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

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.     

Selective media for the quantitation of bacteria in cystic fibrosis sputum

We used selective media together with aerobic and anaerobic incubation for the quantitation of common pathogens in liquefied sputum from children with cystic fibrosis. The accuracy of the technique was verified by reconstruction studies in which laboratory strains with antibiotic-resistance markers were added to sputum from cystic fibrosis patients. Comparison of the numbers of bacteria found on quantitative culture of clinical specimens with the "predominant" organism found on routine culture yielded a poor correlation. When Pseudomonas aeruginosa was the most prevalent on routine culture, it was present in the highest numbers on quantitative culture (mean count = 10(8) cfu/g). However, large numbers of Haemophilus influenzae (mean count = 10(7) cfu/g), Staphylococcus aureus (mean count = 2 X 10(6) cfu/g), and streptococci (mean count = 2 X 10(6) cfu/g) were also present in these cultures. When S. aureus was the predominant organism, H. influenzae and P. aeruginosa were also present in similar numbers (c. 10(7) cfu/g). When H. influenzae was the predominant species on routine culture, the mean count was 7 X 10(6) cfu/g and P. aeruginosa was often completely absent. We conclude that the selective technique permits reliable enumeration of sputum bacteria, and offers a more accurate assessment of the microbial flora of sputum in cystic fibrosis than does simple plating of unhomogenised sputum.     

Bacteriology of sputum in cystic fibrosis: evaluation of dithiothreitol as a mucolytic agent.

Liquefaction and homogenization have been recommended to ensure accurate, representative sputum cultures. We evaluated dithiothreitol (DTT) as mucolytic agent for culturing sputum samples obtained from 79 cystic fibrosis (CF) patients. Liquefaction with DTT was not superior to direct plating of specimens for routine qualitative cultures. Unliquefied sputum cultures failed to direct 3 of 47 Pseudomonas aeruginosa isolates; DTT-treated specimens missed 5 of 13 Candida albicans isolates. Neither treated nor untreated sputum cultures were completely successful in detecting Staphylococcus aureus or Enterobacteriaceae. Since Haemophilus influenzae was recovered from only two qualitative cultures, we could not evaluate the effect of DTT on the receovery of this organism. However, 27 of 29 strains of H. influenzae were inhibited by concentrations of DTT near the recommended final working concentration of 50 micrograms/ml, suggesting that liquefaction might impair isolation of this organism. Liquefaction with DTT permitted quantitative cultures of CF sputum. The predominant pathogen in our CF population was P. aeruginosa; 37 of 43 (86%) patients were colonized with this organism. Median densities of rough and mucoid strains were 3.2 x 10(7) and 4.3 x 10(7) colony-forming units per ml, respectively. Previous oral antistaphylococcal therapy may have accounted for the observed low density of S. aureus (mean density, 3.5 x 10(3) colony-forming units per ml). We conclude that DTT treatment does not improve recovery of organisms from qualitative cultures but does facilitate quantitative studies of S. aureus and P. aeruginosa in CF sputum.     

Cellular immunity to bacteria: impairment of in vitro lymphocyte responses to Pseudomonas aeruginosa in cystic fibrosis patients.

Lymphocyte responses to the mitogens phytohemagglutinin and concanavalin A and to Streptococcus pyogenes, Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa were evaluated in patients with cystic fibrosis and in normal individuals. Lymphocyte proliferation in vitro was stimulated by gentamicin-killed whole bacteria, and the proliferative response was measured by [3H]thymidine incorporation. The in vitro lymphocyte responses to antibiotic-killed bacterial reached maximum thymidine incorporation after 5 days in culture and followed a unimodal dose-response curve for each of the bacteria studied. A significant specific incapacity to respond to P. aeruginosa was detected in cystic fibrosis patients with advanced clinical disease.     

Production and characterisation of mouse monoclonal antibodies reactive with the lipopolysaccharide core of Pseudomonas aeruginosa.

Monoclonal antibodies (MAbs) to the core antigen region of lipopolysaccharide (LPS) of Pseudomonas aeruginosa were produced from mice immunised with whole cells of heat-killed rough mutants of Pseudomonas aeruginosa expressing partial or complete core LPS. MAbs were screened in an enzyme-linked immunosorbent assay (ELISA) against three different antigen cocktails: S-form LPS from three P. aeruginosa strains, R-form LPS from six P. aeruginosa strains and, as a negative control, R-form LPS from Salmonella typhimurium and Escherichia coli. Selected MAbs were subsequently screened against a range of extracted LPS and whole cells from both reference strains and clinical isolates of P. aeruginosa. The antibodies were also screened in ELISA against whole-cell antigens from other Pseudomonas spp. as well as strains of Haemophilus influenzae, Neisseria subflava and Staphylococcus aureus. Five MAbs reacting with the core component of P. aeruginosa LPS were finally selected. Two of these, MAbs 360.7 and 304.1.4, were particularly reactive in immunoblots against unsubstituted core LPS, including that from O-antigenic serotypes of P. aeruginosa. The MAbs also reacted with some of the other Pseudomonas spp., but not with P. cepacia or Xanthomonas (Pseudomonas) maltophilia. Cross-reactivity with whole cells from other bacterial species was minimal or not observed. Reactivity of MAbs with some Staph. aureus strains was observed, and binding to the protein A component was implicated. The reactivity of the MAbs was investigated further by flow cytometry and immunogold electronmicroscopy. The suitability of the MAbs for an immunological assay for detection of P. aeruginosa in respiratory secretions from CF patients is discussed.     

Compliance of clinical microbiology laboratories in the United States with current recommendations for processing respiratory tract specimens from patients with cystic fibrosis

Respiratory tract specimens from patients with cystic fibrosis (CF) require unique processing by clinical microbiology laboratories to ensure detection of all potential pathogens. The present study sought to determine the compliance of microbiology laboratories in the United States with recently published recommendations for CF respiratory specimens. Microbiology laboratory protocols from 150 of 190 (79%) CF care sites were reviewed. Most described the use of selective media for Burkholderia cepacia complex (99%), Staphylococcus aureus (82%), and Haemophilus influenzae (89%) and identified the species of all gram-negative bacilli (87%). Only 52% delineated the use of agar diffusion assays for susceptibility testing of Pseudomonas aeruginosa. Standardizing laboratory practices will improve treatment, infection control, and our understanding of the changing epidemiology of CF microbiology.     

Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients

We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 x 10(5) CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, and P. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.     

Rapid procedure for isolation of plasmid DNA and application to epidemiological analysis.

A rapid and simple plasmid isolation procedure was developed for the epidemiological analysis of plasmid-mediated antimicrobial resistance. By this method, plasmid DNAs ranging in molecular weight between 2.0 and 122 X 10(6) could be detected. Various bacteria, such as strains of the family Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus influenzae, and Staphylococcus aureus, could be analyzed. The plasmid DNA obtained could be directly used for restriction endonuclease analysis without further purification. In addition, this method made it possible to analyze several cultures at the same time.     

Murine models of acute and chronic lung infection with cystic fibrosis pathogens

Animal models of acute and chronic infection, along with mice genetically modified for the Cftr gene, are a key asset in cystic fibrosis (CF) research. Despite some limitations, these models provide valuable resources to mimic the initial and progressive bronchopulmonary infection typical of CF patients. The following review summarizes the strengths and weaknesses of different types of animal models with a major emphasis placed on the significant species differences between mice and humans. Murine models of acute and chronic lung infection with Pseudomonas aeruginosa, Burkholderia cenocepacia, Staphylococcus aureus, and Haemophilus influenzae have been used to study the molecular mechanisms underlying the pathogen virulence and host defense. In addition, they have provided insights in the potential of vaccination to restrict infectious exacerbations, the activity of antibiotics, and the effectiveness of anti-inflammatory therapy in reducing lung damage. Indeed, animal models of infection should allow the validation of future therapeutic interventions for lung infections in patients with CF.     

Profiling of bacterial flora in gastric biopsies from patients with Helicobacter pylori-associated gastritis and histologically normal control individuals by temperature gradient gel electrophoresis and 16S rDNA sequence analysis

The aim of this study was to establish bacterial profiles in gastric biopsy specimens from patients with Helicobacter pylori-associated gastritis by means of temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Specimens from eight patients with asymptomatic gastritis and five histologically normal controls revealed a Helicobacter-specific band in the TTGE profile with increased amounts of Helicobacter-specific DNA in the biopsies from most of the gastritis patients. DNA from other genera including Enterococcus, Pseudomonas, Streptococcus, Staphylococcus and Stomatococcus was also found in the stomach. In the absence of gastric inflammation, Helicobacter spp. appeared to be part of a complex, presumably indigenous microbial flora found in the biopsy specimens from the stomach.     

Sensitivity to cefotiam of 494 hospital bacterial strains. Determination of minimal inhibitory concentration and comparison with other beta-lactams

Susceptibility to cefotiam of 494 bacterial strains was studied by MIC determination using an agar dilution assay. Activity of cefotiam was also compared with that of six other beta-lactams (ampicillin, mezlocillin, ticarcillin, cefalotin, cefotaxime, and moxalactam) using an agar diffusion method. Cefotiam showed a wide antibacterial spectrum including more than 70% of Enterobacteriaceae (except Serratia), beta-lactamase-producing and non-beta-lactamase-producing Haemophilus influenzae, non-enterococcus streptococci, and penicillinase-producing and non-penicillinase-producing staphylococci susceptible to methicillin. Conversely, cefotiam was inactive against Pseudomonas aeruginosa, Acinetobacter anitratum, Streptococcus faecalis, and methicillin-resistant Staphylococcus aureus.     

Antibacterial kinetics of fosfomycin (author's transl)

The authors studied, with the Autobac machine, the kinetics of antibacterial activity of fosfomycin against Staphylococcus aureus, Streptococcus D' Streptococcus pneumoniae, Neisseria meningitidis, Acinetobactor lwoffi, Haemophilus influenzae, Salmonella typhimurium, Escherichia coli, Proteus rettgeri, Klebsiella pneumoniae, Serratia marcescens and Pseudomonas aeruginosa. A correlation appears between the kinetics of fosformycin antibacterial action and the microbial growth rate.     

An evaluation of 12 methods for the demonstration of penicillinase.

Twelve methods for the demonstration of bacterial penicillinase production by strains of Haemophilus influenzae and Staphylococcus aureus are compared, and their suitability for routine clinical laboratory use is evaluated. The acidometric agar plate method is recommended.     

Antimicrobial agent susceptibility patterns of bacteria in hospitals from 1971 to 1982.

Bacterial susceptibility to 16 commonly used antibiotics was analyzed for a 12-year period (from 1971 to 1982, inclusive). Susceptibilities of 5,828,243 strains isolated from a mean of 242 hospitals nationwide and of 194,575 strains isolated at the Massachusetts General Hospital, Boston, Mass., and the Bronx Lebanon Hospital Center, New York, N.Y., were compared. Strains of Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa showed virtually the same susceptibilities to antibiotics throughout the 12-year period, whereas Streptococcus faecalis and Staphylococcus epidermidis showed significant increases in resistance to most antibiotics. The close similarity between antibiotic susceptibilities shown at both the 242 hospitals and the 2 individual hospitals suggests that this analysis accurately reflects trends of bacterial resistance to antibiotics in U.S. hospitals. Since most of the species analyzed produce serious disease and high mortality, their susceptibility to antibiotics is relevant both to physicians treating infectious diseases and to epidemiologists.     

Agglutinin response to bacterial infection in acute exacerbations of chronic bronchitis.

Agglutinin titres to Haemophilus influenzae, Streptococcus pneumoniae, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Proteus vulgaris in the serum of patients with acute exacerbations of chronic bronchitis, patients producing mucoid sputum, and healthy controls were determined. Serological evidence of infection with H. influenzae was found in 38 of 57 patients with acute exacerbations, and Str. pneumoniae infection in 10 of the 57 patients, but was generally absent from healthy control subjects and from patiens producing mucoid sputum. No serological evidence of infection with other organisms named above was found to be associated with exacerbations of chronic bronchitis. Ten patients with acute exacerbations were without serological evidence of infection by any of the bacteria tested.     

Use of PCR with universal primers and restriction endonuclease digestions for detection and identification of common bacterial pathogens in cerebrospinal fluid

We have designed a universal PCR capable of amplifying a portion of the 16S rRNA gene of eubacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecium, Enterococcus faecalis, Mycobacterium tuberculosis, Legionella pneumophila, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Enterobacter cloacae, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Haemophilus influenzae, and Neisseria meningitidis. The sizes of the amplified products from various bacteria were the same (996 bp), but the restriction patterns of most PCR products generated by HaeIII digestion were different. PCR products from S. aureus and S. epidermidis could not be digested by HaeIII but yielded different patterns when they were digested with MnlI. PCR products from S. pneumoniae, E. faecium, and E. faecalis yielded the same HaeIII digestion pattern but could be differentiated by AluI digestion. PCR products from E. coli, K. pneumoniae, S. marcescens, and E. cloacae also had the same HaeIII digestion pattern but had different patterns when digested with DdeI or BstBI. This universal PCR could detect as few as 10 E. coli or 250 S. aureus organisms. Compared with culture, the sensitivity of this universal PCR for detection and identification of bacteria directly from 150 cerebrospinal fluids was 92.3%. These results suggest that this universal PCR coupled with restriction enzyme analysis can be used to detect and identify bacterial pathogens in clinical specimens.     

Intragenomic and intraspecific heterogeneity of the 16S rRNA gene in seven bacterial species from the respiratory tract of cystic fibrosis patients assessed by PCR-Temporal Temperature Gel Electrophoresis

16S rRNA gene-based cultivation-independent methods are increasingly used to study the diversity of microbiota during health and disease. One bias of these methods is the variability of 16S rRNA gene that may exist among strains of a same species (intraspecific heterogeneity) or between rrs copies in a genome (intragenomic heterogeneity). We evaluated the level of intraspecific and intragenomic 16S rDNA variability in seven species frequently encountered in respiratory tract samples in cystic fibrosis (CF). A total of 179 strains were subjected to V3 region 16S rDNA PCR-TTGE. Using this easy-to-perform and rapid method, different levels of V3 region rrs heterogeneity were demonstrated. No intraspecific and intragenomic rrs heterogeneity was demonstrated for Moraxella catarrhalis (n=16), Pseudomonas aeruginosa (n=31) and Streptococcus pneumoniae (n=14) showing a single PCR-TTGE band characteristic of the species. Low level of intraspecific heterogeneity was observed for Staphylococcus aureus (n=30), Stenotrophomonas maltophilia (n=29) and Achromobacter xylosoxidans (n=28), and 17%, 38% and 96% of these strains showed intragenomic heterogeneity (two to four different rrs copies), respectively. Haemophilus influenzae (n=31) displayed the higher level of intraspecific variability with 23 different PCR-TTGE patterns and 61% of the strains showed intragenomic rrs heterogeneity (two to four different rrs copies). Although only one hypervariable region of the 16S rRNA gene was explored, intraspecific and intragenomic rrs heterogeneity was frequently observed in this study and should be taken into consideration for a better interpretation of 16S rRNA gene-based diversity profiles in denaturing gels and to avoid any overestimation of the respiratory microbiota diversity in CF.     

Microbiology of airway disease in patients with cystic fibrosis.

Individuals with cystic fibrosis have abbreviated life spans primarily due to chronic airway infection. A limited number of types of organisms are responsible for these infections, with Staphylococcus aureus and Pseudomonas aeruginosa being of primary importance. In the pre-antibiotic era, greater than 90% of deaths due to infection were caused by S. aureus and death usually occurred in the first 2 years of life. With the advent of effective antistaphylococcal therapy, life spans increased and P. aeruginosa became the pathogen of primary importance. P. aeruginosa isolates recovered from patients with cystic fibrosis have a unique phenotypic characteristic referred to as "mucoid." The mucoid phenotype is due to the production of a mucoid exopolysaccharide. A mucoid exopolysaccharide is believed to play a central role in the establishment of chronic pseudomonal lung infection in these patients. A third organism, Pseudomonas cepacia, has recently been detected in the airways of older patients with cystic fibrosis and is associated with increased mortality. The virulence of P. cepacia is not understood, but the organism is extremely refractory to antimicrobial therapy. Other bacteria, including Haemophilus influenzae and members of the family Enterobacteriaceae, appear to play a secondary role in airway infection. Aspergillus fumigatus is the most important fungal agent causing allergic bronchopulmonary disease. The role of viruses has only recently been examined. At least in some patients with cystic fibrosis, respiratory syncytial virus may be important in predisposing to subsequent bacterial infections.     

ICU患者痰细菌分布特征及药敏分析

目的统计分析2009年1～11月佛山市中医院ICU痰标本细菌培养病原菌分布特征及药敏情况,为临床经验用药提供科学的参考。方法留取经镜检合格的痰标本,经分离培养出病原菌后,以法国梅里埃VITEK2全自动细菌鉴定分析仪鉴定,以K-B法检测药物敏感性,用MICROSOFT OFFICE Excel2003进行数据处理。结果共分离出288株菌,病原菌分布以铜绿假单胞菌最多占20%,其次是金黄色葡萄球菌、鲍曼不动杆菌、肺炎克雷伯菌和大肠埃希菌,分别是14%、13%、10%、7%。药敏结果显示,铜绿假单胞菌和鲍曼不动杆菌对头孢吡肟、头孢他啶、亚胺培南、阿米卡星等的敏感率均达75%以上;阿米卡星、亚胺培南和头孢西丁对肺炎克雷伯菌与大肠埃希菌的抗菌活性也较好;金黄色葡萄球菌对万古霉素、替考拉宁、呋喃妥因等的敏感率较高,均为75%以上,而对青霉素仅有2%的敏感率。结论痰培养病原菌以铜绿假单胞菌和金黄色葡萄球菌为主;对于革兰阴性杆菌,可选用第三代头孢菌素、氨基糖苷类抗生素,对革兰阳性球菌,可用万古霉素、替考拉宁、呋喃妥因。