Double-disk Synergy Test Research Articles

Antimicrobial resistance, biofilm formation, and molecular detection of efflux pump and biofilm genes among Klebsiella pneumoniae clinical isolates from Northern Jordan

The current study aimed to investigate the antimicrobial susceptibility profiles, biofilm production capabilities, and the prevalence of efflux pump and biofilm-associated genes among Klebsiella pneumoniae clinical isolates. One hundred sixty-seven K. pneumoniae isolates were collected from microbiology laboratories in Northern Jordan hospitals. Antimicrobial susceptibility was tested using the Kirby-Bauer method. The double-disk synergy test was used to detect the extended-spectrum beta-lactamase (ESBL) phenotype. PCR was used to detect the frequency of acrAB,tolC, and mdtk efflux pump genes and fimH-1,mrkA, and mrkD biofilm-associated genes among the isolates. The highest nonsusceptibility was observed against azithromycin (87.4 %) and nitrofurantoin (85.0 %). Among the isolates, 75.4 % and 92.2 % were multidrug resistant and produced biofilms, respectively. Efflux pump genes acrAB, tolC, and mdtK were found in 96.4 %, 95.2 %, and 90.4 % of the isolates, respectively. Biofilm-associated genes mrkD, mrkA, and fimH-1 were found in 92.2 %, 89.2 %, and 88.6 % of the isolates, respectively. The presence of the mrkA was significantly associated with biofilm formation. Overall, high percentages of multi-drug resistance, efflux pump, and biofilm-associated genes were observed among the isolates. Subsequent studies are recommended to monitor changes in the prevalence of resistance phenotypes and genotypes of isolates.

Evaluation of Co-production of Colistin Resistance and ESBL Genes among Gram-negative Clinical Isolates from Usmanu Danfodiyo University Teaching Hospital Sokoto, Nigeria

Study’s Excerpt/Novelty This study presents a comprehensive evaluation of colistin-resistant and extended-spectrum beta-lactamase (ESBL) gene co-production among Gram-negative clinical isolates from Usmanu Danfodiyo University Teaching Hospital in Sokoto. Notably, 13.9% of the isolates exhibited phenotypic co-production of colistin resistance and ESBL, with a significant presence of blaCTX-M and CTX-M 8 genes among ESBL producers, although no colistin resistance genes (mcr-1 and mcr-2) were detected via PCR. These findings highlight the necessity for integrated molecular and phenotypic investigations to fully elucidate resistance mechanisms in Gram-negative bacteria and underscore the need for further research to uncover alternative pathways contributing to observed resistance phenotypes. Full Abstract The emergence of antimicrobial resistance (AMR) is a major threat to global health. Its effects include high mortality and morbidity rates, treatment failure, and increased treatment costs. This study aimed to evaluate the co-production of colistin-resistant and extended-spectrum beta-lactamase (ESBL) genes among Gram-negative clinical isolates from Usmanu Danfodiyo University Teaching Hospital in Sokoto. Gram-negative bacteria were isolated from clinical specimens, including urine, feces, and wound aspirates. The Double-Disk Synergy Test and the Colistin Agar Test, respectively, were used to phenotypically validate the existence of colistin resistance and ESBL. Polymerase chain reaction (PCR) was used for molecular characterization. Primers were used to target genes linked to colistin resistance (mcr-1 and mcr-2) and ESBL genes (blaCTX-M, CTX-M 1, CTX-M 2, and CTX-M 8). The findings indicated that 13.9% of the isolates displayed co-production of Colistin and ESBL, and of these isolates, 60% had blaCTX-M genes, and 20% had CTX-M 8 linked to ESBL production. However, the presence of colistin resistance genes was not detected by PCR. Therefore, molecular analysis did not confirm the existence of the colistin resistance genes (mcr-1 and mcr-2) in these isolates. Consequently, the findings showed no molecular co-production of the ESBL and colistin resistance genes. This work emphasizes how crucial it is to look into molecular and phenotypic traits to completely comprehend how colistin resistance and ESBL genes coexist in Gram-negative isolates. More research is required to investigate other mechanisms behind the resistance phenotypes identified.

Prevalence of Multi Drug Resistant Coagulase Negative Staphylococci among some Pregnant Women Attending Antenatal Clinic in Yola, Nigeria

Study’s Novelty/Excerpt This study presents findings on the prevalence and characterization of coagulase-negative staphylococci (CoNS) among pregnant women in public health facilities in Yola, revealing a significant prevalence rate of 26.97%. The research highlights the high susceptibility of CoNS to certain antibiotics and the alarming presence of multi-drug resistant and extended spectrum beta-lactamase (ESBL) producing strains. This underscores the necessity for routine screening of pregnant women for CoNS to prevent potential gestational complications, providing crucial data for public health interventions and antibiotic stewardship programs. Full Abstract Coagulase negative staphylococci have been reported to be a frequent agent of uncomplicated UTI in young sexually active women which can also lead to symptomatic infection, low birth weight (LBW) babies and preterm delivery. This study was undertaken to determine the prevalence and characterize coagulase negative staphylococci among pregnant women in public health facilities in Yola. A total of 250 urine samples were collected from pregnant women attending antenatal clinic at Specialist Hospital and Boshang Clinic and Maternity both in Yola. The samples were cultured on cysteine lactose electrolyte deficient medium and isolates characterized using phenotypic methods. The prevalence of coagulase negative staphylococci from this study was 26.97%, Staphylococcus aureus 16.29% and Escherichia coli 36.51%. The frequency of CoNS was highest among age group of 36-40 years and least among 16-20 years (12.5%). CONS isolated from this study demonstrated high susceptibility to pefloxacin, ciprofloxacin, erythromycin, ceftriaxone and streptomycin. Some isolates showed high resistance to ampiclox, cefuroxime and amoxicillin. About 83% of the isolates had multiple antibiotic resistant indexes greater than 0.2 with about 25% of the antibiotic resistance being plasmid mediated. Furthermore, phenotypic screening using the double disk synergy test showed that 20 (40%) of the isolates were extended spectrum beta lactamase (ESBL) producing. This study revealed the presence of multi-drug resistant and ESBL producing coagulase negative staphylococci among pregnant women in the study area. There is therefore the need for pregnant women to be screened for coagulase negative staphylococci to avoid complications during gestation period.

High prevalence and genetic diversity of multidrug-resistant and extended-spectrum ß-lactamase-producing Escherichia coli and Klebsiella pneumoniae in mothers and neonates in a Cameroonian labor ward

Escherichia coli and Klebsiella pneumoniae rank among the primary bacterial culprits in neonatal infections and fatalities in sub-Saharan Africa. This study characterized the phenotypic and genotypic features of E coli and K pneumoniae in a labor ward in Yaoundé, Cameroon. A prospective and cross-sectional study spanning 5months, from February 21, 2022 to June 30, 2022. Rectovaginal swabs were obtained from expectant mothers, and nasopharyngeal swabs were collected from their babies. Hand swabs of health care workers and environmental samples were also collected. The samples were cultured on eosin methylene blue agar. Extended-spectrum ß-lactamase (ESBL) production was assessed using CHROMAgar ESBL and the double-disk synergy test. A polymerase chain reaction was employed to detect ß-lactamase genes. A total of 93 mothers and 90 neonates were collected. Almost all pregnant women (90%) were colonized by one or more multidrug-resistant (MDR) isolates with 58% being concomitantly ESBL producers. Altogether, 14 of 22 (64%) neonates were colonized by MDR isolates, while out of the 5 workers positive to Enterobacterales, all were colonized by MDR isolates. E coli predominated in pregnant women (55%) and neonates (73%), while K pneumoniae (83%) predominated in health care workers. The blaCTX-M (75%) was the leading ß-lactamase gene detected. Our study suggests that drug-resistant E coli and K pneumoniae are circulating at high prevalence in thelabor ward in Yaoundé and emphasizes the necessity for effective infection prevention and control along with antimicrobial stewardship measures.

Surveillance of Surgical Site Infections in Post-operative Patients and Bacterial Susceptibility in Tanzania

Surgical site infections (SSI) are infections occurring within 30 days of the post-operative procedure. They are common post-operative morbid complications that may cause death if not treated timely. The common causes of SSI include infectious bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and some Enterobacteriaceae. This was a cross-sectional study conducted at St. Francis Referral Hospital, Ifakatra, Tanzania over a period of 12 months to investigate the causes of SSI and antimicrobial susceptibility of the causal agents. The study included consenting patients who developed post-operative wound infections during the study period. Identification of infecting micro-organisms and their antimicrobial susceptibility was done at St Francis Referral Hospital Laboratory. Antibiotic susceptibility tests of the isolates were performed by the Kirby–Bauer (K–B 1966) disc diffusion test, and extended spectrum β-lactamase producing Gram-negative species were tested by using the modified double disc synergy test. A total of 130 patients developed post-operative wound infection. Third and fourth decades were the most affected age groups; females were the dominant group with a 1:1.4 male: female ratio. Out of the 130 specimens, 121 isolates were obtained, and nine specimens were negative for culture. P. aeruginosa was the most commonly isolated agent (42.1%), followed by S. aureus (19.8%), while the least were Streptococcus spp. at 0.8%. The isolates showed the highest resistance to ampicillin (91.7%), and least to ciprofloxacin (1.7%). P. aeruginosa was highly resistant to both amoxicillin + clavulanic acid (98%), and to ampicillin (98.0%). Extended spectrum β-lactamase E. coli producers were 68.4%. The bacteria causing SSI require continuous monitoring to obtain data that will support local and national guidelines in the battle against antimicrobial resistance, and improve therapeutic outcomes following surgical interventions.

Prevalence of blaOXA-10, blaCTX-M-3 and SHV Genes among ESBL-Producing Escherichia coli and Klebsiella pneumoniae Isolated from Clinical samples in Basra city, Iraq

The current study was conducted to determine the prevalence of extended-spectrum β-lactamase (ESBL) in 78 drug-resistant clinical isolates (25 Klebsiella pneumoniae and 53 Escherichia coli strains) using phenotypic and molecular methods. The phenotypic method was performed using a double-disk synergy test (DDST), while the genotypic method screened for the blaSHV, blaCTX-M13U, and blaOXA-10 genes using specific primers. The phenotypic results showed that out of 53 tested strains of E. coli, 17 (32.07%) produced ESBL. Similarly, out of 25 tested strains of K. pneumoniae, 8 (32%) produced ESBL. Genotypic detection showed that in E. coli, the most abundant gene was SHV, present in 24 strains (45.28%), followed by blaOXA-10 in 23 strains (43.39%) and CTX-M-3 in 8 strains (15.09%). In K. pneumoniae, SHV was detected in 12 strains (48%), followed by OXA-10 and CTX-M-3, each found in 5 strains (20%).

Phenotypic identification of different β-Lactamases in intrinsic and acquired colistin resistant uropathogenic gram negative bacteria.

Identification of MBL, AmpC and ESBLs in colistin intrinsic and acquired resistant uropathogenic gram negative bacteria. Urine samples were collected from Hayatabad Medical Complex, Peshawar during 17 January to 30 June 2019. Collected urine samples were aseptically transported microbiology lab of Health Research Institution (HRI), National Institute of Health (NIH), Khyber Medical College, Peshawar and streaked on different media. Positive growth was identified by API-10s. Antibiotic sensitivity profile was done by Modified Kirby Bauer disc diffusion method. Detection of metallo βlactamases (MBL) production by Imipenem EDTA synergy test, Double Disc Synergy Test (DDST) for detection of ESBLs and D-test for the detection of inducible AmpC beta lactamases test was used. Colistin resistance was identified via broth micro dilution according to CLSI manual. Colistin resistant bacteria was divided in two categories; acquired and intrinsic resistant bacteria according to CLSI manual. Out of 2000 urine samples, 281(14%) gram-negative bacteria were isolated. Among positive samples, acquired colistin resistant bacteria were 241 and intrinsic resistant bacteria were 40 isolates. MBL was produce by twenty one (11.7%) E.coli and seventeen (40.5%) Pseudomonas aeruginosa. E. coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae, Serratia Oderifora and Proteus Marblis were ESBLs producing bacteria. AmpC production was prevalent in fourteen (7.8%) E. coli and twelve (28.6%) Pseudomonas aeruginosa. Fifty-five samples showed resistance to colistin out of 241 samples. In colistin resistant bacteria, two E.coli were MBL, ESBLs, while one E.coli was ESBLs, AmpC co-producing bacteria. The most prevalent extended drug resistant bacteria were Pseudomonas aeruginosa (28.6%) and Escherichia coli (6.1%), While 155(86.6%) Escherichia coli, 25 (59.5%) Pseudomonas aeruginosa and 22 (95.7%) Serratia Oderifora was multi drug resistant bacteria. Current study concluded that ESBL, MBL AmpC enzymes and their co-expression was observed with colistin resistance in E.coli and Pseudomonas aeruginosa.

Multidrug-Resistant Bacteria in Surgical Intensive Care Units: Antibiotic Susceptibility and β-Lactamase Characterization.

Multidrug-resistant (MDR) bacteria of the utmost importance are extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Enterobacterales (CRE), carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus spp. (VRE). In this study, an evaluation of MDR bacteria in surgical intensive care units in a tertiary referral hospital was conducted. The study aimed to characterize β-lactamases and other resistance traits of Gram-negative bacteria isolated in surgical intensive care units (ICUs). Disk diffusion and the broth dilution method were used for antibiotic susceptibility testing, whereas ESBL screening was performed through a double disk synergy test and an inhibitor-based test with clavulanic acid. A total of 119 MDR bacterial isolates were analysed. ESBL production was observed in half of the Proteus mirabilis, 90% of the Klebsiella pneumoniae and all of the Enterobacter cloacae and Escherichia coli isolates. OXA-48 carbapenemase, carried by the L plasmid, was detected in 34 K. pneumoniae and one E. coli and Enterobacter cloacae complex isolates, whereas NDM occurred sporadically and was identified in three K. pneumoniae isolates. OXA-48 positive isolates coharboured ESBLs belonging to the CTX-M family in all but one isolate. OXA-23 carbapenemase was confirmed in all A. baumannii isolates. The findings of this study provide valuable insight of resistance determinants of Enterobacterales and A. baumannii which will enhance surveillance and intervention strategies that are necessary to curb the ever-growing carbapenem resistance rates.

Proteome analysis, genetic characterization, and antibiotic resistance patterns of Klebsiella pneumoniae clinical isolates

Klebsiella pneumoniae (K. pneumoniae) is a member of the ESKAPE group and is responsible for severe community and healthcare-associated infections. Certain Klebsiella species have very similar phenotypes, which presents a challenge in identifying K. pneumoniae. Multidrug-resistant K. pneumoniae is also a serious global problem that needs to be addressed. A total of 190 isolates were isolated from urine (n = 69), respiratory (n = 52), wound (n = 48) and blood (n = 21) samples collected from various hospitals in the Al-Qassim, Saudi Arabia, between March 2021 and October 2022. Our study aimed to rapidly and accurately detect K. pneumoniae using the Peptide Mass Fingerprinting (PMF) technique, confirmed by real-time PCR. Additionally, screening for antibiotic susceptibility and resistance was conducted. The primary methods for identifying K. pneumoniae isolates were culture, Gram staining, and the Vitek® 2 ID Compact system. An automated MALDI Biotyper (MBT) instrument was used for proteome identification, which was subsequently confirmed using SYBR green real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Vitek® 2 AST-GN66 cards were utilized to evaluate the antimicrobial sensitivity of K. pneumoniae isolates. According to our results, Vitek® 2 Compact accurately identified 178 out of 190 (93.68%) K. pneumoniae isolates, while the PMF technique correctly detected 188 out of 190 (98.95%) isolates with a score value of 2.00 or higher. Principal component analysis was conducted using MBT Compass software to classify K. pneumoniae isolates based on their structure. Based on the analysis of the single peak intensities generated by MBT, the highest peak values were found at 3444, 5022, 5525, 6847, and 7537 m/z. K. pneumoniae gene testing confirmed the PMF results, with 90.53% detecting entrobactin, 70% detecting 16 S rRNA, and 32.63% detecting ferric iron uptake. The resistance of the K. pneumoniae isolates to antibiotics was as follows: 64.75% for cefazolin, 62.63% for trimethoprim/sulfamethoxazole, 59.45% for ampicillin, 58.42% for cefoxitin, 57.37% for ceftriaxone, 53.68% for cefepime, 52.11% for ampicillin-sulbactam, 50.53% for ceftazidime, 52.11% for ertapenem, and 49.47% for imipenem. Based on the results of the double-disk synergy test, 93 out of 190 (48.95%) K. pneumoniae isolates were extended-spectrum beta-lactamase. In conclusion, PMF is a powerful analytical technique used to identify K. pneumoniae isolates from clinical samples based on their proteomic characteristics. K. pneumoniae isolates have shown increasing resistance to antibiotics from different classes, including carbapenem, which poses a significant threat to human health as these infections may become difficult to treat.

PHENOTYPIC DETECTION OF ESBL AND AMPC BETA LACTAMASES AMONG GRAM-NEGATIVE ISOLATES FROM CLINICAL SAMPLES IN A TERTIARY CARE HOSPITAL

Objectives: Drug-resistant Gram-negative bacilli expressing extended-spectrum beta-lactamases (ESBLs) and AmpC pose a serious therapeutic threat in nosocomial infections. Cost-effective screening methods are a boon to patients. This study aims to detect gram-negative bacilli and their antibiotic sensitivity patterns, as well as detect the ESBL and AmpC-producing isolates among Gram-negative bacilli. Methods: A prospective study was conducted with 150 samples. Gram-negative bacilli were isolated, and their antibiotic sensitivity tests were performed by the Kirby–Bauer disk diffusion method. Potential ESBL producers were screened using Ceftazidime disc, and AmpC producers were screened by Cefoxitin discs by the disc diffusion method. ESBL producers were confirmed by the combined disc diffusion assay method using ceftazidime and ceftazidime/Clavulanic acid disc. AmpC producers were confirmed by the Cefoxitin Cloxacillin Double Disc Synergy Test. Results: About 38% of 150 samples were gram-negative bacilli, of which 40.35% were Escherichia coli, followed by Pseudomonas aeruginosa (35.08%). Maximum sensitivity by E. coli was found toward imipenem, meropenem, and cotrimoxazole. P. aeruginosa showed maximum sensitivity toward piperacillin/tazobactam, imipenem, meropenem, and ceftazidime. 28.07% of Gram-negative isolates were ESBL producers, with E. coli (11 isolates) being the maximum, and 15.78% were AmpC producers, with E. coli (four isolates) being the maximum. Seven isolates were both ESBL and AmpC producers. Conclusion: Routine screening and timely reporting of ESBL and AmpC producers help in preventing the spread of multidrug-resistant strains. Antibiotic resistance surveillance helps in the implementation of strict infection control and prevention practices.

PHENOTYPIC DETECTION AND ANTIMICROBIAL SUSCEPTIBILITY PROFILE OF EXTENDED SPECTRUM BETA LACTAMASE PRODUCING Escherichia coli FROM SUSPECTED CASES OF URINARY TRACT INFECTION IN KANO METROPOLIS, NIGERIA

Urinary tract infections (UTIs) caused by antimicrobial-resistant bacteria, especially ESBL-producing Escherichia coli can be life-threatening as therapeutic options available to treat infected patients are limited. Resistance due to ESBL-producing bacteria poses a peculiar challenge in treating infections because of its association with multidrug resistance. The aim of this study was thus to determine the susceptibility pattern and phenotypic detection of ESBL-producing E. coli from UTI patients. Two hundred and forty-six (246) E.coli isolates obtained from patients with suspected urinary tract infections were studied. The identity of the isolates was confirmed using standard biochemical tests. Antibiotic susceptibility testing was carried out using the Kirby-Bauer Disc Diffusion Technique. Screening for ESBL production was done using the Clinical Laboratory Standards Institute breakpoint. Suspected ESBL producers were subjected to confirmation using the Double Disc Synergy Test. Standard Discs of Augmentin (AMC 30µG Oxoid England), Ceftazidime (CAZ 30µG, Oxoid England) and Cefotaxime (CTX 30µG, Oxoid England) were used for the screening and confirmation. Multidrug-resistant E.coli were found to be 65.4%. Screening for ESBL production showed 67.1% suspected ESBLs producing E.coli. The Double Disc Synergy Test showed 22.4% confirmed ESBLs producing E.coli. Antimicrobial sensitivity of the ESBLs producing organisms showed 100% resistance to augmentin, ceftriaxone, ceftazidime, ciprofloxacin and cefotaxime while resistance to gentamicin was 91.1%, chloramphenicol 89.2%, nitrofurantoin 78.4%, and cotrimoxazole 94.6%. A 100% sensitivity to imipenem was also observed. ESBL-producing E.coli are present in Kano metropolis and are resistant to commonly prescribed antibiotics. We, therefore, suggest screening and confirmation for ESBL, to prevent treatment failure.

MAST® D72C test: a novel option for ESBL, AmpC and carbapenemase detection.

The MAST® D72C test is a phenotypical test which can detect ESBL and AmpC production in Enterobacterales. It can also identify the suspected presence of carbapenemase. The aim of the present study was to assess the sensitivity and specificity of this test and to discuss its usefulness in laboratories, especially those that use only an automated AST system. The performance of the MAST® D72C test was assessed against a collection of 119 non-redundant Enterobacterales isolates characterized for their content in β-lactamases, and compared with that of the reference double disk synergy test. β-lactamase content was established from phenotypic and genotypic analyses to collect a broad diversity of resistance mechanisms and bacterial strains, including 30 ESBL-producing strains, 32 strains overproducing chromosomal AmpC, 10 strains producing plasmid-encoded AmpC, 12 carbapenemase-producing strains, 13 strains combining the production of several β-lactamases, and 22 strains that produced other β-lactamases. The sensitivity and specificity for ESBL-detection were comparable with those of the synergy test, 75 versus 72.5%, and 94.9 versus 93.7%, respectively. The sensitivity and specificity for AmpC-detection were 71.7% and 100%, respectively, and sensitivity reached 78.7% if we excluded carbapenem-resistant isolates. Carbapenemase-detection sensitivity was 90%. These results show that the MAST® D72C test can be a useful tool for the detection of ESBL- and AmpC-production in clinical laboratories.

PCR Detection of Extended Spectrum β-Lactamase from Some Gram Negative Bacteria of Clinical Source

Beta-lactamase-producing microorganisms present distinct challenges in health care systems around the world as a result of the extensive utilization of broad-spectrum antibiotics. The objective of this research endeavor was to ascertain the antibiotic resistance profile and presence of β-lactamase genes in Gram-negative bacteria that had been previously identified and collected from laboratory work benches at the Department of Microbiology and Parasitology, UCH, located in Ibadan, South-West Nigeria. The Kirby-Bauer disc diffusion method was used to assess antibiotic susceptibility on all the isolates, and the double disc synergy test was used to validate extended spectrum beta-lactamase (ESBL) production phenotypically. The PCR techniques were used to detect β-lactamase genes. The sample distribution of the forty clinical isolates were collected from the work benches and the distribution was: urine (17), blood (9), wound (6), sputum (4), amniotic fluid (3) and tracheal aspirate (1). Klebsiella pneumoniae was the most prevalent organism with 17 isolates ( 42.5% ). Out of the 40 isolates that were obtained, 21 were ESBL producers and the distribution was as follows: Klebsiella pneumoniae (9), Escherichia coli (6), Enterobacter cloacae (2), Enterobacter aerogenes (1), Pseudomonas aeruginosa (1), Acinetobacter baumannii (1) and Hafnia alvei (1). The majority of the clinical isolates showed considerable resistance to antibiotic classes that were tested, with amoxicillin-clavulanate showing the highest resistance rate at 65%. Out of all the ESBL producers, 35.3% had blaTEM, 29.4% had blaCTX-M, 23.5% had blaSHV, and 11.8% had blaCMY. AmpC β-lactamases, Antibiotic resistance, Disc diffusion, Gram-negative bacteria, Polymerase chain reaction.

Neonatal Sepsis Due to Multidrug-resistant Bacteria at a Tertiary Teaching Hospital in Ethiopia.

The burden of multidrug-resistant bacterial infections in low-income countries is alarming. This study aimed to identify the bacterial etiologies and antibiotic resistance patterns among neonates in Jimma, Ethiopia. An observational longitudinal study was conducted among 238 presumptive neonatal sepsis cases tested with blood and/or cerebrospinal fluid culture. The bacterial etiologies were confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. The antibiotic resistance patterns were determined using the automated disc diffusion method (Bio-Rad) and the results were interpreted based on the European Committee on Antimicrobial Susceptibility Testing 2021 breakpoints. Extended-spectrum β-lactamases were detected using a double disc synergy test and confirmed by Mast discs (Mast Diagnostica GmbH). A total of 152 pathogens were identified. Of these, Staphylococcus aureus (18.4%) was the predominant isolate followed by Klebsiella pneumoniae (15.1%) and Escherichia coli (10.5%). All the isolates exhibited a high rate of resistance to first- and second-line antibiotics ranging from 73.3% for gentamicin to 93.3% for ampicillin. Furthermore, 74.4% of the Gram-negative isolates were extended-spectrum β-lactamase producers and 57.1% of S. aureus strains were methicillin resistant. The case fatality rate was 10.1% and 66.7% of the deaths were attributable to infections by multidrug-resistant pathogens. The study revealed a high rate of infections with multidrug-resistant pathogens. This poses a significant challenge to the current global and national target to reduce neonatal mortality rates. To address these challenges, it is important to employ robust infection prevention practices and continuous antibiotic resistance testing to allow targeted therapy.

ANTIBIOTIC RESISTANCE OF ESBL-PRODUCING E. COLI AND OTHER GRAM-NEGATIVE BACTERIA ISOLATED FROM STREET-VENDED FOODS IN BANGLADESH

The prevalence and impact of antibiotic-resistant pathogens transmitted through food, particularly street-vended foods, is becoming a major public health concern. Although a significant proportion of the urban population in developing countries consumes street-vended foods, the role of these foods in spreading antibiotic resistance has been rarely investigated. In this study, 50 bacterial isolates were obtained from 25 samples representing five categories of street-vended foods: Phuchka, Chatpati, Sausage, Bun, and Salad. The IMVIC test revealed a notably high occurrence of Escherichia coli (n=32) within the collected samples. Three representative isolates were selected for molecular identification using DNA sequencing of 16S rDNA. They were identified as Klebsiella oxytoca, Burkholderia fungorum, and Serratia nematodiphila. The antibiotic susceptibility of the identified isolates (n=35) was investigated using twelve antibiotics following the Kirby-Bauer disk diffusion method. Around 65.63% of the E. coli isolates (n=21) exhibited multidrug resistance. Double Disc Synergy Test (DDST) and Phenotypic Confirmatory Disk Diffusion Test (PCDDT) confirmed ESBL production of Eight multidrug-resistant E. coli isolates (38.09%). The multiple antibiotic resistance (MAR) index showed that 22 E. coli isolates had MAR above 0.2, with resistance mostly against oxacillin, ampicillin, and cefuroxime. The Klebsiella oxytoca isolate showed multidrug resistance viz., ampicillin, oxacillin, cefuroxime, and kanamycin. The Burkholderia fungorum isolate showed no distinct inhibition zone against ampicillin and chloramphenicol. Additionally, the Serratia nematodiphila isolate showed no distinct inhibition zone against three antibiotics, including ampicillin, oxacillin, and cefuroxime. These findings might contribute to the knowledge of emerging antibiotic-resistant foodborne pathogens and raise concerns about the safety of street-vended foods in Bangladesh.

Hospital Wastes as Potential Sources for Multi-Drug-Resistant ESBL-Producing Bacteria at a Tertiary Hospital in Ethiopia.

The hospital environment is increasingly becoming an important reservoir for multi-drug-resistant (MDR) Gram-negative bacteria, posing serious challenges to efforts to combat antimicrobial resistance (AMR). This study aimed to investigate the role of hospital waste as a potential source of MDR ESBL-producing bacteria. Samples were collected from multiple sources within a hospital and its vicinity, including surface swabs, houseflies, and sewage samples. The samples were subsequently processed in a microbiology laboratory to identify potential pathogenic bacteria and confirmed using MALDI-TOF MS. Bacteria were isolated from 87% of samples, with the predominant isolates being E. coli (30.5%), Klebsiella spp. (12.4%), Providencia spp. (12.4%), and Proteus spp. (11.9%). According to the double disc synergy test (DDST) analysis, nearly half (49.2%) of the bacteria were identified as ESBL producers. However, despite exhibiting complete resistance to beta-lactam antibiotics, 11.8% of them did not test positive for ESBL production. The characterization of E. coli revealed that 30.6% and 5.6% of them carried blaCTX-M group 1 type-15 and blaNDM genes, respectively. This finding emphasizes the importance of proper hospital sanitation and waste management practices to mitigate the spread of AMR within the healthcare setting and safeguard the health of both patients and the wider community.

A Hemagglutination, serum resistance, cell surface hydrophobicity and ESBL production among uropathogenic Escherichia coli

Abstract
 Background
 Bacteria have adapted themselves with increased pathogenesis and drug resistance mechanisms to overcome host defense systems. Uropathogenic E. coli (UPEC) responsible for symptomatic Urinary tract infections (UTIs) carry diverse virulence factors and extended spectrum beta-lactamase (ESBL) production which play pivotal role in pathogenesis and antibiotic resistance. Objective: The purpose of this study was to determine the phenotypic expression of virulence factors; i.e. Hemagglutination, serum resistance, cell surface hydrophobicity and resistance factor ESBL production among UPEC isolates. Methods: In this prospective study 50 drug resistant UPEC strains were evaluated for hemagglutination by slide method, cell surface hydrophobicity by salt aggregation test, serum resistance by serum bactericidal assay and ESBL detection by double disc synergy test. Results: Among 50 UPEC strains 46 (92%) showed mannose resistant hemagglutination, 48 (96%) were found to be serum resistant, 40 (80%) isolates were positive for cell surface hydrophobicity, while 43 (86%) were ESBL producers. Most of the UPEC that expressed virulence factors were ESBL producers as well. Conclusion: Over all, this study indicated that MDR UPEC isolates have expressed a number of virulence factors with ESBL production, which explains how pathogens have increased their virulence and resistance repertoire resulting in treatment failures.

The Abundance of Plasmid-Mediated Quinolone Resistance Genes in Enterobacter cloacae Strains Isolated from Clinical Specimens in Kermanshah, Iran.

Enterobacter cloacae (E. cloacae) is one of the most common Enterobacteriaceae causing nosocomial infections. Plasmid-mediated quinolone resistance (PMQR) determinants have been considered recently. This study evaluated the abundance of PMQR genes in strains of E. cloacae obtained from clinical samples in Kermanshah, Iran. In this descriptive cross-sectional study, after collecting 113 isolates of E. cloacae, their identity was confirmed using specific biochemical tests. After determining their drug resistance patterns using disc diffusion, the phenotypic frequency of extended-spectrum beta-lactamase (ESBL)-producing isolates was measured by the double-disk synergy test (DDST) method. The isolates were examined for the presence of qnrA, qnrB, qnrS, and aac(6')-Ib-cr genes by the polymerase chain reaction (PCR) assay. The antibiotic resistance rate of E. cloacae isolates varied from 9.7% to 60.2%; among them, 78% were multidrug-resistant (MDR). The highest quinolone resistance was observed in ESBL-producing strains of E. cloacae. The frequency of positive isolates for PMQR and ESBL was 79.6% and 57.5%, respectively. The genes aac(6')-ib-cr (70.8%) and qnrB (38.1%) had the highest frequency among other genes. The number of isolates simultaneously carrying 2 and 3 genes was 64 and 5 isolates, respectively. The obtained results indicate a high degree of quinolone resistance among ESBL-producing E. cloacae strains. Nevertheless, there was a significant relationship between the PMQR gene and ESBL-positive isolates. Therefore, special attention should be paid to molecular epidemiological studies on antibiotic resistance to quinolones and beta-lactamases in these strains.

Detection of Extended Spectrum ß-Lactamase-Producing Escherichia coli with Biofilm Formation from Chicken Meat in Istanbul.

Antimicrobial resistance is one of the major public health problems worldwide. This study aimed to detect the presence of extended-spectrum β-lactamase-(ESBL-)producing Escherichia (E.) coli in chicken meat in Istanbul, Türkiye. Raw chicken meat samples (n = 208) were collected from different sale points and analyzed for ESBL-producing E. coli. In total, 101 (48.5%) isolates were confirmed as E. coli by PCR, of which 80/101 (79.2%) demonstrated multiple antibiotic resistance. Resistance against amoxicillin-clavulanic acid was most frequent (87.1%). Eighteen isolates (17.8%) demonstrated phenotypical ESBL resistance, as assessed by the double disc synergy test (DDST). Isolates were tested for the presence of β-lactamase genes and mobilized colistin-resistant genes. The blaTEM group was most frequently detected (97.02%), followed by blaCTX m (45.5%), blaSHV (9.9%), and blaOXA-2 (0.9%). However, mcr genes and blaNDM,blaKPC, blaVIM, and blaOXA-48 genes were not found in any isolate. E. coli strains were tested for biofilm formation in six different media [Nutrient broth, LB broth, Tryptone Soya broth (TSB), TSB containing 1% sucrose, TSB containing 0.6% yeast extract, and BHI]. Biofilm formation by E. coli isolates (44/101, 43.5%) was highest in TSB with 1% sucrose. It is worth noting that all biofilm-producing isolates were found to harbor the blaTEM-1 gene, which can indicate a high level of antibiotic resistance. This is the first report about ESBL-producing E. coli in poultry meat, the exposure of consumers in Istanbul metropolitan areas, and the ability of E. coli from this region to produce biofilms.

Pseudomonas aeruginosa from a Tertiary Teaching Hospital: A Study of AmpC and Biofilm-producing Clinical Isolates

Abstract Introduction: Opportunistic Pseudomonas aeruginosa is one of the most prevalent bacteria with a broad spectrum of human-associated infections. The World Health Organization (WHO) claims that Pseudomonas aeruginosa infection is a typical healthcare-associated disease of critical or high concern. It is well known for being inherently resistant to many antibiotics and has the ability to produce biofilm. In the present study, we aimed to investigate P. aeruginosa with reference to biofilm and AmpC β-lactamases producing isolates in a tertiary care hospital. Materials and Methods: An observational cross-sectional study was performed at the Department of Microbiology, KIMS, KVV, Karad, and Krishna Hospital & Medical Research Center, Karad. (KH & MRC). A total of 180 isolates were subjected to this test to find out which produced the AmpC β -lactamase enzyme by the cefoxitin-cloxacillin double-disc synergy test method and biofilm production by tissue culture plate assay method. Results: Our study found a total of 97 isolates (53.89%) of AmpC production and biofilm production accounted for 151 (83.89%). Conclusion: AmpC- beta-lactamases are responsible for increasing the resistance of P. aeruginosa to several beta-lactam antibiotics and this study also showed that the clinical isolates of P. aeruginosa had a higher propensity to form biofilm and that there was a direct association between biofilm formation and antibiotic resistance.