Mef Gene Research Articles

Comparison study of the measurement of glutamic acid decarboxylase autoantibodies (GADA) and autoantibodies to islet antigen-2 (IA2) by two different methods

A series of novel 11-O-carbamoyl-3-O-descladinosyl clarithromycin derivatives bearing the 1,2,3-triazole group were designed, synthesized, and evaluated for their in vitro antibacterial activity. The antibacterial results indicated that most of the target compounds not only increased their activity against resistant bacterial strains, but also partially retained the activity against sensitive bacterial strains compared with clarithromycin. Among them, 13d had the best antibacterial activity against resistant strains, including Streptococcus pneumoniae B1 expressing the ermB gene (16 µg/mL), Streptococcus pneumoniae AB11 expressing the mefA and ermB genes (16 µg/mL) and Streptococcus pyogenes R1 (16 µg/mL), showing >16, 8 and 16-fold higher activity than that of CAM, respectively. Moreover, 13d and 13g exhibited the best antibacterial activity against sensitive bacterial strains, including Staphylococcus aureus ATCC25923 (4 µg/mL) and Bacillus Subtilis ATCC9372 (1 µg/mL). The MBC results showed that the most promising compounds 13d and 13g exhibited antibacterial activity through bacteriostatic mechanism, while the time-kill kinetic experiment revealed bactericidal kinetics of 13g from microscopic point of view. In vitro antibacterial experiments and molecular docking results further confirmed that it was feasible to our initial design strategy by modifying the C-3 and C-11 positions of clarithromycin to increase the activity against resistant bacteria.

Rapid molecular detection of macrolide resistance

BackgroundEmerging antimicrobial resistance is a significant threat to human health. However, methods for rapidly diagnosing antimicrobial resistance generally require multi-day culture-based assays. Macrolide efflux gene A, mef(A), provides resistance against erythromycin and azithromycin and is known to be laterally transferred among a wide range of bacterial species.MethodsWe use Recombinase Polymerase Assay (RPA) to detect the antimicrobial resistance gene mef(A) from raw lysates without nucleic acid purification. To validate these results we performed broth dilution assays to assess antimicrobial resistance to erythromycin and ampicillin (a negative control).ResultsWe validate the detection of mef(A) in raw lysates of Streptococcus pyogenes, S. pneumoniae, S. salivarius, and Enterococcus faecium bacterial lysates within 7–10 min of assay time. We show that detection of mef(A) accurately predicts real antimicrobial resistance assessed by traditional culture methods, and that the assay is robust to high levels of spiked-in non-specific nucleic acid contaminant. The assay was unaffected by single-nucleotide polymorphisms within divergent mef(A) gene sequences, strengthening its utility as a robust diagnostic tool.ConclusionsThis finding opens the door to implementation of rapid genomic diagnostics in a clinical setting, while providing researchers a rapid, cost-effective tool to track antibiotic resistance in both pathogens and commensal strains.

Azithromycin and erythromycin susceptibility and macrolide resistance genes in Prevotella from patients with periodontal disease.

To study oral Prevotella spp. isolated from patients with chronic periodontitis, to determine their susceptibility to azithromycin and erythromycin and to screen the presence of macrolide resistance genes therein. Isolates with a Prevotella-like morphology were obtained from subgingival samples of 52 patients with chronic periodontitis. Each isolate was identified to the species level by sequencing of the 16S rRNA gene. In 100 Prevotella spp. isolates, azithromycin and erythromycin susceptibility was determined using the E test method, and the screening of erm(A), erm(B), erm(C), erm(F), erm(G), erm(Q) and mef(A) genes was done by PCR. Prevotella intermedia and Prevotella nigrescens were the most identified species (33% each). Minimum inhibitory concentrations (MICs) ranges for both antibiotics were 0.016/0.032 to >256μg/ml. MIC50 values for azithromycin and erythromycin were 1.5 and 1μg/ml, respectively, and MIC90 values were >256μg/ml for both antibiotics. Nineteen per cent of the isolates carried erm(B), and 51% carried erm(F). The MIC values found were high compared to previous studies. erm(F) was greatly prevalent, and we describe for the first time the erm(B) gene in Prevotella spp. The presence of either of the genes seems to be associated with a higher degree of resistance to azithromycin and erythromycin.

Antibacterial Effect of Zinc Oxide Nanoparticles on Group A β-Hemolytic Streptococci with Macrolide Resistance Isolated from University Student Carriers in North of Iran

Background: The increase in the resistance of group A β-hemolytic streptococci (GABHS) to antibiotics is considered as a serious threat to human health. Objectives: The current study aimed at determining the antimicrobial effect of zinc oxide nanoparticles (ZnONPs) on the growth of erythromycin-resistant GABHS harboring mef gene isolated from university student carriers. Methods: The study was conducted on 100 healthy students from Islamic Azad University, Gorgan Branch. Specimens were collected from the nasopharyngeal region using sterile swab. After biochemical tests to identify GABHS, resistance to various antibiotics was determined using agar disk diffusion test (the Kirby-Bauer method). The minimum inhibitory concentration (MIC) of erythromycin was also determined by broth micro-dilution test. The macrolide resistance gene (mefA) was identified by polymerase chain reaction (PCR) using specific primers, and the antibacterial properties of ZnONPs were evaluated by agar-well diffusion method. The data were analyzed by chi-square and ANOVA tests (P < 0.001). Results: In the current study, the frequency of GABHS carriers was reported 11%. The highest amount of bacterial resistance was correlated with erythromycin with the frequency of 45.4%. Also, 91% of the isolated streptococci were susceptible to cefazolin, followed by cefalexin (82%), penicillin (73%), and amoxicillin (46%). Besides, 43% of the erythromycin-resistant isolates had the MIC of ≥ 1 µg/mL. From the five isolates resistant to erythromycin, 60% harbored the mefA gene. The results of agar-well diffusion test showed that 40% of the strains, which were resistant to erythromycin and harbored mefA gene, were inactivated in the concentration of 50 mg/mL of ZnONPs, and the rest were also inactivated in the concentration of 100 mg/mL. Conclusions: The ZnONPs used in the current study had a high antimicrobial activity against all GABHS isolates that were resistant to erythromycin. It was revealed that the bactericidal property increased upon increasing the concentration of the nanoparticles.

Genotypes and Phenotypes of Enterococci Isolated From Broiler Chickens

The objective of this study was to compare the resistance phenotypes to genotypes of enterococci from broiler and to evaluate the persistence and distribution of resistant genotypes in broiler fed bambermycin (BAM), penicillin (PEN), salinomycin (SAL), bacitracin (BAC) or a salinomycin/bacitracin combination (SALBAC) for 35 days. A total of 95 enterococci from cloacal (n=40), cecal (n=38) and litter collected on day 36 (n=17) samples were isolated weekly from day 7 to 36. All isolates were identified by API-20 Strep and their antimicrobial susceptibilities were evaluated using the Sensititre system with the commercially available NARMS’s plates of Gram positive bacteria. Whole genome sequencing (WGS) was used to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics. All isolates were further characterized for hemolysin production (HEM), bile salt hydrolysis (BSH) and gelatinase (GEL) activities. Of the 95 isolates, E. faecium (n = 58) and E. faecalis (n = 24) were the most common Enterococcus species identified. Significant differences in the level of resistance for the E. faecium isolates to ciprofloxacin, macrolide, penicillin and tetracycline were observed among treatments. The bcrR, mefA and aac(6) genes were higher in BAM treatment than the other groups whereas bcrR, ermA, ermB, aphA(3) and tetL were more prevalent in PEN and BAC treatments. Overall, E. faecium isolates showed higher prevalence of antimicrobial resistance, but E. faecalis from litter also exhibited a significant level of resistance. A range of 4 to 15 different virulence genes was detected in E. faecalis. All isolates from litter but one (94.1%) showed BSH activities while 52.9% of them produced GEL. HEM activity was observed only in isolates collected on Day 7 (n= 9) and Day 14 (n= 1). This study confirmed that genetically diverse antimicrobial resistant enterococci harboring virulence factors can be promoted by the use of certain antimicrobials in feed and such enterococci could persist in broiler chickens and their litter, potentially contaminating the soil upon land application. This study underscores the need for ongoing monitoring the AMR enterococci.

1429. Emergence of Multidrug-resistant Serotype 24 Among Children Under 2 Years Old With Invasive Pneumococcal Disease After the Introduction of PCV13 in Argentina

BackgroundIn Argentina PCV13 was included in the National Vaccination Program in January 2012 for children <2 years old Since 1993 S. pneumoniae Surveillance Program (SIREVA II-OPS/WHO) was conducted in the National Reference Laboratory (NRL). Serotype 24 rarely found before 2012, emerged after the introduction of PCV13. We aimed to analyze the trend in serotype 24 distribution and its associated resistance in children <2 years old between 2010 and 2016.MethodsA total of 1,821 Spn isolates (<6 years old) from sterile fluids were received at NRL between January 2010 and December 2016 from 150 hospitals, 24 provinces and Buenos Aires city; 1029 (56.5%) <2 years old. Isolates were serotyped by Quellung, MICs were performed by agar dilution (CLSI) and resistance genes by PCR. Diagnosis: pneumonia (42%), meningitis (28%), sepsis (16%), other (14%). Eighty of 1,029 (7.8%) Spn were serotype 24. Three periods were defined: pre-PCV13 (2010–2011), transitional (2012) and post-PCV13 (2013–2016).ResultsAmong 1,029 Spn isolated in <2 years old, PCV13 serotypes decreased from 86.4% (pre-PCV13) to 33.7% (post-PCV13) related to serotypes: 14, 6A, 6B and 5 (P < 0.05). Non-PCV13 serotypes increased from 13.6% (pre-PCV13) to 66.3%% (post-PCV13), mainly due to serotypes 24, 12F and 23B (P < 0.05). Among 80 serotype 24 Spn, 44 (55%) were <1 years old. Serotype 24 increased from 2.1 to 16.2% (pre-/post-PCV13), and ranks first since 2013. Antimicrobial nonsusceptibility (NS) among serotype 24 in pre-/post-PCV13 periods was: penicillin (PEN) (MIC ≥0.12 µg/mL) 70/91.2%, cefotaxime (MIC ≥ 1 µg/mL) 0/1.5%, erythromycin (ERY) 70/89.7%, tetracycline (TET) 60/83.8% and trimethoprim-sulfamethoxazole (SXT) 60/89.7%. NS to meropenem, chloramphenicol, levofloxacin, rifampicin, ceftaroline, and vancomycin was no detected. 97.1% of ERY-NS isolates carried ermB and 2.9% mefA genes. All the TET-NS isolates carried the tetM gene. Multidrug resistance to PEN, ERY, TET, and SXT increased from 50% in pre-PCV13 to 82% in post-PCV13 period (P < 0.05).ConclusionSerotype 24 represents the main non-PCV13 serotype in <2 years old with IPD after the introduction of PCV13. We observed an increase in prevalence and multidrug resistance in post-PCV13 period. Our results suggest that this emerging serotype could represent a real threat among pneumococcal disease in the near future.Disclosures All authors: No reported disclosures.

Characterization of Streptococcus pluranimalium from a cattle with mastitis by whole genome sequencing and functional validation

BackgroundStreptococcus pluranimalium is a new member of the Streptococcus genus isolated from multiple different animal hosts. It has been identified as a pathogen associated with subclinical mastitis, valvular endocarditis and septicaemia in animals. Moreover, this bacterium has emerged as a new pathogen for human infective endocarditis and brain abscess. However, the patho-biological properties of S. pluranimalium remain virtually unknown. The aim of this study was to determine the complete genome sequence of S. pluranimalium strain TH11417 isolated from a cattle with mastitis, and to characterize its antimicrobial resistance, virulence, and carbon catabolism.ResultsThe genome of S. pluranimalium TH11417, determined by single-molecule real-time (SMRT) sequencing, consists of 2,065,522 base pair (bp) with a G + C content of 38.65%, 2,007 predicted coding sequence (CDS), 58 transfer RNA (tRNA) genes and five ribosome RNA (rRNA) operons. It contains a novel ISSpl1 element (a memeber of the IS3 family) and a Ф11417.1 prophage that carries the mef(A), msr(D) and lnu(C) genes. Consistently, our antimicrobial susceptibility test confirmed that S. pluranimalium TH11417 was resistant to erythromycin and lincomycin. However, this strain did not show virulence in murine pneumonia (intranasal inoculation, 107 colony forming unit – CFU) and sepsis (intraperitoneal inoculation, 107 CFU) models. Additionally, this strain is able to grow with glucose, lactose or galactose as the sole carbon source, and possesses a lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).ConclusionsWe reported the first whole genome sequence of S. pluranimalium isolated from a cattle with mastitis. It harbors a prophage carrying the mef(A), msr(D) and lnu(C) genes, and is avirulent in the murine infection model.

Lack of correlation between reduced outpatient consumption of macrolides and macrolide resistance of invasive Streptococcus pneumoniae isolates in Slovenia during 1997-2017.

The objective of this study was to investigate the correlation between decreased national consumption of macrolides and resistance of invasive Streptococcus pneumoniae isolates in Slovenia during 1997-2017. A total of 4241 invasive S. pneumoniae isolates were collected in Slovenia from 1997 to 2017. The presence of erm(B), mef(E), mef(A) and erm(TR) genes was determined by PCR in 612 erythromycin-resistant isolates. Selected isolates carrying the mef(A) gene were further examined by pulsed-field gel electrophoresis (PFGE). Multilocus sequence typing (MLST) was performed for 161 erythromycin-resistant isolates from 2004 to 2009. Consumption of macrolides decreased by 42.5% between 1997 and 2017, and by 57.0% from the highest consumption during 1999 to 2017. Resistance of S. pneumoniae increased by 120.7% in the same period, from 5.8% in 1997 to 12.8% in 2017. The most prevalent serotypes among macrolide-resistant isolates were 14 (54.9%), 19A (9.0%), 19F (8.3%), 6B (7.2%), 6A (5.2%) and 9V (19; 3.0%). The most prevalent determinant of macrolide resistance in the observed period was erm(B) (43.0%; 263/612), followed by mef(A) (36.3%; 222/612) and mef(E) (14.9%; 91/612). During the study period, an increasing trend in serotype 14, mef(A)-carrying isolates was observed, with a peak in 2011 (P<0.001); 63/71 isolates (88.7%) with the mef(A) gene were clonally related and were related to the international England14-9 clonal cluster. The reason for the observed increase in macrolide resistance among invasive S. pneumoniae in Slovenia despite decreased macrolide consumption was spread of the England14-9 clonal cluster.

Whole-Genome Sequencing of Russian Neisseria Gonorrhoeae Isolates Related to ST 1407 Genogroup

The whole-genome sequencing data of three N. gonorrhoeae strains isolated in the Russian Federation in 2015 are presented. According to the NG-MAST protocol, these strains are related to the globally spread ST 1407 genogroup. The analysis of their resistomes showed the absence of ermA/B/C/F genes and the presence of wild-type alleles of rpsE, rrs, rrl, rplD, rplV, macAB, and mefA genes, and these patterns explain the susceptibility of the sequenced strains to aminocyclitols (spectinomycin) and macrolides (azithromycin). Conjugative resistance determinants (blaTEM, tetM) were absent in the genomes, and the penC/ pilQ, parE, and norM alleles were shown to be wild-type, whereas single or multiple nucleotide substitutions were identified in the genes encoding targets for -lactams (ponA, penA), tetracyclines (rpsJ), and fluoroquinolones (gyrA, parC). The additional mutations were found in porB gene and the promoter of mtrR gene, which nonspecifically reduced the susceptibility to antimicrobials due to the membrane permeability decrease and efflux pump overexpression. The diversity of mutations observed in the analyzed genomes prompted a revision of the phylogenetic relationships between the strains by comparing more than 790 groups of housekeeping genes. A high homology between the N. gonorrhoeae ST 1407 and N. gonorrhoeae ST 12556 genomes was confirmed; the latter had probably diverged from a common ancestor as a result of single mutation events. On the other hand, N. gonorrhoeae ST 12450 was an example of phenotypic convergence which appeared in the emergence of new drug resistance determinants that partially coincide with those of the ST 1407 genogroup.

Synthesis and antibacterial activity of novel 3-O-arylalkylcarbamoyl-3-O-descladinosyl-9-O-(2-chlorobenzyl)oxime clarithromycin derivatives

A novel series of 3-O-arylalkylcarbamoyl-3-O-descladinosyl-9-O-(2-chlorobenzyl)oxime clarithromycin derivatives, were designed, synthesized and evaluated for their in vitro antibacterial activity. These derivatives were found to have strong activity against susceptible and resistant bacteria strains. Among them, compounds 7a and 7q showed the most potent activity (0.125 µg/mL) against erythromycin-resistant S. pneumoniae expressing the mefA gene. Moreover, compounds 7f, 7i, 7p and 7z displayed remarkably improved activity (4 µg/mL) against penicillin-resistant S. aureus ATCC31007, and compounds 7a, 7b, 7f, 7p and 7z showed improved activity (8 µg/mL) against erythromycin-resistant S. pyogenes. In particular, compound 7z exhibited potent and balanced activity against the tested drug-susceptible and -resistant bacterial strains.

Полногеномное секвенирование российских штаммов Neisseria Gonorrhoeae, отнесенных к геногруппе ST 1407

The whole-genome sequencing data of three N. gonorrhoeae strains isolated in the Russian Federation in 2015 are presented. According to the NG-MAST protocol, these strains are related to the globally spread ST 1407 genogroup. The analysis of their resistomes showed the absence of ermA/B/C/F genes and the presence of wild-type alleles of rpsE, rrs, rrl, rplD, rplV, macAB, and mefA genes, and these patterns explain the susceptibility of the sequenced strains to aminocyclitols (spectinomycin) and macrolides (azithromycin). Conjugative resistance determinants (blaTEM, tetM) were absent in the genomes, and the penC/ pilQ, parE, and norM alleles were shown to be wild-type, whereas single or multiple nucleotide substitutions were identified in the genes encoding targets for β-lactams (ponA, penA), tetracyclines (rpsJ), and fluoroquinolones (gyrA, parC). The additional mutations were found in porB gene and the promoter of mtrR gene, which nonspecifically reduced the susceptibility to antimicrobials due to the membrane permeability decrease and efflux pump overexpression. The diversity of mutations observed in the analyzed genomes prompted a revision of the phylogenetic relationships between the strains by comparing more than 790 groups of housekeeping genes. A high homology between the N. gonorrhoeae ST 1407 and N. gonorrhoeae ST 12556 genomes was confirmed; the latter had probably diverged from a common ancestor as a result of single mutation events. On the other hand, N. gonorrhoeae ST 12450 was an example of phenotypic convergence which appeared in the emergence of new drug resistance determinants that partially coincide with those of the ST 1407 genogroup.

Evaluation of enterococci for potential probiotic utilization in dogs.

Some strains of the genus Enterococcus are effective probiotic bacteria if they meet safety and probiotic criteria. In our study, 17 canine enterococci previously selected from a group of 160 isolates based on safety criteria were screened for some functional properties relevant to their use as probiotics. The results of antimicrobial resistance testing showed sensitivity of eleven strains to EFSA recommended antimicrobials. In contrast, the most frequent resistance was observed for cefotaxim (15/17) and oxacillin (13/17). PCR detection of resistance genes (vanA, vanB, vanC, tetM, tetL, ermB, and mefA) revealed the presence of mefA gene in five Enterococcus faecium strains and vanA gene in one strain. The production of enzymes commonly associated with intestinal diseases was in general rare (β-glucosidase 2/17, α-chymotrypsin 1/17, N-acetyl-β-glucosaminidase 0/17, and β-glucuronidase 0/17). The measurement of strain survival rate (%) under the conditions simulating gastric (pH2.5) and bile juices (0.3% bile) showed considerable differences between strains (< 0.01 to 4.7% after 90min for gastric juices, 48.0 to 254.0% after 180min for bile). The concentration of produced L-lactic acid ranged between 83.1 to 119.3mmol/L after 48h cultivation depending on the strain. All strains fermented 16 out of 49 different carbohydrates (range from 17 to 23/49). Antimicrobial activity was recorded for two strains against some species of Listeria sp. and Enterococcus sp. Finally, two E. faecium candidates (IK25 and D7) were selected for testing in dogs, and hereafter they could possibly extend the currently limited range of beneficial bacteria of canine origin used as a dietary supplement for dogs.

Type M Resistance to Macrolides Is Due to a Two-Gene Efflux Transport System of the ATP-Binding Cassette (ABC) Superfamily.

The mef(A) gene was originally identified as the resistance determinant responsible for type M resistance to macrolides, a phenotype frequently found in clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes. MefA was defined as a secondary transporter of the major facilitator superfamily driven by proton-motive force. However, when characterizing the mef(A)-carrying elements Tn1207.1 and Φ1207.3, another macrolide resistance gene, msr(D), was found adjacent to mef(A). To define the respective contribution of mef(A) and msr(D) to macrolide resistance, three isogenic deletion mutants were constructed by transformation of a S. pneumoniae strain carrying Φ1207.3: (i) Δmef(A)–Δmsr(D); (ii) Δmef(A)–msr(D); and (iii) mef(A)–Δmsr(D). Susceptibility testing of mutants clearly showed that msr(D) is required for macrolide resistance, while deletion of mef(A) produced only a twofold reduction in the minimal inhibitory concentration (MIC) for erythromycin. The contribution of msr(D) to macrolide resistance was also studied in S. pyogenes, which is the original host of Φ1207.3. Two isogenic strains of S. pyogenes were constructed: (i) FR156, carrying Φ1207.3, and (ii) FR155, carrying Φ1207.3/Δmsr(D). FR155 was susceptible to erythromycin, whereas FR156 was resistant, with an MIC value of 8 μg/ml. Complementation experiments showed that reintroduction of the msr(D) gene could restore macrolide resistance in Δmsr(D) mutants. Radiolabeled erythromycin was retained by strains lacking msr(D), while msr(D)-carrying strains showed erythromycin efflux. Deletion of mef(A) did not affect erythromycin efflux. This data suggest that type M resistance to macrolides in streptococci is due to an efflux transport system of the ATP-binding cassette (ABC) superfamily, in which mef(A) encodes the transmembrane channel, and msr(D) the two ATP-binding domains.

Characteristics of Streptococcus pneumoniae serotype 19A isolates from children in the pre and post Conjugate Vaccine Era. Single center experience 1986–2015

BackgroundThe present study assessed the prevalence and characteristics of S. pneumoniae serotype 19A isolates from children with pneumococcal disease (PD), before and since introduction of pneumococcal conjugate vaccines (PCVs) in Greece. MethodsS. pneumoniae isolates collected at one large pediatric hospital between 1986 and 2015 were serotyped by the Quellung reaction and MICs determined by Etest. Alterations of pbp genes and the presence of mefA, mefE, ermB genes were detected by polymerase chain reaction. Genotypes were assessed by multilocus sequence typing (MLST). ResultsAmong 1875 isolates, 210 (11.2%) belonged to serotype 19A. The prevalence of PD caused by serotype 19A increased from 4.6% in the pre-PCV7 years (1986–2005) to 19.6% in the post-PCV7 years (2006–2010), peaking at 27% in 2009 (p < 0.001, 95% CI; 2.0, 18.2) with a significant upward trend (p = 0.04, 95% CI; 1.02, 12.66). Following the introduction of PCV13 in 2010, the rate decreased from 22% in 2011 to 11.4% in 2015 (p = 0.08, 95% CI; 0.92, 5.1) with a downward trend of borderline significance (p = 0.05, 95% CI; −6.8, 0.04). The multidrug resistant (MDR) serotype 19A isolates increased from 10.6% in 1986–2005 to 21.2% in 2006–2010 and to 71.8% in 2011–2015 (P < 0.001). Alterations in pbp genes were detected in all penicillin non-susceptible isolates. Of 110 erythromycin resistant isolates, 21 contained the mefE gene, 36 the ermB and 53 both the mefE and ermB genes. MLST analysis of 142 isolates revealed four dominant clonal complexes (CC); CC320, CC172, CC276 and CC199. The majority of CC320 and CC276 isolates displayed MDR phenotypes. ConclusionPD caused by serotype 19A increased significantly after the introduction of PCV7 followed by a decline after PCV13 use. The vast majority of persisting 19A isolates was MDR. Surveillance studies are necessary to monitor the changes in the pneumococcal population.

Azithromycin Resistance in Clinical Isolates of Salmonella enterica Serovars Typhi and Paratyphi in Bangladesh.

Salmonella enterica serovars Typhi and Paratyphi, the causative agents of typhoid and paratyphoid, are major threats in developing countries. The present study aimed to investigate the resistance pattern of 40 clinically isolated Salmonella enterica serovars Typhi (n = 33) and Paratyphi (n = 7) to commonly used antibiotics, particularly azithromycin. The disc diffusion method was used to investigate the resistance pattern of the clinical isolates against selected antibiotics. Minimum inhibitory concentration (MIC) was determined by the broth dilution method. Plate-based assays were used for the detection of efflux pumps. It was observed that 95% of the test isolates were resistant to azithromycin and 100% were resistant to clindamycin. MIC values of azithromycin ranged between 32 and 128 μg mL-1. Although 90% of isolates contained efflux pump, none of the isolates was found to have the mef(A) gene, indicating that some other efflux pump(s) might be present. Macrolide resistance gene, erm(B), was present in 25 isolates (62.5%). Other resistance genes were absent. Plasmids were absent, but class 1 integrons were present in 80% of the isolates. The occurrence of macrolide resistance in clinical Salmonella enterica serovars Typhi and Paratyphi is of particular significance in Bangladesh where azithromycin is a commonly used drug against most diseases.

Synthesis and antibacterial evaluation of novel 11-O-aralkylcarbamoyl-3-O-descladinosylclarithromycin derivatives

A series of novel 11-O-aralkylcarbamoyl-3-O-descladinosylclarithromycin derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity. The results showed that the majority of the target compounds displayed potent activity against erythromycin-susceptible S. pyogenes, erythromycin-resistant S. pneumoniae A22072 expressing the mef gene and S. pneumoniae AB11 expressing the mef and erm genes. Besides, most of the target compounds exhibited moderate activity against erythromycin-susceptible S. aureus ATCC25923 and B. subtilis ATCC9372. In particular, compounds 11a, 11b, 11c, 11e, 11f and 11h were found to exert favorable antibacterial activity against erythromycin-susceptible S. pyogenes with the MIC values of 0.015–0.125 μg/mL. Furthermore, compounds 10e, 11a, 11b and 11c showed superior activity against erythromycin-resistant S. pneumoniae A22072 with the MIC values of 0.25–0.5 μg/mL. Additionally, compound 11c was the most effective against all the erythromycin-resistant S. pneumoniae strains (A22072, B1 and AB11), exhibiting 8-, 8- and 32-fold more potent activity than clarithromycin, respectively.

Extremely Low Prevalence of Erythromycin-Resistant Streptococcus pyogenes Isolates and Their Molecular Characteristics by M Protein Gene and Multilocus Sequence Typing Methods

Background: Group A streptococci (GAS) are notorious bacteria causing a wide variety of clinical manifestations ranging from mild, acute streptococcal pharyngitis to chronic non-suppurative diseases and immunological sequelae. They are further complicated by the global rise on the emergence of macrolide resistance among these bacteria in which several M protein gene (emm) and sequence types are associated with invasive diseases. Objectives: The current study aimed at determining the erythromycin resistance patterns and molecular characteristics of GAS clinical strains by emm and multilocus sequence typing (MLST) methods. Methods: Thirty-five GAS clinical isolates were subjected to antibiotic susceptibility testing by disk diffusion method. The minimum inhibitory concentration (MIC) of erythromycin against GAS by E-test was determined. Clinical and laboratory standards institute (CLSI) guideline was used for the interpretation of results. Detection of ermA, ermB, and mefA genes by polymerase chain reaction (PCR) was performed and emm typing was done by amplification and sequencing of emm genes per standard protocol. Allele and sequence type (ST) of GAS were obtained using the S. pyogenes MLST database. Results: All the isolates were sensitive to erythromycin, penicillin, clindamycin, chloramphenicol, and vancomycin (100%). Resistance to tetracycline was 54.3%. The mefA gene was found in one erythromycin susceptible isolate. No other erythromycin resistance genes were detected in the isolates. Twenty different emm types were found and the most frequent emm types/subtypes detected were emm1, emm18.21, emm28.5, emm97.4, and emm102.2 (each 8.6%). However, no new emm type was detected. A total of 15 sequence types (STs), eight clonal clusters (CCs), and eight singletons were identified among 21 representative isolates. Three isolates exhibited CC1 (ST28/emm1). Conclusions: High susceptibility of GAS isolates against erythromycin could be due to low antibiotic selective pressure in Malaysian clinical settings. High diversity of emm and ST types revealed the heterogenic nature of the strains circulating in Malaysian hospitals. Continuous epidemiological monitoring by molecular typing methods is warranted to improve the management strategies of GAS infections in future.

High prevalence of multidrug-resistant international clones among macrolide-resistant Streptococcus pneumoniae isolates in immunocompromised patients in Tunisia

Macrolide-resistant Streptococcus pneumoniae isolates have increased considerably in the last decade, with important geographical variations in involved phenotypes and genotypes. The aim of this study was to investigate phenotypes, genotypes, serotypes and genetic relatedness of macrolide-resistant S. pneumoniae isolated from immunocompromised patients in Tunisia. Antibiotic susceptibility was determined by disk diffusion, and MICs of erythromycin and clindamycin were determined for macrolide-resistant isolates by Etest. Macrolide-resistant isolates were analysed by PCR for ermB, mefA, tetM, tetO and Int-Tn1545. Serotyping was done by multiplex PCR and the Quellung reaction. Multilocus sequence typing (MLST) was performed for molecular typing. Macrolide resistance was observed in 41 (69.5%) of 59 isolates. Of the 41 isolates, 37 (90.2%) had a macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype, with a predominance of high-level inducible MLSB phenotype, and harboured the ermB gene. All isolates with high-level inducible MLSB phenotype were highly resistant to erythromycin and clindamycin. Four isolates (9.8%) had a macrolide (M) resistance phenotype and harboured the mefA gene. Erythromycin-resistant isolates were multidrug-resistant (MDR) in 97.5% of cases and extensively drug-resistant in 12.2%. The isolates belonged essentially to four serotypes (19F, 23F, 14 and 6B). They were mainly assigned to three sequence types (ST81, ST2918 and ST386). Also, 65.9% of the isolates were grouped in three clonal complexes (CC81, CC838 and CC386). These data indicate a high prevalence of Tn1545 transposon and of three MDR international clones contributing to the high frequency of multidrug resistance among S. pneumoniae isolates in our centre.

Functional Predominance of msr(D), Which Is More Effective as mef(A)-Associated Than mef(E)-Associated, Over mef(A)/mef(E) in Macrolide Resistance in Streptococcus pyogenes.

Although mef(A) and its subclass mef(E) genes have long been considered to play a central role in macrolide efflux-based resistance, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A) gene plays a predominant role in Streptococcus pyogenes macrolide resistance. The mef(A) and mef(E) genes are carried by different genetic elements and the resistance associated with mef(A) was reported to be higher than that associated with mef(E); therefore, we further investigated the functional relevance of mef(A)/mef(E) and its associated msr(D). We established additional mef(A)-, mef(E)-, and their associated msr(D)-knockout strains and confirmed the predominance of msr(D) over mef(A)/mef(E). In addition, we performed experiments introducing mef(A), mef(E), and their associated msr(D) genes into mef(A)/mef(E)-msr(D) double-knockout and mef(A)/mef(E) negative strains. Neither mef(A) nor mef(E) genes had effects on erythromycin resistance. However, both associated msr(D) showed significant effects, and the mef(A)-associated msr(D) exhibited more effect than the mef(E)-associated one. These results suggest that an overall functional predominance of msr(D) over mef(A)/mef(E) is conceivable in efflux-based macrolide resistance in at least some S. pyogenes strains. Furthermore, the higher resistance of mef(A) system over mef(E) system could be derived at least in part from functional differences of mef(A)- and mef(E)-associated msr(D).

Homology and drug resistance gene analysis of Group B Streptococcus resistance induced by clindamycin

Objectives To investigate the homology and drug resistance gene of Group B Streptococcus (GBS) Resistance induced by Clindamycin and provide basic data for clinical prevention and treatment of GBS Resistance infection induced by Clindamycin. Methods 921 strains of GBS were isolated at Obstetrics & Gynecology Hospital of Fudan University from January, 2014 to December, 2015. VITEK2-compact automatic bacterial susceptibility instrument was used to test their sensitivity to 7 antibacterial drugs. 63 positive strains were chosen through D-inhibition zone trial which were drug resistant to Erythromycin and susceptible or intermediary to Clindamycin. The strain′s sequence type was identified by the method of multilocus sequence typing (MLST typing). The drug resistance genes mefA & ermB to Erythromycin were detected by using PCR method. The analysis was carried out to reveal the relevance to drug resistance, multilocus sequence typing and drug resistance gene. Results Among 921 strains of GBS, the drug resistance rate was respectively 53.4% (492/921 strains) to Erythromycin, 50.2% (462/921 strains) to Clindamycin, 34.7% (320/921 strains) to Levofloxacin and 7.5% (69/921 strains) to Nitrofurantoin. The drug resistance rate of Levofloxacin for 63 GBS strains was 27.0% (17/63 strains) and no drug resistant strain was found to Penicillin, Vancomycin & Nitrofurantoin. 12 different ST types were involved in total, including a new ST type: ST1072. The most common ones were ST12 (30.1%) (20/63 strains) & ST19 (25.4%) (16/63 strains). The drug resistance rate of Levofloxacin with ST19 (75.0%) (12/16 strains) was much higher than that of other ST types. The relevance ratio of mefA and ermB among 63 GBS strains was respectively 27.0% (17/63 strains) and 41.3% (26/63 strains). Conclusions The genetic diversity existed in Group B Streptococcus resistance induced by Clindamycin detected in this study. There was significant difference on drug resistance and relevant drug resistant genes among different ST types.(Chin J Lab Med, 2018, 41: 24-28) Key words: GroupB Streptococcus; Multilocus Sequence Typing; Drug Resistance Gene