Occurrence of Pseudomonas spp. in Raw Vegetables: Molecular and Phenotypical Analysis of Their Antimicrobial Resistance and Virulence-Related Traits.

Pseudomonas is characterized by its great capacity to colonize different ecological niches, but also by its antimicrobial resistance and pathogenicity, causing human, animal, or plant diseases. Raw and undercooked food is a potential carrier of foodborne disease. The aim of this study was to determine the occurrence of Pseudomonas spp. among raw vegetables, analysing their antimicrobial resistance, virulence, and molecular typing. A total of 163 Pseudomonas spp. isolates (12 different species) were recovered from 77 of the 145 analysed samples (53.1%) and were classified into 139 different pulsed-field gel electrophoresis patterns. Low antimicrobial resistance levels, but one multidrug-resistant isolate, were found. Among the 37 recovered P. aeruginosa strains, 28 sequence-types and nine serotypes were detected. Eleven OprD patterns and an insertion sequence (ISPa1635) truncating the oprD gene of one imipenem-resistant strain were found. Ten virulotypes were observed, including four exoU-positive and thirty-one exoS-positive strains. The lasR gene was absent in three ST155 strains and was truncated by different insertion sequences (ISPre2, IS1411, and ISPst7) in other three strains. High biofilm, motility, pigment, elastase, and rhamnolipid production were detected. Our study demonstrated a low occurrence of P. aeruginosa (18%) and low antimicrobial resistance, but a high number of virulence-related traits in these P. aeruginosa strains, highlighting their pathological importance.

Characterization of Pseudomonas aeruginosa isolated from various environmental niches: New STs and occurrence of antibiotic susceptible "high-risk clones".

The aim of this study was to investigate the antimicrobial phenotypes, major virulence factors, and the molecular typing of 66 P. aeruginosa isolates collected from various sources: human patients and hospital environment, raw milk, poultry meat, chicken/sheep fecal samples, wastewater, thermal water, and seawater. All isolates, except one, were susceptible to all tested antibiotics. exoA, lasB, rhlR, and lasR genes were harbored by 60 isolates. Forty-six, 18, and 2 isolates amplified exoS, exoU, and exoS+exoU genes, respectively. Twenty-one isolates showed high elastase and pigment production. The PFGE typing identified 26 pulsotypes. Some pulsotypes included isolates from different environmental niches and areas. Twelve selected isolates were typed by MLST and eight different STs were found, three of them were new. Our results highlighted the dissemination of some clones amongst different settings and the occurrence of antibiotic susceptible 'high-risk clones' that might be very harmful when acquiring genes encoding antibiotic resistance.

Macrolide resistance mechanisms in Enterobacteriaceae: Focus on azithromycin.

From its introduction in 1952 onwards, the clinical use of macrolides has been steadily increasing, both in human and veterinary medicine. Although initially designed to the treatment of Gram-positive microorganisms, this antimicrobial family has also been used to treat specific Gram-negative bacteria. Some of them, as azithromycin, are considered in the armamentarium against Enterobacteriaceae infections. However, the facility that this bacterial genus has to gain or develop mechanisms of antibiotic resistance may compromise the future usefulness of these antibiotics to fight against Enterobacteriaceae infections. The present review is focused on the mechanisms of macrolide resistance, currently described in Enterobacteriaceae.

Pseudomonas aeruginosa from river water: antimicrobial resistance, virulence and molecular typing.

Pseudomonas aeruginosa isolates were recovered from surface river water samples in La Rioja region (Spain) to characterise their antibiotic resistance, molecular typing and virulence mechanisms. Fifty-two P. aeruginosa isolates were isolated from 15 different water samples (45.4%) and belonged to 23 different pulsed-field electrophoresis (PFGE) patterns. All isolates were susceptible to all antibiotics tested, except one carbapenem-resistant P. aeruginosa that showed a premature stop codon in OprD porin. Twenty-two sequence types (STs) (six new ones) were detected among 29 selected P. aeruginosa (one strain with a different PFGE pattern per sample), with ST274 (14%) being the most frequent one. O:6 and O:3 were the predominant serotypes (31%). Seven virulotypes were detected, being 59% exoS-exoY-exoT-exoA-lasA-lasB-lasI-lasR-rhlAB-rhlI-rhlR-aprA-positive P. aeruginosa. It is noteworthy that the exlA gene was identified in three strains (10.3%), and the exoU gene in seven (24.1%), exoS in 18 (62.1%), and both exoS and exoU genes in one strain. High motility ranges were found in these strains. Twenty-seven per cent of strains produced more biofilm biomass, 90% more pyorubin, 83% more pyocyanin and 65.5% more than twice the elastase activity compared with the PAO1 strain. These results highlight the importance of rivers as temporary reservoirs and sources of P. aeruginosa transmission, and show the importance of their epidemiological surveillance in the environment.

Spread of Pseudomonas aeruginosa ST274 Clone in Different Niches: Resistome, Virulome, and Phylogenetic Relationship.

Pseudomonas aeruginosa ST274 is an international epidemic high-risk clone, mostly associated with hospital settings and appears to colonize cystic fibrosis (CF) patients worldwide. To understand the relevant mechanisms for its success, the biological and genomic characteristics of 11 ST274-P. aeruginosa strains from clinical and non-clinical origins were analyzed. The extensively drug-resistant (XDR/DTR), the non-susceptible to at least one agent (modR), and the lasR-truncated (by ISPsp7) strains showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity and low motility. Furthermore, the XDR/DTR and modR strains presented low pigment production and biofilm formation, which were very high in the lasR-truncated strain. Their whole genome sequences were compared with other 14 ST274-P. aeruginosa genomes available in the NCBI database, and certain associations have been primarily detected: blaOXA-486 and blaPDC-24 genes, serotype O:3, exoS+/exoU- genotype, group V of type IV pili, and pyoverdine locus class II. Other general molecular markers highlight the absence of vqsM and pldA/tleS genes and the presence of the same mutational pattern in genes involving two-component sensor-regulator systems PmrAB and CreBD, exotoxin A, quorum-sensing RhlI, beta-lactamase expression regulator AmpD, PBP1A, or FusA2 elongation factor G. The proportionated ST274-P. aeruginosa results could serve as the basis for more specific studies focused on better antibiotic stewardship and new therapeutic developments.

Whole Genome Analysis of Environmental Pseudomonas mendocina Strains: Virulence Mechanisms and Phylogeny.

Pseudomonas mendocina is an environmental bacterium, rarely isolated in clinical specimens, although it has been described as producing endocarditis and sepsis. Little is known about its genome. Whole genome sequencing can be used to learn about the phylogeny, evolution, or pathogenicity of these isolates. Thus, the aim of this study was to analyze the resistome, virulome, and phylogenetic relationship of two P. mendocina strains, Ps542 and Ps799, isolated from a healthy Anas platyrhynchos fecal sample and a lettuce, respectively. Among all of the small number of P.mendocina genomes available in the National Center for Biotechnology Information (NCBI) repository, both strains were placed within one of two well-defined phylogenetic clusters. Both P. mendocina strains lacked antimicrobial resistance genes, but the Ps799 genome showed a MOBP3 family relaxase. Nevertheless, this study revealed that P. mendocina possesses an important number of virulence factors, including a leukotoxin, flagella, pili, and the Type 2 and Type 6 Secretion Systems, that could be responsible for their pathogenesis. More phenotypical and in vivo studies are needed to deepen the association with human infections and the potential P. mendocina pathogenicity.

Dissemination of a multidrug resistant CTX-M-65 producer Salmonella enterica serovar Infantis clone between marketed chicken meat and children.

The objective of the present study was to characterize Salmonella enterica serovar Infantis isolated from chicken meat determining their clonal relationships with S. Infantis isolated from children with diarrhea. Fifteen meat-recovered S. Infantis were analyzed. Susceptibility levels to 14 antibacterial agents, the presence of ESBL and that of inducible plasmid-mediated AmpC (i-pAmpC) were determined by phenotypical methods. The presence of ESBL and pAmpC was confirmed by PCR, and detected ESBL-encoding genes were sequenced and their transferability tested by conjugation. The presence of gyrA mutations as well as Class 1 integrons was determined by PCR. Clonal relationships were established by REP-PCR and RAPD. In addition, 25 clinical isolates of S. Infantis were included in clonality studies. All meat-recovered S. Infantis were MDR, showing resistance to ampicillin, nitrofurans and quinolones, while none was resistant to azithromycin, ceftazidime or imipenem. ESBL (blaCTX-M-65) and i-pAmpC (blaDHA) were detected in 2 and 5 isolates respectively (in one case concomitantly), with blaCTX-M-65 being transferable through conjugation. In addition, 1 isolate presented a blaSHV gene. All isolates presented D87Y at GyrA, nalidixic acid active efflux pump and a Class 1 integron of ~1000 bp (aadA1). Clonal analysis showed that all isolates were related. Further they were identical to MDR blaCTX-M-65-producing S. Infantis isolates causing children diarrhea in Lima. The dissemination of MDR blaCTX-M-65-producing S. Infantis between marketed meat and children highlights a public health problem which needs be controlled at livestock level.

The first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant.

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

Nitrofuran resistance in Salmonella enterica isolated from meat for human consumption. / Resistencia a nitrofuranos en Salmonella enterica aisladas de carne para consumo humano.

The mechanisms of resistance to nitrofurans from 18 meat samples with Salmonella enterica (chicken: 15; beef: 2; pork: 1) collected in Lima (Peru) were analyzed. The isolates were serotyped and the susceptibility levels to furazolidone and nitrofurantoin [with and without the efflux pump inhibitor Phenyl-Arginine- ß-naphthylamide (PAßN)], the presence of mutations in the snrA and cnr genes and the transferability of resistance by conjugation were established. Fifteen samples with S. infantis (13 from chicken samples), 2 with S. enteritidis and 1 with S. anatum were identified. All isolates except the S. anatum were resistant to both nitrofurans showing MICs (minimum inhibitory concentration) of furazolidone and nitrofurantoin of 32-64 µg/mL and 128-256 µg/mL, respectively. The addition of PAßN had no effect on the MIC levels. All nitrofuran-resistant isolates showed amino acid codon alterations at both snrA and cnr (S. infantis: snrA STOP-151; cnr STOP-137; S. enteritidis: snrA STOP-180; cnr STOP-179). No transferable mecha nisms of nitrofuran resistance were detected.

En el presente estudio, se analizaron los mecanismos de resistencia a nitrofuranos en 18 muestras cár nicas con Salmonella enterica (15 de pollo, 2 de ternera y 1 de cerdo) de mercados de Lima (Perú). Determinaron los serotipos de los aislamientos y la sensibilidad a furazolidona y nitrofurantoina (con y sin el inhibidor de bombas de expulsión Phenyl-Arginine-ß-Naphthylamide [PAßN]), las mutaciones en los genes snrA y cnr por PCR y la transferabilidad de la resistencia por conjugación. Se identificaron 15 muestras con S. infantis (13 muestras de pollo), 2 con S. enteritidis y 1 con S. anatum. Todos los aisla mientos, excepto S. anatum, fueron resistentes a ambos nitrofuranos (concentración mínima inhibidora [CMI] a furazolidona: 32-64 µg/mL, CMI a nitrofurantoina: 128-256 µg/mL), sin diferencias al adicio narse PAßN. Todos los aislamientos resistentes a nitrofuranos presentaron sustituciones en snrA y cnr (S. infantis: snrA STOP-151; cnr STOP-137; S. enteritidis: snrA STOP-180; cnr STOP-179). No se detectaron mecanismos transferibles de resistencia a nitrofuranos.

Antimicrobial resistance and virulence of Pseudomonas spp. among healthy animals: concern about exolysin ExlA detection.

Pseudomonas is a ubiquitous genus that also causes human, animal and plant diseases. Most studies have focused on clinical P. aeruginosa strains from humans, but they are scarce on animal strains. This study was aimed to determine the occurrence of Pseudomonas spp. among faecal samples of healthy animals, and to analyse their antimicrobial resistance, and pathogenicity. Among 704 animal faecal samples analysed, 133 Pseudomonas spp. isolates (23 species) were recovered from 46 samples (6.5%), and classified in 75 different PFGE patterns. Low antimicrobial resistance levels were found, being the highest to aztreonam (50.3%). Five sequence-types (ST1648, ST1711, ST2096, ST2194, ST2252), two serotypes (O:3, O:6), and three virulotypes (analysing 15 virulence and quorum-sensing genes) were observed among the 9 P. aeruginosa strains. Type-3-Secretion System genes were absent in the six O:3-serotype strains that additionally showed high cytotoxicity and produced higher biofilm biomass, phenazine pigments and motility than PAO1 control strain. In these six strains, the exlAB locus, and other virulence genotypes (e.g. RGP69 pathogenicity island) exclusive of PA7 outliers were detected by whole genome sequencing. This is the first description of the presence of the ExlA exolysin in P. aeruginosa from healthy animals, highlighting their pathological importance.

Azithromycin resistance levels and mechanisms in Escherichia coli.

Despite azithromycin being used in some countries to treat infections caused by Gram-negative pathogens, no resistance breakpoint for Escherichia coli exists. The aim of this study was to analyse the levels and mechanisms of azithromycin resistance in E. coli. The presence of chromosomal (rplD, rplV and 23S rRNA) mutations, 10 macrolide resistance genes (MRGs) and efflux pump overexpression was determined in 343 E. coli isolates. Overall, 89 (25.9%) isolates had MICs ≥ 32 mg/L to azithromycin, decreasing to 42 (12.2%) when assayed in the presence of Phe-Arg-ß-Napthylamide, with 35 of these 42 possessing at least one MRG. Efflux pumps played a role in azithromycin resistance affecting the Minimal Inhibitory Concentration (MIC) levels of 91.2% isolates whereas chromosomal alterations seem to have a minimal role. At least one MRG was found in 22.7% of the isolates with mph(A) being the most commonly found gene. The mph(A) gene plays the main role in the development of azithromycin resistance and 93% of the mph(A)-carrying isolates showed a MIC of 32 mg/L. In the absence of a specific resistance breakpoint our results suggest a MIC of 32 mg/L to be considered in order to detect isolates carrying mechanisms able to confer azithromycin resistance.

[Presence of multidrug resistant Enterobacteriaceae and Escherichia coli in meat purchased in traditional markets of Lima]. / Presencia de Enterobacteriaceae y Escherichia coli multirresistente a antimicrobianos en carne adquirida en mercados tradicionales en Lima.

OBJECTIVE.: The objective of this study was to describe the presence of Enterobacteriaceae in meat samples collected in traditional markets of Lima and to establish the levels of antimicrobial resistance and the presence of extended spectrum beta-lactamases (BLEE) and AmpC in Escherichia coli. MATERIALS AND METHODS.: A total of 138 meat samples, 64 (46.4%) chicken, 44 (31.9%) beef and 30 (21.7%) pork were collected. The isolated bacteria belonged to 17 different genera and, specifically, 14 were classified as Enterobacteriaceae. Sensitivity to ten antimicrobial agents was analyzed using the Kirby-Bauer disc diffusion method, BLEE and AmpC were determined by double disc and imipenem-ceftazidime induction tests, respectively. RESULTS.: Antimicrobial resistance levels were high against trimethoprim-sulfamethoxazole, ampicillin, tetracycline, nalidixic acid, ciprofloxacin and chloramphenicol. There are significant differences in antibiotic resistance levels depending on the type of meat (chicken, beef and pork) (p <0.05). Multiple drug resistance (MDR) levels were particularly high in chicken and pork (98.2% and 86.4%, respectively). In addition, the presence of BLEE in Escherichia coli isolated from chicken meat was 59.4%. CONCLUSIONS.: Multiple drug resistance levels were high compared to antibiotics frequently used in humans; chicken and beef are highlighted as potential reservoirs of BLEE and pAmpC-producing Escherichia coli, respectively.

OBJETIVOS.: El objetivo del presente estudio fue describir la presencia de Enterobacteriaceae en muestras de carne recolectadas en mercados tradicionales de Lima y establecer los niveles de resistencia a antimicrobianos y la presencia de betalactamasas de espectro extendido (BLEE) y AmpC en Escherichia coli. MATERIALES Y MÉTODOS.: Se recolectaron un total de 138 muestras de carne, 64 (46,4 %) de pollo, 44 (31,9 %) de carne de res y 30 (21,7%) de carne de cerdo. Las bacterias aisladas pertenecieron a 17 géneros diferentes, y específicamente 14 fueron clasificados como Enterobacteriaceae. Se analizó la sensibilidad frente a diez agentes antimicrobianos mediante el método de difusión de disco Kirby-Bauer, se determinó la presencia de BLEE y AmpC mediante las pruebas de doble disco y de inducción de imipenem-ceftazidima, respectivamente. RESULTADOS.: Los niveles de resistencia a los antimicrobianos fueron altos frente a trimetoprima-sulfametoxazol, ampicilina, tetraciclina, ácido nalidíxico, ciprofloxacino y cloranfenicol. Existen diferencias significativas en los niveles de resistencia a los antibióticos según el tipo de carne (pollo, carne de res y cerdo) (p <0,05). Los niveles de resistencia a múltiples antimicrobianos (MDR) fueron particularmente altos en pollo y cerdo (98,2 % y 86,4 %, respectivamente). Además, la presencia de BLEE en Escherichia coli aisladas de carne de pollo fue del 59,4 %. CONCLUSIONES.: Los niveles de resistencia a los antimicrobianos fueron altos frente a los antibióticos usados frecuentemente en humanos, se destaca el pollo y la res como potenciales reservorios de Escherichia coli productoras de BLEE y pAmpC, respectivamente.

Differences in tetracycline resistance determinant carriage among Shigella flexneri and Shigella sonnei are not related to different plasmid Inc-type carriage.

OBJECTIVES: The aim of this study was to establish the prevalence of the most common molecular mechanisms involved in tetracycline resistance as well as their relationship with plasmid incompatibility (Inc) groups in a collection of Shigella spp. causing traveller's diarrhoea. METHODS: Tetracycline susceptibility was established in 187 Shigella spp. (74 Shigella flexneri and 113 Shigella sonnei), of which 153 isolates were recovered as a confirmed cause of traveller's diarrhoea. The prevalence of the tet(A), tet(B) and tet(G) genes was analysed by PCR. Eighteen plasmid Inc groups was determined in a subset of 59 isolates. RESULTS: Among 154 tetracycline-resistant isolates, 122 (79.2%) harboured at least tet(A) or tet(B). The tet(B) gene was the most frequently detected, being present in 70 isolates (45.5%), whilst tet(A) was detected in 57 isolates (37.0%). The tet(G) gene was present in only 11 (7.2%) isolates. Moreover, the tet(A) gene was more frequent in S. sonnei (P=0.0007), whilst the tet(B) gene was more frequent in S. flexneri (P<0.0001). Plasmids belonging to Inc group B (P<0.05) were significantly more frequent among S. flexneri, whilst those belonging to groups K, FIC and FIIA (P<0.05) were preferentially detected among S. sonnei. CONCLUSION: The prevalence of the tet(A) and tet(B) genes differed between S. sonnei and S. flexneri. Moreover, the prevalence of plasmid Inc groups in S. flexneri and S. sonnei differed. However, no relationship was found between the two phenomena.

Pseudomonas aeruginosa Isolates from Spanish Children: Occurrence in Faecal Samples, Antimicrobial Resistance, Virulence, and Molecular Typing.

Pseudomonas aeruginosa is a major opportunistic human pathogen, responsible for nosocomial infections and infections in patients with impaired immune systems. Little data exist about the faecal colonisation by P. aeruginosa isolates in healthy humans. The occurrence, antimicrobial resistance phenotype, virulence genotype, and genetic lineages of P. aeruginosa from faecal samples of children from two different Spanish regions were characterised. Seventy-two P. aeruginosa were isolated from 1,443 faecal samples. Low antimicrobial resistance levels were detected: ceftazidime (8%), cefepime (7%), aztreonam (7%), gentamicin (3%), ciprofloxacin (1%), and imipenem (1%); susceptibility to meropenem, amikacin, tobramycin, levofloxacin, and colistin. Four multidrug-resistant strains were found. Important differences were detected between both geographical regions. Forty-one sequence types were detected among the 48 tested strains. Virulence and quorum sensing genes were analysed and 13 virulotypes were detected, being 26 exoU-positive strains. Alteration in protein OprD showed eight different patterns. The unique imipenem-resistant strain showed a premature stop codon in OprD. Intestinal colonisation by P. aeruginosa, mainly by international clones (as ST244, ST253, and ST274), is an important factor for the systemic infections development and the environmental dissemination. Periodic active surveillance is useful to identify these community human reservoirs and to control the evolution of antibiotic resistance and virulence activity.

Development and characterisation of highly antibiotic resistant Bartonella bacilliformis mutants.

The objective was to develop and characterise in vitro Bartonella bacilliformis antibiotic resistant mutants. Three B. bacilliformis strains were plated 35 or 40 times with azithromycin, chloramphenicol, ciprofloxacin or rifampicin discs. Resistance-stability was assessed performing 5 serial passages without antibiotic pressure. MICs were determined with/without Phe-Arg-ß-Napthylamide and artesunate. Target alterations were screened in the 23S rRNA, rplD, rplV, gyrA, gyrB, parC, parE and rpoB genes. Chloramphenicol and ciprofloxacin resistance were the most difficult and easiest (>37.3 and 10.6 passages) to be selected, respectively. All mutants but one selected with chloramphenicol achieved high resistance levels. All rifampicin, one azithromycin and one ciprofloxacin mutants did not totally revert when cultured without antibiotic pressure. Azithromycin resistance was related to L4 substitutions Gln-66 → Lys or Gly-70 → Arg; L4 deletion Δ62-65 (Lys-Met-Tyr-Lys) or L22 insertion 83::Val-Ser-Glu-Ala-His-Val-Gly-Lys-Ser; in two chloramphenicol-resistant mutants the 23S rRNA mutation G2372A was detected. GyrA Ala-91 → Val and Asp-95 → Gly and GyrB Glu474 → Lys were detected in ciprofloxacin-resistant mutants. RpoB substitutions Gln-527 → Arg, His-540 → Tyr and Ser-545 → Phe plus Ser-588 → Tyr were detected in rifampicin-resistant mutants. In 5 mutants the effect of efflux pumps on resistance was observed. Antibiotic resistance was mainly related to target mutations and overexpression of efflux pumps, which might underlie microbiological failures during treatments.

Virulence factors profiles and ESBL production in Escherichia coli causing bacteremia in Peruvian children.

The presence of 25 virulence genes (VGs), genetic phylogroups, quinolone-resistance and Extended Spectrum ß-lactamase (ESBL)-production was assessed in 65 Escherichia coli isolates from blood cultures in children <5 years in Peru. The most frequent VGs were fimA (89.2%), iutA (83.1%), agn43 (72.3%), iucA (67.7%), and fyuA (49.2%). The isolates belonged to D (47.7%), A (26.1%), B1 (21.5%), and B2 (4.6%) phylogroups. D + B2 isolates presented a high number of fimA, hly, papC, sat, and fyuA genes. Quinolone-susceptible (22 isolates - 33.8%) and ESBL-negative (31 isolates - 47.7%) isolates carried more VGs that their respective counterparts (5.7 vs. 4.7 and 5.3 vs. 4.4 respectively); the frequency of the fyuA, aat, aap, and hly genes significantly differed between quinolone-resistant and quinolone-susceptible isolates. Neonatal sepsis isolates tended to be more quinolone-resistant (P = 0.0697) and ESBL-producers (P = 0.0776). Early-onset neonatal sepsis isolates possessed a high number of VGs (5.2 VGs), especially in neonates of ≤1 day (5.9 VGs).

Development and analysis of furazolidone-resistant Escherichia coli mutants.

Furazolidone-resistant mutants were obtained from four clinical isolates of diarrhoeagenic Escherichia coli. The stability of the resistance and the frequency of mutation were established. The minimal inhibitory concentration of furazolidone, nitrofurantoin, nalidixic acid, ampicillin, chloramphenicol and tetracycline was established both in the presence and absence of the efflux pump inhibitor Phe-Arg-ß-Naphtylamyde. The presence of mutations in the nitroreductase genes nfsA and nfsB was analysed by PCR; sequencing and their enzymatic activity was assessed by a spectrophotometric assay. Alterations in outer membrane proteins were studied by SDS-PAGE. The frequency of mutation ranged from <9.6 × 10(-10) to 9.59 × 10(-7) . Neither an effect on efflux pumps inhibited by Phe-Arg-ß-Naphtylamyde nor cross-resistance with the antibiotics studied was observed. Nineteen mutants (52.94%) presented mutations in the nitroreductase-encoding genes: 17 in the nfsA gene (15 mutants with an internal stop codon, 2 with amino acid changes), 2 in the nfsB (all amino acid changes). Alterations in the outer membrane proteins OmpA and OmpW were also observed. Although more studies are necessary to find other resistance mechanisms, present data showed the low potential of selecting furazolidone-resistant mutants, together with the lack of cross-resistance with unrelated antimicrobial agents.

Resistencia a nitrofuranos en Salmonella enterica aisladas de carne para consumo humano / Nitrofuran resistance in Salmonella enterica isolated from meat for human consumption

RESUMEN En el presente estudio, se analizaron los mecanismos de resistencia a nitrofuranos en 18 muestras cár nicas con Salmonella enterica (15 de pollo, 2 de ternera y 1 de cerdo) de mercados de Lima (Perú). Determinaron los serotipos de los aislamientos y la sensibilidad a furazolidona y nitrofurantoina (con y sin el inhibidor de bombas de expulsión Phenyl-Arginine-β-Naphthylamide [PAβN]), las mutaciones en los genes snrA y cnr por PCR y la transferabilidad de la resistencia por conjugación. Se identificaron 15 muestras con S. infantis (13 muestras de pollo), 2 con S. enteritidis y 1 con S. anatum. Todos los aisla mientos, excepto S. anatum, fueron resistentes a ambos nitrofuranos (concentración mínima inhibidora [CMI] a furazolidona: 32-64 µg/mL, CMI a nitrofurantoina: 128-256 µg/mL), sin diferencias al adicio narse PAβN. Todos los aislamientos resistentes a nitrofuranos presentaron sustituciones en snrA y cnr (S. infantis: snrA STOP-151; cnr STOP-137; S. enteritidis: snrA STOP-180; cnr STOP-179). No se detectaron mecanismos transferibles de resistencia a nitrofuranos.

ABSTRACT The mechanisms of resistance to nitrofurans from 18 meat samples with Salmonella enterica (chicken: 15; beef: 2; pork: 1) collected in Lima (Peru) were analyzed. The isolates were serotyped and the susceptibility levels to furazolidone and nitrofurantoin [with and without the efflux pump inhibitor Phenyl-Arginine- β-naphthylamide (PAβN)], the presence of mutations in the snrA and cnr genes and the transferability of resistance by conjugation were established. Fifteen samples with S. infantis (13 from chicken samples), 2 with S. enteritidis and 1 with S. anatum were identified. All isolates except the S. anatum were resistant to both nitrofurans showing MICs (minimum inhibitory concentration) of furazolidone and nitrofurantoin of 32-64 μg/mL and 128-256 μg/mL, respectively. The addition of PAßN had no effect on the MIC levels. All nitrofuran-resistant isolates showed amino acid codon alterations at both snrA and cnr (S. infantis: snrA STOP-151; cnr STOP-137; S. enteritidis: snrA STOP-180; cnr STOP-179). No transferable mecha nisms of nitrofuran resistance were detected.

Presencia de Enterobacteriaceae y Escherichia coli multirresistente a antimicrobianos en carne adquirida en mercados tradicionales en Lima / Presence of multidrug resistant Enterobacteriaceae and Escherichia coli in meat purchased in traditional markets of Lima

RESUMEN Objetivos. El objetivo del presente estudio fue describir la presencia de Enterobacteriaceae en muestras de carne recolectadas en mercados tradicionales de Lima y establecer los niveles de resistencia a antimicrobianos y la presencia de betalactamasas de espectro extendido (BLEE) y AmpC en Escherichia coli. Materiales y métodos. Se recolectaron un total de 138 muestras de carne, 64 (46,4 %) de pollo, 44 (31,9 %) de carne de res y 30 (21,7%) de carne de cerdo. Las bacterias aisladas pertenecieron a 17 géneros diferentes, y específicamente 14 fueron clasificados como Enterobacteriaceae. Se analizó la sensibilidad frente a diez agentes antimicrobianos mediante el método de difusión de disco Kirby-Bauer, se determinó la presencia de BLEE y AmpC mediante las pruebas de doble disco y de inducción de imipenem-ceftazidima, respectivamente. Resultados. Los niveles de resistencia a los antimicrobianos fueron altos frente a trimetoprima-sulfametoxazol, ampicilina, tetraciclina, ácido nalidíxico, ciprofloxacino y cloranfenicol. Existen diferencias significativas en los niveles de resistencia a los antibióticos según el tipo de carne (pollo, carne de res y cerdo) (p <0,05). Los niveles de resistencia a múltiples antimicrobianos (MDR) fueron particularmente altos en pollo y cerdo (98,2 % y 86,4 %, respectivamente). Además, la presencia de BLEE en Escherichia coli aisladas de carne de pollo fue del 59,4 %. Conclusiones. Los niveles de resistencia a los antimicrobianos fueron altos frente a los antibióticos usados frecuentemente en humanos, se destaca el pollo y la res como potenciales reservorios de Escherichia coli productoras de BLEE y pAmpC, respectivamente.

ABSTRACT Objective. The objective of this study was to describe the presence of Enterobacteriaceae in meat samples collected in traditional markets of Lima and to establish the levels of antimicrobial resistance and the presence of extended spectrum beta-lactamases (BLEE) and AmpC in Escherichia coli. Materials and Methods. A total of 138 meat samples, 64 (46.4%) chicken, 44 (31.9%) beef and 30 (21.7%) pork were collected. The isolated bacteria belonged to 17 different genera and, specifically, 14 were classified as Enterobacteriaceae. Sensitivity to ten antimicrobial agents was analyzed using the Kirby-Bauer disc diffusion method, BLEE and AmpC were determined by double disc and imipenem-ceftazidime induction tests, respectively. Results. Antimicrobial resistance levels were high against trimethoprim-sulfamethoxazole, ampicillin, tetracycline, nalidixic acid, ciprofloxacin and chloramphenicol. There are significant differences in antibiotic resistance levels depending on the type of meat (chicken, beef and pork) (p <0.05). Multiple drug resistance (MDR) levels were particularly high in chicken and pork (98.2% and 86.4%, respectively). In addition, the presence of BLEE in Escherichia coli isolated from chicken meat was 59.4%. Conclusions. Multiple drug resistance levels were high compared to antibiotics frequently used in humans; chicken and beef are highlighted as potential reservoirs of BLEE and pAmpC-producing Escherichia coli, respectively.