Unraveling the molecular dynamics of Pseudomonas aeruginosa biofilms at the air-liquid interface.

Aim: The aim of this study was to probe the dynamics of Pseudomonas aeruginosa PA14 air-liquid interface (ALI) biofilms over time through global proteomic analysis. Materials & methods: P. aeruginosa PA14 ALI biofilm samples, collected over 48-144 h, underwent differential expression analysis to identify varying proteins at each time point. Results: A consistent set of 778 proteins was identified, with variable expression over time. Upregulated proteins were mainly linked to 'amino acid transport and metabolism'. Biofilm-related pathways, including cAMP/Vfr and QS, underwent significant changes. Flagella were more influential than pili, especially in early biofilm development. Proteins associated with virulence, transporters and iron showed differential expression throughout. Conclusion: The findings enhance our understanding of ALI biofilm development.

This study looks at how the bacteria Pseudomonas aeruginosa forms a community called a biofilm at the air­liquid interface (ALI), an important environment for bacterial growth. Biofilms at the ALI are resistant to external forces and contribute to antibiotic resistance. Over 48­144 h, we observed a noticeable increase in biofilm thickness. Our data suggested that the flagella, a sort of propeller of the bacterium, plays a crucial role, especially in the initial stages of ALI biofilm formation. Proteins associated with virulence, transporters and iron also showed their significance in ALI biofilms. These findings offer valuable insights into the protein changes and functions involved in P. aeruginosa ALI biofilms, improving our understanding of biofilm development.

Draft genome sequences of nine non-O157 Shiga toxin-producing Escherichia coli in ready-to-eat food from supermarkets in Argentina.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are recognized as an important group of bacterial enteropathogens. Here, we report the draft genome sequence of nine strains of non-O157 STEC isolated from ready-to-eat foods in Argentina. The whole-genome sequence data provide a better understanding of these isolates and will aid epidemiological investigation during outbreaks.

A Single-Laboratory Performance Evaluation of MALDI-TOF MS in Rapid Identification of Staphylococcus aureus, Cronobacter sakazakii, Vibrio parahaemolyticus, and Some Closely Related Bacterial Species of Public Health Importance.

BACKGROUND: Staphylococcus is a genus of Gram-positive bacteria, known to cause food poisoning and gastrointestinal illness in humans. Additionally, the emergence of methicillin-resistant S. aureus (MRSA) strains has caused a major health care burden worldwide. Cronobacter is a group of Gram-negative bacteria that can survive in extreme dry conditions. Cronobacter sakazakii is known to contaminate powdered infant formula and cause life-threatening infections in neonates. Vibrio is a genus of human-pathogenic Gram-negative bacteria that can cause foodborne illness by consuming undercooked or raw seafood. Vibrio parahaemolyticus can cause serious gastrointestinal disease in humans. Thus, rapid identification of Staphylococcus spp., Cronobacter spp., and Vibrio spp. is crucial for the source tracking of contaminated food, as well as to measure the transmission dynamics of these bacterial pathogens causing foodborne diseases and outbreaks. OBJECTIVE: This single-laboratory performance evaluation study used the VITEK MS system to evaluate the potential of MALDI-TOF MS technology for rapid identification of S. aureus-like, C. sakazakii-like, and V. parahaemolyticus-like isolates of public health importance. METHOD: A total of 226 isolates recovered from various food, environmental surveillance samples, and other sources were identified by bioMérieux VITEK 2 and VITEK MS systems as Staphylococcus spp., Cronobacter spp., and Vibrio spp. Five American Type Culture Collection (ATCC) reference Gram-positive and Gram-negative bacterial isolates were also tested to complete the study. In addition, for some Staphylococcus spp. isolates, whole genome sequencing (WGS) and DNA sequencing of 16S rRNA partial region were also performed for species identification. RESULTS: The VITEK MS system was able to provide species identification to all 96 isolates of Staphylococcus spp. and to all 29 isolates of Vibrio spp. examined with a high confidence value (99.9%). Similarly, species identification was observed for the majority of spots (245 of 303) for the 101 Cronobacter spp. isolates (∼82.0%) with a high confidence value (99.9%), and genus level identification was noticed for the rest of the Cronobacter spp. isolates (18.0%; 58 of the 303 spots) analyzed. Species identification data generated by VITEK 2 system were comparable to data obtained by the VITEK MS system. CONCLUSIONS: The VITEK MS system is a reliable high-throughput platform that can rapidly identify Staphylococcus, Vibrio, and Cronobacter to the genus level, as well as S. aureus, C. sakazakii, V. parahaemolyticus, and other closely related foodborne isolates and bacterial isolates from additional sources, in most cases. HIGHLIGHTS: The VITEK MS system can be used in the rapid genus and species identification of human-pathogenic Staphylococcus spp., Cronobacter spp., and Vibrio spp. isolates.

Synthesis of Antibacterial Copper Oxide Nanoparticles by Pulsed Laser Ablation in Liquids: Potential Application against Foodborne Pathogens.

Spherical copper oxide nanoparticles (CuO/Cu2O NPs) were synthesized by pulsed laser ablation in liquids (PLAL). The copper target was totally submerged in deionized (DI) water and irradiated by an infrared laser beam at 1064 nm for 30 min. The NPs were then characterized by dynamic light scattering (DLS) and atomic emission spectroscopy (AES) to determine their size distribution and concentration, respectively. The phases of copper oxide were identified by Raman spectroscopy. Then, the antibacterial activity of CuO/Cu2O NPs against foodborne pathogens, such as Salmonella enterica subsp. enterica serotype Typhimurium DT7, Escherichia coli O157:H7, Shigella sonnei ATCC 9290, Yersinia enterocolitica ATCC 27729, Vibrio parahaemolyticus ATCC 49398, Bacillus cereus ATCC 11778, and Listeria monocytogenes EGD, was tested. At a 3 ppm concentration, the CuO/Cu2O NPs exhibited an outstanding antimicrobial effect by killing most bacteria after 5 h incubation at 25 °C. Field emission scanning electron microscope (FESEM) confirmed that the CuO/Cu2O NPs destructed the bacterial cell wall.

Draft Genome Sequences of 14 Fluoroquinolone-Resistant Escherichia coli Isolates from Imported Shrimp.

We report the draft genome sequences of 14 fluoroquinolone-resistant Escherichia coli strains that were isolated from imported shrimp. All isolates contained multiple point mutations in the quinolone resistance-determining regions (QRDRs) and non-QRDRs of gyrA, parC, and parE genes. The data improve the understanding of fluoroquinolone resistance and indicate resistance mechanisms.

Foodborne Pathogen Biofilms: Development, Detection, Control, and Antimicrobial Resistance.

Bacteria can grow either as planktonic cells or as communities within biofilms [...].

Concentration-Dependent Global Quantitative Proteome Response of Staphylococcus epidermidis RP62A Biofilms to Subinhibitory Tigecycline.

Staphylococcus epidermidis is a leading cause of biofilm-associated infections on implanted medical devices. During the treatment of an infection, bacterial cells inside biofilms may be exposed to sublethal concentrations of the antimicrobial agents. In the present study, the effect of subinhibitory concentrations of tigecycline (TC) on biofilms formed by S. epidermidis strain RP62A was investigated using a quantitative global proteomic technique. Sublethal concentrations of TC [1/8 (T1) and 1/4 minimum inhibitory concentration (MIC) (T2)] promoted biofilm production in strain RP62A, but 1/2 MIC TC (T3) significantly inhibited biofilm production. Overall, 413, 429, and 518 proteins were differentially expressed in biofilms grown with 1/8 (T1), 1/4 (T2), and 1/2 (T3) MIC of TC, respectively. As the TC concentration increased, the number of induced proteins in each Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway increased. The TC concentration dependence of the proteome response highlights the diverse mechanisms of adaptive responses in strain RP62A biofilms. In both COG and KEGG functional analyses, most upregulated proteins belong to the metabolism pathway, suggesting that it may play an important role in the defense of strain RP62A biofilm cells against TC stress. Sub-MIC TC treatment of strain RP62A biofilms led to significant changes of protein expression related to biofilm formation, antimicrobial resistance, virulence, quorum sensing, ABC transporters, protein export, purine/pyrimidine biosynthesis, ribosomes, and essential proteins. Interestingly, in addition to tetracycline resistance, proteins involved in resistance of various antibiotics, including aminoglycosides, antimicrobial peptides, ß-lactams, erythromycin, fluoroquinolones, fusidic acid, glycopeptides, lipopeptides, mupirocin, rifampicin and trimethoprim were differentially expressed. Our study demonstrates that global protein expression profiling of biofilm cells to antibiotic pressure may improve our understanding of the mechanisms of antibiotic resistance in biofilms.

Draft Genome Sequences of Sixteen Fluoroquinolone-Resistant Extraintestinal Escherichia coli Isolates from Human Patients.

We report here the draft genome sequences of 16 fluoroquinolone-resistant extraintestinal Escherichia coli isolates from human patients. These isolates had high MICs (32 to 256 µg/mL) for ciprofloxacin and contained point mutations in the quinolone resistance-determining region (QRDR) of both gyrA and parC that confer resistance to fluoroquinolone. The whole-genome sequence data provide a better understanding of the fluoroquinolone resistance mechanisms in these isolates and would be beneficial in source tracking these pathogens during pandemic outbreaks.

Whole-Genome Sequences of Nine Hospital-Associated Methicillin-Susceptible Staphylococcus aureus Strains.

We present the draft genome sequences of nine hospital-associated methicillin-susceptible Staphylococcus aureus (HA-MSSA) strains. All strains were from Minnesota (eight from blood and one from bone), harbored various virulence genes, and showed diverse multilocus sequence typing and spa types.

Draft Genome Sequences of 27 Hospital-Associated Methicillin-Resistant Staphylococcus aureus Strains Isolated in Minnesota.

Infections caused by hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) strains have higher morbidity and mortality rates and require longer hospital stays than do those caused by hospital-associated methicillin-sensitive Staphylococcus aureus strains. To gain insight into their genomic makeup, antimicrobial resistance, biofilm formation, and virulence potentials, here we present the draft whole-genome sequences of 27 HA-MRSA strains isolated in Minnesota.

Draft Genome Sequences of 12 Panton-Valentine Leucocidin-Positive and Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Strains Isolated from an Intensive Care Unit in Pakistan.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium responsible for difficult-to-treat staphylococcal infections due to multidrug resistance. Twelve Panton-Valentine leucocidin (PVL)-positive and multidrug-resistant clinical MRSA isolates from hospitals in Pakistan were sequenced and annotated to investigate genetic markers associated with antimicrobial resistance, virulence, and biofilm formation.

Draft Genome Sequences of Eight Community-Associated Methicillin-Resistant Staphylococcus aureus Strains.

Here, we report the draft genome sequences of eight community-associated methicillin-resistant Staphylococcus aureus strains that were resistant to cefoxitin, ampicillin, and erythromycin. Three isolates, i.e., CAR1, CAR2, and CAR8, were sequence type 8 (ST8) with staphylococcal cassette chromosome mec (SCCmec) type IVa and were Panton-Valentine leukocidin (PVL) positive, which has been known as a predominant clone in the United States.

Genotypic Characterization of Clinical Isolates of Staphylococcus aureus from Pakistan.

In this study, we compared pulsed-field gel electrophoretic (PFGE), multilocus sequence typing (MLST), Staphylococcal cassette chromosome mec (SCCmec), spa typing, and virulence gene profiles of 19 Panton-Valentine leucocidin (PVL)-positive, multidrug-, and methicillin-resistant clinical Staphylococcus aureus (MRSA) isolates obtained from a hospital intensive care unit in Pakistan. The isolates exhibited 10 pulsotypes, contained eight adhesin genes (bbp, clfA, clfB, cna, fnbA, fnbB, map-eap, and spa), 10 toxin genes (hla, hlb, hld, hlg, pvl, sed, see, seg, seh, and tst), and two other virulence genes (cfb, v8) that were commonly present in all isolates. The spa-typing indicated seven known spa types (t030, t064, t138, t314, t987, t1509, and t5414) and three novel spa types. MLST analysis indicated eight ST types (ST8, ST15, ST30, ST239, ST291, ST503, ST772, and ST1413). All isolates belonged to the agr group 1. Most of the isolates possessed SCCmec type III, but some isolates had it in combination with types SCCmec IV and V. The presence of multidrug-resistant MRSA isolates in Pakistan indicates poor hygienic conditions, overuse of antibiotics, and a lack of rational antibiotic therapy that have led to the evolution and development of hypervirulent MRSA clones. The study warrants development of a robust epidemiological screening program and adoption of effective measures to stop their spread in hospitals and the community.

Dynamic Adaptive Response of Pseudomonas aeruginosa to Clindamycin/Rifampicin-Impregnated Catheters.

Pseudomonas aeruginosa is the most common Gram-negative pathogen causing nosocomial multidrug resistant infections. It is a good biofilm producer and has the potential for contaminating medical devices. Despite the widespread use of antibacterial-impregnated catheters, little is known about the impacts of antibacterial coating on the pathogenesis of P. aeruginosa. In this study, we investigated the adaptive resistance potential of P. aeruginosa strain PAO1 in response to continuous antibiotic exposure from clindamycin/rifampicin-impregnated catheters (CR-IC). During exposure for 144 h to clindamycin and rifampicin released from CR-IC, strain PAO1 formed biofilms featuring elongated and swollen cells. There were 545 and 372 differentially expressed proteins (DEPs) identified in the planktonic and biofilm cells, respectively, by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Both Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the planktonic cells responded to the released antibiotics more actively than the biofilm cells, with metabolism and ribosomal biosynthesis-associated proteins being significantly over-expressed. Exposure to CR-IC increased the invasion capability of P. aeruginosa for Hela cells and upregulated the expression of certain groups of virulence proteins in both planktonic and biofilm cells, including the outer membrane associated (flagella, type IV pili and type III secretion system) and extracellular (pyoverdine) virulence proteins. Continuous exposure of P. aeruginosa to CR-IC also induced the overexpression of antibiotic resistance proteins, including porins, efflux pumps, translation and transcription proteins. However, these upregulations did not change phenotypic minimum inhibitory concentration (MIC) during the experimental timeframe. The concerning association between CR-IC and overexpression of virulence factors in P. aeruginosa suggests the need for additional investigation to determine if it results in adverse clinical outcomes.

Detection of Campylobacter jejuni from Fresh Produce: Comparison of Culture- and PCR-based Techniques, and Metagenomic Approach for Analyses of the Microbiome before and after Enrichment.

ABSTRACT: In this study, we compared the efficiency of culture-based methods with or without membrane filtration, real-time PCR, and digital droplet PCR (ddPCR) for the detection of Campylobacter in fresh produce. Alfalfa sprouts, clover sprouts, coleslaw, and lettuce salad spiked with Campylobacter jejuni were enriched in Bolton broth for 48 h, and enrichment cultures were either directly inoculated onto modified charcoal-cefoperazone-deoxycholate agar or applied on membrane filters placed on the surface of plating media. In parallel, 2-mL Bolton broth cultures were taken to extract DNA for real-time PCR and ddPCR assays and bacterial community analysis. A developed primer set for ddPCR and real-time PCR was evaluated for its inclusivity and exclusivity using pure culture of C. jejuni and non-C. jejuni strains, respectively. In pure culture, the primer set reacted only with C. jejuni strains and showed negative reaction to non-C. jejuni strains. There was no significant difference (P > 0.05) in the detection efficiency of positive Campylobacter isolates from coleslaw and lettuce salad using four detection methods. However, for sprout samples, the detection efficiency of the culture method was significantly (P < 0.05) lower than those of the two PCR assays and the filtration method. The analysis also revealed the presence of Pseudomonas and Acinetobacter as the most prevalent competing microbiota in enriched culture and only Acinetobacter on agar plates in the selective culture step.

Draft Genome Sequences of Two Campylobacter jejuni Strains That Show Significantly Different Colonization Potentials in Chickens.

Here, we report the draft genome sequences of robust (A74/C_24-3) and poor (A74/O_2-2) chicken-colonizing Campylobacter jejuni isolates. Whole-genome sequence analyses of these isolates will be helpful in facilitating further studies to identify genetic factors used in chicken colonization.

Virulence Characteristics of mecA-Positive Multidrug-Resistant Clinical Coagulase-Negative Staphylococci.

Coagulase-negative staphylococci (CoNS) are an important group of opportunistic pathogenic microorganisms that cause infections in hospital settings and are generally resistant to many antimicrobial agents. We report on phenotypic and genotypic virulence characteristics of a select group of clinical, mecA-positive (encoding penicillin-binding protein 2a) CoNS isolates. All CoNS were resistant to two or more antimicrobials with S. epidermidis strain 214EP, showing resistance to fifteen of the sixteen antimicrobial agents tested. Aminoglycoside-resistance genes were the ones most commonly detected. The presence of megaplasmids containing both horizontal gene transfer and antimicrobial resistance genetic determinants indicates that CoNS may disseminate antibiotic resistance to other bacteria. Staphylococcus sciuri species produced six virulence enzymes, including a DNase, gelatinase, lipase, phosphatase, and protease that are suspected to degrade tissues into nutrients for bacterial growth and contribute to the pathogenicity of CoNS. The PCR assay for the detection of biofilm-associated genes found the eno (encoding laminin-binding protein) gene in all isolates. Measurement of their biofilm-forming ability and Spearman's rank correlation coefficient analyses revealed that the results of crystal violet (CV) and extracellular polymeric substances (EPS) assays were significantly correlated (ρ = 0.9153, P = 3.612e-12). The presence of virulence factors, biofilm-formation capability, extracellular enzymes, multidrug resistance, and gene transfer markers in mecA-positive CoNS clinical strains used in this study makes them powerful opportunistic pathogens. The study also warrants a careful evaluation of nosocomial infections caused by CoNS and may be useful in studying the mechanism of virulence and factors associated with their pathogenicity in vivo and developing effective strategies for mitigation.

Comparison of Direct Syringe Filtration and Membrane Filtration for the Selective Isolation of Campylobacter jejuni from Ready-to-Eat Sprouts.

Culture method using enrichment broth and selective agar is one of the most common isolation methods for detecting Campylobacter jejuni from food. However, the overgrowth of competing bacteria in enrichment culture complicates the selective isolation of C. jejuni. In this study, we compared an enrichment/plating method for the isolation of C. jejuni from sprout samples with an enrichment/plating method with syringe or membrane filtration when transferring enriched broths to plates. Four types of sprout samples were artificially contaminated with various levels of C. jejuni and incubated in 100 mL of Bolton broth for 48 h. Enrichment broths were either directly transferred onto modified charcoal-cefoperazone-deoxycholate agar or filtered through membrane or with a syringe. A significantly higher (p < 0.05) isolation rate of Campylobacter positives was obtained with both filtration methods (58-61%) than with the method without filtration (10%). Membrane filtrations yielded 61%, whereas syringe yielded 58% positives. In most cases of unfiltered samples (98%), high competing flora covered most of the plate, making differentiation and picking of suspicious colonies difficult. However, less plates were contaminated with competing flora in both filtration methods. Only 5% of plates were contaminated in the syringe filtration method, whereas no competing flora was observed in membrane filtration (0%).

Genotypic characterization of ESBL-producing E. coli from imported meat in South Korea.

Twenty extended-spectrum ß-lactamase (ESBL)-producing E. coli strains were isolated from imported meat in South Korea. ESBL strains of E. coli were detected in chicken (14/20) more often than in pork (6/20) and beef (0/20); the highest number (12/20) was detected in Brazilian meats. The blaCTX-M genes were predominant in meats from many countries. E. coli from pork imported from France produced the blaCTX-M-58 enzyme, which has never been documented previously in ESBL-producing bacteria from clinical or environmental sources. Additionally, the coexistence of the blaCTX-M-2 and blaOXA-1 enzymes in EC12-5 isolate was found for the first time in an ESBL E. coli isolate. A rare blaCTX-M type, blaCTX-M-25, was found in 40% of ESBL E. coli isolates. Phenotypic susceptibility testing showed that E. coli isolates were resistant to up to eleven antibiotics, including ciprofloxacin. For the first time, a new combination in an integron gene cassette, aacA4-cmlA6-qacEΔ1, was found in an E. coli isolate from poultry imported from Brazil. Three E. coli ST117 isolates, from an avian pathogenic lineage producing CTX-M-94, harbored fimH, fyuA, iutA, papC, rfc, and traT virulence genes and were not susceptible to quinolones. For the first time, rfc and papG virulence factors were detected in ESBL E. coli strains isolated from meat products. Even though E. coli CC21 and CC22 were obtained from meats from the USA and Brazil, respectively, they had a similarity coefficient higher than 99% in rep-PCR and the same MLST type (ST117), phenotypic antibiotic resistance pattern, integron gene (qacEΔ1), and plasmid DNA profile. This study indicates that imported meat products may be a source of ESBL-producing E. coli strains in South Korea.

Prevalence, toxin gene profile, antibiotic resistance, and molecular characterization of Clostridium perfringens from diarrheic and non-diarrheic dogs in Korea.

Clostridium perfringens causes diarrhea and other diseases in animals and humans. We investigated the prevalence, toxin gene profiles, and antibiotic resistance of C. perfringens isolated from diarrheic dogs (DD) and non-diarrheic dogs (ND) in two animal hospitals in Seoul, Korea. Fecal samples were collected from clinically DD (n = 49) and ND (n = 34). C. perfringens was isolated from 31 of 49 DD (63.3%) and 21 of 34 ND dogs (61.8%). All C. perfringens strains were positive for the α toxin gene, but not for the ß, ε, or ι toxin genes; therefore, all strains were identified as type A C. perfringens. All isolates were cpe-negative, whereas the ß2 toxin gene was identified in 83.9% and 61.9% of isolates from DD and ND, respectively. Most isolates were susceptible to ampicillin (94%), chloramphenicol (92%), metronidazole (100%), moxifloxacin (96%), and imipenem (100%). However, 25.0% and 21.2% of isolates were resistant to tetracycline and clindamycin, respectively. Molecular subtyping of the isolated strains was performed by using pulsed-field gel electrophoresis. Fifty-two isolates were classified into 48 pulsotypes based on more than 90% similarity of banding patterns. No notable differences were observed among the isolates from DD and ND.