Clinical Characteristics and Antimicrobial Susceptibility of Mycobacterium intracellulare and Mycobacterium abscessus Pulmonary Diseases: A Retrospective Study

The incidence of nontuberculous mycobacteria (NTM) diseases is increasing every year. The present study was performed to investigate the clinical characteristics, CT findings, and drug susceptibility test (DST) results of patients diagnosed with M. intracellulare or M. abscessus nontuberculous mycobacterial pulmonary disease (NTMPD). This retrospective study included patients diagnosed with NTMPD due to M. intracellulare or M. abscessus for the first time at Anhui Chest Hospital between 01/2019 and 12/2021. The patients were grouped as M. intracellulare-NTMPD group or M. abscessus-NTMPD group. Clinical features, imaging data and DST data, were collected. Patients with M. intracellulare infection had a higher rate of acid-fast smears (66.1% vs. 45.2%, P = 0.032 ) and a higher rate of cavitation based on pulmonary imaging (49.6% vs. 19.4%, P = 0.002 ) than patients with M. abscessus infection, but both groups had negative TB-RNA and GeneXpert results, with no other characteristics significant differences. The results of DST showed that M. intracellulare had high susceptibility rate to moxifloxacin (95.9%), amikacin (90.1%), clarithromycin (91.7%), and rifabutin (90.1%). M. abscessus had the highest susceptibility rate to amikacin (71.0%) and clarithromycin (71.0%). The clinical features of M. intracellulare pneumopathy and M. abscessus pneumopathy are highly similar. It may be easily misdiagnosed, and therefore, early strain identification is necessary. M. intracellulare has a high susceptibility rate to moxifloxacin, amikacin, clarithromycin, and rifabutin, while M. abscessus has the highest susceptibility rate to amikacin and clarithromycin. This study provides an important clinical basis for improving the management of NTMPD.

Whole-genome based strain identification of fowlpox virus directly from cutaneous tissue and propagated virus

Fowlpox (FP) is an economically important viral disease of commercial poultry. The fowlpox virus (FPV) is primarily characterised by immunoblotting, restriction enzyme analysis in combination with PCR, and/or nucleotide sequencing of amplicons. Whole-genome sequencing (WGS) of FPV directly from clinical specimens prevents the risk of potential genome modifications associated with in vitro culturing of the virus. Only one study has sequenced FPV genomes directly from clinical samples using Nanopore sequencing, however, the study didn’t compare the sequences against Illumina sequencing or laboratory propagated sequences. Here, the suitability of WGS for strain identification of FPV directly from cutaneous tissue was evaluated, using a combination of Illumina and Nanopore sequencing technologies. Sequencing results were compared with the sequence obtained from FPV grown in chorioallantoic membranes (CAMs) of chicken embryos. Complete genome sequence of FPV was obtained directly from affected comb tissue using a map to reference approach. FPV sequence from cutaneous tissue was highly similar to that of the virus grown in CAMs with a nucleotide identity of 99.8%. Detailed polymorphism analysis revealed the presence of a highly comparable number of single nucleotide polymorphisms (SNPs) in the two sequences when compared to the reference genome, providing essentially the same strain identification information. Comparative genome analysis of the map to reference consensus sequences from the two genomes revealed that this field isolate had the highest nucleotide identity of 99.5% with an FPV strain from the USA (Fowlpox virus isolate, FWPV-MN00.2, MH709124) and 98.8% identity with the Australian FPV vaccine strain (FWPV-S, MW142017). Sequencing results showed that WGS directly from cutaneous tissues is not only rapid and cost-effective but also provides essentially the same strain identification information as in-vitro grown virus, thus circumventing in vitro culturing.

Comparison of bacteria disintegration methods and their influence on data analysis in metabolomics

Metabolomic experiments usually contain many different steps, each of which can strongly influence the obtained results. In this work, metabolic analyses of six bacterial strains were performed in light of three different bacterial cell disintegration methods. Three strains were gram-negative (Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae), and three were gram-positive (Corynebacterium glutamicum, Bacillus cereus, and Enterococcus faecalis). For extraction, the methanol–water extraction method (1:1) was chosen. To compare the efficiency of different cell disintegration methods, sonication, sand mill, and tissue lyser were used. For bacterial extract metabolite analysis, 1H NMR together with univariate and multivariate analyses were applied. The obtained results showed that metabolite concentrations are strongly dependent on the cell lysing methodology used and are different for various bacterial strains. The results clearly show that one of the disruption methods gives the highest concentration for most identified compounds (e. g. sand mill for E. faecalis and tissue lyser for B. cereus). This study indicated that the comparison of samples prepared by different procedures can lead to false or imprecise results, leaving an imprint of the disintegration method. Furthermore, the presented results showed that NMR might be a useful bacterial strain identification and differentiation method. In addition to disintegration method comparison, the metabolic profiles of each elaborated strain were analyzed, and each exhibited its metabolic profile. Some metabolites were identified by the 1H NMR method in only one strain. The results of multivariate data analyses (PCA) show that regardless of the disintegration method used, the strain group can be identified. Presented results can be significant for all types of microbial studies containing the metabolomic targeted and non-targeted analysis.

First Report of Nosocomial Outbreak of Vancomycin-Resistant Enterococcus Faecium Infection Among COVID-19 Patients Hospitalized in a Non-Intensive Care Unit Ward in Central Europe

Background: The COVID-19 pandemic in 2020 exerted immense pressure on health care systems worldwide, causing substantial resources to be diverted to respond to the pandemic. These changes raise the concern about the potential for reduction in adherence to long-established measures in the prevention of healthcare-associated infections (HAI). Enterococcus species account for most of human enterococcal HAI and multidrug-resistant infections and have become a major threat to modern public health. We examine the rise in the number of vancomycin resistant E. faecium blood stream and urinary tract infections in a COVID-19 department during an epidemiologic outbreak investigation to detect and eliminate nosocomial clusters of the bacteria. Methods: Strain identification was performed by classical isolation and biochemical and cultivation methods. Antibiotic testing results were interpreted according to European committee on antimicrobial susceptibility testing (EUCAST) guidelines. Six isolated samples underwent whole genome sequencing (WGS) during the outbreak investigation. Isolate relatedness was determined using core genome multilocus sequence typing.Results: WGS revealed two genotypically distinct VRE clusters, one of which had genetically closely related patient and environmental isolates. The cluster was terminated by enhanced infection control strategies.Conclusions: This study provides the first description of an outbreak caused by vanA-ST17 E. faecium strains among COVID-19 patients in central Europe, and the first description of an outbreak caused by vanB-ST117 and vanA-ST17 E. faecium strains in Slovakia. This study can help raise awareness about the need for strict adherence to infection control measures and the implementation of rational antimicrobial stewardship as a routine part of COVID-19 management.

Phylogenomic Analysis Substantiates the gyrB Gene as a Powerful Molecular Marker to Efficiently Differentiate the Most Closely Related Genera Myxococcus, Corallococcus, and Pyxidicoccus

Rapid and accurate strain identification of the most closely related genera Myxococcus, Corallococcus, and Pyxidicoccus can enhance the efficiency of the mining of novel secondary metabolites through dereplication. However, the commonly used 16S rRNA gene sequencing cannot accurately differentiate members of the three genera above, and the whole-genome sequencing is unable to rapidly and inexpensively provide species assignation toward a large number of isolates. To overcome the limitations, the gyrB gene was investigated as a candidate genetic marker for exploring the phylogenetic relationships of bacteria within the three genera and for developing the gyrB-based typing method. Here, the bacterial phylogeny and species affiliations of the three genera were determined based on the phylogenomic reconstruction and the analysis of digital DNA–DNA hybridization values among 90 genomes, further confirming nine novel taxa and assigning over one-third of genomes to defined species. The phylogenetic relationships of these strains based on the gyrB gene sequences were congruent with those based on their genome sequences, allowing the use of the gyrB gene as a molecular marker. The gyrB gene-specific primers for the PCR-amplification and sequencing of bacteria within the three genera were designed and validated for 31 isolates from our group collection. The gyrB-based taxonomic tool proved to be able to differentiate closely related isolates at the species level. Based on the newly proposed 98.6% identity threshold for the 966-bp gyrB gene and the phylogenetic inference, these isolates were assigned into two known species and eight additional putative new species. In summary, this report demonstrated that the gyrB gene is a powerful phylogenetic marker for taxonomy and phylogeny of bacteria within the closely related genera Myxococcus, Corallococcus, and Pyxidicoccus, particularly in the case of hundreds or thousands of isolates in environmental studies.

Clinical and Microbiological Characterization of Carbapenem-Resistant Enterobacteriales: A Prospective Cohort Study

Aim: We aim to depict the clinicoepidemiological and molecular information of carbapenem-resistant Enterobacteriales (CRE) in Chongqing, China.Methods: We performed a prospective, observational cohort study, recruiting inpatients diagnosed with CRE infections from June 1, 2018, to December 31, 2019. We carried out strain identification and molecular characterization of CRE. eBURST analysis was conducted to assess the relationships among the different isolates on the basis of their sequence types (STs) and associated epidemiological data using PHYLOViZ. Clinical parameters were compared between the carbapenemase-producing Enterobacteriales (CPE) and non-CPE group.Findings: 128 unique CRE isolates from 128 patients were collected during the study period: 69 (53.9%) CPE and 59 (46.1%) non-CPE. The majority of CPE isolates were blaKPC-2 (56.5%), followed by blaNDM (39.1%) and blaIMP (5.8%). Klebsiella pneumoniae carbapenemase (KPC)–producing clonal group 11 Klebsiella pneumoniae (K. pneumoniae) was the most common CPE. Antibiotic resistance was more frequent in the CPE group than in the non-CPE group. Independent predictors for CPE infection were ICU admission and hepatobiliary system diseases. Although, there was no significant difference in desirability of outcome ranking (DOOR) outcomes between the two groups. At 30 days after index culture, 35 (27.3% ) of these patients had died.Conclusion: CRE infections were related to high mortality and poor outcomes, regardless of CRE subgroups. CPE were associated with prolonged ICU stays and had different clinical and microbiological characteristics than non-CPE. The identification of CPE/non-CPE and CRE resistance mechanisms is essential for better guidance of the clinical administration of patients with CRE infections.

Novel real-time PCR based assays for differentiating fall armyworm strains using four single nucleotide polymorphisms

The fall armyworm, Spodoptera frugiperda, is a polyphagous global pest with a preference for gramineous crops such as corn, sorghum and pasture grasses. This species is comprised of two morphologically identical but genetically distinct host strains known as the corn and rice strains, which can complicate pest management approaches. Two molecular markers are commonly used to differentiate between strains, however, discordance between these markers can lead to inconclusive strain identification. Here, we used double digest restriction site associated DNA sequencing to identify diagnostic single nucleotide polymorphisms (SNPs) with alleles unique to each strain. We then used these strain-specific SNPs to develop four real-time PCR based TaqMan assays to rapidly and reliably differentiate between strains and interstrain hybrids. These assays provide a new tool for differentiating between strains in field-collected samples, facilitating future studies on strain population dynamics and interstrain hybridization rates. Understanding the basic ecology of S. frugiperda strains is necessary to inform future management strategies.

Identification and Antibiotic Profiling of Wohlfahrtiimonas chitiniclastica, an Underestimated Human Pathogen

In the past 12 years, several case reports have clearly demonstrated that Wohlfahrtiimonas chitiniclastica is capable of causing sepsis and bacteremia in humans. However, since most clinicians are not familiar with this species, little is known about its pathogenicity and treatment options while it is as rare but underestimated human pathogen. Therefore, a larger strain collection is required so that methods can be identified that are most suitable to obtain rapid and reliable identification. Moreover, the antimicrobial resistance profile needs to be elucidated in order to explore possible treatment options. Over a period of 6 years, we therefore have collected a total of 14 W. chitiniclastica isolates in routine diagnostics, which now served as the basis for a comprehensive characterization with respect to identification and antibiotic profiling. We compared the accuracy and convenience of several identification techniques in which MALDI-TOF MS and sequencing of the 16S rRNA gene have proven to be suitable for identification of W. chitiniclastica. In addition, whole genome sequencing (WGS)-based digital DNA-DNA hybridization (dDDH) was used as a reference method for strain identification, and surprised with the detection of a novel W. chitiniclastica subspecies. A combination of in silico and in vitro analyses revealed a first insight into the antimicrobial resistance profile and the molecular basis of antimicrobial resistance. Based on our findings, trimethoprim/sulfamethoxazole, levofloxacin, and cephalosporins (e.g., ceftazidime) may be the best antibiotics to use in order to treat infections caused by W. chitiniclastica, while resistance to fosfomycin, amikacin and tobramycin is observed.

Supervivencia intracelular de cepas de Mycobacterium bovis de alta y baja frecuencia probado en un modelo de macrófagos bovinos

Bovine tuberculosis is a disease caused by Mycobacterium bovis that affects cattle and other species, including humans. Mycobacterium bovis resides mainly in macrophages, so bacilli survival within macrophages is related to virulence. Isolation and strain identification are important for disease control. However, little is known about virulence of the circulating strains in cattle populations. Therefore, the aim of this study was to compare the intracellular survival of Mycobacterium bovis strains with high and low frequency genotypes in cattle in Mexico. Four high frequency genotypes and four low frequency genotypes were identified and subjected to intracellular survival assays in bovine macrophages. Results showed that the phagocytosis proportion was approximately 63 % for all strains. There were no significant differences in the average Colony Forming Units (CFUs) in phagocytosis and survival between the high and low frequency groups; however, when the CFU average of phagocytosis was compared with the survival, significant differences were found in both groups. In intracellular growth, a significant difference was observed between low and high frequency strains, and between low frequency strains. Finally, the intracellular growth average of the groups was analyzed showing no significant difference. These results suggest that the frequency of the genotype in cattle population is not related to the intracellular survival and the virulence of the M. bovis strains.

The first isolation and detection of Ornithobacterium rhinotracheale from swollen head syndrome-infected broiler flocks in Iraq

Background and Aim: The swollen head syndrome (SHS) makes up complex diseases that infect the upper respiratory tract in poultry and causes several economic losses. Furthermore, this syndrome is considered one of the multifactorial etiological agents. Therefore, this study isolated and molecularly detected Ornithobacterium rhinotracheale (ORT) in poultry. Materials and Methods: This study was conducted at 67 broiler farms that had birds observed to be infected with the SHS from September 2018 until August 2019. Subsequently, swabs were collected from their trachea, infraorbital sinuses, and lungs, after which obtained samples were treated through two methods: (a) The direct method, by uploading samples on FTA cards, and the indirect method using a transport media. Afterward, reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze the directly treated samples; howeverAQ1, the culture method, followed by PCR, was used to analyze the indirectly treated samples. Next, a partial 16S RNA gene was isolated using four positive PCR products, after which the effect of 16 antibiotics was studied on the seven local ORT strains isolated. Results: The quantity of ORT isolated using the direct method was 28 (41.7%) samples, which were all positive for the strain. Identification was by direct molecular identification (RT-PCR) from samples loaded on FTA cards. Alternatively, 7 (10.4%) ORTs were detected from the indirect method, as obtained using the culture method and biochemical tests. Then, PCR was subsequently used to confirm the results. As observed, 784 bp bands were shown for all seven ORT isolates. Furthermore, results revealed a significant difference in the detection of ORT strains between direct and indirect methods, with p-value (<0.05) and standard deviation of the error ±0.038 for the direct, then ±0.061 for the indirect method. For further analysis on the strain types, four 784 bp PCR products were taken, then partial 16S ribosomal sequence typing was conducted. All these four strains were found to be recorded in NCBI for the 1st time as a local Iraqi strain, with accession numbers (MN931657, MN931656, MN931655, and MN931654). Notably, results also showed that all isolated strains were multidrug-resistant. Conclusion: From the results, ORT is proposed to be implicated as one of the etiological factors that cause SHSs in poultry. Phylogenetic analysis of the current ORT bacterial strains also showed that they are closely related to the Egyptian isolates.