- Volume 71, Issue 10, 2022

Introduction. Enterococcus faecium has emerged as an important nosocomial pathogen, which is increasingly difficult to treat due to the genetic acquisition of vancomycin resistance. Ireland has a recalcitrant vancomycin-resistant bloodstream infection rate compared to other developed countries.

Hypothesis/Gap statement. Vancomycin resistance rates persist amongst E. faecium isolates from Irish hospitals. The evolutionary genomics governing these trends have not been fully elucidated.

Methodology. A set of 28 vancomycin-resistant isolates was sequenced to construct a dataset alongside 61 other publicly available Irish genomes. This dataset was extensively analysed using in silico methodologies (comparative genomics, pangenomics, phylogenetics, genotypics and comparative functional analyses) to uncover distinct evolutionary, coevolutionary and clinically relevant population trends.

Results. These results suggest that a stable (in terms of genome size, GC% and number of genes), yet genetically diverse population (in terms of gene content) of E. faecium persists in Ireland with acquired resistance arising via plasmid acquisition (vanA) or, to a lesser extent, chromosomal recombination (vanB). Population analysis revealed five clusters with one cluster partitioned into four clades which transcend isolation dates. Pangenomic and recombination analyses revealed an open (whole genome and chromosomal specific) pangenome illustrating a rampant evolutionary pattern. Comparative resistomics and virulomics uncovered distinct chromosomal and mobilomal propensity for multidrug resistance, widespread chromosomal point-mutation-mediated resistance and chromosomally harboured arsenals of virulence factors. Interestingly, a potential difference in biofilm formation strategies was highlighted by coevolutionary analysis, suggesting differential biofilm genotypes between vanA and vanB isolates.

Conclusions. These results highlight the evolutionary history of Irish E. faecium isolates and may provide insight into underlying infection dynamics in a clinical setting. Due to the apparent ease of vancomycin resistance acquisition over time, susceptible E. faecium should be concurrently reduced in Irish hospitals to mitigate potential resistant infections.

Introduction. Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae are characterized by the World Health Organization as pathogens for which new antibiotics are urgently needed. Omadacycline and eravacycline are two novel antibacterials within the tetracycline class.

Gap Statement. There are limited data regarding the comparison of the activities of omadacycline, eravacycline and tigecycline against K. pneumoniae isolates with different antimicrobial susceptibility profiles.

Aim. Our objective was to compare the in vitro activities of omadacycline, eravacycline and tigecycline against a collection of K. pneumoniae isolates, including non-ESBL-producing, ESBL-producing and carbapenem-resistant strains.

Methodology. Ninety-four K. pneumoniae isolates, including 30 non-ESBL-producing, 30 ESBL-producing and 34 carbapenem-resistant (22 carrying bla OXA-48, 12 carrying bla NDM) strains were included in the study. ESBL and carbapenemase genes were detected by conventional PCR. Omadacycline, eravacycline and tigecycline MICs were determined by the gradient diffusion method and interpreted using US Food and Drug Administration (FDA)-defined breakpoints.

Results. Overall, the percentage of tigecycline-susceptible strains (97.9 %) was higher than the percentage of omadacyline-susceptible (75.5 %) and eravacycline-susceptible (72.3 %) strains. The omadacycline and eravacycline susceptibility rates were 83.3 % among non-ESBL-producing isolates and 66.7 % among ESBL-producing isolates. The most common ESBL gene detected was blaCTX -M (90 %), followed by bla TEM (50 %) and bla SHV (50 %). The omadacycline and eravacycline susceptibility rate among isolates carrying bla TEM was 33.3 %, whereas it was 100 % among isolates that do not carry bla TEM. The omadacycline and eravacycline susceptibility rates among carbapenem-resistant isolates were 76.5 and 67.6 %, respectively. The omadacycline susceptibility rates among bla OXA-48-positive and bla NDM-positive isolates were 77.3 and 75.0 %, respectively. The eravacycline susceptibility rates among bla OXA-48-positive and bla NDM-positive isolates were 68.2 and 66.7 %, respectively. One carbapenem-resistant isolate was intermediate and one ESBL-producing isolate was resistant to tigecycline.

Conclusion. Overall, tigecycline was the most active tetracycline against isolates. Omadacycline and eravacycline showed excellent activity against ESBL-producing K. pneumoniae isolates that do not carry bla TEM. Omadacycline showed reasonable activity against carbapenem-resistant K. pneumoniae isolates carrying bla OXA-48 or bla NDM.

Background. Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, Pseudomonas aeruginosa .

Objectives. We aimed to develop two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I (gyrA248) and GyrA D87N, D87Y and D87H (gyrA259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa .

Methods. Following assay validation, the gyrA248 and gyrA259 SYBR-MAMAs were tested on 84 Australian clinical P. aeruginosa isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing.

Results. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes.

Conclusions. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa . An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.

Introduction. Stenotrophomonas maltophilia is an important multidrug-resistant Gram-negative pathogen. While largely a hospital-acquired pathogen, there have been increasing reports of the pathogen in the community.

Gap Statement. Trends in S. maltophilia prevalence and resistance rates that include outpatient isolates are unknown.

Aim. We described recent trends in prevalence and resistance of S. maltophilia in the national Veterans Affairs (VA) Healthcare system.

Methodology. The study identified positive S. maltophilia clinical cultures among VA adult patients from 2010 to 2018 across all VA hospitals, long-term care facilities/units, and outpatient settings. Annual S. maltophilia resistance rates were evaluated. Multidrug resistant (MDR) was defined as resistance to sulfamethoxazole/trimethoprim (SMX/TMP) and minocycline or levofloxacin. Time trends were assessed with regression analyses to estimate annual average percent changes (AAPC) with 95 % confidence intervals using Joinpoint software.

Results. Over the 9 year study period, 18 285  S . maltophilia cultures were identified (57 % hospital, 3 % long-term care, 40 % outpatient). The most common source of S. maltophilia cultures were respiratory cultures (34.6 %) followed by urine cultures (30.4 %). In VA hospitals and long-term care facilities, the number of S. maltophilia cultures decreased significantly (by 5.4% and 8.4 % per year respectively). Overall, 3.1 % of isolates were MDR which remained stable over the study period. Resistance to other antibiotics assessed mostly remained stable, except SMX/TMP resistance decreased significantly by 8.5 % (2010, 15 %; 2018, 6 %) per year in VA hospitals.

Conclusion. While previous work has recognized S. maltophilia as primarily a nosocomial pathogen, the present study found that 40 % of cultures collected were among outpatients. Between 2010 and 2018, the number of positive S. maltophilia cultures decreased significantly in the national VA Healthcare System. Resistance to SMX/TMP decreased over the study period in VA hospitals and now more closely reflects previously reported resistance rates worldwide (0–10 %). MDR S. maltophilia remained stable and low in the national VA Healthcare System.

Introduction. Tuberculosis (TB) is a great public health problem in developing countries such as Egypt. Genotyping of Mycobacterium tuberculosis isolates has a prominent role in the field of TB prevention.

Aim. This study aimed to evaluate real-time PCR using Minor Groove Binder (MGB) probes and to identify circulating lineages/sub-lineages of M. tuberculosis and their transmission patterns.

Hypothesis. We hypothesize that MIRU-VNTR technique is efficient in identifying circulating M. tuberculosis lineages in Egypt.

Methodology. Fifty sputum specimens positive for acid-fast bacilli were included. Isoniazid (INH) resistance was detected using the 1 % proportion method. Real-time PCR using MGB-probes was used for simultaneous detection of TB infection and INH resistance. Partial sequencing of the katG gene was used to confirm INH resistance results. A standard 15 Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (15-MIRU-VNTR) approach was used for genotyping through the MIRU-VNTRplus online platform.

Results. Only seven specimens showed phenotypic resistance to INH. M. tuberculosis was detected in all samples, while a mutation in the katG gene codon 315 was detected only in five samples, which were also phenotypically INH-resistant. Sequencing of the katG gene showed codon 315 mutation genotypically and phenotypically in the five INH-resistant isolates. Molecular genotyping of M. tuberculosis isolates revealed that the majority of isolates (26/50, 52 %) belonged to the S family of lineage_4. A low clustering rate (2 %) was observed among our isolates. According to the Hunter-Gaston Discriminatory Index (HGDI), 11 MIRU-VNTR loci were highly or moderately discriminative, while four loci were less polymorphic.

Conclusion. MIRU-VNTR genotyping revealed a low clustering rate with a low recent transmission rate of M. tuberculosis strains in Alexandria, Egypt.

Since the introduction of Haemophilus influenzae (Hi) serotype b (Hib) vaccination, reports of increasing incidence rates of non-Hib serotypes have emerged. A systematic review was performed to investigate whether the Hi serotype f (Hif) incidence rate has increased globally and to describe its associated disease burden. In the post-Hib vaccine era, evidence shows that the incidence rate of Hif infection is increasing worldwide. In total 94 studies including 2 701 patients reported Hif infections. The estimated pooled incidence rate of Hif infection was 0.15/100 000 population per year (range: 0.05–0.40/100 000), with a median case fatality ratio of 14.3 %. Invasive infections most frequently presented as pneumonia (45 %), septicaemia (34 %) and meningitis (20 %). Of 191 Hif isolates, 87 % were ampicillin-susceptible. Multi-locus sequence typing revealed that Hif were relatively clonal, with the majority belonging to clonal complex 124. Hif causes invasive infections of significant variance in both severity and presentation. Globally, the Hif population shows little genetic variability and currently appears to possess low resistance to antimicrobials.

Introduction. The possibility of reinfection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a widely proven fact and may have clinical implications.

Hypothesis /Gap Statement. It is not known whether there have been cases of reinfection by SARS-CoV-2 in La Ribera Health Department.

Aim. To determine whether there have been cases of reinfection by SARS-CoV-2 in La Ribera Health Department and to identify their characteristics.

Methodology. Retrospective cross-sectional observational study of cases of reinfection by SARS-CoV-2 in the population of La Ribera Department between March 2020 and February 2021. The positive baseline cohort includes all cases positive by RT-PCR for SARS-CoV-2, with reinfection cases being those that, after resolution of the first episode according to the World Health Organization (WHO) criteria, presented a new positive RT-PCR result.

Results. Out of a total of 15 687 cases with positive RT-PCR, 40 were considered to be reinfections, which meant a cumulative incidence of 0.255 % and an incidence density of 5.05 cases per 100 000 person-days. Most of the cases occurred during the highest incidence peaks of the pandemic in the department. Seventy-five per cent of the patients in these cases were older than 40 years, 42.5 % were healthcare professionals or nursing home residents and 12.5 % had an immunosuppressive comorbidity. There were no severe, critical or death cases. In the reinfection episodes, with respect to the first episode, there was a tendency to be milder, they required fewer days of hospitalization, their RT-PCR became negative earlier, they developed a greater humoral response and the sick leave period was shorter. The median period between the RT-PCR in the first episode and the RT-PCR in the second episode was 127.5 days (range: 48–301; IQR: 89.5–256.25)

Conclusions. SARS-CoV-2 reinfection cases are rare, tend to be mild and can occur within a median period of 127.5 days.

Introduction. Finding a safe innate immune response stimulator is one of the greatest challenges facing immunologists and vaccine manufacturers.

Gap statement. The role of sterile bacterial secretions (SBSs) of Pseudomonas aeruginosa in stimulating the innate immune response was not investigated previously.

Aim. The comparative effect of SBSs and bacterial cells of P. aeruginosa isolates isolated from freshwater (PAE) and infected wounds (PAC) on the respiratory tract innate immune response.

Methodology. Four test mice groups were instilled intranasally (i.n.) with 106 c.f.u of PAC, 106 c.f.u of PAE, SBS of PAC, and SBS of PAE. Two control groups were given i.n. either LB broth or PBS. Time-course changes in IL-1 beta mRNA, TNF-alpha mRNA, IL-1β and TNF-α, leukocyte count, bacterial uptake, and intracellular bacterial killing by mouse alveolar macrophages (AMs) and histological changes were examined. Lung bacterial burdens were counted in first and second test groups.

Results. The maximum level of IL-1β was seen as early as 2 h (1360±180 pg ml−1) post-instillation (i.n.) with SBS of PAC and 1 h (1910±244 pgml−1) post-instillation with SBS of PAE. The maximum level of TNF-α was seen as early as 4 h (953±192 pg ml−1) post-instillation with SBS of PAC and (1197±298 pg ml−1) post-instillation with SBS of PAE. These values were almost in line with IL-1β and TNF-α gene expression. Moderate infiltration of leukocytes in bronchoalveolar lavage (BAL) and lung sections and moderate activity of AMs (bacterial uptake and bacterial killing) were observed. The above innate immune response parameters in mice instilled i.n. with PAC and PAE were higher (P<0.05) than in the mice groups instilled i.n. with SBSs. The PAC was persistent in the lungs of mice for up to 72 h (3.5±0.22 log10 of c.f.u. g−1) and up to 48 h (2.05±0.21 log10 of c.f.u. g−1) for PAE.

Conclusion. The administration of mice with SBS i.n. stimulates cellular and molecular arms of the innate immune response in the respiratory tract, opening the door to the possibility of using SBS of P. aeruginosa as an adjuvant.

Introduction. Virulence factors (VFs) are the most potent weapon in the molecular armoury of Shigella . In bacteria, the mobile genetic elements (MGEs) are contributors to the evolution of different types of clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-cas) variants and plasmid incompatibility types. The present study explored the virulence potential of Shigella in relation to the CRISPR-cas pattern and incompatibility types among the isolates.

Hypothesis/Gap Statement. The profile of the CRISPR-cas systems among clinical isolates of Shigella in India has not been reported earlier. Limited knowledge is available on the pattern of plasmid incompatibility groups among clinical isolates Shigella . The bias is always towards studying the genetic elements associated with AMR, but the present study highlights CRISPR-cas and incompatibility types among Shigella in association with virulence.

Aim. We aimed to investigate the distribution of virulence factors, CRISPR-cas pattern followed by plasmid incompatibility types among Shigella isolates.

Methodology. Between 2012–2017, a total of 187 isolates of Shigella were included in the study. The virulence genes' distribution was carried out. CRISPR-cas profiling followed by analysis of the repeats and spacers was carried out. PCR-based replicon typing was used to determine the incompatibility types. The interplay was statistically determined using STATA.

Results. The distribution of virulence genes showed varied pattern with ipaH present in all the isolates followed by ompA (93.6 %), virF (66.8 %), ial and sen (60.4 %), set1A (39.6 %) and set1B (39 %). CRISPR 1, CRISPR 3 and Cas6-Cas5 region were dominantly conserved. Twenty-two types of spacers were identified. The CRISPR3 repeat appeared to have a highly conserved sequence. CRISPR2 being the least common CRISPR type showed a strong association with an array of virulence genes (ial-set1A-set1B-virF) while CRISPR1 being the most dominant showed the least association with virulence genes (sen-virF). The dominant plasmids were found to be belonging to the inc FII group. The incompatibility groups FII, IncIγ, U, FIIS, FIIK, K, A/C, I1alpha was found to be associated with a greater number of virulence genes.

Conclusion. The isolates showed increasing diversity in their gene content that contributes to increasing heterogeneity among the isolates, which is a known virulence strategy among pathogens.