The emergence of multi-drug-resistant bacteria causing healthcare-associated infections in COVID-19 patients: a retrospective multi-centre study.

INTRODUCTION: We evaluated the prevalence, aetiologies and antibiotic resistance patterns of bacterial infections in hospitalized patients with laboratory-confirmed SARS-CoV-2. We also investigated comorbidities, risk factors and the mortality rate in COVID-19 patients with bacterial infections. METHODS: This retrospective observational study evaluated medical records of 7249 randomly selected patients with COVID-19 admitted to three clinical centres between 1st January 2021 and 16th February 2022. A total of 6478 COVID-19 patients met the eligibility criteria for analysis. RESULTS: The mean age of the patients with SARS-CoV-2 and bacterial infections was 68.6 ± 15.5 years (range: 24-94 years). The majority of patients (68.7%) were older than 65 years. The prevalence of bacterial infections among hospitalized COVID-19 patients was 12.9%, most of them being hospital-acquired (11.5%). Bloodstream (37.7%) and respiratory tract infections (25.6%) were the most common bacterial infections. Klebsiella pneumoniae and Acinetobacter baumannii caused 25.2% and 23.6% of all bacterial infections, respectively. Carbapenem-resistance in Enterobacterales, A. baumannii and Pseudomonas aeruginosa were 71.3%, 93.8% and 69.1%, respectively. Age >60 years and infections caused by ≥3 pathogens were significantly more prevalent among deceased patients compared with survivors (P<0.05). Furthermore, 95% of patients who were intubated developed ventilator-associated pneumonia. The overall in-hospital mortality rate of patients with SARS-CoV-2 and bacterial infections was 51.6%, while 91.7% of patients who required invasive mechanical ventilation died. CONCLUSIONS: Our results reveal a striking association between healthcare-associated bacterial infections as an important complication of COVID-19 and fatal outcomes.

Typing of macrolide resistant group A streptococci by random amplified polymorphic DNA analysis.

OBJECTIVE: Several studies of group A streptococci (GAS) have revealed that a small number of dominant resistant clones might be responsible for the spread of Streptococcus (S.) pyogenes resistance to macrolides. We aimed to determine the genetic diversity of macrolide resistant group A streptococci (MRGAS), isolated from patients with pharyngitis in Serbia. MATERIALS AND METHODS: The clonal relationships among 76 MRGAS isolates collected during 2008 were studied using two molecular typing methods: emm typing and random amplified polymorphic DNA (RAPD) analysis. Isolates that share the same emm type and RAPD pattern were considered to belong to the same clone. RESULTS: Out of 7 distinct emm types identified, the 3 most frequently occurring overall were emm12, emm75 and emm77 (> 90% of isolates). Although as many as 26 different RAPD patterns were found among the isolates studied, two clones with emm12 and emm77 accounted 32 out of 76 (42%) isolates. CONCLUSIONS: The results indicate a polyclonal spread of erythromycin-resistant Streptococcus pyogenes in our country. Furthermore, predominance of two clones, particularly among emm12 and emm77 strains indicates that erythromycin-resistant GAS of the same clonal origin are widely distributed in Serbia.

Distribution of emm types among group A streptococcal isolates from Serbia.

This is the first study concerning the molecular epidemiology of group A streptococcus in Serbia and includes 145 isolates from patients with various infections during the period 2001-2007. The emm types, superantigen profile and susceptibility pattern were determined. Among 31 emm types identified, the most prevalent were emm 6, emm 12, emm 1, and emm 58. All isolates showed uniform antimicrobial susceptibility to all tested antibiotics, with the exception of tetracycline and erythromycin (41% and 0.7% resistant strains, respectively). Significant heterogeneity of emm types was found, with a high frequency of emm 6 and emm 58, as well as a considerable prevalence of tetracycline resistance, and a low level of macrolide resistance.

Biotypes of group A streptococci from patients with pharyngitis and soft tissue infections.

This study determined the biotypes of group A streptococci (GAS) isolated from 66 pharyngeal and 62 skin and soft-tissue infections. Among all GAS isolates tested, the most common biotypes were 1 and 3, irrespective of the isolation source and the severity of clinical symptoms. However, compared with the pharyngeal group, a more heterogeneous distribution of biotypes was observed among the cutaneous group of isolates, including seven isolates that were non-typeable but had an identical biotype pattern, suggesting that they may represent a new biotype.

Influence of acetylsalicylic acid (aspirin) on biofilm production by Candida species.

Candida spp. are important causative agents of infections associated with biofilm formation. Management of biofilm-related infections is extremely difficult and therefore new therapeutic solutions are needed. This study for the first time explored the possible effect of aspirin on Candida spp. Biofilm-producing capacity. Two strains of C. guilliermondii, and one strain per species of C. kefyr, C. glabrata, C. albicans, and C. parapsilosis were included in the study. The antifungal property of aspirin was tested by the broth microdilution method, while effect of aspirin on biofilm formation was determined by the microtiter-plate test. The minimal inhibitory concentrations of aspirin obtained ranged from 2.17 to 8.67 mM and minimal fungicidal concentrations were from 4.33 to 8.67 mM. The concentrations of aspirin which induced statistically significant decrease in biofilm formation ranged from 0.43 mM to 1.73 mM of aspirin, depending on the tested yeast strain. Therefore, the significant effects of aspirin on growth and biofilm formation of Candida spp. were achieved only with suprapharmacological concentrations of the drug. The influence of the inoculum size on the effect of aspirin on biofilm formation was determined for C. albicans only and a significant decrease was observed also at suprapharmacological concentrations of aspirin, irrespective of the inoculum size. The results obtained in the present study show aspirin to be a drug with the potential to affect and suppress biofilm formation by Candida spp., and provide support for further investigation.

Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface.

AIMS: To investigate the biofilm formation by 122 Salmonella spp. and 48 Listeria monocytogenes strains on a plastic surface. METHODS: Quantification of biofilm formation was performed in brain heart infusion (BHI), trypcase soya broth (TSB), meat broth (MB) and 1/20 diluted trypcase soya broth (1/20-TSB) in plastic microtitre plates. RESULTS: All tested Salmonella spp. and L. monocytogenes strains produced biofilm in a suitable medium. However, the quantities of biofilm produced by Salmonella spp. were greater than those produced by tested L. monocytogenes strains. The nutrient content of the medium significantly influenced the quantity of produced biofilm. Diluted TSB was the most effective in promoting biofilm production by Salmonella spp., followed by TSB, while the least quantity of biofilm was formed in BHI and MB. L. monocytogenes produced the highest quantities of biofilm in BHI, followed by TSA, then MB, and the least quantities of biofilm were produced in 1/20-TSB. CONCLUSIONS: Salmonella spp. produces more biofilm in nutrient-poor medium, while L. monocytogenes produce more biofilm in nutrient-rich medium.

[Streptococcus pneumoniae and beta-lactam antibiotic agents]. / Streptococcus pneumoniae i beta-laktaminski antibiotiski lekovi.

Pneumococcus has been known for over 100 years. Despite an intensive research, the problem of pneumococcal diseases has not yet been solved. During the last few decades, the incidence of pneumococcal pneumonia has declined, but the S. pneumoniae is today the main, or one of the most frequent, causative agents of meningitis, sinusitis, otitis media and conjunctivitis. Besides, cases of pneumococcal appendicitis, tubo-ovarian abscess, haemolytico-uremic syndrome, cellulitis and urinary infections have been described. Therefore, it is very important from medical point of view to follow-up its sensitivity to antibacterial drugs. Unfortunately, during the latest decades, an increase in percentage of resistant clinical isolates has been registered. It is obvious that the investigation of the sensitivity of pneumococci, that is, of their resistance to beta-lactam antibiotics is essential. The examination of the interaction between pneumococci and penicillin has resulted in significant discoveries concerning the mechanism of the effect of penicillin as well as the impact of penicillin-binding proteins, lipoteichoic acid and choline-residues in the cell wall. A particular contribution as regards the effect of penicillin has been achieved by linking murein hydrolases (autolythic enzymes) with the expression of bactericidal effect of penicillin. Besides, the model of pneumococcal resistance to penicillin together with the model of meticillin resistance of staphylococci enabled the perception of the new mechanism of bacteria resistance to beta-lactam antibiotics. Given the pathogenic potential of pneumococci and the increase of clinical isolates resistant to antibiotics, it can be concluded that immunoprophylaxis is of great importance. Although several polyvalent vaccines are being used, there are still unsolved problems whose solution will improve the safety of their application, contribute to a better efficiency and enable a widespread application of antipneumococcal vaccines.

[Is penicillin still the drug of choice in the treatment of diseases caused by beta-hemolytic streptococci?]. / Da li je penicilin jos lek izbora u lecenju oboljenja izazvanih beta-hemolytickim streptokokama?

During the last ten years a new pathomorphosis of streptococci was noticed and described in the USA, Europe and New Zealand. It was expressed by the rise of virulence of beta haemolytic streptococci (BHS) and development of new clinical and epidemiological features. In such circumstances it appears to be very relevant to examine the susceptibility of BHS to penicillin, which is still considered as a drug of choice for the most of streptococcal diseases. Therefore it was decided: 1. to make an analysis of continuous susceptibility testing of BHS to penicillin and 2. to test the possibility of induction and selection of penicillin resistant mutants in vitro. Penicillin susceptibility was examined by broth dilution method Penicillin tolerant strains were separated on the basis of MBC/MIC ratio MBC/MIC > 16 and construction of "killing curves". The possibility of induction and selection of penicillin resistant mutants was tested by subcultivation technique. MIC values for BHS groups: A, B, C and G were: 0.015, 0.060, 0.015 and 0.030 micrograms/ml respectively. The percentage of penicillin tolerant strains was in the range of 3% for group A BHS to 33% for group G BHS. After 60 subcultures in liquid medium containing increasing concentrations of penicillin. MIC values were raised by 2-32 times in comparison with parental strains. As the maximal induced MIC values were 1 and 2 micrograms/ml (one group G and three groups B BHS strains) it can be concluded that BHS at least in vitro expressed penicillin resistance. Although the obtained results are encouraging (there is so far no penicillin resistant clinical isolates), the increasing percent of penicillin tolerant strains and the possibility to induce penicillin resistance in vitro should be considered as a very serious warning. That makes further investigation of the development of penicillin tolerance and resistance mechanisms very current.

[Changes in sensitivity to penicillin and an increase in virulence in beta-hemolytic streptococci followed by changes in the clinical picture of streptococcal syndrome]. / Promena osetljivosti na penicilin i povecanje virulencije beta-hemolitickih streptokoka praceno izmenjenom klinickom slikom streptokoknog sindroma.

According to reports from different geographic areas, the last decade has been characterised with an increasing number of streptococcal diseases. The new streptococcal "pathomorphosis" is presented by alteration of adhesive properties and distribution of various serotypes (domination of M1 and M18 serotypes). It is also expressed by an increased production of pyrogenic exotoxin and necrotic factor. As a consequence, apart from increasing incidence of streptococcal infections, it is observed that the streptococcal syndrome has often grave prognosis followed with a high percentage of mortality. Also a new clinical entity is also described; that of Toxic Shock Like Syndrome. The results of investigation concerning the susceptibility of beta-haemolytic streptococci to penicillin are encouraging. Penicillin resistant strains are not discovered among clinical isolates so far. However, the increased percentage of penicillin tolerant strains, and possibility of induction of penicillin resistance, can be considered as a serious warning. For that reason, further investigation of the mechanisms of developing penicillin tolerance and resistance appears to be highly recommended.

Construction of expression plasmids for the fusion protein of Sendai virus, and their expression in E. coli cells and eucaryotic cells.

To examine the properties and the role of the fusion protein (F) of Sendai virus at the molecular level, a plasmid, pUC-F, was constructed by inserting cDNA for the F protein into a pUC vector. Upon induction of E. coli cells transformed with pUC-F, a new protein was obtained, which was identified as Fo on Western blot analysis. The cDNA fragment for the F gene was excised from pUC-F and inserted into an eucaryotic expression vector, pSVL, to yield pSVL-F. COS-1 cells transfected with pSVL-F gave a band on SDS-gel electrophoresis which corresponded to the size of the Fo proteins.