Antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) abundance in animal faeces, meat, production environments and human faeces in Europe.

BACKGROUND: Real-time quantitative PCR (qPCR) is an affordable method to quantify antimicrobial resistance gene (ARG) targets, allowing comparisons of ARG abundance along animal production chains. OBJECTIVES: We present a comparison of ARG abundance across various animal species, production environments and humans in Europe. AMR variation sources were quantified. The correlation of ARG abundance between qPCR data and previously published metagenomic data was assessed. METHODS: A cross-sectional study was conducted in nine European countries, comprising 9572 samples. qPCR was used to quantify abundance of ARGs [aph(3')-III, erm(B), sul2, tet(W)] and 16S rRNA. Variance component analysis was conducted to explore AMR variation sources. Spearman's rank correlation of ARG abundance values was evaluated between pooled qPCR data and earlier published pooled metagenomic data. RESULTS: ARG abundance varied strongly among animal species, environments and humans. This variation was dominated by between-farm variation (pigs) or within-farm variation (broilers, veal calves and turkeys). A decrease in ARG abundance along pig and broiler production chains ('farm to fork') was observed. ARG abundance was higher in farmers than in slaughterhouse workers, and lowest in control subjects. ARG abundance showed a high correlation (Spearman's ρ > 0.7) between qPCR data and metagenomic data of pooled samples. CONCLUSIONS: qPCR analysis is a valuable tool to assess ARG abundance in a large collection of livestock-associated samples. The between-country and between-farm variation of ARG abundance could partially be explained by antimicrobial use and farm biosecurity levels. ARG abundance in human faeces was related to livestock antimicrobial resistance exposure.

Risk factors for the abundance of antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) in pig and broiler faeces in nine European countries.

OBJECTIVES: The occurrence and zoonotic potential of antimicrobial resistance (AMR) in pigs and broilers has been studied intensively in past decades. Here, we describe AMR levels of European pig and broiler farms and determine the potential risk factors. METHODS: We collected faeces from 181 pig farms and 181 broiler farms in nine European countries. Real-time quantitative PCR (qPCR) was used to quantify the relative abundance of four antimicrobial resistance genes (ARGs) [aph(3')-III, erm(B), sul2 and tet(W)] in these faeces samples. Information on antimicrobial use (AMU) and other farm characteristics was collected through a questionnaire. A mixed model using country and farm as random effects was performed to evaluate the relationship of AMR with AMU and other farm characteristics. The correlation between individual qPCR data and previously published pooled metagenomic data was evaluated. Variance component analysis was conducted to assess the variance contribution of all factors. RESULTS: The highest abundance of ARG was for tet(W) in pig faeces and erm(B) in broiler faeces. In addition to the significant positive association between corresponding ARG and AMU levels, we also found on-farm biosecurity measures were associated with relative ARG abundance in both pigs and broilers. Between-country and between-farm variation can partially be explained by AMU. Different ARG targets may have different sample size requirements to represent the overall farm level precisely. CONCLUSIONS: qPCR is an efficient tool for targeted assessment of AMR in livestock-related samples. The AMR variation between samples was mainly contributed to by between-country, between-farm and within-farm differences, and then by on-farm AMU.

Determinants for antimicrobial resistance genes in farm dust on 333 poultry and pig farms in nine European countries.

Livestock feces with antimicrobial resistant bacteria reaches the farm floor, manure pit, farm land and wider environment by run off and aerosolization. Little research has been done on the role of dust in the spread of antimicrobial resistance (AMR) in farms. Concentrations and potential determinants of antimicrobial resistance genes (ARGs) in farm dust are at present not known. Therefore in this study absolute ARG levels, representing the levels people and animals might be exposed to, and relative abundances of ARGs, representing the levels in the bacterial population, were quantified in airborne farm dust using qPCR. Four ARGs were determined in 947 freshly settled farm dust samples, captured with electrostatic dustfall collectors (EDCs), from 174 poultry (broiler) and 159 pig farms across nine European countries. By using linear mixed modeling, associations with fecal ARG levels, antimicrobial use (AMU) and farm and animal related parameters were determined. Results show similar relative abundances in farm dust as in feces and a significant positive association (ranging between 0.21 and 0.82) between the two reservoirs. AMU in pigs was positively associated with ARG abundances in dust from the same stable. Higher biosecurity standards were associated with lower relative ARG abundances in poultry and higher relative ARG abundances in pigs. Lower absolute ARG levels in dust were driven by, among others, summer season and certain bedding materials for poultry, and lower animal density and summer season for pigs. This study indicates different pathways that contribute to shaping the dust resistome in livestock farms, related to dust generation, or affecting the bacterial microbiome. Farm dust is a large reservoir of ARGs from which transmission to bacteria in other reservoirs can possibly occur. The identified determinants of ARG abundances in farm dust can guide future research and potentially farm management policy.

Antimicrobial Resistance in Salmonella enterica Serovar Paratyphi B Variant Java in Poultry from Europe and Latin America.

Salmonella enterica serovar Paratyphi B variant Java sequence type 28 is prevalent in poultry and poultry meat. We investigated the evolutionary relatedness between sequence type 28 strains from Europe and Latin America using time-resolved phylogeny and principal component analysis. We sequenced isolates from Colombia, Guatemala, Costa Rica, and the Netherlands and complemented them with publicly available genomes from Europe, Africa, and the Middle East. Phylogenetic time trees and effective population sizes (Ne) showed separate clustering of strains from Latin America and Europe. The separation is estimated to have occurred during the 1980s. Ne of strains increased sharply in Europe around 1995 and in Latin America around 2005. Principal component analysis on noncore genes showed a clear distinction between strains from Europe and Latin America, whereas the plasmid gene content was similar. Regardless of the evolutionary separation, similar features of resistance to ß-lactams and quinolones/fluoroquinolones indicated parallel evolution of antimicrobial resistance in both regions.

Incompatibility and phylogenetic relationship of I-complex plasmids.

Plasmid incompatibility is the inability of two plasmids to be stably maintained in one cell, resulting in loss of one of the plasmids in daughter cells. Dislodgement is a phenotypically distinct form of incompatibility, described as an imperfect reproduction, manifesting in rapid exclusion of a resident plasmid after superinfection. The relationship between plasmids of the phenotypic incompatibility groups IncB/O and IncZ is unclear. Their inability to co-exist was initially referred to as dislodgement while other research reached the conclusion that IncB/O and IncZ plasmids are incompatible. In this manuscript we re-evaluated the relationship between IncB/O and IncZ plasmids to settle these conflicting conclusions. We performed dislodgement testing of R16Δ (IncB/O) and pSFE-059 (IncZ) plasmids by electroporation in a bacterial cell and checked their stability. Stability tests of the obtained plasmid pair showed that the IncB/O plasmid was exclusively and almost completely lost from the heteroplasmid Escherichia coli population. Other IncB/O - IncZ pairs could not form a heteroplasmid population, using conjugation or electroporation. Our data supports the previous suggestion that IncB/O and IncZ plasmids may be considered phenotypically incompatible.

Phylogenomic Investigation of IncI1-Iγ Plasmids Harboring blaCMY-2 and blaSHV-12 in Salmonella enterica and Escherichia coli in Multiple Countries.

The objective of this study was to elucidate the genetic and evolutionary relatedness of blaCMY-2- and blaSHV-12-carrying IncI1-Iγ plasmids. Phylogenomic analysis based on core genome alignments and gene presence/absence was performed for different IncI1-Iγ sequence types (STs). Most IncI1-Iγ/ST12 and IncI1-Iγ/ST231 plasmids had near-identical core genomes. The data suggest that widely occurring blaCMY-2-carrying IncI1-Iγ/ST12 plasmids originate from a common ancestor. In contrast, blaSHV-12 was inserted independently into different IncI1-Iγ/ST231-related plasmids.

Novel Carbapenemases FLC-1 and IMI-2 Encoded by an Enterobacter cloacae Complex Isolated from Food Products.

Food for human consumption is screened widely for the presence of antibiotic-resistant bacteria to assess the potential for transfer of resistant bacteria to the general population. Here, we describe an Enterobacter cloacae complex isolated from imported seafood that encodes two carbapenemases on two distinct plasmids. Both enzymes belong to Ambler class A ß-lactamases, the previously described IMI-2 and a novel family designated FLC-1. The hydrolytic activity of the novel enzyme against aminopenicillins, cephalosporins, and carbapenems was determined.

Quantitative and qualitative analysis of antimicrobial usage at farm and flock level on 181 broiler farms in nine European countries.

OBJECTIVES: To control the emerging threat of antimicrobial resistance, international policy appeals for appropriate monitoring of antimicrobial usage (AMU) at supranational, species and farm level. The aim of this study was to quantify AMU in broilers at farm and flock level in nine European countries. METHODS: Antimicrobial treatment data of one flock and purchased antimicrobials over one year were collected at 181 European broiler farms. Afterwards AMU was quantified using treatment incidence (TI) per 100 days based on Defined Daily Dose (DDDvet), Defined Course Dose (DCDvet) or Used Daily Dose (UDDvet) values. Total AMU at flock level was obtained by summing the TIDDDvet of all treatments in the sampled flock (TIDDDvetFl*). RESULTS: The median TIDDDvetFl* was 9.0 (95% CI 5.5-10.8), meaning that broilers were treated with antimicrobials during 9% of their rearing period. TIDDDvetFl* varied considerably within and between countries. However, in every country at least one untreated flock was present. Average TIDDDvetFl* at country level ranged from 3.3 to 36.7. Polymyxins, extended-spectrum aminopenicillins and fluoroquinolones were the most used antimicrobials, accounting for 26%, 26% and 18% of total AMU, respectively. Twenty-six percent of the farms started a treatment on day 1 of production, and 49% of overall AMU was administered within the first week. CONCLUSIONS: Results show that rearing broilers without AMU is feasible. However, a huge variation in AMU in terms of amount, moment of administration and antimicrobial classes was observed. This shows that there is still ground to be covered when it comes to AMU on broiler farms.

Quantitative and qualitative analysis of antimicrobial usage patterns in 180 selected farrow-to-finish pig farms from nine European countries based on single batch and purchase data.

OBJECTIVES: Farm-level quantification of antimicrobial usage (AMU) in pig farms. METHODS: In a cross-sectional study, AMU data on group treatments administered to a single batch of fattening pigs from birth to slaughter (group treatment data) and antimicrobials purchased during 1 year (purchase data) were collected at 180 pig farms in nine European countries. AMU was quantified using treatment incidence (TI) based on defined (DDDvet) and used (UDDvet) daily doses and defined (DCDvet) and used (UCDvet) course doses. RESULTS: The majority of antimicrobial group treatments were administered to weaners (69.5% of total TIDDDvet) followed by sucklers (22.5% of total TIDDDvet). AMU varied considerably between farms with a median TIDDDvet of 9.2 and 7.1 for a standardized rearing period of 200 days based on group treatment and purchase data, respectively. In general, UDDvet and UCDvet were higher than DDDvet and DCDvet, respectively, suggesting that either the defined doses were set too low or that group treatments were often dosed too high and/or administered for too long. Extended-spectrum penicillins (31.2%) and polymyxins (24.7%) were the active substances most often used in group treatments, with the majority administered through feed or water (82%). Higher AMU at a young age was associated with higher use in older pigs. CONCLUSIONS: Collecting farm-level AMU data of good quality is challenging and results differ based on how data are collected (group treatment data versus purchase data) and reported (defined versus used daily and course doses).

The antimicrobial resistome in relation to antimicrobial use and biosecurity in pig farming, a metagenome-wide association study in nine European countries.

OBJECTIVES: Previous studies in food-producing animals have shown associations between antimicrobial use (AMU) and resistance (AMR) in specifically isolated bacterial species. Multi-country data are scarce and only describe between-country differences. Here we investigate associations between the pig faecal mobile resistome and characteristics at the farm-level across Europe. METHODS: A cross-sectional study was conducted among 176 conventional pig farms from nine European countries. Twenty-five faecal samples from fattening pigs were pooled per farm and acquired resistomes were determined using shotgun metagenomics and the Resfinder reference database, i.e. the full collection of horizontally acquired AMR genes (ARGs). Normalized fragments resistance genes per kilobase reference per million bacterial fragments (FPKM) were calculated. Specific farm-level data (AMU, biosecurity) were collected. Random-effects meta-analyses were performed by country, relating farm-level data to relative ARG abundances (FPKM). RESULTS: Total AMU during fattening was positively associated with total ARG (total FPKM). Positive associations were particularly observed between widely used macrolides and tetracyclines, and ARGs corresponding to the respective antimicrobial classes. Significant AMU-ARG associations were not found for ß-lactams and only few colistin ARGs were found, despite high use of these antimicrobial classes in younger pigs. Increased internal biosecurity was directly related to higher abundances of ARGs mainly encoding macrolide resistance. These effects of biosecurity were independent of AMU in mutually adjusted models. CONCLUSIONS: Using resistome data in association studies is unprecedented and adds accuracy and new insights to previously observed AMU-AMR associations. Major components of the pig resistome are positively and independently associated with on-farm AMU and biosecurity conditions.

Associations between antimicrobial use and the faecal resistome on broiler farms from nine European countries.

OBJECTIVES: To determine associations between farm- and flock-level antimicrobial usage (AMU), farm biosecurity status and the abundance of faecal antimicrobial resistance genes (ARGs) on broiler farms. METHODS: In the cross-sectional pan-European EFFORT study, conventional broiler farms were visited and faeces, AMU information and biosecurity records were collected. The resistomes of pooled faecal samples were determined by metagenomic analysis for 176 farms. A meta-analysis approach was used to relate total and class-specific ARGs (expressed as fragments per kb reference per million bacterial fragments, FPKM) to AMU (treatment incidence per DDD, TIDDDvet) per country and subsequently across all countries. In a similar way, the association between biosecurity status (Biocheck.UGent) and the resistome was explored. RESULTS: Sixty-six (38%) flocks did not report group treatments but showed a similar resistome composition and roughly similar ARG levels to antimicrobial-treated flocks. Nevertheless, we found significant positive associations between ß-lactam, tetracycline, macrolide and lincosamide, trimethoprim and aminoglycoside antimicrobial flock treatments and ARG clusters conferring resistance to the same class. Similar associations were found with purchased products. In gene-level analysis for ß-lactams and macrolides, lincosamides and streptogramins, a significant positive association was found with the most abundant gene clusters blaTEM and erm(B). Little evidence was found for associations with biosecurity. CONCLUSIONS: The faecal microbiome in European broilers contains a high diversity of ARGs, even in the absence of current antimicrobial selection pressure. Despite this, the relative abundance of genes and the composition of the resistome is positively related to AMU in European broiler farms for several antimicrobial classes.

The shufflon of IncI1 plasmids is rearranged constantly during different growth conditions.

One of the factors that can affect conjugation of IncI1 plasmids, amongst others, is the genetic region known as the shufflon. This multiple inversion system modifies the pilus tip proteins used during conjugation, thus affecting the affinity for different recipient cells. Although recombination is known to occur in in vitro conditions, little is known about the regulation and the extent of recombination that occurs. To measure the recombination of the shufflon, we have amplified the entire shufflon region and sequenced the amplicons using nanopore long-read sequencing. This method was effective to determine the order of the segments of the shufflon and allow for the analysis of the shufflon variants that are present in a heterogeneous pool of templates. Analysis was performed over different growth phases and after addition of cefotaxime. Furthermore, analysis was performed in different E. coli host cells to determine if recombination is likely to be influenced. Recombination of the shufflon was constantly ongoing in all conditions that were measured, although no differences in the amount of different shufflon variants or the rate at which novel variants were formed could be found. As previously reported, some variants were abundant in the population while others were scarce. This leads to the conclusion that the shufflon is continuously recombining at a constant rate, or that the method used here was not sensitive enough to detect differences in this rate. For one of the plasmids, the host cell appeared to have an effect on the specific shufflon variants that were formed which were not predominant in another host, indicating that host factors may be involved. As previously reported, the pilV-A and pilV-A' ORFs are formed at higher frequencies than other pilV ORFs. These results demonstrate that the recombination that occurs within the shufflon is not random. While any regulation of the shufflon affected by these in vitro conditions could not be revealed, the method of amplifying large regions for long-read sequencing for the analysis of multiple inversion systems proved effective.

Molecular relatedness of ESBL/AmpC-producing Escherichia coli from humans, animals, food and the environment: a pooled analysis.

Background: In recent years, ESBL/AmpC-producing Escherichia coli (ESBL/AmpC-EC) have been isolated with increasing frequency from animals, food, environmental sources and humans. With incomplete and scattered evidence, the contribution to the human carriage burden from these reservoirs remains unclear. Objectives: To quantify molecular similarities between different reservoirs as a first step towards risk attribution. Methods: Pooled data on ESBL/AmpC-EC isolates were recovered from 35 studies in the Netherlands comprising >27 000 samples, mostly obtained between 2005 and 2015. Frequency distributions of ESBL/AmpC genes from 5808 isolates and replicons of ESBL/AmpC-carrying plasmids from 812 isolates were compared across 22 reservoirs through proportional similarity indices (PSIs) and principal component analyses (PCAs). Results: Predominant ESBL/AmpC genes were identified in each reservoir. PCAs and PSIs revealed close human-animal ESBL/AmpC gene similarity between human farming communities and their animals (broilers and pigs) (PSIs from 0.8 to 0.9). Isolates from people in the general population had higher similarities to those from human clinical settings, surface and sewage water and wild birds (0.7-0.8), while similarities to livestock or food reservoirs were lower (0.3-0.6). Based on rarefaction curves, people in the general population had more diversity in ESBL/AmpC genes and plasmid replicon types than those in other reservoirs. Conclusions: Our 'One Health' approach provides an integrated evaluation of the molecular relatedness of ESBL/AmpC-EC from numerous sources. The analysis showed distinguishable ESBL/AmpC-EC transmission cycles in different hosts and failed to demonstrate a close epidemiological linkage of ESBL/AmpC genes and plasmid replicon types between livestock farms and people in the general population.

Chromosome-Based blaOXA-48-Like Variants in Shewanella Species Isolates from Food-Producing Animals, Fish, and the Aquatic Environment.

Carbapenems are considered last-resort antibiotics in health care. Increasing reports of carbapenemase-producing bacteria in food-producing animals and in the environment indicate the importance of this phenomenon in public health. Surveillance for carbapenemase genes and carbapenemase-producing bacteria in Dutch food-producing animals, environmental freshwater, and imported ornamental fish revealed several chromosome-based blaOXA-48-like variants in Shewanella spp., including two new alleles, blaOXA-514 and blaOXA-515 Carbapenemase genes were not associated with mobile genetic elements or Enterobacteriaceae.

Plasmids of Distinct IncK Lineages Show Compatible Phenotypes.

IncK plasmids are some of the main carriers of blaCTX-M-14 and blaCMY-2 genes and show high similarity to other plasmids belonging to the I complex, including IncB/O plasmids. Here, we studied the phylogenetic relationship of 37 newly sequenced IncK and IncB/O plasmids. We show that IncK plasmids can be divided into two compatible lineages named IncK1 and IncK2.

Competitive Exclusion Reduces Transmission and Excretion of Extended-Spectrum-ß-Lactamase-Producing Escherichia coli in Broilers.

Extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (pAmpC) are enzymes able to hydrolyze a large variety of ß-lactam antibiotics, including third-generation cephalosporins and monobactams. Broilers and broiler meat products can be highly contaminated with ESBL- and pAmpC-producing Escherichia coli strains, also known as extended-spectrum cephalosporin (ESC)-resistant E. coli strains, and can be a source for human infections. As few data on interventions to reduce the presence of ESC-resistant E. coli in broilers are available, we used transmission experiments to examine the role of competitive exclusion (CE) on reducing transmission and excretion in broilers. A broiler model to study the transmission of ESC-resistant E. coli was set up. Day-old chickens were challenged with an ESBL-producing E. coli strain isolated from healthy broilers in the Netherlands. Challenged and not challenged chicks were housed together in pairs or in groups, and ESBL-producing E. coli transmission was monitored via selective culturing of cloacal swab specimens. We observed a statistically significant reduction in both the transmission and excretion of ESBL-producing E. coli in chicks treated with the probiotic flora before E. coli challenge compared to the transmission and excretion in untreated controls. In conclusion, our results support the use of competitive exclusion as an intervention strategy to control ESC-resistant E. coli in the field.IMPORTANCE Extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases are a primary cause of resistance to ß-lactam antibiotics among members of the family Enterobacteriaceae in humans, animals, and the environment. Food-producing animals are not exempt from this, with a high prevalence being seen in broilers, and there is evidence pointing to a possible foodborne source for human contamination. We investigated the effect of administration of a commercial probiotic product as an intervention to reduce the amount of ESBL-producing Escherichia coli in broilers. Our results showed a substantial reduction in the level of colonization of broiler intestines by ESBL-producing E. coli after administration of commercial probiotic product. The protective effect provided by these probiotics could be implemented on a larger scale in poultry production. Reductions in the levels of ESBL-producing Enterobacteriaceae in the food chain would considerably benefit public health.

Extended-Spectrum Cephalosporin-Resistant Salmonella enterica serovar Heidelberg Strains, the Netherlands(1).

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains (JF6X01.0022/XbaI.0251, JF6X01.0326/XbaI.1966, JF6X01.0258/XbaI.1968, and JF6X01.0045/XbaI.1970) have been identified in the United States with pulsed-field gel electrophoresis. Our examination of isolates showed introduction of these strains in the Netherlands and highlight the need for active surveillance and intervention strategies by public health organizations.

Molecular Characterization of Extended-Spectrum-Cephalosporin-Resistant Enterobacteriaceae from Wild Kelp Gulls in South America.

Extended-spectrum-cephalosporin-resistant Enterobacteriaceae are a public health concern due to limited treatment options. Here, we report on the occurrence and the molecular characteristics of extended-spectrum-cephalosporin-resistant Enterobacteriaceae recovered from wild birds (kelp gulls). Our results revealed kelp gulls as a reservoir of various extended-spectrum cephalosporinase genes associated with different genetic platforms. In addition, we report for the first time the presence of a known epidemic clone of Salmonella enterica serotype Heidelberg (JF6X01.0326/XbaI.1966) among wild birds.

Quantitative assessment of antimicrobial resistance in livestock during the course of a nationwide antimicrobial use reduction in the Netherlands.

OBJECTIVES: To quantify associations between antimicrobial use and acquired resistance in indicator Escherichia coli over a period of time which involved sector-wide antimicrobial use reductions in broilers and pigs (years 2004-14), veal calves (2007-14) and dairy cattle (2005-14). Prevalence estimates of resistance were predicted for a hypothetical further decrease in antimicrobial use. METHODS: Data reported annually for the resistance surveillance programme in the Netherlands were retrieved. Two multivariate random-effects logistic models per animal sector were used to relate total and class-specific antimicrobial use (as defined daily dosages per animal per year, DDDA/Y) with the probability of E. coli resistance to a panel of 10 antimicrobial agents. RESULTS: Positive dose-response relationships (ORs) were obtained from all models. Specific resistance phenotypes were more often associated with total antimicrobial use than with class-specific use. The most robust associations were found in pigs and veal calves. Resistance to historically widely used antimicrobials (e.g. penicillins, tetracyclines) was, in relative terms, less influenced by drug use changes over time than resistance to newer or less prescribed antimicrobials (e.g. third-/fourth-generation cephalosporins, fluoroquinolones). In pigs and veal calves, prevalence estimates for the most common resistance phenotypes were projected to decline ∼5%-25% during 2014-16 if total antimicrobial use reduction reached 80%; projections for poultry and dairy cows were more modest. CONCLUSIONS: Epidemiological evidence indicated that drug use history and co-selection of resistance are key elements for perpetuation of resistance. Data suggest that recent Dutch policies aimed at reducing total use of antimicrobials have decreased E. coli resistance in the pig and veal calf production sectors while the impact on the dairy cattle and poultry sectors is less clear.

IncI shufflons: Assembly issues in the next-generation sequencing era.

The shufflon is a site-specific recombination system first identified in the IncI1 plasmid R64. The R64 shufflon consists of four segments, separated by short repeats, which are rearranged and inverted by the recombinase protein Rci, generating diversity in the C-terminal end of the PilV protein. PilV is the tip adhesin of the thin pilus structure involved in bacterial conjugation and may play a role in determining recipient cell specificity during liquid mating. The variable arrangements of the shufflon region would be expected to make plasmid assembly difficult, particularly with short-read sequencing technology, but this is not usually mentioned in recent publications reporting IncI plasmid sequences. Here we discuss the issues we encountered with assembly of IncI1 sequence data obtained from the Roche-454 and Illumina platforms and make some suggestions for assembly of the shufflon region. Comparison of shufflon segments from a collection of IncI1 plasmids from The Netherlands and Australia, together with sequences available in GenBank, suggests that the number of shufflon segments present is conserved among plasmids grouped together by plasmid multi-locus sequencing typing but the different reported arrangements of shufflon segments may not be meaningful. This analysis also indicated that the sequences of the shufflon segments are highly conserved, with very few nucleotide changes.