A novel complex class 1 integron found in a Klebsiella pneumoniae isolate from Portugal

Klebsiella pneumoniae Kp1 carrying a novel complex class 1 integron was isolated from an inanimate surface of a female ward sanitary facility in the Hospital Infante D. Pedro, Aveiro, central Portugal. The integron consists of two variable regions (VRs); VR1 was previously described in Escherichia coli and Vibrio cholerae, and VR2 contains an ***ln37-like structure and is located downstream of an ISCR1 element. The integron was found on a plasmid of 225 kb. The qnrB10 gene, although present, is not associated with the complex class 1 integron.     

High prevalence of class 1 integrons in clinical isolates of methicillin-resistant Staphylococcus aureus from India

Introduction: Class1 integrons are one of the prevalent mechanisms of antibiotic resistance gene transfer in Gram-negative organisms, but their prevalence and role in the spread of antibiotic resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) is unexplored. The purpose of this study was to investigate the prevalence of class 1 integrons in clinical isolates of MRSA. Materials and Methods: Total 143 MRSA isolates obtained from two different cities in India (Pune and Mumbai) were characterized by biochemical tests, and the antibiotic sensitivity was performed using the Clinical and Laboratory Standards Institute (CLSI) guidelines. The presence of class 1 integrons, sul1/qacEΔ1 region of class 1 integron and mecA gene among these isolates was determined by polymerase chain reaction (PCR). Results: All 143 isolates were mecA positive and coagulase-positive. Overall, 71% of the MRSA isolates carried class 1 integrons; 58% (45/77) of the isolates obtained from Mumbai and 85% (56/66) of the isolates from Pune carried class 1 integrons. In all, 39% of these isolates carried sul1/qacEΔ1 region, thus confirming the association of class 1 integrons with antibiotic resistance genes. Along with β-lactam antibiotics the MRSA isolates were resistant to several other antibiotics, with resistance to erythromycin, ciprofloxacin and trimethoprim-sulfamethoxazole being observed in 75%, 66% and 60% of the isolates, respectively. Conclusion: To the best of our knowledge, this is the first report of class 1 integrons in MRSA isolates from India. The study provides insights into the prevalence of a novel mechanism adapted by MRSA for the propagation of antibiotic resistance genes.     

An antibiotic-resistant class 3 integron in an Enterobacter cloacae isolate from hospital effluent

Hospital effluents are involved in dissemination of antibiotic-resistant integrons. We describe here a new class 3 integron, In3-5, detected in an Enterobacter cloacae isolate retrieved from a random French hospital effluent sample collected in 2009. In3-5 carries two gene cassettes: the new bla_OXA-256 and an aac(6′)-Ib variant, respectively conferring resistance to β-lactams and aminoglycosides. In3-5 is located on an IncQ-like backbone plasmid. Class 3 integrons could thus be involved in the dissemination of antibiotic resistance in both clinical settings and the environment, and could participate in the exchange of antibiotic-resistance genes between these two ecosystems.     

Association of the blaCMY-10 gene with a novel complex class 1 integron carrying an ISCR1 element in clinical isolates from Korea

The bla_CMY-10 gene responsible for β-lactam resistance was located on a new complex class 1 integron within a conjugative plasmid. The sul1-type class 1 integron, containing an aadA2a gene cassette, was identified upstream of bla_CMY-10. A unique gene array (yqgF–yqgE–gshB–orf97—orf105) was identified downstream of bla_CMY-10.     

Pharmacokinetics/pharmacodynamics of antipseudomonal bacteriophage therapy in rats: a proof-of-concept study

ObjectivesPan-drug-resistant (PDR) Pseudomonas aeruginosa is one of the three top-priority pathogens identified by the WHO, and bacteriophages have been investigated as an alternative therapy. However, knowledge on the pharmacokinetics/pharmacodynamics (PK/PD) of phage therapy is sparse, limiting its clinical applications. This study aimed to evaluate the PK/PD of the antipseudomonal phage øPEV20 in vivo following intravenous administration.MethodsHealthy Sprague-Dawley rats were given øPEV20 as a single intravenous bolus of ~6, 9 and 11-log₁₀PFU/rat. Arterial blood was sampled over 72 h. At 72 h, the animals were killed and multiple tissues were harvested for biodistribution studies. A PK model was developed using the importance sampling algorithm and deterministic simulations with a PD model were performed.ResultsA three-compartment model with non-linear clearance described the exposure of øPEV20 in blood. Model evaluation indicated that the model was robust and parameter estimates were accurate. The median (standard error) values of model-predicted PK parameters for V_C, V_P1, V_P2, Q₁, Q₂, V_m and K_m were 111 mL/rat (8.5%), 128 mL/rat (4.97%), 180 mL/rat (4.59%), 30.4 mL/h/rat (19.2%), 538 mL/h/rat (4.97%), 4.39 × 10¹⁰ PFU/h/rat (10.2%) and 1.64 × 10⁷ PFU/mL/rat (3.6%), respectively. The distribution of øPEV20 was not homogeneous; there was preferential accumulation in the liver and spleen. Deterministic simulations with a PD model confirmed the importance of the host immune system in facilitating phage-mediated bacterial elimination.ConclusionsWe developed a robust PK model to describe the disposition of phages in healthy rats. This model may have significant potential in facilitating future preclinical and clinical PK/PD investigations.     

The effects of topical antibiotics on eradication and acquisition of third-generation cephalosporin and carbapenem-resistant Gram-negative bacteria in ICU patients; a post hoc analysis from a multicentre cluster-randomized trial

ObjectivesThe aim was to quantify the effects of selective digestive tract decontamination (SDD) consisting of a mouth paste and gastro-enteral suspension, selective oropharyngeal decontamination with a mouth paste (SOD) and 1–2% chlorhexidine (CHX) mouthwash on eradication and acquisition of carriage of third-generation cephalosporin-resistant Enterobacterales (3GCR-E) and carbapenem-resistant Gram-negative bacteria (CR-GNB) in Intensive Care Unit (ICU) patients.MethodsThis was a nested cohort study within a cluster-randomized cross-over trial in six European countries and 13 ICUs with 8665 patients. Eradication and acquisition during ICU stay of 3GCR-E and CR-GNB were investigated separately in the rectum and respiratory tract for the three interventions and compared with standard care (SC) using Cox-regression competing events analyses.ResultsAdjusted cause specific hazard ratios (CSHR) for eradication of rectal carriage for SDD were 1.76 (95% CI 1.31–2.36) for 3GCR-E and 3.17 (95% CI 1.60–6.29) for CR-GNB compared with SC. For the respiratory tract, adjusted CSHR for eradication of 3GCR-E were 1.47 (0.98–2.20) for SDD and 1.38 (0.92–2.06) for SOD compared with SC, and for eradication of CR-GNB these were 0.77 (0.41– 1.45) for SDD and 0.81 (0.44–1.51) for SOD, compared with SC. Adjusted CSHRs for acquisition of rectal carriage during SDD (compared with SC) were 0.51 (0.40–0.64) for 3GCR-E and of 0.56 (0.40–0.78) for CR-GNB. Adjusted CSHRs for acquiring respiratory tract carriage with 3GCR-E compared with SC were 0.38 (0.28–0.50) for SDD and 0.55 (0.42–0.71) for SOD, and for CR-GNB 0.46 (0.33–0.64) during SDD and 0.60 (0.44–0.81) during SOD, respectively. SOD was not associated with eradication or acquisition of 3GCR-E and CR-GNB in the rectum.ConclusionsAmong mechanically ventilated ICU patients, SDD was associated with more eradication and less acquisition of 3GCR-E and CR-GNB in the rectum than SC. SDD and SOD were associated with less acquisition of both 3GCR-E and CR-GNB than SC in the respiratory tract.     

第1类整合子中aadA2基因翻译起始位点的确定

目的 确定第1类整合子中aadA2基因能否从上游无核糖体结合位点的密码子ATG起始翻译并合成有功能的蛋白.方法 定点突变含有不同翻译起始密码子的aadA2基因盒,并连同上游的可变区启动子分别克隆入质粒pACYC184中,转化大肠埃希菌JM109,免疫印迹检测含有不同翻译起始密码子的aadA2基因的翻译产物,用微量肉汤稀释法检测链霉素对含有不同翻译起始密码子aadA2基因的大肠埃希菌JM109的最小抑菌浓度.结果 aadA2基因可从上游无核糖体结合位点的密码子ATG及上游具有核糖体结合位点的密码子GTG起始翻译,同时在GTG密码子下游还存在着起始翻译密码子,其翻译产物在免疫印迹中均可与抗氨基糖苷-3″-腺苷酰基转移酶多克隆抗体产生特异性杂交条带,并赋予宿主细菌对链霉素不同水平的耐药.结论 当位于第1类整合子第1位基因盒中,aadA2基因可从上游无核糖体结合位点的密码子ATG起始翻译并合成有功能的蛋白,这一结构特点使得整合入第1类整合子的基因盒,不需带有核糖体结合位点即可起始基因盒中相应读码框的翻译,从而有利于第1类整合子表达从外界环境中捕获的基因.     

Detection of tem-1 and class-1 integrons in multidrug resistant uropathogens from HIV patients with asymptomatic bacteriuria in a Tertiary Care Hospital,SouthWest Nigeria

BackgroundHuman immunodeficiency virus (HIV) infected individuals are at increased risk of asymptomatic bacteriuria (ASB) due to immune suppression. The increasing resistance of uropathogens necessitates the need for regular monitoring of their profile to reduce drug resistance.ObjectivesWe determined the prevalence of ASB and the characteristics of antibiotic-resistant uropathogens isolated from HIV patients.MethodsMid-stream urine samples from 100 HIV positive and 100 HIV negative healthy individuals were cultured for significant bacteriuria. The isolates were identified by standard techniques and their susceptibility patterns determined by the Kirby-Bauer disc diffusion technique. All the Gram-negative isolates were screened for ESBL production by combined disc method, ESBL genes and class 1 integrons by Polymerase chain reaction.ResultsNine (9%) HIV positive individuals and 4 (4%) healthy individuals had ASB yielding a total of 13 (6.5%) uropathogens dominated by Escherichia coli (53.9%). All isolates were multidrug resistant. Five isolates harboured both the blaTEM-1 gene and class 1integrons while Serratia liquefaciens produced ampC.ConclusionThere is a higher burden of ASB characterized by multi-drug resistant uropathogens among HIV patients. Thus emphasizing the need for continuous resistance surveillance and antibiotic stewardship in our environment to reduce drug resistance and prevent treatment failure.     

Novel cassette array in a class 1 integron in clinical isolates of Acinetobacter baumannii from central Iran

Antibiotic resistance in Acinetobacter baumannii is a major problem in the hospital and outbreaks caused by this organism have been reported frequently. The present study aimed at determining the antibiotic susceptibility patterns, the prevalence of different classes of integrons and the characterization of integron class 1 gene cassettes in Iranian A. baumannii isolates. A total of 63 non-duplicate A. baumannii isolates were collected from clinical and environmental specimens in the Vali-Asr hospital in the central province of Iran (March to September, 2011). The antimicrobial susceptibility for 15 antibiotics which are used conventionally was determined by disk diffusion. The presence of different integron classes was investigated by PCR and the size of gene cassettes in class 1 integrons was then determined by PCR as well. Moreover, integron cassette arrays of isolates were delineated by RFLP and sequencing amplicons with different lengths. Of 63 isolates 62 (98.4%) carried a class 1 integron. The prevalence of IntI2 was 15.9% and the length of the amplicons ranged from 500 bp to 3 kb. Sequencing of integrons of class 1 revealed the presence of many resistance genes (aadA, aacA, aacC, dfrA, bla_GES and bla_IMP). We identified a completely new gene cassette which contained aacA7-qacF-aadA5-bla_IMP, this cassette has not been reported previously in A. baumannii.     

Diversity in VIM-2-encoding class 1 integrons and occasional blaSHV2a carriage in isolates of a persistent,multidrug-resistant Pseudomonas aeruginosa clone from Tunis

From 2002 to 2006, 35 of 73 multidrug-resistant Pseudomonas aeruginosa isolates from different wards at Charles Nicolle hospital of Tunis were positive for class B carbapenemase (using the imipenem-EDTA test), owing to a bla_VIM-2 gene cassette in a class 1 integron. Twenty-three isolates additionally produced the extended-spectrum β-lactamase SHV2a. DNA sequences immediately surrounding bla_SHV2a shared extensive identity with a Klebsiella pneumoniae plasmid sequence. Despite belonging to the same chromosomal type, as shown by pulsed-field gel electrophoresis (PFGE), the VIM-2 producing P. aeruginosa isolates prevalent at Charles Nicolle hospital displayed a diversity of VIM-2-carrying integrons.     

New dfr2 gene as a single-gene cassette in a class 1 integron from a trimethoprim-resistant Escherichia coli isolate

Resistance to trimethoprim among Enterobacteriaceae is increasing in spite of a stable or decreasing use of the drug. Integrons are common among these bacteria and many of them contain dfr gene cassettes. A clinical isolate of Escherichia coli from a urine specimen obtained at the Karolinska Hospital in Stockholm was resistant to trimethoprim, ampicillin, sulfonamides, chloramphenicol, streptomycin, and norfloxacin. PCR analysis with primers specific for the 5' and 3' sequences flanking the cassettes in class 1 integrons, detected a very short cassette region of no more than approximately 400 bp. Sequence analysis of the entire integron was performed, revealing a single-gene cassette of 411 bp encoding a new dihydrofolate reductase. The gene cassette comprised all sequences between the flanking conserved sequences and encoded only 78 amino acids. By homology it belongs to the unique group of dfr2 gene cassettes and being the fourth described gene in this group the new gene is called dfr2d. The enzymes encoded by the dfr2 genes are 67% identical and are not related to any other known dfr gene products.     

Establishment and multi drug resistance evolution of ST235 Pseudomonas aeruginosa strains in the intensive care unit of a Colombian hospital

Drug resistant Pseudomonas aeruginosa represents a therapeutic challenge. To assess the diversity of P. aeruginosa antibiotic resistant variants, isolates were recovered from hospital patients in Colombia. Thirty of 60 isolates contained class 1 integrons and five were of Sequence Type ST235 having appeared in a single intensive care unit. All five possessed an unusual integron but showed differences in gene cassette content and the presence/absence of insertion sequence IS26. This showed that differences can arise rapidly, even within a single ICU. Also, the emergence of IS26 in P. aeruginosa is contributing to the evolution of resistance in this bacterium.     

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 β-lactamase

A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besançon, France. Resistance to β-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla_PER-1 gene encoding extended-spectrum β-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2″) enzyme, and aadA11, encoding a new ANT(3″) enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.     

Type 1 diabetes in Jewish Ethiopian immigrants in Israel: HLA class II immunogenetics and contribution of new environment

The interrelationship between human leukocyte antigen immunogenetics and environmental factors and their contribution to the emergence of type 1 diabetes (T1D) were studied in Jewish immigrants from Ethiopia in Israel. This community displays high incidence of T1D, and is unique both by its ethnic segregation and its rapid exposure to a new environment after the immigration. The study population consisted of 152 Ethiopian Jews living in Israel, 33 with T1D and 119 unrelated controls. Human leukocyte antigen class II susceptible and protective alleles in the Jewish Ethiopian patients were similar to those in patients of other ethnic groups in Israel and in non-Jewish Ethiopian patients, with a few exceptions. Three haplotypes were markedly associated with diabetes in Jewish Ethiopian patients: DRB1*0301 DQA1*05 DQB1*02 (OR 4.4, p < 0.001); DRB1*0404 DQA1 03 DQB1*0302 (OR 19.2, p = 0.006), and DRB1*0405 DQA1*03 DQB1*0302 (OR 87.8, p < 0.001). The highly susceptible allele DRB1*0301 was more common in the general Ethiopian population (25.2%) than in all other ethnic groups in Israel, which may render this community prone to the disease. The age at onset of disease in patients with two susceptible haplotypes was negatively correlated with the duration of living in Israel (r = −0.621, p = 0.04). We concluded that ongoing exposure of genetically predisposed immigrants from Ethiopia to diabetogenic environmental factors eventually leads to a high incidence of overt diabetes in this ethnic group.     

PER-1- and OXA-10-like β-lactamases in ceftazidime-resistant Pseudomonas aeruginosa isolates from intensive care unit patients in Istanbul, Turkey

The presence of PER-1- and OXA-10-like beta-lactamases was investigated by PCR in 49 ceftazidime-resistant Pseudomonas aeruginosa isolates from patients hospitalised in the 24-bed general intensive care unit of the Istanbul Faculty of Medicine during a 12-month period between February 1999 and February 2000. The clonal relatedness of the isolates was investigated by random amplified polymorphic DNA (RAPD) analysis, and the sequences of the PER-1 and OXA genes from all isolates were determined. The rates of resistance of the isolates to imipenem, aztreonam and cefepime were 98%, 92% and 96%, respectively, and to piperacillin and piperacillin-tazobactam were 41% and 37%, respectively. Using the double-disk synergy test, 37% (18/49) of the isolates were identified as extended-spectrum beta-lactamase producers. The PER-1 gene was identified in 86% (42/49) and the OXA-10 gene in 55% (27/49) of the ceftazidime-resistant isolates. Of isolates carrying the PER-1 gene, 48% (20/42) also carried the OXA-10 gene. The respective nucleotide sequences were identical for each isolate. Sixteen RAPD patterns were detected among the PER-1-positive isolates, but 60% (25/42) of the PER-1-positive isolates belonged to two distinct patterns, while the remainder exhibited a wide clonal diversity. The results indicated that the prevalence of PER-1- and OXA-10-like beta-lactamases remains high among ceftazidime-resistant P. aeruginosa isolates in Turkey.     

Mononuclear cells from patients recovered from cutaneous leishmaniasis respond to Leishmania major amastigote class I nuclease with a predominant Th1-like response

The Leishmania major amastigote class I nuclease (LmaCIN) is a developmentally regulated protein that is highly expressed in the amastigote stage of L. major. This protein is homologous to the P4 nuclease of L. pifanoi, which has been shown to induce protective immune response in a murine model. To evaluate LmaCIN as a potential human vaccine candidate, cellular immune responses to recombinant LmaCIN were examined in individuals recovered from Old World cutaneous leishmaniasis. Peripheral blood mononuclear cells (PBMC) from patients recovered from L. major infection were cultured either with recombinant LmaCIN or autoclaved L. major (ALM) as control. rLmaCIN induced significant proliferation of PBMC from 90% of recovered patients. Phenotypic analysis of proliferating cells showed that CD8(+) cells were the predominant cell type proliferating in response to rLmaC1N. Screening of culture supernatants for cytokines showed that rLmaCIN induced high levels of interferon (IFN)-gamma (mean +/- s.e.m.: 1398 +/- 179 pg/ml) associated with little interleukin (IL)-10 and little or no IL-5 production. These findings show that LmaCIN is immunogenic in humans during L. major infection and that it can elicit immunological responses relevant to immunoprophylaxis of leishmaniasis.     

Molecular analysis of HLA-DRB1, DQA1, DQB1, DQ promoter polymorphism and extended class I/class II haplotypes in the Seri Indians from Northwest Mexico

The study of the genetics of the Major Histocompatibility Complex (MHC) in Amerindians is of great value in understanding the origins and migrations of these native groups, as well as the impact of immunogenetics on the epidemiology of diseases affecting these populations. We analyzed, using Polymerase Chain Reaction and Sequence Specific Oligonucleotide Probes (PCR-SSOP), DRB1, DQA1, DQB1 alleles and the promoter regions of DQA1 and DQB1 genes in 31 unrelated and 24 related Seri, a Mexican Indian group, from the state of Sonora (Northwest Mexico). The class II genotypes of this population were found to be in genetic equilibrium. The allele frequency (AF) of the prevalent DRB1 alleles were DRB1*0407 (48.4%), DRB1*0802 (33.9%) and DRB1*1402 (16.1%). The most frequent DQA1 and DQB1 alleles were DQA1*03011 (AF = 50.00%), DQA1*0401 (AF = 33.87%) and DQA1*0501 (AF = 16.13%); DQB1*0302 (AF = 50.00%), DQB1*0402 (33.87%) and DQB1*0301 (16.13%); which were in combination with DRB1*0407, DRB1*0802 and DRB1*1402, respectively. Three QAP and three QBP alleles were present (QAP 3.1, 4.1, 4.2; QBP 3.1, 3.21, 4.1) associated with the typical published DQA1 and DQB1 alleles. Four class II haplotypes were present in family members: DRB1*0407-QAP-3.1-DQA1*03011-QBP-3.21-DQB1*0302; DRB1*0802-QAP-4.2-DQA1*0401-QBP-4.1-DQB1*0402; DRB1*1402-QAP-4.1-DQA1*0501-QBP-3.1-DQB1*0301 and DRB1*0701-QAP-2.1-DQA1*0201-QBP-2.1-DQB1*0201. The family data were used to confirm extended haplotypes. A total of 21 haplotypes were found when A* and B* loci were also considered. The three most frequent combinations included A*0201-B*3501-DRB1*0407, A*3101-B*5101-DRB1*0802, and A*0201-B*40-DRB1*1402.     

The inoculum effect of Escherichia coli expressing mcr-1 or not on colistin activity in a murine model of peritonitis

ObjectivesColistin often remains the last resort antibiotic active against carbapenemase-producing Enterobacteriaceae. However, while in vitro inoculum effect has been reported, therapeutic relevance of this phenomenon remains questioned.MethodsTen E. coli strains were used that included the wild-type CFT073 and its transconjugant CFT073-MCR-1 and eight susceptible clinical isolates. Mice with peritonitis were treated for 24 h with colistin sulfate. Bacterial loads were determined in peritoneal fluid (PF) and spleen and colistin-resistant mutants were detected.ResultsMICs of colistin against the eight susceptible clinical strains and CFT073 ranged from 0.125 to 0.5 mg/L with an inoculum of 10⁵ CFU/mL and from 2 to 4 mg/L with a 10⁷ CFU/mL inoculum; 5/9 strains with an MIC of 4 mg/L were considered resistant according to EUCAST breakpoint (resistance, > 2 mg/L). When the bacterial load of wild-type CFT073 inoculated in mice increased from 10⁷ to 10⁸ CFU: i) mean log₁₀ CFU reduction generated by colistin in PF and spleen decreased from 5.8/mL and 3.1/g, respectively, (p < 0.01) to 0.9/mL and 0.8/g, respectively (NS); ii) mice survival rate decreased from 15/15 (100%) to 6/15 (40%) (p = 0.017); and iii) proportion of mice with selection of colistin-resistant mutants increased from 4/15 to 15/15 (p < 0.01). These results were comparable to those obtained when peritonitis was produced with a 10⁷ CFU bacterial load of E. coli CFT073 expressing mcr-1, for which the mean log₁₀ CFU reductions were 3.5/mL and 0.6/g in PF and spleen, respectively (NS), and survival rate was 8/15 (53%) (p < 0.01 versus survival of mice infected with wild-type CFT073).ConclusionsPhenotypic colistin resistance in wild-type E. coli due to an increase in inoculum size had a therapeutic impact in mice with peritonitis that was comparable to that observed when the mcr-1 gene was expressed.