# Patent

## Title
Identification of antibiotic resistance using labelled antibiotics

## Abstract

Subject of the present invention is a method for detection of an antibiotic resistance in a micro-organism comprising the steps of exposing suspected micro-organism to a labelled (fluorescent) antibiotic and observing the differences between it and a non-resistant micro-organism of the same type.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application is a 35 U.S.C. 371 National Phase Entry Application from PCT/EP2009/000621, filed Jan. 30, 2009, which claims the benefit of European Patent Application No. 08001949 filed on Feb. 1, 2008, the disclosure of which is incorporated herein in its entirety by reference.
Subject of the present invention is a method for detection of an antibiotic resistance in a micro-organism.
The characterisation of micro-organisms in routine diagnostic procedures encompasses the determination of a species' identity and its sensitivity towards antibiotics. In order to achieve this, micro-organisms need to be taken from their environment and enriched in a selective environment for the separate identification (ID) and antibiotic sensitivity testing (AST). Currently the AST/ID of micro-organisms is achieved by identifying presence or absence of an array of biochemical features and the (non-) capability to grow in the presence of antibiotics. Alternatively DNA can be extracted from a sample and the then pooled DNA is tested for the presence/absence of specific sequences utilising gene amplification techniques. This can signal the presence of an organism in the sample. Equally, the presence of a gene coding for antibiotic resistance in the sample can be detected. By definition, extracting DNA directly from a sample renders pooled DNA from an unknown mixture of cells. Unequivocal results can only be achieved if the DNA is extracted from a pure colony.
Staphylococcus aureus is one of the most common causes of nosocomial or community-based infections, leading to serious illnesses with high rates of morbidity and mortality. In recent years, the increase in the number of bacterial strains that show resistance to methicillin-resistant Staphylococcus aureus (MRSA) has become a serious clinical and epidemiological problem because this antibiotic (or analogue) is considered the first option in the treatment of staphylococci infections. The resistance to this antibiotic implies resistance to all β-lactam antibiotics. For these reasons, accuracy and promptness in the detection of methicillin resistance is of key importance to ensure correct antibiotic treatment in infected patients as well as control of MRSA isolates in hospital environments, to avoid them spreading.
MRSA strains harbour the mecA gene, which encodes a modified PBP2 protein (PBP2′ or PBP2a) with low affinity for methicillin and all β-lactam antibiotics. Phenotypic expression of methicillin resistance may alter depending on the growth conditions for S. aureus, such as temperature or osmolarity of the medium, and this may affect the accuracy of the methods used to detect methicillin resistance (1). Hetero-resistant bacterial strains may evolve into fully resistant strains and therefore be selected in those patients receiving β-lactam antibiotics, thus causing therapeutic failure. From a clinical point of view they should, therefore, be considered fully resistant.
There are several methods for detecting methicillin resistance (1,9) including classical methods for determining a minimum inhibitory concentration MIC (disc diffusion, Etest, or broth dilution), screening techniques with solid culture medium containing oxacillin, and methods that detect the mecA gene or its protein product (PBP2′ protein) (3,4). Detection of the mecA gene is considered as the reference method for determining resistance to methicillin (1). However, many laboratories throughout the world do not have the funds required, the capacity or the experienced staff required to provide molecular assays for detecting MRSA isolates. It is therefore essential that other, more useful, screening methods are incorporated into routine clinical practice. Moreover, the presence of antibiotic resistance has it's relevance at several levels, all of which are of clinical significance

    1. Presence of a gene conveying resistance, such as mecA, mef(E),
    2. Presence of a repressor gene inhibiting the phenotypic expression of said resistance mechanism; e.g. MecA repressor
    3. Multiple resistance mechanisms; e.g. Macrolide resistance via modification of the ribosomal binding site and presence of efflux mechanism(s).
    4. Level of expression of said resistance mechanism regulated via transcription and translation detectable as the phenotype


Current cultural techniques require the isolation of a discrete colony and the subsequent identification and resistance testing, assuming that a single colony is derived from a single cell and is therefore deemed to be pure. In reality however, the generation of a pure colony from a clinical sample, where pathogens frequently live in bio-film communities, cannot be guaranteed. Equally, using amplification technologies, nucleic acid sequences from multiple cells are extracted and amplified and can therefore render false positive results. Only if identification and resistance can be performed and be read on individual cells, is it possible to a true picture of the invading pathogen.
A wide range of antibiotics carry a primary amino group. It is well known in the art that reagents such as Fluoresceinisothiocyanate (FITC), Fluorecein-N-hydroxysuccinimide ester will readily react with said primary amines.
The increasing spread of antibiotic resistance in both community and healthcare systems necessitates the precision and speed of molecular biology. However, the complexity and cost of these assays prohibits the widespread application in a routine testing environment.
Taking into account the difficulties in identifying a micro-organism and its potential resistance against an antibiotic in a biological sample, it is desirable to be able to quickly identify a pathogen directly from a sample without culturing and without amplification and in addition to be able to detect or exclude the presence of resistance towards an antibiotic of choice.
It is the intention of this invention to provide a solution by enabling the simultaneous identification and resistance testing on the cellular level. This reduces the complexity of the assays so that an unambiguous assignment of a phenotype can be made for individual cells. The assays are designed to reduce handling and turnaround time to enable screening programmes such as the screening of all incoming patients for e.g. MRSA.
A first subject of the present invention is thus a method for the detection of an antibiotic resistance in a predetermined micro-organism in a biological sample, comprising the steps:

    
    
        (a) providing a labelled antibiotic,
        (b) contacting the labelled antibiotic with a biological sample comprising the micro-organism under conditions which allow binding of the labeled antibiotic to its binding site in the micro-organism,
        (c) detecting the labelled antibiotic in the micro-organism, and
        (d) identifying a micro-organism in which the amount of detectable label is altered with respect to the amount of detectable label in the predetermined micro-organism in its non-resistant form,

wherein microorganisms identified in step (d) are microorganism resistant against the antibiotic.

    
    


The underlying principle of the method is that if an organism is sensitive or resistant to an antibiotic, it will markedly differ from its resistant or sensitive counterpart. The antibiotics may bind to their respective binding sites either in the cell lumen, cytoplasm, the cell wall or to secreted proteins such as beta-lactamases. Depending upon the resistance mechanism, resistant organisms may mostly show either reduced or no affinity to the antibiotic due to reduced affinity to e.g. ribosomes or penicillin binding proteins. Conversely, if the resistance mechanism is due to the ab/adsorption to the outer cell membrane, the resistant organism will exhibit highly enhanced fluorescence.
The biological sample may be any sample of biological origin, such as a clinical or food sample, suspected of comprising an antibiotic-resistant microorganism. The micro-organism may be selected from the group consisting of bacteria, yeasts and moulds, in particular from Gram positive and Gram negative bacteria.
Preferably, the predetermined micro-organism is selected from the group consisting of Staphylococcus, Enterococcus, and Streptococcus. 
More preferably, the predetermined micro-organism is selected from the group consisting of Methicillin resistant Staphylococcus, Vancomycin resistant Staphylococcus, Vancomycin resistant Enterococcus, and high level Aminoglycoside resistant Enterococci.
The microroganism is even more preferably selected from the group consisting of Staphylococcus aureus, Methicillin Resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Staphylococcus aureus (VRSA), Vancomycin Resistant Staphylococcus (VRS), Vancomycin Resistant Enterococci (VRE), Streptococcus pneumoniae, drug resistant Streptococcus pneumoniae (DRSP), and Aminoglycoside resistant Enterococci (HLAR).
The antibiotic to be provided in step (a) may be any antibiotic. Preferably, the antibiotic is selected from the group consisting of aminoglycosides, carbacephems, carbapenems, cephalosporins, glycopeptides, macrolides, monobactams, beta-lactam antibiotics, quinolones, bacitracin, sulfonamides, tetracyclines, streptogramines, chloramphenicol, clindamycin, and lincosamide.
More preferably, the antibiotics are selected from beta-lactam antibiotics, macrolides, lincosamide, and streptogramins.
Even more preferably, the antibiotic is selected from the group consisting of Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Loracarbef, Ertapenem, Imipenem, Cilastatin, Meropenem, Cefadroxil, Cefazolin, Cephalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefsulodine, Cefepime, Teicoplanin, Vancomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Aztreonam, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Penicillin, Piperacillin, Ticarcillin, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Norfloxacin, Ofloxacin, Trovafloxacin, Mafenide, Prontosil, Sulfacetamide, Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim, Trimethoprim sulfa, Sulfamethoxazole, Co-trimoxazole, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Chloramphenicol, Clindamycin, Ethambutol, Fosfomycin, Furazolidone, Isoniazid, Linezolid, Metronidazole, Mupirocin, Nitrofurantoin, Platensimycin, Pyrazinamide, Quinupristin/Dalfopristin, Rifampin, Spectinomycin, Amphotericin B, Flucanazole, Fluoropyrimidins, Gentamycin, and clavulanic acid.
Most preferably, the antibiotic is selected from Vancomycin, Methicillin, Clindamycin, Trimethoprim, Trimethoprim sulfa, Gentamycin, and clavulanic acid.
Surprisingly, a modification of an antibiotic with a labelling group does not hinder the binding of an antibiotic to its binding site in the micro-organism.
Preferably, the antibiotic is labelled by a luminescent labelling group. Many fluorophores suitable as labelling groups in the present invention are available. The labelling group may be selected to fit the filters present in the market. The antibiotic may be labelled by any suitable labelling group which can be detected in a micro-organism. Preferably, the labelling group is a fluorescent labelling group. More preferably, the labelling group is selected from Fluorescein and Atto-495-NSI.
The labelling group may be coupled to the antibiotic at a functional group. A wide range of antibiotics carry a primary amino group. For example, a fluorescent compound such as Fluoresceinisothiocyanate (FITC), Fluorecein-N-hydroxysuccinimide ester may be reacted with an amino group of an antibiotic, resulting in a Fluorescein-labelled antibiotic. Other antibiotics such as Clindamycin carry a thio-methyl group which can be coupled to a labelling group. Conditions were found to mildly substitute the methyl group of Clindamycin with a spacer molecule forming a dithio bridge.
The labelling group may be coupled to the antibiotic via a spacer. Many spacers are known in the art and may be applied. Using protein chemistry techniques well known in the art many ways of attaching a spacer and subsequently attaching a fluorophor are feasible. In a preferred embodiment cysteine is chosen as its primary amino group may readily be labelled with a fluorophor. Molecules with longer carbon backbones and other reactive groups well known in the art may also be chosen as linker/spacer between any fluorophor and an antibiotic substance.
A list of antibiotics modified with a fluorophor with or without a spacer is compiled in Table 1. It is preferred that the antibiotics (in particular of Table 1) are labelled with Fluorescein or Atto-495-NSI.
In order to combine the identification with the resistance status, the conditions which allow binding of the labelled antibiotic to its binding site in the micro-organism in step (b) may refer to a binding assay which is not inhibited by the in-situ hybridisation procedure, enabling either a concomitant or subsequent determination of both identification and resistance status in individual cells and cell populations.
A preferred binding site is the PBP2 protein (Penicillin Binding Protein) in Staphylococcus encoded by the mecA gene. In Staphylococcus resistant against beta-lactam antibiotics, the mecA gene encodes a modified PBP2 protein (PBP2′ or PBP2a) with low affinity for methicillin and all β-lactam antibiotics. Thus, in a more preferred embodiment, the micro-organism is a MRSA strain harbour the mecA gene, which encodes a modified PBP2 protein (PBP2′ or PBP2a) with low affinity for methicillin and all β-lactam antibiotics, and the antibiotic is a beta-lactam antibiotic.
A further preferred application is for the determination of resistance due to point mutations in the 23s ribosomal RNA. The point mutations at different position induce resistance to a wide array of antibiotics such as Macrolides, Ketolides, Tetracyclin, Thiazolantibiotics, Lincosamin, Chloramphenicol, Streptogramin, Amecitin, Animosycin, Sparsoycin and Puromycin. Detailed effects of respective point mutations are listed in Table 3. Point mutations at different positions of the 23S rRNA can generate an iso-phenotype. It would require an array of oligo-nucleotide probes to cover all possibilities. This invention offers a cost effective and efficient way of detecting antibiotic resistance irrespective of the position of the mutation.
Another preferred application is the detection of the binding of Vancomycin to surface proteins of Staphylococcus aureus which are anchored to the cell wall peptidoglycan. Vancomycin resistant Staphylococci bind the antibiotic to such an extent that it renders Vancomycin ineffective. Labelled Vancomycin therefore will preferably bind to resistant organisms.
In the present invention, the amount of detectable label in the micro-organism corresponds to the signal of the labelling group of the antibiotic. The amount of detectable label may be directly proportional to the signal obtained from the labeling group.
The method of the present invention may comprise steps to remove labelling groups which have been cleaved off from the antibiotic or/and to remove labelled antibiotic which is not bound to a micro-organism. Such steps may improve the signal-to-noise ratio.
In step (c) of the method of the present invention, the label may be detected by any suitable method known in the art. The reading of the assay may require a resolution down to the individual cell. Preferably, the label is detected via epifluorescence microscopy, flow cytometry, laser scanning devices, time resolved fluorometry, luminescence detection, isotope detection, hyper spectral imaging scanner, Surface Plasmon Resonance or/and another evanscesence based reading technology.
In step (d) of the method of the present invention, alteration of the amount of detectable label may be an increase of detectable label or a decrease of detectable label. In the method of the present invention, the antibiotic resistance to be detected is predetermined by the provision of a labelled antibiotic in step (a). Table 4 indicates resistance mechanisms against commonly known antibiotics in clinically relevant micro-organisms. From the resistance mechanism of a particular predetermined micro-organism, such as indicated in Table 4, is can be deduced which combination of micro-organism/antibiotic resistance are expected to show an increased amount of detectable label in antibiotic resistant cells, and which combination shows a decreased amount of detectable label. For instance, a decrease of the amount of detectable label is expected in micro-organisms resistant against β-lactam antibiotics or macrolides, such as MSRA, ORSA, etc. An increased amount of detectable label is expected in VRSA. A decrease of detectable label is expected in Vancomycin resistant Enterococci, due to the different resistance mechanism as in VRSA. Further details can be found in Table 4.
In the present invention, the predetermined microorganism in its non-resistant form can be employed as a reference to determine if the amount of detectable label is altered (decreased or increased). The predetermined microorganism in its non-resistant form may be added to the sample, or may be presented in a separate preparation. The predetermined microorganism in its non-resistant form may carry at least one further label. Any label as described herein may be employed, provided it is suitable for discrimination from the label of the antibiotic or/and other micro-organisms present in the assay of the present invention. The amount of detectable label in a predetermined microorganism in its non-resistant form may also be provided in the form of specific values or ranges of the amount of detectable label for one or more combinations of the micro-organism, an antibiotic and a labelling group, for instance in the form of a data sheet. In particular, a kit of the present invention may comprise said predetermined micro-organism in its non-resistant form or/and said data sheet.
The method of the present invention may also employ the predetermined micro-organism in its resistant form as a further control, or specific values or ranges of the amount of detectable label in a predetermined microorganism in its resistant form for one or more combinations of the micro-organism, an antibiotic and a labelling group, for instance in the form of a data sheet. The predetermined microorganism in its resistant form may carry at least one further label. Any label as described herein may be employed, provided it is suitable for discrimination from the label of the antibiotic or/and other micro-organisms present in the assay of the present invention. In particular, a kit of the present invention may comprise said predetermined micro-organism in its resistant form or/and said data sheet.
The decrease of the amount of detectable label may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% with respect to the amount of detectable label in the predetermined micro-organism in its non-resistant form. In particular, a micro-organism to be identified may be a micro-organism essentially not carrying the label.
The increase of the amount of detectable label may be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, or at least 200% with respect to the amount of detectable label in the predetermined micro-organism in its non-resistant form.
In yet another embodiment of the present invention, the method comprises identification of the micro-organism in the biological sample. “Identification” in the context of the present invention refers to identification of individual microbial cells as belonging to a particular taxonomic category, such as species, genus, family, class or/and order, etc. Identification can be performed based on morphological or/and biochemical classifications.
A probe may be employed for identifying the micro-organism. It is preferred to identify the predetermined micro-organism by a labelled nucleic acid, in particular a labelled oligonucleotide, capable of specifically hybridising with a nucleic acid in the micro-organism under in-situ conditions. The labelled oligonucleotide may have a length of up to 50 nucleotides. More preferred is identification of the micro-organism by fluorescence in-situ hybridisation (FISH). These preferred and more preferred embodiments allow the detection of the antibiotic phenotype at the molecular level while maintaining in-situ hybridisation conditions to allow the simultaneous and unambiguous identification via in-situ hybridisation and the detection of an antibiotic resistance phenotype within the same cell—even if in a mixed population.
An in-situ hybridisation protocol may be applied as laid down in patent application EP 06 021 267.7, which is incorporated herein by reference. The incubation with a labelled antibiotic may be performed at temperatures below the Tm of the hybridised probe. In a preferred embodiment the temperature is between about 25 and about 65° C., in a more preferred embodiment the temperature is between about 35° C. and about 59° C. In an even more preferred embodiment, the temperature is at about 52° C. The incubation time is preferably between about 1 and about 30 minutes. In a more preferred embodiment the incubation is made for about 15 minutes. After the incubation the slide may be submerged in 50% ethanol followed by a bath in pure ethanol. Both steps may be run for between about 1 and about 10 minutes. The preferred length of incubation is between about 2 and about 6 minutes. It is more preferred to incubate about 4 minutes. The slides may then be air-dried (e.g. on a hot plate) and the cells may be embedded in a balanced salt mounting medium.
The microorganism may be detected by any suitable method known in the art. The reading of the assay may require a resolution down to the individual cell. In particular, the micro-organism is detected via epifluorescence microscopy, flow cytometry, laser scanning devices, time resolved fluorometry, luminescence detection, isotope detection, hyper spectral imaging scanner, Surface Plasmon Resonance or/and another evanscesence based reading technology.
Preferably, in-situ hybridisation is combined with detection of antibiotic resistance. More preferably, FISH is combined with detection of antibiotic resistance.
It is also preferred that the predetermined micro-organism is identified in step (c) of the method of the present invention.
It is preferred that the identification of the micro-organism and the detection of the labelled antibiotic in the micro-organism are run subsequently.
In an alternative preferred embodiment, the identification of the micro-organism and the detection of the labelled antibiotic in the micro-organism are run concurrently. In this embodiment, the labelled antibiotic may be added to the hybridisation buffer. After the incubation the further treatment is performed as disclosed herein for the detection of the micro-organism. Most preferably, in-situ hybridisation and FISH, respectively, and detection of the antibiotic resistance are performed simultaneously.
Preferably, the same detection method, such as epifluorescence microscopy, flow cytometry, laser scanning devices or another method described herein, may be employed for both the identification of the micro-organism and the detection of the labelled antibiotic in the micro-organism.
In-situ hybridisation and enzyme or receptor assays conventionally call for specific environments for their respective assays of the state of the art. It was therefore surprising that it was possible to

    1. prepare the cells for in-situ hybridisation with pores of sufficient size to allow passage of up to 50-mer oligo-nucleotides
    2. make membrane proteins accessible for labelled antibiotics
    3. maintain the integrity of both said proteins and ribosomes to allow the specific binding of antibiotics labelled with fluorophores
    4. Find sufficient binding sites to generate a signal visible under an epifluorescence microscope, in particular under uniform conditions.


It is preferred to use in the method of the present invention an oligo-nucleotide with a fluorophor emitting at a predetermined wavelengths range together with an antibiotic labelled with another fluorophor emitting at a wavelengths range, so that the two fluorophors can be discriminated by luminescence detection. For instance, one of the fluorphors, such as Fluorescein, may emit a green signal, and the other fluorphor may emit a red signal. A list of antibiotics modified with a fluorophor is compiled in Table I.
In a most preferred embodiment, the method of the present invention comprises identification of a Methicillin Resistant Staphylococcus aureus (MRSA) by in-situ hybridisation simultaneously with the detection of the expression of the Penicillin Binding Protein 2 by binding a labelled β-Lactam antibiotic.
In the case of MRSA it is even possible to differentiate between nosocomial and community acquired MRSA by analysing the sensitivity towards Clindamycin or to Trimethoprim sulfa. Community acquired Methicillin resistant Staphylococcus aureus strains maintain their sensitivity towards Clindamycin or to Trimethoprim sulfa. The differentiation is then via the respective binding capacity profiles. In yet another most preferred embodiment, the method of the present invention thus comprises identification of a Methicillin Resistant Staphylococcus aureus (MRSA) by in-situ hybridisation simultaneously with the differentiation between nosocomial and community acquired MRSA via the respective ability to bind labelled Clindamycin or Trimethoprim sulfa.
In yet another most preferred embodiment, the method of the present invention comprises identification of a Vancomycin Resistant Staphylococcus aureus (VRSA) by in-situ hybridisation simultaneously with the ability to bind labelled Vancomycin.
In yet another most preferred embodiment, the method of the present invention comprises identification of a Vancomycin Resistant Staphylococcus (VRS) by in-situ hybridisation simultaneously with the ability to bind labelled Vancomycin.
In yet another most preferred embodiment, the method of the present invention comprises identification of a Vancomycin Resistant Enterococci (VRE) by in-situ hybridisation simultaneously with the ability to bind labelled Vancomycin.
In yet another most preferred embodiment, the method of the present invention comprises detection of a resistance towards β-lactam antibiotics due to the secretion of β-lactamases (ESBL) by the revealing of the presence of said enzyme by the binding of labelled clavulanic acid together with the identification of Gram negative micro-organisms.
Clavulanic acid is a beta-lactamase inhibitor sometimes combined with penicillin group antibiotics to overcome certain types of antibiotic resistance. Specifically, it is used to overcome resistance in bacteria that secrete beta-lactamase enzymes, which otherwise inactivate most penicillins. Most commonly, the potassium salt potassium clavulanate is combined with amoxicillin. Clavulanic acid is a competitive inhibitor of beta-lactam antibiotics. When labelled with a fluorophor it will detect the of presence beta-lactamases.
In yet another most preferred embodiment, the method of the present invention comprises detection of a resistance towards 1′-lactam antibiotics due to the secretion of metalo-β-lactamases (MBL) by the revealing of the presence of said enzyme by the binding of labelled imipenem together with the identification of Gram negative micro-organisms.
In yet another most preferred embodiment, the method of the present invention comprises detection of a resistance to macrolides, lincosamide and streptogramin (MLS) via the binding of labelled erythromycin or/and Clindamycin together with the identification of Streptococci.
In yet another most preferred embodiment, the method of the present invention comprises identification of a drug resistant Streptococcus pneumoniae (DRSP) with FISH together with the respective resistance towards beta-lactams and macrolides.
In yet another most preferred embodiment, the method of the present invention comprises detection of high level Aminoglycoside resistant Enterococci (HLAR) via FISH and labelled Gentamycin.
The biological sample comprising the predetermined micro-organisms may be pretreated in order to facilitate binding of the labelled antibiotic and optionally identification of the micro-organism.
The biological sample may be heat-fixed on a slide according to their designated probes (labelled antibiotic and optionally a probe for detecting the micro-organism), for instance at about 45 to about 65° C., preferably at about 50 to about 55° C., more preferably at about 52° C.
If the micro-organism is a Gram positive bacterium, it may be perforated by a suitable buffer. Gram positive cells may be perforated with a bacteriocin or/and a detergent. In a preferred embodiment a lantibiotic is combined with a biological detergent, and a specially preferred embodiment NISIN is combined with Saponin. In addition lytic enzymes such as Lysozyme and Lysostaphin may be applied. Lytic enzymes may be balanced into the equation. If the sample is treated with ethanol, the concentration of the active ingredients may be balanced with respect to their subsequent treatment in ethanol. In a more preferred embodiment the concentration of Lysozyme, Lysostaphin, Nisin and Saponin is balanced to cover all Gram positive organisms with the exception of Mycobacteria.
An example of a most preferred Gram Positive Perforation Buffer is given in Table 2. It is contemplated that variations of the amounts and concentrations, and application temperatures and incubation times are within the skill in the art.
If the micro-organism is a yeast or a mould, it may be perforated by a suitable buffer. Surprisingly it was found that the cell walls of yeasts and moulds did not form reproducible pores when treated following procedures well known in the art. These procedures frequently rendered both false positive and false negative results. A reliable solution is a preferred buffer comprising a combination of a peptide antibiotic, detergent, complexing agent, and reducing agent. A more preferred buffer comprises the combination of a mono-valent salt generating a specific osmotic pressure, a bacteriocin, a combination of biological and synthetic detergents, a complexing agent for divalent cations, and an agent capable of reducing disulfide bridges. A further surprising improvement was achieved by adding proteolytic enzymes specific for prokaryotes. In an even more preferred buffer, Saponin, SDS, Nisin, EDTA, DTT were combined with Lysozyme and a salt, for in stance in a concentration of about 150 to about 250 mM, more preferably about 200 to about 230 mM, most preferably about 215 mM.
An example of a most preferred Yeast Perforation Buffer is given in Table 2. It is contemplated that variations of the amounts and concentrations, and application temperatures and incubation are within the skill in the art.
In yet another preferred embodiment, the method of the present invention is a diagnostic method.
Yet another aspect of the present invention is a kit suitable for detecting an antibiotic resistance in a predetermined micro-organism, comprising

    (a) a labelled antibiotic, and
    (b) optionally a probe suitable for identification of the micro-organism in the biological sample.


The kit of the present invention is suitable in the method of the present invention. The labelled antibiotic may be a labelled antibiotic as described herein in the context of the method of the present invention.
The probe may be any probe suitable for identification of a microorganism. It is preferred that the probe is a labelled nucleic acid, in particular a labelled oligonucleotide, capable of specifically hybridising with a nucleic acid in the micro-organism under in-situ conditions. The oligonucleotide may have a length up to 50 nucleotides.
As indicated above, the kit may comprise further components, such as a data sheet providing information about the amount of detectable label in at least one combination of micro-organism, antibiotic and label, or a sample of a predetermined micro-organism in its non-resistant or/and resistant form, e.g. for control purposes.
Yet another aspect of the present invention is the use of a labelled antibiotic for the detection of an antibiotic resistance in a predetermined micro-organism in a biological sample.
The present invention is further illustrated by the following examples and the following tables.
Table 1 describes the antibiotics and examples of labelling suitable in the method of the present invention.
Table 2 describes the composition of perforation buffers employed in the present invention.
Table 3: Antibiotic resistance due to mutations on the 23S rRNA.
Table 4: Antibiotic resistance mechanism in micro-organisms and alteration in the amount of labelled antibiotics in resistant micro-organisms.


EXAMPLE 1
The antibiotics of Table I were labelled with FITC and purified as is well known in the art. Clindamycin was modified by substituting the methyl group attached to the X′—S with cysteine via an S—S bond. The attached cysteine was then labelled with Fluorescamin either via an N-hydroxy-succinimide ester or FITC and purified with methods well known in the art.
EXAMPLE 2
An antibiotic resistance, such as a resistance against penicillin, may be detected in a protocol comprising the steps:













1
Apply the biological sample to slide, e.g. 10 μl


2
Dry, for instance at 52° C.


3
Add perforation buffer, e.g. 10 μl


4
Dry


5
Add reconstituted probe mix (e.g. 9 μl)


6
Add antibiotic (e.g. FITC-penicillin)


7
Incubate, e.g. for 15 min at 52° C.


8
EtOH/Stop mix (e.g. 50%:50%), e.g. for 5 min at RT


9
Ethanol, e.g. 99% ethanol for 5 min


10
Dry


11
Balanced Salt Mounting Medium (e.g. one small drop)


12
Read









EXAMPLE 3
Table 4 indicates the alteration of the amount of detectable labelled antibiotics in clinically relevant micro-organisms in its resistant form relative to its non-resistant form. The amount is expressed in % change of fluorescence (decrease and increase, respectively) of an antibiotic which carries a fluorescent label.







TABLE 1







Antibiotics










Generic Name
Examples of Labelling agent













Aminoglycosides











Amikacin
FITC
Atto-495-NSI



Gentamicin
FITC
Atto-495-NSI



Kanamycin
FITC
Atto-495-NSI



Neomycin
FITC
Atto-495-NSI



Netilmicin
FITC
Atto-495-NSI



Streptomycin
FITC
Atto-495-NSI



Tobramycin
FITC
Atto-495-NSI




FITC
Atto-495-NSI









Carbacephem











Loracarbef
FITC
Atto-495-NSI









Carbapenems











Ertapenem
FITC
Atto-495-NSI



Imipenem
FITC
Atto-495-NSI



Cilastatin
FITC
Atto-495-NSI



Meropenem
FITC
Atto-495-NSI









Cephalosporins First generation











Cefadroxil
FITC
Atto-495-NSI



Cefazolin
FITC
Atto-495-NSI



Cephalexin
FITC
Atto-495-NSI









Cephalosporins Second generation











Cefaclor
FITC
Atto-495-NSI



Cefamandole 
FITC
Atto-495-NSI



Cefoxitin
FITC
Atto-495-NSI



Cefprozil
FITC
Atto-495-NSI



Cefuroxime
FITC
Atto-495-NSI









Cephalosporins Third generation











Cefixime
FITC
Atto-495-NSI



Cefdinir
FITC
Atto-495-NSI



Cefditoren
FITC
Atto-495-NSI



Cefoperazone 
FITC
Atto-495-NSI



Cefotaxime
FITC
Atto-495-NSI



Cefpodoxime
FITC
Atto-495-NSI



Ceftazidime
FITC
Atto-495-NSI



Ceftibuten
FITC
Atto-495-NSI



Ceftizoxime
FITC
Atto-495-NSI



Ceftriaxone
FITC
Atto-495-NSI



Cefsulodine
FITC
Atto-495-NSI









Cephalosporins Fourth generation











Cefepime
FITC
Atto-495-NSI









Glycopeptides











Teicoplanin
FITC
Atto-495-NSI



Vancomycin
FITC
Atto-495-NSI









Macrolides











Azithromycin
Coupling of
Coupling of



Clarithromycin
FITC with
Erythro



Dirithromycin
Erythro
mycylamine with



Erythromycin 
mycylamine
Atto-495-NSI



Roxithromycin





Troleandomycin











Monobactam











Aztreonam
FITC
Atto-495-NSI









Penicillins











Amoxicillin
FITC
Atto-495-NSI



Ampicillin
FITC
Atto-495-NSI



Azlocillin





Carbenicillin





Cloxacillin





Dicloxacillin





Flucloxacillin





Mezlocillin





Nafcillin





Penicillin





Piperacillin





Ticarcillin











Polypeptides











Bacitracin
FITC
Atto-495-NSI



Colistin
FITC
Atto-495-NSI



Polymyxin B
FITC
Atto-495-NSI









Quinolones











Ciprofloxacin





Enoxacin





Gatifloxacin





Levofloxacin





Lomefloxacin





Moxifloxacin





Norfloxacin





Ofloxacin





Trovafloxacin











Sulfonamides











Mafenide
FITC
Atto-495-NSI



Prontosil
FITC
Atto-495-NSI



(archaic)





Sulfacetamide
FITC
Atto-495-NSI



Sulfamethizole
FITC
Atto-495-NSI



Sulfanilimide
FITC
Atto-495-NSI



(archaic)





Sulfasalazine
FITC
Atto-495-NSI



Sulfisoxazole
FITC
Atto-495-NSI



Trimethoprim sulfa
FITC
Atto-495-NSI



Trimethoprim
FITC
Atto-495-NSI



Sulfamethoxazole
FITC
Atto-495-NSI



Co-trimoxazole
FITC
Atto-495-NSI



TMP-SMX
FITC
Atto-495-NSI









Tetracyclines











Demeclocycline
FITC
Atto-495-NSI



Doxycycline
FITC
Atto-495-NSI



Minocycline
FITC
Atto-495-NSI



Oxytetracycline
FITC
Atto-495-NSI



Tetracycline
FITC
Atto-495-NSI









Others











Chloramphenicol





Clindamycin
Cysteine + FITC 
Cystein + 





Atto-495-NSI



Ethambutol





Fosfomycin





Furazolidone





Isoniazid





Linezolid





Metronidazole





Mupirocin





Nitrofurantoin





Platensimycin





Pyrazinamide





Quinupristin/





Dalfopristin





Rifampin





Spectinomycin





Amphotericin B
FITC
Atto-495-NSI



Flucanazole





Fluoropyrimidins

















TABLE 2







Gram-Positive Perforation Buffer-












50
μg/ml
Saponin


5
μg/ml
Nisin


20
mM
Tris pH 8


100
μg/m
Lysozym


50
μg/ml
Lysostaphin




H2O










Working soln.


Yeast Perforation Buffer












500
μg/ml
Saponin


10
μg/ml
Nisin


50
mM
Tris pH 8.3


215
mM
NaCl








0.1%
SDS









5
mM
EDTA


10
mM
DTT


100
μg/ml
Lysozyme




H2O
















TABLE 3







Compilation of antibiotic resistance due to mutations on the 23S rRNA












Type







of
Posi-






RNA
tion
Alteration(s)
Phenotype
Organism
Reference





23S
2032
AG to GA
Clr/Azm/Ery-R
Helicobacter
Húlten, K., A. Gibreel, O. Sköld, and L. Engstrand.






pylori
1997. Macrolide resistance in Helicobacter pylori:







mechanism and stability in strains from







clarithromycin-treated patients. Antimicrob. Agents







Chemother. 41: 2550-2553.


23S
2058
A to C
Clr-R
Helicobacter
Stone, G. G., D. Shortridge, R. K. Flamm, J. Versalovic,






pylori
J. Beyer, K. Idler, L. Zulawinski, and S. K. Tanaka.







1996. Identification of a 23S rRNA gene







mutation in clarithromycin-resistant Helicobacter







pylori. Helicobacter. 1: 227-228.


23S
2058
A to C
Mac-R, Lin-R
Helicobacter
Occhialini, A., M. Urdaci, F. Doucet-Populaire,






pylori
C. M. Bébéar, H. Lamouliatte, and F. Mégraud. 1997.







Macrolide resistance in Helicobacter pylori: rapid







detection of point mutations and assays of macrolide







binding to ribosomes. Antimicrob. Agents Chemothe


23S
2058
A to C
MLSB-R
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-specific






pylori
mutations in the 23S rRNA gene of Helicobacter







pylori confer two types of resistance to macrolide-







lincosamide-streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2058
A to C
Cla-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,






pylori
C. M. Vandenbroucke-Grauls, and J. G. Kusters.







1998. Explaining the bias in the 23S rRNA







gene mutations associated with clarithromycin







resistance in clinical isolates of Helicobacter pylori.







Antimi


23S
2058
A to G
Cla-R
Helicobacter
Versalovic, J., D. Shortridge, K. Kibler, M. V. Griffy,






pylori
J. Beyer, R. K. Flamm, S. K. Tanaka. D. Y. Graham, and







M. F. Go. 1996. Mutations in 23S rRNA are







associated with clarithromycin resistance in







Helicobacter pylori. Antimicrob. Agents Chemother.







40: 4


23S
2058
A to G
Mac-R, Lin-R
Helicobacter
Occhialini, A., M. Urdaci, F. Doucet-Populaire,






pylori
C. M. Bébéar, H. Lamouliatte, and F. Mégraud. 1997.







Macrolide resistance in Helicobacter pylori: rapid







detection of point mutations and assays of macrolide







binding to ribosomes. Antimicrob. Agents Chemothe


23S
2058
A to G
MLSB-R
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-specific






pylori
mutations in the 23S rRNA gene of Helicobacter







pylori confer two types of resistance to macrolide-







lincosamide-streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2058
A to G
Cla-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,






pylori
C. M. Vandenbroucke-Grauls, and J. G. Kusters.







1998. Explaining the bias in the 23S rRNA







gene mutations associated with clarithromycin







resistance in clinical isolates of Helicobacter pylori.







Antimi


23S
2058
A to U
MLSB-R
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-specific






pylori
mutations in the 23S rRNA gene of Helicobacter







pylori confer two types of resistance to macrolide-







lincosamide-streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2058
A to U
Cla-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,






pylori
C. M. Vandenbroucke-Grauls, and J. G. Kusters.







1998. Explaining the bias in the 23S rRNA







gene mutations associated with clarithromycin







resistance in clinical isolates of Helicobacter pylori.







Antimi


23S
2059
A to C
Mac-R, Lin-R,
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-specific





SB-S
pylori
mutations in the 23S rRNA gene of Helicobacter







pylori confer two types of resistance to macrolide-







lincosamide-streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2059
A to C
Clr-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,






pylori
C. M. Vandenbroucke-Grauls, and J. G. Kusters.







1998. Explaining the bias in the 23S rRNA







gene mutations associated with clarithromycin







resistance in clinical isolates of Helicobacter pylori.







Antimi


23S
2059
A to G
Clr-R
Helicobacter
Versalovic, J., D. Shortridge, K. Kibler, M. V. Griffy,






pylori
J. Beyer, R. K. Flamm, S. K. Tanaka. D. Y. Graham, and







M. F. Go. 1996. Mutations in 23S rRNA are







associated with clarithromycin resistance in







Helicobacter pylori. Antimicrob. Agents Chemother.







40:4


23S
2059
A to G
Mac-R, Lin-R
Helicobacter
Occhialini, A., M. Urdaci, F. Doucet-Populaire,






pylori
C. M. Bébéar, H. Lamouliatte, and F. Mégraud. 1997.







Macrolide resistance in Helicobacter pylori: rapid







detection of point mutations and assays of macrolide







binding to ribosomes. Antimicrob. Agents Chemothe


23S
2059
A to G
Mac-R, Lin-R,
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-specific





SB-S
pylori
mutations in the 23S rRNA gene of Helicobacter







pylori confer two types of resistance to macrolide-







lincosamide-streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2059
A to G
Cla-R
Helicobacter
Debets-Ossenkopp. Y. J., A. B. Brinkman E. J. Kuipers,






pylori
C. M. Vandenbroucke-Grauls, and J. G. Kusters.







1998. Explaining the bias in the 23S rRNA







gene mutations associated with clarithromycin







resistance in clinical isolates of Helicobacter pylori.







Antimi


23S
 754
“U to A”
Resistant to low
E. coli
Xiong L, Shah S, Mauvais P, Mankin A S.





concentrations of ketolide

1999. A ketolide resistance mutation in





HMR3647; resistant to

domain II of 23S rRNA reveals the





erythromycin b.

proximity of hairpin 35 to the peptidyl







transferase center. Molecular







Microbiology 31 (2): 633-639.


23S
 754
“U to A”
Confers macrolide and
E. coli
Hansen L. H., Mauvais P, Douthwaite S.





ketolide resistance.

1999. The macrolide-ketolide antibiotic







binding site is formed by structures in







domain II and V of 23S ribosomal RNA.







Molecular Microbiology 31 (2): 623-631.


23S
 754
U to A
Ery-LR, Tel-LR
Escherichia
Xiong, L., S. Shah, P. Mauvais, and A. S. Mankin.






coli
1999. A ketolide resistance







mutation in domain II of 23S rRNA reveals







the proximity of hairpin 35 to the peptidyl







transferase centre. Mol. Microbiol.







31: 633-639.


23S
1005
C to G
Slow growth under natural
E. coli
1) Rosendahl, G. and Douthwaite, S.





promoter; (with 2058G and

(1995) Nucleic Acids Res. 23, 2396-2403.





erythromycin) severe growth

2) Rosendahl, G., Hansen, L. H., and





retardation. A

Douthwaite, S. (1995) J. Mol. Biol. 249,







59-68.


23S
1005
C to G
Slow growth under pL
E. coli
1) Rosendahl, G. and Douthwaite, S.





promoter; (with 2058G and

(1995) Nucleic Acids Res. 23, 2396-2403.





erythromycin) Erys. a Double

2) Rosendahl, G., Hansen, L. H., and





mutant (C1005G/C1006U)

Douthwaite, S. (1995) J. Mol. Biol. 249,







59-68.


23S
1006
C to U
Slow growth under pL
E. coli
1) Rosendahl, G. and Douthwaite, S.





promoter; (with 2058G and

(1995) Nucleic Acids Res. 23, 2396-2403.





erythromycin) Erys. a Double

2) Rosendahl, G., Hansen, L. H., and





mutant (C1005G/C1006U)

Douthwaite, S. (1995) J. Mol. Biol. 249,







59-68.


23S
1006
C to U
Lethal under natural
E. coli
1) Rosendahl, G. and Douthwaite, S.





promoter; under pL

(1995) Nucleic Acids Res. 23, 2396-2403.





promoter; (with 2058G and

2) Rosendahl, G., Hansen, L. H., and





erythormycin) Erys. A

Douthwaite, S. (1995) J. Mol. Biol. 249,







59-68.


23S
1056
G to A
Binding of both L11 and
E. coli
Ryan, P. C. and Draper, D. E. (1991) Proc.





thiostrepton is weakened in

Natl. Acad. Sci. USA 88, 6308-6312.





RNA fragments. B




23S
1056
G to A
Stoichiometric L11 binding.b
E. coli
1) Douthwaite, S. and Aagaard, C. (1993)





(with 2058G and

J. Mol. Biol. 232, 725-731. 2) Rosendahl, G.





erythromycin) Reduced

and Douthwaite, S. (1995) Nucleic





growth rate. a

Acids Res. 23, 2396-2403.


23S
1056
G to C
Binding of thiostrepton is
E. coli
Ryan, P. C. and Draper, D. E. (1991) Proc.





weakened in RNA

Natl. Acad. Sci. USA 88, 6308-6312.





fragments. B




23S
1064
C to U
Stoichiometric L11 binding. b
E. coli
1) Douthwaite, S. and Aagaard, C. (1993)





(with 2058G and

J. Mol. Biol. 232, 725-731. 2) Rosendahl, G.





erythromycin) Reduced

and Douthwaite, S. (1995) Nucleic





growth rate. a

Acids Res. 23, 2396-2403.


23S
1067
A to U
Normal growth
E. coli
1) Spahn, C., Remme, J., Schafer, M. and







Nierhaus, K. (1996). J. Biol. Chem. 271:







32849-32856. 2) Spahn, C., Remme, J.,







Schafer, M. and Nierhaus, K. (1996). J.







Biol. Chem. 271: 32857-32862.


23S
1067
A to G
Thiostrepton resistance in
Halobacterium
Hummel, H., and A. Böck. (1987)





Halobacterium sp.

Biochimie 69: 857-861.


23S
1067
A to U
Thiostrepton resistance in
Halobacterium
Hummel, H., and A. Böck. (1987)





Halobacterium sp.

Biochimie 69: 857-861.


23S
1067
A to U
A to C or U confers high
E. coli
1) Thompson, J. and Cundliffe, E. (1991)





level resistance to

Biochimie 73: 1131-1135. 2) Thompson, J.,





thiostrepton, whereas A to G

Cundliffe, E. and Dahlberg, A. E. (1988)





confers intermediate level

J. Mol. Biol. 203: 457-465. 3) Lewicki, B. T. U.,





resistance; drug binding

Margus, T., Remme, J. and





affinity is reduced similarly.

Nierhaus, K. H. (1993) J. Mol. Biol. 231,





a, b Expression by host RNA

581-593. 4) LAST





polymerase results in







formation of active ribosomal







subunits in vivo. A




23S
1067
A to C
A to C or U confers high
E. coli
1) Thompson, J. and Cundliffe, E. (1991)





level resistance to

Biochimie 73: 1131-1135. 2) Thompson, J.,





thiostrepton, whereas A to G

Cundliffe, E. and Dahlberg, A. E. (1988)





confers intermediate level

J. Mol. Biol. 203: 457-465. 3) Lewicki, B. T. U.,





resistance; drug binding

Margus, T., Remme, J. and





affinity is reduced similarly.

Nierhaus, K. H. (1993) J. Mol. Biol. 231,





a, b Expression by host RNA

581-593. 4) LAST





polymerase results in







formation of active ribosomal







subunits in vivo. A




23S
1067
A to G
A to C or U confers high
E. coli
1) Thompson, J. and Cundliffe, E. (1991)





level resistance to

Biochimie 73: 1131-1135. 2) Thompson, J.,





thiostrepton, whereas A to G

Cundliffe, E. and Dahlberg, A. E. (1988)





confers intermediate level

J. Mol. Biol. 203: 457-465. 3) Lewicki, B. T. U.,





resistance; drug binding

Margus, T., Remme, J. and





affinity is reduced similarly.

Nierhaus, K. H. (1993) J. Mol. Biol. 231,





a, b Expression by host RNA

581-593. 4) LAST





polymerase results in







formation of active ribosomal







subunits in vivo. A




23S
1067
“A to U”
Constituted 30% of the total
E. coli
Liiv A, Remme J. 1998. Base-pairing of





23S rRNA pool in the

23S rRNA ends is essential for ribosomal





ribosomes; exhibited 30%

large subunit assembly. J. Mol. Biol. 285:





thiostrepton resistance in

965-975.





poly (U) translation b.




23S
1068
G to A
Reduced L11 binding. b (with
E. coli
1) Rosendahl, G. and Douthwaite, S.





2058G) Lethal when

(1995) Nucleic Acids Res. 23, 2396-2403.





expressed from rrnB or pL

2) Douthwaite, S. and Aagaard, C.





promotor in presence of

(1993) J. Mol. Biol. 232, 725-731.





erythromycin. A




23S
1068
G to A
Suppression of 1068A;
E. coli
Rosendahl, G. and Douthwaite, S.





lethality only in absence of

(1995) Nucleic Acids Res. 23, 2396-2403.





erythromycin. a Double mutant







(G1068A/G1099A)




23S
1072
C to U
Lethal when expressed from
E. coli
Rosendahl, G. and Douthwaite, S.





rrnB or pL promotor in

(1995) Nucleic Acids Res. 23, 2396-2403.





presence of erythromycin. a




23S
1137
G to A
With 2058G and erythromycin,
E. coli
Rosendahl, G., Hansen, L. H., and





lethal when expressed from rrnB

Douthwaite, S. (1995) J. Mol. Biol.





promoter.

249, 59-68.


23S
1137
G to A
Restores normal growth under pL
E. coli
Rosendahl, G., Hansen, L. H., and





promotor; (With 2058G and

Douthwaite, S. (1995) J. Mol. Biol.





erythromycin) Eryr. Double

249, 59-68.





mutant (G1137A/C1006U)




23S
1137
G to A
With 2058G and erythromycin,
E. coli
Rosendahl, G., Hansen, L. H., and





lethal when expressed from rrnB

Douthwaite, S. (1995) J. Mol. Biol. 249,





promoter. Double mutant

59-68.





(G1137A/G1138C)




23S
1138
G to C
With 2058G and erythromycin,
E. coli
Rosendahl, G., Hansen, L. H., and





lethal when expressed from rrnB

Douthwaite, S. (1995) J. Mol. Biol. 249,





promoter.

59-68.


23S
1138
G to C
With 2058G and erythromycin,
E. coli
Rosendahl, G., Hansen, L. H., and





lethal when expressed from rrnB

Douthwaite, S. (1995) J. Mol. Biol. 249,





promoter. Double mutant

59-68.





(G1138C/G1137A)




23S
1207
C to U
Erythromycin resistant. a Double
E. coli
Dam, M., Douthwaite, S., Tenson, T.





mutant (C1207U/C1243U)

and Mankin, A. S. (1996) J. Mol. Biol.







259, 1-6.


23S
1208
C to U
Erythromycin resistant. a Double
E. coli
Dam, M., Douthwaite, S., Tenson, T.





mutant (C1208U/C1243U)

and Mankin, A. S. (1996) J. Mol. Biol.







259, 1-6.


23S
1211
C to U
Erythromycin sensitive. a Double
E. coli
Dam, M., Douthwaite, S., Tenson, T.





mutant (C1211U/C1208U)

and Mankin, A. S. (1996) J. Mol. Biol.







259, 1-6.


23S
1220
G to A
Erythromycin resistant. a Double
E. coli
Dam, M., Douthwaite, S., Tenson, T.





mutant (G1220A/G1239A)

and Mankin, A. S. (1996) J. Mol. Biol.







259, 1-6.


23S
1221
C to U
Erythromycin resistant. a Double
E. coli
Dam, M., Douthwaite, S., Tenson, T.





mutant (C1221U/C1229U)

and Mankin, A. S. (1996) J. Mol. Biol.







259, 1-6.


23S
1221
C to U
Erythromycin resistant. a Double
E. coli
Dam, M., Douthwaite, S., Tenson, T.





mutant (C1221U/C1233U)

and Mankin, A. S. (1996) J. Mol. Biol.







259, 1-6.


23S
1230
1230
Erythromycin sensitive. a Double
E. coli
Douthwaite, S., Powers, T., Lee, J. Y.,





deletion (1230/1231)

and Noller, H. F. (1989) J. Mol. Biol.







209, 655-665.


23S
1231
1231
Erythromycin sensitive. a Double
E. coli
Douthwaite, S., Powers, T., Lee, J. Y.,





deletion (1231/1230)

and Noller, H. F. (1989) J. Mol. Biol.







209, 655-665.


23S
1232
G to A
Erythromycin sensitive. a Double
E. coli
Dam, M., Douthwaite, S., Tenson, T.





mutant (G1232A/G1238A)

and Mankin, A. S. (1996) J. Mol. Biol.







259, 1-6.


23S
1233
C to U
Erythromycin sensitive. a
E. coli
Dam, M., Douthwaite, S., Tenson, T.







and Mankin, A. S. (1996) J. Mol. Biol.







259, 1-6.


23S
1234
“del1234/del1235”
Erythromycin sensitive. a Double
E. coli
Douthwaite, S., Powers, T., Lee, J. Y.,





mutant (U1234C/del1235)

and Noller, H. F. (1989) J. Mol. Biol.







209, 655-665.


23S
1234
U to C
Erythromycin sensitive. a
E. coli
Douthwaite, S., Powers, T., Lee, J. Y.,







and Noller, H. F. (1989) J. Mol. Biol. 209,







655-665.


23S
1243
C to U
Erythromycin resistant. a Double
E. coli
Douthwaite, S., Powers, T., Lee, J. Y.,





mutant (C1243U/C1208U)

and Noller, H. F. (1989) J. Mol. Biol. 209,







655-665.


23S
1243
C to U
Erythromycin resistant. a Double
E. coli
Douthwaite, S., Powers, T., Lee, J. Y.,





mutant (C1243U/C1221U)

and Noller, H. F. (1989) J. Mol. Biol. 209,







655-665.


23S
1243
“C to U”
Erythromycin resistant. a Double
E. coli
Douthwaite, S., Powers, T., Lee, J. Y.,





mutant (C1243U/C1207).

and Noller, H. F. (1989) J. Mol. Biol. 209,







655-665.


23S
1262
A to G
With erythromycin; lethal
E. coli
Aagaard, C., and Douthwaite, S. (1994)







Proc. Natl. Acad. Sci. USA 91, 2989-2993.


23S
1262
A to C
With erythromycin; lethal
E. coli
Aagaard, C., and Douthwaite, S. (1994)







Proc. Natl. Acad. Sci. USA 91, 2989-2993.


23S
1262
A to U
With erythromycin; reduced
E. coli
Aagaard, C., and Douthwaite, S. (1994)





growth rate

Proc. Natl. Acad. Sci. USA 91, 2989-2993.


23S
1262
A to C
With erythromycin; reduced
E. coli
Aagaard, C., and Douthwaite, S. (1994)





growth rate Double mutant

Proc. Natl. Acad. Sci. USA 91, 2989-2993.





(A1262C/U2017G)




23S
1262
A to G
Suppression of growth effects;
E. coli
Aagaard, C., and Douthwaite, S. (1994)





Wild-type growth on

Proc. Natl. Acad. Sci. USA 91, 2989-2993.





erythromycin Double mutant







(A1262G/U2017C)




23S
1262
A to U
Suppression of growth effects;
E. coli
Aagaard, C., and Douthwaite, S. (1994)





Wild-type growth on

Proc. Natl. Acad. Sci. USA 91, 2989-2993.





erythromycin Double mutant







(A1262U/U2017A)




23S
1262
A to U
With erythromycin; reduced
E. coli
Aagaard, C., and Douthwaite, S. (1994)





growth rate Double mutant

Proc. Natl. Acad. Sci. USA 91, 2989-2993.





(A1262U/U2017G)




23S
1423
G to A
Suppressed requirement for
E. coli
O'Connor, M., Brunelli, C. A., Firpo, M. A.,





4.5S RNA in translation of

Gregory, S. T., Lieberman, K. R.,





natural mRNAs by cell extracts. c

Lodmell, J. S., Moine, H., Van Ryk, D. I.







and Dahlberg, A. E. (1995) Biochem.







Cell Biology 73, 859-868.


23S
1698
A to G
Suppresses 2555 mutations
E. coli
O'Connor & Dahlberg, unpublished


23S
2017
“U to G”
Reduced growth
E. coli
Aaagard, C. and Douthwaite, S. (1994) Proc.





rate on

Natl. Acad. Sci. USA 91, 2989-2993.





eyrthomycin.




23S
2017
“U to C”
Reduced growth
E. coli
Aaagard, C. and Douthwaite, S. (1994) Proc.





rate of

Natl. Acad. Sci. USA 91, 2989-2993.





erythromycin.




23S
2017
“U to A”
Reduced growth
E. coli
Aaagard, C. and Douthwaite, S. (1994) Proc.





rate of

Natl. Acad. Sci. USA 91, 2989-2993.





erythromycin.




23S
2017
“U to C”
Reduced growth
E. coli
Aaagard, C. and Douthwaite, S. (1994) Proc.





rate on

Natl. Acad. Sci. USA 91, 2989-2993.





erythromycin.







Double mutation







(U2017C/A1262G)




23S
2017
“U to G”
Reduced growth
E. coli
Aaagard, C. and Douthwaite, S. (1994) Proc.





rate on

Natl. Acad. Sci. USA 91, 2989-2993.





erythromycin.







Double mutation







(U2017G/A1262C)




23S
2017
“U to G”
Reduced growth
E. coli
Aaagard, C. and Douthwaite, S. (1994) Proc.





rate on

Natl. Acad. Sci. USA 91, 2989-2993.





erythromycin.







Double mutation







(U2017G/A1262U)




23S
2032
“G to A”
Lincomycin
Tobbaco
Cseplö, A., Etzold, T., Schell, J., and Schreier, P. H.





resistance.
chloroplasts
(1988) Mol. Genet. 214, 295-299.


23S
2032
“G to A”
EryS, Cds, Cms.
E. coli
1. Douthwaite, S. (1992) J. Bacteriol. 174,





Double mutation

1333-1338. 2. Aaagard, C. and Douthwaite, S.





(G2032A/A2058G)

(1994) Proc. Natl. Acad. Sci. USA 91,







2989-2993. 3. Vester, B., Hansen, L. H., and







Douthwaite, S. (1995) RNA 1, 501-509.


23S
2032
“G to A”
Eryhs, Cds, Cms.
E. coli
1. Douthwaite, S. (1992) J. Bacteriol. 174,





Double mutation

1333-1338. 2. Aaagard, C. and Douthwaite, S.





(G2032A/A2058U)

(1994) Proc. Natl. Acad. Sci. USA 91,







2989-2993. 3. Vester, B., Hansen, L. H., and







Douthwaite, S. (1995) RNA 1, 501-509.


23S
2032
“G to A”
Eryr, Cdr, Cmr.
E. coli
1. Douthwaite, S. (1992) J. Bacteriol. 174,





Double mutation

1333-1338. 2. Aaagard, C. and Douthwaite, S.





(G2032A/G2057A)

(1994) Proc. Natl. Acad. Sci. USA 91,







2989-2993. 3. Vester, B., Hansen, L. H., and







Douthwaite, S. (1995) RNA 1, 501-509.


23S
2032
AG to GA
Clr/Azm/Ery-R
Helicobacter
Húlten, K., A. Gibreel, O. Sköld, and L. Engstrand.






pylori
1997. Macrolide resistance in







Helicobacter pylori: mechanism and stability







in strains from clarithromycin-treated patients.







Antimicrob. Agents Chemother. 41: 2550-2553.


23S
2051
“del A”
Prevents ErmE
E. coli
Vester B, Nielsen A K, Hansen L H, Douthwaite S.





methylation. c

1998. ErmE Methyltransferase Recognition







Elements in RNA Substrates. J. Mol. Biol.







282: 255-264.


23S
2052
“A to C”
Prevents ErmE
E. coli
Vester B, Nielsen A K, Hansen L H, Douthwaite S.





methylation. c

1998. ErmE Methyltransferase Recognition







Elements in RNA Substrates. J. Mol. Biol.







282: 255-264.


23S
2052
“A to G”
Like A2052C. c
E. coli
Vester B, Nielsen A K, Hansen L H, Douthwaite S.







1998. ErmE Methyltransferase Recognition







Elements in RNA Substrates. J. Mol. Biol.







282: 255-264.


23S
2052
“A to U”
Like A2052C. c
E. coli
Vester B, Nielsen A K, Hansen L H, Douthwaite S.







1998. ErmE Methyltransferase Recognition







Elements in RNA Substrates. J. Mol. Biol.







282: 255-264.


23S
2057
“G to A”
Eryr, Clinidamycin
E. coli
1. Ettayebi, M., Prasad, S. M., and Morgan, E. A.





(Cd)s,

(1985) J. Bacteriol. 162, 551-557 2.





Chloramphemicol

Aaagard, C. and Douthwaite, S. (1994) Proc.





(Cm)r; reduces

Natl. Acad. Sci. USA 91, 2989-2993. 3.





methylation of 23S

Vester, B., Hansen, L. H., and Douthwaite, S.





rRNA by ErmE.

(1995) RNA 1, 501-509.


23S
2057
“G to A”
Eyrr.
Chlamydomonas
Harris, E. H., Burkhart, B. D., Gilham, N. W.,






reinhardtii
and Boynton, J. E. (1989) Genetics 123, 281-292.


23S
2057
“G to A”
Slightly Eryr;
E. coli
Vester, B., Hansen, L. H., and Douthwaite, S.





reduced

(1995) RNA 1, 501-509.





methylation.







Double mutation







(G2057A/C2661U)




23S
2057
“G to A”
Eryr, Cdr, Cmr.
E. coli
1. Douthwaite, S. (1992) J. Bacteriol. 174,





Double mutation

1333-1338. 2. Aaagard, C. and Douthwaite, S.





(G2057A/G2032A)

(1994) Proc. Natl. Acad. Sci. USA 91,







2989-2993. 3. Vester, B., Hansen, L. H., and







Douthwaite, S. (1995) RNA 1, 501-509.


23S
2057
G to A
Ery-R, Lin-S
Chlamydomonas
Harris, E. H., B. D. Burkhart, N. W. Gillham,






reinhardtii chl.
and J. E. Boynton. 1989. Antibiotic







resistance mutations in the chloroplast 16S







and 23S rRNA genes of Chlamydomonas







reinhardtii: correlation of genetic and







physical maps of the chloroplast genome.







Genetics.


23S
2057
G to A
Ery-R, M16-S, Lin-
Escherichia coli
Ettayebi, M., S. M. Prasad, and E. A. Morgan.





S, SB-S

1985. Chloramphenicol-







erythromycin resistance mutations in a 23S







rRNA gene of Escherichia coli. J. Bacteriol.







162: 551-557.


23S
2057
G to A
Ery-LR, M16-S
Propionibacteria
Ross, J. I., E. A. Eady, J. H. Cove, C. E. Jones,







A. H. Ratyal, Y. W. Miller, S. Vyakrnam,







and W. J. Cunliffe. 1997.







Clinical resistance to erythromycin and







clindamycin in cutaneous propionibacteria







isolated from acne patients is associated







with mutatio


23S
2057
GG to AA
Ery-R, Lin-R
Escherichia coli
Douthwaite, S. 1992. Functional







interactions within 23S rRNA involving the







peptidyltransferase center. J. Bacteriol.







174: 1333-1338.


23S
2058
“A to G”
Eryr, Lincomycin
Chlamydomonas
Harris, E. H., Burkhart, B. D., Gilham, N. W.,





and clindamycin
reinhardtii
and Boynton, J. E. (1989) Genetics 123,





resistance.

281-292.


23S
2058
“A to G”
Clarithromycin
Helecobacter
Versalovic, J., Shortridge, D., Kibler, K.,





resistance
pylori
Griffy, M. V., Beyer, J., Flamm, R. K.,







Tenaka, S. K., Graham, D. Y., and Go, M. F.







(1996) Antimicrob. Agents Chemother. 40,







477-480.


23S
2058
“A to G”
Eryr, Cdr, Cms;
E. coli
1. Vester, B. and Garrett, R. A. (1988)





abolishes

EMBO J. 7, 3577-3587. 2. Aaagard, C. and





methylation of 23S

Douthwaite, S. (1994) Proc. Natl. Acad. Sci.





rRNA by ErmE.

USA 91, 2989-2993. 3. Vester, B., Hansen, L. H.,







and Douthwaite, S. (1995) RNA 1,







501-509.


23S
2058
“A to G”
Erythromycin
Yeast
Sor, F. and Fukuhara, H. (1982) Nucleic





resistance.
mitochondria
Acids Res. 10, 6571-6577.


23S
2058
“A to G”
Lincomycin
Solanum nigrum
Kavanagh, T. A., O'Driscoll, K. M., McCabe, P. F.,





resistance.

and Dix, P. J. (1994) Mol. Gen. Genet.







242, 675-680.


23S
2058
“A to G”
Lincomycin
Tobacco
Cseplö, A., Etzold, T., Schell, J., and





resistance.
chloroplasts
Schreier, P. H. (1988) Mol. Genet. 214, 295-299.


23S
2058
“A to G”
EryS, Cds, Cms.
E. coli
1. Douthwaite, S. (1992) J. Bacteriol. 174,





Double mutation

1333-1338. 2. Aaagard, C. and Douthwaite, S.





(A2058G/G2032A).

(1994) Proc. Natl. Acad. Sci. USA 91,







2989-2993. 3. Vester, B., Hansen, L. H.,







and Douthwaite, S. (1995) RNA 1, 501-509.


23S
2058
“A to U”
Eryhs, Cds, Cms.
E. coli
1. Douthwaite, S. (1992) J. Bacteriol. 174,





Double mutation

1333-1338. 2. Aaagard, C. and Douthwaite, S.





(A2058U/G2032A).

(1994) Proc. Natl. Acad. Sci. USA 91,







2989-2993. 3. Vester, B., Hansen, L. H.,







and Douthwaite, S. (1995) RNA 1, 501-509.


23S
2058
“A to C”
Confers
E. coli
Hansen L H, Mauvais P, Douthwaite S.





resistance to the

1999. The macrolide-kelotide antibiotic





MLS drugs and

binding site is formed by structures in





chloramphenicol.

domains II and V of 23S ribosomal RNA.







Molecular Microbiology 31 (2): 623-631.


23S
2058
“A to G”
Like A2058C
E. coli
Hansen L H, Mauvais P, Douthwaite S.







1999. The macrolide-kelotide antibiotic







binding site is formed by structures in







domains II and V of 23S ribosomal RNA.







Molecular Microbiology 31 (2): 623-631.


23S
2058
“A to U”
Like A2058C
E. coli
Hansen L H, Mauvais P, Douthwaite S.







1999. The macrolide-kelotide antibiotic







binding site is formed by structures in







domains II and V of 23S ribosomal RNA.







Molecular Microbiology 31 (2): 623-631.


23S
2058
A to G/U
Ery-R, Tyl-R, Lin-R
Brachyspira
Karlsson, M., C. Fellstrom, M. U. Heldtander,






hyodysenteriae
K. E. Johansson, and A. Franklin.







1999. Genetic basis of macrolide







and lincosamide resistance in Brachyspira







(Serpulina) hyodysenteriae. FEMS







Microbiol. Lett. 172: 255-260.


23S
2058
A to G
Ery-R, Lin-R
Chlamydomonas
Harris, E. H., B. D. Burkhart, N. W. Gillham,






reinhardtii chl.
and J. E. Boynton. 1989. Antibiotic







resistance mutations in the chloroplast 16S







and 23S rRNA genes of Chlamydomonas







reinhardtii: correlation of genetic and







physical maps of the chloroplast genome.







Genetics.


23S
2058
A to G
Ery-R, Lin-R
Escherichia coli
Douthwaite, S. 1992. Functional







interactions within 23S rRNA involving the







peptidyltransferase center. J. Bacteriol.







174: 1333-1338. Vester, B., and R. A. Garrett.







1987. A plasmid-coded and site-







directed mutation in Escherichia coli 23S







RNA that confers


23S
2058
A to U
MLSB-R
Escherichia coli
Sigmund, C. D., M. Ettayebi, and E. A. Morgan.







1984. Antibiotic resistance







mutations in 16S and 23S ribosomal RNA







genes of Escherichia coli. Nucl Acids Res.







12: 4653-4663.


23S
2058
A to C
Clr-R
Helicobacter
Stone, G. G., D. Shortridge, R. K. Flamm, J. Versalovic,






pylori
J. Beyer, K. Idler, L. Zulawinski,







and S. K. Tanaka. 1996. Identification of a







23S rRNA gene mutation in clarithromycin-







resistant Helicobacter pylori. Helicobacter.







1: 227-228.


23S
2058
A to C
Mac-R, Lin-R
Helicobacter
Occhialini, A., M. Urdaci, F. Doucet-






pylori
Populaire, C. M. Bébéar, H. Lamouliatte,







and F. Mégraud. 1997. Macrolide







resistance in Helicobacter pylori: rapid







detection of point mutations and assays of







macrolide binding to ribosomes. Antimicrob.







Agents Chemothe


23S
2058
A to C
MLSB-R
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-






pylori
specific mutations in the 23S rRNA gene of







Helicobacter pylori confer two types of







resistance to macrolide-lincosamide-







streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2058
A to C
Cla-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman,






pylori
E. J. Kuipers, C. M. Vandenbroucke-







Grauls, and J. G. Kusters. 1998. Explaining







the bias in the 23S rRNA gene mutations







associated with clarithromycin resistance in







clinical isolates of Helicobacter pylori.







Antimi


23S
2058
A to G
Cla-R
Helicobacter
Versalovic, J., D. Shortridge, K. Kibler, M. V. Griffy,






pylori
J. Beyer, R. K. Flamm, S. K. Tanaka,







D. Y. Graham, and M. F. Go. 1996.







Mutations in 23S rRNA are associated with







clarithromycin resistance in Helicobacter







pylori. Antimicrob. Agents Chemother. 40: 4


23S
2058
A to G
Mac-R, Lin-R
Helicobacter
Occhialini, A., M. Urdaci, F. Doucet-






pylori
Populaire, C. M. Bébéar, H. Lamouliatte,







and F. Mégraud. 1997. Macrolide







resistance in Helicobacter pylori: rapid







detection of point mutations and assays of







macrolide binding to ribosomes. Antimicrob.







Agents Chemothe


23S
2058
A to G
MLSB-R
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-






pylori
specific mutations in the 23S rRNA gene of







Helicobacter pylori confer two types of







resistance to macrolide-lincosamide-







streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2058
A to G
Cla-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman,






pylori
E. J. Kuipers, C. M. Vandenbroucke-







Grauls, and J. G. Kusters. 1998. Explaining







the bias in the 23S rRNA gene mutations







associated with clarithromycin resistance in







clinical isolates of Helicobacter pylori.







Antimi


23S
2058
A to U
MLSB-R
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-






pylori
specific mutations in the 23S rRNA gene of







Helicobacter pylori confer two types of







resistance to macrolide-lincosamide-







streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2058
A to U
Cla-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman,






pylori
E. J. Kuipers, C. M. Vandenbroucke-







Grauls, and J. G. Kusters. 1998. Explaining







the bias in the 23S rRNA gene mutations







associated with clarithromycin resistance in







clinical isolates of Helicobacter pylori.







Antimi


23S
2058
A to G
Clr-R
Mycobacterium
Wallace, R. J., Jr., A. Meier, B. A. Brown, Y. Zhang,






abscessus
P. Sander, G. O. Onyi, and E. C. Böttger.







1996. Genetic basis for







clarithromycin resistance amoung isolates of







Mycobacterium chelonae and







Mycobacterium abscessus. Antimicrob.







Agents Chemother. 40: 1676


23S
2058
A to C/G/U
Clr-R
Mycobacterium
Nash, K. A., and C. B. Inderlied. 1995.






avium
Genetic basis of macrolide resistance in







Mycobacterium avium isolated from patients







with disseminated disease. Antimicrob.







Agents Chemother. 39: 2625-2630.


23S
2058
A to C/G/U
Clr-R
Mycobacterium
Nash, K. A., and C. B. Inderlied. 1995.






avium
Genetic basis of macrolide resistance in







Mycobacterium avium isolated from patients







with disseminated disease. Antimicrob.







Agents Chemother. 39: 2625-2630.


23S
2058
A to C/G
Clr-R
Mycobacterium
Wallace, R. J., Jr., A. Meier, B. A. Brown, Y. Zhang,






chelonae
P. Sander, G. O. Onyi, and E. C. Böttger. 1996.







Genetic basis for







clarithromycin resistance among isolates of







Mycobacterium chelonae and







Mycobacterium abscessus. Antimicrob.







Agents Chemother. 40: 1676


23S
2058
A to C/G/U
Clr-R
Mycobacterium
Meier, A., P. Kirschner, B. Springer, V. A. Steingrube,






intracellulare
B. A. Brown, R. J. Wallace, Jr.,







and E. C. Böttger. 1994. Identification of







mutations in 23S rRNA gene of







clarithromycin-resistant Mycobacterium







intracellulare. Antimicrob. Agents







Chemother. 38: 38


23S
2058
A to U
Clr-R
Mycobacterium
Burman, W. J., B. L. Stone, B. A. Brown, R. J. Wallace,






kansasii
Jr., and E. C. Böttger. 1998.







AIDS-related Mycobacterium kansasii







infection with initial resistance to







clarithromycin. Diagn. Microbiol. Infect. Dis.







31: 369-371.


23S
2058
A to G
Clr-R
Mycobacterium
Sander, P., T. Prammananan, A. Meier, K. Frischkorn,






smegmatis
and E. C. Böttger. 1997. The







role of ribosomal RNAs in macrolide







resistance. Mol. Microbiol. 26: 469-480.


23S
2058
A to G
Ery-HR, Spi-MR,
Mycoplasma
Lucier, T. S., K. Heitzman, S. K. Liu, and P. C. Hu.





Tyl-S, Lin-HR
pneumoniae
1995. Transition mutations in the 23S







rRNA of erythromycin-resistant isolates of







Mycoplasma pneumoniae. Antimicrob.







Agents Chemother. 39: 2770-2773.


23S
2058
A to G
MLSB-R
Propionibacteria
Ross, J. I., E. A. Eady, J. H. Cove, C. E. Jones,







A. H. Ratyal, Y. W. Miller, S. Vyakrnam,







and W. J. Cunliffe. 1997. Clinical







resistance to erythromycin and clindamycin







in cutaneous propionibacteria isolated from







acne patients is associated with mutatio


23S
2058
A to G
MLSB-R
Streptococcus
Tait-Kamradt, A., T. Davies, M. Cronan, M. R. Jacobs,






pneumoniae
P. C. Appelbaum, and J. Sutcliffe.







2000. Mutations in 23S rRNA and







L4 ribosomal protein account for resistance







in Pneumococcal strains selected in vitro by







macrolide passage. Antimicrobial Agents







and


23S
2058
A to G
MLSB-R
Streptomyces
Pernodet, J. L., F. Boccard, M. T. Alegre, M. H. Blondelet-






ambofaciens
Rouault, and M. Guérineau.







1988. Resistance to macrolides,







lincosamides and streptogramin type B







antibiotics due to a mutation in an rRNA







operon of Streptomyces ambofaciens.







EMBO J. 7: 277-282.


23S
2058
A to G
Ery-R
Saccharomyces
Sor, F., and H. Fukuhara. 1982.






cerevisiae mit.
Identification of two erythromycin resistance







mutations in the mitochondrial gene coding







for the large ribosomal RNA in yeast.







Nucleic Acids Res. 10: 6571-6577.


23S
2058
A to G
Ery-R
Treponema
Stamm, L. V., and H. L. Bergen. 2000. A






pallidum
point mutation associated with bacterial







macrolide resistance is present in both 23S







rRNA genes of an erythromycin-resistant







Treponema pallidum clinical isolate [letter].







Antimicrob Agents Chemother. 44: 806-807.


23S
2059
“A to G”
Clarithomycin
Helecobacter
Versalovic, J., Shortridge, D., Kibler, K.,





resistance.
pylori
Griffy, M. V., Beyer, J., Flamm, R. K., Tanaka, S. K.,







Graham, D. Y., and Go, M. F. (1996)







Antimicrob. Agents and Chemother. 40, 477-480.


23S
2059
“A to G”
Lincomycin
Tobacco
Cseplö, A., Etzold, T., Schell, J., and





resistance.
chloroplasts
Schreier, P. H. (1988) Mol. Genet. 214, 295-299.


23S
2059
“A to C”
Conferred
E. coli
Hansen L H, Mauvais P, Douthwaite S. 1999.





resistance to the

The macrolide-kelotide antibiotic binding site





MLS drugs and

is formed by structures in domains II and V





chloramphenicol.

of 23S ribosomal RNA. Molecular







Microbiology 31 (2): 623-631.


23S
2059
“A to G”
Like A2059C
E. coli
Hansen L H, Mauvais P, Douthwaite S. 1999.







The macrolide-kelotide antibiotic binding site







is formed by structures in domains II and V







of 23S ribosomal RNA. Molecular







Microbiology 31 (2): 623-631.


23S
2059
“A to U”
Like A2059C
E. coli
Hansen L H, Mauvais P, Douthwaite S. 1999.







The macrolide-kelotide antibiotic binding site







is formed by structures in domains II and V







of 23S ribosomal RNA. Molecular







Microbiology 31 (2): 623-631.


23S
2059
A to C
Mac-R, Lin-R, SB-S
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-






pylori
specific mutations in the 23S rRNA gene of







Helicobacter pylori confer two types of







resistance to macrolide-lincosamide-







streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2059
A to C
Clr-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,






pylori
C. M. Vandenbroucke-Grauls,







and J. G. Kusters. 1998. Explaining the bias







in the 23S rRNA gene mutations associated







with clarithromycin resistance in clinical







isolates of Helicobacter pylori. Antimi


23S
2059
A to G
Clr-R
Helicobacter
Versalovic, J., D. Shortridge, K. Kibler, M. V. Griffy,






pylori
J. Beyer, R. K. Flamm, S. K. Tanaka,







D. Y. Graham, and M. F. Go. 1996.







Mutations in 23S rRNA are associated with







clarithromycin resistance in Helicobacter







pylori. Antimicrob. Agents Chemother. 40: 4


23S
2059
A to G
Mac-R, Lin-R
Helicobacter
Occhialini, A., M. Urdaci, F. Doucet-






pylori
Populaire, C. M. Bébéar, H. Lamouliatte,







and F. Mégraud. 1997. Macrolide resistance







in Helicobacter pylori: rapid detection of







point mutations and assays of macrolide







binding to ribosomes. Antimicrob. Agents







Chemothe


23S
2059
A to G
Mac-R, Lin-R,
Helicobacter
Wang, G., and D. E. Taylor. 1998. Site-





SB-S
pylori
specific mutations in the 23S rRNA gene of







Helicobacter pylori confer two types of







resistance to macrolide-lincosamide-







streptogramin B antibiotics. Antimicrob.







Agents Chemother. 42: 1952-1958.


23S
2059
A to G
Cla-R
Helicobacter
Debets-Ossenkopp, Y. J., A. B. Brinkman, E. J. Kuipers,






pylori
C. M. Vandenbroucke-Grauls,







and J. G. Kusters. 1998. Explaining the bias







in the 23S rRNA gene mutations associated







with clarithromycin resistance in clinical







isolates of Helicobacter pylori. Antimi


23S
2059
A to C/G
Clr-R
Mycobacterium
Wallace, R. J., Jr., A. Meier, B. A. Brown, Y. Zhang,






abscessus
P. Sander, G. O. Onyi, and E. C. Böttger.







1996. Genetic basis for







clarithromycin resistance among isolates of







Mycobacterium chelonae and







Mycobacterium abscessus. Antimicrob.







Agents Chemother. 40: 1676


23S
2059
A to G
Clr-R
Mycobacterium
Wallace, R. J., Jr., A. Meier, B. A. Brown, Y. Zhang,






chelonae
P. Sander, G. O. Onyi, and E. C. Böttger.







1996. Genetic basis for







clarithromycin resistance among isolates of







Mycobacterium chelonae and







Mycobacterium abscessus. Antimicrob.







Agents Chemother. 40: 1676


23S
2059
A to C
Clr/Azm-R
Mycobacterium
Meier, A., P. Kirschner, B. Springer, V. A. Steingrube,






intracellulare
B. A. Brown, R. J. Wallace, Jr.,







and E. C. Böttger. 1994. Identification of







mutations in 23S rRNA gene of







clarithromycin-resistant Mycobacterium







intracellulare. Antimicrob. Agents







Chemother. 38: 38


23S
2059
A to C
Clr/Azm-R
Mycobacterium
Meier, A., P. Kirschner, B. Springer, V. A. Steingrube,






avium
B. A. Brown, R. J. Wallace, Jr.,







and E. C. Böttger. 1994. Identification of







mutations in 23S rRNA gene of







clarithromycin-resistant Mycobacterium







intracellulare. Antimicrob. Agents







Chemother. 38: 38


23S
2059
A to G
Clr-R
Mycobacterium
Sander, P., T. Prammananan, A. Meier, K. Frischkorn,






smegmatis
and E. C. Böttger. 1997. The







role of ribosomal RNAs in macrolide







resistance. Mol. Microbiol. 26: 469-480.


23S
2059
A to G
Ery-MR, Spi-HR,
Mycoplasma
Lucier, T. S., K. Heitzman, S. K. Liu, and P. C. Hu.





Tyl-LR, Lin-MR
pneumoniae
1995. Transition mutations in the 23S







rRNA of erythromycin-resistant isolates of







Mycoplasma pneumoniae. Antimicrob.







Agents Chemother. 39: 2770-2773.


23S
2059
A to G
Mac-R
Streptococcus
Tait-Kamradt, A., T. Davies, M. Cronan, M. R. Jacobs,






pneumoniae
P. C. Appelbaum, and J. Sutcliffe.







2000. Mutations in 23S rRNA and







L4 ribosomal protein account for resistance







in Pneumococcal strains selected in vitro by







macrolide passage. Antimicrobial Agents







and


23S
2059
A to G
Mac-HR, Lin-LR
Propionibacteria
Ross, J. I., E. A. Eady, J. H. Cove, C. E. Jones,







A. H. Ratyal, Y. W. Miller, S. Vyakrnam,







and W. J. Cunliffe. 1997. Clinical







resistance to erythromycin and clindamycin







in cutaneous propionibacteria isolated from







acne patients is associated with mutatio


23S
2060
“A to C”

E. coli
Vester, B. and Garrett, R. A. (1988) EMBO







J. 7, 3577-3587.


23S
2061
“G to A”
Chloramphenicol
Rat mitochondria
Vester, B. and Garrett, R. A. (1988) EMBO





resistance

J. 7, 3577-3587.


23S
2062
“A to C”
Chloramphenicol
Halobacterium
Mankin, A. S. and Garrett, R. A. (1991) J.





resistance.
halobium
Bacteriol. 173, 3559-3563.


23S
2251
“G to A”
Dominant lethal;
E. coli
Green, R., Samaha, R., and Noller, H.





Abolished both

(1997). J. Mol. Biol. 266, 40-50.





binding of tRNA







and peptidyl







transferase activity.




23S
2251
“G to A”
Dominant lethal;
E. coli
Gregory, S. T. and Dahlberg, A. E.





subunit association

(unpublished).





defect.




23S
2251
“G to C”
Dominant lethal
E. coli
Gregory, S. T. and Dahlberg, A. E.





subunit association

(unpublished).





defect.




23S
2251
“G to U”
Dominant lethal
E. coli
Gregory, S. T. and Dahlberg, A. E.





subunit association

(unpublished).





defect.




23S
2251
“G to U”
Dominant lethal;
E. coli
Green, R., Samaha, R., and Noller, H.





Abolished both

(1997). J. Mol. Biol. 266, 40-50.





binding of tRNA







and peptidyl







transferase activity.




23S
2251
“G to A”
Dominant lethal;
E. coli
Gregory S T, Dahlberg A E, 1999. Mutations





impairs peptidyl

in the Conserved P Loop Perturb the





transferase activity;

Conformation of Two Structural Elements





induces DMS

in the Peptidyl Transferase Center of 23 S





reactivity; induces

Ribosomal RNA. J. Mol. Biol. 285: 1475-1483.





kethoxal reactivity







in G2238, G2409,







G2410, G2529,







and G2532;







enhances CMCT







reactivity in G2238;







induces kethoxal







and CMCT







reactivity in G2269







and G2271;







induces CMCT







reactivity in U2272







and U2408;







enhances kethoxal







reactivity in G2253.




23S
2252
“G to A”
Less than 5% of
E. coli
Porse, B. T., Thi-Ngoc, H. P., and Garrett, R. A.





control level

(1996) J. Mol. Biol. 264: 472-486.





peptidyl transferase







activity.




23S
2252
“G to C”
Less than 5% of
E. coli
Porse, B. T., Thi-Ngoc, H. P., and Garrett, R. A.





control level

(1996) J. Mol. Biol. 264: 472-486.





peptidyl transferase







activity.




23S
2252
“G to U”
Less than 5% of
E. coli
Porse, B. T., Thi-Ngoc, H. P., and Garrett, R. A.





control level

(1996) J. Mol. Biol. 264: 472-486.





peptidyl transferase







activity.




23S
2252
“G to C”
Reduced rate of
E. coli
1. Lieberman, K. R. and Dahlberg, A. E.





peptidyl transferase

(1994) J. Biol. Chem. 269, 16163-16169.





bond formation in

2. Samaha, R. R., Green R., and Noller, H. F.





vitro; severely

(1995) Nature 377, 309-314. 3.





detrimental to cell

O'Connor, M., Brunelli, C. A., Firpo, M. A.,





growth. Double

Gregory, S. T., Lieberman, K. R., Lodmell, J. S.,





mutation

Moine, H., Van Ryk, D. I., and





(G2252C/G2253C).

Dahlberg, A. E. (1995) Cell Biol. 73, 859-868.


23S
2252
“G to A”
Severely detrimental
E. coli
1. Gregory, S. T., Lieberman, K. R., and





to cell growth;

Dahlberg, A. E. (1994) Nucleic Acids Res.





promoted

22, 279-284. 2. Lieberman, K. R. and





frameshifting and

Dahlberg, A. E. (1994) J. Biol. Chem. 269,





readthrough of

16163-16169.





nonsense codons.




23S
2252
“G to C”
Severely detrimental
E. coli
1. Gregory, S. T., Lieberman, K. R., and





to cell growth;

Dahlberg, A. E. (1994) Nucleic Acids Res.





promoted

22, 279-284. 2. Lieberman, K. R. and





frameshifting and

Dahlberg, A. E. (1994) J. Biol. Chem. 269,





readthrough of

16163-16169.





nonsense codons.




23S
2252
“G to U”
Severely detrimental
E. coli
1. Gregory, S. T., Lieberman, K. R., and





to cell growth;

Dahlberg, A. E. (1994) Nucleic Acids Res.





promoted

22, 279-284. 2. Lieberman, K. R. and





frameshifting and

Dahlberg, A. E. (1994) J. Biol. Chem. 269,





readthrough of

16163-16169.





nonsense codons.




23S
2252
“G to A”
Dominant lethal;
E. coli
Gregory S T, Dahlberg A E, 1999.





impairs peptidyl

Mutations in the Conserved P Loop





transferase activity;

Perturb the Conformation of Two





induces DMS

Structural Elements in the Peptidyl





reactivity; induces

Transferase Center of 23 S Ribosomal





kethoxal reactivity in

RNA. J. Mol. Biol. 285: 1475-1483.





G2238, G2409,







G2410, G2529, and







G2532; enhances







CMCT reactivity in







G2238; induces







kethoxal and CMCT







reactivity in G2269







and G2271; induces







CMCT reactivity in







U2272 and U2408;







enhances kethoxal







reactivity in G2253.




23S
2252
“G to A”
Interfere with the
E. coli
Bocchetta M, Xiong L, Mankin A S. 1998.





building of peptidyl-

23S rRNA positions essential for tRNA





tRNA to P site of 50S

binding in ribosomal functional sites. Proc.





subunit.

Natl. Acad. Sci. 95: 3525-3530.


23S
2252
“G to C”
Interferes with the
E. coli
Bocchetta M, Xiong L, Mankin A S. 1998.





binding of peptidyl-

23S rRNA positions essential for tRNA





tRNA to P site of 50S

binding in ribosomal functional sites. Proc.





subunit

Natl. Acad. Sci. 95: 3525-3530.


23S
2252
“G to U”
Interferes with the
E. coli
Bocchetta M, Xiong L, Mankin A S. 1998.





binding of peptidyl-

23S rRNA positions essential for tRNA





tRNA to P site of 50S

binding in ribosomal functional sites. Proc.





subunit

Natl. Acad. Sci. 95: 3525-3530.


23S
2252
“G to U”
Dominant lethal;
E. coli
Nitta I, Ueda T, Watanabe K. 1998.





suppressed AcPhe-

Possible involvement of Escherichia coli





Phe formation;

23S ribosomal RNA in peptide bond





suppressed peptide

formation. RNA 4: 257-267.





bond formation. c




23S
2253
“G to C”
42% control level
E. coli
Porse, B. T., Thi-Ngoc, H. P., and Garrett, R. A.





peptidyl transferase

(1996) J. Mol. Biol. 264: 472-486.





activity.




23S
2253
“G to C”
Slow growth rate.
E. coli
Gregory, S. T., Lieberman, K. R., and







Dahlberg, A. E. (1994) Nucleic Acids Res.







22, 279-284.


23S
2253
“G to C”
Promoted
E. coli
1. Lieberman, K. R. and Dahlberg, A. E.





frameshifting and

(1994) J. Biol. Chem. 269, 16163-16169.





readthrough of

2. Samaha, R. R., Green R., and Noller, H. F.





nonsense codons.

(1995) Nature 377, 309-314. 3.







O'Connor, M., Brunelli, C. A., Firpo, M. A.,







Gregory, S. T., Lieberman, K. R., Lodmell, J. S.,







Moine, H., Van Ryk, D. I., and







Dahlberg, A. E. (1995) Cell Biol. 73, 859-868.


23S
2253
“G to A”
Promoted
E. coli
1. Lieberman, K. R. and Dahlberg, A. E.





frameshifting and

(1994) J. Biol. Chem. 269, 16163-16169.





readthrough of

2. Samaha, R. R., Green R., and Noller, H. F.





nonsense codons.

(1995) Nature 377, 309-314. 3.







O'Connor, M., Brunelli, C. A., Firpo, M. A.,







Gregory, S. T., Lieberman, K. R., Lodmell, J. S.,







Moine, H., Van Ryk, D. I., and







Dahlberg, A. E. (1995) Cell Biol. 73, 859-868.


23S
2253
“G to U”
Promoted
E. coli
1. Lieberman, K. R. and Dahlberg, A. E.





frameshifting and

(1994) J. Biol. Chem. 269, 16163-16169.





readthrough of

2. Samaha, R. R., Green R., and Noller, H. F.





nonsense codons.

(1995) Nature 377, 309-314. 3.







O'Connor, M., Brunelli, C. A., Firpo, M. A.,







Gregory, S. T., Lieberman, K. R., Lodmell, J. S.,







Moline, H., Van Ryk, D. I., and







Dahlberg, A. E. (1995) Cell Biol. 73, 859-868.


23S
2253
“G to A”
19% of control level
E. coli
Porse, B. T., Thi-Ngoc, H. P., and Garrett, R. A.





peptidyl transferase

(1996) J. Mol. Biol. 264: 472-486.





activity.




23S
2253
“G to C”
Severely detrimental
E. coli
1. Lieberman, K. R. and Dahlberg, A. E.





to cell growth;

(1994) J. Biol. Chem. 269, 16163-16169.





reduced rate of

2. Samaha, R. R., Green R., and Noller, H. F.





peptide bond

(1995) Nature 377, 309-314. 3.





formation in vitro.

O'Connor, M., Brunelli, C. A., Firpo, M. A.,





Double mutations

Gregory, S. T., Lieberman, K. R., Lodmell, J. S.,





(C2253C/2252C).

Moine, H., Van Ryk, D. I., and







Dahlberg, A. E. (1995) Cell Biol. 73, 859-868.


23S
2253
“G to U”
Less than 5% control
E. coli
Porse, B. T., Thi-Ngoc, H. P., and Garrett, R. A.





level peptidyl

(1996) J. Mol. Biol. 264: 472-486.





transferase activity.




23S
2253
“G to A”
Induced DMS
E. coli
Gregory S T, Dahlberg A E, 1999.





reactivity; enhanced

Mutations in the Conserved P Loop





CMCT reactivity in

Perturb the conformation of Two





G2238; induced

Structural Elements in the Peptidyl





kethoxal and CMCT

Transferase center of 23 S Ribosomal





reactivity in G2269

RNA. J. Mol. Biol. 285: 1475-1483.





and G2271; induced







CMCT reactivity in







U2272; induced







kethoxal reactivity in







G2409 and G2410.




23S
2253
“G to C”
Induced DMS
E. coli
Gregory S T, Dahlberg A E, 1999.





reactivity; enhanced

Mutations in the Conserved P Loop





CMCT reactivity in

Perturb the Conformation of Two





G2238; induced

Structural Elements in the Peptidyl





kethoxal and CMCT

Transferase Center of 23 S Ribosomal





reactivity in G2269

RNA. J. Mol. Biol. 285: 1475-1483.





and G2271; induced







CMCT reactivity in







U2272; induced







kethoxal reactivity in







G2409 and G2410.




23S
2438
“U to A”
Amicetin resistance
Halobacterium
Leviev, I. G., Rodriguez-Fonseca, C.,





and reduced growth
halobium
Phan, H., Garrett, R. A., Heliek, G., Noller, H. F.,





rate.

and Mankin, A. S (1994) EMBO J. 13,







1682-1686.


23S
2438
“U to C”
Amicetin resistance.
Halobacterium
Leviev, I. G., Rodriguez-Fonseca, C.,






halobium
Phan, H., Garrett, R. A., Heliek, G., Noller, H. F.,







and Mankin, A. S (1994) EMBO J. 13,







1682-1686.


23S
2438
“U to G”
Unstable in presence
Halobacterium
Leviev, I. G., Rodriguez-Fonseca, C.,





or absence of
halobium
Phan, H., Garrett, R. A., Heliek, G., Noller, H. F.,





amicetin

and Mankin, A. S (1994) EMBO J. 13,







1682-1686.


23S
2447
“G to A”
Chloramphenicol
Yeast
Dujon, B. (1980) Cell 20, 185-197.





resistance.
mitochondria



23S
2447
“G to C”
Anisomycin
Halobacterium
Hummel, H. and Böck, A. (1987)





resistance.
halobium
Biochimie 69, 857-861.


23S
2450
“A to C”
Lethal.
E. coli
Vester, B. and Garrett, R. A. (1988) EMBO







J. 7, 3577-3587.


23S
2451
“A to U”
Chloramphenicol
Mouse
Kearsey, S. E. and Craig, I. W. (1981)





resistance.
mitochondria
Nature (London) 290: 607-608.


23S
2451
“A to G”
Like A2451G
E. coli
Bocchetta M, Xiong L, Mankin A S. 1998.







23S rRNA positions essential for tRNA







binding in ribosomal functional sites. Proc.







Natl. Acad. Sci. 95: 3525-3530.


23S
2451
“A to C”
Like A2451G
E. coli
Bocchetta M, Xiong L, Mankin A S. 1998.







23S rRNA positions essential for tRNA







binding in ribosomal functional sites. Proc.







Natl. Acad. Sci. 95: 3525-3530.


23S
2452
“C to A”
Chloramphenicol
Human
Blanc, H., Wright C. T., Bibb M. J., Wallace D. C.,





resistance.
mitochondria
and Clayton D. A. (1981) Proc. Natl.







Acad. Sci. USA 78, 3789-3793.


23S
2452
“C to U”
Animosycin resistance.
Halobacterium
Hummel, H. and Böck, A. (1987) Biochimie







69, 857-861.


23S
2452
“C to U”
Animosycin resistance
Tetrahymena
Sweeney, R., Yao, C. H., and Yao, M. C.






thermophilia
(1991) Genetics 127: 327-334.


23S
2452
“C to U”
Chloramphenicol
Halobacterium
Mankin, A. S. and Garrett, R. A. (1991) J.





resistance.
halobium
Bacteriol. 173: 3559-3563.


23S
2452
“C to U”
Chloramphenicol
Mouse
Slott, E. F., Shade R. O., and Lansman, R. A.





resistance
mitochondria
(1983) Mol. Cell. Biol. 3, 1694-1702.


23S
2452
“C to U”
Low level sparsomycin
Halobacterium
Tan, G. T., DeBlasio, A., and Mankin, A. S.





resistance
halobium
(1996) J. Mol. Biol. 261, 222-230.


23S
2452
C to U
Cbm-R, Lin-R
Sulfolobus
Aagaard, C., H. Phan, S. Trevisanato, and






acidocaldarius
R. A. Garrett. 1994. A spontaneous point







mutation in the single 23S rRNA gene of







the thermophilic arachaeon Sulfolobus







acidocaldarius confers multiple drug







resistance. J. Bacteriol. 176: 7744-7747.


23S
2453
“A to C”
Anisomycin resistance
Halobacterium
Hummel, H. and Böck, A. (1987) Biochimie






halobium
69, 857-861.


23S
2492
“U to A”
Frameshift
E. coli
O'Connor, M. and Dahlberg, A. E. (1996)





suppressors.

Nucleic Acids Res. 24, 2701-2705.


23S
2492
“U to C”
Frameshift
E. coli
O'Connor, M. and Dahlberg, A. E. (1996)





suppressors.

Nucleic Acids Res. 24, 2701-2705.


23S
2493
“del U”
(With A2058G and
E. coli
O'Connor, M. and Dahlberg, A. E. (1996)





erythromycin) Lethal

Nucleic Acids Res. 24, 2701-2705.





growth effects.







Frameshift







suppressors.




23S
2493
“U to A”
(With A2058G and
E. coli
1. Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Lethal

Mol. Biol. 249, 1-10. 2. O'Connor, M.,





growth effects.

Brunelli, C. A., Firpo, M. A., Gregory, S. T.,





Frameshift suppressors

Lieberman, K. R., Lodmell, J. S., Moine, H.,







Van Ryk, D. I., and Dahlberg, A. E. (1995)







Biochem. Cell Biology 73, 859-868.


23S
2493
“U to C”
(With A2058G and
E. coli
1. Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Lethal

Mol. Biol. 249, 1-10. 2. O'Connor, M.,





growth effects.

Brunelli, C. A., Firpo, M. A., Gregory, S. T.,





Frameshift suppressors

Lieberman, K. R., Lodmell, J. S., Moine, H.,







Van Ryk, D. I., and Dahlberg, A. E. (1995)







Biochem. Cell Biology 73, 859-868.







3. O'Connor, M. and Dahlberg, A. E. (1996)







Nucleic Acids Res. 24, 2701-2705


23S
2493
“U to C”
(With A2058G and
E. coli
O'Connor, M. and Dahlberg, A. E. (1996)





erythromycin) Lethal

Nucleic Acids Res. 24, 2701-2705





growth effects.







Frameshift suppressors




23S
2493
“U to G”
(With A2058G and
E. coli
O'Connor, M. and Dahlberg, A. E. (1996)





erythromycin) Lethal

Nucleic Acids Res. 24, 2701-2705





growth effects.







Frameshift suppressors




23S
2493
“U to A”
(With A2058G and
E. coli
O'Connor, M. and Dahlberg, A. E. (1996)





erythromycin) Lethal

Nucleic Acids Res. 24, 2701-2705





growth effects.







Frameshift suppressors




23S
2493
“U to C”
Increased misreading.
E. coli
O'Connor, M. and Dahlberg, A. E. (1996)





Double mutation

Nucleic Acids Res. 24, 2701-2705





(U2493C/G2458A)




23S
2493
“U to C”
Increased misreading.
E. coli
O'Connor, M. and Dahlberg, A. E. (1996)





Double mutation

Nucleic Acids Res. 24, 2701-2705





(U2493C/G2458C)




23S
2497
“A to G”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Reduced

Mol. Biol. 249, 1-10.





growth rate.




23S
2499
“C to U”
Sparsomycin
Halobacterium
Tan, G. T., DeBlasio, A. and Mankin, A. S.





resistance
halobium
(1996) J. Mol. Biol. 261, 222-230


23S
2500
U2500A/C2501A
Inhibits binding of 1A
E. coli
Porse B T, Garrett R A. 1999. Sites of





streptogramin B,

Interaction of Streptogramin A and B





antibiotic pristinamycin

Antibiotics in the Peptidyl Transferase





1A on peptidyl

Loop of 23 S rRNA and the Synergism of





transferase loop

their Inhibitory Mechanisms. J. Mol. Biol.





causing inhibition of

286: 375-387.





peptide elongation. c




23S
2500
U2500A/C2501G
Like U2500A/C2501A. c
E. coli
Porse B T, Garrett R A. 1999. Sites of







Interaction of Streptogramin A and B







Antibiotics in the Peptidyl Transferase







Loop of 23 S rRNA and the Synergism of







their Inhibitory Mechanisms. J. Mol. Biol.







286: 375-387.


23S
2500
U2500A/C2501U
Like U2500A/C2501A. c
E. coli
Porse B T, Garrett R A. 1999. Sites of







Interaction of Streptogramin A and B







Antibiotics in the Peptidyl Transferase







Loop of 23 S rRNA and the Synergism of







their Inhibitory Mechanisms. J. Mol. Biol.







286: 375-387.


23S
2500
U2500G/C2501A
Like U2500A/C2501A. c
E. coli
Porse B T, Garrett R A. 1999. Sites of







Interaction of Streptogramin A and B







Antibiotics in the Peptidyl Transferase







Loop of 23 S rRNA and the Synergism of







their Inhibitory Mechanisms. J. Mol. Biol.







286: 375-387.


23S
2500
U2500G/C2501G
Like U2500A/C2501A. c
E. coli
Porse B T, Garrett R A. 1999. Sites of







Interaction of Streptogramin A and B







Antibiotics in the Peptidyl Transferase







Loop of 23 S rRNA and the Synergism of







their Inhibitory Mechanisms. J. Mol. Biol.







286: 375-387.


23S
2500
U2500G/C2501U
Like U2500A/C2501A. c
E. coli
Porse B T, Garrett R A. 1999. Sites of







Interaction of Streptogramin A and B







Antibiotics in the Peptidyl Transferase







Loop of 23 S rRNA and the Synergism of







their Inhibitory Mechanisms. J. Mol. Biol.







286: 375-387.


23S
2500
U2500C/C2501A
Like U2500A/C2501A. c
E. coli
Porse B T, Garrett R A. 1999. Sites of







Interaction of Streptogramin A and B







Antibiotics in the Peptidyl Transferase







Loop of 23 S rRNA and the Synergism of







their Inhibitory Mechanisms. J. Mol. Biol.







286: 375-387.


23S
2500
U2500C/C2501G
Like U2500A/C2501A. c.
E. coli
Porse B T, Garrett R A. 1999. Sites of







Interaction of Streptogramin A and B







Antibiotics in the Peptidyl Transferase







Loop of 23 S rRNA and the Synergism of







their Inhibitory Mechanisms. J. Mol. Biol.







286: 375-387.


23S
2500
U2500C/C2501A
Like U2500A/C2501A. c
E. coli
Porse B T, Garrett R A. 1999. Sites of







Interaction of Streptogramin A and B







Antibiotics in the Peptidyl Transferase







Loop of 23 S rRNA and the Synergism of







their Inhibitory Mechanisms. J. Mol. Biol.







286: 375-387.


23S
2502
“G to A”
Decreased growth rate
E. coli
Vester, B. and Garrett, R. A. (1988) EMBO







J. 7, 3577-3587


23S
2503
“A to C”
Chloramphenicol
Yeast
Dujon, B. (1980) Cell 20, 185-197





resistance
mitochondria



23S
2503
“A to C”
Decreased growth rate;
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





CAMr

Mol. Biol. 249, 1-10.


23S
2503
“A to G”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Slow

Mol. Biol. 249, 1-10.





growth rate. CAMr




23S
2504
“U to A”
Increased readthrough
E. coli
O'Connor, M., Brunelli, C. A., Firpo, M. A.,





of stop codons and

Gregory, S. T., Lieberman, K. R., Lodmell, J. S.,





frameshifting; lethal

Moine, H., Van Ryk, D. I., and







Dahlberg, A. E. (1995) Biochem. Cell







Biology 73, 859-868.


23S
2504
“U to C”
Increased readthrough
E. coli
O'Connor, M., Brunelli, C. A., Firpo, M. A.,





of stop codons and

Gregory, S. T., Lieberman, K. R., Lodmell, J. S.,





frameshifting; lethal

Moine, H., Van Ryk, D. I., and







Dahlberg, A. E. (1995) Biochem. Cell







Biology 73, 859-868.


23S
2504
“U to C”
Chloramphenicol
Mouse
Blanc, H., Wright, C. T., Bibb, M. J.,





resistance
mitochondria
Wallace, D. C., and Clayton, D. A. (1981)







Proc. Natl. Acad. Sci. USA 78, 3789-3793


23S
2504
“U to C”
Chloramphenicol
Human
Kearsey, S. E., and Craig, I. W. (1981)





resistance
mitochondria
Nature (London) 290, 607-608


23S
2505
“G to A”
14% activity of 70S
E. coli
Porse, B. T., Thi-Ngoc, H. P. and Garrett, R. A.





ribosomes

(1996) J. Mol. Biol. 264, 472-486


23S
2505
“G to C”
(With A1067U and
E. coli
1. Saarma, U. and Remme, J. (1992)





thiostrepton) Temperature

Nucleic Acids Res. 23, 2396-2403. 2.





sensitive growth. a

Saarma, U., Lewicki, B. T. U., Margus, T.,





Hypersensitivity to CAM;

Nigul, S., and Remme, J. (1993) “The





increased sensitivity of in

Translational Apparatus: Structure,





vitro translation. Slight

Function, Regulation and Evolution” 163-172.





increase in sensitivity to







lincomycin. b No effect on







translational accuracy.




23S
2505
“G to C”
Excluded from 70S
E. coli
Porse, B. T., Thi-Ngoc, H. P. and Garrett, R. A.





ribosomes; 17% activity of

(1996) J. Mol. Biol. 264, 472-486





70S ribosomes




23S
2505
“G to U”
<5% activity of 70S
E. coli
Porse, B. T., Thi-Ngoc, H. P. and Garrett, R. A.





ribosomes

(1996) J. Mol. Biol. 264, 472-486


23S
2505
“G to A”
Conferred resistance to
E. coli
Hansen LH, Mauvais P, Douthwaite S.





the MLS drugs and

1999. The macrolide-kelotide antibiotic





chloramphenicol.

binding site is formed by structures in







domains II and V of 23S ribosomal RNA.







Molecular Microbiology 31 (2): 623-631.


23S
2505
“G to C”
Like G2505A.
E. coli
Hansen L H, Mauvais P, Douthwaite S.







1999. The macrolide-kelotide antibiotic







binding site is formed by structures in







domains II and V of 23S ribosomal RNA.







Molecular Microbiology 31 (2): 623-631.


23S
2505
“G to U”
Like G2505A
E. coli
Hansen L H, Mauvais P, Douthwaite S.







1999. The macrolide-kelotide antibiotic







binding site is formed by structures in







domains II and V of 23S ribosomal RNA.







Molecular Microbiology 31 (2): 623-631.


23S
2506
“U to A”
Dominant lethal; 5%
E. coli
Porse, B. T., Thi-Ngoc, H. P. and Garrett, R. A.





activity of 70S ribosomes

(1996) J. Mol. Biol. 264, 472-486


23S
2508
“A to U”
Eryr, Cdr, Cms; abolishes
E. coli
1. Sigmund, C. D., Ettayebi, M., and





methylation of 23S rRNA

Morgan, E. A. (1984) Nucleic Acids Res.





by ErmE.

12, 4653-4663. 2. Vannuffel, P., Di







Giambattista, M., and Cocito, C. (1992)







J. Biol. Chem. 267, 16114-16120. 3.







Douthwaite, S. and Aagaard, C. (1993) J.







Mol. Biol. 232, 725-731. 4. Vester, B.,







Hansen, L. H., and Douthwaite, S. (1995)







RNA 1, 501-509.


23S
2508
“G to U”
Control level peptidyl
E. coli
1. Porse, B. T. and Garrett, R. A. (1995) J.





transferase activity

Mol. Biol. 249, 1-10. 2. Porse, B. T., Thi-







Ngoc, H. P. and Garrett, R. A. (1996) J.







Mol. Biol. 264, 472-486


23S
2514
“U to C”
Control level peptidyl
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





transferase activity

Mol. Biol. 249, 1-10.


23S
2516
“A to U”
Control level peptidyl
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





transferase activity

Mol. Biol. 249, 1-10.


23S
2528
“U to A”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Slow growth

Mol. Biol. 249, 1-10.





rate. Control level peptidyl







transferase activity




23S
2528
“U to C”
Control level peptidyl
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





transferase activity

Mol. Biol. 249, 1-10.


23S
2530
“A to G”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Slow growth

Mol. Biol. 249, 1-10.





rate.




23S
2546
“U to C”
Control level peptidyl
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





transferase activity.

Mol. Biol. 249, 1-10.


23S
2550
“G to A”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Slow growth

Mol. Biol. 249, 1-10.





rate.




23S
2552
“U to A”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Slow growth

Mol. Biol. 249, 1-10.





rate.




23S
2555
“U to A”
Stimulates readthrough of
E. coli
1. O'Connor, M. and Dalhberg, A. E.





stop codons and

(1993) Proc. Natl. Acad. Sci. USA 90,





frameshifting; U to A is

9214-9218 2. O'Connor, M., Brunelli, C. A.,





trpE91 frameshift

Firpo, M. A., Gregory, S. T.,





suppressor; viable in low

Lieberman, K. R., Lodmell, J. S., Moine, H.,





copy number plasmids,

Van Ryk, D. I., and Dahlberg, A. E.





but lethal when expressed

(1995) Biochem. Cell Biology 73, 859-868.





constitutively from lambda







pL promoter




23S
2555
“U to C”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Slow growth

Mol. Biol. 249, 1-10.





rate. Control level peptidyl







transferase activity




23S
2555
“U to C”
no effect
E. coli
O'Connor, M. and Dalhberg, A. E. (1993)







Proc. Natl. Acad. Sci. USA 90, 9214-9218


23S
2557
“G to A”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J. Mol.





erythromycin) Slow growth

Biol. 249, 1-10.





rate. Intermediate decrease







in peptidyl transferase







activity.




23S
2565
“A to U”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J. Mol.





erythromycin) Slow growth

Biol. 249, 1-10.





rate. Very low peptidyl







transferase activity.




23S
2580
“U to C”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J. Mol.





erythromycin) Lethal growth

Biol. 249, 1-10.





effects. No peptidyl







transferase activity.




23S
2581
“G to A”
Dominant lethal inhibition of
E. coli
1. Spahn, C., Reeme, J., Schafer, M. and





puromycin in reaction

Nierhaus, K. (1996) J. Biol. Chem. 271,







32849-32856 2. Spahn, C., Reeme, J.,







Schafer, M. and Nierhaus, K. (1996) J. Biol.







Chem. 271, 32857-32862


23S
2584
“U to A”
Deleterious; 20% activity of
E. coli
Porse, B. T., Thi-Ngoc, H. P. and Garrett, R. A.





70S ribosomes

(1996) J. Mol. Biol. 264, 472-486


23S
2584
“U to C”
Deleterious; 20% activity of
E. coli
Porse, B. T., Thi-Ngoc, H. P. and Garrett, R. A.





70S ribosomes

(1996) J. Mol. Biol. 264, 472-486


23S
2584
“U to G”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J. Mol.





erythromycin) Lethal growth

Biol. 249, 1-10.





effects. No peptidyl







transferase activity.




23S
2589
“A to G”
(With A2058G and
E. coli
Porse, B. T. and Garrett, R. A. (1995) J.





erythromycin) Slow growth

Mol. Biol. 249, 1-10.





rate. Strong reduction in







peptidyl transferase







activity.




23S
2602
A2602C/C2501A
Inhibits binding of 1A
E. coli
Porse B T, Kirillov S V, Awayez M J,





streptogramin B, antibiotic

Garrett R A. 1999. UV-induced





pristinamycin 1A on

modifications in the peptidyl





peptidyl transferase loop

transferase loop of 23S rRNA





causing inhibition of

dependent on binding of the





peptide elongation. c

streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2602
A2602C/C2501U
Like A2602C/C2501A. c
E. coli
Porse B T, Kirillov S V, Awayez M J,







Garrett RA. 1999. UV-induced







modifications in the peptidyl







transferase loop of 23S rRNA







dependent on binding of the







streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2602
A2602C/C2501G
Like A2602C/C2501A. c
E. coli
Porse B T, Kirillov S V, Awayez M J,







Garrett RA. 1999. UV-induced







modifications in the peptidyl







transferase loop of 23S rRNA







dependent on binding of the







streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2602
A2602U/C2501A
Like A2602C/C2501A. c
E. coli
Porse B T, Kirillov S V, Awayez M J,







Garrett R A. 1999. UV-induced







modifications in the peptidyl







transferase loop of 23S rRNA







dependent on binding of the







streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2602
A2602U/C2501U
Like A2602C/C2501A. c
E. coli
Porse B T, Kirillov S V, Awayez M J,







Garrett R A. 1999. UV-induced







modifications in the peptidyl







transferase loop of 23S rRNA







dependent on binding of the







streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2602
A2602U/C2501G
Like A2602C/C2501A. c
E. coli
Porse B T, Kirillov S V, Awayez M J,







Garrett R A. 1999. UV-induced







modifications in the peptidyl







transferase loop of 23S rRNA







dependent on binding of the







streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2602
A2602G/C2501A
Like A2602C/C2501A. c
E. coli
Porse B T, Kirillov S V, Awayez M J,







Garrett R A. 1999. UV-induced







modifications in the peptidyl







transferase loop of 23S rRNA







dependent on binding of the







streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2602
A2602G/C2501U
Like A2602C/C2501A. c
E. coli
Porse B T, Kirillov S V, Awayez M J,







Garrett R A. 1999. UV-induced







modifications in the peptidyl







transferase loop of 23S rRNA







dependent on binding of the







streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2602
A2602G/C2501G
Like A2602C/C2501A. c
E. coli
Porse B T, Kirillov S V, Awayez M J,







Garrett R A. 1999. UV-induced







modifications in the peptidyl







transferase loop of 23S rRNA







dependent on binding of the







streptogramin B antibiotic pristinamycin







IA. RNA 5: 585-595.


23S
2611
“C to G”
Erythromycin and
Chlamydomonas
Gauthier, A., Turmel, M. and Lemieux, C.





spiramycin resistance
reinhardtii
(1988) Mol. Gen. Genet. 214, 192-197.


23S
2611
“C to G”
Erythromycin and
Yeast
Sor, F. and Fukahara, H. (1984)





spiramycin resistance
mitochondria
Nucleic Acids Res. 12, 8313-8318.


23S
2611
“C to U”
Eryr and low level
Chlamydomonas
Harris, E. H., Burkhart, B. D., Gillham, N. W.





lincomycin and clindamycin
reinhardtii
and Boynton, J. E. (1989)





resistance

Genetics 123, 281-292


23S
2611
“C to G”
Eryr and low level
Chlamydomonas
Harris, E. H., Burkhart, B. D., Gillham, N. W.





lincomycin and clindamycin
reinhardtii
and Boynton, J. E. (1989)





resistance

Genetics 123, 281-292


23S
2611
“C to U”
Slightly Eryr; reduced
E. coli
Vester, B., Hansen, L. H., and





methylation Double

Douthwaite, S. (1995) RNA 1, 501-509





mutation (C2611U/







G2057A)




23S
2611
C to G
Ery-R, Spi-LR
Chlamydomonas
Gauthier, A., M. Turmel, and C. Lemieux.






moewusii
1988. Mapping of chloroplast






chl.
mutations conferring resistance to







antibiotics in Chlamydomonas:







evidence for a novel site of







streptomycin resistance in the small







subunit rRNA. Mol. Gen. Genet.







214: 192-197.


23S
2611
C to G/U
Ery-R, Lin-MR
Chlamydomonas
Harris, E. H., B. D. Burkhart, N. W. Gillham,






reinhardtii
and J. E. Boynton. 1989.






chl.
Antibiotic resistance mutations in the







chloroplast 16S and 23S rRNA genes







of Chlamydomonas reinhardtii:







correlation of genetic and physical







maps of the chloroplast genome.







Genetics.


23S
2611
C to U
Ery-R, Spi-S, Tyl-S, Lin-S
Escherichia
Vannuffel, P., M. Di Giambattista, E. A. Morgan,






coli
and C. Cocito. 1992.







Identification of a single base change







in ribosomal RNA leading to







erythromycin resistance. J. Biol. Chem.







267: 8377-8382.


23S
2611
C to A/G
Mac-R, SB-S
Streprococcus
Tait-Kamradt, A., T. Davies, M. Cronan,






pneumoniae
M. R. Jacobs, P. C. Appelbaum, and J. Sutcliffe.







2000. Mutations in 23S rRNA







and L4 ribosomal protein account for







resistance in Pneumococcal strains







selected in vitro by macrolide passage.







Antimicrobial Agents and


23S
2611
C to G
Ery-R, Spi-R
Saccharomyces
Sor, F., and H. Fukuhara. 1984.






cerevisiae
Erythromycin and spiramycin






mit.
resistance mutations of yeast







mitochondria: nature of the rib2 locus







in the large ribosomal RNA gene.







Nucleic Acids Res. 12: 8313-8318.


23S
2611
C to U
Ery-S, Spi-R
Saccharomyces
Sor, F., and H. Fukuhara. 1984.






cerevisiae
Erythromycin and spiramycin






mit.
resistance mutations of yeast







mitochondria: nature of the rib2 locus







in the large ribosomal RNA gene.







Nucleic Acids Res. 12: 8313-8318.


23S
2661
“G to C”
Decreased misreading;
E. coli
1. Tapprich, W. E. and Dalhberg, A. E.





streptomycin dependent

(1990) EMBO J. 9, 2649-2655 2. Tapio, S.





when expressed with Smr,

and Isaksson, L. A. (1991) Eur. J.





hyperaccurate S12

Biochem. 202, 981-984 3. Melancon, P.,





mutation.

Tapprich, W. and Brakier-Gingras, L.







(1992) J. Bacteriol. 174, 7896-7901 4.







Bilgin, N. and Ehrenberg, M. (1994) J.







Mol. Biol. 235, 813-824 5. O'Connor, M.,







Brunelli, C. A., Firpo, M. A., Gregory, S. T.,







Lieberman, K. R., Lodmell, J. S.,







Moine, H., Van Ryk, D. I., and Dahlberg, A. E.







(1995) Biochem. Cell Biology 73,







859-868.


23S
2661
“C to A”
Like C2661
E. coli
Munishkin A, Wool IG. 1997. The







ribosome-in-pieces: Binding of







elongation factor EF-G to







oligoribonucleotides that mimic the







sarcin/ricin and thiostrepton domains of







23S ribosomal RNA. Proc. Natl. Acad.







Sci. 94: 12280-12284.


23S
2661
“C to G”
Like C2661
E. coli
Munishkin A, Wool IG. 1997. The







ribosome-in-pieces: Binding of







elongation factor EF-G to







oligoribonucleotides that mimic the







sarcin/ricin and thiostrepton domains of







23S ribosomal RNA. Proc. Natl. Acad.







Sci. 94: 12280-12284.


23S
2661
“C to U”
Like C2661
E. coli
Munishkin A, Wool IG. 1997. The







ribosome-in-pieces: Binding of







elongation factor EF-G to







oligoribonucleotides that mimic the







sarcin/ricin and thiostrepton domains of







23S ribosomal RNA. Proc. Natl. Acad.







Sci. 94: 12280-12284.


23S
2666
“C to G”
Increased stop codon
E. coli
O'Connor, M. and Dalhberg, A. E. (1996)





readthrough and

Nucleic Acids Res. 24, 2701-2705





frameshifting. a Double







mutation







(C2666G/A2654C)




23S
2666
“C to G”
Minor increase in stop
E. coli
O'Connor, M. and Dalhberg, A. E. (1996)





codon readthrough and

Nucleic Acids Res. 24, 2701-2705





frameshifting. a Double







mutation







(C2666G/A2654U)




23S
2666
“C to U”
Minor increase in stop
E. coli
O'Connor, M. and Dalhberg, A. E. (1996)





codon readthrough and

Nucleic Acids Res. 24, 2701-2705





frameshifting. a Double







mutation (C2666U/A2654C)




23S
2666
“C to U”
Significant increase in stop
E. coli
O'Connor, M. and Dalhberg, A. E. (1996)





codon readthrough and

Nucleic Acids Res. 24, 2701-2705





frameshifting. a Double







mutation







(C2666U/A2654G)




23S
2666
“C to U”
Minor increase in stop
E. coli
O'Connor, M. and Dalhberg, A. E. (1996)





codon readthrough and

Nucleic Acids Res. 24, 2701-2705





frameshifting. a Double







mutation (C2666U/A2654U)






















TABLE 4








Recomended

Effect of resistance 
% Change in



Resistance
Pathogen
Etest
Mechanism
on fluorescence
Fluorescenc







MRSA
Methicillin 
Staphylococcus 

Reduced affinity of PBP2 towards penicillins;
Reduction of 
> to ≈80%



Resistant SA
aureus

nosocomial, Multi-drug-resistant (clindamycin,
Penicillin binding
reduction






gentamicin, FQ); Contain SCCmec type I, II, or III;








Usually PVL-negative; Virulent (esp. skin and lung)




CA-
Community 
Staphylococcus

Multi-drug-resistant (clindamycin, gentamicin, FQ);
Reduction of 
> to ≈80%


MRSA
acquired 
aureus

Usually only resistant to pen, ox ± eryth ± FQs; Usually
Penecillin binding. 
reduction of



MRSA;


produce PVL, especially in the US; In general the
Maintaims binding
penicillin,






organisms remain susceptible to clindamycin and to
capacity for 
clindamycin






trimethoprim sulfa. That's different from a nosocomial
clindamycin and
trimethoprim






pathogen which is usually resistant to one of these
trimethoprim
binding






antibiotics.




PVL-
Panton-
Staphylococcus

Highly abundant toxin, scausing septic shock; large
—
—


MRSA
Valentine
aureus

complications





leukocidin -








MRSA







BORSA
Border.line
Staphylococcus
Oxacillin

Reduction of 
> to ≈80%



oxacillin 
aureus


Penecillin
reduction of



resistant SA



binding.
penicillin


ORSA
Oxacillin
Staphylococcus
Oxacillin

Reduction of 
> to ≈80%



resistant SA
aureus


Penecillin
reduction of







binding.
penicillin


VRSA
Vancomycin
Staphylococcus
Vancomycin; 
Modified phenotypic features, however, include slower
Increased 




resistant SA
aureus
Teicoplanin
growth rates, a thickened cell wall, and increased levels
binding of







of PBP2 and PBP2′ (although the degree of cross-linking
Vancomycin



VRS
Vancomycin
Staphylococci
Vancomycin
within the thick cell wall seems to be reduced) [58].
Increased 
>50%



resistant Staph
(CNS)

Vancomycin resistant strains also seem to have a greater
binding of







ability to absorb the antibiotic from the outside medium,
Vancomycin 







which may be a consequence of the greater availability
>50%







of stem peptides in the thick cell wall. In addition, the








increased amounts of two PBPs may compete with the








antibiotic for the stem peptide substrates, thus








aggravating the resistance profile




VISA
Vancomycin
Staphylococcus
Vancomycin

Increased binding 
20-50%



intermediary 
aureus


of Vancomycin 




SA



20-50%



hVISA
hetero-
Staphylococcus
Vancomycin
very rare, but can be susceptible to methicillin and
Binding of 




(resistant)
aureus

resistant to vancomycin
penicillin and




Vancomycin



vancomycin



VRE
Vancomycin
Enterococci
Vancomycin; 
Reduced affinity to Van by 3 orders of magnitude
Reduction of 
>80%



resistant

Teicoplanin

vancomycin 
reduction







binding



ESBL
Extended
Entero-

Overproduction of β-Lactamases, 
Increased 
>80%



Spectrum β-
bacteriaceae

inhibited by clavulanic acid
binding of
increase



Lactamase



clavulanic acid



ESBL
Extended
Pseudomonas
Ceftazidime
Overproduction of β-Lactamases, 
Increased 
>80%



Spectrum β-
spp.

inhibited by clavulanic acid
binding of
increase



Lactamase



clavulanic acid



ESBL
Extended
Acinetobacter
Ceftazidime
Overproduction of β-Lactamases, 
Increased 
>80%



Spectrum β-


inhibited by clavulanic acid
binding of
increase



Lactamase



clavulanic acid



ESBL
Extended
BCC
Ceftazidime
Overproduction of β-Lactamases, 
Increased 
>80%



Spectrum β-


inhibited by clavulanic acid
binding of
increase







clavulanic acid



ESBL
Extended
Stenotrophomonas
Ceftazidime
Overproduction of β-Lactamases, 
Increased 
>80%



Spectrum β-
maltophilia

inhibited by clavulanic acid
binding of
increase



Lactamase



clavulanic acid



MBL
Metalo-β-

Imipenem

—
—



Lactamase







MLS
macrolide


One mechanism is called MLS, macrolide lincosamide
Reduced 
>50%



lincosamide


streptogramin. And in this situation there is an alteration
binding of




streptogramin


in a target-binding site at the 23-ribosomal RNA level.
macrolides and 







resulting in a point mutation or methylation of 23SRNA
Ketolides







resulting in reduced binding of macrolides (also








Ketolides); organisms with an efflux mechanis will bind








macrolides under FISH conditions. They are also








sensitive to clindamycin




DRSP
drug-resistant
Modify

DR is reported for beta-lactams, macrolides,
Reduction of 
>50%



S. pneumoniae
clavulanic acid,

chloramphenicol, and sulfonamides
macrolides





& single gene








detection for








efflux pump






HLAR
High level
Enterococci
Gentamycin; 

Reduction in 
>80%



Aminoglycoside

Streptomycin

Streptomycin
reduction



Resistance in



binding




Enterococci









REFERENCES


    1. Chambers, H. F. (1997). Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clinical Microbiology Reviews 10, 781-791.
    2. Swenson, J. M., Williams, P., Killgore, G. et al. (2001). Performance of eight methods, including two new methods for detection of oxacillin resistance in a challenge set of Staphylococcus aureus organisms. Journal of Clinical Microbiology 39, 3785-8
    3. Van Leeuwen, W. B., Van Pelt, C., Luijendijk, A. et al. (1999). Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-screen latex agglutination test. Journal of Clinical Microbiology 37, 3029-30.
    4. Louie, L., Majury, A., Goodfellow, J. et al. (2001). Evaluation of a latex agglutination test (MRSA-Screen) for detection of oxacillin resistance in coagulase-negative staphylococci. Journal of Clinical Microbiology 39, 4149-51.
    5. Skov, R., Smyth, R., Clausen, M. et al. (2003). Evaluation of cefoxitin 30 μg disc on Iso-Sensitest agar for detection of methicillin-resistant Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 52, 204-7
    6. Felten, A., Grandry, B., Lagrange, P. H. et al. (2002). Evaluation of three techniques for detection of low-level methicillin-resistant S. aureus (MRSA): a disc diffusion method with cefoxitin and moxalactam, the Vitek 2 system, and the MRSA-screen latex agglutination test. Journal of Clinical Microbiology 40, 2766-71.
    7. Cauwelier, B., Gordts, B., Descheemaecker, P. et al. (2004). Evaluation of a disk diffusion method with cefoxitin (30 μg) for detection of methicillin-resistant Staphylococcus aureus. European Journal of Clinical Microbiology and Infectious Diseases 23, 389-92.
    8. Kluytmans, J., Van Griethuysen, A., Willemse, P. et al. (2002). Performance of CHROM agar selective medium and oxacillin resistance screening agar base for identifying Staphylococcus aureus and detecting methicillin resistance. Journal of Clinical Microbiology 40, 2480-2.
    9. Louie, L., Matsumura, S. O., Choi, E. et al. (2000). Evaluation of three rapid methods for detection of methicillin resistance in S. aureus. Journal of Clinical Microbiology 38, 2170-3.
    10. National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standards M7-A6 and M100-S13. NCCLS, Wayne, Pa., USA.

## Claims

1. A method for the detection of antibiotic resistance in a micro-organism in a biological sample, comprising:
(a) providing a labelled antibiotic,
(b) contacting the labelled antibiotic with a biological sample comprising a micro-organism under conditions which allow binding of the labeled antibiotic to its binding site in the micro-organism or to a protein secreted by the micro-organism,
(c) detecting the labelled antibiotic in the micro-organism,
(d) identifying the micro-organism, and
(e) determining whether the amount of detectable label is altered with respect to the amount of detectable label in the micro-organism in its non-resistant form,
wherein micro-organisms in which the amount of detectable label is altered with respect to the amount of detectable label in the micro-organism in its non-resistant form are microorganisms resistant against the antibiotic.

2. The method according to claim 1, wherein the antibiotic is selected from the group consisting of aminoglycosides, carbacephems, carbapenerns, cephalosporins, glycopeptides, macrolides, monobactams, beta-lactam antibiotics, quinolones, bacitracin, sulfonamides, tetracyclines, streptogramines, chloramphenicol, clindamycin, and lincosamide.

3. The method according to claim 1, wherein the antibiotic is labelled by a luminescent labelling group.

4. The method according to claim 1, wherein the binding site is located in the cell lumen, in the cytoplasm, in the cell wall, or/and in a secreted protein.

5. The method according to claim 1, wherein the antibiotic is a beta-lactam antibiotic.

6. The method according to claim 5, wherein the beta-lactam antibiotic binds to the Penicillin Binding Protein 2.

7. The method according to claim 1, wherein the label in step (c) is detected via epifluorescence microscopy, flow cytometry, laser scanning devices, time resolved fluorometry, luminescence detection, isotope detection, hyper spectral imaging scanner, Surface Plasmon Resonance or another evanescence based reading technology.

8. The method according to claim 1, wherein the microorganism is identified in the biological sample.

9. The method according to claim 8, wherein the microorganism is identified in step (d) by using a labelled nucleic acid capable of specifically hybridizing with a nucleic acid in the micro-organism under in-situ conditions.

10. Method according to claim 8, wherein the microorganism is identified in step (d) by fluorescence in situ hybridization (FISH).

11. The method according to claim 8, wherein the identification of the micro-organism and the detection of the labelled antibiotic in the micro-organism are run concurrently.

12. The method according to claim 8, wherein the microorganism is identified in step (d) using epifluorescence microscopy, flow cytometry, laser scanning devices, time resolved fluorometry, luminescence detection, isotope detection, hyper spectral imaging scanner, Surface Plasmon Resonance or another evanescence based reading technology.

13. The method according to claim 1, wherein said micro-organism is selected from the group consisting of bacteria, yeasts and molds.

14. The method of claim 1, wherein the micro-organism is a gram positive bacterium which is perforated by using a gram Positive Perforation Buffer containing saponin, nisin, tris pH 8, lysozyme, lysostaphin and water.

15. The method according to claim 1, wherein the micro-organism is a yeasts or a mould, which is perforated by the formulation Yeast Perforation Buffer in Table 2.

16. The method according to claim 9, wherein said micro-organism is a Methicillin Resistant Staphylococcus aureus (MRSA) which is identified by in-situ hybridization simultaneously with the detection of the expression of the Penicillin Binding Protein 2 by binding a labelled β-Lactam antibiotic.

17. The method according to claim 9, wherein said micro-organism is a Methicillin Resistant Staphylococcus aureus (MRSA) which is identified by in-situ hybridization simultaneously with screening for resistance to Clindamycin or Trimethoprim sulfa.

18. The method according to claim 9, wherein a Vancomycin Resistant Staphylococcus aureus (VRSA) is identified by in-situ hybridisation simultaneously with the ability to bind labelled Vancomycin.

19. The method according to claim 9, wherein a Vancomycin Resistant Staphylococcus (VRS) is identified by in-situ hybridisation simultaneously with the ability to bind labelled Vancomycin.

20. The method according to claim 9, wherein a Vancomycin Resistant Enterococci (VRE) is identified by in-situ hybridisation simultaneously with the ability to bind labelled Vancomycin.

21. The method according to claim 8, wherein a resistance towards β-lactam antibiotics due to the secretion of β-lactamase (ESBL) is detected by the revealing of the presence of said β-lactamase by the binding of labelled clavulanic acid together with the identification of gram negative micro-organisms.

22. The method according to claim 8, wherein a resistance towards β-lactam antibiotics due to the secretion of metalo-β-lactamases (MBL) is detected by the revealing of the presence of said β-lactamase by the binding of labelled imipenem together with the identification of gram negative micro-organisms.

23. The method according to claim 8, wherein a resistance to macrolides, lincosamide and streptogramin (MLS) is detected via the binding of labelled erythromycin or/and Clindamycin together with the identification of Streptococci.

24. The method according to claim 8, wherein a drug resistant Streptococcus pneumoniae (DRSP) is identified with FSH together with the respective resistance towards beta-lactams and macrolides.

25. The method according to claim 8, wherein high level Aminoglycoside resistant Enterococci (HLAR) are detected via FSH and labelled Gentamycin.

26. The method of claim 1 which is a diagnostic method.

27. The method according to claim 3, wherein the antibiotic is labelled by a fluorescent labelling group.

28. The method according to claim 13, wherein said bacteria is a gram positive or gram negative bacteria.