鼠伤寒沙门菌临床分离株对喹诺酮类药物耐药机制的研究

目的 研究引起儿童腹泻鼠伤寒沙门菌对喹诺酮类抗菌药物的耐药机制.方法 采用K-B法测定62株鼠伤寒沙门菌对常用抗菌药物的敏感性;应用PCR法和DNA测序法检测喹诺酮耐药决定区(QRDRs)和质粒介导的喹诺酮耐药基因;采用接合试验测定耐药基因的转移性;PFGE法测定耐药菌株的同源性.结果 62株肠伤寒沙门菌中有40株对环丙沙星耐药(MIC≥0.5 μg/ml),其中28株为低水平耐药,12株为高水平耐药,且所有对环丙沙星耐药菌株同时对其他多种抗菌药物耐药;40株环丙沙星耐药菌株分属于4种PFGE型,分别是A型7株、B型4株、E型1株、D型28株;两株对环丙沙星高水平耐药菌株同时存在gyrA和parC两个位点的突变,其余环丙沙星耐药菌株仅发现gyrA 1个位点的突变,未检测到qnr和qepA基因;62株鼠伤寒沙门菌中有23株为aac-(6′)-Ib-cr阳性,其中19株PFGE分型为D型.结论 腹泻患儿分离的肠伤寒沙门菌对环丙沙星耐药率高,喹诺耐耐药决定区gyrA基因的点突变和携带aac-(6′) -Ib-cr基因是导致鼠伤寒沙门菌对环丙沙星耐药的主要原因.     

Prevalence and mechanisms of quinolone resistance among selected nontyphoid Salmonella isolated from food animals and humans in Korea

The aim of this study was to investigate the prevalence and mechanism of quinolone resistance among selected nontyphoid Salmonella (NTS) isolates. A total of 1279 NTS isolated from food animals (n=692) and humans (n=587) between 1995 and 2009 were investigated by serotyping, antimicrobial susceptibility testing, screening for plasmid-mediated quinolone resistance (PMQR) genes qnr, aac(6')-Ib-cr, and qepA and mutations in the quinolone resistance-determining region (QRDR) of gyrA and parC by PCR, and DNA sequencing. Three hundred thirty (47.7%) of 692 animal isolates and 177 (30.2%) of 587 human isolates were resistant to nalidixic acid. Most animal (94.8%, 313/330) and human (99.4%, 176/177) NTS exhibited decreased ciprofloxacin susceptibility (minimum inhibitory concentration [MIC]: 0.125-2?mg/L). None of them carried qnr or qepA gene. However, aac(6')-Ib was identified in six animal isolates, of which four carried aac(6')-Ib-cr gene. Based on antimicrobial resistance profile, year of isolation, MIC for quinolones and fluoroquinolones, and isolation frequency of serotype, 114 animal and 83 human isolates were tested for QRDR mutations. All contained a single mutation within the QRDR of gyrA at either codon 87 or 83, and 41 of them contained an additional mutation in parC. The most prevalent mutation was Asp87-Tyr (n=107), followed by Asp87-Gly (n=28), Asp87-Asn (n=26), Ser83-Tyr (n=22), and Ser83-Phe (n=14). Point mutations in parC were observed outside the QRDR, which included 40 isolates with Thr57-Ser substitution and 1 Salmonella Typhimurium with a novel Glu51-Lys substitution. In conclusion, a point mutation within the QRDR of gyrA was primarily responsible for quinolone resistance and reduced susceptibility to fluoroquinolones in NTS in Korea. To our knowledge, this is the first report of occurrence of aac(6')-Ib-cr gene among NTS in Korea. The spread of NTS carrying aac(6')-Ib-cr is of serious concern and should be carefully monitored.     

Cephalosporin and ciprofloxacin resistance in Salmonella, Taiwan

We report the prevalence and characteristics of Salmonella strains resistant to ciprofloxacin and extended-spectrum cephalosporins in Taiwan from January to May 2004. All isolates resistant to extended-spectrum cephalosporins carried blaCMY-2, and all ciprofloxacin-resistant Salmonella enterica serotype Choleraesuis isolates were genetically related.     

Bacteraemia due to ciprofloxacin-resistant Salmonella enterica serotype Choleraesuis in adult patients at a university hospital in Taiwan, 1996-2004

Eighty-one adult patients with Salmonella enterica serotype Choleraesuis (S. Choleraesuis) bacteraemia treated at a university hospital from 1996 to 2004 were evaluated. Multivariate analysis with a logistic regression model was used to characterize risk factors for primary bacteraemia and mycotic aneurysm and to determine the association of clinical characteristics of patients based on ciprofloxacin susceptibility of the causative organism. The incidence per 100,000 discharges was 0.76 in 1996 and 3.9 in 2004. The overall rate of ciprofloxacin resistance among these isolates was 59% (87 isolates) and the annual rate increased with time from 0% prior to 2000 to 80% in 2004. Among these patients, 48 (59%) had primary bacteraemia and 13 (16%) had secondary bacteraemia with mycotic aneurysm. Seventy (86%) patients had fever at presentation, 22 (27%) developed shock during hospitalization, and eight (10%) died of S. Choleraesuis bacteraemia. Patients with immunocompromised conditions had a higher risk of developing primary bacteraemia (OR 18.442, P < 0.001). Hypertension (OR 15.434, P = 0.002) and male gender (OR 7.422, P = 0.039) were associated with mycotic aneurysm. Patients with mycotic aneurysm were more frequently infected with ciprofloxacin-susceptible isolates (P = 0.028) and ciprofloxacin-susceptible isolates were also more frequently associated with recurrent infection than ciprofloxacin-resistant isolates (P = 0.038). The incidence of S. Choleraesuis bacteraemia has increased in the past 8 years, and this increase is associated with the upsurge of ciprofloxacin-resistant isolates.     

Human Salmonella and concurrent decreased susceptibility to quinolones and extended-spectrum cephalosporins

The National Antimicrobial Resistance Monitoring System monitors susceptibility among Enterobacteriaceae in humans in the United States. We studied isolates exhibiting decreased susceptibility to quinolones (nalidixic acid MIC >32 microg/mL or ciprofloxacin MIC > or =0.12 microg/mL) and extended-spectrum cephalosporins (ceftiofur or ceftriaxone MIC > or =2 microg/mL) during 1996-2004. Of non-Typhi Salmonella, 0.19% (27/14,043) met these criteria: 11 Senftenberg; 6 Typhimurium; 3 Newport; 2 Enteridis; and 1 each Agona, Haifa, Mbandaka, Saintpaul, and Uganda. Twenty-six isolates had gyrA mutations (11 at codon 83 only, 3 at codon 87 only, 12 at both). All Senftenberg isolates had parC mutations (S801 and T57S); 6 others had the T57S mutation. The Mbandaka isolate contained qnrB2. Eight isolates contained bla(CMY-2); 1 Senftenberg contained bla(CMY-23). One Senftenberg and 1 Typhimurium isolate contained bla(SHV-12); the Mbandaka isolate contained bla(SHV-30). Nine Senftenberg isolates contained bla(OXA-1) contained bla(OXA-9). Further studies should address patient outcomes, risk factors, and resistance dissemination prevention strategies.     

2010—2011年广东地区人源主要血清型沙门菌喹诺酮耐药特征分析

目的了解广东省沙门菌对喹诺酮耐药特征及其基因突变情况。方法选用2010—2011年广东省非伤寒沙门菌监测收集的4种主要血清型沙门菌,采用半定量药敏检测法测定2010—2011年收集的沙门菌临床株对萘啶酸和环丙沙星的最小抑菌浓度（MIC）;通过PCR扩增鼠伤寒沙门菌株的gyrA和parC的基因,并对序列进行测定,对比野生株,发现突变位点及突变类型。结果共对2010—2011年收集的496株鼠伤寒沙门菌、鼠伤寒沙门菌变种、肠炎沙门菌和斯坦利沙门菌进行耐药性分析,75.4%（374/496）沙门菌对萘啶酸耐药,其中鼠伤寒沙门菌、鼠伤寒沙门菌变种、肠炎沙门菌、斯坦利沙门菌耐药率分别为82.5%（207/251）、84.0%（84/100）、79.5%（70/88）、22.8%（13/57）;5.4%（27/496）沙门菌对环丙沙星耐药,其中鼠伤寒沙门菌、鼠伤寒沙门菌变种、肠炎沙门菌的耐药率分别为8.0%（20/251）、5.0%（5/100）、2.3%（2/88）。71.7%（180/251）鼠伤寒沙门菌发生基因突变,其中86.1%（155/180）发生gyrA上突变,37.2%（67/180）发生parC上突变,23.3%（42/180）发生双基因突变。环丙沙星耐药株、中敏株、敏感株分别有7株（35.0%,7/20）、1株（3.1%,1/32）、6株（3.0%,6/199）发生gyrA Ser83位点突变,3组间Ser83位点突变率差异有统计学意义（P〈0.01）;环丙沙星耐药株、中敏株、敏感株分别有15株（75.0%,15/20）、21株（65.6%,21/32）、111株（55.8%,111/199）发生gyrA Asp87位点突变,3组间突变率差异无统计学意义（P〉0.05）。5株parC Ser80位点突变株均突变为Arg,此突变的菌株均伴有Ser83Phe及Asp87Asn;9株parC Thr57突变株均对萘啶酸耐药。结论广东省沙门菌对萘啶酸普遍耐药,对环丙沙星仍普遍敏感;gyrA和parC中可能影响沙门菌耐药的突变位点应进一步确认其对耐药的影响。     

Increasing ceftriaxone resistance in Salmonellae, Taiwan

In Taiwan, despite a substantial decline of Salmonella enterica serotype Choleraesuis infections, strains resistant to ciprofloxacin and ceftriaxone persist. A self-transferable bla(CMY-2)-harboring IncI1 plasmid was identified in S. enterica serotypes Choleraesuis, Typhimurium, Agona, and Enteritidis and contributed to the overall increase of ceftriaxone resistance in salmonellae.     

耐环丙沙星鲍曼不动杆菌gyrA和parC突变模式分析

目的对临床耐环丙沙星的鲍曼不动杆菌进行gyrA和parC基因突变模式的分子流行病学调查。方法采用E-test法测定环丙沙星对94株临床分离鲍曼不动杆菌的最低抑菌浓度（MIC）；对鲍曼不动杆菌的gyrA和parC基因进行聚合酶链反应（PCR）-限制性内切酶片段长度多态性（RFLP）分析及DNA测序。结果在94株鲍曼不动杆菌中，只有38株（40．43％）对环丙沙星敏感（MIC≤1mg／L），而对环丙沙星的耐药率接近60％；PCR-RFLP分析结果表明，对环丙沙星耐药的56株菌都发生了gyrA和parC基因突变，且parC基因的突变率（87．50％）略高于gyrA基因（82．14％）。结论鲍曼不动杆菌对环丙沙星的耐药与gyrA和parC基因突变相关，其中parC基因突变的明显增长值得重视。     

Antibiotic resistance in Salmonella enterica serotypes Typhimurium, Enteritidis and Infantis from human infections, foodstuffs and farm animals in Italy

The antimicrobial susceptibility of isolates of Salmonella enterica serotypes Typhimurium, Enteritidis, and Infantis isolated from humans, foodstuffs and farm animals in Italy between 1999 and 2001 was examined. All the isolates were susceptible to cefotaxime and ciprofloxacin, but high rates of resistance were observed for several other drugs, especially for S. Typhimurium. The rates of resistance and multiresistance were generally higher among animal and food isolates than in human strains; conversely, no significant difference was observed between animal and food isolates. Among S. Typhimurium, multiresistance was more common in bovine, poultry and rabbit strains than in swine isolates, and was rare in strains from pigeon. Resistance to trimethoprim sulphamethoxazole was mainly found in isolates of swine and human origin. This study confirms the role of livestock as a reservoir of drug-resistant Salmonella spp. and underlines the need for integrated surveillance systems of antibiotic resistance that consider isolates not only from human disease but also from the animal reservoirs and the food vehicles.     

Molecular analysis of fluoroquinolone-resistant Salmonella Paratyphi A isolate, India

Salmonella enterica serovar Paratyphi A is increasingly a cause of enteric fever. Sequence analysis of an Indian isolate showed a unique strain with high-level resistance to ciprofloxacin associated with double mutations in the DNA gyrase subunit gyrA (Ser83-->Phe and Asp87-->Gly) and a mutation in topoisomerase IV subunit parC (Ser80-->Arg).     

鼠伤寒沙门菌耐药株拓扑异构酶基因突变分析

目的　探讨鼠伤寒沙门菌对氟喹诺酮类药物的耐药性与拓扑异构酶基因突变之间的关系。方法　三株鼠伤寒沙门菌分离株X2 ,X7,X11对氟喹诺酮类药物环丙沙星的MIC分别为 32、0 38和 0 0 2 3μg ml,X2为耐药株。利用聚合酶链反应 (PCR)对其拓扑异构酶四个耐药基因gyrA ,gyrB ,parC ,和parE进行扩增和序列分析。结果　发现分离株X2的gyrA基因同时在 2 4 8、2 5 9位点发生点突变 (Ser83→Phe ,Asp87→Asn)。分离株X7gyrA基因在 2 4 8位点发生点突变 (Ser83→Tyr)。分离株X11的gyrA基因没有发生突变。X2parC基因QRDR在 2 38位点发生点突变 (Ser80→Arg)。而分离株X7、X11的parC基因没有发生突变。三株鼠伤寒沙门菌分离株的gyrB和parE基因都没有发现突变。结论　gyrA和parC上同时发生突变会导致鼠伤寒沙门菌对环丙沙星的高耐药性     

氟喹诺酮耐药肠球菌gyrA和parC基因突变特征及其与环丙沙星耐药相关性分析

目的 探讨氟喹诺酮耐药肠球菌的gyrA和parC基因突变特征与环丙沙星耐药程度的相关性。方法 收集2016 - 2018年临床分离59株粪肠球菌和62株屎肠球菌,使用PCR方法特异性扩增gyrA和parC基因,并进行基因测序检测分析。结果 粪肠球菌和屎肠球菌对环丙沙星耐药率分别为59.32%和80.65%。粪肠球菌gyrA基因Ser83突变（χ2 = 51.252, P＜0.001）和parC基因Ser80突变（χ2 = 55.115, P＜0.001）与粪肠球菌对环丙沙星高水平耐药（MIC≥16μg/ml）的相关性差异具有统计学意义;屎肠球菌gyrA基因Ser83突变（χ2 = 42.014, P＜0.01）和parC基因Ser80突变（χ2 = 62.000, P＜0.01）与屎肠球菌对环丙沙星高水平耐药（MIC≥16 μg/ml）的相关性差异具有统计学意义。结论 gyrA基因Ser83突变和parC基因Ser80突变与肠球菌对环丙沙星高水平耐药（MIC≥16 μg/ml）可能相关。     

Tentative colistin epidemiological cut-off value for Salmonella spp

The objective of this research was to determine minimal inhibitory concentration (MIC) population distributions for colistin for Salmonella on subtype level. Furthermore, we wanted to determine if differences in MIC for colistin could be explained by mutations in pmrA or pmrB encoding proteins involved in processes that influence the binding of colistin to the cell membrane. During 2008-2011, 6,583 Salmonella enterica subsp. enterica isolates of human origin and 1931 isolates of animal/meat origin were collected. The isolates were serotyped, and susceptibility was tested towards colistin (range 1-16?mg/L). Moreover, 37 isolates were tested for mutations in pmrA and pmrB by polymerase chain reaction (PCR) and DNA sequencing. MIC distribution for colistin at serotype level showed that Salmonella Dublin (n=198) followed by Salmonella Enteritidis (n=1247) were less susceptible than "other" Salmonella serotypes originating from humans (n=5,274) and Salmonella Typhimurium of animal/meat origin (n=1794). MIC was ≤1?mg/L for 98.9% of "other" Salmonella serotypes originating from humans, 99.4% of Salmonella Typhimurium, 61.3% of Salmonella Enteritidis, and 12.1% of Salmonella Dublin isolates. Interestingly, Salmonella Dublin and Salmonella Enteritidis belong to the same O-group (O:1, 9,12), suggesting that surface lipopolysaccharides (LPS) of the cell (O-antigen) play a role in colistin susceptibility. The epidemiological cut-off value of >2?mg/L for colistin suggested by European Committee on Antimicrobial Susceptibility Testing (EUCAST) is placed inside the distribution for both Salmonella Dublin and Salmonella Enteritidis. All tested Salmonella Dublin isolates, regardless of MIC colistin value, had identical pmrA and pmrB sequences. Missense mutations were found only in pmrA in one Salmonella Reading and in pmrB in one Salmonella Concord isolate, both with MIC of ≤1 for colistin. In conclusion, our study indicates that missense mutations are not necessarily involved in increased MICs for colistin. Increased MICs for colistin seemed to be linked to specific serotypes (Salmonella Dublin and Salmonella Enteritidis). We recommend that Salmonella with MIC of >2?mg/L for colistin be evaluated on the serovar level.     

Molecular analysis of high-level ciprofloxacin resistance in Salmonella enterica serovar Typhi and S. Paratyphi A: need to expand the QRDR region?

Fourteen strains of S. Typhi (n=13) and S. Paratyphi A (n=1) resistant to ciprofloxacin were compared with 30 ciprofloxacin decreased-susceptibility strains on the basis of qnr plasmid analysis, and nucleotide substitutions at gyrA, gyrB, parC and parE. In ciprofloxacin-resistant strains, five S. Typhi and a single S. Paratyphi A showed triple mutations in gyrA (Ser83-->Phe, Asp87-->Asn, Glu133-->Gly) and a novel mutation outside the quinolone resistance determining region (QRDR) (Met52-->Leu). Novel mutations were also discovered in an isolate (minimum inhibitory concentration 8 microg/ml) in gyrA gene Asp76-->Asn and outside the QRDR Leu44-->Ile. Out of 30 isolates with reduced susceptibility, single mutation was found in 12 strains only. Genes encoding qnr plasmid (qnr A, qnr B, AAC1-F) were not detected in ciprofloxacin-resistant or decreased-susceptibility strains. Antimicrobial surveillance coupled with molecular analysis of fluoroquinolone resistance is warranted for reconfirming novel and established molecular patterns of resistance, which is quintessential for reappraisal of enteric fever therapeutics.     

Molecular typing of Salmonella enterica serovars Enteritidis, Corvallis, Anatum and Typhimurium from food and human stool samples in Tunisia, 2001-2004

During the period from 2001 to 2004, a total of 72 isolates of Salmonella enterica serovars: Anatum (n=40), Enteritidis (n=18), Corvallis (n=8), and Typhimurium (n=6), of various origins (mainly food and diarrhoeagenic stool samples), were collected and further characterized by antibiotic resistance, plasmid analysis, and pulsed-field gel electrophoresis (PFGE). Forty-five isolates presented multidrug resistance to antibiotics. Among which one S. enterica serovar Anatum isolate was resistant to 11 antibiotics, and one S. enterica serovar Typhimurium DT104 isolate was resistant to eight antibiotics. Plasmid profiling identified eight plasmid profiles (with 1-5 plasmids) among the isolates, of which one plasmid profile (P01) was predominant. XbaI PFGE analysis revealed the presence of a predominant clone of the four studied Salmonella serovars circulating in Tunisia throughout the years 2001-2004.     

Ciprofloxacin-resistant Salmonella enterica serotype Typhimurium, China

We characterized 44 Salmonella enterica serotype Typhimurium isolates from Tongji Hospital outpatients in Wuhan, China, May 2002-October 2005. All 31 ciprofloxacin-resistant isolates were also resistant to > or = 8 other antimicrobial drugs and carried > or = 2 mutations in GyrA and 1 mutation in ParC. Class 1 integrons were identified in 37 isolates.     

Ciprofloxacin-resistant Salmonella enterica Serotype Typhi, United States, 1999-2008

We report 9 ciprofloxacin-resistant Salmonella enterica serotype Typhi isolates submitted to the US National Antimicrobial Resistance Monitoring System during 1999-2008. The first 2 had indistinguishable pulsed-field gel electrophoresis patterns and identical gyrA and parC mutations. Eight of the 9 patients had traveled to India within 30 days before illness onset.     

大肠埃希菌对氟喹诺酮类的耐药性及其机制研究

目的 探讨大肠埃希菌(ECO)对氟喹诺酮类药物的耐药率及耐药机制.方法 K-B法测定100株ECO对5种氟喹诺酮抗菌药物的耐药性,试管稀释法测定耐环丙沙星和左氧氟沙星的最低抑菌浓度(MIC);错配PCR技术检测耐环丙沙星ECO gyrA基因和parC基因的突变.结果 100株ECO中耐环丙沙星有65株;环丙沙星MIC50、MIC90分别是32、128 μg/ml,左氧氟沙星MIC50、MIC90是16、64 μg/ml;基因位点结果显示,耐环丙沙星的65株中,有60株发生gyrA、parC一个或多个基因位点的突变,有5株未发生突变.结论 gyrA、parC基因突变是该地区ECO,对氟喹诺酮类药物耐药的重要机制,且基因突变位点越多其耐药程度越高.     

The evolution and distribution of phage ST160 within Salmonella enterica serotype Typhimurium

Salmonellosis is an internationally important disease of mammals and birds. Unique epidemics in New Zealand in the recent past include two Salmonella serovars: Salmonella enterica subsp. enterica serovar Typhimurium definitive type (DT) 160 (S. Typhimurium DT160) and S. Brandenburg. Although not a major threat internationally, in New Zealand S. Typhimurium DT160 has been the most common serovar isolated from humans, and continues to cause significant losses in wildlife. We have identified DNA differences between the first New Zealand isolate of S. Typhimurium DT160 and the genome-sequenced strain, S. Typhimurium LT2. All the differences could be accounted for in one cryptic phage ST64B, and one novel P22-like phage, ST160. The majority of the ST160 genome is almost identical to phage SE1 but has two regions not found in SE1 which are identical to the P22-like phage ST64T, suggesting that ST160 evolved from SE1 via two recombination events with ST64T. All of the New Zealand isolates of DT160 were identical indicating the clonal spread of this particular Salmonella. Some overseas isolates of S. Typhimurium DT160 differed from the New Zealand strain and contained SE1 phage rather than ST160. ST160 was also identified in New Zealand isolates of S. Typhimurium DT74 and S. Typhimurium RDNC-April06 and in S. Typhimurium DT160 isolates from the USA. The emergence of S. Typhimurium DT160 as a significant pathogen in New Zealand is postulated to have occurred due to the sensitivity of the Salmonella strains to the ST160 phage when S. Typhimurium DT160 first arrived.     

Antimicrobial susceptibility patterns of Salmonella enterica serotype typhi in eastern Nepal

The aim of the present study was to evaluate antimicrobial susceptibility patterns with special reference to multidrug resistance, susceptibility to ciprofloxacin, and bacteriophage typing of Salmonella enterica serotype Typhi isolated from blood sent for culture in a tertiary-care teaching hospital in eastern Nepal during January 2000-December 2004. In total, 132 strains of S. enterica Typhi, isolated from 2,568 blood culture samples collected from cases of suspected enteric fever, were tested for susceptibility to commonly-used antimicrobials by the disc-diffusion method. There were 35 multidrug-resistant strains. None of the isolates were resistant to ciprofloxacin. Of 52 isolates tested for minimum inhibitory concentration (MIC) of ciprofloxacin, 36 (69.23%) showed reduced susceptibility (MIC >0.25 mg/L). Of 112 strains tested for nalidixic acid susceptibility, 86 (76%) were resistant. Strains with reduced susceptibility to ciprofloxacin and resistance to nalidixic acid could be correlated. The commonest phage type was El. Nalidixic acid susceptibility could be a useful screening test for the detection of decreased susceptibility of S. Typhi to ciprofloxacin, a drug which is commonly used even for minor ailments in this area.