Clavulanic Acid Decreases Cocaine Cue Reactivity in Addiction-Related Brain Areas, a Randomized fMRI Pilot Study.

Background: Preclinical studies show that clavulanic acid (CLAV) inhibits cocaine self-administration. This study investigates the effect of CLAV on regions of brain activation in response to cocaine cues during functional magnetic resonance imaging (fMRI) in participants with cocaine use disorder (CUD). Methods: A double-masked, placebo-controlled clinical trial with thirteen individuals with severe CUD who were randomized to treatment with CLAV (N = 10, 9 completers) 500 mg/day or matched placebo (PBO) (N = 3) for 3 days. fMRI was used to assess brain reactivity to 18 alternating six-second video clips of cocaine or neutral scenes. In this paradigm, participants were exposed to three different stimulus conditions: NEUTRAL, WATCH (passive watching), and DOWN (actively inhibiting craving while watching). Results: Participants who received CLAV demonstrated a significant reduction in brain activity in the anterior cingulate gyrus (p = 0.009) and the caudate (p = 0.018) in response to DOWN cocaine cues. There was a trend toward lessened cue reactivity in other regions implicated in CUD. Conclusion: CLAV reduced the response of the brain regions associated with motivation and emotional response during the DOWN condition compared to PBO, suggesting CLAV may strengthen voluntary efforts to avoid cocaine use. This pilot data supports the use of CLAV for CUD. (Trial registered in ClinicalTrials.gov NCT04411914).

Ethambutol and meropenem/clavulanate synergy promotes enhanced extracellular and intracellular killing of Mycobacterium tuberculosis.

Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy, but further research on their interaction with conventional anti-TB agents is still warranted. Moreover, the complex cell envelope of Mycobacterium tuberculosis (Mtb) may pose an additional obstacle to beta-lactam diffusion. In this context, we aimed to identify synergies between beta-lactams and anti-TB drugs ethambutol (EMB) and isoniazid (INH) by assessing antimicrobial effects, intracellular activity, and immune responses. Checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant strains, exposed that only treatments containing EMB and beta-lactams achieved synergistic effects. Meanwhile, the standard EMB and INH association failed to produce any synergy. In Mtb-infected THP-1 macrophages, combinations of EMB with increasing meropenem (MEM) concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan (PG), confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1ß secretion levels found in infected macrophages after incubation with MEM concentrations above 5 mg/L, indicating an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with EMB. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and PG synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.IMPORTANCEAddressing drug-resistant tuberculosis with existing therapies is challenging and the treatment success rate is lower when compared to drug-susceptible infection. This study demonstrates that pairing beta-lactams with ethambutol (EMB) significantly improves their efficacy against Mycobacterium tuberculosis (Mtb). The presence of EMB enhances beta-lactam access through the cell wall, which may translate into a prolonged contact between the drug and its targets at a concentration that effectively kills the pathogen. Importantly, we showed that the effects of the EMB and meropenem (MEM)/clavulanate combination were maintained intracellularly. These results are of high significance considering that the time above the minimum inhibitory concentration is the main determinant of beta-lactam efficacy. Moreover, a correlation was established between incubation with higher MEM concentrations during macrophage infection and increased IL-1ß secretion. This finding unveils a previously overlooked aspect of carbapenem repurposing against tuberculosis, as certain Mtb strains suppress the secretion of this key pro-inflammatory cytokine to evade host surveillance.

Comprehensive stability analysis of 13 ß-lactams and ß-lactamase inhibitors in in vitro media, and novel supplement dosing strategy to mitigate thermal drug degradation.

Non-clinical antibiotic development relies on in vitro susceptibility and infection model studies. Validating the achievement of the targeted drug concentrations is essential to avoid under-estimation of drug effects and over-estimation of resistance emergence. While certain ß-lactams (e.g., imipenem) and ß-lactamase inhibitors (BLIs; clavulanic acid) are believed to be relatively unstable, limited tangible data on their stability in commonly used in vitro media are known. We aimed to determine the thermal stability of 10 ß-lactams and 3 BLIs via LC-MS/MS in cation-adjusted Mueller Hinton broth at 25 and 36°C as well as agar at 4 and 37°C, and in water at -20, 4, and 25°C. Supplement dosing algorithms were developed to achieve broth concentrations close to their target over 24 h. During incubation in broth (pH 7.25)/agar, degradation half-lives were 16.9/21.8 h for imipenem, 20.7/31.6 h for biapenem, 29.0 h for clavulanic acid (studied in broth only), 23.1/71.6 h for cefsulodin, 40.6/57.9 h for doripenem, 46.5/64.6 h for meropenem, 50.8/97.7 h for cefepime, 61.5/99.5 h for piperacillin, and >120 h for all other compounds. Broth stability decreased at higher pH. All drugs were ≥90% stable for 72 h in agar at 4°C. Degradation half-lives in water at 25°C were >200 h for all drugs except imipenem (14.7 h, at 1,000 mg/L) and doripenem (59.5 h). One imipenem supplement dose allowed concentrations to stay within ±31% of their target concentration. This study provides comprehensive stability data on ß-lactams and BLIs in relevant in vitro media using LC-MS/MS. Future studies are warranted applying these data to antimicrobial susceptibility testing and assessing the impact of ß-lactamase-related degradation.

Positive epistasis drives clavulanic acid resistance in double mutant libraries of BlaC ß-lactamase.

Phenotypic effects of mutations are highly dependent on the genetic backgrounds in which they occur, due to epistatic effects. To test how easily the loss of enzyme activity can be compensated for, we screen mutant libraries of BlaC, a ß-lactamase from Mycobacterium tuberculosis, for fitness in the presence of carbenicillin and the inhibitor clavulanic acid. Using a semi-rational approach and deep sequencing, we prepare four double-site saturation libraries and determine the relative fitness effect for 1534/1540 (99.6%) of the unique library members at two temperatures. Each library comprises variants of a residue known to be relevant for clavulanic acid resistance as well as residue 105, which regulates access to the active site. Variants with greatly improved fitness were identified within each library, demonstrating that compensatory mutations for loss of activity can be readily found. In most cases, the fittest variants are a result of positive epistasis, indicating strong synergistic effects between the chosen residue pairs. Our study sheds light on a role of epistasis in the evolution of functional residues and underlines the highly adaptive potential of BlaC.

Antibiotic Resistance Profile of Clinical Strains of Enterococci from Secondary/Persistent Endodontic Infections: What do We Know? A Systematic Review of Clinical Studies.

INTRODUCTION: Enterococcus faecalis is the most common enterococcal species associated with infective endocarditis and 1 of the most commonly detected bacteria in cases of secondary/persistent endodontic infection (SPEI). Antimicrobial resistance is a global public health concern. This review aimed to answer the following research question: "Is there a change in the antibiotic resistance profile in clinical strains of E. faecalis over the years?". P (population) - patients with SPEI, I (intervention) -endodontic retreatment, C (comparison) -not included, O (outcome) - profile of Enterococci resistance and susceptibility to systemic antibiotics used. METHODS: Two authors independently performed study selection, data extraction, and risk of bias assessment. The literature search was conducted using the following electronic databases: PubMed, Scopus, EMBASE, Web of Science, and Medline. Clinical studies in which Enterococci strains were isolated to assess their antimicrobial resistance were included. RESULTS: Eleven clinical trials were included. Overall, E. faecalis isolated from teeth with SPEI presented an intermediate resistance to 16 antibiotics. In recent years, E. faecalis showed a little resistance to amoxicillin (without clavulanate) and benzylpenicillin. Erythromycin and rifampicin presented an increase in the intermediate-resistance status between the first and the last studies. E. faecium presented intermediate-resistance results. CONCLUSION: The most effective drugs remain the combination of amoxicillin and clavulanate, followed by amoxicillin and benzylpenicillin. In patients allergic to penicillin derivatives, moxifloxacin and azithromycin may be indicated with caution. The antibiotics with the highest pattern of resistance against E. faecalis are clindamycin, gentamicin, metronidazole, and rifampicin and are therefore, contraindicated in cases of SPEI. Very few clinical studies using a microbiological approach in teeth with endodontic failure have been carried out to improve the efficacy of prophylactic regimens. However, as bacteria periodically develop resistance to the main drugs used, regular studies should be carried out on the action of these drugs in infection control.

Clavulanic Acid and its Potential Therapeutic Effects on the Central Nervous System.

Clavulanic acid (CLAV) is a non-antibiotic ß-lactam that has been used since the late 1970s as a ß-lactamase inhibitor in combination with amoxicillin, another ß-lactam with antibiotic activity. Its long-observed adverse reaction profile allows it to say that CLAV is a well-tolerated drug with mainly mild adverse reactions. Interestingly, in 2005, it was discovered that ß-lactams enhance the astrocytic expression of GLT-1, a glutamate transporter essential for maintaining synaptic glutamate homeostasis involved in several pathologies of the central nervous system (CNS). This finding, along with a favorable pharmacokinetic profile, prompted the appearance of several studies that intended to evaluate the effect of CLAV in preclinical disease models. Studies have revealed that CLAV can increase GLT-1 expression in the nucleus accumbens (NAcc), medial prefrontal cortex (PFC), and spinal cord of rodents, to affect glutamate and dopaminergic neurotransmission, and exert an anti-inflammatory effect by modulating the levels of the cytokines TNF-α and interleukin 10 (IL-10). CLAV has been tested with positive results in preclinical models of epilepsy, addiction, stroke, neuropathic and inflammatory pain, dementia, Parkinson's disease, and sexual and anxiety behavior. These properties make CLAV a potential therapeutic drug if repurposed. Therefore, this review aims to gather information on CLAV's effect on preclinical neurological disease models and to give some perspectives on its potential therapeutic use in some diseases of the CNS.

Clavulanic Acid Improves Memory Dysfunction and Anxiety Behaviors through Upregulating Glutamatergic Transporters in the Nucleus Accumbens of Mice Repeatedly Exposed to Khat Extract.

Khat (Catha edulis) is an evergreen shrub whose buds and leaves give a state of delight and euphoria when chewed. Cathinone, an amphetamine-like stimulant that is among the active ingredients in khat, is able to downregulate glutamate transporter subtype I (GLT-1). Neurobehavioral dysfunctions such as altered locomotor activity, anorexia, and nociception have been observed in animals exposed to cathinone. Interestingly, treatment with a ß-lactam antibiotic such as ceftriaxone, which upregulates GLT-1, normalizes cathinone-induced conditioned place preference, and alters repetitive movements in rats. However, little is known about the role of the glutamatergic system in memory dysfunction and anxiety-like behaviors in mice exposed to khat. We found here that clavulanic acid, a ß-lactam-containing compound and GLT-1 upregulator, would modulate the neurobehavioral changes, including memory impairment and anxiety-like behaviors, associated with repeated exposure of mice to khat. Our data supported that clavulanic acid could improve memory impairment and anxiety-like behaviors through upregulating GLT-1 in the nucleus accumbens (NAc), an effect abolished with a selective GLT-1 blocker. This upregulation was associated with restored glutamate/cystine antiporter expression in the NAc using a Western blotting assay. Cathine and cathinone were identified in khat extract using the gas chromatography technique. Our work provides preclinical insight into the efficacy of ß-lactam-containing compounds for the attenuation of neurobehavioral changes induced by khat exposure.

Efficacy of Raphamin against Pneumococcal Infection: a Preclinical Study.

The aim of the study was to evaluate the activity of Raphamin in a model of non-lethal pneumococcal infection caused by Streptococcus pneumoniae 3 in BALB/c mice. The drug or placebo was administered intragastrically 3 days prior to infection, 2 h before and 2 h post infection, and then for 3 full days, alone or in combination with antibiotic (amoxicil-lin/clavulanic acid). Raphamin monotherapy significantly decreased bacterial load in the lungs in comparison with placebo (p<0.05) which was comparable to the effect in antibiotic alone or combined with Raphamin. Raphamin prevented reproduction of Streptococcus pneumoniae in the lower respiratory tract and its combination with the antibiotic was safe and did not reduce the efficacy of amoxicillin/clavulanic acid.

Comparative metabolomics reveal key pathways associated with the synergistic activities of aztreonam and clavulanate combination against multidrug-resistant Escherichia coli.

IMPORTANCE: Multidrug-resistant Escherichia coli is a major threat to the health care system and is associated with poor outcomes in infected patients. The combined use of antibiotics has become an important treatment method for multidrug-resistant bacteria. However, the mechanism for their synergism has yet to be explored.

Cryptic susceptibility to penicillin/ß-lactamase inhibitor combinations in emerging multidrug-resistant, hospital-adapted Staphylococcus epidermidis lineages.

Global spread of multidrug-resistant, hospital-adapted Staphylococcus epidermidis lineages underscores the need for new therapeutic strategies. Here we show that many S. epidermidis isolates belonging to these lineages display cryptic susceptibility to penicillin/ß-lactamase inhibitor combinations under in vitro conditions, despite carrying the methicillin resistance gene mecA. Using a mouse thigh model of S. epidermidis infection, we demonstrate that single-dose treatment with amoxicillin/clavulanic acid significantly reduces methicillin-resistant S. epidermidis loads without leading to detectable resistance development. On the other hand, we also show that methicillin-resistant S. epidermidis is capable of developing increased resistance to amoxicillin/clavulanic acid during long-term in vitro exposure to these drugs. These findings suggest that penicillin/ß-lactamase inhibitor combinations could be a promising therapeutic candidate for treatment of a high proportion of methicillin-resistant S. epidermidis infections, although the in vivo risk of resistance development needs to be further addressed before they can be incorporated into clinical trials.

Multidrug resistant and multivirulent avian bacterial pathogens: tackling experimental leg disorders using phytobiotics and antibiotics alone or in combination.

Locomotor disorders caused by multidrug-resistant (MDR) bacterial pathogens denote one of the most detrimental issues that collectively threaten the poultry industry leading to pronounced economic losses across the world. Hence, searching for effective alternatives, especially those extracted from plant origins became of great priority targeting a partial or complete replacement of chemical antimicrobials to tackle their developing resistance. Therefore, we aimed to determine the prevalence and antimicrobial resistance of Staphylococcus aureus (S. aureus), Salmonella species, Mycoplasma synoviae (M. synoviae), and Escherichia coli (E. coli) recovered from 500 broilers and ducks (250 each) with locomotor disorders in various farms in Dakahlia and Sharkia Governorates, Egypt. Additionally, we assessed, for the first time, the in vitro antimicrobial effectiveness of marjoram, garlic, ginger and cinnamon essential oils (EOs) against MDR and multivirulent bacterial isolates as well as the in vivo efficiency of the most effective antibiotics and EOs either separately or in combination in the treatment of experimentally induced poultry leg disorders. The overall prevalence rates of S. aureus, E. coli, Salmonella species, and M. synoviae were 54, 48, 36, and 2%, respectively. Salmonella species and S. aureus prevailed among ducks and broilers (36 and 76%, respectively). Notably, MDR was observed in 100, 91.7, 81.1, and 78.5% of M. synoviae, E. coli, Salmonella, and S. aureus isolates, respectively. Our in vitro results displayed that marjoram was the most forceful EO against MDR and multivirulent chicken vancomycin-resistant S. aureus (VRSA) and duck S. Typhimurium isolates. The current in vivo results declared that marjoram in combination with florfenicol or amoxicillin/clavulanic acid succeeded in relieving the induced duck and chicken leg disorders caused by S. Typhimurium and VRSA, respectively. This was evidenced by improvement in the clinical and histopathological pictures with a reduction of bacterial loads in the experimental birds. Our encountered successful in vitro and in vivo synergistic effectiveness of marjoram combined with florfenicol or amoxicillin/clavulanic acid recommends their therapeutic application for leg disorders and offers opportunities for reducing the antibiotics usage in the poultry industry.

[In vitro activity of ß-lactamase inhibitors avibanvctam and relebactam in combination with ß-lactams against multidrug-resistant Mycobacterium tuberculosis and mutations of resistance genes].

Objective: To evaluate the activity of six ß-lactams in combination with three ß-lactamase inhibitors against mycobacterium tuberculosis(MTB) in vitro. Methods: A total of 105 multidrug-resistant tuberculosis (MDR-TB) strains from different regions of Henan province from January to September 2020 were included in this study. Drug activity of six ß-lactams (biapenem, meropenem, imipenem, doripenem, ertapenem and tebipenem) alone or in combination with ß-lactamase inhibitors (clavulanic acid, avibactam and relebactam) was examined by minimum inhibitory concentration method (MICs) against 105 clinical isolates. Mutations of blaC, ldtmt1 and ldtmt2 were analyzed by PCR and DNA sequencing. Chi-square test was used to compare the antimicrobial activities of different ß-lactam drugs. Results: Out of the ß-lactams used herein, tebipenem was the most effective against MDR-TB and had an MIC50 value of 8 mg/L(χ2=123.70,P=0.001). Besides, after the addition of ß-lactamase inhibitors, the MICs of most ß-lactam drugs were reduced more evidently in the presence of avibactam and relebactam compared to clavulanic acid.Especially, relebactam decreased both the MIC50 and MIC90 of telbipenem by 16-fold, and diluted the MIC of 23 (21.90%) and 41 (39.04%) isolatesby 32-fold and 16-fold.In addition, a total of 13.33% (14/105) of isolates harbored mutations in the blaC gene, with three different nucleotide substitutions: AGT333AGG, AAC638ACC and ATC786ATT. For the strains with Ser111Arg and Asn213Thr substitution in BlaC, the MIC values of the meropenem-clavulanate combination were reduced compared with a synonymous single nucleotide polymorphism (SNP) group. Conclusions: Both avibactam and relebactam had better synergistic effects on ß-lactams than clavulanic acid. The combination of tebipenem and relebactam showed the most potent activity against MDR-TB isolates. In addition, the Ser111Arg and Asn213Thr substitution of BlaC may be associated with an increased susceptibility of MDR-TB isolates to meropenem in the presence of clavulanate.

Antibiotic Resistance of Hemophilus influenzae Isolated from Children in Southwest China.

BACKGROUND: Hemophilus influenzae (Hi) is one of the major pediatric bacterial pneumonia pathogens that heavily threatens children's lives and global health. With widespread usage as first-line treatment, the prevalence of ß-lactam-resistant strains is increasing sharply. In order to treat Hi more effectively, a systematic study on the antibiotic resistance profiles, ß-lactamase-negative ampicillin-resistant (BLNAR) strains isolation rate, and potential BLNAR resistance mechanism in our region is needed. METHODS: This study analyzed antimicrobial susceptibility of Hi, and clinical data of Hi-infected patients retrospectively. BLNAR and ß-lactamase-positive ampicillin-clavulanate resistant strains (BLPACR) were confirmed by the Kirby-Bauer method and ß-lactamase test. ftsI gene in BLNAR was sequenced to find out whether resistance was induced by penicillin-binding protein mutation. Ampicillin susceptibility test with or without efflux pump inhibitors were done to assess efflux pump contribution in BLNAR. RT-PCR was performed to evaluate the efflux pump genes' transcription levels. RESULTS: A total of 2,561 Hi strains were isolated in our hospital from January 2016 to December 2019. Male to female ratio was 1.52:1. Median age was 10 months. Infant (< 3 years old) infection accounted for 83.72%. Hi resistance rates to sulfamethoxazole-trimethoprim, ampicillin, cefathiamidine, cefaclor, cefuroxime, cephalothin, amoxicillin-clavulanate, tetracycline, chloramphenicol, ofloxacin, cefotaxime, and rifampin were 84.28%, 78.01%, 49.80%, 41.98%, 36.58%, 33.64%, 4.55%, 4.1%, 3.37%, 1.77%, 0.99%, and 0.12%, respectively, while 1.33% were BLNAR. BLNARs were classified into four groups by mutation patterns in ftsI gene and most strains were divided to Group Ⅲ/Ⅲ-like. EmrB, ydeA and norM transcription levels in some ampicillin-resistant strains were higher than their sensitive counterparts. CONCLUSIONS: Ampicillin is not sufficiently effective as a first-line Hi infection treatment. However, ampicillin-clavulanate and cefotaxime may be a better choice. Efflux pumps, emrB, ydeA and norM play roles in the high resistance to ampicillin.

Breaking Antimicrobial Resistance: High-Dose Amoxicillin with Clavulanic Acid for Urinary Tract Infections Due to Extended-Spectrum Beta-Lactamase (ESBL)-Producing Klebsiella pneumoniae.

BACKGROUND Carbapenems are the primary treatment for urinary tract infections (UTIs) caused by extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae. However, the recurrence rate is high, and patients often require rehospitalization. We present the results of an observational study on patients with recurrent UTIs who were treated in an outpatient setting with maximal therapeutic oral doses of amoxicillin with clavulanic acid. MATERIAL AND METHODS All patients had pyuria and ESBL-producing K. pneumoniae in urine culture. The starting dosage was 2875 g of amoxicillin twice daily and 125 mg of clavulanic acid twice daily. We down-titrated the doses every 7-14 days and continued prophylactic therapy with amoxicillin/clavulanic acid at 250/125 mg for up to 3 months. We defined therapeutic failure as ESBL-positive K. pneumoniae in urine culture during therapy and recurrence as positive urine culture with the same strain within 1 month after the end of treatment. RESULTS We included 9 patients: 7 kidney graft recipients, 1 liver graft recipient, and 1 patient with chronic kidney disease. We observed no therapeutic failures and no recurrences in the study group during the study period. In 1 case, the patient experienced a subsequent UTI caused by ESBL-producing K. pneumoniae 4 months after completing the therapy. CONCLUSIONS In conclusion, it is possible to break the resistance of ESBL-producing K. pneumoniae strains with high doses of oral amoxicillin with clavulanic acid. Such treatment could be an alternative to carbapenems in select cases.

In-vitro activity of oral third-generation cephalosporins plus clavulanate against ESBL-producing Enterobacterales isolates from the MERINO trial.

Extended-spectrum-beta-lactamase (ESBL)-producing Enterobacterales as a cause of community-acquired uncomplicated urinary tract infection (UTI) is on the rise. Currently, there are minimal oral treatment options. New combinations of existing oral third-generation cephalosporins paired with clavulanate may overcome resistance mechanisms seen in these emerging uropathogens. Ceftriaxone-resistant Escherichia coli and Klebsiella pneumoniae containing CTX-M-type ESBLs or AmpC, in addition to narrow-spectrum OXA and SHV enzymes, were selected from blood culture isolates obtained from the MERINO trial. Minimum inhibitory concentration (MIC) values of third-generation cephalosporins (cefpodoxime, ceftibuten, cefixime, cefdinir), both with and without clavulanate, were determined. One hundred and one isolates were used with ESBL, AmpC and narrow-spectrum OXA genes (e.g. OXA-1, OXA-10) present in 84, 15 and 35 isolates, respectively. Susceptibility to oral third-generation cephalosporins alone was very poor. Addition of 2 mg/L clavulanate reduced the MIC50 values (cefpodoxime MIC50 2 mg/L, ceftibuten MIC50 2 mg/L, cefixime MIC50 2 mg/L, cefdinir MIC50 4 mg/L) and restored susceptibility (33%, 49%, 40% and 21% susceptible, respectively) in a substantial number of isolates. This finding was less pronounced in isolates co-harbouring AmpC. In-vitro activity of these new combinations may be limited in real-world Enterobacterales isolates co-harbouring multiple antimicrobial resistance genes. Pharmacokinetic/pharmacodynamic data would be useful for further evaluation of their activity.

Antimicrobial susceptibility patterns from urinary isolates obtained from cats (2013-2020).

BACKGROUND: Bacterial urinary tract infections have been associated with comorbidities and increased antimicrobial resistance over time. OBJECTIVE: To identify bacterial species, antimicrobial susceptibility patterns and risk factors associated with antimicrobial resistance. ANIMALS: Three hundred sixty-three positive urine cultures from 308 cats. METHODS: Bacterial species and antimicrobial susceptibility data from positive aerobic bacterial urine cultures from cats with growth of ≥103 colony forming units per milliliter (cfu/ml) were included. Medical records were reviewed, and bacteriuria was classified as sporadic bacterial cystitis, recurrent bacterial cystitis or subclinical bacteriuria (SBU). Multivariable logistic regression analysis was used to evaluate antimicrobial resistance risk factors. RESULTS: A total of 444 bacterial isolates from 363 bacteriuric episodes were identified. Escherichia coli (52%) and SBU (59%) were the most common organism and classification, respectively. When compared to other classifications of bacteriuria, Enterococcus spp. were more likely to be isolated from SBU episodes (P < .001), whereas E. coli was more likely to be isolated from sporadic bacterial cystitis episodes (P < .001). Recurrent bacterial cystitis was associated with an increased risk of antimicrobial resistance to amoxicillin/clavulanic acid (odds ratio [OR], 3.9; 95% confidence interval [CI], 1.3-11.3). The percent susceptibilities of all bacterial isolates to commonly prescribed antimicrobials were amoxicillin/clavulanic acid (72%), cefazolin (49%), enrofloxacin (61%), and trimethoprim/sulfamethoxazole (75%). Multidrug resistance was highest for Enterococcus faecium isolates (65%). CONCLUSIONS AND CLINICAL IMPORTANCE: No antimicrobial achieved >90% susceptible designation to all bacteria isolated highlighting the importance of performing urine culture and susceptibility testing, particularly for cats with recurrent bacterial cystitis.

Evaluation of Phenotypic Tests to Detect Extended-Spectrum ß-Lactamase (ESBL)-Producing Klebsiella oxytoca Complex Strains.

Klebsiella oxytoca complex (KoC) species may overproduce their chromosomal class A OXY ß-lactamases, conferring reduced susceptibility to piperacillin-tazobactam, expanded-spectrum cephalosporins and aztreonam. Moreover, since clavulanate maintains its ability to inhibit these enzymes, the resulting resistance phenotype may falsely resemble the production of acquired extended-spectrum ß-lactamases (ESBLs). In this work, a collection of 44 KoC strains of human and animal origin was characterized with whole-genome sequencing (WGS) and broth microdilution (BMD) susceptibility testing. Comparison of ESBL producers (n = 11; including CTX-M-15 [n = 6] and CTX-M-1 [n = 5] producers) and hyperproducers of OXYs (n = 21) showed certain phenotypic differences: piperacillin-tazobactam (MIC90s: 16 versus >64 µg/mL), cefotaxime (MIC90s: 64 versus 4 µg/mL), ceftazidime (MIC90s: 32 versus 4 µg/mL), cefepime (MIC90s: 8 versus 4 µg/mL) and associated resistance to non-ß-lactams (e.g., trimethoprim-sulfamethoxazole: 90.9% versus 14.3%, respectively). However, a clear phenotype-based distinction between the two groups was difficult. Therefore, we evaluated 10 different inhibitor-based confirmatory tests to allow such categorization. All tests showed a sensitivity of 100%. However, only combination disk tests (CDTs) with cefepime/cefepime-clavulanate and ceftazidime/ceftazidime-clavulanate or the double-disk synergy test (DDST) showed high specificity (100%, 95.5%, and 100%, respectively). All confirmatory tests in BMD or using the MIC gradient strip did not perform well (specificity, ≤87.5%). Of note, ceftazidime/ceftazidime-avibactam tests also exhibited low specificity (CDT, 87.5%; MIC gradient strip, 77.8%). Our results indicate that standard antimicrobial susceptibility profiles can raise some suspicion, but only the use of cefepime/cefepime-clavulanate CDT or DDST can guarantee distinction between ESBL-producing KoC strains and those hyperproducing OXY enzymes.

[Relationship of Hypervirulent Capsular Genotypes of Klebsiella pneumoniae with Antibiotic Susceptibility and Beta-Lactamase Genes]. / Klebsiella pneumoniae Hipervirülan Kapsül Genotiplerinin Antibiyotik Duyarliligi ve Beta-Laktamaz Genleri ile Iliskisi.

Klebsiella pneumoniae which is a causative agent of nosocomial infections, is protected from phagocytosis and the lethal effect of serum bactericidal proteins owing to its capsular polysaccharides (CPS). The important leading problem in treating infections caused by this bacterium that successfully develops antimicrobial resistance, especially via mobile genetic elements, is that it acquires beta-lactamase genes, which are responsible for the resistance against beta-lactam antibiotics. Due to the increase in studies targeting capsular polysaccharides in developing strategies for vaccination and treatment, we aimed to investigate the possible relationship of the capsular genotypes of K.pneumoniae isolates obtained from various clinical specimens with antibiotic susceptibility and beta-lactamase genes. In K.pneumoniae clinical isolates; K1, K2, K5, K20, K54 and K57, which are known as hypervirulent capsular types, were investigated by polymerase chain reaction (PCR) method. In isolates whose capsular genotypes were determined, antibiotic susceptibility was examined by Kirby-Bauer disc diffusion method. Colistin resistance was investigated by the broth microdilution method. Carbapenem resistance was confirmed with the carbapenem inactivation test. The beta-lactamase genes blaCTX-M1, blaCTX-M2, blaCTX-M9, blaCTX-M8/25, blaKPC, blaNDM-1, and blaOXA-48-like were investigated by using PCR. Of the 38 K.pneumoniae isolates whose capsular genotypes were determined, 15 (39.5%), 12 (31.6%), seven (18.4%), two (5.3%), one (2.6%) and one (2.6%) were K5-CPS, K2-CPS, K20-CPS, K1-CPS, K54-CPS and K57-CPS genotypes, respectively. blaOXA-48-like, blaNDM-1, and blaCTX-M1 were detected in 68.4, 10.5, and 7.9%, whereas coexistence of blaOXA-48-like with blaNDM-1, and blaOXA-48-like with blaCTX-M1 were determined in 7.9, and 5.3% of the isolates, respectively. The relationship of the blaCTX-M1 gene, detected only in three K20-CPS isolates, was found to be statistically significant with this genotype. In addition, of the blaNDM-1-positive four isolates, three were K5-CPS, and one was K2-CPS, while blaOXA-48-like was similarly detected mostly in K5-CPS and K2-CPS (10 and 9 isolates, respectively). Except for the two isolates that were resistant to colistin, one K1-CPS and the other K5-CPS, the highest resistance was detected against cefotaxime (36/38) and the lowest resistance was detected against gentamicin (23/38) as a result of antibiotic susceptibility tests. The resistance relationship of K5-CPS isolates with amoxicillin/clavulanate, piperacillin/tazobactam, cefoxitin and ertapenem and the susceptibility relationship of K20-CPs isolates with amoxicillin/clavulanate, piperacillin/tazobactam, imipenem, tetracycline and trimethoprim/sulfamethoxazole were found as statistically significant. Our study is the first to investigate the relationship between K.pneumoniae capsular genotypes and beta-lactamase genes in Turkey. As a result, it is believed that this research will contribute to the determination of vaccine and treatment options by providing data to wider and regional studies that will examine other capsule genotypes and antibiotic resistance genes to clarify the importance of the capsule in virulence.

The Effect of Amoxicillin with Clavulanate on the Microbiota of Tonsillar Tissue in Disease: a Randomized Control Trial.

Despite antibiotics being the primary medical treatment for recurrent tonsillitis, the impact of antibiotics on the tonsillar microbiome is not well understood. This study aimed to determine the effect of amoxicillin with clavulanate on the composition and quantity of bacteria in the tonsils of children with recurrent tonsillitis. A multicenter randomized clinical trial in Auckland, New Zealand was undertaken between August 1, 2017, and June 30, 2018. Sixty children undergoing tonsillectomy for the indication of recurrent tonsillitis were recruited for this study. Following random allocation, 30 participants were prescribed amoxicillin with clavulanate for the week before surgery. The remaining 30 received no antibiotics. Immediately following surgery, the crypts of the right and left tonsils were swabbed. Bacterial 16S rRNA gene-targeted amplicon sequencing and histological techniques were utilized. In the control group, there were significantly higher relative abundances of Haemophilus, Streptococcus, Neisseria, and Porphyromonas. Members from the genera Fusobacterium and Treponema were found to be significantly more abundant in the antibiotic group. There were no significant differences in the absolute quantities of bacteria between the groups. Microscopic examination found fewer bacterial microcolonies present in the tonsillar crypts of participants in the antibiotic group. Streptococcus pyogenes was not present in these bacterial microcolonies. These results suggest that a single course of antibiotics has a significant impact on the tonsil microbiota composition. The duration of this effect and the effect that the altered microbiome has on the course of the condition need to be determined. IMPORTANCE Several studies have identified the presence of multiple pathogenic bacteria in hyperplastic adenoids and palatine tonsils. However, there are currently no studies that utilize this technology to investigate the effect of oral antibiotics in children with recurrent tonsillitis on the tonsillar microbiome. This is the first study to investigate the effect of antibiotics on the microbiome of tonsillar tissue in children with recurrent tonsillitis using molecular techniques. This study has shown that participants who received amoxicillin with clavulanate immediately before tonsillectomy had a significantly reduced number of bacterial taxa commonly associated with recurrent tonsillitis, as well as the number of bacterial microcolonies observed in the tonsillar crypts. This novel finding suggests that either the effect of antibiotics is not sustained or that they are not an effective treatment for recurrent tonsillitis.

Evaluation of antibacterial activity of five biocides and the synergistic effect of biocide/EDTA combinations on biofilm-producing and non-producing Stenotrophomonas maltophilia strains isolated from clinical specimens in Iran.

BACKGROUND: The overuse of biocides in healthcare-facilities poses risk for emergence and spread of antibiotic resistance among nosocomial pathogens. Hospital-acquired infections due to S. maltophilia have been increased in the recent years and with its various resistance mechanisms contribute to patient morbidity and mortality in hospitals. The current study aimed to evaluate the susceptibility of biofilm-producing and non-producing S. maltophilia clinical isolates to five commonly used hospital biocides, alone and in combination with EDTA to examine the synergistic effect of combining EDTA on the bactericidal activity of them by microbroth dilution method. As well as the frequency of efflux genes encoding resistance to biocides among isolates. This study also intended to assess the effect of exposure of S. maltophilia isolates to sub-inhibitory concentrations of sodium hypochlorite upon the antimicrobial susceptibility patterns. RESULTS: Based on minimum inhibitory and bactericidal concentrations of biocides sodium hypochlorite 5% (w/v) and ethyl alcohol 70% (v/v) were the strongest and weakest biocides against S. maltophilia isolates, respectively. The combination of EDTA with biocides significantly increased the effectiveness of the studied biocides. Exposure to sub-inhibitory concentration of sodium hypochlorite showed a significant change in the susceptibility of isolates towards ceftazidime (p = 0.019), ticarcillin/clavulanate (p = 0.009), and chloramphenicol (p = 0.028). As well as among the isolates examined, 94 (95%) were able to produce biofilm. The frequency of sugE1 resistance genes was found in 90.7% of our clinical S. maltophilia isolates. None of the isolates carried qacE and qacEΔ1 gene. CONCLUSIONS: The current study recommended that using the mixture of biocides with EDTA can be effective in reducing nosocomial infections. Also, this study demonstrated that exposure to sub-inhibitory concentrations of sodium hypochlorite leads to reduced antibiotic susceptibility and development of multidrug-resistant S. maltophilia strains.