Double Disk Diffusion Method Research Articles

Frequency of extended spectrum beta lactamase in E.CoIi and klebsiella pneumoniae in bacterial cultures.

Objective: To determine frequency and pattern of extended spectrum beta lactamase in Klebsiella pneumoniae and E. coli in bacterial cultures. Study Design: Cross Sectional study. Setting: Department of Pathology Sahara Medical College Narowal. Period: January to June 2020. Material & Methods: Total 1100 bacterial isolates were studied out of them 655 E.coli and 445 of klebsiella pneumoniae. All samples were subjected to double disc diffusion method using third generation cephalosporin and amoxacillin-clavulanic acid for detection of ESBL. Data was analyzed using SPSS software version 24, and results were calculated in the form of frequency, percentage and standard deviation. P-value ≤0.05 was taken as statistically non-significant. Results: There were 48.9% male and 51.1% female subjects out of 1100 total cases. E.coli was detected in 59.5% and klebsiella in 40.5% samples. Total 44.2% samples were positive for ESBL enzyme. Of 655 E.coli samples 40.8% and of 445 Klebsiella pneumoniae samples 49.2% were positive for ESBL enzyme. Conclusion: It is necessary to detect ESBL positive Klebsiella pneumoniae and E.coli in laboratory workflow to avoid unnecessary use of antibiotics and development of resistance against them.

Screening of Clostridium difficile Isolates for Metallo-Resistant Gene and Extended Spectrum Beta-Lactamases

Clostridium difficile naturally inhabits intestinal tract of animals including humans. Spores of C. difficile are known contaminant in the Environment. Indiscriminate use of antibiotics has led to the emergence of a powerful resistance determinant (known as Metallo resistant genes) in many bacteria worldwide. Multidrug resistant bacteria (MRB) pose threats to health globally. This study aims to determine the distribution of Metallo resistant Clostridium difficile in hospital environment within Yola. A total of 150 surface samples from the hospital wards were collected from Federal Medical Centre, Yola (FMC) and Specialist hospital, Yola (SHY) Adamawa state, Nigeria. Clostridium difficile were recovered by inoculating samples in modified Cycloserine-cefoxitin fructose broth (CCFB) and Cycloserine-cefoxitin fructose agar (CCFA). Screening of isolates for extended spectrum β-lactamase (ESBL) and metallo-β-lactamase enzymes was done by double disc diffusion method and Imipenem-EDTA combined disc test respectively. A total of 18 C. difficile isolates were obtained and found to be extended spectrum β-lactamase (ESBL) positive. 16 (88.89 %) of the Clostridium difficile were found to be metallo-β-lactamase positive. Polymerase chain reaction showed that 11(61.1 %) of the isolates possess bla-IMP metallo resistant gene. This study suggests that multidrug resistant C. difficile with metallo resistant genes are wide spread in hospital settings and there is need for relevant authorities to improve on the sanitization of environment as well as continuous surveillance to hinder C. difficile transmission.

Phenotypic Characterisation of Klebseilla pneumoniae Carbapenemase and Metallo Beta-lactamase in Carbapenem Resistant Gram-negative Bacteria

Background: Antibiotic resistance is an emerging concern in the therapy of clinical infections worldwide. Previous studies conducted in our laboratory have confirmed an increase in the prevalence of extended spectrum beta-lactamase (ESBL) among the Gram-negative bacterial pathogens associated with dogs, which could act as a potential source for the transfer of these resistant pathogens or their genetic determinants to human. Since carbapenems are the last resort drugs against these resistant pathogens, the study was aimed to isolate and characterise carbapenem resistance among Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa) associated with common clinical infections in dogs.Methods: A total of 100 samples were collected from lesional skin, urine and anterior vagina of dogs presented to the Veterinary Hospitals of Kerala Veterinary and Animal Sciences University at Mannuthy and Thrissur. The samples were cultured onto Brain Heart Infusion Agar (BHIA), Eosin Methylene Blue (EMB) and Mac Conkey (MAC) for isolation of bacteria. Identification of the isolates was performed based on cultural, morphological and biochemical characteristics. The isolates were subjected to antimicrobial susceptibility test (ABST) against the 12 commonly used beta-lactam and non–beta-lactam group of antibiotics by disc diffusion method and further subjected to screening for ESBL double disc diffusion method. Carbapenem-resistant isolates were subjected to phenotypic confirmatory test for carbapenemase production employing Imipenem-EDTA and Ertapenem-boronic acid minimum inhibitory concentration (MIC) strip method.Result: Forty four Gram-negative bacterial isolates obtained were viz., E. coli (30), K. pneumonia (11) and P. aeruginosa (3) from the 100 samples. Apart from these, other isolates obtained were Staphylococcus spp. (53) and Bacillus spp. (2). All the Gram-negative isolates were subjected to ABST employing 12 common antibiotics belonging to beta-lactam and non-beta-lactam groups. Multidrug resistance (MDR) could be observed in 28 E. coli, 11 K. pneumoniae and three P. aeruginosa isolates. All the 42 MDR isolates showed positive results for ESBL production. A total of 14 isolates out of the 44 Gram-negative bacilli were found to be resistant to carbapenem either to imipenem, meropenem or ertapenem. Among the 14 Gram-negative isolates, nine turned out to be positive for metallo-beta-lactamase (MBL) and none for K. penumoniae carbapenemase (KPC) on phenotypic confirmatory test for detecting major carbapenemase enzymes. The present study documented that Gram- negative bacteria like E. coli, K. pneumoniae and P. aeruginosa isolated from dogs are showing an increase rate of resistance against carbapenems which are the last resort drugs against ESBL producers. Hence, there is an urgent need to curb the irrational and excessive use of antibiotics in veterinary sector.

Antimicrobial resistance patterns and phylogenetic analysis of Shiga toxin-producing Escherichia coli strains from goats using both Clermont phylogenetic schemes

Foodborne transmission of Shiga toxin-producing Escherichia coli (STEC) poses a threat to public health. The Clermont typing schemes (previous and revised) have been used widely to phylotype E. coli. The present study was conducted to compare the relationship of the Clermont phylogenet-ic schemes in STEC strains isolated from goats and antibiotic resistance patterns in the southeast of Iran. Overall 52 strains carrying the stx gene were used for subsequent analysis. All strains were determined by analysing the genomic DNA with a PCR-based method using the two Clermont et al. (2000, 2013) schemes. Extended spectrum beta-lactamase (ESBL) producing strains were con-firmed by the double disk-diffusion method. STEC strains were also tested for susceptibility to 20 antimicrobials agents. In the original Clermont method, the prevalent phylogroups were B1 (69.2%) and A (28.8%). The significant phylogenetic groups of strains according to the revised Clermont method were B1 (82.7%), A (13.5%) and unknown (3.8%). However, STEC strains underwent changes as noted from A to B1 (17.3%), B1 to unknown (3.8%), B1 to A (1.9%) and D to B1 (1.9%) groupings. Of the 52 stx-positive strains, two ESBL producing strains were detected. Susceptibility data showed that the most frequent resistance phenotype was related to cefazolin (90.4%), streptomycin (88.5%), ampicillin (86.5%) and oxytetracycline (82.7%) respectively. Alt-hough the overall frequency of the reassigned phylotypes was not significant, most changes oc-curred within the A phylotype. Therefore, implementation of the new method on isolates belong-ing to the A phylotype in the old method seems to be necessary to obtain accurate results.

Occurrence and Characteristics of Extended-Spectrum β-Lactamase-Producing Escherichia coli from Dairy Cattle, Milk, and Farm Environments in Peninsular Malaysia.

The emergence and spread of antimicrobial resistance genes and resistant bacteria do not recognize animal, human, or geographic boundaries. Addressing this threat requires a multidisciplinary approach involving human, animal, and environmental health (One Health) sectors. This is because antimicrobial agents used in veterinary medicine have been reported to be the same or similar to those in human medicine use. Extended-spectrum β-lactamase (ESBL) E. coli is a growing public health problem worldwide, and the agri-food industry is increasingly becoming a source of clinically important ESBL bacteria. Accordingly, the aim of this study was to investigate the occurrence and characteristics of ESBL-producing E. coli from dairy cattle, milk, and the farm environment. E. coli isolates were identified by their 16sRNA, and their ESBL production was confirmed using ESBL–CHROMagar media and the double disk diffusion method. Genotypes of ESBL producers were characterized using multiplex polymerase chain reaction (mPCR) assay. It was found that 18 (4.8%) of the total samples were positive for ESBL-producing E. coli. Of these, 66.7% were from milk, and 27.8% and 5.5% were from the farm environment and faecal samples, respectively. Predominant ESBL genotypes identified were a combination of both TEM and CTX-M in eight out of 18 (44.4%) isolates. Four (22.2%) isolates produced the CTX-M gene, two (11.1%) isolates produced the TEM gene, and four (22.2%) remaining isolates produced the ESBL genes other than TEM, SHV, CTX-M, and OXA. The SHV and OXA gene were not detected in all 18 isolates. In all, there were four profiles of genetic similarity. The occurrence of these genotypes in indicator organisms from dairy cattle, milk, and the farm environment further re-enforced the potential of food-animals as sources of ESBL-producing E. coli infection in humans via the food chain. Thus, there is the need for the adoption of a tripartite One Health approach in surveillance and monitoring to control antimicrobial resistance.

Prevalence of inducible clindamycin resistance among Staphylococcus aureus isolates from a tertiary care hospital

Clindamycin is an excellent drug for skin and soft tissue Staphylococcus aureus infections, but resistance mediated by inducible macrolide-lincosamide-streptogramin B (iMLS)phenotype leads to in vivo therapeutic failure even though they may be in vitro susceptible in Kirby–Bauer disk diffusion method (KBDDM). Hence the study was undertaken to detect the prevalence of iMLS phenotype among Staphylococcus aureus isolates by double disk approximation test (D-test) in a tertiary care hospital. A total of 100 consecutive Staphylococcus species isolates were identified by standard microbiological methods and subjected to antimicrobial susceptibility testing by KBDDM. Clindamycin-resistance either in the form of iMLSB or cMLSB was determined through double disk diffusion method or D-test by using erythromycin (2 μg) and clindamycin (15μg) as per the CLSI guidelines. Out of 100 Staphylococcus species studied, 50(50%) were methicillin sensitive Staphylococcus aureus, 30(30%) were Methicillin resistant Staphylococcus aureus and 20 (20%) were Coagulase negative Staphylococci. Out of 80 Staphylococcus aureus studied, iMLSB, cMLSB and MS phenotype were 32.5%, 1.25%, 5% respectively. Inducible resistance and MS phenotype were found to be higher in MRSA as compared to MSSA (60%, 6.66% and 16%, 4% respectively). The study revealed 32.5% of Staphylococcus aureus isolates were inducible clindamycin resistant, which could be easily misidentified as clindamycin susceptible in Kirby–Bauer disk diffusion method. Therefore, clinical microbiology laboratory should routinely perform D-test in all clinically isolated Staphylococcus aureus to guide clinicians for the appropriate use of clindamycin.

Assessment of different methods for diagnosis of Group B streptococci during pregnancy

Objectives: To compare the different diagnostic techniques used to detect GBS colonization in pregnant women in late third trimester after thirty five weeks and to detect the frequency of GBS colonization among a sample of pregnant Egyptian women. Patients and methods: Vaginal swabs from the lower third of vagina were collected from 100 pregnant women in the late third trimester. Isolation of the organism by culture on selective media and confirmation by latex agglutination test and detection of CAMP factor by conventional PCR were compared. GBS isolates were tested by double disk diffusion method and D-zone test simultaneously for susceptibility to erythromycin and clindamycin and inducible clindamycin resistance for intrapartum antibiotic prophylaxis (IAP). Results: 25 participants (25%) were positive for GBS by culture in Lim broth with subculture onto TSA supplemented with 5% defibrinated sheep blood, while 75 participants (75%) were negative. Of the 25 GBS isolates, 19 (76%) were sensitive to erythromycin, 3 (12%) were intermediate and 3 (12%) were resistant. Of the 25 GBS isolates, 15 (60%) were sensitive to clindamycin, 2 (8%) were intermediate and 8 (32%) were resistant. Fourteen isolates (56%) were sensitive to both erythromycin and clindamycin whereas 3 (12%) were resistant to both (cMLSB). Latex agglutination test for GBS detection from the 24 hours incubated Lim broth was positive in 25 cases (25%). GBS was detected in 9 cases (9%) by the conventional PCR assay done directly from vaginal swabs specimens. Sensitivity, specificity, PPV and NPV for latex agglutination from the inoculated broth and PCR assay are 100%, 100%, 100%, 100% and 36%, 100%, 100%, 82.4% respectively. Latex agglutination test from the inoculated broth showed a statistically significant perfect agreement (100.0%) with culture with Kappa value 1.0 and 95% CI (1.0 – 1.0). PCR assay also showed a statistically significant but moderate agreement (84.0%) with culture with Kappa value 0.458 and 95% CI (0.253 – 0.662). Conclusion: Detection of GBS colonization by latex agglutination test from incubated selective broth directly is comparable to the gold standard (culture) as regards accuracy. PCR offers a rapid and highly specific method for detection of GBS colonization especially in intrapartum settings for administration of IAP in non-screened pregnant females; however, sensitivity is low resulting in a low NPV.

High fecal carriage of extended Beta Lactamase producing Enterobacteriaceae among adult patients admitted in referral hospitals in Dar es Salaam, Tanzania

BackgroundMulti-drug resistance pathogens such as Extended-Spectrum Beta-Lactamase (ESBL) producing Enterobacteriaceae (ESBL-PE) are of great global health concern, since they are associated with increased morbidity and mortality. Even in the absence of infections caused by these pathogens, colonization is a great threat and can lead to cross transfer among hospitalized patients. To date data on carriage of these pathogens is still limited in Tanzania. Therefore, this study aimed to determine ESBL-PE fecal carriage rate and associated factors among hospitalized patients at Referral hospitals in Dar es Salaam.MethodsThis was a cross sectional study conducted from May to July 2017 among patients admitted in three referral hospitals in Dar es Salaam, Tanzania. Rectal swabs were collected and screened for ESBL production using MacConkey agar supplemented with Ceftazidime 2 μg/ml. Phenotypic confirmation of ESBL-PE was done by double disk diffusion method. Statistical analysis was performed using Statistical Package for Social Sciences (SPPS) software version 20.ResultsOf the 196 enrolled participants, 59.7% (117/196) were confirmed to carry ESBL-PE. Diarrheic patients (57/79) had statistically significant high prevalence of ESBL colonization compared to those without diarrhea (60/117) (p = 0.01). A total of 131 ESBL-PE were isolated from 117 patients, whereby, Escherichia coli accounted for 68.7%, Klebsiella pneumoniae 28.2% and Citrobacter species 0.8%. ESBL-PE carriage was significantly higher in patients with diarrhea compared to those without diarrhea (72% vs 53.1%, p = 0.01). Recent antibiotic use was independently associated with carriage of ESBL-PE (aOR 14.65, 95%CI 3.07–69.88, p = 0.01).ConclusionsHigh prevalence of fecal carriage of ESBL-PE was observed in patients admitted in tertiary hospitals in Dar es Salaam, Tanzania. The use of antibiotics was associated with carriage of ESBL producers among the study population.

Clinico-microbiological study of community acquired MRSA from skin and soft tissue infections and its antibiogram in a tertiary care hospital in Karnataka

Methicillin resistant staphylococcus aureus (MRSA) has always been a worldwide problem, although its prevalence varies considerably among countries. The epidemiology of MRSA has changed over the years and infections are no longer confined to the hospital setting, but appear in healthy community dwelling individuals with no risk factors. From skin and soft tissue infection, common organism isolated is Community associated MRSA (CA-MRSA). Hence the study was done to know the prevalence of MRSA among community associated skin and soft tissue infections in Basaveshwar teaching and general hospital, attached to M.R. Medical College, Gulbarga. Standard techniques were used to isolate Staphylococcus aureus from clinical specimens. Cefoxitin disc diffusion was used to find MRSA. Antibiogram of MRSA was detected by Kirby Bauer disc diffusion method. Inducible Clindamycin resistance was done by Double Disc Diffusion method (D test). From over 200 cases of CA-MRSA, total of (75.5%) staphylococcus aureus was detected. Out of them, CA-MRSA was 27 (17.9%). These showed high sensitivity to Vancomycin (100%), Linezolid (96.2%),Cindamycin (92.59%), moderately susceptible to TMP-SMX(85.1%), Rifampicin (88.1%), Tetracycline(81.4%),Gentamicin (70.3%), ciprofloxacin(62.9%) and a low susceptibility to Erythromycin (48.14%). 14.8% of CA-MRSA strains were D test positive (inducible MLS positive) and 29.63% were D test negative (MS phenotype). 7.4% of CA-MRSA were positive for constitutive MLS resistance. There is a need for judicious selection of antimicrobial agents, as their indiscriminate use can exert pressure in selecting MRSA and other multi-drug resistant organisms. Further spread of community acquired infections can be done by effective infection control programs.

Clinical Correlation, Isolation, Speciation, and Antibiotic Susceptibility Pattern of Tribe Proteeae from Various Clinical Samples

Introduction: Organisms belonging to Tribe Proteeae are usually present as commensals in the environment but can cause urinary tract and wound infections. There is a need for regular studies on sensitivity pattern of various bacteria causing infections to decide a suitable empirical therapy and there are not many studies on Tribe Proteeae. Aim: To clinically correlate, isolate, speciate and determine the antibiotic susceptibility pattern of Tribe Proteeae from various clinical samples. Materials and Method: All samples were cultured on standard culture media as per routine protocol. Isolates were identified by standard biochemical reactions. Antibiotic sensitivity was done by Kirby Bauer’s disk diffusion method as per CLSI guidelines. ESBL production was detected by double disk diffusion method using ceftazidime (30µg) disk and ceftazidime/clavulanic acid (30/10µg) disk. Clinical data was collected from the record section of the hospital. Results: Sixty samples yielded bacteria belonging to Tribe Proteeae. Most of the patients belonged to male gender 46 (76.7%) and >50 years 38 (63.3%) of age. P mirabilis was the predominant isolate 42 (70%). Maximum rate of isolation of Proteus spp was from diabetic foot ulcer 21 (35%). The predisposing conditions were diabetes mellitus, tracheostomy cases and only one case of carcinoma tongue. Most of our isolates were sensitive to various drugs tested. There were only three (5%) ESBL producers. Most of the isolates of P.mirabilis were sensitive to antibiotics than P vulgaris isolates. Conclusion: Maximum rate of isolation of tribe Proteeae was from diabetic foot ulcer cases. P.mirabilis was the predominant isolate and was more sensitive to the antibiotics tested than P.vulgaris.

Molecular characterization of STEC isolated from Ducks and its relation to ESBL production

The ESBL producing genes are responsible for bacterial resistance to number of antibiotics whereas Shiga toxin producing genes are responsible for bacterial virulence. The association between ESBL producing genes and Shiga toxin producing E. coli (STEC) may pose bigger threat in the battle of antibiotic resistance. This study was conducted to determine the prevalence of Shiga-toxin-producing Escherichia coli (STEC) in ducks reared in organized and unorganized sectors from different agro climatic zones of West Bengal, India and to study their relationship with extended spectrum beta-lactamase (ESBL) production. Total 202 cloacal swab samples were collected from both indigenous ducks reared in backyards sector (110 samples) and Khaki Campbell Ducks reared in organized farm (92 samples). Initially the samples were screened for detection of E. coli on the basis of their cultural, morphological and biochemical properties followed by PCR confirmation for E. coli 16S rRNA. E. coli isolates were subjected to multiplex PCR to detect the presence of shiga toxin producing genes such as stx1, stx2, eaeA and ehxA. STEC isolates were screened phenotypically for production of ESBL and ACBL by double disk diffusion method and subsequently PCR detection for blaCTX-M, blaTEM, blaSHV and blaAmpC genes were performed. Serotyping of all the STEC was also done. Out of 202 samples total 109 were confirmed to be E. coli positive. Out of them total 27 (24.77 %) E. coli isolates were detected to be positive for STEC. Higher prevalence of STEC was observed in unorganized sector compared to the organized sector. Positive association (P < 0.05) was observed between STEC and ESBL production. This study indicates that the duck may play an important role in transmission of Siga-toxin-producing E. coli (STEC) as well as antibiotic resistance genes to human beings, other birds, animals and environment also.

MOLECULAR EPIDEMIOLOGY AND HOSPITAL SPREAD OF CEFTRIAXONE NON‐SUSCEPTIBLE ENTEROBACTERALES IN THE SUNSHINE COAST REGION, AUSTRALIA

Background: Extended spectrum beta‐lactamase (ESBL) and AmpC‐producing Enterobacterales are prevalent worldwide. Poor clinical outcomes, including death, are more common in patients both colonised and infected with these organisms1. Australia is thought to have a comparatively low prevalence of such organisms, however no clear continuous surveillance system exists2. Specific ESBL type has shown significant geographic variation, having different therapeutic and infection control implications for different regions3. Little is known about the prevalence, molecular type and spread of ESBL and AmpC‐producing Enterobacterales among hospitalised patients in the Sunshine Coast region. Aims: To (i) Determine the prevalence and molecular epidemiology of ESBL and AmpC‐producing Enterobacterales, (ii) Develop an in‐house multiplex polymerase chain reaction (PCR) assay to detect common ESBL genes, (iii) Compare our PCR assay to the double disc diffusion method for detection of ESBLs, and to (iv) Establish, with whole genome sequencing (WGS), whether transmission was occurring in our intensive care unit (ICU). Methods: Clinical specimens from adult and paediatric patients culturing Enterobacterales resistant to a third‐generation cephalosporin were collected prospectively from July 2017 to August 2018 at the Sunshine Coast University Hospital laboratory. Antibiotic susceptibility testing was determined using VITEK‐2®. A validated in‐house pentaplex PCR assay was developed and used to detect common beta‐lactamase genes (CTX‐1, CTX‐9, SHV, TEM, CMY). All isolates collected from patients admitted to the ICU were identified and underwent WGS analysis using the Illumina® platform. Results: 295 clinical specimens were identified. Escherichia coli (78.8%), Klebsiella pneumoniae (16.8%) with 1‐2% consisting of Enterobacter spp., Serratia marcescens and Citrobacter freundii. Specimens included urine (53.8%), rectal swab (39.4%), pus (3.0%) and blood (2.4%). Carbapenem resistance was observed in 3.3%. ICU patient specimens made up 18.2%. The majority (87.8%) of isolates had at least one gene identified on PCR testing of which 48.5% produced CTX‐1, 31.2% produced CTX‐9, 15.9% produced SHV, 12.5% produced CMY and 40.6% produced TEM. Approximately forty percent of isolates produced more than one ESBL with the most common being CTX‐1 and TEM coproduction. A small amount (<5%) of isolates were negative on PCR testing. Fourteen (4.7%) isolates that were negative on double disc diffusion had ESBL genes detected by our PCR assay. WGS of non‐identical ICU patient E. coli (n = 44) and K. pneumoniae (n = 10) isolates revealed no strong evidence for transmission of these organisms between patients during this time period, with the smallest single nucleotide polymorphism (SNP) difference being 300 SNPs. Conclusion: Multidrug resistant Gram‐negative organisms are present on the Sunshine Coast and cause significant morbidity. Our real‐time pentaplex PCR assay was able to successfully identify common beta‐lactamase genes, revealing a frequency distribution among our organisms similar to that noted in previous Australian studies (CTX‐1 group most commonly identified). We were not able to identify evidence of ICU transmission, which recognises the success of current infection control practices at our institution.

Detection of Clindamycin Resistance Antibiotic Genes among Staphylococcus Isolates by Using Real Time PCR

STAPHYLOCOCCUS aureus is the most common pathogens and an important nosocomial pathogen. This bacterium has ability to acquire resistance to different antibiotic classes. Erythromycin and clindamycin have been used for treating skin and soft-tissue infections caused by these bacteria. Although, expression of macrolide-lincosamide-streptogramin B resistance (MLSB) can limit the effect of these drugs.This study aimed to detect the presence of genes encoding resistance to macrolides, lincosamides and streptogramins (MLSB) among clinical Staphylococcus strains.A total of 100 Staphylococcus isolates from clinical specimens (blood, sputum and wound) were collected. All these isolates were identified biochemically. They were tested against different antibiotics and double-disk diffusion method and genes were detected using PCR.Results show that 63 isolates were genotypically tested by using real time PCR for detection of Erm (B) and Erm (C) genes. (EryS ClinS) was detected in 50 (50%) of the isolates followed by constitutive phenotype of MLSB resistance (EryR ClinR) 29 (29%) and inducible MLSB resistance (EryRClinInd) 17 (17%), while the MSB phenotype (EryR ClinS) 4(4%) was the least frequent. 51(80.9) isolates have Erm (B) gene positive 33(52.3) strains have Erm (C) gene positive.This study investigate that the double disk diffusion test is a suitable test for detection of staphylococci which are resistant to clindamycin and should be used as fast detection and help in treatment of patients. And thus, the screening procedure using Real time PCR is sensitive in detection of Erm genes which are responsible for inducible resistance to clindamycin.

High clonal diversity of ESBL-producing Klebsiella pneumoniae isolates from clinical samples in a non-outbreak situation. A cohort study

BackgroundKlebsiella pneumoniae has been responsible for a large number of clonal hospital outbreaks. However, some epidemiological changes have been observed since the emergence of CTX-M enzymes in K. pneumoniae.AimTo analyse the transmission dynamics of Extended Spectrum β-Lactamase-producing Klebsiella pneumoniae (ESBL-Kp) in an acute care hospital.MethodsIn 2015 a prospective cohort study was conducted. All new consecutive adult patients with ESBL-Kp isolates in all clinical samples were included. Patients with a previous known infection/colonization by ESBL-Kp and patients in high risk areas (e.g., intensive care units) were excluded. Cross-transmission was defined as the carriage of a clonally-related ESBL-Kp between newly diagnosed patients who shared the same ward for ≥48 h with another case, within a maximum time window of 4 weeks. ESBL-production was confirmed using the double-disk diffusion method and PCR. Clonal relationships were investigated by rep-PCR and multilocus sequence typing (MLST).ResultsSixty ESBL-Kp isolates from 60 patients were included and analysed. Infections and colonizations were classified as hospital-acquired (52%), healthcare-related (40%) or community-acquired (8%).High genetic diversity was detected. When epidemiological clinical data were combined with the rep-PCR, the patterns identified did not show any cases of cross-transmission. ESBL-Kp were detected in 12.5% of environmental samples. No clonal relationship could be established between environmental reservoirs and patients. The genetic mechanism detected in all strains was associated with blaCTX-M genes, and 97% were CTX-M-15.ConclusionsThe dynamics of ESBL-K. pneumoniae isolated in our setting could not be explained by clonal transmission from an index patient. A polyclonal spread of ESBL-Kp was identified.

Limitation of Double Disc Diffusion Method as a phenotypic confirmatory method for ESBL producing organisms in Clinical Microbiology Laboratory

Limitation of Double Disc Diffusion Method as a phenotypic confirmatory method for ESBL producing organisms in Clinical Microbiology Laboratory

Inducible resistance to clindamycin in methicillin-resistant Staphylococcus aureus isolated from Paraguayan children

The double disc diffusion method is an alternative diagnostic that allows the identification of Staphylococcus aureus isolates apparently susceptible to clindamycin but that may develop resistance due to an induction phenomena, mainly asociated to the increase in resistance to methicillin, thus increasing the possibility of failure in the treatment. To determine the frequency of induced clindamycin resistance in methicillin-resistant S. aureus (MRSA) isolated from Paraguayan children. In this cross sectional study, we collected 145 S. aureus isolates that caused skin and soft tissue and osteoarticular infections in pediatric patients of the Central Hospital I.P.S. in the period from December-2012 to November-2013. Resistance to clindamycin was determined by automated methods and double disc diffusion. PCR was performed for ermA, ermB, ermC and msrA genes from representative isolates. The global resistance to methicillin and clindamycin was 67 and 13%, respectively (11% attributable to the inducible mechanism). The ermC and msrA genes were detected individually in 25 and 17% of the isolates respectively while an isolate presented both genes simultaneously. The frequency of inducible resistance to clindamycin indicates the importance of double disc diffusion methods in microbiological practice, as well as being within the cut off points considered acceptable for the use of this antibiotic for skin infections. and osteoarticular caused by MRSA.

Epidemiology of resistance and phenotypic characterization of carbapenem resistance mechanisms in Klebsiella pneumoniae isolates at Sahloul University Hospital-Sousse, Tunisia.

To assess the prevalence of ESBL producing and carbapenem resistant Klebsiella pneumoniae isolated from in-come and out-come patients at Sahloul-university hospital. A retrospective study over a 3 years period (January 2012 and December 2014) focused on 2160 strains of Klebsiella pneumoniae. Statistical analysis was carried out using SPSS program. ESBL detection was performed using a double disc diffusion method and carbapenemase detection was realized by Rosco-Disk kit. A total of 2160 Klebsiella pneumoniae strains were isolated during the period of the study, 26.2% (n=566) were ESBL-producers and 15.8% (n=342) showed resistance to carbapenem. The wards most affected by these strains were basically urology and intensive care units. Eighty four percent of studied strains (203/241) were resistant to temocillin, which correlate with the production of a class D (OXA-48-like) carbapenemase and 7% (17/241) showed sensitivity to EDTA and dipicolinic acid, which indicate the production of metallo-enzyme. The rate of resistance to colistin remains low. Resistance of Enterobacteriaceae, including K. pneumoniae, to third generation cephalosporins (3rd GC) and carbapenem through the mechanism of ESBL and carbapenemases production is becoming increasingly worrying. This suggests a more rational use of antibiotics, as well as the rigorous application of hygiene measurement.

Characterization and Prevalence of Clindamycin Resistance Staphylococcus aureus from Clinical samples of National Medical College and Teaching Hospital, Nepal

Objective: Clindamycin is the drug of choice for the treatment of severe form of skin, soft tissue, and blood infections caused by resistant Staphylococcus aureus in the form of methicillin-resistant S. aureus (MRSA) and erythromycin-resistant S. aureus. In this research, we determine the susceptibility pattern of isolated S. aureus strains against antibiotics and the prevalence of resistant S. aureus in the form of MRSA, inducible clindamycin-resistant S. aureus (inducible macrolide-lincosamide-streptogramin B [iMLSB]) and constitutive clindamycin-resistant S. aureus (cMLSB).&#x0D; Methods: A total of 310 isolated S. aureus among 2000 different clinical samples were subjected to oxacillin (1 μg) as per the Kirby-Bauer disk diffusion method for MRSA. Clindamycin-resistant either in the form of iMLSB or cMLSB was determined through double disk diffusion method or D-test by use erythromycin (2 μg) and clindamycin (15 μg) as per the CLSI guidelines.&#x0D; Results: Out of total S. aureus, MRSA and methicillin-sensitive S. aureus (MSSA) were 78.06% and 20.64%, respectively. This study showed that iMLSB and cMLSB were 34.19% and 23.22%. Both iMLSB and cMLSB were found more among MRSA than MSSA (43.80%, 26.85% and 40.62%, 10.93%), respectively.&#x0D; Conclusion: This study helps for the characterization of different resistant strains of S. aureus along with the determination of the prevalence rate of these mutant forms causing nosocomial infections.

Streptococcus agalactiae from Ethiopian pregnant women; prevalence, associated factors and antimicrobial resistance: alarming for prophylaxis

BackgroundMaternal Streptococcus agalactiae (Group B Streptococcus, GBS) colonization rates and its antibiotic resistance patterns provide important information useful in guiding prevention strategies. There is a paucity of evidence about GBS in the Amhara National Regional State, Ethiopia.ObjectiveTo determine colonization prevalence, associated risk factors, and antibiotics resistance including inducible clindamycin resistance patterns of GBS among Ethiopian pregnant women.MethodsA prospective cross-sectional study was conducted from 1st December 2016 to 30th November 2017 at the University of Gondar Referral hospital delivery ward. Combined recto-vaginal swabs were collected from 385 pregnant women and analyzed at the University of Gondar Bacteriology Laboratory by using LIM broth and 5% defibrinated sheep blood agar culture methods. Isolates were identified by using colony morphology, gram reaction, hemolysis, and CAMP test. Antibiotic susceptibility test was done using the disc diffusion method. Double disc diffusion method was used to identify inducible clindamycin resistance isolates. Data were analyzed by SPSS version 20 software. p ≤ 0.05 was considered as statistically significant.ResultsThe overall prevalence of maternal GBS colonization was 25.5% (95% CI 21–29.5%). Experiencing meconium stained amniotic fluid (AOR = 3.018, 95% CI 1.225, 7.437), and longer duration of premature rupture of membrane (AOR = 1.897, 95% CI 1.014, 3.417) were statistically significant to maternal colonization. Furthermore, GBS resistant to 0 (8.2%), 1 (25.5%) and 3 (39.8%) or more antibiotics were identified. A D-test showed 15.2% inducible clindamycin resistant GBS. Constitutive macrolide lincosamide–streptograminB, L-, and M-phenotypes were also detected.ConclusionsMaternal GBS colonization rate in this study was higher compared to the previous reports in Ethiopia. This much prevalence and antibiotics resistance results are the clue to which attention shall be given to this bacterium during management of pregnant women and the newborns.

Molecular Study of Klebsiella Pneumoniae Virulence Genes from Patients with Hospital Acquired Sepsis.

Klebsiella pneumoniae (K. pneumoniae) is a common pathogen associated with hospital acquired infections. The presence of virulence genes and antibiotic resistance genes are known risk factors for the infection by this bacterial species. The aims of the present study were to i) investigate the coexistence of capsular genes of K. pneumoniae (rmpA and wcaG) with integron genes in clinical isolates of K. pneumoniae by polymerase chain reaction (PCR) from hospital acquired infections and to ii) correlate the presence of these genes with extended spectrum β-lactamase (ESBLs) and carbapenem-resistant phenotypes. The study included K. pneumoniae isolates from 100 patients with hospital acquired sepsis from ICUs. The isolates were subjected to antibiotic susceptibility tests by the disc diffusion method, identification of extended spectrum beta-lactamase detection (ESBLs) and carbapenemase production by specific phenotypic methods. Determination of integrons genes and capsular genes were performed by polymerase chain reaction (PCR). The most common detected virulence genes was wcaG (50%) followed by rmpA (10%). Intl1 was detected in 69% of the isolates, while intl2 and intl3 genes were not detected in any isolate. There was statistically significant association between rmpA and wcaG (p = 0.02), rmpA and intl1 (p = 0.02) and intl1 and wcaG (p = 0.0001). There was statistically significant association between wcaG, rmpA, and intl1 genes and ESBLs production as determined by double disc diffusion method (p = 0.008, p = 0.005, p = 0.05, respectively). On the other hand, only wcaG and intl1 genes had statistically significant association with carbapenemase production. The fatal outcome of the sepsis was significantly associated with the presence of wcaG, rmpA and intl1 genes (p = 0.0001, p = 0.001, p = 0.02 respectively). The study highlights a correlation between wcaG and rmpA virulence genes of K. pneumoniae with integron 1 responsible for antibiotic resistance. There is also an association between phenotypic extended spectrum beta-lactamase and carbapenemase resistance and virulence genes because the genes may be coded on the same transferable genetic elements. There was a correlation between virulence genes and outcome of infection. There is a strict need for compliance of infection control guidelines.