Microaerobic Atmosphere Research Articles

Enhancement of growth media for extreme iron limitation in Escherichia coli.

Iron is an essential nutrient for microbial growth and bacteria have evolved numerous routes to solubilize and scavenge this biometal, which is often present at very low concentrations in host tissue. We recently used a MOPS-based medium to induce iron limitation in Escherichia coli K-12 during the characterization of novel siderophore-conjugated antibiotics. In this study we confirm that growth media derived from commercially available M9 salts are unsuitable for studies of iron-limited growth, probably through the contamination of the sodium phosphate buffer components with over 100 µM iron. In contrast, MOPS-based media that are treated with metal-binding Chelex resin allow the free iron concentration to be reduced to growth-limiting levels. Despite these measures a small amount of E. coli growth is still observed in these iron-depleted media. By growing E. coli in conditions that theoretically increase the demand for iron-dependent enzymes, namely by replacing the glucose carbon source for acetate and by switching to a microaerobic atmosphere, we can reduce background growth even further. Finally, we demonstrate that by adding an exogeneous siderophore to the growth media which is poorly used by E. coli, we can completely prevent growth, perhaps mimicking the situation in host tissue. In conclusion, this short study provides practical experimental insight into low iron media and how to augment the growth conditions of E. coli for extreme iron-limited growth.

Investigating the Significance of Non-jejuni/coli Campylobacter Strains in Patients with Diarrhea.

Campylobacter is one of the most commonly reported foodborne bacteria worldwide. Although Campylobacter jejuni and Campylobacter coli have been reported to be responsible for the great majority of campylobacteriosis, the burden of infections by species other than C. jejuni and C. coli have been increasing as a result of a transition to diagnostic test methods that enable the isolation of emerging species. The aim of the present study was to recover C. jejuni, C. coli, and emerging species from the stool samples of 500 patients with gastroenteritis and 100 healthy subjects via the use of a filtration method and culture techniques using Butzler agar and mCCDA under a microaerobic or hydrogen-enriched atmosphere, identify the species by multiplex PCR methods and assess the significance of emerging species in enteric diseases. Thirty-one (6.2%) Campylobacter spp. were isolated from the stool samples of diarrheic patients but none from healthy individuals. Of 31 isolates, 21 (67.8%), nine (29%), and one (3.2%) were identified as C. jejuni, C. coli, and Campylobacter concisus by multiplex PCR, respectively. The filtration method was superior to the culture technique using mCCDA under a microaerobic atmosphere. C. concisus was evaluated as the etiology of gastroenteritis as a result of laboratory and clinical evaluations. The present study was the first to indicate that emerging Campylobacter species are rarely detected and C. concisus is linked to acute gastroenteritis in Turkey where additional studies are warranted to clarify the significance of emerging species in gastroenteritis.

CAMPYAIR, a New Selective, Differential Medium for Campylobacter spp. Isolation without the Need for Microaerobic Atmosphere.

Campylobacter spp. are considered the most frequent bacterial cause of acute gastroenteritis worldwide. Although the diarrhea produced by these bacteria is self-limiting, the pathogen has been associated with severe long-term sequelae following acute signs and symptoms of the illness. However, research on Campylobacter is hampered by costs and technical requirements for isolating and culturing the bacterium, especially in low and middle-income countries. Therefore, attempts have been made to simplify these culture methods and to reduce costs associated with conducting research on Campylobacter. Recently, a liquid medium which allows selective enrichment of Campylobacter using aerobic incubation has been described. However, a solid medium is also needed for the isolation of pure colonies, enumeration of bacterial populations, and other studies on the pathogen. Therefore, a new medium (CAMPYAIR) was developed, based on the formulation of the liquid medium. CAMPYAIR is a solid chromogenic medium that supports the growth of Campylobacter isolates within 48 h of incubation in aerobic atmospheres. Moreover, CAMPYAIR contains antibiotic supplements with an enhanced ability to recover Campylobacter from environmental samples that may also contain non-campylobacter bacteria. The addition of the indicator 2,3,5-triphenyltetrazolium (TTC) to the medium differentiates Campylobacter from other bacteria growing on the media. The findings from studies on CAMPYAIR suggest that the utilization of the new selective, differential medium could help to reduce the costs, equipment, and technical training required for Campylobacter isolation from clinical and environmental samples.

Bacillus subtilis PS-216 Antagonistic Activities against Campylobacter jejuni NCTC 11168 Are Modulated by Temperature, Oxygen, and Growth Medium.

As the incidence of Campylobacter jejuni and campylobacteriosis grows, so does the need for a better understanding and control of this pathogen. We studied the interactions of C. jejuni NCTC 11168 and a potential probiotic, Bacillus subtilis PS-216, in cocultures at different starting ratios and temperatures (20 °C, 37 °C, 42 °C), under different atmospheres (aerobic, microaerobic), and in different growth media (Mueller–Hinton, chicken litter medium, chicken intestinal-content medium). Under microaerobic conditions, B. subtilis effectively inhibited the growth of C. jejuni at 42 °C (log reduction, 4.19), even when C. jejuni counts surpassed B. subtilis by 1000-fold in the starting inoculum. This inhibition was weaker at 37 °C (log reduction, 1.63), while no impact on CFUs was noted at 20 °C, which is a temperature nonpermissive of C. jejuni growth. Under aerobic conditions, B. subtilis supported C. jejuni survival. B. subtilis PS-216 inhibited the growth of C. jejuni in sterile chicken litter (4.07 log reduction) and in sterile intestinal content (2.26 log reduction). In nonsterile intestinal content, B. subtilis PS-216 was able to grow, to a lesser extent, compared to Mueller–Hinton media, still showing potential as a chicken probiotic that could be integrated into the chicken intestinal microbiota. This study showed the strong influence of environmental parameters on the variability of C. jejuni and B. subtilis interactions. Furthermore, B. subtilis PS-216 antagonism was strongest against C. jejuni NCTC 11168 under conditions that might represent conditions in the chicken environment (42 °C, microaerobic atmosphere, chicken litter medium).

Assessment of biofilm formation by Campylobacter spp. isolates mimicking poultry slaughterhouse conditions

This research aimed to assess the biofilm formation ability of Campylobacter strains under temperature and oxygen stress conditions, similar to those found in the industrial environment, to explain the persistence of this pathogen on the poultry slaughter line. A collection of C. jejuni and C. coli isolates (n = 143) obtained from poultry samples (cecal content and neck skin), collected at slaughterhouse level, from diverse flocks, on different working days, was genotyped by flaA-restriction fragment length polymorphism (RFLP) typing method. A clustering analysis resulted in the assignment of 10 main clusters, from which 15 strains with different flaA-RFLP genotypes were selected for the assessment of biofilm formation ability and antimicrobial susceptibility. Biofilm assays, performed by crystal violet staining method, were conducted with the goal of mimicking some conditions present at the slaughterhouse environment, based on temperature, atmosphere, and contamination levels. Results indicated that many C. jejuni strains with similar flaA-RFLP profiles were present at the slaughterhouse on different processing days. All the strains tested (n = 15) were multidrug-resistant except for one. Biofilm formation ability was strain-dependent, and it appeared to have been affected by inoculum concentration, temperature, and tolerance to oxygen levels. At 10°C, adherence levels were significantly lower than at 42°C. Under microaerobic and aerobic atmospheres, at 42°C, 3 strains (C. jejuni 46E, C. jejuni 61C, and C. coli 65B) stood out, exhibiting significant levels of biofilm formation. C. jejuni strains 46E and 61C were inserted in clusters with evidence of persistence at the slaughterhouse for a long period of time. This study demonstrated that Campylobacter strains from broilers are capable of forming biofilms under conditions resembling the slaughterhouse environment. These results should be seen as a cue to improve the programs of hygiene implemented, particularly in those zones that can promote biofilm formation.

Natural Transformation as a Mechanism of Horizontal Gene Transfer in Aliarcobacter butzleri.

Aliarcobacter butzleri is an emergent enteropathogen, showing high genetic diversity, which likely contributes to its adaptive capacity to different environments. Whether natural transformation can be a mechanism that generates genetic diversity in A. butzleri is still unknown. In the present study, we aimed to establish if A. butzleri is naturally competent for transformation and to investigate the factors influencing this process. Two different transformation procedures were tested using exogenous and isogenic DNA containing antibiotic resistance markers, and different external conditions influencing the process were evaluated. The highest number of transformable A. butzleri strains were obtained with the agar transformation method when compared to the biphasic system (65% versus 47%). A. butzleri was able to uptake isogenic chromosomal DNA at different growth phases, and the competence state was maintained from the exponential to the stationary phases. Overall, the optimal conditions for transformation with the biphasic system were the use of 1 μg of isogenic DNA and incubation at 30 °C under a microaerobic atmosphere, resulting in a transformation frequency ~8 × 10−6 transformants/CFU. We also observed that A. butzleri favored the transformation with the genetic material of its own strain/species, with the DNA incorporation process occurring promptly after the addition of genomic material. In addition, we observed that A. butzleri strains could exchange genetic material in co-culture assays. The presence of homologs of well-known genes involved in the competence in the A. butzleri genome corroborates the natural competence of this species. In conclusion, our results show that A. butzleri is a naturally transformable species, suggesting that horizontal gene transfer mediated by natural transformation is one of the processes contributing to its genetic diversity. In addition, natural transformation can be used as a tool for genetic studies of this species.

Exploring the role of healthy dogs as hosts of enterohepatic Helicobacter species using cultivation-dependent and -independent approaches.

Enterohepatic Helicobacter (EHH) species have been increasingly associated with acute gastroenteritis, inflammatory bowel disease and hepatobiliary diseases in humans. However, their host range and transmission routes are poorly understood. Therefore, the aim of this study was to determine the presence of EHH in healthy dogs using both cultivation-dependent and -independent methods. Three hundred and ninety faecal samples from domestic dogs without gastrointestinal symptoms were analysed between June 2018 and July 2019 in Valdivia (South of Chile). Samples were inoculated on selective medium and in parallel were filtrated over an antibiotic-free blood agar. Both media were incubated in a microaerobic atmosphere at 37°C for 7days. Colonies were identified by PCR and phylogenetic analysis. A subset of 50 samples (half of them positive for EHH by cultivation and the remaining half negative) was analysed by PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) for direct detection. Cultivation method detected EHH in 15.4% (60/390) of the samples, being the most prevalent species H. canis (5.8%, 23/390) and H. canicola (5.1%, 20/390), followed by H. bilis (3.6%, 14/390) and 'H. winghamensis' (1.3%, 5/390). In contrast, PCR-DGGE method detected Helicobacter DNA in almost all (96%, 48/50) tested samples. On the other hand, the method used also allowed to isolate other Campylobacterales, in fact 44.3% (173/390) of the samples were positive for Campylobacter upsaliensis (43.3%, 169/390) followed by C. jejuni (2.0%, 8/390). Moreover, two strains that presented Campylobacter-like morphology were finally identified as Anaerobiospirillum succiniciproducens. Our results indicate that healthy domestic dogs commonly carry EHH and other Campylobacter species. However, further studies are needed to determine whether and how these Helicobacter and Campylobacter species can be transmitted to humans.

Stress resistance associated with multi-host transmission and enhanced biofilm formation at 42 °C among hyper-aerotolerant generalist Campylobacter jejuni

One of the emerging conundrums of Campylobacter food-borne illness is the bacterial ability to survive stressful environmental conditions. We evaluated the heterogeneity among 90 C. jejuni and 21 C. coli isolates from different sources in Egypt with respect to biofilm formation capabilities (under microaerobic and aerobic atmosphere) and resistance to a range of stressors encountered along the food chain (aerobic stress, refrigeration, freeze-thaw, heat, peracetic acid, and osmotic stress). High prevalence (63%) of hyper-aerotolerant (HAT) isolates was observed, exhibiting also a significantly high tolerance to heat, osmotic stress, refrigeration, and freeze-thaw stress, coupled with high biofilm formation ability which was clearly enhanced under aerobic conditions, suggesting a potential link between stress adaptation and biofilm formation. Most HAT multi-stress resistant and strong biofilm producing C. jejuni isolates belonged to host generalist clonal complexes (ST-21, ST-45, ST-48 and ST-206). These findings highlight the potential role of oxidative stress response systems in providing cross-protection (resistance to other multiple stress conditions) and enhancing biofilm formation in Campylobacter and suggest that selective pressures encountered in hostile environments have shaped the epidemiology of C. jejuni in Egypt by selecting the transmission of highly adapted isolates, thus promoting the colonization of multiple host species by important disease-causing lineages.

Microenvironment-Controlled Micropatterned Microfluidic Model (MMMM) for Biomimetic In Situ Studies.

Attachment of trophozoites to the intestine is an indispensable step for Giardia's survival and pathogenicity in almost 280 million infections worldwide each year. However, the analysis of the attachment mechanism is difficult due to the lack of methods that can create a favorable microaerobic atmosphere. Herein, we developed an osmotic-pressure, pH, excretion, nutrition, gas, ionic-strength, flow-rate, and temperature microenvironment-controlled micropatterned microfluidic model to simulate the in vivo microenvironment to study in situ the stress applied to Giardia in the intestinal tract. We designed three nonbiological surfaces with stagger arrangement manners and integrated them with a resistance microfluidic network to split Giardia-attaching forces ingeniously and developed the term "attaching contribution rate" (ACR) to describe their corresponding contributions. Our study shows that the total attaching force measured is 49.58 Pa, with three components being 22.66 Pa (suction force), 12.52 Pa (clutching force), and 14.4 Pa (combined electrostatic and van der Waals force), respectively, with ACRs being 46%, 25%, and 29%, respectively. By decomposing the attaching force and analyzing each force component and their structure and composition basis, whole profiles of the attachment mechanisms were revealed. Our method enables the analysis of the surface attachment mechanisms and their ACRs for Giardia.

In vitro immunomodulatory effect of Bifidobacterium bifidum and Lactobacillus reuteri cell free extracts

Recent studies have shown that alterations of the immune response in the gastrointestinal mucosa are key components of the mechanism of the probiotic action of beneficial bacteria. Most of the beneficial effects of probiotics are due to the action of their structural components and metabolites. Macrophages are first-line defense cells of the immune system, which not only participate in the detection, phagocytosis and destruction of harmful microorganisms, but also determine the nature of the subsequent immune response by presenting antigens to T-cells and initiating inflammation by releasing cytokines. We researched the effect of two types of cell-free extracts (CFEs) containing probiotic derivatives (structural components and metabolites of bacteria) Bifidobacterium bifidum 1 (BbCFE) and Lactobacillus reuteri DSM 17938 (LrCFE) on the activity of mouse peritoneal macrophages and on the ability of peripheral human blood mononuclear cells to produce cytokines. CFEs were obtained by culturing probiotics in their own disintegrates and then removing cells and cell debris by centrifugation and filtration. Peritoneal macrophages were isolated from mice. Some of them were infected in vitro by Salmonella thyphimurium. Uninfected and infected macrophages were incubated in culture medium containing (30% vol) or not containing CFEs at 37 °С in a microaerobic atmosphere (5% СО2) for 18 hours. After incubation, peritoneal macrophages were lysed. The obtained suspensions were centrifuged and supernatants were carefully collected. Macrophages activity was assessed by the nitrites level, superoxide dismutase (SOD), lactate dehydrogenase (LDH) activity and antiinflammatory cytokines levels in supernatants using spectrophotometric method. Peripheral mononuclear cells were isolated from the blood of healthy volunteers. The ability of peripheral mononuclear blood cells to produce antiinflammatory cytokines was evaluated after cell stimulation with lipopolysaccharide (LPS) and incubation with or without CFEs. Cytokine levels in supernatants were determined using enzyme-linked immunosorbent assay (ELISA). After infection with S. thyphimurium in macrophages, nitrite levels increased 5.5-fold, SOD activity 4.8-fold, and LDH 2-fold. Both studied CFEs exerted a similar effect on the macrophages’ activity. Addition of BbCFE to the incubation medium of infected macrophages resulted in a 4-fold decrease in nitrite levels, and the addition of LrCFE was accompanied by a decrease in nitrite levels to levels in intact cells. Under the influence of both CFEs, the activity of SOD and LDH was significantly reduced and did not differ significantly from the activity of these enzymes in intact cells. BbCFE and LrCFE did not have a significant effect on nitrite levels, SOD and LDH activity in intact macrophages. Under the influence of BbCFE, there was a 2-fold decrease in the production of TNF, a 2-fold increase in IL10 production, and a 30% increase in IL6 production by mononuclear cells. LrCFE caused a decrease in TNF production by 26.7% and IL6 by 36%, and IL10 by 1.9 times. Thus, the studied CFEs normalized the nitrite levels in peritoneal macrophages infected with S. thyphymurium and infection-induced activation of SOD and LDH enzymes. This demonstrates their ability to modulate oxidative processes in macrophages. In addition, under the influence of the investigated CFEs, there was a decrease in the production of pro-inflammatory cytokines (TNFα and IL-6) and increased production of anti-inflammatory cytokine (IL-10) by human peripheral mononuclear cells. The results of the study indicate the ability of CFEs by influencing the functions of innate immunity cells to restrict the inflammatory response and oxidative stress. Based on this, CFEs can be considered as promising agents for the treatment of inflammatory diseases.

Rapid Identification of Campylobacter Strains Cultured Under Aerobic Incubation Using Hyperspectral Microscope Imaging

Campylobacter is an organism of concern for food safety and is one of the leading causes of foodborne bacterial gastroenteritis. This pathogen can be found in broiler chickens, and the level of allowable contamination of processed poultry is regulated by federal agency guidelines. Traditional methods for detecting and isolating this pathogen from broiler chicken carcasses require time, expensive reagents, and artificially generated microaerophilic atmospheres. An aerobic medium that simplifies the procedure and reduces the expense of culturing Campylobacter has been recently described, and Campylobacter can be grown in this medium in containers that are incubated aerobically. Hyperspectral microscopic imaging (HMI) has been proposed for early and rapid detection of pathogens at the cellular level. The objective of the present study was to utilize HMI to compare differences between Campylobacter cultures grown under artificially produced microaerobic atmospheres and cultures grown in aerobic medium. Hyperspectral microscopic images of three Campylobacter strains were collected cultures grown for 48 h microaerophilically and for 24 and 48 h aerobically, and a quadratic discriminant analysis was used to characterize the bacterial variability. Microaerobically cultured bacteria were detected with 98.7% accuracy, whereas detection accuracy of cultures grown in the novel medium was slightly reduced (-4.8 and -3.2% for 24 and 48 h, respectfully). The Mahalanobis distance multivariate metric was applied to quantify strain variability under all three treatment conditions. Across all strains and treatments, little cluster variation was present (4.22 to 4.42), indicating the consistency of the images collected from the three strains. The classification and spectral consistency was similar for cultures incubated in the aerobic medium for 24 h and cultures grown for 48 h under microaerobic conditions.

Mucoid and coccoid Helicobacter pylori with fast growth and antibiotic resistance.

In this study, one Helicobacter pylori isolate, from gastric biopsy of a dyspeptic patient that turned into mucoid-coccoid (MC) form upon consecutive subcultures, was identified. The culturability, antibiotic resistance, and lipid contents of MC were compared with those of non-mucoid (NM) spiral Hpylori. Mucoid-coccoid and NM Hpylori were subcultured on Brucella blood agar (BBA) and incubated under aerobic and microaerobic atmospheres at 37°C. Cultures were examined for colony characteristics and bacterial morphology after 1-3days. The isolates were identified by biochemical tests and detection of Hpylori-16S rDNA. Antibiogram was performed with currently used antibiotics for Hpylori eradication. Cellular lipid contents were extracted and analyzed by gas chromatography. Compared with pin-pointed and glistening colonies of NM Hpylori that appeared under microaerobic conditions, MC Hpylori grew well in consecutive subcultures under aerobic and microaerobic atmospheres and produced white patches of mucoid colonies. MC exhibited coccoid and NM spiral morphology. Both isolates were catalase, oxidase, and urease positive and contained 16S rDNA. Compared with NM that was susceptible to almost all the antibiotics, MC was resistant to all the antibiotics. Lipid analyses showed high frequency of unsaturated fatty acids and cholesterol in MC. Coccoid forms with high fatty acid and cholesterol contents that show resistance to antibiotics might resist against other stressful conditions such as gastric acidity and immune response. Moreover, mucoid property may enhance resistance of coccoids to stresses. With mucoid-coccoid lifestyle, Hpylori may establish a chronic infection refractory to antimicrobial therapy.

Consumption of N2O and other N-cycle intermediates by Gemmatimonas aurantiaca strain T-27.

Bacteria affiliated with the phylum Gemmatimonadetes are found in high abundance in many terrestrial and aquatic environments, yet little is known about their metabolic capabilities. Difficulty in their cultivation has prompted interest in identifying better growth conditions for metabolic studies, especially related to their ability to reduce N2O, a potent greenhouse gas. T-27 Gemmatimonas aurantiaca is one of few cultivated strains of Gemmatimonadetes available for physiological studies. Our objective was to test this organism's ability to use nitrite, nitrate, and N2O, and mineral forms of assimilable NH4+ at concentrations not typically used in tests for compound utilization. Cultures incubated under anaerobic conditions with nitrate, nitrite or N2O failed to grow or show depletion of these substrates. Nitrate and nitrite (1 mM) were not used even when cells were grown aerobically with the O2 allowed to deplete first. N2O reduction only commenced in the presence of O2 and continued to be depleted when refed to the culture under anaerobic, microaerobic and aerobic atmospheres. Carbon mineralization was coupled to the electron-accepting processes, with higher reducing equivalents needed for N2O utilization under aerobic atmospheres. N2O was reduced to N2 in the presence of 20% O2, however the rate of this reaction is reduced in the presence of high O2 concentration. This study demonstrated that G. aurantiaca T-27 possesses unique characteristics for assimilative and dissimilative N processes with new implications for cultivation strategies to better assess the metabolic abilities of Gemmatimonadetes.

Comparison of media and growth conditions for culturing enterohepatic Helicobacter species.

This research aims to compare the culturing conditions for enterohepatic Helicobacter, evaluating culture media, incubation atmosphere and susceptibility to antimicrobials used to generate selective conditions. Four common media for the closely related genus Campylobacter (Columbia, Bolton, Brucella and CCDA agar), as well as the need for hydrogen in the microaerobic incubation atmosphere, were evaluated. Serial dilutions of 13 strains belonging to six species (H. apodemus, H. bilis, H. canicola, H. canis, H. equorum and Helicobacter sp.) were inoculated in each media and incubated at 37°C for 48 to 96h using CampyGen (OXOID) and gaseous exchange (including hydrogen) in parallel. Columbia or Brucella agars were the most appropriate for culturing EHH (P<0·05). However, there was no significant difference between the atmospheres evaluated (P=0·13). In addition, minimal inhibitory concentration for six antibiotics showed that all isolates were resistant to trimethoprim, whereas for the rest of the antibiotics (cephalothin, cefoperazone, cefsulodin, teicoplanin and vancomycin) the inhibition range was between 8 and 64μgml- 1 . Our findings suggest that Columbia or Brucella media, regardless of the use of hydrogen, can be used for the EHH isolation. In addition, the concentration of antibiotics included in commercial campylobacteria supplements is suitable for EHH species recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterohepatic Helicobacter (EHH) infections have been associated with several diseases in humans such as acute gastroenteritis, inflammatory bowel disease and hepatobiliary diseases. Although they are frequently detected in clinical samples by molecular methods, only occasionally they are isolated using culture conditions described for the taxonomic related pathogen Campylobacter sp. This is because the optimal conditions for the isolation of EHH have not yet been described, which results in an underestimation of the prevalence and clinical importance of these emerging pathogens. Therefore, this study provides insight for culturing EHH species.

Isolation of emerging Campylobacter species in working farm dogs and their frozen home-killed raw meat diets

We applied 7 culture methods to 50 working farm dog fecal samples and 6 methods to 50 frozen home-killed raw meat diet samples to optimize recovery of a wide range of Campylobacter spp. Culture methods combined filtration, enrichment broths, and agars at 37°C and 42°C in conventional and hydrogen-enriched microaerobic atmospheres. Overall, a prevalence of 62% (31 of 50) and 6% (3 of 50) was detected in dog and meat samples, respectively, based on Campylobacter genus PCR. A total of 356 Campylobacter spp. isolates were recovered from dogs, with successful isolation by individual methods ranging from 2 to 25 dogs. The species detected most commonly were C. upsaliensis and C. jejuni, and less commonly C. coli and C. lari. Species isolated that are rarely reported from dogs included C. rectus, C. lari subsp. concheus, C. volucris, and Helicobacter winghamensis. Six isolates from dogs positive by Campylobacter genus PCR were confirmed, using 16S rRNA sequencing, as Arcobacter cryaerophilus (1) and Arcobacter butzleri (5). C. jejuni multi-locus sequence typing results revealed a diversity of sequence types in working dogs, with several uncommonly reported from other C. jejuni sources in New Zealand. Overall, 20 isolates from 3 meat samples were positive by Campylobacter genus PCR; 1 meat sample was positive for C. jejuni, 1 for C. rectus, and 1 isolate was subsequently identified as A. butzleri. The method using Campylobacter enrichment broth in a hydrogen-enriched environment on nonselective agar resulted in significantly reduced recovery of Campylobacter spp. from both sample types.

2072. Multicenter Evaluation of the Etest vs. Agar Dilution for Susceptibility Testing of Helicobacter pylori

Background Helicobacter pylori is associated with peptic ulcer disease and gastric malignancy. Antimicrobial susceptibility testing (AST) is often requested for patients who fail eradication therapy. The CLSI reference method, agar dilution (AD), is not performed in most laboratories and maintaining organism viability during transit to a reference laboratory is difficult. We assessed the performance of the Etest (bioMerieux) as a method for H. pylori AST in comparison to AD.MethodsFrozen stocks of 82 H. pylori isolates with AD results previously reported by Mayo Clinic were prepared from the same plate for distribution to participating laboratories. Etest was performed at ARUP Laboratories and Cleveland Clinic (CC). For Etest, isolates were incubated for 72 hours in a microaerobic atmosphere. Aged Mueller–Hinton agar with 5% sheep blood plates were inoculated with a three McFarland suspension prepared in brain heart infusion broth. Etest strips were applied and MICs read after 72 hours of microaerobic incubation. Results were interpreted by applying CLSI and EUCAST breakpoints. Categorical agreement (CA), very major, major and minor errors (VME, ME, and mE) were determined for Etest using AD as the reference method. Isolates with errors were repeat tested in duplicate by Etest to determine the final results summarized below.ResultsFor clarithromycin, 65% of isolates were resistant (R) by AD; Etest results at each laboratory showed 97.5% CA (1 mE and 1 ME). For tetracycline, only 2.5% of isolates were R by AD; a single VME occurred at both ARUP and CC (98.8% CA) with the same isolate. The AD dilutions tested for amoxicillin prevented interpretation with EUCAST breakpoints. With one exception, amoxicillin Etest results were susceptible (S, ≤0.125 mg/L) at both laboratories (98.8% of MICs ± one dilution). Applying levofloxacin EUCAST breakpoints (S, ≤1 mg/L) to interpret ciprofloxacin results, 57.8% of isolates were R by AD. ARUP CA was 97.5% (1 ME, 1 VME) and CC CA was 96.3% (1 ME, 2 VMEs).ConclusionClarithromycin, tetracycline, and ciprofloxacin Etest results for H. pylori showed acceptable CA (>95%) at both testing sites compared with the AD reference method. The comparative ease of performance and reproducibility of the Etest may help standardize it as an AST method for H. pylori.Disclosures S. S. Richter, bioMerieux: Grant Investigator, Research grant; BD Diagnostics: Grant Investigator, Research grant; Roche: Grant Investigator, Research grant; Hologic: Grant Investigator, Research grant; Diasorin: Grant Investigator, Research grant; Accelerate: Grant Investigator, Research grant; Biofire: Grant Investigator, Research grant.

A Potential New Human Pathogen Belonging to Helicobacter Genus, Identified in a Bloodstream Infection.

We isolated from aerobic and anaerobic blood culture bottles from a febrile patient, a Helicobacter-like Gram negative, rod-shaped bacterium that MALDI-TOF MS failed to identify. Blood agar cultures incubated in a microaerobic atmosphere revealed a motile Gram negative rod, which was oxidase, catalase, nitrate reductase, esterase, and alkaline phosphatase positive. It grew at 42°C with no detectable urease activity. Antimicrobial susceptibility testing showed that the organism was susceptible to beta-lactams, gentamicin, erythromycin, and tetracycline but resistant to ciprofloxacin. Electronic microscopy analysis revealed a 3 × 0.5 μm curved rod bacterium harboring two sheathed amphitrichous flagella. Whole genome sequencing revealed a genome 1,708,265 base-pairs long with a GC content of 37.80% and a total of 1,697 coding sequences. The genomic analyses using the nucleotide sequences of the 16S rRNA gene, hsp60 and gyrB genes, as well as the GyrA protein sequence, and the results of Average Nucleotide Identity and in silico DNA-DNA hybridization suggest evidence for a novel Helicobacter species close to Helicobacter equorum and belonging to the group of enterohepatic Helicobacter species. As soon as the particular peptide mass fingerprint of this pathogen is added to the spectral databases, MALDI-TOF MS technology will improve its identification from clinical specimens, especially in case of “sterile infection”. We propose to associate the present strain with the Latin name of the place of isolation; Caesarodunum (Tours, France) and suggest “Helicobacter caesarodunensis” for further description of this new bacterium.

Measurement of Giardia lamblia adhesion force using an integrated microfluidic assay.

The mechanisms how Giardias attach to the intestinal epithelium remain unclear. None of the methods currently being used to measure the attachment force could provide a continuous nutrition supply and a micro-aerobic atmosphere to the Giardia. Besides, they are all labor-intensive. In the present research, a microfluidic method based on electric circuit analogy was developed. The input fluid flowed through the inlet channel with different lengths and was distributed in four assay chambers. Shear force gradients were generated in chambers, too. This allowed an easy control of fluids and the shear forces. Most importantly, the shear stress large enough to detach Giardia could be generated in laminar flow regime. Moreover, analysis could be accomplished in one single test. By applying inlet flow rates of 30, 60, and 120μLml-1, shear force gradients ranging from 19.47 to 60.50Pa were generated. The adhesion forces of trophozoites were analyzed and the EC50 of the force that caused 50% trophozoites detachment was calculated as 36.60Pa. This paper presents a novel method for measurement of Giardia adhesion force. Graphical Abstract Measurement of Giardia adhesion force. Various of flow rates were applied to generate different shear forces and Giardia trophozoites remaining attached were counted (a-c). The percentages of attachment vs shear stress were plotted and the EC50 of adhesion force was calculated (d).

Campylobacter jejuni Isolation/Enumeration from Environmental Samples.

Currently, there is no universally accepted standard media or method for the recovery of Campylobacter species. This is likely due to the ubiquity of the organism in nature, the complex sample matrices from which the organism is often recovered, as well as the fragile/viable-but nonculturable state the organism assumes in response to stress. The use of a sterile filter placed upon a nonselective Brucella Agar Blood Plate (BAB), followed by incubation at 37 °C in a hydrogen-containing atmosphere (Campycheck), is one method to recover stressed and emerging Campylobacter spp. from complex environmental matrices; however, this technique does not currently allow for the enumeration of the recovered organisms. Enumeration is performed using serial dilutions of sample homogenate plated onto modified Campy-Cefex media followed by incubation at either 37 °C or 42 °C in a microaerobic atmosphere.

Isolation of Campylobacter spp. from Client-Owned Dogs and Cats, and Retail Raw Meat Pet Food in the Manawatu, New Zealand.

Campylobacter causes acute gastroenteritis in people worldwide and is frequently isolated from food, animals and the environment. The disease is predominately food-borne but many routes of transmission and sources of infection have been described, including contact with pets. The prevalence of Campylobacter spp. in dogs and cats varies widely, and data on New Zealand pets are limited. This study aimed to investigate the prevalence of Campylobacter spp. in dogs, cats and retail raw meat pet food products in New Zealand and to characterize Campylobacter jejuni isolates using multilocus sequence typing (MLST). Ninety dogs and 110 cats examined at the Massey University Veterinary Teaching Hospital for elective procedures, and fifty locally purchased retail raw meat pet diets were sampled. Two culture protocols combining Bolton broth enrichment and mCCDA and CAT agars in a microaerobic atmosphere at 42°C and 37°C with species identification using PCR were performed. The prevalence of Campylobacter spp., C.jejuni, Campylobacter upsaliensis and Campylobacter helveticus was 36%, 13%, 23% and 1% in dogs and 16%, 5%, 5% and 7% in cats, respectively. One dog had Campylobacter lari confirmed, and three dogs and one cat had multiple Campylobacter spp. detected. Significantly more animals tested positive using CAT than mCCDA agar (P<0.001). Being neutered, vaccinated for Bordetella bronchiseptica, fed dry diets and brought in for neutering were protective factors for dogs, whereas attendance for dental treatment was a risk factor for cats. Campylobacter spp. were isolated from 28%, C.jejuni 22%, C.lari 6% and Campylobacter coli 6% of food samples. Six isolates positive by Campylobacter genus PCR were identified as Arcobacter butzleri. Poultry meat was more likely to be positive than non-poultry meat (P=0.006). Of the 13 C.jejuni pet isolates with full MLST profiles, eight were of different sequence types (ST) and all nine food isolates were of different STs.