Bacterial adhesion inhibitor prevents infection in a rodent surgical incision model.

Surgical site infection risk continues to increase due to lack of efficacy in current standard of care drugs. New methods to treat or prevent antibiotic-resistant bacterial infections are needed. Multivalent Adhesion Molecules (MAM) are bacterial adhesins required for virulence. We developed a bacterial adhesion inhibitor using recombinant MAM fragment bound to polymer scaffold, mimicking MAM7 display on the bacterial surface. Here, we test MAM7 inhibitor efficacy to prevent Gram-positive and Gram-negative infections. Using a rodent model of surgical infection, incision sites were infected with antibiotic-resistant bioluminescent strains of Staphylococcus aureus or Pseudomonas aeruginosa. Infections were treated with MAM7 inhibitor or control suspension. Bacterial abundance was quantified for nine days post infection. Inflammatory responses and histology were characterized using fixed tissue sections. MAM7 inhibitor treatment decreased burden of S. aureus and P. aeruginosa below detection threshold. Bacterial load of groups treated with control were significantly higher than MAM7 inhibitor-treated groups. Treatment with inhibitor reduced colonization of clinically-relevant pathogens in an in vivo model of surgical infection. Use of MAM7 inhibitor to block initial adhesion of bacteria to tissue in surgical incisions may reduce infection rates, presenting a strategy to mitigate overuse of antibiotics to prevent surgical site infections.

Bacterial vectors for imaging and cancer gene therapy: a review.

The significant burden of resistance to conventional anticancer treatments in patients with advanced disease has prompted the need to explore alternative therapeutic strategies. The challenge for oncology researchers is to identify a therapy which is selective for tumors with limited toxicity to normal tissue. Engineered bacteria have the unique potential to overcome traditional therapies' limitations by specifically targeting tumors. It has been shown that bacteria are naturally capable of homing to tumors when systemically administered resulting in high levels of replication locally, either external to (non-invasive species) or within tumor cells (pathogens). Pre-clinical and clinical investigations involving bacterial vectors require relevant means of monitoring vector trafficking and levels over time, and development of bacterial-specific real-time imaging modalities are key for successful development of clinical bacterial gene delivery. This review discusses the currently available imaging technologies and the progress to date exploiting these for monitoring of bacterial gene delivery in vivo.

Pyridinyl polythiazole class peptide antibiotic micrococcin P1, secreted by foodborne Staphylococcus equorum WS2733, is biosynthesized nonribosomally.

Recently, foodborne Staphylococcus equorum WS2733 was isolated from a French red smear cheese on account of its strong inhibitory activity against Gram-positive pathogens such as Listeria. The antagonistic substance was identified as macrocyclic peptide antibiotic micrococcin P1, which had previously not been reported for the genus Staphylococcus. Micrococcin P1, also a potent inhibitor of the malaria parasite Plasmodium falciparum, is structurally related to thiostrepton, thiocillins and nosiheptide. Although all of these peptide antibiotics have been known for quite a long time, their mode of biosynthesis had not been determined in detail yet. By using degenerated PCR, a gene fragment encoding a nonribosomal peptide synthetase (NRPS) could be amplified from S. equorum. The corresponding chromosomal locus was disrupted by insertional mutagenesis, and it could be shown that all mutants obtained displayed a micrococcin P1-deficient phenotype. Sequence analysis of a coherent 2.8-kb fragment revealed extensive homology to known NRPSs, and allowed the assignment of the domain organization 'condensation-adenylation-thiolation-condensation'; an arrangement predicted only for two loci within the presumably 14-modular, 1.6-MDa biosynthetic NRPS template. Biochemical characterization of the adenylation domain exhibited selectivity for the substrate amino-acid threonine. All of these data substantiate that the macrocyclic peptide antibiotic is biosynthesized nonribosomally, and provide the basis for the characterization of the entire biosynthetic gene cluster. The biosynthetic machinery of micrococcin will serve as a model system for structurally related, pharmacologically important pyridinyl polythiazole class peptide antibiotics. Furthermore, this knowledge will enable the manipulation of its NRPS template, which in turn may grant the targeted engineering of even more potent anti-listerial and anti-malaria drugs.

Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.

Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r), that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km(r), and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence.

Changes in cspL, cspP, and cspC mRNA abundance as a function of cold shock and growth phase in Lactobacillus plantarum.

An inverse PCR strategy based on degenerate primers has been used to identify new genes of the cold shock protein family in Lactobacillus plantarum. In addition to the two previously reported cspL and cspP genes, a third gene, cspC, has been cloned and characterized. All three genes encode small 66-amino-acid proteins with between 73 and 88% identity. Comparative Northern blot analyses showed that the level of cspL mRNA increases up to 17-fold after a temperature downshift, whereas the mRNA levels of cspC and cspP remain unchanged or increase only slightly (about two- to threefold). Cold induction of cspL mRNA is transient and delayed in time as a function of the severity of the temperature downshift. The cold shock behavior of the three csp mRNAs contrasts with that observed for four unrelated non-csp genes, which all showed a sharp decrease in mRNA level, followed in one case (bglH) by a progressive recovery of the transcript during prolonged cold exposure. Abundance of the three csp mRNAs was also found to vary during growth at optimal temperature (28 degrees C). cspC and cspP mRNA levels are maximal during the lag period, whereas the abundance of the cspL transcript is highest during late-exponential-phase growth. The differential expression of the three L. plantarum csp genes can be related to sequence and structural differences in their untranslated regions. It also supports the view that the gene products fulfill separate and specific functions, under both cold shock and non-cold shock conditions.

Monitoring bioluminescent Staphylococcus aureus infections in living mice using a novel luxABCDE construct.

Strains of Staphylococcus aureus were transformed with plasmid DNA containing a Photorhabdus luminescens lux operon (luxABCDE) that was genetically modified to be functional in both gram-positive and gram-negative bacteria. S. aureus cells containing this novel lux construct, downstream of an appropriate promoter sequence, are highly bioluminescent, allowing the detection of fewer than 100 CFU in vitro (direct detection of exponentially dividing cells in liquid culture). Furthermore, these bacteria produce light stably at 37 degrees C and do not require exogenous aldehyde substrate, thus allowing S. aureus infections in living animals to be monitored by bioluminescence. Two strains of S. aureus 8325-4 that produce high levels of constitutive bioluminescence were injected into the thigh muscles of mice, and the animals were then either treated with the antibiotic amoxicillin or left untreated. Bioluminescence from bacteria present in the thighs of the mice was monitored in vivo over a period of 24 h. The effectiveness of the antibiotic in the treated animals could be measured by a decrease in the light signal. At 8 h, the infection in both groups of treated animals had begun to clear, as judged by a decrease in bioluminescence, and by 24 h no light signal could be detected. In contrast, both groups of untreated mice had strong bioluminescent signals at 24 h. Quantification of CFU from bacteria extracted from the thigh muscles of the mice correlated well with the bioluminescence data. This paper shows for the first time that bioluminescence offers a method for monitoring S. aureus infections in vivo that is sensitive and noninvasive and requires fewer animals than conventional methodologies.

Restart of exponential growth of cold-shocked Yersinia enterocolitica occurs after down-regulation of cspA1/A2 mRNA.

The cellular content of major cold shock protein (MCSP) mRNA transcribed from the tandem gene duplication cspA1/A2 and growth of Yersinia enterocolitica were compared when exponentially growing cultures of this bacterium were cold shocked from 30 to 20, 15, 10, 5, or 0 degrees C, respectively. A clear correlation between the time point when exponential growth resumes after cold shock and the degradation of cspA1/A2 mRNA was found. A polynucleotide phosphorylase-deficient mutant was unable to degrade cspA1/A2 mRNA properly and showed a delay, as well as a lower rate, of growth after cold shock. For this mutant, a correlation between decreasing cspA1/A2 mRNA and restart of growth after cold shock was also observed. For both wild-type and mutant cells, no correlation of restart of growth with the cellular content of MCSPs was found. We suggest that, after synthesis of cold shock proteins and cold adaptation of the cells, MCSP mRNAs must be degraded; otherwise, they trap ribosomes, prevent translation of bulk mRNA, and thus inhibit growth of this bacterium at low temperatures.

Pathogenic Yersinia species carry a novel, cold-inducible major cold shock protein tandem gene duplication producing both bicistronic and monocistronic mRNA.

Inverse PCR was used to amplify major cold shock protein (MCSP) gene families from a diverse range of bacteria, including the psychrotolerant Yersinia enterocolitica, which was found to have two almost identical MCSP coding regions (cspA1 and cspA2) located approximately 300 bp apart. This tandem gene duplication was also found in Y. pestis, Y. pseudotuberculosis, and Y. ruckeri but not in other bacteria. Analysis of the transcriptional regulation of this MCSP gene in Y. enterocolitica, performed by using both reverse transcriptase-PCR and Northern blot assays, showed there to be two cold-inducible mRNA templates arising from this locus: a monocistronic template of approximately 450 bp (cspA1) and a bicistronic template of approximately 900 bp (cspA1/A2). The former may be due to a secondary structure between cspA1 and cspA2 causing either 3' degradation protection of cspA1 or, more probably, partial termination after cspA1. Primer extension experiments identified a putative transcriptional start site (+1) which is flanked by a cold-box motif and promoter elements (-10 and -35) similar to those found in Escherichia coli cold-inducible MCSP genes. At 30 degrees C, the level of both mRNA molecules was negligible; however, upon a temperature downshift to 10 degrees C, transcription of the bicistronic mRNA was both substantial (300-fold increase) and immediate, with transcription of the monocistronic mRNA being approximately 10-fold less (30-fold increase) and significantly slower. The ratio of bicistronic to monocistronic mRNA changed with time after cold shock and was higher when cells were shocked to a lower temperature. High-resolution, two-dimensional protein gel electrophoresis showed that synthesis of the corresponding proteins, both CspA1 and CspA2, was apparent after only 10 min of cold shock from 30 degrees C to 10 degrees C. The data demonstrate an extraordinary capacity of the psychrotolerant Y. enterocolitica to produce major cold shock proteins upon cold shock.

Correlation of 16S ribosomal DNA signature sequences with temperature-dependent growth rates of mesophilic and psychrotolerant strains of the Bacillus cereus group.

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.

Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group.

The Bacillus cereus group comprises the four valid species Bacillus cereus, Bacillus mycoides, Bacillus thuringiensis and bacillus anthracis. Some isolates of B. cereus are known to be psychrotolerant (growth at 7 degrees C or below). Here, specific sequence differences are described between the 16S rDNA, the 23S rDNA, the 16S-23S rDNA spacer region and the genes of the major cold-shock protein homologue cspA in a variety of psychrotolerant and mesophilic B. cereus and B. mycoides strains. Randomly amplified polymorphic DNA analysis using three different primers clearly separated psychrotolerant strains of both species from the rest of the B. cereus group, as did inverse PCR patterns of the rDNA operons. These data strongly support a hitherto unrecognized fifth sub-group within the B. cereus species group comprising psychrotolerant, but not mesophilic, B. cereus strains. Despite the latter finding, the DNA sequences investigated exhibited a high degree of sequence similarity indicating a close relationship between the species of the B. cereus group. Considering the unusual importance of B. cereus in both food poisoning and food spoilage and to avoid merging all species of the group, a new species, Bacillus weihenstephanensis sp. nov., comprising psychrotolerant 'cereus' strains, is proposed. Isolates of the new species grow at 4-7 degrees C but not at 43 degrees C and can be identified rapidly using rDNA or cspA targeted PCR. The type strain is B. weihenstephanensis WSBC 10204T (= DSM 11821T).

Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes.

Detection of psychrotrophic strains (those able to grow at or below 7 degreesC) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7 degreesC) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group.

Identification of the ahp operon of Salmonella typhimurium as a macrophage-induced locus.

Previously, we tagged a macrophage-induced Salmonella typhimurium locus with Mudlux (K. P. Francis and M. P. Gallagher, Infect. Immun. 61:640-649, 1993). The insertion lies within the OxyR-regulated ahpC locus and conveys alkyl peroxide sensitivity. Plasmid-encoded ahp reverses sensitivity but reduces luminescence. This suggests that OxyR is titrated by the multicopy ahp promoter.

Detection and speciation of bacteria through PCR using universal major cold-shock protein primer oligomers.

The detection of bacteria using PCR is a well-established diagnostic technique. However, conventional PCR requires the use of DNA primer oligomers that are specific to the target organism and, as a consequence, a sample can only be tested for the presence of that specific target. A significant advantage would be to probe a sample for the presence of any bacteria, followed by identification. To achieve this it is necessary to identify a DNA sequence common to all bacteria. Here we demonstrate that such a sequence may be that encoding the major cold-shock proteins. Using two universal PCR primer oligomers from conserved regions of these gene homologues, we have amplified a 200 base-pair DNA sequence from more than 30 diverse Gram-positive and Gram-negative bacteria, including representatives from the genera Aeromonas, Bacillus, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Listeria, Pediococcus, Photobacterium, Proteus, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Sequence analysis of the amplified products confirmed a high level of DNA homology. Significantly, however, there are sufficient nucleotide variations to allow the unique allocation of each amplified sequence to its parental bacterium.

Light emission from a Mudlux transcriptional fusion in Salmonella typhimurium is stimulated by hydrogen peroxide and by interaction with the mouse macrophage cell line J774.2.

Hydrogen peroxide is known to induce a multigenic response in Salmonella typhimurium cells. We have used a Mudlux transcriptional reporter system to identify and isolate fusions in the virulent strain SL1344 which respond to hydrogen peroxide in vitro by light production, and one of these fusions, MPG203, has been further characterized. Transient light production was observed from MPG203 at levels of hydrogen peroxide as low as 10 microM. However, high levels of this toxic oxidizing agent resulted in light suppression, particularly at low bacterial densities. This fusion was also shown to produce light following adhesion to cells of the mouse macrophage cell line J774.2. Furthermore, the response was greatly reduced in the presence of catalase, directly implicating hydrogen peroxide as the eliciting agent and suggesting the involvement of the hydrogen peroxide-induced bacterial stress response in the infection process. Chemiluminescence studies also indicated that inhibition of the respiratory burst may occur as the infection ratio is increased. In addition, the level of light produced from bacteria within individual macrophage cells was shown to vary.