High prevalence of plasmid-mediated quinolone resistance determinant aac(6')-Ib-cr amongst Salmonella enterica serotype Typhimurium isolates from hospitalised paediatric patients with diarrhoea in China

In this study, the antimicrobial susceptibilities and prevalence of plasmid-mediated quinolone resistance determinants amongst Salmonella enterica serotype Typhimurium isolates from hospitalised paediatric patients with diarrhoea in China were investigated. In total, 40 (64.5%) of 62 S. Typhimurium isolates were resistant to ciprofloxacin (minimum inhibitory concentration ≥0.5 μg/mL), comprising 28 isolates with low-level resistance and 12 isolates with high-level resistance. All ciprofloxacin-resistant isolates were multiresistant to other antimicrobial agents. Four pulsed-field gel electrophoresis (PFGE) clusters were found amongst the 40 ciprofloxacin-resistant isolates, amongst which PFGE clusters A, B, E and D accounted for 7, 4, 1 and 28 isolates, respectively. Two isolates with high-level ciprofloxacin resistance had two mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The remaining ciprofloxacin-resistant isolates had only one mutation in the QRDR of gyrA. All 62 S. Typhimurium isolates were negative for qnr genes and qepA and 23 (37.1%) of the isolates were positive for aac(6')-Ib-cr. Nineteen isolates harbouring aac(6')-Ib-cr belonged to PFGE cluster D. A high prevalence of ciprofloxacin resistance and aac(6')-Ib-cr was found amongst S. Typhimurium isolates in China from hospitalised paediatric patients with diarrhoea not receiving quinolones. A single mutation in the QRDR of gyrA as well as production of AAC(6')-Ib-cr contributed to ciprofloxacin resistance. Clonal spread was responsible for the dissemination of aac(6')-Ib-cr amongst S. Typhimurium isolates.     

耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变研究

目的　研究临床分离的耐氟喹诺酮类铜绿假单胞菌gyrA及parC基因突变情况。方法　测定临床分离的 5 5株铜绿假单胞菌MIC值 ,从中筛选出 1株敏感菌和 8株耐药菌 ,以标准敏感菌株ATCC2 785 3作为质控菌株。用聚合酶链反应 (PCR)扩增gyrA及parC基因的喹诺酮耐药决定区 (QR DR) ,扩增产物片段长度分别为 35 1bp、397bp。用限制性内切酶SacⅡ消化gyrAPCR产物 ,同时对上述 10株菌的gyrA及parC基因的喹诺酮决定区 (QRDR)进行PCR DNA直接测序分析。结果　有 8株耐菌株的gyrA基因在 83位 (ACC→ATC)有突变 ,导致氨基酸Thr→Ile的改变 ;有 3株高度耐药菌gyrA基因同时在 87位 (GAC→GGC)有突变 ,导致氨基酸Asp→Gly的改变 ;有 4株耐药菌株的parC基因在 87位有TCG→TTG突变 ,导致氨基酸由Ser→Leu的改变。同时具gy rA和parC突变MIC值是仅具gyrA突变菌株MIC值的 2～ 16倍。未发现parC突变单独存在。另外 ,有 6株耐药菌gyrA的 132位有CAC→CAT的突变 ;所有耐药菌株parC基因 115位有GCT→GCG的突变 ,该突变未引起氨基酸的改变。结论　gyrA83、87位突变及parC基因 87位突变都可引起铜绿假单胞菌对氟喹诺酮类药物产生耐药 ,但以gyrA基因 83位突变为主 ,合并gyrA基因 87位及parC基因 87位突变可增加耐药程度。     

Class 1 integrons and virulence genes in Salmonella enterica isolates from pork and humans

In this study, 183 Salmonella enterica isolates were characterised for integrons and virulence genes. Among the isolates, 46% were positive for intI1, but no isolates carried intI2 or intI3. Eighteen class 1 integrons (21%) contained resistance gene cassettes (i.e. dfrA1-orfC, dfrA12-aadA2, bla(PSE-1) and aadA2) and five class 1 integrons with the dfrA12-aadA2 array were conjugally transferable. Two Salmonella pork isolates of serotypes Albany and Kedougou possessed Salmonella genomic island 1 variants SGI1-G and SGI1-F, respectively. Four class 1 integrons contained an atypical 3'-CS linked to the qacH-sul3 domain, and three were not a sul type. Two novel GyrA mutations (Pro-45→Ser and Met-48→Ile) and three novel ParC mutations (Ser-5→Arg, Thr-31→Met and Leu-77→Arg) were identified in ciprofloxacin-resistant isolates. At least 90% of the Salmonella isolates contained pagC, prgH, sitC, sipB or spaN, whereas all isolates harboured invA, msgA, spiA and tolC.     

Detection of integrons and antibiotic-resistance genes in Salmonella enterica serovar Typhimurium isolates with resistance to ampicillin and variable susceptibility to amoxicillin-clavulanate

We characterized 29 antimicrobial-resistant Salmonella enterica serovar Typhimurium strains, including four belonging to the monophasic variant 4,5,12:i:-, mostly isolated from infants. They were selected from 3230 strains isolated in the years 1990-2001 on the basis of resistance to ampicillin and variable susceptibility to the amoxicillin-clavulanate combination. Twenty-three strains were resistant to more than four antibiotics. All the strains carried the bla(TEM) gene and most were able to transfer this gene by conjugation. Sequencing of the gene from one of the amoxicillin-clavulanate-resistant strains allowed identification of the encoded beta-lactamase as TEM-1; all of these strains carried a second gene encoding beta-lactamase production, either pse-1 or oxa1. However, the association of bla(TEM) plus pse-1 genes did not always confer resistance to amoxicillin-clavulanate. The pse-1 gene, found in 17 strains, was located in the Salmonella Genomic Island-1 (SGI1), which carries two integrons and encodes multiple drug-resistance. None of the oxa1-bearing strains had the SGI1, yet this gene was found as part of an integron that also carried the aadA1 gene and was not plasmid-associated. Thirteen of the strains harbouring SGI1 belonged to the definitive phage type (DT) 104, and most of those remaining to DT104b and U302; particularly, strains carrying the oxa1-aadA1 integron belonged to the last two phage types. Pulsed field electrophoresis confirmed the clonal organization of DT104 strains, whereas U302 strains fell into different groups, depending on their resistance determinants.     

Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates

To understand the relationship between mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene and drug resistance to ofloxacin, 85 laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis mutant strains and 110 M. tuberculosis clinical isolates, screened by denaturing high-performance liquid chromatography to contain mutations, were analysed for their mutation patterns by sequencing as well as their ofloxacin minimal inhibitory concentrations (MICs). All mutations detected occurred at the codons Ala74, Ala90, Ser91 and Asp94 in all strains. One of the five different forms of missense mutation in Asp94 occurred in 60% of the laboratory-selected strains and 78% of the clinical isolates. However, 53 clinical isolates (48%) and only 2 laboratory-selected strains (2.4%) harboured double point mutations. The mutation Ala74Ser occurred only in the clinical isolates and only in combination with the Asp94Gly mutation. The ofloxacin MIC for the clinical isolates ranged from 0.5microg/mL to 20microg/mL, whilst the MICs for the laboratory-selected strains were > or =10microg/mL. The differences in gyrA gene mutation patterns and MICs between the laboratory-selected resistant strains and clinically isolated resistant strains identified here might help to understand the mechanisms involved in fluoroquinolone resistance.     

In vitro activity of gemifloxacin against clinical isolates of Neisseria gonorrhoeae with and without mutations in the gyrA gene

The MIC of gemifloxacin and five other quinolones was tested against 31 clinical isolates of Neisseria gonorrhoeae; strains were analyzed for the presence of mutations in both the gyrA and parC genes. Only seven strains were resistant to nalidixic acid due to a mutation in the gyrA gene but not in the parC gene, with six and two considered intermediate to ciprofloxacin and levofloxacin, respectively. The activity of gemifloxacin was similar to that of trovafloxacin and moxifloxacin, but was more active than nalidixic acid, ciprofloxacin or levofloxacin against the gyrA mutant strains. Gemifloxacin is a valid therapeutic alternative to treat infections with N. gonorrhoeae, retaining its activity against strains already presenting a mutation in gyrA.     

聚合酶链反应RFLP与SSCP检测伤寒杆菌DNA旋转酶基因变异

ＤＮＡ旋转酶为喹诺酮类抗菌药物作用靶位,其变异为细菌耐药主要原因之一。本文利用ＰＣＲ－ＲＦＬＰ、ＳＳＣＰ对伤寒杆菌２７５（临床分离敏感株）及其自发耐药突变株ＲＧ１ＤＮＡ旋转酶Ａ亚单位基因（ｇｙｒＡ）耐喹诺酮类决定区进行检测。并以ＤＮＡ序列分析对该变异进行检测。结果表明,与伤寒杆菌２７５相比,ＲＧ１ｇｙｒＡ第２４７位由Ｔ变异为Ｇ,相应于ＤＮＡ旋转酶Ａ亚单位第８３位氨酸由丝氨酸变为丙氨酸,ＨｉｎｆⅠ对     

Clinical isolates of Streptococcus pneumoniae resistant to levofloxacin contain mutations in both gyrA and parC genes.

Thirty Streptococcus pneumoniae clinical isolates resistant to levofloxacin were analyzed for the quinolone resistance-determining DNA sequences to identify point mutations and were tested for in vitro susceptibility to multiple drug classes. Of these isolates, 29 had mutations in both gyrA and parC genes of DNA gyrase and topoisomerase IV, respectively. In GyrA, an amino acid change from Ser-81-->Phe was detected in 27 isolates and a Glu-85-->Lys change was found in the remaining three. Of the 29 isolates for which ParC data were available, Ser-79-->Tyr or Phe were the predominant mutations observed. MICs for levofloxacin were 4-16 mg/l, whereas those for moxifloxacin were 1-2 mg/l. Twenty-four (80%) isolates were susceptible to erythromycin, 25 (83%) to azithromycin, 26 (87%) to clarithromycin, 27 (90%) to clindamycin, 20 (67%) to penicillin, 21 (70%) to ceftriaxone and 30 (100%) to amoxycillin/clavulanate. These results confirm the presence of double mutations among clinical isolates of S. pneumoniae from diverse geographical regions of North America and also suggest that quinolone resistance may develop independently of resistance to other drug classes.     

Molecular study of quinolone resistance mechanisms and clonal relationship of Salmonella enterica clinical isolates

In the last few years, the number of Salmonella enterica strains resistant to nalidixic acid has steadily increased. In a previous study, the quinolone susceptibility phenotype and genotype of 38 S. enterica clinical isolates (19 S. enterica serovar Typhimurium and 19 S. enterica serovar Enteritidis) were determined. Forty-two percent of the isolates showed nalidixic acid resistance associated with a mutation in gyrA together with putative overexpression of efflux pump(s). In this study, mutations in the quinolone resistance-determining region (QRDR) of parE and the regulators of AcrAB (acrR, marRAB, soxRS and ramR) were analysed. Intracellular accumulation of ciprofloxacin and nalidixic acid was determined. Gene expression of the efflux pump components acrB, tolC, acrF and emrB was also assessed. In addition, an epidemiological study of the isolates by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) was performed. No mutations were detected in parE, whereas two amino acid substitutions were found in two susceptible strains in MarR (I84L) and AcrR (N214T) in one strain each, although both were suggested to be polymorphisms. No changes in the gene expression of acrB, tolC, acrF and emrB were detected between nalidixic-acid-resistant and -susceptible strains. Intracellular accumulation was not useful to reveal differences. Epidemiological analysis showed an important clonal relatedness among the S. Enteritidis isolates, whereas major divergence was seen for S. Typhimurium. Altogether, these results suggest the presence of previously undiscovered drug efflux pump(s) and confirm the high clonality of S. Enteritidis and the genetic divergence of S. Typhimurium.     

LightCycler gyrA mutation assay (GAMA) identifies heterogeneity in GyrA in Salmonella enterica serotypes Typhi and Paratyphi A with decreased susceptibility to ciprofloxacin

Mutations in gyrA in strains of Salmonella enterica serotypes Typhi and Paratyphi A have been characterised by a LightCycler-based PCR-hybridisation gyrA mutation assay (GAMA) and by DNA sequencing. Four mutations (Ser-83 to Phe, Asp-87 to Asn, Ser-83 to Tyr and Asp-87 to Gly) have been identified in 13 strains of Typhi and three strains of Paratyphi A resistant to nalidixic acid (=nal^r) and with decreased susceptibility to ciprofloxacin (=Cp_L), with the mutation Ser-83 to Phe predominating. The results have demonstrated heterogeneity in gyrA in nal^r Cp_L strains of Typhi and Paratyphi A and may be useful for epidemiological investigations. No mutations in gyrA were identified in four Cp_L strains of S. Typhi that were sensitive to nalidixic acid. The mechanism of decreased susceptibility to ciprofloxacin in these strains is under investigation.     

Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing

Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.     

临床表葡菌gyrA、gyrB、grlA和grlB基因突变与其对环丙沙星耐药性的关系

用平皿二倍稀释法测定18株表葡菌对环丙沙星的MIC（0．125—128μg／ml）。分别针对表葡菌中gyrA、gyrB、grlA、grlB等四种基因进行引物设计与合成，提取基因组DNA，PCR扩增，对PCR产物进行克隆、鉴定并测序，用DNASIS软件分析测序结果。在表葡菌的GdA中，只有Ser80→Phe、Ser80→Tyr或Asp84→Asn、Asp84→Ala、Asp84→Tyr的改变。在GyrA中，存在ser84→TYr、ser84→Phe、Glu88→Lys的改变；在任一株表葡菌中未见有gyrB或grlB的突变。在被检测的18株临床分离表葡菌中有3个分离株对环丙沙星的MICs≤0．5μg／ml，在GyrA或GrlA的决定区没有变化。对环丙沙星的MICs≥2μg／ml的15个分离株中，GyrA中有Ser84和Glu88及GdA中的Ser80和Asp84的氨基酸改变。1株MIC为2μg／ml的GdA中发生一个氨基酸改变，而GyrA中无变化。说明拓朴异构酶Ⅵ可能是环丙沙星作用于表葡菌的首要靶位酶。在高水平耐药株中，在GdA和GyrA中均存在多位点的突变，提示耐药水平的高低与GdA、GyrA位点的突变直接相关。     

Antibiotic resistance in Salmonella enterica subsp. enterica strains isolated in Poland from 1998 to 1999.

A total of 326 Salmonella enterica subsp. enterica strains representing 29 serotypes, isolated from human stool specimens during 1998-1999 in sanitary-epidemiological units in Poland were tested for antibiotic susceptibility by a standard disk diffusion method. The antibiotics used were ampicillin, cefotaxime, chloramphenicol, tetracycline, streptomycin, gentamicin, kanamycin, nalidixic acid, ciprofloxacin, furazolidone, cotrimoxazole, sulphonamides and trimethoprim. In addition, 201 strains belonging to the five most commonly isolated serotypes (S. Enteritidis, S. Typhimurium, S. Hadar, S. Infantis and S. Virchow) also had minimal inhibitory concentrations (MICs) determined for amoxycillin/clavulanic acid. Selected strains were screened for production of extended spectrum beta-lactamases (ESBLs). There were 49.4% of Salmonella enterica subsp. enterica strains resistant to two or more antibiotics, with the highest prevalence of multiple resistant strains among serotypes Typhimurium, Hadar and Virchow. Resistance to ampicillin, streptomycin, tetracycline, nalidixic acid, furazolidone and sulphonamides occurred most frequently. Over 93% of S. Virchow strains were resistant to furazolidone. No strains resistant to ciprofloxacin by disk-diffusion method were detected but 31.3% of isolates of the 201 strains representing the five most common serotypes had reduced ciprofloxacin susceptibility (MICs ranging 0.125-0.5 mg/l). One strain (S. Mbandaka) was resistant to cefotaxime and produced ESBL.     

淋病奈瑟菌的耐药性与喹诺酮耐药基因突变的相关性分析

目的 分析本地区淋病奈瑟菌临床菌株gyrA基因突变与喹诺酮类药物耐药的相关性.方法 采用琼脂稀释法检测37株淋病奈瑟菌临床菌株对6种抗生素的最小抑菌浓度(MIC),PCR法扩增淋病奈瑟菌临床菌株gyrA基因片段并测序.结果37株淋病奈瑟菌临床菌株对大砚霉素、头孢曲松、四环素、青霉素、氧氟沙星和环丙沙星的耐药率分别为0、...     

沙门氏菌中超广谱β-内酰胺酶的检测

目的:分析石家庄市公共场所从业人员沙门氏菌中产超广谱β-内酰胺酶菌型,确立有效的治疗措施,防止院内感染的爆发流行.方法:采用Etese试条法,对在感染人群中分离的55种沙门氏菌血清型进行了ESBLs产超广谱β-内酰胺酶的检测并观察其对15种抗生素敏感性.结果:检出9种沙门氏菌可产生超广谱β-内酰胺酶.感染的主要菌型分布于9个血清群、55个血清型.结论:分离的沙门氏菌耐药严重,产ESBLS沙门氏菌对新霉素、链霉素、庆大霉素、萘啶酸较敏感.     

Molecular analysis of multiresistant porcine Salmonella enterica subsp. enterica serovar Bredeney isolates from Southern Brazil: identification of resistance genes, integrons and a group II intron

The relationships of 83 porcine Salmonella enterica subsp. enterica serovar Bredeney isolates obtained at two slaughterhouses in Southern Brazil were analysed by XbaI and BlnI macrorestriction analysis, plasmid profiling and determination of antimicrobial resistance patterns. Twenty-nine XbaI and 30 BlnI macrorestriction patterns were identified. The 72 plasmid-bearing isolates exhibited 20 different plasmid profiles. Multiresistance was detected in 49 isolates (59%), of which 39 isolates showed at least resistance to sulfonamides, tetracyclines, chloramphenicol, streptomycin, kanamycin and/or ampicillin. A representative subset of 12 isolates was chosen for identification of resistance genes, their localisation and transferability. The sulfonamide resistance genes sul1, sul2 and sul3, the tetracycline resistance genes tet(A) and tet(B), the phenicol resistance genes catA1 and floR, the streptomycin resistance gene strA, the kanamycin resistance gene aphA1 and the ampicillin resistance gene bla_TEM were detected and found to be located most frequently on plasmids. In addition, class 1 and 2 integrons with the cassette arrangements dfrA21/bla_OXA-129/aadA1 and dfrA1/sat1/aadA1, respectively, were detected. A group II intron was found to be inserted into the 59-base element of an aadA1 gene cassette in a class 1 integron. This study revealed a wide genomic variety among the S. Bredeney isolates, and the high number of multiresistant isolates may point towards the risks that these S. Bredeney isolates can represent to human health.     

Analysis of mechanisms involved in reduced susceptibility to ciprofloxacin in Salmonella enterica serotypes Typhi and Paratyphi A isolates from travellers to Southeast Asia

Owing to multidrug resistance, quinolones and third-generation cephalosporins are currently used as key antibiotics to combat Salmonella organisms. Therapy failure due to reduced ciprofloxacin susceptibility has been reported in endemic areas, but also in imported disease. Different bacterial resistance mechanisms may result in reduced ciprofloxacin susceptibility. In this study, the presence and expression of different resistance mechanisms resulting in reduced minimum inhibitory concentrations (MICs) for ciprofloxacin were evaluated in 23 blood-culture-derived Salmonella enterica serotypes Typhi and Paratyphi A organisms from ill-returned travellers to Asia. The presence of mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene as well as an activated efflux pump and plasmid-mediated quinolone resistance genes was determined. Resistance selection during therapy and the clonal relatedness of all isolates were established. Efflux pump inhibition did not appear to affect the MICs of ciprofloxacin and activity of the efflux pump appeared to be specific for nalidixic acid. Repeated exposure of the isolates to ciprofloxacin did not result in a significant increase in the MICs for ciprofloxacin. Repetitive sequence-based polymerase chain reaction (rep-PCR) profiles identified five different genotypes, but no correlation with resistance was observed. However, a significant relation was found with geographic region; reduced ciprofloxacin susceptibility was only found in travellers returning from India and Pakistan. All isolates with reduced ciprofloxacin susceptibility had a mutation at position 83 in the QRDR region of the gyrA gene. Plasmid-mediated quinolone resistance was not found. These findings confirm that the reduced ciprofloxacin MIC in S. Typhi and S. Paratyphi A is solely due to an amino acid substitution in the QRDR 'cluster' of the gyrA gene.     

耐氟喹诺酮类铜绿假单胞菌的gyrA基因突变研究

目的：研究临床分离的耐氟喹诺酮类药物铜绿假单胞菌gyrA基因突变情况。方法：测定临床分离的55株铜绿假单胞菌的MIC值，从中筛选出1株敏感菌和8株耐药菌。以标准敏感菌株ATCC27853作为质控菌株，用聚合酶链反应（PCR）扩增gyrA基因的喹诺酮耐药决定区（QRDR），扩增产物片段长度为350bp。用限制性内酶SacⅡ消化PCR产物，同时对上述10株菌的gyrA基因的喹诺酮决定区（QRDR）进行PCR－DNA直接测序分析。结果：临床分离铜绿假单胞菌敏感菌株的gyrA基因QRDR经限制性内切酶SacⅡ消化后出现118bp,232bp两条片段，表明该酶切位点未发生突变，此结果经DNA序列析证实。而耐药菌在酶切后均为一条片段，表明该酶切位点消失，经DNA序列分析发现，8株耐药株在83位（ACC→ATC）均有突变，该单位突变引起氨基酸由Thr→Ile的改变；其中有3株高度耐药菌同时发现在87位（GAC→GGC）有突变，该单位点突变引起氨基酸由Asp→Gly的改变，但没有发现87位点突变单独存在，且双位点突变菌株的MIC值与单一位点突变的MIC值相比，有显著的升高，MIC增加4－32倍，除此之外，有6株耐药菌株在132位有一静止突变（CAC→CAT），该 突变未引起氨基酸的改变。结论：gyrA基因突变是铜绿假单胞菌对氟喹酮类药物产生耐药的主要机制之一，铜绿假单胞菌gyrA上83位和87位突变最为常见。