Prevalence of ESBL-producing Pseudomonas aeruginosa isolates in Warsaw, Poland, detected by various phenotypic and genotypic methods.

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-ß-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.

Distribution of carbapenem resistance mechanisms in Pseudomonas aeruginosa isolates among hospitalised children in Poland: Characterisation of two novel insertion sequences disrupting the oprD gene.

This study aimed to analyse the distribution of carbapenem resistance mechanisms among Pseudomonas aeruginosa clinical isolates. Fifty-five P. aeruginosa isolates, resistant both to imipenem and meropenem, from children hospitalised in 2009-2010 were studied. All strains were genotyped by pulsed-field gel electrophoresis (PFGE). Mutations in the oprD gene and the occurrence of insertion sequences (ISs) were determined by DNA sequencing. Mex efflux systems were determined by analysis using the efflux pump inhibitor Phe-Arg ß-naphthylamide. Metallo-ß-lactamase (MBL) production was determined with Etest MBL strips and PCR for blaVIM and blaIMP. PFGE show high genetic diversity among the isolates. Mutations inactivating the oprD gene were detected in 44 strains (80%). Frameshift mutations detected in 20 isolates were the most common cause of inactivation of the oprD gene. Point mutations leading to premature stop codons were found in 12 isolates, and various substitutions were found in 6 isolates. Disruption of the coding sequence of oprD by ISs was found in six isolates. Two novel ISs (ISPa51 and ISPa52) were detected. Increased activity of different Mex systems was observed in 27 isolates (49%). Ten isolates simultaneously overexpressed two (n=3) or three (n=7) types of Mex efflux system. Seven (13%) P. aeruginosa strains were found to have minimum inhibitory concentrations (MICs) of >64mg/L both for imipenem and meropenem (two VIM-4, four VIM-2 and one IMP-1). These results show a significant diversity of P. aeruginosa strategies for resistance development. Noteworthy, a variety of ISs were found to disrupt the oprD gene.

Candidaemia in a Polish tertiary paediatric hospital, 2000 to 2010.

Data on the epidemiology of invasive Candida infections in paediatric patients in Europe are still limited. The aim of this retrospective study was to analyse the epidemiology of candidaemia in a tertiary paediatric hospital in Poland from 2000 to 2010. Using microbiological records, a total of 118 episodes of candidaemia were identified in 114 children, with an annual incidence of 0.35 episodes/1000 discharges. The highest incidences were found in the medical intensive care unit (5.28), and in neonatal intensive care (1.47). The mortality rate was 8.5%. Candida albicans and C. parapsilosis were the most prevalent species (39.8% and 35.6% respectively). The prevalence of non-albicans species increased from 12.5% in 2000 to 70% in 2010. No differences were found between C. albicans and C. non-albicans episodes in terms of demographics, risk factors or mortality. The highest resistance rates (overall 7.6%) were observed for fluconazole (4.3% in C. albicans, 7.1% in C. parapsilosis and 13.8% in other Candida species). Resistance to amphotericin B (2.5%) was limited to non-albicans isolates. The dynamic changes in species distribution and increasing resistance of fungal pathogens confirm the importance of epidemiological surveillance.

Infections caused by RSV among children and adults during two epidemic seasons.

Respiratory Syncytial Virus (RSV) is one of the most common causes of lower respiratory tract infections in young children, immunocompromised patients (children and adults), patients with chronic respiratory diseases and elderly people. Reinfections occur throughout the life, but the severity of disease decreased with subsequent infection. The aim of this study was to analyze the frequency of RSV infections in two selected subpopulations: young children (below 5 y.) and adults with chronic respiratory diseases (25-87 y.). Nasopharyngeal swabs (334) collected from October 2008 to March 2010 were examined. The presence of RSV genome was determined by RT-PCR and the presence of RSV antigen by quick immunochromatographic test. Positive results of RT-PCR were found in 45.2% of all swabs: 48.6% samples in 2008; 41.5% in 2009; 50.8% in 2010. The highest frequency of RSV-positive samples was in fall-winter months, but differences in RSV epidemic seasons were found. In the first season (2008-2009) an increased number of RSV infections was observed from November 2008, but in the second season--from January 2010. Generally, the frequency of RSV-positive RT-PCR among children was 53%, among adults 25%. The highest difference was observed in the first three-month period of 2010. RT-PCR positive samples were found in 68.5% of children and 5.9% of adults. However, the RSV antigen was found in 44.4% of samples collected from adults in this period. Our results indicate that the contribution of RSV infections during epidemic season of respiratory tract infections in Poland was really high among children and adults.

Determination of urinary D-/L-arabinitol ratios as a biomarker for invasive candidiasis in children with cardiac diseases.

A non-invasive, non-culture-based method of determining urinary D-/L-arabinitol (D-/L-ARA) ratios was investigated as a tool for the diagnosis of invasive candidiasis in nosocomial paediatric infection cases. The study encompassed 138 children aged 4 days to 16 years (mean ± SD=1.6 ± 4.2 years) with congenital heart defects (91.4%) or with rhythm disorders or circulatory failure (8.6%). ARA enantiomers were detected by GC using an electron capture detector. Positive D-/L-ARA ratios were found for 11/11 patients with proven candidiasis and 17/19 patients with clinically suspected invasive candidiasis. Thirty children were undergoing antifungal chemotherapy. D-/L-ARA ratios (mean ± SD) were 2.601 ± 0.544 in hospitalized cardiac patients without fungal infection and 5.120 ± 1.253 in those receiving antifungal therapy (P<0.001). The sensitivity of the method was 100%, the specificity 97.2%, the positive predictive value was 78.6% and the negative predictive value was 100%.

[Evaluation of biofilm formation by Staphylococcus aureus isolated from sputum of cystic fibrosis patients]. / Ocena tworzenia biofilmu przez szczepy Staphylococcus aureus wyizolowane z plwociny pacjentów z mukowiscydoza.

The purpose of this study was to evaluate two screening methods for detection of biofilm formation by eighty clinical Staphylococcus aureus isolates from patients with cystic fibrosis, and evaluation of biofilm production on the polystyrene 96-well tissue culture plates, depending on media applied. All clinical strains were incubated in three different media: Luria-Bertani broth (LB), tryptic soy broth supplemented with 2% glucose (TSBglu) and brain heart infusion (BHI). Biofilm production was screened by staining with crystal violet (CV) or with 2,3,5-triphenyltetrazolium chloride (TTC). Both CV and TTC assays showed, that all analyzed isolates created biofilm, in all tested media, however with different intensity. In conclusion, the CV method was found to be more sensitive than the TTC method, when we need information about whole mass of biofilm. The most optimal medium for the biofilm culture was LB medium.

Geographic variation in the frequency of isolation and fluconazole and voriconazole susceptibilities of Candida glabrata: an assessment from the ARTEMIS DISK Global Antifungal Surveillance Program.

Geographic differences in frequency and azole resistance among Candida glabrata may impact empiric antifungal therapy choice. We examined geographic variation in isolation and azole susceptibility of C. glabrata. We examined 23 305 clinical isolates of C. glabrata during ARTEMIS DISK global surveillance. Susceptibility testing to fluconazole and voriconazole was assessed by disk diffusion, and the results were grouped by geographic location: North America (NA) (2470 isolates), Latin America (LA) (2039), Europe (EU) (12 439), Africa and the Middle East (AME) (728), and Asia-Pacific (AP) (5629). Overall, C. glabrata accounted for 11.6% of 201 653 isolates of Candida and varied as a proportion of all Candida isolated from 7.4% in LA to 21.1% in NA. Decreased susceptibility (S) to fluconazole was observed in all geographic regions and ranged from 62.8% in AME to 76.7% in LA. Variation in fluconazole susceptibility was observed within each region: AP (range, 50-100% S), AME (48-86.9%), EU (44.8-88%), LA (43-92%), and NA (74.5-91.6%). Voriconazole was more active than fluconazole (range, 82.3-84.2% S) with similar regional variation. Among 22 sentinel sites participating in ARTEMIS from 2001 through 2007 (84 140 total isolates, 8163 C. glabrata), the frequency of C. glabrata isolation increased in 14 sites and the frequency of fluconazole resistance (R) increased in 11 sites over the 7-year period of study. The sites with the highest cumulative rates of fluconazole R were in Poland (22% R), the Czech Republic (27% R), Venezuela (27% R), and Greece (33% R). C. glabrata was most often isolated from blood, normally sterile body fluids and urine. There is substantial geographic and institutional variation in both frequency of isolation and azole resistance among C. glabrata. Prompt species identification and fluconazole susceptibility testing are necessary to optimize therapy for invasive candidiasis.

Carriage of genes for various extended-spectrum beta-lactamases: a novel resistance strategy of Klebsiella pneumoniae in Poland.

Among 110 randomly sampled strains from a collection of 247 extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae collected from hospitalised children in three paediatric hospitals in Poland, 64 strains (58.2%) with multiple ESBLs were found, including five non-clonal strains (4.5%) harbouring bla genes for ESBLs of three families (CTX-M, SHV and TEM). This is the first report of the emergence of triple ESBL-producing K. pneumoniae in Poland. In addition, K. pneumoniae strains harbouring bla genes for TEM-130 and TEM-132 ESBLs were detected in Poland for the first time. Epidemiological analysis of the multiple ESBL-producing K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) revealed a relatively high genetic diversity between isolates producing the same combination of enzymes. Clonally related strains were uncommon.

Trends in antimicrobial susceptibility of Campylobacter isolates in Poland (2000-2007).

This study analysed the pattern of antibiotic resistance in 251 Campylobacter strains isolated from symptomatic children hospitalized in 4 large paediatric hospitals in Poland from 2000 through 2007. The highest resistance was found for ciprofloxacin (49.5% for C. jejuni and 51.3% for C. coli), followed by tetracycline (17.5% and 18.0%, respectively), and ampicillin (13.2% and 10.2%, respectively). Almost all isolates were susceptible to macrolides. As much as 22.6% of C. jejuni and 25.6% of C. coli were resistant to more than one class of antimicrobial agents. Multidrug resistance (defined as resistance to at least two classes of antimicrobials) rose significantly from 5.1% in 2000-2003 to 34.6% in 2004-2007.

Emergence and persistence of integron structures harbouring VIM genes in the Children's Memorial Health Institute, Warsaw, Poland, 1998-2006.

OBJECTIVES: The aim was to perform a genetically detailed study of the emergence of metallo-beta-lactamase (MBL) genes in Pseudomonas spp. in the Children's Memorial Health Institute over a 9 year period. METHODS: Carbapenem-resistant Pseudomonas spp. isolates were collected from 1998 to 2006 and screened for MBL production. MBL-positive isolates were further investigated by a combination of genetic techniques including PCR, genomic location experiments using pulsed-field gel electrophoresis (PFGE) of I-Ceu1, S1 and SpeI digests, and sequencing. RESULTS: Of the 20 MBL-containing Pseudomonas isolates collected from 1998 to 2006, 16 Pseudomonas aeruginosa isolates contained an identical class 1 integron structure. Two P. aeruginosa isolates contained the bla(VIM-2) gene, and two Pseudomonas putida isolates harboured the bla(VIM-4) gene cassette in different integron structures. PFGE analysis indicated that all bla(VIM-4)-containing P. aeruginosa isolates were closely related, whereas the P. putida isolates were not. All MBL genes in this study were chromosomally encoded, and all isolates harboured only one class 1 integron. The bla(VIM-2) isolates were clonal, and the genetic structure supporting the bla(VIM-2) gene was found in an identical chromosomal position. CONCLUSIONS: MBL gene emergence in this hospital has paralleled a 6-fold increase in carbapenem usage. We have found an increase in MBL gene diversity, MBL host organisms and MBL genetic support structures in the hospital over the 9 year study period. There is also compelling evidence of the persistence of individual strains in the hospital throughout the study period. This suggests that once MBL genes have emerged in a hospital environment, they are difficult to remove.

Trends in antimicrobial susceptibility of Gram-negative isolates from a paediatric intensive care unit in Warsaw: results from the MYSTIC programme (1997-2007).

OBJECTIVES: The Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) programme is a longitudinal global surveillance study to monitor in vitro data on microbial susceptibility in centres that prescribe meropenem. This overview provides data on the susceptibility of Gram-negative bacteria (n = 1300) isolated from clinical specimens of children hospitalized in a paediatric intensive care unit (ICU) during 1997-2007. METHODS: MICs of meropenem and eight other antibiotics were determined using the CLSI agar dilution method. RESULTS: Meropenem, imipenem and ciprofloxacin were most active (>90% susceptibility) against the tested isolates. A greater proportion of Pseudomonas aeruginosa isolates was susceptible to meropenem compared with imipenem. Antibiotic susceptibility of Enterobacteriaceae and Acinetobacter baumannii showed an increase in 2007. Only susceptibility of P. aeruginosa to ceftazidime and cefepime increased. The incidence of extended-spectrum beta-lactamase (ESBL) producers among Enterobacteriaceae isolates decreased from 37% in 1997 to 21.8% in 2007, and AmpC beta-lactamase producers decreased from 24.6% to 5.7%. Consumption of cephalosporins remained the same and piperacillin/tazobactam increased 3-fold. During 11 years, despite an increase in carbapenem consumption, meropenem and imipenem have retained excellent activity against the majority of isolates studied. CONCLUSIONS: The comparison of antibiotic susceptibility of Gram-negative isolates in 1997 and 2007 showed a trend of increase, and the number of beta-lactamase-producing isolates among Enterobacteriaceae showed a trend of decrease possibly related to changes in antibiotic policy.

Comparison of antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from humans and chicken carcasses in Poland.

Campylobacter-associated gastroenteritis remains an important cause of morbidity worldwide, and some evidence suggests that poultry is an important source of this foodborne infection in humans. This study was conducted to analyze the prevalence and genetic background of resistance of 149 Campylobacter jejuni and 54 Campylobacter coli strains isolated from broiler chicken carcasses and from stool samples of infected children in Poland from 2003 through 2005. Nearly all isolates were susceptible to macrolides and aminoglycosides. The highest resistance in both human and chicken strains was observed for ciprofloxacin (more than 40%), followed by ampicillin (13 to 21%), and tetracycline (8 to 29%). Resistance to ampicillin and tetracycline rose significantly between 2003 and 2005. Slight differences in resistance between human and chicken isolates indicate that although chicken meat is not the only source of Campylobacter infection in our population, it can be involved in the transmission of drug-resistant Campylobacter strains to humans.

Enhanced gastric IL-18 mRNA expression in Helicobacter pylori-infected children is associated with macrophage infiltration, IL-8, and IL-1 beta mRNA expression.

OBJECTIVE: To investigate IL-18 mRNA expression in the gastric mucosa in Helicobacter pylori-infected children and its association with macrophage infiltration, IL-8, and IL-1 beta mRNA expression. METHODS: From 39 children, blood samples were taken for IL-1 beta gene polymorphism analysis and antral biopsies were obtained for histology (including macrophage immunostaining), culture and semiquantitative analysis of IL-18, IL-8, IL-1 beta, and CD14 mRNA expression by reverse transcription-PCR (RT-PCR). RT-PCR was used for H. pylori ureA and cagA mRNA detection in gastric tissue. RESULTS: H. pylori-infected patients had significantly higher IL-18, IL-8, and IL-1 beta transcript levels and macrophage numbers in the antral mucosa than H. pylori-negative children. IL-1 beta-511/31 gene polymorphism had no impact on gastric IL-1 beta mRNA levels. IL-18 mRNA expression correlated with mRNA expression of IL-8 and IL-1 beta, and transcript levels of all three cytokines were associated with macrophage infiltration and CD14 mRNA expression in the gastric tissue. Significant correlation was also observed between macrophage numbers and histological parameters of gastritis. CONCLUSION: These results suggest that interleukin(IL)-18 and macrophages may have an important function in gastric inflammatory response to H. pylori infection in children. IL-18, and possibly CD14 receptor signalling pathway, may be involved in macrophage activation and subsequent IL-8 and IL-1 beta release.

[Diagnosis and treatment option strategy for invasive fungal infections in paediatric intensive care]. / Diagnostyka i strategia wyboru postepowania leczniczego inwazyjnych zakazen grzybiczych w intensywnej terapii dzieci.

During the last 20 years rising prevalence of invasive fungal infections, mainly candidiasis or aspergillosis, are observed in paediatric intensive care units. Infections caused by Candida non-albicans species are a particular problem. Results of treatment depend on fast and correct diagnostics, avoidance of risk factors and selected pharmacotherapy. The local epidemiological situation as well as patient factors should be taken into consideration during treatment. Current recommendations for the diagnosis and strategy of choosing treatment options for invasive fungal infections in paediatric intensive care units are presented.

[Phenotype and genotype characterization of Candida albicans strains isolated from patients hospitalized at the Children's Memorial Health Institute]. / Fenotypowa i genotypowa charakterystyka szczepów Candida albicans izolowanych od pacjentów hospitalizowanych w instytucie "Pomnik-Centrum Zdrowia Dziecka".

Phenotype and genotype analysis was prepared from Candida strains causing colonization and fungal infections in children hospitalized at The Children's Memorial Health Institute intensive care unit and oncology unit. We totally analyzed 117 C. albicans strains cultured from different clinical specimens received from 51 patients--oncology unit (26) and intensive care unit (25). Enzymatic activity was assessed by API ZYM bioMerieux. Strains were biotyped according to Williamson, Kurnatowska and Kurnatowski classification. Candida albicans isolates were RAPD typed. C. albicans strains causing colonization and fungal infections on those units released such enzymes like esterase lipasis, waline arylamidasis and phosphohydrolasis. The most frequent biotypes in group of patients oncological unit with fungal infections were biotype E (64.7%) and biotype C (26.6%) while in group of patients with colonized mucosa dominated biotypes: E (28.6%) and A (28.5%). Comparing with intensive care unit group patients, the most frequent biotype isolated among patients with fungal infection symptoms was biotype E (71.7%), while in mucosa colonization group C (35.5%) occurred as a dominating biotype. This study demonstrated clonal character of fungal infections among patients hospitalized in two units and considerable polymorphism among yeast strains: 23 pattern stripes in oncology unit and 28 pattern stripes in intensive care unit. What is more, yeast biotype determination allows showing frequent occurrence of virulence character of isolates received from infected patients in comparison with those received from colonized patients.

[The analyze of capability of biofilm synthesis by Staphylococcus aureus strains isolated from children with cystic fibrosis]. / Ocena wytwarzania biofilmu przez szczepy Staphylococcus a ureus izolowane od dzieci chorych na mukowiscydoze.

The aim of this study was to analyse the capability of biofilm synthesis by S. aureus isolates obtained from the respiratory tract of CF (cystic fibrosis) patients. A total of 297 S. aureus strains isolated from 33 CF patients, and 40 isolates obtained from healthy control children, were analysed. Extracellular slime production was determined using phenotypical methods (Congo red agar, and crystal violet) and molecular techniques (icaA and icaD genes amplification). All S. aureus strains possessed the icaA and icaD genes belonging to the operon responsible for slime synthesis. The isolates obtained from the respiratory tract of CS patients more frequently showed the ability to produce a biofilm than those from healthy individuals.

[The frequency of small colony variants of Staphylococcus aureus strains isolated from children with cystic fibrosis]. / Czestosc wystepowania fenotypów small colony variants wsród szczepów Staphylococcus aureus izolowanych od dzieci chorych na mukowiscydoze.

The aim of this study was to determine the frequency of spontaneous and inducible SCV formation in S. aureus isolates obtained from CF patients. A total of 297 S. aureus strains isolated from 33 CF patients, and 40 isolates obtained from healthy control children, were analysed. S. aureus was cultured on Columbia blood agar, Schaedler agar and Chapman agar under aerophilic and microaerophilic conditions. Subinhibitory gentamicin concentrations (1 mg/L) were used to test S. aureus ability to form SCVs. The study showed that the characteristic feature of S. aureus strains, persistently colonizing the airways of CS patients, was the formation of small colony variants. In the subinhibitory gentamicin concentration, S. aureus strains from CS patients formed SCVs more frequently (55%) than isolates from healthy subjects (20%). SCV formation in CF patients was associated with treatment with inhaled aminoglycosides.

[Immunological response (IgM) to Legionella pneumophila infection in children]. / Odpowiedz odpornosciowa w klasie IgM na zakazenie Legionella pneumophila u dzieci.

Most of the publications report cases of Legionella infection among adults. In our studies the level of IgM to Legionella pneumophila sg 1 was determined by ELISA method in serum samples of 144 children with symptoms of respiratory tract infections. Children were from 5 months to 18 years old. The significant level of IgM was found in 41 sera collected from 34 children aged 2 years and above. There were samples collected from 21 girls and 13 boys. The highest percentage of significant level of IgM was found in the age group 4-10 years (31.8%) and 10-14 years (31.7%). Among the youngest children (below 2 years) the equivocal results were found in 6 samples. The significant difference in IgM level was found between the children age groups. There was not found the significant differences in IgM level depending on gender. Obtained results indicated the importance of such kind of studies and necessity of adjusting cut-off values to age groups of children.

High activity of meropenem against Gram-negative bacteria from a paediatric Intensive Care Unit, 2001-2005.

The susceptibility of Gram-negative bacterial strains (n=620) isolated from clinical specimens of children hospitalized in a paediatric Intensive Care Unit between 2001 and 2005 was tested. Meropenem, imipenem and ciprofloxacin were most active (>90% susceptibility) against the tested isolates, with no observed reduction in activity over 5 years. A decrease in prevalence of extended-spectrum beta-lactamase and AmpC beta-lactamase producing Enterobacteriaceae from 56.3 to 34.1% was found.

Increase of imipenem resistance among Pseudomonas aeruginosa isolates from a Polish paediatric hospital (1993-2002).

Analysis of the in vitro activity of imipenem and 13 other antibiotics against 2485 Pseudomonas aeruginosa isolates obtained from clinical specimens from children hospitalized during 1993-2002 was performed. In 2002, the percentage of P. aeruginosa isolates susceptible to all tested antibiotics, with the exception of imipenem, increased or remained on nearly the same level as in 1993. An increase of resistance to imipenem from 4.3% to 18.3% was observed. The MIC(90) value of imipenem increased from 2mg/L to 16 mg/L. Simultaneously, a four-fold increase of the usage of carbapenems imipenem and meropenem in the hospital was noted. In 2000-2001, a high incidence of imipenem-resistant strains was observed. The imipenem-resistant P. aeruginosa strains of serotype O6 from the general surgery ward and serotype O11 from the intensive care unit were shown to be clonally related by the pulsed-field gel electrophoresis method.