Presumptive Colonies Research Articles

Factors of detection of Bacillus cereus strains in eye cream.

We investigated the detection factors of Bacillus in eye cream preserved with parabens, including the non-ionic surfactants used as neutralizers such as Tween 80, a blend of Tween 60 and Span 80, Tween 20 and selective media. Eye-cream samples were first mixed with neutralizers and individually inoculated with B.cereus strains, B.mycoides, B.subtilis or B.thuringiensis at a final concentration of 5 log CFU per g. The inoculated samples with and without neutralizers were analysed after 30min and during 84-day storage at room temperature. Presumptive colonies of Bacillus were enumerated on the varieties of Bacillus agar by spiral-plating techniques and most probable number (MPN) method. The recovery counts of all Bacillus strains were between 4.10 and 4.58 log CFU per g in samples with Tween 80 and from 3.62 to 4.53 CFU per g in samples with TS after 30min. Tween 20 was the least effective neutralizer. The challenged organisms, in samples without neutralizer, B.subtilis ATCC 15563 and B.cereus 4227A were detected at 1.83 and 1.49 log CFU per g after 30min, respectively. This study showed that Tween 80 was the best neutralizer for reducing the antimicrobial effect of parabens. BACARA® and R&F plating media showed typical reaction of Bacillus cereus strains.

Methicillin-resistant Staphylococcus aureus in the environment of public transport: data from the metropolitan network in Lyon, France

Methicillin-resistant Staphylococcus aureus (MRSA) is involved in community-acquired and nosocomial diseases. The means of MRSA transmission and dissemination in the community remain uncertain. Studies have shown that public transport systems could be a source of MRSA and may serve as a potential source for community-acquired MRSA infections. This study aimed to investigate MRSA contamination on Lyon's metropolitan network (Métro) in France. Hand-touched surfaces were sampled with sterile swabs (Transystem®) during a 1-day transversal study by collecting 50 samples in seven hub stations and two trains for each of the four Métro lines. Then, during a longitudinal study, one sample was collected twice daily for 30 consecutive days in the busiest and most congested hub station. All swabs were incubated in enrichment medium for 24 h and then each suspension was plated onto a chromogenic selective medium for MRSA. After 24 h at 36 °C, all presumptive MRSA colonies were tested using VITEK® MS to confirm identification as S. aureus as well as by Alere™ PBP2a Culture Colony Test and mecA/mecC PCR to check methicillin resistance. Of the 110 swabs tested, 24 presumptive MRSA colonies were isolated, of which 2 were confirmed as S. aureus by VITEK® MS. These two isolates were tested negative using the PBP2a Culture Colony Test and PCR. Unlike other foreign cities such as Lisbon, the current data suggest a low level of MRSA contamination of hand-touched surfaces on Lyon's Métro. This should be put in perspective with the low level of MRSA colonisation in the French community.

Intestinal carriage of multidrug-resistant gram-negative bacteria in preterm-infants during hospitalization in neonatal intensive care unit (NICU)

The prevalence and antibiotic susceptibility of intestinal carriage of Gram-negative bacteria among preterm infants admitted to the neonatal intensive care unit (NICU) in a tertiary teaching hospital in Malaysia were determined. A total of 34 stool specimens were obtained from preterm infants upon admission and once weekly up to two weeks during hospitalization. The presumptive colonies of Escherichia coli and Klebsiella pneumoniae were selected for identification, antibiotic susceptibility testing, and subtyping by using pulsed-field gel electrophoresis (PFGE). Out of 76 Gram-negative isolates, highest resistance was detected for amoxicillin/clavulanate (30.8%, n = 16), ceftriaxone (42.3%, n = 22), ceftazidime (28.8%, n = 15), cefoxitin (28.8%, n = 15), aztreonam (36.5%, n = 19), and polymyxin B (23.1%, n = 12). Three colistin resistant K. pneumoniae have also been detected based on E-test analysis. Thirty-nine isolates of K. pneumoniae and 20 isolates of E. coli were resistant to more than three antimicrobial classes and were categorized as multidrug resistant (MDR). PFGE analysis revealed a higher diversity in pulsotypes for K. pneumoniae (18 pulsotypes) in comparison to E. coli (four pulsotypes). In addition, a total of fifteen pulsotypes was observed from 39 MDR K. pneumoniae. The risk factors for antibiotic resistance were assessed using random forest analysis. Gender was found to be the most important predictor for colistin resistant while length, OFC, and delivery mode were showing greater predictive power in the polymyxin B resistance. This study revealed worrying prevalence rates of intestinal carriage of multidrug-resistant K. pneumoniae and E. coli of hospitalized preterm infants in Malaysia, particularly high resistance to polymyxins.

PM 7/60 (2) Pantoea stewartii subsp. stewartii

Specific scopeThis Standard describes a diagnostic protocol for Pantoea stewartii subsp. stewartii.Specific approval and amendmentApproved in 2005‐09.Revised in 2016‐04.

A Preliminary Assessment of Contamination of Emergency Service Helicopters with MRSA and Multi-Resistant Staphylococcus aureus

Objective: To detect the presence of methicillin-resistant Staphylococcus aureus (MRSA) and multi-resistant S. aureus (Multi-RSA) in emergency service helicopters. Methods: This proof-of-concept study used a convenience sample of two emergency service helicopters located in different Australian towns. Over a three-month period ending January 31 2015, within each helicopter five areas of perceived high contact were sampled by taking swabs on an approximately weekly basis. The precise occasions varied based on timings of cases and maintenance schedules. Swabs were analysed for the presence of MRSA, Multi-RSA and other bacteria. Results: Presumptive MRSA and other colonies were recovered from each helicopter. Of those presumptive colonies tested, 18.7% were identified as Staphylococcus aureus, 76.0% were other staphylococci (such as S. epidermidis), and 5.3% were other genera of bacteria. Further testing of these colonies detected no MRSA or Multi- RSA. Over the period of this study, high total numbers of bacteria were recovered in both helicopters. Typically, the floor of the helicopters had higher counts than the seat belts and the blood pressure cuff containers, whereas the radio and cardiac equipment had comparatively lower counts. Conclusions: Since a total of 94.7% of the colonies tested were identified as Staphylococcus spp. the potential for the existence of MRSA in emergency service helicopters is apparent. Our findings highlight the importance of regular cleaning in emergency service vehicles to promote the reduction of infectious disease transmission. We recommend the implementation of a standard operating procedure for cleaning and disinfection across all emergency service providers, reinforced by appropriate infection control training. For quality control, random intermittent swab testing of selected surfaces within all helicopters is advised. The implications of our findings may assist emergency medical service providers to reduce infectious disease transmission risks, and may help to minimise exposure to pathogenic microorganisms during disaster events including pandemics and bioterrorism.

ESTABLISHING THE STATUS OF CLAVIBACTER MICHIGANENSIS subsp. INSIDIOSUS IN LUCERNE IN SOUTH AFRICA

Clavibacter michiganensis subsp. insidiosus (Cmi) is a seed-borne pathogen that causes bacterial wilt of lucerne (alfalfa, Medicago sativa). The pathogen has a limited distribution worldwide and is included in the A2 pest list of the European and Mediterranean Plant Protection Organisation (OEPP/EPPO). In South Africa, cmi was first reported in 1967 in a few regions (Western-Cape province and in the Eastern Cape), with the last doubtful listing in 1983 also reporting it from the Transvaal region (currently known as Gauteng, Limpopo, North West and Mpumalanga provinces). Subsequently, lucerne plants exhibiting bacterial wilt symptoms have not been observed in any region of South Africa. Nevertheless, for South Africa, Cmi is listed as “present, restricted occurrence” by the OEPP/EPPO. Therefore, the status of Cmi in South Africa was re-investigated through field inspections and seed testing. Field inspections of lucerne seed crops conducted in all production regions from 2006 to 2014 did not reveal any symptomatic plants. Seed testing of 67 representative commercial seed lots showed that Cmi was absent from all of the seed lots. Prior to testing the commercial seed lots, artificially inoculated seed lots were used to show that the serial dilution plate method was sensitive (0.1% detection limit). It was also shown that a published Cmi semi-selective glucose-yeast-carbonate agar medium containing kanamycin and cycloheximide was able to more readily reveal the presence of presumptive Cmi colonies than the media of the EPPO diagnostic protocol, and the International Seed Health Initiative ring test.

Occurrence and characterisation of MRSA and extended-spectrum ß-lactamases producing Escherichia coli isolated from mastitic cows’ milk

Abstract The aim of study was the preliminary evaluation of the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum ß-lactamases (ESBL) - producing Escherichia coli in 650 milk and inflammatory secretions from cows with clinical or subclinical mastitis. One millilitre of the sample was added to Mueller-Hinton broth supplemented with 6.5% NaCl, Tryptone Soya Broth with cefoxitin and aztreonam, and then to MRSA ID agar. Presumptive MRSA colonies were analysed for the presence of mecA gene. Parallel to MRSA identification, the samples were incubated in buffered peptone water, lauryl tryptose broth and McConkey agar supplemented with cefotaxim for ESBL-producing E. coli isolation. These bacteria were identified using API Rapid 32 E and the ability of ESBL production was initially established using disc test D68C and confirmed by MIC technique using Sensititre ESBL plates. The primers (blaCTX, blaTEM, blaSHV, and blaCMY-2-group) for the detection of some of the genes encoding ESBL production were used. The 45 strains of S. aureus with mecA gene and 41 strains of E. coli with blaTEM gene were detected.

Bacteria Isolated from the Oropharynxs of Apparently Healthy Chickens in Jos, Nigeria

There are concerns that apparently healthy chickens may transmit potentially pathogenic pathogens that could threaten not only poultry industry, but also public health. This investigative study was carried out in Jos, Nigeria to determine the prevalent bacterial agents in the oropharynxs of apparently healthy chickens. Four hundred (400) oropharyngeal swabs were collected from 400 apparently healthy chickens for bacteriological analysis. Swab from each sample was cultured on 7% defibrinated sheep blood, Casein Sucrose Yeast (CSY) agar and MacConkey. Oropharyngeal swabs were cultured indirectly by inoculating into 5 ml of brain heart infusion broth (BHI), incubated at 37℃ for 24 h and then streaked unto Casein Sucrose Yeast (CSY) agar, MacConkey agar and Blood agar. Presumptive colonies of bacterial agents were subjected to conventional biochemical characterization. The result of biochemical test identified the following bacteria: Staphylococcus aureus 82 (20.5%), Escherichia coli 53 (13.3%), Klebsiella pneumonia 35 (8.8%) and Proteus species 26 (6.6%), Pasteurella multocida 5 (1.3%) among others. Staphylococcus aureus (20.5%) was the highest followed by Escherichia coli (13.3%), Klebsiella pneumoniae (8.8%) and Pasteurella multocida (1.3%) was the least. It was concluded that the oropharynxs of apparently healthy chickens harbored a lot of bacterial agents which could cause infections when the immune system was compromised.

VYUŽITÍ KULTIVAČNÍCH A BIOCHEMICKÝCH METOD A POLYMERÁZOVÉ ŘETĚZOVÉ REAKCE PRO STANOVENÍ CLOSTRIDIUM TYROBUTYRICUM

Anaerobic spore-forming bacteria of the genusClostridiumare commonly present in raw milk and some milk products. Their spores can survive pasteurization and can provoke so called late blowing defect in cheese caused by butyric acid fermentation. The only species of the genusClostridiumthat is able to provoke late blowing isClostridium tyrobutyricum.In this work, two cultivation methods for detection of butyric acid producing clostridia in raw and pasteurized milk and in cheese samples were compared. The results show that tube method is suitable for route identification (in concentration 102CFU/ml or /g) of clostridia in milk and cheese. The standard cultivation technique is suitable for more sensitive identification (10 CFU/ml or /g). All presumptive colonies grown anaerobically on selective RCM agar with polymyxine B (500 μg/ml) were classified to be of speciesClostridium tyrobutyricumusing PCR only. The confirmation using API tests were different in 50 % cases. The results show, that described PCR method is suitable for rapid screening of the presence ofClostridium tyrobutyricumin milk and cheese. PCR from one colony is possible to use for the analysis.

Comparison of Culture, Conventional and Real-time PCR Methods for Listeria monocytogenes in Foods.

We compared standard culture methods as well as conventional PCR and real-time PCR for the detection of Listeria monocytogenes (L. monocytogenes) in milk, cheese, fresh-cut vegetables, and raw beef that have different levels of background microflora. No statistical differences were observed in sensitivity between the two selective media in all foods. In total, real-time PCR assay exhibited statistically excellent detection sensitivity (p<0.05) and was less time consuming and laborious as compared with standard culture methods. Conventional culture methods showed poor performance in detecting L. monocytogenes in food with high levels of background microflora, generating numerous false negative results. While the detection of L. monocytogenes in fresh cut vegetable by culture methods was hindered only by L. innocua, various background microflora, such as L. innocua, L. welshimeri, L. grayi, and Enterococcus faecalis appeared on the two selective media as presumptive positive colonies in raw beef indicating the necessity of improvement of current selective media. It appears that real-time PCR is an effective and sensitive presumptive screening tool for L. monocytogenes in various types of foods, especially foods samples with high levels of background microflora, thus complementing standard culture methodologies.

Multiplex real-time PCR and culture methods for detection of Shiga toxin-producing Escherichia coli and Salmonella Thompson in strawberries, a lettuce mix and basil

An appropriate approach of high throughput multi-screening was verified for Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp. in strawberries, lettuce and basil. Sample replicates were inoculated with STEC O157 or O26 and Salmonella Thompson (ca. 10–70, 100–700 and 1000–7000cfu/25g) and analysed after 1 and 5days of storage (strawberries and lettuce at 7°C and basil at 10°C). After 18–24h of enrichment at 37°C in buffered peptone water, detection was performed using the GeneDisc multiplex PCR (stx1, stx2, eae and iroB genes) and selective culture media for isolation of STEC (with immunomagnetic separation (IMS)) and Salmonella spp. in parallel. After 1day, the pathogenic strains were recovered from all samples for all inoculum levels, whereas reduced detection rates of STEC O157 and S. Thompson were observed after 5days of storage in case of strawberries, in particular for the lowest inoculums level, suggesting superior survival potential for STEC O26. Overall, this study indicates the ability of PCR based screening methods for reproducible multi-detection of low numbers (10–70cfu/25g) of STEC and Salmonella in this type of foods. However, for the basil samples, PCR needed twofold dilution of the DNA extract to overcome inhibition. It was noted that on several occasions growth of competitive microbiota obstructed finding presumptive colonies on the selective agar media, whereas the use of an additional agar medium such as CHROMagar STEC (without IMS) improved recovery rate of STEC.

Prevalence of Vibrio parahaemolyticus, and Vibrio vulnificus in blue crabs (Callinectes sapidus), seawater and sediments of the Maryland Coastal Bays.

To determine the prevalence of total and pathogenic Vibrio parahaemolyticus (Vp) and V. vulnificus (Vv) in blue crabs, water and sediment from the Maryland Coastal Bays (MCBs), USA. Crab, haemolymph, sediment and seawater samples were collected monthly from four sites in MCBs from February 2012 through October 2012 with environmental parameters recorded. The most-probable-number (MPN) methodology was used to enumerate Vp and Vv with presumptive colonies and the presence of virulence markers confirmed using polymerase chain reaction (PCR). Results indicate that blue crabs contained both Vp and Vv at densities (7·28 and 5·43 log MPN g(-1) , respectively) higher than those reported for bivalves. In addition, markers for clinically relevant strains of both species were detected in over 30% of samples. Haemolymph, sediment and seawater samples were also routinely positive for both species and clinically relevant strains, but generally at lower densities than found in crabs (4·27, 3·28, and 2·39 log MPN g(-1) per ml(-1) Vp, and 4·28, 2·49 and 2·38 log MPN g(-1) per ml(-1) Vv). Blue crabs concentrate Vp and Vv at levels greater than found in water or sediment. While changes in abundance associated with temperature are apparent, there is little evidence to support differences among sampling locations. These results highlight the potential for blue crab related vibriosis and the importance of proper handling, cooking and care of this popular seafood before consumption.

English

This study was undertaken to assess prevalence of virulent and antibiotic-resistant Aeromonas species in chicken meat and fresh water fish washings procured from local market. Isolation was done on three selective agar viz. Aeromonas isolation media, ampicillin dextrin agar and Aeromonas starch DNA agar. Presumptive colonies were directly screened by multiplex polymerase chain reaction (PCR) targeting genus specific 16S rRNA gene, aerolysin (aerA) of Aeromonas hydrophila and hemolysin (asa1) gene of Aeromonas sobria. Of the 200 samples (100 each of chicken and fish washings), 21 isolates were confirmed as Aeromonas species. We could not detect aer A, however, asa1 of A. sobria was detected in six (28.57%) fish isolates. Aeromonas isolates exhibited 100% resistant to amoxicillin, ampicillin and 95.23% to carbenicillin. Moderate sensitivity was observed to kanamycin (90.47%) and neomycin (71.42%). Isolates were 100% sensitive to gentamicin and ciprofloxacin. Maximum sensitivity was recorded with chloramphenicol, tobramycin (95.23% each) and amikacin (80.95%). PCR characterization revealed presence of class 1 integron and Tet (C) genes in six and 10 isolates, respectively. PSE-1 &beta;-lactamase was not detected in any of the isolates. This study demonstrate the incidence of antimicrobial resistant Aeromonas in chicken and fish environment, which may be a potential source of spread of this enteropathogen in food chain. Key words: Aeromonads, antimicrobial resistance, chicken, fish.

Isolation of Enterohaemorrhagic &lt;i&gt;Escherichia coli&lt;/i&gt; O104 Strains from Raw Meat Products in the North West Province, South Africa

Enterohaemorrhagic <i>E. coli</i> (EHEC) are Shiga toxin-producing <i>E. coli</i> strains that possess unique pathogenic properties and are characterized by certain seropathotypes that are frequently associated with outbreaks or sporadic cases of human infections. Although EHEC strains belonging to the serotype O104:H4 has rarely been associated with human diseases in the past, outbreaks of infections caused by this organism resulted to HUS and bloody diarrhoea in humans in countries that have advanced public health facilities. The aim of this study was to isolate <i>E. coli </i>O104 strains from meat samples obtained from supermarkets and retail shops in the North West Province, South Africa. A further objective was to determine the identities of the isolates using Gram staining, preliminary biochemical tests (oxidase test, Triple Sugar Iron (TSI) test, citrate utilization and Sorbitol fermentation test) and confirmatory (API 20E and PCR analysis) assays. A total of nineteen meat samples were collected from four butcheries and shops in the study area and Sorbitol MacConkey agar was used for selective isolation of bacteria. A total of 304 presumptive colonies were subjected to identification tests for <i>E. coli</i>. Large proportions (65.5% to 93%) of these isolates were oxidase negative; fermented the sugars in the TSI medium; utilized citrate and fermented sorbitol. On the contrary only a small proportion (27.3%) of the isolates produced hydrogen sulphide gas. Based on patterns obtained for the biochemical profiles of the isolates 102 (33.6%) were positively identified as <i>E. coli</i>. All the 304 <i>E. coli</i> isolates were subjected to specific PCR designed to amplify the<i>wzx</i><SUB>O104</SUB> gene fragment that facilitates identification of <i>E. coli</i> O104 strains and a total of 52 isolates were positively identified. Despite the fact that not all samples were positive for the pathogen, the presence of <i>E. coli</i> O104 strains in some of the meat samples was of great concern. These bacteria cells have the potential to cause severe health effects on consumers if present in undercooked food products.

Rapid identification of Campylobacter jejuni from poultry carcasses and slaughtering environment samples by real-time PCR

The objective of this study was to develop a real-time PCR assay for rapid identification of Campylobacter jejuni and to apply the method in analyzing samples from poultry processing. A C. jejuni-specific primer set targeting a portion of the C. jejuni hippuricase gene was developed. The specificity of the newly designed primer pair was verified using 5 C. jejuni strains and 20 other bacterial strains. Sensitivity was determined to be as low as 1 genome copy per reaction. A total of 73 samples were collected at different sites along the processing line during 2 visits to a poultry slaughterhouse and were examined by direct plating onto modified charcoal cefoperazone deoxycholate agar or after enrichment in Bolton broth followed by plating on modified charcoal cefoperazone deoxycholate agar. The newly developed real-time PCR assay was used to identify the presumptive colonies as belonging to C. jejuni. A real-time PCR assay targeting 16S ribosomal RNA was also applied to determine Campylobacter spp. prevalence. Results from the real-time PCR analysis indicated considerable variability in Campylobacter contamination, with incidence rates of 72.7 and 27.6% for sampling days A and B, respectively. Campylobacter was isolated from 100% of prescalded and preeviscerated carcasses on sampling day A. In contrast, on sampling day B, the highest number of Campylobacter-positive carcasses was recovered after evisceration (60%). The chilling process significantly reduced (P < 0.05) Campylobacter population, but the percentage of positive samples on sampling day A increased to 80%. All samples collected from the processing environment, except scalding tank 3 and the prechiller and chiller tanks, were 100% positive on day A, whereas no campylobacters were isolated from machinery on sampling day B. Our results revealed the widespread of C. jejuni in poultry processing and proved that the newly developed real-time PCR assay is a simple, specific, and inexpensive method for rapid C. jejuni identification. The newly developed PCR method can be easily used in laboratories for reliable and unambiguous identification of C. jejuni in poultry samples.

Prevalence and Antibiotic Resistance Profile of Salmonella Isolates from Commercial Poultry and Poultry Farm-handlers in Jos, Plateau State, Nigeria

Aim: This study was designed to investigate the prevalence and antibiotic resistance profile of Salmonella serovars from poultry and poultry farm-handlers. Study Design: Investigative Place and Duration of Study: Samples were analyzed at the Central Diagnostic Laboratory, National Veterinary Research Institute Vom and Department of Microbiology, Ahmadu Bello University, Zaria. This work was carried out between August 2012 and April 2013. Methodology: Samples were pre-enriched in buffered peptone water followed by selective enrichment using Selenite Faeces Broth and Rappaport-Vassilidis Broth. Isolation and identification was made by inoculating the selectively enriched sample on to SalmonellaOriginal Research Article British Microbiology Research Journal, 4(4): 462-479, 2014 463 Shigella agar, Xylose Lysine Deoxycholate agar and Brilliant Green agar followed by confirmation of presumptive colonies using different biochemical tests and analytical profile index 20 E. Polyvalent (O) and (H) Salmonella antisera were used for serotyping the Salmonella isolates. The CLSI, 2010 method was used for antimicrobial susceptibility testing Results: A prevalence rate of 10.9% was observed from the 450 samples. Serovars of Salmonella detected were S. Gallinarum 57.2%, S. Typhimurium 8.2%, S. Typhi 20.4%, S. Pullorum 6.1%, S. Enteritidis 6.1% and S. Paratyphi A 2.0%. Statistically, significant difference (p<0.05) was observed between isolates and occurrence at different sample sites. The isolates were 100% resistant to oxacillin, 96.0% to ampicillin, 93.9% tylosin, 83.7 5 ceftazidime and 63.3% oxytetracycline. Five of the isolates were 100% resistant to more than five different antibiotics. There was statistical significant difference (p<0.01) in antimicrobial resistance patterns exhibited by the serovars. However, the isolates showed sensitivity to gentamycin 100%, gendox 83.7%, ciprofloxacin 81.6% and amoxicillinclavulanic acid 57.1%. Conclusion: The study revealed emergence of multiple-drug resistant Salmonella serovars from poultry and poultry farm handlers. We therefore suggest further epidemiological studies.

Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) O104 from sprouts

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O104 have been associated with sporadic cases of illness and have caused outbreaks associated with milk and sprouts. An outbreak that occurred in Europe in 2011 linked to fenugreek sprouts was caused by E. coli O104:H4 that had characteristics of an enteroaggregative E. coli (EAEC) but carried the gene that encoded for Shiga toxin 2. In this study, methods were developed for detection of this enteroaggregative STEC O104, as well as STEC O104 in sprouts. Multiplex PCR assays for enteroaggregative STEC O104:H4 targeted the stx2, aggR, and wzx104 genes, and for STEC O104 targeted the stx1-2, ehxA, and wzx104 genes. After incubating artificially contaminated sprouts at 4°C for 48h and overnight enrichment in modified buffered peptone water with pyruvate supplemented with three antibiotics (mBPWp), the pathogens were detected in all samples inoculated at a level of ca. 100CFU/25g. Several samples inoculated at lower concentrations of ca. 10CFU/25g were negative by the PCR assays, and this could have been due to cells not surviving or not being able to recover after the stress treatment at 4°C for 48h. For isolation of the pathogens, immunomagnetic separation (IMS) using magnetic beads coated with antibodies against O104 were employed, and this was followed by plating the beads onto mRBA and CHROMagar STEC O104 for isolation of E. coli O104:H4 and mRBA and CHROMagar STEC for isolation of E. coli O104:H7. Presumptive colonies were confirmed by agglutination using latex particles attached to antibodies against serogroup O104 and by the multiplex PCR assays. The methodologies described in this study for detection of enteroaggregative STEC O104:H4 and STEC O104 include the use of IMS and latex reagents for serogroup O104, and they enhance the ability to detect and isolate these pathogens from sprouts and potentially other foods, as well.

Prevalence, molecular identification and antimicrobial resistance profile of Salmonella serovars isolated from retail beef products in Mansoura, Egypt

The present study was undertaken to determine the prevalence of Salmonella in 270 raw meat samples (90 each of fresh beef, ground beef, and beef burger) purchased on nine occasions from various supermarkets and butchers' shops in Mansoura city, Egypt. Using conventional biochemical identification, Salmonella species were recovered from 23.3% (21/90), 20% (18/90), and 12.2% (11/90) of fresh beef, ground beef and beef burger samples, respectively with an overall prevalence of 18.5% (50/270) among all the meat products examined. Higher prevalence were obtained based on molecular identification, by detecting gyrB and invA genes, which verified the presence of Salmonella species in 30.0% (27/90), 26.7% (24/90), and 16.7% (15/90) of fresh beef, ground beef, and beef burger samples, respectively with an overall prevalence of 24.4% (66/270) among all the meat products tested. Of the 2635 presumptive colonies tested, 228 were biochemically identified as Salmonella, while 272 were molecularly identified as Salmonella, which were all positive for the enterotoxin (stn) virulent gene. Of the 272 serologically tested strains, 266 were serologically identified into six Salmonella serovars, while 6 strains were untypable. Salmonella Typhimurium and Salmonella Enteritidis were the most prevalent serovars with an incidence of 38.2% (104/272) and 34.6% (94/272), respectively. The other four serovars identified were Salmonella Haifa, Salmonella Muenster, Salmonella Virchow, and Salmonella Anatum were detected at lower prevalences of 11% (30/272), 7.4% (20/272), 4% (11/272) and 2.6% (7/272), respectively. Interestingly, the antimicrobial susceptibility testing indicated that all of the 100 Salmonella serovars tested were multidrug resistant (resistant to three or more antibiotics). Our findings demonstrated that the retail beef products tested were widely contaminated with multidrug-resistant Salmonella and such contamination may constitute a major public health concern.

Multidrug Resistant Pasteurella multocida Strains Isolated from Chickens with Cases of Fowl Cholera in Jos, Nigeria

5 Abstract: Antibiotic resistance is often encountered despite multiple antibiotics being used for the treatment of fowl cholera in Jos. This study was conducted to determine the antibiotic resistant profile of Pasteurella multocida isolated from chickens in Jos. A total of 2000 samples consisting of bone marrow, heart, liver, lung and spleen (400 each) were collected from 400 clinically sick chickens between November, 2010 and October, 2011 for the isolation of P. multocida. Swab from each sample was cultured on 7% defibrinated sheep blood, MacConkey and casein sucrose yeast agar. Presumptive colonies of P. multocida were subjected to biochemical characterization. Isolates identified by biochemical tests were further subjected to Microbact GNB 24E test. Disk diffusion method was employed to test the sensitivity of all the twelve P. multocida isolates confirmed by biochemical and Microbact GNB 24E test. The twelve pure isolates of P. multocida were tested for their sensitivity against fifteen different antibiotics. Drug sensitivity test conducted on P. multocida isolates showed that some of the isolates were resistant to penicillin 11 (73%), microlides 9 (60%), sulfanomides 8 (53.3%), cephalosporins 3 (20%) and other new groups of antibiotics 4 (27%). High resistance of P. multocida was recorded for ampicillin (91.7%) followed by amoxicillin/c lavulanic acid (83.3%), trimethoprim/sulfamethoxazole (66.7%), erythromycin and anicillin (58.3%) each, while tylosin was (33.3%). This study revealed that there is an emergence of multidrug resistance in some P. multocida strains among chickens in Jos, Nigeria. It is therefore recommended that antibiotic sensitivity test should be incorporated on a routine bases as part of measure to control fowl cholera and minimize the emergence of P. multocida resistance.

Performance validation of chromogenic coliform agar for the enumeration of Escherichia coli and coliform bacteria

The performance of chromogenic coliform agar (CCA) for the enumeration of Escherichia coli and coliform bacteria was validated according to ENV ISO 13843 using pure cultures and naturally contaminated water samples. The results indicate that for the detection of E.coli and coliform bacteria, respectively, the method is sensitive (94 and 91%), specific (97 and 94%), selective (selectivity -0·78 and -0·32) and efficient (96 and 92%). Relative recovery of E.coli and coliform bacteria on CCA in comparison with tryptone soy agar (TSA) was good (104 and 94% in mean, >80 and >70% in all cases), and repeatability and reproducibility were sufficient. The linear working range was defined for 10-100 total target colonies per 47-mm membrane filter. A high precision of the method was confirmed by low overdispersion in comparison with Poisson distribution. The robustness of the method with respect to the variable incubation time of 21±3h was found to be low, because an incidental increase in presumptive colonies especially between 18 and 21h was observed. In conclusion, the CCA method was proved as a reliable method for the quantification of E.coli and coliform bacteria. The international standard for the detection and enumeration of E.coli and coliform bacteria by membrane filtration (ISO 9308-1) is currently under revision and will be published in 2014. In the new standard, lactose-triphenyl tetrazolium chloride (TTC) agar will be replaced by a CCA. A performance validation of this revised method according to ENV ISO 13843 is presented in this study to determine fundamental data on its applicability and to provide reference data for secondary validation by users of this method.