Genus-targeted markers for the taxonomic identification and monitoring of coagulase-positive and coagulase-negative Staphylococcus species.

The Staphylococcus genus comprises multiple pathogenic and opportunistic species that represent a risk to public health. Epidemiological studies require accurate taxonomic classification of isolates with enough resolution to distinguish clonal complexes. Unfortunately, 16 S rRNA molecular analysis and phenotypic characterization cannot distinguish all species and do not offer enough resolution to assess intraspecific diversity. Other approaches, such as Multilocus Sequence Tagging, provide higher resolution; however, they have been developed for Staphylococcus aureus and a few other species. Here, we developed a set of genus-targeted primers using five orthologous genes (pta, tuf, tpi, groEs, and sarA) to identify all Staphylococcus species within the genus. The primers were initially evaluated using 20 strains from the Collection of Microorganisms of Interest in Animal Health from AGROSAVIA (CMISA), and their amplified sequences were compared to a set of 33 Staphylococcus species. This allowed the taxonomic identification of the strains even on close species and the establishment of intraspecies diversity. To enhance the scope and cost-effectiveness of the proposed strategy, we customized the primer sets for an Illumina paired-end amplicon protocol, enabling gene multiplexing. We assessed five genes across 177 strains, generating 880 paired-end libraries from the CMISA. This approach significantly reduced sequencing costs, as all libraries can be efficiently sequenced in a single MiSeq run at a fraction (one-fourth or less) of the cost associated with Sanger sequencing. In summary, this method can be used for precise identification and diversity analysis of Staphylococcus species, offering an advancement over traditional techniques in both resolution and cost-effectiveness.

Deciphering the Role of WWTPs in Cold Environments as Hotspots for the Dissemination of Antibiotic Resistance Genes.

Cold environments are the most widespread extreme habitats in the world. However, the role of wastewater treatment plants (WWTPs) in the cryosphere as hotspots in antibiotic resistance dissemination has not been well established. Hence, a snapshot of the resistomes of WWTPs in cold environments, below 5 °C, was provided to elucidate their role in disseminating antibiotic resistance genes (ARGs) to the receiving waterbodies. The resistomes of two natural environments from the cold biosphere were also determined. Quantitative PCR analysis of the aadA, aadB, ampC, blaSHV, blaTEM, dfrA1, ermB, fosA, mecA, qnrS, and tetA(A) genes indicated strong prevalences of these genetic determinants in the selected environments, except for the mecA gene, which was not found in any of the samples. Notably, high abundances of the aadA, ermB, and tetA(A) genes were found in the influents and activated sludge, highlighting that WWTPs of the cryosphere are critical hotspots for disseminating ARGs, potentially worsening the resistance of bacteria to some of the most commonly prescribed antibiotics. Besides, the samples from non-disturbed cold environments had large quantities of ARGs, although their ARG profiles were highly dissimilar. Hence, the high prevalences of ARGs lend support to the fact that antibiotic resistance is a common issue worldwide, including environmentally fragile cold ecosystems.

Whole-Genome Analysis of a High-Risk Clone of Klebsiella pneumoniae ST147 Carrying Both mcr-1 and blaNDM-1 Genes in Peru.

The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other ß-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6')-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies.

Genomic analysis of Burkholderia cenocepacia isolated from a liver abscess in a patient with cystic fibrosis.

Burkholderia cenocepacia complex is associated with high transmissibility, virulence, and poor prognosis in cystic fibrosis (CF) patients. However, extrapulmonary infections are rare. We investigated the genome of a B. cenocepacia IIIA isolated from a liver abscess in a Brazilian CF patient and compared it to strain J2315. The whole genome was sequenced, and contigs were annotated by Rapid Annotation using Subsystem Technology. The Pathosystems Resource Integration Center was used to map antimicrobial and virulence genes. The genomic island (GIs) analysis was performed using two prediction methods, and the presence of putative plasmids and insertion sequences (ISs) was investigated. The isolate was confirmed as B. cenocepacia IIIA to ST-28 (ET12 lineage). A total of 64 genes for antimicrobial resistance and 47 genes related to virulence were identified. Among the virulence factors, there was a predominance of factors related to the invasion mechanism, to the flagellar biosynthesis protein, and to the RNA polymerase sigma factor for flagellar operon (cdpA). Two IS families (IS3 and IS5) and only one plasmid were found. On average 56 GIs were predicted by at least one of the methods applied. Comparative analysis showed resistance mechanisms and virulence factors revealing invasive determinants used by B. cenocepacia IIIA (ET12) in the process of disease spread to other infection sites (extrapulmonary) of highly virulent strains in CF patients.

Climatological and Epidemiological Conditions Are Important Factors Related to the Abundance of blaKPC and Other Antibiotic Resistance Genes (ARGs) in Wastewater Treatment Plants and Their Effluents, in an Endemic Country.

Several physicochemical and season factors have been related to the abundance of antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs), considered hotspots of bacterial resistance. However, few studies on the subject have been carried out in tropical countries endemic for resistance mechanisms such as blaKPC. In this study, the occurrence of ARGs, particularly blaKPC, was determined throughout a WWTP, and the factors related to their abundance were explored. In 2017, wastewater samples were taken from a WWTP in Colombia every 15 days for 6 months, and a total of 44 samples were analyzed by quantitative real-time PCR. sul1, sul2, blaKPC, and ermB were found to be the most prevalent ARGs. A low average reduction of the absolute abundance ARGs in effluent with respect to influent was observed, as well as a greater absolute abundance of ARGs in the WWTP effluent in the rainy season. Factors such as temperature, pH, oxygen, total organic carbon (TOC), chemical oxygen demand (COD), and precipitation were significantly correlated with the absolute abundance of several of the ARGs evaluated. A generalized linear mixed-effects model analysis showed that dissolved oxygen and precipitation in the sampling day were important factors related to the absolute concentration of blaKPC over time. In conclusion, the abundance of ARGs in the WWTP could be influenced by endemic conditions and physicochemical and climatological parameters. Therefore, it is necessary to continuously monitor clinical relevant genes in WWTPs from different global regions, even more so in low-income countries where sewage treatment is limited.

A Trigger Phosphodiesterase Modulates the Global c-di-GMP Pool, Motility, and Biofilm Formation in Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while, its overexpression from an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible promoter showed a positive effect on both swimming and swarming motility and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes, as well as that of the biofilm-related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE c-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, and hence not many examples of these type of signaling proteins have been reported in the literature. Here, we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.

Diversity of ß-lactamase-encoding genes in wastewater: bacteriophages as reporters.

A reservoir of antibiotic resistance genes (ARGs) is present in pathogenic, commensal, and environmental bacteria as well as in mobile genetic elements, including bacteriophages. Wastewater treatment plants (WWTPs) are considered hotspots for the spread of ARGs. The aim of this work was to analyze the diversity of the highly prevalent ARGs blaCTX-M and blaTEM in bacterial and bacteriophage fractions associated with human and animal environments through the study of urban waste and animal residues discharged into WWTPs to provide information about the composition and maintenance of the current resistome in Buenos Aires, Argentina. The results showed that a putative extended-spectrum variant of the blaTEM gene was the most frequently detected, with blaTEM-116 being the most prevalent, while a recently described type, blaTEM-229, was also found. In the bacteriophage fraction, we detected blaCTX-M genes from four out of the five clusters described. The detection of blaCTX- M-9-like and blaCTX-M-25-like genes was unexpected based on surveys of the ARGs from clinical pathogens circulating regionally. The finding of divergent blaCTX-M sequences associated with previously reported environmental genes argues in favor of the natural environment as a reservoir of resistance genes. ARGs were detected in bacteriophages as frequently as in bacterial communities, and furthermore, the blaCTX-M genes were more diverse in the bacteriophage fraction. Bacteriophages might therefore play a role in the spread of ARGs in the environment, but they might also be used as "reporters" for monitoring circulating ARGs.

Identification and characterisation of the lactic acid bacteria associated with the traditional fermentation of dairy fermented product.

The aim of this research was to identify the key lactic acid bacteria associated with the fermentation of dairy traditional fermented products for developing starter cultures for controlled fermentation. A total of 100 lactic acid bacteria (LAB) were isolated from dairy traditional fermented products. Samples were obtained from eight producers in the South East of Nigeria. Isolates were identified by phenotypic and genotypic techniques including rep-PCR genotyping and sequencing of the 16S rRNA, pheS and rpoA genes. Isolates were characterised for antimicrobial activity against foodborne pathogens, exopolysaccharide (EPS) production and survival at low pH and in the presence of bile salts. All isolates clustered into 11 distinct rep-PCR groups and were identified as Lactobacillus fermentum (40%), Lactobacillus delbrueckii (23%), Streptococcus thermophilus (22%), Streptococcus infantarius (10%), Lactobacillus senioris (2%), Leuconostoc pseudomesenteriodes (2%) and Enterococcus thailandicus (1%). Lactobacillus fermentum showed a broad spectrum antimicrobial activity and survival at low pH, while Lactobacillus delbrueckii was able to tolerate low pH and produce EPS. All isolates survived in vitro exposure to 1% (w/v) bile salts over a 3-h period. L. fermentum, L. delbrueckii and S. thermophilus could be used to simulate the fermentation of dairy traditional fermented products.

Characterization of multidrug-resistant avian pathogenic Escherichia coli: an outbreak in canaries.

The canary (Serinus canaria) is appreciated for its beautiful song, colors, and docile temperament and drives a lucrative business. However, diseases caused by avian pathogenic Escherichia coli (APEC) compromise the health of canaries, and the inadequate antimicrobial treatment can lead to the emergence of resistant strains. This study aimed to characterize 21 isolates of E. coli obtained from canaries infected with colibacillosis during an outbreak in northern Paraná State, Brazil. APEC and diarrheagenic E. coli (DEC) virulence genes were screened for by polymerase chain reaction (PCR). All isolates were positive for the hlyF, iss, and ompT genes, which are characteristic of APEC. The iroN gene was found in 95.2% of isolates, and none had the iutA gene. The ipaH gene, characteristic of enteroinvasive E. coli (EIEC), was found in 71.4% of isolates, all belonging to the phylogenetic group B1. High genetic similarity (>95%) was found using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The isolates belonged to serotypes O117:H4 (71.4%) and O1:H20 (23.8%). This is the first report of a clonal colibacillosis outbreak in canaries caused by APEC. All isolates were resistant to ampicillin, nalidixic acid, ciprofloxacin, enrofloxacin, norfloxacin, and tetracycline. The high rate of multidrug resistance in our study shows the importance of avoiding the inadequate antibiotic treatment. We suggest that further studies should be conducted to contribute to the understanding of colibacillosis in canaries since the health of animals is linked to human and environmental health, as defined by the concept of One Health.

Characterization of Third-Generation Cephalosporin-Resistant Escherichia coli Isolated from Pigs in Cuba Using Next-Generation Sequencing.

Resistance to third-generation cephalosporins (3GC) in Escherichia coli has been reported worldwide from humans and animals, but the situation in Cuba is still poorly understood. This study aimed to gain new insights into the phenotypic and genotypic characteristics of third-generation cephalosporin-resistant (3GC-R) E. coli isolated from pigs in Cuba. Rectal swabs from 215 healthy pigs were taken from different municipalities in the western region of Cuba and spread on MacConkey agar supplemented with cefotaxime and ceftazidime. Ninety-six isolates were identified as 3GC-R E. coli and 87.5% of them were resistant to at least three antibiotic classes as determined by the measurement of the minimum inhibitory concentration (MIC) of 14 antibiotics. Twenty-seven different isolates were selected for Illumina next-generation sequencing, and subsequent in silico analysis was performed for the detection of antibiotic resistance and virulence genes, plasmid incompatibility (Inc) groups, multilocus sequence typing (MLST), and core genome MLST (cgMLST). The sequenced isolates contained extended-spectrum ß-lactamase genes blaCTX-M-32 (n = 17), blaCTX-M-15 (n = 5), and blaCTX-M-55 (n = 4) as well as with pAmpC gene blaCMY-2 (n = 2). They also harbored genes for resistance to other clinically important classes of antibiotics, as well as several diverse virulence factors. The 3GC-R E. coli were genetically highly diverse, belonging to 16 different sequence types. IncX1 was the most frequent Inc group. The presence of 3GC-R E. coli in pigs from Cuba containing several different antibiotic resistance mechanisms emphasizes the need for surveillance programs and the establishment of strategies for the prudent use of antibiotics in food-producing animals.

Analysis of genes encoding for proteolytic enzymes and cytotoxic proteins as virulence factors of Piscirickettsia salmonis in SHK-1 cells.

Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic disease generating high mortality rates in farmed salmon cultures of southern Chile. Proteolytic enzymes are important virulence factors since they play a key role in bacterial invasion and proliferation within the host. Bacteria growing in muscle tissues are known to secrete proteases, but no proteolytic enzymes have been described in P. salmonis to date. A battery of putative protease genes was found in the genomes and available strains of P. salmonis by bioinformatics analyses, and their identity was established through comparison with protease genes in databases. The transcript levels of five candidate genes were analysed by in vitro infection and qPCR. All strains were found to generate protease activity to varying degrees, and this was significantly increased when bacteria infected a salmon cell line. Gene expression of several types of proteases was also evidenced, with the highest levels corresponding to the type 1 secretion system (T1SS), which is also involved in the transport of haemolysin A, although transcripts with significant levels of peptidase M4 (thermolysin) and CLP protease were also found.

Letter to the Editor: Escherichia fergusonii Harboring IncHI2 Plasmid Containing mcr-1 Gene-A Novel Reservoir for Colistin Resistance in Brazil.

Emergence of colistin-resistant bacteria harboring mobile colistin resistance genes (mcr genes) pose a threat for food-producing animals and humans. In this article, we aim to highlight the emergence of Escherichia fergusonii as an important new reservoir to mcr-1-harboring plasmid in poultry production. Three strains closely related were isolated from cloacal swabs. Their genome contains four plasmids, including a 182,869 bp IncHI2 plasmid harboring the colistin resistance gene mcr-1. These results will contribute to our understanding of plasmid-mediated mcr-1 gene presence and transmission in E. fergusonii.

Antimicrobial Resistance Profiles and Phylogenetic Analysis of Campylobacter jejuni Strains Isolated in Brazil by Whole Genome Sequencing.

Aims: The objectives of this work were to use whole genome sequencing (WGS) to determine the antimicrobial resistance genotypes of 116 Campylobacter jejuni strains isolated in Brazil and to compare it with the results obtained by antimicrobial susceptibility testing (AST). In addition, WGS was used to uncover the phylogenetic relationship among those strains. Results: By AST, the C. jejuni strains resistant to ciprofloxacin, tetracycline, doxycycline, and erythromycin were 51 (44%), 41 (35.3%), 41 (35.3%), and 6 (5.2%), respectively. By WGS, the genes aph(3')III, aadE, blaOXA-449, blaOXA-184, blaOXA-61, and tet(O) were detected in 6 (5.2%), 3 (2.6%), 1 (0.9%), 10 (8.6%), 55 (47.4%), and 44 (38%) strains, respectively. Fifty-four (46.6%) strains showed the mutation T86I in the gyrA gene, and four (3.4%) strains presented the mutation A2075G in the 23S rRNA gene. The correlation between AST and WGS was 100% for ciprofloxacin, 97.5% for tetracyclines, and 66.7% for erythromycin. The whole genome single nucleotide polymorphism (SNP) tree clustered the C. jejuni strains into two clades comprising strains that were highly related from different sources, places, and years. Conclusion: The high rates of C. jejuni strains resistant to ciprofloxacin and tetracyclines are of concern and may represent a public health problem. WGS has a potential to be a powerful tool for the prediction of resistance of antibiotics used to treat campylobacteriosis. The results obtained by whole genome SNP analysis suggested the potential for transmission between clinical and nonclinical sources and between human and animal sources over the course of 20 years in Brazil.

Dynamic of High-Risk Acinetobacter baumannii Major Clones in a Brazilian Tertiary Hospital During a Short Time Period.

We characterized by whole-genome sequencing (WGS) six carbapenem-resistant Acinetobacter baumannii strains isolated from a Brazilian tertiary hospital during a 14-day period. The ISAba1-blaOXA-23 structure was found in the chromosome of five isolates, whereas blaOXA-72 was inserted in a 16.6-kb plasmid in two isolates. The presence of ISAba1-blaADC-like justified the high broad-spectrum cephalosporins minimal inhibitory concentrations (MICs) (MIC50, > 512 mg/L) verified in all isolates. Only minocycline (MIC50, ≤ 0.5 µg/mL), polymyxin B (MIC50, 0.5 µg/mL), and tigecycline (MIC50, 0.5 µg/mL) were in vitro active against such isolates. A diversity of other antimicrobial resistance determinants (aph(3')-VIa, aadA1, aac(3')-IIa, strA, strB, sul2, drfA1, mph(E), msr(E), tetB, and floR) was also observed, which may confer resistance to at last six distinct antimicrobial classes. Four distinct pulsed-field gel electrophoresis (PFGE) profiles were observed during the study period, which belonged to ST79/ST258 (n = 2; IC5), ST25/ST229 (n = 2; IC7), ST1 (n = 1; IC1), and ST162/ST235 (n = 1; IC4). Although the ST1 isolate that carried blaOXA-23 and blaOXA-72 was introduced in this hospital setting by a transferred patient, two clonally related ST79/ST258 isolates carrying either one of these carbapenemase encoding genes were recovered from two patients who were hospitalized within the same period of time in the same hospital unit. Finally, a good correlation between PFGE/MLST, blaOXA-51 variant, and single nucleotide polymorphisms was also observed. Here we demonstrated that distinct extensively drug-resistant A. baumannii clones can circulate in the same hospital setting during a short time period, illustrating a very complex epidemiological scenario for this priority pathogen.

Antibiotic resistance in wastewater treatment plants: understanding the problem and future perspectives.

Antibiotics residues (AR), antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) are a new class of water contaminants, due to their adverse effects on aquatic ecosystems and human health. Contamination of water bodies occurs mainly by the excretion of antibiotics incompletely metabolized by humans and animals and is considered the main source of contamination of antibiotics in the environment. Given the imminent threat, the World Health Organization (WHO) has categorized the spread of antibiotics as one of the top three threats to public health in the twenty-first century. The Urban Wastewater Treatment Plants (UWWTP) bring together AR, ARB, ARG, making the understanding of this peculiar environment fundamental for the investigation of technologies aimed at combating the spread of bacterial resistance. Several methodologies have been employed focusing on reducing the ARB and ARG loads of the effluents, however the reactivation of these microorganisms after the treatment is widely reported. This work aims to elucidate the role of UWWTPs in the spread of bacterial resistance, as well as to report the efforts that have been made so far and future perspectives to combat this important global problem.

Bacteria with a Potential for Multidrug Resistance in Hospital Material.

The objective of this research was to determine the antimicrobial resistance of bacteria isolated from items related to hygiene and antisepsis, equipment, and instruments used in different hospital wards. Bacterial isolation and identification, phenotypic antimicrobial susceptibility assays, mecA gene detection, and multiple antimicrobial resistance index analysis were performed. In total, 105 bacteria were isolated from 138 items. Of these, 49.52% bacteria were collected from instruments, 43.80% from equipment, and 6.66% from items related to hygiene and antisepsis. All gram-positive bacteria (88 isolates) were identified as coagulase-negative Staphylococcus. Five species of gram-negative bacilli (17 isolates) were isolated, and the prevalence of Enterobacter agglomerans (29.41%), Escherichia coli (11.76%), and Serratia liquefaciens (11.76%) was high. Antimicrobial resistance was reported for 93.33% of the isolates. Gram-positive bacteria were resistant to sulfazotrim (88.64%) and penicillin (82.95%), while gram-negative bacteria showed resistance to sulfazotrim (70.59%) and ampicillin (64.71%). Analysis of multiple antibiotic resistance index showed that 73.33% of the isolates were a high risk to public health. The mecA gene was detected in 23 (71.88%) isolates. The evaluation of microorganisms isolated in the hospital environment revealed their high multidrug resistance index. Thus our study presses the need to pay more attention to the cleanliness of frequently used instruments, which may be potential sources of infections.

Characterization of blaKPC-2-Harboring Klebsiella pneumoniae Isolates and Mobile Genetic Elements from Outbreaks in a Hospital in Ecuador.

Aim: To investigate the mobile genetic elements harboring blaKPC gene in carbapenem-resistant Klebsiella pneumoniae recovered during a 6-month outbreak in a high-complexity hospital from Ecuador. Results: A total of 62 isolates belonging to ST258 pilv-I-positive (n = 45), ST25 serotype K2 (n = 8), ST348 (n = 6), ST42 (n = 1), ST196 (n = 1), and ST1758 (n = 1) were collected from intensive care unit (ICU), neurosurgery, burn unit, internal medicine, pneumology, and neurology. Pulsed-field gel electrophoresis analysis showed two major clusters of ST258 and ST25 related to bloodstream infections and pneumonia circulating in ICU. The PCR assay showed that in non-ST258 isolates, the blaKPC-2 gene were located on the Tn4401a transposon inserted in the transferable pKpQIL-like IncFIIK2 plasmid; the whole-genome sequencing of ST258 clone showed two plasmids, the blaKPC-2 gene was located on nonconjugative IncR plasmid, whereas the IncFIB/IncFII plasmid lacked ß-lactamase genes. We found an IncM plasmid in blaKPC-2-harboring Klebsiella pneumoniae ST1758 clone. Conclusions: These findings highlight the presence of pKpQIL-like plasmids in non-ST258 and nonconjugative plasmids in ST258 isolates causing hospital outbreaks.

Isolation of Harveyi clade Vibrio spp. collected in aquaculture farms: How can the identification issue be addressed?

Aquaculture is a fast growing industry with its development hampered by bacterial diseases. Vibriosis caused by Harveyi clade strains is known for causing heavy loss especially in shrimp aquaculture farms. For farm treatment and pathogen spread management, veterinarians and researchers need reliable bacterial identification tools. A range of identification methods have been presented for Vibrio spp. in recent literature but little feedback on their performance have been made available to this day. This study aims at comparing Vibrio spp. identification methods and providing guidance on their use. Fifty farms were sampled and bacterial colonies were isolated using specific culture media before microscopic analysis and genomic profiling using ERIC-PCR. A preliminary identification step was carried out using MALDI-ToF mass spectrometry. Four methods were compared for strain identification on 14 newly isolated Harveyi clade Vibrio spp. strains: whole genome sequencing (digital DNA DNA Hybridization (dDDH)), 5 MLSA schemes, ferric uptake regulation (fur) and lecithin-dependent haemolysin (ldh) single gene based identification methods. Apart from dDDH which is a reference method, no technique could identify all the isolates to the species level. The other tested techniques allowed a faster, cheaper but sub genus clade identification which can be interesting when absolute precision is not required. In this regard, MALDI-ToF and fur based identification seemed especially promising.

Counter-Selection Method for Markerless Allelic Exchange in Bordetella bronchiseptica Based on sacB Gene From Bacillus subtilis.

Bordetella bronchiseptica is a gram-negative bacterium that causes respiratory tract infections. It is a natural pathogen of a wide variety of mammals, including some used as laboratory models. This makes B. bronchiseptica an ideal organism to study pathogen-host interactions in order to unveil molecular mechanisms behind pathogenic processes. Even though genetic engineering is an essential tool in this area, there are just a few reports about genome manipulation techniques in this organism. In this article we describe an allelic exchange protocol based on double crossover recombination facilitated by the Bacillus subtilis sacB gene that can be applied for partial or complete gene knockouts, single-nucleotide mutations, or even introduction of coding sequences for transcriptional fusions. In contrast to previously employed techniques, this protocol renders genetically manipulated chromosomes without foreign DNA and enables the construction of successive genome manipulation using the same vector backbone. The entire procedure has been developed for fast and reliable manipulations with a total duration of 2 weeks. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Setting up strains Basic Protocol 2: Homologous recombination (first crossing-over) Alternate Protocol: B. bronchiseptica electroporation Basic Protocol 3: Screening for sucrose-sensitive clones Basic Protocol 4: Homologous recombination (second crossing-over) Basic Protocol 5: PCR screening of putative marker-exchange mutants Support Protocol: Electrocompetent cell preparation.

Transcriptional analysis of metabolic and virulence genes associated with biofilm formation in Piscirickettsia salmonis strains.

Piscirickettsia salmonis is a facultative intracellular bacterium that generates piscirickettsiosis affecting salmonids in Chile. The bacterium has the adaptability to survive in the marine environment under multiple stressful conditions. In this sense, this work focused on the analysis of a gene battery associated with biofilm formation under different culture conditions and on the adaptability of this biofilm to different media. The results indicated that the strains LF-89, IBM-034 and IBM-040 were strong biofilm producers, evidencing adaptability to the media by increasing the amount of biofilm through successive growths. Transcript levels of six genes described in various bacteria and P. salmonis, considered to have metabolic functions, and playing a relevant role in biofilm formation, were analyzed to evaluate bacterial functionality in the biofilm. The genes mazE-mazF, implicated in biofilm and stress, were markedly overexpressed in the biofilm condition in the three strains. For its part, gene gltA, an indicator of metabolic activity and related to virulence inhibition in Salmonella typhimurium, also seems to restrain the pathogenesis process in P. salmonis by inhibiting the expression of the virulence-associated genes liso and tcf. Finally, the expression of the glnA gene suggests the use of glutamine as an essential element for the growth of the biofilm.