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.

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).

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.

Occurrence of virulence factors in Cronobacter sakazakii and Cronobacter malonaticus originated from clinical samples.

BACKGROUND: Cronobacter spp. are Gram-negative, facultative-anaerobic, non-spore forming, enteric coliform bacteria, which belongs to the Enterobacteriaceae family. Cronobacter spp. are opportunistic pathogens that have brought rare but life-threatening infections such as meningitis, necrotizing enterocolitis and bloodstream infections in neonates and infants. Information on the diversity, pathogenicity and virulence of Cronobacter species obtained from various sources is still relatively scarce and fragmentary. The aim of this study was to examine and analyse different pathogenicity and virulence factors among C. sakazakii and C. malonaticus strains isolated from clinical samples. METHODS: The thirty-six clinical Cronobacter strains have been used in this study. This bacterial collection consists of 25 strains of C. sakazakii and 11 strains of C. malonaticus, isolated from different clinical materials. Seven genes (ompA, inv, sip, aut, hly, fliC, cpa) were amplified by PCR. Moreover, the motility and the ability of these strains to adhere and invade human colorectal adenocarcinoma (HT-29) and mouse neuroblastoma (N1E-115) cell lines were investigated. RESULTS: Our results showed that all tested strains were able to adhere to both used cell lines, HT-29 and N1E-115 cells. The invasion assay showed that 66.7% (24/36) of isolates were able to invade N1-E115 cells while 83% (30/36) of isolates were able to invade HT-29 cells. On the average, 68% of the C. sakazakii strains exhibited seven virulence factors and only 18% in C. malonaticus. All strains amplified ompA and fliC genes. The other genes were detected as follow: sip 97% (35/36), hlyA 92% (33/36), aut 94% (34/36), cpa 67% (24/36), and inv 69% (25/36). CONCLUSIONS: C. sakazakii and C malonaticus strains demonstrate the diversity of the virulence factors present among these pathogens. It is necessary to permanently monitor the hospital environment to appropriately treat and resolve cases associated with disease. Furthermore, in-depth knowledge is needed about the source and transmission vehicles of pathogens in hospitals to adopt pertinent prevention measures.

[Prevalence of strains of Staphylococcus epidermidis and other coagulase-negative staphylococci with biofilm-forming ability at a department of hemato-oncology].

OBJECTIVES: Staphylococcus epidermidis and coagulase-negative staphylococci generally are important causative agents of hospital-acquired infections. A significant role in this process is played by their common ability to form biofilm, a highly organized community of microorganisms adhering to inert surfaces. The study aimed to determine the prevalence of these bacterial strains and their ability to form biofilm at the Department of Hemato-Oncology, University Hospital Olomouc. MATERIAL AND METHODS: Over a period of 12 months, samples of air and swabs from surfaces and staff members were collected. The samples were subjected to standard microbiology tests; coagulase-negative staphylococci were identified. Staphylococcus epidermidis strains were confirmed by polymerase chain reaction and subsequently tested for biofilm formation. RESULTS AND CONCLUSIONS: Coagulase-negative staphylococci were found in 81 samples, most commonly swabs from staff members. S. epidermidis accounted for 60 % of all positive results; it was most frequently isolated from surface swabs. Almost half of S. epidermidis strains were able to form biofilm. These strains were found in the environment characterized by cleanliness classes FED-STD-209E (USA) - 10 000 and FED-STD-209E (USA) - 100 000. Thus, they pose a risk for immunocompromised patients staying there. Since coagulase-negative staphylococci were also found in healthcare staff of the department, the staff members may play a key role in the transmission of these microorganisms to patients.

[Bacterial pathogens of periprosthetic infections and diagnostic possibilities]. / Bakteriální pùvodci periprotetických infekcí a moznosti jejich diagnostiky.

BACKGROUND: Successful treatment of patients with periprosthetic infection (PPI) requires a correct diagnosis supported by clinical, imaging, microbiological and other laboratory evidence. Equally important is to determine the causative agent of the infection as this may affect the subsequent treatment strategy. The paper aims at presenting cultivation results in a group of PPI patients and their comparison with molecular biology methods. MATERIALS AND METHODS: Material obtained during operations of PPI patients was examined by both the standard culture methods and the PCR technique to identify the etiological agents. The results were statistically compared. RESULTS: In total, the group comprised 49 patients with hip, knee or elbow replacement infection verified during the operation. In 42 cases (85.7 %), the infectious agent was identified by cultivation. The infection was most frequently (62.0 %) caused by coagulase-negative staphylococci and Staphylococcus aureus. Monomicrobial and polymicrobial infections were demonstrated in 35 (71.4 %) and 7 (14.3 %) patients, respectively. The PCR assay aimed at the 16S rRNA segment of bacterial DNA was performed in 35 patients, with positive results in 25 cases (71.4 %). In 82.6 %, the agents detected by specific PCR were consistent with the cultivation results. A statistically non-significant difference in sensitivity between the two methods was found, with higher specificity in the case of PCR. CONCLUSION: Standard cultivation methods remain a highly successful and useful tool for identifying the PPI causative agents.

[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.