Spread of Pseudomonas aeruginosa ST274 Clone in Different Niches: Resistome, Virulome, and Phylogenetic Relationship.

Pseudomonas aeruginosa ST274 is an international epidemic high-risk clone, mostly associated with hospital settings and appears to colonize cystic fibrosis (CF) patients worldwide. To understand the relevant mechanisms for its success, the biological and genomic characteristics of 11 ST274-P. aeruginosa strains from clinical and non-clinical origins were analyzed. The extensively drug-resistant (XDR/DTR), the non-susceptible to at least one agent (modR), and the lasR-truncated (by ISPsp7) strains showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity and low motility. Furthermore, the XDR/DTR and modR strains presented low pigment production and biofilm formation, which were very high in the lasR-truncated strain. Their whole genome sequences were compared with other 14 ST274-P. aeruginosa genomes available in the NCBI database, and certain associations have been primarily detected: blaOXA-486 and blaPDC-24 genes, serotype O:3, exoS+/exoU- genotype, group V of type IV pili, and pyoverdine locus class II. Other general molecular markers highlight the absence of vqsM and pldA/tleS genes and the presence of the same mutational pattern in genes involving two-component sensor-regulator systems PmrAB and CreBD, exotoxin A, quorum-sensing RhlI, beta-lactamase expression regulator AmpD, PBP1A, or FusA2 elongation factor G. The proportionated ST274-P. aeruginosa results could serve as the basis for more specific studies focused on better antibiotic stewardship and new therapeutic developments.

Phenotypic Characteristics and Clonal Relationships of Stenotrophomonas maltophilia Isolates in Hospitalized Adults from a Private Center in Lima, Peru.

Stenotrophomonas maltophilia is an opportunistic pathogen, often associated with nosocomial infections. Ten S. maltophilia were isolated from clinical samples during the period January 2021 and June 2022. Eight (80%) patients had cancer as a background disease and 2 patients had coronavirus disease 2019. A fatal outcome was recorded in 4 cases (40% of patients). All the isolates were susceptible to minocycline and levofloxacin. Trimethoprim/sulfamethoxazole and ceftazidime resistance rates were 20% and 40% respectively. Eight different patterns were observed by Pulsed-Field Gel Electrophoresis, only two isolates being clonally identical. The isolation of S. maltophilia in clinical settings requires the implementation of infection prevention measures.

Development and characterization of anti-biofilm coatings applied by Non-Equilibrium Atmospheric Plasma on stainless steel.

Biofilm-mediated microbial persistence of pathogenic and spoilage bacteria is a serious problem in food industries. Due to the difficulty of removing mature biofilms, great efforts are being made to find new strategies to prevent bacterial adherence to surfaces, the first step for biofilm development. In this study, coatings of (3-aminopropyl)triethoxysilane (APTES), tetraethyl orthosilicate (TEOS) and acrylic acid (AA) were applied by Non-Equilibrium Atmospheric Plasma on stainless steel (SS) AISI 316, the SS most commonly used in food industry equipment. Their anti-biofilm activity was assessed against Listeria monocytogenes CECT911 and Escherichia coli CECT515 after incubation at 37 °C. The best results were obtained for L. monocytogenes, with coatings consisting of a base coating of APTES and a functional coating of TEOS (AP10 + TE6) or AA (AP10 + AA6) that reduced biofilm production by 45% and 74%, respectively, when compared with the uncoated SS. These coatings were further characterized, together with a variation of the best one that replaced the acrylic acid with succinic acid (AP10 + SA6). Their anti-biofilm activity was assessed under different incubation conditions, including two strains of L. monocytogenes isolated from processing environments of a meat industry. The coating AP10 + AA6 reduced the biofilm formation by 90% after incubation at 12 °C, a temperature more representative of those commonly found in food processing environments. The morphological and physico-chemical characterization of the selected coatings showed that the coating with the highest anti-biofilm activity (i.e., AP10 + AA6) had lower surface roughness and higher hydrophilicity. This suggests that the formation of a hydration layer prevents the adherence of L. monocytogenes, an effect that seems to be enhanced by low temperature conditions, when the wettability of the strains is increased.

Caracterización de Escherichia coli D7111 productora de ß-LACTAMASA TEM-176 / Characterization of Escherichia coli D7111 producing the ß-LACTAMASA TEM-176

RESUMEN El presente reporte es la descripción original de bla TEM-176. Se caracterizaron los mecanismos de resistencia a antimicrobianos de un aislamiento de Escherichia coli enterotoxigénica, determinándose la resistencia a 22 antimicrobianos categorizados en 15 grupos diferentes mediante difusión en agar, estableciéndose grupo filogenético, mecanismos de resistencia y presencia de integrones de Clase 1 y 2 mediante PCR. Integrones y genes de resistencia a β-lactámicos fueron secuenciados. El aislamiento del grupo filogenético A, mostró resistencia o sensibilidad disminuida a ampicilina, amoxicilina más ácido clavulánico, ácido nalidíxico, ciprofloxacino, estreptomicina, kanamicina, tetraciclina, trimetoprim, sulfisoxazol, cotrimoxazol, azitromicina y nitrofurantoina, detectándose la presencia de bla TEM, aadA1/2, aphA1, sul3, tet(A) y un integron de Clase 2 conteniendo un gen dfrA1. La resistencia a quinolonas se relacionó con la substitución Ser83Ala. La secuencia de TEM mostró la substitución Ala222Val, la cual a la fecha no había sido descrita, reportándose como una nueva β-lactamasa, con el nombre de bla TEM-176.

ABSTRACT The present report is the original description of bla TEM-176. The mechanisms of resistance to antimicrobial agents were determined in an enterotoxigenic Escherichia coli, determining the susceptibility to 22 antimicrobials classified in 15 different groups by agar diffusion and establishing the phylogenetic group, mechanisms of resistance and presence of Class 1 and 2 integrons. Integrons and β-lactam resistance genes were sequenced. The isolate, belonging to phylogenetic group A, showed the presence of resistance or diminished susceptibility to a ampicillin, amoxicillin plus clavulanic acid, nalidíxic acid, ciprofloxacin, streptomycin, kanamycin, tetracycline, trimethoprim, sulfisoxazole, cotrimoxazole, azithromycin and nitrofurantoin, carrying bla TEM, aadA1/2, aphA1, sul3, tet(A) and a Class 2 integron containing a dfrA1 gene. Quinolone resistance was related to the substitution Ser83Ala. The TEM sequencing showed the presence of the new substitution Ala222Val, which led to the description of the new β-lactamase bla TEM-176.

Caracterización de Escherichia coli D7111 productora de ß-LACTAMASA TEM-176 / Characterization of Escherichia coli D7111 producing the ß-LACTAMASE TEM-176

RESUMEN El presente reporte es la descripción original de bla TEM-176. Se caracterizaron los mecanismos de resistencia a antimicrobianos de un aislamiento de Escherichia coli enterotoxigénica, determinándose la resistencia a 22 antimicrobianos categorizados en 15 grupos diferentes mediante difusión en agar, estableciéndose grupo filogenético, mecanismos de resistencia y presencia de integrones de Clase 1 y 2 mediante PCR. Integrones y genes de resistencia a β-lactámicos fueron secuenciados. El aislamiento del grupo filogenético A, mostró resistencia o sensibilidad disminuida a ampicilina, amoxicilina más ácido clavulánico, ácido nalidíxico, ciprofloxacino, estreptomicina, kanamicina, tetraciclina, trimetoprim, sulfisoxazol, cotrimoxazol, azitromicina y nitrofurantoina, detectándose la presencia de bla TEM, aadA1/2, aphA1, sul3, tet(A) y un integron de Clase 2 conteniendo un gen dfrA1. La resistencia a quinolonas se relacionó con la substitución Ser83Ala. La secuencia de TEM mostró la substitución Ala222Val, la cual a la fecha no había sido descrita, reportándose como una nueva β-lactamasa, con el nombre de bla TEM-176.

ABSTRACT The present report is the original description of bla TEM-176. The mechanisms of resistance to antimicrobial agents were determined in an enterotoxigenic Escherichia coli, determining the susceptibility to 22 antimicrobials classified in 15 different groups by agar diffusion and establishing the phylogenetic group, mechanisms of resistance and presence of Class 1 and 2 integrons. Integrons and β-lactam resistance genes were sequenced. The isolate, belonging to phylogenetic group A, showed the presence of resistance or diminished susceptibility to a ampicillin, amoxicillin plus clavulanic acid, nalidíxic acid, ciprofloxacin, streptomycin, kanamycin, tetracycline, trimethoprim, sulfisoxazole, cotrimoxazole, azithromycin and nitrofurantoin, carrying bla TEM, aadA1/2, aphA1, sul3, tet(A) and a Class 2 integron containing a dfrA1 gene. Quinolone resistance was related to the substitution Ser83Ala. The TEM sequencing showed the presence of the new substitution Ala222Val, which led to the description of the new β-lactamase bla TEM-176.

Antimicrobial Susceptibility Testing in Pseudomonas aeruginosa Biofilms: One Step Closer to a Standardized Method.

The ability of Pseudomonas aeruginosa to form biofilm during a long-term infection makes it difficult to treat patients correctly. The current clinical antimicrobial susceptibility testing methods are based on the study of planktonic strains. A standardized protocol to analyze the antimicrobial susceptibility in biofilms is necessary for routine laboratories. The aims of this study were to develop a simple biofilm model and to study the antimicrobial susceptibility of P. aeruginosa strains in biofilm growth. Different artificial sputum media, and aerobiosis and microaerobiosis conditions were analyzed using a microtiter plate method and P. aeruginosa PAO1 as reference strain. Planktonic and biofilm antimicrobial susceptibility to cefepime, imipenem, azithromycin, gentamicin, tobramycin, and ciprofloxacin were determined in clinical and non-clinical P. aeruginosa strains. The Synthetic Cystic Fibrosis Medium was proposed as a good medium. The biofilm greatly increased the resistance to tested antimicrobials, except for azithromycin. Cefepime and imipenem showed poor anti-biofilm effect while tobramycin, gentamicin, and ciprofloxacin showed good activity in some strains. Azithromycin showed a better activity in biofilm than in planktonic state when aerobic conditions were used. This study establishes useful information to test antimicrobial susceptibility in P. aeruginosa biofilms, and includes possible antimicrobial options to treat long-term infected patients.

Pseudomonas aeruginosa Utilizes Host-Derived Itaconate to Redirect Its Metabolism to Promote Biofilm Formation.

The bacterium Pseudomonas aeruginosa is especially pathogenic, often being associated with intractable pneumonia and high mortality. How P. aeruginosa avoids immune clearance and persists in the inflamed human airway remains poorly understood. In this study, we show that P. aeruginosa can exploit the host immune response to maintain infection. Notably, unlike other opportunistic bacteria, we found that P. aeruginosa alters its metabolic and immunostimulatory properties in response to itaconate, an abundant host-derived immunometabolite in the infected lung. Itaconate induces bacterial membrane stress, resulting in downregulation of lipopolysaccharides (LPS) and upregulation of extracellular polysaccharides (EPS). These itaconate-adapted P. aeruginosa accumulate lptD mutations, which favor itaconate assimilation and biofilm formation. EPS, in turn, induces itaconate production by myeloid cells, both in the airway and systemically, skewing the host immune response to one permissive of chronic infection. Thus, the metabolic versatility of P. aeruginosa needs to be taken into account when designing therapies.

CFTR-PTEN-dependent mitochondrial metabolic dysfunction promotes Pseudomonas aeruginosa airway infection.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor best known for regulating cell proliferation and metabolism. PTEN forms a complex with the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) at the plasma membrane, and this complex is known to be functionally impaired in CF. Here, we demonstrated that the combined effect of PTEN and CFTR dysfunction stimulates mitochondrial activity, resulting in excessive release of succinate and reactive oxygen species. This environment promoted the colonization of the airway by Pseudomonas aeruginosa, bacteria that preferentially metabolize succinate, and stimulated an anti-inflammatory host response dominated by immune-responsive gene 1 (IRG1) and itaconate. The recruitment of myeloid cells induced by these strains was inefficient in clearing the infection and increased numbers of phagocytes accumulated under CFTR-PTEN axis dysfunction. This central metabolic defect in mitochondrial function due to impaired PTEN activity contributes to P. aeruginosa infection in CF.

Great phenotypic and genetic variation among successive chronic Pseudomonas aeruginosa from a cystic fibrosis patient.

BACKGROUND/OBJECTIVES: Different adapted Pseudomonas aeruginosa morphotypes are found during chronic infections. Relevant biological determinants in P. aeruginosa successively isolated from a cystic fibrosis (CF) patient were analyzed in this work to gain insight into P. aeruginosa heterogeneity during chronic infection. METHODS: Seventeen P. aeruginosa isolates collected from a patient over a 3 year period were included, 5 small colony variants (SCV) and 12 mucoids. The following analyses were performed: Pulsed-Field-Gel-Electrophoresis (PFGE)/Multilocus- sequence-typing (MLST)/serotype, antimicrobial susceptibility, growth curves, capacity to form biofilm, pigment production, elastase activity, motility; presence/expression of virulence/quorum sensing genes, and identification of resistance mechanisms. RESULTS: All isolates had closely related PFGE patterns and belonged to ST412. Important phenotypic and genotypic differences were found. SCVs were more resistant to antimicrobials than mucoid isolates. AmpC hyperproduction and efflux pump activity were detected. Seven isolates contained two integrons and nine isolates only one integron. All SCVs showed the same OprD profile, while three different profiles were identified among mucoids. No amino acid changes were found in MutL and MutS. All isolates were slow-growing, generally produced high biofilm, had reduced their toxin expression and their quorum sensing, and showed low motility. Nevertheless, statistically significant differences were found among SCV and mucoid isolates. SCVs grew faster, presented higher biofilm formation and flicA expression; but produced less pyorubin and pyocyanin, showed lower elastase activity and rhlR, algD, and lasB expression than mucoid isolates. CONCLUSION: These results help to understand the molecular behavior of chronic P. aeruginosa isolates in CF patients.

Loss of activity of ceftazidime-avibactam due to MexAB-OprM efflux and overproduction of AmpC cephalosporinase in Pseudomonas aeruginosa isolated from patients suffering from cystic fibrosis.

In Pseudomonas aeruginosa (P. aeruginosa) collected from cystic fibrosis (CF) patients, 24% resistance to ceftazidime-avibactam in isolates negative for carbapenemases and extended-spectrum ß-lactamases (ESBLs) has previously been observed. The current study aimed to unravel the underlying mechanism(s). Using the laboratory strain PAO1 and derivatives thereof, with ampC expression induced by a sub-minimum inhibitory concentration (MIC) of imipenem, a higher MIC of ceftazidime-avibactam was found for those overexpressing MexAB-OprM (quantitative polymerase chain reaction (PCR) of mexA) and, to a lesser extent, MexEF-OprN (PCR of mexE), or without OprD expression (SDS-Page and Coomassie blue staining). This was ascribed to (i) an efflux of avibactam (efflux mutants) and (ii) a lack of avibactam penetration (OprD mutants), respectively. We then used 10 CF clinical isolates resistant to ceftazidime (MIC ≥ 128 mg/L) and with (i) variable basal levels of ampC overexpression, (ii) mutations in mexA or mexB inactivating to variable extent the MexAB-OprM transport capacity (assessed by extrusion of N-phenyl-1-naphthylamine [NPN]), and (iii) expression or not of mexE and of OprD porin. The reduction of ceftazidime MIC in the presence of avibactam was partially lost for isolates with large efflux activity of MexAB-OprM and/or increased ampC expression, but not significantly with mexE expression or lack of OprD (non-parametric and parametric tests). This identified MexAB-OprM as a main avibactam efflux transporter in P. aeruginosa that, together with ampC overexpression, reduced avibactam potency. Since about 30% of CF isolates show mutations in MexAB-OprM compromising efflux (Chalhoub, et al. Sci Reports 2017;7:40208), routine susceptibility testing of CF P. aeruginosa with ceftazidime-avibactam is warranted.

Genotypic and phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) clones with high-level mupirocin resistance.

A high proportion of methicillin-resistant Staphylococcus aureus isolates recovered in one year period showed high-level mupirocin-resistance (HLMUPR-MRSA) in our environment (27.2%). HLMUPR-MRSA isolates were mainly collected from skin and soft tissue samples, and diabetes was the main related comorbidity condition. These isolates were more frequently found in vascular surgery. HLMUPR-MRSA was more resistant to aminoglycosides than mupirocin-susceptible MRSA, linked to the presence of bifunctional and/or nucleotidyltransferase enzymes with/without macrolide resistance associated with the msr(A) gene. Most of HLMUPR-MRSA isolates belonged to ST125/t067. Nine IS257-ileS2 amplification patterns (p3 was the most frequent) were observed in HLMUPR-MRSA isolates, suggesting the presence of several mupirocin-resistance-carrying plasmids in our environment and promoting the emergence of mupirocin resistance. The presence of the same IS257-ileS2 amplification pattern p3 in 65% of HLMUPR-MRSA, all of them ST125/t067, suggests a clonal spread in our hospital and community environment which could explain the high prevalence of HLMUPR-MRSA during the study period. An outbreak situation or an increase in mupirocin consumption was not observed.

High-level resistance to meropenem in clinical isolates of Pseudomonas aeruginosa in the absence of carbapenemases: role of active efflux and porin alterations.

High-level carbapenem resistance is worryingly increasing in clinical isolates and is often attributed to carbapenemase expression. This study aimed to determine the mechanisms leading to high-level meropenem resistance in six carbapenemase-negative Pseudomonas aeruginosa isolated from cystic fibrosis (CF) patients and seven carbapenemase-positive isolates from patients suffering from hospital-acquired pneumonia (HAP). MICs were determined in the absence or presence of l-arginine or glycine-glutamate as competitive substrates for OprD (OccD1) or OpdP (OccD3), respectively, or the efflux pump inhibitor Phe-Arg ß-naphthylamide (PAßN). ß-Lactamases were screened by phenotypic tests and/or PCR. The oprD gene and its promoter were sequenced; protein expression was evidenced by SDS-PAGE. mexA, mexX, mexC and mexE transcripts were evaluated by real-time and semiquantitative PCR. Meropenem/imipenem MICs were 64-128/16-32 mg/L and 128/128-256 mg/L in CF and HAP isolates, respectively; PAßN reduced meropenem MICs to 4-16 mg/L only and specifically in CF isolates; porin competitors had no effect on MICs. All isolates showed an increase in transcription levels of mexA, mexX and/or mexC and mutations in oprD leading to production of truncated proteins. AmpC-type cephalosporinases were overexpressed in CF isolates and VIM-2 was expressed in HAP isolates. Antibiotic exclusion from bacteria by concomitant efflux and reduced uptake is sufficient to confer high-level resistance to meropenem in isolates overexpressing AmpC-type cephalosporinases. As efflux is preponderant in these isolates, it confers a paradoxical phenotype where meropenem is less active than imipenem. Concomitant susceptibility testing of both carbapenems and rapid elucidation of the most probable resistance mechanisms is thus warranted.

Rational design of a Tn antigen mimic.

A novel Tn antigen mimic, in which the natural underlying amino acid has been replaced by the non-natural α-methylserine analogue, is reported. This derivative exhibits a similar affinity for a natural lectin as for the natural Tn and retains the bioactive conformation observed in the Tn-containing glycopeptides with anti-MUC1 antibodies.

Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia.

Characterisation of extended-spectrum beta-lactamase (ESBL) genes and their genetic environments as well as the presence of integrons were analysed in nine Klebsiella pneumoniae and two Escherichia coli ESBL-positive isolates recovered in the Centre of Bone Marrow Transplantation of Tunisia. All strains harboured the bla(CTX-M-15) gene and presented minimum inhibitory concentrations for cefotaxime and ceftazidime of 256-1024 mg L(-1) and 16-512 mg L(-1), respectively, and eight of them showed different pulsed-field gel electrophoresis patterns. The bla(OXA-1) and bla(TEM-1) genes were detected in eight and ten strains, respectively. In addition, bla(SHV-1), bla(SHV-11) and bla(SHV-27) were found in six, one and one K. pneumoniae strains, respectively. The new variant bla(SHV-103) was characterised in one K. pneumoniae strain. The intI1 gene was detected in eight K. pneumoniae strains and the dfrA5+ereA2 and aadA gene cassettes were found in one and five strains, respectively. All strains harboured a 70 kb plasmid, and its transference in addition to bla(CTX-M-15), bla(TEM-1b) and bla(OXA-1) genes was demonstrated from three K. pneumoniae to E. coli. ISEcp1 and orf477 were located upstream and downstream, respectively, of the bla(CTX-M-15) gene in 10 strains. The occurrence of the bla(CTX-M-15) gene in unrelated strains might have originated from the dissemination of mobile genetic elements in which ISEcp1 may have played an important role.