Antibiotics stimulates the development of persistent cells in biofilms of Candida albicans bloodstream isolates.

Candida albicans invasive candidiasis is considered a global health problem. In such cases, biofilm formation on implanted devices represents a therapeutic challenge and the presence of metabolically inactive persistent cells (PCs) in these communities increases their tolerance to fungicidal drugs. This study investigated the influence of amoxicillin, AMX; cefepime, CEF; gentamicin, GEN; amikacin, AMK; vancomycin, VAN; and ciprofloxacin, CIP; on the production of PCs in biofilms of C. albicans bloodstream isolates. 48 h-mature biofilms (n = 6) grown in RPMI-1640 supplemented with antibiotics were treated with 100 µg ml-1 amphotericin B and then evaluated for PCs. Biofilms grown in the presence of antibiotics produced more PCs, up to 10×, when exposed to AMX and CIP; 5 × to CEF; and 6 × to GEN and VAN. The results indicate that antibiotics can modulate PC production in C. albicans biofilms. This scenario may have clinical repercussions in immunocompromised patients under broad-spectrum antibiotic therapy.

Biofilms are microbial communities tolerant to antifungals. Our research showed that antibiotics stimulate the formation of persistent cells within Candida albicans biofilms. These are dormant, metabolically silent cells that resist to therapy and can be related to metastatic and recalcitrant infections.

An approach to combat multidrug-resistant K. pneumoniae strain using synergistic effects of Ocotea diospyrifolia essential oil in combination with amikacin.

The natural antimicrobial properties of essential oils (EOs) have contributed to the battle against multidrug-resistant microorganisms by providing new ways to develop more effective antibiotic agents. In this study, we investigated the chemical composition of Ocotea diospyrifolia essential oil (OdOE) and its antimicrobial properties combined with amikacin (AMK). Through gas chromatography-mass spectrometry (GCMS) analysis, the primary constituents of OdOE were identified as α-bisabolol (45.8 %), ß-bisabolene (9.4 %), γ-elemene (7.6 %), (Z)- ß-farnesene (5.2 %), spathulenol (3.5 %), (Z)-caryophyllene (3.3 %), and (E)-caryophyllene (3.1 %). In vitro assessments showed that the combined administration of OdOE and AMK exerted a synergistic antibacterial effect on the multidrug-resistant K. pneumoniae strain. This synergistic effect demonstrated bacteriostatic action. OdEO combined with amikacin showed protein extravasation within 2 h of treatment, leading to bacterial death, which was determined by a reduction in viable cell count. The effective concentrations showed hemocompatibility. In vivo assessments using Caenorhabditis elegans as a model showed the survival of 85 % of infected nematodes. Therefore, the combination OdEO combined with amikacin exhibited antimicrobial activity against a multidrug-resistant K. pneumoniae strain. Thus, OdOE is a promising agent that may be considered for development of antimicrobial treatment.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

In vitro evaluation of human intravenous immunoglobulin in combination with antimicrobials and human serum against multidrug-resistant isolates of Acinetobacter baumannii.

The high incidence of multidrug-resistant (MDR) Acinetobacter baumannii has been a challenge for health worldwide, due to the reduction of therapeutic options, making the use of antimicrobial combinations necessary for the treatment, such as meropenem, amikacin, and colistin. Antibodies against bacterial species, mainly immunoglobulins G (IgG), are produced for acting as effector mechanisms (neutralization, opsonization, phagocytosis, and complement system activation). Some studies have demonstrated promising results of IgG in combination with antimicrobial preparations against bacterial infections, in which the direct action of IgG has restored the immune system balance. Serious problem caused by the increase of MDR A. baumannii isolates results in a constant search for therapeutic alternatives to defeat these infections. However, this study aims to verify in vitro the phagocytosis rate of the A. baumannii-infected human monocytes, as well as to analyze possible morphological changes induced by intravenous immunoglobulin G (IVIG) with human serum in association with antimicrobials. The phagocytosis rate and bacterial cell binding capacity of IVIG were determined for two A. baumannii isolates submitted to 4 mg/mL of human IVIG alone and in combination with different sub-minimum inhibitory concentrations (sub-MICs) of meropenem, amikacin, and colistin and processed for indirect immunofluorescence. Subsequently, these isolates were resubmitted and coupled with human serum and processed for scanning electron microscopy. There was no statistical difference for phagocytosis rates in the isolates tested. Bacterial isolates showed alterations in cell morphology when exposed to IVIG/human serum alone and in combination with antimicrobials such as alteration in shape, wrinkling, membrane depression, and especially cell rupture with extravasation of cytoplasmic material. The isolates visually differed in the IVIG binding to the bacterial cell, with higher fluorescence intensity, which corresponds to the highest IVIG binding, in the isolate more sensitive to meropenem, amikacin, and colistin. No differences between treatments were observed in the IVIG binding to the bacterial cell. The combined action of IVIG with meropenem, amikacin, and colistin against A. baumannii MDR isolates induced several bacterial cell damages. And when associated with human serum, a massive destruction of cells can be observed. These results may suggest the analysis of the use of IgG preparations for the treatment of A. baumannii MDR infections.

Synergy between amikacin and Protium heptaphyllum essential oil against polymyxin resistance Klebsiella pneumoniae.

AIMS: We investigated the chemical composition and the in vitro and in vivo antibacterial effects of Protium heptaphyllum essential oil (PHEO) alone and in combination with antibiotics against polymyxin-resistant Klebsiella pneumoniae isolates. METHODS AND RESULTS: Hydrodistillation was used to obtain PHEO, and gas chromatography coupled with mass spectrometry revealed α-pinene, δ-3-carene, and ß-pinene as major components present in PHEO. Minimum inhibitory concentration was determined using the broth microdilution technique and ranged from 256 to 512 µg ml-1. The checkerboard method showed synergy with the combination of PHEO and amikacin (AMK) against the polymyxin-resistant K. pneumoniae isolates. In 8 of the 10 isolates tested, the fractional inhibitory concentration indexes (FICIs) ranged from 0.06 to 0.5, while in the remaining two isolates, the combination exerted an additive effect (FICI of 0.6 and 1.0), resulting in AMK dose reduce of range 2- to 16-fold, in the presence of PHEO. Analysis using zero interaction potency revealed high synergy score (63.9). In the in vivo assay, the survival of Caenorhabditis elegans was significantly improved in the presence of PHEO (1 µg ml-1) + AMK (µg ml-1) combination as compared to 32 µg ml-1 AMK alone. Furthermore, PHEO concentrations of 256 and 512 µg ml-1 were found to be non-toxic on the experimental model. CONCLUSION: To our knowledge, this is the first report of such type of synergism demonstrating an antimicrobial effect against polymyxin-resistant K. pneumoniae isolates.

In vitro activity of ceftazidime-avibactam against Gram-negative strains in Chile 2015-2021.

OBJECTIVES: Ceftazidime-avibactam (CAZ-AVI) combines ceftazidime and a reversible ß-lactamase inhibitor that has shown activity against multidrug-resistant (MDR) Enterobacterales and P. aeruginosa. Using data from the Antimicrobial Testing Leadership and Surveillance program (ATLAS), this study examined the in vitro antimicrobial activity of CAZ-AVI and other antibiotics against Gram-negative bacteria collected from Chilean hospitals between 2015 and 2021. METHODS: Clinical isolates of Enterobacterales and P. aeruginosa were collected from three medical centres in Chile. Blood, abdominal fluid, urine, soft tissues, and respiratory tract samples were obtained from infected patients. Minimum inhibitory concentrations using the broth microdilution method were determined for susceptibility testing, and the Clinical and Laboratory Standards Institute (CLSI) breakpoints were used for interpreting the results. Extended-spectrum ß-lactamases (ESBL) and carbapenemase genes were also detected through polymerase chain reaction. RESULTS: A total of 2600 Enterobacterales and 836 P. aeruginosa were analysed. CAZ-AVI was the antibiotic with the highest in vitro activity against Enterobacterales (99.72%). The incidence of carbapenem-resistant Enterobacterales (CRE) was 1.5% (n = 39), and the antibiotics with the best in vitro activity were tigecycline (92.31%), CAZ-AVI (88.57%), and amikacin (79.49%). CAZ-AVI was the antibiotic with the best activity against ESBL-producing Enterobacterales (99.34%) and MDR Enterobacterales (99.31%). For KPC-producing Enterobacterales, susceptibility to amikacin was 100%, whereas susceptibility to CAZ-AVI was 91.67%. Regarding MDR and difficult-to-treat resistance P. aeruginosa, 44.83% and 38.99% were susceptible to CAZ-AVI, respectively. CONCLUSION: CAZ-AVI shows excellent in vitro activity against Enterobacterales in general, CRE, ESBL-producing Enterobacterales, and KPC-producing Enterobacterales. CAZ-AVI is also an option against MDR P. aeruginosa.

In vitro activity of imipenem/relebactam against non-Morganellaceae Enterobacterales and Pseudomonas aeruginosa in Latin America: SMART 2018‒2020.

Carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa are being isolated from patient specimens with increasing frequency in Latin America and worldwide. The current study provides an initial description of the in vitro activity of imipenem/relebactam (IMR) against non-Morganellaceae Enterobacterales (NME) and P. aeruginosa infecting hospitalized patients in Latin America. From 2018 to 2020, 37 clinical laboratories in nine Latin American countries participated in the SMART global surveillance program and contributed 15,466 NME and 3408 P aeruginosa isolates. MICs for IMR and seven comparators were determined using CLSI broth microdilution and interpreted by CLSI M100 (2022) breakpoints. ß-lactamase genes were identified in selected isolate subsets. IMR (96.9% susceptible), amikacin (95.9%), meropenem (90.7%), and imipenem (88.7%) were the most active agents against NME. Among piperacillin/tazobactam-nonsusceptible NME (n = 4124), 90.4% of isolates were IMR-susceptible (range by country, 97.2 [Chile] to 67.0% [Guatemala]) and among meropenem-nonsusceptible NME isolates (n = 1433), 74.0% were IMR-susceptible (94.1% [Puerto Rico] to 5.1% [Guatemala]). Overall, 6.3% of all collected NME isolates carried a KPC (metallo-ß-lactamase [MBL]-negative), 1.8% an MBL, 0.4% an OXA-48-like carbapenemase (MBL-negative), and 0.1% a GES carbapenemase (MBL-negative). Amikacin (85.2% susceptible) and IMR (80.1%) were the most active agents against P. aeruginosa; only 56.5% of isolates were imipenem-susceptible. Relebactam increased susceptibility to imipenem by 22.0% (from 23.9% to 45.9%) in piperacillin/tazobactam-nonsusceptible isolates (n = 1031) and by 35.5% (from 5.5% to 41.0%) in meropenem-nonsusceptible isolates (n = 1128). Overall, 7.6% of all collected P. aeruginosa isolates were MBL-positive and 0.7% carried a GES carbapenemase. In conclusion, in 2018‒2020, almost all NME (97%) and most P. aeruginosa (80%) isolates from Latin America were IMR-susceptible. Continued surveillance of the in vitro activities of IMR and comparator agents against Gram-negative pathogens, and monitoring for ß-lactamase changes (in particular for increases in MBLs), is warranted.

Antibacterial activity of silver nanoparticles functionalized with amikacin applied against multidrug-resistant acinetobacter baumannii.

BACKGROUND: Multidrug-resistant bacteria are one of the world's biggest health problems; therefore, improving the spectrum of action of antibiotics could be necessary to reverse this situation. Amikacin and silver salts have well-known antimicrobial properties. However, both drugs lost their effectiveness against some bacteria, such as Acinetobacter baumannii. This work aims to develop a nanodrug from silver nanoparticles (AgNPs) functionalized with Amikacin against multidrug-resistant Acinetobacter baumannii. METHODS: AgNPs were produced using the bottom-up methodology and functionalized with Amikacin modified by the carbodiimide-based chemistry, forming AgNPs@Amikacin. Susceptibility tests were performed using Amikacin-resistant Acinetobacter baumannii strains to assess the bacteriostatic and bactericidal potential of the developed nanodrug. The clinical strains were induced to form a biofilm, and biomass quantification and the metabolic activity were determined. RESULTS: The AgNPs have a hydrodynamic diameter of the particles with a bimodal distribution, with a size of 37.84 nm. The FT-IR spectrum of AgNPs@Amikacin exhibits vibrational modes corresponding to Amikacin, confirming the conjugation to AgNPs. Susceptibility testing demonstrated a minimal inhibitory and bactericidal concentration of < 0.5 µg/mL. The AgNPs@Amikacin reduced the biofilm metabolic activity of Acinetobacter baumannii at rates ≥ 50%, characterized by the minimal biofilm inhibition concentrations. CONCLUSIONS: Results demonstrate a promising development of a new nanodrug with lower concentrations, less toxicity, and greater efficacy against multidrug-resistant Acinetobacter baumannii.

High Rates of Extensively Drug-Resistant Pseudomonas aeruginosa in Children with Cystic Fibrosis.

Pseudomonas aeruginosa has a high adaptive capacity, favoring the selection of antibiotic-resistant strains, which are currently considered a global health problem. The purpose of this work was to investigate the rate and distribution of extensively drug-resistant (XDR) P. aeruginosa in pediatric patients with cystic fibrosis (CF) with recurrent infections and to distinguish the current efficacy of antibiotics commonly used in eradication therapy at a Mexican institute focused on children. A total of 118 P. aeruginosa isolates from 25 children with CF (2015-2019) underwent molecular identification, antimicrobial sensitivity tests, and Random Amplified Polymorphic DNA genotyping (RAPD-PCR). The bacterial isolates were grouped in 84 RAPD profiles, revealing a cross-infection between two sisters, whose resistance profile remained unchanged for more than 2 years. Furthermore, 77.1% (91/118) and 51.7% (61/118) of isolates showed in vitro susceptibility to ceftazidime and amikacin, respectively, antibiotics often used in eradication therapy at our institution. As well, 42.4% (50/118) were categorized as multi-drug resistant (MDR) and 12.7% (15/118) were XDR. Of these resistant isolates, 84.6% (55/65) were identified from patients with recurrent infections. The high frequency of XDR strains in children with CF should be considered a caution mark, as such resistance patterns are more commonly found in adult patients. Additionally, amikacin may soon prove ineffective. Careful use of available antibiotics is crucial before therapeutic possibilities are reduced and "antibiotic resistance crisis" worsens.

Use of bedaquiline in spinal osteomyelitis and soft tissue abscess caused by multidrug-resistant Mycobacterium tuberculosis: A case report.

INTRODUCTION: Spinal Tuberculosis (STB) represents between 1% and 2% of total tuberculosis cases. STB management remains challenging; the first-line approach consists of medical treatment, while surgery is reserved for patients with complications. No data regarding STB treatment with bedaquiline-containing regimens are available in the literature. CASE DESCRIPTION: Herein, we report the case of a 21-year-old man from Côte d'Ivoire with a multidrug resistance STB with subcutaneous abscess. After approval of the hospital off-label drug committee, we started bedaquiline 400 mg daily for two weeks, followed by 200 mg three times per week, for 22 weeks, associated with linezolid 600 mg daily, rifabutin 450 mg daily, and amikacin 750 mg daily (interrupted after eight weeks). During treatment, we performed a weekly EKG. No QT prolongation was shown, but inverted T waves appeared, requiring several cardiological consultations and cardiac MRI, but no cardiac dysfunction was found. After 24 weeks, bedaquiline was replaced with moxifloxacin 400 mg daily. The patient continued treatment for another year. We performed another computer tomography at the end of treatment, confirming the cure. DISCUSSION: A salvage regimen containing bedaquiline proved effective in treating multidrug-resistance tuberculosis spinal infection without causing severe adverse effects. However, further studies are needed to evaluate better bedaquiline bone penetration and the correct duration of treatment with bedaquiline in MDR spinal tuberculosis.

Potentiation of the Activity of Antibiotics against ATCC and MDR Bacterial Strains with (+)-α-Pinene and (-)-Borneol.

The increasing rates of antimicrobial resistance have demanded the development of new drugs as conventional antibiotics have become significantly less effective. Evidence has identified a variety of phytocompounds with the potential to be used in the combat of infections caused by multidrug-resistant (MDR) bacteria. Considering the verification that terpenes are promising antibacterial compounds, the present research aimed to evaluate the antibacterial and antibiotic-modulating activity of (+)-α-pinene and (-)-borneol against MDR bacterial strains. The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of the compounds and antibiotics and further evaluate the intrinsic and associated antibiotic activity. These analyses revealed that (+)-α-pinene showed significant antibacterial activity only against E. coli (MIC = 512 µg.mL-1), while no significant inhibition of S. aureus and P. aeruginosa growth was observed (MIC ≥ 1024 µg mL-1). However, when combined with antibiotics, this compound induced a significant improvement in the activity of conventional antibiotics, as observed for ciprofloxacin, amikacin, and gentamicin against Staphylococcus aureus, as well as for amikacin and gentamicin against Escherichia coli, and amikacin against Pseudomonas aeruginosa. On the other hand, (-)-borneol was found to inhibit the growth of E. coli and enhance the antibiotic activity of ciprofloxacin and gentamicin against S. aureus. The present findings indicate that (+)-α-pinene and (-)-borneol are phytocompounds with the potential to be used in the combat of antibacterial resistance.

Amikacin prevents platelet aggregation in feline venous blood samples.

INTRODUCTION: Physiologically, feline platelets are more reactive and prone to aggregation, which interferes with platelet counts using automated counters and manual methods. The use of aminoglycoside amikacin in association with EDTA has proven to be efficient in preventing platelet aggregates in cases of pseudo thrombocytopenia (PTP) in people. OBJECTIVES: This study evaluated the efficacy of amikacin in preventing platelet aggregation in EDTA-containing feline blood samples and investigated the possible effects on hematologic measurands. MATERIALS AND METHODS: Blood samples (1.0 mL) collected from 100 healthy cats were stored in two EDTA tubes: 0.5 mL in a microtube containing 10 µL of 250 mg/mL amikacin (EDTA-AMK group) and 0.5 mL in a microtube containing only K2 EDTA 10% (EDTA group). A CBC was executed with an automated impedance blood analyzer, and a microscopic examination of the blood smears was performed. RESULTS: Platelet clumps were observed in 56% of samples from the EDTA group and 5% of samples from the EDTA-AMK group. Platelet counts (PLT), plateletcrit (PCT), and WBC counts were significantly higher (P < .001) in the EDTA-AMK group compared withi the EDTA group. CONCLUSIONS: Amikacin prevents platelet aggregation in feline venous blood samples and does not cause clinically relevant changes in other hematologic measurands. To our knowledge, this is the first report showing the use of amikacin in preventing platelet aggregation in feline blood samples. Based on this study, amikacin could be added to EDTA collection tubes for complete blood counts in cats.

Nickel (II) chloride schiff base complex: Synthesis, characterization, toxicity, antibacterial and leishmanicidal activity.

The use of schiff base complex against microbial agentes a has recently received more attention as a strategy to combat infections caused by multidrug-resistant bacteria and leishmania. This study aimed to evaluate the toxicity, antibacterial and leishmanicidal activities of the nickel (II) chloride schiff base complex ([Ni(L2)] against Leishmania amazonensis promastigote, multi-resistant bacterial strains and evaluate to modulate antibiotic activity against multi-resistant bacterial. The schiff base complex was characterized by the techniques of elemental analysis, Fourier transform infrared spectroscopy (FTIR), UV-vis absorption spectroscopy and thermal analysis (TGA/DTG/DSC). The [Ni(L2)] complex presented moderate toxicity in saline artemia (LC50 = 150.8 µg/mL). In leishmanicidal assay, the NiL2 complex showed values of IC50 of (6.079 µg/mL ± 0.05656 at the 24 h), (0.854 µg/mL ± 0.02474, 48 h) and (1.076 µg/mL ± 0.04039, 72 h). In antibacterial assay, the [Ni(L2)] complex presented significant inhibited the bacterial growth of P. aeruginosa (MIC = 256 µg/mL). However, [Ni(L2)] complex did not present clinically relevant minimum inhibitory concentration (MIC ≥1024 µg/mL) against S. aureus and E. coli. The combination of [Ni(L2)] complex and antibacterial drugs resulted in the increased antibiotic activity of gentamicin and amikacin against S. aureus and E.coli multi-resistant strains. Thus, our results showed that [Ni(L2)] complex is a promising molecule for the development of new therapies associated with aminoglycoside antibiotics and in disease control related to resistant bacteria and leishmaniasis.

Amikacin for the treatment of carbapenem-resistant Klebsiella pneumoniae infections: clinical efficacy and toxicity.

Infections by carbapenem-resistant Klebsiella pneumoniae (CRKp) are an increasing global threat with limited therapeutic options. Our objective was to evaluate clinical and microbiological outcomes of patients treated with amikacin for CRKp infections. We did a retrospective cohort of patients > 18 years old, with CRKp infections treated with amikacin in two tertiary care hospitals in Porto Alegre, Brazil. The impact of clinical factors, antibiotic treatment, and amikacin minimum inhibitory concentration (MIC) on patients' 30-day mortality was assessed. Microbiological clearance and nephrotoxicity (assessed by RIFLE score) were evaluated as secondary outcomes. A Cox regression analysis was done for mortality. We included 84 patients for analysis. Twenty-nine (34.5%) patients died in 30 days. Amikacin MIC values ranged from 0.125 to 8 µg/mL and did not influence on mortality, regardless of the prescribed dose of this antibiotic (P = 0.24). Bacterial clearance occurred in 17 (58.6%) of 29 patients who collected subsequent cultures. Two (16.6%) of the 12 persistently positive cultures changed the amikacin susceptibility profile from susceptible to intermediate. Twenty-nine (37.2%) patients developed acute kidney injury (AKI): risk 13, injury 11, and failure 5. Risk factors for AKI were higher baseline eGFR (P < 0.01) and combination therapy with colistin (P = 0.02). Comparing patients who received combination with colistin vs polymyxin B, AKI occurred in 60.0% vs 20.6%, respectively, P < 0.01. Fifteen of the 16 (16.6%) patients who developed renal injury or failure were receiving colistin. In conclusion, amikacin was an effective treatment for CRKp infections. Within susceptible range, amikacin MIC values did not influence on clinical outcomes. Combination therapy of amikacin and colistin was highly nephrotoxic and should be used with caution.

Resistência aos antimicrobianos e diversidade genética de variantes fenotípicas de Pseudomonas aeruginosa não sensíveis aos carbapenêmicos recuperadas de pacientes com fibrose cística com infecção pulmonar crônica: uma análise temporal / Antimicrobial resistance and genetic diversity of phenotypic variants of carbapenem non susceptible Pseudomonas aeruginosa recovered from patients with cystic fibrosis with chronic lung infection: a temporal analysis

Pseudomonas aeruginosa, bactéria ubíqua e versátil, pode se comportar como um patógeno oportunista, com ampla capacidade adaptativa, por múltiplos fatores de virulência e resistência. Como agente patogênico nas infecções pulmonares em pacientes com fibrose cística (FC), é motivo de prognóstico ruim, aumento de hospitalizações e altas taxas de morbimortalidade, sendo quase impossível a sua erradicação, ao evoluírem para a cronicidade. Globalmente, é notável o aumento nos índices de amostras não sensíveis aos carbapenêmicos e a múltiplos antimicrobianos, essenciais à terapêutica. Assim, avaliamos temporalmente a susceptibilidade aos antimicrobianos e a presença de amostras hipermutáveis (HPM) em P. aeruginosa de diferentes morfotipos, não sensíveis aos carbapenêmicos (PANSC), obtidas de pacientes FC com infecção pulmonar crônica, acompanhados em dois centros de referência no Rio de Janeiro. De 2007 a 2016, a análise retrospectiva, através dos resultados obtidos no teste de disco-difusão (TDD), permitiu selecionar amostras de PANSC incluídas neste trabalho. Usando os resultados obtidos no TDD, foi definida a susceptibilidade a outros antimicrobianos, bem como os fenótipos de resistência, multi-(MDR), extensivo-(XDR) e pandroga resistentes (PDR). Adicionalmente, determinou-se a concentração inibitória mínima (CIM) para imipenem (IPM), meropenem (MEM), doripenem (DOR) e polimixina (POL). Através de teste fenotípico, foi calculada a frequência de mutação espontânea e as amostras hipermutáveis foram caracterizadas. O sequenciamento de genoma total (SGT) foi realizado em seis amostras de diferentes morfotipos, incluindo uma variante fenotípica rara, a small colony variant (SCV). Essas amostras foram recuperadas em dois episódios de exacerbação do paciente. Foram investigadas a clonalidade, resistência a antimicrobianos e virulência. Das 143 amostras, de 18 pacientes (9 pediátricos e 9 adultos), os resultados do TDD apontaram taxas de não susceptibilidade superiores a 44% para gentamicina, amicacina, tobramicina e ciprofloxacina, e maiores de 30 % para POL. Pela determinação da CIM, quase a totalidade (96%) das amostras foram não sensíveis a IMP, seguidos de 56% para MEM e 44% para DOR. Analisando-se a distribuição dos valores da CIM50 e CIM90 nos dois grupos de pacientes, os valores para IMP foram maiores entre as amostras dos pacientes pediátricos, equivalendo a 32 µg/mL e 64 µg/mL, respectivamente. Cerca de 25%, 37% e 6% eram MDR, XDR e PDR, respectivamente. Aproximadamente 12% eram HPM, e mais da metade destas foram XDR. Após o SGT, as seis amostras, recuperadas do caso clínico foram classificadas em um novo sequence type (ST2744), com a presença de genes de resistência adquiridos blaPAO, blaOXA-50, aph(3')-Iib, fosA, catB7 e crpP, apresentando mutações em genes codificadores de porinas e bombas de efluxo. Entretanto, não foram observados marcadores genéticos clássicos exclusivos para os fenótipos SCV e HPM. Este é o primeiro relato de P. aeruginosa SCV na FC, no Brasil. A vigilância epidemiológica de P. aeruginosa é crucial para a conduta terapêutica, bem como para o sucesso da resposta do paciente e erradicação da infecção pulmonar, justificando o uso de técnicas fenotípicas e moleculares na detecção dos mecanismos de resistência e virulência desse microrganismo na FC.

Pseudomonas aeruginosa, a ubiquitous and versatile bacterium, can behave as an opportunistic pathogen, with strong adaptive capacity, due to multiple virulence and resistance factors. As a pulmonary infection pathogen in patients with cystic fibrosis (CF), it is related with poor prognosis, increased hospitalizations and high rates of morbidity and mortality, and the eradication is almost impossible, especially after chronicity. The increase rates of isolates non-susceptible to carbapenem and multiple antimicrobials, essentials to therapy, have been observed worldwide. Therefore, we assessed the antimicrobial susceptibility and the presence of hypermutability (HPM) in non-susceptible to carbapenem P. aeruginosa (PANSC) isolates from different morphotypes, obtained from CF patients with chronic pulmonary infection, followed at two reference centers in Rio de Janeiro. Using the results obtained by disk-diffusion test (DDT) between 2007 to 2016, we select 143 PANSC and susceptibility to other antimicrobials was defined, as well as the resistance phenotypes, multi- (MDR), extensive- (XDR) and pandrug resistant (PDR). Additionally, the minimum inhibitory concentration (MIC) for imipenem (IPM), meropenem (MEM), doripenem (DOR) and polymyxin (POL) was determined. Hypermutable isolates were characterized by determination of mutation frequency. Whole genome sequencing (WGS) was performed in six morphotypes isolates, including the small colony variant (SCV), a rare variant phenotype. These isolates were recovered in two exacerbation episodes. Clonality, antimicrobial resistance and virulence were investigated. Of the total (143 isolates) isolated from 18 patients (9 pediatric and 9 adults), non-susceptibility rates above than 44% for gentamicin, amikacin, tobramycin and ciprofloxacin, and more than 30% for POL were observed. Almost all (96%) of the isolates were non-susceptible to IPM by MIC determination, followed by 56% for MEM and 44% for DOR. MIC50 (32 µg/mL) and MIC90 (64 µg/mL) rates for IPM were higher among pediatric patient isolates and 25%, 37% and 6% were MDR, XDR and PDR, respectively. 12% of all isolates were classified as HPM and more than half were categorized as XDR. Using WGS, the six isolates recovered from the clinical case, were identified as a new sequence type (ST2744). Acquired resistance genes blaPAO, blaOXA-50, aph (3')-Iib, fosA, catB7 and crpP and mutations in encoding genes for porins and efflux pumps, was annotated. None exclusive classic genetic markers related to SCV and HPM phenotypes were not observed. This is the first Brazilian report of P. aeruginosa SCV in CF. Our results highlight the importance of epidemiological surveillance in P. aeruginosa. The application of phenotypic and molecular techniques to investigate resistance and virulence mechanisms, can contribute to therapeutic success in CF.

Effect of the Association and Evaluation of the Induction to Adaptation of the (+)-α-pinene with Commercial Antimicrobials against Strains of Escherichia coli.

BACKGROUND: The increasing and inappropriate use of antibiotics has increased the number of multidrug-resistant microorganisms to these drugs, causing the emergence of infections that are difficult to control and manage by health professionals. As an alternative to combat these pathogens, some monoterpenes have harmful effects on the bacterial cell membrane, showing themselves as an alternative in combating microorganisms. Therefore, the positive enantiomer α -pinene becomes an alternative to fight bacteria, since it was able to inhibit the growth of the species Escherichia coli ATCC 25922, demonstrating the possibility of its use as an isolated antimicrobial or associated with other drugs. AIMS: The aim of this study is to evaluate the sensitivity profile of E. coli ATCC 25922 strain against clinical antimicrobials associated with (+) -α-pinene and how it behaves after successive exposures to subinhibitory concentrations of the phytochemicals. METHODS: The minimum inhibitory concentration (MIC) was determined using the microdilution method. The study of the modulating effect of (+) -α-pinene on the activity of antibiotics for clinical use in strains of E. coli and the analysis of the strain's adaptation to the monoterpene were tested using the adapted disk-diffusion method. RESULTS: The results demonstrate that the association of monoterpene with the antimicrobials ceftazidime, amoxicillin, cefepime, cefoxitin and amikacin is positive since it leads to the potentiation of the antibiotic effect of these compounds. It was observed that the monoterpene was able to induce crossresistance only for antimicrobials: cefuroxime, ceftazidime, cefepime and chloramphenicol. CONCLUSION: It is necessary to obtain more concrete data for the safe use of these combinations, paying attention to the existence of some type of existing toxicity reaction related to the herbal medicine and to understand the resistance mechanisms acquired by the microorganism.

Comparison of methods for the detection of in vitro synergy in multidrug-resistant gram-negative bacteria.

BACKGROUND: The use of combined antibiotic therapy has become an option for infections caused by multidrug-resistant (MDR) bacteria. The time-kill (TK) assay is considered the gold standard method for the evaluation of in vitro synergy, but it is a time-consuming and expensive method. The purpose of this study was to evaluate two methods for testing in vitro antimicrobial combinations: the disk diffusion method through disk approximation (DA) and the agar gradient diffusion method via the MIC:MIC ratio. The TK assay was included as the gold standard. MDR Gram-negative clinical isolates (n = 62; 28 Pseudomonas aeruginosa, 20 Acinetobacter baumannii, and 14 Serratia marcescens) were submitted to TK, DA, and MIC:MIC ratio synergy methods. RESULTS: Overall, the agreement between the DA and TK assays ranged from 20 to 93%. The isolates of A. baumannii showed variable results of synergism according to TK, and the calculated agreement was statistically significant in this species against fosfomycin with meropenem including colistin-resistant isolates. The MIC:MIC ratiometric agreed from 35 to 71% with TK assays. The kappa test showed good agreement for the combination of colistin with amikacin (K = 0.58; P = 0.04) among the colistin-resistant A. baumannii isolates. CONCLUSIONS: The DA and MIC:MIC ratiometric methods are easier to perform and might be a more viable tool for clinical microbiology laboratories.

In vitro activity of polymyxin B in combination with meropenem, amikacin and gentamicin against Klebsiella pneumoniae clinical isolates co-harbouring aminoglycoside-modifying enzymes, blaNDM-1 and blaKPC-2.

OBJECTIVES: Multidrug-resistant Klebsiella pneumoniae carrying blaNDM-1 and blaKPC-2 genes are a worldwide concern for which combination antimicrobial therapy may be the only viable option. The aim of this study was to investigate the in vitro activity of combinations of polymyxin B (PMB) with meropenem (MEM), amikacin (AMK) and gentamicin (GEN) at subinhibitory concentrations against two K. pneumoniae clinical isolates co-harbouring blaNDM-1, blaKPC-2 and aminoglycoside-modifying enzymes and resistant to PMB. METHODS: Synergy and bactericidal activity were evaluated by chequerboard and time-kill assays against two PMB-resistantK. pneumoniae clinical isolates carrying the blaNDM-1, blaKPC-2, aac(3)-IIa, aac(6')-Ib, aph(3')-VI and ant(2'')-Ia genes. Five combinations of PMB, MEM, AMK and GEN were evaluated. RESULTS: The PMB/MEM and PMB/AMK combinations proved to be the best options against isolate K7R2, mainly because they demonstrated bactericidal activity when using subinhibitory concentrations of these antimicrobials. However, none of the studied combinations was bactericidal against isolate K11R2. CONCLUSION: The combinations used in this study showed synergy against NDM-and KPC-producing isolates but, given their bactericidal activity, the combinations of PMB/MEM and PMB/AMK were the most active against one isolate. It can also be concluded that the antimicrobials to which the bacteria were resistant could form part of combination therapy.

Clonal Dissemination of Clinical Isolates of Acinetobacter baumannii Carriers of 16S rRNA Methylase Genes in an Oncological Hospital in Recife, Brazil.

16S rRNA methylases confer high-level resistance to aminoglycosides which are used to treat serious infections caused by gram-negative bacteria, such as Acinetobacter spp. Some genes encoding these enzymes are disseminated worldwide, while others were detected in only some countries. The objective was to characterize the susceptibility profile to aminoglycosides (amikacin and gentamicin) of clinical isolates of Acinetobacter spp. from an oncological hospital in Recife, and given the resistance to both antimicrobials, to characterize minimal inhibitory concentrations (MICs) of amikacin, gentamicin and tobramycin, the occurrence of 16S rRNA methylase genes (armA, rmtB, rmtC and rmtD) and of ß-lactamase gene (blaKPC) and the clonal profile. Isolates resistant to both antimicrobials, amikacin and gentamicin, were selected by disk diffusion technique in Mueller-Hinton agar and identified. Broth microdilution was conducted to determine MICs of amikacin, gentamicin, and tobramycin. These isolates were subjected to polymerase chain reaction and pulsed-field gel electrophoresis. Among 23 analyzed isolates, 12 (52.2%) were resistant to gentamicin and amikacin and identified as Acinetobacter baumannii. Among these, 11 (91.7%), 12 (100%), and 9 (75%) isolates showed respectively MICs > 256 µg/mL of amikacin, > 64 µg/mL of gentamicin, and > 64 µg/mL of tobramycin. The armA gene was found in 12 (100%) isolates and 6 (50%) showed coexistence of armA, rmtB, and rmtC genes. The rmtD and blaKPC genes were not detected. These isolates showed high genetic similarity (92%) and were classified as clone A. Elaboration and fulfillment of measures are thus essential to prevent the spread of this resistance mechanism.