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).     

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 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.     

Detection and characterization of erythromycin-resistant methylase genes in Gram-positive bacteria isolated from poultry litter

The epidemiology of four erythromycin-resistant methylase ( erm) genes, ermA, ermB, ermC and msrA, was determined in erythromycin-resistant staphylococci, enterococci and streptococci isolated from poultry litter. All isolates were resistant to multiple antibiotics. Southern hybridization indicated that 4 of the 20 staphylococci contained the ermC gene on plasmids: on a 2.2 kb plasmid in Staphylococcus hominis and S. sciuri, on a 6.0 kb plasmid in S. xylosus, and on a 7.0 kb plasmid in S. lentus. In 16 of the 20 staphylococci, the ermA gene was harbored exclusively on the chromosome, as a double chromosomal insert on 8.0 and 6.2 kb EcoRI fragments. None of the staphylococci harbored the msrA gene. Dot-blot analysis indicated that all enterococci and streptococci hybridized with a biotinylated ermB gene probe. Southern hybridization indicated that only 2 of the 19 erythromycin-resistant enterococci contained the ermB gene on plasmids. The gene was localized on 4.0 kb and 5.9 kb plasmids, respectively, in two Enterococcus faecium isolates. Results from our studies indicate that the patterns of occurrence of erm genes, the sizes of the plasmids and the copy numbers of the inserts were different from the existing information on the presence of erm genes in clinical strains of Staphylococcus spp.     

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.     

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.     

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.     

Ribosomal RNA methylation in Staphylococcus aureus and Escherichia coli: effect of the "MLS" (erythromycin resistance) methylase

Classical acquired resistance to erythromycin in Staphylococcus aureus ("MLS," or macrolide-lincosamide-streptogramin, resistance) was shown by Weisblum and colleagues to be a direct consequence of the conversion of one or more adenosine residues of 23S rRNA, within the subsequence(s) GA3G, to N6-dimethyladenosine (m62A). The methylation reaction is effected by a class of methylase, whose genes are typically plasmid- or transposon-associated, and whose synthesis is inducible by erythromycin. Using a recently obtained clinical MLS isolate of S. aureus, we have further defined the methylation locus as YGG X m62A X AAGAC; and have shown that this subsequence occurs once in the 23S RNA and that it is essentially completely methylated in all copies of 23S RNA that accumulate in induced cultures. Similar findings were obtained with laboratory S. aureus strains containing two well-characterized evolutionary variants (ermB, ermC) of MLS methylase genes. Analyses of a strain of E. coli containing the ermC gene indicated that the specificity of the methylase gene was unchanged, but that its expression was muted. Even after prolonged periods of induction, the strain manifested only partial resistance to erythromycin, and only about one-third of the copies of the MLS subsequence were methylated in such "induced" cultures. Since the E. coli 23S RNA sequence is known in its entirety, localization of the MLS subsequence is in this case unambiguous; as inferred by homology arguments applied earlier to the S. aureus data, the subsequence is in a highly conserved region of 23S RNA considered to contribute to the peptidyl transferase center of the ribosome.     

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。     

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。     

Rapid identification, virulence analysis and resistance profiling of Staphylococcus aureus by gene segment-based DNA microarrays: application to blood culture post-processing

Up to now, blood culturing systems are the method of choice to diagnose bacteremia. However, definitive pathogen identification from positive blood cultures is a time-consuming procedure, requiring subculture and biochemical analysis. We developed a microarray for the identification of Staphylococcus aureus comprising PCR generated gene-segments, which can reduce the blood culture post-processing time to a single day. Moreover, it allows concomitant identification of virulence factors and antibiotic resistance determinants directly from positive blood cultures without previous amplification by PCR. The assay unambiguously identifies most of the important virulence genes such as tsst-1, sea, seb, eta and antibiotic resistance genes such as mecA, aacA-aphD, blaZ and ermA. To obtain positive signals, 20 ng of purified genomic S. aureus DNA or 2 microg of total DNA extracted from blood culture was required. The microarray specifically distinguished S. aureus from gram-negative bacteria as well as from closely related coagulase negative staphylococci (CoNS). The microarray-based identification of S. aureus can be accomplished on the same day blood cultures become positive in the Bactec. The results of our study demonstrate the feasibility of microarray-based systems for the direct identification and characterization of bacteria from cultured clinical specimens.     

Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis.

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively. The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open reading frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control.     

Naturally occurring Staphylococcus epidermidis plasmid expressing constitutive macrolide-lincosamide-streptogramin B resistance contains a deleted attenuator.

A naturally occurring constitutive macrolide-lincosamide-streptogramin B (MLS) resistance plasmid, pNE131, from Staphylococcus epidermidis was chosen to study the molecular basis of constitutive expression. Restriction and functional maps of pNE131 are presented along with the nucleotide sequence of ermM, the gene which mediates constitutive MLS resistance. Sharing 98% sequence homology within the 870-base-pair Sau3A-TaqI fragment, ermM appears to be almost identical to ermC, the inducible MLS resistance determinant from S. aureus (pE194). The two genes share nearly identical sequences, except in the 5' promoter region of ermM. Constitutive expression of ermM is due to the deletion of 107 base pairs relative to ermC; the deletion removes critical sequences for attenuation, resulting in constitutive methylase expression.     

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。     

Characterisation of virulence genes in methicillin susceptible and resistant Staphylococcus aureus isolates from a paediatric population in a university hospital of Medellín, Colombia

Virulence and antibiotic resistance are significant determinants of the types of infections caused by Staphylococcus aureus and paediatric groups remain among the most commonly affected populations. The goal of this study was to characterise virulence genes of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains isolated from a paediatric population of a Colombian University Hospital during 2009. Sixty MSSA and MRSA isolates were obtained from paediatric patients between zero-14 years. We identified the genes encoding virulence factors, which included Panton-Valentine leucocidine (PVL), staphylococcal enterotoxins A-E, exfoliative toxins A and B and toxic shock syndrome toxin 1. Typing of the staphylococcal chromosome cassette mec (SCCmec) was performed in MRSA strains. The virulence genes were more diverse and frequent in MSSA than in MRSA isolates (83% vs. 73%). MRSA strains harboured SCCmec types IVc (60%), I (30%), IVa (7%) and V (3%). SCCmec type IVc isolates frequently carried the PVL encoding genes and harboured virulence determinants resembling susceptible strains while SCCmec type I isolates were often negative. PVL was not exclusive to skin and soft tissue infections. As previously suggested, these differences in the distribution of virulence factor genes may be due to the fitness cost associated with methicillin resistance.     

The nucleotide sequence of Staphylococcus aureus plasmid pT48 conferring inducible macrolide-lincosamide-streptogramin B resistance and comparison with similar plasmids expressing constitutive resistance

The complete nucleotide sequence of a naturally occurring Staphylococcus aureus plasmid, pT48 (from S. aureus strain T48), has been determined. The 2475 bp plasmid confers inducible resistance to macrolide-lincosamide-streptogramin B (MLS) type antibiotics. It is similar to the constitutive MLS resistance plasmid, pNE131, from Staphylococcus epidermidis and shows homology with S. aureus plasmids pSN2 and pE194. It contains a palA structure homologous to that on S. aureus plasmid pT181. The open reading frame, ORF B, within the pSN2 homologous region has a frameshifted C-terminus, relative to pNE131, resulting in a smaller, 158 amino acid putative polypeptide. The pE194 homologous region has the ermC resistance determinant and retains the leader region, deleted in pNE131, required for inducible expression of an adenine methylase. Another naturally occurring S. aureus strain, J74, shows constitutive resistance to erythromycin and contains a small plasmid, pJ74, which is similar to pNE131 but with a different deletion in the leader sequence. The results are consistent with the translational attenuation model for ermC expression.     

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.     

Virulence and resistance determinants of German Staphylococcus aureus ST398 isolates from nonhuman sources

A series of 100 Staphylococcus aureus isolates ascribed to sequence type 398 (ST398) and recovered from different sources (healthy carrier and diseased pigs, dust from pig farms, milk, and meat) in Germany were investigated for their virulence and antimicrobial resistance genetic background. Antimicrobial resistance was determined by the disk diffusion method. Virulence and resistance determinants (37 and 31 genes, respectively) were tested by PCR. Only two virulence profiles, including the accessory gene regulator agrI and three or four hemolysin-encoding genes, were detected. In contrast, 33 resistance profiles were distinguished (only 11 were shown by more than one isolate). Fifty-nine isolates were multiresistant (four or more antimicrobial classes), and 98 were methicillin resistant (mecA positive). All of the ST398 isolates showed resistance to tetracycline [encoded by tet(M) alone or together with tet(K) and/or tet(L)]. In addition, 98% were resistant to other antimicrobials, including macrolide-lincosamine-streptogramin B (70%, encoded by ermA, ermB, and ermC, alone or in combination), trimethoprim (65%, mostly due to dfrK and dfrG), kanamycin and gentamicin [29% and 14%, respectively, mainly related to aac(6')-Ie-aph(2″)-Ia and/or ant(4')-Ia but also to aph(3')-IIIa], chloramphenicol (9%, fexA or cfr), quinupristin-dalfopristin (9%), ciprofloxacin (8%), and trimethoprim-sulfamethoxazole (4%). The heterogeneity of the resistance profiles underlines the ability of the ST398 clone to acquire multiple antimicrobial resistance genes. However, the virulence gene content of the tested isolates was low. Continuous surveillance is needed to clarify whether its pathogenicity potential for animals and humans will increase over time.     

Assignment of Staphylococcus aureus isolates to clonal complexes based on microarray analysis and pattern recognition

A DNA microarray was designed for the rapid genotyping of Staphylococcus aureus. It covers 185 distinct genes and about 300 alleles thereof, including species-specific controls, accessory gene regulator (agr) alleles, genes encoding virulence factors, and microbial surface components recognizing adhesive matrix molecules, capsule type-specific genes, as well as resistance determinants. It was used to examine 100 clinical isolates and reference strains. Relationships of leukocidin and ssl/set (staphylococcal superantigen-like or exotoxin-like) genes were reviewed considering these experimental results as well as published sequences. A good correlation of overall hybridization pattern and multilocus sequence typing was found. Analysis of hybridization profiles thus allowed not only to assess virulence and drug resistance, but also to assign isolates to strains and to clonal complexes. Hybridization data were used to construct a split network tree and to analyse relationships between strains. Allelic variations of a number of genes indicate a division of S. aureus into three major branches that are not in accordance to agr group or capsule-type affiliations. Additionally, there are some isolated lineages, such as ST75, ST93, or ST152. These strains produce aberrant hybridization profiles, indicating that only a part of the gene pool of S. aureus has been investigated yet.