IncF-type Plasmids Research Articles

Difference analysis and characteristics of incompatibility group plasmid replicons in gram-negative bacteria with different antimicrobial phenotypes in Henan, China

BackgroundMulti-drug-resistant organisms (MDROs) in gram-negative bacteria have caused a global epidemic, especially the bacterial resistance to carbapenem agents. Plasmid is the common vehicle for carrying antimicrobial resistance genes (ARGs), and the transmission of plasmids is also one of the important reasons for the emergence of MDROs. Different incompatibility group plasmid replicons are highly correlated with the acquisition, dissemination, and evolution of resistance genes. Based on this, the study aims to identify relevant characteristics of various plasmids and provide a theoretical foundation for clinical anti-infection treatment.Methods330 gram-negative strains with different antimicrobial phenotypes from a tertiary hospital in Henan Province were included in this study to clarify the difference in incompatibility group plasmid replicons. Additionally, we combined the information from the PLSDB database to elaborate on the potential association between different plasmid replicons and ARGs. The VITEK mass spectrometer was used for species identification, and the VITEK-compact 2 automatic microbial system was used for the antimicrobial susceptibility test (AST). PCR-based replicon typing (PBRT) detected the plasmid profiles, and thirty-three different plasmid replicons were determined. All the carbapenem-resistant organisms (CROs) were tested for the carbapenemase genes.Results21 plasmid replicon types were detected in this experiment, with the highest prevalence of IncFII, IncFIB, IncR, and IncFIA. Notably, the detection rate of IncX3 plasmids in CROs is higher, which is different in strains with other antimicrobial phenotypes. The number of plasmid replicons they carried increased with the strain resistance increase. Enterobacterales took a higher number of plasmid replicons than other gram-negative bacteria. The same strain tends to have more than one plasmid replicon type. IncF-type plasmids tend to be associated with MDROs. Combined with PLSDB database analysis, IncFII and IncX3 are critical platforms for taking blaKPC−2 and blaNDM.ConclusionsMDROs tend to carry more complex plasmid replicons compared with non-MDROs. The plasmid replicons that are predominantly prevalent and associated with ARGs differ in various species. The wide distribution of IncF-type plasmids and their close association with MDROs should deserve our attention. Further investigation into the critical role of plasmids in the carriage, evolution, and transmission of ARGs is needed.

Antimicrobial resistance in food-associated Escherichia coli in Mexico and Latin America.

The World Health Organization (WHO) considers antimicrobial resistance to be one of the critical global public health priorities to address. Escherichia coli is a commensal bacterium of the gut microbiota in humans and animals; however, some strains cause infections and are resistant to antibiotics. One of the most common ways of acquiring pathogenic E. coli strains is through food. This review analyzes multidrug-resistant E. coli isolated from food, emphasizing Latin America and Mexico, and the mobile genetic elements (MGEs) responsible for spreading antibiotic resistance determinants among bacteria in different environments and hosts. We conducted a systematic search of the literature published from 2015 to 2022 in open access databases and electronic repositories. The prevalence of 11 E. coli pathotypes was described, with diarrheagenic E. coli pathotypes being the most frequently associated with foodborne illness in different Latin American countries, highlighting the presence of different antibiotic resistance genes mostly carried by IncF-type plasmids or class 1 integrons. Although the global incidence of foodborne illness is high, there have been few studies in Mexico and Latin America, which highlights the need to generate updated epidemiological data from the "One Health" approach, which allows monitoring of the multidrug-resistance phenomenon in E. coli from a common perspective in the interaction of human, veterinary, and environmental health.

Plasmid genomic epidemiology of blaKPC carbapenemase-producing Enterobacterales in Canada, 2010-2021.

Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant Enterobacterales, but carbapenem resistance due to acquisition of carbapenemase genes is a growing threat that has been reported worldwide. Klebsiella pneumoniae carbapenemase (blaKPC) is the most common type of carbapenemase in Canada and elsewhere; it can hydrolyze penicillins, cephalosporins, aztreonam, and carbapenems and is frequently found on mobile plasmids in the Tn4401 transposon. This means that alongside clonal expansion, blaKPC can disseminate through plasmid- and transposon-mediated horizontal gene transfer. We applied whole genome sequencing to characterize the molecular epidemiology of 829 blaKPC carbapenemase-producing isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short-read and long-read sequencing, we obtained 202 complete and circular blaKPC-encoding plasmids. Using MOB-suite, 10 major plasmid clusters were identified from this data set which represented 87% (175/202) of the Canadian blaKPC-encoding plasmids. We further estimated the genomic location of incomplete blaKPC-encoding contigs and predicted a plasmid cluster for 95% (603/635) of these. We identified different patterns of carbapenemase mobilization across Canada related to different plasmid clusters, including clonal transmission of IncF-type plasmids (108/829, 13%) in K. pneumoniae clonal complex 258 and novel repE(pEh60-7) plasmids (44/829, 5%) in Enterobacter hormaechei ST316, and horizontal transmission of IncL/M (142/829, 17%) and IncN-type plasmids (149/829, 18%) across multiple genera. Our findings highlight the diversity of blaKPC genomic loci and indicate that multiple, distinct plasmid clusters have contributed to blaKPC spread and persistence in Canada.

Broad-Host Dissemination of Plasmids Coharboring the fos Operon for Fructooligosaccharide Metabolism with Antibiotic Resistance Genes.

The fos operon encoding short-chain fructooligosaccharide (scFOS) utilization enables bacteria of the family Enterobacteriaceae to grow and be sustained in environments where they would struggle to survive. Despite several cases of the detection of the fos operon in isolates of avian and equine origins, its global distribution in bacterial genomes remains unknown. The presence of the plasmid-harbored fos operon among resistant bacteria may promote the spread of antibiotic resistance. A collection of 11,538 antimicrobial-resistant Enterobacteriaceae isolates from various sources was screened for the fosT gene encoding the scFOS transporter. Out of 307 fosT-positive isolates, 80% of them originated from sources not previously linked to fosT (humans, wastewater, and animals). The chromosomally harbored fos operon was detected in 163/237 isolates subjected to whole-genome sequencing. In the remaining 74 isolates, the operon was carried by plasmids. Further analyses focusing on the isolates with a plasmid-harbored fos operon showed that the operon was linked to various incompatibility (Inc) groups, including the IncHI1, IncF-type, IncK2, IncI1, and IncY families. Long-read sequencing of representative plasmids showed the colocalization of fos genes with antibiotic resistance genes (ARGs) in IncHI1 (containing a multidrug resistance region), IncK2 (blaTEM-1A), IncI1 [sul2 and tet(A)], and IncY [aadA5, dfrA17, sul2, and tet(A)] plasmids, while IncF-type plasmids had no ARGs but coharbored virulence-associated genes. Despite the differences in the locations and structures of the fos operons, all isolates except one were proven to utilize scFOSs. In this study, we show that the fos operon and its spread are not strictly bound to one group of plasmids, and therefore, it should not be overlooked. IMPORTANCE It was believed that members of the family Enterobacteriaceae are unable to grow under conditions with short-chain fructooligosaccharides as the only source of carbon. Nevertheless, the first Escherichia coli isolate from chicken intestine was able to utilize these sugars owing to the chromosomally harbored fos operon. Studies on E. coli isolates from horses discovered the horizontal transfer of the fos operon on IncHI1 plasmids along with genes for antibiotic resistance. The first plasmid detected was pEQ1, originating from the feces of a hospitalized horse in the Czech Republic. Follow-up studies also revealed the dissemination of the IncHI1 plasmid-harbored fos operon in the Netherlands, Germany, Denmark, and France among healthy horses. Despite several cases of detection of the fos operon, its global distribution in bacterial genomes remains unknown. The fos operon possibly plays a role in the adaptation of plasmids among resistant bacteria and therefore may promote the spread of antibiotic resistance.

Polluted wetlands contain multidrug-resistance plasmids encoding CTX-M-type extended-spectrum β-lactamases

While most detailed analyses of antibiotic resistance plasmids focus on those found in clinical isolates, less is known about the vast environmental reservoir of mobile genetic elements and the resistance and virulence factors they encode. We selectively isolated three strains of cefotaxime-resistant Escherichia coli from a wastewater-impacted coastal wetland. The cefotaxime-resistant phenotype was transmissible to a lab strain of E. coli after one hour, with frequencies as high as 10−3 transconjugants per recipient. Two of the plasmids also transferred cefotaxime resistance to Pseudomonas putida, but these were unable to back-transfer this resistance from P. putida to E. coli. In addition to the cephalosporins, E. coli transconjugants inherited resistance to at least seven distinct classes of antibiotics. Complete nucleotide sequences revealed large IncF-type plasmids with globally distributed replicon sequence types F31:A4:B1 and F18:B1:C4 carrying diverse antibiotic resistance and virulence genes. The plasmids encoded extended-spectrum β-lactamases blaCTX-M-15 or blaCTX-M-55, each associated with the insertion sequence ISEc9, although in different local arrangements. Despite similar resistance profiles, the plasmids shared only one resistance gene in common, the aminoglycoside acetyltransferase aac(3)-IIe. Plasmid accessory cargo also included virulence factors involved in iron acquisition and defense against host immunity. Despite their sequence similarities, several large-scale recombination events were detected, including rearrangements and inversions. In conclusion, selection with a single antibiotic, cefotaxime, yielded conjugative plasmids conferring multiple resistance and virulence factors. Clearly, efforts to limit the spread of antibiotic resistance and virulence among bacteria must include a greater understanding of mobile elements in the natural and human-impacted environments.

The Emerging Nosocomial Pathogen Klebsiella michiganensis: Genetic Analysis of a KPC-3 Producing Strain Isolated from Venus Clam.

The recovery and characterization of a multidrug-resistant, KPC-3-producing Klebsiella michiganensis that was obtained from Venus clam samples is reported in this study. A whole-genome sequencing (WGS) analysis using Illumina and Nanopore technologies of the K. michiganensis 23999A2 isolate revealed that the strain belonged to the new sequence type 382 (ST382) and carried seven plasmid replicon sequences, including four IncF type plasmids (FII, FIIY, FIIk, and FIB), one IncHI1 plasmid, and two Col plasmids. The FIB and FIIk plasmids showed high homology to each other and to multireplicon pKpQIL-like plasmids that are found in epidemic KPC-K. pneumoniae clones worldwide. The strain carried multiple β-lactamase genes on the IncF plasmids: blaOXA-9 and blaTEM-1A on FIB, blaKPC-3 inserted in a Tn4401a on FIIK, and blaSHV-12 on FIIY. The IncHI1-ST11 harbored no resistance gene. The curing of the strain caused the loss of all of the bla genes and a rearrangement of the IncF plasmids. Conjugal transfer of the blaOXA-9, blaTEM-1A and blaKPC-3 genes occurred at a frequency of 5 × 10-7, using K. quasipneumoniae as a recipient, and all of the bla genes were transferred through a pKpQIL that originated from the recombination of the FIB and FIIk plasmids of the donor. A comparison with 31 K. michiganensis genomes that are available in the NCBI database showed that the closest phylogenetic relatives of K. michiganensis 23999A2 are an environmental isolate from soil in South Korea and a clinical isolate from human sputum in Japan. Finally, a pan-genome analysis showed a large accessory genome of the strain as well as the great genomic plasticity of the K. michiganensis species. IMPORTANCE Klebsiella michiganensis is an emerging nosocomial pathogen, and, so far, few studies describe isolates of clinical origin in the environment. This study contributes to the understanding of how the dissemination of carbapenem-resistance outside the hospital setting may be related to the circulation of pKpQIL-like plasmids that are derived from epidemic Klebsiella pneumoniae strains. The recovery of a carbapenem-resistant isolate in clams is of great concern, as bivalves could represent vehicles of transmission of pathogens and resistance genes to humans via the food chain. The study demonstrates the plasticity of K. michiganensis genome, which is probably useful to multiple environment adaptation and to the evolution of the species.

IncFIB plasmids carrying the resistance gene blaCTX-M-15 in ESBL-producing Escherichia coli clones from pediatric patients.

The emergence of extended-spectrum β-lactamases (ESBLs)-producing Escherichia coli clones are a public health concern worldwide. Scarce information does exist about the spread of ESBLs-producing E. coli in pediatric patients from developing countries. E. coli strains were analyzed by multilocus-sequence-typing, pulsed-field-gel-electrophoresis and phylogenetic group. The antimicrobial-resistance genes were detected by PCR, and plasmid content by the PCR-based replicon-typing. Horizontal transfer was tested by conjugation and the location of the blaCTX-M-15 gene by Southern blot hybridization. Thirty-two cefotaxime-resistant E. coli were recovered. Eleven of them were ESBL-producing isolates, which were well characterized and ascribed to seven sequence types and five phylogroups. The ESBL CTX-M-15 was the most prevalent enzyme (9 of 11). Plasmids of variable sizes (40-220 kb) were visualized, and the incompatibility (Inc) group FIB plasmid-replicon was detected in the ESBL strains and transferred by conjugation in 45.45% of them. Plasmid-borne toxin-antitoxin systems were the most frequently detected systems, strongly associated to IncF plasmids. The CTX-M-15-encoding gene was located on IncFIB plasmids. Even though a small number of ESBL-producing strains was recovered, we evidenced that IncFIB plasmids carry the blaCTX-M-15 gene, highlighting the role of IncF-type plasmids in facilitating the spread and maintenance of ESBL-encoding genes, which further favors the rapid increase of the antimicrobial resistance dissemination in disease-causing E. coli strains in pediatric patients.

Interplay between IncF plasmids and topoisomerase mutations conferring quinolone resistance in the Escherichia coli ST131 clone: stability and resistance evolution.

The Escherichia coli ST131 H30-Rx subclone vehicles CTX-M-15 plasmids and mutations in gyrA and parC conferring multidrug resistance successfully in the clinical setting. The aim of this study was (1) to investigate the relationship of specific topoisomerase mutations on the stability of IncF (CTX-M producing) plasmids using isogenic E. coli mutants and (2) to investigate the impact of the IncF-type plasmids present in the E. coli clone ST131 on the evolution of quinolone resistance. E. coli ATCC 25922 (background strain) and derived mutants encoding specific QRDR substitutions were used. Also, NGS-characterized IncFIA and IncFIB plasmids (encoding CTX-M genes) were included. Plasmid stability was evaluated by sequential dilutions into Luria broth medium without antibiotics for 7days. Mutant frequency to ciprofloxacin was also evaluated. Moderate differences in the IncF plasmids stability were observed among E. coli ATCC 25922 and isogenic mutants. Under our experimental conditions, the fluctuation of bacteria harboring plasmids was less than 0.5-log(10) in all cases. In the mutant frequency tests, it was observed that the presence of these IncF plasmids increased this value significantly (10-1000-fold). Quinolone resistance substitutions in gyrA or parC genes, frequently found associated with E. coli clone ST131, do not modify the stability of ST131-associated IncFIA and IncFIB plasmids under in vitro conditions. IncF-type plasmids present in E. coli clone ST131 facilitate the selection of resistance to quinolones. These results are consistent with the clinical scenario in which the combination of resistance to quinolones and beta-lactams is highly frequent in the E. coli clone ST131.

Cross-Border Emergence of Escherichia coli Producing the Carbapenemase NDM-5 in Switzerland and Germany.

A series of clinical NDM-5-producing Escherichia coli isolates obtained from two surveillance networks for carbapenem-producing Enterobacterales from 2018 to 2019, namely, Switzerland (NARA) and Germany (SurvCARE), were analyzed. The 33 NDM-5-producing E. coli isolates were highly resistant to β-lactams, including novel β-lactam/β-lactamase inhibitor combinations (ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam), and remained susceptible to fosfomycin, colistin, and tigecycline. These isolates were assigned to different sequence types (STs) and indicated a predominance of isolates exhibiting ST167 in Switzerland and Germany (n = 10) (phylogenetic group C), followed by ST405 (n = 4) (phylogenetic group E), ST1284 (n = 4) (phylogenetic group C), and ST361 (n = 4) (phylogenetic group C). The blaNDM-5 gene was predominantly present on an IncF-type plasmid (n = 29) and, to a lesser extent, on the narrow-host-range IncX3 plasmid (n = 4). Sequence analyses of eight NDM-5 plasmids indicated that NDM-5-encoding F-type plasmids varied in size between 86 and 132 kb. The two IncX3 plasmids pCH8NDM5 and pD12NDM5 were 46 and 45 kb in size, respectively. The highly conserved blaNDM-5 genetic surrounding structures (ΔISAba125-blaNDM-5-bleMBL-trpT-dsbD-IS26) of both the F-type and IncX3 plasmids suggested a common genetic origin. The emergence of the NDM-5 carbapenemase was evidenced in particular for the E. coli ST167 clone, which is a successful epidemic clone known to be associated with both multiresistance and virulence traits and is therefore of high public health concern. The occurrence of clonally related NDM-5-producing E. coli isolates in Switzerland and Germany further indicates the international spread of this multidrug-resistant superbug at least throughout Europe.

Emergence of NDM-5-Producing Escherichia coli in a Teaching Hospital in Chongqing, China: IncF-Type Plasmids May Contribute to the Prevalence of bla NDM- 5.

The newly emerging NDM-5 confers increased antibiotic resistance and attracts extensive global attention, but the prevalence, dissemination mechanism, and clinical significance of NDM-5 among clinical Escherichia coli (E. coli) infections have not been thoroughly characterized to date. In the present study, 109 unique carbapenem-resistant E. coli (CR-EC) isolates were collected in Southwest China, from 2013 to 2017, among which 41 (37.61%) CR-EC isolates were identified as NDM-5-producers, with most isolates carrying the IncF-type plasmids. Molecular epidemiological studies revealed ST167 being the most common sequence type (ST). Moreover, we described the first report of a clinical CR-EC isolate co-harboring blaKPC–2 and blaNDM–5, which showed a higher level of resistance to carbapenems. In addition, blaNDM–5 plasmid transformation and conjugation indicated that blaNDM–5 itself did confer resistance to carbapenems. Complete sequencing of the blaNDM–5-harboring IncF plasmid revealed highly conserved regions (bleMBL-trpF-tat) and some transposons around blaNDM–5. Our findings revealed a new potential threat of NDM-5-postive CR-EC in mainland China and emphasized an urgent need to control their further spread.

Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli.

Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum β-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.

Characterization of a NDM-7 carbapenemase-producing Escherichia coli ST410 clinical strain isolated from a urinary tract infection in China.

Purpose: The emergence of New Delhi metallo-beta-lactamase (NDM) carbapenemase-producing Escherichia coli leaves few therapeutic options. Infections due to NDM-7 carbapenemase-producing E. coli are infrequent. In this study, we report the whole-genome sequence of an NDM-7 carbapenemase-producing E. coli belonging to sequence type (ST) 410 isolated from a patient with a urinary tract infection in China.Patients and methods: The NDM-7 producing E. coli strain EC25 was isolated from a urine sample of a male patient hospitalized in a tertiary hospital in Zhejiang Province of China. Susceptibility assay of antibiotics was performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). The whole genome of the strain was sequenced, and the blaNDM-7-harboring plasmid was analyzed. The genomic characterization and molecular epidemiology of the strain were further elucidated.Results: E. coli EC25 was resistant to all antimicrobials tested, except tigecycline and colistin. The whole genome of E. coli EC25 was composed of one chromosomal DNA and five plasmids. Four virulence factors and twenty-five antimicrobial resistance genes, including blaNDM-7, were identified. Resistance genes were all located in an IncF-type plasmid (pEC25-1), except blaNDM-7, which was located in an individual IncX3-type plasmid (pEC25_NDM-7). Twenty-one phylogenetically related strains were identified. The phylogenetically related E. coli ST410 strains exist globally. The closest relative strain of EC25 was a strain isolated from Sichuan province of China in 2016, with a similar IncX3-type plasmid that encoded blaNDM-5.Conclusion: Our study reports the emergence of an E. coli ST410 strain harboring blaNDM-7 in China. This strain may evolve as a successful epidemic clone of NDM-producing E. coli in China, and the blaNDM gene is prone to mutate during its dissemination.

Novel variant NDM-11 and other NDM-1 variants in multidrug-resistant Escherichia coli from South India.

Carbapenem resistance mediated by New Delhi metallo-β-lactamase 1 (NDM-1) and its variants has caused a major public-health concern worldwide. Here we report for the first time an Escherichia coli isolate positive for a novel variant (NDM-11). blaNDM genes were investigated in E. coli by PCR and sequencing, and blaNDM variants were further characterised. The susceptibility pattern of novel blaNDM-11 towards different antimicrobials was compared with blaNDM-1 by cloning and expression in E. coli TOP10. A total of 33 carbapenem-resistant E. coli isolates were screened by PCR for the presence of blaNDM, of which 15 (45.5%) were positive. Sequencing of the PCR products revealed 10 isolates with NDM-1 and 5 isolates with NDM variants (one each of NDM-4, NDM-8 and NDM-11 and two NDM-5). Other resistance genes, including blaTEM-1, blaCTX-M-15, blaVIM, plasmid-encoded AmpC blaCMY-2 and 16S methyltransferases (rmtB and rmtC), were also associated with NDM variants in different combinations. The blaNDM variants were located on a transferable IncF-type plasmid of >100kb. Pulsed-field gel electrophoresis (PFGE) showed that all five E. coli isolates were unrelated, and multilocus sequence typing (MLST) revealed that they all belonged to ST131. Expression of the blaNDM-1 and blaNDM-11 genes in E. coli TOP10 showed no significant difference in MICs to various β-lactams, including carbapenems. This study underlines the spread of NDM variants with other antimicrobial resistance genes in E. coli in South India. It also describes a novel NDM variant (blaNDM-11) having an antimicrobial resistance pattern similar to blaNDM-1.

High Prevalence of CTX-M-15-Type ESBL-Producing E. coli from Migratory Avian Species in Pakistan.

The increased presence of clinically relevant multidrug resistant bacteria in natural environments is an emerging challenge for global health care. Little is known regarding the occurrence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli) from environmental sentinels in Pakistan. The goal of the current study was to gain insights into the prevalence and phylogenetic relationships of ESBL-E. coli recovered from wild birds in Pakistan during winter migration. After initial screening of fecal samples on selective chromogenic agar, ESBL-E.coli were analyzed phenotypically using the Vitek-2 automated system. Genotypic characterization was performed using whole genome sequencing (WGS) followed by an in-depth in silico analysis. Of 150 birds screened, 26 (17.3%) were fecal carriers of ESBL-E. coli. Of these, 88.4% isolates exhibited multidrug resistance (MDR) phenotypes. Resistance to cefotaxime, ceftazidime, ampicillin, doxycycline, tetracycline and sulfamethoxazole/trimethoprim (CTX-CAZ-AM-DC-TE-SXT) represented the most common pattern of MDR (76.9%). WGS data analysis found blaCTX-M-15 as the predominant ESBL genotype (92.3%). Other genes encoding resistance to sulfonamides (sul1/sul2/sul3), aminoglycosides (strA, strB, aadA1, aadA2, aadA5, aac(3)-IId-like, aac(3)-IVa-like and aph(4)-Ia), trimethoprim (dfrA14 or dfrA17), tetracyclines [tet(A)/tet(B)], and fluoroquinolones (qnrS1) were detected commonly, often encoded on IncF-type plasmids (76.9%). ESBL-E. coli were assigned to 17 different sequence types (STs) of which ST10 and ST7097 (4 isolates each) were the most abundant followed by ST4720, ST93, and ST1139 (2 isolates each). Core-genome phylogeny of the isolates found low numbers (0–29) of single nucleotide polymorphisms (SNPs) in isolates belonged to ST7097 originated from two different locations (Chashma barrage and Rasul barrage). Similar trends were found among isolates belong to ST1139. In addition, WGS-based plasmid typing and S1-digestion found plasmids of the same pMLST type (IncF[F-:A-:B53]) and similar sizes in different bacterial and avian hosts suggesting horizontal gene transfer as another possibility for the spread of ESBL-E. coli in avian wildlife in Pakistan.

CTX-M-15-Producing E. coli Isolates from Food Products in Germany Are Mainly Associated with an IncF-Type Plasmid and Belong to Two Predominant Clonal E. coli Lineages.

Extended-spectrum beta-lactamases (ESBL) mediating resistance to 3rd generation cephalosporins are a major public health issue. As food may be a vehicle in the spread of ESLB-producing bacteria, a study on the occurrence of cephalosporin-resistantu Escherichia coli in food was initiated. A total of 404 ESBL-producing isolates were obtained from animal-derived food samples (e.g., poultry products, pork, beef and raw milk) between 2011 and 2013. As CTX-M-15 is the most abundant enzyme in ESBL-producing E. coli causing human infections, this study focusses on E. coli isolates from food samples harboring the blaCTX-M-15 gene. The blaCTX-M-15 gene was detected in 5.2% (n = 21) of all isolates. Molecular analyses revealed a phylogenetic group A ST167 clone that was repeatedly isolated from raw milk and beef samples over a period of 6 months. The analyses indicate that spread of CTX-M-15-producing E. coli in German food samples were associated with a multireplicon IncF (FIA FIB FII) plasmid and additional antimicrobial resistance genes such as aac(6)-Ib-cr, blaOXA−1, catB3, different tet-variants as well as a class 1 integron with an aadA5/dfrA17 gene cassette. In addition, four phylogenetic group A ST410 isolates were detected. Three of them carried a chromosomal copy of the blaCTX-M-15 gene and a single isolate with the gene on a 90 kb IncF plasmid. The blaCTX-M-15 gene was always associated with the ISEcp1 element. In conclusion, CTX-M-15-producing E. coli were detected in German food samples. Among isolates of different matrices, two prominent clonal lineages, namely A-ST167 and A-ST410, were identified. These lineages may be important for the foodborne dissemination of CTX-M-15-producing E. coli in Germany. Interestingly, these clonal lineages were reported to be widely distributed and especially prevalent in isolates from humans and livestock. Transmission of CTX-M-15-harboring isolates from food-producing animals to food appears probable, as isolates obtained from livestock and food samples within the same time period exhibit comparable characteristics as compared to isolates detected from human. However, the routes and direction of transmission need further investigation.

Occurrence of Enterobacter hormaechei carrying blaNDM-1 and blaKPC-2 in China

Three carbapenem-resistant clinical isolates of the Enterobacter cloacae complex (ECC) were recovered from different patients in a hospital. All 3 isolates carried 2 carbapenemase genes blaKPC-2 and blaNDM-1. A study was performed to characterize their relatedness and to investigate possible links among the patients. Whole genome sequencing revealed that the isolates were Enterobacter hormaechei and belonged to ST177 of the ECC. There were 19–142 single nucleotide polymorphisms (SNPs) between the isolates, suggesting that the isolates were likely from a central reservoir, which might have existed for some time. blaKPC-2 and blaNDM-1 were carried on 2 different IncF-type plasmids in the isolates. The 3 blaNDM-1-carrying plasmids were almost identical and were self-transmissible, while the blaKPC-2-carrying plasmids were only transmissible in the presence of the blaNDM-1-carrying plasmid. The source of and direct links among them were not identified, suggesting a hospital transmission of a common multidrug resistant strain.

Genetic Background and Expression of the New qepA4 Gene Variant Recovered in Clinical TEM-1- and CMY-2-Producing Escherichia coli.

A new QepA4 variant was detected in an O86:H28 ST156-fimH38 Escherichia coli, showing a multidrug-resistance phenotype. PAβN inhibition of qepA4-harboring transconjugant resulted in increase of nalidixic acid accumulation. The qepA4 and catA1 genes were clustered in a 26.0-kp contig matching an IncF-type plasmid, and containing a Tn21-type transposon with multiple mobile genetic elements. This QepA variant is worrisome because these determinants might facilitate the selection of higher-level resistance mutants, playing a role in the development of resistance, and/or confer higher-level resistance to fluoroquinolones in association with chromosomal mutations.

Antibiotic resistance in Escherichia coli in husbandry animals: the African perspective

In the last few years, different surveillances have been published in Africa, especially in northern countries, regarding antimicrobial resistance among husbandry animals. Information is still scarce, but the available data show a worrying picture. Although the highest resistance rates have been described against tetracycline, penicillins and sulphonamides, prevalence of plasmid-mediated quinolone resistance genes and extended spectrum β-lactamase (ESBL) are being increasingly reported. Among ESBLs, the CTX-M-1 group was dominant in most African surveys. Within this group, CTX-M-15 was the main variant both in animals and humans, except in Tunisia where CTX-M-1 was more frequently detected among Escherichia coli from poultry. Certain blaCTX-M-15 -harbouring clones (ST131/B2 or ST405/D) are mainly identified in humans, but they have also been reported in livestock species from Tanzania, Nigeria or Tunisia. Moreover, several reports suggest an inter-host circulation of specific plasmids (e.g. blaCTX-M-1 -carrying IncI1/ST3 in Tunisia, IncY- and Inc-untypeable replicons co-harbouring qnrS1 and blaCTX-M-15 in Tanzania and the worldwide distributed blaCTX-M-15 -carrying IncF-type plasmids). International trade of poultry meat seems to have contributed to the spread of other ESBL variants, such as CTX-M-14, and clones. Furthermore, first descriptions of OXA-48- and OXA-181-producing E.coli have been recently documented in cattle from Egypt, and the emergent plasmid-mediated colistin resistance mcr-1 gene has been also identified in chickens from Algeria, Tunisia and South Africa. These data reflect the urgent need of a larger regulation in the use of veterinary drugs and the implementation of surveillance programmes in order to decelerate the advance of antimicrobial resistance in this continent.

Transcriptional analysis of blaNDM-1 and copy number alteration under carbapenem stress

BackgroundNew Delhi metallo beta-lactamase is known to compromise carbapenem therapy and leading to treatment failure. However, their response to carbapenem stress is not clearly known. Here, we have investigated the transcriptional response of blaNDM-1 and plasmid copy number alteration under carbapenem exposure.MethodsThree blaNDM-1 harboring plasmids representing three incompatibility types (IncFIC, IncA/C and IncK) were inoculated in LB broth with and without imipenem, meropenem and ertapenem. After each 1 h total RNA was isolated, immediately reverse transcribed into cDNA and quantitative real time PCR was used for transcriptional expression of blaNDM-1. Horizontal transferability and stability of the plasmids encoding blaNDM-1 were also determined. Changes in copy number of blaNDM-1 harboring plasmids under the exposure of different carbapenems were determined by real time PCR. Clonal relatedness among the isolates was determined by pulsed field gel electrophoresis.ResultsUnder carbapenem stress over an interval of time there was a sharp variation in the transcriptional expression of blaNDM-1 although it did not follow a specific pattern. All blaNDM-1 carrying plasmids were transferable by conjugation. These plasmids were highly stable and complete loss was observed between 92nd to 96th serial passages when antibiotic pressure was withdrawn. High copy number of blaNDM-1 was found for IncF type plasmids compared to the other replicon types.ConclusionThis study suggests that the single dose of carbapenem pressure does not significantly influence the expression of blaNDM-1 and also focus on the stability of this gene as well as the change in copy number with respect to the incompatible type of plasmid harboring resistance determinant.

Comparative Phylogenomic Analysis of Escherichia coli ST131 Isolates and IncF-type Plasmids from North Carolina Community Hospitals and Other Regions of the United States

Background Escherichia coli sequence type 131 (ST131), associated with CTX-M-15-type extended-spectrum β-lactamases (ESBLs), predominates globally among multidrug-resistant (MDR) E. coli. Among MDR E. coli ST131 isolates from North Carolina and other US regions, we explored genetic associations of the H30Rx and H30R1 subclones with IncF-type plasmid variants.MethodsSequence data from 144 clinical E. coli isolates, including 39 MDR ST131 isolates from our 7 NC community hospitals and previous 104 ST131 isolates from other US regions, were analyzed and compared. Specific genes were sought using a bioinformatic pipeline at the Center for Genomic Epidemiology and BLAST. Evolutionary analyses were performed using MEGA7. Plasmid incompatibility groups and its STs were identified using PlasmidFinder and pMLST. Comparisons of the completed sequences of 2 ST131 reference plasmids with 144 ST131 draft genome assemblies were performed using CGView Comparison Tool, and circular comparison maps were generated. Among 31 blaCTX-M-containing contigs, blaCTX-M gene insertion sites were examined.ResultsIn the core genome phylogeny, the H30R1 and H30Rx clades were placed within a larger H30R clade (Figure 1). pMLST revealed association of IncF[F2:A1:B-] plasmids (mostly blaCTX-M-15-positive) with H30Rx, and IncF[F1:A2:B20] plasmids (all blaCTX-M-15-negative) with H30R1. The ST131-associated IncF[F2:A1:B-] plasmids from NC generally resembled those from other US regions (Figure 2). However, in contrast to the others, some of the NC plasmids lacked regions previously described as gisland 1 h and/or the gColla-region, h in addition to gisland 2 h (Figure 3). Among the 31 NC ST131 isolates producing CTX-M (26 H30Rx and 5 H30R1 isolates), blaCTX-M-15 was inserted at the same chromosomal site in 3 H30Rx isolates, at different chromosomal sites in 3 H30Rx isolates, and on IncF plasmids in 1 H30Rx and 1 H30R1 isolates, suggesting that the blaCTX-M-containing gene cassette has moved around among and/or within these strains.ConclusionOur genomic and plasmid analysis of community hospital-source ST131 isolates demonstrated fixation and ongoing evolution of specific plasmids within the separate but closely related H30Rx and H30R1 sublineages.Disclosures J. R. Johnson, Crucell/Janssen: Consultant, Consulting fee Merck: Grant Investigator, Grant recipient Tetraphase: Grant Investigator, Research grant Actavis/Allergan: Grant Investigator, Grant recipient