Purulent inflammatory diseases of various types and etiology comprise major causes of death among the HIV-infected individuals. The purpose of this work was to determine a variety of communityacquired pathogens causing pneumonia, their antibiotic resistance profiles, and dependence on the CD4 lymphocyte levels, as well as identification of methicillin-resistant Staphylococcus aureus species and their molecular genetic characteristics in HIV-infected patients from the Krasnoyarsk City. Over the period of 2012 to 2016, we have examined 152 HIV-infected patients at the Clinical Pulmonology Department with a verified diagnosis of community-acquired pneumonia. Sputum specimens, bronchoalveolar lavage, pleural fluid, washings, pleural pus, as well as nasal and pharyngeal smears were studied for microflora, and blood tests for sterility were performed in these patients, by means of bacteriological techniques. Antibiotic sensitivity was determined by the disc diffusion method; drug sensitivity of staphylococci was performed by screening, PCR technique, serial dilution in semi-solid medium, according to the CLSI and EUCAST recommendations. PCR, M-PCR, and gene sequencing were applied for genotyping and determination of their genetic features. The results were processed with WHONET digital program (WHO). The significance level was p < 0.05. During the entire study period, the yeast-like fungi of Candida genus (30.4 and 35.6%) were consistently isolated from HIV-infected patients. These microorganisms were isolated in a pure cultures at etiologically significant amounts from one-third of the HIV-infected cohort. At the same time, they formed active associations, mostly with Enterobacteriaceae family members. At the same time, Candida fungi were most frequently detected in the lower respiratory tract of those HIV-infected persons who showed severe immunodeficiency (CD4 cell levels < 200 cells/µl). We have also isolated non-fermenting Gram-negative bacteria (12.9%), staphylococci (8.9%), and Enterobacteriaceae (4.4%). The microorganisms were characterized by polyresistance to antimicrobial agents. The MRSA clone circulating in the HIV-infected cohort was characterized as ST239/spa3(t037) /agr1/SCCmecIII.1.1.2 (IIIA)/coaIV/tst+ with high virulence and multiresistance levels. Hence, we have found a number of poly-resistant microorganisms playing a role for development of community-acquired pneumonia in HIV-infected patients, i.e., Candida spp, Gram-negative microorganisms, MRSA, often presenting a component of microbial associations. Candida fungi were detected most often in the HIV-infected individuals with severe immunodeficiency, at the CD4 level of < 200 cells/µl. High detection frequency of such microflora requires some modifications of antimicrobial therapy in HIV-infected subjects affected by the community-acquired pneumonia.
государственный медицинский университет имени профессора В. Ф. Войно-Ясенецкого, Красноярск 660022, Российская Федерация 2 Российско-Японский центр микробиологии, метагеномики и инфекционных заболеваний, Красноярск 660022, Российская Федерация 3 Медико-санитарная часть № 24 Федеральной службы исполнения наказаний, Красноярск 660036, Российская Федерация 4 Международный медицинский образовательно-исследовательский центр (IMERC) Ниигата, Япония. Цель исследования. У лиц, инфицированных вирусом иммунодефицита человека, инфекции, вызванные Staphylococcus aureus, составляют значительную долю, при этом особую тревогу вызывают случаи выявления у данных лиц метициллинрезистентных S. aureus (MRSA), т.к. могут вызывать серьезные поражения органов и систем. Необходимо выявлять частоту носительства S. aureus, MRSA среди ВИЧ-инфицированных, в т.ч. находящихся в пенитенциарном учреждении в Красноярском крае, а также изучать молекулярно-генетические особенности выделенных MRSA. Цель-изучение молекулярно-генетических особенностей MRSA, выделенных от ВИЧ-инфицированных, находящихся в пенитенциарном учреждении. Материал и методы. Для выявления частоты носительства S. aureus, MRSA в работе использован бактериологический метод. Для генотипирования и определения молекулярно-генетических особенностей-ПЦР, М-ПЦР, секвенирование. Обработку результатов проводили с использованием компьютерной программы WHONET (ВОЗ).
The problem of microbial antibiotic resistance and investigation of its underlying mechanisms is of paramount importance for all fields of clinical medicine, including oncology. The aim of the study was to examine the mechanisms of antibiotic resistance for major pathogens causing purulent-inflammatory complications in cancer patients. In 2012-2015, there was conducted a prospective examination of 184 cancer patients, including 67 patients at the Department of Surgery no.1 and 117 patients at the Anesthesiology-Intensive Care Unit of the Krasnoyarsk Regional Clinical Oncology Center named after A.I. Kryzhanovsky. For this, we collected bronchoalveolar lavage, wound discharge and investigated by using bacteriological method, as well as MALDI-TOF. Antibiotic sensitivity was studied as follows: disco-diffusion; double disc method; carbapenem inactivation method; staphylococcal sensitivity – by screening method, PCR, E-test method, and serial dilutions in Muller-Hinton broth. Genotyping and antibiotic resistance mechanisms were performed by using PCR, M-PCR, and sequencing. The WHONET program (WHO) was used, with significance level set at p <0.05. Microbiological examination of bronchoalveolar lavage and wound discharge samples allowed to uncover prevalent associations of multi-(MDR) and extremely resistant pathogens (XDR). In the microflora of the lower respiratory tract and in the wound secretion in cancer patients were found to be dominated by non-fermenting Gram-negative bacteria reaching up to 44.5% and 48%, respectively; as well as order Enterobacteriales found in 24% and 34.9%, respectively; Gram-positive bacteria - 24% and 17.1%, respectively. Imipenem- and/or meropenem-resistant P. aeruginosa and A. baumannii, K. pneumoniae strains, were assessed for MBL production phenotypically, as well as the genes of the most common VIM, IMP types, whereas A. baumannii – for OXA-23, OXA-40, and OXA-58; and in K. pneumoniae – for OXA-48. 20 strains and 16 strains of P. aeruginosa and A. baumannii, respectively, were studied by PCR. It was found that A. baumannii strains formed no MBL, but 56.3% of A. baumannii isolates (9 strains) produced OXA-23 and OXA-40 carbapenemases. Among P. aeruginosa strains there were the three of them which possessed VIM (15.0%), whereas the remaining strains formed no MBL, but were resistant to carbapenems being associated with other resistance mechanisms, e.g. efflux, decreased permeability of cell wall etc. Among 6 isolates of K. pneumoniae, 1 strain produced OXA-48. In cancer patients, the percentage of methicillin-resistant strains among all members of order Staphylococcus was 48.9% (4 strains belonged to MRSA). PVL- MRSA strains belonged to the clones ST239/spa3(t037)/SCCmecIIIA/tst,sek,seq+ (75%) and ST8/spa1(t008)/SCCmecIVc/sea+ (25%). MRSA ST239 showed multiple antibiotic resistance: to aminoglycosides (aacA-aphD, aadD genes were detected), linkcosamides/macrolides (the ermA gene was detected), fluoroquinolones (mutations in the GyrA gene - Ser84Leu; in GrlA- Ser80Phe), rifampicin (MIC more than 128 μg/ml; mutations in the rpoB gene are His481Asn, Ile527Met), sulfamethoxazole, tetracycline (tetM gene), and chloramphenicol (66.7% of isolates, the cat gene encoding chloramphenicol acetyl transferase was detected); but sensitive to vancomycin (MIC 1.0 μg/ml), linezolid in 100% of cases. MRSA ST8 are resistant to aminoglycosides (aacA-aphD, aadD genes), lincosamides/macrolides (ermC gene), tetracyclines (tetK gene), chloramphenicol (cat gene); and 100% sensitive to fluoroquinolones, rifampicin (MIC 0.006 μg/ml), sulfamethaxazole, vancomycin (MIC 1.0 μg/ml), daptomycin (MIC 0.094 μg/ml), linezolid (MIC 0.75 μg/ml). Thus, it was found that members of the order Enterobacteriales such as A. baumannii, P. aeruginosa and MRSA retain high resistance to a large number of antibacterial drugs of almost all classes. These data should be taken into account while choosing proper antibiotic therapy, as well as controlling spread of nosocomial infections caused by multiresistant microorganisms.
Изучена роль метициллинрезистентных S. aureus и их молекулярно-генетические особенности в развитии инфекций кожи и мягких тканей (абсцессов, флегмон) у ВИЧ-инфицированных. Материал для исследованиягнойное отделяемое, биоптат. Посев материала осуществляли на комплекс питательных сред по методу Gould, чувствительность определяли стандартными методами дисковой диффузии, скрининга и E-тест. Для генетических исследований MRSA получали суточную культуру и выделяли ДНК. Для генотипирования штаммов (SCCmec, spa; ST, agr) и определения генов вирулентности (42 гена) Summary HIV-infected people are at increased risk of contracting MRSA. The role of MRSA and their molecular genetic features in the development of infections of the skin and soft tissues (abscesses, cellulitis) in HIV-infected individuals were studied. The material for the study-purulent discharge, biopsy. Sowing material was carried out on a complex of culture media. Antibiotic resistance in microorganisms was determined by methods in accordance with international recommendations. For genetic studies, MRSA received a daily culture and DNA was isolated. For the genotyping of strains (SCCmec, spa; ST, agr) and the determination of virulence genes (42 genes),
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