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.

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.

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.

Comparative assessment of acid, alkali and salt tolerance in Listeria monocytogenes virulent and avirulent strains.

Listeria monocytogenes is an opportunistic bacterial pathogen of man and animals that has the capacity to survive under extreme environmental conditions. While our knowledge on L. monocytogenes and its ability to sustain within wide pH and temperature ranges and salt concentrations has been largely built on the virulent strains of this species, relatively little is known about avirulent strains in this regard. In this study, we extend our analysis on avirulent L. monocytogenes strains. By subjecting three virulent (EGD, 874 and ATCC 19196) and three avirulent (ATCC 19114, HCC23 and HCC25) strains to various pH and salt concentrations, it was found that L. monocytogenes recovered well after treatment with 100 mM Tris at pH 12.0, and to a lesser extent at pH 3.0. Interestingly, avirulent L. monocytogenes strains showed a somewhat higher tolerance to alkali than virulent strains. This unique feature of avirulent L. monocytogenes strains may potentially be exploited for the development of a rapid technique for differentiation between avirulent and virulent strains. Furthermore, all L. monocytogenes strains tested were resistant to saturated NaCl (about 7 M, or 40% w/v) for a long period of time (20 h and possibly longer). Together, these results highlight that acid, alkali, and/or salt treatments commonly used in food product processing may not be sufficient to eliminate L. monocytogenes, and therefore stringent quality control measures at the beginning and end of the food manufacturing process is essential to ensure that such food products are free of listerial contamination.

Species-specific PCR determination of Listeria seeligeri.

Listeria seeligeri is a non-pathogenic bacterium coming under the genus Listeria. As this bacterium resembles other Listeria species such as L. monocytogenes and L. ivanovii that are pathogenic to man and animals, it is important that rapid and precise identification techniques be available for L. seeligeri in cases where such determination is desirable. A specific molecular test on the basis of a uniquely present gene region in L. seeligeri will be of particular value under the circumstances. In this report, after comparative screening of genomic DNA from six Listeria species by dot blot hybridization, we isolated one L. seeligeri-specific clone (lse24-315) that contains an insert of 1538 bp. Using primers (lse24-315F and lse24-315R) derived from this clone, we showed that a specific PCR product of 375 bp was generated from genomic DNA of L. seeligeri strains only, but not of other Listeria species or common bacteria. Therefore, the PCR employing primers lse24-315F and lse24-315R provides a rapid, sensitive and specific method for distinguishing L. seeligeri from other Listeria and common bacteria.

Identification of Listeria innocua by PCR targeting a putative transcriptional regulator gene.

Listeria innocua is a common, non-pathogenic bacterial species that shares morphological, biochemical and molecular characteristics with the pathogenic species L. monocytogenes. The presence of L. innocua may cause difficulty or confusion in the laboratory identification of L. monocytogenes or other Listeria spp. In this report, through examining the recently published genome sequence of L. innocua strain CLIP 11262 (serovar 6a), we identified a L. innocua-specific gene (lin0464) encoding a putative transcriptional regulator and evaluated its efficacy for species-specific detection by polymerase chain reaction (PCR). The specificity of the oligonucleotide primers (lin0464F and lin0464R) derived from this gene was confirmed with the formation of a 749-bp fragment in PCR from genomic DNA of L. innocua strains only. We expect that this assay will be useful in confirming identification of L. innocua or in studies where rapid detection of L. innocua is necessary.

Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes.

Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide. However, L. monocytogenes strains demonstrate considerable variation in pathogenic potential. In this report, virulent and avirulent L. monocytogenes isolates were compared by using a comparative screening strategy. Two clones were identified that contained DNA that was only present in virulent L. monocytogenes strains. PCR primers were designed for three genes from these clones and for five other selected L. monocytogenes genes. All eight primer sets predominantly detected virulent L. monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent. Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L. monocytogenes isolates, rather than environmental isolates. The findings from this study suggest that virulent L. monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L. monocytogenes virulence.

Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes.

Pasteurella multocida is an important animal pathogen that may also infect humans through animal bites and scratches. After comparison of transcriptional regulator gene sequences from the P. multocida genome with other DNA sequences at GenBank, we identified two genes (i.e., Pm0762 and Pm1231) uniquely present in P. multocida. By using oligonucleotide primers (Pm0762F/R and Pm1231F/R) designed from these genes in PCR, it was found that specific DNA products of expected sizes were obtained with genomic DNA from P. multocida only, but not from other bacteria. These results indicated that the putative transcriptional regulator genes Pm0762 and Pm1231 are species-specific, and that the PCR methods targeting these genes provide a useful means of rapidly and precisely identifying P. multocida from other bacteria. Further elucidation of the roles and functions of these putative transcriptional regulator genes (Pm0762 and Pm1231) and their protein products may help provide valuable insight into the molecular mechanism of P. multocida virulence and pathogenicity.

Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes.

Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide. In this study, through comparative analysis of L. innocua and L. monocytogenes genomic sequences, we selected a L. monocytogenes specific gene (lmo0733) that has the potential for specific detection of L. monocytogenes. Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L. monocytogenes strains. PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay. Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L. monocytogenes.

PCR detection of a putative N-acetylmuramidase gene from Listeria ivanovii facilitates its rapid identification.

Listeria ivanovii is a Gram-positive bacterial pathogen that is capable of causing abortions and stillbirths in farm animals, particularly sheep and cattle. In terms of morphological, biochemical and molecular characteristics, L. ivanovii resembles other Listeria species such as L. monocytogenes, a pathogen of both man and animals. In this study, through comparative analysis of genomic DNA from the six Listeria species, a L. ivanovii specific clone (liv22-228) containing a 946 bp insert was isolated. This clone contained the 5' ends of two divergently transcribed L. ivanovii genes and an intergenic spacer region, similar in organization to homologous regions from the L. innocua and L. monocytogenes genomes. Regions of low homology in the clone were identified by comparing to the L. innocua and L. monocytogenes genomes, and oligonucleotide primers (liv22-228F and liv22-228R) were designed. These primers amplified a 463 bp band from genomic DNA of L. ivanovii strains only, but not from other Listeria species or common bacteria. Thus, PCR employing L. ivanovii specific primers (liv22-228F and liv22-228R) provides a useful and straightforward method for rapid and precise determination of L. ivanovii.

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.

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.

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.

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.

Isolation and PCR amplification of a species-specific oxidoreductase-coding gene region in Listeria grayi.

Listeria grayi is a nonpathogenic Gram-positive bacterium that demonstrates considerable similarities to other members in the genus Listeria, including the foodborne human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. A rapid diagnostic test to identify and diagnose listeriosis would be valuable, especially in cases where the presence of L. grayi may complicate diagnosis. This test would be based on a unique gene present in L. grayi. In this study, after comparative screening of a recombinant L. grayi DNA library by dot blot hybridization, an L. grayi specific clone (lgr20-246) with an insert of 722 bp was isolated. By applying PCR primers derived from a distinct region of the clone not shared by other bacteria, a specific band of 420 bp was amplified from the genomic DNA of L. grayi only and not of other Listeria species or common bacteria. These results suggest that the PCR assay employing primers lgr20-246F and lgr20-246R provides an independent and precise means of distinguishing L. grayi from other Listeria species and common bacteria. Therefore, it would be another useful technique for laboratory differentiation of Listeria bacteria.