Danuta Dzierzanowska

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 Childrens 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.

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.

Primary resistance to clarithromycin in clinical strains of Helicobacter pylori isolated from children in Poland

Helicobacter pylori resistance to clarithromycin is an important factor in the failure of eradication therapy. The resistance results from point mutations in the 23S rRNA gene of H. pylori. The prevalence of primary resistance of H. pylori to clarithromycin in children and mutations associated with resistance were studied and it was found that 23.5% (23/98) of H. pylori strains isolated in our hospital during 1998-2000 were resistant to clarithromycin. The primary resistance was mainly caused by an A2143G mutation, but the isolates with an A2142G mutation had higher MICs for clarithromycin compared with those with an A2143G mutation: median MIC 256 versus 16 mg/l. Comparison of our data with previous results showed that the prevalence of H. pylori resistance to clarithromycin in children has increased in Poland over the last three years, however the difference was not significant (23.5 vs. 17%, P=0.22).

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.

Enhanced gastric IL-18 mRNA expression in Helicobacter pylori-infected children is associated with macrophage infiltration, IL-8, and IL-1β 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&bgr; mRNA expression. Methods From 39 children, blood samples were taken for IL-1&bgr; gene polymorphism analysis and antral biopsies were obtained for histology (including macrophage immunostaining), culture and semiquantitative analysis of IL-18, IL-8, IL-1&bgr;, 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&bgr; transcript levels and macrophage numbers in the antral mucosa than H. pylori-negative children. IL-1&bgr;-511/31 gene polymorphism had no impact on gastric IL-1&bgr; mRNA levels. IL-18 mRNA expression correlated with mRNA expression of IL-8 and IL-1&bgr;, 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&bgr; release.

Susceptibility patterns of Gram-negative bacteria from a Polish intensive care unit, 1997-2000

The susceptibility of Gram-negative bacterial strains (n=400) isolated from clinical specimens of children hospitalized in a Polish intensive care unit (ICU) between 1997 and 2000 was tested. Meropenem, imipenem and ciprofloxacin were most active (>90% susceptibility) against the tested isolates, with no observed reduction in activity over 4 years. Extended-spectrum beta-lactamases and AmpC beta-lactamase producers among Enterobacteriaceae isolates from this ICU continued to be a serious therapeutic problem, although the carbapenems were highly active against these resistance phenotypes. Resistance to aminoglycosides (gentamicin, tobramycin) and ceftazidime was a characteristic of >40% of tested isolates.

Carriage of genes for various extended-spectrum β-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.

Helicobacter pylori cagA genotype and density of colonization in relation to gastric inflammation in children

Objective To assess the relationship between Helicobacter pylori density determined by quantitative culture, H. pylori cagA status, and gastric histology in children. Methods Children with clinical symptoms indicating pathology in the upper gastrointestinal tract were referred for endoscopy. From each child blood was taken for serology, and antral biopsies were obtained for quantitative culture of H. pylori and histology. Histological assessment was performed according to the updated Sydney System. The cagA status of cultured H. pylori was determined by polymerase chain reaction (PCR) and serum IgG response to CagA by western blotting. Results Adequate antral biopsies were obtained from 41 children with positive H. pylori cultures. CagA IgG antibodies were found in 27 patients (66%), 25 of whom were also cagA positive by the PCR. Two children infected with cagA+ strains as determined by the PCR were CagA seronegative. Infection with cagA+ strains was associated with significantly higher activity of inflammation and denser bacterial colonization in the antrum compared to cagA-negative strains. No correlation was observed between the density of colonization and chronic antral inflammation. Conclusions This study shows that infection of children with cagA+ strains of H. pylori is associated with enhanced activity of antral inflammation and higher density of colonization. There is a good correlation between serum western blot and bacterial PCR cagA positivity in determining cagA status and a positive relationship between histology and quantitative culture in assessing H. pylori density in paediatric patients.

Effect of Pseudomonas aeruginosa exotoxin A on IFN-γ synthesis: expression of costimulatory molecules on monocytes and activity of NK cells

The aim of the study was (1) to evaluate the effect of Pseudomonas aeruginosa Exotoxin A (P-ExA) on the production of IFN-gamma in anti-CD3 induced human peripheral blood mononuclear cells (PBMC) and (2) to establish the effect of P-ExA on the IFN-gamma dependent cellular activities such as the expression of costimulatory molecules on monocytes and cytotoxicity of NK cells. The toxin in a high dose (100 ng/ml) inhibited IFN-gamma synthesis. Inhibitory effect of P-ExA was abolished by IL-1alpha which in a combination with P-ExA exerted a strong synergistic effect on IFN-gamma synthesis. Other monokines such as IL-1beta, IL-6, TNF-alpha neither reversed the inhibitory effect of P-ExA nor induced production of IFN-gamma. P-ExA also inhibited IFN-gamma-induced cellular events: (1) expression of costimulatory molecules on monocytes (CD80, CD86, ICAM-1, HLA-DR); (2) cytotoxic activity of NK cells. Inhibition of NK cells activity by P-ExA was not reversed by cytokines such as IL-2, IFN-alpha and TNF-alpha, which are known to enhance effector functions of NK cells. From these results we conclude that: (1) inhibition of IFN-gamma synthesis, as well as IFN-gamma-induced expression of costimulatory molecules and NK-cell effector functions may lead to suppression of specific and non-specific defense mechanisms, respectively, which are necessary for elimination of PA bacteria; (2) enhancement of IFN-gamma synthesis induced by P-ExA in a combination with IL-1alpha may cause harmful, Th1 cells dependent, inflammatory reactions of the host (septic shock, tissue damage) during infection with Pseudomonas aeruginosa.

EFFECT OF PSEUDOMONAS AERUGINOSA EXOTOXIN A ON CD3-INDUCED HUMAN T-CELL ACTIVATION

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.