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.

Insights into the Resistome and Phylogenomics of a ST195 Multidrug-Resistant Acinetobacter baumannii Clinical Isolate from the Czech Republic.

Increasing antimicrobial resistance in nosocomial pathogens, such as Acinetobacter baumannii, is becoming a serious threat to public health. It is necessary to detect ß-lactamase-producing microorganisms in clinical settings to be able to control the spread of carbapenem resistance. This study was conducted to evaluate the presence of ß-lactamases in a selected clinical isolate of A. baumannii of ST2P/ST195Ox and to characterize possible enzymes, as well as its ß-lactam resistome, using PCR and whole-genome sequencing analysis. PCR and sequencing confirmed that the isolate harbored five bla gene alleles, namely, blaADC-73, blaTEM-1, blaOXA-23, blaOXA-58 and blaOXA-66, as well as aminoglycosides, macrolides, sulfonamides and tetracyclines resistance determinants, which were either chromosomally and/or plasmid located. Furthermore, a gene order comparison using MAUVE alignment showed multiple changes compared with the clinical isolate of Malaysian A. baumannii AC30 genome and 76 regions with high homology. This study suggests that resistance to ß-lactams in this A. baumannii isolate is mainly due to an overproduction of ß-lactamases in combination with other resistance mechanism (efflux pump system).

Detection of clinically important ß-lactamases by using PCR.

Increasing antimicrobial resistance of nosocomial pathogens is becoming a serious threat to public health. To control the spread of this resistance, it is necessary to detect ß-lactamase-producing organisms in the clinical setting. The aims of the study were to design a PCR assay for rapid detection of clinically encountered ß-lactamase genes described in Enterobacteriaceae and Gram-negative non-fermenting bacteria. The functionality of proposed primers was verified using eight reference strains and 17 strains from our collection, which contained 29 different ß-lactamase genes. PCR products of the test strains were confirmed by Sanger sequencing. Sequence analysis was performed using bioinformatics software Geneious. Overall, 67 pairs of primers for detecting 12 members of the class C ß-lactamase family, 15 members of class A ß-lactamases, six gene families of subclass B1, one member each of subclasses B2, B3 and class D ß-lactamases were designed, of which 43 pairs were experimentally tested in vitro. All 29 ß-lactamase genes, including 10 oxacillinase subgroups, were correctly identified by PCR. The proposed set of primers should be able to specifically detect 99.7% of analyzed ß-lactamase subtypes and more than 79.8% of all described ß-lactamase genes.

The molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in the Czech Republic.

OBJECTIVES: To gain data on the current molecular epidemiology and resistance of MRSA in the Czech Republic. METHODS: Between September 2017 and January 2018, a total of 441 single-patient MRSA isolates were collected from 11 Czech hospitals and analysed by spa typing, SCCmec typing, antibiotic susceptibility testing, detection of the PVL toxin and the arcA gene. RESULTS: Of all MRSA isolates, 81.41% (n = 359) belonged to the CC5-MRSA clone represented by the spa types t003 (n = 136), t586 (n = 92), t014 (n = 81), t002 (n = 20) and other spa types (n = 30); a majority of the CC5 isolates (n = 348, 96.94%) carried SCCmec type II. The occurrence of CC5-MRSA was more likely in older inpatients and associated with a healthcare origin (P < 0.001). The CC5-MRSA isolates were resistant to more antimicrobial drugs compared with the other MRSAs (P < 0.001). Interestingly, t586 was detected in blood samples more often than the other spa types and, contrary to other spa types belonging to CC5-MRSA, t586 was not associated with patients of advanced age. Other frequently found lineages were CC8 (n = 17), CC398 (n = 11) and CC59 (n = 10). The presence of the PVL was detected in 8.62% (n = 38) of the MRSA isolates. CONCLUSIONS: The healthcare-associated CC5-MRSA-II lineage (t003, t586, t014) was found to be predominant in the Czech Republic. t586 is a newly emerging spa type in the Czech Republic, yet reported rarely in other countries. Our observations stress the need for MRSA surveillance in the Czech Republic in order to monitor changes in MRSA epidemiology.

The association of a reduced susceptibility to moxifloxacin in causative Clostridium (Clostridioides) difficile strain with the clinical outcome of patients.

OBJECTIVES: To investigate the relationship between Clostridium (Clostridioides) difficile strain characteristics and C. difficile infection (CDI) outcome. METHODS: Between October and December 2017, 16 hospitals collected epidemiological data according to the European Centre for Disease Prevention and Control (ECDC) surveillance protocol for CDI. C. difficile isolates were characterized by ribotyping, toxin genes detection and antibiotic susceptibility testing to metronidazole, vancomycin and moxifloxacin. RESULTS: The overall mean CDI incidence density was 4.5 [95% CI 3.6-5.3] cases per 10,000 patient-days. From the 433 CDI cases, 330 (76.2%) were healthcare-associated, 52 (12.0%) cases were community-associated or of unknown origin and 51 (11.8%) CDI cases recurrent; a complicated course of CDI was reported in 65 cases (15.0%). Eighty-eight (20.3%) of patients died and 59 of them within 30 days after the CDI diagnosis. From the 379 C. difficile isolates, the most prevalent PCR ribotypes were 001 (n = 127, 33.5%) and 176 (n = 44, 11.6%). A total of 186 (49.1%) isolates showed a reduced susceptibility to moxifloxacin (> 4 mg/L) and 96.4% of them had Thr82Ile in the GyrA. Nineteen isolates revealed reduced susceptibility to metronidazole and two isolates to vancomycin (> 2 mg/L). A fatal outcome was associated with a reduced susceptibility to moxifloxacin, the advanced age of the patients and a complicated course of CDI (p<0.05). No association between ribotype, binary toxin and a reduced susceptibility to moxifloxacin and complicated course or recurrent CDI was found. CONCLUSIONS: A reduced susceptibility to moxifloxacin, in causative C. difficile strains was associated with fatal outcome of the patients, therefore it is an important marker in surveillance of CDI.

PCR Detection of Oxacillinases in Bacteria.

Oxacillinases (OXA) have been mostly described in Enterobacteriaceae, Acinetobacter, and Pseudomonas species. Recent years have witnessed an increased prevalence of intrinsic and/or acquired ß-lactamase-producing Acinetobacter in food-producing animals. This study was conducted to assess the prevalence of OXA among selected bacterial species and to characterize these enzymes by in silico analysis. Screening of OXA was performed by PCR amplification using specific pairs of oligonucleotides. Overall, 40 pairs of primers were designed, of which 6 were experimentally tested in vitro. Among 49 bacterial isolates examined, the presence of blaOXA-1-like genes was confirmed in 20 cases (41%; 19 times in Klebsiella pneumoniae and once in Enterobacter cloacae). No OXA were found in animal isolates. The study results confirmed the specificity of the designed oligonucleotide pairs. Furthermore, the designed primers were found to possess the ability to specifically detect 90.2% of all OXA. These facts suggest that the in silico and in vitro tested primers could be used for single or multiplex PCR to screen for the presence of OXA in various bacteria, as well as to monitor their spread. At the same time, the presence of conserved characteristic amino acids and motifs was confirmed by in silico analysis of sequences of representative members of OXA.

[Lethal course of complicated vascular prosthesis infections].

Presented are two cases of vascular prosthesis infections complicated by peritonitis with a lethal course. The authors describe complicated antibiotic therapy with findings, exhausted options for surgical therapy and subsequent decision that the condition was untreatable and palliative care was initiated.

[Clostridium difficile as a potential pathogen in preterm neonates]. / Clostridium difficile jako potenciální patogen u nezralých novorozencu.

OBJECTIVE: To detect the presence of Clostridium difficile toxins A and B in the stools of patients hospitalized in the University Hospital Olomouc who developed diarrhoea or other abdominal symptoms (abdominal pain, tympanites, indigestion, partial intestinal obstruction) related to antibiotic therapy. Given occasional dyspepsia and necrotizing enterocolitis in preterm neonates, to consider the potential role of these toxins, in addition to that of the immature intestine, in the development of the condition. To confirm the hypothesis by stool tests to detect the toxins also in normal neonates or those with no gastrointestinal symptoms. In selected stool samples, to compare detection of the toxins with C. difficile strains confirmed by culture.

Sources and pathways of spread of vancomycin-resistant enterococci in hemato-oncological patients.

The presented study aims at analyzing an increasing prevalence of vancomycin-resistant enterococci (VRE) isolated from various kinds of clinical material obtained from patients in the Department of Hemato-oncology (DHO), University Hospital in Olomouc, Czech Republic. Between January 1 and March 31, 2005, enterococci were isolated by standard microbiological procedures using both clinical material obtained from hospitalized patients and samples from the department environment. Resistance to vancomycin and teicoplanin was determined by a standardized microdilution method. Phenotype determination of resistance to vancomycin was verified by PCR detection of vanA and vanB genes. In VanA Enterococcus faecium, macrorestriction analysis was performed by pulsed-field gel electrophoresis. During the monitored period, a total of 128 Enterococcus sp. strains were isolated, of which 38 (30 %) isolates from 22 different patients were determined as VRE. Dominating were Enterococcus faecium VanA (63 %) and Enterococcus casseliflavus VanC (16 %) strains. At the same time, one Enterococcus faecium VanA strain was acquired from a bed-side table used by a patient in whom a similar strain had been isolated repeatedly from various clinical materials including a rectal swab taken in 2004. Based on the macrorestriction analysis of genome DNA in 24 vancomycin-resistant Enterococcus faecium VanA strains isolated from the patients' clinical material, one strain from the bed-side table surface and one strain isolated from stools in 2004, 8 unique restriction profiles with similarity ranging from 90 % to 100 % were identified, which could be classified into 3 clonal types. Thus, we can assume not only the endogenous origin of the VRE in hemato-oncological patients and their potential selection caused by therapy with broad-spectrum antibiotics but also the ability of the strains to survive in a hospital setting and, subsequently, to be spread clonally by various vectors.

Prevalence of vancomycin-resistant enterococci in hospitalized patients and those living in the community in the Czech Republic.

Between July 1, 2002 and December 31, 2003, rectal swabs from both hospitalized patients and community subjects in the Czech Republic were taken to ascertain the prevalence of vancomycin-resistant enterococci (VRE). The swabs were used for isolating and identifying enterococci and their susceptibility to antibiotics. Vancomycin resistance phenotypes were verified by PCR detection of vanA, vanB, vanC1 and vanC2 genes. A molecular biology analysis was performed in Enterococcus faecium VanA strains. During the observed period, 2691 rectal swabs from the hospitalized patients and 6529 rectal swabs from the subjects in community setting were examined. In total, 31 VRE of hospital origin and 13 community-population strains were isolated. The prevalence of VRE in the gastrointestinal tract was 1.9% in the hospitalized patients and 0.4% in the community subjects. The prevailing strains were Enterococcus faecium VanA (61.3%) in the VRE of hospital origin and Enterococcus gallinarum VanC (46.2%) in the community VRE. Mutual comparison between the hospital and community Enterococcus faecium VanA strains showed no similarity.

Occurrence of vancomycin-resistant enterococci in humans and animals in the Czech Republic between 2002 and 2004.

In the period between September 2002 and May 2004, a total of 6023 rectal swabs from humans in the Czech Republic were evaluated and 821 Enterococcus spp. strains were isolated. Nine strains (1.1 %) were identified as vancomycin-resistant enterococci (VRE). Two strains were VanA Enterococcus faecium, one strain was VanB Enterococcus faecalis and six strains were VanC Enterococcus casseliflavus. In total, 527 Enterococcus spp. strains were isolated from poultry breeds of which 11 (2.1 %) were VRE. Most (54.5 %) were identified as VanA E. faecium. Cluster analysis of SmaI-generated macrorestriction patterns showed high variability in both human and animal VRE strains and no relatedness between strains from the two sources.

[Prevalence of vancomycin-resistant enterococci in hospital and community environment.].

AIM OF THE STUDY: The presented study aimed at determining the prevalence of vancomycin-resistant enterococci (VRE) in rectal swabs taken from both patients in the Teaching Hospital in Olomouc (THO), Czech Republic, and subjects from the community setting of the hospital's catchment area. MATERIALS AND METHODS: Between July 1, 2002 and July 1, 2003, rectal swabs were taken from the THO patients as well as individuals from the community catchment area to be utilized for isolating and identifying enterococci and their suscetibility to antibiotics. Vancomycin resistance phenotypes were verified by PCR detection of vanA, vanB, vanC1 and vanC2 genes. A molecular biology analysis was performed in VanA Enterococcus faecium strains. To determine the relationship of strains, macrorestriction analysis of the total chromosomal DNA digested with SmaI restriction endonuclease was used. RESULTS: During the observed period, 2,157 rectal swabs from the hospitalized patients and 4,874 rectal swabs from the subjects in community setting were examined. In total, 27 VRE of hospital origin and 13 community-population strains were isolated. The prevalence of VRE in the gastrointestinal tract was 2.3 % in the hospitalized patients and 0.6 % in the community subjects. The prevailing strains were Enterococcus faecium VanA (70.4 %) in the VRE of hospital origin and Enterococcus gallinarum VanC (46.2 %) in the community VRE. Mutual comparison between the hospital and community VRE showed no similarity. CONCLUSION: In the Czech Republic, VRE were proved both in community and hospital settings. Their prevalence in rectal swabs is low and does not exceed the values reported in other European countries. The author describes the chief characteristics of the most important quinolone antibiotics, including preparations either in their development stage or whose development has been prematurely interrupted because of adverse side-effects. The list includes all preparations that are or were temporarily registered in the Czech Republic.

[Molecular biology characteristics of ESBL-positive strains of Klebsiella pneumoniae collected in the Neonatal Unit of the Teaching Hospital in Olomouc]. / Molekulárne-biologická analýza ESBL-pozitivních kmenu Klebsiella pneumoniae na novorozeneckém oddelení Fakultní nemocnice Olomouc.

BACKGROUND: One of the problems of contemporary medicine is an increasing number of bacterial strains with hazardous phenotypes of resistance. This is also true for neonatal units where nosocomial infections caused by multiresistant bacteria pose a serious threat to newborns. The feared bacterial pathogens include Klebsiella pneumoniae strains producing AmpA Extended-Spectrum Beta-Lactamases. The study focused on the molecular biology characteristics of ESBL-positive strains of K. pneumoniae collected in the Neonatal Unit of the Teaching Hospital in Olomouc (THO). MATERIALS AND METHODS: Clinical material from newborns hospitalized in the THO Neonatal Unit between January and June 2004 was used to isolate and determine K. pneumoniae strains by standard identification procedures. Their susceptibility to antibiotics was tested using a dilution micromethod. A Double-Disk Synergy Test was used for phenotype determination of ESBL production. The bla gene coding ESBL production was demonstrated by PCR. Molecular biology characteristics of ESBL-positive strains utilized the genomic DNA isolation, XbaI restrictase digestion and PFGE differentiation. The acquired restriction maps of individual isolates were compared using the GelCompare software and their relationship was determined. The selection pressure of antimicrobial agents was assessed according to the absolute number of defined daily doses of individual antibiotics. RESULTS: During the monitored period, 112 K. pneumoniae strains were isolated in total. In 22 of them (19.6%), the TEM-type ESBL production was determined. ESBL-positive strains were only observed in upper respiratory tract and rectal swabs collected from newborns with no signs of infection. The molecular biology analysis showed that 21 ESBL-positive strains had an identical restriction profile, i.e. they were very likely to be identical. The selection pressure of third- and fourth-generation cephalosporins was very low over the observed period and their consumption accounted for 1.9 % of all administered antimicrobial agents. CONCLUSION: The results presented above suggest that ESBL-positive strains of K. pneumoniae occurred in the THO Neonatal Unit due to clonal and horizontal spread from an unidentified source.

Molecular-biological analysis of vancomycin-resistant enterococci isolated from a community in the Czech Republic.

OBJECTIVE: To determine the occurrence of vancomycin-resistant enterococci (VRE) in a community of the Czech Republic and a molecular-biological analysis of the VRE isolated. METHODS: Enterococci were isolated from the rectal swabs of healthy people in the Olomouc region (population 300,000), Czech Republic in the period of January-December 2003. The molecular-biological analysis of VRE was performed by analysis of isolated DNA, which was cleaved by restriction enzyme SmaI and separated by pulse field gel electrophoresis (PFGE). RESULTS: A total number of 5,283 swabs were evaluated and 558 Enterococcus sp. strains were isolated during the follow-up period. 9 strains (1.6%) were identified as VRE. Two strains were E. faecium phenotype VanA, one strain was E. faecalis phenotype VanB, two strains were E. gallinarum phenotype VanC and four strains were E. casseliflavus phenotype VanC. PFGE was used to obtain 9 different restriction profiles of VRE strains. The analysis showed closer a similarity of E. casseliflavus strains (80-95%) than between E. faecium strains (41%). CONCLUSIONS: The presence of VRE in a sample community of the Czech population was confirmed. It is clear that it is necessary to take into account the possibility of VRE spreading from the community into health care facilities.