The ability of bacterial plasmids to adapt to novel hosts and thereby shift their host range is key to their long-term persistence in bacterial communities. Promiscuous plasmids of the incompatibility group P (IncP)-1 can colonize a wide range of hosts, but it is not known if and how they can contract, shift or further expand their host range. To understand the evolutionary mechanisms of host range shifts of IncP-1 plasmids, an IncP-1b mini-replicon was experimentally evolved in four hosts in which it was initially unstable. After 1000 generations in serial batch cultures under antibiotic selection for plasmid maintenance (kanamycin resistance), the stability of the mini-plasmid dramatically improved in all coevolved hosts. However, only plasmids evolved in Shewanella oneidensis showed improved stability in the ancestor, indicating that adaptive mutations had occurred in the plasmid itself. Complete genome sequence analysis of nine independently evolved plasmids showed seven unique plasmid genotypes that had various kinds of single mutations at one locus, namely, the N-terminal region of the replication initiation protein TrfA. Such parallel evolution indicates that this region was under strong selection. In five of the seven evolved plasmids, these trfA mutations resulted in a significantly higher plasmid copy number. Evolved plasmids were found to be stable in four other naive hosts, but could no longer replicate in Pseudomonas aeruginosa. This study shows that plasmids can specialize to a novel host through trade-offs between improved stability in the new host and the ability to replicate in a previously permissive host.

The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance.

The effects of type 2 diabetes mellitus (T2DM) on bone volumetric density, bone geometry, and estimates of bone strength are not well established. We used peripheral quantitative computed tomography (pQCT) to compare tibial and radial bone volumetric density (vBMD, mg/cm3), total (ToA, mm2) and cortical (CoA, mm2) bone area and estimates of bone compressive and bending strength in a subset (n = 1171) of men (≥65 years of age) who participated in the multisite Osteoporotic Fractures in Men (MrOS) study. Analysis of covariance–adjusted bone data for clinic site, age, and limb length (model 1) and further adjusted for body weight (model 2) were used to compare data between participants with (n = 190) and without (n = 981) T2DM. At both the distal tibia and radius, patients with T2DM had greater bone vBMD (+2% to +4%, model 1, p < .05) and a smaller bone area (ToA −1% to −4%, model 2, p < .05). The higher vBMD compensated for lower bone area, resulting in no differences in estimated compressive bone strength at the distal trabecular bone regions. At the mostly cortical bone midshaft sites of the radius and tibia, men with T2DM had lower ToA (−1% to −3%, p < .05), resulting in lower bone bending strength at both sites after adjusting for body weight (−2% to −5%, p < .05) despite the lack of difference in cortical vBMD at these sites. These data demonstrate that older men with T2DM have bone strength that is low relative to body weight at the cortical-rich midshaft of the radius despite no difference in cortical vBMD. © 2010 American Society for Bone and Mineral Research

ABSTRACT:The effect of excess body fat on bone strength accrual is not well understood. Therefore, we assessed bone measures in healthy weight (HW) and overweight (OW) children. Children (9-11 yr) were classified as HW (n ‫ס‬ 302) or OW (n ‫ס‬ 143) based on body mass index. We assessed total (ToD) and cortical (CoD) volumetric BMD and bone area, estimates of bone strength (bone strength index [BSI]; stress-strain index [SSIp]), and muscle cross-sectional area (CSA) at the distal (8%), midshaft (50%), and proximal (66%) tibia by pQCT. We used analysis of covariance to compare bone outcomes at baseline and change over 16 mo. At baseline, all bone measures were significantly greater in OW compared with HW children (+4-15%; p Յ 0.001), with the exception of CoD at the 50% and 66% sites. Over 16 mo, ToA increased more in the OW children, whereas there was no difference for change in BSI or ToD between groups at the distal tibia. At the tibial midshaft, SSIp was similar between groups at baseline when adjusted for muscle CSA, but low when adjusted for body fat in the OW group. At both sites, bone strength increased more in OW because of a greater increase in bone area. Changes in SSIp were associated with changes in lean mass (r ‫ס‬ 0.70, p < 0.001) but not fat mass. In conclusion, although OW children seem to be at an advantage in terms of absolute bone strength, bone strength did not adapt to excess body fat. Rather, bone strength was adapted to the greater muscle area in OW children.

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The purpose of these analyses was to explore whether physical activity score, leg power or grip strength were associated with tibia and radius estimates of bone strength, cortical density, or total bone area. Peripheral quantitative computed tomography (pQCT) was used to compare tibial and radial bone volumetric density (vBMD, mg/cm3), total (ToA, mm2) and cortical (CoA, mm2) bone area, and estimates of bone compressive strength (bone strength index, BSI) and bending strength (polar strength strain index, SSIp) in a subset (n=1171) of men (≥ 65 years) who participated in the multi-site Osteoporotic Fractures in Men (MrOS) study. Physical activity was assessed by questionnaire (PASE), leg power by Nottingham Power Rig, and grip strength by a hand-held Dynamometer. Participants were categorized into quartiles of PASE, grip strength or leg power. The model was adjusted for age, race, clinic, weight, and limb length. In the tibia, BSI (+7%) and SSIp (+4%) were highest in the most active physically quartile compared to the least active (p<0.05). At the 4% site of the tibia, men with the greatest leg power had both greater ToA (+5%, p<0.001) and BSI (+5.3%, p=0.086) compared to men with the least leg power. At the 66% site of the tibia, the men with the highest leg power, compared to the men with the lowest leg power, had greater ToA (+3%, p=0.045) SSIp (+5%, p=0.008). Similar results were found at both the distal and midshaft of the radius. The findings of this study suggest the importance of maintaining levels of physical activity and muscle strength in older men to prevent bone fragility.

These findings suggest that cortical bone strength, cortical area, and MCSA are all lower in runners with a history of stress fracture. However, the lower strength was appropriate for the smaller muscle size, suggesting that interventions to reduce stress fracture risk might be aimed at improving muscle size and strength.

Stress fractures are common injuries with load-bearing activities. Stress fractures have been reported in the scientific literature for over a century; however, the etiology continues to be investigated with important distinctions made between the contributions of the tissue-level processes of bone remodeling and modeling. In response to novel repetitive loading, increased bone remodeling may serve to replace fatigue-damaged bone while at the same time creating temporary porosity. Much attention has been given to the role of remodeling in the etiology of stress fracture; however, the role of bone modeling has received less attention. Modest increases in modeling, via bone formation on the periosteal surface of long bones in response to mechanical loading, greatly increases the fatigue resistance of bone. Thus, enhancing this adaptive bone formation is a promising target for stress fracture prevention, and a focus on adaptive bone formation may reveal novel risk factors for stress fracture.