Aminoglycoside-modifying Enzymes Research Articles

A preliminary study of Aminoglycoside Modifying Enzymes (AMEs) of Multiple Antibiotic Resistance of Methicillin-resistant Staphylococcus aureus (MRSA) isolated from clinical specimens in Al-Diwaniya/Iraq

A preliminary study of Aminoglycoside Modifying Enzymes (AMEs) of Multiple Antibiotic Resistance of Methicillin-resistant Staphylococcus aureus (MRSA) isolated from clinical specimens in Al-Diwaniya/Iraq

Potential for the Development of a New Generation of Aminoglycoside Antibiotics.

The present review summarizes recent publications devoted to aminoglycosides that study the main types of resistance to antibiotics of this class and the main directions of chemical modification aimed at overcoming the resistance or changing the spectrum of biological activity. Conjugates of aminoglycosides with various pharmacophores including amino acids, peptides, peptide nucleic acids, nucleic bases, and several other biologically active molecules and modifications resulting in other types of biological activity of this class of antibiotics are described. It is concluded that a promising research direction aimed at increasing the activity of antibiotics against resistant strains is the search for selective inhibitors of aminoglycoside-modifying enzymes. This would allow renewal of the use of antibiotics already meeting widespread resistance and would increase the potential of a new generation of antibiotics.

Antibiotic Resistance and Pathogenomics of Staphylococci Circulating in Novosibirsk, Russia.

A total of 394 strains of staphylococci found in humans and pets in Novosibirsk, Siberian Russia, were characterized in terms of antibiotic resistance and corresponding genes. Two coagulase-positive and 17 coagulase-negative species were identified. The majority of isolates, with the exception of S. haemolyticus and hospital S. epidermidis isolates, were sensitive to most of the tested antibiotics, and isolates from pets displayed the lowest level of resistance. Nevertheless, methicillin-resistant (MRS) and/or multidrug-resistant (MDR) isolates were found in all prevailed species, including coagulase-negative. A set of genes corresponding to the detected resistance was identified: mecA (beta-lactam resistance), aac(6′)-Ie-aph(2″)-Ia, aph(3′)-IIIa, ant(4′)-Ia (aminoglycoside-modifying enzymes), ermA/ermC, and msrA (macrolide resistance). Complete genome analysis for ten MDR S. epidermidis and five MDR S. haemolyticus isolates revealed additional antibiotic resistance genes mphC, qacA/qacB, norA, dfrC/dfrG, lnuA, BseSR, and fosB. NorA, dfrC, and fosB were present in all S. epidermidis genomes, whereas mphC and msrA were identified in all S. haemolyticus ones. All investigated MDR S. epidermidis and four of five S. haemolyticus strains were moderate or strong biofilm producers, whereas multiple genes responsible for this function and for virulence and pathogenicity were identified mostly in S. epidermidis, but were less frequently represented in S. haemolyticus.

Characterization of Aminoglycoside Modifying Enzymes Producing E. coli and Klebsiella pneumoniae Clinical Isolates

Antimicrobial resistance gene profile characterization and dissemination offer useful detail on the possible challenge in treating bacteria. The development of aminoglycoside modifying enzymes (AMEs) is considered as the primary mechanism of resistance to aminoglycosides, in addition to the 16S rRNA methylases. This study aimed at isolation and characterization of aminoglycosides resistant clinical isolates of enterobacteriaceae family from different clinical samples. Over a period of 24 months, thirty samples were collected and 49 clinical isolates of E. coli [n=25], Klebsiella [n=13], Enterobacter species (n=7) and Proteus species (n=4) were isolated from Egyptian clinical laboratories. The identities of the cultures were confirmed following standard microbiological procedures. Resistance of the isolates to aminoglycosides was determined by the disc diffusion method and isolates with highest resistance (n=9) were selected and investigated for 16S rRNA methylase and AMES encoding genes by polymerase chain reaction (PCR) and sequencing. In general, aminoglycoside resistance was found in 95% of the isolates; the isolates displayed the highest rate of resistance to netilmicin (75%) and kanamycin (55%), while resistance to gentamycin (18%) and tobramycin (16%) was low. A total of 9 isolates have the highest aminoglycoside resistant rate, showed the highest appearance for aac(6′)-Ib as well as ant (3″)-Ia resistant genes, with aac (3)-II (44%) and ant (4′)-IIb (34%) following closely. The high prevalence of AMEs observed among resistant isolates in this study suggests the urgent need for more efficient treatment designs to mitigate the selection burden as well as improved care of patients who have been infected with these drug-resistant organisms.

Draft genome sequence of a carbapenem-resistant clinical Acinetobacter baumannii revealing co-existence of four classes of β-lactamases

Nosocomial carbapenem-resistant Acinetobacter baumannii is a challenge in the treatment of intensive care unit (ICU) patients. The presence of antimicrobial resistance genes (ARGs) and mobile genetic elements can further complicate effects to combat antibiotic resistance in this post-antibiotic era. The aim of this study was to analyse the molecular basis of carbapenem resistance in A. baumannii strain DMC-32a. Strain DMC-32a, isolated from a wound swab of an ICU patient, was screened phenotypically and genotypically for carbapenem resistance. The isolate was subjected whole-genome sequencing (WGS) to investigate the resistance mechanisms. Strain DMC-32a was resistant to all tested antibiotics belonging to seven classes, except for the polymyxins. MICs determined against imipenem and meropenem were 512 mg/L and >512 mg/L, respectively. Gene-specific PCR confirmed the presence of blaNDM-1 and intI1. WGS confirmed the isolate as sequence type ST1053, the presence of four classes of β-lactamases [A (blaPER-7, blaTEM-116), B (blaNDM-1), C (blaADC-26) and D (blaOXA-23, blaOXA-51)], and four genes encoding aminoglycoside-modifying enzymes [aph(6)-Id, aph(3'')-Ib, aph(3')-VI and ant(3'')-IIc] conferring resistance to streptomycin, amikacin, kanamycin and spectinomycin. The analysis also confirmed the presence of a class 1 integron gene cassette harbouring ARGs to rifamycin (arr-2) and phenicols (cmlA5). No plasmid replicon was found from the sequence data. The co-existence of four β-lactamase classes in A. baumannii DMC-32a has not been reported previously from Bangladesh and not in this ST elsewhere. The emergence of such a nosocomial pathogen creates the need for effective therapeutics for critically-ill patients and for controlling hospital-acquired infections.

The First Saudi Study Investigating the Plasmid-borne Aminoglycoside and Sulfonamide Resistance among Acinetobacter baumannii Clinical Isolates Genotyped by RAPD-PCR: the Declaration of a Novel Allelic Variant Called aac(6')-SL and Three Novel Mutations in the sul1 Gene in the Acinetobacter Plasmid (s).

BackgroundAcinetobacter baumannii (A. baumannii) is one of the most important nosocomial pathogens responsible for a wide range of infections.AimThis study aimed to investigate the existence of the plasmidic genes encoding for aminoglycoside modifying enzymes (AMEs), 16S rRNA methyltransferases (RMT), and the altered dihydropetroate synthase (DHPS) encoded by the sul1 gene among A. baumannii clinical isolates collected from Taif, Kingdom of Saudi Arabia (KSA). The mutations in aac(6ʹ)-Ib and sul1 genes were also investigated.MethodsForty A. baumannii clinical isolates were investigated for their susceptibility to ten antibiotics. The plasmid DNA was extracted and screened for nine genes encoding for aminoglycoside resistance in addition to the sul1 gene. The clonal relatedness was determined by random amplified polymorphic DNA (RAPD)-PCR. Mutation in aac(6ʹ)-Ib and the sul1 genes were detected by capillary electrophoresis sequencing (CES).ResultsAll isolates were A. baumannii in which 42.5% of them exhibited a high level of aminoglycoside resistance (HLAR). The most prevalent AMEs and RMT encoding genes were aph(3ʹ)-VI, the two aac(6ʹ) gene variants [aac(6ʹ)-Ib and aac(6ʹ)-SL], ant(3ʹʹ)-I, and armA in which 90%, 87.5%, 85%, and 45% of isolates tested positive, respectively. The other investigated aminoglycoside resistant encoding genes, namely aac(3)-II, aac(6ʹ)-II, and rmtB, were not detected. Only 15% of isolates harbored the sul1 gene. RAPD-PCR classified the 40 isolates into three clusters in which cluster II was the main cluster. DNA sequencing revealed that 34.29% (12/35) of isolates tested positive for aac(6ʹ)-Ib were found to harbor a common missense mutation in position 102 indicating a novel allelic variant named aac(6ʹ)-SL. Also, DNA sequencing revealed three missense mutations in the sul1 gene.ConclusionThis is the first Saudi study to investigate the plasmid borne aminoglycoside and sulfonamide resistance genes among A. baumannii clinical isolates. A novel allelic variant for aac(6ʹ)-Ib was detected in addition to novel mutations in the sul1 gene.

Genome features and antibiotic resistance of Pseudomonas aeruginosa strains isolated in patients with cystic fibrosis in the Russian Federation.

Cystic fibrosis (CF) is a common genetic disease, manifested by airway obstruction and chronic respiratory infection. The most prevalent infectious agent in airways of CF patients is Pseudomonas aeruginosa. This study aimed to determine sequence-types, antimicrobial resistance phenotypes and genes defining adaptive antibiotic resistance in P. aeruginosa isolates recovered from CF patients in Russia. In total, 84 P. aeruginosa strains from 64 CF patients were analyzed. Susceptibility to antibiotics was determined by disk diffusion test. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, PubMLST were used for analysis of WGS data. Examined P. aeruginosa isolates belonged to 53 different sequence-types (STs), including 6 new STs. High-risk epidemic clone ST235 (10%) and clonal CF P. aeruginosa strains ST17, ST242, ST274 (7%) were detected. Non-susceptibility to ticarcillin-clavulanate, cefepime, imipenem was observed in 63%, 12% and 25% of isolates, respectively; to tobramycin - in 24%, to amikacin - in 35%; to ciprofloxacin, levofloxacin - in 35% and 57% of strains, respectively. Multidrug-resistant phenotype was detected in 18% of isolates. In examined strains, genes of beta-lactamases VIM-2 (5 ST235 strains), VEB-1 (two ST2592 strains), GES-1 (1 ST235 strain), PER-1 (1 ST235 strain) were found. Ciprofloxacin-modifying enzyme CrpP gene was detected in 67% of isolates, aminoglycoside-modifying enzymes AAD, ANT, AAC genes - in 7%, 4%, 12% of strains, respectively. P. aeruginosa isolates from CF patients in Russia demonstrate a high clonal diversity, which is similar to other P. aeruginosa infections. The isolates of high-risk clone and clonal CF P. aeruginosa strains are detected.

Metagenomic strategies identify diverse integron-integrase and antibiotic resistance genes in the Antarctic environment.

The objective of this study is to identify and analyze integrons and antibiotic resistance genes (ARGs) in samples collected from diverse sites in terrestrial Antarctica. Integrons were studied using two independent methods. One involved the construction and analysis of intI gene amplicon libraries. In addition, we sequenced 17 metagenomes of microbial mats and soil by high‐throughput sequencing and analyzed these data using the IntegronFinder program. As expected, the metagenomic analysis allowed for the identification of novel predicted intI integrases and gene cassettes (GCs), which mostly encode unknown functions. However, some intI genes are similar to sequences previously identified by amplicon library analysis in soil samples collected from non‐Antarctic sites. ARGs were analyzed in the metagenomes using ABRIcate with CARD database and verified if these genes could be classified as GCs by IntegronFinder. We identified 53 ARGs in 15 metagenomes, but only four were classified as GCs, one in MTG12 metagenome (Continental Antarctica), encoding an aminoglycoside‐modifying enzyme (AAC(6´)acetyltransferase) and the other three in CS1 metagenome (Maritime Antarctica). One of these genes encodes a class D β‐lactamase (blaOXA‐205) and the other two are located in the same contig. One is part of a gene encoding the first 76 amino acids of aminoglycoside adenyltransferase (aadA6), and the other is a qacG2 gene.

Detection of Aminoglycosides Modifying Enzymes (AMEs) genes and mutation in Acinetobacter baumannii isolates along with efflux pump activity

Detection of Aminoglycosides Modifying Enzymes (AMEs) genes and mutation in Acinetobacter baumannii isolates along with efflux pump activity

Role of Aminoglycoside-Modifying Enzymes (AMEs) in Resistance to Aminoglycosides among Clinical Isolates of Pseudomonas aeruginosa in the North of Iran.

In recent years, the prevalence of resistance to aminoglycosides among clinical isolates of Pseudomonas aeruginosa is increasing. The aim of this study was to investigate the role of aminoglycoside-modifying enzymes (AMEs) in resistance to aminoglycosides in clinical isolates of P. aeruginosa. The clinical isolates were collected from different hospitals. Disk agar diffusion test was used to determine the antimicrobial resistance pattern of the clinical isolates, and the minimum inhibitory concentration of aminoglycosides was detected by microbroth dilution method. The PCR was performed for discovery of aminoglycoside-modifying enzyme-encoding genes. Among 100 screened isolates, 43 (43%) isolates were resistant to at least one tested aminoglycosides. However, 13 (13%) isolates were resistant to all tested aminoglycosides and 37 isolates were detected as multidrug resistant (MDR). The resistance rates of P. aeruginosa isolates against tested antibiotics were as follows: ciprofloxacin (41%), piperacillin-tazobactam (12%), cefepime (32%), piperacillin (26%), and imipenem (31%). However, according to the MIC method, 13%, 32%, 33%, and 37% of the isolates were resistant to amikacin, gentamicin, tobramycin, and netilmicin, respectively. The PCR results showed that AAC(6′)-Ib was the most commonly (26/43, 60.4%) identified AME-encoding gene followed by AAC(6′)-IIa (41.86%), APH(3′)-IIb (34.8%), ANT(3″)-Ia (18.6), ANT(2″)-Ia (13.95%), and APH(3″)-Ib (2.32%). However, APH(3′)-Ib was not found in any of the studied isolates. The high prevalence of AME-encoding genes among aminoglycoside-resistant P. aeruginosa isolates in this area indicated the important role of AMEs in resistance to these antibiotics similar to most studies worldwide. Due to the transmission possibility of these genes between the Gram-negative bacteria, we need to control the prescription of aminoglycosides in hospitals.

Capability of Enterococcus faecalis to shield Gram-negative pathogens from aminoglycoside exposure.

Enterococcus faecalis commonly produce aminoglycoside-modifying enzymes (AMEs) and are implicated in polymicrobial infections. To determine if AME-producing E. faecalis is capable of protecting Enterobacteriaceae and Pseudomonas aeruginosa from gentamicin exposure. Two Klebsiella pneumoniae isolates, two Escherichia coli isolates, and two Pseudomonas aeruginosa isolates were investigated in monoculture time-kill experiments, and each Gram-negative organism was also evaluated during co-culture with either AME-producing or AME-deficient E. faecalis. A pharmacokinetic/pharmacodynamics analysis that utilized Log Ratio Areas and a Hill-type mathematical model was used to determine if the maximal killing or potency of gentamicin against the Gram-negative organisms was altered by the presence of the E. faecalis. The maximal killing and potency of gentamicin was the same during monoculture and co-culture experiments for both K. pneumoniae isolates and one E. coli isolate (P > 0.05). In contrast, the maximal killing of gentamicin was attenuated against one E. coli isolate and both P. aeruginosa isolates during co-culture with E. faecalis (P < 0.05). The potency of gentamicin was variable against the three aforementioned isolates. Against the E. coli isolate, the potency of gentamicin was significantly reduced by the presence of either E. faecalis isolate (EC50 95% CI = 4.23-4.43 mg/L monoculture versus 3.86-4.19 mg/L and 3.55-3.96 mg/L during co-culture with AME-producing and AME-deficient E. faecalis, respectively). The potency of gentamicin increased or decreased for P. aeruginosa depending on which E. faecalis isolate was investigated. The AME-producing E. faecalis did not provide a consistent protective effect from aminoglycosides for the Gram-negative pathogens.

Interaction of Acinetobacter baumannii with Human Serum Albumin: Does the Host Determine the Outcome?

Acinetobacter baumannii has become a serious threat to human health due to its extreme antibiotic resistance, environmental persistence, and capacity to survive within the host. Two A. baumannii strains, A118 and AB5075, commonly used as model systems, and three carbapenem-resistant strains, which are becoming ever more dangerous due to the multiple drugs they can resist, were exposed to 3.5% human serum albumin (HSA) and human serum (HS) to evaluate their response with respect to antimicrobial resistance, biofilm formation, and quorum sensing, all features responsible for increasing survival and persistence in the environment and human body. Expression levels of antibiotic resistance genes were modified differently when examined in different strains. The cmlA gene was upregulated or downregulated in conditions of exposure to 3.5% HSA or HS depending on the strain. Expression levels of pbp1 and pbp3 tended to be increased by the presence of HSA and HS, but the effect was not seen in all strains. A. baumannii A118 growing in the presence of HS did not experience increased expression of these genes. Aminoglycoside-modifying enzymes were also expressed at higher or lower levels in the presence of HSA or HS. Still, the response was not uniform; in some cases, expression was enhanced, and in other cases, it was tapered. While A. baumannii AB5075 became more susceptible to rifampicin in the presence of 3.5% HSA or HS, strain A118 did not show any changes. Expression of arr2, a gene involved in resistance to rifampicin present in A. baumannii AMA16, was expressed at higher levels when HS was present in the culture medium. HSA and HS reduced biofilm formation and production of N-Acyl Homoserine Lactone, a compound intimately associated with quorum sensing. In conclusion, HSA, the main component of HS, stimulates a variety of adaptative responses in infecting A. baumannii strains.

Generating Genotype-Specific Aminoglycoside Combinations with Ceftazidime/Avibactam for KPC-Producing Klebsiella pneumoniae.

Antibiotic combinations, including ceftazidime/avibactam (CAZ/AVI), are frequently employed to combat KPC-producing Klebsiella pneumoniae (KPC-Kp), though such combinations have not been rationally optimized. Clinical KPC-Kp isolates with common genes encoding aminoglycoside-modifying enzymes (AMEs), aac(6')-Ib' or aac(6')-Ib, were used in static time-kill assays (n = 4 isolates) and the hollow-fiber infection model (HFIM; n = 2 isolates) to evaluate the activity of gentamicin, amikacin, and CAZ/AVI alone and in combinations. A short course, one-time aminoglycoside dose was also evaluated. Gentamicin plus CAZ/AVI was then tested in a mouse pneumonia model. Synergy with CAZ/AVI was more common with amikacin for aac(6')-Ib'-containing KPC-Kp but more common with gentamicin for aac(6')-Ib-containing isolates in time-kill assays. In the HFIM, although the isolates were aminoglycoside-susceptible at baseline, aminoglycoside monotherapies displayed variable initial killing, followed by regrowth and resistance emergence. CAZ/AVI combined with amikacin or gentamicin resulted in undetectable counts 50 h sooner than CAZ/AVI monotherapy against KPC-Kp with aac(6')-Ib'. CAZ/AVI monotherapy failed to eradicate KPC-Kp with aac(6')-Ib and a combination with gentamicin led to undetectable counts 70 h sooner than with amikacin. A one-time aminoglycoside dose with CAZ/AVI provided similar killing to aminoglycosides dosed for 7 days. In the mouse pneumonia model (n = 1 isolate), gentamicin and CAZ/AVI achieved a 6.0-log10 CFU/lung reduction at 24 h, which was significantly greater than either monotherapy (P < 0.005). Aminoglycosides in combination with CAZ/AVI were promising for KPC-Kp infections; this was true even for a one-time aminoglycoside dose. Selecting aminoglycosides based on AME genes or susceptibilities can improve the pharmacodynamic activity of the combination.

Synthesis and Antibacterial Activity of Propylamycin Derivatives Functionalized at the 5''- and Other Positions with a View to Overcoming Resistance Due to Aminoglycoside Modifying Enzymes.

Propylamycin (4'-deoxy-4'-propylparomomycin) is a next generation aminoglycoside antibiotic that displays increased antibacterial potency over the parent, coupled with reduced susceptibility to resistance determinants and reduced ototoxicity in the guinea pig model. Propylamycin nevertheless is inactivated by APH(3')-Ia, a specific aminoglycoside phosphotransferase isozyme that acts on the primary hydroxy group of the ribofuranosyl moiety (at the 5''-position). To overcome this problem, we have prepared and studied the antibacterial and antiribosomal activity of various propylamycin derivatives carrying amino or substituted amino groups at the 5''-position in place of the vulnerable hydroxy group. We find that the introduction of an additional basic amino group at this position, while overcoming the action of the aminoglycoside phosphoryltransferase isozymes acting at the 5''-position as anticipated, results in a significant drop in selectivity for the bacterial over the eukaryotic ribosomes that is predictive of increased ototoxicity. In contrast, 5''-deoxy-5''-formamidopropylamycin retains the excellent across-the-board levels of antibacterial activity of propylamycin itself, while circumventing the action of the offending aminoglycoside phosphotransferase isozymes and affording even greater selectivity for the bacterial over the eukaryotic ribosomes. Other modifications to address the susceptibility of propylamycin to the APH(3')-Ia isozyme including deoxygenation at the 3'-position and incorporation of a 6',5''-bis(hydroxyethylamino) modification offer no particular advantage.

Characteristics of High-Level Aminoglycoside-Resistant Enterococcus faecalis Isolated from Bulk Tank Milk in Korea.

Simple SummaryAminoglycosides are used to treat various infections in veterinary and human medicine. However, with the emergence of high-level aminoglycoside resistance in human and food-producing animals, the synergism of aminoglycosides with beta-lactam or glycopeptide is being threatened. Moreover, the environmental mastitis-causing agent, enterococci, has emerged as a cause of nosocomial infection due to its antimicrobial resistance. Therefore, the purpose of this study was to investigate the characteristics of high-level aminoglycoside-resistant Enterococcus faecalis isolated from bulk tank milk in Korea. It showed that 185 (61.5%) isolates out of 301 were high-level aminoglycoside resistant, while 149 isolates were multidrug resistant.Enterococci, which are considered environmental mastitis-causing pathogens, have easily acquired aminoglycoside-resistant genes that encode various aminoglycoside-modifying enzymes (AME). Therefore, this study was conducted to compare the distribution of high-level aminoglycoside-resistant (HLAR) and multidrug-resistant (MDR) Enterococcus faecalis (E. faecalis) bacteria isolated from bulk tank milk in four dairy companies in Korea. Moreover, it analyzed the characteristics of their antimicrobial resistance genes and virulence factors. Among the 301 E. faecalis bacteria studied, 185 (61.5%) showed HLAR with no significant differences among the dairy companies. Furthermore, 129 (69.7%) of the 185 HLAR E. faecalis showed MDR without significant differences among companies. In contrast, HLAR E. faecalis from companies A, B, and C were significantly higher in resistance to the four classes than those in company D, which had the highest MDR ability against the three antimicrobial classes (p < 0.05). In addition, in the distribution of AME genes, 72 (38.9%) and 36 (19.5%) of the isolates carried both aac(6′)Ie-aph(2″)-la and ant(6)-Ia genes, and the ant (6)-Ia gene alone, respectively, with significant differences among the companies (p < 0.05). In the distribution of virulence genes, the ace (99.5%), efa A (98.9%), and cad 1 (98.4%) genes were significantly prevalent (p < 0.05). Thus, our results support that an advanced management program by companies is required to minimize the dissemination of antimicrobial resistance and virulence factors.

Drug Resistance Mechanism among Acinetobacter Species

Acinetobacter species cause infections that are difficult to control due to multi-drug resistance and are noted for their intrinsic resistance to antibiotics and for their ability to acquire genes encoding resistance for the production of beta-lactamases and Aminoglycoside-modifying enzymes. MBLs are molecular class B and functional group 3 beta-lactamases which have the capability of hydrolyzing all β-lactams except the Monobactam, Aztreonam. Of several MBLs, only IMP, VIM and SIM types have been detected in these species. To analyze the antibiotic resistance patterns among Acinetobacter isolates and to detect Carbapenemase and MBL among MDR Acinetobacter isolates. The descriptive study of all phenotypically identified strains and multidrug-resistant strains of Acinetobacter species was conducted. A total of 303 isolates were isolated from various samples. They were processed and identified by standard Microbiological procedures. The antibiotics susceptibility testing was performed by Kirby- Bauer disc diffusion method using CLSI guidelines. Carbapenemase production was detected by employing 3 phenotypic test methods (MHT, CDM and DDST). Of 6355 samples processed, 303 were found to be Acinetobacter species, among those 50 were multidrug-resistant strains. The highest isolation of MDR Acinetobacter was from endotracheal tube tip (42%) and pus sample (32%). The majority of MDR Acinetobacter infection was found in male patients 36 (72%) compared to female patients 14 (28%). The majority of the strains were isolated from patients &gt;/ 60 years of age group (%). A number of these isolates were more from ICU wards (30%) followed by Surgery wards (24%). Higher resistance for the Piperacillin/tazobactam ((82%), followed by Ceftazidime (80%), Imipenem (76%) etc. and the most susceptible drug was found to be the Tigecycline (82%) followed by Colistin (80%). Carbapenemase production was detected by MHT and 24 (48%) isolates were MHT positive. MBL production was detected by CDM and 34 (68%) isolates were CDM positive and by DDST 30 (60%) isolates were positive. Acinetobacter species are increasingly important nosocomial pathogens and are capable of rapid adaptation to the hospital environment. The variety of potential source of contamination or infection with these species in the hospital environment makes control of outbreaks caused by these difficult.

Clonal change of carbapenem-resistant Acinetobacter baumannii isolates in a Korean hospital.

The expansion of specific carbapenem-resistant Acinetobacter baumannii (CRAB) clones is a global concern due to its therapeutic difficulty and epidemicity. To understand the prevalence of CRAB isolates in a Korean hospital, we investigated the epidemiological characteristics of 96 CRAB isolates between 2016 and 2018, including the sequence types (STs), antimicrobial susceptibility, and genetic background of resistance to carbapenems and aminoglycosides. Six STs were identified using the Oxford multilocus sequence typing scheme; ST191 (n=8), ST208 (n=12), ST229 (n=11), and ST369 (n=21) were previously identified clones in the study hospital, whereas gpi variants of ST208, ST451 (n=34) and ST784 (n=10), were emerging clones. ST208 isolates exhibited higher resistance rates to minocycline than other ST isolates, whereas ST369 isolates exhibited lower resistance rates to aminoglycosides and trimethoprim/sulfamethoxazole than other ST isolates. All CRAB isolates previously isolated in the study hospital carried ISAbaI-blaOXA-23 for carbapenem resistance, but 10 ST229 isolates carried only ISAbaI-blaOXA-51. The carriage of armA was lower in ST369 isolates (38%) than in other ST isolates (≥83%). The frequency and diversity of aminoglycoside-modifying enzyme genes were decreased among the CRAB isolates between 2016 and 2018 compared with CRAB isolates between 2013 and 2015 at the study hospital. In conclusion, clonal complex 208 CRAB isolates are predominant in the study hospital. This study demonstrates the evolutionary change of CRAB isolates in the study hospital in relation to the emergence of new STs and selection of resistant genes.

Amikacin Combined with Fosfomycin for Treatment of Neonatal Sepsis in the Setting of Highly Prevalent Antimicrobial Resistance.

Antimicrobial resistance (particularly through extended-spectrum β-lactamase and aminoglycoside-modifying enzyme production) in neonatal sepsis is a global problem, particularly in low- and middle-income countries, with significant mortality rates. High rates of resistance are reported for the current WHO-recommended first-line antibiotic regimen for neonatal sepsis, i.e., ampicillin and gentamicin. We assessed the utility of fosfomycin and amikacin as a potential alternative regimen to be used in settings of increasingly prevalent antimicrobial resistance. The combination was studied in a 16-arm dose-ranged hollow-fiber infection model (HFIM) experiment. The combination of amikacin and fosfomycin enhanced bactericidal activity and prevented the emergence of resistance, compared to monotherapy with either antibiotic. Modeling of the experimental quantitative outputs and data from checkerboard assays indicated synergy. We further assessed the combination regimen at clinically relevant doses in the HFIM with nine Enterobacterales strains with high fosfomycin and amikacin MICs and demonstrated successful kill to sterilization for 6/9 strains. From these data, we propose a novel combination breakpoint threshold for microbiological success for this antimicrobial combination against Enterobacterales strains, i.e., MICF × MICA < 256 (where MICF and MICA are the fosfomycin and amikacin MICs, respectively). Monte Carlo simulations predict that a standard fosfomycin-amikacin neonatal regimen would achieve >99% probability of pharmacodynamic success for strains with MICs below this threshold. We conclude that the combination of fosfomycin with amikacin is a viable regimen for the empirical treatment of neonatal sepsis and is suitable for further clinical assessment in a randomized controlled trial.

Rapid and Accurate Detection of Aminoglycoside-Modifying Enzymes and 16S rRNA Methyltransferases by Targeted Liquid Chromatography-Tandem Mass Spectrometry.

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in curbing drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside-modifying enzymes and 16S rRNA methyltransferases in Escherichia coli and Klebsiella pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, AAC(6')-Ib-cr, ANT(2″)-I, APH(3')-VI, ArmA, RmtB, RmtC, and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole-genome sequencing results. Of the 2,460 isolate and resistance mechanism combinations tested, 2,416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin, and amikacin in only the E. coli and K. pneumoniae isolates (n = 191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

Molecular survey of aminoglycoside-resistant Acinetobacter baumannii isolated from tertiary hospitals in Qazvin, Iran

Aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylases (16S RMTase) are two main resistance mechanisms against aminoglycosides. This study aimed to evaluate the frequency of AMEs and 16S rRNA methylase genes among aminoglycoside non-susceptible Acinetobacter baumannii isolates and to assess their clonal relationship using repetitive extragenic palindromic-PCR (rep-PCR). In this cross-sectional study, a total of 192 A. baumannii isolates were collected from the patients hospitalized in Qazvin, Iran (January 2016 to January 2018). Identification of isolates was performed by standard laboratory methods and API 20E strips. Antimicrobial susceptibility was determined by Kirby–Bauer method followed by examination of the genes encoding the AMEs and 16S RMTase by PCR and sequencing methods. The clonal relationship of isolates was carried out by rep-PCR. In total, 98.4% of isolates were non-susceptible to aminoglycosides, 98.4%, 97.9% and 83.9% of isolates were found to be non-susceptible against gentamicin, tobramycin and amikacin, respectively. The frequencies of aph(3′)-VI, aac(6′)-Ib, aac(3)-II, aph(3′)-Ia and armA genes were 59.3%, 39.2%, 39.2%, 31.7% and 69.8%, respectively, either alone or in combination. Rep-PCR results showed that the aminoglycoside non-susceptible isolates belonged to three distinct clones: A (79.4%), B (17.5%) and C (3.2%). The findings of this study showed a high frequency for AMEs with the emergence of armA genes among the aminoglycoside non-susceptible A. baumannii isolates. Rational administration of aminoglycosides as well as using an appropriate infection control policy may reduce the presence of resistance to antibiotics in medical centres.