PCR Screening Research Articles

1884. Evaluating SARS-CoV-2 Surveillance Strategies at the United States Naval Academy: A Comparison of Saliva and Dried Blood Spot Serosurveillance Against Molecular-Confirmed Case Detection

Abstract Background Congregate military populations remain at risk of SARS-CoV-2 outbreaks and the optimal surveillance approach in such settings remains unclear. We enrolled midshipmen at the United States Naval Academy (USNA) in a setting of frequent PCR screening use of prevention strategies. Methods Dried blood spots (DBS) and saliva were collected in August 2020, December 2020, February 2021 (saliva only) and April/May 2021 to measure anti-SARS-CoV-2 spike (S) and nucleoprotein (NP) IgG. COVID-19 vaccine history and records of SARS-CoV-2 PCR tests and routine asymptomatic screening assays were obtained from the USNA Brigade Medical Clinic. Attack rates were compared with cumulative frequencies of infections. Concordance of saliva and DBS anti-NP and anti-S IgG positivity was determined using Cohen’s kappa coefficient. Results The study enrolled 181 midshipmen. COVID-19 vaccinations were administered in March/April 2021. Samples were collected for 101 participants in August, 73 in December, 57 in February (saliva only), and 63 in April/May. In August, 17 (17%) participants showed evidence of SARS-CoV-2 infection based on anti-S IgG values from DBS and/or saliva. By December 2020, anti-S seroconversion was observed for 5 more based on DBS and/or saliva. By May 2021, 100% of participants were anti-S IgG seropositive after vaccination based on DBS and/or saliva; 48% of participants had seroconverted to anti-NP IgG. Among participants with both DBS and saliva samples, a coefficient of 0.64 showed substantial agreement between anti-S IgG results in August and perfect agreement in December (Table 1). DBS and saliva results for anti-NP IgG were in perfect agreement through December and in substantial agreement in May (0.68, Table 2). Prior to vaccination in March/April 2021, 4/48 of participants had at least one documented SARS-CoV-2 PCR positive result (Table 3). Cumulative PCR test positivity concordance with DBS seroconversion was 37.5% and 60% for anti-S IgG and anti-NP IgG, respectively. Conclusion There was a substantive SARS-CoV-2 attack rate before vaccination; all vaccinees mounted an anti-S IgG response in blood. We note high agreement between DBS and saliva for IgG measurement. Serology-based surveillance identified substantially more SARS-CoV-2 infections than PCR screening. Disclosures Jitendrakumar Modi, MD, GlaxoSmithKline: I am a paid speaker for GSK. I do not speak for their flu brand. Timothy H. Burgess, MD, MPH, AstraZeneca: The HJF, in support of the USU IDCRP, was funded to conduct or augment unrelated Phase III Mab and vaccine trials as part of US Govt. COVID19 response Simon Pollett, MBBS, Astra Zeneca: The HJF, in support of the USU IDCRP, was funded to conduct or augment unrelated Phase III Mab and vaccine trials as part of US Govt. COVID19 response Mark P. Simons, PhD, AstraZeneca: The HJF, in support of the USU IDCRP, was funded to conduct or augment unrelated Phase III Mab and vaccine trials as part of US Govt. COVID19 response.

2052. Temporal and Geographical Prevalence of Carbapenem-Resistant Pseudomonas aeruginosa and the In Vitro Activity of Ceftolozane/Tazobactam and Comparators in Taiwan – SMART 2012-2020

Abstract Background Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is listed as a critical pathogen by World Health Organization (WHO). Using clinical isolates collected in Taiwan as part of the global SMART surveillance program, we evaluated the trend of CRPA prevalence in recent nine years (2012-2020) as well as in vitro activity of ceftolozane/tazobactam (C/T) and comparators against CRPA collected from ICU vs non-ICU wards from 2016 to 2020. Methods Between 2012-2020, Pseudomonas aeruginosa (PA) isolates were collected from nine sites in Taiwan (2 in Northern, 3 in Central and 4 in Southern Taiwan). MICs were determined and interpreted based on CLSI broth microdilution guidelines and 2021 CLSI breakpoints. PCR screening and β-lactamase gene sequencing were performed for isolates non-susceptible to imipenem or imipenem/relebactam or C/T. Results During the study period, a total of 2639 PA isolates were collected. Among these, 448 isolates were resistant to either imipenem or meropenem (17.0%). Yearly prevalence rate of CRPA increased from 12.3% in 2012 to 22.8% in 2020: 12.3% in 2012 (14/114), 11.5% in 2013 (16/139), 11.5% in 2014 (13/113), 12.2% in 2015 (41/336), 15.5% in 2016 (57/367), 15.9% in 2017 (68/428), 20.4% in 2018 (73/357), 19.4% in 2019 (75/386) and 22.8% (91/399) in 2020. Hospitals in Northern Taiwan had higher CRPA prevalence rate (22.8%, 131/574) compared to those in Central (15.7%, 126/802) and Southern Taiwan (15.1%, 191/1263). Percentage susceptibility of CRPA isolates to C/T from 2016 to 2020 showed 89.47% (51/57) in 2016, 79.41% (54/68) in 2017, 93.2% (68/73) in 2018, 90.7% (68/75) in 2019 and 98.9% (90/91) in 2020. For ICU vs. Non-ICU CRPA isolates, the susceptibility to C/T were 88.3% vs.93.8%, 77.8% vs. 82.5%, 92.3 % vs. 92.9%, 87.1% vs.93.2% and 100% vs. 98.26%, in 2016,2017, 2018, 2019 and 2020, respectively. Conclusion During the period of 2012-2020, the yearly prevalence of CRPA in Taiwan hospitals increased from 12.3% to 22.8%. The high prevalence and increasing trend of CRPA warrant continuous monitoring of evolving antibiotic resistance in Taiwan. Disclosures Tai-Chin Hsieh, MBBS, MSD Taiwan: Advisor/Consultant|MSD Taiwan: Employee of MSD Taiwan|MSD Taiwan: Stocks/Bonds.

504. Latent HHV7 Infection Attenuates Beneficial Host-Microbe Interactions in the Human Gut

Abstract Background Human Herpes Virus 7 (HHV7) infection results in a lifelong latent infection with over 95% of adults seropositive for HHV7. Lymphocytes are the best understood host cell type for latent HHV7, with the gastric mucosa another site of latent infection. Herpesviridae latency affects host processes like interferon signaling, antigen presentation, and proliferation—the same host pathways are implicated in the beneficial responses to commensal microbes. Methods Adult human colonic organoids with or without latent HHV7 infection were co-cultured with B. longum in asymmetric oxygen conditions that allow for live interaction (F1). The transcriptional state after 24h of interactions was determined with RNA-sequencing. The transcriptional state was compared between axenic (no microbes) or after co-culture and with or without latent HHV7. F1: Establishing oxygen gradients across epithelial monolayers (A) Schematic of oxygen gradient apparatus (B) Closeup view of individual well (C) Growth of the anaerobe B. longum was identical in the apparatus with hypoxic epithelial compared to full anaerobic growth (D) Butyrate administration protects the epithelium from inflammatory effects of deoxycholate. Results Screening human intestinal organoids (that lack lymphocytes and are strictly of the endodermal lineage) we have generated evidence that intestinal stem cells (ISC) and IEC can be a host cell for latent HHV7 infection in some but not all people (T1). RNA-sequencing reveals that this is true latency, with ISC and IEC containing HHV7 without transcripts from any of the protein-coding genes in the HHV7 genome. Further we have observed that latent HHV7 is mosaic in the gut, not affecting all of the ISC in an individual (T1). In organoid-derived human IEC we have noted co-culture with the commensal bacteria B. longum suppresses key executors of apoptosis and pyroptosis (F2). These same effects are no longer observed with the B. longum is interacting with IEC latently infected with HHV7. A similar pattern was observed with NFkB regulators (F3). T1: Latent HHV7 is found in some intestinal organoids derived from healthy adults From the University of Michigan Translational Tissue Modeling Laboratory (TTML) organoid registry. Each donor is assigned a unique numerical ID. Viral panel is from a commercial multiplex PCR screen. Notably, donor 87 has colonic organoids with and without latent HHV7. F2: Co-culture with the commensal microbe B. longum suppresses most apoptosis and pyroptosis executors only in an organoid without latent HHV7 infection. Fragments per kilobase per millions of reads (FPKM) from RNA-sequencing of fully-differentiated organoid-derived human intestinal epithelium after 24 hours of co-culture with B.longum (Orange) or remaining axenic (without microbes; Blue). Results from cell without (left) or with (right) latent HHV7. F3: Stimulation of the NFkB inhibitor NFKBIA by co-culture with the commensal microbe B. longum is absent in organoids with latent HHV7 infection. Fragments per kilobase per millions of reads (FPKM) from RNA-sequencing of fully-differentiated organoid-derived human intestinal epithelium after 24 hours of co-culture with B.longum (Orange) or remaining axenic (without microbes; Blue). Results from cell without (left) or with (right) latent HHV7. Conclusion Latent HHV7 in the human gut epithelium may attenuate the effect of beneficial microbes like B. longum (F4). The evidence for mosaicism of latent HHV7 in the human gut adds a spatial aspect to these attenuating effects that could be relevant for specific disease processes (e.g. skip lesions in Crohn’s disease). Confirmation of these findings, estimating the true prevalence of latent HHV7 in the gut, and identification of the HHV7 latency mechanism will be required to understand the full implications of these findings. F4: A Model of Latent HHV7 Infection Attenuating Responses to Commensal Microbes. Based on our findings, latent HHV7 infection in the intestinal epithelium may leave the gut mucosa at increased risk for injury by impairing the beneficial response to commensal microbes in a spatially complex manner due to the apparent mosaic nature of latent HHV7 infection in the gut. Disclosures Jonathan L. Golob, MD/PhD, Loxo Oncology: Employment of Spouse.

Emergence of Mansonella sp. in free-ranging primates in southern Brazil.

Mansonellosis is a neglected and emerging tropical disease. Among all zoonotic filarial diseases, it is probably the most prevalent and least studied, with approximately 114 million people infected. The parasites of Mansonella spp. are among the most common blood parasitemias and are widely found in Africa and Latin America. Through molecular analysis of blood samples from free-ranging primates Sapajus nigritus (n 33) and Alouatta guariba clamitans (n 5) in the southern states of Brazil (Santa Catarina and Rio Grande do Sul), we identified samples positive for Mansonella perstans in two specimens of A. guariba clamitans. A fragment of 578bp from the ITS intergenic region (5.8S-ITS2-28S) was targeted for an initial PCR screening. Subsequently, positive samples were subjected to other PCR assays targeting a fragment of the 12S and the 18S genes. This is the first record of molecular detection of the agent in this host in the Pampa Biome. With a wide distribution across Brazil and Argentina, these primates may represent a potential wild reservoir for the zoonotic agent of mansonellosis. Entomological and transmission studies are essential to avoid the urbanization of mansonellosis and to understand the cycles of agents in different environmental scenarios.

Finding of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in wild game meat originating from several European countries: predominance of Moellerella wisconsensis producing CTX-M-1, November 2021.

IntroductionMeat can be a vehicle for food-borne transmission of antimicrobial resistant bacteria and antimicrobial resistance genes. The occurrence of extended-spectrum beta-lactamase (ESBL) producing Enterobacterales has been observed in meat from livestock production but has not been well studied in meat from wild game.AimWe aimed to investigate, particularly in central Europe, to what extent ESBL-producing Enterobacterales may be present in wild game meat.MethodsA total of 111 samples of different types of game meat supplied by butchers, hunters, retail stores and a large game-processing establishment in Europe were screened for ESBL-producing Enterobacterales using a selective culture medium. Isolates were genotypically and phenotypically characterised.ResultsThirty-nine samples (35% of the total) yielded ESBL-producing Enterobacterales, with most (35/39) supplied by the game-processing establishment. Isolates included 32 Moellerella wisconsensis, 18 Escherichia coli and one Escherichia marmotae. PCR screening identified bla CTX-M-1 (n = 31), bla CTX-M-32 (n = 8), bla CTX-M-65 (n = 4), bla CTX-M-15 (n = 3), bla CTX-M-8 (n = 1), bla CTX-M-14 (n = 1), bla CTX-M-55 (n = 1), and bla SHV-12 (n = 2). Most E. coli belonged to phylogenetic group A (n = 7) or B1 (n = 9), but several isolates belonged to extraintestinal pathogenic E. coli (ExPEC) sequence types (ST)58 (n = 4), ST68 (n = 1) and ST540 (n = 1). Whole genome sequencing of six selected isolates localised bla CTX-M-1 on megaplasmids in four M. wisconsensis and bla CTX-M-32 on IncN_1 plasmids in one M. wisconsensis and one E. marmotae. Forty-eight isolates (94%) exhibited a multidrug-resistance phenotype.ConclusionWe found a high occurrence of ESBL-producing Enterobacterales in wild game meat, suggesting wildlife habitat pollution and possible microbial contamination events occurring during skinning or cutting carcasses.

Blood Mucorales PCR to track down Aspergillus and Mucorales co-infections in at-risk hematology patients: A case-control study.

Serum Mucorales PCR can precede the final diagnosis of invasive mucormycosis by several days or weeks and could therefore be useful as a non-invasive screening tool. We assessed the performance of a commercial Mucorales PCR assay (MucorGenius®, PathoNostics, Maastricht, The Netherlands) on prospectively collected banked sera from hematology patients at risk for invasive mould infections. We evaluated if there is an underestimated incidence of missed Mucorales co-infections in patients with invasive aspergillosis (IA). We tested Mucorales PCR on the sera of all patients with a diagnosis of at least possible IA (EORTC-MSGERC consensus criteria) before the start of any antifungal therapy, and in a control group of similar high-risk hematology patients without IA (in a 1:4 ratio). When a positive Mucorales PCR was observed, at least 5 serum samples taken before and after the positive one were selected. Mucorales PCR was performed in 46 diagnostic serum samples of cases and in 184 controls. Serum Mucorales PCR was positive in 4 cases of IA (8.7%; 12.9% of probable cases) and in 1 control case (0.5%) (p=0.0061, OR=17.43 (1.90-159.96). Post-mortem cultures of the positive control became positive for Rhizopus arrhizus. Mortality of IA cases with and without a positive Mucorales PCR was not significantly different. Only in the PCR positive control case, serial serum samples before and after the diagnostic sample were also positive. It is not entirely clear what a positive Mucorales PCR in these cases implies since the 4 Mucorales PCR positive cases were treated with antifungals with activity against Mucorales. In addition, PCR was positive only once. This study does not provide enough evidence to implement Mucorales PCR screening. However, our findings emphasize once more the importance of considering the possibility of dual mould infections, even in patients with a positive galactomannan detection.

A new species of Andricus Hartig, 1840 (Hymenoptera, Cynipidae) from China, with references to DNA taxonomy and Wolbachia infection.

In the present paper, a new species of cynipid gall wasp, Andricuselodeoides Liu & Pang, is described from several provinces in southern China. The new species is closely related to the recently redescribed A.mairei (Kieffer, 1906). In addition to differences in adult and gall morphology, the new species is also readily separated by COI sequences, with a 6.2-8.9% genetic distance between populations of the new species and those of A.mairei. A contrasting difference in sex ratios was also observed between the two species, with A.elodeoides extremely female-biased (95.5-97.8% female) while A.mairei male-biased to more balanced (5.4-43.5% female). PCR screening for Wolbachia infection further revealed contrasting infection rates between populations of A.elodeoides and A.mairei: the Wolbachia infection rate was 0% in A.elodeoides and 100% in A.mairei. Cytoplasmic incompatibility induced by Wolbachia is proposed as a potential mechanism of speciation of the sympatric A.elodeoides and A.mairei.

Pre-Endoscopy real-time PCR testing for SARS-CoV2 does not reduce health care workers infection and is associated with a higher reduction of endoscopic activity in an outpatient setting.

The role of pre-procedure SARS-CoV2 testing in digestive endoscopy is still debated. AGA guidelines recommend against pre-procedure testing considering low prevalence of SARS- CoV2 infection in the general population and low incidence of infection among endoscopy units Health Care Workers (HCWs). However, no studies have compared pre-procedure testing associated to symptom screening vs. symptom screening alone in reducing the risk of infection for HCWs. Main aim of the present study is to compare the risk of infection for HCWs in different Endoscopy Units adopting different pre-endoscopy screening and operating in two nearby hospital of the same region in Northern Italy in pre-vaccination period. For outpatients in the Endoscopy Unit of Trento (Unit 1) only pre-procedure symptom screening was performed, while in the Endoscopy Unit of Bolzano (Unit 2) pre-procedure symptom screening and negative pre-procedure real-time PCR were requested. Secondary aims were to assess the impact of pre-procedure real-time PCR testing on endoscopic activity and diagnostic delay. Retrospective data collection on a prospectively maintained database was performed, including outpatient endoscopy procedures performed between June 1st 2020 and February 28th 2021 in Unit 1 and Unit 2. No differences in terms of infection rate in HCWs have been identified in Unit 1 and Unit 2 (9.0 vs. 19.3% P=0.2) over a nine-month period. Moreover, in the unit performing pre- procedure real-time PCR before endoscopy a significantly higher reduction in endoscopic activity has been recorded (61.9% vs. 53.4%; P<0.01). In patients with positive real-time PCR, endoscopy was performed with a mean delay of 61.7 days (range 9-294) and 22.5% of them were lost at follow-up and did not undergo any endoscopic procedure in the following 12 months. This study supports the AGA recommendation suggesting that pre-endoscopy real-time PCR is an expensive and time-consuming procedure without proven benefits in an outpatient setting.

Multi-Locus Sequencing Reveals Putative Novel Anaplasmataceae Agents, 'Candidatus Ehrlichia dumleri' and Anaplasma sp., in Ring-Tailed Coatis (Carnivora: Nasua nasua) from Urban Forested Fragments at Midwestern Brazil.

The Anaplasmataceae family encompasses obligate intracellular α-proteobacteria of human and veterinary medicine importance. This study performed multi-locus sequencing to characterize Ehrlichia and Anaplasma in coati's blood samples in Midwestern Brazil. Twenty-five samples (25/165-15.1%) were positive in the screening PCR based on the dsb gene of Ehrlichia spp. and were characterized using 16S rRNA, sodB, groEL, and gltA genes and the 23S-5S intergenic space region (ITS). Phylogenetic analyses based on all six molecular markers positioned the sequences into a new clade, with a common origin of Ehrlichia ruminantium. Haplotype analyses of 16S RNA sequences revealed the presence of two distinct Ehrlichia genotypes. Six samples (6/165, 3.6%) were positive in the screening nPCR for the 16S rRNA gene of Anaplasma spp. and were submitted to an additional PCR targeting the ITS for molecular characterization. Phylogenetic analyses based on both 16S rRNA gene and ITS positioned the Anaplasma sp. detected in the present study in a large clade with other Anaplasma sp. previously detected in ticks and wild animals and in a clade with 'Candidatus Anaplasma brasiliensis', respectively. Based on distinct molecular markers, the present work described a putative novel Anaplasmataceae agent, namely 'Candidatus Ehrlichia dumleri', and Anaplasma sp. closely related to the previously described 'Candidatus Anaplasma brasiliensis'.

Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin.

CYP105AS1 is a cytochrome P450 from Amycolatopsis orientalis that catalyzes monooxygenation of compactin to 6-epi-pravastatin. For fermentative production of the cholesterol-lowering drug pravastatin, the stereoselectivity of the enzyme needs to be inverted, which has been partially achieved by error-prone PCR mutagenesis and screening. In the current study, we report further optimization of the stereoselectivity by a computationally aided approach. Using the CoupledMoves protocol of Rosetta, a virtual library of mutants was designed to bind compactin in a pro-pravastatin orientation. By examining the frequency of occurrence of beneficial substitutions and rational inspection of their interactions, a small set of eight mutants was predicted to show the desired selectivity and these variants were tested experimentally. The best CYP105AS1 variant gave >99% stereoselective hydroxylation of compactin to pravastatin, with complete elimination of the unwanted 6-epi-pravastatin diastereomer. The enzyme-substrate complexes were also examined by ultrashort molecular dynamics simulations of 50 × 100 ps and 5 × 22 ns, which revealed that the frequency of occurrence of near-attack conformations agreed with the experimentally observed stereoselectivity. These results show that a combination of computational methods and rational inspection could improve CYP105AS1 stereoselectivity beyond what was obtained by directed evolution. Moreover, the work lays out a general in silico framework for specificity engineering of enzymes of known structure.

Group Testing Matrix Design for PCR Screening with Real-Valued Measurements.

Single-step nonadaptive group testing approaches for reducing the number of tests required to detect a small subset of positive samples from a larger set require solving two algorithmic problems. First, how to design the samples-to-tests measurement matrix, and second, how to decode the results of the tests to uncover positive samples. In this study, we focus on the first challenge. We introduce real-valued group testing, which matches the characteristics of existing PCR testing pipelines more closely than combinatorial group testing or compressed sensing settings. We show a set of conditions that allow measurement matrices to guarantee unambiguous decoding of positives in this new setting. For small matrix sizes, we also propose an algorithm for constructing matrices that meet the proposed condition. On simulated data sets, we show that the matrices resulting from the algorithm can successfully recover positive samples at higher positivity rates than matrices designed for combinatorial group testing setting. We use wet laboratory experiments involving SARS-CoV-2 nasopharyngeal swab samples to further validate the approach.

Characteristic of cardiac and neurological forms of non-polio enterovirus infection

Enterovirus infections (EVI) are ubiquitous and generally present with mild symptoms and have a favorable prognosis with full recovery. But sometimes it can be challenging to diagnose mixed forms of EVI which can result in fatal outcomes. An interesting case report on a patient admitted to the Grodno Regional infectious diseases clinical hospital. The patient was diagnosed with enteroviral infection. Histological slides of the brain, heart, lung and other systemic organs were prepared on autopsy and are presented in this scientific paper. Generalized EVI in mixed form can cause primary lesions of the brain (destructive edema), the heart (necrotizing cardiomyopathy), and sepsis while also affecting other organ systems. This can lead to unfavorable outcomes similar to that in our case report. Mixed form EVI (meningitis, myocarditis, and sepsis) can progress rapidly towards an adverse course, with the development of severe life-threatening complications. We strongly suggest that mandatory PCR screening for EVI should be carried out in young individuals with sepsis-like diseases and with a fever of unexplained origin at the time of presentation.

Shiga toxin (stx) encoding genes in sheep and goats reared in Trinidad and Tobago.

Shiga toxin-producing Escherichia coli (STEC) is estimated to cause over two million cases of human disease annually. Trinidad and Tobago is one of the largest livestock producer and consumer of sheep and goat meat in the Caribbean, however, the potential role of these animals in the epidemiology of STEC infections has not been previously described. To fill this critical gap in knowledge, the prevalence of Shiga toxin genes (stx1 and stx2) shed in the faeces of healthy sheep (n = 204) and goats (n = 105) in Trinidad was investigated. Based on PCR screening, goats had a higher stx prevalence than sheep (46% vs 35%, P = 0.06). Most of the recovered STEC isolates were positive for stx1 only; and only three isolates were positive for the eae gene. None of the recovered isolates belonged to the O157 serogroup. In both species, the prevalence of stx was higher in young animals versus older animals. Sheep reared on deep litter flooring (43%) had a higher prevalence than sheep reared other flooring types, however this was not the same for goats. The presence of cows on the same premise was not an associated predictor for STEC carriage in sheep or goats. This study demonstrates that although sheep and goats in Trinidad are reservoirs for stx-positive E. coli isolates, no fecal samples tested positive for O157 STEC, harbored. Furthermore, it appears that non-O157 stx-positive isolates harbored by these animals do not pose a significant threat to human health.

Report and Comparative Genomics of an NDM-5-Producing Escherichia coli in a Portuguese Hospital: Complex Class 1 Integrons as Important Players in blaNDM Spread.

New Delhi metallo-beta-lactamase (NDM) has been spreading across the globe, but the causes of its success are poorly understood. We characterized a blaNDM-5-positive Escherichia coli strain from a Portuguese hospital and conducted comparative genomic analyses to understand the role of clonal background and horizontal gene transfer in blaNDM-5 dissemination. After blaNDM PCR screening and genome sequencing, Ec355340 was subjected to mating, transformation, and plasmid curing assays and MICs determination for several antibiotics. Comparison with data compiled from public databases was performed. blaNDM-5 was in a complex integron co-located in a FIB-FII plasmid (pEc355340_NDM-5). The mating assays were unsuccessful, but plasmid transformation into a susceptible host led to resistance to all beta-lactams and to sulfamethoxazole-trimethoprim. The profile of virulence genes (n = 73) was compatible with extraintestinal pathogenesis. An analysis of genomes from public databases suggested that blaNDM-5 has rarely been associated with ST156 strains (such as Ec355340), while is has frequently been found on strains of the ST10 clonal complex. However, ST156 may play a role in the co-spreading of blaNDM and mcr genes. Regardless, comparative genomics confirmed the presence of blaNDM in similar complex integrons in plasmids (48/100 plasmids most similar to pEc355340_NDM-5) and ST156 genomes (20/41 blaNDM-positive genomes). blaNDM-5 and other blaNDM variants were more frequently associated to complex integrons than previously reported and, therefore, these platforms may be important drivers in their dissemination. The identification of blaNDM-5 for the first time in Portugal could be a game-changer in the current Portuguese antibiotic resistance scenario, as this gene encodes a higher-level resistance phenotype, and its spread may be facilitated due to the association with complex integrons.

Alarming Antibiotic Resistance of Lactobacilli Isolated from Probiotic Preparations and Dietary Supplements.

In this study, we screened eight commercially available brands of Lactobacillus-containing probiotic preparations and dietary supplements for resistance towards commonly administered antibiotics of different classes. According to disc diffusion results, most of the isolates were resistant to vancomycin and susceptible to penicillin-type antibiotics (ampicillin and amoxicillin), carbapenems (imipenem, meropenem, and ertapenem), and inhibitors of protein synthesis (chloramphenicol, erythromycin, tetracycline, clarithromycin, and linezolid). However, based on minimum inhibitory concentration (MIC) values, six strains were reconsidered as resistant to tetracycline. All tested lactobacilli were resistant towards amikacin, ciprofloxacin, and norfloxacin. Resistance to cephalosporins was highly variable and decreased in the following order: ceftazidime/cefepime, ceftriaxone, cefotaxime, cefazolin, and cefoperazone. PCR screening for antibiotic resistance determinants in probiotic lactobacilli revealed a wide occurrence of vancomycin resistance gene vanX, ciprofloxacin resistance gene parC, and extended-spectrum β-lactamase gene blaTEM. We also detected the tetK gene for tetracycline resistance in one isolate. Additionally, we identified discrepancies between the claims of the manufacturers and the identified species composition, as well as the enumerated amount of viable bacteria, for several products. The results of this study raise concerns about the safety of lactobacilli for human consumption as probiotics, as they may act as reservoirs of transferable antibiotic resistance genes.

Asymptomatic Cryptosporidiosis in Children Living with HIV.

Children living with human immunodeficiency virus (HIV) have an increased risk of opportunistic Cryptosporidium infection. Cryptosporidium usually causes chronic diarrhea that may lead to impaired growth and cognitive function in children. This study aimed to estimate the prevalence of cryptosporidiosis in children, describe its clinical characteristics, and the risk factors. A cross-sectional study involving children aged 6 months to 18 years old with confirmed HIV infection was carried out in Sardjito General Hospital, Yogyakarta. Diagnosis of cryptosporidiosis was made by PCR of 18S rRNA after being screened by microscopic examination. The clinical characteristics and risk factors were obtained from medical records and structured questionnaires. A total of 52 participants were included in the final analysis. The prevalence of cryptosporidiosis was 42.3%. Approximately 68% of the HIV children with cryptosporidiosis were asymptomatic, while those who reported symptoms showed weight loss and diarrhea. Independent risk factors of cryptosporidiosis were diarrhea (AOR 6.5; 95% CI 1.16-36.67), well water as drinking water source (AOR 6.7; 95% CI 1.83-24.93), and drink untreated water (AOR 5.8; 95% CI 1.04-32.64). A high prevalence of asymptomatic cryptosporidiosis was observed among children with HIV infection and PCR screening of Cryptosporidium in high-risk children is advisable.

Single Nucleotide Polymorphism-Based Real-Time PCR Screening Assay for Rapid Tracking of Bacterial Infection Clusters To Complement Whole-Genome Sequencing Efforts during Outbreak Investigations.

Infection clusters of multidrug-resistant bacteria increase mortality and entail expensive infection control measures. Whereas whole-genome sequencing (WGS) is the current gold standard to confirm infection clusters, PCR-based assays targeting cluster-specific signatures, such as single nucleotide polymorphisms (SNPs) derived from WGS data, are more suitable to initially screen for cluster isolates within large sample sizes. Here, we evaluated four software tools (SeqSphere+, RUCS, Gegenees, and Find Differential Primers) regarding their efficiency to find SNPs within WGS data sets that were specific for two bacterial monospecies infection clusters but were absent from a WGS reference data set comprising several hundred diverse genotypes of the same bacterial species. Cluster-specific SNPs were subsequently used to establish a probe-based real-time PCR screening assay for in vitro differentiation between cluster and noncluster isolates. SeqSphere+ and RUCS found 2 and 24 SNPs for clusters 1 and 14 and 24 SNPs for cluster 2, respectively. However, some signatures detected by RUCS were not cluster specific. Interestingly, all SNPs identified by SeqSphere+ were also detected by RUCS. In contrast, analyses with the remaining tools either resulted in no SNPs (with Find Differential Primers) or failed (Gegenees). Design of six cluster-specific real-time PCR assays enabled reliable cluster screening in vitro. Our evaluation revealed that SeqSphere+ and RUCS identified cluster-specific SNPs that could be used for large-scale screening in surveillance samples via real-time PCR, thereby complementing WGS efforts. This faster and simplified approach for the surveillance of bacterial clusters will improve infection control measures and will enhance protection of patients and physicians. IMPORTANCE Infection clusters of multidrug-resistant bacteria threaten medical facilities worldwide and cause immense health care costs. In recent years, whole-genome sequencing (WGS) has been increasingly applied to detect and to further control bacterial clusters. However, as WGS is still expensive and time-consuming, its exclusive application for screening and confirmation of bacterial infection clusters contributes to high costs and enhanced turnaround times, which many hospitals cannot afford. Therefore, there is need for alternative methods that can enable further surveillance of bacterial clusters that are initially detected by WGS in a faster and more cost-efficient way. Here, we established a system based on real-time PCR that enables rapid large-scale sample screening for bacterial cluster isolates within 7 days after the initial detection of an infection cluster, thereby complementing WGS efforts. This faster and simplified surveillance of bacterial clusters will improve infection control measures and will enhance protection of patients and physicians.

Nested Real-Time PCR Assessment of Vertical Transmission of Sandalwood Spike Phytoplasma ('Ca. Phytoplasma asteris').

Simple SummaryThe Sandalwood Spike disease (SSD) related to ‘Ca. Phytoplasma asteris’ has almost wiped out the sandalwood population from the forests of southern India. It is known that sap-sucking insect vectors transmit phytoplasmas; however, their transmission through seeds needs thorough investigation. We found that 38.66% and 23.23% of one-month and four-month-old seedlings, respectively, tested positive for SSD phytoplasma screened using modified real-time qPCR assays in insect-free environments. Considering the current efforts to reestablish the healthy sandalwood population and its commercial importance, these findings are worrisome. The role of some other microbes in the high mortality rates of sandalwood seedlings remains unknown and requires further investigation.The Sandalwood Spike disease (SSD)-related to ‘Ca. Phytoplasma asteris’ has threatened the existence of sandalwood in India. The epidemiology of SSD is still poorly understood despite the efforts to understand the involvement of insect vectors in SSD transmission and alternate plant hosts over the last two decades. Apart from the transmission of SSD phytoplasma through insect vectors, the information on vertical transmission is entirely unknown. Over 200 seeds from SSD-affected trees and over 500 seedlings generated using commercially purchased seeds were screened for the presence of SSD phytoplasma to understand the vertical transmission in an insect-free environment. The end-point nested PCR and real-time nested PCR-based screening revealed an alarming rate of 38.66% and 23.23% phytoplasma positivity in one-month and four-month-old seedlings, respectively. These results were further validated by visualizing the phytoplasma bodies in sandalwood tissues using scanning electron microscopy. The presence of phytoplasma DNA in the seeds and seedlings is a concern for the commercial distribution of sandalwood seedlings in the current setup. This also poses a fear of spreading the disease to newer areas and negatively affecting the economy. The seedling mortality was also suspected to be associated with isolated bacterial and fungal isolates such as Erwinia, Curtobacterium, Pseudomonas, Rhodococcus, Aspergillus, Fusarium, and Neofusicoccum isolated using a culture-dependent approach. These findings strongly recommend the accreditation of commercial production of sandalwood seedlings curtailing SSD phytoplasma’s menace. Additionally, a new nested end-point and qRT PCR assays developed in this study proved valuable for the rapid screening of phytoplasma in many plant samples to detect phytoplasmas.

Persistence of transferable oxazolidinone resistance genes in enterococcal isolates from a swine farm in China.

The appearance of transferable oxazolidinone resistance genes poses a major challenge to public health and environmental safety. These genes not only lead pathogenic bacteria to become resistant to linezolid but also reduce sensitivity to florfenicol, which is widely used in the veterinary field. To verify the dissemination of oxazolidinone resistance genes in enterococcal isolates from pigs at different production stages in a swine farm in China, we collected 355 enterococcal isolates that were resistant to florfenicol from 600 (150 per stage) fresh fecal swabs collected from a swine farm. Through initial PCR screening and whole-genome sequencing, 175 isolates harboring different oxazolidinone resistance genes were identified. All isolates carried the optrA gene. A total of 161 (92%, 161/175) isolates carried only the optrA gene. Three (1.71%, 3/175) isolates carried both the optrA and poxtA genes, and 11 (3.1%, 11/175) isolates contained the optrA gene and poxtA2 and cfr(D) variants. A total of 175 isolates that harbored oxazolidinone resistance genes included 161 E. faecalis, 6 E. faecium, and 8 E. hirae. By sequencing the whole genomes, we found that the 161 isolates of E. faecalis belonged to 28 different STs, including 8 new STs, and the 6 isolates of E. faecium belonged to four different STs, including one new ST. The phylogenetic tree based on SNPs of the core genome showed that both clonal spread and horizontal transfer mediated the diffusion of oxazolidone resistance genes in enterococcal isolates at specific stages in pig farms. Moreover, enterococcal isolates carrying oxazolidone resistance genes could spread from breeding pigs to fattening pigs, while transferable oxazolidone resistance genes in enterococcal isolates could persist on a pig farm throughout all production stages. Representative enterococcal isolates with different oxazolidinone resistance genes were further studied through Nanopore sequencing. We identified a novel plasmid, pM4-80 L4 (15,008 bp), carrying the poxtA2 and cfr(D) genes in enterococcal isolates at different stages. We also found three different plasmids harboring the poxtA gene with high genetic variation, and all poxtA genes were flanked by two copies of IS1216E elements. In addition, four genetically distinct plasmids carrying the optrA gene were identified, and Tn554 was found to mediate chromosome-localized optrA gene transfer. Our study highlighted that transferable oxazolidinone resistance genes in enterococcal isolates could persist throughout all production stages on a pig farm, and the prevalence and dissemination of oxazolidinone resistance genes in enterococcal isolates from animal farms should be continually monitored.

Virulence, antimicrobial susceptibility and phylogenetic analysis of Cronobacter sakazakii isolates of food origins from Jordan.

The aim was to characterize a collection of Cronobacter sakazakii isolates collected from various origins in Jordan. The isolates were characterized using 16S rRNA sequencing, DNA microarray, multi-locus sequence typing (MLST), O-serotyping, virulence gene identification and antibiotic susceptibility testing. The identities and phylogenetic relatedness revealed that C. sakazakii sequence type 4 (ST4) and Csak O:1 serotype were the most prevalent STs and serovars amongst these C. sakazakii strains. PCR screening of putative virulence genes showed that the siderophore-interacting protein gene (sip) and iron acquisition gene clusters (eitCBAD and iucABCD/iutA) were the most detected genes with noticeable variability in the type 6 secretion system (T6SS) and filamentous hemagglutinin/adhesion (FHA) gene loci. The antibiotic resistance profiles revealed that the majority of the isolates were susceptible to all antibiotics used despite harbouring a class C β-lactamase resistance gene. The results described in this report provide additional insights about the considerable genotypic and phenotypic heterogeneity within C. sakazakii. The information reported in this study might be of great value in understanding the origins of C. sakazakii isolates, in addition to their diversity and variability, which might be helpful in preventing future outbreaks of this pathogen.