Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae

The mef(A) gene from a clinical isolate of Streptococcus pneumoniae exhibiting the M-type resistance to macrolides was found to be part of the 7,244-bp chromosomal element Tn1207.1, which contained 8 open reading frames. orf2 encodes a resolvase/invertase, and orf5 is a homolog of the macrolide-streptogramin B resistance gene msr(SA).     

Clindamycin resistance among erythromycin-resistant Streptococcus pneumoniae

The increasing proportion of Streptococcus pneumoniae isolates with reduced susceptibility to penicillin has created an urgent need for therapeutic alternatives to some β-lactam agents. Clindamycin is an antimicrobial agent with excellent bioavailability after oral administration which has been considered for the therapy of community-acquired pneumococcal otitis media. Using the Etest methodology, we have studied the in vitro susceptibility of 59 erythromycin-resistant strains of S. pnuemoniae to clindamycin, penicillin, trimethoprim-sulfamethoxazole, and rifampin. The study also addressed the impact of the susceptibility test medium [mueller-Hinton (MH) vs IsoSensitest (Iso), both 5% blood supplement] on the results. A total of 20 isolates (37%) displayed constitutive clindamycin resistance on Iso blood agar, compared with only 11 (22%) on MH blood agar. The remaining nine strains found to be clindamycin susceptible on MH manifested resistance only with erythromycin induction. Resistance to penicillin, rifampin, and trimethoprim-sulfamethoxazole in erythromycin-resistant isolates was 83%, 2%, and 85%–89% (medium dependent), respectively. These results indicate that the choice of susceptibility test medium affects the expression (constitutive or inducible) of macrolide-lincosamide-streptogramin (MLS) resistance in S. pneumoniae. In addition, the common assumption that erythromycin resistance in S. pneumoniae implies clindamycin resistance may need to be reconsidered and routine susceptibility tests (including induction if MH medium is used) should be considered for MLS-class drugs.     

Erythromycin resistance and genetic elements carrying macrolide efflux genes in Streptococcus agalactiae

The macrolide resistance determinants and genetic elements carrying the mef(A) and mef(E) subclasses of the mef gene were studied with Streptococcus agalactiae isolated in 2003 and 2004 from 7,084 vaginorectal cultures performed to detect carrier pregnant women. The prevalence of carriage was 18% (1,276 isolates), and that of erythromycin resistance 11.0% (129 of the 1,171 isolates studied). erm(B), erm(A) subclass erm(TR), and the mef gene, either subclass mef(A) or mef(E), were found in 72 (55.8%), 41 (31.8%), and 12 (9.3%) erythromycin-resistant isolates, while 4 isolates had more than 1 erythromycin resistance gene. Of the 13 M-phenotype mef-containing erythromycin-resistant S. agalactiae isolates, 11 had the mef(E) subclass gene alone, one had both the mef(E) and the erm(TR) subclass genes, and one had the mef(A) subclass gene. mef(E) subclass genes were associated with the carrying element mega in 10 of the 12 mef(E)-containing strains, while the single mef(A) subclass gene found was associated with the genetic element Tn1207.3. The nonconjugative nature of the mega element and the clonal diversity of mef(E)-containing strains determined by pulsed-field gel electrophoresis suggest that transformation is the main mechanism through which this resistance gene is acquired.     

mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae.

Recently, it was shown that a significant number of erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains contain a determinant that mediates resistance via a putative efflux pump. The gene encoding the erythromycin-resistant determinant was cloned and sequenced from three strains of S. pneumoniae bearing the M phenotype (macrolide resistant but clindamycin and streptogramin B susceptible). The DNA sequences of mefE were nearly identical, with only 2-nucleotide differences between genes from any two strains. When the mefE sequences were compared to the mefA sequence from S. pyogenes, the two genes were found to be closely related (90% identity). Strains of S. pneumoniae were constructed to confirm that mefE is necessary to confer erythromycin resistance and to explore the substrate specificity of the pump; no substrates other than 14- and 15-membered macrolides were identified.     

肺炎链球菌对大环内酯类抗生素耐药情况及耐药基因研究

目的调查上海地区肺炎链球菌对红霉素的敏感度，研究肺炎链球菌对大环内酯类抗生素耐药机制。方法对中山医院57株临床分离肺炎链球菌进行红霉素药敏试验；应用聚合酶链反应（PCR）技术对上海4所医院中分离的53株红霉素耐药肺炎链球菌检测耐药基因（ermB，mefA，merE）。结果57株肺炎链球菌中12株（21．0％）敏感，3株（5．3％）中介，42株（73．7％）耐药。53株红霉素耐药肺炎链球菌中，ermB基因、mere基因、mefA基因分别在51株（96．2％）、22株（41．5％）和1株（1．9％）中检测到。其中21株（39．6％）同时检测到ermB基因和mefE基因，1株（1．9％）同时检测到ermB基因和mefA基因，1株（1．9％）未检测到ermB基因、mefE基因或mefA基因。结论上海地区肺炎链球菌对大环内酯类抗生素耐药率较高。ErmB介导的靶位改变是最常见的耐药机制，mef（特别是mefE）介导外排机制引起者也较常见。     

Detection of macrolide resistance in Streptococcus pneumoniae

We determined the susceptibilities of recent clinical isolates of Streptococcus pneumoniae to 19 antibiotics. The frequency of erythromycin nonsusceptibility was high, i.e. 8/13 (61.5%), 10/14 (71.4%) and 11/11 isolates (100%) from 13 penicillin-susceptible, 14 penicillin-intermediate and 11 penicillin-resistant S. pneumoniae, respectively. Macrolide resistance was detected by polymerase chain reaction (PCR) and disk diffusion methods. Of these erythromycin-nonsusceptible pneumococcal isolates, 13/29 (44.8%) isolates contained genomic copies of MEFA and showed non-'D'-shaped zones of inhibition observed around rokitamycin and/or clindamycin disks. Sixteen out of 29 isolates (55.2%) contained copies of ERMB and showed typical 'D'-shaped zones of inhibition, except one isolate. Although the macrolide resistance determinants, MEFA and ERMB, could be identified by PCR and disk diffusion methods, PCR methods were more reliable in elucidating these determinants. The susceptibility pattern to 14-, 15- and 16-membered macrolides and clindamycin differed between the MEFA+ and ERMB+ isolates. All isolates were susceptible to levofloxacin, sparfloxacin and vancomycin. The MICs of sitafloxacin were lowest among the fluoroquinolones examined for 38 isolates.     

Beta-lactam and macrolide resistance in Streptococcus pneumoniae

The resistance of beta-lactam antibiotics in penicillin-resistant Streptococcus pneumoniae (PRSP) is due to alterations in penicillin-binding proteins(PBPs). Recently, highly beta-lactam-resistant strains(MIC: > or = 4 micrograms/ml for penicillin, > or = 8 micrograms/ml for ampicillin, and > or = 4 micrograms/ml for cefotaxime) have been isolated. High resistance of these strains is caused by further alterations in pbp2x and pbp2b genes adjacent to conserved amino acid motifs, in addition to that detected in common PRSP. The resistant mechanisms of macrolides in S. pneumoniae is recognized as production of a 23s rRNA methylase encoded by ermB and efflux system mediated by mefA gene. We detected these genes by PCR in clinical isolates, and showed the relationship between the presence of each gene and the susceptibilities of 14-, 15-, 16-membered macrolides.     

A novel efflux system in inducibly erythromycin-resistant strains of Streptococcus pyogenes

Streptococcus pyogenes strains inducibly resistant (iMLS phenotype) to macrolide, lincosamide, and streptogramin B (MLS) antibiotics can be subdivided into three phenotypes: iMLS-A, iMLS-B, and iMLS-C. This study focused on inducibly erythromycin-resistant S. pyogenes strains of the iMLS-B and iMLS-C types, which are very similar and virtually indistinguishable in a number of phenotypic and genotypic features but differ clearly in their degree of resistance to MLS antibiotics (high in the iMLS-B type and low in the iMLS-C type). As expected, the iMLS-B and iMLS-C test strains had the erm(A) methylase gene; the iMLS-A and the constitutively resistant (cMLS) isolates had the erm(B) methylase gene; and a control M isolate had the mef(A) efflux gene. mre(A) and msr(A), i.e., other macrolide efflux genes described in gram-positive cocci, were not detected in any test strain. With a radiolabeled erythromycin method for determination of the intracellular accumulation of the drug in the absence or presence of an efflux pump inhibitor, active efflux of erythromycin was observed in the iMLS-B isolates as well as in the M isolate, whereas no efflux was demonstrated in the iMLS-C isolates. By the triple-disk (erythromycin plus clindamycin and josamycin) test, performed both in normal test medium and in the same medium supplemented with the efflux pump inhibitor, under the latter conditions iMLS-B and iMLS-C strains were no longer distinguishable, all exhibiting an iMLS-C phenotype. In conjugation experiments with an iMLS-B isolate as the donor and a Rif(r) Fus(r) derivative of an iMLS-C isolate as the recipient, transconjugants which shared the iMLS-B type of the donor under all respects, including the presence of an efflux pump, were obtained. These results indicate the existence of a novel, transferable efflux system, not associated with mef(A) or with other known macrolide efflux genes, that is peculiar to iMLS-B strains. Whereas the low-level resistance of iMLS-C strains to MLS antibiotics is apparently due to erm(A)-encoded methylase activity, the high-level resistance of iMLS-B strains appears to depend on the same methylase activity plus the new efflux system.     

肺炎链球菌对大环内酯类抗生素耐药的研究进展

肺炎链球菌是儿童社区获得性感染的主要病原菌。作为一种条件致病菌,它常定植于鼻咽部黏膜,3岁以内的婴幼儿携带率最高。随着机体免疫力下降,有致病力的肺炎链球菌乘机侵入,引起儿童中耳炎、肺炎、脑膜炎和菌血症等疾病。据估计,发展中国家每年约有814000名5岁以下的儿童死于肺炎链球菌感染。20世纪90年代以来,由于肺炎链球菌对8内酰胺类抗生素,主要是青霉素的耐药逐年增加,再加上大环内酯类抗生素对非典型病原体也有效,因此大环内酯类抗生素被广泛应用于治疗肺炎链球菌感染的疾病。     

Novel mechanism of macrolide resistance in Streptococcus pneumoniae

The mechanism of macrolide resistance was examined in 73 clinical isolates of Streptococcus pneumoniae. Two distinct resistance phenotypes were observed: high-level macrolides-lincosamides-streptogramin B (MLS) resistance and low-level macrolide resistance with lincosamide susceptibility. High-level MLS resistance was associated with the presence of ermAM. Strains with the low-level resistant phenotype (novel) were negative for ermA, ermC, ermAM, ereA, ereB and msrA by polymerase chain reaction (PCR) amplification with genespecific primers. Ribosomes isolated from novel strains bound the same amount of [¹⁴C]-erythromycin as ribosomes from sensitive strains. These novel strains also did not inactivate the macrolide. The novel mechanism was found in 41% of the erythromycin resistant S. pneumoniae examined.     

肺炎链球菌对大环内酯类抗生素耐药的研究进展

肺炎链球菌是社区获得性肺炎（CAP）的重要病原菌。大环内酯类抗生素对敏感的革兰阳性菌有效，且对非典型病原体也有较好疗效，被广泛用于治疗CAP和其他呼吸道感染的经验性治疗。肺炎链球菌对大环内酯类抗生素耐药问题也随之成为全球性问题，近10年来逐年加重，以亚洲国家（地区）的肺炎链球菌对大环内酯类抗生素耐药最为严重。本文就肺炎链球菌对大环内酯类抗生素耐药现状、     

Prevalence and phenotypes of erythromycin-resistant Streptococcus pneumoniae in Shanghai, China

Bacterial resistance of Streptococcus pneumoniae against erythromycin and clindamycin and resistance phenotypes of erythromycin-resistant Streptococcus pneumoniae were investigated. The MICs of erythromycin and clindamycin against 345 strains of Streptococcus pneumoniae were tested with agar dilution method; the phenotypes of erythromycin-resistant Streptococcus pneumoniae were detected by double-disk test. One hundred and eighty-three and 171 of 345 (53.0% and 49.6%) of isolates had MICs > or =1 microg/ml for erythromycin and for clindamycin. Among erythromycin-resistant Streptococcus pneumoniae, the percentage of cMLS, iMLS and M phenotype was 90.3% (159/176), 5.7% (10/176) and 4.0% (7/176), respectively. The incidence of erythromycin-resistant Streptococcus pneumoniae is very high in Shanghai. The main phenotype is cMLS.     

肺炎链球菌对大环内酯类抗生素耐药机制研究

目的了解肺炎链球菌对大环内酯类抗生素的耐药机制和转座子整合酶的流行情况。方法188株红霉素耐药肺炎链球菌，用E试验和K—B纸片扩散法检测其对11种抗菌药物的敏感性；用双纸片法（红霉素和克林霉素）确定其耐药表型；用PCR扩增这些菌株的耐药基因ermB、mefa、mefE、tetM及转座子整合酶基因intTn。结果188株红霉素耐药株中耐药基因ermB总检出率为91．5％（172／188），mefE总检出率为38．3％，未检出mefA基因。97．9Yoo的红霉素耐药株中存在转座子整合酶intTn。耐药基因组合ermB（＋）mefE（-）和ermB（＋）mef（＋），占91．5％，两者均呈cMLSB耐药表型。ermB（-）mefE（＋）占8．5％，耐药表型为M型。结论我院分离的肺炎链球菌大环内酯耐药以errnB介导的cMLS。耐药表型为主。转座子可能在本地区肺炎链球菌耐药基因的水平转移和克隆播散中起重要作用。     

Antimicrobial activity and spectrum of sparfloxacin tested against erythromycin-resistant Streptococcus pneumoniae isolates

Recognition of a worldwide increase of penicillin-resistant Streptococcus pneumoniae and cross-resistance to other classes of antimicrobials have placed a great urgency on the need for new antimicrobial agents. Sparfloxacin, a novel pyridone carboxylic acid fluoroquinolone derivative was evaluated and compared to six other compounds for antimicrobial activity against erythromycin-resistant pneumococci (50 strains). The Etest susceptibility testing method was used to inoculate Mueller-Hinton agar supplemented with 5% sheep blood. There was extensive cross-resistance between erythromycin, clarithromycin (94%), and azithromycin (100%), but no cross-resistance was detected between macrolides/azalides and sparfloxacin (all strains susceptible at ⩽1.0 μg/ml). Sparfloxacin (MIC₉₀, 1 μg/ml) was four-fold more active than ciprofloxacin and ofloxacin (MIC₉₀, 4 μg/ml). Sparfloxacin appears to possess excellent in vitro activity against erythromycin-resistant S. pneumoniae that were often highly resistant to beta-lactams, and further studies are recommended to investigate its in vivo efficacy against these multi-resistant organisms.     

Phenotypic and molecular characterization of tetracycline- and erythromycin-resistant strains of Streptococcus pneumoniae

Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and resistant to both tetracycline (MIC, >or=8 microg/ml) and erythromycin (MIC, >or=1 microg/ml), were investigated. Of these strains, 11% were penicillin resistant and 23% were penicillin intermediate. With the use of the erythromycin-clindamycin-rokitamycin triple-disk test, 14 strains were assigned to the constitutive (cMLS) phenotype of macrolide resistance, 44 were assigned to the partially inducible (iMcLS) phenotype, 1 was assigned to the inducible (iMLS) phenotype, and 6 were assigned to the efflux-mediated (M) phenotype. In PCR assays, 64 of the 65 strains were positive for the tetracycline resistance gene tet(M), the exception being the one M isolate susceptible to kanamycin, whereas tet(K), tet(L), and tet(O) were never found. All cMLS, iMcLS, and iMLS isolates had the erythromycin resistance gene erm(B), and all M phenotype isolates had the mef(A) or mef(E) gene. No isolate had the erm(A) gene. The int-Tn gene, encoding the integrase of the Tn916-Tn1545 family of conjugative transposons, was detected in 62 of the 65 test strains. Typing assays showed the strains to be to a great extent unrelated. Of 16 different serotypes detected, the most numerous were 23F (n = 13), 19A (n = 10), 19F (n = 9), 6B (n = 8), and 14 (n = 6). Of 49 different pulsed-field gel electrophoresis types identified, the majority (n = 39) were represented by a single isolate, while the most numerous type included five isolates. By high-resolution restriction analysis of PCR amplicons with four endonucleases, the tet(M) loci from the 64 tet(M)-positive pneumococci were classified into seven distinct restriction types. Overall, a Tn1545-like transposon could reasonably account for tetracycline and erythromycin resistance in the vast majority of the pneumococci of cMLS, iMcLS, and iMLS phenotypes, whereas a Tn916-like transposon could account for tetracycline resistance in most M phenotype strains.     

肺炎链球菌大环内酯类抗生素的耐药性分析

目的 本研究通过对肺炎链球菌耐药表型检测分析,掌握本地区肺炎链球菌耐药的现状和趋势.方法 本文用E试验和K-B纸片扩散法检测84株肺炎链球菌临床分离株对9种抗生素的敏感性;用双纸片法确定大环内酯类耐药表型.结果 84株肺炎链球菌红霉素耐药占85.7%(72/84),对青霉素不敏感率达57.1%(48/84).左氧氟沙星、阿莫西林/克拉维酸对该组细菌有较好的体外活性,敏感率分别为83.3%(70/84)和88.1%(74/84).结论 肺炎链球菌对大环内酯耐药严重,且表现对四环素、复方磺胺甲噁唑、青霉素等多重耐药;南昌地区大环内酯类耐药表型主要以cMLSB为主.     

In vitro activities of tigecycline against erythromycin-resistant Streptococcus pyogenes and Streptococcus agalactiae: mechanisms of macrolide and tetracycline resistance

The activity of tigecycline was tested against erythromycin-resistant streptococci (107 Streptococcus pyogenes and 98 Streptococcus agalactiae strains). The presence of erythromycin and tetracycline resistance genes was determined by PCR. Among S. pyogenes strains the most prevalent gene was mef(A) (91.6%). The erm(B) gene was the most prevalent (65.3%) among S. agalactiae strains. Tigecycline proved to be very active against all the isolates tested (MIC at which 90% of the isolates tested were inhibited, 0.06 micro g/ml), including those resistant to tetracycline.