The classification of bacterial survival strategies in the presence of antimicrobials.

The survival of bacteria under antibiotic therapy varies in nature and is based on the bacterial ability to employ a wide range of fundamentally different resistance mechanisms. This great diversity requires a disambiguation of the term 'resistance' and the development of a more precise classification of bacterial survival strategies during contact with antibiotics. The absence of a unified definition for the terms 'resistance', 'tolerance' and 'persistence' further aggravates the imperfections of the current classification system. This review suggests a number of original classification criteria that will take into account (1) the bacterial ability to replicate in the presence of antimicrobial agents, (2) existing evolutionary stability of a trait within a species, and (3) the presence or absence of specialized genes that determine the ability of a microorganism to decrease its own metabolism or switch it completely off. This review describes potential advantages of the suggested classification system, which include a better understanding of the relationship between bacterial survival in the presence of antibiotics and molecular mechanisms of cellular metabolism suppression, the opportunity to pinpoint targets to identify a true bacterial resistance profile. The true resistance profile in turn, could be used to develop effective diagnostic and antimicrobial therapy methods, while taking into consideration specific bacterial survival mechanisms.

Stenotrophomonas maltophilia virulence: a current view.

Stenotrophomonas maltophilia is an opportunistic pathogen intrinsically resistant to multiple and broad-spectrum antibiotics. Although the bacterium is considered a low-virulence pathogen, it can cause various severe diseases and contributes significantly to the pathogenesis of multibacterial infections. During the COVID-19 pandemic, S. maltophilia has been recognized as one of the most common causative agents of respiratory co-infections and bacteremia in critically ill COVID-19 patients. The high ability to adapt to unfavorable environments and new habitat niches, as well as the sophisticated switching of metabolic pathways, are unique mechanisms that attract the attention of clinical researchers and experts studying the fundamental basis of virulence. In this review, we have summarized the current knowledge on the molecular aspects of S. maltophilia virulence and putative virulence factors, partially touched on interspecific bacterial interactions and iron uptake systems in the context of virulence, and have not addressed antibiotic resistance.

Genetic Alternatives for Experimental Adaptation to Colistin in Three Pseudomonas aeruginosa Lineages.

Pseudomonas aeruginosa is characterized by a high adaptive potential, developing resistance in response to antimicrobial pressure. We employed a spatiotemporal evolution model to disclose the pathways of adaptation to colistin, a last-resort polymyxin antimicrobial, among three unrelated P. aeruginosa lineages. The P. aeruginosa ATCC-27833 reference strain (Pa_ATCC), an environmental P. aeruginosa isolate (Pa_Environment), and a clinical isolate with multiple drug resistance (Pa_MDR) were grown over an increasing 5-step colistin concentration gradient from 0 to 400 mg/L. Pa_Environment demonstrated the highest growth pace, achieving the 400 mg/L band in 15 days, whereas it took 37 and 60 days for Pa_MDR and Pa_ATCC, respectively. To identify the genome changes that occurred during adaptation to colistin, the isolates selected during the growth of the bacteria (n = 185) were subjected to whole genome sequencing. In total, 17 mutation variants in eight lipopolysaccharide-synthesis-associated genes were detected. phoQ and lpxL/PA0011 were affected in all three lineages, whereas changes in pmrB were found in Pa_Environment and Pa_MDR but not in Pa_ATCC. In addition, mutations were detected in 34 general metabolism genes, and each lineage developed mutations in a unique set of such genes. Thus, the three examined distinct P. aeruginosa strains demonstrated different capabilities and genetic pathways of colistin adaptation.

Clonal diversity, antimicrobial resistance, and genome features among nonfermenting gram-negative bacteria isolated from patients with cystic fibrosis in Russia.

Nonfermenting gram-negative (NFGN) bacteria were isolated from cystic fibrosis (CF) patients and subjected to susceptibility testing and whole-genome sequencing. Among 170 enrolled CF patients, 112 (65.9%) were colonized with at least 1 key NFGN species. The species-specific infection rate was highest for Pseudomonas aeruginosa (40.6%) followed by Stenotrophomonas maltophilia (14.1%), Achromobacter spp. (9.4%), and Burkholderia cepacia complex (Bcc, 8.2%) demonstrating a significant age-dependent increase for P. aeruginosa and Achromobacter spp., but not for S. maltophilia or Bcc. P. aeruginosa sequence types (STs) related to high-risk epidemic and global CF clones were carried by 12 (7.1%) and 13 (7.6%) patients, respectively. In total, 47% NFGN isolates, predominantly P. aeruginosa, harbored at least 1 plasmid-borne resistance gene; 5 ST235 isolates carried blaVIM2. Pathogenicity island-borne virulence genes were harbored by 9% NFGN isolates. These findings in conjunction with frequent early colonization by Bcc raised serious concerns regarding infection control in Russian CF centers.

Analysis of Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis Revealed Novel Groups of Filamentous Bacteriophages.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in humans, especially in hospital patients with compromised host defence mechanisms, including patients with cystic fibrosis. Filamentous bacteriophages represent a group of single-stranded DNA viruses infecting different bacteria, including P. aeruginosa and other human and animal pathogens; many of them can replicate when integrated into the bacterial chromosome. Filamentous bacteriophages can contribute to the virulence of P. aeruginosa and influence the course of the disease. There are just a few isolated and officially classified filamentous bacteriophages infecting P. aeruginosa, but genomic studies indicated the frequent occurrence of integrated prophages in many P. aeruginosa genomes. An analysis of sequenced genomes of P. aeruginosa isolated from upper respiratory tract (throat and nasal swabs) and sputum specimens collected from Russian patients with cystic fibrosis indicated a higher diversity of filamentous bacteriophages than first thought. A detailed analysis of predicted bacterial proteins revealed prophage regions representing the filamentous phages known to be quite distantly related to known phages. Genomic comparisons and phylogenetic studies enabled the proposal of several new taxonomic groups of filamentous bacteriophages.

A case of localized paranasal sinusitis associated with Burkholderia cenocepacia ST 1880 in a cystic fibrosis patient.

Burkholderia cepacia complex (Bcc) bacteria are considered to be very dangerous players in cystic fibrosis (CF) pathogenesis and are a criterion for negative prognosis in CF cases. In this report, a pediatric case of paranasal sinusitis caused by Burkholderia cenocepacia in a CF patient is described. This is an unusual case, since the paranasal sinuses were the only colonization locus of B. cenocepacia in this patient for 5 years (2015-2020). The lungs remained microbiologically clear with no clinical or radiological signs of pulmonary function decrease during this time period. The paranasal sinuses were sanitized by endoscopic sinus surgery on the left side (2020). Although having no local or systemic antibiotic treatment from the time of surgery to 2022, no B. cenocepacia were detected in the samples. The case shows the possibility of a prolonged remission of Bcc-associated paranasal sinusitis in the absence of systemic antibiotic therapy.

The structure of Klebsiella pneumoniae K108 capsular polysaccharide is similar to Escherichia coli colanic acid.

The clinical isolate of Klebsiella pneumoniae 1333/P225 was revealed as containing a KL108 K. pneumoniae K locus for capsule biosynthesis. The gene cluster demonstrated a high level of sequence and arrangement similarity with that of the E. coli colanic acid biosynthesis gene cluster. The KL108 gene cluster includes a gene of WcaD polymerase responsible for joining oligosaccharide K units into capsular polysaccharide (CPS), acetyltransferase, pyruvyltransferasefive and genes for glycosyltransferases (Gtrs), four of which have homologues in genetic units of the colanic acid synthesis. The fifth Gtr is specific to this cluster. The work involved the use of sugar analysis, Smith degradation and one- and two-dimensional 1H and 13C NMR spectroscopy to establish the structure of the K108 CPS. The CPS repetitive K unit is composed of branched pentasaccharide with three monosaccharides in the backbone and a disaccharide side chain. The main chain is the same as for colanic acid but the side chain differs. Two bacteriophages infecting K. pneumoniae strain 1333/P225 were isolated and structural depolymerase genes were determined; depolymerases Dep108.1 and Dep108.2 were cloned, expressed and purified. It was demonstrated that both depolymerases specifically cleave the ß-Glcp-(1→4)-α-Fucp linkage between K108 units in the CPS.

Genetic determinants of virulence and antibiotic resistance are common for Pseudomonas aeruginosa ST235 isolates from cystic fibrosis patients from various geographical regions.

The dissemination of multiple-drug resistant high virulent strains of P. aeruginosa in patients with cystic fibrosis is of concern worldwide. Herein, we describe genomic characteristics of ST235 isolates recovered from cystic fibrosis patients in Russia. Successful core-genome background and acquired resistance determinants provide spreading of high-risk clones in cystic fibrosis populations.

Life-On-Hold: Lanthanoids Rapidly Induce a Reversible Ametabolic State in Mammalian Cells.

Until now, the ability to reversibly halt cellular processes has been limited to cryopreservation and several forms of anabiosis observed in living organisms. In this paper we show that incubation of living cells with a solution containing ~50 mM neodymium induces a rapid shutdown of intracellular organelle movement and all other evidence of active metabolism. We have named this state REEbernation (derived from the terms REE (rare earth elements) and hibernation) and found that the process involves a rapid replacement of calcium with neodymium in membranes and organelles of a cell, allowing it to maintain its shape and membrane integrity under extreme conditions, such as low pressure. Furthermore, phosphate exchange is blocked as a result of non-dissolvable neodymium salts formation, which "discharged" the cell. We further showed that REEbernation is characterized by an immediate cessation of transcriptional activity in observed cells, providing an intriguing opportunity to study a snapshot of gene expression at a given time point. Finally, we found that the REEbernation state is reversible, and we could restore the metabolism and proliferation capacity of the cells. The REEbernation, in addition to being an attractive model to further investigate the basic mechanisms of cell metabolism control, also provides a new method to reversibly place a cell into "on-hold" mode, opening opportunities to develop protocols for biological samples fixation with a minimum effect on the omics profile for biomedical needs.

Mobile Colistin Resistance Genetic Determinants of Non-Typhoid Salmonella enterica Isolates from Russia.

Polymyxin resistance, determined by mcr genes located on plasmid DNA, currently poses a high epidemiological threat. Non-typhoid Salmonella (NTS) are one of the key pathogens causing diarrheal diseases. Here, we report the isolation and whole genome sequencing of multidrug colistin-resistant/susceptible isolates of non-typhoid Salmonella enterica serovars carrying mcr genes. Non-typhoid strains of Salmonella enterica subsp. enterica were isolated during microbiological monitoring of the environment, food, and diarrheal disease patients between 2018 and 2020 in Russia (n = 586). mcr-1 genes were detected using a previously developed qPCR assay, and whole genome sequencing of mcr positive isolates was performed by both short-read (Illumina) and long-read (Oxford Nanopore) approaches. Three colistin-resistant isolates, including two isolates of S. Enteritidis and one isolate of S. Bovismorbificans, carried the mcr-1.1 gene located on IncX4 and IncI2 conjugative plasmids, respectively. The phenotypically colistin-susceptible isolate of S. Typhimurium carried a mcr-9 gene on plasmid IncHI2. In conclusion, we present the first three cases of mcr gene-carrying NTS isolates detected in Russia with both outbreak and sporadic epidemiological backgrounds.

Genotypes, carbapenemase carriage, integron diversity and oprD alterations among carbapenem-resistant Pseudomonas aeruginosa from Russia.

Pseudomonas aeruginosa is a serious opportunistic pathogen demonstrating a high level of resistance to many groups of antibiotics, including carbapenems. This study aimed to characterise the molecular epidemiology and prevalence of mobile genetic elements associated with resistance to carbapenems among P. aeruginosa (CRPA) clinical isolates. Among 145 carbapenem-resistant P. aeruginosa isolates, 34 different sequence types (STs) were detected; the six most common STs were ST654 (24%), ST235 (24%), ST111 (8%), ST446 (6%), ST357 (5%) and ST2592 (a novel single-locus variant of ST357) (4%). A carbapenemase gene was found in 94 isolates (64.8%). The blaVIM-2 gene was harboured by 64 isolates (44.1%) restricted to ST111, ST235 and ST654, and the blaGES-type and blaOXA-10 group genes were each detected in 15 isolates (10.3%); none of other tested carbapenemase genes, including blaIMP, blaNDM and blaGIM, were detected. Among the blaVIM-2-positive isolates, five types of blaVIM-2-containing integrons were discovered, including In56, In559, In59-like, In59 and In249. The oprD gene was disrupted by an insertion sequence (IS) in 15.9% of isolates. Overall, five types of IS elements were found (ISPsme1, ISPa1328, ISPa26, ISPst2 and ISPa195). Observed rearrangements within variable regions of blaVIM-2-carrying integrons in conjunction with the discovery of a novel type of oprD-disrupting IS element illustrate the ongoing evolution of CRPA a, which warrants further investigation.

A rapid method of whole cell sample preparation for scanning electron microscopy using neodymium chloride.

The quality of electron microscopy (EM) visualization of biological objects is constantly improving, primarily with the usage of more complex technologies, such as serial block-face scanning electron microscopy (SEM), focused ion beam scanning electron microscopy, and array tomography. Here we suggest a new rapid method of whole cell sample preparation for scanning EM using neodymium chloride treatment followed by staining with lead acetate. This variant of sample preparation does not require separate fixation, complete dehydration, and metal sputtering. By means of SEM in the back-scattered electron mode, in the neodymium-treated preparations, we visualized various morphological structures in human cells (nuclei with nucleoli, cytoskeleton, mitochondria) and microbial cells (Staphylococcus epidermidis, Streptococcus pneumoniae, Escherichia coli, and Candida albicans) preserving their species-specific shape and size. Thus, the suggested method provides additional information combining capabilities of SEM in visualizing cellular surface and transmission EM in detecting intracellular structures. Moreover, biological sample preparation with neodymium and lead is fast, informative, and cost-saving indicating a potential for its practical use for environmental SEM, and can be effectively combined with optical microscopy.

Inactivation of the oprD porin gene by a novel insertion sequence ISPa195 associated with large deletion in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.

OBJECTIVES: Alteration of the porin-encoding gene oprD by insertion sequences (ISs) is one mechanism conferring carbapenem resistance in Pseudomonas aeruginosa. Here we describe a carbapenem-resistant clinical P. aeruginosa isolate 36-989 harbouring a novel IS (ISPa195) in oprD. METHODS: Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by the broth microdilution method. Carbapenemase activity was assessed using a MALDI-TOF/MS-based assay of meropenem hydrolysis. Efflux-dependent carbapenem resistance was evaluated using an assay with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). The oprD gene and IS sequence were analysed by the Sanger method. Whole-genome sequencing was performed on an Illumina HiSeq 2500 platform. RESULTS: Antimicrobial susceptibility testing demonstrated that P. aeruginosa 36-989 was resistant to imipenem (MIC=32mg/L) and meropenem (MIC=16mg/L). No carbapenemase activity was detected, however an efflux-mediated component of carbapenem resistance was revealed. A new IS element (ISPa195) was found in the oprD gene of P. aeruginosa 36-989. ISPa195 was 1190bp in length, belonging to the IS3 family, and contained two open reading frames that overlapped through a ribosomal slippage to translate the full-size transposase enzyme. There was an IS-associated 284-bp deletion in the oprD gene; no direct repeats at flanking regions of the IS were detected. CONCLUSION: The absence of direct repeats at flanking regions in combination with the IS-associated deletion distinguished ISPa195 from other ISs previously detected in oprD. Carbapenem resistance in P. aeruginosa 36-989 was conferred by a combination of oprD alteration and carbapenem efflux.

Exogenous contaminating DNA in Taq polymerases: A method to avoid false-positive results when detecting the blaTEM gene.

In the course of developing an assay to identify genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that standard (not DNA-free) Taq DNA polymerases were contaminated with blaTEM gene fragments that varied in length and quantities. The complete blaTEM gene sequence was either absent or was detected in infinitesimal amounts. We developed an approach to avoid false-positive findings caused by contaminating blaTEM gene sequences in conventional polymerases. The method is based on selection of a target sequence to be detected within the blaTEM gene in such a way that the chosen sequence is amplified with primers incapable of amplifying contaminating DNA sequences of the polymerase.

Emergence of the Uncommon Clone ST944/ST78 Carrying blaOXA-40-like and blaCTX-M-like Genes Among Carbapenem-Nonsusceptible Acinetobacter baumannii in Moscow, Russia.

Carbapenem-nonsusceptible (Carba-NS) Acinetobacter baumannii has emerged as an important cause of nosocomial infections. In the present study, we characterized 91 Carba-NS A. baumannii isolates collected from patients of surgical departments and intensive care units at three hospitals in Moscow in 2012-2015. Multilocus sequence typing (MLST) using the Oxford (Oxf) scheme identified 16 sequence types (STs) of three clonal complexes (CCs), including CC92Oxf (67%), CC109Oxf (1%), CC944Oxf (29%), and the singleton ST1100Oxf (3%). CC944Oxf was composed of ST944Oxf (n = 16) and two of its newly described single locus variants ST1103Oxf (n = 3) and ST1104Oxf (n = 7); all the three STs were identical to the Pasteur (Pas) MLST scheme ST78. All CC944Oxf/ST78Pas isolates were blaOXA-40-like positive and all but one isolate harbored a blaCTX-M-like gene. ST944Oxf was the only ST found in each of the three study hospitals. Biofilm growth capacity was similar among Carba-NS and nonclonal carbapenem-susceptible isolates. Our data demonstrate the predominance of two clonal lineages among Carba-NS A. baumannii. One of these, the uncommon blaOXA-40-like/blaCTX-M-like-positive clone of CC944Oxf/ST78Pas, seems to be endemic in Russia.

Vesicle formation as a result of interaction between polymorphonuclear neutrophils and Staphylococcus aureus biofilm.

Staphylococcus aureus, a major opportunistic pathogen, is a leading cause of biofilm-related infections in clinical practice. Staphylococcal biofilms are highly resistant to antibacterial medicines and immune effector cells. The main result of our work is the discovery of nano-vesicles in the supernatant of the human neutrophil-S. aureus biofilm system. We also found that phospholipase C treatment causes complete destruction of these vesicles. While the addition of proteinase K led to a partial structural disorganization of the vesicles, DNase treatment did not influence the vesicle structure. These observations allowed us to conclude that phospholipids and proteins play a structure-forming role in the formation of these nano-vesicles. The vesicles demonstrated anti-biofilm activities when tested against Staphylococcus epidermidis (strains 178M and 328/5) biofilms, but were ineffective for S. aureus (strains 5983/2, 5663 and 18A) biofilms.