Comparison of phenotypic and PCR methods for detection of carbapenemases production by Enterobacteriaceae

Dissemination of carbapenem resistance via Enterobacteriaceae, particularly among Klebsiella pneumoniae and Escherichia coli, is a major public health concern. Rapid methods for determining antimicrobial susceptibility are important to ensure adequate and appropriate use of antimicrobial agents and to limit the spread of these bacteria. In the current study, we compared the rapidity, sensitivity and specificity of traditional methods and molecular-based Xpert Carba-R PCR assay to identify sixty isolates, (26 E. coli and 34 K. pneumoniae). The specificity of MicroScan was 100% while sensitivity to ertapenem (ERT), imipenem (IMI), and meropenem (MER) was 93%, 68.9%, and 55.17%, respectively. For the modified Hodge test, the specificity was 96.77% and sensitivity was 89.65%. Although some results of phenotypic assays matched with the definite PCR identification, some results were misleading. Out of the 29 positive PCR samples, three samples of K. pneumoniae were negative for the MHT and one E. coli sample was MHT positive but negative for the PCR. Nine samples were positive for the PCR but were determined as carbapenem sensitive by MicroScan. While MicroScan and MHT requires several hours and multi-steps to obtain results, Xpert Carba-R PCR assay takes less than an hour. Therefore, we recommend using Gene xpert Carba-R assay for the optimal carbapenemnase detection with reducing material, manpower and cost. Also it is important to know the type of carbapenemase is present.     

A real-time PCR antibiogram for drug-resistant sepsis

Current molecular diagnostic techniques for susceptibility testing of septicemia rely on genotyping for the presence of known resistance cassettes. This technique is intrinsically vulnerable due to the inability to detect newly emergent resistance genes. Traditional phenotypic susceptibility testing has always been a superior method to assay for resistance; however, relying on the multi-day growth period to determine which antimicrobial to administer jeopardizes patient survival. These factors have resulted in the widespread and deleterious use of broad-spectrum antimicrobials. The real-time PCR antibiogram, described herein, combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. We have developed a procedure that determines susceptibility by monitoring pathogenic load with the highly conserved 16S rRNA gene in blood samples exposed to different antimicrobial drugs. The optimized protocol removes heme and human background DNA from blood, which allows standard real-time PCR detection systems to be employed with high sensitivity (<100 CFU/mL). Three strains of E. coli, two of which were antimicrobial resistant, were spiked into whole blood and exposed to three different antibiotics. After real-time PCR-based determination of pathogenic load, a ΔC(t)<3.0 between untreated and treated samples was found to indicate antimicrobial resistance (P<0.01). Minimum inhibitory concentration was determined for susceptible bacteria and pan-bacterial detection was demonstrated with 3 gram-negative and 2 gram-positive bacteria. Species identification was performed via analysis of the hypervariable amplicons. In summary, we have developed a universal diagnostic phenotyping technique that assays for the susceptibility of drug-resistant septicemia with the speed of PCR. The real-time PCR antibiogram achieves detection, susceptibility testing, minimum inhibitory concentration determination, and identification in less than 24 hours.     

Bacterial profile and drug susceptibility pattern of urinary tract infection in pregnant women at University of Gondar Teaching Hospital, Northwest Ethiopia

ABSTRACT: BACKGROUND: Urinary tract infection (UTI) is a common health problem among pregnant women. Proper investigation and prompt treatment are needed to prevent serious life threatening condition and morbidity due to urinary tract infection that can occur in pregnant women. Recent report in Addis Ababa, Ethiopia indicated the prevalence of UTI in pregnant women was 11.6 % and Gram negative bacteria was the predominant isolates and showed multi drug resistance. This study aimed to assess bacterial profile that causes urinary tract infection and their antimicrobial susceptibility pattern among pregnant women visiting antenatal clinic at University of Gondar Teaching Hospital, Northwest Ethiopia. METHODS: A cross-sectional study was conducted at University of Gondar Teaching Hospital from March 22 to April 30, 2011. Mid stream urine samples were collected and inoculated into Cystine Lactose Electrolyte Deficient medium (CLED). Colony counts yielding bacterial growth of 105/ml of urine or more of pure isolates were regarded as significant bacteriuria for infection. Colony from CLED was sub cultured onto MacConkey agar and blood agar plates. Identification was done using cultural characteristics and a series of biochemical tests. A standard method of agar disc diffusion susceptibility testing method was used to determine susceptibility patterns of the isolates. RESULTS: The overall prevalence of UTI in pregnant women was 10.4 %. The predominant bacterial pathogens were Escherichia coli 47.5 % followed by coagulase-negative staphylococci 22.5 %, Staphylococcus aureus 10 %, and Klebsiella pneumoniae 10 %. Gram negative isolates were resulted low susceptibility to co-trimoxazole (51.9 %) and tetracycline (40.7 %) whereas Gram positive showed susceptibility to ceftriaxon (84.6 %) and amoxicillin-clavulanic acid (92.3 %). Multiple drug resistance (resistance to two or more drugs) was observed in 95 % of the isolates. CONCLUSION: Significant bacteriuria was observed in asymptomatic pregnant women. Periodic studies are recommended to check the outcome of asymptomatic bacteriuria and also monitor any changes in the susceptibility patterns of urinary tract pathogens in pregnant women.     

407例老年肺癌下呼吸道感染患者病原菌分布及耐药分析

目的 分析老年肺癌下呼吸道感染患者痰培养物的病原菌分布及耐药状况.方法 回顾性分析407例老年肺癌下呼吸道感染患者痰标本细菌培养、鉴定及药敏结果.结果 共分离出病原菌238株,革兰阴性菌163株,占68.49％;革兰阳性菌33株,占13.87％;真菌42株,占17.65％.主要病原菌为肺炎克雷伯菌、大肠埃希菌、铜绿假单胞菌、鲍曼不动杆菌、表皮葡萄球菌和金黄色葡萄球菌.其中,肺炎克雷伯菌和大肠埃希菌ESBLs阳性菌株的比例分别为36.73％、28.85％.结论 老年肺癌患者因长期使用抗生素,呼吸道菌群失调,不同病原菌对抗菌药耐药率均较高,以革兰阴性菌感染为主,肺炎克雷伯菌和大肠埃希菌产ESBLs的菌株有较高比例,二重感染比例较高;革兰阳性菌均对万古霉素敏感,未发现耐药菌株.因此临床医生根据药敏结果合理选用抗生素,对于改善患者肺部微生态失衡,延长生存时间具有重要指导意义.     

食源性相关腹泻肠炎沙门菌感染抗菌药物敏感性

目的 了解食源性相关腹泻非伤寒沙门菌感染的菌种分布及其对抗菌药物敏感性,为控制该类细菌的感染及传播提供技术支持。方法 对2015‒2017年本系统2家社区卫生服务中心就诊的食源性相关腹泻患者粪便（或肛拭子）标本采用直接分离与增菌分离相结合的方法常规培养分离获得42株沙门菌;采用血清凝集法进行快速血清分型,并自动化生化鉴定及抗菌药物敏感性试验;通过现况调查进行流行病学危险因素分析。结果 自动化生化鉴定结果能够覆盖常规血清学快速鉴定结果,同属沙门菌群;42株沙门菌以肠炎沙门菌、鼠伤寒沙门菌和斯坦利沙门菌为主,其中肠炎沙门菌占全部菌株的23.81%,鼠伤寒沙门菌占19.06%,斯坦利沙门菌占14.29%;其中肠炎沙门菌可对氟喹诺酮类、三四代头孢菌素类与碳青霉烯类等敏感（敏感率可达97.00%以上）,而对氨基糖苷类可产生双向耐药。通过现况调查,发现患者有腹痛、腹泻等胃肠道症状,可伴有发热,所有患者48 h内有可疑食物暴露史,无水源性案例,均为散发。结论 菌种鉴定应以常规快速血清学凝集结果为准,自动化生化鉴定仅供参考,并将鉴定菌种与药敏报告相关联,可根据药敏结果合理选用敏感抗菌药物;应对社区居民开展针对性的健康宣教。     

临床病原菌种类及耐药性监测

目的 探讨病原菌种类及其对抗菌药物的耐的耐药状况。方法 收集１９９８年１月－１９９９年１２月临床感染标本分离的病原菌并分析其种类,药敏试验采用Ｋｉｒｂｙ－Ｂａｕｅｒ纸片扩散法。结果 １１８２株病原菌,革兰氏阳性球菌６０４株（５１．１％）,革兰氏阴性杆菌５７８株（４８．９％）。病原菌以金黄一萄球菌、大肠埃希菌、表皮葡萄球菌、铜绿假单胞菌最多见。去甲万古霉素、阿米卡星、新霉素对革兰氏阳性球菌抗菌作用较     

呼吸道与非呼吸道标本主要非发酵菌分离株的耐药性比较

目的对2 229份住院患者标本进行分离培养,并对非发酵菌分离株进行药敏试验,了解呼吸道与非呼吸道主要分离株的耐药率差异,以便指导临床合理使用抗菌药物。方法收集2008年8月至2010年10月分离自患者呼吸道、非呼吸道标本的革兰阴性非发酵菌,采用VITEK-32全自动微生物鉴定药敏仪进行细菌鉴定,药敏测定采用K-B法,并比较呼吸道与非呼吸道标本所检出的主要革兰阴性非发酵菌对抗菌药物的耐药率差异。结果共检出非发酵菌556株,其中呼吸道标本366株,非呼吸道标本190株,前3位非发酵菌分离株分别为铜绿假单胞菌、鲍曼不动杆菌和嗜麦芽窄食单胞菌。非发酵菌菌株ESBLs、AmpC、MBL、同产ESBLs+AmpC和同产ESBLs+MBL检出率分别为17.62%、13.67%、14.39、12.23%和6.12%,其中呼吸道标本分离株铜绿假单胞菌的ESBLs、AmpC检出率明显高于非呼吸道标本(均P<0.05),鲍曼不动杆菌的ESBLs、AmpC检出率明显低于非呼吸道标本(均P<0.05);呼吸道、非呼吸道标本的铜绿假单胞菌、鲍曼不动杆菌和嗜麦芽窄食单胞菌分离株对多种抗生素的耐药率不同,差异均有统计学意义(均P<0.05)。结论呼吸道与非呼吸道标本分离的主要非发酵菌分离株对同一抗菌药物耐药率不同,治疗不同部位非发酵菌引起的感染,要考虑由于感染部位不同而产生的耐药性以及药物有效浓度的差异,根据药敏结果合理使用抗菌药物。     

2011～2013年血液分离葡萄球菌的临床分布及耐药性分析

目的了解临床各科发热患者（怀疑细菌感染）血培养中葡萄球菌分布及耐药性,为临床治疗提供参考依据。方法血培养采用BaeT／Alert3D全自动血培养仪（梅里埃）培养5d,采用MicroScanWalk—Away-96plus全自动微生物鉴定和药敏分析系统（西门子）进行细菌鉴定和药敏试验。结果血培养共检出葡萄球菌185株,检出最多的科室是ICU,耐甲氧西林金黄色葡萄球菌（methicillin—resistant Staphylococcus aureus,MRSA）检出率为30．4％,而耐甲氧西林凝固酶阴性葡萄球菌（methicillin—resistant coagulase-negative Staphylococci,MRCNS）的检出率高达80．7％。还检出了1株万古霉素中介的凝固酶阴性葡萄球菌。结论通过血培养检出葡萄球菌的耐药性分析发现,凝固酶阴性葡萄球菌对苯唑西林、氨苄西林、青霉素、红霉素的耐药率在70％以上,而对氯霉素、利福平、四环素的耐药率在30％以下,因此这三种药物应为我院应对凝固酶阴性葡萄球菌血流感染的常用首选药物。     

胸腹水培养中的病原菌分布及耐药性分析

目的了解胸腹水培养中病原菌的分布和耐药情况,为临床合理用药提供依据。方法通过回顾性分析中山市第二人民医院住院部自2011年1月至2013年11月送检的胸腹水标本细菌培养及药敏资料。结果在494份胸腹水标本中共检出47株病原菌,总阳性率为9.5%;其中326例胸水分离出病原菌28例,阳性率为8.59%,胸水培养中革兰阳性菌占67.86%,革兰阴性菌占32.12%,菌种较为分散;168例腹水分离出病原菌19例,阳性率为11.31%,腹水培养中革兰阴性菌占73.68%,革兰阳性菌占26.32%。检出革兰阴性病原菌对氨苄西林和四环素耐药率较高,对阿米卡星、美罗培南和亚胺培南敏感。检出革兰阳性病原菌对氨苄西林、头孢西丁和红霉素耐药率高,对万古霉素、替考拉宁和吗啉噁酮敏感。结论应重视胸腹水标本的细菌学检查,依据药敏试验合理使用抗菌药物,减少细菌的耐药率。     

儿童医院感染洋葱伯克霍尔德菌三年临床分布特征调查

目的调查三年来儿童医院感染洋葱伯克霍尔德菌的临床分布特点及耐药情况,指导临床合理用药。方法2010年1月-2012年12月住院患儿送检标本采用VITEK-32全自动细菌鉴定仪进行细菌鉴定,药敏试验采用Kirby—Bauer法。结果三年分离到洋葱伯克霍尔德菌分别为28株、36株和51株,共¨5株,呈逐年上升趋势;其来源主要分布在儿童ICU和新生儿病房,以引起儿童呼吸道感染为主要症状;药敏结果显示替卡西林／克拉维酸、庆大霉素、氨苄西林／舒巴坦等耐药率均逐年升高;对哌拉西林／他唑巴坦、头孢他啶和复方新诺明敏感性高。结论儿童医院感染洋葱伯克霍尔德菌呈逐年上升趋势,因其是一种多重耐药菌,天然耐多种抗菌药物,临床监测其分布状况和耐药性具有重要意义。     

恶性肿瘤患者合并感染临床分析

目的：探讨肿瘤患者合并感染常见病原菌的分布及对抗生素的敏感性。方法：回顾性总结分析139例恶性肿瘤患者合并感染的标本细菌培养及药敏情况。结果：112例细菌学检查阳性的患者感染部位以呼吸道最多,占69.6%,153份标本共检出122株细菌,其中真菌48株,占39.3%;G^-杆菌38株,占31.1%;G^ 球菌36株,占29.5%。所检出的葡萄球菌对万古霉素,新霉素较敏感,大肠埃希菌对阿米卡星31.1%;G^ 球菌36株,占29.5%,所检出的葡萄球菌对万古霉素,新霉素较敏感,大肠埃然菌对阿米卡星敏感,铜绿假单胞菌对环丙沙星尚敏感,其余抗生素对病原菌均呈现不同程度的耐药,结论：肿瘤患者合并感染的病原菌主要为条件致病菌,而且有较严重的耐药情况,临床治疗应重视病原学检查,合理使用抗生素,减少耐药菌株的产生和内源性感染。     

Fast and reliable determination of Escherichia coli susceptibility to antibiotics: Infrared microscopy in tandem with machine learning algorithms

Antimicrobial drugs have an important role in controlling bacterial infectious diseases. However, the increasing resistance of bacteria to antibiotics has become a global health care problem. Rapid determination of antimicrobial susceptibility of clinical isolates is often crucial for the optimal antimicrobial therapy. The conventional methods used in medical centers for susceptibility testing are time‐consuming (>2 days). Two bacterial culture steps are needed, the first is used to grow the bacteria from urine on agar plates to determine the species of the bacteria (~24 hours). The second culture is used to determine the susceptibility by growing colonies from the first culture for another 24 hours. Here, the main goal is to examine the potential of infrared microscopy combined with multivariate analysis, to reduce the time it takes to identify Escherichia coli susceptibility to antibiotics and to determine the optimum choice of antibiotic to which the bacteria will respond. E coli colonies of the first culture from patients with urinary tract infections (UTI) were examined for the bacterial susceptibility using Fourier‐transform infrared (FTIR). Our results show that it is possible to determine the optimum choice of antibiotic with better than 89% sensitivity, in the time span of few minutes, following the first culture.      

ICU开放人工气道患者下呼吸道病原菌分布及耐药性分析

目的了解重症监护室（ICU）开放气道患者下呼吸道感染病原菌分布及耐药特点,为临床用药提供依据。方法回顾性分析2007年1月至2008年12月,重症监护室开放气道患者下呼吸道感染者的痰标本418株病原菌进行鉴定及药敏试验。结果革兰阴性杆菌占69．6％,其中铜绿假单胞菌27．02％,其次为鲍曼不动杆菌20．16％。革兰阳性球菌占20．16％,以金黄色葡萄球菌为主占12．5％,真菌10．24％。结论目前重症监护室下呼吸道致病菌以G^-杆菌为主,细菌的耐药性严重,应根据药敏试验选择抗生素。     

346株鲍曼不动杆菌临床分布及耐药率分析

目的了解鲍曼不动杆菌引起医院感染的特点以及对抗菌药物的耐药性的变化趋势。方法应用美国BD公司Phoenix^TM100全自动细菌和药敏系统鉴定仪对所分离的364株鲍曼不动杆菌进行鉴定和药敏试验,并进行统计学分析。结果 346株鲍曼不动杆菌来自痰液、伤口分泌物、中段尿、静脉血及大便,分别占58.96%、30.92%、5.49%、4.34%、0.29%。药敏结果显示鲍曼不动杆菌对多粘菌素和米诺环素敏感性最高,耐药率低于10.4%。结论加强鲍曼不动杆菌的耐药监测,了解其耐药性变迁,可合理指导用药,有效控制鲍曼不动杆菌耐药菌株的产生。     

Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

Background

Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens.

Methods

Identification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx), as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH.

Results

In blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5). Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6). Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9%) as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI ?92.4 to 267.1). Identification by PNA-FISH averaged 16.4 hours (95% CI ?57.3 to 90.0). Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%). For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS), discontinuation of vancomycin could result in savings of $20.00/day.

Conclusions

In this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to conventional culture-based techniques. Species-level identification based on PNA-FISH could contribute to notable cost savings due to adjustments in empiric antimicrobial or antifungal therapy as appropriate to the pathogen identified.     

Guanidylation and tail effects in cationic antimicrobial lipopeptoids

Background

Cationic antimicrobial peptides (CAMPs) are attractive scaffolds for the next generation of antimicrobial compounds, due to their broad spectrum of activity against multi-drug resistant bacteria and the reduced fitness of CAMP-insensitive mutants. Unfortunately, they are limited by poor in vivo performance, including ready cleavage by endogenous serum proteases.

Methodology/Principal Findings

To explore the potential for peptoid residues to replace well studied CAMP scaffolds we have produced a series of antimicrobial lipopeptoids, with sequences similar to previously reported lipopeptides. The activity of the peptoids was assessed against a panel of clinically relevant and laboratory reference bacteria, and the potential for non-specific binding was determined through hemolytic testing and repeating the antimicrobial testing in the presence of added bovine serum albumin (BSA). The most active peptoids displayed good to moderate activity against most of the Gram positive strains tested and moderate to limited activity against the Gram negatives. Antimicrobial activity was positively correlated with toxicity towards eukaryotic cells, but was almost completely eliminated by adding BSA.

Conclusion/Significance

The lipopeptoids had similar activities to the previously reported lipopeptides, confirming their potential to act as replacement, proteolytically stable scaffolds for CAMPs.     

In vitro susceptibility of staphylococci to linezolid and other antimicrobial agents

Linezolid is a member of the new class of antibacterial agents called oxazolidinones that are active against Gram positive organisms and exert their action by protein synthesis inhibition. In this study we investigated the in vitro activity of linezolid versus the other agent against clinical strains of staphylococci: Staphylococcus aureus (n = 82) and S. epidermidis (n = 32) collected in 2002 from hospitalized patients and healthy individuals, isolated from different biological samples. Agar dilution minimum inhibitory concentrations (MICs) were determined by using Mueller-Hinton agar according to the guidelines established by the National Committee for Clinical Laboratory Standards. Linezolid demonstrated excellent in vitro activity against all isolates tested, with MICs values in the range of susceptibility (< or = 8 microg/ml). No associated resistance between linezolid and other agents tested was observed. The resistance among Gram positive bacteria continues to spread and for many patients infected with these resistant organisms antimicrobial therapy is ineffective and linezolid may be a new alternative treatment.     

Rapid identification and antibiotic susceptibility testing of Yersinia pestis using bioluminescent reporter phage

The rapid identification and antibiotic susceptibility testing of Yersinia pestis is paramount for a positive prognosis. We previously engineered a Y. pestis-specific 'bioluminescent' reporter phage for the identification of Y. pestis. In this study, we generated an improved reporter phage and evaluated the ability of this phage to provide direct and rapid susceptibility testing. Compared to the first generation reporter, the second generation reporter exhibited a 100-fold increase in signal strength, leading to a 10-fold increase in assay sensitivity. Y. pestis antimicrobial testing in the presence of the reporter elicited bioluminescent signals that were drug concentration-dependent, and produced susceptibility profiles that mirrored the standard CLSI method. The phage-generated susceptibility profiles, however, were obtained within hours in contrast to days with the conventional method.     

高龄患者血液感染常见革兰阴性杆菌及其耐药性分析

目的分析本院80~100岁高龄患者血液感染常见革兰阴性杆菌的种类及其耐药状况,为本院合理使用抗生素提供依据。方法采用BacT/Alert 3D血培养仪对血液标本进行阳性鉴定;VITEK-2Compact全自动微生物鉴定仪进行鉴定;K-B纸片扩散法对抗菌药物进行敏感性测定;使用WHONET 5.4分析软件分析数据。结果本院高龄患者血液培养阳性标本中共分离出革兰阴性杆菌108株,以肠杆菌科细菌为主,其次为非发酵菌,前者主要为大肠埃希菌52株(48.15%)和肺炎克雷伯菌37株(34.26%),后者主要包括铜绿假单胞菌10株(9.26%)和鲍曼不动杆菌8株(7.41%)。其中大肠埃希菌和肺炎克雷伯菌对亚胺培南的耐药率分别为1.92%和13.51%,两者对氨苄西林、氨苄西林/舒巴坦、头孢唑林、头孢呋辛的耐药率均高于50.00%;铜绿假单胞菌和鲍曼不动杆菌对亚胺培南的耐药率分别为20.00%和25.00%,后者对其他抗菌药物的耐药率均高于前者。结论碳青酶烯类抗生素可作为本院高龄患者常见革兰阴性杆菌所致血液感染的首选药物;但在治疗中应考虑细菌的耐药特点及患者的代谢特点合理选择抗生素。     

解脲脲原体耐药性研究进展

解脲脲原体是条件致病菌。目前对其耐药性的研究主要包括对喹诺酮类、大环内酯类和四环素类3种抗菌药物相关耐药突变位点的检测,以及生物膜对病原体相关药物敏感性的影响,研究方法和检验手段仍较为传统、局限,研究方案也仅停留在对前人实验的重复。近年来,有学者将多位点序列分型技术用于解脲脲原体耐药序列类型的研究。在完善耐药机制研究的基础上,如何实现对耐药株的快速鉴定,从而指导抗菌药物的合理选择等仍需进一步研究。