Metabolic labeling probes for interrogation of the host-pathogen interaction.

Bacterial infections are still one of the leading causes of death worldwide; despite the near-ubiquitous availability of antibiotics. With antibiotic resistance on the rise, there is an urgent need for novel classes of antibiotic drugs. One particularly troublesome class of bacteria are those that have evolved highly efficacious mechanisms for surviving inside the host. These contribute to their virulence by immune evasion, and make them harder to treat with antibiotics due to their residence inside intracellular membrane-limited compartments. This has sparked the development of new chemical reporter molecules and bioorthogonal probes that can be metabolically incorporated into bacteria to provide insights into their activity status. In this review, we provide an overview of several classes of metabolic labeling probes capable of targeting either the peptidoglycan cell wall, the mycomembrane of mycobacteria and corynebacteria, or specific bacterial proteins. In addition, we highlight several important insights that have been made using these metabolic labeling probes.

Potential of mean force and umbrella sampling simulation for the transport of 5-oxazolidinone in heterotetrameric sarcosine oxidase.

The structure of heterotetrameric sarcosine oxidase (HSO) contains a highly complex system composed of a large cavity and tunnels, which are essential for the reaction and migration of the reactants, products, and intermediates. Previous geometrical analysis using the CAVER program has predicted that there are three possible tunnels, T1, T2, and T3, for the exit pathway of the iminium intermediate, 5-oxazolidinone (5-OXA), of the enzyme reaction. Previous molecular dynamics (MD) simulation of HSO has identified the regions containing the water channels from the density distribution of water. The simulation indicated that tunnel T3 is the most probable exit pathway of 5-OXA. In the present study, the potential of mean force (PMF) for the transport of 5-OXA through tunnels T1, T2, and T3 was calculated using umbrella sampling (US) MD simulations and the weighted histogram analysis method. The PMF profiles for the three tunnels support the notion that tunnel T3 is the exit pathway of 5-OXA, and that 5-OXA tends to stay at the middle of the tunnel. The maximum errors of the calculated PMF for the predicted exit pathway, tunnel T3, were estimated by repeating the US simulations using different sets of initial positions. The PMF profile was also calculated for the transport of glycine within T3. The PMF profiles from the US simulations were in good agreement with the previous predictions that 5-OXA escape through tunnel T3 and how glycine is released to the outside of HSO was discussed.

Unveiling the biodiversity of lipid species in Corynebacteria- characterization of the uncommon lipid families in C. glutamicum and pathogen C. striatum by mass spectrometry.

Uncommon lipids in biotechnologically important Corynebacterium glutamicum and pathogen Corynebacterium striatum in genus Corynebacterium are isolated and identified by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high resolution mass measurement. We redefined several lipid structures that were previously mis-assigned or not defined, including cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), (α-d-mannopyranosyl)-(1 â†’ 4)-(α-D-glucuronyl diacyglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), acyl trehalose monomycolate (acyl-TMM). We also report the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in which many isomeric structures are present. The LIT MSn approaches afford identification of the functional group, the fatty acid substituents and their regiospecificity in the molecules, revealing the biodiversities of the lipid species in two Corynebacterium strains that have played very different and important roles in human nutrition and health.

Optimization of l-arginine purification from Corynebacterium crenatum fermentation broth.

l-Arginine has many special physiological and biochemical functions, with wide applications in the food and pharmaceutical industry. Few studies on the purification of l-arginine from fermentation broth have been conducted; however, none of them were systematic enough for industrial scale-up. Therefore, it is necessary to develop a highly efficient and systematic process for the purification of l-arginine from fermentation broth. In this study, we screened out a cation exchange resin, D155, having high exchange capacity, high selectivity, and easy elution capacity, and analyzed its adsorption isotherm, thermodynamics, and kinetics using different models. Further, the process parameters of fixed-bed ion exchange adsorption and elution were optimized, and the penetration curve during the operation was modeled. Based on the fixed-bed ion-exchange parameters, a 30-column continuous ion-exchange system was designed, and the flow velocity in each zone was optimized. Finally, to obtain a high purity of l-arginine, the purification tests were conducted using anion exchange resin 711, and an l-arginine yield of 99.1% and purity of 98.5% was obtained. This effective and economical method also provides a promising strategy for separation of other amino acids from the fermentation broth, which is of great significance to the l-arginine fermentation industry.

Corynebacterium rouxii sp. nov., a novel member of the diphtheriae species complex.

A group of six clinical isolates previously identified as Corynebacterium diphtheriae biovar Belfanti, isolated from human cutaneous or peritoneum infections and from one dog, were characterized by genomic sequencing, biochemical analysis and MALDI-TOF mass spectrometry. The six isolates were negative for the diphtheria toxin gene. Phylogenetic analyses showed that the six isolates (including FRC0190T) are clearly demarcated from C. diphtheriae, Corynebacterium belfantii, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. The average nucleotide identity of FRC0190T with C. diphtheriae NCTC11397T was 92.6%, and was 91.8% with C. belfantii FRC0043T. C. diphtheriae subsp. lausannense strain CHUV2995T appeared to be a later heterotypic synonym of C. belfantii (ANI, 99.3%). Phenotyping data revealed an atypical negative or heterogeneous intermediate maltose fermentation reaction for the six isolates. MALDI-TOF mass spectrometry differentiated the new group from the other Corynebacterium taxa by the presence of specific spectral peaks. rpoB sequences showed identity to atypical, maltose-negative C. diphtheriae biovar Belfanti isolates previously described from two cats in the USA. We propose the name Corynebacterium rouxii sp. nov. for the novel group, with FRC0190T (= CIP 111752T = DSM 110354T) as type strain.

Corynebacterium sanguinis sp. nov., a clinical and environmental associated corynebacterium.

Clinical and environmental-associated strains (n=17), genotypically related to Corynebacterium spp., yet distinct from any species of the genus Corynebacterium with validly published names, have been isolated during the last 20 years and tentatively identified as Corynebacterium sanguinis, although the combination, "Corynebacterium sanguinis" was never validly published. The comprehensive genotypic and phenotypic characterisations and genomic analyses in this study support the proposal for recognizing the species within the genus Corynebacterium, for which the name, Corynebacterium sanguinis sp. nov., is reaffirmed and proposed. Strains of Corynebacterium sanguinis are Gram-positive, non-motile, non-spore-forming, short, pleomorphic and coryneform bacilli, growing aerobically, with CO2. They contain mycolic acids, major respiratory menaquinones, MK-8 (II-H2) and MK-9 (II-H2), and polar lipids, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, phosphoglycolipid, glycolipids and a novel lipid that remains to be characterized and identified. Strains of Corynebacterium sanguinis are genotypically most similar to Corynebacterium lipophiliflavum, with 16S rRNA gene sequence similarities of 98.3% and rpoB sequence similarities of 94.9-95.2%. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis were able to clearly differentiate Corynebacterium sanguinis from the most closely related species. The genome size of Corynebacterium sanguinis is 2.28-2.37Mbp with 65.1-65.5mol% G+C content. A total of 2202-2318 ORFs were predicted, comprising 2141-2251 protein-encoding genes. The type strain is CCUG 58655T (=CCM 8873T=NCTC 14287T).

Autonomously Assembled Synthetic Transmembrane Peptide Pore.

The porinACj is an α-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a 40-amino acid, synthetic peptide, pPorA corresponding to porin PorACj, inserts into the lipid bilayers and forms well-defined pores. By electrical recordings, we measured the single-channel properties that revealed the autonomous assembly of large conductance ion-selective synthetic pores. Further, we characterized the functional properties by blocking the peptide pores by cyclodextrins of different charge and symmetry. We deduced the subunit stoichiometry and putative structure of the pore by site-specific chemical modification in single-channel electrical recordings and gel electrophoresis. On the basis of these findings, we suggest that this is a large functional uniform transmembrane pore built entirely from short synthetic α-helical peptides. Accordingly, we propose a model demonstrating structural assembly of large α-helix-based peptide pores for understanding the action of antimicrobial peptides and for the design of pores with applications in biotechnology.

Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity.

Despite sharing common features, previous studies have shown that gyrases from different species have been modified throughout evolution to modulate their properties. Here, we report two crystal structures of Mycobacterium tuberculosis DNA gyrase, an apo and AMPPNP-bound form at 2.6-Å and 3.3-Å resolution, respectively. These structures provide high-resolution structural data on the quaternary organization and interdomain connections of a gyrase (full-length GyrB-GyrA57)2 thus providing crucial inputs on this essential drug target. Together with small-angle X-ray scattering studies, they revealed an "extremely open" N-gate state, which persists even in the DNA-free gyrase-AMPPNP complex and an unexpected connection between the ATPase and cleavage core domains mediated by two Corynebacteriales-specific motifs, respectively the C-loop and DEEE-loop. We show that the C-loop participates in the stabilization of this open conformation, explaining why this gyrase has a lower ATPase activity. Our results image a conformational state which might be targeted for drug discovery.

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues.

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)-catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne-modified TMM analogue (O-AlkTMM-C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM-based reporters bearing alkyne, azide, trans-cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one- or two-step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell-surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole-cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.

Toll-Like Receptor 2 and Mincle Cooperatively Sense Corynebacterial Cell Wall Glycolipids.

Nontoxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans cause invasive disease in humans and animals. Host sensing of corynebacteria is largely uncharacterized, albeit the recognition of lipoglycans by Toll-like receptor 2 (TLR2) appears to be important for macrophage activation by corynebacteria. The members of the order Corynebacterineae (e.g., mycobacteria, nocardia, and rhodococci) share a glycolipid-rich cell wall dominated by mycolic acids (termed corynomycolic acids in corynebacteria). The mycolic acid-containing cord factor of mycobacteria, trehalose dimycolate, activates the C-type lectin receptor (CLR) Mincle. Here, we show that glycolipid extracts from the cell walls of several pathogenic and nonpathogenic Corynebacterium strains directly bound to recombinant Mincle in vitro Macrophages deficient in Mincle or its adapter protein Fc receptor gamma chain (FcRγ) produced severely reduced amounts of granulocyte colony-stimulating factor (G-CSF) and of nitric oxide (NO) upon challenge with corynebacterial glycolipids. Consistently, cell wall extracts of a particular C. diphtheriae strain (DSM43989) lacking mycolic acid esters neither bound Mincle nor activated macrophages. Furthermore, TLR2 but not TLR4 was critical for sensing of cell wall extracts and whole corynebacteria. The upregulation of Mincle expression upon encountering corynebacteria required TLR2. Thus, macrophage activation by the corynebacterial cell wall relies on TLR2-driven robust Mincle expression and the cooperative action of both receptors.

A chemical reporter strategy for detecting and identifying O-mycoloylated proteins in Corynebacterium.

Protein O-mycoloylation is a unique post-translational lipidation that was recently discovered in Corynebacterium. We describe an alkyne-modified trehalose monomycolate chemical reporter that can metabolically tag O-mycoloylated proteins in C. glutamicum, enabling their detection and identification through click chemistry.

Corynebacterium cutis Lysate Treatment Can Increase the Efficacies of PPR Vaccine.

This study aimed to evaluate the effects of Peste des petits ruminants (PPR) vaccine on cytokine and antibody levels in sheep when administered alone or in combination with Corynebacterium cutis lysate (CCL). The PPR vaccine group received a single subcutaneous axillary injection of the PPR vaccine (1 mL containing tissue culture infectious dose (TCID)50 attenuated live PPRV, n = 6) and the combination treatment (1 mL CCL and 1 mL PPR vaccine, n = 6) groups received a single subcutaneous axillary injection of both CCL and PPR vaccine. Blood samples were collected from sheep before the treatment and at different points after treatment (1, 3, 7, 14, 21, and 28 days). Plasma and serum samples were evaluated for antibody percentage, levels of cytokines IL-6, IL-10, IFN-γ, IL-4, IL-12, and IL-18, oxidative stress marker Thiobarbituric acid reactive substances, and hematological and biochemical parameters. Maximum protective antibody levels reach 3-4 weeks after vaccine administration. The combination treatment resulted in significant changes in IFN-γ, IL-4, IL-12, and IL-18 cytokine levels. These changes were not evident when only the PPR vaccine was administered and antibody percentage against PPRV was short term in PPR vaccine group. In conclusion, combined usage of the PPR vaccine with CCL resulted in a heightened cytokine response, leading to improved antibody level against PPR virus. Repeated CCL treatments can lead to earlier vaccine potency, provide protective efficacy for a longer time, and increase passive immunity.

Discovery, Production and Modification of Five Novel Lantibiotics Using the Promiscuous Nisin Modification Machinery.

To find the right conditions to isolate natively expressed antimicrobial peptides from a wide range of different microorganisms can be a challenge. Here, we exploited a heterologous expression system to produce and characterize several novel lantibiotics. We identified 54 novel putative class I and class II lantibiotics after inspecting all publicly available prokaryotic genomes using the in-house developed mining tool BAGEL3. The genes encoding these new lantibiotics fused to the nisin leader peptide gene sequence were synthesized, and the constructs were plugged into the nisin expression and modification system. Using this approach 30 peptides could be expressed, 27 of which were dehydrated by NisBC on at least 1 predicted position. Good antimicrobial activity against several pathogenic bacteria could be demonstrated for 5 novel heterologously modified lantibiotics. Lantibiotics from Corynebacterium lipophiloflavum DSM 44291 and Streptococcus agalactiae ATCC 13813, named flavucin and agalacticin, respectively, were fully modified and displayed high antimicrobial activity. The efficiency of functional expression was significantly enhanced when we made use of the native nisin leader cleavage site, instead of an artificial factor Xa site. Thus, we describe an efficient way for heterologous production of active lantibiotics, facilitating a rapid identification of promising molecules.

Corynebacterium faecale sp. nov., isolated from the faeces of Assamese macaque.

A Gram-stain-positive, facultatively anaerobic, short rod-shaped, oxidase-negative and non-motile novel strain, designated YIM 101505T, was isolated from the faeces of a primate, Assamese macaque, and was studied to determine its taxonomic position. The cell wall contained meso-diaminopimelic acid and short-chain mycolic acids. Whole cell sugars were mannose, galactose and arabinose as major components. The major fatty acids (>10 %) were C18 : 1ω9c, C16 : 0 and C17 : 1ω8c and the major menaquinone was MK-9(H2). The polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, glycolipid and six unidentified lipids. The new isolate shared most of the typical chemotaxonomic characteristics of members of the genus Corynebacterium. The closest related species was Corynebacterium efficiens based on 16S rRNA gene (98.1 % similarity) and partial rpoB gene (91.4 % similarity) sequences. Similarities with other species of this genus were below 97 % based on the 16S rRNA gene. The DNA-DNA hybridization value between YIM 101505T and C. efficiens DSM 44549T was 47.7±3.6 %. Moreover, the physiological and biochemical characteristics of YIM 101505T and C. efficiens DSM 44549T were different. Thus, strain YIM 101505T is considered to represent a novel member of the genus Corynebacterium, for which the name Corynebacterium faecale sp. nov. is proposed. The type strain is YIM 101505T (=DSM 45971T=CCTCC AB 2013226T).

Corynebacterium accolens Releases Antipneumococcal Free Fatty Acids from Human Nostril and Skin Surface Triacylglycerols.

UNLABELLED: Bacterial interspecies interactions play clinically important roles in shaping microbial community composition. We observed that Corynebacterium spp. are overrepresented in children free of Streptococcus pneumoniae (pneumococcus), a common pediatric nasal colonizer and an important infectious agent. Corynebacterium accolens, a benign lipid-requiring species, inhibits pneumococcal growth during in vitro cocultivation on medium supplemented with human skin surface triacylglycerols (TAGs) that are likely present in the nostrils. This inhibition depends on LipS1, a TAG lipase necessary for C. accolens growth on TAGs such as triolein. We determined that C. accolens hydrolysis of triolein releases oleic acid, which inhibits pneumococcus, as do other free fatty acids (FFAs) that might be released by LipS1 from human skin surface TAGs. Our results support a model in which C. accolens hydrolyzes skin surface TAGS in vivo releasing antipneumococcal FFAs. These data indicate that C. accolens may play a beneficial role in sculpting the human microbiome. IMPORTANCE: Little is known about how harmless Corynebacterium species that colonize the human nose and skin might impact pathogen colonization and proliferation at these sites. We show that Corynebacterium accolens, a common benign nasal bacterium, modifies its local habitat in vitro as it inhibits growth of Streptococcus pneumoniae by releasing antibacterial free fatty acids from host skin surface triacylglycerols. We further identify the primary C. accolens lipase required for this activity. We postulate a model in which higher numbers of C. accolens cells deter/limit S. pneumoniae nostril colonization, which might partly explain why children without S. pneumoniae colonization have higher levels of nasal Corynebacterium. This work narrows the gap between descriptive studies and the needed in-depth understanding of the molecular mechanisms of microbe-microbe interactions that help shape the human microbiome. It also lays the foundation for future in vivo studies to determine whether habitat modification by C. accolens could be promoted to control pathogen colonization.

Structural insights into the synthesis of FMN in prokaryotic organisms.

Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyltransferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure of Corynebacterium ammoniagenes FADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module of CaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-length CaFADS protomer and the quaternary organization.

[Applicability of MALDI Mass Spectrometry for Diagnostics of Phase Variants in Bacterial Populations].

Efficiency of MALDI mass spectrometry for differentiation between phenotypic phase variants (in colony morphology and virulence/avirulence) was investigated.for saprotrophic and opportunistically pathogenic bacteria of five genera (Acinetobacter, Arthrobacter, Rhodococcus, Corynebacterium, and Escherichia). Analysis of MALDI spectra (on the SA and HCCA matrices) included: (1) determination of similarity of the protein spectra as a percentage of the common protein peaks to the total amount of proteins, which reflects the phylogenetic relationships of the objects and has been recommended for identification of closely related species; (2) comparison of intensities of the common peaks; and (3) the presence of specific peaks as determinative characteristics of the variants. Under the standard analytical conditions the similarity between the MALDI profiles was shown to increase in the row: genus-species-strain-variant. Assessment of intensities of the common peaks was most applicable for differentiation between phase variants, especially in the case of high similarity of their profiles. Phase variants (A. oxydans strain K14) with similar colony morphotypes (S, R, M, and S(m)) grown on different media (LB agar, TSA, and TGYg) exhibited differences in their protein profiles reflecting the differences in their physiological characteristics. This finding is in agreement with our previous results on screening of the R. opacus with similar colony morphology and different substrate specificity in decomposition of chlorinated phenols. Analysis of MALDI spectra is probably the only efficient method for detection of such variants.

Isolation and structural characterization of Coryxin, a novel cyclic lipopeptide from Corynebacterium xerosis NS5 having emulsifying and anti-biofilm activity.

Herein we reported the structure and several properties of a new biosurfactants produced by Corynebacterium xerosis strain NS5. This strain was capable of producing a novel lipopeptide biosurfactant that we have named coryxin. The biosurfactant structure was characterized by using Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance spectroscopy (NMR), and Liquid chromatography-mass spectrometry (LC-MS). It contained a hydrophobic moiety of 3-hydroxydecanoic acid and a peptide part predicted as a sequence of seven amino acids including Asn-Arg-Asn-Gln-Pro-Asn-Ser. Coryxin lowered the surface tension of water to 31.4 mN/m, with a critical micelle concentration of 25mg/l. It was a strong emulsifier with an emulsification index of 61% against n-hexane. Coryxin showed antibacterial activity against test organisms belonging to Gram-positive and Gram-negative bacteria and disrupted preformed biofilms of Staphylococcus aureus (82.5%), Streptococcus mutans (80%), Escherichia coli (66%) and Pseudomonas aeruginosa (30%). In conclusion, microbial surfactant from C. xerosis exhibited inhibitory and disruptive activities against biofilm formation that could be of use in biofilm-related menace.

Antimicrobial Treatment Options for Granulomatous Mastitis Caused by Corynebacterium Species.

Corynebacterium species are increasingly recognized as important pathogens in granulomatous mastitis. Currently, there are no published treatment protocols for Corynebacterium breast infections. This study describes antimicrobial treatment options in the context of other management strategies used for granulomatous mastitis. Corynebacterium spp. isolated from breast tissue and aspirate samples stored from 2002 to 2013 were identified and determined to the species level using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 16S RNA sequencing, and rpoB gene targets. The MICs for 12 antimicrobials were performed using Etest for each isolate. Correlations of these with antimicrobial characteristics, choice of antimicrobial, and disease outcome were evaluated. Corynebacterium spp. from breast tissue and aspirate samples were confirmed in 17 isolates from 16 patients. Based on EUCAST breakpoints, Corynebacterium kroppenstedtii isolates (n = 11) were susceptible to seven antibiotic classes but resistant to ß-lactam antibiotics. Corynebacterium tuberculostearicum isolates (n = 4) were multidrug resistant. Two nonlipophilic species were isolated, Corynebacterium glucuronolyticum and Corynebacterium freneyi, both of which have various susceptibilities to antimicrobial agents. Short-course antimicrobial therapy was common (median, 6 courses per subject; range, 1 to 9 courses). Patients with C. kroppenstedtii presented with a hot painful breast mass and underwent multiple surgical procedures (median, 4 procedures; range, 2 to 6 procedures). The management of Corynebacterium breast infections requires a multidisciplinary approach and includes culture and appropriate sensitivity testing to guide antimicrobial therapy. Established infections have a poor outcome, possibly because adequate concentrations of some drugs will be difficult to achieve in lipophilic granulomata. Lipophilic antimicrobial therapy may offer a therapeutic advantage. The role of immunotherapy has not been defined.