Genetic analysis of Tn916-like elements conferring tetracycline resistance in clinical isolates of Clostridium difficile

As an important clinically relevant pathogen, Clostridium difficile has a high multidrug resistance rate. Conjugative transposons play a vital role in its resistance phenotype. In the present study, 34 tetracycline-resistant clinical isolates of C. difficile were studied to detect tetracycline resistance genes and the presence of transposons. Thirty-two isolates were found to harbour Tn916-like elements carrying the tet(M) resistance gene, of which only one copy existed in the genome by Southern blot analysis. To characterise the genetic organisation of the Tn916-like elements, overlap PCR assays were performed with nine primer pairs, revealing three types of elements designated T1 to T3. The prevalent element T1 lacking PCRA (ORF₂₃ to ORF₂₁) and PCRB (ORF₂₁ to ORF₂₀) products, present in the epidemic ST37 clone, was further analysed by genome walking PCR in the left and right end sequences of the novel Tn916-like element. A gene coding for an FtsK/SpoIIIE family protein was found to replace the ORF24 to ORF21 region in Tn916. Moreover, the element could hardly conjugate between cells by filter mating experiments. These findings suggest that the dissemination of Tn916-like elements in epidemic ST37 strains in China was likely to have been conferred by clonal spread, signifying the importance of future surveillance and characterisation of conjugative transposons.     

临床分离铜绿假单胞菌质粒介导的喹诺酮耐药机制研究

目的了解临床分离的铜绿假单胞菌中PMQR基因及ESBLs的分布情况,并对PMQR阳性菌株中染色体介导的喹诺酮耐药机制进行分析。方法采用琼脂稀释法测定菌株对环丙沙星和左氧氟沙星的药物敏感性;PCR检测qnr A、qnr B、qnr C、qnr D、qnr S、aac(6’)-Ib-cr和qep A,对PMQR阳性菌株扩增blaTEM、blaSHV、blaCTX-M和blaPER,同时扩增测序分析染色体基因gyr A、gyr B、par C和par E的突变情况;接合转移试验验证PMQR与bla基因的转移性。结果 106株铜绿假单胞菌中,2株携带qnr S,1株携带qnr D,2株携带aac(6’)-Ib-cr。4株PMQR阳性菌株中有2株接合转移成功,qnr S1和blaTEM-1基因可以通过质粒共同转移。PMQR阳性菌株均在Gyr A亚基和/或Par C亚基存在氨基酸突变,其相应的接合子以及受体菌均未发现突变。结论临床分离的铜绿假单胞菌中存在qnr S、qnr D和aac(6’)-Ib-cr基因,PMQR与bla基因能共同转移,造成多重耐药的传播。     

碳青霉烯类耐药肺炎克雷伯菌临床分离株中质粒介导的喹诺酮类耐药基因检测

目的：探讨质粒介导的喹诺酮类耐药（PMQR）基因在碳青霉烯类耐药肺炎克雷伯菌（CRKP）临床分离株中的分布情况。方法：收集CRKP29株,聚合酶链反应（PCR）扩增并确定产物基因型。结果：CRKP中PMQR基因检出率为48．3％（14／29）,其中qnrB5株,包括qnrB21株、qnrB43株、qnrB101株;qnrS 5株,均为qnrS1;1株同时携带qnrS1和qnrB4;有3株携带aac（6′）-Ib-cr基因。所有PMQR基因阳性菌株均同时携带β-内酰胺酶基因,其中8株同时携带碳青霉烯酶基因,占57．1％（8／14）,以blaKPC-2（4／14）及blaNDM-1（3／14）为主。结论：PMQR基因在临床分离的CRKP中较为普遍,以qnr基因为主,且qnr阳性菌株碳青霉烯酶基因携带率较高,均为多重耐药株。     

ICEGpa1804, a novel integrative and conjugative element carrying eight resistance genes,identified in Glaesserella parasuis

ICEGpa1804 was identified in the genome of a serovar 2, ST279 isolate EHP1804 carrying eight different resistance genes from 200 Glaesserella parasuis strains isolated from swine with lower respiratory tract infection in seven provinces of China. Susceptibility testing for EHP1804 was determined by broth microdilution, and its genetic profile was determined by whole-genome sequencing. The complete ICEGpa1804 was analysed by polymerase chain reaction, conjugation assay and bioinformatics tools. The conjugation assay was performed using EHP1804 as the donor and G. parasuis V43 (rifampicin-resistant) as the recipient. ICEGpa1804 has a size of 71,880 bp and contains 83 genes, including eight resistance genes [tet(B), bla_Rob-1, aphA1, strA, strB, aac(3)-IId, catA3 and sul2]. The conjugation assay showed that ICEGpa1804 could be transferred to G. parasuis V43 with frequencies of 4.3 × 10^?7. To the best of the authors’ knowledge, this is the first study to identify a novel integrative and conjugative element (ICE) carrying eight resistance genes and seven insertion sequence (IS) elements from a G. parasuis isolate. Tn6743, a novel transposon carrying six resistance genes, was identified. Moreover, ISGpa1, a novel IS256 family insertion element, is the first characterized example of a G. parasuis insertion element. Multiple mobile genetic elements involved in resistance genes were located in chromosomal ICEGpa1804, which showed that ICEs may serve as a vital platform for the accumulation of resistance genes.     

Expression of multidrug resistance efflux pump genes in clinical and environmental isolates of Staphylococcus aureus

Increased expression of multidrug resistance efflux pump (MDR-EP) genes in clinical isolates of Staphylococcus aureus occurs frequently, but its temporal and geographic variability is unknown. Such strains may contaminate the hospital environment, posing an infection control problem. Nearly 700 clinical isolates from different geographic locales as well as 91 environmental isolates recovered from two Detroit hospitals were studied. Ethidium bromide (EtBr) minimum inhibitory concentration (MIC), quantitative expression of all characterised chromosomal MDR-EP genes, and the presence of qacA/B and smr were determined for all strains. In addition, for norA- and/or mepA-overexpressing strains, the spa type was established. MDR-EP gene overexpression varied temporally and geographically, and overexpressing strains were present in the hospital environment. Increased expression of norA was associated with meticillin resistance and spa type t002, a rare type among control strains, consistent with widespread dissemination of a norA-overexpressing, meticillin-resistant S. aureus (MRSA) clone. Clonal spread also played a role for spa type t008, mepA-overexpressing, meticillin-susceptible strains. An EtBr MIC of ≤12.5μg/mL was highly specific (>90%) in identifying strains lacking MDR-EP gene overexpression.     

Molecular typing of Staphylococcus aureus clinical isolates by pulsed-field gel electrophoresis, staphylococcal cassette chromosome mec type determination and dissemination of antibiotic resistance genes

Pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of genomic DNA as well as staphylococcal cassette chromosome mec (SCCmec) typing for mecA-carrying isolates were used to study the distribution of clonal types among 177 Staphylococcus aureus clinical isolates recovered in a Spanish hospital between 2000 and 2003. Five major clonal types (P1 to P5) were identified by PFGE, with one of them (P1) comprising the majority of strains (47.5%). According to SCCmec typing, SCCmec type IVA was the most prevalent type, showing increasing prevalence in the hospital setting with respect to other pandemic clones. One SCCmec pattern was detected in different PFGE types, which demonstrates that the latter is a major discriminative typing method. Three novel SCCmec elements or variants were found, each in a different PFGE type. Oxacillin (methicillin)-resistant and -susceptible S. aureus (MRSA and MSSA, respectively) strains were detected showing identical PFGE patterns, suggesting horizontal transfer of mecA to MSSA and/or mecA deletion from MRSA. Persistence of several S. aureus clones throughout the years within the same hospital environment was also observed.     

临床产超广谱β-内酰胺酶细菌的耐药表型与基因型分析

目的 分析临床产超广谱β-内酰胺酶菌株的耐药表型与基因型的关系，指导临床合理应用抗生素，减少耐药菌株的出现。方法 选用TEM和SHV两种特异性引物扩增产ESBLs菌株质粒DNA，并对所有菌株进行药敏试验。结果 180株细菌中有68株产酶，ESBLs检出率37．78％，TEM型占81％，SHV型占29％。结论 ESBLs检出率较高，基因型一致，PCR技术是监控产ESBLs菌株爆发流行的好方法。     

Wide spread of Tn2006 in an AbaR4-type resistance island among carbapenem-resistant Acinetobacter baumannii clinical isolates in Taiwan

The bactericidal activity and resistance selectivity of garenoxacin against Streptococcus pneumoniae with mutations in ParC (S79F) or both GyrA (S81F) and ParC (D83Y and K137N) were investigated using in vitro pharmacokinetic models simulating plasma concentrations for a standard clinical regimen [400mg once daily (q.d.)]. The efficacy of garenoxacin was compared with that of levofloxacin (500 mg q.d.) and moxifloxacin (400mg q.d.). Garenoxacin showed excellent bactericidal activity against S. pneumoniae, including quinolone-resistant S. pneumoniae (QRSP), achieving ratios of area under the plasma concentration-time curve over 24h to minimum inhibitory concentration (AUC(0-24)/MIC) ≥ 26.3, without emerging resistant subpopulations. The area above the killing curves was greater and the time to achieve 99.9% killing was shorter for garenoxacin than the corresponding values for levofloxacin and moxifloxacin. No resistant subpopulations and no additional substitution of amino acids in GyrA or ParC emerged following treatment with garenoxacin. On the other hand, in the parC mutant strain, levofloxacin and moxifloxacin treatment caused an increase in the frequency of the resistant population and an additional substitution of amino acids in GyrA (levofloxacin, S81Y/F/C; moxifloxacin, S81Y or E85K). In QRSP with mutations in GyrA and ParC, levofloxacin had no bactericidal activity, whilst the bactericidal activity of moxifloxacin was less than that of garenoxacin; moreover, an additional substitution of amino acids in ParC (S79Y) was noted. In conclusion, garenoxacin corresponding to an oral dose of 400mg showed excellent bactericidal activity against S. pneumoniae, including QRSP, without the emergence of resistant mutants.     

大肠埃希菌中Ⅰ类整合子耐药基因的分析

目的 对临床分离大肠埃希菌株携带的Ⅰ类整合子及相关耐药基因进行筛选和分析.探讨Ⅰ类整合子在大肠埃希菌耐药中的作用.方法 对43株大肠埃希菌临床分离株做药敏试验;采用PCR扩增、DNA测序、DNA序列比对的方法 对其携带的Ⅰ类整合子相关耐约基因进行分析.结果 43株大肠埃希菌分离株对10种抗菌药物的耐药率依次为亚胺培南4.7%、阿米卡星18.6%、头孢他啶、27.9%、头孢吡肟37.2%、头孢呋辛55.8%、复方磺胺甲嗯唑58.1%、妥布霉素74.4%、庆大霉素79.1%、头孢噻肟81.4%和哌拉两林83.7%.在43株大肠埃希菌分离株中有25株含有Ⅰ类整合子,其中18株携带整合子相关耐药基因,如介导对磺胺类和氨基糖苷类约物的耐药基因等;某些菌株携带的整合子相关耐药基因相同.结论 Ⅰ类整合子在大肠埃希菌中广泛存在,整合子相关耐约基因在该菌耐药性的形成和播散中发挥作用.     

阴沟肠杆菌质粒介导氨基糖苷类耐药基因检测及同源性分析

目的 了解贵阳某三甲医院阴沟肠杆菌的氨基糖苷类耐药机制及其之间的同源性,指导本院阴沟肠杆菌感染的抗菌治疗。方法 采用PCR方法检测110株阴沟肠杆菌的7种氨基糖苷类修饰酶基因AMEs(aph(3')-VI、aac(3)-I、aac(3)-II、aac(3)-III、aac(6')-Ib、ant(2')-I和ant(3')-I),2种16SrRNA甲基化酶基因(armA、rmtB)和整合子基因intI,并分析其与耐药表型之间的相关性;用ERIC-PCR方法分析110株阴沟肠杆菌间的同源性。结果 110株阴沟肠杆菌共检出5种AMEs基因,aac(6')-Ib检出率最高,未检出aph(3')-VI和aac(3)-I,AMEs基因总阳性率为46.36%(51/110),16S rRNA甲基化酶基因总阳性率为8.18%(9/110),intI的阳性率为47.27%(52/110);10株阿米卡星耐药株中有9株检出aac(3)-III,庆大霉素耐药株和妥布霉素耐药株均以aac(6')-Ib检出率最高;82.73%的菌株基因型与耐药表型相符;110株阴沟肠杆菌被分为20个型别。结论 本院阴沟肠杆菌氨基糖苷类耐药基因与氨基糖苷类抗生素的耐药表型间符合率高,部分菌株间存在克隆播散现象。     

Antibiotic resistance genes in phage particles isolated from human faeces and induced from clinical bacterial isolates

Phage particles have emerged as elements with the potential to mobilise antibiotic resistance genes (ARGs) in different environments, including the intestinal habitat. This study aimed to determine the occurrence of ARGs in phage particles present in faecal matter and induced from strains isolated from faeces. Nine ARGs (bla_TEM, bla_{CTX-M-1 group}, bla_{CTX-M-9 group}, bla_OXA-48, qnrA, qnrS, mecA, sul1 and armA) were quantified by qPCR in the phage DNA fractions of 150 faecal samples obtained from healthy individuals who had not received antibiotic treatment or travelled abroad in the 3 months prior to sample collection. On the suspicion that the detected particles originated from bacterial flora, 82 Escherichia coli and Klebsiella pneumoniae isolates possessing at least one identified ARG (bla_TEM, bla_{CTX-M-1 group}, bla_{CTX-M-9 group}, armA, qnrA, qnrS and sul1) were isolated and their capacity to produce phage particles carrying these ARGs following induction was evaluated. Of 150 samples, 72.7% were positive for at least one ARG, with bla_TEM and bla_{CTX-M-9 group} being the most prevalent and abundant. Of the 82 isolates, 51 (62%) showed an increase in the number of copies of the respective ARG in the phage fraction following induction, with bla_TEM, bla_{CTX-M-1 group}, bla_{CTX-M-9 group} and sul1 being the most abundant. Phages induced from the isolates were further purified and visualised using microscopy and their DNA showed ARG levels of up to 10¹⁰ gene copies/mL. This study highlights the abundance of phage particles harbouring ARGs and indicates that bacterial strains in the intestinal habitat could be source of these particles.     

Colony PCR for rapid detection of antibiotic resistance genes in MRSA and enterococci

We examined colony PCR that does not use the prepared template DNA but intact cells as the template DNA source for the rapid, simple and reproducible detection of antibiotic resistance genes in strains of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus. Two factors turned out to be important; i.e. the transfer size of bacterial cells and the use of DNA Polymerase with high performance. In practice, the tip of a toothpick was lightly touched with a colony on agar plates followed by the PCR mixture (20 microliters) containing KOD-plus-DNA Polymerase and multiplex primers. The cell transfer size ranged from 10(3) to 10(4) cfu and 30 cycles of PCR (95 degrees C, 30 sec.-->50 degrees C, 30 sec.-->72 degrees C, 60 sec.) following the first heating (95 degrees C, 3 min.) resulted in good amplification of the target regions of mecA and aac(6')/aph(2") that are responsible for the resistance to methicillin and arbekacin, respectively.     

Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates

DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.     

Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae

A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n = 17); Klebsiellapneumoniae (n = 3); Enterobacter spp. (n = 6); Acinetobacter genospecies 3 (n = 1); Acinetobacterbaumannii (n = 1); Pseudomonasaeruginosa (n = 2); and Stenotrophomonasmaltophilia (n = 2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla_SHV-5 in a bla_OXA-23-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.     

贵阳地区儿童幽门螺杆菌临床分离菌株毒力基因cagA和vacA的表达情况以及与抗生素耐药之间的关系

目的分析贵阳地区儿童幽门螺杆菌临床分离菌株的毒力基因cagA和vacA的表达情况 ,以及探讨其和幽门螺杆菌耐药之间的关系.方法从有上消化道症状的患儿的的胃窦粘膜中分离培养出幽门螺杆菌31株 ,采用聚合酶链式反应检测cagA和vacA的分布情况.用琼脂稀释法检测幽门螺杆菌对阿莫西林、克拉霉素、甲硝唑和左氧氟沙星的耐药性.结果本地区儿童幽门螺杆菌的cagA基因的阳性率为83 .87% .vacA基因亚型s1a、s1b、s1c、m1和m2的检出率分别为83 .87% (26/31)、0% (0/31)、19 .35% (6/31)、12 .90% (4/31)和74 .19% (23/31).优势基因为vacAs1am2型.毒力基因与不同的疾病之间无相关性.cagA基因和vacA基因与阿莫西林、克拉霉素、甲硝唑及左氧氟沙星耐药无相关性(P>0 .05).结论贵阳地区儿童的毒力基因cagA+和vacAs1m2型为主.毒力基因与抗生素的耐药性之间无相关性.     

多重PCR技术检测沙门菌两种四环素耐药基因

应用两对特异性引物同时检测四环素耐药基因tetB和tetC通过对35株沙门菌分离株的四环素耐药性检测，表明所有沙门菌分离株均含tetC基因，与药敏试验结果阳性符合率65．7％，8株同时含有tetB基因，与药敏试验结果阳性符合率100％，tetB基因和tetC基因双阳性的菌株与药敏试验结果阳性符合率也为100％。取其中部分菌株扩增出tetB和tetC基因片段进行序列分析，5株菌的tetB基因扩增产物序列完全相同，与质粒pRT11相应序列同源性达99．7％；14株菌的tetC基因扩增产物与质粒pBR322中的相应序列同源性为100％。证实沙门菌耐药基因普遍存在，且同时含有tetB和tetC基因的菌株表现耐药。多重PCR技术同时检测两种四环素耐药基因，适合大量样本的检测，对开展沙门菌四环素多种耐药基因的分子流行病学监测提供了有效途径。     

多重耐药临床菌株中整合子结构的检测与分析

目的研究广州暨南大学附属第一医院2004年部分临床菌株样本的整合子及其基因盒的分布特性。方法多重PCR检测与细菌耐药关系密切的1、2、3类整合酶基因,进一步对阳性样本可变区的基因盒序列鉴定分析。结果随机抽取109株临床菌株,整合酶阳性检出率为97.2%(106/109),其中1类整合酶阳性菌100株(91.7%),2类整合酶阳性菌1株(0.92%),此外有5株(4.6%)同时检出1、2类整合酶,没有检测到3类整合酶;基因盒鉴定结果显示,插入基因盒以dfrA(甲氧苄氨嘧啶耐药相关)和aadA(氨基糖苷类耐药相关)基因家族为主,也在少数菌株中发现了aacA4、cmlA1、catB3以及sat1基因盒。其中又以dfrA12、orfF和aadA2组合最为常见,耐药基因盒PCR扩增片段为1913bp(64.6%);此外,还发现了同时存在两种整合子结构的菌株。结论整合子普遍存在于临床菌株中,可通过基因水平转移在不同菌属间传播,提示各医药单位必须加强耐药监测及合理选择抗菌药物,以减少多重耐药细菌的发生和发展。     

广东省肇庆市肺炎克雷伯菌临床分离株的耐药性与MLST分型研究

目的 了解广东省肇庆市肺炎克雷伯菌(Klebsiella pneumoniae, KP)临床分离株的抗生素耐药情况及多位点序列分型(multilocus sequence typing, MLST)特征。方法 收集肇庆市两家医院KP临床分离株63株，采用肉汤微量稀释法进行30种抗生素的体外药物敏感性试验，并对所有菌株进行多位点序列分型。结果 63株KP临床分离株的耐药谱特点可分为4类，即全敏感的高毒力肺炎克雷伯菌(hypervirulent K. pneumoniae, hvKP)、产超广谱β-内酰胺酶肺炎克雷伯菌(extended spectrum β-lactamases K. pneumoniae, ESBLsKP)、耐碳青霉烯类肺炎克雷伯菌(carbapenem-resistant K. pneumoniae, CRKP)和其他类型的多重耐药肺炎克雷伯菌(multidrug-resistant K. neumoniae，MDRKP)；MLST分型结果显示63株临床分离株可分成41个ST型。其中，ESBLsKP以ST37型(9株，39.13%)为主，CRKP以ST11型(5株，62.50%)为主，hvKP以ST65(5株，25.00%)型和ST23(3株，15.00%)型为主，而其他类型的多重耐药KP的ST型(12株9种ST型)则呈现明显的多态性。结论 我市肺炎克雷伯菌临床分离株的耐药情况比较严重，要加强院内感染监测，警惕其成为优势菌型并引发院内感染的风险。