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CRISPR-Cas is an adaptive immune system that allows bacteria to inactivate mobile genetic elements. Approximately 50% of bacteria harbor CRISPR-cas, however in the human pathogenStaphylococcus aureus, CRISPR-casloci are less common and often studied in heterologous systems. We analyzed the prevalence of CRISPR-casin genomes of methicillin resistantStaphylococcus aureus(MRSA) isolated in Denmark. Only 2.9 % of the strains carried CRISPR-cassystems, but for strains of sequence type ST630 over half were positive. All CRISPR-casloci were type III-A and located within the staphylococcal chromosomal cassette (SCCmec) type V(5C2&5) conferring β-lactam resistance. Curiously, only 23 different CRISPR spacers were identified in 69 CRISPR-positive strains and almost identical SCCmeccassettes, CRISPR arrays andcasgenes, are present in staphylococcal species other thanaureus, suggesting that these were transferred horizontally. For the ST630 strain 110900, we demonstrate that the SCCmeccassette containing CRISPR-casexcises from the chromosome at high frequency. However, the cassette was not transferable under the conditions investigated. One of the CRISPR spacers targets a late gene in the lytic bacteriophage (phage) virus philPLA-RODI, and we show that the system protects against phage infection by reducing phage burst size. However, CRISPR-Cas can be overloaded or bypassed by CRISPR escape mutants. Our results imply that the endogenous type III-A CRISPR-Cas system inS. aureusis active against targeted phages, albeit with low efficacy. This suggests nativeS. aureusCRISPR-Cas offers only partial immunity, and in nature may work in tandem with other defense systems.ImportanceCRISPR-Cas is an adaptive immune system enabling bacteria and archaea to protect themselves against mobile genetic elements such as phages. In strains ofStaphylococcus aureus, CRISPR-casis rare, but when present, it is located within the SCCmecelement encoding resistance to methicillin and other β-lactam antibiotics. We show that the entire module is excisable, with almost identical versions found in different species ofnon-aureusstaphylococci suggesting that the system only rarely acquires new spacers inS. aureus. Additionally, we show that in its endogenous form, theS. aureusCRISPR-Cas is active but inefficient against lytic phages, with phages being able to form escape mutants or overload the system. This leads us to propose that CRISPR-Cas inS. aureusoffers only partial immunity in native systems, and so may work together with other defense systems to prevent phage-mediated killing.

CRISPR-Cas is an adaptive immune system that protects prokaryotes from the invasion of foreign genetic elements. The components and immunity mechanisms of CRISPR-Cas have been extensively studied, but the regulation of this system inStaphylococciremains unclear. Here, we show that in theS. aureusType III-A CRISPR-Cas system, the Pcasof 300 bp located incas1displays as a critical regulatory node that initiates the transcription ofcasgene clusters. We discovered two transcriptional regulators, SarA and CRP-like ArcR, promote the expression and activity of the CRISPR-Cas system by directly binding to the novel promoter Pcas. Furthermore, we demonstrated that the cell-cell communication, known as quorum sensing (QS), inhibits the activity of CRISPR-Cas system by repressing the positive regulators SarA and ArcR. Bioinformatic analyses suggest that Pcasis conserved in many Type II and III CRISPR-Cas systems in Firmicutes. Our data reveal a new regulatory mechanism for QS-mediated repression of the Type III-A CRISPR-Cas system, which may allowS. aureusto acquire foreign genetic elements encoding antibiotic resistance or virulence factors specifically at high cell density.

CRISPR with its cas genes is an adaptive immune system that protects prokaryotes against foreign genetic elements. The type III-A CRISPR-Cas system is rarely found in Staphylococcus aureus, and little is known about its function in S. aureus. Here, we describe the genome characteristics of the clinical methicillin-resistant S. aureus (MRSA) strain TZ0912, carrying a type III-A CRISPR-Cas system. Phylogenetic analysis of 35 reported CRISPR-Cas-positive S. aureus strains revealed that the CRISPR-Cas system is prevalent in CC8 clones (10/35) and is located in the staphylococcal cassette chromosome mec (SCCmec) V, which confers methicillin resistance. Plasmid transformation and phage infection assays reveal that the type III-A CRISPR-Cas system protects TZ0912 against foreign DNA with sequence homology to the spacers located in the CRISPR array. We observed that the CRISPR-Cas immune system could effectively protect MRSA against phage attacks in both liquid culture and solid medium. In accordance with previous reports, using RNA-seq analysis and plasmid transformation assays, we find that the crRNAs close to the leading sequence of the CRISPR array are more highly expressed and are more effective at directing plasmid elimination compared to the distant spacers. This study established a model for evaluating the efficiency of naive CRISPR-Cas system in MRSA against phage, which could contribute to future research on the function of CRISPR-Cas in clinical MRSA isolates and improve phage therapy against MRSA infections.

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-) derived from S. Typhimurium has become the dominant serotype causing human salmonellosis. Replacement of the fljAB operon in S. Typhimurium by different resistance regions (RRs) can generate Salmonella 4,[5],12:i:- without expressing the second-phase flagellar antigen. However, the generation and evolution of RRs in Salmonella 4,[5],12:i:- across the world remain unknown. In the present study, except for the three reported RRs (RR1–RR3), five novel RRs (RR4–RR8) were identified in Chinese isolates from livestock and humans. The insertion of RR3 into the chromosomal hin-iroB site of S. Typhimurium produced RR3-S. Typhimurium as a primary intermediate strain. Following cis and/or trans intramolecular transpositions mediated by IS26 and/or IS1R elements, Salmonella 4,[5],12:i:- was produced by replacing the fljAB operon and/or its flanking sequences with RRs; the generation process was confirmed by the subcultivation of RR3-S. Typhimurium in vitro and in vivo. In addition, animal experiments revealed that RR3-Salmonella 4,[5],12:i:- displayed more efficient colonisation and survival abilities in mouse tissues than its parent strain, RR3-S. Typhimurium. The present study is the first to demonstrate the molecular mechanism underlying the origin and generation of Salmonella 4,[5],12:i:- from S. Typhimurium through complicated transpositions.

The type VI secretion system (T6SS) encoded by Salmonella Pathogenicity Island 19 (SPI-19) has been confirmed to be involved in bacterial infection or colonization in hosts and in the inhibition of the host T-cell immune response. However, deletion of the core genes (clpV, vgrG, and hcp2) encoding the T6SS apparatus does not affect the phenotypes caused by SPI-19-encoded T6SS. As Salmonella infection in host cells and survival in chickens are closely associated with the type III secretion system (T3SS), RNA-Seq was performed, and the results revealed that most T3SS genes were downregulated in the C79-13ΔSPI-19 mutant. To identify the SPI-19 genes involved in regulating T3SS genes expression, we constructed mutants of genes encoding potential regulators (RS09140 and RS09275) or proteins with Sel1-like motifs (RS09150 and RS09155) and analyzed their associated phenotypes. Deletion of RS09150 and RS09155 caused the decreased bacterial infection in avian cells and bacterial colonization in chicken organs. In addition, qRT-PCR results revealed that both mutants showed decreased expression levels of regulatory genes of T3SS. The present findings demonstrate that the two Sel1-like proteins RS09150 and RS09155 in S. Pullorum SPI-19 contribute to bacterial infection in chickens by mediating the expression of T3SS genes, indicating a potential crosstalk between SPI-19 and T3SS in Salmonella.

Background Polymyxins are currently regarded as a possible last-resort therapy to eradicate multidrug-resistant (MDR) gram-negative bacteria. Meanwhile, the old antimicrobial agent fosfomycin has recently been reintroduced into clinical use for the treatment of extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Enterobacteriaceae. This study investigated a multidrug-resistant Salmonella 4,[5],12:i:- strain from a food catering handler, which had the potential to act as a vehicle for transmitting MDR foodborne pathogens. Methods A Salmonella 4,[5],12:i:- YZU1189 strain was isolated from the fecal sample of a food catering worker according to the standard protocol of the Salmonella detection method from World Health Organization in 2003. Serotyping of YZU1189 was performed according to the Kauffmann-White scheme. The antimicrobial resistance phenotype of the strain was determined by the agar dilution method according to the instruction from Clinical and Laboratory Standards Institute (CLSI). Plasmid conjugation was performed between the donor strain Salmonella 4,[5],12:i:- YZU1189 and the recipient strain Escherichia coli C600. The genetic locations of mcr-1, bla CTX-M-14 and fosA3 genes were determined by the whole genome sequence analysis. Results Salmonella 4,[5],12:i:- YZU1189 was an ESBL-producing stain isolated from a healthy catering worker. The strain displayed resistance to aminoglycosides, beta-lactams, polymyxins, fosfomycins, phenicols, trimethoprims, sulfonamides, tetracyclines and fluoroquinolones. Whole genome sequence analysis and plasmid conjugation revealed that the strain had a transferable IncHI2 plasmid carrying the mcr-1, bla CTX-M-14 and fosA3 genes. Sequence homology analysis showed that this plasmid possessed high sequence similarity to previously reported mcr-1, bla CTX-M-14 and fosA3 positive plasmids in China. Conclusion This study reported a the multidrug-resistant Salmonella 4,[5],12:i:- isolate harboring mcr-1, bla CTX-M-14 and fosA3 from human for the first time in China. The occurrence of mcr-1 and fosA3 genes in the transferable IncHI2 plasmid pYZU1189 from the ESBL-producing Salmonella 4,[5],12:i:- isolate showed a potential threat to public health. Great concern should be taken for the spread of multidrug-resistant ESBL-producing Salmonella isolates from food catering workers to consumers.

In this study, a carbapenem-resistant Salmonella enterica serovar Typhimurium strain W131 (sequence type 34) was isolated from retail pork in Jiangsu province, China. An IncX3 plasmid carrying blaNDM-5 element was identified in this strain by whole-genome sequencing analysis. The conjugation experiment demonstrated that the plasmid can be transferred to Escherichia coli recipient J53 and conferred carbapenem resistance to the recipient strain. This study first reported a Salmonella Typhimurium strain with blaNDM-5 from retail pork, which revealed that retail meat as a potential transmission factor of carbapenem-resistant blaNDM-5 to be a threat human health.

Herein, we report an engineered enzyme that can monooxygenate unprotected tryptophan into the corresponding 3a-hydroxyhexahydropyrrolo[2,3-b]indole-2-carboxylic acid (HPIC) in a single, scalable step with excellent turnover number and diastereoselectivity. Taking advantage of directed evolution, we analyzed the stepwise oxygen-insertion mechanism of tryptophan 2,3-dioxygenases, and transformed tryptophan 2,3-dioxygenase from Xanthomonas campestris into a monooxygenase for oxidative cyclization of tryptophans. It was revealed that residue F51 is vital in determining the product ratio of HPIC to N'-formylkynurenine. Our reactions and purification procedures use no organic solvents, resulting in an eco-friendly method to prepare HPICs for further applications.

Salmonella enterica serovar Typhimurium monophasic variants (Salmonella 4,[5],12:i:-) has increased dramatically, causing human salmonellosis and colonization in pigs. With a difference to S. Typhimurium, the monophasic variants of S. Typhimurium lose the gene cassettes encoding the second phase flagellin. To establish a rapid method to detect and differentiate the two serotypes, we analyzed the published 679 genomes of S. Typhimurium and its monophasic variants and found that no Salmonella 4,[5],12:i:- strains carry both fljB and hin genes. Therefore, we established a novel multiplex PCR method using the fljB-hin region and mdh gene as target sequences to detect and differentiate both serotypes. This method can be used to specifically detect both serotypes with a detection limit for DNA concentration at 10 pg/μL. In addition, the PCR assay successfully differentiated 36 S. Typhimurium isolates from 62 isolates of monophasic variants preserved in our laboratory from 2009 to 2017, which corresponds to the whole-genome-based serotyping results. Application of the multiplex PCR method to 60 fecal samples from a pig farm identified 11.7% (7/60) of S. Typhimurium monophasic variants, which is consistent with the whole-genome-based serotyping results. The multiplex PCR assay is a rapid and precise method for the detection of S. Typhimurium monophasic variants from samples across food production chains.

CRISPR with its

Salmonella enterica serovar Rissen is recognized as one of the most common serotypes in pigs and pig products in many countries and can be transmitted to human through the consumption of contaminated food. To reveal the genetic characteristics of S. Rissen, 39 isolates from human and animal food were subjected to whole-genome sequencing analysis combined with 337 genome sequences downloaded from the NCBI Assembly database. Core genome single nucleotide polymorphism (cgSNP) divided these S. Rissen isolates into two clusters, of which cluster A included 78.3% of American isolates, while cluster B was mainly composed of isolates from China, Asia, and the UK. The 39 S. Rissen isolates located in cluster B were further divided into two subclusters with cluster B-1 of 26 isolates from both humans and food, while cluster B-2 consisted of 13 isolates from animal food, mainly pork. CRISPR typing and cgMLST of the 39 isolates showed perfect correspondence to cgSNP results of their phylogenetic relationship. Virulence factors analysis revealed that ABZJ_0085 and ABZJ_0086 genes presenting in cluster B-2 were lost in cluster B-1. Additionally, antimicrobial resistance gene profiles showed qnrS1, cmlA1, and tet(M) could be detected in cluster B-2 other than cluster B-1. The findings of the divergent difference between cluster B-1 and cluster B-2 demonstrated that S. Rissen was continuously evolved through the pork production chain. In conclusion, pigs are the main reservoir for S. Rissen capable of causing human nontyphoid salmonellosis.

Staphylococcus aureus is a commensal bacterium and an important opportunistic pathogen in humans and animals. The increase in multi-drug resistant (MDR) strains of S. aureus is a growing concern due to their impact on animal health and potential for zoonotic transmission. Increasing evidence has shown that MRSA could be transmitted by faeces. The present study determined the prevalence, antibiotic resistance profile and genotypic characteristics of S. aureus isolated from monkey faecal samples in China. Thirty-eight out of 145 (26.21%) macaque faecal samples were S. aureus positive, which eight (5.5%) isolates were identified as MRSA. Antimicrobial susceptibility tests showed that most of the S. aureus isolates were resistant to tetracycline (TE, 44.74%), followed by penicillin (P, 21.05%), cefoxitin (FOX, 21.05%) and ciprofloxacin (CIP, 18.42%). The predominant spa types were t13638 (44.74%) and t189 (13.16%), which are reported to be closely associated with human infections in China. All MRSA isolates belonged to the SCCmecV type, which six of MRSA isolates were ST3268, while the other two isolates belonged to ST4981. This study for the first time describes the prevalence of S. aureus and MRSA in monkey faeces in China, indicating that faeces could be a potential factor of transmitting S. aureus between humans and monkeys.

CRISPR-based typing was performed to subtype isolates of S. Typhimurium and its monophasic variant Salmonella 4,[5],12:i:- from humans and animals between 2009 and 2017 in China. CRISPR typing classified all isolates into two lineages and four sub-lineages. All isolates from Lineage II and Lineage IB-1 were Salmonella Typhimurium. All of Salmonella 4,[5],12:i: – isolates were distributed in Lineage IA and Lineage IB-2, which all belonged to ST34 by MLST typing. Only Lineage IB-2 contained ST34 isolates from both Salmonella Typhimurium and Salmonella 4,[5],12:i:-. Among the isolates of ST34, TST4 was identified as the most common CRISPR type representing 86.5% of Salmonella 4,[5],12:i:- and 14.5 % of Salmonella Typhimurium mainly from pigs and humans. This study demonstrated that TST4-ST34 isolates were predominant in Salmonella 4,[5],12:i:-, and pig was the main reservoir for Salmonella 4,[5],12:i:- in China, which might have the potential to transmit to humans by pig production.

Salmonella is a worldwide foodborne pathogen causing human disease. Food handlers, who are potential carriers of Salmonella, may transmit the pathogen to consumers through food. To determine the prevalence of Salmonella enterica serovars among food handlers working in the catering industry in Nantong, China, a total of 214,542 food handlers' fecal samples were tested for Salmonella in the Nantong CDC (Centers for Disease Control) from 2012 to 2017. Among those tested, 193 (0.09%) were identified to be positive for Salmonella, and the highest detection rate was 0.16% during the period of July to September. Serotyping analysis showed that Salmonella enterica serovar Typhimurium was the predominant serotype (16.1%), followed by Salmonella Derby (13.5%), Salmonella Enteritidis (11.4%), and Salmonella London (11.4%). The high detection rate of Salmonella Derby was probably closely related to its high prevalence of the serotype in pork, which is the primary meat consumed by the Chinese. Antibiotic susceptibility analysis demonstrated that 73.4% of the isolates were multidrug-resistant (MDR) strains with predominant resistance to ampicillin (AMP, 64.6%), followed by resistance to sulfisoxazole (SUL, 58.1%), nalidixic acid (55.8%), and tetracycline (TET, 44.5%). Therefore, MDR Salmonella strain carriage among food handlers working in the catering industry might be a potential source of human salmonellosis, especially for the predominant MDR genotype isolates (32.3%) with resistance to AMP, SUL, and TET.

To reveal differences in virulence among strains of Salmonella enterica serovar Pullorum (S. Pullorum), we used 2 cell-based infection models and a chicken embryo infection model in this study. S. Pullorum strain S06004 was used to infect 4 different avian cell lines (HD-11, DF-1, LMH, DT-40), and the results showed that the infection of S06004 in both LMH and HD-11 cells was more stable than in DF-1 and DT-40 cells. Therefore, the HD-11 and LMH cell lines were used as the appropriate macrophage and epithelial cell models, respectively, to study the infection of S. Pullorum. Fifty strains isolated during the years 1962 to 2010 were then analyzed to compare their infection rates in HD-11 and LMH cells. The result showed that the infection rates of most strains were very similar to that of S06004, except for S9876 which displayed the highest infection rate among these strains. Based on the cell infection results, 10 strains were selected to be used in the chicken embryo infection model. Sixteen-day-old SPF chicken embryos were infected with the pathogen at a dose of 103 CFU/100 μL via allantoic cavity inoculation. The strains C79-13, 7101, and S06013 caused death of more than 80% embryos, whereas S09C12 and 6703 resulted less than 20% death. Thus, this study established cell-based infection models to screen S. Pullorum strains in vitro, and a chick embryo model to evaluate their in vivo virulence.

Staphylococcus aureus (S. aureus) is one of the most common pathogens causing both human and animal infections. Transmission of S. aureus to humans via contaminated food continues to be a health public concern. In the present study, 104 strains including eight MRSA strains were identified from 507 chicken samples (20.5%) in three cities of China. These strains harbored the highest resistance against penicillin (91.4%), followed by tetracycline (64.4%), erythromycin (53.5%), and kanamycin (32.7%). We used spa typing to classify these isolates into 28 types belonging to two lineages including the predominant type t112 (n = 26) and four newly identified spa types. Among the 104 strains, six carried the CRISPR-Cas system, a prokaryotic immune system which protects against foreign genetic elements. Interestingly, rpoB gene sequencing demonstrated that these six initially designated ST2250 strains were in fact S. argenteus, a novel Staphylococcus species genetically closely related to S. aureus. Three Staphylococcus CRIPSR types containing ten spacers identified in these strains have been reported in CRISPR-positive S. aureus. Additionally, 80% of the spacers showed homology to S. aureus phages demonstrating that these conserved spacers were closely related to the phages in the environment of S. argenteus. We speculated that the identical CRISPR types and spacers in both S. argenteus and S. aureus have resulted via exchange of mobile elements between these two species. Emergence of food-borne ST2250 S. argenteus is a potential threat to human public health.

Salmonella enterica is a major foodborne pathogen causing symptomatic diseases or asymptomatic infections in humans. To reveal the genetic difference of Salmonella strains from patients to that from asymptomatic people, we used CRISPR typing to analyze the phylogenetic relationship of 180 clinical strains during 2017-2018 in Jiangsu, China. The CRISPR typing divided these isolates into 76 CRISPR types with a discriminatory power of 97.6%. S. Typhimurium and its monophasic variants of 6 CRISPR types are the significant serotypes causing both human diseases and asymptomatic infection, while S. Enteritidis mainly resulted in diseases and shared one CRISPR type. The spacer HadB20 displayed as a new molecular marker to differentiate ST34-S. Typhimurium monophasic variant from ST19-S. Typhimurium. S. Derby, S. London, and S. Senftenberg frequently caused asymptomatic infection with diverse CRISPR types, while S. Mbandaka and S. Meleagridis, occasionally isolated from patients, had conserved CRISPR types. Additionally, 30 of 516 newly identified spacers showed homology to sequences in both plasmids and bacteriophages. Interestingly, some spacers from one serotype showed homology to the correspondent prophage or plasmid sequences in another serotype; and more than two spacers identified in one strain showed homology to the sequences located in the identical plasmids or phages, revealing the constant evolution of Salmonella CRISPR arrays during the interactions between bacteria and phages or plasmids.

The monophasic variant of Salmonella Typhimurium has emerged and increased rapidly worldwide during the past two decades. The loss of genes encoding the second-phase flagella and the acquirement of the multi-drug resistance cassette are the main genomic characteristics of the S. Typhimurium monophasic variant. In this study, two Salmonella strains were isolated from the knee effusion and feces of a 4-year-old girl who presented with a case of septic arthritis and fever, respectively. Primary serovar identification did not detect the second-phase flagellar antigens of the strains using the classical slide agglutination test. Whole-genome sequencing analysis was performed to reveal that the replacement of the fljAB operon by a 4.8-kb cassette from E. coli caused the non-expression of phase-2 flagellar antigens of the strains, which were confirmed to be a novel S. Virchow monophasic variant (Salmonella 6,7,14:r:-) by core-genome multi-locus sequence typing (cgMLST). Compared to the 16 published S. Virchow genomes, the two strains shared a unique CRISPR type of VCT12, and showed a close genetic relationship to S. Virchow BCW_2814 and BCW_2815 strains, isolated from Denmark and China, respectively, based on cgMLST and CRISPR typing. Additionally, the acquisition of Salmonella genomic island 2 (SGI2) with an antimicrobial resistance gene cassette enabled the strains to be multidrug-resistant to chloramphenicol, tetracycline, trimethoprim, and sulfamethoxazole. The emergence of the multidrug-resistant S. Virchow monophasic variant revealed that whole-genome sequencing and CRISPR typing could be applied to identify the serovaraints of Salmonella enterica strains in the national Salmonella surveillance system.

Simple Summary The increasing observation of methicillin-resistant Staphylococcus aureus (MRSA) in wildlife species has raised the concern of its impact on the animal health and the potential of zoonotic transmission. The molecular types of MRSA and S. aureus in wild animals has been found to mix with several molecular types from humans and livestock, indicating a dynamics of S. aureus transmission between these animals and humans. Thus, it is important to investigate the epidemiological relationship of MRSA between zoo animals and humans. To investigate the prevalence and transmission of S. aureus in non-human primates and their living environment, we conducted this study on all the non-human primates in a zoo park located in eastern China. Fecal samples from non-human primates and samples from their living environments were selectively enriched and conducted for S. aureus isolation. The whole genome analysis showed that these two MRSA t034/CC398 isolates were found from a monkey fecal sample and its living environment. The virulence factor analysis showed that these two MRSA isolates carried virulence factors relating to the cell adherence, biofilm formation, toxins, and human-associated immune evasion cluster. The further phylogenetic analysis revealed that these two MRSA isolates have a close genomic relationship with the human-associated clinical isolates from China, which indicates a potential risk of bidirectional transfer of MRSA between monkeys and humans. Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important antibiotic resistant pathogens causing infections in humans and animals. The increasing observation of MRSA in wildlife species has raised the concern of its impact on animal health and the potential of zoonotic transmission. This study investigated the prevalence of S. aureus in fecal samples from non-human primates in a zoo located in Jiangsu, China, in which 6 out of 31 (19.4%) fecal samples, and 2 out of 14 (14.3%) indoor room floor swab samples were S. aureus-positive. The antibiotic susceptibility tests of the eight isolates showed that the two isolates were resistant to both penicillin and cefoxitin, the three isolates were resistant only to penicillin, while three isolates were susceptible to all detected antibiotics. The two isolates resistant to cefoxitin were further identified as MRSA by the presence of mecA. Five different spa types were identified including t034 of two MRSA isolates from Trachypithecus francoisi, t189 of two methicillin-susceptible S. aureus (MSSA) isolates from Rhinopithecus roxellana, t377 of two MSSA isolates from Colobus guereza, and two novel spa types t19488 and t19499 from Papio anubis. Whole genome sequencing analysis showed that MRSA t034 isolates belonged to ST398 clustered in clonal complex 398 (CC398) and carried the type B ΦSa3 prophage. The phylogenetic analysis showed that the two MRSA t034/ST398 isolates were closely related to the human-associated MSSA in China. Moreover, two MRSA isolates contained the virulence genes relating to the cell adherence, biofilm formation, toxins, and the human-associated immune evasion cluster, which indicated the potential of bidirectional transfer of MRSA between monkeys and humans. This study is the first to report MRSA CC398 from monkey feces in China, indicating that MRSA CC398 could colonize in monkey and have the risk of transmission between humans and monkeys.

Background Infection with Salmonella enterica usually results in diarrhea, fever, and abdominal cramps, but some people become asymptomatic or chronic carrier as a source of infection for others. This study aimed to analyze the difference in serotype, antimicrobial resistance, and genetic profiles between Salmonella strains isolated from patients and those from asymptomatic people in Nantong city, China. Methods A total of 88 Salmonella strains were collected from patients and asymptomatic people from 2017 to 2018. Serotyping, antimicrobial susceptibility testing, and PFGE analysis were performed to analyze the characteristics of these strains. Results Twenty serotypes belonging to 8 serogroups were identified in the 88 Salmonella strains. S. Typhimurium remained to be the predominant serotype in strains from both patients and asymptomatic people. Among the 27 strains from patients, S. Enteritidis and S. Rissen were shown as the other two major serotypes, while S. London, S. Derby, and S. Meleagridis were demonstrated as the other significant serotypes among the 61 strains from asymptomatic people. Antimicrobial resistance testing revealed that 84.1% of strains from both resources were multi-drug resistant. PFGE displayed a highly discriminative ability to differentiate strains belonging to S. Derby, S. Typhimurium, etc., but could not efficiently differentiate serotypes like S. Enteritidis. Conclusions This study’s results demonstrated that S. Typhimurium could cause human infection in both symptomatic and asymptomatic state; S. London, S. Derby, and S. Meleagridis usually cause asymptomatic infection, while S. Enteritidis infection mainly results in human diseases. The high multi-drug resistance rate detected in the antimicrobial resistance and diverse PFGE profiles of these strains implied that the strains were isolated from different sources, and the increased surveillance of Salmonella from both patients and asymptomatic people should be taken to control the disease.

CRISPR typing is a newly developed method used to reveal the genetic relationship of bacterial isolates from different resources. For Salmonella, CRISPR typing can not only reveal the phylogenic difference among isolates belonging to the identical serotype, but also show good correspondence with Salmonella serotypes. Here we describe the protocol of CRISPR typing method used in Salmonella, and the approaches to analyze the genetic relationship among different strains.

CRISPR typing is a newly developed method used to reveal the genetic relationship of bacterial isolates from different resources. For Salmonella, CRISPR typing can not only reveal the phylogenic difference among isolates belonging to the identical serotype, but also show good correspondence with Salmonella serotypes. Here we describe the protocol of CRISPR typing method used in Salmonella, and the approaches to analyze the genetic relationship among different strains.

Avian salmonellosis caused by Salmonella enterica serovar Enteritidis (S. Enteritidis) and Pullorum (S. Pullorum) remains a big threat to the poultry industry and public hygiene. AvrA is an effector involved in inhibiting inflammation. Compared to AvrA from S. Enteritidis (SE-AvrA), the AvrA from S. Pullorum (SP-AvrA) lacks ten amino acids at the C-terminal. In this study, we compared the anti-inflammatory response induced by SP-AvrA to that of SE-AvrA. Transient expression of SP-AvrA in epithelial cells resulted in significantly weaker inhibition of NF-&kappa, B pathway activation when treated with TNF-&alpha, compared to the inhibition by SE-AvrA. SP-AvrA expression in the S. Enteritidis resulted in weaker suppression of NF-&kappa, B pathway in infected HeLa cells compared to SE-AvrA expression in the cells, while SP-AvrA expressed in S. Pullorum C79-13 suppressed NF-&kappa, B activation in infected HeLa and Caco 2 BBE cells to a greater extent than did SE-AvrA because of the higher expression of SP-AvrA than SE-AvrA in S. Pullorum. Further analysis demonstrated that the inhibition of NF-&kappa, B pathway in Salmonella-infected cells corresponded to the downregulation of the p-JNK and Beclin-1 protein molecules. Our study reveals that AvrA modifies the anti-inflammatory response in a manner dependent on the Salmonella serotype through inhibition of NF-&kappa, B pathway.

Background Polymyxins are currently regarded as a possible last-resort therapy to eradicate multidrug-resistant (MDR) gram-negative bacteria. Meanwhile, the old antimicrobial agent fosfomycin has recently been reintroduced into clinical use for the treatment of extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant Enterobacteriaceae. This study investigated a multidrug-resistant Salmonella 4,[5],12:i:- strain from a food catering handler, which had the potential to act as a vehicle for transmitting MDR foodborne pathogens. Methods A Salmonella 4,[5],12:i:- YZU1189 strain was isolated from the fecal sample of a food catering worker according to the standard protocol of the Salmonella detection method from World Health Organization in 2003. Serotyping of YZU1189 was performed according to the Kauffmann–White scheme. The antimicrobial resistance phenotype of the strain was determined by the agar dilution method according to the instruction from Clinical and Laboratory Standards Institute (CLSI). Plasmid conjugation was performed between the donor strain Salmonella 4,[5],12:i:- YZU1189 and the recipient strain Escherichia coli C600. The genetic locations of mcr-1, blaCTX-M-14 and fosA3 genes were determined by the whole genome sequence analysis. Results Salmonella 4,[5],12:i:- YZU1189 was an ESBL-producing stain isolated from a healthy catering worker. The strain displayed resistance to aminoglycosides, beta-lactams, polymyxins, fosfomycins, phenicols, trimethoprims, sulfonamides, tetracyclines and fluoroquinolones. Whole genome sequence analysis and plasmid conjugation revealed that the strain had a transferable IncHI2 plasmid carrying the mcr-1, blaCTX-M-14 and fosA3 genes. Sequence homology analysis showed that this plasmid possessed high sequence similarity to previously reported mcr-1, blaCTX-M-14 and fosA3 positive plasmids in China. Conclusion This study reported a the multidrug-resistant Salmonella 4,[5],12:i:- isolate harboring mcr-1, blaCTX-M-14 and fosA3 from human for the first time in China. The occurrence of mcr-1 and fosA3 genes in the transferable IncHI2 plasmid pYZU1189 from the ESBL-producing Salmonella 4,[5],12:i:- isolate showed a potential threat to public health. Great concern should be taken for the spread of multidrug-resistant ESBL-producing Salmonella isolates from food catering workers to consumers.

Salmonella enterica serovar Enteritidis remains the most prevalent serotype causing human salmonellosis through the consumption of contaminated foods, especially poultry products. The development of a subunit vaccine against S. Enteritidis can not only protect chickens against Salmonella infection in the poultry industry but also cut the transmission sources. In this study, both the expressed recombinant outer membrane protein F (rOmpF) and extracted outer membrane vesicles (OMVs) were developed as subunit vaccines against S. Enteritidis challenge in chickens. Immunization with the subunit vaccine could induce not only antibody production but also strong cell-mediated immune response. Both rOmpF plus QuilA adjuvant and OMVs alone had highly protective efficacy against S. Enteritidis challenge and rapidly decreased the colonization of bacteria in chicken. These findings revealed the potential application of rOmpF and OMVs as subunit vaccines in the poultry industry.

S. Pullorum and S. Enteritidis are closely related in genetic terms, but they show very different pathogenicity and host range. S. Enteritidis infects many different hosts, usually causing acute gastroenteritis, while S. Pullorum is restricted to avian, where it causes systemic disease in young animals. The reason why they differ in host range and pathogenicity is unknown. The core-genome denotes those genes that are present in all strains within a clade, and in the present work, an automated bioinformatics workflow was developed and applied to identify core-genome differences between these two serovars with the aim to identify genome features associated with host specificity of S. Pullorum. Results showed that S. Pullorum unique coding sequences (CDS) were mainly concentrated in three regions not present in S. Enteritidis, suggesting that such CDS were taken up probably during the separation of the two types from their common ancestor. One of the unique regions encoded Pathogenicity Islands 19 (SPI-19), which encodes a type VI secretion system (T6SS). Single-nucleotide polymorphism (SNP) analysis identified 1791 conserved SNPs in coding sequences between the two serovars, including several SNPs located in a type IV secretion system (T4SS). Analyzing of 100 bp regions upstream of coding sequences identified 443 conserved SNPs between the two serovars, including SNP variations in type III secretion system effector (T3SE). In conclusion, this analysis has identified genetic features encoding putative factors controlling host-specificity in S. Pullorum. The novel bioinformatic workflow and associated scripts can directly be applied to other bacteria to uncover the genome difference between clades.