Carla Andrea Alonso

Characteristics of extended-spectrum β-lactamase (ESBL)- and pAmpC beta-lactamase-producing Enterobacteriaceae of water samples in Tunisia

The presence of extended-spectrum beta-lactamase and plasmid-mediated AmpC beta-lactamase producing Enterobacteriaceae (ESBL-Eb and pAmpC-Eb, respectively) was analyzed in 57 wastewater and 57 surface-water samples in Tunisia. Twenty-four of the 57 wastewater samples (42.1%) and one of the 57 surface-water samples (1.7%, a river that received effluents of a wastewater-treatment-plant) contained ESBL-Eb or pAmpC-Eb; one ESBL/pAmpC-Eb per positive sample was further characterized. Beta-lactamase genes detected were as follows: blaCTX-M-1 (10 Escherichia coli),blaCTX-M-15 (eight E. coli, one Klebsiella pneumoniae, one Citrobacter freundii), blaCTX-M-14 (one E. coli) and blaCMY-2 (four E. coli). The blaTEM-1, blaOXA-1 or blaSHV-1 genes were also found in 72% of these isolates. The ISEcp1, orf477 or IS903 sequences were found upstream or downstream of blaCTX-M genes. Class 1 integrons were present in 16 of the 25 ESBL-Eb/pAmpC-Eb strains (64%), and contained five different gene-cassette arrays. Most of the strains (76%) showed a multiresistant phenotype and qnr genes were identified in four strains. Molecular typing of ESBL/CMY-2-producing E. coli isolates showed 23 different PFGE-patterns and 15 different sequence-types (ST10, ST46, ST48, ST58, ST69, ST101, ST117, ST131, ST141, ST288, ST359, ST399, ST405, ST617, and the new ST4530); these strains were ascribed to phylogroups A (11 isolates), B1 (3 isolates), D (6 isolates) and B2 (3 isolates). From one to five plasmids were detected in each strain (size from 30kb to >240kb) and ESBL or pAmpC genes were transferred by conjugation in 69.5% of the E. coli strains. In conclusion, ESBL-Eb and pAmpC-Eb strains are frequently detected in wastewater samples and they might be a source for dissemination in other environments with repercussion in public health.

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

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 (CTXR) 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): blaSHV-12 (9), blaCTX-M-1 (3), blaCTX-M-14 (2), and blaCMY-2 (1). The last CTXR 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.

Antibiotic resistance in Escherichia coli in husbandry animals: the African perspective

In the last few years, different surveillances have been published in Africa, especially in northern countries, regarding antimicrobial resistance among husbandry animals. Information is still scarce, but the available data show a worrying picture. Although the highest resistance rates have been described against tetracycline, penicillins and sulphonamides, prevalence of plasmid‐mediated quinolone resistance genes and extended spectrum β‐lactamase (ESBL) are being increasingly reported. Among ESBLs, the CTX‐M‐1 group was dominant in most African surveys. Within this group, CTX‐M‐15 was the main variant both in animals and humans, except in Tunisia where CTX‐M‐1 was more frequently detected among Escherichia coli from poultry. Certain blaCTX‐M‐15‐harbouring clones (ST131/B2 or ST405/D) are mainly identified in humans, but they have also been reported in livestock species from Tanzania, Nigeria or Tunisia. Moreover, several reports suggest an inter‐host circulation of specific plasmids (e.g. blaCTX‐M‐1‐carrying IncI1/ST3 in Tunisia, IncY‐ and Inc‐untypeable replicons co‐harbouring qnrS1 and blaCTX‐M‐15 in Tanzania and the worldwide distributed blaCTX‐M‐15‐carrying IncF‐type plasmids). International trade of poultry meat seems to have contributed to the spread of other ESBL variants, such as CTX‐M‐14, and clones. Furthermore, first descriptions of OXA‐48‐ and OXA‐181‐producing E. coli have been recently documented in cattle from Egypt, and the emergent plasmid‐mediated colistin resistance mcr‐1 gene has been also identified in chickens from Algeria, Tunisia and South Africa. These data reflect the urgent need of a larger regulation in the use of veterinary drugs and the implementation of surveillance programmes in order to decelerate the advance of antimicrobial resistance in this continent.

Antimicrobial resistance in faecal Escherichia coli isolates from farmed red deer and wild small mammals. Detection of a multiresistant E. coli producing extended-spectrum beta-lactamase.

Eighty-nine Escherichia coli isolates recovered from faeces of red deer and small mammals, cohabiting the same area, were analyzed to determine the prevalence and mechanisms of antimicrobial resistance and molecular typing. Antimicrobial resistance was detected in 6.7% of isolates, with resistances to tetracycline and quinolones being the most common. An E. coli strain carrying blaCTX-M-1 as well as other antibiotic resistant genes included in an unusual class 1 integron (Intl1-dfrA16-blaPSE-1-aadA2-cmlA1-aadA1-qacH-IS440-sul3-orf1-mef(B)Δ-IS26) was isolated from a deer. The blaCTX-M-1 gene was transferred by conjugation and transconjugants also acquired an IncN plasmid. This strain was typed as ST224, which seems to be well adapted to both clinical and environmental settings. The phylogenetic distribution of the 89 strains varied depending on the animal host. This work reveals low antimicrobial resistance levels among faecal E. coli from wild mammals, which reflects a lower selective pressure affecting these bacteria, compared to livestock. However, it is remarkable the detection of a multi-resistant ESBL-E. coli with an integron carrying clinically relevant antibiotic-resistance genes, which can contribute to the dissemination of resistance determinants among different ecosystems.

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

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

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 stx1, stx2 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 stx2b and stx1c variants, stx2a and stx1a (related with severe disease) were also detected. Additionally, the eae gene was present in one stx2a-positive O145:[H28] STEC from a deer and 11 STEC harbored subAB genes (mainly the subAB2 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.

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

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.

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

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), blaTEM-1 (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.

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

Abstract The clonal diversity of extended‐spectrum‐&bgr;‐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.

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

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 blaCMY-2 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.