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.

Clonal Distribution, Antimicrobial Resistance, and Pilus Islets in S. pneumoniae Isolates from PCV10-Vaccinated Children with Suppurative AOM in Bulgaria (2015‒2020).

Streptococcus pneumoniae is still a leading bacterial pathogen of acute otitis media (AOM), despite the availability of pneumococcal conjugate vaccines (PCVs). We conducted a study on the population structure, antibiotic nonsusceptibility, serotype distribution, and presence of pilus in middle ear fluids ‒ S. pneumoniae isolates recovered from PCV10-vaccinated children with suppurative АОМ in Bulgaria. Non-susceptibility was observed in 68.75% (n = 33) of the isolates, and multidrug resistance (MDR) was detected in 60.4% of the patients. The dual macrolide resistance mechanism was predominant. The most common serotypes were non-PCV10 serotypes 3 (27.1%, n = 13), 19A (25.0%, n = 12), and VT 19F (23.0%, n = 11). Overall, 64.6% were non-PCV10-serotypes. The presence of Pilus type I was observed mostly in the PCV10-serotypes. We found a strong association between clonal complexes (CCs), serotypes, and antimicrobial resistance. Multilocus sequence typing revealed the presence of four CCs: CC320 (39.6%), CC505 (12.5%), CC1377 8.3%), and CC230 (8.3%). The most abundant CC320 comprised MDR 19A and 19F isolates. CC230 clustered MDR isolates from serotypes 19A, 6C, and 14. CC505 and CC1377 were serotype 3 susceptible isolates. The vaccine-induced changes and trends in antimicrobial resistance and clonality must be the focus of systematic investigations.

Relationship between MLSB resistance and the prevalent virulence genotypes among Bulgarian Staphylococcus aureus isolates.

The aim of this study was to investigate the rate of resistance to macrolide-lincosamide-streptogramin B (MLSB) antibiotics, the mechanisms underlying this resistance and to evaluate their relationship with virulence genes profiles of 435 Bulgarian clinical isolates Staphylococcus aureus. The highest resistance was observed to penicillin (96.09%), followed by resistance to erythromycin and clindamycin (34.02 and 22.76%, respectively). Of the tested clinical strains of S. aureus, 96.09% contained the blaZ gene associated with penicillin resistance and 11.03%, the mecA gene responsible for methicillin resistance. The most prevalent were the erm genotypes associated with the presence mainly of ermA and ermC genes followed by ermB. The frequency rates of these genes, alone or in combinations were ermA 41.89%, ermB 27.70%, ermC 43.99%. The majority of Bulgarian macrolide resistant S. aureus exhibited cMLS phenotype, in 58.78% (P = 0.0036). The following virulence genotypes were present significantly more often in the macrolide resistant S. aureus isolates among the studied ones: hlg; hlg,seb; hlg,seb,sec; hlg,seb,seh; hlg,sec; hlg,sec,sei; hlg,sec,sei; hlg,sei; hlg,sei,sej; hlg,sej. This survey found correlation between the virulence profiles with a small number of genes and macrolide resistance among Bulgarian clinical S. aureus isolates, in contrast to sensitive strains, which possessed profiles predominantly with multiple genes.

Rifamycin use for treatment of Helicobacter pylori infection: a review of recent data.

Helicobacter pylori eradication has become increasingly challenging. We focused on recent data about rifamycin resistance and rifamycin-containing regimens. Rifampin (rifampicin) resistance rates were <1-18.8% (often ≤7%), while those to rifabutin were 0-<4%. To detect rifabutin resistance by rifampin, 4 mg/l breakpoint was suggested. Eradication success by rifaximin-based regimens was disappointing (<62%), while that of rifabutin-containing regimens was 54.5->96%, reaching >81% in four studies. Some newer rifamycin analogs like TNP-2092 need further investigation. Briefly, although rifabutin-based regimens carry a risk of adverse effects or increasing mycobacterial resistance, they may be a rational choice for some multidrug-resistant H. pylori strains and as a third-line eradication therapy. Bismuth addition to rifabutin-based therapy and combined rifabutin-containing capsules (Talicia) are promising treatment options.

Delafloxacin against Helicobacter pylori, a potential option for improving eradication success?

Increase in Helicobacter pylori resistance to fluoroquinolones has been reported in many countries. The aim of the study was to compare, for the first time to our knowledge, levofloxacin and delafloxacin activities against H. pylori, including numerous levofloxacin- and multidrug resistant strains. Minimal inhibitory concentrations (MICs) of six antibiotics against 71 consecutive clinical strains were determined. Delafloxacin MIC50 and MIC90 were 0.016 and 0.125 µg/mL versus 0.125 and ≥32 µg/mL, respectively, for levofloxacin. Against the 19 levofloxacin resistant strains, delafloxacin MICs50 and MICs90 were 0.094 and 0.38 µg/mL, respectively. Delafloxacin MICs against the 21 strains with double or multidrug resistance were ≤0.75 µg/mL. The low MICs, the activity against levofloxacin resistant and multidrug resistant H. pylori strains and the increased activity of the agent in acidic conditions make delafloxacin worthy of further investigation, aiming at optimizing fluoroquinolone-based eradication regimens.

Activity of delafloxacin versus that of levofloxacin against anaerobic and microaerophilic isolates.

The aim of the study was to comparatively assess delafloxacin and levofloxacin activities against 96 anaerobic and some microaerophilic isolates. Delafloxacin minimal inhibitory concentrations (MICs) were strikingly lower than those of levofloxacin. Delafloxacin MIC90 against clostridia, other Gram-positive rods, anaerobic/microaerophilic cocci and Gram-negative rods were 0.75, 0.032, 0.38 and 0.5 µg/mL, respectively. The highest (≥4 µg/mL) MICs of the newer fluoroquinolone were found in only 4.2% of isolates versus 46.9% by levofloxacin. The present results and the potency in acidic conditions showed delafloxacin advantages over levofloxacin in terms of usefulness for treatment of mixed anaerobic-aerobic infections and activity against Clostridioides (Clostridium) difficile.

Phenotypic and genotypic characterization of serogroup 6 Streptococcus pneumoniae isolates collected during 10-valent pneumococcal conjugate vaccine era in Bulgaria.

Serogroup 6 remains common in the pneumococcal-conjugated vaccine era in Bulgaria; therefore, we investigated its clonal and serotype dynamics. The antibiotic susceptibilities were assessed by broth microdilution. Strains identified as serogroup 6 with latex agglutination method were subjected to serotype-specific PCRs. Erythromycin-resistant strains were analyzed by PCR for presence of ermB and mefE genes. MLST was performed to define clonal composition of the sequence types (STs). Serogroup 6 was represented by 40 (13.3%) from 301 invasive and non-invasive Streptococcus pneumoniae isolates. Molecular serotyping revealed new emerging serotype 6C (6.6%), not detected in pre-vaccine era. Among unvaccinated patients, mostly we observed serotypes 6А (57.1%) and 6В (28.6%). Serotype 6C was distinctive for vaccinated children (64%), followed by 6A (24%). Penicillin and ceftriaxone non-susceptible serogroup 6 strains were 65% and 5%, respectively; erythromycin- and clindamycin-resistant were 70.0% and 52.5%, respectively. Multidrug-resistant strains were 57.5%. Prevalent genetic determinant for macrolide resistance was ermB gene (75%). MLST revealed 17 STs into 5 clonal complexes and 7 singletons. Predominant genetic lineage was CC386, represented by MDR-6C non-invasive strains. Serotype 6B, principally responsible for invasive diseases in the pre-vaccine era, retreated this position to serotype 6A.

Molecular emm typing of Bulgarian macrolide-resistant Streptococcus pyogenes isolates.

Group A streptococcus (GAS) is a human pathogen causing a broad range of infections, linked with global morbidity and mortality. Macrolide resistance rates vary significantly in different parts of the world. Driving factors of the emergence and spread of resistant clones are not clearly understood. We investigated 102 macrolide-resistant GAS strains collected during the period 2014-2018 from various clinical specimens from Bulgarian patients. Strains were characterized by the presence of mefA/mefE, ermA, and ermB using polymerase chain reaction and sequencing for mefA/mefE. Resistant strains were studied by emm sequence typing and emm-cluster system. Most prevalent emm types among the macrolide-resistant GAS strains were emm28 (22.55%), emm12 (17.65%), and emm4 (16.66%). Almost all (87.25%) of the macrolide-resistant isolates harboring ermB were emm28. The isolates that carried ermA were predominantly emm12 (38.24%) and emm77 (38.24%), with fewer emm89 (23.53%). The isolates harbored predominantly mefE (49 isolates) and only 9 strains carried mefA. The most prevalent emm clusters among the GAS isolates were E4 (40.20%), A-C4 (17.65%), and E1 (16.66%). The study's results suggest that dissemination of specific clones in GAS population may also be the reason for the increasing macrolide-resistance rate in our country.

Multidrug resistance in anaerobes.

Multidrug resistance (MDR) in anaerobes is not a well-known topic. Bacteroides fragilis group isolates have numerous resistance determinants such as multidrug efflux pumps, cfiA and nimB genes and activating insertion sequences, and some isolates exhibited extensive drug-resistant patterns. MDR rates in B. fragilis group were from 1.5 to >18% and up to >71% in cfiA and nimB positive isolates carrying insertion sequences. MDR was present in >1/2 of Clostridioides difficile isolates, most often in epidemic/hypervirulent strains and unusually high metronidazole or vancomycin resistance has been reported in single studies. MDR was found in Prevotella spp. (in ≤10% of isolates), Finegoldia magna, Veillonella spp. and Cutibacterium acnes. Resistance in the anaerobes tends to be less predictable and anaerobic microbiology is required in more laboratories. New hopes may be new antibiotics such as eravacycline, cadazolid, surotomycin, ridinilazol or C. difficile toxoid vaccines; however, more efforts are needed to track the MDR in anaerobes.

Multidrug resistance in Helicobacter pylori: current state and future directions.

Introduction: Helicobacter pylori antibiotic resistance has increased worldwide and multidrug resistance (MDR), which seriously hampers eradication success of the frequent chronic infection, has often been reported. Areas covered: H. pylori MDR rates are discussed, mostly from recent articles published since 2015. Present approaches and future directions to counteract the MDR are outlined. Expert opinion: Alarming presence of triple, quadruple and, in some studies, quintuple and sextuple resistance was detected. Primary MDR rates ranged from <10% in most European countries to >40% in Peru. Post-treatment or overall MDR rates were >23-36% in about half of the studies. MDR prevalence has varied both among and within the countries. Factors linked to the MDR are national antibiotic consumption, antibiotic misuse, treatment failures and bacterial factors such as mutations, efflux pumps, and biofilms. Important directions to counteract the MDR increase can be optimization of present and new eradication regimens, wider use of bismuth-containing regimens, assessment of benefit of vonoprazan, new antibiotics such as newer fluoroquinolones and oxazolidinone analogues, adjuvants involving N-acetylcysteine and probiotics, anti-biofilm approaches using anti-biofilm peptides and rhamnolipid and development of vaccines and non-invasive tests for resistance detection. However, more efforts and studies are required. Strain susceptibility testing is increasingly important.

Relation between emm types and virulence gene profiles among Bulgarian Streptococcus pyogenes clinical isolates.

Background: The Streptococcus pyogenes emm gene, which encodes M protein, is an important epidemiological marker. The aim of this study is to determine the emm genotypes of Bulgarian clinical streptococccal isolates in 2014-2018 and to evaluate their relationship with virulence genes profiling and disease types. Methods: PCR and sequencing were used for emm genotyping of 182 S. pyogenes clinical isolates according to the protocol of the Centre for Disease Control and Prevention. PCR was used to investigate the virulence factors. Results: We identified 15 emm types and eight clusters. Five main clusters with eight emm types were predominant: cluster A-C3 (emm1) - 24.7%, A-C5 (emm3) - 19.2%, E1 (emm4) - 11.0%, A-C4 (emm12) - 11.0% and E4 (emm2,28,77,89) - 20.9%. There were two novel subtypes: emm3.132 and emm3.133. The investigated strains with emm3 genotypes were common in sterile site infections (invasive ones) and types emm4 and emm12, in skin and mucosal infections. More than 60% of the major cluster A-C3 (emm1; emm1.33; emm1.6) members possessed many genes for streptococcal pyrogenic exotoxins that act as super-antigens and bring about potentially higher virulence. Conclusion: The present study described two novel emm3 subtypes. To the best of our knowledge, this study is the first that describe the emm type spectrum of Bulgarian S. pyogenes clinical isolates and associated virulence factors. Monitoring of the S. pyogenes pathogenic potential and epidemiology can lead to better knowledge and higher possibility for prevention and eradication of complications of streptococcal infections.

Quinolone resistance mechanisms among third-generation cephalosporin resistant isolates of Enterobacter spp. in a Bulgarian university hospital.

Background: There have been no reports in Bulgaria about quinolone resistance determinants among Enterobacter spp. Aims: To investigate plasmid and chromosomal quinolone resistance rates among 175 third-generation cephalosporin resistant Enterobacter spp. isolates (167 Enterobacter cloacae complex and eight Enterobacter aerogenes isolates) collected at a university hospital in Varna, Bulgaria, as well as to reveal their association with ESBL/AmpC production and a carriage of specific plasmid replicon types. Methods: PCR, isoelectric focusing, replicon typing, sequencing, and epidemiology typing were carried out. Results: A high level of combined third-generation cephalosporin and quinolone resistant Enterobacter spp. was found - 79.4%. The ESBL production rate was 87%, consisting mainly of CTX-M-15 among E. cloacae complex (in 76%) and CTX-M-3 among E. aerogenes (in 88%). Plasmid mediated quinolone resistance (PMQR) determinants were identified in 57% of the isolates. The most commonly detected PMQR determinants were qnrB (90%), consisting mainly of qnrB1 (in 61%), and qnrB9 (in 27%) of the isolates. Both alleles were transferred with CTX-M-15 genes; transconjugants showed HI2 replicons (for qnrB1 positive transconjugants) and were non-typeable (for qnrB9). One Enterobacter spp. isolate produced qnrB4. QnrA1, qnrS1, and aac(6')-Ib-cr were detected in single isolates only. QnrC, qnrD, qepA, and oqxAB genes were not found. QnrB was associated with CTX-M-15 production, and qnrS1 was linked to CTX-M-3. Alterations in 83 and 87 positions of gyrB in quinolone-resistance determining regions, and 80 position of parC were detected in high level quinolone resistant isolates. Among all the Enterobacter spp. isolates tested, one predominant clone A was identified (53%). Conclusion: Our data showed the necessity of more prudent use of quinolones and third-generation cephalosporins, because of the risk of promoting dissemination, and selection of multiple resistance determinants (ESBL, PMQR) among Enterobacter spp. isolates in Bulgaria.

Recurrent Clostridioides (Clostridium) difficile infection in a patient suffering from inflammatory bowel disease and benefits of resistotyping.

Two-year Clostridioides (Clostridium) difficile recurrences in a boy with ulcerative colitis are described. Isolates were toxin A/B positive and nonhypervirulent, and resistotypes of 2017 isolates differed from those in 2016, suggesting a reinfection, later confirmed by multilocus sequence typing (ST49 and ST92, respectively). Resistotypes may show the need of genotypic analysis.