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.     

Chromosome- and plasmid-mediated gentamicin resistance in Staphylococcus aureus encoded by Tn4001

DNA sequences corresponding to the 4.7-kb gentamicin, tobramycin and kanamycin resistance (GmrTmrKmr) transposon Tn4001 have been detected on a series of nine structurally-related plasmids that mediate this phenotype in Australian isolates of Staphylococcus aureus. Tn4001 sequences have also been demonstrated on the chromosomes of GmrTmrKmr isolates that do not possess these plasmids, and the exhibited diversity of chromosomal sites occupied by this element implies that Tn4001 has transposed to the chromosome on numerous occasions in vivo. These results suggest that the rapid emergence of nosocomial GmrTmrKmr S. aureus in the early 1980s may have been the result of the transposition of Tn4001 from a chromosomal site to a readily disseminated plasmid.     

Real time noninvasive monitoring of contaminating bacteria in a soft tissue implant infection model

Infection is the main cause of biomaterials-related failure. A simple technique to test in-vivo new antimicrobial and/or nonadhesive implant coatings is unavailable. Current in vitro methods for studying bacterial adhesion and growth on biomaterial surfaces lack the influence of the host immune system. Most in vivo methods to study biomaterials-related infections routinely involve implant-removal, preventing comprehensive longitudinal monitoring. In vivo imaging circumvents these drawbacks and is based on the use of noninvasive optical imaging of bioluminescent bacteria. Staphylococcus aureus Xen29 is genetically modified to be stably bioluminescent, by the introduction of a modified full lux operon onto its chromosome. Surgical meshes with adhering S. aureus Xen29 were implanted in mice and bacterial growth and spread into the surrounding tissue was monitored longitudinally from bioluminescence with a highly sensitive CCD camera. Distinct spatiotemporal bioluminescence patterns, extending beyond the mesh area into surrounding tissues were observed. After 10 days, the number of living organisms isolated from explanted meshes was found to correlate with bioluminescence prior to sacrifice of the animals. Therefore, it is concluded that in vivo imaging using bioluminescent bacteria is ideally suited to study antimicrobial coatings taking into account the host immune system. In addition, longitudinal monitoring of infection in one animal will significantly reduce the number of experiments and animals.     

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.     

The novel conjugative transposon tn1207.3 carries the macrolide efflux gene mef(A) in Streptococcus pyogenes

The macrolide efflux gene mef(A) of the Streptococcus pyogenes clinical strain 2812A was found to be carried by a 52-kb chromosomal genetic element that could be transferred by conjugation to the chromosome of other streptococcal species. The characteristics of this genetic element are typical of conjugative transposons and was named Tn1207.3. The size of Tn1207.3 was established by pulsed-field gel electrophoresis (PFGE), and DNA sequencing analysis showed that the 7,244 bp at the left end of Tn1207.3 were identical to those of the pneumococcal Tn1207.1 element. Tn1207.3-like genetic elements were found to be inserted at a single specific chromosomal site in 12 different clinical isolates S. pyogenes exhibiting the M phenotype of resistance to macrolides and carrying the mef(A) gene. Tn1207.3 was transferred from S. pyogenes 2812A to Streptococcus pneumoniae, and sequence analysis carried out on six independent transconjugants showed that insertion of Tn1207.3 in the pneumococcal genome always occurred at a single specific site as in Tn1207.1. Using MF2, a representative S. pneumoniae transconjugant, as a donor, Tn1207.3 was transferred again by conjugation to S. pyogenes and Streptococcus gordonii. The previously described nonconjugative element Tn1207.1 of S. pneumoniae appears to be a defective element, part of a longer conjugative transposon that carries mef(A) and is found in clinical isolates of S. pyogenes.     

A system for generalized mutagenesis of Haemophilus ducreyi.

The lack of a generalized mutagenesis system for Haemophilus ducreyi has hampered efforts to identify virulence factors expressed by this sexually transmitted pathogen. To address this issue, the transposable element Tn1545-delta 3, which encodes resistance to kanamycin, was evaluated for its ability to insert randomly into the H. ducreyi chromosome and produce stable, isogenic mutants. Electroporation of H. ducreyi with 1 microgram of plasmid pMS1 carrying Tn1545-delta 3 resulted in the production of 10(4) kanamycin-resistant transformants; Southern blot analysis of a number of these transformants indicated that insertion of the transposon into the chromosome occurred at a number of different sites. This pMS1-based transposon delivery system was used to produce an H. ducreyi mutant that expressed an altered lipooligosaccharide (LOS). Passage of this mutant in vitro in the presence or absence of kanamycin did not affect the stability of the transposon insertion. To confirm that the observed mutant phenotype was the result of the transposon insertion, a chromosomal fragment containing Tn1545-delta 3 was cloned from this H. ducreyi LOS mutant. Electroporation of the wild-type H. ducreyi strain with this DNA fragment yielded numerous kanamycin-resistant transformants, the majority of which had the same altered LOS phenotype as the original mutant. Southern blot analysis confirmed the occurrence of proper allelic exchange in the LOS-deficient transformants obtained in this backcross experiment. The ability of Tn1545-delta 3 to produce insertion mutations in H. ducreyi should facilitate genetic analysis of this pathogen.     

Urinary tract infections in a South American population: dynamic spread of class 1 integrons and multidrug resistance by homologous and site-specific recombination

One hundred four bacterial strains mediating urinary tract infections in separate individuals from a Uruguayan community were isolated. Forty-six strains conferred a multidrug resistance phenotype. All 104 strains were examined for the presence of class 1, 2, and 3 integrons. Class 1 integrons were found in 21 isolates across four distinct bacterial genera. A large class 1 integron in a Klebsiella pneumoniae strain was fully sequenced and was 29,093 bp in length. This integron probably arose by homologous recombination since it was embedded in a hybrid Tn21-like transposon backbone which comprised a Tn5036-like tnp transposition module at the IRi integron end and a Tn21 mer module at the IRt integron end. The parent integron/transposon that contributed the Tn5036 module was not related to Tn1696 since the integron insertion points in the transposon backbones were 16 bases apart. Examination of the other 20 class 1 integron-containing strains revealed further evidence of genetic exchange. This included a strain that possessed a Tn5036 module at the IRt end but not at the IRi end and another that possessed a tnp module beyond IRi that was a hybrid of Tn21 and Tn5051 and that is presumed to have arisen by site-specific recombination. This study highlights the ability of different genetic elements to act cooperatively to spread and rearrange antibiotic resistance in a community.     

A method for allelic replacement that uses the conjugative transposon Tn916: deletion of the emm6.1 allele in Streptococcus pyogenes JRS4.

The emm6.1 allele of Streptococcus pyogenes JRS4 was deleted by using the conjugative transposon Tn916. The aphA-3 gene, conferring resistance to kanamycin, was cloned between the sequences flanking the structural gene for the type 6 M protein (emm6.1) and inserted into the BstXI site of Tn916 to generate the chimeric transposon Tn916-5K3. Because the BstXI site lies in a nonessential region of Tn916, the chimeric transposon could transfer by conjugation from Bacillus subtilis into JRS4. In some of the transconjugants, Tn916-5K3 replaced the emm6.1 locus of JRS4 by homologous recombination between the cloned emm6.1-flanking regions and the resident chromosome. One recombinant studied in detail, JRS75, was kanamycin resistant and tetracycline sensitive and lacked immunologically detectable M6 protein. Furthermore, by Southern blot analysis, the DNA region encompassing the emm6.1 structural gene was found to have been replaced by aphA-3.     

Rapid and efficient introduction of a foreign gene into bacterial artificial chromosome-cloned varicella vaccine by Tn7-mediated site-specific transposition

Using a rapid and reliable system based on Tn7-mediated site-specific transposition, we have successfully constructed a recombinant Oka varicella vaccine (vOka) expressing the mumps virus (MuV) fusion protein (F). The backbone of the vector was our previously reported vOka-BAC (bacterial artificial chromosome) genome. We inserted the transposon Tn7 attachment sequence, LacZα-mini-attTn7, into the region between ORF12 and ORF13 to generate a vOka-BAC-Tn genome. The MuV-F expressing cassette was transposed into the vOka-BAC genome at the mini-attTn7 transposition site. MuV-F protein was expressed in recombinant virus, rvOka-F infected cells. In addition, the MuV-F protein was cleaved in the rvOka-F infected cells as in MuV-infected cells. The growth of rvOka-F was similar to that of the original recombinant vOka without the F gene. Thus, we show that Tn7-mediated transposition is an efficient method for introducing a foreign gene expression cassette into the vOka-BAC genome as a live virus vector.     

Plasmid-mediated resistance to gentamicin in Staphylococcus aureus: the involvement of a transposon

Resistance to gentamicin, tobramycin and kanamycin (GmrTmrKmr) in strains of Staphylococcus aureus isolated from clinical sources in Australia is mediated by a 4.7 kb transposable element, designated Tn4001. A 2.5 kb HindIII fragment which maps symmetrically within Tn4001, and encompasses the aminoglycoside-resistance coding region, has been shown to hybridise with fragments of identical size in HindIII digests of three different GmrTmrKmr plasmids, two of which were self-transmissible, from strains of S. aureus isolated in the USA. Examination by electronmicroscopy of self-annealed molecules of the North American GmrTmrKmr plasmids revealed the presence of stem and loop structures similar to those produced by Tn4001, but with shorter inverted repeats. These results suggest that GmrTmrKmr in strains of S. aureus isolated in the USA is, or once was, transposable, and that transposable elements analogous to Tn4001 may be found in isolates of GmrTmrKmr S. aureus worldwide.     

Construction of new unencapsulated (rough) strains of Streptococcus pneumoniae

To construct rough strains of Streptococcus pneumoniae in which the capsule locus was completely deleted, a genetic cassette to be used as a donor DNA in transformation was developed. The cassette contained an aphIII gene, conferring kanamycin resistance, flanked by segments of dexB and aliA. Since, in all strains of S. pneumoniae the capsule locus is between dexB and aliA, the DNA segments of these two genes allow insertion of a 1354-bp DNA fragment containing aphIII into the pneumococcal chromosome, determining the deletion of the whole capsule locus. The capsule locus was deleted from the classic type 2 and type 3 Avery's strains, from R6 (whose complete genome sequence is released) and Rx1 (the two most commonly used transformation recipients), from a type 3 clinical strain and type 19F clinical isolate G54 (whose draft genome sequence is annotated). The effect of capsule removal was tested in 4 isogenic pairs. In unencapsulated strains, growth rate increased up to 56% and transformation frequency increased up to 1075-fold. A correlation was observed between the increase in growth rate and an increase in transformation frequency.     

Direct continuous method for monitoring biofilm infection in a mouse model

We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in a mouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters. Two important biofilm-forming bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, were made bioluminescent by insertion of a complete lux operon. These bacteria produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel and highly correlated (S. aureus r = 0.98; P. aeruginosa r = 0.99) and could be maintained for 10 days or longer, provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation of the catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10 (3) to 10 (5) CFU/catheter for S. aureus and P. aeruginosa resulted in a reproducible, localized infection surrounding the catheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the lux constructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could be detected directly on the support matrix, nondestructively, and noninvasively, this method is especially appealing for the study of chronic biofilm infections and drug efficacy studies in vivo.     

Disease symptoms in transgenic tobacco induced by integrated gene VI of cauliflower mosaic virus

A chimeric vector (pKR 612B1) containing the neomycin phosphotransferase (APH) gene from the Tn5 transposon under the control of the gene VI promoter of cauliflower mosaic virus (CaMV) and the cloned gene VI region (SalI-BstEII) of the same virus were used to cotransform tobacco protoplasts. Using the polyethylene glycol transformation procedure, a large number of protoplasts were transformed and proved to be resistant to kanamycin (Km). Whole Km-resistant plants were regenerated and shown to contain the integrated foreign genes. DNA from transformed clones was analyzed by Southern blot hybridization, showing the presence of the Tn5-derived gene and the viral gene. Transgenic plants containing the viral gene show mild mosaic patterns and fasciation. The expression of the gene VI product was detected by immunoblots.     

Cytadherence-deficient mutants of Mycoplasma gallisepticum generated by transposon mutagenesis

Cytadherence-related molecules of Mycoplasma gallisepticum strain R-low were identified by Tn4001 transposon mutagenesis with the hemadsorption (HA) assay as an indicator for cytadherence. Three Gm(r) HA-negative (HA(-)) colonies displaying a stable HA(-) phenotype through several successive generations in which gentamicin selection was maintained were isolated from four independent transformation experiments and characterized. Southern blot analysis showed that the transposon was inserted as a single copy within the genome of each of the HA(-) mutants, suggesting that the transposon insertion was directly responsible for their inability to attach to erythrocytes. Sequence analysis of the transposon insertion sites revealed that in two mutants, the transposon was inserted at two distinct sites within the gapA structural gene. In the third mutant, the insertion was mapped within the crmA gene, which is located immediately downstream of the gapA gene as part of the same operon. In vitro attachment experiments with the MRC-5 human lung fibroblast cell line showed that the cytadherence capabilities of the HA(-) mutants were less than 25% those of original strain R. Experimental infection of chickens, the natural host of M. gallisepticum, with each of the three mutants demonstrated significantly impaired colonization and host responses. These data demonstrate conclusively the role of both GapA and CrmA proteins in the adherence of M. gallisepticum to host cells in model systems and in vivo colonization. Furthermore, these results underscore the relevance of in vitro cytadherence model systems for studying the pathogenesis of natural infections in chickens.     

Genome-wide identification of Streptococcus pneumoniae genes essential for bacterial replication during experimental meningitis

Meningitis is the most serious of invasive infections caused by the Gram-positive bacterium Streptococcus pneumoniae. Vaccines protect only against a limited number of serotypes, and evolving bacterial resistance to antimicrobials impedes treatment. Further insight into the molecular pathogenesis of invasive pneumococcal disease is required in order to enable the development of new or adjunctive treatments and/or pneumococcal vaccines that are efficient across serotypes. We applied genomic array footprinting (GAF) in the search for S. pneumoniae genes that are essential during experimental meningitis. A total of 6,000 independent TIGR4 marinerT7 transposon mutants distributed over four libraries were injected intracisternally into rabbits, and cerebrospinal fluid (CSF) was collected after 3, 9, and 15 h. Microarray analysis of mutant-specific probes from CSF samples and inocula identified 82 and 11 genes mutants of which had become attenuated or enriched, respectively, during infection. The results point to essential roles for capsular polysaccharides, nutrient uptake, and amino acid biosynthesis in bacterial replication during experimental meningitis. The GAF phenotype of a subset of identified targets was followed up by detailed studies of directed mutants in competitive and noncompetitive infection models of experimental rat meningitis. It appeared that adenylosuccinate synthetase, flavodoxin, and LivJ, the substrate binding protein of a branched-chain amino acid ABC transporter, are relevant as targets for future therapy and prevention of pneumococcal meningitis, since their mutants were attenuated in both models of infection as well as in competitive growth in human cerebrospinal fluid in vitro.     

Identification of new genes involved in the virulence of Listeria monocytogenes by signature-tagged transposon mutagenesis

Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. We have adapted signature-tagged transposon mutagenesis to L. monocytogenes to identify new genes involved in virulence in the murine model of infection. We used transposon Tn1545 carried on the integrative vector pAT113. Forty-eight tagged transposons were constructed and used to generate banks of L. monocytogenes mutants. Pools of 48 mutants were assembled, taking one mutant from each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants tested, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to previously unknown genes. The properties of four loci involving putative cell wall components were further studied in vitro and in vivo. The data suggested that these components are involved in bacterial invasion and multiplication in the brain.     

Immunization with polyamine transport protein PotD protects mice against systemic infection with Streptococcus pneumoniae

The human pathogen Streptococcus pneumoniae contains genes for a putative polyamine ABC transporter which are organized in an operon and designated potABCD. Polyamine transport protein D (PotD) is an extracellular protein which binds polyamines and possibly other structurally related molecules. PotD has been shown to contribute to virulence in both a murine sepsis model and a pneumonia model with capsular type 3 pneumococci. The protective efficacy of recombinant PotD was evaluated by active immunization and intravenous challenge with capsular type 3 pneumococci in CBA/N mice. Immunized mice had 91.7% survival following lethal pneumococcal challenge, compared with 100% mortality in the control group. Immunized animals had high-titer anti-PotD antibodies following three immunizations with alum. Protection in a sepsis model was also seen after passive administration of rabbit antiserum raised against PotD (P < 0.004). These results suggest that antibodies to PotD confer protection against invasive pneumococcal disease and that this protein should be studied further as a potential vaccine candidate for protection against invasive pneumococcal infections.     

GAA trinucleotide repeat region regulates M9/pMGA gene expression in Mycoplasma gallisepticum

Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn4001 was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M. gallisepticum. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with a lacZ gene from E. coli, and inserted into the Tn4001-containing plasmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressed lacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M. gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.     

Electroporation of a suicide plasmid bearing a transposon into Brucella abortus

The creation of bacterial mutants by transposon mutagenesis has facilitated the identification of regulatory and structural genes. In the case of B. abortus the number of reported transposon mutants created by mating or P1 infection has been relatively small. We studied the conditions necessary to introduce Tn5 bearing a kanamycin resistance gene (KnR) into B. abortus by electroporation. The highest frequency of Tn5 transposition was obtained using B. abortus 2308 harvested at a density of 5.2 x 10(8) cells/ml; 0.5 microgram of plasmid was electroporated for 10 ms at 625 V (equivalent to 12.5 kV/cm). The frequency of Tn5 transposition obtained under optimum conditions was estimated to be around 18-20 insertions per 10(10) Brucella. The phase of growth (or the number of generations) had a strong influence on the frequency of transposition. Dot blot analysis confirmed that all KnR clones appearing after 4 days of incubation at 37 degrees C carried Tn5 in their genomes. Furthermore, the randomnes of Tn5 insertion was verified by Southern analysis of chromosomal DNA extracted from knR clones and hybridized with labeled Tn5.     

Role of Toll-like receptor 4 in gram-positive and gram-negative pneumonia in mice

To determine the role of Toll-like receptor 4 (TLR4) in the immune response to pneumonia, C3H/HeJ mice (which display a mutant nonfunctional TLR4) and C3H/HeN wild-type mice were intranasally infected with either Streptococcus pneumoniae (a common gram-positive respiratory pathogen) or Klebsiella pneumoniae (a common gram-negative respiratory pathogen). In cases of pneumococcal pneumonia, TLR4 mutant mice showed a reduced survival only after infection with low-level bacterial doses, which was associated with a higher bacterial burden in their lungs 48 h postinfection. In Klebsiella pneumonia, TLR4 mutant mice demonstrated a shortened survival after infection with either a low- or a high-level bacterial dose together with an enhanced bacterial outgrowth in their lungs. These data suggest that TLR4 contributes to a protective immune response in both pneumococcal and Klebsiella pneumonia and that its role is more important in respiratory tract infection caused by the latter (gram-negative) pathogen.