Preliminary snapshot reveals a relationship between multidrug-resistance and biofilm production among enterobacteriaceae isolated from fecal samples of farm-raised poultry in ceará, Brazil.

Antimicrobial resistance and biofilm formation by microbial pathogens pose a significant challenge to poultry production systems due to the persistent risk of dissemination and compromise of bird health and productivity. In this context, the study aimed to investigate the occurrence of different multiresistance phenotypes and the biofilm-forming ability of Enterobacteriaceae isolated from broiler chicken excreta in poultry production units in Ceará, Brazil. Samples were collected from three distinct broiler breeding facilities and subjected to isolation, identification, antibiotic susceptibility testing, phenotypic screening for ß-lactamases enzymes, and biofilm formation evaluation. Seventy-one strains were identified, being Escherichia coli (37 %) and Proteus mirabilis (32 %), followed by Klebsiella pneumoniae (11 %), Providencia stuartii (9 %), Klebsiella aerogenes (6 %), Alcaligenes faecalis (4 %), and Salmonella sp. (1 %). A significant proportion (87 %) of multiresistant strains were detected. For the phenotypic evaluation of ß-lactamases production, strains with resistance to second and third-generation cephalosporins and carbapenems were tested. About 4 of 6 and 10 of 26 were positive for inducible chromosomal AmpC ß-lactamase and extended-spectrum ß-lactamase (ESBL), respectively. Regarding biofilm formation, it was observed that all MDR strains were capable of forming biofilm. In this sense the potential of these MDR bacteria to develop biofilms becomes a significant concern, representing a real threat to both human and animal health, as biofilms offer stability, antimicrobial protection, and facilitate genetic transfer.

Combinatorial effects between aromatic plant compounds and chlorhexidine digluconate against canine otitis-related Staphylococcus spp.

The increasing prevalence of antimicrobial resistance among bacterial pathogens necessitates novel treatment strategies, particularly in veterinary medicine where otitis in dogs is very common in small animals' clinical routines. Considering this challenge, this study explores the efficacy of aromatic plant compounds (APC), including eugenol (EUG), trans-cinnamaldehyde (TC), and geraniol (GER), and their synergistic potential when combined with the antiseptic agent chlorhexidine (CLX), offering insight into alternative therapeutic approaches. The disk diffusion assay revealed differential sensitivity of Staphylococcus spp. strains to the tested compounds, with EUG and GER showing moderate inhibition zones and TC displaying considerably larger inhibition zones. Further analysis through MIC and MBC determinations suggested that EUG required the highest concentrations to inhibit and kill the bacteria, whereas TC and GER were effective at lower concentrations. Combined with CLX, all three plant-derived compounds demonstrated a significant enhancement of antibacterial activity, indicated by reduced MIC values and a predominantly synergistic interaction across the strains tested. GER was the most potent in combination with CLX, presenting the lowest mean FICi values and the highest fold reductions in MIC. This study emphasizes the APC's potential as an adjunct to conventional antimicrobial agents like CLX. The marked synergy observed, especially with GER, suggests that such combinations could be promising alternatives in managing bacterial otitis in dogs, potentially mitigating the impact of antibiotic resistance.

Antibacterial effectiveness of trans-cinnamaldehyde against foodborne Enterobacteriaceae and its adjuvant effect with gentamicin.

The Enterobacteriaceae family is recognized as a primary group of Gram-negative pathogens responsible for foodborne illnesses and is frequently associated with antibiotic resistance. The present study explores the natural-based compound trans-cinnamaldehyde (TC) against drug-resistant Enterobacteriaceae and its synergism with gentamicin (GEN) to address this issue. The research employs three strains of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae, previously isolated from shrimp. The antibacterial activity was evaluated by the disk diffusion method, microdilution test, kinetics of growth, and time-kill curve. In addition, the synergistic effect between TC/GEN was investigated by checkerboard assay. All strains showed sensitivity to TC with an inhibition zone diameter > 35 mm. The TC showed inhibitory and bactericidal action in the most tested bacteria around 625 µg/mL. Sub-inhibitory amounts (1/2 and 1/4 MIC) of TC interfered with the growth kinetics by lag phase extension and decreased the log phase. Time-kill curves show a reduction of viable cells after the first hour of TC treatment at bactericidal concentrations. The synergistic effect between TC/GEN was observed for E. coli and E. cloacae strains with FICi ranging from 0.15 to 0.50. These findings, therefore, suggest TC as a promising alternative in the fight against drug-resistant Enterobacteriaceae that can cause foodborne illnesses.

Effect of Lactobacillus spp. cell-free supernatant against planktonic growth and biofilm formation of foodborne Escherichia coli isolates.

The aim of this work was to evaluate the anti-Escherichia coli effect of cell-free supernatant (CFS) of Lactobacillus spp. against planktonic and biofilm forms of foodborne isolates. Escherichiacoli strains (P12, P25, P35 and P36), previously isolated from fresh filets of fish, were subjected to antimicrobial susceptibility determination by the disc-diffusion agar method. Subsequently, the antagonistic effect between probiotic and pathogenic strains was determined by spot overlay assay. Finally, the CFS activity against pre-established (12 h) biofilms was demonstrated through biomass quantification by crystal violet staining and scanning electron microscopy (SEM). All isolates presented some pattern of resistance, primarily to ampicillin and tetracycline. Probiotic strains presented high antagonistic effects against all E. coli strains, presenting inhibition zones (R) ranging from 15.60 to 20.67 mm. Additionally, the residual biomass of pre-established (12 h) biofilm was drastically reduced about 50% after CFS treatment (P < 0.01). What can be noted by SEM images, which show less surface-attached cells of CFS-treated biofilms of E. coli (P12). Thus, cell-free preparations produced from Lactobacillus spp. may represent a tool in the battle against planktonic cells and biofilm forms of antibiotic-resistant E. coli.

Chemical Composition and Antimicrobial Effectiveness of Ocimum gratissimum L. Essential Oil Against Multidrug-Resistant Isolates of Staphylococcus aureus and Escherichia coli.

The study investigated the antimicrobial activity of the essential oil extract of Ocimum gratissimum L. (EOOG) against multiresistant microorganisms in planktonic and biofilm form. Hydrodistillation was used to obtain the EOOG, and the analysis of chemical composition was done by gas chromatography coupled with mass spectrometry (GC/MS) and flame ionization detection (GC/FID). EOOG biological activity was verified against isolates of Staphylococcus aureus and Escherichia coli, using four strains for each species. The antibacterial action of EOOG was determined by disk diffusion, microdilution (MIC/MBC), growth curve under sub-MIC exposure, and the combinatorial activity with ciprofloxacin (CIP) and oxacillin (OXA) were determined by checkerboard assay. The EOOG antibiofilm action was performed against the established biofilm and analyzed by crystal violet, colony-forming unit count, and SEM analyses. EOOG yielded 1.66% w/w, with eugenol as the major component (74.83%). The MIC was 1000 µg/mL for the most tested strains. The growth curve showed a lag phase delay for both species, mainly S. aureus, and reduced the growth level of E. coli by half. The combination of EOOG with OXA and CIP led to an additive action for S. aureus. A significant reduction in biofilm biomass and cell viability was verified for S. aureus and E. coli. In conclusion, EOOG has relevant potential as a natural alternative to treat infections caused by multiresistant strains.

In vitro resistance of Enterobacter cloacae isolated from fresh seafood to colistin.

INTRODUCTION: Enterobacter cloacae is a clinically important bacterium from the Enterobacteriaceae family. This study evaluated resistance of E. cloacae strains from fish (n=14) and shrimp (n=9) to colistin. METHODS: Biochemical identification and antimicrobial susceptibility tests were carried out in an automated Vitek®2 instrument. RESULTS: Colistin resistance was observed in 21.4% and 66.7% of the strains from fish and shrimp, respectively. We observed minimum inhibitory concentrations of ≥16 mg/L and ≤5 mg/L in 8 and 15 of all strains, respectively. CONCLUSIONS: Fish and shrimp can carry drug-resistant enterobacteria, which can be of clinical interest.

In vitro resistance of Enterobacter cloacae isolated from fresh seafood to colistin

Abstract INTRODUCTION: Enterobacter cloacae is a clinically important bacterium from the Enterobacteriaceae family. This study evaluated resistance of E. cloacae strains from fish (n=14) and shrimp (n=9) to colistin. METHODS: Biochemical identification and antimicrobial susceptibility tests were carried out in an automated Vitek®2 instrument. RESULTS: Colistin resistance was observed in 21.4% and 66.7% of the strains from fish and shrimp, respectively. We observed minimum inhibitory concentrations of ≥16 mg/L and ≤5 mg/L in 8 and 15 of all strains, respectively. CONCLUSIONS: Fish and shrimp can carry drug-resistant enterobacteria, which can be of clinical interest.

Drug resistance, AmpC-ß-lactamase and extended-spectrum ß-lactamase-producing Enterobacteriaceae isolated from fish and shrimp.

The present study aims to detect the production of extended-spectrum beta-lactamases (ESBL) by enterobacteria isolated from samples of fresh shrimp and fish obtained from the retail trade of the city of Sobral, Ceará State, Brazil. All bacterial isolates were submitted to identification and antimicrobial susceptibility testing using aminopenicillin, beta-lactamase inhibitors, carbapenem, 1st, 2nd, 3rd and 4th generation cephalosporins, and monobactam. Three types of beta-lactamases - ESBL, AmpC and KPC - were investigated. 103 strains were identified, and the most frequent species in shrimp and fish samples was Enterobacter cloacae (n = 54). All the strains were resistant to penicillin and more than 50% of the isolates were resistant to ampicillin and cephalothin. Resistance to three 3rd generation cephalosporins (cefotaxime, ceftriaxone and ceftazidime) and one fourth generation cephalosporin (cefepime) was detected in two isolates of E. cloacae from shrimp samples. Phenotypic detection of AmpC was confirmed in seven strains. The ESBL was detected in two strains of E. cloacae from shrimp samples. No strain showed KPC production. These data can be considered alarming, since food (shrimp and fish) may be carriers of enterobacteria resistant to drugs of clinical interest.

Plectranthus amboinicus essential oil and carvacrol bioactive against planktonic and biofilm of oxacillin- and vancomycin-resistant Staphylococcus aureus.

BACKGROUND: The emergence of multidrug-resistant bacteria is a worldwide concern and in order to find an alternative to this problem, the occurrence of antimicrobial compounds in Plectranthus amboinicus essential oil was investigated. Thus, this study aims to determine susceptibility of Staphylococcus aureus isolated from food to antibiotics, P. amboinicus essential oil (PAEO) and carvacrol. METHODS: Leaves and stem of P. amboinicus were used for extraction of essential oil (PAEO) by hydrodistillation technique and EO chemical analysis was performed by gas chromatography coupled to a mass spectrometer. S. aureus strains (n = 35) isolated from food and S. aureus ATCC 6538 were used to evaluate the antimicrobial and antibiofilm activity of PAEO and carvacrol. All strains (n = 35) were submitted to antimicrobial susceptibility profile by disk diffusion method. Determination of MIC and MBC was performed by microdilution technique and antibiofilm activity was determined by microtiter-plate technique with crystal violet assay and counting viable cells in Colony Forming Units (CFU). RESULTS: Carvacrol (88.17%) was the major component in the PAEO. Antibiotic resistance was detected in 28 S. aureus strains (80%) and 12 strains (34.3%) were oxacillin and vancomycin-resistant (OVRSA). From the 28 resistant strains, 7 (25%) showed resistance plasmid of 12,000 bp. All strains (n = 35) were sensitive to PAEO and carvacrol, with inhibition zones ranging from 16 to 38 mm and 23 to 42 mm, respectively. The lowest MIC (0.25 mg mL-1) and MBC (0.5 mg mL-1) values were observed when carvacrol was used against OVRSA. When a 0.5 mg mL-1 concentration of PAEO and carvacrol was used, no viable cells were found on S. aureus biofilm. CONCLUSION: The antibacterial effect of carvacrol and PAEO proves to be a possible alternative against planktonic forms and staphylococcal biofilm.

In Vitro Antibacterial and Antibiofilm Activity of Lippia alba Essential Oil, Citral, and Carvone against Staphylococcus aureus.

In vitro antimicrobial and antibiofilm activities of the Lippia alba essential oil and its major components (citral and carvone) against Staphylococcus aureus were investigated. Essential oils (LA1EO, LA2EO, and LA3EO) were extracted from the aerial parts of three L. alba specimens by hydrodistillation and analyzed by gas chromatography coupled to a mass spectrometer. Minimum Inhibitory Concentrations (MIC) and Minimum Bacterial Concentration (MBC) were determined by the microdilution method. For the antibiofilm assays, the biomass formation in the biofilm was evaluated by the microtiter-plate technique with the crystal violet (CV) assay and the viability of the bacterial cells was analyzed. All oils and their major components presented antibacterial activity, and the lowest MIC and MBC values were 0.5 mg mL-1 when LA1EO and citral were used. Potential inhibition (100%) of S. aureus biofilm formation at the concentration of 0.5 mg mL-1 of all EOs was observed. However, the elimination of biofilm cells was confirmed at concentrations of 1 mg mL-1, 2 mg mL-1, 2 mg mL-1, and 0.5 mg mL-1 for LA1EO, LA2EO, LA3EO, and citral, respectively. The results obtained in the present research point to the promising antibacterial and antibiofilm potential of L. alba EOs against S. aureus, a species of recognized clinical interest.

RAW TROPICAL OYSTERS AS VEHICLES FOR MULTIDRUG-RESISTANT Vibrio parahaemolyticus.

The following study aimed to determine the antimicrobial susceptibility profile of Vibrio parahaemolyticus strains from fresh and frozen oysters Crassostrea rhizophorae sold in Fortaleza-Brazil. An antibiogram was performed on 87 isolates using nine antibiotics: gentamicin (Gen 10 µg), ampicillin (Amp 10 µg), penicillin G (Pen 10U), ciprofloxacin (Cip 5 µg), chloramphenicol (Chl 30 µg), nalidixic acid (Nal 30 µg), tetracycline (Tet 30 µg), vancomycin (Van 30 µg) and erythromycin (Ery 15 µg). All strains were resistant to at least one antibiotic, and 85 (97.7%) were multi-resistant, with predominance of the Van+ Pen+Amp resistance profile (n = 46). Plasmid resistance to Pen, Amp and Ery was detected. Thus, the risk that raw oyster consumption poses to the health of consumers is highlighted, due to the fact that these bivalves may host antibacterial-resistant microorganisms.

RAW TROPICAL OYSTERS AS VEHICLES FOR MULTIDRUG-RESISTANT Vibrio parahaemolyticus / Ostras tropicais cruas como fonte de Vibrio parahaemolyticus multirresistentes

The following study aimed to determine the antimicrobial susceptibility profile of Vibrio parahaemolyticus strains from fresh and frozen oysters Crassostrea rhizophorae sold in Fortaleza-Brazil. An antibiogram was performed on 87 isolates using nine antibiotics: gentamicin (Gen 10 µg), ampicillin (Amp 10 µg), penicillin G (Pen 10U), ciprofloxacin (Cip 5 µg), chloramphenicol (Chl 30 µg), nalidixic acid (Nal 30 µg), tetracycline (Tet 30 µg), vancomycin (Van 30 µg) and erythromycin (Ery 15 µg). All strains were resistant to at least one antibiotic, and 85 (97.7%) were multi-resistant, with predominance of the Van+ Pen+Amp resistance profile (n = 46). Plasmid resistance to Pen, Amp and Ery was detected. Thus, the risk that raw oyster consumption poses to the health of consumers is highlighted, due to the fact that these bivalves may host antibacterial-resistant microorganisms.

O presente estudo objetivou determinar o perfil de suscetibilidade a antimicrobianos de cepas de Vibrio parahaemolyticus oriundas de ostras “in natura” e congeladas comercializadas em Fortaleza-Brasil. Oitenta e sete (87) cepas foram submetidas ao antibiograma com emprego de nove antibióticos: gentamicina (Gen 10 µg), ampicilina (Amp 10 µg), penicilina G (Pen 10U), ciprofloxacin (Cip 5 µg), cloranfenicol (Clo 30 µg), ácido nalidíxico (Nal 30 µg), tetraciclina (Tet 30 µg), vancomicina (Van 30 µg) e eritromicina (Eri 15 µg). Todas as cepas mostram-se resistentes a pelo menos um antibiótico, e 85 (97,7%) apresentaram multirresistência, com predomínio do perfil Van+ Pen+Amp (n = 46). Foi detectada resistência plasmidial a Pen, Amp e Eri. Dessa forma, o risco que o consumo de ostras cruas representa para a saúde dos consumidores merece ser destacado, uma vez que esses bivalves podem ser veículos de transmissão de micro organismos multirresistentes a fármacos antibacterianos.

Multiple enzymatic profiles of Vibrio parahaemolyticus strains isolated from oysters / Multiple enzymatic profiles of Vibrio parahaemolyticus strains isolated from oysters.

The enzymatic characterization of vibrios has been used as a virulence indicator of sanitary interest. The objective of this study was to determine the enzymatic profile of Vibrio parahaemolyticus strains (n=70) isolated from Crassostrea rhizophorae oysters. The strains were examined for the presence of gelatinase (GEL), caseinase (CAS), elastase (ELAS), phospholipase (PHOS), lipase (LIP), amilase (AML) and DNase. All enzymes, except elastase, were detected in more than 60

of the strains. The most recurrent enzymatic profiles were AML + DNase + PHOS + GEL + LIP (n=16; 22.9

) and AML + CAS + DNase + PHOS + GEL + LIP (n=21; 30

). Considering the fact that exoenzyme production by vibrios is closely related to virulence, one must be aware of the bacteriological risk posed to human health by the consumption of raw or undercooked oysters.

Hemolytic and urease activities in vibrios isolated from fresh and frozen oysters.

INTRODUCTION: The present study aimed to survey the Vibrio microbiota of oysters (Crassostrea rhizophorae) obtained from restaurants in Fortaleza, State of Ceará, Brazil, and to identify virulence factors. METHODS: The isolated vibrios were submitted to biochemical identification and were tested for hemolytic and urease activities. RESULTS: The isolated strains belonged to 13 species, with predominance of Vibrio mimicus. Of the strain isolates only from fresh samples, 20.5% and 2.8% showed hemolytic and urease activities, respectively. CONCLUSIONS: The findings support the little-publicized claim that Vibrio species other than V. parahaemolyticus and V. vulnificus can represent a health risk to public health.

Hemolytic and urease activities in vibrios isolated from fresh and frozen oysters

INTRODUCTION: The present study aimed to survey the Vibrio microbiota of oysters (Crassostrea rhizophorae) obtained from restaurants in Fortaleza, State of Ceará, Brazil, and to identify virulence factors. METHODS: The isolated vibrios were submitted to biochemical identification and were tested for hemolytic and urease activities. RESULTS: The isolated strains belonged to 13 species, with predominance of Vibrio mimicus. Of the strain isolates only from fresh samples, 20.5% and 2.8% showed hemolytic and urease activities, respectively. CONCLUSIONS: The findings support the little-publicized claim that Vibrio species other than V. parahaemolyticus and V. vulnificus can represent a health risk to public health.

Multiple enzymatic profiles of Vibrio parahaemolyticus strains isolated from oysters.

The enzymatic characterization of vibrios has been used as a virulence indicator of sanitary interest. The objective of this study was to determine the enzymatic profile of Vibrio parahaemolyticus strains (n=70) isolated from Crassostrea rhizophorae oysters. The strains were examined for the presence of gelatinase (GEL), caseinase (CAS), elastase (ELAS), phospholipase (PHOS), lipase (LIP), amilase (AML) and DNase. All enzymes, except elastase, were detected in more than 60% of the strains. The most recurrent enzymatic profiles were AML + DNase + PHOS + GEL + LIP (n=16; 22.9%) and AML + CAS + DNase + PHOS + GEL + LIP (n=21; 30%). Considering the fact that exoenzyme production by vibrios is closely related to virulence, one must be aware of the bacteriological risk posed to human health by the consumption of raw or undercooked oysters.

Uso de soro de leite bovino para elaboração de meios de cultura para Staphylococcus aureus / Culture media made by bovine whey for Staphylococcus aureus growth

Foi investigada a eficiência do cultivo de Staphylococcus aureus ATCC 25923 em meios experimentais elaborados com soro de leite bovino. Foram preparados meios líquido (caldo soro – CS) e sólido (ágar soro – AS) suplementados com cloreto de sódio (CS+NaCl e AS+NaCl) e extrato de levedura (CS+Lev eAS+Lev). No meio AS, foi testado o enriquecimento com extrato de levedura (0,1%) e diluição em água(40%) (AS+H2O+Lev). Os meios comerciais de caldo infusão cérebro coração (BHI) e ágar Baird-Parker(BP) foram utilizados como controle. A eficiência dos meios experimentais foi avaliada por determinação da biomassa e contagem de colônias, utilizando-se a cepa S. aureus ATCC 25923. Em todos os testes, o crescimento bacteriano foi compatível à quantificação observada nos meios utilizados como controle e houve desenvolvimento de colônias com características morfológicas típicas de S. aureus, principalmente no meio AS+H2O+Lev. Concluiu-se que o soro de leite bovino pode ser utilizado como componente alternativo na elaboração de meio de cultura para S. aureus.

Staphylococcus coagulase-positiva e enterobactérias em camarão Litopenaeus vannamei comercializado in natura / Coagulase-positive staphylococci and enterobacteria in fresh shrimp Litopenaeus vannamei

A qualidade microbiológica de camarões (Litopenaeus vannamei) comercializados in natura em Fortaleza(CE) foi investigada por meio de quantificação de Staphylococcus coagulase-positiva (Sph CP), coliformes totais (CT) e termotolerantes (CTT) e também pela detecção de Salmonella e isolamento de Escherichiacoli. Das 24 amostras adquiridas no comércio varejista, quatro (16,7 por cento) apresentaram índices de Sph CP acima de 103/g. Não foi detectada Salmonella nas amostras analisadas. O número de amostras positivas para CT, CTT e E. coli foi, respectivamente, de 23 (95,8 por cento), 13 (54,2 por cento) e 6 (25 por cento). De acordo com a legislação vigente no Brasil, as quatro amostras com contagens de Sph CP > 103/g são consideradas impróprias para o consumo. Ademais, chama-se a atenção para a ocorrência de E. coli em 25 por cento das amostras analisadas, uma vez que essa bactéria é indicadora de contaminação fecal.

Coliformes, salmonella e staphylococcus coagulase positiva em hortaliças utilizada na merenda escolar da rede pública de ensino de Sobral, CE / Coliforms, salmonella and staphylococcus coagulase positive in vegetables used in school lunches in public schools in Sobral, CE

Foram pesquisadas 24 amostras de hortaliças utilizadas na merenda da rede de escola pública de Sobral – Ceará. A pesquisa retrata a contagem e identificação de bactérias do grupo dos coliformes, Staphylococcus coagulase positiva e pesquisa de Salmonella. Das 24 amostras 62,5% apresentaram contaminação por Coliformes termotolerantes acima da permitida pela ANVISA. Cerca de 14 cepas de Coliformes termotolerantes foram isoladas sendo 14,3% Citrobacter diversus e 85,7% Enterobacter agglomerans. A quantificação de coliformes totais variou de <1,8 a >160 x 10, não sendo detectada a presença de Salmonella. A contagem de Staphylococcus variou de 250 a 20 x 10 UFC/g, desses foram isoladas 32 cepas, sendo 9 de Staphylococcus coagulase positiva e 23 de Staphylococcus coagulase negativa.

In vitro antibacterial effect of aqueous and ethanolic Moringa leaf extracts.

OBJECTIVE: To evaluate the antibacterial effect of aqueous and ethanolic moringa leaf extracts (Moringa oleifera) on the growth of gram-positive and negative bacteria. METHODS: Paper disks were soaked with 100, 200, 300 and 400 µL of extract at 20 g/180 mL and 10 g/190 mL. All extracts were tested against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923), Vibrio parahaemolyticus, Enterococcus faecalis (ATCC29212), Pseudomonas aeruginosa (ATCC27853), Salmonella enteritidis (IH) and Aeromonas caviae. The susceptibility tests were performed using the modified disk diffusion method. RESULTS: The strains E. coli, P. aeruginosa and S. enteritidis (IH) were resistant to all treatments. In general, disks with 400 µL extract were the most efficient against S. aureus, V. parahaemolyticus, E. faecalis and A. caviae. CONCLUSIONS: The study indicates a promising potential for aqueous and ethanolic Moringa leaf extracts as alternative treatment of infections caused by the tested strains.