Laboratory Detection and Gene Cassette Stability of the Novel Extended-Spectrum Beta-Lactamase, GES-2 from Pseudomonas aeruginosa

Extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa tend to be geographically scattered, such as GES-2, which partially compromises the efficacy of imipenem. The G170N mutation, ascribed to a CC to AA base pair substitution on positions 493-494 of the blaGES-2 coding region, distinguishes this ESBL from blaGES-1 and the blaIBC-type genes, making it an ideal target for developing a novel sequence-specific, peptide nucleic acid (PNA)-based, multiplex-PCR detection method. Utilizing two primer pairs in conjunction with a PNA probe, this novel method delivered accurate identification of blaGES-2 compared to standard PCR and gene sequencing techniques, when tested against one hundred (n = 100) P. aeruginosa clinical isolates as well as previously published, well-described control strains. This method has the potential to be used in large-scale, cost-effective screening programmes for specific or geographically restricted ESBLs. To date, in addition to being only described in South Africa, GES-2 is notoriously difficult to identify in P. aeruginosa, using standard methodology. A real-time PCR method using the LightCycler™ was compared to a two-step nested-PCR assay for the detection of blaGES and blaIBC genes from one hundred P. aeruginosa clinical isolates collected over a four-year period from two teaching hospitals in Pretoria, South Africa. Real-time PCR amplification was monitored through hybridisation of fluorescently labelled probes followed by melting curve analysis to detect the relevant G170N mutation occurring in the omega loop region of blaGES-2. Nested-PCR products were subjected to automated DNA sequencing and compared to melting point (Tm) analyses results obtained from the LightCycler assay. Real time and nested-PCR assays detected a blaIBC gene product from 83 and 88 clinical isolates respectively, with the LightCycler thus exhibiting a sensitivity of 94.3% compared to the nested-PCR assay. Comparison of Tm and gene sequencing data however revealed 100% specificity for sequence specific detection of blaGES-2 with the LightCycler. One clinical isolate was found to harbour a blaGES-1 gene, making this the first report of this specific ESBL from South Africa. Selective antibiotic pressure has recently been implicated as a possible driving force behind point mutations observed in blaGES–type genes. This part of the study subjected two well-characterized clinical isolates with class 1 integron-borne blaGES-type genes to five days incubation in the presence of sub-inhibitory concentrations of 15 different antibiotics, including beta-lactams, aminoglycosides and quinolones. Restriction enzyme analysis and DNA sequencing of blaGES-1, blaGES-2 and their immediate upstream genetic environments failed to demonstrate any changes compared to non-exposed controls. Short-term exposure to a sub-inhibitory level of a single antimicrobial agent is thus unlikely to select significant mutations in these beta-lactamase genes or their regulatory mechanisms.Thesis (PhD (Medical Microbiology))--University of Pretoria, 2004.Medical Microbiologyunrestricte

Killing Dynamics of Carbapenems against Pseudomonas aeruginosa Harboring Varied Determinants of Carbapenem Resistance

Background: Ideal dose of the antimicrobials should be decided by considering their killing dynamics since sufficient elimination of the causative microorganisms is critical for proper antimicrobial treatment. In this study, the bactericidal activities of carbapenems by resistance mechanisms were assessed for carbapenem-resistant Pseudomonas aeruginosa. Methods: Minimal inhibition concentrations (MICs) of carbapenems were determined by broth dilution method and the resistance mechanisms were identified by PCR and DNA sequencing. The expression levels of efflux pumps were determined by reverse transcriptase real-time PCR. Time-kill curves were plotted by time-course numeration of the viable cells grown under imipenem and meropenem at 1× and 4× MICs, respectively. Results: One P. aeruginosa strain was susceptible, whereas three were resistant to carbapenems by defective OprD, efflux pump overproduction, and/or IMP-6 production. The susceptible strain had imipenem and meropenem MICs of 2 and 1 mg/L, respectively. The MICs were elevated by eight-fold by defective OprD, 16- and 32-fold by the pump overproduction, and four- and <64-fold by the combination of two determinants and the IMP-6 carbapenemase. While both the carbapenems showed time-dependent bactericidal activity to the susceptible isolate, either of the carbapenem-resistant determinants, such as decreased membrane permeability, carbapenemase production, or the defective OprD,presented concentration-dependent bacteriostatic activity.Conclusion: Different killing dynamics of the carbapenems were observed depending on the resistance determinants, and the results would guide a proper treatment strategy for the patients using these drugs.ope

Epidemiological Study of an Outbreak of KPC-2-producing Klebsiella pneumoniae in a Tertiary Hospital in Korea

Background: The prevalence of carbapenemase-producing Enterobacteriaceae (CPE), especially the KPC-2-producing Klebisella pneumoniae, is rapidly increasing and becoming a menace to global public health. This study aims to present the molecular epidemiology of the KPC-2-producing K. pneumoniae isolates emerged in a tertiary hospital in South Korea and describe its clinical significance. Methods: This study included carbapenem-resistant K. pneumoniae isolates collected from a tertiary hospital from April to December in 2018. Antimicrobial susceptibility of K. pneumoniae isolates was tested using disk diffusion method. PCR and DNA sequence analyses were performed to identify the resistance genotype. In addition, the molecular epidemiology was investigated using pulsed-field gel electrophoresis (PFGE) and multilocus sequencing typing (MLST). Results: Total 100 KPC-2-producing K. pneumoniae isolates were collected, which were mainly classified into two pulsotypes according to the XbaI restriction digestion pattern by PGFE analysis (pulsotype A, n = 31; pulsotype B, n = 63). The isolates exhibiting pulsotype A belonged to ST395 and the remaining isolates exhibiting pulsotype B were attributed to ST307 by MLST analysis. Conclusion: This study investigated clinical information and molecular bacterial profiles for KPC-2-producing K. pneumoniae isolates. These findings indicate that the proper infection control activities are needed to prevent the spread of multidrug-resistant organisms such as CPE, which could cause high mortality in clinical field.ope

The demographic and microbiological profile of cystic fibrosis in public and private sectors in KwaZulu-Natal.

M. Med. Sc. University of KwaZulu-Natal, Durban 2014.Abstract available in hard copy

Characterization of carbapenem-resistant isolates from "Enterobacter cloacae" complex from Spanish hospitals between 2018 and 2022

47 p.El uso masivo e inadecuado de antibióticos en la práctica clínica durante las últimas décadas ha provocado la aparición y diseminación de bacterias multirresistentes. El desarrollo de resistencias frente a carbapenémicos constituye una grave amenaza, al ser considerados antibióticos de última línea para el tratamiento de infecciones provocadas por bacterias resistentes al resto de betalactámicos. El principal mecanismo de resistencia a estos antibióticos es la producción de carbapenemasas y es especialmente preocupante en enterobacterales, ya que dicho mecanismo se ha distribuido rápidamente a nivel global. Esto se debe principalmente a la capacidad que tienen los genes que codifican carbapenemasas de ser transferidos horizontalmente entre bacterias mediante plásmidos. Esta resistencia también puede deberse a una combinación de expresión de AmpC y/o BLEE junto con una disminución en la expresión de porinas. En el presente estudio se caracterizaron los principales mecanismos de resistencia a carbapenémicos y otros antibióticos en 75 aislados de origen clínico del complejo Enterobacter cloacae, procedentes de diferentes hospitales españoles durante 2018-2022. Mediante secuenciación del genoma completo y análisis filogenético por cgMLST, se caracterizaron genotípicamente los principales clones circulantes a nivel de su estructura poblacional, así como los principales genes de resistencia. La detección de genes que codifican carbapenemasas en numerosos aislados, así como otros genes plasmídicos que confieren resistencia frente a otros antibióticos resulta alarmante, al reducir considerablemente las opciones terapéuticas. VIM-1 y OXA-48 fueron las carbapenemasas más comunes y se detectó la presencia en España de clones internacionales de alto riesgo, como el ST114 y el ST78.The massive and inappropriate use of antibiotics in clinical practice over the last decades has led to the emergence and spread of multidrug-resistant bacteria. The development of resistance to carbapenemsis a serious threat as they are considered last-step antibiotics for treating infections caused by bacteria resistant to other beta-lactams. The main mechanism of resistance to these antibiotics is the production of carbapenemases, and it is particularly concerning in Enterobacteriaceae, as this mechanism has rapidly spread globally. This is mainly due to the ability of carbapenemase-encoding genes to be horizontally transferred between bacteria through plasmids. This resistance can also be due to a combination of AmpC and/or ESBL expression along with a decrease in porin expression. In this study, the main mechanisms of resistance to carbapenems and other antibiotics was characterized in 75 clinical isolates of Enterobacter cloacae complex from different Spanish hospitals recovered during 2018-2022. Genotypic characterization of the major circulating clones and the population structure of carbapenem-resistant strains in Spain was performed using whole-genome sequencing and cgMLST (core genome multilocus sequence typing) analysis. The detection of carbapenemase-encoding genes in numerous isolates, as well as other plasmid borne resistance genes related with resistance to other antibiotics, is deeply alarming as it significantly reduces therapeutic options. VIM-1 and OXA-48 were the most common carbapenemases, and the presence of internationally high-risk clones, such as ST114 and ST78, was detected in Spain.Máster Universitario en Microbiología Aplicada a la Salud Pública e Investigación en Enfermedades Infecciosas (M138

Phenotypic and genotypic correlation of carbapenememase-producing Enterobacteriaceae and problems experienced in routine screening

Background. The emergence and transmission of carbapenem-resistant Enterobacteriaceae (CRE) is a concern in both the clinical and public health arenas. Reliable and accurate detection of these organisms is required for patient management and infection prevention and control purposes. In the routine laboratory, phenotypic methods are utilised for identification of CRE.Objectives. To investigate the phenotypic profiles of suspected carbapenemase-producing Enterobacteriaceae (CPE) isolates generated by the automated MicroScan Walkaway system making use of the Clinical and Laboratory Standards Institute (CLSI) guidelines, and correlate these with carbapenemase production by molecular methods.Methods. Antimicrobial susceptibility testing was performed using the MicroScan Walkaway system, and the presence of six carbapenemase genes (blaNDM, blaVIM, blaIMP, blaOXA-48and variants, blaGESand blaKPC) was screened for using a multiplex real-time polymerase chain reaction.Results. A total of 2 678 isolates were evaluated. Klebsiella pneumoniae accounted for 62.9% of the isolates (n=1 685), followed by Enterobacter cloacae (n=361, 13.5%). Carbapenemases accounted for 75.2% of isolates; blaOXA-48 and its variants predominated (n=978, 36.5%), followed by blaNDM (n=904, 33.8%), blaVIM (n=108, 4.0%), blaIMP (n=35, 1.3%), blaGES (n=24, 0.9%) and blaKPC (n=18, 0.7 %).Conclusions. A considerable number of isolates expressing a carbapenemase or carbapenemases (the majority of which were blaOXA-48 producing) were susceptible to third-and fourth-generation cephalosporins and carbapenems, demonstrating that confirmed carbapenemase-producing isolates are not presenting as possible carriers of carbapenemases using routine diagnostic methods. Similar results were obtained when CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints were applied and are suitable for the purpose of patient management. However, since genotyping assays are costly, it is suggested that routine laboratories first perform comprehensive phenotypic screening for CPE

Mobile Carbapenemase Genes in Pseudomonas aeruginosa

Carbapenem-resistant Pseudomonas aeruginosa is one of the major concerns in clinical settings impelling a great challenge to antimicrobial therapy for patients with infections caused by the pathogen. While membrane permeability, together with derepression of the intrinsic beta-lactamase gene, is the global prevailing mechanism of carbapenem resistance in P. aeruginosa, the acquired genes for carbapenemases need special attention because horizontal gene transfer through mobile genetic elements, such as integrons, transposons, plasmids, and integrative and conjugative elements, could accelerate the dissemination of the carbapenem-resistant P. aeruginosa. This review aimed to illustrate epidemiologically the carbapenem resistance in P. aeruginosa, including the resistance rates worldwide and the carbapenemase-encoding genes along with the mobile genetic elements responsible for the horizontal dissemination of the drug resistance determinants. Moreover, the modular mobile elements including the carbapenemase-encoding gene, also known as the P. aeruginosa resistance islands, are scrutinized mostly for their structures.ope

Распространенность генов устойчивости к антибиотикам в составе резистома взрослых жителей Архангельска с учетом тяжести перенесенной COVID-19

Objective: to estimate the prevalence of antibiotic resistance genes in the resistome of adult residents of Arkhangelsk with regard to the severity of the novel coronavirus infection (COVID-19).Materials and methods. A cross-sectional study was conducted between October and November 2022 (2.5 years after the start of the COVID-19 pandemic) on a random sample (N=455) of Arkhangelsk population aged 42-76 years. The data collection involved a questionnaire survey, assessment of immunoglobulins G to S-, S2, N-proteins of SARS-CoV-2 and detection of antibiotic resistance genes in fecal samples by polymerase chain reaction.Results. Almost all participants (98.5%) had at least one antibiotic resistance gene, the resistance determinants to three classes of antibiotics simultaneously were detected in 5.6%. The prevalence of resistance genes to macrolides was 98.5%, to beta-lactams – 29.0%, and to glycopeptides – 16.0%. Antibiotic resistance genes to beta-lactams were more prevalent among participants who had previously been hospitalized for COVID-19 (44.8%) and among those having had frequent acute respiratory infections (50.0%). Individuals vaccinated against SARS-CoV-2 (26.6%) and participants with cardiovascular diseases (17.0%) were less likely to have beta-lactam resistance genes.Conclusion. The high prevalence of antibiotic resistance genes has been revealed in the resistome of adult residents of Arkhangelsk. We determined the association between resistance to beta-lactams and COVID-19 severity. The study results could be used to improve the protocols of antibiotic therapy and to guide a decision-making related to the antibiotic prescription in adults.Цель: оценить распространённость генов устойчивости к антибиотикам в составе резистома взрослых жителей Архангельска с учётом тяжести перенесённой новой коронавирусной инфекции (COVID-19).Материалы и методы: поперечное исследование проведено с октября по ноябрь 2022 г. (через 2,5 года от начала пандемии COVID-19) с участием случайной выборки (N=455) населения Архангельска в возрасте 42–76 лет. Процедура исследования включала опрос, определение иммуноглобулинов G к белкам вируса SARS-CoV-2 и выявление генов устойчивости к антибиотикам в образцах кала методом полимеразной цепной реакции.Результаты: практически все участники (98,5%) имели хотя бы 1 ген устойчивости к антибиотикам, у 5,6% выявлены детерминанты резистентности сразу к 3 классам антибиотиков. Распространённость генов устойчивости к макролидам составила 98,5%, к бета-лактамам – 29,0%, к гликопептидам – 16,0%. Гены резистентности к бета-лактамным антибиотикам чаще выявлялись среди участников, получавших стационарное лечение по поводу COVID-19 (44,8%), и среди лиц, часто болеющих острыми респираторными инфекциями (50,0%). Вакцинированные против SARS-CoV-2 (26,6%) и участники с сердечно-сосудистыми заболеваниями (17,0%) реже имели гены резистентности к бета-лактамам.Заключение: выявлена высокая распространённость генов устойчивости к антибиотикам в составе резистома взрослых жителей Архангельска. Определена связь между резистентностью к бета-лактамам и тяжестью перенесённой COVID-19. Результаты исследования могут быть использованы для оптимизации протоколов стартовой антибиотикотерапии и принятия решений, связанных с назначением антимикробных препаратов различным категориям взрослого населения

Epidemiologia molecular e caracterização da resistência de amostras de Acinetobacter baumannii e Pseudomonas aeruginosa resistentes aos carbapenêmicos provenientes de hospitais da Grande Vitória-ES.

A emergência e a disseminação da resistência aos antimicrobianos entre os bacilos Gram-negativos não fermentadores, tais como Acinetobacter baumannii e Pseudomonas aeruginosa, são um problema mundial. Carbapenêmicos são antimicrobianos beta-lactâmicos indicados para o tratamento de infecções graves causadas por esses agentes, contudo, o surgimento de patógenos multirresistentes ameaça seriamente o uso dessa classe de fármaco no ambiente hospitalar. O presente trabalho teve como objetivo caracterizar amostras clínicas de P. aeruginosa e A. baumannii resistentes aos cabapenêmicos, quanto a presença de genes de resistência aos beta-lactâmicos, perfil epidemiológico e de suscetibilidade aos antimicrobianos utilizados na rotina clínica. Para avaliar a suscetibilidade das amostras aos antimicrobianos foi realizado o método de difusão em ágar a partir do disco e para a determinação da concentração inibitória mínima foi realizado o método do teste de gradiente do antimicrobiano. A pesquisa dos genes codificadores de beta-lactamases foi feita através da técnica de PCR e o polimorfismo genético foi analisado pelas técnicas de eletroforese em gel de campo pulsado (PFGE) e tipagem de sequência de multilocus (MLST). As espécies incluídas no estudo foram: A. baumannii (n=26) e P. aeruginosa (n=15). A maioria das amostras apresentou um perfil de multirresistência aos antimicrobianos testados, destacando-se a resistência à colistina em nove amostras de A. baumannii. Nessa espécie, o gene de carbapenemase prevalente (92,3%) foi o blaOXA-23 e nas amostras de P. aeruginosa o gene blaVIM mostrou-se prevalente (33,3%). Através da análise por PFGE, foi observado a prevalência de dois pulsotipos entre as amostras de A. baumannii: abA (34,6%) e abB (23%) enquanto que as amostras de P. aeruginosa apresentaram pulsotipos distintos, demonstrando a origem policlonal das amostras. Através do MLST realizado em cinco amostras de P. aeruginosa, foram encontrados os STs 357, 2321, 1121, 244 e 227, sendo observados dois complexos clonais de importância mundial o CC235 e o CC244. Adicionalmente, os ST357, ST2321 e ST1121 foram descritos pela primeira vez no Brasil. Palavras-chave: Acinetobacter baumannii. Pseudomonas aeruginosa. Betalactamases. Resistência. Genotipagem

EMERGENCE OF CARBAPENEM RESISTANT ENTEROBACTERIACEAE IN ABU DHABI, 2009-2015

The rapid emergence of carbapenem resistant Enterobacteriaceae (CRE) is a global phenomenon that has not spared the United Arab Emirates. However, unlike in most Western countries with similarly developed health care system, very little data have been available locally on the rate and dynamics of antimicrobial resistance in general, and that of CRE, in particular. Our laboratory has been collecting CRE isolates from Abu Dhabi hospitals since 2009. In the current study we subjected 394 such independent strains, representing 34.0% of the total CRE encountered, to a detailed analysis that included the determination of their antibiotic susceptibility profile, of the type and alleles of the carbapenemases produced, of the pulsotypes and multi locus sequence types of the strains, respectively. 84.0% of the strains produced a carbapenemase. The most common type was OXA-48-like (43.9%) followed by NDM (24.9%). A high rate of double carbapenemase producers (14.5%) was also observed, while no KPC and IMP were encountered. Regarding the specific alleles, OXA-232 and NDM-1 were the most common ones. 16.2% of the strains exhibited non-susceptibility to colistin, while 57.6% to tigecycline, 29.4% exhibited extreme- (XDR), while 5.6% pan-resistance (PDR). Seven major Klebsiella pneumoniae clones were identified: ST14 (n=111), CC147 (n=43), ST231 (n=36), CC101 (n=16), ST11 (n=8), ST45 (n=8) and ST15 (n=5), representing 70.1% of all K. pneumoniae, and 57.6% of all strains encountered. Representatives of these clones were present in majority of the hospitals. The emergence and spread of these clones considerably impacted the local epidemiology of CRE as, in general, they were more likely to be XDR or PDR than the rest of the collection. Our data show that Abu Dhabi is facing a severe CRE problem. Only a continuous surveillance supported with molecular typing data, as those generated by the current study, could offer the background to successfully control the problem