4-4-(Anilinomethyl)-3-[4-(trifluoromethyl)phenyl]-1H-pyrazol-1-ylbenzoic acid derivatives as potent anti-gram-positive bacterial agents.

A collection of potent antimicrobials consisting of novel 1,3-bis-benzoic acid and trifluoromethyl phenyl derived pyrazoles has been synthesized and tested for antibacterial activity. The majority of trifluoromethyl phenyl derivatives are highly potent growth inhibitors of Gram-positive bacteria and showed low toxicity to human cultured cells. In particular, two compounds (59 and 74) were selected for additional studies. These compounds are highly effective against Staphylococcus aureus as shown by a low minimum inhibitory concentration (MIC), a bactericidal effect in time-kill assays, moderate inhibition of biofilm formation as well as biofilm destruction, and a bactericidal effect against stationary phase cells representing non-growing persister cells. Multistep resistance assays showed a very low tendency for S. aureus and Enterococcus faecalis to develop resistance through mutation. Additionally, in vivo mouse model studies showed no harmful effects at doses up to 50 mg/kg using 14 blood plasma organ toxicity markers or TUNEL assay in liver and kidney. Investigations into the mode of action by performing macromolecular synthesis inhibition studies showed a broad range of inhibitory effects, suggesting targets that have a global effect on bacterial cell function.

Synthesis and antimicrobial studies of hydrazone derivatives of 4-[3-(2,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid and 4-[3-(3,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid.

Microbial resistance to antibiotics is an unresolved global concern, which needs urgent and coordinated action. One of the guidelines of the Centers for Disease Control and Preventions (CDC) to combat antibiotic resistance is the development of new antibiotics to treat drug-resistant bacteria. In our effort to find new antibiotics, we report the synthesis and antimicrobial studies of 30 new pyrazole derivatives. These novel molecules have been synthesized by using readily available starting materials and benign reaction conditions. Some of these molecules have shown activity with MIC values as low as 0.78 µg/mL against four bacterial strains; Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Acinetobacter baumannii. Furthermore, active molecules are non-toxic to mammalian cell line.

Expert Elicitation Provides a Rapid Alternative to Formal Case-Control Study of an H7N9 Avian Influenza Outbreak in the United States.

An expert elicitation was staged to rapidly decipher plausible routes and risks of pathogen transmission in the 2017 H7N9 avian influenza (AI) outbreak in the four-state region of Tennessee, Alabama, Georgia, and Kentucky. The process included the identification of risk factors found in a preponderance of commercial broiler breeder case farms over matched controls and an opinion-based weighting of risks and mitigations perceived influential to this outbreak. Although the two highly pathogenic AI case farms had general location and company ownership in common, obvious connections were lacking for the remainder of H7N9-infected (all low pathogenicity) commercial farms. Expert elicitation of differences between known cases and controls suggested a key role for environmental rather than lateral (business network) pathways in the distribution of low pathogenicity AI across commercial broiler breeder operations. Factors with greatest strength as predictors of disease, whether or not they were causal, included mesopredator or rodent incursions, enclosure defects, and habitat disturbance that might attract wildlife to the farm (e.g., feed spills and vacating of neighboring properties). Business affiliations that may have facilitated farm-to-farm transfer, in contrast, were limited. Biosecurity standards varied across this study group but were no more or less stringent among cases over controls. However, results from a parallel hypothetical scenario staged to address field data gaps suggest that uniformity and consistency in the implementation of biosecurity practices may impact risk of disease introduction. Importantly, this study was conducted within a few weeks and with little disruption to emergency response activities. As such, the approach offers an alternative model for interim field investigation of new or emerging high-consequence diseases with immediate decision support needs.

Staphylococcus aureus collagen adhesin contributes to the pathogenesis of osteomyelitis.

To evaluate the role of the Staphylococcus aureus collagen-binding adhesin (Cna) in bone and joint infection, we generated a cna mutant in S. aureus UAMS-1, a strain that was originally isolated from the bone of a patient suffering from osteomyelitis. The mutant (UAMS-237) was unable to bind collagen but bound fibronectin at levels comparable to UAMS-1. The relative virulence of UAMS-1 and UAMS-237 was assessed using a murine model of acute hematogenous osteomyelitis. Specifically, 10(8) colony-forming units (cfu) were introduced into the bloodstream of NIH-Swiss mice via tail-vein injection. After 2 weeks, the left hind limb was harvested and examined histologically for evidence of osteomyelitis and septic arthritis. Osteomyelitis developed in 14 of 20 mice (70%) infected with UAMS-1, but only 1 of 20 (5%) infected with UAMS-237 (p < 0.001). In contrast, septic arthritis was observed in 12 of 20 mice (60%) infected with UAMS-1 and 14 of 20 (70%) infected with UAMS-237 (p < 0.75). These results indicate that Cna is not required to establish joint infection, but does make an important contribution to the ability of S. aureus to establish infection in bone through hematogenous spread.

Multiplex PCR protocol for the diagnosis of staphylococcal infection.

We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection. The protocol was designed to (i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding to Staphylococcus-specific regions of the 16S rRNA genes), (ii) distinguish between S. aureus and the coagulase-negative staphylococci (CNS) (based on amplification of the S. aureus-specific clfA gene), and (iii) provide an indication of the likelihood that the staphylococci present in the specimen are resistant to oxacillin (based on amplification of the mecA gene). The expected fragments were amplified from each of 60 staphylococcal isolates (13 oxacillin-resistant S. aureus isolates, 23 oxacillin-sensitive S. aureus isolates, 17 oxacillin-resistant CNS, and 7 oxacillin-sensitive CNS). No amplification products were observed with template DNA from nonstaphylococcal species, and the efficiency of amplification of staphylococcal targets was not adversely affected by the presence of DNA from other bacterial species in the same sample. The utility of the protocol for the analysis of clinical samples was verified by analysis of aliquots taken directly from BacT/Alert blood culture bottles. Of 77 blood cultures tested, only 7 yielded results inconsistent with those of conventional methods of diagnosis and susceptibility testing. Of those, one was identified as a CNS species by PCR and S. aureus by conventional methods. We also identified two isolates that were mecA positive but were oxacillin sensitive according to conventional methods. The other four samples failed to yield any amplification product even with a control set of primers corresponding to a conserved region of the eubacterial rRNA genes.

Design and performance of a channel reconstruction project in a coastal California gravel-bed stream.

A 0.9 km-reach of Uvas Creek, California, was reconstructed as a sinuous, meandering channel in November 1995. In February 1996, this new channel washed out. We reviewed project documents to determine the basis for the project design and conducted our own historical geomorphological study to understand the processes operating in the catchment and project reach. The project was designed using a popular stream classification system, based on which the designers assumed that a "C4" channel (a meandering gravel-bed channel) would be stable at the site. Our historical geomorphological analysis showed that the reach had been braided historically, typical of streams draining the Franciscan Formation in the California Coast Ranges, with episodic flows and high sand and gravel transport. After the project washed out, Uvas Creek reestablished an irregular, braided sand-and-gravel channel, although the channel here was narrower than it had been historically, probably due to such factors as incision caused by gravel mining. Our study casts doubt on several assumptions common in many stream restoration projects: that channel stability is always an appropriate goal; that channel forms are determined by flows with return periods of about 1.5 years; that a channel classification system is an easy, appropriate basis for channel design; and that a new channel form can be imposed without addressing the processes that determine channel form.

Molecular pathogenesis of staphylcoccal osteomyelitis.

Staphylococcus aureus is the most prominent musculoskeletal pathogen of man and animals. The persistent emergence of antibiotic-resistant strains has prompted renewed efforts to develop alternative protocols for the treatment and prevention of staphylococcal disease. These efforts have included attempts to develop an effective staphylococcal vaccine. Among the potential vaccine candidates are a group of surface proteins that act as adhesins by virtue of their ability to bind host proteins present in plasma and in the extracellular matrix. Because of our interest in the treatment and prevention of musculoskeletal infection, we have focused on adhesins that contribute to the colonization of bone and cartilage. Based on reports suggesting that colonization is a conserved characteristic of S. aureus strains that cause osteomyelitis and septic arthritis, we have paid particular attention to the factors that contribute to the ability to bind collagen. To date, only one collagen-binding adhesin (Cna) has been identified, and the gene encoding this adhesin (cna) is not present in most S. aureus strains. The possibility that a rare phenotype is conserved among isolates that cause a particular form of infection suggests a cause-and-effect relationship in which the phenotype contributes to the pathogenesis of the disease. To further evaluate that hypothesis, we attempted to determine whether Cna is the only collagen-binding adhesin produced by S. aureus and whether strains that encode cna share additional characteristics that distinguish them from other S. aureus strains. We also studied whether immunization with Cna induces a protective immune response. Our results confirm that Cna is the primary and probably the only collagen-binding adhesin and that the genetic element encoding cna does not encode any additional virulence factors. These results strongly suggest that the only consistent difference between cna-positive and cna-negative strains is the ability to bind collagen. We also demonstrated that vaccination with a recombinant fragment of Cna can protect animals against septic death and limit the ability to colonize bone.

Functional analysis of the Staphylococcus aureus collagen adhesin B domain.

The Staphylococcus aureus collagen adhesin (CNA) occurs in at least four forms that differ in the number (one, two, three, or four) of B domains. The B domains contain 187 amino acids and are located between the domains that anchor CNA to the cell envelope and the ligand-binding A domain. To determine whether a B domain is required for functional expression of CNA, we cloned the 2B cna gene from S. aureus strain Phillips and then eliminated both B domains by overlapping PCR. The absence of a B domain did not affect processing of the collagen adhesin to the cell surface or the ability to bind collagen. Based on our recent demonstration that the capsule can mask CNA on the surface of S. aureus cells (A. F. Gillaspy et al., Infect. Immun. 66:3170-3178, 1998), we also investigated the possibility that multiple B domains can extend the ligand-binding A domain outward from the cell surface and thereby overcome the inhibitory effect of the capsule. Specifically, we cloned the naturally occurring 4B CNA variant from S. aureus UAMS-639 and, by successive elimination of B domains, generated 1, 2, and 3B variants that are isogenic with respect to the 4B clone. After introducing each variant into microencapsulated and heavily encapsulated strains of S. aureus and growing cells under conditions known to affect capsule production (e.g., growth on Columbia agar), we correlated capsule production with exposure of CNA on the cell surface and the ability to bind collagen. Under no circumstance was the masking effect of the capsule reduced by the presence of multiple B domains. These results indicate that the B domains do not extend the ligand-binding A domain outward in a fashion that can overcome the inhibition of collagen binding associated with capsule production.

Characterization of the SarA virulence gene regulator of Staphylococcus aureus.

Staphylococcus aureus is a potent human pathogen that expresses a large number of virulence factors in a temporally regulated fashion. Two pleiotropically acting regulatory loci were identified in previous mutational studies. The agr locus comprises two operons that express a quorum-sensing system from the P2 promoter and a regulatory RNA molecule from the P3 promoter. The sar locus encodes a DNA-binding protein that activates the expression of both agr operons. We have cloned the sarA gene, expressed SarA in Escherichia coli and purified the recombinant protein to apparent homogeneity. The purified protein was found to be dimeric in the presence and absence of DNA and to consist mostly of alpha-helices. DNase I footprinting of SarA on the putative regulatory region cis to the agr promoters revealed three high-affinity binding sites composed of two half-sites each. Quantitative electrophoretic mobility shift assays (EMSAs) were used to derive equilibrium binding constants (KD) for the interaction of SarA with these binding sites. An unusual ladder banding pattern was observed in EMSA with a large DNA fragment including all three binding sites. Our data indicate that SarA regulation of the agr operons involves binding to multiple half-sites and may involve other sites located downstream of the promoters.

The Staphylococcal accessory regulator (sar) represses transcription of the Staphylococcus aureus collagen adhesin gene (cna) in an agr-independent manner.

Comparison of Staphylococcus aureus strains carrying mutations inactivating the staphylococcal accessory regulator (sar ) and/or the accessory gene regulator (agr ) suggests that sar is the primary regulatory element controlling transcription of the collagen adhesin gene (cna ) and that the regulatory effect of sar is independent of the interaction between SarA and agr. To test this hypothesis, we cloned the regions encoding each of the overlapping sar transcripts, all of which include the sarA open reading frame (ORF), and introduced each clone into cna-positive sar and agr mutants. The introduction of each clone restored the expected sar transcripts and the temporal pattern of sar transcription. The introduction of each clone also complemented the defect in cna transcription and restored collagen binding to wild-type levels. This was true even when the clones were introduced into a sar/agr double mutant. These results confirm the hypothesis that the sar-mediated regulation of cna transcription occurs via an agr-independent pathway. Direct evidence supporting this hypothesis comes from electrophoretic mobility shift assays demonstrating that SarA exhibits high-affinity binding to cis elements upstream of the cna structural gene. We also examined the correlation between sar transcription and the production of SarA. Western blot analysis of two wild-type strains indicated that SarA was produced in indistinguishable amounts during both the exponential and the post-exponential growth phases.

Mechanisms of Staphylococcus aureus invasion of cultured osteoblasts.

Staphylococcus aureus is a bacterial pathogen causing approximately 80% of all cases of human osteomyelitis. This bacterium can adhere to and become internalized by osteoblasts and previous studies indicate that osteoblasts are active in the internalization process. In the current study, we examined the roles of microfilaments, microtubules and clathrin-dependent receptor-mediated endocytosis in the internalization of S. aureus by MC3T3-E1 mouse osteoblast cells. Microfilament and microtubule polymerization was inhibited with cytochalasin D and colchicine. Clathrin-coated pit formation was examined by using the transaminase inhibitor, monodanslycadaverine. The results of this study indicate that mouse osteoblasts utilize actin microfilaments, microtubules and clathrin-coated pits in the internalization of S. aureus; however, microfilaments seem to play the most significant role in the invasion process.

Rapid identification and typing of Staphylococcus aureus by PCR-restriction fragment length polymorphism analysis of the aroA gene.

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 x 10(2) CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.

Factors affecting the collagen binding capacity of Staphylococcus aureus.

To determine whether the ability of Staphylococcus aureus to bind collagen involves an adhesin other than the collagen adhesin encoded by cna, we examined the collagen binding capacity (CBC) of 32 strains of S. aureus. With only two exceptions, a high CBC corresponded with the presence of cna. Both exceptions involved cna-positive strains with a low CBC. The first was a single strain (ACH5) that encoded but did not express cna. The second were the mucoid strains Smith diffuse and M, both of which encoded and expressed cna but bound only minimal amounts of collagen. Analysis of capsule mutants suggests that the reduced CBC observed in the mucoid strains was due to masking of the collagen adhesin on the cell surface and that this masking effect is restricted to heavily encapsulated strains. Differences in the CBC of the remaining cna-positive strains were correlated to variations in the level of cna transcription and were independent of the number of B domain repeats in the cna gene. In all cna-positive strains other than ACH5, cna transcription was temporally regulated, with cna mRNA levels being highest in cells taken from exponentially growing cultures and falling to almost undetectable levels as cultures entered the post-exponential growth phase. The CBC was also highest with cells taken from exponentially growing cultures. Mutation of agr resulted in a slight increase in cna transcription and a corresponding increase in CBC during the exponential growth phase but did not affect the temporal pattern of cna transcription. Mutation of sar resulted in a more dramatic increase in CBC and a delay in the post-exponential-phase repression of cna transcription. Mutation of both sar and agr had an additive effect on both CBC and cna transcription. We conclude that (i) cna encodes the primary collagen-binding adhesin in S. aureus, (ii) sar is the primary regulatory element controlling expression of cna, and (iii) the regulatory effects of sar and agr on cna transcription are independent of the interaction between sar and agr.

The Staphylococcus aureus collagen adhesin-encoding gene (cna) is within a discrete genetic element.

Although the gene (cna) encoding the Staphylococcus aureus (Sa) collagen adhesin is not present in all strains, the DNA both upstream and downstream of cna is present in all Sa strains. Using oligo primers corresponding to the conserved nt flanking cna and template DNA from Sa strains that do not encode cna, we amplified a 372-bp fragment. These results illustrate that the conserved regions upstream and downstream of cna are contiguous in strains that do not encode cna. Using primers corresponding to the conserved flanking DNA together with primers corresponding to the 5' and 3' ends of cna, we also amplified DNA fragments containing the junctions between the cna genetic element and the conserved flanking sequences. Sequence comparisons of the amplification products from four cna negative and four cna positive strains revealed that cna is within a discrete genetic element that extends 202 bp upstream from the cna start codon and 100 bp downstream of the cna stop codon. Sequence analysis of the ends of the cna element did not reveal any of the repeats characteristic of transposable elements. These results suggest that cna may be part of a larger element (e.g., a phage) that may or may not contain cna. Alternatively, cna may be a subject to a precise excision event resulting in its deletion from the chromosome. Based on sequence analysis of the flanking DNA amplified from strains that do not encode cna, the presence of a cna genetic element does not disrupt an ORF.

Prevalence and chromosomal map location of Staphylococcus aureus adhesin genes.

Using genomic DNA from 25 unrelated strains and probes specific for each gene, we assessed the prevalence of the Staphylococcus aureus (Sa) adhesion genes cna, fnbA, fnbB, fib, clfA, fbpA, ebpS and map. All 25 strains encoded fib, clfA, ebpS, map and at least one of the fnb genes. fbpA and coa appeared to be allelic variants of the same gene with the fbpA variant being present in only four of 25 isolates. cna was present in 10 of 25 strains. Using Southern blot analysis of SmaI-digested genomic DNA resolved by pulsed-field gel electrophoresis, the adhesion genes were mapped to SmaI fragments A (ebpS), B (fib and clfA), C (fnbA/fnbB), E (fbpA), F (map) and G (cna). Despite variations in SmaI restriction profiles, co-localization of adhesin genes with genes known to map to specific SmaI fragments in the Sa 8325-4 chromosome strains suggests that the chromosomal location of each adhesin gene is conserved.

Comparative evaluation of use of cna, fnbA, fnbB, and hlb for genomic fingerprinting in the epidemiological typing of Staphylococcus aureus.

We used a genomic fingerprinting protocol to characterize 59 Staphylococcus aureus strains and a single S. intermedius isolate, all of which were previously typed by 13 different methods (F. C. Tenover et al., J. Clin. Microbiol. 32:407-415, 1994). These 60 strains were divided into three groups of 20 strains each, with each group including internal controls. Two of the three groups (groups SB and SC) included 29 strains from four relatively well-defined outbreaks. The epidemiological relationships of the strains in the third group (group SA) were unclear. Fingerprints were established by Southern blotting with HaeIII-digested genomic DNA and a probe mixture consisting of DNA fragments corresponding to the S. aureus collagen adhesin (cna), fibronectin-binding protein (fnbA and fnbB), and beta-toxin (hlb) genes. An unambiguous fingerprint was obtained for all S. aureus isolates. No hybridization signal was observed with S. intermedius. Twenty-seven of the 29 related strains in the SB and SC groups were correctly identified as belonging to one of the four epidemiologically related groups. Our protocol was less successful with respect to the exclusion of unrelated strains. Specifically, only 6 of 11 unrelated strains in the SB and SC groups had a fingerprint that was distinct by comparison to the fingerprints of the outbreak strains. Nevertheless, our protocol was relatively accurate by comparison to the accuracies of the other methods and was one of only six methods that accurately identified all of the repetitive strains included as internal controls.

Characterization of a rabbit model of staphylococcal osteomyelitis.

We previously described a rabbit osteomyelitis model that involved the direct introduction of Staphylococcus aureus into devascularized bone. To further evaluate the model, we performed experiments aimed at correlating the microbiological, radiographic, and histologic parameters involved in the development of experimental osteomyelitis. Using the strain UAMS-1, we achieved an infection rate of 75% with an inoculum as small as 2 x 10(3) colony-forming units. However, development of significant radiographic and histologic signs of disease required an inoculum of at least 2 x 10(4) colony-forming units. Radiographic signs were minimal 1 week after infection and progressed steadily to a maximum 3 weeks after infection. In contrast, histologic signs of disease were observed within 1 week and remained essentially unchanged throughout the 4-week evaluation period. Unlike the results obtained with UAMS-1, rabbits infected with the heavily encapsulated Staphylococcus aureus strain Smith diffuse exhibited little evidence of disease even when infected with 2 x 10(6) colony-forming units. The reduced virulence of strain Smith diffuse was surprising given its greatly enhanced virulence (relative to UAMS-1) in a murine peritonitis model of staphylococcal disease. These results suggest that UAMS-1 expresses virulence factors that are important in the pathogenesis of osteomyelitis and that some or all of these virulence factors are either absent or are not expressed in strain Smith diffuse. Most importantly, the results suggest that our model may be appropriate for the identification and characterization of these virulence factors.

Transcriptional regulation of the Staphylococcus aureus collagen adhesion gene, cna.

We demonstrate that transcription of the Staphylococcus aureus collagen adhesin gene (cna) is temporally regulated, with expression being highest in exponentially growing cultures and falling to almost undetectable levels as cultures enter the post-exponential-growth phase. The temporal regulation of cna transcription was not affected by mutation of agr. We also demonstrate that the collagen adhesin is expressed by both agr+ and agr-negative S. aureus cells growing in bone.

Genomic fingerprinting for epidemiological differentiation of Staphylococcus aureus clinical isolates.

We used genomic fingerprinting to investigate an outbreak of methicillin-resistant Staphylococcus aureus in the neonatal intensive care units (NICUs) of two hospitals. The hospitals are located in the same city and are part of the same medical care system. Fingerprinting was done by Southern blot hybridization with DNA probes for the genes encoding the S. aureus collagen adhesin (cna), fibronectin-binding proteins (fnbA and fnbB), and beta-toxin (hlb). Genomic DNA was digested with HaeIII (cna and fnbA-fnbB probes) or HindIII (hlb probe). Hybridization patterns could be distinguished on the basis of (i) the presence or absence of cna, (ii) the size of the restriction fragment containing the cna gene, (iii) restriction fragment length polymorphisms within fnbA and fnbB, (iv) the presence of a lysogenic phage within hlb, and (v) the sizes of the restriction fragments containing the phage-bacterial DNA junction fragments. Over a period of 4 months we examined a total of 46 isolates obtained from various wards within each hospital. Among these 46 isolates, we observed a total of 4 cna patterns, 11 fnbA-fnbB patterns, and 11 hlb patterns. Southern blots with HaeIII-digested genomic DNA and a combination of all three gene probes revealed a total of 16 clearly distinguishable patterns. A total of 22 of the 46 isolates were identical with respect to every genomic marker examined. A total of 21 of these 22 isolates were obtained from patients within an NICU. Nineteen of 21 isolates also exhibited identical antibiotic resistance profiles (antibiogram). Although 5 of the remaining 24 strains exhibited an antibiogram identical to those of the NICU isolates, all 24 strains could be distinguished from the NICU isolates by at least one genomic marker. These results suggest that the NICU isolates had a common origin and that genomic fingerprinting with the cna, fnbA, fnbB, and hlb gene probes can provide an important epidemiological tool for the identification of clinical isolates of S. aureus.

Role of the accessory gene regulator (agr) in pathogenesis of staphylococcal osteomyelitis.

To examine the role of the accessory gene regulator (agr) in staphylococcal osteomyelitis, we compared a Staphylococcus aureus osteomyelitis isolate (UAMS-1) with a derivative of the same strain (UAMS-4) carrying an inactivated agr locus. Virulence was assessed with a rabbit model of acute, exogenous osteomyelitis. Bacteria were delivered by microinjection into the midradial region of the forelimb. After 4 weeks, UAMS-1 was identified in the bone of 12 of 13 rabbits infected with > or = 2 x 10(6) CFU and 5 of 6 infected with < or = 2 x 10(5) CFU. In contrast, UAMS-4 was found in 6 of 13 infected with the higher dose and 1 of 6 infected with the lower dose. Additionally, on the basis of a five-point scale assessing radiographic evidence of disease, rabbits infected with UAMS-1 had average scores of 2.64 +/- 0.30 (high dose) and 1.43 +/- 0.39 (low dose) while rabbits infected with UAMS-4 had average scores of 0.95 +/- 0.23 (high dose) and 0.63 +/- 0.20 (low dose). Uninfected controls had an average score of 0.53 +/- 0.08. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < or = 0.047). The results obtained with UAMS-4 were not significantly different from those obtained with the controls at either dose of UAMS-4 (P > or = 0.150). On the basis of a similar five-point scale assessing histopathological evidence of disease, rabbits infected with UAMS-1 had average scores of 2.31 +/- 0.22 (high dose) and 1.96 +/- 0.36 (low dose) while rabbits infected with UAMS-4 had average scores of 1.58 +/- 0.29 (high dose) and 0.83 +/- 0.32 (low dose). Controls had an average score of 0.33 +/- 0.05. The results obtained with UAMS-1 were significantly different from those obtained with UAMS-4 at both doses (P < or = 0.040). However, the results obtained with UAMS-4 were significantly different from the controls only at the high dose of UAMS-4 (P = 0.025). We conclude that mutation of agr reduces the incidence and severity of disease but does not eliminate the ability to colonize bone and cause histopathological evidence of osteomyelitis.