Analysis of Bacterial Pathogens Causing Complicating HAP in Patients with Secondary Peritonitis.

BACKGROUND: Diffuse peritonitis is an acute abdominal condition characterized by high mortality. The main treatment modality is surgery, requiring a subsequent prolonged hospital stay. These patients are, among other things, at risk of developing hospital-acquired pneumonia (HAP), which considerably worsens their treatment outcomes. This study aimed to extend the existing knowledge by providing more detailed microbiological characteristics of complicating HAP in patients with secondary peritonitis, including the identification of isolated bacterial pathogens and their potential sources. METHODS: The 2015-2019 retrospective study comprised all patients with an intraoperatively confirmed diagnosis of secondary diffuse peritonitis who were classified in accordance with the quick Sepsis Related Organ Failure Assessment scoring system. RESULTS: HAP developed in 15% of patients. The 90-day mortality rates were 53% and 24% in patients with and without HAP; respectively. The most frequent pathogens responsible for HAP were Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae complex and Enterococcus faecalis. Multidrug resistance to antibiotics was found in 38% of bacterial pathogens. Clonal spread of these bacterial pathogens among patients was not detected. Rather, the endogenous characteristic of HAP was confirmed. CONCLUSIONS: The initial antibiotic therapy of complicating HAP in patients with secondary peritonitis must be effective mainly against enterobacteria, including strains with the production of ESBL and AmpC beta-lactamases, Pseudomonas aeruginosa and Enterococcus faecalis. The study further highlighted the importance of monitoring the respiratory tract bacterial microflora in patients with secondary peritonitis. The results should be used for initial antibiotic treatment of complicating HAP instances.

Prevalence of Vancomycin-Resistant Enterococci and Antimicrobial Residues in Wastewater and Surface Water.

Due to the extensive use of antimicrobial agents in human and veterinary medicine, residues of various antimicrobials get into wastewater and, subsequently, surface water. On the one hand, a combination of processes in wastewater treatment plants aims to eliminate chemical and biological pollutants; on the other hand, this environment may create conditions suitable for the horizontal transfer of resistance genes and potential selection of antibiotic-resistant bacteria. Wastewater and surface water samples (Morava River) were analyzed to determine the concentrations of 10 antibiotics and identify those exceeding so-called predicted no-effect environmental concentrations (PNECs). This study revealed that residues of five of the tested antimicrobials, namely ampicillin, clindamycin, tetracycline, tigecycline and vancomycin, in wastewater samples exceeded the PNEC. Vancomycin concentrations were analyzed with respect to the detected strains of vancomycin-resistant enterococci (VRE), in which the presence of resistance genes, virulence factors and potential relationship were analyzed. VRE were detected in 16 wastewater samples (11%) and two surface water samples (6%). The PNEC of vancomycin was exceed in 16% of the samples. Since the detected VRE did not correlate with the vancomycin concentrations, no direct relationship was confirmed between the residues of this antimicrobials and the presence of the resistant strains.

Clostridioides difficile and Vancomycin-Resistant Enterococci in COVID-19 Patients with Severe Pneumonia.

Broad-spectrum antibiotics administered to patients with severe COVID-19 pneumonia pose a risk of infection caused by Clostridioides difficile. This risk is reduced mainly by strict hygiene measures and early de-escalation of antibiotic therapy. Recently, oral vancomycin prophylaxis (OVP) has also been discussed. This retrospective study aimed to assess the prevalence of C. difficile in critical COVID-19 patients staying in an intensive care unit of a tertiary hospital department of anesthesiology, resuscitation, and intensive care from November 2020 to May 2021 and the rates of vancomycin-resistant enterococci (VRE) after the introduction of OVP and to compare the data with those from controls in the pre-pandemic period (November 2018 to May 2019). During the COVID-19 pandemic, there was a significant increase in toxigenic C. difficile rates to 12.4% of patients, as compared with 1.6% in controls. The peak rates were noted in February 2021 (25% of patients), immediately followed by initiation of OVP, changes to hygiene precautions, and more rapid de-escalation of antibiotic therapy. Subsequently, toxigenic C. difficile detection rates started to fall. There was a nonsignificant increase in VRE detected in non-gastrointestinal tract samples to 8.9% in the COVID-19 group, as compared to 5.3% in the control group. Molecular analysis confirmed mainly clonal spread of VRE.

[Enterococcal infections and their treatment options].

AIM: The study aimed to characterize enterococcal infections at the University Hospital Olomouc and to define antibiotic treatment options. MATERIAL AND METHODS: The data was obtained from the ENVIS LIMS laboratory information system. Between 1 January 2015 and 31 December 2019, clinically relevant enterococci in the hospital and their resistance to antibiotics were retrospectively evaluated. Until mid-2016, criteria defined by Facklam and Collins and biochemical properties determined with the Encoccus test were used for identification. Subsequently, all enterococci were identified using the MALDI-TOF MS system. The susceptibility to antibiotics was determined using a standard microdilution method according to the EUCAST criteria. RESULTS: A total of 8 239 clinically relevant enterococci were isolated over the 5-year period. The most frequently isolated species were Enterococcus faecalis and Enterococcus faecium, which accounted for more than 90% in the period 2017-2019. Enterococci were most frequently isolated from urine (35 %), surgical wounds (17 %) and urethral/vaginal swabs (17 %). Clinically relevant enterococci were most commonly isolated from patients with oncological diagnoses (22%), those with urinary and genital diseases (15%) and respiratory diseases (9%). Enterococcus faecalis strains showed very low resistance to the antibiotics tested. Enterococcus faecium was shown to have 24 % proportion of vancomycin-resistant strains (VRE). CONCLUSION: Primary antibiotics suitable for treating infections with the etiological role of Enterococcus faecalis include aminopenicillins, in case of severe infections in combination with aminoglycosides, in particular gentamicin. For Enterococcus faecium strains, glycopeptides must be chosen. To treat VRE, linezolid or tigecycline are indicated.

Implementation of Antibiotic Stewardship in a University Hospital Setting.

The article describes activities of an antibiotic center at a university hospital in the Czech Republic and presents the results of antibiotic stewardship program implementation over a period of 10 years. It provides data on the development of resistance of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus to selected antibiotic agents as well as consumption data for various antibiotic classes. The genetic basis of resistance to beta-lactam antibiotics and its clonal spread were also assessed. The study showed significant correlations between aminoglycoside consumption and resistance of Escherichia coli and Klebsiella pneumoniae to gentamicin (r = 0.712, r = 0.869), fluoroquinolone consumption and resistance of Klebsiella pneumoniae to ciprofloxacin (r = 0.896), aminoglycoside consumption and resistance of Pseudomonas aeruginosa to amikacin (r = 0.716), as well as carbapenem consumption and resistance of Pseudomonas aeruginosa to meropenem (r = 0.855). Genotyping of ESBL- positive isolates of Klebsiella pneumoniae and Escherichia coli showed a predominance of CTX-M-type; in AmpC-positive strains, DHA, EBC and CIT enzymes prevailed. Of 19 meropenem-resistant strains of Klebsiella pneumoniae, two were identified as NDM-positive. Clonal spread of these strains was not detected. The results suggest that comprehensive antibiotic stewardship implementation in a healthcare facility may help to maintain the effectiveness of antibiotics against bacterial pathogens. Particularly beneficial is the work of clinical microbiologists who, among other things, approve administration of antibiotics to patients with bacterial infections and directly participate in their antibiotic therapy.

Analysis of Vancomycin-Resistant Enterococci in Hemato-Oncological Patients.

Enterococci are important bacterial pathogens, and their significance is even greater in the case of vancomycin-resistant enterococci (VRE). The study analyzed the presence of VRE in the gastrointestinal tract (GIT) of hemato-oncological patients. Active screening using selective agars yielded VRE for phenotypic and genotypic analyses. Isolated strains were identified with MALDI-TOF MS, (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) their susceptibility to antibiotics was tested, and resistance genes (vanA, vanB, vanC-1, vanC2-C3) and genes encoding virulence factors (asa1, gelE, cylA, esp, hyl) were detected. Pulsed-field gel electrophoresis was used to assess the relationship of the isolated strains. Over a period of three years, 103 VanA-type VRE were identified in 1405 hemato-oncological patients. The most frequently detected virulence factor was extracellular surface protein (84%), followed by hyaluronidase (40%). Unique restriction profiles were observed in 33% of strains; clonality was detected in 67% of isolates. The study found that 7% of hemato-oncological patients carried VRE in their GIT. In all cases, the species identified was Enterococcus faecium. No clone persisted for the entire 3-year study period. However, genetically different clusters were observed for shorter periods of time, no longer than eight months, with identical VRE spreading among patients.

Quinolone-resistant Escherichia coli in Poultry Farming.

Increasing bacterial resistance to quinolone antibiotics is apparent in both humans and animals. For humans, a potential source of resistant bacteria may be animals or their products entering the human food chain, for example poultry. Between July 2013 and September 2014, samples were collected and analyzed in the Moravian regions of the Czech Republic to isolate the bacterium Escherichia coli. As a result, 212 E. coli isolates were obtained comprising 126 environmental isolates from poultry houses and 86 isolates from cloacal swabs from market-weight turkeys. Subsequently, the E. coli isolates were tested for susceptibility to selected antibiotics. Resistance of the poultry isolates to quinolones ranged from 53% to 73%. Additionally, the presence of plasmid-mediated resistance genes was studied. The genes were confirmed in 58% of the tested strains. The data on resistance of isolates from poultry were compared with results of resistance tests in human isolates obtained in the same regions. The high levels of resistance determined by both phenotyping and genotyping methods and reported in the present study confirm the fact that the use of fluoroquinolones in poultry should be closely monitored.

Occurrence of bacteria producing broad-spectrum beta-lactamases and qnr genes in hospital and urban wastewater samples.

The aims were to investigate the level of antibiotic-resistant bacteria in hospital and urban wastewater and to determine the similarity of isolates obtained from wastewater and hospitalized patients. Wastewater samples were collected in September 2013 and 2014. After identification using MALDI-TOF MS, beta-lactamase production was determined by relevant phenotypic tests. Genes responsible for the production of single beta-lactamase groups and Qnr proteins were established. The epidemiological relationship of the isolates from wastewater and hospitalized patients was determined by PFGE. A total of 51 isolates of enterobacteria were obtained. Overall, 45.1% of them produced broad-spectrum beta-lactamases. Genes encoding TEM, SHV, CTX-M, CIT, DHA and EBC types of enzymes and Qnr proteins were detected. No broad-spectrum beta-lactamase production was confirmed in the urban wastewater treatment plant. The most important finding was the detection of two identical isolates of K. pneumoniae in 2013, one from a patient's urinary catheter and the other from a wastewater sample.

Individualized Prophylaxis in Patients with Esophageal Replacement Because of Cancer.

BACKGROUND: Presented are the findings from an analysis of respiratory inflammatory complications in patients undergoing esophageal replacement because of cancer. Respiratory complications are manifested most frequently within five post-operative days, thus, they are likely to be caused by potentially pathogenic micro-organisms originating from the primary or secondary microflora. METHODS: Sputum samples were collected 1-2 wks pre-operatively. In cases of positive microbiologic finding of some potential pathogens, individualized antibiotic prophylaxis was designed. RESULTS: Patients with individualized prophylaxis had fewer respiratory complications (10%) than patients with general antibiotic prophylaxis (41%). CONCLUSIONS: The approach to pre-operative sputum tests should be changed. If culture results are positive for bacteria with high resistance to antimicrobial agents or yeasts, the so-called individualized prophylaxis based on these particular results should be used.

Application of Molecular Diagnostics in Primary Detection of ESBL Directly from Clinical Specimens.

The infections caused by extended-spectrum ß-lactamase (ESBL)-producing organisms are associated with increased mortality. The real-time polymerase chain reaction (PCR) method, which enables detection of ESBLs directly from patients' clinical material, was developed. This study focused on blaCTX-M and blaSHV determination in endotracheal aspirates. Each sample was identified with standard microbiological procedures and simultaneously analyzed for the presence of nucleic acids, which encode CTX-M and SHV ESBL enzymes using real-time PCR. A total of 341 samples were investigated. In the set, 27 ESBL-positive samples were identified by phenotypic methods, while 60 positive samples were identified by the PCR method. Of the 60 PCR-positive samples, 58 were positive for the blaCTX-M. In two samples, the ESBL blaSHV-ESBL gene was detected. One phenotypically positive sample was PCR negative. The real-time PCR assay does not require a cultivation step and therefore enables detection of ESBL in 6 hours. The rapid method is necessary for early and adequate antimicrobial treatment.

The CRZ1/SP1-like gene links survival under limited aeration, cell integrity and biofilm formation in the pathogenic yeast Cryptococcus neoformans.

AIMS: Limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2-arrest in the obligate aerobic pathogenic yeast Cryptococcus neoformans. Also, the ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. The aim of this study was to identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. METHODS: Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The CNAG_00156.2 gene coding for a zinc-finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1-dependent (SP1-like) transcription factors. RESULTS: We have confirmed the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. CONCLUSIONS: Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.

[Bacterial efflux pumps - their role in antibiotic resistance and potential inhibitors]. / Efluxní pumpy bakterií - role v rezistenci na antibiotika a jejich mozné inhibitory.

Efflux pumps capable of actively draining antibiotic agents from bacterial cells may be considered one of potential mechanisms of the development of antimicrobial resistance. The most important group of efflux pumps capable of removing several types of antibiotics include RND (resistance - nodulation - division) pumps. These are three proteins that cross the bacterial cell wall, allowing direct expulsion of the agent out from the bacterial cell. The most investigated efflux pumps are the AcrAB-TolC system in Escherichia coli and the MexAB-OprM system in Pseudomonas aeruginosa. Moreover, efflux pumps are able to export other than antibacterial agents such as disinfectants, thus decreasing their effectiveness. One potential approach to inactivation of an efflux pump is to use the so-called efflux pump inhibitors (EPIs). Potential inhibitors tested in vitro involve, for example, phenylalanyl-arginyl-b-naphthylamide (PAbN), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or agents of the phenothiazine class.

Infected prosthetic dialysis arteriovenous grafts: a single dialysis center study.

BACKGROUND: The prosthetic arteriovenous grafts (AVG) being used increasingly to create hemodialysis access are prone to infections that pose potentially life-threatening infectious and bleeding complications, as well as loss of dialysis access. In this study, we identified the bacteriologic agents of infected AVGs by site swab, blood culture, and prosthesis cultures, and to evaluate the role of microbiological findings in the management of the infection. METHODS: We focused on 51 patients with 53 AVGs operated on in our clinic from January 2006 to December 2009. An infected AVG was identified by clinical, ultrasound, and microbiological findings. Sensitivity to antibiotics was determined for all bacterial strains. Isolates were identified by pulsed-field gel electrophoresis (PFGE) of bacterial DNA. In a few cases, positron emission tomography-computed tomography (PET-CT) examination was performed. RESULTS: Strains of Staphylococcus spp., especially S. aureus, were the most frequent cause of infected AVG. All S. aureus strains were sensitive to methicillin. With the exception of a single case, isolates obtained simultaneously from the skin site and the vascular prosthesis were identical genetically. CONCLUSIONS: Our results suggest that bacterial infectious agents detected in site swab, blood, or graft culture confirm a suspicion of AVG infection. A PET-CT examination can provide confirmation. The combination of microbiologic and radionuclide findings can improve the management of the AVG infection, but surgery remains essential.

Carriage of ESBL- and AmpC-positive Enterobacteriaceae in the gastrointestinal tract of community subjects and hospitalized patients in the Czech Republic.

AIM: The study aimed at analyzing ESBL- and AmpC-positive Enterobacteriaceae in the gastrointestinal tracts of university hospital inpatients and persons from the Olomouc Region community, and comparing the results with data from 2007. METHODS: Bacteria were isolated from rectal swabs inoculated onto the ChromID(TM) ESBL selective medium (bioMérieux). Production of ESBL-type beta-lactamases was confirmed by the modified double-disk synergy test and AmpC enzyme production was detected by the AmpC disk test. ESBL- and AmpC-positive isolates were subjected to basic genetic analysis aimed at detecting the bla(TEM), bla(SHV), bla(CTX-M) and bla(AmpC) genes. RESULTS: Over the study period (1 March 2010 - 1 May 2010), a total of 1,279 rectal swabs (70.4% of community subjects) were analyzed on the above medium. The prevalence rates of ESBL-positive Enterobacteriaceae were 8.2% in hospitalized patients and 3.2% in community subjects. Production of the AmpC enzyme was detected in 1.1% of bacterial isolates from the community and in one (0.3%) hospital isolate. Among ESBL, the most frequent genes encoding enzymes were from the CTX-M-1-like genes. Detected AmpC beta-lactamases belonged to the CIT, DHA and EBC groups. CONCLUSION: When compared with the year 2007, the rates of carriers of ESBL-positive bacteria increased in both hospitalized patients (from 3% to 8%) and community subjects (from 1% to 3%) in 2010. Given the fact that production of extended-spectrum beta-lactamases is clinically significant, knowing the epidemiological situation is very important for selecting adequate antibiotic therapy.

Antibiotic utilization and Pseudomonas aeruginosa resistance in intensive care units.

Pseudomonas aeruginosa is one of the most frequent and dangerous pathogens involved in the etiology of severe nosocomial infections. A retrospective observational study was conducted at all intensive care units of the University Hospital in Olomouc, Czech Republic (155 ICU beds). Complete antibiotic utilization data of the ICUs in the period of 1999 to 2008 were processed according to ATC/DDD system and expressed in defined daily doses per 100 bed-days (DBD). Utilization of meropenem, imipenem, ciprofloxacin, ofloxacin, pefloxacin, gentamicin, amikacin, ceftazidime, cefoperazone, cefoperazone/sulbactam and piperacillin/tazobactam was measured. Pseudomonas aeruginosa strains were isolated from clinical material obtained from patients hospitalized in ICUs. During the ten-year period, utilization of the entire group of antibiotics monitored grew. It increased from 23.52 DBD in 1999 to 27.48 DBD in 2008 with a peak of 33.04 DBD in 2007. P. aeruginosa accounted for as much as 42% of pneumonias and 23% of surgical wound infections. Our results show that P. aeruginosa strains became gradually resistant to all antibiotics used in the treatment of the infections caused by them, with the exception of amikacin and piperacillin/tazobactam.

Using newly developed multiplex polymerase chain reaction and melting curve analysis for detection and discrimination of ß-lactamases in Escherichia coli isolates from intensive care patients.

A total of 78 bacterial strains with known ß-lactamases were used to optimize a rapid detection system consisting of multiplex polymerase chain reaction and melting curve analysis to amplify and identify blaTEM, blaSHV, and blaCTX-M genes in a single reaction. Additionally, to evaluate the applicability of this method, 32 clinical isolates of Escherichia coli displaying an extended-spectrum ß-lactamase phenotype from patients hospitalized at intensive care units were tested. Results were analyzed by the Rotor-Gene operating software and Rotor-Gene ScreenClust HRM Software. The individual melting curves differed by a temperature shift or curve shape, according to the presence of ß-lactamase genes. With the use of this method and direct sequencing, blaCTX-M-15-like was identified as the most prevalent ß-lactamase gene. In conclusion, this novel detection system seems to be a suitable tool for rapid detection of present ß-lactamase genes and their characterization.

Phenotypic detection of broad-spectrum beta-lactamases in microbiological practice.

BACKGROUND: Enterobacteriaceae producing ESBL and AmpC enzymes can be associated with failure of antibiotic therapy and related morbidity and mortality. Their routine detection in microbiology laboratories is still a problem. The aim of this study was to compare the sensitivity of selected phenotypic methods. MATERIAL/METHODS: A total of 106 strains of the Enterobacteriaceae family were tested, in which molecular biology methods confirmed the presence of genes encoding ESBL or AmpC. In ESBL-positive strains, the sensitivity of the ESBL Etest (AB Biodisk) and a modified double-disk synergy test (DDST) were evaluated. AmpC strains were tested by a modified AmpC disk method using 3-aminophenylboronic acid. For simultaneous detection of ESBL and AmpC, the microdilution method with a modified set of antimicrobial agents was used. RESULTS: The sensitivity of the ESBL Etest was 95%; the modified DDST yielded 100% sensitivity for ESBL producers and the AmpC test correctly detected 95% of AmpC-positive strains. The sensitivity of the modified microdilution method was 87% and 95% for ESBL and AmpC beta lactamases, respectively. CONCLUSIONS: The detection of ESBL and AmpC beta lactamases should be based on specific phenotypic methods such as the modified DDST, ESBL Etest, AmpC disk test and the modified microdilution method.

Analysis of ESBL- and AmpC-positive Enterobacteriaceae at the Department of Neonatology, University Hospital Olomouc.

Bacterial infections are an important issue in current clinical medicine. The severity of infectious diseases has increased dramatically in recent years, which is also due to increasing numbers of resistant bacteria, including strains producing broad-spectrum beta-lactamases. The study aimed at determining the prevalence of ESBL- and AmpC-positive Enterobacteriaceae at the Department of Neonatology, University Hospital Olomouc. Enterobacteriaceae were isolated from clinical samples from infants hospitalized at the Department of Neonatology, University Hospital Olomouc over a period of 2 years. ESBL- and AmpC-positive isolates were subjected to basic genetic analysis. In the study period, a total of 1,526 isolates of the Enterobacteriaceae family were identified, including 55 (3.6%) cases of the ESBL phenotype and 17 (1.1%) AmpC-positive isolates. Genetic analysis of ESBL-positive isolates revealed a majority of CTX-M enzymes. Among AmpC beta-lactamases, the EBC, CIT, DHA, and MOX types were detected. An Escherichia coli strain was isolated with mutations in the promoter region of the ampC chromosomal gene that are associated with overproduction of the relevant enzyme.

[Prevalence of ESBL-positive enterobacteriaceae in large moravian hospitals (Czech Republic)]. / Prevalence ESBL-pozitivních enterobakterií ve velkých moravských nemocnicích.

BACKGROUND: bacterial infections have become an important issue in current medicine. Recently, their frequency and severity have significantly increased as a result of the rising number of resistant bacteria. One of important mechanisms of resistance is production of broad-spectrum beta-lactamases, namely the ESBL type. The study aimed at determining the frequency of ESBL-positive Enterobacteriaceae in three large hospitals in Moravia, the eastern part of the Czech Republic. MATERIAL AND METHODS: enterobacteriaceae were isolated from clinical material obtained from patients hospitalized in the University Hospital Olomouc, Teaching Hospital Ostrava and Bata Regional Hospital Zlín throughout 2009. Standard microbiology techniques were used for identification. The production of ESBLs was determined by the modified Double-Disk Synergy Test. ESBL-positive isolates of Escherichia coli from ICU patients were subjected to basic genetic analysis. RESULTS AND CONCLUSION: during the study period, a total of 12,922 strains from the Enterobacteriaceae family were detected. The ESBL phenotype was found in 907 cases, i.e. 7 % of all isolates. The most prevalent species of ESBL-producing Enterobacteriaceae were Klebsiella pneumoniae, Klebsiella oxytoca and Escherichia coli. A comparison of general wards and ICUs revealed a higher percentage of ESBL-positive strains of Klebsiella pneumoniae and a lower proportion of ESBL-positive Escherichia coli isolates in intensive care patients. When assessing the patients' clinical material, ESBL-producing strains were most frequently detected in urine. Genetic analysis of ESBL-positive Escherichia coli strains from ICU patients revealed the CTX-M type of ESBL production in most isolates.