The molecular mechanisms of fluoroquinolone resistance found in rectal swab isolates of Enterobacterales from men undergoing a transrectal prostate biopsy: the rationale for targeted prophylaxis.

BACKGROUND: Transrectal ultrasound-guided prostate biopsy (TRUS-Bx) is considered an essential urological procedure for the histological diagnosis of prostate cancer. It is, however, considered a "contaminated" procedure which may lead to infectious complications. Recent studies suggest a significant share of fluoroquinolone-resistant rectal flora in post-biopsy infections. METHODS: The molecular mechanisms of fluoroquinolone resistance, including PMQR (plasmid-mediated quinolone resistance) as well as mutation in the QRDRs (quinolone-resistance determining regions) of gyrA, gyrB, parC and parE, among Enterobacterales isolated from 32 of 48 men undergoing a prostate biopsy between November 2015 and April 2016 were investigated. Before the TRUS-Bx procedure, all the patients received an oral antibiotic containing fluoroquinolones. RESULTS: In total, 41 Enterobacterales isolates were obtained from rectal swabs. The MIC of ciprofloxacin and the presence of common PMQR determinants were investigated in all the isolates. Nine (21.9%) isolates carried PMQR with qnrS as the only PMQR agent detected. DNA sequencing of the QRDRs in 18 Enterobacterales (E. coli n = 17 and E. cloacae n = 1) isolates with ciprofloxacin MIC ≥ 0.25 mg/l were performed. Substitutions in the following codons were found: GyrA-83 [Ser → Leu, Phe] and 87 [Asp → Asn]; GyrB codon-605 [Met → Leu], ParC codons-80 [Ser → Ile, Arg] and 84 [Glu → Gly, Met, Val, Lys], ParE codons-458 [Ser → Ala], 461 [Glu → Ala] and 512 [Ala → Thr]. Six isolates with ciprofloxacin MIC ≥ 2 mg/l had at least one mutation in GyrA together with qnrS. CONCLUSIONS: This study provides information on the common presence of PMQRs among Enterobacterales isolates with ciprofloxacin MIC ≥ 0.25 mg/l, obtained from men undergoing TRUS-Bx. This fact may partially explain why some men develop post-TRUS-Bx infections despite ciprofloxacin prophylaxis.

Molecular diagnosis of COVID-19 ­ present experiences / Molekularna diagnostyka COVID-19 ­ dotychczasowe doswiadczenia.

Severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), a new highly emerging and pathogenic for human RNA virus, is responsible for the present COVID-19 pandemic. Molecular diagnostic methods, including real-time reverse transcription-PCR (RT-PCR) assay are the recommended methods for the identification and laboratory confirmation of COVID-19 cases. RT-PCR allows for detection the RNA of the virus in clinical specimens from patients suspected of COVID-19 with high specificity and sensitivity. Testing is still crucial for rapid detection of infected persons, implementation of appropriate measures to suppress further virus transmission and mitigate its impact. In response to demand of a molecular diagnostic test for SARS-CoV-2, within a first few months ongoing pandemic many commercial kits has become available on the market. However, these tests have varied in number and type of molecular targets, time of reaction as well as quality. In this study we compared different commercial tests for the detection of SARS-CoV-2 in clinical samples sending to Laboratory of Department of Virology, NIPH-NIH.

Emergence of Enterobacteriaceae co-producing CTX-M-15, ArmA and PMQR in Poland.

BACKGROUND: Plasmid-mediated extended-spectrum ß-lactamases (ESBLs), 16S rRNA methylases and quinolone resistance mechanisms (PMQRs) are well-known agents conferring resistance to more than 1 antimicrobial in its group. The accumulation of these agents poses, therefore, a serious risk to public health. OBJECTIVES: The objective of this study was to investigate the presence of common ß-lactamases and 16S rRNA methylases in Qnr-producing Enterobacteriaceae and their genetic relatedness. MATERIAL AND METHODS: We examined 18 Qnr-producing isolates (Klebsiella pneumoniae n = 8, Enterobacter cloacae n = 6 and Escherichia coli n = 4) selected from a collection of 215 ciprofloxacin-resistant strains obtained from patients in a 1030-bed tertiary hospital from 1 March to 31 August 2010. The antibiotics minimum inhibitory concentration (MIC) was determined by E-test. The detection of common ß-lactamases, 16S rRNA methyltransferases and PMQR genes was performed by polymerase chain reaction (PCR) and sequencing. Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS: All the isolates tested were susceptible to carbapenems and colistin, while 16 were multidrug-resistant. Thirteen, 2 and 2 isolates carried qnrB1, qnrA1 and qnrS1, respectively. Ten of 13 qnrB1-positive Enterobacteriaceae also carried genes encoding for aac(6')-Ib-cr and at least 1 ESBL. The blaCTX-M-15 gene was the most common ESBL. The most prevalent combination of genes was qnrB1+aac(6')-Ib-cr+blaTEM-1+blaCTX-M-15. Two isolates of K. pneumoniae and E. cloacae were found to bear multiple extended range resistance traits: ArmA, CTX-M-15, QnrB1, and AAC (6')-Ib-cr. The PFGE showed that most of the isolates exhibited individual DNA patterns, whilst MLST assigned K. pneumoniae (n = 8) to 5 sequence types (STs) (ST15, ST323, ST336, ST147, and ST525), E. coli (n = 4) to 2 (ST131 and ST1431) and E. cloacae (n = 5) to 4 (ST90, ST89, ST133, and the novel ST407). CONCLUSIONS: Our findings reveal the accumulation of resistance traits and their important role in spreading of multiresistant bacteria among hospitalized patients.

Biodegradable Chitosan Decreases the Immune Response to Trichinella spiralis in Mice.

The purpose of this study was to evaluate the potential of chitosan units released during natural degradation of the polymer to activate the immune system against T. spiralis infection. High molecular weight chitosan was injected intraperitoneally into C57BL/6 mice. Flow cytometry and cytokine concentration, measured by ELISA, were used to characterize peritoneal cell populations during T. spiralis infection. The strong chemo-attractive properties of chitosan caused considerable infiltration into the peritoneal cavity of CD11b⁺ cells, with reduced expression of MHC class II, CD80, CD86, Dectin-1 or CD23 receptors in comparison to T. spiralis-infected mice. After prolonged chitosan biodegradation, cell populations expressing IL-4R, MR and Dectin-1 receptors were found to coexist with elevated IL-6, IL-10, TGF-ß and IgA production. IgA cross-reacted with T. spiralis antigen and chitosan. It was found that chitosan treatment attracted immune cells with low activity, which resulted in the number of nematodes increasing. The glucosamine and N-acetyl-D-glucosamine residues were recognized by wheat germ agglutinin (WGA) lectin and therefore any biodegradable chitosan units may actively downregulate the immune response to the parasite. The findings are relevant for both people and animals treated with chitosan preparations.

[Whole cell protein profiling characterization of Corynebacterium diphtheriae clinical isolates collected from various infections].

INTRODUCTION: Corynebacterium diphtheriae can cause various infections such as diphtheria, wound infections, septic arthritis, bacteraemia and endocarditis. Different virulence properties of the isolates might be related to different virulence factors expressed by the isolates. The objective of this study was to explore whether whole cell protein profiling might be useful in prediction of pathogenic properties of C. diphtheriae isolates. METHODS: C. disphtheriae isolates collected from diphtheria, invasive and local infections and from asymptomatic carriers in Poland, France, New Caledonia and Canada in 1950-2014 were investigated using whole cell protein profile analysis. RESULTS: All the examined isolates were divided into two clades: A and B with similarity about 47%, but clade B was represented by only one isolate. The clade A was divided in two subclades A.I NS .II with similarity 53,2% and then into four groups: A.Ia, A.Ib, A.Ic and A.Id. The comparative analysis did not distinguish clearly toxigenic and nontoxigenic isolates as well as invasive and noninvasive isolates. CONCLUSIONS: Whole cell protein profile analysis of C. diphtheria exhibits good concordance with other genotyping methods but this method is not able to distinguish clearly invasive from non-invasive isolates.

Usefulness of the (GTG)4-PCR for typing of monophasic Salmonella enterica isolates with antigenic shame l,4,[5],12:i:-.

INTRODUCTION: Monophasic Salmonella enterica strains presenting the antigenic shame 1,4,[5],12:i:- are becoming more prevalent. Accurate identification of such strains is hard with routine using biochemical and serological tests. Such strains can be identified with molecular tests. In this study we have tested the usefulness of(GTG)4-PCR for the diagnostic of such monophasic strains. This usefulness of this method was previously confirmed for genoserotyping of S. Enterica, Typhimurium, Infantis, Virchow, Hadar, Newport and Anatum. MATERIALS AND METHODS: 76 strains with antigenic shame l,4,[5],12:i:-, isolated in Poland in years 2007-12 were tested. Additionally (GTG)4-PCR patterns were obtained for reference strains of serotypes S. Lagos, S. Agama, S. Farsta, S. Tsevie, S. Glocester and S. Tumodi. (GTG)4-PCR was performed with DreamTaq DNA polymerase. Obtained patterns were analysed with BioNumerics software. RESULTS: No pattern specific for monophasic pattern was identified. Additionally it was also impossible to differentiate patterns obtained for S. Typhimurium, S. Farsta, S. Tsevie and S. Glocester. Only reference strains of serotypes S. Tumodi, Farsta and Agama has the distinguishable patterns of (GTG)4-PCR. CONCLUSIONS: Analysed (GTG)4-PCR method do not show the ability to distinguish S. enterica serotypes from group 04, H:i, including monophasic strains with the antigenic shame 1,4,[5],12:i:-.

[Development of molecular PCR-RFLP test for identification of the epidemic strain of Y. enterocolitica bioserotype 1B/O8 circulating in Poland since 2004]. / Opracowanie szybkiego testu PCR-RFLP do identyfikacji paleczek Yersinia enterocolitica bioserotypu 1B/O8 z epidemicznego szczepu tych drobnoustrojów izolowanych w Polsce od 2004 roku.

INTRODUCTION: Highly pathogenic Y. enterocolitica bioserotype 1B/O8 is considered to be an important etiological agent of yersiniosis in Poland. Infections caused by Y. enterocolitica 1B/O8 became an important public health problem in Poland, especially because of their high potential of virulence and the unknown source of the bacteria. Y. enterocolitica 1B/O8 isolates recovered in Poland are genetically highly related and constitute single epidemic sensu stricto strain. The aim of the present study was to develop a time- and money-effective molecular assay for rapid identification of pathogenic Y. enterocolitica 1B/O8 isolates belonging to the epidemic strain. METHODS: In the first stage we performed a multiplex-PCR for four genetic markers: ail, ystA, irp1 and 16S rDNA sequence. In the next stage we designed a duplex-PCR-RFLP assay with BtsI endonuclease to detect/identify specific variant of an ysrR gene that is characteristic for epidemic strain of Y. enterocolitica 1B/O8 strain. The assay was tested against a panel of a consisted of a variety Yersinia enterocolitica and Y. pseudotuberculosis strains. RESULTS: All the tested Y. enterocolitica 1B/O8 strains were positive for all the genetic markers in multiplex-PCR assay what distinguished them from other tested Yersinia strains. In duplex-PCR-RFLP test all tested isolates of the epidemic strain were negative for ysrR digestion with BtsI endonuclease, while all tested reference strains of Y. enterocolitica 1B/O8 were positive. CONCLUSIONS: The assay developed in this study was two-stage/two-step molecular test efficiently distinguishing wild-type and the epidemic Y. enterocolitica 1B/O8 strain. Such test can be a useful screening tool for clinical, veterinary and food diagnostics, as well as for the purposes of epidemiological investigation.

[Serum immunoglobulin IgG subclass distribution of antibody responses to Francisella tularensis in patients with tularemia]. / Wystepowanie przeciwcial dla paleczek Francisella tularensis w poszczególnych podklasach IgG u osób z tularemia.

INTRODUCTION: The present study was aimed at determining the IgG subclass distribution against F. tularensis in patients with tularemia. METHODS: The total number of 56 serum samples obtained from patients with serologically confirmed tularemia were tested by in-house ELISA with bacterial sonicate as the antigen for the presence of IgG1, IgG2, IgG3 and IgG4 antibodies to F. tularensis. Based on the results of determining the level of antibodies in the sera of 30 blood donors, the cut-off limit of serum antibodies for each subclass was set at arithmetic mean plus three standard deviations. RESULTS: Antibodies of subclass IgG1 to F. tularensis were diagnosed in 41 (73.2%), IgG2 in 52 (92.9%) and IgG3 in 13 (23.2%) serum samples. The arithmetic mean of OD450 of antibodies IgG2 was over three-times higher than antibodies IgG1 and IgG3 measured in all of tested serum samples. The concentration of IgG4 was below the detection level. CONCLUSION: In conclusion, IgG2 antibodies to F. tularensis are predominating IgG subclass in tularemia. This study showed also that subclasses of IgG1 and IgG3 but not IgG4 antibodies to F. tularensis are produced during natural infection in humans.

[Comparison of usefulness of commercial ELISA Virion/Serion, homemade ELISA and tube agglutination test in serodiagnosis of tularemia]. / Porównanie przydatnosci komercyjnego zestawu ELISA firmy Virion/Serion, zestawu ELISA opracowanego we wlasnym zakresie oraz odczynu aglutynacji probówkowej w serodiagnostyce tularemii.

INTRODUCTION: Tularemia is a highly infectious zoonotic disease caused by Gram-negative bacterium Francisella tularensis. The microbiological diagnosis of tularemia is based on bacteriological, molecular and serological investigations. In the present study we compared of usefulness of commercial ELISA Virion/Serion, home-made ELISA and tube agglutination test in serodiagnosis of tularemia. METHODS: Serum samples from 57 patients with clinical symptoms of tularemia, 13 patients with yersiniosis and 20 blood donors were tested. The cut-off limit of IgA, IgG and IgM serum antibodies in home-made ELISA was set at mean antibody titer determined in sera of healthy blood donors exceeded by the three standard deviations. The cut-off for positivity in tube agglutination test was titers 25. The IgG and IgM antibodies to lipopolysaccharides of F. tularensis in Virion/Serion ELISA were measured and results interpreted according to the instructions by the manufacturer. RESULTS: The results of the study showed that 39 (68.4%) serum samples obtained from the patients suspected for tularemia were positive by tube agglutination test and Virion/Serion ELISA assay for IgG and IgM antibodies. Home-made ELISA was slightly more sensitive and detected the IgA/IgG antibodies in 42 (73.7%) and IgM antibodies in 39 (68.4%) of serum samples. The positive reactions were not detected by the tube agglutination test and home-made ELISA in serum samples from patients with yersiniosis and blood donors. The Virion/Serion ELISA detected IgG antibodies in diagnostically significant level only in one blood donor. CONCLUSIONS: In conclusion, all three serological tests can be successfully used in routine serodiagnosis of tularemia.