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.     

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.     

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.     

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.     

Effects of tylosin use on erythromycin resistance in enterococci isolated from swine

The effect of tylosin on erythromycin-resistant enterococci was examined on three farms; farm A used tylosin for growth promotion, farm B used tylosin for treatment of disease, and farm C did not use tylosin for either growth promotion or disease treatment. A total of 1,187 enterococci were isolated from gestation, farrowing, suckling, nursery, and finishing swine from the farms. From a subset of those isolates (n = 662), 59% (124 out of 208), 28% (80 out of 281), and 2% (4 out of 170) were resistant to erythromycin (MIC >/= 8 microg/ml) from farms A, B, and C, respectively. PCR analysis and Southern blotting revealed that 95% (65 out of 68) of isolates chosen from all three farms for further study were positive for ermB, but all were negative for ermA and ermC. By using Southern blotting, ermB was localized to the chromosome in 56 of the isolates while 9 isolates from farms A and B contained ermB on two similar-sized plasmid bands (12 to 16 kb). Pulsed-field gel electrophoresis revealed that the isolates were genetically diverse and represented a heterogeneous population of enterococci. This study suggests that although there was resistance to a greater number of enterococcal isolates on a farm where tylosin was used as a growth promotant, resistant enterococci also existed on a farm where no antimicrobial agents were used.     

Clonality and Occurrence of Genes Encoding Antibiotic Resistance and Biofilm in Methicillin-Resistant Staphylococcus epidermidis Strains Isolated from Catheters and Bacteremia in Neutropenic Patients

Thirty methicillin-resistant Staphylococcus epidermidis strains isolated from catheters and blood cultures from neutropenic patients were studied. They were classified into 17 multidrug-resistance patterns. Polymerase cahin reaction analysis revealed that methicillin resistance was encoded by the mecA gene in all strains, and aminoglycosides resistance was due to aac(6')-Ie-aph(2')-Ia (23 strains), ant(4')-Ia (13), and aph(3')-IIIa (1) genes. The aac(6')-Ie-aph(2')-Ia gene was detected concomitantly with aph(3')-IIIa, and ant(4')-Ia genes in one and nine strains, respectively. Erythromycin resistance was encoded by the ermC (11 strains), ermA (6), and msrA (2) genes. The ermC gene was inducibly expressed in five strains, whereas the ermA was exclusively constitutively expressed. The icaA and icaC genes were detected in 19 strains; however, biofilm production was observed in only 16 strains. Most strains harbored multiple plasmids of variable sizes ranging from 2.2 to 70 kb, and two strains were plasmid-free. PFGE identified 15 distinct PFGE types, and five predominant genotypes were found. Our study showed the occurrence of complex genetic phenomenons. In unrelated strains, evidence of horizontal transfer of antibiotic-encoding genes and/or ica operon, and in indistinguishable strains, there is a quite good likelihood of independent steps of loss and/or gain of these genes. This genome dynamicity might have enhanced the invasiveness power of these methicillin-resistant S epidermidis strains.     

Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium isolates from hospitals in Tehran

The prevalence of glycopeptides, aminoglycosides and erythromycin resistance among Enterococcus faecalis and Enterococcus faecium was investigated. The susceptibility of 326 enterococcal hospital isolates to amikacin, kanamycin, netilmicin and tobramycin were determined using disk diffusion method. The minimum inhibitory concentration (MIC) of vancomycin, teicoplanin, gentamicin, streptomycin, and erythromycin were determined by microbroth dilution method. The genes encoding aminoglycoside modifying enzymes described as AMEs genes, erythromycin-resistant methylase (erm) and vancomycin-resistant were targeted by multiplex-PCR reaction. High level resistance (HLR) to gentamicin and streptomycin among enterococci isolates were 52% and 72% respectively. The most prevalent of AMEs genes were aac (6')-Ie aph (2") (63%) followed by aph (3')-IIIa (37%). The erythromycin resistance was 45% and 41% of isolates were positive for ermB gene. The ermA gene was found in 5% of isolates whereas the ermC gene was not detected in any isolates. The prevalence of vancomycin resistant enterococci (VRE) was 12% consisting of E. faecalis (6%) and E. faecium (22%) and all of them were VanA Phenotype. The results demonstrated that AMEs, erm and van genes are common in enterococci isolated in Tehran. Furthermore our results show an increase in the rate of vancomycin resistance among enterococci isolates in Iran.     

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.     

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.     

耐甲氧西林凝固酶阴性葡萄球菌中多种抗生素耐药基因的观察

探讨耐甲氧西林凝固酶阴性葡萄球菌(methicillin-resistant coagulase negative staphylococci,MRCNS)中多种抗生素耐药基因的分布情况,采取Kirby Bauer标准纸片扩散法(K-B纸片扩散法)对MRCNS开展药敏实验,并通过PCR检测菌种携带的抗生素耐药基因。药敏实验结果显示,96株MRCNS对庆大霉素、红霉素、莫匹罗星以及四环素的耐药性各有差异,但对氨苄西林的耐药性却达到100%,未发现对万古霉素具有耐药性的菌株;PCR检测结果显示,9种耐药基因merA、ermB、ermC、msrA、tetM、tetK、aac(6’)-aph(2")、aph(3’)-III、mupA的阳性检出率各有差异,没有扩增出vanA、ermA、vanB以及ant(6’)-I基因,但都扩增出mecA基因。结果表明,MRCNS可以同时携带多种抗生素耐药基因,属于一种多重耐药菌。     

Detection of the aminoglycosidestreptothricin resistance gene cluster ant(6)-sat4-aph(3 ')-III in commensal viridans group streptococci.

High-level aminoglycoside resistance was assessed in 190 commensal erythromycin-resistant alpha-hemolytic streptococcal strains. Of these, seven were also aminoglycoside-resistant: one Streptococcus mitis strain was resistant to high levels of kanamycin and carried the aph(3 ')-III gene, four S. mitis strains were resistant to high levels of streptomycin and lacked aminoglycoside-modifying enzymes, and two S. oralis strains that were resistant to high levels of kanamycin and streptomycin harbored both the aph(3 ')-III and the ant(6) genes. The two S. oralis strains also carried the ant(6)-sat4- aph(3 ' ')-III aminoglycoside-streptothricin resistance gene cluster, but it was not contained in a Tn5405-like structure. The presence of this resistance gene cluster in commensal streptococci suggests an exchange of resistance genes between these bacteria and enterococci or staphylococci.     

粪肠球菌对泰利霉素药物敏感性与 耐药基因erm的分布

目的了解粪肠球菌对泰利霉素和其他常用抗菌药物的耐药性,以及泰利霉素耐药与红霉素耐药相关基因ermA、ermB、ermC之间的关系。方法对本院2010-2016年从各种临床标本收集鉴定的320株粪肠球菌,用微量肉汤稀释法测定这些菌株对泰利霉素及8种临床常用抗菌药物的最小抑菌浓度,并用PCR法检测耐药基因ermA、ermB、ermC的分布。结果320株粪肠球菌对泰利霉素中介耐药26株,耐药138株,耐药率达51.3%;对红霉素耐药率达95.6%,泰利霉素抗粪肠球菌效果优于红霉素。对利奈唑胺、万古霉素、呋喃妥因和氨苄西林耐药率分别为15.6%、0.6%、2.2%和0.6%。共10株(3.1%)携带ermA基因,207株(64.7%)携带ermB基因,对泰利霉素中介组中有23株ermB基因阳性,耐药组有131株ermB基因阳性,仅1株(0.3%)ermC基因阳性,该菌同时携带ermB基因。结论粪肠球菌对泰利霉素已有较高耐药率。粪肠球菌对泰利霉素MIC值改变与ermB基因密切相关,与ermA、ermC基因无明显相关性。     

The correlation between phenotypes and genotypes of macrolides, lincosamides and streptogramins B resistance in Staphylococcus aureus

For 31 clinical strains of S. aureus the correlation between phenotype and genotype of resistance to macrolides, lincosamides and streptogramins B (MLSB) was established.. Phenotypes were determined on the basis of: susceptibility to erythromycin and clindamycin and the ability to an induction of the resistance (phenotypes S, susceptible; R , constitutive resistant, D, resistant after induction with erythromycin, D+, resistant after induction with erythromycin and with a presence of the small colonies inside inhibition zone between erythromycin and clindamycin discs), and on the basis of the resistance to spectinomycin (spR, resistant, spS, susceptible). Among examined S. aureus strains eight phenotypes of resistance to MLSB were recognized (the corresponding genotypes are given in brackets). Six phenotypes were typical: SspS (lack of MLS-B resistance genes), NEGspS (msrA/B, 1 strain), D+spS (ermCi, 4 strains),. DspR (ermAi, 11 strains and ermAi + msrA/B, 2 strains), RspR (ermAc, 4 strains and ermA + msrA/B,1 strain and ermA + ermC, 1 strain) and RspS (ermCc, 6 strains and ermB, 1 strain). Two rare phenotypes in two single strains were observed: SspR (ermAi, the strain with altered inducibility, inductor other than erythromycin) and DspS (ermAi, presumably mutation or lack of spc in Tn554).     

6类抗生素耐药基因在耐甲氧西林凝固酶阴性葡萄球菌中的分布及分析

目的了解6类抗生素耐药基因在耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）中的分布情况,为抗生素的合理使用及感染控制提供依据。方法应用K-B纸片扩散法对6种抗生素进行药敏试验,聚合酶链反应（PCR）对6类耐药基因进行检测。结果 MRCNS对氨苄西林、红霉素、四环素、庆大霉素和莫匹罗星的耐药率分别为100%、87.50%、30.21%、61.46%和27.08%,未检测出对万古霉素的耐药菌株;mecA、ermB、ermC、msrA、tetM、tetK、aac（6’）/aph（2＂）、aph（3’）-III和mupA耐药基因的阳性检出率分别为100.0%、10.42%、77.08%、46.86%、5.21%、2.08%、75.00%、39.58%和25.00%,未检测出ermA、ant（6’）-I、vanA和vanB耐药基因。结论 MRCNS能同时携带多种抗生素耐药基因,为多重耐药菌。     

Comparative analysis of staphylococcal plasmids carrying three streptogramin-resistance genes: vat-vgb-vga.

Several staphylococcal plasmids (26-45 kb) carry all three streptogramin-resistance (Sg(R)) genes, vat, vgb, and vga. Seven such plasmids harbored by independent strains belonging to three taxa (Staphylococcus aureus, S. simulans, and S. cohnii subsp. urealyticum) were compared and the deleted derivative of one of them, pIP680 (11.3 kb), carrying the three streptogramin-resistance genes was sequenced. The seven native plasmids had in common a 12.1-kb part cocarrying the three Sg(R) genes. Sequence analysis of pIP680 revealed that the simultaneous presence of these three genes has probably resulted from cointegration of two plasmids: (i) a pAMbeta1-like plasmid harboring vat-vgb and whose replication gene has been inactivated by an IS257 insertion and (ii) a functional vga plasmid whose replication is similar to that of two staphylococcal plasmids, pSX267 and pSK41.     

A Staphylococcus aureus plasmid that specifies constitutive macrolide-lincosamide-streptogramin B resistance contains a novel deletion in the ermC attenuator

A 2.5 kb plasmid, pA22, isolated from a naturally occurring S. aureus strain confers constitutive MLS-resistance. By restriction enzyme analysis, pA22 is indistinguishable from the S. aureus inducible MLS-resistance conferring plasmid, pT48, apart froma small deletion. DNA sequencing showed that the deletion, is in the leader/attenuator region of the ermC (MLS-resistance) gene and removes some of the complementary repeat regions required by the translational attenuation model in pT48 for inducible ermC expression. The deletion in plasmid pA22 is different from that found in similar 2.5kb constitutive MLS-resistance plasmids in other Gram-positive bacteria. It is suggested that plasmids conferring the constitutive phenotype have evolved from an inducible ancestor on several independent occasions.     

Plasmid profile and antibiotic resistance in coagulase-negative staphylococci isolated from polluted water

Four different species of coagulase-negative staphylococci (CNS) were isolated from polluted waters in Fez, Morocco and found to be Staphylococcus simulans, Staph. lenticus, Staph. hyicus and Staph. xylosus . Eight isolates belonging to these four species were analysed for their plasmid content. Southern blot hybridizations were performed to define the resistance determinants of the plasmids harboured by these species. These determinants were found to be carried mainly by Class I staphylococcal plasmids (1–5 kb). A plasmid (4·3 kb) carrying a tetracycline resistance gene was present in five isolates from all identified species. Plasmids carrying a chloramphenicol resistance gene were more frequently encountered and found to be of different sizes. Plasmids carrying erythromycin, neomycin, and streptomycin resistance genes were less frequent and were the same size. The results indicate that the occurrence of multi-resistant CNS in polluted waters may constitute a reservoir for disseminating antibiotic-resistance into the community.     

Dispersal of a plasmid-borne chloramphenicol resistance gene in streptococcal and enterococcal plasmids

Plasmids coding for chloramphenicol resistance, five isolated from streptococci of groups A, B, and G, ten from enterococci (Enterococcus faecalis, Enterococcus faecium), and two from staphylococci, were tested for sequence homology with the chloramphenicol resistance gene of pIP501, a 30-kb plasmid originally isolated from a group B Streptococcus. The 6.3-kb HindIII fragment of pIP501, known to carry the chloramphenicol resistance gene, was cloned into pBR322. A 1.6-kb portion of the cloned fragment, which included most of the chloramphenicol resistance gene, was used as probe in DNA-DNA hybridization experiments. Sequence homology was detected between the probe and four of the streptococcal, seven of the enterococcal, and one of the staphylococcal plasmids. The absence of hybridization between this probe and one plasmid isolated from a group B Streptococcus, as well as three isolated from E. faecalis, indicated that there are at least two different plasmid-borne chloramphenicol resistance determinants in the streptococci and in the enterococci.     

Molecular characterization of the erythromycin resistance plasmid pPV142 from Staphylococcus simulans

The 2.5-kb erythromycin resistance (Em^R) plasmid pPV142 of Staphylococcus simulans 13044 was isolated and characterized. Sequence analysis identified ORF1 and ORF2 encoding a 158-residue replication protein (Rep142) and a 244-residue erythromycin resistance protein (Erm, rRNA adenine N-6-methyltransferase), respectively. Structural analysis and Southern hybridization showed that the rep and ermM genes in pPV142 shared homology with the Em^R plasmid pPV141 (2.4 kb) of S. chromogenes 3688 and other Em^R plasmids known to exist in staphylococci and bacilli. Based on the presence of a 61-bp repeat upstream of the ermM gene, pPV142 is apparently a unique member of the pSN2 family of Em^R plasmids able to express erythromycin resistance constitutively.     

Cloning of erythromycin-resistance determinants and replication origins from indigenous plasmids of Lactobacillus reuteri for potential use in construction of cloning vectors.

Lactobacillus reuteri L1 and N16 strains contain a 7.0-kb plasmid (pTE80) and a 15-kb plasmid (pTE15), respectively, encoding resistance to erythromycin (Em(r)). Physical maps of both plasmids were established. Nucleotide sequences of the genetic determinants encoding Em(r) on pTE80 and pTE15 revealed the existence of a very similar (ca. 99% nucleotide sequence and ca. 98% amino acid sequence identity) open reading frame for an Em(r) transmethylase gene (erm) in both plasmids. These structural erm genes, 753 and 750 bp in length, respectively, were highly related (ca. 98% nucleotide sequence and ca. 97% amino acid sequence identity) to the erm gene of L. fermentum plasmid pLEM3. Sequence analysis showed that these two erm genes from pTE80 and pTE15 could be categorized under the ermB (ermAM) class. These are the first members of the ermB (ermAM) class of Em(r) determinant from L. reuteri to be characterized at the nucleotide sequence level. The Em(r) gene from pTE80 (erm80) was then ligated into pUC18/19 to construct replication origin (RO)-screening vectors pUE80(+) and pUE80(-) (pUE80(+/-)). These plasmids contain the pUC18/19-derived multiple cloning site, ampicillin-resistance trait, and the LacZ' gene, which enable direct screening for recombinants in Escherichia coli. Once the recombinant contains a RO from L. reuteri, the Em(r) trait of erm80 is used as a selection marker for the replication of the chimeric plasmid as it is transformed into L. reuteri using the cloned RO as a replicon. Replication regions from pTE80 and pTE15 were successfully cloned into the constructed vector pUE80(-). The RO cloned from pTE80 was further identified as being highly stable in L. reuteri and also bearing a relatively narrow host range compared with that of pTE15. The Em(r) determinant (erm80) and RO cloned from pTE80 could be used in the future construction of derivatives of cloning vectors for this microbe. Moreover, the pUE80(+/-) and pTE80-RO constructed in this study have the potential to be developed as a suicide vector and an E. coli-L. reuteri shuttle vector, respectively.