Characteristics of ESBL and non-ESBL E. coli carrying qnrB-plasmids
In this study, 33 ESBL-/non-ESBL-EC carrying qnrB on an extrachromosomal element were characterised in detail. This co-occurrence of ESBL and (fluoro)quinolone resistance genes in E. coli poses a threat to public health, as these antimicrobials are highest priority critically important substances in human medicine.
Among the investigated isolates, a broad variety of sources of qnrB-carrying E. coli was found. While poultry seems to be the predominant source for qnr genes , plasmids with PMQR were also found in other sources. The presence of qnrB in ESBL-EC from poultry is frequently reported. The latest summary report of the European Food Safety Authority (EFSA) on AMR in zoonotic and indicator bacteria from humans, animals and food  also addresses this trend. Although the incidence of ESBL-EC was generally low, it was most often detected in broiler isolates (Member State group level of up to 30%). However, EFSA further reported a high level of ciprofloxacin- and nalidixic acid-resistant E. coli especially from broilers (median 73.5% for ciprofloxacin and 64.1% for nalidixic acid) and turkeys. Thus, poultry seems to be a common reservoir for ESBL-EC and (fluoro)quinolone-resistant E. coli. General, the occurrence of MDR E. coli, as characterised in this study, along the food chain poses a risk for a transmission of these bacteria to human via food products.
Interestingly, qnrB-positive ESBL-EC were also detected among isolates of the international high-risk clone ST131  and O89 serotype. ST131 isolates are known to represent a predominant sequence type among extraintestinal pathogenic E. coli, which comprise ESBL-positive as well as (fluoro)quinolone-resistant isolates . E. coli of the serotype O89 are often associated with MDR . Based on the results of this study, a similar association was observed for these E. coli types. The occurrence of qnrB-carrying plasmids in various STs of ESBL-EC further demonstrated that these plasmids exhibit a broad adaptability to E. coli of different ST.
It had been discussed before, that qnrB represents the dominant PMQR group in humans, while qnrS seemed to be more frequent in the environment , the veterinary and food sector [23,24,25,26]. This emphasises the need for a better understanding of the composition and impact of qnrB-carrying plasmids to estimate the transmission possibilities from animal to humans.
Here, the ESBL-EC were mostly phenotypically resistant against penicillins, cephalosporins and (fluoro)quinolones. Further, every isolate carried a bla gene coding for different TEM- or less frequently CTX-enzyme variants. For qnrB, the variant qnrB19 seemed to be predominant. Interestingly, some isolates showed genes encoding penicillinases and ceftazidime resistance. This observation needs to be further verified and characterised in detail to determine, if also other mechanisms like mutations in PBP3 and efflux pumps can cause these effects. However, all isolates of this study further exhibited phenotypical resistance to antimicrobials of other classes and were shown to carry various AMR genes co-occurring in the same isolate.
The detected virulence factors (ompA, csg, fim, chu, iroN, kpsM, irp, iuc, pap, pic and vat) may contribute to an increase in the pathogenic potential of these E. coli. The outer membrane protein A (OmpA) contributes to pathogenesis. The capsular antigen (KpsM) represents a protection factor against phagocytosis. The siderophore aerobactin gene (iuc) as well as irp, iroN and chu are associated with iron uptake often present in uropathogenic E. coli (UPEC). The genes pap (coding for P fimbriae), fim (type 1 fimbriae) and csg (curli fibers) contribute to the adhesion properties of the E. coli. The serine protease autotransporter encoding gene pic and the vacuolating autotransporter encoding gene vat do represent toxins. All detected factors individually contribute to an increase in the pathogenic potential of E. coli . The presence of these virulence factors, in qnr-carrying ESBL-EC demonstrates an aggregated risk. As virulence factors are also frequently present on plasmids , their potential spread can increase the clinical impact of the bacterium dramatically. Further subtyping results, i.e., the phylotype and detected fim variants are given in Supplemental Table 3.
While PMQR genes are the main contributors for horizontal (fluoro)quinolone resistance transmission, alterations in the sequences of the DNA gyrase and topoisomerase IV genes are the main reason for resistance against (fluoro)quinolones in E. coli . We detected previously determined single nucleotide polymorphisms (SNPs) leading to high-level resistances within ten of 33 isolates. The SNPs where mainly identified in the genes gyrA and parC. Especially the mutations in the S83L (in GyrA) and D87N (in ParC), as found here, are common [30, 31]. These mutations, in combination with the carriage of a qnr gene, are responsible for the (fluoro)quinolone resistance phenotype . However, we further detected yet uncharacterised SNPs in the QRDR for every analysed isolate (Supplement Table 4). As the study only focused on (fluoro)quinolone-resistant E. coli, it might be possible that these mutations also contribute to the observed (fluoro)quinolone resistance. Another appropriate interpretation might be a higher contribution of the qnrB genes to (fluoro)quinolone resistance, than commonly expected .
Prevalent qnrB-carrying plasmids in ESBL-/non-ESBL-EC
We identified a 39.5 kb IncN plasmid carrying qnrB2 in combination with blaCTX-M-1 surrounded by IS26 elements as previously described [34, 35]. The blaCTX-M-1 in proximity to IS26 elements was also detected on other plasmid types, suggesting that transmission of this specific region took place via IS26-mediated transfer . The folP gene, identified upstream of the qnrB2 gene, is another characteristic of this plasmid. The folP gene encodes the dihydropteroate synthase (DHPS) enzyme, which is usually encoded on the chromosome and represents the target of sulfonamides [37, 38]. The presence of folP on the plasmid may represent a genetic advantage for E. coli, as it ensures the folate biosynthesis pathway. Furthermore, the MDR transporter, encoded by ermD was detected. It represents a small MDR transporter known to confer resistance to a broad spectrum of disinfectants and quaternary cation compounds . The gene ermD was detected close to the ISSsu9, also suggesting IS-mediated transfer. In this study, we isolated the plasmid from bovine E. coli. However, similar IncN plasmids were identified in isolates of various sources (food, livestock and humans) from the Czech Republic, Poland, Denmark and Italy . Dolejkska et al.  described comparable plasmids also carrying a blaCTX-M-1, in addition to qnrS1 or qnrB19, but to the best of our knowledge not together on the same IncN plasmid. As this plasmid was determined to be conjugative as well as to be a broad-host range plasmid , the risk resulting from this special IncN plasmid and the evolvement of AMR gene accumulation should be further monitored. Another detected plasmid type in this study were plasmids of the IncHI2-IncHI2A incompatibility group. We detected this plasmid type in association with a qnrB1 gene in isolates of different animal sources, which suggests a possible broad dissemination. We identified multiple AMR genes on our qnrB1-carrying plasmids. All IncH plasmids from this study exhibited the following AMR genes: aac(3)-IIe, aac(6′)-Ib-cr5, aadA1, aph(3”)-Ib, aph(6)-Id, blaCTX-M-15, blaOXA-1, blaTEM-1, catA1, catB3, dfrA14, qnrB1, sul2 and tet(A), leading to resistance against aminoglycosides, beta-lactams, chloramphenicol, trimethoprim, (fluoro)quinolones, sulphonamides, and tetracyclines. IncH-like plasmids were previously reported as accumulators, carriers and spreaders of various resistances . The carriage of multiple AMR genes, as present for this plasmid type, probably presents a risk when transmitted. The co-occurrence of genes conferring resistance against two broad antimicrobial classes is alarming. With the presence of blaCTX-M-15, blaOXA-1 and blaTEM-1, three different beta-lactamase genes were located on the same qnrB1-carrying plasmid. The IS26 element in proximity to qnrB1 and blaOXA-1 is known to be responsible for spreading multiple AMR genes . Varani et al. described the importance of IS26 in clinical settings. They mentioned an increased frequency of plasmids carrying IS26 involved in aggregation of antimicrobial resistance genes . Harmer and colleagues described IS26 as key element for the dissemination of AMR genes in Gram-negative bacteria . We further detected hipA on the IncH plasmid. We were not able to detect the antitoxin component hipB, neither in other plasmids nor in the chromosomal DNA of the respective isolates. Thus, it remains unclear how the E. coli copes with the burden of the toxin produced from hipA. We can assume that the presence of this component is a benefit for the plasmid stability within the isolate. The detected terC virulence factor on the plasmid is quite common for IncH plasmid types. It was described as responsible for the control of resistance to infections by some bacteriophages . Thus, it may confer another advantage for the host to retain the respective plasmid. Further, IncH-type plasmids were often detected in animal and human isolates and seemed to be disseminated among different sources contributing to the spread of AMR genes from animals to humans or vice versa . As we determined the self-transmissibility of the IncH-like plasmids characterised within this study, a possible spread of this plasmid carrying multiple AMR genes is indeed given.
The most frequently detected qnrB plasmid type in the ESBL-/non-ESBL-EC in this study belonged to the Col440I-like group. All Col440I plasmids carried a qnrB19 gene and a pspF operon, as well as the gene for the transcription factor sp1. The protein sequence of the hypothetical protein as well as the non-coding regions altered within the different clusters. With the detection of the mobP relaxase gene, this plasmid was categorised as mobilizable, but not self-transmissible. The same plasmid (100% identity) has been described before with exactly the same genome structure but was assigned to a different plasmid type. Karczmarczyk and colleague identified this “ColE-like” plasmids from food samples in Colombia to carry qnrB19 . Pallecchi et al. characterised the same structure as ColE-like plasmid. They found this small qnrB19-carrying plasmid in E. coli from humans around Latin America with a high frequency and suggested a major role of this small plasmid in qnrB dissemination. As the plasmid is small and contains only a few genes, the authors hypothesised that it could have undergone a subsequent excision . This was supported by other studies, describing qnrB19 within a comparable genetic environment in larger plasmids, associated with ISEcp1C-based transposons . Moreno-Switt et al.  described that this small qnrB19-carrying plasmid was reported in Europe, the U.S.A. and South America in Salmonella obtained from food, animals and humans. They demonstrated how this qnrB19-carrying plasmid type was transmitted between different Salmonella serotypes through a P22-mediated transduction, probably explaining the frequent detection of this small plasmid. Although we only detected qnrB19 on the small Col440I plasmids, there are some studies available, presenting the qnrB19 on different plasmids also containing blaTEM-1 or blaSHV-12 . Blasting the Col440I plasmid against the NCBI database, we detected an 11.3 kb plasmid (FDAARGOS_1249) containing the backbone of the qnrB19-carrying plasmid but also additional genes, like the plasmid mobilization gene encoding MOBC.
Overall, we were able to thoroughly determine and characterize the structures of the plasmids carrying the qnrB gene. However, as we investigated the isolates with short-read sequencing the limitations for closing these plasmids has to be mentioned. Although, in silico estimation of the whole plasmid from short reads is getting more reliable, an optimised approach would include long-read sequencing of the plasmids of interest. As previously shown, especially for the determination of large plasmid genomes long read sequencing is necessary . Due to the occurrence of mobile genetic elements or repetitive sequences, short read sequencing techniques represent limitations for addressing this issue.
Risk posed by qnrB-carrying plasmids in ESBL-/non-ESBL-EC
We detected different qnrB genes on plasmids within the E. coli isolates of this study. All investigated isolates were resistant to (fluoro)quinolones. Usually, plasmidic factors were accounted only with a decrease of the susceptibility of the isolate not necessarily resulting in a non-wildtype phenotype of the isolates. However, not all isolates carried a known mutation within the respective QRDRs. Thus, it might be possible that the presence of a qnrB gene without any other yet characterised chromosomal alteration in the PMQR or the presence of other plasmidic factors can lead to a resistance phenotype for (fluoro)quinolones. Different studies had already explained, how the qnr genes are able to alter the resistance against (fluoro)quinolones due to mutations in the chromosomal QRDR regions and how the presence does allow other antimicrobial resistance genes to enter and persist. Thus, Li and colleagues reported on how QnrB promotes DNA replication stress that leads to an increased bacterial mutation risk. In their investigations, they measured a two-fold increase in the mutation rate, when QnrB is expressed. Further, they found how QnrB is responsible for the accumulation of mutations, also including quinolone resistance mutations. Overall, they suggested that QnrB could be in charge for promoting the persistence of plasmids leading to resistance against respective antimicrobial agents . These results let us assume, that the presence of qnrB genes in our isolates are also responsible for an environment allowing some mutations to occur and therewith promote the presence of different resistance profiles.
When the presence of qnrB was investigated for the first time in-depth, the observation of its association with ESBL-producing bacteria was mentioned . Jacoby et al. explained how the qnrB-carrying isolates, primarily detected in the U.S.A. and India, were always present with blaSHV-12 or blaCTX-M-15 genes on the same plasmid . The combined presence of extended-spectrum beta-lactamases encoding bla and qnr genes together within the same isolate, and/or on the same plasmid, is highly unfavourable for medical treatment. Both genes are able to confer resistance against two important classes of antimicrobial agents. Their spread to different sources, as different bacterial species, different environments or to humans is a high risk. Kawamura et al. described how ESBL-EC have become common among healthy people worldwide. They explained how most ESBL-EC usually acquired co-resistance to (fluoro)quinolones and other clinically important antimicrobial agents . As we detected the ESBL and non-ESBL-EC, carrying resistance determinants against (fluoro)quinolone within isolates recovered from livestock and food, one could assume, that the respective plasmids had spread over different areas, thus, demonstrating the necessity of a One-Health approach when estimating the risk especially arising from qnr-carrying ESBL-EC and from non-ESBL-EC.
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