Evaluation of analogs of mutacin 1140 in systemic and cutaneous methicillin-resistant Staphylococcus aureus infection models in mice.

Mutacin 1140 (Mu1140) is a potent antibiotic against Gram-positive bacteria, such as Staphylococcus aureus. The antibiotic is produced by the oral bacterium Streptococcus mutans and is a member of the epidermin family of type AI lantibiotics. The antibiotic exerts its inhibitory activity by binding to the cell wall precursor lipid II, blocking cell wall synthesis, and by disrupting bacterial membranes. In previous studies, the novel K2A and R13A analogs of Mu1140 have been identified to have superior pharmacokinetic properties compared to native Mu1140. In this study, the use of a combined formulation of the Mu1140 K2A and R13A analogs was shown to be more effective at treating MRSA bacteremia than the native Mu1140 or vancomycin. The analogs were also shown to be effective in treating an MRSA skin infection. The use of K2A and R13A analogs may provide a future alternative for treating serious Gram-positive bacterial infections. In a previous study, the Mu1140 analogs were shown to have significantly longer drug clearance times, leading to higher plasma concentrations over time. These properties warranted further testing to determine whether the analogs are effective for the treatment of systemic MRSA and acute skin infections. In this study, Mu1140 analogs were shown to be more effective than currently available treatments for systemic and skin MRSA infections. Further, the study clearly shows that the new analogs are superior to native Mu1140 for the treatment of a systemic MRSA infection. These findings support continued drug product development efforts using the K2A and R13A Mu1140 analogs, and that these analogs may ameliorate the outcome of serious bacterial infections.

Formulation, Pharmacological Evaluation, and Efficacy Studies of Occidiofungin, a Novel Antifungal.

Occidiofungin is a nonribosomally synthesized cyclic lipopeptide that possesses broad-spectrum antifungal properties at submicromolar concentrations. This report explores multiple routes of administration and formulations of occidiofungin, as well as its toxicity in mice. Further, infection studies were performed in mice to assess the application of occidiofungin for treating systemic and intravaginal yeast infections. Formulations for intravenous and intravaginal administration of occidiofungin were prepared. Pharmacokinetic analyses were performed in a murine model, and a liquid chromatography-mass spectrometry (LC-MS) method was developed and used to quantify occidiofungin in mouse plasma samples. Toxicological and histopathological analyses of two repeat-dose studies using occidiofungin were performed. In these animal models, following intravenous administration, a liposomal formulation of occidiofungin improved the half-life and peak plasma drug concentration over that with a liposome-free formulation. Two long-term repeat-dosing toxicity studies of occidiofungin indicated the absence of toxicity in organ tissues. Murine models of a systemic yeast infection and a vulvovaginal yeast infection were performed. The findings of the systemic infection study revealed limitations in the use of occidiofungin that may be alleviated with the development of novel structural analogs or with further formulation studies. The gel formulation of occidiofungin demonstrated improved efficacy over that of the commercial product Monistat 3 in a vulvovaginal candidiasis study. This report outlines the optimal routes of administration of occidiofungin and demonstrates minimal toxicity following chronic exposure. Further, the results of these studies provide a clear indication for the use of occidiofungin for the treatment of recurrent vulvovaginal candidiasis (RVVC), which is a serious and clinically relevant issue.

Draft Genome Sequence of the Lantibiotic-Producing Strain Streptococcus salivarius HS0302.

Streptococcus salivarius is a prevalent commensal species of human oral mucosal surfaces. S. salivarius strain HS0302 produces the type AII lantibiotic salivaricin A2. Here, we report its draft genome sequence, revealing its potential to produce a variety of bacteriocins.

Multidrug resistant Mannheimia haemolytica isolated from high-risk beef stocker cattle after antimicrobial metaphylaxis and treatment for bovine respiratory disease.

Antimicrobial resistance (AMR) in bacterial respiratory pathogens in high-risk stocker cattle has been poorly characterized. The objective of this study was to describe the prevalence of multidrug resistant (MDR; resistance to > 3 antimicrobial classes) respiratory pathogens in 50 conventionally managed stocker cattle over 21 days after arrival. Cattle received tildipirosin metaphylaxis on day 0 and were eligible to receive up to 3 additional antimicrobials for bovine respiratory disease (BRD): florfenicol, ceftiofur and enrofloxacin. Nasopharyngeal swabs were collected on days 0, 7, 14, and 21 for bacterial culture and antimicrobial susceptibility testing using disc diffusion and broth microdilution. Mannheimia haemolytica was isolated from 5 of 48, 27 of 50, 44 of 50, and 40 of 50 cattle on days 0, 7, 14, and 21, respectively. One of 5, 27 of 27, 43 of 44, and 40 of 40 M. haemolytica were MDR on days 0, 7, 14, and 21, respectively. Pasteurella multocida was isolated from 6 of 48 cattle on day 0 and none were MDR; no other pathogens were isolated. Twenty-four cattle required at least one BRD treatment; M. haemolytica was isolated before treatment from 13 of 24 cattle; all were MDR. One hundred-eighteen M. haemolytica isolates were subjected to pulsed-field gel electrophoresis (PFGE); multiple genotypes were identified. Whole genome sequencing of 33 isolates revealed 14 known AMR genes. Multidrug resistant M. haemolytica can be highly prevalent and genetically diverse in stocker cattle; additional research is necessary to determine factors that influence prevalence and the impact on cattle health.

Covalent Structure and Bioactivity of the Type AII Lantibiotic Salivaricin A2.

Lantibiotics are a class of lanthionine-containing, ribosomally synthesized, and posttranslationally modified peptides (RiPPs) produced by Gram-positive bacteria. Salivaricin A2 belongs to the type AII lantibiotics, which are generally considered to kill Gram-positive bacteria by binding to the cell wall precursor lipid II via a conserved ring A structure. Salivaricin A2 was first reported to be isolated from a probiotic strain, Streptococcus salivarius K12, but the structural and bioactivity characterizations of the antibiotic have remained limited. In this study, salivaricin A2 was purified and its covalent structure was characterized. N-terminal analogues of salivaricin A2 were generated to study the importance for bioactivity of the length and charge of the N-terminal amino acids. Analogue salivaricin A2(3-22) has no antibacterial activity and does not have an antagonistic effect on the native compound. The truncated analogue also lost its ability to bind to lipid II in a thin-layer chromatography (TLC) assay, suggesting that the N-terminal amino acids are important for binding to lipid II. The creation of N-terminal analogues of salivaricin A2 promoted a better understanding of the bioactivity of this antibiotic and further elucidated the structural importance of the N-terminal leader peptide. The antibacterial activity of salivaricin A2 is due not only to the presence of the positively charged N-terminal amino acid residues, but to the length of the N-terminal linear peptide.IMPORTANCE The amino acid composition of the N-terminal linear peptide of salivaricin A2 is crucial for function. Our study shows that the length of the amino acid residues in the linear peptide is crucial for salivaricin A2 antimicrobial activity. Very few type AII lantibiotic covalent structures have been confirmed. The characterization of the covalent structure of salivaricin A2 provides additional support for the predicted lanthionine and methyl-lanthionine ring formations present in this structural class of lantibiotics. Removal of the N-terminal Lys1 and Arg2 residues from the peptide causes a dramatic shift in the chemical shift values of amino acid residues 7 through 9, suggesting that the N-terminal amino acids contribute to a distinct structural conformer for the linear peptide region. The demonstration that the bioactivity could be partially restored with the substitution of N-terminal alanine residues supports further studies aimed at determining whether new analogues of salivaricin A2 for novel applications can be synthesized.

Adhesion and Invasion Assay Procedure Using Caco-2 Cells for Listeria monocytogenes.

Listeria monocytogenes is an important Gram-positive foodborne pathogen that is a particular problem in ready-to-eat food. It has an ability to survive in harsh conditions like refrigeration temperatures and high salt concentrations and is known to cross intestinal, placental and blood-brain barriers. Several cancerous cell lines like cervical, liver, dendritic, intestinal and macrophages have been used to study in vitro propagation and survival of listeria in human cells. Human intestinal epithelial cells have been used to study how listeria crosses the intestinal barrier and cause infection. The protocol in this articles describes the procedures to grow Caco-2 cells, maintain cells and use them for adhesion and invasion assays. During adhesion assay the cells are incubated with listeria for 30 min but in invasion assay the cell growth is arrested at several time points after infection to monitor the growth and survival rate of listeria in cells.

Characterization of a novel two-component regulatory system, HptRS, the regulator for the hexose phosphate transport system in Staphylococcus aureus.

Hexose phosphate is an important carbon source within the cytoplasm of host cells. Bacterial pathogens that invade, survive, and multiply within various host epithelial cells exploit hexose phosphates from the host cytoplasm through the hexose phosphate transport (HPT) system to gain energy and synthesize cellular components. In Escherichia coli, the HPT system consists of a two-component regulatory system (UhpAB) and a phosphate sensor protein (UhpC) that tightly regulate expression of a hexose phosphate transporter (UhpT). Although growing evidence suggests that Staphylococcus aureus also can invade, survive, and multiply within various host epithelial cells, the genetic elements involved in the HPT system in S. aureus have not been characterized yet. In this study, we identified and characterized the HPT system in S. aureus that includes the hptRS (a novel two-component regulatory system), the hptA (a putative phosphate sensor), and the uhpT (a hexose phosphate transporter) genes. The hptA, hptRS, and uhpT markerless deletion mutants were generated by an allelic replacement method using a modified pMAD-CM-GFPuv vector system. We demonstrated that both hptA and hptRS are required to positively regulate transcription of uhpT in response to extracellular phosphates, such as glycerol-3-phosphate (G3P), glucose-6-phosphate (G6P), and fosfomycin. Mutational studies revealed that disruption of the hptA, hptRS, or uhpT gene impaired the growth of bacteria when the available carbon source was limited to G6P, impaired survival/multiplication within various types of host cells, and increased resistance to fosfomycin. The results of this study suggest that the HPT system plays an important role in adaptation of S. aureus within the host cells and could be an important target for developing novel antistaphylococcal therapies.

Lactococcosis in Silver Carp.

An adult Silver Carp Hypophthalmichthys molitrix with a focally extensive skin lesion near the caudal peduncle and mild iridial hemorrhage was submitted to the Aquatic Research and Diagnostic Laboratory (ARDL) in Stoneville, Mississippi, as part of a fish kill investigation. Touch impressions of this musculoskeletal lesion revealed small cocci (∼1 µm) in pairs or chains within an inflammatory milieu. A pure Gram-positive cocci isolate was obtained from the brain, while cultures of the kidney and muscle yielded multiple bacterial colony types, including the Gram-positive cocci seen in the brain. This brain isolate was characterized biochemically and identified as Lactococcus spp. Analysis of the near complete 16S small subunit ribosomal DNA (SSU rDNA) and DNA gyrase subunit B (gyrB) gene sequences revealed the bacterium to be L. lactis subsp. lactis (SSU rDNA: 100% identity, 1,372/1,372 bp; gyrB: 99.7% identity, 1,779/1,785 bp). Comparatively, at the gyrB locus the case isolate shared less than 90% similarity to L. lactis subsp. cremoris (1,599/1,781 bp) and less than 80% homology to L. garvieae (1409/1775 bp). Histopathological examination confirmed a severe meningoencephalitis, a moderate mononuclear myositis, and a mild interstitial nephritis. We believe this represents the first report of a natural infection by L. lactis subsp. lactis in Silver Carp.

Nonclinical toxicological evaluation of occidiofungin, a unique glycolipopeptide antifungal.

Occidiofungin, a glycolipopeptide obtained from the liquid culture of Burkholderia contaminans MS14, has been identified as a novel fungicide. The present study was designed to initially assess the in vitro toxicity in a rat hepatoma (H4IIE) cell line and acute toxicological effects of occidiofungin using a mouse model. In vitro toxicity was observed in all variables at 5 µmol/L. B6C3F1 mice were given single and repeat doses of occidiofungin up to 20 mg/kg. Key effects were a reduction in body and organ weights. However, no significant decrease in body weight was noted at a dose of 1 mg/kg, which is comparable to the dose level of other cyclic glycopeptide antifungal agents currently approved for human use. Microscopic examination of treated mice did not identify any signs of organ-specific toxicity at the dose levels tested.

Occidiofungin's chemical stability and in vitro potency against Candida species.

Occidiofungin is a cyclic glyco-lipopeptide produced by Burkholderia contaminans. MICs against Candida species were between 0.5 and 2.0 µg/ml. Occidiofungin retains its in vitro potency in the presence of 5% and 50% human serum with a minimal lethal concentration (MLC) of 2 and 4 µg/ml, respectively. Time-kill and postantifungal effect (PAFE) experiments of occidiofungin against Candida albicans were performed. The results demonstrate that occidiofungin is fungicidal. Occidiofungin was also found to be a very stable molecule. It is resistant to extreme temperatures and pH and maintains its activity following exposure to gastric proteases.

Genome sequence of the solvent-producing bacterium Clostridium carboxidivorans strain P7T.

Clostridium carboxidivorans strain P7(T) is a strictly anaerobic acetogenic bacterium that produces acetate, ethanol, butanol, and butyrate. The C. carboxidivorans genome contains all the genes for the carbonyl branch of the Wood-Ljungdahl pathway for CO(2) fixation, and it encodes enzymes for conversion of acetyl coenzyme A into butanol and butyrate.

Occidiofungin, a unique antifungal glycopeptide produced by a strain of Burkholderia contaminans.

Bacterial strain Burkholderia contaminans MS14 was isolated from soil that suppressed brown patch disease of lawn grass. An antifungal compound was purified from the liquid culture of this bacterium. In this study, complete covalent structures of two purified closely related antifungal compounds were determined by the experiments of TOCSY, NOESY, ROESY, 13C HSQC 2D NMR, and ESI-MS and GC. The analysis of monoisotopic masses of the purified preparation indicated the presence of two related compounds with masses determined to be 1199.543 and 1215.518 Da; the difference corresponds to the mass of an oxygen atom. GC analysis identified a xylose sugar attached to the antifungal compound. NMR experiments revealed that the compound is cyclic and composed of eight amino acids, two of which are beta-hydroxy derivatives of Tyr and Asn, and one being a novel amino acid. The novel amino acid serves as the scaffold for the attachment of the xylose and a short acyl chain. The spectrum and concentration of antifungal activity were determined using a microtiter plate assay. The antifungal compound demonstrated potent antifungal activities against a broad panel of fungal plant and animal pathogens, as well as two Pythium spp. Microscopic observations showed that the antifungal compound disrupts normal membrane morphology. The cells fill with large inclusion bodies and the membrane becomes irregularly shaped and swollen following the exposure to subinhibitory concentrations of the antifungal compound. Our data support the identification of a novel fungicide and the compound has been named occidiofungin, meaning fungal killer.

A multiplex PCR for species- and virulence-specific determination of Listeria monocytogenes.

Listeria monocytogenes internalin gene inlJ has been described previously for differentiation of virulent from avirulent strains. However, a recent report indicated that there exist some unusual lineage IIIB strains (e.g., serotype 7 strain R2-142) that possess no inlJ gene but have the capacity to cause mouse mortality via intraperitoneal inoculation. Therefore, a multiplex PCR incorporating inlA, inlC and inlJ gene primers was developed in this study for rapid speciation and virulence determination of L. monocytogenes. Although inlB gene was also assessed for species-specific recognition, it was not included in the multiplex PCR due to the negative reaction observed between the inlB primers and serotypes 4a-e strains. The species identity of the 36 L. monocytogenes strains under investigation was verified through the amplification of an 800 bp fragment with the inlA primers and the virulence of these strains was ascertained by the formation of 517 bp and/or 238 bp fragments with the inlC and inlJ primers, respectively. Whereas L. monocytogenes pathogenic strains with capacity to cause mortality (showing relative virulence of 30-100%) in A/J mice via the intraperitoneal route were invariably detected by the inlC and/or inlJ primers, naturally non-pathogenic strains (showing relative virulence of 0%) were negative with these primers. While 8 of the 10 L. ivanovii strains reacted with the inlC primers, they could be effectively excluded as non-L. monocytogenes through their negative reactions with the inlA primers in the multiplex PCR. Thus, the use of the multiplex PCR targeting inlA, inlC and inlJ genes facilitates simultaneous confirmation of L. monocytogenes species identity and virulence.

Toward an improved laboratory definition of Listeria monocytogenes virulence.

Listeria monocytogenes is an opportunistic foodborne pathogen that encompasses a diversity of strains with varied virulence. The ability to rapidly determine the pathogenic potential of L. monocytogenes strains is integral to the control and prevention campaign against listeriosis. Early methods for assessing L. monocytogenes virulence include in vivo bioassays and in vitro cell assays. While in vivo bioassays provide a measurement of all virulence determinants of L. monocytogenes, they are not applied routinely due to their reliance on experimental animals whose costs have become increasingly prohibitive. As a low cost alternative, in vitro cell assays are useful for estimating the virulence of L. monocytogenes strains. However, these assays are often slow, and at times variable. Prior attempts to ascertain L. monocytogenes virulence by targeting virulence-associated proteins and genes have been largely unsuccessful, since many of the assay targets are present in both virulent and avirulent strains. Recent identification of novel virulence-specific genes (particularly internalin gene inlJ) has opened a new avenue for rapid, sensitive, and precise differentiation of virulent L. monocytogenes strains from avirulent strains. The application of DNA sequencing technique also offers an additional tool for assessing L. monocytogenes virulence potential. By providing an update on the laboratory methods that have been reported for the determination of L. monocytogenes pathogenicity, this review discusses future research needs that may help achieve an improved laboratory definition of L. monocytogenes virulence.

Characteristics of cell-mediated, anti-listerial immunity induced by a naturally avirulent Listeria monocytogenes serotype 4a strain HCC23.

The characteristics of cell-mediated, anti-listerial immune response initiated by an avirulent Listeria monocytogenes serotype 4a strain HCC23 was assessed. Similar to virulent strain EGD, avirulent strain HCC23 grew readily within macrophage-like J774 cells, but nonhemolytic strain ATCC 15313 did not. Compared with EGD, HCC23 induced a relatively low level of gamma interferon (IFN-gamma) in mice, and ATCC 15313 stimulated no detectable IFN-gamma. The percentages of gated CD4 T cells from mice immunized with EGD and HCC23 showed a notable drop (to 30%) at 21 days post exposure in comparison with that (about 50%) from ATCC 15313-injected or untreated mice; and the percentage of gated NK cells from EGD-immunized group was markedly higher than those from other treatment groups. Mice immunized with HCC23 and EGD developed an equally strong protective immunity against listeriosis that was effective in both short and long terms, but those injected with ATCC 15313 or saline succumbed to listeriosis within 6 days of challenge.

Listeria monocytogenes subgroups IIIA, IIIB, and IIIC delineate genetically distinct populations with varied pathogenic potential.

Listeria monocytogenes lineage III strains belonging to subgroups IIIA (n = 8), IIIB (n = 5), and IIIC (n = 6) were examined along with other known serotype strains (n = 11) by PCR and Southern hybridization using several recently described species-, virulence-, and serotype-specific primers and probes. The virulence of seven representative lineage III strains was then evaluated in mice via the intraperitoneal route. The results suggest that subgroup IIIA consists of typical rhamnose-positive avirulent serotype 4a and virulent serotype 4c strains, subgroup IIIC consists of atypical rhamnose-negative virulent serotype 4c strains, and subgroup IIIB consists of atypical rhamnose-negative virulent non-serotype 4a and non-serotype 4c strains, some of which may be related to serotype 7. It is possible that subgroup IIIB (including serotype 7) may represent a novel subspecies within L. monocytogenes.

PCR detection of pathogenic Leptospira genomospecies targeting putative transcriptional regulator genes.

The genus Leptospira comprises multiple genomospecies that demonstrate varied pathogenic potential. The availability of rapid and precise diagnostic procedures to differentiate pathogenic from nonpathogenic Leptospira spp. is therefore essential to prevent an otherwise easily treatable malaise from developing into a life-threatening disease. In this report, we conducted an investigation on the diagnostic potential of Leptospira genes encoding putative transcriptional regulators. While PCR primers derived from transcriptional regulator gene la1137 recognized all 24 pathogenic Leptospira strains representing seven species, those from la1937, la3231, la3825, and la4130 detected 19 of the 24 Leptospira strains. However, none of these primers reacted with four nonpathogenic Leptospira species or other common bacteria. The putative transcriptional regulator genes la1137, la1937, la3231, la3825, and la4130 are present in pathogenic Leptospira strains, making them potential targets for diagnostic applications. Further characterization of these genes and their proteins may help elucidate the molecular mechanisms of leptospiral virulence and pathogenicity and pave the way for potential development of novel control strategies against leptospirosis.

Listeria monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.

A collection of Listeria monocytogenes serotype 4b strains belonging to lineages I and III were examined by PCR and Southern blot analysis using species-, virulence-, and serotype-specific primers and probes. Whereas four serotype 4b lineage I strains reacted in PCR with the serotype 4b-, 4d-, and 4e-specific ORF2110 and virulence-specific lmo1134 and lmo2821 primers, all nine serotype 4b lineage III strains were negative by ORF2110 and lmo1134 primers. In addition, the nine serotype 4b lineage III strains formed two separate groups through their reactions in PCR with virulence-specific lmo2821 primers. Southern blot analysis using species-specific lmo0733 and virulence-specific lmo2821 gene probes largely confirmed the PCR results. These findings indicate that L. monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.

Rapid identification of Streptococcus pyogenes with PCR primers from a putative transcriptional regulator gene.

Streptococcus pyogenes (GAS) is a common bacterial pathogen that has emerged as an increasingly important health concern in many parts of the world. Although GAS may appear harmless in healthy individuals, the ability of this bacterium to take advantage of a weakened or compromised host defense system is extraordinary. Following the recent publication of the genome sequences of several S. pyogenes strains, we undertook an investigation of a specialized gene group in GAS that encodes transcriptional regulators. By screening S. pyogenes transcriptional regulator genes from the complete genome of M1 strain SF370 against other DNA sequences at GenBank by BLAST searches, we identified a gene (i.e., Spy1258) that is uniquely present in the bacterium. Application of PCR primers (spy1258F and spy1258R) derived from this gene facilitated amplification of a 407-bp DNA fragment from S. pyogenes only, but not from other species of the genus Streptococcus and common bacteria. Apart from offering an additional target for specific confirmation of GAS, further analysis of the putative transcriptional regulator gene Spy1258 and its related protein product may lead to new insights into the molecular mechanisms of S. pyogenes maintenance and pathogenicity.