Potential of ZnO nanoparticles for multi-drug resistant Escherichia coli having CRISPR-Cas from poultry market in Lahore.

BACKGROUND AND OBJECTIVES: Apart from known factors such as irrational use of antibiotics and horizontal gene transfer, it is now reported that clustered regularly interspaced short palindromic repeats (CRISPR) are also associated with increased antimicrobial resistance. Hence, it is critical to explore alternatives to antibiotics to control economic losses. Therefore, the present study aimed to determine not only the association of CRISPR-Cas system with antibiotic resistance but also the potential of Zinc Oxide nanoparticles (ZnO-NPs) for avian pathogenic Escherichia coli (APEC) isolated from poultry market Lahore. MATERIALS AND METHODS: Samples (n = 100) were collected from live bird markets of Lahore, and isolates were confirmed as Escherichia coli (E. coli) using the Remel One fast kit, and APEC was identified using PCR. The antibiotic resistance pattern in APEC was determined using the minimum inhibitory concentration (MIC), followed by genotypic confirmation of antibiotic-resistant genes using the PCR. The CRISPR-Cas system was also identified in multidrug-resistant (MDR) isolates, and its association with antibiotics was determined using qRT-PCR. The potential of ZnO-NPs was evaluated for multidrug-resistant (MDR) isolates by MIC. RESULTS: All isolates of APEC were resistant to nalidixic acid, whereas 95% were resistant to chloramphenicol and 89% were resistant to streptomycin. Nineteen MDR APEC were found in the present study and the CRISPR-Cas system was detected in all of these MDR isolates. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR isolates of APEC. ZnO-NPs inhibited the growth of resistant isolates. CONCLUSIONS: The findings showed the presence of the CRISPR-Cas system in MDR strains of APEC, along with the potential of ZnO-NPs for a possible solution to proceed. This highlights the importance of regulating antimicrobial resistance in poultry to reduce potential health consequences.

Limosilactobacillus fermentum IKP 111 reduces pathogen load and improves immunity of broilers when challenged with Salmonella enteritidis.

In this study, we checked the effectiveness of L. fermentum IKP 111 in treating S. enteritidis infection in an in vivo study. Its oral administration to broiler chicks significantly reduced the colonization of S. enteritidis in the gut and there was a lower bacterial count of S. enteritidis in the droppings after infection. The administration of the probiotic L. fermentum IKP 111 also led to increase in weight gain in the broiler chicks as well as their immunomodulation against avian influenza virus (AIV) and Newcastle disease virus (NDV) as compared to the chicks challenged only with S. enteritidis. Our study provides evidence that the probiotic strain L. fermentum IKP 111 could be an alternate for controlling S. enteritidis infection while enhancing the gut health as well as the immune response of broiler chickens against viral infections.

An in vitro antiviral activity of iodine complexes against SARS-CoV-2.

Since the emergence of COVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In vitro anti-SARS-CoV-2 activity of oral formulations (syrup and capsule)of an Iodine-complex (Renessans). First, cell cytotoxicity of Renessans on the Vero cells was determined using MTT assay. Afterwards, the antiviral activity of Renessans was determined using viral inhibition assays and TCID50. For this, nontoxic concentrations of the Renessans were used. The results showed that Renessans is nontoxic to the cells up to 50 µg/mL. At 1.5 µg/mL concentration, SARS-CoV-2 production was significantly reduced to 101.43 TCID50 and 101.58 TCID50 for the syrup and capsule, respectively, as compare to virus infected control cells 106.08 TCID50 and we found the dose dependent inhibition of virus replication in the presence of Renessans. Renessans inhibited SARS-CoV-2 with an EC50 value of 0.425 µg/mL and 0.505 µg/mL for syrup and capsule, respectively. Furthermore, there was no virus detected at concentration of 50 µg/mL of Renessans. This study indicates that Renessans, containing iodine, have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.

Epidemiological trends of foodborne Campylobacter outbreaks in the United States of America, 1998-2016.

Campylobacter is a major cause of foodborne diarrheal infections in the United States of America (USA). This study aimed to elucidate the patterns of Campylobacter foodborne outbreaks temporally and spatially concerning food vehicles. We collected the data of foodborne outbreaks from 1998 to 2016 reported to the Centers for Disease Control and Prevention. The incidence rate of outbreaks for each food source was calculated and analyzed for each variable including season, food location, and census region. Overall, 465 single-state outbreaks and 8003 cases were reported during 1998-2016. Outbreaks were frequently attributed to dairy products (32%), chicken (17%) and vegetables (6%). Binomial regression analysis showed that compared to chicken items, the highest rate ratio of outbreaks was associated with dairy products (1.86) followed by vegetables (1.35) and meat products (0.76). More outbreaks were reported in the summer (35%) followed by the spring (26%) and fall (22%) season. We found that the highest number of outbreaks occurred in the West 159 (34%) and Midwest 137 (29%) census regions. The study highlights the role of dairy, chicken, and vegetables as food vehicles in Campylobacter outbreaks. Findings from this study can help in devising strategies to mitigate the increasing occurrence of Campylobacter foodborne outbreaks.

Circulating liver-specific microRNAs as noninvasive diagnostic biomarkers of hepatic diseases in human.

CONTEXT: Hepatitis is an endemic disease worldwide leading to chronic and debilitating cancers. The viral agents and hepatotoxic substances lead to damage of hepatocytes and release of damage associated molecules in circulation. The lack of timely and rapid diagnosis of hepatitis results in chronic disease. OBJECTIVE: The present review aimed to describe regulation, release and functions of microRNAs (miR) during human liver pathology and insights into their promising use as noninvasive biomarkers of hepatitis. METHODS: Comprehensive data were collected from PubMed, ScienceDirect and the Web of Science databases utilizing the keywords "biomarkers", "microRNAs" and "hepatic diseases". RESULTS: The miRs are readily released in the body fluids and blood during HBV/HCV associated hepatitis as well as metabolic, alcoholic, drug induced and autoimmune hepatitis. The liver-specific microRNAs including miR-122, miR-130, miR-183, miR-196, miR-209 and miR-96 are potential indicators of liver injury (mainly via apoptosis, necrosis and necroptosis) or hepatitis with their varied expression during acute/fulminant, chronic, liver fibrosis/cirrhosis and hepato-cellular carcinoma. CONCLUSIONS: The liver-specific miRs can be used as rapid and noninvasive biomarkers of hepatitis to discern different stages of hepatitis. Blocking or stimulating pathways associated with miR regulation in liver could unveil novel therapeutic strategies in the management of liver diseases. Clinical significance Liver specific microRNAs interact with cellular proteins and signaling molecules to regulate the expression of various genes controlling biological processes. The circulatory level of liver specific microRNAs is indicator of severity of HBV and HCV infections as well as prognostic and therapeutic candidates. The expression of liver specific microRNAs is strongly associated with infectious, drug-induced, hepatotoxic, nonalcoholic steatohepatitis and nonalcoholic fatty liver diseases.

Review - Lactic acid bacteria in traditional fermented Asian foods.

Lactic acid bacteria play vital roles in various fermented foods in Asia. This paper reviews many types of the world's lactic acid fermented foods and discusses the beneficial effects of lactic acid fermentation of food. The lactic acid bacteria associated with foods now include species of the genera Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella. Lactic acid bacteria (LAB) are involved in many fermentation processes of Asian traditional foods, demonstrating their profound effects on improving food quality and food safety. During the past few decades' interest has arisen in the use of the varied antagonistic activities of LAB to extent the shelf-life of protein-rich products such as meats and fish. This review article outlines the main types of LAB fermentation as well as their typical fermented foods such as idli, kishk, sauerkraut, koumiss, Suan-tsai, stinky tofu, Chinese sausage and kefir. The roles of LAB and the reasons for their common presence are also discussed.

Genomic Diversity and Evolution of SARS-CoV-2 Lineages in Pakistan.

The emergence of SARS-CoV-2 variants has posed a challenge to disease control efforts worldwide. This study explored the genomic diversity and phylogenetic relationship of SARS-CoV-2 variants reported in Pakistan. Our objective was to understand the transmission dynamics of different lineages within the country. We retrieved and analyzed spike protein sequences from Pakistan and compared them with reference sequences reported worldwide. Our analysis revealed the clustering of Pakistan-origin isolates in nine different clades representing different regions worldwide, suggesting the transmission of multiple lineages within the country. We found 96 PANGO lineages of SARS-CoV-2 in Pakistan, and 64 of these corresponded to 4 WHO-designated variants: Alpha, Beta, Delta, and Omicron. The most dominant variants in Pakistan were Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2, AY.108), and Omicron (BA.2.75, BA.5.2), and the N-terminal domain and receptor binding regions were the most hypervariable regions of the spike gene. Compared to the reference strain, characteristic substitutions were found in dominant variants. Our findings emphasize the importance of continuously monitoring and assessing nucleotide and residue substitutions over time to understand virus evolutionary trends better and devise effective disease control interventions.

Design and Assessment of a Novel In Silico Approach for Developing a Next-Generation Multi-Epitope Universal Vaccine Targeting Coronaviruses.

In the past two decades, there have been three coronavirus outbreaks that have caused significant economic and health crises. Biologists predict that more coronaviruses may emerge in the near future. Therefore, it is crucial to develop preventive vaccines that can effectively combat multiple coronaviruses. In this study, we employed computational approaches to analyze genetically related coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, focusing on the spike glycoprotein as a potential vaccine candidate. By predicting common epitopes, we identified the top epitopes and combined them to create a multi-epitope candidate vaccine. The overall quality of the candidate vaccine was validated through in silico analyses, confirming its antigenicity, immunogenicity, and stability. In silico docking and simulation studies suggested a stable interaction between the multi-epitope candidate vaccine and human toll-like receptor 2 (TLR2). In silico codon optimization and cloning were used to further explore the successful expression of the designed candidate vaccine in a prokaryotic expression system. Based on computational analysis, the designed candidate vaccine was found to be stable and non-allergenic in the human body. The efficiency of the multi-epitope vaccine in triggering effective cellular and humoral immune responses was assessed through immune stimulation, demonstrating that the designed candidate vaccine can elicit specific immune responses against multiple coronaviruses. Therefore, it holds promise as a potential candidate vaccine against existing and future coronaviruses.

Temporal study of wastewater surveillance from September 2020 to March 2021: an estimation of COVID-19 patients in Lahore, Pakistan.

The first aim of study was to quantify the viral load in the wastewater samples by RT-qPCR testing in Lahore population to estimate the number of patients affected and predict the next resurgence of COVID-19 wave in the city. The second aim of the study was to determine the hotspot areas of Lahore which remained positive more often for virus with high viral load. In this study, n = 420 sewage samples were collected on an average of two weeks intervals from 30 different sewage water disposal stations (14 sampling events) from Sept 2020 to March 2021. RNA was extracted and quantified by RT-qPCR without concentrating the virus in samples. Number of positive disposal sites (7-93%), viral load from sewage samples (100.296 to 103.034), and estimated patients (660-17,030) ranged from low to high according to the surge and restrain of 2nd and 3rd COVID-19 waves in the country. The viral load and estimated patients were reported high in January 2021 and March 2021 which were similar to the peak of 2nd and 3rd waves in Pakistan. Site 18 (Niaz Baig village DS) showed the highest viral load among all sites. Findings of the present study helped to estimate the number of patients and track the resurgence in COVID-19 waves in Lahore particularly, and in Punjab generally. Furthermore, it emphasizes the role of wastewater-based epidemiology to help policymakers strengthen the quarantine measures along with immunization to overcome enteric viral diseases. Local and national stake holders should work in collaboration to improve the environmental hygiene to control the disease.

Efficient treatment of tannery wastewater through aeration, coagulation, and algal pond.

BACKGROUND: Tannery wastewater effluents contain many toxic and carcinogenic heavy metals and physiochemical parameters that need to be removed before these effluents enter in the main water bodies or rivers. In this study, the effluents from the tannery industry are treated through aeration, coagulation, and Chlorella vulgaris pond treatment processes for the removal of physiochemical: parameters only. METHODS: The effect of removal efficiencies (%) was studied on the physicochemical parameters, including salinity, electrical conductivity (EC), total dissolved solids (TDS), turbidity, total suspended solids (TSS), biochemical oxygen demand (BOD), and chemical oxygen demand (COD). RESULTS: The key results showed that the removal of EC, TDS, turbidity, TSS, BOD, and COD was 80.2%, 67%, 81%, 80.8%, 68.6%, and 100%, respectively, in raw wastewater treatment having 25, 50, and 70 g of algae C. vulgaris doses. The removal efficiencies (%) of salinity, EC, TDS, turbidity, TSS, BOD, and COD were 83%, 87.1%, 77.1%, 80%, 40%, 97%, and 98%, respectively, during coagulated wastewater treatment with three doses of algae. The observed improvement in treated wastewater indicated that the removal efficiencies (%) of salinity, EC, TDS, turbidity, TSS, BOD, and COD were 85.7%, 39.3%, 81.3%, 67.8%, 50.3%, 97%, and 98%, with C. vulgaris. CONCLUSION: This study confirmed that the treatment of tannery wastewater by these processes increased the pollutant removal efficiencies as all the physiochemical parameters were exceeding the permissible limits. RESULTS CONTRIBUTION IN FUTURE: This research will be helpful to treat the industrial wastewaters or effluents before it further mixes up in the main water streams. In this way, water quality will be better, aquatic life will be saved, and further researchers can analyze more ways for efficient treatments as they have a baseline data through this study findings. PRACTITIONER POINTS: One of the most pollutant sources in terms of both physical and chemical parameters is the produced wastewater from tannery industries. The effluents from tannery industry are treated through aeration, coagulation, and algae ponds treatment processes. These treatment made the tannery wastewater as environmental friendly.

One Health Assessment of Bacillus anthracis Incidence and Detection in Anthrax-Endemic Areas of Pakistan.

Anthrax, a severe zoonotic disease, is infrequently reported in anthrax-endemic regions of Pakistan. Despite clinical reports indicating its presence, particularly cutaneous anthrax, there is insufficient laboratory evidence regarding disease occurrence and environmental persistence. The present study aimed to confirm Bacillus anthracis presence, accountable for animal mortality and human infection, while exploring environmental transmission factors. Between March 2019 and July 2021, a total of 19 outbreaks were documented. Of these, 11 affected sheep/goats in Zhob district and 8 affected cattle/sheep in Bajour Agency. Clinical signs suggestive of Bacillus anthracis outbreak were observed in 11 animals. Blood and swab samples were collected for confirmation. The study followed a One Health approach, analyzing animal, environmental (soil/plant), and human samples. Of the 19 outbreaks, 11 were confirmed positive for anthrax based on growth characteristics, colony morphology, and PCR. Soil and plant root samples from the outbreak areas were collected and analyzed microscopically and molecularly. Cutaneous anthrax was observed in six humans, and swab samples were taken from the lesions. Human serum samples (n = 156) were tested for IgG antibodies against PA toxin and quantitative analysis of anthrax toxin receptor 1 (ANTXR1). Bacillus anthracis was detected in 65 out of 570 (11.40%) soil samples and 19 out of 190 (10%) plant root samples from the outbreak areas. Four out of six human samples from cutaneous anthrax lesions tested positive for Bacillus anthracis. Human anthrax seroprevalence was found to be 11% and 9% in two districts, with the highest rates among butchers and meat consumers. The highest ANTXR1 levels were observed in butchers, followed by meat consumers, farm employees, meat vendors, veterinarians, and farm owners. These findings highlight the persistence of anthrax in the region and emphasize the potential public health risks.

Competency of groundwater recharge of irrigated cotton field subjacent to sowing methods, plastic mulch, water productivity, and yield under climate change.

Irrigated agriculture is a foremost consumer of water resources to fulfill the demand for food and fiber with an increasing population under climate changes; cotton is no exception. Depleting groundwater recharge and water productivity is critical for the sustainable cotton crop yield peculiarly in the semiarid region. This study investigated the water productivity and cotton yield under six different treatments: three sowing methods, i.e., flat, ridge, and bed planting with and without plastic mulch. Cotton bed planting without mulch showed maximum water productivity (0.24 kg.m-3) and the highest cotton yield (1946 kg.ha-1). Plastic mulching may reduce water productivity and cotton yield. HYDRUS-1D unsaturated flow model was used to access the groundwater recharge for 150 days under six treatments after model performance evaluation. Maximum cumulative recharge was observed 71 cm for the flat sowing method without plastic mulch. CanESM2 was used to predict climate scenarios for RCP 2.6, 4.5, and 8.5 for the 2050s and 2080s by statistical downscale modeling (SDSM) using historical data from 1975 to 2005 to access future groundwater recharge flux. Average cumulative recharge flux declined 36.53% in 2050 and 22.91% in 2080 compared to 2017 without plastic mulch. Multivariate regression analysis revealed that a maximum 23.78% reduction in groundwater recharge could influence future climate change. Further study may require to understand the remaining influencing factor of depleting groundwater recharge. Findings highlight the significance of climate change and the cotton sowing method while accessing future groundwater resources in irrigated agriculture.

Renessans Helps in Early Clearance of SARS-CoV-2: In-Vivo Activity of the Iodine Complex in Rhesus macaque.

Iodine complexes have known antimicrobial properties along with reported in-vitro antiviral activity for several viruses. Renessans is one such product with iodine complexes and ascorbic acid. The present study was designed to determine its efficacy for SARS-CoV-2 in Rhesus macaque. Rhesus macaque were assigned to: A) prophylactic group (n = 3), (B) treatment group (n = 3), (C) infection control group (n = 4), and (D) negative control group (n = 4). Groups A, B, and C were challenged with 2 × 106 TCID of SARS-CoV-2. The prophylactic group (A) was administered Renessans from 5 days before infection till 8 days postinfection (DPI). The treatment group (B) was administered Renessans from 3 till 8 DPI. Group C was administered water-insoluble fractions only. Nasal swabs from all monkeys of groups A, B, and C remained positive for SARS-CoV-2 till 2 and 7 DPI, while the swabs became negative for groups A and B at 14 DPI. Likewise, fecal matter of monkeys in group A returned negative results during the experiment, while that of group B had significantly decreased viral load (101.5 genome copies/mL) compared to group C (103 genome copies/mL). Hence, it is concluded that Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2.

Fabrication and dialysis performance of functionalized multiwall carbon nanotubes integrated cellulose acetate/poly(vinylpyrrolidone) membranes.

Mixed matrix membranes (MMMs) of cellulose acetate/poly(vinylpyrrolidone) (CA/PVP) infused with acid functionalized multiwall carbon nanotubes (f-MWCNTs) were fabricated by an immersion phase separation technique for hemodialysis application. Membranes were characterized using FTIR, water uptake, contact angle, TGA, DMA and SEM analysis. The FTIR was used to confirm the bonding interaction between CA/PVP membrane matrix and f-MWCNTs. Upon addition of f-MWCNTs, TGA thermograms and glass transition temperature indicated improved thermal stability of MMMs. The surface morphological analysis demonstrated revealed uniform distribution of f-MWCNTs and asymmetric membrane structure. The water uptake and contact angle confirmed that hydrophilicity was increased after incorporation of f-MWCNTs. The membranes demonstrated enhancement in water permeate flux, bovine serum albumin (BSA) rejection with the infusion of f-MWCNTs; whereas BSA based anti-fouling analysis using flux recovery ratio test shown up to 8.4% improvement. The urea and creatinine clearance performance of MMMs were evaluated by dialysis experiment. It has been found that f-MWCNTs integrated membranes demonstrated the higher urea and creatinine clearance with increase of 12.6% and 10.5% in comparison to the neat CA/PVP membrane. Thus, the prepared CA/PVP membranes embedded with f-MWCNTs can be employed for wide range of dialysis applications.

Quaternized Diaminobutane/Poly(vinyl alcohol) Cross-Linked Membranes for Acid Recovery via Diffusion Dialysis.

Diffusion dialysis (DD) using anion exchange membranes (AEM) is an effective process for acid recovery and requires the preparation of suitable materials for AEMs, characterized by unique ions transport properties. In this work, novel AEMs composed of quaternized diaminobutane (QDAB) and poly(vinyl alcohol) (PVA) were cross-linked by tetraethoxysilane (TEOS) via the sol-gel process. The prepared AEMs were systematically characterized by Fourier-transform infrared (FTIR) spectroscopy, ion-exchange capacity (IEC) analysis, thermo gravimetric analysis (TGA), water uptake, linear expansion ratio (LER), and mechanical strength determination, scanning electron microscopy (SEM), and DD performance analysis for acid recovery using a hydrochloric acid/iron chloride (HCl/FeCl2) aqueous mixture and varying the QDAB content. The prepared AEMs exhibited IEC values between 0.86 and 1.46 mmol/g, water uptake values within 71.3 and 47.8%, moderate thermal stability, tensile strength values in the range of 26.1 to 41.7 MPa, and elongation from 68.2 to 204.6%. The dialysis coefficient values were between 0.0186 and 0.0295 m/h, whereas the separation factors range was 24.7-44.1 at 25 °C. The prepared membranes have great potential for acid recovery via diffusion dialysis.

A Cross-Sectional Survey of Knowledge, Attitude, and Practices of University Students in Pakistan Regarding COVID-19.

The COVID-19 pandemic is striking the world with serious public health and socioeconomic complications. The pandemic has influenced all forms of daily life, including educational institutions. Therefore, this cross-sectional survey was conducted to understand knowledge, attitudes, and practices related to COVID-19 among the students of the University of Veterinary and Animal Sciences, Lahore. The data was collected using an online self-directed questionnaire. The survey form includes six items about sociodemographic characteristics, 14 knowledge-based questions, seven questions on attitude, and eight questions on practices. The sample number was calculated using the Raosoft sample size calculator. A total number of 3,854 students, including 1,823 men and 2,031 women, were engaged in this survey, having student representation from all the provinces in the country. The data were analyzed using a chi-square test. A total of 97% of the students knew that the etiological agent of COVID-19 is a virus and that it is a disease of the respiratory system (94%). Many students kept visiting their relatives during the lockdown (45%), and their relatives kept visiting them at home (59%). The responses from the students varied a lot on specific questions about the transmission of the virus. Women tended to have less information regarding precautionary travel measures (p < 0.01), but supplemental knowledge of prevention of disease transmission from positive patients (p < 0.01). Conclusively, the majority of the university students surveyed had imperative knowledge, a good attitude, and active practice in response to the COVID-19 outbreak. Moreover, the KAP scores have varied by demography, gender, and the number of family members. Therefore, continuous awareness of preventative behaviors should be disseminated regularly in emergencies.

Evaluation of synergistic antimicrobial effect of vitamins (A, B1, B2, B6, B12, C, D, E and K) with antibiotics against resistant bacterial strains.

OBJECTIVES: Multidrug-resistant (MDR) superbugs, including Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA), are a challenge for healthcare professionals. In this study, the synergistic activity of vitamins with antibiotics against resistant bacterial strains was evaluated. METHODS: Synergistic effects between antibiotics and stock solutions of vitamins were evaluated by the Kirby-Bauer disk diffusion assay. Distilled water and propylene glycol were used as solvent for water-soluble and fat-soluble vitamins, respectively. Final concentrations of 10mg/mL for each water-soluble vitamin [B1 (thiamine), B2 (riboflavin), B6 (pyridoxine), B12 (methylcobalamin) and C (ascorbic acid)] and 0.1mg/mL for each fat-soluble vitamin [A (retinol), D (cholecalciferol), E (α-tocopherol) and K (menadione)] were used in combination with the antibiotics. RESULTS: The results demonstrated that vitamins K and E had good synergistic activity with piperacillin/tazobactam, imipenem and doripenem against A. baumannii, whilst vitamins B1, B2 and B12 showed remarkable synergistic activity with linezolid against MRSA. Vitamin B1 was further shown to have better synergism with oxacillin, tetracycline, rifampicin and linezolid against MRSA. The fat-soluble vitamins E and K showed good synergism against Gram-negative A. baumannii, whilst the water-soluble vitamins B1, B2 and B12 were effective against MRSA but not against A. baumannii. CONCLUSIONS: This synergistic action of vitamins with antibiotics may be used as a tool to treat MDR superbugs, with further evaluation required at a molecular level.