Combined Disc Method Research Articles

Distribution of blaTEM, blaSHV, blaCTX-M, blaOXA, and blaDHA in Proteus mirabilis Isolated from Diabetic Foot Infections in Erbil, Iraq

Diabetic foot infection is considered to be one of the most important medical, economic, and social problems and a major cause of morbidity and mortality. Proteus mirabilis is a common etiologic agent of diabetic foot infections. This study aimed to determine the prevalence of beta-lactamase genes in P. mirabilis recovered from patients with diabetic foot wounds in Erbil, Iraq. Eighteen P. mirabilis isolated from 84 patients with diabetic foot ulcers were first phenotypically examined for the existence of extended-spectrum beta-lactamases by combined disc method and double-disc synergy method that all isolates showed positive results by both methods. The results were confirmed genetically by PCR to detect beta-lactamase-encoding genes (blaTEM, blaSHV, blaCTX-M, blaOXA, and blaDHA). The results revealed that all isolates contained extended-spectrum beta-lactamase and that 80% of the P. mirabilis isolates contained blaDHA, 60% had blaTEM, 53.3% had blaOXA, and 26.7% had blaCTX-M, whereas no isolates harbored blaSHV. The coexistence of two or more beta-lactamase genes in one isolate was observed. The existence of four genes (blaTEM + blaCTX-M + blaOXA + blaDHA) in the same isolate was documented in two isolates. In conclusion, this is the first study that reports a high prevalence of blaDHA and the coexistence of four resistance genes in the same organism in P. mirabilis isolated from diabetic foot patients in Iraq.

Occurrence of Multidrug-resistant Uropathogens Implicated in Asymptomatic Bacteriuria in Adults with Sickle Cell Disease in Ile-Ife, Southwest Nigeria.

ObjectivesWe sought to determine the prevalence of asymptomatic bacteriuria (ASB) in patients with sickle cell disease (SCD), the susceptibility profile of its agents and their extended-spectrum β-lactamase (ESBL) production.MethodsFifty-nine patients with SCD and 116 healthy controls were investigated. Urine samples were collected and cultured by standard techniques. We used the disc diffusion technique to determine antibiotic susceptibility. ESBL was detected by the combination disc method and detection of blaSHV, blaTEM, and blaCTX-M genes by multiplex-polymerase chain reaction.ResultsThe prevalence of ASB was higher among patients with SCD (8.6%) than controls (0.9%) (p = 0.016), predominantly among females. Coagulase-negative staphylococci (n = 2; 33.3%) predominated among the isolates. Other uropathogens included Stenotrophomonas maltophilia, Acinetobacter baumannii, and Enterobacter cloacae. All isolates were sensitive to meropenem but were resistant to ceftazidime, ampicillin, and tetracycline. blaSHV, blaTEM, and blaCTX-M-15 were detected in Enterobacter cloacae.ConclusionsThe prevalence of ASB is high in patients with SCD predominantly among females. Rare multidrug-resistant uropathogens were implicated. We posit a need for resistance surveillance programs and antibiotic stewardship to prevent treatment failure and reduce drug resistance.

Comparison of Five Methods for Detection of Extended Spectrum β-Lactamases in Gram Negative Enteric Bacteria

The presence of extended-spectrum β-lactamases in 55 isolates of Gram negative enteric bacteria isolated from lower respiratory tract infections, was investigated by using the Clinical Laboratory Standards Institute CLSI method which showed that 41.8% of the isolates produced this type of β-lactamases, and that Klebsiella pneumoniae isolates were the most producing species with a production rate of 61.1%, followed by Escherichia coli isolates 43.75%. Five confirmatory methods were used to detect these enzymes: ceftazidime agar method, double-disk synergy method, combination disk method, modified 3D extract method and enzymatic disks method. The study indicated that ceftazidime agar method was the best method in detecting extended-spectrum β-lactamases as it gave a detection rate of 95.7%, followed by the double-disk synergy method with a rate of 87%, then enzymatic disks method with a rate of 73.9%.

Determination of ESBL Production & Antibiotic Resistance Profile of Escherichia Coli Isolated from Patients with Urinary Tract Infections: A Study from Tertiary Care Hospital

Urinary tract infection (UTI), is defined as a disease caused by invasion of urinary tract by microorganisms. Majority of UTI cases are due to bacterial infection constitute about 95% of total UTI cases. About 80% of UTI cases are caused by E.coli producing extended spectrum ?-lactamase (ESBL) producing isolates. In recent years limitations in treating infections caused by multidrug resistant organisms has increased. This study aims to determine ESBL production of E. coli cases from a tertiary care hospital. Methodology: A total 358 midstream urine samples were collected by random sampling method during March 2015 to June 2018. Identification, antibiotic sensitivity testing, performed according to standard protocol following Clinical and Laboratory Standard Institute (CLSI) guidelines, 2013. Screening for ESBL producing E.coli isolates performed using ceftazidime further confirmation done by phenotypic disc diffusion test using combined disc method using ceftazidime (30µg) & ceftazidime/ clavulanic acid (30/10 µg) as per CLSI guidelines. Results: Total 358 specimens processed for urine culture. Gram negative bacilli isolated from 123(34.35 %), out of which 68 (55.28%) were E.coli, 19 (15.44%) K. pneumoniae, 15 (12.19%), Pseudomonas spp. 08 (6.50%), Citrobacter spp and Acinetobacter spp, 03 (2.43%), Proteus mirabilis, 01 (0.81%) Proteus vulgaris and Enterobacter respectively. Out of 68 isolates of E.coli, 65 (95.58%) were MDR, ESBL was detected in 31 (47.69%) out of these 65 isolates. Out of these 31 cases 19 (61.29%) were female and 12 (38.70%) were male cases. Conclusion: This study concludes 47.69% ESBL producing MDR E. coli were isolated from UTI cases with female predominance.

An investigation of extended-spectrum β-lactamases (ESBLs) in Klebsiella isolated from foodborne outbreaks in Iran

Aims and backgroundsAntibiotic resistance of Enterobacteriaceae such as Klebsiella which usually caused by Extended-Spectrum β-Lactamases (ESBLs) has been an issue for public health. The aim of this study was to evaluate the presence of ESBLs in Klebsiella isolates obtained from foodborne outbreaks diarrheal samples by phenotypic and genotypic methods. Materials and methodsIn this study, 416 diarrheal samples were collected from 120 foodborne outbreaks from March 2017 to March 2018 throughout Iran. Isolation and identification of Klebsiella isolates were performed by phenotypic methods, and antibiotic susceptibility testing was accomplished by disk diffusion method. Production of ESBLs was performed using combined disc method. The presence of blaSHV, blaTEM, and the blaCTX-M genes was investigated by PCR, and the data was analyzed using SPSS version 18. ResultsOf 416 diarrheal samples, 32 isolates (7.69%) were found positive for Klebsiella, of them 24 isolates (75%) were identified as Klebsiella pneumoniae, and 8 isolates (25%) as Klebsiella oxytoca. In Klebsiella pneumoniae isolates, the highest susceptibility was seen to imipenem and piperacillin (91.7%), while the highest resistance was observed to amoxicillin and penicillin (100%). Klebsiella oxytoca isolates were also completely (100%) resistant to amoxicillin and penicillin and completely (100%) susceptible to other antibiotics. Five isolates were identified as ESBLs positive, phenotypically. Of the 32 isolates, 22 isolates (68.7%) were positive for the presence of the blaSHV, 5 isolates (15.6%) for blaCTX-M, and all 32 isolates (100%) for blaTEM genes. ConclusionAlthough Klebsiella isolates are not very common in foodborne outbreaks, but they are important due to the presence of ESBLs introducing high resistance to the common antibiotics.

Detection of ESBLs and NDM-1 genes among urinary Escherichia coli and Klebsiella pneumoniae from healthy students in Niger Delta University, Amassoma, Bayelsa State, Nigeria

INTRODUCTION: Increasing emergence of multidrug-resistant uropathogens among healthy individuals is a serious public health problem capable of causing difficult-to-treat urinary tract infections (UTIs) with limited treatment options. This study therefore, determined the prevalence of asymptomatic bacteriuria (ASB) and molecular characteristics of antibiotic-resistant Escherichia coli and Klebsiella pneumoniae isolated from healthy students of Niger Delta University, Amassoma, Nigeria. METHODS: mid-stream urine samples were collected from 303 healthy students and cultured for significant bacteriuria. Escherichia coli and K. pneumoniae were isolated and identified by conventional methods and Polymerase Chain Reaction (PCR). Antimicrobial susceptibility patterns of the isolates were determined by the disc diffusion technique. The isolates were screened for Extended-Spectrum Beta-Lactamases (ESBLs) production by combined disc method, ESBLs and carbapenemases genes by PCR. RESULTS: the prevalence of ASB was 21.1%, with a significantly higher proportion among the females (P < 0.0001). Escherichia coli and K. pneumoniae isolates were recovered in equal proportion (n=21; 6.9%), with E. coli significantly isolated from the females (P= 0.002). The isolates exhibited 57 - 95% resistance to cefotaxime, ceftazidime, and co-trimoxazole, and 10 - 24% resistance to levofloxacin and imipenem. Multidrug resistance (MDR) was found in 9 (42.9%) E. coli and 18 (85.7%) K. pneumoniae isolates; 60% of E. coli and 72.7% of K. pneumoniae were ESBLs producers. blaTEM (50%) and blaCTX-M (30%) were detected in E. coli while blaSHV (83.3%) and blaTEM (55.6%) were detected in K. pneumoniae. One E. coli and two K. pneumoniae isolates harboured NDM-1 gene. CONCLUSION: the ASB from this study’s healthy individuals was characterized by MDR bacteria that harboured ESBLs and NDM-1 genes. Thus, emphasizing the need for regular surveillance of bacterial resistance and proper hand hygiene to contain the spread of MDR pathogens in the community.

Antimicrobial resistance pattern of extended-spectrum β-lactamase-producing Escherichia coli isolated from fecal samples of piglets and pig farm workers of selected organized farms of India.

Background and Aim:Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are gradually increasing worldwide and carry a serious public threat. This study aimed to determine the antimicrobial resistance pattern of ESBL-producing E. coli isolated from fecal samples of piglets and pig farm workers.Materials and Methods:Fecal samples from <3-month-old piglets (n=156) and farm workers (n=21) were processed for the isolation of ESBL-producing E. coli in MacConkey agar added with 1 µg/mL of cefotaxime. E. coli (piglets=124; farm workers=21) were tested for ESBL production by combined disk method and ESBL E-strip test. Each of the ESBL-positive isolate was subjected to antibiotic susceptibility testing. The ESBL-producing E. coli were further processed for genotypic confirmation to CTX-M gene.Results:A total of 55 (44.4%, 55/124) and nine (42.9%, 9/21) ESBL-producing E. coli were isolated from piglets and farm workers, respectively. Antibiotic susceptibility testing of the ESBL-positive E. coli isolates from piglets and farm workers showed 100% resistance to ceftazidime, cefotaxime, cefotaxime/clavulanic acid, ceftazidime/clavulanic acid, and cefpodoxime. A proportion of 100% (55/55) and 88.9% (8/9) ESBL-positive E. coli were multidrug resistance (MDR) in piglets and farm workers, respectively. On genotypic screening of the ESBL E. coli isolated from piglets (n=55), 15 were positive for the blaCTX-M gene and of the nine ESBL E. coli from farm workers, none were positive for the blaCTX-M gene.Conclusion:Although there was no significant difference in isolation of ESBL-producing E. coli between piglets and farm workers, the ESBL-positive E. coli from piglets showed relatively higher MDR than farm workers.

Extended-Spectrum β-Lactamases-Producing Enterobacteria and Antimicrobial Resistance Pattern among HIV/AIDS Patients in the University of Gondar Specialized Hospital, Ethiopia

There is growing evidence indicating that drug-resistant bacteria notably Extended spectrum β-lactamases producing Enterobacteria are greatly implicated in immunocompromised groups namely HIV-infected patients. Little is known about the burden of ESPL-E in places where HIV infection is rampant. Therefore, the study was aimed to assess the prevalence, antimicrobial resistance pattern of ESPL-E among HIV/AIDS patients. A cross-sectional study was conducted among HIV/AIDS patients seeking Antiretroviral Treatment (ART) service at the University of Gondar Hospital from February–May, 2017. The pretested and -structured questionnaire was used to collect data on sociodemographic and clinicalrelated factors. Clean catch midstream urine samples were collected and cultured in line with standard procedures. The drug susceptibility testing was performed by Kirby Bauer disc diffusion method. Extended-spectrum β-lactamase (ESBL) detection was performed using a doubledisc synergy test and combined disc methods. Data entry and analysis were performed using SPSS version 20. Among a total of 387 HIV/AIDS patients, 42 (10.9%) Enterobacteria uropathogens were identified. Among these isolates, nine (21.4%) were ESBL producers. The highest prevalence of ESBL production was Escherichia coli (44.4%) followed by Klebsiella pneumoniae (22.2%) and Enterobacter spp. (22.2%). Higher drug resistance rates were observed among ESBL-producing isolates compared to ESBL-nonproducing isolates. Amox-clavulanic (100%), ampicillin (95%), cotrimoxazole (74%), cefotaxime (88.9%), and ceftazidime (88.9%) had high resistance rates to Extended spectrum β-lactamases producing Enterobacteria. The overall prevalence of multidrug resistance of all isolates was 92.9%, and all the ESBL isolates were multidrug resistant. Therefore, antimicrobial stewardship programs needed to be promoted for the rational use of drugs especially in the management of HIV/AIDS patients.

Extended-Spectrum β-Lactamase-Producing Escherichia coli and Klebsiella Species in Pediatric Patients Visiting International Friendship Children's Hospital, Kathmandu, Nepal.

Introduction:Emergence and spread of antimicrobial resistance (AMR) is a global threat and significantly affects the treatment options for common infectious diseases. Inappropriate use of antibiotics, particularly third-generation cephalosporins, has contributed to the development of AMR. This study aims to determine the prevalence of extended-spectrum β-lactamase (ESBL) production in Escherichia coli and Klebsiella species isolated from various clinical samples.Methods:This cross-sectional study was conducted at International Friendship Children’s Hospital, Kathmandu, Nepal, from August 2017 to January 2018. A total of 1443 samples that included urine, pus, wound swab, endotracheal tip, catheter tip, and blood were collected from pediatric patients below 15 years and processed by standard microbiological methods. Following sufficient incubation, isolates were identified by colony morphology, gram staining, and necessary biochemical tests. Identified bacterial isolates were then tested for antibiotic susceptibility test by modified Kirby-Bauer disk diffusion method and were subjected to ESBL screening by using 30 µg cefotaxime and ceftazidime. The ESBL production was confirmed by combination disk method.Results:From a total of 103 nonduplicated clinical isolates, E. coli (n = 79), Klebsiella pneumoniae (n = 18), and Klebsiella oxytoca (n = 6) were isolated from different clinical specimens. Of which, 64 (62.1%) exhibited multidrug resistance, and 29 (28.2%) were ESBL producers. All ESBL-producing isolates were resistant toward ampicillin, cefotaxime, ceftriaxone, and ceftazidime. Most ESBL producers were susceptible toward imipenem (89.7%; 26/29), nitrofurantoin (82.8%; 24/29), piperacillin/tazobactam (79.3%; 23/29), and amikacin (72.4%; 21/29).Conclusions:A high prevalence of multidrug-resistant ESBL organisms was found in this study among pediatric patients. Treatment based on their routine identification and susceptibility to specific antibiotics is critical to halt the spread of AMR and ESBL.

Phenotypic characterization of beta-lactamases producing Gram-negative bacteria in a tertiary hospital, Nepal

Infections caused by beta-lactamases producing Gram-negative bacteria are increasing, thus posing a challenge to the management of such infections. The surveillance data of such bacteria is limited in Nepal so this study aimed to detect the beta-lactamase producing Gram-negative bacteria in a tertiary setting. A total of 604 clinical samples, including urine, blood, sputum and body fluids, were cultured and identified by the routine standard laboratory protocols. Antibiotic susceptibility testing was done by Kirby Bauer disc diffusion method following Clinical and Laboratory Standard Institute guidelines (2014). Extended-spectrum beta-lactamases (ESBL) producers were identified by combined disk method and metallo-beta-lactamases (MBL) producers were identified by Imipenem- EDTA combined disk method. Out of 604 samples, 282 (46.7%) samples showed significant growth, of which 229 (81.2%) were Gram-negative bacteria. Of 229 Gram-negative bacteria, 200 (87.3%) were multidrug resistant, 67 (29.3%) were ESBL producers and 16 (7.0%) were MBL producers. Klebsiella pneumoniae were among higher ESBL producers and Pseudomonas aeruginosa were among higher MBL producers. The findings suggest higher antibacterial resistance among Gram-negative bacteria with the added burden of beta-lactamase production. Imipenem was effective against 125 of 229 Gram-negative bacteria tested. Thus, imipenem can be the drug of choice for empirical management. The higher multidrug resistance and higher beta-lactamases production among Gram-negative bacteria warrant the continuous monitoring, surveillance, early detection, and infection control practices of such bacteria

Extended Spectrum Beta-Lactamase and Metallo Beta-Lactamase Producing Pseudomonas Species Isolated From Fish Pond Water in Ibadan, Nigeria

ABSTRACT Gram-negative bacteria that produce Extended-spectrum beta-lactamase (ESBL) and metallo beta-lactamase (MBL) show resistance to beta-lactam antibiotics. This study was to determine Beta-lactamases-producing Pseudomonas species in fish ponds. Pseudomonas species isolated from aquaculture water samples using Pseudomonas base agar were characterised biochemically. The detection of ESBL and MBL was made by double disc synergy and imipenem-EDTA combined disc methods, respectively. Antimicrobial susceptibility was by disc diffusion method. The 94 Pseudomonas species isolated comprised P. aeruginosa (62.8%), P. stutzeri (14.9%), P. putida (9.8%) and P. fluorescens (12.8%). P. aeruginosa 16 (16.3%) and P. stutzeri 4 (4.3%) produced ESBL, but 2 (10%) P. aeruginosa and 1 (5%) P. stutzeri produced MBL. Resistance of ESBL producers to trimethoprim was 55.5% and 7 (35.0%) were multidrug resistant. Detection of ESBL and MBL in Pseudomonas spp. from this study implies environmental health risk. Antibiotics misuse in aquaculture and discharge of untreated aquaculture wastewater into the environment should be discouraged.

Extended Spectrum Beta-lactamase Producing Gram Negative Bacterial Isolates from Urine of Patients Visiting Everest Hospital, Kathmandu, Nepal

Objectives: The study was aimed to determine the prevalence of Extended Spectrum Beta Lactamase (ESBL) producing Gram negative pathogens from urine samples along with their antimicrobial resistance.&#x0D; Methods: This cross-sectional study was conducted from December 2015 to May 2016 at Everest Hospital, Kathmandu. Mid-stream urine samples were collected and processed for culture by standard loop streak method. Identified bacterial isolates were tested for Antibiotic Susceptibility by modified Kirby Bauer disc diffusion method and, were subjected to ESBL screening by using 30µg cefotaxime and ceftazidime. ESBL production was confirmed by combination disc method.&#x0D; Results: Of the three hundred urine samples, 22.7% (67/300) showed significant growth. Four different bacterial species were identified. Among the isolates, E. coli was the most common pathogen (71.64%) followed by Klebsiella pneumoniae (14.92%), Pseudomonas spp (8.95%) and Acinetobacter spp (4.48%). Altogether 92.54% (n=62) isolates were sensitive to gentamicin, 89.55% (n=60) to amikacin, and 79.10% (n=53) to nitrofurantoin. 70.10% (n=47) isolates were resistant to antibiotic ampicillin while 62.68% (n=42) were found as Multi-Drug Resistant (MDR) and 29.8% (n=20) were ESBL producers.&#x0D; Conclusions: The overall prevalence of MDR and ESBL among uropathogens is low in comparison to other studies though it is essential to have a regular monitoring of ESBL producing clinical isolates in laboratory practice.

Extended spectrum \u03b2-lactamase producing uropathogenic Escherichia coli and the correlation of biofilm with antibiotics resistance in Nepal

BackgroundUrinary tract infection (UTI) is one of the frequently diagnosed infectious diseases which is caused mainly by Escherichia coli. E. coli confers resistance against the two major classes of antibiotics due to the production of extended spectrum β-lactamase enzymes (ESBL), biofilm, etc. Biofilm produced by uropathogenic E. coli (UPEC) protects from host immune system and prevent entry of antimicrobial compounds. The main objective of this cross-sectional study was to determine the correlation of biofilm production and antibiotic resistance as well as to characterize the pgaA and pgaC genes responsible for biofilm formation among uropathogenic ESBL producing E. coli.MethodsA total of 1977 mid-stream urine samples were examined and cultured for bacterial strain identification. ESBL was detected by combined disc method following CLSI whereas biofilm formation was analyzed by semi-quantitative method. Furthermore, the pgaA and pgaC genes responsible for biofilm formation in UPEC were detected by multiplex PCR. All the statistical analyses were done via IBM SPSS Statistics 21 where Pearson’s correlation test were used to determine correlation (−1 ≥ r ≤ 1).ResultsE. coli was the predominant causative agent, which accounted 159 (59.3%) of the Gram-negative bacteria, where 81 (50.9%) E. coli strains were found to be ESBL producers. In addition, 86 (54.1%) E. coli strains were found to be biofilm producers. Both the pgaA and pgaC genes were detected in 45 (93.7%) the UPEC isolates, which were both biofilm and ESBL producers. Moreover, there was a positive correlation between biofilm and ESBL production.ConclusionThe analyses presented weak positive correlation between biofilm and ESBL production in which biofilm producing UPEC harbors both pgaA and pgaC genes responsible for biofilm formation.

Extended Spectrem Beta Lactamases among Multi Drug Resistant Gram Negetive Bacilli Causing Urinry Tract Infection

Extended Spectrum Beta Lactamases (ESBL), the main cause of resistance to broad spectrum β-lactams, among uropathogenic bacteria have increased over time raising a global concern in the therapeutic management of infections caused by these organisms. The study was carried out in Janamaitri Hospital, Kathmandu between December 2012 to May 2013 with an objective to determine the status of ESBL producing Gram negative bacilli isolated from the urine sample, collected from patients suspected of urinary tract infection. Gram negative bacilli isolated were tested for the presence of ESBL by combined disk and antibiotic susceptibility by Kirby Bauer disc diffusion method following Clinical and Laboratory Standard Institute guidelines. Among the total 1105 mid-stream urine samples, 256 Gram negative bacilli were isolated. By screening test using third generation cephalosporins, 156 isolates were screened as ESBL producers and 91 isolates were positive for ESBL test by combined disk method. Among the 91 (35.55%) ESBL producers, 70 (39.32%) Escherichia coli, 16 (44.44%) Klebsiella pneumoniae, and 5 (33.33%) Pseudomonas aeruginosa were found to be ESBL producers. Majority of ESBL producer showed resistance to ampicillin, co-trimoxazole, norfloxacin followed by ofloxacin. imipenem, amikacin and nitrofurantoin seemed to be the agent of choice for urinary tract infections when ESBL producers are susceptible to it. ESBL production found in these Gram negative bacilli with resultant microbial resistance to available cephalosporins and other agents may pose difficulties with the choice of therapeutic options for the treatment of severe infections.&#x0D; Keywords: UTI, Extended Spectrum Beta Lactamases, Gram negative bacilli

Phenotypic and genotypic detection of antibiotic resistance among metallo-beta-lactamases producing Klebsiella pneumoniae strains isolated from patients in Intensive Care Units in Shiraz, Iran

Klebsiella pneumoniae is the opportunistic pathogen which is responsible for infections in the hospital intensive care units (ICUs). Increase in the rate of carbapenemase-producing Enterobacteriaceae, is a major concern. The aims of this study were to identify metallo-beta-lactamases (MBL)-producing K. pneumoniae isolates and detect their antibiotic susceptibility pattern. Antibacterial susceptibility testing was performed for 8 antibiotics, using disk diffusion method according to CLSI guidelines. Isolates showing resistance to at least one of the carbapenem antibiotics were then evaluated for production of metallo-beta-lactamase enzymes, using combination disk method. The presence of the blaIMP, blaVIM, and blaSPM genes was assessed by PCR. Out of 150 examined isolates, 24 isolates (16%) were resistant to imipenem and 18 (12%) to meropenem. All of these isolates were resistant to more than three different classes of antibiotics. Of the 24 K. pneumoniae isolates that were resistant to carbapenem, just 3 (12.5%) were positive MBLs. Twenty-one (14%) of 150 isolates had positive results for IMP, 2 (1.3%) for VIM, while none of the isolates were positive for SPM metallo-beta-lactamase gene. MBLs have expanded worldwide and MBL-producing isolates which make the treat of infections associated with high mortality rates difficult; therefore, this study focused on the implementation of infection control practices against enhanced antibiotic resistance.

Detection of Pan drug resistance OXA-48 producing Providencia in an ICU patient for the first time in Nepal

sBackgroundResistance to antimicrobial agents of pathogenic bacteria has become a major problem in routine medical practices. Carbapenem resistance has long been increasing. The production of carbapenem- hydrolysing β-lactamases (carbapenamases), which include NDM, KPC, OXA-48, IMP-1 and VIM is the most common mechanism.Case presentationA 56 years old male presented with fever and mental changes with progressively decreasing sensorium for the last 3 days. He was admitted to Intensive care unit (ICU) with a diagnosis of meningoencephalitis. On day seven, he developed ventilator associated pneumonia due Klebsiella pnemoniae and Acinetobacter baumannii. He was on meropenem, but the isolates were susceptible to colistin, tigecyclin and amikacin solely. Hence, amikacin was started with addition of intravenous and nebulized colistin. Subsequently, vital signs improved with resolution of fever. However, on day 18, he developed fever once again with a drop in blood pressure. Inotropic support was maintained, and echinocandins and tigecycline were added to the regimen.Repeat blood and urine culture grew Providencia species, which were resistant to most of the drugs on phenotypic Kirby-Bauer disk diffusion method and are intrinsically resistant to colistin and tigecycline. Phenotypic detection of ESBL (combined disk method), MBL, KPCs, AmpC and co-producer were tested according to updated CLSI guideline and all were negative. But the Modified Hodges test was found to be positive. Consequenty, OXA-48 drug resistance pattern was brought into action by blank disc method according to A Tsakris et al., which revealed indentation of growth toward both EDTA and EDTA/PBA disk indicating production of OXA-48 carbapenamase. To confirm the resistance pattern we processed the isolated colonies for Xpert Carba-R (Cepheid) assay, which detected blaOXA-48 gene and confirmed the OXA-48 drug resistance pattern. Hence, the infecting organism was not susceptible to any of the antibiotics. The patient was kept under isolation and on 31th day of admission, he died of septic shock.ConclusionsCarbapenamase production along with intrinsic colistin resistance in infecting bacterial pathogens can cause fatal outcomes in the resource limited countries like Nepal where new antibiotic combinations ceftazidime+ Avibactam, or aztreonam +avibactam are not available. Drug resistance patterns including OXA 48 producer should be characterized in all cases by standard phenotypic methods or by Xpert Carba-R assay and larger studies are required to know the exact burden of OXA 48 producer in Nepal.

Generic Salmonella in Asymptomatic Adult Volunteers: Occurrence, Antibiogram, Extended-Spectrum β-Lactamase Production and Carbapenem Resistance

This study was conducted to isolate generic Salmonella from stool of apparently-healthy/asymptomatic adult volunteers in Ankpa North-central Nigeria, and to determine antibacterial susceptibility, extended-spectrum β-lactamase production and carbapenem resistance. About a gram of stool was collected from 295 randomly-selected apparently-healthy/asymptomatic adult volunteers in Ankpa Kogi State, North-central Nigeria. Isolation of Salmonella was done by pre-enrichment with buffered peptone water, enrichment with Rapapport-Vassiliadis and sub-culturing on Salmonella-Shigella and MacConkey agar. Identification of the isolates to genus level was done using standard biochemical tests. Resistance of the isolates was determined by disc diffusion method. Production of ESBL was assessed by combination disc method while carbapenem resistance was determined using meropenem disc screening method. Nine (3.1%) out of 295 samples, gave positive growth of Salmonella. Of these 9 isolates, all were carbapenem (meropenem)-resistant and 1 (11.1%) was ESBL-producer. Among the isolates, all were resistant to ciprofloxacin, ofloxacin, norfloxacin, ampicillin, cefotaxime, ceftazidime, gentamicin and streptomycin, 5 (55.6%) to sulphamethoxazole-trimethoprim, 7 (77.8%) to amoxicillin-clavulanic acid and aztreonam, 6 (66.7%) to nitrofurantoin and 8 (88.9%) to tetracycline. All the isolates were resistant to ≥ 5 classes of antibacterial agents. Mean multiple antibacterial indexes of the isolates were 0.68 (range 0.79 - 1.00). Apparently-healthy/asymptomatic adults in Ankpa North-central Nigeria, are potential reservoirs and disseminators of multi- and extensive-drug resistant salmonellae.

TEM Gene Detection in Clinical Pseudomonas aeruginosa and Escherichia coli Samples

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MOLECULAR EPIDEMIOLOGY OF METALLO BETA LACTAMASES IN ACINETOBACTER BAUMANNII AT A TERTIARY CARE HOSPITAL

Background: Carbapenem resistance mediated by metallo beta lactamases (MBL) in Acinetobacter baumannii is a global challenge due to its rapid spread and limited therapeutic options.&#x0D; Objective: To determine the prevalence of MBL in A. baumannii isolates in hospitalized patients by both phenotypic and genotypic methods.&#x0D; Materials and Methods: The clinical samples were collected from inpatients and subcultured on routine culture media for growth. Identification of bacteria along with antimicrobial sensitivity testing was done by VITEK -2 Compact (bioMerieux). Antibiotics that were not tested by VITEK-2 were tested manually by Kirby-Bauer disk diffusion method according to CLSI 2017 and EUCAST 2016 guidelines. The isolates which were resistant to carbapenem (imipenem and/ or meropenem) were tested by phenotypic (imipenem-EDTA combined disk method) and genotypic method for presence of common metallo beta lactamases genes (blaIMP, blaNDM, blaGIM, blaVIM, blaSPM and blaSIM).&#x0D; Results: 84 non duplicate A.baumannii were isolated out of 947 pathogenic gram negative isolates. Majority (47.6%) of isolates were obtained from tracheostomy/endotracheal/bronchoalveolar lavage (TT/ET/BAL) followed by sputum (21.4%). None of the isolates were found to be resistant to colistin and tigecycline. 73 (86.9%) isolates were found to be carbapenem resistant, among these 60 (82.2%) were found to be MBL positive by phenotypic and 32 (43.2%) by genotypic method. MBL genes detected were blaNDM (39.7%), blaGIM (2.7%) and blaVIM (1.4%). None of the isolates were positive for blaIMP, blaSPM and blaSIM.&#x0D; Conclusion: The prevalence of MBL in carbapenem resistant isolates of A.baumannii was 87.7%. blaNDM was the most common gene detected. No significant difference was found in the ability of phenotypic and genotypic methods for MBL detection. The resistance rate of the A.baumannii is high for most antibiotics except for polymyxins (E&amp;B) and tigecycline.&#x0D; Key words: Metallo beta Lactamases, Acinetobacter baumannii, Carbapenem.

A multicenter study of β-lactamase-producing Klebsiella pneumoniae isolated from university teaching hospitals of Urmia, Iran.

Klebsiella pneumoniae is an opportunistic pathogen accounting for 5-7% of hospital acquired infections. The emergence of carbapenem-resistant Klebsiella pneumoniae has been increasing rapidly over recent years causing many therapeutic problems worldwide. This study aimed to research the antimicrobial resistance profile, detect β-lactamase genes among clinical isolates of K. pneumoniae, and determine their clonal relatedness. All Klebsiella pneumoniae isolates were obtained from teaching hospitals in Urmia, Iran. Antimicrobial susceptibility testing was done by the disk diffusion method. Furthermore, minimum inhibitory concentrations of imipenem were determined by applying Etest strips. Screening of β-lactamase-producing isolates was performed by the combined disk method and modified Hodge test. The detection of β-lactamase genes was conducted by polymerase chain reaction (PCR), and isolates' clonal relatedness was evaluated by random amplified polymorphic DNA (RAPD)-PCR. Overall, 45 out of 182 (24.7%) K. pneumoniae isolates were non-susceptible to imipenem. The combined disk method and modified Hodge test revealed that 93.3% and 71.1% of the imipenem non-susceptible isolates were β-lactamase producers, respectively. The presence of blaVIM, blaNDM, blaKPC, and blaIMP genes was confirmed in 48.9%, 15.6%, 11.1%, and 6.7% of the β-lactamase-producing isolates, respectively. RAPD-PCR revealed that 73% of these isolates were classified into six different clusters. A relatively high prevalence of β-lactamase genes was seen among multidrug-resistant isolates of K. pneumoniae. Most patients infected with β-lactamase-producing isolates had a history of long-term hospitalization and nosocomial infections. The predominance of β-lactamase genes in intensive care unit and internal units alarm clinicians to the growth of hospitalization and mortality rates.