Klebsiella quasipneumoniae subsp. similipneumoniae ST1859 O5:KL35 from Soil: First Report of qnrE1 in the Environment.

A Klebsiella quasipneumoniae subsp. similipneumoniae strain, named S915, belonging to the ST1859 O5:KL35, and harboring the plasmid-mediated quinolone resistance qnrE1 gene, was isolated from a soil sample cultivated with lettuce in Brazil. The core genome multilocus sequence typing analysis revealed that S915 strain was most related to a clinical strain of Brazil. Comparative genomic analysis showed that ST1859 O5:KL35 strains have been circulating in clinical settings and are closely related to multidrug resistance and multimetal tolerance. Strain S915 presented a plasmid contig co-harboring the qnrE1 gene and tellurite tolerance operon. The region harboring the qnrE1 gene (ISEcp1-qnrE1-araJ-ahp) shared high similarity with others from infected humans, ready-to-eat dish, and food-producing animals in Brazil. This is the first report of the plasmid-mediated qnrE1 gene in the environment. Our findings evidence the initial dissemination of the qnrE1 gene in the environment by the introduction of a clinical strain, which may be spread to different sectors, representing a One Health challenge.

Genomic analysis of fluoroquinolone-resistant Leclercia adecarboxylata carrying the ISKpn19-orf-qnrS1-ΔIS3-blaLAP-2 module in a synanthropic pigeon, Brazil.

OBJECTIVES: The aim of this study was to perform a genomic investigation of a multiple fluoroquinolone-resistant Leclercia adecarboxylata strain isolated from a synanthropic pigeon in São Paulo, Brazil. METHODS: Whole-genome sequencing was performed using an Illumina platform, and in silico deep analyses of the resistome were performed. Comparative phylogenomics was conducted using a global collection of publicly available genomes of L. adecarboxylata strains isolated from human and animal hosts. RESULTS: L. adecarboxylata strain P62P1 displayed resistance to human (norfloxacin, ofloxacin, ciprofloxacin, and levofloxacin) and veterinary (enrofloxacin) fluoroquinolones. This multiple quinolone-resistant profile was associated with mutations in the gyrA (S83I) and parC (S80I) genes and the presence of the qnrS gene within an ISKpn19-orf-qnrS1-ΔIS3-blaLAP-2 module, previously identified in L. adecarboxylata strains isolated from pig feed and faeces in China. Genes associated with arsenic, silver, copper, and mercury resistance were also predicted. Phylogenomic analysis revealed clustering (378-496 single nucleotide polymorphism differences) with two L. adecarboxylata strains isolated from human and fish sources in China and Portugal, respectively. CONCLUSIONS: L. adecarboxylata is a Gram-negative bacterium of the Enterobacterales order and is considered an emergent opportunistic pathogen. Since L. adecarboxylata has adapted to human and animal hosts, genomic surveillance is highly recommended, in order to identify the emergence and spread of resistant lineages and high-risk clones. In this regard, this study provides genomic data that can help clarify the role of synanthropic animals in the dissemination of clinically relevant L. adecarboxylata within a One Health context.

Fluoroquinolonas en aves, huevos, alimentos avícolas y su impacto en la microbiota intestinal de aves / Fluoroquinolone in birds, eggs, poultry feeds and their impact on the intestinal microbiota

Se detectó la presencia de fluoroquinolonas en varios alimentos (huevos, alimentos para aves y pechuga de pollo), así como determinar el perfil de susceptibilidad de ácido nalidíxico y ciprofloxacina de las enterobacterias aisladas del contenido intestinal de pollos de Cumaná. Se estudiaron alimentos iniciadores y de engorde (de cinco marcas comerciales) y uno para gallinas ponedoras, así como pechugas de pollos nacionales y de Brasil. I-2 y E-1 fueron los que tuvieron las concentraciones más altas de enrofloxacina. El alimento para las gallinas ponedoras (AP 2,35 µg/mg) tuvo más enrofloxacina que los de los pollos. En los huevos, la mayor acumulación se vio en las yemas. Los pollos nacionales (0,43-0,56 µg/mg) acumularon más ciprofloxacina que los pollos de Brasil (0,14 µg/mg). De los hisopados rectales de los pollos, E. coli fue la principal especie aislada. Por antibiograma, 48% de las cepas fueron resistentes a las quinolonas probadas (ácido nalidíxico y ciprofloxacina). Cuando se determinó la concentración mínima inhibitoria a ciprofloxacina, todas las cepas fueron resistentes (8-128 µg/ml). Todos los alimentos muestreados exceden los límites máximos de fluoroquinolonas permitidos en humanos, lo cual ejerce una presión selectiva importante en las bacterias de la microbiota intestinal de los pollos

To detect the presence of fluoroquinolones in several foods (eggs, poultry food and chicken breast), as well as to determine the susceptibility profile of nalidixic acid and ciprofloxacin of strains of enterobacterias from chicken's intestinal content from Cumaná. Starter and fattening foods (of five commercial marks), and one for laying hens, were studied, as well as domestic chicken's breast (and Brazil. I-2 and E-1 were the ones with the highest concentrations of enrofloxacin. The food for laying hens (AP 2,35 µg/mg) had more enrofloxacin than those for chickens. In eggs, greatest accumulation was seen in the yolks. Domestic chickens (0,43-0,56 µg/mg) accumulated more ciprofloxacin than Brazilian ones (0,14 µg/mg). E. coli was the main specie from chicken rectal swabs. By antimicrobial susceptibility testing, 48% were resistant to both quinolones (nalidixic acid and ciprofloxacin). When the minimum inhibitory concentration of ciprofloxacin was determined, all strains were resistant (8-128 µg/ml). All sampled foods exceeded the maximum limits of fluoroquinolones allowed in humans, which puts significant selective pressure on the bacteria in the chicken gut microbiota

Multiple Introductions of Salmonella enterica Serovar Typhi H58 with Reduced Fluoroquinolone Susceptibility into Chile.

Salmonella enterica serovar Typhi H58, an antimicrobial-resistant lineage, is globally disseminated but has not been reported in Latin America. Genomic analysis revealed 3 independent introductions of Salmonella Typhi H58 with reduced fluoroquinolone susceptibility into Chile. Our findings highlight the utility of enhanced genomic surveillance for typhoid fever in this region.

The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran

Abstract The aim of this study was to examine mutations in the quinolone-resistance-determining region (QRDR) of gyrA and parC genes in Pseudomonas aeruginosa isolates. A total of 100 clinical P. aeruginosa isolates were collected from different university-affiliated hospitals in Tabriz, Iran. Minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were evaluated by agar dilution assay. DNA sequences of the QRDR of gyrA and parC were determined by the dideoxy chain termination method. Of the total 100 isolates, 64 were resistant to ciprofloxacin. No amino acid alterations were detected in gyrA or parC genes of the ciprofloxacin susceptible or ciprofloxacin intermediate isolates. Thr-83 → Ile substitution in gyrA was found in all 64 ciprofloxacin resistant isolates. Forty-four (68.75%) of them had additional substitution in parC. A correlation was found between the number of the amino acid alterations in the QRDR of gyrA and parC and the level of ciprofloxacin and levofloxacin resistance of the P. aeruginosa isolates. Ala-88 → Pro alteration in parC was generally found in high level ciprofloxacin resistant isolates, which were suggested to be responsible for fluoroquinolone resistance. These findings showed that in P. aeruginosa, gyrA was the primary target for fluoroquinolone and additional mutation in parC led to highly resistant isolates.

The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran

The aim of this study was to examine mutations in the quinolone-resistance-determining region (QRDR) of gyrA and parC genes in Pseudomonas aeruginosa isolates. A total of 100 clinical P. aeruginosa isolates were collected from different university-affiliated hospitals in Tabriz, Iran. Minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were evaluated by agar dilution assay. DNA sequences of the QRDR of gyrA and parC were determined by the dideoxy chain termination method. Of the total 100 isolates, 64 were resistant to ciprofloxacin. No amino acid alterations were detected in gyrA or parC genes of the ciprofloxacin susceptible or ciprofloxacin intermediate isolates. Thr-83 Ile substitution in gyrA was found in all 64 ciprofloxacin resistant isolates. Forty-four (68.75%) of them had additional substitution in parC. A correlation was found between the number of the amino acid alterations in the QRDR of gyrA and parC and the level of ciprofloxacin and levofloxacin resistance of the P. aeruginosa isolates. Ala-88 Pro alteration in parC was generally found in high level ciprofloxacin resistant isolates, which were suggested to be responsible for fluoroquinolone resistance. These findings showed that in P. aeruginosa, gyrA was the primary target for fluoroquinolone and additional mutation in parC led to highly resistant isolates.(AU)

The role of gyrA and parC mutations in fluoroquinolones-resistant Pseudomonas aeruginosa isolates from Iran.

The aim of this study was to examine mutations in the quinolone-resistance-determining region (QRDR) of gyrA and parC genes in Pseudomonas aeruginosa isolates. A total of 100 clinical P. aeruginosa isolates were collected from different university-affiliated hospitals in Tabriz, Iran. Minimum inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were evaluated by agar dilution assay. DNA sequences of the QRDR of gyrA and parC were determined by the dideoxy chain termination method. Of the total 100 isolates, 64 were resistant to ciprofloxacin. No amino acid alterations were detected in gyrA or parC genes of the ciprofloxacin susceptible or ciprofloxacin intermediate isolates. Thr-83 → Ile substitution in gyrA was found in all 64 ciprofloxacin resistant isolates. Forty-four (68.75%) of them had additional substitution in parC. A correlation was found between the number of the amino acid alterations in the QRDR of gyrA and parC and the level of ciprofloxacin and levofloxacin resistance of the P. aeruginosa isolates. Ala-88 → Pro alteration in parC was generally found in high level ciprofloxacin resistant isolates, which were suggested to be responsible for fluoroquinolone resistance. These findings showed that in P. aeruginosa, gyrA was the primary target for fluoroquinolone and additional mutation in parC led to highly resistant isolates.

Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli.

Urinary tract infections (UTI) are among the most frequently encountered infections in clinical practice globally. Predominantly a burden among female adults and infants, UTIs primarily caused by uropathogenic Escherichia coli (UPEC) results in high morbidity and fiscal health strains. During pathogenesis, colonization of the urinary tract via fimbrial adhesion to mucosal cells is the most critical point in infection and has been linked to DNA methylation. Furthermore, with continuous exposure to antibiotics as the standard therapeutic strategy, UPEC has evolved to become highly adaptable in circumventing the effect of antimicrobial agents and host defenses. Hence, the need for alternative treatment strategies arises. Since differential DNA methylation is observed as a critical precursor to virulence in various pathogenic bacteria, this body of work sought to assess the influence of the DNA adenine methylase (dam) gene on gene expression and cellular adhesion in UPEC and its potential as a therapeutic target. To monitor the influence of dam on attachment and FQ resistance, selected UPEC dam mutants created via one-step allelic exchange were transformed with cloned qnrA and dam complement plasmid for comparative analysis of growth rate, antimicrobial susceptibility, biofilm formation, gene expression, and mammalian cell attachment. The absence of DNA methylation among dam mutants was apparent. Varying deficiencies in cell growth, antimicrobial resistance and biofilm formation, alongside low-level increases in gene expression (recA and papI), and adherence to HEK-293 and HTB-9 mammalian cells were also detected as a factor of SOS induction to result in increased mutability. Phenotypic characteristics of parental strains were restored in dam complement strains. Dam's vital role in DNA methylation and gene expression in local UPEC isolates was confirmed. Similarly to dam-deficient Enterohemorrhagic E. coli (EHEC), these findings suggest unsuccessful therapeutic use of Dam inhibitors against UPEC or dam-deficient UPEC strains as attenuated live vaccines. However, further investigations are necessary to determine the post-transcriptional influence of dam on the regulatory network of virulence genes central to pathogenesis.

Susceptibility to b-lactams and quinolones of Enterobacteriaceae isolated from urinary tract infections in outpatients

Abstract The antibiotic susceptibility profile was evaluated in 71 Enterobacteriaceae isolates obtained from outpatient urine cultures in July 2010 from two health institutions in Santa Fe, Argentina. The highest rates of antibiotic resistance were observed for ampicillin (AMP) (69%), trimethoprim/sulfamethoxazole (TMS) (33%), and ciprofloxacin (CIP) (25%). Meanwhile, 21% of the isolates were resistant to three or more tested antibiotics families. Thirty integron-containing bacteria (42.3%) were detected, and a strong association with TMS resistance was found. Third generation cephalosporin resistance was detected in only one Escherichia coli isolate, and it was characterized as a blaCMY-2 carrier. No plasmid-mediated quinolone resistance (PMQR) was found. Resistance to fluoroquinolone in the isolates was due to alterations in QRDR regions. Two mutations in GyrA (S83L, D87N) and one in ParC (S80I) were observed in all CIP-resistant E. coli. It was determined to be the main phylogenetic groups in E. coli isolates. Minimum Inhibitory Concentration (MIC) values against nalidixic acid (NAL), levofloxacin (LEV), and CIP were determined for 63 uropathogenic E. coli isolates as MIC50 of 4 μg/mL, 0.03125 μg/mL, and 0.03125 μg/mL, respectively, while the MIC90 values of the antibiotics were determined as 1024 μg/mL, 64 μg/mL, and 16 μg/mL, respectively. An association between the phylogenetic groups, A and B1 with fluoroquinolone resistance was observed. These results point to the importance of awareness of the potential risk associated with empirical treatment with both the families of antibiotics.(AU)

Susceptibility to β-lactams and quinolones of Enterobacteriaceae isolated from urinary tract infections in outpatients

Abstract The antibiotic susceptibility profile was evaluated in 71 Enterobacteriaceae isolates obtained from outpatient urine cultures in July 2010 from two health institutions in Santa Fe, Argentina. The highest rates of antibiotic resistance were observed for ampicillin (AMP) (69%), trimethoprim/sulfamethoxazole (TMS) (33%), and ciprofloxacin (CIP) (25%). Meanwhile, 21% of the isolates were resistant to three or more tested antibiotics families. Thirty integron-containing bacteria (42.3%) were detected, and a strong association with TMS resistance was found. Third generation cephalosporin resistance was detected in only one Escherichia coli isolate, and it was characterized as a blaCMY-2 carrier. No plasmid-mediated quinolone resistance (PMQR) was found. Resistance to fluoroquinolone in the isolates was due to alterations in QRDR regions. Two mutations in GyrA (S83L, D87N) and one in ParC (S80I) were observed in all CIP-resistant E. coli. It was determined to be the main phylogenetic groups in E. coli isolates. Minimum Inhibitory Concentration (MIC) values against nalidixic acid (NAL), levofloxacin (LEV), and CIP were determined for 63 uropathogenic E. coli isolates as MIC50 of 4 μg/mL, 0.03125 μg/mL, and 0.03125 μg/mL, respectively, while the MIC90 values of the antibiotics were determined as 1024 μg/mL, 64 μg/mL, and 16 μg/mL, respectively. An association between the phylogenetic groups, A and B1 with fluoroquinolone resistance was observed. These results point to the importance of awareness of the potential risk associated with empirical treatment with both the families of antibiotics.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

First report of plasmid-mediated fluoroquinolone efflux pump QepA in Escherichia coli clinical isolate ST68, in South America.

This is the first report of the presence of qepA1 efflux pump gene in Escherichia coli clinical isolate from Argentina, which was associated to other plasmid-mediated quinolone resistance determinants, such as aac(6')-Ib-cr and qnrB10 and also quinolone resistance determining regions mutations.

Contribution of different mechanisms to the resistance to fluoroquinolones in clinical isolates of Salmonella enterica

OBJECTIVES: To study the potential factors include gene mutation, efflux pump and alteration of permeability associated with quinolone-resistance of Salmonella enterica strains isolated from patients with acute gastroenteritis and to evaluate the degree of synergistic activity of efflux pump inhibitors when combined with ciprofloxacin against resistant isolates. METHODS: Antimicrobial resistance patterns of fifty-eight Salmonella isolates were tested. Five isolates were selected to study the mechanism of resistance associated with quinolone group, including mutation in topoisomerase-encoding gene, altered cell permeability, and expression of an active efflux system. In addition, the combination between antibiotics and efflux pump inhibitors to overcome the microbial resistance was evaluated. RESULTS: Five Salmonella isolates totally resistant to all quinolones were studied. All isolates showed alterations in outer membrane proteins including disappearance of some or all of these proteins (Omp-A, Omp-C, Omp-D and Omp-F). Minimum inhibitory concentration values of ciprofloxacin were determined in the presence/absence of the efflux pump inhibitors: carbonyl cyanide m-chlorophenylhydrazone, norepinephrin and trimethoprim. Minimum inhibitory concentration values for two of the isolates were 2-4 fold lower with the addition of efflux pump inhibitors. All five Salmonella isolates were amplified for gyrA and parC genes and only two isolates were sequenced. S. Enteritidis 22 had double mutations at codon 83 and 87 in addition to three mutations at parC at codons 67, 76 and 80 whereas S. Typhimurium 57 had three mutations at codons 83, 87 and 119, but no mutations at parC. CONCLUSIONS: Efflux pump inhibitors may inhibit the major AcrAB-TolC in Salmonella efflux systems which are the major efflux pumps responsible for multidrug resistance in Gramnegative clinical isolates.

Contribution of different mechanisms to the resistance to fluoroquinolones in clinical isolates of Salmonella enterica.

OBJECTIVES: To study the potential factors include gene mutation, efflux pump and alteration of permeability associated with quinolone-resistance of Salmonella enterica strains isolated from patients with acute gastroenteritis and to evaluate the degree of synergistic activity of efflux pump inhibitors when combined with ciprofloxacin against resistant isolates. METHODS: Antimicrobial resistance patterns of fifty-eight Salmonella isolates were tested. Five isolates were selected to study the mechanism of resistance associated with quinolone group, including mutation in topoisomerase-encoding gene, altered cell permeability, and expression of an active efflux system. In addition, the combination between antibiotics and efflux pump inhibitors to overcome the microbial resistance was evaluated. RESULTS: Five Salmonella isolates totally resistant to all quinolones were studied. All isolates showed alterations in outer membrane proteins including disappearance of some or all of these proteins (Omp-A, Omp-C, Omp-D and Omp-F). Minimum inhibitory concentration values of ciprofloxacin were determined in the presence/absence of the efflux pump inhibitors: carbonyl cyanide m-chlorophenylhydrazone, norepinephrin and trimethoprim. Minimum inhibitory concentration values for two of the isolates were 2-4 fold lower with the addition of efflux pump inhibitors. All five Salmonella isolates were amplified for gyrA and parC genes and only two isolates were sequenced. S. Enteritidis 22 had double mutations at codon 83 and 87 in addition to three mutations at parC at codons 67, 76 and 80 whereas S. Typhimurium 57 had three mutations at codons 83, 87 and 119, but no mutations at parC. CONCLUSIONS: Efflux pump inhibitors may inhibit the major AcrAB-TolC in Salmonella efflux systems which are the major efflux pumps responsible for multidrug resistance in Gram-negative clinical isolates.