Antibiotic Susceptibility of Staphylococcus Aureus with VanA and MecA Genes.

BACKGROUND: Staphylococcus aureus (S.aureus) is an emerging antibiotic resistant bacterium responsible for various infections in human. Resistance to methicillin and vancomycin are of prime concern in S. aureus. The study aims to determine the minimum inhibitory concentration (MIC) of Vancomycin and evaluate the existence of mecA and vanA genes, associated with antibiotic resistance. METHODS: Clinical specimens from three Kathmandu hospitals were processed and S. aureus was identified using conventional microbiological procedures. MRSA was phenotypically identified with cefoxitin (30µg) disc diffusion, while vancomycin susceptibility was assessed using the Ezy MICTM stripes. The mecA and vanA genes were detected by polymerase chain reaction (PCR). RESULTS: Out of 266 S. aureus samples from various clinical specimen subjected for analysis, 77 (28.9%) were found methicillin-resistant (MRSA) and 10 (3.8%) were observed vancomycin-resistant (VRSA). Vancomycin resistant isolates showed a significant correlation between resistance to ampicillin, chloramphenicol, and cefoxitin. The mecA gene was found in 39 of the MRSA isolates, having 50.64% of MRSA cases, while the vanA gene was detected in 4 of the VRSA cases, constituting 40% of VRSA occurrences. CONCLUSIONS: The strains with higher vancomycin minimum inhibitory concentration values (≥ 1.5 µg/ml) displayed increased resistance rates to various antibiotics compared to strains with lower minimum inhibitory concentration values (< 1.5 µg/ml). The presence of vanA genes was strongly associated (100%) with vancomycin resistance, while the 10.3% mecA gene was identified from MRSA having resistance towards vancomycin also.

Cefoxitin versus carbapenems as definitive treatment for extended-spectrum ß-lactamase-producing Klebsiella pneumoniae bacteremia in intensive care unit: a propensity-matched retrospective analysis.

BACKGROUND: Despite cefoxitin's in vitro resistance to hydrolysis by extended-spectrum beta-lactamases (ESBL), treatment of ESBL-producing Klebsiella pneumoniae (KP) infections with cefoxitin remains controversial. The aim of our study was to compare the clinical efficacy of cefoxitin as definitive antibiotic therapy for patients with ESBL-KP bacteremia in intensive care unit, versus carbapenem therapy. METHODS: This retrospective study included all patients with monomicrobial bacteremia hospitalized in intensive care unit between January 2013 and January 2023 at the University Hospital of Guadeloupe. The primary outcome was the 30-day clinical success defined as a composite endpoint: 30-day survival, absence of relapse and no change of antibiotic therapy. Cox regression including a propensity score (PS) and PS-based matched analysis were performed for endpoint analysis. RESULTS: A total of 110 patients with bloodstream infections were enrolled. Sixty-three patients (57%) received definitive antibiotic therapy with cefoxitin, while forty-seven (43%) were treated with carbapenems. 30-day clinical success was not significantly different between patients treated with cefoxitin (57%) and carbapenems (53%, p = 0.823). PS-adjusted and PS-matched analysis confirmed these findings. Change of definitive antibiotic therapy was more frequent in the cefoxitin group (17% vs. 0%, p = 0.002). No significant differences were observed for the other secondary endpoints. The acquisition of carbapenem-resistant Pseudomonas aeruginosa was significantly higher in patients receiving carbapenem therapy (5% vs. 23%, p = 0.007). CONCLUSIONS: Our results suggest that cefoxitin as definitive antibiotic therapy could be a therapeutic option for some ESBL-KP bacteremia, sparing carbapenems and reducing the selection of carbapenem-resistant Pseudomonas aeruginosa strains.

Screening the NCI diversity set V for anti-MRSA activity: cefoxitin synergy and LC-MS/MS confirmation of folate/thymidine biosynthesis inhibition.

IMPORTANCE: New antibacterial agents are urgently needed to counter increasingly resistant bacteria. One approach to this problem is library screening for new antibacterial agents. Library screening efforts can be improved by increasing the information content of the screening effort. In this study, we screened the National Cancer Institute diversity set V against methicillin-resistant Staphylococcus aureus (MRSA) with several enhancements. One of these is to screen the library before and after microsomal metabolism as means to identify potential active metabolites. A second enhancement is to screen the library in the absence and presence of sub-minimum inhibitory concentration levels of another antibiotic, such as cefoxitin in this study. This identified four agents with synergistic activity with cefoxitin out of 16 agents with good MRSA activity alone. Finally, active agents from this effort were counter-screened in the presence of thymidine, which quickly identified three folate/thymidine biosynthesis inhibitors, and also screened for bactericidal vs bacteriostatic activity.

Antibiotic Resistance Profiles and Molecular Characteristics of blaCMY-2-Carrying Salmonella enterica Serovar Albany Isolated from Chickens During 2013-2020 in South Korea.

The production of ß-lactamase by nontyphoidal Salmonella has become a public health issue throughout the world. In this study, we aimed to investigate the antimicrobial resistance profiles and molecular characteristics of ß-lactamase-producing Salmonella enterica serovar Albany isolates. A total of 434 Salmonella Albany were obtained from feces and carcasses of healthy and diseased food-producing animals [cattle (n = 2), pigs (n = 3), chickens (n = 391), and ducks (n = 38)] during 2013-2020. Among the 434 Salmonella Albany isolates, 3.7% showed resistance to cefoxitin, and all the cefoxitin-resistant isolates were obtained from chickens. Moreover, Salmonella Albany isolates demonstrated high resistance to nalidixic acid (99.3%), trimethoprim/sulfamethoxazole (97.9%), ampicillin (86.6%), chloramphenicol (86.6%), and tetracycline (85.7%), as well as higher rates of multidrug resistance were detected in cefoxitin-resistant isolates compared to cefoxitin-susceptible isolates. All cefoxitin-resistant isolates harbored CMY-2-type ß-lactamase and belonged to seven different pulsotypes, with type IV-b (43.75%) and IV-a (25%) making up the majority. In addition, genes encoding cefoxitin resistant of all blaCMY-2-harboring Salmonella Albany isolates were horizontally transmitted to a recipient Escherichia coli J53 by conjugation. Furthermore, 93.75% (15/16) of conjugative plasmids harboring blaCMY-2 genes belong to ST12/CC12-IncI1. Genetic characteristics of transmitted blaCMY-2 genes were associated with ISEcp1, which can play an essential role in the effective mobilization and expression of these genes. Salmonella Albany containing blaCMY-2 in chickens can potentially be transferred to humans. Therefore, it is necessary to restrict antibiotic use and conduct continuous monitoring and analysis of resistant bacteria in the poultry industry.

Remarkable antibiofilm activity of ciprofloxacin, cefoxitin, and tobramycin, by themselves or in combination, against enteroaggregative Escherichia coli in vitro.

Enteroaggregative Escherichia coli (EAEC), a biofilm forming pathogen, causes acute and persistent diarrhea worldwide, requiring antimicrobial therapy in severe or persistent cases. To determine the susceptibility of EAEC biofilm to antimicrobials, as single-agent or combined therapy, biofilm formation was investigated using EAEC clinical strains via peg lid. Of the 78 initially analyzed strains, 35 could form biofilms, 15 (42.9%; 15/35) were resistant to at least 1 tested antimicrobial and 20 (57.1%) were susceptible to all of them in the planktonic form. The biofilms of these susceptible strains were challenged against chosen antimicrobials, and displayed resistance to tetracycline, trimethoprim-sulfamethoxazole, chloramphenicol, ampicillin, cefotaxime, ceftriaxone (85%-100%), tobramycin (25%), cefoxitin (20%), and ciprofloxacin (5%). Moreover, ciprofloxacin combined with ampicillin, and tobramycin eradicated the biofilm of 2 of the 4 tested strains. Ciprofloxacin, cefoxitin, and tobramycin maintained their activity well against EAEC biofilm, suggesting their possible effectiveness to treat diarrhea caused by biofilm-forming EAEC strains.

Detection of Genes Encoding Microbial Surface Component Recognizing Adhesive Matrix Molecules in Methicillin-Resistant Staphylococcus aureus Isolated from Pyoderma Patients.

Pyoderma is a common skin infection predominantly caused by Staphylococcus aureus. In addition to methicillin resistance, this pathogen is resistant to many other antibiotics, which ultimately limits the available treatment options. Therefore, the present study aimed to compare the antibiotic-resistance pattern, to detect the mecA gene and the genes encoding microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) in S. aureus isolates. A total of 116 strains were isolated from patients suffering with pyoderma. Disk diffusion assay was opted to perform antimicrobial susceptibility testing of the isolates. Out of the isolates tested, 23-42.2% strains appeared susceptible to benzylpenicillin, cefoxitin, ciprofloxacin and erythromycin. While linezolid was found to be the most effective anti-staphylococcal drug, followed by rifampin, chloramphenicol, clindamycin, gentamicin and ceftaroline. Out of 116 isolates, 73 (62.93%) were methicillin-resistant S. aureus (MRSA). Statistically significant (p ≤ 0.05) differences in antibiotic resistance patterns between MRSA and methicillin-susceptible S. aureus (MSSA) were found. A significant association of resistance to ceftaroline, rifampin, tetracycline, ciprofloxacin, clindamycin, trimethoprim-sulfamethoxazole and chloramphenicol was found in MRSA. However, no significant difference was observed between MRSA and MSSA for resistance against gentamicin, erythromycin or linezolid. All cefoxitin-resistant S. aureus, nonetheless, were positive for the mecA gene. femA was found in all the MRSA isolates. Among other virulence markers, bbp and fnbB were found in all the isolates, while can (98.3%), clfA and fnbA (99.1%) were present predominately in MRSA. Thus, this study offers an understanding of antibiotic resistance MSCRAMMs, mecA, and femA gene patterns in locally isolated strains of S. aureus.

Geographic patterns of antimicrobial susceptibilities for Bacteroides spp. worldwide: Results from the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2007-2020.

Antimicrobial susceptibilities of 4973 Bacteroides spp. isolates recovered from various sources of patients from 12 countries (99.6% from European countries) in the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2007-2020, were investigated. The minimum inhibitory concentrations (MICs) of the isolates with six commonly used agents were determined using the agar dilution method. Among the isolates, 10 Bacteroides spp. were included: B. fragilis (n=3180, 64.0%) was encountered most frequently, followed by B. thetaiotaomicron (n=675) and B. ovatus (n=409). During the 14 years, the proportion of B. fragilis declined, but the proportion of non-fragilis Bacteroides spp. increased. More than 90% of the isolates tested were susceptible to piperacillin-tazobactam, meropenem and tigecycline. Significantly lower susceptibility rates to cefoxitin (P<0.001), clindamycin (P<0.001), piperacillin/tazobactam (P<0.001) and tigecycline (P=0.006) were observed among non-fragilis Bacteroides spp. isolates than among B. fragilis isolates. Moreover, the susceptibility rates to clindamycin (P=0.003) and tigecycline (P=0.044) decreased significantly among non-fragilis Bacteroides spp. over time. Clindamycin susceptibility rates >80% were found in Greece (100%), Sweden (86.3%) and the UK (80.7%), and the lowest susceptibility rates were found in the USA (42.9%) and Japan (53.9%). In conclusion, the susceptibility of Bacteroides spp. to commonly used antibiotics varied geographically. Empirical antibiotic therapy for suspected anaerobic infections with clindamycin and cefoxitin should be avoided due to high resistance rates. Piperacillin-tazobactam, meropenem, metronidazole and tigecycline could be considered favourable options for the treatment of infections caused by Bacteroides spp.

[Evaluation of Antimicrobial Susceptibilities of Rapidly Growing Mycobacteria]. / Hizli Üreyen Mikobakterilerin Antimikrobiyal Ilaçlara Duyarliliklarinin Belirlenmesi.

Infections related to the rapidly growing mycobacteria (RGM), which are common in the environment, have clinical significance as they can affect both immunocompromised and immunocompetent patients. Treatment of RGM related infections is difficult, because they are resistant to many of the first-line tuberculosis agents, require a long-term multiple drug regimen, which is costly, and is associated with drugrelated toxicities. The aim of this study was to investigate the in vitro antimicrobial susceptibility profiles of RGM isolated in Dokuz Eylül University Hospital and also to reveal epidemiological data. A total of 58 isolates [(Mycobacterium fortuitum (n= 35), Mycobacterium abscessus (n= 19) and Mycobacterium chelonae (n= 4)], which were isolated in Dokuz Eylül University Hospital between 2013 and 2018, were subjected to in vitro testing for nine antimicrobial agents (amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, moxifloxacin and tobramycin) with the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI). For M.abscessus; 73.68% of the isolates were found susceptible to amikacin; 73.68% of isolates were susceptible to clarithromycin at early reading and only 21.05% of them remained susceptible at late reading time. No resistance to imipenem were observed. M.abscessus isolates were highly resistant to tobramycin, doxycycline and fluoroquinolones. Antibiotic susceptibility testing of M.chelonae isolates demonstrated 100% susceptibility for amikacin, clarithromycin and tobramycin. No resistance to linezolid, imipenem and moxifloxacin were observed. None of the isolates were susceptible to cefoxitin. Ciprofloxacin and doxycycline also showed poor in vitro activity against M.chelonae isolates. For M.fortuitum clarithromycin susceptibility decreased from 32.35% to 2.94% after an additional incubation until 14 days. All tested isolates of the M.fortuitum were susceptible to amikacin, ciprofloxacin and moxifloxacin. None of the M.fortuitum isolates exhibited resistance to cefoxitin and imipenem. Most of the M.fortuitum isolates were resistant to tobramycin and doxycycline. When the results were evaluated together, RGM isolates in this study were highly susceptible to amikacin; and were highly resistant to doxycycline. In conclusion, this study supported that the status of antimicrobial susceptibilities were different between species and also showed the importance for hospitals to know susceptibility patterns of isolates in their region. It should be noted that accurate species determination is critical for treatment as well as susceptibility status of rapidly growing mycobacteria to the antimicrobials in use.

Community acquired methicillin resistant Staphylococci (CA-MRS) in fecal matter of wild birds - A 'one health' point of concern.

BACKGROUND: Antibiotic resistance in Staphylococci, particularly methicillin resistance is a major public health concern. While this problem has been reported from the clinical settings, its presence in non-clinical settings also needs to be investigated. The role of wildlife in carrying and disseminating the resistant strains has been established in different studies but its role in Pakistani environment has not been explored yet. To evaluate this, we investigated the carriage of antibiotic resistant Staphylococci in wild birds from Islamabad region. METHODOLOGY: Birds fecal matter were collected during September 2016-August 2017 from eight different environmental settings of Islamabad. Prevalence of Staphylococci, their susceptibility profile against eight classes of antibiotics through disc diffusion method, their SCCmec types, co-resistance of macrolide and cefoxitin through PCR assay and biofilm formation through microtitre plate assay were studied. RESULTS: Out of 320 birds feces collected, 394 Staphylococci were isolated, where 165 (42%) were resistant to at least one or two classes of antibiotics. High resistance was found against erythromycin (40%) and tetracycline (21%) while cefoxitin resistance was 18% and vancomycin resistance was only in 2%. One hundred and three (26%) isolates exhibited multi-drug resistance (MDR) pattern. mecA gene was detected in 45/70 (64%) cefoxitin resistant isolates. Community acquired methicillin resistant Staphylococci (CA-MRS) were 87% while Hospital acquired methicillin resistant Staphylococci (HA-MRS) were 40%. In the MRS isolates showing co-resistance to macrolides, mefA (69%) and ermC (50%) genes were more prevalent. Strong biofilm formation was observed in 90% of the MRS, of which 48% were methicillin resistant Staphylococcus aureus (MRSA) isolates while 52% were methicillin resistant coagulase negative Staphylococci (MRCoNS). CONCLUSION: Occurrence of methicillin resistant strains of Staphylococci in wild birds suggests their role in the carriage and dissemination of resistant strains into the environment. The findings of the study strongly recommend the monitoring of resistant bacteria in wild birds and wildlife.

Probiotic disruption of quorum sensing reduces virulence and increases cefoxitin sensitivity in methicillin-resistant Staphylococcus aureus.

Therapies which target quorum sensing (QS) systems that regulate virulence in methicillin-resistant Staphylococcus aureus (MRSA) are a promising alternative to antibiotics. QS systems play a crucial in the regulation of MRSA antibiotic resistance, exotoxin production, antioxidant protection and immune cell evasion, and are therefore attractive therapeutic targets to reduce the virulence of a pathogen. In the present work the the effects of bioactive peptides isolated from two strains of lactic acid bacteria were tested against antibiotic resistance, carotenoid production, resistance to oxidative killing and biofilm structure in two clinical MRSA isolates. The results obtained from fractional-inhibitory concentration assays with bulk and semi-purified bioactive molecules showed a significant synergistic effect increasing cefoxitin mediated killing of MRSA. This was coupled to a six-fold decrease of the major membrane pigment staphyloxanthin, and a 99% increase in susceptibility to oxidative stress mediated killing. Real-time quantitative PCR analysis of the QS-genes agrA and luxS, showed differential expression between MRSA strains, and a significant downregulation of the hemolysin gene hla. Light microscopy and scanning electron microscopy revealed alteration in biofilm formation and clustering behavior. These results demonstrate that bioactive metabolites may be effectively applied in tandem with beta-lactam antibiotics to sensitize MRSA to cefoxitin. Moreover, these results shown that several key QS-controlled virulence mechanisms are diminished by probiotic metabolites.

Piperacillin/tazobactam versus cefepime or carbapenems for cefoxitin-non-susceptible Enterobacter cloacae, Klebsiella aerogenes, Citrobacter freundii, Serratia marcescens and Morganella morganii bacteraemia in immunocompromised patients.

BACKGROUND: The role of piperacillin/tazobactam for treatment of serious infections due to AmpC-producing organisms remains debatable, particularly in immunocompromised patients. METHODS: This was a retrospective cohort study in immunocompromised patients that investigated the effect of definitive treatment with either piperacillin/tazobactam versus cefepime or carbapenems for bacteraemia caused by cefoxitin-non-susceptible Enterobacterales. The primary endpoint was a composite of clinical and microbiological failure. A logistic regression model was constructed to assess the impact of definitive treatment choice on the primary endpoint. RESULTS: A total of 81 immunocompromised patients with blood cultures positive for cefoxitin-non-susceptible Enterobacterales were included for analysis. There was more microbiological failure in the piperacillin/tazobactam arm compared with the cefepime/carbapenem arm (11.4% versus 0.0%, P = 0.019). Definitive treatment with cefepime or a carbapenem was associated with a decreased odds of clinical or microbiological failure (OR 0.303, 95% CI 0.093-0.991, P = 0.048) when controlling for baseline characteristics. CONCLUSIONS: In immunocompromised patients with bacteraemia due to cefoxitin-non-susceptible Enterobacterales, definitive treatment with piperacillin/tazobactam was associated with an increased risk of microbiological failure and higher odds of clinical or microbiological failure compared with cefepime or carbapenems.

Brazilian red propolis in combination with ß-lactams exerts an efficient antibacterial action over methicillin-resistant Staphylococcus aureus (MRSA) strains.

AIMS: The antibacterial activity of red propolis extract (RPE) and brown propolis extracts (BPE) and the synergistic effect of RPE with cefoxitin (CEFO), imipenem (IMI), and ertapenem (ERTA) was evaluated in vitro against methicillin-resistant Staphylococcus aureus (MRSA) strains. METHODS AND RESULTS: MRSA ATCC 33591, community-associated (CA-MRSA) USA300, and four clinical isolates were used. A broth microdilution assay was performed to obtain inhibitory and bactericidal concentrations of BPE, RPE, CEFO, IMI, and ERTA. RPE in combination with CEFO, IMI, and ERTA was evaluated on the formation or eradication of biofilm. The bacterial relative membrane conductivity of the strains was assessed after RPE and combinations exposition. Surface/binding computational analyzes between RPE compounds and penicillin binding protein 2a (PBP2a) were performed. BPE samples had no activity against MRSA (MICs 3.2-5 g l-1; MBCs 10-15 g l-1), so the subsequent assays were carried out only with RPE and antimicrobials. RPE exerted a bacteriostatic action (MICs 0.0156-0.125 g l-1; MBCs 0.5-2 g l-1) but the combinations with IMI and ERTA showed the highest inhibition, as observed in the time-kill curve. However, the FICI index showed synergism (≥0.5) only to RPE + IMI. This combination was the most effective in inhibiting the biofilm and showed the highest values of membrane conductivity. Computational predictions indicated that RPE constituents may interact with PBP2a. CONCLUSION: RPE and RPE + IMI exerted an antibacterial and antibiofilm activity on MRSA strains probably due to membrane/wall damage and interactions with PBP2a.

Identification of staphylococcal cassette chromosome mec in Staphylococcus aureus and non-aureus staphylococci from dairy cattle in Belgium: Comparison of multiplex PCR and whole genome sequencing.

The present study compared multiplex PCR (mPCR) and Whole Genome Sequencing (WGS) using the SCCmecFinder database to identify the Staphylococcal Cassette Chromosome (SCC) mec in five Staphylococcus aureus (SA) and nine non-aureus staphylococci (NAS) isolated from dairy cattle. mPCR identified an SCCmecIV in four SA and one NAS, but could not differentiate between SCCmecII and IV in the fifth SA, that all harbored the mecA gene and were phenotypically resistant to cefoxitin. SCCmecFinder confirmed the presence of an SCCmecIVc(2B) in four SA and of the SCCmecIVa(2B) in the fifth SA and the one NAS. Both methods also detected one untypeable SCCmec in another cefoxitin-resistant NAS harboring the mecA gene and a pseudo SCCmec in one cefoxitin-sensitive NAS harboring one mecC-related gene. No SCCmec elements were identified either in one cefoxitin-sensitive NAS harboring the mecA2 gene, or in five NAS (one resistant and four sensitive to cefoxitin) harboring the mecA1 gene. SCCmecFinder could even not identify the presence of any mecA1 gene in these five NAS, whose presence was nevertheless confirmed by ResFinder. The conclusions of this study are: (i) mPCR and WGS sequencing using SCCmecFinder are complementary methodologies to identify SCCmec; (ii) SCCmecFinder and ResFinder to a lesser extent cannot identify all mec gene allotypes; (iii) a specific classification of the SCCmec in NAS would be epidemiologically helpful; (iv) presence of a mecA gene and a complete SCCmec is linked to cefoxitin resistance, whereas presence of other mec genes and of pseudo or no SCCmec is not.

MIC Discrepancies between Parenteral and Oral Anti-Staphylococcal Beta-Lactams among MSSA.

INTRODUCTION: Recent evidence has shown that oral antibiotic therapy is not inferior to IV antibiotic therapy in the treatment of complicated Staphylococcus aureus infections. Therefore, oral antibiotic therapy is now frequently prescribed in clinical practice due to cost benefit, ease of administration, decreased complication rate, and lack of need for IV access. In vitro susceptibility testing for ß-lactam oral antibiotics is not routinely performed as the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) recommend using oxacillin and cefoxitin as surrogate markers. Hence, oral antibiotic susceptibilities for cephalexin and dicloxacillin are not reported and implied based on oxacillin and cefoxitin. The objective of the current study was to determine whether susceptibilities among S. aureus isolates are predictable when comparing commonly used IV and oral beta-lactams. METHODS: Cefazolin, cephalexin, dicloxacillin, and oxacillin broth microdilution minimum inhibitory concentrations (MICs) were determined for 100 clinical isolates of methicillin-sensitive S. aureus by broth microdilution following CLSI guidelines. RESULTS: Among these isolates, median MICs for cephalexin were eight-fold higher than cefazolin MICs and median MICs for dicloxacillin were four-fold less than oxacillin MICs. Ten percent of more strains studied had a major or very major error in its susceptibility reporting when cephalexin was compared to its surrogate marker oxacillin. DISCUSSIONS/CONCLUSIONS: The variations in MICs observed compounded with the dosing and pharmacokinetic differences of oral versus IV ß-lactam suggests that establishing breakpoints for oral ß-lactam antibiotics is necessary to ensure adequate therapy is selected for the treatment of complex S. aureus infections.

Unraveling antibiotic resistance mechanisms in Mycobacterium abscessus: the potential role of efflux pumps.

OBJECTIVES: Mycobacterium abscessus is an opportunistic respiratory pathogen in patients with underlying lung disease. It is infamously known for its low treatment success rates because of its resistance to multiple classes of antibiotics. Further insight into M. abscessus resistance mechanisms is needed to improve treatment options. In this in vitro study, the role of efflux pumps in reaction to antibiotic stress is explored, as well as the ability of the putative efflux inhibitors, thioridazine and verapamil, to potentiate the activity of guideline-recommended antibiotics. METHODS: To evaluate the effects of antibiotic stress on mycobacterial efflux pumps, M. abscessus subspecies abscessus was exposed to amikacin, cefoxitin, clarithromycin, clofazimine, and tigecycline for 24 hours. Transcriptomic responses were measured by RNA sequencing to gain insight into upregulation of efflux pump encoding genes. Subsequently, in time-kill kinetics assays, the above-mentioned antibiotics were combined with thioridazine and verapamil to evaluate their potentiating capacity. RESULTS: All five antibiotics led to a fold change of ≥2 Log2 in expression of one or more genes encoding transporter systems. This effect was most pronounced for the ribosome-targeting antibiotics amikacin, clarithromycin, and tigecycline. Time-kill kinetics assays demonstrated synergy between amikacin, tigecycline, clofazimine, cefoxitin, and both thioridazine and verapamil. CONCLUSION: Antibiotic stressors induce expression of efflux pump encoding genes in M. abscessus, especially antibiotics that target the ribosome. Putative efflux inhibitors thioridazine and verapamil show synergy with various guideline-recommended antibiotics, making them interesting candidates for the improvement of M. abscessus treatment.

Prevalence and distribution of ampc beta-lactamase producing escherichia coli and klebsiella pneumoniae isolates obtained from urine samples at a tertiary care hospital in the caribbean.

Introduction: The aim of this study was to investigate the prevalence and distribution of AmpC beta-lactamases (BLs) in uropathogens (E. coli and K. pneumoniae) at the University Hospital of the West Indies Jamaica (UHWI). Method: De-duplicated consecutive urine samples, collected from January to March 2020 at the UHWI, were analyzed. Screening and phenotypic confirmatory tests were conducted using resistance to cefoxitin and the Disc Approximation Test (DAT) respectively, for isolates of interest. Multiplex PCR was performed on cefoxitin resistant (CR) isolates for the detection of bla CIT, bla MOX, bla FOX, bla ACC, and bla DHA genes. Whole genome sequencing (WGS) was used to further detect AmpC BL genes in PCR negative isolates with indeterminate phenotypic results. Results: Sixty-four Gram negative isolates were obtained from 61 patients (55% female), aged 18 months to 88 years old. At least 35% (26) had complicated urinary tract infections. Only 7 out of 64 isolates were E. coli or K. pneumoniae, had antibiograms suggestive of possible AmpC BL production and were CR. DATs confirmed AmpC BL in two of these (1 K. pneumoniae; 1 E. coli), one tested negative (E. coli) and four had inconclusive results (K. pneumoniae). PCR detected bla DHA and bla CIT in two CR isolates. WGS further detected bla CMY-42 in one isolate. The prevalence of screened CR isolates with AmpC BL is 57.14% (4 of 7), representing 6.25% of the sample. AmpC BL producers tested had 100% susceptibility to meropenem and nitrofurantoin. Conclusion: AmpC BL prevalence among E. coli and K. pneumoniae, common urinary pathogens, in the studied isolates is low. Although cefoxitin screening is helpful, phenotypic screening using the DAT can yield indeterminate results best clarified by molecular testing.

A simple label-free method reveals bacterial growth dynamics and antibiotic action in real-time.

Understanding the response of bacteria to environmental stress is hampered by the relative insensitivity of methods to detect growth. This means studies of antibiotic resistance and other physiological methods often take 24 h or longer. We developed and tested a scattered light and detection system (SLIC) to address this challenge, establishing the limit of detection, and time to positive detection of the growth of small inocula. We compared the light-scattering of bacteria grown in varying high and low nutrient liquid medium and the growth dynamics of two closely related organisms. Scattering data was modelled using Gompertz and Broken Stick equations. Bacteria were also exposed meropenem, gentamicin and cefoxitin at a range of concentrations and light scattering of the liquid culture was captured in real-time. We established the limit of detection for SLIC to be between 10 and 100 cfu mL-1 in a volume of 1-2 mL. Quantitative measurement of the different nutrient effects on bacteria were obtained in less than four hours and it was possible to distinguish differences in the growth dynamics of Klebsiella pneumoniae 1705 possessing the BlaKPC betalactamase vs. strain 1706 very rapidly. There was a dose dependent difference in the speed of action of each antibiotic tested at supra-MIC concentrations. The lethal effect of gentamicin and lytic effect of meropenem, and slow bactericidal effect of cefoxitin were demonstrated in real time. Significantly, strains that were sensitive to antibiotics could be identified in seconds. This research demonstrates the critical importance of improving the sensitivity of bacterial detection. This results in more rapid assessment of susceptibility and the ability to capture a wealth of data on the growth dynamics of bacteria. The rapid rate at which killing occurs at supra-MIC concentrations, an important finding that needs to be incorporated into pharmacokinetic and pharmacodynamic models. Importantly, enhanced sensitivity of bacterial detection opens the possibility of susceptibility results being reportable clinically in a few minutes, as we have demonstrated.

Detection of mecA gene and methicillin-resistant Staphylococcus aureus (MRSA) isolated from milk and risk factors from farms in Probolinggo, Indonesia.

Background: Staphylococcus aureus is commonly found in dairy cows and is a source of contamination in milk. S. aureus that are resistant to beta-lactam antibiotics are referred to as methicillin-resistant Staphylococcus aureus (MRSA). The spread of MRSA cannot be separated from sanitation management during milking; it can originate from milk collected from the udder or from the hands of farmers during the milking process. The purpose of this study was to examine the level of MRSA contamination in dairy cow's milk and farmer's hand swabs. Methods: A total of 109 samples of dairy cow's milk and 41 samples of farmers' hand swabs were collected at a dairy farm in Probolinggo, East Java, Indonesia. Samples were cultured and purified using mannitol salt agar (MSA). The profile of S. aureus resistance was established by disk diffusion test using a disk of beta-lactam antibiotics, namely oxacillin and cefoxitin. Results: The S. aureus isolates that were resistant to oxacillin and cefoxitin antibiotics were then tested for oxacillin resistance screening agar base (ORSAB) as a confirmation test for MRSA identity. S. aureus isolates suspected to be MRSA were then tested genotypically by polymerase chain reaction (PCR) method to detect the presence of the mecA gene. The results of the isolation and identification found 80 isolates (53.33%) of S. aureus. The results of the resistance test found that 42 isolates (15%) of S. aureus were resistant to oxacillin and 10 isolates (12.5%) were resistant to cefoxitin. The ORSAB test found as many as 20 isolates (47.62%) were positive for MRSA. In PCR testing to detect the presence of the mecA gene, three isolates (30%) were positive for the mecA gene. Conclusions: This study shows that several S. aureus isolates were MRSA and had the gene encoding mecA in dairy farms.

Biogenic synthesis of CuO-NPs as nanotherapeutics approaches to overcome multidrug-resistant Staphylococcus aureus (MDRSA).

Due to the misuse of antibiotics, the multidrug-resistant Staphylococcus aureus (MDRSA) has caused serious infections and become more difficult to deal with. Here we propose to synthesise copper oxide nanoparticles (CuO-NPs) using a cell-free filter of Streptomyces rochei to enhance antibiotics activity against (MDRSA) and kill them. Characterisation of CuO-NPs using ultraviolet, dynamic light scattering, zeta potential, transmission electron microscopic (TEM), and X-ray diffraction, were investigated. The antibacterial action of the CuO-NPs was tested against standard strain and clinical isolates using the agar well diffusion method and the microdilution assay. The results showed the monodispersed spherical shape CuO-NPs with a mean diameter of 10.7 nm and were found to be active against (MDRSA). By a combination of CuO-NPs with different antibiotics, the highest synergistic effect was observed with cefoxitin, the minimum inhibitory concentration (MIC) was reduced to 6.5 for CuO-NPs, and 19.5 for cefoxitin. Time-kill assay showed the highest reduction in log10 colony-forming unit (CFU)/ml of initial inoculum of MRSA after 24 h. The HFB-4 cells cultured in the presence of CuO-NPs showed normal morphology with 100% viability at 8 µg/ml. TEM showed that combination (1/4 MIC cefoxitin +1/16 MIC CuO-NPs) highly damages bacterial cells' shape. The biosynthesis CuO-NPs showed antibacterial activity against S. aureus suggesting a promising alternative in clinical.