Identical Sequence Types Research Articles

Outbreak detection in Harar town and Kersa district, Ethiopia using phylogenetic analysis and source attribution

BackgroundFoodborne diseases (FBDs) represent a significant risk to public health, with nearly one in ten people falling ill every year globally. The large incidence of foodborne diseases in African low- and middle-income countries (LMIC) shows the immediate need for action, but there is still far to a robust and efficient outbreak detection system. The detection of outbreak heavily relies on clinical diagnosis, which are often delayed or ignored due to resource limitations and inadequate surveillance systems.MethodsIn total, 68 samples of non-typhoidal Salmonella isolates from human, animal and environmental sources collected between November 2021 and January 2023 were analyzed using sequencing methods to infer phylogenetic relationships between the samples. A source attribution model using a machine-learning logit-boost that predicted the likely source of infection for 20 cases of human salmonellosis was also run and compared with the results of the cluster detection.ResultsThree clusters of samples with close relation (SNP difference < 30) were identified as non-typhoidal Salmonella in Harar town and Kersa district, Ethiopia. These three clusters were comprised of isolates from different sources, including at least two human isolates. The isolates within each cluster showed identical serovar and sequence type (ST), with few exceptions in cluster 3. The close proximity of the samples suggested the occurrence of three potential outbreaks of non-typhoidal Salmonella in the region. The results of the source attribution model found that human cases of salmonellosis could primarily be attributed to bovine meat, which the results of the phylogenetic analysis corroborated.ConclusionsThe findings of this study suggested the occurrence of three possible outbreaks of non-typhoidal Salmonella in eastern Ethiopia, emphasizing the importance of targeted intervention of food safety protocols in LMICs. It also highlighted the potential of integrated surveillance for detecting outbreak and identifying the most probable source. Source attribution models in combination with other epidemiological methods is recommended as part of a more robust and integrated surveillance system for foodborne diseases.

Clean clothes or dirty clothes? Outbreak investigation of carbapenem-resistant Acinetobacter baumannii related to laundry contamination through multilocus sequence typing (MLST).

To investigate the source in an outbreak of carbapenem-resistant Acinetobacter baumannii (CRA) in a general hospital due to contamination of a laundry evaporative cooler and the laundry environment using multilocus sequence typing (MLST). For CRA culture, clinical samples were collected from infected patients and close contacts, and environmental sampling was performed in patient surroundings and laundry facilities. MLST was used for the molecular typing of representative CRA isolates. Bacterial isolates with identical sequence types were considered epidemiologically linked and attributable to the same source. OXA genes in Acinetobacter baumannii were detected using polymerase chain reaction (PCR). In total, 58 patients were affected in this outbreak. The mean patient age was 75.3, and 50% were female. The most common diagnoses at admission were skin and soft-tissue infection (n = 12, 20.7%) and pneumonia (n = 12, 20.7%). OXA-23 was positive in 64.7% of isolates. A CRA isolate from the evaporative cooler in the laundry was identical to that of 11 patients across 3 wards, belonging to ST345. Isolates from 3 laundry linen racks were identical to those of 7 patients from 3 wards, classified as ST1145. Isolates found on another linen rack and a pajama shelf were identical to isolates from 3 other patients from 2 wards, belonging to ST2207. There was no significant difference between sequence type distributions of clinical and environmental isolates (P = .12), indicating high likelihood of CRA originating from the same source. MLST confirmed that contamination of the laundry evaporative cooler and surrounding environment caused a polyclonal CRA hospital outbreak. Hospital laundry is an important area for infection control and outbreak investigations of CRA.

Suspected chlamydial foetal loss highlights the need for standardised on-farm protocols.

Chlamydia psittaci is a recognised cause of late-term equine foetal loss and poses a zoonotic risk in Australia. However, a management strategy is lacking to protect at-risk humans handling infected aborted material and pregnant mares. This study proposes a protocol for approaching C. psittaci foetal loss after investigating four foetal losses that occurred on a horse stud in the Hunter Valley, Australia in 2021. Swabs from the foetal loss cases (n=4), close contact mares (n=59), and foals of the close contact mares (n=33) were collected and tested for C. psittaci using both isothermal points of care and quantitative polymerase chain reaction (qPCR) laboratory-based testing. Genotyping was performed utilising C. psittaci multilocus sequence typing and ompA sequencing from C. psittaci positive pooled foetal and placental (n=3) DNA. Foetal and placental samples from the four foetal loss cases were all positive for C. psittaci with 100% agreement between the isothermal swab testing on the farm and qPCR DNA testing at an external laboratory. Genotyping revealed the clonal and identical sequence type 24 (ST24) C. psittaci strains in all samples. C. psittaci was not detected in close contact with mares or their foals. There was no statistically significant difference in foal survival between the close contact mare groups that did and did not receive antimicrobial intervention (P > 0.05). The proposed protocol is intended to raise awareness and begin a discussion for guidelines around handling of chlamydial foetal loss cases in late pregnant mares which pose a zoonotic threat to farm workers and veterinarians.

Microbiological and Molecular Features Associated with Persistent and Relapsing Staphylococcus aureus Prosthetic Joint Infection.

Background: Persistent and relapsing prosthetic joint infection (PJI) due to Staphylococcus aureus presents a clinical challenge. This study aimed to provide an extensive description of phenotypic and genomic changes that could be related to persistence or relapse. Methods: Initial and second S. aureus isolates from 6 cases of persistent and relapsing PJI, along with clinical isolates from 8 cases, with favorable outcome were included. All isolates were studied by phenotypic and genotypic approaches. Results: Recurrent S. aureus isolates exhibited a significant increase in adhesive capacity, invasion and persistence compared to resolved isolates. No association was found for the presence or absence of certain genes with the persistence or relapse of PJI. All sequential isolates showed identical sequence type (ST). Resistance gene loss during the infection and a great diversity of variants in different virulence genes between the pair of strains, mainly in genes encoding adhesins such as fnbA, were observed. Conclusions: S. aureus-caused relapse and persistence PJI is associated with bacterial phenotypical and genotypical adaptation. The main paths of adaptation were persistence in the intracellular compartment, and the loss of antibiotic resistance genes and variant acquisition, especially in genes encoding adhesins.

Streptococcus pneumoniae Strains Isolated From a Single Pediatric Patient Display Distinct Phenotypes.

Streptococcus pneumoniae is the leading cause of bacterial paediatric meningitis after the neonatal period worldwide, but the bacterial factors and pathophysiology that drive pneumococcal meningitis are not fully understood. In this work, we have identified differences in raffinose utilization by S. pneumoniae isolates of identical serotype and sequence type from the blood and cerebrospinal fluid (CSF) of a single pediatric patient with meningitis. The blood isolate displayed defective raffinose metabolism, reduced transcription of the raffinose utilization pathway genes, and an inability to grow in vitro when raffinose was the sole carbon source. The fitness of these strains was then assessed using a murine intranasal infection model. Compared with the CSF isolate, mice infected with the blood isolate displayed higher bacterial numbers in the nose, but this strain was unable to invade the ears of infected mice. A premature stop codon was identified in the aga gene in the raffinose locus, suggesting that this protein likely displays impaired alpha-galactosidase activity. These closely related strains were assessed by Illumina sequencing, which did not identify any single nucleotide polymorphisms (SNPs) between the two strains. However, these wider genomic analyses identified the presence of an alternative alpha-galactosidase gene that appeared to display altered sequence coverage between the strains, which may account for the observed differences in raffinose metabolic capacity. Together, these studies support previous findings that raffinose utilization capacity contributes to disease progression, and provide insight into a possible alternative means by which perturbation of this pathway may influence the behavior of pneumococci in the host environment, particularly in meningitis.

Genomic Characterization of Multidrug-Resistant Salmonella Serovars Derby and Rissen From the Pig Value Chain in Vietnam.

Nontyphoidal Salmonella (NTS) is the most reported cause of bacterial foodborne zoonoses in Vietnam, and contaminated pork is one of the main sources of human infection. In recent years, the prevalence of NTS carrying multiple antimicrobial resistance genes (ARGs) have been increased. The genomic characterization along the pig value chain and the identification of ARGs and plasmids have the potential to improve food safety by understanding the dissemination of ARGs from the farm to the table. We report an analysis of 13 S. Derby and 10 S. Rissen isolates, collected in 2013 at different stages in Vietnamese slaughterhouses and markets. VITEK 2 Compact System was used to characterize the phenotypical antimicrobial resistance of the isolates. In addition, whole-genome sequencing (WGS) was used to detect ARGs and plasmids conferring multidrug resistance. Whole genome single nucleotide polymorphism typing was used to determine the genetic diversity of the strains and the spread of ARGs along the pig value chain. Altogether, 86.9% (20/23) of the samples were resistant to at least one antibiotic. Resistance to ampicillin was most frequently detected (73.9%), followed by piperacillin and moxifloxacin (both 69.6%). At least one ARG was found in all strains, and 69.6% (16/23) were multidrug-resistant (MDR). The observed phenotype and genotype of antimicrobial resistance were not always concordant. Plasmid replicons were found in almost all strains [95.6% (22/23)], and the phylogenetic analysis detected nine clusters (S. Derby, n = 5; S. Rissen, n = 4). ARGs and plasmid content were almost identical within clusters. We found six MDR IncHI1s with identical plasmid sequence type in strains of different genetic clusters at the slaughterhouse and the market. In conclusion, high rates of multidrug resistance were observed in Salmonella strains from Vietnam in 2013. Genomic analysis revealed many resistance genes and plasmids, which have the potential to spread along the pig value chain from the slaughterhouse to the market. This study pointed out that bioinformatics analyses of WGS data are essential to detect, trace back, and control the MDR strains along the pig value chain. Further studies are necessary to assess the more recent MDR Salmonella strains spreading in Vietnam.

Genome assisted molecular typing and pathotyping of rice blast pathogen, Magnaporthe oryzae, reveals a genetically homogenous population with high virulence diversity

Genome sequence-driven molecular typing tools have the potential to uncover the population biology and genetic diversity of rapidly evolving plant pathogens like Magnaporthe oryzae. Here, we report a new molecular typing technique -a digitally portable tool for population genetic analysis of M. oryzae to decipher the genetic diversity. Our genotyping tool exploiting allelic variations in housekeeping and virulence genes coupled with pathotyping revealed a prevalence of genetically homogenous populations within a single-field and plant niches such as leaf and panicle. The M. oryzae inciting leaf-blast and panicle-blast were confirmed to be genetically identical with no or minor nucleotide polymorphism in 17 genomic loci analyzed. Genetic loci such as Mlc1, Mpg1, Mps1, Slp1, Cal, Ef-Tu, Pfk, and Pgk were highly polymorphic as indicated by the haplotype-diversity, the number of polymorphic sites, and the number of mutations. The genetically homogenous single field population showed high virulence variability or diversity on monogenic rice differentials. The study indicated that the genetic similarity displayed by the isolates collected from a particular geographical location had no consequence on their virulence pattern on rice differentials carrying single/multiple resistance genes. The data on virulence diversity showed by the identical Sequence Types (STs) is indicative of no congruence between polymorphic virulence genes-based pathotyping and conserved housekeeping genes-based genotyping.

First Report of blaNDM-1 Bearing IncX3 Plasmid in Clinically Isolated ST11 Klebsiella pneumoniae from Pakistan.

The New Delhi Metallo-β-lactamase (NDM) is among the most threatening forms of carbapenemases produced by K. pneumoniae, well-known to cause severe worldwide infections. The molecular epidemiology of blaNDM-1-harboring K. pneumoniae is not well elucidated in Pakistan. Herein, we aim to determine the antibiotics-resistance profile, genes type, molecular type, and plasmid analysis of 125 clinically isolated K. pneumoniae strains from urine samples during July 2018 to January 2019 in Pakistan. A total of 34 (27.2%) K. pneumoniae isolates were carbapenemases producers, and 23 (18.4%) harbored the blaNDM-1 gene. The other carbapenemases encoding genes, i.e., blaIMP-1 (7.2%), blaVIM-1 (3.2%), and blaOXA-48 (2.4%) were also detected. The Multi Locus Sequence Typing (MLST) results revealed that all blaNDM-1-harboring isolates were ST11. The other sequence types detected were ST1, ST37, and ST105. The cluster analysis of Xbal Pulsed Field Gel Electrophoresis (PFGE) revealed variation amongst the clusters of the identical sequence type isolates. The blaNDM-1 gene in all of the isolates was located on a 45-kb IncX3 plasmid, successfully transconjugated. For the first time, blaNDM-1-bearing IncX3 plasmids were identified from Pakistan, and this might be a new primary vehicle for disseminating blaNDM-1 in Enterobacteriaceae as it has a high rate of transferability.

Genomics of an endemic cystic fibrosis Burkholderia multivorans strain reveals low within-patient evolution but high between-patient diversity.

Burkholderia multivorans is a member of the Burkholderia cepacia complex (Bcc), notorious for its pathogenicity in persons with cystic fibrosis. Epidemiological surveillance suggests that patients predominantly acquire B. multivorans from environmental sources, with rare cases of patient-to-patient transmission. Here we report on the genomic analysis of thirteen isolates from an endemic B. multivorans strain infecting four cystic fibrosis patients treated in different pediatric cystic fibrosis centers in Belgium, with no evidence of cross-infection. All isolates share an identical sequence type (ST-742) but whole genome analysis shows that they exhibit peculiar patterns of genomic diversity between patients. By combining short and long reads sequencing technologies, we highlight key differences in terms of small nucleotide polymorphisms indicative of low rates of adaptive evolution within patient, and well-defined, hundred kbps-long segments of high enrichment in mutations between patients. In addition, we observed large structural genomic variations amongst the isolates which revealed different plasmid contents, active roles for transposase IS3 and IS5 in the deactivation of genes, and mobile prophage elements. Our study shows limited within-patient B. multivorans evolution and high between-patient strain diversity, indicating that an environmental microdiverse reservoir must be present for this endemic strain, in which active diversification is taking place. Furthermore, our analysis also reveals a set of 30 parallel adaptations across multiple patients, indicating that the specific genomic background of a given strain may dictate the route of adaptation within the cystic fibrosis lung.

Genomic data reveals the emergence of an IncQ1 small plasmid carrying blaKPC-2 in Escherichia coli of the pandemic sequence type 648

ObjectivesThe global success of carbapenem-resistant pathogens has been attributed to large plasmids carrying blaKPC genes circulating among high-risk clones. In this study, we sequenced the genome of a carbapenem-resistant Escherichia coli strain (Ec351) isolated from a human infection. Phylogenomic analysis based on single nucleotide polymorphisms (SNPs) as well as the comparative resistome and plasmidome of globally disseminated blaKPC-2-positive E. coli strains with identical sequence type (ST) were further investigated. MethodsTotal DNA was sequenced using an Illumina NextSeq 500 platform and was assembled using Unicycler. Genomic data were evaluated through bioinformatics tools available from the Center of Genomic Epidemiology and by in silico analysis. ResultsGenomic analysis revealed the convergence of a wide resistome and virulome in E. coli ST648, showing a high-level phylogenetic relationship with a KPC-2-positive ST648 cluster identified in the USA and association with international clade 2. Additionally, the emergence of an IncQ1 small plasmid (pEc351) carrying blaKPC-2 (on an NTEKPC-IId element), aph(3')-VIa, and plasmid regulatory and replication genes in the pandemic clone ST648 is reported. ConclusionIdentification of a blaKPC-2-positive IncQ1 plasmid in a high-risk E. coli clone represents rapid adaptation and expansion of these small plasmids encoding carbapenemases to novel bacterial hosts with global distribution, which deserves continued monitoring.

Phenotypic and genetic characteristics of Streptococcus mutans isolates from site-specific dental plaque in China.

Introduction. Streptococcus mutans is an important cariogenic microbe.Hypothesis/Gap Statement. The potential characteristics of S. mutans isolates from site-specific dental plaque are still not clear.Aim. This study aimed to investigate the phenotypic and genetic characteristics of S. mutans isolates from site-specific dental plaque in China.Methodology. We used S. mutans isolated from children with early-childhood caries (ECC) and caries-free children to compare the phenotypic and genetic characteristics of S. mutans from site-specific dental plaque samples. The ECC subjects presented two sites: a cavitated lesion and a sound surface. The caries-free subjects presented one sound surface. Growth pattern, biofilm, decrease in pH, extracellular polysaccharide, expression levels of virulence-related genes, multilocus sequence typing (MLST) and phylogenetic trees were evaluated among these three sites.Results. The phenotypes detected between the cavitated and sound surfaces of ECC children were similar. However, the capacity for biofilm formation, pH drop and expression levels of genes (gtfB and spaP) of S. mutans in the caries-free group were lower compared with those of the ECC group. We identified 44 new alleles and 77 new sequence types. More than 90 % of the children with ECC shared an identical sequence type. The distribution of sequence types among different subjects showed diversity, and child-to-child transmission was detected.Conclusions. This is the first report of MLST on site-specific dental plaques in a single subject, and indicates that S. mutans isolated from site-specific dental plaque of a single subject showed similar phenotypes as a result of the isolates were closely related.

ANTIBIOTIC RESISTANCE IN ESCHERICHIA COLI AND ENTEROCOCCUS SPP. ISOLATED FROM UNGULATES AT A ZOOLOGICAL COLLECTION IN THE UNITED KINGDOM.

Increase of antimicrobial resistance (AMR) is a global threat to health. The AMR profile of bacteria isolated from domesticated animals and free-ranging wildlife has been studied, but there are relatively few studies of bacteria isolated from captive wild animals. Understanding the dynamics of AMR in different populations is key to minimizing emergence of resistance and to preserve the efficacy of antimicrobials. In this study, fecal samples were collected from 17 species of healthy ungulates from a zoological collection in southeast England, which yielded 39 Escherichia coli and 55 Enterococcus spp. isolates for further analysis. Antibiotic sensitivity was investigated using agar disk diffusion. Escherichia coli isolates were resistant to a range of antibiotics, with resistance to ampicillin being the most common (28%). All E. coli isolates were susceptible to apramycin, enrofloxacin, chloramphenicol, and florfenicol. None tested positive for extended-spectrum beta-lactamase or AmpC activity. Seven of 39 (18%) E. coli isolates were resistant to three or more antibiotic classes. The E. coli isolates were further analyzed using multilocus sequence typing, which identified four pairs of identical sequence type isolates and 27 diverse strains. The Enterococcus spp. isolates were resistant to a range of antibiotics, with resistance to cefpodoxime seen in 95% of isolates. All Enterococcus spp. isolates were susceptible to ampicillin, gentamicin, chloramphenicol, and vancomycin. This study identified multidrug-resistant phenotypes in enterobacterial isolates that were like those commonly found in domestic ungulates. There was no apparent spatial clustering of the resistance profiles within the zoo. Review of the medical records of individual animals showed no direct relation to the AMR profiles observed. Observed resistance to antibiotics rarely or never used may have been due to coselection or directly acquired from other sources.

679. Predominance of Acinetobacter baumannii Sequence Type 2 (ST2) among Clinical Isolates from Two Detroit Hospitals using Whole Genome Sequencing

BackgroundHealthcare-associated infections (HAIs) caused by Acinetobacter baumannii, an opportunistic gram-negative pathogen, often contribute to morbidity and mortality among hospitalized patients. To determine molecular epidemiology of circulating hospital Acinetobacter strains in Detroit area in a non-outbreak situation, we performed whole genome sequencing (WGS) and whole genome multilocus sequence typing (wgMLST) analysis to characterize genomic diversity.MethodsWe performed WGS on patient isolates from two disparate, geographically distinct tertiary care Detroit hospitals admitted to 16 intensive care units (ICU) and non-ICU wards between 2017-2019. The samples were obtained 48 hours following admission and WGS was performed using the Illumina NextSeq instrument. The contigs were de novo assembled using SPAdes and WgMLST analysis was performed using BioNumerics software v7.6. Minimum spanning tree (MST) was constructed to demonstrate the clusters in each hospital and their wards.ResultsWgMLST analysis was performed on a total of 62 Acinetobacter baumannii patient isolates, 33 of which were from hospital # 1 (H1) and 29 were from hospital # 2 (H2). ST2 (52%) was the predominant sequence type for both hospitals. In addition to ST2, ST93 (8%) and ST406 (8%) clusters were also found in H1. Most of the isolates were predominantly clustered in the intensive care unit (ICU) setting. Several identical sequence types of ST93 (2 identical in H1), ST36 (2 identical in H1) and ST2 (5 identical for H1 and 2 identical for H2) were also observed from different patients in both hospitals. Unlike H1, H2 only has ST2 as a predominant cluster. Other sporadic sequence types such as ST119, ST36, ST49, ST212, ST667 for H1 and ST395, ST214, ST427 and ST268 for H2 were also observed.Figure 1. MInimum Spanning Tree of Acinetobacter strains in hospitals (H1 and H2) and different wards (U1-U12). For identical strains circles are marked with dividing lines. Different sequence types (STs) are marked om the side bar. ConclusionOur data suggests that ST2 is the predominant sequence type circulating in both the geographically distinct hospitals and is endemic to the ICU. There was in hospital spread of this distinct Acinetobacter sequence type from one patient to the other, raising concerns about infection control practices and the role of environment in the spread of these infections. Early detection of these endemic strains within the hospital may be critical in preventing healthcare-associated infections in the future.Disclosures Chetan Jinadatha, MD, MPH, AHRQ (Research Grant or Support)Department of Veterans Affairs (Other Financial or Material Support, Owner: Department of Veterans Affairs. Licensed to: Xenex Disinfection System, San Antonio, TX)Inventor (Other Financial or Material Support, Methods for organizing the disinfection of one or more items contaminated with biological agents)NiH/NINR (Research Grant or Support)NSF (Research Grant or Support)Xenex Healthcare Services (Research Grant or Support) Mark Stibich, PhD MHS, Xenex Disinfection Services, Inc (Board Member, Employee)

Decrease of Mycoplasma gallisepticum seroprevalence and introduction of new genotypes in Dutch commercial poultry during the years 2001–2018

ABSTRACT Almost two decades ago, in addition to a compulsory M. gallisepticum (Mg) monitoring programme of breeding stock based on European Union regulations, the Dutch poultry industry added national regulations to further reduce the Mg prevalence in Dutch commercial poultry. Currently, all commercial chicken and turkey flocks except broilers are monitored for Mg. All breeding flocks on a farm where one or more flocks tested Mg positive are culled. Mg positive layer pullets are channelled and layer pullets placed on Mg positive multi-age farms are vaccinated. The monitoring data obtained were analysed covering a period of 17 years. Moreover, 31 Dutch Mg isolates from the same period were analysed by multilocus sequence typing (MLST) and compared to available PubMLST data. The results show that in breeding stock the seroprevalence decreased from 1.6% to 0.0%, in commercial layers from 6.3% to 1.9%, and in meat turkeys from 17.6% to 2.4%. The MLST results showed the presence of closely related and identical sequence types (STs) within the different Dutch poultry types. Similar STs were found in Northern and Southern Europe only. The results show a fast decline in the Mg prevalence since 2001, although in layers the Mg prevalence has stabilized and suggests backyard poultry might pose a risk for commercial poultry. The need for Mg control across poultry sectors and in trade was confirmed by the similarity in STs found in different types of poultry and regions. These results from the Dutch poultry industry can be extrapolated to Mg control in general.

Rapid Increase of CTX-M-Producing Shigella sonnei Isolates in Switzerland Due to Spread of Common Plasmids and International Clones.

The Swiss Centre for Antibiotic Resistance (ANRESIS) has recently noted an increase of extended-spectrum cephalosporin-resistant (ESC-R) Shigella sonnei isolates nationwide (3.8% in 2016 versus 37.5% in 2019). To understand this phenomenon, we analyzed 25 representative isolates (of which 14 were ESC-R) collected in Switzerland during 2016 to 2019. Whole-genome sequencing was achieved using both the Illumina and the Nanopore platforms. Both ESC-R and extended-spectrum cephalosporin-susceptible isolates belonged to sequence type 152 (ST152). The ESC-R isolates carried blaCTX-M-3 in IncI1-pST57 (n = 5), blaCTX-M-15 in IncFII (F2:A-:B-) (n = 5), blaCTX-M-15 in IncI1-pST16, and blaCTX-M-27, blaCTX-M-55, or blaCTX-M-134 in other IncFII plasmids (n = 1 each). Plasmids having the same bla and Inc group exhibited high degrees of genetic identity to each other but also to plasmids previously reported in other Enterobacterales Core-genome analysis showed that there were 4 main clusters, each of which included strains that differed by <58 single nucleotide variants (SNVs) and that consisted of both blaCTX-M-positive and blaCTX-M-negative isolates. Moreover, most isolates belonging to the same cluster shared an identical core-genome sequence type (cgST). For instance, cluster 1 included 4 isolates of cgST113036, of which only 3 harbored the IncI1-pST57 blaCTX-M-3-positive plasmid. The 25 S. sonnei isolates were also subjected to phylogenetic comparison with deposited international strains. As a result, matching isolates (isolates that had the same cgST and that differed by <8 SNVs) have been reported in the United Kingdom, the United States, France, and the Netherlands. Overall, our results suggest that some common S. sonnei clusters can spread between continents and can be imported into other nations after international trips. Such clusters include, in part, isolates that do not possess blaESBL-harboring plasmids, indicating their tendency to acquire them from other Enterobacterales.

Management of an outbreak of postpartum Streptococcus pyogenes emm75 infections

Streptococcus pyogenes is a well-known cause of postpartum infections and is causing significant morbidity and mortality. To describe measures taken to control an outbreak of postpartum infections caused by S.pyogenes emm75 on a maternity ward. Patients presenting postpartum with signs and symptoms of infection were cultured for β-haemolytic streptococci with cervical swabs and blood cultures, and bacterial isolates were species-determined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and emm-typed. Pharyngeal swabs were taken from healthcare workers (HCWs) at the ward. Bacterial isolates were subjected to whole-genome sequencing (WGS). The multi-locus sequence type and the number of single nucleotide polymorphisms (SNPs) compared to an index genome were determined. During a three-month period, six cases of postpartum infection with S.pyogenes emm75 were identified on the maternity ward. By comparing delivery dates with duty rotas, one HCW was identified as a possible source of infection in five cases. After repeated pharyngeal swabs from this individual, an S.pyogenes emm75 was isolated. The five isolates from patients epidemiologically linked to the HCW and the two isolates of the family members had an identical sequence type (ST49) and 0-2 SNPs difference compared to the HCW isolate, whereas the sixth patient had an unrelated isolate. Eradication antibiotic therapy with clindamycin and rifampicin was given to the carrier. All patients received intravenous antibiotic treatment and recovered. A three-month outbreak was stopped when a carrier was identified and treated. Source identification and WGS proved vital for outbreak control.

Interventions designed to control postpasteurization contamination in high-temperature, short-time-pasteurized fluid milk processing facilities: A case study on the effect of employee training, clean-in-place chemical modification, and preventive maintenance programs

Postpasteurization contamination (PPC) with gram-negative bacteria adversely affects the quality and shelf-life of milk through the development of flavor, odor, texture, and visual defects. Through evaluation of milk quality at 4 large fluid milk processing facilities in the northeast United States, we examined the efficacy of 3 strategies designed to reduce the occurrence of PPC in fluid milk: (1) employee training (focusing on good manufacturing practices) alone and (2) with concurrent implementation of modified clean-in-place chemistry and (3) preventive maintenance (PM) focused on replacement of wearable rubber components. Despite increases in employee knowledge and self-reported behavior change, microbiological evaluation of fluid milk before and after interventions indicated that neither training alone nor training combined with modified clean-in-place interventions significantly decreased PPC. Furthermore, characterization of gram-negative bacterial isolates from milk suggested that specific bacterial taxonomic groups (notably, Pseudomonas sequence types) continued to contribute to PPC even after interventions and that no major changes in the composition of the spoilage-associated microbial populations occurred as a consequence of the interventions. More specifically, in 3 of 4 facilities, gram-negative bacteria with identical 16S rDNA sequence types were isolated on multiple occasions. Evaluation of a PM intervention showed that used rubber goods harbored PPC-associated bacteria and that PPC may have been less frequent following a PM intervention in which wearable rubber goods were replaced (reduction from 3/3 samples with PPC before to 1/3 samples after). Overall, our findings suggest that commonly used "broad stroke interventions" may have a limited effect on reducing PPC. Our case study also demonstrates the inherent complexities of identifying and successfully addressing sanitation problems in large and complex fluid milk processing facilities. For example, broad changes to sanitation practices without improvements in PM and sanitary equipment design may not always lead to reduced PPC. Our data also indicate that although short-term evaluations, such as pre- and post-tests for employee training, may suggest improvements after corrective and preventive actions, extensive microbial testing, ideally in combination with isolate characterization, may be necessary to evaluate return on investment of different interventions.

Polyphasic phenotypic and genetic analysis reveals clonal nature of Xanthomonas axonopodis pv. punicae causing pomegranate bacterial blight

Yellow‐pigmented bacteria isolated from blight‐affected pomegranate leaves and fruit across seven Indian states in epidemics during the years 2008–2016 were characterized and identified using phenotypic and genotypic tools. All bacterial isolates shared phenotypic traits such as colony morphology, NaCl and pH sensitivity and fuscan production, and caused typical lesions on pomegranate plants upon artificial inoculation. Analysis of 16S ribosomal DNA and 16S–23S rDNA intergenic spacer sequences confirmed their identity as Xanthomonas axonopodis pv. punicae. The new isolates collected after 2000 were compared with an old isolate from the 1950s using polyphasic taxonomic approaches including multilocus sequence analysis (MLSA). Nucleotide polymorphism in 24 isolates for nine genomic loci (dnaK, fyuA, gyrB (Young), gyrB (Almeida), rpoD, fusA, gapA, gltA and lepA) showed minor variations in loci fyuA and gyrB. Isolates were grouped into four nearly identical sequence types, ST1, ST2, ST3 and ST4, based on their allelic profiles, ST3 being widespread in Indian states. Molecular phylogenetic analysis of concatenated 5690 bp with other Xanthomonas pathovars revealed its close genetic similarity with the X. citri group. The blight outbreak in diverse geographical locations is attributed to a re‐emerged clonal population of X. axonopodis pv. punicae on a genetically homogenous pomegranate cultivar. The latently infected vegetative planting material of elite pomegranate cultivars contributed to the dissemination of the bacterial inoculum. This study highlights and forewarns of the role played by the clonally propagated elite pomegranate cultivars in disseminating and sustaining clonal populations of this bacterial plant pathogen in many Indian states.

Prediction of Persistence of Listeria monocytogenes ST451 in a Rabbit Meat Processing Plant in the Czech Republic

This study was focused on characterization of the genetic diversity of Listeria monocytogenes isolated from packed fresh rabbit meat obtained from one producer via retail outlets. The partial aim was to compare the characteristics of a suspect persistent strain with strains from human cases. The occurrence of L. monocytogenes in vacuum-packed rabbit meat was monitored during 2013 to 2016. All strains were characterized by serotyping, pulsed-field gel electrophoresis, and multilocus sequence typing (MLST). Selected strains, which represented each year, were analyzed using the whole genome sequencing method. L. monocytogenes was detected in 21 (38%) of 56 originally packed rabbit meat samples from one food producer during the whole monitored period. All strains showed the identical serotype (1/2a), AscI/ApaI pulsotype (735/2), and sequence type (ST451). The clonal similarity of strains from rabbit meat was also confirmed on the basis of core genome MLST (on 1,701 loci). This fact suggests the occurrence of a suspect persistent strain in the meat processing plant. Results of core genome MLST enabled us to unambiguously exclude rabbit meat as a source of listeriosis in humans caused by the indistinguishable AscI/ApaI pulsotype and sequence type, although all strains carried all genes important for the virulence of L. monocytogenes. No specific genes that may be associated with its persistence in the food processing environment were detected among the tested strains of ST451.

Characterization of Emetic and Diarrheal Bacillus cereus Strains From a 2016 Foodborne Outbreak Using Whole-Genome Sequencing: Addressing the Microbiological, Epidemiological, and Bioinformatic Challenges.

The Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) is not yet routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group isolates were obtained from human cases (n = 7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (group III) and B. cereus sensu stricto (group IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely related and formed a well-supported clade distinct from publicly available emetic group III genomes with an identical sequence type (ST 26). The 30 emetic group III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly available emetic group III ST 26 B. cereus group genomes by a mean of 301.7–528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. We hypothesize that the outbreak was a single source outbreak caused by emetic group III B. cereus belonging to the B. paranthracis species, although food samples were not tested for presence of the emetic toxin cereulide. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.