Double-disk Synergy Test Research Articles

Prevalence of antibiotic-resistant gram-negative bacteria among patients with urinary tract infection at a facility in Ado Ekiti, Ekiti-State, Nigeria

Urinary Tract Infections (UTIs) constitute a huge public health burden especially in developing countries. This study was carried out to determine the current incidence of antibiotic resistance among Gram-negative bacteria recovered from urine of patients with UTI. This study used a cohort study design wherein one hundred and twenty one (121) mid-stream urine samples were collected and cultured onto sterile Cysteine Lactose Electrolyte Deficient medium (CLED) and MacConkey agar plates. All bacteria recovered were tested for resistance to the selected antibiotics as follows: ceftazidime (30 μg), ceftriaxone (30 μg), gentamicin (1 0μg), ampicillin (10 μg), meropenem (10 μg), ertapenem (10 μg), tetracycline (30 μg), norfloxacin (10 μg) and pefloxacin (5 μg). Twenty (20) isolates were chosen for detection of extended spectrum beta-lactamase producers using the double disk synergy test (DDST). All the bacteria were tested for biofilm production and haemolysis. Among the one hundred and twenty-one (121) mid-stream urine samples collected from patients, forty-six (46) samples grew significant bacteria. The bacteria recovered were Klebsiella pneumonia17 (37.0%), Klebsiella oxytoca 2 (4.3%), Escherichia coli (32.6%) and Proteus mirabilis (26.0%). Overall, the bacteria showed the highest resistance to pefloxacin (97.8%) and least resistance to meropenem (34.7%). The bacteria also showed high multiple resistance. All the bacteria subjected to DDST were confirmed as extended-spectrum beta-lactamase ESBL producers. Forty (43.6%) bacteria were designated as strong biofilm producers while 17.4 % bacteria showed hemolysis. The findings obtained in this study have ramifications for clinical management of UTI and public health public health in general. There should be strict enforcement of antibiotic stewardship.

Poultry slaughterhouse wastewater as reservoirs for spreading extended-spectrum beta-lactamase-producing Escherichia coli in Abakaliki, Nigeria

Abstract. Ugbo EN, Jacob JI, Effendi MH, Witaningrum AM, Agumah BN, Ugbo AI, Moses BI. 2023. Poultry slaughterhouse wastewater as reservoirs for spreading extended-spectrum beta-lactamase-producing Escherichia coli in Abakaliki, Nigeria. Biodiversitas 24: 4960-4966. The emergence of antimicrobial and multi-resistance extended-spectrum beta-lactamase-producing organisms (ESBL) from the environment and hospitals is a serious global health concern. This research focused on the role of poultry slaughterhouse wastewater as a reservoir for spreading extended-spectrum beta-lactamase-producing Escherichia coli. Exactly 110 poultry slaughterhouses wastewater was collected using sterile universal containers from abattoirs in Abakaliki. The samples were analyzed microbiological for the presence of E. coli using standard techniques. The E. coli isolates found were subjected to a sensitivity test to antibiotics using the Kirby-Bauer disc diffusion method and further tested for ESBL-producing E. coli using a double disc synergy test and chromogenic media Brilliance ESBL agar. Of 110 wastewater samples analyzed, 55 (50.0%) were positive for E. coli contamination. The E. coli isolates showed antibiotic resistance ranging between 21.8% (amoxicillin clavulanic acid) to 69.1% (tetracycline), and good susceptibility was observed on cefepime (72.7%). Thus, among 55 E. coli isolates, 11(20.0%) were ESBL-producing E. coli with resistance patterns as follows: TET-STX-CAZ-CTX-FEP (four isolates); TET-STX-CAZ-CTX (four isolates); TET-STX-CAZ-CTX-FEP-CRO (three isolates), and average multidrug resistance index of 0.7. This study revealed that poultry slaughterhouse wastewater was a reservoir for multidrug resistance ESBL-producing E. coli. Therefore, properly treating wastewater from poultry production or farms before disposing into the drainage channel or water bodies is highly recommended to avoid spreading ESBL-producing organisms from animal waste products to humans and their environment.

Antimicrobial resistance and biofilm formation of Escherichia coli in a Vietnamese Pangasius fish processing facility

This study aimed to investigate the occurrence, antibiotic resistance, and biofilm formation of Escherichia coli in the Vietnamese Pangasius fish processing facility. Among 144 samples including Pangasius fish, wash water, food contact surfaces, and personnel gloves, 18 E. coli isolates was detected and characterized. The E. coli was detected most frequently in wash water samples (22%, 8/36), followed by Pangasius fish (18%, 8/45). According to the antibiotic susceptibility test by the disc diffusion method, isolates showed the highest resistance against sulfamethoxazole/trimethoprim (45%), followed by tetracycline (39%), whereas all the E. coli isolates were susceptible to meropenem and fosfomycin. Notably, 39% of the isolates (7/18) were found to be multidrug resistant while no E. coli isolates were confirmed as extended-spectrum β-lactamase producers by the double-disk synergy test. The potency to form biofilm on the polystyrene surface of E. coli isolates indicated that 44% of the isolates (8/18) were classified as weak, 39% (7/18) as moderate, and 17% (3/18) as strong biofilm formers. Interestingly, multidrug resistant E. coli isolates were observed in moderate and strong biofilm producers. Additionally, either slightly acidic hypochlorous water with 40 mg/L of available chlorine or sodium hypochlorite with 100 mg/L of available chlorine exhibited a significant reduction in biofilm mass and biofilm cells of E. coli isolates. This study may provide helpful information about the actual state of E. coli isolates for effective control in the fish processing plant.

Characterization of carbapenemase-producing Gram-negative bacilli: first report of blaNDM-1 in Enterobacter cloacae.

The spread of multidrug-resistant bacteria, particularly carbapenem-resistant Gram-negative bacilli (CR-GNB), has become a serious challenge for clinicians due to limited therapeutic options. The aim of the study was to investigate the prevalence of carbapenemase production among clinical isolates recovered from 352 samples collected in Tebessa hospital, Algeria. Bacterial isolates were identified by 16S RNA gene sequencing and susceptibility to antibiotics was determined by disk diffusion method. Carbapenem-resistant isolates were screened for carbapenemase production using modified carba Nordmann-Poirel test, modified Hodge test and imipenem-EDTA combined disc test. Extended-spectrum β-lactamases (ESBL) were detected using double-disk synergy test. Molecular characterization of carbapenemases and ESBL genes was performed by polymerase chain reaction (PCR) and sequencing. A total of 85 Gram-negative bacilli isolates were recovered mainly from urine samples and were identified as: Klebsiella pneumoniae (17.65%), Serratia odorifera (15.29%), Escherichia coli (12.94%), Raoultella ornithinolytica, Enterobacter cloacae (11.76%), Serratia marcescens (10.59%), Morganella morganii (7.06%), Proteus mirabilis (5.88%), Acinetobacter baumannii (4.70%) and Pseudomonas aeruginosa (2.35%). All strains were resistant or intermediate to imipenem and/or ertapenem. ESBL, carbapenemase and metallo-beta-lactamases (MBL) phenotypes were detected in 19 (22.35%), 9 (10.59%) and 2 (2.35%) GNB isolates, respectively. PCR results in nine carbapenemase-producing GNB strains chosen showed the presence of one to four carbapenemase genes (blaGES, blaSME, blaNDM-1, blaVIM, blaGIM, blaSPM, blaOXA-48) in four strains; however, seven strains had at least one ESBL gene (blaTEM-1, blaCTXM-15, blaSHV). In this study, we report the first incidence of blaNDM-1 gene in Enterobacter cloacae isolated from urine sample in Algeria.

Fecal carriage of extended spectrum beta-lactamase and fluoroquinolone resistant gene in non-typhoidal Salmonella enterica isolates from food-producing animals and humans

This study seeks to determine the feacal carriage of extended spectrum beta-lactamase and fluoroquinolone resistant non-typhoidal Salmonella enterica isolates from food-producing animals and humans. A total of three hundred (300) fecal samples were collected using sterile universal containers from food-producing animals namely (Chicken [100], Pig [100] and humans (100) from Onicha Local Government Area of Ebonyi State and analyzed for the presence of non-typhoidal Salmonella enterica using standard microbiological techniques. Phenotypic detection of extended-spectrum beta-lactamase (ESBL) were done by disc diffusion and Double Disk Synergy Test. Molecular characterization for ESBL and fluoroquinolone-resistant genes were done by PCR with specific primers. The result shows that non-typhoidal Salmonella species (NTS) accounted for 25 % and 17 % in poultry and pig fecal sample respectively while 60 % and 40% were phenotypic ESBL producers respectively. When compared statistically there is significant difference among isolates confirmed ESBL-positive (P˂ 0.05). Also, none of the 16 (58 %) NTS isolated from humans harbored ESBL phenotype. PCR analysis with β-lactam specific primer detected the presence of blaOXA 50 % and 50 %, blaSHV 36 %, and 64 %, blaTEM 43 % and 57 %, blaCTX-M 36 % and 64 % in poultry and pig respectively. Fluoroquinolone resistant gene QnrA was present in 0 and 100 % of poultry and pig respectively. QnrB was 40 % and 60 % in poultry and pig isolates respectively. QnrS was present in 64 % isolates of poultry and 13 % isolates in pig. The high prevalence of genes encoding beta-lactamases and fluoroquinolone resistance (TEM, SHV, CTX-M and OXA, (qnrA, qnrB and qnrS) were present more in poultry and pig than in humans and demonstrate a significant public health threat from consumption of food-producing animal harboring such pathogenic resistant genotype if not properly controlled.
 Keywords: Extended spectrum beta-lactamase, Fluoroquinolone, non-typhoidal Salmonella enterica, feacal carriage

Characterization of beta-lactamase producing Enterobacterales isolated from an urban community wastewater treatment plant in Iran.

he occurrence and characteristics of Extended Spectrum- and AmpC-β-lactamase producing Enterobacterales (ESBL-PE and AmpC-PE) in an urban wastewater treatment plant (WWTP) were investigated. A total of 30 wastewater samples were collected from all sections of WWTP. Enterobacterales were isolated and identified using standard microbiological tests. The antibiotic resistance profile was determined by the Kirby-Bauer disk diffusion method. Phenotypic screening for ESBL-PE and AmpC-PE isolates was performed by double-disk synergy and boronic acid disk potentiation tests, respectively. The isolates were examined for AmpC- and ESBL-encoding genes by PCR and sequencing methods. Among 146 Enterobacterales isolates, 8.9% (n=13) [ESBL-only; 5.48% (n=8) and ESBL + AmpC; 3.42% (n=5)] were ESBL-producers and 15.75% (n=23) [AmpC-only; 12.33% (n=18) and ESBL + AmpC; 3.42% (n=5)] AmpC-producers. Hafnia spp. with 33.33% (n=1/3) and E. coli with 20.58% (n=7/34) [ESBL-only; 17.64% (n=6/34) and ESBL + AmpC; 2.94% (n=1/34)] were the most common ESBL-producing bacteria. Enterobacter spp. with 37.50% (n=6/16) of isolates were the most common AmpC-producing organisms. ESBL- and/or AmpC-producing isolates were identified in all parts of the WWTP including 80% (n=8/10) of samples taken from effluent. Among ESBL-producing isolates, bla CTX-M , bla TEM, and bla SHV ESBL-encoding genes were found in 61.5% (n=8), 15.3% (n=2), and 7.7% (n=1) of isolates, respectively. All CTX-M-type enzymes belonged to the CTX-M-1 group and CTX-M-15 subgroup. bla TEM and bla SHV type genes belonged to bla TEM-20 and bla HSV-12 subtypes, respectively. bla DHA with 73.9% (n=17/23), and bla CIT and bla FOX with 30.4% (n=7/23) each, were the most common AmpC-encoding genes among AmpC-producing isolates. Overall, 75% of ESBL-producing and 55.5% of AmpC-producing isolates exhibited multi-drug resistance phenotypes. The organisms were most resistant against ampicillin (82.2%) nalidixic acid (43.8%) and cephalexin (41.1%). ESBL- and AmpC-producing Enterobacterales spp. with diverse genetic resistance backgrounds in WWTP effluent poses a significant risk to public health.

Synergistic Effects of Gentamicin, Cefepime, and Ciprofloxacin on Biofilm of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen involved in number of hospital-acquired infections such as catheter-associated urinary tract infections, bacteremia, septicemia, skin infections, and ventilator-associated pneumoniae. Biofilm formation is an important trait implicated in chronic infections, such as cystic fibrosis and chronic pulmonary obstruction. We evaluated effects of gentamicin, cefepime, and ciprofloxacin on biofilm of P. aeruginosa. A total of 266 isolates were collected from the Armed Forces Institute of Pathology (AFIP). Antibiotic susceptibility was assessed by double disk synergy testing. ESBL and carbapenemase detection was performed by phenotypic testing. Molecular screening of the genes was done by PCR. Micro-dilution broth method was used to determine minimum inhibitory concentrations of antibiotics. Biofilm formation was done by micro-titer plate assay. Overall, 20% of the P. aeruginosa isolates were extensively drug-resistant (XDR-PA), and 25% were multi-drug-resistant (MDR-PA). Likewise, 43% of the isolates were ESBL producers, and carbapenemase production was detected in 40% of the isolates. Molecular analysis confirmed occurrence of different resistant factors in ESBL-positive isolates; 67% carried blaTEM, 62% blaCTXM-15, 41% blaSHV, 34% blaCTXM-14, and 33% blaOXA-1. In addition, 68% of the carbapenem-resistant isolates were positive for blaNDM-1, 25% for blaOXA-48, and 22% for blaKPC-2. Biofilm formation was assessed for 234 isolates, out of which 28% were strong biofilm formers. Moderate and weak biofilm formers constituted 46% and 23%, respectively. Overall, ciprofloxacin, levofloxacin, and cefepime showed inhibitory effects on P. aeruginosa biofilms. Antibiotics in combination showed strong synergistic effects (ciprofloxacin and cefepime), while gentamicin and cefepime resulted in complete eradication of P. aeruginosa biofilm. We confirm strong synergistic effects of gentamicin and cefepime that completely eradicated P. aeruginosa biofilm. We further confirm inhibitory effects of ciprofloxacin, levofloxacin, and cefepime on P. aeruginosa biofilms. Hence, combination therapy can be more effective against biofilm-associated infections.

Antimicrobial resistance patterns of Gram-negative bacteria in an Iranian referral pediatric hospital: A present danger of New Delhi metallo-β-lactamase.

Antimicrobial resistance among gram-negative bacteria has been growing, particularly in developing countries, like Iran. The emergence and spread of carbapenem-resistance mechanisms is a major public health concern because no definite treatments have yet been established for this problem. This study aimed to evaluate antibiotic susceptibility of gram-negative bacteria, metallo-β-lactamases (MBLs) and carbapenemase-producing genes, including bla NDM, bla VIM, and bla IMP in patients referred to Children's Medical Center, Tehran, Iran. In this cross-sectional study, a total of 944 gram-negative isolates were tested in the study, and antimicrobial susceptibility testing was performed. Moreover, MBL production of carbapenem-resistant isolates, as well as the presence of bla NDM, bla VIM, and bla IMP, was investigated. The most common gram-negative isolated bacteria were Escherichia coli (489 samples, 52%), followed by Klebsiella pneumoniae (167 samples, 18%), Pseudomonas aeruginosa (101 samples, 11%), Enterobacter spp. (64 samples, 7%), Pseudomonas spp. (35 samples, 4%), Acinetobacter baumannii (18 samples, 2%), and Burkholderia cepacia (17 samples, 2%). Imipenem-resistant was found in 75%, 61%, and 60% of Stenotrophomonas maltophilia, Enterobacter spp., and A. baumannii isolates, respectively. Moreover, the highest resistance to meropenem was observed in S. maltophilia, A. baumannii, P. aeruginosa, and B. cepacia (100%, 96%, 83%, and 61.5%, respectively). Double disk synergy test (DDST) results showed that 112 out of 255 carbapenem-resistant isolates (44%) were MBL-producing ones. The presence of the bla NDM gene was identified in 32 (29%) of MBL-producing isolates, 13 of which were K. pneumoniae, 7 P. aeruginosa, and 7 E. coli, 3 Enterobacter spp., and 2 Klebsiella spp, respectively. The presence of the bla IMP and bla VIM genes was detected in 2 (2%) and 1 (1%) of MBL-producing isolates. These genes were detected in only MBL-producing P. aeruginosa isolates. Our findings suggest the emergence of NDM-producing strains in our hospital, and bla NDM was the most frequently detected carbapenemase gene in MBL-producing P. aeruginosa, K. pneumoniae, and Klebsiella spp. Since such bacteria can easily spread among patients in the hospital, a strong infection control and prevention plan is highly recommended.

Epidemiological study of antimicrobial-resistant bacteria in healthy free-ranging bantengs (Bos javanicus) and domestic cattle.

Antimicrobial-resistant microorganisms (ARMs) have been increasing among wild animals. Interactions occurring at the interface between wildlife, humans, and livestock can lead to the transmission of ARMs. Thus, the prevalence of ARMs in wild and domestic animals should be determined to address and prevent this issue. This study aimed to determine the resistance patterns of cefotaxime (CTX)-resistant Escherichia coli and identify the presence of extended-spectrum beta-lactamase (ESBL) genes in ESBL-producing E. coli among a population of wild banteng (Bos javanicus) and domestic cattle kept on farms located close to the Lam Pao non-hunting area, Kalasin province, Thailand. Forty-five fecal samples were taken from wild bantengs inhabiting the Lam Pao non-hunting area in Thailand, alongside 15 samples from domestic cattle. Bacterial culture, triple sugar iron, and motile indole lysine tests were conducted to identify E. coli. A polymerase chain reaction (PCR) was conducted for specific confirmation. MacConkey agar supplemented with 2 μg/mL of CTX was used to identify CTX-resistant E. coli, which would be used to identify ESBL production based on a double-disk synergy test. Extended-spectrum beta-lactamase-producing samples were subjected to disk diffusion tests to determine resistant patterns, and the sizes of PCR bands and DNA sequencing were used to differentiate ESBL gene types. All samples tested positive for E. coli. Forty-five isolates from 15 banteng samples and three isolates from one domestic cattle sample displayed CTX-resistant and ESBL-producing traits. The banteng and domestic cattle populations exhibited nine and three distinct resistant patterns, respectively. The PCR results indicated that the banteng isolates harbored the following genes: Cefotaxime-M1 (n = 38), CTX-M9 (n = 5), and the SHV group (n = 2). All three isolates from the domestic cattle sample contained the CTX-M1 gene. Classification of ESBL genes based on the DNA sequences of the banteng isolates showed the characteristics of CTX-M15 (n = 20), CTX-M55 (n = 6), CTX-M14 (n = 5), and CTX-M79 (n = 1). The three domestic cattle isolates exhibited the characteristics of CTX-M15, CTX-M55, and CTX-M79. Despite no previous antibiotic applications, approximately one-third of the banteng samples displayed CTX resistance, indicating ARM contamination within the ecosystem. The similarity in ESBL genes between the banteng and domestic cattle populations suggests potential gene transmissions between these animal groups. However, the initial source of ARMs remains unclear, as the banteng population exhibited more ESBL genes than the domestic cattle, suggesting the possibility of multiple ARM sources. These findings raise concerns because the banteng population inhabits an area that is an important source of freshwater and nourishes the entire north-east region of Thailand and other South-east Asian countries, including Laos, Cambodia, and Southern Vietnam.

Molecular identification of blaTEM and blaCTX-M genes in multidrug-resistant Escherichia coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia.

Escherichia coli is an opportunistic bacteria that can grow easily, produce toxins, and resist antibiotics. The phenomenon of E. coli developing multidrug resistance is currently the subject of extensive research. The objective of this study was to molecularly identify blaTEM and blaCTX-M genes in multidrug-resistant E. coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia. One hundred and ten milk samples were collected from 45 dairy cattle farms in Tulungagung, Indonesia. Indole, methyl red, Voges-Proskauer and in citrate tests and triple iron sugar agar tests were used to identify E. coli. Multidrug resistance was determined in isolates through antibiotic sensitivity tests using tetracycline, streptomycin, trimethoprim, chloramphenicol and aztreonam. Extended-spectrum beta lactamase enzyme production was confirmed by double-disc synergy test (DDST). Molecular identification was performed to confirm the blaTEM and blaCTX-M genes. One hundred and one (91.82%) E. coli strains were isolated from the samples. The antibiotic sensitivity test showed four (3.96%) multidrug-resistant (MDR) and one (0.99%) ESBL-positive E. coli by DDST confirmation. There were three (77.78%) blaTEM genes and one (0.99%) blaCTX-M gene discovered in the MDR E. coli isolates using PCR for molecular identification. The findings of the blaTEM and blaCTX-M genes encoding ESBL E. coli in dairy cattle milk in Tulungagung, Indonesia is concerning and argues for prompt action to stop the emergence of antibiotic resistance which has an impact on public health.

Detection of extended-spectrum β-lactamase-producing Escherichia coli genes isolated from cat rectal swabs at Surabaya Veterinary Hospital, Indonesia.

Escherichia coli causes a bacterial illness that frequently affects cats. Diseases caused by E. coli are treated using antibiotics. Because of their proximity to humans, cats possess an extremely high risk of contracting antibiotic resistance genes when their owners touch cat feces containing E. coli that harbor resistance genes. This study was conducted to identify multidrug-resistant E. coli and extended-spectrum β-lactamase (ESBL)-producing genes from cat rectal swabs collected at Surabaya City Veterinary Hospital to determine antibiotic sensitivity. Samples of cat rectal swabs were cultured in Brilliant Green Bile Lactose Broth medium and then streaked on eosin methylene blue agar medium for bacterial isolation, whereas Gram-staining and IMViC tests were conducted to confirm the identification results. The Kirby-Bauer diffusion test was used to determine antibiotic sensitivity, and the double-disk synergy test was used to determine ESBL-producing bacteria. Molecular detection of the genes TEM and CTX-M was performed using a polymerase chain reaction. Based on morphological culture, Gram-staining, and biochemical testing, the results of sample inspection showed that of the 100 cat rectal swab samples isolated, 71 (71%) were positive for E. coli. Furthermore, 23 E. coli isolates (32.39%) demonstrated the highest resistance to ampicillin. Four isolates were confirmed to be multidurg-resistant and ESBL-producing strains. Molecular examination revealed that three E. coli isolates harbored TEM and CTX-M. In conclusion, pet owners must be educated on the use of antibiotics to improve their knowledge about the risks of antibiotic resistance.

Phenotypic methods for detection of metallo-β-lactamase (MBL) production in multidrug resistant gram negative bacilli – comparative study

Introduction: One of the most common mechanism of resistance of bacterial pathogens to β-lactam antibiotics is production of β-lactamase, there are different types of Beta lactamases, which are expressed by drug resistant gram-negative bacteria. Carbapenemases (Metallo beta lactamases/MBL) are the β-lactamases with the widest spectrum of activity. Early detection of MBL-producing organisms is crucial to establish appropriate antimicrobial therapy and to prevent their interhospital and intrahospital dissemination. Several phenotypic methods are available for the detection of MBL producing bacteria. As there is no standardized method present study was done to screen MDR gram negative bacilli isolated from clinical samples for MBL-production by a low cost, convenient and sensitive procedure. Methods: All non-duplicate MDR gram negative isolates obtained from various clinical samples were screened for carbapenam resistance. All carbapenam resistant bacteria were screened for production of MBL by 3 phenotypic tests (Double disc synergy, combined disk test, Hodge test). The results were compared and analyzed on the basis of results obtained by E test. Results: During Study Period, 988 non duplicate gram negative bacilli were isolated, 70.64% (698) were multi drug resistant. Amongst Total number of MDR Isolates to carbapenam resistance was seen in 62(9.28%). These 62 isolates that were resistant to carbapenam were tested for MBL production. 54 (87%) of these 62 isolates showed MBL production by disc potentiation test whereas 41 isolates (66%) gave positive result by DDST. By Modified Hodge test, out of 62, 48 isolates (77.4%) were MBL positive. Compared to E- test, the Sensitivity Specificity and Accuracy for Disc potentiation test was 90%,100% and 90.32%, for Modified Hodge test was 80%,100% and 80.6% and for Double disc synergy test was 68.3%,100% and 69.3%. Conclusion: In our study, in comparison to MBL E test, disc potentiation test is more sensitive than double disc synergy test and Modified Hodge test for detection of MBL phenotypically

Detection and Phylogenetic Analysis of Extended-Spectrum β-Lactamase (ESBL)-Genetic Determinants in Gram-Negative Fecal-Microbiota of Wild Birds and Chicken Originated at Trimmu Barrage.

Extended-spectrum β-lactamases (ESBL) give rise to resistance against penicillin and cephalosporin antibiotics in multiple bacterial species. The present study was conducted to map genetic determinants and related attributes of ESBL-producing bacteria in three wild aquatic bird species and chickens at the "Trimmu Barrage" in district Jhang, Punjab province, Pakistan. To study the prevalence of ESBL-producing bacteria, a total of 280 representative samples were collected from wild bird species; cattle egrets (Bubulcus ibis), little egrets (Egretta garzetta) and common teals (Anas crecca) as well as from indigenous chickens (Gallus gallus domesticus) originating from a local wet market. The isolates were confirmed as ESBL producers using a double disc synergy test (DDST) and bacterial species were identified using API-20E and 20NE strips. A polymerase chain reaction (PCR) was used to detect ESBL genetic determinants and for genus identification via 16S rRNA gene amplification. A phenotypic antimicrobial susceptibility test was performed for ESBL-producing isolates against 12 clinically relevant antibiotics using the Kirby-Bauer disk diffusion susceptibility test. A phylogenetic tree was constructed for the sequence data obtained in this study and comparative sequence data obtained from GenBank. The overall prevalence of ESBL-producing bacteria was 34.64% (97/280). The highest percentage (44.28%; 31/70) of ESBL-producing bacteria was recovered from chickens (Gallus gallus domesticus), followed by little egrets (Egretta garzetta) (41.43%; 29/70), common teal (Anas crecca) (28.57%; 20/70) and cattle egrets (Bubulcus ibis) (24.28%; 17/70). Five different ESBL-producing bacteria were identified biochemically and confirmed via 16S rRNA gene sequencing, which included Escherichia coli (72; 74.23%), Enterobacter cloacae (11; 11.34%), Klebsiella pneumoniae (8; 8.25%), Salmonella enterica (4; 4.12%) and Pseudomonas aeruginosa (2; 2.06%). Based on PCR, the frequency of obtained ESBL genes in 97 isolates was blaCTX-M (51.55%), blaTEM (20.62%), blaOXA (6.18%) and blaSHV (2.06%). In addition, gene combinations blaCTX-M + blaTEM, blaTEM + blaOXA and blaCTX-M + blaSHV were also detected in 16.49%, 2.06% and 1.03% of isolates, respectively. The ESBL gene variation was significant (p = 0.02) in different bacterial species while non-significant in relation to different bird species (p = 0.85). Phylogenetic analysis of amino acid sequence data confirmed the existence of CTX-M-15 and TEM betalactamases. The average susceptibility of the antibiotics panel used was lowest for both Klebsiella pneumoniae (62.5% ± 24.42) and Salmonella enterica (62.5% ± 31.08) as compared to Enterobacter cloacae (65.90% ± 21.62), Pseudomonas aeruginosa (70.83% ± 33.42) and Escherichia coli (73.83% ± 26.19). This study provides insight into the role of aquatic wild birds as reservoirs of ESBL-producing bacteria at Trimmu Barrage, Punjab, Pakistan. Hence, active bio-surveillance and environment preservation actions are necessitated to curb antimicrobial resistance.

In vitro effectiveness of ceftazidime-avibactam in combination with aztreonam on carbapenemase-producing Enterobacterales

ObjectiveThis work aimed to describe the in vitro performance of the combined activity of ceftazidime-avibactam (CZA) plus aztreonam (ATM) against carbapenemase-producing Enterobacterales (CPE). MethodsWe studied 44 CPE clinical isolates: NDM-1 (31), KPC-2 (5), KPC-3 (3), VIM-2 (2), NDM-1+KPC-2 (2), and OXA-48 (1). The efficacy of CZA in combination with were determined by two methods: (i) Kirby-Bauer's double disk synergy test and; (ii) Determination of the minimum inhibitory concentration to CZA by E-test, in either Mueller-Hinton agar alone or, supplemented with ATM 4 mg/L. Additionally, the Fractional inhibitory concentration index (FICI) was determined; values of ≤ 0.5 were interpreted as synergistic, while FICI > 0.5 were considered indifferent. ResultsAll isolates were carbapenem-resistant, 14 were resistant to CZA and ATM, 15 were only CZA resistant, 12 were only ATM resistant, and three were susceptible to both. 34/44 isolates presented positive double disk synergy tests between CZA and ATM regardless of their susceptibility profile, the isolates with negative synergy tests were susceptible to at least one of the agents. On the other hand, the 21 isolates selected to compare the MIC to CZA alone and CZA plus 4 mg/L ATM of exhibited FICI values between 0.016 and 0.125, indicating a synergistic effect. ConclusionsThis method is available to clinical laboratories and would provide valuable information to guide the treatment of infections with CZA and ATM. In this sense, the use of CZA together with ATM is a potentially suitable combination for the treatment of carbapenemase-producing microorganisms.

Antimicrobial susceptibility and molecular characteristics of beta-lactam- and fluoroquinolone-resistant E. coli from human clinical samples in Nigeria

This study determined the antimicrobial resistance profiles and molecularly characterized beta-lactam- and fluoroquinolone-resistant E. coli recovered from human clinical samples. Three hundred (300) clinical samples collected from two major tertiary healthcare institutions were analyzed using standard microbiological techniques. Antimicrobial susceptibility testing and phenotypic detection of extended-spectrum beta-lactamase (ESBL) were done by disc diffusion and Double Disk Synergy Test. Molecular characterization for ESBL and fluoroquinolone-resistant genes were done by PCR with specific primers. In total, 44 (14.7%) ESBL-producing E. coli were recovered from human clinical specimens. The recovered isolates were multidrug-resistant with multiple antibiotics resistance index values ranging from 0.5–0.8. Isolates exhibited resistance to sulfamethoxazole/trimethoprim 44 (100%), cefoxitin 42 (95.5%), tetracycline 42 (95.5 %), cefuroxime 41 (93.2%), ciprofloxacin 40 (90.9%), cefotaxime 37 (84.1%), amoxicillin 37 (84.1%), amoxicillin/clavulanic acid 37 (84.1), and ceftazidime 35 (79.5%). BlaTEM 41 (93.2%) ESBL gene was the most predominant gene among the isolates, followed by blaCTX-M 9 (20.5%), and blaSHV 1 (2.3%) genes. Co-existence of blaTEM +blaSHV, blaTEM + blaCTX-M, and blaTEM +blaSHV + blaCTX-M genes was observed in 1 (2.3%), 7 (15.9%), and 4 (9.1%) isolates, respectively. All the fluoroquinolone-resistant isolates harbored aac-lb-6-cr gene while Qnr gene was absent. Association of fluoroquinolone resistance- and β-lactam resistance genes; aac-lb-6-cr + blaTEM + blaSHV + blaCTM was observed in 5 (11.4%) isolates. Our study showed high frequency of ESBL-producing E. coli harboring ESBL and fluoroquinolone resistance genes from human clinical samples in Ebonyi State, Nigeria. The high frequency of multidrug-resistant E. coli in our study is very worrisome and could have significant public health impact such as treatment failures, and possibly death, if not properly managed. It is therefore imperative to establish strong regulative measures and guidelines that would help in curtailing the increasing dissemination of these superbugs in healthcare institutions in Nigeria.

COMPARISON OF DOUBLE-DISC SYNERGY, COMBINED DISC DIFFUSION, AND E TEST FOR METALLO-BETA-LACTAMASE DETECTION IN PSEUDOMONAS AERUGINOSA IN A TERTIARY CARE HOSPITAL

Objective: Pseudomonas aeruginosa is an important opportunistic pathogen associated with nosocomial infections. The metallo-beta-lactamases (MBLs) are an important class of carbapenemases which are predominantly produced by P. aeruginosa. This study is aimed to study the prevalence of MBLs among imipenem (IMP)-resistant P. aeruginosa and to evaluate three different methods of screening and detecting MBLs produced by P. aeruginosa. Methods: 100 isolates of P. aeruginosa were obtained from various clinical specimens, including pus, wound, sputum, urine, body fluids, and ET tips received at the laboratory from July 2021 to December 2021. Screening for MBL production among IMP-resistant P. aeruginosa was done by double-disc synergy test and combined disc test. Confirmation of MBL production was done by E-test. Results: Of the 100 P. aeruginosa isolates, 16 were IMP resistant, of which 16 are MBL producers. Most of the samples were obtained from 50 to 60 years followed by 40–50 and 60–70 were pus (50%) followed by sputum (20%), urine (15%), and body fluids (5%). Of the 100 isolates, 66 were isolated from females and 34 from males. Pus samples showed most MBL-producing isolates, accounting for 60.5%. MBL producers accounted for 90% of IMP-resistant cases using combined disc method and 100% using MBL E-strip test. By comparison, double-disc synergy test (DDST) retrieved 45% of Pseudomonas MBL producers. Conclusion: This study found moderate prevalence of Pseudomonas MBL producers (16/100). The study supports the use of combined disc-diffusion test and DDST for screening and confirming MBL producers of P. aeruginosa by E test where polymerase chain reaction is not available.

High frequency and molecular characterization of ESBL-producing Enterobacteriaceae isolated from wound infections in North Lebanon

ABSTRACT Background Extended-spectrum beta-lactamases producing Enterobacteriaceae (ESBL-PE) represent a major problem in wound infections. Here, we investigated the prevalence and molecular characterization of ESBL-PE associated with wound infections in North Lebanon. Research design and methods A total of 103 non-duplicated E. coli and K. pneumoniae strains isolated from 103 patients with wound infections, were collected from seven hospitals in North Lebanon. ESBL-producing isolates were detected using a double-disk synergy test. In addition, multiplex polymerase chain reaction (PCR) was used for the molecular detection of ESBLs genes. Results E. coli was the predominant bacteria (77.6%), followed by K. pneumoniae (22.3%). The overall prevalence of ESBL-PE was 49%, with a significantly higher rate among females and elderly patients. K. pneumoniae was the common MDR and ESBL-producer bacteria (86.95% and 52.17%) compared to E. coli (77.5% and 47.5%). Most of the isolated ESBL producers harbored multiple resistant genes (88%), where blaCTX-M was the most predominant gene (92%), followed by blaTEM (86%), blaSHV (64%), and blaOXA genes (28%). Conclusions This is the first data on the ESBL-PE prevalence associated with wound infections in Lebanon, showing the emergence of multidrug-resistant ESBL-PE, the dominance of multiple gene producers, and the widespread dissemination of blaCTX-M and blaTEM genes.

Detection of Inflammatory Biomarkers Among Patients with Sepsis of Gram-Negative Bacteria: A Cross-Sectional Study.

Sepsis is a highly mixed ailment that affects patients with numerous conditions of infectious sources and can lead to multi-organ failure with dysregulated host immune response. To determine inflammatory biomarkers in patients with sepsis caused by Gram-negative bacteria and compare their role in the early detection of sepsis. This cross-sectional study was conducted on patients with sepsis admitted to the intensive care unit at different hospitals in Sulaimaniyah, Iraq, from May to December 2021. Patients (n=147) were enrolled in this study according to the primary diagnosis of sepsis by Sequential Organ Failure Assessment scores. Blood samples were taken from patients to investigate white blood cells, inflammatory biomarkers (pentraxin-3, procalcitonin, adrenomedullin, lipopolysaccharide binding protein, interleukin-17A, lactate dehydrogenase, and C-creative protein), blood culture, antibiotic susceptibility test, and coagulation biomarkers (Prothrombin time, activated partial thromboplastin time, and international normalized ratio). Then, isolated Gram-negative bacteria were tested for extended-spectrum β-lactamase enzymes production by screening and combined disc tests. A total of 51.7% samples were blood culture positive for different Gram-negative bacteria, and P. aeruginosa (51.95%) was a more isolated bacterium. Both males and females were affected by sepsis in a ratio of 1.23:1 with different age groups. Extended-spectrum β-lactamase was estimated to be 77.2% by antibiotic profile, and the rate decreased using two double-disc synergy tests. This was confirmed by combined disc test at a rate of 41.35%. The most prevalent biomarkers were procalcitonin (88.16%), adrenomedullin (84.21%), pentraxin-3 (22.37%), and lipopolysaccharide binding protein (11.84%). Sepsis is a life-threatening condition that can be diagnosed early by several blood biomarkers such as procalcitonin, adrenomedullin, and pentraxin-3 combined with a standard blood culture technique to improve the patient outcome.

Distribution and Antibiotic Resistance Profile of Extended Spectrum Beta-Lactamase Producing Escherichia coli from Fish Farms within Abakaliki Metropolis

Background and Objectives: The emergence of antibiotic resistant determinant in fish farms and its spread is on the increase and has evolved into strains that are resistant to many classes of antibiotics. Thus, it is critical to identify the distribution and antibiotic resistance profile of Extended Spectrum Beta-lactamase (ESBL) producing Escherichia coli from fish farms within Abakaliki Metropolis.
 Methodology: Aseptically, fifty (50) milliliters of fishpond water was collected from twenty locations in fifteen (15) fish farms and were analyze using standard microbiological culture and identification of Escherichia coli. Detection of phenotypic extended spectrum β-lactamases production was performed using Double-Disk Synergy Test (DDST). Antibiotic susceptibility studies of extended spectrum β-lactamases producing Escherichia coli was determined using the Kirby–Bauer disk diffusion method and the results were construed using the Clinical Laboratory Standard Institute (CLSI) zone diameter breakpoints.
 Results: Extended spectrum beta-lactamase producing Escherichia coli distribution from fishpond water revealed overall occurrence rate of 34(11.3 %). The proportion of ESBL producing Escherichia coli was 5(25.0 %) from fish farm L followed by Farm A, Farm E, Farm G, which both accounted for 20.0 % respectively while the least occurrence of 1(5.0 %) was recorded against Farm I. ESBL-producing E. coli were resistant to cephalosporin particularly Ceftriaxone (88.2%), Ceftazidime (91.2 %), Cefotaxime (94.1) and Cefepime (85.3 %). This was followed by Amoxicillin-Clavulanate (91. 2 %), Azetronam (97.1 %). In all, Ciprofloxacin (82.4 %), Imipenem (97.1 %), and Meropenem (100 %) were the most effective antibiotic against ESBL-producing E. coli isolate.
 Conclusion: This study reveals the prevalence of the ESBL phenotype in fish farms. The increasing prevalence of resistance to routinely used antibiotics in medical and veterinary therapies among the study isolates from aquaculture products poses a significant challenge to the treatment of human and animal diseases. As a result, adequate antibiotic intervention is essential to ensure the continued efficacy of antibiotics for aquaculture and human health, as well as the industry's viability.

Comparison of Antibiotic-Resistant Pattern of Extended Spectrum Beta-Lactamase and Carbapenem-Resistant Escherichia Coli Isolates from Clinical and Non-Clinical Sources

The increasing rate of antibiotic resistance among E. coli especially those mediated by extended spectrum beta-lactamases and carbapenem-resistant (CR) presents a major threat to public health and healthcare delivery globally. The aim of this research was to compare the antibiotic resistant pattern of extended spectrum beta-lactamase and carbapenem-resistant Escherichia coli isolates from clinical and non-clinical sources. A total of two hundred and fifteen (215) clinical and non-clinical samples were collected for the study. The collected samples were analysis using Standard Microbiological protocol for isolation and identification. Phenotypic detection of ESBL Production and carbapenem resistant was performed using Double Disk Synergy Test (DDST) and Modified Hodge Test (MHT) respectively. Antibiogram studies of ESBL producing and Carbapenem-resistant E. coli was determined using the Kirby–Bauer disk diffusion method. The result of isolation and characterization revealed higher occurrence rate of clinical isolates of E. coli 66.1 % over non-clinical sample 54.0%. Phenotypic ESBL screening of isolated E. coli revealed overall detection rate of 35(30.4 %) and 13(13.0 %) in clinical and non-clinical source respectively while overall detection rate of carbapenem resistant ESBL producers accounted 9.6 % and 8.0 % in clinical and non-clinical isolates respectively. The isolates exhibited high percentage of resistance to nitrofurantion 100 %, cefepime 100 % colistin 66.7 %, amikacin 50.0 % and also exhibit MDR with MARI value of ≥ 0.5. Comparison of antibiotic resistant pattern of carbapenem-resistant extended spectrum beta-lactamase producing E. coli isolates from clinical and non-clinical sources showed no statistical significant difference P-value < 0.05 but were 100 % susceptible to ciprofloxacin and gentamicin. The pattern of similar MDR portrayed by clinical and non-clinical isolate should not be overlooked due to its hyper-motility and virulence domination leading to possible AMR transfer to other bacterial organisms and also, appropriate measures such as pretreatment of animal excrement before being used as fertilizers and the quality of irrigation water, hospital effluent discharge need to be taken into consideration to prevent the spread of resistant determinant.
 Keywords: Escherichia coli, Extended Spectrum Beta-Lactamase, Carbapenem-Resistant, clinical, non-clinical