Młynarczyk G

Molecular Characteristics of KPC-Producing Enterobacteriaceae at the Early Stage of Their Dissemination in Poland, 2008–2009

ABSTRACT After the first report in May 2008, the National Reference Center for Susceptibility Testing confirmed 113 cases of infection or colonization by KPC-producing members of the family Enterobacteriaceae in Poland by the end of 2009. The vast majority of patients were found in 18 hospitals; three patients were diagnosed at outpatient clinics. Most of the institutions were in the Warsaw area, including three hospitals with the highest numbers of cases. When available, the data on previous hospitalizations often indicated that these hospitals were the probable acquisition sites; one patient arrived from New York. The group of 119 unique isolates consisted of Klebsiella pneumoniae (n = 114), followed by Klebsiella oxytoca (n = 3), and Escherichia coli (n = 2). The K. pneumoniae isolates were dominated by the clone sequence type 258 (ST258) (n = 111); others were ST11 and ST23. The ST258 group was heterogeneous, with 28 pulsed-field gel electrophoresis (PFGE) subtypes, ∼25 plasmid profiles, and nine β-lactamase patterns differing by KPC variants (KPC-2 mainly), and SHV-12, CTX-M-3, and TEM-1-like enzymes. Plasmids carrying blaKPC genes varied in size (∼48 to 250 kb), structure, and conjugation potential. Transferable IncFIIK plasmids of ∼110 to 160 kb, probably pKpQIL or its derivatives, were observed in all K. pneumoniae clones and in K. oxytoca. Also prevalent were nontypeable pETKp50-like plasmids of ∼50 kb, found in K. pneumoniae ST258 and E. coli isolates (ST93 and ST224). Two K. pneumoniae-E. coli pairs from single patients might represent the in vivo transfer of such plasmids. The striking diversity of KPC producers at the early stage of dissemination could result from several introductions of these bacteria into the country, their multidirectional evolution during clonal spread, and transfer of the plasmids.

Antibiotic resistance in Gram-positive cocci

Gram-positive cocci still predominate as a cause of nosocomial- and community-acquired infections. These organisms frequently reveal a high natural, intrinsic resistance to antimicrobials. Additionally, these bacteria are able to acquire resistance to frequently used drugs rapidly through selective pressure of the environment and via the genetic evolution of bacteria. The wide application of antimicrobials in medical and veterinary practice, usage of antibiotics in agriculture and common usage of antiseptics and disinfectants result in selective pressure. The use of antibiotics directly selects resistant variants to different antibiotics or disinfectants. The same genetic element (e.g. qac or smr) conferring resistance to some disinfectants are often present on the same plasmid conferring resistance to antibiotics. Selection of resistant variants occurs most frequently in the hospital environment. Staphylococcus aureus and enterococci are the most commonly isolated bacteria causing nosocomial infections. Among those giving therapeutic problems are methicillin-resistant staphylococci and vancomycin-resistant enterococci. Resistance to high levels of aminoglycosides or penicillins among hospital enterococcal strains can completely abolish synergism of the drugs. In these cases glycopeptides will be the drugs of choice in the treatment of serious infections. Recently S. aureus strains with decreased susceptibility to vancomycin has appeared. A mechanism for this elevated resistance, although intensively investigated, still remains unknown.

Urinary tract infections in the early posttransplant period after kidney transplantation: etiologic agents and their susceptibility.

OBJECTIVE Urinary tract infection (UTI) is among the most common infections in solid organ transplantation, especially in kidney transplantation. PATIENTS AND METHODS This study included 295 adult patients undergoing KTx between September 2001 and December 2007. All patients were followed prospectively for UTI during the first 4 weeks after surgery. Samples of urine were investigated by bacteriological cultures to identify microorganisms in accord with standard procedures. Susceptibility testing was performed using Clinical and Laboratory Standards Institute procedures. RESULTS Urine specimens (n=582) were obtained from 84.5% of 245 recipients during the first month after transplantation. Among the isolated bacterial strains (n=291), the most common were Gram-negative bacteria (56.4%) predominantly Serratia marcescens (32.3%) and Enterobacter cloacae (14.6%). Extended- spectrum beta-lactamase (ESBL+) strains were isolated in 52.5% of cases. Gram-positive bacteria comprised 35.7%; most commonly, high-level aminoglycoside resistant (HLAR; 87.8%) and vancomycin-resistant (VRE; 11%) Enterococci. There were fungal strains in 23 cases (7.9%). CONCLUSION Our study showed predominantly Gram-negative rods from the Enterobacteriaceae family comprising (84.8%) of Gram-negative isolates: 52.5% ESBL and resistant enterococci (87.5%) in Gram-positive isolates. The increased proportion of isolates of multi-drug-resistant bacterial agents which can cause severe UTIs may be due to our frequent use of ceftriaxone for perioperative bacterial prophylaxis.

Clostridium difficile infection in Polish pediatric outpatients with inflammatory bowel disease

The prevalence of Clostridium difficile infection (CDI) in pediatric patients with inflammatory bowel disease (IBD) is still not sufficiently recognized. We assessed the prevalence of CDI and recurrences in outpatients with IBD. In addition, the influence of IBD therapy on CDI and antimicrobial susceptibility of the potentially causative C. difficile strains was assessed. This was a prospective, single-center, observational study. All specimens were obtained between January 2005 and January 2007 from the IBD outpatient service and screened for C. difficile and its toxins. C. difficile isolates were genotyped by PCR ribotyping. Diagnosis of Crohn’s disease (CD) and ulcerative colitis (UC) was based on Porto criteria. Severity of disease was assessed using the Hyams scale (for Crohn’s disease) and the Truelove–Witts scale (for ulcerative colitis). One hundred and forty-three fecal samples from 58 pediatric IBD patients (21 with Crohn’s disease and 37 with ulcerative colitis) were screened. The risk of C. difficile infection was 60% and was independent of disease type (CD or UC) (χ2 = 2.5821, df = 3, p = 0.4606). About 17% of pediatric IBD patients experienced a recurrence of CDI. All C. difficile strains were susceptible to metronidazole, vancomycin and rifampin. A high prevalence of C. difficile infection and recurrences in pediatric outpatients with IBD was observed, independent of disease type. There was no significant correlation between C. difficile infection and IBD therapy. PCR ribotyping revealed C. difficile re-infection and relapses during episodes of IBD in pediatric outpatients.

Epidemiological aspects of antibiotic resistance in respiratory pathogens.

Abstract Respiratory infections are the most frequent reason for primary health care consultation. The main causes of respiratory tract infections in children are viruses and the most common types are upper respiratory tract infections: common cold, pharyngitis, otitis media and sinusitis. Pneumonia is much more serious. As well as viruses, bacteria are often involved in respiratory tract infections. Three bacterial species are most commonly isolated: Streptococcus pneumoniae, non-encapsulated Haemophilus influenzae and Moraxella (Branhamella) catarrhalis. The most common bacterial cause of pharyngitis is Streptococcus pyogenes. Bacteria isolated from community-acquired infection usually are sensitive to the majority of suitable drugs, but during the past two decades, significant antibiotic resistance has emerged. Resistance to penicillins has spread among H. influenzae and S. pneumoniae. The mechanism of penicillin resistance in H. influenzae is mainly by production of β-lactamases TEM-1 and ROB-1, whereas in S. pneumoniae resistance is an effect of the changes in penicillin binding proteins. Among respiratory pathogens, resistance to tetracyclines, macrolides, trimethoprim–sulphamethoxazole and fluoroquinolones has also appeared. Several mechanisms depending on changes in target, active efflux and modifying enzymes are involved.

Hospital-based Clostridium difficile infection surveillance reveals high proportions of PCR ribotypes 027 and 176 in different areas of Poland, 2011 to 2013

As part of the European Clostridium difficile infections (CDI) surveillance Network (ECDIS-Net), which aims to build capacity for CDI surveillance in Europe, we constructed a new network of hospital-based laboratories in Poland. We performed a survey in 13 randomly selected hospital-laboratories in different sites of the country to determine their annual CDI incidence rates from 2011 to 2013. Information on C. difficile laboratory diagnostic testing and indications for testing was also collected. Moreover, for 2012 and 2013 respectively, participating hospital-laboratories sent all consecutive isolates from CDI patients between February and March to the Anaerobe Laboratory in Warsaw for further molecular characterisation, including the detection of toxin-encoding genes and polymerase chain reaction (PCR)-ribotyping. Within the network, the mean annual hospital CDI incidence rates were 6.1, 8.6 and 9.6 CDI per 10,000 patient-days in 2011, 2012, and 2013 respectively. Six of the 13 laboratories tested specimens only on the request of a physician, five tested samples of antibiotic-associated diarrhoea or samples from patients who developed diarrhoea more than two days after admission (nosocomial diarrhoea), while two tested all submitted diarrhoeal faecal samples. Most laboratories (9/13) used tests to detect glutamate dehydrogenase and toxin A/B either separately or in combination. In the two periods of molecular surveillance, a total of 166 strains were characterised. Of these, 159 were toxigenic and the majority belonged to two PCR-ribotypes: 027 (n=99; 62%) and the closely related ribotype 176 (n=22; 14%). The annual frequency of PCR-ribotype 027 was not significantly different during the surveillance periods (62.9% in 2012; 61.8% in 2013). Our results indicate that CDIs caused by PCR-ribotype 027 predominate in Polish hospitals participating in the surveillance, with the closely related 176 ribotype being the second most common agent of infection.

A comparison of Api 20A vs MALDI-TOF MS for routine identification of clinically significant anaerobic bacterial strains to the species level.

Adequate identification of anaerobic bacteria still presents a challenge for laboratories conducting microbiological diagnostics. The aim of this study was to compare the use of Api 20A and MALDI-TOF MS techniques for identification of obligate anaerobes. The results indicate that MALDI-TOF MS ensures a rapid and accurate identification of the species isolated from patients.

A Threat of the Klebsiella Pneumoniae Carbapenemase–Producing Strains Among Transplant Recipients

BACKGROUND Infections due to Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae are associated with increased therapeutic failure and mortality. Our laboratory recognized several strains producing KPC, most of which originated from transplantation ward patients. MATERIALS AND METHODS All strains of K pneumoniae resistant to at least 1 carbapenem isolated in 2010 were examined for KPC production by disc diffusion and then verified by molecular methods. RESULTS All positive strains originated from 7 patients. Six of them were from transplantation wards. None of the KPC-producing strains was isolated from the patients blood. CONCLUSIONS A quick, accurate diagnosis of KPC-producing strains enabled immediate isolation of carriers or infected persons. Isolation prevented spread of dangerous strains among immunocompromised patients and reduced the possibility of serious infections.

Bacterial and fungal infections in the early post-transplantation period after liver transplantation: etiologic agents and their susceptibility.

OBJECTIVE It has been reported in many studies that one of the main factors influencing morbidity and mortality in patients receiving transplants is infection after transplantation. PATIENTS AND METHODS The study included 190 adult patients undergoing orthotopic liver transplantation (OLT) between September 2001 and December 2007. All the patients were followed prospectively for infections from the OLT date and during the first 4 weeks after surgery. Immunosuppression consisted of steroids and tacrolimus. Antimicrobial prophylaxis included piperacillin/tazobactam, fluconazole, and selective bowel decontamination (SBD) was performed. Samples of clinical materials were investigated for microbiological cultures. The micro-organisms were cultured and identified in accordance with standard bacteriological procedures. Susceptibility testing was performed using Clinical and Laboratory Standards Institute procedures. RESULTS From 190 OLT recipients, 2213 clinical samples were obtained for microbiological examination. Positive cultures were found in 27.2% (n = 603) of all samples tested; 1252 strains were collected. Gram-positive bacteria were found in 64.1% (n = 802), Gram-negative bacteria were found in 31.6% (n = 396), and fungal strains were isolated in 4.3% (n = 54). Surgical site specimens (n = 1031) were obtained from 190 recipients during the first month after transplantation. Positive cultures accounted for 29.2% (n = 301) of all samples tested. Among the isolated microbial strains (n = 677), most common were Gram-positive bacteria (73.7%; n = 499). Gram-negative bacteria comprised 25.1% (n = 170). There were fungal strains in 1.2% (n = 8). There were 539 urine specimens. Positive cultures accounted for 16.7% (n = 90) of those. Among the isolated microbial strains (n = 210), most common were Gram-negative bacteria (62.4%; n = 131). Gram-positive bacteria comprised 28.6% (n = 60) and fungi 9% (n = 19). There were 549 blood specimens. Positive cultures were found in 30.6% (n = 168) of all samples tested. Among the isolated microbial strains (n = 263), most common were Gram-positive bacteria in 72.3% (n = 190); Gram-negative bacteria were found in 26.2% (n = 69), and fungal strains were isolated in 1.5% (n = 4). There were 69 respiratory tract specimens. Positive cultures were found in 46.4% (n = 32) of all samples tested. Among the isolated microbial strains (n = 84), most common were Gram-positive bacteria (51.2%; n = 43); Gram-negative bacteria comprised 27.4% (n = 23) and fungi 21.4% (n = 18). CONCLUSIONS (1) Surgical site samples were predominated samples after LTx. (2) Our study showed Gram-positive bacteria were 64.1% (n = 802), Gram-negative bacteria, 31.6% (n = 396) and fungal strains isolated in 4.3% (n = 54). (3) The increased proportion of isolates of multi-drug-resistant bacterial strains (methicillin resistant coagulase negative Staphylococcus, vancomycin-resistant Enterococcus, high-level aminoglycoside resistance, and extended- spectrum β-lactamase). (4) These data indicate strict cooperation infection control procedures in these patients.

Incidence, pattern and clinical relevance of microbial contamination of preservation fluid in liver transplantation

BACKGROUND Transmission of pathogens via preservation fluid (PF) is a potential cause of infection among liver transplant recipients. Here, we evaluated the incidence and pattern of microbial contamination of PF and its impact on postoperative graft function after liver transplantation. MATERIAL/METHODS This longitudinal study included data from 41 primary liver transplantations and 5 re-transplantations performed between December 2010 and September 2011. Results of microbiological analyses of 92 PF samples collected before and after the back-table procedure were evaluated in order to establish the incidence and pattern of contamination. The impact of positive PF cultures on early graft function and rate of pathogen transmission was assessed. Post-transplant antibiotic protocol was based on piperacillin/tazobactam administration for a minimum of 10 days. RESULTS The incidence of contamination was 84.8% (39/46), both for samples collected before and after the back-table procedure. Gram-positive low-virulence organisms typical for superficial saprophytic flora, mainly coagulase-negative staphylococci, were predominant. There were no cases of pathogen transmission from PF to the recipient. Positive cultures of PF samples obtained after the back-table procedure were associated with significant elevation of aspartate (p=0.034) and alanine aminotransferase (p=0.048) on the first 5 postoperative days. No significant differences were found regarding serum bilirubin concentration (p=0.335) and international normalized ratio (p=0.137). CONCLUSIONS Despite high incidence of PF contamination, infections caused by pathogens isolated from PF were not observed. However, presence of pathogens in PF might lead to temporary impairment of graft function.