Molecular epidemiology of carbapenem-resistant Enterobacteriaceae isolated from patients in COVID-19 wards and ICUs in a Bulgarian University Hospital.

Many studies report an increase in antimicrobial resistance of Gram - negative bacteria during the COVID-19 pandemic. Our aim was to evaluate the epidemiological relationship between carbapenem-resistant (CR) Enterobacteriaceae isolates from patients in COVID-19 wards and to investigate the main mechanisms of carbapenem resistance in these isolates during the period April 2020-July 2021. A total of 45 isolates were studied: Klebsiella pneumoniae (n = 37), Klebsiella oxytoca (n = 2), Enterobacter cloacae complex (n = 4) and Escherichia coli (n = 2). Multiplex PCR was used for detection of genes encoding carbapenemases from different classes (blaKPC, blaIMP, blaVIM, blaNDM, blaOXA-48). For epidemiological typing and analysis, ERIC PCR was performed. Two clinical isolates of E. cloacae, previously identified as representatives of two dominant hospital clones from the period 2014-2017, were included in the study for comparison. In the CR K. pneumoniae group, 23 (62.2%) carried blaKPC, 13 (35.1%) blaNDM, 10 (27.0%) blaVIM, and 9 (24.3%) were positive for both blaKPC and blaVIM. The blaKPC was identified also in the two isolates of K. oxytoca and blaVIM in all E. cloacae complex isolates. The two CR isolates of E. coli possessed blaKPC and blaOXA-48 genes. Epidemiological typing identified 18 ERIC profiles among K. pneumoniae, some presented as clusters of identical and/or closely related isolates. The carbapenem resistance in the studied collection of isolates is mediated mainly by blaKPC. During the COVID-19 pandemic intrahospital dissemination of CR K. pneumoniae, producing carbapenemases of different molecular classes, as well as continuing circulation of dominant hospital clones of multidrug-resistant E. cloacae complex was documented.

Phenotypic and Molecular Characteristics of Carbapenem-Resistant Acinetobacter baumannii Isolates from Bulgarian Intensive Care Unit Patients.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is designated as an urgent public health threat, both due to its remarkable multidrug resistance and propensity for clonal spread. This study aimed to explore the phenotypic and molecular characteristics of antimicrobial resistance in CRAB isolates (n = 73) from intensive care unit (ICU) patients in two university hospitals in Bulgaria (2018-2019). The methodology included antimicrobial susceptibility testing, PCR, whole-genome sequencing (WGS), and phylogenomic analysis. The resistance rates were as follows: imipenem, 100%; meropenem, 100%; amikacin, 98.6%; gentamicin, 89%; tobramycin, 86.3%; levofloxacin, 100%; trimethoprim-sulfamethoxazole, 75.3%; tigecycline, 86.3%; colistin, 0%; and ampicillin-sulbactam, 13.7%. All isolates harbored blaOXA-51-like genes. The frequencies of distribution of other antimicrobial resistance genes (ARGs) were: blaOXA-23-like, 98.6%; blaOXA-24/40-like, 2.7%; armA, 86.3%; and sul1, 75.3%. The WGS of selected extensively drug-resistant A. baumannii (XDR-AB) isolates (n = 3) revealed the presence of OXA-23 and OXA-66 carbapenem-hydrolyzing class D ß-lactamases in all isolates, and OXA-72 carbapenemase in one of them. Various insertion sequencies, such as ISAba24, ISAba31, ISAba125, ISVsa3, IS17, and IS6100, were also detected, providing increased ability for horizontal transfer of ARGs. The isolates belonged to the widespread high-risk sequence types ST2 (n = 2) and ST636 (n = 1) (Pasteur scheme). Our results show the presence of XDR-AB isolates, carrying a variety of ARGs, in Bulgarian ICU settings, which highlights the crucial need for nationwide surveillance, especially in the conditions of extensive antibiotic usage during COVID-19.

Dissemination of High-Risk Clones Enterobacterales among Bulgarian Fecal Carriage Isolates.

The gastrointestinal tract is an important reservoir of high-risk Enterobacteria clones and a driver of antimicrobial resistance in hospitals. In this study, patients from six hospitals in four major Bulgarian towns were included in this study. Overall, 205 cefotaxime-resistant isolates (35.3%) of Enterobacterales order were detected in fecal samples among 580 patients during the period of 2017-2019. ESBL/carbapenemase/plasmidic AmpC producer rates were 28.8%, 2.4%, and 1.2%, respectively. A wide variety of ESBLs: CTX-M-15 (41%), CTX-M-3 (24%), CTX-M-27 (11%), and CTX-M-14 (4%) was found. The carbapenemases identified in this study were New Delhi metalo-ß-lactamase (NDM)-1 (5.4%) and Klebsiella carbapenemase (KPC)-2 (1.5%). Most NDM-1 isolates also produced CTX-M-15/-3 and CMY-4 ß-lactamases. They belonged to ST11 Klebsiella pneumoniae clone. The epidemiology typing revealed three main high-risk K. pneumoniae clones (26%)-ST11, ST258, and ST15 and five main Escherichia coli clones-ST131 (41.7%), ST38, ST95, ST405, and ST69. Sixty-one percent of ST131 isolates were from the highly virulent epidemic clone O25b:H4-ST131. Phylotyping revealed that 69% of E. coli isolates belonged to the virulent B2 and D groups. Almost all (15/16) Enterobacter isolates were identified as E. hormaechei and the most common ST type was ST90. Among all of the isolates, a high ESBL/carbapenemases/plasmid AmpC (32.4%) prevalence was observed. A significant proportion of the isolates (37%) were members of high-risk clones including two pan-drug-resistant K. pneumoniae ST11 NDM-1 producing isolates. Due to extensive antibiotic usage during COVID-19, the situation may worsen, so routine screenings and strict infection control measures should be widely implemented.

First report of rhino-orbital-cerebral mucormycosis after COVID-19 infection in Bulgaria.

Mucormycosis is a relatively rare infection but with a high mortality rate due to the difficult and time-consuming diagnostic and therapeutic process. The authors present the first case of rhino-orbital-cerebral mucormycosis, histologically and microbiologically proven, in a patient after COVID-19 infection in Bulgaria.

Dealing with inconclusive SARS-CoV-2 PCR samples-Our experience.

PURPOSE: Early confirmation of SARS-CoV-2 is a key point in the timely management of infected patients and contact persons. Routine diagnostics of COVID-19 cases relies on RT-PCR detection of two or three unique sequences of the virus. A serious problem for the laboratories is how to interpret inconclusive samples which are positive for only one of the SARS-CoV-2 specific genes. MATERIALS AND METHODS: A total of 16364 naso-oropharyngeal swabs were collected and tested with SARS-CoV-2 Real-TM kit (Sacace Biotechnologies, Italy) between May and September 2020. We retrospectively analyzed their amplification plots to determine the number of inconclusive samples. We also reviewed the medical records to summarize the patient's COVID-19 testing history and basic demographic characteristics. RESULTS: We obtained 136 (0.8%) inconclusive samples with amplification signal only for the N-gene. Thirty-nine of the samples were excluded from further analysis as no additional data were available for them. Of the rest of the samples, the majority- 48% (95% CI 38-59%) had a previous history of SARS-CoV-2 positivity, 14% (95% CI 8-23%)-a subsequent history of positivity and 37% (95% CI 28-48%) were considered as false positive. CONCLUSION: A substantial proportion of the inconclusive results should be considered as positive samples at the beginning or the end of the infection. However, the number of false-positive results is also significant and each patient's result should be analyzed separately following the clinical symptoms and epidemiological data.

Dealing with inconclusive SARS-CoV-2 PCR samples—Our experience

Purpose Early confirmation of SARS-CoV-2 is a key point in the timely management of infected patients and contact persons. Routine diagnostics of COVID-19 cases relies on RT-PCR detection of two or three unique sequences of the virus. A serious problem for the laboratories is how to interpret inconclusive samples which are positive for only one of the SARS-CoV-2 specific genes. Materials and methods A total of 16364 naso-oropharyngeal swabs were collected and tested with SARS-CoV-2 Real-TM kit (Sacace Biotechnologies, Italy) between May and September 2020. We retrospectively analyzed their amplification plots to determine the number of inconclusive samples. We also reviewed the medical records to summarize the patient’s COVID-19 testing history and basic demographic characteristics. Results We obtained 136 (0.8%) inconclusive samples with amplification signal only for the N-gene. Thirty-nine of the samples were excluded from further analysis as no additional data were available for them. Of the rest of the samples, the majority– 48% (95% CI 38–59%) had a previous history of SARS-CoV-2 positivity, 14% (95% CI 8–23%)–a subsequent history of positivity and 37% (95% CI 28–48%) were considered as false positive. Conclusion A substantial proportion of the inconclusive results should be considered as positive samples at the beginning or the end of the infection. However, the number of false-positive results is also significant and each patient’s result should be analyzed separately following the clinical symptoms and epidemiological data.

Fecal Carriage and Epidemiology of Extended-Spectrum Beta-Lactamase/Carbapenemases Producing Enterobacterales Isolates in Bulgarian Hospitals.

The gastrointestinal tract is an important reservoir of extended spectrum beta-lactamase (ESBL)/carbapenemase-producing Enterobacterales isolates. This study included patients from two Bulgarian hospitals. Overall, 98 ESBL producers (including 68 Escherichia coli and 20 Klebsiella pneumoniae isolates) were detected among 99 hospitalized patients, 212 patients at admission, and 92 hospital staff in 42.4%, 24.5%, and 4%, respectively. We observed blaCTX-M-15 in 47% of isolates, blaCTX-M-3 in 39% and blaCTX-M-14 in 11%. Three blaCTX-M-15 positive isolates were also blaKPC-2 positive. High transferability was detected for blaCTX-M-3 carrying plasmids (55%) with L/M and I1 replicon plasmids, followed by CTX-M-14 (36.4%) and CTX-M-15 (27.9%) with IncF plasmids. BlaKPC-2 was carried by FIIAs plasmids. Epidemiology typing revealed 8 K. pneumoniae ST types-ST15(8/20), ST17(4/20), ST37(2/20) and 9 E. coli ST types-ST131 (30.9%, 21/68), ST38 (8/68), ST95(7/68) and ST316(7/68). All ST131 isolates but one was from the highly virulent epidemic clone O25bST131. This is the first report in Bulgaria about ESBL/carbapenemase faecal carriage. We observed high ESBL/carbapenemases prevalence. A predominant number of isolates were members of highly epidemic and virulent PanEuropean clones ST15 K. pneumoniae and O25bST131 E. coli. High antibiotics usage during the COVID pandemic will worsen the situation. Routine screenings and strict infection control measures should be widely implemented.