Antimicrobial Resistance Patterns in Patients with Vaginal Discharge: A 2019-2022 Analysis at the National Health Laboratory in Eritrea.

Background: Antimicrobial resistance poses a significant global threat to the treatment of bacterial infections, particularly in low- and middle-income regions such as Africa. This study is aimed at analyzing antimicrobial resistance patterns in vaginal swab samples from patients at the National Health Laboratory from 2019 to 2022. Methods: This retrospective study examined patient records from vaginal swab analyses performed at the National Health Laboratory between January 1, 2019, and December 31, 2022. Ethical approval was obtained from the Ministry of Health Research Ethical Approval and Clearance Committee on 15/02/2023. Results: Of the 622 samples, 83% underwent microbial isolation and identification. Citrobacter spp. exhibited high resistance (>43%) to antibiotics such as cephalexin, ceftazidime, nalidixic acid, ampicillin, gentamicin, and tetracycline. E. coli showed resistance rates of more than 50% to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Klebsiella spp. and Proteus spp. exhibited resistance rates that exceeded 47% to specific antibiotics. Gram-positive bacteria have resistance rates of more than 49% with ampicillin, trimethoprim-sulfamethoxazole, tetracycline, oxacillin, vancomycin, and penicillin G. In particular, S. aureus demonstrated no resistance to rifampicin or clindamycin, while Streptococcus spp. showed 100% resistance to rifampicin and vancomycin. Several species, including Proteus species, Streptococcus spp., S. aureus, and Klebsiella spp. exhibited multidrug resistance. Conclusion: Most gram-negative bacteria displayed higher resistance of >45% to ampicillin, trimethoprim-sulfamethoxazole, and tetracycline. Among gram-positive bacteria, a higher resistance rate with ampicillin, trimethoprim-sulfamethoxazole, tetracycline, oxacillin, vancomycin, and penicillin G was recorded. S. aureus showed no resistance to rifampicin and clindamycin, and Strep. spp. indicated 100% resistance to rifampicin and vancomycin. This study highlights critical gaps and areas for further exploration. Expanding the spectrum of antibiotics tested and investigating underlying multidrug resistance mechanisms would provide a more comprehensive understanding of resistance patterns.

Synergism with Shikimic Acid Restores ß-Lactam Antibiotic Activity against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved into a dangerous pathogen resistant to beta-lactam antibiotics (BLAs) and has become a worrisome superbug. In this study, a strategy in which shikimic acid (SA), which has anti-inflammatory and antibacterial activity, is combined with BLAs to restart BLA activity was proposed for MRSA treatment. The synergistic effects of oxacillin combined with SA against oxacillin resistance in vitro and in vivo were investigated. The excellent synergistic effect of the oxacillin and SA combination was confirmed by performing the checkerboard assay, time-killing assay, live/dead bacterial cell viability assay, and assessing protein leakage. SEM showed that the cells in the control group had a regular, smooth, and intact surface. In contrast, oxacillin and SA or the combination treatment group exhibited different degrees of surface collapse. q-PCR indicated that the combination treatment group significantly inhibited the expression of the mecA gene. In vivo, we showed that the combination treatment increased the survival rate and decreased the bacterial load in mice. These results suggest that the combination of oxacillin with SA is considered an effective treatment option for MRSA, and the combination of SA with oxacillin in the treatment of MRSA is a novel strategy.

Identification and biological characteristics of Enterococcus casseliflavus TN-47 isolated from Monochamus alternatus.

With the widespread use of antibiotics, the incidence of antibiotic resistance in microorganisms has increased. Monochamus alternatus is a trunk borer of pine trees. This study aimed to investigate the in vitro antimicrobial and biological characteristics of Enterococcus casseliflavus TN-47 (PP411196), isolated from the gastrointestinal tract of M. alternatus in Jilin Province, PR China. Among 13 isolates obtained from the insects, five were preliminarily screened for antimicrobial activity. E. casseliflavus TN-47, which exhibited the strongest antimicrobial activity, was identified. E. casseliflavus TN-47 possessed antimicrobial activity against Staphylococcus aureus USA300 and Salmonella enterica serovar Pullorum ATCC 19945. Furthermore, E. casseliflavus TN-47 was sensitive to tetracyclines, penicillins (ampicillin, carbenicillin, and piperacillin), quinolones and nitrofuran antibiotics, and resistant to certain beta-lactam antibiotics (oxacillin, cefradine and cephalexin), macrolide antibiotics, sulfonamides and aminoglycosides. E. casseliflavus TN-47 could tolerate low pH and pepsin-rich conditions in the stomach and grow in the presence of bile acids. E. casseliflavus TN-47 retained its strong auto-aggregating ability and hydrophobicity. This strain did not exhibit any haemolytic activity. These results indicate that E. casseliflavus TN-47 has potential as a probiotic. This study provides a theoretical foundation for the future applications of E. casseliflavus TN-47 and its secondary metabolites in animal nutrition and feed.

Prevalence and antimicrobial resistance profiles of mastitis causing bacteria isolated from dairy goats in Mukurweini Sub-County, Nyeri County, Kenya.

BACKGROUND: Ruminant mastitis continues to be a cause of economic losses in the dairy industry and remains a major public health hazard globally. OBJECTIVES: This cross-sectional study was carried out in Mukurweini Sub-County of Nyeri County, Kenya, to investigate the prevalence of bacteria causing mastitis, risk factors associated with goat mastitis and the antibiotic resistance profiles of bacteria isolated from the goat milk. METHODS: Farm level data on risk factors for mastitis was obtained from 56 farmers using a semi structured questionnaire. A total of 189 goat milk samples were collected. The goat's udder was observed for signs of clinical mastitis and the California Mastitis Test (CMT) used to test the milk for sub-clinical mastitis. All samples were then cultured for morphological identification of bacteria and strain typing by Matrix Assisted Laser Desorption/Ionization (MALDI)-Time of Flight (ToF) technique. Antimicrobial susceptibility of the isolated Staphylococcus aureus, coagulase-negative Staphylococcus (CoNS), Escherichia coli, Klebsiella oxytoca, Pseudomonas spp., Enterobacter spp., Proteus vulgaris and Escherichia vulneris to eight commonly used antibiotics was done by the disc diffusion method and validated by determining the presence of antibiotic resistance genes (mecA and blaTEM) using polymerase chain reaction method. RESULTS: The prevalence of clinical mastitis was 1.1% (2/189) while that of sub-clinical mastitis was 84.7% (160/189). Higher (p < 0.05) prevalence of mastitis was observed in goats whose houses were cleaned fortnightly and in cases where farmers used same towel to dry different does' udders during the milking process. Thirteen different bacterial species were isolated from the milk samples and identified by MALDI-ToF, and these included S. aureus (22.0%), CoNS (20.3%), E. coli (18.1%), Pseudomonas spp. (14.3%), Enterobacter spp. (10.4%), K. oxytoca (6.0%), E. vulneris (1.7%), P. vulgaris (1.7%), Raoutella ornithinolytica (1.7%), Stenotrophomonas maltophilia (1.1%), Pantoea agglomerans (1.1%), Serratia marcescens (1.1%) and Cedeceas spp. (0.6%). One hundred pathogenic bacterial isolates were randomly selected and tested for antibiotic sensitivity to eight antibiotics out of which S. aureus were 97.5% resistant to Oxacillin and 100% sensitive to Ciprofloxacin. The CoNSs were 100% resistant to Oxacillin and 100% sensitive to Ciprofloxacin. E. coli were 93.9% resistant to Oxacillin, 69.7% sensitive to Ciprofloxacin and 87.9% sensitive to both Amoxicillin/Clavulanic acid and Meropenem. The antimicrobial resistant genes detected in S. aureus and E. coli were mecA [66.7%, 0%], and blaTEM [20% and 78.3%], respectively. CONCLUSION: In conclusion, the study showed that most of the does were affected by subclinical mastitis with the main causative bacteria being Staphylococci spp. and coliforms. Farmers need to be trained on improved control of mastitis by adoption of good milking practices and use of CMT kit for early detection of mastitis. Occurrence of multidrug resistance by key mastitis causing pathogens was shown to be prevalent and therefore there is need for development of intervention strategies.

Antimicrobial and antibiofilm activity of highly soluble polypyrrole against methicillin-resistant Staphylococcus aureus.

AIMS: The purpose was to evaluate the antimicrobial activity of highly soluble polypyrrole (Hs-PPy), alone or combined with oxacillin, as well as its antibiofilm potential against methicillin-resistant Staphylococcus aureus strains. Furthermore, the in silico inhibitory mechanism in efflux pumps was also investigated. METHODS AND RESULTS: Ten clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two reference strains were used. Antimicrobial activity was determined by broth microdilution, and the combination effect with oxacillin was evaluated by the checkerboard assay. The biofilm formation capacity of MRSA and the interference of Hs-PPy were evaluated. The inhibitory action of Hs-PPy on the efflux pump was evaluated in silico through molecular docking. Hs-PPy showed activity against the isolates, with inhibitory action between 62.5 and 125 µg ml-1 and bactericidal action at 62.5 µg ml-1, as well as synergism in association with oxacillin. The isolates ranged from moderate to strong biofilm producers, and Hs-PPy interfered with the formation of this structure, but not with mature biofilm. There was no in silico interaction with the efflux protein EmrD, the closest homolog to NorA. CONCLUSIONS: Hs-PPy interferes with biofilm formation by MRSA, has synergistic potential, and is an efflux pump inhibitor.

Antimicrobial resistance markers distribution in Staphylococcus aureus from Nsukka, Nigeria.

BACKGROUND: Multidrug resistance in Staphylococcus aureus continues to influence treatment complications in clinical settings globally. Multidrug-resistant-S. aureus (MDR-SA) is often genetically driven by resistance markers transferable in pathogenic strains. This study aimed to determine the distribution of resistance markers in clinical isolates of S. aureus in Nsukka, Nigeria. METHODS: A total of 154 clinical samples were cultured on mannitol salt agar. Isolates were characterized using conventional cultural techniques and confirmed by PCR detection of S. aureus-specific nuc gene. Antibiotic resistance profiles of the isolates were determined against selected antibiotics using the disk-diffusion method, while screening for antibiotic resistance genes (Mec A, Erm A, Erm B, Erm C, Van A, and Van B) was by PCR. RESULTS: A total of 98 isolates were identified as S. aureus by conventional methods. Of these, 70 (71.43%) were confirmed by PCR. Phenotypically, the isolates exhibited high degrees of resistance to oxacillin (95.72%), erythromycin (81.63%), and ertapenem (78.57%) and 75.51% and 47.30% against methicillin and vancomycin, respectively. Multiple antibiotic resistance indexes of the isolates ranged from 0.3 to 1, and the most prevalent pattern of resistance was oxacillin-ertapenem-vancomycin-erythromycin-azithromycin-clarithromycin-ciprofloxacin- cefoxitin-amoxicillin-clavulanic acid. PCR screening confirmed the existence of various antibiotic resistance makers among the strains, with the most common resistance genes found in the isolates being Mec A (32.14%), Van A (21.43%), Van B (10.71%), Erm B (10.71%), and Erm C (17.86%). None possessed the Erm A gene. CONCLUSION: The study supports the need for necessary action, including rational drug use, continuous surveillance, and deployment of adequate preventive and curative policies and actions.

Biological characteristics of mechanosensitive channels MscS and MscL in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is an important respiratory pathogen that can cause porcine contagious pleuropneumonia (PCP), resulting in significant economic losses in swine industry. Microorganisms are subjected to drastic changes in environmental osmolarity. In order to alleviate the drastic rise or fall of osmolarity, cells activate mechanosensitive channels MscL and MscS through tension changes. MscL not only regulates osmotic pressure but also has been reported to secrete protein and uptake aminoglycoside antibiotic. However, MscL and MscS, as the most common mechanosensitive channels, have not been characterized in A. pleuropneumoniae. In this study, the osmotic shock assay showed that MscL increased sodium adaptation by regulating cell length. The results of MIC showed that deletion of mscL decreased the sensitivity of A. pleuropneumoniae to multiple antibiotics, while deletion of mscS rendered A. pleuropneumoniae hypersensitive to penicillin. Biofilm assay demonstrated that MscL contributed the biofilm formation but MscS did not. The results of animal assay showed that MscL and MscS did not affect virulence in vivo. In conclusion, MscL is essential for sodium hyperosmotic tolerance, biofilm formation, and resistance to chloramphenicol, erythromycin, penicillin, and oxacillin. On the other hand, MscS is only involved in oxacillin resistance.IMPORTANCEBacterial resistance to the external environment is a critical function that ensures the normal growth of bacteria. MscL and MscS play crucial roles in responding to changes in both external and internal environments. However, the function of MscL and MscS in Actinobacillus pleuropneumoniae has not yet been reported. Our study shows that MscL plays a significant role in osmotic adaptation, antibiotic resistance, and biofilm formation of A. pleuropneumoniae, while MscS only plays a role in antibiotic resistance. Our findings provide new insights into the functional characteristics of MscL and MscS in A. pleuropneumoniae. MscL and MscS play a role in antibiotic resistance and contribute to the development of antibiotics for A. pleuropneumoniae.

P-type ATPase zinc transporter Rv3270 of Mycobacterium tuberculosis enhances multi-drug efflux activity.

Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn2+ and Zn2+ metal ion toxicity in Mycobacterium tuberculosis, in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of Escherichia coli and Mycobacterium smegmatis. Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn2+ resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.

Doxycycline post-exposure prophylaxis could theoretically select for resistance to various antimicrobials in 19 pathobionts: an in silico analysis.

OBJECTIVES: Doxycycline post exposure prophylaxis (PEP) has been shown to reduce the incidence of bacterial STIs. However, if there is genetic linkage between resistance to tetracycline and other antimicrobials, then it could also select for resistance to these other antimicrobials. We therefore undertook to evaluate if there is an association between the minimum inhibitory concentrations (MICs) of tetracycline and other antimicrobials in 19 clinically important bacterial species. METHODS: Mixed-effects linear regression was used to assess if minocycline MICs were associated with the MICs of eight other antimicrobials (ceftriaxone, ampicillin, oxacillin, vancomycin, erythromycin, levofloxacin, amikacin, and trimethoprim-sulfamethoxazole) in 19 bacterial species in the Antimicrobial Testing Leadership and Surveillance (ATLAS) database. RESULTS: With the notable exception of vancomycin, where no association was found, strong positive associations were typically found between the MICs of minocycline and each of the eight antimicrobials in each of the species assessed. For example, the minocycline MICs of all the Gram-positive species were positively associated with ampicillin, ceftriaxone, oxacillin and erythromycin MICs (all P-values < 0.001). The only exceptions were ampicillin for Streptococcus pyogenes and ceftriaxone for S. dysgalactiae, where no significant associations were found. Similarly in the Gram-negative species, the minocycline MICs of all the species except Haemophilus influenzae and Stenotrophomonas maltophilia were positively associated with the MICs of ceftriaxone, ampicillin, levofloxacin and amikacin (all P-values < 0.001). CONCLUSIONS: There is a theoretical risk that doxycycline PEP could select for resistance not only to tetracyclines but to a range of other antimicrobials in each of the 19 pathobionts assessed.

Staphylococcus aureus AbcA transporter enhances persister formation under ß-lactam exposure.

We evaluated the role of Staphylococcus aureus AbcA transporter in bacterial persistence and survival following exposure to the bactericidal agents nafcillin and oxacillin at both the population and single-cell levels. We show that AbcA overexpression resulted in resistance to nafcillin but not oxacillin. Using distinct fluorescent reporters of cell viability and AbcA expression, we found that over 6-14 hours of persistence formation, the proportion of AbcA reporter-expressing cells assessed by confocal microscopy increased sixfold as cell viability reporters decreased. Similarly, single-cell analysis in a high-throughput microfluidic system found a strong correspondence between antibiotic exposure and AbcA reporter expression. Persister cells grown in the absence of antibiotics showed neither an increase in nafcillin MIC nor in abcA transcript levels, indicating that survival was not associated with stable mutational resistance or abcA overexpression. Furthermore, persister cell levels on exposure to 1×MIC and 25×MIC of nafcillin decreased in an abcA knockout mutant. Survivors of nafcillin and oxacillin treatment overexpressed transporter AbcA, contributing to an enrichment of the number of persisters during treatment with pump-substrate nafcillin but not with pump-non-substrate oxacillin, indicating that efflux pump expression can contribute selectively to the survival of a persister population.

Antimicrobial susceptibility of mastitis pathogens isolated from North American dairy cattle, 2011-2022.

A total of 10,890 bacterial isolates of Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Escherichia coli isolated as etiological agents from dairy cows with mastitis by 29 veterinary laboratories across North America between 2011 and 2022 were tested for in vitro antimicrobial susceptibility by broth microdilution to ampicillin, cefoperazone, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin-novobiocin and pirlimycin according to CLSI standards. Using available clinical breakpoints, antimicrobial resistance among S. dysgalactiae (n = 2406) was low for penicillin-novobiocin (0% resistance), ceftiofur (0.1%), erythromycin (3.2%) and pirlimycin (4.6%). Among S. uberis (n = 2398), resistance was low for ampicillin (0%) and ceftiofur (0.2%) and moderate for erythromycin (11.9%) and pirlimycin (18.4%). For S. aureus (n = 3194), resistance was low for penicillin-novobiocin (0%), ceftiofur (0.1%), oxacillin (0.2%), erythromycin (0.7%), cefoperazone (1.2%) and pirlimycin (2.8%). For E. coli (n = 2892), resistance was low for ceftiofur (2.8%) and cefoperazone (3.4%) and moderate for ampicillin (9.2%). Overall, the results indicate that mastitis pathogens in the United States and Canada have not shown any substantial changes in the in vitro susceptibility to antimicrobial drugs over the 12 years of the study, or among that of the proceeding survey from 2002-2010. The data support the conclusion that resistance to common antimicrobial drugs among mastitis pathogens, even to drugs that have been used in dairies for mastitis management for many years, continues to remain low.

Combination antimicrobial therapy: in vitro synergistic effect of anti-staphylococcal drug oxacillin with antimicrobial peptide nisin against Staphylococcus epidermidis clinical isolates and Staphylococcus aureus biofilms.

The ability of Staphylococcus epidermidis and S. aureus to form strong biofilm on plastic devices makes them the major pathogens associated with device-related infections (DRIs). Biofilm-embedded bacteria are more resistant to antibiotics, making biofilm infections very difficult to effectively treat. Here, we evaluate the in vitro activities of anti-staphylococcal drug oxacillin and antimicrobial peptide nisin, alone and in combination, against methicillin-resistant S. epidermidis (MRSE) clinical isolates and the methicillin-resistant S. aureus ATCC 43,300. The minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) of oxacillin and nisin were determined using the microbroth dilution method. The anti-biofilm activities of oxacillin and nisin, alone or in combination, were evaluated. In addition, the effects of antimicrobial agents on the expression of icaA gene were examined by quantitative real-time PCR. MIC values for oxacillin and nisin ranged 4-8 µg/mL and 64-128 µg/mL, respectively. Oxacillin and nisin reduced biofilm biomass in all bacteria in a dose-dependent manner and this inhibitory effect was enhanced with combinatorial treatment. MBEC ranges for oxacillin and nisin were 2048-8192 µg/mL and 2048-4096 µg/mL, respectively. The addition of nisin significantly decreased the oxacillin MBECs from 8- to 32-fold in all bacteria. At the 1× MIC and 1/2× MIC, both oxacillin and nisin decreased significantly the expression of icaA gene in comparison with untreated control. When two antimicrobial agents were combined at 1/2× MIC concentration, the expression of icaA were significantly lower than when were used alone. Nisin/conventional oxacillin combination showed considerable anti-biofilm effects, including inhibition of biofilm formation, eradication of mature biofilm, and down-regulation of biofilm-related genes, proposing its applications for treating or preventing staphylococcal biofilm-associated infections, including device-related infections.

Liquid chromatography-tandem mass spectrometry methods for quantification of total and free antibiotic concentrations in serum and exudate from patients with post-sternotomy deep sternal wound infection receiving negative pressure wound therapy.

BACKGROUND: Systemically administered antibiotics are thought to penetrate the wounds more effectively during negative pressure wound therapy (NPWT).To test this hypothesis total and free antibiotic concentrations were quantified in serum and wound exudate. METHODS: UHPLC-MS/MS methods were developed and validated for the determination of ceftazidime, cefepime, cefotaxime, cefuroxime, cefazolin, meropenem, oxacillin, piperacillin with tazobactam, clindamycin, ciprofloxacin, sulfamethoxazole/trimethoprim (cotrimoxazole), gentamicin, vancomycin, and linezolid. The unbound antibiotic fraction was obtained by ultrafiltration using a Millipore Microcon-30kda Centrifugal Filter Unit. Analysis was performed on a 1.7-µm Acquity UPLC BEH C18 2.1 × 100-mm column with a gradient elution. RESULTS: The validation was performed for serum, exudates and free fractions. For all matrices, requirements were met regarding linearity, precision, accuracy, limit of quantitation, and matrix effect. The coefficient of variation was in the range of 1.2-13.6%.and the recovery 87.6-115.6%, respectively. Among the 29 applications of antibiotics thus far, including vancomycin, clindamycin, ciprofloxacin, oxacillin, cefepime, cefotaxime, cotrimoxazole, and gentamicin, total and free antibiotic concentrations in serum and exudate were correlated. CONCLUSION: This method can accurately quantify the total and free concentrations of 16 antibiotics. Comparison of concentration ratios between serum and exudates allows for monitoring individual antibiotics' penetration capacity in patients receiving NPWT.

High prevalence of heteroresistance in Staphylococcus aureus is caused by a multitude of mutations in core genes.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

Resistance and virulence in Staphylococcus aureus by whole-genome sequencing: a comparative approach in blaZ-positive isolates.

Mastitis caused by Staphylococcus aureus is a worldwide problem in dairy farms, in part because of the pathogenicity of the bacteria, biofilm formation, and mechanisms of antimicrobial resistance that make the disease difficult to diagnose and treat, which is typically done with the use of beta-lactam antibiotics. The aim of the present study was to determine the virulence and resistance factors of S. aureus isolates from subclinical mastitis, blaZ + /mecA - /mecC - , resistant and sensitive to oxacillin. All isolates were classified as CC97 by MLST analysis, a clonal complex well adapted to the mammary gland and although STAU23 and STAU73 were resistant to oxacillin while STAU32 and STAU78 were sensitive, the genomic analysis identified only the blaZ operon corresponding to resistance to beta-lactams. However, the presence of the sdrC gene was revealed exclusively in resistant isolates, an important adhesin in the colonization process that potentiates pathogenicity in S. aureus. In addition, resistance islands (REIs) were identified in these isolates, suggesting more conserved REIs. In the analysis of SNPs throughout the genome, mutations were found in the trmB and smpB genes of the resistant isolates and in the murD and rimM genes of the sensitive isolates. This study highlights the potential benefit of genome-wide characterization tools to identify molecular mechanisms of S. aureus in bovine mastitis.

Acetylenic spiroketal enol ethers from Artemisia rupestris and their synergistic antibacterial effects on methicillin-resistant Staphylococcus aureus.

Synergistic bioassay-guided isolation of the extracts of Artemisia rupestris L, which belongs to the family Asteraceae, afforded two acetylenic spiroketal enol ethers, namely rupesdiynes A (1) and B (2). Their structures were determined based on spectroscopic analysis and experimental and calculated ECD investigations. The two compounds exhibited synergistic activity and were able to reduce the minimum inhibitory concentration (MIC) of oxacillin four-fold, with a fractional inhibitory concentration index (FICI) of 0.5 in combination with oxacillin against the oxacillin-resistant EMRSA-16. Biofilm formation inhibitory and Ethidium bromide (EtBr) efflux assay were further employed to verify the possible mechanism of the synergistic antibacterial effect. Additionally, molecular docking studies were conducted to investigate the binding affinities of the two compounds with penicillin-binding protein 2a (PBP2a) of EMRSA-16. Taken together, rupesdiynes A (1) and rupesdiyne B (2) showed moderate synergistic activity against EMRSA-16 with oxacillin via inhibiting biofilm formation and efflux pump activity, respectively.

Genetic and Phenotypic Diversity of Listeria monocytogenes in Pig Slaughterhouses in Korea.

Listeria monocytogenes is a foodborne pathogen that has variable subtypes associated with human listeriosis and occurs in food and processing environments. This study was conducted to provide the genetic and phenotypic characterization of L. monocytogenes in pig carcasses and environments of slaughterhouses in Korea. A total of 22 L. monocytogenes were isolated from eight of 26 pig slaughterhouses between 2020 and 2022, and the most common serotype was 1/2c (40.9%), followed by serotypes 1/2b (31.8%) and 1/2a (27.3%). The isolates showed a significantly high prevalence of virulence genes located in Listeria pathogenicity island-1 (LIPI-1) and internalins (90.9-100%; p < 0.05). However, the prevalence rates of llsX, ptsA, and stress survival islet-1 (SSI-1) located in LIPI-3, LIPI-4, and SSI were only 9.1%, 22.7%, and 31.8%, respectively. In addition, among the epidemic clones (EC), ECI, ECII, ECIII, and ECV, only one isolate was represented as ECV. Isolates identified from the same slaughterhouses were divided into two or more pulsotypes, except for two slaughterhouses. Furthermore, the seven STs were classified into seven clonal complexes (CCs) (CC8, CC9, CC37, CC87, CC121, CC155, and CC288), and all CCs belonged to lineages I (31.8%) and II (68.1%). Interestingly, the isolates showed a high prevalence of oxacillin resistance (59.1%), and most isolates of the serotypes 1/2a and 1/2b exhibited oxacillin resistance, whereas only one of nine serotype 1/2c isolates exhibited oxacillin resistance. These results provide the genetic diversity of L. monocytogenes in pig carcasses and environments of slaughterhouses, and continuous monitoring will be helpful in predicting food safety risks.

Simultaneous determination of oxacillin and cloxacillin in plasma and CSF using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry: Application in therapeutic drug monitoring.

Cloxacillin and oxacillin are group M penicillins. The therapeutic monitoring of plasma concentrations of these antibiotics and those of their hydroxymethylated metabolites is of great clinical interest, especially in the choice of an adequate dosage allowing an effective treatment while limiting the occurrence of undesirable effects and the development of bacterial resistance. In this context, we conducted this work aiming at developing and validating a method allowing the determination of cloxacillin and oxacillin as well as the identification of their active metabolites in different biological matrices (CSF and plasma) using turbulent flow liquid chromatography coupled to high-resolution mass spectrometry. To do this, we carried out several optimisation tests. Subsequently, we validated our method according to the latest bioanalytical validation recommendations of the European Medicines Agency. The validation results showed that our method is specific and sensitive. We obtained good linearity in the range 0.5 to 100 µg/mL with correlation coefficients above 0.995. The lower limit of quantification was 0.5 µg/mL for each analyte. The method was found to be accurate with repeatability and reproducibility coefficients of variation below 15 %. Our method is also accurate with bias values below 15 %. Recovery values ranged from 87 % to 95 %. Finally, we were able to apply our method to the therapeutic monitoring of the analysed molecules and to identify their active metabolites. Our results suggest that LC-MS shows superiority in the therapeutic monitoring of these antibiotics due to the superiority of specificity shown by this method. This assay method can be routinely used for the daily plasma assays of patients treated with these antibiotics in the context of therapeutic monitoring.

Synergistic Potential of α-Melanocyte Stimulating Hormone Based Analogues with Conventional Antibiotic against Planktonic, Biofilm-Embedded, and Systemic Infection Model of MRSA.

The repotentiation of the existing antibiotics by exploiting the combinatorial potential of antimicrobial peptides (AMPs) with them is a promising approach to address the challenges of slow antibiotic development and rising antimicrobial resistance. In the current study, we explored the ability of lead second generation Ana-peptides viz. Ana-9 and Ana-10, derived from Alpha-Melanocyte Stimulating Hormone (α-MSH), to act synergistically with different classes of conventional antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). The peptides exhibited prominent synergy with ß-lactam antibiotics, namely, oxacillin, ampicillin, and cephalothin, against planktonic MRSA. Furthermore, the lead combination of Ana-9/Ana-10 with oxacillin provided synergistic activity against clinical MRSA isolates. Though the treatment of MRSA is complicated by biofilms, the lead combinations successfully inhibited biofilm formation and also demonstrated biofilm disruption potential. Encouragingly, the peptides alone and in combination were able to elicit in vivo anti-MRSA activity and reduce the bacterial load in the liver and kidney of immune-compromised mice. Importantly, the presence of Ana-peptides at sub-MIC doses slowed the resistance development against oxacillin in MRSA cells. Thus, this study highlights the synergistic activity of Ana-peptides with oxacillin advocating for the potential of Ana-peptides as an alternative therapeutic and could pave the way for the reintroduction of less potent conventional antibiotics into clinical use against MRSA infections.

Diagnostic and commensal Staphylococcus pseudintermedius genomes reveal niche adaptation through parallel selection of defense mechanisms.

Staphylococcus pseudintermedius is historically understood as a prevalent commensal and pathogen of dogs, though modern clinical diagnostics reveal an expanded host-range that includes humans. It remains unclear whether differentiation across S. pseudintermedius populations is driven primarily by niche-type or host-species. We sequenced 501 diagnostic and commensal isolates from a hospital, veterinary diagnostic laboratory, and within households in the American Midwest, and performed a comparative genomics investigation contrasting human diagnostic, animal diagnostic, human colonizing, pet colonizing, and household-surface S. pseudintermedius isolates. Though indistinguishable by core and accessory gene architecture, diagnostic isolates harbor more encoded and phenotypic resistance, whereas colonizing and surface isolates harbor similar CRISPR defense systems likely reflective of common household phage exposures. Furthermore, household isolates that persist through anti-staphylococcal decolonization report elevated rates of base-changing mutations in - and parallel evolution of - defense genes, as well as reductions in oxacillin and trimethoprim-sulfamethoxazole susceptibility. Together we report parallel niche-specific bolstering of S. pseudintermedius defense mechanisms through gene acquisition or mutation.