Characterization of phage evolution and phage resistance in drug-resistant Stenotrophomonas maltophilia.

Phage therapy has become a viable antimicrobial treatment as an alternative to antibiotic treatment, with an increase in antibiotic resistance. Phage resistance is a major limitation in the therapeutic application of phages, and the lack of understanding of the dynamic changes between bacteria and phages constrains our response strategies to phage resistance. In this study, we investigated the changing trends of mutual resistance between Stenotrophomonas maltophilia (S. maltophilia) and its lytic phage, BUCT603. Our results revealed that S. maltophilia resisted phage infection through mutations in the cell membrane proteins, while the evolved phage re-infected the resistant strain primarily through mutations in structure-related proteins. Compared with the wild-type strain (SMA118), the evolved phage-resistant strain (R118-2) showed reduced virulence, weakened biofilm formation ability, and reduced resistance to aminoglycosides. In addition, the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant S. maltophilia in vitro and significantly improve the survival rate of S. maltophilia-infected mice. Altogether, these results suggest that in vitro characterization of bacteria-phage co-evolutionary relationships is a useful research tool to optimize phages for the treatment of drug-resistant bacterial infections.IMPORTANCEPhage therapy is a promising approach to treat infections caused by drug-resistant Stenotrophomonas maltophilia (S. maltophilia). However, the rapid development of phage resistance has hindered the therapeutic application of phages. In vitro evolutionary studies of bacteria-phage co-cultures can elucidate the mechanism of resistance development between phage and its host. In this study, we investigated the resistance trends between S. maltophilia and its phage and found that inhibition of phage adsorption is the primary strategy by which bacteria resist phage infection in vitro, while phages can re-infect bacterial cells by identifying other adsorption receptors. Although the final bacterial mutants were no longer infected by phages, they incurred a fitness cost that resulted in a significant reduction in virulence. In addition, the combination treatment with phage and aminoglycoside antibiotics could prevent the development of phage resistance in S. maltophilia in vitro. These findings contribute to increasing the understanding of the co-evolutionary relationships between phages and S. maltophilia.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

A 20-year trend of prevalence and susceptibility to trimethoprim/sulfamethoxazole of Stenotrophomonas maltophilia in a single secondary care hospital in Korea.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can cause serious infection. We aimed to analyze the prevalence and susceptibility rates to trimethoprim/sulfamethoxazole of S. maltophilia. We conducted a retrospective study of S. maltophilia isolates from a university hospital from 2001 to 2020. Clinical information, the numbers of isolates and susceptibility rates were analyzed by year. Susceptibility rates and changes in respiratory and non-respiratory samples were compared. 1805 S. maltophilia isolates were identified, of which 81.4% (1469/1805) were from respiratory samples. There was a male predominance and 52% of the isolates were from general wards. The average susceptibility rate was 87.7% and there was no significant annual trend (P = .519). The susceptibility rate was 88.7% in respiratory samples and 84.1% in non-respiratory samples (P = .018). Susceptibility analyses using clinical data over long periods can guide the choice of antimicrobials especially for pathogen whose treatment options are limited.

Review on Stenotrophomonas maltophilia: An Emerging Multidrug- resistant Opportunistic Pathogen.

Stenotrophomonas maltophilia is an opportunistic pathogen that results in nosocomial infections in immunocompromised individuals. These bacteria colonize on the surface of medical devices and therapeutic equipment like urinary catheters, endoscopes, and ventilators, causing respiratory and urinary tract infections. The low outer membrane permeability of multidrug-resistance efflux systems and the two chromosomally encoded ß- lactamases present in S. maltophilia are challenging for arsenal control. The cell-associated and extracellular virulence factors in S. maltophilia are involved in colonization and biofilm formation on the host surfaces. The spread of antibiotic-resistant genes in the pathogenic S. maltophilia attributes to bacterial resistance against a wide range of antibiotics, including penicillin, quinolones, and carbapenems. So far, tetracycline derivatives, fluoroquinolones, and trimethoprim-sulfamethoxazole (TMP-SMX) are considered promising antibiotics against S. maltophilia. Due to the adaptive nature of the intrinsically resistant mechanism towards the number of antibiotics and its ability to acquire new resistance via mutation and horizontal gene transfer, it is quite tricky for medicinal contribution against S. maltophilia. The current review summarizes the literary data on pathogenicity, quorum sensing, biofilm formation, virulence factors, and antibiotic resistance of S. maltophilia.

Fosfomycin Resistance Evolutionary Pathways of Stenotrophomonas maltophilia in Different Growing Conditions.

The rise of multidrug-resistant Gram-negative pathogens and the lack of novel antibiotics to address this problem has led to the rescue of old antibiotics without a relevant use, such as fosfomycin. Stenotrophomonas maltophilia is a Gram-negative, non-fermenter opportunistic pathogen that presents a characteristic low susceptibility to several antibiotics of common use. Previous work has shown that while the so-far described mechanisms of fosfomycin resistance in most bacteria consist of the inactivation of the target or the transporters of this antibiotic, as well as the production of antibiotic-inactivating enzymes, these mechanisms are not selected in S. maltophilia fosfomycin-resistant mutants. In this microorganism, fosfomycin resistance is caused by the inactivation of enzymes belonging to its central carbon metabolism, hence linking metabolism with antibiotic resistance. Consequently, it is relevant to determine how different growing conditions, including urine and synthetic sputum medium that resemble infection, could impact the evolutionary pathways towards fosfomycin resistance in S. maltophilia. Our results show that S. maltophilia is able to acquire high-level fosfomycin resistance under all tested conditions. However, although some of the genetic changes leading to resistance are common, there are specific mutations that are selected under each of the tested conditions. These results indicate that the pathways of S. maltophilia evolution can vary depending on the infection point and provide information for understanding in more detail the routes of fosfomycin resistance evolution in S. maltophilia.

Infectious Diseases Society of America Guidance on the Treatment of AmpC ß-Lactamase-Producing Enterobacterales, Carbapenem-Resistant Acinetobacter baumannii, and Stenotrophomonas maltophilia Infections.

The Infectious Diseases Society of America (IDSA) is committed to providing up-to-date guidance on the treatment of antimicrobial-resistant infections. A previous guidance document focused on infections caused by extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E), carbapenem-resistant Enterobacterales (CRE), and Pseudomonas aeruginosa with difficult-to-treat resistance (DTR-P. aeruginosa). Here, guidance is provided for treating AmpC ß-lactamase-producing Enterobacterales (AmpC-E), carbapenem-resistant Acinetobacter baumannii (CRAB), and Stenotrophomonas maltophilia infections. A panel of 6 infectious diseases specialists with expertise in managing antimicrobial-resistant infections formulated questions about the treatment of AmpC-E, CRAB, and S. maltophilia infections. Answers are presented as suggested approaches and corresponding rationales. In contrast to guidance in the previous document, published data on the optimal treatment of AmpC-E, CRAB, and S. maltophilia infections are limited. As such, guidance in this document is provided as "suggested approaches" based on clinical experience, expert opinion, and a review of the available literature. Because of differences in the epidemiology of resistance and availability of specific anti-infectives internationally, this document focuses on the treatment of infections in the United States. Preferred and alternative treatment suggestions are provided, assuming the causative organism has been identified and antibiotic susceptibility results are known. Approaches to empiric treatment, duration of therapy, and other management considerations are also discussed briefly. Suggestions apply for both adult and pediatric populations. The field of antimicrobial resistance is highly dynamic. Consultation with an infectious diseases specialist is recommended for the treatment of antimicrobial-resistant infections. This document is current as of 17 September 2021 and will be updated annually. The most current version of this document, including date of publication, is available at www.idsociety.org/practice-guideline/amr-guidance-2.0/.

Subtractive genomics and molecular docking approach to identify drug targets against Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a multidrug resistant pathogen associated with high mortality and morbidity in patients having compromised immunity. The efflux systems of S. maltophilia include SmeABC and SmeDEF proteins, which assist in acquisition of multiple-drug-resistance. In this study, proteome based mapping was utilized to find out the potential drug targets for S. maltophilia strain k279a. Various tools of computational biology were applied to remove the human-specific homologous and pathogen-specific paralogous sequences from the bacterial proteome. The CD-HIT analysis selected 4315 proteins from total proteome count of 4365 proteins. Geptop identified 407 essential proteins, while the BlastP revealed approximately 85 non-homologous proteins in the human genome. Moreover, metabolic pathway and subcellular location analysis were performed for essential bacterial genes, to describe their role in various cellular processes. Only two essential proteins (Acyl-[acyl-carrier-protein]-UDP-N acetyl glucosamine O-acyltransferase and D-alanine-D-alanine ligase) as candidate for potent targets were found in proteome of the pathogen, in order to design new drugs. An online tool, Swiss model was employed to model the 3D structures of both target proteins. A library of 5000 phytochemicals was docked against those proteins through the molecular operating environment (MOE). That resulted in to eight inhibitors for both proteins i.e. enterodiol, aloin, ononin and rhinacanthinF for the Acyl-[acyl-carrier-protein]-UDP-N acetyl glucosamine O-acyltransferase, and rhazin, alkannin beta, aloesin and ancistrocladine for the D-alanine-D-alanine ligase. Finally the ADMET was done through ADMETsar. This study supported the development of natural as well as cost-effective drugs against S. maltophilia. These inhibitors displayed the effective binding interactions and safe drug profiles. However, further in vivo and in vitro validation experiment might be performed to check their drug effectiveness, biocompatibility and their role as effective inhibitors.

The role of MfsR, a TetR-type transcriptional regulator, in adaptive protection of Stenotrophomonas maltophilia against benzalkonium chloride via the regulation of mfsQ.

A gene encoding the TetR-type transcriptional regulator mfsR is located immediately downstream of mfsQ and is transcribed in the same transcriptional unit. mfsQ encodes a major facilitator superfamily (MFS) efflux transporter contributing to the resistance of Stenotrophomonas maltophilia towards disinfectants belonging to quaternary ammonium compounds (QACs), which include benzalkonium chloride (BAC). Phylogenetic analysis revealed that MfsR is closely related to CgmR, a QAC-responsive transcriptional regulator belonging to the TetR family. MfsR regulated the expression of the mfsQR operon in a QAC-inducible manner. The constitutively high transcript level of mfsQ in an mfsR mutant indicated that MfsR functions as a transcriptional repressor of the mfsQR operon. Electrophoretic mobility shift assays showed that purified MfsR specifically bound to the putative promoter region of mfsQR, and in vitro treatments with QACs led to the release of MfsR from binding complexes. DNase I protection assays revealed that the MfsR binding box comprises inverted palindromic sequences located between motifs -35 and -10 of the putative mfsQR promoter. BAC-induced adaptive protection was abolished in the mfsR mutant and was restored in the complemented mutant. Overall, MfsR is a QACs-sensing regulator that controls the expression of mfsQ. In the absence of QACs, MfsR binds to the box located in the mfsQR promoter and represses its transcription. The presence of QACs derepresses MfsR activity, allowing RNA polymerase binding and transcription of mfsQR. This MfsR-MsfQ system enables S. maltophilia to withstand high levels of QACs.

Clinical outcomes of Stenotrophomonas maltophilia infection treated with trimethoprim/sulfamethoxazole, minocycline, or fluoroquinolone monotherapy.

OBJECTIVES: The historical treatment of choice for Stenotrophomonas maltophilia infection is trimethoprim/sulfamethoxazole and this is primarily based on preclinical studies. The objective of this study was to examine the clinical outcomes of patients receiving monotherapy with different agents. METHODS: This was a retrospective study of adult patients receiving monotherapy for S. maltophilia infection with trimethoprim/sulfamethoxazole (TMP/SMX), a fluoroquinolone, or minocycline from 2010 to 2016. The primary outcome was clinical failure, a composite of recurrence, alteration of therapy due to adverse reaction or concern for clinical failure, or 30-day in-hospital mortality. The secondary outcome was 30-day in-hospital mortality. To account for treatment selection bias, multivariate regression and propensity score weighting were conducted. RESULTS: 284 patients were included (217 received TMP/SMX, 28 received a fluoroquinolone, and 39 received minocycline). The TMP/SMX and minocycline groups appeared to include similar patients whereas the fluoroquinolone group appeared to represent a slightly less severely ill population. Clinical failure was similar between groups (36%, 29%, and 31% in the TMP/SMX, fluoroquinolone, and minocycline groups, respectively, P=0.69) as was 30-day mortality (15%, 7%, and 5% in the TMP/SMX, fluoroquinolone, and minocycline groups, respectively, P=0.16). After controlling for confounding factors, receipt of minocycline (adjusted odds ratio [OR]=0.2 [0.1-0.7]) but not a fluoroquinolone (adjusted OR=0.3 [0.1 to 2.1]) was associated with lower mortality compared with TMP/SMX. This association persisted after propensity score weighting. CONCLUSIONS: Outcomes were similar or better with alternatives to TMP/SMX monotherapy, which indicates this may not be the treatment of choice for infections caused by S. maltophilia.

Emergence of fluoroquinolone resistance and possible mechanisms in clinical isolates of Stenotrophomonas maltophilia from Iran.

Stenotrophomonas maltophilia exhibits wide spectrum of fluoroquinolone resistance using different mechanisms as multidrug efflux pumps and Smqnr alleles. Here, the role of smeDEF, smeVWX efflux genes and contribution of Smqnr alleles in the development of fluoroquinolone resistance was assessed. Ciprofloxacin, levofloxacin and moxifloxacin resistance were found in 10.9%, 3.5%, and 1.6% of isolates, respectively. More than four-fold differences in ciprofloxacin MICs were detected in the presence of reserpine and smeD, F, V expression was significantly associated with ciprofloxacin resistance (p = 0.017 for smeD, 0.003 for smeF, and 0.001 for smeV). Smqnr gene was found in 52% of the ciprofloxacin-resistant isolates and Smqnr8 was the most common allele detected. Fluoroquinolone resistance in S. maltophilia clinical isolates was significantly associated with active efflux pumps. There was no correlation between the Smqnr alleles and ciprofloxacin resistance; however, contribution of the Smqnr genes in low-level levofloxacin resistance was revealed.

Antimicrobial resistance and multilocus sequence types of Stenotrophomonas maltophilia isolated from dogs and cats in Japan.

As the representative multidrug-resistant pathogen, Stenotrophomonas maltophilia has multiple intrinsic and acquired resistances, including carbapenem resistance. In companion animals, the antimicrobial susceptibility and sequence types (STs) of S. maltophilia are not well understood due to its limited isolation rate. We investigated the antimicrobial susceptibilities and multilocus sequence types (MLSTs) of 38 S. maltophilia strains isolated from dogs and cats in Japan. Prevalence of resistance was detected for imipenem (100 %), aztreonam (94.7 %), piperacillin (65.8 %), trimethoprim-sulfamethoxazole (65.8 %), and ceftazidime (60.5 %). Rates of resistances to chloramphenicol, minocycline, and levofloxacin were low (2.6-5.3 %). MLST analysis revealed that all 38 strains were assigned to 34 STs, including 11 previously reported STs and 23 newly identified STs. Phylogenetic analysis of MLSTs enabled categorization of 13 isolates (34.2 %) into genogroup 6, which is a major genogroup of human isolates. Multinational surveillance would be needed to clarify the significance of antimicrobial-resistant S. maltophilia isolates from companion animals.

Stenotrophomonas maltophilia: An emerging opportunistic nosocomial pathogen in a tertiary care hospital in Al Batinah North Governorate, Oman.

OBJECTIVES: Stenotrophomonas maltophilia, a Gram-negative non-fermentative bacillus, has emerged as an important nosocomial pathogen in recent years. It is intrinsically resistant to many antibiotics and has the ability to acquire antibiotic resistance by multiple mechanisms. Treating Stenotrophomonas infections, therefore, is a serious challenge for physicians. This study aimed to investigate the antibiotic susceptibility patterns and risk factors contributing to S. maltophilia infections. METHODS: A retrospective cross-sectional study was conducted at Sohar Hospital in Sohar, Oman. The demographic, clinical and microbiological data of individuals from whom S. maltophilia was isolated between September 2016 and August 2019 were reviewed. Descriptive statistics were presented as frequencies and percentages. RESULTS: A total of 41 S. maltophilia isolates from clinical specimens of 41 patients were studied. Infection occurred predominantly in males (73%) and the majority of patients (88%) were either ≤5 years old or >60 years old. All inpatients had at least one comorbidity while 50% had more than one. All inpatients were exposed to various medical interventions such as intensive care (44%), mechanical ventilation (41%), haemodialysis (25%), Foley's catheterisation (13%) and central venous lines (6%). Most patients (81%) were in hospital longer than two weeks. The susceptibility rates of S. maltophilia to minocycline (97%), trimethoprim-sulfamethoxazole (93%) and levofloxacin (92%) were high; the rate was lowest for ceftazidime (50%). CONCLUSION: S. maltophilia was found to be an important nosocomial opportunistic pathogen. Prolonged hospital stay and exposure to various medical interventions were key factors contributing to the development of infection. Minocycline and ceftazidime were found to be the most and least susceptible drugs, respectively.

Avibactam potentiated the activity of both ceftazidime and aztreonam against S. maltophilia clinical isolates in vitro.

BACKGROUND: Treatment options for Stenotrophomonas maltophilia (S. maltophilia) infections were limited. We assessed the efficacy of ceftazidime (CAZ), ceftazidime-avibactam (CAZ-AVI), aztreonam (ATM), and aztreonam-avibactam (ATM-AVI) against a selection of 76 S. maltophilia out of the 1179 strains isolated from the First Affiliated Hospital of Chongqing Medical University during 2011-2018. METHODS: We investigated the antimicrobial resistance profiles of the 1179 S. maltophilia clinical isolates from the first affiliated hospital of Chongqing Medical University during 2011-2018, a collection of 76 isolates were selected for further study of microbiological characterization. Minimum inhibitory concentrations (MICs) of CAZ, CAZ-AVI, ATM and ATM-AVI were determined via the broth microdilution method. We deemed that CAZ-AVI or ATM-AVI was more active in vitro than CAZ or ATM alone when CAZ-AVI or ATM-AVI led to a category change from "Resistant" or "Intermediate" with CAZ or ATM alone to "Susceptible" with CAZ-AVI or ATM-AVI, or if the MIC of CAZ-AVI or ATM-AVI was at least 4-fold lower than the MIC of CAZ or ATM alone. RESULTS: For the 76 clinical isolates included in the study, MICs of CAZ, ATM, CAZ-AVI and ATM-AVI ranged from 0.03-64, 1-1024, 0.016-64, and 0.06-64 µg/mL, respectively. In combined therapy, AVI was active at restoring the activity of 48.48% (16/33) and 89.71% (61/68) of S. maltophilia to CAZ and ATM, respectively. Furthermore, CAZ-AVI showed better results in terms of the proportion of susceptible isolates (77.63% vs. 56.58%, P < 0.001), and MIC50 (2 µg/mL vs. 8 µg/mL, P < 0.05) when compared to CAZ. According to our definition, CAZ-AVI was more active in vitro than CAZ alone for 81.58% (62/76) of the isolates. Similarly, ATM-AVI also showed better results in terms of the proportion of susceptible isolates (90.79% vs.10.53%, P < 0.001) and MIC50 (2 µg/mL vs. 64 µg/mL, P < 0.001) when compared to ATM. According to our definition, ATM-AVI was also more active in vitro than ATM alone for 94.74% (72/76) of the isolates. CONCLUSIONS: AVI potentiated the activity of both CAZ and ATM against S. maltophilia clinical isolates in vitro. We demonstrated that CAZ-AVI and ATM-AVI are both useful therapeutic options to treat infections caused by S. maltophilia.

Effect of antimicrobials on Stenotrophomonas maltophilia biofilm.

Aim: To evaluate the activity of five antimicrobials against young and mature Stenotrophomonas maltophilia biofilms. Materials & methods: Nineteen clinical strains from hemoculture of hemodialysis patients were tested for biofilm kinetics, MIC and minimum biofilm inhibitory concentration (MBIC) in young and mature biofilms. Results: All strains were moderate biofilm producers. MIC showed total susceptibility to levofloxacin and trimethoprim-sulfamethoxazole and partial resistance to ceftazidime (63.2%) and gentamicin (21%). Young and mature biofilms showed the lowest MBIC/MIC ratio for gentamicin, chloramphenicol and levofloxacin, respectively. The highest MBIC/MIC was for trimethoprim-sulfamethoxazole (young) and ceftazidime (mature). Conclusion: Gentamicin displayed surprising activity against S. maltophilia biofilms. Chloramphenicol was indicated as a good option against young S. maltophilia biofilms, and trimethoprim-sulfamethoxazole showed limited antibiofilm activity.

mfsQ encoding an MFS efflux pump mediates adaptive protection of Stenotrophomonas maltophilia against benzalkonium chloride.

The persistence of Stenotrophomonas maltophilia, especially in hospital environments where disinfectants are used intensively, is one of the important factors that allow this opportunistic pathogen to establish nosocomial infections. In the present study, we illustrated that S. maltophilia possesses adaptive resistance to the disinfectant benzalkonium chloride (BAC). This BAC adaptation was abolished in the ΔmfsQ mutant, in which a gene encoding an efflux transporter belonging to the major facilitator superfamily (MFS) was deleted. The ΔmfsQ mutant also showed increased susceptibility to BAC and chlorhexidine gluconate compared with a parental wild type. The expression of mfsQ increased upon exposure to quaternary ammonium compounds, including BAC. Thus, the results of this study suggest that mfsQ plays a role in both adaptive and nonadaptive protection of S. maltophilia from the toxicity of the disinfectant BAC.

The Antibiotic Fosfomycin Mimics the Effects of the Intermediate Metabolites Phosphoenolpyruvate and Glyceraldehyde-3-Phosphate on the Stenotrophomonas maltophilia Transcriptome.

Stenotrophomonas maltophilia is an opportunistic pathogen with an environmental origin, which presents a characteristically low susceptibility to antibiotics and is capable of acquiring increased levels of resistance to antimicrobials. Among these, fosfomycin resistance seems particularly intriguing; resistance to this antibiotic is generally due to the activity of fosfomycin-inactivating enzymes, or to defects in the expression or the activity of fosfomycin transporters. In contrast, we previously described that the cause of fosfomycin resistance in S. maltophilia was the inactivation of enzymes belonging to its central carbon metabolism. To go one step further, here we studied the effects of fosfomycin on the transcriptome of S. maltophilia compared to those of phosphoenolpyruvate-its structural homolog-and glyceraldehyde-3-phosphate-an intermediate metabolite of the mutated route in fosfomycin-resistant mutants. Our results show that transcriptomic changes present a large degree of overlap, including the activation of the cell-wall-stress stimulon. These results indicate that fosfomycin activity and resistance are interlinked with bacterial metabolism. Furthermore, we found that the studied compounds inhibit the expression of the smeYZ efflux pump, which confers intrinsic resistance to aminoglycosides. This is the first description of efflux pump inhibitors that can be used as antibiotic adjuvants to counteract antibiotic resistance in S. maltophilia.

Stenotrophomonas maltophilia phenotypic and genotypic features through 4-year cystic fibrosis lung colonization.

Introduction. Stenotrophomonas maltophilia has emerged as one of the most common multi-drug-resistant pathogens isolated from people with cystic fibrosis (CF). However, its adaptation over time to CF lungs has not been fully established.Hypothesis. Sequential isolates of S. maltophilia from a Brazilian adult patient are clonally related and show a pattern of adaptation by loss of virulence factors.Aim. To investigate antimicrobial susceptibility, clonal relatedness, mutation frequency, quorum sensing (QS) and selected virulence factors in sequential S. maltophilia isolates from a Brazilian adult patient attending a CF referral centre in Buenos Aires, Argentina, between May 2014 and May 2018.Methodology. The antibiotic resistance of 11 S. maltophilia isolates recovered from expectorations of an adult female with CF was determined. Clonal relatedness, mutation frequency, QS variants (RpfC-RpfF), QS autoinducer (DSF) and virulence factors were investigated in eight viable isolates.Results. Seven S. maltophilia isolates were resistant to trimethoprim-sulfamethoxazole and five to levofloxacin. All isolates were susceptible to minocycline. Strong, weak and normomutators were detected, with a tendency to decreased mutation rate over time. XbaI PFGE revealed that seven isolates belong to two related clones. All isolates were RpfC-RpfF1 variants and DSF producers. Only two isolates produced weak biofilms, but none displayed swimming or twitching motility. Four isolates showed proteolytic activity and amplified stmPr1 and stmPr2 genes. Only the first three isolates were siderophore producers. Four isolates showed high resistance to oxidative stress, while the last four showed moderate resistance.Conclusion. The present study shows the long-time persistence of two related S. maltophilia clones in an adult female with CF. During the adaptation of the prevalent clones to the CF lungs over time, we identified a gradual loss of virulence factors that could be associated with the high amounts of DSF produced by the evolved isolates. Further, a decreased mutation rate was observed in the late isolates. The role of all these adaptations over time remains to be elucidated from a clinical perspective, probably focusing on the damage they can cause to CF lungs.

Polypyridine ligands as potential metallo-ß-lactamase inhibitors.

Bacteria have developed multiple resistance mechanisms against the most used antibiotics. In particular, zinc-dependent metallo-ß-lactamase producing bacteria are a growing threat, and therapeutic options are limited. Zinc chelators have recently been investigated as metallo-ß-lactamase inhibitors, as they are often able to restore carbapenem susceptibility. We synthesized polypyridyl ligands, N,N'-bis(2-pyridylmethyl)-ethylenediamine, N,N,N'-tris(2-pyridylmethyl)-ethylenediamine, N,N'-bis(2-pyridylmethyl)-ethylenediamine-N-acetic acid (N,N,N'-tris(2-pyridylmethyl)-ethylenediamine-N'-acetic acid, which can form zinc(II) complexes. We tested their ability to restore the antibiotic activity of meropenem against three clinical strains isolated from blood and metallo-ß-lactamase producers (Klebsiella pneumoniae, Enterobacter cloacae, and Stenotrophomonas maltophilia). We functionalized N,N,N'-tris(2-pyridylmethyl)-ethylenediamine with D-alanyl-D-alanyl-D-alanine methyl ester with the aim to increase bacterial uptake. We observed synergistic activity of four polypyridyl ligands with meropenem against all tested isolates, while the combination N,N'-bis(2-pyridylmethyl)-ethylenediamine and meropenem was synergistic only against New Delhi and Verona integron-encoded metallo-ß-lactamase-producing bacteria. All synergistic interactions restored the antimicrobial activity of meropenem, providing a significant decrease of minimal inhibitory concentration value (by 8- to 128-fold). We also studied toxicity of the ligands in two normal peripheral blood lymphocytes.

Detection of multidrug resistant environmental isolates of acinetobacter and Stenotrophomonas maltophilia: a possible threat for community acquired infections?

Acinetobacter spp. and Stenotrophomonas maltophilia are bacteria commonly associated with infections at the clinical settings. Reports of infections caused by environmental isolates are rare. Therefore, this study focused on determination of the antibiotic resistance patterns, antibiotic resistance genes, efflux pumps and virulence signatures of Acinetobacter spp. and S. maltophilia recovered from river water, plant rhizosphere and river sediment samples. The isolates were identified and confirmed using biochemical tests and PCR. The antimicrobial resistance profiles of the isolates were determined using Kirby Bauer disk diffusion assay and presence of antibiotic resistance and virulence genes were detected using PCR. S. maltophilia was more frequent in plant rhizosphere and sediment samples than the water samples. Acinetobacter spp. were mostly resistant to trimethoprim-sulfamethoxazole (96% of isolates), followed by polymyxin b (86%), cefixime (54%), colistin (42%), ampicillin (35%) and meropenem (19%). The S. maltophilia isolates displayed total resistance (100%) to trimethoprim- sulfamethoxazole, meropenem, imipenem, ampicillin and cefixime, while 80% of the isolates were resistant to ceftazidime. Acinetobacter spp. contained different antibiotic resistance genes such as sul1 (24% of isolates), sul2 (29%), blaOXA 23/51 (21%) and blaTEM (29%), while S. maltophilia harbored sul1 (8%) and blaTEM (20%). Additionally, efflux pump genes were present in all S. maltophilia isolates. The presence of multidrug resistant Acinetobacter spp. and Stenotrophomonas maltophilia in surface water raises concerns for community-acquired infections as this water is directly been used by the community for various purposes. Therefore, there is the need to institute measures aimed at reducing the risks of these infections and the resulting burden this may have on the health care system within the study area.