Publications by authors named "Carla Andrea Alonso"

33 Publications

Frequency and Characterization of Antimicrobial Resistance and Virulence Genes of Coagulase-Negative Staphylococci from Wild Birds in Spain. Detection of -Carrying Isolates.

Microorganisms 2020 Aug 29;8(9). Epub 2020 Aug 29.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain.

The objective of this study was to determine the prevalence and diversity of coagulase-negative staphylococci (CoNS) species from wild birds in Spain, as well as to analyze the antimicrobial resistance phenotype/genotype and the virulence gene content. During 2015-2016, tracheal samples of 242 wild birds were collected in different regions of Spain for staphylococci recovery. The species identification was performed using MALDI-TOF. The antimicrobial resistance phenotype and genotype was investigated by the disk diffusion method and by PCR, respectively. The presence of the virulence genes /-PV, , , , and was investigated by PCR. Moreover, CoNS carrying the gene were subjected to SCC typing. Of the tested animals, 60% were CoNS-carriers, and 173 CoNS isolates were recovered from the 146 positive animals, which belonged to 11 species, with predominance of ( = 118) and ( = 25). A total of 34% of CoNS isolates showed a multidrug resistance phenotype, and 42 -positive methicillin-resistant CoNS (MRCoNS) were detected. The isolates showed resistance to the following antimicrobials (percentage of resistant isolates/antimicrobial resistance genes detected): penicillin (49/ , ), cefoxitin (24/ ), erythromycin and/or clindamycin (92/ (B), (C), (43), (A), (C), (A), (B), (A) and (A)), gentamicin and/or tobramycin (5/ (6')-Ie-(2″)-Ia, (4')-Ia), streptomycin (12/), tetracycline (17/ (K), (L), (M)), ciprofloxacin (4), chloramphenicol (1/ ), fusidic acid (86/ , ) and trimethoprim-sulfamethoxazole (1/ ). None of the isolates harbored the /-PV, , , and genes, but two isolates (1%) carried the gene. Wild birds are frequently colonized by CoNS species, especially . We identified scavenging on intensively produced livestock and feeding on landfills as risk factors for CoNS carriage. High proportions of MRCoNS and multidrug resistant CoNS were detected, which coupled with the presence of important virulence genes is of concern.
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http://dx.doi.org/10.3390/microorganisms8091317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564563PMC
August 2020

Mechanisms of Linezolid Resistance Among Enterococci of Clinical Origin in Spain-Detection of - and (D)-Carrying .

Microorganisms 2020 Jul 30;8(8). Epub 2020 Jul 30.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain.

The mechanisms of linezolid resistance among 13 and 6 isolates, recovered from six Spanish hospitals during 2017-2018, were investigated. The presence of acquired linezolid resistance genes and mutations in 23S rDNA and in genes encoding for ribosomal proteins was analyzed by PCR and amplicon sequencing. Moreover, the susceptibility to 18 antimicrobial agents was investigated, and the respective molecular background was elucidated by PCR-amplicon sequencing and whole genome sequencing. The transferability of the linezolid resistance genes was evaluated by filter-mating experiments. The gene was detected in all 13 isolates; and one -positive isolate also carried the recently described (D) gene. Moreover, one isolate displayed the nucleotide mutation G2576T in the 23S rDNA. This mutation was also present in all six isolates. All linezolid-resistant enterococci showed a multiresistance phenotype and harbored several antimicrobial resistance genes, as well as many virulence determinants. The gene was located upstream of the gene in 12 of the isolates. Moreover, an (A)-like gene was located downstream of in two isolates recovered from the same hospital. The gene was transferable in all but one isolates, in all cases along with the gene. The (D) gene was not transferable. The presence of and mutations in the 23S rDNA are the main mechanisms of linezolid resistance among and , respectively. We report the first description of the (D) gene in . The presence of the and (D) genes in Spanish hospitals is a public health concern.
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http://dx.doi.org/10.3390/microorganisms8081155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464793PMC
July 2020

Mechanisms of Linezolid Resistance Among Clinical spp. in Spain: Spread of Methicillin- and Linezolid-Resistant ST2.

Microb Drug Resist 2021 Feb 22;27(2):145-153. Epub 2020 May 22.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain.

This study aimed at determining the mechanisms of linezolid resistance and the molecular characteristics of clinical ( = 2) and coagulase-negative staphylococci ( = 15) isolates obtained from four Spanish hospitals. The detection of linezolid resistance mechanisms (mutations and acquisition of resistance genes) was performed by PCR/sequencing. The antimicrobial resistance and virulence profile was determined, and the isolates were typed by different molecular techniques. Moreover, the genetic environment of the gene was determined by whole-genome sequencing. The gene was detected in one methicillin-resistant (MRSA) that also displayed the amino acid change Val118Ala in the ribosomal protein L4. The second isolate was methicillin susceptible and showed different alterations in the ribosomal protein L4. All remaining linezolid-resistant ( = 14) and isolates ( = 1) showed the mutation G2576T ( = 14) or C2534T ( = 1) in the 23S rRNA. Moreover, different amino acid changes were detected in the ribosomal proteins L3 and L4 in isolates. All isolates belonged to the multilocus sequence type ST2. Linezolid-resistant staphylococci (LRS) showed a multiresistance phenotype, including methicillin resistance that was detected in all isolates but one, and was mediated by the gene. The gene in the MRSA isolate was located together with the gene on a conjugative 38,864 bp plasmid. Linezolid- and methicillin-resistant ST2 showing mutations in the 23S rRNA and in the ribosomal proteins L3 and L4 are spread among Spanish hospitals, whereas LRS carrying acquired linezolid resistance genes are rarely detected.
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http://dx.doi.org/10.1089/mdr.2020.0122DOI Listing
February 2021

Extended-Spectrum Beta-Lactamase-Producing Isolated from Healthy and Sick Dogs in Portugal.

Microb Drug Resist 2020 Jun 31;26(6):709-715. Epub 2019 Dec 31.

MicroART-Antibiotic Resistance Team, Department of Veterinary Sciences, Universidade de Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal.

Extended-spectrum beta-lactamase (ESBL)- and carbapenemase (CP)-producing isolates are a public health concern at clinical level, mainly in Southern European countries. However, there are scarce data on the role of companion animals in the emergence of resistance to clinically relevant antibiotics. Therefore, our study aimed to determine the presence of with relevant beta-lactamases in fecal samples from healthy dogs (kennel and house dogs) and sick dogs in seven different hospitals in Portugal. Fecal samples from 125 healthy dogs and 231 sick dogs (one per animal) were collected during April-August 2017. Samples were screened on MacConkey agar supplemented with meropenem, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was used for identification. Genotypic detection of ESBLs or CPs was carried out by PCR/sequencing. Moreover, the presence of other antimicrobial resistance genes and multilocus sequence typing was tested by PCR/sequencing. isolates were obtained from 16 tested samples (4.4%), and 3 of them were ertapenem and/or meropenem intermediate/resistant (all of them imipenem susceptible and negative for CP genes). Fifteen isolates were ESBL producers, and they carried the following beta-lactamase genes: + (four isolates, in three cases associated with ), + (five isolates, associated with TEM-1 in three cases), and + (six isolates). Three ESBL-producing isolates of different origins and beta-lactamase genotypes (CTX-M-15+SHV-28, CTX-M-15+SHV-28+TEM-1, or SHV-28+TEM-1) belonged to the lineage ST307, and one isolate was identified as ST15 (CTX-M-15+SHV-1). These findings highlight that dogs are frequent carriers of ESBL-producing isolates, harboring mostly genes encoding CTX-M-15 or SHV-28, associated in some cases with the high-risk clones ST307 and ST15.
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http://dx.doi.org/10.1089/mdr.2019.0205DOI Listing
June 2020

Corrigendum to "Molecular diversity and conjugal transferability of class 2 integrons among Escherichia coli isolates from food, animal and human sources" [International Journal of Antimicrobial Agents 51 (2018) 905-911].

Int J Antimicrob Agents 2019 12 16;54(6):834. Epub 2019 Nov 16.

Instituto de Microbiología y Parasitología Médica, Facultad de Medicina, Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Tecnológicas (IMPaM, UBA-CONICET), Ciudad Autónoma de Buenos Aires, Argentina. Electronic address:

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http://dx.doi.org/10.1016/j.ijantimicag.2019.10.016DOI Listing
December 2019

First Report of KPC-2 and KPC-3-Producing Enterobacteriaceae in Wild Birds in Africa.

Microb Ecol 2020 Jan 5;79(1):30-37. Epub 2019 May 5.

Laboratoire des Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Université de Tunis El Manar, 2092, Tunis, Tunisia.

The increased incidence of antibiotic-resistant Enterobacteriaceae is a public health problem worldwide. The aim of this study was to analyze the potential role of wild birds, given their capacity of migrating over long distances, in the spreading of carbapenemase, extended-spectrum β-lactamase (ESBL), and acquired-AmpC beta-lactamase-producing Enterobacteriaceae in the environment. Fecal and pellet samples were recovered from 150 wild birds in seven Tunisian regions and were inoculated in MacConkey-agar plates for Enterobacteriaceae recovery (one isolate/animal). Ninety-nine isolates were obtained and acquired resistance mechanisms were characterized in the five detected imipenem-resistant and/or cefotaxime-resistant isolates, by PCR and sequencing. The following ESBL, carbapenemase, and acquired-AmpC beta-lactamase genes were detected: bla (two Escherichia fergusonii and one Klebsiella oxytoca isolates), bla (one K. oxytoca), bla (one E. fergusonii), bla, and bla (two K. oxytoca, four E. fergusonii, and two E. coli). The IncFIIs, IncF, IncFIB, IncK, IncP, and IncX replicons were detected among these beta-lactamase Enterobacteriaceae producers. The bla, tetA, sul3, qnrB, and cmlA determinants were co-transferred by conjugation from K. oxytoca strain to E. coli J153, in association with IncK and IncF replicons. Our results support the implication of wild birds as a biological vector for carbapenemase, ESBL, and acquired-AmpC-producing Enterobacteriaceae.
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http://dx.doi.org/10.1007/s00248-019-01375-xDOI Listing
January 2020

Detection of MRSA of Lineages CC130-mecC and CC398-mecA and Staphylococcus delphini-lnu(A) in Magpies and Cinereous Vultures in Spain.

Microb Ecol 2019 Aug 29;78(2):409-415. Epub 2019 Jan 29.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006, Logroño, Spain.

The aim of this study was to determine the carriage rate of coagulase-positive staphylococci (CoPS) in wild birds and to characterize recovered isolates. Tracheal samples from 324 wild birds, obtained in different Spanish regions during 2015-2016, were screened for CoPS carriage. The antimicrobial resistance profile and the virulence gene content were investigated. Molecular typing was performed by spa, agr, MLST, SCCmec, and S. delphini group classification. CoPS were recovered from 26 samples of wild birds (8.3%), and 27 isolates were further characterized. Two CoPS species were detected: S. aureus (n = 15; eight cinereous vultures and seven magpies) and S. delphini (n = 12; 11 cinereous vultures and one red kite). Thirteen S. aureus were methicillin-resistant (MRSA) and the remaining two strains were methicillin-susceptible (MSSA). Twelve MRSA were mecC-positive, typed as t843-ST1583/ST1945/ST1581/ST1571 (n = 11) and t1535-ST1945 (n = 1) (all of clonal-complex CC130); they were susceptible to the non-β-lactams tested. The remaining MRSA strain carried the mecA gene, was typed as t011-ST398-CC398-agrI-SCCmec-V, and showed a multiresistance phenotype. MSSA isolates were ascribed to lineages ST97-CC97 and ST425-CC425. All S. aureus lacked the studied virulence genes (lukS/F-PV, tst, eta, etb, and etd), and the IEC type E (with scn and sak genes) was detected in four mecC-positive and one MSSA isolates. S. delphini strains were methicillin-susceptible but showed resistance to at least one of the antimicrobials tested, with high penicillin (75%, with blaZ gene) and tetracycline [58%, with tet(K)± tet(L)] resistance rates. All S. delphini isolates presented the virulence genes lukS-I, siet, and se-int, and four carried the clindamycin-resistance lnu(A) gene.
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http://dx.doi.org/10.1007/s00248-019-01328-4DOI Listing
August 2019

Identification of Enterococci, Staphylococci, and Enterobacteriaceae from Slurries and Air in and around Two Pork Farms.

J Food Prot 2018 11;81(11):1776-1782

2 Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain (ORCID: http://orcid.org/0000-0001-6873-1940 [S.S.]).

In this study, we investigated the airborne dissemination of bacteria from the inside of two very different pork farms (an intensively confined farm and an open-range farm) to the immediate environment. Samples were taken from the slurry, from the air inside the farms (area 0), and from their immediate surroundings at a distance of 50, 100, and 150 m in four directions (north, south, east, and west). A control sample in the air of a zone far away from human or animal activity was also taken. Identification of isolates was made by means of the matrix-assisted laser desorption-ionization time of flight system. A total of 1,063 isolates were obtained, of which a mere 7 came from the air of the control area. Staphylococci, enterococci, and Enterobacteriaceae were selectively targeted for isolation and represented 48.6, 27.2, and 21.6% of the isolates, respectively. The species identified from the air of surrounding areas ( Enterococcus faecalis, Enterococcus hirae, and Staphylococcus arlettae, mainly) were also present inside the farms studied. The results suggest that air is involved in bacterial dissemination, and pork farms should be considered a potential source of foodborne bacteria that might contaminate surrounding areas, including vegetable orchards. Wind direction appears as a factor involved in bacterial dispersion through the air, but its effect may be conditioned by existing vegetation and orographic conditions.
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http://dx.doi.org/10.4315/0362-028X.JFP-18-098DOI Listing
November 2018

Antimicrobial Resistance in spp. of animal origin.

Microbiol Spectr 2018 07;6(4)

Department of Microbiology, Ramón y Cajal University Hospital, Ramón y Cajal Health Research Institute (IRYCIS), Madrid, Spain.

Enterococci are natural inhabitants of the intestinal tract in humans and many animals, including food-producing and companion animals. They can easily contaminate the food and the environment, entering the food chain. Moreover, is an important opportunistic pathogen, especially the species and , causing a wide variety of infections. This microorganism not only contains intrinsic resistance mechanisms to several antimicrobial agents, but also has the capacity to acquire new mechanisms of antimicrobial resistance. In this review we analyze the diversity of enterococcal species and their distribution in the intestinal tract of animals. Moreover, resistance mechanisms for different classes of antimicrobials of clinical relevance are reviewed, as well as the epidemiology of multidrug-resistant enterococci of animal origin, with special attention given to beta-lactams, glycopeptides, and linezolid. The emergence of new antimicrobial resistance genes in enterococci of animal origin, such as and , is highlighted. The molecular epidemiology and the population structure of and isolates in farm and companion animals is presented. Moreover, the types of plasmids that carry the antimicrobial resistance genes in enterococci of animal origin are reviewed.
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http://dx.doi.org/10.1128/microbiolspec.ARBA-0032-2018DOI Listing
July 2018

Metallo-β-lactamases and class D carbapenemases in south-east Tunisia: Implication of mobile genetic elements in their dissemination.

Int J Antimicrob Agents 2018 Dec 15;52(6):871-877. Epub 2018 Jun 15.

Laboratoire des Microorganismes et Biomolécules Actives, Faculté des Sciences de Tunis, Université de Tunis El Manar, 2098 El-Manar II, Tunisia; Laboratoire de Recherche Sciences et Technologies de l'Environnement, Institut Supérieur des Sciences et Technologies de l'Environnement de Borj-Cedria, Université de Carthage, Technopôle de Borj-Cedria, BP-1003, Hammam-Lif, Tunisia. Electronic address:

Carbapenem resistance in Gram-negative bacteria constitutes a major clinical problem. We characterized molecular features among carbapenem-resistant Gram-negative clinical isolates collected from Southeastern Tunisian Island Hospital. Eighteen carbapenem-resistant clinical isolates (13 Klebsiella pneumoniae, 1 Proteus mirabilis, 1 Enterobacter cloacae, 3 Acinetobacter baumannii) were recovered during April 2015-August 2016. Molecular characterization of antimicrobial resistance was performed using polymerase chain reaction (PCR) and sequencing. Molecular typing of carbapenemase-producing K. pneumoniae was performed by pulsed-field gel electrophoresis (PFGE) after XbaI digestion and multilocus sequence typing (MLST). Conjugation experiments were conducted and type/number/size of plasmids were characterized by PCR-Based-Replicon-Typing and PFGE after S1 digestion. Carbapenemase genes were detected in K. pneumoniae [bla(8), bla+bla(1), bla(4)], P. mirabilis [bla(1)], E. cloacae [bla(1)] and A. baumannii [bla(3)]. K. pneumoniae isolates were typed as ST15, ST1412 and ST147 and showed seven different pulsotypes. The genetic structure surrounding bla was composed of ISAba125 and ble. The bla carried by E. cloacae was located within the variable region of a class1 integron and bla gene was inserted into Tn1999.2. IncA/C and IncFIIA replicons were implicated in dissemination of bla and a non-typeable 48.5 kb plasmid in the propagation of bla. The emergence of carbapenemase-producing Gram-negative species in a Tunisian hospital shows the need for preventive strategies and hygiene measures to minimize their spread. Although conjugative plasmids play an important role in rapid carbapenemase genes dissemination, other mobile genetic elements, such as insertion sequences, transposons and integrons, are involved in acquisition of these resistances.
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http://dx.doi.org/10.1016/j.ijantimicag.2018.06.002DOI Listing
December 2018

Detection and molecular characterisation of extended-spectrum β-lactamase-producing enteric bacteria from pigs and chickens in Nsukka, Nigeria.

J Glob Antimicrob Resist 2018 12 5;15:36-40. Epub 2018 Sep 5.

Biochemistry and Molecular Biology, University of La Rioja, Logroño, Spain.

Objectives: This study screened chickens and pigs slaughtered for human consumption for the presence and characteristics of extended-spectrum β-lactamase (ESBL)- and plasmid-encoded AmpC (pAmpC) β-lactamase-producing enteric bacteria.

Methods: Faecal samples from 410 broiler chickens and 100 pigs were cultured on MacConkey agar supplemented with 2μg/mL cefotaxime. Antimicrobial resistance phenotypes of the recovered isolates were determined by disk diffusion. PCR and sequencing were performed to identify the ESBL and pAmpC gene variants and other associated resistance determinants. Genetic diversity of the isolates was analysed by phylotyping and multilocus sequence typing.

Results: ESBL-producing Escherichia coli, Klebsiella pneumoniae, Enterobacter asburiae and Providencia spp. were isolated from 17 (4.1%) and 2 (2.0%) of the samples from chickens and pigs, respectively. One pAmpC-producing E. coli isolate was obtained from a chicken. Resistance to tetracycline, trimethoprim/sulfamethoxazole, chloramphenicol and gentamicin was exhibited by 95%, 80%, 60% and 55% of the ESBL/pAmpC-producing strains, respectively. tet(A) and aac(3)-II were the predominant genes detected in tetracycline- and aminoglycoside-resistant strains, respectively. bla, encoding CTX-M-15 (15 isolates) or CTX-M-1 variants (3 isolates), was present in all but one ESBL-producer, either alone or in combination with bla and/or bla. The remaining ESBL-producer, a Providencia spp. recovered from a chicken, harboured bla. The only pAmpC-positive E. coli strain carried bla. The 11 ESBL-producing E. coli strains belonged to five lineages (ST226-A, ST3625-B1, ST10-A, ST46-A and ST58-B1).

Conclusions: Healthy chickens and pigs act as reservoirs of ESBL/pAmpC-producing enterobacteria that can potentially be transmitted to humans through direct contact or ingestion of contaminated meat.
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http://dx.doi.org/10.1016/j.jgar.2018.06.002DOI Listing
December 2018

NDM-1- and OXA-23-producing Acinetobacter baumannii isolated from intensive care unit patients in Tunisia.

Int J Antimicrob Agents 2018 Dec 14;52(6):910-915. Epub 2018 Apr 14.

University of Tunis El Manar, Faculty of Medicine of Tunis-LR99ES09 Research Laboratory 'Antimicrobial resistance', 15 Rue Djebel Akhdhar, La Rabta, 1007 Tunis, Tunisia; Charles Nicolle Hospital, Laboratory of Microbiology, 1006 Tunis, Tunisia.

Gastrointestinal colonisation by carbapenem-resistant Acinetobacter baumannii (CRAB) is a critical step before nosocomial infection. This study evaluated CRAB intestinal carriage in patients admitted to a Tunisian ICU and determined the antimicrobial resistance mechanisms involved. From December 2014 to February 2015, all 63 patients admitted to the ICU were screened for rectal CRAB colonisation upon admission and once weekly thereafter. ICU patients who acquired a CRAB nosocomial infection were also included. β-Lactamases and associated resistance genes were screened by PCR sequencing, and molecular typing was performed by PFGE and MLST. The CRAB faecal carriage rate at admission was 4.8% (3/63). The CRAB acquisition rate during ICU stay was analysed in 39 of the remaining 60 patients and the rate of acquired CRAB faecal carriage was 15.4% (6/39); 4 patients also showed an ICU-acquired CRAB infection (one patient was a faecal carrier and suffered infection). Overall, 13 CRAB isolates were collected from 12 patients, of which 11 isolates showed resistance to all antibiotics tested except colistin. bla and bla were detected in 11 and 2 isolates, respectively. All OXA-23-producing strains carried armA, tetB, sul1 and catB, and some of them carried aph(3')-VIa, bla, aph(3')-Ia and ant(2'')-Ia. The bla-positive isolates harboured aph(3')-VIa and catB. Three PFGE patterns and two STs were identified [ST195 (n = 11), ST1089 (n = 2, NDM-1-positive)]. Whether imported or acquired during ICU stay, CRAB colonisation is a major risk factor for the occurrence of serious nosocomial infection. Systematic screening of faecal carriage is mandatory to prevent their spread.
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http://dx.doi.org/10.1016/j.ijantimicag.2018.04.008DOI Listing
December 2018

Molecular diversity and conjugal transferability of class 2 integrons among Escherichia coli isolates from food, animal and human sources.

Int J Antimicrob Agents 2018 Jun 8;51(6):905-911. Epub 2018 Feb 8.

Instituto de Microbiología y Parasitología Médica, Facultad de Medicina, Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Tecnológicas (IMPaM, UBA-CONICET), Ciudad Autónoma de Buenos Aires, Argentina.

Integrons are genetic platforms able to excise, integrate and express antibiotic resistance gene cassettes (GCs). Here we investigated the complete genetic organisation, genetic environment, location and conjugative transferability of a collection of class 2 integrons carried by Escherichia coli strains from different sources (poultry/pork meat, animals and humans). PCR cartography was conducted to determine the genetic arrangement of the integrons, their physical linkage to Tn7 and chromosomal insertion at the attTn7 site. Clonal relatedness of specific isolates was determined by MLST and DO-PCR. Transferability of class 2 integrons was tested by conjugation. The resulting transconjugants were characterised by antimicrobial resistance genotyping, S1-PFGE and replicon typing. Although a limited diversity of GCs was shown, a high percentage of novel structures was identified owing to the integration of insertion sequence (IS) elements at different sites (IS3/IS4/IS5/IS21 families). Insertion of IS10 in the attI2 site of a class 2 integron, between Pc2B and Pc2C promoters, was likely mediated by a site-specific transposition event. Chromosomal insertion of integrons at attTn7 was confirmed in 80% of the isolates. Conjugation experiments demonstrated that 29% of class 2 integrons could be mobilised to E. coli CHS26, demonstrating that they can be located in conjugative/mobilisable elements at a low frequency. Reported structures evidence how class 2 integrons have evolved by the activity of integron integrases and the invasion of ISs. Since most of them are chromosomally located, dispersion is predominantly vertical, although conjugation events also contribute to the spread of class 2 integrons among bacterial communities.
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http://dx.doi.org/10.1016/j.ijantimicag.2018.02.001DOI Listing
June 2018

Emergence of plasmid-mediated colistin-resistance in CMY-2-producing Escherichia coli of lineage ST2197 in a Tunisian poultry farm.

Int J Food Microbiol 2018 Mar 31;269:60-63. Epub 2018 Jan 31.

Universidad de La Rioja, Area de Bioquímica y Biología Molecular, Logroño, Spain.

Our study aimed to investigate colistin resistance and the mechanisms involved in a collection of 35 extended-spectrum beta-lactamase (ESBL) and 13 CMY-2-producing E. coli strains which were previously recovered from chicken gut microbiota in Tunisia, as well as to determine the genetic location of mcr genes. Forty-eight ESBL and CMY-2-producing E. coli strains were obtained from 137 fecal samples of healthy chickens during 2013. These strains were tested for colistin resistance by the broth microdilution method, and screened for mcr-1 and mcr-2 genes by PCR. Two of these strains were colistin-resistant (MIC = 8 mg/L). Both harbored the mcr-1 gene, were CMY-2 producers, and were additionally resistant to tetracycline, ciprofloxacin, chloramphenicol, gentamicin, tobramycin and trimethoprim-sulfamethoxazole. They shared phylogroup A, the same pulsed-field gel electrophoresis (PFGE)-pattern, and were typed as ST2197. In both strains, ISApl1 and pap2 were detected upstream and downstream of mcr-1 gene, respectively. The analysis of the two mcr-1-positive strains and their transconjugants by PCR-based replicon typing and S1-PFGE, demonstrated that mcr-1 gene is linked to an IncP plasmid (~242 kb), and bla to an IncI1 plasmid (97 kb). The occurrence of E. coli harboring mcr-1 gene among intestinal microbiota in poultry and its location on a conjugative plasmid could represent a risk for public health. The evolution of this type of resistant microorganisms should be evaluated in the future.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2018.01.017DOI Listing
March 2018

Diversity of Ochrobactrum species in food animals, antibiotic resistance phenotypes and polymorphisms in the blaOCH gene.

FEMS Microbiol Lett 2017 Sep;364(17)

Department of Veterinary Pathology and Microbiology, University of Nigeria, Nsukka 410001, Nigeria.

Twenty-six lactose non-fermenting, oxidase, urease and citrate-positive Gram-negative rods, isolated from broiler chickens, pigs and cattle at slaughter, were subjected to the matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry and 16S rDNA sequencing for identification. Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Ochrobactrum isolates resistant to third-generation cephalosporins were PCR-screened for the presence of the Ochrobactrum anthropi ampC gene (blaOCH). A 547-bp internal segment of blaOCH in the Ochrobactrum spp isolates was amplified with a newly designed primer set, and a phylogenetic reconstruction based on the complete amino acid sequence of blaOCH obtained from nine Ochrobactrum strains in our collection and 20 O. anthropi available in the GenBank was undertaken. All the Ochrobactrum isolates were resistant to the expanded-spectrum beta-lactams and streptomycin. None of the isolates was resistant to imipenem while 41.7% to 50.0% of them were resistant to fluoroquinolones. The blaOCH gene was detected in 16 (66.7%) and 20 (83.3%) of the 24 Ochrobactrum isolates (O. intermedium/O. tritici species), using primers designed for O. anthropi and the newly designed primer set, respectively. Six blaOCH variants grouped into two divergent clusters were identified. This is the first report of the complete nucleotide sequence of the blaOCH gene in non-antropi Ochrobactrum species.
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http://dx.doi.org/10.1093/femsle/fnx178DOI Listing
September 2017

Novel sequence types of extended-spectrum and acquired AmpC beta-lactamase producing Escherichia coli and Escherichia clade V isolated from wild mammals.

FEMS Microbiol Ecol 2017 08;93(8)

Unit of Biochemistry and Molecular Biology, University of La Rioja, 26006 Logroño, Spain.

The closer contact with wildlife due to the growing human population and the destruction of natural habitats emphasizes the need of gaining insight into the role of animals as source of antimicrobial resistance. Here, we aim at characterizing the antimicrobial resistance genes and phylogenetic distribution of commensal Escherichia coli from 62 wild mammals. Isolates exhibiting resistance to ≥1 antibiotic were detected in 25.8% of the animals and 6.4% carried an extended-spectrum beta-lactamase (ESBL)/AmpC-producing E. coli. Genetic mechanisms involved in third-generation cephalosporin resistance were as follows: (i) hyperproduction of chromosomal AmpC (hedgehog), (ii) production of acquired CMY-2 β-lactamase (hedgehog), (iii) production of SHV-12 and CTX-M-14 ESBLs (n = 2, mink and roe-deer). ESBL genes were transferable by conjugation, and blaCMY-2 was mobilized by a 95kb IncI1 plasmid. The distribution of the phylogenetic groups in the E. coli collection studied was B1 (44.6%), B2 (24.6%), E (15.4%), A (4.6%) and F (3.1%). Five isolates (7.7%) were cryptic Escherichia clades (clade IV, 4 mice; clade V, 1 mink). ESBL/AmpC-E. coli isolates showed different sequence types (STs): ST1128/B1, ST4564/B1 (new), ST4996/B1 (new) and a non-registered ST. This study contributes to better understand the E. coli population and antimicrobial resistance flow in wildlife and reports new AmpC-E. coli STs and a first described ESBL-producing Escherichia clade V isolate.
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http://dx.doi.org/10.1093/femsec/fix097DOI Listing
August 2017

Occurrence and characterization of stx and/or eae-positive Escherichia coli isolated from wildlife, including a typical EPEC strain from a wild boar.

Vet Microbiol 2017 Aug 1;207:69-73. Epub 2017 Jun 1.

Área Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain. Electronic address:

Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains are food-borne pathogens associated with acute diarrhea. Haemolytic-uremic syndrome (HUS) is often a complication of STEC infection. In order to examine the occurrence, serotypes, virulence and antimicrobial-resistance profiles of STEC and EPEC in wildlife, 326 faecal E. coli strains from 304 clinically healthy animals were analyzed. For this approach stx, stx and eae genes, as well as accessory virulence determinants (ehx, hlyA, saa, tia, bfp, subAB) were PCR-screened and sequenced. Serotyping was performed employing all available O (O1-O185) and H (H1-H56) antisera. Genetic diversity was analyzed by XbaI-PFGE and phylotyping. Thirteen STEC (4.3%) and 10 EPEC (3.3%) were identified among 12 deer, 3 mouflon, 6 wild boars and 2 birds. Nine STEC showed seropathotypes B (O145:[H28]) and C (O22:H8, O128:[H2]) associated with HUS, and D (O110:H28, O146:H21, O146:[H28], ONT:H8) associated with human diarrhea. Although most isolates harbored stx and stx variants, stx and stx (related with severe disease) were also detected. Additionally, the eae gene was present in one stx-positive O145:[H28] STEC from a deer and 11 STEC harbored subAB genes (mainly the subAB variant). EPEC isolates showed 7 different intimin variants (β1, β2, γ1, ε1, ζ1, ι1-A, κ). Interestingly, the O49:[H10] eae-κ EPEC isolated from a wild boar was bfpA-positive showing a combination of serotype/virulence profile previously detected among human clinical tEPEC. Based on present results, wild ruminants, wild boars and to a lesser extent birds would be carriers of potentially pathogenic STEC and EPEC strains.
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http://dx.doi.org/10.1016/j.vetmic.2017.05.028DOI Listing
August 2017

Extended-spectrum β-lactamase-producing Escherichia coli isolated from healthy humans in Mexico, including subclone ST131-B2-O25:H4-H30-Rx.

J Glob Antimicrob Resist 2017 06 25;9:130-134. Epub 2017 May 25.

Benemérita Universidad Autónoma de Puebla, Posgrado en Microbiología, Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Ciudad Universitaria, San Manuel, 72570 Puebla, Mexico. Electronic address:

Objectives: The resistance mechanisms, molecular type and plasmid content of cefotaxime-resistant Escherichia coli isolated from faecal samples of healthy volunteers in Puebla, Mexico, were characterised.

Methods And Results: Cefotaxime-resistant E. coli were recovered from 11 (18%) of 60 healthy volunteers. The isolates (one per sample) were characterised as multidrug-resistant and phenotypically extended-spectrum β-lactamase (ESBL)-producing strains. Genes encoding resistance to β-lactams (bla, bla, bla, bla, bla), quinolones [aac(6')-Ib-cr, qnrB19], aminoglycosides [aac(3')-II] and tetracycline [tet(A), tet(B)] were detected among the 11 ESBL-producing E. coli by PCR and sequencing, as well as gene cassette arrays in class 1 (dfrA17-aadA5) and class 2 (dfrA1-sat2-aadA1) integrons. Seven pulsotypes were identified by XbaI PFGE and the strains were distributed into phylogroups (number of isolates) A (2), B2 (4) and D (5). Seven sequence types were identified, four of them novel (ST5060, ST5079, ST5080 and ST5081), associated with phylogroups A-D. Transfer of a 140-kb IncFIA plasmid carrying the bla gene was evidenced in the ST5060 strain. Four CTX-M-15-producing E. coli strains of phylogroup B2 belonged to the ST131 complex, and IncFIB plasmids of 130kb and 155kb were detected in two of them. Multiple plasmid addiction systems were also found. Serotyping and fimH subtyping of ST131-B2 strains identified the ST131-B2-O25:H4-H30-Rx subclone. Additionally, this subclone and CTX-M-14-producing isolates were detected among residents living in the same household, suggesting clonal dissemination.

Conclusions: This study reports the detection of E. coli ST131-B2-O25:H4-H30-Rx subclone in healthy humans in Mexico, highlighting its dissemination in the community setting.
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http://dx.doi.org/10.1016/j.jgar.2017.02.014DOI Listing
June 2017

High frequency of B2 phylogroup among non-clonally related fecal Escherichia coli isolates from wild boars, including the lineage ST131.

FEMS Microbiol Ecol 2017 03;93(3)

Área Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain.

Wild boars are worldwide distributed mammals which population is increasing in many regions, like the Iberian Peninsula, leading to an increased exposition to humans. They are considered reservoirs of different zoonotic pathogens and have been postulated as potential vectors of antimicrobial-resistant (AMR) bacteria. This study aimed to determine the prevalence of antimicrobial resistance and phylogenetic distribution of Escherichia coli from wild boar feces. Antimicrobial resistance and integron content was genetically characterized and E. coli of B2 phylogroup was further analyzed by molecular typing and virulence genotyping. The prevalence of AMR E. coli was low, with only 7.5% of isolates being resistant against at least one antimicrobial, mainly ampicillin, tetracycline and/or sulfonamide. An unexpected elevated rate of B2 phylogroup (47.5%) was identified, most of them showing unrelated pulsed-field-gel-electrophoresis patterns. ST131/B2 (fimH 22 sublineage), ST28/B2, ST1170/B2, ST681/B2 and ST625/B2 clones, previously described in extraintestinal infections in humans, were detected in B2 isolates, and carried one or more genes associated with extraintestinal pathogenic E. coli (ExPEC). This study demonstrated a low prevalence of antimicrobial resistance in E. coli from wild boars, although they are not exempt of AMR bacteria, and a predominance of genetically diverse B2 phylogroup, including isolates carrying ExPEC which may contribute to the spread of virulence determinants among different ecosystems.
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http://dx.doi.org/10.1093/femsec/fix016DOI Listing
March 2017

Clonal diversity of extended-spectrum beta-lactamase producing Escherichia coli isolates in fecal samples of wild animals.

FEMS Microbiol Lett 2017 03;364(5)

Research Unit on Applied Molecular Biosciences (UCIBIO-REQUIMTE), University NOVA of Lisboa, Lisboa, 2829-516 Caparica, Portugal.

The clonal diversity of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolates from nine different species of wild animals from distinct regions of Portugal and Spain and their content in replicon plasmids were analyzed. Among the initial 53 ESBL-producing E. coli isolates that were studied (from previous studies), 28 were selected, corresponding to different animal origins with distinct ESBL types and pulsed-field gel electrophoresis (PFGE) patterns. These 28 isolates produced different ESBLs ascribed to the following families: CTX-M, SHV and TEM. The isolates were classified into three phylogenetic groups: B1 (n = 11), A (n = 10) and D (n = 7). The seven E. coli of phylogroup D were then typed by multilocus sequence typing and ascribed to four distinct sequence types: ST117, ST115, ST2001 and ST69. The clonal diversity and relationship between isolates was studied by PFGE. Lastly, the plasmids were analyzed according to their incompatibility group using the PCR-based-replicon-typing scheme. A great diversity of replicon types was identified, with up to five per isolate. Most of the CTX-M-1 and SHV-12 producing E. coli isolates carried IncI1 or IncN replicons. The diversity of ESBL-producing E. coli isolates in wild animals, which can be disseminated in the environment, emphasizes the environmental and health problems that we face nowadays.
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http://dx.doi.org/10.1093/femsle/fnx039DOI Listing
March 2017

Analysis of blaSHV-12-carrying Escherichia coli clones and plasmids from human, animal and food sources.

J Antimicrob Chemother 2017 06;72(6):1589-1596

Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), Neustadt-Mariensee, Germany.

Objectives: This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective bla SHV-12 -carrying plasmids and sequencing one of these plasmids completely.

Methods: Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed bla SHV-12 -carrying plasmids were examined by replicon typing, S1-nuclease, conjugation, EcoRI-HindIII-BamHI digests and plasmid MLST. Co-located resistance genes and integrons as well as the bla SHV-12 genetic environment were analysed by PCR and sequencing. One IncI1 plasmid was sequenced completely using HiSeq 2500 and gap closure by PCRs and Sanger sequencing.

Results: Among the 23 SHV-12-positive E. coli , some isolates from different sources showed the same characteristics: ST23/phylogroup A (human, dog, livestock), ST57/D (wild bird, chicken meat) and ST117/D (chicken meat, chicken). All bla SHV-12 genes were horizontally transferable via 30-120 kb plasmids of incompatibility groups IncI1 ( n  = 17), IncK ( n  = 3), IncF ( n  = 1), IncX3 ( n  = 1) and a non-typeable plasmid. IncK plasmids, indistinguishable in size and restriction patterns, were found in isolates from different sources (ST57/D, meat; ST131/B2, meat; ST57/B1, dog). The IncI1- bla SHV-12 -carrying plasmids were mostly assigned to plasmid ST (pST) 26 and pST3. Three plasmids showed novel pSTs (pST214, pST215). The majority of the IncI1 transformants exhibited resistance to β-lactams, chloramphenicol and streptomycin (in relation with a class 1 integron containing an estX - psp - aadA2 - cmlA1 - aadA1 - qacI gene cassette array), and to tetracycline. A novel bla SHV-12 environment was detected and whole plasmid sequencing revealed a Tn 21 -derived- bla SHV12 -ΔTn 1721 resistance complex.

Conclusions: Results from this study suggest that the dissemination of bla SHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids.
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http://dx.doi.org/10.1093/jac/dkx024DOI Listing
June 2017

Community fecal carriage of broad-spectrum cephalosporin-resistant Escherichia coli in Tunisian children.

Diagn Microbiol Infect Dis 2017 Feb 8;87(2):188-192. Epub 2016 Mar 8.

Université de Tunis El Manar, Faculté de Médecine de Tunis, LR99ES09 Laboratoire de Résistance aux antimicrobiens, 1007, Tunis, Tunisie; CHU Charles Nicolle, Service de Microbiologie, 1006, Tunis, Tunisie.

The spread of extended spectrum β-lactamases (ESBL) and plasmid mediated AmpC β-lactamases (pAmpC) was evaluated in Escherichia coli strains collected from the intestinal microbiota of healthy children in Tunisia. The carriage rate of CTXE. coli was 6.6% (7 of 105 samples) and one strain/sample was further characterized (7 isolates). These isolates harbored bla (n = 4), bla (n = 2), and bla gene (n = 1), which were usually located on FIB replicon type and carried class 1 integrons. The acc(6')-Ib-cr variant was identified in one isolate that harbored bla. CTXE. coli isolates were genetically unrelated and belonged to B1 (n = 3/ST155/ST398/ST58), D (n = 2/ST117/ST493), B2 (n = 1/ST127), and A (n = 1/ST746) phylogroups. Strain virulence scores varied from 3 to 12, and frequently harbored the pathogenicity island PAI IV. The intestinal tract of healthy children constitute an important reservoir of ESBL producing E. coli. Thus, improvement of hygiene measures mainly in the school environment and rational use of antibiotics would be of great help in preventing selection and diffusion of resistant strains from intestinal microbiota.
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http://dx.doi.org/10.1016/j.diagmicrobio.2016.03.008DOI Listing
February 2017

Wild Birds, Frequent Carriers of Extended-Spectrum β-Lactamase (ESBL) Producing Escherichia coli of CTX-M and SHV-12 Types.

Microb Ecol 2016 11 21;72(4):861-869. Epub 2015 Dec 21.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006, Logroño, Spain.

To get a better insight into the role of birds as reservoirs of extended-spectrum β-lactamase (ESBL) and plasmidic AmpC β-lactamase (pAmpC) Escherichia coli producers, 100 fecal samples belonging to 15 different wild avian species from Northern Spain were analyzed. Cefotaxime-resistant (CTX) E. coli isolates were identified in 16 of the 100 tested birds, which corresponded to 9 animal species (Gyps fulvus-griffon vulture, Larus michahellis-yellow-legged gull, Milvus migrans-black kite, Milvus milvus-red kite, Ciconia ciconia-white stork, Sturnus unicolor-spotless starling, Aquila chrysaetos-golden eagle, Cuculus canorus-common cuckoo, Tyto alba-barn owl). Fifteen isolates harbored ESBL or pAmpC-encoding genes (number of isolates): bla (9), bla (3), bla (2), and bla (1). The last CTX isolate presented a -42-point-mutation in the chromosomal ampC promoter. Eleven out of 15 ESBL/pAmpC E. coli isolates were multiresistant (most common resistance phenotype: β-lactams-quinolones-tetracycline-sulfamethoxazole/trimethoprim). A plasmid-mediated quinolone resistance determinant (qnrS1) was identified in one E. coli from a barn owl. High genetic diversity was observed among ESBL/pAmpC E. coli isolates, with 12 different sequence types (STs), including several strains of STs frequently detected among human clinical isolates (ST38/D, ST131/B2, ST155/B1, ST10/A). The ST131 isolate belonged to the emergent ciprofloxacin-resistant H30R subclone. This study reveals a high percentage of bird as carriers of ESBL/pAmpC E. coli isolates in Spain, highlighting the elevated rate among storks, kites, and vultures. Wild birds can contribute to the global spread of ESBL/pAmpC-producing E. coli in natural ecosystems.
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http://dx.doi.org/10.1007/s00248-015-0718-0DOI Listing
November 2016

First Description of KPC-2-Producing Escherichia coli and ST15 OXA-48-Positive Klebsiella pneumoniae in Tunisia.

Microb Drug Resist 2017 Apr 18;23(3):365-375. Epub 2016 Oct 18.

2 Service des Laboratoires, Centre National de Greffe de Moelle Osseuse , Tunis, Tunisie.

The aim of this study was to investigate the molecular features among Klebsiella pneumoniae and Escherichia coli strains showing a resistant/intermediate-resistant phenotype to ertapenem (R/IR-ERT), implicated in colonization/infection in patients of the Hematology and Graft Units of the National Bone Marrow Transplant Center of Tunisia (3-year period, 2011-2014). The major carbapenemase, extended-spectrum beta-lactamase, and plasmidic AmpC beta-lactamase genes were analyzed and characterized by PCR and sequencing. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) using XbaI and multilocus sequencing typing. The bla and bla carbapenemase genes were detected among R/IR-ERT isolates. All R/IR-ERT K. pneumoniae strains (n = 19) had bla gene, and 14/19 strains also harbored the bla gene. Eight different PFGE patterns were detected among these K. pneumoniae isolates, and they showed eight different sequences types, ST11 and ST15 being the most prevalent ones. Two out of three R/IR-ERT E. coli isolates carried bla and one coproduced the bla gene. One E. coli strain, ascribed to the new sequence type ST5700, harbored the bla gene. E. coli isolates were not clonally related and belonged to different sequence types (ST5700, ST227, and ST58). To our knowledge, this is the first report in Tunisia of either KPC-2 carbapenemase in E. coli or OXA-48 carbapenemase in K. pneumoniae of lineage ST15.
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http://dx.doi.org/10.1089/mdr.2016.0090DOI Listing
April 2017

Characterization of extended-spectrum β-lactamase (ESBL)-producing Klebsiella, Enterobacter, and Citrobacter obtained in environmental samples of a Tunisian hospital.

Diagn Microbiol Infect Dis 2016 Oct 15;86(2):190-3. Epub 2016 Jul 15.

Area de Bioquímica y Biología Molecular, Universidad de La Rioja, 26006 Logroño, Spain. Electronic address:

The assessment of the hospital environment as a reservoir of ESBL-producing Enterobacteriaceae in Tunisian hospitals is scarcely analyzed, except for Escherichia coli. The aim of this study was to evaluate the presence of ESBL-producing non-E. coli Enterobacteriaceae (ESBL-EbNoEc) in 300 samples of abiotic surfaces and the hands of patients and staff of a Tunisian Hospital, and to characterize the ESBL genes of the recovered isolates. ESBL-EbNoEc were recovered in 28 of 300 (9.3%) analyzed samples and were identified as Klebsiella pneumoniae (n= 11), Enterobacter cloacae (n=11), Citrobacter freundii (n=4) and Klebsiella oxytoca (n=2). The bla genes identified by PCR and sequencing among the strains were as follows: 11 K.pneumoniae strains [blaCTX-M-15+ blaTEM-1+ blaSHV-11 (n=6); blaCTX-M-15+ blaTEM-1+ blaSHV-28 (n=3); blaCTX-M-15+ blaTEM-1+ blaSHV-1 (n=2)], 11 E. cloacae strains [blaCTX-M-15 (n=6); blaCTX-M-15+ blaTEM-1b (n=2); blaCTX-M-15+ blaTEM-1b+ blaOXA-1 (n=1);blaCTX-M-15+ blaOXA-1 (n=1);blaSHV-12 (n=1)], 4 C. freundii strains [blaCTX-M-15] and 2 K. oxytoca strains [blaCTX-M-15 (n=1); blaSHV-12 (n=1)]. The ISEcp1 and orf477 sequences were identified upstream and downstream of the blaCTX-M-15 gene, respectively, in 3 K. pneumoniae and 3 E. cloacae isolates. The PFGE analysis demonstrated three unrelated pulsotypes in K. pneumoniae strains and five pulsotypes in E. cloacae. The uncontrolled dissemination of ESBL-producing bacteria, even in the hospital environment, has become a real problem and new strategies and hygienic rules are needed to stop this bacterial dissemination.
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http://dx.doi.org/10.1016/j.diagmicrobio.2016.07.013DOI Listing
October 2016

High prevalence of extended-spectrum and plasmidic AmpC beta-lactamase-producing Escherichia coli from poultry in Tunisia.

Int J Food Microbiol 2016 Aug 3;231:69-75. Epub 2016 May 3.

University of Tunis El Manar, Faculty of Medicine of Tunis-Research Laboratory «Antimicrobial resistance», Tunis, Tunisia; Charles Nicolle Hospital, Laboratory of Microbiology, Tunis, Tunisia.

This study was conducted to detect extended spectrum beta-lactamases (ESBLs) and plasmidic AmpC beta-lactamase (pAmpC-BL)-producing Escherichia coli isolates in industrial poultry samples were collected from healthy chickens of the three farms. Samples were inoculated onto desoxycholate-lactose-agar plates supplemented with cefotaxime (2mg/L). E. coli was identified by biochemical and molecular methods and antibiotic susceptibility testing by the disk diffusion method. Genes encoding ESBLs and pAmpC-BL were detected by PCR and sequencing. Phylogenetic groups were determined by triplex PCR. The molecular typing of strains was done by pulsed field gel electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) in those isolates showing different PFGE patterns. Cefotaxime-resistant E. coli isolates were recovered in 48 of 137 fecal samples (35%), and one isolate/sample was further studied. The following beta-lactamase genes were detected: blaCTX-M-1 (29 isolates, isolated in all three farms), blaCTX-M-15 (5 isolates, confined in farm II), blaCTX-M-14 and blaCMY-2 (one isolate and 13 isolates, respectively, in farm III). The 48 cefotaxime-resistant isolates were distributed into phylogroups: B1 (n=21), A (n=15) and D (n=12). PFGE analysis revealed 19 unrelated patterns: 15 different profiles among ESBL-positive strains and 4 among the CMY-2-positive isolates. The following sequence types-associated phylogroups were detected: a) CTX-M-1-positive strains: lineages ST542-B1, ST212-B1, ST58-B1, ST155-B1 and ST349-D; b) CTX-M-15-positive strain: lineage ST405-D; c) CTX-M-14-positive strain: lineage ST1056-B1; d) CMY-2-positive strains: lineages ST117-D, ST2197-A, and ST155-B1. Healthy chickens constitute an important reservoir of ESBL- and pAmpC-BL-producing E. coli isolates that potentially could be transmitted to humans via the food chain or by direct contact.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2016.05.001DOI Listing
August 2016

Persistence of a ST6 clone of Enterococcus faecalis genotype vanB2 in two Hospitals in Aragon (Spain).

Enferm Infecc Microbiol Clin 2017 Nov 5;35(9):578-581. Epub 2016 Apr 5.

Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, España. Electronic address:

Introduction: In order to study the evolution of the outbreak that occurred between 2009 and 2010 in 3 hospitals in Zaragoza, all vancomycin-resistant clinical Enterococcus faecalis isolates identified between 2011 and 2013 at these hospitals were characterised.

Methods: Molecular characterisation of the isolates and analysis of their clonal relationships was performed using pulsed field electrophoresis, along with a retrospective review of the patient records.

Results: A total of 79 vancomycin-resistant E.faecalis isolates with genotype vanB2 of 73 patients were recovered in 2 of the 3 hospitals, most of them from urine specimens. About 46% of the cases were nosocomial. Distribution of the isolates among hospital services demonstrated high variability, making it difficult to predict a common source of infection. All the strains were multiresistant (vancomycin, erythromycin, tetracycline, ciprofloxacin, streptomycin, gentamicin, kanamycin) and belonged to lineage ST6. Seventy-four isolates (93.7%) were identical or closely related to the dominant one in the origin of the outbreak.

Conclusion: The outbreak remains constant over three years after being initially described, indicating the need to implement an active control in order to limit the emergence and spread of vancomycin-resistant clones.
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http://dx.doi.org/10.1016/j.eimc.2016.02.020DOI Listing
November 2017

Molecular characterization of antibiotic resistance in Escherichia coli strains from a dairy cattle farm and its surroundings.

J Sci Food Agric 2017 Jan 15;97(1):362-365. Epub 2016 Apr 15.

Departamento de Agricultura y Alimentación, Universidad de La Rioja, Logroño, Spain.

Background: This study describes the phenotypic and genotypic characteristics of 78 genetically different Escherichia coli recovered from air and exudate samples of a dairy cattle farm and its surroundings in Spain, in order to gain insight into the flow of antimicrobial resistance through the environment and food supply.

Results: Antimicrobial resistance was detected in 21.8% of the 78 E. coli isolates analyzed (resistance for at least one of the 14 agents tested). The highest resistance rates were recorded for ampicillin, nalidixic acid, trimethoprim/sulfamethoxazole and tetracycline. The resistance genes detected were as follows (antibiotic (number of resistant strains), gene (number of strains)): ampicillin (9), bla (6); tetracycline (15), tet(A) (7), tet(B) (4), tet(A) + tet(B) (1); chloramphenicol (5), cmlA (2), floR (2); trimethoprim/sulfamethoxazole (10), sul2 (4), sul1 (3), sul3 (2), sul1 + sul2 (1); gentamicin-tobramycin (1), ant(2″) (1). About 14% of strains showed a multidrug-resistant phenotype and, of them, seven strains carried class 1 integrons containing predominantly the dfrA1-aadA1 array. One multidrug-resistant strain was found in both inside and outside air, suggesting that the airborne spread of multidrug-resistant bacteria from the animal housing facilities to the surroundings is feasible.

Conclusions: This study gives a genetic background of the antimicrobial resistance problem in a dairy cattle farm and shows that air can act as a source for dissemination of antimicrobial-resistant bacteria. © 2016 Society of Chemical Industry.
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http://dx.doi.org/10.1002/jsfa.7709DOI Listing
January 2017

Detection of CTX-M-15-Producing Escherichia coli Isolates of Lineages ST131-B2 and ST167-A in Environmental Samples of a Tunisian Hospital.

Microb Drug Resist 2016 Jul 9;22(5):399-403. Epub 2016 Mar 9.

2 Área de Bioquímica y Biología Molecular, Universidad de La Rioja , Logroño, Spain .

To investigate the possible role of the hospital environment in the dissemination of extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli isolates, 300 samples were taken during 2013 from abiotic surfaces (n = 250), healthcare worker hands (n = 27), and hands of patients (n = 23) in a Tunisian Hospital. ESBL-producing E. coli isolates were recovered in 3.7% of analyzed samples (4% abiotic surfaces; 4.3% hands of patients; 0% in healthcare worker hands), and one isolate/sample was further studied. The characterization of beta-lactamase genes, as well as the genetic environment of blaCTX-M gene, was performed by PCR and sequencing. The ESBL genes found were as follows: blaCTX-M-15 (eight isolates), blaCTX-M-15+blaSHV-12 (two isolates), and blaSHV-12 (one isolate). The blaTEM-1b gene was detected in seven ESBL-positive isolates. The orf477 was found downstream of blaCTX-M-15 gene in 10 strains, whereas the ISEcp1 sequence was identified upstream of this gene in two isolates. The analysis of class 1 integrons by PCR and sequencing revealed five positive isolates with the following gene cassette arrangements: dfrA1-aadA1 (two isolates), aadA1 (two isolates), and aadA2 (one isolate). The virulence-encoding genes aer, eae, bfp, and hly were detected by PCR in six, four, four, and three isolates, respectively. The following sequence types and associated phylogroups were detected among ESBL-producing strains: ST167-phylogroup-A (six isolates) and ST131-phylogroup-B2 (two isolates). In conclusion, the hospital environment could be a reservoir of multiresistant bacteria, including ESBL-positive E. coli isolates, which could be acquired by the patient population, and strict control measures should be established to minimize this problem.
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http://dx.doi.org/10.1089/mdr.2015.0354DOI Listing
July 2016