Microaerophilic Incubation Research Articles

Regulation of iron metabolism is critical for the survival of Salmonella Typhimurium in pasteurized milk

Salmonella is known to survive in raw/pasteurized milk and cause foodborne outbreaks. Lactoferrin, present in milk from all animal sources, is an iron-binding glycoprotein that limits the availability of iron to pathogenic bacteria. Despite the presence of lactoferrins, Salmonella can grow in milk obtained from different animal sources. However, the mechanism by which Salmonella overcomes iron scarcity induced by lactoferrin in milk is not evaluated yet. Salmonella employs the DNA binding transcriptional regulator Fur (ferric update regulator) to mediate iron uptake during survival in iron deplete conditions. To understand the importance of Fur in Salmonella milk growth, we profiled the growth of Salmonella Typhimurium Δfur (ST4/74Δfur) in both bovine and camel milk. ST4/74Δfur was highly inhibited in milk compared to wild-type ST4/74, confirming the importance of Fur mediated regulation of iron metabolism in Salmonella milk growth. We further studied the biology of ST4/74Δfur to understand the importance of iron metabolism in Salmonella milk survival. Using increasing concentrations of FeCl3, and the antibiotic streptonigrin we show that iron accumulates in the cytoplasm of ST4/74Δfur. We hypothesized that the accumulated iron could activate oxidative stress via Fenton’s reaction leading to growth inhibition. However, the inhibition of ST4/74Δfur in milk was not due to Fenton’s reaction, but due to the ‘iron scarce’ conditions of milk and microaerophilic incubation conditions which made the presence of the fur gene indispensable for Salmonella milk growth. Subsequently, survival studies of 14 other transcriptional mutants of ST4/74 in milk confirmed that RpoE-mediated response to extracytoplasmic stress is also important for the survival of Salmonella in milk. Though we have data only for fur and rpoE, many other Salmonella transcriptional factors could play important roles in the growth of Salmonella in milk, a theme for future research on Salmonella milk biology. Nevertheless, our data provide early insights into the biology of milk-associated Salmonella.

Head-to-head comparison of CAMPYAIR aerobic culture medium versus standard microaerophilic culture for Campylobacter isolation from clinical samples.

Campylobacter spp. are considered the most frequent cause of acute gastroenteritis worldwide. However, outside high-income countries, its burden is poorly understood. Limited published data suggest that Campylobacter prevalence in low- and middle-income countries is high, but their reservoirs and age distribution are different. Culturing Campylobacter is expensive due to laboratory equipment and supplies needed to grow the bacterium (e.g., selective culture media, microaerophilic atmosphere, and a 42°C incubator). These requirements limit the diagnostic capacity of clinical laboratories in many resource-poor regions, leading to significant underdiagnosis and underreporting of isolation of the pathogen. CAMPYAIR, a newly developed selective differential medium, permits Campylobacter isolation without the need for microaerophilic incubation. The medium is supplemented with antibiotics to allow Campylobacter isolation in complex matrices such as human feces. The present study aims to evaluate the ability of the medium to recover Campylobacter from routine clinical samples. A total of 191 human stool samples were used to compare the ability of CAMPYAIR (aerobic incubation) and a commercial Campylobacter medium (CASA, microaerophilic incubation) to recover Campylobacter. All Campylobacter isolates were then identified by MALDI-TOF MS. CAMPYAIR showed sensitivity and specificity values of 87.5% (95% CI 47.4%-99.7%) and 100% (95% CI 98%-100%), respectively. The positive predictive value of CAMPYAIR was 100% and its negative predictive value was 99.5% (95% CI 96.7%-99.9%); Kappa Cohen coefficient was 0.93 (95% CI 0.79-1.0). The high diagnostic performance and low technical requirements of the CAMPYAIR medium could permit Campylobacter culture in countries with limited resources.

Evaluation of a commercial loop-mediated isothermal amplification assay, 3MTM Molecular Detection Assay 2 – Campylobacter, for the detection of Campylobacter from poultry matrices

ABSTRACT 1. The objective of this study was to evaluate performance of a commercial loop-mediated isothermal amplification (LAMP) method as an alternative method for the detection of Campylobacter spp. in primary production samples, poultry rinses and raw poultry products, as compared to the US Department of Agriculture Food Inspection Service Microbiology Laboratory Guide Book PCR reference method, MLG 41A. 2. The Campylobacter spp. LAMP was used in conjunction with a ready-to-use enrichment broth that does not require microaerophilic incubation. After enrichment, boot swabs from poultry farms, carcase rinses and raw poultry products were tested by the LAMP method and the MLG 41A PCR method. 3. The ready-to-use enrichment broth enabled the growth of Campylobacter spp. within 22 to 28 hours under aerobic incubation conditions. The LAMP method enabled Campylobacter detection in the enriched samples of various poultry matrices and had equivalent sensitivity and specificity to the MLG 41A PCR method. 4. No significant difference (95% confidence interval) was found between the alternative and the MLG 41A PCR method, as determined by probability of detection analysis, except for neutralising buffered peptone water post-chill rinsates. For the post-chill neutralising buffered peptone water rinsates, the LAMP method had significantly higher confirmed portions.

Gangrenous mastitis in sheep caused by multidrug-resistant Staphylococcus haemolyticus

ABSTRACT: Mastitis is a multifactorial disease and considered one of the most critical problems in the dairy industry worldwide. The condition is characterized by reduced milk and several abnormalities in the mammary gland. This study aimed to report an outbreak of gangrenous mastitis caused by multidrug-resistant Staphylococcus haemolyticus in a Santa Inês sheep herd. Eighteen sheep were affected, and five of them with severe clinical pictures were examined. The clinical and pathological picture were variable and characterized by apathy, anorexia, emaciation, opaque and brittle hair, apparent and congested episcleral vessels, and hyperthermia. These ewes had enlarged, firm, and painful mammary glands. Macroscopically, these lesions consisted of severe gangrenous mastitis, and microscopically, the primary lesions consisted of necrosis, thrombosis, and fibrosis of the mammary parenchyma. Milk samples from one of the five severely affected ewes were collected and cultured under aerobic or microaerophilic incubation at 37°C for 24 hours on sheep blood agar. The obtained colonies were then submitted to MALDI-TOF for speciation. The colonies were also submitted to an antimicrobial susceptibility test, genotyping of virulence factors and resistance genes were also performed. The isolates showed antimicrobial multiresistance since they were resistant to seven out of 13 tested antibiotics. The isolates were also positive for two staphylococcal enterotoxigenic genes (sec and see) and fibronectin-binding protein B (fnbB).

A protocol for culturing environmental strains of the Buruli ulcer agent, Mycobacterium ulcerans

Contaminations and fastidiousness of M. ulcerans may have both hamper isolation of strains from environmental sources. We aimed to optimize decontamination and culture of environmental samples to circumvent both limitations. Three strains of M. ulcerans cultured onto Middlebrook 7H10 at 30 °C for 20 days yielded a significantly higher number of colonies in micro-aerophilic atmosphere compared to ambient atmosphere, 5% CO2 and anaerobic atmosphere. In a second step, we observed that M. ulcerans genome uniquely encoded chitinase, fucosidase and A-D-GlcNAc-diphosphoryl polyprenol A-3-L-rhamnosyl transferase giving M. ulcerans the potential to metabolize chitine, fucose and N-acetyl galactosamine (NAG), respectively. A significant growth-promoting effect of 0.2 mg/mL chitin (p < 0.05), 0.01 mg/mL N-acetyl galactosamine (p < 0.05), 0.01 mg/mL fucose (p < 0.05) was observed with M. ulcerans indicating that NAG alone or combined with fucose and chitin could complement Middlebrook 7H10. Finally, the protocol combining 1% chlorhexidine decontamination with micro-aerophilic incubation on Middlebrook 7H10 medium containing chitin (0.2%), NAG (0.01%) and fucose (0.01%) medium and auto-fluorescence detection of colonies allowed for the isolation of one mycolactone-encoding strain from Thryonomys swinderianus (aulacode) feces specimens collected near the Kossou Dam, Côte d’Ivoire. We propose that incubation of chlorhexidine-decontaminated environmental specimens on Middlebrook 7H10-enriched medium under micro-aerophilic atmosphere at 30 °C may be used for the tentative isolation of M. ulcerans strains from potential environmental sources.

Hypoxia stimulates neural stem cell proliferation by increasing HIF‑1α expression and activating Wnt/β-catenin signaling.

Evidence indicates that after brain injury, neurogenesis is enhanced in regions such as hippocampus, striatum, and cortex. To study the role of hypoxia-inducible factor-1 (HIF‑1α) and Wnt signaling in cerebral ischemia/hypoxia-induced proliferation of neural stem cells (NSCs), we investigated the proliferation of NSCs, expression of HIF‑1α, and activation of Wnt signaling under conditions of pathologic hypoxia in vitro. NSCs were isolated from 30-day-old Sprague-Dawley rats and subjected to 0.3% oxygen in a microaerophilic incubation system. Cell proliferation was evaluated by measuring the diameter of neurospheres and by bromodeoxyuridine incorporation assays. Real-time quantitative PCR and Western blotting were used to detect mRNA and protein levels of HIF-1α, β-catenin, and cyclin D1 in the NSCs. The results showed that hypoxia increased NSC proliferation and the levels of HIF-1α, β‑catenin, and cyclin D1 (p &lt; 0.05). Blockade of the Wnt signaling pathway decreased hypoxia-induced NSC proliferation, whereas activation of this pathway increased hypoxia-induced NSC proliferation (p &lt; 0.05). Knockdown of HIF-1α with HIF-1α siRNA decreased β‑catenin nuclear translocation and cyclin D1 expression, and inhibited proliferation of NSCs (p &lt; 0.05). These findings indicate that pathologic hypoxia stimulates NSC proliferation by increasing expression of HIF-1α and activating the Wnt/β-catenin signaling pathway. The data suggest that Wnt/β-catenin signaling may play a key role in NSC proliferation under conditions of pathologic hypoxia.

Exploring the potential of fungal-bacterial consortium for low-cost biodegradation and detoxification of textile effluent

AbstractIn the present study, the enrichment and isolation of textile effluent decolorizing bacteria were carried out in wheat bran (WB) medium. The isolated bacterium Providencia rettgeri strain HSL1 was then tested for decolorization of textile effluent in consortium with a dyestuff degrading fungus Aspergillus ochraceus NCIM 1146. Decolorization study suggests that A. ochraceus NCIM 1146 and P. rettgeri strain HSL1 alone re moves only 6 and 32% of textile effluent American Dye Manufacturing Institute respectively in 30 h at 30 ±0.2°C of microaerophilic incubation, while the fungal-bacterial consortium does 92% ADMI removal within the same time period. The fungal-bacterial consortium exhibited enhanced decolorization rate due to the induction in activities of catalytic enzymes laccase (196%), lignin peroxidase (77%), azoreductase (80%) and NADH-DCIP reductase (84%). The HPLC analysis confirmed the biodegradation of textile effluent into various metabolites. Detoxification studies of textile effluent before and after treatment with fungal-bacterial consortium revealed reduced toxicity of degradation metabolites. The efficient degradation and detoxification by fungal-bacterial consortium pre-grown in agricultural based medium thus suggest a promising approach in designing low-cost treatment technologies for textile effluent.

Biodegradation of Azo Dye Disperse Orange S-RL by a Newly Isolated Strain Acinetobacter sp. SRL8.

The strain SRL8, which could decolorize the azo dye disperse orange S-RL (S-RL), was first isolated from sludge and identified as Acinetobacter sp. through physiobiochemical identification and 16S rRNA gene sequences. The effects of temperature, pH, dye concentration, O2, and glucose concentration on S-RL decolorization by the strain SRL8 were studied. The optimal conditions were 30 °C, pH 7.0, 4g·L(-1) of inoculation (wet cells), and microaerophilic incubation. The decolorization percentage for S-RL by the strain SRL8 could reach 90.2% under optimal conditions. The strain SRL8 was highly tolerant to the azo dye SRL up to 300 mg·L(-1) and it had a broad decolorizing spectrum. According to the Monod equation, kinetic parameters of decolorization by SRL8 were calculated. The vmax and Km were 5.57×10(-3) h(-1) and 14.53 mg·L(-1), respectively.

Low Recovery Rate of Helicobacter Pylori from Positive CLO Test Patients Suffering from Dyspepsia

Method: Gastroscopy and gastric biopsy were performed on 53 patients with dyspepsia from January 2012 to January 2013; all were subjected urease CLO test and culture. The CLO-positive biopsies were cultured using brain-heart infusion agar with added blood (7%), and Skirrow's supplement was used for isolating Helicobacter pylori. Inoculated plates were incubated at 37°C for 7–10 days in a microaerophilic incubation environment and examined for suspected Helicobacter pylori colonies. Helicobacter pylori cultures were confirmed by the positive urease, oxidase and rapid antigen test. Cultures of non-Helicobacter pylori bacteria were identified using few phenotypic tests then confirmed by VITEK 2 automated system.

Low-Cost Biodegradation and Detoxification of Textile Azo Dye C.I. Reactive Blue 172 byProvidencia rettgeriStrain HSL1

Present study focuses on exploitation of agricultural waste wheat bran (WB) as growth medium for degradation of textile azo dye C.I. Reactive Blue 172 (RB 172) using a single bacteriumP. rettgeristrain HSL1 (GenBank accession numberJX853768.1). The bacterium was found to completely decolorize 50 mg L−1of dye RB 172 within 20 h at 30 ± 0.2°C under microaerophilic incubation conditions. Additionally, significant reduction in COD (85%) and TOC (52%) contents of dye decolorized medium was observed which suggested its mineralization. Induction in the activities of azoreductase (159%) and NADH-DCIP reductase (88%) provided an evidence for reductive cleavage of dye RB 172. The HPLC, FTIR, and GC-MS analysis of decolorized products confirmed the degradation of dye into various metabolites. The proposed metabolic pathway for biodegradation of RB 172 has been elucidated which showed the formation of 2 intermediate metabolites, namely, 4-(ethenylsulfonyl) aniline and 1-amino-1-(4-aminophenyl) propan-2-one. The acute and phytotoxicity evaluation of degraded metabolites suggests that bacterial strain favors the detoxification of dye RB 172. Thus, WB could be utilized as a low-cost growth medium for the enrichment of bacteria and their further use for biodegradation of azo dyes and its derivatives containing wastes into nontoxic form.

Hypoxia stimulates proliferation of rat neural stem cells with influence on the expression of cyclin D1 and c-Jun N-terminal protein kinase signaling pathway in vitro

Ischemia/hypoxia is known to induce the neural stem cells proliferation and neural differentiation in rodent and human brain; however its mechanisms remain largely unknown. In this study we investigated the effect of hypoxia on neural stem cells (NSCs) proliferation with the expression of cyclin D1 and the phosphorylation of mitogen-activated protein kinases (MAPK) signaling molecules. NSCs were cultured from cortex of fetal Sprague–Dawley rats on embryonic day 5.5. The hypoxia was made using a microaerophilic incubation system. The NSCs proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, diameter measurement of neurospheres, bromodeoxyuridine (BrdU) incorporation assay and cell cycle analysis. The cell death of NSCs was evaluated by terminal dUTP nick-end labeling (TUNEL) assay. The expression of cyclin D1, phosphorylated extracellular signal regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38 were analyzed by immunoblotting assay. The results showed that hypoxia increased NSCs proliferation in cell amount, diameter of neurospheres, BrdU incorporation and cell division, and the highest proliferation of the NSCs was observed with 12 h hypoxic treatment; hypoxia did not decrease cell death of NSCs; after hypoxic treatment, the expression of cyclin D1 increased, meanwhile P-JNK2 level increased, P-p38 decreased, and no significant change in P-ERK2 level compared to normoxic cultures. JNK inhibitor SP600125 attenuated the increase of cyclin D1 induced by hypoxia. These findings propose that hypoxia increases cyclin D1 expression through activation of JNK in NSCs of rat in vitro, suggesting a novel possible mechanism for hypoxia-induced proliferation of NSCs.

GROWTH RESPONSE OF LACTOBACILLUS RHAMNOSUS TO MICROAEROPHILIC INCUBATION AND ADDITION OF REDUCTANTS IN A RIBOFLAVIN MICROTITER PLATE ASSAY

ABSTRACT Given the importance of riboflavin in human diets, a rapid riboflavin assay is needed by the food industry to allow simultaneous determination of riboflavin in numerous food sources. The growth responses of Lactobacillus rhamnosus to microaerophilic conditions and three reducing agents were examined as an attempt to reduce riboflavin assay time. Ascorbic acid, dithiothreitol and thioglycollate were applied at three concentrations. None of the concentrations of reducing agents caused a further increase of bacterial optical density (OD) or had a significant effect on the growth rates. More optimal microaerophilic conditions were achieved through the use of an anaerobic chamber. The growth rate of L. rhamnosus under microaerophilic conditions (0.30 ± 0.05/h) was not significantly different (P &gt; 0.05) from the growth rate of the bacteria under aerobic conditions (0.34 ± 0.07/h). However, after 14 h of bacterial growth, the OD of the bacterial culture under anaerobic hood incubation (0.71 ± 0.09) was significantly higher (P &lt; 0.05) than under aerobic incubation conditions (0.58 ± 0.05). Additional research is needed involving more controlled atmospheres for incubation during growth and a wider range of reductants to optimize and shorten the time required for riboflavin microtiter plate assay.PRACTICAL APPLICATIONSQuantifying riboflavin in foods and food ingredients is important to the food industry. Lactobacillus rhamnosus growth response to varying levels of riboflavin is used to estimate the amount of this vitamin. In an effort to optimize growth on microtiter plates and decrease assay time, microaerophilic conditions were examined. Although rate of growth was not altered, cell concentration measured as optical density (OD) did increase. Microaerophilic incubation of microtiter plates represents a potential approach to decrease assay time and provide the food industry with more timely results.

Media for the Aerobic Resuscitation of Campylobacter jejuni

The microaerophilic nature of Campylobacter jejuni has complicated its recovery from human and animal sources. In this study, enhancement of the growth and aerotolerance of C. jejuni ATCC 35921 in nutrient broth no. 2 (NB2) was investigated. The efficiency of recovery of C. jejuni in NB2 containing FBP (0.025% [each] ferrous sulfate, sodium metabisulfite, and sodium pyruvate), 5% laked horse blood, hemin, Oxyrase, or activated charcoal in an aerobic atmosphere was compared with that obtained under microaerophilic incubation. The shortest lag time (λ) for cells grown aerobically was observed with NB2 supplemented with FBP, 5% laked horse blood, 0.01 g/liter of hemin, or 0.15 U/ml of Oxyrase. The efficacy of these media to resuscitate C. jejuni cells in late exponential phase, as well as cells subjected to stress induced by cold, heat, starvation, or acid, was determined in aerobic or microaerobic atmospheres. The λ of cells grown aerobically in NB2 containing both FBP and blood was similar to that obtained in the same medium incubated in a microaerobic environment (P > 0.05). However, the λ was longer during aerobic growth when low numbers of cells (approximately 1 log CFU/ml) in late exponential phase were used as the initial inoculum. The best recovery of stressed C. jejuni was observed in NB2 supplemented with FBP and blood and incubated aerobically. Enrichment in media incorporating FBP and 5% laked horse blood is a simple, convenient, and time-saving method to replace microaerophilic incubation methods for the resuscitation of C. jejuni.

Survival of mixed oral bacterial flora in bottled soft drinks by drinking directly in microaerophilic incubation and the differences between individuals in aerobic incubation

Survival of mixed oral bacterial flora in bottled soft drinks by drinking directly in microaerophilic incubation and the differences between individuals in aerobic incubation

Nutrient Shock and Incubation Atmosphere Influence Recovery of Culturable Helicobacter pylori from Water

Three different media-Columbia agar, Wilkins-Chalgren agar, and Helicobacter pylori special peptone agar-were prepared in a diluted version and compared to the standard medium formulation in order to study a possible nutrient shock effect observed when recovering H. pylori from water by counting the number of CFU. This same parameter was subsequently used to evaluate the influence of the incubation atmosphere by using a modular atmosphere-controlled system to provide different atmospheres and by employing an established gas generation kit as a control. Both a low nutrient content of the media and a rapidly achieved microaerophilic incubation atmosphere proved to increase the numbers of environment-stressed H. pylori organisms recovered. An atmosphere of 5% CO(2), 5% O(2), and 3% H(2) is recommended, although other atmospheres with a low oxygen concentration are also acceptable. Besides highlighting and assessing the importance of several factors in the culturability of H. pylori, this paper demonstrates the potential ability to develop an optimized technique for recovery of this pathogen from water.

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SIRS, The purpose of testing for nitroimidazole resistance in Helicobacter pylori is that in vitro resistance is significantly related to reduced efficacy of nitroimidazole-containing eradication regimens1234 – these data have been obtained from microaerophilic incubation of samples. Furthermore, a recent study compared the effects of microaerophilic incubation with anaerobic incubation followed by microaerophilic incubation on the assessment of metronidazole resistance in 102 isolates and found little difference in the evaluation of resistance prevalence.5 In addition, alterations in the H. pylori gene rdxA have been identified as responsible for the vast majority of metronidazole resistance.6 Failure to produce the protein product of this gene can be assessed by immunoblotting with antisera to RdxA. This was confirmed in 25 out of 27 microaerophilically cultured metronidazole-resistant isolates7 supporting the currently recommended culture condition as being able to identify correctly metronidazole resistance in H. pylori. Current methods of sensitivity testing of H. pylori have been validated in the identification of resistance. Resistance to antimicrobials is a frequent cause of eradication failure. Specific resistance profiles of individual patients' samples will aid in the selection of effective and microbiologically sound eradication regimens.

Tongue Microflora in Edentulous Geriatric Denture-Wearers

The aim of this study was to investigate the bacterial composition of tongue plaque of healthy edentate geriatric individuals wearing dentures. One male of 67 years-old and four females ranging from 68 to 73 years-old were involved. Plaque was obtained from an area of 10 mm 2 on the dorsal surface at the anterior two-thirds of the tongue. Total numbers of colony-forming units (CFU) were determined and the predominant bacteria were isolated for identification. Significantly more bacteria (p<0.05) were recovered after anaerobic incubation (mean; 2.0 x 10 7 CFU/mg) than after aerobic (mean; 1.1 x 10 7 CFU/mg) or microaerophilic (mean; 1.0 x 10 7 CFU/mg) incubation. Out of 210 predominant strains isolated, 33% were obligate anaerobes and 66% were facultatively anaerobic. Veillonella (8% of total isolates) and Streptococcus (35% of total isolates) were the most major genera identified among obligate and facultative anaerobes, respectively. Actinomyces strains represented 27% of total isolates. Gram negative anaerobic rods and asaccharolytic Eubacterium were also detected, comprising 14% and 3% of total isolates, respectively. The bacterial composition of the tongue was somewhat similar to that of saliva and denture plaque reported in geriatric edentulous persons, suggesting that tongue plaque in geriatric edentulous persons who wear dentures may function as a major bacterial reservoir. Keywords: tongue plaque, microflora, edentulous, geriatric.

Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions.

To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H. pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37 degrees C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days. Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation. By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid. However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation. These results suggest that H. pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H. pylori cultured for a prolonged time under anaerobic conditions.

Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions

To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H. pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37°C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days. Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation. By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid. However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation. These results suggest that H. pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H. pylori cultured for a prolonged time under anaerobic conditions.

Comparison of the effects of anaerobic and microaerophilic incubation on resistance of Helicobacter pylori to metronidazole

To assess the influence of incubation conditions on the resistance of Helicobacter pylori this study compared the effect of micro-aerophilic and anaerobic incubation followed by micro-aerophilic incubation on the measurement of metronidazole resistance of 102 H. pylori isolates, by both disk diffusion and Epsilometer (E)-tests. Anaerobic incubation for 24 h before micro-aerophilic incubation for 48 h consistently increased metronidazole activity in both assay methods. Although statistically significant, this was microbiologically less significant, as only 4 of 102 isolates gave discrepant readings (all four were resistant in micro-aerophilic conditions but susceptible in anaerobic/micro-aerophilic conditions). In all four cases variation was by a few millimeters in zone size (i.e., all were close to the cut-off point). There was 100% agreement between disk diffusion and E-test results. Of 104 observations (52 duplicate assays: 13 strains, two atmospheric conditions, two methods of determining resistance) there was 100% intra-observer and inter-observer agreement with regard to susceptibility and resistance status for both E-test and disk diffusion methods. Anaerobic incubation followed by micro-aerophilic incubation had little effect on the estimation of prevalence of metronidazole resistance and seemed to add little, if any, significant advantage over micro-aerophilic incubation alone.