Double Disk Synergy Test Method Research Articles

Detection and prevalence of extended spectrum β-lactamases production among Enterobacteriaceae isolated from urinary tract infections

Urinary tract infections (UTIs) by extended-spectrum β-lactamase producing Enterobacteriaceae (ESBL-E) have become an important public health problem with a substantial economic burden, as they lead to more complicated infections, longer duration of treatment, and increases in patient mortality. A total of 1267 urine samples were collected from patients during the period of 2018 to 2020. The isolates were identified based on colony morphology and biochemical tests. Antimicrobial susceptibility testing was done by modified Kirby Bauer disc diffusion method. Resistant isolates to third generation cephalosporins were tested for ESBL production by double-disk synergy test (DDST), DDST methods on MH agar plates supplemented with cloxacillin and by confirmatory double disk test (DDT). 400 strains causing UTIs were identified and classified as members of Enterobacteriaceae. The results of antimicrobial susceptibility tests showed that 68.5% (274/400) of the Enterobacteriaceae isolates were multi-drug resistant (MDR) strains. The overall prevalence of ESBL-E was 59.5% (238/400). The highest frequencies of ESBL-E were observed in internal medicine devices (68/400) and the highest ESBL production was observed in E. coli (85/238). The ESBL producing isolates were significantly resistant to penicillins and cephalosporins compared to non-ESBL producers. However, those ESBL-E strains were sensitive to imipenem (80.2%) and nitrofurantoin (87.8%). High levels of MDR strains and ESBL-E in our study highlights the need for applying specific infection control measures, and accordingly we urge physicians to opt for specific strategies for regular surveillance of uropathogenic bacteria.

The Prevalence of Extended Spectrum Beta-Lactamase Producing Uropathogenic Escherichia coli from Mouau Female Hostel Students

Aim: The study is aimed at determining the antimicrobial susceptibility pattern and production of Extended-spectrum Βeta-Lactamase (ESBLs) among Escherichia coli isolates from urine samples.
 Methods: A total of 35 samples of early morning mid-stream urine samples of about 10-15 ml were collected from Michael Okpara University of Agriculture Umudike (MOUAU) female hostel students; using sterile plastic containers. Each urine sample was inoculated on MacConkey Agar (MCA). Isolates were identified based on morphological features, Gram staining, and biochemical characterization. The antibiotic susceptibility test was done by the Kirby Bauer Disc diffusion method on Muller Hinton agar. ESBL detection was done as per Clinical and Laboratory Standard Institute (CLSI) guidelines, using the double disc synergy test method.
 Results: The study reviewed a total of 12 isolates of Escherichia coli of which is (34.29%) of which were obtained from urine samples. The susceptibility test results showed that most isolates were susceptible to Ofloxacin with 83.3% susceptibility, followed by Levofloxacin (66.7%), whereas gentamicin, nalidixic acid, cefuroxime, and cefotaxime exhibited a susceptibility rate of 33.3%. On the other hand, the highest resistance rate was to imipenem (96.7%), followed by Amoxicillin and ampiclox with 83.3%, while the least percentage resistance was observed in Ofloxacin with (8.3%) and Levofloxacin with (16.7%). Extended-spectrum Βeta-Lactamase (ESBLs) production among E. coli isolates was detected in 25.0% of the potential ESBL producers. The result of the multiple antibiotic resistance index showed that the isolate (E20) was significantly higher than other isolates with p<0.05, whereas E3, E7, E10 and E12 had same resistance pattern.
 Conclusion: So drug resistance due to ESBL production is a severe threat in UTIs, narrowing down the choice of antibiotics for treatment. So, there is a need to introduce routine screening for ESBL production for all uropathogenic Escherichia coli, causing urinary tract infections.

Detection and Dissemination of Extented-Spectrum Beta-Lactamases Genes (CTX-M-15 and SHV-187) Isolated in Multi-Drug Resistant Uropathogenic Klebsiella pneumoniae and Escherichia coli in Cote D'ivoire

Enterobacteriaceae are ubiquitous commensal bacteria of humans that have become major causative agents of hospital infections. The objective of the study was to characterized the extended-spectrum beta-lactam resistance genes from multi-drug uropathogenic clinical strains in Côte d'Ivoire. Bacterial strains isolated from various biological products were collected from January 2011 to June 2016 at the Observatoire de la résistance des micro-organismes aux anti-infectieux en Côte d'ivoire (ORMICI). Two hundred and sixty six (266) enterobacterial strains were collected during this study. Antibacterial susceptibility and the presence of bla genes were determined by the solid-state diffusion method and by PCR, respectively. The presence of ESBL was confirmed by the double disc synergy test method and Sequencing was performed. Of the strains collected, the most isolated were Escherichia coli 53 (39.25%) and Klebsiella pneumoniae 36 (26.66%). Antibiotic resistance of more than 50% was observed for gentamicin, norfloxacin, third generation cephalosporins and tobramicin in E coli. Only imipenem had a low resistance rate of 5.6%. However, apart from norfloxacin, the Klebsiella pneumoniae strains tested expressed resistance to aztreonam and second generation cephalosporins in excess of 50%. The rate of resistance to aztreonam and cefotaxin was statistically different between E. coli and Klebsiella pneumoniae strains (p-value ₌ 0,008 and p-value ₌ 0,032 respectively). The genes blaCTX-M, blaSHV, blaTEM, qnr B and a class I integron were detected. After sequencing, the SHV-1, SHV-28, SHV-187 and CTX-M15 variants were detected. Although these results are of low proportions, this may be considered critical for the future, hence the need for a better antibiotic surveillance strategy in Abidjan.

Molecular identification of blaTEM gene of extended-spectrum beta-lactamase-producing Escherichia coli from healthy pigs in Malang district, East Java, Indonesia.

The increase and prevalence of multidrug-resistant bacteria in livestock animals are serious public health concerns. This study aimed to identify the presence of the blaTEM gene in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from rectal swabs of apparently healthy pigs in Malang District, East Java, Indonesia. A total of 120 rectal swab samples were collected from the pigs. The rectal swabs were screened for the presence of E. coli using standard microbiological identification procedures. The Kirby-Bauer disk diffusion method identified multidrug-resistant E. coli. Five different classes of antibiotics were used to identify multidrug-resistant isolates, including Ciprofloxacin, Trimethoprim, Tetracycline, Streptomycin, and Aztreonam. Multidrug-resistant E. coli isolates were characterized for the presence of ESBL using double-disk synergy test methods. The presence of blaTEM genes was determined using polymerase chain reaction methods. The results of this study indicated that 107 (89.2%) out of 120 samples analyzed were positive for E. coli isolates. A total of 32 (29.9%) E. coli isolates were identified to be multidrug-resistant and further subjected to molecular testing. The molecular analysis revealed (5; 15.6%) E. coli isolates to harbor the blaTEM gene. The results of this study revealed that pigs and products of pork origin must be considered a source of transmission of ESBL-producing E. coli to public health important under the food chain.

Prevalence of extended-spectrum β-lactamase-producing Escherichia coli in companion dogs in animal clinics, Surabaya, Indonesia

Background and Aim: The practice of keeping animals as pets is becoming increasingly common. The upsurge of extended-spectrum β-lactamase (ESBL)-producing organisms of animal origin is a health threat globally. This study aimed to identify the presence of extended-spectrum β-lactamase-producing Escherichia coli in companion dogs in animal clinics in Surabaya, Indonesia. Materials and Methods: A total of 85 rectal swab samples were collected from companion dogs at five animal clinics in different regions of Surabaya, Indonesia. The presence of E. coli was identified from the samples using standard methods, followed by antibiotic sensitivity testing. The resistant isolates were examined for the presence of ESBL using the double-disk synergy test method. The phenotypically identified ESBL-producing E. coli was further confirmed with an automated system using Vitek-2. Results: The rectal swab samples (n=85) tested were 100% positive for E. coli isolates. Eight (9.41%) out of the 85 E. coli obtained from rectal swabs were extended-spectrum β-lactamase producers. All eight ESBL-producing E. coli were identified by automated Vitek-2 confirmatory tests. Conclusion: This study provides insight into the prevalence of ESBL-producing organisms isolated from companion dogs in Indonesia. This work indicates the need for the general public to be more aware of the role of companion animals in disseminating pathogenic organisms, since they serve as potential reservoirs in the spread of antibiotic resistance affecting human health.

Evaluation of different phenotypic diffusion methods in the identification of extended spectrum beta lactamase producing uropathogenic

: The study was aimed to identify the occurrence of extended spectrum of Beta lactamases (ESBLs), to compare different phenotypic methods used for the confirmation and to evaluate the antibiotic resistance pattern in ESBL producing uropathogenic : The strains were isolated from urine and the isolates resistance to at least one of the three representative cephalosporins (cefotaxime, cefpodoxime and ceftazidime) was tested for ESBL production by Double disc synergy test (DDST), Inhibitory potentiated disc diffusion (IPDD) test and quantitative E-strip method.: Of 120strains isolated, 62(51.6%) were resistant to at least one of the three cephalosporins and 28 (45.1%) were positive for ESBL by IPDD and E-strip test. However, 9 (14.5%) strains were positive by DDST method. Among third generation cephalosporins, cefpodoxime was (45.8%) better screening indicator followed by ceftazidime (40.0%) and cefotaxime (37.5%). Most of the ESBL producers (97.3%) were resistant to three or more drugs, compared to (51.2%) non-ESBL producers. : The acceptable method for detection of ESBL producing were IPDD and E-strip tests compared to DDST with better sensitivity (100%), specificity (95.8%) and positive predictive value (96.5%). ESBL producers showed significantly (p<0.05) higher resistance to tobramycin, amoxyclav and amikacin compared to non ESBL producers.

Phenotypic methods for detection of metallobetalactamase in Meropenem resistant Pseudomonas aeruginosa and molecular detection of blaIMP & blaVIM genes carrying strains

Introduction and Objectives: Pseudomonas aeruginosa is one of the major organisms responsible for nosocomial infections such as pneumonia, UTIs, surgical site infections, and bloodstream infections.1 High intrinsic and acquired resistance to many antimicrobial agents have allowed P. aeruginosa to persist in both community and hospital settings. This study was done to find the prevalence rate of Meropenem resistant Pseudomonas aeruginosa carrying metallobeta lactamase producing genes. Materials and Methods: The study included a total of 100 non-duplicate clinically significant, random bacterial isolates obtained from a North Indian rural tertiary care hospital (Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar, India) from various clinical specimens, over a period of one year (January2012 to December 2012) . Identification of organisms was done using the standard microbiological techniques as described by Colle et al 1996.7 The antimicrobial susceptibility testing was performed by Kirby Bauer disc diffusion method. To detect MBL producing isolates phenotypicaly, Disc potentiation test (DPT) and Double disc synergy test (DDST) were performed. PCR was performed on Meropenem resistant isolates to detect blaIMP and blaVIM genes. Results: Out of 100 examined isolates, 36% isolates were resistant to Meropenem. Out of these Meropenem resistant strains, 44.4%(16) P. aeruginosa isolates were MBL producers as detected by DDST method and 69.44%(25) isolates were MBL producers phenotypically by DPT method . PCR revealed prescence of bla VIM gene in 16.66%(6) of Meropenem resistant Pseudomonas aeruginosa isolates and 38.88% (14) isolates were found to be carrying bla IMP gene . Three isolate were shown to carry both blaIMP and blaVIM genes. Conclusion: Resistance of P. aeruginosa isolates to Meropenem due to MBL enzymes is increasing in rural area. Resistance due to MBL has clinical significance, rapid detection of MBL producing strains followed by

Molecular investigation of extended-spectrum β-lactamases (ESBLs) genes in the Salmonella isolates obtained from children with acute diarrhea

Salmonellosis is an important public health concern among children in worldwide. Extended-spectrum β-lactams (ESBLs) cause resistance to clinically important beta-lactams which are generally used to treat invasive Salmonella infections. Therefore, the aim of this study was to investigate the presence of SHV , TEM and CTX-M genes in different strains of Salmonella isolated from children with acute diarrhea and to determine their resistance profile. In this cross-sectional study, 300 fecal samples were collected from children referred to the Amirkola Children's Hospital, Babol, Iran. Antibiotic susceptibility testing was done according to the CLSI guideline. ESBLs-producing strains were identified using double disk synergy test method on the Mueller-Hinton agar plates. Multiplex-PCR was performed using oligonucleotide specific primers to detect of SHV , TEM and CTX-M genes. In total, 7% (n; 21/300) salmonella were isolated, which 61.9%, 28.6% and 9.5% were Salmonella typhimurium , Salmonella enteritidis and Salmonella typhi , respectively. The prevalence of the ESBL-producing isolates were 52.4%. M-PCR results showed that 42.8%, 38.1% and 14.3% of isolates were carried CTX-M , TEM and SHV genes, respectively. Also, 18.2% of isolates harbored CTX-M , and TEM genes, simultaneously. The high rate of ESBLs-producing Salmonella strains in the pediatric patients is an alarm. It is also recommended that alternative drugs be used with less resistance, which requires further investigation.

IDENTIFIKASI BAKTERI Escherichia coli PENGHASIL Extended Spectrum β-Lactamase DARI SWAB RECTAL SAPI PERAH MENGGUNAKAN METODE VITEK-2 DI KUD TANI WILIS SENDANG KABUPATEN TULUNGAGUNG

Antibiotic resistance in animals and humans has become a global problem that needs attention. The use of antibiotics in inappropriate on food-producing animals can lead to resistance many of the pathogenic bacteria to the various types of antibiotics, one of which is the Escherichia coli (E. coli) which produces extended spectrum β-lactamase (ESBL). The aim of this study was to isolate and identified ESBL- E. coli isolate from dairy cow rectal swabs in Sendang, Tulungagung district using the Vitek-2 method. The number of rectal swab samples used in the present study was 50. The result of the study showed that from all of the samples could be isolated and indentified E. coli, based on the colony characteristics on EMBA and biochemical test. Based on the double disc synergy test method using antibiotic disc amoxicylyn-clavulanate, ceftriaxone, aztreonam, ceftazidime and cefotaxime, 10 isolates could be identify els ESBL- E. coli. furthermore 3 out of 10 isolates DDST positives were confirmed ESBL- E. coli using Vitek-2 method.

Emerge of NDM-1-Producing Multidrug-Resistant Pseudomonas aeruginosa and Co-harboring of Carbapenemase Genes in South of Iran

New Delhi metallo-beta-lactamase-1 (NDM-1) is one of the most important emerging antibiotic resistance. Co-harboring three or four carbapenemases is rare and only a few reports exist in the literature. We described the characteristics of the large epidemic outbreaks and reports co-producing blaNDM-1 with the other carbapenemase genes in P. aeruginosa isolates. This present cross-sectional research was conducted on 369 P. aeruginosa isolates obtained from burn and general hospitals within years 2013 to 2016. Beta-lactamase classes A, B and D genes were identified by PCR method. Modified hodge test (MHT), double-disk potentiation tests (DDPT) and double disk synergy test (DDST) were performed for detection carbapenemase and metallo beta-lactamase (MBL) production of blaNDM-1 positive P. aeruginos isolates. From 236 carbapenem-resistant P. aeruginosa (CRPA), 116 isolates have had MBL genes and twenty-nine isolates were found positive for blaNDM-1 . In CRPA isolates, blaIMP-1 , blaVIM-2 and blaOXA-10 were identified in 27.5%, 21.1% and 32.2% of isolates respectively, while co-producing blaNDM-1 , blaIMP-1 , blaOXA-10 , co-producing blaNDM-1 , blaVIM-2 , blaOXA-10 and co-producing blaIMP-1 , blaVIM-2 were determined in 11 (4.6%), 8 (3.4%) and 27 (11.4%) of isolates respectively. The finding of this co-existence of multiple carbapenemase resistance genes is threating for public health. Dipicolinic acid is a superior MBL inhibitor in DDPT antique than EDTA in DDST method for the detection of MBL-blaNDM-1 producing P. aeruginosa.

First phenotypic and molecular characterization of clinical isolates of drug resistant Providencia rettgeri in Imo state, South-Eastern Nigeria.

Background: Nosocomial outbreak of drug resistant Enterobacteriaceae including those mediated by Providencia species has become a serious public health challenge across the globe – owing to the difficulty in selecting antibiotics for proper treatment especially in the face of resistance. The morbidity and mortality rate of bacteremia due to Providencia species could be high, especially among the elderly and those with pre-existing medical conditions. This study was aimed at determining the occurrence of multidrug resistance phenotypes in clinical isolates of Providencia rettgeri using both phenotypic and genotypic techniques. Methods: Clinical isolates were identified to be isolates of P. rettgeri using standard microbiological and biochemical techniques; and these were characterized with respect to antibiotic resistant patterns. Overall, 143 clinical isolates of non-duplicate P. rettgeri isolates were bacteriologically recovered from the clinical specimens which included blood/wound swab samples (n=108) and swabs from surgical instruments and suction devices (n=35). Antimicrobial susceptibility test (AST) was done using the modified Kirby-Bauer disk diffusion techniques as per the Clinical Laboratory Standard Institute (CLSI) criteria. The isolates were tested phenotypically for ESBL production by the double disk synergy test (DDST) method and the β-lactamase genes were identified using polymerase chain reaction. The strains were sequenced with ABI3500XL analyzer with a 50 cm array, using POP7. Sequences data generated were analyzed with Geneious version 9. 0. 5. Results: The result of AST showed that Providencia rettgeri isolates were resistant to ceftazidime, cefotaxime, ampicillin and tetracycline. Based on the DDST method, some of the Providencia rettgeri isolates were phenotypically confirmed to be ESBL positive (11%) and MBL positive (6%). The results of the molecular analysis showed that the P. rettgeri isolates harbored SHV genes (n=6; 12. 5%) which mediates ESBL production. However, none of the isolates harboured the CTX-M or TEM gene. Conclusion: Our study shows that the P. rettgeri isolates analyzed were multidrug resistant, and they also harbour some important phenotypes and/or genotypes that mediate the production of ESBL (extended spectrum beta-lactamase). This is the first report of ESBL detection in clinical isolates of P. rettgeri in Imo state, southeast Nigeria.

Multi-Antibiotic Resistance and Factors Affecting Carriage of Extended Spectrum β-Lactamase-Producing Enterobacteriaceae in Pediatric Population of Enugu Metropolis, Nigeria.

Extended-spectrum β-lactamase (ESBL)-producing organisms have become a serious challenge in healthcare delivery globally. The prevalence of ESBL carriage in healthy and sick children in Enugu, Nigeria, was bacteriologically investigated in this study. Four hundred and twenty-two biological samples (mid-stream urine and feces) were bacteriologically analyzed. The isolates were screened for ESBL production using Clinical and Laboratory Standards Institute (CLSI) breakpoints. The suspected ESBL producers were confirmed using double disc synergy test method. Out of the 162 isolates screened, 32 (19.8%) were confirmed as ESBL positive, with a prevalence of 25.32% among sick children in Enugu State University Teaching Hospital (ESUTH), Parklane, Enugu and 13.89% in apparently healthy children in a community setting. Klebsiella spp. and Escherichia coli had the highest prevalence of 34.6% and 28.6%, respectively; Citrobacter spp. and Enterobacter spp. were 18.2% and 16.7%, respectively. The ESBL positive isolates were resistant to sulfamethoxazole/trimethoprim (100%), tetracycline (100%), kanamycin (96.9%), nitrofurantoin (84.4%), ciprofloxacin (68.6%), and chloramphenicol (62.5%) but susceptible to meropenem (100%), colistin (56.3%), and gentamicin (50%). Klebsiella spp. had the highest ESBL occurrence among sick children while E. coli had the highest ESBL occurrence among healthy children in Enugu. All ESBL-positive isolates were multiply resistant to conventional antibiotics. The emergence and spread of β-lactamase-producing Enterobacteriaceae in hospital and community environments highlight the possibility for an infection outbreak if not checked.

Emergence of bla TEM resistance gene in ESBL-producing Escherichia coli clinical isolates from Health facilities in Makurdi, Benue State Nigeria

The emergence of extended-spectrum-β-lactamases (ESBLs)-producing Escherichia coli represents a serious clinical concern in healthcare. β-lactamases produced by these strains of E. coli render ineffective cephalosporins and other β-lactam antibiotics used to treat infections caused by Gram–negative bacteria. We determined the presence of ESBL in 400 clinical isolates of E. coli isolated from various clinical specimens (urine, stool, blood, sputum, throat and wound swabs) from 216 female and 184 male patients with mean age of 28.1 ± 16.8 years (age range: 2 – 71 years), who were attending 6 selected health facilities in Makurdi, Benue State Nigeria. Antibiotic susceptibility test was carried out on the isolates using Kirby Bauer diffusion method. Presence of ESBLs was determined by the double disc synergy test (DDST). Specific primers were employed to characterize the ESBL gene using PCR. The isolates showed high level of resistance to all the antibiotics tested except mipenem. Highest resistance was to penicillin 392(98.0%) followed by ceftriaxone 385(96.3%). Out of the 400 isolates, 64 (16.0%) tested positive for ESBLs by DDST method, while PCR technique confirmed 47(11.8%) to harbour bla TEM genes. Isolates from blood specimens harboured highest percentage of ESBL genes 5(26.3%) and also plasmid-mediated bla TEM genes 5(26.3%), followed by wound swabs 9(17.3). The least percentage of plasmid-mediated bla TEM genes was carried by isolates from sputum specimens 1(8.3). Age group 45 to 58 years harboured the highest percentage of bla TEM genes 15(14.6%), while female patients, 27(12.5%) carried more bla TEM resistance genes than the male patients 20(10.9%). A prevalence of 11.8% (n=47) of bla TEM resistance gene has been reported for the present study. In view of multidrug resistant ESBL-producing Escherichia coli bacteria circulating in the study location, prescription of antibiotics, especially cephalosporins should be based on laboratory results of antibiotic susceptibility tests that are carried out along with ESBL detection. Infection prevention and control strategies should be stepped up in the health facilities under study.

Extended-spectrum β-lactamases producing multidrug resistance Escherichia coli, Salmonella and Klebsiella pneumoniae in pig population of Assam and Meghalaya, India.

Aim:The present study was conducted to record the prevalence of extended spectrum β-lactamases (ESBLs) producing Escherichia coli, Salmonella spp., and Klebsiella pneumoniae from pig population of Assam and Meghalaya and to record the ability of the resistant bacteria to transfer the resistance genes horizontally.Materials and Methods:Fecal samples (n=228), collected from pigs of Assam (n=99) and Meghalaya (n=129), were processed for isolation and identification of E. coli and Salmonella spp. All the isolates were tested for ESBLs production by double disc synergy test (DDST) followed by screening for ESBLs producing genes (blaTEM, blaSHV, blaCTX-M, and blaCMY) by polymerase chain reaction (PCR). Possible transfer of resistance encoding genes between enteric bacterial species was carried out by in vitro and in vivo horizontal gene transfer (HGT) method.Results:A total of 897 enteric bacteria (867 E. coli and 30 Salmonella) were isolated and identified. Altogether 25.41% isolates were confirmed as ESBL producers by DDST method. Majority of the isolates were E. coli followed by Salmonella. By PCR, 9.03% isolates were found positive for at least one of the target resistance genes. blaSHV was absent in all the isolates. blaCMY was the most prevalent gene. All the E. coli isolates from Assam were negative for blaTEM. A total of 2.76% isolates were positive for blaTEM + blaCMY. On the other hand, 0.67% isolates were positive for blaCTX-M + blaCMY genes. Only 0.33% isolates carried all the three genes. Altogether, 4.68% bacteria carried the resistance encoding genes in their plasmids. blaTEM gene could be successfully transferred from Salmonella (donor) to E. coli (recipient) by in vitro (5.5-5.7×10−5) and in vivo (6.5×10−5 to 8.8×10−4) methods. In vivo method was more effective than in vitro in the transfer of resistance genes.Conclusion:The pig population of Assam and Meghalaya are carrying multidrug resistance and ESBLs producing E. coli and Salmonella. The isolates are also capable to transfer their resistance trait to other bacterial species by HGT. The present finding could be considered as a serious public health concern as similar trait can also be transmitted to the human commensal bacteria as well as pathogens.

Characterization of extended-spectrum β-lactamases (ESBLs)-producing Salmonella in retail raw chicken carcasses

Extended-spectrum β-lactamases (ESBLs)-producing Salmonella is considered a serious concern to public health worldwide. However, limited information is available on ESBLs-producing Salmonella in retail chicken products in China. The objective of this study was to characterize ESBLs-producing Salmonella isolates from retail chickens in China. A total of 890 Salmonella isolates from retail chicken carcasses collected from 4 provinces were firstly screened for ESBLs-production phenotype via the double-disk synergy test method. A total of 96 (10.8%, n=890) ESBLs-producing Salmonella were identified and subjected to PFGE analysis, characterization for the presence of ESBLs encoding genes, transposons, carbapenemase and virulence genes. A total of 59 PFGE profiles were detected in these 96 isolates, among which 57.3% were found to harbor blaTEM-1, whereas 30.2%, 24.0%, 18.8% and 7.3% were carrying blaOXA-1, blaCTX-M-15, blaCTX-M-3 and blaPSE-1 genes, respectively. Moreover, 42 (43.8%) isolates co-carried 2 ESBLs-producing genes, and two (2.1%) isolates co-carried 3 genes. Furthermore, 24 (25.0%) ESBLs-producing isolates carried VIM and 10 (10.4%) carried KPC encoding genes that closely associated with carbapenems resistance. Eighty-eight isolates harbored transposons ranging from 4.2% for Tn903 to 76.0% for Tn21. Out of the 88 Salmonella that harbored transposons, 25%, 22.7%, 23.9%, 10.2% and 1.1% of isolates were found to carry 2, 3, 4, 5 and 6 transposons, respectively. The minimum inhibitory concentration (MIC) values for cephalosporins (ceftriaxone, cefoperazone and cefoxitin) to ESBLs-producing isolates were from 4 to 1024μg/mL, for nalidixic acid were from 64 to 512μg/mL, for fluoroquinolones (ciprofloxacin, levofloxacin and gatifloxacin) were from 4 to 256μg/mL. Twenty-nine virulence genes were detected in the 96 ESBLs-producing isolates with 2.1% harbored spvR (lowest) and 90.6% harbored marT and steB (highest). All isolates carried at least one virulence gene, 83.3% of the isolates co-carried ≥10, 17.7% co-carried ≥15, and 1.0% co-carried 23 virulence genes. Interestingly, 16.7% of the isolates resistant to >12 antibiotics tested and shown to carry >4 transposons and 10 virulence genes. Our findings indicated that ESBLs-producing Salmonella isolated from retail chicken meat in China were highly resistant to antibiotics, frequently harbored transposons, virulence genes, carbapenems hydrolysis enzymes and ESBLs encoding genes. These isolates can pose a significant public health risk.

Occurrence of extended-spectrum and AmpC β-lactamases in multiple drug resistant Salmonella isolates from clinical samples in Lagos, Nigeria.

PurposeSalmonella spp. are important foodborne pathogens exhibiting increasing resistance to antimicrobial drugs. Resistance to broad-spectrum β-lactams, mediated by extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase enzymes is fast spreading and has had negative impacts on the clinical outcomes, particularly on third-generation cephalosporins. This study investigated the carriage of AmpC gene among multidrug-resistant Salmonella spp. from Lagos, Nigeria.MethodsForty Salmonella spp. from clinical samples (S. typhi = 13; S. typhimurium = 10; S. enteritidis = 8; S. choleraesuis = 5; S. paratyphi = 4) were subjected to in vitro susceptibility test by disk diffusion methods. Isolates that were resistant to cefoxitin and third-generation cephalosporins were screened for ESBL (Double Disk Synergy Test Method) and AmpC enzyme (AmpC disk test) production. Detection of AmpC fox gene was carried out by polymerase chain reaction.ResultsThirty-two (80%) of the Salmonella isolates were cefoxitin resistant. Plasmid-mediated AmpC β-lactamase and ESBL enzymes were recorded in 10/40 (25%) and 16/40 (40%) of the Salmonella isolates, respectively. Specifically, 16/40 (40%) of the Salmonella isolates possessed 380 bp AmpC fox gene, with the highest occurrence found in S. typhi strains (43.8%) followed by S. typhimurium (25%). There was no AmpC fox gene detected in S. paratyphi strains. Interestingly, coproduction of enzymes occurred in some of the isolates, raising fears of resistance to a multitude of antibiotics in the treatment of bacterial infections.ConclusionEmergence of AmpC β-lactamase–producing Salmonella isolates in our environment was recorded for the first time, raising concern on increased antibiotic resistance among strains of Salmonella serovars in Lagos. Further genotypic study of the isolates could answer the questions on strain sources, clonal relatedness, and mechanism of spread.

Antibiotic susceptibility pattern and analysis of plasmid profiles of Pseudomonas aeruginosa from human, animal and plant sources.

Multidrug resistant organisms (MDROs) constitute a major public health threat globally. Clinical isolates of Pseudomonas aeruginosa remains one of the most studied MDROs however there is paucity of information regarding the susceptibility of its animal and plants isolates to antipseudomonas drug in Nigeria. From a total of 252 samples consisting of plants, animals and clinical samples, 54, 24 and 22 P. aeruginosa were isolated from vegetables, animals and clinical sources respectively. All the isolates were identified by standard biochemical methods. Antimicrobial susceptibility testing (AST) of the 100 P. aeruginosa isolates against 7 antipseudomonal drugs was carried out by disk diffusion method, the phenotypic detection of ESBL was done by double disk synergy test (DDST) while plasmid extraction on 20 selected isolates based on their resistance to 2 or more classes of antibiotics was carried out by alkaline lysis method and analysed with Lambda DNA/Hind lll marker respectively. The AST results revealed highest resistance of 91 and 55 % to ceftazidime and carbenicillin respectively while highest susceptibilities of 99 % for piperacillin–tazobactam and imipenem were recorded in overall assay. Fifteen out of 100 isolates specifically (10) from vegetables, (3) clinical and (2) poultry isolates showed synergy towards the beta-lactamase inhibitor indicating production of ESBL by DDST method. Detection of plasmids was among vegetable (n = 4), poultry (n = 4), cow (n = 3) and clinical isolates (n = 1). Plasmid profile for the selected isolates revealed 6 of the strains had one plasmids each while 5 strains possessed 2–4 plasmids and 1 strain had 5 plasmids. The sizes of the plasmid range from <1 to ≥23kbp. Detection of ESBL and Plasmids among the investigated isolates is suggestive of multiple interplay of resistance mechanism among the isolates. Plants and animal isolates of P. aeruginosa harbouring multiple mechanisms of resistance is of concern due to the danger it poses on the public health.

Kinetics activity of Yersinia Intermedia Against ZnO Nanoparticles Either Synergism Antibiotics by Double-Disc Synergy Test Method.

Bacterial resistance to the commonly used antibacterial agents is an increasing challenge in the medicine, and a major problem for the health care systems; the control of their spread is a constant challenge for the hospitals. In this study, we have investigated the antimicrobial activity of the Zinc Oxide nanoparticles against clinical sample; Yersinia intermedia bacteria. Nanoparticle susceptibility constants and death kinetic were used to evaluate the antimicrobial characteristics of the Zinc Oxide (ZnO) against the bacteria. Antimicrobial tests were performed with 108 cfu.mL-1 at baseline. At first, Minimum Inhibitory Concentration (MIC) of ZnO was determined and then nanoparticle suspension at one and two times of the MIC was used for death kinetic and susceptibility constant assay at 0 to 360 min treatment time. ZnO nanoparticles with size ranging from 10 to 30 nm showed the highest susceptibility reaction against Y. intermedia (Z=39.06 mL.μg-1). The process of Y. intermedia death in ZnO suspension was assumed to follow the first-order kinetics and the survival ratio of bacteria decreased with the increasing treatment time. An increased concentration of the nanoparticle was seen to enhance the bactericidal action of the nanoparticle. Then we performed the best ratio of the nanoparticles on semi-sensitive and resistance antibiotic for the bacteria. However, based on experimental results, synergy of ZnO nanoparticles and Oxacilin was determined and Y. intermedia showed a higher sensitivity compared to the ZnO nanoparticles alone. The results of the present study illustrates that ZnO has a strong antimicrobial effect and could potentially be employed to aid the bacterial control. It could also improve- antibacterial effects in combination with the antibiotics.

A study on extended spectrum β lactamase &amp; metallo β lactamase producing bacteria from drinking water sources in and around Kelambakkam

Introduction: Water is essential to life and many people do not have access to clean safe drinking water. Poor sanitation is the major cause for water contamination. Death due to infection like cholera, typhoid fever, bacillary dysentery are transmitted through contaminated water. Aims & Objectives: To study the bacteriological profile of drinking water samples, to perform antimicrobial sensitivity testing of the isolates and genotypic identification of resistant gene. Materials and Methodology: Drinking water sample were collected from in and around Kelambakkam for a period of 1year. Antimicrobial susceptibility testing was carried out for the isolates according to standard guidelines. ESBL producers were detected using double disc synergy test method. MBL producer were detected by combined disk test. Screening for bla TEM, bla VIM gene among the drug resistance isolates were done. Result: Among 161 samples from various sources, most common isolates were Escherichia coli (n=41) 25.4%, followed by Klebsiella spp. Out of 41 isolates of E.coli were 23 and 10 were ESBL and MBL producer respectively. Among 10 isolates 8 were both ESBL and MBL producer, isolates positive for bla TEM gene & bla VIM gene were 5 & 2 respectively. Keywords: E.coli, Drinking water, ESBL and MBL

Phenotypic Characterization and Comparative Study on ESBL-producing E. coli of Clinical Origin

Extended-spectrum β-lactamases (ESBLs) continue to be a major challenge in clinical setups worldwide, conferring resistance to the expanded-spectrum cephalosporins. The present study focused on the prevalence of ESBL-producing E. coli clinical isolates among patients diagnosed of Wound and Urinary Tract Infections attending Federal Teaching Hospital Abakaliki. A total of one hundred and ninety two clinical isolates of E. coli was studied for their susceptibility patterns to cephalosporin antibiotics and detection of ESBL producers was carried out by double disc synergy test (DDST) and Brilliance ESBL Agar. Of the 192 isolates tested for their antibiogram, 19(9.9%), 41(21.4%), 132(68.7%); 48(25.0%) and 144(75.0%) isolates were from wound, high virginal swab, urine, male and female respectively. The isolates showed higher susceptibility to cefepime (a 4th-generation cephalosporin) with percentage susceptibility of 78.9, 85.4 and 73.5 to the isolates from wound, HVS and urine respectively. Higher resistance was recorded among the 3rd-generation cephalosporins which include Cefotaxime (63.4%), Ceftriaxone (57.9%), and Cefpodoxime (73.7%). Among the resistant isolates of E. coli, 20 isolates were phenotypically confirmed ESBL producers by the DDST and Brilliance ESBL Agar methods. Ten (10) ESBL producing E. coli was confirmed using DDST method whereas 15 ESBL producing E. coli was confirmed using the Brilliance ESBL Agar. Brilliance ESBL Agar was found to be better than DDST in the detection of ESBLs. Continuous monitoring of drug resistance and regulating the use of cephalosporin drugs in our hospitals is vital for proper infectious disease management and treatment.