Combined Disk Diffusion Method Research Articles

Characterisation of uropathogenic E.coli by detecting the virulence factors and its drug resistance pattern in a tertiary care hospital in India

Urinary tract infections (UTIs) are among the most prevalent nosocomial and community-acquired bacterial diseases in humans, with E.coli being the most typical pathogen isolated. To detect the prevalence of virulence factors like haemolysin, haemagglutination of human erythrocytes with its effect of D-mannose, and cell surface hydrophobicity, the antibiotic sensitivity pattern and ESBL production in urinary isolates of E.coli obtained from clinical samples. We included the E.coli isolates obtained from a midstream urine sample for the study. Virulence factors like haemolysin, hemagglutination and salt aggregation were detected as per standard protocols. Antibiotic sensitivity testing was performed by the Kirby Bauer disc diffusion method. Extended-spectrum beta-lactamase (ESBL) production was seen by the combined disc diffusion method on Muller Hinton agar as per CLSI guidelines. A total of 103 E.coli isolates were tested, and among them, 24(23.30%) produced haemolysin, 65(63.10%) produced hemagglutination and 38(36.89%) had salt aggregation properties. Most isolates obtained were resistant to beta-lactam antibiotics but showed high sensitivity towards antibiotics like chloramphenicol, meropenem, amikacin, imipenem and nitrofurantoin. Around 48% of them were ESBL producers. The common virulence factors associated with UTI were P-fimbriae (MRHA), haemolysin production, cell surface hydrophobicity and type-1 fimbriae. Because of the emerging drug resistance among UPEC, therapy should be advocated as far as possible after obtaining the culture and sensitivity results to determine exact aetiology and susceptibility patterns. The sensitivity to nitrofurantoin is very high, suggesting that antibiotic recycling will help clinicians treat UPEC.

Determination of antibiotic resistance rates of Escherichia coli and Klebsiella pneumoniae isolates, which are the causative agents of urinary tract infection in pregnant women

Aim: Urinary tract infections are common infections during pregnancy. Infections seen during pregnancy have a spectrum ranging from asymptomatic bacteriuria to cystitis, pyelonephritis and, urosepsis. In this study, it was aimed to determine the antibiotic resistance rates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) isolates isolated from urinary cultures of pregnant women who applied to the Ankara Training and Research Hospital. 
 Material and Method: The identification and antibiotic susceptibility of E. coli and K. pneumoniae isolates isolated from urinary samples of pregnant women who applied to the Ankara Training and Research Hospital between January 2021 and December 2022 were investigated with VITEK-2 (Biomerioux, France) fully automated system, and the presence of extended-spectrum beta-lactamase (ESBL) was investigated by combined disc diffusion method. The obtained data were analysed retrospectively.
 Results: Bacterial growth was detected in 1090 (1.2%) out of a total of 8923 urine samples over a two-year period. 480 (4.4%) of the microbial agents reproducing in urine culture were E. coli and 105 (0.96%) were K. pneumoniae. The rate of extended-spectrum beta-lactamase (ESBL) in E. coli strains was 16.04% (77/480), and the rate of ESBL in K. pneumoniae strains was 20.9% (22/105). Resistance rates of amoxicillin-clavulanic acid, ceftriaxone, phosphomycin, ciprofloxacin, piperacillin-tazobactam, nitrofurantoin, imipenem, meropenem, ertapenem in ESBL negative E. coli strains were 15.9%, 8.82%, 20%, 11.1%, 5.88%, 0%, 0%, 0% and 0%, respectively. Resistance rates of amoxicillin-clavulanic acid, ceftriaxone, phosphomycin, ciprofloxacin, piperacillin-tazobactam, nitrofurantoin, imipenem, meropenem, ertapenem in ESBL positive E. coli strains were determined as 66.5%, 100%, 2.2%, 33.8%, 11.5%, 0%, 0%, 0% and, 0%, respectively. Resistance rates of amoxicillin-clavulanic acid, ceftriaxone, phosphomycin, ciprofloxacin, piperacillin-tazobactam, nitrofurantoin, imipenem, meropenem, ertapenem in ESBL negative K. pneumoniae strains were 53%, 100%, 12.5%, 28.5%, 2.2%, 3.5%, 0%, 0% and, 4.5%, respectively. Resistance rates of amoxicillin-clavulanic acid, ceftriaxone, phosphomycin, nitrofurantoin, ciprofloxacin, piperacillin-tazobactam, imipenem, meropenem, ertapenem in ESBL positive K. pneumoniae strains were 62.5%, 100%, 12.5%, 35%, 28.5%, 22.7%, 0%, 0% and, 4.5%, respectively.
 Conclusion: According to the antibiotic susceptibility data in our hospital, phosphomycin or carbapenems may be preferred due to the low resistance rate in the empirical treatment of E. coli-related urinary tract infections in pregnant women. In the treatment of urinary tract infections due to K. pneumoniae, phosphomycin, piperacillin-tazobactam or carbapenems may be preferred due to low resistance rates.

CORRELATION OF DRUG RESISTANCE PATTERN WITH LIPASE PRODUCTION IN CLINICAL ISOLATES OF KLEBSIELLA PNEUMONIAE

Aim:The aim of our study is to correlate drug resistance patterns with Lipase production in clinical isolates of Klebsiella pneumoniae. Materials and Methods:Atotal of 150 clinical isolates of Klebsiella pneumoniae from various clinical samples collected during the period from July 2020 to December 2020 was included in this study. Lipase production was detected by tween 80 agar and an antibiotic susceptibility test has been performed by Kirby-Bauer disk diffusion method as per CLSI guidelines [10]. Extended Spectrum Beta-lactamase (ESBL) producers were detected by combined disc diffusion method and Carbapenemase production was detected by using the E-test strip method. Results and Discussion: Out of 150 consecutive non-duplicate isolates of Klebsiella pneumoniae, 74 isolates from Exudate, 7 from Blood, and 69 were from urine. Among 150 isolates, 85(56.6%) isolates produced Lipase production, 43(57.3%) were ESBL producers and 20(32.25%) isolates were positive for Carbapenemase production. Conclusion: Showing virulence in clinical isolates of Klebsiella pneumoniae, and the antibiotic susceptibility pattern with lipase production in Klebsiella pneumoniae were noticed. If the virulence is increased in Klebsiella pneumoniae, a drug-resistant pattern also shows more resistance in number. And nally, to correlate the ESBL and Carbapenemase producer with lipase production, in that also the ESBL and Carbapenemase producer shows more number with lipase production. And also noted, when compared with outpatients, inpatients show more lipase production. And hence ampicillin is an intrinsic resistance to Klebsiella pneumoniae. In this study, to correlate the drug-resistant pattern, ESBL, and carbapenemase producer with lipase production in clinical isolates of Klebsiella pneumoniae.

Multidrug-Resistant and Extended-Spectrum β-Lactamase (ESBL) - Producing Enterobacterales Isolated from Carriage Samples among HIV Infected Women in Yaoundé, Cameroon.

The exacerbation of antimicrobial resistance (AMR) is a major public health threat worldwide. In sub-Saharan Africa, there is a scarcity of data regarding multidrug-resistant (resistance to at least one antibiotic of three or more families of antibiotics) as well as extended spectrum β-lactamase-producing Enterobacterales (ESBL-PE), isolated among clinical and asymptomatically healthy patients, especially in women living with HIV (WLHIV) despite their immunocompromised status. The overarching aim of this study was set to determine the prevalence and characterize genotypically multi-drug resistant Enterobacterales (MDR-E) and ESBL- PE isolated from vaginal swabs of WLHIV attending the Yaoundé Central Hospital, Yaoundé, Cameroon. A cross-sectional study was conducted among WLHIV during a four-month periods from 1 February to 31 May 2021. A total of 175 WLHIV, of childbearing age and under antiretroviral treatment were contacted. One hundred and twenty participants (120) were recruited and vaginal swabs were collected from them. After culture on Eosine-Methylen Blue (EMB) agar, the identification of Enterobacterales was performed using API 20E kit. A double-screening of ESBL-PE was performed using a combined disc diffusion method and ROSCO Diagnostica kits. An antibiotic susceptibility test was carried out by disc diffusion as per the Kirby-Bauer method and the β-lactamase resistance genes, blaCTX-M, blaCTX-M-group1-2-9, blaTEM were molecularly characterized using a conventional Polymerase Chain Reaction (PCR). Overall, 30.83% (37/120) of the included WLHIV were colonized with Enterobacterales and the prevalence of vaginal carriage of MDR Enterobacterales among them was 62.16% (23/37). Among MDR-E isolates, the most prevalent species were E. coli (56.0%; 14/25) and K. pneumoniae (20.0%; 5/25). High rates of resistance to trimethoprim-sulfamethoxazole (96.0%; 24/25), amoxicillin-clavulanic acid (88.0%; 22/25) and gentamicin (72%; 18/25) were observed. The resistance mechanisms detected among these isolates were ESBL (48.0%; 12/25), ESBL+ porin loss (8.0%; 2/25), ESBL+AmpC (24%; 6/25), with blaCTX-M, blaCTX-M-group-1,2,9 being identified at 48.0% (12/25) for each of them and blaTEM at 72.0% (18/25). Our findings confirm the high-prevalence of MDR as well as ESBL-PE isolated in WLHIV, and suggest that a real time monitoring system of antimicrobial resistant bacteria coupled with the reinforcement of infection prevention control (IPC) strategies are needed to sustainably contain these life-threatening pathogens especially in the most vulnerable populations.

Plasmid-mediated quinolone resistance determinants in clinical bacterial pathogens isolated from the Western Region of Ghana: a cross-sectional study.

quinolones are critically important antibiotics that are reserved for treating very severe infections caused by multidrug-resistant bacterial pathogens. However, their indiscriminate uses have resulted in an increased number of resistant strains in many parts of the world including Ghana. We determined the quinolone resistance profile of Gram-negative bacterial pathogens and characterized the underlying molecular determinants of resistance. Gram-negative pathogens obtained from clinical specimens at three hospital laboratories were tested for resistance to quinolones and other commonly used antibiotics. ESBL production among the Enterobacterial isolates was confirmed using the combined disc diffusion method. We then used PCR to determine seven types of plasmid-mediated quinolone resistance genes present in the isolates resistant to nalidixic acid and ciprofloxacin. in this study, 29.5% of the isolates were resistant to ciprofloxacin, with the highest of 50% among E. coli resistance to the other quinolones was levofloxacin (24.4%), norfloxacin (24.9%), and nalidixic acid (38.9%). Significant proportions of the quinolone-resistant isolates were ESBL producers (P-values < 0.001). The aac(6´)-Ib-cr, qnrS, oqxA, and qepA genes were present in 43 (89.6%), 27 (56.3%), 23 (47.9%), and one (2.1%) of the isolates, respectively. None of the isolates tested positive to qnrA, qnrB, and oqxB genes. The presence of the aac(6´)-Ib-cr gene positively correlated with resistance to ceftriaxone, cefotaxime, and gentamicin (P-values < 0.05). high proportions of Gram-negative bacterial isolates were resistant to quinolones and most of these isolates possessed multiple PMQR genes. There is a need to implement measures to limit the spread of these organisms.

Antimicrobial resistance in patients with suspected urinary tract infections in primary care in Assam, India.

ObjectivesWe investigated the prevalence and diversity of antimicrobial resistance in bacteria isolated from urine samples of community-onset urinary tract infection (UTI) patients in southern Assam, India.MethodsFreshly voided midstream urine samples were collected from patients attending primary healthcare centres, with the patients’ epidemiological data also recorded. Species identification was confirmed using a VITEK 2 compact automated system. Phenotypic confirmation of ESBLs was performed using the combined disc diffusion method (CLSI 2017) and carbapenemase production was phenotypically characterized using a modified Hodge test. Common ESBLs and carbapenem-resistance mechanisms were determined in Escherichia coli isolates using PCR assays. Incompatibility typing of the conjugable plasmids was determined by PCR-based replicon typing; the phylotypes and MLSTs were also analysed.ResultsA total of 301 (59.7%) samples showed significant bacteriuria along with symptoms of UTI and among them 103 isolates were identified as E. coli of multiple STs (ST3268, ST3430, ST4671 and others). Among them, 26.2% (27/103) were phenotypically ESBL producers whereas 12.6% (13/103) were carbapenemase producers. This study describes the occurrence of diverse ESBL genes—blaCTX-M-15, blaSHV-148, blaPER-1 and blaTEM—and two E. coli isolates carrying the blaNDM-1 carbapenemase gene. ESBL genes were located within transconjugable plasmids of IncP and IncF type whereas blaNDM-1 was carried in an IncFrepB type plasmid.ConclusionsThis study illustrates the high rate of MDR in E. coli causing UTI in primary care in rural Assam. UTIs caused by ESBL- or MBL-producing bacteria are very difficult to treat and can often lead to treatment failure. Thus, future research should focus on rapid diagnostics to enable targeted treatment options and reduce the treatment failure likely to occur with commonly prescribed antibiotics, which will help to combat antimicrobial resistance and the burden of UTIs.

Detection of blaNDM-1 gene in ESBL producing Escherichia coli and Klebsiella pneumoniae isolated from urine samples.

Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the most prominent bacterial species resistant to almost all commonly used antibiotics. Carbapenem is one of the last resort drugs for treating such emerging multidrug-resistant bacteria. This study aimed to detect carbapenem-resistant blaNDM-1 gene in ESBL producing E. coli and K. pneumoniae isolates. A total of 190 E. coli and 350 K. pneumoniae isolates were screened for extended spectrumβ-lactamase (ESBL), carbapenemase and metallo β-lactamase (MBL) production via double-disk synergy test (DDST), modified Hodge test and combined-disk diffusion method. The blaNDM-1 gene was detected by PCR and confirmed via Sanger sequencing method. Of the 540 isolates tested, 71.8% were found to be multidrug-resistant. Overall rate of ESBL-positive isolates were 57.89% E. coli and 31.42% K. pneumoniae. Among ESBL positive isolates, 49.09% E. coli and 40% K. pneumoniae were positive for carbapenemase production whereas MBL production was detected in 29% E. coli and 22% K. pneumoniae isolates. In MBL positive isolates, (37%) E. coli and (40%) K. pneumoniae isolates harboured blaNDM-1 gene. The pair-wise DNA was aligned with the NDM-1 sequence from GenBank. The alignment score was 243 and the blast nucleotide sequencing results showed 97% sequence similarity with the sequences in GenBank for the blaNDM-1 gene. The blaNDM-1 gene was found to be the most prevalent in urine samples. There is a dire need to conduct screening tests in hospitals and communities to find out the exact prevalence of the blaNDM-1 spread in our population.

Detection of Extended Spectrum Beta Lactamases (ESBLs) Producing Enterobacteriaceae Family from Urinary Tract Infection (UTI) Patients

Background: Extended spectrum beta lactamase (ESBLs) are betalactamase capable of conferring bacterial resistance to the first, second and third-generation Cephalosporin’s but these are inhibited by lactamase inhibitors such as clavulanic acid. The ESBLs are typically plasmidmediated enzymes, mostly produced by E. coli and Klebsiella pneumonia Enterobacteriaceae family. ESBLs enzymes were discovered in Germany in 1983 from Klebsiella pneumonia. ESBL-producing gram negative bacteria have been responsible for numerous outbreaks of infection throughout the several counties and region challenging infection control issues. Different phenotypic test for ESBL detection have been developed, which are easy to use and cost effective. Materials and Methods: In this study, 10 Urine samples were collected from Government Hospital, Dharmapuri, Tamil Nadu. Collected clinical samples were isolated and identified by standard microbiological method. The antibiotic susceptibility testing was performed by the disc-diffusion method. The preliminary phenotypic detection of ESBLs was carried out by combination disc-diffusion test (CDDT) Performed using Kirby- bauer disc diffusion method on muller hinton agar. Results: Totally 10 clinical samples were collected and investigated Among 7 clinical samples of positive of Urinary tract infection (UTI), 20 micro-organisms belonging to 3 genus were isolated. Of the patients, 7 were female and 3 were male. E. coli and Klebsiella pneumonia were most prevalent species. E. coli and Klebsiella pneumonia were multidrug-resistant (resistance to five or more antibiotics) among the isolates. Of the isolated in Uro pathogens such as E. coli and Klebsiella pneumonia were ESBLs positive. Conclusion: Based on the results, E. coli and Klebsiella pneumonia predominated in the samples, giving the maximum frequency of ESBLs producing followed by E. coli (70%) and Klebsiella pneumonia (30%) from the combined disk diffusion method. Herbal medicine should be perform for UTI patients to prevent the spread of more. Only limited therapeutically options for ESBL-PE.

Characterization of extended-spectrum beta-lactamase-producing Enterobacterales from organic and conventional chicken meats.

This study was conducted to isolate and identify extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales in conventional and organic chicken meats, which were sold in Turkey. A total of 200 raw chicken meat sample (100 conventional and 100 organic) were used as material. Classic culture technique based on chromogenic method was used for the isolation of bacteria, and the identification was performed with VITEK MS. Phenotypic ESBL production was detected by combined disc diffusion method. Gene regions responsible for ESBL production were determined by PCR. MIC values of isolates were detected by VITEK 2. Phenotypic ESBL-producing Enterobacterales were detected in 46% of conventional chicken meats and in 22% of organic chicken meats. Of the 115 isolates obtained, 97 (84%) were Escherichia coli, 12 (10%) were Klebsiella pneumoniae, four (3·48%) were Serratia fonticola, one (0·87%) was Rahnella aquatilis, and one (0·87%) was Serratia liquefaciens. PCR analysis revealed that 109 of 115 isolates (94·78%) contained at least one of the blaCTX-M , blaTEM , and blaSHV genes. Of the 115 ESBL-producing isolates, 103 (89·57%) were found resistant to at least one antibiotic except for the β-lactam group. The contamination level of ESBL-producing Enterobacterales was higher in conventional chicken meats (P<0·001).

Bacteriological Profile and Antibiotic Susceptibility Pattern of Isolates of Wound Infection In Children Visiting Kanti Children Hospital

Objectives: The objectives of this study was to isolate and identify the bacterial etiological agent of wound infection and explore the status of methicillin-resistant Staphylococcus aureus ( MRSA), multidrug Resistant (MDR) and extended spectrum β-lactamase (ESBL) producers’ strains in clinical specimens and to find the antibiotic susceptibility pattern.&#x0D; Methods: A prospective cross sectional study design was conducted from period of February 2014 to October 2014 at Kanti Children Hospital, Kathmandu. The organisms were isolated and identified from pus sample by standard microbiological methods. Antimicrobial susceptibility test was performed by modified the Kirby Bauer disc diffusion method to evaluate the status of MRSA and MDR. ESBL detection was performed by the combined disc diffusion method.&#x0D; Results: Out of 365 specimens collected between the age group below &lt; 2 to 15 years, 210 (57.73%) samples from male patients and 155 (42.47%) from female patients. In the total samples processed, Gram-positive organisms were found to be more prevalent in which Staphylococcus aureus accounts for 135(47.20%), followed by P. aeruginosa 62 (21.67%), E. coli 29 (10.20%), K. pneumoniae 27 (9.44%), Acinetobacter spp. 20 (6.70%), P. vulgaris 7 (2.44%) and CoNS 6 (2.10%). Among the S. aureus isolates, 29 (21.48%) were found MRSA. Of the total Gram-negative organisms isolated, 74 (51.03%) were MDR and 14 (100%) ESBL producer, (P&lt;0.01). S. aureus was found to be the most important and leading cause of wound infection in this study.&#x0D; Conclusion: Thus, routine antibiotic susceptibility testing is recommended for empirical drug therapy and proper management of disease.

Phenotypic Characterization of Extended-Spectrum Beta-Lactamases and Metallo-Beta-Lactamase of Multi Drug Resistant Acinetobacter baumannii Causing Nosocomial Infections in Erbil City

Background: Resistance to broad‑spectrum beta‑lactams, mediated by extended‑spectrum beta‑lactamases, and metallo‑beta‑lactamase enzymes, is an increasing problem worldwide. The main aim is to study phenotypic characterization of extended‑spectrum beta‑lactamases and metallo‑beta‑lactamase multidrug resistant Acinetobacter baumannii in Erbil City. Materials and Methods: A total of 112 Acinetobacter baumannii isolations were collected from patients of all age groups from clinical specimens sputum, blood, pus, wound swab, urine and body fluids (Pleural fluid and cerebrospinal fluid) collected from different medical wards and intensive care unit departments of hospitals in Erbil City for a period of one year from march 2018--march 2019. Isolates were tested for the presence of extended‑spectrum beta‑lactamases and metallo‑beta‑lactamase. Detection of extended‑spectrum beta‑lactamases was done by the combined disk diffusion method according to Clinical and Laboratory Standards Institute guidelines, while metallo‑beta‑lactamase was detected by meropenem and imipenem combined with ethylenediaminetetraacetic acid disk method. Results: 25% (28) Acinetobacter baumannii isolates were positive for extended‑spectrum beta‑lactamases, while 100 % (112) were metallo‑beta‑lactamase producers. Conclusion: Acinetobacter baumannii is becoming a global medical challenge due to the emergence of multi-drug resistance. Newer beta lactamase is a matter of concern as they are developing rapidly and lead to treatment failure. Carbapenems are known to be effective therapeutic agents for Acinetobacter baumannii infections and its resistance limits the use to polymyxins and colistin. Several new medicines are still in research and combination of drug therapy is being currently used in the hospitals together with ours to treat multidrug resistant Acinetobacter baumannii infections.

Molecular ty­ping of cephalosporin resistant serovars of Salmonella enterica from poultry and farm animals

In this study, the susceptibility to cephalosporins of 74 Salmonella isolates, including 16 isolates from farm animals and 58 isolates from poultry was assessed by the disc diffusion method. ESBL production was evaluated by combined disc diffusion method (CDDM) and double disc synergy test (DDST). The genetic relatedness of isolates was investigated by the Rep-PCR method. The highest prevalence of resistance was observed against cefotaxime (27%) and the least – against cefixime (4%). None of the isolates was ESBL positive. The Rep-PCR generated 54 reproducible fingerprint patterns for all isolates and grouped them in four clusters and six singletons. Due to public health risk of cross contamination, it is important to have sufficient information on the occurrence of these resistant isolates.

Which antibiotics should we prefer empirical treatment of urinary tract infections in elderly patients?

Aim: Urinary tract infection (UTI) is a major cause of mortality and morbidity in elderly patients. In this cross-sectional study, we aimed to determine the frequency and antibiotic resistance profile of the bacteria causing UTI and contribute to the empirical treatment options in elderly patients.Methods: This study included urine culture results from 347 elderly outpatients who were referred to Karabuk Training and Research Hospital between January 2018 and June 2019. The identification and antibiotic susceptibilities of microorganisms were determined using the BD-Phoenix 100 fully automated system, and the extended-spectrum beta-lactamase (ESBL) positivity was analyzed using the combined disk diffusion method. The results were retrospectively analyzed. Results: The most common pathogens were Escherichia coli (58%), Enterococcus spp. (18%) and Klebsiella pneumoniae (11%). The rate of resistance to ampicillin, amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole (TMP-SMX), cefixime, and ciprofloxacin, which are oral antibiotics used in the treatment of UTI, was between 30% and 70%. The rate of resistance to nitrofurantoin was 3%. The gentamicin and piperacillin-tazobactam resistance were 12% and 9%, respectively. The ESBL positivity for E. coli and K. pneumoniae were 29% and 49%, respectively (P=0.03).Conclusion: The rates of resistance to oral antibiotics such as ampicillin, amoxicillin-clavulanic acid, TMP-SMX, cefixime, and ciprofloxacin, which are used treatment of UTI, were more than 20%. Therefore, these antibiotics should not be used in the empirical treatment of UTI. Instead, nitrofurantoin may be preferred in the empirical treatment of uncomplicated UTI, or gentamicin and piperacillin-tazobactam, which are parenteral antibiotics that may be used depending on the patient’s clinical condition.

Exploring the phenotype and genotype of multi-drug resistant Klebsiella pneumoniae harbouring blaCTX-M group extended-spectrum \u03b2-lactamases recovered from paediatric clinical cases in Shenzhen, China

BackgroundEmergence and spread of β-lactamase resistant Klebsiella pneumoniae have posed a serious threat, especially in paediatric patients globally. The present study focuses on explore drug resistance profile and molecular characterization of carbapenemase and extended-spectrum β-lactamase producing K. pneumoniae isolated from paediatric patients in Shenzhen, China.MethodsPresent study, a total of 31 isolates of multi-drug resistant K. pneumoniae were collected from Shenzhen Children’s Hospital, China during Jan 2014 to December 2015. ESBLs production was confirmed by using the combination disc diffusion method followed by antimicrobial susceptibility. In addition, β-lactamase encoding genes were determined by PCR assay and sequencing. The genotypic diversity and phylogenetic relationship were determined by multi-locus sequence typing (MLST) method and pulsed-field gel electrophoresis (PFGE).ResultsWe examined 31, unique K. pneumoniae isolates collected from 2014 and 2015 in Shenzhen Children’s Hospital, China. All the 31 isolates 100% were resistant to ceftazidime, ertapenem, ampicillin, cefazolin and ampicillin-sulbactam followed by ceftriaxone 94% (n = 29), aztreonam 89% (n = 26), cefepime 84% (n = 26), nitrofurantoin 75% (n = 24), piperacillin 52% (n = 16), and levofloxacin 49% (n = 15). Of the 31 β-lactamase gene coding isolates, blaCTX-M was mainly detected in about 100% (n = 31), followed by blaKPC 71% (n = 22), blaSHV 61% (n = 19), blaNDM 25% (n = 8), blaCYM 13% (n = 4), blaOXA-48 9% (n = 3), blaGES 9% (n = 3) and blaTEM 6% (n = 2). Seventeen distinct sequences type were observed with ST20 being mostly identified 16% (n = 5). Pulsed-field gel electrophoresis (PFGE) typing showed that identical profile for the isolates recovered from the Department of Intensive Care Unit and Department of Neurology of our hospital. Plasmid replicon typing result indicates the presence of IncFIS type as highest in all isolates as 61% (n = 19), followed by IncFIB 23% (n = 7), IncFIA and IncFIC 16% (n = 5) each.ConclusionOur study reports the occurrence and spread of extended β-lactamase K. pneumoniae ST20 and ST2407 for the first time, in Shenzhen, particularly in paediatric patients. To prevent and control the infection by limiting the spread of infection-causing organisms it is very crucial to detect the presence of resistant genes at an early stage.

Antimicrobial Resistance and Resistance Determinant Insights into Multi-Drug Resistant Gram-Negative Bacteria Isolates from Paediatric Patients in China.

IntroductionThe emergence of multi-drug-resistant Gram-negative bacteria (GNB) is a concern in China and globally. This study investigated antimicrobial resistance traits and resistance determinant detection in GNB isolates from paediatric patients in China.MethodsIn the present study, a total of 170 isolates of GNB including the most prevalent Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii were collected from Shenzhen Children’s Hospital, China. ESBLs production was confirmed by using the combination disc diffusion method, and carbapenemase production was confirmed by using a carbapenem inactivation method followed by antimicrobial susceptibility. In addition, β-lactamase-encoding genes and co-existence of plasmid-borne colistin resistance mcr-1 gene were determined by PCR and sequencing.ResultsOverall, 170 etiological agents (GNB) were recovered from 158 paediatric patients. The most prevalent species was E. coli 40% (n=68), followed by K. pneumoniae 17.64% (n=30), and Enterobacter cloacae 14.11% (n=24). Of 170 GNB, 71.76% (n=122) were multi-drug-resistant, 12.35% (n=21) extreme-drug resistant, and 7.64% (n=13) single-drug-resistant, while 8.23% (n=14) were sensitive to all of the studied antibiotics. The prevalence of ESBLs and carbapenemase producers were 60% and 17%, respectively. blaCTX-M was the most prevalent resistance gene (59.42%), followed by blaTEM (41.17%), blaSHV (34.270%), blaKPC (34.11%), blaOXA-48 (18.82%) and blaNDM-1 (17.64%).ConclusionThe present study provides insights into the linkage between the resistance patterns of GNB to commonly used antibiotics and their uses in China. The findings are useful for understanding the genetics of resistance traits and difficulty in tackling of GNB in paediatric patients.

Phenotypic and genotypic characterization of multi-drug-resistant Escherichia coli isolates harboring blaCTX-M group extended-spectrum β-lactamases recovered from pediatric patients in Shenzhen, southern China.

Aims and Objectives: The emergence and spread of extended-spectrum β-lactamases (ESBLs) particularly CTX-M producing multi-drug-resistant (MDR) Escherichia coli (E. coli) is one of the greatest challenges for community health globally. The study investigated the phenotypic and genotypic characteristics of ESBLs-producing E. coli recovered from pediatric patients from Shenzhen Children’s Hospital, China.Materials and methods: Present study, a total of 2,670 isolates of E. coli were collected from Shenzhen Children’s Hospital, China of which 950 were ESBLs producer. ESBLs production was confirmed by using the combination disc diffusion method, and antimicrobial susceptibility test was detected. In addition, β-lactamase-producing genes and co-existence of carbapenem/colistin resistance genes were determined by PCR assay and sequencing. The diversity and phylogenetic relationship were determined by multi-locus sequence typing method.Results: Thirty-five percent (n=950) prevalence of ESBLs-producing E. coli we reported in Shenzhen, China of which 50 ESBLs producing E. coli were randomly selected for a further characterization. All 50 ESBLs- producing E. coli isolates revealed MDR phenotype and 100% were resistant to Ampicillin/sulbactam, Ampicillin, Cefazolin, and Ceftriaxone. All 50 ESBLs producers harbored at least one type of β-lactamase gene particular blaCTX-M. The PCR and sequencing revealed the most common CTX-M subtype was blaCTX-M-15 (n=18), followed by blaCTX-M-14 (n=16), blaCTX-M-90 (n=9), blaCTX-M-55 (n=3), blaCTX-M-27, blaCTX-M-101, and blaCTX-M-211 each (n=1). Co-existence of blaCTX-M with blaTEM, blaSHV, blaGES, and blaVEB was detected in few isolates. Among identified sequence types, ST131 (12%) was more dominant in ESBLs-producing E. coli. Phylogenetic group A was the most prominent group among the ESBLs-producing E. coli based on multiplex PCR.Conclusion: Our study shows the prevalence of blaCTX-M gene in ESBLs-producing E. coli in pediatric patients in Shenzhen, China. We highlight the importance to monitor the emergence and trends of ESBLs-producing isolates in a pediatric healthcare setting.

Detection of different &amp;beta;- lactamases and their co-existence in gram negative bacteria isolated from clinical samples at a Tertiary care centre

Detection of different β- lactamases and their co-existence in gram negative bacteria isolated from clinical samples at a Tertiary care centre - IJMR- Print ISSN No: - 2394-546X Online ISSN No:- 2394-5478 Article DOI No:- 10.18231/2394-5478.2019.0013, Indian Journal of Microbiology Research-Indian J Microbiol Res

CHARACTERIZATION OF VARIOUS VIRULENCE PROPERTIES OF DRUG-RESISTANT KLEBSIELLA ISOLATES: AN IN VITRO STUDY AMONG “SUPERBUGS”

Objective: The objective of this study was to evaluate the various virulence factors among all extended-spectrum beta-lactamase (ESBL)-producing Klebsiella isolates.Methods: Klebsiella species (n=201) isolated from various non-repeated clinical samples from October 2016 to May 2017 were collected and identified by biochemical method and confirmed by BD Phoenix. Antibiotic susceptibility test was performed by Kirby-Bauer disc diffusion method, and ESBL detection was done by combined disc diffusion method. Capsule detection was done by nigrosin dye staining, hypermucoviscosity was done by string test, biofilm was performed by microtiter plate assay, and hemagglutination was performed with human erythrocytes treated with tannic acid.Results: Among 201 ESBL-producing Klebsiella isolates, 63.2 % were multidrug resistance and 24.4% were extensively drug-resistant. Among these isolates, Klebsiella infection is more in older patients. All isolates were (100%) positive for capsule and siderophores production. Of all 201 isolates, 14 (7%) were strong, 61 (30.3%) were moderate, and 104 (51.8%) were weak biofilm producers. Among these isolates, 36 (17.9%) were type 1 fimbriae, 140 (69.7%) were type 3 fimbriae, and only 17 (8.5%) were positive for hypermucoviscosity.Conclusion: Several virulence factors were studied, in which biofilm with fimbriae is the main cause for the resistance of antibiotics. Bacterial resistance profile found to be high with ESBL production and multiple drug resistance in the majority of the Klebsiella isolates, which is a result of improper antibiogram schedule and their virulence property.

An Outbreak of NDM-1-Producing Klebsiella pneumoniae, Associated with OmpK35 and OmpK36 Porin Loss in Tunisia.

To describe clinical and molecular characteristics of an outbreak due to metallo-β-lactamases (MBLs) producing Klebsiella pneumoniae collected at Charles Nicolle Hospital of Tunis and to analyze the impact of outer membrane porin (OMP) loss on carbapenem resistance levels. Between 2010 and 2015, 178 carbapenem-resistant Enterobacteriaceae were isolated. Screening for MBL production was performed using combined disk diffusion method, with imipenem and ethylene diamine tetraacetic acid (EDTA) as inhibitors. Resistance genes and virulence factors were identified by polymerase chain reaction (PCR) and sequencing. Genotyping was performed by pulsed-field gel electrophoresis and multilocus sequence typing. Genetic environment of carbapenemase genes was determined by PCR mapping. Conjugation assays were performed, and plasmids were assigned to incompatibility groups by PCR-based replicon typing. OMPs were profiled by sodium dodecyl sulfate-polyacrilamide gel electrophoresis, and porin genes were sequenced. Nineteen K. pneumoniae (10.6%) showing MBL activity were isolated from patients hospitalized on four different wards. NDM-1 was the only MBL identified, in association with blaOXA-48. All strains lacked at least one OMP, and carbapenem resistance levels were remarkably elevated in strains lacking OmpK35 and OmpK36. blaNDM-1 was located in IncFIA-type conjugative plasmid, with the same genetic context in all strains. The epidemiological diffusion of blaNDM-1 was due to two clones, one major clone belonging to sequence type (ST) 147 (n = 16) and the other clone belonging to ST307 (n = 3). This study describes an outbreak of NDM-1-producing K. pneumoniae strains, isolated from a Tunisian hospital, caused by two clones belonging to ST147 and ST307; and highlights the role of OMPs loss, in combination with β-lactamase expression, in conferring high carbapenem resistance.

Prevalence of Extended Spectrum Beta-Lactamase Producing Escherichia coli and Klebsiella species from Urinary Specimens of Children attending Friendship International Children’s Hospital

Extended-spectrum β-lactamase producing E. coli and K. pneumoniae is a serious threat to the patients. These organisms are major extended spectrum beta lactamase (ESBL) producers. The objective of this study was to determine the prevalence of Extended spectrum β- lactamase producing strains of Escherichia coli and Klebsiella spp isolates from the urine sample of children visiting International Friendship Children Hospital. During the seven months, between June 2016 to December 2016, 1018 mid-stream urine samples(MSU) were collected from patients suspected of having UTI. The samples were investigated by conventional semi-quantitative culture technique and identification of E. coli and Klebsiella spp. was done by microscopy and biochemical test. Antibiotic susceptibility test of isolates was performed by modified Kirby Bauer Disc diffusion test. ESBL screening test was done by using 3rd generation Cephalosporin and confirmation done by combination disc diffusion method. Out of total 1018 MSU samples investigated, 200(19.64%) isolates of E. coli and 28(2.7%) isolates of Klebsiella spp. making a total of 228(22.39%) were found to cause significant bacteriuria. 76(33.33%) isolates, from those causing significant bacteriuria, were Multi-drug resistant organisms. Out of 228 isolates, 54(23.68%) were ESBL producers, that includes 51(25.5%) Escherichia coli and 3(12.5%) Klebsiella pneumoniae. ESBL producers were more common in in-patient (36.17%) than out-patient (20.44%). Most of the ESBL producers were resistance to amoxicillin, followed by Cotrimoxazole and Ciprofloxacin respectively. They were highly sensitive to Imipenem, Tigecycline, Amikacin, Piperacillin-Tazobactam, and Nitrofurantoin. High prevalence of ESBL producing E. coli and Klebsiella pneumoniae was found among children. Regular and routine monitoring of ESBL producing isolates is essential.Nepal Journal of Biotechnology. Dec. 2017 Vol. 5, No. 1: 32-38