HK1148785B - Identification of antibiotic resistance using labelled antibiotics - Google Patents

Description

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 ß-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 ß-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 ß-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. 1. Presence of a gene conveying resistance, such as mecA, mef(E),
2. 2. Presence of a repressor gene inhibiting the phenotypic expression of said resistance mechanism; e.g. MecA repressor
3. 3. Multiple resistance mechanisms; e.g. Macrolide resistance via modification of the ribosomal binding site and presence of efflux mechanism(s).
4. 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 particular micro-organism in a biological sample, comprising the steps:

1. (a) providing a labelled antibiotic,
2. (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,
3. (c) detecting the labelled antibiotic in the micro-organism and identifying this micro-organism in the same biological sample, and
4. (d) determining whether the amount of detectable label is altered with respect to the amount of detectable label in the particular micro-organism in its non-resistant form,

wherein microorganisms in which the amount of detectable label is altered with respect to the amount of detectable label in the particular micro-organism in its non-resistant form 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/adsorbtion 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 particular micro-organism is selected from the group consisting of Staphylococcus, Enterococcus, and Streptococcus.

More preferably, the particular 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.

Ball et al. (Biochem.Biophys.Res. 93(1), 74-81, 1980) discloses using tetracycline as a naturally fluorescent antibiotic efflux marker.

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 ß-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 ß-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 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 ß-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 particular 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 particular microorganism in its non-resistant form may be added to the sample, or may be presented in a separate preparation. The particular 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 particular 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 particular micro-organism in its non-resistant form or/and said data sheet.

The method of the present invention may also employ the particular micro-organism in its resistant form as a further control, or specific values or ranges of the amount of detectable label in a particular 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 particular 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 particular 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 particular 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 particular micro-organism in its non-resistant form.

In step (c) of the method 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 particular 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 . The incubation with a labelled antibiotic may be performed at temperatures below the T_m 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 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. 1. prepare the cells for in-situ hybridisation with pores of sufficient size to allow passage of up to 50-mer oligo-nucleotides
2. 2. make membrane proteins accessible for labelled antibiotics
3. 3. maintain the integrity of both said proteins and ribosomes to allow the specific binding of antibiotics labelled with fluorophores
4. 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.

The biological sample comprising the particular 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.

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. 10µl

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.

	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	Coupling of FITC with	Coupling of Erythro
	Erythro	mycylamine with
	mycylamine	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
	FITC	Atto-495-NSI
Cysteine +FITC	Cystein + Atto-495-NSI
	FITC	Atto-495-NSI

50	Saponin
5	Nisin
20	mM	Tris pH 8
100	Lysozym
50	Lysostaphin

500	Saponin
10	Nisin
50	mM	Tris pH 8,3
215	mM	NaCl
0.1	%	SDS
5	mM	EDTA
10	mM	DTT
100	Lysozyme

23S	2032	AG to GA	Clr/Azm/Ery-R	Helicobacter pylori
23S	2058	A to C	Clr-R	Helicobacter pylori
23S	2058	A to C	Mac-R, Lin-R	Helicobacter pylori
23S	2058	A to C	MLSB -R	Helicobacter pylori
23S	2058	A to C	Cla-R	Helicobacter pylori
23S	2058	A to G	Cla-R	Helicobacter pylori
23S	2058	A to G	Mac-R, Lin-R	Helicobacter pylori
23S	2058	A to G	MLSB -R	Helicobacter pylori
23S	2058	A to G	Cla-R	Helicobacter pylori
23S	2058	A to U	MLSB -R	Helicobacter pylori
23S	2058	A to U	Cla-R	Helicobacter pylori
23S	2059	A to C	Mac-R, Lin-R, SB-S	Helicobacter pylori
23S	2059	A to C	Clr-R	Helicobacter pylori
23S	2059	A to G	Clr-R	Helicobacter pylori
23S	2059	A to G	Mac-R, Lin-R	Helicobacter pylori
23S	2059	A to G	Mac-R, Lin-R, SB-S	Helicobacter pylori
23S	2059	A to G	Cla-R	Helicobacter pylori

23S	754	"U to A"	Resistant to low concentrations of ketolide HMR3647; resistant to erythromycin b.	E. coli
23S	754	"U to A"	Confers macrolide ketolide resistance.	E. coli
23S	754	U to A	Ery-LR, Tel-LR	Escherichia coli
23S	1005	C to G	Slow growth under natural promoter; (with 2058G and erythromycin) severe growth retardation. A	I E. coli
23S	1005	C to G	Slow growth under pL promoter; (with 2058G and erythromycin) Erys. a Double mutant (C1005G/C1006U)	E. coli
23S	1006	C to U	Slow growth under pL promoter; (with 2058G and erythromycin) Erys. a Double mutant (C1005G/C1006U)	E. coli
23S	1006	C to U	Lethal under natural promoter; under pL promoter; (with 2058G and erythormycin) Erys. A	E. coli
23S	1056	G to A	Binding of both L11 and thiostrepton is weakened in RNA fragments. B	E. coli
23S	1056	G to A	Stoichiometric L11 binding.b (with 2058G and erythromycin) Reduced growth rate. a	E. coli
23S	1056	G to C	Binding of thiostrepton is weakened in RNA fragments. B	E. coli
23S	1064	C to U	Stoichiometric L11 binding. b (with 2058G and erythromycin) Reduced growth rate. a	E. coli
23S	1067	A to U	Normal growth	E. coli
23S	1067	A to G	Thiostrepton resistance in Halobacterium sp.	Halobacteri um
23S	1067	A to U	Thiostrepton resistance in Halobacterium sp.	Halobacteri um
23S	1067	A to U	A to C or U confers high level resistance to thiostrepton, whereas A to G confers intermediate level resistance; drug binding affinity is reduced similarly. a, b Expression by host RNA polymerase results in formation of active ribosomal subunits in vivo. A	E. coli
23S	1067	A to C	A to C or U confers high level resistance to thiostrepton, whereas A to G confers intermediate level resistance; drug binding affinity is reduced similarly. a, b Expression by host RNA polymerase results in formation of active ribosomal subunits in vivo. A	E. coli
23S	1067	A to G	A to C or U confers high level resistance to thiostrepton, whereas A to G confers intermediate level resistance; drug binding affinity is reduced similarly. a, b Expression by host RNA polymerase results in formation of active ribosomal subunits in vivo. A	E. coli
23S	1067	"A to U"	Constituted 30% of the total 23S rRNA pool in the ribosomes; exhibited 30% thiostrepton resistance in poly (U) translation b.	E. coli
23S	1068	G to A	Reduced L11 binding. b (with 2058G) Lethal when expressed from rrnB or pL promotor in presence of erythromycin. A	E. coli
23S	1068	G to A	Suppression of 1068A; lethality only in absence of erythromycin, a Double mutant (G1068A/G1099A)	E. coli
23S	1072	C to U	Lethal when expressed from rrnB or pL promotor in presence of erythromycin. a	E. coli

23S	1137	G to A	With 2058G and erythromycin, lethal when expressed from rrnB promoter.	E. coli
23S	1137	G to A	Restores normal growth under pL promotor; (With 2058G and erythromycin) Eryr. Double mutant (G1137A/C1006U)	E. coli
23S	1137	G to A	With 2058G and erythromycin, lethal when expressed from rrnB promoter. Double mutant (G1137A/G1138C)	E. coli
23S	1138	G to C	With 2058G and erythromycin, lethal when expressed from rrnB promoter.	E. coli
23S	1138	G to C	With 2058G and erythromycin, ethal when expressed from rrnB promoter. Double mutant (G1138C/G1137A)	E. coli
23S	1207	C to U	Erythromycin resistant. a Double mutant (C1207U/C1243U)	E. coli
23S	1208	C to U	Erythromycin resistant. a Double mutant (C1208U/C1243U)	E. coli
23S	1211	C to U	Erythromycin sensitive. a Double mutant (C1211U/C1208U)	E. coli
23S	1220	G to A	Erythromycin resistant. a Double mutant (G1220A/G1239A)	E. coli
23S	1221	C to U	Erythromycin resistant. a Double mutant (C1221U/C1229U)	E. coli
23S	1221	C to U	Erythromycin resistant. a Double mutant (C1221 U/C1233U)	E. coli
23S	1230	1230	Erythromycin sensitive. a Double deletion (1230/1231)	E. coli
23S	1231	1231	Erythromycin sensitive. a Double deletion (1231/1230)	E. coli
23S	1232	G to A	Erythromycin sensitive. a Double mutant (G1232A/G1238A)	E. coli
23S	1233	C to U	Erythromycin sensitive. a	E. coli
23S	1234	"del1234/del 1235"	Erythromycin sensitive. a Double mutant (U1234C/del1235)	E. coli

23S	1234	C to U	Erythromycin sensitive. a	E. coli
23S	1243	C to U	Erythromycin resistant. a Double mutant (C1243U/C1208U)	E. coli
23S	1243	C to U	Erythromycin resistant. a Double mutant (C1243U/C1221U)	E. coli
23S	1243	"C to U"	Erythromycin resistant. a Double mutant (C1243U/C1207).	E. coli
23S	1262	A to G	With erythromycin; lethal	E. coli
23S	1262	A to C	With erythromycin; lethal	E. coli
23S	1262	A to U	With erythromycin; reduced growth rate	E. coli
23S	1262	A to C	With erythromycin; reduced growth rate Double mutant (A1262C/U2017G)	E. coli
23S	1262	A to G	Suppression of growth effects; Wild-type growth on erythromycin Double mutant (A1262G/U2017C)	E. coli
23S	1262	A to U	Suppression of growth effects; Wild-type growth on erythromycin Double mutant (A1262U/U2017A)	E. coli
23S	1262	A to U	With erythromycin; reduced growth rate Double mutant (A1262U/U2017G)	E. coli
23S	1423	G to A	Suppressed requirement for 4.5S RNA in translation of natural mRNAs by cell extracts c	E. coli
23S	1698	A to G	Suppresses 2555 mutations	E. coli	O'Connor & Dahlberg, unpublished

23S	2017	"U to G"	Reduced growth rate on eyrthomycin.	E. coli
23S	2017	"U to C"	Reduced growth rate of erythromycin.	E. coli
23S	2017	"U to A"	Reduced growth rate of erythromycin.	E. coli
23S	2017	"U to C"	Reduced growth rate on erythromycin. Double mutation (U2017C/A1262G)	E. coli
23S	2017	"U to G"	seduced growth rate on erythromycin. Double mutation (U2017G/A1262C)	E. coli
23S	2017	"U to G"	Reduced growth rate on erythromycin. Double mutation (U2017G/A1262U)	E. coli
23S	2032	"G to A"	Lincomycin resistance.	Tobbaco chloroplasts
23S	2032	"U to G"	EryS, Cds, Cms. Double mutation (G2032A/A2058G)	E. coli
23S	2032	"G to A"	Eryhs, Cds, Cms. Double mutation (G2032A/A2058U)	E. coli
23S	2032	"G to A"	Eryr, Cdr, Cmr. Double mutation (G2032A/G2057A)	E. coli
23S	2032	AG to GA	Clr/Azm/Ery-R	Helicobacter pylori
23S	2051	"del A"	Prevents ErmE methylation. c	E. coli
23S	2052	"A to C"	Prevents ErmE methylation. c	E. coli
23S	2052	"A to G"	Like A2052C c	E. coli
23S	12052	"A to U"	LikeA2052C. c	E. coli
23S	2057	"G to A"	Eryr, Clinidamycin (Cd)s, Chloramphemicol (Cm)r; reduces methylation of 23S rRNA by ErmE.	E. coli
23S	2057	"G to A"	Eyrr.	Chlamydomonas reinhardtii
23S	2057	"G to A"	Slightly Eryr; reduced methylation. Double mutation (G2057A/C2661 U)	E. coli
23S	2057	"G to A"	Eryr, Cdr, Cmr. Double mutation (G2057A/G2032A)	E. coli
23S	2057	G to A	Ery-R, Lin-S	Chlamydomonas reinhardtii chl.
23S	2057	G to A	Ery-R, M16-S, Lin-S, SB-S	Escherichia coli
23S	2057	G to A	Ery-LR, M16-S	Propionibacteria
23S	2057	GG to AA	Ery-R, Lin-R	Escherichia coli
23S	2058	"A to G"	Eryr, Lincomycin and clindamycin resistance.	Chlamydomonas reinhardtii
23S	2058	"A to G"	Clarithromycin resistance	Helecobacter pylori
23S	2058	"A to G"	Eryr, Cdr, Cms; abolished methylation of 23S rRNA by ErmE.	E.coli
23S	2058	"A to G"	Erythromycin resistance.	Yeast mitochondria
23S	2058	"A to G"	Lincomycin resistance.	Solanum nigrum
23S	2058	"A to G"	Lincomycin resistance.	Tobacco chloroplasts
23S	2058	"A to G"	EryS, Cds, Cms. Double mutation (A2058G/G2032A)	E. coli
23S	2058	"A to U"	Eryhs, Cds, Cms. Double mutation (A2058U/G2032A)	E. coli
23S	2058	"A to C"	Confers resistance to the MLS drugs and chloramphenicol.	E. coli
23S	2058	"A to G"	Like A2058C	E. coli
23S	2058	"A to U"	Like A2058C	E. coli
23S	2058	A to G/U	Ery-R, Tyl-R, Lin-R	Brachyspira hyodysenteriae
23S	2058	A to G	Ery-R, Lin-R	Chlamydomonas reinhardtii chl.
23S	2058	A to G	Ery-R, Lin-R	Escherichia coli
23S	2058	A to U	MLSB-R	Escherichia coli
23S	2058	A to C	Clr-R	Helicobacter pylori
23S	2058	A to C	Mac-R, Lin-R	Helicobacter pylori
23S	2058	A to C	MLSB -R	Helicobacter pylori
23S	2058	A to C	Cla-R	Helicobacter pylori
23S	2058	A to G	Cla-R	Helicobacter pylori
23S	2058	A to G	Mac-R, Lin-R	Helicobacter pylori
23S	2058	A to G	MLSB -R	Helicobacter pylori
23S	2058	A to G	Cla-R	Helicobacter pylori
23S	2058	A to U	MLSB -R	Helicobacter pylori
23S	2058	A to U	Cla-R	Helicobacter pylori
23S	2058	A to G	Clr-R	Mycobacterium abscessus

23C	2058	A to C/G/U	Clr-R	Mycobacterium avium

23S	2058	A to C/G/U	Clr-R	Mycobacterium avium
23S	2058	A to C/G	Clr-R	Mycobacterium chelonae
23S	2058	A to C/G/U	Clr-R	Mycobacterium intracellulare
23S	2058	A to U	Clr-R	Mycobacterium kansasii
23S	2058	A to G	Clr-R	Mycobacterium smegmatis
23S	2058	A to G	Ery-HR, Spi-MR, Tyl-S, Lin-HR	Mycoplasma pneumoniae
23S	2058	A to G	MLSB -R	Propionibacteria
23S	2058	A to G	MLSB -R	Streptococcus pneumoniae
23S	2058	A to G	MLSB -R	Streptomyces ambofaciens
23S	2058	A to G	Ery-R	Saccharomyces cerevisiae mit.
23S	2058	A to G	Ery-R	Treponema pallidum
23S	2059	"A to G"	Clarithomycin resistance.	Helecobacter pylori
23S	2059	"A to G"	Lincomycin resistance.	Tobacco chloroplasts
23S	2059	"A to C"	Conferred resistance to the MLS drugs and chloramphenicol.	E. coli
23S	2059	"A to G"	Like A2059C	E. coli
23S	2059	"A to U"	Like A2059C	E. coli
23S	2059	A to C	Mac-R, Lin-R, SB-S	Helicobacter pylori
23S	2059	A to C	Clr-R	Helicobacter pylori
23S	2059	A to G	Clr-R	Helicobacter pylori
23S	2059	A to G	Mac-R, Lin-R	Helicobacter pylori
23S	2059	A to G	Mac-R, Lin-R, SB-S	Helicobacter pylori
23S	2059	A to G	Cla-R	Helicobacter pylori
23S	2059	A to C/G	Clr-R	Mycobacterium abscessus
23S	2059	A to G	Clr-R	Mycobacterium chelonae
23S	2059	A to C	Clr/Azm-R	Mycobacterium intracellulare
23S	2059	A to C	Clr/Azm-R	Mycobacterium avium
23S	2059	A to G	Clr-R	Mycobacterium smegmatis
23S	2059	A to G	Ery-MR, Spi-HR, Tyl-LR, Lin-MR	Mycoplasma pneumoniae
23S	2059	A to G	Mac-R	Streptococcus pneumoniae
23S	2059	A to G	Mac-HR, Lin-LR	Propionibacteria

23S	2060	"A to C"		E. coli
23S	2061	"G to A"	Chloramphenicol resistance	Rat mitochondria
23S	2062	"A to C"	chloramphenicol resistance.	Halobacterium halobium
23S	2251	"G to A"	Dominant lethal; Abolished both binding of tRNA and peptidyl transferase activity.	E. coli
23S	2251	"G to A"	Dominant lethal; subunit association defect.	E. coli	Gregory, S.T. and Dahlberg, A.E. (unpublished).
23S	2251	"G to C"	Dominant lethal subunit association defect.	E. coli	Gregory, S.T. and Dahlberg, A.E. (unpublished).
23S	2251	"G to U"	Dominant lethal subunit association defect.	E. coli	Gregory, S.T. and Dahlberg, A.E. (unpublished).
23S	2251	"G to U"	Dominant lethal; Abolished both binding of tRNA and peptidyl transferase activity.	E. coli
23S	2251	"G to A"	Dominant lethal; impairs peptidyl transferase activity; induces DMS reactivity; induces 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.	E. coli
23S	2252	"G to A"	Less than 5% of control level peptidyl transferase activity.	E. coli
23S	2252	"G to C"	Less than 5% of control level peptidyl transferase activity.	E. coli
23S	2252	"G to U"	Less than 5% of control level peptidyl transferase activity.	E. coli
23S	2252	"G to C"	Reduced rate of peptidyl transferase bond formation in vitro; severely detrimental to cell growth. Double mutation (G2252C/G2253C).	E. coli
23S	2252	"G to A"	Severely detrimental to cell growth; promoted frameshifting and readthrough of nonsense codons.	E. coli
23S	2252	"G to C"	Severely detrimental to cell growth; promoted frameshifting and readthrough of nonsense codons.	E. coli
23S	2252	"G to U"	Severely detrimental to cell growth; promoted frameshifting and readthrough of nonsense codons.	E. coli
23S	2252	"G to A"	Dominant lethal; impairs peptidyl transferase activity; induces DMS reactivity; induces 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.	E. coli
23S	2252	"G to A"	Interfere with the building of peptidyl-tRNA to P site of 50S subunit.	E. coli
23S	2252	"G to C"	Interferes with the binding of peptidyl-tRNA to P site of 50S subunit	E. coli
23S	2252	"G to U"	Interferes with the binding of peptidyl-tRNA to P site of 50S subunit	E. coli
23S	2252	"G to U"	Dominant lethal; suppressed AcPhe-Phe formation; suppressed peptide bond formation. c	E. coli
23S	2253	"G to C"	42% control level peptidyl transferase activity.	E. coli
23S	2253	"G to C"	Slow growth rate.	E. coli
23S	2253	"G to C"	Promoted frameshifting and readthrough of nonsense codons.	E. coli
23S	2253	"G to A"	Promoted frameshifting and readthrough of nonsense codons.	E. coli
23S	2253	"G to U"	Promoted frameshifting and readthrough of nonsense codons.	E. coli
23S	2253	"G to A"	19% of control level peptidyl transferase activity.	E. coli
23S	2253	"G to C"	Severely detrimental to cell growth; reduced rate of peptide bond formation in vitro. Double mutations (C2253C/2252C).	E. coli
23S	2253	"G to U"	Less than 5% control level peptidyl transferase activity.	E. coli
23S	2253	"G to A"	Induced DMS reactivity; enhanced CMCT reactivity in G2238; induced kethoxal and CMCT reactivity in G2269 and G2271; induced CMCT reactivity in U2272; induced kethoxal reactivity in G2409 and G2410.	E. coli
23S	2253	"G to C"	Induced DMS reactivity; enhanced CMCT reactivity in G2238; induced kethoxal and CMCT reactivity in G2269 and G2271; induced CMCT reactivity in U2272; induced kethoxal reactivity in G2409 and G2410.	E. coli
23S	2438	"U to A"	Amicetin resistance and reduced growth rate.	Halobacterium halobium
23S	2438	"U to C"	Amicetin resistance.	Halobacterium halobium
23S	2438	"U to G"	Unstable in presence or absence of amicetin	Halobacterium halobium
23S	2447	"G to A"	Chloramphenicol resistance.	Yeast mitochondria
23S	2447	"G to C"	Anisomycin resistance.	Halobacterium halobium
23S	2450	"A to C"	Lethal.	E. coli
23S	2451	"A to U"	Chloramphenicol resistance.	Mouse mitochondria
23S	2451	"A to G"	Like A2451G	E. coli

23S	2451	"A to C"	Like A2451G	E. coli
23S	2452	"C to A"	Chloramphenicol resistance.	Human mitochondria
23S	2452	"C to U"	Animosycin resistance.	Halobacterium
23S	2452	"C to U"	Animosycin resistance	Tetrahymena thermophilia
23S	2452	"C to U"	Chloramphenicol resistance.	Halobacterium halobium
23S	2452	"C to U"	Chloramphenicol resistance	Mouse mitochondria
23S	2452	"C to U"	Low level sparsomycin resistance	Halobacterium halobium
23S	2452	C to U	Cbm-R, Lin-R	Sulfolobus acidocaldarius
23S	2453	"A to C"	Anisomycin resistance	Halobacterium halobium

23S	2492	"U to A"	Frameshift suppressors.	E. coli
23S	2492	"U to C"	Frameshift suppressors.	E. coli
23S	2493	"del U"	(With A2058G and erythromycin) Lethal growth effects. Frameshift suppressors.	E. coli
23S	2493	"U to A"	(With A2058G and erythromycin) Lethal growth effects. Frameshift suppressors	E. coli
23S	2493	"U to C"	(With A2058G and erythromycin) Lethal growth effects. Frameshift suppressors	E. coli
23S	2493	"U to C"	(With A2058G and erythromycin) Lethal growth effects. Frameshift suppressors	E. coli
23S	2493	"U to G"	(With A2058G and erythromycin) Lethal growth effects. Frameshift suppressors	E. coli
23S	2493	"U to A"	(With A2058G and erythromycin) Lethal growth effects. Frameshift suppressors	E. coli
23S	2493	"U to C"	Increased misreading. Double mutation (U2493C/G2458A)	E. coli
23S	2493	"U to C"	Increased misreading. Double mutation "(U2493C/G2458C)	E. coli
23S	2497	"A to G"	(With A2058G and erythromycin) Reduced growth rate.	E. coli
23S	2499	"C to U"	Sparsomycin resistance	Halobacterium halobium
23S	2500	U2500A/C250 1A	Inhibits binding of 1A streptogramin B, antibiotic pristinamycin 1A on peptidyl transferase loop causing inhibition of peptide elongation. c	E. coli
23S	2500	U2500A/C250 1G	Like U2500A/C2501A. c	E. coli
23S	2500	U2500A/C250 1U	Like U2500A/C2501A. c	E. coli
23S	2500	1A	Like U2500A/C2501A. c	E. coli
23S	2500	IU2500G/C250 1G	Like U2500A/C2501 A. c	E. coli
23S	2500	U2500G/C250 1U	Like U2500A/C2501A. c	E. coli
23S	2500	U2500C/C250 1A	Like U2500A/C2501A. c	E. coli
23S	2500	U2500C/C250 1G	Like U2500A/C2501 A. C.	E. coli
23S	2500	U2500C/C250 1A	Like U2500A/C2501A.	E. coli
23S	2502	"G to A"	Decreased growth rate	E. coli
23S	2503	"A to C"	Chloramphenicol resistance	Yeast mitochondria
23S	2503	"A to C"	Decreased growth rate; CAMr	E. coli
23S	2503	"A to G"	(With A2058G and erythromycin) Slow growth rate. CAMr	E. coli
23S	2504	"U to A"	Increased readthrough of stop codons and frameshifting; lethal	E. coli
23S	2504	"U to C"	Increased readthrough of stop codons and frameshifting; lethal	E. coli
23S	2504	"U to C"	Chloramphenicol resistance	Mouse mitochondria
23S	2504	"U to C"	Chloramphenicol resistance	Human mitochondria
23S	2505	"G to A"	14% activity of 70S ribosomes	E. coli

23S	2505	"G to C"	(With A1067U and thiostrepton) Temperature sensitive growth. a Hypersensitivity to CAM; increased sensitivity of in vitro translation. Slight ,increase in sensitivity to lincomycin. b No effect on translational accuracy.	E. coli
23S	2505	"G to C"	Excluded from 70S ribosomes; 17% activity of 70S ribosomes	E. coli
23S	2505	"G to U"	<5% activity of 70S ribosomes	E. coli
23S	2505	"G to A"	Conferred resistance to the MLS drugs and chloramphenicol.	E. coli
23S	2505	"G to C"	Like G2505A.	E. coli
23S	2505	"G to U"	Like G2505A	E. coli
23S	2506	"U to A"	Dominant lethal; 5% activity of 70S ribosomes	E. coli
23S	2508	"A to U"	Eryr, Cdr, Cms; abolishes methylation of 23S rRNA by ErmE.	E. coli
23S	2508	"G to U"	Control level peptidyl activity	E. coli
23S	2514	"U to C"	Control level peptidyl transferase activity	E. coli
23S	2516	"A to U"	Control level peptidyl transferase activity	E. coli
23S	2528	"U to A"	(With A2058G and erythromycin) Slow growth rate. Control level peptidyl transferase activity	E. coli
23S	2528	"U to C"	Control level peptidyl transferase activity	E. coli
23S	2530	"A to G"	(With A2058G and erythromycin) Slow growth rate.	E. coli
23S	2546	"U to C"	Control level peptidyl transferase activity.	E. coli
23S	2550	"G to A"	(With A2058G and erythromycin) Slow growth rate.	E. coli
23S	2552	"U to A"	(With A2058G and erythromycin) Slow growth rate.	E. coli
23S	2555	"U to A"	Stimulates readthrough of stop codons and frameshifting; U to A is trpE91 frameshift suppressor; viable in low copy number plasmids, but lethal when expressed constitutively from lambda pL promoter	E. coli
23S	2555	"U to C"	(With A2058G and erythromycin) Slow growth rate. Control level peptidyl transferase activity	E. coli
23S	2555	"U to C"	no effect	E. coli

23S	2557	"G to A"	(With A2058G and erythromycin) Slow growth rate. Intermediate decrease in peptidyl transferase activity.	E. coli
23S	2565	"A to U"	(With A2058G and erythromycin) Slow growth rate. Very low peptidyl transferase activity.	E. coli
23S	2580	"U to C"	(With A2058G and erythromycin) Lethal growth effects. No peptidyl transferase activity.	E. coli
23S	2581	"G to A"	Dominant lethal inhibition of puromycin in reaction	E. coli
23S	2584	"U to A"	Deleterious; 20% activity of 70S ribosomes	E. coli
23S	2584	"U to C"	Deleterious; 20% activity of 70S ribosomes	E. coli
23S	2584	"U to G"	(With A2058G and erythromycin) Lethal growth effects. No peptidyl transferase activity.	E. coli

23S	2589	"A to G"	(With A2058G and erythromycin) Slow growth rate. Strong reduction in peptidyl transferase activity.	E. coli
23S	2602	A2602C/C250 1A	Inhibits binding of 1A streptogramin B, antibiotic pristinamycin 1 A on peptidyl transferase loop causing inhibition of peptide elongation. c	E. coli
23S	2602	A2602C/C250 1U	Like A2602C/C2501A. c	E. coli
23S	2602	A2602C/C250 1G	Like A2602C/C2501A. c	E. coli
23S	2602	A2602U/C250 1A	Like A2602C/C2501A. c	E. coli
23S	2602	A2602U/C250 1U	Like A2602C/C2501A. c	E. coli
23S	2602	A2602U/C250 1G	Like A2602C/C2501A. c	E. coli
23S	2602	A2602G/C250 1A	Like A2602C/C2501A. c	E. coli
23S	2602	A2602G/C250 1U	Like A2602C/C2501A. c	E. coli
23S	2602	A2602G/C250 1G	Like A2602C/C2501A. c	E. coli
23S	2611	"C to G"	Erythromycin and spiramycin resistance	Chlamydomon as reinhardtii
23S	2611	"C to G"	Erythromycin and spiramycin resistance	Yeast mitochondria
23S	2611	"C to U"	Eryr and low level lincomycin and clindamycin resistance	Chlamydomon as reinhardtii
23S	2611	"C to G"	Eryr and low level lincomycin and clindamycin resistance	Chlamydomon as reinhardtii
23S	2611	"C to U"	Slightly Eryr; reduced methylation Double mutation (C2611U/ G2057A)	E. coli
23S	2611	C to G	Ery-R, Spi-LR	Chlamydomon chl.
23S	2611	C to G/U	Ery-R, Lin-MR	Chlamydomon as reinhardtii chl.
23S	2611	C to U	Ery-R, Spi-S, Tyl-S, Lin-S	Escherichia coli
23S	2611	C to A/G	Mac-R, SB-S	Streptococcus pneumoniae
23S	2611	C to G	Ery-R, Spi-R	Saccharomyce s cerevisiae mit.
23S	2611	C to U	Ery-S, Spi-R	Saccharomyce s cerevisiae mit.
23S	2661	"G to C"	Decreased misreading; streptomycin dependent when expressed with Smr, hyperaccurate S12 mutation.	E. coli
23S	2661	"C to A"	Like C2661	E. coli
23S	2661	"C to G"	Like C2661	E. coli
23S	2661	"C to U"	Like C2661	E. coli
23S	2666	"C to G"	Increased stop codon readthrough and frameshifting. a Double mutation (C2666G/A2654C)	E. coli
23S	2666	"C to G"	Minor increase in stop codon readthrough and frameshifting. a Double mutation (C2666G/A2654U)	E. coli
23S	2666	"C to U"	Minor increase in stop codon readthrough and frameshifting. a Double mutation (C2666U/A2654C)	E. coli
23S	2666	"C to U"	Significant increase in stop codon readthrough and frameshifting. a Double mutation (C2666U/A2654G)	E. coli
23S	2666	"C to U"	Minor increase in stop codon readthrough and frameshifting. a Double mutation (C2666U/A2654U)	E. coli

Methicillin Resistant SA	Staphylococcus aureus	Reduced affinity of PBP2 towards penicillins; nosocomial, Multi-drug-resistant (clindamycin, gentamicin, FQ); Contain SCCmec type I, II, or III; Usually PVL-negative; Virulent (esp. skin and lung)	Reduction of Penicillin binding	> to ≈ 80% reduction
Community acquired MRSA;	Staphylococcus aureus	Multi-drug-resistant (clindamycin, gentamicin, FQ); Usually only resistant to pen, ox ± eryth ± FQs; Usually produce PVL, especially in the US; In general the organisms remain susceptible to clindamycin and to trimethoprim sulfa. That's different from a nosocomial pathogen which is usually resistant to one of these antibiotics.	Reduction of Penecillin binding. Maintaims binding capacity for clindamycin and trimethoprim	> to ≈ a 80% reduction of penicillin, clindamycin trimethoprim binding
Panton-Valentine leukocidin - MRSA	Staphylococcus aureus	Highly abundant toxin, scausing septic shock; large complications	--	--
Border.line oxacillin resistant SA	Staphylococcus aureus	Oxacillin	Reduction of Penecillin binding.	> to ≈ 80% reduction of penicillin
Oxacillin resistant SA	Staphylococcus aureus	Oxacillin	Reduction of Penecillin binding.	> to ≈ 80% reduction of penicillin
Vancomycin resistant SA	Staphylococcus aureus	Vancomycin; Teicoplanin	Modified phenotypic features, however, include slower growth rates, a thickened cell wall, and increased levels of PBP2 and PBP2' (although the degree of cross-linking within the thick cell wall seems to be reduced) [58]. Vancomycin resistant strains also seem to have a greater ability to absorb the antibiotic from the outside medium, which may be a consequence of the greater availability 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	Increased binding of Vancomycin
Vancomycin resistant Staph	Staphylococci (CNS)	Vancomycin		Increased binding of Vancomycin >50%	>50%
Vancomycin intermediary SA	Staphylococcus aureus	Vancomycin	Increased binding of Vancomycin 20 - 50%	20 - 50%
helero-(resistant) Vancomycin	Staphylococcus aureus	Vancomycin	very rare, but can be susceptible to methicillin and resistant to vancomycin	Binding of penicillin and vancomycin
Vancomycin resistant	Enterococci	Vancomycin; Teicoplanin	Reduced affinity to Van by 3 orders of magnitude	Reduction of vancomycin binding	>80% reduction
Extended Spectrum ß-Lactamase	Enterobacteriaceae	Overproduction of ß-Lactamases, inhibited by clavulanic acid	Increased binding of clavulanic acid	>80% increase
Extended Spectrum ß-Lactamase	Pseudomonas spp.	Ceftazidime	Overproduction of ß-Lactamase, inhibited by clavulanic acid	Increased binding of clavulanic acid	>80% increase
Extended Spectrum ß-Lactamase	Acinetobacter	Ceftazidime	Overproduction of ß-Lactamases, inhibited by clavulanic acid	Increased binding of clavulanic acid	>80% increase
Extended Spectrum ß-	BCC	Ceftazidime	Overproduction of ß-Lactamases, inhibited by clavulanic acid	Increased binding of clavulanic acid	>80% increase
Extended Spectrum ß-Lactamase	Stenotrophomon as maltophilia	Ceftazidime	Overproduction of ß-Lactamase, inhibited by clavulanic acid	Increased binding of clavulanic acid	>80% increase
Metalo-ß-Lactamase	Imipenem	- -	-
macrolide lincosamide streptogramin	One mechanism is called MLS, macrolide lincosamide streptogramin. And in this situation there is an alteration in a target-binding site at the 23-ribosomal RNA level. resulting in a point mutation or methylation of 23SRNA 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	Reduced binding of macrolides and Ketolides	>50%
drug-resistant S. pneumoniae	Modify clavulanic acid, & single gene detection for efflux pump	DR is reported for beta-lactams, macrolides, chloramphenicol, and sulfonamides	Reduction of macrolides	>50%
High level Aminoglycoside Resistance in Enterococci	Enterococci	Gentamycin; Streptomycin	Reduction in Streptomycin binding	>80% reduction

Claims (12)

1. Method for the detection of an antibiotic resistance in a particular 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 labelled antibiotic to its binding site in the micro-organism,

   (c) detecting the labelled antibiotic in the micro-organism and identifying this micro-organism in the same biological sample, and

   (d) determining whether the amount of detectable label is altered with respect to the amount of detectable label in the particular 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 particular micro-organism in its non-resistant form are microorganism resistant against the antibiotic.
2. The method according to claim 1, wherein 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.
3. The method according to claim 1 or 2, wherein the antibiotic is labelled by a luminescent labelling group, in particular by a fluorescent labelling group.
4. The method according to any of the preceding claims, 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 any of the preceding claims, wherein the antibiotic is a beta-lactam antibiotic, which preferably binds to the PBP2 binding protein and not to PBP2a.
6. The method according to any of the preceding claims, 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 evanscesence based reading technology.
7. The method according to any of the preceding claims, wherein the particular micro-organism is identified by a labelled nucleic acid capable of specifically hybridising with a nucleic acid in the micro-organism under in-situ conditions.
8. Method according to claim 7, wherein the particular micro-organism is identified by FISH.
9. The method according to claim 7 or 8, wherein 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 another evanscesence based reading technology.
10. The method according to any of the preceding claims, wherein the micro-organism is selected from the group consisting of bacteria, yeasts and moulds, in particular from Gram positive and Gram negative bacteria.
11. The method of any of the preceding claims, wherein the micro-organism is a Gram positive bacterium which is perforated by the formulation Gram Positive Perforation Buffer in Table 2, or wherein the micro-organism is a yeasts or a mould, which is perforated by the formulation Yeast Perforation Buffer in Table 2.
12. The method of any of the preceding claims which is a diagnostic method.