Identification of the Causative Bacteria in Musculoskeletal Infections Using 16s rDNA - Denaturing Gradient Gel Electrophoresis Analysis

Musculoskeletal infections are infections of the bone and surrounding tissues. They are currently diagnosed based on culture analysis, which is the gold standard for pathogen identification. However, these clinical laboratory methods are frequently inadequate for the identification of the causative agents, because a large percentage (25-50%) of confirmed musculoskeletal infections are false negatives in which no pathogen is identified in culture. My data supports these results. The goal of this project was to use PCR amplification of a portion of the 16S rRNA gene to test an alternative approach for the identification of these pathogens and to assess the diversity of the bacteria involved. The advantages of this alternative method are that it should increase sample sensitivity and the speed of detection. In addition, bacteria that are non-culturable or in low abundance can be detected using this molecular technique. However, a complication of this approach is that the majority of musculoskeletal infections are polymicrobial, which prohibits direct identification from the infected tissue by DNA sequencing of the initial 16S rDNA amplification products. One way to solve this problem is to use denaturing gradient gel electrophoresis (DGGE) to separate the PCR products before DNA sequencing. Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on their melting point, which is determined by their DNA sequence. This analytical technique allows a mixture of PCR products of the same length that electrophoreses through agarose gels as one band, to be separated into different bands and then used for DNA sequence analysis. In this way, the DGGE allows for the identification of individual bacterial species in polymicrobial-infected tissue, which is critical for improving clinical outcomes. By combining the 16S rDNA amplification and the DGGE techniques together, an alternative approach for identification has been used. The 16S rRNA gene PCR-DGGE method includes several critical steps: DNA extraction from tissue biopsies, amplification of the bacterial DNA, PCR product separation by DGGE, amplification of the gel-extracted DNA, and DNA sequencing and analysis. Each step of the method was optimized to increase its sensitivity and for rapid detection of the bacteria present in human tissue samples. The limit of detection for the DNA extraction from tissue was at least 20 Staphylococcus aureus cells and the limit of detection for PCR was at least 0.05 pg of template DNA. The conditions for DGGE electrophoreses were optimized by using a double gradient of acrylamide (6 – 10%) and denaturant (30-70%), which increased the separation between distinct PCR products. The use of GelRed (Biotium) improved the DNA visualization in the DGGE gel. To recover the DNA from the DGGE gels the gel slices were excised, shredded in a bead beater, and the DNA was allowed to diffuse into sterile water overnight. The use of primers containing specific linkers allowed the entire amplified PCR product to be sequenced and then analyzed. The optimized 16S rRNA gene PCR-DGGE method was used to analyze 50 tissue biopsy samples chosen randomly from our collection. The results were compared to those of the Memorial Hermann Hospital Clinical Microbiology Laboratory for the same samples. The molecular method was congruent for 10 of the 17 (59%) culture negative tissue samples. In 7 of the 17 (41%) culture negative the molecular method identified a bacterium. The molecular method was congruent with the culture identification for 7 of the 33 (21%) positive cultured tissue samples. However, in 8 of the 33 (24%) the molecular method identified more organisms. In 13 of the 15 (87%) polymicrobial cultured tissue samples the molecular method identified at least one organism that was also identified by culture techniques. Overall, the DGGE analysis of 16S rDNA is an effective method to identify bacteria not identified by culture analysis.

Relationship between enterotoxigenic Escherichia coli and travelers' diarrhea in Mexico, from 1992 to 1997

The purpose of this study was to examine the relationship between enterotoxigenic ETEC and travelers' diarrhea over a period of five years in Guadalajara, Mexico. Specifically, this study identified and characterized ETEC from travelers with diarrhea. The objectives were to study the colonization factor antigens, toxins and antibiotic sensitivity patterns in ETEC from 1992 to 1997 and to study the molecular epidemiology of ETEC by plasmid content and DNA restriction fragment patterns. ^ In this survey of travelers' diarrhea in Guadalajara, Mexico, 928 travelers with diarrhea were screened for enteric pathogens between 1992 and 1997. ETEC were isolated in 195 (19.9%) of the patients, representing the most frequent enteric pathogen identified. ^ A total of 31 antimicrobial susceptibility patterns were identified among ETEC isolates over the five-year period. ^ The 195 ETEC isolates contained two to six plasmids each, which ranged in size from 2.0 to 23 kbp. ^ Three different reproducible rRNA gene restriction patterns (ribotypes R-1 to R-3) were obtained among the 195 isolates with the enzyme, HindIII. ^ Colonization factor antigens (CFAs) were identified in 99 (51%) of the 195 ETEC strains studied. ^ Cluster analysis of the observations seen in the four assays all confirmed the five distinct groups of study-year strains of ETEC. Each group had a >95% similarity level of strains within the group and <60% similarity level between the groups. In addition, discriminant analysis of assay variables used in predicting the ETEC strains, reveal a >80% relationship between both the plasmid and rRNA content of ETEC strains and study-year. ^ These findings, based on laboratory observations of the differences in biochemical, antimicrobial susceptibility, plasmid and ribotype content, suggest complex epidemiology for ETEC strains in a population with travelers' diarrhea. The findings of this study may have implications for our understanding of the epidemiology, transmission, treatment, control and prevention of the disease. It has been suggested that an ETEC vaccine for humans should contain the most prevalent CFAs. Therefore, it is important to know the prevalence of these factors in ETEC in various geographical areas. ^ CFAs described in this dissertation may be used in different epidemiological studies in which the prevalence of CFAs and other properties on ETEC will be evaluated. Furthermore, in spite of an intense search in near 200 ETEC isolates for strains that may have clonal relationship, we failed to identify such strains. However, further studies are in progress to construct suitable live vaccine strains and to introduce several of CFAs in the same host organism by recombinant DNA techniques (Dr. Ann-Mari Svennerholm's lab). (Abstract shortened by UMI.)^

Clinical and microbiological aspects of linezolid resistance mediated by the cfr gene encoding a 23S rRNA methyltransferase.

The cfr (chloramphenicol-florfenicol resistance) gene encodes a 23S rRNA methyltransferase that confers resistance to linezolid. Detection of linezolid resistance was evaluated in the first cfr-carrying human hospital isolate of linezolid and methicillin-resistant Staphylococcus aureus (designated MRSA CM-05) by dilution and diffusion methods (including Etest). The presence of cfr was investigated in isolates of staphylococci colonizing the patient's household contacts and clinical isolates recovered from patients in the same unit where MRSA CM-05 was isolated. Additionally, 68 chloramphenicol-resistant Colombian MRSA isolates recovered from hospitals between 2001 and 2004 were screened for the presence of the cfr gene. In addition to erm(B), the erm(A) gene was also detected in CM-05. The isolate belonged to sequence type 5 and carried staphylococcal chromosomal cassette mec type I. We were unable to detect the cfr gene in any of the human staphylococci screened (either clinical or colonizing isolates). Agar and broth dilution methods detected linezolid resistance in CM-05. However, the Etest and disk diffusion methods failed to detect resistance after 24 h of incubation. Oxazolidinone resistance mediated by the cfr gene is rare, and acquisition by a human isolate appears to be a recent event in Colombia. The detection of cfr-mediated linezolid resistance might be compromised by the use of the disk diffusion or Etest method.

Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Accessing novel developmental mechanisms in the mouse by gene trapping

Genetic analysis is a powerful method for analyzing the function of specific genes in development. I sought to identify novel genes in the mouse using a genetic analysis relying on the expression pattern and phenotype of mutated genes. To this end, I have conducted a gene trap screen using the vector $\rm SA\beta geo,$ a promoterless DNA construct that encodes a fusion protein with lacZ and neomycin resistance activities. Productive integration and expression of the $\beta$geo protein in embryonic stem (ES) cells requires integration into an active transcription unit. The endogenous regulatory elements direct reporter gene expression which reflects the expression of the endogenous gene. Of eight mouse lines generated from gene trap ES cell clones, four showed differential regulation of $\beta$geo activity during embryogenesis. These four were analyzed in more detail.^ Three of the lines RNA 1, RNA2 and RNA 3 had similar expression patterns, within subsets of cells in sites of embryonic hematopoiesis. Cloning of the trapped genes revealed that all three integrations had occurred within 45S rRNA precursor transcription units. These results imply that there exists in these cells some mechanism responsible for the efficient production of the $\beta$geo protein from an RNA polymerase I transcript that is not present in most of the cells in the embryo.^ The fourth line, GT-2, showed widespread, dynamic expression. Many of the sites of expression were important classic embryonic induction systems. Cloning of the sequences fused to the $5\sp\prime$ end of the $\beta$geo sequence revealed that the trapped gene contained significant sequence homology with a previously identified human sequence HumORF5. An open reading frame of this sequence is homologous to a group of eukaryotic proteins that are members of the RNA helicase superfamily I.^ Analysis of the gene trap lines suggests that potentially novel developmental mechanisms have been uncovered. In the case of RNA 1, 2 and 3, the differential production of ribosomal RNAs may be required for differentiation or function of the $\beta$geo positive hematopoietic cells. In the GT-2 line, a previously unsuspected temporal and spatial regulation of a putative RNA helicase implies a role for this activity during specific aspects of mouse development. ^

Functional studies of the homeobox gene {\it Goosecoid\/} during mouse embryogenesis

A fundamental question in developmental biology is to understand the mechanisms that govern the development of an adult individual from a single cell. Goosecoid (Gsc) is an evolutionarily conserved homeobox gene that has been cloned in vertebrates and in Drosophila. In mice, Gsc is first expressed during gastrulation stages where it marks anterior structures of the embryo, this pattern of expression is conserved among vertebrates. Later, expression is observed during organogenesis of the head, limbs and the trunk. The conserved pattern of expression of Gsc during gastrulation and gain of function experiments in Xenopus suggested a function for Gsc in the development of anterior structures in vertebrates. Also, its expression pattern in mouse suggested a role in morphogenesis of the head, limbs and trunk. To determine the functional requirement of Gsc in mice a loss of function mutation was generated by homologous recombination in embryonic stem cells and mice mutant for Gsc were generated.^ Gsc-null mice survived to birth but died hours after delivery. Phenotypic analysis revealed craniofacial and rib cage abnormalities that correlated with the second phase of Gsc expression in the head and trunk but no anomalies were found that correlated with its pattern of expression during gastrulation or limb development.^ To determine the mode of action of Gsc during craniofacial development aggregation chimeras were generated between Gsc-null and wild-type embryos. Chimeras were generated by the aggregation of cleavage stage embryos, taking advantage of two different Gsc-null alleles generated during gene targeting. Chimeras demonstrated a cell-autonomous function for Gsc during craniofacial development and a requirement for Gsc function in cartilage and mesenchymal tissues.^ Thus, during embryogenesis in mice, Gsc is not an essential component of gastrulation as had been suggested in previous experiments. Gsc is required for craniofacial development where it acts cell autonomously in cartilage and mesenchymal tissues. Gsc is also required for proper development of the rib cage but it is dispensable for limb development in mice. ^

Functional analysis of SOX9 in chondrogenesis by targeted gene inactivation

Sox9 is a Sry-related HMG-domain containing transcription factor. Lines of evidence suggest that Sox9 has a potential role in skeletal development. During mouse development, Sox9 is predominantly expressed in all chondroprogenitors and differentiated chondrocytes, throughout the deposition of cartilage matrix. Mutations in one allele of SOX9 in humans result in campomelic dysplasia (CD), a skeletal dysplasia. syndrome characterized by the bowing of long bones. Moreover, Sox9 binds to and activates chondrocyte-specific enhancers in Col2a1 and Col11a2 genes. To further investigate the function of Sox9 in chondrogenesis, we analyzed chimeras derived from Sox9 heterozygous and homozygous null embryonic stem (ES) cells. In mouse chimeras, Sox9 −/− cells were excluded from all cartilages and did not express chondrocyte-specific genes. The segregation occurred during mesenchymal condensation. No cartilages developed in teratocarcinomas derived from Sox9 −/− ES cells. Mice heterozygous for the Sox9 mutation died neonatally and exhibited skeletal abnormalities resembling those of the CD patients. The Sox9 +/− mutants had a cleft palate and hypoplasia of scapula, pelvis and other skeletal structures derived by endochondral ossification. Bending of the radius, ulna and tibia cartilage was prominent at embryonic day 14.5 (E14.5). At E12.5 many pre-cartilaginous condensations were already defective. Advanced ossification was observed and the hypertrophic zone was enlarged in the growth plates, suggesting that Sox9 also regulates hypertrophic chondrocyte differentiation. Our results identify Sox9 as the first essential regulator of chondrocyte differentiation, which plays multiple roles in chondrogenesis. ^