Ten years of molecular epidemiology surveillance of Listeria monocytogenes in Chile 2008-2017.

Listeria monocytogenes causes severe diseases in humans, including febrile gastroenteritis and systemic infections that has a high mortality despite antibiotic treatment. This pathogen may cause massive outbreaks associated to the consumption of contaminated food products, which highlight its importance in public health. In the last decade, L. monocytogenes has emerged as a foodborne pathogen of major importance in Chile. A previous work showed that in Chile during 2008 and 2009, L. monocytogenes serotypes 1/2a, 1/2b and 4b were the most frequently identified in food and clinical strains. Here we report the molecular characterization of L. monocytogenes strains isolated from 2008 to 2017 in the country. Our results indicate that serotypes 1/2a, 1/2b and 4b continue to be the most commonly found in food products. In addition, we identify persistent and widespread PFGE subtypes. This study reports ten years of epidemiological surveillance ofL. monocytogenes in Chile.

Selection of Biodegrading Phytosterol Strains.

The phytosterol-biotransforming strains can be selected from Mycobacterium sp. using a high concentration of ß-sitosterol. The selection is made by culturing the strains in a medium enriched with 14 g/L of ß-sitosterol as the unique source of carbon. During 2 months, the bacterial cultures are transferred successively. The extraction of the biotransformation products is made with methanol and ethyl acetate. The qualitative and quantitative analyses are made by means of thin-layer chromatography, gas-liquid chromatography (GLC), and GLC-mass spectrometry. Under these conditions, it is observed that after seven transfers, the strains Mycobacterium sp. MB-3683 and Mycobacterium fortuitum B-11045 increase their biotransformation capacity from 20% to 64% and from 34% to 55%, respectively. The products in the highest proportion identified for each trial are androstenedione and androstadienedione. The results suggest that the high substrate concentration could be a selective mechanism to obtain strains more efficient in the biotransformation of ß-sitosterol into steroidal bases.

Genomic characterization of the non-O1/non-O139 Vibrio cholerae strain that caused a gastroenteritis outbreak in Santiago, Chile, 2018.

Vibrio cholerae is a human pathogen, which is transmitted by the consumption of contaminated food or water. V. cholerae strains belonging to the serogroups O1 and O139 can cause cholera outbreaks and epidemics, a severe life-threatening diarrheal disease. In contrast, serogroups other than O1 and O139, denominated as non-O1/non-O139, have been mainly associated with sporadic cases of moderate or mild diarrhea, bacteremia and wound infections. Here we investigated the virulence determinants and phylogenetic origin of a non-O1/non-O139 V. cholerae strain that caused a gastroenteritis outbreak in Santiago, Chile, 2018. We found that this outbreak strain lacks the classical virulence genes harboured by O1 and O139 strains, including the cholera toxin (CT) and the toxin-coregulated pilus (TCP). However, this strain carries genomic islands (GIs) encoding Type III and Type VI secretion systems (T3SS/T6SS) and antibiotic resistance genes. Moreover, we found these GIs are wide distributed among several lineages of non-O1/non-O139 strains. Our results suggest that the acquisition of these GIs may enhance the virulence of non-O1/non-O139 strains that lack the CT and TCP-encoding genes. Our results highlight the pathogenic potential of these V. cholerae strains.

Occurrence and antibiogram of Listeria species in raw pork, beef, and chicken meats marketed in Enugu State, Southeast Nigeria.

AIM: This study was undertaken to isolate Listeria (L.) species from raw meats sold in markets in Enugu State, Southeast Nigeria, and to determine the antibacterial resistance profile. MATERIALS AND METHODS: Twenty-five grams of beef (n=144), chicken meat (n=144), and pork (n=144) were collected randomly from supermarkets and general markets in Enugu State. Isolation of Listeria was done using half and full Fraser broths, and polymyxin acriflavine lithium chloride ceftazidime aesculin mannitol agar. Identification of isolates was done using an analytical profile index kit specific for Listeria. Confirmation of the genus Listeria was done by a polymerase chain reaction. The resistance of the isolates was determined using the disk diffusion method. RESULTS: Listeria was isolated from 39/144 (27.1%) chicken meat, 19/144 (13.2%) pork, and 66/144 (45.8%) beef samples cultured. Listeria innocua was the predominant species in chicken meat (52.6%) and beef (81.8%) samples. Listeria grayi, Listeria welshimeri, and Listeria ivanovii were also isolated from the beef and chicken meat samples. More than 65% of the isolates were resistant to penicillin, rifampicin, ciprofloxacin, sulfamethoxazole-trimethoprim, and cephalothin. All the isolates from beef and pork samples and 23 (92%) from chicken meat samples, were resistant to ≥3 classes of antibacterial agents. Mean multiple antibiotic resistance index (MARI) was 0.77 (range=0.42-1.00), 0.58 (range=0.25-0.83), and 0.79 (range=0.58-0.92) for the isolates from beef, chicken meat, and pork samples, respectively. All the isolates had MARI >0.2. CONCLUSION: Multidrug-resistant Listeria strains contaminate raw beef, pork, and chicken meats marketed in Enugu State, Southeast Nigeria.

Selection of Biodegrading Phytosterol Strains.

The phytosterol-biotransforming strains were selected from Mycobacterium sp., using a high concentration of ß-sitosterol. The selection was made by culturing the strains in a medium enriched with 14 g ß-sitosterol/L as the unique source of carbon. During 2 months, the bacterial cultures were transferred successively. The extraction of the biotransformation products was made with methanol and ethyl acetate. The qualitative and quantitative analysis was made by means of thin-layer chromatography, gas-liquid chromatography (GLC), and GLC-mass spectrometry. Under these conditions, it was observed that after seven transfers, the strains Mycobacterium sp. MB-3683 and the Mycobacterium fortuitum B-11045 increased their biotransformation capacity from 20% to 64% and from 34% to 55%, respectively. The products in the highest proportion identified for each trial were androstenedione and androstadienedione. The results suggest that the high substrate concentration could be a selective mechanism to obtain strains more efficient in the biotransformation of ß-sitosterol into steroidal bases.

Characterization of enterotoxigenic Escherichia coli strains isolated from the massive multi-pathogen gastroenteritis outbreak in the Antofagasta region following the Chilean earthquake, 2010.

In March 2010, a massive outbreak of gastroenteritis started in the region of Antofagasta (northern Chile). The outbreak was mainly attributed to Norovirus genogroup II although ETEC strains were also isolated with high frequency from clinical samples. We review this outbreak and determined that ETEC was an underestimated etiologic agent.

Characterization of Antimicrobial Susceptibility and Its Association with Virulence Genes Related to Adherence, Invasion, and Cytotoxicity in Campylobacter jejuni and Campylobacter coli Isolates from Animals, Meat, and Humans.

The aim of this research was to statistically analyze the association between antimicrobial susceptibility/resistance to erythromycine, gentamicin, ciprofloxacin, and tetracycline and 11 virulence genes associated with adherence, invasion, and cytotoxicity in 528 isolates of Campylobacter coli and Campylobacter jejuni obtained from retail meat and fecal samples from food-producing animals and human patients. A high percentage of Campylobacter strains were resistant to antimicrobials, specifically ciprofloxacin and tetracycline. Moreover, we observed a wide distribution of virulence genes within the analyzed strains. C. jejuni strains were more susceptible to antimicrobials, and showed greater number of virulence genes than C. coli strains. Genes related to invasion capability, such as racR, ciaB, and pldA, were associated with antimicrobial-susceptible strains in both species. The genes cdtA and dnaJ, a citotoxin unit and an adherence-related gene, respectively, were associated with antimicrobial-resistant strains in both species. In conclusion, Campylobacter strains show a statistically significant association between antimicrobial susceptibility and the presence of virulence genes.

Molecular epidemiology and genetic diversity of Listeria monocytogenes isolates from a wide variety of ready-to-eat foods and their relationship to clinical strains from listeriosis outbreaks in Chile.

Listeria monocytogenes is a pathogen transmitted through food that can cause severe infections in high-risk groups such as pregnant women, elderly, young children and immunocompromised individuals. It is a ubiquitous bacterium that can survive in harsh conditions, such as dry environments, at low temperatures, in brine conditions and at low pH values. It also has the capacity to form biofilms, which makes it particularly successful even in colonizing surfaces within food processing plants. This study analyzed the presence of L. monocytogenes in ready-to-eat food (RTE) such as sausage, cheese, fresh salads, and other types of raw food. 850 samples of refrigerated and packaged food collected in 2008 and 2009 were analyzed. It was found that 25% of these samples were contaminated with L. monocytogenes strains. Serotyping and virulence genes detection by polymerase chain reaction (PCR) identified that strains belonging to serotype 4b, and containing one or more genes encoded by pathogenicity island (LIPI-1), were significantly associated with specific food types. Furthermore, using pulse field gel electrophoresis (PFGE), it was possible to associate isolates from cheese with strains from clinical cases of listeriosis outbreaks that occurred during the same time period within the same geographic regions. In addition, a strong correlation was observed between isolates from frozen seafood and from clinical strains obtained from sporadic cases of listeriosis. In agreement with reports described in other countries, our results shown that Chilean strains of L. monocytogenes from food products include the most virulent serotypes, encoding for the main virulence genes of the LIPI-1, and were clonally related to clinical isolates from sporadic cases and outbreaks of listeriosis. In conclusion, we show that Chilean isolates of L. monocytogenes from RTE and raw food products can cause disease in humans, representing a public health risk that justifies permanent surveillance.

Subtractive hybridization and identification of putative adhesins in a Shiga toxin-producing eae-negative Escherichia coli.

Adherence to epithelial cells by specific adhesins is a characteristic of Shiga toxin-producing Escherichia coli (STEC) strains. The eae-encoded protein intimin is the main adhesin implicated in intestinal colonization in vivo. We recently showed that STEC strains isolated in Chile displayed a wide variety of adhesins; here we demonstrate that some of these STEC strains are eae-negative and still adhere to epithelial cells at a level 100-fold higher than enterohaemorrhagic E. coli (EHEC) O157 : H7 prototype strain EDL933. This phenotype is associated with the presence of adherence factors different from the intimin protein. Subtractive hybridization between EHEC EDL933 and STEC eae-negative strain 472-1 was used to identify regions implicated in adhesion. In addition to the saa gene, we identified 18 specific genes in STEC 472-1, 16 of which had nucleotide identity to Salmonella ST46 phage genes; the two remaining ones shared identity to a gene encoding a hypothetical protein of uropathogenic E. coli. The DNA sequence of the STEC 472-1 psu-int region identified five open reading frames with homology to phage genes. We constructed mutant strains in the saa gene and the psu-int region to study the participation of these genes in the adherence to epithelial cells and our results demonstrated that STECDeltasaa and STECDeltapsu-int mutants displayed a 10-fold decrease in adherence as compared to the STEC 472-1 wild-type strain. Overall, our results suggest that STEC strain 472-1 adheres to epithelial cells in an eae-independent matter and that saa and psu-int participate in this adhesion process.

Single multiplex PCR assay to identify simultaneously the six categories of diarrheagenic Escherichia coli associated with enteric infections.

We designed a multiplex PCR for the detection of all categories of diarrheagenic Escherichia coli. This method proved to be specific and rapid in detecting virulence genes from Shiga toxin-producing (stx(1), stx(2), and eae), enteropathogenic (eae and bfp), enterotoxigenic (st II and lt), enteroinvasive (vir F and ipa H), entero-aggregative (aaf II), and diffuse adherent (daa E) Escherichia coli in stool samples.

[Evaluation of molecular and immunoenzymatic assays for detecting Enterohemorrhagic E coli in food borne outbreaks]. / Evaluación de técnicas moleculares e inmunoenzimáticas para la detección de E coli enterohemorrágico en brotes de toxi-infecciones alimentarias.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC), is an emergent pathogen that causes sporadic infections and outbreaks of gastroenteritis associated with consumption of contaminated food products. Because detection of EHEC in diarrhea patients is not routinely performed, infection is most probably underestimated. AIM: To compare three techniques to detect EHEC: Colony hybridization with DNA probes, polymerase chain reaction (PCR) for the detection of stx1 and stx2 genes and immunoenzymatic detection by ELISA (Premier EHEC) of Stx1 and Stx2 toxins. MATERIAL AND METHODS: Four outbreaks of food-borne gastroenteritis were studied including 16 patients and 78 strains of E coli. Twenty-one (26.9%) strains, hybridized with the stx1 probe, 1 (1.3%) hybridized only with the stx2 probe and 36 (46.1%) with both probes. PCR amplification for cytotoxin genes was observed in 6 strains (7.7%) from the second outbreak studied. The immunoenzymatic assay detected the cytotoxins in 18 (23.0%), of the 78 studied strains. Agreement between probes and ELISA was 44.8%, between PCR and probes 34.7% and 82.4% between ELISA and PCR. CONCLUSIONS: These results indicate a variable yield among different EHEC detection techniques. Considering PCR as the gold standard, ELISA technique showed a better sensitivity and specificity than probes.

Multiplex PCR for diagnosis of enteric infections associated with diarrheagenic Escherichia coli.

A multiplex PCR for detection of three categories of diarrheagenic Escherichia coli was developed. With this method, enterohemorrhagic E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli were identified in fecal samples from patients with hemorrhagic colitis, watery diarrhea, or hemolytic-uremic syndrome and from food-borne outbreaks.