Genomic Analysis of Corynebacterium diphtheriae Strains Isolated in the Years 2007-2022 with a Report on the Identification of the First Non-Toxigenic tox Gene-Bearing Strain in Poland.

Infections caused by non-toxigenic Corynebacterium diphtheriae have been reported every year in Poland since 2004, with the ST8 biovar gravis strains being most commonly isolated. This study analyzed thirty strains isolated between 2017 and 2022 and six previously isolated strains. All the strains were characterized using classic methods in terms of species, biovar level, and diphtheria toxin production, as well as by means of whole genome sequencing. The phylogenetic relationship based on SNP analysis was determined. The number of C. diphtheriae infections has been rising in Poland every year with a maximum of 22 cases in the year 2019. Since 2022, only the non-toxigenic gravis ST8 (most common) and mitis ST439 (less common) strains have been isolated. An analysis of the genomes of the ST8 strains showed that they had many potential virulence factors, such as adhesins and iron-uptake systems. The situation rapidly changed in 2022 and strains from different STs were isolated (ST32, 40, and 819). The ST40 biovar mitis strain was found to be non-toxigenic tox gene-bearing (NTTB), with the tox gene inactivated due to a single nucleotide deletion. Such strains were previously isolated in Belarus. The sudden appearance of new C. diphtheriae strains with different STs and the isolation of the first NTTB strain in Poland indicate that C. diphtheriae should be classified as a pathogen of special public health concern.

The Mechanisms Involved in the Fluoroquinolone Resistance of Salmonella enterica Strains Isolated from Humans in Poland, 2018-2019: The Prediction of Antimicrobial Genes by In Silico Whole-Genome Sequencing.

Salmonellosis remains the second most common zoonosis in Europe. Resistance to fluoroquinolones (FQs) in Salmonella has been increasing worldwide, with WHO considering FQ-resistant Salmonella spp. as high-priority pathogens. The aim of this study was a retrospective analysis of the molecular mechanisms of FQ resistance, detected among clinical ciprofloxacin-resistant Salmonella enterica belonging to the most common serotypes. The whole genome sequences (WGS) of tested isolates were also analysed for the occurrence of other antimicrobial resistance determinants. Out of a total of 1051 Salmonella collected in the years 2018-2019, 447 strains belonging to the most common serotypes in Poland were selected were screened for FQ resistance using the pefloxacin disc test according to EUCAST recommendations. All pefloxacin-resistant isolates were confirmed as ciprofloxacin-resistant using the E-test. A total of 168 (37.6%) Salmonella enterica, which belonged to seven serotypes, were resistant to ciprofloxacin (mostly Hadar, Virchow and Newport). A hundred randomly selected Salmonella were investigated by WGS. A total of 127 QRDR mutations in GyrA and ParC were identified in 93 isolates. The qnr genes were the only PMQR determinants detected and were found in 19% of the sequenced isolates. Moreover, 19 additional resistance genes (including: bla,,tet, sul, aad, aac-, ant-, aph-, floR, cmlA) were identified among the FQ-resistant Salmonella tested that confer resistance to clinically important antibiotics such as ß-lactams, tetracyclines, sulphonamides, aminoglycosides and phenicol, respectively). In conclusion, FQ resistance of human Salmonella in Poland is rising towards a critical level and needs to be tightly monitored.

Prevalence of Plasmid-Mediated Quinolone Resistance (PMQRs) Determinants and Whole Genome Sequence Screening of PMQR-Producing E. coli Isolated from Men Undergoing a Transrectal Prostate Biopsy.

Fluoroquinolones (FQs) are recommended as prophylaxis for men undergoing transrectal prostate biopsy (TRUS-Bx). Recent studies suggest a significant share of FQ-resistant rectal flora in post-TRUST-Bx infections. METHODS: 435 Enterobacterales isolates from 621 patients attending 12 urological departments in Poland were screened by PCR for PMQR genes. PMQR-positive isolates were tested for quinolone susceptibility and investigated by whole genome sequencing (WGS) methods. RESULTS: In total, 32 (7.35%) E. coli strains with ciprofloxacin MIC in the range 0.125-32 mg/L harbored at least one PMQR gene. qnrS and qnrB were the most frequent genes detected in 16 and 12 isolates, respectively. WGS was performed for 28 of 32 PMQR-producing strains. A variety of serotypes and sequence types (STs) of E. coli was noticed. All strains carried at least one virulence gene. AMR genes that encoded resistance against different classes of antibiotics were identified. Additionally, five of 13 ciprofloxacin-susceptible E. coli had alterations in codon 83 of the GyrA subunits. CONCLUSION: This study provides information on the common presence of PMQRs among E. coli, which may explain the cause for development of post-TRUS-Bx infections. High numbers of virulence and antimicrobial resistance genes detected show a potential for analysed strains to develop infections.

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.

Antimicrobial Resistance and Whole-Genome Characterisation of High-Level Ciprofloxacin-Resistant Salmonella Enterica Serovar Kentucky ST 198 Strains Isolated from Human in Poland.

BACKGROUND: Salmonella Kentucky belongs to zoonotic serotypes that demonstrate that the high antimicrobial resistance and multidrug resistance (including fluoroquinolones) is an emerging problem. To the best of our knowledge, clinical S. Kentucky strains isolated in Poland remain undescribed. METHODS: Eighteen clinical S. Kentucky strains collected in the years 2018-2019 in Poland were investigated. All the strains were tested for susceptibility to 11 antimicrobials using the disc diffusion and E-test methods. Whole genome sequences were analysed for antimicrobial resistance genes, mutations, the presence and structure of SGI1-K (Salmonella Genomic Island and the genetic relationship of the isolates. RESULTS: Sixteen of 18 isolates (88.9%) were assigned as ST198 and were found to be high-level resistant to ampicillin (>256 mg/L) and quinolones (nalidixic acid MIC ≥ 1024 mg/L, ciprofloxacin MIC range 6-16 mg/L). All the 16 strains revealed three mutations in QRDR of GyrA and ParC. The substitutions of Ser83 → Phe and Asp87 → Tyr of the GyrA subunit and Ser80→Ile of the ParC subunit were the most common. One S. Kentucky isolate had qnrS1 in addition to the QRDR mutations. Five of the ST198 strains, grouped in cluster A, had multiple resistant determinants like blaTEM1-B, aac(6')-Iaa, sul1 or tetA, mostly in SGI1 K. Seven strains, grouped in cluster B, had shorter SGI1-K with deletions of many regions and with few resistance genes detected. CONCLUSION: The results of this study demonstrated that a significant part of S. Kentucky isolates from humans in Poland belonged to ST198 and were high-level resistant to ampicillin and quinolones.

The influence of a swab type on the results of point-of-care tests.

Most point-of-care tests (POCT) use swabs for sampling and/or for applying a sample on the test. A variety of swabs differing in tip materials is commercially available. Different tip materials have different chemical and physical characteristics which might influence the specimen collection and release. We investigated properties of various types of swabs used in clinical diagnostics with focusing on two kinds of analytes, DNA and proteins, which are most often used targets in POCT. As the model samples we used diphtheria toxoid NIBSC 69/017 for investigating recovery of protein analytes such as antigens and bacterial strains of Escherichia coli ATCC 25922, diphtheria toxin-producing Corynebacterium diphtheriae NCTC 10648, and the clinical isolate nontoxigenic C. diphtheriae 5820/15 for investigating the recovery of nucleic acids. We investigated four types of swabs most commonly used in clinical diagnostics in terms of absorption capacity and efficiency of release of nucleic acids and proteins. Volume uptake was measured in milligrams. For DNA release various washing out buffers were used and the amount of released DNA was measured spectrophotometrically. The amount of protein released from the swabs were examined using the Lowry assay. We observed statistically significant differences (p < 0.05) in the mean weights of absorbed liquid, in the DNA recovery and protein recovery by the four variety of swab examined. However, the efficiency of DNA and protein release was not correlated to the absorbed volume of a sample, but rather to the properties of swabs. The swab composition and structure can have a significant impact on the collection and release efficiency of a sample. Therefore, validation of POCT in relation to the used swabs for sampling is really important. The use of inappropriate swabs could lead to false negative or misleading analysis results.

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.

Clinical outcomes for cystic fibrosis patients with Pseudomonas aeruginosa cross-infections.

INTRODUCTION: Pseudomonas aeruginosa cross-infections are related to increased morbidity and mortality in cystic fibrosis (CF). OBJECTIVES: The aim of the study was to evaluate the incidence of cross-infections with P. aeruginosa in children with CF. METHODOLOGY: CF patients from whom at least one P. aeruginosa strain had been isolated were included in the study. The strain genotyping was performed using pulse-field gel electrophoresis. The history of contacts between patients was established based on questionnaires. RESULTS: The study group consisted of 75 patients (aged 1.0-19.2 years) and the material included 170 P. aeruginosa strains. Cross-infections occurred in a group of 26 patients. In this group, the risk of the predicted occurrence of forced expiratory volume in 1 second ≤ 70% was five times greater and the risk of longer cumulative hospitalization time for intravenous antibiotic therapy (>14 days/year) was almost five times greater. In the clonal groups of strains, the multidrug-resistance rate was significantly higher than in other groups. In 2011, all tested strains were susceptible to colistin, whereas in 2012, three strains from the largest clonal group showed high levels of resistance to colistin. CONCLUSION: Cross-infections with P. aeruginosa occurred in our group of patients and were associated with poor clinical outcomes. Antimicrobial resistance rate in the strains isolated from such infections was significantly higher, and this included three strains resistant to colistin.

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.

Draft genome sequence of an Escherichia coli ST410 isolate co-harbouring blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes with gyrA and parC mutations isolated from a paediatric patient in Poland.

OBJECTIVES: Escherichia coli is one of the major causative agents of nosocomial infections. Here we report the first draft genome sequence of an E. coli strain (no. 158) isolated in Poland carrying blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes together with mutations in the gyrA and parC genes. METHODS: Total DNA was sequenced using an Illumina NextSeq 500 platform. The draft genome of E. coli strain 158 was assembled using SPAdes 3.9 assembler. Contigs were annotated using the Prokka v.1.12 algorithm. Species confirmation, multilocus sequence typing (MLST), serotyping, molecular virulence and resistance traits, and plasmid replicons were analysed using appropriate bioinformatics tools available at the Centre for Genomic Epidemiology website. Additional in silico analyses were also conducted. RESULT: The genome size was estimated at 4883487bp, with 4601 predicted coding sequences. The presence of blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes was detected in addition to other antimicrobial resistance genes as well as mutations in the gyrA (Ser83Leu and Asp87Asn) and parC (Ser80Ile) genes. The investigated strain E. coli 158 belongs to ST410. CONCLUSION: To our knowledge, this is the first draft genome of an E. coli strain co-harbouring blaCTX-M-15, blaCMY-42, blaOXA-1, aac(3)-IIa and aac(6')-Ib-cr genes with mutations in gyrA and parC reported in Poland. The reported genome sequence contains valuable information on genetic features of antimicrobial resistance mechanisms of E. coli in Poland.

Isothermal DNA amplification combined with lateral flow dipsticks for detection of biothreat agents.

The recently developed methods of nucleic acids isothermal amplification are promising tools for point-of-care diagnostics and in the field detection of pathogenic microorganisms. However, application of these methods outside a laboratory faces some challenges such as the rapid and sensitive detection of amplified products and the absence of cross-reactivity with genetically related microorganisms. In the presented study we compared three methods of isothermal DNA amplification loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) and thermophilic helicase-dependent isothermal DNA amplification (tHDA), for detection of highly dangerous pathogens, such as Bacillus anthracis, Francisella tularensis and Yersinia pestis, and combined them with lateral flow dipsticks for the rapid visualization of amplified products. We observed low specificity of the three methods for B. antharcis, medium for Y. pestis and high for F. tularensis detection. Sensitivity and the detection limit were high and comparable for all the methods. We concluded that the lateral flow dipsticks have been a very useful tool for product detection of the isothermal amplification methods and enable reading the results without the use of any equipment. However, our results showed that the use of isothermal amplification methods is strongly related to the risk of false positive results.

Distribution of 16S rRNA Methylases Among Different Species of Aminoglycoside-Resistant Enterobacteriaceae in a Tertiary Care Hospital in Poland.

BACKGROUND: Aminoglycosides are a group of antimicrobial agents still the most commonly used in the treatment of life-threatening bacterial infections in human and animals. The emergence and spread of 16S rRNA methylases, which confer high-level resistance to the majority of clinically relevant aminoglycosides, constitute a major public health concern. OBJECTIVES: Our goal was to evaluate the distribution of 16S rRNA methylases among different species of Enterobacteriaceae during a five month-long survey in a tertiary hospital in Warszawa, Poland. MATERIAL AND METHODS: In the survey, a total of 1770 non-duplicate clinical isolates were collected from all hospital wards in a tertiary hospital in Warszawa, Poland. The survey was conducted between 19 April and 19 September 2010. The ability to produce 16S rRNA methylase was examined by determining MICs for gentamicin, kanamycin, amikacin by means of the agar dilution method. The isolates resistant to high concentration of aminoglycosides were PCR tested for genes: armA, rmtA, rmtB and rmtC. PCR products were subjected to DNA sequencing by the Sanger method. The genetic similarity of the ArmA-producing isolates was analysed by pulsed-filed gel electrophoresis (PFGE). RESULTS: ArmA was the only 16S rRNA methylase detected in 20 of 1770 tested isolates. The overall prevalence rate of ArmA was 1.13%. In K. pneumoniae (n = 742), P. mirabilis (n = 130), and E. cloacae (n = 253) collected in the survey, the prevalence of ArmA was 0.4%, 0.8% and 5.9%, respectively. The PFGE revealed both horizontal and clonal spread of the armA gene in the hospital. CONCLUSIONS: The prevalence of 16S rRNA methylase ArmA reported in this study is significantly higher than observed in other countries in Europe.

[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.

Characterisation of Yersinia Secretion Apparatus--Pathogenicity Island (Ysa-PI) of Yersinia enterocolitica 1B/O8 in Poland: an Idle Ysa is a Specific Hallmark of the Epidemic Sensu Stricto Strain.

Yersinia secretion apparatus (Ysa), the chromosomal type three secretion system (T3SS) is considered to contribute to virulence of high-pathogenicity Yersina enterocolitica biovar 1B. DNA-sequence of Ysa pathogenicity island was determined for clinical isolate DM0110 of Y enterocolitica 1B/08 with origin in Poland. We found a premature stop-codon in the regulatory gene ysrR (mutation at position 269). Altered ysrR was detected in all tested 78 isolates of Y enterocolitica 1B/O8 collected from clinical samples in Poland from 2004 to 2013. Since aberrations in YsrR are considered to inactivate Ysa, our findings may suggest Ysa is not indispensable for Y enterocolitica 1B/O8 to infect humans.

Cross-infections with Pseudomonas aeruginosa in patients with cystic fibrosis attending the Warsaw Centre.

AIM: 1. To assess the prevalence of cross-infections with P. aeruginosa in order to evaluate the epidemiological situation of this infection in patients with cystic fibrosis attending our centre; 2. To correlate the clinical features of the patients carrying a potentially transmissible strain with the entire study group in order to determine the risk factors and possible effects of its acquisition. MATERIAL AND METHODS: 170 Pseudomonas aeruginosa strains obtained from the respiratory tract of 75 cystic fibrosis patients attending the Warsaw Centre in 2011 and 2012 were typed using restriction enzyme analysis-pulsed field gel electrophoresis (Spe I restriction enzyme was used). Simultaneously, the information concerning contacts between patients, as well as several clinical data regarding the course of the disease were collected. RESULTS: Twenty four clusters of strains were detected. The main cluster included 49 isolates derived from 21 patients. The other detected clusters included 2 to 12 isolates derived from 1 to 7 patients. Three clusters comprised the isolates derived from three pairs of siblings. There were 15 clusters containing 2 to 7 strains belonging to the same patient. The remaining 24 patients were infected with their own strains, not fitting any clonal group. Several clinical parameters showed that the 21 patients whose strains constituted the main cluster, were in worse clinical condition than the other patients in the study group. Moreover, the total duration of their hospitalizations in order to perform intravenous antibiotic treatment was longer. CONCLUSIONS: 1. Frequent hospitalizations of CF patients with a more severe course of the disease seem to be a risk factor of cross-infections with P. aeruginosa. 2. Intensification of measures to prevent cross-infection, such as hygienic precautions, patient segregation, introduction of home intravenous antibiotic therapy programme, as well as further education of patients and their parents should lead to the improvement of the epidemiological situation in our centre. .

Co-existence of plasmid-mediated quinolone resistance determinants and mutations in gyrA and parC among fluoroquinolone-resistant clinical Enterobacteriaceae isolated in a tertiary hospital in Warsaw, Poland.

Plasmid-mediated quinolone resistance (PMQR) determinants and the distribution of mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC were investigated in 215 ciprofloxacin-resistant (MIC>1mg/L) clinical Enterobacteriaceae collected during a 6-month prospective study in a tertiary hospital in Warsaw, Poland. PMQR determinants were present in 49 isolates (22.8%), among which aac(6')-Ib-cr and qnrB1 predominated (85.7% and 26.5%, respectively). Mutations in gyrA and parC QRDRs were detected among 89.8% of isolates (MIC≥4mg/L). Changes in Ser-83, Ala-84 and Asp-87 in GyrA and Ser-80 and Glu-84 in ParC were detected. Five isolates with ciprofloxacin MICs in the range 1.5-16 mg/L were found to have unaltered QRDRs, with PMQR as the only fluoroquinolone (FQ) resistance trait detected. The remaining 44 PMQR-positive isolates were found to carry altered QRDRs. Three substitutions (two in GyrA and one in ParC) were detected in 23 isolates, whilst 8 isolates carried four mutations (two in GyrA and two in ParC). One isolate of Klebsiella pneumoniae with two amino acid substitutions in the ParC QRDR in the presence of aac(6')-Ib-cr and qnrB1 had a ciprofloxacin MIC of 16mg/L. The results presented here show that FQ resistance in these clinical Enterobacteriaceae is a complex interplay between PMQR determinants and mutations in gyrA and parC rather than a single stepwise accumulation of mutations in the gyrase and topoisomerase subunits. In addition, these results show the role of PMQR determinants in promoting QRDR mutations and the acquisition of high-level FQ resistance in clinical settings.

Probable Interspecies Transfer of the bla(VIM-4) Gene between Enterobacter cloacae and Klebsiella pneumoniae in a Single Infant Patient.

We report the interspecies transfer of the bla(VLM-4) gene in MBL-producing Enterobacter cloacae and Klebsiella pneumoniae isolates from a newborn patient who had received meropenem therapy. We show evidence that gene bla(VIM-4) was transmitted as a part of the class-1 integron on a ca. -90 kb conjugative plasmid. High homology of nucleotide sequence was observed between the integron found in VIM-4 producing E. cloacae and K. pneumoniae strains tested and class-1 integrons previously reporteded in Pseudomonas aeruginosa from Hungary and Poland. This finding may suggest P. aeruginosa as a potential source of acquired VIM-4 in Enterobacteriaceae.

[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.

[Occurrence and characterisation of aac(6')-Ib-cr gene encoding fluoroquinolone-modifying enzyme in clinical ciprofloxacin-resistant Enterobacteriaceae strains isolated in Poland]. / Wystepowanie i charakterystyka genu aac(6')-Ib-cr kodujacego enzym modyfikujacy fluorochinolony u opornych na ciprofloksacyne szczepów Enterobacteriaceae wyizolowanych od ludzi.

INTRODUCTION: The aac(6')-Ib-cr gene encodes a variant of aminoglycoside acetyltransferase that confers reduced susceptibility to hydrophilic fluoroquinolones such as ciprofloxacin and norfloxacin. AAC(6')-Ib-cr has two amino acid changes, Trp 102Arg and Asp179Tyr, which together are necessery and sufficient for the enzyme's ability to reduce the activity of fluoroquinolones, including ciprofloxacin and norfloxacin. The aim of this study was to evaluate the prevelance of aac(6')-Ib-cr determinant among 15 Enterobacteriaceae isolates randomly chosen from 215 fluorochinolone resistant strains recovered during the 6 months of 2010. METHODS: The aac(6')-Ib was detected by PCR. The presence of aac(6')-Ib-cr gene variant was futher identified by digestion with BseGI (BtsCI) and sequencing. RESULTS: 11/15 of the resistant (MIC CIP 2-1024 microg/ml) Enterobacteriaceae strains carried aac(6')-Ib-cr variant. CONCLUSION: This is the first study identifying the variant of aminoglycoside acetyltransferase determinant in Poland. Our results demonstrate that this enzym may be even more widespread than Qnr determinants among fluoroquinolone resistant Enterobacteriaceae in Poland.