Macrolide resistance in Streptococcus species.

BACKGROUND: The Streptococci are Gram-positive spherical bacteria (cocci) that characteristically form pairs and chains during growth. Some macrolide-resistant bacteria lack the proper receptor on the ribosome (through methylation of the rRNA). This may be under plasmid or chromosomal control. AIM AND
OBJECTIVES: The aim was to study the prevalence of macrolide resistance among the isolate and evaluate the degree of resistance by minimum inhibitory concentration (MIC) method. And also to detect the phenotypic pattern of macrolide resistance.
MATERIALS AND METHODS: All age group attending general medicine OPD and pediatric OPD with symptoms of respiratory and pyogenic infections are included in the study. Various samples are collected with detailed case history and processed for macrolide resistance among beta hemolytic Streptococci MIC method and D-test.
RESULTS: According to our studies resistance pattern in Group A Streptococci by D-test, cMLS was 27.85%, iMLS was 13.92%, M-type was 55.69%, in GCS, cMLS was 17.6%, M-type was 82.35% In GGS, cMLS was 31.58%, iMLS was 10.53% and M-type was 57.89%.
CONCLUSIONS: Therefore by this study, we would like to highlight the necessity to do antibiotic sensitivity testing for all isolates, and limit the usage of antibiotics, whenever necessary and select the appropriate antibiotics for resistant strains.

Streptococcus pyogenes was first described by Billroth in 1874 in patients with Erysipelas and wound infections. The Streptococci is Gram-positive spherical bacteria (cocci) that characteristically form pairs and chains during growth. They are widely distributed in nature. Some are members of the normal human flora for, e.g. commensal Streptococci of the oral cavity, are common causes of sub-acute bacterial endocarditis, others are associated with important human disease attributable in part to injection by Streptococci, in part to sensitization to them. Streptococci elaborates a variety of extracellular substances and enzymes. These drugs (erythromycin, azithromycin, clarithromycin, roxythromycin, ketolide and telithromycin) bind to the 50 s subunit of the ribosome, and the binding site is a 23S rRNA. They may interfere with formation of initiation complexes for peptide chain synthesis or may interfere with aminoacyl translocation reactions. Some Macrolide-resistant bacteria lack the proper receptor on the ribosome (through methylation of the rRNA). This may be under plasmid or chromosomal control. Streptococcus species is uniformly susceptible to penicillin. However, macrolides remain drug of choice for patient allergic to penicillin. S. pneumoniae is known in medical microbiology as the Pneumococcus, referring to its morphology and its consistent involvement in pneumococcal pneumonia.[1]

Over the last several decades, there has been increased resistance to Macrolides in several countries. European surveillance in Italy identified that 32% of Group A Streptococci (GAS) isolates exhibits resistance to macrolides. France has reported a steady escalation of erythromycin reaching 23% to date. Portugal identified 11% of GAS isolates as resistant to macrolides. Resistance in other European countries during 1990's fell between 1% and 7%.[2] The chemical structure of macrolides is characterized by a large lactone ring containing from 12 to 16 atoms to which are attached, via glycosidic bonds, one or more sugars. The only compound with a 15-membered ring contains a tertiary amino group.[2] The classification of Streptococci is based on (i) colony morphology and hemolytic reactions on Blood agar, (ii) serologic specificity of the cell wall, group-specific substance and other cell wall or capsular antigens (iii) Biochemical reactions and resistance to physical and chemical factors, and (iv) Ecologic features. The epidemiological studies of the mid-1900's helped establish the link between GAS infection and acute rheumatic fever (ARF) and acute glomerulonephritis.[3]

Group A Streptococci or S. pyogenes is a form of beta hemolytic Streptococci responsible for most cases of Streptococcal illness. Other types like (B, C, and G) also cause infections. They cause a wide variety of human infections ranges from mild skin infections or a sore throat to severe, life-threatening condition.[4] M-protein is a stable dimer, is anchored to the cell membrane and traverses and penetrates the cell wall. Streptococci isolated from chronic pharyngeal carriers contain little or no M protein and are also relatively avirulent.[5] Serum–Opacity Factor, T-protein, R-protein, Streptococcal pyogenic exotoxins and emm Typing are the various antigenic properties of the Streptococci to determine.

Another typing scheme that characterizes and measures the genetic diversity among isolates of S. pyogenes is emm typing.[6] This is based on sequence at the 5´ end of a locus (emm) that is present in all isolates. More than 150 emm types have been described. The emm gene encodes the M protein, which forms the basis of a serological typing scheme.

There are four major subfamilies of emm genes, which are defined by sequence difference within 3´ end, encoding the peptidoglycan-spanning domain. The chromosomal arrangement of emm subfamily genes reveals 5 major emm patterns, designated as emm patterns A to E. 14 genetically unrelated clones were obtained from patients showing genetic diversity of S. pyogenes. However 2 clones belonging to M1 and M12 serotypes represented about 70% of isolates in patients.[7]

emm patterns A-C were strongly associated with throat. This is in agreement with reports from temperate regions. In Europe and USA, a strong association was reported between Type emm 75 and erythromycin resistance.[8] GAS serotypes M1, 3, 5, 6, 14, 18, 19, and 24 were associated with rheumatic fever in temperate regions.

Epidemiological surveys from Europe, USA, Caribbean Islands have isolated M serotypes 2, 4, 12, 15, 25, 49, 55, 56, 59, 60 with acute glomerulonephritis.

The pathogenicity of Streptococcus may be localized as tonsillitis or may involve the pharynx more diffusely (pharyngitis). From the throat, Streptococci may spread to the surrounding tissues leading to suppurative complications such as otitis media, mastoiditis, quinsy, Ludwig's angina and a suppurative adenitis. It may rarely lead to meningitis.[9]

The poststreptococcal infections, shows morbidity from ARF is directly proportional to the rate of Streptococcal infections. The mortality rate has declined steadily over the last three decades.[10] Acute glomerulonephritis is a disease characterized by the sudden appearance of edema; hematuria, proteinuria, and hypertension with or without oliguria. It can follow Streptococcal infections. Urea-nitrogen retention, high blood pressure, low levels of serum complement is the symptoms. Majority recover completely whereas some develop chronic glomerulonephritis and ultimate kidney failure.[11]

The other Streptococcus species causes sore throat, its other symptoms include low to high fever, neck swelling, enlarged tonsils and hoarseness and even acute to moderate nausea. More severe symptoms included arthritis, pneumonia and even bacteremia all of which could lead to toxic shock.[12] In recent years, GGS have been reported to cause a variety of human infections, such as, sore throat, pharyngitis, cellulitis, meningitis, infection of the heart valves (endocarditis) and sepsis. Reported mortality rates for patients with GGS bacteremia also vary ranging from 5% to 30%.[13] It is an important cause of endocarditis, septicemia and septic arthritis. The heterogeneity of GCS and GGS may obscure the role of certain subtypes, such as the large-colony-forming strains of group C (Streptococcus dysgalactiae subsp. equisimilis) or group G in endemic pharyngitis.[14] They cause isolated exudative or common source epidemic pharyngitis and cellulitis, indistinguishable clinically from GAS disease.[15] Beta hemolytic GCS and GGs and hemolysin deficient variants cause epidemics of exudative pharyngitis pharyngotonsillitis.

Materials and Methods

All age group attending general medicine OPD and pediatric OPD with symptoms of respiratory and pyogenic infections are included in the study. Exclusion criteria: Patients already on antibiotics. Inclusion criteria: Patients with respiratory tract infections like cold, cough, sore throat, running nose and bacteremia, sepsis, wound infections. Throat swab, sputum, blood, pus, tracheal aspirate, bronchial wash, bronchio-alveolar lavage etc., are collected with detailed case history. Materials required: A sterile throat swab, tongue depressor, sterile sputum container, sterile syringe and needle. Samples are collected by aseptic procedure. E.g., Sterile swab and tongue depressor in case of case of collection of throat swab. Gram stain: The samples were subjected to Gram staining, Gram-positive cocci in short chains and pairs were observed in smear. Culture: They were then plated onto Blood agar plates-to observe hemolysis and Chocolate agar plates to observe bleaching and MacConkey agar plates. Biochemical tests for identification: Catalase, Bacitracin sensitivity, Pyrase test, Optochin sensitivity, Bile solubility rest, Inulin Fermentation, Latex agglutination test and Antibiotic susceptibility of the isolates was done by Kirby-Bauer method of disk diffusion. The direct colony suspension method is used. A direct broth suspension of the colonies from Blood agar plates that has been incubated at 18-24 h was done. Inoculum is adjusted to the standard density.

Hydrochloric acid extraction method

The isolates are grown in Todd Hewitt broth overnight. It is then centrifuged, and the deposit is harvested in a small tube. The deposit is resuspended thoroughly in 0.4 ml of 0.5 mol/l HCL. The tube is then placed in a boiling water bath for 10 min. It is then cooled. One drop of 0.02% phenol red is added. It is then neutralized with 0.5 mol/l and then 0.2 mol/l of NaOH. It is centrifuged again. The clear supernatant is the extract for testing. Group specific antisera are used. A small volume of group A antiserum is added to the antigen extract. In the presence of group A antigen a white precipitate is seen at the interface within 5 min at room temperature. Antisera for groups B, C and G were done in a similar manner.

Double disk diffusion test (D-test)

Testing of Streptococcal isolates with erythromycin and clindamycin disks applied closed together can often yield phenotypic information, although it is not always possible to differentiate between phenotypes using this method. D-test is performed for the detection of inducible clindamycin resistance. The emm genes that encode resistance to the macrolides and lincosamides. The gold standard for diagnosis of inducible resistance is the genotyping.

The Clinical and Laboratory Standard Institute recommends disk diffusion or broth microdilution testing for susceptibility testing of Streptococci.[16] The swab should be rotated several times and pressed firmly on the inside wall of the tube above the fluid level. Use the swab to inoculate the entire surface of a plate of Mueller-Hinton agar with 5% sheep blood. After the plate is dry, use sterile forceps to place a clindamycin (2 μg) disk and an erythromycin (15 μg) disk 12 mm apart for D-zone testing (note-this differs from recommended 15-26 mm for Staphylococci and a disk dispenser cannot be used to place disks on the plate for Streptococci testing.[17] Incubate inoculated agar plate at 35°C in 5% CO2 for 20-24 h.

Isolates with blunting of the inhibition zone around the clindamycin disk adjacent to the erythromycin disk (D-zone positive) should be considered to have inducible clindamycin resistance and are presumed to be resistant.[18]

Interpretation

There are three types the zone were seen in this. They are iMLSB-the clindamycin zone is blunted toward the erythromycin because the erythromycin induces clindamycin resistance. cMLSB – No zone around either erythromycin or clindamycin because erm gene is fully expressed all times. M-type-no change in the clindamycin zone induced by erythromycin because mef does not pump out clindamycin regardless of erythromycin presence [Figures 1–3].

Figure 1

iMLS type of resistance

Figure 3

M type of resistance

cMLS type of resistance

Minimum inhibitory concentration

Minimum inhibitory concentration by agar dilution technique

It is a quantitative method for determining the minimum inhibitory concentration (MIC) of the antibiotic against a given organism. It is mainly useful in testing isolates from serious infections like bacterial endocarditis or to verify equivocal results.

Procedure

Prepare a stock solution containing 2,000 μg/ml of the antibiotic to be tested, e.g. weigh 200 mg of the antibiotic powder and dissolve in 5 ml of distilled water/appropriate solvent.

Preparation of the agar plate with different concentration of the antibiotic

Dispense 2 ml of the diluted antibiotic solution into each of the marked sterile screw capped bottle. It is advisable to start will the highest dilution and hence that single pipette can be used to dispense all the dilutions prepared. Sterile Mueller–Hinton agar is cooled and maintained at 50-55°C in a water bath. This medium (18 ml) is poured into the screw capped bottle containing the different concentration of antibiotic, and adds 2 ml of 5% sheep blood, shaken well and poured into sterile petridish. By this method, exact volume of medium is delivered into the screw capped bottles without the danger of the molten agar jellifying during transfer into dilution of the antibiotic. Poured plates after setting can be kept at 4°C.

Observation and interpretation

Check the control plate for growth. Read the test plate. The concentration at which growth is completely inhibited is considered as the MIC. The organisms are reported Sensitive, Intermediate or Resistant by comparing the test MIC values with that given in the NCCLS table[19] [Figures 4 and 5].

Figure 4

MIC no growth at 128 dl

Figure 5

MIC plate shows growth at 1 dl

E-test for minimum inhibitory concentration determination

E-test consists of a nonporous plastic strip calibrated with MIC values covering 15 twofold dilutions. A predefined antibiotic gradient is immobilized on the surface opposite the MIC scale. When transferred to the agar. The continuous antibiotic gradient established under the strip remains stable an over period covering the critical times of most microorganism subjected to susceptibility testing. This method produces a means for producing MIC data in those situations in which the level of resistance can be clinically important.

Inoculate Todd–Hewitt or BHI broth with test organism and incubate at 37°C for 3-4 h. Adjust the turbidity to 0.5 McFarland's for aerobes and 1 McFarland's for fastidious organisms that die rapidly and have encapsulated strains. Ensure that the agar surface is dry before swabbing it. Dip a swab in the inoculums, remove excess fluid and swab the entire agar surface evenly in three directions. Allow the agar surface to dry for 10-15 min on the bench or in the incubator. Open the E-test package and place the strips in a dry petridish. Apply the strips to the agar surface with a forceps. Always apply the strip with the MIC scale facing the opening of the plate. Do not apply it upside down. Once applied, do not move the strip. Incubate 37°C with 10% CO2 for 24 h.

Observations and interpretation

Read plates after the recommended incubation period only if sufficient growth is seen and the inhibition eclipse is clearly visible. Read the MIC where the ellipse intersects the scale. Always read the end point at complete inhibition of growth including hazes and isolated colonies. Always round up these values o the next two-fold dilution before interpretation. For e.g. If Ampicillin breakpoints are given as S = 1, I = 2, R = 4 μg/ml and the category reported as Intermediate (I) [Figure 6].

Figure 6

Clindamycin

Results

Total sample - 366

Total number of beta-hemolytic colonies collected - 297

Total number of positive by serotyping - 201

GAS - 123

GCS - 34

GGS - 44

Total number of Pneumococci collected - 69.

Positive group A Streptococci

Total number of resistance and intermediate strains=119.

Discussion

The prevalence of pathogenic Streptococci causing various infections, like respiratory tract infections, bacteremia, skin infections, etc., macrolide resistance among the isolated Streptococcus strains were detected and the phenotypic pattern of the macrolide resistance was studied by D-test. All positive cases of Streptococcal and Pneumococcal infection were included in this study. A detailed case history was collected for all the positive cases. Antibiotic resistance pattern of each isolate were detected.

Total samples collected was 366, 270 were positive, out of which 49.63% were sputum, 36.3% were throat swab, 8.89% were pus, 2.59% were blood, 2.22% were tracheal aspirate, 0.37% were bronchial wash. Out of 366 strains collected, beta hemolytic Streptococci were 297(81.15%), which when serotyped with antisera, 123(41.41%) were positive with Group A antisera, 34 (11.45%) were positive with Group C antisera, and 44 (14.82%) with Group G antisera, others were nontypeable. Nontypeable strains were (32.32%) not included in our studies. All alpha hemolytic Streptococci sensitive to Optochin were included as Pneumococci, which was 69 (18.85%) [Figures 7 and 8]. All the strains were stocked for further studies. Antibiotic sensitivity tests were done for all isolates on Mueller Hinton agar with 5% sheep Blood. All erythromycin intermediately sensitive and resistant strains were grouped as resistant strains and stocked for further study. A D-test was done by using erythromycin (15 μg) and clindamycin (2 μg) disc kept at distance of 15 mm apart to detect the phenotypes of macrolide resistance among the isolates of all Beta Hemolytic Streptococcus species [Table 1] [Figure 9]. Epsilon (E-test) test were done for selected resistant strains with erythromycin, azithromycin, clindamycin, quinopristin and dalfopristin. MIC was done for all the erythromycin resistant strains for antibiotics like erythromycin, clindamycin and penicillin (with concentration ranging from 0.25 to 128 μg/ml).[Figure 10] The break points for erythromycin resistant were ≥1 μg/ml, for penicillin ≥4.0 μg/ml and for clindamycin ≥1 μg/ml.[20] For Pneumococcus, antibiotic sensitivity testing is done by disc diffusion method and resistance pattern is identified. Increasing antibiotic resistance in streptococcus [Table 2].[21]

Figure 7

Diagrammatic representation of the positive samples

Figure 8

Resistance pattern in Streptococcus pneumoniae

Table 1

Antibiotic disks for beta-hemolytic Streptococci

Figure 9

Resistance pattern in Streptococcus species (D-test)

Figure 10

Graphical representation of minimum inhibitory concentration resultss

Table 2

For Streptococcus pneumoniae

In D-test done to detect the phenotypic pattern of the erythromycin resistance to detect cMLS,[22] iMLS and M-type, studies done by Melo Crustino 1999, in Portugal shows cMLS as 79.6%, M-type as 16.7%. The previous studies shows the percentage of erythromycin resistance strain showed cMLS as 26.3%, M-type as 73.6%. Our study we included both intermediate and resistance strain for typing, According to our studies resistance pattern in GAS, cMLS was 27.85%, iMLS was 13.92%, M-type was 55.69%. In GCS, cMLS was 17.6%, M-type was 82.35%. In GGS, cMLS was 31.58%, iMLS was 10.53% and M-type was 57.89%. Our results were similar to the finding done by Thangam Menon, which showed higher rates of M-type among macrolide-resistant isolates. However, this was opposite to the finding of Melo crustino who showed high percentage of cMLS. Our study also show a high level of iMLS, among GAS and GGS which was not reported in many of the earlier studies. Thus, according to our studies there is an alarming rise in GAS infection, followed by Pneumococcal infection, which is of great concern. Macrolide resistances among the isolates are also higher compared to many studies. This can be explained by the over usage of macrolide for respiratory infections without proper antibiotic policies [Tables 3 and 4].

Table 3

Distribution of positives in different samples

Table 4

Minimum inhibitory concentration results