呼吸道产超广谱β-内酰胺酶分离株耐药基因初步分型

目的了解产超广谱β-内酰胺酶 (ESBLs)呼吸道分离株的主要基因型分布特点.方法用表型确证试验确定临床呼吸道标本中产ESBLs的大肠埃希菌和肺炎克雷伯菌.应用聚合酶链反应(PCR)方法扩增产ESBLs株的bla(TEM)、bla(SHV)和bla(CTX-M)基因.结果 PCR结果显示bla(TEM)、bla(SHV)和bla(CTX-M)基因的总阳性率分别为40 .7%、45.7%和75.3%,其中大肠埃希菌分别为:64.9%、2.7%和91.9%,肺炎克雷伯菌分别为:20.5%、81.8%和61.4%.67.6%的大肠埃希菌和95.5%的肺炎克雷伯菌同时携带多个基因.结论深圳市人民医院呼吸道分离的产ESBLs大肠埃希菌的主要基因型为CTX-M,肺炎克雷伯菌主要基因型为SHV.大多数菌株同时携带多个基因.     

Resistance to broad-spectrum antibiotics in aquatic systems: anthropogenic activities modulate the dissemination of bla(CTX-M)-like genes

We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular bla(CTX-M). Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a bla(CTX-M) gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. ISEcp1 was found in the upstream regions of all isolates examined. Culture-independent bla(CTX-M)-like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as bla(RAHN-1). The results demonstrate that the occurrence and diversity of bla(CTX-M) genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments.     

大肠埃希菌临床分离株CTX—M型超广谱β-内酰胺酶基因型研究

目的了解我院临床分离大肠埃希菌产CTX-M型超广谱β-内酰胺酶（ESBLs）的基因型分布情况。方法收集我院临床分离大肠埃希菌200株,按美国临床和实验室标准协会2006年制订的标准确定ESBLs表型。PCR扩增CTX—M耐药基因,扩增产物测序,测序结果与GenBank比对,确定CTX．M基因型。结果200株大肠埃希菌中有94株ESBLs阳性,其中92株CTX-M基因扩增阳性。测序结果表明,大肠埃希菌CTX．M基因型以CTX．M14为主,占67．4％,同时CTX—M3占27．2％,5株未分型。结论大肠埃希菌中产CTX．M型ESBLs的检出率较高,其基因型以CTX．M14和CTX．M3型为主。     

Antimicrobial Resistance and Molecular Epidemiology of Escherichia coli Causing Bloodstream Infections in Three Hospitals in Shanghai,China

Escherichia coli (E. coli) is one of the most frequent and lethal causes of bloodstream infections (BSIs). We carried out a retrospective multicenter study on antimicrobial resistance and phylogenetic background of clinical E. coli isolates recovered from bloodstream in three hospitals in Shanghai. E. coli isolates causing BSIs were consecutively collected between Sept 2013 and Sept 2014. Ninety isolates randomly selected (30 from each hospital) were enrolled in the study. Antimicrobial susceptibility testing was performed by disk diffusion. PCR was used to detect antimicrobial resistance genes coding for β-lactamases (TEM, CTX-M, OXA, etc.), carbapenemases (IMP, VIM, KPC, NDM-1 and OXA-48), and phylogenetic groups. eBURST was applied for analysis of multi-locus sequence typing (MLST). The resistance rates for penicillins, second-generation cephalosporins, fluoroquinolone and tetracyclines were high (>60%). Sixty-one of the 90 (67.8%) strains enrolled produced ESBLs and no carbapenemases were found. Molecular analysis showed that CTX-M-15 (25/61), CTX-M-14 (18/61) and CTX-M-55 (9/61) were the most common ESBLs. Phylogenetic group B2 predominated (43.3%) and exhibited the highest rates of ESBLs production. ST131 (20/90) was the most common sequence type and almost assigned to phylogenetic group B2 (19/20). The following sequence types were ST405 (8/90) and ST69 (5/90). Among 61 ESBL-producers isolates, B2 (26, 42.6%) and ST131 (18, 29.5%) were also the most common phylogenetic group and sequence type. Genetic diversity showed no evidence suggesting a spread of these antimicrobial resistant isolates in the three hospitals. In order to provide more comprehensive and reliable epidemiological information for preventing further dissemination, well-designed and continuous surveillance with more hospitals participating was important.     

Molecular characteristics of extended-spectrum beta-lactamases and qnr determinants in Enterobacter species from Japan

The incidence of extended-spectrum β-lactamases (ESBLs) has been increasing worldwide, but screening criteria for detection of ESBLs are not standardized for AmpC-producing Enterobacteriaceae such as Enterobacter species. In this study, we investigated the prevalence of ESBLs and/or AmpC β-lactamases in Japanese clinical isolates of Enterobacter spp. and the association of plasmid-mediated quinolone resistance (PMQR) determinants with ESBL producers. A total of 364 clinical isolates of Enterobacter spp. collected throughout Japan between November 2009 and January 2010 were studied. ESBL-producing strains were assessed by the CLSI confirmatory test and the boronic acid disk test. PCR and sequencing were performed to detect CTX-M, TEM, and SHV type ESBLs and PMQR determinants. For ESBL-producing Enterobacter spp., pulsed-field gel electrophoresis (PFGE) was performed using XbaI restriction enzyme. Of the 364 isolates, 22 (6.0%) were ESBL producers. Seven isolates of Enterobacter cloacae produced CTX-M-3, followed by two isolates producing SHV-12. Two isolates of Enterobacter aerogenes produced CTX-M-2. Of the 22 ESBL producers, 21 had the AmpC enzyme, and six met the criteria for ESBL production in the boronic acid test. We found a significant association of qnrS with CTX-M-3-producing E. cloacae. The 11 ESBL-producing Enterobacter spp. possessing bla(CTX-M), bla(SHV), or bla(TEM) were divided into six unique PFGE types. This is the first report about the prevalence of qnr determinants among ESBL-producing Enterobacter spp. from Japan. Our results suggest that ESBL-producing Enterobacter spp. with qnr determinants are spreading in Japan.     

重症监护病房产ESBLs菌的基因分型

目的了解深圳市人民医院重症监护病房分离菌超广谱β-内酰胺酶（ESBLs）的检出率及其基因型分布情况。方法收集来自重症监护病房大肠埃希菌和肺炎克雷伯菌分离株48株,采用CLSI推荐的表型确证方法筛选出ESBLs株,并利用PCR及DNA测序法分析产酶菌株的ESBL基因型。结果（1）48分离株菌中共检出产ESBLs菌24株,阳性率为50.0%。（2）产酶菌中93.8%（15/16）的大肠埃希菌和87.5%（7/8）的肺炎克雷伯菌分别检出CTX-M基因;其中72.7%（16/22）为CTX-M-14。6株肺炎克雷伯菌检出SHV基因,其中3株为SHV-11型,另3株为SHV-12型,6株含SHV基因的肺炎克雷伯菌中5株合并CTX-M基因。而所有大肠埃希菌株均未检出SHV基因。所有产酶菌中,分别有10株大肠埃希菌和2株肺炎克雷伯菌检出TEM-1基因,其中1株大肠埃希菌只检出TEM-1基因,未检出SHV型或CTX-M型基因。结论重症监护病房分离菌ESBLs检出率高,以CTX-M-14为主要基因型。     

产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌blaCTX-M基因的检测

目的了解产CTX-M型超广谱β-内酰胺酶 (ESBLs) 大肠埃希菌和肺炎克雷伯菌在深圳市人民医院的分布情况.方法应用美国国家临床实验室标准化委员会(NCCLS)推荐的表型确证试验,从2000年6月～2003年8月该院临床标本分离株中筛选出不重复的产ESBLs菌株215株,其中大肠埃希菌151株,肺炎克雷伯菌64株,应用聚合酶链反应(PCR)检测所有产ESBLs株的bla(CTX-M)基因.结果 PCR结果显示,大肠埃希菌bla(CTX-M)基因阳性率为92.1%(139/151),肺炎克雷伯菌的阳性率为65.6%(42/64).bla(CTX-M)基因阳性菌株主要来源于临床送检尿和痰标本,并广泛分布于20多个临床科室.结论该院临床分离的大肠埃希菌和肺炎克雷伯菌产生的ESBL大多数为CTX-M型,该类酶广泛分布于各临床科室,需引起重视.     

亚抑菌浓度头孢他啶对大肠埃希菌生物膜形成的影响与其耐药性相关性研究

目的:探讨亚抑菌浓度头孢他啶对大肠埃希菌生物膜形成的影响与细菌耐药性、超广谱β-内酰胺酶(ESBLs)产生及ESBLs基因分型的相关性,为临床生物膜感染的治疗和抗生素的合理使用提供理论依据。方法:大肠埃希菌最低抑菌浓度(MIC)检测采用琼脂平板倍比稀释法,超广谱β-内酰胺酶(ESBLS)表型确证实验采用双纸片协同法,大肠埃希菌ESBLs基因检测采用PCR扩增,生物膜形成能力检测采用96孔板结晶紫染色法。结果:50株大肠埃希菌临床株对青霉素类、氟喹诺酮类、头孢哌酮及复方新诺明具有较高的耐药性,而对阿米卡星、哌拉西林/他唑巴坦敏感性较高。所有菌株均对碳青霉烯类抗菌药物敏感。31株大肠埃希菌为ESBLs阳性菌株。CTX-M、TEM、OXA、SHV和VEB基因阳性率分别为93.5%、83.9%、19.4%、16.1%和3.2%。亚-MIC头孢他啶对9株(18.0%)大肠埃希菌生物膜形成具有抑制作用。亚-MIC头孢他啶对大肠埃希菌生物膜形成的影响与细菌耐药性和ESBLs均无相关性(P0.05)。结论:亚-MIC头孢他啶对大肠埃希菌生物膜形成的调控作用与细菌耐药性、产ESBLs及ESBLs基因分型均无相关性。     

Extended-Spectrum β-Lactamases and/or Carbapenemases-Producing Enterobacteriaceae Isolated from Retail Chicken Meat in Zagazig,Egypt

Objectives

The aim of the present study was to determine the prevalence and to characterize extended-spectrum β-lactamases- and/or carbapenemases-producing Enterobacteriaceae among Enterobacteriaceae isolated from retail chicken meat in Zagazig, Egypt.

Methods

One hundred and six Enterobacteriaceae isolates were collected from retail chicken meat samples purchased in Zagazig, Egypt in 2013. Species identification was done by MALDI-TOF MS. Screening for ESBL-E was performed by inoculation of isolates recovered from meat samples onto the EbSA (Cepheid Benelux, Apeldoorn, the Netherlands) selective screening agar. ESBL production was confirmed by combination disc diffusion test with clavulanic acid (Rosco, Taastrup, Denmark). Carbapenemases production was confirmed with double disk synergy tests. Resistance genes were characterized by PCR with specific primers for TEM, SHV, and CTX-M and carbapenemases (KPC, NDM, OXA-48, IMP and VIM). PCR products of CTX-M genes were purified and sequenced. Phylogenetic grouping of E. coli was performed by a PCR-based method.

Results

Of these 106 isolates 69 (65.09%) were ESBL producers. Twelve (11.32%) of these isolates were also phenotypically class B carbapenemases producer. TEM genes were detected in 61 (57.55%) isolates. 49 (46.23%) isolates harbored CTX-M genes, and 25 (23.58%) carried genes of the SHV family. All CPE belonged to the NDM group. The predominant CTX-M sequence type was CTX-M-15 (89.80%). The majority (80%) of the ESBL-EC belonged to low virulence phylogroups A and B1.

Conclusions

This is the first study from Egypt reporting high rates of ESBLs and carbapenemases (65.09% and 11.32%, respectively) in Enterobacteriaceae isolated from retail chicken meat. These results raise serious concerns about public health and food safety as retail meat could serve as a reservoir for these resistant bacteria which could be transferred to humans through the food chain.     

社区和医院获得性尿道致病性大肠埃希菌耐药性分析

目的了解浙江省社区和医院获得性尿道致病性大肠埃希菌的耐药现状及产超广谱β-内酰胺酶(ESBLs)菌株基因型,为临床合理用药提供科学依据。方法对2014年1月至2014年12月浙江省社区和医院获得性尿道致病性大肠埃希菌耐药情况进行分析,并随机各抽取20株菌株进行CTX-M基因型检测。结果社区和医院获得性大肠埃希菌共收集到93株和992株,产ESBLs菌株的阳性率分别为47.7%和68.5%,差异有统计学意义(P0.05);社区获得性菌株CTX-M基因检出率为30.0%(6/20),其中又以CTX-M-9为主(4/6)。医院获得性菌株CTX-M基因检出率50.0%(10/20),其中CTX-M-9占7/10。医院获得性菌株对头孢呋辛、头孢唑啉、头孢曲松、环丙沙星、左旋氧氟沙星、甲氧苄氨嘧啶/磺胺甲恶唑、呋喃妥因和阿米卡星的敏感率明显低于社区获得性菌株(P0.05)。结论浙江省医院获得性尿道致病性大肠埃希菌对部分抗菌药物的耐药率明显高于社区获得性菌株,ESBLs基因以CTX-M-9为主。     

Molecular characteristics of extended-spectrum β-lactamase-producing Escherichia coli in Riyadh: emergence of CTX-M-15-producing E. coli ST131

Background

The prevalence of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) has increased recently. The aim of this study was to further characterise and to assess the occurrence of ESBL-EC in Riyadh, to use pulsed field gel electrophoresis (PFGE) typing to investigate the epidemiology of ESBL-EC and to determine the prevalence of ST131 in ESBL-EC.

Methods

A total of 152 E. coli isolates were collected at a tertiary hospital in Riyadh from September 2010 to June 2011. Genotypic and phenotypic methods were used to characterise ESBLs. PFGE was used to determine genetic relatedness. Detection of ST131 and CTX-M-like ESBLs was performed using real-time PCR.

Results

Of 152 strains, 31 were positive for ESBLs by phenotypic methods. The bla _CTX-M-15 gene was highly prevalent (30/31 strains, 96.77%) among the 31 ESBL-positive E. coli strains. The bla _CTX-M-27 gene was detected in one strain. Twenty (64.5%) out of 31 of ESBL-EC were ST131. PFGE revealed 29 different pulsotypes.

Conclusions

Our study documented the high prevalence of ESBLs in E. coli isolates, with CTX-M-15 as the predominant ESBL gene. ST131 clone producing CTX-M-15 has a major presence in our hospital. The high prevalence of CTX-M producers was not due to the spread of a single clone. To the best of our knowledge, this study represents the first report of CTX-M-15 and CTX-M-27 β-lactamases and the detection of the ST131 clone in Saudi E. coli isolates.     

产ESBLs大肠埃希菌和肺炎克雷伯菌呼吸道分离株的基因分型及耐药性分析

目的了解深圳市人民医院大肠埃希菌和肺炎克雷伯菌呼吸道分离株超广谱β-内酰胺酶(ESBLs)的基因型特点及耐药性。方法采用临床实验室标准化协会(CLSI)推荐的表型确证试验筛选出该院呼吸道分离株产ESBLs大肠埃希菌和肺炎克雷伯菌共78株。应用PCR及DNA测序法分析产酶株的TEM、SHV及CTX-M3种β-内酰胺酶基因,用琼脂稀释法测定细菌最低抑菌浓度(MIC)。结果 37株产ESBLs大肠埃希菌中,28株(75.7%)检出CTX-M-14基因,4株(10.8%)检出CTX-M-9基因,其他型较少见。41株肺炎克雷伯菌中,25株(61.0%)检出SHV-12基因,4株(9.8%)检出SHV-11基因,其他SHV型较少。20株(48.8%)检出CTX-M-14基因,5株(12.2%)检出CTX-M-3基因,其他型较少。产ESBL菌株均对亚胺培南敏感,对氨苄西林/舒巴坦的耐药率最高(90%),对其他抗生素有不同程度耐药。结论深圳市人民医院呼吸道分离的产ESBLs大肠埃希菌以CTX-M-14型为主,产酶肺炎克雷伯菌以SHV-12和CTX-M-14型为最常见。     

Prevalence of ESBL and MBL encoding genes in Acinetobacter baumannii strains isolated from patients of intensive care units (ICU)

The aim of this study was to investigate the prevalence of ESBL and MBL encoding genes among A. baumannii isolates. In this cross sectional study, 100 A. baumannii strains were isolated from ICU wards of 3 educational hospitals of Hamadan City, Iran in 2011. Phenotypic identification of the production of ESBLs and MBLs has been carried out by using E-test and DDST methods, respectively. PCR technique was used for amplification of the ESBL and MBL encoding genes, namely: CTX-M, SHV, TEM, OXA-51, VIM-Family, IMP-Family, SPM-1, SIM-1, and GIM-1. Eighty seven (87%), 95 (95%), 98 (98%) and 95 (95%) out of 100 A. baumannii isolates were resistant to imipenem, meropenem, ceftazidime and cefotaxime, respectively. Also, 99% and 7% of the isolates were MBLs and ESBLs produced phenotypically. Thirty (30%), 20 (20%) and 58 (58%) out of 100 A. baumannii isolates have been confirmed to harbor the bla_VIM-family, TEM and SHV genes, respectively. Our results show no significant relationship between the detected gens with production of MBLs and ESBLs in spite of high prevalence of MBL encoding and drug resistant A. baumannii. Probably some other genes rather than what we studied are involved in phenotypic production of MBLs and ESBLs and subsequent drug resistance in Hamadan area, Iran.     

Genetic environments of bla(CTX-M) genes located on conjugative plasmids of Enterobacteriaceae nosocomial isolates collected in Russia within 2003-2007

The study showed that bla(CTX-M) genes were present in the genomes of 71% of cephalosporin resistant Enterobacteriaceae nosocomial isolates (n=833) collected in Russian hospitals within 2003-2007, including 91% of E.coli, 90% of Klebsiella spp., 38% of Enterobacter spp., 31% of Citrobacter spp. (n=9), and 36% of the other Enterobacteriaceae species. The genes belonging to the following subtypes (clusters) were identified: bla(CTX-M-1) (529 bla(CTX-M-15) genes; 25 bla(CTX-M-3) genes; 1 bla(CTX-M-22) gene, 1 bla(CTX-M-23) gene, and 1 bla(CTX-M-34) gene); bla(CTX-M-2) (1 bla(CTX-M-2) gene, and 4 bla(CTX-M-5) genes), and bla(CTX-M-9) (2 bla(CTX-M-9) genes, and 28 bla(CTX-M-14) genes). It was shown that bla(CTX-M) genes were located on high-molecular weight (60-160 bp) conjugative plasmids belonging mainly to the incompatibility groups IncF, IncL/M and IncA/C (bla(CTX-M-15) gene); IncL/M(bla(CTX-M-3) gene); and IncF, IncL/Mand IncI1-ly (CTX-M-14 gene). The gene environments of bla(CTX-M) genes were shown specific for the subtype of the genes. A mobile genetic element ISEcp1 (in some cases deleted or inserted by IS26, IS1, IS10, resTn2, or resTn3 sequences, in direct or reverse position) were detected upstream of bla(CTX-M-3), bla(CTX-M-14), and bla(CTX-M-15) genes. A special characteristic was the sequence between ISEcp1 and bla(CTX-M) gene: 48 bp for bla(CTX-M-15) (except 1 E.coli isolate having such a sequence deleted by 3 bp); 127 bp for bla(CTX-M-3); 42 bp for bla(CTX-M-14). Downstream of bla(CTX-M) and bla(CTX-M-15) genes in the major bacterial isolates orf477 mucA and Delta orf477-Delta mucA sequences were detected respectively. Two isolates had additional Delta orf3 insertion inside of Delta orf477-Delta mucA sequence. Insertion sequence IS903 (intact or deleted) was detected downstream of bla(CTX-M-14) gene. Unlike the others, bla(CTX-M-2) and bla(CTX-M-9) genes were located inside of ISCR1 mobile element, downstream of class 1 integron and orf513 sequence.     

Pathogenic and commensal Escherichia coli from irrigation water show potential in transmission of extended spectrum and AmpC β‐lactamases determinants to isolates from lettuce

There are few studies on the presence of extended-spectrum β-lactamases and AmpC β-lactamases (ESBL/AmpC) in bacteria that contaminate vegetables. The role of the production environment in ESBL/AmpC gene transmission is poorly understood. The occurrence of ESBL/AmpC in Escherichia coli (n = 46) from lettuce and irrigation water and the role of irrigation water in the transmission of resistant E. coli were studied. The presence of ESBL/AmpC, genetic similarity and phylogeny were typed using genotypic and phenotypic techniques. The frequency of β-lactamase gene transfer was studied in vitro. ESBLs/AmpC were detected in 35 isolates (76%). Fourteen isolates (30%) produced both ESBLs/AmpC. Prevalence was highest in E. coli from lettuce (90%). Twenty-two isolates (48%) were multi-resistant with between two and five ESBL/AmpC genes. The major ESBL determinant was the CTX-M type (34 isolates). DHA (33% of isolates) were the dominant AmpC β lactamases. There was a high conjugation efficiency among the isolates, ranging from 3.5 × 10⁻² to 1 × 10⁻² ± 1.4 × 10⁻¹ transconjugants per recipient. Water isolates showed a significantly higher conjugation frequency than those from lettuce. A high degree of genetic relatedness between E. coli from irrigation water and lettuce indicated possible common ancestry and pathway of transmission.     

Silver Resistance Genes Are Overrepresented among Escherichia coli Isolates with CTX-M Production

Members of the Enterobacteriaceae with extended-spectrum beta-lactamases (ESBLs) of the CTX-M type have disseminated rapidly in recent years and have become a threat to public health. In parallel with the CTX-M type expansion, the consumption and widespread use of silver-containing products has increased. To determine the carriage rates of silver resistance genes in different Escherichia coli populations, the presence of three silver resistance genes (silE, silP, and silS) and genes encoding CTX-M-, TEM-, and SHV-type enzymes were explored in E. coli isolates of human (n = 105) and avian (n = 111) origin. The antibiotic profiles were also determined. Isolates harboring CTX-M genes were further characterized, and phenotypic silver resistance was examined. The silE gene was present in 13 of the isolates. All of them were of human origin. Eleven of these isolates harbored ESBLs of the CTX-M type (P = 0.007), and eight of them were typed as CTX-M-15 and three as CTX-M-14. None of the silE-positive isolates was related to the O25b-ST131 clone, but 10 out of 13 belonged to the ST10 or ST58 complexes. Phenotypic silver resistance (silver nitrate MIC > 512 mg/liter) was observed after silver exposure in 12 of them, and a concomitant reduced susceptibility to piperacillin-tazobactam developed in three. In conclusion, 12% of the human E. coli isolates but none of the avian isolates harbored silver resistance genes. This indicates another route for or level of silver exposure for humans than that caused by common environmental contamination. Since silE-positive isolates were significantly more often found in CTX-M-positive isolates, it is possible that silver may exert a selective pressure on CTX-M-producing E. coli isolates.     

Extended-Spectrum β-Lactamase- and Carbapenemase-Producing Enterobacteriaceae Isolated from Egyptian Patients with Suspected Blood Stream Infection

ObjectivesThe aim of the study was to investigate the prevalence of extended-spectrum β-lactamase and carbapenemase production among Enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection.MethodsNinety-four Enterobacteriaceae blood culture isolates from Egyptian patients with suspected blood stream infection were collected, one isolate per patient. Identification of bacterial isolates was performed with MALDI-TOF (MS-based Vitek MS system, bioMerieux). Screening for ESBLs and carbapenemases production was done with the Vitek 2 system (bioMérieux). ESBL production was confirmed using the combined disk diffusion method for cefotaxime, ceftazidime, and cefepime, all with and without clavulanic acid (Rosco). Real-time PCR and sequencing were used to characterize the resistance genes. The phylogenetic groups of E. coli were identified by a PCR-based method.ResultsOf the 94 Enterobacteriaceae isolates 46 (48.93%) showed an ESBL phenotype. One Enterobacter spp isolate was ESBL-producer and meropenem-resistant. The genetic analysis showed that CTX-M was present in 89.13% (41/46) of the ESBL-producing Enterobacteriaceae, whereas TEM and SHV were detected in 56.52% (26/46) and 21.74% (10/46) respectively (47.83%) of the ESBL-producing isolates were multidrug resistant (MDR). Eleven out of 30 ESBL-producing E-coli isolates were assigned to phylogroup B2, followed by groups B1 (8 isolates), A (6 isolates) and D (5 isolates).ConclusionsThe high ESBL-E rates (48.93%) found in this study together with the identification of one carbapenem-resistant Enterobacter spp isolate is worrisome. Our results indicate that systems for monitoring and detection of ESBL-producing bacteria in Egyptian hospitals have to be established. Also strict hospital infection control policies with the restriction of the consumption of extended-spectrum cephalosporins are necessary.     

Molecular epidemiology of antibiotic-resistant genes and potent inhibitors against TEM,CTX-M-14, CTX-M-15, and SHV-1 proteins of Escherichia coli in district Peshawar,Pakistan

The Extended Spectrum Beta-Lactamases (ESBLs) producing bacteria is an issue of concern for clinicians resulting in minimize the treatment options. To overcome resistance mechanisms, novel inhibitors with good Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties must inhibit the ESBLs resistant genes. The current study aimed to identify the antibiotic resistance genes of ESBLs producing E. coli and a single inhibitor was designed to inhibit all the resistant proteins. The results showed that 42.9% ESBL producers had CTX-M (69.9%), TEM (63.4%), SHV (34.5%) and CTX-M-14 (17.5%) genes. The ESBLs producing isolates were resistant to cephalosporins, quinolones, and sulfonamide with Minimum Inhibitory Concentration (MICs) ranging from 64 to >256 μg/ml. To design multi inhibitory ligands, RECAP synthesis was used for the de-novo discovery of 1000 inhibitors database. Protein crystal structures were retrieved from Protein Data Base (PDB). Lipinski’s rules of five were applied to the novel inhibitors database to improve the ADMET properties. The novel inhibitors database was selected for docking simulations. Placement of the ligand was used by the London dG algorithm implemented in Molecular Operating Environment (MOE), while GBVI/WSA dG algorithm was used for final refinement. Based on docking score, visual inspection of ligands interaction with key residues, binding affinity, and binding energy of ligands with proteins, ten compounds were selected for ESBLs proteins with best ADMET properties, binding energy, and binding affinity the reported ones. These hits compounds have unique scaffolds and are predicted to be a starting point for developing potent inhibitors against antibiotic-resistant proteins.     

Phylogenetic grouping and virulence potential of extended-spectrum-β-lactamase-producing Escherichia coli strains in cattle

In line with recent reports of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of highly virulent serotypes, such as O104:H4, we investigated the distribution of phylogroups (A, B1, B2, D) and virulence factor (VF)-encoding genes in 204 ESBL-producing E. coli isolates from diarrheic cattle. ESBL genes, VFs, and phylogroups were identified by PCR and a commercial DNA array (Alere, France). ESBL genes belonged mostly to the CTX-M-1 (65.7%) and CTX-M-9 (27.0%) groups, whereas those of the CTX-M-2 and TEM groups were much less represented (3.9% and 3.4%, respectively). One ESBL isolate was stx(1) and eae positive and belonged to a major enterohemorrhagic E. coli (EHEC) serotype (O111:H8). Two other isolates were eae positive but stx negative; one of these had serotype O26:H11. ESBL isolates belonged mainly to phylogroup A (55.4%) and, to lesser extents, to phylogroups D (25.5%) and B1 (15.6%), whereas B2 strains were quasi-absent (1/204). The number of VFs was significantly higher in phylogroup B1 than in phylogroups A (P = 0.04) and D (P = 0.02). Almost all of the VFs detected were found in CTX-M-1 isolates, whereas only 64.3% and 33.3% of them were found in CTX-M-9 and CTX-M-2 isolates, respectively. These results indicated that the widespread dissemination of the bla(CTX-M) genes within the E. coli population from cattle still spared the subpopulation of EHEC/Shiga-toxigenic E. coli (STEC) isolates. In contrast to other reports on non-ESBL-producing isolates from domestic animals, B1 was not the main phylogroup identified. However, B1 was found to be the most virulent phylogroup, suggesting host-specific distribution of virulence determinants among phylogenetic groups.     

A Five-Year Experience of Carbapenem Resistance in Enterobacteriaceae Causing Neonatal Septicaemia: Predominance of NDM-1

Treatment of neonatal sepsis has become a challenge with the emergence of carbapenemase-producing bacteria. This study documents the trend of carbapenem susceptibility in Enterobacteriaceae that caused septicaemia in neonates over a five year period (2007–2011) and the molecular characterisation of Enterobacteriaceae resistant to carbapenems and cephalosporins. Hundred and five Enterobacteriaceae including Escherichia coli (n = 27), Klebsiella pneumoniae (n = 68) and Enterobacter spp. (n = 10) were isolated from blood of septicaemic neonates followed by antibiotic susceptibility tests, determination of MIC values, phenotypic and genotypic detection of β-lactamases. Carbapenem was the most active antimicrobial tested after tigecycline. CTX-M type was the most prevalent ESBL throughout the period (82%). New Delhi Metallo-β-lactamase-1 (NDM-1), which is a recent addition to the carbapenemase list, was the only carbapenemase identified in our setting. Fourteen percent of the isolates possessed bla_NDM-1. Carbapenem non-susceptibility was first observed in 2007 and it was due to loss of Omp F/Ompk36 in combination with the presence of ESBLs/AmpCs. NDM-1 first emerged in E. coli during 2008; later in 2010, the resistance was detected in K. pneumoniae and E. cloacae isolates. NDM-1-producing isolates were resistant to other broad-spectrum antibiotics and possessed ESBLs, AmpCs, 16S-rRNA methylases, AAC(6′)-Ib-cr, bleomycin resistant gene and class 1 integron. Pulsed field gel electrophoresis of the NDM-1-producing isolates indicated that the isolates were clonally diverse. The study also showed that there was a significantly higher incidence of sepsis caused by NDM-1-harbouring isolates in the male sex, in neonates with low birth weight and neonates born at an extramural centre. However, sepsis with NDM-1-harbouring isolates did not result in a higher mortality rate. The study is the first to review the carbapenem resistance patterns in neonatal sepsis over an extended period of time. The study highlights the persistence of ESBLs (CTX-Ms) and the emergence of NDM-1 in Enterobacteriaceae in the unit.