High-level Aminoglycoside Resistance Research Articles

Occurrence of Antimicrobial-Resistant Enterococcus spp. in Healthy Chickens Never Exposed to Antimicrobial Agents in Central Italy

Enterococci are part of the natural flora of the gastrointestinal tract of mammals, including humans, birds and invertebrates. They can cause infection, mainly among hospitalized patients, as well as acquire and transfer antimicrobial resistance genes. The present study allowed the isolation of 98 Enterococcus (73.47% E. faecium, 23.47% E. faecalis, 3.06% E. avium) strains from 120-day-old healthy chickens that had never been treated with antimicrobials. Their antimicrobial resistance was evaluated by the agar disk diffusion method; high-level aminoglycoside (streptomycin and gentamicin) and vancomycin resistance were established using the microbroth dilution method. The highest percentages of resistant isolates were detected with quinupristin–dalfopristin (88.78%), rifampicin (64.29%), tetracyclines (45.92%), and enrofloxacin (41.84%). High percentages of susceptible strains were found with teicoplanin (100%), amoxicillin–clavulanic acid (97.96%), nitrofurantoin (94.90%), ampicillin (92.86%), chloramphenicol (90.82%), and linezolid (88.78%). About 60% of the strains were classified as MDR (multidrug-resistant). Moreover, PCR was carried out to investigate genes encoding for tetracyclines resistance determinants: tet(M), tet(L), tet(O), tet(K), and Int-Tn. Genes were detected in 68 (69.38%) strains: 36 were shown to be resistant with the agar disk diffusion method, while 28 were intermediate, and 2 were susceptible. The present study showed that chickens never treated with antimicrobials potentially harbor enterococci having phenotypic and genotypic characters of antimicrobial resistance.

When it rains it pours: An increased prevalence of intestinal carriage of vancomycin-resistant Enterococcus faecium related to higher use of oral vancomycin in a tertiary care Hungarian clinical centre during the years of the COVID-19 pandemic

ObjectivesThis study aims to investigate the association between oral vancomycin consumption and intestinal vancomycin-resistant Enterococcus carriage in the pre- and COVID era in the clinical centre of the University of Szeged, Hungary. MethodsThis retrospective microbiological examination was carried out using electronically collected data, corresponding to the period between 1 January 2018 and 31 December 2022, at the Department of Medical Microbiology. Data included isolated species and the according antimicrobial susceptibility patterns. Annual consumption data for oral vancomycin consumption were exported from the database of the central pharmacy of the clinical centre. As a strain typing procedure, Fourier transform infrared spectroscopy analysis was used. ResultsThere was a significant increase in the number of faecal vancomycin-resistant Enterococcus isolates throughout the study. The prevalence increased significantly during the years of the pandemic. The use of orally administered vancomycin in the clinical centre increased significantly. A strong positive correlation existed between the two phenomena. Several strains with different resistance patterns spread in the clinical centre. Two of these occurred in greater numbers, differing in their high-level aminoglycoside resistance. However, the overall resistance of these strains was stagnating. FTIR analysis revealed that 59 of the 62 strains were also divided into 2 large clusters differing partially in their high-level aminoglycoside resistance. ConclusionsDuring the pandemic, intestinal VRE carriage among clinical centre patients increased significantly, linked to increased oral vancomycin use. Different strains spread, with aminoglycoside resistance being the primary distinction. This highlights the negative impact of the pandemic on VRE carriage.

Presence of genes encoding aminoglycoside-modifying enzyme (AME) and virulence factors in high-level aminoglycoside-resistant (HLAR) Enterococcus strains isolated from retail chicken meat in Turkey

In this study, the presence of aminoglycoside-modifying enzyme (AME) and virulence factor genes were investigated in previously isolated 32 high-level aminoglycoside-resistant (HLAR) Enterococcus strains isolated from retail chicken meat in Turkey. At least one AME-encoding gene was detected in HLAR enterococci by polymerase chain reaction (PCR). The ant(6ʹ)-Ia was identified as the most prevalent (87.5%, 28/32) AME gene. The aph(3ʹ)-IIIa (78.13%, 25/32), ant(4ʹ)-Ia (68.75%, 22/32), aph(2ʹʹ)-Ib (62.5%, 20/32), aac(6ʹ)-Ie-aph(2ʹʹ)-Ia (21.88%, 7/32) and aph(2ʹʹ)-Ic (9.38%, 3/32) are the other detected AME-encoding genes in strains. The aph(2ʹʹ)-Id was found in none of the HLAR strains. The aph(2ʹʹ)-Ib and ant(6ʹ)-Ia were identified as the most frequently AME-encoding genes in high-level gentamicin-resistant (HLGR) and high-level streptomycin-resistant (HLSR) strains, respectively. Among the 32 HLAR strains, only E. faecalis MSE61.1 and E. avium MSE63.1 were found capable of hydrolyzing gelatine. All HLAR strains showed α-hemolytic activity except E. durans MG13.4 and E. casseliflavus MGM111.1, which were exhibited β- and γ-hemolytic activity, respectively. It was determined that all HLAR strains, except E. durans MGE13.1 and MGE63.1, contain at least one virulence factor gene. The efaAfm (87.5%, 28/32), acm (65.63%, 21/32) and gelE (37.5%, 12/32) were found to be the most prevalent virulence factor genes. HLAR enterococci strains that have the virulence factor genes may pose a risk to consumer health.

Activity of Cinnamic Acid Derivatives with 4-Chloro-2-mercaptobenzenesulfonamide Moiety against Clinical HLAR and VRE Enterococcus spp.

The rapid increase in strains that are resistant to antibiotics requires new active compounds to be found whose mechanism of action on bacteria is different to those that are currently known. Of particular interest are compounds that occur in plants as secondary metabolites. The focus of this study concerns the examination of the effects of synthetic cinnamic acid derivatives, with 4-chloro-2-mercaptobenzenesulfonamide moiety on Enterococcus spp. with HLAR (high-level aminoglycoside resistance) and VRE (vancomycin-resistant Enterococcus) mechanisms. The minimum inhibitory concentration (MIC) values of the tested compounds were determined using the serial dilution method for Enterococcus spp. groups, and the most active compounds were as follows: 16d, 17c, 16a, 16c and 16f (2-4 µg/mL). These compounds, at a concentration of 4 × MIC, inhibited the biofilm formation of HLAR strains (70 to 94%). At concentrations of 2 × MIC and 4 × MIC, they also inhibited the growth of VRE strains (42 to 96%). The best effect produced on the formed biofilm was demonstrated by compound 16f (from 62% MIC concentration to 89% 4 × MIC concentration) on the tested HLAR strains. In vitro studies, using the peripheral blood of domestic sheep, demonstrated the stable bacteriostatic activity of the tested compounds against Enterococcus spp. The compounds 16a, 16c, 16d, 16f and 17c showed synergism and additivity with ampicillin, streptomycin, gentamicin and vancomycin against resistant strains of Enterococcus spp. The tested compounds, when combined, reduce the MIC for antibiotics by 800 to 10,000 times for HLAR strains and by 8 to 10,000 times for VRE strains. The MIC of the tested compounds, in combination with antibiotics, is reduced 2-16-fold for HLAR strains and 2-32-fold for VRE strains. These studies demonstrate the potential for the therapeutic use of synthetic, cinnamic acid derivatives, with 4-chloro-2-mercaptobenzenesulfonamide moiety, to work against clinical strains of Enterococcus spp.

An insight into genes responsible for fosfomycin resistance among uropathogens of asymptomatic bacteriuria during pregnancy: A North Indian study.

Asymptomatic bacteriuria (ASB) is a common finding during pregnancy. Effective antibiotic treatment could reduce its adverse effects on both mother and fetus. However, emerging antimicrobial resistance limits the treatment options. Fosfomycin might be a promising drug in this regard, as its resistance is still low. The aim of the study was to determine the antimicrobial susceptibility pattern of fosfomycin in isolates causing ASB by disc diffusion and agar dilution (in selected isolates), determine minimum inhibitory contribution (MIC) by agar dilution in isolates resistant by disc diffusion and detect the genes responsible for fosfomycin resistance. This was a 2-year study carried in the Department of Microbiology, Jawaharlal Nehru Medical College and Hospital (JNMCH), Aligarh Muslim University (AMU), Aligarh. A total of 10 252 urine samples from asymptomatic pregnant females (18-45 years) attending the antenatal care (ANC) outpatient department (OPD) were submitted. Identification of pathogen and antimicrobial susceptibility testing (AST) was carried out as per standard methods of CLSI. There was phenotypic detection of methicillin-resistant Staphylococcus aureus (MRSA) and other Staphylococcus species (MRSS), high-level aminoglycoside resistance (HLAR), vancomycin resistant Enterococci (VRE) and S. aureus (VRSA), extended spectrum β-lactamase (ESBL) and carbapenem-resistant Enterobacterales (CRE). All the fosfomycin-resistant isolates (by disk diffusion) were tested by agar dilution. Conventional PCR was performed for murA, fosA, uhpT and glpT genes on all resistant isolates. In this study, the prevalence of ASB among pregnant females was 1173(11.4 %), in which Escherichia coli 495(42 %) was the predominant organism. The overall sensitivity of fosfomycin among Gram-positive cocci (GPC) and Gram-negative bacilli (GNB) was 99 % and 97.6 %, respectively. MRSA and MRSS accounted for 50 (66.6 %) and 71 (76 %), respectively. The highest rates of MIC >2048 µg ml-1 were shown by most isolates (mainly E. coli ) on agar dilution. PCR studies revealed four E. coli strains possessed both murA (also present in one K. pneumoniae strain) and glpT genes. While only one isolate ( E. faecalis ) was positive for fosA gene. But none of the strain possessed the uhpT gene. According to this study, murA and glpT genes were more frequent than fosA. We cannot comment on the prevalence and regional distribution of fosfomycin-resistant genes based on this preliminary study. Therefore, more Indian studies should be carried out to create awareness about the presence of genes in a particular area.

The transmutation of Escherichia coli ATCC 25922 to small colony variants (SCVs) E. coli strain as a result of exposure to gentamicin

BackgroundSmall colony variants (SCVs) are biotypes of bacteria that have a size of approximately one-tenth or less of the wild types and has distinct characteristics comparing to the wild type strains. Clinical SCVs are usually associated with persistent infection and require a long-term treatment program with antibiotics. In Saudi Arabia, there are few studies about SCVs Escherichia coli for this reason, this study is aimed to investigate the ability of gentamicin to mutate E. coli ATCC 25922 to produce small SCVs and investigate the genotypes and phenotypes changes and stress tolerance comparing to clinical SCVs E. coli and normal clinical E. coli Isolated from blood samples. MethodsIn this investigation, four clinical blood samples were collected ted from patients and the cultivation and isolation were carried out in KFMC between December 2019 and February 2021. The identification of positive blood culture samples was done using phoenix MD. Non-SCV E. coli ATCC25922 were mutated to SCV using exposure to increasing gradual concentrations of gentamicin at 100-generation intervals. Biochemical features and susceptibility to standard antibiotics using automated Phoenix MD 50 and. The survival assays were done using several stresses including heat shock, low pH, high osmotic pressure, and oxidative pressure. Virulence genes screening included the detection of genes that encoded to α-haemolysin, CS12 fimbriae, F17-like fimbrial adhesion, P-related fimbriae, yersiniabactin siderophore system, P-fimbriae, aerobactin, iron-regulated genes using PCR and gel electrophoresis. ResultsThe data from the mutating E. coli ATCC 25922 small colony test with gentamicin revealed that the first emergence of the multidrug resistance (MDR) SCV E. coli strain occurred at generation number 250, corresponding to a gentamicin concentration of 57 g/ml. Pathogenicity islands detection revealed that all tested E. coli strains have PAI IV 536 genes on their chromosomes furthermore, mutated SCV E. coli ATCC 25922 acquired PAII CFT073 and PAI IV 536. Survival tests showed no significant differences changes in tolerance of mutated SCVs comparing to parental strain. ConclusionThe present work concluded that gentamicin sub-MIC concentration gradual exposure can induce mutation responsible for SCV formation and evolving of MDR E. coli strains. The mutated SCVs evolved high-level aminoglycoside resistance for gentamicin and resistance to amikacin, it also developed resistance to 2 cephalosporin antibiotics cefuroxime, and cephalothin and a resistance to aztreonam.

Phenotypic and Genotypic Analysis of Bacterial Pathogens Recovered from Patients Diagnosed with Fever of Unknown Origin in Egypt.

Fever of unknown origin (FUO) is a medical term describing fever that lasts for at least three weeks without a diagnosis being reached after extensive diagnostic evaluation. Therefore, this study aimed to identify the common pathogens causing FUO in patients admitted to Abbasia Fever Hospital in Egypt from January 2020 to December 2022, their antimicrobial susceptibility profiles, and associated resistance genes. The study also aimed to investigate the burden of multidrug-resistant (MDR) pathogens and the priority pathogens nominated by the World Health Organization (WHO) for posing the greatest threat to human health due to antibiotic resistance. During the study period, about 726 patients were diagnosed with FUO. After extensive investigations, the cause of the FUO was found to be infectious diseases in 479/726 patients (66.0%). Of them, 257 patients had positive bacterial cultures, including 202 Gram-negative isolates that comprised Klebsiella pneumoniae (85/202; 42.1%), Escherichia coli (71/202; 35.1%), Acinetobacter baumannii (26/202; 12.9%), and Pseudomonas aeruginosa (14/202; 6.9%) and 55 Gram-positive isolates, including Staphylococcus aureus (23/55; 41.8%), Streptococcus pneumoniae (7/55; 12.7%), and Enterococcus spp. (25/55; 45.5%). The MDR phenotype was shown by 68.3% and 65.5% of the Gram-negative and Gram-positive isolates, respectively. Carbapenem resistance (CR) was shown by 43.1% of the Gram-negative isolates. Of the 23 S. aureus isolates obtained from research participants, 15 (65.2%) were methicillin-resistant S. aureus (MRSA). A high-level aminoglycoside resistance (HLAR) phenotype was found in 52.0% of the Enterococcus sp. isolates. The PCR screening of resistance genes in the MDR isolates showed that blaOXA-48 was the most prevalent (84%) among the carbapenemase-coding genes, followed by blaVIM (9%) and then blaIMP (12%). The ESBL-coding genes blaTEM, blaCTX-M,aac(6')-Ib, and blaSHV, were prevalent in 100%, 93.2%, 85,% and 53.4% of the MDR isolates, respectively. This study updates the range of bacteria that cause FUO and emphasizes the burden of multidrug resistance and priority infections in the region. The obtained data is of relevant medical importance for the implementation of evidence-based antimicrobial stewardship programs and tailoring existing empirical treatment guidelines.

Contribution of different mechanisms to aminoglycoside resistance in clinical isolates of Acinetobacter baumannii

The antibiotics overuse for infection treatment was the sparkle in the spreading of multi-drug resistance Acinetobacter baumannii in hospitals. In our study, we evaluated the contribution of the aminoglycoside resistance mechanisms of A. baumannii to the resistance surge in some selected Egyptian hospitals with a checkerboard assay application to retrieve the aminoglycoside activity. The resistance profile analysis of collected 200 A. baumannii isolates revealed a multidrug-resistant pattern with limited susceptibilities to aminoglycosides. Analysis of the prevalence of aminoglycoside-modifying enzyme (AMEs) genes revealed the presence of the six AMEs genes either singly or in combination in selected isolates and aph (3)-VIa gene was the predominant one. At the same time, four efflux pump genes of AdeABC and AdeKJL family showed significant (P < 0.001) up-regulation levels. Moreover, the implementation of combination strategy showed fourteen synergistic activities against two high-level aminoglycoside-resistance (HLAR) A. baumannii isolates. The findings highlighted the alarming levels of aminoglycoside resistance in A. baumannii isolates, which proved that a common enzymatic modification mechanism acts synergistically with decreased antibiotic accumulation in acquiring aminoglycoside resistance. Additionally, the study provides useful information for the promising synergistic combination therapy that reduces the therapeutic dose of aminoglycosides used and subsequently increases their clinical application.

The emergence of Klebsiella pneumoniae sequence type 395 non-susceptible to carbapenems and colistin from Turkey

PurposeOXA-48 producing Klebsiella pneumoniae is an emerging threat and outbreaks due to specific sequence types have been commonly reported. Here, we report an outbreak due to multidrug-resistant ST395 K. pneumoniae ST395. To the best of our knowledge, this is the first outbreak of K. pneumoniae ST395 harbouring blaOXA-48 genes in our country. MethodsThe strains were characterized by antimicrobial susceptibility, extended-spectrum β-lactamase (ESBL) and carbapenemase production, plasmid-mediated colistin, high-level aminoglycoside, and quinolone resistance. Also multidrug efflux pumps and porin coding genes were investigated. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), wzi typing and plasmid analysis were used for the epidemiological relationships. ResultsAll strains were positive for blaOXA-48 with at least one of the β-lactamase genes (blaCTX-M, blaTEM, blaSHV) and harboured IncL plasmids. 16 of 20 (80%) isolates carried qnrA. All isolates were positive for aac(6’)-1b, acrAB-tolC, ompK35, and ompK36 genes but none of them harboured 16s rRNA methyltransferase, mcr-1-5, qepA, oqxAB, and mdtK genes. All strains had the same PFGE pattern, that is, wzi type K2 and found to be ST395 with MLST. ConclusionThe association of ST395 with OXA-48-producers could be an emerging threat for Turkey and continuous monitoring is crucial to prevent the spread of these powerful strains.

Investigation of High-level Aminoglycoside Resistance in Enterococcus spp. Isolated from Caspian Horse Feces in Guilan Province, Northern Iran

Investigation of High-level Aminoglycoside Resistance in <i>Enterococcus spp.</i> Isolated from Caspian Horse Feces in Guilan Province, Northern Iran

Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6′)Ie-aph(2″)Ia and aph(3′)-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6′)-Ie-aph(2″)-Ia or aph(3’)-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

Antibiogram Pattern and Virulence Trait Characterization of Enterococcus Species Clinical Isolates in Eastern India: A Recent Analysis.

Objective We aimed to evaluate the current antimicrobial susceptibility pattern and characterize putative virulence traits among Enterococcus species isolates from various clinical specimens in view of their increased isolation rates in both community-related and serious nosocomial infections, as well as resistance to many antibiotics. Methods Study (April 2017–March 2018) included consecutive, nonrepeated, discrete, and clinically significant isolates of enterococci. Susceptibility testing included detection of high-level aminoglycoside-resistant (HLAR) and glycopeptide-resistant enterococci (GRE). All screen-positive GRE isolates were investigated by polymerase chain reaction for species confirmation and presence of vanA/vanB genes. Virulence genes ace , asa1 , cyt , efa , esp , gelE , and hyl were investigated by molecular methods. Hemolysin and biofilm production were studied using phenotypic methods. Results Of 111 isolates, 89 (80.1%), 16 (14.4%), and 6 (5.4%) were from urine, pus, and blood, respectively, consisting predominantly of E. faecalis (67, 60.4%) and E. faecium (32, 28.8%). E. hirae (5, 4.5%) was the predominant non- E. faecalis non- E. faecium isolate. Other species were E. durans (4, 3.6%), E. avium (2, 1.8%), and E. mundtii (1, 0.9%). Seven (6.3%) out of the 111 isolates were GRE, all vanA genotype. HLAR was observed in 70 (63.1%) isolates, significantly higher in E. faecium than E. faecalis (81.2 vs. 58.2%; p < 0.05). All were susceptible to daptomycin. Hemolysin activity and biofilm production were observed in 38 (34.2%) and 36 (32.4%) isolates. Most frequent virulence genes were efa (77, 69.4%), ace (71, 63.9%), asa1 (67, 60.3%), and gelE (66, 59.4%). There was a predominant association of esp and hyl genes with E. faecium and that of the other genes with E. faecalis . Conclusion The study will contribute to the existing limited data on virulence trait characterization of clinical E. spp. isolates in India. At the same time, it will help to serve as a guide in the choice of empirical therapy in enterococcal infections leading to favorable clinical outcomes by decreasing the clinical failure, microbiological persistence, and associated mortality, and will lead to future studies on controlling the spread of virulent and multiresistant isolates.

MICROBIOLOGICAL ISOLATES AND IT’S RESISTOTYPE FROM CLINIC OF VASCULAR SURGERY FOR THE FIRST QUATER OF 2022

One of the most common complications of surgical exposures is the surgical site infection (SSI). Although it varies between different surgical profiles, it can reach up to one third of all complications. In vascular surgery patients ischemic ulcers are very common, as well as factors, compromising the immune system such as diabetes, chronic kidney disease etc. One of the main surgical exposures in vascular surgery is inguinal, providing access to the femoral artery and its bifurcation. Although it allows a wide range of reconstructions, implanting different types of prosthetic materials, stents and providing anastomosis site, it contains lymph nodes, which can contaminate the reconstruction and cause SSI with severe consequences. Patients, prone to SSI due to concomitant diseases, are threatened by sepsis, limb loss and even death, which makes prevention of those type of complications essential. Aim: To investigate etiological spectrum of microbiological isolates and their resistance against most common antimicrobials among vascular surgery patients. Materials and methods: The study is retrospective, conducted in the period 01 January 2022 – 31 March 2022. All of the samples were obtained from patients of Clinic of Vascular Surgery. After isolation of pure culture from the samples, the strains were identified by MALDI TOF MS and Vitec – 2 Compact. Antibiotic resistance was determined with Bauer-Kirby disk diffusion method. Results: From all 419 of the patients, hospitalized in the Clinic of Vascular surgery for this period, 28 isolates from 26 (6,21%) patients were obtained, of which Gram-negative were 19 (67,86%) and Gram-positive - 9 (32,14%). From Gram-negative - enterobacteria – 14 (73,68%), and non-fermenting gram-negative bacteria (NFGNB) were 5 (26,32%). Only 3 (21,43%) from all enterobacteria were extended spectrum beta-lactamases producing strains (ESBLs). No strains, resistant to carbapenems (RCP) were isolated. Five (55,55%) of the Gram-positive isolates were Staphylococcus aureus, 4 (80%) of which were methicillin resistant Staphylococcus aureus (MRSA). Two of the Gram-positive species isolated were Enterococcus faecalis, of which 1 with a high-level aminoglycoside resistance (HLAR). No Vancomycin resistant enterococci (VRE) were discovered. There were no colistin-resistant Acinetobacter baumannii and Pseudomonas aeruginosa strains. Conclusion: From all 28 isolates 8 (28,57%) were with acquired types of antimicrobial resistance. With almost one third of the isolates that are problematic in terms of antibiotic susceptibility, treatment of those patients can be challenging. Prevention of in hospital contamination with polyresistant strains, associated with medical care it is crucial for reducing the number of severe complications, decreasing of hospital stay and cost for treatment.

Novel 16S rRNA methyltransferase RmtE3 in Acinetobacter baumannii ST79.

Introduction. The 16S rRNA methyltransferase (16S RMTase) gene armA is the most common mechanism conferring high-level aminoglycoside resistance in Acinetobacter baumannii, although rmtA, rmtB, rmtC, rmtD and rmtE have also been reported.Hypothesis/Gap statement. The occurrence of 16S RMTase genes in A. baumannii in the UK and Republic of Ireland is currently unknown.Aim. To identify the occurrence of 16S RMTase genes in A. baumannii isolates from the UK and the Republic of Ireland between 2004 and 2015.Methodology. Five hundred and fifty pan-aminoglycoside-resistant A. baumannii isolates isolated from the UK and the Republic of Ireland between 2004 and 2015 were screened by PCR to detect known 16S RMTase genes, and then whole-genome sequencing was conducted to screen for novel 16S RMTase genes.Results. A total of 96.5 % (531/550) of isolates were positive for 16S RMTase genes, with all but 1 harbouring armA (99.8 %, 530/531). The remaining isolates harboured rmtE3, a new rmtE variant. Most (89.2 %, 473/530) armA-positive isolates belonged to international clone II (ST2), and the rmtE3-positive isolate belonged to ST79. rmtE3 shared a similar genetic environment to rmtE2 but lacked an ISCR20 element found upstream of rmtE2.Conclusion. This is the first report of rmtE in A. baumannii in Europe; the potential for transmission of rmtE3 to other bacterial species requires further research.

Prevalence of High Level Gentamicin Resistance among the Clinical Isolates of Enterococci Species

Enterococci infections, have drawn attention of clinicians due to rapid increase in high level aminoglycoside resistance (HLAR). This resistance predicts failure of β-lactam antibiotic and aminoglycoside combination, which is the current treatment of choice for serious enterococci infections. This led us to investigate about the prevalence of HLAR enterococci in our hospital setting in Shimoga, Karnataka. All the clinical samples received at Microbiology laboratory, McGan hospital, Shivamogga between June 2016 and December 2016 was considered for the study. Enterococci were identified and further speciated by following conventional method. Antibiotic susceptibility pattern of all the isolates was determined according to CLSI guidelines. High level gentamicin resistance was detected by disc diffusion method using 120µg gentamicin disc. A total of 64 Enterococci species were isolated from the clinical specimens. On speciation, Enterococcus faecalis (E. faecalis) accounted for 72% (46) of the total isolates, 24%(16) of the isolates were Enterococcus faecium (E. faecium)and 4% (2) isolates were Enterococcus durans (E. durans). High level gentamicin resistance was noted in 42% (27) among the total isolates. E. faecium was found to be more resistant compared to E. faecalis. E. durans was a sensitive strain. Our study reconfirms the high prevalence of HLAR in the clinical setting. The study stresses on the need for regular screening of HLAR in all Enterococci infections and revise the battery of drugs for its treatment according to the report.

Molecular Characterization of High-Level Aminoglycoside Resistance among Enterococcus Species.

Background Enterococci are nosocomial pathogen. They can develop high-level resistance to aminoglycoside by producing aminoglycoside modifying enzymes (AMEs). In enterococci, high level resistance to aminoglycosides is mediated by acquisition of plasmid mediated genes encoding for aminoglycoside modifying enzymes (AMEs). High level gentamicin resistance (MIC ≥ 500μg /mL) is predominantly mediated by aac(6′)-Ie-aph(2″)-Ia, encoding the bifunctional aminoglycoside modifying enzyme AAC(6′)-APH(2″). This enzyme eliminates the synergistic activity of gentamicin when combined with a cell wall active agent. Other AME genes such as aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id and ant(4′)-1a have also been detected in enterococci. Objective This study was carried out to determine the diverse prevalence of AME and their pattern of occurrence in the clinical isolates of Enterococci . Materials and Methods A total number of 150 clinical isolates were included in this study. Susceptibility to various antibiotics was determined by disc diffusion. Minimum Inhibitory Concentration (MIC) was ascertained by agar dilution method. Polymerase chain reaction was done to screen the following AMEs (aac(6′)-Ie-aph(2″)-Ia; aph(2″)-Ib; aph(2″)-Ic; aph(2″)-Id and aph(3′)- IIIa genes) . Results 51.3% of the study isolates exhibited high level gentamicin resistance. Polymerase chain reaction revealed that aph(3′)-111a is the most prevalent AME, followed by aac(6′)-1e-aph(2″)-1a . The combination of both the genes were detected in 44.1% of the study isolates. The rest of the AMEs and their combinations were not encountered in this study. 8.6% of the study isolates did not harbour any AME genes screened for, but was phenotypically resistant to gentamicin. In contrast 31.3% anchored the AME genes but phenotypically appeared susceptible to gentamicin. Conclusion This study indicates the high- level aminoglycoside resistance disseminated among Enterococci in our geographical region. It also emphasizes the detection of AMEs by PCR is mandatory because strains that appear susceptible by disc diffusion and/or MIC method may harbour one or more AMEs genes leading to therapeutic failure.

Detection and characterisation of 16S rRNA methyltransferase-producing Pseudomonas aeruginosa from the UK and Republic of Ireland from 2003–2015

16S rRNA methyltransferase (16S RMTase) genes confer high-level aminoglycoside resistance, reducing treatment options for multidrug-resistant Gram-negative bacteria. Pseudomonas aeruginosa isolates (n=221) exhibiting high-level pan-aminoglycoside resistance (amikacin, gentamicin and tobramycin MICs ≥64, ≥32 and ≥32 mg/L, respectively) were screened for 16S RMTase genes to determine their occurrence among isolates submitted to a national reference laboratory from December 2003 to December 2015. 16S RMTase genes were identified using two multiplex PCRs, and whole-genome sequencing (WGS) was used to identify other antibiotic resistance genes, sequence types (STs) and the genetic environment of 16S RMTase genes. 16S RMTase genes were found in 8.6% (19/221) of isolates, with rmtB4 (47.4%; 9/19) being most common, followed by rmtD3 (21.1%; 4/19), rmtF2 (15.8%; 3/19) and single isolates harbouring rmtB1, rmtC and rmtD1. Carbapenemase genes were found in 89.5% (17/19) of 16S RMTase-positive isolates, with blaVIM (52.9%; 9/17) being most common. 16S RMTase genes were found in 'high-risk' clones known to harbour carbapenemase genes (ST233, ST277, ST357, ST654 and ST773). Analysis of the genetic environment of 16S RMTase genes identified that IS6100 was genetically linked to rmtB1; IS91 to rmtB4, rmtC or rmtD3; ISCR14 to rmtD1; and rmtF2 was linked to Tn3, IS91 or Tn1721. Although 16S RMTase genes explained only 8.6% of pan-aminoglycoside resistance in the P. aeruginosa isolates studied, the association of 16S RMTase genes with carbapenemase-producers and 'high-risk' clones highlights that continued surveillance is required to monitor spread as well as the importance of suppressing the emergence of dually-resistant clones in hospital settings.

Analysis of the phenotypic and genotypic antimicrobial resistance profiles of clinically significant enterococci isolated in the Provincial Specialist Hospital in Lublin, Poland

Abstract The increasing significance of enterococci as healthcare-associated pathogens can be linked to their limited susceptibility to antibiotics. In this study, phenotypic and genotypic resistance profiles of 35 [n=18 E. faecium (Efm); n=17 E. faecalis (Efs)] invasive isolates cultured from hospitalized patients were analysed. Phenotypic identification was verified by the multiplex PCR targeting the 16S rDNA and the ddl genes encoding for the Efs and Efm – specific ligases. Antimicrobial susceptibility was determined using the disc diffusion method and E-tests. The high-level streptomycin resistance (HLSR), high-level gentamicin resistance (HLGR) and glycopeptide resistance was verified by amplification of the ant(6)-Ia, aac(6’)-Ie-aph(2’’)-Ia, as well as vanA and vanB genes, respectively. More than 70% of all isolates were cultured from patients in the Intensive Care and Internal Medicine Units. Blood was the predominant (77%) site of isolation. All Efm isolates were resistant to ampicillin, imipenem, and norfloxacin; 17 isolates demonstrated high-level aminoglycoside resistance (HLAR), including 27.7% with HLSR, 38.8% with HLGR and 27.7% with both phenotypes. HLAR was also common in Efs (HLSR&gt;70%, HLGR&gt;50%), followed by norfloxacin (64.7%) and ampicillin (11.7%) resistance. The ant(6)-Ia and aac(6’)-Ie-aph(2’’)-Ia genes were detected in &gt;90% of the HLSR and HLGR isolates, respectively. Glycopeptide resistance was detected in 4 (22.2%) Efm isolates and mediated by the vanA gene. 19 (54.3%) isolates were multidrug resistant, including 17 (89.5%) Efm. All isolates were susceptible to linezolid. The study constitutes a contribution to the analysis of enterococcal antimicrobial resistance in Polish hospitals. The monitoring of enterococcal prevalence and antimicrobial resistance is crucial to control and prevent infections.

Analysis of incidence and risk factors of the multidrug resistant gastrointestinal tract infection in children and adolescents undergoing allogeneic and autologous hematopoietic cell transplantation: a nationwide study

The aim of this multi-center study was to evaluate the incidence, clinical course, and risk factors for bacterial multidrug-resistant (MDR) gastrointestinal tract infections (GTI) among children undergoing allogeneic and autologous hematopoietic cell transplantation. A total number of 175 pediatric patients (aged 1–18 years), transplanted between January 2018 and December 2019, who were tested for bacterial colonization/infection were enrolled into this multi-center analysis. Episodes of MDR GTI occurred in 77/175 (44%) patients. In multivariate analysis for higher GTI incidence, the following factors were significant: matched-unrelated donor (MUD) transplantation, HLA mismatch, presence of graft-versus-host disease (GVHD), and gut GVHD. The most common GTI were Clostridium difficile (CDI), multidrug-resistant Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli extended-spectrum β-lactamase), and Enterococcus HLAR (high-level aminoglycoside-resistant). No MDR GTI–attributed deaths were reported. MDR GTI is a frequent complication after HCT among children, causes prolonged hospitalization, but rarely contributes to death. We identified risk factors of MDR GTI development in children, with focus on GVHD and unrelated donor and HLA mismatch. We conclude that the presence of Clostridiales plays an important anti-inflammatory homeostatic role and decreases incidence of GVHD or alleviate its course.

High-Level Aminoglycoside and Vancomycin Resistance in Enterococcus spp. Isolated from Hospital Acquired Infections, Ismailia, Egypt

High-Level Aminoglycoside and Vancomycin Resistance in Enterococcus spp. Isolated from Hospital Acquired Infections, Ismailia, Egypt