Detection of Inducible Resistance to Clindamycin among Methicillin Resistant and Sensitive strains of Staphylococcus aureus from India

The resistance to MLSB antibiotics, i.e. Macrolide-Lincosamide-Streptogramin B (MLSB), is an increasing problem among Methicillin-resistant Staphylococci. The resistance to macrolides can be by efflux mechanism or via inducible or constitutive resistance. Unfortunately, routine clindamycin susceptibility testing fails to detect the inducible resistance, which commonly results in treatment failure and necessitates incorporating a simple D-test to detect such resistance. A retrospective observational study was performed on S. aureus isolates from patients. The strains were subjected to antibiotic susceptibility testing followed by detection of mecA gene by a polymerase chain reaction and, the ‘D-test’ was performed to know the inducible resistance to clindamycin. A total of 235 isolates were identified as S. aureus. Antibiotic susceptibility test indicated 190 MRSA and 45 are sensitive to MLSB (MS). Inducible clindamycin resistance was found among 48 (20.4%) isolates and constitutive resistance in 104 (44.2%). MRSA strains had higher inducible and constitutive resistance than MSSA strains (22.1%, 51.6% and 13.3%, 13.3%, respectively). Clindamycin is a commonly used antibiotic in patients with MRSA infections to spare higher-end anti-MRSA antibiotics like linezolid and vancomycin. To detect inducible clindamycin to avoid treatment failures; the study showed the importance of incorporating the D-test in routine testing.

New update on molecular diversity of clinical Staphylococcus aureus isolates in Iran: Antimicrobial resistance, adhesion and virulence factors, biofilm formation and SCCmec typing

Staphylococcus. aureus is often considered as a potential pathogen and resistant to a wide range of antibiotics. The pathogenicity of this bacterium is due to the presence of multiple virulence factors and ability to form biofilm. SCCmec types I, II and III are mainly attributed to HA-MRSA, while SCCmec types IV and V have usually been reported in CA-MRSA infections. In this study, we performed a cross-sectional study in order to determine the antimicrobial resistance, adhesion and virulence factors, biofilm formation and SCCmec typing of clinical S. aureus isolates in Iran. S. aureus isolate was identified using microbiological standard methods and antibiotic susceptibility test was performed as described by the Clinical and Laboratory Standards Institute (CLSI) guidelines. Inducible resistance phenotype and biofilm formation were determined using D-test and tissue culture plate methods, respectively. Multiplex-PCRs were performed to detect adhesion and virulence factors, antibiotic resistance genes, biofilm formation and SCCmec typing by specific primers. Among 143 clinical samples, 67.8% were identified as MRSA. All isolates were susceptible to vancomycin. The prevalence of cMLSB, iMLSB and MS phenotypes were 61.1%, 22.2% and 14.8%, respectively. The TCP method revealed that 71.3% of isolates were able to form biofilm. The predominant virulence and inducible resistance genes in both MRSA and MSSA isolates were related to sea and ermC respectively. SCCmec type III was the predominant type. Data show the high prevalence rates of virulence elements among S. aureus isolates, especially MRSA strains. This result might be attributed to antibiotic pressure, facilitating clonal selection.

Evaluation of the MGIT 960/EpiCenter TB eXiST system for drug susceptibility testing for Mycobacterium abscessus complex

Background: Drug susceptibility testing (DST) of the Mycobacterium abscessus complex (MABSC) and other rapidly growing nontuberculous mycobacteria by conventional broth microdilution (BMD) is complicated due to inducible resistance to clarithromycin and other technical factors. The goals of this study were to develop a protocol for performing DST of MABSC clinical isolates on the BACTEC MGIT 960/Epicenter TB eXiST and provide an initial assessment of its reliability and utility.Methods: M. abscessus ATCC19977 was used as the reference strain for developing the protocol and as the internal control for DST done by BMD method per CLSI guidelines plus resazurin. Both methods were applied to 31 MABSC clinical isolates submitted to our reference center. Genotyping was performed for the rrl and erm(41) genes known to impact clarithromycin resistance phenotype.Results: The 31 MABSC isolates included 14 M. abscessus subsp. abscessus, 8 M. abscessus subsp. massiliense, and 9 M. abscessus subsp. bolletii. By standard BMD method, a high percentage of the isolates were resistant to cefoxitin (93.5%) and imipenem (100%), and sensitive to amikacin (96.7%). Comparing microplate and MGIT 960 results across those 93 pairs of results (31 isolates x 3 antibiotics), 75 (80.6%) were concordant and the remaining 18 (19.4%) represented minor errors; there were no major or very major errors. Concordance was 100% for amikacin, 84% for imipenem, and 58% for cefoxitin. Clarithromycin DST by microplate and MGIT 960 both identified 14 (45.2%) isolates as susceptible. Microplate identified 3 (9.6%) isolates as resistant after 3 days incubation, with 14 (45.2%) demonstrating inducible resistance from Day 5 to 14. Among those 17 isolates, the MGIT 960 protocol, without modifications, reported 9 (29.0%) as resistant and 8 (25.8%) as intermediate. For all isolates, the observed clarithromycin susceptibility phenotypes were consistent with the genotypes. Conclusion: This study details the development of a DST protocol for MABSC isolates using the MGIT 960/Epicenter TB eXiST system. Based on direct comparison with the standard BMD, the protocol provided highly reliable results, including, without further modification, detection of isolates with inducible-resistance to clarithromycin. These findings support proceeding to the multi-laboratory collaborative study required for validation of the protocol.

Inducible clindamycin resistance and erm genes in Staphylococcus aureus in school children in Kathmandu, Nepal

Aim: Resistance to methicillin and Macrolide–Lincosamide and Streptogramins B and their association with erm genes in Staphylococcus aureus are unknown in Nepal. Materials & methods: Nonduplicate nasal swabs from 160 school children were collected from April to September 2018 and processed using standard microbiological procedures. Results: Out of 160 samples, 64 (40%) were S. aureus in which 17 (26.6%) were methicillin-resistance Staphylococcus aureus (MRSA). D-test identified 15 (23.4%) as inducible clindamycin-resistant, which were more prevalent in MRSA (76.4%) than methicillin-sensitive S. aureus (MSSA; 4.2%). 18.7% of isolates harbored the ermC gene followed by ermA (15.6%) and ermB (3.1%), and were more in MRSA than MSSA. Conclusion: To prevent treatment failure by inducible resistance, D-test must be performed on erythromycin-resistant and/or clindamycin-sensitive isolates.

Constitutive and Inducible Resistance to Thrips Do Not Correlate With Differences in Trichome Density or Enzymatic-Related Defenses in Chrysanthemum

Western flower thrips (WFT), Frankliniella occidentalis, is a serious insect pest of Chrysanthemum [Chrysanthemum × morifolium Ramat. (Asteraceae)]. Here we have investigated whether genotypic variation in constitutive and inducible resistance to WFT correlates with phenotypic differences in leaf trichome density and the activity of the defense-related enzyme polyphenol oxidase (PPO) in chrysanthemum. Non-glandular and glandular leaf trichome densities significantly varied among ninety-five chrysanthemum cultivars. Additional analyses in a subset of these cultivars, differing in leaf trichome density, revealed significant variation in PPO activities and resistance to WFT as well. Constitutive levels of trichome densities and PPO activity, however, did not correlate with chrysanthemum resistance to WFT. Further tests showed that exogenous application of the phytohormone jasmonic acid (JA) increased non-glandular trichome densities, PPO activity and chrysanthemum resistance to WFT, and that these effects were cultivar dependent. In addition, no tradeoff between constitutive and inducible resistance to WFT was observed. JA-mediated induction of WFT resistance, however, did not correlate with changes in leaf trichome densities nor PPO activity levels. Taken together, our results suggest that chrysanthemum can display both high levels of constitutive and inducible resistance to WFT, and that leaf trichome density and PPO activity may not play a relevant role in chrysanthemum defenses against WFT.

Evaluation of the MGIT 960/EpiCenter TB eXiST system for drug susceptibility testing for Mycobacterium abscessus group

BackgroundDrug susceptibility test (DST) of the Mycobacterium abscessus group (MAG) and other rapidly growing nontuberculous mycobacteria by conventional microplate techniques is complicated due to inducible resistance to clarithromycin and other technical factors. This study evaluated the application of the BACTEC MGIT 960/Epicenter TB eXiST for DST of MAG clinical isolates. MethodsM. abscessus ATCC19977 was used as the reference strain for the standardizing the DST by MGIT 960 and as the internal control for testing of 31 clinical isolates tests submitted to a reference laboratory for DST and confirmed as MAG. Clarithromycin genotyping was performed for the loci in the rrl and erm (41) genes known to impact resistance phenotype. ResultsThe 31 MAG isolates included 14 M. abscessus , 8 M. massiliense , and 9 M. bolletii . Using conventional microplate technique according to CLSI guideline, the isolates had a high percentage of resistance for cefoxitin (93.5%) and imipenem (100%), and sensitivity for amikacin (96.7%). Comparing microplate and MGIT 960 results across those 93 pairs of results (31 isolates x 3 antibiotics), 73 (80.6%) were concordant and the remaining 18 (19.4%) represented minor errors; there were no major or very major errors. Concordance was 100% for amikacin, 84% for imipenem and 58% for cefoxitin. Clarithromycin DST by microplate identified 14 isolates as susceptible (all susceptible by MGIT 960), 3 isolates as resistant after 3 days incubation, and 14 isolates demonstrating inducible resistance from Day 5 through 14. Among the latter isolates, MGIT 960 reported 8 as resistant and 6 as intermediate, without modifications to the protocol developed. For all isolates, the observed clarithromycin susceptibility phenotypes were consistent with the genotypes. ConclusionThe present study is the first description of a DST protocol for MAG isolates using the MGIT 960/Epicenter TB eXiST system. The protocol developed provided highly reliable results based on direct comparison with the conventional microplate method, including, without further modification, detection of isolates with inducible-resistance to clarithromycin. Laboratories using MGIT 960 for DST of other mycobacteria may find benefit to incorporating MAG into their routine.

Assessment of inducible clindamycin resistance and Hyper Variable Region (HVR) of mecA gene in clinical staphylococci

Objective: To study the prevalence of inducible clindamycin along with vancomycin and methicillin resistance and assessment of hyper variable region (HVR) of mecA gene among different clinical isolates of Staphylococcus spp. Methods: A total of 176 clinical isolates of Staphylococci were collected from Pakistan Institute of Medical Sciences (PIMS), Islamabad during 2014-2015. The sample sources were pus, blood, urine, sputum, tracheal secretions and tissue fluids. Bacterial identification was done by colony morphology and biochemical tests. Kirby-Bauer disc-diffusion method was carried out to assess the susceptibility against different antibiotics. Minimal inhibitory concentrations (MICs) were done for vancomycin resistance. Double Disk Diffusion test (D-test) was used to detect the clindamycin inducible resistance. PCR was performed to detect erm(C), mecA and HVR genes. Results: Clindamycin inducible resistance among Staphylococcal isolates was found to be 7%, whereas in S. aureus it was 4%, and in coagulase negative Staphylococci (CoNS) it was 11%. The highest resistance was observed against fosfomycin, fusidic acid and cefoxitin. Vancomycin resistance was observed in 23 isolates (13%) of Staphylococci. erm(C), mecA and HVR genes were found in 18%, 50% and 42% respectively. Conclusions: D-test must be performed routinely to avoid clindamycin failure. A high level of resistance against vancomycin in Staphylococcal isolates is a concern for public health. doi: https://doi.org/10.12669/pjms.36.2.665 How to cite this:Khan AA, Farooq J, Abid M, Zahra R. Assessment of inducible clindamycin resistance and Hyper Variable Region (HVR) of mecA gene in clinical staphylococci. Pak J Med Sci. 2020;36(2):---------. doi: https://doi.org/10.12669/pjms.36.2.665 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Antibiograms and Molecular Characterization of Drug Resistance of Mycobacterium abscessus complex from Patients with Multidrug-Resistant Pulmonary Tuberculosis (MDR TB) Infection

ABSTRACTIn the Philippines, acid fast bacilli positive sputum samples commonly treated as TB due to Mycobacterium tuberculosis (MTB) complex. However, Mycobacterium abscessus (MAB) complex is often found in MTB cultures, or in patients confirmed negative for TB through sputum microscopy and culture. Hence, patients with MAB infections are mistakenly prescribed six-month anti-TB treatments. In this study, MAB complex isolates from MDRTB patients were identified and further sub-speciated using the mass 3210 gene. Antimicrobial susceptibility was tested using broth microdilution and resistance genes erm(41), rrs, rrl, gyrA, and gyrB were studied for mutations. Majority were susceptible to amikacin, azithromycin, clarithromycin, and moxifloxacin [MAB: 100%, 100%, 100%, 81.8%, respectively; M. massiliense (MAM): 100%, 100%, 100%, 60%, respectively]. 50% MAM and 63.6% MAB were susceptible to cefoxitin; 60% MAM and 45.5% MAB were susceptible to ciprofloxacin; 72.7% MAB, and 10%MAM were susceptible to doxycycline. Inducible resistance to azithromycin and clarithromycin was found in 27.3%MAB and 30% MAM. 42.9% MAB complex isolates were MDR. Macrolide resistant MAB and MAM had T28 sequevar, showing functional erm(41) responsible for inducible resistance. Unexpectedly, full length erm(41) was found in MAM. Therrl gene in these isolates showed no point mutations, indicating T28 sequevar as cause of inducible resistance. All fluoroquinolone resistant isolates showed Ala-83 in gyrA fluoroquinolone resistant-dependent region (QRDR) and Arg-447 and Asn-464 in gyrB QRDR. These are associated with resistance to the drug.