KCNJ11 or ABCC8 can be treated by oral sulfonylurea drugs instead of insulin therapy. This diagnosis is currently based on Sanger sequencing of at least 42 PCR fragments from the KCNJ11, ABCC8, and INS genes. Here, we assessed the feasibility of using the next-generation whole exome sequencing (WES) for the NDM molecular diagnosis.Accurate molecular diagnosis of monogenic non-autoimmune neonatal diabetes mellitus (NDM) is critical for patient care, as patients carrying a mutation in KCNJ11, ABCC8 and INS and abnormalities in chromosome 6q24 had been previously excluded. A solution hybridization selection was performed to generate WES in 76 bp paired-end reads, by using two channels of the sequencing instrument. WES quality was assessed using a high-resolution oligonucleotide whole-genome genotyping array. From our WES with high-quality reads, we identified a novel non-synonymous mutation in ABCC8 (c.1455G>C/p.Q485H), despite a previous negative sequencing of this gene. This mutation, confirmed by Sanger sequencing, was not present in 348 controls and in the patient's mother, father and young brother, all of whom are normoglycemic.We carried out WES for a patient presenting with permanent NDM, for whom mutations in de novo ABCC8 mutation in a NDM patient. Compared to the current Sanger protocol, WES is a comprehensive, cost-efficient and rapid method to identify mutations in NDM patients. We suggest WES as a near future tool of choice for further molecular diagnosis of NDM cases, negative for chr6q24, KCNJ11 and INS abnormalities.WES identified a novel  ATP channel genes KCNJ11 and ABCC8, and in the preproinsulin gene, NDM has been linked to numerous other genetic causes including point mutations in GCK, GLIS3, EIF2AK3, PDX1, PTF1A, SLC2A2, HNF1B or FOXP3Neonatal diabetes mellitus (NDM) is a rare monogenic form of non-autoimmune diabetes which affects 1 in ∼300,000 live births and is diagnosed before six months of age e.g. relatively late age of onset, pancreas agenesis, developmental delay, renal failure, anaemia, thyroid disease, cardiac disorders…) or a family history of diabetes or consanguinity may suggest potential molecular aetiology(ies) for NDM, a molecular genetic diagnosis is crucial as it can predict the most appropriate treatment and genuinely improve quality of life ATP channel genes, who can be treated effectively with oral sulfonylureas that directly bind the SUR1 regulatory subunit of the channel, rather than requiring life-long insulin therapy which usually provides poor glycemic contol Even if the presence of specific clinical features , it is suggested to first search for a chromosome 6q24 abnormality or for a ABCC8 by a hospital laboratory, we identified a novel non-synonymous mutation in this gene through WES. We show that this cutting-edge novel technology is more comprehensive, less labour intensive and thus cheaper for NDM diagnosis than standard sequencing protocols.In the present study, we demonstrate the feasibility of next-generation whole exome sequencing (WES) for the molecular diagnosis of a patient with NDM without any extrapancreatic features or family history of diabetes. Despite previous negative Sanger sequencing of The patient's clinical record reported that he has developed severe hyperglycemia, ketoacidosis and weight loss at two months of age. HLA typing showed neutral alleles for type 1 diabetes mellitus susceptibility. Pancreas ultrasound scan was normal and the patient did not show any specific extra-pancreatic clinical features. He was firstly treated with continuous subcutaneous insulin infusion during two years with rather low dose of insulin (<0.5 units/kg/day) for a pretty good metabolic control (A1C <8.5%). He was then switched with a basal-bolus scheme for technical adverse outcome. The patient is currently 20 years old. He is treated with 1.1 units/kg/day of insulin, with A1C values ranging between 8 and 9%. He always had an attention disorder and a learning disability without obvious motor symptoms or epilepsy.After target enrichment, the whole exome DNA library from the patient was sequenced in 76 base-pairs (bp) paired-end reads, using two channels of the sequencing instrument. The WES generated 34,600,000 bp of nucleotide sequences mapping once to the targeted exome, which achieved a mean coverage of 122×of the target exome. WES identified 55,202 targeted DNA variants from the reference human genome sequence NCBI36/hg18, of which 4,463 were novel compared to the public database dbSNP130 and the eight HapMap exomes sequenced by Ng et al. To validate our WES data and analysis protocol, the patient's DNA was genotyped using an Illumina Human1M-Duo array, which contains 8,500 single nucleotide polymorphisms (SNPs) located in exons captured by the Agilent SureSelect technology. WES identified 7,969 exomic SNPs present on the genotyping array (93.8%), and showed high concordance rates for homozygous and heterozygous calls (100% and 99.5% and respectively). By using only one channel of the sequencing instrument, the generated sequence achieved a mean coverage of 65×of the target genome and 88.9% of the exomic SNPs present on the genotyping array were identified. These results would suggest that our WES based on the use of two channels of the genome analyzer has a low false negative rate for detecting exomic mutations, which is necessary to achieve accurate molecular diagnosis for patients presenting with monogenic disorders.ABCC8. Also, no abnormalities in KCNJ11, INS and chromosome 6q24 were reported by the molecular diagnostic laboratory of Robert Debré Hospital . Since these assessments were performed six years ago, we checked again for mutations in these three genes as well as in all other genes known to be involved in monogenic forms of diabetes. Unexpectedly, we identified a novel heterozygous non-synonymous mutation c.1455G>C/p.Q485H in the 9th exon of ABCC8. Based on this startling result, we used a standard Sanger sequencing protocol, to study the patient's DNA sample available in the CNRS-UMR8199 unit  as well as the sample stored at the Robert Debré hospital . In both cases, we identified the p.Q485H mutation in ABCC8, confirming our WES results. In addition, retrospective re-examination of the data generated six years ago indicated that the p.Q485H mutation was present in the original ABCC8 exon 9 sequences (chromogram). The ABCC8 p.Q485H mutation was not found in 348 French nondiabetic individuals or in the patient's mother, father and young brother, all of whom are normoglycemic. The p.Q485H mutation affects an amino acid that is located in the transmembrane domain 1 (TMD1) of the ABCC8/SUR1 core; and that is highly conserved across species  according to the UCSC (NCBI/hg18) comparative genomics alignment pipeline (http://www.bx.psu.edu/miller_lab/). We evaluated the possible functional significance of the p.Q485H mutation by the PolyPhen-2 (Polymorphism Phenotyping v2) software which uses sequence- and structure-based criteria to predict the putative impact of point mutations on the structure and function of human proteins ABCC8 mutation in the patient's DNA, a switch from insulin to oral sulfonylurea treatment will be tried soon at Necker Hospital .In 2004, single-stranded Sanger sequencing analysis of the patient's DNA by a hospital unit showed no evidence for coding mutations in In the present study, we demonstrate for the first time that WES can be seen as a relevant alternative for molecular diagnosis of NDM. Since an accurate molecular diagnosis for this condition can lead to very dramatic improvements in patient care, development of reliable and cost efficient methods for quick and accurate DNA analysis are of major interest.KCNJ11, ABCC8 and INS alone, which requires 42 PCR fragments is 2,835€ . In comparison, the all inclusive cost of WES for NDM, which will detect mutations in KCNJ11, ABCC8 and INS as well as rarer genetic aetiologies of NDM, is currently 3,274€  per sample, by performing a sequencing on two channels and in 76 bp paired-end configuration . Moreover, it is very likely that WES cost will fall in next months towards 2,000€  or even less.Currently, patients with NDM are evaluated using Sanger sequencing, which is far more expensive per sequenced base-pairs than WES. Indeed, the standard sequencing of a single PCR fragment costs 67.50€ ($82) for the only French hospital laboratory specialized in NDM molecular diagnosis . This price includes consumables, equipment amortization, personnel salary and hospital overhead costs. Therefore, the total cost for the French National Insurance for the sequencing of ABCC8 only with its 39 exons. The p.Q485H mutation was missed six years ago by the research assistant in charge of the sequence reading. Although we can assume that mutation detection bio-informatics tools were less efficient a few years ago  and that current methods are more accurate, the Sanger protocol and specially the semi-automated reading of sequence traces is always laborious and demanding (thus expensive), and a double-check of sequence readings by two different persons is performed in several French diagnostic laboratories in order to avoid any errors in the mutation identification process.We believe that the WES protocol is less labour intensive and time-consuming than the standard Sanger protocol for genetically heterogeneous disorders requiring several large genes to be screened. A WES run involving four DNA samples can be completed in two weeks, including the time required to analyse the data, which is comparable to the time required by current Sanger sequencing of de novo or are not fully penetrant. Most NDM genes have been found via candidate gene analyses but this approach has now reached its limits i.e. not yet present in human genome variants databases). Therefore, the identification of truly causal variants would be strongly facilitated by the development of a high quality WES database of novel mutations found in both elucidated cases or in cases of unknown aetiology as well as in controls coming from same ethnicity. WES would also permit the identification of putative NDM modifier genes, a very challenging task for targeted gene analysis.WES method is not only a cost-effective tool for molecular diagnosis; it should be also seen as an excellent tool for further genetic research and identification of novel causal mutations. Indeed, in the French NDM cohort, half of PNDM cases are still not elucidated ABCC8 mutation (e.g. de novo mutation associated with very early-onset of the disease and attention disorder) We are quite confident that the p.Q485H mutation is likely to be functional given the non ambiguous prediction of its putative damaging effect. In addition, the clinical data from the patient fit well with the features of PNDM linked to a We believe that other NDM patients should be assessed with the same protocol as DNA quality may change the WES accuracy. Also, our DNA capture was not totally perfect as we could miss approximately 6% of exomic SNPs present in the high-resolution oligonucleotide genotyping array. Furthermore, the exomic coverage was not homogeneous between NDM genes , thus weKCNJ11 and INS using Sanger sequencing as these two genes can be easily and quickly sequenced. If negative, we propose a WES analysis of the patient's DNA which is the most comprehensive way to fully explore the molecular causes of this NDM case.Knowing that the capture technology is improving day after day (by enriching exomic loci poorly captured with the previous kits), our present study suggests that it will be possible to soon update the protocols for molecular diagnosis of NDM KCNJ11, ABCC8 and INS, and for chromosome 6q24 abnormalities was negative. DNA samples from the patient's parents and his young brother were available for genetic testing.For WES, we selected a patient of European origin, diagnosed with PNDM who was referred to the French Network for the Study of Neonatal Diabetes Mellitus The study was approved by the local ethics committees , and both parents gave written informed consent for the genetic testing of their child.Approximately 187,000 coding exons from 3 µg of genomic DNA from the patient were captured using the Agilent SureSelect Human All Exon kit, following the manufacturer's protocols. Briefly, DNA was sheared by acoustic fragmentation (Covaris) and purified using the QIAquick PCR Purification Kit (Qiagen). The quality of the fragmentation and purification was assessed with the Agilent 2100 Bioanalyzer. The fragment ends were repaired and adaptors were ligated to the fragments . The resulting DNA library was purified using the QIAquick PCR Purification Kit, amplified by PCR and captured by hybridization to the biotinylated RNA library “baits” (Agilent). Bound genomic DNA was purified with streptavidin coated magnetic Dynal beads (Invitrogen) and re-amplified. The whole-exome DNA library was sequenced on the Illumina Genome Analyzer IIx in 76 bp paired-end reads and using two channels.Sequence reads were mapped to the reference human genome (UCSC NCBI36/hg18) using the ELANDv2 software (Illumina). Variant detection was performed with the CASAVA software  and filtered to fit a CASAVA quality threshold ≥10 and depth of ≥8× CASAVA filters duplicate reads and reads without matched pairs.A genomic DNA sample from the patient was genotyped on the Illumina Human 1M-Duo DNA Analysis BeadChips  as previously described th exon of ABCC8 was sequenced using a standard protocol and the automated 3730xl DNA Analyser (Applied Biosystems). Electrophoregram reads were assembled and analysed with the Variant Reporter software (Applied Biosystems).The p.Q485H mutation identified via WES was confirmed using the Sanger method. Primer sequences and PCR conditions are available upon request to authors. The PCR fragment fitting the 9


Determinants of adaptation in the capsid protein were studied using different maximum likelihood approaches to identify sites subject to diversifying or directional selection and sites that co-evolved. While a number of the computationally determined adaptively evolving sites were on the surface of the capsid and possible subject to immune selection, we also detected sites that were subject to constrained or compensatory evolution due to secondary RNA structures, relevant in virus-replication. We highlight codons that may prove useful in identifying emerging novel variants, and, using these, indicate that the novel 2008 variant is more likely to cause a future epidemic than the 2007 variant. While norovirus infections are generally mild and self-limiting, more severe outcomes of infection frequently occur in elderly and immunocompromized people, and no treatment is available. The observed pattern of continually emerging novel variants of GII.4, causing elevated numbers of infections, is therefore a cause for concern.Noroviruses are the most common cause of viral gastroenteritis. An increase in the number of globally reported norovirus outbreaks was seen the past decade, especially for outbreaks caused by successive genogroup II genotype 4 (GII.4) variants. Whether this observed increase was due to an upswing in the number of infections, or to a surveillance artifact caused by heightened awareness and concomitant improved reporting, remained unclear. Therefore, we set out to study the population structure and changes thereof of GII.4 strains detected through systematic outbreak surveillance since the early 1990s. We collected 1383 partial polymerase and 194 full capsid GII.4 sequences. A Bayesian MCMC coalescent analysis revealed an increase in the number of GII.4 infections during the last decade. The GII.4 strains included in our analyses evolved at a rate of 4.3–9.0×10 Noroviruses, known as the viruses that cause the ‘stomach flu’ or as the ‘cruise ship virus’, cause sporadic cases and large outbreaks of gastrointestinal illness in humans. An increase in norovirus outbreaks was reported globally around 2002. Doubts remained as to whether this increase was real, or caused by improved detection-techniques and increased awareness. This study was performed to address this ambiguity, and to determine the possible virological causes for such changes. Using a population genetic approach, we studied sequences of epidemic norovirus strains collected through time and we indeed demonstrated expanding epidemic dynamics. Global epidemics were caused by subsequent variants of norovirus, observed in 2002, 2004 and 2006 and at a smaller scale in 1996, whereas no evidence for such epidemic evolutionary patterns occurring previous to these peaks. Based on the sequences analyzed the strains of the genotype under study here were shown to have circulated at least since the early 1980s, and likely earlier. We showed that not only surface exposed sites on the outside of the virus shell were under selective pressure, involved in avoiding host immune responses, but also codons that are apparently conserved for the purpose of virus replication. Their 7.5 kb genome includes three open reading frames (ORFs). The first ORF encodes a polyprotein that is post-translationally processed to form the non-structural proteins, the second and third ORFs encode the major and minor structural proteins; VP1 or the capsid protein and VP2. The viral capsid is formed by 180 copies of the major capsid protein, and governs antigenicity, host-specificity and environmental stability.NoVs are a genetically diverse group of positive sense single-stranded RNA viruses from the NoVs are classified into five distinct genogroups, which are further subdivided into genotypes, based on their amino acid capsid sequence. Molecular epidemiological studies have shown that in recent years approximately 70% of NoV outbreaks among humans have been caused by one dominant genotype, GII.4 With continuous surveillance systems in place in some countries since the mid-1990s, it has become apparent that the number of reported NoV outbreaks, and especially those caused by GII.4 strains has risen since the appearance of the 2002 variant of GII.4 in vitro and in silico analyses The pattern of continuous lineage turnover, referred to as epochal evolution http://www.noronet.nl/fbve/) and from the global NoV surveillance network Noronet http://www.noronet.nl/).In this study, we aimed to provide a rigorous measurement of NoV GII.4 diversity through time, and we investigated viral population expansions in relationship to the increased numbers of infections reported in recent years. Evolutionary and population dynamics of GII.4 NoVs were estimated by a Bayesian coalescent approach, using two different datasets of sequences from strains with known detection dates, between 1987 and 2008. One set of sequences contained full capsid sequences, the other short partial polymerase sequences, which had been obtained for standard-procedure genotyping in NoV surveillance in Europe in silico techniques. To identify the molecular characteristics of NoV GII.4 strain replacement, we investigated both directional and diversifying selection and elucidated capsid protein positions showing evidence for co-evolutionary dynamics acting between sites.We also tested whether these dynamics differed from neutral expectations, so whether and how they were shaped by selective pressure, and we attempted to further elucidate the molecular determinants of NoV evolutionary and epidemiological dynamics using To examine the extent to which recombination has shaped NoV evolution, we analyzed an alignment of 20 GII.4 sequences, two for each variant, spanning the genome from region A , and posterior predictive simulation FD than expected (P<0.01), suggesting a selective process that generated a significant excess of mutations on terminal branches. The same was true using an exponential growth prior, but a Bayes factor test did not support an exponential growth scenario . A Bayes factor comparison also favored a constant population size model over an exponential growth model for the matched polymerase data set (ln BF = −2.23). In this case, we did not observe a significant difference in FD (P = 0.15). Because the polymerase sequences were considerably shorter, they provided less information to evaluate branch length properties. To counteract the loss of power we increased the number of sequences, and indeed, an analysis of the complete polymerase sequences dataset  rejected the model of neutral evolution (P = 0.001). As previously introduced FD values than expected under neutrality were observed for the capsid data set (P = 0.019) and the complete polymerase data set (P = 0.018), whereas the matched polymerase lacked power in rejecting neutrality (P = 0.029).To test whether GII.4 evolution deviated from selective neutrality, we applied a genealogical neutrality test that involved Bayesian coalescent inference, a tree-based summary statistic  and the matched polymerases set (4.32×10−3 substitutions per site per year) are lower than the rate found for the large partial polymerase dataset (8.98×10−3 substitutions per site per year). The estimated MRCA for these GII.4 strains lies in the first half of the 1980s  were borderline significant (p = 0.06 and p = 0.10). The rates of false positives for FEL analyses at p = 0.05 was approximately 0.04 and 0.08 at p = 0.1, based on dataset-matched neutral simulations, suggesting that the putatively selected sites were not due to elevated rates of false positives at given nominal significance values.In order to apply a codon model based on a general bivariate discrete distribution (GBDD) of dN and dS p≤0.05.To uncover population level selection processes, FEL analysis may be more appropriately applied to internal branches (iFEL) Codon models are powerful tools to detect an unusually high rate of nonsynonymous replacement, which generally occurs under a scenario of diversifying selection. However selection of episodic nature, e.g. directional selection or frequency-dependent selection is more difficult to detect and involves the question of which residues are being selected for or against In folded proteins amino acids are not arranged linearly; many functionally interact, making their evolution dependant on that of others. Various types of interactions exist, and interacting sites are not necessarily direct neighbors in either the protein sequence or in the 3D protein structure. We used Bayesian graphical models (BGM) to detect co-evolving sites. The sites identified, are shown as a network in All sites for which molecular adaptation was detected are listed presented in AT (Asn) to AGT (Ser) changes. The signal for codon 9 is solely attributable to substitutions at the second codon position of this site; AAC (Asn), ACC (Thr) and AGC (Ser). The N-terminal region of ORF2 was otherwise highly conserved. Because contrary to the other amino acids that were identified to be under selective pressure, the part of the protein encoded by these two amino acids is located on the inside of the virus capsid structure, and not surface exposed, we investigated the potential RNA secondary structure encoded by this region. In silico replacement of nucleotides at position 17 (codon 6) did not lead to secondary structure changes (not shown). Secondary structure predictions of the RNA encoding the 5′end of ORF2 were performed with all four possible nucleotides modeled at position 26  in the polymerase dataset was seen just before January 1996, around the time the first global GII.4 epidemic was noted eτ increased continuously through January 2000. Surveillance data from around the world identified relatively few GII.4 outbreaks among NoV positive outbreaks in the period between 1997 and spring 2002 eτ observed for this period is congruent with the long branch-lengths in the MCC tree during this period. Given the prolonged circulation time of the 1996 variant compared to the later variants, it seems likely that the BSP in this instance is a better representative of the relatively high genetic diversity built up in an extended period of co-circulation of the 1996 variant with other genotypes, rather than of the number of infections with this particular variant. The three most recent GII.4 epidemics caused by emerging GII.4 variants were clearly visible in the polymerase BSP. The 2002 epidemic was preceded by a sharp decline in Neτ, indicative of a purifying selection event against the previously dominating variant, in which the diversity that had been gradually built up by the long circulation of the 1996 variant collapsed. Next, coinciding with the off-seasonal peak observed in public health surveillance systems in the northern hemisphere, Neτ in the BSP rose sharply. After a brief and insignificant decline another peak occurred in the winter season of 2002–03. During the epidemic caused by the 2004 variant Neτ peaked slightly less high and less long than during the 2002 epidemic peak. During the 2006–07 epidemic, with two distinct GII.4 variants circulating, Neτ was the highest  in the BSP is lower than the estimates between subsequent epidemic peaks. This suggests an increase in the number of NoV GII.4 infections, which is further reinforced by a recent study of archival stool samples from the Children's Hospital, Washington, DC (1974 to 1991) . Although this study identified GII.4 strains in the early seventies, this was not the predominant genotype before 1991 . An increase of the GII.4 variant therefore seems to provide a plausible explanation for the coincident increase in the number of norovirus infections.While Bayesian coalescent analyses of the large partial polymerase dataset reflected seasonal epidemic dynamics, the analysis of the longer capsid sequences offered little detail about the phylodynamic patterns of NoV. To investigate the nature of this difference, we performed an analysis of polymerase sequences matching the capsid sequences genotypically as well as temporally, which showed that a lack of phylodynamic detail can generally be attributed to a lower sampling density  corresponded to the values recently reported for NoV GII.4 −3 and 8.5×10−4 for HMPV complete genomes −3 substitutions per site per year The estimated substitution rates  NoV these are only sparsely available. Instead, we showed that Bayesian coalescent demographic analyses of a large dataset containing very short sequences offered important and reliable insights into GII.4 variant dynamics. For the GII.4 NoV datasets presented here, the densely sampled but short polymerase sequences provided data that better defined the epidemic history than fewer longer capsid sequences. Perhaps additional to the limited size of the capsid dataset, strong selective pressure on the capsid protein confounded analysis of the capsid gene. We were also aware of the possibility that our capsid sequences dataset may have been biased in sampling. Sequencing of full capsid genes is not standard practice; the viruses of which sequences were available have all previously been selected by various researchers as ‘interesting enough to sequence’. Thus, relatively many sequences of strains belonging to the 1996 variant are present in the dataset, especially compared to strains belonging to the younger variants, 2004, 2006a and 2006b.p<0.05 with the iFEL approach. Of these sites, four  are located in the protruding regions of the protein. These sites have also been identified previously as ‘informative’ sites  et al, (296-297-298) et al.Our genealogical test clearly rejected neutral evolution for the NoV capsid dataset. Although P-values were somewhat higher using a Bayesian skyline plot model in the posterior predictive simulation compared to more restricted demographic functions, we arrived at the same conclusions for all analyses. This demonstrates that the neutrality test is not overly sensitive to the demographic detail in the analysis. Nevertheless, through the advances made here, we demonstrate that this test can now be performed under any complex demographic scenario, a generalization that may further promote its use. To investigate the selective forces in more detail, we fitted different evolutionary models to identify sites in the capsid under selective pressure. We examined an extension of previously performed dN/dS analyses et al. also identified sites under positive selection, using fewer sequences. They used three different methods, single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL) and random effects likelihood (REL), under the Tamura-Nei model of evolution. More codons under positive selection were identified in this study, but less strict nominal significance values were used. Bok et al.Lindesmith et al.Codon 333 was previously identified as an informative site th codon, e.g. at the third nucleotide position, nor at other nucleotides in this genomic area, apart from the previously mentioned nucleotide 17, in an RNA loop. Tentative analyses of other NoV genotypes demonstrated that the 5′ region is equally conserved within the different genotypes and yielded similar secondary RNA structures, which allow for point mutations in the loops of the structures (data not shown). To further substantiate this hypothesis site-directed mutagenesis studies are required, which go beyond the scope of this study.Codons 6 and especially 9 have consistently been identified as sites under strong selective pressure, and involved in defining the distinction between variants, not only in this study but in others as well All of the substitutions described above were associated with at least one variant transition; they appear at branches that give rise to new variants . This inet al, and 2007 is also identical in site 393–395, whereas the 2008 variant has two substitutions on those sites . Thus, considering the sites listed above, the 2008 variant would have the best chance of becoming the next dominant strain, whereas, when considering the full capsid sequence, the 2008 variant is more similar to the 2006b variant than is the 2007 variant.When amino acids 6, 9, 294, 333, 352, 368, 372, 407 and 534, identified here as under positive, directional or co-evolutionary pressure, are added to these six amino acids, all currently identified GII.4 variants  are separated by at least two amino acid differences . Thus, sUsing Bayesian phylodynamic techniques we showed that since 2002 the number of GII.4 infections has experienced expansion dynamics. Additionally we further substantiated the evidence for signature sites for variant transition, which may aid in the early recognition of potential new epidemic variants, although we stress that examining pre-defined amino acids does not enable certain identification of GII.4 variants, for which full capsid sequences should be determined. We showed that it is important to select the genomic region to analyze by phylodynamic, coalescent methods with care, and our different datasets illustrated that for the phylodynamic analysis of pathogens undergoing repeated selective bottlenecks a considerable sampling density through time is required.www.fbve.nl) and of the Noronet (www.noronet.nl) networks, the contributing institutions of which are listed in the acknowledgements. Sequences of sufficient length  were included, generating a dataset of 1383 taxa, 247 nt in length. Strains originated from systematic surveillance collections, and form the best representative reflection of the circulating strains currently available.We compiled two different NoV GII.4 datasets. First, partial polymerase gene sequences with known detection month and year were collected. These sequences, encoding a short genomic region commonly referred to as Region A, have been collected for genotyping purposes, as an essential part of the ongoing surveillance practice in institutions around the world Second, complete capsid sequences of GII.4 NoV strains with known sampling date were collected. This resulted in a dataset of 194 taxa, 1623 nt long. To allow comparison of results from capsid based versus polymerase based analysis, a set of 172 partial polymerase gene sequences matching the sequences in the capsids dataset  were selected from the total polymerase dataset. The 2003Asia variant was excluded from this mirror-dataset, since it was identified as recombinant and ORF1 does not belong to the GII.4 genotype.Details on the nature of the strains comprised by the two generated datasets are provided in the Supporting Materials For recombination analyses, a set comprising 20 sequences, two of each GII.4 variant, spanning the genome region between Region A, in ORF1, and the complete capsid sequence was collected.Sequences were aligned using the Clustal W algorithm implemented in Bioedit (version 7.0.9.0) and edited where necessary. Sequence alignments can be obtained from the authors on request.Recombination within the genomic area under study invalidates the use of phylogenetic approaches. Therefore, we checked for possible recombination signal by analyzing 20 sequences (two for each variant) spanning Region A through the complete capsid protein (2404 nt). Different evolutionary histories across this genome region were inferred using the genetic algorithm for recombination detection (GARD) 4 model of substitution and the uncorrelated lognormal relaxed clock model to accommodate variation in substitution rates among different branches Evolutionary dynamics were estimated using a Bayesian Markov chain Monte Carlo (MCMC) approach implemented in BEAST (BEAST version 1.4.7 FD), which compares the length of terminal branches to the total length of the coalescent genealogy. Strongly negative values for this statistic indicate terminal branch lengths being larger than expected, which reflects an excess of slightly deleterious mutations on these branches. This statistic has proven to be most sensitive in uncovering non-neutral evolution, and has for example rejected neutrality for human IAV hemagglutinin genes To test selective neutrality of GII.4 molecular evolution, we adopted the genealogical framework presented by Drummond and Suchard e) and generation time (τ), Ne τ, through time. To obtain a detailed measurement of NoV GII.4 diversity through time, given the large dataset, we specified 40 groups in the piecewise constant population size function. All chains were run sufficiently long to achieve stationarity after burn-in, as checked using TRACER (http://tree.bio.ed.ac.uk/software/tracer/).To reconstruct the NoV GII.4 demographic history in more detail, we employed the Bayesian skyline plot (BSP) model, which generates piecewise constant population size trajectories Additionally, the polymerase dataset was split up into separate subsets, each comprising all available sequences from a major GII.4 variant, and these were analyzed individually using the same model and settings as was used for the whole polymerase dataset.To examine how different sampling densities through time can impact our demographic estimates, we performed an additional analysis of a subset of the polymerase data, containing sequences selected to best mirror the sequences in the capsid dataset, using the same specifications as described above.dN/dS) in a codon model framework  nonsynonymous substitutions have accumulated faster than synonymous substitutions. We considered a generalization of the random effects Dual model D rates estimates 3D-2 parameters: a pair of rates (synonymous and non-synonymous) and a weight for each class. Two constraints that must be satisfied by the parameters are: the weights must sum to one, and the mean for synonymous rates must also be one to avoid confounding the mean of substitution rates and the length of the tree , where the fit of models with D and D+1 rate classes was compared using small sample AIC. Posterior distributions of substitution rate parameters were approximated using sampling importance resampling. We identified sites under diversifying or positive selection using an empirical Bayes procedure. We chose an empirical Bayes Factor cut-off of 20 to classify positively selected sites Selective pressure in the capsid genes was analyzed using the nonsynonymous/synonymous rate ratio  in a phylogenetic framework http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) Secondary structure predictions of the 5′end of the ORF2 encoding RNA were generated using the web based RNA Fold Webserver Click here for additional data file.Figure S2Sites identified to be under directional selection by DEPS analysis. Sites are depicted by MCC trees colored for which amino acid was present on each branch.(0.25 MB TIF)Click here for additional data file.Figures S3Co-evolving sites. S3A) Sites 238 and 504. S3B) Sites 231 and 509.(0.28 MB TIF)Click here for additional data file.Figure S4Minimum Spanning Tree (MST) of amino acids 6, 9, 294, 296-297-298, 333, 352, 368, 372, 393-394-395, 407, 534. Thick lines represent distances of 1 or 2 amino acids, thin lines 3 or more. An MST connects all samples in such a manner that the summed distance between all samples or branches is minimized. Different colors indicate the different variants. Different variants are separated by at least 2 amino acids.(0.52 MB TIF)Click here for additional data file.Figures S5Detection dates of NoV GII.4 strains included in the study. S5A) The polymerase sequence detection dates. S5B) The capsid sequence detection dates.(0.18 MB TIF)Click here for additional data file.Table S1Background information on strains comprised by the two datasets. NA: Not Assigned, C'well: Camberwell.(0.09 MB DOCX)Click here for additional data file.


In energized cells, FLC accumulation correlates inversely with expression of ATP-dependent efflux pumps. In de-energized cells, all strains accumulate FLC, suggesting that FLC import is not ATP-dependent. The kinetics of import in de-energized cells displays saturation kinetics with a mK of 0.64 uM and maxV of 0.0056 pmol/min/108 cells, demonstrating that FLC import proceeds via facilitated diffusion through a transporter rather than passive diffusion. Other azoles inhibit FLC import on a mole/mole basis, suggesting that all azoles utilize the same facilitated diffusion mechanism. An analysis of related compounds indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. Import of FLC by facilitated diffusion is observed in other fungi, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei, indicating that the mechanism of transport is conserved among fungal species. FLC import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered facilitated diffusion may be a previously uncharacterized mechanism of resistance to azole drugs.Despite the wealth of knowledge regarding the mechanisms of action and the mechanisms of resistance to azole antifungals, very little is known about how the azoles are imported into pathogenic fungal cells. Here the  Azole antifungals are used to treat a wide variety of fungal infections of humans, animals and plants. A great deal is known about how the azoles interact with their target enzyme within fungal cells and how the azoles are exported from the fungal cell through various efflux pumps. Altered interactions with the target enzyme and altered efflux pump expression are common mechanisms of azole resistance in fungi. However, the mechanism by which azoles enter a fungal cell is not clear—many have assumed that azoles passively diffuse into the cell. This study demonstrates that azoles are not passively diffused, or actively pumped, into the cell. Instead, azoles are imported by facilitated diffusion, mediated by a transporter. Facilitated diffusion of azoles is saturable. All clinically important azoles, and many structurally related compounds, compete for FLC import, while structurally unrelated drugs do not compete. Azole import by facilitated diffusion is shown in four species of fungi, suggesting that it is common for most if not all fungi. Altered facilitated diffusion is observed in a collection of clinical isolates, suggesting that altered import is a previously uncharacterized mechanism of resistance. Candida species are the most common invasive fungal pathogens, with Candida albicans accounting for more than 50% of all infections C. albicans causes oral, vaginal and systemic disease, with the highest morbidity rate (30%–50%) occurring with systemic Candida infections in neutropenic transplant patients The incidence of invasive fungal disease has increased over 200% in the US in the last 25 years ERG11 gene. The significant increase in invasive fungal infections and the prolonged and repeated treatment of AIDS patients has resulted in a marked increase in the emergence of FLC-resistant C. albicans isolates C. albicans, several mechanisms of resistance have been well characterized  by inhibiting 14α lanosterol demethylase, the product of the C. albicansMDR1, and the ABC transporter efflux pump genes CDR1 and CDR2Several studies have investigated the accumulation of drugs in resistant clinical isolates of C. albicans cell remain unstudied. Defects in drug import are a common mechanism of drug resistance in other pathogenic organisms, but to date, there have been no reports that C. albicans utilizes altered import as a resistance mechanism. Azoles are widely assumed to enter the fungal cell via passive diffusion Surprisingly, the mechanism(s) by which FLC enters the C. albicans cells. The results suggest that the FLC enters the cell by energy-independent facilitated diffusion in C. albicans and other pathogenic fungi. In addition, import levels vary among resistant clinical isolates, suggesting that import is a previously uncharacterized mechanism of resistance to azole drugs in C. albicans.This study biochemically characterized the mechanism by which FLC is taken into 3H]-FLC was analyzed in energized cells in the presence of glucose , a susceptible clinical isolate (#1), a matched resistant clinical isolate that overexpresses CDR1, CDR2 and MDR1 . This suggests that accumulation does occur in de-energized cells and that efflux pumps do not have an effect on FLC accumulation in the absence of glucose.Since FLC accumulation in the presence of glucose will be the sum of both FLC import and export with ATP-dependent efflux pumps, the strains were tested after glucose starvation for 2 h . If impolar rate . The add3H]-FLC did not accumulate when SC5314 cells were killed by heat . Germ tubes (hyphae) exhibited a 4 fold increase in [3H]-FLC accumulation when compared to yeast cells , voriconazole (VRC), and itraconazole (ITC). As seen in 3H]-FLC at 10 fold and 100 fold excess, suggesting that the same transporter is involved in the uptake of all four azole drugs.To determine whether other azole drugs utilize the same transporter as FLC, import of [50) values were calculated for KTC and ITC by measuring the rate of [3H]-FLC import in the presence of excess unlabeled KTC or ITC. The calculated IC50 values are 65 nM and 48 nM for KTC and ITC respectively .50% inhibitory concentration  and the fluorescent dye rhodamine 6G (R6G) have been hypothesized to share the same import mechanism as FLC 3H]-FLC . These oAs FLC is unlikely to be the natural substrate for the import mechanism, additional compounds were tested as competitors for FLC import. The following types of compounds were tested in molar excess and were shown not to compete with FLC: sugars, nucleosides, amino acids, unrelated antifungals, salts and unrelated drugs (complete list of compounds is in Saccharomyces cerevisiae3H]-FLC import in this assay (data not shown).It is known that uptake of ergosterol increases under low-oxygen conditions in In analyzing the competition data with FLC, ITC, KTC and VRC , it becaFour other drugs were tested because they are active against the sterol biosynthetic pathway and have antimicrobial activities, including Tipifarnib, the Tipifarnib derivative 2g To analyze these two components separately, the compounds difluoro-benzene and imidazole were tested in the import assay independently and together  and the aromatic ring resembles phenylalanine (F). These two amino acids alone do not compete for FLC import . None ofCryptococcus neoformans, S. cerevisiae and C. krusei, which is an intrinsically azole resistant Candida species, were all tested for import of [3H]-FLC and import levels were compared to [3H]-FLC import of C. albicans (SC5314). As shown in 3H]-FLC at levels similar to C. albicans. Similar experiments with equivalent OD units of E. coli cells did not detect FLC import above background (data not shown), supporting the conclusion that FLC import is not by passive diffusion. Collectively, these data suggest that azoles are imported by a specific transporter that may be conserved across fungal species.FLC and other azoles are used in the treatment of many pathogenic fungi. De-energized S. cerevisiae shows FLC import similar to C. albicans  that is involved in recycling or degrading membrane proteins. DOA4 is a de-ubiquitination enzyme involved in the same process and physically interacts with SNF7. As these proteins are both cytoplasmic and are involved in surface protein processing, they are not likely to be directly involved in import, but might be involved in subsequent processing of the import protein. Other ESCRT proteins in the screen did not have a significant loss of FLC import (data not shown).A collection of over 5,000 SNF7 and DOA4 in FLC import is that they may interfere with degradation of efflux pumps, resulting in increased efflux. To test for efflux pump activity in snf7 and doa4 strains, R6G efflux was monitored over time . This suggests that the role of SNF7 and DOA4 is not conserved between the two species. It is not unusual for genes to have different functions between the two species  or MDR1. The isolate with significantly increased import had no known mechanism of resistance. It is possible that these strains have altered import as well as other mechanisms of azole resistance, as it is not uncommon for clinical isolates to have multiple mechanisms of resistance To date, changes in azole import have not been reported as a mechanism of antifungal resistance in C. albicans. This is achieved using biochemical analyses. To date, FLC interactions with its target enzyme and FLC efflux from the cell have been studied in detail, but it was not known how FLC enters the fungal cell. With increasing resistance to azole therapies, it is increasingly important to understand mechanisms of FLC import and the contribution of that import to clinical resistance.This study is the first to demonstrate and characterize facilitated diffusion of FLC into C. albicans is not the result of passive diffusion but the result of facilitated diffusion. The biochemical evidence includes the following:The biochemical analyses clearly demonstrate that FLC import into Inactivated cells do not accumulate FLC, no matter the method of inactivation: heat killing or methanol treatment . PassivemK of 0.64 uM (0.2 ug/ml) and maxV of 0.0056 pmol/min/1×108 cells, strongly indicating that FLC import proceeds via facilitated diffusion. At low concentrations, the importer transports FLC into the cell at an increasing rate as the FLC concentration increases, but at higher FLC concentrations, the transporter becomes saturated and the rate of import levels off. The mK of 0.2 ug/ml is below the minimum inhibitory concentration (MIC) of the strain to FLC (1 ug/ml).The kinetics of FLC accumulation  are not 3H]-FLC does not exceed the extracellular concentration. This is consistent with facilitated diffusion, and inconsistent with active transport, which can accumulate drug to levels above the extracellular concentration. Intracellular levels were estimated using the results from this study (Saccharomyces cells 3H]-FLC is 50 nM (data not shown), which is the extracellular concentration that is used in the assay.The final intracellular concentration of -FLC. It was shown that C. neoformans, S. cerevisiae and C. krusei import [3H]-FLC with similar kinetics (data not shown) and to final levels similar to C. albicans  are routinely treated with fluquinconazole, penconazole, tebuconazole or triadimenol. A significant portion of the isolates from different species that are resistant to these agricultural azoles were cross resistant to medical azoles including FLC, ITC, KTC or VRC. The cross-resistance could be the result of over-expression of the efflux pumps, but it is also possible that these isolates contain an alteration in an azole importer that is conserved across various fungal species and confers cross resistance to all azoles since all clinically significant azoles compete for FLC import . SNF7 encodes one of the four subunits of the ESCRT III complexes, and DOA4 encodes ubiquitin isopeptidase that is closely associated with the complex. However, the C. albicans snf7 and doa4 mutants did not exhibit a reduction in import, suggesting that the role of SNF7 and DOA4 in S. cerevisiae is not central to the import mechanism. The two gene deletion strains, snf7 and doa4, do not have an altered MIC to FLC. However, the efflux pump PDR5 is highly active in Saccharomyces wild type strains, and may mask any effect of snf7 and doa4 on FLC MIC. It is curious that other ESCRT proteins do not have an altered FLC import. Further work awaits the identification of the FLC import protein.The biochemical screen did identify 3H]-FLC import. One of these four has no known mechanism(s) of resistance, while the other three are known to overexpress ERG11, MDR1, CDR1 and/or CDR2 or contain a mutation in ERG11. However, it is common for clinical isolates to exhibit multiple mechanisms of resistance 3H]-FLC import, in addition to other mechanisms of resistance. These data suggest that loss, reduction, or alteration of azole import may be a previously unknown mechanism of antifungal resistance.To date, there has been no report of altered FLC import as a mechanism of antifungal resistance. The data in this study suggests that all azoles utilize the same import mechanism mediated by a transporter. Therefore, it is possible that a mutation in the putative transporter would lead to azole cross-resistance. 35 unmatched clinical isolates in which known mechanisms of resistance had been documented C. albicans. This work also demonstrates that azoles share a common transport mechanism and azole import is conserved between several pathogenic fungi. Future directions will be focused on identifying and characterizing the protein responsible for this newly identified FLC facilitated diffusion.In conclusion, this study uses biochemical analyses to demonstrate that FLC import is not via passive diffusion but is in fact via facilitated diffusion. The data presented here represents the first comprehensive analysis of FLC import in Candida albicans SC5314  and #17 (12–99) are a matched set of FLC susceptible and resistant clinical isolates in which #17 overexpresses ERG11, CDR1, CDR2 and MDR1. #1 and #17 are from a series of 17 oral isolates from a single HIV positive patient C. albicans DSY1050 .Strains were maintained on YEPD , or on CSM complete medium . All isolates were stored at −80°C in CSM or YEPD containing 10% glycerol. Overnight cultures were inoculated from a single colony on a YEPD agar plate and inoculated into YEPD broth and grown overnight at 30°C, 180 rpm, unless otherwise noted.www.neobiosci.com).Medium components were obtained from Fischer Scientific  or Bio 101 . General chemicals were obtained from Fisher Scientific, or Sigma-Aldrich . Itraconazole, voriconazole, ketoconazole, paclobutrazol, azaconazole, flucytosine and R6G were obtained from Sigma-Aldrich . FLC was a generous donation from Pfizer, New York, NY. Fluorescein-labeled ITC was the generous gift of J. Lui and collaborators (Johns Hopkins U). POS, benznidazole, Tipifarnib, Tipifarnib 2g, and STN54 were the generous gifts of Fred Buckner, (U Washington). Oligopeptides were obtained from Neo BioScience . Cells were grown overnight in CSM complete medium at 30°C to a density typically between OD600 6.0 to 8.0, unless otherwise noted. Cells were subsequently harvested by centrifugation  and washed three times with YNB complete  without glucose (for starvation) and without supplementation, unless otherwise noted. Cells were resuspended at an OD600 of 75 in YNB for 2–3 h for glucose starvation. Reaction mixes consisted of 250 uL of YNB, 200 uL of cells (75 OD) and 50 uL of [3H]-FLC (1/100 dilution of stock). The resulting [3H]-FLC concentration is 50 nM (0.015 ug/ml), which is significantly below the MIC for all strains. Samples (100 ul) were removed at various time points and placed into 5 ml stop solution (YNB +20 µM [6 ug/ml] FLC), filtered on glass fibre filters  pre-wetted with stop solution and washed with 5 ml of stop solution. Filters were transferred to 20 ml scintillation vials. Scintillation cocktail  was added (15 ml) and the radioactivity associated with the filter was measured with a liquid scintillation analyzer  and normalized to CPM/1×108 cells. Rate of [3H]-FLC uptake was determined by incubating samples in the presence of increasing concentrations of unlabeled FLC (unless otherwise noted) and samples were analyzed for [3H]-FLC accumulation at designated time points. These data were analyzed using linear regression to determine the rate of [3H]-FLC uptake. GraphPad Prism 4.0 was used to determine linear regression, Michaelis-Menten import kinetics (maxV and mK) and 50% inhibitory concentration (IC50) values.FLC uptake was determined using [3H]-FLC by a collection of 35 Candida clinical isolates, and by the Saccharomyces gene deletion library was determined by following the above protocol with the exception that cells (1.5 ml) were grown for 48 h in 2.0 ml 96-deep well plates . The reactions were half of the size: 125 uL of YNB, 100 uL of cells, and 25 uL of [3H]-FLC (1/100 dilution of stock). Samples (100 ul) from the reaction were removed 24 h post incubation and filtered over 96-well multiscreen HTS filter plates , wells were dried and the bottoms were sealed with sealing tape . Scintillation fluid (150 ul) was added to each well, the tops were sealed with sealing tape and the plates were counted on a 96-well liquid scintillation counter using both top and bottom counting .Uptake of -FLC was determined in cells inactivated (killed) by heat (70°C for 45 m) and methanol (95% methanol for 45 m). These conditions decrease colony forming units greater than 100 fold compared to the untreated culture. To assess permeability, cells were stained using the Live/Dead FungaLight Yeast Viability Kit (Invitrogen), which contains SYTO9 and propidium iodide. The stained cells were analyzed by FACS (fluorescent activated cell sorting). After killing, cells were harvested by centrifugation, washed with YNB complete and resuspended at original density of OD600 75. Samples were compared to live SC5314 cells.Uptake of [Cells grown overnight as previously discussed, then incubated at 4°C, 25°C, 30°C or 37°C for various times and samples were removed and processed as described.pH: Cells were incubated in YNB without supplementation adjusted to pH 3, 5, 7 or 9 with potassium hydroxide (to increase pH) or hydrochloric acid (to decrease pH) and processed at various timepoints as outlined previously. Samples were compared to pH 5 and recorded as relative change.600 0.4, late log at OD600 2.0 and post diauxic shift at OD600 8–10. Samples were harvested by centrifugation  and processed as previously described. All samples were compared to the post diauxic shift sample and recorded as relative change. Post diauxic shift samples were used for most of the analyses as it reduces the sample volumes that must be processed for the analyses.For each growth phase tested  cells were inoculated into YEPD broth at varying concentrations and grown overnight. After overnight growth, log phase cultures were harvested at OD20. Cells were diluted to a concentration three logs below their overnight YEPD density in spider medium  pre-warmed to 37°C. Cells were then incubated at 37°C without shaking until germ tubes formed (approximately 1 h). Hyphae were washed two times with sterile, distilled H20 and resuspended in YNB complete (no glucose) for 2 h until they were processed and analyzed for uptake. Samples were compared to SC5314 yeast cells.SC5314 cells were grown overnight in YEPD . Cells were then washed three times with sterile, distilled HSC5314 cells were grown over night in YEPD. In order to create a microaerophilic environment, a layer of mineral oil was added to the surface of the sample and cultures grown at 30°C without shaking, as previously described Figure S1Structures of compounds from (0.22 MB TIF)Click here for additional data file.


Rutilus rutilus L.) and bream (Abramis brama L.) is well documented throughout Europe, however hybrids in Ireland occur at an unprecedented frequency, often exceeding that of both parental species. Utilising an integrated approach, which incorporates geometric morphometrics, life history and molecular genetic analyses we identify the levels and processes of hybridisation present, while also determining the direction of hybridisation, through the analysis of mitochondrial DNA.The potential role hybridisation in adaptive radiation and the evolution of new lineages has received much recent attention. Hybridisation between roach (The presence of F2 hybrids was found to be unlikely from the studied populations, although significant levels of backcrossing, involving both parental taxa was observed in some lakes. Hybridisation represents a viable conduit for introgression of genes between roach and bream. The vast majority of hybrids in all populations studied exhibited bream mitochondrial DNA, indicating that bream are maternal in the majority of crosses.The success of roach × bream hybrids in Ireland is not due to a successful self reproducing lineage. The potential causes of widespread hybridisation between both species, along with the considerations regarding the role of hybridisation in evolution and conservation, are also discussed. Rutilus rutilus L., and bream, Abramis brama L., in many Irish lakes. Hybridisation between members of the cyprinidae is more widespread than in any other group of freshwater fish [A. brama and Abrmis bjoerkna (L.) [Chondrostroma nasus (L.) and Parachondrostroma toxostoma (L.) [Barbus barbus (L.) and Barbus meridionalis (L.) [Hybridisation is recognised as a potentially powerful mechanism of diversification among vertebrates ,2. The iter fish  and exteter fish ,15. Althkna (L.) , Chondrooma (L.)  or Barbulis (L.)  is facillis (L.) , roach aet al. [Neither fish is native to Ireland ,21: the et al.  estimateet al. -37.et al. [et al. [Although studies of natural populations are scarce, captive breeding studies have demonstrated that F1 hybrids are fertile and progeny have been produced both as F2 (hybrid × hybrid) as well as through back-crossing with either parental species ,31,38,39 [et al.  observedUnlike meristic counts, providing information restricted to particular skeletal elements, the development of geometric morphometric techniques allowed quantification and visualization of overall body shape changes -43 and tGiven the limitations of exclusively phenotype-based methods, their integration with molecular genetic markers represents a significant opportunity to study hybridisation ,5,10,39.a) investigate the nature of hybridisation in selected environments, b) assess the likely ecological processes underlying the observed patterns and c) discuss the conservation and evolutionary implications of this phenomenon.By conducting an interdisciplinary analysis, using geometric morphometrics, two types of genetic markers and life-history traits (age structure and growth rates), we attempt to Geometric morphometrics was carried out on 225 fish in Lough Ramor. Of these, 101, 57 and 67 had been visually identified as roach, bream and roach × bream hybrids respectively. In Ross Lake, 233 fish consisting of 68, 112 and 53 putative roach, bream and roach × bream hybrids were analysed.The first relative warp RW1) explained the majority of the variation recorded in both Lough Ramor (75.4%) and Ross Lake (77.6), consequently the three taxa could be separated into clearly definable groups along this axis . Similarly, a positive correlation was observed between bream and roach in Lough Corrib .Age and growth analysis was carried out on 697 fish in total, 185, 164, 119 and 229 fish were analysed from Lough Corrib, Lough Ramor, Ross Lake and Leixlip Reservoir respectively Fig. . In the b at 670 bp, indicating that bream were maternal during hybridisation. In Ross L. and L. Ramor all hybrids examined exhibited bream mtDNA, while four hybrids in Leixlip Res. and one hybrid in L. Corrib exhibited roach mtDNA . No homozygous fish with hybrid morphology were recorded, while the DFA of morphometric results suggest that most of hybrids are F1 . Allied with geometric morphometric results, whereby the majority of samples were assigned to one of three distinct groups, indicating limited hybrid phenotypic diversity, this indicates that roach × bream hybrids in these habitats are primarily produced as F1 crosses of the parental species, whereby roach males fertilize bream eggs. This finding discounts the view that hybrid swarming is the cause for the extremely high proliferation of roach × bream hybrids. It is worth noting that any heterozygous F2 progeny with a hybrid phenotype, in the sample would not be detectable using this methodology, however to account for the possibility of their existence one would have to assume lethality of F2 individuals intermediate between F1 and parental morphologies, which is not a parsimonious explanation in a scenario where hybrids are so plentiful .b, indicating that they are likely the progeny of a backcross between a bream and a roach × bream hybrid. However, due to the predominance of bream cytochrome b in the hybrid population any attempt to ascertain the direction of the cross using this methodology would be futile.Despite such an apparent dominance of first generation crosses, there were varying examples of back-crossed hybrids. Morphometric analysis in Lough Ramor identified two fish with morphologies intermediate between roach and roach × bream hybrids, one of these was heterozygous and the other was not available for genetic analyses. The heterozygote exhibited bream mtDNA and is typical of back-crossed fish recorded in previous laboratory rearing experiments ,31. In Rb. Considering that the vast majority of roach × bream hybrids analysed contained bream mitochondrial DNA it is probable that these fish are the offspring of a female roach × bream hybrid and a male roach. Thus while it appears that hybrid swarming is not evident in Lough Ramor, introgression of bream genes into the roach population as a result of backcrossing is a feature of the hybrid zone.Two putative back-crossed hybrids with roach morphology were observed in Lough Ramor, though one was heterozygous for ITS1 the other was homozygous for roach ITS1 while both amplified bream cytochrome The presence of two homozygous fish with a phenotype intermediate bream and hybrids in Ross Lake either reflects a very unlikely non-recombinant F2/backcross, or perhaps more likely, the action of concerted evolution, such as biased gene conversion which can homogenise nr DNA to that of one parental species after a relatively small number of generations . This trInstances of back-crossing between bream and roach × bream hybrids were also observed in Lough. Ramor. Out of 20 bream morphotypes on which molecular analysis was carried out, four were heterozygous. Predictably, all four fish exhibited bream mitochondrial DNA, which, given the prevalence of this in roach × bream hybrids, makes any prediction of the direction of back-crossing between bream and hybrids impossible. Following Hardy-Weinberg assumptions, an approximately equal number of homozygote bream morphotypes bearing bream mtDNA could arguably be back-crosses between bream and F1 hybrids; therefore the extent of introgressive hybridisation in the lake is probably greater than that which the test can detect directly: eight out of 20 bream morphotypes (40% of the sample) are likely to be back-crosses.There was also evidence of some back-crossing in Lough Corrib. Two fish visually identified as roach were heterozygous. One of these amplified bream mtDNA while the other exhibited roach mtDNA, showing that natural back-crossing between roach and roach × bream hybrids can occur in either direction.Heterosis of the hybrid population was evident in both the growth rate and maximum length attained by the roach × bream in all lakes. The F1 hybrid phenotype, as portrayed by morphometric analysis, was intermediate between both parental phenotypes; consequently it can be assumed that the recorded intermediate mean 'length at age' size of the F1 hybrids is indicative of a healthy population. Similarly, the hybrid exhibited a growth rate which could not be significantly distinguished from that of either parent taxa and as such the intermediate hybrid must be recognised as a successful phenotype in Irish lakes. Similar levels of hybrid vigour where observed in three of the four lakes studied however it should be noted that in Ross Lake the growth characteristics of the F1 hybrid most closely resembled that of roach. While this may be due to a relatively small sample size (n = 23) of hybrids which was dominated by one year class it is also possible that the hybrid was less successful in the mesotrophic conditions found in Ross Lake, the least productive of the four lakes studied.et al. [The overall success of the F1 hybrid is in marked contrast to that of post-F1 generations. The absence of F2 progeny may be explained by poor early development of the offspring. Wood and Jordan  provide et al.  demonstret al. ,31. Morpet al. [et al. [Bream was the maternal species in the vast majority of hybrids. This is in agreement with Wyatt et al.  who founet al. ,31,38, a [et al. ,24,48. L [et al. . The oppet al. [et al. [Salmo salar L.) and trout  when sneaking male salmon fertilised eggs of female trout and a similar strategy may explain the increased presence of roach mitochondrial DNA in roach × bream hybrids in Leixlip.A small number of hybrids in Lough Corrib and Leixlip Reservoir did exhibit roach mitochondrial DNA. Hence, while there may be ecological barriers to the hybridisation between female roach and male bream, these are not insurmountable. The highest incidence of roach mitochondrial DNA in hybrids was recorded in Leixlip Reservoir, whereby the bream population was comprised of predominantly younger fish than in the other three lakes. Poncin et al.  report y [et al.  recordedWhile this study rejects the hypothesis of a stable and self-sustaining roach × bream hybrid swarm in Irish waters, it does raise interesting questions regarding the possible impact of the roach × bream hybrid on resident fish populations. The population structure of fish communities in Irish lakes changed dramatically following the invasion of roach into Irish waters with numbers of bream declining rapidly . While tet al. [Given that hybridisation occurs to a small extent in the native ranges of both parental species, the situation observed in the Irish lakes is certainly peculiar. The key to understand what is occurring perhaps lies in the fact that the studied habitats are relatively novel for both species . As both species spawn on similar substrates, preferring shallow areas rich in vegetation ,48,51,52et al.  both recet al. ,52,54, aet al. ,56 and bet al.  recordedMoreover, Ireland is species-poor relative to mainland Europe , hence tInterestingly, the nature and levels of hybridisation in the lakes studied appears to vary significantly in both space and time. For instance, Lough Ramor exhibits significant levels of back-crossing, especially for bream; whereas data on Leixlip Reservoir exhibits a complete absence of any post-F1 reproduction. Levels of hybridisation also vary annually; the hybrid population in Ross Lake is dominated by one year class, whereas in Lough Ramor annual hybrid recruitment appears to be more stable than that of either parental species. Such variations most likely reflect the interplay between colonisation history and environmental fluctuations, which results in a wide variety of hybridisation scenarios that are likely to respond dynamically to climatic changes. Species will change their spawning time depending on environmental factors -56 and tThe Irish roach × bream hybrid zone represents a dynamic scenario whereby a vigorous hybrid coexists with both parental species and acts as a conduit for introgression of genes between both species. In the absence of a self sustaining lineage the hybrid population consists primarily of F1 individuals, the offspring of hybridisation between female bream and male roach, though small but significant numbers of back-crossed progeny are also present. The proliferation of hybrids following colonisation of waters by invasive populations of roach highlights potential secondary impacts of species introductions while also shedding light on ecological factors which facilitate widespread hybridisation.2) spans two geological basins (granite and limestone) and contains a wide variety of macrophyte habitats suitable for spawning [2) situated near the Corrib catchment, on limestone bedrock, and is dominated by roach and perch but also contains populations of bream and hybrids [Sampling was carried out in four cyprinid dominated lakes in Ireland, selected in order to represent the range of environmental conditions found in Irish freshwater systems Fig. . Leixlipspawning . In conj hybrids . All samFish were initially identified on the basis of their visual appearance and the number of rays in the anal fin. Roach have 8-14 rays in the anal fin and bream have 23-30, while studies of both laboratory reared F1 and natural populations of roach × bream hybrids, which may contain F2 or later generations, report between 16 and 21 anal fin rays ,31. A suGeometric morphometric analysis was carried out on a subsample of specimens from Lough Ramor and Ross Lake, the same analyses could not be performed on fish from Leixlip Reservoir or Lough Corrib, as the lakes were sampled prior to Lough Ramor and Ross Lake digital images were not available for these fish. A sample of 458 fish, comprised of roach, bream and putative roach × bream hybrids, were analysed (225 from Lough Ramor and 233 from Ross Lake). Each fish was photographed in lateral aspect and digital images were processed as follows: On the right side of each specimen, 17 landmarks , were used to identify parental species and hybrids.Molecular analysis was carried out on a sub-sample of 80 fish, 40 individuals visually identified as hybrids and 20 of each parental species, from each lake. Fin clips were taken from the caudal fin of each fish and stored in 200 proof (>99.5%) molecular grade ethanol. DNA isolation was carried out using a modified chloroform method ,74. Follet al. , the ITSet al. [20, 2.5 μl NH4+ buffer, 2.5 μl dNTP's, 1 μlMgCL2, 1 μl of both species specific forward primers (10 pmol), 1 μl universal reverse primer (10 pmol), 0.2 μl Taq DNA polymerase and 4 μl DNA template. PCR was performed following the thermocycle described by Wyatt et al. [A multiplex PCR containing two species-specific forward primers, outlined in Wyatt et al. , and a ut et al. , althougb amplification took place in a mixture of 9.8 μl H20, 2.5 μl NH4+ buffer, 2.5 μl dNTP's, 1 μlMgCL2, 1 μl of both species specific forward primers (10 pmol), 1 μl universal reverse primer (10 pmol), 0.2 μl Taq DNA polymerase and 5 μl DNA template. PCR was performed following the thermocycle described by Wyatt et al. [Cytochrome t et al. . PCR proCombining the results of both methodologies allowed the estimation of the incidence of post-F1 hybridisation and back-crossing. Geometric morphometric analysis was used to visualise the morphology of each specimen while molecular analysis was used to ascertain its genotype and haplotype. Controlled breeding studies have shown most back-crossed hybrids to have morphologies intermediate between an F1 hybrid and the parent involved in the cross ,27,31. SIn order to identify post-F1 hybrids information from biparentally and maternally inherited molecular markers was used. Mitochondrial DNA was used to discern which species was maternal during hybridisation, while the nuclear marker was used to assign the genotype of the fish . FollowiBH carried out molecular analysis, contributed to morphometric and growth analysis and drafted the manuscript. DP performed morphometric analysis, contributed to statistical analysis and drafted relevant sections of the manuscript. AMcG carried out fish ageing analysis. MO'G, JC and MKQ conceived the study, contributed in its design, and helped to draft the manuscript. SM orchestrated both the geometric morphometric and genetic aspects of the study, coordinated laboratory based aspects of the study and was involved in drafting the manuscript. All authors read and approved the final manuscript.


This study aimed at detecting the presence of antibiotic-resistant Gram-negatives in samples of meals delivered at the University General Hospital of Palermo, Italy. Antibiotic resistant Gram negatives were isolated in July—September 2007 ffrom cold dishes and food contact surfaces and utensils. Bacterial strains were submitted to susceptibility test and subtyped by random amplification of polymorphic DNA (RAPD). Forty-six of 55 (83.6%) food samples and 14 of 17 (82.3%) environmental swabs were culture positive for Gram negative bacilli resistant to at least one group of antibacterial drugs. A total of 134 antibiotic resistant strains, 51 fermenters and 83 non-fermenters, were recovered. Fermenters and non-fermenters showed frequencies as high as 97.8% of resistance to two or more groups of antibiotics and non fermenters were 28.9% resistant to more than three groups. Molecular typing detected 34 different profiles among the fermenters and 68 among the non-fermenters. Antibiotic resistance was very common among both fermenters and non-fermenters. However, the wide heterogeneity of RAPD patterns seems to support a prominent role of cross-contamination rather than a clonal expansion of a few resistant isolates. A contribution of commensal Gram negatives colonizing foods to a common bacterial resistance pool should not been overlooked. Resistance to various antibacterial drugs is rapidly emerging and posing a major challenge to Public Health. A comprehensive understanding of the most important dissemination routes of antimicrobial resistant bacterial (ARB) strains and resistance encoding genetic sequences is crucial to effectively control and minimize the problem . Food appears to be an effective source for the acquisition by humans of drug resistant bacteria and drug resistance genes, but the extension and the actual consequences of this exposure are still insufficiently investigated –5. MoreoSuch transfers will likely be most successful when the host is simultaneously submitted to a selective pressure by an antimicrobial substance to which the involved organisms are resistant . From thHospital food service systems are considered one of the most critical segment of hospitality industry, where the client base is often a vulnerable group, and a strict and systematic monitoring of foodborne hazards has to be consistently applied . This study aimed at detecting the presence of multidrug resistant Gram-negatives (MDR-GN) in food samples delivered through a plated service at the University general hospital “Azienda Ospedaliero-Universitaria Policlinico” (AOUP), Palermo, Italy. Food contact surfaces of equipment and utensils at the Hospital caterer food premise, where food was prepared, portioned and placed into the personalized trays were also sampled during an inspective visit. Patterns of antibacterial drug susceptibility and genetic heterogeneity of the MDR-GN were assessed.This investigation was conducted at the University general hospital “Azienda Ospedaliero-Universitaria Policlinico” (AOUP), Palermo, Italy, in the period July–September 2007. At this hospital, the food service was contracted out to an external caterer who was employing a traditional cook and serve production scheme and a plated meal distribution system. In particular, food was being ordered according to the patient's choice up to 24 hours in advance. At the caterer plant, meals were prepared, assembled, and then plated using a conveyor belt with food handlers standing either side and serving appropriate portions into plates. Hot dishes were then placed into trays that were being in turn stacked into preheated cabinets, with cold dishes being placed into separate compartments, before transport and delivery to the hospital wards.For the purpose of the study, cold dishes only that had not been submitted to thermal treatment were selected. Samples of 50 g approximately were daily collected at receipt in the hospital wards from lunch meals, by taking them from a preordered tray similar to that of a patient. Samples were immediately placed in a refrigerated container and kept at 4°C until transferred to the laboratory for testing (approximately 15 minutes). When delivered to the laboratory, 10 g of each food sample were aseptically weighted into a sterile Stomacher bag, and 90 mL of peptone water were then added. Samples were homogenised for 60 seconds and incubated for 24 hours at 37°C.The inspection and the environmental sampling were carried out on September 2007. For food contact surfaces and utensils of the caterer premise, a swab sampling technique was used. The tip of each sterile cotton swab was moistened with sterile saline, pH 7.0, and then rolled repeatedly over each 10 cmq surface area. After the sampling, the swabs were placed aseptically into 10 mL of peptone water and transferred to the laboratory at chilled temperature. After the swabs were delivered to the laboratory, each tube containing the swab was vortexed 10 seconds to assure mixture of the sample and then incubated for 24 hours at 37°C.μg), ceftazidime (30 μg), gentamicin (10 μg), and nalidixic acid (30 μg), were placed on each plate before incubation, as previously described [One MacConkey agar plate was inoculated with 0.2 mL of the 24-hour enrichment culture to obtain a continuous lawn after overnight incubation in ambient air at 37°C. Four antibiotic disks, containing amoxicillin-clavulanic acid (20/10 escribed .β-lactamase-(ESBL-) producing Gram negative fermenters. Biochemical characterization to the species level of the remaining isolates was thought to be unable to provide useful additional information, because of the largely unreliable results that are generally obtained from food isolates by the commercial phenotypic systems, including the API system, according to recent findings [After incubation, plates were examined, and all colonies of different morphology growing into each antibiotic inhibition halo were Gram stained and subcultured for purity. All isolates were submitted to a biochemical screening by testing for oxidase and catalase activity and glucose fermentation and classified as Gram negative fermenters or non-fermenters. A complete biochemical identification by the system API 20E or API 20NE  was deserved to some clustered isolates and to two extended-spectrum findings .μg), cefotaxime, Ctx (30 μg), ceftazidime, Caz (30 μg), ceftriaxone, Cro (30 μg), gentamicin, Cn (10 μg), netilmycin, Net (30 μg), nalidixic acid, Na (30 μg), ciprofloxacin, Cip (5 μg), and tetracycline, Te (30 μg). Susceptibility of each isolate to a panel of nine antimicrobial substances was assessed by disk diffusion on Mueller-Hinton agar plates, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . The folESBL production was detected by decreased susceptibility or resistance to third-generation cephalosporins and the synergy between disks containing cefotaxime, ceftazidime, cefepime, and aztreonam and a disk containing amoxicillin-clavulanic acid .For the purpose of the study, a Gram negative organism resistant to at least two different groups of antimicrobial agents  was defined as MDR-GN.μL of sterile deionized water in a microcentrifuge tube. DNA extracts were prepared by boiling the suspensions for 10 minutes. After a quick spin to pellet the cell debris, the supernatants were used as DNA templates for subsequent amplifications or stored at −20°C until PCR was applied.Single colonies growing on solid media were removed with a sterile plastic tip and resuspended in 100 μM each dNTP, 150 ng primer, buffer , 2 U Taq DNA polymerase , and 1 μL whole-cell DNA in a total volume of 50 μL. The amplification protocol consisted of the following steps: initial denaturation at 94°C for 5 minutes, followed by 40 cycles of denaturation (1 minute at 94°C), annealing (1 minute at 36°C) and extension (72°C for 2 minutes), and a final extension step (72°C for 10 minutes). RAPD was performed as previously described , 20. TheThe primers used in this study were ERIC2 (5′-ATGTAAGCTCCTGGGGATTCAC-3′) and M13 (5′-GAGGGTGGCGGTTCT-3′). Primer ERIC2 was primarily used, whereas M13 was used for confirmation of clustering .Amplified PCR products were separated using 2% agarose gels, visualized by UV transillumination and photographed. A 1 Kbp or a 100 bp DNA ladder (Promega) was used as molecular weight. DNA fingerprints were compared by visual inspection and considered unique when they differed by at least one band, irrespectively of band intensity.P ≤ .05.Data were analyzed by the EpiInfo software . Frequency analysis was performed with the chi-square test. Cross tabulation and chi-square or Fisher exact tests were performed to determine the relationship between resistance and some characteristics of isolates and food products processed for isolation of resistant Gram negatives. For some statistical analysis, susceptibility to antibiotics was categorized as a dichotomous variable by interpreting intermediate susceptibility as resistance. In all analyses, differences were considered statistically significant at During the period July–September 2007, 55 food samples and 17 swabs from food contact surfaces and utensils were examined. Forty-six of 55 (83.6%) food samples and 14 of 17 (82.3%) environmental swabs proved to be culture positive for Gram negative bacilli resistant to at least one group of antibacterial drugs. A total of 115 different isolates of Gram negative bacilli resistant to at least one antimicrobial group were identified from the following food products: 22 from 18 samples of soft cheese, three from three samples of sliced ham, 20 from 12 samples of mixed ham and cheese dishes, and 70 from 24 samples of vegetables. The 19 resistant environmental isolates were, respectively, six from countertops, a centrifuge, a sink, and a conveyor belt in the washing area of fresh vegetables, nine from cutting boards, trays, and knives in the vegetables processing area, and four from a slicing machine and food contact surfaces in the cured meat working area.P = .74).The 134 Gram negative isolates were biochemically categorized as 51 fermenters and 83 non-fermenters. Prevalence of resistant Gram negative fermenters versus non-fermenters did not significantly differed in the four groups of food products examined  with a higher percentage of resistance to three or more than three groups of antibiotics.Figures Enterobacter cloacae.Two fermentative isolates that proved to be ESBL producing by the modified double-disk synergy test were identified as Klebsiella oxytoca and Pseudomonas aeruginosa. Molecular typing of the resistant Gram negative bacilli by RAPD with the ERIC2 primer detected 34 different profiles among the 51 fermenters and 68 among the 83 non-fermenters Figures . Eight RE. cloacae showed unique RAPD profiles.All clusters were confirmed by using the M13 primer. The two ESBL positive P = .02) more likely to be resistant to three antibiotic groups and less likely (P = .01) to be resistant to more than three antibiotic groups. No association was found between clustering and resistance to at least two antibiotic groups (P = .25). Among the 10 K. oxytoca isolates with RAPD pattern F26, eight were resistant to Na, Cip, and Te and two to Na and Te only. The eight P. aeruginosa with pattern NF28 had more heterogeneous resistance patterns: three were Amc Cro Ctx Na, two Amc Cro Ctz Na Te, and one each Amc Caz Cro Ctx Na, and Amc Ctx Na, Amc Na Te, respectively.Clustered isolates were significantly (P = .43). Clustering was not significantly associated to any food product (K. oxytoca with RAPD pattern F26 had been recovered from seven leaf lettuce salads and three dishes containing mixed ham and soft cheese distributed in different days. The seven P. aeruginosa isolates RAPD pattern NF28 were also sampled in different days: they were, respectively, five from leaf green salads, one from a tomato salad, and one from a soft cheese based dish. The interval of time between the first and the last isolation was in both case of 50 days.Except for the two larger ones, the remaining clusters included isolates recovered from food samples in an interval of time ranging from 0, when their isolation was made from different food products sampled in the same date, to 10 days. The 10 isolates of Salmonella, towards acquiring resistance to fluoroquinolone and 3rd and 4th generation cephalosporins [The role of food within the overall framework of human exposure to drug resistant bacteria has been until now insufficiently investigated from a Public Health perspective. Many studies have focused on the contribute of antimicrobial resistance to the severity of the hazard posed by foodborne pathogens and the specific measures to be adopted along the food chain to minimize the current trend of some pathogenic bacteria, such as osporins –23. Howeosporins , 24. To contribute additional information about food-mediated exposure to Gram negative ARB, we investigated the occurrence of these organisms in food products processed in a catering premise and delivered to hospitalized patients. Indeed, a considerable portion of these subjects belongs to a population subgroup of consumers where colonization by resistant bacteria and selective pressure due to use of antibacterial drugs may interact within a supportive environment and generate more severe health risks , 13. Enterobacteriaceae isolated from minced meat and vegetables, but with a less sensitive detection method lacking of the preliminary step on a selective culture medium [A proportion as high as 83.6% of food samples tested positive for Gram negative resistant to one or more groups of antibiotics, with a great heterogeneity of resistance patterns and RAPD patterns among both fermenters and nonfermenters. Moreover, both Gram negative groups showed frequencies as high as 97.8% of resistance to two or more groups of antibiotics. Nonfermenters, in particular, were 28.9% resistant to more than three groups. Markedly lower resistance prevalences have been previously described in e medium , 26. Hene medium . Enterobacteriaceae, a family where fluoroquinolone resistance is emerging in opportunistic and pathogen members in both hospital and community settings [β-lactamases producing bacteria and codifying sequences is a further worrying feature in the complex epidemiology of drug resistance [E. cloacae isolates confirms previous results by other Authors [Of particular concern appears the high prevalence of resistance to nalidixic acid and, to a less extent, cefotaxime. Literature suggests that resistance to nalidixic acid determined by the disk diffusion method may be a reliable indicator of decreased susceptibility to ciprofloxacin . Resistasettings , 22, 27.sistance . Detecti Authors .A further finding that deserves consideration is the large heterogeneity of RAPD patterns, that excludes clonal expansion as a possible reason of the high prevalence of antibiotic resistances in foods. Moreover, identification of some clusters of fermenter and non-fermenter isolates in intervals of time ranging between 0 and 50 days proves the persistence in the food processing environment of resistant organisms and, consequently, the potential effectiveness of Good Hygienic Practices in minimizing their diffusion. β-lactames or tetracycline resistance in various Enterobacteriaceae, could have likely overestimated the multiresistance prevalence. Finally, no evidence has been searched for of a possible relationship with colonizing isolates among patients staying in the hospital during the study.Our study has some limits. Firstly, sampling has been carried out in a single food catering premise. Consequently, the results could have been heavily influenced by the hygienic conditions of the plant, and their generalizability could be questionable. Furthermore, the issue of location and horizontal transferability of resistance genetic determinants have not been addressed. Intrinsic resistances to some antibiotics, for example, presence of AmpC mediated Based on our results, a contribution of commensal ARB Gram negatives colonizing processed foods consumed without further thermal treatment or processing to a common resistance pool should not been overlooked. This is a disturbing finding when considering the possible impact of a daily administration of resistant bacteria to a susceptible population, particularly those with defective immune systems or comorbidities and those receiving antibiotic treatment. Understanding the routes connecting the resistant bacteria and genetic resistance determinants to humans, including the role of food vehicles, is critical to define effective strategies to control this problem. The consistent and effective application of good food hygiene practices is a key issue in the prevention and control of food contamination with antimicrobial-resistant pathogenic and commensal bacteria.