Microaerobic Atmosphere Research Articles

The influence of dissolved oxygen level and medium on biofilm formation by Campylobacter jejuni

Campylobacter jejuni survival in aerobic environments has been suggested to be mediated by biofilm formation. Biofilm formation by eight C. jejuni strains under both aerobic and microaerobic conditions in different broths (Mueller-Hinton (MH), Bolton and Brucella) was quantified. The dissolved oxygen (DO) content of the broths under both incubation atmospheres was determined. Biofilm formation for all strains was highest in MH broth under both incubation atmospheres. Four strains had lower biofilm formation in MH under aerobic as compared to microaerobic incubation, while biofilm formation by the other four strains did not differ under the 2 atm. Two strains had higher biofilm formation under aerobic as compared to microaerobic atmospheres in Bolton broth. Biofilm formation by all other strains in Bolton, and all strains in Brucella broth, did not differ under the 2 atm. Under aerobic incubation DO levels in MH > Brucella > Bolton broth. Under microaerobic conditions levels in MH = Brucella > Bolton broth. Levels of DO in MH and Brucella broth were lower under microaerobic conditions but those of Bolton did not differ under the 2 atm. Experimental conditions and especially the DO of broth media confound previous conclusions drawn about aerobic biofilm formation by C. jejuni.

Epithelial Coculture and l-Lactate Promote Growth of Helicobacter cinaedi under H2-Free Aerobic Conditions.

H. cinaedi is an increasingly recognized pathogen in people with compromised immune systems. Atypical among other members of its bacterial class, H. cinaedi has been associated with infections of diverse anatomic sites. Growing H. cineadi in the laboratory is quite difficult, due in large part to the need for a specialized atmosphere. The suboptimal growth of H. cinaedi is an obstacle to clinical diagnosis, and it also limits investigation into the organism's biology. The current work shows that H. cinaedi has more flexible atmospheric requirements in the presence of host cells and a common host-derived molecule. This nutritional interplay raises new questions about how the organism behaves during human infections and provides insights for how to optimize its laboratory cultivation.

Effect of dietary sugars on dual-species biofilms of Streptococcus mutans and Streptococcus sobrinus – a pilot study

Abstract Introduction Frequent consumption of sugars and the presence of Streptococcus mutans and Streptococcus sobrinus are correlated with higher caries experience. Objective The aim of this pilot study was to elucidate the effect of different fermentable carbohydrates on biomass formation and acidogenicity of S. mutans and S. sobrinus biofilms. Material and method Single and dual-species biofilms of S. mutans ATCC 25175 and S. sobrinus ATCC 27607 were grown at the bottom of microtiter plates at equal concentrations for 24 h at 37 °C under micro-aerobic atmosphere. Carbohydrates were added at 2% concentration: maltose, sucrose, glucose and lactose. BHI Broth (0.2% glucose) was used as negative control. Acidogenicity was assessed by measuring the pH of spent culture medium after 24 h, immediately after refreshing the culture medium and for the next 1 h and 2 h. Crystal violet staining was used as an indicator of the total attached biofilm biomass after 24 h incubation. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. Significance level was set at 5%. Result All carbohydrates resulted in higher biomass formation in single- and dual-species biofilms when compared to the control group. Sucrose, lactose and maltose showed higher acidogenicity than the control group in both single- and dual-species biofilms after 24 h. Conclusion These findings indicate that the type of biofilm (single- or dual-species) and the carbohydrate used may influence the amount of biomass formed and rate of pH reduction.

Defining the metabolic requirements for the growth and colonization capacity of Campylobacter jejuni.

During the last decade Campylobacter jejuni has been recognized as the leading cause of bacterial gastroenteritis worldwide. This facultative intracellular pathogen is a member of the Epsilonproteobacteria and requires microaerobic atmosphere and nutrient rich media for efficient proliferation in vitro. Its catabolic capacity is highly restricted in contrast to Salmonella Typhimurium and other enteropathogenic bacteria because several common pathways for carbohydrate utilization are either missing or incomplete. Despite these metabolic limitations, C. jejuni efficiently colonizes various animal hosts as a commensal intestinal inhabitant. Moreover, C. jejuni is tremendously successful in competing with the human intestinal microbiota; an infectious dose of few hundreds bacteria is sufficient to overcome the colonization resistance of humans and can lead to campylobacteriosis. Besides the importance and clear clinical manifestation of this disease, the pathogenesis mechanisms of C. jejuni infections are still poorly understood. In recent years comparative genome sequence, transcriptome and metabolome analyses as well as mutagenesis studies combined with animal infection models have provided a new understanding of how the specific metabolic capacity of C. jejuni drives its persistence in the intestinal habitat of various hosts. Furthermore, new insights into the metabolic requirements that support the intracellular survival of C. jejuni were obtained. Because C. jejuni harbors distinct properties in establishing an infection in comparison to pathogenic Enterobacteriaceae, it represents an excellent organism for elucidating new aspects of the dynamic interaction and metabolic cross talk between a bacterial pathogen, the microbiota and the host.

Isolation of amylolytic, xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO₂ or CO₂/H₂) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacillus subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce β-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed β-glucosidase, amylase, cellulase, and xylanase activities. Finally, B. subtilis strain ABGx produced xylanase and amylase.

Evaluation of fecal calprotectin in Campylobacter concisus and Campylobacter jejuni/coli gastroenteritis

Calprotectin (CP) is a calcium-binding cytosolic neutrophil protein and the concentration in feces reflects the migration of neutrophils into the gut lumen. Testing for fecal CP (f-CP) in patients with negative cultures for enteric pathogens is widely accepted as a useful screening tool for identifying patients who are most likely to benefit from endoscopy for suspected inflammatory bowel disease (IBD) with the assumption that a negative f-CP is compatible with a functional disorder. Campylobacter concisus has recently been reported to have a high incidence in the Danish population almost equal to Campylobacter jejuni and Campylobacter coli and has been reported to cause prolonged watery diarrhea. However, isolation of C. concisus from feces requires the filter method in a hydrogen-enriched microaerobic atmosphere, which is not commonly used in the laboratory, and the diagnosis may consequently be missed. The aim of this study was to evaluate the f-CP levels, as a marker for the intestinal inflammation in C. jejuni/coli- and C. concisus-infected patients. The authors found a high concentration of f-CP (median 631: IQR 221–1274) among 140 patients with C. jejuni/coli infection, whereas the f-CP level among 99 C. concisus-infected patients was significantly lower (median 53: IQR 20–169). The data correlate to the severe inflammatory gastroenteritis seen in patients infected with C. jejuni/coli, whereas C. concisus-infected patients have a much lower intestinal inflammation which could be compared with viral gastroenteritis. Nevertheless, clinicians should be aware of C. concisus infection, especially in patients with prolonged mild diarrhea, in the differential diagnosis to IBD.

Multidrug Efflux Systems in Helicobacter cinaedi.

Helicobacter cinaedi causes infections, such as bacteremia, diarrhea and cellulitis in mainly immunocompromised patients. This pathogen is often problematic to analyze, and insufficient information is available, because it grows slowly and poorly in subculture under a microaerobic atmosphere. The first-choice therapy to eradicate H. cinaedi is antimicrobial chemotherapy; however, its use is linked to the development of resistance. Although we need to understand the antimicrobial resistance mechanisms of H. cinaedi, unfortunately, sufficient genetic tools for H. cinaedi have not yet been developed. In July 2012, the complete sequence of H. cinaedi strain PAGU 611, isolated from a case of human bacteremia, was announced. This strain possesses multidrug efflux systems, intrinsic antimicrobial resistance mechanisms and typical mutations in gyrA and the 23S rRNA gene, which are involved in acquired resistance to fluoroquinolones and macrolides, respectively. Here, we compare the organization and properties of the efflux systems of H. cinaedi with the multidrug efflux systems identified in other bacteria.

Resistance rates of metronidazole and other antibacterials in Helicobacter pylori from previously untreated patients in Norway

The aim of the study was to describe the antimicrobial resistance rate of Helicobacter pylori isolated from previously untreated patients in Norway, including the application of two different methods for the determination of metronidazole susceptibility. Altogether 102 isolates obtained in 2008 and 2009 from previously untreated patients suspected of H. pylori related disease, were examined applying a standardized European study protocol. The activity of amoxicillin, tetracycline, clarithromycin, metronidazole, rifabutin and levofloxacin was recorded after an incubation period of 72-96 h in a microaerobic atmosphere. Strains resistant to metronidazole were re-examined for metronidazole resistance applying anaerobic conditions for the first 24 h. None of the isolates were resistant to amoxicillin or tetracycline, whereas 5, 9% were resistant to clarithromycin and 22, 5% resistant to metronidazole tested conventionally. Applying local standards the metronidazole resistance rate fell to 7, 8%, highlighting the importance of the methodology applied for metronidazole susceptibility testing.

Identification of Gallibacterium species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry evaluated by multilocus sequence analysis

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) whole-cell fingerprinting was used for characterization of 66 reference strains of Gallibacterium. The 4 recognised Gallibacterium species and Gallibacterium genomospecies 1 yielded reproducible and unique mass spectrum profiles, which were confirmed with Bruker Biotyper reference database version 3. The reproducibility of MALDI-TOF MS results were evaluated varying the age and storage of the cultures investigated. Reliable species identification was possible for up to 8 days of storage at 4 °C and less reliable if the bacteria were stored at room temperature (20 °C). However, if the strains were grown longer than 48 h at 37 °C under microaerobic atmosphere, poor identification results were obtained, due to changes in protein profile. The MALDI-TOF MS results of all 66 strains demonstrated 87.9% concordance with results based upon biochemical/physiological characterization. In addition, diversities outlined by MALDI-TOF MS were verified by sequencing the rpoB ( n = 43), 16S rRNA ( n = 28), infB ( n = 14), and recN ( n = 14) genes (multilocus sequence analysis, MLSA). In addition, discrepancies were observed between some of the genes sequenced. Results obtained demonstrated that MALDI-TOF MS fingerprinting represents a fast and reliable method for identification and differentiation of the 4 recognised Gallibacterium species and possible a fifth species Gallibacterium genomospecies 1, with applications in clinical diagnostics.

Profound differences in the transcriptome of Campylobacter jejuni grown in two different, widely used, microaerobic atmospheres

It was noted that quantitative and qualitative differences occurred between the growth of Campylobacter in microaerobic atmospheres provided by a gas replacement jar and that in a modular atmosphere controlled system cabinet, despite the fact that oxygen levels were comparable. Hydrogen was, however, only present in the replacement mixture (3%). Investigations were therefore carried out to examine any accompanying physiological or transcriptional differences. Growth curves and motility studies using Campylobacter jejuni HPC5 showed that cultures growing in the cabinet were impaired, but only in the early stages of growth compared to growth in the jar. However, transcriptome studies highlighted profound changes in the transcript profiles of exponential cultures grown in the cabinet compared to the jar, including genes indicative of oxidative stress. Genes involved in detoxification, synthesis and modification of macromolecules, probable prophage genes and genes associated with inhibition of natural transformation showed relative increases in expression in the cabinet. Conversely, genes that function in energy metabolism, chaperones, heat shock and motility were increased in the jar, which was indicative of balanced growth. This work highlights the need to carefully annotate the different methods of atmosphere generation in the description of experiments in microarray databases; the assessment of these experimental details is crucial to overcome difficulties in comparing transcriptomic studies of campylobacter cultures between different laboratories.

Simple and rapid detection of Campylobacter spp. in naturally contaminated chicken-meat samples by combination of a two-step enrichment method with an immunochromatographic assay

A simple and rapid method to detect Campylobacter spp. in chicken-meat samples was established. This method consisted of a combination of a two-step enrichment method with a commercially available immunochromatographic assay, named NH Immunochromato Campylobacter (NH IC Campy, Nippon Meat Packers, Ibaraki, Japan), which is able to detect Campylobacter antigen in an enrichment culture within 15 min. The enrichment method did not require much blood or a particular system of generating a microaerobic atmosphere, in contrast to the standard method of enriching Campylobacter spp. in chicken-meat samples. The sensitivity of a combination of the two-step enrichment method with NH IC Campy for detection of non- and freeze-stressed Campylobacter spp. in spiked chicken meat was determined using bacterial cells of Campylobacter jejuni and Campylobacter coli. The detection sensitivities for non-stressed C. jejuni and C. coli were found to range from 5.5 to 1.3 × 10 1 CFU per 25 g of chicken meat, and those for freeze-stressed C. jejuni and C. coli were found to range from 9.2 × 10 1 to 1.5 × 10 2 CFU per 25 g of chicken meat. When a total of 68 chicken-meat samples were tested, the combination method determined that 61 samples were positive for Campylobacter spp. This method was more sensitive than a bacterial culture test, which consists of standard enrichment culturing and plating onto selective agars. Because the combination could be conducted in approximately 48 h, from the beginning of the enrichment culture to final determination, it was more rapid than the bacterial culture test, which requires four to five days. Moreover, the combination was simple to perform. These results suggest that combining the two-step enrichment method with NH IC Campy is useful as a simple and rapid alternative to the conventional bacterial culture test for detecting Campylobacter spp. in naturally contaminated chicken meat samples.

Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells

The Helicobacter pylori protein CagA may undergo tyrosine phosphorylation following its entry into human gastric epithelial cells with downstream effects on signal transduction. Disruption of the gp130 receptor that modulates the balance of the SHP2/ERK and JAK/STAT pathways enhanced peptic ulceration and gastric cancer in gp130 knock-out mice. In this study, we evaluated the effect of translocated CagA in relation to its tyrosine phosphorylation status on the gp130-mediated signal switch between the SHP2/ERK and JAK/STAT3 pathways. We showed that in the presence of CagA, SHP2 was recruited to gp130. Phosphorylated CagA showed enhanced SHP2 binding activity and ERK1/2 phosphorylation, whereas unphosphorylated CagA showed preferential STAT3 activation. These findings indicate that the phosphorylation status of CagA affects the signal switch between the SHP2/ERK and JAK/STAT3 pathways through gp130, providing a novel mechanism to explain H. pylori signaling.

Heme oxygenase 2 of the cyanobacterium Synechocystis sp. PCC 6803 is induced under a microaerobic atmosphere and is required for microaerobic growth at high light intensity

Cyanobacteria, red algae, and cryptomonad algae utilize phycobilin chromophores that are attached to phycobiliproteins to harvest solar energy. Heme oxygenase (HO) in these organisms catalyzes the first step in phycobilin formation through the conversion of heme to biliverdin IXalpha, CO, and iron. The Synechocystis sp. PCC 6803 genome contains two open reading frames, ho1 (sll1184) and ho2 (sll1875), whose products have in vitro HO activity. We report that HO2, the protein encoded by ho2, was induced in the cells growing under a microaerobic atmosphere [0.2% (v/v) O(2)], whereas HO1 was constitutively expressed under both aerobic and microaerobic atmospheres. Light intensity did not have an effect on the expression of both the HOs. Cells, in which ho2 was disrupted, were unable to grow microaerobically at a light intensity of 40 micromol m(-2) s(-1), but did grow microaerobically at 10 micromol m(-2) s(-1) light intensity. These cells grew normally aerobically at both light intensities. Comparative analysis of complete cyanobacterial genomes revealed that possession of two HOs is common in cyanobacteria. In phylogenetic analysis of their amino acid sequences, cyanobacterial HO1 and HO2 homologs formed distinct clades. HO sequences of cyanobacteria that have only one isoform were most similar to HO1 sequences. We propose that HO2 might be the more ancient HO homolog that functioned under low O(2) tension, whereas the derived HO1 can better accommodate increased O(2) tension in the environment.

Factors affecting the recovery ofCampylobacterspp. from retail packs of raw, fresh chicken using ISO 10272-1:2006

This study sought to determine the most effective protocol for the detection of Campylobacter spp. in retail packs of fresh, raw chicken based on ISO 10272-1:2006. Three sample preparation protocols were studied; two based on excision and one combining excision with a rinse of the remaining sample. Enrichment cultures were incubated both in closed bottles and microaerobically, and sub-cultured at 24 and 48 h. Packs of chicken (110) were analysed and only two yielded no Campylobacter spp. Subculturing enrichment broths at 24 h gave the same prevalence as at 48 h, P > 0.4 but microaerobic incubation yielded approximately 50% more positive samples than did incubation in closed bottles. Sampling based on excision plus rinsing gave the highest Campylobacter prevalence (92.7%). To isolate Campylobacter spp. from retail packs of chicken, enrichment cultures must be incubated in a microaerobic atmosphere and sub-cultured at 24 h and, possibly, 48 h. Sampling packs by excision plus rinsing maximized recoveries. ISO 10272-1:2006 permits the use of inefficient protocols which markedly underestimate the true prevalence of Campylobacter spp. in retail, fresh chicken. Equivalent results could be obtained 24 h earlier, with consequent savings. Its revision is essential.

SIMPLIFIED CAPACITANCE MONITORING FOR THE DETERMINATION OF CAMPYLOBACTER SPP. GROWTH RATES

ABSTRACT Capacitance monitoring is commonly used as an efficient means to generate growth curves of bacterial pathogens. However, the use of capacitance monitoring with Campylobacter spp. was previously determined to be difficult due to the complexity of the required media. We investigated capacitance monitoring using a simplified medium for the efficient and reproducible construction of growth curves for Campylobacter spp. It was determined that Campylobacter spp. should initially be propagated on Mueller Hinton plates in a microaerobic atmosphere (5% O2, 10% CO2, 85% N2) at 37C for 24 h, followed by transfer to Mueller Hinton biphasic cultures for 6 h at 37C in a microaerobic atmosphere. Serial dilutions of the Campylobacter spp. cultures should be used for inoculation of Bactometer wells that contain 1 mL Mueller Hinton broth supplemented with 0.1 M sodium pyruvate for the completion of Campylobacter spp. growth curves with the Bactometer.PRACTICAL APPLICATIONSGrowth rates can vary greatly among Campylobacter isolates; therefore, the rapid and reproducible methods described will prove useful in studies where the generation of growth curves is critical for subsequent experimental analyses.

Enrichment culture for the isolation of Campylobacter spp: Effects of incubation conditions and the inclusion of blood in selective broths

Isolation of Campylobacter spp. using enrichment culture is time consuming and complex. Reducing the time taken to confirm the presence or absence of Campylobacter spp. would have many advantages for diagnostic, commercial and research applications. Rapid techniques such as real-time PCR can detect campylobacters from complex samples but blood in enrichment culture can inhibit the PCR reaction, if applied directly to enriched samples. The aim of this study was to investigate the effect of blood in enrichment culture on the isolation of campylobacters from chicken caeca, carcass rinses and bootsock (gauze sock walked through a broiler chicken house) samples using Bolton broth. The effect of incubation temperature (37 °C or 41.5 °C for 48 h, or 37 °C for 4 h then transfer to 41.5 °C for 44 h) and method of generating atmosphere (incubation of container in jar gassed with microaerobic atmosphere or incubation of container with small headspace and tightly screwed lid in an aerobic atmosphere) with and without blood on isolation from chicken carcass rinses and chicken faeces was also investigated. The presence of blood in enrichment culture did not improve the isolation of campylobacters from chicken faeces or bootsock samples but significantly improved recovery from chicken carcass rinse samples. There was no significant effect of the method used to generate incubation atmosphere. Isolation rates did also not depend significantly on whether broths were incubated at 37 or 41.5 °C for 24 or 48 h. Overall, the presence of blood in such media is not essential, although isolation can vary depending on sample type and enrichment method used.

Infected Aneurysms of Bilateral Deep Femoral Arteries due to Campylobacter fetus Subspecies fetus

Infected aneurysms due to Campylobacter fetus subspecies fetus have rarely been reported. Here, we describe the first case of infected aneurysm of bilateral deep femoral arteries due to C. fetus fetus. We successfully treated this case by administration of antibiotics effective for C. fetus fetus and bilateral obturator bypass with complete resection of the infected aneurysms. The aneurysmal wall culture disclosed the presence of C. fetus fetus in a microaerobic atmosphere after the operation. A distinctive culture condition was necessary to detect C. fetus fetus. In the case of infected aneurysms, we should be aware of the possibility of infection with C. fetus fetus, and an appropriate culture for this organism may be needed.

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.

Effects of Helicobacter hepaticus on the proteome of HEp-2 cells

Helicobacter hepaticus infects the bowel and biliary tree of several animals, producing inflammation. Colonisation of mouse livers can induce hepatocellular carcinomas. The effects of H. hepaticus on the proliferation and global protein expression of human HEp-2 cells were studied by examining the changes in the protein profiles of cells exposed to the bacterium. HEp-2 cells were grown for four days under a microaerobic atmosphere or under the same conditions in co-cultures with H. hepaticus at various inoculum densities. Enlargement, distension and elongation of HEp-2 cells were observed in co-cultures with H. hepaticus. The number of live cells declined by only an order of magnitude at bacterial inocula of approximately 10(9)cfu/ml, but were reduced to less than 10(3)cells/ml at approximately 10(10)cfu/ml bacteria inocula. Protein expression by HEp-2 cells was investigated employing two-dimensional gel electrophoresis. In cells grown with or without bacteria, 17 differentially expressed proteins were identified by tandem mass spectrometry. These proteins participated in several biological functions including amino acid metabolism, cell growth and proliferation, stress response, protein translation and modification, etc. The onset of a catastrophic killing of HEp-2 cells at a bacterial density of approximately 10(9)cfu/ml suggested a multimodal action for H. hepaticus infection, and the modulation of the expression of proteins involved in different biological functions showed that the presence of H. hepaticus has broad effects on the physiology of HEp-2 cells.

Frequency and Enumeration of Campylobacter Species from Processed Broiler Carcasses by Weep and Rinse Samples

Frequency and numbers of Campylobacter spp. were assessed per freshly processed, contaminated broiler carcass. Campylobacter-positive flocks were identified by cecal sample analysis at slaughter. These flocks had been tested as Campylobacter negative at 4.1 +/- 0.9 d prior to slaughter. Levels of contamination were estimated using 2 sampling approaches per carcass: (1) free weep fluids and (2) whole-carcass, 100 mL of distilled water rinses. Estimations of counts were determined by directly plating dilutions of weeps and rinses onto Campy-Cefex agar and incubating the plates at 41.5 degrees C under microaerobic atmosphere. Confirmation was provided by latex agglutination to quantify levels per milliliter of weep and per 100 mL of rinse. Thirty-two slaughter groups ( approximately 20 carcasses per group) were compared from 2003 to 2004. The Campylobacter-positive weep frequency was 84.8%, whereas the frequency for rinse samples was 74.4% (P < 0.001). Enumeration of Campylobacter spp. on positive samples ranged from 0.70 to 6.13 log(10) cfu/mL of weep (geometric mean of 2.84) and from 2.30 to 7.72 log(10) cfu/100 mL of rinse (geometric mean of 4.38). The correlations between weep and rinse were 0.814 with 0.5 mL of rinse and 0.6294 when applying 0.1 mL of rinse The quantitative regression analyses for these 2 corresponding tests were log(10) rinse (for 0.5 mL of inoculum) = 1.1965 log(10) weep + 0.4979, and log(10) rinse (for 0.1 mL of inoculum) = 1.322 log(10) weep - 0.1521. FlaA SVR sequencing of isolates indicated that the same genotypes were found in weep and rinse samples. Weep and rinse sampling led to different proportions of Campylobacter-positive carcasses detection, but we demonstrated that this difference was reduced by increasing the amount of rinse fluid used for plating.