Adriana Bentancor

Emerging types of Shiga toxin-producing E. coli (STEC) O178 present in cattle, deer, and humans from Argentina and Germany

More than 400 serotypes of Shiga toxin-producing Escherichia coli (STEC) have been implicated in outbreaks and sporadic human diseases. In recent years STEC strains belonging to serogroup O178 have been commonly isolated from cattle and food of bovine origin in South America and Europe. In order to explore the significance of these STEC strains as potential human pathogens, 74 German and Argentinean E. coli O178 strains from animals, food and humans were characterized phenotypically and investigated for their serotypes, stx-genotypes and 43 virulence-associated markers by a real-time PCR-microarray. The majority (n = 66) of the O178 strains belonged to serotype O178:H19. The remaining strains divided into O178:H7 (n = 6), O178:H10 (n = 1), and O178:H16 (n = 1). STEC O178:H19 strains were mainly isolated from cattle and food of bovine origin, but one strain was from a patient with hemolytic uremic syndrome (HUS). Genotyping of the STEC O178:H19 strains by pulsed-field gel electrophoresis revealed two major clusters of genetically highly related strains which differ in their stx-genotypes and non-Stx putative virulence traits, including adhesins, toxins, and serine-proteases. Cluster A-strains including the HUS-strain (n = 35) carried genes associated with severe disease in humans (stx2a, stx2d, ehxA, saa, subAB1, lpfAO113, terE combined with stx1a, espP, iha). Cluster B-strains (n = 26) showed a limited repertoire of virulence genes (stx2c, pagC, lpfAO113, espP, iha). Among O178:H7 strains isolated from deer meat and patients with uncomplicated disease a new STEC variant was detected that is associated with the genotype stx1c/stx2b/ehxA/subAB2/espI/[terE]/espP/iha. None of the STEC O178 strains was positive for locus of enterocyte effacement (LEE)- and nle-genes. Results indicate that STEC O178:H19 strains belong to the growing group of LEE-negative STEC that should be considered with respect to their potential to cause diseases in humans.

Clade 8 and clade 6 strains of Escherichia coli O157:H7 from cattle in Argentina have hypervirulent-like phenotypes

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.

Profile of Shiga toxin-producing Escherichia coli strains isolated from dogs and cats and genetic relationships with isolates from cattle, meat and humans.

Pets can be reservoirs of Shiga toxin-producing Escherichia coli (STEC) strains. The aim of this study was to examine nine strains belonging to several serotypes (O91:H21, O91:H16, O178:H19, O8:H19, O22:H8, O22:HNT, ONT:H8), previously recovered from cats or dogs. To this end, we assessed a set of additional virulence genes (stx(2) subtype, subAB, ehxA, eae and saa), cytotoxic activity, and genetic relationships with strains isolated from cattle, meat and humans using pulsed-field gel electrophoresis (PFGE). Most of the isolates carried the stx(2) and/or stx(2vh-b) sequences, while only the O91:H21 isolate presented the mucus-activatable stx(2d) variant, as confirmed by sequencing the genes of subunits A and B. All the strains showed cytotoxic activity in cultured cells. One of the two O178:H19, selected for its high level of cytotoxicity in Vero cells, showed the ability to cause functional alterations in the human colon mucosa in vitro. None of the strains possessed the subAB, eae or saa genes and only the strains belonging to serotype O8:H19 carried the ehxA gene. The isolates shared 90-100% similarity by PFGE to epidemiologically unrelated strains of the corresponding serotypes recovered from cattle, meat or humans. Our results demonstrate that dogs and cats may have a role in the infection of humans by STEC, probably serving as a vehicle for bovine strains in the cycle of human infection, and thus emphasize the health risks for owners and their families.

Characterization of non -Shiga toxin- producing Escherichia coli O157 strains isolated from dogs

Shiga toxin-negative Escherichia coli O157 strains of various H types have been associated with diarrhea in children and are considered potentially pathogenic for humans. In this study, we describe non-Shiga toxin-producing E. coli O157 E. coli strains previously obtained from dogs in Argentina. Different E. coli phylogenetic lineages corresponding to flagellar types H16, H29 and H45 were identified. E. coli serotypes O157:H16 and O157:H45 contained intimin subtypes epsilon and alpha 1, respectively. Serotype O157:H45 carried the bfp gene encoding the bundle-forming pilus. Localized adherence-like patterns to HEp-2 cells were observed in O157:H16 strains, while O157:H45 adhered in a typical localized pattern. A total of eight different XbaI-pulse field electrophoresis patterns with more than 74 % similarity were identified among the nine E. coli O157:H16 strains. Our data emphasized the fact that dogs may harbor human pathogenic E. coli O157 which do not correspond to Shiga toxin-producing strains and whose potential human health hazard should not be underestimated.

Characterization of Shiga Toxin-Producing Escherichia coli Isolated from Ground Beef Collected in Different Socioeconomic Strata Markets in Buenos Aires, Argentina

Consumption of raw/undercooked ground beef is the most common route of transmission of Shiga toxin-producing E. coli (STEC). The aim of the study was to determine the STEC contamination level of the ground beef samples collected in 36 markets of different socioeconomic strata in Buenos Aires, Argentina, and the characterization of the isolated strains. Ninety-one out of 252 (36.1%) samples were stx+. Fifty-seven STEC strains were recovered. Eleven STEC strains belonged to O157 serogroup, and 46 to non-O157 serogroups. Virulence markers of the 57 STEC were stx1, 5.3% (3/57); stx2, 86.0% (49/57); stx1/stx2, 8.8% (5/57); ehxA, 61.4% (35/57); eae, 26.3% (15/57); saa, 24.6% (14/57). Shiga toxin subtypes were stx2, 31.5% (17/54); stx2c-vhb, 24.1% (13/54); stx2c-vha, 20.4% (11/54); stx2/stx2c-vha, 14.8% (8/54); stx2/stx2c-vhb, 5.6% (3/54); stx2c-vha/vhb, 3.7% (2/54). Serotypes O178:H19 and O157:H7 were prevalent. Contamination rate of STEC in all strata was high, and the highest O157 contamination was observed at low strata at several sampling rounds. Persistence of STEC was not detected. Sixteen strains (28.1%) were resistant to ampicillin, streptomycin, amikacin, or tetracycline. The STEC contamination level of ground beef could vary according to the sociocultural characteristics of the population.

Synanthropic rodents as possible reservoirs of shigatoxigenic Escherichia coli strains

Shigatoxigenic Escherichia coli (STEC) strains are worldwide zoonotic pathogen responsible for different cases of human disease including hemolytic uremic syndrome (HUS). Transmission of STEC to humans occurs through the consumption of food and water contaminated by faeces of carriers and by person-to-person contact. The objective of this study was two-fold: (1) to investigate whether synanthropic rodents are possible reservoirs of STEC in the urban area and (2) whether a particular genus out of synanthropic rodent is the principal carrier of STEC. One hundred and forty-five rodents were captured in Buenos Aires City. Screening for stx1/stx2 and rfbO157 was done by PCR from the confluence zone. STEC isolates were further characterized with biochemical tests by standard methods. Additional virulence factors (eae, ehxA, and saa) were also determined by PCR. Forty-one of the rodents were necropsied and sample of kidney and small and large intestine were taken for histopathological diagnosis. The samples sections were stained with hematoxylin-eosin, and observed by light microscopy to evaluate the systemic involvement of these species in natural infections. STEC was isolated from seven out of 27 suspect animals at screening. The following genotypes were found in the STEC strains: stx1/stx2/ehxA (1), stx2 (4), stx2/ehxA (1), stx2/ehxA/eae (1). Neither gross nor microscopic lesions compatible with those produced by Shiga toxin were observed in the studied organs of necropsied rodents. The bivariate analysis including the 145 rodents data showed that the isolation of STEC is associated positively to Rattus genus. This synanthropic species may play a role in the transmissibility of the agent thus being a risk to the susceptible population. Their control should be included specifically in actions to dismiss the contamination of food and water by STEC in the urban area, as additional strategies for epidemiological control.

Clonal relations of atypical enteropathogenic Escherichia coli O157:H16 strains isolated from various sources from several countries.

Atypical enteropathogenic Escherichia coli (aEPEC) is comprised of a large heterogeneous group of strains and serotypes that carry the intimin gene (eae) but no other EPEC virulence factors. In a previous study, we examined a few aEPEC strains of O157:H16 serotype from the U.S. and France and found these to be nearly homologous, and speculated that the same strain had been disseminated or perhaps they are part of a large clonal group that exists worldwide. To test that hypothesis, we examined additional 45 strains isolated from various sources from 4 other countries and determined that although there are a few eae-negative O157:H16 strains, most are aEPEC that carried eae and specifically, the ε-eae allele. Analysis by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing showed that as a whole, O157:H16 strains are phylogenetically diverse and have different sequence types and PFGE profiles. But the aEPEC strains within the O157:H16 serotype, regardless of the eae allele carried, are a highly conserved and homologous group of sequence type (ST)-171 strains that shared similar PFGE profiles. These aEPEC strains of O157:H16 serotype are not closely related to any of the major EPEC and enterohemorrhagic E. coli clonal lineages and appear to be part of a large clonal group that are prevalent worldwide.

The icaA gene in staphylococci from bovine mastitis

INTRODUCTION Staphylococcus aureus and coagulase-negative staphylococci (CNS) are frequently isolated from cows with mastitis. A main virulence factor of CNS is the ability to adhere and form biofilms. The intercellular gene cluster adhesion (ica) operon is one factor involved in biofilm production although ica-independent factors are also involved. Previous reports based on the results of S. epidermidis and S. aureus suggested that ica is highly conserved between species, but this detection decreases in other CNS biofilm producers. In this study we evaluated the presence of the icaA gene in strains of Staphylococcus spp. isolated from the milk of bovines with mastitis. METHODOLOGY Thirty-seven staphylococci strains were evaluated by detecting the icaA gene. A new set of PCR primers was designed by consensus region of eight staphylococci from GenBank. Species characterization was performed using the Kloos and Schleifer scheme. RESULTS We identified the presence of the gene in S. aureus (n:4), S. chromogenes (n:4), and S. sciuri strains (n:2). We also, identified the presence of the gene in S. xylosus (n:5) for the first time. The icaA gene was not detected in S. capitis (n:1), S. epidermidis (n:2), S. hominis (n:2), S. saccharolyticus (n:1), S. simulans (n:4) and S. saprophyticus (n:3). The icaA gene was detected in 40.54% (15/37) of the CNS evaluated. CONCLUSIONS Our results confirm the presence of the ica operon in various species of CNS pointing to polysaccharide intercellular adhesin (PIA) as the most important component for the formation of biofilms.

Molecular identification of coagulase-negative staphylococci isolated from bovine mastitis and detection of β-lactam resistance.

INTRODUCTION Bovine mastitis is a frequent cause of economic loss in dairy herds. Coagulase-negative staphylococci (CoNS) are increasing in importance as causeof bovine intramammary infection (IMI) throughout the world in recent years. CoNShave been isolated from milk samples collected from cows with clinical andsubclinical mastitis in several countries. Identification of mastitis pathogensis important when selection appropriate antimicrobial therapy. METHODOLOGY A total of 93 strains of Staphylococcusspp isolated from bovine clinical andsubclinical mastitis in Argentina during 2010-2013 were identified by Restriction Fragment Length Polymorphism (RFLP)-PCR using the gap gene. The isolates were tested by PCR for the presence of blaZ gene and mecA gene and were tested by disk diffusion for the susceptibilityto penicillin and cefoxitin. RESULTS The most common CoNS species was S.chromogenes 46.2% (43/93), followed by S. devriesei 11.8% (11/93) and S. haemolyticus 9.7% (9/93). The blaZ gene was detected in 19 (20.4%) but only 16 (17.2%) isolates were resistant to penicillin; the mecA was detected in6 (6.5%) isolates but only 4 (4.3) were resistant to cefoxitin. The 6 mecA-positive isolates showed oxaxillinMICs ≤ 0.5 μg/ml. DISCUSSION CoNSare important minor mastitis pathogens and can be the cause of substantial economic losses. Despite the low resistance to PEN in Argentina, the presenceof MR isolates found in this study emphasize the importance of identificationof CoNS when an IMI is present because of the potentially risk of lateraltransfer of resistance genes between staphylococcal species.

Escherichia coli in Animals

Escherichia coli is the most widely studied bacterium over the world. It is well-known that E. coli is the predominant non-pathogenic microbiota of warm blood species; however, some strains have developed the ability to cause severe diseases. Taking into account the diversity in American countries, this chapter examines the complex situation of puzzling intestinal pathogenic E. coli, also called diarrheagenic, (enteropathogenic E. coli, Shiga toxin-producing E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, enteroinvasive E. coli, diffusely adherent E. coli), and extra-intestinal E. coli (uropathogenic E. coli, neonatal meningitis-associated E. coli, avian pathogenic E. coli, sepsis-associated E. coli, mammary pathogenic E. coli, endometrial pathogenic E. coli, and necrotoxigenic E. coli) in animals. In addition to E. coli-associated animal diseases, the role of carriers and reservoirs is presented, including the last regional references from synanthropic and wild animals. Findings of the last 5 years are discussed and data of the eco-epidemiology of E. coli is also included. Considering the concept of One Health, which recognizes that health of humans is connected to health of animals and the environment, the strategies to diminish illness in human population cannot exclude control and vigilance of pathogenic strains in animals. However, in E. coli control, strategies distinguish between those strains that produce animal illness and those that affect humans and have an animal reservoir. The different proposed ways to E. coli control are also discussed.