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In August 2023, the Shanghai CDC laboratory received diarrheal stool specimens from two pediatric hospitals in close succession. These specimens were identified and serotyped as Salmonella enterica serotype Grumpensis (S. Grumpensis). The rarity of this serotype was confirmed upon consulting the local Chinese Salmonella genome database (1), which contains no recorded instances, suggesting that it is an infrequent occurrence in China. Commonly, symptoms of salmonellosis emerge anywhere from 6 hours to 6 days following infection. The discovery of two instances of this unusual serotype within a 24-hour period signals a red flag for a possible outbreak and underscores the pathogen’s transmission capability.
Two male children, aged 1 and 2 years, presented to the hospital on August 1and 2 with similar clinical symptoms of bloody diarrhea (>3 episodes in 24 hours) and abdominal pain (Table 1). Initially treated with cefdinir, patient G2’s symptoms persisted despite a 5-day course, leading to a switch to azithromycin, which resulted in gradual improvement and ful recovery.
Case number Date of admission Hospital name Age of patient Gender Presented symptoms G2 August 1, 2023 SCMC 1 year Male Bloody diarrhea and abdominal pain G5 August 2, 2023 SCH 2 years Male Bloody diarrhea and abdominal pain Abbreviation: SCMC=Shanghai Children’s Medical Center; SCH=Shanghai Children’s Hospital. Table 1. Medical history of pediatric diarrhea cases.
Epidemiological investigations play a crucial role in managing cases related to uncommon pathogens. Despite the initial findings showing no evidence of typical sources of infection such as dining out, travel, contact with symptomatic individuals, consumption of raw water, undercooked foods, or owning pets, it posed a challenge in determining the origin of the infection.
CDC laboratory personnel collected specimens from the household of individual G2. Adhering to the procedures specified in GB4789.4-2016, a diverse set of samples, including stool from family members, uneaten food, and environmental swabs, were gathered. Maintaining sterility and a constant temperature of 4 ℃, samples were transported to the lab for pathogen examination within two hours. Despite these precautions, no Salmonella was detected in any of the samples. Additionally, there were no further cases involving this particular Salmonella serotype reported at the same hospital. In our continued investigation of the two S. Grumpensis strains, we conducted a comprehensive analysis that included both antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS). The AST employed the broth microdilution technique to assess the resistance against 22 antibiotics encompassing 11 classes, as listed in
Supplementary Table S1 . This method was strictly in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines and the National Antimicrobial Resistance Monitoring System (NARMS) protocol, aiming to determine the Minimum Inhibitory Concentrations (MICs) for each antibiotic (2-3). The AST findings, as presented in Supplementary Table S1, indicated that the strains were susceptible to the full array of antibiotics tested.Meanwhile, WGS results, detailed in Table 2, classified both isolates as Salmonella Grumpensis S.I (13, 23: d: 1,7). They shared multilocus sequence type (ST) 2060 and core genome multilocus sequence type (cgST) 175517, differing in only one allele, suggesting a strong genetic similarity. Their matching phenotypic and genotypic resistance patterns, absence of plasmid replicons, and common genetic features indicate a close genetic relationship, typical for strains involved in outbreaks, pointing to a common source or transmission chain.
Feature Isolates G2 G5 Serotype Salmonella Grumpensis (13, 23: d: 1,7) Salmonella Grumpensis (13, 23: d: 1,7) Multilocus Sequence Type 2060 2060 Core Genome Multilocus Sequence Type 175517 175517 Antibiotic Resistance Phenotype All Sensitive All Sensitive Genotype acc(6’)-Iaa, fosA7 acc(6’)-Iaa, fosA7 Plasmid Replicons Detected None None Note: Core genome multilocus sequence type is 1 allele difference. Table 2. Genomic characteristics of the two Salmonella Grumpensis isolates.
The study analyzed the genomes of 51 S. Grumpensis available in the NCBI database (
Supplementary Table S2 ), revealing its widespread across 11 countries and regions globally, with the highest numbers in Spain (n=20), the United Kingdom (n=15), and the United States (n=7). The strains were isolated between 2005 and 2023, with a surge from 2017 to 2023. Various sample types were identified, including human (n=42), food (n=2), plant (n=1), poultry (n=1), and unknown sources (n=7). Human samples primarily consisted of fecal specimens (n=39), as well as blood (n=2) and cerebrospinal fluid (n=1).Phylogenetic analysis (Figure 1) identified ST2060 (n=37) and ST751 (n=13) as the predominant global STs among S. Grumpensis isolates. Most isolates harbored acc(6’)-Iaa (98.1%) and fosA7 (96.2%) genes. ST751 has been observed since 2016 in the UK, Canada, the USA, and Brazil, from both humans and poultry, notably lacks of multidrug resistance. Initially reported in 2006, ST2060 is mainly present in human samples (97.3%) and comprised of two genetic clades: 2060.1 and 2060.2, with the latter branching into three sub-clades (2060.2-1, 2060.2-2, 2060.2-3). The study conducted hierarchical single linkage clustering based on pairwise single nucleotide polymorphism (SNP) differences at different thresholds (100, 50, 25, 10, 5, 0). Two isolates from the study belonged to the 2060.2-1 sub-clade, genetically close (0–80 SNPs) to isolates from the UK, USA, and Senegal, and highly similar (0–4 SNPs) to a 2023 USA strain (SRR26351730). An intriguing finding was an isolate from Senegal, in the 2060.2-1 sub-clade, having 14 antibiotic resistance genes (ARGs) and originating from a cerebrospinal fluid sample.
Figure 1.Genetic evolution and antibiotic resistance dissemination of 53 global S. Grumpensis isolates.
Image source: https://itol.embl.de/tree/1391771982423341698626779#. -
This study highlights the crucial contribution of public health laboratories in identifying and addressing outbreaks of uncommon Salmonella serotypes. It underscores the common dilemma faced by public health departments in confirming outbreaks that exhibit typical characteristics but are challenging to confirm through regular surveillance due to few cases or unidentified sources.
A foodborne disease outbreak is defined when two or more cases of similar illness caused by the consumption of a common food (4). In the investigation, two young boys with identical clinical symptoms within a 24-hour period were involved. The pathogens isolated from their diarrheal stool samples were conclusively identified as S. Grumpensis, a serotype not been previously recorded in the Chinese local Salmonella genome database, highlighting its rarity (1). Unfortunately, the epidemiological investigation did not yield a probable source of infection due to limited data. Nevertheless, the absence of additional cases suggests that the event might have been a confined occurrence, unlikely to have propagated further.
WGS is an essential tool for investigating outbreaks. In our study, both strains were found to have the same multilocus sequence type and core genome multilocus sequence type, differing by only one allele or four SNPs, indicating a close genetic relationship. Despite the absence of a direct epidemiological link, several factors strongly suggest that the cases were part of an outbreak of S. Grumpensis stemming from a single source. These factors encompass the temporal proximity of the cases, the high genetic similarity among isolates, consistent antibiotic resistance patterns, and the rarity of the S. Grumpensis serotype.
In the management of patient G2, a discernible divergence was observed between the ineffectiveness of cefdinir and the subsequent clinical improvement with azithromycin. This contrasted with the results indicated by AST and WGS. This variance could be due to a confluence of factors, such as drug pharmacokinetics, pharmacodynamics, and individual patient characteristics like immune function or microbiota. We also considered the possibility that adverse effects of cefdinir, rather than its antimicrobial ineffectiveness, might have contributed to the patient’s condition. Commonly, cefdinir is associated with diarrhea in pediatric patients at therapeutic doses (5). Additionally, occurrences of reddish stools have been noted, which are thought to result from a nonabsorbable complex forming between cefdinir or its metabolites and dietary iron in the gut (5). The absence of follow-up stool testing complicates the ability to discern whether the continuing symptoms were due to an uncontrolled infection or a reaction to cefdinir. This underscores the importance of thorough follow-up testing to accurately assess treatment outcomes.
In this study, we identified the presence of the acc(6')-Iaa and fosA7 genes in S. Grumpensis isolates, illuminating their potential contribution to antibiotic resistance. The acc(6')-Iaa gene codes for an aminoglycoside acetyltransferase, which can confer resistance to aminoglycoside antibiotics, frequently observed in Salmonella species (6–7). It is vital to note that this gene usually remains inactive in Salmonella genus (7). Thus, the mere presence of the acc(6')-Iaa gene does not necessarily result in aminoglycoside resistance. On the other hand, the fosA7 gene codes for a metal-dependent glutathione-S-transferase, conferring fosfomycin resistance (8). Originating chromosomally, possibly through gene islands or prophages, it aids in spreading fosA7 among various Salmonella serovars (8). In China, the frequency of the fosA7 gene ranges from 10% to 15% (9–10), with its presence being inconsistent among certain serotypes. Furthermore, most fosA7-positive Salmonella isolates demonstrate only low-level resistance to fosfomycin (8). Notably, our study discovered a higher prevalence of the fosA7 gene in S. Grumpensis isolates.
The limited availability of information on S. Grumpensis globally highlights a gap in understanding its sources and transmission patterns. Utilizing WGS data and comparing it with international databases can help identify similar strains worldwide, aiding in pinpointing common origins. In our study, we combined publicly accessible genomes from the NCBI database with our own strains to conduct a thorough analysis of the global distribution and genetic characteristics of S. Grumpensis. This serotype has been prevalent in 11 countries and regions globally since 2005, with sources ranging from animal products to plant-based items like chili powders. However, the scarcity of non-human-origin genomes in the phylogenetic tree indicates a significant surveillance gap, especially in the food chain. Therefore, enhancing surveillance to encompass diverse sources beyond human clinical samples is essential.
For S. Grumpensis, two STs are notable: ST2060 and ST751. ST2060 has been prevalent in human samples since 2006 and has evolved into two genetic clades: 2060.1 and 2060.2, with the latter further dividing into three sub-clades. The two strains in this study are categorized under sub-clade 2060.2-1, similar to strains found in the UK, USA, and Senegal. Comparing the genetic profiles, the G5 isolate matches strains from the USA, while the G2 isolate varies by only 4 SNPs, suggesting a close genetic relationship and a possible common origin or shared transmission chain that might span international boundaries. However, the origin of the USA strain remains undisclosed, highlighting the necessity for robust genomic surveillance to monitor the global spread and evolution of pathogens like S. Grumpensis. Additionally, an isolate from Senegal within sub-clade 2060.2-1 was observed to contain 14 ARGs and show fewer than 100 SNP disparities compared to other ST2060.2-1 strains. The global spread of sub-clade 2060.2-1 indicates genetic adaptations influenced by various local conditions, leading to differences in ARG prevalence across regions. This highlights the crucial role of global cooperation and the implementation of comprehensive genomic surveillance networks.
In essence, identifying two cases of a rare Salmonella serotype in close proximity, both geographically and temporally, highlights the vital role of public health laboratories in early outbreak detection and response. By utilizing advanced diagnostics and genomics, these laboratories produce crucial scientific data essential for robust surveillance and efficient alert systems, crucial in densely populated urban areas where outbreaks can rapidly escalate, leading to significant public health threats. Embracing the “One Health” approach underscores the importance of breaking down barriers between sectors like human health, veterinary science, agriculture, and food production to enhance communication and collaboration. Additionally, establishing a comprehensive WGS database is key to fortifying a more vigilant and responsive public health infrastructure. The integration of cutting-edge laboratory techniques with a collaborative public health strategy forms the cornerstone of an effective surveillance and response mechanism, setting the stage for a healthier future.
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No conflicts of interest.
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All members at all participating CDCs and hospitals.
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