Infrequent transmission of HIV-1 drug-resistant variants.

Transmission of drug-resistant variants is influenced by several factors, including the prevalence of drug resistance in the population of HIV-1-infected patients, HIV-1 RNA levels and transmission by recently infected patients. In order to evaluate the impact of these factors on the transmission of drug-resistant variants, we have defined the population of potential transmitters and compared their resistance profiles to those of newly infected patients. Sequencing of pol gene was performed in 220 recently infected patients and in 373 chronically infected patients with HIV-1 RNA >1000 copies/ml. Minimal and maximal drug-resistance profiles of potential transmitters were estimated by weighting resistance profiles of chronically infected patients with estimates of the Swiss HIV-1-infected population, the prevalence of exposure to antiviral drugs and the proportion of infections attributed to primary HIV infections. The drug-resistance prevalence in recently infected patients was 10.5% (one class drug resistance: 9.1%; two classes: 1.4%; three classes: 0%). Phylogenetic analysis revealed significant clustering for 30% of recent infections. The drug-resistance prevalence in chronically infected patients was 72.4% (one class: 29%; two classes: 27.6%; three classes: 15.8%). After adjustment, the risk of transmission relative to wild-type was reduced both for one class drug resistance (minimal and maximal estimates: odds ratio: 0.39, P<0.001; and odds ratio: 0.55, P=0.011, respectively), and for two to three class drug resistance (odds ratios: 0.05 and 0.07, respectively, P<0.001). Neither sexual behaviour nor HIV-1 RNA levels explained the low transmission of drug-resistant variants. These data suggest that drug-resistant variants and in particular multidrug-resistant variants have a substantially reduced transmission capacity.

Preventive and therapeutic strategies in critically ill patients with highly resistant bacteria.

The antibiotic pipeline continues to diminish and the majority of the public remains unaware of this critical situation. The cause of the decline of antibiotic development is multifactorial and currently most ICUs are confronted with the challenge of multidrug-resistant organisms. Antimicrobial multidrug resistance is expanding all over the world, with extreme and pandrug resistance being increasingly encountered, especially in healthcare-associated infections in large highly specialized hospitals. Antibiotic stewardship for critically ill patients translated into the implementation of specific guidelines, largely promoted by the Surviving Sepsis Campaign, targeted at education to optimize choice, dosage, and duration of antibiotics in order to improve outcomes and reduce the development of resistance. Inappropriate antimicrobial therapy, meaning the selection of an antibiotic to which the causative pathogen is resistant, is a consistent predictor of poor outcomes in septic patients. Therefore, pharmacokinetically/pharmacodynamically optimized dosing regimens should be given to all patients empirically and, once the pathogen and susceptibility are known, local stewardship practices may be employed on the basis of clinical response to redefine an appropriate regimen for the patient. This review will focus on the most severely ill patients, for whom substantial progress in organ support along with diagnostic and therapeutic strategies markedly increased the risk of nosocomial infections.

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

Biofilm Forming Multi Drug Resistant Staphylococcus spp. Among Patients with Conjunctivitis

Biofilm forming multidrug resistant Staphylococcus spp. are major reservoirs for transmission of ophthalmic infections. They were isolated from ocular patients suffering from conjunctivitis. In this study we analyzed biofilm forming ability, antibiotic resistance profile of the Staphylococcus spp. isolated from clinical ocular patients, and their phylogenetic relationship with other community MRSA. Sixty Staphylococcus spp. strains isolated from clinical subjects were evaluated for their ability to form biofilm and express biofilm encoding ica gene. Among them 93% were slime producers and 87% were slime positive. Staphylococcus aureus and S. epidermidis were dominant strains among the isolates obtained from ocular patients. The strains also exhibited a differential biofilm formation quantitatively. Antibiotic susceptibility of the strains tested with Penicillin G, Ciprofloxacin, Ofloxacin, Methicillin, Amikacin, and Gentamicin indicated that they were resistant to more than one antibiotic. The amplicon of ica gene of strong biofilm producing S. aureus strains, obtained by polymerase chain reaction, was sequenced and their close genetic relationship with community acquired MRSA was analyzed based on phylogenetic tree. Our results indicate that they are genetically close to other community acquired MRSA

Colonization pressure and risk factors for acquisition of imipenem-resistant Acinetobacter baumannii in a medical surgical intensive care unit in Brazil

We studied a nonconcurrent cohort of 582 patients admitted to a medical-surgical intensive care unit. Use of antimicrobials (imipenem and metronidazole) was a risk factor for acquisition of imipenem-resistant Acinetobacter baumannii only for the subcohort of patients admitted in months in which colonization pressure was lower than the median value. © 2013 by the Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Methicillin-resistant Staphylococcus aureus of lineage ST398 as cause of mastitis in cows

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Risk factors for the acquisition of imipenem-resistant Acinetobacter baumannii in a burn unit: An appraisal of the effect of colonization pressure

Imipenem-resistant Acinetobacter baumannii (IRAB) is a major threat for critically ill patients, including those admitted to burn units. Recent studies have suggested that colonization pressure (the proportion of patients or patient-days harbouring the pathogen of interest) is an important driver of the risk for acquisition of multidrug-resistant organisms. With that in mind, we conducted a cohort study, enrolling 208 patients admitted to a burn unit from November 2008 through December 2009. The outcome of interest was the acquisition of IRAB. In addition to the usual risk factors, we assessed the impact of colonization pressure. The number of wound excisions (odds ratio (OR) 12.06, 95% confidence interval (CI) 2.82-51.64) and the number of antimicrobials used (OR 22.82, 95% CI 5.15-101.19) were significant risk factors for the outcome of interest. On the other hand, colonization pressure (measured for whole time of exposure or up to the last 14, 7, or 3 days) was not associated with the risk for IRAB acquisition.

Comparison of disc diffusion, Etest and broth microdilution for testing susceptibility of carbapenem-resistant P. aeruginosa to polymyxins

Abstract Background Considering the increasing use of polymyxins to treat infections due to multidrug resistant Gram-negative in many countries, it is important to evaluate different susceptibility testing methods to this class of antibiotic. Methods Susceptibility of 109 carbapenem-resistant P. aeruginosa to polymyxins was tested comparing broth microdilution (reference method), disc diffusion, and Etest using the new interpretative breakpoints of Clinical and Laboratory Standards Institute. Results Twenty-nine percent of isolates belonged to endemic clone and thus, these strains were excluded of analysis. Among 78 strains evaluated, only one isolate was resistant to polymyxin B by the reference method (MIC: 8.0 μg/mL). Very major and major error rates of 1.2% and 11.5% were detected comparing polymyxin B disc diffusion with the broth microdilution (reference method). Agreement within 1 twofold dilution between Etest and the broth microdilution were 33% for polymyxin B and 79.5% for colistin. One major error and 48.7% minor errors were found comparing polymyxin B Etest with broth microdilution and only 6.4% minor errors with colistin. The concordance between Etest and the broth microdilution (reference method) was respectively 100% for colistin and 90% for polymyxin B. Conclusion Resistance to polymyxins seems to be rare among hospital carbapenem-resistant P. aeruginosa isolates over a six-year period. Our results showed, using the new CLSI criteria, that the disc diffusion susceptibility does not report major errors (false-resistant results) for colistin. On the other hand, showed a high frequency of minor errors and 1 very major error for polymyxin B. Etest presented better results for colistin than polymyxin B. Until these results are reproduced with a large number of polymyxins-resistant P. aeruginosa isolates, susceptibility to polymyxins should be confirmed by a reference method.

CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age

Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is associated with the expression of the multidrug resistance (MDR) proteins P-glycoprotein, encoded by the MDR1/ABCB1 gene, multidrug resistant-related protein (MRP/ABCC1), the lung resistance-related protein (LRP), or major vault protein (MVP), and the breast cancer resistance protein (BCRP/ABCG2). The clinical value of MDR1, MRP1, LRP/MVP, and BCRP messenger RNA (mRNA) expression was prospectively studied in 154 newly diagnosed AML patients >or=60 years who were treated in a multicenter, randomized phase 3 trial. Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count (respectively, p < 0.05, p < 0.001, p < 0.001 and p < 0.001). Higher BCRP mRNA was associated with secondary AML (p < 0.05). MDR1 and BCRP mRNA were highly significantly associated (p < 0.001), as were MRP1 and LRP mRNA (p < 0.001) expression. Univariate regression analyses revealed that CD34 expression, increasing MDR1 mRNA as well as MDR1/BCRP coexpression, were associated with a lower complete response (CR) rate and with worse event-free survival and overall survival. When adjusted for other prognostic actors, only CD34-related MDR1/BCRP coexpression remained significantly associated with a lower CR rate (p = 0.03), thereby identifying a clinically resistant subgroup of elderly AML patients.

Clinical and molecular features of methicillin-resistant, coagulase-negative staphylococci of pets and horses

OBJECTIVES To determine the antibiotic resistance and fingerprint profiles of methicillin-resistant coagulase-negative staphylococci (MRCoNS) from animal infections among different practices and examine the history of antibiotic treatment. METHODS Isolates were identified by mass spectrometry and tested for antimicrobial resistance by broth dilution, microarrays and sequence analysis of the topoisomerases. Diversity was assessed by PFGE, icaA PCR and staphylococcal cassette chromosome mec (SCCmec), arginine catabolic mobile element (ACME) and multilocus sequence typing. Clinical records were examined retrospectively. RESULTS MRCoNS were identified as Staphylococcus epidermidis (n=20), Staphylococcus haemolyticus (n=17), Staphylococcus hominis (n=3), Staphylococcus capitis (n=1), Staphylococcus cohnii (n=1) and Staphylococcus warneri (n=1). PFGE identified one clonal lineage in S. hominis isolates and several in S. haemolyticus and S. epidermidis. Fourteen sequence types were identified in S. epidermidis, with sequence type 2 (ST2) and ST5 being predominant. Ten isolates contained SCCmec IV, seven contained SCCmec V and the others were non-typeable. ACMEs were detected in 11 S. epidermidis isolates. One S. hominis and 10 S. epidermidis isolates were icaA positive. In addition to mecA-mediated β-lactam resistance, the most frequent resistance was to gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia, aph(3')-III] (n=34), macrolides/lincosamides [erm(C), erm(A), msr, lnu(A)] (n=31), tetracycline [tet(K)] (n=22), streptomycin [str, ant(6)-Ia] (n=20), trimethoprim [dfr(A), dfr(G)] (n=17), sulfamethoxazole (n = 34) and fluoroquinolones [amino acid substitutions in GyrA and GrlA] (n=30). Clinical data suggest selection through multiple antibiotic courses and emphasize the importance of accurate diagnosis and antibiograms. CONCLUSIONS MRCoNS from animal infection sites are genetically heterogeneous multidrug-resistant strains that represent a new challenge in the prevention and therapy of infections in veterinary clinics.

Extended-spectrum cephalosporin-resistant Gram-negative organisms in livestock: an emerging problem for human health?

Escherichia coli, Salmonella spp. and Acinetobacter spp. are important human pathogens. Serious infections due to these organisms are usually treated with extended-spectrum cephalosporins (ESCs). However, in the past two decades we have faced a rapid increasing of infections and colonization caused by ESC-resistant (ESC-R) isolates due to production of extended-spectrum-β-lactamases (ESBLs), plasmid-mediated AmpCs (pAmpCs) and/or carbapenemase enzymes. This situation limits drastically our therapeutic armamentarium and puts under peril the human health. Animals are considered as potential reservoirs of multidrug-resistant (MDR) Gram-negative organisms. The massive and indiscriminate use of antibiotics in veterinary medicine has contributed to the selection of ESC-R E. coli, ESC-R Salmonella spp. and, to less extent, MDR Acinetobacter spp. among animals, food, and environment. This complex scenario is responsible for the expansion of these MDR organisms which may have life-threatening clinical significance. Nowadays, the prevalence of food-producing animals carrying ESC-R E. coli and ESC-R Salmonella (especially those producing CTX-M-type ESBLs and the CMY-2 pAmpC) has reached worryingly high values. More recently, the appearance of carbapenem-resistant isolates (i.e., VIM-1-producing Enterobacteriaceae and NDM-1 or OXA-23-producing Acinetobacter spp.) in livestock has even drawn greater concerns. In this review, we describe the aspects related to the spread of the above MDR organisms among pigs, cattle, and poultry, focusing on epidemiology, molecular mechanisms of resistance, impact of antibiotic use, and strategies to contain the overall problem. The link and the impact of ESC-R organisms of livestock origin for the human scenario are also discussed.

Occurrence and Genetic Characteristics of Third-Generation Cephalosporin-Resistant Escherichia coli in Swiss Retail Meat

Prevalence and genetic relatedness were determined for third-generation cephalosporin-resistant Escherichia coli (3GC-R-Ec) detected in Swiss beef, veal, pork, and poultry retail meat. Samples from meat-packing plants (MPPs) processing 70% of the slaughtered animals in Switzerland were purchased at different intervals between April and June 2013 and analyzed. Sixty-nine 3GC-R-Ec isolates were obtained and characterized by microarray, PCR/DNA sequencing, Multi Locus Sequence Typing (MLST), and plasmid replicon typing. Plasmids of selected strains were transformed by electroporation into E. coli TOP10 cells and analyzed by plasmid MLST. The prevalence of 3GC-R-Ec was 73.3% in chicken and 2% in beef meat. No 3GC-R-Ec were found in pork and veal. Overall, the blaCTX-M-1 (79.4%), blaCMY-2 (17.6%), blaCMY-4 (1.5%), and blaSHV-12 (1.5%) β-lactamase genes were detected, as well as other genes conferring resistance to chloramphenicol (cmlA1-like), sulfonamides (sul), tetracycline (tet), and trimethoprim (dfrA). The 3GC-R-Ec from chicken meat often harbored virulence genes associated with avian pathogens. Plasmid incompatibility (Inc) groups IncI1, IncFIB, IncFII, and IncB/O were the most frequent. A high rate of clonality (e.g., ST1304, ST38, and ST93) among isolates from the same MPPs suggests that strains persist at the plant and spread to meat at the carcass-processing stage. Additionally, the presence of the blaCTX-M-1 gene on an IncI1 plasmid sequence type 3 (IncI1/pST3) in genetically diverse strains indicates interstrain spread of an epidemic plasmid. The blaCMY-2 and blaCMY-4 genes were located on IncB/O plasmids. This study represents the first comprehensive assessment of 3GC-R-Ec in meat in Switzerland. It demonstrates the need for monitoring contaminants and for the adaptation of the Hazard Analysis and Critical Control Point concept to avoid the spread of multidrug-resistant bacteria through the food chain.

Genetic Relatedness, Antimicrobial and Biocide Susceptibility Comparative Analysis of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius from Portugal

Forty methicillin-resistant and -susceptible Staphylococcus pseudintermedius (MRSP and MSSP, respectively) from colonization and infection in dogs and cats were characterized for clonality, antimicrobial, and biocide susceptibility. MSSP were genetically more diverse than MRSP by multi-locus sequence typing and pulsed-field gel electrophoresis. Three different spa types (t06, t02, t05) and two SCCmec types (II-III and V) were detected in the MRSP isolates. All MRSP and two MSSP strains were multidrug-resistant. Several antibiotic resistance genes (mecA, blaZ, tet(M), tet(K), aac(6')-Ie-aph(2')-Ia, aph(3')-III, ant(6)-Ia, sat4, erm(B), lnu(A), dfr(G), and catpC221) were identified by microarray and double mutations in the gyrA and grlA genes and a single mutation in the rpoB gene were detected by sequence analysis. No differences were detected between MSSP and MRSP in the chlorhexidine acetate (CHA) minimum inhibitory concentrations (MICs). However, two MSSP had elevated MIC to triclosan (TCL) and one to benzalkonium chloride and ethidium bromide. One MSSP isolate harboured a qacA gene, while in another a qacB gene was detected. None of the isolates harboured the sh-fabI gene. Three of the biocide products studied had high bactericidal activity (Otodine(®), Clorexyderm Spot Gel(®), Dermocanis Piocure-M(®)), while Skingel(®) failed to achieve a five log reduction in the bacterial counting. S. pseudintermedius have become a serious therapeutic challenge in particular if methicillin- resistance and/or multidrug-resistance are involved. Biocides, like CHA and TCL, seem to be clinically effective and safe topical therapeutic options.

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. RIF-resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes ≤2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader dependent and limits its clinical use. The aim of this study was to develop an objective, reader-independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from 2 regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: the Texas–Mexico border and Colombia. Using coded DNA specimens, mutations within an 81-bp hot spot region of rpoB were established by qPCR with 5 beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF-resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.^