Antimicrobial Resistance and Molecular Typing of Salmonella Stanley Isolated from Humans, Foods, and Environment.

Salmonella enterica serovar Stanley is an important serovar that has been increasingly identified in human salmonellosis. The present study aimed to investigate the antimicrobial resistance and molecular typing of 88 Salmonella Stanley strains isolated from humans (diarrhea patients, n = 64; and healthy carrier, n = 1), foods (aquatic products, n = 16; vegetable, n = 1; and pork, n = 1), and environment (waste water, n = 2; and river water, n = 3) in Shanghai, China from 2006 to 2012. Nearly half of the strains were resistant to sulfafurazole (43/88, 48.9%), and many were resistant to streptomycin (35/88, 39.8%), tetracycline (22/88, 25%), and nalidixic acid (19/88, 21.6%). Approximately a quarter of the strains (24/88, 27.3%) were resistant to more than three antimicrobials, and five had ACSSuT resistance type. Six clusters (A-F) were identified by pulsed-field gel electrophoresis (PFGE) with 80% similarity. Interestingly, strains in the same cluster identified by PFGE possessed similar antibiotic resistance patterns. PFGE typing also indicated that aquatic products might serve as a transmission reservoir for Salmonella Stanley infections in humans.

Antimicrobial susceptibility and molecular typing of salmonella agona isolated from humans and other sources.

Salmonella enterica serotype Agona (Salmonella Agona) has been among the top 10 serotypes that cause human diarrheal diseases in China. A total of 95 Salmonella Agona (67 from humans, and 28 from animals, food of animal origins, and environmental sources) recovered in Shanghai, China from 2005 to 2011 were subjected to antimicrobial susceptibility testing and molecular subtyping using pulsed-field gel electrophoresis (PFGE). Approximately 68.4% of the Salmonella Agona isolates were pansusceptible to 15 antimicrobial agents tested, and 4 isolates (4.21%) were resistant to at least 3 antimicrobials. PFGE analysis resulted in 41 unique patterns, of which 4 major PFGE patterns (X3, X4, X5, and X6) were grouped together at 96.1% similarity. Isolates of the four patterns included those from food (pork, beef, and chicken) and humans. Our findings showed that the same clones of Salmonella Agona were recovered from human patients and food, and that food of animal origin was potentially a major vehicle of Salmonella Agona in human salmonellosis in Shanghai.

Role of alternative sigma factor 54 (RpoN) from Vibrio anguillarum M3 in protease secretion, exopolysaccharide production, biofilm formation, and virulence.

The sigma factor σ(54) (RpoN) is an important regulator of bacterial response to environmental stresses. Here, we demonstrate the roles of RpoN in Vibrio anguillarum M3 by comparative investigation of physiological phenotypes and virulence of the wild-type, an rpoN mutant, and an rpoN complemented strain. Disruption of rpoN was found to decrease biofilm formation, production of exopolysaccharides, and production of the metalloproteases EmpA and PrtV. Injection experiments in fish showed that the M3 ΔrpoN mutant was attenuated in virulence when administrated either by intramuscular injection or by immersion challenge. Slower proliferation of the mutant in fish was also observed. Complementation of the mutant strain with rpoN restored some of the phenotypes to wild-type levels. RpoN was involved in regulation of some virulence-associated genes, as shown by real-time quantitative reverse PCR analysis. These results revealed a pleiotropic regulatory role of RpoN in biofilm formation, production of proteases and exopolysaccharides, and virulence in V. anguillarum M3.

Characterization of internalin genes in Listeria monocytogenes from food and humans, and their association with the invasion of Caco-2 cells.

BACKGROUND: Internalins are surface proteins that are utilized by Listeria monocytogenes to facilitate its invasion into human intestinal epithelial cells. The expression of a full-length InlA is one of essential virulence factors for L. monocytogenes to cross the intestinal barrier in order to invade epithelial cells. RESULTS: In this study, the gene sequences of inlA in 120 L. monocytogenes isolates from food (n = 107) and humans (n = 13) were analyzed. Premature stop codon (PMSC) mutations in inlA were identified in 51 isolates (50 from food and 1 from human). Six mutation types of PMSCs were identified. Among the 51 isolates with PMSCs in inlA, there were 44 serogroup 1/2c, 3c isolates from food, of which seven belonged to serogroups 1/2a, 3a. A total of 153,382 SNPs in 2247 core genes from 42 genomes were identified and used to construct a phylogenetic tree. Serotype 1/2c isolates with inlA PMSC mutations were grouped together. Cell culture studies on 21 isolates showed that the invasion to Caco-2 cells was significantly reduced among isolates with inlA PMSC mutations compared to those without PMSC mutations (P < 0.01). The PMSC mutations in inlA correlated with the inability of the L. monocytogenes isolates to invade Caco-2 cells (Pearson's coefficient 0.927, P < 0.01). CONCLUSION: Overall, the study has revealed the reduced ability of L. monocytogenes to invade human intestinal epithelial cells in vitro was linked to the presence of PMSC mutations in inlA. Isolates with PMSC mutations shared the same genomic characteristics indicating the genetic basis on the potential virulence of L. monocytogenes invasion.

Evolution and Diversity of Listeria monocytogenes from Clinical and Food Samples in Shanghai, China.

Listeria monocytogenes is a significant foodborne pathogen causing severe systemic infections in humans with high mortality rates. The objectives of this work were to establish a phylogenetic framework of L. monocytogenes from China and to investigate sequence diversity among different serotypes. We selected 17 L. monocytogenes strains recovered from patients and foods in China representing serotypes 1/2a, 1/2b, and 1/2c. Draft genome sequences were determined using Illumina MiSeq technique and associated protocols. Open reading frames were assigned using prokaryotic genome annotation pipeline by NCBI. Twenty-four published genomes were included for comparative genomic and phylogenetic analysis. More than 154,000 single nucleotide polymorphisms (SNPs) were identified from multiple genome alignment and used to reconstruct maximum likelihood phylogenetic tree. The 41 genomes were differentiated into lineages I and II, which consisted of 4 and 11 subgroups, respectively. A clinical strain from China (SHL009) contained significant SNP differences compared to the rest genomes, whereas clinical strain SHL001 shared most recent common ancestor with strain SHL017 from food. Moreover, clinical strains SHL004 and SHL015 clustered together with two strains (08-5578 and 08-5923) recovered from an outbreak in Canada. Partial sequences of a plasmid found in the Canadian strain were also present in SHL004. We investigated the presence of various genes and gene clusters associated with virulence and subgroup-specific genes, including internalins, L. monocytogenes pathogenicity islands (LIPIs), L. monocytogenes genomic islands (LGIs), stress survival islet 1 (SSI-1), and clustered regularly interspaced short palindromic repeats (CRISPR)/cas system. A novel genomic island, denoted as LGI-2 was identified. Comparative sequence analysis revealed differences among the L. monocytogenes strains related to virulence, survival abilities, and attributes against foreign genetic elements. L. monocytogenes from China were genetically diverse. Strains from clinical specimens and food related closely suggesting foodborne transmission of human listeriosis.

Prevalence and Antimicrobial Resistance Patterns of Diarrheagenic Escherichia coli in Shanghai, China.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) is a major cause of bacterial gastroenteritis in children. Also, antibiotic resistance among DEC is becoming a critical area of concern in clinical settings. METHODS: This study was conducted in 4 hospitals in Shanghai from June 2012 to October 2013. DEC isolates from stool samples of patients with diarrhea were examined to determine their antimicrobial susceptibilities and presence of virulence genes, in order to identify high risk clones. RESULTS: A total of 735 (10.2%) DEC isolates were identified from 7204 stool samples from patients with diarrhea, including 374 enteropathogenic E. coli, 318 enterotoxigenic E. coli, 36 Shigella/enteroinvasive E. coli and 7 Shiga toxin-producing E. coli (STEC). Among the 735 DEC isolates, 299 (40.7%) were isolated from children less than 5 years old. High resistance rates were observed to streptomycin (90.7%), ampicillin (63.4%), nalidixic acid (61.1%), sulfisoxazole (49.1%), tetracycline (41.2%), trimethoprim (35.6%), trimethoprim-sulfamethoxazole (35.4%), followed by amoxicillin-clavulanic acid (27.2%), cefotaxime (24.5%), cefepime (23.5%), gentamicin (16.7%), ceftazidime (12.4%), chloramphenicol (10.6%), ciprofloxacin (7.2%) and ofloxacin (3.4%). All the isolates were susceptible to imipenem. In addition, potential virulence genes were screened by polymerase chain reaction. A total of 15 enterotoxigenic E. coli belonging to the same clone were identified to be associated with nosocomial neonatal diarrhea and resistant to greater than 10 antimicrobials. CONCLUSION: Our findings suggest that active surveillance programs combining both phenotypic and genetic data would help identify disease outbreaks and strengthen antibiotic management.

Molecular Characterization, Antimicrobial Resistance and Caco-2 Cell Invasion Potential of Campylobacter jejuni/coli from Young Children with Diarrhea.

BACKGROUND: Campylobacter is a major cause of bacterial gastroenteritis worldwide. Young children represent a particular age group affected by Campylobacter infection because of their limited diets and weak immune systems. METHODS: In this study, a total of 110 Campylobacter (80 Campylobacter jejuni and 30 Campylobacter coli) isolated from children younger than 5 years of age with diarrhea in Shanghai, China in 2011 were examined for their genetic relationship and antimicrobial susceptibility. The presence of virulence genes and its association with invasion potential in Caco-2 cell were also determined. RESULTS: Multilocus sequence typing revealed 62 sequence types (STs) under 14 clonal complexes from C. jejuni and 15 STs under 2 clonal complexes from C. coli. High resistance rates among the 110 isolates were observed to nalidixic acid (88.2%), ciprofloxacin (87.3%) and tetracycline (87.3%), followed by ampicillin (30.9%), gentamicin (28.2%), clindamycin (21.8%), erythromycin (21.8%) and chloramphenicol (8.2%). Compared with that of C. jejuni (32.5%), a larger proportion of C. coli (83.3%) were resistant to multiple antimicrobials, including 16 isolates of ST-828 complex resistant to 6 antimicrobials: ciprofloxacin, clindamycin, erythromycin, gentamicin, nalidixic acid and tetracycline. Furthermore, 57 Campylobacter isolates were selected based on their distinct STs and the presence of virulence genes to determine their abilities to adhere to and invade Caco-2 cells. The level of invasion varied widely among isolates and had relatively weak correlation with the genotype data. CONCLUSION: Our findings provided baseline data on Campylobacter among young children. Active surveillance of Campylobacter is needed to better understand the epidemiology and antimicrobial resistance trends of this significant pathogen to help control and protect young children from such infections.

Antimicrobial susceptibility, virulence gene profiles and molecular subtypes of Salmonella Newport isolated from humans and other sources.

Salmonella Newport (S. Newport) is a major serotype associated with human salmonellosis. A total of 79 S. Newport recovered from humans and other sources in China were characterized for antimicrobial susceptibility, virulence gene profiles and molecular subtypes using pulsed field gel electrophoresis (PFGE). Approximately 63.3% of the isolates were susceptible to all of 16 antimicrobials tested. Nearly one third of the isolates (31.6%) were resistant to sulfisoxazole, 20.3% to tetracycline and 13.9% to nalidixic acid. Twelve isolates (15.2%) were resistant to three or more antimicrobials. Among 10 virulence genes detected, Salmonella pathogenicity island genes avrA, ssaQ, mgtC, siiD, and sopB and fimbrial gene bcfC were present in most of the isolates (93.7% to 100%). Overall, we observed nine distinct virulence gene profiles, three of which (VP1, VP2 and VP3) were most common (86.1%). A total of 56 PFGE patterns were identified and mainly grouped into seven clusters (A to G) with 80% pattern similarity. Isolates from aquatic product shared a high similarity with those from humans in several clusters, highlighting a potential risk of aquatic product as a source of S. Newport that infect humans. Furthermore, there was a strong association between certain PFGE clusters and virulence gene profiles, suggesting virulence subtyping can be a useful epidemiological tool to discriminate S. Newport isolates.

Whole-Genome Sequences of 12 Clinical Strains of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen of global concern due to the high mortality rate among immunocompromised patients. Whole-genome sequences of 12 strains of L. monocytogenes from humans were reported. The availability of these genomes should provide useful information on the evolutionary history and genetic diversity of L. monocytogenes.

Molecular analysis and antimicrobial susceptibility of enterotoxigenic Escherichia coli from diarrheal patients.

A total of 123 enterotoxigenic Escherichia coli (ETEC) isolates from diarrheal patients from June to December 2012 in Shanghai, China, were examined to determine their genetic relatedness using multilocus sequence typing and pulsed-field gel electrophoresis (PFGE) and for the presence of virulence genes and antimicrobial susceptibility. Twenty-nine sequence types (STs) and 63 PFGE patterns were identified, and results from the 2 subtyping methods correlated well. The 12 isolates of PFGE cluster B all belonged to ST-2332 and were associated with nosocomial neonatal diarrhea. Isolates of a cluster usually had the same set of virulence factors, whereas isolates of different PFGE clusters carried diverse combinations of virulence determinants. Isolates belonging to ST-2332 and ST-182 (n=9) were resistant to at least 6 antimicrobials. Our findings highlighted the need of active surveillance programs for infectious diseases collecting data at both epidemiological and genetic levels that can detect high-risk lineages of pathogens in order to rapidly identify disease outbreaks.

Whole-Genome Sequences of Four Salmonella enterica Serotype Newport Strains from Humans.

Salmonellosis contributes significantly to the public health burden globally. Salmonella enterica serotype Newport is among Salmonella serotypes most associated with food-borne illness in the United States and China. It was thought to be polyphyletic and to contain different lineages. We report draft genomes of four S. Newport strains isolated from humans in China.