Resistant Enterobacteriaceae Research Articles

Effect of single parenteral administration of marbofloxacin on bacterial load and selection of resistant Enterobacteriaceae in the fecal microbiota of healthy pigs

BackgroundAntimicrobial resistance (AMR) is a global concern impacting both humans, animals and their environment. The use of oral antimicrobials in livestock, particularly in pigs, has been identified as a driver in the selection of AMR bacteria. The aim of the present study was to evaluate the effects of a single intramuscular (IM) dose of marbofloxacin (8 mg/kg) on Enterobacteriaceae and E. coli populations, as well as on fluoroquinolone resistance within the fecal microbiota of pigs. Twenty healthy pigs, 60-days old, were divided into two groups: a treated group (n = 13) and a control group (n = 7) and were monitored over a 28-day experimental period. Fecal samples were collected from all animals for the isolation of E. coli and Salmonella strains. The minimum inhibitory concentration (MIC) of marbofloxacin for the isolates recovered on MacConkey agar supplemented with 1 or 4 µg/mL of marbofloxacin and for some generic E. coli isolates (recovered from MacConkey agar not supplemented with marbofloxacin) was determined using the broth microdilution method. Genomic DNA was extracted from the confirmed bacterial strains and sequenced using the Sanger method to identify mutations in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes.ResultsThe single IM administration of marbofloxacin resulted in a significant decrease in Enterobacteriaceae and E. coli fecal populations from days 1 to 3 post- treatment. No Salmonella isolates were detected in either group, and no marbofloxacin-resistant E. coli isolates were identified. The MIC of the selected generic E. coli strains (n = 100) showed an increase to up to 0.5 µg/mL between days 1 and 3 post-treatment but remained below the clinical breakpoint of marbofloxacin resistance (4 µg/mL). Sequencing of these isolates revealed no mutations in gyrA and parC genes.ConclusionsThe present study showed that this dosing regimen of marbofloxacin significantly decreases the fecal shedding of Enterobacteriaceae and E. coli populations in pigs, while limiting the selection of marbofloxacin-resistant E. coli isolates. These findings warrant validation in sick pigs to support the selective use of this antibiotic solely in cases of clinical disease, thereby minimizing the reliance on conventional (metaphylactic) group treatments in pigs.

Community Healthcare-Associated Infections: Enhancing Control with Software and Tailored Protocols

Abstract Issue/Problem Healthcare-associated infections are major global public health challenges. In Portugal, the prevalence of resistant Enterobacteriaceae is particularly alarming. Studies typically focus on hospitals and long-term care facilities, neglecting community settings where patients, particularly the elderly with multiple comorbidities, often transition between hospital and home. Description of the problem Effective control measures are vital across all healthcare environments. In home care, sporadic implementation of Contact Precautions, due to the lack of specific guidelines and challenges in accessing colonization information, has been a major gap. This project aimed to close this gap by integrating a software, previously used only in hospitals, to enhance infection control in domiciliary care. The intervention adapted contact transmission-based precautions for home settings and used data from the previous year’s infections and colonization to tailor recommendations for each patient, preventing transmission between homes and enhancing caregiver education. Results Preliminary data from a two-month monitoring period involved 40 patients under the care of three domiciliary teams. Eleven had records of infections involving 19 microorganisms, including 7 Enterobacteriaceae, five of which were carbapenemase producers. This led to five customized recommendations for improved precautions. The intervention also heightened nurses’ awareness of patients’ microbiological histories, evidenced by increased inquiries to the infection control team. Lessons Adapting measures to the unique dynamics of home care, distinct from hospital settings, is critical. Involvement of care teams in decision-making ensures practical adherence and enhances measure effectiveness. Regular monitoring is crucial for compliance, making this approach a model for other settings. Key messages • Adapting hospital protocols to community settings and offering close support to caregiving teams increases compliance. • Implementing software that facilitates communication about infection and colonization enhances infection control effectiveness in community settings.

An Investigation Into Carbapenem Resistance in Enterobacteriaceae Among Outpatients With Urinary Tract Infection in Southwestern Uganda

An Investigation Into Carbapenem Resistance in Enterobacteriaceae Among Outpatients With Urinary Tract Infection in Southwestern Uganda

Epidemiology of Infections Caused by ESBL-Producing Enterobacteriaceae and Antibiotic Resistance Profile of Strains Isolated at Meknes-Based Moulay Ismail Military Hospital: A Retrospective Study from January 2018 to December 2020

Introduction: Infections caused by ESBL-producing Enterobacteriaceae (ESBL-PE) pose a major public health challenge. In addition to their widespread presence and growing resistance to several categories of antibiotics, the medical management of these bacteria becomes intricate, potentially resulting in a therapeutic impasse. Our study's goal is to examine the regional epidemiological characteristics of ESBL-PE infections and present a current assessment of their antibiotic resistance. Methods: The retrospective investigation undertaken at the bacteriology laboratory of the Meknes-based Moulay Ismail Military Hospital, covering a period of three years (January 2018 to March 2021), specifically examines the isolates of ESBL-PE. Results: Among the 2596 strains of Enterobacteriaceae described, 16.4% were found to produce ESBL, with E. coli being accountable for 63.6% of these strains. 85.1% of these ESBL-PE were isolated from urine samples. Surgical services were the largest source of these ESBL-PE (54.3%), followed by the emergency department (22%), medical services (14.5%), and intensive care unit (9.2%). The antibiotic resistance of these ESBL-PE has been studied and found to be high for gentamicin (63%), tobramycin (66%), cotrimoxazole (85%), and ciprofloxacin (91%). Only 7% exhibited good sensitivity to amikacin, 4% to imipenem, and 5% to fosfomycin. Conclusion: The advent of ESBL-PE in our institution is a big challenge. To limit the use of carbapenems and prevent the spread of strains causing carbapenemases, other kinds of antibiotics can be utilized. The partnership between clinicians and the bacteriology laboratory is vital for efficiently preventing and monitoring the spread of these multidrug-resistant bacteria.

B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae

Abstract Background In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.). Methods We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA). Results The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1). Conclusions The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.

Effect of X-ray radiation on Multi-drug Resistant (MDR) Enterobacteriaceae & P.aeruginosa isolated from burn unit patient. The phenotypic and molecular study, Biofilm formation

Background: Multi-drug-resistant (MDR) bacteria are a significant global health concern due to their elevated rates of morbidity and mortality in burn patients. In This study investigated the presence of multidrug-resistant pathogenic bacteria in individuals with burn injuries, the formation of biofilms, and the impact of X-ray radiation. Method: For this study Clinical swabs about120 burn patients were obtained between December 2023 and June 2024 from burn units at Ramadi and Falluja Teaching Hospitals. Both conventional and Vitek2(bioMérieux) methods were used to identify the bacterial isolates. The isolates were then divided into three multi-drug resistance groups. The Colorimetric method (MTP) detected biofilms. To detect blaOXA-23,blaOXA-48, Omp, and blaTEM resistance genes, multiplex PCR was used. This research used two lumbosacral spine X-ray levels. Two levels of radiation were used 40 (80KV) mAs and 51.2 (85KV). Result: Results of burn isolation revealed 30(27%) P.aeruginosa, 18(16%) k.pneumoniae, 7(6%) E.coli, 7(6%) P.mirabilis. Of the all isolates, 100% produced biofilm. The Multiplex-PCR analysis revealed that the blaTEM genes were the predominant ones, constituting 30% of the samples. Subsequently, blaOXA-23 constituted 24.5% of the cases, whereas Omp accounted for 22.7% and blaOXA-48 for 13.6%. The radiation impact of X-rays produced by two dosages, 40(80KV)mAs for 125msec and 51,2(85KV) mAs for 160msec, demonstrates that there is no effect or alteration in the resistance capacity of all the bacterial isolates tested. Conclusion: This study showed that burn unit isolates produce ESBLs, which is a major concern in the intensive care unit due to its high morbidity and potential mortality. These data match antibiotic phenotypic test results showing substantial cefepime resistance. All isolates forming biofilms, this may association with high pathogenicity. This demands quick intervention to manage nosocomial outbreaks. No X-ray effect was seen on susceptibility a burn unit bacterial isolate.

In Vitro Inhibitory Effect of Four Essential Oils against Fluoroquinolone- Resistant Enterobacteriaceae Responsible for Community-Acquired Urinary Tract Infections

In Vitro Inhibitory Effect of Four Essential Oils against Fluoroquinolone- Resistant Enterobacteriaceae Responsible for Community-Acquired Urinary Tract Infections

Systematic review and meta-analysis on antimicrobial resistance and drug resistance in Saudi Arabia

BackgroundAntimicrobial resistance (AMR) and drug resistance have emerged as major global health concerns, threatening the effectiveness of antimicrobial agents and compromising patient outcomes [21]. The rapid spread of resistant pathogens poses significant challenges to healthcare systems worldwide, leading to increased morbidity, mortality, and healthcare costs [20]. Methods Search StrategyTo find pertinent studies released up until September 2023, a thorough search was done in electronic databases like PubMed, Embase, and Scopus. The search method combined terms linked to Saudi Arabia, drug resistance, and antimicrobial resistance (AMR). The specific search terms and Boolean operators used were tailored to the requirements and functionalities of each database. ResultsThe systematic review includes 25 papers in total, representing various Saudi Arabian healthcare environments and patient groups. The prevalence of AMR varied across different pathogens and antimicrobial agents. The most resistant Enterobacteriaceae were carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum -lactamase-producing Enterobacteriaceae (ESBL), and methicillin-resistant Staphylococcus aureus (MRSA). The full manuscript will report the pooled prevalence rates and 95% CIs for each resistance type. The rates for such bacteria were 38.7%, 26.4%, and 15.2% respectively suggesting an imminent need for improvement in surveillance measures & interventions to reduce the burden of AMR (Antimicrobial Resistance). ConclusionThe prevalence of antibiotic resistance (AMR) is alarmingly high in Saudi Arabia. The aim of this study was to examine the prevalence of AMR across different types of pathogens and antimicrobial agents. A meta-analysis was conducted to quantify the pooled prevalence of each type of resistance across hospitals, environments, and food samples from Saudi Arabian locations. The study identified three types of resistance: carbapenem-resistant Enterobacteriaceae (CRE), extended-spectrum -lactamaseproducing Enterobacilli (ESBL), and methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, the study identified several risk factors associated with AMR, including prior antibiotic use, healthcare-associated infections, and prolonged hospital stays.

Antibacterial Resistance and Extended-Spectrum Beta-Lactamase (ESBL) Phenotypes in Enterobacteriaceae Isolated from Fecal Samples of Humans and Animals in Selected Local Government Areas of Nasarawa State, Nigeria

It is quite alarming the increasing rate of antibacterial resistance all over the world considering the public health threat and the re-emergence of multi-drug resistant Enterobacteriaceae. The aim of this study is Antibacterial resistance and phenotypic detection of Extended Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae isolated from human and animal fecal samples in selected local government areas of Nasarawa state, Nigeria was carried out in the study. Hundred (100) samples comprising human and animal (goats, cattle, and chicken) were collected and 55 samples were multidrug resistant. A commercial biochemical kit (Eneterosystem 18R) was used for the isolation and identification of Enterobacteriaceae. Kirby Bauer Disk Diffusion Method was used for antibacterial susceptibility testing of Enterobacteriaceae isolates. The Double Disc Synergy Test (DDST) method was also used for the phenotypic confirmation test of Extended Spectrum Beta Lactamase (ESBL). The occurrence of multidrug-resistant isolates shows that Escherichia coli (100.00%) which is the highest, Proteus mirabilis (14.54%), Klebsiella pneumoniae, and Salmonella enterica (10.90%), while the occurrence of Shigella flexneri (9.09%) was the lowest. The Enterobacteriaceae isolates were more resistant to Cefuroxime, Cefexime, Amoxicillin Clavulanate, and Imipenem/Cilastatin with percentage resistance ranges from 66.6% - 100%. The occurrence of ESBL producers shows that Escherichia coli (60.00%) and Proteus mirabilis (62.5%) were high while Shigella flexneri (20.0%) had a low occurrence of ESBL. The sale and in-discriminate use of antibiotics without a prescription is an important regulatory issue in the abuse of antibiotics for both humans and animals. The Beta-Lactam and gentamycin antibiotics were not effective against the Multi-Drug Resistant (MDR) isolates and most of the isolates were ESBL producers.

Extended-spectrum beta-lactamase and carbapenemase producing Klebsiella Pneumoniae isolated from patient with suspected urinary tract infection

Urinary tract infection (UTI) is the most common bacterial infections in humans and serious health problem in many parts of the world. Carbapenem resistant Enterobacteriaceae (CRE) are increasingly reported from healthcare facilities in Nepal and around the world. This study is carried out with an objective to assess the Prevalence of ESBL and carbapenemase producing Klebsiella pneumoniae isolate from patient with suspected of UTI. Total 880 urine sample was collected during 6-month period and processed and identified by using conventional microbiological procedure and biochemical test. ESBL screening isolates were done by using Ceftazidime and Ceftazidime with clavulanic acid. Production of ESBL was determine by combination Disc Test. For the confirmation of carbapenemase producing K. pneumoniae Modified Hodge Method, Carbapenem Inactivation Method and EDTA Impregnated Disc test (MBL) was performed. Out of total sample, 9.30% (82) showed significant growth. Prevalence of UTI seen more in male as compare to female patients. Out of positive isolates, 37.8% were K. pneumoniae. K. pneumoniae were resistance with Ceftriaxone (54.8%) followed by 51.61% Ceftazidime, Cotrimoxazole. Multi drug resistance (MDR) was found to be 54.83%. Out of 31 isolates, 10(32%) were confirmed as ESBL positive by Combination Disc Assay. 14 isolates were screened as carbapenemase producers but 2(14.28%) showed positive through Modified Hodge Method, 4(28.57%) Modified Carbapenem Inactivation Method and 7(50%) EDTA impregnated Disc Test (MBL). Analytically, the sensitivity of MHT, CIM, and MBL tests was 42.81%, 58.10%, and 76.96%, respectively, all with 100% specificity. Colistin was the drug of choice, with 93.54% of isolates showing sensitivity to it. Early detection of ESBL and MBL-producing isolates is crucial for reducing mortality rates and preventing the intra-hospital spread of these strains.

Impact of Discontinuing Contact Precautions for Multidrug-resistant Gram-negative Enterobacteriaceae in a Large Health System

Background: Contact precautions (CP) to prevent transmission of multidrug-resistant gram-negative (MDRGN) Enterobacteriaceae are recommended, although studies of discontinuation of CP (DcCP) have found no change in healthcare associated infections (HAI) due to extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae. Limited data exists on DcCP for MDRGN in a large health system. Methods: We performed a retrospective observational study analyzing the relationship between use of CP and HAI due to two definitions of MDRGN Enterobacteriaceae: ESBL, and non-susceptibility to ≥3 drug classes (3DC-GNR), with carbapenem resistant Enterobacteriaceae (CRE) serving as control. The study included all inpatient admissions from 2/2017 through 9/2022 at 21 acute care hospitals. Hospitals had latitude to determine CP practices based on local risk assessment, but in 2/2018, system-wide transmission-based precautions guidance was updated to recommend DcCP for MDRGN Enterobacteriaceae and in 12/2019 was updated to clarify DcCP specifically for ESBL and 3DC-GNR while continuing CP for carbapenem-resistant organism carriage. We interviewed infection preventionists to define when CP were used for CRE, ESBL, and 3DC-GNR Enterobacteriaceae. HAI were defined using National Healthcare Safety Network criteria including all HAI categories. We compared the incidence rate of HAI attributable to the two MDRGN types in hospital months with and without use of CP, with HAI due to CRE as a comparison group since all hospitals used CP for CRE throughout the study period. Results: The periods of CP use, by hospital, are shown in Figure 1. Throughout the study period, there were 987 HAI attributed to ESBL Enterobacteriaceae, 579 due to 3DC-GNR Enterobacteriaceae, and 329 due to CRE. Figure 2 shows the unadjusted aggregate rate of HAI for each of the three MRGN types, including among hospitals with and without CP in each month, for ESBL and 3DC-GNR. In months with and without CP, the rate of HAI was 1.482/10,000 and 1.093/10,000 patient days (incidence rate ratio [IRR], 1.356 [95% confidence interval, 1.195-1.540]) for ESBL Enterobacteriaceae. In months with and without CP, the rate of HAI was 1.071/10,000 and 0.493/10,000 patient days (IRR,2.173[95% confidence interval, 1.838-2.569]) for 3DC-GNR Enterobacteriaceae. Conclusion: DcCP was not associated with an increase in HAI due to ESBL and 3DC-GNR Enterobacteriaceae in aggregated facilities that self-selected for DcCP. Facilities that used CP were associated with significantly higher rates of ESBL and 3DC-GNR Enterobacteriaceae, a relationship that did not change as hospitals DcCP for these MDRGN. Further analyses are necessary to assess for a causal relationship.

The scourge of carbapenem resistant Enterobacteriaceae: how to fight back

The Enterobacteriaceae species cause both community acquired and health care associated infections like bloodstream infections, ventilator-associated pneumonia, intra-abdominal infections and urinary tract infections. Carbapenem-resistant Enterobacteriaceae (CRE) have been included in the list of global priority pathogens (GPP) declared by WHO in 2017. These infections pose a serious threat due to the associated significant morbidity and mortality. The mechanisms of antimicrobial resistance in these organisms are numerous; however, β-lactamase genes carried on mobile genetic elements are a key mechanism for the rapid spread of these antibiotic-resistant strains on a global scale. The carbapenem resistance (CR) in Enterobacteriaceae has been recognized for the past two decades, but the carbapenemase-producing Enterobacteriaceae (CPE) has been a more recent issue and is spreading at an alarming pace worldwide. In this article, we conducted a systematic literature search of the PubMed, Embase, Web of Science and Cochrane Library databases to identify relevant studies that reported the epidemiology and the outcomes for hospitalised patients with confirmed infections due to CRE and carbapenem-susceptible Enterobacteriaceae (CSE) published between 1 January 2010 and 30 August 2023.The results emphasize that patients with CRE infection still face a greater risk of mortality and need an urgent need for newer antibiotics and appropriate treatment regimens to reduce the risk of morbidity and mortality.

Antibiotic Resistance in Enterococci and Enterobacteriaceae from Laboratory-Reared Fresh Mealworm Larvae (Tenebrio molitor L.) and Their Frass.

The occurrence of antibiotic-resistant bacteria in foodstuff involves a human health risk. Edible insects are a precious resource; however, their consumption raises food safety issues. In this study, the occurrence of antibiotic resistant bacteria in laboratory-reared fresh mealworm larvae (Tenebrio molitor L.) and frass was assessed. Antibiotics were not used during the rearing. Enterobacteriaceae and enterococci were isolated from 17 larvae and eight frass samples. In total, 62 and 69 isolates presumed to belong to Enterobacteriaceae and Enterococcus spp., respectively, were obtained and tested for antibiotic susceptibility via disk diffusion. Based on the results, isolates were grouped, and representative resistant isolates were identified at species level through 16S rRNA gene sequencing. For enterococci resistance, percentages higher than 15% were observed for vancomycin and quinupristin-dalfopristin, whereas Enterobacteriaceae resistance higher than 25% was found against cefoxitin, ampicillin, and amoxicillin-clavulanic acid. Based on the species identification, the observed resistances seemed to be intrinsic both for enterococci and Enterobacteriaceae, except for some β-lactams resistance in Shigella boydii (cefoxitin and aztreonam). These could be due to transferable genetic elements. This study suggests the need for further investigations to clarify the role of edible insects in the spreading of antibiotic resistance determinants through the food chain.

Detection of Colistin Resistant Enterobacteriaceae among Backyard Farm Swine and Swine Raisers in Tanay, Rizal Swine Farm

Addressing difficulties in susceptibility testing of colistin and the emergence of colistin-resistant Enterobacteriaceae became a huge burden to medical practitioners due to the complexity of the method and interpretation of the minimum inhibitory concentration of antibiotics. These circumstances are not only rampant among human infection but also in the animal farm industry – particularly, the swine farms, which have the greatest number of cases of Enterobacteriaceae infection. This study aims to determine the prevalence of colistin-resistant Enterobacteriaceae from 40 fecal samples of backyard farm swine (30) and swine raisers (10) in Tanay, Rizal, the Philippines via culture, biochemical testing, and antimicrobial susceptibility testing for colistin. Enterobacteriaceae from fecal samples were isolated, singly picked, and subjected to biochemical tests. Susceptibility testing was performed to determine the minimum inhibitory concentration. The isolates recovered from fecal samples of backyard swine were Escherichia coli (77%), Salmonella species (16%), Citrobacter freundii (7%), and two non-Enterobacteriaceae Pseudomonas species, whereas all fecal samples from swine raisers were positive for E. coli (100%). Moreover, 13.04% of E. coli isolates from backyard farm swine exhibited resistance to colistin with a minimum inhibitory concentration of 4ug/mL, following the international guidelines for interpretation of colistin resistance, whereas the rest of the isolates were susceptible to colistin. E. coli, Salmonella, and C. freundii were present in fecal samples of backyard swine and three E. coli were resistant to colistin. There is a 13.04% prevalence of colistin-resistant E. coli isolated from pooled fecal samples of a backyard swine farm in Tanay, Rizal, the Philippines. The increasing cases of colistin resistance should be strictly monitored in animal farms to come up with a robust solution to fight against antimicrobial resistance.

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.

Development of a multiplex real-time PCR assay for simultaneous detection of common colistin and carbapenemase genes

Carbapenem and colistin are often used as last-resort treatment for Gram-negative multi-drug resistant (MDR) bacteria. Nevertheless, co-resistance of these drugs is threatening the global healthcare system. Rapid and accurate detection of carbapenem and colistin resistant bacteria is critical for adequate antibiotic therapy and infection control, particularly in the context of an outbreak. The presence of blaNDM, blaKPC, blaIMP-1 and blaOXA-48 is responsible for greater than 95% phenotypic resistance in carbapenem-resistant Enterobacteriaceae, while mcr-1 is the most prevalent and well disseminated of all mcr genes in colistin-resistant strains. In this study, we aim to develop a multiplex real time-PCR assay for simultaneous detection of the five genes blaNDM, blaKPC, blaIMP-1, blaOXA-48 and mcr-1. The melting curve-based multiplex real time PCR assay was established with the dissociation temperature range extended from 76°C to 87°C. The whole process is completed within one hour and half, allowing rapid screening of the five genes in cultured bacteria samples with a limit of detection of 10 CFU/ml. The proposed multiplex real-time PCR assay is a robust, reliable and rapid method for the detection of bacterial strains carrying blaOXA-48, blaIMP, blaNDM, blaKPC and mcr-1 gene individually or in cocktail of genes. This assay will be a valuable tool for surveillance and monitoring of MDR bacteria additionally resistant to either carbapenem or colistin or both drugs.

High rates of intestinal colonization with carbapenemase producing Enterobacteriaceae inhematopoietic stem cell transplant recipients.

Carbapenem resistant Enterobacteriaceae (CRE) are major human pathogens because, these cause high number of difficult-to-treat infections. Allogeneic hematopoietic stem cell transplant (AHSCT) recipients are highly exposed to these type of bacteria. The aim of our study was to investigate prevalence of CRE colonization in AHSCT patients and to determine genes encoding carbapenem resistance. Aretrospective study conducted between January 2015 and December 2019, involved 55 patients colonized with CRE strains. We determined the rate of antibiotic resistance according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the carbapenem resistance genes by PCR assays for genes encoding most frequent β-lactamases namely, blaGES, blaKPC, blaIMI, blaNDM, blaVIM, blaIMP and blaOXA-48. Eighty-one episodes of CRE colonization were recorded in 55 patients, mainly suffering from acute leukaemia (30%) and aplastic anemia (26%). History of hospitalization was noted in 80 episodes. Prior antibiotic treatment, severe neutropenia and corticosteroid therapy were respectively found in 94%, 76% and 58% of cases. Among the 55 patients, six patients (11%) developed a CRE infection. The CRE responsible for colonization were carbapenemase producers in 90% of cases. They belonged mostly to Klebsiella pneumoniae (61/81) and Escherichia coli species (10/81). Antibiotic resistance rates were 100% for ertapenem, 53% for imipenem, 42% for amikacin, 88% for ciprofloxacin and 27% for fosfomycin. Molecular study showed that blaOXA-48 gene was the most frequent (60.5%), followed by blaNDM (58%). Thirty-five (43%) strains were co-producers of carbapenemases. In our study, we report a high rate of CRE intestinal colonization in AHSCT recipients of our center.

Sub-MIC Antibiotics Modulate Productions of Outer Membrane Vesicles in Tigecycline-Resistant Escherichia coli.

Antimicrobial resistance (AMR) has been recognized as one of the most important crises affecting global human health in the 21st century. Tigecycline is one of the last resort antibiotics for treating severe infections caused by multi-drug resistant Enterobacteriaceae. However, the mobile resistance gene tet(X4), which could mediate high-level tigecycline resistance, was discovered in 2019. The outer membrane vesicle (OMV) has been recognized as a new route for horizontal gene transfer; antimicrobial resistant bacteria also have the ability to secret OMVs, while little is known about the impact of antibiotics on the secretion and characteristics of OMVs from tigecycline resistant bacteria till now. This study aimed to investigate the effects of antibiotics on the production and traits of a tigecycline resistant Escherichia coli strain of 47EC. The results showed that sub-inhibitory (1/2 MIC or 1/4 MIC) concentrations of gentamicin, meropenem, ceftazidime, chloramphenicol, tigecycline, ciprofloxacin, polymycin, rifaximin and mitomycin C could significantly increase the secretion of OMVs (0.713 ± 0.05~6.333 ± 0.15 mg/mL) from E. coli 47EC compared to the respective untreated control (0.709 ± 0.03 mg/mL). In addition, the particle sizes of OMVs were generally larger, and the zeta potential were lower in the antibiotics-treated groups than those of the antibiotic-free group. The copy numbers of the tigecycline resistance gene of tet(X4) in the OMVs of most antimicrobial-treated groups were higher than that of the control group. Moreover, transcriptome analysis on ciprofloxacin-treated E. coli 47EC indicated that the SOS response and prophage activation might participate in the ciprofloxacin-induced OMV formation. In conclusion, the clinical application of antibiotics in treating bacterial infections, especially multi-drug resistant bacteria, might lead to the increased secretion of bacterial OMVs and the enrichment of antimicrobial-resistant genes in the OMVs.

A systematic review and meta-analysis of carbapenem resistance and its possible treatment options with focus on clinical Enterobacteriaceae: Thirty years of development in Pakistan

BackgroundCarbapenem resistance is epidemic worldwide, these last resort antimicrobials are listed in the WHO ‘watch group’ with higher resistance potential. During the years 2017-18 Pakistan Antimicrobial Resistance Surveillance System reported an increase in carbapenem resistance. However, a comprehensive information on prevalence and molecular epidemiology of carbapenem resistance in Pakistan is not available. This systematic review and meta-analysis is aimed to report the current carbapenem resistance situation in Pakistan and its treatment options. MethodsIn this systematic review and meta-analysis, we investigated the pooled prevalence (PPr) of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae by organizing available data, from Web of Science and PubMed by April 2, 2020, in various groups and subgroups including species, years, provinces, extended spectrum β-lactamase production, clinical presentation, carbapenemase and metallo-β-lactamase production, and New Delhi metallo-β-lactamase (NDM) prevalence. Literature review was updated for the studies publisehd by December 07, 2023. Moreover, we descriptively reviewed the molecular epidemiology of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae in Pakistan. Lastly, we statistically explored different treatment options available for carbapenem resistant infections. We used R package ‘metafor’ for performing meta-analysis and influence diagnostics and determining treatment options. ResultsFrom two academic databases Web of Science and PubMed we identified 343 studies. Eighty-eight studies were selected for the systematic review and meta-analysis. Seventy-four studies were selected for phenotypic analysis, 36 for genotypic analysis, and 31 for available treatment options. PPr-ID of 12% [0.12 (0.07, 0.16)] was observed for phenotypic carbapenem resistance in Enterobacteriaceae with more prevalence recorded in Klebsiella pneumoniae 24% [0.24 (0.05, 0.44)] followed by 9% [0.09 (−0.03, 0.20)] in Escherichia coli. During the last two decades we observed a striking increase in carbapenem resistance PPr i.e., from 0% [0.00 (−0.02, 0.03)] to 36% [0.36 (0.17, 0.56)]. blaNDM with PPr 15% [0.15 (0.06, 0.23)] in naive isolates was found to be the fundamental genetic determinant for carbapenem resistance in Enterobacteriaceae in Pakistan. Polymyxin B, colistin, tigecycline, and fosfomycin were identified as the suggested treatment options available for multidrug resistant infections not responding to carbapenems. Various studies reported carbapenem resistance from human, animal, and environment sources. ConclusionIn conclusion, we found that NDM-1 producing carbapenem resistant Enterobacteriaceae are increasing in Pakistan. Meta-analysis showed that metallo-β-lactamases producing E. coli ST405 and K. pneumoniae sequence type11 are the major resistant clones. Number of reported studies in various subgroups and inconsistency in following CLSI guidelines are the potential limitations of this meta-analysis. A National antimicrobial resistance (AMR) surveillance strategy based on One Health is urgently needed to check any future AMR crisis in Pakistan.

Antibacterial and antibiofilm activities of Thymus leptobotrys and Lavandula mairei essential oils against extended spectrum β-lactamase producing Enterobacteriaceae

The aim of the study was to investigate the antibacterial and antibiofilm activities of Lavandula mairei and Thymus leptobotrys essential oils against Extended spectrum β-lactamase producing Enterobacteriaceae, responsible of nosocomial infections. PCR and sequencing methods detected three different resistance genes (blaSHV-42, blaSHV-12 et blaCTXM-15) in the studied strains. Gas chromatography-mass spectrometry analysis highlighted the predominance of carvacrol in both Lavandula mairei and Thymus leptobotrys essential oils. Their antibacterial activity was conducted by disk diffusion and microdilution methods. Great sensitivity of the multidrug resistant strains was noted for the two essential oils with inhibition zones’ diameters ranging from 13.3±2.3 to 32.3±1.4 mm (Thymus leptobotrys) and from 22 to 34.66±0.57 mm (Lavandula mairei), and low minimal and bactericidal concentrations varying from 1.56 to 6.25 mg/mL (CMI) and from 1.56 to 12.5 mg/mL (CMB) for both essential oils. Four strains out of six, considered as the strongest biofilm producers by congo red agar and crystal violet methods, were chosen to evaluate biofilm inhibition and eradication effects of Lavandula mairei and Thymus leptobotrys essential oils. In most cases, these latter inhibited considerably the biofilm formation of the targeted strains with proportions greater than 80%. The eradication effect was lower, with rates not higher than 50%. Thus, these essential oils could be used as an effective alternative approach to prevent biofilm formation of multidrug resistant Enterobacteriaceae. To our knowledge, no previous works have reported the antibiofilm activity of Lavandula mairei and Thymus leptobotrys essential oils against these multidrug resistant bacteria.