Isolation, antimicrobial susceptibility patterns, and risk factors assessment of non-typhoidal Salmonella from apparently healthy and diarrheic dogs

Background Dogs are one of the important asymptomatic carriers of antimicrobial resistant and potentially pathogenic strains of Salmonella. They can harbor large bacterial load in the intestines and mesenteric lymph nodes which can be shed in their feces with the possibility of transmission to humans. Therefore, a cross-sectional study was conducted with the objectives of estimating the prevalence of non-typhoidal Salmonella, assessing the risk factors for dog’s Salmonella carriage, and profiling the antimicrobial resistance pattern of Salmonella isolates among housed dogs in Harar town, Eastern Ethiopia. A total of 415 rectal swab samples were collected from randomly selected dogs. Samples were examined for non-typhoidal Salmonella using standard bacteriologic culture and biochemical tests. The disk diffusion method (Kirby-Bauer test) was employed to evaluate the isolates for their susceptibility against five antimicrobials. Results Non-typhoidal Salmonella were isolated from 26 (6.3%) of the rectal swab samples, with significantly higher occurrence in diarrheic (15.2%) than non-diarrheic (5.5%) dogs. The risk of Salmonella harboring was significantly higher in female dogs than in male dogs (OR = 2.5, p = 0.027). Dogs fecal shedding of Salmonella was relatively higher in households who used offal as a main feed type for their dogs (23.1%; 95% CI = 5–53.8) than those who used leftover food (10.1%; 95% CI = 5.7–16.1) and practiced mixed feeding system (17%; 95% CI = 7.6–30.8). Salmonella isolates showed higher resistance to ampicillin (41.7%), while all isolates were fully susceptible to gentamicin. Moreover, 58.3% of Salmonella isolates showed resistance to at least one of the tested antimicrobials. Majorities (72.7%) of the dog owners had no awareness on the risk of zoonotic salmonellosis from dog and all of the respondents use bare hand to clean dog kennel. Conclusion Our study reveals the importance of both diarrheic and apparently healthy housed dogs in the harboring and shedding of antimicrobial resistant non-typhoidal Salmonella. The risk of non-typhoidal Salmonella spread among pet owners is not negligible, especially in households who use offal as main feed type. Therefore, an integrated approach such as: proper dog handling practices; continuous evaluation of antimicrobial resistance; and rational use of antimicrobials in the field of veterinary sector are necessary to tackle the problem.

Molecular surveillance reveals widespread colonisation by carbapenemase and extended spectrum beta-lactamase producing organisms in neonatal units in Kenya and Nigeria

Objectives Neonatal sepsis, a major cause of death amongst infants in sub-Saharan Africa, is often gut derived. Impairments in immunity and the gut barrier in sick neonates allow colonisation by opportunistic pathogens such as Enterobacteriaceae to progress to blood stream infection. Colonisation by Enterobacteriaceae producing extended spectrum beta-lactamase (ESBL) or carbapenemase enzymes is particularly problematic and can lead to antimicrobial-resistant (AMR) or untreatable infections. We sought to explore the rates of colonisation by ESBL or carbapenemase producers and their genotypes in two neonatal units (NNUs) in West and East Africa. Methods Stool and rectal swab samples were taken at multiple timepoints from newborns admitted to the NNUs at the University College Hospital, Ibadan, Nigeria and the Jaramogi Oginga Odinga Teaching and Referral Hospital, Kisumu, western Kenya. Samples were tested for ESBL and carbapenemase genes using a previously validated qPCR assay with high resolution melt analysis. Kaplan-Meier survival analysis was used to examine colonisation rates at both sites. Results A total of 119 stool and rectal swab samples were taken from 42 infants admitted to the two NNUs. Six (14.3%) infants were extremely preterm (gestation <28 weeks), 19 (45.2%) were born by Caesarean section and 3 (8.6%) mothers were HIV positive. Median (IQR) duration of admission was 12.5 (5-26) days and 12 (28.6%) infants died. Overall, colonisation with ESBL (37 infants, 89%) was more common than with carbapenemase producers (26, 62.4%; P = 0.093). Median survival time before colonisation with ESBL organisms was 7 days and with carbapenemase producers 16 days (P=0.035). The majority of ESBL genes detected belonged to the CTX-M-1 (36/38; 95%), and CTX-M-9 (2/36; 5%) groups. The most prevalent carbapenemase was blaNDM (27/29, 93%). Single blaVIM (1/32, 3%) and blaOXA-48 genes (1/32, 3%) were also detected. Conclusions Gut colonisation of neonates by AMR organisms was common and occurred rapidly in NNUs in Kenya and Nigeria. Active surveillance of colonisation will improve the understanding of AMR in these settings and guide infection control and antibiotic prescribing practice to improve clinical outcomes.

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.

Molecular Epidemiology and Genetic Evolution of Canine Parvovirus Type 2 in Diarrheic Dogs in Serbia From 2008 To 2020

Canine Parvovirus 2 (CPV2) is a causal agent of an infectious disease with the highest fatality rate among dogs. However, in Serbia, it has never been investigated thoroughly. This study was conducted on samples originating from dogs with diarrhea in anamnesis, stored in the sample bank, submitted for various reasons to the Institute of Veterinary Medicine of Serbia. In total, 50 rectal swab samples were collected from the period 2008 to 2020, and consequently tested. Out of 50 rectal swab samples, the CPV2 genome was detected in 14 (28%). This retrospective study showed the presence of three different variants of CPV2 in diarrheic dogs during the last 12 years in Serbia. CPV2a was the most prevalent variant (60%) followed by CPV2b (30%), and CPV2c (10%). Interestingly, CPV2a had been the predominantly detected variant up until 2018. Nevertheless in 2019, there was the first detected occurrence of the CPV2b variant, followed by the first detection of the CPV2c in 2020. This study reports the evidence and distribution of CPV2 throughout the time-lapse from 2008 to 2020, providing new information about the presence and the prevalence of virus strains in Serbia.

Undetectable Production of the VIM-1 Carbapenemase in an Atlantibacter hermannii Clinical Isolate

The differential expression of VIM-1 in Atlantibacter hermannii WEB-2 and Enterobacter hormaechei ssp. hoffmannii WEB-1 clinical isolates from a rectal swab of a hospitalized patient in France was investigated. A. hermannii WEB-2 was resistant to all β-lactams except carbapenems. It produced ESBL SHV-12, but the Carba NP test failed to detect any carbapenemase activity despite the production of VIM-1. Conversely, E. hormaechei WEB-1, previously recovered from the same patient, was positive for the detection of carbapenemase activity. The blaVIM–1 gene was located on a plasmid and embedded within class 1 integron. Both plasmids were of the same IncA incompatibility group and conferred the same resistance pattern when electroporated in Escherichia coli TOP10 or Enterobacter cloacae CIP7933. Quantitative RT-PCR experiments indicated a weaker replication of pWEB-2 in A. hermannii as compared to E. hormaechei. An isogenic mutant of A. hermannii WEB-2 selected after sequential passages with increased concentrations of imipenem possessed higher MICs for carbapenems and cephalosporins including cefiderocol, higher levels of the blaVIM–1 gene transcripts, and detectable carbapenemase activity using the Carba NP test. Assessment of read coverage demonstrated that a duplication of the region surrounding blaVIM–1 gene occurred in the A. hermannii mutant with detectable carbapenemase activity. The lack of detection of the VIM-1 carbapenemase activity in A. hermannii WEB-2 isolate was likely due to a weak replication of the IncA plasmid harboring the blaVIM–1 gene. Imipenem as selective pressure led to a duplication of this gene on the plasmid and to the restoration of a significant carbapenem-hydrolyzing phenotype.

The evaluation of vancomycin-resistant enterococci and carbapenamase producing Klebsiella colonization among ICU-Hospitalized Patients

Background: Multi-drug resistant organisms, especially Vancomycin-Resistant Enterococcus (VRE) and Carbapenam Resistant Klebsiella pneumoniae (KPC), are serious health threat. Early detection of resistant bacteria colonization amongpatients in intensive care units (ICUs) not only enables effective treatment but more importantly prevents disease and limits transmission. Therefore, we aimed to to assess the frequency of VRE and KPC colonization via rectal swab sampling. Methods: The study was carried out in ICUs of a tertiary hospital. Two rectal swab samples were collected within the first 24 hours of admission and another one was taken every subsequent 15 days to test for for VRE and KPC carriage. Results: A total 316 rectal swab samples taken from 230 patients. Forty-seven patients were screened at least 2 times. 183 patients were not further screened due to discharge, exitus or transfer to other wards. Thirty-six patients (16%) were determinedto be VRE (+). The most frequently isolated strain was E. faecium (80.5%) and its most common genotype was VanA (87.5%). Seven patients (3%) were identified as KPC (+). OXA-48 type crbapenamase was confirmed in all KPC isolates. Conclusion: This study shows that VRE and KPC colonization continues to be a serious threat in ICUs. Keywords: Carbapenam resistant klebsiella pneumoniae; vancomycin-resistant enterococci; intensive care units.

Prevalence of extended-spectrum β-lactamase-producing Escherichia coli in companion dogs in animal clinics, Surabaya, Indonesia

Background and Aim: The practice of keeping animals as pets is becoming increasingly common. The upsurge of extended-spectrum β-lactamase (ESBL)-producing organisms of animal origin is a health threat globally. This study aimed to identify the presence of extended-spectrum β-lactamase-producing Escherichia coli in companion dogs in animal clinics in Surabaya, Indonesia. Materials and Methods: A total of 85 rectal swab samples were collected from companion dogs at five animal clinics in different regions of Surabaya, Indonesia. The presence of E. coli was identified from the samples using standard methods, followed by antibiotic sensitivity testing. The resistant isolates were examined for the presence of ESBL using the double-disk synergy test method. The phenotypically identified ESBL-producing E. coli was further confirmed with an automated system using Vitek-2. Results: The rectal swab samples (n=85) tested were 100% positive for E. coli isolates. Eight (9.41%) out of the 85 E. coli obtained from rectal swabs were extended-spectrum β-lactamase producers. All eight ESBL-producing E. coli were identified by automated Vitek-2 confirmatory tests. Conclusion: This study provides insight into the prevalence of ESBL-producing organisms isolated from companion dogs in Indonesia. This work indicates the need for the general public to be more aware of the role of companion animals in disseminating pathogenic organisms, since they serve as potential reservoirs in the spread of antibiotic resistance affecting human health.

Rapid detection of Enterococcus and vancomycin resistance using recombinase polymerase amplification

Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes (vanA and vanB) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium, vanA, and vanB, and it has the potential to be used as a point-of-care device for VRE therapy and prevention.

Retrospective analysis of Clostridioides difficile and other intestinal infections in patients with Crohn’s disease and ulcerative colitis in the tertiary hospital in Poland. POLIBD survey results

Background There are several studies which evaluated the number of infections caused by enteric pathogens, including Clostridioides difficile in patients with inflammatory bowel disease (IBD). Our aim was to assess the prevalence of intestinal infections among patients suffering from IBD, when admitted to the hospital due to exacerbation of the disease. Results The performed, retrospective analysis covered test results for C. difficile toxins A and B along with rectal swab cultures sampled from patients, treated in a tertiary IBD center in Poland, between 2017 and 2019. Main objective was to estimate the presence of any infection, which could imitate or co-exist along with the exacerbation of the IBD. All in all 1471 patients had microbiological tests performed, including 1112 tested for C. difficile toxins A and B; and 359 patients who had rectal swab culture. Positive test results for C. difficile toxins A and B were reported in 358 cases, positive results from rectal swab culture were confirmed altogether in case of 25 samples. As far as patients with IBD are concerned, positive results for C. difficile toxins A and B were detected in 82 cases, positive results in rectal swab culture from patients with IBD were reported in 20 cases. Conclusion Intestinal infections were reported in 14.9% of patients (102/685) with IBD symptoms. Positive test results for C. difficile toxins A and B and rectal swab cultures among patients without IBD symptoms were reported in 35.7% of cases (281/786). Intestinal superinfections may complicate the clinical picture of IBD patients, increasing the diagnostic and therapeutic burden. Appropriate early procedures are thus needed in these patients.

17. Comparative Assessment of a Machine Learning Model and Rectal Swab Surveillance to Predict Hospital Onset Clostridioides difficile

Background Hospital onset Clostridioides difficile infection (HO-CDI) is associated with significant morbidity and mortality. Screening individuals at risk could help limit transmission, however swab-based surveillance for HO-CDI is resource intensive. Applied to electronic health records (EHR) data, machine learning (ML) models present an efficient approach to assess patient risk. We compare the effectiveness of swab surveillance against daily risk estimates produced by a ML model in detecting patients who will develop HO-CDI. Methods Patients presenting to Michigan Medicine’s ICUs and oncology wards between June 6th and October 8th 2020 had rectal swabs collected on admission, weekly, and at discharge from the unit, as part of VRE surveillance. We performed anaerobic culture on the residual media followed by a custom, multiplex PCR on isolates to identify toxigenic C. difficile. Risk of HO-CDI was calculated daily for each patient using a previously validated EHR-based ML model. Swab results and model risk scores were aggregated for each admission and assessed as predictors of HO-CDI. Holding sensitivity equal, we evaluated both approaches in terms of accuracy, specificity, and positive predictive value (PPV). Results Of 2,044 admissions representing 1,859 patients, 39 (1.9%) developed HO-CDI. 23.1% (95% CI: 11.1–37.8%) of HO-CDI cases had at least one positive swab. At this sensitivity, model performance was significantly better than random but worse compared to swab surveillance—accuracy: 87.5% (86.0–88.9%) vs. 94.3% (93.3–95.3%), specificity: 88.7% (87.3–90.0%) vs. 95.7% (94.8–96.6%), PPV: 3.8% (1.6–6.4%) vs. 9.4% (4.3–16.1%). Combining swab AND model yielded lower sensitivity 2.6% (0.0–8.9%) compared to combining swab OR model at 43.6% (27.3–60.0%), and yielded PPV 7.1% (0.0–25.0%) vs. 43.6% (27.3–60.0%) respectively (Figure 1). Figure 1. Surveillance & risk score performance. Binary classification performance metrics of ML model (Model), toxigenic C. difficile rectal swab surveillance (Swab), and combination approaches (Model AND Swab and Model OR Swab), reported in terms of percentage points. Bold numbers highlight the best performing approach for a given performance metric. The combined approach of monitoring the Model AND Swab yielded the highest accuracy 97.5% (95% confidence interval: 96.8%, 98.1%), it also had the highest specificity 99.4% (99.0%, 99.7%). The combined approach of monitoring the Model OR Swab yielded the highest sensitivity 43.6% (27.3%, 60.0%) and negative predictive value (NPV) 98.7% (98.2, 99.2%). Using the Swab alone yielded the highest PPV 9.4% (4.3%, 16.1%) and F1 score 13.3% (6.2%, 21.8%). These results highlight the complementarity of the model and swab-based approaches. Conclusion Compared to swab surveillance using a ML model for predicting HO-CDI results in more false positives. The ML model provides daily risk scores and can be deployed using different thresholds. Thus, it can inform varied prevention strategies for different risk categories, without the need for resource intensive swabbing. Additionally, the approaches may be complimentary as the patients with HO-CDI identified by each approach differ. Disclosures Vincent B. Young, MD, PhD, American Society for Microbiology (Other Financial or Material Support, Senior Editor for mSphere)Vedanta Biosciences (Consultant) Krishna Rao, MD, MS, Bio-K+ International, Inc. (Consultant)Merck & Co., Inc. (Grant/Research Support)Roche Molecular Systems, Inc. (Consultant)Seres Therapeutics (Consultant)