Several pathogens associated with chronic infections, including Pseudomonas aeruginosa in cystic fibrosis pneumonia, Haemophilus influenzae and Streptococcus pneumoniae in chronic otitis media, Staphylococcus aureus in chronic rhinosinusitis and enteropathogenic Escherichia coli in recurrent urinary tract infections, are linked to biofilm formation. Biofilms are usually defined as surface-associated microbial communities, surrounded by an extracellular polymeric substance (EPS) matrix. Biofilm formation has been demonstrated for numerous pathogens and is clearly an important microbial survival strategy. However, outside of dental plaques, fewer reports have investigated biofilm development in clinical samples. Typically biofilms are found in chronic diseases that resist host immune responses and antibiotic treatment and these characteristics are often cited for the ability of bacteria to persist in vivo. This review examines some recent attempts to examine the biofilm phenotype in vivo and discusses the challenges and implications for defining a biofilm phenotype

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1462-5822.2009.01323.x

Evolving concepts in biofilm infections

In this age of modern era, the use of internet must be maximized. Yeah, internet will help us very much not only for important thing but also for daily activities. Many people now, from any level can use internet. The sources of internet connection can also be enjoyed in many places. As one of the benefits is to get the on-line analysis of human genetic linkage book, as the world window, as many people suggest.

Analysis of human genetic linkage

In a recent study, immunoglobulin G in human plasma was identified as a major inhibitor of diagnostic PCR (W. Abu Al-Soud, L. J. Jonsson, and P. Radstrom. J. Clin. Microbiol. 38:345–350, 2000). In this study, two major PCR inhibitors in human blood cells were purified using size exclusion and anion-exchange chromatographic procedures. Based on N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptides, hemoglobin and lactoferrin were identified as PCR-inhibitor components in erythrocytes and leukocytes, respectively. When different concentrations of hemoglobin or lactoferrin were added to PCR mixtures of 25 μl containing 10 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, AmpliTaq Gold, Pwo, and Ultma were inhibited in the presence of ≤1.3 μg of hemoglobin and ≤25 ng of lactoferrin, while rTth and Tli were found to resist inhibition of at least 100 μg of hemoglobin. In addition, the quantitative effects of seven low-molecular-mass inhibitors, present in blood samples or degradation products of hemoglobin, on real-time DNA synthesis of rTth using the LightCycler Instrument were investigated. A reaction system based on a single-stranded poly(dA) template with an oligo(dT) primer annealed to the 3′ end was used. It was found that the addition of 0.25 to 0.1 mg of bile per ml, 2.5 mM CaCl2, 0.25 mM EDTA, 5 μM FeCl3, and 0.01 IU of heparin per ml reduced the fluorescence to approximately 76, 70, 46, 17, and 51%, respectively. Finally, the effects of nine amplification facilitators were studied in the presence of hemoglobin and lactoferrin. Bovine serum albumin (BSA) was the most efficient amplification facilitator, so that the addition of 0.4% (wt/vol) BSA allowed AmpliTaq Gold to amplify DNA in the presence of 20 instead of 1 μg of hemoglobin and 500 instead of 5 ng of lactoferrin. Including 0.02% (wt/vol) gp32, a single-stranded-DNA binding protein, in the reaction mixture of AmpliTaq Gold was also found to reduce the inhibitory effects of hemoglobin and lactoferrin.

Purification and Characterization of PCR-Inhibitory Components in Blood Cells

ContextChronic otitis media (OM) is a common pediatric infectious disease. Previous studies demonstrating that metabolically active bacteria exist in culture-negative pediatric middle-ear effusions and that experimental infection with Haemophilus influenzae in the chinchilla model of otitis media results in the formation of adherent mucosal biofilms suggest that chronic OM may result from a mucosal biofilm infection.ObjectiveTo test the hypothesis that chronic OM in humans is biofilm-related.Design, Setting, and PatientsMiddle-ear mucosa (MEM) biopsy specimens were obtained from 26 children (mean age, 2.5 [range, 0.5-14] years) undergoing tympanostomy tube placement for treatment of otitis media with effusion (OME) and recurrent OM and were analyzed using microbiological culture, polymerase chain reaction (PCR)-based diagnostics, direct microscopic examination, fluorescence in situ hybridization, and immunostaining. Uninfected (control) MEM specimens were obtained from 3 children and 5 adults undergoing cochlear implantation. Patients were enrolled between February 2004 and April 2005 from a single US tertiary referral otolaryngology practice.Main Outcome MeasuresConfocal laser scanning microscopic (CLSM) images were obtained from MEM biopsy specimens and were evaluated for biofilm morphology using generic stains and species-specific probes for H influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis. Effusions, when present, were evaluated by PCR and culture for evidence of pathogen-specific nucleic acid sequences and bacterial growth, respectively.ResultsOf the 26 children undergoing tympanostomy tube placement, 13 (50%) had OME, 20 (77%) had recurrent OM, and 7 (27%) had both diagnoses; 27 of 52 (52%) of the ears had effusions, 24 of 24 effusions were PCR-positive for at least 1 OM pathogen, and 6 (22%) of 27 effusions were culture-positive for any pathogen. Mucosal biofilms were visualized by CLSM on 46 (92%) of 50 MEM specimens from children with OME and recurrent OM using generic and pathogen-specific probes. Biofilms were not observed on 8 control MEM specimens obtained from the patients undergoing cochlear implantation.ConclusionDirect detection of biofilms on MEM biopsy specimens from children with OME and recurrent OM supports the hypothesis that these chronic middle-ear disorders are biofilm-related.

https://jamanetwork.com/journals/jama/articlepdf/211068/JPC60005.pdf

Direct Detection of Bacterial Biofilms on the Middle-Ear Mucosa of Children With Chronic Otitis Media

Recognition of the fact that bacterial biofilm may play a role in the pathogenesis of disease has led to an increased focus on identifying diseases that may be biofilm-related. Biofilm infections are typically chronic in nature, as biofilm-residing bacteria can be resilient to both the immune system, antibiotics, and other treatments. This is a comprehensive review describing biofilm diseases in the auditory, the cardiovascular, the digestive, the integumentary, the reproductive, the respiratory, and the urinary system. In most cases reviewed, the biofilms were identified through various imaging technics, in addition to other study approaches. The current knowledge on how biofilm may contribute to the pathogenesis of disease indicates a number of different mechanisms. This spans from biofilm being a mere reservoir of pathogenic bacteria, to playing a more active role, e.g., by contributing to inflammation. Observations also indicate that biofilm does not exclusively occur extracellularly, but may also be formed inside living cells. Furthermore, the presence of biofilm may contribute to development of cancer. In conclusion, this review shows that biofilm is part of many, probably most chronic infections. This is important knowledge for development of effective treatment strategies for such infections.

https://www.mdpi.com/2079-6382/9/2/59/pdf

Bacterial Biofilm and its Role in the Pathogenesis of Disease.

Objective. —To determine if the polymerase chain reaction (PCR) can detect bacterial DNA in pediatric middle ear effusions that are sterile by standard cultural methods. Design. —Single-center, blinded, comparative study of diagnostic assays. The PCR-based detection systems for Moraxella catarrhalis, Haemophilus influenzae , and Streptococcus pneumoniae were designed and validated using a battery of DNAs obtained from cultured bacteria. Chronic middle ear effusion specimens were collected and comparatively analyzed by culture and the PCR. Setting. —Tertiary care pediatric hospital. Patients. —A total of 97 middle ear effusions were collected from pediatric outpatients at Children's Hospital of Pittsburgh (Pa) during myringotomy and tube placement for chronic otitis media with effusion (duration >3 months). All patients had failed multiple courses of antimicrobial therapy and were diagnosed by a combination of validated otoscopy and tympanograms. Main Outcome Measure. —Differences in the percentage of positive test results between PCR-based assays and culture for M catarrhalis, H influenzae , and S pneumoniae . Results. —Of the 97 specimens of otitis media with effusion, 28 (28.9%) tested positive by both culture and PCR for M catarrhalis, H influenzae , or S pneumoniae . An additional 47 specimens (48%) were PCR positive/culture negative for these three bacterial species. Thus, 75 (77.3%) of the 97 specimens tested PCR positive for one or more of the three test organisms. The minimum number of bacterial genomic equivalents present in the average culture-negative ear was estimated to be greater than 10 4 based on dilutional experiments. Conclusions. —The PCR-based assay systems can detect the presence of bacterial DNA in a significant percentage of culturally sterile middle ear effusions. While this finding is not proof of an active bacterial infectious process, the large number of bacterial genomic equivalents present in the ears is suggestive of an active process. ( JAMA . 1995;273:1598-1604)

Molecular Analysis of Bacterial Pathogens in Otitis Media With Effusion

Streptococcus pneumoniae is a major cause of bacteremia in both children and adults. Currently, the diagnosis of pneumococcal bacteremia relies on the isolation and identification of the bacteria from blood cultures. We have developed a sensitive assay for the detection of S. pneumoniae in whole blood by the PCR. A specific primer-probe set (JM201 and JM202 primers with JM204 probe) designed from the penicillin-binding protein 2B gene was demonstrated to reproducibly detect between 10 and 100 fg of input purified S. pneumoniae DNA. This assay system was shown to be inclusive for all strains of S. pneumoniae evaluated, including 15 different serotypes and a battery of penicillin-resistant and -sensitive strains. The specificity of this PCR-based assay was demonstrated by its inability to support amplification from a series of human, bacterial, and yeast genomic DNAs. A general specimen preparation method which should be suitable for the purification of DNA from any pathogens in whole blood was developed. With this protocol it was possible to detect S. pneumoniae-specific DNA from whole blood specimens inoculated with as little as 4 CFU/ml. Copurified human blood DNA, ranging from 0 to 4.5 micrograms per PCR, did not affect the sensitivity of S. pneumoniae detection by PCR. A blinded clinical trial was used to compare the PCR-based assay with standard microbiological blood culture for the detection of S. pneumoniae bacteremia in 36 specimens obtained from pediatric patients seen in the emergency room of Children's Hospital of Pittsburgh.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Streptococcus pneumoniae in whole blood by PCR.

ContextGastroesophageal reflux (GER) has not previously been widely regarded
as a hereditary disease. A few reports have suggested, however, that a genetic
component may contribute to the incidence of GER, especially in its severe
or chronic forms.ObjectiveTo identify a genetic locus that cosegregates with a severe pediatric
GER phenotype in families with multiple affected members.DesignA genome-wide scan of families affected by severe pediatric GER using
polymorphic microsatellite markers spaced at an average of 8 centimorgans
(cM), followed by haplotyping and by pairwise and multipoint linkage analyses.SettingGeneral US community, with research performed in a university tertiary
care hospital.SubjectsAffected and unaffected family members from 5 families having multiple
individuals affected by severe pediatric GER, identified through a patient
support group.Main Outcome MeasuresDetermination of inheritance patterns and linkage of a genetic locus
with the severe pediatric GER phenotype by logarithm-of-odds (lod) score analysis,
considering a lod score of 3 or greater as evidence of linkage.ResultsIn these families, severe pediatric GER followed an autosomal dominant
hereditary pattern with high penetrance. A gene for severe pediatric GER was
mapped to a 13-cM region on chromosome 13q between microsatellite markers
D13S171 and D13S263. A maximum multifamily 2-point lod score of 5.58 and a
maximum multifamily multipoint lod score of 7.15 were obtained for marker
D13S1253 at map position 35 cM when presumptively affected persons were modeled
as unknown (a maximum multipoint score of 4.88 was obtained when presumptively
affected persons were modeled as unaffected).ConclusionThese data suggest that a gene for severe pediatric GER maps to chromosome
13q14.

https://jamanetwork.com/journals/jama/articlepdf/192899/joc91307.pdf

Mapping of a gene for severe pediatric gastroesophageal reflux to chromosome 13q14.

Identification of a patient withStreptococcus pneumoniaebacteremia and meningitis by the polymerase chain reaction (PCR)

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Molecular Analysis of Bacterial Pathogens in Otitis Media With Effusion

Detection of Streptococcus pneumoniae in whole blood by PCR.

Mapping of a gene for severe pediatric gastroesophageal reflux to chromosome 13q14.

Identification of a patient withStreptococcus pneumoniaebacteremia and meningitis by the polymerase chain reaction (PCR)