Optimized Design of Degenerate Primers for PCR Based on DNA or Protein Sequence Comparisons.

PCR with degenerate primers can be used to identify the coding sequence of an unknown protein or to detect a genetic variant within a gene family. These primers, which are complex mixtures of slightly different oligonucleotide sequences, can be optimized to increase the efficiency and/or specificity of PCR in the amplification of a sequence of interest by the introduction of mismatches with the target sequence and balancing their position toward the primers 5'- or 3'-ends. In this work, we explain in detail examples of rational design of primers in three different applications, including the use of specific determinants at the 3'-end, to (i) improve PCR efficiency with related sequences for members of a protein family by complete degeneration at a core box of conserved genetic information at the 3'-end with the reduction of degeneration at the 5'-end, (ii) optimize specificity of allelic discrimination of closely related DNA sequences of orthologous by 5'-end fully degenerate primers, and (iii) increase the PCR efficiency of primers by targeting DNA sequences belonging to specific phylogenetic groups, within a large and diverse gene family, allowing the use of multiplex/degenerate PCR.

Plasmid-mediated Kluyvera-like arnBCADTEF operon confers colistin (hetero)resistance to Escherichia coli.

The use of colistin as a last resort antimicrobial is compromised by the emergence of resistant enterobacteria with acquired determinants like mcr genes, mutations that activate the PmrAB system and by still unknown mechanisms. This work analyzed 74 E. coli isolates from healthy swine, turkey or bovine, characterizing their colistin resistance determinants. The mcr-1 gene, detected in 69 isolates, was the main determinant found among which 45% were carried by highly mobile plasmids, followed by four strains lacking previously known resistance determinants or two with mcr-4 (one in addition to mcr-1), whose phenotypes were not transferred by conjugation. Although a fraction of isolates carrying mcr-1 or mcr-4 genes also presented missense polymorphisms in pmrA or pmrB, constitutive activation of PmrAB was not detected, in contrast to strains with mutations that confer colistin resistance. The expression of mcr genes negatively controls the transcription of the arnBCADTEF operon itself, a down-regulation that was also observed in the four isolates lacking known resistance determinants, three of them sharing the same macrorestriction and plasmid profiles. Genomic sequencing of one of these strains, isolated from a bovine in 2015, revealed a IncFII plasmid of 62.1 Kb encoding an extra copy of the arnBCADTEF operon closely related to Kluyvera ascorbata homologs. This element, called pArnT1, was cured by ethidium bromide and the cells lost resistance to colistin in parallel. Furthermore, a susceptible E. coli strain acquired heteroresistance after transformation with pArnT1 or pBAD24 carrying the Kluyvera-like arnBCADTEF operon, revealing it as a new colistin resistance determinant.

Longitudinal study of the mcr-1 gene prevalence in Spanish food-producing pigs from 1998 to 2021 and its relationship with the use of polymyxins.

BACKGROUND: Resistance to colistin was an uncommon phenomenon traditionally linked to chromosome point mutations, but since the first description of a plasmid-mediated colistin-resistance in late 2015, transmissible resistance to colistin has become a Public Health concern. Despite colistin is considered as a human last resort antibiotic, it has been commonly used in swine industry to treat post-weaning diarrhoea in piglets. However, the progressively increase of colistin resistance during the last decade led to the Spanish Medicines and Healthcare Products Agency (AEMPS) to launch a strategic and voluntary plan aimed to reduce colistin consumption in pig production. Our longitudinal study (1998-2021) aimed to evaluate the trend of colistin resistance mediated through the mcr-1 mobile gene in Spanish food-producing pig population and compare it with published polymyxin sales data in veterinary medicine to assess their possible relationships. RESULTS: The first mcr-1 positive sample was observed in 2004, as all samples from 1998 and 2002 were mcr-1 PCR-negative. We observed a progressive increase of positive samples from 2004 to 2015, when mcr-1 detection reached its maximum peak (33/50; 66%). From 2017 (27/50; 54%) to 2021 (14/81; 17%) the trend became downward, reaching percentages significantly lower than the 2015 peak (p < 0.001). The abundance of mcr-1 gene in PCR-positive samples showed a similar trend reaching the highest levels in 2015 (median: 6.6 × 104 mcr-1 copies/mg of faeces), but decreased significantly from 2017 to 2019 (median 2.7 × 104, 1.2 × 103, 4.6 × 102 mcr-1 copies/mg of faeces for 2017, 2018 and 2019, respectively), and stabilizing in 2021 (1.6 × 102 mcr-1 copies/mg of faeces) with similar values than 2019. CONCLUSIONS: Our study showed the decreasing trend of colistin resistance associated to mcr-1 gene, after a previous increase from among 2004-2015, since the European Medicines Agency and AEMPS strategies were applied in 2016 to reduce colistin use in animals, suggesting a connection between polymyxin use and colistin resistance. Thus, these plans could have been effective in mcr-1 reduction, reaching lower levels than those detected in samples collected 17 years ago, when resistance to colistin was not yet a major concern.

Dissemination of antimicrobial-resistant isolates of Salmonella spp. in wild boars and its relationship with management practices.

Antimicrobial resistance (AMR) is a global concern and controlling its spread is critical for the effectiveness of antibiotics. Members of the genus Salmonella are broadly distributed, and wild boar may play an important role in its circulation between peri-urban areas and the environment, due to its frequent interactions both with livestock or human garbage. As the population of these animals is rising due to management on certain hunting estates or the absence of natural predators, the aim of the present work is to identify the mechanisms of AMR present and/or expressed in Salmonella spp. from wild boar populations and to determine the possible role of management-related factors applied to different game estates located in central Spain. The detection of Salmonella spp. was carried out in 121 dead wild boar from 24 game estates, and antimicrobial resistance traits were determined by antibiotic susceptibility testing and screening for their genetic determinants. The effects of feeding supplementation, the proximity of livestock, the existence of a surrounding fence and the density of wild boar on the AMR of the isolates were evaluated. The predominant subspecies and serovar found were S. enterica subsp. enterica (n = 69) and S. choleraesuis (n = 33), respectively. The other subspecies found were S. enterica subsp. diarizonae, S. enterica subsp. salamae and S. enterica subsp. houtenae. AMR was common among isolates (75.2%) and 15.7% showed multi drug resistance (MDR). Resistance to sulphonamides was the most frequent (85.7%), as well as sul1 which was the AMR determinant most commonly found. Plasmids appeared in 38.8% of the isolates, with IncHI1 being the replicon detected with the highest prevalence. The AMR of the isolates increased when the animals were raised with feeding supplementation and enclosed by fences around the estates.

The D165H Polymorphism of QiMYB-like-1 Is Linked to Interactions between Tannin Accumulation, Herbivory and Biogeographical Determinants of Quercus ilex.

The accumulation in the leaves and young stems of phenolic compounds, such as hydrolyzable and condensed tannins, constitutes a defense mechanism of plants against herbivores. Among other stressing factors, chronic herbivory endangers Quercus ilex, a tree playing a central role in Mediterranean forests. This work addressed the connections between the chemical defenses of Q. ilex leaves and their susceptibility to herbivory, quantitative traits whose relationships are modulated by environmental and genetic factors that could be useful as molecular markers for the selection of plants with improved fitness. A search for natural variants detected the polymorphism D165H in the effector domain of QiMYB-like-1, a TT2-like transcription factor whose family includes members that control the late steps of condensed tannins biosynthesis in different plant species. QiMYB-like-1 D165H polymorphism was screened by PCR-RFLP in trees from six national parks in Spain where Q. ilex has a relevant presence, revealing that, unlike most regions that match the Hardy-Weinberg equilibrium, homozygous plants are over-represented in "Monfragüe" and "Cabañeros", among the best examples to represent the continental Mediterranean (cM) ecosystem. Accordingly, the averages of two stress-related quantitative traits measured in leaves, herbivory index and accumulation of condensed tannins, showed asymmetric distributions depending on the clustering of trees based on ecological and genetic factors. Thus, the impact of herbivory was greater in managed forests with a low density of trees from the cM region, among which QiMYB-like-1 D165 homozygotes stand out, whereas condensed tannins accumulation was higher in leaves of QiMYB-like-1 H165 homozygotes from low-density forests, mainly in the Pyrenean (Py) region. Besides, the correlation between the contents of condensed tannins and total tannins vanished after clustering by the same factors: the cM region singularity, forest tree density, and QiMYB-like-1 genotype, among which homozygous shared the lowest link. The biogeographical and genetic constraints that modulate the contribution of condensed tannins to chemical defenses also mediated their interactions with the herbivory index, which was found positively correlated with total phenolics or tannins, suggesting an induction signal by this biotic stress. In contrast, a negative correlation was observed with condensed tannins after tree clustering by genetics factors where associations between tannins were lost. Therefore, condensed tannins might protect Q. ilex from defoliation in parks belonging to the cM ecosystem and carrying genetic factor(s) linked to the QiMYB-like-1 D165H polymorphism.

Colistin Selection of the Mcr-1 Gene in Broiler Chicken Intestinal Microbiota.

Colistin has a long story of safe use in animals for the treatment and prevention of certain bacterial diseases. Nevertheless, the first description of the mcr-1 gene showed that colistin resistance can spread by horizontal gene transfer and changed the landscape. This study aimed to assess the effect of colistin administration on the dispersion of resistance in the microbiota of day-old broiler chicks and how the presence of mcr-1 genes influences the spread of colistin resistance determinants. In this study, 100 one-day-old chicks were divided into four groups of 25 animals (G1, G2, G3, and G4). Animals from G3/G4 were challenged with mcr-1-carrying Salmonella (day 7), while colistin (600 mg/L) was administered daily to G2/G4 animals through drinking water (from day 8 to day 15). Two quantitative PCR assays were performed to compare the amount of Salmonella and mcr-1 that were present in the caecal samples. We observed that levels of mcr-1 were higher in G3/G4 animals, especially G4, due to the spread of mcr-1-carrying Salmonella. On day 21, Salmonella levels decreased in G4, reaching similar values as those for G3, but mcr-1 levels remained significantly higher, suggesting that colistin may accelerate the spreading process when mcr-1-carrying bacteria reach the gut.

Smartphone App (2kmFIT-App) for Measuring Cardiorespiratory Fitness: Validity and Reliability Study.

BACKGROUND: There is strong evidence suggesting that higher levels of cardiorespiratory fitness (CRF) are associated with a healthier metabolic profile, and that CRF can serve as a powerful predictor of morbidity and mortality. In this context, a smartphone app based on the 2-km walk test (UKK test) would provide the possibility to assess CRF remotely in individuals geographically distributed around a country or continent, and even between continents, with minimal equipment and low costs. OBJECTIVE: The overall aim of this study was to evaluate the validity and reliability of 2kmFIT-App developed for Android and iOS mobile operating systems to estimate maximum oxygen consumption (VO2max) as an indicator of CRF. The specific aims of the study were to determine the validity of 2kmFIT-App to track distance and calculate heart rate (HR). METHODS: Twenty participants were included for field-testing validation and reliability analysis. The participants completed the UKK test twice using 2kmFIT-App. Distance and HR were measured with the app as well as with accurate methods, and VO2max was estimated using the UKK test equation. RESULTS: The validity results showed the following mean differences (app minus criterion): distance (-70.40, SD 51.47 meters), time (-0.59, SD 0.45 minutes), HR (-16.75, SD 9.96 beats/minute), and VO2max (3.59, SD 2.01 ml/kg/min). There was moderate validity found for HR (intraclass correlation coefficient [ICC] 0.731, 95% CI -0.211 to 0.942) and good validity found for VO2max (ICC 0.878, 95% CI -0.125 to 0.972). The reliability results showed the following mean differences (retest minus test): app distance (25.99, SD 43.21 meters), app time (-0.15, SD 0.94 seconds), pace (-0.18, SD 0.33 min/km), app HR (-4.5, 13.44 beats/minute), and app VO2max (0.92, SD 3.04 ml/kg/min). There was good reliability for app HR (ICC 0.897, 95% CI 0.742-0.959) and excellent validity for app VO2max (ICC 0.932, 95% CI 0.830-0.973). All of these findings were observed when using the app with an Android operating system, whereas validity was poor when the app was used with iOS. CONCLUSIONS: This study shows that 2kmFIT-App is a new, scientifically valid and reliable tool able to objectively and remotely estimate CRF, HR, and distance with an Android but not iOS mobile operating system. However, certain limitations such as the time required by 2kmFIT-App to calculate HR or the temperature environment should be considered when using the app.

Complementarity of Selective Culture and qPCR for Colistin Resistance Screening in Fresh and Frozen Pig Cecum Samples.

Retrospective studies involving the screening of frozen stored collections of samples are commonplace when a new threat emerges, but it has been demonstrated that the freeze-thaw process can affect bacterial viability. The study of colistin-resistant bacteria in human and animal samples is an example of this issue. In this study, we compared culture-based and PCR-based methods for analyzing relative occurrence and diversity of colistin-resistant bacteria in caecal samples to determine the most appropriate method for frozen samples. Thus, 272 samples from the caecal contents of healthy pigs were tested before and after a 6-month freezing period. A selective medium was used when traditional isolation of colistin-resistant bacteria was tested, while a real-time SYBR® Green I PCR assay was applied for mcr-1 quantification. The number of samples with colistin-resistant isolates was higher in fresh samples (247/272) than in frozen ones (67/272) and showed a higher diversity of colistin-resistant genera. PCR identification of mcr colistin resistance genes evidenced that mcr-1 was the most prevalent mcr gene and mcr-2 was detected for the first time in pigs from Spanish animal production. The number of samples with mcr-1-carrying bacteria after a freezing period decreased, while real-time quantitation of the mcr-1 gene showed similar values in frozen and fresh samples. Therefore, when frozen cecal samples need to be analyzed, molecular detection of DNA could be the best option to provide a highly representative frame of the initial population present in the sample, and culture-based methods might be a useful complement to study colistin resistance levels.

Occurrence of tet(O/M/O) Mosaic Gene in Tetracycline-Resistant Campylobacter.

Campylobacter is one of the most important microorganisms responsible for foodborne diseases in the EU. In this study, we investigated resistance to tetracycline in 139 Campylobacter jejuni and Campylobacter coli samples isolated from human clinical cases. From these, 110 were resistant to tetracycline, with MIC (minimal inhibitory concentration) varying in a range of 1 to >512 µg/mL, and 109 (78.4%) carried tet(O), a gene that confers resistance to tetracycline through the expression of a protein that confers protection to the ribosome. Amongst the tetracycline-resistant isolates, one C. jejuni (HCC30) was the only tet(O)-negative sample, presenting an MIC of 256 µg/mL. Instead, the mosaic gene tet(O/M/O) was found in HCC30 and, as far as we know, this is the first description of this chimeric gene originating from homologous recombination between tet(O) and tet(M). The previously described mosaic gene tet(O/32/O), also found in Campylobacter, presents a chimeric structure very similar to that of tet(O/M/O), affecting domains II and III of encoded proteins distantly related to the elongation factor G (EF-G). The tet(O/M/O) mosaic gene has been found in nucleotide databases in several genomes of Campylobacter isolated from different origins, indicating its frequent acquisition, even though it can be undetected through screening by PCR with specific tet(O) primers. In this work, we address the improvement of classical PCR to efficiently diagnose the most prevalent tetracycline resistance determinants in Campylobacter, including tet(O/M/O), which should be taken into account in the optimization of campylobacteriosis treatments.

Spread of Antimicrobial Resistance by Salmonella enterica Serovar Choleraesuis between Close Domestic and Wild Environments.

The Salmonellaenterica serovar Choleraesuis affects domestic pig and wild boar (WB), causing clinical salmonellosis. Iberian swine production is based on a free-range production system where WB and Iberian pig (IP) share ecosystems. This study focuses on the negative impact on the pork industry of infections due to this serotype, its role in the spread of antibiotic resistance, and its zoonotic potential. Antibiotic resistance (AR) and genetic relationships were analyzed among 20 strains of S. Choleraesuis isolated from diseased WB and IP sampled in the southwest region of the Iberian Peninsula. AR was studied using the Kirby-Bauer method with the exception of colistin resistance, which was measured using the broth microdilution reference method. Resistance and Class 1 integrase genes were measured using PCR, and the genetic relationship between isolates and plasmid content by pulsed field gel electrophoresis. The results show a higher incidence of AR in isolates from IP. Phylogenetic analysis revealed seven profiles with two groups containing isolates from IP and WB, which indicates circulation of the same clone between species. Most pulsotypes presented with one plasmid of the same size, indicating vertical transmission. AR determinants blaTEM and tetA were routinely found in IP and WB, respectively. One isolate from IP expressed colistin resistance and presented the mcr-1 gene carried by a plasmid. This study suggests that S. Choleraesuis circulates between WB and IP living in proximity, and also that the mobilization of AR genes by plasmids is low. Furthermore, the detection of plasmid-mediated colistin resistance in bacteria from IP is alarming and should be monitored.

Involvement of hpap2 and dgkA Genes in Colistin Resistance Mediated by mcr Determinants.

Plasmid-mediated colistin resistance (mcr) determinants are challenging the efficacy of polymyxins against Gram-negative pathogens. Among 10 mcr genes described so far, the major determinants mcr-1 and mcr-3 are found closely linked to hpap2 or dgkA genes, encoding a hypothetical phosphatidic acid phosphatase of type 2 (PAP2) and a diacylglycerol kinase, respectively, whose functions are still unknown. In this study, mcr-1, mcr-1-hpap2, mcr-3, and mcr-3-dgkA were expressed in Escherichia coli, and recombinant strains were analyzed to detect antimicrobial susceptibility and changes in the expression of genes involved in phospholipid metabolism. The mcr-1 or mcr-3 single genes were enough to drive growth on colistin selective media, although co-expression of linked genes conferred maximal antibiotic resistance. Expression of mcr determinants downregulated endogenous genes involved in lipopolysaccharide (LPS) modification or phospholipid recycling, although to different extents of repression: strong for arnB, ybjG, and pmrR; medium for eptA, lpxT, and dgkA; small for bacA and pgpB. Four of these genes (bacA, lpxT, pgpB, and ybjG) encode undecaprenyl pyrophosphate (UPP) phosphatases. In these conditions, cells presented resistance against bacitracin, an antibiotic that sequesters UPP from PAP2 enzymes. The hpap2 and dgkA genes might play a role in colistin resistance by compensating for phospholipid metabolism functions altered during LPS modification by colistin resistance determinants.

Carbapenemase-Producing Elizabethkingia Meningoseptica from Healthy Pigs Associated with Colistin Use in Spain.

Carbapenems are considered last-resort antimicrobials, especially for treating infections involving multidrug-resistant Gram-negative bacteria. In recent years, extended-spectrum ß-lactamase (ESBL) and carbapenemase-producing Gram-negative bacteria have become widespread in hospitals, community settings, and the environment, reducing the range of effective therapeutic alternatives. The use of colistin to treat infection caused by these multi-drug bacteria may favour the selection and persistence of carbapenem-resistant bacteria. In this study, it is described, for the first time to our knowledge, a carbapenemase-producing isolate of Elizabethkingia meningoseptica from healthy pigs in Spain. The isolate we report was recovered during a study to detect colistin-resistant bacteria from faecal samples of healthy food-production animals using a chromogenic selective medium. Unexpectedly, we found an isolate of Elizabethkingia meningoseptica with high Minimum Inhibitory Concentration (MIC) values for several antibiotics tested. Molecular analysis did not show any mcr family genes related with colistin resistance, but two carbapenemase genes, blaB-12_1 and blaGOB-17_1, were detected. This finding in healthy animals could suggest that colistin may favour the selection and persistence of carbapenem-resistant bacteria.

Nasal shedding of Mycobacterium tuberculosis in wild boar is related to generalised tuberculosis and concomitant infections.

BACKGROUND: Wild boar is an important reservoir of Mycobacterium tuberculosis variant bovis, the main causative agent of bovine tuberculosis (bTB). A proportion of tuberculosis (TB)-affected wild boars shed M tuberculosis by nasal route, favouring the maintenance of bTB in a multihost scenario. The aim of this work was to assess if M tuberculosis nasal excretion is influenced by factors commonly associated with high TB prevalence in wild boar. METHODS: TB diagnosis and M tuberculosis isolation were carried out in 112 hunted wild boars from mid-western Spain. The association between the presence of M tuberculosis DNA in nasal secretions and explanatory factors was explored using partial least squares regression (PLSR) approaches. RESULTS: DNA from M tuberculosis was detected in 40.8 per cent nasal secretions of the TB-affected animals. Explanatory factors provided a first significant PLSR X's component, explaining 25.70 per cent of the variability observed in M tuberculosis nasal shedding. The presence of M tuberculosis in nasal secretions is more probable in animals suffering from generalised TB and mainly coinfected with Metastrongylus species and porcine circovirus type 2, explaining nearly 90 per cent of the total variance of this model. CONCLUSION: Measures aiming to control these factors could be useful to reduce M tuberculosis shedding in wild boar.

Gene pool transmission of multidrug resistance among Campylobacter from livestock, sewage and human disease.

The use of antimicrobials in human and veterinary medicine has coincided with a rise in antimicrobial resistance (AMR) in the food-borne pathogens Campylobacter jejuni and Campylobacter coli. Faecal contamination from the main reservoir hosts (livestock, especially poultry) is the principal route of human infection but little is known about the spread of AMR among source and sink populations. In particular, questions remain about how Campylobacter resistomes interact between species and hosts, and the potential role of sewage as a conduit for the spread of AMR. Here, we investigate the genomic variation associated with AMR in 168 C. jejuni and 92 C. coli strains isolated from humans, livestock and urban effluents in Spain. AMR was tested in vitro and isolate genomes were sequenced and screened for putative AMR genes and alleles. Genes associated with resistance to multiple drug classes were observed in both species and were commonly present in multidrug-resistant genomic islands (GIs), often located on plasmids or mobile elements. In many cases, these loci had alleles that were shared among C. jejuni and C. coli consistent with horizontal transfer. Our results suggest that specific antibiotic resistance genes have spread among Campylobacter isolated from humans, animals and the environment.

Regulation by biotic stress of tannins biosynthesis in Quercus ilex: Crosstalk between defoliation and Phytophthora cinnamomi infection.

Sustainability of the Mediterranean forest is threatened by oak decline, a disease of holm oak and other Quercus species that is initiated by infection with the oomycete Phytophthora cinnamomi. Focusing on the role of tannins in the chemical defense of plants, this work investigated whether tannins content in Quercus ilex is regulated by biotic stress. Screening of published genomes allowed the identification of Quercus sequences encoding enzymes for early steps of the biosynthesis of phenolic compounds, like hydrolysable tannins and condensed tannins (CT) among others, plus genes involved in the late steps of CT biosynthesis. Four days after treatment of Q. ilex seedlings by mechanical defoliation, P. cinnamomi infection and both stressors simultaneously, mRNA concentrations for tannins biosynthesis enzymes were measured in leaves. Among the transcript amount for shikimate dehydrogenase (SDH, EC 1.1.1.25), anthocyanidin reductase (EC 1.3.1.77), anthocyanidin synthase (EC 1.14.11.19) and leucoanthocyanidine reductase (EC 1.17.1.3), defoliation induced gene expression for SDH2 isoenzyme. About 4 days after infection of roots by P. cinnamomi, this up-regulation was canceled and SDH enzyme activity decreased. Furthermore, during this late stage of biotrophic interaction the pathogen switched off the correlation engaged by defoliation between the expression of SDH1 and SDH2 encoding genes and chemical defenses corresponding to total tannins, which were down-regulated. Thus, tannins biosynthesis in seedlings of Q. ilex is induced after mechanical defoliation whereas infection by the pathogen interferes with this regulation, potentially increasing the susceptibility of plants to herbivory and aggravating the impact of biotic stress.

ant(6)-I Genes Encoding Aminoglycoside O-Nucleotidyltransferases Are Widely Spread Among Streptomycin Resistant Strains of Campylobacter jejuni and Campylobacter coli.

Thermotolerant Campylobacter species C. jejuni and C. coli are actually recognized as the major bacterial agent responsible for food-transmitted gastroenteritis. The most effective antimicrobials against Campylobacter are macrolides and some, but not all aminoglycosides. Among these, susceptibility to streptomycin is reduced by mutations in the ribosomal RPSL protein or by expression of ANT(6)-I aminoglycoside O-nucleotidyltransferases. The presence of streptomycin resistance genes was evaluated among streptomycin-resistant Campylobacter isolated from humans and animals by using PCR with degenerated primers devised to distinguish ant(6)-Ia, ant(6)-Ib and other ant-like genes. Genes encoding ANT(6)-I enzymes were found in all possible combinations with a major fraction of the isolates carrying a previously described ant-like gene, distantly related and belonging to the new ant(6)-I sub-family ant(6)-Ie. Among Campylobacter isolates, ant(6)-Ie was uniquely found functional in C. coli, as shown by gene transfer and phenotype expression in Escherichia coli, unlike detected coding sequences in C. jejuni that were truncated by an internal frame shift associated to RPSL mutations in streptomycin resistant strains. The genetic relationships of C. coli isolates with ANT(6)-Ie revealed one cluster of strains presented in bovine and humans, suggesting a circulation pathway of Campylobacter strains by consuming contaminated calf meat by bacteria expressing this streptomycin resistance element.

Genome Comparison of Erythromycin Resistant Campylobacter from Turkeys Identifies Hosts and Pathways for Horizontal Spread of erm(B) Genes.

Pathogens in the genus Campylobacter are the most common cause of food-borne bacterial gastro-enteritis. Campylobacteriosis, caused principally by Campylobacter jejuni and Campylobacter coli, is transmitted to humans by food of animal origin, especially poultry. As for many pathogens, antimicrobial resistance in Campylobacter is increasing at an alarming rate. Erythromycin prescription is the treatment of choice for clinical cases requiring antimicrobial therapy but this is compromised by mobility of the erythromycin resistance gene erm(B) between strains. Here, we evaluate resistance to six antimicrobials in 170 Campylobacter isolates (133 C. coli and 37 C. jejuni) from turkeys. Erythromycin resistant isolates (n = 85; 81 C. coli and 4 C. jejuni) were screened for the presence of the erm(B) gene, that has not previously been identified in isolates from turkeys. The genomes of two positive C. coli isolates were sequenced and in both isolates the erm(B) gene clustered with resistance determinants against aminoglycosides plus tetracycline, including aad9, aadE, aph(2″)-IIIa, aph(3')-IIIa, and tet(O) genes. Comparative genomic analysis identified identical erm(B) sequences among Campylobacter from turkeys, Streptococcus suis from pigs and Enterococcus faecium and Clostridium difficile from humans. This is consistent with multiple horizontal transfer events among different bacterial species colonizing turkeys. This example highlights the potential for dissemination of antimicrobial resistance across bacterial species boundaries which may compromise their effectiveness in antimicrobial therapy.

Co-occurrence of colistin-resistance genes mcr-1 and mcr-3 among multidrug-resistant Escherichia coli isolated from cattle, Spain, September 2015.

Colistin resistance genes mcr-3 and mcr-1 have been detected in an Escherichia coli isolate from cattle faeces in a Spanish slaughterhouse in 2015. The sequences of both genes hybridised to same plasmid band of ca 250 kb, although colistin resistance was non-mobilisable. The isolate was producing extended-spectrum beta-lactamases and belonged to serotype O9:H10 and sequence type ST533. Here we report an mcr-3 gene detected in Europe following earlier reports from Asia and the United States.