C. A. Hart

A Cystic Fibrosis Epidemic Strain of Pseudomonas aeruginosa Displays Enhanced Virulence and Antimicrobial Resistance

The Liverpool epidemic strain (LES) of Pseudomonas aeruginosa is a transmissible aggressive pathogen of cystic fibrosis (CF) patients. We compared transcriptome profiles of two LES isolates with each other and with a laboratory and genetic reference strain (PAO1) after growth to late exponential phase and following exposure to oxidative stress. Both LES isolates exhibited enhanced antimicrobial resistances linked to specific mutations in efflux pump genes. Although transcription of AmpC beta-lactamase was up-regulated in both, one LES isolate contained a specific mutation rendering the ampC gene untranslatable. The virulence-related quorum-sensing (QS) regulon of LES431, an isolate that caused pneumonia in the non-CF parent of a CF patient, was considerably up-regulated in comparison to either isolate LES400, associated with a chronic CF infection, or strain PAO1. Premature activation of QS genes was detected in isolates from both non-CF parents and the CF patient in a previously reported infection episode. LES isolates lacking the up-regulated QS phenotype contained different frameshift mutations in lasR. When fed to Drosophila melanogaster, isolate LES431 killed the fruit flies more readily than either isolate LES400 or strain PAO1, indicating that virulence varies intraclonally. The LES may represent a clone with enhanced virulence and antimicrobial resistance characteristics that can vary or are lost due to mutations during long-term colonization but have contributed to the successful spread of the lineage throughout the CF population of the United Kingdom.

Increased morbidity associated with chronic infection by an epidemic Pseudomonas aeruginosa strain in CF patients

Background: Chronic pulmonary infection with transmissible Pseudomonas aeruginosa strains in individuals with cystic fibrosis (CF) has been reported, raising issues of cross infection and patient segregation. The first such strain to be described (the Liverpool epidemic strain, LES) is now widespread in many UK CF centres. However, whether such infection carries a worse prognosis is unknown. To address this, the clinical course of a group of CF patients chronically infected by LES was compared with that in patients harbouring unique strains. Methods: Using P aeruginosa strain genotyping, two cohorts of CF patients attending the Liverpool CF service were identified who were LES positive or negative in 1998 and remained so until 2002. From these, two groups of 12 patients were matched in 1998 for age, spirometric parameters, and nutritional state and their clinical course was followed for 5 years. Patients chronically infected with Burkholderia cepacia were excluded. Results: Patients chronically infected with LES had a greater annual loss of lung function than those not chronically infected by LES (mean difference between groups −4.4% (95% CI −8.1 to −0.9; p<0.02)), and by 2002 their percentage predicted forced expiratory volume in 1 second (FEV1) was worse (mean 65.0% v 82.6%, p<0.03). Their nutritional state also deteriorated over the study period (mean difference between groups in body mass index −0.7 (95% CI −1.2 to −0.2; p<0.01)), such that by 2002 they were malnourished compared with LES negative patients (mean BMI 19.4 v 22.7, p<0.02). Conclusions: Chronic infection with the Liverpool epidemic P aeruginosa strain in CF patients confers a worse prognosis than infection with unique strains alone, confirming the need for patient segregation. Since this strain is common in many CF units, strain identification in all CF centres is essential. This can only be carried out using genomic typing methods.

Bacterial Diversity in Cases of Lung Infection in Cystic Fibrosis Patients: 16S Ribosomal DNA (rDNA) Length Heterogeneity PCR and 16S rDNA Terminal Restriction Fragment Length Polymorphism Profiling

ABSTRACT The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections. Many bacterial species have been cultured from CF specimens and so are associated with lung disease. Despite this, much remains to be determined. In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples. Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities. Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands. Nine distinct LH-PCR profiles were identified containing between one and four bands. T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae. In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa. A total of 103 16S rRNA gene clones were examined from five patients. P. aeruginosa was the most commonly identified species (59% of clones). Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones. In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients.

Predominance of Rotavirus P(4)G2 in a Vaccinated Population, Brazil

We identified 21 rotaviruses in 129 patients with diarrhea in a Brazilian city with high rotavirus vaccine coverage. All rotaviruses were genotype P[4]G2 with 1 mixed infection with P[NT]G9. Although virus predominance could have occurred randomly, the vaccine may be less protective against P[4]G2. Prospective surveillance is urgently needed.

Rotavirus strain diversity in Blantyre, Malawi, from 1997 to 1999.

ABSTRACT In a 2-year study of viral gastroenteritis in children in Blantyre, Malawi, the diversity of rotavirus strains was investigated by using electropherotyping, reverse transcription-PCR amplification of the VP7 and VP4 genes (G and P genotyping), and nucleotide sequencing. Of 414 rotavirus strains characterized, the following strain types were identified: P[8], G1 (n = 111; 26.8%); P[6], G8 (n = 110; 26.6%); P[8], G3 (n = 93; 22.5%); P[4], G8 (n = 31; 7.5%); P[8], G4 (n = 21; 5.1%); P[6], G3 (n = 12; 2.9%); P[6], G1 (n = 7; 1.7%); P[6], G9 (n = 3; 0.7%); P[6], G4 (n = 3; 0.7%); P[4], G3 (n = 1; 0.2%); and mixed (n = 15; 3.6%). While all strains could be assigned a G type, seven strains (1.7%) remained P nontypeable. The majority of serotype G8 strains and all serotype G9 strains had short electropherotype profiles. All remaining typeable strains had long electropherotypes. Divergent serotype G1 rotaviruses, which contained multiple base substitutions in the 9T-1 primer binding site, were commonly identified in the second year of surveillance. Serotype G2 was not identified. Overall, G8 was the most frequently identified VP7 serotype (n = 144; 34.8%) and P[8] was the most frequently detected VP4 genotype (n = 227; 54.8%). Partial sequence analysis of the VP4 gene of genotype P[8] rotaviruses identified three distinct clusters, which predominantly (but not exclusively) comprised strains belonging to a distinct VP7 serotype (G1, G3, or G4). As a result of mutations in the 1T-1 primer binding site, strains belonging to each cluster required a separate primer for efficient typing. One cluster, represented by P[8], G4 strain OP354, was highly divergent from the established Wa and F45 VP4 P[8] lineages. As is the case for some other countries, the diversity of rotaviruses in Malawi implies that rotavirus vaccines in development will need to protect against a wider panel of serotypes than originally envisioned.

Effect of respiratory virus infections including rhinovirus on clinical status in cystic fibrosis.

One hundred and eight patients with cystic fibrosis were investigated over one year to determine whether an association existed between rhinovirus or other respiratory virus infection and clinical status. Forced expiratory volume in one second (FEV1), forced vital capacity (FVC), Shwachman score, Chrispin-Norman chest radiograph score, and percentage weight for height were recorded at the beginning and end of the study; days of intravenous antibiotics were noted. Nasopharyngeal aspirates were taken for viral studies during respiratory exacerbations. Serum was collected at enrollment and 2-6 weeks after each respiratory exacerbation. One hundred and fifty seven exacerbations occurred in 76 patients. Respiratory virus infection was detected in 44 exacerbations and rhinovirus was present in 16% (25/157) of exacerbations. Patients with one or more respiratory virus infections were compared with those who had none. When all respiratory virus infections were considered, patients had a significantly greater deterioration in Shwachman score and received significantly more days of intravenous antibiotics. When rhinovirus was considered separately, patients received significantly more days of intravenous antibiotics, but showed no deterioration in clinical status. However, patients infected with another respiratory virus had a significant decline in FEV1, with trends towards significance for decline in FVC and Shwachman score.

Antibiotic resistance found in wild rodents

Resistance to antibiotics is an increasingly common problem in both veterinary and human medicine, and its management is the subject of urgent debate. Efforts to reduce this resistance are based on the assumption that it is maintained in bacterial populations as a result of exposure to antibiotics, and that restricting the use of antibiotics should therefore restrain the spread of resistance. But we have found that antibiotic resistance is prevalent in populations of wild rodents that have not been exposed to antibiotics, indicating that approaches to control it based on this assumption may be overoptimistic.

Procalcitonin as a marker of sepsis

Prompt diagnosis and treatment with appropriate antimicrobial chemotherapy is of paramount importance to reduce morbidity and mortality associated with sepsis. Inflammatory markers currently in use, such as C-reactive protein (CRP) do not reliably differentiate between the systemic inflammatory response and sepsis. Procalcitonin (PCT), a precursor of calcitonin, is a 116 amino acid protein that has been proposed as a marker of disease severity in conditions such as septicaemia, meningitis, pneumonia, urinary tract infection (UTI) and fungal and parasitic infection. In particular, serial measurements are useful in order to monitor response to therapy. Together with good clinical judgement and judicious use of antimicrobial agents, PCT should serve as a valuable adjunct in the diagnosis and management of sepsis.

Placental antibody transfer: influence of maternal HIV infection and placental malaria

AIM To determine the influence of placental malaria, maternal HIV infection, and maternal hypergammaglobulinaemia on transplacental IgG antibody transfer. METHODS One hundred and eighty materno-neonatal pairs from a Malawian population were assessed. Cord and maternal serum samples were tested for total serum IgG antibody titres using nephelometry, and for specific IgG antibody titres toStreptococcus pneumoniae, measles, and tetanus toxoid antibodies using an enzyme linked immunsorbent assay (ELISA). RESULTS Multiple regression analyses showed that placental malaria was associated with a decrease in placental IgG antibody transfer to S pneumoniae and measles to 82% and 81%, respectively. Maternal HIV infection was associated with a reduction in IgG antibody transfer to S pneumoniae to 79%; raised maternal total serum IgG titres were correlated withS pneumoniae and measles IgG antibody transfer reduction to 86% and 87%, respectively. No effect was seen with tetanus toxoid antibody transfer. CONCLUSION The combined influence of placental malaria, maternal HIV infection, and maternal hypergammaglobulinaemia seems to be linked to the low transplacental antibody transfer observed in the Malawian population.

Meningococcal bacterial DNA load at presentation correlates with disease severity

Aims: To determine bacterial loads in meningococcal disease (MCD), their relation with disease severity, and the factors which determine bacterial load. Methods: Meningococcal DNA quantification was performed by the Taqman PCR method on admission and sequential blood samples from patients with MCD. Disease severity was assessed using the Glasgow Septicaemia Prognostic Score (GMSPS, range 0–15, severe disease ≥8). Results: Median admission bacterial load was 1.6 × 106 DNA copies/ml of blood (range 2.2 × 104 to 1.6 × 108). Bacterial load was significantly higher in patients with severe (8.4 × 106) compared to milder disease (1.1 × 106, p = 0.018). This difference was greater in septicaemic patients (median 1.6 × 107 versus 9.2 × 105, p < 0.001). Bacterial loads were significantly higher in patients that died (p = 0.017). Admission bacterial load was independent of the duration of clinical symptoms prior to admission, with no difference between the duration of symptoms in mild or severe cases (median, 10.5 and 11 hours respectively). Bacterial loads were independent of DNA elimination rates following treatment. Conclusion: Patients with MCD have higher bacterial loads than previously determined with quantitative culture methods. Admission bacterial load is significantly higher in patients with severe disease (GMSPS ≥8) and maximum load is highest in those who die. Bacterial load is independent of the duration of clinical symptoms or the decline in DNA load.