Phage therapy as strategy to face post-antibiotic era: a guide to beginners and experts.

Interest in the therapeutic use of bacteriophages (phages) has emerged in recent years, driven mainly by the antimicrobial resistance crisis. This review aimed to summarize some important studies addressing the use of phages as a therapeutic alternative for multiresistant bacterial infections. To this end, a literature search was conducted to address the efficacy and versatility of phage therapy, the advantages and disadvantages of its use, and potential limitations for the application of phage therapy that need to be overcome, especially in Western countries. Thus, this review highlights that phage therapy may be a promising route in the treatment of infections caused by multidrug-resistant pathogens and that a combined approach has the potential to prolong the life of the current available antimicrobials. In addition, standardized clinical trials using monoclonal or polyclonal phages, alone or in combination with antimicrobials, are crucial to determine the real potential of these treatments in clinical practice.

Novel ST1465/CC216 Nosocomial Lineage of Carbapenem-Resistant Acinetobacter baumannii Harboring an Unusual Plasmid Carrying blaNDM-1 Gene.

This study used whole-genome sequencing to analyze the first case of NDM-1-producing Acinetobacter baumannii belonging to the novel sequence type 1465/CC216 recovered in Brazil. The study identified an unusual plasmid carrying blaNDM-1 gene, in which some genes of the Tn125 transposon were lost. Besides, on the chromosome, the strain reported here presented blaOXA-106 gene, a variant of blaOXA-51 gene, and blaADC-25 with ISAba1 upstream. The isolation of new STs of A. baumannii carrying blaNDM-1 genes elicits our concerns about the possible spread of these genes among clinically relevant bacteria.

Gram-negative bacilli bacteremia: a 7 year retrospective study in a referral Brazilian tertiary-care teaching hospital.

Introduction. Bloodstream infection is one of the most frequent and challenging hospital-acquired infections and it is associated with high morbidity, mortality and additional use of healthcare resources.Hypothesis/Gap Statement: Bloodstream infections have consequences for the patient, such as the evolution to mortality and inappropriate empirical antibiotic prescription, especially when caused by multidrug-resistant Gram-negative bacilli.Objective. To assess the impact of bloodstream infection and the status of multidrug resistance (MDR) in the evolution of patients who received inappropriate initial antibiotic therapy.Methods. A retrospective surveillance was conducted on nosocomial bloodstream infections caused by Gram-negative bacilli (GNB) from January 2012 to December 2018 in an adult intensive care unit of a Brazilian tertiary teaching hospital.Results. We identified 270 patients with GNB nosocomial bacteremia. Non-survivors were older (with an average age of 58.8 years vs 46.9 years, P=<0.0001), presented more severe illnesses, were immunosuppressed (73.7 vs 37.6%, P=<0.0001), were more likely to have septic shock (55.8 vs 22.4%, P=<0.0001) and had an increased usage of mechanical ventilators (98.6 vs 89.6%, P=0.0013) than survivors. In a logistic regression model, inappropriate empirical antibiotic therapy was not an independent predictor of mortality, different from mechanical ventilator (P=<0.0001; OR=28.0; 95% CI=6.3-123.6), septic shock (P=0.0051; OR=2.5; 95% CI=1.3-4.9) and immunosuppression (P=0.0066; OR=2.6; 95% CI=1.3-5.2). In contrast, in a separate model, MDR was strongly associated with the prescription of inappropriate initial antibiotic therapy (P=0.0030; OR=5.3; 95% CI=1.7-16.1). The main isolated pathogens were Acinetobacter baumannii (23.6 %) and Klebsiella pneumoniae (18.7 %). The frequency of MDR organisms was high (63.7 %), especially among non-fermenting bacilli (60.9 %), highlighting A. baumannii (81.6 %) and Pseudomonas aeruginosa (41.8 %).Conclusion. Illness severity (septic shock and immunosuppression) and mechanical ventilation were identified as predictors of mortality. Additionally, MDR was a major determinant of inappropriate antibiotic empirical therapy, but not associated with mortality, and both characteristics were not statistically associated with death.

Incidence of infections caused by carbapenem-resistant Acinetobacter baumannii.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CR-Ab) has become a worrying health care problem, mainly in developing countries, such as Brazil. The objective was to investigate the prevalence and prognostic factors for CR-Ab infections at a Brazilian university hospital and examine the impact of inappropriate antimicrobial therapy on patient outcome. METHODS: A retrospective study on hospitalized patients with CR-Ab infections was carried out from January 2013 to December 2017. An epidemiologic analysis was carried out to determine the frequency of infections, the epidemiologic indicators by year, the risk factors for 30-day mortality, and the impact of inappropriate therapy. RESULTS: A total of 489 patients were included in the study. A rate of 0.7 per 1,000 patient-day CR-Ab infections was observed, mostly in the lungs (54.7%), and predominantly in the adult intensive care unit. The occurrence of infections by CR-Ab per 1,000 patient-days in November 2014 exceeded the established control limit, confirming an outbreak. CONCLUSIONS: The prevalence of CR-Ab increased in the investigated hospital, passing to an endemic pathogen with a direct impact on mortality and the control of these strains.

Molecular Detection of Class 1 Integron-Associated Gene Cassettes in KPC-2-Producing Klebsiella pneumoniae Clones by Whole-Genome Sequencing.

The dissemination of antimicrobial resistance genes and the bacterium that harbor them have increasingly become a public concern, especially in low- and middle-income countries. The present study used whole-genome sequencing to analyze 10 KPC-2-producing Klebsiella pneumoniae isolates obtained from clinical specimens originated from Brazilian hospitals. The study documents a relevant "snapshot" of the presence of class 1 integrons in 90% of the strains presenting different gene cassettes (dfrA30, dfrA15, dfrA12, dfrA14, aadA1, aadA2, and aac(6')Iq), associated or not with transposons. Two strains presented nonclassical integron (lacking the normal 3'conserved segment). In general, most strains showed a complex resistome, characterizing them as highly resistant. Integrons, a genetically stable and efficient system, confer to bacteria as highly adaptive and low cost evolution potential to bacteria, even more serious when associated with high-risk clones, indicating an urgent need for control and prevention strategies to avoid the spread of resistance determinants in Brazil. Despite this, although the class 1 integron identified in the KPC-2-producing K. pneumoniae clones is important, our findings suggest that other elements probably have a greater impact on the spread of antimicrobial resistance, since many of these important genes were not related to this cassette.

Molecular characterization and clonal dynamics of nosocomial blaOXA-23 producing XDR Acinetobacter baumannii.

The emergence of infections associated to new antimicrobial resistance in Acinetobacter baumannii (Ab) genotypes represents a major challenge. In this context, this study aimed to determine the diversity of resistance mechanisms and investigate clonal dissemination and predominant sequence types (STs) in multidrug-resistant Ab strains of clinical (tracheal aspirate, n = 17) and environmental (surface, n = 6) origins. Additionally, the major clones found in clinical (A) and environmental (H) strains had their complete genomes sequenced. All strains were submitted to polymerase chain reactions (PCR) for the detection of the ISAba1/blaOXA-51-like and ISAba1/blaOXA-23-like genes, while the expression of genes encoding the carO porin, AdeABC (adeB), AdeFGH (adeG), and AdeIJK (adeJ) efflux pumps was determined by real time PCR (qPCR). Most of the strains were characterized as extensively drug-resistant (XDR) with high minimal inhibitory concentrations (MICs) detected for tigecycline and carbapenems. Associations between ISAba1/OXA-51 and ISAba1/OXA-23 were observed in 91.3% and 52.2% of the strains, respectively. Only the adeB gene was considered hyper-expressed. Furthermore, most of the strains analyzed by the MuLtilocus Sequence-Typing (MLST) were found to belong to the clonal complex 113 (CC113). In addition, a new ST, ST1399, belonging to CC229, was also discovered herein. Strains analyzed by whole genome sequencing presented resistance genes linked to multidrug-resistance phenotypes and confirmed the presence of Tn2008, which provides high levels carbapenem-resistance.

Association of Colistin-Resistant KPC Clonal Strains with Subsequent Infections and Colonization and Biofilm Production.

Carbapenemase-producing organisms are pandemic and a significant threat to public health. We investigated the clonal relatedness of colistin-resistant Klebsiella pneumoniae strains producing KPC-type carbapenemase (KPC-KP) causing subsequent infections or colonization. Moreover, we aimed to gain insight into the ability of biofilm production in K. pneumoniae strains producing carbapenemase. Twenty-two consecutive KPC-KP and one KPC-negative strain was identified from an adult intensive care unit in Brazil. Seventy-five percent of isolates that harbored the blaKPC gene exhibited genetic relatedness by pulsed-field gel electrophoresis, and none presented the plasmid-mediated mcr-1 and blaNDM genes. This study showed that the majority of repeated KPC infections in adults were caused by a clone that caused the previous infections/colonizations even after a long period of time and illustrates the capacity of multiple clones producing biofilms to coexist in the same patient at the same time, becoming a reservoir of KPC-KP in the hospital environment.

Hypervirulence and biofilm production in KPC-2-producing Klebsiella pneumoniae CG258 isolated in Brazil.

In this study, we describe the frequency of virulence genes in Klebsiella pneumoniae carbapenemase-2-producing Klebsiella pneumoniae (KPC-KP), including hypervirulent (hv) and hypermucoviscous (hm) strains by whole-genome sequencing. We also evaluate the capacity for biofilm formation by using phenotypic techniques. The occurrence of several virulence genes (fimABCDEFGHIK, mrkABCDFHJ, ecpA, wabG, entB, ugE, irp1, irp2, traT, iutA and ureADE) and a high frequency of hvhmKPC-KP isolates was found. Most hospital-associated lineages of KPC-KP belong to the international clonal group 258 (CG258). Biofilm formation was a constant feature among 90.9 % of KPC-KP strains. This report suggests a close relationship between ST437 and weak biofilm production, given that all weakly biofilm-producing strains belonged to this sequence type. This also supports the dissemination of KPC-KP containing numerous virulence determinants belonging to the biofilm-producing CG258 type in Brazil, including hv and hm strains. These factors allow this pathogen to cause infections, leading to its rapid expansion and persistence in hospital settings.

High frequency of the combined presence of QRDR mutations and PMQR determinants in multidrug-resistant Klebsiella pneumoniae and Escherichia coli isolates from nosocomial and community-acquired infections.

Plasmid-mediated quinolone resistance (PMQR) determinants combined with mutations in quinolone resistance-determining regions (QRDRs) and clonal dissemination were investigated in 40 fluoroquinolone-resistant Klebsiella pneumoniae and Escherichia coli isolates from nosocomial and community-acquired infections. We observed nucleotide substitutions in gyrA (Ser83Ile, Val37Leu, Lys154Arg, Ser171Ala, Ser19Asn, Ile198Val, Ser83Tyr, Ser83Leu, Asp87Asn and Asp87Gly) and parC genes (Ser80Ile, Glu84Lys, Ala129Ser, Val141Ala and Glu84Gly). Two novel substitutions were detected in the gyrA gene (Val37Leu and Ile198Val). The presence of PMQR genes predominated in community isolates (55.5 %). In addition to the frequent presence of the class 1 integron in isolates from community-acquired infections, the genetic similarity results obtained by PFGE showed high genomic diversity. This study suggests that management of multidrug-resistant Enterobacteriaceae isolates from the community are a possible source of genetic mobile elements that carry genes that confer resistance to fluoroquinolones. More attention should be paid to the surveillance of community-acquired infections.

Molecular epidemiological survey of bacteremia by multidrug resistant Pseudomonas aeruginosa: the relevance of intrinsic resistance mechanisms.

The bacterial factors associated with bacteremia by multidrug-resistant and extensively drug-resistant P. aeruginosa, including overexpression of efflux pumps, AmpC overproduction, and loss/alteration of the OprD porin in isolates that are non-Metallo-ß-Lactamase producing were analyzed in a retrospective study. Molecular analyses included strain typing by Pulsed Field Gel Electrophoresis and identification of key genes via qualitative and quantitative PCR-based assays. Previous use of carbapenems and tracheostomy was independently associated with the development of bacteremia by extensively drug-resistant and multidrug-resistant strains of P. aeruginosa. A high consumption of antimicrobials was observed, and 75.0% of the isolates contained amplicons with the blaSPM-1 and blaVIM genes. Of the 47 non-Metallo-ß-Lactamase isolates, none had another type of carbapenemase. However, the isolates exhibited high rates of hyperproduction of AmpC, loss of the OprD porin (71.4%) and the presence of MexABOprM (57.1%) and MexXY (64.3%). This study suggests that in non-Metallo-ß-Lactamase isolates, the association of intrinsic resistance mechanisms could contributes to the expression of multidrug-resistant/extensively drug-resistant phenotypes.

Clinical and Molecular Epidemiology of Multidrug-Resistant P. aeruginosa Carrying aac(6')-Ib-cr, qnrS1 and blaSPM Genes in Brazil.

We described a comprehensive analysis of the molecular epidemiology of multidrug-resistant (MDR) P. aeruginosa. Molecular analysis included typing by Pulsed Field Gel Electrophoresis, identification of genes of interest through PCR-based assays and sequencing of target genes. Case-control study was conducted to better understand the prognostic of patients and the impact of inappropriate therapy in patients with bacteremia, as well as the risk factors of MDR infections. We observed a high rate of MDR isolates (40.7%), and 51.0% of them was independently associated with inappropriate antibiotic therapy. Bacteremia was detected in 66.9% of patients, and prolonged hospital stay was expressive in those resistant to fluoroquinolone. Plasmid-mediated quinolone resistance genes (PMQR), qnrS1 and aac(6')Ib-cr, were detected in two different nosocomial isolates (5.3%), and the aac(6')-Ib7 variant was detected at a high frequency (87.5%) in those negative to PMQR. The presence of mutations in gyrA and parC genes was observed in 100% and 85% of selected isolates, respectively. Isolates harboring PMQR genes or mutations in gyrA and parC were not closely related, except in those containing SPM (São Paulo metallo-ß-lactamase) clone. In addition, there is no study published in Brazil to date reporting the presence of Pseudomonas aeruginosa isolates harboring both qnrS1 and aac(6')Ib-cr genes, with alarming frequency of patients with inappropriate therapy.

Multidrug Resistance Related to Biofilm Formation in Acinetobacter baumannii and Klebsiella pneumoniae Clinical Strains from Different Pulsotypes.

The emergence of Acinetobacter baumannii and Klebsiella pneumoniae strains in the hospital environment has been associated with the presence of multiple genetic elements, virulence factors and the ability to form biofilms. This study evaluated the biofilm formation ability of clinical and environmental A. baumannii and K. pneumoniae strains, isolated from various sources and presenting different molecular characteristics, resistance profiles and pulsed-field gel electrophoresis patterns. Fifty-three isolates were recovered from 2009 to 2014 in a Brazilian university hospital. Investigation of biofilm formation was performed for 10 strains of each species assessed by an initial adhesion assay, biofilm cell concentration and biofilm biomass, evaluated by quantitative assays in replicates, in three independent experiments. All strains of A. baumannii were able to attach to polystyrene plates, although two strains adhered to a lesser degree than the control. K. pneumoniae strains showed opposite behaviour, where only three strains adhered significantly when compared to the control. Quantitative evaluation revealed that in five A. baumannii and four K. pneumoniae isolates the biomass production could be characterised as moderate. None of the isolates were strong biofilm producers. Our results demonstrate: (1) biofilm formation is a heterogeneous property amongst A. baumannii and K. pneumoniae clinical strains and it was not associated with certain clonal types; (2) no relationship between multidrug resistance and biofilm production was observed; (3) more virulent K. pneumoniae strains tended to present higher production of biofilm.

Biofilm formation of Brazilian meticillin-resistant Staphylococcus aureus strains: prevalence of biofilm determinants and clonal profiles.

Biofilms plays an important role in medical-device-related infections. This study aimed to determine the factors that influence adherence and biofilm production, as well as the relationship between strong biofilm production and genetic determinants in clinical isolates of meticillin-resistant Staphylococcus aureus (MRSA). Fifteen strains carrying different chromosomal cassettes recovered from hospitalized patients were selected; five SCCmecII, five SCCmecIII and five SCCmecIV. The SCCmec type, agr group and the presence of the virulence genes (bbp, clfA, icaA, icaD, fnbB, bap, sasC and IS256) were assessed by PCR. PFGE and multilocus sequence typing (MLST) techniques were also performed. The initial adhesion and biofilm formation were examined by quantitative assays. The surface tension and hydrophobicity of the strains were measured by the contact angle technique to evaluate the association between these parameters and adhesion ability. SCCmecIII and IV strains were less hydrophilic, with a high value for the electron acceptor parameter and higher adhesion in comparison with SCCmecII strains. Only SCCmecIII strains could be characterized as strong biofilm producers. The PFGE showed five major pulsotypes (A-E); however, biofilm production was related to the dissemination of one specific PFGE clone (C) belonging to MLST ST239 (Brazilian epidemic clonal complex). The genes agrI, fnbB and IS256 in SCCmecIII strains were considered as genetic determinants associated with strong biofilm-formation by an ica-independent biofilm pathway. This study contributes to the understanding of biofilm production as an aggravating factor potentially involved in the persistence and severity of infections caused by multidrug-resistant MRSA belonging to this genotype.

Spread of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clones in patients with ventilator-associated pneumonia in an adult intensive care unit at a university hospital

Background:In Brazil, ventilator-associated pneumonia (VAP) caused by carbapenem resis- tant Acinetobacter baumanniiand Pseudomonas aeruginosaisolates are associated with significant mortality, morbidity and costs. Studies on the clonal relatedness of these isolates could lay the foundation for effective infection prevention and control programs.Objectives: We sought to study the epidemiological and molecular characteristics of A. baumannii vs. P. aeruginosaVAP in an adult intensive care unit (ICU).Methods: It was conducted a cohort study of patients with VAP caused by carbapenem resistant A. baumanniiand P'. aeruginosaduring 14 months in an adult ICU. Genomic studies were used to investigate the clonal relatedness of carbapenem resistant OXA-23-producing A. baumanniiand P. aeruginosaclinical isolates. The risk factors for acquisition of VAP were also evaluated. Clinical isolates were collected for analysis as were samples from the environment and were typed using pulsed field gel electrophoresis.Results: Multivariate logistic regression analysis identified trauma diagnosed at admission and inappropriate antimicrobial therapy as independent variables associated with the development of A. baumanniiVAP and hemodialysis as independent variable associated with P. aeruginosaVAP. All carbapenem resistant clinical and environmental isolates of A. baumanniiwere OXA-23 producers. No MBL-producer P. aeruginosawas detected. Molecular typing revealed a polyclonal pattern; however, clone A (clinical) and H (surface) were the most frequent among isolates of A. baumanniitested, with a greater pattern of resistance than other isolates. In P. aeruginosathe most frequent clone I was multi-sensitive.Conclusion: These findings suggest the requirement of constant monitoring of these microor- ganisms in order to control the spread of these clones in the hospital environment.

Spread of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa clones in patients with ventilator-associated pneumonia in an adult intensive care unit at a university hospital.

BACKGROUND: In Brazil, ventilator-associated pneumonia (VAP) caused by carbapenem resistant Acinetobacter baumannii and Pseudomonas aeruginosa isolates are associated with significant mortality, morbidity and costs. Studies on the clonal relatedness of these isolates could lay the foundation for effective infection prevention and control programs. OBJECTIVES: We sought to study the epidemiological and molecular characteristics of A. baumannii vs. P. aeruginosa VAP in an adult intensive care unit (ICU). METHODS: It was conducted a cohort study of patients with VAP caused by carbapenem resistant A. baumannii and P. aeruginosa during 14 months in an adult ICU. Genomic studies were used to investigate the clonal relatedness of carbapenem resistant OXA-23-producing A. baumannii and P. aeruginosa clinical isolates. The risk factors for acquisition of VAP were also evaluated. Clinical isolates were collected for analysis as were samples from the environment and were typed using pulsed field gel electrophoresis. RESULTS: Multivariate logistic regression analysis identified trauma diagnosed at admission and inappropriate antimicrobial therapy as independent variables associated with the development of A. baumannii VAP and hemodialysis as independent variable associated with P. aeruginosa VAP. All carbapenem resistant clinical and environmental isolates of A. baumannii were OXA-23 producers. No MBL-producer P. aeruginosa was detected. Molecular typing revealed a polyclonal pattern; however, clone A (clinical) and H (surface) were the most frequent among isolates of A. baumannii tested, with a greater pattern of resistance than other isolates. In P. aeruginosa the most frequent clone I was multi-sensitive. CONCLUSION: These findings suggest the requirement of constant monitoring of these microorganisms in order to control the spread of these clones in the hospital environment.