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The efflux pumps, beside the class D carbapenem-hydrolysing enzymes (CHLDs), are being increasingly investigated as a mechanism of carbapenem resistance in Acinetobacter baumannii. This study investigates the contribution of efflux mechanism to carbapenem resistance in 61 acquired blaCHDL-genes-carrying A. baumannii clinical strains isolated in Warsaw, Poland. Studies were conducted using phenotypic (susceptibility testing to carbapenems ± efflux pump inhibitors (EPIs)) and molecular (determining expression levels of efflux operon with regulatory-gene and whole genome sequencing (WGS)) methods. EPIs reduced carbapenem resistance of 14/61 isolates. Upregulation (5–67-fold) of adeB was observed together with mutations in the sequences of AdeRS local and of BaeS global regulators in all 15 selected isolates. Long-read WGS of isolate no. AB96 revealed the presence of AbaR25 resistance island and its two disrupted elements: the first contained a duplicate ISAba1-blaOXA-23, and the second was located between adeR and adeA in the efflux operon. This insert was flanked by two copies of ISAba1, and one of them provides a strong promoter for adeABC, elevating the adeB expression levels. Our study for the first time reports the involvement of the insertion of the ΔAbaR25-type resistance island fragment with ISAba1 element upstream the efflux operon in the carbapenem resistance of A. baumannii. [ABSTRACT FROM AUTHOR]

Simple Summary: Fluoroquinolones, mainly levofloxacin, are considered an alternative treatment option of Stenotrophomonas maltophilia infections to trimethoprim/sulfamethoxazole. However, an increase in the number of levofloxacin-resistant strains is observed worldwide. The fluoroquinolone resistance in S. maltophilia is usually caused by an overproduction of various multidrug efflux pumps, which are able to extrude antibiotics and chemotherapeutics from the bacterial cells. The purpose of the study was to analyze the contribution of efflux systems to levofloxacin resistance in S. maltophilia clinical strains, isolated in Warsaw, by phenotypic and molecular methods. Previously, the occurrence of genes encoding various ten efflux pumps was shown in 94 studied isolates. Additionally, 44 of 94 isolates demonstrated reduction in susceptibility to levofloxacin. In this study, in the presence of efflux pump inhibitors, an increase in levofloxacin susceptibility was observed in 13 isolates. The overexpression of genes encoding two efflux pump system, such as SmeDEF and Sme VWX (in five and one isolate, respectively), was demonstrated. Sequencing analysis revealed an amino acid change in the local regulators of these efflux pump operons. Our data indicate that the overproduction of the SmeVWX efflux system, unlike SmeDEF, plays a significant role in the levofloxacin resistance of the clinical isolates. Levofloxacin is considered an alternative treatment option of Stenotrophomonas maltophilia infections to trimethoprim/sulfamethoxazole. The fluoroquinolone resistance in S. maltophilia is usually caused by an overproduction of efflux pumps. In this study, the contribution of efflux systems to levofloxacin resistance in S. maltophilia clinical isolates was demonstrated using phenotypic (minimal inhibitory concentrations, MICs, of antibiotics determination ± efflux pump inhibitors, EPIs) and molecular (real-time polymerase-chain-reaction and sequencing) methods. Previously, the occurrence of genes encoding ten efflux pumps was shown in 94 studied isolates. Additionally, 44/94 isolates demonstrated reduction in susceptibility to levofloxacin. Only 5 of 13 isolates (with ≥4-fold reduction in levofloxacin MIC) in the presence of EPIs showed an increased susceptibility to levofloxacin and other antibiotics. The overexpression of smeD and smeV genes (in five and one isolate, respectively) of 5 tested efflux pump operons was demonstrated. Sequencing analysis revealed 20–35 nucleotide mutations in local regulatory genes such as smeT and smeRv. However, mutations leading to an amino acid change were shown only in smeT (Arg123Lys, Asp182Glu, Asp204Glu) for one isolate and in smeRv (Gly266Ser) for the other isolate. Our data indicate that the overproduction of the SmeVWX efflux system, unlike SmeDEF, plays a significant role in the levofloxacin resistance. [ABSTRACT FROM AUTHOR]

Knowledge of the prevalence of ESBL enzymes among P. aeruginosa strains compared to the Enterobacteraiceae family is limited. The phenotypic tests recommended by EUCAST for the detection of ESBL-producing Enterobacteriaceae are not always suited for P. aeruginosa strains. This is mainly due to the presence of other families of ESBLs in P. aeruginosa isolates more often than in Enterobacteriaceae, production of natural AmpC cephalosporinase and its overexpression, and co-production of metallo-β-lactamases. The aim of this study was to determine the occurrence of ESBLs in P. aeruginosa isolated from patients from hospitals in Warsaw, to evaluate the ESBL production of these isolates using currently available phenotypic tests, their modifications, multiplex PCR and molecular typing of ESBL-positive isolates by PFGE. Clinical isolates of P. aeruginosa were collected in 2000-2014 from four Warsaw hospitals. Based on the data obtained in this study, we suggest using three DDST methods with inhibitors, such as clavulanic acid, sulbactam and imipenem, to detect ESBL-producing P. aeruginosa strains. Depending on the appearance of the plates, we suggest a reduction in the distance between discs with antibiotics to 15 mm and the addition of boronic acid at 0.4 mg per disc. The analysed isolates carried genes encoding ESBL from the families VEB (69 isolates with VEB-9), GES (6 with GES-1, 1 GES-5, 5 GES-13 and 2 with GES-15), OXA-2 (12 with OXA-15, 1 OXA-141, 1 OXA-210, 1 OXA-543 and 1 with OXA-544) and OXA-10 (5 isolates with OXA-74 and one with OXA-142). The most important result of this study was the discovery of three new genes, blaGES-15, blaOXA-141 and blaOXA-142; their nucleotide sequences have been submitted to the NCBI GenBank. It is also very important to note that this is the first report on the epidemiological problem of VEB-9-producing bacterial strains, not only in Poland but also worldwide. [ABSTRACT FROM AUTHOR]

Simple Summary: Nosocomial infections caused by Stenotrophomonas maltophilia have been increasing worldwide. These bacteria are intrinsically resistant to most antibiotics. The underestimated resistance mechanism of Gram-negative rods is an overexpression of multidrug-resistant (MDR) efflux pumps. The aim of this study was to analyze the genetic diversity of isolates derived from various clinical materials, including blood, and the prevalence of MDR efflux pump genes and susceptibility profiles to the anti-S. maltophilia drugs. The research was conducted on 94 S. maltophilia isolates derived from hospitalized patients and outpatients in Warsaw, Poland. All isolates were susceptible to trimethoprim-sulfamethoxazole and minocycline, while 44/94 isolates demonstrated reduction in susceptibility to levofloxacin. A large genetic variation was observed among these isolates. However, a clonal relationship was revealed among two groups of bloodstream isolates from one hospital ward: (1) nine isolates, (2) six isolates. Moreover, the presence of genes encoding ten different efflux pumps from the resistance-nodulation-division family and the ATP-binding cassette family was shown in the majority of the 94 isolates. The obtained knowledge about the prevalence of efflux pump genes in clinical S. maltophilia strains makes it possible to predict the scale of the risk of resistance emergence in strains as a result of gene overexpression. An increase of nosocomial infections caused by Stenotrophomonas maltophilia strains has recently been observed all over the world. The isolation of these bacteria from the blood is of particular concern. In this study we performed the phenotypic and genotypic characterization of 94 S. maltophilia isolates, including isolates from patients hospitalized in a tertiary Warsaw hospital (n = 79) and from outpatients (n = 15). All isolates were found to be susceptible to trimethoprim-sulfamethoxazole and minocycline, while 44/94 isolates demonstrated a reduction in susceptibility to levofloxacin. A large genetic variation was observed among the isolates tested by pulsed-field gel electrophoresis. A clonal relationship with 100% similarity was observed between isolates within two sub-pulsotypes: the first included nine bloodstream isolates and the second involved six. Multilocus sequence typing showed two new sequence types (ST498 and ST499) deposited in public databases for molecular typing. Moreover, the presence of genes encoding ten different efflux pumps from the resistance-nodulation-division family and the ATP-binding cassette family was shown in the majority of the 94 isolates. The obtained knowledge about the prevalence of efflux pump genes in clinical S. maltophilia strains makes it possible to predict the scale of the risk of resistance emergence in strains as a result of gene overexpression. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is an important cause of nosocomial infections worldwide. The elucidation of the carbapenem resistance mechanisms of hospital strains is necessary for the effective treatment and prevention of resistance gene transmission. The main mechanism of carbapenem resistance in A. baumannii is carbapenemases, whose expressions are affected by the presence of insertion sequences (ISs) upstream of blaCHDL genes. In this study, 61 imipenem-nonsusceptible A. baumannii isolates were characterized using phenotypic (drug-susceptibility profile using CarbaAcineto NP) and molecular methods. Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were utilized for the genotyping. The majority of isolates (59/61) carried one of the following acquired blaCHDL genes: blaOXA-24-like (39/59), ISAba1-blaOXA-23-like (14/59) or ISAba3-blaOXA-58-like (6/59). Whole genome sequence analysis of 15 selected isolates identified the following intrinsic blaOXA-66 (OXA-51-like; n = 15) and acquired class D β-lactamases (CHDLs): ISAba1-blaOXA-23 (OXA-23-like; n = 7), ISAba3-blaOXA-58-ISAba3 (OXA-58-like; n = 2) and blaOXA-72 (OXA-24-like; n = 6). The isolates were classified into 21 pulsotypes using PFGE, and the representative 15 isolates were found to belong to sequence type ST2 of the Pasteur MLST scheme from the global IC2 clone. The Oxford MLST scheme revealed the diversity among these studied isolates, and identified five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). CHDL-type carbapenemases and insertion elements upstream of the blaCHDL genes were found to be widespread among Polish A. baumannii clinical isolates, and this contributed to their carbapenem resistance. [ABSTRACT FROM AUTHOR]