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Escherichia coli carrying IncK-blaCMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK-blaCMY-2-positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates (n = 46) confirmed the presence of the blaCMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the blaCMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0–60 and 13–90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates (n = 28) allowed further characterization and comparison of the IncK-blaCMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK-blaCMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK-blaCMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.

We describe a case of carbapenem resistant E. coli isolated from urine in an 87-year-old woman with recurrent urinary tract infections. Using whole genome sequencing (WGS), we identified the carbapenem resistance mechanism to be a combination of ompC porin loss and plasmid-mediated AmpC gene blaCMY-2 , which was not detected by routine molecular and phenotypic carbapenemase assays. Our case raises a concern for the limitation of current CRE screening tools for emerging resistance mechanisms and demonstrates the utility of WGS as a better tool for characterization of CRE in the clinical setting.

Salmonella is a leading cause of foodborne illness worldwide and is a common concern in food safety. Salmonella enterica displaying resistance to extended-spectrum cephalosporins (ESCs) and fluoroquinolone (FQs) has been deemed a high-priority pathogen by the World Health Organization. Co-resistance to ESCs and FQs has been reported in S. enterica serovar Thompson (S. Thompson). However, the genetic context of ESCs and FQs resistance genes in S. Thompson lacks sufficient characterization. In this study, we characterized a multi-drug resistant (MDR) S. Thompson isolate recovered from a retail ready-to-eat (RTE) pork product in China. Short- and long-read sequencing (HiSeq and MinION) of the genome identified the presence of blaCMY-2, qnrS1, and qepA8, along with 11 additional acquired antimicrobial resistance genes, residing on a 152,940 bp IncA/C plasmid. Specifically, the blaCMY-2, qnrS1, and qepA8 genes were located in insertion sequences (ISs) and integron mediated mobile genetic structure, sugE-blc-blaCMY-2-ISEc9, IS26-orf6 -qnrS1-orf5-ISKpn19, and intl1-qepA8-orf10-IS91-orf1-dfrA12-orf11-aadA2-qacE11-sul1, respectively. Each gene was identified in various bacteria species, indicating their high transfer ability. The plasmid was found to be transferable to Escherichia coli J53 by conjugation and resulted in the acquiring of multiple resistances in the transconjugants. The plasmid is closely related to plasmids from two human S. Thompson strains isolated in different regions and years in China. Moreover, core-genome Multi Locus Sequence Typing (cgMLST) and phylogenetic analysis based on global 1,868 S. Saintpaul isolates showed that the S. Thompson isolate was highly epidemiologically linked to a human isolate in China. Our findings suggest that Chinese RTE pork products are a possible source of human pathogenic ESCs and FQs co-resistant S. Thompson. Furthermore, the results underline the important role of conjugative plasmids in acquiring and transmission of ESCs and FQs resistance in S. Thompson isolates, which need continuous investigation. [ABSTRACT FROM AUTHOR]

At a time when antibiotic resistance is seemingly ubiquitous worldwide, understanding the mechanisms responsible for successful emergence of new resistance genes may provide insights into the persistence and pathways of dissemination for antibiotic-resistant organisms in general. For example, Escherichia coli strains harboring a class A b-lactamase-encoding gene (blaCTX-M-15) appear to be displacing strains that harbor a class C b-lactamase gene (blaCMY-2) in Washington State dairy cattle. We cloned these genes with native promoters into low-copy-number plasmids that were then transformed into isogenic strains of E. coli, and growth curves were generated for two commonly administered antibiotics (ampicillin and ceftiofur). Both strains met the definition of resistance for ampicillin ($32 mg/mL) and ceftiofur ($16 mg/mL). Growth of the CMY-2-producing strain was compromised at 1,000 mg/mL ampicillin, whereas the CTX-M-15-producing strain was not inhibited in the presence of 3,000 mg/mL ampicillin or with most concentrations of ceftiofur, although there were mixed outcomes with ceftiofur metabolites. Consequently, in the absence of competing genes, E. coli harboring either gene would experience a selective advantage if exposed to these antibiotics. Successful emergence of CTX-M-15-producing strains where CMY-2-producing strains are already established, however, requires high concentrations of antibiotics that can only be found in the urine of treated animals (e.g., .2,000 mg/mL for ampicillin, based on literature). This ex vivo selection pressure may be important for the emergence of new and more efficient antibiotic resistance genes and likely for persistence of antibiotic-resistant bacteria in food animal populations. [ABSTRACT FROM AUTHOR]

Salmonella Thompson is a prevalent foodborne pathogen and a major threat to food safety and public health. This study aims to reveal the dissemination mechanism of S. Thompson with co-resistance to ceftriaxone and ciprofloxacin. In this study, 181 S. Thompson isolates were obtained from a retrospective screening on 2118 serotyped Salmonella isolates from foods and patients, which were disseminated in 12 of 16 districts in Shanghai, China. A total of 10 (5.5 %) S. Thompson isolates exhibited resistance to ceftriaxone (MIC ranging from 8 to 32 μg/mL) and ciprofloxacin (MIC ranging from 2 to 8 μg/mL). The AmpC β-lactamase gene bla CMY-2 and plasmid-mediated quinolone resistance (PMQR) genes of qnr S and qep A were identified in the 9 isolates. Conjugation results showed that the co-transfer of bla CMY-2 , qnr S, and qep A occurred on the IncC plasmids with sizes of ∼150 (n = 8) or ∼138 (n = 1) kbp. Three typical modules of IS Ecp1 - bla CMY-2 - blc-sugE , IS 26 -IS 15DIV-qnr S-IS Kpn19 , and IS CR3 - qep A-intl1 were identified in an ST3 IncC plasmid pSH11G0791. Phylogenetic analysis indicated that IncC plasmids evolved into Lineages 1, 2, and 3. IncC plasmids from China including pSH11G0791 in this study fell into Lineage 1 with those from the USA, suggesting their close genotype relationship. In conclusion, to our knowledge, it is the first report of the co-existence of bla CMY-2 , qnr S, and qep A in IncC plasmids, and the conjugational transfer contributed to their dissemination in S. Thompson. These findings underline further challenges for the prevention and treatment of Enterobacteriaceae infections posed by IncC plasmids bearing bla CMY-2 , qnr S, and qep A. • Ceftriaxone and ciprofloxacin co-resistance was found in Salmonella Thompson. • Co-transfer of blaCMY-2, qnrS and qepA occurred on the IncC plasmids. • ISEcp1-blaCMY-2-blc-sugE, IS26-IS15DIV-qnrS-ISKpn19 and ISCR3-qepA-intl1 were found. • IncC plasmids from China fell into Lineage 1 with those from the USA. [ABSTRACT FROM AUTHOR]

Overall, 836 Escherichia coli isolates (695 isolates from dogs and 141 from cats) were recovered from the diarrhea, skin/ear, urine, and genitals of dogs and cats between 2018 and 2019. Cefovecin and enrofloxacin resistance were noted in 17.1% and 21.2% of E. coli isolates, respectively. The cefovecin and enrofloxacin resistance rates were higher in dog isolates (18.1% and 22.9%) compared with the rates in cat isolates (12.1%, 12.8%). Interestingly, resistance to both antimicrobials was noted in 10.8% (90/836) of the isolates, predominantly in isolates from dogs. blaCTX-M-14, blaCTX-M-15, and blaCMY-2 were the most frequent extended-spectrum β-lactamase/plasmid-mediated AmpC β-lactamase (ESBL/AmpC)- gene types. The co-existence of blaCTX-M andblaCMY-2 was noted in six E. coli isolates from dogs. Sequencing analysis demonstrated that S83L and D87N in gyrA and S80I in parC were the most frequent point mutations in the quinolone resistance-determining regions of the cefovecin and enrofloxacin-resistant isolates. A total of 11 isolates from dogs carried the plasmid-mediated quinolone resistance genes (six aac(6’)-Ib-cr, four qnrS, and one qnrB), while only two cat isolates carried the qnrS gene. Multilocus sequence typing of the cefovecin and enrofloxacin-resistant isolates revealed that sequence type (ST)131 E. coli carrying blaCTX-M-14 and blaCTX-M-15 genes and ST405 E. coli carrying blaCMY-2 gene were predominant among the isolated E. coli strains. The majority of the ESBL/AmpC-producing isolates displayed diverse pulsed-field gel electrophoresis profiles. This study demonstrated that third-generation cephalosporin- and fluoroquinolone-resistant E. coli were widely distributed in companion animals. The detection of the pandemic ST131 clone carrying blaCTX-M-14/15 in companion animals presented a public health threat.

Objectives: Salmonella enterica subsp. enterica serovar Kentucky has been associated with the worldwide ciprofloxacin-resistant (CIPR) Salmonella Kentucky sequence type 198 (ST198) epidemic clone, mostly recovered from poultry farms and products. The aim of this study was to examine whether this expanding clone exists in the Lebanese broiler chain. Methods: Eight CIPR and extended-spectrum cephalosporin-resistant Salmonella Kentucky isolates previously recovered from Lebanese broilers were genetically characterised by whole-genome sequencing. Results: Seven of the eight isolates belonged to ST198 and were phylogenetically closely related. They all harboured mutations in the chromosomal quinolone resistance genesgyrA and parC with double and single substitutions, respectively. The blaTEM-1B and blaCMY-2 genes were both detected in six isolates. Insertion sequence ISEcp1 was located upstream of blaCMY-2, harboured by IncI1 plasmids in four strains. An IS10 transposition coupled to homologous recombination at transposition sites mediated CMY-2 plasmid integration into the chromosome of one strain. Resistance genes to aminoglycosides [aadA7 and aac(3)-Id], tetracyclines [tet(A)] and sulfonamides (sul1) were detected in five strains, among which four were positive for the presence of Salmonella genomic island 1 (SGI1) variant SGI1-K. All studied isolates harboured a variety of Salmonella pathogenicity islands (SPIs) as well as common regulatory and virulence genes. Conclusion: Here we report for the first time in Lebanon the detection and dissemination of the emerging highly drug-resistantSalmonella Kentucky ST198. Our findings shed new light on this clone as a potential public-health threat.

Although the spread of plasmid-mediated antibiotic-resistant bacteria is a public health concern, food contamination with plasmid-mediated antibiotic-resistant Escherichia coli in Vietnam has not been well investigated. This study aimed to describe the prevalence of colistin-resistant, carbapenem-resistant, and endemic blaCTX-M in extended-spectrum β-lactamase (ESBL) producing E. coli isolates. Colistin and carbapenem-resistant ESBL-producing E. coli were isolated from chickens in Vietnam and Japan. Colistin-resistant and AmpC/ESBL-producing E. coli (52% and 93%, respectively) were detected in chickens from Vietnam, in comparison to 52.7%, AmpC/ESBL-producing E. coli found in chicken from Japan. Carbapenem-resistant E. coli has not been isolated in Vietnam and Japan. Genotyping revealed that colistin-resistant E. coli harboured mcr-1, and most of the AmpC/ESBL-related genes were blaCTX-M-55 and blaCTX-M-65 together with blaTEM in Vietnamese chickens and blaCMY-2 in Japanese chickens. Multi-drug resistance analysis showed that ESBL-producing E. coli isolates had greater resistance to quinolones, streptomycin, and chloramphenicol than colistin-resistant E. coli isolates from Vietnam, suggesting the selection of multiple antibiotic resistance genes in ESBL-producing E. coli. In conclusion, colistin-resistant E. coli was detected in approximately half of the chicken samples, the majority of which harboured mcr-1. The high prevalence of ESBL-producing E. coli has remained constant in the last 5 years. The predominant blaCTX-M in ESBL-producing E. coli was blaCTX-M-55 or blaCTX-M-65, with the coexistence of blaTEM in Vietnam. These results can be implemented in monitoring systems to overcome the development of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Colistin is a last-resort antibiotic in fighting severe infections caused by multidrug resistant Gram negative pathogens in hospitals. Zoonotic bacteria acquire colistin resistance in animal reservoirs and mediate its spread along the food chain. This is the case of non-typhoid serovars of Salmonella enterica. Colistin-resistant S. enterica in foods represents a threat to human health. Here, we assessed the prevalence of colistin-resistance in food-borne isolates of S. enterica (2014–2019; Asturias, Spain), and established the genetic basis and transferability of this resistance. Five out of 231 isolates tested (2.2%) were resistant to colistin. Four of them, belonging to the European monophasic ST34 clone of S. Typhimurium, were characterized in the present study. They were collected from pork or pork and beef meat-derived products, either in 2015 (three isolates) or 2019 (one isolate). Molecular typing with XbaI-PFGE and plasmid profiling revealed distinct patterns for each isolate, even though two of the 2015 isolates derived from the same sample. The MICs of colistin ranged from 8 to 16 mg/L. All isolates carried the mcr-1.1 gene located on conjugative plasmids of the incompatibility groups IncX4 (2015 isolates) or IncHI2 (2019 isolate). Apart from colistin resistance, the four isolates carried chromosomal genes conferring resistance to ampicillin, streptomycin, sulfonamides and tetracycline [blaTEM–1, strA-strB, sul2, and tet(B)] and heavy metals, including copper and silver (silESRCFBAGP and pcoGE1ABCDRSE2), arsenic (arsRSD2A2BCA1D1) ± mercury (merEDACPTR), which are characteristically associated with the European ST34 monophasic clone. The 2019 isolate was also resistant to other antibiotics, comprising third generation cephalosporins and cephamycins. The latter phenotype was conferred by the blaCMY–2 gene located on an IncI1-I(α)-ST2 plasmid. Results in the present study identified meat-derived products as a reservoir of a highly successful clone harboring transferable plasmids which confer resistance to colistin and other clinically important antibiotics. An important reduction in the number of food-borne S. enterica detected during the period of the study, together with the low frequency of colistin resistance, underlines the success of One Health initiatives, such as those implemented at the UE, to control zoonotic bacteria along the food chain and to halt the spread of antimicrobial resistance.

Dissemination of extended-spectrum cephalosporin (ESC)-resistant Salmonella is a public health concern in the egg production industry. ESC-resistant Salmonella often acquires the bla gene via insertion sequences (ISs). Therefore, this study aimed to assess antimicrobial resistance in Salmonella from Japanese layer breeding chains and egg processing chains, and determine the genetic profiles of IS-like elements in ESC-resistant Salmonella. Antimicrobial susceptibility testing was performed on 224 isolates from 49 facilities involving layer breeder farms, hatcheries, pullet-rearing farms, and layer farms in breeding chains along with egg processing chains. ESC-resistant Salmonella strains were whole-genome sequenced. Among them, 40 (17.9%) were resistant to at least streptomycin, tetracycline, ampicillin, chloramphenicol, cefpodoxime, nalidixic acid, ciprofloxacin, and/or kanamycin despite lacking resistance to azithromycin and meropenem. Moreover, 15 were ESC-resistant Salmonella harboring blaCMY-2 (Salmonella enterica serovar Ohio, n=12; S. Braenderup, n=1; untypeable with O7:b:-, n=1) and blaCTX-M-14 (S. Cerro, n=1). IncA/C2 plasmids containing ISEcp1, IS26, and multiple antimicrobial resistance genes (including blaCMY-2) were identified in S. Ohio isolates from pullet-rearing and layer farms belonging to the same company. Chromosomal integration of partial or whole IncA/C2 plasmids was seen with two S. Ohio isolates via ISEcp1 or IS26, respectively. Antimicrobial resistance genes such as blaCMY-2 might be transmitted among the upper and the lower levels of layer breeding chains via the replicon type IncA/C2 plasmids containing ISEcp1 and IS26. [ABSTRACT FROM AUTHOR]

The growth rate of the population aging is increasing worldwide. To assess antimicrobial susceptibility of extended-spectrum β-lactamase- (ESBL-) producing Klebsiella pneumoniae Isolated from Patients aging in Rasool Akram, Hospital, as well as to identify AmpC genes. 100 K. pneumoniae strain isolated from different clinical samples. Isolates resistant to oxyimino-cephalosporins and to cefoxitin evaluated to phenotypic ESBL production and to phenotypic AmpC production, respectively. Detection of resistance genes was then performed using primers specific for AmpC genes. Piperacillin/tazobactam and carbapenems remained the active β-lactam antibiotic against K. pneumoniae. ESBLs were detected among 40 (40%) of K. pneumoniae isolates. CMY-1 gene was detected in 34.3% of all AmpC-positive isolates, whereas CMY-2 and FOX genes were 14.2% and 28.6%, respectively. The consumption of Carbapenem family drugs is high in Iranian hospitals which are used as a first line of treatment without antibiotic susceptibility testing. Therefore, increase in antibiotic resistance to this family drugs is unavoidable in the near future. Therefore, it is necessary to take the necessary measures to modify the administration and use of antibiotics. [ABSTRACT FROM AUTHOR]

Thobeka P Mthembu,1 Oliver T Zishiri,1 Mohamed E El Zowalaty2,3 1School of Life Sciences, University of KwaZulu-Natal, Durban, 4000, South Africa; 2Virology and Microbiology Research Group, School of Health Sciences, University of KwaZulu-Natal, Durban, 4000, South Africa; 3Infectious Diseases and Anti-Infective Research Group, College of Pharmacy, University of Sharjah, Sharjah, 27272, UAECorrespondence: Mohamed E El ZowalatyDepartment of Pharmacy, City University College of Ajman, Ajman 18484, UAEEmail elzow001@gmail.comBackground: Antibiotic-resistant bacterial pathogens associated with livestock remain a major concern worldwide as they get transmitted from animals to humans and cause foodborne and zoonotic diseases.Methods: Antimicrobial resistance in livestock-associated Salmonella spp in South Africa was investigated using molecular DNA methods. Three hundred and sixty-one environmental faecal samples were randomly collected from avian (chicken and ducks), cows, pigs, goats, and sheep. Salmonella spp. were isolated on selective media and were confirmed using the polymerase chain reaction. Antimicrobial susceptibility testing against ampicillin, chloramphenicol, ciprofloxacin, ceftriaxone, azithromycin, tetracycline, amoxicillin-clavulanate and trimethoprim-sulfamethoxazole was determined using the Kirby–Bauer disk diffusion method. Isolates were screened for the presence of blaTEM-1, blaCMY-2, tetA, tetC, sul2 and dfrA7 resistance genes by PCR.Results: Most of the isolates were resistant to ampicillin (64%), tetracycline (63%), amoxicillin-clavulanate (49%), trimethoprim-sulfamethoxazole (38%), and ceftriaxone (20%). Eight percent of the tested isolates were ciprofloxacin-resistant Salmonella spp. Multidrug resistance was observed with the mean multiple antibiotic resistance (MAR) index of 0.31. The study demonstrated that 43% of the isolates were multiple drug resistant. The prevalence rates of resistance genes were 44% for blaTEM-1, 35% for blaCMY-2, 21% for sul2, 18% for tetC, 14% for dfrA7 and 8% for tetA.Conclusion: Resistance to ceftriaxone, detection of blaCMY-2 gene and the high level of intermediate susceptibility (33%) against ciprofloxacin suggested that livestock carry problematic Salmonella spp. This study used the global one-health initiative to report the potential public health risks of livestock-associated pathogens and highlights the importance of monitoring the trends of antimicrobial resistance for sustainability of antibiotics.Keywords: Salmonella, food-borne pathogens, zoonotic, animals, antimicrobial, resistance, blaTEM-1, blaCMY-2, bacteria, ciprofloxacin

The accelerated dispersion of multidrug-resistant (MDR) Escherichia coli due to the production of extended-spectrum β-lactamases (ESBLs) or AmpC enzymes has been noted in Egypt, presenting a serious treatment challenge. In this study, we investigate the prevalence of ESBLs and AmpC enzymes among 48 E. coli isolates collected from patients with urinary tract infections admitted to a teaching hospital in Alexandria. Phenotypic and genotypic methods of detection are conducted. Isolates producing both enzymes are tested for the mobilization of their genes by a broth mating experiment. Whole genome sequencing (WGS) is performed for isolate EC13655. The results indicate that 80% of the isolates are MDR, among which 52% and 13% were ESBL and AmpC producers, respectively. Conjugation experiments fail to show the mobilization of blaCMY-2 in EC13655, which was chosen for WGS. In silico analysis reveals that the isolate belongs to a ST410-H24Rx high-risk clone. It coharbors the ESBL-encoding genes blaCTX-M-15, blaTEM-1, blaOXA-1 and blaNDM-5 on an IncFIA/IncFIB/IncFII/IncQ1 multireplicon plasmid. The chromosomal location of blaCMY-2 is detected with a flanking upstream copy of ISEcp1. This chromosomal integration of blaCMY-2 establishes the stable maintenance of the gene and thus, necessitates an imperative local surveillance to reduce further spread of such strains in different clinical settings.

Horizontal gene transfer is an important mechanism which facilitates bacterial populations in overcoming antimicrobial treatment. In this study, a total of 120 Escherichia coli and 62 Salmonella enterica subsp. enterica isolates were isolated from broiler chicken farms in Alberta. Fourteen serovars were identified among Salmonella isolates. Thirty one percent of E. coli isolates (37/120) were multiclass drug resistant (resistant to ≥ 3 drug classes), while only about 16% of Salmonella isolates (10/62) were multiclass drug resistant. Among those, eight E. coli isolates had an AmpC-type phenotype, and one Salmonella isolate had an extended-spectrum beta-lactamase (ESBL)-type beta-lactamase phenotype. We identified both AmpC-type (blaCMY-2) and ESBL-type (blaTEM) genes in both E. coli and Salmonella isolates. Plasmids from eight of nine E. coli and Salmonella isolates were transferred to recipient strain E. coli J53 through conjugation. Transferable plasmids in the eight E. coli and Salmonella isolates were also transferred into a lab-made sodium azide-resistant Salmonella recipient through conjugation. The class 1 integrase gene, int1, was detected on plasmids from two E. coli isolates. Further investigation of class 1 integron cassette regions revealed the presence of an aadA gene encoding streptomycin 3’’-adenylyltransferase, an aadA1a/aadA2 gene encoding aminoglycoside 3’’-O-adenyltransferase, and a putative adenylyltransferase gene. This study provides some insight into potential horizontal gene transfer events of antimicrobial resistance genes between E. coli and Salmonella in broiler chicken production.

Chromosomal and transferable AmpC β-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when bla DHA-1 was expressed in an ampG -defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro , showing slight or insignificant virulence attenuations together with high-level resistance to β-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most β-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable β-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC β-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for β-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies., Competing Interests: The authors declare no conflict of interest.

Escherichia coli carrying IncK- bla CMY-2 plasmids mediating resistance to extended-spectrum cephalosporins (ESC) has been frequently described in food-producing animals and in humans. This study aimed to characterize IncK- bla CMY-2 -positive ESC-resistant E. coli isolates from poultry production systems in Denmark, Finland, and Germany, as well as from Danish human blood infections, and further compare their plasmids. Whole-genome sequencing (Illumina) of all isolates ( n = 46) confirmed the presence of the bla CMY-2 gene. Minimum inhibitory concentration (MIC) testing revealed a resistant phenotype to cefotaxime as well as resistance to ≥3 antibiotic classes. Conjugative transfer of the bla CMY-2 gene confirmed the resistance being on mobile plasmids. Pangenome analysis showed only one-third of the genes being in the core with the remainder being in the large accessory gene pool. Single nucleotide polymorphism (SNP) analysis on sequence type (ST) 429 and 1286 isolates showed between 0-60 and 13-90 SNP differences, respectively, indicating vertical transmission of closely related clones in the poultry production, including among Danish, Finnish, and German ST429 isolates. A comparison of 22 ST429 isolates from this study with 80 ST429 isolates in Enterobase revealed the widespread geographical occurrence of related isolates associated with poultry production. Long-read sequencing of a representative subset of isolates ( n = 28) allowed further characterization and comparison of the IncK- bla CMY-2 plasmids with publicly available plasmid sequences. This analysis revealed the presence of highly similar plasmids in ESC-resistant E. coli from Denmark, Finland, and Germany pointing to the existence of common sources. Moreover, the analysis presented evidence of global plasmid transmission and evolution. Lastly, our results indicate that IncK- bla CMY-2 plasmids and their carriers had been circulating in the Danish production chain with an associated risk of spreading to humans, as exemplified by the similarity of the clinical ST429 isolate to poultry isolates. Its persistence may be driven by co-selection since most IncK- bla CMY-2 plasmids harbor resistance factors to drugs used in veterinary medicine.

Liang-Xing Fang,1,2,* Xing-Ping Li,1,2,* Liang Li,1,2 Mu-Ya Chen,1,2 Cai-Yan Wu,3 Lu-Lu Li,4 Xiao-Ping Liao,1,2 Ya-Hong Liu,1,2 Jian Sun1,2 1National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, South China Agricultural University, Guangzhou, People’s Republic of China; 2Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, People’s Republic of China; 3Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou, People’s Republic of China; 4Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan, People’s Republic of China *These authors contributed equally to this work Background: CMY-2 is the most prevalent pAmpC β-lactamase, but the chromosomal blaCMY-2 gene transfer via horizontal transmission has been seldom reported. This study aimed to describe an ISEcp1-mediated transposition of a chromosomal blaCMY-2 gene from Escherichia coli into a small endogenous ColE1-like plasmid, resulting in elevated resistance to extended-spectrum cephalosporins. Methods: Three ESCs-resistant ST641 E. coli strains EC6413, EC4103 and EC5106 harbored the blaCMY-2 gene. S1- PFGE, I-ceu I-PFGE, Southern blotting and electroporation experiments were performed to investigate the location and transferability of blaCMY-2. The genetic context and gene expression of blaCMY-2 in the original isolates and the corresponding electroporants were explored by PCR mapping, primer walking strategy and RT-qPCR. Results: The blaCMY-2-containing region (ISEcp1-blaCMY-2-∆blc-∆yggR-∆tnp1-orf7-orf8-orf9-∆tnp2-∆hsdR) was transposed into endogenous ColE1-like plasmid pSC137 in the process of electroporation at very low frequencies (10–8–10–9). The transpositions resulted in novel larger blaCMY-2-harboring ColE1-like plasmids with size of 14,845 bp, enabling increase in MICs of 2 to 8-fold for cefotaxime, ceftiofur, and ceftazidime in recipient strains over their respective original counterparts. Transcriptional level analysis revealed that the increased blaCMY-2 expression was correlated with elevated MIC values of cephalosporins. The blaCMY-2 transposition unit was identical to that in a clinical isolate E. coli TN44889 from France isolated in 2004. Conclusions: Our results firstly demonstrated that ISEcp1 mediated a transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid by electroporation. Amplification of the blaCMY-2 gene facilitates the strain adaptation to a changed environment with an elevated antibiotic pressure. Keywords: blaCMY-2, chromosome-borne, ColE1-like plasmid, ISEcp1-mediated transposition, extended-spectrum cephalosporin

Sweden has a long tradition of monitoring occurrence of antibiotic resistant bacteria in both animals and humans, but there currently is no organised and harmonized monitoring on carriage of Enterobacteriaceae producing extended-spectrum beta-lactamase (ESBL), plasmid-mediated AmpC beta-lactamase (pAmpC), or methicillin-resistant coagulase positive staphylococci e.g. methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs. The aim of the current study was therefore to determine the prevalence of ESBL/pAmpC producing Enterobacteriaceae and methicillin-resistant coagulase positive staphylococci in healthy dogs in Sweden, and to phenotypically and genotypically characterize any identified isolates. It was shown that 0.9% (95% confident interval 0.3–2.7%) of the dogs (n = 325) carried multi-resistant ESBL-producing Escherichia coli, but that no methicillin-resistant coagulase positive staphylococci could be detected. In conclusion, the occurrence of multi-drug resistant bacteria remains rare among healthy dogs in Sweden. In addition, the ESBL-producing E. coli identified showed genetic characteristics related to those reported from humans. [ABSTRACT FROM AUTHOR]

This study was carried out to determine the antimicrobial resistance (AMR) genes and mobile genetic elements of 4 fecal bla CMY-2 -producing Escherichia coli isolated from Holstein dairy calves on the same farm using whole-genome sequencing. Genomic analysis revealed that 3 of the 4 isolates shared similar genetic features, including sequence type (ST), serotype, plasmid characteristics, insertion ST, and virulence genes. In addition to genes encoding for complex multidrug resistance efflux systems, all 4 isolates were carriers of genes conferring resistance to β-lactams (bla CMY-2 , bla TEM-1B), tetracyclines (tetA , tetB , tetD), aminoglycosides [ aadA1 , aph (3") -lb , aph (6) -ld ], sulfonamides (sul2), and trimethoprim (dfrA1). We also detected 4 incompatibility plasmid groups: Inc.F, Inc.N, Inc.I, and Inc.Q. A novel ST showing a new purA and mdh allelic combination was found. The 4 isolates were likely enterotoxigenic pathotypes of E. coli , based on serotype and presence of the plasmid Inc.FII(pCoo). This study provides information for comparative genomic analysis of AMR genes and mobile genetic elements. This analysis could give some explanation to the multidrug resistance characteristics of bacteria colonizing the intestinal tract of dairy calves in the first few weeks of life. [ABSTRACT FROM AUTHOR]

A total of 136 Salmonella isolates from chicken feces and meat samples of the top 12 integrated chicken production companies throughout Korea were collected. Among the 17 ESC-resistant Salmonella; blaCTX-M-15 was the most prevalent gene and two strains carried blaTEM-1/blaCTX-M-15 and blaCMY-2, respectively. The transferable blaCTX-M-15 gene was carried by IncFII plasmid in three isolates and the blaCMY-2 gene carried by IncI1 plasmid in one isolate. blaCMY-2 gene-harboring strain was selected as the donor based on the high frequency of blaCMY-2 gene transfer in vitro and its transfer frequencies were determined at 10−3 transconjugants per recipient. The transfer of blaCMY-2 gene-harboring plasmid derived from chicken isolate into a human pathogen; enteroinvasive Escherichia coli (EIEC), presented in mouse intestine with about 10−1 transfer frequency without selective pressure. From the competition experiment; blaCMY-2 gene-harboring transconjugant showed variable fitness burden depends on the parent strains. Our study demonstrated direct evidence that the blaCMY-2 gene harboring Salmonella from chicken could frequently transfer its ESC-resistant gene to E. coli in a mouse intestine without antimicrobial pressure; resulting in the emergence of multidrug resistance in potentially virulent EIEC isolates of significance to human health; which can increase the risk of therapeutic inadequacy or failures.

Ceftiofur, a third-generation cephalosporin antimicrobial, was used in Canadian hatcheries for many years to prevent early mortality in chicks, leading to a high prevalence of cephalosporin resistance in Escherichia coli in chickens. Preventive use of ceftiofur in hatcheries ceased in 2014. We examined the effect of ceftiofur cessation (n = 40 flocks with ceftiofur and n = 28 flocks without antimicrobial at hatchery) and its replacement with an antimicrobial combination, lincomycinspectinomycin (n = 32), at the hatchery on the proportion of samples with E. coli positive for extended-spectrum-β-lactamase (ESBL) and AmpC β-lactamase-related genes, and on the multidrug resistance profiles of ESBL/AmpC-positive E. coli in broilers and their associated breeders (n = 46 samples), at 1 year postcessation. For indicator E. coli from nonenriched media, a significant decrease postcessation in the proportion of samples harboring E. coli isolates positive for blaCMY-2 and/or blaCTX-M was observed. In contrast, following enrichment in medium containing ceftriaxone (1 mg/liter) to facilitate recovery of ESBL/AmpC β-lactamase-producing E. coli colonies, both pre- and postcessation, 99% of the samples harbored E. coli positive for blaCMY-2 or blaCTX-M. Among the 15 tested antimicrobial agents, flocks receiving lincomycin-spectinomycin after cessation of ceftiofur showed a significantly greater nonsusceptibility to aminoglycosides, folate inhibitors, phenicols, and tetracyclines and a greater proportion of possible extensively drug-resistant E. coli than those receiving ceftiofur or no antimicrobial at hatchery. This study clearly demonstrates an initial decrease in ESBL/AmpC-positive E. coli following the cessation of ceftiofur in the hatchery but an increase in antimicrobial non-β-lactam resistance of ESBL/ AmpC-positive E. coli following replacement with lincomycin-spectinomycin. [ABSTRACT FROM AUTHOR]

Extended-spectrum cephalosporin-resistant Escherichia coli (ESCR E. coli) with plasmids carrying the blaCMY-2 resistance gene have been isolated from the Norwegian broiler production chain through the Norwegian monitoring program for antimicrobial resistance in animals, food and feed, NORM-VET. The aim of the present study was to investigate the biofilm forming abilities of these strains, and in particular to see whether these might be influenced by the carriage of blaCMY-2 plasmids. The ESCR E. coli from the broiler production chain displayed relatively low biofilm forming abilities in the crystal violet biofilm assay as compared to quinolone-resistant E. coli (QREC) from the same population (mean ± SD = 0.686 ± 0.686 vs. 1.439 ± 0.933, respectively). Acquisition of two different blaCMY-2 plasmids by QREC strains reduced their biofilm production in microtiter plates, but not their biofilm production on Congo Red agar plates. Furthermore, motility was reduced, but not planktonic growth. We hypothesize that genes carried by these plasmids may have caused the observed reduction in biofilm formation, possibly mediated through changes in flagellar expression or function. Furthermore, this may help explain the different biofilm forming abilities observed between ESCR E. coli and QREC. The results also indicate that the risk of biofilm reservoirs of antimicrobial resistant E. coli on in the broiler production is lower for ESCR E. coli than for QREC.

Whole-genome sequencing of 24 Proteus mirabilis isolates revealed the clonal expansion of two cefoxitin-resistant strains among patients with community-onset infection. These strains harboured blaCMY-2 within a chromosomally located integrative and conjugative element and exhibited multidrug resistance phenotypes. A predominant strain, identified in 18 patients, also harboured the PGI-1 genomic island and associated resistance genes, accounting for its broader antibiotic resistance profile. The identification of these novel multidrug-resistant strains among community-onset infections suggests that they are endemic to this region and represent emergent P. mirabilis lineages of clinical significance.

Distinguishing epidemiologically related and unrelated plasmids is essential to confirm plasmid transmission. We compared IncI1–pST12 plasmids from both human and livestock origin and explored the degree of sequence similarity between plasmids from Enterobacteriaceae with different epidemiological links. Short-read sequence data of Enterobacteriaceae cultured from humans and broilers were screened for the presence of both a blaCMY-2 gene and an IncI1–pST12 replicon. Isolates were long-read sequenced on a MinION sequencer (OxfordNanopore Technologies). After plasmid reconstruction using hybrid assembly, pairwise single nucleotide polymorphisms (SNPs) were determined. The plasmids were annotated, and a pan-genome was constructed to compare genes variably present between the different plasmids. Nine Escherichia coli sequences of broiler origin, four Escherichia coli sequences, and one Salmonella enterica sequence of human origin were selected for the current analysis. A circular contig with the IncI1–pST12 replicon and blaCMY-2 gene was extracted from the assembly graph of all fourteen isolates. Analysis of the IncI1–pST12 plasmids revealed a low number of SNP differences (range of 0–9 SNPs). The range of SNP differences overlapped in isolates with different epidemiological links. One-hundred and twelve from a total of 113 genes of the pan-genome were present in all plasmid constructs. Next generation sequencing analysis of blaCMY-2-containing IncI1–pST12 plasmids isolated from Enterobacteriaceae with different epidemiological links show a high degree of sequence similarity in terms of SNP differences and the number of shared genes. Therefore, statements on the horizontal transfer of these plasmids based on genetic identity should be made with caution.

We aimed to identify and characterize extended-spectrum β-lactamase (ESBL)-and/or plasmid-mediated AmpC β-lactamase (pAmpC)-producing Escherichia coli isolated from healthy broiler chickens slaughtered for human consumption in Korea. A total of 332 E. coli isolates were identified from 339 cloacal swabs in 2019. More than 90% of the isolates were resistant to multiple antimicrobials. ESBL/pAmpC-production was noted in 14% (46/332) of the isolates. Six of the CTX-M-β-lactamase-producing isolates were found to co-harbor at least one plasmid-mediated quinolone resistance gene. We observed the co-existence of blaCMY-2 and mcr-1 genes in the same isolate for the first time in Korea. Phylogenetic analysis demonstrated that the majority of blaCMY-2-carrying isolates belonged to subgroup D. Conjugation confirmed the transferability of blaCTX-M and blaCMY-2 genes, as well as non-β-lactam resistance traits from 60.9% (28/46) of the ESBL/pAmpC-producing isolates to a recipient E. coli J53. The ISECP, IS903, and orf477 elements were detected in the upstream or downstream regions. The blaCTX-M and blaCMY-2 genes mainly belonged to the IncI1, IncHI2, and/or IncFII plasmids. Additionally, the majority of ESBL/pAmpC-producing isolates exhibited heterogeneous PFGE profiles. This study showed that healthy chickens act as reservoirs of ESBL/pAmpC-producing E. coli that can potentially be transmitted to humans.

The production of β-lactamase by nontyphoidal Salmonella has become a public health issue throughout the world. In this study, we aimed to investigate the antimicrobial resistance profiles and molecular characteristics of β-lactamase-producing Salmonella enterica serovar Albany isolates. A total of 434 Salmonella Albany were obtained from feces and carcasses of healthy and diseased food-producing animals [cattle ( n  = 2), pigs ( n  = 3), chickens ( n  = 391), and ducks ( n  = 38)] during 2013-2020. Among the 434 Salmonella Albany isolates, 3.7% showed resistance to cefoxitin, and all the cefoxitin-resistant isolates were obtained from chickens. Moreover, Salmonella Albany isolates demonstrated high resistance to nalidixic acid (99.3%), trimethoprim/sulfamethoxazole (97.9%), ampicillin (86.6%), chloramphenicol (86.6%), and tetracycline (85.7%), as well as higher rates of multidrug resistance were detected in cefoxitin-resistant isolates compared to cefoxitin-susceptible isolates. All cefoxitin-resistant isolates harbored CMY-2-type β-lactamase and belonged to seven different pulsotypes, with type IV-b (43.75%) and IV-a (25%) making up the majority. In addition, genes encoding cefoxitin resistant of all bla CMY-2 -harboring Salmonella Albany isolates were horizontally transmitted to a recipient Escherichia coli J53 by conjugation. Furthermore, 93.75% (15/16) of conjugative plasmids harboring bla CMY-2 genes belong to ST12/CC12-IncI1. Genetic characteristics of transmitted bla CMY-2 genes were associated with IS Ecp1 , which can play an essential role in the effective mobilization and expression of these genes. Salmonella Albany containing bla CMY-2 in chickens can potentially be transferred to humans. Therefore, it is necessary to restrict antibiotic use and conduct continuous monitoring and analysis of resistant bacteria in the poultry industry.

Abstract IncI1 plasmids play a central role in the transfer of antimicrobial resistance genes among Enterobacteriaceae in animals and humans. Knowledge on the dynamics of IncI1 plasmid transfer is limited, mainly due to lack of culture-independent methods that can quantify donor strain survival and plasmid transfer in complex microbial communities. The aim of this study was to develop a culture-independent method to study the dynamics of IncI1 plasmids transfer by fluorescence-activated cell sorting. We genetically modified three wild-type Escherichia coli of animal (n = 2) and human (n = 1) origin carrying bla CMY-2 or bla CTX-M-1 on two epidemic IncI1 plasmids (pST12 and pST7). Non-coding regions on the chromosome and on the IncI1 plasmid of each strain were tagged with mCherry (red) and GFPmut3 (green) fluorescent proteins, respectively, using lambda recombineering. A gene cassette expressing mCherry and lacI q was inserted into the chromosome, whereas the plasmid was marked with a GFPmut3 cassette with LacI q repressible promoter. Therefore, gfpmut3 was repressed in donor strains but expressed in recipient strains acquiring the plasmids. We demonstrated that genetic engineering of the strains did not affect the growth rate and plasmid transfer-ability in filter and broth matings. A proof-of-concept experiment using the CoMiniGut, an in vitro model of the colon, proved the validity of our method for studying the survival of wild-type E. coli and horizontal transfer of IncI1 plasmids under different pH and oxygen conditions. The dual-labeling method by fluorescent proteins is useful to determine persistence of exogenous E. coli and transfer dynamics of IncI1 plasmids in microbial communities. Highlights • A culture-independent method to study IncI1 plasmid transfer and donor survival • Dual-labeling of wild-type E. coli not affecting cell biology was developed • The fluorescence-based system allowed high-throughput study of plasmid transfer. [ABSTRACT FROM AUTHOR]

Background: CMY-2 is the most prevalent pAmpC β-lactamase, but the chromosomal blaCMY-2 gene transfer via horizontal transmission has been seldom reported. This study aimed to describe an ISEcp1-mediated transposition of a chromosomal blaCMY-2 gene from Escherichia coli into a small endogenous ColE1-like plasmid, resulting in elevated resistance to extended-spectrum cephalosporins. Methods: Three ESCs-resistant ST641 E. coli strains EC6413, EC4103 and EC5106 harbored the blaCMY-2 gene. S1- PFGE, I-ceu I-PFGE, Southern blotting and electroporation experiments were performed to investigate the location and transferability of blaCMY-2. The genetic context and gene expression of blaCMY-2 in the original isolates and the corresponding electroporants were explored by PCR mapping, primer walking strategy and RT-qPCR. Results: The blaCMY-2-containing region (ISEcp1-blaCMY-2-∆blc-∆yggR-∆tnp1-orf7-orf8-orf9-∆tnp2-∆hsdR) was transposed into endogenous ColE1-like plasmid pSC137 in the process of electroporation at very low frequencies (10–8–10–9). The transpositions resulted in novel larger blaCMY-2-harboring ColE1-like plasmids with size of 14,845 bp, enabling increase in MICs of 2 to 8-fold for cefotaxime, ceftiofur, and ceftazidime in recipient strains over their respective original counterparts. Transcriptional level analysis revealed that the increased blaCMY-2 expression was correlated with elevated MIC values of cephalosporins. The blaCMY-2 transposition unit was identical to that in a clinical isolate E. coli TN44889 from France isolated in 2004. Conclusions: Our results firstly demonstrated that ISEcp1 mediated a transposition of chromosome-borne blaCMY-2 into an endogenous ColE1-like plasmid by electroporation. Amplification of the blaCMY-2 gene facilitates the strain adaptation to a changed environment with an elevated antibiotic pressure. [ABSTRACT FROM AUTHOR]

Overall, 836 Escherichia coli isolates (695 isolates from dogs and 141 from cats) were recovered from the diarrhea, skin/ear, urine, and genitals of dogs and cats between 2018 and 2019. Cefovecin and enrofloxacin resistance were noted in 17.1% and 21.2% of E. coli isolates, respectively. The cefovecin and enrofloxacin resistance rates were higher in dog isolates (18.1% and 22.9%) compared with the rates in cat isolates (12.1%, 12.8%). Interestingly, resistance to both antimicrobials was noted in 10.8% (90/836) of the isolates, predominantly in isolates from dogs. bla CTX-M-14 , bla CTX-M-15 , and bla CMY-2 were the most frequent extended-spectrum β-lactamase/plasmid-mediated AmpC β-lactamase (ESBL/AmpC)- gene types. The co-existence of bla CTX-M and bla CMY-2 was noted in six E. coli isolates from dogs. Sequencing analysis demonstrated that S83L and D87N in gyrA and S80I in parC were the most frequent point mutations in the quinolone resistance-determining regions of the cefovecin and enrofloxacin-resistant isolates. A total of 11 isolates from dogs carried the plasmid-mediated quinolone resistance genes (six aac(6')-Ib-cr , four qnrS , and one qnrB ), while only two cat isolates carried the qnrS gene. Multilocus sequence typing of the cefovecin and enrofloxacin-resistant isolates revealed that sequence type (ST)131 E. coli carrying bla CTX-M-14 and bla CTX-M-15 genes and ST405 E. coli carrying bla CMY-2 gene were predominant among the isolated E. coli strains. The majority of the ESBL/AmpC-producing isolates displayed diverse pulsed-field gel electrophoresis profiles. This study demonstrated that third-generation cephalosporin- and fluoroquinolone-resistant E. coli were widely distributed in companion animals. The detection of the pandemic ST131 clone carrying bla CTX-M - 14/15 in companion animals presented a public health threat.

Abstract During a regular monitoring of antimicrobial resistance in a farrowing farm in Southern China, 117 Escherichia coli isolates were obtained from sows and piglets. Compared with the isolates from piglets, the isolates from sows exhibited higher resistance rates to the tested cephalosporins. Correspondingly, the total detection rate of the blaCMY-2/blaCTX-M genes in the sow isolates (34.2%) was also significantly higher than that of the piglet isolates (13.6%) (p

The aim of this study was to investigate the evolution with time of ceftiofur-resistant Escherichia coli clinical isolates from pigs in Québec, Canada, between 1997 and 2012 with respect to pathotypes, clones and antimicrobial resistance. Eighty-five ceftiofur-resistant E. coli isolates were obtained from the OIE (World Organisation for Animal Health) Reference Laboratory for Escherichia coli . The most prevalent pathovirotypes were enterotoxigenic E. coli (ETEC):F4 (40%), extraintestinal pathogenic E. coli (ExPEC) (16.5%) and Shiga toxin-producing E. coli (STEC):F18 (8.2%). Susceptibility testing to 15 antimicrobial agents revealed a high prevalence of resistance to 13 antimicrobials, with all isolates being multidrug-resistant. bla CMY-2 (96.5%) was the most frequently detected β-lactamase gene, followed by bla TEM (49.4%) and bla CTX-M (3.5%). Pulsed-field gel electrophoresis (PFGE) applied to 45 representative E. coli isolates revealed that resistance to ceftiofur is spread both horizontally and clonally. In addition, the emergence of extended-spectrum β-lactamase-producing E. coli isolates carrying bla CTX-M was observed in 2011 and 2012 in distinct clones. The most predominant plasmid incompatibility (Inc) groups were IncFIB, IncI1, IncA/C and IncFIC. Resistance to gentamicin, kanamycin and chloramphenicol as well as the frequency of bla TEM and IncA/C significantly decreased over the study period, whereas the frequency of IncI1 and multidrug resistance to seven antimicrobial categories significantly increased. These findings reveal that extended-spectrum cephalosporin-resistant porcine E. coli isolates in Québec belong to several different clones with diverse antimicrobial resistance patterns and plasmids. Furthermore, bla CMY-2 was the major β-lactamase gene in these isolates. From 2011, we report the emergence of bla CTX-M in distinct clones. [ABSTRACT FROM AUTHOR]

During a regular monitoring of antimicrobial resistance in a farrowing farm in Southern China, 117 Escherichia coli isolates were obtained from sows and piglets. Compared with the isolates from piglets, the isolates from sows exhibited higher resistance rates to the tested cephalosporins. Correspondingly, the total detection rate of the blaCMY-2/blaCTX-M genes in the sow isolates (34.2%) was also significantly higher than that of the piglet isolates (13.6%; p < 0.05). The blaCMY-2 gene had a relatively high prevalence (11.1%) in the E. coli isolates. MLST and PFGE analysis revealed the clonal spread of ST1121 E. coli in most (7/13) of the blaCMY-2-positive isolates. An indistinguishable IncHI2 plasmid harboring blaCMY-2 was also identified in each of the seven ST1121 E. coli isolates. Complete sequence analysis of this IncHI2 plasmid (pEC5207) revealed that pEC5207 may have originated through recombination of an IncHI2 plasmid with a blaCMY-2-carrying IncA/C plasmid like pCFSAN007427_01. In addition to blaCMY-2, pEC5207 also carried other resistance determinants for aminoglycosides (aacA7), sulfonamides (sul1), as well as heavy metals ions, such as Cu and Ag. The susceptibility testing showed that the pEC5207 can mediate both antibiotic and heavy metal resistance. This highlights the role of pEC5207 in co-selection of blaCMY-2-positive isolates under the selective pressure of heavy metals, cephalosporins, and other antimicrobials. In conclusion, clonal spread of an ST1121 type E. coli strain harboring an IncHI2 plasmid contributed to the dissemination of blaCMY-2 in a farrowing farm in Southern China. We also have determined the first complete sequence analysis of a blaCMY-2-carrying IncHI2 plasmid. [ABSTRACT FROM AUTHOR]

The use of antimicrobials in food animals is the major determinant for the propagation of resistant bacteria in the animal reservoir. The objective of this study was to investigate the presence and distribution of third-generation cephalosporin (3GC) -resistant and plasmid-mediated AmpC (pAmpC)-producing Escherichia coli isolated from food animals in Southern China. In total, 744 3GC-resistant and 40 bla CMY-2 -positive E. coli strains were recovered from 1656 food animal fecal samples across five rearing regions. The bla CMY-2 genes were located on IncC, IncFIB or IncI1 type plasmids in 12 E. coli isolates. In the other 22 isolates, S1-PFGE and hybridization analyses revealed that the bla CMY-2 gene was chromosomally located and demonstrated a high prevalence of the chromosomally encoded bla CMY-2 gene in E. coli. Plasmid stability and growth curve experiments demonstrated that IncI1, IncC and IncFIB plasmids can exist stably in the host bacteria and with a low growth burden and may be the reason these plasmids can be widely disseminated in breeding environments. Whole genome sequencing indicated that IS Ecp1 and IS 1294 played important roles in bla CMY-2 transfer to both plasmids and the chromosome. Our study confirmed that bla CMY-2 mediated resistance of food animal-derived E. coli to 3GC and highlights the urgent need for appropriate monitoring programmes. • We identified 40 3GC-resistant E. coli isolates that lacked ESBL genes and all carried bla CMY-2 located on both plasmids (IncC, IncI1, IncFIB) and the chromosome. • Plasmid stability and growth curve experiments demonstrated that IncI1, IncC and IncFIB plasmids can exist stably in the host bacteria and with a low growth burden. • Our study confirmed that bla CMY-2 mediated resistance of food animal-derived E. coli to 3GC and highlights the urgent need for appropriate monitoring programmes. [ABSTRACT FROM AUTHOR]

Objectives In Sweden the prevalence of Enterobacteriaceae with transferable resistance to extended-spectrum cephalosporins (ESCs) is low. However, in broilers ESC-resistant Escherichia coli is common, with a clear dominance of blaCMY-2. Antimicrobials are rarely used in broiler production in Sweden and cephalosporins are never used. Introduction through imported breeding stock and subsequent vertical transmission of the bacteria through the production pyramid could be one explanation for this high prevalence. Methods To test this hypothesis, paper linings from imported flocks of grandparent animals were screened for the presence of ESC-resistant E. coli and a positive flock, together with its progeny, was followed longitudinally through the production pyramid using boot swabs. The relationship of isolated ESC-resistant E. coli was investigated using multiple-locus variable number tandem repeat analysis (MLVA). Results ESC-resistant E. coli carrying blaCMY-2 was isolated from six out of eight imported flocks of grandparent animals. One clone of E. coli carrying blaCMY-2 occurred in all levels of the production pyramid and in flocks of imported grandparent animals. Conclusions E. coli carrying blaCMY-2 is frequently present among grandparent animals imported to Sweden for breeding purposes. The occurrence of one clone in all levels of the production pyramid indicates that its introduction through imported breeding stock and vertical transmission through the production pyramid could be one explanation for the high proportion of Swedish broilers colonized with ESC-resistant E. coli. [ABSTRACT FROM PUBLISHER]

The accelerated dispersion of multidrug-resistant (MDR) Escherichia coli due to the production of extended-spectrum β-lactamases (ESBLs) or AmpC enzymes has been noted in Egypt, presenting a serious treatment challenge. In this study, we investigate the prevalence of ESBLs and AmpC enzymes among 48 E. coli isolates collected from patients with urinary tract infections admitted to a teaching hospital in Alexandria. Phenotypic and genotypic methods of detection are conducted. Isolates producing both enzymes are tested for the mobilization of their genes by a broth mating experiment. Whole genome sequencing (WGS) is performed for isolate EC13655. The results indicate that 80% of the isolates are MDR, among which 52% and 13% were ESBL and AmpC producers, respectively. Conjugation experiments fail to show the mobilization of bla CMY-2 in EC13655, which was chosen for WGS. In silico analysis reveals that the isolate belongs to a ST410-H24Rx high-risk clone. It coharbors the ESBL-encoding genes bla CTX-M-15 , bla TEM-1 , bla OXA-1 and bla NDM-5 on an IncFIA/IncFIB/IncFII/IncQ1 multireplicon plasmid. The chromosomal location of bla CMY-2 is detected with a flanking upstream copy of IS Ecp1 . This chromosomal integration of bla CMY-2 establishes the stable maintenance of the gene and thus, necessitates an imperative local surveillance to reduce further spread of such strains in different clinical settings.

The article discusses a study on the molecular and phenotypic characteristics of Salmonella enterica serovar Typhimurium isolated from cattle in two farms in Fukushima Prefecture, Japan. A brief discussion on Salmonella infections and the emergence of extended-spectrum cephalosporin (ESC)-resistant Salmonella is provided. In the study, an AmpC &beta-lactamase encoded by the blaCMY-2 gene was used to mediate the resistance of the two isolates from the antimicrobial ESC. It was found that the blaCMY-2-carrying plasmids were different in size. The isolates seem to be closely related based on the findings from macrorestriction analysis and phage typing.

Sixty-nine Escherichia coli and 10 Salmonella isolates, recovered from retail chicken meat in Hiroshima prefecture, Japan, were assayed for antimicrobial susceptibility, the presence of integrons and antimicrobial resistance genes. Twenty-eight out of 69 (40.6%) of E. coli and all 10 Salmonella isolates were exhibited multidrug resistance phenotypes. The most commonly reported resistance phenotypes were against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, and trimethoprim/sulfamethoxazole. PCR screening for integrons showed that 8 (11.6%) of the E. coli isolates were positive for the class 1 integrons and 1 isolate (1.4%) was positive for the class 2 integrons. Among the 10 Salmonella isolates, 9 were positive for class 1 integrons and none was positive for class 2 integrons. The identified antibiotic resistance gene cassettes within the class 1 integrons were dfrA1, dfrA7, aadA1, aadB, and catB3, while dfrA1, sat2, and aadA1 were identified within class 2 integron. The β-lactamase resistance gene blaTEM-1 was identified in 12 (17.3%) of E. coli isolates and in only one of the Salmonella isolates. The blaCMY-2 gene, encoding AmpC β-lactamase, was detected in 16 (23.2%) of the E. coli isolates only. Conjugation experiments demonstrated that there was plasmid-mediated transfer of blaCMY-2 and blaTEM-1. These results highlighted the role of retail chicken meat as a potential source for multidrug-resistant strains of E. coli and Salmonella. To the best of our knowledge, this is the 1st report of isolation and molecular characterization of multidrug-resistant strains of E. coli from retail chicken meat in Japan. [ABSTRACT FROM AUTHOR]