- Volume 70, Issue 8, 2021

Introduction. There are several β-lactamase genes described for Bacteroide s strains, of which cepA and cfiA are specific for Bacteroides fragilis and define two genetic divisions. The expression and phenotypic effects of these genes are usually regulated by insertional activation.

Hypotheses/Gap Statement. Information is lacking about how cepA is regulated for most of the B. fragilis strains and whether there could be a genetic element for it.

Aim. We aimed to investigate the molecular background of ampicillin (and other β-lactam) resistance among Bacteroides strains as mediated mainly by cepA and also to find a genetic element for it as known for cfiA.

Methodology. Various PCR methods were used for β-lactamase-resistance gene and insertion sequence (IS) element detection in 42 Bacteroides strains. β-Lactamase activity measurements and antimicrobial-susceptibility testing using agar dilution were also applied. Further molecular experiments involved sequencing, gene targeting, Southern blotting and bioinformatic analyses.

Results. We found that high antibiotic resistance and β-lactamase levels are brought about by insertional activation of the cepA gene or by similar or dissimilar activation of cfxA or cfiA, or by the newly described pbbA genes. Non-activated cepA genes produced low levels of specific β-lactamase activities that did not correlate with ampicillin resistance. We found a genetic element for cepA and another region close to it that are characteristic for division I B. fragilis strains, which are replaced by other sequences in division II B. fragilis strains.

Conclusion. cepA usually is not activated by IS elements and usually produces low β-lactamase activities that do not correlate with the ampicillin MICs; therefore, it probably involves some non-β-lactamase-mediated resistance mechanism(s). pbpA is a newly described, effective β-lactamase gene that is located on a plasmid, and cepA resides on a well-defined chromosomal segment that is mutually replaced in division II B. fragilis strains. This latter finding demonstrates the genetic dichotomy of cepA–cfiA in B. fragilis and requires further investigation.

Introduction. Fluoroquinolone (FQ) resistant Salmonella are classified as high priority pathogens by WHO. FQ resistance among Salmonella Typhi has emerged rapidly and is predominantly mediated by mutations in the topoisomerase genes gyrA, and parC. Mutations in GyrA result in classical FQ resistance (DCS-NAR) i.e. decreased susceptibility to ciprofloxacin (MIC of 0.12 to 0.5 µg ml−1) (DCS) and resistance to nalidixic acid (NAR). Previously a nalidixic acid disc test was proposed for detection of DCS. Recently isolates with non-classical FQ resistance caused by plasmid-mediated quinolone resistance (PMQR) and mutations in GyrB have emerged. These mechanisms also result in DCS but are nalidixic acid susceptible (NAS) and thus pose diagnostic challenges. CLSI and EUCAST have recommended use of 5 µg pefloxacin discs for detection of DCS in Salmonella .

Hypothesis. The CLSI and EUCAST recommendations for use of 5 µg pefloxacin for detection of DCS has not been validated on typhoidal Salmonella and resistance mediated by GyrB mutation in Salmonella species.

Aim. The aim of the present study was to validate the performance of the 5 µg pefloxacin discs to detect isolates of S. Typhi with DCS with special reference to GyrB mutations.

Methodology. A total of 180 clinical isolates of Salmonella Typhi (2005–2014) were investigated for genetic mechanisms of resistance. Zone diameters for nalidixic acid (30μg), ciprofloxacin (5μg) and pefloxacin (5µg) and minimum inhibitory concentration (MIC) for ciprofloxacin were determined using CLSI guidelines. Performance of the three discs was evaluated to detect FQ resistance in S. Typhi.

Results. Topoisomerase mutations in GyrB +/ ParC and GyrB were detected in 112 and 34 isolates respectively. Different mutations have a varied effect on the MIC for ciprofloxacin. The current breakpoints for susceptible (≤0.06 µg ml−1) and non-susceptible (≥0.125 µg ml−1), failed to detect all isolates with a resistance mechanism. Performance of both ciprofloxacin and pefloxacin discs were excellent compared to nalidixic acid in differentiating isolates with non-classical resistance mediated by GyrB from wild-type.

Conclusion. The pefloxacin disc can be used to detect FQ resistance among S. Typhi. This is the first report of validation of pefloxacin for detection of FQ resistance in S. Typhi mediated by GyrB mutation.

Introduction. Resistance rates to azoles and echinocandins of Candida spp. increased over the last decade.

Hypothesis/Gap Statement. Widespread use of antifungals could lead to development and dissemination of resistant Candida spp.

Aim. To identify risk factors for isolation of Candida spp. non-susceptible to either fluconazole or echinocandins.

Methodology. All patients hospitalized in the Intensive Care Unit (ICU) of the University General Hospital of Patras, Greece with Candida spp. isolated from clinical specimens during a ten-year period (2010–19) were included. Candida isolates were identified using Vitek-2 YST card. Consumption of antifungals was calculated.

Results. During the study period, 253 isolates were included. C. non-albicans predominated (64.4 %) with C. parapsilosis being the most commonly isolated (42.3 %) followed by C. glabrata (nomenclatural change to Nakaseomyces glabrataa; 8.7 %) and C. tropicalis (11.9 %). Among all isolates, 45.8 and 28.5 % were non-susceptible and resistant to fluconazole, respectively. Concerning echinocandins, 8.7 % of isolates were non-susceptible to at least one echinocandin (anidulafungin or micafungin) and 3.1 % resistant. Multivariate analysis revealed that hospitalization during 2015–19, as compared to 2010–14, isolate being non-albicans or non-susceptible to at least one echinocandin was associated with isolation of fluconazole non-susceptible isolate. Administration of echinocandin, isolate being C. glabrata or C. tropicalis, or Candida spp. non-susceptible to fluconazole were independently associated with isolation of Candida spp. non-susceptible to at least one echinocandin. Fluconazole’s administration decreased during the study period, whereas liposomal-amphotericin B’s and echinoncandins’ administration remained stable.

Conclusion. Fluconazole’s non-susceptibility increased during the study period, despite the decrease of its administration. Although echinocandins’ administration remained stable, non-susceptibility among Candida spp. increased.

A previous study conducted in Gabon, Central Africa, in 2010/11 found a high colonization rate with extended-spectrum β-lactamase-producing enterobacterales (ESBL-E) among children of ~34 %. Eight years later, we aimed to reassess the ESBL-E rate and previously identified risk factors for colonization in children from Gabon. We conducted a cross-sectional cohort study in 2018 on 92 outpatients under 5 years of age with diarrhoea in Lambaréné, Gabon, in whom a rectal swab was obtained at the initial medical encounter (baseline). Fifty-eight of these provided a further rectal swab 1 week afterwards. ESBL-E colonization was assessed [following the European Committee on Antimicrobial Susceptibility Testing (EUCAST)], and in confirmed ESBL-E isolates the susceptibility to meropenem and the prevalence of the most abundant ESBL genes, bla CTX-M, bla SHV, and bla TEM, were investigated. At baseline, the ESBL-E colonization rate was 57 % (52/92; 95 % CI: 46–67). Hospitalization during the previous year, chicken consumption in the past week and young age were identified as independent risk factors for ESBL-E colonization at baseline. On day 7, the ESBL-E carriage rate was 72 % (42/58; 95 % CI: 59–83). All ESBL-E isolates (n=293) were susceptible to meropenem and bla CTX-M was the most frequently detected β-lactamase gene. The ESBL-E colonization rate among children from Gabon is alarmingly high, with indications of further increase over recent years. While all ESBL-E strains remain currently susceptible to meropenem, in practice no adequate treatment is available locally for severe infections with such isolates. It is thus of the utmost importance to invest in improved hospital infection prevention and control measures to combat ESBL-E effectively.

Following prolonged hospitalization that included broad-spectrum antibiotic exposure, a strain of Providencia rettgeri was cultured from the blood of a patient undergoing extracorporeal membrane oxygenation treatment for hypoxic respiratory failure due to COVID-19. The strain was resistant to all antimicrobials tested including the novel siderophore cephalosporin, cefiderocol. Whole genome sequencing detected ten antimicrobial resistance genes, including the metallo-β-lactamase bla NDM-1, the extended-spectrum β-lactamase bla PER-1, and the rare 16S methyltransferase rmtB2.

Introduction. The current severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) pandemic has stressed the global supply chain for specialized equipment, including flocked swabs.

Hypothesis. Saliva could be a potential alternative specimen source for diagnosis of SARS-CoV-2 infection by reverse-transcriptase PCR (RT-PCR).

Aim. To compare the detection efficiency of SARS-CoV-2 RNA in saliva and oro-nasopharyngeal swab (ONPS) specimens.

Methodology. Patients recruited from hospital provided paired saliva and ONPS specimens. We performed manual or automated RT-PCR with prior proteinase K treatment without RNA extraction using the Seegene Allplex 2019 nCoV assay.

Results. Of the 773 specimen pairs, 165 (21.3 %) had at least one positive sample. Additionally, 138 specimens tested positive by both sampling methods. Fifteen and 12 cases were detected only by nasopharyngeal swab and saliva, respectively. The sensitivity of ONPS (153/165; 92.7 %; 95 % CI: 88.8–96.7) was similar to that of saliva (150/165; 90.9 %; 95 % CI: 86.5–95.3; P=0.5). In patients with symptoms for ≤ 10 days, the sensitivity of ONPS (118/126; 93.7 %; 95 % CI: 89.4–97.9) was similar to that of saliva (122/126; 96.8 %; 95 % CI: 93.8–99.9 %; P=0.9). However, the sensitivity of ONPS (20/22; 95.2 %; 95 % CI: 86.1–100) was higher than that of saliva (16/22; 71.4 %; 95 % CI: 52.1–90.8) in patients with symptoms for more than 10 days.

Conclusions. Saliva sampling is an acceptable alternative to ONPS for diagnosing SARS-CoV-2 infection in symptomatic individuals displaying symptoms for ≤ 10 days. These results reinforce the need to expand the use of saliva samples, which are self-collected and do not require swabs.

Introduction. Probiotic supplementation of preterm infants may prevent serious morbidities associated with prematurity.

Aim. To investigate the impact of probiotic supplementation on the gut microbiota and determine factors associated with detection of probiotic species in the infant gut.

Hypothesis/Gap Statement. Probiotic supplementation increases the long-term colonization of probiotic species in the gut of preterm infants.

Methodology. Longitudinal stool samples were collected from a cohort of very preterm infants participating in a blinded randomized controlled trial investigating the impact of probiotic supplementation (containing Bifidobacterium longum subsp. infantis BB-02, Bifidobacterium animalis subsp. lactis BB-12 and Streptococcus thermophilus TH-4) for prevention of late-onset sepsis. The presence of B. longum subsp. infantis , B. animalis subsp. lactis and S. thermophilus was determined for up to 23 months after supplementation ended using real-time PCR. Logistic regression was used to investigate the impact of probiotic supplementation on the presence of each species.

Results. Detection of B. longum subsp. infantis [odds ratio (OR): 53.1; 95 % confidence interval (CI): 35.6–79.1; P < 0.001], B. animalis subsp. lactis (OR: 89.1; 95 % CI: 59.0–134.5; P < 0.001) and S. thermophilus (OR: 5.66; 95 % CI: 4.35–7.37; P < 0.001) was increased during the supplementation period in infants receiving probiotic supplementation. Post-supplementation, probiotic-supplemented infants had increased detection of B. longum subsp. infantis (OR: 2.53; 95 % CI: 1.64–3.90; P < 0.001) and B. animalis subsp. lactis (OR: 1.59; 95 % CI: 1.05–2.41; P=0.030). Commencing probiotic supplementation before 5 days from birth was associated with increased detection of the probiotic species over the study period ( B. longum subsp. infantis, OR: 1.20; B. animalis subsp. lactis, OR: 1.28; S. thermophilus, OR: 1.45).

Conclusion. Probiotic supplementation with B. longum subsp. infantis BB-02, B. animalis subsp. lactis BB-12 and S. thermophilus TH-4 enhances the presence of probiotic species in the gut microbiota of very preterm infants during and after supplementation. Commencing probiotic supplementation shortly after birth may be important for improving the long-term colonization of probiotic species.

Introduction. Staphylococcus coagulans (formerly Staphylococcus schleiferi subsp. coagulans ) is a common commensal and opportunistic pathogen of companion dogs. It carries a range of antimicrobial resistance genes and is an occasional zoonotic pathogen.

Hypothesis/Gap Statement. Despite the potential insight offered by genome sequencing into the biology of S. coagulans , few genomes are currently available for study.

Aim. To sequence and analyse S. coagulans genomes to improve understanding of this organism’s molecular epidemiology, antimicrobial resistance and bacterium–host interactions.

Methodology. Twenty-five genomes of clinical isolates collected at a veterinary referral hospital in Scotland, UK, were sequenced with Illumina technology. These genomes were analysed by a series of bioinformatics tools along with 16 previously sequenced genomes.

Results. Phylogenetic comparison of the 41 genomes shows that the current S. coagulans phylogeny is dominated by clades of closely related isolates, at least one of which has spread internationally. Ten of the 11 methicillin-resistant S. coagulans genomes in this collection of 41 encoded the mecA promoter and gene mutations that are predicted to render the isolates susceptible to penicillins in the presence of clavulanic acid, a feature only described to date in methicillin-resistant Staphylococcus aureus . Seven such isolates were from the current study and, in line with the genome-based prediction, all were susceptible to amoxicillin/clavulanic acid in vitro. S. coagulans shared very few highly conserved virulence-associated genes with Staphylococcus pseudintermedius , another common commensal and opportunistic canine pathogen.

Conclusion. The availability of a further 25 genome sequences from clinical S. coagulans isolates will aid in better understanding the epidemiology, bacterial–host interactions and antimicrobial resistance of this opportunistic pathogen.