Comparative Evaluation of Culture and PCR for the Detection and Determination of Persistence of Bacterial Strains and DNAS in the Chinchilla Laniger Model of Otitis Media

University of Pittsburgh, Pittsburgh, Pennsylvania, United States
The Annals of otology, rhinology, and laryngology (Impact Factor: 1.09). 07/1998; 107(6):508-13. DOI: 10.1177/000348949810700609
Source: PubMed


This study was designed to determine the persistence of culturable bacteria versus DNA in the presence of a middle ear effusion in a chinchilla model of otitis media. Cohorts of animals were either infected with an ampicillin-resistant Haemophilus influenzae strain or injected with a tripartite inoculum consisting of freeze-thawed Streptococcus pneumoniae; pasteurized Moraxella catarrhalis; and DNA from H influenzae. The H influenzae-infected animals displayed culture positivity and polymerase chain reaction positivity through day 35. In the chinchillas infected with the low-copy number inocula of S pneumoniae, DNA was not detectable after day 1 from the co-inoculated pasteurized M catarrhalis bacteria or the purified H influenzae DNA; however, amplifiable DNA from the live low-copy number bacteria persisted through day 21 even though they were not culture-positive past day 3. These results demonstrate that DNA, and DNA from intact but nonviable bacteria, does not persist in an amplifiable form for more than a day in the presence of an effusion; however, live bacteria, while not culturable, persist in a viable state for weeks.

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    • "In addition to specific pathways that respond to and deal with each of the damaging physical or chemical stressors present within the various environments the bacteria may encounter, many bacteria have a capacity to switch their lifestyle such that these stresses no longer cause damage to their cell. One key lifestyle adaptation is the switch from planktonic (or “free-living”) cells to a biofilm [3-5]. This process is based upon numerous features of the bacterial cell including alterations in their metabolism and physiology, the presence and nature of surface structures, and the general physical properties of the bacterial cell. "
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    ABSTRACT: Haemophilus influenzae colonizes the nasopharynx as a commensal. Strain-specific factors allow some strains to migrate to particular anatomical niches, such as the middle ear, bronchi or blood, and induce disease by surviving within the conditions present at these sites in the body. It is established that H. influenzae colonization and in some cases survival is highly dependent on their ability to form a biofilm. Biofilm formation is a key trait in the development of chronic infection by certain isolates. This is exemplified by the contrast between the biofilm-forming strains found in middle ear infections and those isolates that survive within the blood and are rarely associated with biofilm development. Screening a group of H. influenzae strains revealed only slight variations in their growth across a range of pH conditions. However, some isolates responded to a pH of 8.0 by the formation of a biofilm. While the type b capsular blood isolate Eagan did not form a biofilm and grew at the same rate regardless of pH 6.8-8.0, transcriptomic analyses demonstrated that at pH 8.0 it uniquely induced a gluconate-uptake and metabolism pathway, which concurrently imports H+. A non-typeable H. influenzae, isolated from the middle ear, induced biofilm formation at pH 8.0, and at this pH it induced a series of iron acquisition genes, consistent with previous studies linking iron homeostasis to biofilm lifestyle. Different strains of H. influenzae cope with changes in environmental factors using strain-specific mechanisms. These pathways define the scope and mode of niche-survival for an isolate. The pH is a property that is different from the middle ear (at least pH 8.0) compared to other sites that H. influenzae can colonize and infect. The transcriptional response to increasing pH by H. influenzae varies between strains, and pH is linked to pathways that allow strains to either continue free-living growth or induction of a biofilm. We showed that a biofilm-forming isolate induced iron metabolism pathways, whereas a strain that does not form biofilm at increasing pH induced mechanisms for growth and pH homeostasis based on sugar acid transport.
    Full-text · Article · Feb 2014 · BMC Microbiology
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    • "Several factors may have contributed to the low A. otitidis isolation rate. Firstly, PCR-positive but culture-negative results may indicate detection of non-viable bacteria; however, it is generally accepted that DNA is quickly cleared from the middle ear [39-41] suggesting that a positive PCR is indicative of viable cells. Secondly, A. otitidis viability may have been affected by storage at -70°C for up to five years; however, viability of S. pneumoniae and H. influenzae has been demonstrated in swabs stored for up to 12 years at -70°C [42] suggesting good overall bacterial survival despite the prolonged storage. "
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    ABSTRACT: Background Otitis media is endemic in remote Indigenous communities of Australia’s Northern Territory. Alloiococcus otitidis is an outer ear commensal and putative middle ear pathogen that has not previously been described in acute otitis media (AOM) in this population. The aims of this study were to determine the presence, antibiotic susceptibility and bacterial load of A. otitidis in nasopharyngeal and ear discharge swabs collected from Indigenous Australian children with AOM with perforation. Methods Paired nasopharyngeal and ear discharge swabs from 27 children with AOM with perforation were tested by A. otitidis quantitative PCR (qPCR). Positive swabs were cultured for 21 days. Total and respiratory pathogen bacterial loads in A. otitidis-positive swabs were determined by qPCR. Results A. otitidis was detected by qPCR in 11 ear discharge swabs from 10 of 27 (37%) children, but was not detected in paired nasopharyngeal swabs. A. otitidis was cultured from 5 of 11 qPCR-positive swabs from four children. All A. otitidis isolates had minimum inhibitory concentrations consistent with macrolide resistance. All A. otitidis qPCR-positive swabs were culture-positive for other bacteria. A. otitidis bacterial load ranged from 2.2 × 104-1.1 × 108 cells/swab (median 1.8 × 105 cells/swab). The relative abundance of A. otitidis ranged from 0.01% to 34% of the total bacterial load (median 0.7%). In 6 of 11 qPCR-positive swabs the A. otitidis relative abundance was <1% and in 5 of 11 it was between 2% and 34%. The A. otitidis bacterial load and relative abundance measures were comparable to that of Haemophilus influenzae. Conclusions A. otitidis can be a dominant species in the bacterial communities present in the ear discharge of Indigenous children with AOM with perforation. The absence of A. otitidis in nasopharyngeal swabs suggests the ear canal as the likely primary reservoir. The significance of A. otitidis at low relative abundance is unclear; however, at higher relative abundance it may be contributing to the associated inflammation. Further studies to better understand A. otitidis as a secondary otopathogen are warranted, particularly in populations at high-risk of progression to chronic suppurative otitis media and where macrolide therapies are being used.
    Full-text · Article · Oct 2012 · BMC Ear Nose and Throat Disorders
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    • "While NTHi and other bacteria may be frequently isolated from patients with OM, middle-ear effusions from a large proportion of these patients do not yield culturable bacteria. However, in a series of important early studies, Post and colleagues showed that NTHi and other bacteria could be detected in these samples by PCR-based methods (Post et al., 1995, 1996a,b; Aul et al., 1998; Bakaletz et al., 1998; Liederman et al., 1998). Similarly, middle-ear effusion samples were shown to contain bacterial components (Dingman et al., 1998) and transcripts, indicating bacterial metabolic activity (Rayner et al., 1998). "
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    ABSTRACT: Like many pathogens inhabiting mucosal surfaces, nontypeable Haemophilus influenzae (NTHi) forms multicellular biofilm communities both in vitro and in various infection models. In the past 15 years much has been learned about determinants of biofilm formation by this organism and potential roles in bacterial virulence, especially in the context of chronic and recurrent infections. However, this concept has not been without some degree of controversy, and in the past some have expressed doubts about the relevance of NTHi biofilms to disease. In this review, I will summarize the present information on the composition and potential role(s) of NTHi biofilms in different clinical contexts, as well as highlight potential areas for future work.
    Full-text · Article · Jul 2012 · Frontiers in Cellular and Infection Microbiology
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