Detection of methicillin- and aminoglycoside-resistant genes and simultaneous identification of S. aureus using triplex real-time PCR Taqman assay

In this study we describe a triplex real-time PCR assay that enables the identification of S. aureus and detection of two important antibiotic resistant genes simultaneously using real-time PCR technology in a single assay. In this triplex real-time PCR assay, the mecA (methicillin resistant), femA (species specific S. aureus) and aacA-aphD (aminoglycoside resistant) genes were detected in a single test using dual-labeled Taqman probes. The assay gives simultaneous information for the identification of S. aureus and detection of methicillin and aminoglycoside resistance in staphylococcal isolates. 152 clinical isolates were subjected to this triplex real-time PCR assay. The results of the triplex real-time PCR assay correlated with the results of the phenotypic antibiotic susceptibility testing. The results obtained from triplex real-time PCR assay shows that the primer and probe sets were specific for the identification of S. aureus and were able to detect methicillin- and aminoglycoside-resistant genes. The entire assay can be performed within 3 h which is a very rapid method that can give simultaneous information for the identification of S. aureus and antibiotic resistance pattern of a staphylococcal isolate. The application of this rapid method in microbiology laboratories would be a valuable tool for the rapid identification of the S. aureus isolates and determination of their antibiotic resistance pattern with regards to methicillin and aminoglycosides.     

Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan)

Real-time PCR assays were developed for the quantifiable detection of the antibiotic-resistance genes vanA of enterococci, ampC of Enterobacteriaceae, and mecA of staphylococci in different municipal wastewater samples. Primer and probe designs for these resistance genes were constructed and optimised for application in standardised TaqMan PCR assays. Using reference strains, the linear measurement ranges of the assays were defined and covered concentration ranges of five to seven exponential values. Wastewater isolates of vancomycin-resistant enterococci (VRE) and beta-lactam-resistant Enterobacteriaceae were cultivated from municipal wastewaters in order to verify the specificity and sensitivity of the primer-probe systems. Additionally, clinical strains of staphylococci resistant to methicillin (MRSA) confirmed the applicability of the mecA-specific detection system. Total DNAs were extracted from five different wastewater treatment plants and used for direct TaqMan PCR detection of the resistance genes without prior cultivation. In municipal wastewater, the resistance gene vanA was detected in 21% of the samples, and ampC in 78%. The gene mecA was not found in municipal wastewater, but in two clinical wastewater samples.     

Rapid detection of methicillin resistance in teicoplanin-resistant coagulase-negative staphylococci by a penicillin-binding protein 2' latex agglutination method

Thirty-five clinical isolates of coagulase-negative staphylococci with decreased glycopeptide sensitivity were examined by a penicillin-binding protein (PBP2′) latex agglutination (LA) test and were compared to the detection of the mecA gene by PCR, and oxacillin susceptibility determined minimum inhibitory concentrations. The latex test demonstrated high sensitivity and specificity for detecting methicillin resistance in coagulase-negative staphylococci after PBP2′ induction with oxacillin.     

Staphylococcus aureus resistance to antibiotics: key points in 2010

Staphylococcus aureus has a strong adaptive capacity and thus acquired various types of resistance to antistaphylococcal agents. More than 90% of isolates produce a penicillinase. Oxacillin remains active against these strains, but hospital associated staphylococci and more recently community acquired staphylococci have developed crossed resistance between methicillin (MRSA), oxacillin and other beta-lactams by production of a penicillin binding protein (PBP) with low affinity for beta-lactams, PBP2a. The gene encoding PBP2a, mecA is carried by a chromosomal element which also contains other resistance genes to heavy metals and other antibiotics thus explaining the multiresistant profile of hospital associated MRSA. By contrast, community acquired MRSA (CA-MRSA) are only resistant to kanamycin, fusidic acid and tetracycline, in addition to methicillin. This profile is specific of the European CA-MRSA ST80 clone which also encodes for a very particular virulence factor, the Panton-Valentine leukocidin. Glycopeptides, vancomycin and teicoplanin, are alternatives to oxacillin in case of resistance or intolerance. Strains with decreased susceptibility to glycopeptides have been reported. Their detection is difficult but necessary because vancomycin MIC creep seems linked to poor outcome in patients.     

Simultaneous PCR detection of ica cluster and methicillin and mupirocin resistance genes in catheter-isolated Staphylococcus.

Recent data show that more than 50% of catheter-associated bloodstream infections are caused by staphylococci. Staphylococcal infections produced by intercellular-adhesion cluster (ica) carriers can be even more problematic due to the presence of methicillin and mupirocin resistance genes. In the present study, a multiplex PCR protocol that allows the simultaneous identification of staphylococci and detection of both the ica and methicillin and/or mupirocin resistance genes was developed. Furthermore, the method allows differential detection of the ica locus from Staphylococcus aureus and Staphylococcus epidermidis.     

Adhesion of a Staphylococcus aureus strain to biomaterials does not select methicillin-resistant mutants

Bacterial adhesion to polymethylmethacrylate and to silicon elastomer, materials frequently used in clinical applications, has been investigated to assess whether adhesion selects methicillin-resistant mutants in the bacterial population in contact with the materials. The methicillin susceptibility of a susceptible Staphylococcus aureus (ATCC 25923) was measured by a modification of plate antibiogram Kirby-Bauer method, which allows optimised detection of small variations in antibiotic susceptibility. In both adherent and non-adherent bacterial subpopulations, the presence of mecA gene, which encodes for the protein PBP 2a responsible for methicillin resistance was searched for by Polymerase Chain Reaction (PCR). The contact with the two polymers did not induce in the bacteria population any phenotypic increase in methicillin resistance, or the selection of mutants carrying the mecA gene.     

Investigation of aminoglycoside modifying enzyme genes in methicillin-resistant staphylococci

Methicillin-resistant staphylococci may also be resistant to some other antibiotics as well as beta-lactams. In this study, co-existence of resistance to methicillin and aminoglycosides was genetically investigated in staphylococci. A total of 50 staphylococci from in-patients, 17 Staphylococcus aureus and 33 coagulase negative staphylococci (CNS) that contained mecA (gene encoding PBP 2a, an altered penicillin-binding protein) determined by polymerase chain reaction (PCR) were included in the study. Aminoglycoside modifying enzyme (AME) genes were investigated using multiplex-PCR. Aminocyclitol-6'-acetyltransferase-aminocyclitol-2'-phosphotransferase [aac(6')/aph(2')] gene (encoding bifunctional acetyltransferases/phosphotransferases) was determined in 66% of the isolates, aminocyclitol-4'-adenylytransferase (ant(4')-Ia) gene (encoding phosphotransferases) in 24%, and aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene (encoding nucleotidyltransferases) in 8%. Two isolates contained all these three genes. Thirty-six (72%) isolates had at least one of these genes. Three CNS and one S. aureus isolates sensitive to oxacillin had the mecA gene. In conclusion, a high rate of aminoglycoside resistance was determined in methicillin-resistant staphylococci. The aac(6')/aph(2') was the most frequently detected.     

耐甲氧西林金黄色葡萄球菌染色体盒的研究进展

耐甲氧西林金黄色葡萄球菌（MRSA）的产生是由甲氧西林敏感的金黄色葡萄球菌（MSSA）获得外源性的SCCmec所致。MRSA菌株可以产生一种新的青霉素结合蛋白PBP2a,PBP2a降低了与β-内酰胺类抗生素的亲合力,从而对β-内酰胺类抗生素产生耐药性。PBP2a由mecA基因编码,mecA基因存在于葡萄球菌盒式染色体（Staphylococcal cassette chromosome mec,SCCmec）中,SCCmec是一种可移动的遗传元件,该元件还携带除mecA基因外的其他抗菌药物的耐药基因,造成多重耐药（Multidrug-resistance,MDR）。SCCmec目前主要分为8型,其中又分为若干亚型。SCCmec的基因型与MRSA的流行背景有关,不同地区的SCCmec基因分型分布可能不同。     

Detection of methicillin resistance in Staphylococcus aureus isolates using two-step triplex PCR and conventional methods

A two-step triplex PCR assay targeting the mecA, femA, and nuc genes was developed for the detection of methicillin resistance genes harbored by some Staphylococcus aureus isolates and for the simultaneous identification of such isolates at the species level. The triplex PCR revealed the presence of the femA and nuc genes in all the S. aureus isolates examined (n=105). Forty-four clinical isolates were mecA positive and no foodborne isolates were mecA positive. The PCR results had a 98 or 99% correlation with the results of PBP2a latex agglutination tests or oxacillin susceptibility tests, respectively.     

Factors influencing methicillin resistance in staphylococci

Methicillin resistance in staphylococci is due to an acquired penicillin-binding protein, PBP2' (PBP2a). This additional PBP, encoded by mecA, confers an intrinsic resistance to all beta-lactams and their derivatives. Resistance levels in methicillin-resistant Staphylococcus aureus (MRSA) depend on efficient PBP2' production and are modulated by chromosomal factors. Depending on the genetic background of the strain that acquired mecA, resistance levels range from phenotypically susceptible to highly resistant. Characteristic for most MRSA is the heterogeneous expression of resistance, which is due to the segregation of a more highly resistant subpopulation upon challenge with methicillin. Maximal expression of resistance by PBP2' requires the efficient and correct synthesis of the peptidoglycan precursor. Genes involved in cell-wall precursor formation and turnover, regulation, transport, and signal transduction may determine the level of resistance that is expressed. At this stage, however, there is no information available on the functionality or efficacy of such factors in clinical isolates in relation to methicillin resistance levels.     

Intestinal colonization of newborns treated in intensive care units by multiple drug resistant microorganisms

The aim of the study was to examine the digestive tract colonisation of the newborns by multiple drug resistant bacteria during hospitalization. On the day of admission, after 5 days of hospitalization and at the day of discharge swabs from the anus of the 31 newborns hospitalized in OITiPN were taken and cultured on nutrient and selective media for staphylococci, enterococci, gram negative bacilli and fungi. Susceptibility to antibiotics of bacteria was determined, with giving attention to such resistance mechanisms as: methicillin resistant staphylococci (MRS), high level aminoglycoside resistant (HLAR) and vancomycin resistant enterococci (VRE) and the production of extended spectrum beta-lactamases (type ESBL) by gram negative bacilli. On the day of admission in 7 newborns methicillin resistant staphylococci (MRSCN) were grown in 24 no multiple drug resistant bacteria were found. Among those in 23 already after 5 days of hospitalization, colonization by multiple drug resistant strains was determined: coagulase-negative methicillin resistant staphylococci (MRSCN) were found in 16 children, strains of enterococci (HLAR) in 3 newborns and gram negative ESBL (+) bacilli also in 3 cases. On the day of discharge from hospital (after 13-141 days) in 23 out of 24 newborns enteric tract colonization by multiple drug resistant strains was assessed. In the enteric tract of 3 newborns hospitalized up to 2 weeks coagulase-negative methicillin resistant staphylococci (MRSCN) and/or HLAR enterococci were found; gram negative bacilli that produce ESBL appeared in newborns hospitalized for longer than 14 days. They were isolated in 12 out of 21 newborns. Forming of the enteric tract bacterial flora of the long hospitalized newborns depends on the time of hospitalization as well as on the used therapy.     

Low-affinity penicillin-binding protein associated with beta-lactam resistance in Staphylococcus aureus

Methicillin resistance in Staphylococcus aureus has been associated with alterations in the penicillin-binding proteins (PBPs). An intriguing property of all methicillin-resistant staphylococci is the dependence of resistance on the pH value of the growth medium. Growth of such bacteria at pH 5.2 completely suppressed the expression of methicillin resistance. We have examined the PBP patterns of methicillin-resistant staphylococci grown at pH 7.0. We detected a high-molecular-weight PBP (PBP-2a; approximate size, 78,000 daltons) that was only present in the resistant bacteria but not in the isogenic sensitive strain. In cultures grown at pH 5.2, the extra PBP was not detectable.     

Methicillin resistance study in clinical isolates of coagulase-negative staphylococci and determination of their susceptibility to alternative antimicrobial agents

AIMS: To achieve reliable detection of methicillin resistance in clinical isolates of coagulase-negative staphylococci. METHODS AND RESULTS: Strains (105) were evaluated by normatized antimicrobial susceptibility methods, and for the presence of the methicillin resistance-determining mecA gene, using the polymerase chain reaction. Correlation between phenotypic and genotypic methods was obtained in 87.6% of the samples. Six strains, classified as methicillin-susceptible by phenotypic assays, revealed the presence of the mecA gene, indicating that methicillin resistance expression was probably repressed. Another seven isolates failed to show mecA amplification after displaying methicillin resistance in phenotypic evaluations. The susceptibility of the methicillin-resistant isolates to other antimicrobial agents was variable. CONCLUSION: Genotypic determination of the mecA gene proved to be the most reliable method for detection of methicillin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Correct assessment of methicillin resistance, such as that attained through genotyping, is essential for defining therapeutic strategies, particularly when treating severely compromised patients.     

Enterococcal Infective Endocarditis following Periodontal Disease in Dogs

In humans, one of the major factors associated with infective endocarditis (IE) is the concurrent presence of periodontal disease (PD). However, in veterinary medicine, the relevance of PD in the evolution of dogs’ endocarditis remains poorly understood. In order to try to establish a correlation between mouth-associated Enterococcus spp. and infective endocarditis in dogs, the present study evaluated the presence and diversity of enterococci in the gum and heart of dogs with PD. Samples were collected during necropsy of 32 dogs with PD and visually diagnosed with IE, which died of natural causes or euthanasia. Enterococci were isolated, identified and further characterized by Pulsed-Field Gel Electrophoresis (PFGE); susceptibility to antimicrobial agents and pathogenicity potential was also evaluated. In seven sampled animals, PFGE-patterns, resistance and virulence profiles were found to be identical between mouth and heart enterococci obtained from the same dog, allowing the establishment of an association between enterococcal periodontal disease and endocarditis in dogs. These findings represent a crucial step towards understanding the pathogenesis of PD-driven IE, and constitute a major progress in veterinary medicine.     

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.     

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.     

Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis

Abstract Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in β-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in β-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not stictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.     

Composition of AME encoding gene in Gram positive Cocci--study on spread of aminoglycoside resistance

Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Composition of six genes encoding AMEs (including lately described aph 2"-genes) was investigated by PCR for 16 clinical isolates of Enterococcus faecalis, 16 clinical isolates of coagulase-positive (S. aureus) and 13 clinical isolates of coagulase negative staphylococci (S. haemolyticus, S. epidermidis) collected in Gdańsk region (Northern Poland) in the years 1998-2001. Diversity of AME encoding gene profiles (composition) was used to analyze spread of AME encoding gene among and within studied group of cocci. According to presence of particular genes we distinguish eleven different AME encoding gene profiles: seven profiles were unique for particular species while the most common was shared among S. aureus, coagulase negative staphylococci and enterococci. Regarding profile frequency statistical analysis (Fstat, AMOVA, cluster analysis UPGMA) shows: the difference between S. aureus and enterococci and coagulase-negative staphylococci, lack of difference between enterococci and coagulase-negative staphylococci, higher variability within than between studied species and presence of multispecies cluster. On the basis of the reports about ability of staphylococci to synthesis enterococcal pheromones, this finding lets assume that spread of aminoglycoside resistance gene among gram (+) cocci is limited only by the ability of stains to synthesis or induction of synthesis conjugation protein.     

Donor activity of methicillin resistant staphylococci in transduction experiments]

Antibiotic resistance gene transmission from methicillin resistant strains of Staphylococcus aureus (MRSA), isolated in a burn care unit, was studied in transduction experiments with type phages 29, 52, 52A and in experiments with prophage induction. The results of the experiments demonstrated high donor activity of MRSA. Recombinants with different antibiotic resistance phenotypes were revealed, but there were no methicillin resistant staphylococci among them. Stability of cloramphenicol resistance gene transmission in the experiments on specific transduction with the prophage induction could be indicative of the prophage localization near the chloramphenicol resistance genes. Variety of the antibiotic resistance combinations in the transductants from the clinical strains of MRSA could prove heterogeneity of the strains even under conditions of one hospital.     

Distribution of antimicrobial resistance and virulence genes in Enterococcus spp. and characterization of isolates from broiler chickens

Enterococci are now frequent causative agents of nosocomial infections. In this study, we analyzed the frequency and distribution of antibiotic resistance and virulence genotypes of Enterococcus isolates from broiler chickens. Fecal and cecal samples from nine commercial poultry farms were collected to quantify total enterococci. Sixty-nine presumptive enterococci were isolated and identified by API 20 Strep, and their susceptibilities to antibiotics were determined. Genotypes were assessed through the use of a novel DNA microarray carrying 70 taxonomic, 17 virulence, and 174 antibiotic resistance gene probes. Total enterococcal counts were different from farm to farm and between sample sources (P < 0.01). Fifty-one (74%) of the isolates were identified as E. faecium, whereas nine (13%), seven (10%), and two (3%) isolates were identified as E. hirae, E. faecalis, and E. gallinarum, respectively. Multiple-antibiotic resistance was evident in E. faecium and E. faecalis isolates. The most common multiple-antibiotic resistance phenotype was Bac Ery Tyl Lin Str Gen Tet Cip. Genes conferring resistance to aminoglycoside (aac, aacA-aphD, aadB, aphA, sat4), macrolide (ermA, ermB, ermAM, msrC), tetracycline (tetL, tetM, tetO), streptogramin (satG_vatE8), bacitracin (bcrR), and lincosamide (linB) antibiotics were detected in corresponding phenotypes. A range of 9 to 12 different virulence genes was found in E. faecalis, including ace, agg, agrB(Efs) (agrB gene of E. faecalis), cad1, the cAM373 and cCF10 genes, cob, cpd1, cylAB, efaA(Efs), and gelE. All seven E. faecalis isolates were found to carry the gelE gene and to hydrolize gelatin and bile salts. Results from this study showed the presence of enterococci of public and environmental health concerns in broiler chicken farms and demonstrated the utility of a microarray to quickly and reliably analyze resistance and virulence genotypes of Enterococcus spp.