Development of Nested PCR for SARS-CoV-2 Detection and Its Application for Diagnosis of Active Infection in Cats.

SARS-CoV-2 emerged in 2019 and found diagnostic laboratories unprepared worldwide. To meet the need for timely and accurate virus detection, laboratories used rapid Ag tests and PCR kits based on costly multi-channel real-time techniques. This study aimed to develop a conventional nested PCR based on the SARS-CoV-2 N gene, validate it against some approved assays, and apply it to samples from six cats with respiratory symptoms obtained in early 2020 during the first COVID-19 wave in humans in Bulgaria. The nested PCR technique showed 100% sensitivity and specificity; it could detect extracted SARS-CoV-2 RNA at concentrations as low as 0.015 ng/µL. The results identified the six tested cat samples as positive. Sequence analysis performed in two of them confirmed this. The presented technique is reliable, easy to implement and inexpensive, and can be successful in strategies for the prevention and control of SARS-CoV-2 in humans, cats and other susceptible species.

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.

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.

Multicentre investigation of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in Bulgarian hospitals - Interregional spread of ST11 NDM-1-producing K. pneumoniae.

AIM: The aim of this study was to investigate the mechanisms of beta-lactam-resistance and the clonal relatedness of carbapenem-nonsusceptible Klebsiella pneumoniae and Escherichia coli isolates, collected consecutively in eight centers in five Bulgarian cities from November 2014 to March 2018. Carbapenemase-producing enterobacteria were detected in all but one centers. Overall, 104 K. pneumoniae and one E. coli were analysed. MATERIALS AND METHODS: Antimicrobial susceptibility and beta-lactamases were analysed. Conjugation experiments, plasmid fingerprinting and replicon typing, as well as MLST and ERIC-PCR were carried out. RESULTS: KPC-2 (51%) and NDM-1 (47%) were the main carbapenemases identified. KPC-2 producing K. pneumoniae were classified into 10 MLST-types. The four dominating MLST-types ST29, ST15, ST336 and ST902 comprised 79% of the KPC-2 producers. All but one of the NDM-1 producing isolates belonged to the MLST-type ST11 and were found in seven centers. Furthermore, single K. pneumoniae isolates producing VIM-1 (ST147) and OXA-48 (ST15) were identified. In addition to the carbapenemases, the ESBLs CTX-M-15, CTX-M-3, and SHV-12 as well as AmpC enzyme CMY-4 were found. The FIIAs-replicon-type was found in all KPC-2 producers while the A/C-replicons dominated in NDM-1 producing isolates. The single NDM-1 producing E. coli was determined as MLST-Type ST10 (Warwick scheme). CONCLUSION: The interregional clonal expansion of NDM-1 producing ST11 K. pneumoniae and the dissemination of blaKPC-2 carrying plasmids were responsible for the spread of carbapenemase-producing K. pneumoniae in Bulgaria. Our findings highlight the urgency to prevent dissemination of these highly transmissible and dangerous lineages.

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in Bulgarian hospitals.

The aim of the study was to describe the emergence, the spread, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria. Over eight years (1996-2003), 442 ESBL-screen-positive isolates were collected in nine medical institutions in four Bulgarian towns. Class A ESBLs of the SHV, TEM, and CTX-M groups were identified in seven species. SHV-type enzymes persisted during the whole study period, TEM-ESBLs appeared first in 1999, and CTX-M-types appeared first in 2001. The rate of CTX-M enzyme producers increased rapidly between 2001 and 2003, while the rate of SHV producers decreased. Six different ESBL-types were identified, namely, SHV-2, -5, and -12, CTX-M-3 and -15, and a new TEM-3-like variant (TEM-139). The most widespread enzymes were SHV-12, CTX-M-15, and CTX-M-3 found in seven centers. TEM-139 was identified mainly in one center. A trend for strains harboring more than one ESBL gene, for example, CTX-M + SHV, was observed since 2002. Plasmid fingerprinting and random amplified polymorphic DNA analysis typing revealed wide dissemination of identical plasmids among different bacterial species and hospitals, as well as clonal spread of ESBL producers. Our data contribute to clarify the dynamics in the prevalence of ESBLs in Bulgaria and demonstrate the importance of molecular procedures for their analysis.

Dissemination of a Multidrug-Resistant VIM-1- and CMY-99-Producing Proteus mirabilis Clone in Bulgaria.

The aim of this study was to analyze the beta-lactamases and the molecular epidemiology of 19 clinically significant isolates of Proteus mirabilis with decreased susceptibility to imipenem, which have been collected from seven hospitals, located in different Bulgarian towns (Sofia, Varna, and Pleven). The isolates were obtained from blood, urine, tracheal and wound specimens. One additional isolate from hospital environment was included. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta-lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and PFGE. Integron mapping was performed by PCR and sequencing. All isolates showed a multidrug-resistance profile, but remained susceptible to piperacillin/tazobactam, cefepime, meropenem, and fosfomycin. They produced identical beta-lactamases, namely: TEM-1, VIM-1, and CMY-99. PCR mapping revealed that the blaVIM-1 gene was part of a class 1 integron that additionally included the aac(6')-I, dhfrA1, and ant(3″)-Ia genes. In addition, 17 of the isolates carried the armA gene. Conjugation experiments and plasmid replicon typing were unsuccessful. The isolates were clonally related according to RAPD and PFGE typing. This study reveals the nationwide distribution of a multidrug-resistant P. mirabilis clone producing VIM-1 and CMY-99 along with the presence of different aminoglycoside resistance mechanisms.

Dissemination of successful international clone ST15 and clonal complex 17 among Bulgarian CTX-M-15 producing K. pneumoniae isolates.

A total of 82 extended spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae and 4 Klebsiella oxytoca isolates were collected in 2014 from four geographical areas in Bulgaria and their multilocus sequence type (MLST) and transferability of the ESBL encoding genes were investigated. The predominant type was CTX-M-15 (87%), followed by CTX-M-3 (9%), SHV-12 or SHV-2 (2%) and CTX-M-14 (1%). The CTX-M-15 producers belonged to ST15 (34.1%) and to a lesser extent to CC17 (ST16, ST17, ST336). The CTX-M-15 transconjugants showed a presence of R, A/C2 and F replicons. The CTX-M-3 producers were assigned to ST29, ST70, ST432, ST542 and ST15 types and the transconjugants carried M2 replicons. To the best of our knowledge, this is the first report that fully describes the MLST types among Bulgarian ESBL producing K. pneumoniae and the first report of the detection of IncR plasmid replicon type in our country.

Predominance of IncL/M and IncF plasmid types among CTX-M-ESBL-producing Escherichia coli and Klebsiella pneumoniae in Bulgarian hospitals.

Our objective was to investigate the plasmid replicon-types involved in spread of ESBLs among Bulgarian Klebsiella pneumoniae and Escherichia coli. Sixty-three isolates, with transferable beta-lactam resistance determinants, collected between 2007 and 2009 in six medical institutions, were analysed with respect to their antimicrobial susceptibility, ESBL-, RAPD-, and plasmid replicon-type. Phylogenetic typing and screening for the O25b-ST131 lineage were carried out for E. coli. The predominant ESBLs were CTX-M-15 (81%) among E. coli and CTX-M-3 (58%) among K. pneumoniae. Other sporadically found ESBLs were SHV-12 and TEM-139, and for the first time in Bulgaria, CTX-M-1 and CTX-M-14. Replicon typing revealed that plasmids carrying blaCTX-M-3 exclusively belonged to IncL/M-type, while blaCTX-M-15 was predominantly (94%) associated with IncF-type plasmids. Among E. coli, 59% of the isolates were clonally related. Isolates of that cluster produced CTX-M-15, belonged to the O25b-ST131 lineage, predominantly harboured plasmids with the FIA replicon, and were found in five centres. Among CTX-M-3-producing K. pneumoniae, two prevailing RAPD-types were found, one remained restricted to one centre and the second was found in three centres. The incompatibility groups IncN and IncA/C linked with blaSHV-12 respectively blaTEM-139 were found only once. To the best of our knowledge, this is the first detailed investigation of plasmids carrying ESBL genes among Bulgarian isolates demonstrating wide distribution of conjugative IncF plasmids among CTX-M-15-producing E. coli and IncL/M plasmids among CTX-M-3 positive K. pneumoniae isolates.

High prevalence of CTX-M-15-producing O25b-ST131 Escherichia coli clone in Bulgarian hospitals.

According to the European Antimicrobial Resistance Surveillance System project results, Bulgaria has become one of the European countries with dramatically increasing rates of extended-spectrum beta-lactamase (ESBL) producers. The aim of this work was to investigate the epidemiology of ESBL-producing Escherichia coli clinical isolates in Bulgaria, collected from seven clinical centers in three towns, during two study periods: 2002-2003 and 2006-2009. For 193 ESBL-producing E. coli isolates random amplified polymorphic DNA (RAPD) analyses, phylogenetic typing, and screening for O25b-ST131 isolates were carried out. Antimicrobial susceptibility, ESBL-type and transferability of resistance determinants were analyzed. Four different ESBL-types, namely TEM-139, SHV-12, CTX-M-3, and CTX-M-15 were found. CTX-M-15 dominated, being found in 88% of the isolates. RAPD-typing revealed 35 types, among which type A dominated, comprising 65% of the isolates. Sixty-eight percent of the 193 isolates belonged to the O25b-ST131 clone, to the phylogenetic group B2, mostly showed RAPD-type A (92%) and were found in all participating hospitals. O25b-ST131 isolates predominantly produced CTX-M-15 (96%), and less SHV-12 (n=3) or TEM-139 (n=2). In conclusion, this study demonstrated for the first time the country-wide dissemination of a highly resistant B2 O25b-ST131 CTX-M-15 producing E. coli clone in Bulgaria.