Phenotypic and genetic characterization of antimicrobial resistance in Salmonella enterica serovar Choleraesuis isolates from humans and animals in Spain from 2006 to 2021.

OBJECTIVES: While an increase in the levels of MDR in Salmonella enterica sevorar Choleraesuis has been reported in Europe, little is known about the situation in Spain. Therefore, we first aimed to assess the phenotypic resistance profile and to determine the presence of genetic determinants of resistance of S. Choleraesuis isolates collected in animal and human. Our second objective was to identify and characterize clusters of highly related isolates. METHODS: We analysed 50 human and 45 animal isolates retrieved from 2006 to 2021 using the disc diffusion method and performed WGS followed by analyses of genetic determinants and phylogenetic analysis. RESULTS: All isolates were of ST145 and corresponded to the variant Kunzendorf. Swine isolates harboured a significantly higher number of antimicrobial resistance genes than human isolates, and often carried plasmid replicons of the IncHI2/IncHI2A type (42% of all animal isolates). In addition, we identified several MDR S. Choleraesuis strains circulating in humans and swine between 2006 and 2021. The phylogenetic analyses identified four clades associated with specific patterns of resistance genes and plasmid replicons. The clades also included isolates that differed in terms of year and region of isolation as well as host of origin. CONCLUSIONS: This One Health approach highlights that reducing human MDR S. Choleraesuis infections may require the adoption of strategies that not only seek to prevent cases in humans but also to characterize and reduce the infection burden in swine.

Mycobacterium tuberculosis Sub-Lineage 4.2.2/SIT149 as Dominant Drug-Resistant Clade in Northwest Ethiopia 2020-2022: In-silico Whole-Genome Sequence Analysis.

Introduction: Drug resistance (DR) in Mycobacterium tuberculosis complex (MTBC) is mainly associated with certain lineages and varies across regions and countries. The Beijing genotype is the leading resistant lineage in Asia and western countries. M. tuberculosis (Mtb) (sub) lineages responsible for most drug resistance in Ethiopia are not well described. Hence, this study aimed to identify the leading drug resistance sub-lineages and characterize first-line anti-tuberculosis drug resistance-associated single nucleotide polymorphisms (SNPs). Methods: A facility-based cross-sectional study was conducted in 2020-2022 among new and presumptive multidrug resistant-TB (MDR-TB) cases in Northwest Ethiopia. Whole-genome sequencing (WGS) was performed on 161 isolates using Illumina NovaSeq 6000 technology. The SNP mutations associated with drug resistance were identified using MtbSeq and TB profiler Bioinformatics softwares. Results: Of the 146 Mtb isolates that were successfully genotyped, 20 (13.7%) harbored one or more resistance-associated SNPs. L4.2.2.ETH was the leading drug-resistant sub-lineage, accounting for 10/20 (50%) of the resistant Mtb. MDR-TB isolates showed extensive mutations against first-line anti-TB drugs. Ser450Leu/(tcg/tTg) for Rifampicin (RIF), Ser315Thr/(agc/aCc) for Isoniazid (INH), Met306Ile/(atg/atA(C)) for Ethambutol (EMB), and Gly69Asp for Streptomycin (STR) were the leading resistance associated mutations which accounted for 56.5%, 89.5%, 47%, and 29.4%, respectively. The presence of both clustered and non-clustered drug resistance (DR) isolates indicated that the epidemics is driven by both new DR development and acquired resistance. Conclusion: The high prevalence of drug-resistant TB due to geographically restricted sub-lineages (L4.2.2.ETH) indicates the ongoing local micro epidemics. The Mtb drug resistance surveillance system must be improved. Further evolutionary analysis of L4.2.2.ETH strain is highly desirable to understand evolutionary forces that leads L4.2.2.ETH in to high level DR and transmissible sub-lineage.

Genetic Characterization of Extensively Drug-Resistant Shigella sonnei Infections, Spain, 2021-2022.

In 2022, the United Kingdom reported an increase in drug resistance in Shigella sonnei isolates. We report 33 cases in Spain genetically related to the UK cases and 4 cases with similar antimicrobial resistance profiles infected with genetically distant strains. Our results suggest circulation of multiple genetic clusters of multidrug-resistant S. sonnei in Spain.

A highly-safe live auxotrophic vaccine protecting against disease caused by non-typhoidal Salmonella Typhimurium in mice.

BACKGROUND: Salmonella enterica serovar Typhimurium (S. Typhimurium) has become an important intestinal pathogen worldwide and is responsible for lethal invasive infections in populations at risk. There is at present an unmet need for preventive vaccines. METHODS: IRTA GN-3728 genome was sequenced by Illumina and d-glutamate and d-glutamate/d-alanine knockout-auxotrophs were constructed. They were characterized using electron microscopy, growth/viability curves, reversion analysis, and motility/agglutination assays. Their potential as vaccine candidates were explored using two BALB/c mouse models for Salmonella infections: a systemic and an intestinal inflammation. Clinical signs/body weight and survival were monitored, mucosal lactoferrin and specific/cross-reactive IgA/IgG were quantified by enzyme-linked-immunosorbent assays and bacterial shedding/burden in fecal/tissues were evaluated. RESULTS: The d-glutamate auxotroph, IRTA ΔmurI, is highly attenuated, immunogenic and fully protective against systemic infection. The IRTA ΔmurI Δalr ΔdadX double auxotroph, constructed to reinforce vaccine safety, showed a higher level of attenuation and was 100% effective against systemic disease. In the intestinal model, it proved to be safe, yielding a low-degree of mucosal inflammation, short-term shedding and undetectable invasiveness in the long-term, while eliciting cross-reactive fecal IgA/serum IgG against clinically relevant multidrug-resistant (MDR) S. Typhimurium strains. It also conferred protection against homologous oral challenge, and protected mice from local and extra-intestinal dissemination caused by one MDR strain responsible for an international outbreak of highly severe human infections. Additionally, oral vaccination promoted extended survival after lethal heterologous infection. CONCLUSION: This study yielded a very safe S. Typhimurium vaccine candidate that could be further refined for mucosal application against disease in humans.

Comparative whole-genome sequence analysis of Mycobacterium tuberculosis isolated from pulmonary tuberculosis and tuberculous lymphadenitis patients in Northwest Ethiopia.

Background: Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex (MTBC), is a chronic infectious disease with both pulmonary and extrapulmonary forms. This study set out to investigate and compare the genomic diversity and transmission dynamics of Mycobacterium tuberculosis (Mtb) isolates obtained from tuberculous lymphadenitis (TBLN) and pulmonary TB (PTB) cases in Northwest Ethiopia. Methods: A facility-based cross-sectional study was conducted using two groups of samples collected between February 2021 and June 2022 (Group 1) and between June 2020 and June 2022 (Group 2) in Northwest Ethiopia. Deoxyribonucleic acid (DNA) was extracted from 200 heat-inactivated Mtb isolates. Whole-genome sequencing (WGS) was performed from 161 isolates having ≥1 ng DNA/µl using Illumina NovaSeq 6000 technology. Results: From the total 161 isolates sequenced, 146 Mtb isolates were successfully genotyped into three lineages (L) and 18 sub-lineages. The Euro-American (EA, L4) lineage was the prevailing (n = 100; 68.5%) followed by Central Asian (CAS, L3, n = 43; 25.3%) and then L7 (n = 3; 2.05%). The L4.2.2.ETH sub-lineage accounted for 19.9%, while Haarlem estimated at 13.7%. The phylogenetic tree revealed distinct Mtb clusters between PTB and TBLN isolates even though there was no difference at lineages and sub-lineages levels. The clustering rate (CR) and recent transmission index (RTI) for PTB were 30 and 15%, respectively. Similarly, the CR and RTI for TBLN were 31.1 and 18 %, respectively. Conclusion and recommendations: PTB and TBLN isolates showed no Mtb lineages and sub-lineages difference. However, at the threshold of five allelic distances, Mtb isolates obtained from PTB and TBLN form distinct complexes in the phylogenetic tree, which indicates the presence of Mtb genomic variation among the two clinical forms. The high rate of clustering and RTI among TBLN implied that TBLN was likely the result of recent transmission and/or reactivation from short latency. Hence, the high incidence rate of TBLN in the Amhara region could be the result of Mtb genomic diversity and rapid clinical progression from primary infection and/or short latency. To validate this conclusion, a similar community-based study with a large sample size and better sampling technique is highly desirable. Additionally, analysis of genomic variants other than phylogenetic informative regions could give insightful information. Combined analysis of the host and the pathogen genome (GXG) together with environmental (GxGxE) factors could give comprehensive co-evolutionary information.

Shigelosis atendidas en el servicio de urgencias de un hospital regional del sudeste de España: Desde su presencia a la multirresistencia / Shigellosis attended in the emergency department of a regional hospital in southeastern Spain: from their presence to multiresistance

Introduction. In the bacterial etiology of severe acute infectious diarrhea, except that caused by Clostridioides difficile, most of them have an invasive character and antibiotic treatment will be necessary in specific situations. Shigella is a classic pathogen, in which it is crucial to know the sensitivity to different classic and alternative antimicrobials. The objective of this work was to analyze the presence of shigellosis and the rate of antibiotic resistance.Methods. A descriptive-retrospective study of the reports of shigellosis of stool cultures issued between January 2016 and April 2022 was conducted.Results. A total of 34 episodes (16 -47.1%- by Shigella sonnei) were observed, as of 2018. There were only 2 pediatric cases. The overall resistance rate to azithromycin, trimethoprim-sulfamethoxazole and ciprofloxacin was 52.9%, 64.7% and 44.1%, respectively. 26.5% were resistant to the 3 groups of antibiotics. There was a higher rate of resistance for S. sonnei. The emergence of resistance to cephalosporins in recent years stands out. Episodes of multidrug-resistant shigellosis were detected between 2020 (1 by S. flexneri) and 2022 (4 by S. sonnei).Conclusions. The episodes of shigellosis are emerging in our environment with a higher rate of multi-resistance. In this context, current empirical treatments for acute enteroinvasive enteritis are at risk of failure, if necessary.

Introducción: En la etiología bacteriana de la diarrea infecciosa aguda grave, exceptuando la causada por Clostridioides difficile, la mayor parte presentan un carácter invasor y el tratamiento antibiótico será preciso en situaciones concretas. Shigella es un patógeno clásico, en el que es crucial conocer la sensibilidad a distintos antimicrobianos clásicos y alternativos. El objetivo de este trabajo fue analizar la presencia de shigelosis y la tasa de resistencia a los antibióticos. Métodos: Se realizó un estudio descriptivo-retrospectivo de los informes de shigelosis de los coprocultivos emitidos entre enero de 2016 y abril de 2022. Resultados: Se observó un total de 34 episodios (16-47,1%- por Shigella sonnei), a partir del 2018. Sólo hubo 2 casos pediátricos. La tasa de resistencia global a azitromicina, trimetoprim-sulfametoxazol y ciprofloxacino fue de 52,9%, 64,7% y 44,1%, respectivamente. El 26,5% fueron resistentes a los 3 grupos de antibióticos. Hubo mayor tasa de resistencia por S. sonnei. Destaca la aparición de resistencia a cefalosporinas en los últimos años. Los episodios de shigelosis multirresistente se detectaron entre 2020 (1 por S. flexneri) y 2022 (4 por S. sonnei). Conclusiones: Los episodios de shigelosis importada están emergiendo en nuestro medio con una mayor tasa de multirresistencia. En este contexto, los tratamientos empíricos actuales para las enteritis agudas enteroinvasivas corren el riesgo de fracasar, en caso de ser necesarios. (AU)

Cluster of cases due to Shigella flexneri producing CTX-M-15 in Spain.

The aim of our study was to delineate an outbreak of gastroenteritis caused by Shigella flexneri and affecting sixteen persons between May and June 2014 in Bilbao, Spain. All patients exhibited symptoms after consuming kebab in the same kebab shop.The outbreak is described through the clinical cases, the microbiological and molecular genetic diagnosis, and the epidemiologic investigation. Minimum inhibitory concentrations for ampicillin, amoxicillin plus clavulanic acid, third and fourth generation cephalosporins, carbapenems, monobactams, aminoglycosides, fluoroquinolones, co-trimoxazole, colistin and tigecycline were measured. The S. flexneri strains were screened by PCR for TEM, SHV, CTX-M beta-lactamases and plasmidic AmpCs and aac(6')-Ib gene. Serotyping, pulsed field gel-electrophoresis, conjugation assay, plasmid sizing by S1 enzyme digestion and Southern blot hybridization were accomplished.All the S. flexneri isolates proved to be serotype 2 and produced extended-spectrum beta-lactamase (ESBL). Carbapenems, fluoroquinolones, tigecycline, colistin, and co-trimoxazole remained active antibiotics. All the strains harboured blaCTX-M-15 and blaOXA-1 genes. The strains hosted two high-molecular weight plasmids of 100 and 230 kb, respectively. According to the hybridization assay blaCTX-M-15 was located on the plasmid of 230 kb. The identical pulsotype verified the presence of outbreak.Remarkable, that one of the food handlers has travelled recently to Pakistan, where ESBL-producing Shigella strains had been reported previously. To the best of our knowledge, this is the first outbreak caused by CTX-M-15-expressing S. flexneri in Spain and as well as in Europe.

Prediction of Antibiotic Susceptibility Profiles of Vibrio cholerae Isolates From Whole Genome Illumina and Nanopore Sequencing Data: CholerAegon.

During the last decades, antimicrobial resistance (AMR) has become a global public health concern. Nowadays multi-drug resistance is commonly observed in strains of Vibrio cholerae, the etiological agent of cholera. In order to limit the spread of pathogenic drug-resistant bacteria and to maintain treatment options the analysis of clinical samples and their AMR profiles are essential. Particularly, in low-resource settings a timely analysis of AMR profiles is often impaired due to lengthy culturing procedures for antibiotic susceptibility testing or lack of laboratory capacity. In this study, we explore the applicability of whole genome sequencing for the prediction of AMR profiles of V. cholerae. We developed the pipeline CholerAegon for the in silico prediction of AMR profiles of 82 V. cholerae genomes assembled from long and short sequencing reads. By correlating the predicted profiles with results from phenotypic antibiotic susceptibility testing we show that the prediction can replace in vitro susceptibility testing for five of seven antibiotics. Because of the relatively low costs, possibility for real-time data analyses, and portability, the Oxford Nanopore Technologies MinION sequencing platform-especially in light of an upcoming less error-prone technology for the platform-appears to be well suited for pathogen genomic analyses such as the one described here. Together with CholerAegon, it can leverage pathogen genomics to improve disease surveillance and to control further spread of antimicrobial resistance.

Zoonotic Transmission of Diphtheria from Domestic Animal Reservoir, Spain.

Toxigenic Corynebacterium ulcerans is as an emerging zoonotic agent of diphtheria. We describe the zoonotic transmission of diphtheria caused by toxigenic C. ulcerans from domestic animals in Spain, confirmed by core-genome multilocus sequence typing. Alongside an increasing number of recent publications, our findings highlight the public health threat posed by diphtheria reemergence.

Investigation of an international outbreak of multidrug-resistant monophasic Salmonella Typhimurium associated with chocolate products, EU/EEA and United Kingdom, February to April 2022.

An extensive multi-country outbreak of multidrug-resistant monophasic Salmonella Typhimurium infection in 10 countries with 150 reported cases, predominantly affecting young children, has been linked to chocolate products produced by a large multinational company. Extensive withdrawals and recalls of multiple product lines have been undertaken. With Easter approaching, widespread product distribution and the vulnerability of the affected population, early and effective real-time sharing of microbiological and epidemiological information has been of critical importance in effectively managing this serious food-borne incident.

Considerations for COVID-19 management in reception centers for refugees, asylum seekers, and migrants, Spain 2020.

Shortly after the declaration of the COVID-19 pandemic, governments around the world were urged to leave no population behind. Following a COVID-19 risk evaluation in a refugee and asylum seekers reception center, in September 2020, we considered the priorities of managing COVID-19 in these settings. We encourage actions on the following four fronts to reduce the COVID-19-associated burden among these vulnerable populations based on our interviews, observations, and recommendations: (i) decongestion, (ii) facilitated testing, (iii) screening for symptoms, and (iv) targeted public health and risk communication.

Molecular and epidemiologic characterization of the diphtheria outbreak in Venezuela.

In 2016, Venezuela faced a large diphtheria outbreak that extended until 2019. Nasopharyngeal or oropharyngeal samples were prospectively collected from 51 suspected cases and retrospective data from 348 clinical records was retrieved from 14 hospitals between November 2017 and November 2018. Confirmed pathogenic Corynebactrium isolates were biotyped. Multilocus Sequence Typing (MLST) was performed followed by next-generation-based core genome-MLST and minimum spanning trees were generated. Subjects between 10 and 19 years of age were mostly affected (n = 95; 27.3%). Case fatality rates (CFR) were higher in males (19.4%), as compared to females (15.8%). The highest CFR (31.1%) was observed among those under 5, followed by the 40 to 49 age-group (25.0%). Nine samples corresponded to C. diphtheriae and 1 to C. ulcerans. Two Sequencing Types (ST), ST174 and ST697 (the latter not previously described) were identified among the eight C. diphtheriae isolates from Carabobo state. Cg-MLST revealed only one cluster also from Carabobo. The Whole Genome Sequencing analysis revealed that the outbreak seemed to be caused by different strains with C. diphtheriae and C. ulcerans coexisting. The reemergence and length of this outbreak suggest vaccination coverage problems and an inadequate control strategy.

Molecular and Epidemiological Characterization of Toxigenic and Nontoxigenic Corynebacterium diphtheriae, Corynebacterium belfantii, Corynebacterium rouxii, and Corynebacterium ulcerans Isolates Identified in Spain from 2014 to 2019.

This study examines the microbiological and epidemiological characteristics of toxigenic and nontoxigenic Corynebacterium isolates submitted to the national reference laboratory in Spain, between 2014 and 2019, in order to describe the current situation and improve our knowledge regarding these emerging pathogens. Epidemiological information was extracted from the Spanish Surveillance System. Microbiological and molecular characterization was carried out using phenotypic methods, multilocus sequence typing (MLST), whole-genome sequencing (WGS), and core genome MLST (cgMLST). Thirty-nine isolates were analyzed. Twenty-one isolates were identified as Corynebacterium diphtheriae (6 toxigenic), 14 as C. belfantii, 4 as C. ulcerans (3 toxigenic), and 1 as C. rouxii One C. diphtheriae isolate was identified as nontoxigenic tox gene bearing (NTTB). Ages of patients ranged from 1 to 89 years, with 10% (3/30) of nontoxigenic and 22% (2/9) of toxigenic isolates collected from children less than 15 years. Twenty-five of the patients were males (17/30 in nontoxigenic; 8/9 in toxigenic). MLST identified 28 sequence types (STs), of which 7 were described for the first time in Spain. WGS analysis showed that 10 isolates, including 3 toxigenic isolates, harbored a variety of antibiotic resistance genes in addition to the high prevalence of penicillin resistance phenotypically demonstrated. Phylogenetic analysis revealed one cluster of isolates from family members. Risk information was available for toxigenic isolates (9/39); 3 patients reported recent travels to countries of endemicity and 3 had contact with cats/dogs. One unvaccinated child with respiratory diphtheria had a fatal outcome. Including nontoxigenic Corynebacterium infections in disease surveillance and using WGS could further improve current surveillance.

Management of a COVID-19 outbreak in a hotel in Tenerife, Spain.

Since the first accounts of SARS-CoV-2, authorities have encountered numerous unprecedented situations threatening public health. This rapid communication addresses events that led to the quarantining of a hotel in Tenerife, Spain and the effectiveness of the rapidly implemented control measures. In total, eight cases have been associated with the hotel. Due to the international nature of the guests, had these timely precautions not been in place, a multinational cluster might have formed.

Traveller's diarrhoea by Vibrio cholerae in patients returning from the Dominican Republic / Diarrea del viajero por Vibrio cholerae en pacientes que regresan de República Dominicana

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Epidemiología descriptiva de infección por Yersinia enterocolitica en un área de alta incidencia durante 8 años, 2006-2013. Proyecto EDICS / Descriptive epidemiology of Yersinia enterocolitica infection in a high-incidence area over an 8-year period, 2006-2013. EDICS Project

Epidemiología descriptiva de la infección por Yersinia enterocolitica en un área de Castellón (España) entre 2006 y 2013 a partir de las cepas de Yersinia enterocolitica aisladas en el área y confirmadas por el laboratorio de referencia nacional. Total 144 casos. La incidencia estimada fue de 9,7 casos 105 persona-año. El grupo de edad más afectado fue el de 0-4 años (tasa 110,3 por 105 persona-año), con una máximo en lactantes de 6 a 11 meses de edad (190,4 por 105 persona-año). La duración media de la enfermedad fue de 15,5 días. El 7% de los pacientes fueron hospitalizados. Solo se detectaron 2 brotes, de carácter familiar relacionados con el consumo de carne de cerdo. La evolución temporal refleja mayor incidencia en invierno (enero). El factor de exposición más frecuente entre los casos fue el consumo de longaniza seca de cerdo (el 50% de los casos entrevistados). Las 58 cepas tipadas fueron todas del biotipo 4, serotipo O:3, excepto una O:9. Se distinguieron 21 pulsotipos agrupados en 8 clusters con similitud del 97%. A lo largo de algunos años se observó una sustitución de unos pulsotipos por otros.La yersiniosis tiene una incidencia alta en nuestra área, con una estacionalidad clara de predominio invernal. Afecta sobre todo a niños muy pequeños. Las cepas son del mismo serotipo, pero la variedad de pulsotipos cambió a lo largo del tiempo. Como factor de exposición para ulteriores estudios analíticos se propone el consumo de algunos productos del cerdo, sin descartar otros factores

Descriptive epidemiology of Yersinia enterocolitica infection in an area of Castellón (Spain) between 2006 and 2013 from Yersinia enterocolitica strains isolated in the area and confirmed by the Spanish national reference laboratory. There were a total of 144 cases. The estimated incidence was 9.7 cases per 105 person-year. The age group most affected was 0-4 years (rate 110.3 per 105 p-y), with a maximum in infants aged 6 to 11 months of age (190.4 per 105 p-y). The average duration of the disease was 15.5 days. 7% of the patients were hospitalised. Only 2 outbreaks of a family nature related to the consumption of pork were detected. The temporal evolution reflects higher incidence during the winter season (January). The most common exposure factor among the cases was the consumption of dried pork sausage (50% of the cases interviewed). The 58 typed strains were all of the biotype 4, serotype O:3, except one O:9. We distinguished 21 pulsotypes grouped in 8 clusters with a similarity of 97%. Over a number of years, a substitution of some pulsotypes for others was observed. Yersiniosis has a high incidence in our area, with a clear seasonality of winter predominance. It affects very young children, in particular. The strains are of the same serotype, but the variety of pulsotypes changed over time. As an exposure factor for further analytical studies, the consumption of some pork products is proposed, without ruling out other factors

Outbreak of Salmonella enterica serotype Poona in infants linked to persistent Salmonella contamination in an infant formula manufacturing facility, France, August 2018 to February 2019.

We describe a Salmonella Poona outbreak involving 31 infant cases in France. Following outbreak detection on 18 January 2019, consumption of rice-based infant formula manufactured at a facility in Spain was identified as the probable cause, leading to a recall on 24 January. Whole genome sequencing analysis linked present outbreak isolates to a 2010-11 S. Poona outbreak in Spain associated with formula manufactured in the same facility, indicating a persistent source of contamination.

Evaluation of the SHIGA TOXIN QUIK CHEK after overnight enrichment as screening tool for Shiga toxin-producing Escherichia coli detection in human fecal samples.

We evaluated the SHIGA TOXIN QUIK CHEK (STQC) on its suitability for Shiga toxin-producing Escherichia coli (STEC) testing on human fecal samples after overnight enrichment. Our in-house PCR-based protocol for STEC detection was used as the standard for comparison. STQC detected all described Shiga toxin subtypes with the only exception of Stx2f. In comparison to PCR, STQC performed with an overall sensitivity of 55.4%, specificity of 100.0%, positive predictive value of 100.0%, negative predictive value of 73.0%, infinite positive likelihood ratio, and negative likelihood ratio of 0.45. We conclude that STQC may not be considered a suitable screening tool for STEC detection in human fecal samples, although it could be useful for laboratories where PCR is not a routine tool for STEC screening yet, subject to the confirmation of negative samples by a reference laboratory with full diagnostic capabilities.