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Characterisation of Methicillin-Resistant Staphylococcus aureus from Alexandria, Egypt
The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt’s second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.
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.
Characterisation of a novel composite SCCmec-SCCfus element in an emerging Staphylococcus aureus strain from the Arabian Gulf region
Fusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that—to the best of our knowledge—has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.Fusidic acid is a steroid antibiotic known since the 1960s. It is frequently used in topical preparations, i.e., ointments, for the treatment of skin and soft tissue infections caused by Staphylococcus aureus. There is an increasing number of methicillin-resistant S. aureus (MRSA) strains that harbour plasmid-borne fusB/far1 or fusC that is localised on SCC elements. In this study we examined a series of related CC30-MRSA isolates from the Arabian Gulf countries that presented with SCCmec elements and fusC, including a variant that—to the best of our knowledge—has not yet formally been described. It consisted of a class B mec complex and ccrA/B-4 genes. The fusidic acid resistance gene fusC was present, but contrary to the previously sequenced element of HDE288, it was not accompanied by tirS. This element was identified in CC30 MRSA from Kuwait, Saudi Arabia and the United Arab Emirates that usually also harbour the Panton-Valentin leukocidin (PVL) genes. It was also identified in CC8 and ST834 isolates. In addition, further CC30 MRSA strains with other SCCmec VI elements harbouring fusC were found to circulate in the Arabian Gulf region. It can be assumed that MRSA strains with SCCmec elements that include fusC have a selective advantage in both hospital and community settings warranting a review of the use of topical antibiotics and indicating the necessity of reducing over-the-counter sale of antibiotics, including fusidic acid, without prescription.
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.
Characterisation of S. aureus/MRSA CC1153 and review of mobile genetic elements carrying the fusidic acid resistance gene fusC
While many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.
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.
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.
Genotyping of methicillin resistant Staphylococcus aureus from the United Arab Emirates
Reports from Arabian Gulf countries have demonstrated emergence of novel methicillin resistant Staphylococcus aureus (MRSA) strains. To address the lack of data from the United Arab Emirates (UAE), genetic characterisation of MRSA identified between December 2017 and August 2019 was conducted using DNA microarray-based assays. The 625 MRSA isolates studied were grouped into 23 clonal complexes (CCs) and assigned to 103 strains. CC5, CC6, CC22 and CC30 represented 54.2% (n/N = 339/625) of isolates with other common CCs being CC1, CC8, CC772, CC361, CC80, CC88. Emergence of CC398 MRSA, CC5-MRSA-IV Sri Lanka Clone and ST5/ST225-MRSA-II, Rhine-Hesse EMRSA/New York-Japan Clone in our setting was detected. Variants of pandemic CC8-MRSA-[IVa + ACME I] (PVL+) USA300 were detected and majority of CC772 strains were CC772-MRSA-V (PVL+), “Bengal- Bay Clone”. Novel MRSA strains identified include CC5-MRSA-V (edinA+), CC5-MRSA-[VT + fusC], CC5-MRSA-IVa (tst1+), CC5-MRSA-[V/VT + cas + fusC + ccrA/B-1], CC8-MRSA-V/VT, CC22-MRSA-[IV + fusC + ccrAA/(C)], CC45-MRSA-[IV + fusC + tir], CC80-MRSA-IVa, CC121-MRSA-V/VT, CC152-MRSA-[V + fusC] (PVL+). Although several strains harboured SCC-borne fusidic acid resistance (fusC) (n = 181), erythromycin/clindamycin resistance (ermC) (n = 132) and gentamicin resistance (aacA-aphD) (n = 179) genes, none harboured vancomycin resistance genes while mupirocin resistance gene mupR (n = 2) and cfr gene (n = 1) were rare. An extensive MRSA repertoire including CCs previously unreported in the region and novel strains which probably arose locally suggest an evolving MRSA landscape. © 2020, The Author(s).
An epidemic CC1-MRSA-IV clone yields false-negative test results in molecular MRSA identification assays: a note of caution, Austria, Germany, Ireland, 2020
We investigated why a clinical meticillin-resistant Staphylococcus aureus (MRSA) isolate yielded false-negative results with some commercial PCR tests for MRSA detection. We found that an epidemic European CC1-MRSA-IV clone generally exhibits this behaviour. The failure of the assays was attributable to a large insertion in the orfX/SCCmec integration site. To ensure the reliability of molecular MRSA tests, it is vital to monitor emergence of new SCCmec types and junction sites.
Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics
Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.