Characterization of commensal Escherichia coli isolates from slaughtered sheep in Mexico.

INTRODUCTION: Commensal Escherichia coli is defined as bacteria without known virulence factors that could be playing a specific role in some diseases; however, they could be responsible to disseminate antimicrobial resistance genes to other microorganisms. This study aimed to characterize the commensal E. coli isolates obtained from slaughtered sheep in the central region of Mexico. METHODOLOGY: Isolates were classified as commensal E. coli when distinctive genes related to diarrheagenic pathotypes (stx1, stx2, eae, bfp, LT, stp, ipaH, and aggR) were discarded by PCR. Identification of serotype, phylogenetic group, and antimicrobial resistance was also performed. RESULTS: A total of 41 isolates were characterized. The phylogenetic groups found were B1 in 37 isolates (90.2%), A in 2 (4.8%), and 1 isolate (2.4%) for C and D groups. Serotypes associated with diarrhea in humans (O104:H2 and O154:NM) and hemolytic uremic syndrome (O8:NM) were detected. Thirty-three isolates (80%) were resistant to ceftazidime, 23 (56%), to tetracycline 8 (19.5%) to ampicillin, and 1 to amikacin. Six isolates (14.6%) were multidrug-resistant. CONCLUSIONS: This study provides new information about commensal E. coli in slaughtered sheep, high percentages of resistance to antibiotics, and different profiles of antimicrobial resistance were found, their dissemination constitute a risk factor towards the consuming population.

Identification, HPG2 Sequence Analysis, and Antimicrobial Susceptibility of Avibacterium paragallinarum Isolates Obtained from Outbreaks of Infectious Coryza in Commercial Layers in Sonora State, Mexico.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Nota de investigación­Análisis de secuencias de la región HPG2 y susceptibilidad antimicrobiana de aislamientos de Avibacterium paragallinarum obtenidos de brotes de coriza infecciosa en aves de postura comerciales en el estado de Sonora, México. Este es el primer informe extenso sobre la identificación y caracterización de aislamientos de Avibacterium paragallinarum (AVP) obtenidos de brotes de coriza infecciosa (IC) de parvadas de ponedoras vacunadas con coriza infecciosa en el estado de Sonora en México. Los aislamientos obtenidos de los brotes de coriza infecciosa durante los años 2007, 2014, 2015, 2017 y 2019 se identificaron mediante una prueba de PCR convencional y el análisis del gene de ARNr 16S, se serotipificaron mediante el método de Page y se genotipificaron mediante el análisis parcial de secuencias descrito recientemente de la región HPG2. Además, se determinaron los perfiles de susceptibilidad a los antimicrobianos mediante la prueba de concentración mínima inhibitoria (MIC) que ha sido mejorada recientemente. La prueba de PCR convencional y los análisis de secuencias del gene ARNr 16S confirmaron que los aislados eran A. paragallinarum. Los resultados de la serotipificación mostraron la participación de aislamientos pertenecientes a los serotipos A, B y C en los brotes de coriza infecciosa. La genotipificación de la región HPG2 reveló la presencia de secuencias del tipo (ST) 1, ST4 y ST11, de los cuales este último también ha sido identificada en Europa. La prueba de susceptibilidad por concentración mínima inhibitoria mostró que todos los aislados analizados eran susceptibles a la mayoría de los antimicrobianos analizados, incluida la eritromicina y la tetraciclina, que son antibióticos importantes para el tratamiento contra la coriza infecciosa. La situación de coriza infecciosa en el estado de Sonora, México, es compleja por la presencia de los serotipos A, B y C. Este hallazgo enfatiza la importancia de la bioseguridad en combinación con la aplicación de los programas de vacunación óptimos en el control de la coriza infecciosa en el estado de Sonora, México.

In vitro transduction of antimicrobial resistance genes into Escherichia coli isolates from backyard poultry in Mexico.

The transmission of multidrug-resistant pathogens and antimicrobial resistance genes is an emerging problem involving multiple factors (humans, domestic animals, wildlife). The aim of this study was to investigate the presence of Escherichia coli isolates with different antimicrobial resistance genes from backyard poultry and to demonstrate the in vitro transduction phenomenon of these genes between phages from migratory wild birds and poultry E. coli isolates. We collected 197 E. coli isolates from chickens, turkeys, and ducks in backyard production units (northern region of the State of Mexico). Isolates were resistant to ampicillin (80.7%), tetracycline (64.4%), carbenicillin (56.3%), and nalidixic acid and trimethoprim-sulfamethoxazole (both, 26.9%). Moreover, the genes blaTEM (56.3%), tetB (20.8%), tetA (19.2%), sulI (7.6%), sulII (10.1%), qnrA (9.6%), and qnrB (5.5%) were found. In vitro transduction using phages from migratory wild birds sampled in the wetland Chimaliapan (State of Mexico) was successfully achieved. It was possible to transduce qnrA, tetB, blaTEM, and sulII genes to E. coli isolates from poultry. This is the first report that describes the transduction of antimicrobial resistance genes from phages of migratory wild birds to poultry and suggests the possible transmission in backyard production units.

Haemoparasites in endemic and non-endemic passerine birds from central Mexico highlands.

Haemosporidian parasites of birds are found worldwide and include the genera Haemoproteus, Plasmodium and Leucocytozoon. Infection with haemosporidian parasites can affect host physical condition and reproductive success. The aim of this study was to identify the blood parasites and parasitaemia in endemic and non-endemic passerine birds from central Mexico highlands. This study included 157 passerines representing 29 species from 17 families. Overall, 30.6% (48/157) of the birds were infected with blood parasites. Of those, Haemoproteus spp. were found in 14.0% (n = 22), Leucocytozoon spp. 12.1% (n = 19) and microfilariae 0.6% (n = 1). Blood parasites were found in 71.4% (5/7) of endemic bird species and 45.4% (10/22) of non-endemic species. Medium to high parasitaemia (number of parasites/number erythrocytes) was observed in birds with infections of Haemoproteus spp. and Leucocytozoon spp. Co-infections 3.8% (n = 6) were observed in two species of endemic birds. This study contributes to the knowledge of haemoparasites in endemic and non-endemic passerine birds from central Mexico highlands. Additional investigation on the molecular identification of haemosporidian parasites, pathogenicity and health status of these birds is necessary.

Serotyping, Genotyping, and Antimicrobial Susceptibility of Ornithobacterium rhinotracheale Isolates from Mexico.

The bacterium Ornithobacterium rhinotracheale is associated with respiratory disease and septicemia in poultry. In this study, 9 reference strains and a total of 23 isolates of O. rhinotracheale from respiratory diseased poultry from Mexico were serotyped and genotyped. Furthermore, the antimicrobial susceptibility of isolates and reference strains of O. rhinotracheale were determined. All isolates belong to serotype A and showed a clonal relationship. All reference strains and isolates were resistant to colistin, fosfomycin, gentamicin, kanamycin, streptomycin, and trimethoprim-sulfamethoxazole. These results should eventually be helpful in planning strategies for the control of O. rhinotracheale infections in poultry in Mexico.

Distribution of lymphocytes, immunoglobulin-containing cells, macrophages, and dendritic cells in the accessory sex glands of rams experimentally infected with actinobacillus seminis / Distribuição de linfócitos, células carreadoras de imunoglobulinas, macrófagos e células dendríticas em glândulas sexuais acessórias de carneiros infectados experimentalmente com actinobacillus seminis

The distribution of cells involved in the immune response in accessory sex glands of rams experimentally infected with Actinobacillus seminis was studied. Twelve one-year old rams were experimentally infected by intraurethral (IU) (n=4) and intraepididymal (IE) (n=4) route, and four control (CON) animals were used. The animals were slaughtered 35 days post-inoculation, samples were taken from accessory sex glands, and bacteriology and histopathology tests were performed. The presence of CD4, CD8 and TCRγδ (WC1) lymphocytes, CD45RO cells, macrophages (CD14), dendritic cells (CD1b), IgA-, IgG- and IgM-containing cells (IgCC) was determined. Animals of the IE group developed clinical epididymitis. No lesions were seen in rams of the IU group; two of the intraepididymal inoculated CON developed small lesions in the epididymis. A. seminis isolates were achieved from 6:16 (37.5%) accessory sex glands in the IE group, but not in the IU and CON groups. In the CON group, IgA- and IgM- containing cells predominated in the bulbourethral glands and the disseminated prostate, and they were scarce or null in the vesicles and ampullae. A significant increase of IgA-, IgG- and IgM- containing cells was confirmed in the seminal vesicles, the ampullae and the bulbourethral glands in the IE group. In the IE and IU groups, an increase in CD4, CD8, WC1, CD45RO and CD14 was evidenced in the vesicles and ampullae. CD1b dendritic cells were present in the ampullae and vesicles with inflammatory processes. A. seminis triggered a local immune response in the IE and IU groups. These results indicate a different pattern of infiltrating immune cells in the accessory sex glands of infected A. seminis rams.

A distribuição das células envolvidas na resposta imune em glândulas sexuais acessórias de carneiros experimentalmente infectados com Actinobacillus seminis foi estudada. Doze carneiros de um ano de idade foram experimentalmente infectados via intrauretral (IU) (n=4) e via intraepididimal (IE) (n=4) e quatro animais controles (CON) foram utilizados. Os animais foram abatidos 35 dias após a inoculação, amostras foram retiradas das glândulas sexuais acessórias e testes bacteriológicos e histopatológicos foram realizados. A presença de linfócitos CD4, CD8 e TCRγδ (WC1), células CD45RO, macrófagos (CD14), células dendríticas (CD1b) e células contendo IgA, IgG and IgM (IgCC) foi determinada. Os animais do grupo IE desenvolveram epididimite clínica. Não foram visualizadas lesões nos carneiros do grupo IU, dois dos CON inoculados intraepididimalmente desenvolveram pequenas lesões no epidídimo. Isolados de A. seminis foram obtidos de 6:16 (37,5%) nas glândulas sexuais acessórias no grupo IE mas não nos grupos IU e CON. No grupo CON células contendo IgA and IgM predominaram nas glândulas bulbouretrais e na próstata e foram escassas ou ausentes nas vesículas e na ampola. Um incremento significativo de células contendo IgA, IgG and IgM foi confirmado nas vesículas seminais, na ampola e nas glândulas bulbouretrais no grupo IE. Nos grupos IE e IU foi evidenciado um aumento em CD4, CD8, WC1, CD45RO e CD14 nas vesículas e ampola. As células dendríticas CD1b estavam presentes na ampola e nas vesículas com processo inflamatório. A. seminis induziu uma resposta imune local nos grupos IE e IU. Estes resultados indicam um padrão diferente de células imunes infiltrantes nas glândulas sexuais acessórias de carneiros infectados por A. seminis.

Determination of extended spectrum ß-lactamases/AmpC ß-lactamases and plasmid-mediated quinolone resistance in Escherichia coli isolates obtained from bovine carcasses in Mexico.

Food-borne bacterial infections have worldwide importance, and a great variety of antibiotic resistance mechanisms, mainly of the chromosome type, have rapidly developed. Antimicrobial resistance was determined in this study in terms of the presence of extended-spectrum ß-lactamases (ESBLs), plasmid AmpC ß-lactamases (pAmpC), and plasmid-mediated quinolone resistance (PMQR) from 155 Escherichia coli isolates obtained from bovine carcasses from two states in Mexico (states of Mexico and Jalisco). Isolates were challenged with ß-lactam antimicrobials (ampicillin, ceftazidime, and cefotaxime) and quinolones (nalidixic acid and ciprofloxacin). The presence of the bla TEM, bla SHV, bla CTX-M, bla OXA , bla CMY, bla MOX, bla LAT, bla BIL, qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA genes was examined by PCR. Clonal relationship was determined using pulsed-field gel electrophoresis (PFGE). The highest resistance was found to be to nalidixic acid (64 %), followed by ampicillin (32 %), ciprofloxacin (10 %), and ceftazidime and cefotaxime (both 1.3 %). bla CMY (n = 1), bla TEM (n = 24), qnrB (n = 9), and qnrS (n = 7) genes were detected. PFGE analysis showed that the majority of isolates had a different genotypic profile. To our knowledge, this is the first report of the presence of the qnrB, qnrS, and bla CMY genes in E. coli isolated from bovine meat in Mexico.

Genetic characterization of Escherichia coli isolated from cattle carcasses and feces in Mexico state.

Meat of bovine origin is one of the major vehicles in the transmission of verotoxigenic Escherichia coli (VTEC) to human consumers. This pathogen can produce serious human illness, including bloody diarrhea and hemolytic uremic syndrome. The aim of the current study was to characterize E. coli isolates (mainly VTEC strains) belonging to several serotypes in samples from cattle carcasses and feces of three municipal slaughter plants from Mexico State. The genetic diversity and molecular relatedness among the isolates was evaluated with multiple-locus variable-number tandem repeat analysis (MLVA). To our knowledge, and with the exception of E. coli O157:H7, this is the first time that serotypes analyzed here have been subtyped by MLVA in Mexico. MLVA typing grouped the 37 strains from this study into 30 distinct genotypes, 26 of which were unique. These findings indicate that cattle carcasses and feces from slaughter plants in Mexico are a source of VTEC that are genetically diverse in terms of serotypes and virulence profiles. The presence of these pathogens in carcasses indicates the high probability of the spread of VTEC strains during slaughter and processing.

Presencia de integrones clase I en Escherichia coli aislada de productos cárnicos en plantas Tipo Inspección Federal (TIF) en el Estado de México / Presence of class I integrons in Escherichia coli isolated from meat products in Federal Inspection Type (TIF) plants in the Estado de Mexico

Meat foods are the main vehicle of foodborne diseases as a result of poor handling during processing. The objective of this study was to determine the frequency and antibiotic resistance factors of Escherichia coli in TIF plants of the Estado de Mexico. For this, 3 Federal Inspection Type (TIF) plants in Mexico were analyzed, with n = 90 samples, 10 raw meat product (beef, pork and turkey meat), 10 finished meat product and 70 work tools. Eighteen (20%) E. coli strains were isolated (3 raw meat product, 2 finished meat products and 13 work tools (P > 0.05). The E. coli isolates showed high levels of resistance to ampicillin (88.8%), cephalothin (88.8%), carbenicillin (83.3%) and chloramphenicol (61.1%). There was a relationship between E. coli strains resistant to ampicillin and chloramphenicol and presence of resistance genes Pse-1 4/18 (22%) and floR 4/18 (22%). Five (55.5%) positive isolates to Pse-1 and floR, also exhibit the Cs3 Cs5 genes for the class I integrons. The results indicate that antimicrobial resistance and genetic resistance factors are present in Escherichia coli isolated from food processing plants, suggesting that they can be transmitted to the intestine microbiota of human population by contamination and consumption of improperly processed products and become a risk factor for public health.

Los alimentos cárnicos constituyen uno de los principales vehículos de enfermedades transmitidas por alimentos, como consecuencia de un manejo deficiente durante su procesamiento. El objetivo del presente trabajo fue determinar la frecuencia de algunos factores de resistencia antibiótica de Escherichia coli en plantas Tipo Inspección Federal (TIF) del Estado de México. Para este fin se analizaron muestras de tres plantas TIF en el Estado de México (n = 90), 10 de materia prima (carne de bovino, cerdo y pavo), 10 de producto terminado y 70 de utensilios de trabajo. Se aislaron 18 (20%) cepas de E. coli, 3 de materia prima, 2 de producto terminado y 13 de utensilios de trabajo (P > 0.05). Las E. coli aisladas presentaron una frecuencia alta de resistencia a ampicilina (88.8%), cefalotina (88.8%), carbencilina (83.3%) y cloranfenicol (61.1%). Se encontró una relación entre las cepas de E. coli resistentes a ampicilina y cloranfenicol y la presencia de genes de resistencia Pse-1 4/18 (22%) y floR 4/18 (22%). Cinco (55.5%) aislamientos positivos a Pse-1 y floR también presentaron el gen Cs3 Cs5 del integrón clase I. Los resultados indican que la resistencia antimicrobiana y los factores de resistencia genéticos están presentes en Escherichia coli aislada de plantas procesadoras de alimentos, lo que sugiere que estos elementos pueden transmitirse a la microbiota intestinal de la población humana a través de la contaminación y consumo de productos mal procesados, y ser un factor de riesgo para la salud pública.

Phenotypic-genotypic resistance in Salmonella spp. isolated from cattle carcasses from the north central zone of the State of Mexico.

Salmonella is a public and animal health problem due to the generation of strains multiresistant to antimicrobial products. The objective of this study was to determine prevalence and antimicrobial phenotypic and genotypic resistance of Salmonella spp. isolated from beef cattle carcasses killed in slaughterhouses of the north central zone of the State of Mexico. Sampling was carried out according to the European Directive 2001/471/EC; isolation and identification of the strain was carried out according to the Mexican Official Standard NOM-114-SSA1-1994; resistance was established by CMI according to the National Committees for Clinical Laboratory Standards (NCLS) and multiplex PCR according to Ahmed et al. (Journal of Applied Microbiology 106:402-409, 2009) with PSE-1, tetG, qnrS, FloR, STR, and sul1 oligonucleotides. Twenty-seven strains of Salmonella spp. were obtained from 327 samples (prevalence of 0.083); 19 strains (70 %) were resistant to 10 µg/ml of ampicillin, 15 of these (79 %) had the PSE-1 gene; 22 strains (84 %) were resistant to 30 µg/ml streptomycin, 14 of these (63.6 %) had the STR gene. Genes PSE-1 and STR were factors in the presence of resistance, the rest of the genes (tetG, qnrS, FloR, and sul1) were not factors of resistance in the studied strains.

Resistencia antibiótica de genotipos de cepas de Salmonella spp de cerdos sacrificados en rastros del Estado de México / Antibiotic resistance of Salmonella spp strain genotypes isolated from pigs slaughtered at abattoirs in the Estado de Mexico

Multiresistant Salmonella serovar Typhimurium strains are a worldwide problem in animal and human health. The aim of this study was to determine the frequency of some Salmonella spp resistance genes (cmlA/tetR, PSE-1, TEM, Sip B/C) in strains isolated from pigs slaughtered at abattoirs in the Estado de Mexico. Of 87 analyzed strains 22 (25.28%) had phenotypical resistance to chloramphenicol (30 μg), 15 (17.24%) to ampicillin (10 μg) and 54 (62.07%) to sulfamethoxazole (60 μg). The phenotypical and genotypical relation of the 87 strains was: of the 22 chloramphenicol resistant strains only 14 (63.63%) expressed the cmlA/tetR resistance gene, and of the 65 strains non-resistant to chloramphenicol only 36 (55.38%) expressed the cmlA/tetR resistance gene. Regarding the 15 ampicillin resistant strains only 2 (13.33%) were carriers of the PSE-1 gene and 7 (46.66%) presented the TEM gene; both genes confer genotypical ampicillin resistance. Of 72 non-resistant ampicillin strains, 11 (15.27%) carried the TEM gene which confers ampicillin resistance. Two Salmonella strains (2.28%) belonged to phagotype DT104. Strains not showing phenotypical resistance but carrying resistance genes have not been exposed to selection by competition, although they possess the mechanism to express such resistance.

La aparición de cepas multirresistentes de Salmonella Typhimurium es un problema mundial, tanto en salud animal como en salud pública. El objetivo del presente trabajo fue determinar la frecuencia de algunos genes de resistencia (cmlA/tetR, PSE-1, TEM, Sip B/C) en cepas de Salmonella spp aisladas de cerdos en rastros del Estado de México. De las 87 cepas analizadas, 22/87 (25.28%) mostraron resistencia al cloranfenicol (30 μg), 15/87 (17.24%) a la ampicilina (10 μg) y 54/87 (62.07%) fueron resistentes al sulfametoxazol (60 μg). La relación fenotípica y genotípica de las 87 cepas analizadas fue: de las 22 cepas que presentaron resistencia fenotípica al cloranfenicol, sólo 14/22 (63.63%) expresaron el gen de resistencia cmlA/tetR, y de las 65 cepas que manifestaron sensibilidad al cloranfenicol, 36/65 (55.38%) expresaron el gen de resistencia cmlA/tetR. De las 15 cepas que expresaron resistencia a la ampicilina, sólo 2/15 (13.33%) mostraron el gen PSE-1, y 7/15 (46.66%) presentaron el gen TEM, ambos genes confieren resistencia genotípica a la ampicilina. De las 72 cepas que manifestaron sensibilidad a la ampicilina, 11 (15.27%) mostraron el gen TEM, el cual da resistencia a la ampicilina. De las 87 cepas de Salmonella sólo 2/87 (2.28%) expresaron el fagotipo DT104. Las cepas que son portadoras de genes de resistencia, pero no la manifiestan fenotípicamente, no han sido expuestas a una selección por competencia, por lo tanto, no expresan la resistencia fenotípica, pero cuentan con el mecanismo necesario para expresarla.

In vitro evaluation of the binding capacity of Saccharomyces cerevisiae Sc47 to adhere to the wall of Salmonella spp.

Saccharomyces cerevisiae Sc47 (Biosaf) is a commercially available baker's yeast strain (Lesaffre, France) that has been used as a probiotic in animal nutrition. It has been previously reported that animals fed with the yeast showed an improved resistance to several enteric infectious diseases. Some of the S. cerevisiae strains adhere potentially pathogenic bacteria such as Escherichia coli and Salmonella spp. This could be a mechanism through which animals fed with the yeast may become more resistant to infections caused by these microorganisms. In this paper, the adhesion of forty-five Salmonella spp. isolates to Sc47 was assessed by sedimentation and agglutination tests, and by light and electron microscopy. Results showed that 57.7% (26/45) of the isolates and 66.6% (6/9) of the Salmonella serovars tested adhered to the Sc47 cell wall.