/608µg/mL for TMP/STX. For tetracycline MRSA, isolates with SCCmec III showed MIC₉₀ = 32µg/mL, and all isolates with SCCmec IV presented MIC between ≤ 0.125 and 0.5µg/mL for this antimicrobial. Type II isolates showed MIC ranging from ≤ 0.125 to 8µg/mL.

The MIC test performed with oxacillin showed values ranging from 2 to > 256µg/mL. Among type III isolates, the MIC₉₀ was ≥ 256µg/mL, whereas type IV isolates showed MIC₉₀ = 32µg/mL for oxacillin. The only strain with MIC = 256µg/mL to oxacillin was also resistant to TMP/STX. The MIC₉₀ of type II isolates was 128µg/mL ( Table 2 ).

To ensure a reliable therapy and to control MRSA strains in the hospital environment, a better characterization of isolates presenting different types of SCCmec became necessary. We evaluated the susceptibility profiles of 140 MRSA clinical isolates to verify the possibility of using them as phenotypic markers. The disk diffusion method found that all type IV isolates were susceptible to tetracycline and 98% were susceptible to TMP/STX. Additionally, the MIC tests confirmed these results. Despite this, all type III isolates were resistant to both drugs. Moreover, type III isolates were more resistant to ciprofloxacin, clindamycin, cloramphenicol, erythromycin, gentamicin, and rifampin than type IV ones (p < 0.05). Similar results have been found in Brazil².

Among all type IV MRSA isolates, only one showed high MIC to TMP/STX (≥ 32µg/mL), but it was susceptible to tetracycline (MIC = 0.5µg/mL). Trindade et al.¹¹ did not find any type IV isolate resistant to TMP/STX and suggested the use of this drug as susceptibility marker for these strains. On the other hand, Amorim et al.¹² proposed the use of TMP/STX as resistance marker among isolates with SCCmec III. A recent study involving strains carrying SCCmec IV from hospitalized patients has shown emergence of multiresistant variants among them⁴. The authors found about 7% isolates resistant to TMP/STX or tetracycline. However, only 2% of the isolates presented resistance to both antibiotics. Moreover, these isolates presented equal profiles of antimicrobial susceptibility, demonstrating that they belonged to the same genotypic profiles.

Some authors have suggested the use of TMP/STX and tetracycline as therapeutic options in CA-MRSA infections^13,14. However, MIC evaluation for these drugs analyzing type IV and III MRSA isolates has not been performed. In this study, we found very low MIC values to TMP/STX and tetracycline, ≤ 0.125/2.375 and ≤ 0.5, respectively. This fact can be related to the success of the therapy with these drugs. Moreover, Sievert et al.¹⁵ analyzed 7 MRSA isolates from wound infections presenting full resistance to vancomycin (VRSA) and found that all isolates were susceptible only to STX and linezolid, indicating that STX could also be an alternative treatment for infections by VRSA.

Susceptibility to tetracycline and TMP/STX was also observed for type II isolates, which showed susceptibility similar to type IV isolates. However, in Brazil, isolates carrying SCCmec II are rare^2-4.

In conclusion, this study showed that detection of specific antimicrobial susceptibility profiles among MRSA isolates, mainly for TMP/STX and/or tetracycline in regions where types III and IV of SCCmec are prevalent, could help to predict them, mainly when there are no technical conditions in identifying the type of mec cassette in MRSA isolates.