Katalin Kristóf

Extended-spectrum beta-lactamase-producing Klebsiella spp. in a neonatal intensive care unit: risk factors for the infection and the dynamics of the molecular epidemiology

The extended-spectrum beta-lactamase (ESBL)-producing Klebsiella spp. cause worldwide problems in intensive care units. The aim of this study was to investigate the molecular epidemiology of ESBL-producing Klebsiella pneumoniae and K. oxytoca strains in a neonatal intensive care unit (NICU) in Budapest, Hungary and to determine the risk factors of the infections and the epidemiological features. Infections with Klebsiella spp. were analyzed retrospectively by reviewing the medical records between January 2001 and December 2005. Antibiotic susceptibility tests, isoelectric focusing, pulsed field gel electrophoresis, plasmid analysis, PCR for blaTEM and blaSHV and DNA sequencing analysis were performed on ESBL-producing Klebsiella isolates. A total of 45 babies were found to be infected with non-ESBL-producing Klebsiella spp. and 39 with ESBL-producing Klebsiella spp. Of the parameters analyzed, including sex, gestational age, twin pregnancy, birth weight, presence of central vascular catheter, mechanical ventilator use, parenteral nutrition, polymicrobial infection, caesarean section, transfusion and mortality, we found no statistically significant difference between the ESBL and the non-ESBL groups, or between the K. pneumoniae and K. oxytoca species. Further characterization of the ESBL-producing K. pneumoniae and K. oxytoca strains isolated between February 2001 and January 2003 revealed three distinct PFGE patterns of SHV-5-producing K. pneumoniae (A, B, E) and two distinct patterns of SHV-12-producing K. oxytoca (C,D) isolates; these had different plasmid profiles. From July to November 2005, a new SHV-5 producing K. oxytoca (F) was isolated. The molecular epidemiology of ESBL-producing organisms in a NICU over time shows substantial shifts in predominant strains. The ESBL production of the infected organisms has an impact on the survival of newborn babies with infections caused by Klebsiella spp.

Bacterial contamination in the anterior chamber after povidone–iodine application and the effect of the lens implantation device

PURPOSE: To assess the incidence of anterior chamber bacterial contamination during cataract surgery, and compare results of injector implantation and forceps implantation of foldable intraocular lenses (IOLs). SETTING: Department of Ophthalmology and Institute of Medical Microbiology, Semmelweis University, Budapest, Hungary. METHODS: This prospective randomized controlled clinical study comprised 97 eyes of 96 patients. Antibiotic eyedrops were not used; however, povidone–iodine 10% solution was used to prepare the eyebrow and eyelids and povidone–iodine 5% to disinfect the ocular surface. A Steri‐Drape (3M) was used to surround the eye. Aqueous fluid samples were aspirated from the anterior chamber at the beginning and the end of surgery. The samples were cultured for 14 days under aerobic and anaerobic conditions simultaneously. Cataract surgery was performed using a sutureless, superotemporal, clear corneal phacoemulsification technique. The IOL was implanted with an injector (n = 47) or a forceps (n = 50), with the instrument randomly selected. The frequency of positive bacterial cultures with each implantation method was compared using the Fisher exact test. RESULTS: Bacteria were found in the conjunctival samples in 21 eyes (21.65%) before povidone–iodine application and in 4 eyes (4.12%) after disinfection. The anterior chamber sample before surgery was culture positive for Staphylococcus epidermidis in 2 eyes and for Micrococcus luteus in 1 eye. After surgery, the culture was positive for S epidermidis in 1 eye (2.15%) in the injector group and 1 eye (2.00%) in the forceps group (P = .74). Neither sample came from an eye that had a positive culture preoperatively. There were no intraoperative complications. CONCLUSIONS: In uneventful clear corneal phacoemulsification, meticulous technique can prevent antibiotic use during surgery. No difference in anterior chamber bacterial contamination was found between IOL implantation using an injector or a forceps.

Identification of a blaVIM-4 gene in the internationally successful Klebsiella pneumoniae ST11 clone and in a Klebsiella oxytoca strain in Hungary

1 Clermont O, Lavollay M, Vimont S et al. The CTX-M-15-producingEscherichia coli diffusing clone belongs to a highly virulent B2phylogenetic subgroup. J Antimicrob Chemother 2008; 61: 1024–8.2 Nicolas-Chanoine MH, Blanco J, Leflon-Guibout V et al. Intercontinentalemergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15.J Antimicrob Chemother 2008; 61: 273–81.3 Johnson JR, Menard M, Johnston B et al. Epidemic clonal groups ofEscherichia coli as a cause of antimicrobial-resistant urinary tractinfections in Canada, 2002 to 2004. Antimicrob Agents Chemother2009; 53: 2733–9.4 Clinical and Laboratory Standards Institute. Performance Standardsfor Antimicrobial Susceptibility Testing: Seventeenth InformationalSupplement M100-17. CLSI, Wayne, PA, USA, 2007.5 Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination ofthe Escherichia coli phylogenetic group. Appl Environ Microbiol 2000; 66:4555–8.6 Clermont O, Dhanji H, Upton M et al. Rapid detection of theO25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J Antimicrob Chemother 2009; 64: 274–7.7 Sidjabat HE, Paterson DL, Adams-Haduch JM et al. Molecularepidemiology of CTX-M-producing Escherichia coli isolates at a tertiarymedical center in western Pennsylvania. Antimicrob Agents Chemother2009; 53: 4733–9.8 Lau SH, Cheesborough J, Kaufmann ME et al. Rapid identification ofuropathogenic Escherichia coli of the O25:H4-ST131 clonal lineageusing the DiversiLab repetitive sequence-based PCR system. ClinMicrobiol Infect 2009; 16: 232–7.

Fitness cost associated with resistance to fluoroquinolones is diverse across clones of Klebsiella pneumoniae and may select for CTX-M-15 type extended-spectrum β-lactamase

Lowered fitness cost associated with resistance to fluoroquinolones was recently demonstrated to influence the clonal dynamics of methicillin-resistant Staphylococcus aureus (MRSA) in the health care setting. We investigated whether or not a similar mechanism impacts Klebsiella pneumoniae. The fitness of K. pneumoniae isolates from major international hospital clones (ST11, ST15, ST147) already showing high-level resistance to fluoroquinolones and of strains from three minor clones (ST25, ST274, ST1028) in which fluoroquinolone resistance was induced in vitro was tested in a propagation assay. Strains from major clones showed significantly less fitness cost than three of four fluoroquinolone-resistant derivatives of minor clone isolates. In addition, plasmids with CTX-M-15 type extended-spectrum β-lactamase (ESBL) genes were all retained in both major and minor clone isolates, irrespective of the strains’ level of fluoroquinolone resistance, while each plasmid harboring SHV-type ESBLs had been lost during the induction of resistance. Major clone K. pneumoniae strains harbored more amino acid substitutions in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes than minor clone isolates. The presence of an active efflux system could be demonstrated in all fluoroquinolone-resistant derivatives of originally SHV-producing minor clone isolates but not in any CTX-M-15-producing strain. Further investigations are needed to expand and confirm our findings on a larger sample. In addition, a long-term observation of our ciprofloxacin-resistant minor clone isolates is required in order to elucidate whether or not they are capable of restoring their fitness while concomitantly retaining high minimum inhibitory concentration (MIC) values.

CR3 is the dominant phagocytotic complement receptor on human dendritic cells.

Dendritic cells (DCs) play a decisive role in immunity; they interact with various pathogens via several pattern recognition and different opsonophagocytotic receptors, including Fc- and complement-receptors. β2-integrins, including complement receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) participate in many immunological processes, especially those involving cell migration, adherence, and phagocytosis. Human monocyte derived dendritic cells (MDCs) are known to express CR3 as well as CR4, however possible differences regarding the role of these receptors has not been addressed so far. Our aim was to explore whether there is a difference between the binding and uptake of various complement-opsonized microorganisms, mediated by CR3 and CR4. Studying the expression of receptors during differentiation of MDCs we found that the appearance of CD11b decreased, whereas that of CD11c increased. Interestingly, both receptors were present in the cell membrane in an active conformation. Here we demonstrate that ligation of CD11b directs MDCs to enhanced phagocytosis, while the maturation of the cells and their inflammatory cytokine production are not affected. Blocking CD11c alone did not change the uptake of opsonized yeast or bacteria by MDCs. We confirmed these results using siRNA; namely downregulation of CD11b blocked the phagocytosis of microbes while silencing CD11c had no effect on their uptake. Our data clearly demonstrate that complement C3-dependent phagocytosis of MDCs is mediated mainly by CR3.

Significance of methicillin–teicoplanin resistant Staphylococcus haemolyticus in bloodstream infections in patients of the Semmelweis University hospitals in Hungary

The purpose of this study was to quantify the impact of Staphylococcus haemolyticus in the epidemiology of the blood stream infection (BSI) and to characterize the rates and quantitative levels of resistance to antistaphylococcal drugs. During an eight-year period, 2967 BSIs of the patients hospitalized in different clinical departments of the Semmelweis University, Budapest, Hungary were analyzed. One hundred eighty-four were caused by S. haemolyticus, amounting to 6% of all infections. The antibacterial resistance of S. haemolyticus isolates was investigated by the broth microdilution method, vancomycin agar screen, population analysis profile and PCR for mecA, vanA and vanB genes detection. Epidemiological investigation was processed by determining phenotypic antibiotic resistance patterns and PFGE profiles. Extremely high MIC levels of resistance were obtained to oxacillin, erythromycin, clindamycin, gentamicin and ciprofloxacin. The incidence of teicoplanin reduced susceptibility revealed 32% without possessing either the vanA or vanB gene by the strains. PFGE revealed 56 well-defined genotypes indicating no clonal relationship of the strains. The propensity of S. haemolyticus to acquire resistance and its pathogenic potential in immunocompromised patients, especially among preterm neonates, emphasise the importance of species level identification of coagulase-negative staphylococci and routinely determine the MIC of proper antibacterial agents for these isolates.

Molecular Analysis of the Simultaneous Production of Two SHV-Type Extended-Spectrum Beta-Lactamases in a Clinical Isolate of Enterobacter cloacae by Using Single-Nucleotide Polymorphism Genotyping

ABSTRACT Bacteria that simultaneously produce multiple extended-spectrum beta-lactamases are frequently isolated. We report an Enterobacter cloacae isolate, ES24, producing four different beta-lactamases (AmpC type beta-lactamase, TEM-1, SHV-7, and a novel extended-spectrum beta-lactamase, SHV-30). Direct sequencing of blaSHV gene products gave a “double peak” at position 703, suggesting the presence of more than one allele. Using fluorescence resonance energy transfer real-time PCR to detect single-nucleotide polymorphisms, we were able to distinguish two different blaSHV genes in a single isolate. This may prove to be a useful technique in surveys of beta-lactamase production in contemporary clinical isolates.

Clinical microbiology of early-onset and late-onset neonatal sepsis, particularly among preterm babies.

Prematurity has got special challenge for clinicians and also other medical staff, such as microbiologists. Immature host defense mechanisms support early-onset sepsis, which can be very serious with very high mortality. While the past decade has been marked by a significant decline in early-onset group B streptococcal (GBS) sepsis in both term and preterm neonates, the overall incidence of early-onset sepsis has not decreased in many centers, and several studies have found an increase in sepsis due to gram-negative organisms. With increasing survival of these more fastidious preterm infants, late-onset sepsis or specially nosocomial bloodstream infection (BSI) will continue to be a challenging complication that affects other morbidities, length of hospitalization, cost of care, and mortality rates. Especially the very low birthweight (VLBW) infants sensitive to serious systemic infection during their initial hospital stay. Sepsis caused by multiresistant organisms and Candida spp. are also increasing in incidence, has become the most common cause of death among preterm infants. This review focuses on the clinical microbiology of neonatal sepsis, particularly among preterm babies, summarizing the most frequent bacterial and fungal organisms causing perinatally acquired and also nosocomial sepsis.

First detection of plasmid-mediated, quinolone resistance determinants qnrA, qnrB, qnrS and aac(6′)-Ib-cr in extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in Budapest, Hungary

Sir, Quinolone resistance in Enterobacteriaceae usually results from mutations in genes coding for chromosomally encoded type II topoisomerases, efflux pumps or porin-related proteins. Recently, transferable, plasmid-borne, quinolone resistance genes—qnrA, qnrB, qnrS, cr variant of aac(60)-Ib, qepA and oxqB—have been observed in clinical isolates, more frequently among strains producing plasmid-mediated, extended-spectrum b-lactamases (ESBLs). The objective of this study was to determine the prevalence of qnrA, qnrB, qnrS and aac(60)-Ib-cr genes in ESBL-producing clinical isolates of 70 Escherichia coli, 101 Klebsiella pneumoniae, 5 Citrobacter freundii and 61 Enterobacter spp., isolated in two microbiological laboratories in Budapest by collecting samples from seven different hospitals and clinics from 2002 until 2006. The prevalence of ESBL production among Enterobacteriaceae was between 1% and 28% during this period. All of the ESBL-producing organisms were tested for the presence of qnrA, qnrB, qnrS and aac(60)-Ib genes by PCR. To distinguish the qnr gene alleles, restriction enzymes were used and the result was further confirmed by sequencing. The aac(60)-Ib-cr was identified by digestion with BstF5I. The prevalence of qnr determinants was relatively high among K. pneumoniae (8%) isolates and low in the case of E. coli (1.4%) and in Enterobacter spp. (0%). One ESBL-producing C. freundii isolate (out of five) was qnr-positive. qnrA1, qnrB2 and qnrS1 were detected and qnrA1 was the most prevalent (3%) (Table 1). Sixty-three (26.6%) of all ESBL-producing isolates were positive for aac(60)-Ib, of which 19 (8% of all)—16 K. pneumoniae and 3 E. coli—had the aac(60)-Ib-cr variant. None of the qnr-positive strains harboured the aac(60)-Ib-cr variant. The MICs of quinolones were determined by a broth microdilution method according to the CLSI. Conjugation assays were performed for all of the qnr-positive isolates. The qnrA1-positive strains—six K. pneumoniae and one E. coli— were resistant to nalidixic acid, norfloxacin, ciprofloxacin and levofloxacin with very high MIC values (Table 1). The conjugation assay, which was successful in three qnrA1-positive strains, confirmed that the presence of qnrA1 gene alone causes only low-level quinolone resistance. The MIC values for the transconjugants were more than 8-fold higher for nalidixic acid, 132-fold higher for norfloxacin and 66to 132-fold higher for ciprofloxacin, compared with E. coli J53 Rif (nalidixic acid, 4 mg/L; norfloxacin, 0.06 mg/L; and ciprofloxacin, 0.12 mg/L). However, the transconjugants still showed only low-level quinolone resistance, suggesting that the high-level quinolone resistance in the original isolates may be related to the coordinating action of several resistance mechanisms. The qnrB gene was detected in two isolates: one K. pneumoniae and one C. freundii. K. pneumoniae M95 harboured both qnrA1 and qnrB2 genes, but only qnrA1 was transferable by conjugation, suggesting that the two genes have different genetic backgrounds. The ciprofloxacin and levofloxacin MIC values were the highest in K. pneumoniae M95, compared with the other qnrA1-positive strains, which might be explained by the parallel qnrA1 and qnrB2 production of the original isolate. The transfer of qnrA1 alone did not result in transconjugants with greatly altered quinolone susceptibility. The C. freundii M141 harbouring only the qnrB2 gene was susceptible to the fluoroquinolones tested with MICs at least 8-fold lower than the susceptibility breakpoints recommended by the CLSI. Other authors have also observed that QnrB is present in isolates with a wide range of quinolone MIC values, including full susceptibility. The wide range of MIC values for the qnr-positive strains highlights that the detection of the qnr genes in clinical strains might be very difficult phenotypically. The qnrS1 gene was detected only in a K. pneumoniae strain, which was resistant to most of the tested fluoroquinolones. However, the norfloxacin (16 mg/L) and ciprofloxacin (4 mg/L) MIC values were the breakpoints recommended by the CLSI. The levofloxacin MIC value (4 mg/L) was between the two CLSI breakpoints (8 and 2 mg/L). Of the 19 aac(60)-Ib-cr-positive isolates, 6 (32%) were susceptible to both ciprofloxacin and levofloxacin. The presence of aac(60)-Ib-cr does not result in large increases in MIC values of quinolones; however, it substantially increases the frequency of selection of chromosomal mutants. The characteristics of the b-lactamase enzymes—isoelectric points, PCR and sequencing data—produced by the qnr-positive strains were determined. In our study, the presence of qnrA1 gene was connected to SHV-5, SHV-12 and CTX-M-15 enzymes (Table 1). The presence of qnrB2 was connected with SHV-12. We could not determine the genotype of the ESBL enzyme in the qnrB2-positive C. freundii strain. The qnrS1 gene was found together with SHV-2 ESBL. Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkn206

Set a thief to catch a thief: Self-reactive innate lymphocytes and self tolerance

Self-reactive lymphocytes form part of the peripheral repertoire in healthy individuals. Some of these cells are anergic classical lymphocytes, but a remarkable subset of self-reactive clones is related to innate immunity and many of them bear a partially activated phenotype. In the past few years growing evidence has pointed out the importance of this physiological autoimmunity in self tolerance, with special regard to the role of periportal innate lymphocytes. This population is involved in a wide range of immunoregulatory processes including immune privilege and oral tolerance, providing systemic tolerance to highly tissue-specific antigens as well as microbial epitopes cross-reactive to self. This kind of self-protection is dominantly mediated by self-reactive clones, which commonly play a dual role by acting as potent effectors and regulators at the same time. Here we provide an overview of the field.