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.

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.

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.

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.

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.

Dietary supplementation with fermented defatted "alperujo" induces modifications of the intestinal mucosa and cecal microbiota of broiler chickens.

Nutraceuticals are not only nutritionally beneficial for animals but also their use as feed supplements may reduce environmental contamination. The effect of fermented defatted "alperujo," an olive oil by-product, supplementation on the intestinal health of broiler chickens was assessed by analyzing the intestinal mucosal morphology of the duodenum and the cecum. The microbiota of the cecum was also characterized by analyzing the V3-V4 region of the 16S rRNA gene on days 7, 14, 21, 28, 35, and 42. Supplemented broilers from 14 to 35 D of age showed an increase in villus height in the duodenum. This increase likely improved digestibility and absorption capacity during growth, leading to the observed increase in BW at day 35 of life. A progressive increase in crypt depth in both the duodenum and the cecum was also observed. This modification likely enhanced epithelial renewal, thus safeguarding the turnover capacity of the intestinal mucosa. Our molecular analysis of cecal microbiota suggests that this dietary supplement may favor the growth of certain bacteria and may control the spread of pathogenic bacteria by means of competitive exclusion.

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.

Reduction of Salmonella Typhimurium Cecal Colonisation and Improvement of Intestinal Health in Broilers Supplemented with Fermented Defatted 'Alperujo', an Olive Oil By-Product.

Salmonella spp. contaminates egg and poultry meat leading to foodborne infections in humans. The emergence of antimicrobial-resistant strains has limited the use of antimicrobials. We aimed to determine the effects of the food supplement, fermented defatted 'alperujo' (FDA), a modified olive oil by-product, on Salmonella Typhimurium colonisation in broilers. One hundred and twenty 1-day-old broilers were divided into four experimental groups-two control groups and two treated groups, and challenged with S. Typhimurium at day 7 or 21. On days 7, 14, 21, 28, 35, and 42 of life, duodenum and cecum tissue samples were collected for histopathological and histomorphometric studies. Additionally, cecum content was collected for Salmonella spp. detection by culture and qPCR, and for metagenomic analysis. Our results showed a significant reduction of Salmonella spp. in the cecum of 42-day-old broilers, suggesting that fermented defatted 'alperujo' limits Salmonella Typhimurium colonization in that cecum and may contribute to diminishing the risk of carcass contamination at the time of slaughter. The improvement of the mucosal integrity, observed histologically and morphometrically, may contribute to enhancing intestinal health and to limiting Salmonella spp. colonisation in the host, mitigating production losses. These results could provide evidence that FDA would contribute to prophylactic and therapeutic measures to reduce salmonellosis prevalence in poultry farms.

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.