A Nanopore Sequencing-based Pharmacogenomic Panel to Personalize Tuberculosis Drug Dosing.

Rationale: Standardized dosing of antitubercular drugs leads to variable plasma drug levels, which are associated with adverse drug reactions, delayed treatment response, and relapse. Mutations in genes affecting drug metabolism explain considerable interindividual pharmacokinetic variability; however, pharmacogenomic assays that predict metabolism of antitubercular drugs have been lacking. Objectives: We sought to develop a Nanopore sequencing panel and validate its performance in patients with active tuberculosis (TB) to personalize treatment dosing. Methods: We developed a Nanopore sequencing panel targeting 15 SNPs in five genes affecting the metabolism of antitubercular drugs. For validation, we sequenced DNA samples (n = 48) from the 1,000 Genomes Project and compared the variant calling accuracy with that of Illumina genome sequencing. We then sequenced DNA samples from patients with active TB (n = 100) from South Africa on a MinION Mk1C and evaluated the relationship between genotypes and pharmacokinetic parameters for isoniazid (INH) and rifampin (RIF). Measurements and Main Results: The pharmacogenomic panel achieved 100% concordance with Illumina sequencing in variant identification for the samples from the 1,000 Genomes Project. In the clinical cohort, coverage was more than 100× for 1,498 of 1,500 (99.8%) amplicons across the 100 samples. Thirty-three percent, 47%, and 20% of participants were identified as slow, intermediate, and rapid INH acetylators, respectively. INH clearance was 2.2 times higher among intermediate acetylators and 3.8 times higher among rapid acetylators, compared with slow acetylators (P < 0.0001). RIF clearance was 17.3% (2.50-29.9) lower in individuals with homozygous AADAC rs1803155 G→A substitutions (P = 0.0015). Conclusions: Targeted sequencing can enable the detection of polymorphisms that influence TB drug metabolism on a low-cost, portable instrument to personalize dosing for TB treatment or prevention.

Detection of Salmonella Typhi bacteriophages in surface waters as a scalable approach to environmental surveillance.

BACKGROUND: Environmental surveillance, using detection of Salmonella Typhi DNA, has emerged as a potentially useful tool to identify typhoid-endemic settings; however, it is relatively costly and requires molecular diagnostic capacity. We sought to determine whether S. Typhi bacteriophages are abundant in water sources in a typhoid-endemic setting, using low-cost assays. METHODOLOGY: We collected drinking and surface water samples from urban, peri-urban and rural areas in 4 regions of Nepal. We performed a double agar overlay with S. Typhi to assess the presence of bacteriophages. We isolated and tested phages against multiple strains to assess their host range. We performed whole genome sequencing of isolated phages, and generated phylogenies using conserved genes. FINDINGS: S. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river and 6.3% (1/16) drinking water samples from the Kathmandu Valley and Kavrepalanchok. Water samples collected within or downstream of population-dense areas were more likely to be positive (72.6%, 193/266) than those collected upstream from population centers (5.3%, 5/95) (p=0.005). In urban Biratnagar and rural Dolakha, where typhoid incidence is low, only 6.7% (1/15, Biratnagar) and 0% (0/16, Dolakha) river water samples contained phages. All S. Typhi phages were unable to infect other Salmonella and non-Salmonella strains, nor a Vi-knockout S. Typhi strain. Representative strains from S. Typhi lineages were variably susceptible to the isolated phages. Phylogenetic analysis showed that S. Typhi phages belonged to the class Caudoviricetes and clustered in three distinct groups. CONCLUSIONS: S. Typhi bacteriophages were highly abundant in surface waters of typhoid-endemic communities but rarely detected in low typhoid burden communities. Bacteriophages recovered were specific for S. Typhi and required Vi polysaccharide for infection. Screening small volumes of water with simple, low-cost (~$2) plaque assays enables detection of S. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance.

A Nanopore sequencing-based pharmacogenomic panel to personalize tuberculosis drug dosing.

Rationale: Standardized dosing of anti-tubercular (TB) drugs leads to variable plasma drug levels, which are associated with adverse drug reactions, delayed treatment response, and relapse. Mutations in genes affecting drug metabolism explain considerable interindividual pharmacokinetic variability; however, pharmacogenomic (PGx) assays that predict metabolism of anti-TB drugs have been lacking. Objectives: To develop a Nanopore sequencing panel and validate its performance in active TB patients to personalize treatment dosing. Measurements and Main Results: We developed a Nanopore sequencing panel targeting 15 single nucleotide polymorphisms (SNP) in 5 genes affecting the metabolism of isoniazid (INH), rifampin (RIF), linezolid and bedaquiline. For validation, we sequenced DNA samples (n=48) from the 1000 genomes project and compared variant calling accuracy with Illumina genome sequencing. We then sequenced DNA samples from patients with active TB (n=100) from South Africa on a MinION Mk1C and evaluated the relationship between genotypes and pharmacokinetic parameters for INH and RIF. Results: The PGx panel achieved 100% concordance with Illumina sequencing in variant identification for the samples from the 1000 Genomes Project. In the clinical cohort, coverage was >100x for 1498/1500 (99.8%) amplicons across the 100 samples. One third (33%) of participants were identified as slow, 47% were intermediate and 20% were rapid isoniazid acetylators. Isoniazid clearance was significantly impacted by acetylator status (p<0.0001) with median (IQR) clearances of 11.2 L/h (9.3-13.4), 27.2 L/h (22.0-31.7), and 45.1 L/h (34.1-51.1) in slow, intermediate, and rapid acetylators. Rifampin clearance was 17.3% (2.50-29.9) lower in individuals with homozygous AADAC rs1803155 G>A substitutions (p=0.0015). Conclusion: Targeted sequencing can enable detection of polymorphisms influencing TB drug metabolism on a low-cost, portable instrument to personalize dosing for TB treatment or prevention.

Global diversity and antimicrobial resistance of typhoid fever pathogens: Insights from a meta-analysis of 13,000 Salmonella Typhi genomes.

Background: The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods: This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results: Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions: The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding: No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

Salmonella Typhi (Typhi) is a type of bacteria that causes typhoid fever. More than 110,000 people die from this disease each year, predominantly in areas of sub-Saharan Africa and South Asia with limited access to safe water and sanitation. Clinicians use antibiotics to treat typhoid fever, but scientists worry that the spread of antimicrobial-resistant Typhi could render the drugs ineffective, leading to increased typhoid fever mortality. The World Health Organization has prequalified two vaccines that are highly effective in preventing typhoid fever and may also help limit the emergence and spread of resistant Typhi. In low resource settings, public health officials must make difficult trade-off decisions about which new vaccines to introduce into already crowded immunization schedules. Understanding the local burden of antimicrobial-resistant Typhi and how it is spreading could help inform their actions. The Global Typhoid Genomics Consortium analyzed 13,000 Typhi genomes from 110 countries to provide a global overview of genetic diversity and antimicrobial-resistant patterns. The analysis showed great genetic diversity of the different strains between countries and regions. For example, the H58 Typhi variant, which is often drug-resistant, has spread rapidly through Asia and Eastern and Southern Africa, but is less common in other regions. However, distinct strains of other drug-resistant Typhi have emerged in other parts of the world. Resistance to the antibiotic ciprofloxacin was widespread and accounted for over 85% of cases in South Africa. Around 70% of Typhi from Pakistan were extensively drug-resistant in 2020, but these hard-to-treat variants have not yet become established elsewhere. Variants that are resistant to both ciprofloxacin and ceftriaxone have been identified, and azithromycin resistance has also appeared in several different variants across South Asia. The Consortium's analyses provide valuable insights into the global distribution and transmission patterns of drug-resistant Typhi. Limited genetic data were available fromseveral regions, but data from travel-associated cases helped fill some regional gaps. These findings may help serve as a starting point for collective sharing and analyses of genetic data to inform local public health action. Funders need to provide ongoing supportto help fill global surveillance data gaps.

Population structure and antimicrobial resistance patterns of Salmonella Typhi and Paratyphi A amid a phased municipal vaccination campaign in Navi Mumbai, India.

We performed whole-genome sequencing of 174 Salmonella Typhi and 54 Salmonella Paratyphi A isolates collected through prospective surveillance in the context of a phased typhoid conjugate vaccine introduction in Navi Mumbai, India. We investigate the temporal and geographical patterns of emergence and spread of antimicrobial resistance. We evaluated the relationship between the spatial distance between households and genetic clustering of isolates. Most isolates were non-susceptible to fluoroquinolones, with nearly 20% containing ≥3 quinolone resistance-determining region mutations. Two H58 isolates carried an IncX3 plasmid containing blaSHV-12, associated with ceftriaxone resistance, suggesting that the ceftriaxone-resistant isolates from India independently evolved on multiple occasions. Among S. Typhi, we identified two main clades circulating (2.2 and 4.3.1 [H58]); 2.2 isolates were closely related following a single introduction around 2007, whereas H58 isolates had been introduced multiple times to the city. Increasing geographic distance between isolates was strongly associated with genetic clustering (odds ratio [OR] = 0.72 per km; 95% credible interval [CrI]: 0.66-0.79). This effect was seen for distances up to 5 km (OR = 0.65 per km; 95% CrI: 0.59-0.73) but not seen for distances beyond 5 km (OR = 1.02 per km; 95% CrI: 0.83-1.26). There was a non-significant reduction in odds of clustering for pairs of isolates in vaccination communities compared with non-vaccination communities or mixed pairs compared with non-vaccination communities. Our findings indicate that S. Typhi was repeatedly introduced into Navi Mumbai and then spread locally, with strong evidence of spatial genetic clustering. In addition to vaccination, local interventions to improve water and sanitation will be critical to interrupt transmission. IMPORTANCE Enteric fever remains a major public health concern in many low- and middle-income countries, as antimicrobial resistance (AMR) continues to emerge. Geographical patterns of typhoidal Salmonella spread, critical to monitoring AMR and planning interventions, are poorly understood. We performed whole-genome sequencing of S. Typhi and S. Paratyphi A isolates collected in Navi Mumbai, India before and after a typhoid conjugate vaccine introduction. From timed phylogenies, we found two dominant circulating lineages of S. Typhi in Navi Mumbai-lineage 2.2, which expanded following a single introduction a decade prior, and 4.3.1 (H58), which had been introduced repeatedly from other parts of India, frequently containing "triple mutations" conferring high-level ciprofloxacin resistance. Using Bayesian hierarchical statistical models, we found that spatial distance between cases was strongly associated with genetic clustering at a fine scale (<5 km). Together, these findings suggest that antimicrobial-resistant S. Typhi frequently flows between cities and then spreads highly locally, which may inform surveillance and prevention strategies.

Antimicrobial activity of cinnamaldehyde against multidrug-resistant Klebsiella pneumoniae: an in vitro and in vivo study.

The emergence and spread of multidrug-resistant (MDR) Klebsiella pneumoniae strains have increased worldwide, posing a significant health threat by limiting the therapeutic options. This study aimed to investigate the antimicrobial potential of cinnamaldehyde against MDR-K. pneumoniae strains in vitro and in vivo assays. The presence of resistant genes in MDR- K. pneumoniae strains were evaluated by Polymerase Chain Reaction (PCR) and DNA sequencing. Carbapenem-resistant K. pneumoniae strains show the blaKPC-2 gene, while polymyxin-resistant K. pneumoniae presented blaKPC-2 and alterations in the mgrB gene. Cinnamaldehyde exhibited an inhibitory effect against all MDR- K. pneumoniae evaluated. An infected mice model was used to determine the in vivo effects against two K. pneumoniae strains, one carbapenem-resistant and another polymyxin-resistant. After 24 h of cinnamaldehyde treatment, the bacterial load in blood and peritoneal fluids decreased. Cinnamaldehyde showed potential effectiveness as an antibacterial agent by inhibiting the growth of MDR-K. pneumoniae strains.

Genetic Diversity of Virulent Polymyxin-Resistant Klebsiella aerogenes Isolated from Intensive Care Units.

This study evaluated the scope and genetic basis of polymyxin-resistant Klebsiella aerogenes in Brazil. Eight polymyxin-resistant and carbapenemase-producing K. aerogenes strains were isolated from patients admitted to the ICU of a tertiary hospital. Bacterial species were identified by automated systems and antimicrobial susceptibility profile was confirmed using broth microdilution. The strains displayed a multidrug resistant profile and were subjected to whole-genome sequencing. Bioinformatic analysis revealed a variety of antimicrobial resistance genes, including the blaKPC-2. No plasmid-mediated colistin resistance gene was identified. Nonetheless, nonsynonymous mutations in mgrB, pmrA, pmrB, and eptA were detected, justifying the colistin resistance phenotype. Virulence genes encoding yersiniabactin, colibactin, and aerobactin were also found, associated with ICEKp4 and ICEKp10, and might be related to the high mortality observed among the patients. In fact, this is the first time ICEKp is identified in K. aerogenes in Brazil. Phylogenetic analysis grouped the strains into two clonal groups, belonging to ST93 and ST16. In summary, the co-existence of antimicrobial resistance and virulence factors is deeply worrying, as it could lead to the emergence of untreatable invasive infections. All these factors reinforce the need for surveillance programs to monitor the evolution and dissemination of multidrug resistant and virulent strains among critically ill patients.

The international and intercontinental spread and expansion of antimicrobial-resistant Salmonella Typhi: a genomic epidemiology study.

BACKGROUND: The emergence of increasingly antimicrobial-resistant Salmonella enterica serovar Typhi (S Typhi) threatens to undermine effective treatment and control. Understanding where antimicrobial resistance in S Typhi is emerging and spreading is crucial towards formulating effective control strategies. METHODS: In this genomic epidemiology study, we sequenced the genomes of 3489 S Typhi strains isolated from prospective enteric fever surveillance studies in Nepal, Bangladesh, Pakistan, and India (between 2014 and 2019), and combined these with a global collection of 4169 S Typhi genome sequences isolated between 1905 and 2018 to investigate the temporal and geographical patterns of emergence and spread of antimicrobial-resistant S Typhi. We performed non-parametric phylodynamic analyses to characterise changes in the effective population size of fluoroquinolone-resistant, extensively drug-resistant (XDR), and azithromycin-resistant S Typhi over time. We inferred timed phylogenies for the major S Typhi sublineages and used ancestral state reconstruction methods to estimate the frequency and timing of international and intercontinental transfers. FINDINGS: Our analysis revealed a declining trend of multidrug resistant typhoid in south Asia, except for Pakistan, where XDR S Typhi emerged in 2016 and rapidly replaced less-resistant strains. Mutations in the quinolone-resistance determining region (QRDR) of S Typhi have independently arisen and propagated on at least 94 occasions, nearly all occurring in south Asia. Strains with multiple QRDR mutations, including triple mutants with high-level fluoroquinolone resistance, have been increasing in frequency and displacing strains with fewer mutations. Strains containing acrB mutations, conferring azithromycin resistance, emerged in Bangladesh around 2013 and effective population size of these strains has been steadily increasing. We found evidence of frequent international (n=138) and intercontinental transfers (n=59) of antimicrobial-resistant S Typhi, followed by local expansion and replacement of drug-susceptible clades. INTERPRETATION: Independent acquisition of plasmids and homoplastic mutations conferring antimicrobial resistance have occurred repeatedly in multiple lineages of S Typhi, predominantly arising in south Asia before spreading to other regions. FUNDING: Bill & Melinda Gates Foundation.

High lethality rate of carbapenem-resistant Acinetobacter baumannii in Intensive Care Units of a Brazilian hospital: An epidemiologic surveillance study.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a growing threat to public health. METHODS: A 3-year retrospective study was conducted to evaluate the prevalence and lethality of multidrug-resistant (MDR) A. baumannii isolated from Brazilian patients. RESULTS: In this study, 219 Acinetobacter baumannii isolates were identified, of which 70.8% (155/219) were isolated from patients hospitalized in intensive care units. Of these, 57.4% (n = 89/155) were assessed, of which 92.1% (82/89) were carbapenem-resistant, and 49 were classified as infected. The lethality rate was 79.6% (39/49). CONCLUSIONS: We highlight the need of an effective epidemiological surveillance measure to contain the dissemination of CRAB in the hospital environment.

Overview of polymyxin resistance in Enterobacteriaceae.

Polymyxin antibiotics are disfavored owing to their potential clinical toxicity, especially nephrotoxicity. However, the dry antibiotic development pipeline, together with the increasing global prevalence of infections caused by multidrug-resistant (MDR) gram-negative bacteria, have renewed clinical interest in these polypeptide antibiotics. This review highlights the current information regarding the mechanisms of resistance to polymyxins and their molecular epidemiology. Knowledge of the resistance mechanisms and epidemiology of these pathogens is critical for the development of novel antibacterial agents and rapid treatment choices.

High lethality rate of carbapenem-resistant Acinetobacter baumannii in Intensive Care Units of a Brazilian hospital: An epidemiologic surveillance study

ABSTRACT Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a growing threat to public health. Methods: A 3-year retrospective study was conducted to evaluate the prevalence and lethality of multidrug-resistant (MDR) A. baumannii isolated from Brazilian patients. Results: In this study, 219 Acinetobacter baumannii isolates were identified, of which 70.8% (155/219) were isolated from patients hospitalized in intensive care units. Of these, 57.4% (n = 89/155) were assessed, of which 92.1% (82/89) were carbapenem-resistant, and 49 were classified as infected. The lethality rate was 79.6% (39/49). Conclusions: We highlight the need of an effective epidemiological surveillance measure to contain the dissemination of CRAB in the hospital environment.

Overview of polymyxin resistance in Enterobacteriaceae

ABSTRACT Polymyxin antibiotics are disfavored owing to their potential clinical toxicity, especially nephrotoxicity. However, the dry antibiotic development pipeline, together with the increasing global prevalence of infections caused by multidrug-resistant (MDR) gram-negative bacteria, have renewed clinical interest in these polypeptide antibiotics. This review highlights the current information regarding the mechanisms of resistance to polymyxins and their molecular epidemiology. Knowledge of the resistance mechanisms and epidemiology of these pathogens is critical for the development of novel antibacterial agents and rapid treatment choices.

Antisense peptide nucleic acid inhibits the growth of KPC-producing Klebsiella pneumoniae strain.

Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance (MDR) and mortality. In this study, we investigated the ability of the antisense peptide nucleic acids (PNA) conjugated to the (KFF)3K cell-penetrating peptide (CPP) to target the gyrA KPC-producing K. pneumoniae and inhibit bacterial growth in vitro. The inhibitory effect on gyrA gene was evaluated by measuring 16s gene amplification in KPC-producing K. pneumoniae treated with the antisense PNA conjugate. The hemolytic property of the antisense PNA conjugate was accessed toward mice red blood cells. Finally, molecular modeling and dynamics simulations analyses in aqueous solutions were performed to predict the PNA conformation alone in contact with DNA (gyrA gene sequence). PNA was capable of inhibiting bacterial growth at 50 µM, also reducing 16S gene amplification in 96.7%. Besides, PNA presented low hemolytic activity (21.1% hemolysis) at this same concentration. Bioinformatics analysis demonstrated that the structure of the PNA is stable in water without major changes in its secondary structure. The ability of PNA and its conjugated CPP ((KFF)3K) to inhibit bacterial growth demonstrates the potential of this new class of antibacterial agents, encouraging further in vivo studies to confirm its therapeutic efficacy.

Three challenging cases of infections by multidrug-resistant Serratia marcescens in patients admitted to intensive care units.

The occurrence of multidrug-resistant Serratia marcescens strains represents a serious public health threat. The purpose here is to report three cases of carbapenem-resistant S. marcescens infections with unfavorable clinical outcomes and provide a molecular description of the antibiotic resistance determinants at a genomic level. We performed bacterial identification by VITEK 2 and MALDI-TOF. The minimal inhibitory concentrations of antimicrobials were determined according to the Clinical and Laboratory Standards Institute guidelines, except for tigecycline, for which they were determined using Etest strips. Preliminary screening for the presence of carbapenemases was performed by ertapenem hydrolysis using MALDI-TOF MS. Whole-genome sequencing was provided to identify genes responsible for virulence and antimicrobial resistance. Here we report three challenging cases of S. marcescens that were resistant to the most commonly used antibiotics. Otherwise, we performed a genome description, which includes several genes involved in the resistance and virulence. These cases illustrate serious infection due to multidrug-resistant organisms and the complexity of treatment. Our results highlight the need to evaluate isolates regularly during long-term hospital stay to achieve optimal quality of clinical care and thus improve patient outcomes.

Epidemiological study in Brazilian women highlights that syphilis remains a public health problem.

Syphilis, an infectious disease considered a global public health concern, can cause stillbirths and neonatal deaths. This highlights the importance of continuous surveillance studies among women of reproductive age. A cross-sectional study was carried out to analyze the prevalence and risk factors associated with Treponema pallidum infection in women assisted by primary health care units in Dourados, a city located in Mato Grosso do Sul State, Brazil, which borders Paraguay. A questionnaire was applied to a population-based sample, blood samples were collected for syphilis testing and multivariable analyses were performed to screen associations with T. pallidum infection. The prevalence of T. pallidum infection was 6.04%. Bivariate analysis showed that women referring multiple sexual partners (c2: 6.97 [p=0.014]), income less 2 minimal wages (c2: 15.93 [p=0.003]), who did not have high school (c2: 12.64 [p=0.005]), and reporting history of STIs (c2: 7.30 [p=0.018]) are more likely to have syphilis. In the multivariate analysis, a highest prevalence ratio was observed in women with income less than 2 minimal wages (PR: 0.96 [95% CI: 0.85 - 0.97]), and who did not have high school (PR: 0.94 [95% CI: 0.90 - 0.98]). In addition, 80% of the women reported irregular use of condoms and 63.89% declared having sexual intercourses with multiple partners, which creates more opportunities for the transmission of the infection. These results highlight the need for healthcare systems to implement initiatives to monitor syphilis screening and the commitment of patients and their sexual partners to the treatment in order to achieve a decrease of new cases.

Multidrug-resistant Acinetobacter baumannii outbreaks: a global problem in healthcare settings.

INTRODUCTION: The increase in the prevalence of multidrug-resistant Acinetobacter baumannii infections in hospital settings has rapidly emerged worldwide as a serious health problem. METHODS: This review synthetizes the epidemiology of multidrug-resistant A. baumannii, highlighting resistance mechanisms. CONCLUSIONS: Understanding the genetic mechanisms of resistance as well as the associated risk factors is critical to develop and implement adequate measures to control and prevent acquisition of nosocomial infections, especially in an intensive care unit setting.

Antibacterial activity of Cinnamomum cassia L. essential oil in a carbapenem- and polymyxin-resistant Klebsiella aerogenes strain.

INTRODUCTION: Essential oils can serve as novel sources of antibiotics for multidrug-resistant bacteria. METHODS: The multidrug-resistance profile of a Klebsiella aerogenes strain was assessed by PCR and sequencing. The antibacterial activity of Cinnamomum cassia essential oil (CCeo) against K. aerogenes was assessed by broth microdilution and time-kill methods. RESULTS: K. aerogenes showed high antibiotic resistance. The genes bla KPC-2, ampC, bla CTX-M-15, bla OXA-1, and bla TEM were present. CCeo exhibited an inhibitory effect with a minimum inhibitory concentration of 17.57 µg/mL. CONCLUSIONS: The antibacterial activity of CCeo makes it a potential candidate for treating carbapenem- and polymyxin-resistant K. aerogenes strains.

Molecular and epidemiological surveillance of polymyxin-resistant Klebsiella pneumoniae strains isolated from Brazil with multiple mgrB gene mutations.

The prevalence of polymyxin-resistant Enterobacteriaceae is increasing worldwide. Their emergence is worrisome and limits therapeutic options for severely ill patients. We aimed to investigate the molecular and epidemiological characteristics of polymyxin-resistant Klebsiella pneumoniae circulating in Brazilian hospitals. Polymyxin-resistant K. pneumoniae isolates from two Brazilian healthcare facilities were characterized phenotypically and subjected to whole genome sequencing (WGS). Using the WGS data we determined their sequence type, resistance gene content (resistome), their composition of virulence genes and plasmids. ST11 was the most common (80 %) sequence type among the isolates followed by ST345, ST15 and ST258. A resistome analysis revealed the common presence of blaKPC-2 and less frequently blaSHV-11, blaTEM-1, blaCTX-M-15, and blaOXA-9. Genes conferring resistance to aminoglycosides, fluoroquinolones, phenicols, sulphonamides, tetracyclines, trimethoprim and macrolide-lincosamide-streptogramin were also detected. We observed a clonal spread of polymyxin-resistant K. pneumoniae isolates, with polymyxin-resistance associated with various alterations in the mgrB gene including inactivation by an insertion sequence and nonsense point mutations. We additionally identified a novel 78-bp repeat sequence, encoding a MgrB protein with 26 amino acids duplicated in six isolates. This is the first observation of this type of alteration being associated with polymyxin resistance. Our findings demonstrate that mgrB alterations were the most common source of polymyxin-resistance in Brazilian clinical settings. Interestingly, distinct genetic events were identified among clonally related isolates, including a new amino acid alteration. The clinical implications and investigation of the resistance mechanisms is of great importance to patient safety and control of these infections, particularly in long-term care facilities.

Antimicrobial activity of plant extracts against carbapenem-producing Klebsiella pneumoniae and in vivo toxicological assessment.

The global spread of multidrug-resistant strains has prompted the scientific community to explore novel sources of chemicals with antimicrobial activity. The aim of the study was to examine the antimicrobial activity in vitro of 28 extracts against carbapenem-producing Klebsiella pneumoniae, individually and in combination with antibiotics and in vivo toxicological assessment of the most active product. The multi-resistant K. pneumoniae strain was submitted for phenotypic and molecular characterization. The antibacterial activity of 28 plant extracts was evaluated alone and in combination with antibiotics against this strain through the agar disk diffusion. Of these, 16 extracts showed synergism against carbapenem-producing K. pneumoniae, being that B. crassifolia extract exhibited synergism with three antibiotics. Based on this assessment, B. crassifolia-extract-induced toxicity on Swiss male mice was evaluated by administering this extract and subsequently determining apoptosis and splenic phagocytosis using the comet and micronucleus assays. The results of this study showed that B. crassifolia extract had synergistic activity promising and groups treated with B. crassifolia exhibited no genotoxic or mutagenic activity, indicating that B. crassifolia extract exerted beneficial effects and appeared safe to use at the studied concentrations.

Synergistic effects of Cinnamomum cassia L. essential oil in combination with polymyxin B against carbapenemase-producing Klebsiella pneumoniae and Serratia marcescens.

Multidrug resistance prompts the search for new sources of antibiotics with new targets at bacteria cell. To investigate the antibacterial activity of Cinnamomum cassia L. essential oil (CCeo) alone and in combination with antibiotics against carbapenemase-producing Klebsiella pneumoniae and Serratia marcescens. The antimicrobial susceptibility of the strains was determined by Vitek® 2 and confirmed by MALDI-TOF/TOF. The antibacterial activity of CCeo and its synergism with antibiotics was determined using agar disk diffusion, broth microdilution, time-kill, and checkboard methods. The integrity of the bacterial cell membrane in S. marcescens was monitored by protein leakage assay. CCeo exhibited inhibitory effects with MIC = 281.25 µg.mL-1. The association between CCeo and polymyxin B showed a decrease in terms of viable cell counts on survival curves over time after a 4 hour-treatment with a FIC index value of 0.006. Protein leakage was observed with increasing concentrations for CCeo and CCeo + polymyxin B treatments. CCeo showed antibacterial activity against the studied strains. When associated with polymyxin B, a synergistic effect was able to inhibit bacterial growth rapidly and consistently, making it a potential candidate for the development of an alternative treatment and drug delivery system for carbapenemase-producing strains.