Analysis of the gyrA Gene of Clinical Yersinia ruckeri Isolates with Reduced Susceptibility to Quinolones

Antimicrobial susceptibility of seven clinical strains of Yersinia ruckeri representative of those isolated between 1994 and 2002 from a fish farm with endemic enteric redmouth disease was studied. All isolates displayed indistinguishable pulsed-field gel electrophoresis restriction patterns, indicating that they represented a single strain. However, considering both inhibition zone diameters (IZD) and MICs, the isolates recovered in 2001-2002 formed a separate cluster with lower levels of susceptibility to all the quinolones tested, especially nalidixic acid (NA) and oxolinic acid (OA), compared with the isolates recovered between 1994 and 1998. Analysis of the PCR product of the quinolone resistance-determining region of the gyrA gene from clinical isolates of Y. ruckeri with reduced susceptibility to OA and NA revealed a single amino acid substitution, Ser-83 to Arg-83 (Escherichia coli numbering). Identical substitution was observed in induced OA-resistant mutant strains, which displayed IZD and MICs of quinolones similar to those of the clinical isolates of Y. ruckeri with reduced susceptibility to these antimicrobial agents. These data indicate in that for Y. ruckeri, the substitution of Ser by Arg at position 83 of the gyrA gene is associated with reduced susceptibility to quinolones.     

Analysis of gyrA and parC mutations in enterococci from environmental samples with reduced susceptibility to ciprofloxacin

The quinolone resistance determining regions of gyrA and parC in four species of enterococci from environmental samples with reduced susceptibility to ciprofloxacin were sequenced. The nucleotide sequence variations of parC could be related to the different enterococcal species. Mutations in Enterococcus faecalis and Enterococcus faecium related to reduced susceptibility were identical to mutations detected in E. faecalis and E. faecium of clinical origin. A minimal inhibitory concentration of 8 microg ml(-1) to ciprofloxacin was not associated with any mutations in the gyrA and parC gene of Enterococcus casseliflavus and Enterococcus gallinarum. These two species may be intrinsically less susceptible to ciprofloxacin.     

Nucleotide sequence of the gyrA gene of Arcobacter species and characterization of human ciprofloxacin-resistant clinical isolates

The nucleotide sequence of the gyrA gene of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter skirrowii was determined. The deduced GyrA proteins are closely related to those of Wolinella succinogenes and Helicobacter pullorum, whereas those of Campylobacter species showed less sequence identity. The phylogenetic analysis of GyrA sequences provides a result similar to 16S rRNA gene sequence phylogenetic analysis and allows the discrimination among A. butzleri species. In addition, a Thr-->Ile mutation at amino acid 85 in the quinolone resistance-determining region was associated with ciprofloxacin resistance for two A. butzleri and one A. cryaerophilus ciprofloxacin-resistant strains.     

Searching for Staphylococcus aureus strains with reduced susceptibility to glycopeptides among clinical isolates obtained during the year of 2002

The methicillin-resistant strains of S. aureus (MRSA) isolated in our laboratory were searched for strains with decreased susceptibility to glycopeptides, GISA (glycopeptide intermediate Staphylococcus aureus) and hetero-GISA strains. 103 strains isolated in 2002 were investigated. Five h-VISA and none VISA strain were found. There was not observed any increase in the frequency of VISA and h-VISA comparing to the former years. In the case of some clinical as well as standard strains (susceptible, GISA and especially h-GISA) the start inoculum significantly influenced a shape of the growth curve obtained as a result of the population analysis.     

Comparison of human and soil Candida tropicalis isolates with reduced susceptibility to fluconazole

Infections caused by treatment-resistant non-albicans Candida species, such as C. tropicalis, has increased, which is an emerging challenge in the management of fungal infections. Genetically related diploid sequence type (DST) strains of C. tropicalis exhibiting reduced susceptibility to fluconazole circulated widely in Taiwan. To identify the potential source of these wildly distributed DST strains, we investigated the possibility of the presence in soil of such C. tropicalis strains by pulsed field gel electrophoresis (PFGE) and DST typing methods. A total of 56 C. tropicalis isolates were recovered from 26 out of 477 soil samples. Among the 18 isolates with reduced susceptibility to fluconazole, 9 belonged to DST149 and 3 belonged to DST140. Both DSTs have been recovered from our previous studies on clinical isolates from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) program. Furthermore, these isolates were more resistant to agricultural azoles. We have found genetically related C. tropicalis exhibiting reduced susceptibility to fluconazole from the human hosts and environmental samples. Therefore, to prevent patients from acquiring C. tropicalis with reduced susceptibility to azoles, prudent use of azoles in both clinical and agricultural settings is advocated.     

Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda

Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83 --> Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83 --> Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42 degrees C.     

Culture media for the differentiation of isolates of Yersinia ruckeri, based on detection of a virulence factor

M.D. FURONES, M.L. GILPIN AND C.B. MUNN. 1993. Strains of the bacterial fish pathogen Yersinia ruckeri were identified with the API 20E system and distinguished on the basis of whole cell agglutination with antisera, sorbitol fermentation and polymyxin B sensitivity. Strains which were shown to possess the virulence-associated heat-sensitive factor (HSF) were shown to grow preferentially on culture media containing sodium dodecyl sulphate (SDS) and to produce a creamy deposit around the colonies. By contrast, strains lacking this factor (HSF^-) grew poorly and without forming a deposit. Enhancement of the differentiation between the two types was shown by the incorporation of Coomassie brilliant blue dye into agar containing 1% SDS, and the uptake of Coomassie blue and Congo red was shown to be temperature-dependent. Most strains tested were shown to belong to serotype I, and were sensitive to polymyxin and did not ferment sorbitol. With the medium developed most serotype I strains but not those of other serotypes were shown to possess HSF. It is suggested that the medium is used in epidemiological studies of Y. ruckeri.     

Analysis of regions determining resistence to fluoroquinolones in genes gyrA and parC in clinical isolates of Mycoplasma hominis

Fifteen strains of M. hominis isolated from patients with urogenital inflammations were analyzed. Variations in the quinolone resistance-determining regions (QRDR) have been found in fluoroquinolone-resistant M. hominis clinical isolates in comparison with the reference PG21 strain. In one isolate, parC had Asn substitute at position 91.     

临床分离纹带棒状杆菌喹诺酮类耐药株gyrA和parC基因突变的研究

目的 探讨gyrA 、parC基因的改变与纹带棒状杆菌耐喹诺酮类抗生素的关系.方法 采用微量稀释法测试纹带棒状杆菌对环丙沙星、左旋氧氟沙星、万古霉素的敏感性.PCR扩增检测gyrA和parC基因喹诺酮类耐药决定区相关片段并测序,在GenBank中进行Blast及BlastX分析并观察氨基酸突变位点,用NcoI酶进行PCR-RFLP.结果 37株纹带棒状杆菌对环丙沙星和左旋氧氟沙星的耐药率为94.6％,30株菌发生双突变(87位Ser突变为phe,91位Asp突变为Ala),5株菌单点突变(87位Ser突变为phe).PCR-RFLP经NcoI酶切后显示,敏感株和耐药株均产生3个条带.结论 纹带棒状杆菌对环丙沙星、左旋氧氟沙星的耐药率较高,对万古霉素全部敏感.纹带棒状杆菌对喹诺酮类抗生素的耐药机制主要是gyrA突变引起的,尤以双突变占优势.纹带棒状杆菌不携带parC基因,不编码拓扑异构酶Ⅳ.     

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.     

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.     

BamHI restriction endonuclease analysis of Yersinia ruckeri plasmids and their relatedness to the genus Yersinia 42- to 47-megadalton plasmid

The plasmid profile and BamHI restriction pattern of 17 sorbitol-negative and 1 sorbitol-positive French Yersinia ruckeri strain of the American type strain were studied. The 17 sorbitol-negative strains and the American strain harbored a 62-megadalton (MDa) plasmid with an identical BamHI restriction pattern. Southern hybridization indicated that this 62-MDa plasmid is common among these various strains. The sorbitol-positive strain had four plasmid bands (70, 62, 32, and 25 MDa), and there was no comigration of the DNA fragments of these cleaved plasmids with the fragments of the 62-MDa plasmid. Hybridization of these restricted plasmids with the common 62-MDa plasmid showed a weak DNA homology. The Y. ruckeri plasmid (62 MDa) had a different molecular weight than the virulence plasmid (42 to 47 MDa) of the genus Yersinia, and they had different BamHI restriction patterns. Furthermore, no sequence of the Y. ruckeri plasmid DNA was recognized after Southern hybridization when the 47-MDa plasmid of Y. enterocolitica was used as a probe.     

CIP耐药的铜绿假单胞菌两种分子耐药机制关系的研究

目的探讨环丙沙星（CIP）耐药的铜绿假单胞菌临床分离株主动外排药物与gyrA、parC基因突变的关系。方法联合碳酰氰基-对-氯苯腙（CCCP）和CIP对CIP耐药的铜绿假单胞菌株进行主动外排阳性株和阴性株的筛选,并对这些菌株的gyrA,parC基因进行聚合酶链式反应-限制性片段长度多态性分析（PCR—RFLP）。结果57％（55／97）的CIP耐药菌株最小抑菌浓度（MIC）可被逆转,gyrA单基因突变率为65％,gyrA和pa-C双基因突变率为35％,未发现parC单基因突变的菌株。主动外排阳性组与阴性组gyrA、parC基因突变情况差异无显著性。结论在本地区铜绿假单胞菌对CIP的耐药机制中,主动外排系统表达上调与抗菌药物作用靶位的改变均占有重要的地位,两者可能是并存的两种相对独立的机制。     

Investigation of virulence genes in clinical isolates of Yersinia enterocolitica

In this study, we aimed to investigate the distribution of virulence genes in clinical isolates of pathogenic Yersinia enterocolitica. Two thousand six hundred stool samples were collected from 2600 patients with diarrhea, and were tested using the culture method and real-time PCR. Then, all isolates of pathogenic Y. enterocolitica cultured from the culture method were examined for virulence genes (inv, ail, ystA, ystB, ystC, yadA, virF) by PCR and for the presence of plasmid by four phenotypic tests. As a result, 160 pathogenic strains were successfully detected by the culture method, including bio/serotype 1A/unknown (4), 1B/unknown (8), 2/O:9 (39), 2/unknown (7), 3/O:3 (22), 3/unknown (6), 4/O:3 (55), 4/unknown (10) and 5/unknown (9). The positive rate of virulence genes tested in 160 isolates was inv (100%), ail (94%), ystA (93%), ystB (7.5%), ystC (5%), yadA (89%) and virF (82%) while the phenotypic test included autoagglutination (87%), binding of crystal violet (89%), calcium-dependent growth (74%) and Congo red absorption (78%), respectively. Finally, we found that not all pathogenic Y. enterocolitica necessarily carry all traditional virulence genes in both chromosomes and plasmids to cause illness. Perhaps, some of them, lacking some traditional virulence genes, contain other unknown virulence markers that interact with each other and play an important role in the diverse pathogenesis of pathogenic Y. enterocolitica.     

Multilocus Electrophoretic Assessment of the Genetic Structure and Diversity of Yersinia ruckeri

Multilocus isoenzyme electrophoresis was used to screen 47 field isolates of Yersinia ruckeri for electrophoretic variation at 15 enzyme loci. Only four electrophoretic types were observed, thus indicating that the genetic structure of Y. ruckeri is clonal. Forty-two isolates were of one electrophoretic type, a reflection of the low amount of genetic diversity extant in this species. Although sorbitol fermentation has been considered to be indicative of a second biotype, no significant gene frequency differences were found between the group of 20 isolates that readily used sorbitol as the sole carbon source and the group of 27 that did not.     

BamHI restriction endonuclease analysis of Yersinia ruckeri plasmids and their relatedness to the genus Yersinia 42- to 47-megadalton plasmid.

The plasmid profile and BamHI restriction pattern of 17 sorbitol-negative and 1 sorbitol-positive French Yersinia ruckeri strain of the American type strain were studied. The 17 sorbitol-negative strains and the American strain harbored a 62-megadalton (MDa) plasmid with an identical BamHI restriction pattern. Southern hybridization indicated that this 62-MDa plasmid is common among these various strains. The sorbitol-positive strain had four plasmid bands (70, 62, 32, and 25 MDa), and there was no comigration of the DNA fragments of these cleaved plasmids with the fragments of the 62-MDa plasmid. Hybridization of these restricted plasmids with the common 62-MDa plasmid showed a weak DNA homology. The Y. ruckeri plasmid (62 MDa) had a different molecular weight than the virulence plasmid (42 to 47 MDa) of the genus Yersinia, and they had different BamHI restriction patterns. Furthermore, no sequence of the Y. ruckeri plasmid DNA was recognized after Southern hybridization when the 47-MDa plasmid of Y. enterocolitica was used as a probe.     

Analysis of point mutations in the ygeD, gyrA and parC genes in fluoroquinolones resistant clinical isolates of Chlamydia trachomatis

Resistance of 14 clinical isolates of C. trachomatis to fluoroquinolones, i.e. of ciprofloxacin, pefloxaxin and ofloxacin, was assayed. Three isolates with a high resistance degree to all 3 drugs (MIC equal or above 64 microg/ml) were detected. MIC was found to be equal to or below 4 microg/ml for 3 isolates. The remaining isolates had an intermediate resistance level. The nucleotide sequence was established for the Quinolone-Resistance Determining Region (QRDR) genes coding the DNA-gyrase subunit A (gyrA) and DNA-topoisomerase IV subunit C (parC) as well as for the 3'-region of ygeD coding, presumably, the efflux protein. In none of the isolates, the gyrA and gyrC QRDR differed from the corresponding regions in the published C. trachomatis genome sequence. Several silent mutations and mutations resulting in amino acid substitutions were observed in the ygeD 3' region of 2 isolates resistant to high FQ concentrations and in 1 isolate with the intermediate resistance level.     

Antibiotic susceptibility of clinical isolates of Pseudomonas aeruginosa

Three hundred and twenty two clinical isolates of Pseudomonas aeruginosa collected in Morelia, México, were analyzed for in vitro susceptibility to five antibiotics by agar dilution tests. Antibiotic resistance was shown by 50% of total isolates. Frequencies of resistance were: streptomycin, 47%; gentamicin, 13%; tobramycin, 8%; and carbenicillin, 7%; no amikacin resistance was found. The more common resistance patterns were streptomycin, gentamicin-streptomycin, and tobramycin-gentamicin-streptomycin. Resistance to either tobramycin, gentamicin or carbenicillin was found mainly in pyocin type 10 isolates. The proportion of antibiotic resistant isolates ranged from 37 to 75% in four hospitals, and amounted 24% in three clinical laboratories.     

Immobilization induces alterations in the outer membrane protein pattern of Yersinia ruckeri

We compared the outer membrane protein (OMP) pattern of 2-day-old immobilized Yersinia ruckericells (IC) with that of early (FC24) and late (FC48) stationary-phase planktonic counterparts. Fifty-five OMPs were identified. Principal component analysis discriminated between the protein maps of FC and IC. Some OMPs involved in bacterial adaptation were accumulated by both FC48 and IC but the expression of other proteins was controlled by the sessile mode of growth.     

Characteristics of the MRSA clone with reduced susceptibility to vancomycin

The MIC of vancomycin was determined for all S. aureus strains isolated during 1997 in one hospital. MIC values for most isolates were in the range of 0.5-2 mg/l. In 18 strains, MIC was = 6 mg/L. All these strains were MRSA. Recently described VISA strains possessed MIC values for vancomycin equal or higher than 8 mg/l and such strains were not detected in the investigated group. Although strains with MIC = 6 mg/l are not VISA, but they are candidate for reduced vancomycin susceptibility, e.g. during therapy in compromised patients. Analysis of DNA of these strains by pulsed-field gel electrophoresis (PFGE) revealed that 15 of them shared a significant similarity, allowing to place them in the same group. The comparison data of phage patterns as well as antibiotic resistance patterns strongly suggest that all these strains were derivatives of a single clone.