Rsp activates expression of the Cnt system in Staphylococcus aureus.

BACKGROUND: The Cnt system is crucial for the optimal import of essential metals in metal-limiting conditions and contributes to virulence in S. aureus. In a screen for regulators of efflux pumps in a phage-based ultra-high-density transposon library, we identified Rsp as a candidate regulator of the cntE gene. RESULTS: A two-fold decrease in expression of all genes of the cnt operon was observed by RT-qPCR in the rsp mutant compared to the parental strain, indicating that Rsp acts as an activator of the cnt operon. To determine whether the Rsp activation depends on iron, we compared mutant and parent cnt expression under varying metal conditions. A 2-fold reduction in cnt gene expression was detected in the rsp mutant in TSB, and a slightly smaller decrease (1.9, 1.7, and 1.5-fold changes for cntK, cmtA, and cntE respectively) was observed after addition of dipyridyl. The greatest decrease was seen with addition of FeSO4 (4.1, 5.3 and 6.3-fold changes for cntK, cmtA and cntE respectively). These findings suggest that Rsp activates the cnt operon in low and high iron conditions. To study the relationship between Rsp and the cnt repressors Fur and Zur, we created single and double mutants. Both fur and zur single mutants had significant increases in cnt gene expression compared to the parental strain, as did the fur rsp double mutant. The zur rsp double mutant also had a significant increase in cntK expression and a trend in increases in cntA and cntE expression just below statistical significance. Thus, the ability of Fur and Zur to repress cnt gene expression are not eliminated by the presence of Rsp. However, there were significantly smaller increases in cnt gene expression in the double mutants compared to single mutants, suggesting that Rsp activation can still occur in the absence of these repressors. To determine if Rsp directly modulates expression of cnt genes, incubation of purified Rsp caused a DNA-specific band shift for the cntK and cntA promoters. CONCLUSIONS: Rsp activation may act to maintain basal cellular levels of staphylopine to scavenge free metals when needed, in addition to metal dependent regulation by Fur and Zur.

Plasmids and genes contributing to high-level quinolone resistance in Escherichia coli.

INTRODUCTION: The importance of plasmid-mediated quinolone resistance (PMQR) in Enterobacterales and its high incidence has been emphasised many times. However, a clinical strain carrying more than two PMQR genes is rare. This study sequenced plasmid transconjugants from a donor strain carrying four different PMQR genes to establish their genetic locations. METHODS: An Escherichia coli clinical strain displayed remarkable quinolone resistance with a ciprofloxacin MIC of 1024 mg/L carrying four PMQR genes: qnrA1, qepA1, aac(6')1b-cr and oqxAB. When outcrossed to Escherichia coli J53 AziR, different PMQR genes were transferred and the resulting strains 7C and 8C were chosen for further characterisation. Plasmids were extracted and sequenced by the Illumina and Oxford Nanopore Technologies platforms. S1 nuclease-PFGE was used to estimate the number and size of plasmids. RESULTS: The parental strain had three plasmid bands, as determined by PFGE. Transconjugant 8C obtained three plasmids: pMG336 (162 647 bp, F18:A-:B1:C4) carrying oqxAB; pMG335 carrying qepA1 (73 874 bp, F2:A-:B-); and pMG334 (59 724 bp, IncN (ST5)) with qnrA1 and aac(6')1b-cr. Interestingly, strain 7C obtained plasmid pMG333 (134 435 bp), which was not present in the parental strain but was an IncN-IncF cointegrate of plasmids pMG334 and pMG335 linked via insertion sequence IS26. CONCLUSION: This study described the complete nucleotide sequence of three plasmids carrying four PMQR genes in a single strain and the plasmid profile obtained after outcrosses. In addition, it described a cointegrate of two plasmids formed with flanking insertion sequences.

Cold shock induces chromosomal qnr in Vibrio species and plasmid-mediated qnrS1 in Escherichia coli.

qnr genes are found in aquatic bacteria and preceded the development of synthetic quinolones. Their natural functions are unknown. We evaluated the expression of chromosomal qnr in Vibrio species in response to environmental stresses and DNA damaging agents. Sub-inhibitory concentrations of quinolones, but not other DNA damaging agents, induced the expression of chromosomal qnr by more than five times in Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio mytili Cold shock also induced the expression of qnr in V. parahaemolyticus, V. vulnificus, and V. mytili, as well as qnrS1 in Escherichia coli qnrS1 induction by cold shock was not altered in ΔihfA or ΔihfB mutants or in a strain over-expressing dnaA, that otherwise directly modulate qnrS1 induction by ciprofloxacin. In contrast, qnrS1 induction by cold shock was reduced in a ΔcspA mutant in the cold shock regulon compared to the wild type. In conclusion, cold shock as well as quinolones induce chromosomal qnr in Vibrio species, and the related qnrS1 in E. coli.

Multiple Copies of qnrA1 on an IncA/C2 Plasmid Explain Enhanced Quinolone Resistance in an Escherichia coli Mutant.

In a previous study, mutants with enhanced ciprofloxacin resistance (Cipr) were selected from Escherichia coli J53/pMG252 carrying qnrA1 Strain J53 Cipr 8-2 showed an increase in the copy number and transcription level of qnrA1 We sequenced the plasmids on Illumina and MinION platforms. Parental plasmid pMG252 and plasmid pMG252A from strain J53 Cipr 8-2 were almost identical, except for the region containing qnrA1 that in pMG252A contained 4 additional copies of the qnrA1-qacEΔ1-sul1-ISCR1 region.

Chromosomal mutations that accompany qnr in clinical isolates of Escherichia coli.

We examined 13 qnr-positive and 14 qnr-negative clinical isolates of Escherichia coli for mutations previously seen in a qnr-containing laboratory strain exposed to supra minimum inhibitory concentrations (MICs) of ciprofloxacin. Among the qnr-positive strains, those with ciprofloxacin MICs of ≥ 2 µg/mL had at least one mutation in gyrA. Mutations in parC were present in strains with a ciprofloxacin MIC of ≥ 128 µg/mL. The 6 most ciprofloxacin-resistant strains contained additional plasmid-mediated quinolone resistance determinants. aac(6')-Ib-cr was found in 5 of the 6 strains. Eleven of the 13 strains had alterations in MarR, 9 in SoxR, and 5 had mutations in AcrR. All had elevated expression of at least one efflux pump gene, predominantly acrA (92% of the strains), followed by mdtE (54%) and ydhE (46%). Nine had functionally silent alterations in rfa, two had mutations in gmhB, and one of these also had a mutation in surA. An E. coli with ciprofloxacin MIC of 1024 µg/mL contained 4 different plasmid-mediated quinolone resistance determinants as well as gyrA, parC, parE and pump overexpression mutations. Nine of the 14 qnr-negative strains had mutations in topoisomerase genes with a ciprofloxacin MIC of 0.25 to 256 µg/mL. The three most resistant strains also had mutations in parE. Twelve had alterations in MarR, 10 in SoxR and 5 in AcrR. Ten of the 14 strains had elevated expression of efflux pumps with acrA (71.4%), followed by ydhE (50%) and mdtE (14.3%). A diversity of resistance mechanisms occurs in clinical isolates with and without qnr genes.

A simple technique for suppressor detection in Escherichia coli.

To study the viability of a gyrA S83 stop mutation found in an Escherichia coli J53 ciprofloxacin-resistant strain (J53 CipR27), a pBR322 derivative was constructed with a TAG mutation in the bla gene knocking out ampicillin resistance. Ampicillin resistance was restored, suggesting that the strain contains tRNA suppressor activity able to suppress the UAG codon gyrA and allow viability. The method was applied to 22 unique clinical E. coli isolates, and all were found to have low-level suppressor activity.

Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli with qnrA1.

Plasmid-mediated qnr genes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia coli strain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrA were absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marR and soxR associated with increased expression of marA and soxS and, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhE pump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaD and rfaE in the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrA that persisted when pMG252 from these strains was outcrossed. gyrA mutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coli J53 with aac(6')-Ib-cr or qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. coli strain carrying qnrA1, aac(6')-Ib-cr, or qepA because these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrA to predominate.

Characterization of Pc promoter variants of class 1 integrons in Escherichia coli isolates from poultry meat.

Integrons are important genetic elements implicated in acquisition and expression of antimicrobial resistance genes. Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant. Thirty-four Escherichia coli isolates (carrying 38 class 1 integrons), recovered from poultry meat in previous studies in Tunisia and selected by their specific traits, were further characterized in this study. Integron promoter variants and the pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of isolates were determined. Three types of promoter variants were identified among the 38 class 1 integrons (PcW, PcH1, and PcS); the weak promoters were the most predominant. A high clonal diversity of the E. coli strains was demonstrated by PFGE or by MLST. Fifteen PFGE profiles were detected among 19 integron-positive isolates of phylogroup B2, and 12 different sequence types were identified by MLST among the remaining 15 isolates (ST48, ST88, ST101, ST117, ST155, ST189, ST351, ST359, ST410, ST641, ST665, and one new ST). These data reflect that the presence of integrons in these isolates is not due to the clonal dispersion but to dissemination of genetic structures carrying integrons in different clones. To the best of our knowledge, this is the first report examining the gene cassette promoter variants in class 1 integrons of E. coli isolates from poultry meat origin. The predominance of promoters implicated in weak expression/high excision activity of gene cassette arrays is of interest because they could theoretically enhance the capacity of integrons to adapt to antibiotic pressure.

The 216-bp marB gene of the marRAB operon in Escherichia coli encodes a periplasmic protein which reduces the transcription rate of marA.

The marRAB operon is conserved in seven genera of enteric bacteria (Escherichia, Shigella, Klebsiella, Enterobacter, Salmonella, Cronobacter, and Citrobacter). MarA is a transcriptional regulator affecting many genes involved in resistance to stresses, and MarR is an autorepressor of the operon, but a role for the marB gene has been unclear. A recent work reported that deletion of marB causes resistance to certain stresses and increases the amount of marA transcript. We show here that the small (216 bp) marB gene encodes a protein, not an sRNA, because two different stop codons within the predicted open reading frame of marB prevented plasmid-borne marB from complementing ΔmarB::Kan. The ΔmarB::Kan mutation did not increase the stability of the marA transcript, suggesting that MarB does not destabilize the marA transcript but rather reduces its rate of transcription. Placing the putative signal sequence of MarB upstream of signal-sequence-less alkaline phosphatase guided the phosphatase to its normal periplasmic location. We conclude that MarB is a small periplasmic protein that represses the marRAB promoter by an indirect mechanism, possibly involving a signal to one of the cytoplasmic regulators of that promoter.

Molecular characterization of multiresistant Escherichia coli producing or not extended-spectrum ß-lactamases.

BACKGROUND: The prevalence and type of plasmids, resistance genes and integrons carried by two collections of multiresistant E. coli producing or not extended-spectrum ß-lactamases have been compared. Rep-PCR was used to determine the clonal relationship of the organisms. Plasmids were classified according to their incompatibility. Class 1 and Class 2 integrons and antibiotic resistance genes were analysed by PCR and sequencing. RESULTS: Both collections of organisms contained a large diversity of unrelated strains with some clones distributed in both groups of isolates. Large plasmids were identified in the two groups of organisms. Plasmids with replicons repK and repColE were more frequent among ESBL-producing isolates, while repFIA, repFII and repA/C replicons were more frequent in isolates lacking ESBL. Conjugative plasmids with repK and repA/C replicons coded for CTX-M-14 and CMY-2 ß-lactamases, respectively. No significant differences were observed in the distribution of class 1 and class 2 integrons among multiresistant E. coli producing or not ESBL, and dfrA17-ant(3")-Ie was the cassette arrangement most commonly found. CONCLUSIONS: In the concrete temporal and geographical context of this study, multiresistant E. coli producing ESBL or other mechanisms of resistance were largely clonally diverse and present some differences in the types of harboured plasmids. Still, some clones were found in both ESBL-producing and -lacking isolates.

Lineages and virulence gene content among extended-spectrum ß-lactamase-producing Escherichia coli strains of food origin in Tunisia.

Nineteen extended-spectrum ß-lactamase (ESBL)-positive Escherichia coli strains recovered from food samples in Tunisia were characterized by multilocus sequence typing and phylogenetic typing, and the virulence gene and plasmid content were also determined. These strains presented unrelated pulsed-field gel electrophoresis patterns and contained genes coding for the following ESBLs (the number of strains is in parentheses): CTX-M-1 (15), CTX-M-14 (2), CTX-M-8 (1), and SHV-5 (1). Twelve different sequence types (STs) were identified among the 19 ESBL-positive strains, which included two new STs (ST2022 in 2 bla(CTX-M-14)-containing strains and ST1970 in 2 bla(CTX-M-1)-containing strains). ST155 and ST602 were detected in four and three bla(CTX-M-1)-containing strains, respectively, and ST405 was detected in one bla(CTX-M-8)-producing strain. All ESBL-positive strains were ascribed to the phylogenetic groups A and B1. Most of the bla(CTX-M-1)-containing strains harbored an IncI1 plasmid, except for the four bla(CTX-M-1)-positive strains of beef origin and ST155, which harbored an IncN plasmid. The two bla(CTX-M-14)-containing strains contained an IncI1 plasmid. The virulence gene fimA was detected in all strains. Most strains also carried the aer gene, and six strains carried the eae gene. All strains were negative for the virulence genes sxt, papG-III, papC, hly, cnf1, and bfp. We conclude that ESBL-producing E. coli strains of food origin in Tunisia show high diversity and that plasmids harboring ESBL genes could be implicated in the dissemination of this resistance phenotype.

Change of integrons over time in Escherichia coli isolates recovered from healthy pigs and chickens.

The aims of this study were (a) to perform a time-related quantitative analysis of relative integron frequencies in intestinal Escherichia coli isolates from food animals (pigs and chickens) and (b) to analyse putative relationships between integrons, antimicrobial resistance and phylogenetic groups. The E. coli collection of the Spanish Veterinary Antimicrobial Resistance Surveillance Network was used to extract 393 intestinal isolates from healthy pigs and chickens belonging to the oldest (1998/99) and the latest (2006) available surveillance programs, and their quantitative antimicrobial resistance data. PCR and sequencing were used for detection and characterisation of integrons. Integron overall relative frequencies ranged between 80% and 49%, being higher in pig than in chicken E. coli isolates in both periods. Time-related analysis showed no variations when considering overall frequencies (80% versus 75% in pig E. coli isolates and 49% versus 51% in E. coli chicken isolates). Apart from the 3'-integron sul gene, six different antimicrobial-related gene cassettes (with different variants) were detected in the sequenced integron variable regions: aadA, dfrA, and sat in classes 1 and 2, and cmlA, linF and aadB only in class 1. Multiresistance profiles showed a high association between antimicrobial resistance and integron presence for those antimicrobials corresponding to the antimicrobial-related gene cassettes detected (streptomycin, trimethoprim, chloramphenicol, plus sulphonamides). However, the presence of integrons was also associated with resistance to amoxicillin and tetracycline, two antimicrobials that are widely used in animals but not linked to these genetic elements.

Phenotypic and genotypic characterization of Salmonella enterica recovered from poultry meat in Tunisia and identification of new genetic traits.

Thirty-seven Salmonella enterica isolates obtained from poultry meat in Tunisia were included in this study for characterization of antibiotic resistance mechanisms. High percentages of resistance were detected to ampicillin, sulfonamides, tetracycline, nalidixic acid, and streptomycin (32.4%-89.2%), and lower percentages to amoxicillin-clavulanic acid, kanamycin, amikacin, trimethoprim-sulfamethoxazol, and chloramphenicol (2.7%-18.9%). All strains showed susceptibility to ceftazidime, cefotaxime, gentamicin, and ciprofloxacin. Class 1 integrons were detected in 30% of Salmonella isolates, and four different gene cassette arrangements were detected, including genes implicated in resistance to aminoglycosides (aadA1 and aadA2) and trimethoprim (dfrA1). Four different Pc variants (PcW, PcH1, PcH1(TTN-10), PcW(TGN-10)) with inactive P2 have been found among these isolates. Integron-positive isolates were ascribed to eight different serotypes. A Salmonella Schwarzengrund isolate harbored a new class 1 integron containing the qacH-dfrA1b-aadA1b-catB2 gene cassette arrangement, with the very unusual PcH1(TTN-10) promoter, which has been registered in GenBank (accession no. HQ874651). Different plasmid replicon types were demonstrated among integron-positive isolates: IncI1 (8 isolates), IncN (8), IncP (2), IncFIB (2), and IncFII (2). Ten different pulsed-field gel electrophoresis profiles were detected among the 11 integron-positive isolates and 8 different sequence types were identified by multilocus sequence typing, one of them (registered as ST867) was new, detected in 3 Salmonella Zanzibar isolates. A high diversity of clones is observed among poultry Salmonella isolates and a high proportion of them show a multiresistant phenotype with very diverse mobile genetic structures that could be implicated in bacterial dissemination in different environments.

Diversity of class 1 integron gene cassette Pc promoter variants in clinical Escherichia coli strains and description of a new P2 promoter variant.

Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant as well as occasionally from a second promoter located downstream of Pc, named P2. So far, the distribution of the variants has only been described in an in silico study. In this study, the prevalence of these variants in vivo was analysed in a population of 85 Escherichia coli strains from a variety of phylogenetic groups isolated from healthy subjects and clinical samples in Spain and France from 2004 to 2007. The weakest variants (PcW and PcH1) prevailed (variants associated with the integrase having the most efficient excision activity), whilst the two strongest variants, PcW(TGN-10) and PcS, were less frequent. Furthermore, a new variant of P2 associated with PcW was characterised in one integron (harbouring the gene cassette bla(OXA-1)-aadA1) from a French strain of a healthy subject. This variant was hereafter named P2m3 and shows a G→A substitution in its -10 element (TACAGT to TACAAT), a mutation that doubled the strength of P2 and approached the level of expression of the strong PcW(TGN-10) variant. When the correlation between the Pc variants and the origin of the strains was analysed, no significant difference (P<0.05) was observed in the Pc variant distribution according to the geographic origin or clinical setting.

Escherichia coli of poultry food origin as reservoir of sulphonamide resistance genes and integrons.

The antimicrobial resistance phenotype and genotype, the flanking regions of sulphonamide resistance genes and the integrons were analyzed in 166 Escherichia coli isolates recovered from poultry meat in Tunisia. High percentages of resistance were detected to ampicillin, streptomycin, nalidixic acid, sulphonamide and tetracycline (66-95%), and lower percentages to gentamicin, amoxicillin-clavulanic acid and cefoxitin (1-4%). The bla(TEM), tet(A)/tet(B), aph(3')-Ia, aac(6')-Ib-cr, aac(3)-II and cmlA genes were identified in 92, 82, 29, 2, 2 and 7 isolates, respectively. Class 1 and/or class 2 integrons were detected in 52% of E. coli isolates and five different gene cassette arrangements were identified in the variable regions of class 1 integrons, which included antimicrobial resistance determinants. Sixty-eight isolates contained the sul1 gene and 37 of them presented this gene into a class 1 integron structure. The sul3 gene was detected associated with non-classic class 1 integrons in 4 out of 46 sul3-positive isolates. The sul2 gene was detected in 66 isolates, 51 of them were linked to strA/B genes in seven different genetic structures. Seventy-three-per-cent of integron-positive isolates presented resistance to at least five different antimicrobial families versus 38.7% of integron-negative isolates. Our study highlights the role of commensal E. coli isolates from poultry meat as an important reservoir for sulphonamide resistance genes and integrons carrying antimicrobial resistance genes.

Genetic characterization of extended-spectrum beta-lactamases in Escherichia coli isolates of pigs from a Portuguese intensive swine farm.

There is a great concern by the emergence and the wide dissemination of extended-spectrum beta-lactamases (ESBLs) among animal Escherichia coli isolates. We intended to determinate the carriage level and type of ESBLs in E. coli obtained from fecal samples from pigs raised on an intensive pig farm in Portugal; further to characterize other associated resistance genes and their plasmid content, the phylogenetic groups, and the clonal relationship of ESBL-positive isolates. Sixty-five fecal samples were seeded in Levine media supplemented with cefotaxime for E. coli recovery. Susceptibility to 16 antimicrobial agents was performed by disk diffusion agar. ESBL-phenotypic detection was carried out by double-disk test; and the presence of the genes encoding TEM, OXA, SHV, and CTX-M type beta-lactamases was studied by polymerase chain reaction and sequencing. Other mechanisms of antimicrobial resistance and phylogenetic groups were also determined. Clonal relationship was performed by pulsed-field gel electrophoresis. ESBL-producing E. coli isolates were detected in 16 fecal samples, and one isolate per sample was studied. The CTX-M-1 type ESBL was detected in the 16 isolates. The gene encoding TEM-1 was identified to be associated with eight CTX-M-1-positive isolates. The tet(A) gene was found in 12 of 14 tetracycline-resistant isolates, and the aadA or strA-strB genes were found in the streptomycin-resistant isolates. Fourteen and two ESBL-containing isolates belonged to A and B1 phylogenetic groups, respectively. Clonal relationship of ESBL-containing isolates identified seven unrelated patterns. Swine represent an important reservoir of ESBL-containing E. coli isolates, especially of the CTX-M-1 type.

In vivo selection of aac(6')-Ib-cr and mutations in the gyrA gene in a clinical qnrS1-positive Salmonella enterica serovar Typhimurium DT104B strain recovered after fluoroquinolone treatment.

OBJECTIVES: To characterize the mechanisms implicated in the in vivo selection of quinolone and aminoglycoside resistance in a faecal Salmonella enterica serovar Typhimurium DT104B strain recovered after ciprofloxacin treatment of a hospitalized elderly patient with acute gastroenteritis. METHODS: Two Salmonella Typhimurium isolates were obtained before (Se6) and after (Se20) treatment and they were typed by PFGE and multilocus sequence typing (MLST). Antimicrobial susceptibility was determined by disc diffusion and agar dilution methods. Class 1, 2 and 3 integrons and resistance mechanisms were studied by PCR and sequencing. Plasmids were typed. RESULTS: Both Salmonella Typhimurium strains were resistant to tetracycline, streptomycin and sulphonamides, while Se20 was also resistant to nalidixic acid, ciprofloxacin, norfloxacin, levofloxacin, ofloxacin, amikacin, tobramycin, kanamycin and trimethoprim. PFGE and MLST showed a clonal relationship between the strains, which belonged to the sequence type ST36. Both strains contained the repC-sul2-strA-strB structure and tet(A) and qnrS1 genes, and strain Se20 also contained the aac(6')-Ib-cr gene, the Ser83-->Tyr substitution in GyrA and one class 1 integron with the dfrA17 + aadA5 gene cassette arrangement lacking qacEDelta1 + sul1. Two different transconjugants from Salmonella Se20 (TCSe20B and TCSe20L) harboured qnrS1 and sul2 genes and the class 1 integron. The TCSe20B strain also acquired the aac(6')-Ib-cr gene located on a non-typeable plasmid. qnrS1 was identified on a ColE-type plasmid and the class 1 integron on an IncI1-type plasmid. CONCLUSIONS: This is the first report of in vivo selection of the aac(6')-Ib-cr gene and the Ser83-->Tyr change in GyrA in a qnrS1-positive Salmonella Typhimurium strain after ciprofloxacin treatment; the in vitro transfer of both plasmid-mediated quinolone resistance genes was also demonstrated.

Detection of CTX-M-14 and TEM-52 extended-spectrum beta-lactamases in fecal Escherichia coli isolates of captive ostrich in Portugal.

The main aim of this study was to determine the frequency of antibiotic resistance among Escherichia coli isolates recovered in Levine agar plates from 54 fecal samples of captive ostriches from a farm in the South of Portugal. Fifty-four nonselected E. coli isolates were obtained (one/sample) and the phenotypes and genotypes of antibiotic resistance were characterized. The following numbers of isolates showed antibiotic resistance: ampicillin (nine), tetracycline (seven), streptomycin (three), amoxicillin-clavulanic acid, cefoxitin, or gentamicin (one), and cefotaxime, ceftazidime, azthreonam, imipenem, nalidixic acid, ciprofloxacin, and trimethoprim/sulfamethoxazole (zero). The bla(TEM) gene was identified in six out of nine ampicillin-resistant isolates, and the tet(A) or tet(B) genes in five out of seven tetracycline-resistant isolates. Mutations at positions -42, -18, -1, and +58 of ampC promoter region were identified in one cefoxitin-resistant isolate. Further, the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates was estimated in the 54 fecal samples of ostriches using cefotaxime-supplemented Levine agar plates for ESBL-positive E. coli recovery. Three samples contained ESBL-positive E. coli isolates of which one isolate/sample was characterized, leading to the detection of the following beta-lactamases: bla(CTX-M-14a) + bla(TEM-1b) (two isolates) and bla(TEM-52c) (one isolate). The three ESBL-positive isolates were classified into the phylogroup B1, and contained class 1 integrons with the gene cassettes dfrA17 + aadA5 (one isolate) and aadA1 (two isolates). This study adds to our knowledge about the wide dissemination of ESBL-producing E. coli isolates in different ecosystems, including captive ostriches, that could be transferred to humans through the food chain.