Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages

A systematic review of the diagnostic accuracy of physical examination for the detection of cirrhosis

BACKGROUND: We conducted a review of the diagnostic accuracy of clinical examination for the diagnosis of cirrhosis. The objectives were: to identify studies assessing the accuracy of clinical examination in the detection of cirrhosis; to summarize the diagnostic accuracy of reported physical examination findings; and to define the effects of study characteristics on estimates of diagnostic accuracy. METHODS: Studies were identified through electronic literature search of MEDLINE (1966 to 2000), search of bibliographic references, and contact with authors. Studies that evaluated indicants from physical examination of patients with known or suspected liver disease undergoing liver biopsy were included. Qualitative data on study characteristics were extracted. Two-by-two tables of presence or absence of physical findings for patients with and without cirrhosis were created from study data. Data for physical findings reported in each study were combined using Summary Receiver Operating Characteristic (SROC) curves or random effects modeling, as appropriate. RESULTS: Twelve studies met inclusion criteria, including a total of 1895 patients, ranging in age from 3 to 90 years. Most studies were conducted in referral populations with elevated aminotransferase levels. Ten physical signs were reported in three or more studies and ten signs in only a single study. Signs for which there was more study data were associated with high specificity (range 75–98%), but low sensitivity (range 15–68%) for histologically-proven cirrhosis. CONCLUSIONS: Physical findings are generally of low sensitivity for the diagnosis of cirrhosis, and signs with higher specificity represent decompensated disease. Most studies have been undertaken in highly selected populations

In vitro influence of dietary protein and fructooligosaccharides on metabolism of canine fecal microbiota

BACKGROUND: The present in vitro study investigated whether the utilization of fructooligosaccharides (FOS) may influence canine fecal microbial population in presence of diets differing in their protein content and digestibility. Fresh fecal samples were collected from five adult dogs, pooled, and incubated for 24 h with the undigested residue of three diets: 1, Low protein high digestibility diet (LP HD, crude protein (CP) 229 g/kg); 2, High protein high digestibility diet (HP HD, CP 304 g/kg); 3, High protein low digestibility diet (HP LD, CP 303 g/kg) that had been previously subjected to enzymatic digestion. In the in vitro fermentation study, there were six treatments: 1) LP HD; 2) HP HD 3) HP LD; 4) LP HD + FOS; 5) HP HD + FOS; 6) HP LD + FOS. Fructooligosaccharides were added at the final concentration of 1.5 g/L. Samples of fermentation fluid were collected at 6 and 24 h of incubation. RESULTS: Values of pH were reduced by FOS at 6 and 24 h (P < 0.001); conversely, low protein digestibility and high dietary protein level resulted in higher pH at both sampling times (P < 0.001). At 24 h, FOS lowered ammonia (−10 %; P < 0.001) and resulted (P < 0.05) in higher concentrations of total volatile fatty acids (VFA) (+43 %), acetic acid (+14 %), propionic acid (+75 %) and n-butyric acid (+372 %). Conversely, at 24 h, low protein digestibility resulted (P < 0.01) in lower concentrations of acetic acid (−26 %), propionic acid (−37 %) and total VFA (−21 %). Putrescine concentrations were increased at 6 and 24 h of fermentation by low protein digestibility (+21 and 22 %, respectively; P < 0.05) and FOS (+18 and 24 %, respectively; P < 0.01). After 24 h of fermentation, high dietary protein level resulted in lower counts of lactobacilli and enterococci (−0.5 and −0.7 log cells/mL, respectively; P < 0.05) whereas low protein digestibility tended to increase counts of C. perfringens (+0.2 log cells/mL; P = 0.07). CONCLUSIONS: Results from the present study showed that diets rich in protein may exert negative influences on the canine intestinal ecosystem, slightly increasing the presence of ammonia and reducing counts of lactobacilli and enterococci. Moreover, the presence of poorly digestible protein resulted in lower concentrations of VFA. Conversely, administration of FOS may improve metabolism of canine intestinal microbiota, reducing ammonia concentrations and enhancing VFA production