Jan Patzer

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.

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.

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.

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.

Characterization of Antibody Response to Pseudomonas aeruginosa in Patients with Wound Infections

ELISA was used to measure the amount and avidity of IgG antibodies to exotoxin A (ExA) and 7 Fishers immunotypes of lipopolysaccharide (LPS) in the sera of 13 patients with mild or moderate Pseudomonas aeruginosa infections. Changes in the specificity of tested sera during the course of infection were demonstrated. A statistically significant increase was seen in the amount and avidity of the antibodies to ExA in a majority of the sera, and an increase was seen in amount of antibodies to LPS immunotype 4 in the sera of patients with moderate infections.

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 β-naphthylamide. Metallo-β-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.

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-β-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.

Evaluation of Therapeutic Efficacy of Ureidopenicillins in Comparison to Sisomicin and Cephalotin in Experimental Infections of Rabbits

The therapeutic efficacy of azlocillin, mezlocillin, cephalotin, and sisomicin was evaluated by experimental infection in rabbits. After one hour following infection with Escherichia coli, Klebsiella pneumoniae or Pseudomonas aeruginosa, antibiotics were applied intramuscularly. Colony-forming units were counted before the infection and every day thereafter. Leukocytosis was determined before infection and 3 and 7 days after initiation of therapy. Therapy with antibiotics was continued for seven days, rabbits sacrificed and CFU/g tissue determined. It was shown that in experimental infection caused by E. coli or K. pneumoniae in rabbits, sisomicin was most effective, followed by mezlocillin, azlocillin, and cephalotin. Efficacy of therapy against P. aeruginosa was as follows: sisomicin, azlocillin, mezlocillin.