Erythromycin Resistance Gene Research Articles

A novel erythromycin resistance plasmid from Bacillus sp. strain HS24, isolated from the marine sponge Haliclona simulans.

A better understanding of the origin and natural reservoirs of resistance determinants is fundamental to efficiently tackle antibiotic resistance. This paper reports the identification of a novel 5.8 kb erythromycin resistance plasmid, from Bacillus sp. HS24 isolated from the marine sponge Haliclona simulans. pBHS24B has a mosaic structure and carries the erythromycin resistance gene erm(T). This is the first report of an erythromycin resistance plasmid from a sponge associated bacteria and of the Erm(T) determinant in the genus Bacillus.

Detection of the efflux-mediated erythromycin resistance transposon in Streptococcus pneumoniae.

BackgroundThe present analysis focuses on phenotypic and genotypic characterizations of efflux-mediated erythromycin resistance in Streptococcus pneumoniae due to an increase in macrolide resistance in S. pneumoniae worldwide.MethodsWe investigated the prevalence of efflux-mediated erythromycin resistance and its relevant genetic elements from 186 specimens of S. pneumonia isolated from clinical and normal flora from Tehran, Iran. The presence of erythromycin resistance genes was tested by PCR with two sets of primers, specific for erm(B) and mef(A/E), and their genetic elements with tetM, xis, and int genes. Isolates were typed with the BOX PCR method and tested for resistance to six antibiotics.ResultsAntibiotic susceptibility tests revealed that 100% and 47% isolates were resistant to tetracycline and erythromycin, respectively. The erythromycin and clindamycin double-disc diffusion test for macrolide-lincosamide-streptograminB (MLSB) resistance phenotype showed 74 (84%) isolates with the constitutive MLSB phenotype and the remaining with the M phenotype. BOX PCR demonstrated the presence of 7 types in pneumococci with the M phenotype. Fourteen (16%) isolates with the M phenotype harbored mef(A/E), tetM, xis, and int genes.ConclusionsThe present results suggest dissemination of polyclonal groups of S. pneumoniae with the M phenotype carrying resistance genes attributed to transposon 2009.

A type 2 A/C2 plasmid carrying the aacC4 apramycin resistance gene and the erm(42) erythromycin resistance gene recovered from two Salmonella enterica serovars.

To determine the relationships between RepA/C2 plasmids carrying several antibiotic resistance genes found in isolates of Salmonella enterica serovars Ohio and Senftenberg from pigs. Illumina HiSeq was used to sequence seven S. enterica isolates. BLAST searches identified relevant A/C2 plasmid contigs and contigs were assembled using PCR. Two serovar Ohio isolates were ST329 and the five Senftenberg isolates were ST210. The A/C2 plasmids recovered from the seven isolates belong to type 2 and contain two resistance islands. Their backbones are closely related, differing by five or fewer SNPs. The sul2-containing resistance island ARI-B is 19.9 kb and also contains the kanamycin and neomycin resistance gene aphA1, the tetracycline resistance gene tetA(D) and an erythromycin resistance gene, erm(42), not previously seen in A/C2 plasmids. A second 30.3 kb resistance island, RI-119, is in a unique location in the A/C2 backbone 8.2 kb downstream of rhs. RI-119 contained genes conferring resistance to apramycin, netilmicin and tobramycin (aacC4), hygromycin (hph), sulphonamides (sul1) and spectinomycin and streptomycin (aadA2). In one of the seven plasmids, this resistance region contained two IS26-mediated deletions. A discrete 5.7 kb segment containing the aacC4 and hph genes and bounded by IS26 on one side and the inverted repeat of Tn5393 on the other was identified. The presence of almost identical A/C2 plasmids in two serovars indicates a common origin. Type 2 A/C2 plasmids continue to evolve via addition of new resistance regions such as RI-119 and evolution of existing ones.

Development of an antibiotic-free plasmid selection system based on thymine auxotrophy in Lactococcus lactis

Lactococcus lactis has become the best studied species of the lactic acid bacteria (LAB) clade and an ideal cell factory for heterogenous proteins. We have constructed an antibiotic-free expression vector, pMG-thyA, using thymidine synthase gene thyA as the selection marker. The thyA gene was cloned from the food industry strain Streptococcus thermophilus St-JY and was used to replace the erythromycin resistance genes on L. lactis expression vector pMG36e in order to construct pMG-thyA. The construction of the new vector and thyA-null host L. lactis MG1363-TT yielded an antibiotic-free expression system. The α-amylase gene (amy) was cloned onto the multiple cloning site of the vector pMG-thyA as a reporter gene, yielding the recombinant plasmid pMGthyA-amy. This plasmid was electroporated into L. lactis MG1363-TT, and the recombinant strain grown on SA plates containing 0.5 % (w/v) soluble starch formed distinct bacterial colonies and clear zones (halo) around the colonies following the addition of iodine solution. These research findings lay the foundation for food-grade expression in L. lactis.

Erythromycin-resistant genes in group A β-haemolytic Streptococci in Chengdu, Southwestern China.

The management of Group A β-haemolytic Streptococci (Streptococcus pyogenes or GAS) infection include the use of penicillins, cephalosporins or macrolides for treatment. A general increase in macrolides resistance in GAS has been observed in recent years. Differences in rates of resistance to these agents have existed according to geographical location and investigators. To investigate the antibiotic pattern and erythromycin-resistant genes of GAS isolates associated with acute tonsillitis and scarlet fever in Chengdu, southwestern China. To assess the macrolide resistance, phenotype, and genotypic characterization of GAS isolated from throat swabs of children suffering from different acute tonsillitis or scarlet fever between 2004 and 2011 in the city of Chengdu, located in the southwestern region of China. Minimal inhibitory concentration with seven antibiotics was performed on 127 GAS isolates. Resistance phenotypes of erythromycin-resistant GAS isolates were determined by the double-disk test. Their macrolide-resistant genes (mefA, ermB and ermTR) were amplified by PCR. A total of 98.4% (125/127) of the isolates exhibited resistance to erythromycin, clindamycin and tetracycline. All isolates were sensitive to penicillin G and cefotaxime. Moreover, 113 ermB-positive isolates demonstrating the cMLS phenotype of erythromycin resistance were predominant (90.4%) and these isolates showed high-level resistance to both erythromycin and clindamycin (MIC 90>256 μg/ml); 12 (9.6%) isolates demonstrating the MLS phenotype of erythromycin resistance carried the mefA gene, which showed low-level resistance to both erythromycin (MIC 90=8 μg/ml) and clindamycin (MIC 90=0.5 μg/ml); and none of the isolates exhibited the M phenotype. The main phenotype is cMLS, and the ermB gene code is the main resistance mechanism against macrolides in GAS. Penicillin is the most beneficial for treating GAS infection, and is still used as first-line treatment. And macrolide antibiotics are not recommended for treatment of GAS infection in children because of the high rates of antimicrobial resistance in mainland China.

A mariner transposon vector adapted for mutagenesis in oral streptococci.

This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication repATs-pWV01, a selectable kanamycin resistance gene, a Himar1 transposase gene regulated by a xylose-inducible promoter, and an erythromycin resistance gene flanked by himar inverted repeats. The pMN100 plasmid was transformed into Streptococcus mutans UA159 and transposon mutagenesis was performed via a protocol established to perform high numbers of separate transpositions despite a low frequency of transposition. The distribution of transposon inserts in 30 randomly picked mutants suggested that mariner transposon mutagenesis is unbiased in S. mutans. A generated transposon mutant library containing 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen.

Prevalence and Antimicrobial Resistance of Enterococcus Species: A Hospital-Based Study in China

Objective: to investigate the prevalence and antimicrobial resistance of Enterococcus species isolated from a university hospital, and explore the mechanisms underlying the antimicrobial resistance, so as to provide clinical evidence for the inappropriate clinical use of antimicrobial agents and the control and prevention of enterococcal infections. Methods: a total of 1,157 enterococcal strains isolated from various clinical specimens from January 2010 to December 2012 in the General Hospital of Ningxia Medical University were identified to species level with a VITEK-2 COMPACT fully automated microbiological system, and the antimicrobial susceptibility of Enterococcus species was determined using the Kirby-Bauer disc diffusion method. The multiple-drug resistant enterococcal isolates were screened from the clinical isolates of Enterococcus species from the burns department. The minimal inhibitory concentration (MIC) of Enterococcus species to the three fluoroquinolones, including ciprofloxacin, gatifloxacin and levofloxacin was determined with the agar dilution method, and the changes in the MIC of Enterococcus species to the three fluoroquinolones following reserpine treatment were evaluated. The β-lactam, aminoglycoside, tetracycline, macrolide, glycopeptide resistance genes and the efflux pump emeA genes were detected in the enterococcal isolates using a polymerase chain reaction (PCR) assay. Results: the 1,157 clinical isolates of Enterococcus species included 679 E. faecium isolates (58.7%), 382 E. faecalis isolates (33%), 26 E. casseliflavus isolates (2.2%), 24 E. avium isolates (2.1%), and 46 isolates of other Enterococcus species (4%). The prevalence of antimicrobial resistance varied significantly between E. faecium and E. faecalis, and ≤1.1% of these two Enterococcus species were found to be resistant to vancomycin, teicoplanin or linezolid. In addition, the Enterococcus species isolated from different departments of the hospital exhibited various resistances to the same antimicrobial agent, while reserpine treatment reduced the resistance of Enterococcus species to ciprofloxacin, gatifloxacin and levofloxacin. The β-lactamase gene TEM, aminoglycoside-modifying-enzyme genes aac(6')-aph(2"), aph(3')-III, ant(6)-I and ant(2")-I, tetracycline resistance gene tetM, erythromycin resistance gene ermB, vancomycin resistance gene vanA and the enterococcal multidrug resistance efflux emeA gene were detected in 77%, 62%, 26%, 13%, 36%, 31%, 66%, 5% and 55% of the 100 multiple-drug resistant enterococcal isolates. Conclusions: similar to previous findings, E. faecium and E. faecalis are predominant conditionally pathogenic bacteria that cause hospital-acquired infections that can cause urinary and respiratory system infections. Multiple and high-level antimicrobial resistance is highly prevalent in the hospital isolates of Enterococcus species. Reserpine treatment inhibits the active efflux of Enterococcus species to ciprofloxacin, gatifloxacin and levofloxacin in vitro and reduces the MIC of Enterococcus species to these three fluoroquinolones. The presence of the enterococcal multidrug resistance efflux emeA gene is associated with the resistance to antibiotics in Enterococcus species. The monitoring of the prevalence and antimicrobial resistance of Enterococcus species is of great significance to guide the control and prevention of enterococcal infections.

Development of a genetic system for the deep-sea psychrophilic bacterium Pseudoalteromonas sp. SM9913

BackgroundPseudoalteromonas species are a group of marine gammaproteobacteria frequently found in deep-sea sediments, which may play important roles in deep-sea sediment ecosystem. Although genome sequence analysis of Pseudoalteromonas has revealed some specific features associated with adaptation to the extreme deep-sea environment, it is still difficult to study how Pseudoalteromonas adapt to the deep-sea environment due to the lack of a genetic manipulation system. The aim of this study is to develop a genetic system in the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913, making it possible to perform gene mutation by homologous recombination.ResultsThe sensitivity of Pseudoalteromonas sp. SM9913 to antibiotic was investigated and the erythromycin resistance gene was chosen as the selective marker. A shuttle vector pOriT-4Em was constructed and transferred into Pseudoalteromonas sp. SM9913 through intergeneric conjugation with an efficiency of 1.8 × 10-3, which is high enough to perform the gene knockout assay. A suicide vector pMT was constructed using pOriT-4Em as the bone vector and sacB gene as the counterselective marker. The epsT gene encoding the UDP-glucose lipid carrier transferase was selected as the target gene for inactivation by in-frame deletion. The epsT was in-frame deleted using a two-step integration–segregation strategy after transferring the suicide vector pMT into Pseudoalteromonas sp. SM9913. The ΔepsT mutant showed approximately 73% decrease in the yield of exopolysaccharides, indicating that epsT is an important gene involved in the EPS production of SM9913.ConclusionsA conjugal transfer system was constructed in Pseudoalteromonas sp. SM9913 with a wide temperature range for selection and a high transfer efficiency, which will lay the foundation of genetic manipulation in this strain. The epsT gene of SM9913 was successfully deleted with no selective marker left in the chromosome of the host, which thus make it possible to knock out other genes in the same host. The construction of a gene knockout system for Pseudoalteromonas sp. SM9913 will contribute to the understanding of the molecular mechanism of how Pseudoalteromonas adapt to the deep-sea environment.

Molecular identification and quantification of tetracycline and erythromycin resistance genes in Spanish and Italian retail cheeses.

Large antibiotic resistance gene pools in the microbiota of foods may ultimately pose a risk for human health. This study reports the identification and quantification of tetracycline- and erythromycin-resistant populations, resistance genes, and gene diversity in traditional Spanish and Italian cheeses, via culturing, conventional PCR, real-time quantitative PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). The numbers of resistant bacteria varied widely among the antibiotics and the different cheese varieties; in some cheeses, all the bacterial populations seemed to be resistant. Up to eight antibiotic resistance genes were sought by gene-specific PCR, six with respect to tetracycline, that is, tet(K), tet(L), tet(M), tet(O), tet(S), and tet(W), and two with respect to erythromycin, that is, erm(B) and erm(F). The most common resistance genes in the analysed cheeses were tet(S), tet(W), tet(M), and erm(B). The copy numbers of these genes, as quantified by qPCR, ranged widely between cheeses (from 4.94 to 10.18log⁡10/g). DGGE analysis revealed distinct banding profiles and two polymorphic nucleotide positions for tet(W)-carrying cheeses, though the similarity of the sequences suggests this tet(W) to have a monophyletic origin. Traditional cheeses would therefore appear to act as reservoirs for large numbers of many types of antibiotic resistance determinants.

Antibiotic resistance in lactic acid bacteria isolated from some pharmaceutical and dairy products.

A total of 244 lactic acid bacteria (LAB) strains were isolated from 180 dairy and pharmaceutical products that were collected from different areas in Minia governorate, Egypt. LAB were identified phenotypically on basis of morphological, physiological and biochemical characteristics. Lactobacillus isolates were further confirmed using PCR-based assay. By combination of phenotypic with molecular identification Lactobacillus spp. were found to be the dominant genus (138, 76.7%) followed by Streptococcus spp. (65, 36.1%) and Lactococcus spp. (27, 15%). Some contaminant organisms such as (Staphylococcus spp., Escherichia coli, Salmonella spp., mould and yeast) were isolated from the collected dairy samples but pharmaceutical products were free of such contaminants. Susceptibility of LAB isolates to antibiotics representing all major classes was tested by agar dilution method. Generally, LAB were highly susceptible to Beta-lactams except penicillin. Lactobacilli were resistant to vancomycin, however lactococci and streptococci proved to be very susceptible. Most strains were susceptible to tetracycline and showed a wide range of streptomycin MICs. The MICs of erythromycin and clindamycin for most of the LAB were within the normal range of susceptibility. Sixteen Lactobacillus, 8 Lactococcus and 8 Streptococcus isolates including all tetracycline and/or erythromycin resistant strains were tested for the presence of tetracycline and/or erythromycin resistant genes [tet(M) and/or erm(B)]. PCR assays shows that some resistant strains harbor tet(M) and/or erm(B) resistance genes.

Presence of Vancomycin, Aminoglycosides, and Erythromycin Resistance Genes in Enterococci Isolated from Clinical Samples in Turkey

Emergence of high-level aminoglycoside and glycopeptide resistance causes more severe prognosis, higher mortality, and recurrence in enterococcal infections. The present study was conducted to investigate the antimicrobial resistance patterns, prevalence of AMEs, erm and van genes of enterococci isolated from various clinical samples at Istanbul University, Cerrahpasa Medical School hospital. During the period of 2012-2013, a total of 128 enterococcal isolates collected from various clinical samples were typed with the API 20 strep test, and antibiotic sensibilities were analysed with the disc diffusion and E-test methods. The detection of resistant genes was determined by a multiplex PCR method. Forty-nine percent E. faecalis, 46% E. faecium, 4 E. avium and 2 E. durans were detected. E. faecium resistance rates were significantly higher than E. faecalis (p < 0.001). The resistant genes were analysed in 50 enterococcus strains. The vanA gene was found in 29 of the 30 VRE strains. The most prevalent AMEs genes were aac (6')-Ie-aph (2")-Ia gene (72%), followed by aph (3')-IIIa gene (30%). The ermB gene was found in 49 of a total of 50 strains. This study shows that aac (6')-Ie-aph (2")-Ia, ermB, and vanA genes are common among enterococci isolates in our hospital and spread of VRE along with acquisition of resistance to most of the antibiotics used in therapy.

Phenotypic expression, molecular characterization and transferability of erythromycin resistance genes in Enterococcus spp. isolated from naturally fermented food

To evaluate phenotypic resistance to macrolides-lincosamides and streptogramin B (MLSB ) antibiotics and to determine their localization as well as transferability of erythromycin resistance genes in enterococcal isolates of naturally fermented food-Idli batter. Diverse MLSB phenotypes observed among the enterococcal spp. (n=32) were analysed through double disc and triple disc test. Standard minimum inhibitory concentration tests along with induction studies displayed synergistic or cross-resistance among MLSB antibiotics. Plasmid profiling and Southern hybridization revealed that erm(B) and msr(C) genes were localized either onchromosome or on high molecular weight plasmids and showed co-localization of these genes with lnu(B), tet(L) and tet(W) in one of the isolate. In vitro conjugation experiments demonstrated plasmid-mediated transfer of erm(B) gene from three Enterococcus durans strains to Enterococcus faecalis JH2-2. The study illustrated diverse MLSB phenotypes, multiple resistance genes and transferable plasmids among enterococci isolated from naturally fermented foods. From a public health point of view, the study identified that naturally fermented foods could represent a source of antibiotic resistance enterococci that can spread through foods. These results also suggest that vigilance may be exercised with the use of combination or novel MLSB antibiotics in treating enterococcal infections.

Genetic determinants of antimicrobial resistance in Gram positive bacteria from organic foods

Bacterial biocide resistance is becoming a matter of concern. In the present study, a collection of biocide-resistant, Gram-positive bacteria from organic foods (including 11 isolates from genus Bacillus, 25 from Enterococcus and 10 from Staphylococcus) were analyzed for genes associated to biocide resistance efflux pumps and antibiotic resistance. The only qac-genes detected were qacA/B (one Bacillus cereus isolate) and smr (one B. cereus and two Staphylococcus saprophyticus isolates). Efflux pump genes efrA and efrB genes were detected in Staphylococcus (60% of isolates), Bacillus (54.54%) and Enterococcus (24%); sugE was detected in Enterococcus (20%) and in one Bacillus licheniformis; mepA was detected in Staphylococcus (60%) and in one Enterococcus isolate (which also carried mdeA), and norE gene was detected only in one Enterococcus faecium and one S. saprophyticus isolate. An amplicon for acrB efflux pump was detected in all but one isolate. When minimal inhibitory concentrations (MICs) were determined, it was found that the addition of reserpine reduced the MICs by eight fold for most of the biocides and isolates, corroborating the role of efflux pumps in biocide resistance. Erythromycin resistance gene ermB was detected in 90% of Bacillus isolates, and in one Staphylococcus, while ereA was detected only in one Bacillus and one Staphyloccus, and ereB only in one Staphylococcus. The ATP-dependent msrA gene (which confers resistance to macrolides, lincosamides and type B streptogramins) was detected in 60% of Bacillus isolates and in all staphylococci, which in addition carried msrB. The lincosamide and streptogramin A resistance gene lsa was detected in Staphylococcus (40%), Bacillus (27.27%) and Enterococcus (8%) isolates. The aminoglycoside resistance determinant aph (3_)-IIIa was detected in Staphylococcus (40%) and Bacillus (one isolate), aph(2_)-1d in Bacillus (27.27%) and Enterococcus (8%), aph(2_)-Ib in Bacillus (one isolate), and the bifunctional aac(6_)1e-aph(2_)-Ia in Staphylococcus (20%), Enterococcus (8%) and Bacillus (one isolate). Chloramphenicol resistance cat gene was detected in Enterococcus (8%) and Staphylococcus (20%), and blaZ only in Staphylococcus (20%). All other antibiotic or biocide resistance genes investigated were not detected in any isolate. Isolates carrying multiple biocide and antibiotic determinants were frequent among Bacillus (36.36%) and Staphylococcus (50%), but not Enterococcus. These results suggest that biocide and antibiotic determinants may be co-selected.

Improvement of ClosTron for successive gene disruption in Clostridium cellulolyticum using a pyrF-based screening system

Clostridium includes a number of species, such as thermophilic Clostridium thermocellum and mesophilic Clostridium cellulolyticum, producing biofuels and chemicals from lignocellulose, while genetic engineering is necessary to improve wild-type strains to fulfill the requirement of industrialization. ClosTron system is widely used in the gene targeting of Clostridium because of its high efficiency and operability. However, the targetron plasmid present in cell hinders the successive gene disruption. To solve this problem, a pyrF-based screening system was developed and implemented in C. cellulolyticum strain H10 in this study for efficient targetron plasmid curing. The screening system was composed of a pyrF-deleted cell chassis (H10ΔpyrF) constructed via homologous recombination and a PyrF expression cassette located in a targetron plasmid containing an erythromycin resistance gene. With the screening system, the gene targeting could be achieved following a two-step procedure, including the first step of gene disruption through targetron transformation and erythromycin selection and the second step of plasmid curing by screening with 5-fluoroorotic acid. To test the developed screening system, successive inactivation of the major cellulosomal exocellulase Cel48F and the scaffoldin protein CipC was achieved in C. cellulolyticum, and the efficient plasmid curing was confirmed. With the assistance of the pyrF-based screening system, the targetron plasmid-cured colonies can be rapidly selected by one-plate screening instead of traditional days' unguaranteed screening, and the successive gene disruption becomes accomplishable with ClosTron system with improved stability and efficiency, which may promote the metabolic engineering of Clostridium species aiming at enhanced production of biofuels and chemicals.

Genetic engineering techniques for lactic acid bacteria: construction of a stable shuttle vector and expression vector for β-glucuronidase

The shuttle vector, pUL6erm, was constructed by using a replicon from pL2, a multiple cloning site, colE1 ori, the ori of Gram-negative bacteria from vector pUC19, and the erythromycin resistance gene from pVA838 as a selection marker. pUL6erm could be transformed easily and maintained stably in Lactococcus lactis, Streptococcus thermophilus, Lactobacillus plantarum and Lactobacillus casei. Transformation assays of pUL6erm indicated that it had a narrow host range. β-Glucuronidase was induced in the presence of 0.3M NaCl and 50mM glutamate and expressed at 2.4Umg(-1) with the expression vector (pUL6erm-gadR-GUS) constructed based on pUL6erm carrying β-glucuronidase gene wuth a chloride-inducible (gadR) expression cassette using Pgad as promoter. Therefore, pUL6erm and pUL6erm-gadR-GUS might be a safe and useful genetic tool for the improvement of lactic acid bacteria.

The trend of macrolide resistance and emm types of group A streptococci from children at a medical center in southern Taiwan

Group A streptococcus (GAS) is a common pathogen in children. Macrolide resistance in GAS has been described worldwide. The aims of this study are to analyze macrolide resistance of GAS isolates in southern Taiwan and to clarify the relationship of emm typing and macrolide resistance in the past decade. All GAS isolated from patients younger than 18 years at a single tertiary center in southern Taiwan were collected from 2000 to 2012. Antibiotics susceptibility to erythromycin, azithromycin, and clindamycin were determined by agar dilution method, and were interpreted by Clinical and Laboratory Standards Institute (CLSI) standards. emm typing was performed by polymerase chain reaction (PCR). A total of 301 isolates were collected during the period of 13 years. Scarlet fever (38.5%) and acute pharyngitis (32.2%) were the most common diagnosis. Decreased resistance rate of erythromycin from 53.1% in 2000 to 0% in 2010 was found, but it increased rapidly to 65% in 2011. The resistance rate of azithromycin was the lowest (4.2%) in 2005, but was higher than 15% after 2006. The involvement of the erythromycin resistance genes were mefA (53.1%), ermB (35.9%), and ermTR (10.9%). The resistance of clindamycin also increased since 2011. emm12 was the most common serotype and accounted for 44.9% of all isolates. Compared with the non-emm12 group, resistance to erythromycin, azithromycin, and clindamycin were more frequently detected in the emm12 group. Increased resistance of GAS to macrolide and clindamycin was found in recent years. emm12 was the main serotype for macrolide resistance.

Antimicrobial resistance determinants in antibiotic and biocide-resistant gram-negative bacteria from organic foods

In the present study, a collection of 29 Gram-negative bacteria selected from organic foods according to antibiotics and biocide resistance were screened for multidrug efflux pumps and specific antibiotic resistance genes. The acrB pump gene of the AcrAB–TolC system was detected in all isolates. EfrAB determinants efrA and efrB were frequently detected (Enterobacter, Pantoea, Salmonella), while other efflux pump genes (norE, mepA mdeA, norC) were detected only in some Enterobacter and Pantoea isolates. Genes encoding resistance to quaternary ammonium compounds qacE and qacJ were detected only in Enterobacter isolates, while sugE was detected in Enterobacter and in one Pantoea isolate. The aminoglycoside-resistance ant(4_)-Ia gene was detected in Enterobacter, Pantoea agglomerans and Klebsiella oxytoca. The antibiotic resistance determinant most frequently found was lsa gene (Enterobacter, Pantoea and Salmonella isolates). Erythromycin resistance genes (ereA, ereB) were detected among Enterobacter, Klebsiella or Salmonella. The macrolide efflux pump msrA and the phosphotransferase type I mphA genes were detected in Salmonella. Other resistance determinants analyzed (mdfA, yhiUV, evgA, 455emeA, yvcC, qacA/B, qacC, qacG, qacH, norA, norB, norC, sdrM, sepA, mecA, emrE, smr, bla, cat, aac(6_)-Ie-aph(2_)-Ia, aph(2_)-Ib, aph(2_)-Ic, aph(2_)-Id, aph(3_)-IIIa, ermA, ermB, ermC, and mefA genes) were not detected in any isolate. Results from the present study suggest that the main mechanisms of antibiotic and biocide resistance in Gram-negative bacteria from organic foods rely on different kinds of efflux pumps of broad substrate specificity. The indiscriminate use of biocides in the food chain should be revised in order to minimize persistence of reservoirs of antimicrobial resistance in the food chain.

High Prevalence of Erythromycin- and Tetracycline-Resistant Clinical Isolates of Streptococcus pneumoniae in Iran

Background With respect to clinical burden of pneumococcal infections and the role of erythromycin in the treatment of these infections, this study was performed to characterize phenotypic and genotypic patterns of erythromycin resistance and distribution of tetracycline resistance genes among clinical isolates of Streptococcus pneumoniae. Methods Clinical isolates of S. pneumoniae were collected from 15 hospital and clinical laboratories in Tehran for 1 year during 2011. Identification was confirmed by biochemical and molecular tests. Erythromycin- and tetracycline-resistant isolates were selected by standard disc diffusion method. Minimal inhibitory concentration was determined by E-test method for erythromycin. Erythromycin resistance phenotype was determined by double-disc test. Finally, 3 erythromycin resistance genes were detected by a multiplex polymerase chain reaction (PCR) for erm(A), erm(B), and mef(A/E), then 4 tetracycline resistance genes were detected by multiplex PCR with the specific primers for tet(M), tet(O), tet(K), and tet(L). Results A total of 88 pneumococcal isolates were collected from clinical samples. Fifty-two percent of the isolates were found to be resistant to erythromycin, and 62%, to tetracycline by disc diffusion method. E-test method showed that the minimal inhibitory concentration ranges of the isolates were from 2 up to greater than 256 μg/mL. Erythromycin-clindamycin double-disc test exhibited that 79% of resistant isolates were constitutive MLS (cMLS) phenotype, and the remaining isolates were M phenotype. erm(B)and mef(A/E) genes were detected in all cMLS and M phenotypes, respectively, by multiplex PCR. Six isolates were double positive for mef(A/E)and erm(B)genes. Eighty-five percent of erythromycin-resistant isolates were resistant to tetracycline as well, and only tet(M) genes were detected in these isolates. Conclusions To the best of our knowledge as a first report about the phenotypes and genotypes of erythromycin resistance in clinical pneumococci isolates in Iran, the high prevalence of erythromycin resistance with predominant cMLS phenotype together with coresistance to tetracycline can be alarming and need more attentions in Iran. It seems necessary to design a national multicenter study to characterize macrolide-resistant pneumococci in various geographical areas of Iran and determine if either these resistant isolates belong to the dominant serotypes and clones or not.

Nasopharyngeal carriage, serotype distribution and antimicrobial resistance of Streptococcus pneumoniae among children from Brazil before the introduction of the 10-valent conjugate vaccine

BackgroundStreptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide. Nasopharyngeal colonization plays an important role in the development and transmission of pneumococcal diseases, and infants and young children are considered to be the main reservoir of this pathogen. The aim of this study was to evaluate the rates and characteristics associated with nasopharyngeal carriage, the distribution of serotypes and the antimicrobial resistance profiles of Streptococcus pneumoniae among children in a large metropolitan area in Brazil before the introduction of the 10-valent pneumococcal conjugate vaccine.MethodsBetween March and June 2010, nasopharyngeal swabs were collected from 242 children aged <6 years attending one day care center and the emergency room of a pediatric hospital. Pneumococcal isolates were identified by conventional methods and serotypes were determined by a sequential multiplex PCR assay and/or the Quellung reaction. The antimicrobial susceptibilities of the pneumococci were assessed by the disk diffusion method. MICs for erythromycin and penicillin were also performed. Erythromycin resistance genes were investigated by PCR.ResultsThe overall colonization rate was 49.2% and it was considerably higher among children in the day care center. Pneumococcal carriage was more common among day care attenders and cohabitants with young siblings. The most prevalent serotypes were 6B, 19F, 6A, 14, 15C and 23F, which accounted for 61.2% of the isolates. All isolates were susceptible to clindamycin, levofloxacin, rifampicin and vancomycin. The highest rate of non-susceptibility was observed for sulphamethoxazole-trimethoprim (51.2%). Penicillin non-susceptible pneumococci (PNSP) accounted for 27.3% of the isolates (MICs of 0.12-4 μg/ml). Penicillin non-susceptibility was strongly associated with serotypes 14 and 23F. Hospital attendance and the presence of respiratory or general symptoms were frequently associated with PNSP carriage. The two erythromycin-resistant isolates (MICs of 2 and 4 μg/ml) belonged to serotype 6A, presented the M phenotype and harbored the mef(A/E) gene.ConclusionsCorrelations between serotypes, settings and penicillin non-susceptibility were observed. Serotypes coverage projected for the 10-valent pneumococcal conjugate vaccine was low (45.5%), but pointed out the potential reduction of PNSP nasopharyngeal colonization by nearly 20%.

Antimicrobial resistance and virulence traits of Enterococcus faecalis from primary endodontic infections

ObjectivesTo determine the phenotypic and molecular characteristics of Enterococcus faecalis recovered from primary endodontic infections in Brazilian patients. MethodsTwenty isolates of E. faecalis recovered from 43 Brazilian patients with primary endodontic infections were identified by biochemical profiling (API20Strep) and 16S rDNA sequencing. Antimicrobial susceptibility was ascertained by agar dilution, using the recommended protocol of the Clinical and Laboratory Standards Institute (CLSI). PCR with validated primers was used to detect genes associated with antibiotic resistance and specific virulence factors. ResultsAll isolates were deemed susceptible to penicillin G, erythromycin and vancomycin. However, nine isolates had a minimum inhibitory concentration of 4μg/mL to vancomycin (the resistance breakpoint). Fourteen isolates (70% of isolates) were also resistant to tetracycline with MICs of >64μg/mL. PCR products for tetracycline resistance genes were detected in test isolates, while erythromycin and vancomycin resistance genes were not evident. Gelatinase, aggregation substance and enteroccocal surface protein genes were detected in 20, 18 and 12 isolates, respectively. ConclusionsEndodontic E. faecalis isolates exhibit high level of resistance to tetracycline, an antibiotic that has use in local treatment of dental infections. This opens up a much-needed debate on the role and efficacy of this antibiotic for oral infections. Furthermore, these isolates were shown to possess genes that could contribute to pathogenicity in the pulp cavity.