The details of all steps involved in the quantification of
biofilm formation in microtiter plates are described. The
presented protocol incorporates information on assessment of
biofilm production by staphylococci, gained both by direct
experience as well as by analysis of methods for assaying
biofilm production. The obtained results should simplify
quantification of biofilm formation in microtiter plates, and
make it more reliable and comparable among different
laboratories.

Quantification of biofilm in microtiter plates: overview oftesting conditions and practical recommendations for assessmentof biofilm production by staphylococci.

Colistin is considered to be an antimicrobial of last-resort for the treatment of multidrug-resistant Gram-negative bacterial infections. The recent global dissemination of mobilized colistin resistance (mcr) genes is an urgent public health threat. An accurate estimate of the global prevalence of mcr genes, their reservoirs and the potential pathways for human transmission are required to implement control and prevention strategies, yet such data are lacking. Publications from four English (PubMed, Scopus, the Cochrane Database of Systematic Reviews and Web of Science) and two Chinese (CNKI and WANFANG) databases published between 18 November 2015 and 30 December 2018 were identified. In this systematic review and meta-analysis, the prevalence of mcr genes in bacteria isolated from humans, animals, the environment and food products were investigated. A total of 974 publications were identified. 202 observational studies were included in the systematic review and 71 in the meta-analysis. mcr genes were reported from 47 countries across six continents and the overall average prevalence was 4.7% (0.1–9.3%). China reported the highest number of mcr-positive strains. Pathogenic Escherichia coli (54%), isolated from animals (52%) and harboring an IncI2 plasmid (34%) were the bacteria with highest prevalence of mcr genes. The estimated prevalence of mcr-1 pathogenic E. coli was higher in food-animals than in humans and food products, which suggests a role for foodborne transmission. This study provides a comprehensive assessment of prevalence of the mcr gene by source, organism, genotype and type of plasmid.

https://biblio.ugent.be/publication/8645004/file/8645009.pdf

Global Burden of Colistin-Resistant Bacteria: Mobilized Colistin Resistance Genes Study (1980-2018).

The emergence of antimicrobial-resistant (AR) Salmonella has a major concern worldwide. This study was designed to determine the AR profiles and serovars distribution of Salmonella enterica isolated from different breeds of breeder chickens in the province of Henan, China. For this, 2,139 dead embryo samples were collected from 28 breeder chicken hatcheries, representing two domestic and four foreign breeds. The samples were subjected to the isolation and identification of Salmonella by PCR. The confirmed strains were serotyped according to the Kauffmann-White scheme and their AR profiles against 20 antimicrobial agents were determined by Kirby-Bauer (K-B) disc diffusion method. The results of this study showed the prevalence of Salmonella in 504 strains (23.56%) with a high abundance in southern regions of Yellow River (28.66%, n = 495, N = 1,727) compared to the northern regions (2.18%, n = 9, N = 412) (p < 0.0001). The domestic breeds were more contaminated than imported breeds (p < 0.0001). However, the contamination rate of samples recovered from M-hatcheries was the highest (p < 0.0001). Serotyping method identified 12 serovars, with the dominance of S. Pullorum (75.79%), followed by S. Enteritidis (7.14%). The AR assay showed high resistant to ciprofloxacin (77.00%), sulfisoxazole (73.00%), and ampicillin (55.60%), as well as 98.81% (n = 498) of the isolated strains, were resistant to at least one antimicrobial and 69.64% (n = 351) were resistant to three or more antimicrobials. Among them, one strain of S. Thompson was resistant to 15 antimicrobial agents belonging to eight different classes. In conclusion, Salmonella strains isolated in this study were multidrug-resistant (MDR), presenting a serious problem for human and animal health. Therefore, it is necessary to monitor, control, and rationalize the use of antimicrobials agents in chicken farms in order to limit the increasing resistance against the recent antimicrobial agents.

/pdf/epidemiological-investigation-and-antimicrobial-resistance-2crt47q5ht.pdf

Epidemiological Investigation and Antimicrobial Resistance Profiles of Salmonella Isolated From Breeder Chicken Hatcheries in Henan, China.

Salmonella enterica serovar 4,[5],12:i:-, so-called Typhimurium monophasic variant, has become one of the most frequently isolated serovars both in humans and in animals all over the world. The increasing prevalence of mcr-1-carrying Salmonella poses significant global health concerns. However, the potential role of Salmonella 4,[5],12:i:- in mcr-1 gene migration through the food chain to the human remains obscure. Here, we investigated 337 Salmonella isolates from apparently healthy finishing pigs, which is rarely studied, obtained from pig farms and slaughterhouses in China. The mcr-1 gene was found in four colistin-resistant S. enterica 4,[5],12:i:- isolates. Notably, all four isolates belonged to sequence type 34 (ST34) with multidrug resistance phenotype. Further genomic sequencing and antimicrobial resistance characterization confirmed that mcr was responsible for the colistin resistance, and the conjugation assay demonstrated that three of four isolates carried mcr-1 in IncHI2 plasmid. Importantly, mcr-1 and class-1 integron were found to co-localize in two strains with IncHI2 plasmid. By collecting all the mcr-1-carrying Typhimurium and monophasic variant strains across the food chain (farm animals, animal-origin food, and humans), our phylogenomic analysis of available 66 genomes, including four strains in this study, demonstrated an independent phylogenetic cluster of all eight Chinese swine-originated isolates and one human isolate. Together, this study provides direct evidence for clonal and pork-borne transmission of mcr-1 by Salmonella 4,[5],12:i:- ST34 in China and highlighted a domestication pathway by acquisition of additional antimicrobial resistance determinants in Chinese ST34 isolates.

/pdf/genomic-characterization-of-mcr-1-carrying-salmonella-2jmzi4yzte.pdf

Genomic Characterization of mcr-1-carrying Salmonella enterica Serovar 4,[5],12:i:- ST 34 Clone Isolated From Pigs in China.

Salmonella enterica subsp. enterica serovar Dublin (S. Dublin), a cattle adapted serovar causes enteritis, and systemic disease in bovines. The invasive index of this serovar far exceeds that of the other serovars and human infections often present as fatal or highly resistant infections. In this, observational study, phenotypic properties of human and bovine-derived isolates of S. Dublin along with antibiogram of common antimicrobials were evaluated. The multiplex PCR confirmed isolates were genotyped using 7-gene legacy MLST. MIC assay was done by broth microdilution method. Previously published protocols were used to assess the motility, biofilm formation and morphotype. Vi antigen was agglutinated using commercial antiserum. Caenorhabditis elegans infection model was used to evaluate the virulence potiential. Phenotyping experiments were done in duplicates while virulence assay was done in triplicates. Whole-genome sequencing was used to predict the genes responsible for acquired resistance and a genotype-phenotype comparison was made. We evaluated 96 bovine and 10 human isolates in this study. All the isolates belonged to ST10 in eBG53 and were negative for Vi-antigen. The swarming motility, biofilm formation and morphotype were variable in the isolates of both groups. Resistance to sulfamethoxazole, ampicillin, chloramphenicol, tetracycline was > 90% in animal isolates whereas resistance to sulfamethoxazole was > 70% in human isolates. MDR was also higher in animal isolates. Human isolates were significantly (P < 0.0001) more virulent than animal isolates on C. elegans infection model. The genomic comparison based on the core SNPs showed a high degree of homogeneity between the isolates. The carriage of IncA/C2 plasmid was seen as a typical feature of isolates from the bovine hosts. Human isolates showed more diversity in the phenotypic assays. Animal isolates showed a higher degree of antimicrobial resistance with greater MDR but human isolates formed more biofilm and had greater swarming motility as well as increased virulence to the nematode C. elegans. The carriage of IncA/C2 plasmid could contribute to the distinguishing feature of the bovine isolates. The tandem use of genotypic-phenotypic assays improves the understanding of diversity and differential behaviour of the same serovar from unrelated host sources.

/pdf/characterization-of-salmonella-dublin-isolated-from-bovine-wgsz3h37m9.pdf

Characterization of Salmonella Dublin isolated from bovine and human hosts.

With the increase in commercial pig farming, there is a simultaneous increase in the use of antibiotics for prophylaxis as well as therapeutics in China. In this study, we evaluated the prevalence and resistance diversity of salmonellae isolated from feces of asymptomatic, live and slaughtered pigs. We analyzed 1,732 pig fecal samples collected over 8 months, at Henan province of China. The salmonellae were isolated and identified by PCR. They were serotyped using commercial antisera and assayed for the MIC of 16 antibiotics by broth microdilution method. The average prevalence of Salmonella was 19.4% (95% CI: 17.6-21.4). Large farms (herd size ≥1,000) were found to have a higher prevalence as compared to the small- and medium-scale farms (p 80%) was recorded toward ciprofloxacin (100%), tetracycline (99.4%), doxycycline (97%), sulfamethoxazole (85.8%), ampicillin (81.6%), and amoxicillin (80.4%). Multidrug resistance (MDR) to four, five, and seven classes of antibiotics was recorded to be approximately 25% in the most prevalent serovar like Derby. We conclude that the presence of alarmingly high resistance, toward the critical antibiotics such as fluoroquinolones and beta-lactams, in large swine farms in China, should draw public attention. These results highlight the need for continued antibiotic stewardship programs for judicious use of critical antibiotics in animal health as well as for producing safe pork.

/pdf/antibiotic-resistance-profiles-of-salmonella-recovered-from-2fxbs3f8ep.pdf

Antibiotic Resistance Profiles of Salmonella Recovered From Finishing Pigs and Slaughter Facilities in Henan, China.

Prevalence and genomic investigation of Salmonella isolates recovered from animal food-chain in Xinjiang, China.

Salmonella is composed of a wide variety of serovars, causing human self-limited gastrointestinal illnesses or invasive infections Invasive non-typhoidal Salmonella (iNTS) is well documented, with high mortality for children and immunocompromised adults in sub-Saharan Africa and has recently been reported in Southeast Asia However, iNTS in China remains unknown In May 2019, a case of invasive infection caused by Salmonella enterica serovar Uzaramo (S Uzaramo) was reported for the first time in China Phylogenomic analysis was performed by genomic sequencing the available contextualized isolates, which separated the two Chinese strains into different sublineages Both phenotypic and genomic characterization demonstrated that the S Uzaramo isolates showed in general low antimicrobial resistance potential, except one isolated from lake-water in China Additional comparative genomic analysis and Caenorhabditis elegans killing assays suggested a unique combination of virulence factors, including typhoid toxin and tcf fimbrial adhesin, which might play a role in the invasive infection This study highlights that the transparency of global surveillance genomic data could accelerate understanding of virulence and antimicrobial resistance makeup of a previously unknown threat

Genomic characterization of Salmonella Uzaramo for human invasive infection.

Probiotic bacteria exhibit beneficial effects on human and/or animal health, and have been widely used in foods and fermented products for decades. Most probiotics consist of lactic acid bacteria (LAB), which are used in the production of various food products but have also been shown to have the ability to prevent certain diseases. With the expansion of applications for probiotic LAB, there is an increasing concern with regard to safety, as cases with adverse effects, i.e., severe infections, transfer of antimicrobial resistance genes, etc., can occur. Currently, in vitro assays remain the primary way to assess the properties of LAB. However, such methodologies are not meeting the needs of strain risk assessment on a high-throughput scale, in the context of the evolving concept of food safety. Analyzing the complete genetic information, including potential virulence genes and other determinants with a negative impact on health, allows for assessing the safe use of the product, for which whole-genome sequencing (WGS) of individual LAB strains can be employed. Genomic data can also be used to understand subtle differences in the strain level important for beneficial effects, or protect patents. Here, we propose that WGS-based bioinformatics analyses are an ideal and cost-effective approach for the initial in silico microbial risk evaluation, while the technique may also increase our understanding of LAB strains for food safety and probiotic property evaluation.

Xianqi Peng

Papers

Antibiotic Resistance Profiles of Salmonella Recovered From Finishing Pigs and Slaughter Facilities in Henan, China.

Characterization of Salmonella Dublin isolated from bovine and human hosts.

Prevalence and genomic investigation of Salmonella isolates recovered from animal food-chain in Xinjiang, China.

Genomic characterization of Salmonella Uzaramo for human invasive infection.

Whole genome sequencing for the risk assessment of probiotic lactic acid bacteria.