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Comparison of Different Label-Free Raman Spectroscopy Approaches for the Discrimination of Clinical MRSA and MSSA Isolates
Methicillin-resistant Staphylococcus aureus (MRSA) is classified as one of the priority pathogens that threaten human health. Resistance detection with conventional microbiological methods takes several days, forcing physicians to administer empirical antimicrobial treatment that is not always appropriate. A need exists for a rapid, accurate, and cost-effective method that allows targeted antimicrobial therapy in limited time. In this pilot study, we investigate the efficacy of three different label-free Raman spectroscopic approaches to differentiate methicillin-resistant and -susceptible clinical isolates of S. aureus (MSSA). Single-cell analysis using 532 nm excitation was shown to be the most suitable approach since it captures information on the overall biochemical composition of the bacteria, predicting 87.5% of the strains correctly. UV resonance Raman microspectroscopy provided a balanced accuracy of 62.5% and was not sensitive enough in discriminating MRSA from MSSA. Excitation of 785 nm directly on the petri dish provided a balanced accuracy of 87.5%. However, the difference between the strains was derived from the dominant staphyloxanthin bands in the MRSA, a cell component not associated with the presence of methicillin resistance. This is the first step toward the development of label-free Raman spectroscopy for the discrimination of MRSA and MSSA using single-cell analysis with 532 nm excitation. IMPORTANCE Label-free Raman spectra capture the high chemical complexity of bacterial cells. Many different Raman approaches have been developed using different excitation wavelength and cell analysis methods. This study highlights the major importance of selecting the most suitable Raman approach, capable of providing spectral features that can be associated with the cell mechanism under investigation. It is shown that the approach of choice for differentiating MRSA from MSSA should be single-cell analysis with 532 nm excitation since it captures the difference in the overall biochemical composition. These results should be taken into consideration in future studies aiming for the development of label-free Raman spectroscopy as a clinical analytical tool for antimicrobial resistance determination.
The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates
Escherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.
The Dissemination and Molecular Characterization of Clonal Complex 361 (CC361) Methicillin-Resistant Staphylococcus aureus (MRSA) in Kuwait Hospitals
Methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex 361 (CC361-MRSA) is rare among patients' populations globally. However, CC361-MRSA has been isolated with an increasing trend among patients in Kuwait hospitals since 2010. This study investigated the molecular characteristics of CC361-MRSA isolated from patients in Kuwait hospitals in 2016-2018 to understand their genetic relatedness and virulence determinants. Of 5,223 MRSA isolates investigated by DNA microarray, 182 (3.4%) isolates obtained in 2016 (N = 55), 2017 (N = 56), and 2018 (N = 71) were identified as CC361-MRSA. The CC361-MRSA isolates were analyzed further using antibiogram, spa typing and multi locus sequence typing (MLST). Most of the isolates were resistant to fusidic acid (64.8%), kanamycin (43.4%), erythromycin (36.3%), and clindamycin (14.3%) encoded by fusC, aphA3, and erm(B)/erm(C) respectively. Nine isolates (4.9%) were resistant to linezolid mediated by cfr. The isolates belonged to 22 spa types with t3841 (N = 113), t315 (N = 16), t1309 (N = 14), and t3175 (N = 5) constituting 81.3% of the spa types, four genotypes (strain types), CC361-MRSA-[V/VT + fus] (N = 112), CC361-MRSA-IV, WA MRSA-29 (N = 36), CC361-MRSA-V, WA MRSA-70/110 (N = 33) and CC361-MRSA-[V + fus] variant (N = 1). MLST conducted on 69 representative isolates yielded two sequence types: ST361 (11/69) and ST672 (58/69). All CC361-MRSA isolates were positive for cap8, agr1, and the enterotoxin egc gene cluster (seg, sei, selm, seln, selo, and selu). The tst1 was detected in 19 isolates. The immune evasion cluster (IEC) genes type B (scn, chp, and sak) and type E (scn and sak) were detected in 20 and 152 isolates, respectively. The CC361-MRSA circulating in Kuwait hospitals consisted of two closely related sequence types, ST361 and ST672 with ST672-MRSA [V/VT + fus] as the dominant genotype. The dissemination of these newly emerged clones and the emergence of linezolid resistance limits therapeutic options, as well as present significant challenges for the control of MRSA infections in Kuwait hospitals.
Exploring the evolution and epidemiology of European CC1-MRSA-IV: tracking a multidrug-resistant community-associated meticillin-resistant Staphylococcus aureus clone
This study investigated the evolution and epidemiology of the community-associated and multidrug-resistant Staphylococcus aureus clone European CC1-MRSA-IV. Whole-genome sequences were obtained for 194 European CC1-MRSA-IV isolates (189 of human and 5 of animal origin) from 12 countries, and 10 meticillin-susceptible precursors (from North-Eastern Romania; all of human origin) of the clone. Phylogenetic analysis was performed using a maximum-likelihood approach, a time-measured phylogeny was reconstructed using Bayesian analysis, and in silico microarray genotyping was performed to identify resistance, virulence-associated and SCCmec (staphylococcal cassette chromosome mec) genes. Isolates were typically sequence type 1 (190/204) and spa type t127 (183/204). Bayesian analysis indicated that European CC1-MRSA-IV emerged in approximately 1995 before undergoing rapid expansion in the late 1990s and 2000s, while spreading throughout Europe and into the Middle East. Phylogenetic analysis revealed an unstructured meticillin-resistant S. aureus (MRSA) population, lacking significant geographical or temporal clusters. The MRSA were genotypically multidrug-resistant, consistently encoded seh, and intermittently (34/194) encoded an undisrupted hlb gene with concomitant absence of the lysogenic phage-encoded genes sak and scn. All MRSA also harboured a characteristic ~5350 nt insertion in SCCmec adjacent to orfX. Detailed demographic data from Denmark showed that there, the clone is typically (25/35) found in the community, and often (10/35) among individuals with links to South-Eastern Europe. This study elucidated the evolution and epidemiology of European CC1-MRSA-IV, which emerged from a meticillin-susceptible lineage prevalent in North-Eastern Romania before disseminating rapidly throughout Europe.
Long-Term Sinonasal Carriage of Staphylococcus aureus and Anti-Staphylococcal Humoral Immune Response in Patients with Chronic Rhinosinusitis
We investigated Staphylococcus aureus diversity, genetic factors, and humoral immune responses against antigens via genome analysis of S. aureus isolates from chronic rhinosinusitis (CRS) patients in a long-term follow-up. Of the 42 patients who provided S. aureus isolates and serum for a previous study, 34 could be included for follow-up after a decade. Clinical examinations were performed and bacterial samples were collected from the maxillary sinus and nares. S. aureus isolates were characterized by whole-genome sequencing, and specific anti-staphylococcal IgG in serum was determined using protein arrays. S. aureus was detected in the nares and/or maxillary sinus at both initial inclusion and follow-up in 15 of the 34 respondents (44%). Three of these (20%) had S. aureus isolates from the same genetic lineage as at inclusion. A low number of single-nucleotide polymorphisms (SNPs) were identified when comparing isolates from nares and maxillary sinus collected at the same time point. The overall change of antibody responses to staphylococcal antigens over time showed great variability, and no correlation was found between the presence of genes encoding antigens and the corresponding anti-staphylococcal IgG in serum; thus our findings did not support a role, in CRS, of the specific S. aureus antigens investigated.
Sequence Analysis of Novel Staphylococcus aureus Lineages from Wild and Captive Macaques
Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species (Macaca mulatta, M. assamensis, and M. sylvanus) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2/etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton–Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.
ConsensusPrime—A Bioinformatic Pipeline for Ideal Consensus Primer Design
Background: High-quality oligonucleotides for molecular amplification and detection procedures of diverse target sequences depend on sequence homology. Processing input sequences and identifying homogeneous regions in alignments can be carried out by hand only if they are small and contain sequences of high similarity. Finding the best regions for large and inhomogeneous alignments needs to be automated. Results: The ConsensusPrime pipeline was developed to sort out redundant and technical interfering data in multiple sequence alignments and detect the most homologous regions from multiple sequences. It automates the prediction of optimal consensus primers for molecular analytical and sequence-based procedures/assays. Conclusion: ConsensusPrime is a fast and easy-to-use pipeline for predicting optimal consensus primers that is executable on local systems without depending on external resources and web services. An implementation in a Docker image ensures platform-independent executability and installability despite the combination of multiple programs. The source code and installation instructions are publicly available on GitHub.
Characterization of PVL-Positive MRSA Isolates in Northern Bavaria, Germany over an Eight-Year Period
Purpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton–Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period. Methods: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa-typing. Results: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites. Conclusions: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.
Presence of β-Lactamase-producing Enterobacterales and Salmonella Isolates in Marine Mammals
Marine mammals have been described as sentinels of the health of marine ecosystems. Therefore, the aim of this study was to investigate (i) the presence of extended-spectrum β-lactamase (ESBL)- and AmpC-producing Enterobacterales, which comprise several bacterial families important to the healthcare sector, as well as (ii) the presence of Salmonella in these coastal animals. The antimicrobial resistance pheno- and genotypes, as well as biocide susceptibility of Enterobacterales isolated from stranded marine mammals, were determined prior to their rehabilitation. All E. coli isolates (n = 27) were screened for virulence genes via DNA-based microarray, and twelve selected E. coli isolates were analyzed by whole-genome sequencing. Seventy-one percent of the Enterobacterales isolates exhibited a multidrug-resistant (MDR) pheno- and genotype. The gene blaCMY (n = 51) was the predominant β-lactamase gene. In addition, blaTEM-1 (n = 38), blaSHV-33 (n = 8), blaCTX-M-15 (n = 7), blaOXA-1 (n = 7), blaSHV-11 (n = 3), and blaDHA-1 (n = 2) were detected. The most prevalent non-β-lactamase genes were sul2 (n = 38), strA (n = 34), strB (n = 34), and tet(A) (n = 34). Escherichia coli isolates belonging to the pandemic sequence types (STs) ST38, ST167, and ST648 were identified. Among Salmonella isolates (n = 18), S. Havana was the most prevalent serotype. The present study revealed a high prevalence of MDR bacteria and the presence of pandemic high-risk clones, both of which are indicators of anthropogenic antimicrobial pollution, in marine mammals.
Characterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt
This study aimed to investigate the prevalence and antimicrobial resistance of enterococci- and ESBL-producing E. coli isolated from milk of bovine mastitis cases in Egypt. Fifty milk samples of dairy animals were collected from localities in the Nile Delta region of Egypt. Isolates were identified using MALDI-TOF MS, and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Seventeen Enterococcus isolates and eight coliform isolates could be cultivated. Vancomycin resistance rate was high in Ent. faecalis. The VITEK 2 system confirmed all E. coli isolates as ESBL-producing. All Ent. faecalis isolates harbored erm(B), tetL and aac-aphD genes. The vanA gene was detected in Ent. faecalis isolate, vanB was found in other Enterococcus, while one isolate of E. casseliflavus exhibited the vanA gene. E. coli isolates exhibited high prevalence of erm(B) and tetL. E. coli isolates were analyzed by DNA microarray analysis. Four isolates were determined by O-serotyping as O8 (n = 1), O86 (n = 2) and O157 (n = 1). H-serotyping resulted in H11, H12, H21 (two isolates each) and one was of H16 type. Different virulence-associated genes were detected in E. coli isolates including lpfA, astA, celB, cmahemL, intI1 and intI2, and the iroN gene was identified by DNA microarray analysis.