Mef Gene Research Articles

Phenotypic and molecular characterization of Streptococcus pneumoniae in pre-conjugate vaccine era: A Chinese hospital-based retrospective study

BackgroundStreptococcus pneumoniae (S. pneumoniae) is an important pathogen in causing global morbidity and mortality among children. This study aimed to determine phenotypic and molecular characteristics of S. pneumoniae causing infections in children under five years in China. MethodsA hospital-based retrospective study was conducted. All 537 S. pneumoniae isolates were tested for antimicrobial susceptibility by E-test method, molecular characteristics including resistance genes, virulence genes and serotypes by multiplex polymerase chain reaction (PCR) method, and sequence types (STs) by sequencing seven housekeeping genes. Minimum spanning tree and correspondence analysis were used to reveal the potential relationship between serotypes and STs. ResultsMost of S. pneumoniae isolates were resistant to erythromycin (93.9%) and tetracycline (86.4%), with the predominant resistance genes being erm(B) (92.6%) and tet(M) (95.5%). The prevalent serotypes were 19F, 6B, 19A, 23F and 14, the coverage rate of PCV13 was high in 85.8%, and the predominant STs were ST271, ST320, ST3173, ST81 and ST876. A significant correlation existed between STs and serotypes, with ST271/19F and ST320/19A as the most prevalent clones. Notably, ST271/19F and ST320/19A isolates were associated with resistance to specific antibiotics and carrying of mef(A/E), rlrA and sipA genes. ConclusionsOur findings suggest the introduction of PCV13 vaccine to Chinese children, and underscore the value of monitoring multiple characteristics to detect new epidemiologic trends and provide implications for the formulation of multivalent pneumococcal vaccines.

Synthesis and SARs of novel lincomycin derivatives Part 5: optimization of lincomycin analogs exhibiting potent antibacterial activities by chemical modification at the 6- and 7-positions.

In order to modify lincomycin at the C-6 and C-7 positions, we prepared target molecules, which have substituted pipecolinic acid at the 6-amino group and a para-substituted phenylthio group at the C-7 position, in application of palladium-catalyzed cross-coupling as a key reaction. As the result of structure-activity relationship (SAR) studies at the 6-position, analogs possessing 4'-cis-(cyclopropylmethyl)piperidine showed significantly strong antibacterial activities against Streptococcus pneumoniae and Streptococcus pyogenes with an erm gene. On the basis of SAR, we further synthesized novel analogs possessing 4'-cis-(cyclopropylmethyl)piperidine by transformation of a C-7 substituent. Consequently, novel derivatives possessing a para-heteroaromatic-phenylthio group at the C-7 position exhibited significantly strong activities against S. pneumoniae and S. pyogenes with an erm gene even when compared with those of telithromycin. Finally, in vivo efficacy of selected two derivatives was evaluated in a rat pulmonary infection model with resistant S. pneumoniae with erm + mef genes. One of them exhibited strong and constant in vivo efficacy in this model, and both compounds showed strong in vivo efficacy against resistant S. pneumoniae with a mef gene.

Nghiên cứu độ nhạy cảm kháng sinh của các chủng Streptococus nhóm A, C, G phân lập từ nhầy họng học sinh tiểu học giai đoạn 2012 – 2015

Antibiotic resistance is a global problem, and serious threat to human health. Study on antibiotic susceptibility in streptococci strains group A, C, G isolated from primary school pupils in Viet Nam during 2012÷2015 showed that all streptococci strains were susceptible to cefalosporins and vancomycin as well, simultaneous resistance to amikacin. The resistant rate of GAS to tetracycline and doxycillin were 70% and 75%, respectively. The rate of erythromycin tolerance of GAS was 52.5% and 40.74% in that of GCS/GGS. The third and fourth generation cephalosporin antibiotic group and vancomycin are considered as essential drugs for treating group A, C and G streptococcal infections. The correlation between mefA and ermTR genes in streptococci genome in the macrolide resistant mechanism were not detected. A tetracycline resistant gene in the jumping gene of the GAS genome was found.

Distribution of species and antimicrobial resistance among enterococci isolated from the fecal microbiota of captive blue-fronted parrot (Amazona aestiva) in Rio de Janeiro, Brazil

Enterococcal strains recovered from fecal samples of captive blue-fronted parrots (Amazona aestiva) assisted at two wild animal screening centers in Rio de Janeiro, Brazil, were identified as Enterococcus hirae (the predominant species; 75.3%), followed by Enterococcus faecalis (17.3%), Enterococcus casseliflavus (4.8%), Enterococcus gallinarum (1.7%), and Enterococcus hermanniensis (0.9%). All strains were susceptible to linezolid and teicoplanin. Rates of nonsusceptibility (including resistant and intermediate categories) to other 16 antimicrobials tested varied from 69.3% to 0.4%, A considerable proportion (48.0%) of the strains was multidrug-resistant and diverse genetic determinants associated with antimicrobial resistance were identified. Tetracycline-resistant strains carried the tet(M) and/or tet(L) genes. Macrolides resistance was associated with the erm(B), erm(A) and mefA genes, while 43.2% of the isolates were negative for the investigated genes. High-level resistance to gentamicin associated with the aac(6′)-le-aph(2″)-la gene was detected in one E. faecalis strain. The two strains presenting high-level resistance to streptomycin were negative for the ant(6′)-Ia, ant(3′)-Ia, ant(9′)-Ia and ant(9′)-Ib genes. The vat(D) gene was found in all the 47 quinupristin/dalfopristin resistant strains identified as non-E. faecalis. Analysis of PFGE profiles of E. hirae strains after restriction with SmaI demonstrated the occurrence of five clonal groups. The predominant E. hirae clone was distributed among birds in the two institutions, suggesting that this clone was well adapted to the host and environments investigated. The four clonal groups identified among E. faecalis were composed by small numbers of strains and, generally, restricted to birds in the same sector. The occurrence of enterococcal strains exhibiting antimicrobial resistance traits and carrying genetic determinants that represent potential threats to the health of both humans and animals, in the intestinal microbiota of A. aestiva, highlights the need for additional monitoring studies to elucidate the population structure and the dynamics of transmission of these microorganisms among animals, humans and the environment.

Distribution of macrolide resistance mechanisms in Bulgarian clinical isolates of Streptococcus pyogenes during the years of 2013-2016.

Objectives of the present study was to determine and evaluate the distribution of genetic mechanisms associated with certain phenotypes of macrolide resistance in Bulgarian GAS isolated during the years of 2013-2016. All GAS strains were screened for the macrolide resistance genes erm(A), erm(B) and mef(A), using multiplex polymerase chain reaction (PCR). The minimal inhibitory concentrations (MICs) of erythromycin, azithromycin, clarithromycin, clindamycin were determined by E-tests. Almost 23% of GAS isolates obtained in 2013-2014 and near 40% of them in 2015-2016 contained various elements of resistance. The predominant gene was mef(A), which encodes an efflux pump (M-phenotype), identified in 57.84% of the macrolide-resistant strains. The next frequently prevalent mechanism was a combination of mef(A) and erm(B) in 22.55%, which determined high-level inducible or constitutive resistance to macrolides, lincosamides and streptogramins (iMLSB or cMLSB). The highest MIC value (>256mg/L) was detected in association with erm(B) (p<0.05). The MIC range was observed to be much higher in the isolates with combinations of resistance genes vs. those with mef genes alone (p<0.05). The data about the distribution and prevalence of macrolide resistance mechanisms obtained in this study can help in the treatment of persistent and recurrent GAS infections and in the correct choice of empiric therapy.

The novel mef(C)-mph(G) macrolide resistance genes are conveyed in the environment on various vectors.

The novel tandem genes mef(C) and mph(G) have been reported in marine bacteria in Japan. This paper aimed to characterise the extent of environmental distribution of mef(C) and mph(G) as well as their dissemination and persistence in aquatic bacterial communities. Erythromycin-resistant bacteria were isolated from Japan, Taiwan and Thailand aquaculture sites. The mef(C)-mph(G) genes were detected by PCR. The size of mobile genetic elements conveying mef(C) and mph(G) was examined by Southern blotting. The conjugation rate was assessed by filter mating. The mef(C)-mph(G) tandem genes were distributed in erythromycin-resistant isolates from aquaculture seawater in Japan and northern Taiwan and in animal farm wastewater in Thailand. A total of 29 bacterial isolates were positive for mef(C)-mph(G). The genes were found on vectors of various sizes. Partial sequencing of the traI relaxase gene revealed homology with a pAQU1-like plasmid, an IncA/C-type plasmid and an SXT/R391 family integrative conjugative element (SRI) as vectors. Thirteen isolates (45%) were positive for traI(pAQU-IncA/C-SRI), whereas the others were negative. The traI(pAQU-IncA/C-SRI)-positive isolates exhibited a higher transfer frequency (10-4-10-5 transconjugants/donor) than traI(pAQU-IncA/C-SRI)-negative isolates (<10-9). These results suggest that mef(C)-mph(G) are coded on various vectors and are distributed among marine and wastewater bacteria in Asian countries. Vectors with traI(pAQU-IncA/C-SRI) play a role in the spread of mef(C)-mph(G).

Synthesis and antibacterial evaluation of novel 11-O-carbamoyl clarithromycin ketolides

A series of novel 11-O-carbamoyl clarithromycin ketolides were designed, synthesized and evaluated for their in vitro antibacterial activity. The results showed that the majority of the target compounds displayed improved activity compared with references against erythromycin-resistant S. pneumoniae A22072 expressing the mef gene, S. pneumoniae B1 expressing the erm gene and S. pneumoniae AB11 expressing the mef and erm genes. In particular, compounds 9, 18, 19 and 22 showed the most potent activity against erythromycin-resistant S. pneumoniae A22072 with the MIC values of 0.5μg/mL. Furthermore, compounds 11, 18, 19, 24 and 29 were also found to exhibit favorable antibacterial activity against erythromycin-susceptible S. pyogenes with the MIC values of 0.125–1μg/mL, and moderate activity against erythromycin-susceptible S. aureus ATCC25923 and B. subtilis ATCC9372.

Synthesis and antibacterial activity of novel 4″-O-(1-aralkyl-1,2,3-triazol-4-methyl-carbamoyl) azithromycin analogs

Three novel structural series of 4″-O-(1-aralkyl-1,2,3-triazol-4-methyl-carbamoyl) azithromycin analogs were designed, synthesized and evaluated for their in vitro antibacterial activity. All the target compounds exhibited excellent activity against erythromycin-susceptible Streptococcus pyogenes, and significantly improved activity against three phenotypes of erythromycin-resistant Streptococcus pneumoniae compared with clarithromycin and azithromycin. Among the three series of azithromycin analogs, the novel series of 11,4″-disubstituted azithromycin analogs 9a–k exhibited the most effective and balanced activity against susceptible and resistant bacteria. Among them, compound 9j showed the most potent activity against Staphylococcus aureus ATCC25923 (0.008µg/mL) and Streptococcus pyogenes R2 (1µg/mL). Besides, all the 11,4″-disubstituted azithromycin analogs 9a–k except 9f shared the identical activity with the MIC value <0.002µg/mL against Streptococcus pyogenes S2. Furthermore, compounds 9g, 9h, 9j and 9k displayed significantly improved activity compared with the references against all the three phenotypes of resistant S. pneumoniae. Particularly, compound 9k was the most effective (0.06, 0.03 and 0.125µg/mL) against all the erythromycin-resistant S. pneumoniae expressing the erm gene, the mef gene and the erm and mef genes, exhibiting 2133, 133 and 2048-fold more potent activity than azithromycin, respectively.

Phenotypic and genotypic antimicrobial susceptibility pattern of Streptococcus spp. isolated from cases of clinical mastitis in dairy cattle in Poland.

Mastitis of dairy cattle is one of the most frequently diagnosed diseases worldwide. The main etiological agents of mastitis are bacteria of the genus Streptococcus spp., in which several antibiotic resistance mechanisms have been identified. However, detailed studies addressing this problem have not been conducted in northeastern Poland. Therefore, the aim of our study was to analyze, on phenotypic and genotypic levels, the antibiotic resistance pattern of Streptococcus spp. isolated from clinical cases of mastitis from dairy cattle in this region of Poland. The research was conducted using 135 strains of Streptococcus (Streptococcus uberis, n = 53; Streptococcus dysgalactiae, n = 41; Streptococcus agalactiae, n = 27; other streptococci, n = 14). The investigation of the antimicrobial susceptibility to 8 active substances applied in therapy in the analyzed region, as well as a selected bacteriocin (nisin), was performed using the minimum inhibitory concentration method. The presence of selected resistance genes (n = 14) was determined via PCR. We also investigated the correlation between the presence of resistance genes and the antimicrobial susceptibility of the examined strains in vitro. The highest observed resistance of Streptococcus spp. was toward gentamicin, kanamycin, and tetracycline, whereas the highest susceptibility occurred toward penicillin, enrofloxacin, and marbofloxacin. Additionally, the tested bacteriocin showed high efficacy. The presence of 13 analyzed resistance genes was observed in the examined strains [gene mef(A) was not detected]. In most strains, at least one resistance gene, mainly responsible for resistance to tetracyclines [tet(M), tet(K), tet(L)], was observed. However, a relationship between the presence of a given resistance gene and antimicrobial susceptibility on the phenotypic level was not always observed.

Pneumococcal carriage among children after four years of routine 10-valent pneumococcal conjugate vaccine use in Brazil: The emergence of multidrug resistant serotype 6C

BackgroundIn 2010, the 10-valent pneumococcal conjugate vaccine (PCV10) was introduced free of charge in Brazil as part of the public immunization program. Here we investigated the carriage prevalence, colonization risk factors, capsular types, and antimicrobial resistance among pneumococcal isolates obtained from children in Brazil four years after routine PCV10 use. MethodsBetween September and December 2014, we conducted a cross-sectional study among children<6years old who attended one public and two private clinics in Niterói, RJ, Brazil to evaluate pneumococcal nasopharyngeal carriage. Antimicrobial susceptibility and capsular types were determined for all isolates. ResultsOf 522 children, 118 (22.6%) were pneumococcal carriers. Being≥2years old, attending childcare center, presenting with any symptoms, having acute or chronic respiratory disease, and residing in a slum were associated with pneumococcal carriage. The most prevalent capsular types were 6C (14.5%), 15B/C (11.5%), 11A/D (9.2%), and 6A (7.6%). PCV10 serotypes represented 2.5%. All isolates were susceptible to levofloxacin, rifampicin, and vancomycin. Penicillin non-susceptible pneumococci (PNSP) comprised 39%, with penicillin and ceftriaxone MICs ranging from 0.12–8.0μg/ml and 0.012–1.0μg/ml, respectively. The 33 (28%) erythromycin-resistant isolates (MICs of 1.5 to >256μg/ml) displayed the cMLSB (72.7%) or M (27.3%) phenotypes, harboring the erm(B) and/or mef(A/E) genes. High non-susceptibility rates (>20%) to clindamycin, erythromycin, penicillin, and tetracycline were largely explained by the prevalence of multidrug resistant (MDR) serotype 6C isolates. ConclusionsEffects of universal childhood PCV10 use on carriage were evident, with the near elimination of PCV10 serotypes. The emergence of MDR serotype 6C isolates, however, is a concern. Ongoing surveillance to monitor serotype 6C increase in invasive diseases is warranted.

Emergence of Extensively Drug-resistant Shigella sonnei in Bangladesh

The objective of the study was to investigate current species distribution and growing antimicrobial resistance (AMR) of Shigella isolates for proper treatment. Shigellae, isolated from faecal samples in International Centre for Diarrhoeal Disease research, Bangladesh, Dhaka hospital in 2015, were tested for antimicrobial susceptibility by disc diffusion method to ampicillin, co-trimoxazole, ciprofloxacin, azithromycin, mecillinam, ceftriaxone/cefixime and meropenem. Extensively drug-resistant (XDR, resistant to 5 or 6 of 7 useful anti-Shigella drugs tested) Shigella isolates resistant to 6 drugs were analyzed for ESBL and AmpC phenotypes, plasmid profiles, R-plasmids transfer, blaSHV, blaTEM, blaCTX-M, blaOXA; and mphA, mphB, ermA, ermB, ermC, ereA, ereB, mefA and msrA genes by PCR; and clonality of S. sonnei by PFGE. Of 134 isolates cultured from 3722 (3.6%) diarrhoeal faecal samples, 46% were S. sonnei, 37% S. flexneri, 4% S. boydii, 5% S. dysenteriae and 7% non-typeable. Multidrug-resistant (MDR, resistant simultaneously to ≥3 drugs) S. sonnei were 95% compared to 66% (P<0.01) MDR S. flexneri including 18% & 14% XDR types, respectively. All isolates were susceptible to meropenem. Four (6%) S. sonnei, 2 (4%) S. flexneri and 1 (17%) S. boydii (total of 7 isolates) were six-drugs XDR; 5 of them had ESBL phenotypes. Three S. sonnei and 1 S. flexneri had blaTEM and blaCTX-M; 1 S. boydii had blaSHV, blaTEM and blaCTX-M; 1 S. sonnei had blaTEM β-lactamase. All but one S. flexneri had only mphA gene on 62-MDa conjugative-R-plasmid coding azithromycin resistance. PFGE identified MDR-S. sonnei Global III clade. Thus, MDR-S. sonnei replaced S. flexneri as predominant isolate in Dhaka, Bangladesh; many emerged as XDR strains requiring treatment by meropenem. The findings demand judicial use of antibiotics to contain emergence and spread of resistance locally and globally. Physicians should be informed about MDR and XDR Shigella for judicious prescribing of antimicrobial therapy.

Short-read whole genome sequencing for determination of antimicrobial resistance mechanisms and capsular serotypes of current invasive Streptococcus agalactiae recovered in the USA

ObjectivesOur objective was to evaluate and exploit a whole genome sequence (WGS) bioinformatics pipeline for predicting antimicrobial resistance and capsular serotypes from invasive group B streptococci (iGBS). MethodsFor 1975 iGBS recovered during 2015 from CDC's Active Bacterial Core surveillance, we compared pipeline predictions with broth dilution testing. Fifty-six isolates from earlier surveillance were included for testing β-lactams. Conventional serotyping was compared to WGS-based assignments for 302 isolates. ResultsAll 28 isolates with reduced susceptibility to β-lactam antibiotics harboured one of 19 rare PBP2x types. Resistances to erythromycin/clindamycin (808/1975 isolates, 41.0%), erythromycin (235/1975, 11.9%) and lincosamide/streptogramin A/pleuromutilins (56/1975, 2.8%) were predicted by the presence of erm-methylase, mef and lsa determinants, respectively (41 of 56 lsa gene-positive isolates also contained lnu, erm and/or mef genes). Presence of both erm and lsa determinants (25 isolates) predicted non-susceptibility to quinupristin/dalfopristin. Most isolates (1680/1975, 85.1%) were tet gene-positive, although 41/1565 (2.6%) tetM-positive isolates were tetracycline-susceptible. All 53 fluoroquinolone-resistant isolates contained ParC and/or GyrA substitutions. Resistances to rifampin (eight isolates), trimethoprim, chloramphenicol and vancomycin (two isolates each) were predicted by the pipeline. Resistance to macrolides/lincosamides without pipeline prediction was rare and correlated to divergent resistance genes or rRNA A2062G substitution. A selection of 267 isolates assigned WGS-based serotypes were also conventionally serotyped. Of these, 246 (92.1%) were in agreement, with the remaining 21 (7.8%) conventionally non-serotypeable. For 32 of 1975 isolates (1.6%), WGS-based serotypes could not be assigned. ConclusionThe WGS-based assignment of iGBS resistance features and serotypes is an accurate substitute for phenotypic testing.

Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon.

In recent years, the increased use of macrolides was linked with the emergence of resistance Streptococcus pneumoniae worldwide. The main aim of this study was to determine the prevalence of S. pneumoniae resistant to macrolides and to identify the macrolide resistance genotypes among clinical isolates collected in North Lebanon. Disk diffusion susceptibility method was performed for 132 strains of S. pneumoniae isolated over a period of 5 years in North Lebanon. Polymerase Chain Reaction followed by pyrosequencing was carried out for confirmation of phenotypic diagnosis. The macrolide resistance genotypes were also identified by using PCR amplification of genes implicated in this resistance: erm(A), erm(B), erm(C), msr(A), lin(A) and mef(A/E). Macrolide resistance was found in 34.1% of S. pneumoniae isolates. We observed that the cMLSB phenotype (31/45, 68.9%) was the most common in these pneumococci and erm(B) was the most common resistance gene (32/45, 71.1%). This study shows that macrolide resistance in S. pneumoniae in North Lebanon is mainly related to target site modification with predominance of cMLSb phenotype but is also mediated by efflux pumps. lin(A) gene was reported for the first time in one S. pneumoniae strain in combination with erm(B) and mef(A/E) genes.

Fluoroquinolone, Macrolide, and Ketolide Resistance in Haemophilus parainfluenzae from South Africa.

Fluoroquinolones and ketolides are among the drugs of choice for the treatment of Haemophilus parainfluenzae infections. There has been a report of an emerging fluoroquinolone and telithromycin resistance in H. parainfluenzae isolates from the private sector of KwaZulu-Natal Province of South Africa that necessitates molecular investigation. The aim of this study is to characterize these resistance delineating mutations in genes commonly associated with reduced susceptibility. Ten H. parainfluenzae isolates retrieved from the sputum of 10 patients with H. parainfluenzae pneumonia were subjected to sensitivity testing by the disc diffusion and CLSI broth microdilution methods, polymerase chain reaction (PCR) and DNA sequencing of selected genes associated with resistance were carried out, while repetitive extragenic palindromic PCR (REP-PCR) was used to ascertain clonality. Fluoroquinolone resistance was attributed to the following amino acid substitutions: S84F, D88Y in GyrA, and S84Y/L, S138T, and M198 L change in ParC of the isolates. The plasmid-mediated quinolone resistance gene aac-(6')-Ib-cr was detected for the first time in four isolates of H. parainfluenzae and D420 N change was observed in ParE in one isolate. Macrolide and ketolide resistance were ascribed to the resistance genes mef (A), msr (D), and erm (B) detected in the isolates. REP-PCR analysis showed that the isolates were not clonal. All the observed resistance mechanisms are first reports in Africa. There is an emerging fluoroquinolone and macrolide resistance in H. parainfluenzae in South Africa that is attributable to known/novel resistance mechanisms, necessitating the monitoring of this pathogen as a potential opportunistic pathogen in a country with a high HIV and AIDS prevalence.

Characteristics of Streptococcus pneumoniae Serotype 19A Isolates from Children in the Pre- and Post-Conjugate Vaccine Era

Background The aim of the present study was to assess the prevalence and characteristics of S. pneumoniae serotype 19A isolates from invasive and non-invasive pneumococcal disease (IPD) in children, before and after the introduction of pneumococcal conjugate vaccines (PCVs) in Greece.Methods All S. pneumoniae isolates collected between 1986 and 2015 were re-cultured for further testing. Serotyping was performed using the quellung reaction and antibiotic susceptibilities were determined by E-test. The altered pbp genes (pbp2b, pbp2x, and pbp1a) conferring resistance to penicillin and the macrolide-resistant genes [mef(A), mef(E), and erm(B)] were detected by polymerase chain reaction (PCR) using specific primers. The sequence types (ST) and clonal complexes were determined by multilocus sequence typing (MLST).ResultsAmong 1,875 isolates examined, 210(11.2%) (belonged to serotype 19A. The proportion of serotype 19A isolates increased from 4.6% (47/1018) in the pre-PCV7 years (1986–2005) to 19.6% (88/449) in the post-PCV7 years (2006–2010) and to 18.4% (75/408) in the post PCV-10 and PCV13 years (2011–2015).The resistance frequencies to penicillin, cefotaxime, erythromycin, clindamycin, and tetracycline increased significantly after 2005 as compared with the preceding period (1986–2005) from 8.5% to 31.9%, 2.1% to 20.8%, 10.6% to 65.6%, 4.2% to 47.7% and 19.1% to 46.0%, respectively (P < 0.01). Moreover, 94 (57.7%) of 163 isolates after 2005 exhibited multidrug-resistant (MDR) phenotype, whereas only seven (15.9%) of 47 isolates from period 1986–2005 were MDR (P <0.001). All the penicillin non-susceptible isolates examined (N = 136), had altered one or more of the pbp genes. Of 112 erythromycin-resistant isolates, 22 contained the mefE gene, 39 the ermB and 51 both the mefE and ermB genes. MLST analysis of 31 invasive isolates revealed four clonal complexes (CC); CC172 (n = 11), CC199 (n = 8), CC320 (n = 5), and CC276 (n = 3), and four isolates belonged to CC2669, CC177, CC81, and CC63. The CC172 and CC199 were present throughout the study period, whereas the CC320 and CC276 appeared after 2009.Conclusion S. pneumoniae serotype 19A increased significantly after the introduction of PCV7 and a substantial proportion of these isolates exhibited MDR phenotype. The majority of the examined isolates belonged to limited number of CC.Disclosures V. Syriopoulou, Hellenic Center for Disease Control and Prevention: Grant Investigator, Research grant. Pfizer Inc.Pharmaceutical Company: Grant Investigator, Research grant

Quintuplex PCR to Detect Antibiotic Resistance Genes in Streptococcus pneumoniae

Introduction: Streptococcus pneumoniae is an important bacterial pathogen, causing respiratory infection. Penicillin resistance in S. pneumoniae is associated with alterations in the penicillin binding proteins, while resistance to macrolides is conferred either by the modification of the ribosomal target site or efflux mechanism. This study aimed to characterize S. pneumoniae and its antibiotic resistance genes using 2 sets of multiplex PCRs. Methods: A quintuplex and triplex PCR was used to characterize the pbp1A, ermB, gyrA, ply, and the mefE genes. Fifty-eight penicillin sensitive strains (PSSP), 36 penicillin intermediate strains (PISP) and 26 penicillin resistance strains (PRSP) were used. Results: Alteration in pbp1A was only observed in PISP and PRSP strains, while PCR amplification of the ermB or mefE was observed only in strains with reduced susceptibility to erythromycin. The assay was found to be sensitive as simulated blood cultures showed the lowest level of detection to be 10cfu. Conclusions: As predicted, the assay was able to differentiate penicillin susceptible from the non-susceptible strains based on the detection of the pbp1A gene, which correlated with the MIC value of the strains.

Molecular Characteristics of Erythromycin-Resistant Streptococcus pyogenes Strains Isolated from Children Patients in Tunis, Tunisia.

The aims of our study were to characterize phenotypically and genotypically erythromycin-resistant Streptococcus pyogenes or group A streptococci (ERGAS) isolates, to evaluate macrolide resistance and to analyze the association between emm types and virulence factors. Included in this study were all ERGAS strains isolated from 2000 to 2013 at the Children's hospital of Tunis. Antimicrobial susceptibility was performed according to the CA-SFM guidelines. Macrolide resistance genes were revealed by polymerase chain reaction (PCR) method. Virulence factor genes (pyrogenic exotoxin genes and superantigen gene) were detected by PCR, and the emm types were defined by the sequencing of the variable 5' end of the emm gene. Among the 289 GAS isolates collected, 15 (5.2%) were resistant to erythromycin; 7 of the strains were assigned to the cMLSB phenotype (46.6%); 5 harbored ermB gene alone (33.3%); and 2 strains coharbored ermB and mefA (13.3%). The remaining (53.4%) were assigned to the M phenotype and harbored the mefA gene. The frequency of detection of each toxin gene among ERGAS was 13.4% for speA (2 strains), 53.4% for speC (8 strains), and 13.4% for ssa (2 strains). Emm types 1, 58, 11, and 78 were the most frequent among ERGAS strains. The distribution of the cMLSB and M phenotypes changed over the period of investigation with a decrement of cMLSB phenotype and ermB gene that predominated between 2000 and 2006 and an increase of M phenotype and mefA gene between 2007 and 2013, but this difference was nonstatistically significant because of the low number of resistant strains. Emm types 1, 58, and 4 were only present among strains assigned to the M phenotype. However strains assigned to the cMLSB phenotype were associated to emm11, emm22, emm28, emm78, or emm76. There was diversity in emm distribution in ERGAS between the two study periods. There was diversity in emm distribution among ERGAS particularly in 2000-2006. Indeed, from 2000 to 2006, the 6 ERGAS belonged to 5 different emm types (22, 28, 76, 11, and 4), while between 2007 and 2013, seven among the nine ERGAS belonged to only 2 emm types 58 and 1. The speA gene was present only among emm1 isolates, and the ssa gene was associated with emm4 and emm78 types. All emm78, emm28, and emm11 strains harbored speC gene. Our study revealed a low frequency of ERGAS and few emm types were associated with these strains.

Serotypes, antimicrobial resistance and genotypes of Streptococcus pneumoniae associated with infections in cancer patients in Brazil

We sought to characterize pneumococcal isolates associated with bacteremia, pneumonia and meningitis in cancer patients and to estimate the coverage of the available pneumococcal vaccines. Fifty isolates recovered from 49 patients attending a cancer reference center over a 1-year period were analyzed. The prevalent serotypes were: 23F (12%), 6A (8%), 3, 4, 20, and 23A (6% each). All isolates were susceptible to chloramphenicol, levofloxacin, rifampicin, and vancomycin. Resistance or reduced susceptibility to penicillin made up 14%, and one isolate was also intermediately resistant to ceftriaxone. The three (6%) erythromycin-resistant isolates presented the M or cMLSB phenotypes and harbored the mef(A/E) gene exclusively or along with the erm(B) gene. Twenty-two (44%) isolates were closely related to 11 international clones, being strongly associated with penicillin non-susceptibility. Combined immunization with the 13-valent conjugate and the 23-valent polysaccharide vaccines might contribute to reduce (76%) the burden of the pneumococcal infections in the population investigated.

Synthesis and antibacterial activity of novel 3-O-descladinosylazithromycin derivatives

Novel series of novel 3-O-arylalkylcarbamoyl descladinosylazithromycin derivatives with the 2′-O-acetyl and 11,12-cyclic carbonate groups, the 11,12-cyclic carbonate group and the 11-O-arylalkylcarbamoyl side chain, and 2′-O-arylalkylcarbamoyl descladinosylazithromycin with the 11,12-cyclic carbonate group were designed, synthesized and evaluated for their antibacterial activity using broth microdilution method. The results showed that the majority of the target compounds showed moderate to favorable activity against six kinds of susceptible strains and almost all of them displayed significantly improved activity compared with references against three erythromycin-resistant strains of S. pneumoniae B1 expressing the ermB gene, S. pneumoniae AB11 expressing the ermB and mefA genes, and S. pyogenes R1. In particular, compound 6h exhibited the most potent activity against susceptible B. subtilis ATCC9372 (0.5 μg/mL), penicillin-resistant S. epidermidis (0.125 μg/mL), and erythromycin-resistant S. pneumoniae B1 (1 μg/mL) and S. pneumoniae AB11 (1 μg/mL), which were 2-, 2-, 256-, 256-fold better activity than azithromycin, respectively. Additionally, compounds 6f (0.5 μg/mL) and 6g (0.25 μg/mL) were the most active against S. pneumoniae A22072, which were 8- and 16-fold better activity than azithromycin (4 μg/mL). As for erythromycin-resistant S. pyogenes R1, compound 5a presented the most excellent activity (8 μg/mL), showing 32- and 32-fold higher activity than azithromycin (256 μg/mL) and clarithromycin (256 μg/mL).

Inducible Expression of both ermB and ermT Conferred High Macrolide Resistance in Streptococcus gallolyticus subsp. pasteurianus Isolates in China.

Streptococcus gallolyticus subsp. pasteurianus is an under-recognized pathogen and zoonotic agent causing opportunistic infections in humans. Despite increasing recognition of this subspecies as a cause for human infectious diseases, limited information is known about its antibiotic resistance mechanism. In this study, we aim to identify the molecular mechanism underlying the high macrolide resistance of six S. gallolyticus subsp. pasteurianus isolates from dead ducklings collected in several natural outbreaks in China during 2010–2013. All isolates exhibited multi-drug resistance including high macrolide resistance (MIC ≥ 1024 mg/L for erythromycin, and 512 mg/L for clarithromycin). Efflux-encoding mefA and mefE genes were not detectable in these isolates. The presence of 23S rRNA mutations in specific isolates did not significantly change macrolide MICs. No nucleotide substitutions were found in genes encoding ribosomal proteins L4 or L22. The ermB and ermT genes were found in the genomes of all isolates. These two genes were acquired independently in one highly virulent isolate AL101002, and clustered with Tn916 and IS1216, respectively. The expression of both ermB and ermT in all isolates was erythromycin inducible and yielded comparable macrolide MICs in all six isolates. Taken together, inducible expression of both ermB and ermT conferred high macrolide resistance in these S. gallolyticus subsp. pasterianus isolates. Our findings reveal new macrolide resistance features in S. gallolyticus subsp. pasteurianus by both ermB and ermT.