Molecular Epidemiological Investigation Research Articles

Molecular characterization and epidemiological investigation of colistin resistance in carbapenem-resistant Klebsiella pneumoniae in a tertiary care hospital in Tehran, Iran

BackgroundCarbapenemase-producing Klebsiella pneumoniae (CRKP) presents a significant challenge to antimicrobial therapy, especially when compounded by resistance to colistin. The objective of this study was to explore molecular epidemiological insights into strains of clinical K. pneumoniae that produce carbapenemases and exhibit resistance to colistin. Eighty clinical isolates of CRKP were obtained from Milad Hospital in Tehran, Iran. Antimicrobial susceptibility and colistin broth disk elution were determined. PCR assays were conducted to examine the prevalence of resistance-associated genes, including blaKPC, blaIMP, blaVIM, blaOXA−48, blaNDM and mcr-1 to -10. Molecular typing (PFGE) was used to assess their spread.ResultsColistin resistance was observed in 27 isolates (33.7%) using the Broth Disk Elution method. Among positive isolates for carbapenemase genes, the most frequent gene was blaOXA−48, identified in 36 strains (45%). The mcr-1 gene was detected in 3.7% of the obtained isolates, with none of the other of the other mcr genes detected in the studied isolates.ConclusionTo stop the spread of resistant K. pneumoniae and prevent the evolution of mcr genes, it is imperative to enhance surveillance, adhere rigorously to infection prevention protocols, and implement antibiotic stewardship practices.

A Duplex PCR Assay for Rapid Detection of Klebsiella pneumoniae and Chryseobacterium in Large Yellow Croaker Fish.

Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 μM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.

Molecular epidemiological investigation and recombination analysis of Cachavirus prevalent in China.

Chaphamaparvovirus carnivoran1 (canine Chaphamaparvovirus, also known as Cachavirus [CachaV]) is a novel parvovirus first reported in dog feces collected from the United States in 2017 and China in 2019. To continuously track its infection and evolution status, 276 canine anal swabs were obtained from pet hospitals in central, northern, and eastern China between 2021 and 2023 and screened via polymerase chain reaction; subsequently, a systematic study was conducted. Of these samples, nine (3.3%) were positive for CachaV. Using polymerase chain reaction, whole genome sequences of the nine CachaV-positive strains were amplified. The NS1 amino acid sequence identity between CachaV strains from China and other countries was 96.23-99.85%, whereas the VP1 protein sequence identity was 95.83-100%. CHN230521 demonstrated the highest identity for NS1 amino acids (99.85%) and VP1 amino acids (100%) with NWT-W88 and CP-T015. According to the model prediction of CHN220916-VP1 protein, Met64Thr, Thr107Ala, and Phe131Ser mutations may cause tertiary structural changes in VP1 protein. Interestingly, each of the nine CachaV strains harbored the same site mutations in NS1 (Ser252Cys, Gly253Leu, and Gly254Thr). Although no explicit recombination events were predicted, the clustering and branching of the phylogenetic tree were complicated. Based on the evolution trees for VP1 and NS1, the nine CachaV strains identified from 2021 to 2023 were closely related to those identified in gray wolves and cats. This study may be beneficial for evaluating the prevalence of CachaVs in China, thereby understanding the evolution trend of CachaVs.

Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China.

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/μL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

A molecular epidemiological investigation of contagious caprine pleuropneumonia in goats and captive Arabian sand gazelle (Gazella marica) in Oman

BackgroundContagious caprine pleuropneumonia (CCPP) is a fatal WOAH-listed, respiratory disease in small ruminants with goats as primary hosts that is caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). Twelve CCPP outbreaks were investigated in 11 goat herds and a herd of captive Arabian sand gazelle (Gazella marica) in four Omani governorates by clinical pathological and molecular analysis to compare disease manifestation and Mccp genetic profiles in goats and wild ungulates.ResultsThe CCPP forms in diseased and necropsied goats varied from peracute (5.8%), acute (79.2%) and chronic (4.5%) while all of the five necropsied gazelles showed the acute form based on the clinical picture, gross and histopathological evaluation. Colonies of Mccp were recovered from cultured pleural fluid, but not from lung tissue samples of one gazelle and nine goats and all the isolates were confirmed by Mccp-specific real time PCR. Whole genome-single nucleotide polymorphism (SNP) analysis was performed on the ten isolates sequenced in this study and twenty sequences retrieved from the Genbank database. The Mccp strains from Oman clustered all in phylogroup A together with strains from East Africa and one strain from Qatar. A low variability of around 125 SNPs was seen in the investigated Omani isolates from both goats and gazelles indicating mutual transmission of the pathogen between wildlife and goats.ConclusionRecent outbreaks of CCPP in Northern Oman are caused by Mccp strains of the East African Phylogroup A which can infect goats and captive gazelles likewise. Therefore, wild and captive ungulates should be considered as reservoirs and included in CCPP surveillance measures.

Rapid molecular epidemiology investigations into two recent measles outbreaks in Israel detected from October 2023 to January 2024.

Between late 2023 and early 2024, two measles outbreaks occurred in Israel, each caused by importation of measles virus strains of respective B3 and D8 genotypes. In this study, we validate transmission pathways uncovered by epidemiological investigations using a rapid molecular approach, based on complete measles virus genomes. The presented findings support this rapid molecular approach in complementing conventional contact tracing and highlight its potential for informing public health interventions.

Complete Mitogenome sequencing of the fish louse Argulus japonicus (Crustacea: Branchiura): Comparative analyses and phylogenetic implications.

The fish louse Argulus japonicus, a branchiuran crustacean of the Argulidae family, is attracting increasing attention because of its parasitic tendencies and significant health threats to global fish farming. The mitogenomes can yield a foundation for studying epidemiology, genetic diversity, and molecular ecology and therefore may be used to assist in the surveillance and control of A. japonicus. In this study, we sequenced and assembled the complete mitogenome of A. japonicus to shed light on its genetic and evolutionary blueprint. Our investigation indicated that the 15,045-bp circular genome of A. japonicus encodes 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), and 2 ribosomal RNAs (rRNAs) with significant AT and GC skews. Comparative genomics provided an evolutionary scenario for the genetic diversity of 13 PCGs: all were under purifying selection, with cox1 and nad6 having the lowest and highest evolutionary rates, respectively. Genome-wide phylogenetic trees established a close relationship between species of the families Argulidae (Arguloida) and Armilliferidae (Porocephalida) within Crustacea, and further, A. japonicus and Argulus americanus were determined to be more closely related to each other than to others within the family Argulidae. Single PCG-based phylogenies supported nad1 and nad6 as the best genetic markers for evolutionary and phylogenetic studies for branchiuran crustaceans due to their similar phylogenetic topologies with those of genome-based phylogenetic analyses. To sum up, these comprehensive mitogenomic data of A. japonicus and related species refine valuable marker resources and should contribute to molecular diagnostic methods, epidemiological investigations, and ecological studies of the fish ectoparasites in Crustacea.

Epidemiological investigation of coccidiosis and associated risk factors in broiler chickens immunized with live anticoccidial vaccines in China.

Coccidiosis is a costly intestinal disease of chickens caused by Eimeria species. This infection is associated with high mortality, reduced feed efficiency, and slowed body weight gain. The diagnosis and control of coccidiosis becomes challenging due to the fact that chickens can be infected by seven different Eimeria species and often occur mixed-species co-infections. Grasping the epidemiology of Eimeria species is crucial to estimate the efficiency of poultry management. This study aimed to explore the distribution of Eimeria species in broiler chickens in China after administering live anticoccidial vaccines. A total of 634 samples were obtained, and the survey results showed that the prevalence of Eimeria was 86.12% (546/634), and the most common species were E. acervulina (65.62%), E. necatrix (50.95%), E. mitis (50.79%), E. tenella (48.42%), and E. praecox (41.80%). Most samples indicated mixed-species infections (an average of 3.29 species per positive sample). Notably, 63.98% of samples contain 3 to 5 Eimeria species within a single fecal sample. The most prevalent combinations were E. acervulina-E. tenella (38.96%) and E. acervulina-E. necatrix (37.22%). Statistical analysis showed that flocks vaccinated with trivalent vaccines were significantly positive for E. necatrix in grower chickens (OR = 3.30, p < 0.05) compared with starter chickens, and tetravalent vaccinated flocks showed that starter chickens demonstrated a higher susceptibility to E. tenella-E. brunetti (OR = 2.03, p < 0.05) and E. acervulina-E. maxima (OR = 2.05, p < 0.05) compared with adult chickens. Geographically, in the case of tetravalent vaccine-immunized flocks, a substantial positive association was observed between E. necatrix infection rates and flocks from eastern (OR = 3.88, p < 0.001), central (OR = 2.65, p = 0.001), and southern China (OR = 3.17, p < 0.001) compared with southwestern China. This study also found a positive association between E. necatrix (OR = 1.64, p < 0.05), E. acervulina (OR = 1.59, p < 0.05), and E. praecox (OR = 1.81, p < 0.05) infection and coccidiosis occurrence compared with non-infected flocks in tetravalent vaccinated flocks. This molecular epidemiological investigation showed a high prevalence of Eimeria species in the field. The emergent species, E. brunetti and E. praecox, might be incorporated into the widely-used live vaccines in the future. These insights could be useful in refining coccidiosis control strategies in the poultry industry.

Molecular epidemiological investigation of Cryptococcus spp. carried by captive koalas (Phascolarctos cinereus) in Japan.

Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a systemic mycosis that infects a wide range of species. Recent molecular biological investigations have allowed for the genotyping of these species, providing more detailed information on their pathogenicity and infection routes. Koalas (Phascolarctos cinereus) are frequently colonized by Cryptococcus spp., but molecular epidemiological studies have yet to be conducted in Japan. Here, we conducted multi-locus sequence typing (MLST) analysis on Cryptococcus spp. colonization isolates obtained from all koalas kept in seven parks across Japan. Out of 46 koalas examined, 10 (22%) were positive for C. gattii and 3 (6.5%) were positive for C. neoformans. All C. gattii isolates belonged to molecular type VGI and were either sequence type (ST) 51 or a novel ST, and all C. neoformans isolates belonged to molecular type VNI and ST23. Despite the frequent movement of koalas between parks, the STs were relatively park-specific, suggesting that the floor of the rearing barns is a source of infection and may act as a reservoir. MLST analysis confirmed that C. gattii was transported, established, and spread by koalas in areas where C. gattii was not originally present. MLST analysis is considered useful in assessing the pathogenicity and tracing the transmission routes of Cryptococcus spp. carried by koalas.IMPORTANCEThis is the first study to conduct a multi-locus sequence typing analysis on Cryptococcus spp. carried by captive koalas in Japan. Cryptococcosis remains a globally high-fatality fungal infection in humans, and captive koalas are known to carry a high percentage of Cryptococcus spp. Through this research, the molecular types and transmission routes of Cryptococcus spp. carried by koalas have been elucidated, revealing the potential role of enclosure flooring as a reservoir. It has been confirmed that Cryptococcus gattii, which is not endemic in Japan, has become established through koalas and is spreading to new individuals in Japan. This study is believed to provide valuable insights into koala conservation and contribute to the One Health approach for Cryptococcosis, a zoonotic infection.

Serological detection of Newcastle disease virus in backyard poultry production system in Woliso District South West Shewa, Central Ethiopia.

Newcastle disease (ND)is one of the most important respiratory viral diseases. The disease is endemic in many parts of Ethiopia. However, there is no clear record about the introduction of the virus to the country (Ethiopia). Hence, detail about the ND is very important in its (ND) control and prevention. Despite these facts, there is no available research work done on ND in the current research area that would help either as references for researchers or that could help in the control and prevention of the disease. Therefore, the objective of this study was to detect the ND virus (NDV), using serological methods in from December 2018 to November 2019. A cross-sectional type of study was conducted to detect the NDV. The convenience sampling method was used for sample data collection. Before data collection, chicken with previous history of vaccination against the NDV was excluded from the sampling animals. Then, a total of 348 blood samples of 2 mLwere collected from the brachial vein in 3 mLdisposable syringes. The serum was collected in labeled 2 mL cryovial tubes. Indirect enzyme-linked immunosorbent assay(ELISA) tests were performed to detect antibodies against NDV and to determine its antibody titer. The test was performed using (ID.vetinnovative version 2) procedure. In the indirect ELISA test, 37.64% (131/348) were positive and antibody titer mean value of (1761.9088) was scored. The standard deviation of 2592.42160 and a percentage coefficient of variation of147% was scored. From the finding, we conclude that indirect ELISA test detected the presence of the NDV in the study area and the heterogeneousity of antibody titer in the study area. Therefore, further molecular characterization and epidemiological investigation should be carried and vaccination of animals is critical in the study area.

Serological and molecular epidemiological investigation of Mediterranean spotted fever in Yunnan Province, China

ObjectivesGiven the limited research and its potential hazards, the study aimed to determine the prevalence of Mediterranean spotted fever (MSF) caused by Rickettsia conorii (R. conorii), a tick-borne disease, in Yunnan Province, China. MethodsThrough stratified sampling across five distinct regions in Yunnan, 5358 blood samples were obtained from the general healthy population. Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and Polymerase chain reaction (PCR) were employed for analysis. ResultsIFA identified 27 (0.50%) subjects with immunoglobulin G (IgG) positivity; none were positive for immunoglobulin M (IgM) via ELISA. PCR detected one individual with R. conorii outer membrane protein A (ompA). Significant seroprevalence variation was observed, particularly in Southern Yunnan (P = 0.032), with R. conorii subsp. conorii confirmed in the PCR-positive sample. ConclusionsThis research reveals a correlation between MSF prevalence, geography, and climate in Yunnan. The paucity of prior studies underscores MSF's potential diagnostic challenges in the region. Comprehensive understanding of the pathogen's distribution is pivotal for intervention. Given the study's scope and Yunnan's unique setting, additional research is advocated.

Nosocomial surveillance of multidrug-resistant Acinetobacter baumannii: a genomic epidemiological study.

Acinetobacter baumannii is a major opportunistic pathogen causing hospital-acquired infections, and it is imperative to comprehend its evolutionary and epidemiological dynamics in hospitals to prevent and control nosocomial transmission. Here, we present a comprehensive genomic epidemiological study involving the genomic sequencing and antibiotic resistance profiling of 634 A. baumannii strains isolated from seven intensive care units (ICUs) of a Chinese general hospital over 2 consecutive years. Our study reveals that ST2 is highly dominant (90.54%) in the ICUs, with 98.90% of the ST2 exhibiting multidrug resistant or extensively drug resistant. Phylogenetic analyses of newly sequenced genomes and public data suggest that nosocomial isolates originated outside the hospital but evolved inside. The major lineages appear to be stable, with 9 of the 28 identified nosocomial epidemic clones infecting over 60% of the affected patients. However, outbreaks of two highly evolved clones have been observed in different hospitals, suggesting significant inter-hospital transmission chains. By coupling patient medical records and genomic divergence of the ST2, we found that cross-ward patient transfer played a crucial role in pathogen's nosocomial transmission. Additionally, we identified 831 potential adaptive evolutionary loci and 44 associated genes by grouping and comparing the genomes of clones with different prevalence. Overall, our study provides a comprehensive and contemporary survey on the epidemiology and genomic evolution of A. baumannii in a large Chinese general hospital. These findings shed light on the nosocomial evolution and transmission of A. baumannii and offers valuable information for transmission prevention and antibiotic therapy.IMPORTANCEThis study delved into the genomic evolution and transmission of nosocomial Acinetobacter baumannii on a large scale, spanning both an extended time period and the largest sample size to date. Through molecular epidemiological investigations based on genomics, we can directly trace the origin of the pathogen, detecting and monitoring outbreaks of infectious diseases in a timely manner, and ensuring public health safety. In addition, this study also collects a large amount of genomic and antibiotic resistance detection data, which is helpful for phenotype prediction based on genomic sequencing. It enables patients to receive personalized antibiotic treatment quickly, helps doctors select antibiotics more accurately, and contributes to reducing the use of antibiotics and lowering the risk of antibiotic resistance development.

Molecular epidemiological investigation of G6PD deficiency in Yangjiang region, western Guangdong province.

Objectives: The prevalence of G6PD deficiency has not been reported in Yangjiang, a western city in Guangdong province. This study aims to investigate the molecular characteristics of G6PD deficiency in this region. Methods: Blood samples were collected from adults at a local hospital to screen for G6PD deficiency. The deficient samples were subjected to further analysis using PCR and reverse dot blot to determine the specific G6PD variants. Results: Among the 3314 male subjects, 250 cases of G6PD deficiency were found using the G6PD enzyme quantitative assay, resulting in a prevalence of 7.54% (250/3314) in the Yangjiang region. The prevalence of G6PD deficiency in females was 3.42% (176/5145). Out of the 268 cases of G6PD deficiency tested for G6PD mutations, reverse dot blot identified 20 different G6PD variants. The most common G6PD variant was c.1388G>A (81/268), followed by c.1376G>T (48/268), c.95A>G (32/268), c.1024C>T (9/268), c.392G>T (7/268), and c.871G>A/c.1311C>T (6/268). It was observed that c.871G>A was always linked to the polymorphism of c.1311C>T in this population. Conclusion: This investigation into G6PD deficiency in this area is expected to significantly improve our understanding of the prevalence and molecular characterization of this condition.

Phylogenetic relationship of Prohemistomum vivax to other trematodes based on the internal transcribed spacer region and mitochondrial genes.

Prohemistomum vivax is a zoonotic small cyathocotylid trematode that inhabits the intestines of fish-eating birds and mammals. Here, we amplified the internal transcribed spacer (ITS) sequence and six mitochondrial protein-coding genes (PCGs) from P. vivax. The ITS region was 1389 base pairs long and had a partial 18S ribosomal RNA gene, a full ITS1, 5.8S rRNA, and ITS2 sequence, and a partial 28S rRNA gene. The ITS region of P. vivax showed a minimum pairwise distance (0.3-0.6%) from the ITS sequences of Cyathocotylidae sp. 1 and 2 metacercariae from Clarias gariepinus. This result suggests that these metacercariae belong to P. vivax metacercariae. We first amplified mitochondrial genes from P. vivax, including cytochrome c oxidase subunit III (cox3) partial sequence; tRNA-His, cytochrome b (cytb), and NADH dehydrogenase subunit 4L (nad4L) complete sequences; and NADH dehydrogenase subunit 4 (nad4), cytochrome c oxidase I (cox1), and NADH dehydrogenase subunit 5 (nad5) partial sequences. P. vivax was most closely related to Cyathocotyle prussica (NC_039780) and Holostephanus sp. (OP082179), with cox1, cox3, and cytb genes conserved among the three trematodes. The ML phylogenetic tree of ITS sequences supports the order Diplostomida, divided into two main clades (the superfamily Diplostomoidea and Schistosomatoidea). The phylogeny of concatenated amino acid sequences of P. vivax six PCGs revealed that diplostomoids and Clinostomum sp. evolved in a clade with Plagiorchiida members, away from Schistosoma species. These results may yield ribosomal and mitochondrial genetic markers for molecular epidemiological investigations of cyathocotylid intestinal flukes.

Investigating a pulmonary Mycobacterium abscessus infection outbreak among elderly inpatients in the intensive care ward.

Mycobacterium abscessus is an opportunistic nontuberculous mycobacteria pathogen; however, the prevalence of nosocomial and community infections is increasing. In January 2016, several bedridden inpatients in the intensive care unit of a hospital had positive sputum smears for acid-fast bacilli, suggesting a mycobacteria outbreak. Acid-fast bacilli smear microscopy, isolation, and culturing were performed twice using sputa from each suspected intensive care unit inpatient (n = 13); in addition, medical history was obtained for each inpatient with suspected infection. Furthermore, environmental specimens were surveyed, collected, and cultured. We used DNA microarray chip analysis to identify positive mycobacterial isolates at the species level and performed whole-genome sequencing and phylogenetic tree construction. Seven inpatients had M. abscessus pulmonary infection, confirmed by 2 positive cultures; five of the inpatients had only one positive culture, while one had two negative cultures. Six of 13 ventilator condensate samples were mycobacterial culture-positive, identified as M. abscessus; the other environmental samples were negative. The M. abscessus isolates (15 sputa and 4 environmental samples) clustered together in the phylogenic analysis with only one single-nucleotide polymorphism difference. All patients were symptom-free after 8 months of multi-drug treatment. We confirmed a pulmonary M. abscessus outbreak among 12 bedridden patients in the intensive care unit through microbiological, molecular epidemiological, and environmental investigations. The possible infection source was contaminated ventilator condensate. This outbreak reemphasizes the importance of standardized ventilator maintenance and disinfection for preventing ventilator-associated pneumonia and is a reminder that nontuberculous mycobacteria-related ventilator-associated pneumonia is possible.

Genomic and epidemiological insight of an outbreak of carbapenemase-producing Enterobacterales in a Portuguese hospital with the emergence of the new KPC-124

BackgroundCarbapenemase-producing Enterobacterales (CPE) is an increasing problem in healthcare settings. This study aimed to identify the source of a CPE outbreak that occurred in 2022, in a tertiary hospital in the North of Portugal, to identify exposed patients, and to assess the risk of becoming CPE-positive following hospital admission. MethodsA multi-disciplinary investigation was conducted including descriptive, analytical, and molecular epidemiology, environmental screening, and assessment of infection control measures. Clinical and environmental isolates were analyzed using whole-genome sequencing and phylogenetic analysis. Additionally, a prospective observational cohort study was conducted to further investigate the risk factors associated with the emergence of new cases in cohorts of CPE-negative admitted patients. ResultsWe observed the presence of multispecies KPC-, IMP-, and/or NDM-producing isolates. Genetically indistinguishable clinical and environmental isolates were found on the same room/ward. The ST45 KPC-3-producing Klebsiella pneumoniae clone was the responsible for the outbreak. During patients’ treatment, we detected the emergence of resistance to ceftazidime-avibactam, associated with mutations in the blaKPC-3 gene (blaKPC-46, blaKPC-66 and blaKPC-124, the last variant never previously reported), suggesting a vertical evolutionary trajectory. Patients aged ≥ 75 years, hygiene/feeding-care dependent, and/or subjected to secretion aspiration were risk factors for CPE colonization after hospital admission. Additionally, cases with previous admission to the emergency department suggest that CPE dissemination may occur not only during hospitalization but also in the emergency department. ConclusionOverall, the study highlights that selection pressure with antibiotics, like ceftazidime-avibactam, is a contributing factor to the emergence of new β-lactamase variants and antibiotic resistance. It also shows that the hospital environment can be a significant source of CPE transmission, and that routine use of infection control measures and real-time molecular epidemiology investigations are essential to ensure the long-term termination of CPE outbreaks and prevent future resurgences.

Contamination of Plant Foods with Bacillus cereus in a Province and Analysis of Its Traceability.

Bacillus cereus is an important zoonotic foodborne conditional pathogen. It is found in vegetables, dairy products, rice, and other foods, thereby greatly endangering human health. Investigations on B. cereus contamination in China primarily focus on raw milk, dairy products, meat, and others, and limited research has been conducted on plant-based foodstuffs. The rapid development of sequencing technology and the application of bioinformatics-related techniques means that analysis based on whole-genome sequencing has become an important tool for the molecular-epidemiology investigation of B. cereus. In this study, we investigated the contamination of B. cereus in six types of commercially available plant foods from eight regions of a province. The molecular epidemiology of the isolated B. cereus was analyzed by whole-genome sequencing. We aimed to provide fundamental data for the surveillance and epidemiology analysis of B. cereus in food products in China. The rapid traceability system of B. cereus established in this study can provide a basis for rapid molecular epidemiology analysis of B. cereus, as well as for the prevention and surveillance of B. cereus. Moreover, it can also be expanded to monitoring and rapid tracing of more foodborne pathogens.

Molecular epidemiological investigation of carbapenem resistant Klebsiella pneumoniae isolated from intensive care unit patients of six geographical regions of Turkey.

Klebsiella pneumonia causes serious infections in hospitalized patients. In recent years, carbapenem-resistant infections increased in the world. The molecular epidemiological investigation of carbapenem-resistant K. pneumoniae isolates was aimed in this study. Fifty carbapenem-resistant K. pneumoniae isolates from six geographical regions of Turkey between September 2019-2020 were included in the study. The disk diffusion method was used for the antibiotic susceptibility testing. The microdilution confirmed colistin susceptibility. Genetic diversity was investigated by MLST (Multi-Locus Sequence Typing). The resistance rates were as follows: 49 (98%) for meropenem, 47 (94%) imipenem, 50 (100%) ertapenem, 30 (60%) colistin and amoxicillin-clavulanate, 49 (98%) ceftriaxone, 48 (96%) cefepime, 50 (100%) piperacillin-tazobactam, 47 (94%) ciprofloxacin, 40 (80%) amikacin, 37 (74%) gentamicin. An isolate resistant to colistin by disk diffusion was found as susceptible to microdilution. ST 2096 was the most common (n:16) sequence type by MLST. ST 101 (n:7), ST14 (n:6), ST 147 and ST 15 (n:4), ST391 (n:3), ST 377 and ST16 (n:2), ST22, ST 307, ST 985, ST 336, ST 345, and ST 3681 (n:1) were classified in other isolates. In İstanbul and Ankara ST2096 was common. Among Turkey isolates, the most common clonal complexes (CC) were CC14 (n:26) and CC11 (n = 7). In Turkey, a polyclonal population of CC14 throughout the country and inter-hospital spread were indicated. The use of molecular typing tools will highlight understanding the transmission dynamics.

Dissemination and characteristics of carbapenem-resistant Klebsiella pneumoniae in nine district hospitals in southwestern China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is epidemically transmitted globally, but few studies focused on the prevalence in district-level hospitals. In this study, we investigated CRKP strains collected from nine district hospitals from September 2019 to September 2020, aiming to determine the resistance mechanisms, virulence profiles, and molecular epidemiological characteristics of CRKP in district hospitals in Southwest China. A total of 51 CRKP strains were collected from 9 district-level hospitals. Matrix-assisted laser desorption/ionization-time of flight mass spectrometer was used for strain identification review, and the micro-broth dilution method was used for antibiotic sensitivity detection. Molecular epidemiological investigation of strains was performed by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) methods. PCR and efflux pump inhibition tests were used to detect CRKP resistance mechanisms. PCR and serum killing tests were used to detect capsular serotype, virulence-related genes, and virulence validation. The CRKP strains in district hospitals presented high levels of MIC50 and MIC90 in carbapenem antibiotics especially ertapenem and meropenem. A total of 90.2% (46/51) CRKP strains were detected as carbapenemase producers, and the proportion of strains co-expressing carbapenemases was 11.8% (6/51). All CRKP strains were grouped into eight MLST types, and ST11 was the most prevalent genotype. A total of 11.8% (6/51) CRKP isolates were positive for the string test, and three strains of hypervirulent and carbapenem-resistant K. pneumoniae (HV-CRKP) were positive in serum killing test. The molecular typing of all the CRKP isolates was grouped into 29 different PFGE patterns, and 40 ST11 isolates belonged to 20 different PFGE clusters. CRKP strains showed high-level antibiotic resistance and virulence phenotype in district hospitals in Southwest China, which suggested that we should immediately pay attention to the rapid dissemination of the CRKP in regional hospitals. Our study will provide new insights into the epidemiology of CRKP in regional hospitals, which will help regional hospitals develop nosocomial infection prevention and control policies tailored to local conditions.

Molecular epidemiological investigation of piroplasms carried by pet cats and dogs in an animal hospital in Guiyang, China.

Piroplasmosis is a zoonotic disease mainly caused by the Babesia and Theileria parasites. Piroplasmosis is often a subclinical infection in dogs and cats that is difficult to detect and is often suspected when clinical signs such as anemia are present. It has been reported to be prevalent in China. However, molecular evidence of the disease has not been reported in pet dogs and cats in Guiyang. In this study, we collected 307 anticoagulated blood samples from an animal hospital in the Wudang District of Guiyang during the period March 2021 to November 2021 and extracted DNA from the samples. The 18S rDNA gene was amplified using PCR, and the positive amplification product was sequenced. The sequences were then analyzed for homology and phylogeny. Of the 307 samples collected, 164 were feline and 143 were canine, with a total of 23 amplifying a target band of approximately 400 bp. The percentage of positives of piroplasms infection in pet cats was 4.27% (7/164), with the pathogens being T. uilenbergi (3) and T. luwenshuni (4). One Colpodella sp. and two undetermined species were also detected in the cat samples. The percentage of positives of piroplasms infection in pet dogs was 7.69% (11/143), with the pathogen being T. uilenbergi (11). One Colpodella sp. was also detected in the dog samples. The results confirmed that T. uilenbergi and T. luwenshuni are prevalent in pet cats and dogs in this area. In addition, the study found a rare zoonotic pathogen, Colpodella sp., in cats and dogs. Therefore, this study is expected to serve as a valuable reference for decision-making regarding animal health management and public health work.