Epidemiological characteristics of human- and chicken-derived CTX-M-type extended-spectrum ß-lactamase-producing Escherichia coli from China.

Bacterial resistance to ß-lactams is mainly attributed to CTX-M-type extended-spectrum ß-lactamases (ESBLs). However, the predominant sequence type (ST) of blaCTX-M-carrying Escherichia coli (blaCTX-M-Ec) in chickens, an important food animal, in China and its contribution to human ß-lactam resistance are not investigated. In this study, approximately 1808 chicken-derived strains collected from 10 provinces from 2012 to 2020 were screened for blaCTX-M-Ec, and 222 blaCTX-M-Ec were identified. Antimicrobial susceptibility tests, whole genome sequencing and conjugation experiment were performed. All quality-controlled 136 chicken-derived blaCTX-M-Ec and 1193 human-derived blaCTX-M-Ec genomes were downloaded from NCBI and EnteroBase to comprehensively analyze the prevalence of blaCTX-M-Ec in China. blaCTX-M-55 (153/358, 42.7% in chicken isolates; 312/1193, 26.2% in human isolates) and blaCTX-M-14 (92/358, 25.7% in chicken isolates; 450/1193, 37.7% in human isolates) were dominant in blaCTX-M-Ec. The STs of blaCTX-M-Ec were diverse and scattered, with ST155 (n = 21) and ST152 (n = 120) being the most abundant in chicken- and human-derived isolates, respectively. Few examples indicated that chicken- and human-derived blaCTX-M-Ec have 10 or less core genome single nucleotide polymorphisms (cgSNPs). Genetic environment analysis indicated that ISEcp1, IS26 and IS903B were closely associated with blaCTX-M transfer. The almost identical pc61-55 and pM-64-1161 indicated the possibility of plasmid-mediated transmission of blaCTX-M between humans and chickens. Although the genomes of most blaCTX-M-Ec isolated from chickens and humans were quite different, the prevalence and genetic environment of blaCTX-M variants in both hosts were convergent. CTX-M-mediated resistance is more likely to spread through horizontal gene transmission than bacterial clones.

Increased mutations in lipopolysaccharide biosynthetic genes cause time-dependent development of phage resistance in Salmonella.

Understanding how bacteria evolve resistance to phages has implications for phage-based therapies and microbial evolution. In this study, the susceptibility of 335 Salmonella isolates to the wide host range Salmonella phage BPSELC-1 was tested. Potentially significant gene sets that could confer resistance were identified using bioinformatics approaches based on phage susceptibility phenotypes; more than 90 potential antiphage defense gene sets, including those involved in lipopolysaccharide (LPS) biosynthesis, DNA replication, secretion systems, and respiratory chain, were found. The evolutionary dynamics of Salmonella resistance to phage were assessed through laboratory evolution experiments, which showed that phage-resistant mutants rapidly developed and exhibited genetic heterogeneity. Most representative Salmonella hosts (58.1% of 62) rapidly developed phage resistance within 24 h. All phage-resistant mutant clones exhibited genetic heterogeneity and observed mutations in LPS-related genes (rfaJ and rfaK) as well as other genes such as cellular respiration, transport, and cell replication-related genes. The study also identified potential trade-offs, indicating that bacteria tend to escape fitness trade-offs through multi-site mutations, all tested mutants increased sensitivity to polymyxin B, but this does not always affect their relative fitness or biofilm-forming capacity. Furthermore, complementing the rfaJ mutant gene could partially restore the phage sensitivity of phage-resistant mutants. These results provide insight into the phage resistance mechanisms of Salmonella and the complexity of bacterial evolution resulting from phage predation, which can inform future strategies for phage-based therapies and microbial evolution.

Whole-Genome Sequencing Analysis of Non-Typhoidal Salmonella Isolated from Breeder Poultry Farm Sources in China, 2020-2021.

Non-typhoidal salmonellosis is a dangerous foodborne disease that causes enormous economic loss and threatens public health worldwide. The consumption of food, especially poultry or poultry products, contaminated with non-typhoidal Salmonella (NTS) is the main cause of human salmonellosis. To date, no research has identified the molecular epidemiological characteristics of NTS strains isolated from breeder chicken farms in different provinces of China. In our study, we investigated the antimicrobial resistance, phylogenetic relationships, presence of antimicrobial resistance and virulence genes, and plasmids of NTS isolates recovered from breeder chicken farms in five provinces of China between 2020 and 2021 by using a whole-genome sequencing (WGS) approach and phenotypic methods. All sequenced isolates belonged to six serovars with seven sequence types. Nearly half of the isolates (44.87%) showed phenotypic resistance to at least three classes of antimicrobials. Salmonella enterica serotype Kentucky harbored more antimicrobial resistance genes than the others, which was highly consistent with phenotypic resistance. Furthermore, the carried rate of 104 out of 135 detected virulence genes was 100%. Overall, our WGS results highlight the need for the continuous monitoring of, and additional studies on, the antimicrobial resistance of NTS.

Comparative Analysis of Phylogenetic Relationships and Virulence Factor Characteristics between Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates Derived from Clinical Sites and Chicken Farms.

Antimicrobial resistance in bacteria is the most urgent global threat to public health, with extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-E. coli) being one of the most documented examples. Nonetheless, the ESBL-E. coli transmission relationship among clinical sites and chicken farms remains unclear. Here, 408 ESBL-E. coli strains were isolated from hospitals and chicken farms in Sichuan Province and Yunnan Province in 2021. We detected blaCTX-M genes in 337 (82.62%) ESBL-E. coli strains. Although the isolation rate, prevalent sequence type (ST) subtypes, and blaCTX-M gene subtypes of ESBL-E. coli varied based on regions and sources, a few strains of CTX-ESBL-E. coli derived from clinical sites and chicken farms in Sichuan Province displayed high genetic similarity. This indicates a risk of ESBL-E. coli transmission from chickens to humans. Moreover, we found that the high-risk clonal strains ST131 and ST1193 primarily carried blaCTX-M-27. This indicates that drug-resistant E. coli from animal and human sources should be monitored. As well, the overuse of ß-lactam antibiotics should be avoided in poultry farms to ensure public health and build an effective regulatory mechanism of "farm to fork" under a One Health perspective. IMPORTANCE Bacterial drug resistance has become one of the most significant threats to human health worldwide, especially for extended-spectrum ß-lactamase-producing E. coli (ESBL-E. coli). Timely and accurate epidemiological surveys can provide scientific guidance for the adoption of treatments in different regions and also reduce the formation of drug-resistant bacteria. Our study showed that the subtypes of ESBL-E. coli strains prevalent in different provinces are somewhat different, so it is necessary to individualize treatment regimens in different regions, and it is especially important to limit and reduce antibiotic use in poultry farming since chicken-derived ESBL-E. coli serves as an important reservoir of drug resistance genes and has the potential to spread to humans, thus posing a threat to human health. The use of antibiotics in poultry farming should be particularly limited and reduced.

Widespread Dissemination of Plasmid-Mediated Tigecycline Resistance Gene tet(X4) in Enterobacterales of Porcine Origin.

The emergence of the plasmid-mediated high levels of the tigecycline resistance gene has drawn worldwide attention and has posed a major threat to public health. In this study, we investigated the prevalence of the tet(X4)-positive Enterobacterales isolates collected from a pig slaughterhouse and farms. A total of 101 tigecycline resistance strains were isolated from 353 samples via a medium with tigecycline, of which 33 carried tet(X4) (9.35%, 33/353) and 2 carried tet(X6) (0.57%, 2/353). These strains belong to seven different species, with Escherichia coli being the main host bacteria. Importantly, this report is the first one to demonstrate that tet(X4) was observed in Morganella morganii. Whole-genome sequencing results revealed that tet(X4)-positive bacteria can coexist with other resistance genes, such as blaNDM-1 and cfr. Additionally, we were the first to report that tet(X4) and blaNDM-1 coexist in a Klebsiella quasipneumoniae strain. The phylogenetic tree of 533 tet(X4)-positive E. coli strains was constructed using 509 strains from the NCBI genome assembly database and 24 strains from this study, which arose from 8 sources and belonged to 135 sequence types (STs) worldwide. We used Nanopore sequencing to interpret the selected 21 nonclonal and representative strains and observed that 19 tet(X4)-harboring plasmids were classified into 8 replicon types, and 2 tet(X6) genes were located on integrating conjugative elements. A total of 68.42% of plasmids carrying tet(X4) were transferred successfully with a conjugation frequency of 10-2 to 10-7. These findings highlight that diverse plasmids drive the widespread dissemination of the tigecycline resistance gene tet(X4) in Enterobacterales of porcine origin. IMPORTANCE Tigecycline is considered to be the last resort of defense against diseases caused by broad-spectrum resistant Gram-negative bacteria. In this study, we systematically analyzed the prevalence and genetic environments of the resistance gene tet(X4) in a pig slaughterhouse and farms and the evolutionary relationship of 533 tet(X4)-positive Escherichia coli strains, including 509 tet(X4)-positive E. coli strains selected from the 27,802 assembled genomes of E. coli from the NCBI between 2002 and 2022. The drug resistance of tigecycline is widely prevalent in pig farms where tetracycline is used as a veterinary drug. This prevalence suggests that pigs are a large reservoir of tet(X4) and that tet(X4) can spread horizontally through the food chain via mobile genetic elements. Furthermore, tetracycline resistance may drive tigecycline resistance through some mechanisms. Therefore, it is important to monitor tigecycline resistance, develop effective control measures, and focus on tetracycline use in the pig farms.

Outer Membrane Vesicles of Avian PathogenicEscherichia coli Mediate the Horizontal Transmission of blaCTX-M-55.

The CTX-M-55 type extended-spectrum ß-lactamase (ESBL) producing Enterobacteriaceae is increasing in prevalence worldwide without the transmission mechanism being fully clarified, which threatens public and livestock health. Outer membrane vesicles (OMVs) have been shown to mediate the gene horizontal transmission in some species. However, whether blaCTX-M-55 can be transmitted horizontally through OMVs in avian pathogenic Escherichia coli (APEC) has not been reported yet. To test this hypothesis, an ESBL-producing APEC was isolated and whole-genome sequencing (WGS) was performed to analyze the location of blaCTX-M-55. Ultracentrifugation and size exclusion chromatography was used to isolate and purify OMVs, and the transfer experiment of blaCTX-M-55 via OMVs was performed finally. Our results showed that the blaCTX-M-55 was located on an IncI2 plasmid. The number and diameter of OMVs secreted by ESBL-producing APEC treated with different antibiotics were significantly varied. The transfer experiment showed that the OMVs could mediate the horizontal transfer of blaCTX-M-55, and the frequency of gene transfer ranged from 10-5 to 10-6 CFU/mL with the highest frequency observed in the Enrofloxacin treatment group. These findings contribute to a better understanding of the antibiotics in promoting and disseminating resistance in the poultry industry and support the restrictions on the use of antibiotics in the poultry industry.

Clostridium butyricum Inhibits Fat Deposition via Increasing the Frequency of Adipose Tissue-Resident Regulatory T Cells.

SCOPE: Clostridium butyricum (CB) exerts beneficial actions in several disorders. However, the impact and molecular cues of CB in fat metabolism remain elusive. This study demonstrates the CB inhibition of fat deposition by increasing the relative number of adipose tissue-resident Treg cells (aTregs). METHODS AND RESULTS: CB is administered orally to wild type (WT) mice fed with chow diet, which decrease fat deposition and adipogenic gene expression, associating with elevated serum levels of butyrate. Sodium butyrate (SB) feeding mimics the CB suppression of fat accumulation. Of note, the frequency of aTregs in both the CB and SB treatments, analyzed by flow cytometry, is markedly increased, accompanied by activated Wnt10b expression in white adipose tissues. However, CB and SB fail to inhibit fat deposition in Wnt10b-KO mice. Intriguingly, CB and SB are able to alleviate the obesity, fatty liver, and glucose abnormalities in high fat diet (HFD)-fed WT mice. CONCLUSIONS: These findings suggest that CB, through its metabolite butyrate, inhibits fat deposition via potentiating aTreg cell generation, and support the option of CB and SB for therapeutic interventions in obesity and related disorders.

Technological and Safety Characterization of Kocuria rhizophila Isolates From Traditional Ethnic Dry-Cured Ham of Nuodeng, Southwest China.

Nuodeng ham is known for its unique processing techniques and flavor. In the present study, proteolytic microorganisms from cured artisanal Nuodeng ham were investigated in order to identify and select potential starter cultures for its faster and safer fermentation. Eight isolates, accounting for 57% of proteolytic microorganisms, were found to be related to Kocuria rhizophila. Relevant properties of K. rhizophila as potential starter culture were evaluated in vitro for the first time. Intra-species diversities were found in phylogenetic and physiological properties of K. rhizophila isolates. Nevertheless, desirable attributes, such as halo-tolerance, nitrate reductase and protease activity, as well as the absence of antimicrobial resistance and amino acid decarboxylase activity, were observed in selected isolates. Moreover, genome analysis of isolates K24 and K45 confirmed their lack of typical genes for virulence, antimicrobial resistance and amino acid decarboxylase. K. rhizophila may thus represent a novel starter candidate of coagulase-negative cocci group and contribute to color and flavor development of fermented meats.

Prevalence and Antimicrobial Resistance of Salmonella Isolated From Dead-in-Shell Chicken Embryos in Shandong, China.

The present study was designed to explore the Salmonella prevalence and antimicrobial resistance characteristics in the context of chick mortality at hatching in China. Between December 2015 and August 2017, 1,288 dead-in-shell chicken embryos were collected from four breeder chicken hatcheries in Tai'an, Rizhao, Jining, and Heze, China. Salmonella isolates were successfully recovered from 6.7% of these embryos (86/1,288) and were evaluated for serotype, antimicrobial susceptibility, Class 1 integron prevalence, antimicrobial resistance gene expression, and multilocus sequence typing (MLST). Salmonella Thompson (37.2%), and Salmonella Infantis (32.6%) were the two most prevalent isolates in these chicken embryos, and 66.3% of isolates exhibited robust ampicillin resistance, while 55.8% of isolates exhibited multi-drug resistance (MDR). The majority of isolates harbored the bla TEM gene (74.4%), with the qnrS gene also being highly prevalent (50.0%). In contrast, just 27.9% of these isolates carried Class 1 integrons. These 86 isolates were separated into four sequence types (STs), whereby ST26 (32.2%) was the most prevalent. Overall, these results suggested that Salmonella infections may be an important cause of chicken embryo mortality in China, and that efforts to support the appropriate use of antibiotics in managing poultry populations are essential.

Clostridium butyricum Ameliorates Salmonella Enteritis Induced Inflammation by Enhancing and Improving Immunity of the Intestinal Epithelial Barrier at the Intestinal Mucosal Level.

This study was aimed to investigate the effects of Clostridium butyricum (C. butyricum) immunity and intestinal epithelial barrier function at the intestinal mucosal level, by using Salmonella enteritidis (S. enteritidis) to infect specific-pathogen-free (SPF) chickens and intestinal epithelial cells (IEC). We found that C. butyricum could decrease cytokine levels (IFN-γ, IL-1ß, IL-8, and TNF-α) via the TLR4-, MyD88-, and NF-κB-dependent pathways in intestinal tissues and intestinal epithelial cells. Additionally, C. butyricum could attenuate bacteria-induced intestinal damage and increase the expression level of muc-2 and ZO-1 in the intestine and intestinal epithelial cells. Furthermore, C. butyricum altered the intestinal microbial composition, increased the diversity of the bacterial communities in the cecum of Salmonella-infected chickens. In conclusion, C. butyricum effectively attenuated inflammation and epithelial barrier damage, altered the intestinal microbial composition, increased the diversity of the bacterial communities in the intestine of Salmonella-infected chickens. The result suggests that C. butyricum might be an effective and safe therapy for the treatment of Salmonella infection.

Serotype, antimicrobial susceptibility and genotype profiles of Salmonella isolated from duck farms and a slaughterhouse in Shandong province, China.

BACKGROUND: Salmonella has been considered as one of the most important foodborne pathogens that threatened breeding industry and public health. To investigate the prevalence and characterization of Salmonella isolated from duck farms and a slaughterhouse in Shandong province, a total of 49 Salmonella strains were isolated from 2342 samples from four duck farms and one duck slaughterhouse in Jinan and Tai'an, Shandong province, China. RESULTS: Among the isolates, S. Enteritidis (20/49, 40.8%) and S. Anatum (10/49, 20.4%) were the most prevalent, and high resistance rates were detected for erythromycin (49/49, 100.0%) and nalidixic acid (47/49, 95.9%). Class I integrons were detected in 17 isolates (34.7%17/49), which contained gene cassettes aadA7 + aac3-Id(15/17) and aadA5 + dfrA17 (2/17). Eleven different kinds of resistance genes were detected while blaTEM(36/49, 73.5%) was the most prevalent, followed by sul2(14/49, 28.6%). Thirteen virulence genes were tested, and all of the strains carried invA, hilA and sipA. Multilocus sequence typing (MLST) results showed that seven sequence types (STs) were identified; ST11 was the most prevalent ST (20/49, 40.8%), followed by ST2441 (10/49, 20.4%). There was a strong correlation between STs and serovars. The results of pulsed field gel electrophoresis(PFGE) showed that 39 PFGE patterns were generated from 49 Salmonella strains. PFGE patterns were mostly diverse and revealed high similarity between the isolates from the same sampling sites. CONCLUSIONS: The presence of Salmonella infections among duck farms revealed that ducks could also be potential reservoirs for Salmonella. The high resistance rates against commonly used antimicrobials suggested a need for more reasonable use of antimicrobials, as well as for investigating substitutes for antimicrobials.

Molecular and Phenotypic Characteristics of Escherichia coli Isolates from Farmed Minks in Zhucheng, China.

In this study, the prevalence, phenotypes, and clonal relationships of Escherichia coli (E. coli) strains isolated from minks were investigated. In July 2017, a total of 62 fresh faecal swab samples were randomly collected from one large-scale mink farm in Zhucheng, Shandong Province, China. In all the samples, 50 E. coli strains were isolated and then assigned to serotyping, antimicrobial susceptibility test, detection of antimicrobial resistance genes and the Class 1 integrons, and multilocus sequence typing (MLST). Four pathogenic serotypes were identified among all the isolates, while the most common serotype was enterohemorrhagic E. coli O104:H4 (6.0 %). Antimicrobial sensitivity testing revealed that most isolates were susceptible to cefoxitin (96.0 %) and amikacin (82.0 %), while most isolates were resistant to ampicillin (92.0 %) and tetracycline (90.0 %). An analysis of the nucleotide sequences revealed that 7 isolates (14.0%) carried 4 types of Class 1 integron cassette, including dfrA27+aadA2+qnrA (57.1%), dfrA17+aadA5 (14.3%), dfrA12+aadA2 (14.3%), and dfrA1+aadA1 (14.3%). PCR screening showed that 14 antibiotic resistance genes were presented in 50 isolates, while the most prevalent resistance gene was qnrS, which was detected in 60.0 % of isolates, followed by sul2 (40.0%) and oqxA (38.0%). MLST analysis showed that 32 sequence types (STs) were identified, while ST46 was the predominant genotype among all isolates. Clonal complex 3 (CC3) was dominant. Compared with 340 human E. coli STs reported in China, the ST10 clonal complex, known as the largest human clonal complex, was also found in the 50 mink E. coli isolates. Meanwhile, mink-derived strain ST206 formed a new clonal complex, CC206, which was different from human ST strains. Our results showed that farmed minks could be reservoirs of antimicrobial-resistant E. coli with Class 1 integron cassettes and resistance genes, which were likely to pose a threat to public health. Therefore, continuous inspections and monitoring of E. coli in minks are essential for detecting and controlling emerging E. coli with different serovars as well as antibiotic resistance.

Molecular Characterization of Antimicrobial Resistance in Escherichia coli from Rabbit Farms in Tai'an, China.

To investigate the prevalence and resistance against antimicrobials of Escherichia coli (E. coli) in Tai'an, March 2016, a total of 55 E. coli strains were isolated from 60 faecal samples of diarrheic rabbits collected from three rabbit farms in Tai'an. The E. coli isolates were assayed for antimicrobial susceptibility and prevalence of resistance genes and Class I integrons and genotyped using Multilocus Sequence Typing (MLST). All the E. coli isolates were sensitive to ceftazidime, ceftriaxone, imipenem, and amikacin, while 78.2% of the isolates showed resistance against tetracycline, and 65.5% were resistant against ampicillin. The most common resistance gene detected was blaTEM, present in 98.2% of isolates, followed by blaCTX-M (94.6%) and sul2 (58.2%). Class I integrons were detected in 17 out of the 55 (30.9%) E. coli strains. Seven kinds of gene cassette were detected: dfrA17 + aadA5, dfrA1 + catB3 + aacA4, aadA2 + LinF, dfrA1 + aadA1, aadA22, dfrA12 + orfF + addA2, and aadA16 + dfrA27 + arr-3. All the 55 E. coli strains were identified and classified as 13 sequence types (STs); ST302 (22/55, 40.0%) was the most prevalent type, followed by ST370 (12/55, 21.8%). This study showed that E. coli isolated from diarrheic farmed rabbits in the Tai'an area exhibit sometimes very frequent resistance to antimicrobials important to human medicine, which further highlights the need for reasonable use of antibiotics.