Resistance to macrolide, lincosamide and streptogramin antibiotics in staphylococci isolated in Istanbul, Turkey

The purpose of this study was to investigate the prevalence and genetic mechanisms of erythromycin resistance in staphylococci. A total of 102 erythromycin resistant non-duplicate clinical isolates of staphylococci [78 coagulase negative stapylococci (CNS), 24 Staphylococcus aureus] were collected between October 2003 and August 2004 in Istanbul Faculty of Medicine in Turkey. The majority of the isolates were from blood and urine specimens. Antimicrobial susceptibilities were determined by the agar dilution procedure and the resistance phenotypes by the double disk induction test. A multiplex PCR was performed, using primers specific for erm(A), erm(B), erm(C), and msrA genes. Among the 78 CNS isolates, 57.8% expressed the MLSB-constitutive, 20.6% the MLSB-inducible, and 21.6% the MSB phenotypes. By PCR, 78.2% of these isolates harbored the erm(C) gene, 8.9% erm(A), 6.4% erm(B), and 11.5% msrA genes. In S. aureus, the constitutive MLSB (58.3%) was more common than the inducible phenotype (20.8%). erm(A) was detected in 50% and erm(C) in 62.5% of the isolates, while 37.5% contained both erm(A) and erm(C). erm(C)-associated macrolide resistance was the most prevalent in CNS, while erm(C) and erm(A, C) was the most prevalent in S. aureus.     

Microarray analysis of erythromycin resistance determinants

AIMS: To develop a DNA microarray for analysis of genes encoding resistance determinants to erythromycin and the related macrolide, lincosamide and streptogramin B (MLS) compounds. METHODS AND RESULTS: We developed an oligonucleotide microarray containing seven oligonucleotide probes (oligoprobes) for each of the six genes (ermA, ermB, ermC, ereA, ereB and msrA/B) that account for more than 98% of MLS resistance in Staphylococcus aureus clinical isolates. The microarray was used to test reference and clinical S. aureus and Streptococcus pyrogenes strains. Target genes from clinical strains were amplified and fluorescently labelled using multiplex PCR target amplification. The microarray assay correctly identified the MLS resistance genes in the reference strains and clinical isolates of S. aureus, and the results were confirmed by direct DNA sequence analysis. Of 18 S. aureus clinical strains tested, 11 isolates carry MLS determinants. One gene (ermC) was found in all 11 clinical isolates tested, and two others, ermA and msrA/B, were found in five or more isolates. Indeed, eight (72%) of 11 clinical isolate strains contained two or three MLS resistance genes, in one of the three combinations (ermA with ermC, ermC with msrA/B, ermA with ermC and msrA/B). CONCLUSIONS: Oligonucleotide microarray can detect and identify the six MLS resistance determinants analysed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that microarray-based detection of microbial antibiotic resistance genes might be a useful tool for identifying antibiotic resistance determinants in a wide range of bacterial strains, given the high homology among microbial MLS resistance genes.     

Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

Multiplex PCR detection of the antibiotic resistance genes in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains isolated from auricular infections

Thirty-five Staphylococcus aureus strains from auricular infections were isolated. The identification of strains was confirmed by Api ID 32 Staph strips, the antibiotic susceptibility test was performed using ATB Staph kit. PCR assay was used to detect the oxacillin resistance gene (mecA) and the erythromycin genes (ermA, ermB, ermC, msrA and mef). The susceptibility profile of all strains revealed a low resistance level to oxacillin and erythromycin. The PCR results show that 60 % of the strains are mecA positive. The frequency of erythromycin genes was: ermA (+) 22.8 %, ermB (+) 45.7, ermC (+) 17.1, msrA (+) 28.6. The mef gene was not detected in any strain. No correlations between genotypic and phenotypic methods for the determination of oxacillin and erythromycin resistance was found. However, multiplex PCR technique was shown to be a fast, practical and economic technique for the detection of methicillin-and erythromycin-resistant staphylococci.     

The prevalence of genotypes that determine resistance to macrolides, lincosamides,and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, can be regarded as potential reservoirs of resistance genes for pathogenic strains, e.g., Staphylococcus aureus. The aim of this study was to assess the prevalence of different resistance phenotypes to macrolide, lincosamide, and streptogramins B (MLSB) antibiotics among erythromycin-resistant S. epidermidis, together with the evaluation of genes promoting the following different types of MLSB resistance:ermA, ermB, ermC,msrA, mphC, and linA/A’. Susceptibility to spiramycin was also examined. Among 75 erythromycin-resistantS. epidermidis isolates, the most frequent phenotypes were macrolides and streptogramins B (MSB) and constitutive MLSB (cMLSB). Moreover, all strains with the cMLSB phenotype and the majority of inducible MLSB (iMLSB) isolates were resistant to spiramycin, whereas strains with the MSB phenotype were sensitive to this antibiotic. The D-shape zone of inhibition around the clindamycin disc near the spiramycin disc was found for some spiramycin-resistant strains with the iMLSB phenotype, suggesting an induction of resistance to clindamycin by this 16-membered macrolide. The most frequently isolated gene was ermC, irrespective of the MLSB resistance phenotype, whereas the most often noted gene combination wasermC, mphC, linA/A’. The results obtained showed that the genes responsible for different mechanisms of MLSB resistance in S. epidermidis generally coexist, often without the phenotypic expression of each of them.     

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ) that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE), serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85% of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.     

Nasal carriage of methicillin resistant Staphylococcus aureus and their antibiotic susceptibility patterns in children attending day-care centers

Nasal colonization with community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) is being increasingly reported, especially in places where people are in close contact and in reduced hygiene, such as day-care centers. In this study we investigated the frequency of MRSA colonization and their antibiotic susceptibility patterns in 1-6 years old children of day-care centers in Hamadan, West of Iran.Five hundred nasal swabs were collected from children of 27 day-care centers that had no risk factors for colonization by S. aureus. The specimens were cultured for isolation of S. aureus by standard methods. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. For evaluation of the frequency of erythromycin induced clindamycin resistance, disk approximation test (D-test) was applied.Totally, 148 (29.6%) children were colonized by S. aureus. Out of 260 male, 94 (36.2%) and of 240 female, 54 (22.5%) cases were nasal carriers of S. aureus (P value = 0.001). Six (4.1%) of the 148 S. aureus isolated from children were MRSA strains. None of MRSA and methicillin susceptible S. aureus (MSSA) was resistant to vancomycin and clindamycin. Three of the 6 strains of MRSA and 7 (4.9%) of the 142 MSSA strains were resistant to erythromycin, and D-test was positive in all of them.We conclude that the rate of colonization by S. aureus is high in children attending day-care centers but colonization with MRSA is not common in our areas. Clindamycin or trimethoprim-sulfamethoxazol could be used in mild to moderataly severe diseases caused by CA-MRSA. However, if the CA-MRSA isolates are erythromycin resistant, D-test should be carried out for detection of inducible clindamycin resistance.     

The frequency of the occurrence of genes ermA, ermB, ermC and msrA/B among methicillin-resistant Staphylococcus aureus strains resistant to erythromycin

The group of 50 clinical MRSA strains resistant to MLS-B was examined for the presence of ermA, ermB, ermC, msrA/B genes by using PCR. Gene ermA was found in 43 strains (86%). 20 of ermA strains demonstrated inducible whereas 23 constitutive type of expression. The gene ermC was present in 15 of examined MRSA strains (30%). The expression of the gene was inducible in the case of 9 and constitutive in the case of 6 of the strains. The msrA/B gene was present in the case of 5 strains (10%). The ermB gene was not detected among the investigated strains.     

High rates of macrolide resistance among clinical isolates of Streptococcus agalactiae in Tunisia

One hundred sixty non duplicate erythromycin resistant Streptococcus agalactiae isolates were collected in Tunisia from January 2005 to December 2007 They were investigated to determine their resistance level to different macrolides and the mechanisms involved. Most erythromycin resistant S. agalactiae isolates were isolated from urinary specimens (38.75%, 62/160). The constitutive MLSB phenotype (cMLS) showed in 84.3% (135/160) with high MICs of macrolides and lincosamides (MIC90>256 microg/mL) and 8.2% (13/160) inducible MLSB phenotype (iMLS) with high MICs of macrolides (MIC90>256 microg/mL) and moderately increased MICs of lincosamides (MIC90=8 microg/mL). The M phenotype showed in 7.5% (12/160) with moderately increased MICs of macrolides (MIC90=32 microg/mL) and low MICs of lincosamides (MIC90=0.75 microg/mL). All strains were susceptible to quinupristun-dalfopristin association and linezolid (MIC90: 05 and 0.38 microg/mL respectively). Strains with MLSB phenotype harboured erm(B) gene with 825% (n=132), erm(TR) gene with 8.12% (n=13) and erm(B) plus mef (A) with 1.88% (n=3). All strains categorized as M phenotype carried the mef(A) gene (75%, n=12). cMLSB phenotype conferring cross resistance to macrolides, lincosamides and streptogramins B with high level of resistance was the most prevalent.     

Genetic basis of macrolide and lincosamide resistance in Brachyspira (Serpulina) hyodysenteriae

Macrolide antibiotic resistance is widespread among Brachyspira hyodysenteriae (formerly Serpulina hyodysenteriae) isolates. The genetic basis of macrolide and lincosamide resistance in B. hyodysenteriae was elucidated. Resistance to tylosin, erythromycin and clindamycin in B. hyodysenteriae was associated with an A-->T transversion mutation in the nucleotide position homologous with position 2058 of the Escherichia coli 23S rRNA gene. The nucleotide sequences of the peptidyl transferase region of the 23S rDNA from seven macrolide and lincosamide resistant and seven susceptible strains of Brachyspira spp. were determined. None of the susceptible strains were mutated whereas all the resistant strains had a mutation in position 2058. Susceptible strains became resistant in vitro after subculturing on agar containing 4 micrograms ml-1 of tylosin. Sequencing of these strains revealed an A-->G transition mutation in position 2058.     

Antimicrobial resistance profiles and genetic characterisation of macrolide resistant isolates of Streptococcus agalactiae

In this study, 100 clinical isolates of Streptococcus agalactiae recovered from genitourinary tract specimens of non-pregnant individuals living in Rio de Janeiro were submitted for antimicrobial susceptibility testing, detection of macrolide resistance genes and evaluation of the genetic diversity of erythromycin-resistant isolates. By agar diffusion method, all isolates were susceptible to ceftazidime, penicillin and vancomycin. Isolates were resistant to levofloxacin (1%), clindamycin (5%), erythromycin (11%) and tetracycline (83%) and were intermediated to erythromycin (4%) and tetracycline (6%). Erythromycin-resistant and intermediated isolates presented the following phenotypes: M (n = 3), constitutive macrolide-lincosamide-streptogramin B (MLS B, n = 5) and inductive MLS B (n = 7). Determinants of macrolide resistance genes, erm and mef, were detected in isolates presenting MLS B and M phenotypes, respectively. Randomly amplified polymorphic DNA profiles of erythromycin-resistant isolates were clustered into two major groups of similarity.     

Staphylococcus aureus nosocomial infections: the role of a rapid and low-cost characterization for the establishment of a surveillance system

Continuous surveillance on resistance patterns and characterization of Staphylococcus aureus represent simple and low-cost techniques to understand and evaluate the effectiveness of infection control and antimicrobial prescribing measures. In this study we analyzed the antibiotic susceptibility and trends for S. aureus strains collected from bacteraemia cases in a five year period. Between 2004 and 2008 we noted a progressive decrease in the number of S. aureus isolates compared to all pathogens from clinical specimens and S. aureus bloodstream infections (BSI) reflected a similar trend. In particular we analyzed 185 isolates from blood cultures: 89 isolates were MSSA and 96 isolates were MRSA. Molecular SCCmec typing of these strains showed an absolute prevalence of types I and II, whereas five spa types from 96 isolates were obtained. Resistance pattern analysis allowed us to place MRSA strains into 12 antibiotypes and the major antibiotype was resistant to penicillin, gentamicin, erythromycin, clindamycin and ciprofloxacin. The predominant antibiotype among the MSSA isolates was resistant only to penicillin. In addition, 19.1% of MSSA are susceptible to all antibiotics tested. We also found a close association between antibiotyping 1 and genotyping t002/SCCmecI of MRSA strains, suggesting a nosocomial scenario dominated by a few particular clones.     

长沙地区临床分离金黄色葡萄球菌的耐药监测

目的了解长沙地区临床分离金黄色葡萄球菌（以下简称金葡菌）对常用抗菌药物的耐药现状,探讨金黄色葡萄球菌对甲氧西林的耐药水平。方法收集长沙地区11家医院2009年11月至2010年11月临床分离的非重复金葡菌279株,应用Vitek-2全自动微生物分析系统进行鉴定,K-B法检测金葡菌对24种药物的敏感性,产色头孢菌素试验检测β-内酰胺酶以及D试验检测诱导型克林霉素耐药。应用头孢西丁和苯唑西林纸片扩散法筛查耐甲氧西林的金葡菌（MRSA）,琼脂稀释法检测头孢西丁和苯唑西林的最低抑菌浓度（MIC）。结果在被检测的24种药物中,敏感率〉50%的药物为9种,未发现对万古霉素、替考拉宁和利奈唑胺耐药菌株;耐药率〉50%的抗菌药物有11种,其中以青霉素和氨苄西林的耐药率最高（均为97.1%）。MRSA的分离率达54.5%,且对常用的16种抗菌药物的耐药率均显著高于甲氧西林敏感金黄色葡萄球菌（MSSA）。279株金葡菌中,β-内酰胺酶阳性250株（89.6%）;红霉素耐药而克林霉素敏感或中介的30株中,D试验阳性22株（73.3%）。苯唑西林（OXA）和头孢西丁（FOX）MIC范围分别为0.125～〉256μg/mL和2～〉256μg/mL,苯唑西林的MIC50和MIC90分别为128μg/mL和256μg/mL,头孢西丁的MIC50和MIC90分别为64μg/mL和256μg/mL。结论长沙地区临床分离金葡菌对常用抗菌药物呈多重耐药;MRSA不仅分离率高,而且对甲氧西林呈高水平耐药。     

Characteristics of macrolide-resistant Mycoplasma pneumoniae strains isolated from patients and induced with erythromycin in vitro

Some patients with Mycoplasma pneumoniae infection are clinically resistant to antibiotics such as erythromycin, clarithromycin, or clindamycin. We isolated M. pneumoniae from such patients and found that one of three isolates showed a point mutation in the 23S rRNA gene. Furthermore, 141 EM-sensitive clinical isolates of M. pneumoniae were cultured in broth medium containing 100 microg/ml of erythromycin (EM). Among 11 EM-resistant strains that grew in the medium, point mutations in the 23S rRNA were found in 3 strains at A2063G, 5 strains at A2064G and 3 strains at A2064C. The relationship between the point mutation pattern of these EM-resistant strains and their resistance phenotypes to several macrolide antibiotics was investigated.     

Characterization and Transfer of Antibiotic Resistance in Lactic Acid Bacteria from Fermented Food Products

The study provides phenotypic and molecular analyses of the antibiotic resistance in lactic acid bacteria (LAB) from fermented foods in Xi'an, China. LAB strains (n = 84) belonging to 16 species of Lactobacillus (n = 73), and Streptococcus thermophilus (n = 11) were isolated and identified by sequencing their 16S rRNA gene. All strains were susceptible to ampicillin, bacitracin, and cefsulodin, and intrinsically resistant to nalidixic acid, kanamycin, and vancomycin (except L. bulgaricus, L. acidophilus, and S. thermophilus, which were susceptible to vancomycin). Some strains had acquired resistance for penicillin (n = 2), erythromycin (n = 9), clindamycin (n = 5), and tetracycline (n = 14), while resistance to gentamycin, ciprofloxacin, streptomycin, and chloramphenicol was species dependent. Minimum inhibitory concentrations presented in this study will help to review microbiological breakpoints for some of the species of Lactobacillus. The erm(B) gene was detected from two strains of each of L. fermentum and L. vaginalis, and one strain of each of L. plantarum, L. salivarius, L. acidophilus, L. animalis, and S. thermophilus. The tet genes were identified from 12 strains of lactobacilli from traditional foods. This is the first time, the authors identified tet(S) gene from L. brevis and L. kefiri. The erm(B) gene from L. fermentum NWL24 and L. salivarius NWL33, and tet(M) gene from L. plantarum NWL22 and L. brevis NWL59 were successfully transferred to Enterococcus faecalis 181 by filter mating. It was concluded that acquired antibiotic resistance is well dispersed in fermented food products in Xi'an, China and its transferability to other genera should be monitored closely.     

Changes in antibiotic sensitivity in strains of Staphylococcus aureus, 1952-78.

Two hundred strains of Staphylococcus aureus isolated from outpatients with infections of the skin and subcutaneous tissues were tested for sensitivity to penicillin, erythromycin, tetracycline, sodium fusidate, methicillin, clindamycin, chloramphenicol, and gentamicin. One hundred and sixty-three (81.5%) of the strains were resistant to penicillin and 16 (8%) resistant to tetracycline. Incidence of resistance to other antibiotics was low. No strain was resistant to chloramphenicol, gentamicin, or methicillin. When compared with results of earlier studies, there was an increase in the incidence of resistance to penicillin and tetracycline, but no appreciable increase in resistance to other antibiotics.     

Pneumococci resistant to erythromycin.

Susceptibility to erythromycin was determined for all pneumococci isolated in one laboratory from clinical specimens between 1969 and 1977. All 4724 isolates examined prior to October 1973 were susceptible to erythromycin. From October 1973 to December 1977, 64 (0.71%) of 8995 pneumococcus isolates were resistant to erythromycin. The resistant strains were isolated from 38 patients living in six widely separated communities in Alberta. The erythromycin-resistant strains were of nine capsular types, including six that often cause bacteremic disease and five for which resistance to erythromycin has not been reported hitherto. Certain strains of type 33 and of type 15 were highly resistant, the minimum inhibitory concentration (MIC) of erythromycin being 2000 microgram/mL; these strains were also highly resistant to lincomycin and clindamycin. Resistance in strains of other types was much lower, the MIC of erythromycin being 0.6 to 20 microgram/mL, and all but one of these strains were susceptible to lincomycin and clindamycin. All the erythromycin-resistant pneumococci were suspectible to penicillin.     

Serotypes and Patterns of Antibiotic Resistance in Strains Causing Invasive Pneumococcal Disease in Children Less than 5 Years of Age

Objective

The serotypes and patterns of antibiotic resistance of Streptococcus pneumoniae (S. pneumoniae) strains that cause invasive pneumococcal disease (IPD) in infants were analyzed to provide guidance for clinical disease prevention and treatment.

Methods

The clinical features of confirmed IPD were evaluated in 61 patients, less than 5 years of age, who were admitted to our hospital between January 2009 and December 2011. The serotypes and antibiotic resistance of strains of S.pneumoniae were determined using the capsular swelling method and the E-test.

Results

A total of 61 invasive strains were isolated. The serotype distribution of those isolates were 19A (41.0%), 14 (19.7%), 19F (11.5%), 23F (9.8%), 8 (4.9%), 9V (4.9%), 1 (3.3%), and 4, 6B, and 20 (each 1.6%). The percentage of S. pneumoniae strains resistant to erythromycin, clindamycin, and cotrimoxazole were 100%, 86.9%, and 100%, respectively. The percentage of S. pneumoniae strains resistant to penicillin, amoxicillin/clavulanic acid, cefuroxime, ceftriaxone, cefotaxime, cefepime, and meropenem were 42.6%, 18.0%, 82.0%, 18.0%, 13.1%, 13.1%, and 36.1%, respectively. The percentage of multidrug-resistant strains was 95.6%. Strains of all serotypes isolated in this study were highly resistant to erythromycin, cotrimoxazole, and clindamycin. Strains with serotype 19A had the highest rates of resistance.

Conclusions

Serotype 19A strains were most frequently isolated from children with IPD treated in our hospital. The strains causing IPD are highly resistant to antibiotics.