Mechanisms of fluoroquinolone resistance in Escherichia coli isolates from food-producing animals

Eleven multidrug-resistant Escherichia coli isolates (comprising 6 porcine and 5 bovine field isolates) displaying fluoroquinolone (FQ) resistance were selected from a collection obtained from the University Veterinary Hospital (Dublin, Ireland). MICs of nalidixic acid and ciprofloxacin were determined by Etest. All showed MICs of nalidixic acid of >256 μg/ml and MICs of ciprofloxacin ranging from 4 to >32 μg/ml. DNA sequencing was used to identify mutations within the quinolone resistance-determining regions of target genes, and quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the major porin, OmpF, and component genes of the AcrAB-TolC efflux pump and its associated regulatory loci. Decreased MIC values to nalidixic acid and/or ciprofloxacin were observed in the presence of the efflux pump inhibitor phenylalanine-arginine-β-naphthylamide (PAβN) in some but not all isolates. Several mutations were identified in genes coding for quinolone target enzymes (3 to 5 mutations per strain). All isolates harbored GyrA amino acid substitutions at positions 83 and 87. Novel GyrA (Asp87 → Ala), ParC (Ser80 → Trp), and ParE (Glu460 → Val) substitutions were observed. The efflux activity of these isolates was evaluated using a semiautomated ethidium bromide (EB) uptake assay. Compared to wild-type E. coli K-12 AG100, isolates accumulated less EB, and in the presence of PAβN the accumulation of EB increased. Upregulation of the acrB gene, encoding the pump component of the AcrAB-TolC efflux pump, was observed in 5 of 11 isolates, while 10 isolates showed decreased expression of OmpF. This study identified multiple mechanisms that likely contribute to resistance to quinolone-based drugs in the field isolates studied.     

Mercury resistance among clinical isolates of Escherichia coli

The use of organomercurials in liquid detergents and disinfectants promoted resistance to mercury among bacteria. Dental amalgam and industries using mercury are the main source of human exposure to mercury vapor. Release of mercury from dental amalgam contributes to the enrichment of the intestinal flora with mercury resistance plasmids which may be associated with antibiotic resistance. The aim of our study was to evaluate the frequency of E. coli strains resistant to mercury and other antimicrobial agents currently used in therapy. The bacterial mercury and ampicillin, cephalexin, cefotaxime, gentamicin, tetracycline and chloramphenicol resistance was tested against 363 E. coli strains obtained from faeces and urine between 1999-2000. According to the guidelines suggested by NCCLS (1998), minimum inhibitory concentrations (MICs) were determined on Mueller-Hinton agar, using the dilution technique with an inoculum of about 10(5) CFU. The MICs were read after 18 h incubation at 37 degrees C as the lowest concentration that inhibited the development of visible growth. Plasmids in enterobacteria may carry genes encoding resistance to both mercury and antibiotics. Among the tested E. coli strains, mercury resistance rose to 29.2%. Mercury resistance in E. coli is significantly linked to multiresistance to antimicrobial agents. Between 91.5-23.6 of mercury chloride resistant isolates were also resistant to the tested antibiotics. The increased use of non antibiotic antimicrobial agents is a possible selection factor for antibiotic-resistant strains in clinical and domestic environments.     

Fluoroquinolone resistance in clinical and environmental isolates of Escherichia coli in Mexico City

Aims:  To assess the different phenotypes and mechanisms of fluoroquinolone (FQ) resistance in clinical and environmental isolates of Escherichia coli .
Methods and Results:  We compared FQ-resistant E. coli isolates, measuring minimal inhibitory concentrations (MIC) of ciprofloxacin, along with susceptibility to other antibiotics. We also searched for the presence of efflux pumps, using efflux inhibitors, and for plasmid-borne FQ-resistance by PCR. We found that, aside from the higher FQ-resistance prevalence among clinical strains, environmental ones resist much lower concentrations of ciprofloxacin. Efflux pumps mediate fluoroquinolone resistance as frequently among environmental isolates than in clinical strains. Plasmid-borne qnrA genes were not detected in any resistant strain.
Conclusions:  Environmental FQ-resistant strains may have a nonclinical origin and/or a selective pressure different from the clinical use of FQs.
Significance and Impact of the Study:  The identification of the source of low-level FQ-resistant strains (ciprofloxacin MIC c . 8 μg ml⁻¹) in the environment could be important to curb the rapid emergence and spread of FQ-resistance in clinical settings, as these strains can easily become fully resistant to FQ concentrations achievable in fluids and tissues during therapy.     

The frequency of in vitro resistance to fluoroquinolones among clinical isolates of Escherichia coli

The aim of this study was to investigate the activity of new quinolones against clinical isolates of E. coli, obtained from hospitalized patients between 1999-2000, in Eastern Romania. We tested 411 strains, isolated from urine. Susceptibility of the isolates to norfloxacin, ciprofloxacin, nalidixic acid and ofloxacin was performed using the dilution method in Mueller-Hinton agar. Susceptibility was reported using the NCCLS breakpoints. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of antimicrobials that inhibited growth of the bacteria. Based on MIC breakpoints for defining susceptibility, between 12.4-13.7% of isolates were resistant to modern quinolones. Resistance to nalidixic acid significantly diminished the clinical use: level of the susceptible strains was only 12.9%.     

Aerobactin genes in clinical isolates of Escherichia coli.

The location of the aerobactin gene complex on either the chromosome or plasmid was determined in eight aerobactin-positive clinical isolates of Escherichia coli by Southern hybridization analysis, using as probes the cloned aerobactin genes from the ColV-K30 plasmid. The aerobactin genes were in two cases detected on large plasmids, whereas in the other strains the aerobactin genes are most likely located on the chromosome. Restriction mapping revealed only slight variations in the structural genes and an at least 3.4-kilobase-long upstream region conserved in all three plasmid-coded systems. A 7.7-kilobase HindIII fragment upstream and adjacent to the 16.3-kilobase HindIII fragment carrying the complete aerobactin system was cloned from the ColV-K30 plasmid. Fine-structure restriction mapping identified the left insertion sequence in the upstream region as IS1, in inverted orientation to the IS1 element downstream from the aerobactin operon. The upstream and downstream sequences of IS1 appear to have perfect homology, as indicated by S1 nuclease resistance of a 760-base-pair DNA duplex formed by both IS1 elements.     

High presence of extended-spectrum beta-lactamases and resistance to quinolones in clinical isolates of Escherichia coli

A study was conducted to detect the presence of extended-spectrum β-lactamases (ESBLs) in 706 isolates of Escherichia coli, largely from outpatients (75.2%). The Clinical and Laboratory Standards Institute (formerly NCCLS)-recommended disk diffusion procedure was used to detect ESBL presence; the VITEK 2 system (bioMérieux, Marcy L’Etoile, France) was used to determine the susceptibility to antibiotics of clinical interest, and the ESBLs were characterized by biochemical study, determining the isoelectric point, and by molecular study with PCR. Clonal distribution was studied in eight hospital isolates. There were 115 ESBL-producing isolates (16.3%), with a predominance of CTX-M9 type (58.3%). We draw attention to the high resistance to quinolones (>70%) in CTX-M9 and SHV enzyme producing isolates and the lower aminoglycoside activity in the latter.     

Genetic analysis of chromosomal resistance to trimethoprim derived from clinical isolates of Escherichia coli

Chromosomal genes conferring resistance to trimethoprim were transferred from three independently isolated thy+ clinical strains of Escherichia coli to Escherichia coli K12 by using P1 transduction. Trimethoprim-resistant transductants were obtained less frequently than transductants of other chromosomal markers, suggesting that there were problems related to the expression of the trimethoprim resistance genes in E. coli K12. Mapping studies revealed that one of the resistance determinants was located at a similar position on the chromosome (1 min) to the fol-type mutations previously described in E. coli K12. The two remaining resistance determinants mapped at separate positions between 2.5 and 3 min on the chromosome. The presence of one of these determinants reduced the efficiency with which either donor or recipient cells carrying it could participate in conjugation mediated by the sex factor F and also resulted in phenotypic interaction with the azi gene. The mechanisms of trimethoprim resistance in the three clinical E. coli isolates studied were more complex and diverse than was expected from previous studies of E. coli K12 mutants.     

产超广谱β-内酰胺酶大肠埃希菌对喹诺酮类抗菌药物的耐药性检测

目的 了解临床分离产超广谱β-内酰胺酶（ESBLs）大肠埃希菌对喹诺酮类等抗菌药物的耐药性。方法 NCCLS表型确证试验（纸片增强法）检测出临床分离大肠埃希菌中产ESBLs菌株,琼脂稀释法测定产ESBLs菌株对喹诺酮类等抗菌药物的耐药性。结果 临床分离大肠埃希菌中产ESBLs菌株的检出率为40．2％（92／229）,产ESBLs菌株以尿标本多见,对6种喹诺酮类抗菌药物的耐药率均在89％以上,哌拉西林的耐药率为100％;对头孢噻肟、头孢他啶和哌拉西林-三唑巴坦的耐药率分别为77．2％、1．1％和21．7％,对亚胺培南极其敏感,耐药率为0％。结论 产ESBLs大肠埃希菌发生率较高,对喹诺酮类抗菌药物耐药显著,临床应加强检测和监测。     

Aerobactin production and plasmid distribution in Escherichia coli clinical isolates

The distribution of plasmids as a function of aerobactin production, antibiotic resistance and antimicrobial agents production was studied in 139 Escherichia coli strains obtained from clinical sources. Ninety eight per cent of the strains analyzed presented plasmids with a median value of 2.97 plasmids per cell. Differences in the number of plasmids were observed for aerobactin production (3.52 for aerobactin producing strains, 2.56 (for non-producing ones) and antibiotic resistance (3.19 for antibiotic resistant strains and 2.58 for the sensitive ones). But this was not the case for antibacterial agent production (2.96 for the producing strains, 2.98 for the non-producing ones. Ecological implications of these results are discussed.     

Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii

We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C. freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones. Our results suggest that in C. freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C. freundii clinical isolates.     

Antimicrobial resistance of Escherichia coli isolates from broiler chickens and humans

Background

Inguinal hernias are usually caused by a congenital defect, which occurs as a weakness of the inguinal canal. Porcine β-glucuronidase gene (GUSB) was chosen as functional candidate gene because of its involvement in degradation of hyaluronan within gubernacular tissue during descent of testes. Since a genome-wide linkage analysis approach has shown evidence that two regions on porcine chromosome 3 (SSC 3) are involved in the inheritance of hernia inguinalis/scrotalis in German pig breeds, GUSB also attained status as a positional candidate gene by its localization within a hernia-associated chromosomal region.

Results

A contig spanning 17,157 bp, which contains the entire GUSB, was assembled. Comparative sequence analyses were conducted for the GUSB gene locus. Single nucleotide polymorphisms (SNPs) located within the coding region of GUSB were genotyped in 512 animals. Results of transmission disequilibrium test (TDT) for two out of a total of five detected SNPs gave no significant association with the outcome of hernia in pigs.

Conclusion

On the basis of our studies we are able to exclude the two analyzed SNPs within the porcine GUSB gene as causative for the transmission of inguinal hernia.     

Antimicrobial resistance patterns among different Escherichia coli isolates in the Kingdom of Saudi Arabia

Antimicrobial resistance patterns among different Escherichia coli isolates in the Kingdom of Saudi Arabia. This study aimed to investigate the patterns of antimicrobial resistance in E. coli isolated from different samples, and to identify potential pathogenic isolates in Riyadh, Kingdom of Saudi Arabia (KSA). In total, 51 bacterial isolates were recovered from 113 samples of human urine, food (raw meat, raw chicken, raw egg surface, and fresh vegetables), water, and air. Twenty-four E. coli isolates were tested for susceptibility to 26 antibiotics. The air sample isolates were most resistant to amoxicillin, ampicillin, amoxicillin/clavulanic acid, amoxicillin/sulbactam, piperacillin/tazobactam, cefalotin, cefuroxime, cefoxitin, cefixime, nitrofurantoin, and trimethoprim/sulfamethoxazol. The isolates from vegetable samples were resistant to amoxicillin, ampicillin, amoxicillin/clavulanic acid, amoxicillin/sulbactam, cefalotin, cefuroxime, cefoxitin, and cefixime. By contrast, the isolates from the water samples were resistant only to amoxicillin and ampicillin. The isolates from the human urine samples were most frequently resistant to norfloxacin (80%) followed by amoxicillin and ampicillin (70%), trimethoprim/sulfamethoxazole (55%), ciprofloxacin and ofloxacin (50%), cefalotin (30%), cefuroxime, cefixime and cefotaxime (25%), ceftazidime, ceftriaxone, cefepime and aztreonam (20%), amoxicillin/clavulanic acid, piperacillin/tazobactam and gentamicin (10%), and amoxicillin/sulbactam and cefoxitin (5%). Almost all (23/25, 95.8%) (n = 23) of the isolates were multi-drug resistant (MDR) (i.e., resistant to 3 or more classes of antibiotics), and 16.7% (n = 4) of those were positive for extended spectrum β-lactamase (ESBL). Of the 4 ESBL-producers, 3 were positive for blaCTX_-M-15 and blaCTX_-M1_group, 2 were positive for blaCMY_-2, and 1 each was positive for blaCTX_{-M-2 group,} blaSHV, and blaOXA_-47. The quinolone resistance gene qnrS was detected in 25% (n = 6) of the E. coli strains isolated from urine (N = 5) and air (N = 1) samples. The considerable number of antimicrobial resistance genes detected among E. coli isolates tested here is alarming and should raise public health concern.     

Association of antibiotic resistance in agricultural Escherichia coli isolates with attachment to quartz

Surface water can be contaminated by bacteria from various sources, including manure from agricultural facilities. Attachment of these bacteria to soil and organic particles contributes to their transport through the environment, though the mechanism of attachment is unknown. As bacterial attachment to human tissues is known to be correlated with antibiotic resistance, we have investigated here the relationship between bacterial attachment to environmental particles and antibiotic resistance in agricultural isolates. We evaluated 203 Escherichia coli isolates collected from swine facilities for attachment to quartz, resistance to 13 antibiotics, and the presence of genes encoding 13 attachment factors. The genes encoding type I, EcpA, P pili, and Ag43 were detected, though none was significantly related to attachment. Quartz attachment was positively and significantly (P < 0.0038) related to combined resistance to amoxicillin/streptomycin/tetracycline/sulfamethazine/tylosin/chlortetracycline and negatively and significantly (P < 0.0038) related to combined resistance to nalidixic acid/kanamycin/neomycin. These results provide clear evidence for a link between antibiotic resistance and attachment to quartz in agricultural isolates. We propose that this may be due to encoding by the responsible genes on a mobile genetic element. Further exploration of the relationship between antibiotic resistance and attachment to environmental particles will improve the understanding and modeling of environmental transport processes, with the goal of preventing human exposure to antibiotic-resistant or virulent microorganisms.     

Antimicrobial resistance and virulence genes of Escherichia coli isolates from swine in Ontario

A total of 318 Escherichia coli isolates obtained from diarrheic and healthy pigs in Ontario from 2001 to 2003 were examined for their susceptibility to 19 antimicrobial agents. They were tested by PCR for the presence of resistance genes for tetracycline, streptomycin, sulfonamides, and apramycin and of 12 common virulence genes of porcine E. coli. Antimicrobial resistance frequency among E. coli isolates from swine in Ontario was moderate in comparison with other countries and was higher in isolates from pigs with diarrhea than in isolates from healthy finisher pigs. Resistance profiles suggest that cephamycinases may be produced by > or = 8% of enterotoxigenic E. coli (ETEC). Resistance to quinolones was detected only in enterotoxigenic E. coli (< or = 3%). The presence of sul3 was demonstrated for the first time in Canada in porcine E. coli isolates. Associations were observed among tetA, sul1, aadA, and aac(3)IV and among tetB, sul2, and strA/strB, with a strong negative association between tetA and tetB. The paa and sepA genes were detected in 92% of porcine ETEC, and strong statistical associations due to colocation on a large plasmid were observed between tetA, estA, paa, and sepA. Due at least in part to gene linkages, the distribution of resistance genes was very different between ETEC isolates and other porcine E. coli isolates. This demonstrates that antimicrobial resistance epidemiology differs significantly between pathogenic and commensal E. coli isolates. These results may have important implications with regards to the spread and persistence of resistance and virulence genes in bacterial populations and to the prudent use of antimicrobial agents.     

重庆地区儿童感染患者11 039株大肠埃希菌的临床分布及耐药性

目的 分析2010—2015年重庆地区儿童感染的11039株大肠埃希菌的临床分布特征及耐药性,为合理应用抗菌药物和预防控制医院感染提供依据。方法 分析大肠埃希菌对19种抗菌药物的耐药性,采用BD Phoenix 100 MIC法结合K-B纸片扩散法进行药敏试验,按美国临床实验室标准化委员会（CLSI）标准判断结果。结果 大肠埃希菌总检出率为13.53%;大肠埃希菌在呼吸病房和新生儿病房的检出率高于其他病房;以痰标本多见,占62.22%（6869/11039）;大肠埃希菌对氨苄青霉素耐药率最高（90.33%）,对美罗培南、亚胺培南的耐药率低,分别为0.81%和0.78%。ESBLs平均检出率为50.98%（5628/11039）。结论 大肠埃希菌的耐药性呈上升趋势,呼吸病房和新生儿病房是预防控制的重点科室。     

Multiple antibiotic resistance patterns of Escherichia coli isolates from swine farms.

Antibiotic resistance of Escherichia coli from sows and pigs was determined to compare patterns between pigs of various ages and degrees of antibiotic use. Resistance patterns differed between farm types and pigs of differing ages, indicating that pig age and degree of antibiotic use affect resistance of fecal E. coli.     

Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.     

traT gene sequences, serum resistance and pathogenicity-related factors in clinical isolates of Escherichia coli and other gram-negative bacteria

The R6-5 plasmid-specified outer membrane protein, TraT protein, has previously been shown to mediate resistance to bacterial killing by serum. Colony hybridization with a 700 bp DNA fragment carrying most of the traT gene was used to examine the prevalence of traT in Gram-negative bacteria, particularly strains of Escherichia coli, isolated from clinical specimens. traT was found in isolates of E. coli, Salmonella, Shigella and Klebsiella, but not in Pseudomonas, Aeromonas or Plesiomonas, nor in the few isolates of Enterobacter, Proteus, Acinetobacter, Citrobacter, Serratia or Yersinia that were examined. It was detected in a significantly higher proportion of the E. coli strains isolated from the blood of patients with bacteraemia/septicaemia or from faeces of patients with enteric infections (50-70%) than in that of strains isolated from normal faeces (20-40%). The incidence of traT in strains isolated from cases of urinary tract infections was variable. traT was found to be frequently associated with production of the K1 capsule and with the carriage of ColV plasmids, but not with the carriage of R plasmids, nor with serum resistance or the production of haemolysin.