Dinesh M. Fernando

Growth phase-dependent expression of RND efflux pump- and outer membrane porin-encoding genes in Acinetobacter baumannii ATCC 19606

OBJECTIVES To analyse the expression of resistance-nodulation-division (RND) efflux pumps and outer membrane porins during various growth phases of Acinetobacter baumannii ATCC 19606. METHODS Expression of five different RND pump-encoding genes (adeB, adeG, adeJ, AciBau_2436 and AciBau_2746) and two outer membrane porin-encoding genes (carO and oprD) was analysed at four growth timepoints, representing early, mid and late log phases and the stationary phase using quantitative RT-PCR. RESULTS The adeB and adeJ RND pump genes were expressed at higher levels than all of the other RND pumps, though their expression was reduced at higher cell density. The adeG gene was found to be constitutively expressed, albeit at much lower levels than those seen for adeB or adeJ. The previously uncharacterized AciBau_2436 was found to be expressed in the early exponential phase. The expression level of oprD was found to be inversely proportional to cell density, while that of carO was found to increase in the mid-log phase but then decrease in the later stages of the growth phase. CONCLUSIONS This work shows cell density-dependent expression of adeB, adeJ, AciBau_2436, carO and oprD genes, suggesting a role of global regulatory mechanisms in the expression of these genes in A. baumannii ATCC 19606.

Adjuvants Based on Hybrid Antibiotics Overcome Resistance in Pseudomonas aeruginosa and Enhance Fluoroquinolone Efficacy

The use of adjuvants that rescue antibiotics against multidrug-resistant (MDR) pathogens is a promising combination strategy for overcoming bacterial resistance. While the combination of β-lactam antibiotics and β-lactamase inhibitors has been successful in restoring antibacterial efficacy in MDR bacteria, the use of adjuvants to restore fluoroquinolone efficacy in MDR Gram-negative pathogens has been challenging. We describe tobramycin-ciprofloxacin hybrid adjuvants that rescue the activity of fluoroquinolone antibiotics against MDR and extremely drug-resistant Pseudomonas aeruginosa isolates in vitro and enhance fluoroquinolone efficacy in vivo. Structure-activity studies reveal that the presence of both tobramycin and ciprofloxacin, which are separated by a C12 tether, is critical for the function of the adjuvant. Mechanistic studies indicate that the antibacterial modes of ciprofloxacin are retained while the role of tobramycin is limited to destabilization of the outer membrane in the hybrid.

Detection of Antibiotic Resistance Genes in Source and Drinking Water Samples from a First Nations Community in Canada

ABSTRACT Access to safe drinking water is now recognized as a human right by the United Nations. In developed countries like Canada, access to clean water is generally not a matter of concern. However, one in every five First Nations reserves is under a drinking water advisory, often due to unacceptable microbiological quality. In this study, we analyzed source and potable water from a First Nations community for the presence of coliform bacteria as well as various antibiotic resistance genes. Samples, including those from drinking water sources, were found to be positive for various antibiotic resistance genes, namely, ampC, tet(A), mecA, β-lactamase genes (SHV-type, TEM-type, CTX-M-type, OXA-1, and CMY-2-type), and carbapenemase genes (KPC, IMP, VIM, NDM, GES, and OXA-48 genes). Not surprisingly, substantial numbers of total coliforms, including Escherichia coli, were recovered from these samples, and this result was also confirmed using Illumina sequencing of the 16S rRNA gene. These findings deserve further attention, as the presence of coliforms and antibiotic resistance genes potentially puts the health of the community members at risk. IMPORTANCE In this study, we highlight the poor microbiological quality of drinking water in a First Nations community in Canada. We examined the coliform load as well as the presence of antibiotic resistance genes in these samples. This study examined the presence of antibiotic-resistant genes in drinking water samples from a First Nations Community in Canada. We believe that our findings are of considerable significance, since the issue of poor water quality in First Nations communities in Canada is often ignored, and our findings will help shed some light on this important issue.

Triclosan Can Select for an AdeIJK-Overexpressing Mutant of Acinetobacter baumannii ATCC 17978 That Displays Reduced Susceptibility to Multiple Antibiotics

ABSTRACT In order to determine if triclosan can select for mutants of Acinetobacter baumannii ATCC 17978 that display reduced susceptibilities to antibiotics, we isolated a triclosan-resistant mutant, A. baumannii AB042, by serial passaging of A. baumannii ATCC 17978 in growth medium supplemented with triclosan. The antimicrobial susceptibility of AB042 was analyzed by the 2-fold serial dilution method. Expression of five different resistance-nodulation-division (RND) pump-encoding genes (adeB, adeG, adeJ, A1S_2818, and A1S_3217), two outer membrane porin-encoding genes (carO and oprD), and the MATE family pump-encoding gene abeM was analyzed using quantitative reverse transcriptase (qRT) PCR. A. baumannii AB042 exhibited elevated resistance to multiple antibiotics, including piperacillin-tazobactam, doxycycline, moxifloxacin, ceftriaxone, cefepime, meropenem, doripenem, ertapenem, ciprofloxacin, aztreonam, tigecycline, and trimethoprim-sulfamethoxazole, in addition to triclosan. Genome sequencing of A. baumannii AB042 revealed a 116G→V mutation in fabI, the gene encoding the target enzyme for triclosan. Expression analysis of efflux pumps showed overexpression of the AdeIJK pump, and sequencing of adeN, the gene that encodes the repressor of the adeIJK operon, revealed a 73-bp deletion which would cause a premature termination of translation, resulting in an inactive truncated AdeN protein. This work shows that triclosan can select for mutants of A. baumannii that display reduced susceptibilities to multiple antibiotics from chemically distinct classes in addition to triclosan resistance. This multidrug resistance can be explained by the overexpression of the AdeIJK efflux pump.

Antibiotic resistance and expression of resistance-nodulation-division pump- and outer membrane porin-encoding genes in Acinetobacter species isolated from Canadian hospitals.

BACKGROUND Bacterial pathogens belonging to the genus Acinetobacter cause serious infections in immunocompromised individuals that are very difficult to treat due to their extremely high resistance to many antibiotics. OBJECTIVE To investigate the role of resistance-nodulation-division (RND) pumps and porins in the antibiotic resistance of Acinetobacter species collected from Canadian hospitals. METHODS Clinical isolates of Acinetobacter species collected from Canadian hospitals were analyzed for the expression of genes encoding RND pumps (adeB, adeG, adeJ, AciBau_2746 and AciBau_2436) and outer membrane porins (carO, 33 kDa porin and oprD) using quantitative reverse transcription (qRT) polymerase chain reaction. Species identification of the isolates was performed using a multiplex polymerase chain reaction method for gyrB. RESULTS The expression of RND pump-encoding genes was widespread in the clinical isolates of Acinetobacter species, with each of the isolates expressing at least one RND pump. adeG was found to be overexpressed in all of the isolates, while adeB was found to be overexpressed in only two isolates. Among the porin-encoding genes, the expression of carO was considerably downregulated among the majority of isolates. CONCLUSION The present study was the first to analyze the expression of RND pump- and porin-encoding genes in the clinical isolates of Acinetobacter species from Canadian hospitals. The overexpression of genes encoding RND pumps and the downregulation of genes encoding porins was common in clinical isolates of Acinetobacter species from Canadian hospitals, with the AdeFGH pump being the most commonly expressed RND pump.

Resistance-Nodulation-Division Multidrug Efflux Pumps in Gram-Negative Bacteria: Role in Virulence

Resistance-Nodulation-Division (RND) efflux pumps are one of the most important determinants of multidrug resistance (MDR) in Gram-negative bacteria. With an ever increasing number of Gram-negative clinical isolates exhibiting MDR phenotypes as a result of the activity of RND pumps, it is clear that the design of novel effective clinical strategies against such pathogens must be grounded in a better understanding of these pumps, including their physiological roles. To this end, recent evidence suggests that RND pumps play an important role in the virulence of Gram-negative pathogens. In this review, we discuss the important role RND efflux pumps play in different facets of virulence including colonization, evasion of host defense mechanisms, and biofilm formation. These studies provide key insights that may ultimately be applied towards strategies used in the design of effective therapeutics against MDR Gram negative bacterial pathogens.

KatG and KatE confer Acinetobacter resistance to hydrogen peroxide but sensitize bacteria to killing by phagocytic respiratory burst.

AIMS Catalase catalyzes the degradation of H2O2. Acinetobacter species have four predicted catalase genes, katA, katE, katG, and katX. The aims of the present study seek to determine which catalase(s) plays a predominant role in determining the resistance to H2O2, and to assess the role of catalase in Acinetobacter virulence. MAIN METHODS Mutants of Acinetobacter baumannii and Acinetobacter nosocomialis with deficiencies in katA, katE, katG, and katX were tested for sensitivity to H2O2, either by halo assays or by liquid culture assays. Respiratory burst of neutrophils, in response to A. nosocomialis, was assessed by chemiluminescence to examine the effects of catalase on the production of reactive oxygen species (ROS) in neutrophils. Bacterial virulence was assessed using a Galleria mellonella larva infection model. KEY FINDINGS The capacities of A. baumannii and A. nosocomialis to degrade H2O2 are largely dependent on katE. The resistance of both A. baumannii and A. nosocomialis to H2O2 is primarily determined by the katG gene, although katE also plays a minor role in H2O2 resistance. Bacteria lacking both the katG and katE genes exhibit the highest sensitivity to H2O2. While A. nosocomialis bacteria with katE and/or katG were able to decrease ROS production by neutrophils, these cells also induced a more robust respiratory burst in neutrophils than did cells deficient in both katE and katG. We also found that A. nosocomialis deficient in both katE and katG was more virulent than the wildtype A. nosocomialis strain. SIGNIFICANCE Our findings suggest that inhibition of Acinetobacter catalase may help to overcome the resistance of Acinetobacter species to microbicidal H2O2 and facilitate bacterial disinfection.

Genome Sequence of an Extremely Drug-Resistant Clinical Isolate of Acinetobacter baumannii Strain AB030

ABSTRACT We report the 4.3-Mbp genome sequence of a blood isolate of Acinetobacter baumannii strain AB030.

Genome Sequence of a Tigecycline-Resistant Clinical Isolate of Acinetobacter baumannii Strain AB031 Obtained from a Bloodstream Infection

ABSTRACT We report here the 3.8-Mbp genome sequence of a blood isolate of Acinetobacter baumannii strain AB031.

Isolation and Characterization of Acinetobacter baumannii Recovered from Campylobacter Selective Medium

Acinetobacter baumannii, a Gram-negative opportunistic pathogen, is known to cause multidrug resistant infections. This organism has primarily been isolated from clinical environments and its environmental reservoirs remain largely unknown. In the present study, we recovered seven isolates of A. baumannii growing under conditions selective for Campylobacter spp. (microaerophilic at 42°C and in the presence of antibiotics) from dairy cattle manure storage tank or surface water impacted by livestock effluents. Antibiotic susceptibility tests revealed that all of these isolates were less susceptible to at least two different clinically relevant antibiotics, compared to the type strain A. baumannii ATCC17978. Expression of resistance-nodulation-division efflux pumps, an important mechanism of intrinsic resistance in these organisms, was analyzed, and adeB was found to be overexpressed in one and adeJ was overexpressed in three isolates. Comparison of these isolates using genomic DNA Macro-Restriction Fragment Pattern Analysis (MRFPA) revealed relatively low relatedness among themselves or with some of the clinical isolates from previous studies. This study suggests that A. baumannii isolates are capable of growing under selective conditions for Campylobacter spp. and that this organism can be present in manure and water.