Spectrofluorimetric quantification of antibiotic drug concentration in bacterial cells for the characterization of translocation across bacterial membranes.

The efficacy of antibacterial molecules depends on their capacity to reach inhibitory concentrations in the vicinity of their target. This is particularly challenging for drugs directed against Gram-negative bacteria, which have a complex envelope comprising two membranes and efflux pumps. Precise determination of the bacterial drug content is an essential prerequisite for drug development. Here we describe three approaches that have been developed in our laboratories to quantify drugs accumulated in intact cells by spectrofluorimetry, microspectrofluorimetry, and kinetics microspectrofluorimetry (KMSF). These different procedures provide complementary results that highlight the contribution of membrane-associated mechanisms, including influx through the outer membrane (OM) and efflux, and the importance of the physicochemical properties of the transported drugs for the intracellular concentration of a given antibiotic in a given bacterial population. The three key stages of this protocol are preparation of the bacterial strains in the presence of the antibiotic; preparation of the whole-cell lysates (WCLs) and fluorescence readings; and data analysis, including normalization and quantitation of the intracellular antibiotic fluorescence relative to the internal standard and the antibiotic standard curve, respectively. Fluorimetry is limited to naturally fluorescent or labeled compounds, but in contrast to existing alternative methods such as mass spectrometry, it uniquely allows single-cell analysis. From culture growth to data analysis, the protocol described here takes 5 d.

Fluoroquinolone structure and translocation flux across bacterial membrane.

Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed "Structure Intracellular Concentration Activity Relationship" (SICAR) concept.

Porin flexibility in Providencia stuartii: cell-surface-exposed loops L5 and L7 are markers of Providencia porin OmpPst1.

Epidemiologically unrelated Providencia stuartii strains isolated in hospitals in the south of France were investigated for their porin sequences and profiles. Noticeable resistance to ß-lactams was found to be associated with production of extended spectrum ß-lactamases or AmpC overproduction, but not metallo-ß-lactamases. At the same time, the expression level of outer membrane porins was unmodified in these isolates. The identity of the amino acid sequences of the major porin OmpPst1 was less than 90% in the tested clinical strains, whereas sequences of the second major porin OmpPst2 were found to be identical in all isolates. Sequence diversity identified in the OmpPst1 porins was mainly located in two cell-surface-exposed loops (L5 and L7): these loops were found to be responsible for 80% of the main movements of the protein. Parallel tempering MD simulations indicated possible coordinated movement of these loops that might affect the electrostatic interaction of the porin with membrane components (e.g. LPS) or with external molecules/surfaces. This suggests that such flexibility of surface-exposed domains of OmpPst1 may participate in bacterial adaptation to the environment.

Microspectrofluorimetry to dissect the permeation of ceftazidime in Gram-negative bacteria.

A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to understand membrane permeation and intracellular concentration of antibiotics in clinical isolates: passing the membrane barrier to reach the threshold concentration inside the bacterial periplasm or cytoplasm is the pivotal step of antibacterial activity. Ceftazidime (CAZ) is a key molecule of the combination therapy for treating resistant bacteria. We designed and synthesized different fluorescent CAZ derivatives (CAZ*, CAZ**) to dissect the early step of translocation-accumulation across bacterial membrane. Their activities were determined on E. coli strains and on selected clinical isolates overexpressing ß-lactamases. The accumulation of CAZ* and CAZ** were determined by microspectrofluorimetry and epifluorimetry. The derivatives were properly translocated to the periplasmic space when we permeabilize the outer membrane barrier. The periplasmic location of CAZ** was related to a significant antibacterial activity and with the outer membrane permeability. This study demonstrated the correlation between periplasmic accumulation and antibiotic activity. We also validated the method for approaching ß-lactam permeation relative to membrane permeability and paved the way for an original matrix for determining "Structure Intracellular Accumulation Activity Relationship" for the development of new therapeutic candidates.

New amphiphilic neamine conjugates bearing a metal binding motif active against MDR E. aerogenes Gram-negative bacteria.

Structure of bacterial envelope is one of the major factors contributing to Gram negative bacterial resistance. To develop new agents that target the bacterial membranes, we synthesized, by analogy with our previous peptide conjugates, new amphiphilic 3',4',6-trinaphthylmethylene neamines functionalized at position 5 through a short spacer by a chelating group, tris(2-pyridylmethyl)amine (TPA) and di-(picolyl)amine (DPA) and tetraazacyclotetradecane (Cyclam). ESI+ mass spectrometry analyses showed that neither Zn(II)(NeaDPA) nor Cu(II)(NeaCyclam) were stable in the Mueller Hinton (MH) medium used for antibacterial assays. In contrast Zn(NeaTPA) was stable in the MH medium. Interestingly, in MH, the free ligand NeaTPA was found bound to zinc, the zinc salt being the most abundant salt in this medium. Thus, the antibacterial activities of all compounds were evaluated as free ligands against E. coli strains, wild type AG100 and E. aerogenes EA289 (a clinical MDR strain that overexpresses AcrAB-TolC efflux pump), as well as AG100A an AcrAB- E. coli strain and EA298 a TolC- derivative. NeaCyclam and Zn(NeaTPA) were by far the most efficient compounds active against resistant isolate EA289 with MICs in the range 16-4 and 4 µM, respectively, while usual antibiotics such as ß-lactams and phenicols were inactive (MICs > 128) and ciprofloxacin was at 64 µM. Zn(NeaTPA) and NeaCyclam were shown to target and permeabilize the outer membrane of EA289 by promoting the cleavage of nitrocefin by periplasmic ß-lactamase. Moreover, all the neamine conjugates were able to block the efflux of 1,2'-dinaphthylamine in EA289, by acting on the efflux transporter located in the inner membrane. These membranotropic properties contribute to explain the activities of these neamine conjugates toward the MDR EA289 strain.

A unique peptide deformylase platform to rationally design and challenge novel active compounds.

Peptide deformylase (PDF) is considered an excellent target to develop antibiotics. We have performed an extensive characterization of a new PDF from the pathogen Streptococcus agalactiae, showing properties similar to other known PDFs. S. agalactiae PDF could be used as PDF prototype as it allowed to get complete sets of 3-dimensional, biophysical and kinetic data with virtually any inhibitor compound. Structure-activity relationship analysis with this single reference system allowed us to reveal distinct binding modes for different PDF inhibitors and the key role of a hydrogen bond in potentiating the interaction between ligand and target. We propose this protein as an irreplaceable tool, allowing easy and relevant fine comparisons between series, to design, challenge and validate novel series of inhibitors. As proof-of-concept, we report here the design and synthesis of effective specific bacterial PDF inhibitors of an oxadiazole series with potent antimicrobial activity against a multidrug resistant clinical isolate.

New insight into the structural, electrochemical and biological aspects of macroacyclic Cu(II) complexes derived from S-substituted dithiocarbazate schiff bases.

Copper (II) complexes synthesized from the products of condensation of S-methyl- and S-benzyldithiocarbazate with 2,5-hexanedione (SMHDH2 and SBHDH2 respectively) have been characterized using various physicochemical (elemental analysis, molar conductivity, magnetic susceptibility) and spectroscopic (infrared, electronic) methods. The structures of SMHDH2, its copper (II) complex, CuSMHD, and the related CuSBHD complex as well as a pyrrole byproduct, SBPY, have been determined by single crystal X-ray diffraction. In order to provide more insight into the behaviour of the complexes in solution, electron paramagnetic resonance (EPR) and electrochemical experiments were performed. Antibacterial activity and cytotoxicity were evaluated. The compounds, dissolved in 0.5% and 5% DMSO, showed a wide range of antibacterial activity against 10 strains of Gram-positive and Gram-negative bacteria. Investigations of the effects of efflux pumps and membrane penetration on antibacterial activity are reported herein. Antiproliferation activity was observed to be enhanced by complexation with copper. Preliminary screening showed Cu complexes are strongly active against human breast adenocarcinoma cancer cell lines MDA-MB-231 and MCF-7.

Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level.

Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake.

In Vivo Evolution of Bacterial Resistance in Two Cases of Enterobacter aerogenes Infections during Treatment with Imipenem.

Infections caused by multidrug resistant (MDR) bacteria are a major concern worldwide. Changes in membrane permeability, including decreased influx and/or increased efflux of antibiotics, are known as key contributors of bacterial MDR. Therefore, it is of critical importance to understand molecular mechanisms that link membrane permeability to MDR in order to design new antimicrobial strategies. In this work, we describe genotype-phenotype correlations in Enterobacter aerogenes, a clinically problematic and antibiotic resistant bacterium. To do this, series of clinical isolates have been periodically collected from two patients during chemotherapy with imipenem. The isolates exhibited different levels of resistance towards multiple classes of antibiotics, consistently with the presence or the absence of porins and efflux pumps. Transport assays were used to characterize membrane permeability defects. Simultaneous genome-wide analysis allowed the identification of putative mutations responsible for MDR. The genome of the imipenem-susceptible isolate G7 was sequenced to closure and used as a reference for comparative genomics. This approach uncovered several loci that were specifically mutated in MDR isolates and whose products are known to control membrane permeability. These were omp35 and omp36, encoding the two major porins; rob, encoding a global AraC-type transcriptional activator; cpxA, phoQ and pmrB, encoding sensor kinases of the CpxRA, PhoPQ and PmrAB two-component regulatory systems, respectively. This report provides a comprehensive analysis of membrane alterations relative to mutational steps in the evolution of MDR of a recognized nosocomial pathogen.

Novel synthetic bis-indolic derivatives with antistaphylococcal activity, including against MRSA and VISA strains.

OBJECTIVES: We report the synthesis, antibacterial activity and toxicity of 24 bis-indolic derivatives obtained during the development of new ways of synthesis of marine bis-indole alkaloids from the spongotine, topsentin and hamacanthin classes. METHODS: Innovative ways of synthesis and further structural optimizations led to bis-indoles presenting either the 1-(1H-indol-3'-yl)-1,2-diaminoethane unit or the 1-(1H-indol-3-yl)ethanamine unit. MIC determination was performed for reference and clinical strains of Staphylococcus aureus and CoNS species. MBC, time-kill kinetics, solubility, hydrophobicity index, plasma protein-binding and cytotoxicity assays were performed for lead compounds. Inhibition of the S. aureus NorA efflux pump was also tested for bis-indoles with no antistaphylococcal activity. RESULTS: Lead compounds were active against both S. aureus and CoNS species, with MICs between 1 and 4 mg/L. Importantly, the same MICs were found for MRSA and vancomycin-intermediate S. aureus strains. Early concentration-dependent bactericidal activity was observed for lead derivatives. Compounds with no intrinsic antibacterial activity could inhibit the S. aureus NorA efflux pump, which is involved in resistance to fluoroquinolones. At 0.5 mg/L, the most effective compound led to an 8-fold reduction of the ciprofloxacin MIC for the SA-1199B S. aureus strain, which overexpresses NorA. However, the bis-indole compounds displayed a high hydrophobicity index and high plasma protein binding, which significantly reduced antibacterial activity. CONCLUSIONS: We have synthesized and characterized novel bis-indole derivatives as promising candidates for the development of new antistaphylococcal treatments, with preserved activity against MDR S. aureus strains.

Conjugation of a new series of dithiocarbazate Schiff base Copper(II) complexes with vectors selected to enhance antibacterial activity.

A new series of six Schiff bases derived from S-methyldithiocarbazate (SMDTC) and S-benzyldithiocarbazate (SBDTC) with methyl levulinate (SMML, SBML), levulinic acid (SMLA, SBLA), and 4-carboxybenzaldehyde (SM4CB, SB4CB) were reacted with copper(II), producing complexes of general formula ML2 (M = Cu(II), L = ligand). All compounds were characterized using established physicochemical and spectroscopic methods. Crystal structures were determined for three Schiff bases (SMML, SBML, SBLA) and two Cu(II) complexes (Cu(SMML)2 and Cu(SMLA)2). In order to provide more insight into the behavior of the complexes in solution, electron paramagnetic resonance (EPR) and electrochemical experiments were performed. The parent ligands and their respective copper(II) complexes exhibited moderate antibacterial activity against both Gram-negative and Gram-positive bacteria. The most active ligand (SB4CB) and its analogous S-methyl derivative (SM4CB) were conjugated with various vector moieties: polyarginines (R1, R4, R9, and RW9), oligoethylene glycol (OEG), and an efflux pump blocker, phenylalanine-arginine-ß-naphthylamide (PAßN). Nonaarginine (R9) derivatives showed the most encouraging synergistic effects upon conjugation and complexation with copper ion including enhanced water solubility, bacteria cell membrane permeability, and bioactivity. These Cu(II)-R9 derivatives display remarkable antibacterial activity against a wide spectrum of bacteria and, in particular, are highly efficacious against Staphylococcus aureus with minimum inhibitory concentration (MIC) values of 0.5-1 µM. This pioneer study clearly indicates that the conjugation of cell-penetrating peptides (CPPs) to dithiocarbazate compounds greatly enhances their therapeutic potential.

New peptides with metal binding abilities and their use as drug carriers.

Many new designed molecules that target efficiently in vitro bacterial metalloproteases were completely inactive in cellulo against Gram negative bacteria. Their activities were limited by the severe restriction of the penetration/diffusion rate through the outer membrane barrier. To bypass this limitation, we have assayed the strategy of metallodrugs, to improve the delivery of hydroxamic acid inhibitors to peptide deformylase. In this metal-chaperone, to facilitate bacterial uptake, the ancillary ligand tris(2-pyridylmethyl)amine (TPA) or di(picolyl)amine (DPA) was functionalized by a tetrapeptide analogue of antimicrobial peptide, RWRW(OBn) (AA08 with TPA) and/or an efflux pump modulator PAßN (AA09 with TPA and AA27 with DPA). We prepared Co(III), Zn(II), and Cu(II) metallodrugs. Using a fluorescent hydroxamic acid, we showed that, in contrast to Cu(II) metallodrugs, Co(III) metallodrugs were stable in the Mueller Hinton (MH) broth during the time required for bacterial assays. The antibacterial activities were determined against E. coli strain wild-type (AG100) and E. coli strain deleted from acrAB efflux pump (AG100A). While none of the PDFinhs used in this study (SMP289 with an indole scaffold, AT015 and AT019 built on a 1,2,4-oxadiazole scaffold) displayed activity higher than 128 µM, all the metallodrugs were active with MICs around 8 µM both against AG100 and AG100A. However, compared to the activities of equimolar combinations of PDFinhs and the free chelating peptides (AA08, AA09, or AA27), they showed similar activities. A synergistic association between AT019 and AA08 or AA09 was determined using the fractional inhibitory concentration with AG100 and AG100A. Combinations of peptides lacking the chelating group with PDFinhs were inefficient. LC-MS analyses showed that the chelating peptides bind Zn(II) cation when incubated in MH broth. These results support the in situ formation of a zinc metallodrug, but we failed to detect it by LC-MS in MH. Nevertheless, this chelating peptides metalated with zinc act as permeabilizers which are more efficient than PAßN to facilitate the uptake of PDFinhs by Gram(-) bacteria.

1-(1H-indol-3-yl)ethanamine derivatives as potent Staphylococcus aureus NorA efflux pump inhibitors.

The synthesis of 37 1-(1H-indol-3-yl)ethanamine derivatives, including 12 new compounds, was achieved through a series of simple and efficient chemical modifications. These indole derivatives displayed modest or no intrinsic anti-staphylococcal activity. By contrast, several of the compounds restored, in a concentration-dependent manner, the antibacterial activity of ciprofloxacin against Staphylococcus aureus strains that were resistant to fluoroquinolones due to overexpression of the NorA efflux pump. Structure-activity relationships studies revealed that the indolic aldonitrones halogenated at position 5 of the indole core were the most efficient inhibitors of the S. aureus NorA efflux pump. Among the compounds, (Z)-N-benzylidene-2-(tert-butoxycarbonylamino)-1-(5-iodo-1H-indol-3-yl)ethanamine oxide led to a fourfold decrease of the ciprofloxacin minimum inhibitory concentration against the SA-1199B strain when used at a concentration of 0.5 mg L(-1) . To the best of our knowledge, this activity is the highest reported to date for an indolic NorA inhibitor. In addition, a new antibacterial compound, tert-butyl (2-(3-hydroxyureido)-2-(1H-indol-3-yl)ethyl)carbamate, which is not toxic for human cells, was also found.

New Peptide-based antimicrobials for tackling drug resistance in bacteria: single-cell fluorescence imaging.

New peptide molecules with metal binding abilities proved to be active against multidrug resistant clinical isolates. One of them labeled with a dansylated lysine has been imaged inside single-multidrug resistant bacteria cells by deep ultraviolet fluorescence, showing a heterogeneous subcellular localization. The fluorescence intensity is clearly related to the accumulation of the drug inside the bacteria, being dependent both on its concentration and on the incubation time with cells.

Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution.

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack.

Hydroxamic acids as potent inhibitors of Fe(II) and Mn(II) E. coli methionine aminopeptidase: biological activities and X-ray structures of oxazole hydroxamate-EcMetAP-Mn complexes.

New series of acids and hydroxamic acids linked to five-membered heterocycles including furan, oxazole, 1,2,4- or 1,3,4-oxadiazole, and imidazole were synthesized and tested as inhibitors against the Fe(II) , Co(II) , and Mn(II) forms of E. coli methionine aminopeptidase (MetAP) and as antibacterial agents against wild-type and acrAB E. coli strains. 2-Aryloxazol-4-ylcarboxylic acids appeared as potent and selective inhibitors of the Co(II) MetAP form, with IC(50) values in the micromolar range, whereas 5-aryloxazol-2-ylcarboxylic acid regioisomers and 5-aryl-1,2,4-oxadiazol-3-ylcarboxylic acids were shown to be inefficient against all forms of EcMetAP. Regardless of the heterocycle, all the hydroxamic acids are highly potent inhibitors and are selective for the Mn(II) and Fe(II) forms, with IC(50) values between 1 and 2 µM. One indole hydroxamic acid that we previously reported as a potent inhibitor of E. coli peptide deformylase also demonstrated efficiency against EcMetAP. To gain insight into the positioning of the oxazole heterocycle with reversed substitutions at positions 2 and 5, X-ray crystal structures of EcMetAP-Mn complexed with two such oxazole hydroxamic acids were solved. Irrespective of the [metal]/[apo-MetAP] ratio, the active site consistently contains a dinuclear manganese center, with the hydroxamate as bridging ligand. Asp 97, which adopts a bidentate binding mode to the Mn2 site in the holoenzyme, is twisted in both structures toward the hydroxamate bridging ligand to favor the formation of a strong hydrogen bond. Most of the compounds show weak antibacterial activity against a wild-type E. coli strain. However, increased antibacterial activity was observed mainly for compounds with a 2-substituted phenyl group in the presence of the nonapeptide polymyxin B and phenylalanine-arginine-ß-naphthylamide as permeabilizer and efflux pump blocker, respectively, which boost the intracellular uptake of the inhibitors.

Synthesis and evaluation of 1-(1H-indol-3-yl)ethanamine derivatives as new antibacterial agents.

A collection of 3-substituted indole derivatives was prepared using nucleophilic addition of indoles to nitrones. The compounds were then tested for their antibacterial activity against almost thirty bacterial strains representative of common human pathogens. Two types of indolic molecules inhibit the growth of Staphylococcus aureus, including MRSA and VISA strains, with MIC values ranging from 8 to 16 mg/L.

Identification and distribution of genetic markers in three closely related taxa of the Mycoplasma mycoides cluster: refining the relative position and boundaries of the Mycoplasma sp. bovine group 7 taxon (Mycoplasma leachii).

Mycoplasmas belonging to the Mycoplasma mycoides phylogenetic cluster are all important ruminant pathogens that are genetically closely related but differ in terms of severity and prevalence of the associated diseases. They are distributed among six taxa, the description of which has recently been amended. In the present study, DNA fragments that diverge between the type strains of three taxa were enriched using suppression subtractive hybridization. Of the three taxa, two were representative of the well-established species M. mycoides and M. capricolum, while the third one, Mycoplasma sp. bovine group 7 (Mbg7), has only recently been proposed as a separate species, Mycoplasma leachii. Specific DNA fragments were further characterized by sequencing and used as markers to assess the genetic diversity within and between taxa. The data indicate that the selected markers are unequally distributed within their own taxon but also across taxa. The patterns observed suggest the occurrence of a genetic continuum of strains within the M. mycoides cluster that may compromise the boundaries between taxa and, in turn, diagnosis outcomes. For Mbg7, the overall nature and distribution of the markers indicate a rather homogeneous group that is distinct from the M. capricolum and M. mycoides species and might be considered as a genomic chimera between these two species.

Suppression-subtractive hybridization as a strategy to identify taxon-specific sequences within the Mycoplasma mycoides Cluster: design and validation of an M. capricolum subsp. capricolum-specific PCR assay.

The phylogenetically related Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony are two small-ruminant pathogens involved in contagious agalactia. Their respective contributions to clinical outbreaks are not well documented, because they are difficult to differentiate with the current diagnostic techniques. In order to identify DNA sequences specific to one taxon or the other, a suppression-subtractive hybridization approach was developed. DNA fragments resulting from the reciprocal subtraction of the type strains were used as probes on a panel of M. capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony strains to assess their intrataxon specificity. Due to a high intrataxon polymorphism and important cross-reactions between taxa, a single DNA fragment was shown to be specific for M. capricolum subsp. capricolum and to be present in all M. capricolum subsp. capricolum field isolates tested in this study. A PCR assay targeting the corresponding gene (simpA51) was designed that resulted in a 560-bp amplification only in M. capricolum subsp. capricolum and in M. capricolum subsp. capripneumoniae (the etiological agent of contagious caprine pleuropneumonia). simpA51 was further improved to generate a multiplex PCR (multA51) that allows the differentiation of M. capricolum subsp. capripneumoniae from M. capricolum subsp. capricolum. Both the simpA51 and multA51 assays accurately identify M. capricolum subsp. capricolum among other mycoplasmas, including all members of the M. mycoides cluster. simpA51 and multA51 PCRs are proposed as sensitive and robust tools for the specific identification of M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae.