Aspirin, sodium benzoate and sodium salicylate reverse resistance to colistin in Enterobacteriaceae and Pseudomonas aeruginosa.

BACKGROUND: MDR bacterial infections are currently a serious problem for clinicians worldwide. Klebsiella pneumoniae and Enterobacter spp., among Enterobacteriaceae, and Pseudomonas aeruginosa, are part of the group of ESCAPE pathogens or bacteria that 'escape' from common antibacterial treatments. The lack of effectiveness of the first common line of antibiotics has led to the search for new therapies based on older antibiotics, such as colistin. OBJECTIVES: We searched for new enhancers of the action of colistin against MDR Gram-negative bacteria that can be easily applicable to clinical treatments. METHODS: Colistin MICs were determined alone and with the protonophores CCCP, sodium benzoate, sodium salicylate and aspirin using the broth microdilution method and FIC indexes were calculated to assess synergy between colistin and each chemical. Time-kill assays of colistin with and without protonophores were performed to determine the bactericidal action of combinations of colistin with protonophores. Likewise, the effect of sucrose, l-arginine and l-glutamic acid on the MICs of colistin alone and combined with each protonophore was assessed. RESULTS: It was found that sodium benzoate, sodium salicylate and aspirin, at concentrations allowed for human and animal use, partially or totally reversed resistance to colistin in P. aeruginosa and highly resistant enterobacterial strains. The mechanism of action could be related to their negative charge at a physiological pH along with their lipid-soluble character. CONCLUSIONS: Sodium benzoate, sodium salicylate and aspirin are good enhancers to use in antibiotic therapies that include colistin.

Antimicrobial susceptibility profiles of porcine mycoplasmas isolated from samples collected in southern Europe.

BACKGROUND: Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. hyosynoviae are significant pathogens for the porcine industry worldwide. The aim of the present study was to determine the antimicrobial susceptibility of six key antimicrobials (tylosin, tilmicosin, tylvalosin, lincomycin, tiamulin and valnemulin) routinely used for treating infections caused by these pathogens. Twenty-seven M. hyopneumoniae, 48 M. hyorhinis and 40 M. hyosynoviae field strains isolated from clinical samples from different Southern European countries between 2013 and 2018 using broth microdilution method were evaluated. RESULTS: Tylvalosin exhibited the highest in vitro activity among the macrolides assayed, with MIC90 values 4 to 5 two-fold dilutions lower than those of tylosin and tilmicosin. The pleuromutilin valnemulin showed one of the highest in vitro activities against the three mycoplasma species. On the contrary, lincomycin exhibited the highest MIC values of the antimicrobials tested. CONCLUSIONS: The data obtained in the present study supports the use of pleuromutilins and macrolides for the control of infections caused by porcine mycoplasmas. The use of lincomycin for the treatment of porcine mycoplasma infections should be carefully evaluated due to the presence of circulating field isolates with decreased susceptibility to this antimicrobial.

General phytoplasma detection by a q-PCR method using mycoplasma primers.

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field-infected plant samples showed a single peak at 82.5 °C (±0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: 'Candidatus Liberibacter solanacearum', Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except 'Ca. L. solanacearum' that was amplified with a melting temperature at 85 °C. Absolute quantification of phytoplasma titer was calculated using standard curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 106 to 103 according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10-6 for conventional PCR and 10-7 for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material.

Mycoplasma detection and isolation from one-humped camels (Camelus dromedarius).

In scientific literature, a small amount of information is found concerning mycoplasmosis in camel species. Mycoplasma (M.) arginini, Acholeplasma (A.) laidlawii, and Acholeplasma oculi have been reported to be isolated from these host species. Serologically positive results have been reported for Mycoplasma mycoides subsp. mycoides SC type, Mycoplasma capricolum subsp. capripneumoniae, and M. mycoides subsp. capri. The aims of this study were to detect, isolate, and identify mycoplasmas from camels (Camelus dromedarius). Initially, saliva and ear smears plus conjunctival and vaginal secretions were taken from five female animals, but only conjunctival secretions in three male animals, all belonging to the same farm. An unknown mycoplasma was isolated from one of the vagina samples. Additionally, another unknown and uncultured mycoplasma was detected with molecular biology in the same sample. In the second stage, 23 vaginal secretions were taken from the same farm plus another secretion from a different one. Ten isolates of the same unknown and previously isolated mycoplasma were detected, nine of them recovered from the vagina of female camels. Some mycoplasmas have been related to reproductive disorders; however, there is no evidence that the isolated mycoplasmas are related to such disorders.

Differential Gene Expression in Porcine Lung Compartments after Experimental Infection with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhyo) is the causative agent of porcine enzootic pneumonia (EP), as well as one of the main pathogens involved in the porcine respiratory disease complex. The host-pathogen interaction between Mhyo and infected pigs is complex and not completely understood; however, improving the understanding of these intricacies is essential for the development of effective control strategies of EP. In order to improve our knowledge about this interaction, laser-capture microdissection was used to collect bronchi, bronchi-associated lymphoid tissue, and lung parenchyma from animals infected with different strains of Mhyo, and mRNA expression levels of different molecules involved in Mhyo infection (ICAM1, IL-8, IL-10, IL-23, IFN-α, IFN-γ, TGF-ß, and TNF-α) were analyzed by qPCR. In addition, the quantification of Mhyo load in the different lung compartments and the scoring of macroscopic and microscopic lung lesions were also performed. Strain-associated differences in virulence were observed, as well as the presence of significant differences in expression levels of cytokines among lung compartments. IL-8 and IL-10 presented the highest upregulation, with limited differences between strains and lung compartments. IFN-α was strongly downregulated in BALT, implying a relevant role for this cytokine in the immunomodulation associated with Mhyo infections. IL-23 was also upregulated in all lung compartments, suggesting the potential involvement of a Th17-mediated immune response in Mhyo infections. Our findings highlight the relevance of Th1 and Th2 immune response in cases of EP, shedding light on the gene expression levels of key cytokines in the lung of pigs at a microscopic level.

Microbiological Survey and Evaluation of Antimicrobial Susceptibility Patterns of Microorganisms Obtained from Suspect Cases of Canine Otitis Externa in Gran Canaria, Spain.

A retrospective study of microbiological laboratory results from 2020 to 2022, obtained from a veterinary diagnostic laboratory of the island of Gran Canaria, Spain, focused on canine otitis cases, was performed. The objective of this study was to analyze the pathogen distribution, antimicrobial susceptibility, prevalence of multidrug resistant phenotypes and the role of coinfections in otitis cases in order to provide up-to-date evidence that could support effective control strategies for this prevalent pathology. A total of 604 submissions were processed for the diagnosis of canine external otitis. Of the samples analyzed, 472 were positive for bacterial or fungal growth (78.1%; 95% CI: 74.8-81.4%). A total of 558 microbiological diagnoses were obtained, divided in 421 bacterial (75.4%; 95% CI: 71.8-79.0%) and 137 fungal (24.6%; 95% CI: 20.9-28.1%) identifications. Staphylococcus pseudintermedius, Malassezia pachydermatis and Pseudomonas aeruginosa were the most prevalent microorganisms detected in clinical cases of otitis. High level antimicrobial resistance was found for Pseudomonas aeruginosa (30.7%), Proteus mirabilis (29.4%), Staphylococcus pseudintermedius (25.1%) and Escherichia coli (19%). Multidrug-resistant phenotypes were observed in 47% of the bacteria isolated. In addition, a 26.4% prevalence of methicillin-resistant Staphylococcus pseudintermedius was detected. The high prevalence of antimicrobial resistant phenotypes in these bacteria highlights the current necessity for constant up-to-date prevalence and antimicrobial susceptibility data that can support evidence-based strategies to effectively tackle this animal and public health concern.

Vaccination Upregulates Th1 Cytokines in the Lung of Pigs Experimentally Infected with Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (Mhy) is the causative agent of enzootic pneumonia, characterized by high morbidity and low mortality rates in intensive swine production systems. To better understand the mechanisms underlying the protection of an inactivated whole cell vaccine, we investigated the immunohistochemical differences in the cytokine expression in vaccinated and non-vaccinated pigs experimentally infected with Mhy. Four-week-old Mhy-negative pigs (n = 24) were allocated to negative control (n = 8), or one of two Mhy-infected groups: vaccinated (n = 8) and non-vaccinated (n = 8). Infection was carried out by a combination of trans-tracheal and aerosol route. Lung samples were processed for histopathological and immunohistochemical studies, by using antibodies against Mhy, IL1-α, IL1-ß, IL-2, IL-4, IL-5, IL-6, Il-8, IL-10, IL-12p35, IL-13, IL-17A, TNF-α, IFN-γ, and CD-4 lymphocytes. Although all cytokines increased in both infected groups, IL-2, IL-12, and IFN-γ were significantly overexpressed in vaccinated pigs. These findings, in conjunction with the decrease of macroscopic and histological lesions in vaccinated animals, indicate the importance to enhance Th1 response in the immunization strategies to control Mhy infection.

Mycoplasma bradburyae sp. nov. isolated from the trachea of sea birds.

In the search for mollicutes in wild birds, six Mycoplasma strains were isolated from tracheal swabs taken from four different species of seabirds. Four strains originated from three Yellow-legged gulls (Larus michahellis) and a Cory's shearwater (Calonectris borealis) from Spain, one from a South African Kelp gull (Larus dominicanus), and one from an Italian Black-headed gull (Chroicocephalus ridibundus). These Mycoplasma strains presented 99 % 16S rRNA gene sequence similarity values with Mycoplasma (M.) gallisepticum. Phylogenetic analyses of marker genes (16S rRNA gene and rpoB) confirmed the close relationship of the strains to M. gallisepticum and M. tullyi. The seabirds' strains grew well in modified Hayflick medium, and colonies showed typical fried egg morphology. They produced acid from glucose and mannose but did not hydrolyze arginine or urea. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas, presenting spherical to flask-shaped cells with an attachment organelle. Gliding motility was also observed. Furthermore, serological tests, MALDI-ToF mass spectrometry and genomic studies demonstrated that the strains were different to any known Mycoplasma species, for which the name Mycoplasma bradburyae sp. nov. is proposed; the type strain is T158T (DSM 110708 = NCTC 14398).

Mycoplasmas hyorhinis in different regions of cuba. diagnosis.

M. hyorhinis is considered one of the etiological agents of arthritis in sucking pigs, but recently as seen, some strains can produce pneumonia that could not be distinguished from the mycoplasmosis caused by M. hyopneumoniae. The study was conducted to research the presence of Mycoplasma hyorhinis (M. hyorhinis ) in different regions of the country from exudates of pig lungs with typical EP lesions. Exudates from 280 pig lungs with typical EP lesions were studied using molecular techniques such as PCR, real time PCR and amplification of the 16S-23S rRNA. It was detected that the 66% of the samples studied resulted positive to M. hyorhinis, and the presence of this species was detected in all the provinces. Amplification and studies on the intergenic region 16S-23S of M. hyorhinis rRNA demonstrated the existing variability among strains of a same species. This study is the first report on M. hyorhinis detection in Cuba.

Pathological and Immunohistochemical Studies of Experimental Mycoplasma pneumoniae in Gerbils (Meriones unguiculatus).

Mycoplasma pneumoniae (Mp) is a leading cause of human community-acquired pneumonia. To investigate the pathogenesis of the infection, 36 gerbils were intranasally inoculated with Mp culture (30 animals) or sterile mycoplasma broth (6 animals) and euthanized from 1 to 5 weeks post infection. A morphological and immunohistochemical study was carried out in all animals to determine the cellular populations present in lung parenchyma. Polyclonal and monoclonal antibodies were used to detect antigens of Mp and CD3, CD4, CD8 and CD79 lymphocytes, as well as cells containing S100 and major histocompatibility complex class II (MHC-II) antigens. There was progressive infiltration of mononuclear cells in the lamina propria of bronchi and bronchioles, and hyperplasia of the bronchus-associated lymphoid tissue (BALT) in the infected animals. BALT contained dendritic cells immunopositive to S100 and MHC-II and numerous CD3, CD4 and CD79 lymphocytes. The immunohistochemical results showed that T lymphocytes, particularly CD4 and CD79 cells, may play a role in the immune response of gerbils against Mp. This experimental model is valuable for investigation of the pathogenesis of Mp infection and may assist in the development of therapeutic strategies.

Resistance to 16-Membered Macrolides, Tiamulin and Lincomycin in a Swine Isolate of Acholeplasma laidlawii.

Acholeplasma (A.) laidlawii is an opportunistic pathogen with the ability to disseminate resistance determinants to antibiotics; however, its resistance to macrolides has been less studied. The aim of the present study was to characterize the mechanisms responsible for the resistance to macrolides, tiamulin and lincomycin found in a strain of A. laidlawii isolated from a pig with pneumonia. MICs of erythromycin, 15- and 16-membered macrolides, tiamulin and lincomycin were determined by microdilution method with and without reserpine, an inhibitor of ABC efflux pumps and regions of the genome were sequenced. Reserpine only decreased lincomycin MIC but it did not change the MICs of macrolides and tiamulin. The analysis of the DNA sequence of 23S rRNA showed nucleotide substitutions at eight different positions, although none of them were at positions previously related to macrolide resistance. Five mutations were found in the L22 protein, one of them at the stop codon. In addition, two mutations were found in the amino acid sequence of L4. The combination of multiple mutations in the ribosomal proteins L22 and L4 together with substitutions in 23S rRNA DNA sequence was associated with the resistance to macrolides, the pleuromutilin and lincomycin in the studied A. laidlawii strain.

Quorum-sensing regulator sdiA and marA overexpression is involved in in vitro-selected multidrug resistance of Escherichia coli.

OBJECTIVES: The role of sdiA in the acquisition of low-level multidrug resistance (MDR) was analysed and compared with that of marA and soxS in two Escherichia coli clinical isolates and two in vitro-selected mutants. METHODS: The mutants were developed by growth in lomefloxacin and ceftazidime. The sdiA, marA, soxS, ftsI, tolC and acrB gene transcript levels were determined by RT-PCR. Analyses of 2,4-dinitrophenol susceptibility, the effect of an active efflux inhibitor on antibiotic and mitomycin C susceptibility, beta-lactamase hydrolytic activity, outer and inner membrane proteins and acrR gene sequencing were also performed. RESULTS: Both mutants showed elevated marA and sdiA gene transcript levels, which were associated with increased susceptibility to 2,4-dinitrophenol; soxS overexpression was only seen in the mutant selected with ceftazidime. The two mutants showed MDR phenotypes in which ceftazidime, cefpirome and aztreonam MICs increased 4- to 128-fold, in addition to decreased susceptibility to quinolones, chloramphenicol and mitomycin C. The highest ceftazidime MIC in one of the mutants coincided with a frameshift mutation in acrR and the highest transcript level of ftsI (penicillin-binding protein 3), but not with a higher beta-lactamase activity. Likewise, active efflux associated with increased levels of acrB and tolC and decreased OmpF expression contributed to low-level MDR in both mutants. CONCLUSIONS: marA and sdiA overexpression was a common feature of multidrug-resistant mutants selected by growth in lomefloxacin and ceftazidime. To our knowledge, this report is the first to describe in vitro selection with a fluoroquinolone or ceftazidime triggering sdiA overexpression in E. coli isolates.

First description of two moderately halophilic and psychrotolerant Mycoplasma species isolated from cephalopods and proposal of Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov.

Two moderately halophilic and psychrotolerant new Mycoplasma species were isolated from common cephalopods. Three strains were isolated in pure culture from two individual European flying squid (Todarodes sagittatus), and two individual octopuses (Octopus vulgaris). The strains showed optimal growth at 25 °C and a salinity of 3% (w/v) NaCl. Molecular analyses revealed that the isolates belonged to two new, but phylogenetically related species, divergent from all previously described Mollicutes, representing the first marine isolates of the class, and also the first Mycoplasma strains for which NaCl requirement has been demonstrated. A genome search against all available marine metagenomes and 16S rRNA gene databases indicated that these two species represent a novel non-free-living marine lineage of Mollicutes, specifically associated with marine animals. Morphology and physiology were compatible with other members of this group, and genomic and phenotypic analyses demonstrated that these organisms represent two novel species of the genus Mycoplasma, for which the names Mycoplasma marinum sp. nov. and Mycoplasma todarodis sp. nov. are proposed; the type strains are PET (DSM 105487T, CIP 111404T) and 5HT (DSM 105,488T, CIP 111405T), respectively.

A semi-defined medium without serum for small ruminant mycoplasmas.

The composition of the medium used to cultivate Mycoplasma species is very important. Serum is one of the most important additives as it contains lipids (cholesterol) and serum proteins, which are essential for the growth of the organisms. This work reports the development of a semi-defined medium, called MWS (Medium Without Serum) produced without animal serum and bovine serum albumin. MWS seems to be suitable for cultivating several species of caprine mycoplasma, especially M. mycoides subsp. mycoides (LC) and M. mycoides subsp. capri.

Applications of flow cytometry to mycoplasmology.

Flow cytometry has become a valuable tool in different fields of microbiology, such as clinical microbiology, aquatic and environmental microbiology, food microbiology, and biotechnology. It combines direct and rapid assays to determine numbers, biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population. This review focuses on the applications of flow cytometry to the field of mycoplasmology. It tries to give a scope of the important breakthroughs which occurred in this field in the last decades, and in the advantages of introducing flow cytometry in research and routine diagnostic procedures of mycoplasmas.

Detection of mycoplasmas in goat milk by flow cytometry.

The detection of mycoplasma in milk can be performed by either culture techniques or polymerase chain reaction (PCR) based methods. Although PCR can reduce the average diagnostic time to 5 h in comparison with the several days for the isolation of the agent, there is still a need to develop methods, which could give earlier results. For this purpose, we tested the ability of flow cytometry (FC) to detect mycoplasmas in milk samples. Milk samples inoculated with four different mycoplasmas, Mycoplasma agalactiae, Mycoplasma putrefaciens, Mycoplasma capricolum subsp. Capricolum, or Mycoplasma mycoides subsp. mycoides large-colony type, known to cause contagious agalactia in goats, were stained with the DNA stain SYBR Green I and analyzed by FC. Three goat milk samples, from which mycoplasmas have been isolated in broth medium were also analyzed. All mycoplasmas were easily distinguished from debris of milk samples, but it was not possible to distinguish between the different mycoplasma species. In our conditions, the detection limit of the technique was of the order of 10(3)-10(4) cells ml(-1). Furthermore, mycoplasmas were also distinguished from Staphylococcus aureus. FC together with SYBR Green I was able to distinguish between mycoplasma cells and debris present in milk samples and gave results in 20-30 min. This is an important first step in developing a robust, routine flow cytometric method for the detection of mycoplasmas in milk samples.

Field trial of a combined vaccine against caprine contagious agalactia: humoral immune response in lactating goats.

Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) were developed using inactivated strains selected in previous characterization studies. Formaldehyde and phenol were used as the inactivating agents for vaccines A and B, respectively. Aluminium hydroxide plus purified saponin (Quil-A) were added to both vaccines as adjuvant. The field trial was designed to evaluate the specific humoral immune response to the two mycoplasma species in lactating goats over a period of 7 months. The vaccines were tested on 120 goats randomly assigned to three groups of 40 animals each. Two groups received two injections of vaccine A or B respectively, and a third group remained in the herd as control. Antibody titres determined by ELISA indicated a significant difference between both vaccines and the control group over a 6-month period. Immunoblotting assays also revealed the production of antibodies against the two mycoplasma species. Further field trials are underway to evaluate the efficacy and protection conferred to the animals by these specific antibodies.

Quantification of mycoplasmas in broth medium with sybr green-I and flow cytometry.

Mycoplasmas are the smallest and simplest organisms known. They form a large group of bacteria that can infect humans, animals, and plants. Even though several techniques have been proposed to enumerate mycoplasmas in broth medium, the determination of mycoplasma growth still remains a difficult task. The potential of using flow cytometry (FC) for rapidly estimating several species of mycoplasmas, M. agalactiae (Ma), M. putrefaciens (Mp), M. capricolum subsp. capricolum (Mcc), M. bovis (Mb), M. capricolum subsp. capripneumoniae (Mccp) and M. hyopneumoniae (Mh) in broth medium was examined. The FC analysis was performed by staining the mycoplasma cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count (Colony Forming Units--CFU) or Colour Changing Units (CCU) methods, depending on the mycoplasma species. There was a good correlation between mycoplasma counts determined by FC (cells ml(-1)) and by traditional plate count (CFU) or CCU methods. A correlation of 0.841, 0.981, 0.960, 0.913, 0.954, and 0.844 was obtained for Ma, Mp, Mcc, Mb, Mccp and Mh, respectively. FC method allowed results in 20-30 min, while 24-72 h was necessary for plate count method and 15 days for CCU method. FC was found to be a very useful, practical and fast technique to count mycoplasmas. These findings suggest that FC can be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasmas in broth medium.

Mechanisms involved in quinolone resistance in Mycoplasma mycoides subsp. capri.

Mycoplasma mycoides subsp. capri is a causative agent of contagious agalactia in goats. In this study, M. mycoides subsp. capri mutants were selected for resistance to fluoroquinolones (norfloxacin, enrofloxacin and ciprofloxacin) by serial passes in broth with increasing concentrations of antibiotic. Mutations conferring cross-resistance to the three fluoroquinolones were found in the quinolone resistance determining regions of the four genes encoding DNA gyrase and topoisomerase IV. Different mutations in the DNA gyrase GyrA subunit suggest a different mechanism of inhibition between norfloxacin and the other tested fluoroquinolones. The presence of an adenosine triphosphate-dependent efflux system was suggested through the use of the inhibitor orthovanadate.

Expression of cyclooxygenase-2 in the mammary gland tissue of goats affected with caprine contagious agalactia.

Caprine contagious agalactia is a syndrome most frequently caused by Mycoplasma agalactiae. The pathogenic mechanisms that allow M. agalactiae to persist in the mammary gland tissues following infection, despite a prominent inflammatory response, are yet to be fully established. The aim of the present study was to investigate cyclooxygenase (COX)-2 in the mammary gland of goats during M. agalactiae infection. COX-2 expression was evaluated by immunohistochemistry in the inflammatory lesions of 10 goats affected with M. agalactiae-induced mastitis (five naturally infected and five experimentally infected). Epithelial cells, macrophages, endothelial cells, fibroblasts and, to a lesser extent, neutrophils demonstrated positive immunostaining for COX-2, associated with areas of mastitis and with the presence of M. agalactiae antigen. These research findings suggest that COX-2 is involved in the inflammatory response that occurs in caprine contagious agalactia.