From Farms to Markets: Gram-Negative Bacteria Resistant to Third-Generation Cephalosporins in Fruits and Vegetables in a Region of North Africa.

The role of food in human exposure to antimicrobial-resistant bacteria is a growing food safety issue. The contribution of fruits and vegetables eaten raw to this exposure is still unclear. The evaluation of contamination levels of fruits, vegetables and the agricultural environment by third-generation cephalosporin (3GC)-resistant Gram-negative bacteria was performed by analyzing 491 samples of fruits and vegetables collected from 5 markets and 7 farms in Bejaia area, north-eastern Mediterranean coast of Algeria. Ninety soil samples and 45 irrigation water samples were also sampled in farms in order to assess them as potential inoculum sources. All samples were investigated at the same time on ceftazidime-containing selective media for 3GC-resistant Gram-negative bacteria detection and on Hektoen media, for Salmonella spp. presence. The bacteria isolated (n = 30) from fruits and vegetables, soil and irrigation water collected in the farms were almost all non-fermenting bacterial species (Stenotrophomonas, Acinetobacter, Pseudomonas, Ochrobactrum) except one strain of Enterobacter cloacae and two strains of Citrobacter murliniae, isolated on one cucumber and two tomato samples in the same farm. Greater diversity in bacterial species and antimicrobial resistance profiles was observed at markets: Enterobacteriaceae (n = 41) were as strongly represented as non-fermenting bacteria (n = 37). Among Enterobacteriaceae, E. cloacae (n = 21), and Klebsiella pneumoniae (n = 13) were the most common isolates. Most of the K. pneumoniae isolates were extended-spectrum beta-lactamase (ESBL) producers (n = 11). No Salmonella spp. was recovered in any sample. This study showed that fruits and vegetables including those which may be eaten up raw constitute a reservoir of 3GC-resistant Gram-negative bacteria and multi-drug resistant-bacteria in general that can be transferred to humans through food. The general public should be informed of this hazard for health in order to encourage good domestic hygiene practices. In addition, further investigation is needed throughout the production chain to enrol professionals in actions to reduce this contamination.

MLVA for Salmonella enterica subsp. enterica Serovar Dublin: Development of a Method Suitable for Inter-Laboratory Surveillance and Application in the Context of a Raw Milk Cheese Outbreak in France in 2012.

Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) figures among the most frequently isolated Salmonella strains in humans in France. This serovar may affect production and animal health mainly in cattle herds with corresponding high economic losses. Given that the current gold standard method, pulsed-field gel electrophoresis (PFGE), provides insufficient discrimination for epidemiological investigations, we propose a standard operating procedure in this study for multiple-locus variable number tandem repeat analysis (MLVA) of S. Dublin, suitable for inter-laboratory surveillance. An in silico analysis on the genome of S. Dublin strains CT_02021853 was performed to identify appropriate microsatellite regions. Of 21 VNTR loci screened, six were selected and 401 epidemiologically unrelated and related strains, isolated from humans, food and animals were analyzed to assess performance criteria such as typeability, discriminatory power and epidemiological concordance. The MLVA scheme developed was applied to an outbreak involving Saint-Nectaire cheese for which investigations were conducted in France in 2012, making it possible to discriminate between epidemiologically related strains and sporadic case strains, while PFGE assigned only a single profile. The six loci selected were sequenced on a large set of strains to determine the sequence of the repeated units and flanking regions, and their stability was evaluated in vivo through the analysis of the strains investigated from humans, food and the farm environment during the outbreak. The six VNTR selected were found to be stable and the discriminatory power of the MLVA method developed was calculated to be 0.954 compared with that for PFGE, which was only 0.625. Twenty-four reference strains were selected from the 401 examined strains in order to represent most of the allele diversity observed for each locus. This reference set can be used to harmonize MLVA results and allow data exchange between laboratories. This original MLVA protocol could be used easily and routinely for monitoring of serovar Dublin isolates and for conducting outbreak investigations.

Contribution of Avian Salmonella enterica Isolates to Human Salmonellosis Cases in Constantine (Algeria).

An epidemiological investigation was carried out on one hundred Salmonella isolates from broiler farms, slaughterhouses, and human patients in the Constantine region of Algeria, in order to explore the contribution of avian strains to human salmonellosis cases in this region over the same period of time. The isolates were characterized by phenotypic as well as genotypic methods. A large variety of antimicrobial resistance profiles was found among human isolates, while only seven profiles were found among avian isolates. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR), Insertion Sequence 200-PCR (IS200-PCR), and Pulsed Field Gel Electrophoresis (PFGE) resulted in the allocation of the isolates to 16, 20, and 34 different profiles, respectively. The 3 genotyping methods led to complementary results by underlining the clonality of some serovars with the diffusion and persistence of a single clone in the Constantine area as well as stressing the polymorphism present in isolates belonging to other serovars, indicating the diversity of potential reservoirs of nontyphoidal Salmonella. Altogether, our results seem to indicate that nontyphoidal avian Salmonella may play an important role in human salmonellosis in the Constantine region.

Are Salmonella-Induced Gastroenteritis Neglected in Developing Countries? Feedback from Microbiological Investigations in N'Djamena Hospitals, Chad.

Salmonella is considered to be one of the main pathogens causing human gastroenteritis worldwide. Looking for Salmonella in Africa in patients suffering from gastroenteritis is rather unusual, and the use of antibiotics is not subject to any regulation. This study intends for stressing the possible prominent importance of Salmonella in digestive diseases in Africa as well as identifying antimicrobial resistance of Salmonella isolates from faeces samples of human origin. All samples were collected from five N'Djamena hospitals, from patients suffering from diarrhoea. The collecting was undertaken over two periods of six months each: from August 2010 to January 2011 and from September 2011 to February 2012. Salmonella isolates were obtained by standard cultivation and serotyping methods. A total of 43 Salmonella isolates were identified, belonging to 21 different serovars. The most prevalent serovar was Salmonella Stanleyville (n = 7), followed by S. Anatum (n = 4) and S. Kottbus (n = 3). The other serovars were under-represented. The majority of these isolates were susceptible to all antibiotics tested (CLSI Standards), except two S. Enteritidis isolates that exhibited resistance to fluoroquinolones. The different serovars and antibiotic resistance profiles that were observed highlight the substantial diversity of Salmonella in N'Djamena, Chad. Roughly, one out of ten patients who consulted for gastroenteritis was shedding Salmonella spp. and none of them would have been diagnosed outside the context of this research program. This study may encourage local clinicians to explore more often salmonellosis suspicion in their daily practice.

Foodborne outbreak and nonmotile Salmonella enterica variant, France.

We report a food-related outbreak of salmonellosis in humans caused by a nonmotile variant of Salmonella enterica serotype Typhimurium in France in 2009. This nonmotile variant had been circulating in laying hens but was not considered as Typhimurium and consequently escaped European poultry flock regulations.

Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-ß1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeß1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeß1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.

Semi-automated repetitive-sequence-based polymerase chain reaction compared to pulsed-field gel electrophoresis for Listeria monocytogenes subtyping.

Listeriosis is a severe infection that mainly affects pregnant women, neonates, and immuno-compromised adults. The commercially available semi-automated repetitive-sequence-based polymerase chain reaction assay system, DiversiLab, has been successfully used for subtyping several species of bacteria. In this article we compare the DiversiLab System with macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), which is currently the gold standard for molecular subtyping of Listeria monocytogenes. We used a panel of 116 human and food L. monocytogenes isolates for the comparative evaluation. Among these isolates, there were 4 pairs of duplicates, 13 strains were epidemiologically related, and the remaining food isolates were epidemiologically unrelated. The isolates of different serotypes represented distinct DiversiLab types (DTs) and ApaI/AscI-PFGE types except for one DT-containing isolates of two serotypes, 4b and 1/2b. The four duplicates displayed the same DT and ApaI/AscI PFGE type demonstrating the good reproducibility of the two methods. The epidemiologically related strains were clustered in the same DT and PFGE type. The Simpson's index of diversity was 0.954; 0.988; 0.994; and 0.998 for DiversiLab, AscI-PFGE, ApaI-PFGE, and AscI/ApaI-PFGE, respectively. Thus, PFGE was more discriminating than DiversiLab. However, for 1/2a serotype strains, six AscI-PFGE, three ApaI-PFGE, and one ApaI/AscI PFGE type were divided into different DTs. DiversiLab enabled a good discrimination between serotype 1/2a strains. DiversiLab is less labor intensive than PFGE and provides results in <24 hours compared with 30 hours to 3 days for PFGE from the time a pure culture of the bacteria has been obtained. On the basis of these results, DiversiLab may be useful for tracking the source of contamination in food-processing facilities and their environments. Also, DiversiLab may be more appropriate for long-term epidemiological studies where less discrimination is needed.

Evaluation of a multiplex PCR assay as an alternative method for Listeria monocytogenes serotyping.

Listeria monocytogenes serotyping is commonly used as the first level of characterisation in the epidemiological surveillance of food and clinical isolates and is therefore widely accepted. The aim of this study was to define a scheme for multiplex molecular serotyping of L. monocytogenes based on a previously described PCR assay and then to evaluate and compare this new procedure with conventional serotyping by agglutination. The study included 1204 Listeria strains collected from food products in France, from March 2005 to October 2006. Two multiplex PCR assays were designed to cluster L. monocytogenes strains into five molecular serogroups: IIa, IIb, IIc, IVa, IVb in agreement with the most commonly encountered serotypes. Amplification of the prfA gene was added to the multiplex PCR to check for L. monocytogenes species; forty-eight (4%) of the isolates tested belonged to the genus Listeria but were not L. monocytogenes. Using this first multiplex PCR, the concordance between conventional and molecular methods was 90.6%, 97.8%, 100% and 100%, for 1/2a, 1/2c, 1/2b and 4b serotypes respectively. False results were observed for some atypical 1/2a, 3a and 1/2c strains. Therefore, this lack of specificity was resolved by using an additional PCR assay based on amplification of the flaA gene, a specific target of 1/2a and 3a strains. When applying the second PCR assay to IIa and IIc molecular serogroup strains, total agreement was obtained between molecular and conventional serotyping methods with a lower level of discrimination for the molecular one. This study proposes to define a strategy for molecular serotyping using both PCR assays: a multiplex and the flaA PCR in order to assign the atypical 1/2a, 3a and 1/2c strains. Moreover, prs gene detection was added for Listeria genus recognition as a positive control in association with flaA detection. Indeed, this molecular serotyping scheme could be considered as a useful and rapid method for first-level characterisation of the most frequently encountered L. monocytogenes serotypes.

Epidemiological analysis of Salmonella enterica from beef sampled in the slaughterhouse and retailers in Dakar (Senegal) using pulsed-field gel electrophoresis and antibiotic susceptibility testing.

Seventy-eight isolates of Salmonella spp. isolated from beef sampled from the official city slaughterhouse and from retailers in Dakar, Senegal were analyzed using serotyping, antimicrobial testing and macrorestriction profiling by Pulsed-Field Gel Electrophoresis (PFGE). These analyses were done to identify clonal relationships and potential transmission routes in beef channel. XbaI macrorestriction allowed defining 17 genotypes among the six main analyzed serotypes: Salmonella bredeney (3 genotypes), S. muenster (6), S. waycross (1), S. corvallis (3), S. kentucky (1) and S. brandenburg (3). The cross analysis of PFGE profiles and origin of the beef samples reveals a wide range of contamination sources in the beef channel in Dakar. Comparison of PFGE and antimicrobial resistance types shows that the Salmonella contamination sources are equally shared by the slaughterhouse (56% of the isolates) and by the distribution channel (44% of the isolates) by handlings and houseflies.

Pulsed-field gel electrophoresis subtyping database for foodborne Salmonella enterica serotype discrimination.

Nontyphoid Salmonella is one of the main causes of bacterial gastroenteritis worldwide and is responsible for 65% of reported outbreaks of foodborne diseases in France. Serotyping is widely used for isolate preliminary identification, but it poorly discriminates strains. Rapid, efficient molecular subtyping tools have therefore been developed for the investigation of outbreaks. We evaluated the performance of the pulsed-field gel electrophoresis (PFGE) method for discrimination of 31 Salmonella serotypes frequently isolated in France. We set up a genomic database of Salmonella strains isolated from food, animals, the environment, and humans to improve the management of contamination and reactions to foodborne disease outbreaks. We studied 1128 isolates by PFGE, according to the standardized PulseNet protocol. We identified 452 PFGE patterns, 67.5% of which corresponded to a single isolate. The ability of this method to distinguish between isolates was estimated by calculating the Simpson index and the 95% confidence interval. Values obtained ranged between 0.33 (0.11-0.54) to 0.99 (0.96-1.00), depending on serotype. Epidemiological information about isolates was used for analyses of intra- and interserotype diversity results and for determining whether PFGE patterns were linked to the source of the isolate. Clustering analysis of the PFGE patterns obtained confirmed that serotype and PFGE genotype were closely linked. Some PFGE patterns were identified as major patterns, each of these patterns being found in at least 10 isolates. The database generated has already proved its effectiveness in epidemiological investigations in livestock production and foodborne outbreaks.