Activity of N-Acetylcysteine Alone and in Combination with Colistin against Pseudomonas aeruginosa Biofilms and Transcriptomic Response to N-Acetylcysteine Exposure.

Chronic colonization by Pseudomonas aeruginosa is critical in cystic fibrosis (CF) and other chronic lung diseases, contributing to disease progression. Biofilm growth and a propensity to evolve multidrug resistance phenotypes drastically limit the available therapeutic options. In this perspective, there has been growing interest in evaluating combination therapies, especially for drugs that can be administered by nebulization, which allows high drug concentrations to be reached at the site of infections while limiting systemic toxicity. Here, we investigated the potential antibiofilm activity of N-acetylcysteine (NAC) alone and in combination with colistin against a panel of P. aeruginosa strains (most of which are from CF patients) and the transcriptomic response of a P. aeruginosa CF strain to NAC exposure. NAC alone (8,000 mg/L) showed a limited and strain-dependent antibiofilm activity. Nonetheless, a relevant antibiofilm synergism of NAC-colistin combinations (NAC at 8,000 mg/L plus colistin at 2 to 32 mg/L) was observed with all strains. Synergism was also confirmed with the artificial sputum medium model. RNA sequencing of NAC-exposed planktonic cultures revealed that NAC (8,000 mg/L) mainly induced (i) a Zn2+ starvation response (known to induce attenuation of P. aeruginosa virulence), (ii) downregulation of genes of the denitrification apparatus, and (iii) downregulation of flagellar biosynthesis pathway. NAC-mediated inhibition of P. aeruginosa denitrification pathway and flagellum-mediated motility were confirmed experimentally. These findings suggested that NAC-colistin combinations might contribute to the management of biofilm-associated P. aeruginosa lung infections. NAC might also have a role in reducing P. aeruginosa virulence, which could be relevant in the very early stages of lung colonization. IMPORTANCE Pseudomonas aeruginosa biofilm-related chronic lung colonization contributes to cystic fibrosis (CF) disease progression. Colistin is often a last-resort antibiotic for the treatment of such P. aeruginosa infections, and it has been increasingly used in CF, especially by nebulization. N-acetylcysteine (NAC) is a mucolytic agent with antioxidant activity, commonly administered with antibiotics for the treatment of lower respiratory tract infections. Here, we show that NAC potentiated colistin activity against in vitro biofilms models of P. aeruginosa strains, with both drugs tested at the high concentrations achievable after nebulization. In addition, we report the first transcriptomic data on the P. aeruginosa response to NAC exposure.

Relevant increase of CTX-M-producing Escherichia coli carriage in school-aged children from rural areas of the Bolivian Chaco in a three-year period.

OBJECTIVES: The aim of this study was to perform two cross-sectional surveys on the fecal carriage of CTX-M-producing Enterobacterales in school-aged children from rural areas of the Bolivian Chaco (2016 vs 2019). METHODS: A total of 757 fecal samples were collected from school-aged children living in nine indigenous communities (n=337, 2016; n=420, 2019). After a first passage onto MacConkey agar (MCA), samples were plated onto MCA plus cefotaxime 2 µg/mL (MCA-CTX), and a loopful of the bacterial growth was used as a template for the detection of group 1, 2, 8/25, and 9 blaCTX-M variants by multiplex reverse transcriptase polymerase chain reaction . Positive samples were tested again for detecting, identifying, and characterizing CTX-M-positive isolates. RESULTS: Growth onto MCA-CTX was obtained with 208 samples (27.5%; 62/337, 2016; 146/420, 2019), of which 201 (96.6%) were positive for blaCTX-M genes. Overall, a relevant increase of fecal carriage of CTX-M-producing Enterobacterales was observed in the study period: 17,5% (59/337) in 2016 compared with 33,8% (142/420) in 2019, p<0.01. Nonetheless, the relative group distribution of CTX-M groups remained stable, with group 1 being the prevalent, followed by group 9 and group 8/25. Group 2 was not detected. CONCLUSIONS: The present study demonstrated an alarming spread of CTX-M enzymes in rural areas of the Bolivian Chaco, where antibiotics consumption is limited. Further studies are encouraged to better understand the dissemination dynamics of such relevant resistance determinants.

Activity of fosfomycin/colistin combinations against planktonic and biofilm Gram-negative pathogens.

OBJECTIVES: To investigate the in vitro activity of fosfomycin, colistin and combinations thereof against planktonic and biofilm cultures of Gram-negative pathogens, mostly showing MDR phenotypes, at concentrations achievable via inhalation of aerosolized drugs. METHODS: Activity against planktonic cultures was tested by the chequerboard assay with 130 strains, including 52 Pseudomonas aeruginosa, 47 Klebsiella pneumoniae, 19 Escherichia coli, 7 Stenotrophomonas maltophilia and 5 Acinetobacter baumannii. Activity against biofilm cultures was tested by biofilm chequerboard and quantitative antibiofilm assays with a subset of 20 strains. In addition, 10 of these strains were tested in mutant prevention concentration (MPC) assays. RESULTS: Against planktonic cultures, synergism between fosfomycin and colistin was detected with a minority (10%) of strains (eight K. pneumoniae and five P. aeruginosa), while antagonism was never observed. Synergism between fosfomycin and colistin against biofilms was observed with the majority of tested strains (16/20 in biofilm chequerboard assays, and 18/20 in the quantitative antibiofilm assays), including representatives of each species and regardless of their resistance genotype or phenotype. Furthermore, combination of fosfomycin and colistin was found to significantly reduce the MPC of individual drugs. CONCLUSIONS: Fosfomycin and colistin in combination, at concentrations achievable via inhalation of nebulized drugs, showed notable synergy against MDR Gram-negative pathogens grown in biofilm, and were able to reduce the emergence of fosfomycin- and colistin-resistant subpopulations.

High prevalence of carriage of mcr-1-positive enteric bacteria among healthy children from rural communities in the Chaco region, Bolivia, September to October 2016.

BackgroundThe mcr-1 gene is a transferable resistance determinant against colistin, a last-resort antimicrobial for infections caused by multi-resistant Gram-negatives.AimTo study carriage of antibiotic-resistant bacteria in healthy school children as part of a helminth control and antimicrobial resistance survey in the Bolivian Chaco region.MethodsFrom September to October 2016 we collected faecal samples from healthy children in eight rural villages. Samples were screened for mcr-1- and mcr-2 genes. Antimicrobial susceptibility testing was performed, and a subset of 18 isolates representative of individuals from different villages was analysed by whole genome sequencing (WGS).ResultsWe included 337 children (mean age: 9.2 years, range: 7-11; 53% females). The proportion of mcr-1 carriers was high (38.3%) and present in all villages; only four children had previous antibiotic exposure. One or more mcr-1-positive isolates were recovered from 129 positive samples, yielding a total of 173 isolates (171 Escherichia coli, 1 Citrobacter europaeus, 1 Enterobacter hormaechei). No mcr-2 was detected. Co-resistance to other antimicrobials varied in mcr-positive E. coli. All 171 isolates were susceptible to carbapenems and tigecycline; 41 (24.0%) were extended-spectrum ß-lactamase producers and most of them (37/41) carried blaCTX-M-type genes. WGS revealed heterogeneity of clonal lineages and mcr-genetic supports.ConclusionThis high prevalence of mcr-1-like carriage, in absence of professional exposure, is unexpected. Its extent at the national level should be investigated with priority. Possible causes should be studied; they may include unrestricted use of colistin in veterinary medicine and animal breeding, and importation of mcr-1-positive bacteria via food and animals.

In vitro activity of N-acetylcysteine against Stenotrophomonas maltophilia and Burkholderia cepacia complex grown in planktonic phase and biofilm.

Stenotrophomonas maltophilia and Burkholderia cepacia complex (Bcc) have been increasingly recognized as relevant pathogens in hospitalized, immunocompromised and cystic fibrosis (CF) patients. As a result of complex mechanisms, including biofilm formation and multidrug resistance phenotype, S. maltophilia and Bcc respiratory infections are often refractory to therapy, and have been associated with a worse outcome in CF patients. Here we demonstrate for the first time that N-acetylcysteine (NAC), a mucolytic agent with antioxidant and anti-inflammatory properties, may exhibit antimicrobial and antibiofilm activity against these pathogens. The antimicrobial and antibiofilm activity of high NAC concentrations, potentially achievable by topical administration, was tested against a collection of S. maltophilia (n = 19) and Bcc (n = 19) strains, including strains from CF patients with acquired resistance traits. Minimum Inhibitory Concentrations (MICs) and Minimum Bactericidal Concentrations (MBCs) ranged from 16 to 32 mg/ml and from 32 to >32 mg/ml, respectively. Sub-MIC concentrations (i.e., 0.25 × MIC) slowed down the growth kinetics of most strains. In time-kill assays, 2-day-old biofilms were more affected than planktonic cultures, suggesting a specific antibiofilm activity of NAC against these pathogens. Indeed, a dose- and time-dependent antibiofilm activity of NAC against most of the S. maltophilia and Bcc strains tested was observed, with a sizable antibiofilm activity observed also at 0.5 and 1 × MIC NAC concentrations. Furthermore, at those concentrations, NAC was also shown to significantly inhibit biofilm formation with the great majority of tested strains.

In vitro synergism of colistin in combination with N-acetylcysteine against Acinetobacter baumannii grown in planktonic phase and in biofilms.

Objectives: To investigate the potential synergism of colistin in combination with N-acetylcysteine against Acinetobacter baumannii strains grown in planktonic phase or as biofilms. Methods: Sixteen strains were investigated, including nine colistin-susceptible (MIC range 0.5-1 mg/L) and seven colistin-resistant (MIC range 16-256 mg/L) strains. Synergism of colistin in combination with N-acetylcysteine was investigated by chequerboard assays. The activity of colistin/N-acetylcysteine combinations was further evaluated by time-kill assays with planktonic cultures (three colistin-resistant strains and one colistin-susceptible strain) and by in vitro biofilm models (three colistin-resistant and three colistin-susceptible strains). Results: Chequerboard assays revealed a relevant synergism of colistin/N-acetylcysteine combinations with all colistin-resistant strains, whereas no synergism was observed with colistin-susceptible strains. Time-kill assays showed a concentration-dependent potentiation of colistin activity by N-acetylcysteine against colistin-resistant strains, with eradication of the culture by combinations of N-acetylcysteine at 8000 mg/L plus colistin at 2 or 8 mg/L. A static effect during the first 8 h of incubation was demonstrated with the colistin-susceptible strain exposed to 0.25 × MIC colistin plus 8000 mg/L N-acetylcysteine. A remarkable antibiofilm synergistic activity of 8 mg/L colistin plus 8000 mg/L N-acetylcysteine was demonstrated with all colistin-resistant and colistin-susceptible strains. The effects were greater with colistin-resistant strains (marked reduction of viable biofilm cells was observed at sub-MIC colistin concentrations). Conclusions: N-acetylcysteine, at concentrations achievable by topical administration, was shown to revert the colistin-resistant phenotype in A. baumannii, and to exert a relevant activity against biofilms of colistin-susceptible and colistin-resistant A. baumannii strains.

Effect of High N-Acetylcysteine Concentrations on Antibiotic Activity against a Large Collection of Respiratory Pathogens.

The effect of high N-acetylcysteine (NAC) concentrations (10 and 50 mM) on antibiotic activity against 40 strains of respiratory pathogens was investigated. NAC compromised the activity of carbapenems (of mostly imipenem and, to lesser extents, meropenem and ertapenem) in a dose-dependent fashion. We demonstrated chemical instability of carbapenems in the presence of NAC. With other antibiotics, 10 mM NAC had no major effects, while 50 mM NAC sporadically decreased (ceftriaxone and aminoglycosides) or increased (penicillins) antibiotic activity.

Antimicrobial susceptibility and emerging resistance determinants (blaCTX-M, rmtB, fosA3) in clinical isolates from urinary tract infections in the Bolivian Chaco.

BACKGROUND: Bolivia is among the lowest-resourced South American countries, with very few data available on antibiotic resistance in bacterial pathogens. The phenotypic and molecular characterization of bacterial isolates responsible for urinary tract infections (UTIs) in the Bolivian Chaco are reported here. METHODS: All clinical isolates from UTIs collected in the Hospital Basico Villa Montes between June 2010 and January 2014 were analyzed (N=213). Characterization included susceptibility testing, extended-spectrum beta-lactamase (ESBL) detection, identification of relevant resistance determinants (e.g., CTX-M-type ESBLs, 16S rRNA methyltransferases, glutathione S-transferases), and genotyping of CTX-M producers. RESULTS: Very high resistance rates were observed. Overall, the lowest susceptibility was observed for trimethoprim-sulphamethoxazole, tetracycline, nalidixic acid, amoxicillin-clavulanic acid, ciprofloxacin, and gentamicin. Of E. coli and K. pneumoniae, 11.6% were ESBL producers. Resistance to nitrofurantoin, amikacin, and fosfomycin remained low, and susceptibility to carbapenems was fully preserved. CTX-M-15 was the dominant CTX-M variant. Four E. coli ST131 (two being H30-Rx) were identified. Of note, isolates harbouring rmtB and fosA3 were detected. CONCLUSIONS: Bolivia is not an exception to the very high resistance burden affecting many South American countries. Optimization of alternative approaches to monitor local antibiotic resistance trends in resource-limited settings is strongly encouraged to support the implementation of effective empiric treatment guidelines.

Characterization of IncI1 sequence type 71 epidemic plasmid lineage responsible for the recent dissemination of CTX-M-65 extended-spectrum ß-lactamase in the Bolivian Chaco region.

During the last decade, a significant diffusion of CTX-M-type extended-spectrum ß-lactamases (ESBLs) was observed in commensal Escherichia coli from healthy children in the Bolivian Chaco region, with initial dissemination of CTX-M-2, which was then replaced by CTX-M-15 and CTX-M-65. In this work, we demonstrate that the widespread dissemination of CTX-M-65 observed in this context was related to the polyclonal spreading of an IncI1 sequence type 71 (ST71) epidemic plasmid lineage. The structure of the epidemic plasmid population was characterized by complete sequencing of four representatives and PCR mapping of the remainder (n = 16). Sequence analysis showed identical plasmid backbones (similar to that of the reference IncI1 plasmid, R64) and a multiresistance region (MRR), which underwent local microevolution. The MRR harbored genes responsible for resistance to ß-lactams, aminoglycosides, florfenicol, and fosfomycin (with microevolution mainly consisting of deletion events of resistance modules). The bla CTX-M-65 module harbored by the IncI1 ST71 epidemic plasmid was apparently derived from IncN-type plasmids, likely via IS26-mediated mobilization. The plasmid could be transferred by conjugation to several different enterobacterial species (Escherichia coli, Cronobacter sakazakii, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, and Salmonella enterica) and was stably maintained without selective pressure in these species, with the exception of K. oxytoca and S. enterica. Fitness assays performed in E. coli recipients demonstrated that the presence of the epidemic plasmid was apparently not associated with a significant biological cost.

Methicillin-resistant Staphylococcus aureus in hospitalized patients from the Bolivian Chaco.

OBJECTIVES: Information is lacking on the methicillin-resistant Staphylococcus aureus (MRSA) clonal lineages circulating in Bolivia. We investigated the prevalence and molecular epidemiology of S. aureus colonization in hospitalized patients from the Bolivian Chaco, and compared their features with those of the few clinical isolates available from that setting. METHODS: S. aureus nasal/inguinal colonization was investigated in 280 inpatients from eight hospitals in two point prevalence surveys (2012, n=90; 2013, n=190). Molecular characterization included genotyping (spa typing, multilocus sequence typing, and pulsed-field gel electrophoresis), detection of virulence genes, and SCCmec typing. RESULTS: Forty-one inpatients (14.6%) were S. aureus nasal/inguinal carriers, of whom five were colonized by MRSA (1.8%). MRSA isolates mostly belonged to spa-type t701, harboured SCCmec IVc, and were negative for Panton-Valentine leukocidin (PVL) genes. However, a USA300-related isolate was also detected, which showed the characteristics of the USA300 Latin American variant (USA300-LV; i.e., ST8, spa-type t008, SCCmec IVc, presence of PVL genes, absence of arcA). Notably, all the available MRSA clinical isolates (n=5, collected during 2011-2013) were also identified as USA300-LV. CONCLUSIONS: Overall, MRSA colonization in inpatients from the Bolivian Chaco was low. However, USA300-LV-related isolates were detected in colonization and infections, emphasizing the importance of implementing control measures to limit their further dissemination in this resource-limited area.

Efficacy and toxicity of the antimicrobial peptide M33 produced with different counter-ions.

The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015-1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5-30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.

[In vitro synergism of ulifloxacin plus piperacillin/tazobactam against clinical isolates of Enterobacteriaceae producing extended-spectrum or AmpC-type ß-lactamases]. / Sinergismo in vitro tra ulifloxacina e piperacillina/tazobactam verso isolati clinici di enterobatteri produttori di beta-lattamasi a spettro esteso e AmpC.

Extended spectrum beta-lactamase (ESBL) and ampicillinase C (AmpC) producing Enterobacteriaceae are nowadays frequently isolated in clinical practice. Carbapenems are generally the drugs of choice in such a case and resistance to these molecules is on the rise. Rationalizing their use is to be considered essential, possibly identifying alternative regimens. We thus examined 10 strains of Enterobacteriaceae isolated from patients previously unsuccessfully treated with a beta-lactam or a quinolone; eight strains were either ESBL or AmpC producers. Ulifloxacin showed minimum inhibitory concentrations (MICs) lower than ciprofloxacin and levofloxacin. Tested with the checkerboard method, the association of ulifloxacin and piperacillin/tazobactam proved fully synergistic on five strains and partially synergistic on three. The above association was fully synergistic towards three strains resistant to piperacillin/tazobactam and one strain resistant to ulifloxacin, with MICs in association easily obtainable at standard doses. Our in vitro study demonstrates a synergistic activity of ulifloxacin and piperacillin/tazobactam in association towards ESBL and AmpC-producing Enterobacteriaceae. Clinical studies are needed to confirm in vivo the effectiveness of this regimen.

Antimicrobial activity of Inga fendleriana extracts and isolated flavonoids.

The EtOAc and n-BuOH extracts of Inga fendleriana inhibited Gram-positive, but not Gram-negative bacteria; a narrow spectrum of activity against Staphylococcus epidermidis was detected. The MIC values of the extracts ranged from 125 to 850 microg/mL. Quercetin 3-methylether, myricetin 3-O-rhamnoside and tricetin showed antibacterial activity against the same bacterial strains with MICs in the range from 31 to 250 microg/mL. In time-kill kinetic studies, the flavonoids showed bactericidal effects at the concentrations corresponding to four times the MICs.

Rapid dissemination and diversity of CTX-M extended-spectrum beta-lactamase genes in commensal Escherichia coli isolates from healthy children from low-resource settings in Latin America.

A survey carried out in 2005 among members of a healthy population of children living in Bolivia and Peru revealed that fecal carriage of Escherichia coli strains resistant to expanded-spectrum cephalosporins was remarkably increased compared to that observed in the same settings in 2002 (1.7% in 2005 versus 0.1% in 2002). In this work, we demonstrated that this phenomenon was mainly related to the dissemination of CTX-M-type extended-spectrum beta-lactamase (ESBL) determinants among commensal E. coli strains. Of 50 ESBL-producing isolates collected in the 2005 survey, 44 harbored a CTX-M-type and 6 an SHV-type (SHV-2 or SHV-12) ESBL. Compared to 2002 results, an increased diversity of CTX-M-type ESBLs was also observed: members of the CTX-M-1 group (CTX-M-15) emerged in Bolivia (where only CTX-M-2 was observed in 2002), while members of the CTX-M-9 group (CTX-M-14 and CTX-M-24) emerged in Peru (where only CTX-M-15 and CTX-M-2 were observed in 2002). A new CTX-M-2 variant named CTX-M-56 was also detected. Molecular characterization of the CTX-M-producing isolates and gene transfer experiments suggested that different mechanisms could be involved in the spreading of different CTX-M group determinants and revealed that additional resistance determinants for non-beta-lactam antibiotics were preferentially carried by plasmids encoding certain CTX-M variants (CTX-M-15 and variants of the CTX-M-2 group). Three CTX-M-15-encoding conjugative plasmids from Peruvian isolates carried the new fluoroquinolone resistance gene aac(6')-Ib-cr. To our best knowledge, this is the first report of the detection of aac(6')-Ib-cr in Latin America.

Chemical and antibacterial evaluation of Hypericum triquetrifolium Turra.

Five extracts and pure compounds from the aerial parts of Hypericum triquetrifolium were tested for their antibacterial activity against 31 gram-positive and gram-negative strains using the agar dilution method (Lorian). The ethyl acetate extract exhibited a weak antibacterial activity against Staphylococcus strains, and pure constituents such as quercetin and I3,II8-biapigenin were the active components of this extract.