Research progress of loop-mediated isothermal amplification in the detection of Salmonella for food safety applications.

Salmonella, the prevailing zoonotic pathogen within the Enterobacteriaceae family, holds the foremost position in global bacterial poisoning incidents, thereby signifying its paramount importance in public health. Consequently, the imperative for expeditious and uncomplicated detection techniques for Salmonella in food is underscored. After more than two decades of development, loop-mediated isothermal amplification (LAMP) has emerged as a potent adjunct to the polymerase chain reaction, demonstrating significant advantages in the realm of isothermal amplification. Its growing prominence is evident in the increasing number of reports on its application in the rapid detection of Salmonella. This paper provides a systematic exposition of the technical principles and characteristics of LAMP, along with an overview of the research progress made in the rapid detection of Salmonella using LAMP and its derivatives. Additionally, the target genes reported in various levels, including Salmonella genus, species, serogroup, and serotype, are summarized, aiming to offer a valuable reference for the advancement of LAMP application in Salmonella detection. Finally, we look forward to the development direction of LAMP and expect more competitive methods to provide strong support for food safety applications.

One-tube detection of Salmonella Typhimurium using LAMP and CRISPR-Cas12b.

Salmonella enterica serovar Typhimurium (ST) is a predominant serovar causing foodborne illnesses worldwide. Traditional detection methods often face challenges, including the need for specialized equipment, skilled operators, and lengthy procedures. To address these limitations, we developed a rapid, sensitive, and specific ST detection method by integrating loop-mediated isothermal amplification (LAMP) with the clustered regularly interspaced short palindromic repeats and associated protein 12b (CRISPR/Cas12b) system, all within a single tube. Our results indicate that the LAMP-CRISPR/Cas12b reaction can be completed isothermally in under 1 h without requiring specialized instruments. The platform's limit of detection (LoD) is 12.5 copies per reaction. Additionally, the system demonstrated 100% inclusivity and exclusivity when tested against 30 reference strains, highlighting its specificity. In practical applications, the LoDs for ST in pure nucleic acid and contaminated fecal samples were 2.32 and 23.2 CFU/mL, respectively, with higher sensitivity observed in pure nucleic acid samples. Overall, our findings underscore the potential of the one-tube LAMP-CRISPR/Cas12b platform as a rapid, sensitive, and specific tool for ST detection, particularly in resource-limited settings. IMPORTANCE: Here, we have provided a novel one-step method for Salmonella Typhimurium detection in one pot by integrating the LAMP assay with the CRISPR/Cas12b system, offering significant advantages in terms of simplicity, speed, and accuracy.

A Retrospective Analysis of Salmonella Isolates across 11 Animal Species (1982-1999) Led to the First Identification of Chromosomally Encoded blaSCO-1 in the USA.

Antimicrobial resistance (AMR) in non-typhoidal Salmonella is a pressing public health concern in the United States, necessitating continuous surveillance. We conducted a retrospective analysis of 251 Salmonella isolates from 11 animal species recovered between 1982 and 1999, utilizing serotyping, antimicrobial susceptibility testing, and whole-genome sequencing (WGS). Phenotypic resistance was observed in 101 isolates, with S. Typhimurium, S. Dublin, S. Agona, and S. Muenster prevailing among 36 identified serovars. Notably, resistance to 12 of 17 antibiotics was detected, with ampicillin being most prevalent (79/251). We identified 38 resistance genes, primarily mediating aminoglycoside (n = 13) and ß-lactamase (n = 6) resistance. Plasmid analysis unveiled nine distinct plasmids associated with AMR genes in these isolates. Chromosomally encoded blaSCO-1 was present in three S. Typhimurium and two S. Muenster isolates from equine samples, conferring resistance to amoxicillin/clavulanic acid. Phylogenetic analysis revealed three distinct clusters for these five isolates, indicating evolutionary divergence. This study represents the first report of blaSCO-1 in the USA, and our recovered isolates harboring this gene as early as 1989 precede those of all other reports. The enigmatic nature of blaSCO-1 prompts further research into its function. Our findings highlight the urgency of addressing antimicrobial resistance in Salmonella for effective public health interventions.

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection.

Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 °C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5'-/6-FAM/CCCCCCCC/BHQ1/-3') presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection.

Genomic Characterization of a Plasmid-Free and Highly Drug-Resistant Salmonella enterica Serovar Indiana Isolate in China.

The emergence of multi-drug resistant (MDR) Salmonella enterica serovar Indiana (S. Indiana) strains in China is commonly associated with the presence of one or more resistance plasmids harboring integrons pivotal in acquiring antimicrobial resistance (AMR). This study aims to elucidate the genetic makeup of this plasmid-free, highly drug-resistant S. Indiana S1467 strain. Genomic sequencing was performed using Illumina HiSeq 2500 sequencer and PacBio RS II System. Prodigal software predicted putative protein-coding sequences while BLASTP analysis was conducted. The S1467 genome comprises a circular 4,998,300 bp chromosome with an average GC content of 51.81%, encompassing 4709 open reading frames (ORFs). Fifty-four AMR genes were identified, conferring resistance across 16 AMR categories, aligning closely with the strain's antibiotic susceptibility profile. Genomic island prediction unveiled an approximately 51 kb genomic island housing a unique YeeVU toxin-antitoxin system (TAS), a rarity in Salmonella species. This suggests that the AMR gene cluster on the S1467 genomic island may stem from the integration of plasmids originating from other Enterobacteriaceae. This study contributes not only to the understanding of the genomic characteristics of a plasmid-free, highly drug-resistant S. Indiana strain but also sheds light on the intricate mechanisms underlying antimicrobial resistance. The implications of our findings extend to the broader context of horizontal gene transfer between bacterial species, emphasizing the need for continued surveillance and research to address the evolving challenges posed by drug-resistant pathogens.

Progress in methods for the detection of viable Escherichia coli.

Escherichia coli (E. coli) is a prevalent enteric bacterium and a necessary organism to monitor for food safety and environmental purposes. Developing efficient and specific methods is critical for detecting and monitoring viable E. coli due to its high prevalence. Conventional culture methods are often laborious and time-consuming, and they offer limited capability in detecting potentially harmful viable but non-culturable E. coli in the tested sample, which highlights the need for improved approaches. Hence, there is a growing demand for accurate and sensitive methods to determine the presence of viable E. coli. This paper scrutinizes various methods for detecting viable E. coli, including culture-based methods, molecular methods that target DNAs and RNAs, bacteriophage-based methods, biosensors, and other emerging technologies. The review serves as a guide for researchers seeking additional methodological options and aiding in the development of rapid and precise assays. Moving forward, it is anticipated that methods for detecting E. coli will become more stable and robust, ultimately contributing significantly to the improvement of food safety and public health.

Genome degradation promotes Salmonella pathoadaptation by remodeling fimbriae-mediated proinflammatory response.

Understanding changes in pathogen behavior (e.g. increased virulence, a shift in transmission channel) is critical for the public health management of emerging infectious diseases. Genome degradation via gene depletion or inactivation is recognized as a pathoadaptive feature of the pathogen evolving with the host. However, little is known about the exact role of genome degradation in affecting pathogenic behavior, and the underlying molecular detail has yet to be examined. Using large-scale global avian-restricted Salmonella genomes spanning more than a century, we projected the genetic diversity of Salmonella Pullorum (bvSP) by showing increasingly antimicrobial-resistant ST92 prevalent in Chinese flocks. The phylogenomic analysis identified three lineages in bvSP, with an enhancement of virulence in the two recently emerged lineages (L2/L3), as evidenced in chicken and embryo infection assays. Notably, the ancestor L1 lineage resembles the Salmonella serovars with higher metabolic flexibilities and more robust environmental tolerance, indicating stepwise evolutionary trajectories towards avian-restricted lineages. Pan-genome analysis pinpointed fimbrial degradation from a virulent lineage. The later engineered fim-deletion mutant, and all other five fimbrial systems, revealed behavior switching that restricted horizontal fecal-oral transmission but boosted virulence in chicks. By depleting fimbrial appendages, bvSP established persistent replication with less proinflammation in chick macrophages and adopted vertical transovarial transmission, accompanied by ever-increasing intensification in the poultry industry. Together, we uncovered a previously unseen paradigm for remodeling bacterial surface appendages that supplements virulence-enhanced evolution with increased vertical transmission.

siRNA targeting Atp5a1 gene encoding ATPase α, the ligand of Peg fimbriae, reduced Salmonella Enteritidis adhesion.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a zoonotic pathogen that can infect both humans and animals. Among the 13 types of fimbrial operons in S. Enteritidis, the highly conserved Peg fimbriae play a crucial role in the adhesion and invasion of S. Enteritidis into host cells but are not well studied. In this study, we identified the ATP synthase subunit alpha (ATPase α) as a ligand of Peg fimbriae using ligand blotting and mass spectrometry techniques. We confirmed the in vitro binding of ATPase α to the purified adhesion protein (PegD). Furthermore, we used siRNA to suppress the expression of ATPase α gene Atp5a1 in Leghorn male hepatoma (LMH) cells, which resulted in a significant reduction in the adhesion rate of S. Enteritidis to the cells (P < 0.05). The findings in this study provide insight into the mechanism of S. Enteritidis infection through Peg fimbriae and highlight the importance of ATPase α in the adhesion process.RESEARCH HIGHLIGHTS Ligand blotting was performed to screen the ligand of S. Enteritidis Peg fimbriae.Binding assay confirmed that ATPase α is the ligand of the Peg fimbriae.siRNA targeting ATPase α gene (Atp5a1) significantly reduced S. Enteritidis adhesion.

Advances in detection methods for viable Salmonella spp.: current applications and challenges.

Salmonella is a common intestinal pathogen that can cause food poisoning and intestinal disease. The high prevalence of Salmonella necessitates efficient and sensitive methods for its identification, detection, and monitoring, especially of viable Salmonella. Conventional culture methods need to be more laborious and time-consuming. And they are relatively limited in their ability to detect Salmonella in the viable but non-culturable status if present in the sample to be tested. As a result, there is an increasing need for rapid and accurate techniques to detect viable Salmonella spp. This paper reviewed the status and progress of various methods reported in recent years that can be used to detect viable Salmonella, such as culture-based methods, molecular methods targeting RNAs and DNAs, phage-based methods, biosensors, and some techniques that have the potential for future application. This review can provide researchers with a reference for additional method options and help facilitate the development of rapid and accurate assays. In the future, viable Salmonella detection approaches will become more stable, sensitive, and fast and are expected to play a more significant role in food safety and public health.

Graphene oxide-assisted optimized narrow-thermal-cycling amplification for accurate detection of Salmonella spp.

Salmonella is a rod-shaped, Gram-negative zoonotic pathogen that poses a serious global socioeconomic and public health threat. Rapid and accurate detection of Salmonella spp. is critical for effective control of its infection. In this study, an accurate, sensitive and specific graphene oxide-assisted accelerated strand exchange amplification (GO-ASEA) method for rapid detection of Salmonella spp. was developed and validated. The detection limit of the GO-ASEA method was 8.6 × 101 fg µL-1 of Salmonella genomic DNA or 1 × 101 CFU g-1 of Salmonella in spiked chicken faeces free of pre-enrichment. And the GO-ASEA method could specifically detect Salmonella spp. without cross-reactivity with other enteric pathogens. In addition, the novel method achieved Salmonella detection within 30 min and was validated using 209 clinical samples, showing its good clinical applicability. Therefore, the GO-ASEA method is a new optional tool for the rapid detection of pathogenic microorganisms, which is ideal for food safety monitoring and high-throughput detection.

Comprehensive Assessment of Subtyping Methods for Improved Surveillance of Foodborne Salmonella.

High-resolution and efficient typing for the bacterial pathogen is essential for tracking the sources, detecting or diagnosing variants, and conducting a risk assessment. However, a systematic in-field investigation of Salmonella along the food chain has not been documented. This study assessed 12 typing methods, such as antimicrobial-resistance (AMR) gene profile typing, Core Genome Multilocus Sequence Typing (cgMLST), and CRISPR multi-virulence locus sequence typing (CRISPR-MVLST), to evaluate their effectiveness for use in routine monitoring of foodborne Salmonella transmission along the poultry production chain. During 2015-16, a total of 1,064 samples were collected from poultry production chain, starting from breeding farms and slaughterhouses to the markets of Zhejiang province in China. A total of 61 consecutive unique Salmonella isolates recovered from these samples were selected for genome sequencing and further comparative typing analysis. Traditional typing methods, including serotyping, AMR phenotype-based typing, as well as modern genotyping approaches, were evaluated and compared by their discrimination index (DI). The results showed that the serotyping method identified nine serovars. The gold standard cgMLST method indicated only 18 different types (DI = 0.8541), while the CRISPR-MVLST method detected 30 types (DI = 0.9628), with a higher DI than all examined medium-resolution WGS-based genotyping methods. We demonstrate that the CRISPR-MVLST might be used as a tool with high discriminatory power, comparable ease of use, ability of tracking the source of Salmonella strains along the food chain and indication of genetic features especially virulence genes. The available methods with different purposes and laboratory expertise were also illustrated to assist in rational implementation. IMPORTANCE In public health field, high-resolution and efficient typing of the bacterial pathogen is essential, considering source-tracking and risk assessment are fundamental issues. Currently, there are no recommendations for applying molecular characterization methods for Salmonella along the food chain, and a systematic in-field investigation comparing subtyping methods in the context of routine surveillance was partially addressed. Using 1,064 samples along a poultry production chain with a considerable level of Salmonella contamination, we collected representative isolates for genome sequencing and comparative analysis by using 12 typing techniques, particularly with whole-genome sequence (WGS) based methods and a recently invented CRISPR multi-virulence locus sequence typing (CRISPR-MVLST) method. CRISPR-MVLST is identified as a tool with higher discriminatory power compared with medium-resolution WGS-based typing methods, comparable ease of use and proven ability of tracking Salmonella isolates. Besides, we also offer recommendations for rational choice of subtyping methods to assist in better implementation schemes.

Tri-primer-enhanced strand exchange amplification combined with rapid lateral flow fluorescence immunoassay to detect SARS-CoV-2.

The novel coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world, which has exposed humanity to unprecedented economic, social and health impacts. To achieve efficient and accurate detection of SARS-CoV-2 on site, we developed and verified a rapid and sensitive fluorescence lateral flow immunoassay based on the innovative enhanced strand exchange amplification (ESEA-LFIA) in this study. With good amplification efficiency for short-sequence targets, ESEA is an ideal choice for the point-of-care testing of SARS-CoV-2 with a high mutation rate. ESEA reaction can be completed in one step and verified by restriction enzyme digestion. The design consisting of three working primers greatly improved the amplification efficiency. Amplification of the target sequences of the RdRP and N genes can be accomplished under the same reaction conditions, and does not require expensive instruments. The sensitivity of the ESEA-LFIA assay targeting the RdRP and N genes was 90 copies per µL and 70 copies per µL, respectively. Specificity tests showed that the novel assay can specifically detect SARS-CoV-2, and had no cross-reactivity with 9 closely-related human pathogenic coronaviruses and other common respiratory pathogens with similar clinical manifestations. The cutoff values of the RdRP and N gene assays are 11 and 12, respectively, and the assays can be completed within 1 h. The novel strategy proposed in this study is a sensitive and specific method for the rapid detection of SARS-CoV-2, and is suitable as an effective potential bioanalytical tool to respond to future regional or global outbreaks of emerging infectious pathogens with high mutation rates.

[Construction and evaluation of a pUC-type prokaryotic promoter reporter system based on lacZ gene].

To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.

Hydroxymethylbilane synthase (HMBS) gene-based endogenous internal control for avian species.

With PCR becoming one of the most important and widely-used diagnostic tools for infectious diseases of poultry, an urgent need has developed for an endogenous internal control (EIC) that monitors the quality and quantity of poultry DNA in test samples. In this study we developed a SYBR-qPCR to target the poultry homolog of the hydroxymethylbilane synthase (HMBS) gene as an EIC for avian species. The avian HMBS-based qPCR was very sensitive, detecting one HMBS gene copy in a 20 µL reaction, and is highly specific for avian species. It amplified DNA from 11 organs and tissues of chickens showing it can be used as an EIC on a large variety of samples. The application of the established EIC on clinically and experimentally infected samples demonstrated that false negativity and result variations could result from samples being collected using different operators, techniques, preservatives, and storage times. The high sensitivity and specificity of the avian HMBS-based qPCR, its ability to quantify DNAs extracted from a wide range of tissues and poultry species along with its usefulness in reducing false negativity in PCR results associated with inadequate sampling and storage degradation makes it an ideal EIC for poultry DNA and RNA PCR diagnostics. The study also highlights the importance of appropriate sampling and storage of samples in ensuring accuracy of molecular diagnostic testing.

Complete Genome Sequence of a Colistin-Resistant Klebsiella pneumoniae Isolate from Houseflies (Musca domestica) in a Trash Disposal Truck in the United States.

A Klebsiella pneumoniae strain isolated from houseflies in a trash disposal truck in the United States was resistant to colistin, a last-resort drug for treating infections caused by multidrug-resistant Gram-negative bacteria. Complete genome sequencing resulted in a total genome size of 5,337,408 bp for this isolate with a plasmid of 224,442 bp.

Lateral flow fluorescent immunoassay based on isothermal amplification for rapid quantitative detection of Salmonella spp.

Salmonella spp. are zoonotic pathogens of substantial public health concern. To enable detection in the field or under instrument-free conditions, we developed a rapid and robust lateral flow fluorescent immunoassay based on strand exchange amplification (SEA-LFIA) for the quantitative detection of Salmonella spp. As far as we know, this work is the first report regarding the use of Bst DNA polymerase-assisted SEA for fluorescence sensing to detect Salmonella spp. The SEA method was further confirmed by enzymatic digestion and Sanger dideoxy sequencing. The specificity of SEA-LFIA assay was verified by 89 Salmonella strains (18 Salmonella reference strains and 71 clinical isolates) and 15 non-Salmonella reference strains (different genera). The sensitivity of SEA-LFIA assay was 6 × 100 CFU mL-1 of Salmonella pure culture or 3 × 104 CFU 25 g-1 of artificially spiked raw chicken meat. Using this assay, it was found that 37 (16%) of the 236 samples collected were positive, which was consistent with the results of conventional PCR. The cutoff value is 15 and SEA-LFIA assay only takes ∼30 min without high equipment and reagent cost. In addition, the proposed strategy can be easily extended by redesigning the corresponding amplification primers to detect target analytes. In conclusion, the optimized SEA-LFIA assay is an efficient and specific method for the detection of Salmonella spp., and can potentially serve as a new on-site diagnostic tool in life sciences.

Lsr operon is associated with AI-2 transfer and pathogenicity in avian pathogenic Escherichia coli.

The function of Autoinducer-2 (AI-2) which acts as the signal molecule of LuxS-mediated quorum sensing, is regulated through the lsr operon (which includes eight genes: lsrK, lsrR, lsrA, lsrC, lsrD, lsrB, lsrF, and lsrG). However, the functions of the lsr operon remain unclear in avian pathogenic Escherichia coli (APEC), which causes severe respiratory and systemic diseases in poultry. In this study, the presence of the lsr operon in 60 APEC clinical strains (serotypes O1, O2, and O78) was investigated and found to be correlated with serotype and has the highest detection rate in O78. The AI-2 binding capacity of recombinant protein LsrB of APEC (APEC-LsrB) was verified and was found to bind to AI-2 in vitro. In addition, the lsr operon was mutated in an APEC strain (APEC94Δlsr(Cm)) and the mutant was found to be defective in motility and AI-2 uptake. Furthermore, deletion of the lsr operon attenuated the virulence of APEC, with the LD50 of APEC94Δlsr(Cm) decreasing 294-fold compared with wild-type strain APEC94. The bacterial load in the blood, liver, spleen, and kidneys of ducks infected with APEC94Δlsr(Cm) decreased significantly (p < 0.0001). The results of transcriptional analysis showed that 62 genes were up-regulated and 415 genes were down-regulated in APEC94Δlsr(Cm) compared with the wild-type strain and some of the down-regulated genes were associated with the virulence of APEC. In conclusion, our study suggests that lsr operon plays a role in the pathogenesis of APEC.

Development of a multiplex PCR assay for the simultaneous and rapid detection of six pathogenic bacteria in poultry.

Escherichia coli, Pasteurella multocida, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella spp. and Staphylococcus aureus are six bacterial pathogens of avian. However, these pathogens may cause many similar pathological changes, resulting in clinical isolates that are difficult to quickly and simultaneously detect and identify. Here, a multiplex polymerase chain reaction (m-PCR) assay is reported to rapidly identify targets genes (phoA, KMT1, ureR, toxA, invA, and nuc) of these six pathogens in clinical samples. Six pairs of specific primers were designed. The optimal reaction conditions, specificity, and sensitivity of the m-PCR assay were investigated. The results showed that betaine remarkably improved amplification of the target genes. Specific test results showed that all six pathogens were detected by the proposed m-PCR protocol without cross-amplification with viruses or parasites. Sensitivity test results showed that the m-PCR system could amplify the six target genes from bacterial genomes or cultures with template amounts of 500 pg or 2.8-8.6 × 103 colony forming units, respectively. Furthermore, the six bacterial pathogens isolated from the infected tissue samples were successfully identified. The proposed m-PCR assay is a useful tool to monitor and diagnose bacterial infection in birds with high specificity, sensitivity and throughput.

Efficient fecal-oral and possible vertical, but not respiratory, transmission of emerging Chlamydia gallinacea in broilers.

Chlamydia gallinacea is an endemic Chlamydia agent in poultry with a worldwide distribution. The aim of this study was to investigate whether C. gallinacea can be transmitted via fecal-oral, respiratory and vertical routes. After co-housing with C. gallinacea-inoculated broilers (n = 10) for 15 days, over 90.0% of SPF broilers (n = 10) became C. gallinacea-positive in their oropharyngeal and cloacal swabs. Connection of isolators with ventilation tubing resulted in transmission of infectious bronchitis virus, but not of C. gallinacea, from infected broilers in one isolator to uninfected ones in the other isolator. Chlamydia-qPCR determined that 97.6% of shells of embryonated eggs (287/294) from a breeding farm were positive for C. gallinacea. C. gallinacea positivity in egg albumen increased significantly from 7.6% (10/128) before incubating to 44.4% (8/18) of 7-day incubation, and from 5.5% (7/128) to 38.9% (7/18) in egg yolk. After incubating for 19 days, C. gallinacea DNA was detected in heart (5/55, 9.1%), liver (3/55, 5.5%), spleen (7/55, 12.7%), lung (6/55, 10.1%), kidney (8/55; 14.5%) and intestine (4/55, 7.3%) of chicken embryos. Taken together, our data indicate that C. gallinacea can be efficiently transmitted by the fecal-oral route, but not via aerosol. Additionally, vertical transmission can occur via penetration of C. gallinacea from eggshell to albumen, yolk, and the growing embryo. Our findings provide essential information for the control of C. gallinacea in poultry farms.

Characterization of the emerging multidrug-resistant Salmonella enterica serovar Indiana strains in China.

Emergence of multidrug-resistant (MDR) Salmonella enterica serovar Indiana (S. Indiana), a dominant Salmonella serovar in China, has raised global awareness because the MDR S. Indiana also was rapidly emerged in other countries recently. To improve our understanding of underlying MDR mechanism and evolution of this emerging zoonotic pathogen, here we examined the standard ATCC51959 strain together with 19 diverse and representative Chinese S. Indiana strains by performing comprehensive microbiological, molecular, and comparative genomics analyses. The findings from S1-PFGE, plasmid origin analysis and Southern blotting suggested the MDR phenotype in the majority of isolates was associated with large integron-carrying plasmids. Interestingly, further in-depth analyses of two recently isolated, plasmid-free MDR S. Indiana revealed a long chromosomal class I integron (7.8 kb) that is not linked to the Salmonella Genome Island 1 (SGI1), which is rare. This unique chromosomal integron shares extremely high similarity to that identified in a MDR E. coli plasmid pLM6771 with respect to both genomic organization and sequence identity. Taken together, both plasmid and chromosomal integron I exist in the examined MDR S. Indiana strains. This timely study represents a significant step toward the understanding of molecular basis of the emerging MDR S. Indiana.