William A. Falkler

Bacteria in the apical 5 mm of infected root canals

Ten freshly extracted teeth which had carious pulpal exposures and periapical lesions contiguous with the root apex were placed inside an anaerobic chamber and the apical 5 mm of the root canals cultured. In addition to anaerobic incubation, duplicate cultures were incubated aerobically. Fifty strains of bacteria from the 10 root canals were isolated and identified. The most prominent bacteria cultured from the 10 root canals were Actinomyces, Lactobacillus, black-pigmented Bacteroides, Peptostreptococcus, nonpigmented Bacteroides, Veillonella, Enterococcus faecalis, Fusobacterium nucleatum, and Streptococcus mutans. Of the 50 bacterial isolates, 34 (68%) were strict anaerobes. This study demonstrates the presence of predominantly anaerobic bacteria in the apical 5 mm of infected root canals in teeth with carious pulpal exposures and periapical lesions.

Fungal biofilms and drug resistance.

Candida species, including the novel opportunistic pathogen Candida dubliniensis, are now emerging as major agents of nosocomial infections. Many such manifestations of infections associated with the formation of Candida biofilms include those occurring on devices such as indwelling intravascular catheters. Fungal biofilm-associated infections are frequently refractory to conventional therapy because of resistance to antimicrobial agents. This resistance could be in part due to the surface-induced upregulation of drug efflux pumps. Biofilm-associated Candida show uniform resistance to a wide spectrum of the currently available conventional antifungal agents, which implies that antimicrobial drugs that specifically target biofilm-associated infections are needed. The novel classes of antifungal agents, the lipid formulation of amphotericins, and the echinocandins have demonstrated unique antifungal activity against the resistant Candida biofilms, providing a breakthrough in the treatment of life-threatening invasive systemic mycoses. The use of drugs effective in combating biofilm-associated infections could lead to major developments in the treatment of fungal implant infections.

Prevalent Bacterial Species and Novel Phylotypes in Advanced Noma Lesions

ABSTRACT The purpose of this study was to determine the bacterial diversity in advanced noma lesions using culture-independent molecular methods. 16S ribosomal DNA bacterial genes from DNA isolated from advanced noma lesions of four Nigerian children were PCR amplified with universally conserved primers and spirochetal selective primers and cloned into Escherichia coli. Partial 16S rRNA sequences of approximately 500 bases from 212 cloned inserts were used initially to determine species identity or closest relatives by comparison with sequences of known species or phylotypes. Nearly complete sequences of approximately 1,500 bases were obtained for most of the potentially novel species. A total of 67 bacterial species or phylotypes were detected, 25 of which have not yet been grown in vitro. Nineteen of the species or phylotypes, including Propionibacterium acnes, Staphylococcus spp., and the opportunistic pathogens Stenotrophomonas maltophilia and Ochrobactrum anthropi were detected in more than one subject. Other known species that were detected included Achromobacter spp., Afipia spp., Brevundimonas diminuta, Capnocytophaga spp., Cardiobacterium sp., Eikenella corrodens, Fusobacterium spp., Gemella haemoylsans, and Neisseria spp. Phylotypes that were unique to noma infections included those in the genera Eubacterium, Flavobacterium, Kocuria, Microbacterium, and Porphyromonas and the related Streptococcus salivarius and genera Sphingomonas and Treponema. Since advanced noma lesions are infections open to the environment, it was not surprising to detect species not commonly associated with the oral cavity, e.g., from soil. Several species previously implicated as putative pathogens of noma, such as spirochetes and Fusobacterium spp., were detected in at least one subject. However, due to the limited number of available noma subjects, it was not possible at this time to associate specific species with the disease.

Noma (cancrum oris)

Noma is an opportunistic infection promoted by extreme poverty. It evolves rapidly from a gingival inflammation to grotesque orofacial gangrene. It occurs worldwide, but is most common in sub-Saharan Africa. The peak incidence of acute noma is at ages 1-4 years, coinciding with the period of linear growth retardation in deprived children. Noma is a scourge in communities with poor environmental sanitation. It results from complex interactions between malnutrition, infections, and compromised immunity. Diseases that commonly precede noma include measles, malaria, severe diarrhoea, and necrotising ulcerative gingivitis. The acute stage responds readily to antibiotic treatment. The sequelae after healing include variable functional and aesthetic impairments, which require reconstructive surgery. Noma can be prevented through promotion of national awareness of the disease, poverty reduction, improved nutrition, promotion of exclusive breastfeeding in the first 3-6 months of life, optimum prenatal care, and timely immunisations against the common childhood diseases.

Oro-Facial Gangrene (Noma/Cancrum Oris): Pathogenetic Mechanisms

Cancrum oris (Noma) is a devastating infectious disease which destroys the soft and hard tissues of the oral and para-oral structures. The dehumanizing oro-facial gangrenous lesion affects predominantly children ages 2 to 16 years, particularly in sub-Saharan Africa, where the estimated frequency in some communities varies from 1 to 7 cases per 1000 population. The risk factors are poverty, malnutrition, poor oral hygiene, residential proximity to livestock in unsanitary environments, and infectious diseases, particularly measles and those due to the herpesviridae. Infections and malnutrition impair the immune system, and this is the common denominator for the occurrence of noma. Acute necrotizing gingivitis (ANG) and oral herpetic ulcers are considered the antecedent lesions, and ongoing studies suggest that the rapid progression of these precursor lesions to noma requires infection by a consortium of micro-organisms, with Fusobacterium necrophorum (Fn) and Prevotella intermedia (Pi) as the suspected key players. Additional to production of a growth-stimulating factor for Pi, Fn displays a classic endotoxin, a dermonecrotic toxin, a cytoplasmic toxin, and a hemolysin. Without appropriate treatment, the mortality rate from noma is 70-90%. Survivors suffer the two-fold afflictions of oro-facial mutilation and functional impairment, which require a time-consuming, financially prohibitive surgical reconstruction.

Microbiological studies of carious dentine from human teeth with irreversible pulpitis

Aerobic and anaerobic cultures were made from 29 extracted teeth with irreversible pulpitis to identify the predominant flora in different parts of deep carious lesions. Most isolates were Gram-positive rods, in which lactobacilli were the most frequent organisms, then other Gram-positive, non-branching rods. Gram-positive cocci were the next most common; only a low number of Streptococcus mutans was recovered. Two types of carious lesions were found, one with high numbers of lactobacilli, the other with low. In the 15 lesions with high numbers, lactobacilli constituted 91.9% of the total flora at the pulpal site and gradually decreased in number as the sampling moved away from the pulp. Strep. mutans and alpha-haemolytic streptococci were not recovered from pulpal or deep carious sites in this group. In the 14 lesions with low numbers of lactobacilli, the flora was diverse. Gram-positive cocci, anaerobic Gram-positive non-branching rods, branching rods and/or Bacteroides were the main isolates in a few of this group.

A study of T and B cells in pulpal pathosis

A study was undertaken using monoclonal antibodies to determine the types of lymphocytes present in pulpal tissues. Pulps were extirpated from teeth clinically diagnosed as normal, reversibly inflamed, or irreversibly inflamed and stained with hematoxylin and eosin and an indirect immunoperoxidase technique using monoclonal antibodies reactive to pan-B lymphocytes (B), pan-T lymphocytes (T1), and helper (T4) and suppressor (T8) T lymphocytes. T and/or B lymphocytes were observed in normal pulpal tissues with T8 lymphocytes being predominant. The pulpal tissue in the reversible group demonstrated that more than 90% of the lymphocyte population were T lymphocytes, with a T4/T8 ratio of 0.56. Higher numbers of T1, T4, T8; and B lymphocytes were observed in the pulp from teeth in the irreversible group. A ratio of 1.14 of T4/T8 lymphocytes was observed in the irreversible group. A B/T1 lymphocyte ration of 1.60 suggested this ratio might be used as an index in the immunohistological diagnosis of irreversible pulpal pathosis. There appeared to be no association between the periodontal status of the teeth and the number of immunocompetent cells observed in the pulps. An hypothesis on the regulatory functions of T4 and T8 lymphocytes as well as the interaction of T and B lymphocytes and their products in the pathogenesis of pulpal disease is presented.

Evaluation of a Reformulated CHROMagar Candida

ABSTRACT CHROMagar Candida is a differential culture medium for the isolation and presumptive identification of clinically important yeasts. Recently the medium was reformulated by Becton Dickinson. This study was designed to evaluate the performance of the new formula of CHROMagar against the original CHROMagar Candida for recovery, growth, and colony color with stock cultures and with direct plating of clinical specimens. A total of 90 stock yeast isolates representing nine yeast species, including Candida dubliniensis, as well as 522 clinical specimens were included in this study. No major differences were noted in growth rate or colony size between the two media for most of the species. However, all 10 Candida albicans isolates evaluated consistently gave a lighter shade of green on the new CHROMagar formulation. In contrast, all 26 C. dubliniensis isolates gave the same typical dark green color on both media. A total of 173 of the 522 clinical specimens were positive for yeast, with eight yeast species recovered. The recovery rates for each species were equivalent on both media, with no consistent species-associated differences in colony size or color. Although both media were comparable in performance, the lighter green colonies ofC. albicans isolates on the new CHROMagar made it easier to differentiate between C. albicans and C. dubliniensis isolates. In conclusion, the newly formulated Becton Dickinson CHROMagar Candida medium is as equally suited as a differential medium for the presumptive identification of yeast species and for the detection of multiple yeast species in clinical specimens as the original CHROMagar Candida medium.

Recovery of bdellovibrios from submerged surfaces and other aquatic habitats.

The distribution of bdellovibrios was investigated over a wide geographical area of the Chesapeake Bay including some tributaries and subestuaries. Bdellovibrios were recovered from five aquatic habitats; water, sediment, oyster shell surface biofilm, zooplankton, and plants over a wide range of temperature and salinity measurements. Consistently, the greatest number of the predators was recovered from samples of biofilm irrespective of temperature and salinity. A decrease in the numbers and frequency of predators recovered from all habitats was observed at temperatures below 10°C. Only the shell surface biofilm samples yielded bdellovibrios 100% of the time. The organisms were recovered from 79% of water samples and 44% of sediment samples. The results reveal that bdellovibrios are surface-associated organisms and that this association appears to provide some protection for the predators at low temperatures.

Enhanced Interleukin-1β, Interleukin-6 and Tumor Necrosis Factor-α Production by LPS Stimulated Human Monocytes Isolated from HIV + Patients

Abstract Periodontal disease and tooth loss is a common finding among advanced HIV+ patients. In addition to local oral lipopolysaccharide (LPS) stimulation, systemic up-regulation of monocyte pro-inflammatory cytokine secretion may also be involved in the pathogenesis of HIV disease. A study was undertaken to investigate IL-1β, IL-6 and TNF-α production by resting and LPS stimulated monocytes isolated from HIV + patients and also to investigate the relationship of the patients HIV viral load status to the cytokine production. Whole blood samples in EDTA were collected from 39 HIV-1 infected patients and 20 age and sex matched uninfected controls. Plasma was separated by centrifugation. Viral load was determined using a quantitative RT-PCR. Monocytes were isolated by Ficoll-hypaque gradient separation followed by overnight plastic adherence. Cultured monocytes (1 × 106ml) were stimulated with LPS (1 μg/ml) of either P. gingivalis or F. nucleatum for 2, 8, 24 and 48 h and supernatant fluids were collected. IL-1β, IL-6, and TNF-α levels in supernatant fluids were estimated by ELISA. Increased overall production of IL-1β, IL-6 and TNF-α by LPS stimulated monocytes isolated from HIV-1 infected patients was observed when compared to HIV-1 uninfected controls. LPS stimulated monocytes from HIV-1 infected patients with high viral load (HVL) produced significant (p<0.05) elevations in these pro-inflammatory cytokines when compared to HIV-1 uninfected controls. Both LPS of P. gingivalis and F. nucleatum produced a comparable cytokine production by monocytes after 8 h of stimulation. These data suggest that enhanced IL-1β, IL-6 and TNF-α is produced by monocytes/macrophages isolated from HVL HIV + patients and may be involved in the overall pathogenesis of HIV-1 infection.