Classical Bacteriological Methods Research Articles

Genomic analysis of <i>Klebsiella pneumoniae</i> strains virulence and antibiotic resistance

Recently, Klebsiella pneumoniae strains have become widespread both in community-acquired infectious processes and in nosocomial infections. There are two pathotypes of K. pneumoniae: classical (cKp) and hypervirulent (hvKp). Representatives of any pathotype are prone to acquire and further transmit genetic factors of antibiotic resistance and virulence. This combination accounts for severity of the infectious process. Therefore, information about whether the strain belongs to either pathotype can help in prescribing proper therapy. Since there is no consensus upon hypervirulence marker, we attempted to find the most significant combinations of genetic markers of virulence and antibiotic resistance in K. pneumoniae strains. The study was aimed to conduct a genomic analysis of virulence and antibiotic resistance of K. pneumoniae clinical isolates. Materials and methods. There were examined 85 strains of K. pneumoniae isolated from diverse clinical material samples from patients in large St. Petersburg hospitals. In our work, we used classical bacteriological methods, including determination of the hypermucoviscous type using the “string test”, the mass spectrometric method (MALDI-ToF MS) for identifying bacteria, molecular methods for studying markers of virulence and antibiotic resistance (multilocus sequence typing, genome sequencing of K. pneumoniae strains). Results. Among the studied K. pneumoniae strains, the most common carbapenemase genes were OXA-48 (18.7%) and NDM-1 genes — 17.3% of strains; in 6.7% of strains, NDM-1 and OXA-48 genes were found simultaneously. The percentage of strains with β-lactamase genes CTX-M-15 was 54.7%, OXA-1 — 17.3%, TEM-1D — 13.3%, and in 17.3% of cases the OXA-1 and TEM-1D genes were simultaneously present in bacterial strains. Quinolone resistance genes were found in 68.4% of strains. The most common genes were qnrS1 (40% of strains) and qnrB1 (22.7%). Phenotypic antimicrobial susceptibility testing showed that 23.5% and 64.7% strains were resistant to colistin and carbapenems, respectively. 32.9% K. pneumoniae strains, isolated in patients with phlegmon, pneumonia, sepsis, and peritonitis, had a hypermucoid phenotype. The most common sequence types were: ST395 (24.3%), ST23 (17.6%) and ST512 (9.5%). 8% and 25.3% of strains belonged to capsule types K1 and K2, respectively. The polyketide synthesis locus ybt, which characterizes virulent strains, was detected in 69.3% isolates, and the clb locus was present in 10.7% of strains. In 73.3% and 14.7% strains, the plasmid-associated virulence loci iuc and iro were identified, which encode the biosynthesis of the siderophores aerobactin and salmochelin. We described 44 cases (58.7% of strains) of genotypic convergence of virulence and antibiotic resistance, as shown by simultaneously detected the aerobactin (iuc) locus and β-lactamase or carbapenemase genes. Thus, identification of hypervirulence may provide valuable information for the clinical management of patients with hvKp infections. Therefore, it is is obviously necessary to develop comprehensive diagnostic test for simultaneous screening of multidrug-resistant hypervirulent K. pneumoniae strains.

Seasonality of the conjunctival microbiota in cattle from a microregion in midwestern Brazil

This study aimed to identify the bacterial and fungal organisms in the conjunctival fornix of healthy cattle in a microregion located in midwestern Brazil. Additionally, we evaluated whether seasonality and cattle category (dairy vs. beef) play a role in our findings. The conjunctival fornix of 74 Holstein x Gyr and 70 Nelore cattle were sampled from February to March (rainy season) and August to September (dry season) of 2017. Bacterial and fungal strains were isolated by classical bacteriological and mycological methods. Some bacterial species were also identified by polymerase chain reaction. From the 144 animals included in the study, a total of 12 bacterial genera were identified, with Bacillus cereus being the most prevalent agent in both seasons and cattle categories. The total number of bacteria was not influenced by seasonality or cattle category (P = 0.25). The number of Moraxella bovoculi identified in dairy (5/74) and beef cattle (7/70) did not differ statistically (P = 0.76). However, seasonality played a role in M. bovoculi identification, being found only during the rainy season in the dairy cattle (P = 0.01), and only during the dry season in the beef cattle (P = 0.04). During the dry season, the number of gram-positive bacteria was significantly higher than the number of gram-negative bacteria in beef cattle (P = 0.009). Fungi were isolated in 20/144 samples. The number of fungi isolated in dairy cattle (16/144) was significantly higher than that isolated in beef cattle (4/144) (P = 0.007). Although a higher number of fungi were isolated in both cattle categories during the rainy season, this finding was not significant (P = 0.53). In this microregion located in midwestern Brazil, Bacillus cereus was the most prevalent and Corynebacterium spp. and Streptococcus spp. the least prevalent bacteria found in the conjunctival fornix of healthy cattle. The prevalence of the conjunctival fungal microbiota (11.8%) was lower than previously reported, with a higher number of isolates found in dairy cattle. Trichosporon spp. (41.2%) and Aspergillus spp. (35.3%) were the most frequent genera, being most commonly isolated in the rainy season.

Comparison of bacteriological culture method and multiplex real-time PCR for detection of mastitis

This study includes the evaluation of multiplex real-time PCR (rPCR) kit, which was developed to provide rapid diagnosis of mastitis infections, by working with milk samples of 2 different sources of mastitis and comparing the results with the classical bacteriological culture method (BC). A total of 273 bacteria were isolated in 226 samples (47.88%) out of 472 samples by BC. These were 139 (50.91%) Staphylococcus spp., 61 (22.34%) Streptococcus spp., 15 (5.49%) E. coli, 8 (2.93%) Enterococcus spp., 50 (18.31%) other bacteria. When we look at the multiplex rPCR results; 1052 positive were obtained for the gene regions of 14 different bacteria, 1 yeast, and 1 β-lactamase gene examined in 472 samples. While no searched gene region was found by rPCR in 78 (16.5%) of the 472 samples studied, at least 1 gene was detected in 394 (83.5%) samples. These 1052 positive samples by rPCR were; 263 (28.43%) Staphylococcus spp., 51 (5.51%) S. aureus, 57 (6.16%) Enterococcus spp., 49 (5.29%) C. bovis, 16 (1.73%) S. dysgalactiae, 84 (9.08%) S. agalactiae, 71 (7.67%) S. uberis, 73 (7.89%) E. coli, 14 (1.51%) Prototheca spp., 39 (4.21%) T. pyogenes/P. indolicus, 5 (0.54%) S. marcescens, 15 (1.62%) K. oxytoca/pneumonia, 117 (12.64%) Mycoplasma spp., 31 (3.35%) M. bovis, 40 (4.32%) yeast, and 127 samples (26.90%) were β-lactamase positive. When the antibiotic resistance of the isolates was evaluated, 78 (31.96%) tetracycline, 72 (29.5%) penicillin, and 60 (24.59%) clindamycin resistance were observed predominantly in Gram-positive isolates, while 6 (23.07%) tigecycline, 6 (23.07%) netilmicin, 6 (23.07%) pipercillin resistance was found in gram-negative isolates.While a bacteria and/or yeast gene was found by rPCR in 187 of 246 (76.01%) samples with no bacterial growth, a bacterium was isolated with BC in only 20 (8.84%) samples whose gene region was not found by rPCR. As a result, the multiplex rPCR system used in the diagnosis of mastitis has been found to be quite reliable as it can detect a large number of bacteria in a very short time compared to classical methods. Therefore, we advise the use of rPCR and/or culture for confirmation of clinical signs in mastitis and at routine mastitis surveillance.

Characteristics of <i>Streptococcus pneumoniae</i> carriage in the pediatric population

Objective: to investigate the regional peculiarities of Streptococcus pneumoniae carriage in the pediatric population and characterize the dominant serotypes of the pathogen.
 Materials and methods. The clinical study group consisted of 509 healthy children attending preschool institutions. Examination of nasopharyngeal samples for the detection of S. pneumonae was carried out by classical bacteriological and molecular biological methods. The serotype was determined by real-time PCR. Genome-wide sequencing of the serogroups 15 and 11 isolates and bioinformatic analysis were performed.
 Results. The S. pneumoniae bacterial carriers in the group of healthy children was detected in 207 children (40.7%), while the frequency of detection of S. pneumoniae in urban children living in Kazan was significantly higher than in children living in rural area and amounted to 53.4 and 31.1%, respectively (p 0.05). Among children vaccinated with the 13-valent pneumococcal conjugate vaccine (PCV-13), S. pneumoniae carriers were not detected in 57.5% of cases. There were no significant differences in the degree of nasopharyngeal contamination depending on the vaccination status. Analysis of the serotype composition indicates the predominance of vaccine serotypes (57.7%), while the share of serotypes included in the PСV-13 vaccine accounts for only 24.7%, the share of non-vaccine serotypes was 32.1%, untyped — 10.2%. In unvaccinated children, vaccine serotypes that are part of the PCV-13 and 23-valent polysaccharide pneumococcal vaccine prevailed (PPSV-23): 6ABCD (21%), 11 AD (15%), 14 (13%). In vaccinated children, serotypes not included in the active vaccines dominated: 15AF (17.4%), 23A (19.2%), as well as 11AD (19.6%) (11А is included in PPSV-23). The 27 Kz isolate (serotype 15C) belonged to one of the most common sequence types ST1025. The 105_Kz isolate (serotype 11D) belonged to another common sequence type ST 62.
 Conclusion. In order to improve epidemiological surveillance of pneumococcal infection, it is necessary to introduce the monitoring of circulating clonal complexes of dominant S. pneumoniae serogroups and analyze the genetic determinants of antibiotic resistance and virulence depending on the sequence type.

Antibiotic resistance of infectious agents associated with prior hospitalization

Introduction: One of the frequent causes of re-hospitalization is infectious complications due to previous colonization of patient loci by microorganisms circulating in the hospital environment. In the conditions of real clinical practice among hospital-acquired infections (HAI), it is advisable to distinguish a special group of diseases – infections associated with previous hospitalization (IPAH). Of particular scientific interest is the study of the antibiotic resistance profile of IPAH pathogens in order to determine the further strategy of empirical antibiotic therapy. Materials and Methods: A two-center descriptive study was conducted in Tomsk region. We analyzed 170 cases of IPAH according to the medical records of patients receiving medical care in inpatient settings (form N 003/u) in the period from 2019 to 2023. Identification of microorganisms was carried out by classical bacteriological method. Results and Discussion: Gram-negative bacteria (95.3%) predominated in the etiology of pneumonia associated with prior hospitalization. Among Gram-negative microorganisms, the most frequent were K. pneumoniae, P. aeroginosa and K. oxytoca. Representatives of the families Enterobacteriaceae (48.2%), Staphylococcaceae (28.9%) and Enterococcaceae (10.8%) predominated in the etiology of surgical infection associated with previous hospitalization. In the species structure, the key pathogens were K. pneumoniae, S. aureus and E. coli. IPAH pathogens were characterized by an unfavorable resistance profile. Conclusion: Despite the fact that the etiological structure and antibiotic resistance profile of IPAH are similar to those of classical nosocomial infections, IPAH has important features that should certainly be taken into account when organizing medical care for this cohort of patients.

Микробный пейзаж респираторного тракта детей с муковисцидозом: результаты мониторинга

Background. The article presents the results of microbiological monitoring of microorganisms of the respiratory tract of patients with cystic fibrosis (CF), changes in the spectrum, palette, and antibacterial resistance during repeated hospitalizations in a specialized hospital. Purpose of the study. To analyze the results of microbiological examination of respiratory tract biomaterial of children with CF hospitalized in a multidisciplinary pediatric hospital to optimize epidemiological surveillance at the local level. Materials and methods. The material for the study was the results of a microbiological study of smears from the pharynx, nose, or induced sputum of patients with cystic fibrosis observed at the Children's Center for Cystic Fibrosis (CF). Identification of isolated cultures was carried out by classical bacteriological methods. Subsequently, the isolated microorganisms were systematized to species. The analysis was carried out for 2019-2023. Determination of sensitivity to antibacterial drugs was carried out using the disk diffusion method. Indicators of the frequency (%) of isolation of various microorganisms and the frequency (%) of resistant microorganisms to the antimicrobial drugs used in a medical organization (MO) were calculated. A comparison was made of the frequency (%) and spectrum of isolated cultures during initial and subsequent hospitalizations at the Children's Cystic Fibrosis Center. Confidence intervals CI (95%) were calculated for relative values. Calculations were carried out using the epidemiological calculator Confidence Interval Calculator. Results. In the structure of isolated cultures from the biomaterial of the respiratory tract of children with cystic fibrosis, 45.4% were gram-negative microorganisms. During initial and subsequent hospitalizations, the highest frequency of isolation were Pseudomonas aeruginosa, Klebsiella pneumoniae, Stenotrophomonas maltophilia. Gram-positive microorganisms in the spectrum of isolated cultures amounted to 37.9%. The majority of the isolated gram-positive microorganisms are represented by Staphylococcus aureus (75.7%). During repeated hospitalizations to the Children's CF Center, based on the monitoring results, changes in the spectrum, frequency of isolation, and spectrum of resistance to antimicrobial drugs were determined. An increase in the frequency (p<0.05) of the isolation of Pseudomonas aeruginosa and Staphylococcus aureus was determined. During repeated hospitalizations, the frequency of isolation of associations of microorganisms increased from 50.5% CI [55.7; 45.3] to 81.4% CI [83.8; 79.0] (p<0.05). An increase in the frequency (p<0.05) of isolation of Pseudomonas aeruginosa strains resistant to reserve antibacterial drugs was determined by 3.2 times during subsequent hospitalizations. Conclusion. The revealed changes in the spectrum, frequency, palette and antibiotic resistance of microorganisms of the respiratory tract of CF patients at the local level of epidemiological surveillance indicate the need to develop algorithms of action in outpatient and inpatient management of CF patients to ensure epidemiologic safety of medical activity and improve the quality of medical care.

Epizootic situation of bacterial kidney disease (BKD)

Relevance. Bacterial kidney disease (BKD, corineal renal infection, Di disease), the causative agent of which is the gram–positive non-spore motionless bacterium Renibacterium.salmoninarum, is a common disease of salmon fish (Salmonidae), which causes significant damage to fish production in aquaculture and the development of the industry, as well as reduces the number of wild populations.The purpose of this review was to analyze the epizootic situation of bacterial renal disease (BKD) among wild salmon and fish populations in aquaculture on the territory of the Russian Federation, as well as in other countries.The article presents information on the incidence of BKD in the world and on the territory of the Russian Federation for the period 1933–2023, shows data on the spread of the disease among wild populations and farmed fish in aquaculture, describes possible transmission routes, native and suspected vectors of infection.Methods. The authors consider measures to control the growth of the incidence of BKD in the world and on the territory of the Russian Federation. Due to the lack of an effective vaccine and a method of treatment, the most affordable method of controlling the spread of the disease is timely diagnosis. The article describes the methods developed for fast and effective diagnostics by the staff of the Laboratory of Ichthyopathology of the FSC VIEV: microagglutination reaction (PMA) and polymerase chain reaction in real time (real-time PCR).Results. Unlike classical bacteriological methods, these reactions allow for the indication and identification of the causative agent of BKD within 24 hours.

Isolation of a rare pathogen <i>Chryseobacterium indologenes</i> in patients with pneumonia

Introduction. Chryseobacterium indologenes bacteria are rarely detected, poorly studied opportunistic bacteria of the nonfermenting gram-negative bacilli group that are characterized by high intrinsic and acquired multidrug resistance and involvement in healthcare associated infections.Aim. To analyze detection rate of C. indologenes strains from various biomaterials obtained from patients with pneumonia, as well as levels of pathogen drug resistance to antimicrobial drugs.Materials and methods. The research included 1740 clinical samples obtained from patients with pneumonia hospitalized in in-patient facilities of the Khabarovsk city during the COVID-19 pandemic. Clinical samples included 1305 respiratory samples (744 sputum samples and 561 nasopharyngeal swabs), 435 samples of autopsy material (lung tissue) obtained from patients with a fatal outcome of the disease. The study was performed via classical bacteriological method. Pathogens were identified with Vitek 2 bacteriological analyzer and Vitek MS mass spectrometer. Sensitivity to antimicrobial drugs was determined by disk-diffusion method and in a bacteriological analyzer.Results. A comparative analysis of detection of the entire nonfermenting gram-negative bacteria (NFGNB) group and C. indologenes from various clinical samples was carried out. Detection rate of NFGNB equaled 17.3%. NFGNB were isolated from autopsy material most often ‒ in 31.7% of cases, less often ‒ from sputum and nasopharyngeal smears (13.8% and 10.7%, respectively). C. indologenes was detected in 0.5% of the cases (8 isolates out of 1740 samples). Isolation of this pathogen was most frequent in nasopharyngeal smears – in 1.1% (6 cases out of 561 samples), least frequent ‒ from sputum, which totaled 0.3% (2 isolates from 744 samples). The detection rate of the pathogen in respiratory samples equaled 0.6% (8 isolates out of 1305 samples). This pathogen was not found in the autopsy material. Carbapenem-resistant variants of C. indologenes were isolated in 75% (6 cases out of 8) of the cases.Conclusion. Further intensifying of diagnostic methods will result in more frequent detection of these unusual pathogens, clinical significance of which needs more justification.

Regional Features of the Serotype Composition of Streptococcus pneumoniae isolated from Bacterial Carriers of Preschool Age in the Republic of Tatarstan

Relevance. Vaccination of the child population against pneumococcal infection (PI) has been carried out in the Republic of Tatarstan according to the national calendar of preventive vaccinations since 2014. The vaccination scheme includes vaccination with PCV-13 conjugated pneumococcal vaccine.Aim. Study of the the frequency of bacterial transmission and the serotype landscape of S. pneumoniae isolated from healthy children- bacterial carriers of preschool age in the Republic of Tatarstan (RT).Materials and methods. During the period from 2016 to 2022, 1,426 children from Kazan and the districts of the Republic of Tatarstan were examined. Examination of nasopharyngeal smears was carried out by the classical bacteriological method. Serotyping was performed using molecular genetic methods (PCR).Results. The detection rate of S. pneumoniae among healthy preschool children varied in different years from 29.5 to 63%, averaging 38.5%. In urban children, the incidence of pneumococcal transmission was significantly higher than in rural children (p<0.01). Also, when analyzing the serotype landscape, mixed colonization by several serotypes was observed. As of January 1, 2021, the immune layer to the pathogen PI among preschool children was 81.7%. Monitoring of the serotype landscape of S.pneumoniae strains circulating in the RT showed the dominance of vaccine serotypes (67.2%), of which 44.4% are PCV13 serotypes. The proportion of non–vaccinated serotypes is 26%, untyped - 6.8%. Unvaccinated serotypes 35B (21.3%) and 23A (13.6%) dominated in vaccinated children, as well as serotypes not included in the PKV-13 vaccine cocktail, but included in the PPSV-23 polysaccharide vaccine not used for vaccination of children, namely 11AD (15.3%) 9LN (9.6%). In unvaccinated children, on the contrary, vaccine serotypes included in PCV-13 prevailed: 6ABCD (17.3%), 19F (20.9%), and unvaccinated serotypes 11AD, 9LN, 35B, 23A were detected with lower frequency 11,8%, 10,0%, 4,2%, 7,3% accordingly.Conclusion. Data on the regional features of the pneumococcal serotype landscape can be the basis for expanding the vaccine cocktail due to the dominant serotypes: 9LN,11 AD, 35B, 23A.

Prevalence of Salmonella Typhimurium and Salmonella Enteritidis isolated from poultry meat: virulence and antimicrobial-resistant genes

Salmonellosis, a zoonotic disease, is one of the leading causes of foodborne illness worldwide. It is responsible for most infections caused by consumption of contaminated food. In recent years, a significant increase in the resistance of these bacteria to common antibiotics has been observed, posing a serious threat to global public health. The aim of this study was to investigate the prevalence of virulent antibiotic-resistant Salmonella spp. strains in Iranian poultry markets. A total of 440 chicken meat samples were randomly selected from meat supply and distribution facilities in Shahrekord and tested for bacteriological contamination. After culturing and isolating the strains, identification was performed using the classical bacteriological method and PCR. To determine antibiotic resistance, a disc diffusion test was performed according to the recommendations of the French Society of Microbiology. PCR was used to detect resistance and virulence genes. Only 9% of the samples were positive for Salmonella. These were Salmonella typhimurium isolates. All Salmonella typhimurium serotypes tested positive for the rfbJ, fljB, invA and fliC genes. Resistance to TET, cotrimoxazole, NA, NIT, piperacillin/tazobactam and other antibiotics was found in 26 (72.2%), 24 (66.7%), 22 (61.1%) and 21 (58.3%) isolates, respectively. The sul1, sul2 and sul3 genes were present in 20, 12 and 4 of 24 cotrimoxazole-resistant bacteria, respectively. Chloramphenicol resistance was found in six isolates, but more isolates tested positive for the floR and cat two genes. In contrast, 2 (33%) of the cat three genes, 3 (50%) of the cmlA genes and 2 (34%) of the cmlB genes were all positive. The results of this investigation showed that Salmonella typhimurium is the most common serotype of the bacterium. This means that most of the antibiotics commonly used in the livestock and poultry industries are ineffective against most Salmonella isolates, which is important for public health.

Comparison of pcr diagnostic method and mass spectrometry of microbial markers method as applied to the evaluation of oral microbiota.

Rapid informative methods for assessing the species and quantitative composition of the microflora of the periodontal and oropharyngeal covering tissues are necessary for operative diagnostics, including those of the dentofacial system. The use of classical bacteriological methods, including seeding, incubation, counting and identification of microorganisms takes up to 5 days, resulting in a significant delay in obtaining the necessary information, which makes it difficult to carry out operative treatment measures. Therefore, the search for means and methods of operative microbiological control is urgent. The present work is devoted to substantiation of MSMM (mass spectrometry of microbial markers) technology application as a means of microbiological control of periodontal and other oropharyngeal biotopes.

Gut microbiota in patients with arterial hypertension and metabolic syndrome

In recent years, attention has been growing to assessing the role of the state of intestinal microbiocenosis in the development of cardiovascular diseases, including arterial hypertension (AH) and metabolic syndrome (MS), the individual components of which are more or less associated with the state of the microbiota. In this regard, we made an attempt to clarify the features of the intestinal microbiota in hypertensive patients with MS in a comparative aspect with those with isolated hypertension. A microbiological study of feces was carried out in 86 patients, of which 46 with AH (comparison group) and 40 with AH and MS (main group). The microbiota of patients’ faeces was investigated using the classical bacteriological method and the biochemical method for express diagnostics of dysbiosis. For fecal microbiota in AH with MS, in comparison with AH, a lower release of representatives of normal microfl ora (bacteroids, latobacilli, bifi dobacteria), with the exception of Escherichia coli, is characteristic, more frequent excretion of facultative microfl ora (enterococci, Klebsiella, peptostreptococcus, staphylococcus protrusion)., II and III degree of dysbiosis (versus I and II with hypertension). The study revealed that the composition of the intestinal microbiota in patients with hypertension is associated with MS both in terms of a decrease in individual representatives of normal microfl ora and in terms of the greater presence and appearance of new representatives of opportunistic microfl ora, forming a high degree of dysbiosis in hypertensive patients with MS.

MRSA as an indicator of infection control measures in Turaif General Hospital, Northern Area-Saudi Arabia.

Saudi Arabia can be considered a hot spot for Methicillin-resistant Staphylococcus aureus (MRSA) infections with significant regional variations. As far as we know, this is the first study to evaluate the prevalence of MRSA in clinical samples obtained from Turaif general hospital (TGH), Northern Area-Saudi Arabia, and screening the resistance profile to the most regularly used antimicrobials as an indicator for evaluation of the implemented infection control measures. Totally, 410 Samples were collected from patients in TGH with clinically suspected nosocomial infections. MRSA isolates were identified by the classical bacteriological, biochemical, and cefoxitin-based methods as recommended by the Clinical Laboratory Standard Institute. Confirmation of isolates and testing of their antimicrobial susceptibilities were performed by the automated Vitek 2 compact system. Totally, 130 nosocomial isolates were detected. Staphylococcus aureus (29.23%) was the most frequently isolated Gram-positive pathogen. MRSA represented 39.47% of Staphylococcus aureus and 11.54% of all isolates. MRSA-causing surgical site infections were the most predominant type of MRSA nosocomial infections representing (25.00%). Recent antibiotic therapy, prolonged hospital stays, and indwelling devices were significant risk factors for the development of MRSA infections. Although all MRSA isolates were sensitive to vancomycin, teicoplanin, linezolid, Fosfomycin, and tigecycline, many isolates were resistant to other tested antimicrobials. Hospital administrators should strengthen the ideal use of antibiotics according to the local hospital policy to control the selective drug pressure on Staphylococcus aureus strains with minimizing exposure to the risk factors by implementing the proper infection control policies.

Express detection of Escherichia coli and bacteria of the Escherichia coli group at the oral cavity.

Currently, in the diagnosis of diseases, a decisive place is given to laboratory methods, which should be informative, relatively simple to perform and rapid. The article describes the approbation of a method for rapid detection of Escherichia coli and bacteria of Escherichia coli group in the oral cavity. Research involved 44 volunteers, who were sampled from the oral cavity, followed by incubation in Koda's medium. The study used oral (n=11) and gingival fluids (n=11); smears-prints from the oral mucosa (n=11); dental biofilm (n=11). After 24 hours, the change in color and transparency of the medium was assessed. The preservation of the initial green color and transparency by the medium meant the absence of E. coli and bacteria of Escherichia coli group in the sample. A change in the color of the medium to yellow, turbidity and / or the formation of bubbles indicated the presence of E. coli and bacteria of Escherichia coli group. In parallel, the material was inoculated onto Endo agar, followed by identification of strains to species. As a result of the study, a complete coincidence of the results of the classical bacteriological method and the method using Koda medium was shown. In the latter case, a significant advantage is the speed of obtaining the result (18-20 hours), in contrast to the classical method, the interpretation of the results of which is available only after 72 hours or more. All of this is in line with the state of the art in clinical microbiology and rapid diagnosis based on «point-of-care testing / doctor's office» diagnostic principle. The presented method can be successfully applied in clinical practice for topical diagnosis of microorganisms E. coli and bacteria of Escherichia coli group in the oral cavity.

Species identification and antimicrobial susceptibility profile of bacteria associated with cow mastitis in southern Brazil

ABSTRACT: Bovine mastitis is the most common disease in dairy cattle and responsible for economic losses in the milk industry. The present study aimed to identify the main species and to evaluate the antimicrobial susceptibility of bacterial isolates from cow herds with mastitis in dairy farms from southern Brazil. A total of 107 milk samples were collected from different cow herds in one important dairy producing region in southern Brazil, including farms located in ten cities from the Northeast region in the Rio Grande do Sul state. Bacterial strains were isolated and submitted to presumptive identification by classical bacteriological methods. Bacterial species were also identified by MALDI-TOF MS and antimicrobial susceptibility testing was performed with 12 antimicrobials commonly used in dairy farms. Fifty-one bacterial strains were isolated and the presumptive identification demonstrated the occurrence of Staphylococcus spp. (82.3%), Bacillus spp. (3.9%), Klebsiella spp. (3.9%), Streptococcus spp. (3.9%), Corynebacterium sp. (2%), Enterococcus sp. (2%) and Serratia sp. (2%). Forty-one isolates were successfully identified in the MALDI-TOF analysis, including 35 isolates from eleven different bacterial species. Importantly, there were eight different Staphylococcus species, with a high frequency of Staphylococcus chromogenes (48.6%) and Staphylococcus aureus (20%). Overall, bacterial isolates demonstrated resistance to penicillin (46.3%), tetracycline (39%), amoxicillin (36.6%), ampicillin (34.1%) and sulfamethoxazole/trimethoprim (31.7%). Enrofloxacin was the unique antimicrobial that all isolates were susceptible. In addition, there were six multidrug resistant isolates (five S. chromogenes and one S. aureus). This study highlights that bacterial pathogens with resistance to several antimicrobials were identified in cows from dairy farms in a very important milk producing region located in southern Brazil. Microbial identification of the bovine mastitis pathogens and determination of the antimicrobial profile is necessary for the rational use of the medicines.

CHARACTERISTICS OF THE COMPOSITION OF GINGIVAL POCKETS MICRFLORA ISOLATED FROM PATIENTS WITH PURULENT INFLAMMATORY DISEASES IN THE PERIODONTAL TISSUES

The problem of the purulent inflammatory disorders of periodontal tissues (PIDPT) at the present time is extremely prevalent. According to the World Health Organization (WHO), around 80% of people suffer from periodontal diseases, and the disease incidence among young people increases significantly and reaches up to 80-95 %. The aim of the present research was to study the composition of microflora of gingival pockets in patients with purulent inflammatory periodontal diseases (PIDPT) in order to establish the specific weight of the determined microbial associations.
 Materials and methods. Microbiological researches included isolation and identification of bacteria that were present in the gingival pockets of patients with different types of PIDPT. Identification of anaerobic microorganisms and associated microflora was carried out with the help of commonly used classical bacteriological methods, with determination of biochemical properties of the microorganisms cultures using APІ and ANAEROtest 23.
 Results. There were 192 patients studied in total, all aged older than 20 with diagnoses acute and chronical periodontitis, aggressive periodontitis, hypertrophic pulpitis, granulating periodontitis, parodontopathy, and local periodontitis with a presence of fistula. During the analysis of composition of the microflora isolated from periodontal tissue in patients with PIDPT, the following pathogens were isolated most frequently during the bacteriological research: Streptococcus spp., Staphylococcus spp., Neisseria spp., Stomatococcus spp., and Enterococcus spp. (58%). The quantity of periodontally pathogenic microorganisms was constituted 19,3% from the total quantity of isolated streains (81 isolates). The most numerous group was determined to be the “orange” complex (59,3%) – isolates of Prevotella melaninogenica, Fusobacterium nucleatum, Parvimonas micra. The least numerous was the group of “green” complex - (2,5 %) – Streptococcus mitis. Periodontally pathogenic representatives of microbiota in all cases were isolated as a part of associations with other bacteria.
 The presented materials signified that the periodontally pathogenic agents of the “red” and “orange” complexes were isolated together with representatives of Streptococcus spp - 33 (56,0 %) associations; Staphylococcus spp - 11(18,6%) – associations. Also in 13 cases (22,1 %) of associations the associated flora was represented by Candida spp. fungi.
 Conclusions. In course of the research, 4 species of “red” and “orange” complexes were identified, and the quantity of different associations there were present in was significant. There were 22 associations determined. The obtained date concerning microbial associates in the content of gingival pockets in PIDPT show the necessity of individual approach during treatent of this patient group, with taking into account the microbial diversity.

Epidemiology of Fire Blight in Fruit Crops in Kazakhstan

A survey on 13 regions of Kazakhstan was carried out to identify foci of fire blight, the extent of its spread, the range of affected crops, and the identification of infection reserves. The disease was mainly concentrated in the main fruit growing zone.at the south and southeast of Kazakhstan. Symptoms of fire blight were characteristic of two bacterial diseases: namely, necrosis, by Pseudomonas syringae Van. Hall. and fire blight by Erwinia amylovora (Burrill) Winslow et al. The authors performed bacteriological analyses to isolate and identify the causative agent of fire blight from various fruit and wild cultures of the Rosaceae family, using classical bacteriological and molecular genetic methods. Two types of bacteria were isolated from the samples affected by the disease, namely E. amylovora, the causative agent of fire blight, and P. syringae, the causative agent of bacterial necrosis. The results of the studies on the identification of bacterial species E. amylovora and P. syringae were confirmed by Swiss scientists from the Agroscope research center based on an immuno-chromatographic test and by Russian scientists at the All-Russian Research Institute of Plant Quarantine using enzyme-linked immunosorbent assay, FLASH polymerase chain reaction, and polymerase chain reaction in real-time.

Retrospective Analysis of Antibiotic Therapy and Sensitivity to Antifungals of Fungi of the Genus Candida, Secreted from Patients with Viral-Bacterial Pneumonia Associated with COVID-19

The appointment of antibacterial agents for the treatment of pneumonia that develops with COVID-19 is one of the treatment regimens. Antibacterial agents are prescribed only in the case of presence of confirmed bacterial co-infection, but can be appointed empirically. This approach promotes the development of antibiotic resistance of opportunistic and saprophytic microflora of almost all habitats, including the oropharynx that can lead to dysbiotic lesions with activation of fungal flora. The aim of the study was to analyze the composition of the oropharyngeal microbiome of patients with viral and bacterial pneumonia who took antibiotics, as well as the sensitivity of fungi of the genus Candida to antifungal drugs for effective treatment of the underlying disease. Methods. The results of bacteriological examination of 113 inpatients treated with a diagnosis of COVID-19 were analyzed. Microbiological examination of oropharyngeal swabs was performed by the classical bacteriological method with dosed seeding of suspended material on differential diagnostic media (in particular Saburo medium was used for selection of fungi of the genus Candida) and genus identification by morphological, cultural, biochemical properties. Results. PCR tests were performed for all patients in the clinical trial to confirm the diagnosis of viral and bacterial pneumonia. According to the results of bacteriological examination, fungi of the genus Candida were found in 52 (46.0%) patients with pneumonia associated with COVID-19. The analysis of prescriptions showed that only 14 (26.9%) patients were treated with one antibiotic, two antibiotics were prescribed to 31 (59.6%) patients, and three – 7 (13.5%). In the structure of antibiotic therapy, the lion’s share were cephalosporins of the third generation (ceftriaxone, hepacef) – 33 (63.5%), and macrolides (azithromycin) – 16 (30.8%) patients. In the structure of the oropharyngeal microbiome, according to the results of bacteriological research, fungi of the genus Candida significantly prevailed, which were found in 52 (46.0%), and in 29 patients (25.7%) S. pneumoniae was found. The sensitivity of fungi of the genus Candida to antifungal agents was analyzed, the maximum number of resistant strains was detected to nystatin and amphotericin – 38.5% and 26.9%, with only 8 (15.3%) fungi of the genus Candida sensitive to nystatin. Conclusions. All patients with viral-bacterial pneumonia associated with COVID-19 received antibiotic therapy, the lion’s share were third-generation cephalosporins (63.5%), and macrolides – (30.8%). According to the results of bacteriological examination of the oropharyngeal microbiome after antibiotic therapy, fungi of the genus Candida predominated (46.0%), followed by S. pneumoniae (25.7 %). Isolated strains of fungi of the genus Candida showed resistance to nystatin (38.5%) and amphotericin (26.9%). Antifungal agents of the imidazole subgroup have shown high efficiency and a low percentage of resistant strains, which allows us to recommend them for the treatment of complications of COVID-19 caused by fungi of the genus Candida.

Современные методы видовой идентификации термофильных бактерий рода Campylobacter

Emergent thermophilic Campylobacter hepaticus is the causative agent of Spotty Liver Disease (SLD) in laying hens. C. hepaticus is difficult to cultivate because commercial media for the isolation and cultivation of Campylobacter contain cefoperazone, which inhibits many isolates of the C. hepaticus species. Campylobacter was isolated using modified Preston broth, incubated at 37 °C under microaerophilic conditions for 7 days and then subcultured onto selective Preston agar, erythritol agar with Oxoid selective additives and 5–7% defibrinated horse blood. Commercial test systems (API Campy) were used for identification. The use of the classical bacteriological diagnostic method, which is considered the 'gold' standard, is limited due to the difficulties of cultivation. The identification of new Campylobacter species requires revision of phenotypic identification algorithms. Specific primers for the identification of new Campylobacter species also need to be developed. In our studies, using the KAM-BAC kit, we detected Campylobacter jejuni DNA in clinically healthy birds. Consequently, the carriage of Campylobacter is massive. 30 samples of test material were examined using the molecular-biological method, and 60 samples using the bacteriological method. Analyzing the results of Campylobacter detection, it should be noted that thermophilic Campylobacteria were isolated from 60 clinical samples by the bacteriological method in 5,0% (3 Campylobacter cultures), and from 30 samples by the molecular-biological method in 27,0% (8 positive samples). Based on the analysis of the study results, it is necessary to conduct an in-depth study of the natural sources of Campylobacter hepaticus distribution, virulence factors, pathogenesis and mechanisms of infections caused by these emergent pathogens. The most promising research in the study of the causative agents of Campylobacteriosis in birds will be based on the application of innovative genomic technologies based on multiplex polymerase chain reactions and genome sequencing of Campylobacter hepaticus.

Modern Aspects of Etiological Diagnostics, Clinical Picture and Treatment of Severe Community-Acquired Pneumonia in Soldiers

The problem of community-acquired pneumonia is one of the most relevant for military medicine. The relevance of community-acquired pneumonia is determined by the high incidence of conscription by military personnel, the severity of the clinical course, the presence of severe complications, the duration of labor losses, the tendency to epidemic spread, and the risk of deaths. It is necessary to improve laboratory research methods with the introduction of express methods for verifying bacterial and viral agents, determining the clinical features of the viral-bacterial pneumonia, and justifying the inclusion of antiviral agents in the etiotropic treatment regimen. An expanded complex of microbiological diagnosis of pneumonia has been developed, combining classical bacteriological methods with express methods (polymerase chain reaction, enzyme- linked immunosorbent assay, immunochromatography), which made it possible to determine atypical pathogens and viruses in addition to agents of a bacterial nature. Using these techniques, the modern etiological structure of community-acquired pneumonia in the military has been established, the prevalence of viral-bacterial pneumonia has been revealed. Among viruses, the leadership of adenovirus infection has been established, clinical and laboratory features of the disease are shown depending on the pathogens identified, the feasibility of additional prescribing of antiviral agents in addition to antibiotics in the treatment of viral-bacterial pneumonia is justified.