Could a nonfunctional adrenal incidentaloma be a risk factor for increased carotid intima-media thickness and 10-year cardiovascular mortality based on the SCORE algorithm? A study from a single centre in Poland.

INTRODUCTION: Adrenal incidentaloma (AI) secreting small amounts of glucocorticoids may cause morphological and functional changes in the blood vessels. Early stages of cardiovascular remodeling may be observed among asymptomatic patients with AI. But it is unclear whether the nonfunctional adrenal incidentalomas (NFAI) may also be a risk factor for cardiovascular diseases. The aim of this study was to determine the relationship between NFAI, carotid intima-media thickness (CIMT), and cardiovascular risk (CVR) based on Systematic Coronary Risk Evaluation (SCORE) prediction models for Europe. MATERIAL AND METHODS: This study from a single centre in Poland included 48 NFAI patients and 44 individuals in the control group matched for age, sex, and body mass index (BMI). All participants underwent adrenal imaging, biochemical evaluation, measurement of CIMT, and assessment of the 10-year risk of cardiovascular mortality based on the SCORE algorithm. Hormonal evaluation was conducted in AI patients. RESULTS: The NFAI group showed significantly higher sodium (p = 0.02) and glucose levels in the 2-h oral glucose tolerance test (OGTT) (p = 0.04), a higher CIMT (p < 0.01), and a higher CVR calculated according to the SCORE algorithm (p = 0.03). The estimated glomerular filtration rate (eGFR) was higher in the NFAI group (p = 0.015). Hypertension (p < 0.01) and IGT (p = 0.026) were more common in the NFAI group. Statistically significant positive correlations were found between CIMT and age (r = 0.373, p = 0.003), waist circumference (r = 0.316, p = 0.029), diastolic blood pressure (r = 0.338, p = 0.019), and CVR based on the SCORE algorithm (r = 0.43, p = 0.004). There was a statistically significant positive correlation between CIMT and serum cortisol levels after 1 mg dexamethasone suppression test (r = 0.33, p = 0.02). CONCLUSION: Non-functional adrenal adenomas are associated with increased CIMT and CVR. Early stages of cardiovascular remodelling can be observed in asymptomatic NFAI patients.

Do Non-Functional Adrenal Adenomas Affect Metabolic Profile and Carotid Intima-Media Thickness? A Single Centre Study from Poland.

Background: Compared to the general population, among people with adrenal incidentalomas (AIs) the diagnosis of obesity, hypertension, impaired carbohydrate and lipid metabolism is more common. The aformentioned disorders represent typical cardiovascular remodeling risk factors. The study was designed to assess the association between NFAIs, metabolic profile and carotid intima-media thickness (CIMT) as the predictive factor of atherosclerosis. Material: The study included 48 patients with NFAI (16 men, 32 women, mean age 58.6 +/- 9 years) and 44 control participants (15 men, 29 women, mean age 57 +/- 7 years). Both groups were matched for age, gender and BMI. Subjects with history of myocardial infarction, stroke or diabetes mellitus (DM) were excluded. Participants underwent adrenal imaging, biochemical evaluation, and measurement of CIMT. Hormonal evaluation was conducted in AI patients. Results: The NFAI group had significantly higher waist circumference (p < 0.01), higher systolic (p < 0.01) and diastolic blood pressure (p < 0.01), fasting insulin (p = 0.03) and glucose in the 2 h OGTT (p = 0.04) as well as higher CIMT (p < 0.01). Hypertension (p < 0.01) and IGT (p = 0.026) were more common in this group as well. There was a positive correlation between CIMT and cortisol levels in 1 mg dexamethasone suppression test (r = 0.33, p = 0.02). Conclusions: Patients diagnosed with NFAIs, despite normal cortisol inhibition in the 1 mg dexamethasone test, still presented a number of metabolic abnormalities. The assessment of IMT may proove valuable in indicate the presence of early vascular remodelling in asymptomatic patients. The underlying mechanisms of these findings are still unknown, hence further studies are required.

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.

Pertactin-deficient Bordetella pertussis isolates in Poland-a country with whole-cell pertussis primary vaccination.

The introduction of pertussis vaccination in the 1950s resulted in a significant decrease in the incidence of disease. However, since the 1990s many highly vaccinated countries have observed the re-emergence of the disease. One of the causes of this phenomenon might be related to the adaptation of Bordetella pertussis to vaccination. The purpose of the presented study was an investigation of the emergence and spread of vaccine antigen-deficient B. pertussis isolates in Poland and genomic characterization of the currently circulating pathogen population using PFGE, MLVA and MAST. The results revealed that all tested isolates expressed Ptx, FHA and ACT antigens but 15.4% (4/26) of isolates from 2010 to 2016 were Prn-deficient. Moreover, one TcfA-deficient isolate was collected in 2015. The genotyping showed a genetic distinction between the isolates circulating in 2010-2016 and isolates from previous periods. The majority of currently circulating isolates belonged to PFGE group IV (96.2%), type MT27 (73.1%), and carried ptxA1-ptxC2-ptxP3-prn2-tcfA2-fim2-1-fim3-1 alleles (61.5%). The unique genetic structure of the B. pertussis population in Poland has changed since 2010 and became similar to that observed in countries with aP vaccination. This could be a result of increasing use of aP vaccines (60% of primary vaccination in 2013) over wP vaccines, which have been broadly used for primary vaccination in Poland for decades.

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.

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.

[Advantages and disadvantages of microbiological methods used in the diagnosis of Mycoplasmapneumoniae infections in selected clinical situation]. / Wady i zalety mikrobiologicznych metod diagnostycznych stosowanych w rozpoznaniu zakazenia wywolanego przez Mycoplasma pneumoniae na przykladzie wybranej sytuacji klinicznej.

Mycoplasma pneumoniae is one of the most common causes of community-acquired pneumonia in children and adults. Correct and rapid laboratory diagnosis of M pneumoniae infections is important to introduce appropriate antibiotic treatment. Diagnosis for M. pneumoniae usually relies on serological tests and/or molecular investigations. Both methods have some advantages but also limitations. This paper presents advantages and disadvantages of microbiological methods used in M. pneumoniae infection an example of case of patient with mycoplasmosis.

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.

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.

Usefulness of laboratory methods in diagnosis of pertussis in adult with paroxysmal cough.

INTRODUCTION: Pertussis is an acute, highly contagious bacterial infection of respiratory system caused by Bordetella pertussis. Principally, disease affects young children, however, recently it is also reported in adolescents and adults. Symptoms of pertussis in adults are non-specific, i.e. dry, paroxysmal and protracted cough. Thus, it is rarely diagnosed in this group. AIM: This paper aimed at evaluating the usefulness of the laboratory methods in diagnosis of pertussis in adults based on a case presenting with dry, paroxysmal and chronic cough. MATERIAL AND METHODS: Sputum (collected on 25th January 2013) and paired serum samples (collected on 13th February and 19 April 2013) were tested. Pertussis diagnostics involved culture, in-house PCR, real-time PCR and ELISA. RESULTS: Sputum culture, using commercial medium Bordetella Selective Medium by Oxoid did not reveal the presence of B. pertussis. Real-time PCR and PCR, however, confirmed the presence of insertion sequence IS481 and pertussis toxin promoter sequence ptx-Pr, markers indicative of B. pertussis infection. Serological testing revealed the high titres of IgA, IgG and IgM antibodies to B. pertussis in the first sample. In the second sample, collected 2 months following the first one, a significant decrease in IgA antibodies was reported. CONCLUSIONS: These data suggest a high usefulness of the laboratory methods in the diagnosis of pertussis in adults with chronic cough. Application of such methods ensures adequate diagnosis of disease, quick introduction of proper treatment and implementation of procedures preventing the spread of infection.

[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.

[Sequence-based typing--molecular typing of Legionella pneumophila strains within the framework of an international external quality assessment]. / Sequence-based typing--molekularne typowanie szczepów Legionella pneumophila w ramach miedzynarodowego sprawdzianu external quality assessment.

INTRODUCTION: The aim of the study was evaluation results of molecular typing L. pneumophila strains that was carried by using SBT (Sequence-Based Typing) method, obtained by laboratory of Department of Bacteriology NIZP-PZH within the framework of the ninth international external quality assessment (ELDSNet Legionella pneumophila DNA SBT) and their comparision with the results obtained by other reference laboratories in EU. MATERIAL AND METHODS: The panel of five coded isolates of L. pneumophila was investigated. Genomic DNA of Legionella were extracted and defined regions of seven genes were amplified by PCR and sequenced. Then, consensus sequence of the correct length were generated. In order to determine the complete allelic profile and Seqence Type (ST) forward and reverse sequence for each allele were submitted online by using the L. pneumophila database. RESULTS: All of L. pneumoniae isolates sent to genotyping by SBT method were correctly identified in our laboratory. CONCLUSIONS: Results of the ninth international external quality assessment have confirmed competences of laboratory of Department of Bacteriology NIZP-PZH in typing of L. pneumophila isolates accordance with the requirements of the international classification.

[Detection of bordetella within the framework of the Eupert-labnet Bordetella PCR EQA]. / Wykrywanie paleczek Bordetella w ramach miedzynarodowego sprawdzianu Eupert-labnet Bordetella PCR EQA.

INTRODUCTION: The aim of this study was evaluation of molecular identification results of samples including genomic DNA of Bordetella by using PCR method, obtained by laboratory of Department of Bacteriology NIZP-PZH and their comparison with the results obtained by other reference laboratories in EU. The study was conducted within the framework of the first external quality assessment (Eupert-labnet Bordetella PCR EQA). METHODS: The panel of ten coded samples of purified genomic DNA was investigated. The panel was designed to include dilution of genomic DNA from B. pertussis at the three concentrations 2 pg/microl (high), 0,2 pg/microl (medium) and 0,02 pg/microl (low). The panel included as well DNA of other Bordetella species (B. parapertussis, B. holmesii, B. bronchiseptica) and H. influenzae at concentrations 2 pg/microl. There was also two ,,blank" samples containing only Tris Buffet (10mM, pH 8,0). Presence or absence of B. pertussis DNA in the tested samples was determined by using four PCR assays: conventional in-house PCR (detection of IS481 B. pertussis and IS1001 B. parapertussis), commercial multiplex PCR (detection of DNA B. pertussis), conventional in-hause real-time PCR (detection of IS481 B. pertussis) and commercial real-time PCR (detection of IS1001 B. parapertussis). RESULTS: All but one samples were correctly identified in our laboratory. Laboratory of Department of Bacteriology NIZP-PZH correctly detected DNA ofB. pertussis at both the ,,high" and ,,medium" dilution. In addition, the distinction between B. pertussis and other Bordetella species was correctly obtained by our laboratory. The negative samples, the two blank samples and one containing H. influenzae were correctly detected. CONCLUSIONS: Results of the first international external quality assessment have confirmed competences of laboratory of Department of Bacteriology NIZP-PZH in molecular identification of Bordetella pertussis.

[Detection and identification of highly pathogenic bacteria within the framework of the EQADeBa project--Part II: samples containing inactivated pathogens]. / Wykrywanie i identyfikacja wysoce patogennych bakterii w ramach miedzynarodowego projektu EQADeBa-- Czesc II: próbki zawierajace inaktywowane patogeny.

THE AIM: The aim of the studies was analysis of methods applied and results of detection and identification of Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella sp., Bulkholderia mallei and B. pseudomallei in inactivated samples obtained within the framework of the third external quality assessment exercise (EQAE) in the project ,,Establishment of Quality Assurances for Detection of Highly Pathogenic Bacteria of Potential Bioterrorism Risk (EQADeBa)". MATERIAL AND METHODS: Fifteen samples in the form of water, calf serum and milk spiked with bacteria mentioned above were investigated. Detection and identification of highly pathogenic bacteria were carried out using a PCR technique. RESULTS. Y. pestis, F. tularensis ssp. holarctica, B. anthracis, B. mallei and B. pseudomallei were detected in the investigated samples. The most problematic were samples of milk, probably because of presence of PCR inhibitors such as milk proteins and calcium ions and a low number of bacterial cells contained in the samples. CONCLUSIONS. Inactivation of samples spiked with highly pathogenic microorganisms ensure safety of labora- tory workers, but investigation of such samples needs very precise selection and validation diagnostics methods due to possibility of obtaining false negative results. Results of the third international external quality assessment exercise have confirmed competences of laboratory of Department of Bacteriology NIZP-PZH in the area of detection and identification highly pathogenic bacteria covered by the EQAE.

[Occurence of pili genes in Corynebacterium diphtheriae strains]. / Wystepowanie genów z wyiazanych z wytwarzaniem pili u szczepów Corynebacterium diphtheriae.

INTRODUCTION: Adhesion of pathogenic bacteria to host cells is a crucial step during infection. The presence of specific adhesion factors on the bacterial cell surface determines the tropism of the pathogen to the tissues expressing certain surface receptors. The adhesion is mediated primarily by filamentous structures called pili. Three distinct pilus structures can be produced by Corynebacterium diphtheriae: SpaA-, SpaD- and SpaH-type pili. Pilus genes encode a total of nine pilus proteins, named SpaA through SpaI, and six sortases, named SrtA through SrtF. All the pilus genes are located on pathogenicity islands and can be acquired and lost by different strains. The aim of presented studies was assessment of occurence of pili genes among C. diphtheriae strains isolated from different infections, including invasive infections, in Poland. METHODS: Thirty-one toxigenic and nontoxigenic C. diphtheriae strains isolated from wounds, blood, nose and pharynx were investigated for presence of 15 pili genes. The studies were conducted using PCR. Gene specific primers were designed on the basis of the complete genome sequence of C. diphtheriae NCTC 13129. RESULT: All the nontoxigenic C. diphtheriae strains isolated from invasive infections possess every tested genes in contrast to toxigenic strains that revealed highly mosaic structure of pili gene clusters. Differences in gene content were detected in SpaA- and SpaH-type pili gene clusters. Complete set of genes in SpaD-type pili gene cluster was detected in all but two strains. The two strains did not possess any of SpaD-type pili genes. CONCLUSIONS: Invasiveness of C. diphtheriae strains could be related to adhesive factors. Results of our studies suggest that ability to express all types of pili is indispensable for causing invasive infections by nontoxigenic C. diphtheriae. Whereas full set ofpili genes is not necessary for causing classical diphtheria.

N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars.

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.

[Prevalence of qnr genes in clinical Enterobacteriaceae non-susceptible to fluoroquinolone in Poland]. / Wystepowanie genów qnr u niewrazliwych na fluorochinolony paleczek z rodziny Enterobacteriaceae izolowanych z materialu klinicznego.

INTRODUCTION: Fluoroquinolone are broad-spectrum antimicrobial agents extensively used by physicians. This widespread use has been associated with increased level ofquinolone resistance strains, particularly in Enterobacteriaceae. Plasmid-mediated quinolone resistance (PMQR) including Qnr determinants with the potential for horizontal transfer confer to quinolone resistance. Plasmid harboring qnr genes may also encode extended-spectrum beta-lactamases (ESBLs) such as CTX-M, SHV and TEM type. The prevalence ofplasmid-mediated quinolone resistance (PMQR) determinants like qnrA, qnrB and qnrS was investigated in a collection of 215 Enterobacteriaceae strains with reduced susceptibility to fluoroquinolone. METHODS: The isolates (n=215) were collected from 1 March to 31 September, 2010 in a regular hospital in Warsaw, Poland. The resistance to nalidixic acid, norfloxacin and ciprofloxacin was determinated by twofold agar dilution method, while MICs of moxifloxacin were examined by using E-test. The prevalence of qnrA, qnrB, qnrS, blaCTX-M, blaSHV and blaiTEM was evaluated by PCR. All PCR-products for qnr were sequenced. The epidemiological relationship between positive isolates was studied by PFGE method. RESULTS: Eighteen isolates (8,3%) carried the qnr gene encoding the QnrA, QnrB or QnrS. The coexistence of both qnrA and qnrS genes was noted in one isolate of E. coli. The qnrB gene was the most common qnr type found. All the Qnr-producing strains were simultaneously resistant to naldixic acid and different - level non-susceptible fluoroquinolone (MIC CIP 1.5-1024 microg/ml). Most of qnr-positive strains (88.9%) were extended-spectrum beta-lactamase (ESBL) producers of CTX-M and TEM types predominantly. CONCLUSIONS: The present study highlights the wide spread of Qnr-like determinants in clinical Enterobacteriaceae non-susceptible to fluoroquinolone in Poland, with an association with the ESBL.