The Impact of Intraspecies Variability on Growth Rate and Cellular Metabolic Activity of Bacteria Exposed to Rotating Magnetic Field

Majority of research on the influence of magnetic fields on microorganisms has been carried out with the use of different species or different groups of microorganisms, but not with the use of different strains belonging to one species. The purpose of the present study was to assess the effect of rotating magnetic fields (RMF) of 5 and 50 Hz on the growth and cellular metabolic activity of eight species of bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter cloacae, Moraxella catarrhalis, and Bacillus cereus. However, contrary to the research conducted so far, each species was represented by at least four different strains. Moreover, an additional group of S. aureus belonging to a single clonal type but representing different biotypes was also included in the experiment. The results showed a varied influence of RMF on growth dynamics and cellular metabolic activity, diversified to the greatest extent in dependence on the bacterial strain exposed to the RMF and to a lesser extent in dependence on the frequency of the generated magnetic field. It was found that, with regard to the exposed strain of the same species, the effect exerted by the RMF may be positive (i.e., manifests as the increase in the growth rate or/and cellular metabolic activity) or negative (i.e., manifests as a reduction of both aforementioned features) or none. Even when one clonal type of S. aureus was used, the results of RMF exposure also varied (although the degree of differentiation was lower than for strains representing different clones). Therefore, the research has proven that, apart from the previously described factors related primarily to the physical parameters of the magnetic field, one of the key parameters affecting the final result of its influence is the bacterial intraspecies variability.

EPIDEMIOLOGY OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS IN ARAB COUNTRIES OF THE MIDDLE EAST AND NORTH AFRICAN REGION

Available data suggests a high burden of methicillin-resistant staphylococcus aureus (MRSA) in Arab countries of the Middle East and North Africa (MENA). To review the MRSA prevalence and molecular epidemiology in this region, we used PubMed search engine to identify relative articles published from January 2005 to December 2019. Great heterogeneity in reported rates was expectedly seen. Nasal MRSA colonization ranged from 0-32% in healthy volunteers but 4-73% in healthcare workers. Infective MRSA rates ranged between 6%-66% in Saudi Arabia, 12%-29% in United Arab of Emirates, 13%-21% in Qatar, 14%-37% in Oman, 20%-72% in Lebanon, 56% in Gaza, 32%-68% in Jordan, 34%-88% in Iraq and Iraqi Kurdistan, 47%-77% in Egypt, 19-86% in Algeria, and 18-40% in Tunisia. In the GCC, [PVL-] ST239-III, [PVL+] ST80-IV, [PVL+] ST30-IV, [PVL-] ST22-IV and its [PVL+] and [tst1+] variants, [PVL-] CC6-IV, [PVL-] CC5-IV, and [PVL-] ST5-II had a significant presence. In the Levant region, [PVL+] ST80-IV, [PVL+] ST30-IV, [PVL-] ST239-III, [PVL-] ST22-IV were prevalent in Lebanon, Jordan, and Gaza; [PVL-] ST22-IVa-t223 (“Gaza strain”) was prevalent in Gaza and Jordan; USA300, and USA800 were prevalent in Iraq. In North Africa, [PVL+] ST80-IV and [PVL-] ST239-III was commonly reported in Egypt, Algeria, and Tunisia. Finally, significant antimicrobial resistance was seen in the region with variation in patterns depending on location and clonal type. For a more accurate assessment of MRSA epidemiology and burden, the Arab countries need to implement national surveillance systems.

Increased capsule thickness and hyper-motility are traits of carbapenem-resistant Acinetobacter baumannii ST3 strains causing fulminant infection

Acinetobacter baumannii is a successful nosocomial pathogen, causing severe, life-threatening infections in hospitalized patients, including pneumonia and bloodstream infections. The spread of CRAB (carbapenem-resistance Acinetobacter baumannii) strains is a major world-health threat. The successful spread of CRAB is mostly due to it's highly plasticity genome. Although some virulence factors associated with CRAB have been uncovered, many mechanisms contributing to its success are not fully understood. Here we describe strains of CRAB that were isolated from fulminant cases in two hospitals in Israel. These isolates show a rare, hyper mucoid (HM) phenotype and were investigated using phenotypic assays, comparative genomics and in vivo Galleria Mellonella model. The three isolates belonged to the ST3 international clonal type and closely related to each-other as shown by Fourier Transform Infrared Spectroscopy (FTIR) and phylogenetic analyses. These isolates possessed thickened capsules and dense filamentous extracellular polysaccharides (EPS) matrix as shown by transmission electron microscopy (TEM), and overexpress capsule polysaccharide synthesis pathway-related wzc gene. The HM isolates possessed a unique combination of virulence genes involved in iron metabolism, protein secretion, adherence and membrane glycosylation. HM strains were more virulent than control strains in two G. mellonella infection models. In conclusion, our findings demonstrated several virulence factors, all present in three CRAB isolates with rare hyper mucoid phenotypes.

Evidence for the Dissemination to Humans of Methicillin-Resistant Staphylococcus aureus ST398 through the Pork Production Chain: A Study in a Portuguese Slaughterhouse

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 was recovered from infections in humans exposed to animals, raising public health concerns. However, contact with food producing chain as a means of transmission of LA-MRSA to humans remains poorly understood. We aimed to assess if pork production chain is a source of MRSA ST398 for human colonization and infection. MRSA from live pigs, meat, the environment, and slaughterhouse workers were analyzed by Pulsed-Field Gel Electrophoresis (PFGE), spa, MLST typing, SNPs and for antibiotic resistance and virulence gene profiles. We compared core and accessory genomes of MRSA ST398 isolated from slaughterhouse and hospital. We detected MRSA ST398 (t011, t108, t1451) along the entire pork production chain (live pigs: 60%; equipment: 38%; meat: 23%) and in workers (40%). All MRSA ST398 were multidrug resistant, and the majority carried genes encoding biocide resistance and enterotoxins. We found 23 cross-transmission events between live pigs, meat, and workers (6–55 SNPs). MRSA ST398 from infection and slaughterhouse environment belonged to the same clonal type (ST398, t011, SCCmec V), but differed in 321–378 SNPs. Pork production chain can be a source of MRSA ST398 for colonization of human slaughterhouse workers, which can represent a risk of subsequent meat contamination and human infection.

Isolation and genetic characterization of Toxoplasma gondii in Spanish sheep flocks

Background Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain. Methods Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes. Results As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms. Conclusions The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity.

Molecular characterization of carbapenem-resistant Acinetobacter baumannii using WGS revealed missed transmission events in Germany from 2012–15

Background Infection and colonization with multi-resistant Acinetobacter baumannii causes therapeutic and economic problems in the nosocomial setting. Due to the sensitivity issue of screening schemes for A. baumannii, it is difficult to implement adequate transmission prevention measures. The high discriminatory power of WGS for transmission-chain analysis provides us with the necessary tool to study and identify transmission events. We retrospectively sequenced and analysed 39 A. baumannii isolates from 2012–15 to search for possible missed transmission events. Methods Molecular typing by WGS was performed for non-repetitive (n=39) carbapenem-resistant A. baumannii. Retrospective assessment of patient records was performed to investigate and confirm possible transmission events. Results Between July 2012 and September 2015, A. baumannii was isolated from 268 patients, of which 16% (42/268) were carbapenem resistant. Thirty-nine of these isolates were recoverable and sequenced. Fifteen percent (6/39) of these were resistant to all antibiotics tested. Most isolates belong to the circulating IC2 clonal type. SNP analysis revealed four potential outbreak clusters. Two of these clusters showed high concordance with the local spatio-temporal epidemiology, suggesting that transmission events were very likely. Conclusions Our data suggest that there were two independent transmission events, which would have been missed by conventional MLST owing to high clonality. The routine implementation of WGS can optimize surveillance and initiation of suitable containment measures. In addition, emerging resistance to salvage therapy is a major therapeutic problem and should be monitored closely.

Emergence of NDM-1-producing Klebsiella pneumoniae in Greece: evidence of a widespread clonal outbreak

ObjectivesNDM-producing Enterobacteriaceae clinical isolates remain uncommon in the European region. We describe the emergence and broad dissemination of one successful NDM-1-producing Klebsiella pneumoniae clone in Greek hospitals.MethodsDuring a 4 year survey (January 2013–December 2016), 480 single-patient carbapenem non-susceptible K. pneumoniae isolates, phenotypically MBL positive, were consecutively recovered in eight Greek hospitals from different locations and subjected to further investigation. Antimicrobial susceptibility testing, combined-disc test, identification of resistance genes by PCR and sequencing, molecular fingerprinting by PFGE, plasmid profiling, replicon typing, conjugation experiments and MLST were performed.ResultsMolecular analysis confirmed the presence of the blaNDM-1 gene in 341 (71%) K. pneumoniae isolates. A substantially increasing trend of NDM-1-producing K. pneumoniae was noticed during the survey (R2 = 0.9724). Most blaNDM-1-carrying isolates contained blaCTX-M-15, blaOXA-1, blaOXA-2 and blaTEM-1 genes. PFGE analysis clustered NDM-1 producers into five distinct clonal types, with five distinct STs related to each PFGE clone. The predominant ST11 PFGE clonal type was detected in all eight participating hospitals, despite adherence to the national infection control programme; it was identical to that observed in the original NDM-1 outbreak in Greece in 2011, as well as in a less-extensive NDM-1 outbreak in Bulgaria in 2015. The remaining four ST clonal types (ST15, ST70, ST258 and ST1883) were sporadically detected. blaNDM-1 was located in IncFII-type plasmids in all five clonal types.ConclusionsThis study gives evidence of possibly the largest NDM-1-producing K. pneumoniae outbreak in Europe; it may also reinforce the hypothesis of an NDM-1 clone circulating in the Balkans.

Virulence of atypical Toxoplasma gondii strains isolated in French Guiana in a murine model

Background. Toxoplasma gondii is an obligate intracellular protozoan parasite of warm-blooded vertebrates. Most infections in immunocompetent patients are asymptomatic. However, since 2000s, strains with particular genetic profiles that differ from the known clonal type (type I, II, III), have been described. In French Guiana, these strains are highly pathogenic in immunocompetent patients. They have defined a new clinical entity called Amazonian Toxoplasmosis. The present study aims to further improve our knowledge on the pathogenicity of these Amazonian T. gondii strains in comparison with three reference strains using Swiss strain mice. With these data, we tried to establish a predictive virulence score to classify these strains, but also to correlate this virulence with the severity of the disease in infected patients. Results. All the virulence indicators revealed that the Amazonian strains isolated in French Guiana presented a high virulence profile, but lower than the highly virulent type I reference RH strain. The findings reveal differences in virulence between human and animal strains, but also between anthropized and wild strains. Conclusion. In addition to being a clinically relevant animal model of Amazonian Toxoplasmosis, this model could also provide a solid experimental basis for future studies aiming to investigate the underlying mechanisms of Amazonian Toxoplasmosis disease.

A novelqacAallele results in an elevated chlorhexidine glu conate minimum inhibitory concentration in cutaneousStaphylococcus epidermidisisolates

Chlorhexidine gluconate (CHG) is a topical antiseptic widely used in healthcare settings. InStaphylococcusspp., the pump QacA effluxes CHG, while the closely related QacB cannot due to a single amino acid substitution. We characterized 1,050 cutaneousStaphylococcusisolates obtained from 173 pediatric oncology patients enrolled in a multicenter CHG bathing trial. CHG susceptibility testing revealed 63 (6%) of these isolates had elevated CHG MICs (≥ 4 μg/mL). Screening of all 1,050 isolates forqacA/Bby restriction fragment length polymorphism (RFLP) yielded 56 isolates with a novelqacA/BRFLP pattern,qacAB273. The CHG MIC was significantly higher forqacAB273-positive isolates (MIC50: 4 μg/mL, [range: 0.5 – 4 μg/mL]) compared to otherqacgroups:qacA-positive (n=559, 1 μg/mL, [0.5 – 4 μg/mL]),qacB-positive (n=17, 1 μg/mL, [0.25 – 2 μg/mL]), andqacA/B-negative (n=418, 1 μg/mL, [0.125 – 2 μg/mL], p=0.001). TheqacAB273-positive isolates also displayed a high proportion of methicillin resistance (96.4%) compared to otherqacgroups (24.9 – 61.7%, p=0.001). Whole genome sequencing revealed thatqacAB273-positive isolates encoded a variant of QacA with 2 amino acid substitutions. This new allele, namedqacA4, was carried on the novel plasmid pAQZ1. TheqacA4-carrying isolates belonged to the highly resistantS. epidermidisclone ST2 and were collected from multiple centers across the United States and Canada. Curing an isolate ofqacA4resulted in a four-fold decrease in the CHG MIC, confirming the role ofqacA4in the elevated CHG MIC. Our results highlight the importance of further studyingqacA4and its functional role in clinical staphylococci.ImportanceStaphylococcus epidermidisis an important cause of infections in patients with implanted devices. Bathing with chlorhexidine gluconate (CHG), a topical antiseptic, has been shown to reduce rates of device-associated infections, especially those caused byS. epidermidis. InS. epidermidis, reduced susceptibility to CHG is associated with carriage of theqacAgene. As part of a multicenter CHG bathing trial, we obtained cutaneousStaphylococcusisolates from pediatric oncology patients across the United States and Canada. We identified a group of isolates capable of surviving in higher concentrations of CHG and determined a novel allele ofqacA, termedqacA4and carried on the novel plasmid pAQZ1, was responsible for the isolates’ survival in higher CHG concentrations. TheqacA4-carryingS. epidermidisisolates belonged to the highly resistant and virulent ST2 clonal type. Our results highlight the need to understand the global distribution of novelqacAalleles, includingqacA4, and their mechanistic effect on efflux.