Coli Clinical Isolates Research Articles

Evaluation of antimicrobial activity of β-lactam antibiotics by Etest against clinical isolates from 100 medical centers in Japan (2004)

This antimicrobial resistance surveillance study was performed in 100 medical centers. The susceptibility of 9347 strains including Escherichia coli (997 strains), Klebsiella spp. (997 strains), Enterobacter spp. (988 strains), Citrobacter spp. (834 strains), indole-positive Proteae spp. (855 strains), Serratia spp. (925 strains), Acinetobacter spp. (902 strains), Pseudomonas aeruginosa (996 strains), oxacillin-susceptible Staphylococcus aureus (992 strains), and coagulase-negative staphylococci (861 strains) to 7 β-lactam antibiotics, cefepime, cefpirome, ceftazidime, cefoperazone/sulbactam, imipenem and piperacillin (for Gram negatives), or oxacillin (for Gram positives) was tested. No strain resistant to these β-lactams except for ceftazidime was found in oxacillin-susceptible S. aureus and coagulase-negative staphylococci. E. coli (16.5%) clinical isolates were resistant to piperacillin, whereas 1.5% or less (cefpirome = 1.5%) was resistant to other β-lactams. Klebsiella spp. strains were more susceptible to imipenem (99.7%), cefepime (98.4%), and cefpirome (97.3%). Isolates of Enterobacter spp., Citrobacter spp., indole-positive Proteae, and Serratia spp. were susceptible to imipenem, cefepime, and cefpirome, as well. Acinetobacter spp. strains were most susceptible to cefoperazone/sulbactam (0.8% resistance), imipenem (3.2%), ceftazidime (6.0%), and cefepime (7.0%) than other β-lactam antibiotics tested. Isolates of P. aeruginosa were more susceptible to ceftazidime (9.9% resistance), cefoperazone/sulbactam (14.9%), and cefepime (11.2%) than piperacillin (15.5%), cefpirome (19.1%), and imipenem (19.3%). The percentage of imipenem-resistant P. aeruginosa is around 20% in clinical isolates in Japan.

CTX-M-2 and a New CTX-M-39 Enzyme Are the Major Extended-Spectrum Beta-Lactamases in Multiple Escherichia coli Clones Isolated in Tel Aviv, Israel

The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was bla(CTX-M); 16 isolates carried bla(CTX-M-2) and three carried a new ESBL gene designated bla(CTX-M-39). Three strains carried bla(SHV) (two bla(SHV-12) and one bla(SHV-5)), and two strains carried inhibitor-resistant ESBL genes, bla(TEM-33) and bla(TEM-30); 18 strains carried bla(TEM-1) and eight strains carried bla(OXA-2). Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that bla(CTX-M-2) is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated bla(CTX-M-39), which revealed 99% homology with bla(CTX-M-26), with a substitution of arginine for glutamine at position 225.

Sequence Typing and Comparison of Population Biology of Campylobacter coli and Campylobacter jejuni

A multilocus sequence typing (MLST) scheme that uses the same loci as a previously described system for Campylobacter jejuni was developed for Campylobacter coli. The C. coli-specific primers were validated with 53 isolates from humans, chickens, and pigs, together with 15 Penner serotype reference isolates. The nucleotide sequence of the flaA short variable region (SVR) was determined for each isolate. These sequence data were compared to equivalent information for 17 C. jejuni isolates representing the known genetic diversity of this species. C. coli and C. jejuni share approximately 86.5% identity at the nucleotide sequence level within the MLST loci. There is evidence of genetic exchange of the housekeeping genes between the two species, but at a very low rate; only one sequence type from each species showed evidence of imported DNA. The flaA gene was more variable and has been exchanged many times between the two species, making it an unreliable marker for species identification but useful for distinguishing closely related strains. All but 3 of 21 human C. coli clinical isolates were distinct, according to the combined MLST and SVR sequences. The use of a common MLST scheme allows direct comparisons of the population biology and molecular epidemiology of these two closely related human pathogens.

Virulence Genotype and Phylogenetic Origin in Relation to Antibiotic Resistance Profile among Escherichia coli Urine Sample Isolates from Israeli Women with Acute Uncomplicated Cystitis

To clarify the virulence and phylogenetic implications of antimicrobial agent resistance in Escherichia coli, 100 E. coli isolates from urine samples of Israeli women with acute uncomplicated cystitis were analyzed by molecular phylotyping and virulence genotyping for comparison with resistance phenotypes. The differences between the isolates that were resistant and susceptible to trimethoprim-sulfamethoxazole and ampicillin were minimal. In contrast, ciprofloxacin resistance was associated with greatly reduced inferred virulence and categorical shifts away from the highly virulent phylogenetic group B2, which explained much of the virulence effect. The results of amplification fingerprinting suggested that most ciprofloxacin-resistant isolates represented unique clonal groups and were not derived from clonal groups with more highly virulent susceptible isolates. These findings suggest that virulence and antimicrobial resistance are not mutually exclusive in E. coli clinical isolates. Instead, the relationship between virulence and antimicrobial resistance varies according to the particular resistance phenotype; for ciprofloxacin resistance, the relationship is strongly influenced by phylogenetic background. The basis for the concentration of ciprofloxacin resistance in non-B2 phylogenetic groups remains unknown.

Spontaneous zygogenesis in Escherichia coli, a form of true sexuality in prokaryotes.

A new type of mating, differing from classic conjugation and previously observed in a certain strain of Escherichia coli K-12, has also been found in strains derived from ordinary F- cells of E. coli K-12 exposed to an exogenous factor originating in an E. coli clinical isolate. Immunofluorescence and electron microscopy after single and double labelling of DNA were used to produce evidence in favour of a novel mating mechanism by cell contact at the poles of the bacterial rod. These findings are supported by genetic analyses indicating complete genetic mixing. Unstable complementing diploids were formed, which throw off phenotypically haploid cells, of which some showed a parental phenotype and some were true genetic recombinants. Recombination was observed even when one parent was a UV-inactivated F- RecA- strain. The name 'spontaneous zygogenesis' (Z-mating, for short) is proposed for this kind of mating.

Genomic variability of O islands encoding tellurite resistance in enterohemorrhagic Escherichia coli O157:H7 isolates.

Strains of Escherichia coli causing enterohemorrhagic colitis belonging to the O157:H7 lineage are reported to be highly related. Fifteen strains of E. coli O157:H7 and 1 strain of E. coli O46:H(-) (nonflagellated) were examined for the presence of potassium tellurite resistance (Te(r)). Te(r) genes comprising terABCDEF were shown previously to be part of a pathogenicity island also containing integrase, phage, and urease genes. PCR analysis, both conventional and light cycler based, demonstrated that about one-half of the Te(r) E. coli O157:H7 strains (6 of 15), including the Sakai strain, which has been sequenced, carried a single copy of the Te(r) genes. Five of the strains, including EDL933, which has also been sequenced, contained two copies. Three other O157:H7 strains and the O46:H(-) strain did not contain the Te(r) genes. In strains containing two copies, the Te(r) genes were associated with the serW and serX tRNA genes. Five O157:H7 strains resembled the O157 Sakai strain whose sequence contained one copy, close to serX, whereas in one isolate the single copy was associated with serW. There was no correlation between Te(r) and the ability to produce Shiga toxin ST1 or ST2. The Te(r) MIC for most strains, containing either one or two copies, was 1,024 micro g/ml, although for a few the MIC was intermediate, 64 to 128 micro g/ml, which could be increased to 512 micro g/ml by pregrowth of strains in subinhibitory concentrations of potassium tellurite. Reverse transcriptase PCR analysis confirmed that in most strains Te(r) was constitutive but that in the rest it was inducible and involved induction of terB and terC genes. Only the terB, -C, -D, and -E genes are required for Te(r). The considerable degree of homology between the ter genes on IncH12 plasmid R478, which originated in Serratia marcescens, and pTE53, from an E. coli clinical isolate, suggests that the pathogenicity island was acquired from a plasmid. This work demonstrates diversity among E. coli O157:H7 isolates, at least as far as the presence of Te(r) genes is concerned.

In vitro antibacterial activity of enrofloxacin and ciprofloxacin in combination against Escherichia coli and staphylococcal clinical isolates from dogs

SUMMARYThe objective of the study was to determine the in vitro interaction between enrofloxacin and ciprofloxacin against Escherichia coli and staphylococcal isolates from dogs. The microdilution checkerboard assay was used to determine the interaction of the drugs against 50 E coli and 50 β-haemolytic staphylococcal clinical isolates. The checkerboard assay revealed that the activity of enrofloxacin and ciprofloxacin was additive against E coli and staphylococcal clinical isolates. It was concluded that for bacterial species against which ciprofloxacin is more potent than enrofloxacin, the in vivo transformation of enrofloxacin to ciprofloxacin may enhance the efficacy of enrofloxacin, if additivity of the drugs is confirmed in vivo.

Ciprofloxacin and the fluoroquinolones: New concepts on the mechanism of action and resistance

Ciprofloxacin, a new fluoroquinolone, is a potent, broad-spectrum antibacterial agent. It rapidly blocks bacterial deoxyribonucleic acid (DNA) replication by inhibiting DNA gyrase, an essential prokaryotic enzyme that catalyzes chromosomal DNA supercoiling. Molecular genetic approaches have been used to study the interaction of 4-quinolones with DNA gyrase from quinolone-sensitive strains and from uropathogenic quinolone-resistant clinical isolates of Escherichia coli. An important mutational locus in the gyrase. A gene that confers resistance to ciprofloxacin and other quinolones has been identified, and a new, rapid method to examine clinical isolates for the presence of mutations at this position has been devised. A quinolone resistant gyrA gene has been previously cloned and sequenced from an E. coli clinical isolate. Genetic analysis indicated that resistance resulted from a Ser-83→ Trp change in the 875 residue gyrase A protein: two other changes observed in the protein, Asp-678→ Glu and Ala-828→ Ser, were neutral. GyrA genes carrying these mutations have now been expressed, corresponding mutant gyrase A proteins purified, and their quinolone resistance properties tested by complementing with gyrase B protein and studying the resulting gyrase activity in an adenosine triphosphate-dependent DNA supercoiling assay. The in vitro DNA supercoiling activity of the A (Ser-83→ Trp) mutant subunit complemented with wild-type gyrase B subunit was highly resistant to ciprofloxacin and other 4-quinolones. In contrast, A subunit carrying codon 678 and 828 changes reconstituted a quinolone-sensitive gyrase activity. Thus, quinolone-resistant gyrase A proteins may be readily obtained for study by using high-copy gyrA plasmids. In addition, other quinolone-resistant strains of E. coli have been examined for the presence of mutations at gyrase A codons 82 and 83 using a new analytical method based on a restriction fragment length polymorphism (RFLP). This analysis revealed that seven of eight resistant clinical isolates of E. coli examined carried gyrA mutations at codon 82 or 83, whereas five sensitive strains appeared to possess wild-type sequence. Thus, mutations at codon 83 (and possibly 82) in the gyrA gene frequently confer resistance to 4-quinolones, including ciprofloxacin. The RFLP method described should prove useful in examining strains for such mutations. These results are discussed with regard to the mode of interaction of the 4-quinolones with gyrase.

General method for site-directed mutagenesis in Escherichia coli O18ac:K1:H7: deletion of the inducible superoxide dismutase gene, sodA, does not diminish bacteremia in neonatal rats

A defined deletion in the Escherichia coli K-12 sodA gene (encoding manganese-superoxide dismutase) linked to a nontransposable selectable marker was generated by transposon Tn5 insertion in combination with in vitro mutagenesis. This mutant allele was used to replace the wild-type sodA gene in an E. coli clinical isolate of serotype O18ac:K1:H7 by bacteriophage P1 transduction. The O18ac:K1:H7 sodA mutant contained no manganese-superoxide dismutase and no hybrid manganese-iron-superoxide dismutase. The sodA mutant was more sensitive to paraquat toxicity than were the parental strain and an isogenic mutant bearing an analogously constructed sodA+ Tn5 insertion allele. In a suckling rat model for bacteremia following oral inoculation of E. coli K1, the sodA mutant was undiminished in its capabilities both to colonize the gastrointestinal tract and, surprisingly, to cause bacteremia. In conjunction with the rat model for E. coli K1 pathogenesis, the method for site-directed mutagenesis described in this paper permits determination of the role played in colonization and bacteremia by any K1 gene which either has a homolog in E. coli K-12 or can be cloned and manipulated therein.

Analysis of the plasmid content of an E. coli clinical isolate: Characterization of pPJ3a, pPJ3b, and of the transposon Tn2301

Analysis of the plasmid content of an E. coli clinical isolate: Characterization of pPJ3a, pPJ3b, and of the transposon Tn2301