Bacterial Resistance to Antibiotics and Clonal Spread in COVID-19-Positive Patients on a Tertiary Hospital Intensive Care Unit, Czech Republic.

This observational retrospective study aimed to analyze whether/how the spectrum of bacterial pathogens and their resistance to antibiotics changed during the worst part of the COVID-19 pandemic (1 November 2020 to 30 April 2021) among intensive care patients in University Hospital Olomouc, Czech Republic, as compared with the pre-pandemic period (1 November 2018 to 30 April 2019). A total of 789 clinically important bacterial isolates from 189 patients were cultured during the pre-COVID-19 period. The most frequent etiologic agents causing nosocomial infections were strains of Klebsiella pneumoniae (17%), Pseudomonas aeruginosa (11%), Escherichia coli (10%), coagulase-negative staphylococci (9%), Burkholderia multivorans (8%), Enterococcus faecium (6%), Enterococcus faecalis (5%), Proteus mirabilis (5%) and Staphylococcus aureus (5%). Over the comparable COVID-19 period, a total of 1500 bacterial isolates from 372 SARS-CoV-2-positive patients were assessed. While the percentage of etiological agents causing nosocomial infections increased in Enterococcus faecium (from 6% to 19%, p < 0.0001), Klebsiella variicola (from 1% to 6%, p = 0.0004) and Serratia marcescens (from 1% to 8%, p < 0.0001), there were significant decreases in Escherichia coli (from 10% to 3%, p < 0.0001), Proteus mirabilis (from 5% to 2%, p = 0.004) and Staphylococcus aureus (from 5% to 2%, p = 0.004). The study demonstrated that the changes in bacterial resistance to antibiotics are ambiguous. An increase in the frequency of ESBL-positive strains of some species (Serratia marcescens and Enterobacter cloacae) was confirmed; on the other hand, resistance decreased (Escherichia coli, Acinetobacter baumannii) or the proportion of resistant strains remained unchanged over both periods (Klebsiella pneumoniae, Enterococcus faecium). Changes in pathogen distribution and resistance were caused partly due to antibiotic selection pressure (cefotaxime consumption increased significantly in the COVID-19 period), but mainly due to clonal spread of identical bacterial isolates from patient to patient, which was confirmed by the pulse field gel electrophoresis methodology. In addition to the above shown results, the importance of infection prevention and control in healthcare facilities is discussed, not only for dealing with SARS-CoV-2 but also for limiting the spread of bacteria.

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.

Bacterial Pathogens and Evaluation of a Cut-Off for Defining Early and Late Neonatal Infection.

Bacterial infections are an important cause of mortality and morbidity in newborns. The main risk factors include low birth weight and prematurity. The study identified the most common bacterial pathogens causing neonatal infections including their resistance to antibiotics in the Neonatal Department of the University Hospital Olomouc. Additionally, the cut-off for distinguishing early- from late-onset neonatal infections was assessed. The results of this study show that a cut-off value of 72 h after birth is more suitable. Only in case of early-onset infections arising within 72 h of birth, initial antibiotic therapy based on gentamicin with ampicillin or amoxicillin/clavulanic acid may be recommended. It has been established that with the 72-h cut-off, late-onset infections caused by bacteria more resistant to antibiotics may be detected more frequently, a finding that is absolutely crucial for antibiotic treatment strategy.

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.

Antibiotic Susceptibility of Cronobacter spp. Isolated from Clinical Samples.

Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for bla TEM, bla SHV, and bla CTX-M ß-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted.Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for bla TEM, bla SHV, and bla CTX-M ß-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted.

Antibiofilm activity of bioactive hop compounds humulone, lupulone and xanthohumol toward susceptible and resistant staphylococci.

Bacterial biofilms pose a serious medical problem due to their significant resistance to antimicrobials, and staphylococci are recognized as the most frequent cause of biofilm-associated infections. The hop plant (Humulus lupulus L.) contains substances that have been determined to act as anti-infective agents against bacteria, mainly in planktonic form. Therefore, we decided to investigate the antibiofilm properties of H. lupulus L.-derived compounds (humulone, lupulone and xanthohumol) against a selected group of Staphylococcus spp., including methicillin-susceptible and resistant strains. All tested hop compounds were shown to possess antimicrobial properties against all tested staphylococci, both planktonic and biofilm-dwelling, with no significant difference between resistant and susceptible strains. All compounds lowered the number of bacterial cells released from the biofilm, with the strongest effect seen for lupulone, followed by xanthohumol. Moreover, lupulone and xanthohumol were not only able to penetrate the biofilm and reduce the number of bacteria within it, but their higher concentrations (∼60 µg/mL for xanthohumol and ∼125 µg/mL for lupulone) reduced the number of surviving bacterial cells to zero.

Bacterial resistance to silver nanoparticles and how to overcome it.

Silver nanoparticles have already been successfully applied in various biomedical and antimicrobial technologies and products used in everyday life. Although bacterial resistance to antibiotics has been extensively discussed in the literature, the possible development of resistance to silver nanoparticles has not been fully explored. We report that the Gram-negative bacteria Escherichia coli 013, Pseudomonas aeruginosa CCM 3955 and E. coli CCM 3954 can develop resistance to silver nanoparticles after repeated exposure. The resistance stems from the production of the adhesive flagellum protein flagellin, which triggers the aggregation of the nanoparticles. This resistance evolves without any genetic changes; only phenotypic change is needed to reduce the nanoparticles' colloidal stability and thus eliminate their antibacterial activity. The resistance mechanism cannot be overcome by additional stabilization of silver nanoparticles using surfactants or polymers. It is, however, strongly suppressed by inhibiting flagellin production with pomegranate rind extract.

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.

Primer Evaluation for PCR and its Application for Detection of Carbapenemases in Enterobacteriaceae.

BACKGROUND: During the last decade, the prevalence of carbapenem-resistant Enterobacteriaceae in human patients has increased. Carbapenemase-producing bacteria are usually multidrug resistant. Therefore, early recognition of carbapenemase producers is critical to prevent their spread. OBJECTIVES: The objective of this study was to develop the primers for single and/or multiplex PCR amplification assays for simultaneous identification of class A, class B, and class D carbapenem hydrolyzing ß-lactamases in Enterobacteriaceae and then to evaluate their efficiency. MATERIALS AND METHODS: The reference sequences of all genes encoding carbapenemases were downloaded from GenBank. Primers were designed to amplify the following 11 genes: bla KPC, bla OXA, bla VIM, bla NDM, bla IMP, bla SME, bla IMI, bla GES , bla GIM, bla DIM and bla CMY . PCR conditions were tested to amplify fragments of different sizes. Two multiplex PCR sets were created for the detection of clinically important carbapenemases. The third set of primers was included for detection of all known carbapenemases in Enterobacteriaceae. They were evaluated using six reference strains and nine clinical isolates. RESULTS: Using optimized conditions, all carbapenemase-positive controls yielded predicted amplicon sizes and confirmed the specificity of the primers in single and multiplex PCR. CONCLUSIONS: We have reported here a reliable method, composed of single and multiplex PCR assays, for screening all clinically known carbapenemases. Primers tested in silico and in vitro may distinguish carbapenem-resistant Enterobacteriaceae and could assist in combating the spread of carbapenem resistance in Enterobacteriaceae.

Silver nanoparticles strongly enhance and restore bactericidal activity of inactive antibiotics against multiresistant Enterobacteriaceae.

Bacterial resistance to conventional antibiotics is currently one of the most important healthcare issues, and has serious negative impacts on medical practice. This study presents a potential solution to this problem, using the strong synergistic effects of antibiotics combined with silver nanoparticles (NPs). Silver NPs inhibit bacterial growth via a multilevel mode of antibacterial action at concentrations ranging from a few ppm to tens of ppm. Silver NPs strongly enhanced antibacterial activity against multiresistant, ß-lactamase and carbapenemase-producing Enterobacteriaceae when combined with the following antibiotics: cefotaxime, ceftazidime, meropenem, ciprofloxacin and gentamicin. All the antibiotics, when combined with silver NPs, showed enhanced antibacterial activity at concentrations far below the minimum inhibitory concentrations (tenths to hundredths of one ppm) of individual antibiotics and silver NPs. The enhanced activity of antibiotics combined with silver NPs, especially meropenem, was weaker against non-resistant bacteria than against resistant bacteria. The double disk synergy test showed that bacteria produced no ß-lactamase when treated with antibiotics combined with silver NPs. Low silver concentrations were required for effective enhancement of antibacterial activity against multiresistant bacteria. These low silver concentrations showed no cytotoxic effect towards mammalian cells, an important feature for potential medical applications.

Characteristics of Quinolone Resistance in Escherichia coli Isolates from Humans, Animals, and the Environment in the Czech Republic.

Escherichia coli is a common commensal bacterial species of humans and animals that may become a troublesome pathogen causing serious diseases. The aim of this study was to characterize the quinolone resistance phenotypes and genotypes in E. coli isolates of different origin from one area of the Czech Republic. E. coli isolates were obtained from hospitalized patients and outpatients, chicken farms, retailed turkeys, rooks wintering in the area, and wastewaters. Susceptibility of the isolates grown on the MacConkey agar with ciprofloxacin (0.05 mg/L) to 23 antimicrobial agents was determined. The presence of plasmid-mediated quinolone resistance (PMQR) and ESBL genes was tested by PCR and sequencing. Specific mutations in gyrA, gyrB, parC, and parE were also examined. Multilocus sequence typing and pulsed-field gel electrophoresis were performed to assess the clonal relationship. In total, 1050 E. coli isolates were obtained, including 303 isolates from humans, 156 from chickens, 105 from turkeys, 114 from the rooks, and 372 from wastewater samples. PMQR genes were detected in 262 (25%) isolates. The highest occurrence was observed in isolates from retailed turkey (49% of the isolates were positive) and inpatients (32%). The qnrS1 gene was the most common PMQR determinant identified in 146 (56%) followed by aac(6')-Ib-cr in 77 (29%), qnrB19 in 41 (16%), and qnrB1 in 9 (3%) isolates. All isolates with high level of ciprofloxacin resistance (>32 mg/L) carried double or triple mutations in gyrA combined with single or double mutations in parC. The most frequently identified substitutions were Ser(83)Leu; Asp(87)Asn in GyrA, together with Ser(80)Ile, or Glu(84)Val in ParC. Majority of these isolates showed resistance to beta-lactams and multiresistance phenotype was found in 95% isolates. Forty-eight different sequence types among 144 isolates analyzed were found, including five major clones ST131 (26), ST355 (19), ST48 (13), ST95 (10), and ST10 (5). No isolates sharing 100% relatedness and originating from different areas were identified. In conclusion, our study identified PMQR genes in E. coli isolates in all areas studied, including highly virulent multiresistant clones such as ST131 producing CTX-M-15 beta-lactamases.

[Validity comparison of various biological samples from lower airway and their contribution for the detection of nosocomial pneumonia etiological agents]. / Porovnání validity ruzných vzorku biologického material z dýchacích cest a jejich prínosu v detekci puvodcu nozokomiální pneumonie.

BACKGROUND: The aim of the study was to compare the validity of bronchial secretion sampling and bronchoscopy-assisted protected specimen brushing (PSB) in patients with hospital-acquired pneumonia (HAP). MATERIALS AND METHODS: In patients with HAP, bronchial secretion samples (aspiration of lower airway secretions from an orotracheal tube with a suctioning catheter) and PSB (bronchoscopy-assisted sampling from the most affected area of the lung, verified by CT scan) were taken at the same time. Both samples were processed by semiquantitative routine microbiological techniques. Identification of microorganisms was performed by standard microbiological techniques using the MALDI-TOF automated system. For similarity or identity determination of bacterial isolates from bronchial secretion sampling and PSB, pulsed-field gel electrophoresis was used. RESULTS: Thirty patients were enrolled into the study. Thirty pairs of bronchial secretion samples and PSB samples were obtained and processed. The samples were positive in 23 patients (77 %) and 15 patients (50 %), respectively. In 15 cases, the same pathogen was determined in both samples, and in all those cases, the isolates were genetically identical. CONCLUSION: The results of the study show that bronchial secretion samples analysis enabled identification of all pathogens that were identified by PSB. Given the high sensitivity of the bronchial secretion aspiration technique and genetic identity of isolates in both samples, bronchial secretion sampling may be recommended for determining HAP etiological agents as the samples are much easier to obtain from patients.

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.

Incidence of fecal Enterobacteriaceae producing broad-spectrum beta-lactamases in patients with hematological malignancies.

AIM: Given the steadily increasing numbers of resistant bacteria, the frequency and severity of infections are on the rise. In patients with hematological malignancies, the treatment itself increases the risk of complicating bacterial infections. One important mechanisms of resistance is production of broad-spectrum beta-lactamases, increasingly detected not only in bacterial pathogens but also in bacteria contained in the normal microflora of the human body. The objectives of this study were determination and analysis of the prevalence of multiresistant ESBL- and AmpC-positive Enterobacteriaceae in the gastrointestinal tract (GIT) of patients with hematological malignancies. METHODS: For 3 months, rectal swabs were taken from patients with hematological malignancies and analyzed using chromogenic screening plates to isolate ESBL- and AmpC-producing Enterobacteriaceae. Beta-lactamase production was determined by phenotype tests and confirmed by detecting genes encoding ESBL and AmpC types. At the same time, ESBL- and AmpC-positive Enterobacteriaceae were isolated from clinical samples collected from patients with bacterial infection. RESULTS: Over the study period, fifteen patients (21%) of all patients treated at the Department of Hemato-Oncology were shown to have ESBL- or AmpC-positive Enterobacteriaceae in their GIT. Most frequently identified were ESBL-positive strains of Klebsiella pneumoniae and AmpC-positive strains of Citrobacter freundii. The ESBL enzymes were mainly of the CTX-M type. Isolates producing AmpC were found to contain genes for enzymes mainly from the CIT and DHA groups. CONCLUSION: The study identified patients diagnosed with urinary tract and bloodstream infections caused by ESBL-positive strain of Klebsiella pneumoniae and AmpC-positive strain of Enterobacter cloacae contained in the GIT microflora.

[New technologies for antibiotic susceptibility testing of bacteria].

The past two decades have witnessed increasing infections due to multidrug-resistant bacteria. Therefore, transmission of these pathogens could limit the antibiotic therapy options. Many reports suggest that initiation of appropriate antimicrobial therapy can be lifesaving. Physicians rely on combination of clinical, epidemiological and demographic data to guide empirical therapy because results of culture and antimicrobial susceptibility testing may require 48 hours or longer. Therefore, an ongoing effort for the development of earlier and more sensitive detection of resistant bacteria is inevitable. This review presents a summary of the most advanced methods (e.g. PCR-based techniques, flow cytometry, mass spectrometry, microarrays and others) that are able to rapidly detect antibiotic resistance in bacterial pathogens which have the potential to become valuable alternatives to the existing methods (standard phenotypic resistance testing) in the very near future.