The Prevalence and Clinical Implications of Rectal SARS-CoV-2 Shedding in Danish COVID-19 Patients and the General Population

Background: SARS-CoV-2 has resulted in a global pandemic since its outbreak in Wuhan, 2019. Virus transmission primarily occurs through close contact, respiratory droplets, and aerosol particles. However, since SARS-CoV-2 has been detected in fecal and rectal samples from infected individuals, the fecal-oral route has been suggested as another potential route of transmission. This study aimed to investigate the prevalence and clinical implications of rectal SARS-CoV-2 shedding in Danish COVID-19 patients.Methods: Hospitalized and non-hospitalized adults and children who were recently tested with a pharyngeal COVID-19 test, were included in the study. A rectal swab was collected from all participants. Hospitalized adults and COVID-19 positive children were followed with both pharyngeal and rectal swabs until two consecutive negative results were obtained. RT-qPCR targeting the envelope gene was used to detect SARS-CoV-2 in the samples. Demographic, medical, and biochemical information was obtained through questionnaires and medical records.Results: Twenty-eight of 52 (53.8%) COVID-19 positive adults and children were positive for SARS-CoV-2 in rectal swabs. Seven of the rectal positive participants were followed for more than 6 days. Two of these (28.6%) continued to test positive in their rectal swabs for up to 29 days after the pharyngeal swabs had turned negative. Hospitalized rectal positive and rectal negative adults were comparable regarding demographic, medical, and biochemical information. Furthermore, no difference was observed in the severity of the disease among the two groups.Conclusions: We provided evidence of rectal SARS-CoV-2 shedding in Danish COVID-19 patients. The clinical importance of rectal SARS-CoV-2 shedding appears to be minimal.

The bacterial density of clinical rectal swabs is highly variable, correlates with sequencing contamination, and predicts patient risk of extraintestinal infection

Background In ecology, population density is a key feature of community analysis. Yet in studies of the gut microbiome, bacterial density is rarely reported. Studies of hospitalized patients commonly use rectal swabs for microbiome analysis, yet variation in their bacterial density—and the clinical and methodologic significance of this variation—remains undetermined. We used an ultra-sensitive quantification approach—droplet digital PCR (ddPCR)—to quantify bacterial density in rectal swabs from 118 hospitalized patients. We compared bacterial density with bacterial community composition (via 16S rRNA amplicon sequencing) and clinical data to determine if variation in bacterial density has methodological, clinical, and prognostic significance. Results Bacterial density in rectal swab specimens was highly variable, spanning five orders of magnitude (1.2 × 104–3.2 × 109 16S rRNA gene copies/sample). Low bacterial density was strongly correlated with the detection of sequencing contamination (Spearman ρ = − 0.95, p < 10−16). Low-density rectal swab communities were dominated by peri-rectal skin bacteria and sequencing contaminants (p < 0.01), suggesting that some variation in bacterial density is explained by sampling variation. Yet bacterial density was also associated with important clinical exposures, conditions, and outcomes. Bacterial density was lower among patients who had received piperacillin-tazobactam (p = 0.017) and increased among patients with multiple medical comorbidities (Charlson score, p = 0.0040) and advanced age (p = 0.043). Bacterial density at the time of hospital admission was independently associated with subsequent extraintestinal infection (p = 0.0028), even when controlled for severity of illness and comorbidities. Conclusions The bacterial density of rectal swabs is highly variable, and this variability is of methodological, clinical, and prognostic significance. Microbiome studies using rectal swabs are vulnerable to sequencing contamination and should include appropriate negative sequencing controls. Among hospitalized patients, gut bacterial density is associated with clinical exposures (antibiotics, comorbidities) and independently predicts infection risk. Bacterial density is an important and under-studied feature of gut microbiome community analysis.

Intra-Host SARS-CoV-2 Evolution in the Gut of Mucosally-Infected Chlorocebus aethiops (African Green Monkeys)

In recent months, several SARS-CoV-2 variants have emerged that enhance transmissibility and escape host humoral immunity. Hence, the tracking of viral evolutionary trajectories is clearly of great importance. Little is known about SARS-CoV-2 evolution in nonhuman primate models used to test vaccines and therapies and to model human disease. Viral RNA was sequenced from rectal swabs from Chlorocebus aethiops (African green monkeys) after experimental respiratory SARS-CoV-2 infection. Two distinct patterns of viral evolution were identified that were shared between all collected samples. First, mutations in the furin cleavage site that were initially present in the virus as a consequence of VeroE6 cell culture adaptation were not detected in viral RNA recovered in rectal swabs, confirming the necessity of this motif for viral infection in vivo. Three amino acid changes were also identified; ORF 1a S2103F, and spike D215G and H655Y, which were detected in rectal swabs from all sampled animals. These findings are demonstrative of intra-host SARS-CoV-2 evolution and may identify a host-adapted variant of SARS-CoV-2 that would be useful in future primate models involving SARS-CoV-2 infection.

Characterization of 2 Klebsiella pneumoniae carbapenemase–producing Enterobacterales isolated from canine rectal swabs

Globally, carbapenemase-producing Enterobacterales (CPE) cause life-threatening, hospital-acquired infections in people, and have been reported recently among veterinary patients. Organisms that produce a Klebsiella pneumoniae carbapenemase (KPC) are one of the most common CPE isolated from people but have been reported only rarely in animals. We characterized 2 KPC-producing Enterobacterales isolated from companion animal rectal swabs during the response to an outbreak caused by a strain of blaNDM-5 Escherichia coli. Both isolates were characterized by whole-genome sequencing (WGS) and analysis. The first isolate (case A) was from an immunosuppressed 6-y-old Yorkshire Terrier and was identified as E. coli (ST372) with a blaKPC-18 gene and an IncFII plasmid. The second isolate (case B) was from a 3-y-old Labrador Retriever with acute diarrhea and was identified as Citrobacter koseri with a blaKPC-2 gene, multiple plasmids (ColRNAI, pKPC-CAV1193), and a putative enterotoxin gene ( senB). Further research is needed to determine what role animals might play in the epidemiology of CPE in communities. It is imperative that all CPE isolated from companion animals be fully characterized by WGS and the associated case examined. All veterinary isolates should be sequenced and shared for surveillance, monitoring, and investigation purposes.

Clonal Dissemination of Plasmid-Mediated Carbapenem and Colistin Resistance in Refugees Living in Overcrowded Camps in North Lebanon

Carbapenem and colistin-resistant bacteria represent a global public health problem. Refugees carrying these bacteria and living in inadequate shelters can spread these microorganisms. The aim of this study was to investigate the intestinal carriage of these bacteria in Syrian refugees in Lebanon. Between June and July 2019, 250 rectal swabs were collected from two refugee camps in North Lebanon. Swabs were cultured on different selective media. Antibiotic susceptibility testing was performed using the disk diffusion method. Carbapenemase-encoding genes and mcr genes were investigated using real-time polymerase chain reaction (RT-PCR) and standard polymerase chain reaction (PCR). Epidemiological relatedness was studied using multilocus sequence typing (MLST). From 250 rectal swabs, 16 carbapenem-resistant, 5 colistin-resistant, and 4 colistin and carbapenem-resistant Enterobacteriaceae were isolated. The isolates exhibited multidrug-resistant phenotypes. Seven Klebsiella pneumoniae isolates harboured the blaOXA-48 gene, and in addition four K. pneumoniae had mutations in the two component systems pmrA/pmrB, phoP/phoQ and co-harboured the blaNDM-1 gene. Moreover, the blaNDM-1 gene was detected in six Escherichia coli and three Enterobacter cloacae isolates. The remaining five E. coli isolates harboured the mcr-1 gene. MLST results showed several sequence types, with a remarkable clonal dissemination. An urgent strategy needs to be adopted in order to avoid the spread of such resistance in highly crowded underserved communities.

In-vitro antibiotic resistance phenotypes of respiratory and enteric bacterial isolates from weaned dairy heifers in California

Antimicrobial drug (AMD) use for bovine respiratory disease (BRD) continues to be concerning for development of antimicrobial resistance (AMR) in respiratory and enteric bacteria of cattle. This study aimed to provide data regarding AMR in respiratory isolates, and identify relationships between respiratory and enteric AMD susceptibility, in weaned dairy heifers. A cross-sectional study was performed between June of 2019 and February 2020, on 6 calf rearing facilities in California. Deep nasopharyngeal and rectal swabs were collected from 341 weaned heifers and submitted for selective bacterial culture and AMR testing. Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni were selectively isolated from respiratory samples; Escherichia coli and Enterococcus spp. were selectively isolated from rectal swabs. Minimum inhibitory concentrations (MIC) were determined for selected isolates against 19 AMD. The proportion of resistant isolates was calculated using Clinical Laboratory Standards Institute (respiratory) or USDA NARMS (enteric) breakpoints; when no applicable breakpoint was available, the distribution of MIC was described and compared. Association between AMR in a calf’s respiratory isolate and a higher or lower MIC of the matched enteric isolates was determined. More than 50% of P. multocida isolates were resistant to each of 7 AMD commonly used to treat BRD (florfenicol, gamithromycin, tildipirosin, tilmicosin, danofloxacin, enrofloxacin and tetracycline). Resistance in respiratory isolates was only associated with higher matched enteric MIC for gamithromycin and tulathromycin. Multidrug resistance was reported in >70% of P. multocida and M. haemolytica isolates. Antimicrobial resistance, including multidrug resistance, in respiratory isolates appears to be widespread in weaned dairy heifers; this finding has not previously been reported and raises concern for the future efficacy of AMD used to treat respiratory diseases in weaned dairy heifers. Enteric bacterial MIC appear to have limited direct association with respiratory isolate AMR classification.

Molecular characterisation and antibiotic susceptibility of meningococcal isolates from healthy men who have sex with men

ObjectivesTo evaluate and characterise meningococcal carriage among healthy men who have sex with men (MSM) within a screening programme for Neisseria gonorrhoeae infection at the San Gallicano Dermatological Institute, Italy.MethodsA total of 441 MSM attending the STI/HIV Centre of the San Gallicano Institute, Rome, Italy, in 2016 were routinely screened for N. gonorrhoeae infection by pharyngeal and rectal swabs. N. meningitidis isolates were evaluated for antibiotic susceptibility and characterised by whole genome sequencing. Genetic relationships among the meningococcal carriage isolates were determined using core genome multilocus sequence typing analysis. The soluble domain of AniA (sAniA) protein expression by western blotting was also evaluated.ResultsA total of 62 (14.1%, 95% CI 11.1 to 17.6) carriage meningococci were found among 441 MSM. Forty-three viable N. meningitidis isolates were cultivated (42 from pharyngeal and 1 from rectal swabs). All the viable isolates were susceptible to cefotaxime, ceftriaxone, ciprofloxacin and rifampicin. Four isolates were penicillin G-resistant and 73% of those penicillin G-susceptible showed a minimum inhibitory concentration from 0.064 μg/mL to 0.25 μg/mL. Serogroup B was the most frequent (44.2%), followed by Z (16.3%), E (9.3%), and Y and W (2.3%), respectively. Multilocus sequence typing analysis identified 29 sequence types belonging to 12 clonal complexes. The sAniA protein was expressed in 8 out of 28 (29%) screened meningococcal carriage isolates.ConclusionsSerogroup B meningococcal carriage identified from oral and anal specimens among healthy MSM was the most frequent serogroup identified in this study. Molecular evaluation revealed a degree of similarity among strains belonging to the same clonal complex.

Prolonged rectal shedding of SARS-CoV-2 in a 22-day-old-neonate: a case report

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the novel coronavirus disease 2019 (COVID-19), which is characterized by a diverse clinical picture. Children are often asymptomatic or experience mild symptoms and have a milder disease course compared to adults. Rectal shedding of SARS-CoV-2 has been observed in both adults and children, suggesting the fecal-oral route as a potential route of transmission. However, only a few studies have investigated this in neonates. We present a neonate with a mild disease course and prolonged rectal SARS-CoV-2 shedding. Case presentation A 22-day old neonate was admitted to the hospital with tachycardia and a family history of COVID-19. The boy later tested positive for COVID-19. His heart rate normalized overnight without intervention , but a grade 1/6 heart murmur on the left side of the sternum was found. After excluding signs of heart failure, the boy was discharged in a habitual state after three days of admission. During his admission, he was enrolled in a clinical study examining the rectal shedding of SARS-CoV-2. He was positive for SARS-CoV-2 in his pharyngeal swabs for 11 days after initial diagnosis and remained positive in his rectal swabs for 45 days. Thereby, the boy remained positive in his rectal swabs for 29 days after his first negative pharyngeal swab. Conclusions The presented case shows that neonates with a mild disease course can shed SARS-CoV-2 in the intestines for 45 days. In the current case, it was not possible to determine if fecal-oral transfer to the family occurred, and more research is needed to establish the potential risk of the fecal-oral transmission route.

Variants of ctxB alleles of Vibrio cholerae O1 caused sequential cholera outbreaks in the tribal areas of Odisha, India

Cholera localized outbreaks/epidemics accounting for high morbidity and mortality have been reported in different years both from the coastal and tribal districts of Odisha. In the present study, the emergence and spread of two sequential cholera outbreaks reported in July to October 2012 from Rayagada and Kalahandi districts of Odisha was investigated. Environmental water samples from different sources and rectal swabs from diarrhoea patients were analysed for identification, antibiogram profiles and molecular studies using DMAMA-PCR assays. The pulsed field gel electrophoresis (PFGE) was done on some selected Vibrio cholerae O1 strains isolated from these cholera outbreak areas. Results showed 42% of rectal swabs and 2.3% of water samples collected from both the districts were positive for Vibrio cholerae O1 Ogawa biotype El Tor carrying both ctxB1 and ctxB7 genotypes. The common resistance profile of V. cholerae O1 strains was ampicillin, nalidixic acid, furazolidone and co-trimoxazole. The PFGE analysis on selected V. cholerae O1 strains of ctxB1 and ctxB7 genotypes showed three pulsotypes with 96% similarity matrix exhibiting the relationship with their respective water sources. Hence, continuous surveillance is highly essential to monitor the antibiogram profile and changing pattern of ctxB genotypes of V. cholerae O1 in this region.