Comprehensive evaluation of abattoirs with no Hazard Analysis Critical Control Point plan in Tucumán,Argentina / Evaluación integral de frigoríficos sin un plan de análisis de peligros y puntos críticos de control en Tucumán, Argentina

Abstract This work focused on the comprehensive study of two provincial transit abattoirs inTucumán, Argentina, with no Hazard Analysis Critical Control Point (HACCP) plan. Visits (n = 20)were conducted between 2016 and 2018 during the operational and post-operational processes.Risk was estimated and the bacteriological analysis of carcass and environmental samples wasperformed. Risk estimation showed the predominance of high risk in both abattoirs. The maindeviations from the HACCP plan were: deficient building conditions, deficient workflow, lack of sectorization of changing rooms and bathrooms, lack of implementation of Standardized Sanitary Operational Procedures, and no food safety training of workers. The counts of indi-cator microorganisms from both abattoirs were not significant. Salmonella spp. was isolated from 7.5% carcass and 7.3% environmental samples. The Salmonella serovars identified were Cerro, Corvallis, Havana and Agona. Shiga toxin (stx) genes were detected in 24.4% carcass and 30.9% environmental samples. The isolates were characterized as Escherichia coli O8:H7/stx1, O116:H49/stx2 and O136:H40/stx2. Based on these results, it would be possible to implement an improvement plan in Tucumán abattoirs together with the local health authorities. Still, the need to work jointly with the sanitary authority in search of a unique sanitary standard for Argentina remains unaddressed.

Resumen Este trabajo se centró en el estudio integral de dos frigoríficos de tránsito provincial en Tucumán, Argentina, carentes de un plan de análisis de peligros y puntos críticos de control (HACCP, por sus siglas en inglés). Las visitas (n = 20) se realizaron entre 2016 y 2018 durante los procesos operativos y posoperativos. Se realizó la estimación del riesgo y el análisis bacteriológico de medias reses y muestras ambientales. La estimación del riesgo demostró un predominio de riesgo alto en ambos frigoríficos. Las principales desviaciones del plan HACCP fueron las deficientes condiciones edilicias, un inadecuado flujo de trabajo, la falta de sectorización de vestuarios y banños, una implementación nula de procedimientos operativos estandarizados de saneamiento y una insuficiente capacitación en seguridad alimentaria de los operarios. Los recuentos de microorganismos indicadores de ambos frigoríficos no presentaron diferencias significativas. Salmonella spp. se aisló del 7,5% de muestras de medias reses y del 7,3% de muestras ambientales. Se identificaron las siguientes serovariedades de Salmonella: Cerro, Corvallis, Havana y Agona. Se detectaron genes de toxina Shiga (sfx) en el 24,4% de las muestras de medias reses y en el 30,9% de las muestras ambientales. Los aislamientos se caracterizaron como Escherichia coli O8:H7/sfx1, O116:H49/sfx2 y O136:H40/sfx2. Teniendo en cuenta estos resultados, sería posible implementar un plan de mejoramiento en frigoríficos de Tucumán conjuntamente con las autoridades locales de salud. Aun así, sigue sin abordarse la necesidad de trabajar en vinculación con las autoridades sanitarias en la búsqueda de una norma integrada única para Argentina.

Occurrence and genetic characterization of Shiga toxin-producing Escherichia coli on bovine and pork carcasses and the environment from transport trucks.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens causing severe diseases. The ability of STEC to produce disease is associated with Shiga toxin (Stx) production. We investigated the occurrence of STEC on bovine and pork carcasses and walls of trucks where they were transported, and we characterized virulence genes and serotypes of STEC strains. We compared the whole genomic sequencing of a STEC O157:H7 strain isolated from a bovine carcass in this work and a STEC O157:H7 strain isolated from a child with HUS, both isolated in 2019. We studied the relationship between these isolates and others collected in the database. The results show a 40% of STEC and two different serogroups were identified (O130 and O157). STEC O157:H7 were isolated from bovine carcasses and harbored stx2, eae, ehxA, katP, espP, stcE, ECSP_0242/1773/2687/2870/2872/3286/3620 and were classified as lineage I/II. In STEC non-O157 isolates, three isolates were isolated from bovine carcasses and harbored the serogroup O130 and one strain isolated from pork carcasses was O-non-typeable. All STEC non-O157 harbored sxt1 gene. The analysis from the whole genome showed that both STEC O157:H7 strains belonged to the hypervirulent clade 8, ST11, phylogroup E, carried the allele tir 255 T > A T, and they were not clonal. The analysis of information allows us to conclude that the STEC strains circulate in pork and bovine carcasses arriving in transport. This situation represents a risk for the consumers and the need to implement an integrated STEC control in the food chain.

Comprehensive evaluation of abattoirs with no Hazard Analysis Critical Control Point plan in Tucumán, Argentina.

This work focused on the comprehensive study of two provincial transit abattoirs in Tucumán, Argentina, with no Hazard Analysis Critical Control Point (HACCP) plan. Visits (n=20) were conducted between 2016 and 2018 during the operational and post-operational processes. Risk was estimated and the bacteriological analysis of carcass and environmental samples was performed. Risk estimation showed the predominance of high risk in both abattoirs. The main deviations from the HACCP plan were: deficient building conditions, deficient workflow, lack of sectorization of changing rooms and bathrooms, lack of implementation of Standardized Sanitary Operational Procedures, and no food safety training of workers. The counts of indicator microorganisms from both abattoirs were not significant. Salmonella spp. was isolated from 7.5% carcass and 7.3% environmental samples. The Salmonella serovars identified were Cerro, Corvallis, Havana and Agona. Shiga toxin (stx) genes were detected in 24.4% carcass and 30.9% environmental samples. The isolates were characterized as Escherichia coli O8:H7/stx1, O116:H49/stx2 and O136:H40/stx2. Based on these results, it would be possible to implement an improvement plan in Tucumán abattoirs together with the local health authorities. Still, the need to work jointly with the sanitary authority in search of a unique sanitary standard for Argentina remains unaddressed.

Carbapenemase-Producing Extraintestinal Pathogenic Escherichia coli From Argentina: Clonal Diversity and Predominance of Hyperepidemic Clones CC10 and CC131.

Extraintestinal pathogenic Escherichia coli (ExPEC) causes infections outside the intestine. Particular ExPEC clones, such as clonal complex (CC)/sequence type (ST)131, have been known to sequentially accumulate antimicrobial resistance that starts with chromosomal mutations against fluoroquinolones, followed with the acquisition of bla CTX-M-15 and, more recently, carbapenemases. Here we aimed to investigate the distribution of global epidemic clones of carbapenemase-producing ExPEC from Argentina in representative clinical isolates recovered between July 2008 and March 2017. Carbapenemase-producing ExPEC (n = 160) were referred to the Argentinean reference laboratory. Of these, 71 were selected for genome sequencing. Phenotypic and microbiological studies confirmed the presence of carbapenemases confirmed as KPC-2 (n = 52), NDM-1 (n = 16), IMP-8 (n = 2), and VIM-1 (n = 1) producers. The isolates had been recovered mainly from urine, blood, and abdominal fluids among others, and some were from screening samples. After analyzing the virulence gene content, 76% of the isolates were considered ExPEC, although non-ExPEC isolates were also obtained from extraintestinal sites. Pan-genome phylogeny and clonal analysis showed great clonal diversity, although the first phylogroup in abundance was phylogroup A, harboring CC10 isolates, followed by phylogroup B2 with CC/ST131, mostly H30Rx, the subclone co-producing CTX-M-15. Phylogroups D, B1, C, F, and E were also detected with fewer strains. CC10 and CC/ST131 were found throughout the country. In addition, CC10 nucleated most metalloenzymes, such as NDM-1. Other relevant international clones were identified, such as CC/ST38, CC155, CC14/ST1193, and CC23. Two isolates co-produced KPC-2 and OXA-163 or OXA-439, a point mutation variant of OXA-163, and three isolates co-produced MCR-1 among other resistance genes. To conclude, in this work, we described the molecular epidemiology of carbapenemase-producing ExPEC in Argentina. Further studies are necessary to determine the plasmid families disseminating carbapenemases in ExPEC in this region.

Identification of Two New Sequences of Flagellin-Encoding Gene in Escherichia coli Using PCR and Sequencing-Based Methods.

Escherichia coli has traditionally been serotyped using antisera against the O and H antigens. However, a proportion of E. coli isolates are nonmotile and, in addition, some isolates do not react with the currently available H-typing sera. Alternative molecular methods have been developed based on the detection of genes encoding for H antigens. In this study, we studied 13 serologically nontypable H antigen E. coli strains using polymerase chain reaction (PCR) and sequencing-based methods. We found two new sequences of flagellin-encoding gene, for each of which a specific antiserum was produced to confirm their expression. Sequencing of the flagellin gene offers a rapid determination of E. coli H antigens and could be used to detect potential novel flagellar antigens.

Diversity of Potentially Pathogenic Escherichia coli O104 and O9 Serogroups Isolated before 2011 from Fecal Samples from Children from Different Geographic Regions.

In 2011, an outbreak of hemorrhagic colitis and hemolytic uremic syndrome (HUS) was reported in Europe that was related to a hybrid STEAEC of Escherichia coli (E. coli) O104:H4 strain. The current study aimed to analyze strains of E. coli O104 and O9 isolated before 2011. The study included 47 strains isolated from children with and without diarrhea between 1986 and 2009 from different geographic regions, as well as seven reference strains. Serotyping was carried out on 188 anti-O and 53 anti-H sera. PCR was used to identify DEC genes and phylogenetic groups. Resistance profiles to antimicrobials were determined by diffusion in agar, while PFGE was used to analyze genomic similarity. Five serotypes of E. coli O104 and nine of O9 were identified, as well as an antigenic cross-reaction with one anti-E. coli O9 serum. E. coli O104 and O9 presented diarrheagenic E. coli (DEC) genes in different combinations and were located in commensal phylogenetic groups with different antimicrobial resistance. PFGE showed that O104:H4 and O9:(H4, NM) strains from SSI, Bangladesh and México belong to a diverse group located in the same subgroup. E. coli O104 and O9 were classified as commensal strains containing DEC genes. The groups were genetically diverse with pathogenic potential making continued epidemiologic surveillance important.

Characterization and molecular subtyping of Shiga toxin-producing Escherichia coli strains in provincial abattoirs from the Province of Buenos Aires, Argentina, during 2016-2018.

We characterized Shiga toxin-producing Escherichia coli (STEC) O157 (n = 20) and non-O157 (n = 68) isolated from carcasses (n = 54), the environment (n = 20), head meat (n = 3) and viscera washing and chilling water (n = 11) in provincial abattoirs before and after implementing improvement actions. The strains were tested for eae, saa, ehxA and fliCH7 genes. Variants stx1 and stx2 were also determined. Pulsed-field gel electrophoresis (PFGE) was carried out with restriction enzymes XbaI and BlnI. All twenty O157 STEC strains [H7; H21; HNM] carried genes rfbO157 and ehxA; 90.0 % were positive for eae and 15.0 % were negative for fliCH7 and positive for saa. Results of PFGE showed 17 XbaI patterns, of which 14 were unique and three formed clusters. From the 68 non-O157 STEC strains, 66.2 %, 55.9 % and 2.9 % were positive for ehxA, saa and eae genes, respectively. Fifty-three XbaI patterns were obtained (49 unique and four forming clusters). Cross-contamination between products and between the environment and products was confirmed in all abattoirs. While the proposed improvements reduced the risk of contamination, Good Hygiene Practices and Good Manufacturing Practices should be implemented in provincial abattoirs, stressing the importance of having a uniform national food safety standard.

Comprehensive evaluation and implementation of improvement actions in bovine abattoirs to reduce pathogens exposure.

The slaughter process plays an important role in animal welfare, meat quality, safety and public health through the meat production chain. In this study, we performed a three-stage evaluation: I) comprehensive evaluation, II) implementation of improvement actions and III) verification of the success of the actions implemented in three abattoirs from Argentina during 2016-2018. Risk was estimated using two checklists, quantified on a 1-100 scale and classified as high (1-40), moderate (41-70) and low (71-100). In stages I and III, Salmonella spp., E. coli O157:H7 and non-O157 STEC were detected and isolated in samples from carcasses (n = 252), the environment (n = 252); head meat (n = 21) and viscera washing and chilling water (n = 105). Carcass samples were analyzed for mesophilic aerobic organisms, coliforms and E. coli enumeration. Of 201 water samples taken, 42.0-75.6 % were non-potable quality. After the implementation of improvement actions in stage II (building, processes, systems for water purification and training), the estimation of risk of contamination was reduced from high to moderate in all three abattoirs, the count of indicator microorganisms decreased in two abattoirs, and the presence of pathogens significantly decreased. Salmonella spp. was not isolated from any of the samples collected in two abattoirs. Isolation of E. coli O157:H7 decreased in carcass and was not isolated from viscera washing and chilling water. Isolation of non-O157 STEC decreased in carcass but not in environmental samples. Finally, 75.0-95.0 % of water samples were of potable quality. Although this was only the first step in the process of change and improvement of abattoirs, the assessment of the situation and the proposal of solutions to correct deviations in a joint effort with the health authorities helped to implement a work model for enhancing food safety before meat reaches consumers.

Design of Two Multiplex PCR Assays for Serotyping Shigella flexneri.

Shigella flexneri is a major health problem in developing countries. There are 19 serotypes recognized based on O-antigen structure and its typing is important for epidemiological purposes. However, the diversity of serotypes and the difficulties presented by phenotypic serotyping, for example, unavailable antisera for less common antigens, require the implementation of molecular techniques. In this study, we developed two multiplex PCR assays targeting the O-antigen synthesis genes and the O-antigen modification genes, for the rapid identification of S. flexneri serotypes 1/7, 2, 4, 5, and 6 (PCR A) and serotype 7 and group antigenic factors (3,4; 6; 7,8; E1037) (PCR B). A total of 73 S. flexneri strains representing 18 serotypes, except serotype 1d, were used in the study. Specific amplification patterns were obtained for each of the different serotypes. All strains tested had concordant results with phenotypic and genotypic serotyping; therefore, its implementation in the microbiology clinical laboratory will significantly improve S. flexneri serotyping.

Molecular Characterization and Cluster Analysis of Field Isolates of Avian Infectious Laryngotracheitis Virus from Argentina.

Avian infectious laryngotracheitis (ILT) is a worldwide infectious disease that causes important economic losses in the poultry industry. Although it is known that ILT virus (ILTV) is present in Argentina, there is no information about the circulating strains. With the aim to characterize them, seven different genomic regions (thymidine kinase, glycoproteins D, G, B, C, and J, and infected cell polypeptide 4) were partially sequenced and compared between field samples. The gJ sequence resulted to be the most informative segment, it allowed the differentiation among field sample strains, and also, between wild and vaccine viruses. Specific changes in selected nucleotidic positions led to the definition of five distinct haplotypes. Tests for detection of clustering were run to test the null hypothesis that ILTV haplotypes were randomly distributed in time in Argentina and in space in the most densely populated poultry region of this country, Entre Rios. From this study, it was possible to identify a 46 km radius cluster in which higher proportions of haplotypes 4 and 5 were observed, next to a provincial route in Entre Rios and a significant decline of haplotype 5 between 2009 and 2011. Results here provide an update on the molecular epidemiology of ILT in Argentina, including data on specific genome segments that may be used for rapid characterization of the virus in the field. Ultimately, results will contribute to the surveillance of ILT in the country.

AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri.

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100% correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological data.

AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri. / AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri.

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100

correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological data.

Laboratory-based prospective surveillance for community outbreaks of Shigella spp. in Argentina.

BACKGROUND: To implement effective control measures, timely outbreak detection is essential. Shigella is the most common cause of bacterial diarrhea in Argentina. Highly resistant clones of Shigella have emerged, and outbreaks have been recognized in closed settings and in whole communities. We hereby report our experience with an evolving, integrated, laboratory-based, near real-time surveillance system operating in six contiguous provinces of Argentina during April 2009 to March 2012. METHODOLOGY: To detect localized shigellosis outbreaks timely, we used the prospective space-time permutation scan statistic algorithm of SaTScan, embedded in WHONET software. Twenty three laboratories sent updated Shigella data on a weekly basis to the National Reference Laboratory. Cluster detection analysis was performed at several taxonomic levels: for all Shigella spp., for serotypes within species and for antimicrobial resistance phenotypes within species. Shigella isolates associated with statistically significant signals (clusters in time/space with recurrence interval ≥365 days) were subtyped by pulsed field gel electrophoresis (PFGE) using PulseNet protocols. PRINCIPAL FINDINGS: In three years of active surveillance, our system detected 32 statistically significant events, 26 of them identified before hospital staff was aware of any unexpected increase in the number of Shigella isolates. Twenty-six signals were investigated by PFGE, which confirmed a close relationship among the isolates for 22 events (84.6%). Seven events were investigated epidemiologically, which revealed links among the patients. Seventeen events were found at the resistance profile level. The system detected events of public health importance: infrequent resistance profiles, long-lasting and/or re-emergent clusters and events important for their duration or size, which were reported to local public health authorities. CONCLUSIONS/SIGNIFICANCE: The WHONET-SaTScan system may serve as a model for surveillance and can be applied to other pathogens, implemented by other networks, and scaled up to national and international levels for early detection and control of outbreaks.

Characterization of lipid A profiles from Shigella flexneri variant X lipopolysaccharide.

RATIONALE: In developing countries, Shigella flexneri (Sf) is the major causative agent of the endemic shigellosis (bacillary dysentery) responsible annually for one million fatalities mostly among infants. Lipopolysaccharides (LPSs) are characteristic components of the outer membrane of the overwhelming majority of Gram-negative bacteria. Since lipid A is essential for the viability of the Gram-negative bacteria, it is subject to extensive chemical studies with new analytical techniques. METHODS: Lipid A was released by mild acid hydrolysis from the lipopolysaccharide which was obtained via the phenol/water extraction, purified and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and matrix-assisted laser desorption/ionization laser-induced dissociation tandem mass spectrometry (MALDI-LID-MS/MS). RESULTS: A detailed structural study of the whole lipid A obtained from S. flexneri variant X was carried out for the first time. Thus, we have shown that lipid A is a heterogeneous mixture having different numbers of acylated and phosphoethanolamine groups attached to the diglucosamine backbone. Furthermore, we found in the phenol phase an unusual hepta-acylated lipid A species, although the abundance was very low. CONCLUSIONS: MALDI-TOF-MS allowed us to unravel the lipid A heterogeneity, which was not previously reported in Sf LPS. It is well known that slight variations of the chemical structure of lipid A may change its biological activity. Thus, the knowledge of the detailed chemical structure represents an essential step for further development of new preventive or therapeutically active compounds.