First detection of a plasmid located carbapenem resistant bla(VIM-1) gene in E. coli isolated from meat products at retail in Belgium in 2015

Carbapenemase-producing Enterobacteriaceae (CPE) confer resistance to antibiotics that are of critical importance to human medicine. There have only been a few reported cases of CPEs in the European food chain. We report the first detection of a carbapenemase-producing Escherichia coli (ST 5869) in the Belgian food chain. Our aim was to characterize the origin of the carbapenem resistance in the E. coli isolate. The isolate was detected during the screening of 178 minced pork samples and was shown to contain the carbapenemase gene bla(VIM-1) by PCR and Sanger sequencing. Whole genome short and long read sequencing (MiSeq and MinION) was performed to characterize the isolate. With a hybrid assembly we reconstructed a 190,205 bp IncA/C2 plasmid containing bla(VIM-1) (S15FP06257_p), in addition to other critically important resistance genes. This plasmid showed only low similarity to plasmids containing bla(VIM-1) previously reported in Germany. Moreover, no sequences existed in the NCBI nucleotide database that completely covered S15FP06257_p. Analysis of the bla(VIM-1) gene cassette demonstrated that it likely originated from an integron of a Klebsiella plasmid reported previously in a clinical isolate in Europe, suggesting that the meat could have been contaminated by human handling in one of the steps of the food chain. This study shows the relevance of fully reconstructing plasmids to characterize their genetic content and to allow source attribution. This is especially important in view of the potential risk of antimicrobial resistance gene transmission through mobile elements as was reported here for the of public health concern bla(VIM-1)

Combining short and long read sequencing to characterize antimicrobial resistance genes on plasmids applied to an unauthorized genetically modified Bacillus

Antimicrobial resistance (AMR) is a major public health threat. Plasmids are able to transfer AMR genes among bacterial isolates. Whole genome sequencing (WGS) is a powerful tool to monitor AMR determinants. However, plasmids are difficult to reconstruct from WGS data. This study aimed to improve the characterization, including the localization of AMR genes using short and long read WGS strategies. We used a genetically modified (GM) Bacillus subtilis isolated as unexpected contamination in a feed additive, and therefore considered unauthorized (RASFF 2014.1249), as a case study. In GM organisms, AMR genes are used as selection markers. Because of the concern of spread of these AMR genes when present on mobile genetic elements, it is crucial to characterize their location. Our approach resulted in an assembly of one chromosome and one plasmid, each with several AMR determinants of which five are against critically important antibiotics. Interestingly, we found several plasmids, containing AMR genes, integrated in the chromosome in a repetitive region of at least 53 kb. Our findings would have been impossible using short reads only. We illustrated the added value of long read sequencing in addressing the challenges of plasmid reconstruction within the context of evaluating the risk of AMR spread

SARS-CoV-2 Catalonia contact tracing program : evaluation of key performance indicators

Background: Guidance on SARS-CoV-2 contact tracing indicators have been recently revised by international public health agencies. The aim of the study is to describe and analyse contact tracing indicators based on Catalonia's (Spain) real data and proposing to update them according to recommendations. Methods: Retrospective cohort analysis including Catalonia's contact tracing dataset from 20 May until 31 December 2020. Descriptive statistics are performed including sociodemographic stratification by age, and differences are assessed over the study period. Results: We analysed 923,072 contacts from 301,522 SARS-CoV-2 cases with identified contacts (67.1% contact tracing coverage). The average number of contacts per case was 4.6 (median 3, range 1-243). A total of 403,377 contacts accepted follow-up through three phone calls over a 14-day quarantine period (84.5% of contacts requiring follow-up). The percentage of new cases declared as contacts 14 days prior to diagnosis evolved from 33.9% in May to 57.9% in November. All indicators significantly improved towards the target over time (p < 0.05 for all four indicators). Conclusions: Catalonia's SARS-CoV-2 contact tracing indicators improved over time despite challenging context. The critical revision of the indicator's framework aims to provide essential information in control policies, new indicators proposed will improve system delay's follow-up. The study provides information on COVID-19 indicators framework experience from country's real data, allowing to improve monitoring tools in 2021-2022. With the SARS-CoV-2 pandemic being so harmful to health systems and globally, is important to analyse and share contact tracing data with the scientific community

Azithromycin resistance in Escherichia coli and Salmonella from food-producing animals and meat in Europe.

OBJECTIVES To characterize the genetic basis of azithromycin resistance in Escherichia coli and Salmonella collected within the EU harmonized antimicrobial resistance (AMR) surveillance programme in 2014-18 and the Danish AMR surveillance programme in 2016-19. METHODS WGS data of 1007 E. coli [165 azithromycin resistant (MIC > 16 mg/L)] and 269 Salmonella [29 azithromycin resistant (MIC > 16 mg/L)] were screened for acquired macrolide resistance genes and mutations in rplDV, 23S rRNA and acrB genes using ResFinder v4.0, AMRFinder Plus and custom scripts. Genotype-phenotype concordance was determined for all isolates. Transferability of mef(C)-mph(G)-carrying plasmids was assessed by conjugation experiments. RESULTS mph(A), mph(B), mef(B), erm(B) and mef(C)-mph(G) were detected in E. coli and Salmonella, whereas erm(C), erm(42), ere(A) and mph(E)-msr(E) were detected in E. coli only. The presence of macrolide resistance genes, alone or in combination, was concordant with the azithromycin-resistant phenotype in 69% of isolates. Distinct mph(A) operon structures were observed in azithromycin-susceptible (n = 50) and -resistant (n = 136) isolates. mef(C)-mph(G) were detected in porcine and bovine E. coli and in porcine Salmonella enterica serovar Derby and Salmonella enterica 1,4, [5],12:i:-, flanked downstream by ISCR2 or TnAs1 and associated with IncIγ and IncFII plasmids. CONCLUSIONS Diverse azithromycin resistance genes were detected in E. coli and Salmonella from food-producing animals and meat in Europe. Azithromycin resistance genes mef(C)-mph(G) and erm(42) appear to be emerging primarily in porcine E. coli isolates. The identification of distinct mph(A) operon structures in susceptible and resistant isolates increases the predictive power of WGS-based methods for in silico detection of azithromycin resistance in Enterobacterales

High-pressure inactivation and sublethal injury of pressure-resistant Escherichia coli mutants in fruit juices.

The potential of high-pressure-resistant mutants of Escherichia coli to survive high-pressure pasteurization in fruit juices and in low-pH buffers was investigated. Treatments with up to 500 MPa of pressure caused only a limited direct inactivation of the mutants but resulted in an accelerated low-pH inactivation during subsequent storage.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Livestock veterinarians at high risk of acquiring methicillin-resistant Staphylococcus aureus ST398.

The prevalence and risk factors associated with livestock-associated MRSA (LA-MRSA) carriage was examined in Danish and Belgian veterinarians. The MRSA and LA-MRSA carriage rates were 9·5% (95% CI 5·3-15·6) and 7·5% (95% CI 3·8-13·1) for MRSA and LA-MRSA, respectively, in Belgium and 1·4% (95% CI: 0·17-5·05) in Denmark (all Danish MRSA isolates belonged to the LA-MRSA genotype). All LA-MRSA isolates were resistant to tetracycline and 53·4% (7/13) showed a multi-resistant phenotype. LA-MRSA was significantly associated with veterinarians in contact with livestock (P=0·046). In the multivariable analysis, working with small animals in a veterinary clinic seems to be negatively associated (OR 0·15, 95% CI 0-1·0, P=0·05) and a strong direct association was found for LA-MRSA acquisition and exposure to live pigs (OR 12·1, 95% CI 1·6-548·5, P=0·01). Since carriage of MRSA ST398 may increase the risk of complications during hospitalization, our results underline that preventive measures may need to be developed for veterinary professionals, particularly for livestock veterinarians.Journal ArticleResearch Support, Non-U.S. Gov'tReviewSCOPUS: re.jinfo:eu-repo/semantics/publishe

First Belgian Report of Ertapenem Resistance in an ST11 Klebsiella Pneumoniae Strain Isolated from a Dog Carrying blaSCO-1 and blaDHA-1 Combined with Permeability Defects

Klebsiella pneumoniae of sequence type (ST) 11 is a hyper-epidemic nosocomial clone, which is spreading worldwide among humans and emerging in pets. This is the first report, to the best of our knowledge, of multidrug-resistant (MDR) K. pneumoniae ST11 carrying blaSCO-1 and blaDHA-1, isolated from a four-month-old dog in Belgium. Antimicrobial susceptibility testing (AST) of the isolate, performed via broth microdilution following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, revealed resistance to eight different classes of antimicrobials, including carbapenems, in particular ertapenem, third-generation cephalosporins and fluoroquinolones. A hybrid approach, combining long- and short-read sequencing, was employed for in silico plasmid characterization, multi-locus sequence typing (MLST) and the identification and localization of antimicrobial resistance (AMR) and virulence-associated genes. Three plasmids were reconstructed from the whole-genome sequence (WGS) data: the conjugative IncFIB(K), the non-mobilizable IncR and the mobilizable but unconjugative ColRNAI. The IncFIB(K) plasmid carried the blaSCO-1 gene, whereas IncR carried blaDHA-1, both alongside several other antimicrobial resistance genes (ARGs). No virulence genes could be detected. Here, we suggest that the resistance to ertapenem associated with susceptibility to imipenem and meropenem in K. pneumoniae could be related to the presence of blaSCO-1 and blaDHA-1, combined with permeability defects caused by point mutations in an outer membrane porin (OmpK37). The presence of the blaSCO-1 gene on a conjugative IncFIB(K) plasmid is worrisome as it can increase the risk of transmission to humans, to animals and to the environment

High abundance and diversity of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faeces and tonsils of pigs at slaughter

This cross-sectional study investigates the abundance of cefotaxime-resistant Escherichia coil (CREC) in the faeces and tonsils of 96 pigs during slaughter. Moreover, different isolates from a selected number of pigs were tested to study the diversity of biaESBL genes within E. coil isolates from one pig. Cefotaxime-resistant bacteria (based on enumeration results on MacConkey agar supplemented with 1 mg/L cefotaxime) were found in the faeces of 77 pigs (80%; 95% CI: 70-87%) and the tonsils of 91 pigs (95%; 95% CI: 88%-98%). Cefotaxime-resistant E. coil (based on enumeration results on Tryptone Bile X-glucuronide agar supplemented with 1 mg/L cefotaxime) were detected in 72 faecal samples (75%; 95% CI: 64-83%) and 45 tonsil samples (47%; 95% CI: 35-59%), in numbers up to 5.5 and 5.6 logio CFU/g, respectively. On average, around 1/10,000 E. coll. in both faeces and tonsils were cefotaxime-resistant, though large variations were observed between pigs. Within one sample, CREC isolates with up to five different combinations of ESBL genes were observed. In three out of 16 faecal samples and six out of 14 tonsil samples, only one ESBL gene profile was found. The high numbers of CREC that are occasionally found in the faeces and tonsils of pigs during slaughter may represent an important source of contamination of carcasses and subsequently pork