Effect of Sodium Nitrite, Nisin and Lactic Acid on the Prevalence and Antibiotic Resistance Patterns of Listeria monocytogenes Naturally Present in Poultry.

The impact of treating minced chicken meat with sodium nitrite (SN, 100 ppm), nisin (Ni, 10 ppm) and lactic acid (LA, 3000 ppm) on the levels of some microbial groups indicating hygiene quality were investigated. Specifically, aerobic plate counts and culture-based counts of psychrotrophic microorganisms and enterobacteria were obtained. Additionally, the prevalence of Listeria monocytogenes and the resistance of 245 isolates from this bacterium to 15 antibiotics were documented. L. monocytogenes was isolated using the ISO 11290-1:2017 method and confirmed with polymerase chain reaction using the lmo1030 gene. Antibiotic resistance was established using the disc diffusion technique (EUCAST and CLSI criteria). Twenty-four hours after treatment, the microbial load (log10 cfu/g) was reduced (p < 0.05) relative to controls in those samples treated with LA, with counts of 5.51 ± 1.05 (LA-treated samples) vs. 7.53 ± 1.02 (control) for APC, 5.59 ± 1.14 (LA) vs. 7.13 ± 1.07 (control) for psychrotrophic microorganisms and 2.33 ± 0.51 (LA) vs. 4.23 ± 0.88 (control) for enterobacteria. L. monocytogenes was detected in 70% (control samples), 60% (samples receiving SN), 65% (Ni) and 50% (LA) (p > 0.05) of samples. All strains showed resistance to multiple antimicrobials (between 3 and 12). In all, 225 isolates (91.8%) showed a multi-drug resistant (MDR) phenotype, and one isolate (0.4%) showed an extensively drug-resistant (XDR) phenotype. The mean number of resistances per strain was lower (p < 0.01) in the control samples, at 5.77 ± 1.22, than in those receiving treatment, at 6.39 ± 1.51. It is suggested that the use of food additives might increase the prevalence of resistance to antibiotics in L. monocytogenes, although additional studies would be necessary to verify this finding by analyzing a higher number of samples and different foodstuffs and by increasing the number of antimicrobial compounds and concentrations to be tested.

Viability and Virulence of Listeria monocytogenes in Poultry.

The prevalence of Listeria monocytogenes in 30 samples of poultry was determined using culture-dependent (isolation on OCLA and confirmation by conventional polymerase chain reaction -PCR-, OCLA&PCR) and culture-independent (real-time polymerase chain reaction, q-PCR) methods. L. monocytogenes was detected in 15 samples (50.0%) by OCLA&PCR and in 20 (66.7%) by q-PCR. The concentrations (log10 cfu/g) of L. monocytogenes (q-PCR) ranged from 2.40 to 5.22 (total cells) and from <2.15 to 3.93 (viable cells). The two methods, q-PCR using a viability marker (v-PCR) and OCLA&PCR (gold standard), were compared for their capacity to detect viable cells of L. monocytogenes, with the potential to cause human disease. The values for sensitivity, specificity and efficiency of the v-PCR were 100%, 66.7% and 83.3%, respectively. The agreement between the two methods (kappa coefficient) was 0.67. The presence of nine virulence genes (hlyA, actA, inlB, inlA, inlC, inlJ, prfA, plcA and iap) was studied in 45 L. monocytogenes isolates (three from each positive sample) using PCR. All the strains harbored between six and nine virulence genes. Fifteen isolates (33.3% of the total) did not show the potential to form biofilm on a polystyrene surface, as determined by a crystal violet assay. The remaining strains were classified as weak (23 isolates, 51.1% of the total), moderate (one isolate, 2.2%) or strong (six isolates, 13.3%) biofilm producers. The strains were tested for susceptibility to a panel of 15 antibiotics. An average of 5.11 ± 1.30 resistances per isolate was observed. When the values for resistance and for reduced susceptibility were taken jointly, this figure rose to 6.91 ± 1.59. There was a prevalence of resistance or reduced susceptibility of more than 50.0% for oxacillin, cefoxitin, cefotaxime, cefepime ciprofloxacin, enrofloxacin and nitrofurantoin. For the remaining antibiotics tested, the corresponding values ranged from 0.0% for chloramphenicol to 48.9% for rifampicin. The high prevalence and level of L. monocytogenes with numerous virulence factors in poultry underline how crucial it is to follow correct hygiene procedures during the processing of this foodstuff in order to reduce the risk of human listeriosis.

Estimation by flow cytometry of percentages of survival of Listeria monocytogenes cells treated with tetracycline, with or without prior exposure to several biocides.

In certain circumstances, disinfectants are used at sublethal concentrations. The aim of this research work was to determine whether contact of Listeria monocytogenes NCTC 11994 with subinhibitory concentrations of three disinfectants widely used in food processing environments and in the health-care system, benzalkonium chloride (BZK), sodium hypochlorite (SHY) and peracetic acid (PAA), can cause the adaptation of the strain to the biocides and increase its resistance to tetracycline (TE). The minimum inhibitory concentrations (MIC; ppm) were 2.0 (BZK), 3500.0 (SHY) and 1050.0 (PAA). On exposure to increasing subinhibitory concentrations of the biocides, the maximum concentrations (ppm) of the compounds that allowed the strain to grow were (ppm) 8.5 (BZK), 3935.5 (SHY) and 1125.0 (PAA). Both the control cells (non-exposed) and the cells that had been in contact with low doses of biocides were treated with different concentrations of TE (0 ppm, 250 ppm, 500 ppm, 750 ppm, 1000 ppm and 1250 ppm) for 24, 48 and 72 h, and the survival percentages determined using flow cytometry, following dying with SYTO 9 and propidium iodide. The cells previously exposed to PAA presented higher survival percentages (P < 0.05) than the rest of the cells for most of the concentrations of TE and treatment times trialled. These results are worrying because TE is sometimes used to treat listeriosis, highlighting the importance of avoiding the use of disinfectant at subinhibitory doses. Furthermore, the findings suggest that flow cytometry is a fast and simple technique to obtain quantitative data on bacterial resistance to antibiotics.

Quantification of Total and Viable Cells and Determination of Serogroups and Antibiotic Resistance Patterns of Listeria monocytogenes in Chicken Meat from the North-Western Iberian Peninsula.

Twenty samples of minced chicken meat procured from butcher's shops in León (Spain; 10 samples) and Vila Real (Portugal; 10 samples) were analyzed. Microbial concentrations (log10 cfu/g) of 7.53 ± 1.02 (viable aerobic microbiota), 7.13 ± 1.07 (psychrotrophic microorganisms), and 4.23 ± 0.88 (enterobacteria) were found. The detection method described in the UNE-EN ISO 11290-1 standard (based on isolation from the chromogenic medium OCLA) with confirmation by the polymerase chain reaction (PCR; lmo1030) (OCLA−PCR), revealed Listeria monocytogenes in 14 samples (70.0% of the total), nine of Spanish origin and five of Portuguese (p > 0.05). The levels of viable and inactivated L. monocytogenes in the samples were determined with a q-PCR using propidium monoazide (PMAxx) as a viability marker. Seven samples tested positive both with the OCLA−PCR and with the q-PCR, with estimated concentrations of viable cells varying between 2.15 log10 cfu/g (detection limit) and 2.94 log10 cfu/g. Three samples tested negative both with the OCLA−PCR and with the q-PCR. Seven samples were positive with the OCLA−PCR, but negative with the q-PCR, and three samples tested negative with the OCLA−PCR and positive with the q-PCR. The percentage of viable cells relative to the total ranged between 2.4% and 86.0%. Seventy isolates of L. monocytogenes (five from each positive sample) were classified in PCR serogroups with a multiplex PCR assay. L. monocytogenes isolates belonged to serogroups IIa (52 isolates; 74.3%), IIc (7; 10.0%), IVa (2; 2.9%), and IVb (9; 12.9%). The susceptibility of the 70 isolates to 15 antibiotics of clinical interest was tested. The strains presented resistance to between three and eight antibiotics. The average number of resistances was greater (p < 0.001) among strains isolated from Spanish samples (6.20 ± 1.08), than in those from Portugal (5.00 ± 1.08). In both groups of strains, a prevalence of resistance higher than 95% was observed for oxacillin, cefoxitin, cefotaxime, and cefepime. The need to handle minced chicken meat correctly, taking care to cook it sufficiently and to avoid cross-contamination, so as to reduce the danger of listeriosis, is emphasized. A combination of culture-dependent and culture-independent methods offers complementary routes for the detection in food of the cells of L. monocytogenes in various different physiological states.

Exploring the Biofilm Formation Capacity in S. pseudintermedius and Coagulase-Negative Staphylococci Species.

The ability of biofilm formation seems to play an important role in the virulence of staphylococci. However, studies reporting biofilm formation of coagulase-negative staphylococci isolated from animals are still very scarce. Thus, we aimed to evaluate the biofilm-forming capacity of CoNS and S. pseudintermedius isolated from several animal species and to investigate the effect of conventional antimicrobials on biofilm reduction. A total of 35 S. pseudintermedius and 192 CoNS were included. Biofilm formation was accessed by the microtiter plate assay and the biofilms were stained by crystal violet. Association between biofilm formation and staphylococci species and antimicrobial resistance was also performed. Biofilm susceptibility testing was performed with tetracycline and amikacin at the minimum inhibitory concentration (MIC) and 10 × MIC. The metabolic activity of the biofilm cells after antimicrobial treatment was accessed by the XTT assay. All isolates formed biofilm, with S. urealyticus producing the most biofilm biomass and S. pseudintermedius producing the least biomass. There was a positive association between biofilm formation and multidrug resistance as well as resistance to individual antimicrobials. Neither tetracycline nor amikacin were able to eradicate the biofilm, not even at the highest concentration used. This study provides new insights into biofilm formation and the effects of antimicrobials on CoNS species.

Biofilm Formation of Staphylococcus aureus from Pets, Livestock, and Wild Animals: Relationship with Clonal Lineages and Antimicrobial Resistance.

This study aimed to compare the biofilm formation ability of Staphylococcus aureus isolated from a wide range of animals and study the association between biofilm formation and antimicrobial resistance and genetic lineages. A total of 214 S. aureus strains isolated from pets, livestock, and wild animals were evaluated regarding their ability to form biofilms by the microtiter biofilm assay and their structure via confocal scanning laser microscopy. Statistical analysis was used to find an association between biofilm formation and antimicrobial resistance, multidrug resistance, sequence types (STs), spa and agr-types of the isolates. The antimicrobial susceptibility of 24 h-old biofilms was assessed against minimum inhibitory concentrations (MIC) and 10× MIC of amikacin and tetracycline, and the biomass reduction was measured. The metabolic activity of biofilms after antimicrobial treatment was evaluated by the XTT assay. All isolates were had the ability to form biofilms. Yet, significant differences in biofilm biomass production were detected among animal species. Multidrug resistance had a positive association with biofilm formation as well as methicillin-resistance. Significant differences were also detected among the clonal lineages of the isolates. Both tetracycline and amikacin were able to significantly reduce the biofilm mass. However, none of the antimicrobials were able to eradicate the biofilm at the maximum concentration used. Our results provide important information on the biofilm-forming capacity of animal-adapted S. aureus isolates, which may have potential implications for the development of new biofilm-targeted therapeutics.

Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa.

In recent years, the effectiveness of antimicrobials in the treatment of Pseudomonas aeruginosa infections has gradually decreased. This pathogen can be observed in several clinical cases, such as pneumonia, urinary tract infections, sepsis, in immunocompromised hosts, such as neutropenic cancer, burns, and AIDS patients. Furthermore, Pseudomonas aeruginosa causes diseases in both livestock and pets. The highly flexible and versatile genome of P. aeruginosa allows it to have a high rate of pathogenicity. The numerous secreted virulence factors, resulting from its numerous secretion systems, the multi-resistance to different classes of antibiotics, and the ability to produce biofilms are pathogenicity factors that cause numerous problems in the fight against P. aeruginosa infections and that must be better understood for an effective treatment. Infections by P. aeruginosa represent, therefore, a major health problem and, as resistance genes can be disseminated between the microbiotas associated with humans, animals, and the environment, this issue needs be addressed on the basis of an One Health approach. This review intends to bring together and describe in detail the molecular and metabolic pathways in P. aeruginosa's pathogenesis, to contribute for the development of a more targeted therapy against this pathogen.

Characterization of ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical Samples in a Northern Portuguese Hospital: Predominance of CTX-M-15 and High Genetic Diversity.

BACKGROUND: Enterobacteriaceae are major players in the spread of resistance to ß-lactam antibiotics through the action of CTX-M ß-lactamases. We aimed to analyze the diversity and genetic characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolates from patients in a Northern Portuguese hospital. METHODS: A total of 62 cefotaxime/ceftazidime-resistant E. coli (n = 38) and K. pneumoniae (n = 24) clinical isolates were studied. Identification was performed by MALDI-TOF MS. Antimicrobial susceptibility testing against 13 antibiotics was performed. Detection of ESBL-encoding genes and other resistance genes, phylogenetic grouping, and molecular typing (for selected isolates) was carried out by PCR/sequencing. RESULTS: ESBL activity was detected in all 62 E. coli and K. pneumoniae isolates. Most of the ESBL-producing E. coli isolates carried a blaCTX-M gene (37/38 isolates), being blaCTX-M-15 predominant (n = 32), although blaCTX-M-27 (n = 1) and blaCTX-M-1 (n = 1) were also detected. Two E. coli isolates carried the blaKPC2/3 gene. The lineages ST131-B2 and ST410-A were detected among the ESBL-producing blood E. coli isolates. Regarding the 24 ESBL-producing K. pneumoniae isolates, 18 carried a blaCTX-M gene (blaCTX-M-15, 16 isolates; blaCTX-M-55, 2 isolates). All K. pneumoniae isolates carried blaSHV genes, including ESBL-variants (blaSHV-12 and blaSHV-27, 14 isolates) or non-ESBL-variants (blaSHV-11 and blaSHV-28, 10 isolates); ten K. pneumoniae isolates also carried the blaKPC2/3 gene and showed imipenem-resistance. ESBL-positive E. coli isolates were ascribed to the B2 phylogenetic group (82%), mostly associated with ST131 lineage and, at a lower rate, to ST410/A. Regarding K. pneumoniae, the three international lineages ST15, ST147, and ST280 were detected among selected isolates. CONCLUSIONS: Different ESBL variants of CTX-M (especially CTX-M-15) and SHV-type (specially SHV-12) were detected among CTX/CAZRE. coli and K. pneumoniae isolates, in occasions associated with carbapenemase genes (blaKPC2/3 gene).

Antimicrobial Resistance Genes and Diversity of Clones among Faecal ESBL-Producing Escherichia coli Isolated from Healthy and Sick Dogs Living in Portugal.

The purpose of this study was to analyse the prevalence and genetic characteristics of ESBL and acquired-AmpC (qAmpC)-producing Escherichia coli isolates from healthy and sick dogs in Portugal. Three hundred and sixty-one faecal samples from sick and healthy dogs were seeded on MacConkey agar supplemented with cefotaxime (2 µg/mL) for cefotaxime-resistant (CTXR) E. coli recovery. Antimicrobial susceptibility testing for 15 antibiotics was performed and the ESBL-phenotype of the E. coli isolates was screened. Detection of antimicrobial resistance and virulence genes, and molecular typing of the isolates (phylogroups, multilocus-sequence-typing, and specific-ST131) were performed by PCR (and sequencing when required). CTXRE. coli isolates were obtained in 51/361 faecal samples analysed (14.1%), originating from 36/234 sick dogs and 15/127 healthy dogs. Forty-seven ESBL-producing E. coli isolates were recovered from 32 sick (13.7%) and 15 healthy animals (11.8%). Different variants of blaCTX-M genes were detected among 45/47 ESBL-producers: blaCTX-M-15 (n = 26), blaCTX-M-1 (n = 10), blaCTX-M-32 (n = 3), blaCTX-M-55 (n = 3), blaCTX-M-14 (n = 2), and blaCTX-M-variant (n = 1); one ESBL-positive isolate co-produced CTX-M-15 and CMY-2 enzymes. Moreover, two additional CTXR ESBL-negative E. coli isolates were CMY-2-producers (qAmpC). Ten different sequence types were identified (ST/phylogenetic-group/ß-lactamase): ST131/B2/CTX-M-15, ST617/A/CTX-M-55, ST3078/B1/CTX-M-32, ST542/A/CTX-M-14, ST57/D/CTX-M-1, ST12/B2/CTX-M-15, ST6448/B1/CTX-M-15 + CMY-2, ST5766/A/CTX-M-32, ST115/D/CMY-2 and a new-ST/D/CMY-2. Five variants of CTX-M enzymes (CTX-M-15 and CTX-M-1 predominant) and eight different clonal complexes were detected from canine ESBL-producing E. coli isolates. Although at a lower rate, CMY-2 ß-lactamase was also found. Dogs remain frequent carriers of ESBL and/or qAmpC-producing E. coli with a potential zoonotic role.

Multidrug-resistant Klebsiella pneumoniae harboring extended spectrum ß-lactamase encoding genes isolated from human septicemias.

Klebsiella pneumoniae is a major pathogen implicated in nosocomial infections. Extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae isolates are a public health concern. We aim to characterize the type of ß-lactamases and the associated resistance mechanisms in ESBL-producing K. pneumoniae isolates obtained from blood cultures in a Portuguese hospital, as well as to determine the circulating clones. Twenty-two cefotaxime/ceftazidime-resistant (CTX/CAZR) K. pneumoniae isolates were included in the study. Identification was performed by MALDI-TOF MS and the antimicrobial susceptibility testing by disk-diffusion. The screening test for ESBL-production was performed and ESBL-producer isolates were further characterized. The presence of different beta-lactamase genes (blaCTX-M, blaSHV, blaTEM, blaKPC, blaNDM, blaVIM, blaOXA-48, blaCMY-2, blaDHA-1, blaFOX, blaMOX, and blaACC) was analyzed by PCR/sequencing in ESBL-producer isolates, as well as the presence of other resistance genes (aac(6')-Ib-cr, tetA/B, dfrA, qnrA/B/S, sul1/2/3) or integron-related genes (int1/2/3). Multilocus-sequence-typing (MLST) was performed for selected isolates. ESBL activity was detected in 12 of the 22 CTX/CAZR K. pneumoniae isolates and 11 of them carried the blaCTX-M-15 gene (together with blaTEM), and the remaining isolate carried the blaSHV-106 gene. All the blaCTX-M-15 harboring isolates also contained a blaSHV gene (blaSHV-1, blaSHV-11 or blaSHV-27 variants). Both blaSHV-27 and blaSHV-106 genes correspond to ESBL-variants. Two of the CTX-M-15 producing isolates carried a carbapenemase gene (blaKPC2/3 and blaOXA-48) and showed imipenem resistance. The majority of the ESBL-producing isolates carried the int1 gene, as well as sulphonamide-resistance genes (sul2 and/or sul3); the tetA gene was detected in all eight tetracycline-resistant isolates. Three different genetic lineages were found in selected isolates: ST348 (one CTX-M-15/TEM/SHV-27/KPC-2/3-producer isolate), ST11 (two CTX-M-15/TEM/SHV-1- and CTX-M-15-TEM-SHV-11-OXA-48-producer isolates) and ST15 (one SHV-106/TEM-producer isolate). ESBL enzymes of CTX-M-15 or SHV-type are detected among blood K. pneumoniae isolates, in some cases in association with carbapenemases of KPC or OXA-48 type.

Topical Application of Ozonated Oils for the Treatment of MRSA Skin Infection in an Animal Model of Infected Ulcer.

Diabetic foot ulcers are a common cause of morbidity in diabetic patients. One of the main pathogens found in these ulcers is methicillin-resistant Staphylococcus aureus (MRSA). MRSA often carries resistance to several classes of antibiotics and their infections are becoming harder to treat. Therefore, new alternatives are urgently needed. Thus, this study aimed to investigate the capacity of topical ozonated oil application on the treatment of early-stage skin infected with MRSA in an animal model. Ozonated oil was prepared from a mixture of oils subjected to a gas stream of O2/O3 mixture. Sixteen Wistar rats were inoculated by an intradermic injection of MRSA suspension, producing an abscess lesion. After 3 days, the skin epidermis was removed to open the wound. Group 1 received an application of oil mixture without ozone treatment and Group 2 received an application of ozonated oil. After the treatment period, skin was collected, colony-forming units (CFU) of bacteria were quantified and the histological analysis of the skin was carried out. Skin samples from the control 1 and 2 had a bacterial load was of 1.1 × 105 and 5.7 × 103 CFU/mL, respectively. Group 2 showed better wound healing from mild to moderate epidermal regeneration. Topical application of ozonated vegetable oil in MRSA-infected skin in rats showed a small reduction of the bacterial load and better wound healing.

Survey of the Knowledge and Use of Antibiotics among Medical and Veterinary Health Professionals and Students in Portugal.

Antimicrobial resistance (AMR) is an urgent and complex problem worldwide, exacerbated by the frequently inappropriate use of antibiotics. The purpose of this study was to survey the levels of knowledge and awareness about antibiotic use and stewardship, among human and veterinary health professionals or students in Portugal, and the associations between antibiotic knowledge factors and socio-professional groups. In cross-sectional survey design, a total of 449 online structured questionnaires were completed in 2018-2019. The statistical analysis was performed dividing the respondents into four groups, A (undergraduate students), B (PhD students and researchers), C (lecturers), and D (technicians and other occupation). Among all respondents, 17% (n = 75) revealed some gap in knowledge about antibiotic resistance and the antibiotics that should be administered for different infection types (bacterial, viral, or fungal). Of the 159 pet owners among the respondents, only half had administered antibiotics to their animal and 64% (n = 102) knew that veterinary prescription is mandatory when administering antibiotics to animals. All groups statistically agreed that the AMR is a major public health problem and the antibiotics should be administrated for bacterial infections and used until the whole pack has been finished (p = 0.00). As expected, only groups B and C demonstrated a higher level of knowledge to recognize the antibiotic name and their active ingredient than undergraduate students (p = 0.00). About the antibiotic use on pets, only group B was statistically significant to no used antibiotics on their pets (p = 0.00). However, groups A, C, and D were statistically significant for the knowledge about the mandatory veterinarian prescription and groups C and D were significantly statistics for fully aware of the transmission of bacteria between animals and humans. In conclusion, in matters related to AMR, the behavior, education, and training of the general public and health professionals, including those who prescribe antibiotics for humans and animals, need to be improved.

Clonal Diversity and Antimicrobial Resistance of Methicillin-Resistant Staphylococcus pseudintermedius Isolated from Canine Pyoderma.

The emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) antimicrobial resistance and epidemic genetic lineages is posing a challenge in veterinary medicine due to the limited therapeutical options. MRSP has been identified as an important canine pyoderma pathogen. Thus, we aimed to characterize the antimicrobial resistance and clonal lineages of MRSP isolated from canine cutaneous pyoderma. Thirty-one MRSP isolates recovered from pyoderma were further characterized. The antimicrobial susceptibility testing of the isolates was performed by the Kirby-Bauer disc diffusion method against 14 antimicrobial agents. The presence of antimicrobial and virulence genes was carried out by PCR. Multilocus sequence typing was performed in all isolates. All strains had a multidrug-resistant profile showing resistance mainly to penicillin, macrolides and lincosamides, aminoglycosides, tetracycline and trimethoprim-sulfamethoxazole, which was encoded by the blaZ, ermB, msr(A/B), aac(6')-Ie-aph(2'')-Ia, aph(3')-IIIa, ant(4')-Ia, tetM, tetK and dfrG genes. All isolates harbored the lukS-I/lukF-I virulence factors. Isolates were ascribed to nine previously described sequence types (STs): ST123, ST339, ST727, ST71, ST537, ST45, ST1029, ST118 and ST1468; and to five STs first described in this study: ST2024, ST2025, ST2026, ST2027 and ST2028. In this study, most isolates belonged to ST123 (n = 16), which belongs to CC71 and is the most common clone in Europe. All isolates were multidrug-resistant, which may impose a serious threat to animal health.

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) for Twelve Antimicrobials (Biocides and Antibiotics) in Eight Strains of Listeria monocytogenes.

When selecting effective doses of antimicrobials, be they biocides or antibiotics, it is essential to know the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these substances. The present research determined the MICs and MBCs for three biocides, sodium hypochlorite (SH), benzalkonium chloride (BC), and peracetic acid (PAA), and nine antibiotics in eight strains of Listeria monocytogenes of varying serotypes. Marked intra-species differences were observed in the resistance of L. monocytogenes to the biocides and antibiotics. The MICs (ppm) for the biocides ranged between 1750 and 4500 for SH, 0.25 and 20.00 for BC, and 1050 and 1700 for PAA. Their MBCs (ppm) ranged from 2250 to 4500 for SH, 0.50 to 20.00 for BC, and 1150 to 1800 for PAA. The MICs (ppm) for antibiotics lay between 1 and 15 for ampicillin, 8 and 150 for cephalothin, 20 and 170 for cefoxitin, 0.05 and 0.20 for erythromycin, 4 and 50 for chloramphenicol, 3 and 100 for gentamicin, 2 and 15 for tetracycline, 2 and 80 for vancomycin, and 160 and 430 for fosfomycin. The corresponding MBCs (ppm) were from 5 to 20 for ampicillin, 9 to 160 for cephalothin, 70 to 200 for cefoxitin, 4 to 5 for erythromycin, 9 to 70 for chloramphenicol, 5 to 100 for gentamicin, 3 to 30 for tetracycline, 3 to 90 for vancomycin, and 160 to 450 for fosfomycin. Notably, erythromycin showed considerable efficacy, demonstrated by the low values for both MIC and MBC. Based on EUCAST and the CLSI criteria, all strains were susceptible to erythromycin. All strains were resistant to cephalothin, cefoxitin, gentamicin, and fosfomycin. Further values for resistance were 87.50% for ampicillin and vancomycin, 75.00% for tetracycline, and 62.50% for chloramphenicol. The high prevalence of antibiotic resistance is a matter for concern. A positive correlation was found between MIC and MBC values for most of the biocides and antibiotics. The higher the hydrophobicity of the cell surface, the higher the susceptibility to biocides, suggesting that surface characteristics of bacterial cells influence resistance to these compounds.

Biovolume and spatial distribution of foodborne Gram-negative and Gram-positive pathogenic bacteria in mono- and dual-species biofilms.

The objective of this study was to characterize the biofilms formed by Salmonella enterica serotype Agona, Listeria monocytogenes, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) after 12, 48, 72, 120 and 240 h of incubation at 10 °C. Biofilms containing a single species, together with dual-species biofilms in which S. enterica and a Gram-positive bacterium existed in combination, were formed on polystyrene and evaluated by using confocal laser scanning microscopy (CLSM). All strains were able to form biofilm. The greatest biovolume in the observation field of 14,161 µm2 was observed for mono-species biofilms after 72 h, where biovolumes of 94,409.0 µm3 ± 2131.0 µm3 (S. enterica), 58,418.3 µm3 ± 5944.9 µm3 (L. monocytogenes), 68,020.8 µm3 ± 5812.3 µm3 (MRSA) and 59,280.0 µm3 ± 4032.9 µm3 (VRE) were obtained. In comparison with single-species biofilms, the biovolume of S. enterica was higher in the presence of MRSA or VRE after 48, 72 and 120 h. In dual-species biofilms, the bacteria showed a double-layer distribution pattern, with S. enterica in the top layer and Gram-positive bacteria in the bottom layer. This spatial disposition should be taken into account when effective strategies to eliminate biofilms are being developed.

Antibiotic Resistance and Biofilm-Forming Ability in Enterococcal Isolates from Red Meat and Poultry Preparations.

This study investigated the resistance to antibiotics and the capacity to form a biofilm of 200 isolates of enterococci isolated from raw preparations of beef (51 strains), pork (47), chicken (50), and turkey (52) acquired in north-western Spain. Fifteen antimicrobials of clinical importance were tested by the disc diffusion method. The average number of resistances per strain was 4.48 ± 1.59. If resistant strains were taken together with those showing reduced susceptibility, the total number of resistances per strain was 6.97 ± 2.02. Two isolates (1.0% of strains) were resistant to a single antibiotic, twenty-two isolates (11.0%) presented resistance to two, one strain (0.5%) was resistant to three, and 175 isolates (87.5%) showed a multiple drug-resistant phenotype (MDR; defined as no susceptibility to at least one agent from each of three or more antimicrobial categories). The prevalence of resistance varied between 0.5% (gentamicin) and 100% (kanamycin). All strains produced biofilm on polystyrene microwell plates, determined using crystal violet assay. Isolates were classified as having a weak (51 strains; average optical density at 580 nanometers -OD580- = 0.206 ± 0.033), moderate (78 strains; average OD580 = 0.374 ± 0.068), or strong (71 strains; average OD580 = 1.167 ± 0.621) ability to produce biofilm (p < 0.05). Isolates from beef preparations produced the most substantial (p < 0.05) biofilms. The results of this study indicate that meat and poultry preparations are major reservoirs of antibiotic-resistant enterococcal strains capable of forming a biofilm. In order for food-borne infections to be prevented, the importance of careful handling of these foodstuffs during preparation, avoiding cross-contamination, and ensuring thorough cooking, is stressed.

High Efficacy of Ozonated Oils on the Removal of Biofilms Produced by Methicillin-Resistant Staphylococcus aureus (MRSA) from Infected Diabetic Foot Ulcers.

Ozone has a high wound healing capacity and antibacterial properties and can be used as a complementary treatment in infections. Methicillin-resistant S. aureus (MRSA) is the most common pathogen found in infected diabetic foot ulcers. Most of MRSA are resistant to several classes of antibiotics and, therefore, there is a need for new, effective, and well-tolerated agents. Thus, we aimed evaluate the antimicrobial and antibiofilm potentials of ozonated vegetable oils against MRSA strains isolated from diabetic foot ulcers. Six ozonated oils were produced with concentrations of ozone ranging from 0.53 to 17 mg of ozone/g of oil. The peroxide values were determined for each oil. Ozonated oils content on fatty acid was determined by gas chromatography equipped with a flame ionization detector. The antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method and the effect of ozonated oils on biofilm formation ability and on established biofilms was investigated. In general, the content in identified unsaturated fatty acid in oils decreased with the increase of ozonation time and, consequently, the peroxide value increased. Most bacterial strains were inhibited by ozonated oil at a concentration of 4.24 mg/g. Ozonated oils had moderate to high ability to remove adhered cells and showed a high capacity to eradicate 24 h old biofilms. Our results show promising use of ozonated oils on the treatment of infections, in particular those caused by multidrug-resistant MRSA strains.

Implications of antibiotics use during the COVID-19 pandemic: present and future.

COVID-19 is caused by the SARS-CoV-2 virus, which has infected more than 4 million people with 278 892 deaths worldwide as of 11 May 2020. This disease, which can manifest as a severe respiratory infection, has been declared as a public health emergency of international concern and is being treated with a variety of antivirals, antibiotics and antifungals. This article highlights the administration of antimicrobials in COVID-19 patients worldwide, during the 2019-20 pandemic. It is imperative to be aware of the unreported amounts of antibiotics that have been administered worldwide in just a few months and a marked increase in antimicrobial resistance should therefore be expected. Due to the lack of data about antimicrobial use during this pandemic, the global impact on the emergence of new antimicrobial resistance is as yet unknown. This issue must be at the forefront of public health policymaking and planning in order that we are prepared for the potentially severe consequences for human and animal health and the environment.

Diversity, Antibiotic Resistance, and Biofilm-Forming Ability of Enterobacteria Isolated from Red Meat and Poultry Preparations.

A total of 44 samples of beef, pork, and poultry preparations were tested. Average counts (log cfu/g) of enterobacteria were 1.99 ± 0.99 (beef preparations), 1.96 ± 1.44 (pork), 2.09 ± 0.92 (chicken), and 2.17 ± 1.06 (turkey) (p > 0.05). Two hundred enterobacterial strains were identified and 13 genera (21 species) were distinguished, including species that are a significant cause of infection. The most common genera were Escherichia (32.5% of strains), Serratia (17.0%), Hafnia (12.5%), and Salmonella (12.0%). Isolates were screened by disc diffusion for susceptibility to 15 antibiotics. A total of 126 strains (63% of the isolates) were multirresistant (having resistance to two or more antibiotics), 46 (23%) were resistant to one antibiotic, and 28 (14%) were sensitive to all antibiotics. The average number of resistances per strain was 2.53 ± 2.05. A higher (p < 0.05) average number of resistances was observed in strains from turkey (3.14 ± 2.55) than in strains from beef (2.15 ± 1.22), pork (2.16 ± 1.39), or chicken (2.44 ± 2.22). At least 50% of strains showed resistance or reduced susceptibility to ampicillin, cefotaxime, ceftazidime, or streptomycin, considered to be "critically important" antimicrobial agents in human medicine. Seventy-nine strains (39.5%), 60 strains (30.0%), and 46 strains (23.0%) were weak, moderate, and strong biofilm producers (crystal violet assay), respectively. This investigation provides evidence that bacteria from red meat and poultry preparations pose major potential risk to consumers.

Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Human Bloodstream Infections: Detection of MLSB Resistance.

In this study we aimed to characterize antimicrobial resistance in methicillin-resistant Staphylococcus aureus (MRSA) isolated from bloodstream infections as well as the associated genetic lineages of the isolates. Sixteen MRSA isolates were recovered from bacteremia samples from inpatients between 2016 and 2019. The antimicrobial susceptibility of these isolates was tested by the Kirby-Bauer disk diffusion method against 14 antimicrobial agents. To determine the macrolide-lincosamide-streptogramin B (MLSB) resistance phenotype of the isolates, erythromycin-resistant isolates were assessed by double-disk diffusion (D-test). The resistance and virulence genes were screened by polymerase chain reaction (PCR). All isolates were characterized by multilocus sequence typing (MLST), spa typing, staphylococcal chromosomal cassette mec (SCCmec) typing, and accessory gene regulator (agr) typing. Isolates showed resistance to cefoxitin, penicillin, ciprofloxacin, erythromycin, fusidic acid, clindamycin, and aminoglycosides, confirmed by the presence of the blaZ, ermA, ermC, mphC, msrA/B, aac(6')-Ie-aph(2'')-Ia, and ant(4')-Ia genes. Three isolates were Panton-Valentine-leukocidin-positive. Most strains (n = 12) presented an inducible MLSB phenotype. The isolates were ascribed to eight spa-types (t747, t002, t020, t1084, t008, t10682, t18526, and t1370) and four MLSTs (ST22, ST5, ST105, and ST8). Overall, most (n = 12) MRSA isolates had a multidrug-resistance profile with inducible MLSB phenotypes and belonged to epidemic MRSA clones.