Prevalence of inducible clindamycin resistance in methicillin-resistant Staphylococcus aureus : the first study in Jordan

Introduction: A high rate of infections with methicillin-resistant Staphylococcus aureus (MRSA) has been documented, in both hospital(HAMRSA) and community-acquired (CA-MRSA) diseases in Jordan. Erythromycin and clindamycin are considered treatments of choice. However, resistance to erythromycin with false susceptibility to clindamycin in vitro may lead to therapeutic failure. Hence, it is mandatory to study the prevalence of inducible resistance to macrolide-lincosamide-streptogramin B (iMLSB) antibiotics conferred by erm genes in those bacteria. Methodology: S. aureus isolates were identified morphologically and biochemically, and MRSA were appraised using standard procedures. Induction in resistance to MLSB antibiotics among MRSA isolates was detected phenotypically using the D-test, and the presence of erm genes was revealed by polymerase chain reaction (PCR). Results: Of 126 collected Staphylococcus isolates, 71 (56.3%) isolates were S. aureus, of which 55 (77.5%) were MRSA. A total of 43 (78.2%) MRSA-discordant isolates were resistant to erythromycin, of which 33 (76.7%) exhibited the iMLSB (D-test positive), 2 (4.7%) the MSB (Dtest negative), and 8 (18.6%) the constitutive resistant (cMLSB) phenotypes. Induction of clindamycin resistance was 1.6 times greater in CAMRSA than in HA-MRSA. Furthermore, ermA and ermC were significantly prevalent in HA-MRSA and CA-MRSA, respectively. Conclusions: Continuous surveillance of the MLSB resistance is important and required before the prescription of clindamycin to treat MRSA infections.

A false susceptibility to clindamycin in iMLSB MRSA phenotypes may lead to therapeutic failure [5].Therefore, accurate detection of iMLSB-resistant isolates of S. aureus in vivo is a priority concern in therapeutic strategies.Herein, as the first study in Jordan to our best knowledge, this study reports the prevalence of iMLSB, cMLSB, and MS phenotypes with detection of erm genes in clinical and nasal carriage MRSA isolates.

Methodology
A total of 126 non-duplicated Staphylococcus isolates were obtained from different sources of hospitalized adult Jordanian patients (Al-Karak Hospital and Prince Ali Hospital) in Al-Karak Governorate, Jordan.In addition, nasal swabs of carrier individuals were collected from the same area.Each participant signed a written informed consent document, and the study was approved by the ethics and scientific committees of the Faculty of Medicine and Faculty of Graduate studies, Mu'tah University, Jordan.
Different MLSB phenotypes were perceived for erythromycin-resistant MRSA isolates using the double diffusion test (D-test) based on CLSI guidelines [9].Meanwhile, genotypic detection of erm genes was carried out for iMLSB phenotypes.Genomic DNA from iMLSB-MRSA was isolated following a standard protocol [10], adopting the rapid lysis method recommended by Al-Talib et al. [11].DNA amplification was carried out by PCR (XP Thermal cycler, Bioer Technology, Binjiang, China) using specific primer pairs for the ermB [4], ermA, ermC, sau, and methicillin-specific resistance (mecA) genes as described previously [8].Single PCR reactions were employed using 2x master mix (i-MAX II, iNtRON Biotechnology, Gyeonggi-do, Korea), while multiplex PCR reactions were employed using 2x master mix (Master/MultiMAX, iNtRON Biotechnology, Gyeonggi-do, Korea) (Table 1).Genomic DNA from MRSA (S. aureus ATCC 43300) was used as a control.
The amplified PCR fragments were resolved by electrophoresis through a 1.5% agarose gel containing 0.5 µg/mL ethidium bromide and visualized under UV light (gel documentation, Transilluminator UVP, Upland, USA).

Statistical analysis
Data were analyzed using the Chi-squared test (χ 2 ) and Fisher's exact test.P values ≤ 0.05 were considered statistically significant.

Results
A total of 71 isolates from the collected samples were identified as S. aureus (56%), of which 55 (77.5%) isolates were MRSA and 16 (22.5%)were methicillinsusceptible S. aureus (MSSA).Moreover, resistance to Table 1.Polymerase chain reaction (PCR) components and their conditions used in detection of erm and methicillin resistance genes.

PCR reactions components
Conditions of PCR reactions Single PCR: A total volume of 20 µL containing 10 µL of master mix, 100 ng of template DNA, 1.5 μL of each primer (10 pmol/μL, Midland Company/Midland, USA) and nucleic acid free water.Initial denaturation step of 3 min at 95°C, followed by 35 cycles at 95°C for 15 s of denaturation, 15 s of annealing at 54°C, 30 s of elongation at 72°C, and a final extension step of 5 min at 72°C.An annealing temperature of 47°C was applied in case of ermB amplification.Multiplex PCR: A total volume of 20 µL containing 10 µL of master mix, 100 ng of template DNA, 6 μL of primer mixture (10 pmol/μL, Midland Company, Midland, USA), and nucleic acid free water.Primer mixture included 1 μL from each ermC primer, 1.5 μL from each of sau and mecA primers, and 2 μL from each ermA primer.Initial denaturation step of 5 min at 95°C, followed by 35 cycles at 95°C for 30 s of denaturation, 1 min of annealing at 54°C, 1 min of elongation at 72°C, and a final extension step of 5 min at 72°C.methicillin was confirmed by detecting a 532 bp PCR product of the mecA gene.MRSA were highly resistant to erythromycin (78%), kanamycin (80%), and tetracycline (63.6%).Meanwhile, 82% and 100% of MRSA samples were susceptible to clindamycin and to vancomycin, respectively.MRSA prevailed in 67.3% of hospital-acquired (HA) infections and 32.7% of community-acquired (CA) diseases.A high rate of resistance to methicillin was detected in 24.6% and 32.7% of nasal and wound isolates, respectively.Furthermore, all isolates of CA-MRSA were susceptibility to clindamycin versus 70% of HA-MRSA.
Statistically, it was noticed that ermA was significantly detected 2.55 more times in HA-MRSA (p = 0.01) than in CA-MRSA, with insignificant differences in the prevalence of ermB or ermC between the two MRSA groups.However, within the same group, ermC was significantly detected in CA-MRSA   (p = 0.007) (Table 3).Interestingly, ermB was detected at relatively high frequency in both HA-MRSA and CA-MRSA (56.3% and 58.8%, respectively).

Discussion
The emergence of MRSA led to difficulties in treating S. aureus infections, especially in developing countries [12].Therefore, clindamycin has been used to treat soft tissue and pediatric infections and is used in patients allergic to β-lactams due to its pharmacokinetics and low cost when compared to other newer agents used to treat MRSA infections [13].However, harboring erm genes may lead to therapeutic failure due to inducible resistance to clindamycin in those patients.
Coinciding with our results, a high prevalence rate (57%-70%) of MRSA was documented among Jordanian hospitalized adults [14]; the MRSA samples and were recovered mainly from upper respiratory tract and wound swabs [15].Moreover, herein we reported a higher incidence of inducible clindamycin resistance in MRSA (76.7%) with a higher rate of iMLSB phenotypes in CA-MRSA than in HA-MRSA.Conversely, in studies conducted in Europe, Turkey, Japan, and India, the incidence of iMLSB was 24%-39% in MRSA, and inducible phenotypes were detected more frequently in HA-MRSA [16][17][18].This could be attributed to differences in drug usage recommendations in each country and inconsistent use of erythromycin in different infectious cases.
High prevalence of ermA was documented previously in HA-MRSA [19].In addition, presence of ermA on the transposon Tn554 within SCCmec (staphylococcal cassette chromosome mec) I, II, and III, and absence on SCCmec IV explains the low prevalence of this gene in CA-MRSA [20].The occurrence of the ermB gene, originally identified from Streptococcus species isolated from animals [21], in high frequency in this study may reflect the high capacity of this gene to be horizontally transferred from Streptococcus species to S. aureus.

Conclusions
The demonstrated high incidence of iMLSB in clinical practice and community supports limiting the use of erythromycin for prophylaxis and treatment of MRSA.Performing the D-test on isolates conferring erythromycin-resistant and clindamycin-susceptible phenotypes is prudent to exclude inducible clindamycin resistance.

Table 3 .
Frequency and overall prevalence of erm genes detection in erythromycin-resistant iMLSB-MRSA.