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

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 ß-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: blaCTX-M-15 (two Escherichia fergusonii and one Klebsiella oxytoca isolates), blaKPC-2 (one K. oxytoca), blaKPC-3 (one E. fergusonii), blaACT-36, and blaACC-2 (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 blaKPC-2, 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.

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

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 ß-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.

Persistence of a ST6 clone of Enterococcus faecalis genotype vanB2 in two Hospitals in Aragon (Spain). / Persistencia de un clon ST6 de Enterococcus faecalis con genotipo vanB2 en dos hospitales de Aragón.

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.

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. © 2016 Society of Chemical Industry.

Wild Birds, Frequent Carriers of Extended-Spectrum ß-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 ß-lactamase (ESBL) and plasmidic AmpC ß-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): bla SHV-12 (9), bla CTX-M-1 (3), bla CTX-M-14 (2), and bla CMY-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: ß-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.

Phenotypic Characteristics and Clonal Relationships of Stenotrophomonas maltophilia Isolates in Hospitalized Adults from a Private Center in Lima, Peru.

Stenotrophomonas maltophilia is an opportunistic pathogen, often associated with nosocomial infections. Ten S. maltophilia were isolated from clinical samples during the period January 2021 and June 2022. Eight (80%) patients had cancer as a background disease and 2 patients had coronavirus disease 2019. A fatal outcome was recorded in 4 cases (40% of patients). All the isolates were susceptible to minocycline and levofloxacin. Trimethoprim/sulfamethoxazole and ceftazidime resistance rates were 20% and 40% respectively. Eight different patterns were observed by Pulsed-Field Gel Electrophoresis, only two isolates being clonally identical. The isolation of S. maltophilia in clinical settings requires the implementation of infection prevention measures.

Antimicrobial Resistance and Antimicrobial Activity of Staphylococcus lugdunensis Obtained from Two Spanish Hospitals.

Staphylococcus lugdunensis is a coagulase-negative-staphylococci (CoNS) that lately has gained special attention in public health as a human pathogen and also as a bacteriocin-producer bacteria. In this study, we characterized 56 S. lugdunensis isolates recovered from human samples in two Spanish hospitals. Antimicrobial susceptibility testing was performed and antimicrobial resistance and virulence genotypes were determined. Antimicrobial activity (AA) production was evaluated by the spot-on-lawn method against 37 indicator bacteria, including multidrug-resistant (MDR) isolates, and the presence of the lugD gene coding for lugdunin bacteriocin was analyzed by PCR. The antibiotic resistance detected was as follows (% resistance/genes detected): penicillin (44.6%/blaZ), oxacillin (1.8%/mecA on SCCmec-V), erythromycin-clindamycin inducible (7.1%/erm(C), msrA), tetracycline (5.3%/tetK), gentamicin and/or tobramycin (3.6%/ant(4')-Ia, acc(6')-aph(2″)), and fosfomycin (21.4%). A MDR phenotype was detected in 5% of isolates. Twenty-one of the S. lugdunensis isolates showed susceptibility to all 20 antibiotics tested (37.5%). The screening for AA revealed 23 antimicrobial producer (AP) isolates with relevant inhibition against coagulase-positive-staphylococci (CoPS), including both methicillin-susceptible and -resistant S. aureus. The lugD gene was detected in 84% of the 56 S. lugdunensis isolates. All of the AP S. lugdunensis isolates (n = 23) carried the lugD gene and it was also detected in 24 of the non-AP isolates, suggesting different gene expression levels. One of the AP isolates stood out due to its high antimicrobial activity against more than 70% of the indicator bacteria tested, so it will be further characterized at genomic and proteomic level.

IncFIB plasmids carrying the resistance gene blaCTX-M-15 in ESBL-producing Escherichia coli clones from pediatric patients.

INTRODUCTION: The emergence of extended-spectrum ß-lactamases (ESBLs)-producing Escherichia coli clones are a public health concern worldwide. Scarce information does exist about the spread of ESBLs-producing E. coli in pediatric patients from developing countries. METHODOLOGY: E. coli strains were analyzed by multilocus-sequence-typing, pulsed-field-gel-electrophoresis and phylogenetic group. The antimicrobial-resistance genes were detected by PCR, and plasmid content by the PCR-based replicon-typing. Horizontal transfer was tested by conjugation and the location of the blaCTX-M-15 gene by Southern blot hybridization. RESULTS: Thirty-two cefotaxime-resistant E. coli were recovered. Eleven of them were ESBL-producing isolates, which were well characterized and ascribed to seven sequence types and five phylogroups. The ESBL CTX-M-15 was the most prevalent enzyme (9 of 11). Plasmids of variable sizes (40-220 kb) were visualized, and the incompatibility (Inc) group FIB plasmid-replicon was detected in the ESBL strains and transferred by conjugation in 45.45% of them. Plasmid-borne toxin-antitoxin systems were the most frequently detected systems, strongly associated to IncF plasmids. The CTX-M-15-encoding gene was located on IncFIB plasmids. CONCLUSIONS: Even though a small number of ESBL-producing strains was recovered, we evidenced that IncFIB plasmids carry the blaCTX-M-15 gene, highlighting the role of IncF-type plasmids in facilitating the spread and maintenance of ESBL-encoding genes, which further favors the rapid increase of the antimicrobial resistance dissemination in disease-causing E. coli strains in pediatric patients.

Genomic Insights into Drug Resistance and Virulence Platforms, CRISPR-Cas Systems and Phylogeny of Commensal E. coli from Wildlife.

Commensal bacteria act as important reservoirs of virulence and resistance genes. However, existing data are generally only focused on the analysis of human or human-related bacterial populations. There is a lack of genomic studies regarding commensal bacteria from hosts less exposed to antibiotics and other selective forces due to human activities, such as wildlife. In the present study, the genomes of thirty-eight E. coli strains from the gut of various wild animals were sequenced. The analysis of their accessory genome yielded a better understanding of the role of the mobilome on inter-bacterial dissemination of mosaic virulence and resistance plasmids. The study of the presence and composition of the CRISPR/Cas systems in E. coli from wild animals showed some viral and plasmid sequences among the spacers, as well as the relationship between CRISPR/Cas and E. coli phylogeny. Further, we constructed a single nucleotide polymorphisms-based core tree with E. coli strains from different sources (humans, livestock, food and extraintestinal environments). Bacteria from humans or highly human-influenced settings exhibit similar genetic patterns in CRISPR-Cas systems, plasmids or virulence/resistance genes-carrying modules. These observations, together with the absence of significant genetic changes in their core genome, suggest an ongoing flow of both mobile elements and E. coli lineages between human and natural ecosystems.

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

This study aimed at determining the mechanisms of linezolid resistance and the molecular characteristics of clinical Staphylococcus aureus (n = 2) and coagulase-negative staphylococci (n = 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 cfr gene was determined by whole-genome sequencing. The cfr gene was detected in one methicillin-resistant S. aureus (MRSA) that also displayed the amino acid change Val118Ala in the ribosomal protein L4. The second S. aureus isolate was methicillin susceptible and showed different alterations in the ribosomal protein L4. All remaining linezolid-resistant Staphylococcus epidermidis (n = 14) and Staphylococcus hominis isolates (n = 1) showed the mutation G2576T (n = 14) or C2534T (n = 1) in the 23S rRNA. Moreover, different amino acid changes were detected in the ribosomal proteins L3 and L4 in S. epidermidis isolates. All S. epidermidis 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 mecA gene. The cfr gene in the MRSA isolate was located together with the fexA gene on a conjugative 38,864 bp plasmid. Linezolid- and methicillin-resistant S. epidermidis 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.

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

Extended-spectrum beta-lactamase (ESBL)- and carbapenemase (CP)-producing Klebsiella pneumoniae 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 K. pneumoniae 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 K. pneumoniae 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. K. pneumoniae 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 K. pneumoniae isolates were ESBL producers, and they carried the following beta-lactamase genes: blaCTX-M-15+blaSHV-28 (four isolates, in three cases associated with blaTEM-1), blaCTX-M-15+blaSHV-1 (five isolates, associated with TEM-1 in three cases), and blaSHV-28+blaTEM-1 (six isolates). Three ESBL-producing K. pneumoniae 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 K. pneumonia isolates, harboring mostly genes encoding CTX-M-15 or SHV-28, associated in some cases with the high-risk clones ST307 and ST15.

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

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 lukF/S-PV, tst, eta, etb, etd and scn was investigated by PCR. Moreover, CoNS carrying the mecA gene were subjected to SCCmec 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 S. sciuri (n = 118) and S. lentus (n = 25). A total of 34% of CoNS isolates showed a multidrug resistance phenotype, and 42 mecA-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/ blaZ, mecA), cefoxitin (24/ mecA), erythromycin and/or clindamycin (92/ erm(B), erm(C), erm(43), msr(A), mph(C), lnu(A), lsa(B), vga(A) and sal(A)), gentamicin and/or tobramycin (5/ aac(6')-Ie-aph(2″)-Ia, ant(4')-Ia), streptomycin (12/str), tetracycline (17/ tet(K), tet(L), tet(M)), ciprofloxacin (4), chloramphenicol (1/ fexA), fusidic acid (86/ fusB, fusD) and trimethoprim-sulfamethoxazole (1/ dfrK). None of the isolates harbored the lukF/S-PV, eta, etb, etd and scn genes, but two S. sciuri isolates (1%) carried the tst gene. Wild birds are frequently colonized by CoNS species, especially S. sciuri. 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.

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

The mechanisms of linezolid resistance among 13 E. faecalis and 6 E. faecium 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 optrA gene was detected in all 13 E. faecalis isolates; and one optrA-positive isolate also carried the recently described cfr(D) gene. Moreover, one E. faecalis isolate displayed the nucleotide mutation G2576T in the 23S rDNA. This mutation was also present in all six E. faecium isolates. All linezolid-resistant enterococci showed a multiresistance phenotype and harbored several antimicrobial resistance genes, as well as many virulence determinants. The fexA gene was located upstream of the optrA gene in 12 of the E. faecalis isolates. Moreover, an erm(A)-like gene was located downstream of optrA in two isolates recovered from the same hospital. The optrA gene was transferable in all but one E. faecalis isolates, in all cases along with the fexA gene. The cfr(D) gene was not transferable. The presence of optrA and mutations in the 23S rDNA are the main mechanisms of linezolid resistance among E. faecalis and E. faecium, respectively. We report the first description of the cfr(D) gene in E. faecalis. The presence of the optrA and cfr(D) genes in Spanish hospitals is a public health concern.

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

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.

Antimicrobial Resistance in Enterococcus spp. of animal origin.

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, Enterococcus is an important opportunistic pathogen, especially the species E. faecalis and E. faecium, 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 optrA and cfr, is highlighted. The molecular epidemiology and the population structure of E. faecalis and E. faecium 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.

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

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 [blaNDM-1(8), blaNDM-1+blaOXA-48(1), blaOXA-48(4)], P. mirabilis [blaOXA-48(1)], E. cloacae [blaVIM-2(1)] and A. baumannii [blaOXA-23(3)]. K. pneumoniae isolates were typed as ST15, ST1412 and ST147 and showed seven different pulsotypes. The genetic structure surrounding blaNDM-1 was composed of ISAba125 and ble. The blaVIM-2 carried by E. cloacae was located within the variable region of a class1 integron and blaOXA-48 gene was inserted into Tn1999.2. IncA/C and IncFIIA replicons were implicated in dissemination of blaNDM-1 and a non-typeable 48.5 kb plasmid in the propagation of blaOXA-48. 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.

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

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.

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.