Gentamicin Susceptibility Research Articles

Yersinia pseudotuberculosis growth arrest during type-III secretion system expression is associated with altered ribosomal protein expression and decreased gentamicin susceptibility.

It has been long appreciated that expression of the Yersinia type-III secretion system (T3SS) in culture is associated with growth arrest. Here we sought to understand whether this impacts expression of ribosomal protein genes, which were among the most highly abundant transcripts in exponential phase Yersinia pseudotuberculosis based on RNA-seq analysis. To visualize changes in ribosomal protein expression, we generated a fluorescent transcriptional reporter with the promoter upstream of rpsJ/S10 fused to a destabilized gfp variant. We confirmed reporter expression significantly increases in exponential phase and decreases as cells transition to stationary phase. We then utilized a mouse model of systemic Y. pseudotuberculosis infection to compare T3SS and S10 reporter expression during clustered bacterial growth in the spleen, and found that cells expressing high levels of the T3SS had decreased S10 levels, while cells with lower T3SS expression retained higher S10 expression. In bacteriological media, growth inhibition with T3SS induction and a reduction in S10 expression were observed in subsets of cells, while cells with high expression of both T3SS and S10 were also observed. Loss of T3SS genes resulted in rescued growth and heightened S10 expression. To understand if clustered growth impacted bacterial gene expression, we utilized droplet-based microfluidics to encapsulate bacteria in spherical agarose droplets, and also observed growth inhibition with high expression of T3SS and reduced S10 levels that better mirrored phenotypes observed in the mouse spleen. Finally, we show that T3SS expression is sufficient to promote tolerance to the ribosome-targeting antibiotic, gentamicin. Collectively, these data indicate that the growth arrest associated with T3SS induction leads to decreased expression of ribosomal protein genes, and this results in reduced antibiotic susceptibility.

Implementing Updated Intraamniotic Infection Guidelines at a Large Academic Medical Center.

Intraamniotic infection (IAI) affects 2%-5% of pregnancies, causing significant neonatal and maternal morbidity. The American College of Obstetrics and Gynecology suggests ampicillin and gentamicin as first-line IAI treatment. Due to potential drug toxicity, changes in gentamicin susceptibility cutoff points, and rising Enterobacterales gentamicin and ampicillin resistance, changes in IAI antibiotic treatment were implemented at Vanderbilt University Medical Center. Combination ampicillin, gentamicin, and clindamycin were replaced by piperacillin-tazobactam in institutional IAI treatment. Implementation strategies included repeated education sessions to gain stakeholder trust and buy-in and changing preexisting electronic clinical decision support tools (eCDSTs) to a default selection of piperacillin-tazobactam, capitalizing on highly reliable intervention strategies of forcing function and automatization/computerization. Change in antibiotic use, measured in days of therapy (DOT)/1000 patient-days present (1000PDP) by week initiated, before and after eCDST changes, was analyzed with interrupted time series analysis. Effects on hospital length of stay, repeat antibiotics within 14 days, and 30 day readmission were evaluated using multivariable linear and logistic regression. After updated eCDST go-live, piperacillin-tazobactam use increased by 1.9 DOT/1000PDP (95% CI, 0.7 to 3.1) by week initiated, and ampicillin, gentamicin, and clindamycin use decreased by -2.5 DOT/1000PDP (95% CI, -3.8 to -1.2) by week initiated. Hospital length of stay, repeat antibiotics within 14 days, and 30-day readmission rate did not significantly change. Forced function changes to existing eCDSTs, supported by stakeholder education, successfully changed IAI empiric antibiotic use without unintended patient safety consequences.

Gentamicin Susceptibility and Comparison of Adhesion and Invasion of Caco-2 and HD11 Cell Lines by Salmonella enterica Serotypes

Foodborne Salmonella serovars are important facultative intracellular pathogens that cause gastroenteritis in humans. Four strains from three of the more predominant Salmonella serovars in poultry were studied: Typhimurium, Enteritidis, and Heidelberg. Gentamicin susceptibility was determined using an agar disc diffusion test and minimum inhibitory concentration (MIC) assays for S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14. Both strains were susceptible to gentamicin in disc diffusion. The MIC of gentamicin was approximately 125 mg/ml for all strains tested. These strains’ adhesion and invasion abilities were determined with two different cell lines, a human intestinal epithelial cell line (Caco-2) as well as a chicken macrophage cell line (HD11). Attachment percentages for each Salmonella strain were greater than the strain’s ability to invade cells. Similar attachment percentages to Caco-2 cells were observed for S. Typhimurium and S. Heidelberg. Attachment percentages were lower in HD11 cells than in Caco-2 cells, although Salmonella exhibited higher apparent HD11 invasion, likely from HD11 phagocytosis. Salmonella Enteritidis showed lower rates of adhesion and invasion in HD11 cells compared to Salmonella Typhimurium. Developing a better understanding of Salmonella virulence mechanisms is critical to reducing Salmonella infections.

Characterization of Antibiotic Resistance in Select Tertiary Hospitals in Uganda: An Evaluation of 2020 to 2023 Routine Surveillance Data.

Antimicrobial resistance (AMR) is a public health concern in Uganda. We sought to conduct an extended profiling of AMR burden at selected Ugandan tertiary hospitals. We analyzed routine surveillance data collected between October 2020 and March 2023 from 10 tertiary hospitals. The analysis was stratified according to the hospital unit, age, gender, specimen type, and time. Up to 2754 isolates were recovered, primarily from pus: 1443 (52.4%); urine: 1035 (37.6%); and blood: 245 (8.9%). Most pathogens were Staphylococcus aureus, 1020 (37%), Escherichia coli, 808 (29.3%), and Klebsiella spp., 200 (7.3%). Only 28% of Escherichia coli and 42% of the other Enterobacterales were susceptible to ceftriaxone, while only 44% of Staphylococcus aureus were susceptible to methicillin (56% were MRSA). Enterococcus spp. susceptibility to vancomycin was 72%. The 5-24-year-old had 8% lower ampicillin susceptibility than the >65-year-old, while the 25-44-year-old had 8% lower ciprofloxacin susceptibility than the >65-year-old. The 0-4-year-old had 8% higher ciprofloxacin susceptibility. Only erythromycin susceptibility varied by sex, being higher in males. Escherichia coli ciprofloxacin susceptibility in blood (57%) was higher than in urine (39%) or pus (28%), as was ceftriaxone susceptibility in blood (44%) versus urine (34%) or pus (14%). Klebsiella spp. susceptibility to ciprofloxacin and meropenem decreased by 55% and 47%, respectively, during the evaluation period. During the same period, Escherichia coli ciprofloxacin susceptibility decreased by 40%, while Staphylococcus aureus gentamicin susceptibility decreased by 37%. Resistance was high across the Access and Watch antibiotic categories, varying with time, age, sex, specimen type, and hospital unit. Effective antimicrobial stewardship targeted at the critical AMR drivers is urgently needed.

Identification and mechanism determination of the efflux pump subunit amrB gene mutations linked to gentamicin susceptibility in clinical Burkholderia pseudomallei from Malaysian Borneo.

The bacterium Burkholderia pseudomallei is typically resistant to gentamicin but rare susceptible strains have been isolated in certain regions, such as Thailand and Sarawak, Malaysia. Recently, several amino acid substitutions have been reported in the amrB gene (a subunit of the amrAB-oprA efflux pump gene) that confer gentamicin susceptibility. However, information regarding the mechanism of the substitutions conferring the susceptibility is lacking. To understand the mechanism of amino acid substitution that confers susceptibility, this study identifies the corresponding mutations in clinical gentamicin-susceptible B. pseudomallei isolates from the Malaysian Borneo (n = 46; Sarawak: 5; Sabah: 41). Three phenotypically confirmed gentamicin-susceptible (GENs) strains from Sarawak, Malaysia, were screened for mutations in the amrB gene using gene sequences of gentamicin-resistant (GENr) strains (QEH 56, QEH 57, QEH20, and QEH26) and publicly available sequences (AF072887.1 and BX571965.1) as the comparator. The effect of missense mutations on the stability of the AmrB protein was determined by calculating the average energy change value (ΔΔG). Mutagenesis analysis identified a polymorphism-associated mutation, g.1056T > G, a possible susceptible-associated in-frame deletion, Delta V412, and a previously confirmed susceptible-associated amino acid substitution, T368R, in each of the three GENs isolates. The contribution of Delta V412 needs further confirmation by experimental mutagenesis analysis. The mechanism by which T368R confers susceptibility, as elucidated by in silico mutagenesis analysis using AmrB-modeled protein structures, is proposed to be due to the location of T368R in a highly conserved region, rather than destabilization of the AmrB protein structure.

Addition of gentamicin for antibiotic prophylaxis in hip hemiarthroplasty does not decrease the rate of surgical site infection.

The addition of Gram-negative coverage to antibiotic prophylaxis protocols prior to elective total hip arthroplasty (THA) has been reported to reduce periprosthetic joint infection (PJI). However, it is unknown whether adding a Gram-negative-targeted antibiotic agent improves outcomes in the trauma population. This study aimed to investigate whether the addition of a single, pre-operative dose of Gentamicin is associated with lower rates of PJI in patients undergoing hemiarthroplasty (HA) as treatment for a hip fracture. We retrospectively reviewed cases of patients who underwent HA as treatment for a hip fracture from January 2011 to January 2022, and had a minimum 1-year of follow-up. Patients were divided into two groups based on the antibiotic prophylaxis they received during surgery: cefazolin (control group) or cefazolin with addition of Gentamicin (case group). The primary outcome was the rate of surgical site infections (SSI), and secondary outcomes included rates of prosthetic joint infection (PJI) and superficial SSIs. The final study population consisted of 1521 patients. 336 patients (22.1%) were in the case group and 1185 (77.9%) patients were in the control group. Rates of SSI were comparable between the groups (3.8% for the case group vs. 2.8% in the control group, p = 0.34). This held true for both PJIs (3.5 vs. 2.5%, p = 0.3) and superficial SSIs (0.29 vs. 0.33%, p = 0.91). The distribution of the causing pathogen was similar between the groups (p = 0.84). Gentamicin susceptibility rates of the Gram-negative bacteria associated with PJI were similar between the cohorts (p = 0.51). The addition of a single, pre-operative dose of Gentamicin to the antibiotic prophylaxis protocol of patients undergoing HA as treatment for a hip fracture was not associated with lower rates of SSI, PJI or superficial SSI. The findings of this study indicate that the prophylactic benefits of Gentamicin may not apply to HA as they do to THA.

Dynamics of antimicrobial resistance and virulence of staphylococcal species isolated from foods traded in the Cape Coast metropolitan and Elmina municipality of Ghana

The impact of staphylococci on food poisoning and infections could be higher than previously reported. In this study, we characterised the occurrence and coexistence of antimicrobial resistance and virulence genes of staphylococci isolates in foods. Staphylococci were isolated from 236 samples of selected street-vended foods and identified. The pattern of antimicrobial resistance and virulence genes in the staphylococci were assessed using disc diffusion, PCR and analysis of next-generation sequencing data. The food samples (70.76 %) showed a high prevalence of staphylococci and differed among the food categories. Forty-five Staphylococcus species were identified and comprised coagulase-negative and positive species. Staphylococcus sciuri (now Mammaliicoccus sciuri), S. aureus, S. kloosii, S. xylosus, S. saprophyticus, S. haemolyticus and S. succinus were the most abundant species. The staphylococcal isolates exhibited resistance to tetracycline, levofloxacin, ciprofloxacin, norfloxacin, gentamicin and amikacin and susceptibility to nitrofurantoin. Antimicrobial susceptibilities were also reported for cefoperazone, ceftriaxone, cefotaxime, nalidixic acid and piperacillin-tazobactam. The antimicrobial resistance and virulence genes commonly detected consisted of tet, arl, macB, van, gyr, nor, optrA, bcrA, blaZ, taeA and S. aureus lmrS. The isolates frequently exhibited multiple resistance (30.42 %) of up to eight antimicrobial drug classes. The isolates predominantly harboured genes that express efflux pump proteins (50.53 %) for antibiotic resistance compared with inactivation (10.05 %), target alteration (26.72 %), protection (7.67 %) and replacement (3.17 %). The virulence determinants comprised genes of pyrogenic toxin superantigens (eta, etb, tst), adhesions (clf, fnbA, fnbB, cna, map, ebp, spA, vWbp, coa) and genes that express exoproteins (nuclease, metalloprotease, γ-hemolysin, hyaluronate lyase). There was a statistically significant difference in the prevalence of staphylococci isolates and their antimicrobial resistance and virulence profile as revealed by the phenotypic, PCR and next-generation sequencing techniques. The findings suggest a higher health risk for consumers. We recommend a critical need for awareness and antimicrobial susceptibility and anti-virulence strategies to ensure food safety and counteract the spread of this clinically relevant genus.

The SlyD metallochaperone targets iron-sulfur biogenesis pathways and the TCA cycle.

Correct folding of proteins represents a crucial step for their functions. Among the chaperones that control protein folding, the ubiquitous PPIases catalyze the cis/trans-isomerization of peptidyl-prolyl bonds. Only few protein targets of PPIases have been reported in bacteria. To fill this knowledge gap, we performed a large-scale two-hybrid screen to search for targets of the Escherichia coli and Helicobacter pylori SlyD PPIase-metallochaperone. SlyD from both organisms interacts with enzymes (i) containing metal cofactors, (ii) from the central metabolism tricarboxylic acid (TCA) cycle, and (iii) involved in the formation of the essential and ancestral Fe-S cluster cofactor. E. coli and H. pylori ∆slyD mutants present similar phenotypes of diminished susceptibility to antibiotics and to oxidative stress. In H. pylori, measurements of the intracellular ATP content, proton motive force, and activity of TCA cycle proteins suggest that SlyD regulates TCA cycle enzymes by controlling the formation of their indispensable Fe-S clusters.

The canonical Brucella species-host dependency is changing, however, the antibiotic susceptibility profiles remain unchanged

Brucellosis is a chronic disease caused by Brucella species with a wide range of hosts, from marine mammals to terrestrial species, but with strict host preferences. With the zoonotic character, the prevalence of human brucellosis cases is a reflection of animal infections. This study aimed to identify 192 Brucella isolates obtained from various sources by Bruce-ladder PCR and to determine their antibiotic susceptibilities by gradient diffusion method (E-test). As a result of the PCR, all human isolates (n = 57) were identified as B. melitensis. While 58 (82.9%) of the cattle isolates were identified as B. abortus, 59 (90.8%) of the sheep isolates were identified as B. melitensis. In addition, 12 (17.1%) of the cattle isolates and 6 (9.2%) of the sheep isolates were determined as B. melitensis and B. abortus, respectively. The primary host change behavior of B. melitensis was 1.9 times higher than that of B. abortus. While gentamicin and ciprofloxacin susceptibilities of Brucella isolates were 100%, tetracycline, doxycycline, streptomycin, trimethoprim/sulfamethoxazole and rifampicin susceptibilities were 99%, 99%, 97.4%, 91.7% and 83.9%, respectively. The lowest sensitivity of the isolates was determined against to cefoperazone as 26%. A triple-drug resistance was detected in 1 B. abortus isolate that included simultaneous resistance to cefoperazone, rifampicin, and trimethoprim/sulfamethoxazole. The high susceptibility profiles we found against to antibiotics such as tetracycline, doxycycline gentamicin and ciprofloxacin, used widely in treatment, are encouraging. However, the change in the canonical Brucella species-primary host preference suggests the need to reconsider eradication program, including updating vaccine formulations.

Carbonic Anhydrase Inhibition as a Target for Antibiotic Synergy in Enterococci.

Enterococcus faecalis is a hospital-associated opportunistic pathogen that can cause infections with high mortality, such as infective endocarditis. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. We isolated a gentamicin-hypersusceptible E. faecalis strain from a patient with infective endocarditis that carried a mutation in the alpha-carbonic anhydrase (α-CA) and investigated how disruption of α-CA sensitized E. faecalis to killing with gentamicin. The gentamicin-hypersusceptible α-CA mutant strain showed increased intracellular gentamicin uptake in comparison to an isogenic strain encoding full-length, wild-type α-CA. We hypothesized that increased gentamicin uptake could be due to increased proton motive force (PMF), increased membrane permeability, or both. We observed increased intracellular ATP production in the α-CA mutant strain, suggesting increased PMF-driven gentamicin uptake contributed to the strain's gentamicin susceptibility. We also analyzed the membrane permeability and fatty acid composition of isogenic wild-type and α-CA mutant strains and found that the mutant displayed a membrane composition that was consistent with increased membrane permeability. Finally, we observed that exposure to the FDA-approved α-CA inhibitor acetazolamide lowered the gentamicin MIC of eight genetically diverse E. faecalis strains with intact α-CA but did not change the MIC of the α-CA mutant strain. These results suggest that α-CA mutation or inhibition increases PMF and alters membrane permeability, leading to increased uptake of gentamicin into E. faecalis. This connection could be exploited clinically to provide new combination therapies for patients with enterococcal infections. IMPORTANCE Enterococcal infections can be difficult to treat, and new therapeutic approaches are needed. In studying an E. faecalis clinical strain from an infected patient, we found that the bacteria were rendered hypersusceptible to aminoglycoside antibiotics through a mutation that disrupted the α-CA. Our follow-on work suggested two different ways that α-CA disruption causes increased gentamicin accumulation in E. faecalis: increased proton motive force-powered uptake and increased membrane permeability. We also found that a mammalian CA inhibitor could sensitize a variety of E. faecalis strains to killing with gentamicin. Given that mammalian CA inhibitors are frequently used to treat conditions such as glaucoma, hypertension, and epilepsy, our findings suggest that these "off-the-shelf" inhibitors could also be useful partner antibiotics for the treatment of E. faecalis infections.

Prevalence and antimicrobial susceptibility of high-level gentamicin resistant enterococci isolated from urine at a hospital in Pune, India

Introduction: Enterococci are one of the common organisms isolated from hospitalized patients with urinary tract infections. Guidelines recommend testing enterococcifor susceptibility to high-level gentamicin (HLG) and streptomycin. The present study was planned to determine the susceptibility of uropathogenic enterococci to high-level gentamicin in a tertiary care hospital. Materials and Methods: Prospective observational research was carried out at a tertiary care hospital for two years on all isolated enterococci from urine specimens. Identification and antibiotic susceptibility were performed as per standard methods. All the isolated enterococci were tested for high level gentamicin ((120µg) resistance and susceptibility to other recommended antimicrobial agents by standard methods. Results: A total of 320 uropathogenic enterococci were isolated and tested for antibiotic susceptibility. The majority of enterococci were isolated from elderly (34.06%) and admitted patients (69.06%). A total of 68.4% isolated enterococci were HLG resistant. HLG resistant enterococci were highly resistant to erythromycin (96.3%), ciprofloxacin (96.8%) and nalidixic acid (97.7%). Enterococci sensitive to HLG were significantly (p <0.05) less resistant to the other antimicrobial agents except nalidixic acid. Only 20.5% isolated Enterococci were resistant to vancomycin. All isolated enterococci were susceptible to linezolid. Conclusion: The study demonstrated high prevalence of HLG resistant enterococci causing UTI in our hospital setting. Compared to HLG sensitive enterococci, HLG resistant enterococci were more resistant to other antimicrobial agents tested. The findings highlight the need for mandatory testing of enterococci for HLG resistance to determine effective antimicrobials for treatment. IMC J Med Sci. 2023; 17(2):008. DOI: https://doi.org/10.55010/imcjms.17.018 *Correspondence: Dr. Sahjid Mukhida, Department of Microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharashtra, India. E-mail: drssmukhida@rediffmail.com

Molecular screening of oxacillinases beta-lactamase among gentamicin-resistantEscherichia coliisolates

Expansion of β-lactamases in Gram-negative rods has been documented as most severe threat to the management of infectious diseases. The ever-increasing use of antibiotics with the evolution of intrinsic and acquired resistance has led to the development of resistance mechanism in Gram-negative rods contributing to the expansion of several multi-drug resistance epidemics in hospital environment. So this study aimed to investigate genes responsible for Oxacillinases production among gentamicin- resistantEscherichia coliisolates. The results showed that out of 573 specimens, 270 (56.13%) showed bacterial growth versus 264(46.07%) showed no bacterial growth 309(53.92%), among 573 clinical specimens 102(17.80%) were male patients while 471(82.19%) were female patients. According to result of the vitek-2 system recorded 110 isolates asE. coli. However result of gentamicin susceptibility demonstrated that 29 (26.36%)E. coliisolates were resistance to gentamicin compared with 39 (35.45%) and 42 (38.18%) of isolates were intermediate and sensitive to this antibiotic respectively. Results of antibiotic susceptibility showed that the highest bacterial resistance was Tobramycin 27(93.1%) and Ciprofloxacin 29(100 %),while Nitrofurantoin 3(10.3%) had the least resistance. Results of polymerase chain reaction (PCR ) amplification showed thatOXA, OXA-1 and OXA-9,OXA-10were 29(100%),26/(89.65%),29(100%) and 20(68.96%) respectively While OXA-2 did not detect.

High prevalence of ArmA-16S rRNA methyltransferase among aminoglycoside-resistant Klebsiella pneumoniae bloodstream isolates.

Introduction. Aminoglycosides are used for the treatment of carbapenemase-producing Klebsiella pneumoniae (CPK) infections. 16S rRNA methyltransferases (RMTs) confer resistance to all aminoglycosides and are often cocarried with NDM.Hypothesis/Gap Statement. There is a dart of studies looking at the aminoglycoside resistance mechanisms for invasive CPK isolates, particularly in OXA-48 endemic settings.Aim. We aimed to determine the prevalence of RMTs and their association with beta lactamases and MLSTs amongst aminoglycoside-resistant CPK bloodstream isolates in an OXA-48 endemic setting.Methodology. CPK isolates (n=181), collected as part of a multicentre cohort study, were tested for amikacin, gentamicin and tobramycin susceptibility using custom-made sensititre plates (GN2XF, Thermo Fisher Scientific). All isolates were previously subjected to whole-genome sequencing. Carbapenemases, RMTs, MLSTs and plasmid incompatibility groups were detected on the assembled genomes.Results. Of the 181 isolates, 109(60 %) were resistant to all three aminoglycosides, and 96 of 109(88 %) aminoglycoside-resistant isolates carried an RMT (85 ArmA, 10 RmtC, 4 RmtF1; three isolates cocarried ArmA and RmtC). Main clonal types associated with ArmA were ST2096 (49/85, 58 %) and ST14 (24/85, 28 %), harbouring mainly OXA-232 and OXA-48 +NDM, respectively. RmtC was cocarried with NDM (5/10) on ST395, and NDM +OXA-48 or NDM +KPC (4/10) on ST14, ST15 and ST16. All RMT producers also carried CTX-M-15, and the majority cocarried SHV-106, TEM-150 and multiple other antibiotic resistance genes. The majority of the isolates harboured a combination of IncFIB, IncH and IncL/M type plasmids. Non-NDM producing isolates remained susceptible to ceftazidime-avibactam.Conclusion. Aminoglycoside resistance amongst CPK bloodstream isolates is extremely common and mainly driven by clonal spread of ArmA carried on ST2096 and ST14, associated with OXA-232 and OXA48 +NDM carriage, respectively.

Antibacterial effect and evaluation of the inhibitory effect against efflux pump in Staphylococcus aureus by abietic acid: In vitro and in silico assays

Abietic acid is a diterpene found in resins mainly from diverse species of genus Pinus. The present study evaluated the antibacterial and inhibitory effect against the NorA and MepA efflux pump of Staphylococcus aureus by abietic acid using in vitro and in silico assays. The microdilution bacterial assay was used to evaluate antibacterial activity in standard bacteria (SA 25923 and EC 25922) and clinical isolates bacteria multiresistant (SA-10 and EC-06). Their association with antibiotics ampicillin, gentamicin, and ciprofloxacin was also estimated. Staphylococcus aureus (SA-1199B and SA-K2068) was used with a NorA and MepA pump machine, respectively, to verify the inhibitory effect using MIC methodology proposed by CLSI and Ethidium Bromide, an indicator of an efflux pump. Molecular dynamics and molecular docking calculations were used to evaluate and validate the interaction of abietic acid with NorA and MepA efflux pumps. The results demonstrated a significate reduction of MIC values to EC 25922 and SA 10 and showed a synergistic effect when combined with increased gentamicin susceptibility against multiresistant strains. The abietic acid showed direct activity against Staphylococcus aureus overexpressing gene of efflux pump, demonstrating the possibility of interference in the efflux pump NorA (SA 1199B) and MepA (SA K2068) mediated by hydrogen bonds and hydrophobic interactions. Together, these findings are promisors validating the potential antimicrobial activity and the possibility of using abietic acid as antibiotic adjuvant resistance breakers (ARBs) to treat infections caused by multiresistant bacteria. However, other studies are necessary to confirm this potential using the in vivo model.

Spread of armA 16S rRNA Methylase Gene in a Tertiary Hospital and Errors in Aminoglycoside Susceptibility Results of Rapid Susceptibility Test Systems

Aminoglycosides (AGs) are actively used in combination therapies against carbapenem resistant gram negative species in recent years. Spread of 16S rRNA methylases which can cause high-level resistance to AG antibiotics, limits this treatment choice. Although there are some studies showing that errors in determining AG susceptibility in automated systems may be related to the armA gene, one of the 16S rRNA methylase genes, the exact reason for these errors is not yet known. In our study, we aimed to investigate the relevance of 16S rRNA methylases to the discrepancies between VITEK 2.0 and disc diffusion test results for amikacin (AK) and gentamicin (GEN) susceptibility of Acinetobacter baumannii and Klebsiella pneumoniae isolates. All K.pneumoniae and A.baumannii isolates from 1st January-10th February 2018 were collected prospectively and included in the study. Additionally, two initial isolates from July 2017 (one K.pneumoniae and one A.baumannii isolate) for which first discrepant susceptibility results were determined, were also included. Amikacin and gentamicin susceptibility results of 37 isolates [A. baumannii (n= 20) and K.pneumoniae (n= 17)] were evaluated together with VITEK 2.0 system, disc diffusion and gold standard broth microdilution methods and minor error (mE), major error (ME) and very major error (VME) rates were calculated. The rmtB, rmtC and armA genes in isolates were investigated by polymerase chain reaction (PCR) and the relationship between the presence of 16S rRNA methylases and false susceptibility results were examined. In addition, disc diffusion test results were evaluated at the end of four, six, eight hours and one night incubation periods to examine the effect of the double zone phenotype observed in 13 of the study isolates on rapid susceptibility tests. All disc diffusion test results were found to be compatible with broth microdilution test results. When the VITEK 2.0 system and the broth microdilution test were compared, 10.3% and 12.1% VME and 8.1% and 5.4% mE were detected for AK and GEN susceptibility results, respectively. While rmtB and rmtC genes were not detected in the study isolates, armA gene was positive in eight (47.1%) of 17 K.pneumoniae isolates and in 15 (75%) of 20 A.baumannii isolates. All three VMEs in A.baumannii isolates were detected in AK susceptibility results. Two of those were armA gene positive and one was armA gene negative isolates. All four VMEs in K. pneumoniae isolates were detected in GEN susceptibility results only, and all of these isolates were armA gene positive. No direct correlation was found between the errors detected in the VITEK 2.0 system susceptibility results and the double zone phenotype. When the isolates were evaluated in the 4-16 hours incubation time interval, it was observed that resistant colonies could be detected after a minimum of six hours of incubation period in the inhibition zone surrounding the aminoglycoside discs. To the best of our knowledge this is the first report of armA producing A.baumannii from Turkey. The high rate of armA gene positivity detected in our isolates suggested that the prevalence of armA gene increased in our country or at least in our region, in recent years. In the AG susceptibility results of the VITEK 2.0 system, the rate of VME above the acceptance criterion has shown that the errors occurred were not directly related to armA gene positivity or double zone phenotype. Finally, our study results indicated that AG susceptibility results should be evaluated minimum six hours later of incubation while implementing rapid susceptibility tests.

Australian Gonococcal Surveillance Programme, 1 July to 30 September 2021.

The National Neisseria Network (NNN), Australia, comprises reference laboratories in each state and territory established in 1979. The NNN has reported data on susceptibility profiles for all Neisseria gonorrhoeae isolated from each jurisdiction for an agreed group of antimicrobial agents for the Australian Gonococcal Surveillance Programme (AGSP) since 1981. The antibiotics reported represent current or potential agents used for the treatment of gonorrhoea and include ceftriaxone; azithromycin; ciprofloxacin; and penicillin. More recently, gentamicin susceptibilities are included in the AGSP Annual Report. Ceftriaxone, combined with azithromycin, is the recommended treatment regimen for gonorrhoea in the majority of Australia. However, there are substantial geographic differences in susceptibility patterns in Australia, with certain remote regions of the Northern Territory and Western Australia having low gonococcal antimicrobial resistance rates. In these regions, an oral treatment regimen comprising amoxycillin, probenecid, and azithromycin is recommended for the treatment of gonorrhoea.

Evaluation of three different methods for susceptibility testing of gentamicin in carbapenem resistant Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) have become a growing problem worldwide in recent years. Options for the treatment of CRE are limited and one of these options is gentamicin. For this reason, gentamicin susceptibility should be properly determined. In a recently reported study, it is recommended to review the results of automated systems for assessing gentamicin susceptibility in carbapenem-resistant isolates. In this study, we aimed to determine gentamicin susceptibility using three different methods and compare the methods. The study included 107 CRE isolates from different samples. Gentamicin susceptibility was determined using Vitek 2 Compact (bioMérieux, France), Microscan Walkaway Plus (Beckman Coulter, USA) automatic systems, and disk diffusion (DD) method. The broth microdilution method (BMD) was used as reference method. Minor, major, and very major errors and categorical agreement rates were determined for each method. Aminoglycoside-modifying enzymes (aac(6')Ib and aph(2″)Ia) were assayed in discrepant isolates. According to BMD results, 90.7%, 1,8 %, and 7.5 % of the isolates were determined as susceptible, intermediate, and resistant to gentamicin, respectively. Compared to the results of the BMD for detecting gentamicin susceptibility, disk diffusion method showed the highest categorical agreement (98.1%), and Vitek 2 Compact showed the lowest categorical agreement (90.6%). The very major error rates were determined 7.5%, 0.9%, and 0.9% for Vitek 2 Compact, Microscan Walkaway Plus, and DD method, respectively. In addition, aac(6')Ib and aph(2″)Ia genes were detected in 8 discrepant isolates. For gentamicin susceptibility, the DD showed the most compatible results. The DD can be used as a reliable method for determining gentamicin susceptibility. Compatibility of automated systems with BMD was acceptable, although lower than DD. The discrepancies detected in the Vitek 2 Compact results could be due to the presence of aac(6')Ib and/or aph(2″)Ia aminoglycoside-modifying enzymes.

Antimicrobial Susceptibility Profiles Among Neonatal Early-onset Sepsis Pathogens.

Empiric administration of ampicillin and gentamicin is recommended for newborns at risk of early-onset sepsis (EOS). There are limited data on antimicrobial susceptibility of all EOS pathogens. Retrospective review of antimicrobial susceptibility data from a prospective EOS surveillance study of infants born ≥22 weeks' gestation and cared for in Neonatal Research Network centers April 2015-March 2017. Nonsusceptible was defined as intermediate or resistant on final result. We identified 239 pathogens (235 bacteria, 4 fungi) in 235 EOS cases among 217,480 live-born infants. Antimicrobial susceptibility data were available for 189/239 (79.1%) isolates. Among 81 Gram-positive isolates with ampicillin and gentamicin susceptibility data, all were susceptible in vitro to either ampicillin or gentamicin. Among Gram-negative isolates with ampicillin and gentamicin susceptibility data, 72/94 (76.6%) isolates were nonsusceptible to ampicillin, 8/94 (8.5%) were nonsusceptible to gentamicin, and 7/96 (7.3%) isolates were nonsusceptible to both. Five percent or less of tested Gram-negative isolates were nonsusceptible to each of third or fourth generation cephalosporins, piperacillin-tazobactam, and carbapenems. Overall, we estimated that 8% of EOS cases were caused by isolates nonsusceptible to both ampicillin and gentamicin; these were most likely to occur among preterm, very-low birth weight infants. The vast majority of contemporary EOS pathogens are susceptible to the combination of ampicillin and gentamicin. Clinicians may consider the addition of broader-spectrum therapy among newborns at highest risk of EOS, but we caution that neither the substitution nor the addition of 1 single antimicrobial agent is likely to provide adequate empiric therapy in all cases.

Gentamicin Susceptibility in Neisseria gonorrhoeae and Treatment Outcomes for Urogenital Gonorrhea After 25 Years of Sustained Gentamicin Use in Malawi.

Gentamicin has been used for the treatment of gonorrhea in Malawi since 1993. However, declining clinical cure rates have been suspected. We evaluated current Neisseria gonorrhoeae susceptibility to gentamicin in vitro and clinically. Men with acute urethritis were recruited at the Bwaila District Hospital STI Clinic in Lilongwe, Malawi, between January 2017 and August 2019. All men provided urethral swabs for etiological testing at enrollment and test of cure (TOC), 1 week later, using Gram-stained microscopy and culture. We used Etest to determine minimum inhibitory concentrations (MICs) of gentamicin, azithromycin, cefixime, ceftriaxone, ciprofloxacin, and spectinomycin; disc diffusion for tetracycline susceptibility; and whole-genome sequencing (WGS) to verify/refute treatment failure. Among 183 N. gonorrhoeae culture-positive men enrolled, 151 (82.5%) had a swab taken for TOC. Of these 151 men, 16 (10.6%) had a positive culture at TOC. One hundred forty-one baseline isolates were tested for gentamicin susceptibility using Etest: 2 (1.4%), MIC = 2 μg/mL; 111 (78.7%), MIC = 4 μg/mL; and 28 (19.9%), MIC = 8 μg/mL. All isolates were susceptible to azithromycin, cefixime, ceftriaxone, and spectinomycin, whereas 63.1% had intermediate susceptibility or resistance to ciprofloxacin. Almost all (96.1%) isolates were resistant to tetracycline. All examined isolates cultured at TOC (n = 13) had gentamicin MICs ≤8 μg/mL. Ten men had pretreatment and posttreatment isolates examined by whole-genome sequencing; 2 (20%) were verified new infections (4119 and 1272 single-nucleotide polymorphisms), whereas 8 (80%) were confirmed treatment failures (0-1 single-nucleotide polymorphism). Gentamicin MICs poorly predict gonorrhea treatment outcome with gentamicin, and treatment failures are verified with gonococcal strains with in vitro susceptibility to gentamicin. The first-line treatment of gonorrhea in Malawi should be reassessed.

Benzalkonium chloride antagonises aminoglycoside antibiotics and promotes evolution of resistance.

BackgroundBiocide disinfectants are essential tools in infection control, but their use can inadvertently contribute to emergence of antibiotic-resistant bacteria. In this study we systematically examine the effect of the biocide benzalkonium chloride, which is primarily used for surface disinfection but is also present as a preservative in many consumer products, on the activity of aminoglycoside antibiotics in Acinetobacter baumannii.MethodsThe effect of subinhibitory BAC on aminoglycoside treatment of A. baumannii ATCC17978 was investigated using time-to-kill assays, MIC determination, directed evolution experiments, fluctuation tests and labelled gentamicin accumulation assays. Further MIC determinations and directed evolution experiments were performed with additional Gram-negative ESKAPE pathogens.FindingsIn A. baumannii ATCC17978, BAC prevents gentamicin killing and drastically increases the frequency at which resistant mutants emerge, through reducing intracellular antibiotic accumulation. BAC also increases the MIC of multiple aminoglycoside antibiotics (kanamycin, tobramycin, streptomycin, gentamicin and amikacin). BAC promotes the emergence of mutants with reduced gentamicin susceptibility in other Gram-negative ESKAPE pathogens but does not always alter the MIC. These effects occur at BAC concentrations which are similar to residual levels in high-use environments, and just below the concentration range for BAC when used as a preservative in eye drops and ear drops.InterpretationOur results suggest that subinhibitory BAC has the potential to antagonise aminoglycoside activity and promote the emergence of bacterial mutants with reduced susceptibility. We suggest that the extremely widespread use of BAC in clinical and home settings and its long half-life mean there is potential for these interactions to occur in the environment, or in patients who use BAC-containing products while taking aminoglycosides to treat skin, eye or ear infections, although such co-exposure is likely to be rare. We suggest that biocide stewardship is needed to prevent the types of exposure that can contribute to antibiotic resistance.FundingThis work was funded by the National Health and Medical Research Council of Australia. The funders had no role in study design, interpretation or decision to publish.