Selective Agar Plates Research Articles

Quarter- and cow-level risk factors for intramammary infection with coagulase-negative staphylococci species in Swiss dairy cows

Bacteriological status, evaluation of udder symmetry, udder hygiene, and teat end scores of 92 dairy cows were assessed on 3 Swiss dairy farms in a longitudinal 1-yr study to determine risk factors for intramammary infection (IMI) with coagulase-negative staphylococci (CNS) species. Farm visits were performed monthly including sterile quarter milk sampling and udder evaluation of all lactating cows. Milk samples were evaluated for the presence of staphylococci using selective agar plates. Species identification was performed using MALDI-TOF mass spectrometry. Intramammary infection was defined as milk samples having ≥100 cfu per mL of milk according to culture results. Overall, 3,151 quarter samples were included in the statistical analysis. Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus xylosus, and a Staphylococcus warneri-like species were the 4 most prevalent CNS species found. Hierarchical multivariable logistic regression models were built to evaluate risk factors for species-specific CNS IMI. Risk factors for Staph. chromogenes IMI were presence in herd B, the period from June 2014 to August 2014 and December 2014 to February 2015, and presence of udder edema. For Staph. haemolyticus, the relevant risk factor included coinfection with Staph. xylosus coinfection with other than the above-mentioned CNS species ("others") and the period from June 2014 to November 2014. Coinfection with Staph. haemolyticus and "others," the periods from June 2014 to August 2014 and December 2014 to February 2015, early phase of lactation (1-60 d in milk), and belonging to herd B were significantly associated with Staph. xylosus IMI. Mid and late lactation, coinfection with Staph. xylosus, and the period September 2014 to May 2015 were identified as significant risk factors for Staph. warneri-like IMI. For Staph. chromogenes, 60.6 and 26% of the variance was observed at the quarter and cow level, respectively, whereas for the other investigated species the highest variance was observed at the sample level. The predominant species within herds differed and was most pronounced for the Staph. warneri-like species.

Shift in Oral Commensals During A 5-Day Metronidazole Therapy

Background: Antibiotic treatment induces great ecological impact on normal flora of the human body. Suppression of growth on these commensal microorganisms may give rise to opportunistic infection especially to those who are immuno-compromised. In the mouth, oral thrush represents a common indication of an opportunistic infection by fungus of the genus Candida. This study aimed to assess the effect of a 5-day metronidazole therapy on oral microbes to enable evaluation of its influence on the candidal, streptococci and other bacterial inhabitants of the oral cavity. Methods: Tongue scrape specimens from ten patients who were due for minor oral surgery were collected and processed using conventional microbiological procedures involving spreading of specimens on selective agar plates. The specimens were collected on the first, second and a day after the 5-day metronidazole therapy. Results: Distribution of oral candida, streptococci and other bacterial inhabitants experienced changes over the duration of treatment. An increased candidal population from 34.2% before to 45.3% during, and a contrasting decreased in streptococcal presence from 41% before to 28% during the therapy were observed. Similar trend of reduction was obtained on other bacteria from 37% before to 24% over the antibiotic therapy. The influence of metronidazole seem to slowly diminish upon completion of the 5-day therapy as all three microbial groups adapt to regain the original distribution pattern. Conclusion: The course of a 5-day metronidazole treatment affected microbial balance of the oral residents that lead to a significant rise of opportunistic species to take advantage of the disturbed ecological balance.

Simultaneous Detection and Serotyping of Salmonellae by Immunomagnetic Separation and Label-Free Surface-Enhanced Raman Spectroscopy

Current detection and characterization techniques for Salmonellae are time consuming, and rapid methods could benefit investigation and control of foodborne outbreaks. In this study, the potential of surface-enhanced Raman spectroscopy (SERS) in label-free detection and serotyping of Salmonella was evaluated. After immunomagnetic separation (IMS) and overnight culture, SERS spectra were collected from multiple replicates and experiments and analyzed by chemometrics. The detection/characterization accuracies were evaluated in real unknown mixture samples, which were confirmed by plating on selective agar plates and anti-sera agglutination tests. Prediction accuracies were found between 93 and 100%, 87 and 100%, and 67 and 100% for detecting Salmonella from other species, characterization of Salmonella serotypes, and simultaneous detection and characterization, respectively. When validated in mixture samples consisting of six bacteria, accuracies were 65–100% with increased misclassification. Overall, the approach may provide an inexpensive alternative within similar or slightly longer periods of time, but further improvement in spectral reproducibility and accuracy is needed.

68 The bifidobacterium longum 35624® culture transits in high numbers through the human gut

Background Most probiotic products rely on in vitro laboratory tests to assess transit and lack data on survival through the human gastrointestinal tract (GIT). However, in vitro tests are not representative of the multiple physiological states of the gut. Aim To confirm transit of the Bifidobacterium longum35 624 strain, found in Alflorex®, in different formats in humans. Method 35 624 strain was administered in a capsule, straw, sachet or milk format to volunteers for 7 days at doses ranging from 1x107–1 × 1010 CFU/day (n=1–6) followed by a wash-out period. The transit of viable 35 624 strain through the human GIT was assessed from stool collected at various timepoints by bifidobacteria selective agar plates and/or typing representative colonies using strain-specific PCR. Results Independent of form, the 35 624 strain transited in humans to high numbers, ranging from 1 × 104 – 1 × 108 CFU/g of stool in a dose-dependent manner at Day 7. Furthermore, despite antibiotic use in a 10-year-old cystic fibrosis sufferer (250 mg Zithromax 3 times/week), the 35 624 strain transited well (≥1 × 106 CFU/g). Co-administration of the antibiotic and probiotic resulted in a 1 log decrease in levels recovered from stool compared to administration of the probiotic 12 hour post antibiotic. Conclusion Independent of delivery format, the 35 624® strain was detected in stool at day 7 in a viable form at levels greater than 1×104 CFU/g of stool. Transit of viable 35 624® strain in a patient with long term antibiotic use was confirmed demonstrating that the Zithromax did not affect the 35 624® viability under the conditions of the experiment.

Postnatal changes in the relative abundance of intestinal Lactobacillus spp. in newborn calves.

The intestinal microbiota of newborn calves changes during the early postnatal period and influences their health and immune function. We studied the compositional changes in the intestinal microbiome of newborn calves during the first week after birth by metagenomic analysis. In feces from newborn calves, we identified 4 bacterial phyla, namely, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. The relative abundance of Lactobacillaceae significantly increased from day 1 to day 7. We evaluated Lactobacillus spp. colony numbers using selective agar plates and confirmed that the abundance of Lactobacillus spp. significantly increased during the first 7 days after birth. In conclusion, Lactobacillus spp. colonized the intestinal tract of calves during the first 7 days after birth.

Salmonella Contamination in Poultry—Are We Missing a Potential Vector?

ObjectivesIn the third quarter of 2015, USDA Salmonella prevalence in young chicken carcasses and chicken parts was 1.4 and 22.1%, respectively. These data indicate that efforts to control carcass contamination are effective; however, the relatively high prevalence in chicken parts suggests the pathogen is somehow evading carcass decontamination strategies. Thus, the objectives of this study were to assess the presence of Salmonella enterica in an alternative carcass location, namely joint synovial fluid, and further, to characterize any recovered Salmonella isolates.Materials and MethodsThe synovial fluid of 3 unique true joints (shoulder, coxofemoral, and stifle) of 500 broiler carcasses were individually sampled (1500 total samples) and analyzed for Salmonella presence. Broiler carcasses were collected immediately post chilling from 3 conventional and 2 antibiotic free broiler processing facilities located in the Southeast and Western United States. Each processing location was sampled twice during the study period. Broiler carcasses were subjected to a decontamination protocol to reduce the potential of cross-contamination of the joint synovial fluid from the carcass surface. The decontamination protocol included immersing the carcass in ethanol, flame sterilization, removing the carcass skin around the joint to be sampled and finally, immersion in boiling water (10 s). The joints to be sampled were then aseptically exposed using a sterile scalpel. The synovial fluid of the 3 joints was individually sampled, using a sterile swab, and the swab was enriched (35°C, 22 h) in buffered peptone water. Enriched synovial fluid samples from each bird (n = 3 joints per bird) were composited and subjected to rapid-based PCR Salmonella detection. If the composite sample was deemed a presumptive positive, the individual enriched synovial samples were further subjected to rapid based PCR Salmonella detection. Individual samples deemed a presumptive positive were subjected to secondary enrichment and selective agar plating (Brilliant Green Sulfa Agar and XLT4 Agar) to facilitate isolation of Salmonella. Presumptive Salmonella isolates were serotyped prior to determination of antimicrobial susceptibility.ResultsOverall, the prevalence of presumptive positive Salmonella among all joints for all birds was 0.47% (7 out of 1500 samples) with a 95% confidence interval of 0.20 to 1.00%. The prevalence of presumptive positive Salmonella among the antibiotic free and conventionally raised broilers was 0.17 and 0.67%, respectively. Among regions, presumptive positive Salmonella prevalence tended to be greater in the Southeast (0.83%) versus the Western region (0.22%). Among joint types, prevalence was greater in the shoulder joint (0.80%) when compared to the coxofemoral (0.40%) and stifle (0.20%) joints, respectively.ConclusionTo our knowledge, no previous assessments of Salmonella in the synovial fluid of broilers exists. However, as the presence of Salmonella in ground poultry and poultry parts remains problematic, alternative vectors for Salmonella should be evaluated. These results suggest that Salmonella may be present in the synovial fluid of broilers. Although prevalence is relatively low, when extrapolated to the scale of broilers produced, this information provides valuable insight into potential poultry contamination pathways. Further evaluation of this pathway is warranted.

Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

Population Screening Using Sewage Reveals Pan-Resistant Bacteria in Hospital and Community Samples.

The presence of pan-resistant bacteria worldwide possesses a threat to global health. It is difficult to evaluate the extent of carriage of resistant bacteria in the population. Sewage sampling is a possible way to monitor populations. We evaluated the presence of pan-resistant bacteria in Israeli sewage collected from all over Israel, by modifying the pour plate method for heterotrophic plate count technique using commercial selective agar plates. This method enables convenient and fast sewage sampling and detection. We found that sewage in Israel contains multiple pan-resistant bacteria including carbapenemase resistant Enterobacteriacae carrying blaKPC and blaNDM-1, MRSA and VRE. blaKPC carrying Klebsiella pneumonia and Enterobacter cloacae were the most common Enterobacteriacae drug resistant bacteria found in the sewage locations we sampled. Klebsiella pneumonia, Enterobacter spp., Escherichia coli and Citrobacter spp. were the 4 main CRE isolated from Israeli sewage and also from clinical samples in our clinical microbiology laboratory. Hospitals and Community sewage had similar percentage of positive samplings for blaKPC and blaNDM-1. VRE was found to be more abundant in sewage in Israel than MRSA but there were more locations positive for MRSA and VRE bacteria in Hospital sewage than in the Community. Therefore, our upgrade of the pour plate method for heterotrophic plate count technique using commercial selective agar plates can be a useful tool for routine screening and monitoring of the population for pan-resistant bacteria using sewage.

Detection of <italic>L. Monocytogenes</italic> in Enrichment Cultures by Immunoseparation and Immunosensors

There is a need to identify the presence of bacterial pathogens in foods earlier than it is currently carried out. At present, pre-enrichment culture followed by selective agar plating is standard microbiological methods applied to detect Listeria monocytogenes and other bacterial pathogens with zero tolerance in food products. PCR and biosensor methods may shorten the time required to individuate contamination by L. monocytogenes in a pre-enrichment culture. In this paper, a protein chip immunosensor with a sensitivity of 100 CFU/mL was developed for Listeria spp. detection. The method provides a ten-fold increase in sensitivity compared with published work on bacteria immunosensing coupled to fluorescence detection. When 40 mL of Listeria spp. pre-enrichment culture were processed by immunoseparation, the limit of detection was lowered to 2.5 CFU/mL. The results obtained from the real food samples were confirmed by qPCR. We also showed the potential for multiplexing of the protein chip method demonstrating the selective detection of Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., Escherichia coli, and Campylobacter spp. A future development is the integration into a system for rapid screening of pathogens contaminating food productions.

Prevalence of Campylobacter jejuni and Campylobacter coli in raw milk and some dairy products.

Aim:This study was accomplished to test raw milk and certain dairy products sold in local markets of Qena, Egypt, for the presence of Campylobacter coli and Campylobacter jejuni.Materials and Methods:A total of 150 samples of raw milk, kareish cheese, and yoghurt (50 samples each) were subjected first to enrichment in Bolton broth at 42°C for 2 days under a microaerobic condition, subsequently campylobacter blood free selective agar plates were cultured and incubated in the same condition of the broth. Based on the morphological and biochemical themes of the growing colonies, it was further classified into Campylobacter spp. The identified isolates were later affirmed by polymerase chain reaction using primers that were designed to locate hipO genes in C. jejuni and glyA in C. coli.Results:Of the total 150 examined samples of raw milk and soft cheese samples; 37 (24.6%) samples were contaminated with Campylobacter spp. C. jejuni was dominating in this study in 20%, 14%, and 8% of the examined raw milk, kareish cheese, and yoghurt samples, respectively. No sample harbored C. coli.Conclusion:Campylobacter spp. could be detected in 24.6% of the investigated samples. C. jejuni isolated from 14% of the total tested samples, while C. coli could not be detected from the examined samples. Campylobacter spp. is rampant in the areas of poor hygienic conditions making products made from raw milk of public health hazard.

A four‐quadrant sequential streak technique to evaluate Campylobacter selective broths for suppressing background flora in broiler carcass rinses

AbstractA semi‐quantitative ecometric technique was used to evaluate efficacy of selective culture media. It involved a standardized chronological streaking technique, leading to ever‐decreasing number of colonies. The objective was to evaluate the efficacy of this technique (four‐quandrant sequential streak [FQS]) with two commonly used Campylobacter broths (Bolton's and Tecra) with whole carcass rinses of post‐pick broilers (n = 30) obtained from a commercial processing plant. Ten ?L from each sample type was streaked (five individual streak lines) onto one quarter of a Campy‐cefex agar plate and sequentially into the next three quadrants. Growth of extraneous bacteria was expressed as an absolute growth index (AGI). A significant (P < 0.05) difference in background microflora suppression was observed using this procedure. A positive correlation between decreasing levels of background microflora within the broths was observed as the AGI declined. FQS can be used for rapid semi‐quantitative estimation of an enrichment broth's efficacy.Practical applicationsThe heavy presence of extraneous background microorganisms on a selective agar plate is a serious deterrent to the accurate detection of a target organism. This study confirmed sequential streaking as an appropriate and easily applied method to evaluate the relative efficiency of selective enrichment broths for Campylobacter in the suppression of background microflora on selective plates. The four‐quadrant sequential streak (FQS) ecometric technique is an inexpensive, rapid, semi‐quantitative method to evaluate the effectiveness of various selective enrichment broths.

Prevalence of Fusobacterium necrophorum in tonsils from patients with chronic tonsillitis

Conclusion: There was a high prevalence of Fusobacterium necrophorum (FN) in patients with chronic tonsillitis in the age group 15–23 years. This indicates that FN might play an important role in the pathogenesis of chronic tonsillitis in this age group, which is also the age group in which chronic or recurrent tonsillitis is most common.Objectives: The role of FN in patients with acute and chronic tonsillitis is unclear. Thus, this study investigated the occurrence of FN in tonsils of patients with chronic tonsillitis. The aim of the study was to determine the prevalence of FN in patients that underwent tonsillectomy due to chronic tonsillitis. This study also investigated if FN was found at different areas in the tonsils.Method: One hundred and twenty-six consecutive patients undergoing tonsillectomy due to chronic tonsillitis were included from the ENT clinics at Sunderby Hospital and Gällivare Hospital, Sweden. Both children and adults were included to encompass various age groups (age =2–57 years). Culture swabs were taken from three different levels of the tonsils – the surface, the crypts, and the inner core of the tonsils. Selective agar plates for detecting FN were used for culture. Culture was also made for detecting β-hemolytic streptococci, Haemophilus influenzae, and Arcanobacterium.Results: FN was the most common pathogen (19%). The highest prevalence of FN was found in the age group 15–23 years (in 34% of the patients). FN was detected both at the surface and in the core of the tonsils. Furthermore, in the few patients where FN was not detected in all three areas, FN was always detected at the tonsillar surface, in spite of being an anaerobic bacterium. Streptococci group G and C also occurred most frequently (30%) in the same age group as FN (15–23 years), whereas Streptococci group A was more evenly spread among the age groups.

Variation of Burkholderia cenocepacia virulence potential during cystic fibrosis chronic lung infection

ABSTRACTDuring long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces multiple selective pressures in this highly stressful and fluctuating environment. As a consequence, the initial infecting strain undergoes genetic changes that result in the diversification of genotypes and phenotypes. Whether this clonal expansion influences the pathogenic potential is unclear. The virulence potential of 39 sequential B. cenocepacia (recA lineage IIIA) isolates, corresponding to 3 different clones retrieved from 3 chronically infected CF patients was compared in this study using the non-mammalian infection hosts Galleria mellonella and Caenorhabditis elegans. The isolates used in this retrospective study were picked randomly from selective agar plates as part of a CF Center routine, from the onset of infection until patients' death after 3.5 and 7.5 y or the more recent isolation date after 12.5 y of chronic infection. The infection models proved useful to assess virulence potential diversification, but for some isolates the relative values diverged in C. elegans and G. mellonella. Results also reinforce the concept of the occurrence of clonal diversification and co-existence of multiple phenotypes within the CF lungs, also with respect to pathogenicity. No clear trend of decrease (or increase) of the virulence potential throughout long-term infection was found but there is an apparent tendency for a clone/patient-dependent decrease of virulence when the G. mellonella model was used. The sole avirulent variant in both infection hosts was found to lack the small third replicon previously associated to virulence. Although possible, the in vivo loss of this nonessential megaplasmid was found to be a rare event (1 among a total of 64 isolates examined).

The housefly Musca domestica as a mechanical vector of Clostridium difficile

Clostridium difficile is a bacterial healthcare-associated infection that may be transferred by houseflies (Musca domestica) due to their close ecological association with humans and cosmopolitan nature. To determine the ability of M.domestica to transfer C.difficile both mechanically and following ingestion. M.domestica were exposed to independent suspensions of vegetative cells and spores of C. difficile, then sampled on to selective agar plates immediately postexposure and at 1-h intervals to assess the mechanical transfer of C. difficile. Fly excreta was cultured and alimentary canals were dissected to determine internalization of cells and spores. M.domestica exposed to vegetative cell suspensions and spore suspensions of C.difficile were able to transfer the bacteria mechanically for up to 4h upon subsequent contact with surfaces. The greatest numbers of colony-forming units (CFUs) per fly were transferred immediately following exposure (mean CFUs 123.8+/-66.9 for vegetative cell suspension and 288.2+/-83.2 for spore suspension). After 1h, this had reduced (21.2+/-11.4 for vegetative cell suspension and 19.9+/-9 for spores). Mean C.difficile CFUs isolated from the M.domestica alimentary canal was 35+/-6.5, and mean C.difficile CFUs per faecal spot was 1.04+/-0.58. C.difficile could be recovered from fly excreta for up to 96h. This study describes the potential for M.domestica to contribute to environmental persistence and spread of C.difficile in hospitals, highlighting flies as realistic vectors of this micro-organism in clinical areas.

Detection of plasmid-borne extended-spectrum β-lactamase (ESBL) genes in Escherichia coli isolates from bovine mastitis

Extended-spectrum β-lactamase (ESBL)-producing isolates have been increasingly reported during recent years. The aims of this study were to characterize ESBL-producing Escherichia coli from bovine mastitis as well as their ESBL gene-carrying plasmids. A culture collection of E. coli isolated from bovine quarter milk samples (2009–2013), was screened for ESBL production using ESBL selective agar plates. Putative ESBL producers (n=16) were investigated by phenotypic confirmatory tests and were characterized by the detection/sequencing of ESBL genes, XbaI macrorestriction analysis, multilocus sequence typing (MLST), phylotyping and antimicrobial susceptibility testing. ESBL gene-carrying plasmids were investigated by transfer experiments, PCRs for the detection of co-located antimicrobial resistance genes, PCR-based replicon typing and S1-nuclease pulsed-field gel electrophoresis. Twelve ESBL-producing isolates were found. They showed eleven different XbaI patterns and were distributed among eight MLST types [ST10 (n=3), ST117 (n=2), ST361 (n=1), ST362 (n=1), ST540 (n=1), ST1431 (n=2), ST1508 (n=1), and the novel ST5447 (n=1)] and the phylogenetic groups A (n=6), B1 (n=2), B2 (n=1) and D (n=3). ESBL genes blaCTX-M-1 (n=5), blaCTX-M-2 (n=2), blaCTX-M-14 and blaCTX-M-15 (n=4) were found on conjugative plasmids (35–225kb) of diverse incompatibility groups (e.g. IncF, IncI1 or HI2+P). Co-located resistance to sulfonamides, tetracycline, trimethoprim, and chloramphenicol/florfenicol was detected on five ESBL gene-carrying plasmids, but seven plasmids conferred solely resistance to β-lactam antibiotics. The presence of additional resistance genes on the ESBL gene-carrying plasmids suggests that co-selection of ESBL genes may occur even in the absence of β-lactam antibiotics.

Characterisation of class 3 integrons with oxacillinase gene cassettes in hospital sewage and sludge samples from France and Luxembourg

In this study, antibiotic resistance class 3 integrons in Gram-negative bacteria isolated from hospital sewage and sludge and their genetic contents were characterised. Two samples of hospital effluent from France and Luxembourg and one sample of sludge from a wastewater treatment plant in France were collected in 2010 and 2011. Bacteria were cultured on selective agar plates and integrons were detected in colonies by quantitative PCR. Integron gene cassette arrays and their genetic environments were analysed by next-generation sequencing. Three class 3 integron-positive isolates were detected, including Acinetobacter johnsonii LIM75 (French hospital effluent), Aeromonas allosaccharophila LIM82 (sludge) and Citrobacter freundii LIM86 (Luxembourg hospital effluent). The gene cassettes were all implicated in antibiotic (aminoglycoside and β-lactam) or antiseptic resistance. An oxacillinase gene cassette (blaOXA-10, blaOXA-368 or blaOXA-2) was found in each integron. All of the class 3 integrons were located on small mobilisable plasmids. This study highlights the role of class 3 integrons in the dissemination of clinically relevant antibiotic resistance genes, notably oxacillinase genes, in hospital effluent.

Travel to Asia and traveller's diarrhoea with antibiotic treatment are independent risk factors for acquiring ciprofloxacin-resistant and extended spectrum β-lactamase-producing Enterobacteriaceae—a prospective cohort study

Travel to (sub)tropical countries is a well-known risk factor for acquiring resistant bacterial strains, which is especially of significance for travellers from countries with low resistance rates. In this study we investigated the rate of and risk factors for travel-related acquisition of extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E), ciprofloxacin-resistant Enterobacteriaceae (CIPR-E) and carbapenem-resistant Enterobacteriaceae. Data before and after travel were collected from 445 participants. Swabs were cultured with an enrichment broth and sub-cultured on selective agar plates for ESBL detection, and on plates with a ciprofloxacin disc. ESBL production was confirmed with the double-disc synergy test. Species identification and susceptibility testing were performed with the Vitek-2 system. All isolates were subjected to ertapenem Etest. ESBL and carbapenemase genes were characterized by PCR and sequencing. Twenty-seven out of 445 travellers (6.1%) already had ESBL-producing strains and 45 of 445 (10.1%) travellers had strains resistant to ciprofloxacin before travel. Ninety-eight out of 418 (23.4%) travellers acquired ESBL-E and 130 of 400 (32.5%) travellers acquired a ciprofloxacin-resistant strain. Of the 98 ESBL-E, predominantly Escherichia coli and predominantly blaCTX-M-15, 56% (55/98) were resistant to gentamicin, ciprofloxacin and co-trimoxazole. Multivariate analysis showed that Asia was a high-risk area for ESBL-E as well as CIPR-E acquisition. Travellers with diarrhoea combined with antimicrobial use were significantly at higher risk for acquisition of resistant strains. Only one carbapenemase-producing isolate was acquired, isolated from a participant after visiting Egypt. In conclusion, travelling to Asia and diarrhoea combined with antimicrobial use are important risk factors for acquiring ESBL-E and CIPR-E.

Protein Chips for Detection of Salmonella spp. from Enrichment Culture.

Food pathogens are the cause of foodborne epidemics, therefore there is a need to detect the pathogens in food productions rapidly. A pre-enrichment culture followed by selective agar plating are standard detection methods. Molecular methods such as qPCR have provided a first rapid protocol for detection of pathogens within 24 h of enrichment culture. Biosensors also may provide a rapid tool to individuate a source of Salmonella contamination at early times of pre-enrichment culture. Forty mL of Salmonella spp. enrichment culture were processed by immunoseparation using the Pathatrix, as in AFNOR validated qPCR protocols. The Salmonella biosensor combined with immunoseparation showed a limit of detection of 100 bacteria/40 mL, with a 400 fold increase to previous results. qPCR analysis requires processing of bead-bound bacteria with lysis buffer and DNA clean up, with a limit of detection of 2 cfu/50 μL. Finally, a protein chip was developed and tested in screening and identification of 5 common pathogen species, Salmonella spp., E. coli, S. aureus, Campylobacter spp. and Listeria spp. The protein chip, with high specificity in species identification, is proposed to be integrated into a Lab-on-Chip system, for rapid and reproducible screening of Salmonella spp. and other pathogen species contaminating food productions.

Determination of the extent of Clostridium difficile colonisation and toxin accumulation in sows and neonatal piglets

Clostridium difficile is an important spore-forming, opportunistic pathogen in animal husbandry and health care. In pig farming, only neonatal piglets are affected, and diarrhoea and necrotising lesions are common symptoms leading to dehydration and in some cases death. This study aimed at the assessment of the quantitative development of C. difficile colonisation in neonatal piglets by determining the shedding of spores and C. difficile toxins A (TcdA) and B (TcdB) concentrations in sow (n = 5–6) and piglet pen faeces (n = 5–6) at different time points. Spores were quantified on selective agar plates and toxins using ELISA method. C. difficile was not detected in the faeces of all but one sow during the perinatal period. Faeces of 2- and 4-day-old piglets contained 0.65 log cells/g and 5.88 log cells/g of C. difficile, respectively. Toxins were detected on day 4 at a concentration of 2.13 log ng/g (TcdA) and 2.06 log ng/g (TcdB). On day 6, concentration of C. difficile reached 6.14 log CFU/g and toxins 2.02 log ng/g (TcdA) and 2.20 log ng/g (TcdB). Two-week-old piglets showed 4.72 log CFU/g of C. difficile but toxins could not be detected. At 21 days of age, both C. difficile and toxins were undetectable. The concentration and the prevalence of C. difficile were positively associated with the prevalence of toxins in piglets. A very short time window for colonisation by C. difficile, including toxin-producing strains can be observed in neonatal piglets. The significance for animal health and the risk of a carrier status need to be addressed in future studies.

Anticlostridial agent 8-hydroxyquinoline improves the isolation of faecal bifidobacteria on modified Wilkins-Chalgren agar with mupirocin.

The need for suitable selective cultivation media for the isolation of Bifidobacterium spp. continues to be a real concern in the field of intestinal microbiology. Isolation of bifidobacteria from human and animal faecal samples using selective agar plating may be problematic especially in samples with increased clostridial counts than bifidobacterial counts. Due to the absence of anticlostridial agents in existing selective media, clostridia can displace bifidobacteria resulting in incorrect estimation of their counts. Therefore, we supplemented the existing selective medium 'modified Wilkins Chalgren agar with mupirocin' (MWM) with 90mgl(-1) of 8-hydroxyquinoline (8HQ), which was recently proved to act selectively against clostridia. The newly composed 'modified Wilkins-Chalgren agar with 8HQ' (MWMQ) was tested on pure bifidobacterial and clostridial strains, their mixtures, and using faecal samples of mammalian origin; its selectivity was evaluated by genus-specific identification of isolates. The results demonstrated that the presence of 8HQ in this agar eliminated the growth of nonbifidobacterial strains on MWMQ compared to that on MWM, whereas the recovery of bifidobacterial counts was at satisfactory levels. In conclusion, MWMQ could be recommended for bifidobacterial isolation from human and animal faeces especially when bifidobacteria are not numerically dominant and there are chances of clostridial contamination. Routine isolation of bifidobacteria from mammalian faeces does not use a reliable selective agar with an anticlostridial agent. Overgrowth of clostridia may result in incorrect estimation of bifidobacterial counts. Thus, in order to improve the selectivity of existing media for bifidobacterial isolation, we chose the modified Wilkins-Chalgren agar with mupirocin and supplemented it with 8-hydroxyquinoline (8HQ), a molecule that shows anticlostridial activity without affecting the growth of bifidobacteria. This newly composed medium showed enhanced selectivity and specificity compared to the original medium and therefore, can be recommended for the isolation of bifidobacteria from mammal faeces.