Carbapenems Research Articles

Characterization of the novel broad-spectrum lytic phage Phage_Pae01 and its antibiofilm efficacy against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is present throughout nature and is a common opportunistic pathogen in the human body. Carbapenem antibiotics are typically utilized as a last resort in the clinical treatment of multidrug-resistant infections caused by P. aeruginosa. The increase in carbapenem-resistant P. aeruginosa poses an immense challenge for the treatment of these infections. Bacteriophages have the potential to be used as antimicrobial agents for treating antibiotic-resistant bacteria. In this study, a new virulent P. aeruginosa phage, Phage_Pae01, was isolated from hospital sewage and shown to have broad-spectrum antibacterial activity against clinical P. aeruginosa isolates (83.6%). These clinical strains included multidrug-resistant P. aeruginosa and carbapenem-resistant P. aeruginosa. Transmission electron microscopy revealed that the phage possessed an icosahedral head of approximately 80 nm and a long tail about 110 m, indicating that it belongs to the Myoviridae family of the order Caudovirales. Biological characteristic analysis revealed that Phage_Pae01 could maintain stable activity in the temperature range of 4~ 60°C and pH range of 4 ~ 10. According to the in vitro lysis kinetics of the phage, Phage_Pae01 demonstrated strong antibacterial activity. The optimal multiplicity of infection was 0.01. The genome of Phage_Pae01 has a total length of 93,182 bp and contains 176 open reading frames (ORFs). The phage genome does not contain genes related to virulence or antibiotic resistance. In addition, Phage_Pae01 effectively prevented the formation of biofilms and eliminated established biofilms. When Phage_Pae01 was combined with gentamicin, it significantly disrupted established P. aeruginosa biofilms. We identified a novel P. aeruginosa phage and demonstrated its effective antimicrobial properties against P. aeruginosa in both the floating and biofilm states. These findings offer a promising approach for the treatment of drug-resistant bacterial infections in clinical settings.

Molecular study of Klebsiella pneumoniae antibiotic resistance genes isolates from wound infections

K. pneumoniae, which produces Carbapenemases, is of public health importance because of its resistance to antimicrobials. K. Pneumoniae is identified phenotypically by its large pink mucus appearance on MacConkey agar because of lactose fermentation and by bacteriological, microscopic, and biochemical tests using the VITK 2 device. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion. Klebsiella pneumoniae represented 5.3% (n = 16). It is highly resistant to antibiotics used to treat wound infections and shows the highest rate of resistance with cefepime 11(68.8%), followed by Ertapenem 9(56.3%), Doripenem and Imipenem 7(43.8%), and Meropenem 6(37.5%). Carbapenemases resistance was identified by phenotype through the Hodge test and also detection of Carbapenemases genes by polymerase chain reaction. The results showed the presence of the bla SHV 16 gene (100%) in K. pneumoniae isolates. The results showed that there were 4 isolates (25%) positive for the bla AMPC gene. The results also showed the presence of the bla KPC gene in two isolates (12.5%). The bla GES gene was not found in K. pneumoniae isolates. In general, the isolates were resistant to carbapenem and cefepime antibiotics. Five random samples were examined by publishing the results of the sequence analysis by BLAST in (NCBI) and the Mega 7 polymorphism detection program: it was found that there was no difference between the sequences, but rather the match was with the bacterium Klebsiella pneumoniae.

Detection of Beta-lactamase Resistance in Klebsiella pneumoniae Isolated from Different Clinical Sources

186 samples were collected from various pathogenic specimens, including 75 urine samples, 46 cerebrospinal fluid samples, 23 sputum samples, 9 body fluid samples, 14 blood samples, 4 discharge samples, 11 fluid samples from wounds and burns, and 4 bone marrow samples. Accordingly, 23 isolates of K. pneumoniae were identified as follows: 12 from urine samples, three isolates from cerebrospinal fluid, two each from catheters, wounds, and sputum, and one isolate from burns and bronchitis. The 23 isolates under study were tested for their susceptibility to 11 penicillin, cephalosporin, and carbapenem antibiotics, including beta-lactam and other antibiotics. The results showed high resistance of K. pneumoniae to piperacillin at a rate of 91%, and then to ampicillin at a rate of 86.9%, while were sensitive to both imipenem and meropenem, at a rate of 78%. The results showed that 100% of the isolates showed their ability to produce beta-lactamase enzymes as confirmed by the acid and mineral methods. The high percentage of enzymatic production indicates the presence of enzymatic resistance among bacterial isolates under study, which is why the bacteria gave high resistance to antibiotics. The results of the study showed the ability of 14 isolates to produce broad-spectrum beta-lactamase enzymes at a rate of 60%.

Urinary Tract Infections with Carbapenem-Resistant Klebsiella pneumoniae in a Urology Clinic-A Case-Control Study.

The aim of our study was to analyze the factors associated with the increased risk of urinary tract infection (UTI) with carbapenem-resistant (CR) Klebsiella pneumoniae (Kpn) and the antibiotic resistance spectrum of the strains in patients. As secondary objectives, we elaborated the profile of these patients and the incidence of different types of carbapenemases. We conducted a retrospective case-control study in which we compared a group of 62 patients with urinary tract infections with CR Kpn with a control group consisting of 136 patients with urinary tract infections with multidrug-resistant (MDR), but carbapenem-sensitive (CS), Kpn, who were hospitalized between 1 January 2022 and 31 March 2024. Compared to patients with urinary tract infections with CS Kpn, patients with urinary tract infections with CR Kpn were preponderant in rural areas (62.9% vs. 47.1%, p = 0.038) and more frequently had an upper urinary tract infection (69.4% vs. 36.8%, p < 0.01). Among the risk factors examined, patients in the study group had a higher presence of urinary catheters inserted for up to one month (50% vs. 34.6%, p = 0.03), rate of hospitalization in the last 180 days (96.8% vs. 69.9%, p < 0.01) and incidence of antibiotic therapy in the last 180 days (100% vs. 64.7%, p < 0.01). They also had a higher rate of carbapenem treatment in the last 180 days (8.1% vs. 0%, p < 0.01). Patients in the study group had a broader spectrum of resistance to all antibiotics tested (p < 0.01), with the exception of sulfamethoxazole-trimethoprim, where the resistance rate was similar in both groups (80.6% vs. 67.6%, p = 0.059). In the multivariate analysis, transfer from other hospitals (OR = 3.51, 95% and CI: 1.430-8.629) and treatment with carbapenems in the last 180 days (OR = 11.779 and 95% CI: 1.274-108.952) were factors associated with an increased risk of disease compared to the control group. The presence of carbapenemases was observed in all patients with CR Kpn, in the order of frequency New Delhi metallo-ß-lactamase (NDM) (52.2%), Klebsiella pneumoniae carbapenemase (KPC) (32.6%), and carbapenem-hydrolyzing oxacillinase (Oxa-48) (15.2%). The environment of origin and previous treatment with carbapenems appear to be the factors associated with an increased risk of urinary tract infection with CR Kpn compared to patients with urinary tract infections with CS Kpn. CR Kpn exhibits a broad spectrum of antibiotic resistance, among which is resistance to carbapenem antibiotics.

Combinations of Bacteriophage Are Efficacious against Multidrug-Resistant Pseudomonas aeruginosa and Enhance Sensitivity to Carbapenem Antibiotics.

The Gram-negative ESKAPE bacterium Pseudomonas aeruginosa has become a pathogen of serious concern due its extensive multi-drug resistance (MDR) profile, widespread incidences of hospital-acquired infections throughout the United States, and high occurrence in wound infections suffered by warfighters serving abroad. Bacteriophage (phage) therapy has received renewed attention as an alternative therapeutic option against recalcitrant bacterial infections, both as multi-phage cocktails and in combination with antibiotics as synergistic pairings. Environmental screening and phage enrichment has yielded three lytic viruses capable of infecting the MDR P. aeruginosa strain PAO1. Co-administration of each phage with the carbapenem antibiotics ertapenem, imipenem, and meropenem generated enhanced overall killing of bacteria beyond either phage or drug treatments alone. A combination cocktail of all three phages was completely inhibitory to growth, even without antibiotics. The same 3× phage cocktail also disrupted PAO1 biofilms, reducing biomass by over 75% compared to untreated biofilms. Further, the phage cocktail demonstrated broad efficacy as well, capable of infecting 33 out of 100 diverse clinical isolate strains of P. aeruginosa. Together, these results indicate a promising approach for designing layered medical countermeasures to potentiate antibiotic activity and possibly overcome resistance against recalcitrant, MDR bacteria such as P. aeruginosa. Combination therapy, either by synergistic phage-antibiotic pairings, or by phage cocktails, presents a means of controlling mutations that can allow for bacteria to gain a competitive edge.

Unveiling the Secrets of Acinetobacter baumannii: Resistance, Current Treatments, and Future Innovations.

Acinetobacter baumannii represents a significant concern in nosocomial settings, particularly in critically ill patients who are forced to remain in hospital for extended periods. The challenge of managing and preventing this organism is further compounded by its increasing ability to develop resistance due to its extraordinary genomic plasticity, particularly in response to adverse environmental conditions. Its recognition as a significant public health risk has provided a significant impetus for the identification of new therapeutic approaches and infection control strategies. Indeed, currently used antimicrobial agents are gradually losing their efficacy, neutralized by newer and newer mechanisms of bacterial resistance, especially to carbapenem antibiotics. A deep understanding of the underlying molecular mechanisms is urgently needed to shed light on the properties that allow A. baumannii enormous resilience against standard therapies. Among the most promising alternatives under investigation are the combination sulbactam/durlobactam, cefepime/zidebactam, imipenem/funobactam, xeruborbactam, and the newest molecules such as novel polymyxins or zosurabalpin. Furthermore, the potential of phage therapy, as well as deep learning and artificial intelligence, offer a complementary approach that could be particularly useful in cases where traditional strategies fail. The fight against A. baumannii is not confined to the microcosm of microbiological research or hospital wards; instead, it is a broader public health dilemma that demands a coordinated, global response.

An enantioselective formal synthesis of thienamycin

Thienamycin is a carbapenem antibiotic with potent activity against gram-negative and gram-positive bacteria. Due to its promising activity but lack of chemical stability, thienamycin serves as inspiration for new synthetic antibiotic scaffolds. In this study, we report a nine-step enantioselective formal synthesis of thienamycin. Our route utilizes an asymmetric reduction, enabled by NaBH4 and d-tartaric acid, followed by a series of diastereoselective reactions to access the key azetidinone precursor to thienamycin. This azetidinone precursor could be used as an intermediate to further develop and expand the scope of next-generation beta-lactam antibiotic scaffolds.

Genomic Characterization of Multidrug-Resistant Enterobacteriaceae Clinical Isolates from Southern Thailand Hospitals: Unraveling Antimicrobial Resistance and Virulence Mechanisms.

The emergence and spread of antimicrobial resistance (AMR) among Enterobacteriaceae pose significant threats to global public health. In this study, we conducted a short-term surveillance effort in Southern Thailand hospitals to characterize the genomic diversity, AMR profiles, and virulence factors of Enterobacteriaceae strains. We identified 241 carbapenem-resistant Enterobacteriaceae, of which 12 were selected for whole-genome sequencing (WGS) and genome analysis. The strains included Proteus mirabilis, Serratia nevei, Klebsiella variicola, Klebsiella aerogenes, Klebsiella indica, Klebsiella grimontii, Phytobacter ursingii, Phytobacter palmae, Kosakonia spp., and Citrobacter freundii. The strains exhibited high levels of multidrug resistance, including resistance to carbapenem antibiotics. Whole-genome sequencing revealed a diverse array of antimicrobial resistance genes (ARGs), with strains carrying genes for ß-lactamase, efflux pumps, and resistance to other antibiotic classes. Additionally, stress response, metal tolerance, and virulence-associated genes were identified, highlighting the adaptability and pathogenic potential of these strains. A plasmid analysis identified several plasmid replicons, including IncA/C2, IncFIB(K), and Col440I, as well as several plasmids identical to those found globally, indicating the potential for the horizontal gene transfer of ARGs. Importantly, this study also identified a novel species of Kosakonia spp. PSU27, adding to the understanding of the genetic diversity and resistance mechanisms of Enterobacteriaceae in Southern Thailand. The results reported in this study highlight the critical importance of implementing effective antimicrobial management programs and developing innovative treatment approaches to urgently tackle AMR.

Comparison of ERIC carbapenem-resistant Enterobacteriaceae test, BD Phoenix CPO detect panel, and NG-test CARBA 5 for the detection of main carbapenemase types of carbapenem-resistant Enterobacterales

BackgroundThis study aimed to assess the performance of three commercial panels, the ERIC Carbapenem-Resistant Enterobacteriaceae Test (ERIC CRE test), the NG-Test CARBA 5 (NG CARBA 5), and the BD Phoenix CPO Detect Panel (CPO panel), for the detection of main types of carbapenemases among carbapenem-resistant Enterobacterales (CRE). MethodsWe collected 502 isolates of carbapenem-resistant Enterobacterales (CRE) demonstrating intermediate or resistant profiles to at least one carbapenem antibiotic (ertapenem, imipenem, meropenem, or doripenem). Carbapenemase genes and their specific types were identified through multiplex PCR and sequencing methods. Subsequently, the ERIC CRE test, CPO panel, and NG CARBA 5 assay were conducted on these isolates, and the results were compared with those obtained from multiplex PCR. ResultsThe results indicated that the ERIC CRE test exhibited an overall sensitivity and specificity of 98.1% and 93.6%, respectively, which were comparable to 99.1% and 90.6% for the NG CARBA 5. However, the CPO panel demonstrated a sensitivity of only 56.2% in identifying Ambler classes, exhibiting the poorest sensitivity for class A. Moreover, while the ERIC CRE test outperformed the NG CARBA 5 in identifying multi-gene isolates with multiple carbapenemase-encoding genes, the CPO panel failed to accurately classify these isolates. ConclusionsOur findings support the utilization of the ERIC CRE test as one of the methods for detecting carbapenemases in clinical laboratories. Nonetheless, further optimization is imperative for the CPO panel to enhance its accuracy in determining carbapenemase classification and address limitations in detecting multi-gene isolates.

Characterization of carbapenemase-producing Enterobacterales from rectal swabs of patients in the intensive care units of a tertiary hospital in Cali-Colombia

BackgroundCarbapenemase-producing Enterobacterales (CPE) represents a significant threat to global health. This study aimed to characterize clinically and molecularly the CPE isolated from rectal swabs of patients in the intensive care units (ICUs) of a tertiary hospital in Cali, Colombia. MethodsThis was a cross-sectional observational study. Rectal swabs from patients admitted to the ICUs were collected. Bacterial identification and carbapenemase production were determined using phenotypic and molecular methods. Demographic and clinical data were extracted from electronic medical records. ResultsThe study included 223 patients. Thirty-six patients (36/223, 16.14 %) were found to be colonized or infected by CPE. Factors such as prolonged stay in the ICU, previous exposure to carbapenem antibiotics, use of invasive procedures, and admission due to trauma were associated with CPE. Klebsiella pneumoniae (52.5 %) was the most prevalent microorganism, and the dominant carbapenemases identified were KPC (57.8 %) and NDM (37.8 %). ConclusionDistinguishing carbapenemase subtypes can provide crucial insights for controlling dissemination in ICUs in Cali, Colombia.

Exploring the Antibacterial Potential of Artemisia judaica Compounds Targeting the Hydrolase/Antibiotic Protein in Klebsiella pneumoniae: In Vitro and In Silico Investigations.

Carbapenem antibiotic resistance is an emerging medical concern. Bacteria that possess the Klebsiella pneumoniae carbapenemase (KPC) protein, an enzyme that catalyzes the degradation of carbapenem antibiotics, have exhibited remarkable resistance to traditional and even modern therapeutic approaches. This study aimed to identify potential natural drug candidates sourced from the leaves of Artemisia judaica (A. judaica). The phytoconstituents present in A. judaica dried leaves were extracted using ethanol 80%. A reasonable amount of the extract was used to identify these phytochemicals via gas chromatography/mass spectrometry (GC/MS). One hundred twenty-two bioactive compounds from A. judaica were identified and subjected to docking analysis against the target bacterial protein. Four compounds (PubChem CID: 6917974, 159099, 628694, and 482788) were selected based on favorable docking scores (-9, -7.8, -7.7, and -7.5 kcal/mol). This computational investigation highlights the potential of these four compounds as promising antibacterial candidates against the specific KPC protein. Additionally, in vitro antibacterial assays using A. judaica extracts were conducted. The minimum inhibitory concentration (MIC) against the bacterium K. pneumonia was 125 μg/mL. Well-disk diffusion tests exhibited inhibition zones ranging from 10.3 ± 0.5 mm to 17 ± 0.5 mm at different concentrations, and time-kill kinetics at 12 h indicated effective inhibition of bacterial growth by A. judaica leaf extracts. Our findings have revealed the pharmaceutical potential of Artemisia judaica as a natural source for drug candidates against carbapenem-resistant pathogens.

Elizabethkingia meningoseptica Carbapenem Resistance in Cerebrospinal Fluid Cultures of Patients Suspected of Bacterial Meningitis: A Case Report

Background: Elizabethkingia meningoseptica is a Gram-negative bacterium that rarely causes meningitis in children. These infections are often resistant to carbapenem antibiotics, making diagnosis and treatment more complicated. Case presentation: A 1-month-old male patient was treated to the PICU of Dr. M. Djamil General Hospital Padang with symptoms of decreased consciousness, seizures, and fever. The patient was diagnosed with bacterial meningitis and was referred from Sungai Dareh Regional General Hospital with previous therapy for 4 days. A cerebrospinal fluid examination showed increased protein and decreased glucose, and a CT scan showed hydrocephalus. The cerebrospinal fluid culture showed growth of Elizabethkingia meningoseptica which was resistant to imipenem and meropenem. The patient was treated with a combination of non-carbapenem antibiotics and recovered from meningitis. Conclusion: Carbapenem-resistant Elizabethkingia meningoseptica is a rare causative agent of meningitis in children. Early diagnosis and treatment with appropriate non-carbapenem antibiotics are essential to increase the chance of cure.

Uncovering the growing burden of enteric fever: A molecular analysis of Salmonella Typhi antimicrobial resistance

Enteric fever, a persistent public health challenge in developing regions, is exacerbated by suboptimal socioeconomic conditions, contaminated water and food sources, and insufficient sanitation. This study delves into the antimicrobial susceptibility of Salmonella Typhi, uncovering the genetic underpinnings of its resistance. Analyzing 897 suspected cases, we identified a significant prevalence of typhoid fever, predominantly in males (58.3 %) and younger demographics. Alarmingly, our data reveals an escalation in resistance to both primary and secondary antibiotics, with cases of multi-drug resistant (MDR) and extensively drug-resistant (XDR) S. Typhi reaching 14.7 % and 43.4 %, respectively, in 2021. The Multiple Antibiotic Resistance (MAR) index exceeded 0.2 in over half of the isolates, signaling widespread antibiotic misuse. The study discerned 47 unique antibiotic resistance patterns and pinpointed carbapenem and macrolide antibiotics as the remaining effective treatments against XDR strains, underlining the critical need to preserve these drugs for severe cases. Molecular examinations identified blaTEM, blaSHV, and blaCTX-M genes in ceftriaxone-resistant strains, while qnrS was specific to ciprofloxacin-resistant variants. Notably, all examined strains exhibited a singular mutation in the gyrA gene, maintaining wild-type gyrB and parC genes. The erm(B) gene emerged as the primary determinant of azithromycin resistance. Furthermore, a distressing increase in resistance genes was observed over three years, with erm(B), blaTEM and qnrS showing significant upward trends. These findings are a clarion call for robust antimicrobial stewardship programs to curtail inappropriate antibiotic use and forestall the burgeoning threat of antibiotic resistance in S. Typhi.

Molecular detection of blaNDM and blaOXA-48 genes in Carbapenem-Resistant Klebsiella pneumoniae isolates from a tertiary care hospital

BackgroundThe incidence of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections is increasing globally. In India, around 65% of K. pneumoniae isolates are resistant to carbapenem antibiotics. The blaNDM is the predominant carbapenem-resistant gene in CRKP isolates. However, blaOXA-48 has also been reported to be increasing in recent days. MethodsK. pneumoniae strains isolated from the clinical specimens of patients attending a tertiary care hospital in Hyderabad from June 2021 to May 2022 were included in this study. Resistance to carbapenems and other antibiotics was screened using Kirby-Bauer’s disc diffusion method. Phenotypic tests such as the Carbapenemase Nordmann Poirel (CarbaNP) test, Modified Carbapenem Inactivation Method (mCIM), and EDTA Carbapenem Inactivation Method (eCIM) were used to detect the carbapenemase producers. The genes responsible for carbapenemase production were detected by the real-time polymerase chain reaction (RT-PCR) method. ResultsOut of 1265 K. pneumoniae strains isolated, 241 (19 %) isolates showed resistance to any one of the carbapenem antibiotics tested. Phenotypic tests such as CarbaNP, mCIM, and eCIM revealed that 94.6 %, 97.1 %, and 95.4 % of CRKP isolates were carbapenemase producers, respectively. About 66.4 % of CRKP isolates harbored blaNDM, 17.4 % harbored blaOXA-48, and 16.2 % harbored both blaNDM and OXA-48 genes. None of the isolates tested positive for blaKPC, blaVIM, and blaIMP genes. ConclusionIn this study, blaNDM is the most prevalent carbapenemase gene in CRKP isolates. Furthermore, blaOXA-48 and the co-existence of both blaNDM and blaOXA-48 genes were also found. CRKP is emerging as a serious threat among drug-resistant bacterial pathogens which would complicate the treatment of bacterial infections with available antibiotics.

Carbapenem use in extended-spectrum cephalosporin-resistant Enterobacterales infections in US hospitals and influence of IDSA guidance: a retrospective cohort study

Disparate and rapidly changing practice recommendations from major professional infectious diseases societies for managing non-severe infections caused by extended-spectrum β-lactamase-producing Enterobacterales might hamper carbapenem stewardship. We aimed to understand the real-world management of extended-spectrum cephalosporin-resistant (ECR) Enterobacterales infections in US hospitals and factors influencing preference for carbapenems over alternative treatments. This retrospective cohort study included adults (aged ≥18 years) admitted to hospital with ECR Enterobacterales infections in the PINC AI database. Antibiotic regimens were assessed during empirical and targeted treatment periods and by infection severity and site. Likelihood of receiving targeted carbapenems over time and before or after initial release of the Infectious Diseases Society of America (IDSA) guidance on Sept 8, 2020, was established with generalised estimating equations controlling for patient, hospital, and temporal confounders. Between Jan 1, 2018, and Dec 31, 2021, 30 041 inpatient encounters with ECR Enterobacterales infections were identified at 168 US hospitals, of which 16 006 (53·3%) encounters were in women and 14 035 (46·7%) were in men, with a mean age of 67·3 years (SD 15·1). Although few patients received carbapenems empirically (5324 [17·7%] of 30 041), many did so as targeted treatment (17 518 [58·3%] of 30 041), including subgroups of patients without septic shock (3031 [45·6%] of 6651) and patients with urinary tract infections without septic shock (1845 [46·8%] of 3943) in whom specific narrower-spectrum alternatives were active. Transitions from non-carbapenem to carbapenem antibiotics occurred most often on the day that the ECR phenotype was reported, regardless of illness severity. Carbapenems were the predominant choice to treat ECR Enterobacterales infections over time (adjusted odds ratio 1·00 [95% CI 1·00-1·00]), with no additional immediate change (1·07 [0·95-1·20]) or sustained change (0·99 [0·98-1·00]) after IDSA guidance release. High carbapenem use in targeting non-severe ECR Enterobacterales infections in US hospitals predates 2020 IDSA guidance and has persisted thereafter. Efforts to increase awareness and implementation of recommendations among clinicians to use carbapenem-sparing alternatives in ECR Enterobacterales infections might decrease global carbapenem selective pressure. US National Institutes of Health Intramural Research Program, National Institute of Allergy and Infectious Diseases, and US Food and Drug Administration.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-β-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum β-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of β-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-β-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and β-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Culture-free detection of β-lactamase-Producing bacteria in urinary tract infections using a paper sensor

Developing simple, inexpensive, fast, sensitive, and specific probes for antibiotic-resistant bacteria is crucial for the management of urinary tract infections (UTIs). We here propose a paper-based sensor for the rapid detection of β-lactamase-producing bacteria in the urine samples of UTI patients. By conjugating a strongly electronegative group –N+(CH3)3 with the core structures of cephalosporin and carbapenem antibiotics, two visual probes were achieved to respectively target the extended-spectrum/AmpC β-lactamases (ESBL/AmpC) and carbapenemase, the two most prevalent factors causing antibiotic resistance. By integrating these probes into a portable paper sensor, we confirmed 10 and 8 cases out of 30 clinical urine samples as ESBL/AmpC- and carbapenemase-positive, respectively, demonstrating 100% clinical sensitivity and specificity. This paper sensor can be easily conducted on-site, without resorting to bacterial culture, providing a solution to the challenge of rapid detection of β-lactamase-producing bacteria, particularly in resource-limited settings.

Antimicrobial resistance of Enterobacteriaceae and Staphylococcus spp. isolated from raw cow's milk from healthy, clinical and subclinical mastitis udders

Mastitis is the most common disease of dairy cattle and can be manifested in clinical and subclinical forms. The overuse of antimicrobials in the treatment and prevention of mastitis favours antimicrobial resistance and milk can be a potential route of dissemination. This study aimed to evaluate the biological quality of bulk tank milk (BTM) and the microbiological quality and signs of mastitis of freshly milked raw milk. In addition, to evaluate antimicrobial resistance in Enterobacteriaceae and Staphylococcus spp. isolated from freshly milked raw milk. None of the farms were within the official Brazilian biological quality limits for BTM. Freshly milked raw milk with signs of clinical (CMM), subclinical (SCMM) and no signs (MFM) of mastitis were detected in 6.67%, 27.62% and 65.71% samples, respectively. Most samples of freshly milked raw milk showed acceptable microbiological quality, when evaluating the indicators total coliforms (78.10%), Escherichia coli (88.57%) and Staphylococcus aureus (100%). Klebsiella oxytoca and S. aureus were the most prevalent microorganisms in SCMM and MFM samples. Antimicrobial resistance and multidrug resistance (MDR) were observed in 65.12% and 13.95% of Enterobacteriaceae and 84.31% and 5.88% of Staphylococcus spp., respectively, isolated from both SCMM and MFM samples. Enterobacteriaceae resistant to third-generation cephalosporin (3GCR) (6.98%) and carbapenems (CRE) (6.98%) and methicillin-resistant S. aureus (MRSA) (4.88%) were observed. Antimicrobial-resistant bacteria can spread resistance genes to previously susceptible bacteria. This is a problem that affects animal, human and environmental health and should be evaluated within the one-health concept.

Antibacterial activity of deer musk and Ziziphus spina-christi against carbapebem resis-tant gram negative bacteria isolated from patients with burns and wounds

Bacteria were isolated from 250 specimens obtained from patients attending the Plastic Reconstructive and Burn Surgery Hospital in the Sulaymaniyah (Kurdistan region) and the burn and wound care units of the Azadi Teaching Hospital (Kirkuk), Iraq. Gram-negative bacteria were isolated from 100 (40%) of the samples which identified by BD phoenix, 66 isolates (66%) were carbapenem-resistant species, Rapidec® Carba NP test and sensitivity tests revealed 17 different genera and species of carbapenem-resistant bacteria. The BD Phoenix system was used to evaluate the susceptibility test of the isolates to 18 different antibiotics. The number of isolates that exhibited resistance to the carbapenem antibiotics, ertapenem, imipenem and meropenem, was 40 (61%), 35 (54%) and 28 (42%) respectively. Meanwhile, the number of isolates resistant to the penicillin antibiotics, ampicillin, amoxicillin-clavulanic acid and piperacillin-tazobactam, was 61 (92%), 54 (82%) and 24 (36%) respectively. The isolates were also evaluated for their resistance to the cephalosporins, cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, and ceftolozane-tazobactam; the respective number of resistant isolates were 60 (91%), 60 (91%), 39 (59%), 48 (73%), 42 (64%) and 27(41%). In contrast, a fraction of Ziziphus spina-christi leaves that was extracted using ethyl acetate inhibited all of the carbapenem-resistant isolates studied. The zone of inhibition (ZoI) Ø was between 19 and 24 mm. The ZoI Ø of black deer musk was 11–19 mm. The minimum inhibitory concentration (MICs) of ethyl acetate extract of Ziziphus spina-christi leaves for Acinetobacter baumannii (PDR), CR-Citrobacter farmeri, CR-Escherichia coli, CR-Proteus mirabilis, CR-Pseudomonas fluorescens, CR-Escherichia vulneris, CR-Kluyvera ascorbata, CR-Pantoea agglomerans, CR-Pseudomonas putida and CR-Serratia marcescens, was 6.25 mg/mL. Meanwhile, the MIC for Acinetobacter calcoaceticus–baumannii complex, Citrobacter freundii, Enterobacter cloacae, Klebsiella pneumoniae, Morganella morganii, Pseudomonas aeruginosa and Stenotrophomonas maltophilia was 12.5 mg/mL. High-performance liquid chromatography (HPLC) was used to analyse the components of the ethyl acetate-extract of Ziziphus spina-christi leaves. The analysis found gallic acid (760.3 ppm/mL), caffeine (84.0 ppm/mL) and quercetin (2.2 ppm/mL); the total phenolic content was 846.5 ppm/mL. The evidence obtained from this study indicates that leaves of this plant (named sidr in the Middle East) have the potential to be used as a natural antibiotic against carbapenem-resistant Gram-negative bacteria. Thus, the leaves of this tree present an important opportunity in the development of novel therapeutic agents. The study found the MIC of deer musk for CR-Citrobacter farmeri, CR-Citrobacter freundii, CR-Enterobacter cloacae, CR-Escherichia vulneris, CR-Klebsiella pneumoniae, CR-Kluyvera ascorbata, CR-Morganella morganii, CR-Pantoea agglomerans, CR-Proteus mirabilis, CR-Pseudomonas fluorescens, CR-Pseudomonas putida and CR-Serratia marcescens to be 50 mg/mL. In contrast, the MIC of deer musk was 100 mg/mL for Acinetobacter baumannii (PDR), CR-Acinetobacter calcoaceticus–baumannii complex, CR-Escherichia coli, CR-Pseudomonas aeruginosa and CR-Stenotrophomonas maltophilia. The results of the gas chromatography–mass spectrometry (GC–MS) indicate that the primary constituents of musk were1,4,4-tetramethyltetralin, 7-acetyl-6-ethyl-1, diethyl phthalate and tonalid; their contribution to the whole ranged from 12.2% to 19.6%. Black musk exhibits considerable antibacterial activity, able to inhibit seventeen different species of carbapenem-resistant Gram-negative bacteria. The non-natural form of black musk can be used to scent detergent.

Based on molecular docking and real-time PCR technology, the two-component system Bae SR was investigated on the mechanism of drug resistance in CRAB.

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein-ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at - 5.81kcal/mol, while the docking energy for meropenem was - 4.92kcal/mol. For Bae R, imipenem had a maximum docking energy of - 4.28kcal/mol, compared with - 4.60kcal/mol for meropenem. The highest binding energies for Bae S-levofloxacin and Bae R-levofloxacin were - 3.60 and - 3.65kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16µg/mL, whereas levofloxacin-resistant strains had MIC values of 8µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2h under the action of 8µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.