Strain differences in the susceptibility and resistance of Pasteurella multocida to phagocytosis and killing by rabbit polymorphonuclear neutrophils. - Related Documents




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621201.0000Strain differences in the susceptibility and resistance of Pasteurella multocida to phagocytosis and killing by rabbit polymorphonuclear neutrophils. The interactions of 2 capsular serotype A and 4 serotype D strains of Pasteurella multocida with rabbit polymorphonuclear neutrophils (PMN) were compared in vitro, using a PMN phagocytic and bactericidal assay. Bacteria and rabbit PMN were incubated for 15 minutes. The suspensions were subjected to differential centrifugation and the percentage of phagocytosis (cell association) was determined from the number of viable noncell-associated bacteria. The cell pellets and the associated bacteria were resuspended and PMN bactericidal activity was calculated from the number of remaining viable cell-associated bacteria at 45 and 75 minutes after the start of the assay. Test bacteria were not opsonized or were opsonized with immune serum containing active complement. One type A strain was ingested and killed by PMN in the presence and absence of opsonins. The 5 remaining strains were resistant to PMN killing, but only the type A strain resisted phagocytosis. Resistance of the type A strain was attributed to the hyaluronic acid capsule, since pretreatment of the bacteria with hyaluronidase rendered opsonized bacteria susceptible to ingestion and killing. The pattern of resistance of the 4 type D strains was different from that of the resistant type A strain. Both opsonized and nonopsonized type D bacteria became cell associated, but none were killed by PMN. The mechanism of resistance of these 4 strains to PMN bactericidal activity is currently unknown.19846742581
617110.9995Host response to infection with a temperature-sensitive mutant of Salmonella typhimurium in a susceptible and a resistant strain of mice. The inoculation of a temperature-sensitive mutant of Salmonella typhimurium induced a long-lasting infection in susceptible (C57BL/6) and resistant (A/J) mice. During week 1 of infection, the number of bacteria in the spleens was similar in both mouse strains. Then, the decrease of bacteria was more rapid in the resistant strain. Splenomegaly and granulomatous hepatitis were more severe in the susceptible strain. The immune response induced by this infection was studied. In both mouse strains delayed-type hypersensitivity to Salmonella antigens was present, and resistance to reinfection with a virulent strain of S. typhimurium or with Listeria monocytogenes appeared with the same kinetics. Thus, it does not seem that the gene(s) controlling natural resistance to S. typhimurium act(s) on acquired immunity.19853897053
617020.9995Resistance and susceptibility of mice to bacterial infection. IV. Functional specificity in natural resistance to facultative intracellular bacteria. The effect of opsonic antibody on resistance of susceptibility of three strains of mice, C57Bl/10, BALB/c, and CBA to the intracellular bacteria Listeria monocytogenes, Salmonella typhimurium, and Brucella abortus was tested. Bacteria were opsonized by serum treatment before their injection into mice, or the mice were preimmunized by injection with alcohol killed bacteria which induces antibody without macrophage activation. Antibody did not increase the rate of clearance of Listeria from the bloodstream, nor did it affect the subsequent growth of that organism in the spleen and liver. Blood clearance of S. typhimurium and of B. abortus was increased by preopsonization with specific antibody, indicating that opsonins were a limiting factor in resistance to these two bacteria. However, neither opsonization before infection nor immunization with alcohol killed vaccines had any effect on the strain distribution of resistance/susceptibility, which differs for each of the three intracellular pathogens. Thus, even in the presence of adequate opsonization the three strains of mice showed different patterns of resistance/susceptibility to Listeria, S. typhimurium, and B. abortus. This implies that each has a unique cellular mechanism of early nonspecific resistance.19836413682
480830.9994Key Roles of Human Polymorphonuclear Cells and Ciprofloxacin in Lactobacillus Species Infection Control. Lactobacilli have the potential to act as reservoirs of antibiotic resistance genes similar to those found in human pathogens, with the risk of transferring these genes to many pathogenic bacteria. In this study, we investigated the role of human polymorphonuclear cells (PMNs) against Lactobacillus spp. both resistant and susceptible to ciprofloxacin (a fluoroquinolone) and the effect of ciprofloxacin on the interaction between PMNs and three Lactobacillus spp. with different patterns of susceptibility to this drug. Hence, the primary functions of PMNs, such as phagocytosis and bacterial intracellular killing, against lactobacilli were investigated. The rate of PMN phagocytosis was high for ciprofloxacin-sensitive and ciprofloxacin-resistant strains. The patterns of intracellular killing of ciprofloxacin-sensitive and ciprofloxacin-resistant strains by PMNs underline that PMNs alone were able to kill lactobacilli. The addition of ciprofloxacin to PMNs did not result in a significant increase in the bacterial uptake by phagocytes. On the contrary, ciprofloxacin had a marked effect on PMN intracellular killing, resulting in increased numbers of killed ciprofloxacin-sensitive bacteria in comparison with antibiotic-free controls. Our data show that by itself, the profile of antibiotic resistance does not constitute an intrinsic factor of greater or lesser pathogenicity toward the host. The ability of PMNs to kill a diverse array of bacterial pathogens is essential for human innate host defense, primarily in immunocompromised patients.201526711767
617240.9994Resistance and susceptibility of mice to bacterial infection. IV. Genetic and cellular basis of resistance to chronic infection with Brucella abortus. The number of Brucella abortus strain 19 organisms in the spleens of CBA/H mice peaked two weeks after intravenous injection of 5 X 10(6) organisms. With the onset of specific cell-mediated immunity, 90% of the bacteria were killed, but approximately 10(6) bacteria persisted up to seven weeks after infection. In contrast, in BALB/c, C57BL/10, and B10Br mice, bacterial numbers peaked at two weeks but decreased steadily with the onset of bactericidal activity. In all strains, clearance of bacteria from the liver was relatively efficient. The course of infection in (CBA/H X BALB/c) F1 mice was similar to that in CBA/H mice, indicating that the mechanism(s) leading to slower recovery from infection was dominant. The H-2 haplotype of the mice did not influence the rate of recovery from infection. The use of backcross mice showed that multiple genes were involved. In bone marrow-chimeric mice, resistance was determined by the genome of the bone marrow donor, not that of the host.19826809847
478750.9992Strain Specific Variations in Acinetobacter baumannii Complement Sensitivity. The complement system is required for innate immunity against Acinetobacter baumannii, an important cause of antibiotic resistant systemic infections. A. baumannii strains differ in their susceptibility to the membrane attack complex (MAC) formed from terminal complement pathway proteins, but the reasons for this variation remain poorly understood. We have characterized in detail the complement sensitivity phenotypes of nine A. baumannii clinical strains and some of the factors that might influence differences between strains. Using A. baumannii laboratory strains and flow cytometry assays, we first reconfirmed that both opsonization with the complement proteins C3b/iC3b and MAC formation were inhibited by the capsule. There were marked differences in C3b/iC3b and MAC binding between the nine clinical A. baumannii strains, but this variation was partially independent of capsule composition or size. Opsonization with C3b/iC3b improved neutrophil phagocytosis of most strains. Importantly, although C3b/iC3b binding and MAC formation on the bacterial surface correlated closely, MAC formation did not correlate with variations between A. baumannii strains in their levels of serum resistance. Genomic analysis identified only limited differences between strains in the distribution of genes required for serum resistance, but RNAseq data identified three complement-resistance genes that were differentially regulated between a MAC resistant and two MAC intermediate resistant strains when cultured in serum. These data demonstrate that clinical A. baumannii strains vary in their sensitivity to different aspects of the complement system, and that the serum resistance phenotype was influenced by factors in addition to the amount of MAC forming on the bacterial surface.202235812377
621960.9992Isolation and characterization of bacteriophage-resistant mutants of Vibrio cholerae O139. Vibrio cholerae O139 strains produce a capsule which is associated with complement resistance and is used as a receptor by bacteriophage JA1. Spontaneous JA1-resistant mutants were found to have several phenotypes, with loss of capsule and/or O-antigen from the cell surface. Determination of the residual complement resistance and infant mouse colonization potential of each mutant suggested that production of O-antigen is of much greater significance than the presence of capsular material for both of these properties. Two different in vitro assays of complement resistance were compared and the results of one shown to closely reflect the comparative recoveries of bacteria from the colonization experiments. Preliminary complementation studies implicated two rfb region genes, wzz and wbfP, as being essential for the biosynthesis of capsule but not O-antigen.200111312617
42170.9992Effect of pap copy number and receptor specificity on virulence of fimbriated Escherichia coli in a murine urinary tract colonization model. Escherichia coli FN506 containing pap genes that encode two different P fimbriae adherence specificity types were tested for virulence in a murine urinary colonization model. Strains containing adherence genes on either high copy or low copy plasmids were compared. Bacteria that harbored the adherence genes on high copy plasmids colonized mouse kidneys less well than bacteria with the same adherence genes in low copy even though the high copy strains exhibited greater hemagglutination capacity. Bacteria with either type of P fimbriae were able to colonize but pap-2+ bacteria showed increased colonizing capacity when strains containing pap-1 or pap-2 genes on low copy plasmids were compared. Bacteria containing plasmids with both adherence specificities had a similar colonizing capacity as bacteria with either type separately.19947861959
622780.9992Lactoferrin Disaggregates Pneumococcal Biofilms and Inhibits Acquisition of Resistance Through Its DNase Activity. Streptococcus pneumoniae colonizes the upper airways of children and the elderly. Colonization progresses to persistent carriage when S. pneumoniae forms biofilms, a feature required for the development of pneumococcal disease. Nasopharyngeal biofilms are structured with a matrix that includes extracellular DNA (eDNA), which is sourced from the same pneumococci and other bacteria. This eDNA also allows pneumococci to acquire new traits, including antibiotic resistance genes. In this study, we investigated the efficacy of lactoferrin (LF), at physiological concentrations found in secretions with bactericidal activity [i.e., colostrum (100 μM), tears (25 μM)], in eradicating pneumococcal biofilms from human respiratory cells. The efficacy of synthetic LF-derived peptides was also assessed. We first demonstrated that LF inhibited colonization of S. pneumoniae on human respiratory cells without affecting the viability of planktonic bacteria. LF-derived peptides were, however, bactericidal for planktonic pneumococci but they did not affect viability of pre-formed biofilms. In contrast, LF (40 and 80 μM) eradicated pneumococcal biofilms that had been pre-formed on abiotic surfaces (i.e., polystyrene) and on human pharyngeal cells, as investigated by viable counts and confocal microscopy. LF also eradicated biofilms formed by S. pneumoniae strains with resistance to multiple antibiotics. We investigated whether treatment with LF would affect the biofilm structure by analyzing eDNA. Surprisingly, in pneumococcal biofilms treated with LF, the eDNA was absent in comparison to the untreated control (∼10 μg/ml) or those treated with LF-derived peptides. EMSA assays showed that LF binds S. pneumoniae DNA and a time-course study of DNA decay demonstrated that the DNA is degraded when bound by LF. This LF-associated DNase activity inhibited acquisition of antibiotic resistance genes in both in vitro transformation assays and in a life-like bioreactor system. In conclusion, we demonstrated that LF eradicates pneumococcal-colonizing biofilms at a concentration safe for humans and identified a LF-associated DNAse activity that inhibited the acquisition of resistance.201931681240
631790.9992O-specific polysaccharide confers lysozyme resistance to extraintestinal pathogenic Escherichia coli. Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bloodstream and other extraintestinal infections in human and animals. The greatest challenge encountered by ExPEC during an infection is posed by the host defense mechanisms, including lysozyme. ExPEC have developed diverse strategies to overcome this challenge. The aim of this study was to characterize the molecular mechanism of ExPEC resistance to lysozyme. For this, 15,000 transposon mutants of a lysozyme-resistant ExPEC strain NMEC38 were screened; 20 genes were identified as involved in ExPEC resistance to lysozyme-of which five were located in the gene cluster between galF and gnd, and were further confirmed to be involved in O-specific polysaccharide biosynthesis. The O-specific polysaccharide was able to inhibit the hydrolytic activity of lysozyme; it was also required by the complete lipopolysaccharide (LPS)-mediated protection of ExPEC against the bactericidal activity of lysozyme. The O-specific polysaccharide was further shown to be able to directly interact with lysozyme. Furthermore, LPS from ExPEC strains of different O serotypes was also able to inhibit the hydrolytic activity of lysozyme. Because of their cell surface localization and wide distribution in Gram-negative bacteria, O-specific polysaccharides appear to play a long-overlooked role in protecting bacteria against exogenous lysozyme.201829405825
6213100.9992Use of a Dictyostelium model for isolation of genetic loci associated with phagocytosis and virulence in Klebsiella pneumoniae. Phagocytosis resistance is an important virulence factor in Klebsiella pneumoniae. Dictyostelium has been used to study the interaction between phagocytes and bacteria because of its similarity to mammalian macrophages. In this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2,500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium. In the 29 mutants, six loci were identified; three were capsular synthesis genes. Of the other three, one was related to carnitine metabolism, one encoded a subunit of protease (clpX), and one encoded a lipopolysaccharide O-antigen transporter (wzm). Deletion and complementation of these genes showed that only ΔclpX and Δwzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae. Furthermore, we demonstrated that clpP, which exists in an operon with clpX, was also involved in resistance to phagocytosis. The transcriptional profile of ΔclpX was examined by microarray analysis and revealed a 3-fold lower level of expression of capsular synthesis genes. Therefore, we have identified genes involved in resistance to phagocytosis in K. pneumoniae using Dictyostelium, and this model is useful to explore genes associated with resistance to phagocytosis in heavily encapsulated bacteria.201121173313
8928110.9992Increased survival of antibiotic-resistant Escherichia coli inside macrophages. Mutations causing antibiotic resistance usually incur a fitness cost in the absence of antibiotics. The magnitude of such costs is known to vary with the environment. Little is known about the fitness effects of antibiotic resistance mutations when bacteria confront the host's immune system. Here, we study the fitness effects of mutations in the rpoB, rpsL, and gyrA genes, which confer resistance to rifampin, streptomycin, and nalidixic acid, respectively. These antibiotics are frequently used in the treatment of bacterial infections. We measured two important fitness traits-growth rate and survival ability-of 12 Escherichia coli K-12 strains, each carrying a single resistance mutation, in the presence of macrophages. Strikingly, we found that 67% of the mutants survived better than the susceptible bacteria in the intracellular niche of the phagocytic cells. In particular, all E. coli streptomycin-resistant mutants exhibited an intracellular advantage. On the other hand, 42% of the mutants incurred a high fitness cost when the bacteria were allowed to divide outside of macrophages. This study shows that single nonsynonymous changes affecting fundamental processes in the cell can contribute to prolonged survival of E. coli in the context of an infection.201323089747
6244120.9992Whole-genome sequencing reveals a link between β-lactam resistance and synthetases of the alarmone (p)ppGpp in Staphylococcus aureus. The overwhelming majority of methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates exhibit a peculiar heterogeneous resistance to β-lactam antibiotics: in cultures of such strains, the majority of cells display only a low level of methicillin resistance--often close to the MIC breakpoint of susceptible strains. Yet, in the same cultures, subpopulations of bacteria exhibiting very high levels of resistance are also present with variable frequencies, which are characteristic of the particular MRSA lineage. The mechanism of heterogeneous resistance is not understood. We describe here an experimental system for exploring the mechanism of heterogeneous resistance. Copies of the resistance gene mecA cloned into a temperature-sensitive plasmid were introduced into the fully sequenced methicillin-susceptible clinical isolate S. aureus strain 476. Transductants of strain 476 expressed methicillin resistance in a heterogeneous fashion: the great majority of cells showed only low MIC (0.75 μg/ml) for the antibiotic, but a minority population of highly resistant bacteria (MIC >300 μg/ml) was also present with a frequency of ∼10(-4). The genetic backgrounds of the majority and minority cells were compared by whole-genome sequencing: the only differences detectable were two point mutations in relA of the highly resistant minority population of bacteria. The relA gene codes for the synthesis of (p)ppGpp, an effector of the stringent stress response. Titration of (p)ppGpp showed increased amounts of this effector in the highly resistant cells. Involvement of (p)ppGpp synthesis genes may explain some of the perplexing aspects of β-lactam resistance in MRSA, since many environmental and genetic changes can modulate cellular levels of (p)ppGpp.201323659600
6328130.9991Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis. Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis.201828847541
6250140.9991High prevalence of heteroresistance in Staphylococcus aureus is caused by a multitude of mutations in core genes. Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.202438175839
3805150.9991De Novo Characterization of Genes That Contribute to High-Level Ciprofloxacin Resistance in Escherichia coli. Sensitization of resistant bacteria to existing antibiotics depends on the identification of candidate targets whose activities contribute to resistance. Using a transposon insertion library in an Escherichia coli mutant that was 2,000 times less susceptible to ciprofloxacin than its parent and the relative fitness scores, we identified 19 genes that contributed to the acquired ciprofloxacin resistance and mapped the shortest genetic path that increased the antibiotic susceptibility of the resistant bacteria back to a near wild-type level.201627431218
4785160.9991Study of MazEF, sam, and phd-doc putative toxin-antitoxin systems in Staphylococcus epidermidis. Today, to replace the antibacterial targets to overcome antibiotic resistance, toxin-antitoxin (TA) system is noticeable, where the unstable antitoxin neutralizes the stable toxin and protects the bacteria against the toxic effects. The presence and expression of TA genes in clinical and non-clinical strains of Staphylococcus epidermidis were investigated in this study. After identification of three TA pairs (mazEF, sam, and phd-doc) via existing databases (earlier, there has been no information in the case of S. epidermidis isolates), the presence and expression of these pairs were investigated by PCR and q-PCR, respectively. We detected three TA modules in all antibiotic sensitive and resistant isolates. In addition, q-PCR analysis revealed that the transcripts were produced from the three TA modules. This study showed the significant prevalence of these systems in pathogenic bacteria and they were equally found in both oxacillin-resistant and oxacillin-susceptible bacteria. The high prevalence of three systems can make them suitable as potential targets for antibiotic therapy.201829471693
4495170.9991Mutations in the bacterial ribosomal protein l3 and their association with antibiotic resistance. Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin.201525845869
6040180.9991Investigating the antibacterial effects of some Lactobacillus, Bifidobacterium and acetobacter strains killed by different methods on Streptococcus mutans and Escherichia coli. Although there are many health advantages assigned to different live bacteria such as probiotics, some health threatening effects have also been reported. For example, live bacteria can transfer antibiotic resistance genes to other commensal and opportunistic bacteria of gastrointestinal tract. Recently, it was shown that using killed bacteria have some advantages over live ones. In this research, heat, paraformaldehyde and ozone killing methods were used to kill the bacteria. Acetobacter cerevisiae, Lactobacillus acidophilus, Bifidobacterium lactis and traditional vinegar and fermented dairy product (Kumeh) derived bacteria were killed and their antibacterial activity against Streptococcus mutans and Escherichia coli was investigated. To identify the bacteria isolated from the traditional products, 16S rDNA gene was partially sequenced. The gene analysis showed vinegar and Kumeh derived bacteria were Acetobacter pasteurianus and Lactobacillus crustorum (LcK) strains respectively. The S. mutans growth inhibition was detected in the all concentrations of all killed samples. However, generally, E. coli showed more resistant to the killed bacteria than S. mutans and the antibacterial effect of heat-killed bacteria against E. coli was not observed in the all concentrations for some killed bacteria. Among the pathogenic bacteria, S. mutans was the most sensitive one to the killed bacteria with 70% of reduction in its viability. In conclusion, this research showed that different killed bacteria had different effects on other bacteria and the killing method showed an impact on these effects. Overall, paraformaldehyde-killed L.crustorum (LcK) showed the best antibacterial activity against S. mutans; about 70% decrease in bacterial viability.201931998811
6295190.9991Anaerobiosis and Mutations Can Reduce Susceptibility of Pseudomonas aeruginosa to Tobramycin Without Reducing the Cellular Concentration of the Antibiotic. Chronic infections of Pseudomonas aeruginosa are commonly treated with tobramycin. During infections, the bacteria can exist under conditions of oxygen deprivation that render them less susceptible to this antibiotic. The aims of this research were to investigate the genetic basis of tobramycin resistance under anaerobic conditions, and to investigate the effects of anaerobiosis and mutations on the cellular concentration of tobramycin. Ten mutants with lowered susceptibility to tobramycin than wild-type bacteria were evolved from a laboratory reference strain under anaerobic conditions. Mutations were identified by genome sequencing. Mutations had arisen most frequently in the fusA1 gene that encodes elongation factor EF-G1A and in genes involved in twitching motility. Cellular concentrations of tobramycin were then measured. Mutations in fusA1 or absence of the MexXY efflux pump that is associated with tobramycin resistance did not alter the cellular tobramycin concentration under either anaerobic or aerobic conditions. Anaerobic growth reduced the cellular concentration of tobramycin, relative to aerobically grown bacteria, in some but not all of five tested P. aeruginosa isolates. Overall, our findings indicate that anaerobiosis and mutations that reduce aminoglycoside effectiveness do not lower the cellular concentration of antibiotic but instead reduce susceptibility through other mechanisms.202540005562