Pharmacodynamic Attainment of the Synergism of Meropenem and Fosfomycin Combination against Pseudomonas aeruginosa Producing Metallo-ß-Lactamase.

Fosfomycin combined with other antimicrobials has shown good efficacy against multidrug-resistant (MDR) bacteria in both in vitro and clinical studies; however, the activity of fosfomycin combined with other antimicrobials against metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa strains has not been tested. The objective of this study was to determine the synergism and optimal intravenous dosing regimens of fosfomycin with meropenem against MDR and MBL-producing P. aeruginosa strains. The MICs of both antimicrobials were determined by the checkerboard method and analyzed by two synergism tests with 19 clones of P. aeruginosa isolates, 10 of which were MBL producers. A pharmacodynamic (PD) analysis was performed for meropenem (administered at 1 g every 8 h [q8h], 1.5 g every 6 h [q6h], and 2 g q8h) and fosfomycin (administered at 4 g q8h, 4 g q6h, 6 g q8h, and 8 g q8h) regimens with a dose reduction for renal impairment by determining the probability of target attainment (PTA) for target PD indices of meropenem (the percentage of the time in a 24-h duration at which the free drug concentration remains above the MIC [fT>MIC], ≥40%) and fosfomycin (the ratio of the area under the free drug concentration-versus-time curve over 24 h and the MIC [fAUC/MIC], ≥40.8). The combination reduced the MIC50 and MIC90 by 8-fold. Seven (44%) isolates with MICs in the intermediate or resistant ranges became sensitive to meropenem. For the MBL-producing isolates, the combination resulted in 40% of isolates becoming sensitive to meropenem. The meropenem regimens reached a PTA of ≥90% (MIC = 4 µg/ml) in 6 (32%) isolates when they were used as monotherapy and 13 (68%) isolates when they were combined with fosfomycin. None of the fosfomycin monotherapy regimens reached the PTA of ≥90% (MIC = 16 µg/ml). When combined with meropenem, the fosfomycin regimens reached the PTA of ≥90% in 14 (74%) isolates. The increase in pharmacodynamic activities resulting from the synergistic action of meropenem with fosfomycin demonstrates the potential relevance of this combination to fight infections caused by MDR and MBL-producing P. aeruginosa strains.

Development of a melting-curve based multiplex real-time PCR assay for simultaneous detection of Streptococcus agalactiae and genes encoding resistance to macrolides and lincosamides.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) remains the leading cause of infections in newborns worldwilde. Prenatal GBS screening of pregnant women for vaginal-rectal colonization is recommended in many countries to manage appropriate intrapartum antimicrobial prophylaxis for those identified as carriers. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of GBS and macrolide and lincosamide resistance markers was developed. The usefulness of the assay was evaluated for rapid and accurate prenatal GBS screening. METHODS: One hundred two pregnant women who were at 35-37 weeks of gestation were enrolled in this study. The analytical performance of the multiplex real-time PCR was first tested using a panel of reference and clinical bacterial and fungal strains. To test the clinical performance, vaginal-rectal swabs were obtained from pregnant women who were seen at the teaching hospital for regular prenatal care. The results of real-time were compared with those obtained from microbiological analyses. RESULTS: The real-time PCR assay showed 100% specificity and a limit of detection of 104 colony forming units equivalent per reaction. The prevalence of GBS colonization among the population studied was 15.7% (16/102) based on a positive culture and the real-time PCR results. Agreement between the two assays was found for 11 (68.75%) GBS colonized women. Using the culture-based results as a reference, the multiplex real-time PCR had a sensitivity of 91.7% (11/12, CI 59.7-99.6%), a specificity of 95.5% (86/90, CI 89.8-98.7%), a positive predictive value of 73.3% (11/15, CI 44.8-91.1%) and a negative predictive value of 98.9% (86/87, CI 92.9-99.9%). CONCLUSION: The multiplex real-time PCR is a rapid, affordable and sensitive assay for direct detection of GBS in vaginal-rectal swabs.

Pharmacodynamic Evaluation of Fosfomycin against Escherichia coli and Klebsiella spp. from Urinary Tract Infections and the Influence of pH on Fosfomycin Activities.

Fosfomycin is widely used for the treatment of uncomplicated urinary tract infection (UTI), and it has recently been recommended that fosfomycin be used to treat infections caused by multidrug-resistant (MDR) Gram-negative bacilli. Whether urine acidification can improve bacterial susceptibility to fosfomycin oral dosing regimens has not been analyzed. The MIC of fosfomycin for 245 Gram-negative bacterial isolates, consisting of 158 Escherichia coli isolates and 87 Klebsiella isolates which were collected from patients with urinary tract infections, were determined at pH 6.0 and 7.0 using the agar dilution method. Monte Carlo simulation of the urinary fosfomycin area under the concentration-time curve (AUC) after a single oral dose of 3,000 mg fosfomycin and the MIC distribution were used to determine the probability of target attainment (PTA). Fosfomycin was effective against E. coli (MIC90 ≤ 16 µg/ml) but not against Klebsiella spp. (MIC90 > 512 µg/ml). Acidification of the environment increased the susceptibility of 71% of the bacterial isolates and resulted in a statistically significant decrease in bacterial survival. The use of a regimen consisting of a single oral dose of fosfomycin against an E. coli isolate with an MIC of ≤64 mg/liter was able to achieve a PTA of ≥90% for a target pharmacodynamic index (AUC/MIC) of 23 in urine; PTA was not achieved when the MIC was higher than 64 mg/liter. The cumulative fractions of the bacterial responses (CFR) were 99% and 55% against E. coli and Klebsiella spp., respectively, based on simulated drug exposure in urine with an acidic pH of 6.0. A decrease of the pH from 7.0 to 6.0 improved the PTA and CFR of the target pharmacodynamic index in both E. coli and Klebsiella isolates.

Pharmacodynamic Evaluation of the Potential Clinical Utility of Fosfomycin and Meropenem in Combination Therapy against KPC-2-Producing Klebsiella pneumoniae.

KPC-producing Klebsiella pneumoniae causes serious infections associated with high death rates worldwide. Combination therapy consisting of fosfomycin and a carbapenem is better than monotherapy to combat multidrug-resistant microorganisms, but no dosages for the combination have been defined. The MICs of meropenem and fosfomycin were evaluated against 18 clinical isolates of KPC-2-producing K. pneumoniae The activities of combination antimicrobials were also determined by the checkerboard method. The MIC50 and MIC90 of each agent alone and in combination were challenged against short (1.5-h) or prolonged (3-h) infusion regimens of meropenem (1 g every 8 h [q8h], 1.5 g q6h, 2 g q8h) and fosfomycin (4 g q8h, 6 g q6h, 8 g q8h) by Monte Carlo simulation to evaluate the time above the MIC of the free drug concentration as a percentage of the dosing interval (fT>MIC). The monotherapy MIC50s and MIC90s were 32 and 256 mg/liter for meropenem and 64 and 512 mg/liter for fosfomycin, respectively. Antimicrobial combination increased bacterial susceptibility to 1/4 the MIC50s and to 1/8 to 1/16 the MIC90s of monotherapy. The antimicrobial combination demonstrated a synergistic effect for at least two-thirds of the isolates. In combination therapy, fosfomycin regimens of 6 g q6h and 8 g q8h as a 3-h infusion against the MIC50 and MIC90 had better chances of achieving ≥90% probability of target attainment (PTA) of 70% fT>MIC. Meropenem regimens of 1.5 g q6h and 2 g q8h in prolonged infusion can achieve close to 90% PTA of 40% fT>MIC for MIC50 but not MIC90 The significant reduction in the MIC values and the achievement of appropriate PTA demonstrated that regimens containing fosfomycin with meropenem can be effective against KPC-2-producing K. pneumoniae.

Whole Genome Sequencing of an Extensively Drug-Resistant Raoultella planticola Isolate Containing blaKPC-2, blaNDM-1, and blaCTX-M-15.

Raoultella planticola harboring genes that confer resistance to antimicrobials, such as carbapenems, have been associated with severe infections in immunocompromised patients. In this study, we reported the first whole genome sequence of a Brazilian isolate of R. planticola and the genomic context of antibiotic resistance markers. By whole-genome sequencing (WGS) of a carbapenem-resistant R. planticola isolate, RpHUM1, we found 23 resistance-encoding genes belonging to 9 classes of antibiotics (aminoglycosides, ß-lactams, fluoroquinolones, fosfomycin, macrolides, phenicols, sulfonamides, tetracycline, and diaminopyrimidine derivatives) and 3 plasmids (RpHUM1pEaer-4382s, RpHUM1_pFDAARGOS_440, and RpHUM1pRSF1010). This isolate coharbored the genes blaKPC-2, which is carried by the plasmid RpHUM1pEaer-4382s, and blaNDM-1 and blaCTX-M-15 all located in the accessory genome. In addition, these genes were associated with, at least, one mobile genetic element. This comprehensive knowledge is of great importance for implementation of control measures to prevent the rapid dissemination of this neglected microorganism and their genetic resistance background.

Development of a Melting-Curve-Based Multiplex Real-Time PCR Assay for the Simultaneous Detection of Viruses Causing Respiratory Infection.

The prompt and accurate identification of the etiological agents of viral respiratory infections is a critical measure in mitigating outbreaks. In this study, we developed and clinically evaluated a novel melting-curve-based multiplex real-time PCR (M-m-qPCR) assay targeting the RNA-dependent RNA polymerase (RdRp) and nucleocapsid phosphoprotein N of SARS-CoV-2, the Matrix protein 2 of the Influenza A virus, the RdRp domain of the L protein from the Human Respiratory Syncytial Virus, and the polyprotein from Rhinovirus B genes. The analytical performance of the M-m-qPCR underwent assessment using in silico analysis and a panel of reference and clinical strains, encompassing viral, bacterial, and fungal pathogens, exhibiting 100% specificity. Moreover, the assay showed a detection limit of 10 copies per reaction for all targeted pathogens using the positive controls. To validate its applicability, the assay was further tested in simulated nasal fluid spiked with the viruses mentioned above, followed by validation on nasopharyngeal swabs collected from 811 individuals. Among them, 13.4% (109/811) tested positive for SARS-CoV-2, and 1.1% (9/811) tested positive for Influenza A. Notably, these results showed 100% concordance with those obtained using a commercial kit. Therefore, the M-m-qPCR exhibits great potential for the routine screening of these respiratory viral pathogens.

Metallo-ß-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread.

The aim of this study was to characterize two metallo-ß-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Prevalence of HPV infection and genotypes in women with normal cervical cytology in the state of Paraná, Brazil.

OBJECTIVE: We investigated the prevalence of the human Papillomavirus (HPV) and its genotypes in women with normal cervical cytology in the state of Paraná, Brazil, and also epidemiological characteristics. METHODS: The enrolled patients were seen at six primary health-care units in Paiçandú City, Paraná. Through polymerase chain reaction (PCR) and PCR-RFLP (restriction fragment length polymorphism) techniques, 40 HPV genotypes were found, including 15 high risk, 3 undetermined risk and 22 low risk. Socio-demographic characteristics and sexual behavior were also recorded by interviews based on a structured questionnaire completed at the time of enrollment. RESULTS: Among 418 patients examined, HPV was detected in 6.7 %, mainly in women aged <25 years. The overall prevalence of high-risk, low-risk and undetermined-risk HPV types was 42.9, 45.7, and 11.7 %, respectively. HPV-16 was the most common type detected (14.3 %), followed by types 66 (11.4 %) and 31 and 70 (8.6 % each). Detection of HPV DNA was positively associated with the number of sexual partners within the previous 12 months (p < 0.031; OR = 5.4; CI = 0.98-29.8). CONCLUSION: When considering the lack of studies in Paraná on women with normal cytology, the results of this study will improve estimates of HPV DNA populations, and provide baseline values against which the impact of vaccination can be assessed in the future.

Carriage frequency, phenotypic and genotypic characteristics of Staphylococcus aureus isolated from dialysis and kidney tranplant patients at a hosptial in northern paraná.

The objective of the present study was to determine the frequency of Staphylococcus aureus nasal carriage among dialysis and kidney transplant patients, to identify the antimicrobial resistance profile of these strains and to verify their genetic profiles with the RW3A primer. The study included 159 individuals, comprising 111 dialysis and 48 kidney transplant patients. Of the 48 transplant patients, 75% were positive for S. aureus, whereas 49% of the 111 dialysis patients were carriers. Two samples yielded conflicting results for oxacillin sensitivity between the disk diffusion and minimum inhibitory concentration (MIC) assays: both were sensitive by the disk diffusion assay and resistant by MIC (4 µg/ml). In the antibiogram by disk diffusion, ten samples were resistant to cefoxitin, among which eight were also resistant to oxacillin. The resistance of the ten samples to cefoxitin by the disk diffusion assay was confirmed by MIC. Of the ten oxacillin-resistant samples, eight harbored the mecA gene. All samples were sensitive to vancomycin, and most were resistant to penicillin and demonstrated high rates of resistance to the other antimicrobials tested. The samples from dialysis patients exhibited a more homogenous genetic profile. Among the samples with a high percent similarity, no correlation with sensitivity or resistance to oxacillin was observed. According to the results of this study, the implementation of prevention and control measures, such as increased restrictions on prescriptions for antimicrobial drugs and nasal decontamination prior to high-risk procedures, is recommended.

Evaluation of phenotypic methods for detection of polymyxin B-resistant bacteria.

Determination of sensitivity to polymyxins has always been a challenge, especially in clinical laboratory routines. This study evaluated two rapid, simple, and inexpensive phenotypic methods to test polymyxin B (PMB) susceptibility in Enterobacterales and non-fermenting Gram-negative bacilli. One hundred isolates were used in the tests. The isolates were collected in three hospitals in southern and southeastern Brazil from 1995 to 2019. We compared broth microdilution (reference method) with the broth disk elution test and modified drop test, using polymyxin B -disk or PMB -powder in 2 concentrations (12 and 16 µg/ml). For the broth disk elution and modified drop test with the concentration of 12 µg/ml, categorical agreement values exceeded 90%. The modified drop test with a concentration of 12 µg/ml and broth disk elution may be excellent for initial screening of polymyxin-resistance in laboratory routines. Moreover, these methods are simple and use inexpensive supplies, and may optimize therapeutic decisions.

Genomic insights of Acinetobacter baumannii ST374 reveal wide and increasing resistome and virulome.

WGS-based surveillance has significantly improved the ability to track global spread and emergence of multidrug-resistant clones of clinically relevant pathogens. In this study, we performed the genomic characterization and comparative analysis of an Acinetobacter baumannii (strain Ac56) belonging to the sequence type ST374, which was isolated for the first time in Brazil, in 1996. Genomic analysis of Ac56 predicted a total of 5373 genes, with 3012 being identical across nine genomes of A. baumannii isolates of ST374 from European, Asian, North and South American countries. GoeBURST analysis grouped ST374 lineages into clonal complex CC3 (international clone IC-III). Resistome analysis of ST374 clone predicted genes associated with resistance to heavy metals and clinically relevant beta-lactams and aminoglycosides antibiotics. In this regard, in two closely related A. baumannii strains, the intrinsic blaADC gene was linked to the insertion sequence ISAba1; including the Ac56 strain, where it has been possibly associated with intermediate susceptibility to meropenem. Other four carbapenem-resistant A. baumannii strains carried the ISAba1/blaOXA-23 gene array, which was associated with the transposon Tn2008 or with Tn2006 in an AbaR4-type resistance island. While most virulence genes were shared for A. baumannii strains of ST374, three isolates from Thailand harbored KL49 capsular loci, previously identified in the hypervirulent A. baumannii LAC-4 strain. Analysis of thirty-four predicted plasmids showed eight major groups, of which GR-6 (LN-1) and GR-2 (LN-2) were prevalent. All strains, including the earliest isolate Ac56 harbored at least one complete prophage, whereas none CRISPR-associated (cas) gene was detected. In summary, genomic data of A. baumannii ST374 reveal a potential of this lineage to become a successful clone.

Polymyxin B Activity Rescue by (-)-Camphene-Based Thiosemicarbazide Against Carbapenem-Resistant Enterobacterales.

Due to the significant shortage of therapeutic options for carbapenem-resistant Enterobacterales (CRE) infections, new drugs or therapeutic combinations are urgently required. We show in this study that (-)-camphene-based thiosemicarbazide (TSC) may act synergistically with polymyxin B (PMB) against CRE, rescuing the activity of this antimicrobial. With the specific aim of a better molecular understanding of this effect caused by the presence of TSC, theoretical calculations were also performed in this study. Based on these findings, it is concluded that the presence of TSC moieties contributes to significant changes in the hydrogen atom charge of PMB structure, which trend more positives for the PMB/TSC system studied. This could lead to the formation of stronger hydrogen bonds in the Enterobacterales active site and, thus contribute to a molecular understanding of the PMB rescue of activity promoted by the presence of TSC moiety. As such, the clinical potential of these drug combinations requires further evaluation.

Mutant prevention concentration (MPC) of ciprofloxacin against Salmonella enterica of epidemic and poultry origin.

Salmonella isolates resistant or with reduced susceptibility to quinolones increased in recent years. The mutant prevention concentration (MPC) is a new alternative that can prevent the selection and multiplication of resistant Salmonella spp. strains. The MPC of ciprofloxacin (CipMPC) was evaluated for 312 Salmonella enterica strains of epidemic and poultry origin susceptible and resistant to nalidixic acid (NAL). The CipMPC for NAL-susceptible strains were in the range from 0.002 to 4 µg/ml and for NAL-resistant strains, it ranged from 0.004 to 16 µg/ml. The average MPC/MIC ratio for NAL-resistant strains was higher than NAL susceptible. S. Enteritidis showed the highest CipMPC and the highest MPC/MIC ratio also for NAL-resistant strains and with mutations in gyrA. Serovar Corvallis, a NAL-resistant strain without mutations, and of poultry origin showed the highest CipMPC value. The lowest value was observed for epidemic NAL-susceptible strains serovars Typhimurium and London. The average MPC/MIC ratio for strains with mutations in Aspartate 87 was higher than that mutated in Serine 83. The results show the importance of MPC in determining the correct dosage of Cip for treatment of Salmonella spp.

Detection of quinolone-resistance mutations in salmonella spp. Strains of epidemic and poultry origin.

Mutations into codons Aspartate-87 (62%) and Serine-83 (38%) in QRDR of gyrA were identified in 105 Salmonella strains resistant to nalidixic acid (94 epidemic and 11 of poultry origin). The results show a high incidence of mutations associated to quinolone resistance but suggest association with others mechanisms of resistance.

Pharmacodynamic evaluation of suppression of in vitro resistance in Acinetobacter baumannii strains using polymyxin B-based combination therapy.

The emergence of polymyxin resistance in Gram-negative bacteria infections has motivated the use of combination therapy. This study determined the mutant selection window (MSW) of polymyxin B alone and in combination with meropenem and fosfomycin against A. baumannii strains belonging to clonal lineages I and III. To evaluate the inhibition of in vitro drug resistance, we investigate the MSW-derived pharmacodynamic indices associated with resistance to polymyxin B administrated regimens as monotherapy and combination therapy, such as the percentage of each dosage interval that free plasma concentration was within the MSW (%TMSW) and the percentage of each dosage interval that free plasma concentration exceeded the mutant prevention concentration (%T>MPC). The MSW of polymyxin B varied between 1 and 16 µg/mL for polymyxin B-susceptible strains. The triple combination of polymyxin B with meropenem and fosfomycin inhibited the polymyxin B-resistant subpopulation in meropenem-resistant isolates and polymyxin B plus meropenem as a double combination sufficiently inhibited meropenem-intermediate, and susceptible strains. T>MPC 90% was reached for polymyxin B in these combinations, while %TMSW was 0 against all strains. TMSW for meropenem and fosfomycin were also reduced. Effective antimicrobial combinations significantly reduced MSW. The MSW-derived pharmacodynamic indices can be used for the selection of effective combination regimen to combat the polymyxin B-resistant strain.

First report of oxacillin-susceptible mecA-positive Staphylococcus aureus in healthy dogs and their owners in southern Brazil.

Oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) isolates have been described worldwide, but data regarding dogs and their owners have not been reported. This study investigated the occurrence of OS-MRSA and MRSA isolates in the nasal mucosa of 241 healthy dogs and 208 owners in the community. S. aureus isolates were characterized by susceptibility testing, detection of the mecA and the Panton-Valentine leukocidin (PVL) genes, staphylococcal chromosome cassette (SCC)mec typing and rep-PCR-RW3A. We report an unprecedented detection of nasal carriage of OS-MRSA in 5.3 % (2/38) of healthy dogs and 1.75 % (1/57) of their owners. We also found MRSA in 2.6 % (1/38) of the dogs and 3.5 % (2/57) of the owners. Only the human isolate was SCCmec IV and PVL-positive. Molecular typing revealed that the same cluster of S. aureus was present in owners and dogs from the same or different families attended at the same veterinary clinic. The three OS-MRSA isolates did not show genetic similarity to each other. Detection of OS-MRSA in this context alerts us to the role of dogs and owners as possible silent reservoirs of this microorganism in the community, which may potentially be misidentified as methicillin-sensitive S. aureus (MSSA) in the laboratory routine, representing an additional threat in antimicrobial therapy for staphylococcal infections.

Consecutive outbreaks of Burkholderia cepacia complex caused by intrinsically contaminated chlorhexidine mouthwashes.

BACKGROUND: We report 2 consecutive outbreaks of the Burkholderia cepacia complex (Bcc) in an intensive care unit (ICU) and describe its characteristics and consequences. METHODS: Over a 72-day period, a multidisciplinary ICU team detected 2 distinct periods of high and unusual incidence of Bcc isolates that were recovered from cultures of endotracheal aspirate. Cultures of tap water, ultrasound gel and mouthwash (opened and unopened bottles) were performed. Bcc was identified with the BD-Phoenix and MALDI-TOF MS systems, with molecular typing using the enterobacterial repetitive intergenic consensus-polymerase chain reaction technique. RESULTS: In both outbreak 1 (6 patients) and outbreak 2 (5 patients), the point sources of Bcc were chlorhexidine mouthwashes of 2 different brands, both of them intrinsically contaminated. All patients had a clinical diagnosis of ventilator-associated pneumonia (VAP), and 6 died. MALDI-TOF MS identified 2 species of Bcc (B. cenocepacia and B. cepacia). Enterobacterial repetitive intergenic consensus-polymerase chain reaction typing confirmed 100% genetic similarity between patient and mouthwash isolates from each period. The first outbreak was controlled in 20 days and the second in 6 days. CONCLUSIONS: The surveillance program for multidrug-resistant organisms, especially in high-risk patients, with the active participation of a multidisciplinary team, was crucial for success in controlling these outbreaks.

Synergistic activity of polymyxin B combined with vancomycin against carbapenem-resistant and polymyxin-resistant Acinetobacter baumannii: first in vitro study.

PURPOSE: The effect of a combination of polymyxin B (PMB) and vancomycin (VAN) was assessed against six Acinetobacter baumannii clinical isolates belonging to six different clusters (three PMB-susceptible and three PMB-resistant). METHODOLOGY: The synergistic effect of the PMB-VAN combination was determined with the checkerboard, time-kill, disk-diffusion and M.I.C.Evaluator assays. PMB-resistance was investigated with mcr-1 gene amplification and a mutant frequency assay. RESULTS: In the checkerboard assay, all PMB-resistant isolates showed a synergistic effect. The time-kill assay demonstrated that the PMB-VAN combination had a bactericidal effect at 24 h against isolates with a high mutant rate for PMB, suggesting that this combination may block the hypermutation of some isolates. No antagonism was detected. All PMB-resistant isolates also showed synergism in the disk-diffusion test, and a significant decrease in VAN MICs in the M.I.C.Evaluator assay. CONCLUSION: Our findings indicate that the PMB-VAN combination has a synergistic effect on A. baumannii, especially against PMB-resistant isolates.

Multidrug- and Extensively Drug-Resistant Acinetobacter baumannii in a Tertiary Hospital from Brazil: The Importance of Carbapenemase Encoding Genes and Epidemic Clonal Complexes in a 10-Year Study.

This study aimed to characterize the main mechanisms of acquired antimicrobial resistance of 103 multidrug-resistant Acinetobacter baumannii isolated from bloodstream from 2006 to 2016 from a hospital in Londrina, Brazil. All 103 isolates were identified as A. baumannii by amplification of the blaOXA-51-like and rpoB genes. Mortality was observed in the majority (81.6%) of the patients. High non-susceptibility rates (100.0-10.7%) were obtained for the evaluated antimicrobials, including colistin, polymyxin B, and tigecycline, and most isolates were classified as extensively drug-resistant (78.6%). Carbapenemase production was observed in 92.2% of the isolates. All carbapenem-resistant isolates showed a carbapenem-hydrolyzing class D ß-lactamase being either blaOXA-23-like (97.9%) or blaOXA-143-like (2.1%). None of the isolates had the genes blaOXA-24-like, blaOXA-58-like, blaOXA-48, blaKPC, blaNDM, blaSPM-1, blaSIM-1, blaVIM, blaIMP, blaGIM, blaGES, mcr-1, qnrA, qnrB, qnrC, qnrS, and qnrVc. As a genetic context of the blaOXA-23-like gene, Tn2006 was predominated (86.0%), and Tn2008 was less frequent (12.9%). Isolates harboring the blaOXA-143-like gene showed the blaOXA-253-like variant. A polyclonal profile was observed among the A. baumannii isolates. The presence of the international clonal complexes CC113/79, CC109/1, CC110/25, and CC103/15 was detected, with prevalence of CC113/79 (38.8%). This study provides essential information to understand the antimicrobial resistance patterns of A. baumannii and can be used to strengthen infection control measures in our hospital. Also, the study reinforces the urgent need to develop stewardship programs to avoid the spread and potential outbreaks by this pathogen.

Pharmacodynamic Effects of Sulbactam/Meropenem/Polymyxin-B Combination Against Extremely Drug Resistant Acinetobacter baumannii Using Checkerboard Information.

Aim: The aims of the study are to evaluate the activity of sulbactam, meropenem, and polymyxin B alone and in combination against six isolates of extremely drug resistant Acinetobacter baumannii and to determine dosing regimens that achieve a sufficient joint probability of target attainment (PTA) based on combination antimicrobial pharmacodynamics. Materials and Methods: The combinations were evaluated by the checkerboard method and were considered synergistic when the fractional inhibitory concentration index (FICI) ≤0.5. Pharmacodynamic analyses were carried out by evaluating dosing regimens that achieve ≥90% joint PTA at the percentage of time over a 24-h period wherein the free drug concentration is above the minimum inhibitory concentration (%fT> MIC) of 40% and 60% for meropenem and sulbactam, respectively, and 20 for the ratio of the area under the free drug concentration-time curve over MIC (fAUC/MIC) for polymyxin B. Results: For both polymyxin B-resistant and susceptible isolates, the addition of sulbactam in combination with meropenem and subinhibitory concentration of polymyxin B showed important synergistic activity (five isolates; FICI ≤0.281); the recommended dosing regimens were 2/4 g meropenem/sulbactam q8 hours and 0.5 mg/kg polymyxin B q12 hours. Conclusion: This in vitro study showed that sulbactam can significantly improve the action of meropenem and polymyxin B in OXA-producing A. baumannii isolates, especially when there are no new treatment options available for infections caused by these microorganisms.