María Ugarte-Ruiz

Detection of plasmid mediated colistin resistance (MCR-1) in Escherichia coli and Salmonella enterica isolated from poultry and swine in Spain.

Recent findings suggest that use of colistin as a last resort antibiotic is seriously threatened by the rise of a new plasmid mediated mechanism of resistance (MCR-1). This work identifies, for the first time in Southern Europe, the gene mcr-1 in nine strains from farm animals (poultry and swine) corresponding to five Escherichia coli and four Salmonella enterica, among which three belong to serovar Typhimurium and one to Rissen. The MCR-1 was found encoded by a plasmid highly mobilizable by conjugation to the E. coli J53 strain. Two E. coli strains carried two determinants, mcr-1 plus pmrA or pmrB mutations, known to confer colistin resistance.

Evaluation of four protocols for the detection and isolation of thermophilic Campylobacter from different matrices

Aims:  To identify the optimal method for detection of thermophilic Campylobacter at various stages in the food chain, three culture‐dependent (direct plating, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n = 38), neck skin (n = 38) and packed fresh meat (n = 38).

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.

Description of an erm(B)-carrying Campylobacter coli isolate in Europe

Sir, Campylobacter jejuni and Campylobacter coli are the principal cause of foodborne zoonoses in humans. Campylobacteriosis is caused by C. jejuni and C. coli, and the infection is produced by ingesting contaminated food. The main reservoir is the intestinal microbiota of birds, especially poultry. Although the treatment for campylobacteriosis is generally oral rehydration therapy, the drugs of choice are macrolides, such as erythromycin. Thus, macrolide resistance poses a serious public health threat. Bacteria carrying the erm(B) gene are resistant to macrolides, lincosamide and streptogramin B (MLSB phenotype) via methylation of the 23S rRNA gene. The erm(B) gene has been described in highly erythromycin-resistant Campylobacter isolates in China, located in an MDR genomic island (MDRGI). Transference to erythromycin-susceptible C. jejuni by transformation has been observed. The aims of our study were to detect erm(B) in erythromycin-resistant isolates of our collection and to compare these findings with the presence of other known mechanisms of erythromycin resistance (target mutations of the 23S rRNA gene and ribosomal proteins L4 and L22, and antibiotic efflux pumps). Of 555 isolates obtained from food animals in Spain during 2008–11, 74 isolates with an erythromycin MIC ≥32 mg/L (broth microdilution by UNE-EN ISO20776 –1) were randomly selected (broiler: n1⁄429; fattening pigs: n1⁄434; young cattle: n1⁄411). Additionally, 14 Campylobacter isolates from urban effluents (MIC ≥32 mg/L) were also selected. Isolates were not epidemiologically related. Agar dilution susceptibility testing (CLSI methodology) was performed to increase the antimicrobial range (0.25– 1024 mg/L). The assay was carried out using Mueller –Hinton 2 agar supplemented with erythromycin and 5% lysed horse blood. Isolates with the highest level of resistance (MIC ≥1024 mg/L) were tested to detect the erm(B) gene as well as target mutations in the 23S gene, target modifications in ribosomal proteins L4 and L22 and the effect of PABN as an efflux pump inhibitor. – 9 WGS (Ion Torrent PGM) was performed to determine the location of the methylase gene erm(B) and its genetic environment. Raw data were aligned to C. coli RM4661 (NZ_CP007181) using CLC Genomics Workbench 7.5.1. The BLAST application was used to study similarities. Five isolates of 88 revealed a high level of resistance to erythromycin (MIC ≥1024 mg/L), all of them C. coli isolated from broilers. Only the C. coli ZTA09/02204 isolate was positive for the erm(B) gene; additional erythromycin-resistance mechanisms were not detected. The C. coli ZTA09/02204 isolate presented resistance to nalidixic acid, ciprofloxacin, tetracycline and streptomycin, and was susceptible to gentamicin (data not shown). The MLSB phenotype could not be confirmed, as lincosamide and streptogramin B were not included in our panel. No effect of PABN was observed on the MIC for isolate ZTA09/ 02204, which suggests that the active efflux mediated by CmeABC makes no contribution to the phenotype. Although overexpression of cmeABC could have led to the results, previous studies describe no synergy between methylase erm(B) gene and efflux pumps. The erm(B) gene is 738 bp and shows 100% nucleotide identity to erm(B) of Enterococcus faecium e389 (JN899587), Clostridium difficile 200785596 (FN665654) and Streptococcus suis 2 – 22 (EU047808). When the erm(B) gene from this study is compared with the erm(B) gene described in Campylobacter, the nucleotide identity is 99% (G299A). The erm(B) gene is located in the chromosome of C. coli ZTA09/ 02204 within a gene cluster with other antimicrobial resistance genes constituting an MDRGI (Figure 1). The MDRGI (GenBank accession no. KT953380) is 11769 bp, and it was inserted in one hypothetical protein (YSS_00750 gene). The average guanine and cytosine content is 38.5%, higher than the guanine and cytosine content in the genome (31.1%). The MDRGI contains 12 ORFs, five of them antibiotic resistance genes (Table S1, available as Supplementary data at JAC Online). The MDRGI of C. coli ZTA09/ 02204 seems to be a new type, as the organization of genes and its location in the chromosome differ from those in other types. Therefore, the MDRGI of this study might be designated type VIII. When the MDRGI C. coli ZTA09/02204 is compared with those already described, its similarity is higher to types V, VI and VII, as they present a complete tet(O) coding region. However, our isolate contains a full-length pnp gene, in contrast to the truncated forms carried by types V to VII (Figure 1). The different structures of the ZTA09/02204 MDRGI and the MDRGI previously published likely reflect a different origin. According to the guanine and cytosine percent content, the MDRGI have been divided into three different regions (Figure 1 and Table S1). Region B had 99% identity with Eggerthella sp. YY918 (AP012211) and Bacteroides uniformis WH207 (AY345595). Regions A and C presented 99% identity with plasmid pN29710– 1 from C. coli CVM N29710 (CP004067) (Figure 1), which would suggest a plasmid as the insertion vehicle of the erm(B) gene

The effect of different isolation protocols on detection and molecular characterization of Campylobacter from poultry

We determined whether different methods to isolate Campylobacter (including the ISO standard 10272:2006‐1) affected the genotypes detectable from poultry, at three points during slaughter: caecal content, neck skin and meat. Carcasses from 28 independent flocks were thus sampled (subset A). In addition, ten neck skin samples from four flocks, ten caecal samples from ten different flocks and ten unrelated meat samples obtained from local supermarkets were collected (subset B). Campylobacter was isolated using eight different protocols: with and without enrichment using Bolton broth, Preston broth or Campyfood broth (CFB), followed by culture on either modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA) or Campyfood agar (CFA). All obtained isolates were genotyped for flaA‐SVR, and over half of the isolates were also typed by MLST. The strain richness, as a measure of number of detected fla‐genotypes, obtained from subset A neck skin and caecal samples was higher than that of meat samples. In half of the cases, within a flock, at least one identical fla‐genotype was obtained at all three slaughter stages, suggestive of autologous contamination of carcasses. Enrichment reduced the observed richness of isolates, while CFA plates increased richness compared to mCCDA plates, irrespective of inclusion of an enrichment step. Because the isolation protocol used influences both the yield and the fla‐genotype richness obtained from poultry, this variable should be taken into account when different studies are being compared.

Prevalence of Type VI Secretion System in Spanish Campylobacter jejuni Isolates

Infections from Campylobacter jejuni pose a serious public health problem and are now considered the leading cause of foodborne bacterial gastroenteritis throughout the world. Sequencing of C. jejuni genomes has previously allowed a number of loci to be identified, which encode virulence factors that aid survival and pathogenicity. Recently, a Type VI secretion system (T6SS) consisting of 13 conserved genes was described in C. jejuni strains and recognised to promote pathogenicity and adaptation to the environment. In this study, we determined the presence of this T6SS in 63 Spanish C. jejuni isolates from the food chain and urban effluents using whole‐genome sequencing. Our findings demonstrated that nine (14%) strains harboured the 13 ORFs found in prototype strain C. jejuni 108. Further studies will be necessary to determine the prevalence and importance of T6SS‐positive C. jejuni strains.

Method Comparison for Enhanced Recovery, Isolation and Qualitative Detection of C. jejuni and C. coli from Wastewater Effluent Samples

Seeking a sensitive protocol, culture-dependent methods were compared to detect thermophilic Campylobacter species in untreated urban effluents. We evaluated various combinations of selective media, with and without an enrichment steps, as well as an extra filtration step. Culture-independent real-time quantitative PCR was also included and all detected isolates underwent antimicrobial susceptibility testing. All tested water samples contained Campylobacter DNA, but only 64% were positive after culture. Although enrichment using Preston broth resulted in better recovery of potentially stressed Campylobacter than Bolton or Campyfood broth (CFB), there was no significant increase in efficiency compared to direct plating. The type of selective agar media used, on the other hand, had a significant effect, with CASA plates performing better than mCCDA or CFA ones. Inclusion of an enrichment step increased the ratio of C. coli vs. C. jejuni being isolated. Resistances against all antimicrobials tested were observed in C. coli, but fewer instances of resistance were found in C. jejuni isolates. Whether this difference was the result of selection during the enrichment step could not be determined. The presence of Campylobacter in urban effluents can be considered as a valuable proxy for Campylobacter populations present in urban environments.

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.

Identification of the main quinolone resistance determinant in Campylobacter jejuni and Campylobacter coli by MAMA-DEG PCR.

Among zoonotic diseases, campylobacteriosis stands out as the major bacterial infection producing human gastroenteritis. Antimicrobial therapy, only recommended in critical cases, is challenged by resistance mechanisms that should be unambiguously detected for achievement of effective treatments. Quinolone (ciprofloxacin) resistance of Campylobacter jejuni and Campylobacter coli, the 2 main Campylobacter detected in humans, is conferred by the mutation gyrA C-257-T, which can be genotyped by several methods that require a previous identification of the pathogen species to circumvent the sequence polymorphism of the gene. A multiplex PCR, based on degenerated oligonucleotides, has been designed for unambiguous identification of the quinolone resistance determinant in Campylobacter spp. isolates. The method was verified with 249 Campylobacter strains isolated from humans (141 isolates) and from the 3 most important animal sources for this zoonosis: poultry (34 isolates), swine (38 isolates), and cattle (36 isolates). High resistance to ciprofloxacin, MIC above 4μg/mL, linked to the mutated genotype predicted by MAMA-DEG PCR (mismatch amplification mutation assay PCR with degenerated primers) was found frequently among isolates from the different hosts.

Escherichia coli ST167 carrying plasmid mobilisable mcr-1 and bla CTX-M-15 resistance determinants isolated from a human respiratory infection

Please cite this article as: Rosario Sánchez-Benito, M. Rocío Iglesias, Narciso M. Quijada, Maria J. Campos, María Ugarte-Ruiz, Marta Hernández, Carmen Pazos, David Rodríguez-Lázaro, Eugenio Garduño, Lucas Domínguez, Alberto Quesada, Escherichia coli ST167 carrying plasmid mobilisable mcr-1 and blaCTX-M-15 resistance determinants isolated from a human respiratory infection, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.05.005.