Research Note: Synergistic effect of isopropoxy benzene guanidine and colistin against mcr-1-positive Escherichia coli in vitro and in duck intestine infection models.

Colistin (CST) is considered as "agent of last resort" against gram-negative bacteria as feed additive. Its clinical effectiveness has reduced since the emergence of mcr-1 gene in ducks. Isopropoxy benzene guanidine (IBG), a new guanidine derivative, showed positive effects on improving animal weights and alleviating intestinal pathogens, therefore, the objective of this study was to evaluate the effect of this compound supplement with CST in ducks and explore the possibilities in feed additive. A total of fifteen duck-origin Escherichia coli carrying the mcr-1 gene were included in this study. A checkerboard microdilution assay was used to evaluate the in vitro antibacterial activity of IBG combined with CST against mcr-1-positive E. coli. A 3-by-2 time-kill array of IBG (16, 32, and 64 µg/mL) and CST (1/2 MIC and 1/4 MIC) over 24 hours was utilized to characterize the activity of the agents alone and in combination against E. coli strain 1 in vitro. The intestinal colonization model was used to evaluate the in vivo effect of IBG combined with CST. These results indicated that the combination of IBG plus CST showed a synergistic effect against all clinical isolates (FICI < 0.5). The bacterial burden was reduced by more than 2 log10 CFU/mL when E. coli strain 1 was tested with 1/2 MIC CST plus 64 µg/mL IBG for 24 h. Further experiments in vivo demonstrated that the CST combined with IBG was able to increase duck weights, reduced intestinal pathogenic E. coli and showed a synergistic antibacterial effect. Combination of CST (4 mg/kg b.w.) plus IBG (32 or 64 mg/kg b.w.) achieved 1.84 to 3.29 log10 CFU/g killing after 7 d of therapy, which was significantly different from that in the challenge control group (p<0.05). In summary, our study demonstrated the potential use of IBG as feed additive for veterinary purposes in ducks and provided new insights into overcoming resistance in the future.

Rapid Dissemination of blaNDM-5 Gene among Carbapenem-Resistant Escherichia coli Isolates in a Yellow-Feather Broiler Farm via Multiple Plasmid Replicon.

Although carbapenems have not been approved for animal use, carbapenem-resistant Escherichia coli (CREC) strains are increasingly being detected in food-producing animals, posing a significant public health risk. However, the epidemiological characteristics of CREC isolates in yellow-feather broiler farms remain unclear. We comprehensively investigated the genetic features of carbapenem-resistance genes among E. coli isolates recovered from a yellow-feather broiler farm in Guangdong province, China. Among the 172 isolates, 88 (51.2%) were recovered from chicken feces (88.5%, 54/61), the farm environment (51.1%, 24/47), and specimens of dead chickens (15.6%, 41/64). All CREC isolates were positive for the blaNDM-5 gene and negative for other carbapenem-resistance genes. Among 40 randomly selected isolates subjected to whole-genome sequencing, 10 belonged to distinct sequence types (STs), with ST167 (n = 12) being the most prevalent across different sources, suggesting that the dissemination of blaNDM-5 was mainly due to horizontal and clonal transmission. Plasmid analysis indicated that IncX3, IncHI2, and IncR-X1-X3 hybrid plasmids were responsible for the rapid transmission of the blaNDM-5 gene, and the genetic surrounding of blaNDM-5 contained a common mobile element of the genetic fragment designated "IS5-△ISAba125-blaNDM-5-bleMBL-trpF-dsbC". These findings demonstrate a critical role of multiple plasmid replicons in the dissemination of blaNDM-5 and carbapenem resistance.

Molecular epidemiology and transmission of rmtB-positive Escherichia coli among ducks and environment.

This study aimed to investigate the transmission and molecular epidemiological characteristics of the rmtB gene in Escherichia coli (E. coli) strains isolated from duck farms in Guangdong Province of China from 2018 to 2021. A total of 164 (19.4%, 164/844) rmtB-positive E. coli strains were recovered from feces, viscera, and environment. We performed antibiotic susceptibility tests, pulsed-field gel electrophoresis (PFGE), and conjugation experiments. We obtained the genetic context of 46 rmtB-carrying E. coli isolates and constructed a phylogenetic tree via whole genome sequencing (WGS) and bioinformatic analysis. The isolation rate of rmtB-carrying E. coli isolates in duck farms increased yearly from 2018 to 2020 but decreased in 2021. All rmtB-harboring E. coli strains were multidrug resistant (MDR), and 99.4% of the strains were resistant to more than 10 drugs. Surprisingly, duck- and environment-associated strains similarly showed high MDR. Conjugation experiments revealed that the rmtB gene horizontally cocarried blaCTX-M and blaTEM gene dissemination via IncFII plasmids. Insertion sequences IS26, ISCR1, and ISCR3 were closely associated with the spread of rmtB-harboring E. coli isolates. WGS analysis indicated that ST48 was the most prevalent sequence type. The results of single nucleotide polymorphism (SNP) differences revealed potential clonal transmission between ducks and the environment. Based on One Health principles, we need to strictly use veterinary antibiotics, monitor the distribution of MDR strains, and evaluate the impact of plasmid-mediated rmtB gene on human, animal, and environmental health.

Emergence of IncHI2 Plasmid-Harboring blaNDM-5 from Porcine Escherichia coli Isolates in Guangdong, China.

Carbapenem resistance has posed potential harmful risks to human and animals. The objectives of this study were to understand the prevalence of blaNDM-5 in pigs and investigate the molecular characteristics of NDM-5-producing Escherichia coli isolates in Guangdong province in China. Carbapenem-resistant E. coli isolates were isolated from pigs and obtained using MacConkey plates containing 0.5 mg/L meropenem. Conjugation assay and antimicrobial susceptibility testing were conducted for the isolates and their transconjugants. Whole-genome sequence (WGS) was used to analyze the plasmid genetic feature. A total of five blaNDM-5-carrying E. coli isolates were obtained in the present investigations. They belonged to five ST types. The blaNDM-5 genes were found to be in IncX3 and IncHI2 plasmid. The IncX3 plasmid was 46,161 bp in size and identical to other reports. IncHI2 plasmid was 246,593 bp in size and similar to other IncHI2-ST3 plasmids. It consisted of a typical IncHI2 plasmid backbone region and a multiresistance region (MRR). The blaNDM-5 was closely associated with the IS3000-ISAba125-blaNDM-5-bleMBL-trpF-tat-IS26 unit. We first reported the blaNDM-5-carrying IncHI2 in E. coli isolates recovered from pigs and revealed the molecular characterization. Continued surveillance for the dissemination of blaNDM-5 among food-producing animals is required.

Identification of the Multiresistance Gene poxtA in Oxazolidinone-Susceptible Staphylococcus haemolyticus and Staphylococcus saprophyticus of Pig and Feed Origins.

Previous studies on the prevalence and transmission mechanism of oxazolidinone resistance gene poxtA in CoNS are lacking, which this study addresses. By screening 763 CoNS isolates from different sources of several livestock farms in Guangdong, China, 2018-2020, we identified that the poxtA was present in seven CoNS isolates of pig and feed origins. Species identification and multilocus sequence typing (MLST) confirmed that seven poxtA-positive CoNS isolates were composed of five ST64-Staphylococcus haemolyticus and two Staphylococcus saprophyticus isolates. All poxtA-positive Staphylococcus haemolyticus isolates shared similar pulsed-field gel electrophoresis (PFGE) patterns. Transformation assays demonstrated all poxtA-positive isolates were able to transfer poxtA gene to Staphylococcus aureus RN4220. S1-PFGE and whole-genome sequencing (WGS) revealed the presence of poxtA-carrying plasmids in size around 54.7 kb. The plasmid pY80 was 55,758 bp in size and harbored the heavy metal resistance gene czcD and antimicrobial resistance genes, poxtA, aadD, fexB and tet(L). The regions (IS1216E-poxtA-IS1216E) in plasmid pY80 were identified in Staphylococcus spp. and Enterococcus spp. with different genetic and source backgrounds. In conclusion, this was the first report about the poxtA gene in Staphylococcus haemolyticus and Staphylococcus saprophyticus, and IS1216 may play an important role in the dissemination of poxtA among different Gram-positive bacteria.

Rapid Increase in the IS26-Mediated cfr Gene in E. coli Isolates with IncP and IncX4 Plasmids and Co-Existing cfr and mcr-1 Genes in a Swine Farm.

This paper aimed to investigate the molecular epidemiological features of the cfr gene in E. coli isolates in a typical swine farm during 2014-2017. A total of 617 E. coli isolates were screened for the cfr gene using PCR amplification. A susceptibility test, pulsed-field gel electrophoresis (PFGE), S1-PFGE, southern blotting hybridization, and the genetic context of the cfr gene were all used for analyzing all cfr-positive E. coli isolates. A conjugation experiment was conducted with the broth mating method using E. coli C600 as the recipient strain and 45 mcr-1-cfr-bearing E. coli isolates as the donor strain. Plasmids pHNEP124 and pHNEP129 were revealed by Illumina Miseq 2500. Eighty-five (13.7%) E. coli isolates were positive for the cfr gene and the prevalence of the cfr gene had significantly increased from 1.6% in 2014 to 29.1% in 2017. The Pulsed-Field Gel Electrophoresis (PFGE) analysis indicated that the spread of the cfr gene among E. coli isolates was mainly due to horizontal transfer. In addition, the cfr gene was primarily located on the plasmids between 28.8-kb to 60-kb in size, and the cfr gene was flanked by two copies of IS26 with the same orientation. Sequence analysis suggested that the plasmids pHNEP124 and pHNEP129 co-harboring the cfr and mcr-1 genes belonged to the plasmids IncP plasmid and IncX4 plasmid, respectively. In conclusion, this is the first study to report the high prevalence of the cfr gene among E. coli isolates and the first report of the complete genome sequence of IncP and IncX4 plasmids carrying the mcr-1 and cfr genes. The occurrence and dissemination of the cfr/mcr-1-carrying plasmids among E. coli isolates need further surveillance.

Reversal of mcr-1-Mediated Colistin Resistance in Escherichia coli by CRISPR-Cas9 System.

PURPOSE: The plasmid-borne mobilized colistin resistance gene (mcr-1) was discovered in 2015. Subsequently, the rapid horizontal transfer of mcr-1 gene to diverse bacterial species poses a serious threat to public health, which urgently needs the introduction of novel antimicrobial strategies. Therefore, the purpose of this study is to sensitize bacteria to colistin and reduce the propagation of mcr-1 gene by curing mcr-1-harboring plasmid in Escherichia coli (E. coli) using the CRISPR-Cas9 system. METHODS: Two sgRNAs specific to mcr-1 gene were designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-mcr was transformed into E. coli carrying pUC19-mcr-1 or pHNSHP45, separately. The elimination efficiency in strains was evaluated by PCR and quantitative real-time PCR (qPCR). The antimicrobial susceptibility test was performed using the broth microdilution method. RESULTS: In this study, we constructed the high copy number plasmid pUC19-mcr-1 and recombinant plasmid pCas9-m1 or pCas9-m2, which contain 20 nt or 30 nt sgRNA sequences targeted to mcr-1, respectively. PCR and qPCR results showed that mcr-1-harboring plasmids could be efficiently eliminated, and there was no significant correlation between sgRNA lengths and curing efficiency. However, when comparing restructured high copy number plasmid (pUC19-mcr-1) to natural resistance plasmid (pHNSHP45) in eliminating efficiency, we found that the content of plasmid backbone had an influence on efficiency. Furthermore, the conjugation assays verified that the engineered CRISPR-Cas9 system in bacteria or in bacteria genome can protect the recipient from plasmid-borne mcr-1 transfer via conjugation. Additionally, sequence analysis showed that three different types of defects in CRISPR-Cas9 system lead to escape mutants. CONCLUSION: We presented a method that only one plasmid-mediated CRISPR-Cas9 system can be used to efficiently resensitize E. coli to colistin. Moreover, this system provided a great potentiality to counteract the propagation of mcr-1 among bacterial pathogens.

Biofilm Production Ability, Virulence and Antimicrobial Resistance Genes in Staphylococcus aureus from Various Veterinary Hospitals.

: Staphylococcus aureus (S. aureus) is one of the most clinically important zoonotic pathogens, but an understanding of the prevalence, biofilm formulation ability, virulence, and antimicrobial resistance genes of S. aureus from veterinary hospitals is lacking. By characterizing S. aureus in different origins of veterinary hospitals in Guangzhou, China, in 2019, we identified with the presence of S. aureus in pets (17.1%), veterinarians (31.7%), airborne dust (19.1%), environmental surfaces (4.3%), and medical device surfaces (10.8%). Multilocus sequence typing (MLST) and Staphylococcus protein A (spa) typing analyses demonstrated methicillin-sensitive S. aureus (MSSA) ST398-t571, MSSA ST188-t189, and methicillin-resistant S. aureus (MRSA) ST59-t437 were the most prevalent lineage. S. aureus with similar pulsed-field gel electrophoresis (PFGE) types distributed widely in different kinds of samples. The crystal violet straining assays revealed 100% (3/3) of MRSA ST59 and 81.8% (9/11) of MSSA ST188 showed strong biofilm formulation ability, whereas other STs (ST1, ST5, ST7, ST15, ST88, ST398, ST3154 and ST5353) showed weak biofilm production ability. Polymerase chain reaction (PCR) confirmed the most prevalent leucocidin, staphylococcal enterotoxins, ica operon, and adhesion genes were lukD-lukE (49.0%), sec-sel (15.7%), icaA-icaB-icaC-icaR (100.0%), and fnbB-cidA-fib-ebps-eno (100.0%), respectively. Our study showed that the isolates with strong biofilm production ability had a higher prevalence in clfA, clfB, fnbA and sdrC genes compared to the isolates with weak biofilm production ability. Furthermore, 2 ST1-MRSA isolates with tst gene and 1 ST88-MSSA isolate with lukS/F-PV gene were detected. In conclusion, the clonal dissemination of S. aureus of different origins in veterinary hospitals may have occurred; the biofilm production capacity of S. aureus is strongly correlated with ST types; some adhesion genes such as clfA, clfB, fnbA, and sdrC may pose an influence on biofilm production ability and the emergence of lukS/F-PV and tst genes in S. aureus from veterinary hospitals should raise our vigilance.

Clonal Spread of Escherichia coli ST93 Carrying mcr-1-Harboring IncN1-IncHI2/ST3 Plasmid Among Companion Animals, China.

The purpose of this study was to investigate the occurrence of plasmid-mediated colistin resistance gene mcr-1 in Enterobacteriaceae isolates from companion animals in Guangzhou, China. Enterobacteriaceae isolated from 180 samples collected from cats and dogs were screened for mcr-1 by PCR and sequencing. MCR-1-producing isolates were further characterized by multilocus sequence typing and pulsed-field gel electrophoresis (PFGE). Plasmid characterization was performed by conjugation, replicon typing, S1-PFGE, and Southern blot hybridization. Plasmid pHN6DS2 as a representative IncN1-IncHI2/ST3 plasmid from ST93 E. coli was fully sequenced. pHN6DS2-like plasmids were screened by PCR-mapping and sequencing. The mcr-1 gene was detected in 6.25% (8/128) Escherichia coli isolates, of which, five belonged to E. coli ST93 and had identical PFGE patterns, resistance profiles and resistance genes. mcr-1 genes were located on ∼244.4 kb plasmids (n = 6), ∼70 kb plasmids, and ∼60 kb plasmids, respectively. Among them, five mcr-1-carrying plasmids were successfully transferred to recipient by conjugation experiments, and were classified as IncN1-IncHI2/ST3 (∼244.4 kb, n = 4, all obtained from E. coli ST93), and IncI2 (∼70 kb, n = 1), respectively. Plasmid pHN6DS2 contained a typical IncHI2-type backbone, with IncN1 segment (ΔrepA-Iterons I-gshB-ΔIS1294) inserted into the multiresistance region, and was similar to other mcr-1-carrying IncHI2/ST3 plasmids from Enterobacteriaceae isolates of various origins in China. The remaining five mcr-1-bearing plasmids with sizes of ∼244.4 kb were identified to be pHN6DS2-like plasmids. In conclusion, clonal spread of ST93 E. coli isolates was occurred in companion animals in Guangzhou, China.

Evolution and Comparative Genomics of F33:A-:B- Plasmids Carrying blaCTX-M-55 or blaCTX-M-65 in Escherichia coli and Klebsiella pneumoniae Isolated from Animals, Food Products, and Humans in China.

To understand the underlying evolution process of F33:A-:B- plasmids among Enterobacteriaceae isolates of various origins in China, the complete sequences of 17 blaCTX-M-harboring F33:A-:B- plasmids obtained from Escherichia coli and Klebsiella pneumoniae isolates from different sources (animals, animal-derived food, and human clinics) in China were determined. F33:A-:B- plasmids shared similar plasmid backbones comprising replication, leading, and conjugative transfer regions and differed by the numbers of repeats in yddA and traD and by the presence of group II intron, except that pHNAH9 lacked a large segment of the leading and transfer regions. The variable regions of F33:A-B- plasmids were distinct and were inserted downstream of the addiction system pemI/pemK, identified as the integration hot spot among F33:A-B- plasmids. The variable region contained resistance genes and mobile elements or contained segments from other types of plasmids, such as IncI1, IncN1, and IncX1. Three plasmids encoding CTX-M-65 were very similar to our previously described pHN7A8 plasmid. Four CTX-M-55-producing plasmids contained multidrug resistance regions related to that of F2:A-B- plasmid pHK23a from Hong Kong. Five plasmids with IncN and/or IncX replication regions and IncI1-backbone fragments had variable regions related to those of pE80 and p42-2. The remaining five plasmids with IncN replicons and an IncI1 segment also possessed closely related variable regions. The diversity in variable regions was presumably associated with rearrangements, insertions, and/or deletions mediated by mobile elements, such as IS26 and IS1294IMPORTANCE Worldwide spread of antibiotic resistance genes among Enterobacteriaceae isolates is of great concern. F33:A-:B- plasmids are important vectors of resistance genes, such as blaCTX-M-55/-65, blaNDM-1, fosA3, and rmtB, among E. coli isolates from various sources in China. We determined and compared the complete sequences of 17 F33:A-:B- plasmids from various sources. These plasmids appear to have evolved from the same ancestor by mobile element-mediated rearrangement, acquisition, and/or loss of resistance modules and similar IncN1, IncI1, and/or IncX1 plasmid backbone segments. Our findings highlight the evolutionary potential of F33:A-:B- plasmids as efficient vectors to capture and diffuse clinically relevant resistance genes.

The role of wildlife (wild birds) in the global transmission of antimicrobial resistance genes.

Antimicrobial resistance is an urgent global health challenge in human and veterinary medicine. Wild animals are not directly exposed to clinically relevant antibiotics; however, antibacterial resistance in wild animals has been increasingly reported worldwide in parallel to the situation in human and veterinary medicine. This underlies the complexity of bacterial resistance in wild animals and the possible interspecies transmission between humans, domestic animals, the environment, and wildlife. This review summarizes the current data on expanded-spectrum ß-lactamase (ESBL), AmpC ß-lactamase, carbapenemase, and colistin resistance genes in Enterobacteriaceae isolates of wildlife origin. The aim of this review is to better understand the important role of wild animals as reservoirs and vectors in the global dissemination of crucial clinical antibacterial resistance. In this regard, continued surveillance is urgently needed worldwide.