Susan E. H. West

Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa.

The nucleotide sequence of the 1.9-kb PstI fragment from pRO1614, that allows stable maintenance of pMB1 (ColE1)-based cloning vectors in Pseudomonas, was determined. This fragment encodes a putative origin of replication (ori), a replication-controlling protein, and the C terminus of the Tn3 beta-lactamase-encoding gene. Improved versions of the broad-host-range plasmid vectors, pUCP18 and pUCP19, were constructed by deletion of nonessential DNA or replacement of nonessential DNA with an antibiotic-resistance cassette.

Quorum sensing in Pseudomonas aeruginosa controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide.

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm‐grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI‐1 (N‐(3‐oxododecanoyl)‐l‐homoserine lactone) and PAI‐2 (N‐butyryl‐l‐homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn‐SOD and Fe‐SOD, and the major catalase, KatA. The expression of sodA (encoding Mn‐SOD) was particularly dependent on PAI‐1, whereas the influence of autoinducers on Fe‐SOD and KatA levels was also apparent but not to the degree observed with Mn‐SOD. β‐Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI‐1/2‐deficient double mutant partially restored KatA activity, while the addition of PAI‐1 only was sufficient for full restoration of Mn‐SOD activity. In biofilm studies, catalase activity in wild‐type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild‐type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Acceleration of lung disease in children with cystic fibrosis after Pseudomonas aeruginosa acquisition

As part of the ongoing Wisconsin Cystic Fibrosis (CF) Neonatal Screening Project, we had the unique opportunity to study the longitudinal relationship between Pseudomonas aeruginosa (Pa) acquisition and infection and developing lung disease in children with CF. The primary objective was to determine whether acquisition of Pa was associated with a measurable change in the progression of lung disease. Two outcome measures were used to study 56 patients who were diagnosed through newborn screening: 1) Wisconsin additive chest radiograph score (WCXR), based on the average of scores from a pulmonologist and a radiologist, and 2) the highest forced expired volume in 1 sec (FEV1)/forced vital capacity (FVC) ratio. We used two measures of Pa acquisition: 1) time of first positive protocol‐determined oropharyngeal (with cough) culture, and 2) the magnitude of antibody titer detected by ELISA assays, using as antigen a crude cell lysate, purified exotoxin A, or an elastase toxoid prepared from three Pa strains. Other predictor variables included age, pancreatic status, height‐for age, and weight‐for‐age‐percentiles.

Effect of vfr mutation on global gene expression and catabolite repression control of Pseudomonas aeruginosa

Vfr of Pseudomonas aeruginosa is 91% similar to the cAMP receptor protein (CRP) of Escherichia coli. Based on the high degree of sequence homology between the two proteins, the question arose whether Vfr had a global regulatory effect on gene expression for P. aeruginosa as CRP did for E. coli. This report provides two-dimensional polyacrylamide gel electrophoretic evidence that Vfr is a global regulator of gene expression in P. aeruginosa. In a vfr101::aacC1 null mutant, at least 43 protein spots were absent or decreased when compared to the proteome pattern of the parent strain. In contrast, 17 protein spots were absent or decreased in the parent strain when compared to the vfr101::aacC1 mutant. Thus, a mutation in vfr affected production of at least 60 proteins in P. aeruginosa. In addition, the question whether Vfr and CRP shared similar mechanistic characteristics was addressed. To ascertain whether Vfr, like CRP, can bind cAMP, Vfr and CRP were purified to homogeneity and their apparent dissociation constants (K(d)) for binding to cAMP were determined. The K(d) values were 1.6 microM for Vfr and 0.4 microM for CRP, suggesting that these proteins have a similar affinity for cAMP. Previously the authors had demonstrated that Vfr could complement a crp mutation and modulate catabolite repression in E. coli. This study presents evidence that Vfr binds to the E. coli lac promoter and that this binding requires the presence of cAMP. Finally, the possible involvement of Vfr in catabolite repression control in P. aeruginosa was investigated. It was found that succinate repressed production of mannitol dehydrogenase, glucose-6-phosphate dehydrogenase, amidase and urocanase both in the parent and in two vfr null mutants. This implied that catabolite repression control was not affected by the vfr null mutation. In support of this, the cloned vfr gene failed to complement a mutation in the P. aeruginosa crc gene. Thus, although Vfr is structurally similar to CRP, and is a global regulator of gene expression in P. aeruginosa, Vfr is not required for catabolite repression control in this bacterium.

Construction of Actinobacillus pleuropneumoniae-Escherichia coli shuttle vectors: expression of antibiotic-resistance genes

We constructed several cloning vectors, designated pGZRS-18/19 and pGZRS-38/39, which were based on an endogenous Actinobacillus pleuropneumoniae (Apl) 4.3-kb plasmid. They carry the lacZ alpha-complementation fragment and MCS from pUC18/19, and either the bla gene under the control of a putative Apl promoter or the KmR gene from Tn903. These vectors replicate in representative strains of Apl serotypes 1 and 7, Escherichia coli, Pasteurella haemolytica (Ph) and Haemophilus (Actinobacillus) actinomycetemcomitans. We also found that Apl and Ph did not express genes under the control of the lacZ or bla promoters, suggesting that their RNA polymerases may not utilize these promoters.

Temporal Associations Among Energy Intake, Plasma Linoleic Acid, and Growth Improvement in Response to Treatment Initiation After Diagnosis of Cystic Fibrosis

OBJECTIVE. It is unclear why some patients with cystic fibrosis (CF) succeed (“responders”) in recovering from malnutrition and growth faltering after treatment initiation whereas others fail to do so (“nonresponders”). We conducted a study to test the hypothesis that sustained high energy intake (↑EN) and normal plasma essential fatty acid status are critical determinants of treatment responsiveness within 2 years after diagnosis of CF. METHODS. A total of 71 CF children who had pancreatic insufficiency but not meconium ileus and were enrolled in the Wisconsin CF Neonatal Screening Project were studied. Responders were defined by having achieved adequate weight gain, as indicated by a recovery of weight z score (Wtz) comparable to Wtz at birth (WtzBR) within 2 years of diagnosis. ↑EN and sustained normal plasma linoleic acid level (↑pLA) were defined by achieving energy intake ≥120% of estimated requirement for ≥75% of the time and maintaining plasma LA ≥26% of total fatty acids for ≥75% of the time, respectively. RESULTS. Thirty-two (68%) screened patients and 13 (54%) patients whose CF was diagnosed conventionally recovered WtzBR within 2 years of diagnosis. Screened patients responded at significantly younger ages (mean/median: 6.3/4.3 months) than patients whose CF was diagnosed conventionally (mean/median: 15.8/11.8 months). Proportionately fewer screened patients (33%) achieved ↑EN compared with patients whose CF was diagnosed conventionally (73%). However, more screened patients responded to ↑EN and recovered WtzBR (91%) than patients whose CF was diagnosed conventionally (56%), although this difference was of borderline significance. Compared with having neither ↑EN nor ↑pLA, the likelihood of being a responder was greatest with combined ↑EN and ↑pLA, followed by ↑EN only. The positive associations between ↑EN and ↑pLA to treatment responsiveness remained significant after adjustment for neonatal screening status, baseline height and weight status, and indices of pulmonary disease severity. CONCLUSION. ↑EN and ↑pLA are critical in promoting adequate weight gain in children with newly diagnosed CF.

Pseudomonas aeruginosa Serological Analysis in Young Children with Cystic Fibrosis Diagnosed Through Newborn Screening

With newborn screening (NBS) for cystic fibrosis (CF), eradication of Pseudomonas aeruginosa (PA) is possible if PA detection occurs early. A serological response to infection likely precedes culture positivity in CF patients, so PA serological testing is very appealing in this population. However, controversies continue to exist about serology testing, titer cutoffs for enzyme‐linked immunosorbent assay (ELISA) antibody tests, and their value in children with CF.

Antimicrobial Susceptibility Profiles of Multidrug-Resistant Salmonella Anatum Isolated from Horses

Salmonella anatum is a common cause of salmonellosis, an important infectious disease in horses of all ages. These infections range in severity from asymptomatic colonization to serious life-threatening septicemia. In horses, the most common forms of clinical disease are acute enterocolitis with severe diarrhea and septicemia with or without diarrhea. Stress, transportation, hospitalization, antimicrobial therapy, colic, and surgery are risk factors associated with the development of clinical disease. Salmonella typhi-, murium is the serotype isolated most frequently from horses; other frequently isolated serotypes include S. agona, S. anatum, S. krefeld, S. newport, and S. typhimurium var. copenhagen. In the spring of 1991, multidrug-resistant (MDR) S. anatum was cultured from 2 septicemic foals referred for treatment to the University of Wisconsin Veterinary Medical Teaching Hospital (UWVMTH). Subsequently, 7 more isolates were cultured from horses treated at the UWVMTH and/or an Illinois veterinary clinic. The present report includes the antimicrobial susceptibility profiles of these MDR S. anatum. A total of 20 strains of S. anatum were examined for this study. Between April 1991 and August 1992, S. anatum was isolated from 3 septicemic foals and 3 adult horses referred for treatment to the UWVMTH. These animals were referred either from a clinic located in Illinois or by veterinarians from other locations in Illinois and Wisconsin. Two S. anatum isolates were obtained directly from the National Veterinary Service Laboratory (NVSL), Ames, IA. These isolates were from 2 additional horses treated at the Illinois clinic during the spring of 1991. In June 1991, 6 isolates of S. anatum were cultured from an environmental study of the Illinois clinic. Five isolates were obtained from stalls, 1 from a mineral oil bucket, and 1 from a horse being treated at the clinic. In addition, 5 different strains of S. anatum that were obtained at the UWVMTH between August 1986 and May 1991 from horses, cows, and the necropsy room drain were included in this study. These 20 isolates were the only strains of S. anatum cultured at the UWVMTH between August 1986 and August 1992. All strains were stored at -70 C. The isolates were identified using a commercial biochemical strip and were serotyped by NVSL and/or the Central Animal Health Laboratory, Madison, WI. All isolates were serotyped as S. anatum. An automated microbiology system was used to determine minimal inhibitory concentrations (MIC) of tetracy-

Pseudomonas aeruginosa in children with cystic fibrosis diagnosed through newborn screening: Assessment of clinic exposures and microbial genotypes

Chronic pulmonary infection with Pseudomonas aeruginosa (PA) is responsible for significant morbidity and mortality in cystic fibrosis (CF). Because of the limited studies evaluating early exposure and the progression of genetic variability of PA, our goal was to assess PA in young children with CF followed in two clinic types.

Construction of an Actinobacillus pleuropneumoniae-Escherichia coli cosmid cloning vector [Gene 160 (1995) 81–86]

We constructed a cosmid vector, pSW206, which should be useful for the construction of genomic libraries of Actinobacillus pleuropneumoniae (Apl) DNA. pSW206 is based on the broad-host-range plasmid RK2 and can be introduced into Apl by conjugation.